Chemoreception: A Consideration of Olfactory Signalling in Non-Olfactory Systems

Yuliya Makeyeva

A thesis in fulfillment of the requirements for the degree of

Doctor of Philosophy

School of Women’s and Children’s Health

Faculty of Medicine

November 2018 Thesis/Dissertation Sheet Australia's Global UNSWSYDNEY University

Surname/Family Name Makeyeva Given Name/s Yuliya Abbreviation for degree as given in the University calendar PhD Faculty Faculty of Medicine School School of Women's and Children's Health Thesis Title Chemoreception: A Consideration of Olfactory Signalling Proteins in Non­ Olfactory Systems

Abstract 350 words maximum: Chemoreception is a biological process whereby cells and/or organisms are stimulated by chemicals in their environment. The detectibn of chemicals triggers a response that can be the attraction or aversion by a single II cell or a highly integrative process that initiates a complex behaviour. Olfactory receptors (ORs) are a multigene family of molecules used to monitor extracellular chemical cues. They were originally described in the olfactory system as mediators of the but have since been observed in a number of non-olfactory tissues. OR-mediated chemoreception in the olfactory system involves a G (Gait), Ill (AC3), and OMP. ORs are difficultto study so little is known about their functions and expression profiles in these tissues. I,

Olfactory marker protein (OMP) is a highly expressed protein found in mature olfactory sensory neurons of all vertebrates, co-labels with individual ORs, and is involved in intracellular . OMP expression may indicate OR-mediated chemoreception in non-olfactory systems. Cells that are mobile are optimal candidates because they might use chemoreception to guide their movements. My thesis used OMP, Gait, and AC3 to identify chemoreception in non-olfactory cells: spermatozoa, mast cells, and Leydig cells.

The distribution of these proteins was investigated using immunohistochemistry and microscopic image analysis. In rats, expression of these proteins was documented in spermatozoa, mast cells, and Leydig cells. In humans, expression was investigated in spermatozoa in three modes of activation: control, activated, and hyper-activated. The results showed that OMP, Gait, and AC3 were co-expressed in spermatozoa, mast cells, and Leydig cells. Due to the adverse reaction of tissues to a high-fat diet, the response of mast cells, the I olfactory epithelium, and olfactory bulb was tested and found to be rapidly altered. This presence of an OR-mediated signalling pathway suggests chemoreception and guidance in diverse cells of non-olfactory tissues. A high-fat diet, even in the short term, adversely affected immunity and chemoreception. My data reveal the widespread and complex interactions among diverse systems that are mediated by chemoreception.

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iii Supervisors

Supervisor: Professor William Ledger

School of Women’s and Children’s Health

UNSW Sydney

Co-Supervisor: Professor David Ryugo

Hearing Research

Garvan Institute of Medical Research and

UNSW Sydney

Co-Supervisor: Dr Steven Leach

School of Women’s and Children’s Health

UNSW Sydney

iv Dedication

For my father Valentine, my mother Elmira, and my daughter

Kseniya—for their unflagging love and support

v Acknowledgements

I wish to thank the following people for their assistance with my PhD.

My sincere thanks go: to Professor Laura Poole-Warren for her support through my PhD despite considerable difficulties; to my primary supervisor, Professor William Ledger, for inspiring my interest in reproduction and for his support and guidance during the final stages of my PhD. He gave me the strength and motivation to complete what has been an arduous journey; to my co-supervisor, Professor David Ryugo, for allowing me to complete my PhD in his laboratory, for his encouragement and feedback. I am grateful for his gentle guidance and the freedom he gave me to pursue my aims and the confidence to keep going. I also thank him for helpful comments and discussions on my thesis; to Dr Steven Leach, my co-supervisor, for his helpful assistance in interpreting the results of my metabolic and studies and for assistance with my work at Westfield Laboratory; to Professor Margaret Morris and Dr Amy Reichelt, for their co-operation with my metabolic project and for providing access to animals for the experiments described in Chapter 6; to Dr Louise Cole (Bosch Institute) for providing technical support and assisting me with the creation of a program to image analyse OMP intensity; to my colleagues in Professor Ryugo’s lab:

• Dr Michael Muniak for providing technical support and assisting me with the creation of a program for cell counting, described in Chapter 6 and for assistance during the final phase of my thesis preparation; • Tan Pongstaporn, for assisting me with electron microscopy and with the analysis of my results; • Kiera Grierson, for always being ready to assist me with laboratory work;

vi to my colleagues from the NSW Health Pathology Andrology Laboratory at the Royal Hospital for Women:

• Chris Nicol for assisting me to conduct my human sperm study and for innovative suggestions for experiments; • Dr Harleen Kaur and Sukran Ozsoy for providing technical support for these experiments; to Dr Fei Sheng (Histology and Microscopy Unit) for her amazing work in histology and to Dr Iveta Slapetova (Biomedical Imaging Facility) for her assistance with image analysis; to Arne Muller (ZEISS Australasia) for assisting me in image analysis of human sperm; to Professor Mechyslav R Gzhegotskyi (First Vice-Rector) and Professor Antonina Yashchenko (Department of Histology, Cytology and Embryology), colleagues and mentors from Lviv National Medical University in the Ukraine, who moulded me and encouraged my development as a scientist;

To A/Professor Taras Gutor (Department of Public Health Management), from Lviv National Medical University in the Ukraine, for his mentoring in statistical analysis; to my husband Kostya and my mother-in-law Marilena for their financial and moral support and their patience—even during some difficult times; to my cousins Serge and Alex Kyrmanov for their constant encouragement throughout my studies; to Lyn McLean, my friend, editor, and advisor, for an amazing job in proofreading, for always finding time for me at her house;

To Dr John Feller whose advice and encouragement set me on track to undertake my PhD; to my friend Vlad Kanashkevich who motivated me to apply for the PhD program and supported me through those first difficult steps on this journey;

Finally, to Dr Tala Kaplinovsky, who taught me the rudiments of laboratory procedures and has been a valuable confidant.

The thesis work was directly supported by APA and Top-Up scholarships. Additional support was provided by the School of Women’s and Children’s Health and funds from Professor Ryugo’s research that included NHMRC Grant no. 1080652 and a donation from Alan and Lynne Rydge. vii Table of Contents

Thesis/Dissertation Sheet ...... ii

Originality Statement ...... iii

Supervisors ...... iv

Dedication ...... v

Acknowledgements ...... vi

Table of Contents ...... viii

List of Abbreviations ...... xii

Chapter 1: Introduction to Chemoreception ...... 1 1.1 Olfactory System ...... 3 1.1.1 Olfactory epithelium...... 3 1.1.2 Olfactory bulb ...... 14 1.1.3 Olfactory receptors, G proteins, and adenylyl cyclase in olfactory system 22 1.1.4 Olfactory signal transduction pathway ...... 32 1.1.5 ORs, G proteins, and ACs in non-olfactory tissues ...... 32 1.2 Expression and functions of olfactory marker protein (OMP) in olfactory system and non-olfactory tissues ...... 38 1.2.1 OMP in olfactory system ...... 38 1.2.2 OMP in non-olfactory tissues ...... 43 1.3 Reproductive System ...... 43 1.3.1 Structure and function of testes ...... 43 1.3.2 Structure and function of epididymis...... 50 1.3.3 Structure and function of spermatozoa...... 60 1.4 Mast cells ...... 66 1.4.1 Mast cell types ...... 69 1.4.2 Mast cell mediators ...... 71 1.4.3 Mast cell activation ...... 74 1.4.4 Receptors for mast cell activation ...... 74 1.4.5 Mast cell ...... 78 1.4.6 GPCR-mediated pathway in mast cell activation ...... 79 1.4.7 Mast cells in brain ...... 81 1.5 Hypothesis and Aims ...... 84

Chapter 2: Expression of OMP, Golf, and AC3 in olfaction ...... 86 2.1 Abstract ...... 87

viii 2.2 Introduction ...... 87 2.3 Materials and methods ...... 89 2.3.1 Animals ...... 89 2.3.2 Perfusion ...... 89 2.3.3 Histological methods ...... 89 2.3.4 Photography and image processing ...... 91 2.4 Results ...... 92 2.4.1 Expression of OMP in olfactory epithelium and olfactory bulb ...... 92

2.4.2 Expression of Golf in olfactory epithelium and olfactory bulb ...... 94 2.4.3 Expression of AC3 in olfactory epithelium and olfactory bulb ...... 95 2.5 Discussion ...... 96 2.6 Conclusion ...... 98

Chapter 3: Expression of olfactory signalling proteins in spermatozoa of humans and rats ...... 99 3.1 Abstract ...... 100 3.2 Introduction ...... 100 3.3 Expression of OMP in human spermatozoa ...... 103 3.3.1 Materials and methods ...... 103 3.3.2 Photography and image processing ...... 106 3.3.3 Results...... 108

3.4 Expression of OMP, Golf, and AC3 in epididymal spermatozoa of rats ...... 115 3.4.1 Materials and methods ...... 115 3.4.2 Photography and image processing ...... 115 3.4.3 Results...... 116 3.5 Discussion ...... 118 3.6 Conclusion ...... 121

Chapter 4: Expression of OMP, Golf, and AC3 in mast cells of rats ...... 122 4.1 Abstract ...... 123 4.2 Introduction ...... 123 4.3 Materials and methods ...... 125 4.3.1 Animals ...... 125 4.3.2 Perfusion ...... 125 4.3.3 Histological methods ...... 125 4.3.4 Photography and image processing ...... 128 4.4 Results ...... 130

4.4.1 Expression of OMP, Golf, and AC3 in mast cells of tongue ...... 131

4.4.2 Expression of OMP, Golf, and AC3 in mast cells of liver ...... 138

ix 4.4.3 Expression of OMP, Golf, and AC3 in mast cells of white adipose tissue . 144 4.5 Discussion ...... 150 4.6 Conclusion ...... 152

Chapter 5: Expression of OMP, Golf, and AC3 in Leydig cells of interstitial tissue of rat testes ...... 153 5.1 Abstract ...... 154 5.2 Introduction ...... 154 5.3 Materials and methods ...... 157 5.3.1 Animals ...... 157 5.3.2 Perfusion ...... 157 5.3.3 Histological methods ...... 157 5.3.4 Photography and image processing ...... 160 5.4 Results ...... 161 5.4.1 Mouse testes...... 161 5.4.2 Rat testes ...... 162 5.5 Discussion ...... 167 5.6 Conclusion ...... 173

Chapter 6: Adverse effects of a short-term, high-fat diet on metabolic metrics in rats ...... 174 6.1 Abstract ...... 175 6.2 Introduction ...... 175 6.3 Materials and methods ...... 177 6.3.1 Animals ...... 177 6.3.2 Animal groups and diet ...... 178 6.3.3 Experimental ante-mortem measurements ...... 180 6.3.4 Perfusion ...... 181 6.3.5 Experimental post-mortem measurements ...... 181 6.3.6 Histological methods ...... 181 6.3.7 Tissue evaluation and image analysis ...... 182 6.3.8 Photography and image processing ...... 190 6.3.9 Statistics ...... 190 6.4 Results ...... 191 6.4.1 Body weight measurements ...... 191 6.4.2 Parameters of white adipose tissue ...... 191 6.4.3 Structure of white adipose tissue and area of adipose cells ...... 193 6.4.4 Structure of liver and lipid droplet accumulations ...... 194 6.4.5 Evaluation of blood serum chemistry parameters ...... 197 6.4.6 Mast cells assessment ...... 198

x 6.4.7 Analysis of mOSNs in olfactory epithelium (OE) ...... 211 6.4.8 Analysis of juxtaglomerular (JG) cells in olfactory bulb (OB)...... 212 6.5 Discussion ...... 219 6.6 Conclusion ...... 225

Conclusions and Future Directions...... 226

References ...... 228

xi List of Abbreviations AC adenylyl cyclase AC3 adenylyl cyclase 3 AdC adipose cell AG Alexa488-DαG Alexa488-conjugated donkey anti-goat IgG Alexa488-DαR Alexa488-conjugated donkey anti-rabbit IgG Alexa594-DαG Alexa594-conjugated donkey anti-goat IgG Alexa594-DαR Alexa594-conjugated donkey anti-rabbit IgG AMP antimicrobial peptide AR acrosome reaction ATP adenosine 5'-triphosphate BBB blood-brain barrier BMMC mast cell BV blood vessel cAMP cyclicadenosine 3',5'-monophosphate CB calbindin D-28k CD cytoplasmic droplet CGRP calcitonin related peptide CNG cyclic-nucleotide gated CNS central nervous system CP connecting piece CPA3 carboxypeptidase A3 CR complement CRF corticotropin-releasing factor CRMP collapsing response mediator protein CT connective tissue CTMC connective tissue mast cell DA dopamine DAB 3,3'-diaminobenzidine DAG diacylglycerol DAPI 4',6'-diamidino-2-phenylindole DCX doublecortin DIC differential interference contrast microscopy DNA deoxyribonucleic acid EM electron microscopy

xii ENK enkephalin EOG electro-olfactogram EP endorphin EPL external plexiform layer ER ES equatorial segment ET external tufted cell FcɛR I Fc epsilon receptor I FSH follicle-stimulating GABA γ-aminobutyric acid GAD glutamic acid decarboxylase GAP43 growth associated protein 43 GBC globose basal cell GCL granule cell layer GDP guanosine 5'-diphosphate GFAP glial fibrillary acidic protein GL glomerular layer

Golf olfactory specific G protein GO gene ontology GPCR G protein-coupled receptor GTP guanosine 5'-triphosphate HA hyper-activated spermatozoa HBC horizontal basal cells HDL high-density lipoprotein HE Haematoxylin and Eosin HEPES N-[2-hydroxyethyl] piperaxine-N'-[2-ethanesulphonic acid] Hes1 hairy and enhancer of split 1 HF high-fat HMC human mast cell HR histamine receptor HSA human serum albumin ICC immunocytochemistry IF immunofluorescent histochemistry IgE immunoglobulin E IHC immunohistochemistry ImR immediate reaction IL interleukin

xiii IMC intermediate type of mast cells imOSN immature olfactory sensory neuron IFN interferon IP immunoperoxidase histochemistry

IP3 inositol 1,4,5-trisphosphate IPL internal plexiform layer ISH in situ hybridisation IT interstitial tissue IVF in vitro fertilisation JG juxtaglomerular cell LD lipid droplet LH luteinizing hormone LPR late phase reaction LPS lipopolysaccharide (endotoxin) LT M muscle MAP-2 microtubule-associated protein MAPK mitogen-activated protein Mash 1 mammalian achete-scute homologue1 MC mast cell MCL mitral cell layer MCP chemoattractant protein MIF inhibitory factor MLD medium positive LD accumulations MMC mucosal mast cell MMP matrix metalloproteinase mOSN mature olfactory sensory neuron Mrg Mas-related gene mRNA messenger ribonucleic acid NCAM neuronal cell adhesion molecule NDS normal donkey serum NGF NGS next generation sequencing NMU neuromedin U NO nitric oxide NOS nitric oxide synthases NPY Y

xiv NS normal serum ns not significant NSF N-ethylmaleimide-sensitive factor NST neuron-specific beta- type 3 NT neurotensin OB olfactory bulb OE olfactory epithelium OEC olfactory ensheathing cell OMP olfactory marker protein ON olfactory nerve ONL olfactory nerve layer OR olfactory receptor ORO Oil Red O staining OSN olfactory sensory neuron p probability value p75 low affinity neurotrophin receptor p75NTR PAF activating factor PAR2 protease-activate receptor 2 Pax paired box protein PBS phosphate buffered saline PCR polymerase chain reaction PDE phosphodiesterase PFA paraformaldehyde PG periglomerular cell

PIP2 phosphatidylinositol 4-phosphate PKA PKC protein kinase C PLC POMC pro-opiomelanocortin PV portal vein QAM Quinn's Advantage Medium rER rough endoplasmic reticulum R rat Rcf relative centrifugal force rER rough endoplasmic reticulum RMS rostral migratory stream RNA ribonucleic acid

xv RT-PCR reverse transcription PCR S-100 -binding protein SA short cell SCF stem cell factor SD standard deviation SOM somatostatin SP substance P SSE stratified squamous epithelium SUS sustentacular cell SVZ subventricular zone TB Toluidine Blue TG triglycerides TGF transforming growth factor TH tyrosine hydroxylase TLR toll-like receptors TNF tumour necrosis factor TR taste receptor TRC taste chemoreceptor cell TRH thyrotropin-releasing hormone VEGF vascular endothelial growth factor VIP vasoactive intestinal peptide VNO vomeronasal organ VW vein wall WAT white adipose tissue WB Western blot WLD weak positive LD accumulations WM white matter ZP zona pellucida

xvi

Chapter 1: Introduction to Chemoreception

1 The detection of chemicals by smell, taste, or other means is generally known as chemoreception. It is the physiological response of a sensory organ to a chemical stimulus. Chemoreception is the most ancient sensory ability, allowing organisms to react to environmental cues and to communicate with each other (Buck, 2000). The ability to detect these cues is crucial for survival of an individual and a species (Mombaerts, 2004; Keller and Vosshall, 2008). Chemoreception is a process that occurs widely in nature, from the simple chemotaxis of a motile bacterium towards food to the more complex interpretative pathways associated with an animal’s ability to smell and taste. The presence of chemoreception in unicellular organisms suggests this is an ancient process and may have played an important role in evolution. Chemoreceptors are proteins on the plasma membranes of receptor cells that detect specific molecules. Chemoreceptors that respond to chemical stimuli in the internal environment are called interceptors. Those that respond to chemical stimuli in the external environment are exteroceptors and include olfactory receptors (ORs) and taste receptors (TRs). Olfaction is the sense of smell and is mediated by ORs. They detect and distinguish an enormous diversity of volatile chemicals (odorants) with a large variety of structures (Beets, 1970; Shepherd, 1988; Malnic et al., 1999). They are also responsible for sensing the , chemicals that act on conspecifics, to evoke physiological or behavioural responses (Wilson, 1963; Shepherd, 1988). ORs are located in the nasal cavity, in the main olfactory epithelium (OE) and in the vomeronasal organ (VNO).

Taste—the sensory component of the gustation system—provides a sense of flavour perceived in the throat and mouth upon contact with a substance. This sensory system uses taste chemoreceptor cells (TRCs) present in taste buds in taste papillae of the tongue, soft palate, epiglottis, and mucous membranes of the larynx and pharynx. TRs respond to chemicals contained in food or drink. The tongue is an important organ associated with the sense of taste because its chemoreceptors detect chemical compounds dissolved in saliva. These chemical compounds can either trigger an appetitive response for nutrients or a defensive response against toxins, depending on which receptors are activated. These are five commonly-recognised, “basic” taste modalities (Breslin, 2013):

§ sweet is pleasant and indicates carbohydrates that provide energy; § umami or savoury indicates proteins such as L-glutamate and other L-amino acids; § salty indicates sodium and other salts essential for ion balance and blood circulation; § bitter has a sharp pungent or astringent quality and often signals toxic food;

2 § sour indicates dietary acids and guards against spoiled food. § A sixth taste modality has been proposed: the ability to taste fat, mediated by G protein-coupled receptors (GPCRs) (Galindo et al., 2011; Chevrot et al., 2014).

Sweet, umami, and bitter tastes are also mediated by GPCRs; sweet and umami by T1Rs and bitter by T2Rs. The senses of olfaction and taste are closely related. The ability to taste food and drink, often associated solely with taste, is a combination of the gustatory and olfactory processes.

Chemoreception leading to movement of a cell or organism is known as chemotaxis. Chemotaxis occurs when a motile bacterium follows a chemoattractant towards a food source or moves away from the presence of a repelling (repellent) chemical. It also directs chemically-directed cell migration during vertebrate development, sperm guidance towards the oocyte, microglia convergence on degenerative debris, and immune cell mobilisation on invading bacteria. Chemotaxis also occurs when a cell simply changes its shape. For example, non-motile yeast cells mate by elongating towards each other when they detect complementary pheromones. Chemotaxis also occurs in chemically-directed cell migration during mammalian development, sperm guidance towards the oocyte, microglia convergence on degenerative debris, and immune cell mobilisation on invading bacteria.

Chemoreception occurs in a number of diverse systems. My thesis has studied some of the features of chemoreception, and so it becomes important to introduce the reader to the systems involved and to reveal some of the basic cellular units.

1.1 Olfactory System

1.1.1 Olfactory epithelium The olfactory system is one of the sensory systems—including vision, hearing, touch, and taste—and is involved in the perception of smell (Fig. 1.1). It represents one of the oldest sensory modalities in vertebrates serving as a chemical sensor to detect food, predators, and potential mates. Activation of olfaction occurs when odorant molecules come into contact with specialised receptors.

3

Figure 1.1: Odorant detection and pathway for transmission to olfactory bulb. Humans can perceive many thousands of odorous molecules, termed odorants. Perception of different odours is determined by specific chemical structure of odorant. In mammals, ability to discriminate odours is connected with several anatomical sites. Olfactory epithelium of nasal cavity contains olfactory sensory neurons (OSNs), which detect external odours. Olfactory nerve transmits information from OSNs to olfactory bulb, after which information is relayed via lateral olfactory tract to olfactory cortex for processing and perceiving odour. Odours are detected through two pathways. First pathway is orthonasal pathway, which involves odours that are sniffed through nose: odours enter nasal passages and are detected by receptors there. Second pathway is retronasal pathway, which connects top of throat to nasal cavity and detects aromas from foods. These odours travel through throat to nasal cavity, where they are detected by receptors in nose. Adapted from Dorling Kindersley/Getty Images.

§ Cell types in olfactory mucosa The olfactory sense organ is a mucosal membrane that consists of two layers: the olfactory epithelium (OE) and a subepithelial lamina propria of connective tissue, blood vessels, and glands. The OE is a pseudostratified structure composed of cell types that can be characterised by their morphology, immunoreactivity, and location within the epithelium. These cells types are: non-sensory sustentacular supporting (SUS) cells, olfactory sensory neurons (OSNs), and two types of basal cells: horizontal basal cells (HBCs) and globose basal cells (GBCs) (Menco and Morrison, 2003) (Figs. 1.2 & 1.5). The OE also contains leucocytes (Suzuki et al., 1995) and other cells that have microvilli

4

Figure 1.2: Cross section of rat olfactory epithelium. Olfactory epithelium (OE) is composed of many cell types. Sustentacular cell bodies are located apically and possess microvilli with narrow descending process that attached on epithelial base. OE contains two types of olfactory sensory neurons (OSNs)—mature olfactory neurons and immature olfactory neurons— located in middle compartment of OE. They possess cilia that sample odorants from lumen of nasal cavity. Basal cells are located at bottom of OE. They are a heterogeneous population that house active and reserve stem cells—globose (basal) cells and horizontal (basal) cells. Lamina propria contains olfactory ; olfactory ensheathing cells (OECs) and Bowman’s gland cells, which extend to apical layer of OE. Left panel: Y. Makeyeva, present work; right panel: adapted from Milho et al. (2012).

(Moran et al., 1982b; Menco, 1997; Pixley et al., 1997). The lamina propria mainly contains olfactory axons, olfactory ensheathing cells (OECs), Bowman’s glands, blood vessels, and connective tissue (Menco and Morrison, 2003) (Fig. 1.2).

§ Olfactory sensory neurons (OSNs) OSNs comprise roughly 80% of the entire cellular population of the OE. Their cell bodies span the middle compartment of the OE (Fig. 1.2). OSNs are truly bipolar neurons

5

Figure 1.3: Morphology of olfactory sensory neuron. Schematic drawing (left panel) and immunoflourescent image (right panel) show bipolar morphology of olfactory sensory neuron (OSN) (left: violet; right: green). Single thick dendrite projects from apical pole of cell body and ends in dendritic knob that gives rise to cilia (right: yellow). Cilia contain olfactory receptors that interact with odorants (left: red dots). Unbranched axon projects from basal pole of cell body to lamina propria (top of drawing). Adapted from Coren (2003).

and have three major components: an apical dendrite, a cell body (soma), and a single axon (Fig. 1.3).

§ Structure of OSNs A single dendrite arises from the apical pole of the cell body. On its way to the mucosal surface of the OE, this dendrite weaves around the cell bodies of neighbouring OSNs, other dendrites, and supporting cells (Fig. 1.3). The dendrite is thickest towards the cell body. It contains a Golgi body, smooth and rough endoplasmic reticulum (rER), mitochondria, microtubules, and vesicles. The length of the dendrites varies from short to almost the entire depth of the OE, depending on the position of the OSN.

The dendrite possesses a thickened, club-like ending, which extends to the epithelial surface and is known as the olfactory dendritic knob. This olfactory dendritic knob contains basal bodies, which produce non-motile cilia (Fig. 1.3). An individual OSN can project up to 50 olfactory cilia. These cilia project from dendritic knob perpendicularly into the mucus layer and sometimes overlap with the cilia of adjacent neurons. Olfactory cilia of humans and most other mammals measure approximately 50 µm in length. However, in non-mammalian vertebrates, the cilia can measure up to 200 µm.

6 The plasma membrane of olfactory cilia contains numerous intramembranous particles, which may represent protein entities important in olfaction. Immunochistochemical (IHC) studies, employing light microscopy and thin-section transmission electron microscopy (EM), showed that olfactory cilia contain ORs and other vital components of the olfactory transduction pathway, involving Gs type G proteins. These proteins include Gsa, Golfa (Golf), adenylyl cyclase III (AC3), and olfactory cyclic-nucleotide gated (CNG) channels. The majority of these proteins are mostly localised in the distal segments of olfactory cilia.

The dendrite joins the cell body (soma) beneath the epithelial surface. The cell body of an OSN houses a nucleus and , which contain all the machinery associated with intense macromolecular synthesis. At the ultrastructural level, the OSN appears to be a rather heterogeneous population of cells based on nuclear and cytoplasmic characteristics. The OSNs located close to the basal elements of OE have nuclei with clear chromatin patterns, and electro-lucent cytoplasm containing free ribosomes, Golgi profiles, and sparse rER.

The OSNs located in the middle compartment of the OE have irregularly rounded nuclei with clumped chromatin. Their cytoplasm contains extensive Golgi apparati located predominantly above the distal poles of the nuclei. The rER of these OSNs is arranged in orderly stacks and ribosomes are scattered in the cytoplasm. OSNs located in the upper portion of the middle compartment feature electron dense cytoplasm, swollen Golgi apparati, and rER. They also contain dense, membrane-bound bodies with the characteristics of lipofuscin granules (Graziadei and Graziadei, 1979; Moran et al., 1982b).

The olfactory axon originates in the basal pole of the OSN cell body and transmits information about odorant intensity and quality to the brain (Fig. 1.3). Olfactory axons are unmyelinated and unbranched. They are 0.1 – 0.7 µm in diameter and are among the thinnest fibres in the central nervous system (CNS). Axons project into the lower region of the OE and are surrounded by SUS cells. Olfactory axons then pass through the basal lamina into the lamina propria (Fig. 1.5). Inside the lamina propria, they converge into axon bundles called fila olfactoria, which collectively comprise the olfactory nerve (cranial nerve I). The olfactory nerve passes through the cribriform plate of the ethmoid bone where it projects to the olfactory bulb (OB), from which information is transmitted to the brain (Fig. 1.4).

OSNs are identified immunologically by a number of . These include

PGP9.5, putative ORs, Golf, and AC3 (Rogers et al., 1987; Jones and Reed, 1989;

7

Figure 1.4: Lateral view of frontal skull locating olfactory nerve in nose. Each OSN projects an unbranched axon. Inside lamina propria, axons converge into axon bundles called fila olfactoria, which collectively comprise olfactory nerve. Olfactory bulb receives olfactory signals, processes information and transmits it via olfactory tract to cerebral cortex. Cortex refines this information to discriminate thousands of different odours.

Bakalyar and Reed, 1990; Buck and Axel, 1991; Holbrook et al., 1995). Similar to other neurons, OSNs express NCAM and NST. NCAM and NST label the cell bodies, dendrites and axons of all neurons of the OE (Calof and Chikaraishi, 1989; Calof et al., 1991; Roskams et al., 1998) (Table 1.1).

Because of the constant turnover of sensory neurons in the OE, there are two stages of OSNs: mature OSNs (mOSNs) and immature OSNs (imOSNs) (Figs. 1.2 & 1.5). mOSNs are located more superficially throughout the OE. Their dendrites protrude apically and terminate in a knob from which cilia project into the nasal cavity. The axons pass through the basal lamina to make synaptic contact within the OB (Figs. 1.2 & 1.5). mOSNs are characterised by the expression of olfactory marker protein (OMP). OMP staining is observed in cell bodies of OSNs, their dendrites including vesicles and throughout most of the length of the olfactory cilia, and their axons (Farbman and Margolis, 1980; Baker et al., 1989) (Table 1.1 & Fig. 1.13).

imOSNs are located deep in the OE above the basal cells near the basal lamina and comprise more or less one single cell row (Fig. 1.2). imOSNs usually have dendrites without cilia and their axons do not make synaptic contacts within the OB (Fig. 1.5). imOSNs can be identified by GAP43, also known as B-50. GAP43 staining is observed

8

Figure 1.5: Diagram of cell types and position in olfactory epithelium. Cell morphology and position are used to identify cell types in olfactory epithelium (OE) in vivo. The Bowman’s gland spans OE from its basal to its apical layer. There are two types of basal cells. Horizontal basal cells (HBCs) are flattened and sit on basal lamina. Globose basal cells (GBCs) are globular and are positioned between HBCs and olfactory sensory neurons (OSNs). OSNs span middle compartment of OE. Mature OSNs (mOSNs) are located apically throughout OE, their dendrites protruding apically and terminating in knob, projecting cilia into nasal cavity. Axons of mOSNs pass through basal lamina. Immature OSNs (imOSNs) are located above basal cells near basal lamina. SUS cell extends from basal lamina to epithelial surface. Adapted from Farbman (1992) and Schwob (2002).

in apical dendrites, cell bodies, axons and axon bundles of OSNs (Gispen et al., 1989) (Table 1.1).

§ Sustentacular supporting (SUS) cells These non-neuronal cells are tall, columnar cells extending from the basal lamina to the epithelial surface (Figs. 1.2 & 1.5). They are goblet shaped and taper towards the base where their foot-like projections attach to the basal lamina. SUS cells are closely associated with OSNs (Breipohl et al., 1974). They partially surround perikarya and dendrites of sensory neurons and extend to the epithelial surface of OE. In the lower epithelial region, SUS cells form sleeve-like extensions that surround exiting OSN axons.

9 Cell Type Marker References

Horizontal basal cells Keratin 5 and 14 Calof and Chikaraishi (1989) Pixley (1992a)

Globose basal cells GBC-1 Goldstein and Schwob (1996)

Neuronal progenitor cell Mash1 Gordon et al. (1995) Cau et al. (1997)

Immediate neuronal precursor Ngn1 Cau et al. (1997)

Olfactory sensory neurons NCAM Calof and Chikaraishi (1989) NST Roskams et al. (1998)

Immature GAP 43 Gispen et al. (1989)

Mature OMP Farbman and Margolis (1980)

Supporting/Sustentacular SUS-1 Hempstead and Morgan (1983) SUS-4 Goldstein and Schwob (1996) Keratin 8 and 18 Holbrook et al. (1995) Sox2, Pax6 Guo et al. (2010)

Olfactory ensheathing cells GFAP Barber and Dahl (1987) S 100 Pixley (1992b) p75 Au and Roskams (2003)

Bowman's gland SUS-1 Hempstead and Morgan (1983) SUS-4 Goldstein and Schwob (1996) IF4 Pixley et al. (1997)

Table 1.1: Summary of olfactory cell types and their corresponding markers.

The apical part of the SUS cell is covered with microvilli. These microvilli extend into and terminate at the mucosal surface, where they intermingle with the olfactory cilia (Seifert, 1970; Naessen, 1971; Bannister and Dodson, 1992). The apical cytoplasm of a SUS cell contains many organelles (mitochondria, free ribosomes, Golgi apparati, and vesicles), indicative of their secretory function. Cytoplasmic vesicles fuse with apical SUS cell surface membranes, suggesting that these cells release materials into and/or absorb materials from the mucus.

10 SUS cells are highly enriched for cytochrome p450s and glutathione-S- . These proteins are involved in metabolising foreign compounds, suggesting a role in detoxifying tissue from harmful compounds (Yu et al., 2005). SUS may also be involved in removing debris from the OE, primarily when neurons turnover and act as phagocytes (Suzuki et al., 1996). SUS cells are derived from non-nervous ectoderm of the olfactory placode. These cells divide slowly to renew their own population during the mammal’s lifetime.

SUS cells functionally resemble glial cells. However, unlike glial cells, they do not express GFAP, distinguishing them from glial ensheathing cells (Vollrath et al., 1985; Ophir and Lancet, 1988).

SUS cells express SUS-1 and SUS-4 markers, although the nature of these antigens has not been clarified (Hempstead and Morgan, 1983; Goldstein and Schwob, 1996). They also express cytochrome p450 isoforms, Sox, Pax6, Hes1, K 8/18 and cell adhesion protein E-cadherin (E-Cad). Sox2 and Pax6 labels the nuclei of SUS cells densely (Guo et al., 2010). Hes1 labels nuclei of the SUS cells at the apical layer of the OE in humans, rats and mice K 8/18 and cell adhesion protein E-Cad label the basal region of SUS cells (Holbrook et al., 1995) (Table 1.1).

§ Basal cells There are two types of basal cells: horizontal basal cells (HBCs) and globose basal cells (GBCs) (Graziadei and Graziadei, 1979; Vollrath et al., 1985; Yamagishi et al., 1989). Both are roughly 4 – 7 µm in diameter and have a round, centrally-located nucleus (Figs. 1.2 & 1.5). Morphologically, the HBCs are flattened and sit on the basal lamina, whereas the GBCs are more globular in shape and positioned between the OSNs and the HBCs.

Horizontal basal cells (HBCs) are attached as a single-cell layer directly to the basal lamina by hemi-desmosomes (Gutièrrez-Mecinas et al., 2005) and span the entire length of the basal lamina (Figs. 1.2 & 1.5). A fate-mapping study of mice with lesions caused by olfactotoxic reagents showed that HBCs regenerated neuronal and non- neuronal differentiated cells. In response to extensive lesions, when the GBC population, and probably other mature components, were compromised by olfactotoxic reagents, HBCs proliferated and produced multiple differentiated neuronal and glial progeny, which fully reconstituted the OE. However, in intact mice, HBCs remained largely inactive and their behaviour was not affected by olfactory bulbectomy, which selectively depleted mOSNs. These results suggest that only GBC progenitors were adequate for OE regeneration under normal neuronal turnover conditions as well as after selective

11 neuronal loss, and HBCs only became active in regenerating the OE in the case of extensive damage (Carter et al., 2004; Leung et al., 2007). On the other hand, a mouse study, using a constitutively active Cre system showed that HBCs, even during normal neuronal turnover, actively generated a significant number of OSNs, SUS cells, and Bowman’s gland cells throughout the animals’ adult life. An initial wave of HBC-derived neurogenesis was observed after birth and a second wave at four months of age. HBCs, upon selective depletion of mOSNs caused by olfactory bulbectomy, substantially increased the number of HBC-derived neuronal and non-neuronal cells. This observation suggests that HBCs may contain multipotent progenitor cells, which are active during normal neuronal turnover in the OE and at the site of injury (Iwai et al., 2008). HBCs express a range of specific markers (Table 1.1).

Globose basal cells (GBCs) are round and have a very large nucleus compared to their cytoplasm. GBCs are the major proliferating population in the OE (Caggiano et al., 1994) (Figs. 1.2 & 1.5). It has been suggested that OE stem cells reside among the GBC population (Huard et al., 1998; Jang et al., 2003) or GBCs themselves may be stem cells (Chen et al., 2004). GBCs are a functionally heterogeneous cell population (Huard and Schwob, 1995; Goldstein et al., 1998; Chen et al., 2004; Manglapus et al., 2004; Jang et al., 2007). In the OE they are identified as neuronal precursors of OSNs (Graziadei and Graziadei, 1979; Calof and Chikaraishi, 1989; Caggiano et al., 1994; Schwob et al., 1994; Huard et al., 1998), subdivided into transit-amplifying progenitors: Mash 1+ and immediate neuronal progenitor (INP), which later become postmiotic OSNs (DeHamer et al., 1994; Gordon et al., 1995; Calof et al., 2002; Guo et al., 2010). For the most part, GBCs are identified by their non-immunoreactivity to keratins and , distinguishing them from other cells of the OE. A GBC-series of antibodies, including the antibody, GBC-1, whose antigens are of unknown function, can be used for GBC identification (Goldstein and Schwob, 1996) (Table 1.1).

GBCs actively regenerate OSNs in the OE of normal and bulbectomised animals, according to lineage studies in vivo (Calof and Chikaraishi, 1989; Caggiano et al., 1994; Schwob et al., 1994). However, some components of the GBCs within the OE of normal animals are bipotential and regenerate both OSNs and SUS cells when the need arises (Huard et al., 1998; Chen et al., 2004). Retroviral lineage analyses, including some from models of injury-induced OE regeneration, indicate that GBCs are multipotent progenitors and give rise to all neuronal and non-neuronal cells in the OE, including themselves (Caggiano et al., 1994; Schwob et al., 1994; Schwob et al., 1995; Huard et al., 1998; Chen et al., 2004).

12 § Microvillous cells There are at least five cell types that line the nasal cavity with microvilli. The first type is the brush cell, which has more rigid microvilli than SUS cells and occurs in olfactory and respiratory epithelia (Jeffery and Reid, 1975; Jourdan, 1975; Menco and Jackson, 1997). The second type is the infrequent microvillous cell. The microvilli of these cells have a more uniform diameter and length than those of SUS cells and are positioned parallel to each other. The cytoplasm of these cells tends to be more electron- opaque (Agasandyan, 1990; Carr et al., 1991; Johnson et al., 1993) or more electron- lucent than the cytoplasm of the SUS cells that surrounds them, depending on the fixation method (Menco, 1992, 1994; Pixley et al., 1997). The third type of microvillous cells have microvilli, which are more compacted than those of the infrequent microvillous cell type and are more electron-lucent than supporting cells in conventionally fixed tissue (Miller et al., 1995). The fourth type of microvillous cells are found in humans and represent approximately 10% of their neuronal population (Moran et al., 1982b; Moran et al., 1982a; Morrison and Costanzo, 1990). These cells are electron-lucent, flask- shaped and possess short microvilli and a sub-nuclear, pole-like process (Morrison and Costanzo, 1990). The fifth type of microvillous cells look similar to apex hair cells of the ear and these are only present during development. These cells are very sparse and are zonally distributed (Menco and Jackson, 1997).

§ Lamina propria The olfactory mucosa resides on the lamina propria that contains axon bundles, blood vessels, connective tissue, and Bowman’s glands (Figs. 1.2 & 1.5).

Axons of OSNs fasciculate to form small intraepithelial bundles, surrounded by a line of OECs (Doucette, 1991; Bartolomei and Greer, 2000). The axons leave the OE, cross the basal lamina, and enter the lamina propria to bundle together into fascicles and nerves. These OE fascicles pass through the 15 to 20 foramina of each cribriform plate to synapse within the OB (Fig. 1.4) (Hadley et al., 2004).

Olfactory ensheathing cells (OECs) are heterogeneous, resembling astrocytes and Schwann cells. They are located in the lamina propria between the OE and OB (Au and Roskams, 2003) (Fig. 1.2). Instead of surrounding individual axon fibres, they project cytoplasm that encloses bundles between 5 and 200 olfactory axons (Doucette, 1993). OECs bind the axons together and form the unique packaging of axons. This connection allows fibres to interact for metabolism, ionic flux, and transduction of electrical currents (Gesteland, 1986; Eng and Kocsis, 1987; Zhang et al., 2000). OECs may also be involved in organising the developing axons, supporting re-innervation of the OB in the event of traumatic injury. OECs can be identified by GFAP (Barber and 13 Dahl, 1987), S-100 (Pixley, 1992), and p75 (Au and Roskams, 2003) (Fig. 1.2 & Table 1.1).

Bowman’s glands are present in the OE of all vertebrates except fish. These glands are found throughout the OE and extend from basal to apical layers (Figs. 1.2 & 1.5). The glandular acini of Bowman’s glands are surrounded by myoepithelial cells, which contain actin filaments. Myoepithelial cells squeeze secretory cells and thereby help to move their secretory products toward the mucous surface of the OE. Mucous creates the microenvironment around the olfactory cilia. It prevents the epithelial surface from becoming dry (Farbman and Buchholz, 1996), protects from dust and other debris (Polyzonis et al., 1979), clears the cilia, and facilitates access for new odoriferous substances (Frisch, 1967; Ohno et al., 1981; Bhandawat et al., 2005). Bowman’s gland cells may share a common lineage with SUS cells since they are both immunoreactive to SUS-1 (Hempstead and Morgan, 1983), SUS-4, and IF4, the latter which specifically stains microvilli of SUS cells and Bowman’s gland cells (Goldstein and Schwob, 1996; Pixley et al., 1997). They only express Pax6 and lack Sox2 (Guo et al., 2010) (Table 1.1).

1.1.2 Olfactory bulb In most vertebrates, the OBs are a pair of ovoid-shaped structures located in the rostral part of the brain dedicated to odour information processing. The OB is divided into multiple concentric layers comprised of specific cell types: olfactory nerve layer (ONL); glomerular layer (GL), containing juxtaglomerular cells (JG); external plexiform layer (EPL), containing tufted cells; mitral cell layer (MCL), containing mitral cells; internal plexiform layer (IPL); granule cell layer (GCL), containing granule cells; and white matter (WM) (Price and Powell, 1970a, b; Pinching and Powell, 1971b; Nagayama et al., 2014) (Figs. 1.6 – 1.7).

Olfactory glomeruli are complex multicellular spherical structures located near the surface of the OB. Glomeruli vary in diameter from 30 to 200 μm and range in number from 1,000-2,400, depending on the species (Pinching and Powell, 1971b; Kratskin and Belluzzi, 2003). They are arranged in one or several rows, forming the GL of the OB (Fig. 1.6). Glomeruli are the functional units for processing odour information and the initial sites of synaptic integration in the olfactory signal transduction pathway (Leveteau and MacLeod, 1966; Shepherd, 1994). Each glomerulus can be divided into two zones: the olfactory nerve (ON) zone and non-ON zone. The ON zone comprises pre-terminals and terminals of OSN axons. The non-ON zone contains olfactory bulbar neuron processes (Kosaka and Kosaka, 2005; Baltanás et al., 2007) (Fig. 1.7).

14

Figure 1.6: Cross section of left and right olfactory bulb. Image shows seven layers of olfactory bulb (OB): olfactory nerve layer, located outside OB, contains unmyelinated afferent axons of olfactory sensory neurons; glomerular layer contains glomeruli; external plexiform layer; mitral cell layer; thin internal plexiform layer; large and cell-dense granular layer; and white matter. Adapted from Ng et al. (2005).

Glomeruli are one of the structures in the nervous system with the highest density of synapses and the first synapse made by OSN axons occurs in glomeruli (Fig. 1.7). Each glomerulus can be divided into two zones: the olfactory nerve (ON) zone and non- ON zone (Fig. 1.8). The ON zone comprises pre-terminals and terminals of OSN axons, which establish excitatory synapses with the dendrites of both interneurons and projection neurons. The non-ON zone comprises dendritic processes of interneurons that establish inhibitory synapses with the mitral cells, tufted cells, and PG interneurons (Kosaka and Kosaka, 2005; Baltanás et al., 2007).

1.1.2.1 Cellular components of glomerular layer Neurons in the GL are morphologically heterogeneous. There are three types of JG neurons: periglomerular (PG) cells, short axon (SA) cells and external tufted (ET) cells (Pinching and Powell, 1971b). These cells can be distinguished by their morphology, intrinsic and synaptic properties and physiological characteristics (Price and Powell, 1970a; Pinching and Powell, 1971b, a, 1972b, a). JG cells form at least two major circuits: the intraglomerular circuit, consisting of ET cells and PG cells, and an interglomerular circuit, comprised of SA cells (Kiyokage et al., 2010). However, when the morphological type of neurons in the GL is not clear, they may be referred to as JG cells.

15

Figure 1.7: Schematic drawing of olfactory bulb, showing neuronal layers, major cell types, and basic neuronal circuitry. Olfactory sensory neuron (OSN) axons from olfactory epithelium form olfactory nerve layer on surface of olfactory bulb (OB). These projections terminate in olfactory glomeruli, located in glomerular layer of OB. Glomerulus is globular structure of neuropil, comprised of OSN (green) axons and dendrites of mitral and tufted cells. Small interneurons (red), juxtaglomerular (JG) cells, are located adjacent to glomerulus. Glomerular layer receives input from other areas of CNS through centrifugal afferents (grey) that use wide variety and neuromodulators. Output of glomerular layer is mediated by efferent neurons (violet) of OB and forms central projections. External plexiform layer is formed primarily by neuropil and contains tufted cells and other interneurons (red). Thin mitral cell layer contains mitral cells. Large and cell dense granular layer contains many small interneurons such as granule cells. Adapted from Simpson and Sweazey (2006).

Most JG cells are interneurons because they do not innervate brain regions outside the OB (Nagayama et al., 2014) (Fig. 1.8).

§ Periglomerular cells (PG cells) PG cells are the most abundant type of neurons in the GL (Parrish‐Aungst et al., 2007). Of the three types of JG cells, PG cells have the smallest cell body,

16 approximately 5-10 µm in diameter. PG cells generally project their dendrites to a single glomerulus but, in some cases, project them into 2 glomeruli (Kiyokage et al., 2010). PG cells generally project a single axon, whose length can vary (Pinching and Powell, 1971b), but axonless subtypes may also exist (Kosaka and Kosaka, 2011). Axons can connect 5 or 6 glomeruli (Pinching and Powell, 1971b) and terminate in the interglomerular space. PG cells can be divided into diverse subtypes according to their synaptic organisation, neuronal transmitters and molecular markers (Kosaka et al., 1998; Kosaka and Kosaka, 2007; Parrish‐Aungst et al., 2007; Whitman and Greer, 2007; Kiyokage et al., 2010; Nagayama et al., 2014). Based on synaptic organisation, there are two types of PG cells: Type 1 PG cells and Type 2 PG cells. Type 1 PG cells extend their dendrites into both the ON zone and the non-ON zone. They receive synaptic inputs from OSNs and neurons of the OB. Type 2 PG cells extend their dendrites only to the non-ON zone, suggesting that they do not receive synaptic inputs from OSNs (Kosaka et al., 1998) (Fig. 1.8). PG cells can be categorised according to their neuronal transmitters. The main markers are tyrosine hydroxylase (TH) and two isoforms of glutamic acid decarboxylase (GAD): GAD65 and GAD67 (Kosaka et al., 1998). Expression of GAD65 and GAD67 in PG cells implies these cells function as GABAergic neurons (Parrish‐Aungst et al., 2007; Whitman and Greer, 2007). Expression of TH in PG cells implies these cells function as dopamine- (DA) expressing neurons. TH-positive (TH(+)) cells account for approximately 10% of all JG cells. Most, and perhaps all, TH(+) PG cells are also positive for GAD67. Just a small percentage is positive for both GAD65 and GAD67, or possibly for GAD65 alone. However, recent studies revealed that some TH(+) neurons are morphologically similar to SA cells (Kiyokage et al., 2010; Kosaka and Kosaka, 2011; Nagayama et al., 2014). PG cells can also be categorised according to molecular markers of their cytoplasm (Kosaka et al., 1998; Panzanelli et al., 2007; Parrish‐Aungst et al., 2007). These cells express TH, calretinin or calbindin (CB). TH(+) cells are Type 1 PG cells (Kosaka and Kosaka, 2005; Kosaka and Kosaka, 2007). CB(+) and calretinin(+) cells are Type 2 PG cells (Kosaka and Kosaka, 2005; Kosaka and Kosaka, 2007). Nearly all TH(+), CB(+) and calretinin(+) PG cells also express GAD (Kosaka and Kosaka, 2007; Sawada et al., 2011). TH, CB or calretinin co-express with both GAD65 and GAD67 and, therefore, are not specifically associated with any one GAD isoform (Nagayama et al., 2014). The molecular diversity of PG cells is also shown by the expression neurochemical markers such as neurocalcin, parvalbumin and GABAA receptor α5 subunit (Panzanelli et al., 2007; Parrish‐Aungst et al., 2007; Whitman and Greer, 2007). However, there is no evidence that these markers co-express with TH, CB or calretinin, so they may be distinct subtypes (Nagayama et al., 2014). The great

17

Figure 1.8: Schematic drawing of cellular components of glomerular layer Juxtaglomerular neurons of glomerular layer (GL) consist of three morphologically distinct cell types: periglomerular cells (PG cells; blue), short axon cells (SA cells; purple) and external tufted cells (ET cells; red). Two subtypes of PG cells, based on synaptic connections, are Type 1 PG cells and Type 2 PG cells. Type 1 PG cells receive synaptic inputs from olfactory sensory neurons (OSNs; green) and neurons of olfactory bulb. Type 2 PG cells only receive inputs from neurons of olfactory bulb. There are two subtypes of SA cells: classic SA cells and SA cells that are positive for tyrosine hydroxylase (TH). SA cells have axon and dendrites. Classic SA cells have axon that extends into single glomerulus or 2 glomeruli and its dendrites avoid glomeruli. TH-positive (TH(+)) SA cells have axon that connects to tens of glomeruli. Subtypes of ET cells are determined by morphology: ET cells without (w/o) dendrites and ET cells with (w/) secondary dendrites. Abbreviations: ONL, olfactory nerve layer; EPL, external plexiform layer. Adapted from Nagayama et al. (2014).

diversity of PG cells makes it difficult to reliably estimate the total number of PG cells or the proportion of their subtypes. It is likely that PG cells will be categorised into further subtypes in the future (Nagayama et al., 2014).

§ Short axon cells (SA cells) There are two subtypes of SA cells: classic SA cells and TH(+)GAD(+) SA cells (Pinching and Powell, 1971b; Kiyokage et al., 2010; Kosaka and Kosaka, 2011). Both types of SA cells have larger cell bodies (8-12 µm in diameter) than PG cells and have axons and dendrites. There are morphological differences between the two subtypes. A TH(+)GAD(+) SA cell has an axon that extends for approximately 1 mm and its dendrites contact up to 50 glomeruli. A classic SA cell has an axon that extends into a single

18 glomerulus or into 2 glomeruli and its dendrites avoid glomeruli (Nagayama et al., 2014) (Fig. 1.8).

§ External tufted cells (ET cells) Of the three types of JG cells, the ET cells have the largest cell body, approximately 10-15 µm in diameter (Pinching and Powell, 1971b). The primary dendrites of ET cells generally project to a single glomerulus but a small subpopulation of ET cells project their dendrites into 2 glomeruli (Ennis and Hayar, 2008). There are two morphologically distinct subtypes of ET cells: those without secondary dendrites, called non-basal-dendrite-bearing ET cells and those with secondary dendrites, called basal-dendrite-bearing ET cells (Macrides and Schneider, 1982; Schoenfeld et al., 1985). Basal-dendrite-bearing ET cells are sometimes referred to as superficial tufted cells (Ezeh et al., 1993; Aungst et al., 2003; Kiyokage et al., 2010) (Fig. 1.8). ET cells can also be categorised according to their neurochemical markers. While previously all ET cells were considered to be glutamatergic, new subtypes expressing vesicular transporter (VGLUT) 3, have been identified and called VGLUT(+) ET cells (Tatti et al., 2014). Two other subtypes of ET cells are categorised by virtue of their release of the peptide cholecystokinin and vasopressin. Cholecystokinin is released by superficial tufted cells that are involved in intrabulbar connections (Liu and Shipley, 1994). Vasopressin is released by some ET cells that are involved in processing olfactory signals related to social recognition (Tobin et al., 2010).

1.1.2.2 Adult subventricular zone and olfactory bulb neurogenesis: migration of Type-A cells In addition to its function in signal processing, the olfactory pathway is of general interest because of the presence of neurogenesis. This was found firstly for the OSNs, which arise from basal cells in the OE (Graziadei and Graziadei, 1985). Continual generation of new neurons was then found in the subventricular zone (SVZ), with migration of neuron precursors through the rostral migratory stream (RMS) into the OB (Luskin, 1993).

In the adult mammalian brain, new neurons are added to the OB throughout life. In rodents, the adult germinal region for OB neurogenesis is the SVZ, a layer of cells found along the walls of the brain lateral ventricles (Alvarez-Buylla et al., 2001). Neuroblasts from the SVZ migrate to the OB where they disperse radially and differentiate into interneurons. Most of these new OB neurons integrate into local functional circuits (Petreanu and Alvarez-Buylla, 2002; Belluzzi et al., 2003; Carleton et al., 2003).

19 Of the four cell types of SVZ in the adult mouse, type-A cells are of most interest in SVZ-OB neurogenesis because of their ability to migrate (Lois et al., 1996; Doetsch et al., 1997). From the SVZ, where they are generated, chains of type-A cells migrate over considerable distances at high speeds (20 – 40 mm/hrs) interspersed with low speeds or stationary periods through the RMS, a restricted pathway, that leads type-A cells into the OB (Lois and Alvarez-Buylla, 1994; Luskin and Boone, 1994; Doetsch and Alvarez-Buylla, 1996; Doetsch et al., 1997; Wichterle et al., 1997; Bovetti et al., 2007a). The migration of these cells is remarkable, not only for the distance that they travel, but also for the highly directed nature of their migration (Luskin, 1993; Lois and Alvarez- Buylla, 1994). This migration, known as tangential migration, occurs parallel to the surface of the brain as well as glia- and axon-independent (Kishi et al., 1990). During their migration, Type-A cells appear to be guided and do not deviate from the restricted path into the OB. During their migration, chains of type-A cells are completely surrounded by glia cells and their processes (“glial tubes”) (Jankovski and Sotelo, 1996; Lois et al., 1996; Peretto et al., 1997). This glial tube, found in adult mice, not only protects type-A cells, but is also a source of migration-inducing activity (Mason et al., 2001), which in vitro, increases the number of migrating cells and the distance they migrate (Hu, 1999; Wu et al., 1999). Doublecortin (DCX), a microtubule-associated protein important for neuronal migration in the embryo, and collapsing response mediator protein (CRMP)-4, which is involved in axon guidance, are both expressed in type-A cells (Francis et al., 1999; Gleeson et al., 1999; Nacher et al., 2000). However, how DCX and CRMP-4 contribute to the internal molecular machinery of neuroblast migration is not resolved. GABA, which participates in SVZ cell proliferation, also reduces the speed of neuroblast migration by activating GABAα receptors which interfere with the release of Ca2+ from intracellular calcium stores, reducing migration (Bolteus and Bordey, 2004).

This sort of neuroblast migratory activity suggests the presence of chemotactic agents that may drive and guide the cells along the RMS to the OB. It appears that SLIT protein, which is expressed by the septum and choroid plexus, may be a critical chemorepulsive factor for directional SVZ neuroblast migration (Hu, 1999; Wu et al., 1999). The OB has also been suggested to be sources of chemoattractants for type-A cells. Some researchers have suggested that type-A cells were attracted in vitro by Prokineticin-2 (Ng et al., 2005) and -1 (Astic et al., 2002; Murase and Horwitz, 2002) and that both are expressed in the OB. However, others have not confirmed this (Hu et al., 1996; Mason et al., 2001).

After migrating cells reach the OB, they depart from the RMS and migrate radially along blood vessels to reach their final destination in different layers of the OB (Bovetti

20 et al., 2007b). Here, they differentiate into mature OB neurons: granular or periglomerular cells. The molecular determinants of these changes in mode of migration are still unresolved. A number of proteins may be involved. Reelin is expressed in mitral cells of the OB and is necessary for the separation of type-A cells from tangentially oriented chains and initiating radial migration (Ogawa et al., 1995; Hack et al., 2002). Tenascin-R and Prokineticin-2 also induce detachment of type-A cells from RMS chains and also appear to attract radially migrating neuroblasts to appropriate OB layers (Saghatelyan et al., 2004; Ng et al., 2005).

There is evidence that neurogenesis exists in the adult human OB (Bédard and Parent, 2004; Curtis et al., 2007), suggesting that cell migration between the SVZ and OB may continue in the adult human brain (Curtis et al., 2007).

PG cells and granule cells are continually produced throughout the life span of mammals (Altman, 1969). Despite the elimination of some, more than 30,000 newly generated interneurons reach the OB circuit every day and are integrated into the local inhibitory network of the OB throughout life, resulting in a high degree of plasticity in OB microcircuits (Petreanu and Alvarez-Buylla, 2002). It may be that one of the functions of adult neurogenesis is to improve the plasticity of neuronal networks. This hypothesis was supported by the findings that sensory deprivation substantially decreases the number, the dendritic length, and the spine density of newly generated interneurons, leaving all pre-existing interneurons intact (Saghatelyan et al., 2004). Thus, newly generated interneurons that migrated into the OB enable the OB circuit to adapt to novel sensory challenges (Lledo et al., 2008). SVZ-OB neurogenesis occurs in a neuronal network in which sensory afferent inputs are subjected to a continual replacement as well. Mature OSNs in the OE have limited life span (60 days in rodent) and are tightly regulated by many factors. SVZ-OB neurogenesis and cell migration might therefore be a mechanism for modulation of sensory information processing in the brain in response to imbalance of highly-sensitive OSNs.

Significant progress has been made in the understanding of SVZ-OB neurogenesis, however new experiments studying progenitor cell-fate specification, cell migration, and cell differentiation are necessary. More experiments are needed to elucidate whether sensory neurogenesis and SVZ-OB neurogenesis are tightly concerted and whether OSNs generation influences changes in OB microcircuits in control and under pathology (Gage et al., 2008).

21

Figure 1.9: Structure of olfactory receptor. Olfactory receptor passes through plasma membrane seven times, with N-terminus located extracellularly and C-terminus intracellularly. Adapted from Liu and Rao (2003) and Siegel et al. (2006).

1.1.3 Olfactory receptors, G proteins, and adenylyl cyclase in olfactory system

1.1.3.1 Olfactory receptors Olfactory receptor (OR) were first identified in 1991 in the rat (Buck and Axel, 1991) and have since been identified in other species, mainly by PCR amplification with degenerate oligonucleotide primers derived from conserved motifs. ORs belong to a large superfamily of seven-transmembrane GPCRs (Buck and Axel, 1991) and share with them a number of stereotypical motifs, including an apparent seven transmembrane domain topology (Fig. 1.9). However, ORs share a unique sequence motif, not seen in other GPCRs, which define them as members of a novel, unique receptor family. The OR gene family in mammals is extremely large and probably reflects the specific olfactory requirements of each species. Genomic analyses have revealed approximately 700 genes in humans (of which approximately 350 are functional) and over 1,200 genes in rodents (of which two-thirds are functional) (Glusman et al., 2001; Zozulya et al., 2001; Young et al., 2002; Zhang and Firestein, 2002).

ORs can be divided, according to conservation of protein sequences, into two phylogenetic classes, known as Class 1 and Class 2. Class 1 ORs comprise approximately 10% of mammalian OR genes (Ngai et al., 1993), whereas Class 2 genes comprise the majority of mammalian OR genes. Class 1 ORs recognise moderately hydrophobic volatile odorants, whereas Class 2 ORs recognise hydrophobic

22 compounds. Despite the tremendous diversity of OR genes, they can be divided into subfamilies based on the similarity of their sequences. Within a subfamily, members exhibit a sequence conservation of more than 40% amino-acid identity (Zhang and Firestein, 2002). Different subfamilies of OR genes may recognise different structural classes of odorants. Closely related members of a subfamily may possibly recognise particular odorants or may recognise tiny differences between structurally-related odorants.

§ OR protein structure OR proteins are 300 – 350 amino-acids long and are devoid of N-terminal signal sequences. Sequence analyses reveal that they contain structural features common to all GPCRs (Gat et al., 1994; Strader et al., 1994; Pierce et al., 2002; Vaidehi et al., 2002). They have seven hydrophobic stretches (19 – 26 amino acids each) representing the transmembrane domains; a potential disulphide bond between the highly conserved cysteines in extracellular loops 1 and 2; a conserved NXS/T consensus for glycosylation in the N-terminal region; several potential phosphorylation sites in intracellular regions; and finally, numerous conserved short sequences. However, OR families have specific characteristics, such as an unusually long second extracellular loop, two conserved cysteines in this loop, and conserved amino acid motifs (Probst et al., 1992). These consensus motifs include LHTPMY in intracellular loop 1; MAYDRYVAIC at the end of TM3 and the beginning of intracellular loop 2; SY at the end of TM 5; FSTCSSH at the beginning of TM6; and PMLNPF in TM7. In the second and third intracellular loops of ORs, highly conserved olfactory-specific amino-acid sequence motifs may comprise the G protein recognition epitope (Pilpel and Lancet, 1999; Fuchs et al., 2001). The OR has highly variable TM domains 4, 5, and parts of 3, which contain seventeen highly variable residues. These domains are part of the odorant-binding pocket, which allow different ORs to recognise different chemical odorants (Buck and Axel, 1991).

§ Specific features of ORs: “One neuron-one receptor” rule There are approximately 1,000 types of OSNs, each expressing only one type of OR. This monogenic and monoallelic mode of OR gene expression is known as the “one neuron-one receptor” rule. This rule has been demonstrated by RNA in situ hybridisation (ISH), genetic labelling, and single-cell RT-PCR (Malnic et al., 1999; Serizawa et al., 2003). For example, a study using complementary DNA analyses of single OSNs showed that only one OR gene is expressed in each cell (Malnic et al., 1999). Another study found that assay of OR transcripts for polymorphisms by PCR showed that the OR gene is exclusively expressed from one maternal or paternal allele (Chess et al., 1994). Experiments on gene-targeted mice, in which both alleles were differentially tagged, 23 using RNA- and DNA fluorescence ISH, confirmed the monoallelic expression of the OR gene within OSN nuclei (Strotmann et al., 2000; Ishii et al., 2001). However, experiments such as these were not able to probe all 1,000 or so ORs in one cell at a time nor were they able to show expression of these OR genes in a single cell at different stages of development (Malnic et al., 1999; Serizawa et al., 2003; Tietjen et al., 2003; Li et al., 2004; Shykind et al., 2004; Tian and Ma, 2008). It has been hypothesised that each cell may transiently express multiple ORs and then eliminates all but one during development (Mombaerts, 2004). Evidence for this theory comes from a number of studies using single-cell transcriptomics of the OE in newborn and adult mice which showed co- expression of multiple distinct ORs in a subset of cells, primarily in immature neurons (Hanchate et al., 2015; Tan et al., 2015). These studies revealed that, during development, a single OR is selected by supressing a few transiently expressed ORs in a single cell of the olfactory system (Tan et al., 2015). Regardless of developmental dynamics, it seems that the “one neuron-one receptor” rule is maintained in the adult cell in which only one OR is expressed at any given time (Li et al., 2004) or after switching between a few ORs (Shykind et al., 2004; Dalton et al., 2013).

§ Specific features of ORs: Combinatorial receptor codes for odours The OR family discriminate odorants in a combinatorial manner. Odorants bind to an OR and this mediates the transduction signal in the cell. A number of studies have shown that a specific odour is associated with specific ORs. Moreover, an OR may recognise more than one odorant because different odorants could contain a molecular sequence fragment of the primary epitope. The result is that a single OR may be activated by numerous odorants. Different concentrations of a particular odorant or combinations of smells may also activate different sets of ORs, creating the perception of a different odour (Krautwurst et al., 1998; Zhao et al., 1998; Malnic et al., 1999; Wetzel et al., 1999; Araneda et al., 2000; Kajiya et al., 2001; Gaillard et al., 2002). Thus, the pattern of glomerular activation is thought to underlie the perception of different odours.

§ Specific features of ORs: Axon targeting Axons from OSNs expressing the same OR gene converge onto specific glomeruli in the OB thereby creating a topographic map of odour quality (Ressler et al., 1994a, b; Mombaerts et al., 1996; Wang et al., 1998; Bozza et al., 2002) (Fig. 1.10). Two techniques have been used in mouse and rat studies to show the olfactory bulb glomeruli and converging axonal projections of OSNs express the same OR. Analysis of ISH- sections was used to detect the presence of minute amounts of OR mRNA within axon terminals. The results identified a small number of spatially defined glomeruli for an OR (Ressler et al., 1994a, b). 24

Figure 1.10: Wiring of axons from olfactory sensory neurons to glomeruli. This figure shows three types of olfactory sensory neurons (OSNs) (red, blue, and green), each expressing different olfactory receptor (OR). Red OSNs project red axons that connect to red glomeruli in olfactory bulb (OB). Similarly, other OSNs (blue and green) are wired to specific glomeruli (blue and green), forming olfactory sensory map. In glomeruli, axons of OSNs synapse with dendrites of mitral and tufted cells and interneurons. Adapted from Buck and Axel (1991).

Researchers created a mutant mouse model in which a defined OR was coupled to the axonal marker tau-lacZ (Callahan and Thomas, 1994). They found that labelled axons typically project to either of two glomeruli in a spatially invariant pattern (Ressler et al., 1994a, b; Vassar et al., 1994; Mombaerts et al., 1996; Mori et al., 1999). The glomerulus is a convergent site of axonal projections from OSNs. Glomeruli that express a certain OR reside at stereotyped and symmetrical positions in each OB. This situation means that OR-specific neurons typically innervate one glomerulus in the OB’s medial hemisphere and another in its lateral hemisphere (Ressler et al., 1994a; Vassar et al., 1994; Mombaerts, 1996; Nagao et al., 2000).

The dendrites of the projection neurons of the OB—the mitral and tufted cells— in turn innervate the glomeruli and carry information along their axons to the olfactory

25 cortex. The spatial patterns of activity elicited in the OB appear to be represented, not as a corresponding spatial map in the olfactory cortex, but rather in a sparse and distributed manner at this level (Poo and Isaacson, 2009). ORs may have dual roles: in odorant reception at the level of their dendrite and in contributing in axon guidance to the OB (Gierer, 1998).

§ Specific features of ORs: Zonal expression of ORs in OE The OE is divided into four distinct zones (Ressler et al., 1993; Vassar et al., 1994). Each zone occupies approximately 25% of the surface area of the OE (Fig. 1.11). The zones are symmetrically distributed along the dorsal-ventral axis of the OE. Zone 1 is located in the dorsal region of the OE and zone 4 in the ventral region (Sullivan et al., 1996). Many different members of the OR gene family are expressed within each zone. However, each individual OR gene may be expressed only within a single zone (Siegel et al., 2006) (Fig. 1.11).

1.1.3.2 G proteins There are two major groups of mammalian G proteins: heterotrimeric G proteins and small G proteins. Heterotrimeric G proteins are involved in transmembrane signalling in the nervous system. They also participate in numerous cellular processes, such as membrane vesicle transport, cytoskeletal assembly, cell growth and protein synthesis.

The nervous system contains numerous forms of G proteins. Originally three types of heterotrimeric G proteins were identified: Gt, Gs and Gi. Subsequently, over 35 subunits have been found in studies that used a combination of biochemical and molecular cloning techniques (Gilman, 1995; Neer, 1995; Wickman and Clapham, 1995; Preininger and Hamm, 2004). Other types of G protein in brain include

Go, Golf, Ggust, Gz, Gq, and G11-16.

G proteins are divided into four main families: the Gs family (which includes: Gαs1,

Gαs2, Gαs3, Gαs4, Gαolf) stimulates adenylyl cyclase (AC); the Gi family (which includes Gi/o, + Ggust, Gt, Gz) can inhibit AC, activate a certain type of K channel, inhibit voltage-gated Ca2+ channels, activate the MAP-kinase pathway, or activate phosphodiesterase (PDE); the Gq family activates phospholipase C-β (PLCβ); and the G12/13 family activates a group of proteins termed Rho-GEFs (guanine nucleotide exchange factors).

Olfactory G protein (Gαolf) is a heterotrimeric Gs protein, comprising Gαolf, β1, and γ13 (Jones and Reed, 1989; Kerr et al., 2008). In mOSNs, this heterotrimer provides the link between the ORs and AC3. Gαs is also expressed in OSNs. The phenotypic switch from Gαs to Gαolf occurs later in olfactory cilia with the maturation of the OE (Menco, 1994; Belluscio et al., 1998; Siegel et al., 2006).

26

Figure 1.11: Zone-to-zone axonal connections between olfactory epithelium and olfactory bulb. Schematic diagram illustrates pattern of connection between axons of olfactory sensory neurons (OSNs) and olfactory bulb (OB). Olfactory receptors (ORs) are classified into four groups, according to their expression patterns in olfactory epithelium (OE). A given type of OR is expressed in one of four zones in OE: zones 1 (violet), 2 (pink), 3 (light blue), or 4 (green). OSNs in each zone of OE project to glomeruli in same corresponding zone of OB. OSNs (red) in zone 1 of OE (violet), which express same OR, project axons to specific glomerulus in zone 1 (violet) of OB. Same wiring pattern applies to OSNs in zone 3 (blue). Adapted from Mori et al. (1999).

In the , each G proteins exists as a heterotrimer, composed of single α, β, γ subunits. In the unstimulated, resting state, the α subunit has GDP bound and the G protein is inactive. A ligand binds to and activates the receptor, producing a conformational change in the receptor, which is associated with the α subunit. This, in turn, triggers a dramatic conformational change in the α subunit of the G protein. This conformational change decreases the affinity of the α subunit for GDP. GDP dissociates from the α subunit and GTP binds to the α subunit instead. This binding generates a free α subunit by the dissociating α subunit from βγ subunit dimer and the receptor. Free α subunits (still bound to GTP) and free βγ subunit dimers are both functionally active. They directly regulate a number of effector proteins, including ion channels, AC, PLCβ, phospholipase A2, and PDE.

27 GTP-ase activity in the α subunit degrades GTP to GDP. This leads the free α subunit to reassociate with the βγ subunit and combined with the dissociation of the ligand from the receptor, restores the resting state. G proteins operate through several signalling pathways and regulate intracellular concentrations of second messengers.

1.1.3.3 G proteins activate cAMP signalling pathway through adenylyl cyclase G proteins control intracellular cAMP concentration by mediating the ability of neurotransmitters to activate or inhibit AC. Activation of the receptors that couple to Gs results in the generation of free Gas subunits that bind to and directly activate AC. In addition, free βγ-subunit complexes activate certain subtypes of AC. A similar mechanism occurs with Gaolf, the type of G protein structurally related to Gas, that is enriched in OE and striatum. Another G protein, Gi, inhibits AC. When a neurotransmitter receptor couples to Gi, free Gai subunits are generated and these bind to and directly inhibit certain subtypes of AC. For other AC isoforms, free βγ-subunit complexes, generated by the release of Gai, appear to mediate inhibition of the enzyme.

As well as Gi, Gaz, a subtype of the Gi family, can also mediate neurotransmitter inhibition of AC.

§ G proteins activate inositol signalling pathway through phospholipase C-β

The transducing family of G proteins (Gt), enriched in , mediate signal transduction by regulating specific forms of PDE that catalyse the metabolism of cyclic nucleotides. Gt activates PDE by binding directly to the enzyme. G protein

(Ggust) shares a high degree of homology with Gt. Ggust enriched in taste epithelium and is believed to mediate signal transduction in this tissue through the activation of a distinct form of PDE (Siegel et al., 2006).

PLCβ acts on a phosphorylated inositol phospholipid called PIP2. Receptors that activate this inositol phospholipid signalling pathway are mediated by G protein-Gq that activates PLCβ in much the same way that Gs activate AC. The activated PLCβ then cleaves the PIP2 to generate two products: inositol 1,4,5-trisphosphate (IP3) (a small intracellular mediator) and diacylglycerol (DAG) (Siegel et al., 2006).

1.1.3.4 Adenylyl cyclase Nine separate and unique forms of AC, which comprise a distinct enzyme family, referred to as AC1 – AC9, have been identified by biochemical and molecular cloning studies (Cooper et al., 1995; Sunahara et al., 1996). These nine members of the AC superfamily are all membrane-bound AC (mAC) and are activated by the stimulatory G protein Gαs. All, with the exception of AC9, are stimulated by forskolin. All forms of AC

28 are inhibited by P-site inhibitors. The different forms of AC exhibit distinct patterns of expression in brain and peripheral tissue. They are differentially regulated by Ca2+- calmodulin, by the G protein subunits Gαi and Gβγ, and by phosphorylation.

Multiple forms of AC exist in the nervous system. AC1, the only neural-specific AC, is expressed in the brain (Feinstein et al., 1991; Mons et al., 1993; Cali et al., 1994). High levels of AC1 are expressed in the hippocampus, cerebral cortex, cerebellum, olfactory system, and pineal gland. High levels of AC2 are expressed in many brain regions, including the hippocampus, hypothalamus, cerebellum, neocortex, piriform cortex, and amygdala. Lower levels of AC2 are found in skeletal muscles, the lungs, and heart (Feinstein et al., 1991; Mons et al., 1993). AC4 is widely distributed in the brain and in most peripheral tissues, including the heart, intestines, kidneys, liver, and lungs (Gao and Gilman, 1991). AC2 and AC4 are present in olfactory cilia, but their function in olfaction has not been elucidated (Wong et al., 2000). AC5 is expressed in the brain, especially in the striatum, nucleus accumbens, and olfactory tubercule. It is also expressed in the heart and kidneys (where it is associated with blood vessels), the anterior lobe of the pituitary, and the retina. AC6, structurally similar to AC5, is expressed in the brain and heart but low levels of expression are seen in testes, muscles, kidneys, and lungs (Kwon et al., 2010). AC7 is most prevalent in the lungs and spleens; is moderately-expressed in the heart; and expressed at low levels in the brain, kidneys, and skeletal muscles (Watson et al., 1994; Hellevuo et al., 1995). AC8 is expressed in the brain—in the OB, thalamus, habenula, cerebral cortex, and hypothalamic nuclei— and in the lungs and parotid glands. AC9 is expressed in the brain and skeletal muscles.

AC3 was first cloned by Reed and colleagues in 1990 (Bakalyar and Reed, 1990). It is a Ca2+-calmodulin-stimulated isoform of AC that is responsible for the elevation of intracellular cAMP in the cilia of OSNs (Bakalyar and Reed, 1990; Choi et al., 1992). AC3 is the major form of AC in OSNs and is expressed predominantly in the cilia. EM confirmed that AC3 immunoreactivity was confined to the ciliary structure of the OE in rats and this localisation is nearly identical with that of Golf, suggesting that AC3 and Golf interact at this site to mediate olfaction (Bakalyar and Reed, 1990).

Many other tissues have been found to express AC3 at mRNA and protein levels. These include brain regions, spinal cord, adrenal medulla, adrenal cortex, heart, lungs, and retina. IHC showed that AC3 is expressed predominantly in neuronal primary cilia throughout the adult mouse brain, including the cortex, hippocampus, hypothalamus, amygdala, nucleus accumbens, and dorsal raphe nucleus (Bishop et al., 2007). AC3 proteins are also present in the primary cilia of astrocytes (Bishop et al., 2007) and in epithelial cells of the plexus in the adult brain (Gonçalves et al., 2016).

29 Outside the brain, AC3 is expressed in the primary cilia of kidneys (Pluznick et al., 2009), the pancreas (Portela-Gomes et al., 2002), and brown and white adipose tissue (WAT) (Wang et al., 2009). In addition, AC3 expression has been detected in tumours (Hong et al., 2013), vascular (Wong et al., 2001) and bronchial smooth muscles (Jourdan et al., 2001), male germ cells (Defer et al., 1998), and hepatic cells (Liang et al., 2016).

Polymorphisms in AC3, which is expressed in brown adipose tissue and the hypothalamus, are associated with obesity, suggesting that AC3 may play a role in weight control (Wang et al., 2009; Qiu et al., 2016). Wang et al. (2009) found that AC3 knock-out mice exhibit pronounced obesity and that this was primarily due to higher fat weight and higher levels of leptin and leptin resistance compared to their control mice. The AC3 knock-out mice were less active throughout their circadian cycle and consumed more food. These mice achieved hyperphagia and obesity was not due to a loss of AC3 from WAT but by ablation of AC3 levels in the hypothalamus. Therefore, the presence of AC3 in primary cilia of hypothalamic neurons may trigger cAMP signals in the hypothalamus and this may be involved in the regulation of energy balance (Wang et al., 2009; Qiu et al., 2016).

A study by Liang et al. (2016) also demonstrated that AC3 is an anti-obesity gene and plays an important role in the regulation of body weight. The authors found that AC3 expressed at mRNA and protein levels in the liver of mice using RT-PCR and Western blot (WB). In mice with high-fat, diet-induced obesity and diabetes, body weight and insulin resistance were decreased and AC3 expression levels were unregulated compared to those in non-diabetic, lean mice (Gu, 2010; Liang et al., 2016).

1.1.3.5 Expression of ORs, G proteins, and AC3 in OSNs of olfactory system OR proteins were first identified in the ciliary layer of the rat OE (Krieger et al., 1997). Antibodies to two putative odor receptors (D3 and M4) in both rat and mice bound equally well to proximal and distal parts of cilia (Menco et al., 1997). OR proteins were reported in the dendritic knobs and soma of OSNs and in axonal bundles in the OE submucosa (Menco, 1997; Menco et al., 1997).

In addition to ORs, olfactory cilia contain other important components of the olfactory transduction cascade, involving Gs type G proteins. These proteins include Gαs

(Mania-Farnell and Farbman, 1990), Gαolf (Jones and Reed, 1989), AC3, (Bakalyar and Reed, 1990), and CNG channels (Menco, 1997; Menco et al., 1997). IHC showed that

Gαs was located in the olfactory cilia and its β subunits were found in cilia, but also in dendrites and dendritic endings. Light microscopy showed that Gαolf (Jones and Reed,

30 1989) and AC3 (Bakalyar and Reed, 1990) were located in the ciliated epithelial surface. EM and post-embedding IHC on rapidly frozen, freeze-substituted specimens showed that Gαolf and AC3 and the CNG channel were present in the same OSN compartments in the distal parts of the olfactory cilia (Menco, 1992; Menco et al., 1994; Menco, 1994; Menco, 1995).

IHC at high magnification and EM have shown that IP3 receptors are located in OSNs in the cilia, in the dendritic knobs, within some cell bodies, and in axon bundles beneath the basal lamina. IP3 receptors are also located in the apical ER of SUS cells.

Double-labelling IHC at high resolution showed that IP3 and Golf were strongly co- expressed across the ciliary layer of all OSNs in the OE (Cunningham et al., 1993). This finding shows that there are two possible pathways that ORs may utilise for olfactory signal transduction. They can use the secondary messengers: cAMP or IP3. However, patch clamp techniques have not located IP3-gated channels in mammalian OSNs (Firestein et al., 1991; Lowe and Gold, 1993; Brunet et al., 1996; Gold, 1999). This suggests that only cAMP acts as a second messenger (Elsaesser et al., 2005).

The cilia and dendritic knobs of OSNs also contain G protein-coupled β adrenergic receptor : GRK3 (βARK2) and β-arrestin2. These kinases have been found to take part in agonist-dependent desensitisation in olfaction (Dawson et al., 1993).

OR proteins are present in the axonal processes and nerve terminals of OSNs, with higher OR protein concentration in the distal axonal segments (Strotmann et al., 2004). These proteins are also found in the dendritic knob, in the glomerulus, and in the axon fascicles immediately adjacent to the glomerulus (Barnea et al., 2004). OR mRNA translation was found in OSN axons in vivo (Dubacq et al., 2009). Most members of the olfactory transduction cascade (including G proteins and AC3) were present on the OSN axonal projections at mRNA level.

Golf is present in sensory axons and in glomeruli (Belluscio et al., 1998). The

OSNs of Golf-deficient mice displayed profound reductions in their electrophysiological ability to respond to odors. However, this did not affect the topographical map and the OSN projections converged normally into the OB (Belluscio et al., 1998).

AC3 was expressed in both OSN cilia and axons during the period of active glomerular formation in neonatal animals, as shown with ISH and IHC. AC3 was also present in axonal projections that comprised the ONL and glomeruli (Dal Col et al., 2007). AC3 is thought to play a role in the formation and position of glomeruli because AC3 deletion leads to drastic modifications of the wiring diagram in the OB. Examination of OR-tagged AC3 knock-out mice showed that the absence of AC3 perturbed the

31 formation of representative glomeruli (Zou et al., 2007). AC3-deficient OSNs projected aberrantly to the OB (Dal Col et al., 2007). AC3-deficient mice are anosmic and did not respond to odorants in electro-olfactogram (EOG) studies (Wong et al., 2000).

AC3 deficiency in mammals is associated with behavioral disorders and causes abnormal IP3 signal production in the OE of mice (Chen et al., 2000). AC3 deletion has also been shown to impair male fertility in mice. AC3-deficient males, who demonstrated normal anogenital sniffing and mounting behavior with females, failed to reproduce (Wong et al., 2000; Zou et al., 2007).

1.1.4 Olfactory signal transduction pathway The olfactory signalling cascade begins when an odorant binds to the OR on the cilia of an OSN to initiate the odorant detection pathway (Fig. 1.12). The OR activates the olfactory G protein that activates Ca2+-calmodulin-sensitive AC3, increasing the local concentration of cAMP. cAMP opens CNG channels. The activation of tens to hundreds of these channels and the subsequent influx of cations, including both Na+ and Ca2+, leads to the depolarisation of the cell membrane. Membrane depolarisation extends to the dendrite and soma, triggering an action potential that travel along the axon of the OSN and transmits a signal to the OB (Jones and Reed, 1989; Bakalyar and Reed, 1990; Dhallan et al., 1990; Choi et al., 1992; Kleene, 1994; Brunet et al., 1996; Buck, 1996; Belluscio et al., 1998; Schild and Restrepo, 1998; Wong et al., 2000).

1.1.5 ORs, G proteins, and ACs in non-olfactory tissues It was initially thought that ORs were exclusively expressed in the olfactory system. Subsequent studies, however, demonstrated “ectopic” expression of some ORs in a variety of other tissues (Kang and Koo, 2012; Foster et al., 2014) including sperm (Parmentier et al., 1992; Vanderhaeghen et al., 1993; Flegel et al., 2015), tongue (Thomas et al., 1996; Gaudin et al., 2001; Durzyński et al., 2005; Gaudin et al., 2006), spleen, pancreas (Blache et al., 1998; Nakagawa et al., 2009), (Itakura et al., 2006), and enterochromaffin cells of the gut (Braun et al., 2007), variously in dogs, humans, rats, and mice. Ectopic expression of OR transcripts was also found in the heart using RT-PCR and ISH analyses, and this expression was developmentally regulated (Drutel et al., 1994; Weber et al., 2002). The OR subtype MOR2.3 was found in the transgenic mouse line MOL2.3-IGITL using X-gal analysis in a small segment of the aorta of the thoracic region (Weber et al., 2002). Additionally, ectopic expression of human OR51E2, also known as prostate-specific GPCR, was found and physiologically characterised in prostate tissue. OR51E2 can be activated in prostate cells by the

32

Figure 1.12: Olfactory signalling through G protein-coupled pathway.

When odorants bind to OR, initial transduction events in cell are mediated via Golfα and AC3 producing cAMP. Intracellular cAMP triggers opening of CNG channel. Flow of Na+ and Ca2+ ions ultimately depolarises plasma membrane. Depolarisation extends to dendrite and soma, triggering action potential that travels along axon of OSN and transmits signal to olfactory bulb. Abbreviations: OR, olfactory receptor; CNG, olfactory- specific cyclic nucleotide-gated channel; cAMP, cyclic adenosine 3’,5’-monophosphate; PDE, phosphodiesterase; ATP, adenosine 5’-triphosphate; AMP, adenosine 5’- monophosphate; CaM, serine/threonine protein kinase. Adapted from Pifferi et al. (2006).

specific OR ligand, β-ionone, which has a characteristic odour of violets and this activation, inhibits the proliferation of prostate cells (Neuhaus et al., 2009).

Several OR genes were reported in epithelial cells of the human airway, where they have a chemosensory function, sensing the inhalation of noxious bitter compounds (Shah et al., 2009; Ashmole and Bradding, 2013). OR transcripts and their olfactory transduction components, AC3 and Gαolf, were shown to be ectopically-expressed in the mouse kidney by IHC (Pluznick et al., 2009). Their presence suggests that they may play a role in the modulation of renin secretion, glomerular filtration rate, and in regulating blood pressure (Pluznick et al., 2013). Additionally, ORs, AC3, and Golf, as well as OR

33 transporters, were found in neurons of the cerebral cortex and other regions in the adult human brain with the use of functional genomic analysis (Garcia-Esparcia et al., 2013).

Next Generation Sequencing (NGS) analysis has been used to identify ectopically-expressed ORs in a wide range of human tissue. Apart from the olfactory system, the testes contain the highest number of ectopically-expressed OR transcripts (55 different OR transcripts) in comparison to the other 15 human tissues investigated (Flegel et al., 2013). This finding strengthens the idea that ORs may play an especially important role in the reproductive system, although the nature of this function remains elusive (Flegel et al., 2013).

1.1.5.1 ORs and their signalling components in reproductive system Parmentier et al. (1992) used homology cloning, low-stringency PCR, and Northern blot analysis to demonstrate the presence of approximately 20 human OR transcripts in dog germ cells and suggested that they might encode receptors involved in the chemotaxis of sperm cells during fertilisation. In other research, OR cDNA fragments were cloned from dog, mouse, and rat testes, using RT-PCR with primers specific for the OR gene family (Vanderhaeghen et al., 1997). The presence of 66 OR genes was reported in mouse testes using oligonucleotide microarray (Zhang et al., 2004). In a comprehensive RNA-Seq study, 55 different OR transcripts were reported in human testes (Flegel et al., 2013).

Attempting to address the functional role of the ORs in the reproductive system, Spehr et al. (2003) cloned a human testicular OR, hOR17-4 (synonymous with OR1D2), and examined its ligand preferences in a heterologous cell expression system. These authors found that bourgeonal acts as a powerful agonist of this receptor and human spermatozoa showed functional activation and chemotaxis towards bourgeonal, which was blocked by undecanal, a strong bourgeonal inhibitor. Moreover, an AC3 inhibitor blocked the Ca2+ flux by bourgeonal, suggesting that an OR-mediated signalling pathway is important in sperm attraction to the oocyte (Spehr et al., 2003).

Fukuda et al. (2004) showed that MOR23, a mouse OR expressed in the OE and testes (Asai et al., 1996), functions as a chemosensing receptor in spermatogenic cells and mature spermatozoa. MOR23 was functionally cloned from single OSNs that responded to the floral odorant lyral (Touhara et al., 1999). Lyral, one of the cognate ligands for MOR23, specifically activated MOR23 in both homologous and heterologous expression systems (Touhara et al., 1999). Spermatozoa migrated towards an increasing gradient of lyral, with directional changes that were caused by Ca2+ inducing flagellar beating asymmetry (Spehr et al., 2003).

34 Fukuda and Touhara (2006) reported that other types of testicular OR genes could regulate spermatogenesis or epididymal spermatozoal maturation. These authors observed that ORs (such as MOR248-11) were expressed in premeiotic germ cells and concluded that these types of ORs might recognise some hormonal factors that are required for germ cell differentiation.

Walensky et al. (1995) used antibodies OD1 and OD2 raised against two peptide sequences conserved among ORs (Walensky et al., 1995). They reported OD1 and OD2 expression in the adluminal late spermatids in rat testes; OD1 and OD2 were also expressed on the middle piece of mature spermatozoa and OD2 was expressed on the connecting piece.

Vanderhaeghen et al. (1993), used purified serum in dog testes to detect the OR gene product (DTMT receptor). They found OR expression in the late stage of round spermatids; in elongated spermatids; in cytoplasmic droplets (CDs) of spermatozoa in the lumen of some testes; and in the content of epididymal tubes. ORs were also expressed on the connecting and principal pieces of the tail. TOR9 (a testicular OR) was expressed in 90% of seminiferous tubules of mouse testes at the mRNA level. This expression occurred from panchytene spermatocytes to elongated spermatids (Tatsura et al., 2001).

The first comprehensive analysis of OR transcripts in human spermatozoa found the expression of a large panel of approximately 90 putative OR transcripts (Flegel et al., 2015). The authors also found a strong overlapping pattern of OR expression in spermatozoa and testes and suggested that most of the OR transcripts detected in the testes were derived from spermatozoa or precursor sperm cells. They used IHC and showed that different OR proteins were expressed on the head, middle piece, and tail in a compartment-specific manner. OR6B2 was strongly and specifically expressed on the equatorial segment of the head and the principal piece of the flagella. The OR3A2 protein was expressed on the middle piece. OR2W3 was expressed on the flagella. The finding that the different OR proteins were detected in all human spermatozoa and that their locations were compartment-specific indicates that one spermatozoon expresses multiple ORs that may perform distinct functions. Single-cell calcium imaging showed that a panel of various OR ligands induced intracellular Ca2+ signals in human spermatozoa, which can be inhibited by mibefradil, a known Ca2+ channel blocker.

AC3 is significantly expressed in the male reproductive system. In rat and mouse testes, AC3 expression was found on the acrosomal membrane of round spermatids and elongated spermatids by RNAase-protection assays, ISH, and IHC (Defer et al., 1998;

35 Gautier-Courteille et al., 1998; Defer et al., 2000; Sinclair et al., 2000; Hanoune and Defer, 2001; Livera et al., 2005). In mouse spermatozoa, AC3 was found on the acrosomal cap, neck, and middle piece (Baxendale and Fraser, 2003a). In human spermatozoa, it was found on the head, on the equatorial segment, and on the middle piece (Spehr et al., 2004b). Importantly, AC3 knockout mice were subfertile. Spermatozoa from these mice produced few embryos and displayed a greatly reduced ability to fertilise oocytes in vitro. Despite an apparently normal structure, these spermatozoa had decreased motility, showed an increase in spontaneous acrosome reaction (AR), and a weak ability to penetrate oocyte complexes, resulting in sterility (Livera et al., 2005). Zhang et al. (2017) proposed a mechanism that explained the sterility in male AC3 knockout mice. Using TUNEL staining, they found that sperm cell apoptosis in the testicular tissue of AC3 knockout mice (AC-/-) was significantly higher than in the wild-type (AC+/+) mice. To clarify the mechanism for this increase, the authors used gene chip hybridization on all RNA in the testicular tissue of AC-/- and AC+/+ mice. They found that the expression of 693 genes was altered in AC-/- mice, with expression of 330 genes upregulated and 363 downregulated. The authors used gene ontology (GO) functional annotation analysis to determine how the differentially expressed genes affected AC3 activity. Significant alterations were found in 7 genes involved in the Ca2+ signalling pathway; in 28 genes involved in the OR signalling pathway; in 6 genes involved in the axon guidance signalling pathway and in 25 genes involved in signalling pathways involved in cell junctions. All these genes play important roles in spermatogenesis, sterility, chemotaxis, fertilisation, and male hormone synthesis – all functions affecting the AC-/- mice (Zhang et al., 2017).

Mammalian somatic cells and spermatozoa contain several different heterotrimeric G protein α subunits (Clapham and Neer, 1993; Downes and Gautam, 1999; Vanderbeld and Kelly, 2000). Each α subunit has a unique pattern of subcellular localisation in testes and mature spermatozoa. IHC showed that Gaolf expression was restricted to pachytene spermatocytes, spermatids, and residual bodies (Defer et al., 1998). G protein α subunits were identified in mouse spermatocytes and spermatids by immunoprecipitation and indirect immunofluorescent analysis: Gai was present in pachytene spermatocytes, round and elongated spermatids, and epididymal spermatozoa in association with the developing acrosome. Gao was present in spermatocytes and spermatids but was not detected in mature spermatozoa (Karnik et al., 1992). G proteins were also associated with the acrosomal region of the sperm cells in mice, guinea pigs, and cows. Additionally, low levels were present on the principal piece of the tail of mouse and human spermatozoa l (Garty et al., 1988; Glassner et al.,

36 1991; Hinsch et al., 1995). The Gaq/11 subunit was found on the mouse acrosome, using indirect immunofluorescence, with the highest expression occurring on the equatorial segment (Merlet et al., 1999) and on the middle piece of the tail (Walensky and Snyder,

1995). Ga12 was found on the acrosome and middle piece of the tail, and Ga13 was found on the post-nuclear cap and middle piece of the tail (Merlet et al., 1999).

In the testes of rats, AC3 and Golf were both expressed at the same stage of germ cell differentiation, from pachytene spermatocytes to spermatids, and in residual bodies, as shown by Northern blot analysis, PCR analysis, WB analysis, and IHC (Defer et al.,

1998). In human spermatozoa, Golf and AC3 were both expressed on the middle piece and tail (Spehr et al., 2004b).

Several other components of olfactory signal transduction pathway were found in mammalian spermatozoa and these include receptor protein kinases (GRK3/βARK2), β- arrestin2 (Walensky et al., 1995; Neuhaus et al., 2006), β1 isoform of PLC and IP3 (Walensky and Snyder, 1995), G protein α subunit gustducin (Fehr et al., 2007), CNGA3, and a CNGA2 subunit of CNG channels (Weyand et al., 1994; Wiesner et al., 1998; Flegel et al., 2013). The proteins GPK3/βARK2 and β-arrestin2, implicated in olfactory desensitisation, were found in sperm cytosolic extracts using WB. Confocal microscopy found these proteins on the middle piece of rat spermatozoa, with higher immunoreactivity occurring at the proximal part of the middle piece immediately subjacent to the connecting piece. IHC co-localised ORs, GPK3/βARK2, and β-arrestin2 in rat elongated spermatids and on the middle piece of the spermatozoa, suggesting that these proteins could regulate chemoreceptor responses in mature spermatozoa (Walensky et al., 1995). In mature human spermatozoa, β-arrestin2 and hOR17-4 were co-localised on the middle piece of the tail as well. However, odorant stimulation of the G protein-coupled hOR17-4 in human spermatozoa led to PKA-dependent translocation of cytosolic protein β-arrestin2 to the nucleus and to nuclear accumulation of phosphorylated mitogen-activated protein kinases (MAPKs). In the nucleus, β-arrestin2 was involved in transcriptional regulation, as shown by the use of a Ga14-based transactivation assay. The authors speculated that the nuclear translocation of β- arrestin2 could be important for the regulation of gene expression during the early stages of fertilisation (Neuhaus et al., 2006).

Gaq/11 and PLCβ1, signalling elements which are related to the phosphoinositide system in mammalian spermatozoa (Lee et al., 1992), were expressed on the anterior acrosomal region of the head and middle piece in mice (Walensky and Snyder, 1995). 2+ IP3-gated Ca channels were found in the testes and mature sperm of rats with the use of IHC. IP3 was found on the developing acrosome of round spermatids.

37 In mature spermatozoa of rats, mice, hamsters, and dogs, IP3 was found on the outer membrane of the acrosomal cap and, in rats, on the proximal middle piece. The authors suggested that the AR may be activated by releasing Ca2+ from an acrosomal

IP3-gated calcium store (Walensky and Snyder, 1995).

It is remarkable that many of the components of the OR signalling transduction pathway that are used for sensing olfactory cues are also expressed in the reproductive system. It has been suggested that an olfactory-like signalling pathway may be used by the male gamete (Parmentier et al., 1992; Walensky et al., 1995; Livera et al., 2005). The putative in vivo stimulus for guiding mammalian spermatozoa remains to be identified (Babcock, 2003).

1.2 Expression and functions of olfactory marker protein (OMP) in olfactory system and non-olfactory tissues

1.2.1 OMP in olfactory system Olfactory marker protein (OMP) is a small, abundant, soluble acidic protein of 19 kDa with an isoelectric point of about 5 (Margolis, 1972; Keller and Margolis, 1975; Brunet et al., 1996; Buck, 1996; Belluscio et al., 1998; Wong et al., 2000). Immunological studies have demonstrated that this protein is phylogenetically conserved and widely distributed, being expressed in the olfactory system of virtually every vertebrate species including humans (Margolis, 1972; Hartman and Margolis, 1975; Keller and Margolis, 1975; Margolis, 1980; Buiakova et al., 1994).

The OMP protein and its gene have been isolated, sequenced, and cloned from OBs of mice, rats, and humans and its genomic regulatory motifs identified (Margolis, 1972; Sydor et al., 1986; Rogers et al., 1987; Danciger et al., 1989; Kudrycki et al., 1993). The OMP gene, in all species, lacks canonical TATA and CAAT motifs and introns (Buiakova et al., 1994). The rat OMP gene promoter consists of at least two types of regulatory motifs: Olf-1 elements, which bind olfactory-specific transcription factor(s), and the upstream binding region (UBE) motif, which binds to nuclear proteins present in many tissues (Kudrycki et al., 1993; Buiakova et al., 1994). The promoter regions of other genes, preferentially expressed in rat olfactory neurons, also contain Olf-1 motifs. The promoters of the olfactory CNG channel and AC3 contain both Olf-1 motifs and the UBE motif (Wang and Reed, 1993; Wang et al., 1993). The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG (Kudrycki et al., 1993). Studies with transgenic mice showed that a 0.3 kb fragment of the OMP gene containing one Olf-1

38 motif was sufficient for olfactory tissue-specific expression. The reporter gene, together with other identified motifs, may interact with olfactory nuclear protein extracts. This raises the possibility that Olf-1 represents an olfactory, neuron-specific trans-acting factor, or complex of factors, capable of interacting with other neuron-specific genes and playing a role in regulating the expression of genes associated with odour transduction or neuronal regeneration (Kudrycki et al., 1993). The amino acid sequence of OMP is more than 50% identical in fish (Çelik et al., 2002), Xenopus laevis (Rössler et al., 1998), rodents, and humans (Danciger et al., 1989; Buiakova et al., 1994).

OMP is neither glycosylated nor phosphorylated and shows no endogenous enzymatic activity. Additionally, it contains no obvious structural motifs, such as zinc or calcium domains (Wright et al., 2005). The soluble structure of OMP has been observed by multi-dimensional nuclear magnetic resonance spectroscopy, which showed that OMP is a monomeric globular protein. The crystal structure of OMP was identified by X- ray crystallography at a 2.3 Å resolution. Its core is made up of eight β-strands, composing a β-clamshell domain with a prominent α-helical projection that comprises the vertex of the structure (Baldisseri et al., 2002; Smith et al., 2002; Wright et al., 2005). The soluble structure is very similar to the crystal structure except for a dramatic reorientation of α-helix 1 and the adjacent loop connecting its C terminus to β-strand 2.

These conformational differences between structures could potentially correspond to a physiological change in conformation, which allow OMP to function as a molecular switch. The surface of OMP is convex, with three highly conserved regions, which have been identified as possible protein-protein interaction sites. These sites can take part in modulating the olfactory signal transduction cascade (Smith et al., 2002). It adopts a static conformation by binding to one of the membrane proteins or enzymes in the cascade and acting as an allosteric modulator of its activity. OMP could also act as a dynamic switch in the transduction cascade by undergoing a significant conformational change involving reorientation of the α-helical projection. Changes in the surface topography of OMP could alter its ability to interact with different protein partners. Interaction of OMP with one protein co-factor could stabilise the protein in one of its two different conformational states and thereby control its ability to form other protein-protein interactions. OMP may play a role, therefore, as a conformational controlled molecular adaptor protein (Smith et al., 2002).

1.2.1.1 OMP expression OMP concentrations in OSNs are developmentally regulated and only produced in fully mOSNs (Fig. 1.13). In rat OSNs, OMP was observed, using IHC staining, at

39

Figure 1.13: Expression of OMP in olfactory epithelium. Image shows olfactory epithelium (OE) stained with olfactory marker protein (OMP) (green), marker for mature olfactory sensory neurons. Courtesy of Oboti et al. (2011).

embryonic day 18 (Farbman and Margolis, 1980), and in mice at embryonic day 14 (Farbman and Margolis, 1980). This time coincides with establishment of initial synapses with their target cells in the OB and the appearance of the characteristic cellular complement of olfactory cilia (Farbman and Margolis, 1980; Graziadei et al., 1980; Menco and Farbman, 1985). From embryonic day 21 in rats, OMP staining in the OE is more extensive and is evident in the outer fibrous layer and in some glomeruli of the OB. OMP expression increases progressively and reaches adult levels approximately one month postnatally (Farbman and Margolis, 1980; Graziadei et al., 1980).

In the adult animal, where OSNs are undergoing a process of continuous replacement (Moulton and Fink, 1972; Graziadei, 1973), OMP is not found in the basally- located stem cells nor in neurons still in the intermediate stages of differentiation. OMP is found only in mOSNs in the more superficial layers of the OE in mice and rats (Monti- Graziadei et al., 1977; Farbman and Margolis, 1980). OMP is unique to the primary olfactory pathway. It is synthesised by primary OSNs in the perikaryon and transported both to the dendrite and along the axon to its termination in the GL of the OB (Margolis and Tarnoff, 1973). OMP is expressed almost exclusively and abundantly in maturing and mOSNs and is used as a biochemical marker for recognising this cell type (Keller and Margolis, 1975; Farbman and Margolis, 1980; Menco, 1989; Buiakova et al., 1994). OMP accumulation in OSNs is one of the main criteria for their maturation. OMP is expressed in the OE, septal organ of Masera, and VNO of Jacobson (Johnson et al., 1993; Matsuoka et al., 2002; Ma et al., 2003; Fuss et al., 2005; Weiler and Benali, 2005; Breer et al., 2006; Fleischer et al., 2006).

40 In the rat OE, OMP expression was reported within the cytoplasm of the body, dendrites, axons, and cilia of mOSNs. OMP immunoreactivity was reported consistently in the cilia of the OSNs of the OE. The cilia appeared to be labelled along their lengths, suggesting that OMP is found throughout these structures (Johnson et al., 1993). These findings are consistent with results reported by Menco (1989). This researcher showed OMP expression within and throughout dendrites, dendritic knobs (only in those with cilia), and proximal and distal parts of the olfactory cilia of OSNs by using EM IHC (Menco, 1989). In the mOSNs, OMP is expressed abundantly in the cytosol, throughout the most of cytoplasm. However, it was not found in organelles, such as mitochondria, or in cytoskeletal structures (Menco, 1989; Johnson et al., 1993). Some expression of OMP was reported over the nucleus of OSNs in the OE and this expression was dependent on the physiological state of the cell (Koo et al., 2004).

Intense OMP expression is seen along axons of OSNs and in glomeruli in the OB. ISH experiments with rats demonstrated that OMP RNA is found in axons and terminals of the OE in the region of the OB that contains arriving axons and their synaptic terminals (Wensley et al., 1995). These results are comparable with findings described by Vassar et al. (1994) and Ressler et al. (1994a) that OMP and/or OR RNAs were found in the OB. The presence of functional OMP mRNA in the OB was confirmed by in vitro translation and immunoprecipitation and by other biochemical studies (Rogers et al., 1987; Ehrlich et al., 1990; Grillo and Margolis, 1990). However, the evidence that OMP is synthesised in axons or terminals is lacking. There have been no reports documenting ribosomes in distal segments of mature mammalian axons.

Low levels of OMP are expressed in the cytoplasm of mitral and tufted cells of the OB (Weiler and Benali, 2005) and the protein is reported to be present in neurons of several brain areas (Baker et al., 1989). OMP, by its selective expression in OSNs and its phylogenetic conservation, may serve a common function in vertebrate chemosensory transduction. There is evidence that OMP can play a physiological role in olfactory processing as a novel modulatory component with involvement of OMP in protein-protein interactions and in neurogenesis in olfactory tissue, suggesting a developmental role for OMP (Buiakova et al., 1994; Carr et al., 1998; Farbman et al., 1998; Youngentob and Margolis, 1999). Experiments with knockout animals have provided insights into the function of the OMP protein. With the use of EOG, it was observed that the OE of OMP-deficient mice in vivo exhibited a slow response to odour stimuli, alteration kinetics of response generation and recovery, and a reduced ability to respond to the second stimuli of a pair (Buiakova et al., 1996).

41 1.2.1.2 OMP functions The OMP-null mouse phenotype is characterised by a dramatic reduction in the OE projection to the OB (Buiakova et al., 1996). Infection of OMP-null mice with a recombinant adenovirus restored the kinetics of electrophysiological responses of OMP- null mice to those of the control phenotype in vivo, suggesting that the EOG deficit is due to an acute absence of OMP rather than a developmental role of this protein (Ivic et al., 2000). OMP-null mice also have deficits in their ability to detect and discriminate odours, likewise reversible by adenoviral delivery of OMP cDNA. Behaviourally, OMP-null animals demonstrated a more than 50-fold decrease in sensitivity to odours (Youngentob and Margolis, 1999), alteration in odorant quality perception (Youngentob et al., 2001), and alterations in odorant-induced mucosal activity patterns (Youngentob et al., 2003). Constant, albeit low, levels of activity as that observed in the OMP-null mice can desensitise OSNs quite effectively, leading to a shift of the odour dose-response relation to higher odour concentration and even complete failure to generate action potentials in response to odour stimulation. OMP is reportedly involved in clearing the elevated Ca2+ that follows olfactory transduction (Reisert et al., 2007). Using suction-pipette recordings from isolated mice OSNs, it has been shown that single OSNs substantially slowed their response to odorants in the absence of OMP (Reisert et al., 2007).

Experiments with isobutylmethylxanthine (IBMX) and low-Ca2+ solution indicated that OMP acts very early in the transduction cascade, possibly at the level of the AC3 where its absence leads to a delayed response to odour stimulation (Reisert et al., 2007). Using patch-clamp technique on mouse OSNs, expressing a defined OR (MOR23) with a known ligand—lyral—demonstrated that OSNs exhibit functional maturation during the first month of postnatal life. MOR23 neurons from P30 mice show faster response kinetics, higher sensitivity, and higher selectivity than those from P0 mice and MOR23 neurons become selective detectors for the cognate odorant within two weeks. OMP is required for the maturation of MOR23 neurons, suggesting that OMP plays a critical role in maturation of OSNs (Lee et al., 2011a). Altered odorant-induced phosphorylation of AC3 may provide molecular and cellular mechanisms underlying the slow decay observed in OMP-null neurons. Although total AC3 level was upregulated in OMP-null mice, odorant-induced phosphorylation of AC3 was reduced, which potentially leads to slower termination of the responses. A behavioural assay, in which mouse pups were given a choice between their biological mother and another unfamiliar lactating female, showed that wild-type pups preferred the biological mother, whereas OMP knock-out pups failed to show preference. These results suggest that OMP is included in the

42 functional maturation of OSNs, involves the postnatal development of smell, and is important for the survival of altricial mice (Lee et al., 2011a).

1.2.2 OMP in non-olfactory tissues OMP expression has been reported in non-olfactory tissues. RT-PCR analysis was used to identify OMP genes in 13 tissues: the liver, skeletal muscle, bladder, pancreas, stomach, duodenum, testes, spleen, heart, , kidney, thyroid and lung. Conventional Western blot analysis using a specific OMP antibody, failed to detect OMP expression (Kang et al., 2015). High-resolution double immunoassay showed a low level of OMP expression throughout all tissues listed above, except for the kidney, with different levels of expression seen in various tissues. This method found that the highest levels of OMP expression occurred in skeletal muscle, heart, thymus, and thyroid and that the lowest levels of expression occurred in the liver, bladder, pancreas, stomach, duodenum, testes, spleen, and lung (Kang et al., 2015). IHC with goat anti-OMP serum detected the expression of OMP in only five non-olfactory tissues: bladder, thyroid, thymus, heart, and testes (Kang et al., 2015). In the bladder, OMP-positive (OMP(+)) cells were found in the submucosal layer. In the thymus, OMP(+) cells were detected in the medullar area. In the testes, OMP was found in a Leydig-like cell population within the interstitial tissue. In the tissue in which OMP(+) cells were found, the authors studied co-expression of OMP with some available ORs (anti-olfr1386, anti-olfr544, anti- OR51E1). They found that OMP was strongly co-localised with ORs and only a few OMP expressed singly. OMP expression has also been reported in the tongue of rats. The authors used light-microscopic IHC with polyclonal rabbit antibody raised against bacterially expressed recombinant OMP. In this study, selective OMP immunoreactivity was revealed in the distinct area of circumvallate papillae of the tongue where it was confined to the taste bud (Budanova and Bystrova, 2010). Collectively, these results suggest that OMP expression can be an indicator of OR-mediated chemoreception in non-olfactory tissue and IHC analysis of OMP can be a useful tool for determining the cellular location of ectopically-expressed ORs (Kang et al., 2015).

1.3 Reproductive System

1.3.1 Structure and function of testes The testes are a pair of ovoid glands, each of which consists of two parts or “compartments”: the tubular compartment, consisting of the seminiferous tubules (Figs. 1.14 – 1.15) where spermatogenesis occurs, and the interstitial compartment (interstitial

43

Figure 1.14: Morphology of rat testes. Photomicrograph of cross section of testes stained with Haematoxylin-Eosin show seminiferous tubules and interstitial tissue (IT). Seminiferous tubule is covered by seminiferous epithelium, which consists of spermatogonia (S), primary spermatocytes (PS), round spermatids (RS), elongated spermatids (ES), and Sertoli cells (SC) (arrowhead). IT predominately contains Leydig cells (LCs) (arrows). Y. Makeyeva, present work.

tissue [IT]), located between the seminiferous tubules (Fig. 1.14). The IT is a thin web of connective tissue containing Leydig cells (Fox, 1996; Darszon et al., 2011) (Fig. 1.16). The IT and tubular compartments are anatomically separate but closely connected to each other. They interact with each other in complex ways to produce healthy sperm (Saez, 1994).

The testes have a dual function: the production of male gametes and male sexual hormones (). Spermatogenesis includes all the processes involved in the production of gametes (sperm) (Fig. 1.15), whereas steroidogenesis refers to the enzymatic reactions leading to the production of male hormones. Testicular function is determined by endocrine regulation through the hypothalamus and pituitary glands of the CNS. It is also regulated by local paracrine and autocrine factors (Heindel and Treinen, 1989; Haider, 2004).

44

Figure 1.15: Diagram of mammalian male reproductive organs and spermatogenesis. Diagram on left shows stages of spermatogenesis and location of germ cells. Spermatogenesis advances from base of lumen (basement membrane) of seminiferous tubule where spermatogonia reside and proliferate. Spermatogonia differentiate into primary spermatocytes (leptotene), which transverse blood-testis barrier, forming tight junction with adjacent Sertoli cells. Primary spermatocytes undergo first meiosis, forming secondary spermatocytes (pachytene and diplotene). They are converted into round spermatids by second meiosis. Round spermatids differentiate into elongated spermatids that undergo spermiation. In final stage of spermiation, fully developed spermatozoa are formed. Sertoli cells phagocytise most of cytoplasm (residual bodies) of elongated spermatids, leaving only small remnant of spermatid’s cytoplasm— cytoplasmic droplet. Middle diagram shows cross section of seminiferous tubule. Newly- formed spermatozoa are transported towards epididymis through lumen of tubule (L). Diagram on right shows testis and epididymis. Epididymis consists of single, highly- coiled tubule. Epididymis is divided into three regions: caput, corpus, and cauda. Sperm movement through these epididymal regions is required for their maturation. Courtesy of Darszon et al. (2011).

1.3.1.1 Testicular structure: Tubular compartment (seminiferous tubules) The tubular compartment represents approximately 60 – 80% of the total testicular volume and contains the germ cells and two different types of somatic cells: the peritubular cells and the Sertoli cells (Figs. 1.14 – 1.16).

45

Figure 1.16: Schematic representation of anatomical structure of testes. Sertoli cells are located on basement membrane and their tips point towards cavity in middle of tubule. Undifferentiated spermatogenic stem cells and Sertoli cells that surround them are located on basement membrane. Seminiferous tubule is divided into basal compartment, adluminal compartment, and lumen. Basal compartment contains spermatogonia and Sertoli cells. Adluminal compartment contains differentiating germ cells: spermatocytes, round spermatids, and maturing spermatozoa. From lumen, mature spermatozoa are released into epididymis. Close to lumen, most of spermatid’s cytoplasm (residual body) is pinched off and ingested by Sertoli cells. Only small remnant of the spermatid’s cytoplasm—cytoplasmic droplet—is left on spermatozoa. Leydig cells are often seen to lie adjacent to blood vessels. Adapted from Kerr and De Kretser (2006).

§ Peritubular cells (myofibroblasts) The seminiferous tubules are covered by the lamina propria, which consists of a basement membrane (Figs. 1.15 – 1.16), a layer of collagen, and the peritubular cells. Peritubular cells, together with the layer of collagen, form the lamina propria that covers the seminiferous tubules. Peritubular cells are stratified around the tubules and form concentric layers that are separated by collagen. This structure differentiates the human from that of most other mammals, whose seminiferous tubules are surrounded

46 by only 2 to 4 layers of myofibroblasts. Peritubular cells produce panactin, desmin, gelsolin, smooth muscle , and actin, which are reportedly involved in cellular contractility (Holstein et al., 1996). It is proposed that the contraction of peritubular cells initiates the movement of mature sperm towards the exit of the seminiferous tubules.

§ Sertoli cells Sertoli cells are somatic columnar cells which rest on the basement membrane of the seminiferous tubule (Figs. 1.14 – 1.16). The Sertoli cells have an extensive cytoplasm, which extends through the width of the germinal epithelium and envelops the developing germ cells throughout spermatogenesis. Sertoli cells are bound to one another by junctional complexes containing extensive tight junctions. Sertoli cells occupy approximately 17 – 19% of the volume of the seminiferous epithelium of adult rats (Wong and Russell, 1983; Russell et al., 1990) and one Sertoli cell sustains approximately 50 germ cells (Wong and Russell, 1983).

There are two types of Sertoli cells: Type A and type B (Morales and Clermont, 1993; Russell, 1993a, b). Type A Sertoli cells transform during the 14 stages of the epithelial cycle into type B Sertoli cells. This enables them to adapt to the cellular changes occurring during the development and movement of germ cells (Fawcett, 1975; Parvinen, 1982). In adulthood, these cells are mitotically inactive.

Sertoli cells have multiple functions, including: a) providing structural support; b) creating an impermeable and immunological barrier; c) secreting factors that regulate spermatogenesis and spermiogenesis; d) secreting factors that regulate the function of Leydig cells and peritubular cells; e) secreting tubular fluid; and f) the phagocytosis of discarded spermatid cytoplasm—the residual body (Fawcett, 1975; Bardin et al., 1988; Jégou, 1992; Griswold, 1998; Zhu et al., 2000).

Sertoli cells produce a number of specialised proteins, , growth factors, opioids, , and that may have specific roles in the process of spermatogenesis. Some compounds are secreted from the apical (luminal) face of the Sertoli cells and they may have a role in regulating epididymal function or may be involved in the process of sperm maturation. Other secretions that occur through the basal surface of Sertoli cells may affect the function of the anterior pituitary gland and Leydig cells (Amann, 1989). Sertoli cells express the receptor in a stage- specific manner and produce androgen binding protein, which binds and concentrates testosterone inside the tubules (Bremner et al., 1994b; Vornberger et al., 1994). Sertoli cells may be the primary mediators of -mediated regulation of spermatogenesis because testosterone withdrawal causes retention of mature

47 spermatids and premature release of round spermatids (Russell and Clermont, 1977; Ghosh et al., 1991; Kerr et al., 1993; O’Donnell et al., 1996). Sertoli cells contain follicle- stimulating hormone (FSH) receptors. Therefore, the effects of FSH on the tubules must be mediated through the action of Sertoli cells. These effects include the FSH-induced stimulation of spermiogenesis and the autocrine interactions between Sertoli and Leydig cells (Griswold, 1998). Sertoli cells secrete the hormone inhibin in response to FSH. Inhibin facilitates the Leydig cells’ response to luteinizing hormone (LH).

§ Germinal cells Spermatogonia, immature spermatogenic cells (Figs. 1.14 – 1.16), are situated adjacent to the basement membrane (Fig. 1.16) of the seminiferous tubules. There are three types of spermatogonia: spermatogonia type A pale (Ap), spermatogonia type A dark (Ad) and type B spermatogonia. The Ap spermatogonia have a pale cytoplasm and a round or ovoid nucleus with finely granular chromatin and peripheral nucleoli. The Ad spermatogonia have dispersed chromatin and central nucleoli. Type A spermatogonia serve as stem cells for the germinal epithelium and go through several cycles of mitosis to produce further type A spermatogonia and type B spermatogonia (Ehmcke and Schlatt, 2006). Type B spermatogonia undergo final mitotic division and produce primary spermatocytes (Figs. 1.14 – 1.15). Primary spermatocytes migrate to the adluminal compartment (Fig. 1.16) of the seminiferous tubule before commencing their first meiotic division. They are the largest germ cells in the seminiferous tubules and occupy the middle region of the germinal epithelium. Their cytoplasm contains large nuclei with coarse clumps or thin threads of chromatin. The first meiotic division of the diploid primary spermatocytes produces two haploid secondary spermatocytes (Fig. 1.15) with less-dense nuclear chromatin. At the end of the second meiotic division, each of two secondary spermatocytes produces two haploid spermatids. Spermatids are smaller than the primary or secondary spermatocytes. There are two types of spermatids: early or round spermatids and late or elongated spermatids (Figs. 1.14 – 1.15). The spermatids differentiate into sperm by spermiogenesis, which has four phases: Golgi, cap, acrosomal, and maturation phases. The small, dark-staining heads of the maturing spermatids are embedded in the cytoplasm of Sertoli cells and their tails extend into the lumen of the seminiferous tubule (Fox, 1996) (Fig. 1.16).

1.3.1.2 Testicular structure: Interstitial compartment The interstitial compartment of the testes contains the Leydig cells, fibroblasts, , mast cells, blood, lymph vessels, and nerves (Connell, 1976; Pinart et al., 2001) (Figs. 1.14 & 1.16). This compartment comprises about 2.6% of the total testicular

48 volume of animals. However, this compartment occupies approximately 12 – 15% of the total testicular volume of humans and 10 – 20% of this is occupied by Leydig cells.

§ Leydig cells Leydig cells are large, polygonal, and eosinophilic cells (Fig. 1.16). They are the principal cell type found in the interstitium. They occur singly or in clumps and are generally found near blood and lymph capillaries, which surround the seminiferous tubules (Schulze and Rehder, 1984). There are different types of Leydig cells. In rodents, there are two generations of Leydig cells, fetal and adult, and in humans, there is an additional middle generation of neonatal Leydig cells (Prince, 2001). Fetal Leydig cells originate from embryonic stem cells and remain in the testes after birth but do not proliferate or contribute significantly to androgen levels. They are gradually lost from the testes by attrition (Faria et al., 2003).

Adult Leydig cells form during pubertal development and are derived from interstitium undifferentiated stem cells. Leydig cells develop in four stages: stem, progenitor, immature, and adult (Ge et al., 1996; Lo et al., 2004; Ge et al., 2005). Stem Leydig cells are spindle-shaped and do not express Leydig cell markers (Lo et al., 2004). However, they are capable of self-renewal and differentiate into progenitor Leydig cells. Progenitor Leydig cells are also spindle-shaped, have a low capacity for steroidogenesis, and mainly produce androsterone (Shan and Hardy, 1992; Shan et al., 1993; Ge et al., 1996; Ge and Hardy, 1998). Progenitor Leydig cells develop into immature Leydig cells, which are round and have a well-developed smooth ER and abundant lipid droplets (LDs) (Benton et al., 1995). Immature Leydig cells develop into adult Leydig cells during pubertal development, when testosterone is the main androgen (Ge and Hardy, 1998). An adult Leydig cell contains a round nucleus with dispersed chromatin and one or two nucleoli at the periphery. Its cytoplasm is rich in smooth ER and mitochondria with tubular cristae. It generally contains lipofuscin pigment, which is the final product of endocytosis and lysosomal degradation. Adult Leydig cells also contain LDs, in which testosterone synthesis begins.

The main function of Leydig cells is to differentiate and secrete testosterone, which is important for embryonic development, sexual maturation, and reproduction. Testosterone either associates with androgen binding protein and moves into the seminiferous tubule lumen to regulate spermatogenesis or it further metabolises into different steroids. Leydig cells express the androgen receptor, as do the Sertoli cells. Leydig cell dysfunction is associated with spermatogenic damage in rodents (Rich and De Kretser, 1977; Rich et al., 1979). Rodent data have demonstrated that selective

49 elimination of Leydig cells, interruption of testicular testosterone transport, and specific Sertoli cell androgen receptor knockout models all provoke profound alterations in germ cell maturation (Takamiya et al., 1998).

In the cytoplasm of Leydig cells a number of marker substances for neural and neuroendocrine cells have been reported in human and rat testes. These include neurofilament protein 200, , NCAM, MAP-2, S-100, CB, calretinin, and parvalbumin (Michetti et al., 1985; Dráberová et al., 1986; Tähkä, 1986; Schulze et al., 1987; Kägi et al., 1988; Vernon and Sage, 1989; Mayerhofer et al., 1992; Davidoff et al., 1993).

§ Other cells of interstitial compartment In addition to Leydig cells, other cell types are present in the interstitial compartment, including fibroblasts, macrophages, and a small number of mast cells. Macrophages represent approximately 25% of all interstitium cells. They are scattered in the interstitial tissue, closely attached to and mixed with the Leydig cells. One macrophage is present for approximately 10 – 50 Leydig cells. Although the function of macrophages is not clear, they secrete cytokines, which may affect the function of Leydig cells, including their proliferation, differentiation, and steroid production (Hales, 2007). It is thought that testicular macrophages might have a role in the regulation of capillary permeability in the testes, similar to macrophage function in the kidney (Niemi et al., 1986). Mast cells are present in the interstitial compartment and their numbers differ in different mammalian species (Anton et al., 1998). They are abundant in humans, swine, and stallions (Anton et al., 1998; Meinhardt et al., 1998). Mast cells are absent in the testicular parenchyma of rats, dogs, cats, bulls, and deer (Anton et al., 1998).

1.3.2 Structure and function of epididymis The epididymis is a single, highly convoluted long tubule that links the efferent ducts to the vas deferens. The length varies from 3 to 4 m in humans, 80 m in horses, and approximately 1.8 m in rats (Maneely, 1959; Turner, 1979). The epididymis can be divided into three regions: the caput (head), corpus (body), and cauda (tail) (Cornwall, 2009) (Fig. 1.15). The epididymis is made up of two major compartments: the pseudostratified epithelium (wall), surrounded by a smooth muscles, and the lumen (Robaire and Hermo, 1988) (Fig. 1.17).

1.3.2.1 Epididymal cell types The epididymal epithelium contains several different types of cells that vary in morphology, relative distribution, and functions (Robaire and Hermo, 1988). As identified

50

Figure 1.17: Morphology of rat epididymis. Photomicrograph of cross section of epididymis stained with Haematoxylin-Eosin shows lumen and wall, surrounded by connective tissue (CT). Epididymal wall is lined by pseudostratified columnar epithelium, cells (star) of which possess non-motile cilia (arrowhead). Lumen contains sperm cells and luminal fluid. Y. Makeyeva, present work.

in light microscopic preparations, these cells are: principal, basal, clear, apical, narrow, and halo cells (Cleland, 1957; Orgebin-Crist et al., 1975; Cooper, 1986; Robaire and Hermo, 1988) (Fig. 1.18).

The epididymis plays an important role in sperm transport, concentration, protection, storage and maturation. The maturation of sperm in the epididymis results in sperm cell motility and their ability to fertilise an oocyte (Caballero et al., 2010).

§ Principal cells Principal cells (Fig. 1.18) are the primary cell type in the epididymis and comprise approximately from 65 – 80% of the total epithelial cell population of the epididymis (Trasler et al., 1988; Cornwall, 2009). These cells appear along the entire length of the tubule (Kierszenbaum and Laura, 2007). However, they are structurally different in each region in a number features such as: height, incidence, size, intracellular distribution of vacuoles, shape of nuclei, and the distribution of chromatin within them (Robaire and Hermo, 1988). These differences are reflected in the appearance and organisation of their secretory apparatus (ER, Golgi apparatus, and secretory granules) and endocytic organelles (coated pits, endosomes, multivesicular bodies and lysosomes). They are tall, columnar cells that extend the full thickness of the epithelium, from basement lamina to lumen, and vary in height from 30 to 60 µm. They have a single round or elliptical nucleus and their free surfaces have long stereocilia. These stereocilia are of variable diameters

51

Figure 1.18: Schematic representation of cell types and position in epididymis. This schematic diagram represents cellular organisation of rat epididymis. Adult epididymis consists of pseudostratified epithelium of several cell types, such as: principal, clear, basal, narrow, apical, and halo cells. Lumen contains spermatozoa. Adapted from Cornwall (2009).

and have a tendency to branch (Horstmann, 1962; Nicander, 1965; Hoffer et al., 1973). The rat epididymis consists of tall principal cells (45 µm) with long stereocilia in the proximal portion of the head. In the tail of the epididymis, the principal cells diminish in size (10 µm) and become cuboidal with short stereocilia (Clermont and Flannery, 1970). The free surface of the principal cells between the bases of the stereocilia has highly irregular contours, numerous shallow depressions, and deeper, pit-like invaginations. These invaginations become coated vesicles and multivesicular bodies, located on the apical and supernuclear cytoplasm of principal cells. In other cells this process reflects pinocytotic uptake of protein-rich fluid (Roth and Porter, 1964; Droller and Roth, 1966; Friend and Farquhar, 1967). Principal cells contain an elaborate Golgi apparatus, abundant rER, and long and occasionally-branched mitochondria (Hoffer et al., 1973). In addition, the multivesicular bodies of the principal cells contain a large number of small vesicles (Hermo et al., 1988).

Principal cells exhibit the morphological features of cells that are actively involved in secretion and absorption (Burgos, 1964; Brooks and Higgins, 1980; Brooks, 1981). They contain a well-developed endocytic apparatus, including coated pits on the apical cell surface, pinocytic and coated vesicles, endosomes, multivesicular bodies, and

52 lysosomes (Robaire et al., 2002). As well as their absorptive features, the principal cells contain the organelles involved in the classical pathway of protein secretion referred to as “merocrine secretion” (Robaire et al., 2002). Principal cells secrete a variety of substances such as ions, small organic molecules, and glycoproteins (Hamilton et al., 1969; Bedford, 1975; Orgebin-Crist et al., 1975; Robaire and Hermo, 1988). Evidence that principal cells secrete glycoproteins was obtained from a radioautographic study and from EM using radioactive amino acids or sugars (Neutra and Leblond, 1966; Flickinger, 1979, 1981; Flickinger et al., 1984; Flickinger, 1985). Principal cells of the caput epididymis also secrete immobilin, a glycoprotein responsible for immobilising epididymal spermatozoa in rats (Turner and Giles, 1982; Usselman and Cone, 1983; Usselman et al., 1985). Principal cells are also involved in secretion of glycerylphosphorylcholine, carnitine, sialic acid, and steroids (Dawson et al., 1957; Marquis and Fritz, 1965; Hamilton et al., 1969; Rajalakshmi and Prasad, 1969; Riar et al., 1973; Brown-Woodman et al., 1976; Robaire and Hermo, 1988). Another of their functions is absorption of fluid and particulate matter. They are found to absorb a variety of absorptive and fluid phase markers, injected into the rete testis or microinjected directly into epididymal tubules (Mason and Shaver, 1952; Grant, 1958; Burgos, 1964; Nicander, 1965; Sedar, 1966; Hoffer et al., 1973).

§ Basal cells Basal cells (Fig. 1.18) are small, flat, hemispherical cells found throughout the epididymis (Robaire and Viger, 1995). These cells reside at the base of the epithelium in direct contact with the basement membrane (Robaire and Hermo, 1988). They constitute 12% of the total epithelial cell numbers in the initial segment of the epididymis and 21% in the corpus and cauda epididymis (Trasler et al., 1988). Basal cells possess the cytoplasmic projections that extend along the base of the epididymal epithelium, making contact with adjacent principal cells (Veri et al., 1993; Gregory et al., 2001). Morphological studies provide evidence that basal cells are equipped with the Golgi apparatus and possess abundant secretory granules (Hermo and Robaire, 2002).

There is limited understanding of the functions of basal cells. It has been suggested that they have a supportive role for the epithelial structure on the basis of the abundant presence of microfilaments in basal cells that have been observed in ductus deference and epididymal epithelium of humans (Hoffer, 1976; Vendrely, 1981) and mice (Abe and Takano, 1989).

Basal cells express an Yf protein (Yf subunit of glutathione S- P) along the base of the epididymal tubule in the corpus and proximal cauda epididymis of rats.

53 This finding suggests that basal cells may provide a protective system against oxygen stress for the epididymal epithelium and the maturing and stored spermatozoa (Veri et al., 1993). Basal cells in the human epididymis have a high level of copper-zinc superoxide dismutase, a scavenger of superoxide anion radicals (Nonogaki et al., 1992). Therefore, they could play role in local defence mechanisms against tissue damage mediated through superoxide anion radicals.

§ Clear cells Clear cells (Fig. 1.18) are large, active endocytic cells that present only in the caput, corpus, and cauda regions of the epithelium and are found in many species, including humans (Brown and Montesano, 1981; Cooper, 1986; Robaire and Hermo, 1988). They have numerous vesicles of various sizes in the apical region, which includes: clear vesicles, larger vacuoles, and dense, multivesicular bodies that are located in the super-nuclear region. These cells have a fine flocculent material with 35-nm particles and membranous profiles that have an irregular, flattened or spherical form. The mid- region of clear cells contains secondary lysosomes and various quantities of LDs near their bases (Robaire and Hermo, 1988). In the middle or basal third of the cell, a slightly indented pale nucleus is present. The microvilli of these cells are short and irregularly spaced and this distinguishes them from the uniformly-spaced, tall stereocilia of the neighbouring epithelial principal cells. The Golgi apparatus of a clear cell is formed from several short, well-dispersed stacks of saccules that are located in the supernuclear region. Cisternae of the ER are sparse and occasionally studded with ribosomes.

While the principal cells are involved primarily, but not exclusively, in secretion, the clear cells are involved chiefly in absorption (Moore and Bedford, 1979b; Robaire and Hermo, 1988). The view that clear cells are specialised for absorption is supported by their fine structure, characterised by prominent lysosomal inclusions and apical canaliculi, which are typical of epithelial cells absorbing proteins and carbohydrates. The clear cells absorb horseradish peroxidise introduced into the lumen of the cauda epididymis of rats and are actively involved in absorptive endocytosis of cationic ferritin (Moore and Bedford, 1979b; Hermo et al., 1988).

Clear cells take part in the uptake and disposal of the contents of CDs originating from spermatozoa (Hermo et al., 1988). The vacuolar H+ATPase (V-ATPase), a multi- subunit enzyme, that couples ATP hydrolysis to proton pumping across the membrane, as well as carbonic anhydrase ІІ and soluble AC, are selectively expressed in clear cells and underscores the role of these cells in acidifying the lumen of the epididymis. CIC-5, a member of the voltage-gated CIC chloride channel family, is also expressed

54 exclusively in clear cells and partially co-localises with the H+ATPase in their apical region (Brown and Breton, 1996; Pietrement et al., 2006). Breton’s group proposed that, in clear cells, apical membrane accumulation of V-ATPase is triggered by a soluble AC- dependent rise in cAMP in response to alkaline luminal pH (Jensen et al., 1999; Isnard- Bagnis et al., 2003; Pastor-Soler et al., 2003).

§ Apical and narrow cells The initial segment of the rat epididymis contains cells that differ from principal, basal, and halo cells by virtue of the fact that their nuclei are located in the upper half of their cytoplasm (Moore and Bedford, 1979b; Brown and Montesano, 1981; Abou-Haïla and Fain-Maurel, 1984; Cooper, 1986; Robaire and Hermo, 1988). These cells have been known by many names including: holocrine cells (Martan and Risley, 1963; Edmonds and Nagy, 1973; Feuchter et al., 1988), dark cells (Humar et al., 1990; Morales and Cavicchia, 1991), apical cells (Hoffer et al., 1973; Moore and Bedford, 1979b, a; Goyal, 1985; Goyal and Williams, 1991), narrow columnar cells (Abou-Haïla and Fain- Maurel, 1984; Arya and Vanha-Perttula, 1986; Morales and Cavicchia, 1991), mitochondria goblet cells (Abou-Haïla and Fain-Maurel, 1984), flask cells (Burkett et al., 1987), and apical mitochondria-rich cells (Palacios et al., 1991; Martínez-García et al., 1995). Apical and narrow cells in the adult rat epididymis are distinct cell types, differing from each other as well as from principal and basal cells in their structure, distribution, and expression of different proteins (Adamali and Hermo, 1996) (Fig. 1.18).

§ Halo cells Halo cells (Fig. 1.18) are small cells with a narrow rim of clear cytoplasm. These cells are found throughout the epididymis and in various positions within the epithelium, but not spanning it. Halo cells are usually located at the base of the epithelium (Robaire and Hermo, 1988). In the adult albino rat, the halo cells appear rounded or polygonal in shape with nuclei that are round to oval and slightly indented, are located centrally, and have prominent nucleoli (Ahmed et al., 2009). The nucleus is surrounded by a large quantity of electron lucent cytoplasm. The cytoplasm is scanty and between it and the membrane of the surrounding epithelial cells lies an area called the “halo” (Cleland, 1957). This cytoplasm contains a few organelles and some electron-dense granules in close proximity to the Golgi apparatus (Serre and Robaire, 1998; Ahmed et al., 2009). Adult albino rats with ischemia reperfusion injury have more halo cells than controls, together with leukocyte migration from blood vessels (Ahmed et al., 2009). This suggests that these cells play a role in the immunological barrier of the male reproductive tract (Palacios et al., 1993; Veri et al., 1993; Ahmed et al., 2009).

55 1.3.2.2 Epididymal luminal environment Sperm are not capable of biosynthesis (Hammerstedt, 1981) and their maturation depends on the unique interaction between spermatozoa and the luminal fluid in the surrounding epithelium (Fig. 1.19). The luminal fluids provide the necessary biocatalysts and ions (Dacheux et al., 1989; Jones, 1989; Turner, 1991).

The epididymal lumen contains one of the most complex fluids within the endocrine glands. This fluid is highly concentrated and constantly changing (Dacheux and Dacheux, 2002). More than 95% of the fluid that carries the spermatozoa out of the testes through the efferent ducts is reabsorbed into the caput epididymis (Clulow et al., 1998). Moreover, 62% of the fluid remaining in the caput is reabsorbed as the luminal fluid travels along the epididymal tubule to the distal cauda epididymis (Turner, 1991).

This progression leads to a sharp increase in the spermatocrit (the volume of sperm cells as a percentage of the volume of the fluid) and to changes in the composition of the luminal fluid throughout the length of the epididymal duct (Crabo, 1964; Jenkins et al., 1980; Turner, 1991). The mammalian epididymis is reported to be relatively acidic, pH 5.5 to 6.8 (Levine and Kelly, 1978; Caflisch and DuBose, 1990; Rodriguez-Martinez et al., 1990; Caflisch, 1992). In the rat, the pH of testicular fluid is 7.4 while in the proximal epididymis, it is 6.6 (Levine and Kelly, 1978). In the ram the pH of the fluid leaving the rete testis is pH 7.81 and when it arrives in the cauda epididymis it is more acidic with pH 6.05 (Cosentino and Cockett, 1986).

The epididymal plasma contains a wide range of ions and different organic constituents such as sialomuco-proteins, lipoproteins, enzymes, and lipids. These substances are not only produced in the epididymal epithelium, but also absorbed from the circulation by trancytosis through the epithelium (Dacheux et al., 2003) or enter the epididymis from the testes (Cosentino and Cockett, 1986).

The concentration of intraluminal proteins between the caput and cauda of the epididymis remains quite constant, being about 33% of protein concentration of serum (Turner, 1991). Luminal fluid proteins are thought to be synthesised post-testicularly by epithelial cells lining the epididymal duct, such as clusterin (SGP-2), immobilin, acrosomal stabilising factor, α-lactalbumin-like protein, and several enzymes important in carbohydrate steroid and protein biochemistry (Turner, 1991). These proteins are then secreted apically into the lumen, where they contact the surface of spermatozoa (Kirchhoff, 1998). Schematic representation of the potential interactions between sperm and epididymal luminal proteins is presented in Fig. 1.19.

56

Figure 1.19: Schematic representation of potential interactions between spermatozoa and epididymal environment. This diagram represents proteins and mechanisms by which they interact with spermatozoa. 1) Proteins, such as hydrophobic or sperm binding proteins interact with spermatozoa membrane. 2) Secretory proteins are processed before interacting with spermatozoa. 3) Proteins, such as enzymes, directly affect spermatozoa’s surface. 4) Secreted proteins with reduced affinity for surface of spermatozoa prevent sperm agglutination or peroxidation. 5) Proteins, released from spermatozoa surface, influence epididymal regulation. Any changes to the protein microenvironment in intraluminal fluid may be relevant to development of maturing spermatozoa. Adapted from Dacheux et al. (2003).

In the rabbit, there are no fertile spermatozoa in the caput or proximal corpus epididymis and fertility is acquired when the spermatozoa pass through the distal region of the corpus epididymis (Orgebin-Crist, 1967). In the rat, spermatozoa acquire their fertilising ability in the proximal cauda epididymis (Dyson and Orgebin-Crist, 1973) and this indicates that spermatozoa need the special lumen environment created by the epithelium of the corpus epididymis for maturation (Orgebin-Crist, 1967). For spermatozoa to mature, they need not only to acquire fertilising ability, but also the ability to recognise the zona pellucida (ZP) in order to bind to and penetrate ova. This ability is associated with surface changes on the spermatozoon head in the rat (Orgebin-Crist and Fournier-Delpech, 1982) and mouse (Saling, 1982).

Intraluminal proteins are involved not only in spermatozoa maturation, but also in spermatozoa storage. Spermatozoa in the cauda lumen of the epididymis of rats, hamsters, guinea pigs, and humans are immotile, being in a quiescent state in their native fluids. Cauda spermatozoa of hamsters and rats are kept immotile by a

57 proteinaceous factor in luminal fluid and this contributes to the high viscoelasticity of this fluid (Turner and Reich, 1985). This proteinaceous factor that inhibits spermatozoa motility was first found in the rat cauda epididymis (Turner and Giles, 1982) and later isolated and termed “immobilin” (Usselman and Cone, 1983). In contrast, the luminal fluid in the human epididymis is not highly viscous (Turner and Reich, 1985). Nevertheless, spermatozoa are essentially immotile in the cauda epididymis. This situation is also the case in bulls. This quiescent state of human spermatozoa is a result of interactions between proteinaceous factors, such as immobilin, with acidic intracellular pH.

The luminal fluid of the epididymis contains epididymosomes, which are small membranous vesicles released by principal epithelial cells through apocrine secretion (Aumüller et al., 1997; Aumüller et al., 1999). They were first reported by Yanagimachi et al. (1985). These vesicles have a similar structure to exosomes with diameters of 50 – 500 nm. Epididymosomes have been found in humans, mice, sheep, rats, hamsters, and bulls (Yanagimachi et al., 1985; Fornes et al., 1995; Légaré et al., 1999; Eickhoff et al., 2001; Frenette and Sullivan, 2001; Gatti et al., 2002; Frenette et al., 2005; Gatti et al., 2005; Frenette et al., 2006; Rejraji et al., 2006). Epididymosomes are characterised by a very high ratio of and phospholipid with sphingomyelin as the major phospholipid constituent (Rejraji et al., 2006). Epididymosomes are associated with complex patterns of proteins. The protein composition of epididymosomes varies from one epididymal segment to another and selected proteins from epididymosomes are transferred to spermatozoa during the epididymal maturation (Sullivan et al., 2007). While the function of these epididymosomes is not fully understood, they possibly assist in delivering some proteins to the spermatozoa. They may also serve as signalling centres in the luminal of the epididymis. Epididymosomes may also bind to proteins to remove them from the lumen and deliver them to epithelial cells for their endocytosis (Cornwall, 2009).

1.3.2.3 Cytoplasmic droplet of spermatozoa The cytoplasmic droplet (CD) (Figs. 1.15 – 1.16) is a small remnant of the original cytoplasm of the spermatids, phagocytised as the residual body by the Sertoli cells (Cooper and Yeung, 2003). The CD remains with the spermatozoa after spermiation has occurred and is attached to the connecting piece (proximal droplet) of the spermatozoa in the seminiferous tubules of the testes, rete testes, efferent ducts, and initial segment of epididymis (Hermo et al., 1988; Robaire and Hermo, 1988; Oko et al., 1993; Kerr et al., 2006). Following this, in the caput epididymis, the CD gradually migrates along the middle piece of the tail up to the annulus and occupies position next to the annulus of

58 the tail. In the corpus epididymis, the CD is laterally displaced (distal droplet) (Hermo et al., 1988; Oko et al., 1993). The CDs in the cauda epididymis become dissociated and eventually detach from the spermatozoa. The migration of the CD from the spermatozoon connecting piece along the middle piece starts within the efferent ducts of the epididymis and finishes in the caput or corpus epididymis.

The morphology of CDs has been investigated in studies that have used light and EM immunolabelling, subcellular fractionation, and exogenous and endogenous glycosylation. These have found that CDs contain loosely organised saccules, which have been identified as Golgi elements with endogenous glycosylation capability. These saccules spiral inside the CD as it moves along the flagellum and maintain a close association with the plasma membrane of the spermatozoa. Plasma membrane modifications mostly occur during the distal migration of the CD along the spermatozoon tail and before its loss in the cauda epididymis. This suggests that the CD may be involved in spermatozoa plasma membrane modification and this is an important step in maturation (Oko et al., 1993). A CD also contains numerous vesicular elements and small particles derived from the Golgi apparatus, sperm histones, and ubiquitin (Moreno et al., 2000b; Sutovsky, 2003; Kuster et al., 2004). These components are present during spermiation. They are not detected in mature, ejaculated spermatozoa, indicating that the CDs are lost during epididymal transit (Moreno et al., 2000a).

The function of the CD is not fully understood. However, it has been suggested that the shedding of the CD may regulate the volume of sperm (Cooper, 2011). After spermatozoa are ejaculated, the detachment of the CD, filled with permeant osmolytes, would reduce the sperms’ uptake of water and thus keep its flagellum straight (Cooper, 2011). On the other hand, if the CD is still attached to the spermatozoa in the female reproductive tract, it swells, causing spermatozoa flagellum to bend like hairpins (Yeung et al., 1999; Sipilä et al., 2002). The retention of CDs on ejaculated spermatozoa is associated with infertility in many mammalian species (Waberski et al., 1994; Yeung et al., 1999; Amann et al., 2000; Thundathil et al., 2001; Kuster et al., 2004; Lovercamp et al., 2007).

Human spermatozoa are different from that of other species because the CD remains attached to it in the female reproductive tract (Mortimer et al., 1982; Abraham- Peskir et al., 2002; Cooper et al., 2004). In this case, the CD may continue to regulate fluid intake as spermatozoa travel through the female duct. When spermatozoa reach the oocyte, the CD may release all its osmolytes, causing the spermatozoa to swell. This may activate their motility and penetration of the ZP (Yeung et al., 2003; Cooper, 2011).

59 1.3.2.4 Epididymal stereocilia The epithelial cells of the epididymis possess numerous apical, non-motile, microvillus-like projections that are referred to as stereocilia (Fig. 1.17) (Reichel, 1921; Fawcett and Porter, 1954). The stereocilia are approximately 80 µm in the head region of the epididymal duct decreasing to 40 µm in the epididymal tail and ductus deferens (Horstmann, 1962; Popovic et al., 1973; Dym, 1983).

The stereocilia contain a variety of membrane pumps, channels, and transporter systems and can maintain their proper volume of as well as the appropriate pH for both the principal cell and epididymal lumen (Primiani et al., 2007). Stereocilia provide the cell with a large surface area to interact with molecules present in the lumen, a situation which may facilitate the uptake and transport of substances by pinocytosis (Ikeda, 1906; Burgos, 1964). Stereocilia are actively engaged in phagocytosis. Membrane-bound vesicles were found in the lumen of the rat caput and cauda epididymis, which were possibly formed by exocytosis, suggesting that vesicles may originate from stereocilia tips. Some of these membrane-bound vesicles were found to be in contact with the head or the tail of maturing spermatozoa (Păunescu et al., 2014).

1.3.3 Structure and function of spermatozoa The spermatozoon is an elongated cell (about 65 µm long) and consists of three main components: head, neck (connecting piece), and tail (flagellum). The tail is subdivided into three segments: the middle, principal, and end pieces. The structure of the spermatozoon head varies between different species of mammals: bull sperm have paddle-shaped heads, rodent sperm have hook-shaped heads, and chicken sperm have spindle-shaped heads. In humans, the spermatozoa head has a flattened, pear shape (Fig. 1.20).

The spermatozoon head is almost entirely occupied by the nucleus, which contains condensed chromatin. The nucleus of human spermatozoon head contains nuclear vacuoles that are areas of dispersed chromatin. The anterior two thirds of the nucleus are covered by the acrosomal cap (acrosome). This is a large secretory-granule- like organelle, containing glycoproteins, hydrolytic enzymes, and oocyte-interacting proteins for penetrating the oocyte (Friend, 1977; Yanagimachi, 1994; Kopf, 2002). The neck (connecting piece) of sperm contains vestiges of centrioles and one of these gives rise to the axoneme of the flagellum. In human spermatozoa, a significant amount of cytoplasm—CD—often remains in the connecting piece after ejaculation (Fig. 1.20). The middle piece of the spermatozoa tail contains elongated mitochondria, which are wrapped helically around the coarse fibres and the axonemal complex. The mitochondria

60 are prevented from sliding into the principal piece of spermatozoa tail by the annulus, a fibrous thickening, which is located beneath the plasma membrane. The mitochondria in the middle piece are the source of energy for the movement of the spermatozoa’s tail and sperm motility. In rodents, the middle piece of the spermatozoa tail is longer than the spermatozoa head and has a relatively small CD (Cooper, 2005, 2006, 2007). On the other hand, the middle piece of human spermatozoa is as long as the spermatozoa head and has a relatively larger CD at the neck, compared to that of rodents (Cooper, 2011). The principal piece occupies most of the spermatozoa tail. It consists of a central core, which contains the axoneme and coarse fibres continuing from the middle piece. The end piece of the spermatozoa tail is a short, tapered part that only contains the axoneme.

Spermatozoa leave the lumen of the seminiferous tubule after completing spermatogenesis and spermiation as immotile cells, unable to fertilise oocytes. During their passage through the epididymis, spermatozoa become fully mature, with the ability to swim and to recognise and fertilise eggs. However, freshly ejaculated spermatozoa have not yet acquired a state of readiness to fertilise the oocyte (Jaiswal and Eisenbach, 2002).

1.3.3.1 Spermatozoa activation in female reproductive tract When mammalian sperm enters the female reproductive tract, the change in pH and osmolarity of surrounding fluid causes them to mature through the stages of capacitation, hyper-activation, and the acrosome reaction (AR) (Cohen-Dayag et al., 1995; Jaiswal et al., 1998; Jaiswal and Eisenbach, 2002; Breitbart, 2003).

Capacitation takes about 5 – 6 hrs in humans and is completed only when the sperm arrive in the oviduct. Spermatozoa achieve capacitation only once in their lifetime and those in which capacitation had ceased did not achieve capacitation again (Cohen- Dayag et al., 1995). During capacitation, spermatozoa undergo extensive biochemical and functional changes, including changes in glycoproteins, lipids, and ion channels in the sperm plasma membrane and a significant change in the resting potential of this membrane. Capacitation alters two crucial aspects of spermatozoa behaviour: it increases the motility of the flagellum and allows them to undergo the AR. A form of spermatozoa motility, called hyper-activation typically increases the amplitude of the flagellar bend asymmetry of flagellar beat (Yanagimachi, 1994; Ho and Suarez, 2001). Only spermatozoa that are capacitated and hyper-activated can penetrate the oocyte (Demott and Suarez, 1992; Cohen-Dayag et al., 1995; Jaiswal and Eisenbach, 2002; Eisenbach and Giojalas, 2006).

61

Figure 1.20: Diagram of rat and human spermatozoa. Diagram on left shows structure of spermatozoa of rats and humans. Rat spermatozoon has hook-shaped head, whereas human spermatozoon has rounded, pear-shaped head. Spermatozoa heads are covered by acrosomal membrane and contain equatorial and post-acrosomal segments. Connecting piece contains centriole. Flagellum is divided into middle, principal, and end pieces. Middle piece is separated from principal piece by annulus. In rats, cytoplasmic droplet is present only in testicular and epididymal spermatozoa and migrates along middle piece to occupy position next to annulus of tail, as shown in diagram, before detaching from tail before ejaculation. In humans, cytoplasmic droplet is present in testicular and epididymal spermatozoa. Cytoplasmic droplet often remains in connecting piece after ejaculation. Diagram on right shows human spermatozoon from two different angles (90° rotations). Nucleus occupies most of head space. Centriole and redundant nuclear envelope are located in connecting piece at base of head. In middle piece, axoneme is surrounded by outer dense fibres and mitochondria; in principal piece, it is surrounded only by fibrous sheath. Adapted from Darszon et al. (2011).

On encountering an egg, capacitated spermatozoa first must penetrate the layers of granulosa cells, making use of a hyaluronidase enzyme on the surface of the sperm. It can then bind to the ZP, which causes spermatozoa to undergo the AR. This is a specialised form of exocytosis because it includes loss of membrane and acrosomal content (Russell et al., 1979; Yudin et al., 1988; Gerton, 2002; Meizel, 2004). However,

62 acrosomal exocytosis, an essential prerequisite for fertilisation, differs from other exocytotic processes: each sperm has a only one secretory vesicle (the acrosome); it occurs only once in a spermatozoon’s lifetime; multiple fusion points form between the outer acrosomal membrane and the overlying plasma membrane; acrosomal exocytosis is irreversible; and the spermatozoon loses membrane when the outer acrosomal membrane and plasma membrane fuse to form hybrid membrane vesicles and particles that are shed during exocytosis (Mayorga et al., 2007).

Capacitation and AR depend on Ca2+-signalling which comes from extracellular and intracellular sources (Florman and Ducibella, 2006; Darszon et al., 2011). The AR may be initiated by progesterone (which activate CatSper channels) and ZP sperm- binding protein 3 (ZP3) (Florman and Storey, 1982; Bleil and Wassarman, 1983; Kopf, 1991; S Melendrez et al., 1994; Yanagimachi, 1994; Walensky and Snyder, 1995; Jia et al., 1997; O'Toole et al., 2000; Jungnickel et al., 2001; Kopf, 2002; Costello et al., 2009; Litscher et al., 2009; Darszon et al., 2011). After a spermatozoon has undergone the AR and penetrated the ZP, it binds to the egg plasma membrane by overlying the tips of microvilli on the egg surface and fusing with the egg.

1.3.3.2 Spermatozoa chemotaxis Chemotaxis in the reproductive system is a guidance mechanism in which chemoattractants orient spermatozoa to the oocyte (Cosson, 1990; Eisenbach and Tur- Kaspa, 1999; Eisenbach and Giojalas, 2006; Kaupp et al., 2008; Teves et al., 2009). Because chemotaxis is a “local” mechanism, it is likely to act in the immediate surroundings of the oocyte-cumulus complex. Chemotaxis appears to be a two-step process: guidance first towards the cumulus cells that surround the oocyte, and then guidance within the cumulus mass towards the oocyte. Spermatozoa chemotaxis is well established in marine invertebrates with fertilisation in species like sea urchins, starfish and ascidians and it has also been demonstrated in mammals, such as humans, mice, rabbits, rats, horses, and pigs (Ralt et al., 1991; Ralt et al., 1994; Giojalas, 1998; Navarro et al., 1998; Oliveira et al., 1999; Serrano et al., 2001; Fabro et al., 2002; Eisenbach and Giojalas, 2006).

The number and identity of spermatozoa chemoattractants, secreted in the female genital tract, have not been fully identified. However, several possible putative chemoattractants have been proposed. One of them—follicular fluid—is a chemoattractant that causes spermatozoa chemotactic activity in humans in vitro (Ralt et al., 1991; Ralt et al., 1994; Sun et al., 2003). Chemoattractants may be secreted by the mature oocyte and the surrounding cumulus cells. These chemoattractants are

63 secreted within the follicle before ovulation and outside the follicle after oocyte maturation (Sun et al., 2005). The chemoattractant secreted by the cumulus cells is probably progesterone (Teves et al., 2006; Oren-Benaroya et al., 2008). In human and rabbit spermatozoa, progesterone is chemotactically active at concentrations as low as 10-11 – 10-10 M (Teves et al., 2006; Oren-Benaroya et al., 2008; Baldi et al., 2009; Guidobaldi et al., 2012).

The identity of the chemoattractant secreted from the oocyte is not yet known. However, it has been shown to be more potent than progesterone (Sun et al., 2005). Several possible oocyte-derived chemoattractants have been reported to attract human spermatozoa in vitro. These include CCL5b (Isobe et al., 2002), allurin (Burnett et al., 2011), CCL20 (Caballero-Campo et al., 2014), and A-type natriuretic peptide (Zamir et al., 1993). Alternatively, hydrophobic non-peptide molecule, which is associated with a carrier protein, has been proposed as an oocyte-derived chemoattractant (Armon et al., 2014).

Chemotaxis is characterised by directional changes in spermatozoa movement towards the source of the chemoattractant. It requires a fine-turned interplay of spermatozoa motility and chemoreception. In species with external fertilisation, most, if not all, of the sperm population is chemotactically responsive (Cosson, 1990; Eisenbach and Tur-Kaspa, 1999). However, in humans, only a small fraction of the sperm population (about 5 – 10%) is chemotactically responsive at any given time (Cohen-Dayag et al., 1994; Cohen-Dayag et al., 1995). The chemotactically responsive human sperm subpopulation consists of capacitated cells (Cohen-Dayag et al., 1995). Sperm chemotaxis is highly-dependent on spermatozoa hyperactive motility and Ca2+ ions (Ward et al., 1985). As was described in Section 1.3.2.2, proteins secreted by the epididymal epithelium together with the acidic pH of the luminal fluid keep sperm in an immotile state. When spermatozoa reach the female reproductive tract, the sperm’s inner Ca2+ levels increase, which initiates the symmetrical beat pattern of spermatozoa flagella that heralds motility (Visconti, 2009). The Ca2+ influx increases further during spermatozoa capacitation, and causes spermatozoa to hyper-activate, more detailed described in Section 1.3.3.1 (Suarez et al., 1993). The release of oviduct-binding proteins together with hyper-activation causes sperm to detach from their storage reservoir and move toward the oocyte (Demott and Suarez, 1992; Pacey et al., 1995; Gwathmey et al., 2003; Gwathmey et al., 2006; Ignotz et al., 2007; Ho et al., 2009; Ishijima, 2011). In vitro studies show that hyperactivity changes the motility path of spermatozoa from a straight-line trajectory to circular, figure-eight, or zig-zag movements, depending on the species (Yanagimachi, 1972; Fraser, 1977; Cooper et al.,

64 1979). During chemotaxis, spermatozoa react to chemoattractants by increased asymmetrical flagellar bends and sharp turning movements, similar to those of hyperactive motility (Spehr et al., 2003; Fukuda et al., 2004; Armon and Eisenbach, 2011). For spermatozoa to swim in the right direction, their movement must be coupled to the chemoattractant gradient, i.e., flagellum steering must be in register or in phase with the stimulus. When human spermatozoa swim up the ascending chemoattractant gradient, their flagella beat symmetrically and they swim in the same direction to the source of the chemoattractant (Spehr et al., 2004a). Moreover, the swimming speed of the spermatozoa increases because their flagella beat more frequently (Spehr et al., 2004a). On the other hand, when human spermatozoa swim away from the chemoattractant, the reduction in signal causes hyperactive motility, their flagella beat asymmetrically, and this causes them to turn around sharply and then swim towards the source of chemoattractant (Böhmer et al., 2005; Eisenbach and Giojalas, 2006; Friedrich and Jülicher, 2007). This activation of motility is caused by increased Ca2+ influx into the spermatozoa, either directly, through the activation of the sperm-specific cation channel, CatSper (Strünker et al., 2011; Brenker et al., 2012), or indirectly, by the activation of G protein-coupled ORs (Spehr et al., 2003; Fukuda et al., 2004).

1.3.3.3 “Neuronal” receptors in spermatozoa The spermatozoon plasma membrane expresses different types of “neuronal” receptors, including GABAA, GABAB, glycine, nicotinic acetylcholine, adrenergic, muscarinic acetylcholine, metabotropic glutamate, serotonin, and olfactory receptors (described in Section 1.1.5.1). Both spermatozoa and neurons can utilise certain

“neuronal” receptors for exocytosis, including GABAA, GABAB, adrenergic, muscarinic acetylcholine, nicotinic acetylcholine, serotonin, purinergic, ǁ, and receptors. Spermatozoa also can use specific “neuronal” receptors for capacitation, including GABAA, adrenergic, and cannabinoid receptors. They utilise other receptors for motility, fertilisation, and sperm “communication”, including nicotinic acetylcholine, purinergic, angiotensin ǁ, cannabinoid, and olfactory receptors.

Some types of “neuronal” receptors present in spermatozoa can influence fertilisation more than others. Ligand binding to several different types of neuronal receptors can initiate capacitation or AR in vitro or motility in vivo. These spermatozoa receptors may be activated via their specific ligands or via receptor cross talk. Some ligands present in the female reproductive tract may stimulate fertilisation. Others might be antagonistic, inhibiting fertilisation until the optimal time for spermatozoon-egg communication. Ligands could cause such inhibition by blocking some stages of

65 spermatozoa maturation, such as capacitation, or by stimulating the AR at an unsuitable time (Yanagimachi, 1994; Meizel, 2004).

1.4 Mast cells Mast cells are important effector cells in the (Metcalfe et al., 1997; Nilsson and Metcalfe, 2000; Metcalfe and Boyce, 2006; Theoharides and Kalogeromitros, 2006; Metz and Maurer, 2007; Galli et al., 2008). Mast cells occur in many organs and tissues, including the CNS, of mammals and avians (Dropp, 1972, 1976; Theoharides, 1990). In vascularised tissue, mast cells occur near small blood vessels, epithelia, smooth muscles, and peripheral nerves and usually are diffused through these tissues (Rakusan et al., 1990; Johnson and Krenger, 1992; Bienenstock et al., 1993; Marshall et al., 1993) (Fig. 1.21). In the brain, mast cells reside in the blood brain barrier (BBB) and their mediators interact with neurons, glia, microglia, blood vessels, the extracellular matrix and other hematopoietic cells (Florenzano and Bentivoglio, 2000; Khalil et al., 2007; Dong et al., 2014).

Mast cells are distinguished by their high content of electron-dense secretory granules, which are filled with large quantities of preformed and pre-activated immunomodulatory compounds. When activated, mast cells degranulate, releasing their granule compounds (mediators) into their surroundings (Wernersson and Pejler, 2014). Two types of mast cell degranulation have been described: “anaphylactic exocytosis” and “piecemeal exocytosis”. Both have been observed in vivo, ex vivo, and in vitro in mast cells of humans (Dvorak and Kissell, 1991; Dvorak et al., 1991; Dvorak et al., 1992; Dvorak and Morgan, 1997), mice (Dvorak et al., 1994), and rats (Chyczewski et al., 1996).

During the process of anaphylactic exocytosis, granules translocate from the interior of the cell towards the plasma membrane (Nishida et al., 2005). The process begins with extensive granule-granule and/or granule-plasma membrane fusions. A “degranulation channel” is generated and this is followed by an explosive release of the granules’ contents or entire granules to the outside of cells (Nishida et al., 2005). Unlike anaphylactic exocytosis, piecemeal exocytosis is characterised by the selective release of a few granules or portions of the granules’ contents at a time, without granule-granule and/or granule-plasma membrane fusions. In this process, granules largely retain their morphology, although some ultrastructural changes occur (Dvorak and Morgan, 1997; Dvorak, 2005b; Wernersson and Pejler, 2014). Alternatively, mast cells may be induced to elaborate cytokines and with no release of granule contents. Thus,

66 although the mast cell is well equipped to release large volumes of mediators, it is equally capable of a response tailored to the activating stimulus (Nigrovic and Lee, 2004).

The process of degranulation is divided into two events: the Ca2+-independent, microtubule-dependent translocation of granules to the plasma membrane and Ca2+- dependent membrane fusion and exocytosis (Nishida et al., 2005). The activation and degranulation of mast cells affect many physiological and pathological conditions. With respect to normal physiological functions, mast cells are known to regulate vasodilatation, vascular homeostasis, innate and adaptive immune responses, angiogenesis, and venom detoxification. On the other hand, mast cells have also been implicated in the pathophysiology of many diseases, such as asthma, atopic dermatitis, coronary artery disease, inflammatory bowel disease, chronic prostatitis, chronic rhinitis, fibrosis, fibromyalgia, migraine, multiple sclerosis, neurofibromatosis, osteoarthritis, rheumatoid arthritis, and coronary artery disease (Enerbäck, 1965; Claman, 1985; Sarin et al., 1987; Metcalfe et al., 1997; Castells, 1999; Nilsson and Metcalfe, 2000; Theoharides and Kalogeromitros, 2006; Gross and Pothoulakis, 2007; Balakumar et al., 2008; Galli et al., 2008; Galli and Tsai, 2008).

§ Mast cell distribution in rats In rats, mast cells are widely distributed in mesenteric lymph nodes, tongue and in the brain. Some are present in the sciatic nerve, and small numbers are found in other lymph nodes, in the heart, salivary glands, thymus, pancreas, cervix, vagina, uterus, epididymis, liver, skeletal muscle, ovaries, prostate, seminal vesicle, harderian glands, parathyroid, thyroid, mammary glands, skin, adipose tissue, preputial gland, and bone marrow. Occasional mast cells are scattered in the submucosa and serosa of the stomach, oesophagus, small and large intestine, and urinary bladder. Mast cells are rarely present in the lung, spleen, or adrenals (Majeed, 1994) and are not found in the parenchyma of testes (Gaytan et al., 1989; Majeed, 1994; Anton et al., 1998). There was a difference in mast cell numbers in young and older rats. Mast cells appear to be more abundant in Wistar rats than in Sprague-Dawley rats (Majeed, 1994).

§ Identification of mast cells In tissue sections stained with Haematoxylin and Eosin (HE), normal mast cells usually display a round-to-oval nucleus with clumped chromatin and indistinct or no nucleoli. They have moderately abundant cytoplasm and are oval, spindle, or polygonal in shape. The cytoplasm is amphophilic, and sometimes small slightly eosinophilic granules may be visible. HE staining is not a specific or reliable method for detecting

67

Figure 1.21: Rat tongue with mast cells. Photomicrograph of cross section of tongue stained with Haematoxylin and Eosin (HE) shows mast cells (MCs), indicated by arrows, and located between muscles (M) and blood vessels (BV). Some MCs have round-to-oval nuclei that stain dark blue. Cytoplasm stains pink and contains violet granules. In some MCs, nuclei are not visible, being covered with clumps of granules (arrowhead). Y. Makeyeva, present work.

mast cells in tissue sections because of variable cellular morphology (Shukla et al., 2005) (Fig. 1.21).

The most distinguishing morphological feature of mast cells is their high content of electron-dense lysosome–like secretory granules that occupy a major proportion of the cytoplasm of mature mast cells. The main criterion for the identification of mast cells is the presence of secretory granules, which are easily visualised with the use of various cationic dyes that produce the classical metachromatic staining of mast cells (Shukla et al., 2005; Krishnaswamy and Chi, 2006) (Fig. 1.22).

The most powerful method of detecting mast cells is EM. Studies of this sort have shown that mast cells possess a small monolobed nucleus, round-to-oval in shape. The mast cell surface has slender filiform cytoplasmic projections or narrow elongated folds. Its cytoplasm contains numerous membrane-bound granules, rER, Golgi vesicles, free ribosomes, mitochondria lysosomes, filaments, microtubules, and lipid bodies. Mast cells can be divided into two subtypes, depending on the scroll-rich or scroll-poor morphologies of their granules. The scroll-rich granules contain multiple discrete complete membranous scroll formations that are usually loosely wound and may enclose

68

Figure 1.22: Mast cells in rat tongue stained with Toluidine Blue. Photomicrograph shows metachromatic granules in TB-positive mast cells (TB(+) MCs) (arrows) and light-blue orthochromatic background. MC granules are seen as violet masses inside intact MCs. Y. Makeyeva, present work.

cores of central electron-dense material. The scroll-poor granules are more numerous, larger and uniform in shape (Shukla et al., 2005; Krishnaswamy and Chi, 2006) (Fig. 1.23).

1.4.1 Mast cell types Mast cells are characterised by their morphological and pharmacological heterogeneity, best described in rodents and humans (Pearce et al., 1985; Le Trong et al., 1987; Miller et al., 1989).

Rodents have different types of mast cells. Connective tissue mast cells (CTMC) are found in skin, muscle, and the peritoneal cavity. Mucosal mast cells (MMC) predominate in the mucosa of the gastrointestinal tract. The intermediate type of mast cells (IMC) is also found in rodents (Enerback, 1981; Dimitriadou et al., 1997). Rat and mouse CTMCs and MMCs differ in size, histamine content, and proteoglycan and neutral protease composition, the latter determining their differences in function. The differences in CTMC and MMC types of mast cells in rodents can be determined by their response to fixation solutions (Strobel et al., 1981). Intestinal MMC displayed metachromatic staining after fixation in Carnoy’s solution, but never when fixed in neutral buffered

69

Figure 1.23: Ultrastructure of mature liver mast cell. Electron micrograph shows mast cell in connective tissue. Mast cell is filled with monolobed nucleus (N) and granules (Grs) that vary in size, shape and electron density. Collagen fibres (Co) are seen in the surrounding background. Y. Makeyeva, present work.

Formalin solution. Conversely, CTMC showed metachromatic staining properties regardless of the fixative solution (Enerback, 1981; Newlands et al., 1984).

An alternative approach to identifying mast cell types is by examining the distribution of their cytoplasmic granule protease content. The granule-associated neutral proteases are restricted to mast cells and may provide important insights into their functions (Lowman et al., 1988). Rat mast cell protease I (RMCP I and RMCP II) is highly specific mast cell granule markers in rats. RMCP I is found in CTMCs of skin, ear pinna, tongue, tracheal submucosa, and lung parenchyma. RMCP II is found in the MMCs of the intestinal mucosa and in other tissue, including the thymus, mesenteric lymph node, liver, bone marrow, heart, kidney, and spleen (Gibson and Miller, 1986; Le Trong et al., 1987; Huntley et al., 1990; Matsson, 1992).

Human mast cells (HMCs) differ according to neutral protease granules’ content and are divided into different types. HMCs which contain measurable levels of tryptase, chymase, a cathepsin G-like protease, and carboxypeptidase (Harvima et al., 1993) are

70 named as MCTC for their tryptase and chymase content. The MCTC type predominates in the skin and in small intestinal submucosa and corresponds to CTMC in rodents. The human MCT cell type contains only tryptase and predominates in the alveolar septa of the lung and in small intestinal mucosa. It corresponds most closely to MMC in rodents.

The human MCC type contains only chymase. It is less abundant and is found in the intestinal submucosa and nasal mucosa (Castells, 1999; Nilsson and Metcalfe, 2000; Gurish and Boyce, 2006; Krishnaswamy and Chi, 2006; Theoharides and Kalogeromitros, 2006; Balakumar et al., 2008; Galli and Tsai, 2008).

Mast cells have been shown to be functionally multifaceted cells. A single mast cell can behave as an immune cell, an endocrine cell, and as a sensorial neuron (Galli, 1987; Wershil et al., 1989; Tore and Tuncel, 2009).

1.4.2 Mast cell mediators Mast cells store a wide variety of biologically active molecules in their granules and can also de novo synthesise additional mediators. Mast cells have numerous receptors that can trigger a wide spectrum of cellular responses, some of which can be programmed against specific pathogens (Metcalfe et al., 1997; Nilsson and Metcalfe, 2000; Krishnaswamy and Chi, 2006; Theoharides and Kalogeromitros, 2006; Caughey, 2007; Balakumar et al., 2008; Galli et al., 2008). Mast cell mediators are both pleiotropic and redundant, that is, each mediator has more than one function and mediators may overlap in their biological effects.

Mast cell-dependent mediators can be divided into three groups: preformed secretory granule mediators, lipid-derived mediators, and cytokines. Some mediators released by mast cells are preformed and stored in granules inside the mast cells and released upon mast cell degranulation. They include histamine, serotonin (5-HT), renin, adenosine, heparin, tryptase, chymase, elastase, carboxypeptidase A and B (CPA and CPB), cathepsin, β-galactosidase, β-glucuronidase, matrix metalloproteinase (MMPs), chemotactic factors for and . Other mediators undergo de novo synthesis. They include various lipid mediators such as platelet activating factor (PAF), D2, leukotriene (LT): LTB4, LTC4, LTD4, A2 and B2, and tumor necrosis factor α (TNF-α). In addition, mast cells release nitric oxide (NO) and various cytokines such as TNF-α, interferon (IFN)-γ, interleukins (ILs): ILs-1-16, transforming growth factor (TGF)-β and other growth factors including vascular endothelial growth factor (VEGF), colony stimulating factor (CSF), granulocyte-monocyte colony stimulating factor (GM-CSF), nerve growth factor (NGF), and basic fibroblast growth factor (b-FGF) (Norrby, 2002; Dvorak, 2005a; Vliagoftis and Befus, 2005; Reid et

71 al., 2007). Mast cells also produce such as corticotropin-releasing factor (CRF), endorphins (β-EP), kinins (bradykinin), somatostatin (SOM), undecapeptide substance P (SP), calcitonin gene related peptide (CGRP), galanin, neurotensin, vasoactive intestinal peptide (VIP), urocortin (UCN), and neuromedin U (NMU) (Metcalfe et al., 1997; Luger and Lotti, 1998; Nilsson and Metcalfe, 2000; Groneberg et al., 2003; Kempuraj et al., 2004; Moriyama et al., 2005; Slominski, 2006; Theoharides and Kalogeromitros, 2006; Reid et al., 2007; Akaishi et al., 2008; Galli et al., 2008).

Mast cell activation causes degranulation, which leads to multiple biological effects. Some of these effects are shown in Fig. 1.24.

Mast cells release granules that contain various mediators, including: prostaglandin D2, LTB4, LTC4, and PAF, all of which cause microvascular responses, such as venular permeability and leucocyte adherence, and tryptase. Prostaglandin D2 mobilises leukocytes and stimulates chemokinesis. It also augments venular permeability, pulmonary bronchoconstriction, and acts as a peripheral vasodilator. LTC4 is rapidly metabolised to LTB4 and, subsequently, to LTE4. All three mediators are responsible for smooth muscle contraction, prolonged bronchoconstriction, and bronchial mucus secretion and are considered to be slow-reacting substances of anaphylaxis. LTB4 also causes neutrophil chemotaxis and adherence. PAF is a powerful chemotactic agent for eosinophils and neutrophils and causes platelet aggregation, bronchoconstriction, and goblet cell mucus production in the lungs. Tryptase increases vascular permeability, upregulates adhesion molecules, attracts and activates eosinophils, and augments epithelial and fibroblast proliferation (Stevens and Austen, 1989; Walls et al., 1995; Metcalfe et al., 1997; Moon et al., 2014; Wernersson and Pejler, 2014).

Mast cells also release histamine, serglycin, and heparin mediators, which can be detected by metachromatic staining (Wernersson and Pejler, 2014).

Histamine, or β-aminoethylimidazole, can mediate several physiological and pathological processes within and outside the nervous system and is present in all subtypes of mast cells in all species (Dachman et al., 1994; Moon et al., 2014). Endogenous histamine regulates gastric-acid secretion, CNS neurotransmission, cardiovascular function, and sympathetic and/or afferent nerve activity. Histamine also participates in and in the regulation of immune responses. Histamine is an autacoid, is released during anaphylactic reactions, and is likely to act at or near the site of its release (Metcalfe et al., 1997). Histamine stimulates a host of smooth muscles and

72

Figure 1.24: Biological effects of activated mast cells. Diagram shows examples of various biological effects that are attributable to granule compounds that mast cells release upon activation. Mast cell degranulation leads to powerful pro-inflammatory response, which is partly mediated by preformed granule compounds (such as histamine), various cytokines (such as TNF) and mast cell-specific proteases (such as chymases, tryptases, and CPA3). Histamine binds to histamine receptors to produce multiple effects such as stimulating afferent nerve cells, stimulating smooth muscle contraction, and causing vascular permeability and vasodilation. Cytokines and proteases generally contribute to pro-inflammatory response. Their granule compounds (particularly proteases) affect extracellular matrix, protecting it against noxious compounds. Granule remnants also released by mast cells are transported through to draining lymph nodes, carrying various bioactive compounds. Abbreviation: PAR2, protease-activate receptor 2; TNF, tumour necrosis factor; CPA3, carboxypeptidase A3 enzyme; MMP, matrix metalloproteinase enzyme. Adapted from Wernersson and Pejler (2014).

induces vasodilation, vascular permeability, and hypotension (Dachman et al., 1994; Siegel et al., 2006; Ebeigbe and Talabi, 2014) (Fig. 1.24).

Serglycin is a proteoglycan with heparin or chondroitin sulphate side chains (Metcalfe et al., 1997; Wernersson and Pejler, 2014). Serglycin and histamine contribute to the maturation of mast cell granules. Serglycin also stores granule compounds, including chymases, tryptases, histamine, and serotonin (Åbrink et al., 2004; Braga et al., 2007).

73 Heparin is a member of the polyanionic polysaccharides family. This glycosaminoglycan, which is synthesised exclusively by mast cells, stores various mediators (Forsberg et al., 1999; Humphries et al., 1999). Heparin is also a potent anticoagulant. Heparin-initiated bradykinin formation increases vascular permeability and promotes angiogenesis in vivo in rodents (Metcalfe et al., 1997; Rajangam et al., 2006; Oschatz et al., 2011).

1.4.3 Mast cell activation Mast cells may be activated by the interaction of a multivalent antigen (allergen) with its specific IgE, attached to the cell membrane, via the high affinity Fc epsilon receptor I (FcɛR I). Mast cells may also be activated by non-immunologic substances such as neuropeptides, basic or complement compounds, and even certain drugs. In both cases, mast cell degranulation appears to be morphologically similar. However, there may be differences in the biochemical processes involved and their biological effects.

Mast cells possess a great number and diversity of surface receptors that that can initiate a wide spectrum of cellular responses (Fig. 1.24). Some receptors mediate activation of mast cells, others are inhibitory.

1.4.4 Receptors for mast cell activation

1.4.4.1 IgE-dependent pathway

§ Fc epsilon receptor I FcɛR I is a receptor participating in IgE-dependent immediate hypersensitivity reactions (Metcalfe et al., 1997; Theoharides and Kalogeromitros, 2006; Galli et al., 2008). FcɛR I belongs to the multi-chain immune recognition class of receptors (MIRRs) (Keegan and Paul, 1992). It is a tetrameric protein complex, consisting of the IgE-binding α-chain, a single β-chain, and two disulphide-linked γ-chains.

The binding of antigen to FcɛR I triggers mast cell activation, degranulation, and release of vasoactive, pro-inflammatory, and nociceptive mediators. Mediator release occurs within minutes and causes an immediate reaction (ImR). Mediators interact with surrounding tissue and cause inflammation. Many, but not all, hypersensitivity reactions also involve a second reaction, the late phase reaction (LPR). This reaction begins 2 – 6 hrs after the original challenge and peaks between 8 – 12 hrs afterwards (Theoharides et al., 1982; Lemanske and Kaliner, 1983; Benoist and Mathis, 2002; Woolley, 2003) (Fig. 1.25).

74

Figure 1.25: Schematic representation of mast cell-dependent immediate-and late- phase reactions. Antigen (AG) induces mast cell activation that leads to inflammation. There are two types of clinically-distinctive inflammation reactions. Immediate reaction (ImR) starts within minutes. This can be followed by late phase reaction (LPR), which develops after 2 – 6 hrs and peaks after 8 – 12 hrs. ImR is caused by release of histamine and other preformed or de novo synthetised mediators by degranulation. These mediators cause vasodilation, increase vascular permeability with oedema, and acute functional changes in affected organs. Some of released mediators also promote local recruitment and activation of leucocytes, contributing to develop of LFR. LFR is also caused by induced and secretion of cytokines and chemokines from activated mast cells, which are locally recruited. Adapted from Metcalfe et al. (1997).

Ca2+ plays a key role in IgE-dependent activation of mast cells and is essential for exocytosis of mediators and for synthesis of , cytokines, and chemokines. K+ and Cl- ions also play an important role in mast cell activation. They affect cell membrane potential and, thus, the influx of Ca2+ (Ashmole and Bradding, 2013).

75 1.4.4.2 IgE-independent pathway

§ KIT receptor KIT receptor (KITR or CD117s) is a receptor for stem cell factor (SCF) and is located on the plasma membrane of mast cells. KITR activation is crucial for mast cell growth, survival, differentiation, and homing into target tissue. SCF via KITR increases mast cell degranulation and potentiates and prolongs Ca2+ signals and also influences mast cell chemotaxis and adhesion (Vosseller et al., 1997).

§ Toll-like receptors Toll-like receptors (TLRs) enable mast cells to respond to products of Gram- negative and Gram-positive bacteria. TLR2 and TLR4 act synergistically with an antigen to enhance de novo synthesis and release cytokines (McCurdy et al., 2001; Pundir and Kulka, 2010).

§ Interleukin receptors There are a variety of receptors for interleukins (ILs), including interleukin receptors (ILRs): IL-3R-R5, IL-10R, IL-15, IL-18, IL-33.

Interleukins that bind to their receptors play a role in leukocyte communication. IL-3 and IL-4, when bound to their receptors, increase FcɛRI-dependent histamine release (Bischoff et al., 1999; Gebhardt et al., 2002). IL-10 inhibited the mast cell release of TNF-α, IL-8, and histamine (Royer et al., 2001). Activation of IL-15R induced migration of both mouse bone marrow mast cells (BMMC) and human BMMC in a dose-responsive and biphasic manner (Jackson et al., 2005). Activation of IL-33R induced mast cell degranulation and production and release of some pro-inflammatory cytokines (Hsu et al., 2010; Liew et al., 2010).

§ Complement receptor Complement receptors (CRs) are activated by anaphylatoxins C3a, C4a, and C5a, which are mast cell chemoattractants (Nilsson et al., 1994). Anaphylatoxins cause mast cell degranulation and elicit anaphylactic reactions through the complement receptor CD88 (Hugli and Müller-Eberhard, 1978; Gorski et al., 1979).

§ Dextran and lectin receptors Dextrans release histamine from mast cells by creating a multipoint interaction with the cell membrane and cross-linking glucose receptors on the membrane without cross-linking with IgE or FcɛRI receptors. Lectins induce exocytosis by interacting with specific sugar moieties within the Fc region of IgE (Lagunoff et al., 1983; Foreman, 1993).

76 1.4.4.3 “Neuronal” receptors in mast cells Mast cells express various “neuronal” receptors, such as those expressed for histamine, serotonin and for various neuropeptides.

§ Histamine receptors The actions of histamine are mediated through the four subclasses of histamine receptors (HRs): H1R, H2R, H3R and H4R (Black et al., 1972; Arrang et al., 1983; Leurs et al., 1995; Rangachari, 1998). The activation of H1R and H2R receptors mediates vasodilation in most vascular beds and mobilises intracellular Ca2+ (Tilly et al., 1990). However, the vessels’ responses depend on their sensitivity to histamine and the duration and mechanisms of activation (Hudgins and Weiss, 1968; Leurs et al., 1995).

Activation of H3R auto-regulates histamine release (Ohkubo et al., 1994). H4R in mouse 2+ mast cells affects chemotaxis and mobilises intracellular Ca . The activation of H4R does not cause mast cells to degranulate. However, it causes mast cells to release histamine, leading to the recruitment of effector cells, especially eosinophils (Hofstra et al., 2003; Nordlind et al., 2008).

§ Serotonin receptors Serotonin (5-hydroxytryptamine) receptors have been identified on mast cells. Their activation induces inflammation, due to mast cell migration to the site of inflammation, but they are not involved in mast cell degranulation (Nordlind et al., 2008).

§ Neuropeptide receptors Mast cells also express receptors for various neuropeptides such as: VIP, neuropeptide Y (NPY), SP, CGRP, pro-opiomelanocortin (POMC), galanin, NMU, and neurotensin (NT) (Metcalfe et al., 1997; Nilsson and Metcalfe, 2000; Groneberg et al., 2003; Kempuraj et al., 2004; Krishnaswamy and Chi, 2006; Theoharides and Kalogeromitros, 2006; Gomariz et al., 2007; Gross and Pothoulakis, 2007; Talero et al., 2007; Galli and Tsai, 2008; Kulka et al., 2008). Most neuropeptide receptors are seven- transmembrane GPCRs. Neuropeptides are produced both in neurons of the peripheral and central nervous systems and also in mast cells and affect their effector cells, including mast cells (Siegel et al., 2006). They release, in turn, other bioactive factors, acting back on nerve terminals and fibres. This extends the flexibility of neurogenic signalling pathway via reciprocity. Neuropeptides have dual function in inflammation and can adjust balance between continuation/chronicity and resolution of inflammation. On one hand, they modulate plasma extravasation, oedema, release of pro-inflammatory cytokines, and recruit inflammatory cells. They also exert trophic influences on healthy

77 tissue and during tissue repair (Metcalfe et al., 1997; Metcalfe and Boyce, 2006; Metz and Maurer, 2007; Tore and Tuncel, 2009; Krystel-Whittemore et al., 2016).

1.4.5 Mast cell chemotaxis Mast cells can react to chemotactic stimuli and migrate to target sites of inflammation. These cells derive from multipotent hematopoietic stem cells and their myeloid precursors that are released into the blood from bone marrow before migrating to target tissue (Okayama and Kawakami, 2006; Halova et al., 2012). During their migration, mast cells undergo maturation and differentiation and these processes are controlled by chemotactic agents and various growth factors present in blood and in the target tissue in which they reside. Once the mast cell progenitors reach their target tissue, they differentiate into two classes of mature mast cells—connective and mucosal—and this process is also directed by growth factors and various chemoattractants produced by cells in surrounding tissue (Halova et al., 2012).

Mast cells, themselves, can also produce and secrete various chemoattractants which can attract other mast cells and/or their progenitors in an autocrine/paracrine fashion (Halova et al., 2012). Mast cells possess numerous surface receptors for various ligands that are potent chemoattractants. These ligands include: a) SCF; b) highly cytokinergic IgE, recognised by FcɛRI; c) eicosanoids [PGE2 and PGD2, LTB4, LTD4, and

LTC4]; d) chemokines (CC [or β-], CXC, C, and CX3C); e) TGF β1-3; f) adenosine; g) C1q, C3a, and C5a; h) 5-hydroxytryptamine; i) catestatin - neuroendocrine peptide; and j) TNF-α.

The key chemoattractant for mast cells and their precursors is SCF, also known as steel factor or c-Kit-ligand (Chabot et al., 1988; Meininger et al., 1992; Nilsson et al., 1994; Nilsson et al., 1998; Okayama and Kawakami, 2006; Jensen et al., 2008). SCF recruits mast cell progenitors into tissue and contributes to their maturation and activation (Halova et al., 2012). Chemotaxis of HMCs towards SCF was found to be dose-dependent, reaching a maximum at 50 ng/ml. The activity of SCF could be blocked by anti-SCF antibodies. SCF acts not only as a growth and differentiation factor for HMCs in vitro but also as a potent chemoattractant for mast cells and mast cell progenitors (Nilsson et al., 1994).

Anaphylotoxins C3a and C5a are chemoattractants for the human mast cell line HMC-1, human cord blood-derived mast cells, and cutaneous mast cells in vitro. Migration of mast cells towards C3a and C5a was reported to be dose-dependent, peaking at 1 µg/ml. Pre-treatment with pertussis toxin inhibited the anaphylotoxin- mediated migration of HMC-1 cells, indicating that Gi proteins are involved in

78 complement-activated signal transduction pathways in HMCs. Both C3a and C5a also mobilised intracellular free Ca2+ in mast cells (Hartmann et al., 1997).

1.4.6 GPCR-mediated pathway in mast cell activation There is limited scientific evidence about the GPCR-mediated pathway in mast cell activation. However, some studies have shown that a number of mediators activate mast cells and cause degranulation though GPCR-mediated pathway. They include mastoparan, compound 48/80, and several others. Evidence that G proteins might be involved in activating mast cells first came from a study that found pertussis toxin (a selective inhibitor of Gαi protein) blocks histamine secretion by inhibiting mastoparan (Higashijima et al., 1988). Mastoparan from wasp venom is an amphiphilic tetradecapeptide. Mastoparan binds to Gαi proteins and this promotes a GDP/GTP exchange, mast cell degranulation and release of mast cell mediators, such as cytokines. This process is likely to be calcium dependent (Okano et al., 1985; Higashijima et al., 1990).

Compound 48/80, which is mixture of different-sized polymers, directly activates G proteins. The activation involves insertion of the aromatic rings of compound 48/80 into the mast cell membrane and interaction of the positively-charged domain of the molecule with the COOH-terminal portion of the α subunit of G protein (Mousli et al., 1990c; Mousli et al., 1990b). This would explain the activation of signal transduction pathways by compound 48/80 and other polybasic compounds.

Substance P (SP), which is a sensory undecapeptide of the tachykinin family, induces histamine release from rat peritoneal mast cells. Pertussis toxin and benzalkonium chloride (a compound 48/80 inhibitor) (Mousli et al., 1990a) inhibit SP and it has been proposed that the site of SP’s binding is a Gi-like G protein (Mousli et al., 1990c; Mousli et al., 1990a).

Mas-related gene (Mrg) receptors belong to the GPCR family. They are also known as the sensory neuron-specific GPCRs (Tatemoto et al., 2006). Mrg receptors are expressed in a specific subset of nociceptive sensory neurons, suggesting they play a role in regulating nociceptor function, including the sensation or modulation of pain. Mrg receptors in mast cells are activated by corticostatin, SOM, neuropeptide FF, oxytocin, and SP and this activation leads to mast cell degranulation (Dong et al., 2001; Robas et al., 2003).

Antimicrobial peptides (AMPs) are a unique and diverse group of gene-encoded molecules, which are highly-conserved in their structure, function, and mechanism. They

79 are grouped into four main classes according to similarities in charge, sequence homology, structure, and function. AMPs exhibit microbicide activity against invading microbes and may act as important mediators of innate immune responses. AMPs regulate inflammation and promote wound healing, tissue repair, angiogenesis, vascularisation, and epithelisation. AMPs are chemoattractants for leukocytes and other immune cells and they influence cell proliferation and direct T helper immune responses (Brown and Hancock, 2006). AMPs are present in the immediate microenvironment surrounding mast cells. After pathogen invasion, epithelial surfaces release AMPs, which can activate nearby mast cells through their receptors. Mast cells respond by releasing pro-inflammatory mediators, which protect the host by limiting microbial invasion. Mast cells also clear pathogens by phagocytosis and/or through the secretion of the antimicrobial peptide LL-37 from cathelicidin family (Diamond et al., 2000). AMPs may be a component of the extracellular net that mast cells can form and which also contains a chromatin-DNA backbone; this net traps and kills microbes (von Köckritz-Blickwede et al., 2008).

Under specific conditions, AMP may activate mast cells through GPCR signalling and this can lead to mast cell degranulation. The majority of AMPs are believed to act through GPCRs because pertussis toxin inhibited the cellular functions induced by AMPs. Many GPCRs have been theorised to bind and be activated by AMPs. However, the nature of this protein-receptor interaction in the context of mast cell activation is largely unknown.

The schematic diagram of the GPCR signalling cascade in mast cell activation by AMPs is presented in Fig. 1.26. Ligand binds to GPCRs to activate Gα and Gβγ dimers. Gα-GTP activates PLCβ and PKC, resulting in a massive influx of extracellular Ca2+ (Vig and Kinet, 2009). High Ca2+ concentrations cause mast cell granule translocation and granule docking (Puri et al., 2003). Integral membrane proteins, called soluble N-Ethylmaleimide-sensitive Factors (NSFs), interact with attachment proteins (SNAREs), which are present on both granules and the plasma membrane. This interaction ultimately leads to fusion of granules to the cell membrane and to exocytosis of granules (Puri et al., 2003). PKC activates substances such as LTC4 and prostaglandin D2 through the phosphorylation of cytosolic phospholipases’ A2 pathway (Fujishima et al., 1999). This pathway is Ca2+-dependent.

Ligand-GPCR binding activates Gβγ dimers. This activates the RAS-RAF pathway, which phosphorylates and activates MAPKs, extracellular-signal-regulated kinases (ERKs), and c-Jun N-terminal kinases (JNKs). Together with PLC-β and

80

Figure 1.26: Schematic representation of GPCR signalling cascade in mast cells.

Active Gα-GTP subunit stimulates phospholipase C-β (PLCβ), which hydrolyse

phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and

diacylglycerol (DAG) (Kuehn and Gilfillan, 2007). IP3 binds IP3 receptor present on surface of internal calcium stores, mainly smooth endoplasmic reticulum, and opens Ca2+ channels. Releasing of intracellular calcium leads to mast cell degranulation (Vig

and Kinet, 2009). Protein kinase C (PKC) activates substances such as LTC4 and PGD2

through phosphorylation of cytosolic phospholipases’ A2 pathway (Gilfillan and Tkaczyk,

2006). Gβγ dimer activates RAS-RAF pathway, which phosphorylates MEPKs, extracellular-signal-regulated kinases (ERKs) and c-Jun N-terminal kinases. Together with PLCβ and phosphinositide 3-kinase (PI3Kγ) it leads to cytokine and chemokine generation. Adapted from Pundir and Kulka (2010).

phosphinositide 3-kinase (PI3Kγ) it leads to the generation of cytokines and chemokines. This pathway is also Ca2+-dependent.

1.4.7 Mast cells in brain Mast cells are found intracranially. Generally, the brain is an immunologically privileged organ (Fabry et al., 1994; Owens et al., 1994), protected by the BBB from infiltration by cells of the immune system and toxins (Zhuang et al., 1999). However, the immunologically privileged status of the brain is not absolute because this organ contains immunocompetent cells, including mast cells, microglia (which are resident brain

81 macrophages) and astrocytes (Johnson and Krenger, 1992; Purcell and Atterwill, 1995; Williams et al., 1995; Silver et al., 1996; Xanthos and Sandkühler, 2014). Mast cells are resident cells in the of many species. They enter brain during development and migrate within it (Silver et al., 1992). They are most numerous in the leptomeninges, thalamus, hypothalamus, and in the dura matter of the spinal cord. They are also found in the infundibulum, pineal gland, area postrema, choroid plexus, and in the leptomeninges surrounding the pineal gland and infundibulum. Mast cells are occasionally found in the supraoptic crest, the subfornical organ and the ventricles (Nelissen et al., 2013; Polyzoidis et al., 2015; Ribatti, 2015).

Exact numbers of mast cells in the brain are difficult to ascertain because numbers vary between species (Persinger, 1977). In humans, less than five cells were found in the meninges and in the perivascular area and only a few mast cells were found in the brain. Mice had higher numbers of mast cells in the brain, ranging from 150 to 500 during development (Nautiyal et al., 2012).

The number of mast cells in the brain is influenced by a variety of factors. Most CNS structures show a decrease in mast cell numbers with age (Kelsall, 1966; Dropp, 1976). In three one-hour-old rats, an average of 7,356 mast cells was counted, while three 1-week-old rats averaged 38,037 mast cells. Three 1-month-old rats averaged approximately 10,000 mast cells, while 2-year-old animals averaged approximately 1,200 mast cells (Dropp, 1976). Behavioural factors also play a role in mast cell numbers (Asarian et al., 2002). Immobilisation and handling stresses reduced mast cell numbers in rats (Persinger, 1980; Theoharides et al., 1995). Social isolation induced stress in rats, resulting in a 90% reduction of mast cells during the first day of isolation compared to group-housed rats (Bugajski et al., 1994). Additionally, hormonal factors influence mast cell numbers. Higher numbers of thalamic mast cells were found in male mice that had mated and cohabitated with females as compared to male mice cohabitating with other males (Yang et al., 1999). More mast cells were found in the brain of post-partum female rats than in virgin rats (Silverman et al., 2000). More mast cells were found in dove brains after two hours of courtship behaviour (Zhuang et al., 1993), or when treated with estradiol, testosterone or dihydrotestosterone, compared with doves housed in isolation (Wilhelm et al., 2000).

Mast cell numbers in different brain structures vary according to developmental stage (Persinger, 1981; Lambracht-Hall et al., 1990; Michaloudi et al., 2003). Mast cells enter the developing brain by migrating along the abluminal wall of penetrating blood vessels, with which they continue to be associated (Skaper et al., 2014). The first mast cells appear in the pia, the deepest layer of the meninges, where they are surrounded a

82 mesh of astroglial processes. It is likely that these first mast cells originate from circulating precursors and that they are attracted by chemoattractant(s) (Wendling et al., 1985). In rats, the pial mast cells increase in number up to postnatal day 11. From postnatal days 11 to 15, their numbers decline rapidly, with a loss of approximately 840 cells per day. The loss of mast cells in the pia can be accounted for by two possible mechanisms: apoptosis and/or cell emigration to other brain regions (Khalil et al., 2007). Mast cells are also found in the thalamus, where they are surrounded by astroglial endfeet (Asarian et al., 2002). Numbers of thalamic mast cells increase from postnatal day 8 to maximum (adult) levels at postnatal day 30, possibly as a result of migration from the pia (Lambracht-Hall et al., 1990; Michaloudi et al., 2003). Within the thalamus, mast cells migrate from one area to another. Thalamic mast cells enter in the anterior part and gradually migrate posteriorly, where they reside, at maximum (adult) levels by postnatal day 30 (Khalil et al., 2007). How mast cells populate the thalamus is not fully understood. However, it is possible that mast cells migrate in response to chemotactic signals in an organ-specific manner that involves both constitutive and inducible factors (Gurish and Boyce, 2006).

The increased numbers of mast cells in the adult brain may be explained by their division in situ or migration from external sources (Cunha and Vitković, 1992; Esposito et al., 2001; Asarian et al., 2002). The rapid change in numbers of mature mast cells in the adult brain suggests that mature mast cells can migrate from the blood into the brain. For example, it took only one hour for mature donor peritoneal mast cells, injected intravenously into rats, to cross the BBB and enter the brain parenchyma (Silverman et al., 2000; Nautiyal et al., 2011). It is thought that chemotaxis is the more likely mechanism for this mast cell migration. Numerous chemoattractants for mast cells are produced by cells in the brain, including NGF, TGF-β1, chemokines, CRF, neuropeptides, such as SP and somatostatin, and IL-3. NGF is synthesised by mast cells (Manova et al., 1992), can induce chemotaxis of these cells in the brain (Woolf et al., 1994) and stimulate hypertrophy of mast cells (Aloe and Levi-Montalcini, 1977). TGF-β1 is a mast-cell mediator that produced by mast cells and astrocytes (Gruber et al., 1994).

Mast cells perform a wide range of functions – both beneficial and pathological - in the brain (Silver et al., 1996; Theoharides, 1996; Benoist and Mathis, 2002). They also contribute to cognitive and emotional behaviour (Dong et al., 2014). Mast cells initiate and magnify the immune response. They cooperate with glial cells and neurons to degranulate and release mediators such as cytokines, proteases and reactive oxygen mediators (Hendriksen et al., 2017). These mediators activate the innate immune system, leading to inflammation, a protective response that initiates the healing process.

83 However, protracted inflammation upregulates inflammatory mediators released from mast cells and microglia, causing pathogenic neuroinflammation. Prolonged neuroinflammation results in neurodegeneration, BBB alterations, neuronal hyper- excitability and neuronal death (Banuelos-Cabrera et al., 2014; Dong et al., 2014; Lyman et al., 2014). Because mast cells play such important roles in immune-related diseases, it is important to develop a greater understanding of their characteristics and functions. This could be critical for the development of new approaches and strategies for treating inflammation-related diseases.

1.5 Hypothesis and Aims The work described in this thesis was performed to investigate chemoreception in a variety of non-olfactory systems in rats and humans, and in rats with experimentally- induced metabolic disorders. I hypothesise that OR-mediated chemoreception can occur in non-olfactory systems and that this can be demonstrated by the expression of the olfactory signalling proteins, OMP, Golf, and AC3. Cells that are motile are optimal candidates because they might use chemoreception to guide their movements. Such cells can be found in the reproductive and immune systems. A special case for perturbing chemoreception was examined by studying the response of the immune system to a short-term, high-fat diet, an intervention, which is known to adversely affect the olfactory system. As a result of these considerations, the specific objectives of my research are as follows:

1) To determine the expression of OMP, Golf, and AC3 in the olfactory epithelium of rats to confirm what is already known so that the staining can serve as a positive control for studies conducted in the other chapters (Chapter 2).

2) To determine the expression of OMP and ORs in human spermatozoa in three functional modes and to determine the expression of OMP, Golf, and AC3 in rat epididymal spermatozoa. I hypothesise that ORs may interact with molecules secreted by the ovum to guide the spermatozoa to the target (Chapter 3).

3) To determine the expression of OMP, Golf, and AC3 in mast cells in rat tongue, liver, and white adipose tissue. Mast cells are migratory cells that evoke immune responses. I hypothesise that they may use ORs to navigate their way to areas of inflammation (Chapter 4).

4) To determine the expression of OMP, Golf, and AC3 in the Leydig cells of interstitial tissue in testes in rats. Mast cells are not found in the interstitial tissue of the

84 rat testis. Leydig cells, which inhabit the interstitial tissue, serve as a paracrine cell, and may use ORs to trigger their response (Chapter 5).

5) To determine the effects of a short-term, high-fat diet on the blood chemistry and histology of tongue, liver, and white adipose tissue. Due to the inflammatory reaction of tissues to unhealthy diet, I tested the response of mast cells and certain olfactory neurons characteristics of the olfactory epithelium and olfactory bulb to a high fat diet (Chapter 6).

85

Chapter 2: Expression of OMP, Golf, and AC3 in olfaction

86 2.1 Abstract Olfactory receptors (ORs), first identified in 1991, are expressed by olfactory sensory neurons (OSNs) and function as chemosensory receptors, capable of detecting volatile odorants. Although it was initially postulated that ORs were exclusively expressed in the olfactory system, subsequent studies have demonstrated non-olfactory (ectopic) expression of ORs in various tissues. These tissues are not considered classical chemosensory tissue and it has been proposed that ORs may be involved in monitoring extracellular chemical cues.

Olfactory marker protein (OMP), a small, abundant, soluble, acidic protein, is a tag for maturing and mature olfactory sensory neurons (mOSNs). The phylogenetic conservation of cellular localisation and amino acid sequence of OMP in vertebrates suggests an important role for OMP in the function of OSNs. OMP and other molecules involved in the olfactory signal transduction pathway, such as Golf and AC3, have also been found in non-olfactory cells and tissues. Expression of OMP, Golf, and AC3 may indicate potential OR-mediated chemoreception in non-olfactory systems. In order to assess the expression of OMP, Golf, and AC3 in non-olfactory tissues, the current study aimed to confirm their expression in the olfactory epithelium (OE).

2.2 Introduction OMP is expressed by mOSNs located in the apical region of the OE and is used as a biochemical marker for recognising this cell type (Keller and Margolis, 1975; Farbman and Margolis, 1980; Menco, 1989; Buiakova et al., 1994). OMP is distributed throughout most of the cytoplasm of dendrites, soma, and axons of mOSNs. With the use of immuno-electron microscopy, OMP was found within the cytoplasm of olfactory chemoreceptor cell dendrites, vesicles, cilia, dendritic knobs (only in those with cilia), proximal and distal parts of the olfactory cilia, and cell bodies (Menco, 1989; Johnson et al., 1993). Some OMP expression has also been found in the nuclei of mOSNs and this expression may depend on the physiological state of cells (Koo et al., 2004). Intense OMP expression was observed along the axons of OSNs. Consequently, the olfactory nerve layer (ONL) and glomeruli of the olfactory bulb (OB) were found to be intensely OMP-immunoreactive (Weiler and Benali, 2005).

Golf and AC3 are known to be located in the cilia of the OE, according to studies that used light microscopy (Jones and Reed, 1989; Bakalyar and Reed, 1990). Electron microscopy (EM) and post-embedding immunohistochemistry (IHC) on rapidly frozen, freeze-substituted specimens showed that Golf and AC3 were present in the same OSN

87 compartments in the distal parts of olfactory cilia (Menco, 1992; Menco et al., 1994;

Menco, 1994; Menco, 1995). Golf was evident in sensory axons and glomeruli of the OB (Belluscio et al., 1998). AC3 was abundant in the dendritic endings of OSNs but was barely detected in the soma of OSNs. IHC showed that AC3 was present, although at lower levels, in axonal projections comprising the ONL and glomeruli of the OB (Dal Col et al., 2007).

OMP expression has been reported in non-olfactory tissues (Kang et al., 2015). OMP mRNA was found in various tissues and the protein is reported to be present in some neurons of the brain (Baker et al., 1989). OMP genes and proteins were found, with the use of conventional RT-PCR analysis and double immunoassay techniques, in a wide range of non-olfactory mouse tissues, such as skeletal muscle, thymus, duodenum, testes, and thyroid (Kang et al., 2015). OMP was expressed in specific cell types of the bladder, thyroid, thymus, heart, and testes. In the testes, OMP expression was found in a Leydig cell-like population of interstitial tissue (Kang et al., 2015). OMP,

Golf, AC3 were co-expressed in the bladder and thymus (Kang et al., 2015). Three ORs (olfr544, olfr558, and olfr1386) were co-expressed with OMP in the bladder and thyroid (Kang et al., 2015). The authors suggested that it may be useful to use IHC with OMP to identify ectopically-expressed ORs, rather than using ORs themselves (Kang and Koo, 2012; Kang et al., 2015).

Although the functional roles of ORs in non-olfactory tissues have been intensively studied in the last decade, details about their expression and physiological functions remain unclear and further systematic investigation is required (Kang and Koo, 2012; Flegel et al., 2013). Kang et al. (2015) suggested that such research be directed towards identifying expression of ORs and OR-associated proteins in additional cell types in chemosensory and non-chemosensory tissues. Such studies would clarify and expand awareness about the physiological functions of ORs in olfactory and non- olfactory systems (Kang and Koo, 2012; Foster et al., 2014).

The aim of this chapter is to establish the expression of OMP, Golf, and AC3 in the OE of adult rats. The expression of these proteins will provide a positive control for studies conducted in later chapters.

88 2.3 Materials and methods

2.3.1 Animals Adult male Sprague-Dawley rats were used, with the approval of the University of New South Wales’ Animal Care and Experimentation Committee and in strict accordance with the NHMRC Animal Experimentation Guidelines and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1990). All rats were obtained from the Animal Resources Centre (Perth, WA) and handled with care prior to experimentation.

2.3.2 Perfusion Rats (150 – 250 g) were given an overdose (80 mg/kg) of intraperitoneal (i.p.) Lethabarb (pentobarbitone sodium, Vibrac Animal Health, Pty. Ltd., Australia), transcardially perfused with ice-cold 0.1 M phosphate buffered saline (PBS), pH 7.2, followed by a solution of 4% paraformaldehyde (PFA) in 0.1 M PBS.

2.3.3 Histological methods

2.3.3.1 Tissue preparation Olfactory turbinates containing OE, and OBs were dissected for histologic examination. 10% neutral buffered Formalin solution (Sigma-Aldrich Pty Ltd., Australia) was used for post-fixation of olfactory turbinates for 12 – 14 hrs. Tissue was immersed overnight in 70% (v/v) ethanol and embedded using standard paraffin embedding procedures. Tissue was sectioned serially at 5 µm on a motorised microtome (Leica RM 2155, Leica Microsystems Pty Ltd). Consecutive sections were mounted on electrostatic glass slides (Menzel-Glaser, Braunschweig, Germany).

2.3.3.2 Haematoxylin and Eosin staining Haematoxylin and Eosin (HE) staining was performed to evaluate tissue structure and quality. Sections were dewaxed in Histo-Clear (National Diagnostic Products Pty Ltd., Australia) and rehydrated in a graded series of decreasing alcohol dilutions (100%,

70%, 50%, H2O). Sections were then stained in Harris Haematoxylin (Fronine Pty Ltd, Australia), differentiated in acid alcohol, and immersed in Scott’s Blueing Solution (Fronine Pty Ltd, Australia). Sections were dehydrated in 70% alcohol, then counterstained with alcoholic eosin and again dehydrated in a series of alcohol concentrations. Sections were cleared in Histo-Clear (National Diagnostic Products Pty

89 Primary Antibody (name, source) Antibody Dilution

Olfactory marker protein (OMP), goat antiserum, raised 1:50 (IP) with rodent OMP as the immunogen, diluted 1:1 with 1:1000 (IF) glycerol, containing 0.05% sodium azide (019-22291; Wako Pure Chemical Industries, Ltd., Japan)

Gaolf (Golf), goat polyclonal, P-15, raised against a 1:50 (IP) peptide mapping near the N-terminus of Gaolf of human 1:350 (IF) origin (sc-26763; Santa Cruz Biotechnology, CA, USA)

Adenylyl cyclase III (AC3), rabbit polyclonal, C-20, 1:50 (IP) raised against a peptide mapping at the C-terminus of 1:350 (IF) adenylyl cyclase III of mouse, rat, and human origin (sc-588; Santa Cruz Biotechnology, CA, USA)

Table 2.1: Primary antibodies used in Chapter 2. Abbreviations: immunoperoxidase histochemistry, IP; immunofluorescent histochemistry, IF.

Ltd., Australia), and cover-slipped using Aqua Mount (BDH Laboratories Supplies, England).

2.3.3.3 Immunohistochemistry methods

§ Immunoperoxidase histochemistry Histological sections of OE and OBs were dewaxed in xylene twice for 5 mins, rehydrated in a graded series of alcohol dilutions (100% and 70% ethanol) and placed in water for 1 min. Sections were subjected to heat-induced antigen retrieval with citrate buffer (10 mM, pH 6.0) using a microwave oven for 5 mins. Sections were then rinsed in a buffer solution (Tris buffer, pH 7.4). Endogenous peroxidase activity was blocked by exposing sections to 0.3% hydrogen peroxide (H2O2; Merck Millipore, Australia) for 5 mins. After rinsing in the buffer solution, sections were incubated at room temperature for 1 hr with one of the primary antibodies listed in Table 2.1. Sections were then rinsed three times in the buffer solution for 2 mins each and left in a post-primary solution (Bond Polymer Refine, Leica, DS 9800) for 8 mins. They were rinsed three times in the buffer solution and placed in polymer (Bond Polymer Refine, Leica, DS 9800) for 8 mins. Sections were then rinsed twice in the buffer solution for 2 mins each and again in water once. Colorimetric visualisation was performed using 3,3’-diaminobenzidine (DAB; DAKO liquid DAB+Substrate Chromogen System, DAKO Australia Pty Ltd., Australia).

90 Reactions were stopped by washing slides in water. Sections were counterstained with haematoxylin for 5 mins, and then rinsed once in water, once in the buffer solution, and again in water. They were dehydrated four times in 100% ethanol, passed through xylene, and cover-slipped with Aqua mount (BDH Laboratories Supplies, England). Primary antibodies used for immunoperoxidase histochemistry (IP IHC), their sources, and working concentrations are listed in Table 2.1.

§ Immunofluorescent histochemistry Histological sections were dewaxed in Histo-Clear (National Diagnostic Products Pty Ltd., Australia) and rehydrated in a graded series of alcohol dilutions (100%, 75%,

50%, H2O). Sections were subjected to heat-induced antigen retrieval with citrate buffer (10 mM, pH 6.0). After cooling, sections were washed three times in PBS. Non-specific staining was blocked with 10% normal donkey serum (NDS; D9663, Sigma-Aldrich, Australia) for 20 mins at room temperature in a humidified chamber. Tissue sections were incubated in 2% NDS in PBS containing primary antibody and then placed in a humidified chamber for either 1 hr at room temperature or overnight at 4 °C. Sections were rinsed three times with PBS and incubated with appropriate Alexa secondary antibodies: Alexa488-DαR, Alexa488-DαG, Alexa594-DαR, and Alexa594-DαG (Molecular Probes, Invitrogen, Carlsbad, CA, USA). Sections were left for 30 mins at room temperature in the dark and then washed three times with PBS. Nuclei were stained with DAPI using an anti-fading fluorescence mounting medium (Vectashield Hard+Set Mounting Medium with DAPI, Vector Labs, Fluoroshield with DAPI, Sigma- Aldrich, Australia). Sections were then cover-slipped. Negative control slides were incubated with 1% normal serum (NS) without the primary antibody. Primary antibodies used for immunofluorescent histochemistry (IF IHC), their sources, and working concentrations are listed in Table 2.1.

2.3.4 Photography and image processing Brightfield and fluorescent images were captured using Axioplan HR and MC digital cameras and a motorised Axioplan2 microscope (Zeiss) with AxioVision software. Additional brightfield images were captured using a ProgRes C5 digital camera (Jenoptik) mounted on an Axioskop microscope (Zeiss). Final images were imported into CorelDRAW (X3; Corel Corporation) or Photoshop (CS6; Adobe Systems). Slight adjustments were made to image balance, contrast, and evenness. Tissues stained with HE and DAB were scanned, viewed, and analysed with Aperio ImageScope (Leica). Slides were scanned at the Biomedical Imaging Facility, Mark Wainwright Analytical Centre, UNSW.

91 2.4 Results

2.4.1 Expression of OMP in olfactory epithelium and olfactory bulb OMP was densely expressed in the OE, along the ONL, and in the glomerular layer (GL) of the OB (Fig. 2.1A). In the negative control slides, no OMP expression was observed (Fig. 2.1B). At higher magnification, OMP expression was seen to be restricted to the upper band of neuronal cells in the OE (Figs. 2.2 – 2.3). OMP particularly stained mOSNs, located above the lamina propria and rows of OMP-unstained cells in the OE (Fig. 2.3). The cytoplasm of mOSNs showed diffuse patterns of OMP expression, with occasional dense accumulations (Fig. 2.3). OMP was also expressed in dendrites of mOSNs and throughout most of the olfactory cilia. Intense OMP expression was observed along axons and within axon bundles (Figs. 2.2 – 2.3).

Figure 2.1: Expression of OMP in olfactory epithelium and olfactory bulb using immunoperoxidase histochemistry. Photomicrographs show cross sections of olfactory turbinate and olfactory bulbs (OBs) stained with OMP (OMP(+)) (A) and OMP-negative control (B). (A) OMP (brown) is expressed in olfactory epithelium (OE), olfactory nerve layer (ONL), and glomerular layer (GL) of OB. (B) In negative control slide (OMP(-)), no OMP expression is seen.

92

Figure 2.2: Expression of OMP in olfactory epithelium using immunoperoxidase histochemistry. Photomicrograph shows cross section of olfactory epithelium (OE) stained with OMP (OMP(+)). OMP (brown) is expressed in mature olfactory sensory neurons (mOSNs) located in upper band of OE. OMP expression is also apparent in cilia (arrow) and axons of mOSNs that converge to form OSN axon bundles.

Figure 2.3: Expression of OMP in olfactory epithelium using immunofluorescent histochemistry. Photomicrograph shows olfactory epithelium (OE) and lamina propria stained with OMP. OMP (red) is expressed in cell bodies of mature olfactory sensory neurons (mOSNs), their cilia, dendrites, and axons. OMP-positive mOSNs are located above lamina propria and rows of neuronal cells that show no OMP expression. DAPI stains cell nuclei (blue). 93 2.4.2 Expression of Golf in olfactory epithelium and olfactory bulb

Golf was expressed in the OE, ONL, and GL of the OB (Fig. 2.4A). No Golf expression was observed in the negative control slides (Fig. 2.4B). At higher

Figure 2.4: Expression of Golf in olfactory epithelium and olfactory bulb using immunoperoxidase histochemistry. Photomicrographs show cross sections of olfactory turbinate and olfactory bulbs (OBs),

stained with Golf (Golf(+)) (A) and Golf-negative control (B). (A) Golf (brown) is expressed in olfactory epithelium (OE), olfactory nerve layer (ONL), and glomerular layer (GL) of

OB. (B) In negative control slide (Golf(-)), no Golf expression is seen.

Figure 2.5: Expression of Golf in olfactory epithelium using immunoperoxidase histochemistry.

Photomicrograph shows cross section of olfactory epithelium (OE) stained with Golf

(Golf(+)). Golf (brown) is expressed in cilia (arrow) of OE and in axons that converge to form OSN axon bundles.

94 magnification, Golf was expressed in the ciliated epithelial surface of the OE and along the axons of OSNs (Fig. 2.5).

2.4.3 Expression of AC3 in olfactory epithelium and olfactory bulb AC3 was expressed in the OE, ONL, and GL of the OB (Fig. 2.6). At higher magnification, prominent expression of AC3 was seen in the ciliated epithelial surface of the OE. Expression of AC3 was also observed in some cell bodies of OSNs. Faint AC3 expression was found in OSN axon bundles (Fig. 2.7A). No AC3 expression was observed in negative control slides (Fig. 2.7B).

Figure 2.6: Expression of AC3 in olfactory epithelium and olfactory bulb using immunoperoxidase histochemistry. Photomicrograph shows cross section of olfactory turbinate and olfactory bulb (OB) stained with AC3 (AC3(+)). AC3 (brown) is expressed in olfactory epithelium (OE), olfactory nerve layer (ONL), and glomerular layer (GL) of OB.

95

Figure 2.7: Expression of AC3 in olfactory epithelium using immunoperoxidase histochemistry. Photomicrographs show cross sections of olfactory epithelium (OE) stained with AC3 (AC3(+)) (A) and AC3-negative control (B). (A) AC3 (brown) is prominently expressed in ciliated epithelial surface (arrow) of OE. Faint expression of AC3 is seen in cell bodies of some OSNs and in axons that converge to form axon bundles. (B) In negative control

slide (AC3(-)), no AC3 expression is seen.

2.5 Discussion

The results presented in this chapter indicate that significant levels of OMP, Golf, and AC3 are present in the OE of adult rats. All three proteins were expressed in the ciliated epithelial surface of the OE and, to some extent, in axons of OSNs. OMP was expressed prominently in cell bodies of mOSNs and in their axons and may be used as a marker for detecting mOSNs. The results are consistent with those of previous studies, as discussed in Sections 1.1.3.4 & 1.2.1.1 (Margolis, 1972; Margolis, 1980; Jones and

96 Reed, 1989; Menco, 1989; Bakalyar and Reed, 1990; Johnson et al., 1993). The current study found that antibodies for OMP, Golf, and AC3 could be used to detect OMP, Golf, and AC3 in olfactory tissue. IHC with these antibodies was used to identify the expression of OMP, Golf, and AC3 in non-olfactory tissues in experiments that will be described in subsequent chapters. Co-expression of these three proteins suggests that OR-mediated chemoreception may occur in non-olfactory systems. The cells selected to investigate OMP, Golf, and AC3 expression include spermatozoa, mast cells, and Leydig cells. These cells were selected because they share many common features with each other and with neurons (Blank and Rivera, 2004; Meizel, 2004). These cells express and utilise “neuronal” receptors, including several neurotransmitters (Meizel, 2004; Siegel et al., 2006), as discussed in Sections 1.3.3.3 & 1.4.4.3. Furthermore, spermatozoa, mast cells, and neurons can activate other cells. For example, neurons activate somatic effector cells and other neurons; spermatozoa activate the egg; and mast cells activate other somatic effector cells and nerve endings (Meizel, 2004; Siegel et al., 2006). Additionally, spermatozoa, mast cells, and neurons all perform the function of exocytosis. Exocytosis is important for intercellular communication and is characterised by the secretion of vesicular content (Moon et al., 2014). Exocytosis in neurons releases neurotransmitters at chemical synapses. In mast cells, it releases mediators through degranulation, as described in Section 1.4. In spermatozoa, a special type of exocytosis, called the acrosome reaction (AR), releases hydrolytic enzymes that are essential for fertilisation (Meizel, 1984; Gerton, 2002; Blank and Rivera, 2004; Meizel, 2004; Tore and Tuncel, 2009; Wernersson and Pejler, 2014), as discussed in Section 1.3.3.1.

Spermatozoa, mast cells and neurons share the characteristic of cell migration towards a chemoattractant. In spermatozoa, this migration, known as chemotaxis, takes place in the female reproductive tract to “communicate” (interact) with the oocyte, as described in Section 1.3.3.2 (Cosson, 1990; Eisenbach and Tur-Kaspa, 1999; Eisenbach and Giojalas, 2006; Kaupp et al., 2008; Teves et al., 2009). In mast cells, chemotaxis occurs towards sites of inflammation (Okayama and Kawakami, 2006; Halova et al., 2012; Draber et al., 2016), as described in Section 1.4.5. In the case of nervous system development, immature neurons migrate from zones of proliferation to their final destinations in the OB, guided by chemical and electrical signals (Kishi et al., 1990; Wichterle et al., 1997; Hu, 1999; Wu et al., 1999; Mason et al., 2001; Astic et al., 2002; Murase and Horwitz, 2002; Saghatelyan et al., 2004; Ng et al., 2005), as discussed in Section 1.1.2.2.

The interstitial tissue of rat testes was selected because Kang et al. (2015) had previously found OMP expression in the Leydig cell-like population of interstitial tissue in

97 mice, and this observation constituted evidence that OMP was ectopically-expressed in the reproductive system of mice. The authors did not find expression of AC3 or Golf, but suggested that this may have been due to low levels of protein expression and/or antibody sensitivity. The current study sought to expand the observations of Kang et al. (2015) and provide a better understanding of immunity and chemoreception in olfactory and non-olfactory tissues.

2.6 Conclusion

The current chapter demonstrated that OMP, Golf, and AC3 are expressed in the olfactory system of adult rats. Their expression in the OE can now serve as a positive control for studies in non-olfactory tissues.

98

Chapter 3: Expression of olfactory signalling proteins in spermatozoa of humans and rats

99 3.1 Abstract Ectopic expression of olfactory receptors (ORs) has been widely reported over the last 20 years in non-chemosensory tissues, including mammalian testes and spermatozoa. In humans, an OR was found to be expressed in both the olfactory epithelium (OE) and reproductive system (testes and sperm cells), where they were functional and responded to the odorant bourgeonal. Stranded mRNA-Seq analysis revealed 91 different OR transcripts in human spermatozoa. A variety of OR proteins was expressed at the mRNA and protein levels on the head, connecting piece, and tail of human spermatozoa, indicating that a single cell expresses multiple ORs. Single-cell Ca2+ imaging showed that a panel of various OR ligands induced intracellular Ca2+ signals in human spermatozoa, which could be inhibited by the Ca2+ channel blocker, mibefradil.

OMP, a marker for mature olfactory sensory neurons (mOSNs) in the OE, co- expresses with ORs. I hypothesised that OMP would be expressed in human spermatozoa in compartment-specific locations. Indeed, the present study determined that the chemosensory proteins OMP, Golf, and AC3 were expressed in compartment- specific locations in spermatozoa of rats and humans. Further, expression of OMP was observed in control, activated, and hyper-activated human spermatozoa, and OMP was co-expressed with Golf and AC3 in the rat spermatozoa. It is concluded that OMP expression indicates OR-mediated chemoreception in non-olfactory tissue and that ORs may act as receptors in the reproductive system via their olfactory signalling partners

(OMP, Golf, and AC3).

3.2 Introduction Years of research have shown that ORs are expressed in and beyond the olfactory system. Expression of many ORs in various non-olfactory tissues of different mammalian species has been described. ORs have been found in spermatozoa (Parmentier et al., 1992; Vanderhaeghen et al., 1997), tongue (Thomas et al., 1996; Gaudin et al., 2001; Durzyński et al., 2005; Gaudin et al., 2006), spleen, pancreas (Blache et al., 1998; Nakagawa et al., 2009), placenta (Itakura et al., 2006), and enterochromaffin cells of the gut (Braun et al., 2007).

Kang and Koo (2012) conducted a study on OR gene expression in non-olfactory tissue and concluded that ORs function as two groupings. The first group is involved in secreting hormones, such as serotonin and renin, triggered by external chemical stimuli (Braun et al., 2007; Pluznick et al., 2009; Pluznick et al., 2013). The second group

100 controls cell adhesion (Griffin et al., 2009), proliferation (Neuhaus et al., 2009), cytokinesis (Zhang et al., 2012), and cell migration (Spehr et al., 2003; Fukuda et al., 2004). Some ectopically-expressed ORs were conserved in different species (Gilad et al., 2005; de La Cruz et al., 2009), whereas other ORs were not related to each other (Feldmesser et al., 2006). A number of studies identified a novel role for ectopically- expressed ORs in the testes and spermatozoa (Parmentier et al., 1992; Walensky et al., 1995; Vanderhaeghen et al., 1997; Walensky et al., 1998; Spehr et al., 2003; Fukuda et al., 2004; Fukuda and Touhara, 2006; Veitinger et al., 2011). Parmentier et al. (1992) used homologous cloning, PCR, and Northern blot analysis to demonstrate the presence of approximately 20 human OR transcripts in dog germ cells. The authors suggested that these might encode receptors involved in the chemotaxis of spermatozoa during fertilisation. Flegel et al. (2013) used Next Generation Sequencing (NGS) analysis to identify ectopically-expressed ORs in a wide range of human tissues, showing that outside the olfactory system, the testes contained the highest number of ORs. This finding strengthened the idea that ORs may perform an important function in reproduction (Flegel et al., 2013).

Spehr et al. (2003) cloned a human testicular OR, hOR17-4 (synonymous with OR1D2), and examined its ligand preferences in a heterologous cell expression system in an attempt to address the functional role of ORs in the reproductive system. These authors found that bourgeonal acts as a powerful agonist of this receptor and human spermatozoa showed functional activation and chemotaxis towards bourgeonal, which was blocked by undecanal, a strong bourgeonal inhibitor. In a further study, Spehr et al. (2004a) found that hOR17-4 was expressed not just in the testes, but also in the nose. The authors briefly exposed a large number of human participants to undecanal and found that it significantly decreased their olfactory perception of bourgeonal (Spehr et al., 2004a). The authors concluded that hOR17-4 behaved in a similar manner in the testes and OE and that the receptor may play a role in fertilisation (Spehr et al., 2004a; Vosshall, 2004). Ottaviano et al. (2013) showed that normozoospermiatic men with idiopathic infertility had a lower bourgeonal threshold than controls and their spermatozoa were less sensitive to bourgeonal attraction, showing reduced chemotaxis. These results confirmed the role of hOR17-4 in fertilisation and connected bourgeonal sensitivity to male infertility (Ottaviano et al., 2013).

In the olfactory system, each OR is expressed by only one of many hundreds of OR genes. In the OE, each OSN expresses primarily one OR (“one neuron-one receptor” rule), as described in Section 1.1.3.1 (Chess et al., 1994; Ressler et al., 1994a). However, in other tissues, this rule does not apply (Flegel et al., 2013). For example,

101 more than one OR was expressed in one germ cell of the testes and in muscle (Fukuda and Touhara, 2006; Griffin et al., 2009). Similarly, more than one OR mRNA was found in round spermatids in the testes of mice (Fukuda and Touhara, 2006).

Flegel et al. (2015) did the first comprehensive analysis of OR transcripts in human spermatozoa. Using mRNA-Seq, they found 91 different ORs transcripts that could be aligned to annotated OR genes in an antisense orientation. Using IHC, they showed that ORs were expressed in compartment-specific locations on the head, middle piece, and tail of human spermatozoa and that each OR had a distinct pattern of expression. This result suggested that one spermatozoon expresses multiple ORs, each of which may perform a different function. For example, OR3A2 was expressed on the middle piece, whereas OR2W3 was expressed along the tail. OR6B2 was expressed on the equatorial segment of the head, suggesting that this type of OR may be involved in acrosomal exocytosis and spermatozoa to egg binding. The authors further investigated 10 known ligands for ORs to see how they affected human spermatozoa. They used single-cell Ca2+ imaging, which showed that seven of the ten OR ligands induced Ca2+ signals and that these ligands could be inhibited by the Ca2+ channel blocker mibefradil (Flegel et al., 2015). Among these ligands were dimetol, coumarin, bourgeonal, methional, myrac, nonanoic acid and β-ionone (Spehr et al., 2003; Neuhaus et al., 2009; Saito et al., 2009; Veitinger et al., 2011; Adipietro et al., 2012; Flegel et al., 2015).

Flegel et al. (2015) concluded that because human spermatozoa express multiple OR transcripts and proteins, each OR might be involved in different physiological processes as spermatozoa make their way to the egg. For example, some ORs on the tail may be involved in chemotaxis and chemokinesis (Flegel et al., 2015), whereas ORs localised to the head may be involved in acrosomal exocytosis, capacitation, spermatogenesis, and epididymal maturation (Fukuda and Touhara, 2006). Nevertheless, it is still not entirely clear just how ORs and the odorant-induced signal transduction cascade function in mammalian spermatozoa.

The study of ORs in non-chemosensory tissues is fraught with difficulties. Firstly, non-olfactory tissues express comparatively lower levels of OR transcripts compared to that of olfactory tissue (Parmentier et al., 1992; Vanderhaeghen et al., 1993; Drutel et al., 1994; Walensky et al., 1995; Asai et al., 1996; Vanderhaeghen et al., 1997; Walensky et al., 1998; Gaudin et al., 2001; Yuan et al., 2001; Durzyński et al., 2005; Itakura et al., 2006; Veitinger et al., 2011). Secondly, there are insufficient high-quality, commercially available, specific OR antibodies to study ORs. Thirdly, less than 100 ligands have been isolated for ORs (Matarazzo et al., 2005; Sanz et al., 2005; Saito et al., 2009; Nara et al., 2011). To overcome all these difficulties, Flegel et al. (2013) suggested that research

102 on ectopic expression of ORs should diversify beyond study of the ORs. The authors proposed that Golf and AC3 may be useful for the study of ORs because, when they are found in non-olfactory tissues, they may indicate the presence of ORs and an OR-like signalling pathway. The authors further suggested an alternative approach may be to use olfactory-specific proteins to identify ORs and an OR-like signalling pathway in non- olfactory tissues.

One promising olfactory-specific protein is olfactory marker protein (OMP). Kang et al. (2015) found that OMP, a marker for mOSNs in the OE, was expressed in specific cell types of the bladder, thyroid, thymus, heart, and testes of mice. Using refined microarray analysis, RT-PCR, and IHC with commercially available antibodies, they found that ORs, such as olfr544 and olfr558 (a human OR51E1 ortholog), were co- expressed with OMP in the interstitial cells of the bladder. More than 80% of the OMP(+) cells co-expressed with one of the tested ORs. Only a small number of OMP(+) cells were observed that did not co-express an OR. The co-expression of ORs and OMP indicated that OMP could be used to identify most but not all ectopically-located ORs. This approach can be both economical and practical in cases where it is difficult to detect ORs. Thus, OMP appears to be a powerful indicator of OR-mediated chemoreception in non-olfactory systems.

The present study aimed to determine the extent to which OMP is expressed in the spermatozoa of humans and rats. Based on evidence that various ORs were expressed in compartment-specific locations in human spermatozoa (Flegel et al., 2015),

I hypothesised that OMP, Golf, and AC3 would be expressed in human spermatozoa in compartment-specific locations and might co-express with some ORs. IHC with commercially available antibodies was used to investigate expression of OMP and co- expression of OMP and ORs in control, activated, and hyper-activated ejaculated human spermatozoa.

3.3 Expression of OMP in human spermatozoa

3.3.1 Materials and methods

3.3.1.1 Human semen sample collection Men attending the NSW Health Pathology Andrology Laboratory at the Royal Hospital for Women for semen-analysis tests were asked to provide samples of sperm for the current study. Consenting donors provided samples by masturbating after 2 – 5 days of abstinence and without the use of lubricants. Anonymous samples were placed into a non-toxic specimen container and processed within 1 hr of collection. Samples

103 were examined for seminal parameters, according to the World Health Organisation criteria (WHO, 2010). Only samples of semen with normal spermatozoa morphology were used for analysis (15 samples from 15 individual donors, labelled 001 – 015). Sperm concentration ranged from 42.0 x106 mL-1 to 68.9 x106 mL-1 (mean 57.4 x106 mL-1), sperm motility (grade a: progressive motility) ranged from 36% to 65% (mean 50.3%). Morphologically normal spermatozoa exhibit a smooth, oval head, a middle piece, and a long tail (Fig. 1.20). A well-defined acrosomal area occupies approximately 40 – 70% of the head. The heads are approximately 3.7 – 4.7 µm in length with a width of 2.5 – 3.2 µm. The midpiece is approximately 0.6 µm in width and 3.3 – 5.2 µm in length, connecting the head to the tail. The principal piece of the tail is unbroken, without kinks or coils and is approximately 45 µm long (James et al., 2004; Coward and Wells, 2013).

3.3.1.2 Spermatozoa preparation Three functional modes of spermatozoa preparation, described below, were used. Initially, a general procedure was used for all three modes of spermatozoa preparation. Following this, an additional procedure was used for each of the three modes of spermatozoa.

§ Initial procedure Spermatozoa from 15 semen samples (001-015) were separated from the seminal plasma by centrifugation using SAGE PureCeption Media (In-vitro Fertilization Inc., Cooper Surgical, USA). In an 11 mL Nunc IVF centrifuge tube (Thermo Fisher Scientific, Denmark, 137860), 2 mL of liquefied semen was layered in a single column over an Upper Phase Gradient (silane-coated colloidal silica in HEPES-Buffered HTF, ART-2040) (40%, v/v, 2 mL) and Lower Phase Gradient (ART-2080) (80%, v/v, 2 mL) and centrifuged (Eppendorf 5702 RH) for 18 mins at 244 rcf (relative centrifugal force) (at 30 oC). The seminal plasma and waste phase gradients were removed. The lower pellet of spermatozoa was removed and washed in a clean 11 mL Nunc tube by inversion in 10 mL Quinn’s Advantage Medium (QAM) with HEPES (SAGE, ART-1024) supplemented with 10 mg/mL human serum albumin (HSA) (SAGE, 100mg/mL, ART- 3003). The spermatozoa were then centrifuged for 8 mins at 244 rcf (at 30 oC). The wash solution was removed and the remaining spermatozoa pellet resuspended in 0.5 mL in the same QAM wash solution.

§ Preparation of mode 1: control/washed 0 min spermatozoa Two μl aliquots of washed spermatozoa from all samples (001-015) were placed inside a defined circle on a plain Superfrost Plus microscope slide (Menzel-Glaser,

104 Thermo Fisher Scientific, 4951PLUS4). Spermatozoa were air-dried and fixed with cold AR-Methanol (Fronine Pty Ltd, Australia) at -20 °C for 10 mins. The slides were labelled “control/washed 0 min” with the sample number added and stored in PBS at 4°C until staining.

§ Preparation of mode 2: activated/washed 1.5 hrs incubated spermatozoa The spermatozoa from all samples (001-015) were activated by incubating in the fresh QAM wash solution at 30 °C for 1.5 hrs. On a plain Superfrost Plus microscope slide (Menzel-Glaser, Thermo Fisher Scientific, 4951PLUS4) a circular area was defined into which 2 μl aliquots of the activated spermatozoa was placed. They were air-dried and fixed with cold AR-Methanol (Fronine Pty Ltd, Australia) at -20 °C for 10 mins. The slides with fixed-spermatozoa were labelled “activated/washed 1.5 hrs incubation” with the sample number added and stored in PBS at 4 °C until staining.

§ Preparation of mode 3: hyper-activated (HA) spermatozoa 0.5 ml of “washed 0 min” spermatozoa from 2 samples (014 and 015) were placed in an 11 mL Nunc IVF centrifuge tube and 0.5 mL of HAmax reagent was added. The HAmax reagent contained: 7.2 mM Pentoxifylline (Sigma P1784), HSA (Sigma A1653), and 2 μg/mL Progesterone (Sigma P6149) and was dissolved in bicarbonate buffered Earle’s Balanced Salt Solution (Sigma E2888), a spermatozoa medium that supports human spermatozoa capacitation. The 11mL Nunc tubes were then incubated at 37 °C for 60 mins in a CO2 incubator. On a plain Superfrost Plus microscope slide (Menzel- Glaser, Thermo Fisher Scientific, 4951PLUS4) a circular area was defined into which 2 μl aliquots of the hyper-activated spermatozoa were placed. They were air-dried and fixed with cold AR-Methanol (Fronine Pty Ltd, Australia) at -20 °C for 10 mins. The slides with fixed-spermatozoa were labelled “HA” with the sample number added and stored in PBS at 4 °C until staining.

3.3.1.3 Immunocytochemistry

§ Single-labelling immunofluorescent cytochemistry Spermatozoa-fixed samples were washed three times in PBS. Non-specific staining was blocked with 10% NDS (D9663, Sigma-Aldrich, Australia) for 20 mins at room temperature in a humidified chamber. Spermatozoa were incubated in 2% NDS in PBS containing primary antibodies, listed in Table 3.1, and then placed in a humidified chamber for overnight at 4 °C. Spermatozoa were rinsed three times with PBS and then were incubated with appropriate Alexa secondary antibodies: Alexa488-DαR, Alexa488- DαG, Alexa594-DαR, and Alexa594-DαG (Molecular Probes, Invitrogen, Carlsbad, CA, USA). Sections were left for 30 mins at room temperature in the dark and then washed 105 three times with PBS. Nuclei were stained with DAPI using an anti-fading fluorescence mounting media (Vectashield Hard+Set Mounting Medium with DAPI, Vector Labs, Fluoroshield with DAPI, Sigma-Aldrich, Australia). In the negative control slides, spermatozoa were incubated with 1% NS without the primary antibody.

Prior to the study, the optimal antibody dilution (working concentration) for OMP and OR6B2 was determined. OMP antibody dilution optimisation was performed using 2 serial slides (one of which was a negative control) at dilutions of 1:1200, 1:1000 and 1:800. It was determined that the optimal dilution for OMP was 1:800. OR6B2 antibody dilution optimisation was performed using 2 serial slides (one of which was a negative control) at dilutions of 1:120, 1:100 and 1:80. It was determined that the optimal dilution for OR6B2 was 1:100. Single labelling immunofluorescent cytochemistry (IF ICC) was conducted on spermatozoa from all 15 samples (001-015). From each functional mode, 5 randomly selected slides were stained with OMP and 2 were stained with OR6B2 and analysed.

§ Double-labelling immunofluorescent cytochemistry Double-labelling IF ICC was performed, using the same protocol as described for single-labelling IF ICC, with the exception that two primary antibodies (OMP and OR6B2) were mixed in PBS containing 2% NDS. The same method was used with the Alexa- labelled secondary antibodies. Double-labelling IF ICC was conducted on spermatozoa from all 15 samples (001-015). From each functional mode, 5 randomly selected slides were used for OMP and OR6B2 staining.

3.3.2 Photography and image processing Slides of stained spermatozoa from each of the 3 functional modes (control, activated and HA) were examined under 40x and 63x lens objectives and photographed. OMP and OR6B2 showed a similar pattern of expression in each mode so images were randomly selected for analysis.

Additional analysis was conducted on control and activated spermatozoa using Z-stack image analysis. For this task, a spermatozoon was randomly selected from a control mode image (from samples 011 and 012) and another from activated mode image (from sample 012).

Photography and image processing were conducted as described in Section 2.3.4.

106 Antibody Dilution

Primary Antibody (name, source) Spermatozoa

Rats Humans

Olfactory marker protein (OMP), goat antiserum, raised 1:800 (IF) 1:800 (IF) with rodent OMP as the immunogen, diluted 1:1 with glycerol, containing 0.05% sodium azide (019-22291; Wako Pure Chemical Industries, Ltd., Japan)

Gaolf (Golf), goat polyclonal, P-15, raised against a 1:250 (IF) n/a peptide mapping near the N-terminus of Gaolf of human origin (sc-26763; Santa Cruz Biotechnology, CA, USA)

Adenylyl cyclase III (AC3), rabbit polyclonal, C-20, 1:250 (IF) n/a raised against a peptide mapping at the C-terminus of adenylyl cyclase III of mouse, rat, and human origin (sc-588; Santa Cruz Biotechnology, CA, USA)

OR6B2, rabbit polyclonal, developed against n/a 1:100 (IF) Recombinant Protein corresponding to amino acids: ENVTKVSTFILVGLPTAPGLQYLL (NBP2-13711; Novus Biologicals, CA, USA)

Table 3.1: Primary antibodies used in Chapter 3. Abbreviations: immunofluorescent histochemistry, IF; not applicable, n/a.

107 3.3.3 Results

3.3.3.1 Expression of OMP in human spermatozoa In human spermatozoa, the three main components are evident: the head containing the anterior acrosome and equatorial segment, the connecting piece, and the tail, as described in Section 1.3.3 (see Fig. 1.20) and shown in Fig. 3.1A. Expression of OMP was observed on the head, connecting piece, and tail of control human spermatozoa (Fig. 3.1B-C). Expression was lighter on the acrosomal region than on the post-acrosomal sheath or tail. When the head was viewed in the classical orientation, expression of OMP was prominently observed along the peripheral edges of the equatorial segment (where they fold back), often appearing as a pair of lateral blotches with dense reaction product (Fig. 3.1B). Prominent expression of OMP was also evident on the connecting piece, where the centriole is located. Along the tail, OMP was expressed in discontinuous, punctate and seemingly irregular patterns (Fig. 3.1B-C).

Z-stack image analysis was conducted to evaluate the staining on control human spermatozoa (functional mode 1: control/washed 0 min) stained with OMP. Image analysis showed that OMP was expressed prominently in a dot-like pattern along the equatorial segment and on the anterior part of the head. OMP was also expressed on the connecting piece and in discontinuous and punctate patterns along the entire length of the tail, except for the middle piece (Fig. 3.2).

3.3.3.2 Co-expression of OMP and an OR in human spermatozoa Double-labelling IF ICC was applied to human spermatozoa to investigate whether OMP and/or OR(s) were co-expressed. Flegel et al. (2015) had previously demonstrated that OR6B2 was the only OR to be expressed on the equatorial segment of the head—where it was strongly expressed—and on the principal piece of the tail.

In control spermatozoa double-labelling IF ICC showed that OMP co-expressed with OR6B2 (Fig. 3.3A). All control spermatozoa showed strong OMP and OR6B2 co- expression along the equatorial segment on the head and along the tail, except for the middle piece (Fig. 3.3B-C). OR6B2 expression was continuous and more prominent along the tail (Fig. 3.3C) than expression of OMP and was in agreement with observations by Flegel et al. (2015).

108

Figure 3.1: Expression of OMP in human spermatozoa using immunofluorescent cytochemistry. Photomicrographs show spermatozoa (mode 1: control/washed 0 min) stained with OMP. (A) DIC optic show structure of human spermatozoa, consisting of head, connecting piece, and tail. (B) Immunofluorescence shows expression of OMP (red) in spermatozoa. OMP is prominently expressed on equatorial segment (arrows) of head and connecting piece (arrowheads), and faintly expressed on acrosomal cap region of head (crossed arrows). (C) DAPI stains spermatozoa nuclei (blue) (arrow).

109

Figure 3.2: Expression of OMP in two human spermatozoa using immunofluorescent cytochemistry. Photomicrograph shows Z-stack image of spermatozoa (mode 1: control/washed 0 min) stained with OMP. OMP (red) is expressed in prominent, dot-like pattern on equatorial segment of head (arrows) and faintly on acrosomal cap (crossed arrow) of head. OMP is expressed prominently on connecting piece (arrowhead) and in discontinuous and punctate pattern along tail except for middle piece (double-headed arrow). DAPI stains spermatozoa nuclei (blue).

Figure 3.3 (next page): Co-expression OMP with OR6B2 in human spermatozoa. Photomicrographs show spermatozoa (mode 1: control/washed 0 min) stained with OMP and OR6B2. (A) Co-expression (yellow) of OMP and OR6B2 is observed on equatorial segment of head (arrows) and tail, except for middle piece. DAPI stains spermatozoa nuclei (blue). (B) OMP (red) is expressed on equatorial segment, acrosomal cap (faintly), and tail, except for middle piece. OMP is expressed on connecting piece. (C) OR6B2 (green) is prominently expressed on tail, except for middle piece, and on equatorial segment of head (faintly).

110

111 In activated spermatozoa (functional mode 2: activated/washed 1.5 hrs incubation) double-labelling IF ICC showed that OMP co-expressed with OR6B2 (Fig. 3.4A). Two types of co-expression were observed in activated spermatozoa. In most activated spermatozoa, co-expression occurred along the equatorial segment of the head, and along the tail, with OR6B2 expressing more strongly on the tail (Fig. 3.4A-C) and OMP expressing more strongly on the equatorial segment (Fig. 3.4A-B). These patterns of co-expression were similar to those at described in control spermatozoa (Fig. 3.4A). However, in a few grouped spermatozoa no co-expression was observed on the equatorial segment, but prominent co-expression was observed on the acrosomal cap of the head instead (Fig. 3.4A).

Z-stack image analysis was conducted to analyse the activated human spermatozoa stained with OMP and OR6B2. Image analysis showed that OMP and OR6B2 prominently co-expressed on the acrosomal cap of the head. Co-expression was observed in a punctate pattern along the tail, except for the middle piece. OMP was singly expressed on the connecting piece (Fig. 3.5).

In hyper-activated (HA) spermatozoa (functional mode 3: hyper-activation) double-labelling IF ICC showed that OMP co-expressed with OR6B2 (Fig. 3.6A). In all HA spermatozoa, prominent co-expression was seen only on the acrosomal cap of the head (Fig. 3.6A). OR6B2 and OMP were faintly co-expressed along the tail, except for the middle piece. In all spermatozoa, OMP was singly expressed on the connecting piece (Fig. 3.6A).

Figure 3.4 (next page): Co-expression OMP with OR6B2 in human spermatozoa. Photomicrographs show spermatozoa (mode 2: activated/washed 1.5 hrs incubation) stained with OMP and OR6B2. (A) Co-expression (yellow) of OMP and OR6B2 is seen on head and on tail, except for middle piece. On head, prominent co-expression (arrowheads) is seen on acrosomal cap. DAPI stains spermatozoa nuclei (blue). (B) OMP (red) is expressed on equatorial segment (arrow) and on acrosomal cap (arrowheads) of head. OMP is expressed on connecting piece and only faintly on tail and no expression is visible on middle piece. (C) OR6B2 (green) is expressed on acrosomal cap (arrowheads) of head. OR6B2 is also expressed prominently on tail and no expression is visible on middle piece.

112

113

Figure 3.5: Co-expression OMP with OR6B2 in human spermatozoon. Photomicrograph shows Z-stack image of spermatozoon (mode 2: activated/washed 1.5 hrs incubation) stained with OMP and OR6B2. Co-expression (yellow) of OMP and OR6B2 is seen on acrosomal cap of head and in punctate pattern on tail, except for middle piece. OMP (red) is singly expressed on connecting piece. DAPI stains spermatozoa nucleus (blue).

Figure 3.6: Co-expression OMP with OR6B2 in human spermatozoa. Photomicrograph shows spermatozoa (mode 3: hyper-activation) stained with OMP and OR6B2. Strong co-expression (yellow) (arrowheads) is seen on acrosomal cap of all spermatozoa. OMP is singly expressed on connecting piece. DAPI stains spermatozoa nuclei (blue).

114 3.4 Expression of OMP, Golf, and AC3 in epididymal spermatozoa of rats

3.4.1 Materials and methods

3.4.1.1 Animals Adult male Wistar rats (n=16) were used in accordance with the NHMRC Animal Experimentation Guidelines and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1990) and with the approval of the Animal Care and Experimentation Committee for the University of New South Wales. All animals were obtained from the Animal Resources Centre (Perth, WA).

3.4.1.2 Spermatozoa collection and preparation Rats were euthanised and spermatozoa were collected from the epididymis. The epididymis was separated from the testes, cut along its length, and placed in Dulbecco’s Phosphate Buffered Saline (PBS, Sigma-Aldrich, D8537). Spermatozoa were allowed to diffuse into PBS at room temperature for 10 mins. Diluted spermatozoa were aliquoted into 10 – 20 µL batches, smeared, and air-dried on Superfrost microscope slides (Menzel-Glaser, Thermo Fisher Scientific, 4951PLUS4). Spermatozoa preparations were then fixed by immersion in AR-Methanol (Fronine Pty Ltd, Australia) at -20 °C for 10 mins, then washed three times and stored in PBS at 4 °C until staining.

3.4.1.3 Immunocytochemistry methods

§ Single-labelling immunofluorescent cytochemistry IF ICC was performed, following the procedure outlined in Section 3.3.1.3. Primary antibodies used for ICC on rat spermatozoa, their sources, and working concentrations are listed in Table 3.1.

3.4.2 Photography and image processing Photography and image processing were conducted, as described in Section 2.3.4.

115 3.4.3 Results

To determine expression of OMP, Golf, and AC3, IF ICC was conducted on intact epididymal spermatozoa of rats. Only intact spermatozoa with normal histological structure, as described in Section 1.3.3 were chosen for image analysis.

Figure 3.7: Expression of OMP in rat spermatozoa using immunofluorescent cytochemistry. Photomicrographs show epididymal spermatozoa stained with OMP. (A) DIC optics shows typical structure of rat spermatozoa, consisting of the head (bracket), connecting piece, and tail. (B) Expression of OMP (red) is observed on the acrosome and equatorial segment (ES) (arrowheads) of the head, on the connecting piece (arrow), and in a discontinuous and punctate pattern along length of the tail with strongest expression on the middle piece. (C) Expression of OMP (red) is seen on the acrosome (arrow). DAPI stains spermatozoa nucleus (blue) (arrow).

116 3.4.3.1 Expression of OMP, Golf, and AC3 in rat epididymal spermatozoa Expression of OMP was observed on the head, connecting piece, and tail of rat spermatozoa (Fig. 3.7B-C). On the head, OMP was expressed on the acrosome (located on the apical part of the head), and on the equatorial segment. OMP was expressed prominently on the connecting piece where the centriole is located. OMP was expressed along the plasma membrane of the tail in a discontinuous fashion, with strongest expression observed on the middle piece (Fig. 3.7B-C). The anatomic location of OMP expression was determined by comparing to published images of rat spermatozoa in the literature (Oko et al., 1993; Darszon et al., 2011).

Expression of Golf and AC3 was observed on the head, connecting piece, and tail of rat spermatozoa (Fig. 3.8A-B). On the head, Golf and AC3 were expressed on the

Figure 3.8: Expression of Golf and AC3 in rat spermatozoa using immunofluorescent cytochemistry.

Photomicrographs show epididymal spermatozoa stained with Golf (A) and AC3 (B).

Expression of both proteins is seen in similar locations. On head, Golf (red) and AC3 (green) are expressed on acrosome (white arrow) and equatorial segment (ES)

(arrowhead). Golf and AC3 are expressed on connecting piece (CP) (yellow arrow) and in discontinuous and punctate pattern on tail, with strongest expression on middle piece. DAPI stains spermatozoa nuclei (blue).

117 acrosome (apical part) and equatorial segment (Fig. 3.8A-B). Golf and AC3 were simultaneously expressed along the plasma membrane of the tail, in a discontinuous fashion, with strongest expression observed on the middle piece and gradually diminishing towards the principle piece of the tail. These patterns of Golf and AC3 expression were similar to those of OMP (Fig. 3.8A-B).

3.5 Discussion The current study is the first to show OMP expression in ejaculated human spermatozoa. OMP was expressed on the equatorial segment and anterior acrosome of the head, on the connecting piece, and along the plasma membrane of the tail, except for the middle piece. The current study is also the first to show expression of OMP in rat epididymal spermatozoa and to compare it with expression of the chemosensory proteins: Golf and AC3. OMP was expressed on the acrosome and equatorial segment of the head, connecting piece, and along the tail. Along the tail, the strongest OMP expression was observed on the middle piece. Golf and AC3 were expressed in locations similar to those in which OMP was expressed: on the acrosome and equatorial segment of the head, on the connecting piece, and along the tail in a discontinuous pattern, with strongest expression evident on the middle piece.

These observations extend results reported in previous publications (Vanderhaeghen et al., 1993; Walensky et al., 1995; Baxendale and Fraser, 2003a, b; Spehr et al., 2004b; Livera et al., 2005). For example, antibodies OD1 and OD2, raised against two conserved OR peptide sequences were found in late spermatids and the middle piece of mature spermatozoa from the cauda epididymis in rats (Walensky and Snyder, 1995). OD2 was also found on the connecting piece of spermatozoa (Walensky and Snyder, 1995). The DTMT receptor (recombinant protein encoded by the major OR transcript found in dog testes) was found in late round and elongated spermatids—in the cytoplasmic droplet (CD) and on the middle piece of the tail in epididymal and ejaculated spermatozoa of dogs (Vanderhaeghen et al., 1993). AC3 was found on the acrosomal cap, neck, and middle piece of spermatozoa in mice (Baxendale and Fraser, 2003a; Livera et al., 2005).

OMP was expressed prominently on the acrosome and on the equatorial segment of the head, on the connecting piece, and along the tail of all human and rat spermatozoa. OMP expression on the acrosomal cap was quite consistent and may imply the existence of a guidance mechanism to direct spermatozoa to the oocyte. The expression of OMP on the tail occurred in a discontinuous and more variable punctate

118 pattern that was nevertheless common across human and rat spermatozoa. These results suggest a role for chemoreception in the reproductive system.

Alternatively, OMP may contribute to sperm development and maturation or may participate in the regulation of speed and direction of the spermatozoa movement by recognising chemicals, such as hormones, proteins or lipids. This process may involve epididymosomes, which are located in the lumen of the epididymis. The protein composition varied in epididymosomes from different epididymal regions (Fig. 1.15). Epididymosomes deliver specific proteins to spermatozoa as they pass through the epididymis as discussed in Section 1.3.2.2. Although the current study showed that OMP expressed in dot-like patterns on the tail of both human and rat spermatozoa, some differences in expression were observed, on the middle piece of the tail, between species.

In rats, OMP was expressed prominently on the middle piece of the tail, together with Golf and AC3. However, in humans, no OMP expression was observed on the middle piece. This raises questions about what these differences imply for chemoreception and sperm function. These species-specific differences in OMP expression on the middle piece of the tail may be explained in several ways. The differences may be due to the nature of the spermatozoa studied. In humans, fully-mature ejaculated spermatozoa were studied. In contrast, samples of rat spermatozoa were collected from different parts of the epididymis and the sperm may have been in different stages of maturation. Further study on ejaculated rat spermatozoa is recommended in order to compare OMP expression between epididymal and ejaculated spermatozoa to assess developmental status.

Differences in OMP expression on the middle piece of the tail in rat and human spermatozoa may be due to differences in their morphology, as described in Section 1.3.3. For example, in many species, including rats, CDs are shed from spermatozoa after ejaculation, as described in Section 1.3.2.3. However, unlike CDs of other species, human CDs remain attached to spermatozoa in the female reproductive tract and may regulate fluid intake as sperm travel (Mortimer et al., 1982; Abraham-Peskir et al., 2002; Cooper et al., 2004). Finally, the differences in OMP expression may be the due to different species-specific challenges that spermatozoa face as they negotiate the female genital tract. For example, differences between mammalian species have been observed in the expression of ion channels and signalling in the spermatozoa (Strünker et al., 2011; Brenker et al., 2012; Lishko et al., 2012; Strünker et al., 2015). Human spermatozoa, but not mouse spermatozoa, contain the proton channel Hv1 (Lishko et al., 2010). Mouse spermatozoa, but not human spermatozoa, contains purinergic P2X

119 channels (Navarro et al., 2011; Brenker et al., 2012). Progesterone activates CatSper in humans (Lishko et al., 2011; Strünker et al., 2011), but not in mice (Lishko et al., 2011). 2+ Human Slo3 is activated by Ca rather than pH1, whereas mouse Slo3 is activated only by pH1 (Brenker et al., 2014).

The current study examined the co-expression of OMP and OR6B2 in human spermatozoa. OR6B2 is one of the many ORs in human spermatozoa identified by Flegel et al. (2015). This OR was chosen for the current study because of its defined, compartment-specific expression on the equatorial segment of the head and along the tail. Flegel et al. (2015) suggested that such a pattern of expression could indicate that OR6B2 participates in acrosomal exocytosis, binding of the spermatozoa to egg and perhaps chemotaxis. The pattern of OR6B2 expression in control spermatozoa, reported in the current study, replicates the pattern of expression reported by Flegel et al. (2015). Co-expression of OMP and OR6B2 was studied in three experimentally-created functional modes of spermatozoa:

mode 1 - control/washed 0 min;

mode 2 - activated/washed 1.5 hrs incubation; and

mode 3 - hyper-activation (HA).

Co-expression occurred in all three modes. In all control spermatozoa, OMP and OR6B2 were strongly co-expressed on the equatorial segment of the head and along the tail, except for the middle piece. In activated spermatozoa, OMP and OR6B2 were strongly co-expressed and two types of co-expression were observed. Co-expression of OMP and OR6B2 occurred in most activated spermatozoa, similar to the co-expression observed in control spermatozoa – on the equatorial segment of the head and along the tail, except for the middle piece. Strong co-expression of OMP and OR6B2 occurred, in a small number of activated spermatozoa, on the acrosomal cap of the head and along the tail, except for the middle piece. In all hyper-activated spermatozoa, OMP and OR6B2 were strongly co-expressed on the acrosomal cap of the head and along the tail, except for the middle piece. However, the co-expression of OMP and OR6B2 that was observed on the tail did not change in activated or hyper-activated spermatozoa and did not differ from control spermatozoa.

OMP did not co-express with OR6B2 in only one location: the connecting piece. This finding was consistent with the results of the Kang et al. (2015) study on co- expression of OMP and ORs in the bladder and thyroid of mice. The authors observed that a small number of OMP(+) cells did not co-express with ORs. Even though, in the current study, OMP did not co-express with OR6B2 on the connecting piece, it may be

120 that OMP may co-express with other ORs reported by Flegel et al. (2015)—such as OR3A2, OR2H1/2, OR10J1, or OR51E2—in this location and this could be the subject of future investigation.

3.6 Conclusion The current study found that OMP was expressed in compartment-specific locations in human and rat spermatozoa. OMP co-expressed with an OR (OR6B2) in control, activated, and hyper-activated human spermatozoa. Activation and hyper- activation of spermatozoa affected co-expression locations. OMP, Golf, and AC3 were expressed in similar, compartment-specific locations in epididymal rat spermatozoa. These observations demonstrate that OMP expression is a reliable indicator of OR- mediated chemoreception in non-olfactory tissues and that OMP may be used to identify ectopically-expressed ORs.

ORs may be involved in different physiological processes that occur when spermatozoa travel to the oocyte to complete fertilisation. ORs may enable spermatozoa to detect and respond to a variety of chemotactic or chemokinetic factors that could influence the speed and direction of their movement. It remains to be determined whether these functions are performed by all ORs in spermatozoa or whether they are specific to particular ORs located in specific compartments, for example OR6B2.

Much remains to be learned about the nature of the ligands that activate ORs. It is unlikely that floral ligands such as bourgeonal or lyral themselves would be released by oocytes, but endogenous mimics of these ligands may exist. Future studies involving oocyte extracts and analysis of follicular fluid are needed to identify the nature of ligand(s). The resulting knowledge could have direct importance for a better understanding of infertility.

121

Chapter 4: Expression of OMP, Golf, and AC3 in mast cells of rats

122 4.1 Abstract Olfactory receptor- (OR-) associated events are mediated by conserved signal transduction molecules of olfactory sensory neurons (OSNs), involving olfactory G protein (Golf), adenylyl cyclase 3 (AC3), and olfactory marker protein (OMP). OMP is a tag for maturing and mature OSNs (mOSNs) in the olfactory epithelium (OE). ORs, OMP,

Golf, and AC3 are co-expressed in non-olfactory tissues in mice. This suggests that OMP may serve as an indicator of OR-mediated chemoreception in non-olfactory systems. I hypothesised that mast cells—mobile cells of the immune system—might rely on an OR- mediated sensing mechanism for guidance. Expression of OMP, Golf, and AC3 in mast cells of the tongue, liver, and white adipose tissue (WAT) of rats was investigated using

IHC. I showed that OMP, Golf, and AC3 were expressed in mast cells of the tongue, liver, and WAT. OMP, Golf, and AC3 showed identical patterns of expression and co- expressed in the mast cells of the tongue. These observations suggest that an OR- mediated signalling pathway is present in mast cells and is involved in chemoreception and cellular guidance.

4.2 Introduction Chemoreception is a process by which a cell or an organism responds to a chemical substance in its environment (Malnic et al., 1999). Living organisms interact with and respond to environmental molecules by expressing chemoreceptors in the form of specific ORs (Giorgi et al., 2011). Deciphering odorant signals presents a common challenge to all animals. Environmental odorants serve to communicate a diverse array of complex information that often requires immediate behavioural reactions (Ache and Young, 2005). For example, they help the animals to locate desirable items (food, water), avoid danger (fire, predators), mate and care for their offspring (Ache and Young, 2005). Additionally, they allow animals to establish a comprehensive image of the environment and to appreciate change by monitoring ambient olfactory patterns (Wilson and Leon, 1988; Sullivan and Wilson, 2003). The mechanism by which ORs interact with specific environmental odorants is still the subject of numerous investigations (Giorgi et al., 2011). ORs possess several hypervariable transmembrane domains with high binding affinity for aliphatic or hydrophobic hydrocarbon chains, as discussed in Section 1.1.3.1 (Abaffy et al., 2007). An environmental odorant may bind specifically to its respective receptor only if it expresses a conformational structure comprised of a number of routable carbon bonds. However, ORs have a number of adaptable binding sites within their transmembrane regions to interact with each other and with the incoming odorant (Peterlin et al., 2008). This type of odorant (ligand) - OR interaction means that individual

123 OR responds to a particular odorant but can also recognise different substances with similar binding characteristics. This means that a single OR can recognise several odorants and a single odorant to be recognised by multiple ORs, as discussed in Section 1.1.3.1. Thus, this type of interactions may result in combinatorial codes, giving rise to the recognition of new patterns of odorant activity (Dreyer, 1998; Galizia and Menzel, 2000; Stensmyr et al., 2003).

Several studies have reported that olfactory-mediated sensing mechanisms may also monitor extracellular chemical cues in non-olfactory tissues (Kang and Koo, 2012;

Kang et al., 2015). The principal components of olfactory signalling (ORs, Golf, and AC3) have been found in various non-olfactory tissues in studies using microarray and RNA- Seq expression analyses (Zhang et al., 2004; Feldmesser et al., 2006; Kang and Koo, 2012). OMP expression was first reported in various non-olfactory tissues in mice (Kang et al., 2015). With the use of conventional quantitative RT-PCR analysis and double immunoassay techniques, the authors showed high levels of OMP expression in skeletal muscle, the heart, thymus, and thyroid. Lower levels of expression were found in the liver, bladder, pancreas, stomach, duodenum, testes, spleen, and lung (Kang et al., 2015). Using IHC, they detected significant levels of OMP expression in five non- olfactory tissues: the bladder, thyroid, thymus, heart, and testes. Moreover, they determined that OMP was expressed in certain cell types rather than indiscriminately throughout the tissue. For example, OMP expression was found in the interstitial cells of the submucosal layer of the bladder, in the parafollicular cells of the thymus, in the epithelial cells of the medullar area of the thyroid, and in a Leydig cell-like population of the interstitial tissue of the testes (Kang et al., 2015).

The authors suggested that OMP expression could be used to infer the presence of ORs and that an OR-mediated signalling pathway might be present in several non- olfactory tissues. In the reproductive system, the authors found lower levels of OMP expression in the interstitial tissue and no evidence for the presence of Golf or AC3 (Kang et al., 2015). These results suggest low or zero levels of expression or insensitivity of the antibodies. Kang et al. (2015) findings suggest that OMP is expressed in various non- olfactory tissues with or without components of the olfactory transduction cascade—OR,

Golf, and AC3. This raises the question of whether olfactory-mediated sensing molecules work alone or in conjunction with each other to mediate mechanisms involving chemosensing guidance in mobile cells. It also raises questions about what molecules might be utilised in other mammals.

To answer these questions, I aimed to determine whether olfactory-mediated sensing mechanisms might be present in mast cells of the rat. I hypothesised that mast

124 cells may use ORs to navigate their way to areas of inflammation using the chemosensory proteins OMP, Golf, and AC3. IHC was used to show the presence of these proteins in mast cells of the tongue, liver, and WAT.

4.3 Materials and methods

4.3.1 Animals Six adult Sprague-Dawley male rats were used, with the approval of the University of New South Wales’ Animal Care and Experimentation Committee and in strict accordance with the NHMRC Animal Experimentation Guidelines and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1990). All rats were obtained from the Animal Resources Centre (Perth, WA) and handled with care prior to experimentation.

4.3.2 Perfusion Perfusion was performed, as described in Section 2.3.2.

4.3.3 Histological methods

4.3.3.1 Tissue preparation From each rat, the following tissues were prepared and postfixed for 12-14 hours in 10% neutral buffered formalin (Sigma-Aldrich Pty Ltd., Australia): the olfactory turbinate with OBs (as positive control); the tongue (from apex to root); three pieces of liver that were randomly selected; and white adipose tissue (WAT). Tissue was immersed overnight in 70% (v/v) ethanol and embedded using standard paraffin embedding procedures. Tongue was positioned longitudinally, dorsal surface down, on the mould. Three pieces of liver were positioned parallel to each other on one mould. Tissue was sectioned at 5 μm thickness on a motorised microtome (Leica RM 2155, Leica Microsystems Pty Ltd). Tongue, liver and WAT were serially cut, mounted consecutively and numbered on electrostatic slides (Menzel-Glaser, Braunschweig, Germany). Slides of each tissue type were randomly selected from each experimental animal. Experimental samples were selected from different points in the numbering spectrum. The exception was for experiments with a negative control staining, for which adjacent slides were selected.

125 4.3.3.2 Haematoxylin and Eosin staining Haematoxylin and Eosin (HE) staining was performed, as outlined in Section 2.3.3.2.

4.3.3.3 Toluidine Blue staining Toluidine Blue (TB) is a basic, weak cationic, hydrophilic thiazine metachromatic dye. TB has a high affinity for acidic tissue components (sulphates, carboxylates, and phosphate radicals). Because it also has a high affinity for nucleic acids, it stains tissue rich in DNA and RNA (Epstein et al., 1992; Epstein et al., 1997). Metachromatic stains are strongly recommended as routine stains for mast cells. TB stains mast cells red- purple (metachromatic staining) and the background blue (orthochromatic staining) (Krishnaswamy and Chi, 2006).

§ First application of TB staining Staining with TB was used to detect the presence of TB-positive (TB(+)) mast cells in tongue, liver, and WAT.

Preparatory to the study, the optimal time for TB staining was determined. Two slides of tongue, liver and WAT (6 slides) from 2 randomly selected rats were stained for 30, 45 or 60 secs. It was observed that the optimal staining time was 60 secs and this time was used for subsequent experiments. TB staining was performed on 5 randomly selected slides of tongue, 5 slides of WAT and 2 slides of liver from each of 6 rats. The first slide in the numbering spectrum was stained with TB and called the “TB control slide”. Subsequent slides in the numbering spectrum were stained with the appropriate primary antibodies (as described below) using IHC. All slides were scanned, photographed and image processed to detect TB(+) mast cells.

TB staining was performed by dewaxing paraffin sections and hydrating them with distilled water and TB (working solution of 1% TB in 70% ethanol, 5 ml and 1% sodium chloride in distilled water, 45 ml mixed well, pH around 2.3) for 60 secs. Sections were washed three times in distilled water and dehydrated quickly once in 95% ethanol and twice in 100% ethanol. They were then cleared twice in xylene for 3 mins each and cover-slipped with DPX mounting medium.

§ Second application of TB staining Slides to which IHC had been applied were restained with TB to determine co- expression of TB with OMP, Golf or AC3 in the cells of tongue, liver tissue and WAT. This co-expression was an indicator that these immunopositive cells were TB(+) mast cells.

126 Slides with tongue tissue, liver tissue, and WAT were left in xylene until coverslips were removed. Slides were washed three times in xylene for 60 secs each, washed three times in 100% ethanol, then three times in 70% ethanol for 60 secs each. Slides were hydrated with distilled water, stained with TB (working solution of 1% TB in 70% ethanol and 1% sodium chloride in distilled water, pH around 2.3) for 60 secs, washed three times in distilled water and dehydrated quickly, once in 95% ethanol and twice in 100% ethanol. They were then cleared twice in xylene for 3 mins each and cover slipped with DPX mounting medium.

4.3.3.4 Immunohistochemistry methods

§ Immunoperoxidase histochemistry Prior to the study, the optimal antibody dilution (working concentration) for OMP,

Golf and AC3 was determined by testing a number of different dilutions. OMP antibody dilution optimisation was performed on 2 serial slides (one of which was a negative control) from tongue, liver and WAT from 2 randomly selected rats at dilutions of 1:100, 1:50 and 1:30. It was determined that the optimal dilution for OMP was 1:50 for all tissues. Golf antibody dilution optimisation was performed on 2 serial slides (one of which was a negative control) from tongue, liver and WAT from 2 randomly selected rats at dilutions of 1:100, 1:80 and 1:50. It was determined that the optimal dilution for Golf was 1:50 for tongue and liver tissues and 1:80 for WAT. AC3 antibody dilution optimisation was performed on 2 serial slides (one of which was a negative control) from tongue, liver and WAT from 2 randomly selected rats at dilutions of 1:100, 1:50 and 1:25. It was determined that the optimal dilution was 1:25 for all tissues. Immunoperoxidase histochemistry (IP IHC) was performed on 10 OMP-stained OMP(+) and 10 negative control OMP(-) slides, 10 Golf(+) and 10 Golf(-) slides and another 10 AC3(+) and 10 AC3(-) slides of each tissue of each rat. IP IHC was performed, following the procedure outlined in Section 2.3.3.3. Primary antibodies used for IP IHC, their sources and working concentrations are listed in Table 4.1.

Ten slides each were stained for OMP(+), Golf(+) and AC3(+), and of these, 4 from each tissue cohort were randomly selected for restaining with TB. This method was performed following the procedure, outlined in Section 4.3.3.3.

§ Single-labelling immunofluorescent histochemistry

Prior to the study, the optimal antibody dilution was determined for OMP, Golf and AC3 on tongue tissue. OMP antibody dilution optimisation was performed on 2 serial slides (one of which was a negative control) from tongue tissue of 2 randomly selected rats at dilutions of 1:1200, 1:1100, 1:1000, 1:900 and 1:800. It was determined that the

127 optimal dilution for OMP was 1:1100. Golf antibody dilution optimisation was performed on 2 serial slides (one of which was a negative control) from tongue tissue of 2 randomly selected rats at dilutions of 1:350, 1:300, 1:250 and 1:200. It was determined that the optimal dilution for Golf was 1:300. AC3 antibody dilution optimisation was performed on 2 serial slides (one of which was a negative control) from tongue tissue of 2 randomly selected rats at dilutions of 1:350, 1:300, 1:250 and 1:200. It was determined that the optimal dilution for AC3 was 1:300. For single-labelling immunoflourescent histochemistry (IF IHC), the number of tongue slides for each rat was as follows: 10

OMP(+), 10 Golf(+) and 10 AC3(+), with 1 negative control slide for each staining. Single- labelling IF IHC was performed, following the procedure outlined in Section 2.3.3.3. Primary antibodies used for IF IHC, their sources and working concentrations are listed in Table 4.1.

§ Double-labelling immunofluorescent histochemistry Double-labelling IF IHC was performed using the same protocol as described for single-labelling IF with the exception that two primary antibodies (OMP and Golf; Golf and AC3) were mixed in PBS containing 2% NDS. The same method was used with the Alexa-labelled secondary antibodies. For double-labelling IF IHC, 5 randomly selected slides of tongue tissue were used for OMP and Golf staining and 5 slides for Golf and AC3 staining.

4.3.3.5 Co-expression of OMP, Golf, or AC3 with TB in mast cells of tongue, liver and white adipose tissue

Co-expression of OMP, Golf or AC3 with TB was assessed in mast cells of the tongue, liver and WAT from 2 randomly selected rats (R21 and R24). IP IHC using OMP,

Golf or AC3 was applied to tissue slides, which were subsequently restained with TB and scanned (Aperio ImageScope, Leica). Mast cells were manually counted at 40x magnification. In each rat, mast cells were counted as follows: in the liver - around three randomly selected portal triads; in the tongue - within 5 randomly selected areas; in WAT - within 15 randomly selected areas.

4.3.4 Photography and image processing Slides of tongue, liver and WAT were all examined under 2x, 10x, 20x, 40x and 100x lens objectives. The tissue was remarkably similar in appearance between animals so for photography, after eliminating rat 23 (R 23) because of artifactual folding in all of its tongue slides, rat R21 was randomly selected to represent the group and photographs of tongue, liver tissue, and WAT were collected. Photography and image processing were conducted, as described in Section 2.3.4.

128 Antibody Dilution

Primary Antibody (name, source) Tissue

Tongue Liver WAT

Olfactory marker protein (OMP), goat 1:50 (IP) 1:50 (IP) 1:50 (IP) antiserum, raised with rodent OMP as the 1:1100 (IF) immunogen, diluted 1:1 with glycerol, containing 0.05% sodium azide (019-22291; Wako Pure Chemical Industries, Ltd., Japan)

Gaolf (Golf), goat polyclonal, P-15, raised 1:50 (IP) 1:50 (IP) 1:80 (IP) against a peptide mapping near the N- terminus of Gaolf of human origin (sc-26763; Santa Cruz Biotechnology, CA, USA)

Gaolf (Golf), rabbit polyclonal, K-19, raised 1:300 (IF) n/a n/a against an epitope mapping within a highly divergent domain of Gaolf of rat origin (sc- 385; Santa Cruz Biotechnology, CA, USA)

Adenylyl cyclase III (AC3), rabbit polyclonal, 1:25 (IP) 1:25 (IP) 1:25 (IP) C-20, raised against a peptide mapping at 1:300 (IF) the C-terminus of adenylyl cyclase III of mouse, rat, and human origin (sc-588; Santa Cruz Biotechnology, CA, USA)

Table 4.1: Primary antibodies used in Chapter 4. Abbreviations: immunoperoxidase histochemistry, IP; immunofluorescent histochemistry, IF; WAT, white adipose tissue; not applicable, n/a.

129 4.4 Results Tongue, liver, and WAT of six rats were examined for the presence of mast cells expressing OMP, Golf, and AC3. Results of these experiments on a representative rat are shown in photomicrographs below at increasing magnification.

Figure 4.1: Rat tongues stained with Haematoxylin and Eosin and Toluidine Blue. Photomicrographs of cross sections show tongue stained with Haematoxylin and Eosin (A) and tongue stained with Toluidine Blue (TB control) (B) at low magnification, tongue structure, and mast cell distribution. (A) Photomicrograph shows classical structures of tongue in longitudinal orientation. (B) Photomicrograph shows TB control slide. TB- positive mast cells, violet in colour, are observed on tongue surface, widely and evenly distributed across tongue body in dot-like pattern. Square box highlights a section of tongue examined at higher magnifications (in Figs. 4.2 – 4.4).

130 4.4.1 Expression of OMP, Golf, and AC3 in mast cells of tongue Haematoxylin and Eosin (HE) staining, Toluidine Blue (TB) staining and IHC were performed to determine expression of OMP, Golf, and AC3 in the mast cells of rat tongue.

4.4.1.1 Toluidine Blue control staining of tongue Toluidine Blue (TB) staining showed that TB-positive (TB(+)) mast cells were present in the tongue. These cells, at low magnification, were observed as dark blue/violet dots distributed evenly across the entire body of the tongue (Fig. 4.1A-B).

At higher magnification, TB(+) mast cells were observed in the connective tissue and between the muscle fibres of the tongue. Denser cell accumulations were observed around blood vessels. Mast cells displayed different sizes and shapes and their granules appeared as deep blue/violet masses. Some mast cells displayed pale blue nuclei, while the nuclei of others were obscured by granules (Fig. 4.2).

Figure 4.2: Rat tongue stained with Toluidine Blue. Photomicrograph of cross section of Toluidine Blue- (TB-) stained tongue shows TB- positive mast cells (TB(+) MCs) (arrows), violet in colour, located between muscle fibres (M) and throughout connective tissue (CT) around blood vessel (BV). TB(+) MCs are heterogeneous in size. In some cells, nucleus is visible (black arrow), while in others, granule-filled cytoplasm obscures nuclei (red arrow).

131 4.4.1.2 Expression of OMP, Golf, and AC3 in tongue

IP IHC showed expression of OMP, Golf, and AC3 in certain cells of the tongue.

When the OMP(+), Golf(+), and AC3(+) cell phenotypes were compared with the corresponding morphological phenotype of TB(+) mast cells in the tongue, it was apparent that OMP, Golf, and AC3 were expressed in mast-like cells. As was the case for mast cells that expressed TB, mast-like cells that expressed OMP, Golf, or AC3 were

Figure 4.3: Expression of OMP, Golf, and AC3 in tongue using immunoperoxidase histochemistry. Photomicrographs of cross sections of tongue show (A) OMP-stained tongues. OMP (brown) is expressed in mast-like cells (OMP(+) MCs) (arrows) located between muscle fibres (M) and around blood vessel (BV); (B) in negative control slide (OMP(-)), no OMP

expression is seen; (C) Golf-stained tongue. Golf (brown) is expressed in mast-like cells

(Golf(+) MCs) (arrows) located between muscle fibres (M); (D) in negative control slide

(Golf(-)), no Golf expression is seen; (E) AC3-stained tongue. AC3 (brown) is expressed in mast-like cells (AC3(+) MCs) (arrows) located between muscle fibres (M) and around blood vessel (BV); (F) in negative control slide (AC3(-)), no AC3 expression is seen.

132 distributed across the body of the tongue. These cells were located between muscle fibres, in connective tissue, and around blood vessels similar to TB(+) mast cells. Both

TB(+) mast cells and mast-like cells that expressed OMP, Golf, or AC3 varied in size and shape. Some cells had visible nuclei, whereas the nuclei of others were obscured by granules (Fig. 4.3A,C,E).

In the mast-like cells of the tongue, OMP was expressed strongly at a dilution of

1:50 (Fig. 4.3A). Golf was expressed only faintly at a dilution of 1:50 (Fig. 4.3C). No expression of AC3 was observed at the same dilution (1:50); however, expression occurred at a lower dilution of 1:25 (Fig. 4.3E). In the negative control slides no expression of OMP, Golf, or AC3 was observed (Fig. 4.3B,D,F).

Microscopy at high magnification showed that OMP, Golf, and AC3 were expressed in the cytoplasm of mast-like cells of the tongue. OMP, Golf, and AC3 expression was observed as dark brown dots, occasionally clustered together, on the light brown cytoplasm. The expression pattern was similar in each of these proteins. No

OMP, Golf, or AC3 expression was observed in the nuclei, which appeared bright blue in some mast-like cells (Fig. 4.4A-C). In other cells, no nuclei were visible and expression of OMP, Golf, or AC3 was observed on the cell surface.

Figure 4.4 (next page): Expression of OMP, Golf, and AC3 in tongue using immunoperoxidase histochemistry. Photomicrographs of cross sections of tongue at high magnification show (A) OMP (dark brown) expression in cytoplasm of mast-like cells (OMP(+) MCs) (arrows). OMP

accumulation in dot-like patterns is observed in cytoplasm of MCs; (B) Golf (dark brown)

expression in cytoplasm of mast-like cells (Golf(+) MCs) (arrows). Golf accumulation in dot-like patterns is observed in cytoplasm of MCs; (C) AC3 (dark brown) expression in cytoplasm of mast-like cells (AC3(+) MCs) (arrows). AC3 accumulation in dot-like patterns is observed in cytoplasm of MCs.

133

134 4.4.1.3 Identification of OMP(+), Golf(+), and AC3(+) mast-like cells in tongue

Tongue tissue in which mast-like cells had expressed OMP, Golf, or AC3 were restained with TB. The results showed that OMP(+), Golf(+), or AC3(+) cells were positive for TB. Co-expression of OMP and TB; Golf and TB; or AC3 and TB was observed as prominent, dark brown/black, dot-like patterns within the cell cytoplasm, as shown in Fig.

4.5A (for OMP and TB), Fig. 4.5B (for Golf and TB) and Fig. 4.5C (for AC3 and TB). In the study of OMP and TB co-expression, 69 mast cells were counted. All 69 cells (100%) co-expressed OMP and TB. No mast cells were observed to express OMP or TB separately. In the study of Golf and TB co-expression, 63 mast cells were counted.

All 63 cells (100%) co-expressed Golf and TB. No mast cells were observed to express

Golf or TB separately. In the study of AC3 and TB co-expression, 59 mast cells were counted. All 59 cells (100%) co-expressed AC3 and TB. No mast cells were observed to express AC3 or TB separately. These results demonstrate 100% co-expression of OMP,

Golf, or AC3 with TB in mast cells of the tongue.

4.4.1.4 Co-expression OMP, Golf, and AC3 in mast cells of tongue Double-labelling IF IHC was applied to tongue tissue to investigate whether OMP,

Golf, and AC3 were co-expressed in mast cells of the tongue. The results showed that

OMP co-expressed with Golf (Fig. 4.6A) and Golf also co-expressed with AC3 (Fig. 4.6B). In both cases, co-expression was observed as prominent dot-like patterns within the cytoplasm of the mast cells (Fig. 4.6A-B). These data indicate that all three proteins—

OMP, Golf, and AC3—were generally co-localised in the cytoplasm of mast cells of the tongue.

Figure 4.5 (next page): Identification of OMP(+), Golf(+), and AC3(+) cells in tongue restained with Toluidine Blue (TB). Photomicrographs of cross sections of tongue at high magnification show (A) OMP(+) and TB(+) mast cells (OMP(+)TB(+) MCs) (arrows). OMP and TB co-expression (dark

brown/black) is observed in dot-like patterns in cytoplasm of mast cells; (B) Golf(+) and

TB(+) mast cells (Golf(+)TB(+) MCs) (arrows). Golf and TB co-expression (dark brown/black) is observed in dot-like patterns in cytoplasm of mast cells; (C) AC3(+) and TB(+) mast cells (AC3(+)TB(+) MCs) (arrows). AC3 and TB co-expression (black) is observed in dot-like patterns or in clusters in cytoplasm of mast cells.

135

136

Figure 4.6: Co-expression OMP with Golf and AC3 in tongue using double-labelling immunofluorescent histochemistry. Photomicrographs of cross sections of tongue show (A) co-expression of OMP (red) with Golf (green) in mast cells (MCs) (arrows); (B) co-expression of Golf (red) with AC3

(green) in MCs (arrows). Co-expression (yellow) of OMP, Golf, and AC3 are seen in dot- like patterns in MC cytoplasm. DAPI stains spermatozoa nuclei (blue) (arrow).

137 4.4.2 Expression of OMP, Golf, and AC3 in mast cells of liver Haematoxylin and Eosin (HE) staining, Toluidine Blue (TB) staining, and IHC were performed to determine expression of OMP, Golf, and AC3 in the mast cells of rat liver.

Figure 4.7: Rat livers stained with Haematoxylin and Eosin and Toluidine Blue. Photomicrographs of cross sections show liver stained with Haematoxylin and Eosin (A) and liver stained with Toluidine Blue (TB control) (B) at low magnification. (A) Photomicrograph shows classical structure of portal triad: portal vein (PV), branches of hepatic artery, and bile ducts, and also hepatocytes in liver parenchyma; (B) photomicrograph shows TB-positive (TB(+)) mast cells (arrows), violet in colour, located around PV of portal triad. No mast cells are observed between hepatocytes. Square box highlights section of liver examined at higher magnifications (in Figs. 4.8 – 4.9).

138 4.4.2.1 Toluidine Blue control staining of liver Toluidine Blue (TB) staining showed that TB-positive (TB(+)) mast cells were present in the liver. TB(+) mast cells were only located around the portal triad, comprised of the portal vein, branches of hepatic artery, and liver bile ducts (Fig. 4.7A). These cells, at low magnification, were observed as dark blue/violet dots (Fig. 4.7B). No TB(+) mast cells were observed in the hepatocytes of the parenchyma of the liver (Fig. 4.7B).

4.4.2.2 Expression of OMP, Golf, and AC3 in liver

IP IHC showed that OMP, Golf, and AC3 were expressed in certain cells of the liver. When the OMP(+), Golf(+), and AC3(+) cell phenotypes were compared with the corresponding morphological phenotype and location of TB(+) mast cells in the liver, it was apparent that OMP, Golf, and AC3 were expressed in mast-like cells. As was the case for TB(+) mast cells, mast-like cells expressing OMP, Golf, or AC3 were also located only in the connective tissue surrounding the portal triad. Both TB(+) mast cells and mast-like cells expressing OMP, Golf, or AC3 exhibited various sizes and shapes. Some of them had visible nuclei, whereas the nuclei of others were obscured by granules (Fig. 4.8A,C,E).

OMP and Golf were expressed strongly in mast-like cells at a dilution of 1:50. No AC3 expression was observed in mast-like cells at a dilution of 1:50; however, expression was observed at the lower dilution of 1:25 (Fig. 4.8E). Unlike OMP and Golf (Fig. 4.8A,C), AC3 was also expressed, at different intensities, in hepatocytes (Fig.

4.8E). In the negative control slides no expression of OMP, Golf, or AC3 was observed (Fig. 4.8B,D,F).

Microscopy at high magnification showed that OMP, Golf, and AC3 were expressed in the cytoplasm of mast-like cells of the liver. Expression appeared as dark brown dots, occasionally clustered together, on the light brown cytoplasm (Fig. 4.9A-C).

The expression pattern was similar in each of these proteins. No OMP, Golf, or AC3 expression was observed in the nuclei, which appeared bright blue in some cells. In other cells, no nuclei were visible and expression of OMP (Fig. 4.9A), Golf, and AC3 was observed on the cell surface.

139

Figure 4.8: Expression of OMP, Golf, and AC3 in liver using immunoperoxidase histochemistry. Photomicrographs of cross section of liver show (A) liver stained with OMP. OMP (brown) is expressed in mast-like cells (OMP(+) MCs) (arrows) located in connective tissue (CT) around portal vein (PV); (B) in negative control slide (OMP(-)), no OMP expression is seen; (C) liver stained with Golf. Golf (brown) is expressed in mast-like cells

(Golf(+) MCs) (arrows) located in connective tissue (CT) around portal vein (PV); (D) in negative control slide (Golf(-)), no Golf expression is seen; (E) liver stained with AC3. AC3 (brown) is expressed in mast-like cells (AC3(+) MCs) (arrows) located in connective tissue (CT) around portal vein (PV). Strong AC3 expression is also observed in hepatocytes; (F) in negative control slide (AC3(-)), no AC3 expression is seen.

140

141 Figure 4.9 (previous page): Expression of OMP, Golf, and AC3 in liver using immunoperoxidase histochemistry. Photomicrographs of cross section of liver at high magnification show (A) OMP (brown) expression in cytoplasm of mast-like cells (OMP(+) MCs) (arrows). OMP accumulation

in dot-like patterns is seen in cytoplasm of MCs; (B) Golf (dark brown/brown) expression

in cytoplasm of mast-like cells (Golf(+) MCs) (arrows). Some granules are darker in colour than others; (C) AC3 (dark brown/brown) expression in cytoplasm of mast-like cells (AC3(+) MCs) (arrows). Some granules are darker in colour than others.

4.4.2.3 Identification of OMP(+), Golf(+), and AC3(+) mast-like cells in liver

Liver tissue in which mast-like cells had expressed OMP, Golf, or AC3 were restained with TB. The results showed that OMP(+), Golf(+), or AC3(+) cells were positive for TB. Co-expression of OMP and TB; Golf and TB; or AC3 and TB was observed as prominent, dark brown/black, dot-like patterns within the cell cytoplasm, as shown in Fig.

4.10A (for OMP and TB), Fig. 4.10B (for Golf and TB) and Fig. 4.10C (for AC3 and TB).

In the study of OMP and TB co-expression, 82 mast cells were counted. All 82 cells (100%) co-expressed OMP and TB. No mast cells were observed to express OMP or TB separately. In the study of Golf and TB co-expression, 90 mast cells were counted.

All 90 cells (100%) co-expressed Golf and TB. No mast cells were observed to express

Golf or TB separately. In the study of AC3 and TB co-expression, 87 mast cells were counted. All 87 cells (100%) co-expressed AC3 and TB. No mast cells were observed to express AC3 or TB separately. These results demonstrate 100% co-expression of OMP,

Golf, or AC3 with TB in mast cells of the liver.

Figure 4.10 (next page): Identification of OMP(+), Golf(+), and AC3(+) cells in liver restained with Toluidine Blue. Photomicrographs of cross section of liver at high magnification show (A) OMP(+) and TB(+) mast cells (OMP(+)TB(+) MCs) (arrows). OMP and TB co-expression (dark

brown/black) is observed in dot-like patterns in cytoplasm of mast cells; (B) Golf(+) and

TB(+) mast cells (Golf(+)TB(+) MCs) (arrows). Golf and TB co-expression (black) is observed in dot-like patterns in cytoplasm of mast cells; (C) AC3(+) and TB(+) mast cells (AC3(+)TB(+) MCs) (arrows). AC3 and TB co-expression (black) is observed in dot-like patterns or in clusters in cytoplasm of mast cells. No co-expression is seen in hepatocytes.

142

143 4.4.3 Expression of OMP, Golf, and AC3 in mast cells of white adipose tissue Haematoxylin and Eosin (HE) staining, Toluidine Blue (TB) staining, and IHC were performed to determine expression of OMP, Golf, and AC3 in the mast cells of rat WAT.

4.4.3.1 Toluidine Blue control staining of white adipose tissue Toluidine Blue (TB) staining showed that TB-positive (TB(+)) mast cells were present in WAT. TB(+) cells were observed in two locations: occasionally between adipose cells (Fig. 4.11A) and in the connective tissue around blood vessels (Fig. 4.11B).

Figure 4.11: White adipose tissue stained with Toluidine Blue. Photomicrographs of cross sections of Toluidine Blue- (TB-) stained white adipose tissue (WAT) shows TB-positive mast cells (TB(+) MCs) (arrows), violet in colour, in two locations: between adipose cells (AdCs) (A) and around blood vessels (BVs) (B). (A) TB(+) mast cells (arrows) are seen between AdCs. (B) TB(+) mast cells (arrows) are located around BV.

144 4.4.3.2 Expression of OMP, Golf, and AC3 in white adipose tissue

IP IHC showed OMP, Golf, and AC3 were expressed in certain cells of WAT.

When the OMP(+), Golf(+), and AC3(+) cell phenotypes were compared with the corresponding morphological phenotype and locations of TB(+) mast cells in WAT, it was apparent that OMP, Golf, and AC3 were expressed in mast-like cells. As was the case

Figure 4.12: Expression of OMP, Golf, and AC3 in white adipose tissue using immunoperoxidase histochemistry. Photomicrographs of cross sections of WAT show (A) WAT stained with OMP (OMP(+)). OMP (brown) is expressed in mast-like cell (OMP(+) MC) (arrow) located between blood vessels (BVs) and adipose cells (AdCs); (B) in negative control slide

(OMP(-)), no OMP expression is seen; (C) WAT stained with Golf (Golf(+)). Golf (brown)

is expressed in mast-like cell (Golf(+) MC) (arrow) located near blood vessel (BV) and

adipose cells (AdCs); (D) in negative control slide (Golf(-)), no Golf expression is seen; (E) WAT stained with AC3 (AC3(+)). AC3 (brown) is expressed in a mast-like cell (AC3(+) MC) (arrow) located between adipose cells (AdCs); (F) in negative control slide (AC3(-)), no AC3 expression is seen.

145 for TB(+) mast cells, mast-like cells expressing OMP, Golf, or AC3 were seen in two locations: between adipose cells and around blood vessels (Fig. 4.12A,C,E). OMP was expressed strongly at a dilution of 1:50 in mast-like cells located between adipose cells or around blood vessels (Fig. 4.12A); Golf was expressed strongly at a dilution of 1: 80 (Fig. 4.12C); AC3 was expressed strongly only at a dilution of 1:25 (Fig. 4.12E). In the negative control slides no expression of OMP, Golf, or AC3 was observed (Fig. 4.12B,D,F).

Microscopy at high magnification showed that OMP, Golf, and AC3 were expressed in the cytoplasm of mast-like cells of WAT. Expression was observed as brown dots, occasionally clustered together, on the light brown cytoplasm. The expression pattern was similar in each of these proteins. No OMP, Golf, or AC3 expression was observed in the nuclei, which appeared bright blue in some cells (Fig.

4.13A-B). In other cells, no nuclei were visible and OMP, Golf, or AC3 expression was observed on the cell surface.

Figure 4.13 (next page): Expression of OMP, Golf, and AC3 in white adipose tissue using immunoperoxidase histochemistry. Photomicrographs of cross section of WAT at high magnification show (A) OMP (brown) expression in cytoplasm of mast-like cell (OMP(+) MC) (arrow). OMP accumulation in dot-like pattern is observed in cytoplasm of MC. Nucleus is visible and unstained; (B)

Golf expression (dark brown/brown) in dot-like pattern in cytoplasm of mast-like cell

(Golf(+) MC) (arrow). Nucleus is visible and unstained; (C) AC3 expression (dark brown/brown) in cytoplasm of mast-like cell (AC3(+) MCs) (arrow). Some granules are darker in colour than others. Nucleus is visible and unstained.

146

147 4.4.3.3 Identification of OMP(+), Golf(+), and AC3(+) mast-like cells in white adipose tissue

WAT in which mast-like cells had expressed OMP, Golf, or AC3 were restained with TB. The results showed that OMP(+), Golf(+), or AC3(+) cells were positive for TB.

Co-expression of OMP and TB; Golf and TB; or AC3 and TB was observed as prominent, dark brown/black, dot-like patterns within the cell cytoplasm, as shown in Fig. 4.14A (for

OMP and TB), Fig. 4.14B (for Golf and TB) and Fig. 4.15C (for AC3 and TB).

In the study of OMP and TB co-expression, 7 mast cells were counted. All 7 cells (100%) co-expressed OMP and TB. No mast cells were observed to express OMP or TB separately. In the study of Golf and TB co-expression, 5 mast cells were counted. All 5 cells (100%) co-expressed Golf and TB. No mast cells were observed to express Golf or TB separately. In the study of AC3 and TB co-expression, 7 mast cells were counted. All 7 cells (100%) co-expressed AC3 and TB. No cells were observed to express AC3 or TB separately. This co-expression indicates that OMP, Golf, and AC3 are expressed in TB(+) mast cells of the WAT.

Figure 4.14 (next page): Identification of OMP(+), Golf(+), and AC3(+) cells in white adipose tissue restained with Toluidine Blue. Photomicrographs of cross sections of WAT at high magnification show (A) OMP(+) and TB(+) mast cell (OMP(+)TB(+) MC) (arrow). OMP and TB co-expression (black) is

observed in dot-like patterns in cytoplasm of mast cell; (B) Golf(+) and TB(+) mast cell

(Golf(+)TB(+) MC) (arrow). Golf and TB co-expression (black) is observed in dot-like patterns in cytoplasm of mast cell; (C) AC3(+) and TB(+) mast cell (AC3(+)TB(+) MC) (arrow). AC3 and TB co-expression (black) is observed on mast cell surface.

148

149 4.5 Discussion Mast cells are important effector cells in the immune system, as discussed in Section 1.4. They are particularly prominent in tissues that are in contact with the external environment, such as the skin, the gastrointestinal tract, and the airways. Mast cells are present in the brain from birth and reside in specific brain regions in many species, as discussed in Section 1.4.7. They are generally present in greater numbers at sites of inflammation in many pathophysiological conditions and this may be due to directed migration, as described in Section 1.4.5 and 1.4.7. It has been suggested that mast cell recruitment depends on the presence of chemoattractants, which are produced locally at sites of inflammation by various cell types. Mast cells contain granules that are filled with a range of inflammatory mediators, including histamine, proteases, lipid mediators, and cytokines, as described in Section 1.4.2. At sites of inflammation, mast cells are activated by chemoattractants, such as allergens and non-immunologic substances (Blank and Benhamou, 2013) that include chemokines (Halova et al., 2012), sex hormones (Zierau et al., 2012), tetraspanins (Köberle et al., 2012), and bacterial products (such as endotoxin lipopolysaccharide [LPS]) (Leal-Berumen et al., 1994). Once activated, mast cells degranulate and release inflammatory mediators (Leal- Berumen et al., 1994; Blank and Benhamou, 2013; Wernersson and Pejler, 2014), as described in Sections 1.4.3 & 1.4.4. When mature mast cells are activated and degranulated, more mast cells progenitors are recruited to the site of inflammation (Collington et al., 2011). Thus, mast cells both produce and respond to chemoattractants.

The current study is the first to demonstrate the expression of OMP with Golf and AC3 in mast cells of the tongue, liver, and WAT in rats. Mast cells were identified by staining tissue with Toluidine Blue (TB), which has an affinity for mast cell granules, as discussed in Sections 1.4 & 4.3.3.3. TB-positive (TB(+)) mast cells were observed in the tongue, liver, and WAT of rats. In the body of the tongue, mast cells were dispersed between muscles and connective tissue and were grouped around blood vessels. In the liver, they were confined only to the connective tissue of the portal triad, consistent with locations reported by Chan et al. (2001). In the WAT, they were grouped around blood vessels in the connective tissue and occasionally seen between adipose cells.

Single-labelling IP IHC was used to determine the expression of OMP, Golf, and

AC3. The results showed that OMP, Golf, and AC3 were expressed in mast-like cells in the tongue, liver, and WAT. They were labelled OMP(+), Golf(+), and AC3(+) mast-like cells. In the tongue, all of these mast-like cells were located between the connective tissue and muscles and were grouped around the blood vessels. No OMP, Golf, or AC3 expression was observed in myocytes or fibroblasts of the tongue. In the liver, OMP(+),

150 Golf(+), and AC3(+) mast-like cells were located only in the connective tissue of the portal triads. In the WAT, the OMP(+), Golf(+), and AC3(+) mast-like cells were located around blood vessels and between the adipose cells. No OMP, Golf, or AC3 expression was observed in adipose cells. OMP(+), Golf(+), and AC3(+) mast-like cells were found in the tongue, liver, and WAT at similar locations to those in which TB(+) mast cells had been found. Mast cells also showed morphological similarity to TB(+) cells.

Finally, mast-like cells that expressed OMP, Golf, or AC3 were restained with TB to clarify the nature of these cells. The results showed that all OMP(+), Golf(+), and AC3(+) cells were positive for TB and displayed a high density of intracellular granules. This co-expression showed that these cells were, in fact, mast cells and suggests that they may use OMP, Golf, and AC3 in the olfactory signal transduction pathway.

The results of the current study provide evidence that ectopically-expressed ORs are likely to be functional in migratory cells — mast cells and spermatozoa — via their olfactory signal transduction cascade, possibly responding to molecules of the local environment. It should not, therefore, be surprising that key molecular mechanisms are conserved between the reproductive system, immune system and olfaction. Whether ORs are expressed in mast cells, spermatozoa, at the synapse or in sensory cells, they serve the same function: the detection of ligands - such as hormones, neurotransmitters, peptides or growth factors – from the environment (Dryer, 2000).

Future studies are needed to identify potential ligands and determine the functional significance of ORs in cell migration. One complication that may be encountered is that migratory cells, as they negotiate their chemotactic pathways, may recognise more the one signal at a particular location and/or recognise different signals at different locations (Goto et al., 2001). Mast cells and spermatozoa share many features with each other and with neurons, as discussed in Chapter 1 and Chapter 2. It is known that ORs play a dual role in OSNs of the olfactory system. On the one hand, they define the odorant specificity expressed by ORs of OSNs and, on the other hand, they play a role in axon guidance by recognising internal cues in the olfactory bulb (Giorgi et al., 2011). I speculated that ORs in mast cells may also play a dual role: promoting cell migration to sites of inflammation and/or stimulating the release of mediators.

The fact that ORs are expressed in both migratory cells — mast cells and spermatozoa — suggests that they may be important for recognising ligands that trigger chemotaxis. This suggests that further investigation of the OR gene family may be a promising area of future research.

151 4.6 Conclusion The current study showed that OMP was expressed in mast cells of the tongue, liver, and WAT in rats. OMP, Golf, and AC3 showed similar patterns of individual expression in these cells and they co-localised in mast cells of the tongue. These observations strengthen the argument that OR-mediated mechanisms are involved in chemoreception and cellular guidance in mast cells. It is possible that OR-mediated signals (OMP, Golf, and AC3) may be involved in attracting mast cells to sites of inflammation and be additionally involved in releasing mediators. The current study provides evidence that allows us to more confidently assert that ORs act as chemoreceptors in mast cells via their olfactory signalling partners. It is suggested that future studies be conducted to identify the ligands that activate this signalling pathway and determine the physiological functions they mediate in different organs and tissues, especially the testes, brain and WATs. Particular attention should be paid to the interaction between mast cells and other cells that are thought to participate in chemoreception. This knowledge will be critical for developing clinical approaches to preventing and treating immune-mediated inflammatory diseases.

152

Chapter 5: Expression of OMP, Golf, and AC3 in Leydig cells of interstitial tissue of rat testes

153 5.1 Abstract Olfactory receptors (ORs) are expressed in olfactory sensory neurons (OSNs) as well as in non-olfactory (ectopic) tissues. Next Generation Sequencing (NGS) analysis found that the testes contained the highest number of ectopically-expressed ORs outside the olfactory system, implying involvement in diverse physiological processes of the reproductive system, such as chemotaxis, acrosomal exocytosis, capacitation, spermatogenesis, and epididymal spermatozoal maturation. OMP, a known marker for ORs, is expressed in the Leydig cell-like population of the mouse testes. Because Leydig cells have many functions, I hypothesised that the OR- mediated sensing mechanism in rat testes would involve OMP, Golf and AC3 and facilitate morphological maturation, differentiation, or secretory activity. In fact, the current study demonstrated that OMP,

Golf and AC3 were expressed in Leydig cells of the interstitial tissue. These observations suggest that an OR-mediated signalling pathway is involved in chemoreception in the Leydig cells. .

5.2 Introduction ORs are not only expressed in OSNs but also in non-olfactory tissues. Approximately 20 mammalian OR genes have been found to be expressed in male dog germ cells, using homology cloning, low-stringency PCR, and Northern blot analysis (Parmentier et al., 1992). Human testes were found to contain the highest number of ectopically-expressed ORs outside of the olfactory system, using NGS analysis (Flegel et al., 2013). The expression of about 90 putative OR transcripts in human spermatozoa and testes was found using olfactory transcriptome analysis (Flegel et al., 2015). Moreover, the human spermatozoa and testes expressed the highest number of different putative OR transcripts compared to the brain, colon, liver, and skeletal muscle (Flegel et al., 2015). This finding suggests that ORs may play an important role in the reproductive system where certain ORs may be involved in diverse physiological processes such as chemotaxis, acrosomal exocytosis, capacitation, spermatogenesis, and epididymal spermatozoal maturation (Vanderhaeghen et al., 1993, 1997; Fukuda and Touhara, 2006; Flegel et al., 2015).

Olfactory marker protein (OMP) is highly expressed in primary OSNs and is considered to be an identifying feature of mature OSNs (mOSNs) in the olfactory epithelium (OE) (Keller and Margolis, 1975; Monti-Graziadei et al., 1977; Farbman and Margolis, 1980; Fleischer et al., 2006). OMP plays a pivotal role in one of the earliest steps of olfactory transduction in the OSNs and participates in the OR-associated signal transduction cascade (Youngentob et al., 2003; Reisert et al., 2007; Kwon et al., 2009).

154 OMP gene expression in non-olfactory mouse tissues, such as skeletal muscle, the thymus, duodenum, kidney, testes, and thyroid has received considerable attention (Kang et al., 2015).

OMP was expressed in the Leydig cell-like population of the testes, parafollicular cells of the thyroid, and the bladder's interstitial cells of Cajal (Kang et al., 2015). IHC showed that several components of the olfactory signal transduction pathway—OMP,

Golf, AC3—were co-expressed with ORs (Kang et al., 2015). Likewise, OMP, Golf, and AC3 were strongly co-expressed in cells of the bladder and thymus, suggesting that these three proteins might be employed as a chemosensory signalling system in these tissues. In the thyroid, OMP was co-expressed with AC3 but not with Golf (Kang et al.,

2015). In the heart and testes, OMP was expressed singly, but not with AC3 or Golf. Failure to detect co-expression, however, may have been due to technical difficulties and the authors suggested it could be valuable to repeat the experiment (Kang et al., 2015).

Leydig cells are a heterogeneous cell population located in the interstitial tissue of mammals, as described in Section 1.3.1.2. There are significant differences in the organisation, size, number, and shape of these cells among different mammalian species (Pudney, 1996; Russell, 1996; Prince, 2007). These cells are generally located in clusters close to blood and/or lymph vessels (Fawcett et al., 1973; Heindel and Treinen, 1989; Saez, 1994; Amrani et al., 1996). Leydig cells are part of a complex testicular network in which different cell types communicate with each other through endocrine and exocrine activities (Heindel and Treinen, 1989). Leydig cell differentiation, proliferation, endocrine function, and regulation can be modulated by an endocrine mechanism (through luteinizing hormone [LH]) and by various growth factors (including transforming growth factors [TGFs], interleukins [ILs]: IL-1 and IL-6, insulin-like growth factors [IGFs], corticotropin-releasing factor [CRF], and vascular endothelial growth factor [VEGF]) (Sharpe, 1990; Skinner, 1991; Spiteri-Grech and Nieschlag, 1993; Saez, 1994; Haider, 2004). The Leydig cell itself synthesises steroids, particularly testosterone, and neuropeptides as well as various proteins, including POMC-derived peptise, undecapeptide substance P (SP), nitric oxide synthases (NOSs), insulin-like protein 3 (INSL3), oxytocin, and macrophage inhibitory factor (MIF), all of which are involved in regulating steroidogenesis and communicating between testicular cells (Huhtaniemi and Toppari, 1995; Jégou and Pineau, 1995; Lejeune et al., 1996; Saez and Lejeune, 1996; Diemer et al., 2003; Ge et al., 2009; Coward and Wells, 2013). Leydig cells may influence the contractile activity of smooth muscle cells in blood vessels and the tunica albuginea, and peritubular myofibroblasts (Davidoff et al., 1995; Davidoff et al., 1996). Leydig cells have a number of functions: a) they are known to produce several types of neuronal

155 signalling molecules, including γ-aminobutyric acid (GABA) and histamine and, in this respect, they resemble mast and germ cells; b) they have receptors for these neuronal signalling molecules, suggesting that Leydig cells have an autocrine signalling function (Safina et al., 2002; Albrecht et al., 2005; Mayerhofer, 2007); c) they modulate local inflammatory responses in the testes (Haider, 2004; Hedger et al., 2005). Human Leydig cells and Sertoli cells secrete pro-inflammatory substances, such as IL-1 and IL-6 and exogenous factors, such as lipopolysaccharides (LPS) and latex beads (Cudicini et al., 1997); d) they are responsible for recruiting and controlling the population of testicular macrophages and leucocytes (Hedger, 2002); e) they produce various immunoregulatory cytokines, TGF-β1, MIF and interferon (IFN)-γ, either during testicular development or in the adult (Teerds and Dorrington, 1993; Dejucq et al., 1995; Meinhardt et al., 1996); f) they, as well as Sertoli cells, produce pro-inflammatory mediators - such as the 2 IL-1 isoforms, IL-6 and NOS - during testicular inflammation (Meinhardt et al., 1996; Stéphan et al., 1997; O’Bryan et al., 2000b; Söder et al., 2000). Leydig cells also decrease testosterone production in response to moderate testicular inflammation. Moderate inflammation increased testicular macrophages, attenuated production of the pro-inflammatory mediators IL-1β and of nitric oxide by Leydig cells, and reduced volume of testicular interstitial fluid (O’Bryan et al., 2000b; O’Bryan et al., 2000a; Gow et al., 2001; Gerdprasert et al., 2002a; Gerdprasert et al., 2002b).

The purpose of the current study was to determine whether Leydig cells expressed OMP, Golf, and AC3 in the reproductive system of the rat. Based on evidence that OMP was expressed in the interstitial tissue of mice (Kang et al., 2015), I hypothesised that OMP, Golf and AC3 would be expressed in the interstitial tissue of rat testes. IHC was used to identify expression of OMP, Golf and AC3. The results showed that OMP, Golf and AC3, were simultaneously expressed in Leydig cells of the interstitial tissue of rat testes. This observation suggests that an OR-mediated signalling transduction pathway is present in Leydig cells.

156 5.3 Materials and methods

5.3.1 Animals Four adult male CBA/Ce mice and six adult male Sprague-Dawley rats were used, with the approval of the University of New South Wales’ Animal Care and Experimentation Committee and in strict accordance with the NHMRC Animal Experimentation Guidelines and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1990). Mice were obtained from the Animal Resources Centre (Moss Vale, NSW); rats were obtained from the Animal Resources Centre (Perth, WA). All animals were handled with care prior to experimentation.

5.3.2 Perfusion Perfusion was performed as described in Section 2.3.2.

5.3.3 Histological methods

5.3.3.1 Tissue selection and preparation Testes from rats and mice were dissected. 10% neutral buffered Formalin solution was used for post-fixation of testes (12 – 14 hrs). Tissue was immersed overnight in 70% (v/v) ethanol and embedded in paraffin using standard embedding procedures. Tissue was sectioned at 5 μm on a motorised microtome (Leica RM 2155, Leica Microsystems Pty Ltd). Testes tissue from each mouse and rat was serially cut and mounted in consecutive order on electrostatic slides (Menzel-Glaser, Braunschweig, Germany). Slides were randomly selected from each experimental animal from different points in the numbering spectrum. The exception was for experiments with negative control staining, for which adjacent slides were selected.

5.3.3.2 Haematoxylin and Eosin staining Haematoxylin and Eosin (HE) staining was performed as outlined in Section 2.3.3.2.

5.3.3.3 Immunohistochemistry methods

§ Immunoperoxidase histochemistry

The optimal antibody dilution (working concentration) for OMP, Golf and AC3 was determined by testing a number of different dilutions. The testes of four mice were studied. OMP antibody dilution optimisation was reached using 2 serial slides (one of which was a negative control) at dilutions of 1:100, 1:50 and 1:25. It was determined that

157 the optimal dilution for OMP was 1:25. For immunoperoxidase histochemistry (IP IHC), 4 OMP-positive OMP(+) and 4 negative control OMP(-) slides of testes tissue were used from each mouse.

The testes of three randomly selected rats were studied. OMP antibody dilution optimisation was determined using 2 serial slides (one of which was a negative control) at dilutions of 1:50 and 1:25. It was determined that the optimal dilution for OMP was

1:25. Golf antibody dilution optimisation was determined using 2 serial slides (one of which was a negative control) at dilutions of 1:20, 1:40, 1:80 and 1:160. It was determined that the optimal dilution for Golf was 1:160. AC3 antibody dilution optimisation was determined using 2 serial slides (one of which was a negative control) at dilutions of 1:50 and 1:25. It was determined that the optimal dilution was 1:25. For IP IHC, 10

OMP(+) and 10 OMP(-) slides, 10 Golf(+) and 10 Golf(-) slides and another 10 AC3(+) and 10 AC3(-) slides were used. IP IHC was performed, following the procedure outlined in Section 2.3.3.3. Primary antibodies used for IP IHC, their sources and working concentrations are listed in Table 5.1.

§ Single-labelling immunofluorescent histochemistry Calretinin is a 29-kDa neuronal calcium-binding protein originally purified from guinea pig brains (Winsky et al., 1989). Calretinin has been found in distinct subsets of neurons and fibres in the central and peripheral nervous systems (Winsky et al., 1989; Arai et al., 1991; Brookes et al., 1991). Expression of calretinin has also been found in some non-neural human tissues and such as paratesticular adenomatoid tumor, ameloblastoma, mesothelioma, and pulmonary adenocarcinoma (Ichikawa et al., 1991; Jacobowitz and Winsky, 1991; Résibois and Rogers, 1992; Altini et al., 2000; Delahunt et al., 2000). Calretinin expression has also been found in rat ovaries (Pohl et al., 1992) and in steroid-producing cells in human ovaries, particularly in the cells of the germinal epithelium and the androgen-secreting cells of the theca interna (Bertschy et al., 1997). Calretinin mRNA and calretinin protein have been found in the Leydig cells of interstitial tissue of rat testes (Strauss et al., 1994). In human testes, prominent and diffuse calretinin expression has been observed in all Leydig cells in the interstitial tissue. The expression was cytoplasmic and sometimes nuclear. No expression of calretinin was observed in the germ cells of seminiferous tubules. Based on those findings, calretinin has been suggested as a valuable marker for detecting Leydig cells in the testes (Augusto et al., 2002). In the current study, calretinin was used to identify Leydig cells in the interstitial tissue of rat testes. Single- and double-labelling immunofluorescent histochemistry (IF IHC) was used.

158 Antibody Dilution

Primary Antibody (name, source) Testes

Rats Mice

Olfactory marker protein (OMP), goat antiserum, raised 1:25 (IP) 1:25 (IP) with rodent OMP as the immunogen, diluted 1:1 with 1:1000 (IF) glycerol, containing 0.05% sodium azide (019-22291; Wako Pure Chemical Industries, Ltd., Japan)

Gaolf (Golf), goat polyclonal, P-15, raised against a 1:160 (IP) n/a peptide mapping near the N-terminus of Gaolf of human origin (sc-26763; Santa Cruz Biotechnology, CA, USA)

Adenylyl cyclase III (AC3), rabbit polyclonal, C-20, 1:250 (IP) n/a raised against a peptide mapping at the C-terminus of adenylyl cyclase III of mouse, rat, and human origin (sc-588; Santa Cruz Biotechnology, CA, USA)

Calretinin, rabbit polyclonal, full-length calretinin protein 1:400 (IF) n/a (ab702; Abcam, Australia)

Table 5.1: Primary antibodies used in Chapter 5. Abbreviations: immunoperoxidase histochemistry, IP; immunofluorescent histochemistry, IF; not applicable, n/a.

The optimal antibody dilution (working concentration) for OMP and calretinin was determined. The testes of 2 randomly selected rats were studied. OMP antibody dilution optimisation was determined using 2 serial slides (one of which was a negative control) at dilutions of 1:1200, 1:1000 and 1:800. It was determined that the optimal dilution for OMP was 1:1000. Calretinin antibody dilution optimisation was determined using 2 serial slides (one of which was a negative control) at dilutions of 1:800, 1:400 and 1:200. It was determined that the optimal dilution for calretinin was 1:400. For single-labelling IF IHC, number of testes slides from each rat was as follows: 10 OMP(+) and 10 calretinin- positive (calretinin(+)) with 1 negative control slide for each staining. Single-labelling IF IHC was performed, following the procedure outlined in Section 2.3.3.3. Primary antibodies used for IF IHC, their sources and working concentrations are listed in Table 5.1.

159 § Double-labelling immunofluorescent histochemistry Double-labelling IF IHC was performed using OMP and calretinin. Because calretinin is a known marker for Leydig-like cells, we proposed to determine using IHC whether OMP(+) Leydig-like cells in the interstitial tissue were actually OMP(+) Leydig cells. Five randomly selected slides of testes tissue from each rat were used for OMP and calretinin staining. Double-labelling IF IHC was performed using the same protocol as described for single-labelling IF IHC with the exception that two primary antibodies (OMP and calretinin) were mixed in PBS containing 2% NDS. The same method was used with the Alexa-labelled secondary antibodies.

5.3.3.4 Co-expression of OMP and calretinin in Leydig cells of interstitial tissue The proportion of Leydig cells co-expressing OMP and calretinin was determined using slides containing interstitial tissue (IT) from the testes of 5 randomly selected rats. Double-labelling IF IHC with OMP and calretinin was performed. Slides were visually evaluated with 63x objective lens and photographed. Images were opened in Photoshop (CS6; Adobe Systems). Each image was separated into three channels - blue (for nuclei), green (for calretinin(+) cells) and red (for OMP(+) cells) – that were adjusted for brightness and contrast. These images were converted to black and white to emphasise OMP(+) and calretinin(+) cells. These cells were counted manually across the IT. Total number of calretinin(+) Leydig cells was compared to the total number of OMP(+) Leydig cells per IT and data were expressed as percentages.

5.3.3.5 Toluidine Blue staining Toluidine Blue (TB) staining was chosen for reasons described in Section 4.3.3.3 to identify TB-positive (TB(+)) mast cells in the testes of 6 rats. Two slides from each rat were TB-stained and analysed. Staining was done by dewaxing paraffin sections, hydrating them with distilled water and TB (working solution of 1% TB in 70% ethanol, 5 ml and 1% sodium chloride in distilled water, 45 ml mixed well, pH around 2.3) for 60 secs. Sections were washed three times in distilled water and dehydrated quickly once in 95% ethanol and twice in 100% ethanol. They were then cleared twice in xylene for 3 mins each and cover-slipped with DPX mounting medium. Slides were scanned, photographed and image processed.

5.3.4 Photography and image processing Photography and image processing were conducted, as described in Section 2.3.4.

160 5.4 Results

5.4.1 Mouse testes

5.4.1.1 Expression of OMP in interstitial tissue of mouse testes IHC showed that OMP was expressed in certain cells of the IT of mouse testes: the Leydig-like cells (Fig. 5.1A). In the negative control slides of the testes, no OMP expression was observed in the interstitial tissue (Fig. 5.1B).

Figure 5.1: Expression of OMP in interstitial tissue of mouse testes using immunoperoxidase histochemistry. Photomicrographs show cross section of testes stained with OMP (OMP(+)) (A) and OMP-negative control (B), comprised of interstitial tissue (IT) and seminiferous tubules (STs). (A) OMP (brown) is expressed in cytoplasm of Leydig-like cells (OMP(+) cells) (star) of IT. (B) In negative control slide (OMP(-)), no OMP expression is seen.

161

5.4.2 Rat testes Haematoxylin and Eosin (HE), Toluidine Blue (TB), and immunohistochemistry (IHC)—IP IHC, and single- and double-labelling IF IHC—were performed to determine expression of OMP, Golf, and AC3 in the interstitial tissue of rat testes.

Figure 5.2: Expression of OMP, Golf, and AC3 in interstitial tissue of rat testes using immunoperoxidase histochemistry. Photomicrographs show cross sections of rat testes, comprised of interstitial tissue (IT) and seminiferous tubules (STs). (A) OMP (brown) is expressed in Leydig-like cells (OMP(+) cells) (star) of IT. (B) In negative control slide (OMP(-)), no OMP expression

is seen. (C) Golf (brown) is expressed in Leydig-like cells (Golf(+) cells) (star) of IT. (D)

In negative control slide (Golf(-)), no Golf expression is seen. (E) AC3 (brown) is expressed in Leydig-like cells (AC3(+) cells) (star) of IT. (F) In negative control slide (AC3(-)), no AC3 expression is seen.

162 5.4.2.1 Expression of OMP, Golf, and AC3 in interstitial tissue of rat testes Single-labelling IP IHC and microscopy at low magnification showed that OMP,

Golf, and AC3 were expressed in certain cells of the interstitial tissue of rat testes: Leydig- like cells (Fig. 5.2A,C,E). These cells were primarily oval or round in shape, located singly or in clumps, and were generally grouped near the blood and lymph capillaries

(Fig. 5.2A). In the negative control slides of the testes, no expression of OMP, Golf, or AC3 was observed (Fig. 5.2B,D,F).

Microscopy at high magnification showed OMP expression in the cytoplasm of the Leydig-like cells of the interstitial tissue. This expression varied in intensity between cells (Fig 5.3A). In each cell, two variations of Golf expression were observed: a) faint (light brown) expression occurred across the cell cytoplasm and b) strong (dark brown) expression was accumulated in dot-like or punctate-patterns in different locations inside the cytoplasm. No detectable specific pattern of Golf expression was observed between cells (Fig. 5.3B). Strong expression of Golf was observed in the Sertoli cells of seminiferous tubules (Fig. 5.3B). Expression of AC3 was observed in the cytoplasm of the Leydig-like cells of the interstitial tissue (Fig. 5.3C) and in some cells over the nuclei. Expression varied in intensity between cells (Fig. 5.3C).

Figure 5.3 (next page): Expression of OMP, Golf, and AC3 in Leydig-like cells of interstitial tissue using immunoperoxidase histochemistry. Photomicrographs of cross sections of rat testes at high magnification show (A) OMP (brown) is expressed in cytoplasm of Leydig-like cells (star) at various intensities; (B)

Golf (brown) is expressed in cytoplasm of Leydig-like cells (star). Expression varies from faint (light brown) to intense (dark brown) in different locations, with no detectable

pattern of intensity. Golf is prominently expressed on borders of Sertoli cells (SCs) (arrowhead); (C) AC3 (brown) is expressed in cytoplasm of Leydig-like cells (star) at various intensities.

163

164

165 Figure 5.4 (previous page): Co-expression OMP with calretinin in Leydig cells using double-labelling immunofluorescent histochemistry. Photomicrographs show cross section of rat testes stained with OMP and calretinin. (A) Co-expression (yellow) of OMP and calretinin is observed in cytoplasm of Leydig cells (star) of interstitial tissue (IT) (arrow). No co-expression is observed in seminiferous tubule (ST). (B) OMP (red) is expressed in cytoplasm of Leydig cells, at various intensities. (C) Calretinin (green) is expressed in cytoplasm of Leydig cells. DAPI stains cell nuclei (blue).

5.4.2.2 Identification of OMP(+) cells in interstitial tissue of rat testes Double-labelling IF IHC was performed to examine co-expression of OMP and calretinin in the rat testes. The results showed that both OMP and calretinin were frequently expressed in the same cells of the interstitial tissue. More than 81% of OMP(+) cells were calretinin(+) and less than 19% of cells were only calretinin(+). No cells were observed to singly express OMP. OMP and calretinin co-expression indicates that OMP is expressed in Leydig cells (Fig. 5.4).

5.4.2.3 Identification of mast cells in rat testes TB staining did not reveal the presence of any TB(+) mast cells in the interstitial tissue (Fig. 5.5).

Figure 5.5: Toluidine Blue- (TB-) stained rat testes. Photomicrograph shows absence of TB(+) mast cells in interstitial tissue (IT) and in seminiferous tubules (STs).

166 5.5 Discussion The current study determined OMP expression in rat testes. The results showed that OMP was expressed in Leydig-like cells of the interstitial tissue similar to what was observed in mice (Kang et al., 2015). OMP was expressed in similar patterns in the interstitial tissue of both species. More significantly, the current study demonstrated expression of Golf and AC3 in the interstitial tissue of rats. Three proteins—OMP, Golf, and AC3—were found in Leydig-like cells. The expression of these proteins was observed throughout the cytoplasm, in no detectable specific pattern, and at various intensities. It is suggested that further studies, using double labelling IF IHC, are required to clarify distribution of these proteins in the interstitial tissue and patterns of co- localisation in the cytoplasm of individual cells. Kang et al. (2015) looked for but did not find expression of Golf, and AC3 in the mouse testes. The differences between results of the current study and those of Kang et al. (2015) could be due to species differences or variations in the sensitivities of the antibodies.

The current study also aimed to identify the OMP(+) cells in the interstitial tissue. Double-labelling IF IHC was applied with calretinin, a specific marker for normal and neoplastic Leydig cells of the testes (Strauss et al., 1994; Augusto et al., 2002). The results showed that OMP and calretinin were co-expressed throughout the cytoplasm of cells. This finding confirmed that OMP is expressed in the Leydig cells of interstitial tissue. The presence of OMP, Golf, and AC3 in Leydig cells suggests that these cells might use these proteins in the OR-signalling cascade. Kang et al. (2015) speculated that the presence of OMP(+) cells in three non-chemosensory tissues located distantly from each other (testes, bladder, and thyroid) linked these tissues to the neuroendocrine system. The authors further speculated that OMP, Golf, and AC3 may be involved in the OR-mediated signalling transduction cascade between the nervous and endocrine systems (Kang et al., 2015). Leydig cells express numerous neuronal markers that characterise neuroendocrine cells, such as neuron-specific enolase (NSE), GAP 43 neuronal nuclei antigen (NeuN), transcription factor neuro D1, and protein gene product 9.5 (Davidoff et al., 1993; Bakalska et al., 2001; Davidoff et al., 2002). All of these markers have been identified in Leydig cells of different species of mammals, although with species-related differences (Angelova and Davidoff, 1989; Angelova et al., 1991; Davidoff et al., 1993; Davidoff et al., 1996; Davidoff et al., 2009).

Just as Leydig cells express OMP, Golf, and AC3, so do mast cells as described in Chapter 4. The current study investigated whether mast cells were also present in the interstitial tissue of rat testes. The results showed that no TB(+) mast cells were present in the interstitial tissue or in seminiferous tubules of rat testes. These findings are

167 consistent with reports that mast cells and eosinophils were absent from the testicular parenchyma of the rat, dog, cat, bull and deer, but present only around blood vessels in the tunica albluginea (Gaytan et al., 1989; Gaytan et al., 1990a; Anton et al., 1998). However, in other studies, mast cells have been found in the interstitium and peritubular areas of human testes (Nistal et al., 1984; Anton et al., 1998; Fijak and Meinhardt, 2006; Pérez et al., 2013).

One exciting result of the current study was the discovery that both Leydig cells and mast cells express OMP, Golf and AC3. This finding suggests that both these cell types may participate in chemoreception using an OR-mediated signalling pathway. This result raises questions as to what role ORs may play in Leydig cells, given that adult Leydig cells do not exhibit chemotaxis within the interstitial tissue. However, fetal Leydig cells do exhibit chemotaxis during the process of gonad formation. The results suggest that the presence of mast cells in interstitial tissue is species-specific. It found that mast cells were not present in the interstitial tissue or seminiferous tubules of rat testes, yet chemoreception occurs in this tissue. Therefore, chemoreception may involve the participation of Leydig cells, such as triggering the release of hormones or cytokines. Clearly there remain questions about the functional nature of Leydig cells and mast cells in the testes.

The relationship between mast cells and Leydig cells in the testes has been the focus of considerable research (Gaytan et al., 1989; Pinart et al., 2001; Haider, 2004; Albrecht et al., 2005). However, it is far from fully elucidated. It is possible that there is an inverse relationship between mast cells and Leydig cells in the testes. It has been shown that the prevention of Leydig cell differentiation in pubertal animals or depletion of the existing Leydig cells in adult rats leads to the appearance of large numbers of mast cells (Gaytan et al., 1990a). In the adult rat testes, mast cells and Leydig cells developed simultaneously after Leydig cell depletion (Gaytan et al., 1990b; Gaytan et al., 1992). The establishment of a new population of Leydig cells inhibited the development of both mast cells and Leydig cell precursors, suggesting that dynamic interactions might occur between these two cell types and also that common regulatory factors may be involved in both cell types (Gaytan et al., 1992).

The present data raise the possibility that interactions between mast cells and Leydig cells may help maintain the immune environment of the testes. Mammalian testes possess a special immunological environment consisting of two defence mechanisms: immune privilege and local innate immunity (Pérez et al., 2013; Zhao et al., 2014). Testicular immune privilege protects immunogenic germ cells from systemic immune attack (Fijak and Meinhardt, 2006) and varies from species to species (Head and

168 Billingham, 1985; Statter et al., 1988; Setchell et al., 1995; Li et al., 2012). Testicular immune privilege is maintained by the coordination of systemic immune tolerance and immunosuppressive properties of local cells (Meinhardt et al., 1998; Meinhardt and Hedger, 2011; Pérez et al., 2013; Zhao et al., 2014). Testicular cells express and secret numerous immunoregulatory molecules that play important roles in regulating immune responses in the testes (Li et al., 2012).

Local innate immunity protects the testes against invading microbial pathogens. Toll-like receptors (TLRs) are crucial triggers of local innate immunity (Kawai and Akira, 2006), as described in Section 1.4.4.2. Several types of TLRs were identified in the testes (Hedger, 2011). Adult human testes express TLR2 and TLR4 (Nishimura and Naito, 2005). Rat testes express TLR1-TLR10 (Palladino et al., 2007). TLRs were found in Leydig cells, Sertoli cells and germ cells in murine, mice, rats and humans (Riccioli et al., 2006; Palladino et al., 2007; Bhushan et al., 2008; Starace et al., 2008; Wu et al., 2008). Numerous paracrine and autocrine cytokines, produced by cells in the testes, are critical for normal testicular function. Four pro-inflammatory cytokines - TNF-β, IL-1, IL-6 and TNF-α - can be induced by TLR activation in Leydig cells, Sertoli cells and germ cells. The upregulation of these cytokines has been linked to inflammation in the testes. Leydig cells, Sertoli cells and germ cells also produce a number of antiviral cytokines including IFN-α, and -β, by activating TLRs (Wu et al., 2008; Sun et al., 2010; Shang et al., 2011; Wang et al., 2012). These cytokines could defend the host against pathogens by regulating immune responses or killing invading pathogens in vivo but could impair spermatogenesis under the inflammatory conditions (Li et al., 2012).

Mast cells are actively involved in testicular immunity (Frungieri et al., 2002; Fijak and Meinhardt, 2006; Iosub et al., 2006; Li et al., 2012). Mast cells increase in number during inflammation and infiltrate the parenchyma and seminiferous tubules of the testes where they are not usually found. Mast cells degranulate and secrete serine protease tryptase into the interstitial spaces of the testes. This action triggers further cell proliferation and the production of inflammatory mediators, including TGF-β, cyclooxygenase-2 and monocyte chemoattractant protein (MCP)-1 (Apa et al., 2002; Iosub et al., 2006) (Fig.1.24). Upregulation of MCP-1 is responsible for the massive infiltration of macrophages into the testes (Li et al., 2012). Mast cells, together with macrophages and germ cells, synthesise TNF-α, one of the most potent pro- inflammatory cytokines, in the testes (De et al., 1993; Xiong and Hales, 1993). At physiologically-low concentrations, TNF-α protects germ cells from apoptosis in normal testes. Conversely, in inflamed testes, TNF-α is upregulated and induces apoptosis and germ cell death (Theas et al., 2008).

169 The role of mast cells in inflammation has been studied primarily in relation to orchitis and orchi-epididymis, which are co-factors in human subfertility and infertility (Pérez et al., 2013). Orchitis is frequently caused by the mumps virus (Wu et al., 2016). It starts when macrophages and move from the bloodstream into the testes. This invasion breaks the interaction between germ cells and Sertoli cells and triggers neutrophil and infiltration into the testes, causing infertility from death of developing sperm (Kohno et al., 1983; Zhou et al., 1989).

The role of mast cells in testicular immune privilege has not been fully investigated to date (Li et al., 2012). It has been suggested that the relatively low numbers and restricted distribution of mast cells could determine immune privilege status of mammalian testes (Li et al., 2012). Numerous studies have found a correlation between the number and/or distribution of mast cells and the development of these diseases (Johnson et al., 1988; Brenner et al., 1994; Ibrahim et al., 1996; Secor et al., 2000; Brown et al., 2002). Abnormal spermatogenesis (Apa et al., 2002; Hussein et al., 2005) and infertility have all been associated with increased numbers of testicular mast cells (Agarwal et al., 1987; Hashimoto et al., 1988; Banek et al., 1999; Meineke et al., 2000).

In contrast to the situation with mast cells, much is known about how Leydig cells contribute to testicular immune privilege through their ability to produce testosterone and secret cytokines (Li et al., 2012; Zhao et al., 2014; Itoh, 2017). Testosterone had an immunosuppressive function, downregulating pro-inflammatory cytokines and increasing production of anti-inflammatory cytokines via the androgen receptor expressed in Leydig cells, Sertoli cells and myoid peritubular cells (Li et al., 1993; Bremner et al., 1994a; Vornberger et al., 1994; Schlatt et al., 1995; D'agostino et al., 1999; Gornstein et al., 1999; Li et al., 2004; Page et al., 2006; Rettew et al., 2008; Zhao et al., 2014). Testosterone inhibits experimental autoimmune orchitis in rats (Fijak et al., 2011). Leydig cells decrease testosterone production in response to moderate inflammation in the testes (O’Bryan et al., 2000b; O’Bryan et al., 2000a) and regulate testicular immunity via their effects on immune cells. Leydig cells, Sertoli cells and macrophages regulate activities of dendritic cells, T lymphocytes and mast cells by secreting immunosuppressive molecules (Hedger and Meinhardt, 2000; Diemer et al., 2003; Li et al., 2012).

Testicular immune privilege and local innate immunity vary from species to species. In the testes of small laboratory animals, such as rats, mice and guinea pigs, allografts and xenografts survive for prolonged periods of time (Head et al., 1983; Head and Billingham, 1985). However, in larger species, such as rams and monkeys, allografts

170 and xenografts fail to survive (Maddocks and Setchell, 1988; Setchell et al., 1995). In mice, allografts of testes grafted to the kidney capsule survived for a prolonged time, but in adult rat testes those grafted to the kidney were rejected (Statter et al., 1988; Bellgrau et al., 1995). The testes’ ability to respond to viral infections is also species-specific (Dejucq et al., 1998; Melaine et al., 2003; Le Tortorec et al., 2008). Human testes have weaker antiviral abilities and delayed responses to viral infection compared to the testes of murine, rats and mice. It is of interest that the strong antiviral innate immunity of the rat testes correlates to the absence of naturally occurring viral orchitis in these species. In contrast, orchitis frequently occurs in humans (Zhao et al., 2014). The differences in innate immunity in rat and human testes may be explained by the differences in levels of IFNs and antiviral proteins produced by Leydig cells. In rats, Leydig cells produced high levels of IFNs-α, -β and -γ and antiviral proteins in response to the Sendai virus (Dejucq et al., 1995; Melaine et al., 2003). In humans, Leydig cells did not produce detectable level of IFNs-α, -β and -γ normally or even when exposed to mumps, poly I:C, herpes simplex virus or human immunodeficiency virus 2 (Le Tortorec et al., 2008). Patients with mumps-related orchitis benefitted from treatment with IFN-α which prevented subsequent testicular atrophy and infertility (Erpenbach, 1991; Rüther et al., 1995; Ku et al., 1999).

The current study showed that the interstitial tissue of the testes is a complex network in which different cell types communicate with each other. Interactions between Leydig cells and mast cells are an important part of this communication, contributing to testicular immunity. The current study suggests a new role for Leydig cells in chemoreception. It is possible that Leydig cells may assume some functions performed by mast cells in the rat testes, contributing to the higher antiviral ability of the rat testes. This action may involve the components of the OR-mediated signalling transduction pathway - OR, OMP, Golf and AC3. This hypothesis is in line with studies showing that drugs stabilising mast cell activation were beneficial in treating some types of male infertility in humans (Yamamoto et al., 1995; Matsuki et al., 2000; Hibi et al., 2002). Additionally, mast cell blockers have been shown to ameliorate the severity of experimental autoimmune encephalomyelitis (Dietsch and Hinrichs, 1989; Seeldrayers et al., 1989; Brosnan et al., 1990). Prevention of mast cell activation may be an important factor in the maintenance of an immunosuppressive phenotype, not only in the testes but also in other immune-privileged sites and this possibility could be investigated in future studies.

The current study found the presence of OMP, Golf and AC3 in Leydig cells. This suggests that Leydig cells may use these proteins in an OR-mediated signalling

171 cascade. This result raises questions as to what role ORs may play in Leydig cells, given that adult Leydig cells do not exhibit chemotaxis within the interstitial tissue. However, fetal Leydig cells do exhibit chemotaxis during the process of gonad formation. Because germ cell-specific OR genes have been found in primordial germinal cells, it’s possible that ORs may play an important role in primordial germ cell migration (Goto et al., 2001).

Thus, fetal Leydig cells may use OR-mediated signals (OMP, Golf and AC3) in developmental migration during gonad formation.

Leydig cells appear prenatally in the testes and mature into adult Leydig cells postnatally (Griswold and Behringer, 2009), as described in Section 1.3.1.2. The number of fetal Leydig cells rapidly declines after birth. Some of these immature cells persist in the postnatal interstitium, but are eventually replaced by adult Leydig cells, which multiply at puberty. Fetal Leydig cells migrate during the process of gonad formation. They are the primary source of androgens and are responsible for driving male sexual differentiation in developing mammals. Adult Leydig cells appear at puberty. Unlike fetal Leydig cells, they do not exhibit chemotaxis inside the interstitial tissue of the testes and produce androgen throughout the rodent’s adult life (Griswold and Behringer, 2009).

While much is known about the origin and development of adult Leydig cells, little is known about that of fetal Leydig cells. There are a number of reasons for this. Firstly, it is very difficult to study these cells because they develop almost entirely in utero. Secondly, it is difficult to observe the sexual development of the embryo, as it occurs so rapidly. The developing testes undergo a dynamic series of cellular and organisational changes that transform the genital ridge into a highly structured organ called the testes cord. Formation of the testes cord depends on cell migration (Combes et al., 2009). It is thought that Leydig cells arise from different progenitors (Roosen-Runge and Anderson, 1959; Lording and De Kretser, 1972; Buehr et al., 1993; Nef and Parada, 1999; Schmahl et al., 2000; Brennan et al., 2003; O’Shaughnessy et al., 2003; Val et al., 2006). However, this idea has not yet been confirmed. There appear to be several sources of fetal Leydig cells: neural crest cells, mesonephros, the coelomic epithelium, or unspecified somatic cells of the gonads. From these sources, Leydig cells migrate to the interstitial tissue. Migration may be governed by unknown chemotactic signals (Griswold and Behringer, 2009). The role of several molecules, such as TGF-β1, integrin receptors and c-kit, in migration has been investigated at the molecular level in mice (Buehr, 1997). However, much remains to be elucidated. Further light may be shed on this process by studies on embryonic cells using OMP, Golf and AC3.

172 5.6 Conclusion

In summary, this study has provided evidence that OMP, Golf, and AC3 are expressed in Leydig cells of rats, suggesting that Leydig cells, like mast cells, may use these proteins as chemosensory signals. This observation supports the argument that OR-mediated signalling mechanisms may be involved in chemoreception and also in fetal Leydig cell migration. It is possible that OR-mediated signals may be involved in maintaining the species-specific immune privilege and local innate immunity of the testes. Mast cells are not found in the interstitial tissue of the rat testes. Leydig cells serve as paracrine cells and might use ORs to trigger their response.

173

Chapter 6: Adverse effects of a short-term, high-fat diet on metabolic metrics in rats

174 6.1 Abstract High-fat diets cause metabolic alterations that lead to obesity, diabetes, heart disease, colon cancer, and cognitive loss. These metabolic disorders perturb general CNS function, including altering olfactory sensation and participating in the pathogenesis of inflammatory diseases. The current study examined the hypothesis that a short-term, high-fat (HF) diet would lead to metabolic changes, mast cell imbalances, and olfactory abnormalities in rats. Rats were fed a HF lard-based diet for seven weeks. The results showed that overconsumption of fat, even in the short-term, caused weight gain, changed blood chemistry, and structural abnormalities in tongue, liver, and white adipose tissue (WAT). This HF diet, additionally, caused a reduction of OMP expression in mature olfactory sensory neurons (mOSNs) in the olfactory epithelium (OE), reduction of juxtaglomerular (JG) cells in the olfactory bulb (OB), reduction in mast cell numbers, and mast cell degranulation in tongue, liver, WAT, and the olfactory system. Mast cells evoked inflammatory responses in these tissues and could be implicated in the early stages of metabolic, gustatory and olfactory disorders. Even in the short-term, overconsumption of fat via a HF diet can impair chemoreception in the olfactory system, alter olfactory-driven behaviours, and create poor food choices, possibly contributing to further overconsumption of obesogenic foods.

6.2 Introduction Obesity is a metabolic disorder characterised by energy input that exceeds energy output, increased body weight, and glucose intolerance (Akiyama et al., 1996). It is a significant public health problem, exemplified by an unhealthy diet and reduced exercise (Abu-Abid et al., 2002). Some studies have suggested that genetic, physiological, and behavioural factors also play a role in the etiology of obesity (Jacobson, 2002; Wilborn et al., 2005; Farooqi and O’Rahilly, 2007; Bouchard, 2009). The consequences of obesity include a higher risk of cardiovascular disease, type 2 diabetes, sleep apnoea, pulmonary dysfunction, stroke, and venous insufficiency as well as diseases of the gallbladder, liver, and musculoskeletal system (Abu-Abid et al., 2002; Pi-Sunyer, 2009). Obesity may also be an underlying cause of of the breast, endometrium, colon, and prostate (Abu-Abid et al., 2002; Matafome et al., 2013; Ramos- Nino, 2013). With respect to reproductive biology, obesity causes impairment in sperm motility, morphology, concentration, and DNA (Aitken et al., 2004; Kasturi et al., 2008; Du Plessis et al., 2010). Obesity and related metabolic disorders are associated with chronic inflammation, initiated by nutrient overload (Ramos et al., 2003; Hotamisligil, 2006; Hildebrandt et al., 2009).

175 White adipose tissue (WAT) is an important site of inflammatory events in obesity (van der Heijden et al., 2015). In addition to regulating fat mass and nutrient homeostasis, WAT synthesises and releases leptin and inflammatory factors, including tumour necrosis factor- (TNF) α and interleukin- (IL) 6, which are elevated in proportion to the degree of obesity (Ramos et al., 2003). Diet-induced obesity in mice has been associated with elevated plasma concentrations of endotoxin lipopolysaccharide (LPS). Release of LPS into the circulation results in a condition termed “metabolic endotoxemia” (Cani et al., 2007; Cani et al., 2008). The endotoxemia initiates downstream inflammatory events, including release of IL-6 and TNF-α, through binding to Toll-like receptor- (TLR-) 4 and may be the stimulus for the inflammatory processes associated with obesity and insulin resistance (Sweet and Hume, 1996; Fernández-Real et al., 2003; Cani et al., 2007). The liver is a strong contributor to the development of metabolic inflammation in obesity (Shoelson et al., 2006; Glass and Olefsky, 2012; Sheedfar et al., 2013). The production of pro-inflammatory cytokines that are released by resident macrophages in the liver is linked to disruption of hepatic insulin signalling (Tilg and Moschen, 2008) and reduced insulin sensitivity (Cai et al., 2005; Lee et al., 2011b).

Obesity-related inflammation of WAT, which promotes metabolic dysregulation, has been linked with increased numbers of mast cells. A study of obesity in humans found that obese humans had higher numbers of mast cells in WAT and significantly higher tryptase concentrations in their serum, compared to lean individuals (Liu et al., 2009). The authors suggested that mast cells may contribute to diet-induced obesity by releasing the inflammatory cytokines IL-6 and interferon- (INF-) γ. In addition to releasing cytokines, mast cells may also contribute to obesity by promoting angiogenesis. Mast cells are often localised next to microvessels in WAT. It has been shown that the increase in numbers of microvessels can be correlated with the increase in numbers of mast cells in obesity (Liu et al., 2009). It has also been suggested that high numbers of mast cells in WAT of obese mice may be the result of enhanced mast cell proliferation, reduced mast cell apoptosis, or increased mast cell recruitment, all of which may contribute to angiogenesis in WAT (Liu et al., 2009).

In the liver of healthy rats, mast cells were found primarily around the portal triads and their numbers were associated with the size of the portal vein (Chan et al., 2001). In the livers of mice fed with a Western-type diet, mast cells were more abundant and expressed c-Kit CD117+ and livers exhibited steatosis (Liu et al., 2009). These pathologies might be the consequence of chronic inflammation and there are hints that the nervous system might be involved (Niissalo et al., 2000). Mast cells and neuropeptide-containing nerve fibres in the tongue have been investigated in several

176 rodent studies on experimentally-induced diabetes. Following a week of induced diabetes, rats had a decreased number of immunoreactive neuropeptide-containing nerve fibres in the tongue (Batbayar et al., 2003; Batbayar et al., 2004; Hevér et al., 2013; Kispélyi et al., 2014).

HF diet-induced obesity has been found to cause neuropathology, although it is unclear how and the extent to which obesity impacts the sensory systems (Fadool et al., 2011; Palouzier-Paulignan et al., 2012). Depending upon the severity and duration of obesity, an individual can undergo many endocrinological and physiological changes, including hyperglycemia, hyperleptinemia, hyperinsulinemia, insulin resistance, and leptin resistance (West and York, 1998; Kadowaki et al., 2003; Neary and Batterham, 2009). At present, human studies collectively indicate that obesity increases the detection threshold of odours; severe obesity raises the risk of anosmia and those higher cognitive changes may affect the ability to identify odours. These symptoms change with degree of adiposity, gender, and age (Palouzier-Paulignan et al., 2012).

Mice with long-term (six-month duration) HF diet-induced obesity exhibited marked loss of OMP-expressing mOSNs and their axonal projections (Thiebaud et al., 2014). This loss of OSNs and associated circuitry was linked to changes in proliferation of neuronal cells and their apoptotic cycles. It also induced proliferation of basal cells, activated microglia, and increased the number of pro-inflammatory cells in OE (Thiebaud et al., 2014). These alterations in the OE could underlie the impaired sense of smell directly or initiate multiple changes at higher levels of the olfactory system that cause the impairment.

Given that a long-term HF diet has been shown to cause dysfunction in a wide range of tissues, the current study aimed to evaluate the effects of a short-term (seven weeks) HF diet. It particularly focus on metabolic, structural and inflammatory changes in the blood, tongue, liver, WAT, mast cells, and olfactory system.

6.3 Materials and methods

6.3.1 Animals Adult Sprague-Dawley male rats were used, with the approval of the University of New South Wales’ Animal Care and Experimentation Committee and in strict accordance with the NHMRC Animal Experimentation Guidelines and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1990). All rats were obtained from the Animal Resources Centre (Perth, WA) and handled with care prior to and during experimentation. Rats were individually-housed in conventional open

177 cages and in a controlled environment with a 12-hrs light/12-hrs dark cycle. After the commencement of the experiment, all participating rats were monitored weekly to ensure good health.

6.3.2 Animal groups and diet

§ Experimental groups Twenty-four experimental rats, four weeks of age, were randomly divided into two groups of 12: control diet (control) group (labelled R5-8, R13-16 and R21-24) and high- fat diet (HF) group (labelled R1-4, R9-12 and R17-20).

§ Diets Rats were fed ab libitum for a duration of seven weeks. Control rats received Standard Laboratory Diet (11 MJ/Kg digestible energy; 14% from fat, 65% from carbohydrates, and 21% from protein; Gordon’s Specialty Stockfeeds, NSW, Australia). HF rats received Diet SF04-025 (Table 6.1) (18.4 MJ/Kg digestible energy; 35% from fat, 46% from carbohydrates, and 19% from protein; Speciality Feeds, WA, 6071). The HF diet is a high-lard modification of semi-pure high-fat diet AIN 76 for laboratory rats and mice in which 20% lard has been added to the formulation in place of canola oil, starch, and sucrose (Table 6.1).

178 Calculated Nutritional Parameters

Protein 19.40% Total Fat 20.00% Crude Fibre 4.70% AD Fibre 4.70% Digestible Energy 18.4 MJ / Kg % Total calculated digestible energy from lipids 36.00% % Total calculated digestible energy from protein 19.00%

Ingredients g/Kg

Casein (Acid) 200 Sucrose 396 Lard 200 Cellulose 50 Wheat Starch 100 Dextrinised Starch 117 L Methionine 3.0 Calcium Carbonate 11.3 Sodium Chloride 2.6 AIN93 Trace Minerals 1.6 Potassium Citrate 2.5 Potassium Dihydrogen Phosphate 5.3 Potassium Sulphate 1.6 Choline Chloride (75%) 1.3 Mono-Calcium Phosphate 6.7 Magnesium Oxide 1.2 Sodium Cholate 1.0 Cholesterol 5.0 AIN93 Vitamins 10

Table 6.1: Nutritional parameters, diet ingredients, and composition of high- fat lard-based diet (SF04-025). Data are taken from booklet provided by Speciality Feeds (3150 Great Eastern Hwy, Glen Forrest, WA 6071).

179 Fatty Acid Composition % of total

Saturated Fats C12:0 or less 0.10 Myristic Acid 14:0 0.30 Palmitic Acid 16:0 5.30 Stearic Acid 18:0 3.40 Palmitoleic Acid 16:1 0.30 Oleic Acid 18:1 6.80 Gadoleic Acid 20:1 0.20 Linoleic Acid 18:2 n6 2.90 a Linolenic Acid 18:3 n3 0.30 EPA 20:5 n3 No data DHA 22:6 n3 No data Total n6 2.90 Total Cholesterol 0.50 Total Mono Unsaturated Fats 7.32 Total Polyunsaturated Fats 3.32 Total Saturated Fats 9.30

Table 6.1 (continued): Nutritional parameters, diet ingredients, and fatty acid composition of high-fat lard-based diet (SF04-025). Data are taken from booklet provided by Speciality Feeds (3150 Great Eastern Hwy, Glen Forrest, WA 6071).

6.3.3 Experimental ante-mortem measurements

§ Body weight measurements All 24 rats were weighed prior to the start of the experiments, every 3-4 days during the experiment, and at the end of the experiment. Measurements were recorded in grams (g). Number of rats—control: n=12, HF: n=12.

§ Blood serum chemistry measurements Blood samples were taken at completion of the experiment from all 24 animals after 12 hrs of fasting. Glucose levels were determined using a commercially available glucose monitoring system (Accu-Chek Performa) immediately after the perfusions of the rats. Number of rats—control: n=12, HF: n=12. Levels of triglycerides (TG) and high-

180 density lipoprotein (HDL) cholesterol were measured using a Cardio-Chek Blood Testing Device. Number of rats—control: n=11, HF: n=12 (data from one rat lost for technical reasons). All data recorded in mmol/L.

6.3.4 Perfusion Perfusion was performed, as described in Section 2.3.2.

6.3.5 Experimental post-mortem measurements

§ White adipose tissue weight and percentage WAT was removed from the abdominal fat pad of 23 rats and weighed. Number of rats—control: n=11, HF: n=12. Results were recorded in grams (g). WAT percentage was calculated using the formula: (WAT weight (g) / Body weight (g) x 2) x 100. Results were recorded as WAT percentage. Of the 24 rats from whom samples were taken, 8 were randomly selected (4 controls and 4 HF) for histological analysis.

6.3.6 Histological methods

6.3.6.1 Tissue selection and preparation Histological methods were applied to tissues obtained from 4 controls (R21-24) and 4 HF rats (R17-20). From each rat, the following tissues were taken: OE with OBs; tongue from apex to root; three pieces randomly selected from liver; WAT from the abdominal fat pad. A solution of 10% neutral buffered Formalin (Sigma-Aldrich Pty Ltd., Australia) was used to post-fix tissue for 12 – 14 hrs. Postfixed tissue was immersed overnight in 70% (v/v) ethanol. Liver tissue for cryohistology was also harvested and frozen rapidly with Ultrafreeze Freeze Gel (Clear; Cancer Diagnostics Inc.) and stored at -80 °C. Tissue was prepared using standard paraffin embedding procedures. Three sections of liver were placed parallel on one mould. Tongues were positioned longitudinally, dorsal surface down on the mould. Tissue was sectioned at 5 μm on a motorised microtome (Leica RM 2155, Leica Microsystems Pty Ltd). OE with OBs, tongue, liver and WAT from each rat was serially-cut and mounted on electrostatic slides (Menzel-Glaser, Braunschweig, Germany). These slides were numbered consecutively for each tissue.

6.3.6.2 Haematoxylin and Eosin staining Haematoxylin and Eosin (HE) staining was performed, as outlined in Section 2.3.3.2.

181 6.3.7 Tissue evaluation and image analysis

6.3.7.1 Characteristics of white adipose tissue (WAT)

§ Image analysis of adipose cell area WAT was taken from the abdominal cavity of 4 rats: 2 controls (R21 and R24) and 2 HF rats (R18 and R20) for the measurement of adipose cell area. These tissues were serially cut and mounted on numbered slides. Six randomly chosen slides were taken from the control group (3 slides from R21 and 3 slides from R24) and 6 slides from the HF group (3 slides from R18 and 3 slides from R20), stained with HE and analysed. HE-stained WAT slides from each group (control and HF) were coded and examined by a "blinded" observer. Each slide was viewed at 20x magnification where fields were randomly selected and photographed. Each field was represented by a montage of six photomicrographs, saved as a .psd file (Photoshop CS6; Adobe Systems). Only images of adipose cells with nuclei and intact membranes were selected for further study. The inside circumference of each cell membrane was traced and the enclosed area measured using FIJI software (Schindelin et al., 2012). The contents were subsequently referred to as the “area” of the adipose cells. One particle represented one adipose cell. All measurements were collected and the results saved in an Excel (Microsoft) file. Data are presented as “adipose cell area” (μm2). Number of rats—control: n=2, HF: n=2; number of slides—control: n=6, HF: n=6; number of calculations (area)—control: 452, HF: 298.

6.3.7.2 Characteristic of liver tissue

§ Oil Red O staining

Oil Red O (ORO) (C26H24N4O) is a fat-soluble, hydrophobic, diazol dye with a maximum absorption at 518 nm. ORO is only marginally soluble and its solubility decreases when it is diluted in water before use (Mehlem et al., 2013). ORO stains only the most hydrophobic and neutral lipids, such as triglycerides, diacylglycerols, and cholesterol esters, but not biological membranes (Ramirez-Zacarias et al., 1992). ORO staining has been found to be the most accurate method for detecting and quantifying hepatic steatosis in the liver.

Liver tissue was taken from 8 rats: 4 controls (R21-24) and 4 HF rats (R17-20) to detect the neutral lipid accumulations. These tissues were serially cut and mounted on numbered slides, three sections per slide, as described in Section 6.3.6.1. Four randomly selected slides were taken from the control group (one slide from each rat) and 4 slides from the HF group (one slide from each rat), stained with ORO and analysed. Lipid droplet (LD) accumulations appeared as red droplets.

182 § Image analysis of lipid droplet accumulations Frozen liver sections were thawed for 10 mins at room temperature and were then stained with ORO working solution (60 mL stock solution [ORO, 1 g and 99% isopropanol, 1000 mL] and 40 mL distilled water) for 15 mins. The stained sections were washed in tap water and counterstained in Mayer Haematoxylin for 1 min. Sections were washed again in tap water and stained in Scott’s Blueing solution for 15 secs. Sections were washed in tap water and cover-slipped in a water-soluble medium.

Liver sections, stained with ORO, were "coded" for analysis and scanned using Aperio ImageScope (Leica). Images were analysed using ImageScope software (Leica). Images were colour deconvoluted and positive pixels were calculated per “area of liver” (µm2). Medium positive accumulations were called “medium positive LD accumulations” (MLD) and presented as “mean percentage (%) area of MLD” per area of liver (µm2). Weak positive accumulations were called “weak positive LD accumulations” (WLD) and presented as “mean % area of WLD” per area of liver (µm2). Number of rats—control: n=4, HF: n=4; number of slides—control: n=4, HF: n=4; number of calculations (area)— control: 4, HF: 4.

6.3.7.3 Mast cells assessment

§ Toluidine Blue staining Toluidine Blue (TB) staining was chosen to identify mast cells. As discussed in Section 4.3.3.3, TB, a classic metachromatic stain, is strongly recommended as a routine stain for mast cells (Krishnaswamy and Chi, 2006). The cytoplasm of mast cells contains granules composed of biologically-active mediators, including acidic mucopolysaccharides and glycosaminoglycans, such as heparin and histamine, which bind TB. The use of this staining also shows mast cell degranulation, the release of mast cell granules into surrounding tissue (Overman et al., 2012; Chauhan et al., 2015). Once the mast cells have degranulated, no granules will be detected in the cells with the use of TB staining (D’mello et al., 2016).

TB staining was applied to sections of tongue, liver, WAT and the OE of rats from control and HF groups. Paraffin sections were dewaxed and hydrated with distilled water and stained by TB (working solution of 1% TB in 70% ethanol, 5 ml and 1% sodium chloride in distilled water, 45 ml mixed well, pH around 2.3) for 30 secs. Sections were washed three times in distilled water and dehydrated by soaking once in 95% ethanol and two times in 100% ethanol. They were then cleared by two 3-min soakings in xylene and cover-slipped with DPX mounting medium.

183 § Density of mast cells in tongue Tongue tissue was taken from 6 rats: 3 controls (R21, 22 and R24) and 3 HF rats (R17, 18 and R20) to detect the density of TB-positive (TB(+)) mast cells. These tissues were serially cut and mounted on numbered slides. Twelve randomly selected slides were taken from the control group (4 slides from each rat) and 12 slides from the HF group (4 slides from each rat), stained with TB and analysed. The density of TB(+) mast cells in scanned and blinded tissue sections from both groups (control and HF) was calculated using Aperio ImageScope (Leica). An area of interest was defined by drawing a line along the lamina propria and separating the overlying stratified squamous epithelium (SSE) from the submucosa and muscle layers. The selected area was calculated and called “area” (µm2). A positive pixel count algorithm in ImageScope software (Leica) was applied, based on intensity and shape thresholding, to identify TB(+) mast cells. The total area of TB(+) mast cells was calculated and called “A-total” (mm2). The ratio of A-total divided by area gave the mean percentage area of TB(+) mast cells per area of tongue and was recorded as “density of TB(+) mast cells” per area of tongue. Number of rats—control: n=3, HF: n=3; number of slides—control: n=12, HF: n=12; number of calculations (area)—control: 12, HF: 12.

§ Density of mast cells in liver Liver tissue was taken from 8 rats: 4 controls (R21-24) and 4 HF rats (R17-20) to detect the density of TB(+) mast cells. These tissues were serially cut and mounted on numbered slides, three sections per slide, as described in Section 6.3.6.1. Twelve randomly selected slides were taken from the control group (three slides from each rat) and 12 slides from the HF group (three slides from each rat), stained with TB and analysed. These 24 slides were later used to calculate the area and number of portal veins in the liver. The total liver area in each animal and in each group (control and HF) was calculated and called “total area” (µm2). The number of TB(+) mast cells inside total area was calculated and recorded as “number of TB(+) mast cells”. The ratio of number of TB(+) mast cells divided by total area was recorded as the “density (number) of TB(+) mast cells” per total area of liver. Number of rats—control: n=4, HF: n=4, number of slides—control: n=12, HF: n=12; number of calculations (area)—control: 12, HF: 12.

§ Area of portal vein per total area of liver Liver tissue was taken from 8 rats: 4 controls (R21-24) and 4 HF rats (R17-20) to detect the area of portal veins. These tissues were serially cut and mounted on numbered slides, three sections per slide, as described in Section 6.3.6.1. Twelve randomly selected slides were taken from the control group (three slides from each rat) and 12 slides from the HF group (three slides from each rat), stained with TB and 184 analysed. The total liver area in each animal and group (control and HF) was measured and called “total area” (µm2). The total area of the portal veins in the portal triads in each animal and in each group was also determined by measuring the circumference of the portal vein and calculating the area within. This measurement was called “area of portal vein” (µm2). The ratio of area of portal vein divided by total area was recorded as “area of portal vein per total area” of liver. Number of rats —control: n=4, HF: n=4; number of slides—control: n=12, HF: n=12; number of calculations (area)—control: n=189, HF: n=174.

§ Number of portal veins per total area of liver Liver tissue was taken from 8 rats: 4 controls (R21-24) and 4 HF rats (R17-20) to detect the number of portal veins. These tissues were serially cut and mounted on numbered slides, three sections per slide, as described in Section 6.3.6.1. Twelve randomly selected slides were taken from the control group (three slides from each rat) and 12 slides from the HF group (three slides from each rat), stained with TB and analysed. The total liver area for each rat and for each group (control and HF) was measured and called “total area” (µm2). The number of portal veins in each animal and in each group was counted and called “number of portal veins”. The ratio of number of portal veins divided by total area was recorded as “number of portal veins per total area” of liver. Number of rats—control: n=4, HF: n=4; number of slides—control: n=12, HF: n=12; number of calculations—control: n=189, HF: n=174.

§ Correlation between number of TB(+) mast cells and portal vein area in liver Analysis was performed on the correlation between the number of TB(+) mast cells and area of the portal veins in the livers of 8 rats: 4 controls (R21-24) and 4 HF rats (R17-20). Liver slides, stained with TB, were scanned using Aperio ImageScope (Leica). In each liver section, all portal triads that contained a portal vein, an artery, and a bile duct were identified and analysed. The analysis was done on all identified portal triads whether or not mast cells were also located in proximity to the triad. In each triad the number of TB(+) mast cells were counted and the area of the portal vein calculated by measuring the circumference of the portal vein and calculating the area within. This measurement was called “area of portal vein” (µm2). The number of TB(+) mast cells in each triad was manually counted and the results presented as “number of TB(+) mast cells” per portal triad. If no TB(+) mast cells were observed in the portal triad, the number of the mast cell per total triad was recorded as 0. The area of the portal vein and number of TB(+) mast cells in the liver of (a) each animal and (b) each group of animals (control and HF) were correlated using Pearson’s correlation coefficient.

185 § Density of mast cells in WAT WAT was taken from 6 rats: 4 controls (R21-24) and 2 HF rats (R18 and R20) to detect the density of TB(+) mast cells. These tissues were serially cut and mounted on numbered slides. Sixteen randomly selected slides were taken from the control group (four slides from each) and 13 slides from the HF group (six slides from R18 and seven from R20), stained with TB and analysed. FIJI software (Schindelin et al., 2012), along with custom Python scripts, was used to count TB(+) mast cells in WAT. The original scanned-slide files (TB-stained WAT in Aperio SVS format) were imported into FIJI and converted to RGB images. Each image was subsequently deconvoluted using the “Colour Deconvolution” macro (vector: “H DAB”) to produce an 8-bit image of TB(+) staining separated from the counterstain. The remaining two channels were discarded. However, as the colour deconvolution algorithm tended to underestimate the full size of TB(+) cells, the original RGB image was also converted to 8-bit grayscale for analysis. The total area of WAT in the RGB image was manually outlined to limit the region of analysis. 8-bit images were normalised, processed with a median filter (radius: 2 px), and converted to binary images with the “Auto Threshold” macro (settings: RGB-8bit, ‘Intermodes’; TB(+)-8bit, ‘IsoData’) to produce discrete particles/cells. Binary images were then despeckled, segmented with the watershed algorithm (RGB-8bit only), and any holes were filled. Particles with an area <10 µm2 were excluded. To ensure only TB(+) particles were assessed from the thresholded RGB-8bit image, only those particles whose bounds also contained a particle in the thresholded TB(+)-8bit image were included. In addition, these remaining particles were overlaid upon the source RGB image and manually reviewed to exclude obvious artefactual labelling (e.g., debris on slide). Each analysed particle represented one TB(+) mast cell. The summed results of particle counting were recorded as “total area of particles”. The total area of manually segmented WAT on the slide was called “total area” (µm2). The ratio of total area of particles divided by total area was recorded as the “density (number) of TB(+) mast cells” per total area of WAT. Number of rats—control: n=4, HF: n=2; number of slides—control: n=16, HF: n=13 number of calculations (area)—control: 16, HF: 13.

6.3.7.4 Analysis of mOSNs in olfactory epithelium (OE)

§ Immunofluorescent histochemistry with OMP Single-labelling immunofluorescent histochemistry (IF IHC) was applied to the OE of rats from both control and HF groups to assess mOSNs. OMP was chosen for reasons described in Section 1.2 to detect mOSNs in the OE. Olfactory turbinate tissue was taken from 6 rats: 3 controls (R21, 22 and R24) and 3 HF rats (R17-19). These

186 tissues were serially cut and mounted on numbered slides. Sixteen randomly selected slides were taken from the control group and 12 slides from the HF group.

Single-labelling IF IHC was performed following the procedure outlined in Section 2.3.3.3. The primary antibody for OMP—goat antiserum, raised with rodent OMP as the immunogen, diluted 1:1 with glycerol, containing 0.05% sodium azide (code N 019- 22291, Wako Pure Chemical Industries, Ltd)—was used at dilution: 1: 1,100.

§ Image analysis of OMP intensity in mOSNs FIJI software (via the Bioformats plugin) (Schindelin et al., 2012) was used to open the original files (ZEISS AxioVision format, .zvi), separate the channels, and export the images (16-bit .tif). Images were opened in MetaMorph (Molecular Devices), converted to 8-bit (0-255 dynamic range) and calibrated. MetaMorph software was then used to select the area of analysis in the OE, measure fluorescence intensity of OMP, and count the nuclei of mOSNs. The area selected for analysis (ROI) was located beneath the ciliary layer and encompassed the position of mOSNs (red-channel of the image). The ROI was recorded, duplicated, and saved separately as a .tif file. The ROI was saved as an .rgn file and transferred to the blue-channel of the image. Integrated Morphometric Analysis (IMA) was used to filter objects greater than 5 µm2 and the standard ROI was 40 µm2 (“area”). The threshold levels of the red-channel were set up (inclusive, 30-255) to measure intensity of OMP within the ROI. The threshold levels of the blue-channel were set up (inclusive, 41-255) to highlight and calculate the nuclei. All recorded data were saved in Excel (Microsoft). All ROI measurements are recorded in µm2 and intensity of OMP recorded as greyscale values. Results for both control and HF groups were recorded and graphed as “mean intensity values for OMP expression” in mOSNs and the “number of nuclei per area” in OE. Number of rats—control: n=3, HF: n=3; number of slides—control: n=16, HF: n=12; number of calculations (area)—control: 137, HF: 332.

6.3.7.5 Analysis of juxtaglomerular (JG) cells in olfactory bulb (OB)

§ Immunoperoxidase histochemistry with calbindin D-28k Calbindin D-28k (CB) is a soluble, intracellular calcium-binding protein (CaBP) (Parmentier, 1990). It is expressed in all layers of the rat OB and the highest number of CB-positive (CB(+)) neurons is found in the glomerular layer (GL) (Garcia-Segura et al., 1984; Celio, 1990; Brinon et al., 1992). CaBP-expressing GABAergic and DAergic JG neurons are considered to be PG cells (Gall et al., 1987; Kosaka et al., 1995; Parrish- Aungst et al., 2007). GABAergic GAD65(+) JG cells are categorised as uniglomerular and generally project their dendrites to a single glomerulus (Kiyokage et al., 2010). Their

187 axons connect 5 or 6 glomeruli and terminate in the interglomerular space (Pinching and Powell, 1971b). They receive inputs from many cells: ET cells, PG cells, SA cells, tufted cells, mitral cells and OSNs (Kiyokage et al., 2010; Nagayama et al., 2014). GABAergic GAD65(+) PG cells have an inhibitory function within the glomerulus (Kiyokage et al., 2010).

OB tissue was taken from 6 rats: 3 controls (R21, 22 and R24) and 4 HF rats (R17-20). These tissues were serially cut and mounted on numbered slides. Eight randomly selected slides were taken from the control group (two slides from R21 and 24 and 4 slides from R22) and 8 slides from the HF group (two slides from each rat).

Immunoperoxidase histochemistry (IP IHC) was performed following the procedure outlined in Section 2.3.3.3. CB was used to detect and analyse CB(+) JG cells in the GL of the rat OB in control and HF groups. The primary antibody for calbindin D- 28k—rabbit lyophilised antiserum, produced against recombinant rat CD-28k (code No: CB-38a, SWANT, Switzerland)—was used at dilution: 1:2,000.

§ Density of CB(+) JG cells in OB The density of CB(+) cells in the GL of the OB was calculated for control and HF groups using Aperio ImageScope (Leica). In each section, an area of interest was defined by outlining the GL, thus separating the glomeruli from the other layers of the OB, and called “area” (μm2). An algorithm for counting positive pixels was applied to identify CB(+) cells in the GL, based on their intensity and shape thresholding using ImageScope software (Leica). The areas occupied by CB(+) cells were calculated, summed, and called “A-total” (mm2). The ratio of A-total divided by area gave the mean percentage area of CB(+) cells and was recorded as the “density of CB(+) JG cells” per GL area in the OB. Number of rats—control: n=3, HF: n=4; number of slides—control: n=8, HF: n=8; number of calculations (area)—control: 53, HF: 53.

§ Immunoperoxidase histochemistry with tyrosine hydroxylase Tyrosine hydroxylase (TH) is a rate-limiting enzyme involved in DA synthesis (Halasz et al., 1976; Versteeg et al., 1976). DA is the most abundant neurotransmitter in the rat OB and inhibits the OSN axons at the presynaptic level by activating the D2- receptor (Koster et al., 1999; Ennis et al., 2001; Puopolo et al., 2005). In the OB, TH is expressed almost entirely in the GL. However, weakly TH-immunostained neurons can occasionally be detected in other OB layers (Halasz et al., 1976; Halász et al., 1981; Toida et al., 2000). TH-positive (TH(+)) cells account for approximately 10% of all JG cells and most of these, if not all, co-express with glutamic acid decarboxylase (GAD). TH expression depends upon olfactory sensory inputs (Baker et al., 1983). Previously,

188 TH(+) JG cells were regarded as DAergic and GABAergic PG cells (Kosaka et al., 1998; Kosaka and Kosaka, 2005). However, these days, with the use of retrograde tracing analysis, these cells have been shown to be SA cells (Kiyokage et al., 2010). Thus, DAergic TH(+)GAD67(+) SA cells are categorised as interglomerular, possessing long axons and dendrites that contact up to 50 glomeruli (Kiyokage et al., 2010). They receive inputs from ET cells from the same glomerulus and have an inhibitory function across glomeruli (Kiyokage et al., 2010).

OB tissue was taken from 8 rats: 4 controls (R21-24) and 4 HF rats (R17-20). These tissues were serially cut and mounted on numbered slides. Four randomly selected slides were taken from the control group (one slide from each rat) and 4 slides from the HF group (one slide from each rat).

Immunoperoxidase histochemistry (IP IHC) was performed following the procedure outlined in Section 2.3.3.3. TH was used to detect and analyse TH(+) JG cells in the GL of the rat OB in control and HF groups. The antibody for anti-tyrosine hydroxylase—mouse monoclonal ascites fluid (clone TH-16, product number: T2928, Sigma, USA)—was used at a dilution of 1:60,000.

§ Density of TH(+) JG cells in OB Original image files (OBs stained with TH) were imported into FIJI software (Schindelin et al., 2012) to count TH(+) JC cells in the GL of the OB. The images were converted to RGB and colours deconvoluted to separate the HE and DAB channels. The DAB channel image was adjusted for brightness and contrast to decrease staining background and emphasise the DAB-positive TH(+) cells. The same brightness/contrast settings were used throughout the image analysis process. Threshold settings on the images were adjusted to highlight the DAB-positive TH(+) cells and to prepare them for particle analysis. A trial analysis on a TH(+) control slice was conducted to determine the settings for particle analysis (including minimum and maximum sizes of particles to be counted). Particles <10 pixels and >350 pixels in area were excluded from analysis. Each analysed particle represented one TH(+) cell. The summed area of all counted particles was recorded as “total area of particles”. These parameters were applied to the analysis of all sets of slides. The ratio of total area of particles divided by total area of the GL of the OB gave the mean percentage area of TH(+) JG cells and was recorded as the “density of TH(+) JG cells” per GL area of the OB. Number of rats—control: n=4, HF: n=4; number of slides—control: n=4, HF: n=4; number of calculations (particles)— control: 10,043, HF: 12,201.

189 6.3.8 Photography and image processing Photography and image processing were conducted as described in Section 2.3.4.

6.3.9 Statistics The Mann-Whitney U test was used for two independent samples when the data were not normally distributed. Data for this test were presented as “mean ± SD” (median, [25th percentile; 75th percentile]; p-value” in the text. Statistical analysis with p<0.05 (two-tailed) was considered significant (*).For normally distributed data, one of two tests was applied: Student’s paired t-test (one-sample population studies) and t-test (two- sample population studies). Data for these tests were presented as “mean ± SD”; p- value”. Statistical analyses with p<0.05 were considered significant (*).

Growth rate was calculated according to the formula: past value ÷ present value. Depending on the results, different analyses were conducted. Where growth rate was in the range 0 – 1, the following formula was used: (past value ÷ present value - 1) x 100. The results indicated negative growth (-) and were presented as a percentage (%). Where growth rate was in the range 1 – 2, the following formula was used: (past value ÷ present value - 1) x 100. The results indicated positive growth (+) and were presented as a percentage (%). Where growth rate was 2 or more, the number of times by which it had increased was shown and this was calculated according to the formula: past value ÷ present value.

The Pearson correlation coefficient method was applied to determine the linear relationship between the number of TB(+) mast cells and area of portal veins in the rat liver. The strength of the correlation between these two variables was determined by r (correlation coefficient), where r=1 indicates a perfect linear relationship. The significance of the correlation was determined by p-value, where p<0.05 was considered significant.

All tabled and graphed values are shown as the mean ± SD and p-value and labelled as significant (*) or not significant (ns).

190 6.4 Results

6.4.1 Body weight measurements Before the start of the HF diet (start), there was no difference in total body weight between rats of the control and HF groups (control start: 111.08 ± 5.84 g; HF start: 111.92 ± 8.37 g; p=0.78ns; t-test) (Table 6.2).

At the end of the experiment the control group gained a total body weight of 309.68% (control start: 111.08 ± 5.84 g; control end: 455.08 ± 33.51 g; p<0.0001*; paired t-test). The HF group had gained 372.38% (i.e., 416.75 g) (HF start: 111.92 ± 8.37 g; HF end: 528.67 ± 61.34 g; p<0.0001*; paired t-test) (Table 6.2). The total body weight of rats in the HF group was 16.17% higher than that of controls (control end: 455.08 ± 33.51 g; HF end: 528.67 ± 61.34 g; p=0.002*; t-test) (Table 6.2).

6.4.2 Parameters of white adipose tissue At the end of the experiment, the HF group had 2.46 times more WAT weight than the control group (control: 4.20 ± 2.17 g, (4.10, [3.75; 4.78]); HF: 10.32 ± 2.80 g, (10.30, [7.98; 12.63]); p<0.0001*; Mann-Whitney U test).

Further, the percentage of WAT in the HF group was 2.1 times higher than in controls (control: 1.85 ± 0.37%, (1.84, [1.58; 2.19]); HF: 3.88 ± 0.73%, (3.90, [3.29; 4.36]); p<0.0001*; Mann-Whitney U test) (Table 6.3).

control HF p-value growth rate

Before diet 111.08 ± 5.84 111.92 ± 8.37 0.375ns +0.75% commenced (n=12) (n=12) (start)

After 7 weeks 455.08 ± 33.51 528.67 ± 61.34 0.002* +16.17% (end) (n=12) (n=12) weight gain 344.00 416.75 p-value < 0.0001* < 0.0001* growth rate +309.68% +372.38% Table 6.2: Metabolic parameters: body weight (g) in control and HF groups. Short-term HF diet leads to excessive weight gain. Data are expressed as mean ± SD; p < 0.05 versus control (*). Abbreviations: HF, high-fat group; ns, not significant.

191 WAT weight (g) WAT percentage control 4.20 ± 0.89 (n=12) 1.85 ± 0.37 (n=12)

HF 10.32 ± 2.80 (n=12) 3.88 ± 0.73 (n=12) p-value < 0.0001* < 0.0001* growth rate 2.46´ 2.10´ Table 6.3: Metabolic parameters: organ adiposity in control and HF groups. Short-term HF diet leads to adiposity. Data are expressed as mean ± SD; p < 0.05 versus control (*). Abbreviation: HF, high-fat group; WAT, white adipose tissue.

Figure 6.1: Morphology of white adipose tissue (WAT) of control and HF groups. Photomicrographs show HE-stained WAT of control (A) and HF (B) groups. In HF group, WAT is structurally disorganised and adipose cells (AdCs) have thinner walls and flattened nuclei (B).

192 6.4.3 Structure of white adipose tissue and area of adipose cells Histological analysis of HE-stained WAT showed that the HF group had mild changes in tissue structure as compared with controls (control: Fig. 6.1A). In the HF group, WAT was structurally disorganised and had adipose cells of different sizes, most of them with disrupted cell membranes (HF: Fig. 6.1B). Adipose cells had thinner walls and flattened nuclei (HF: Fig. 6.1B). In the HF group, erythrocytes clustered between adipose cells (HF: Fig. 6.2A). The stroma of the WAT exhibited enlarged vessels, most of which contained blood and inflammatory cells (HF: Fig. 6.2B).

Figure 6.2: Inflammation of white adipose tissue (WAT) in rats fed a HF diet for 7 weeks. Photomicrographs of HE-stained WAT show (A) cluster of erythrocytes (arrow) located between adipose cells (AdCs); (B) blood vessel (BV) filled with erythrocytes and inflammatory cells (arrows).

193

Figure 6.3: Area of adipose cells of control and HF groups. Graph shows differences in total area of adipose cells (µm2) in control and HF groups. The area of adipose cells in HF group was larger than that in controls. Data are expressed as mean ± SD; p < 0.05 versus control (*). Abbreviation: HF, high-fat group.

Image analysis of WAT showed that the HF group had 31.7% larger area of adipose cells than the control group (control: 3313.60 ± 1777.72 µm2, (2946.03, [2043.73; 4251.17]); HF: 4364.01 ± 2163.70 µm2, (3917.27, [2848.92; 5425.38]); p<0.00001*; Mann-Whitney U test) (Fig. 6.3).

6.4.4 Structure of liver and lipid droplet accumulations Histological analysis of HE-stained livers showed that the HF group had changes in tissue structure associated with inflammation, unlike controls (control: Fig. 6.4A). In the HF liver, erythrocytes clustered in blood vessels, infiltrated liver parenchyma and clustered between the hepatocytes (HF: Fig. 6.4B). Vacuolised liver tissue contained ballooned hepatocytes and inflammatory cell aggregates (HF: Fig. 6.4C). Fibrosis was observed in some liver areas (HF: Fig. 6.4D).

Histological analysis of ORO-stained livers showed that the HF group had many more LDs in the cytoplasm of hepatocytes than did the control group (control: Fig. 6.5A; HF: Fig. 6.5B). Moreover, the HF group had 143.83 times more MLD accumulations than the control group (control: 0.001 ± 0.002%, (0.0005, [0.0002; 0.0014]); HF: 0.157 ± 0.108%, (0.181, [0.103; 0.235]); p=0.01*; Mann-Whitney U test) and had 23.33 times more WLD accumulations than the control group (control: 0.507 ± 0.641%, (0.280,

194 [0.173; 0.615]); HF: 11.834 ± 5.720%, (11.552 [8.878; 14.508]); p=0.01*; Mann-Whitney U test) (Table 6.4).

Figure 6.4: Hepatic steatosis, steatohepatitis, and fibrosis in livers of rats fed HF diet for 7 weeks. Photomicrograph of HE-stained liver from a control rat (A) shows parenchyma (P) containing hepatocytes and blood vessel (BV). Hepatocytes are linked by sinusoids (arrows) or other hepatocytes. Photomicrographs of HE-stained liver from rats fed HF diet show (B) erythrocytes in blood vessel (BV) (arrow) and clusters of erythrocytes (arrowhead) between hepatocytes of liver parenchyma (P); (C) pronounced vacuolisation in liver tissue, “balloons” of hepatocytes (arrow), and clusters of focal inflammatory cells (arrowhead); (D) fibrosis of liver tissue (arrow).

195

Figure 6.5: Lipid droplet accumulations in livers of control and HF groups. Photomicrographs show Oil Red O-stained livers of control (A) and HF (B) groups. (A) In control group, small amount of lipid droplet accumulations (LDs) (red) (arrow) are observed in hepatocytes. (B) In HF group, large amount of micro- and macro-vesicular accumulation of LDs (red) (arrows) are observed in hepatocytes.

196 MLDs (%) WLDs (%) control 0.001 ± 0.002 (n=4) 0.507 ± 0.641 (n=4)

HF 0.157 ± 0.108 (n=4) 11.834 ± 5.720 (n=4) p-value 0.01* 0.01* growth rate 143.83´ 23.33´ Table 6.4: Lipid metabolism parameters in control and HF groups. Short-term HF diet leads to elevation of lipid droplet accumulations and development of hepatic steatosis. Data are expressed as mean ± SD; p < 0.05 versus control (*). Abbreviations: HF, high-fat group; LDs, lipid droplet accumulations; MLDs (%), mean percentage of medium intensity LDs per area (µm2); WLDs (%), mean percentage of weak intensity LDs per area (µm2).

6.4.5 Evaluation of blood serum chemistry parameters At the conclusion of the experiment, glucose levels in the HF group were 22.53% higher than in the control group (control: 9.28 ± 2.17 mmol/L, (10.10, [9.10; 10.38]); HF: 11.38 ± 1.54 mmol/L, (11.25, [10.20; 12.93]); p=0.01*; Mann-Whitney U test). TGs in the HF group were 62.95% higher than in controls (control: 105.09 ± 48.70 mmol/L, (118.00, [54.50; 147.50]); HF: 171.25 ± 46.79 mmol/L; 150.00, [143.00; 175.50]); p=0.01*; Mann- Whitney U test). Levels of HDLs in the HF group were 21.61% lower than in controls (control: 39.55 ± 10.94 mmol/L, (36.00, [32.50; 40.50]); HF: 31.0 ± 4.71 mmol/L, (32.00, [28.50; 34.00]); p=0.02*; Mann-Whitney U test) (Table 6.5).

glucose (mmol/L) TG (mmol/L) HDL (mmol/L) control 9.28 ± 2.17 105.09 ± 48.70 39.55 ± 10.94 (n=12) (n=11) (n=11)

HF 11.38 ± 1.54 171.25 ± 46.79 31.00 ± 4.71 (n=12) (n=12) (n=12) p-value 0.01* 0.01* 0.02* growth rate +22.53% +62.95% -21.61% Table 6.5: Metabolic parameters: blood chemistry of control and HF groups. Short-term HF diet elevated glucose and triglyceride levels and reduced high density lipoproteins in blood serum. Blood parameters were measured at the end of experiment. Data are expressed as mean ± SD; p < 0.05 versus control (*). Abbreviations: HF, high-fat diet group; TG, triglycerides; HDL, high-density lipoproteins.

197 6.4.6 Mast cells assessment

§ Analysis of TB(+) mast cells in tongue Histological analysis of TB-stained tongue sections showed that mast cells were distributed across the anterior surface of the tongue from apex to root. A HF diet did not cause any observable differences in TB(+) mast cell distribution or density, in comparison with controls. In both groups, TB(+) mast cells were observed as dark blue/violet cells, which were evenly distributed throughout the lamina propria, in the connective tissue, and between the muscle fibres of the tongue (control: Figs. 6.6A & 6.7A; HF: Figs. 6.6B & 6.7B). Denser accumulations were observed around blood

Figure 6.6: Toluidine Blue- (TB-) stained tongues of control and HF groups. Photomicrographs show TB(+) mast cells in tongues of control (A) and HF (B) groups. In both groups, TB(+) mast cells (MCs) (arrows) are evenly distributed between muscle fibres (M) and throughout connective tissue (CT) that surround blood vessels (stars) (A) and (B). More blood vessels (stars) are observed in the HF group (B) than in controls (A).

198 vessels in both groups, however, and more and larger blood vessels were observed in the HF group (control: Fig. 6.6A; HF: Fig. 6.6B).

No TB(+) mast cells were detected in SSE or taste buds in tongues of either control or HF groups (control: Fig. 6.7A; HF: Fig. 6.7B).

In the control group, at higher magnification, granules of the intact TB(+) mast cells were observed as deep blue/violet masses. The pale blue nuclei of some mast cells were visible, whereas granules obscured the nuclei in others (control: Fig. 6.8A). However, a HF diet caused degranulation in most of the TB(+) mast cells of the tongues. In some cases, degranulation produced only a few granules, located at various distances from the cell. In other cases, profound degranulation occurred, as seen in Fig. 6.8B (HF).

Figure 6.7: Toluidine Blue- (TB-) stained tongues of control and HF groups. Photomicrographs show TB(+) mast cells in tongues of control (A) and HF (B) groups. In both groups, TB(+) mast cells (MCs) (arrows) are observed in the lamina propria (arrows), but not in stratified squamous epithelium (SSE) or taste buds (A-B).

199 Image analysis showed that the density (mean percentage area) of TB(+) mast cells in tongues of the HF group was 13.4% less than that in tongues of controls (control: 0.57 ± 0.10% per mm2; HF: 0.49 ± 0.03% per mm2; p=0.02*; t-test) (Table 6.6) (Fig. 6.9).

Figure 6.8: Toluidine Blue- (TB-) stained tongues of control and HF groups. Photomicrographs show TB(+) mast cells in tongues of control (A) and HF (B) groups. (A) In control group, TB(+) mast cells (MCs) (arrows) are located between muscles (M) and blood vessels (BVs). Granules are observed as dark blue/violet masses within cells. In some cells pale-blue nuclei are visible; in others they are hidden by granules. (B) In HF group, profound degranulation of MCs (arrows) is seen. Granules (arrowhead) are dispersed around MCs.

200

Figure 6.9: Density of TB(+) mast cells in tongues of control and HF groups. Graph shows differences in density of TB(+) mast cells in tongues of both groups. TB(+) mast cells were significantly less densely distributed in tongues of HF group than in those of controls. Data are expressed as mean ± SD; p < 0.05 versus control (*). Abbreviations: HF, high-fat group; TB(+) cells, Toluidine Blue-positive mast cells.

§ Analysis of TB(+) mast cells in WAT Histological analysis of TB-stained WAT showed that in both control and HF groups, TB(+) mast cells were generally located around blood vessels (control: Fig. 6.10A; HF: Fig. 6.10B), and occasionally between adipose cells (control: Fig. 6.11A; HF: Fig. 6.11B).

TB(+) mast cells were degranulating in both the control group (control: Fig. 6.11A) and HF group (HF: Fig. 6.11B-C). In controls, TB(+) mast cells found to release only a few granules. In the HF group, most TB(+) mast cells were in the process of profound degranulation (HF: Fig. 6.11B). Some TB(+) mast cells were completely degranulated and their granules were observed both close to and at some distance from the host cells (HF: Fig. 6.11C).

Image analysis of TB(+) mast cells in WAT showed that the density (number) of TB(+) mast cells in the WAT of the HF group was 9.62% less than in those of the control group (control: 1.86 ± 0.65 per mm2; HF: 1.68 ± 0.32 per mm2; p=0.90ns; t-test) (Table 6.6).

201

Figure 6.10: Toluidine Blue- (TB-) stained white adipose tissue (WAT) of control and HF groups. Photomicrographs show TB(+) mast cells in WAT of control (A) and HF (B) groups. In both groups, TB(+) mast cells (MCs) (arrows) are located in connective tissue (CT) that surround blood vessels (BVs).

Figure 6.11 (next page): Toluidine Blue- (TB-) stained white adipose tissue (WAT) of control and HF groups. Photomicrographs show TB(+) mast cell in WAT of control (A) and HF (B-C) groups. (A) In control group, TB(+) mast cells (MC) (arrow) are observed between adipose cells (AdCs). Some degranulation has occurred and single granules (arrowhead) are evident. (B) In HF group, TB(+) mast cells (MCs) (arrows) are found between adipose cells (AdCs). Profound degranulation has occurred and some granules (arrowhead) are observable inside adipose cells. (C) In HF group, TB(+) mast cells MCs (arrows) are also observed in connective tissue (CT). Profound degranulation has occurred and one MC (red arrow) has released all of its granules and become almost invisible.

202

203 TB(+) mast cells

tongue liver WAT control 0.571 ± 0.096 (n=3) 0.79 ± 0.25 (n=4) 1.86 ± 0.65 (n=4)

HF 0.494 ± 0.034 (n=3) 0.73 ± 0.21 (n=4) 1.68 ± 0.32 (n=2) p-value 0.02* 0.50ns 0.28ns growth rate -13.44% -7.03% -9.62% Table 6.6: Density of Toluidine Blue- (TB-) stained mast cells in tongue, liver, and white adipose tissue (WAT) of control and HF groups. Short-term HF diet reduced density of TB(+) mast cells in tongue (*), liver (ns), and WAT (ns). Data are expressed as mean ± SD; p < 0.05 versus control (*). Data are presented as density (number) of TB(+) mast cells per total area (mm2). Abbreviations: HF, high-fat diet group; TB(+),Toluidine Blue-positive mast cells; ns, not significant.

§ Analysis of TB(+) mast cells in liver Histological analysis of TB-stained control liver sections showed that TB(+) mast cells were located exclusively around the portal triad, comprised of the portal vein, branches of hepatic artery, and liver bile ducts (control: Fig. 6.12A). Similarly, in the HF group, TB(+) mast cells were also only located around the portal triads (HF: Fig. 6.12B). In both groups, no mast cells were observed in the parenchyma of the liver (control: Fig. 6.12A; HF: Fig. 6.12B). The portal veins of the HF group, unlike those of controls, had irregular circumferences and their vein walls were thicker and consisted of loose connective tissue fibres (HF: Fig. 6.12B).

Mast cells in livers were examined under higher magnification. In the control group, TB(+) mast cells were located in the connective tissue of the portal triad. Granules are observed as deep blue/violet orbs packed inside the cell (control: Fig. 6.13A). Similarly, TB(+) mast cells were located in the connective tissue of the portal triad of the HF group but these cells showed evidence of degranulation. TB(+) mast cells released granules both locally and remotely (HF: Fig. 6.13B). Some profoundly degranulated mast cells had released so many of their granules that they became almost invisible. Where these granules were observed, blood cells were also found in vessels and liver parenchyma (HF: Fig. 6.13C).

204

Figure 6.12: Toluidine Blue-(TB-) stained liver tissue of control and HF groups. Photomicrographs show TB(+) mast cells in liver tissue of control (A) and HF (B) groups. (A) In control group, TB(+) mast cells (MCs) (arrows) are located around portal triad. Portal vein (PV) is observed as round to oval in shape and vein wall (VW), composed of connective tissue (CT) (line), is of high integrity. No mast cells can be seen between hepatocytes. (B) In HF group, TB(+) mast cells (MCs) (arrows) are located around portal triad. Portal vein (PV) is irregular in shape, and vein wall (VW), composed of connective tissue (CT) (line), is thick and less firmly attached to liver parenchyma. No mast cells can be seen among hepatocytes.

205

206 Figure 6.13 (previous page): Toluidine Blue- (TB-) stained liver tissue of control and HF groups. Photomicrographs show TB(+) mast cells in liver tissue of control (A) and HF (B-C) groups. (A) In control group, intact TB(+) mast cells (MCs) (arrows) are observed in connective tissue (CT) of portal vein (PV) wall (VW). (B) In HF group, TB(+) mast cells (MCs) (arrows) are located in connective tissue (CT) of portal vein (PV) wall (VW). MCs are in the process of degranulating where granules (arrowheads) are seen at various distances away. (C) In HF group, profound degranulation of TB(+) mast cells (MCs) has occurred. Two MCs have released most of their granules and are almost invisible. These MCs are in close proximity to blood cells (arrowheads) located in blood vessel (BV) and in liver parenchyma.

§ Density (number) of TB(+) mast cells in liver The density of TB(+) mast cells in liver tissue (number of mast cells per total area), showed that mast cells of the HF group were 7.03% less than that observed in the control group (control: 0.79 ± 0.25 per mm2; HF: 0.73 ± 0.21 per mm2; p=0.74ns; t-test) (Tables 6.6 – 6.7).

§ Area of portal veins in liver The area of portal veins was on average 13.46% greater in the HF group than in the control group (control: 4.90ˣ10-3 ± 1.21ˣ10-3 per µm2; HF: 5.56ˣ10-3 ± 1.63ˣ10-3 per µm2; p=0.32ns; t-test) (Table 6.7).

§ Number of portal veins in liver The number of portal veins was 11.37% less in the HF group than in control group (control: 2.19ˣ10-7 ± 0.35ˣ10-7 per µm2; HF: 1.94ˣ10-7 ± 0.73 ˣ10-7 per µm2; p=0.56ns; t- test) (Table 6.7).

207 liver

mast cell density portal vein area portal vein number control 7.9ˣ10-7 ± 2.5ˣ10-7 4.90ˣ10-3 ± 1.21ˣ10-3 2.19ˣ10-7 ± 0.35ˣ10-7 (n=4) (n=4) (n=4)

HF 7.3ˣ10-7 ± 2.1ˣ10-7 5.56ˣ10-3 ± 1.63ˣ10-3 1.94ˣ10-7 ± 0.73ˣ10-7 (n=4) (n=4) (n=4) p-value 0.50ns 0.19ns 0.56ns growth rate -7.03% +13.46% -11.37% Table 6.7: Relationship between Toluidine Blue- (TB-) stained mast cells and portal vein area and number in livers of control and HF groups. Short-term HF diet reduced density of TB(+) mast cell, increased total area of portal veins, and reduced number of portal veins. Data are expressed as mean ± SD; p=ns. Mast cell density: data are presented per total area of liver (µm2). Portal vein area: data are presented per total area of liver (µm2). Portal vein number: data are presented per total area of liver (µm2). Abbreviations: HF, high-fat diet group; TB(+), Toluidine Blue-positive mast cells; ns, not significant.

§ Correlation between the number of TB(+) mast cells and area of portal veins in liver There was a strong correlation between the number of TB(+) mast cells and area of the portal veins. The number of mast cells significantly increased in proportion to the area of the portal veins. This observation was true for both control and HF groups (Fig. 6.14).

§ Analysis of TB(+) mast cell distribution in OE and OB Histological analysis of TB-stained OE and OB showed only a few TB(+) mast cells and these were located in the lamina propria. In both control and HF groups, these mast cells were distributed around small blood vessels and occasionally between the axon bundles (control: Fig. 6.15A; HF: Fig. 6.15B). In the HF group, TB(+) mast cells had begun to degranulate (HF: Fig. 6.15B).

208

Figure 6.14: Correlation between number of Toluidine Blue- (TB-) stained mast cells and area of portal vein in livers of control and HF groups. In control (blue) and HF (red) groups, the number of TB(+) mast cells increased in proportion to area of the portal vein. In control group, association between number of TB(+) mast cells and area of portal vein is strong (r=0.84) and significant p<0.05. In HF group, association between number of TB(+) mast cells and area of portal vein is strong (r=0.88) and significant p<0.05. Pearson’s correlation coefficient method was applied.

209

Figure 6.15: Toluidine Blue- (TB-) stained olfactory epithelium of control and HF groups. Photomicrographs show TB(+) mast cells in lamina propria of control (A) and HF (B) groups. (A) In control group, intact TB(+) mast cell (MC) (arrow) is located between the axon bundles of OSNs and small blood vessel (BV) in lamina propria. (B) In HF group, MC (arrow) has begun to degranulate and some granules (arrowhead) have been released.

210 6.4.7 Analysis of mOSNs in olfactory epithelium (OE) Light microscopic analysis of OMP-stained OE showed that OMP labelled the cilia, dendrites, bodies of mOSNs, and axon bundles (control: Fig. 6.16A). However, in the HF group, the intensity of OMP expression in many mOSNs was less than in controls. Some mOSNs did not express any OMP (HF: Fig. 6.16B).

Figure 6.16: Expression of OMP in olfactory epithelium of control and HF groups. Photomicrographs show OMP-stained olfactory epithelium (OE) of control (A) and HF (B) groups. In both control and HF groups, expression of olfactory marker protein (OMP) (red) is observed in cilia, dendrites, cell bodies, and axons that cross lamina propria of mature olfactory sensory neurons (mOSNs) (A-B). In HF group, OMP immunoreactivity is dim compared to that in controls, implying reduced OMP expression in mOSNs (B). DAPI stains cell nuclei (blue).

211

Figure 6.17: Intensity of OMP expression in mOSNs of control and HF groups. Graph shows differences in mean intensity of OMP expression in mOSNs of OE of both groups. Intensity of OMP expression was significantly less in mOSNs of HF group than in those of control group. Data are expressed as mean ± SD; p < 0.05 versus control (*). Abbreviations: HF, high-fat group; OE, olfactory epithelium; OMP, olfactory marker protein; mOSNs, mature olfactory sensory neurons.

Image analysis of mOSNs in control and HF groups showed that the mOSNs in the OE of the HF group expressed 23.85% less OMP than those of controls (control: 50.63 ± 13.60, (47.18, [39.18; 62.27]); HF: 38.55 ± 2.82, (37.41, [36.78; 39.35]); p=0.006*; Mann-Whitney U test) (Fig. 6.17). This analysis also showed that there was less OMP expression in mOSNs in the OE of all individual animals from the HF group than there was in all individual rats from the control group (Table 6.8). The number of nuclei per area in the OE was equivalent in both control and HF groups (control: 229.19 ± 90.03 per mm2, (215.50, [191.25; 258.50]); HF: 227.75 ± 107.68 per mm2, (209.50, [157.75; 239.50]); p=0.39ns; Mann-Whitney U test).

6.4.8 Analysis of juxtaglomerular (JG) cells in olfactory bulb (OB)

6.4.8.1 CB(+) JG cells in OB Histological analysis of OBs showed that calbindin D-28k (CB) was expressed in cells of the glomerular layer (GL) in control and HF groups (control: Fig. 6.18A; HF: Fig. 6.18B). In both groups, no CB expression was observed in the olfactory nerve layer (ONL) or in the external plexiform layer (EPL) of the OB (control: Fig. 6.18A; HF: Fig.

212 HF control p-value growth rate

R 17 (37.02±0.53) vs. R 21 (53.96±15.39) 0.049* -31.40%

R 22 (45.0±8.36) 0.042* -17.74%

R 24 (47.38±12.97) 0.322ns -21.87%

R 21,22,24 (50.63±13.60) 0.061ns -26.89%

R 18 (38.63±2.51) vs. R 21 (53.96±15.39) 0.015* -28.41%

R 22 (45.0±8.36) 0.044* -14.16%

R 24 (47.38±12.97) 0.285ns -18.47%

R 21,22,24 (50.63±13.60) 0.019* -23.71%

R 19 (39.41±4.61) vs. R 21 (53.96±15.39) 0.083ns -26.96%

R 22 (45.0±8.36) 0.138ns -12.42%

R 24 (47.38±12.97) 0.240ns -16.82%

R 21,22,24 (50.63±13.60) 0.090ns -22.17%

R17,18,19 (38.55±2.82) vs. R 21 (53.96±15.39) 0.006* -28.54%

R 22 (45.0±8.36) 0.030* -14.32%

R 24 (47.38±12.97) 0.233ns -18.63%

R 21,22,24 (50.63±13.60) 0.006* -23.85% Table 6.8: Mean intensity of OMP expression in mOSNs in individual rats of control and HF groups. All rats from HF group expressed less OMP in mOSNs of their OE than rats from control group. Data are expressed as mean ± SD; p < 0.05 versus control (*). Abbreviations: HF, high-fat diet group; OE, olfactory epithelium; OMP, olfactory marker protein; mOSNs, mature olfactory sensory neurons; ns, not significant.

6.18B). In negative control slides, no CB expression was observed in any cells in the GL of the OB (Fig. 6.18C).

Glomeruli were further examined under higher magnification. In control and HF groups, CB(+) JG cells were found in each glomerulus. CB strongly expressed in the body of JG cells but with variable intensity, labelled “strong”, “medium” and “weak” (control: Fig. 6.19A; HF: Fig. 6.19B).

213

Figure 6.18: Calbindin- (CB-) stained olfactory bulbs (OBs) of control and HF groups. Photomicrographs show CB-positive (CB(+)) juxtaglomerular (JG) cells in glomerular layer (GL) of OBs of control (A) and HF (B) groups and CB-negative control (C). In both groups, CB(+) JG cells (brown) (arrows) are seen in GL (double-headed arrow) of OBs (A-B). CB(+) JG cells are dispersed between unstained cells of each glomerulus (A-B). No CB(+) cells are seen in olfactory nerve layer (ONL) or in external plexiform layer (EPL) of OB (A-B). In negative control slide (CB(-)), no CB expression is seen (C).

214

Figure 6.19: Calbindin- (CB-) stained olfactory bulbs (OBs) of control and HF groups. Photomicrographs show CB-positive (CB(+)) juxtaglomerular (JG) cells in glomerular layer (GL) of OBs of control (A) and HF (B) groups. In both groups, CB(+) JG cells (arrows) show different intensities of CB expression in cell bodies: “strong” (dark brown, black arrow), “medium” (brown, magenta arrow) and “weak” (light brown, green arrow) (A-B). CB(+) JG cells are dispersed between unstained cells of each glomerulus (G) (A- B).

Image analysis showed that, in the HF group, density of CB(+) JG cells in the GL was 12.83% less than in the control group (control: 1.06 ±0.27 per μm2, (1.08, [0.91; 1.18]); HF: 0.92 ± 0.31 per μm2, (0.86, [0.71; 1.05]); p=0.0007*; Mann-Whitney U test) (Fig. 6.20).

215

Figure 6.20: Density of CB(+) JG cells in OB of control and HF groups. Graph shows differences in density of CB(+) JG cells in GL (μm2) of OB. Density of CB(+) JG cells was significantly reduced in HF group compared to controls. Data are expressed as mean ± SD; p < 0.05 versus control (*). Abbreviations: HF, high-fat group; CB(+), calbindin-positive; JG cells, juxtaglomerular cells; OB, olfactory bulb.

6.4.8.2 TH(+) JG cells in OB Histological analysis of OBs showed that tyrosine hydroxylase (TH) was expressed in cells of the GL in control and HF groups (control: Fig. 6.21A; HF: Fig. 6.21B). In both groups, weak TH expression was observed in olfactory nerve layer (ONL) in close proximity to glomeruli (HF: Fig. 6.22B). Some cells in the external plexiform layer (EPL) and olfactory nerve layer (ONL) of the OB expressed TH. A dot-like pattern of TH expression was observed within the neuropil of each glomerulus (control: Fig. 6.21A; HF: Fig. 6.21B). No TH expression was observed in any cells of the glomeruli of the OB in the negative control slides (Fig. 6.21C).

Glomeruli were examined under higher magnification. In control and HF groups, TH(+) JG cells were found in each glomerulus. TH strongly expressed in the body of JG cells but with variable intensity, labelled “strong”, “medium” and “weak” (control: Fig. 6.22A; HF: Fig. 6.22B).

Image analysis showed that in the HF group, density of TH(+) JG cells in the GL was 21.9% less than in the control group (control: 0.59 ± 0.77, (0.27, [0.12; 0.66]); HF: 0.46 ± 0.62, (0.22, [0.08; 0.60]); p=0.386ns; Mann-Whitney U test) (Fig. 6.23).

216

Figure 6.21: Tyrosine hydroxylase- (TH-) stained olfactory bulbs (OBs) of control and HF groups. Photomicrographs show TH-positive (TH(+)) juxtaglomerular (JG) cells in glomerular layer (GL) of OBs of control (A) and HF (B) groups and TH-negative control (C). In both groups, TH(+) JG cells (brown) (arrows) are seen in GL (double-headed arrow) of OBs (A-B). TH (+) JG cells are dispersed between unstained cells of each glomerulus (G) (A-B). Some TH(+) cells are visible in the external plexiform layer (EPL) of the OB (A-B) and in the olfactory nerve layer (ONL) (B). In negative control slide (TH(- )), no TH expression is seen (C). 217

Figure 6.22: Tyrosine hydroxylase- (TH-) stained olfactory bulbs (OBs) of control and HF groups. Photomicrographs show TH-positive (TH(+)) juxtaglomerular (JG) cells in glomerular layer (GL) of OBs of control (A) and HF (B) groups. In both groups, TH(+) JG cells (arrows) show different intensities of TH expression in cell bodies: “strong” (dark brown, black arrow), “medium” (brown, magenta arrow), and cells with only cytoplasmic TH expression “weak” (green arrow) (A-B). TH(+) JG cells are dispersed between unstained cells of each glomerulus (G) (A-B).

218

Figure 6.23: Density of TH(+) JG cells in OB of control and HF groups. Graph shows differences in density of TH(+) JG cells in GL (μm2) of OB. TH(+) JG cells were less densely distributed in GL of HF group than in controls. Data are expressed as mean ± SD; p=ns. Abbreviations: HF, high-fat group; OB, olfactory bulb; TH(+), tyrosine hydroxylase positive cells; JG cells, juxtaglomerular cells; HF, high-fat group; ns, not significant.

6.5 Discussion The present study showed that overconsumption of fat, even in the short term, caused excessive weight gain, changes to blood chemistry, and structural abnormalities in tongue, liver, and WAT, consistent with previous studies (Winzell and Ahrén, 2004; Lee et al., 2011b). In the nervous system, a HF diet caused a reduction of OMP expression in mOSNs in the OE and reduction of JG cells in the GL of the OB. It also reduced mast cell numbers and led to mast cell degranulation in tongue, liver, WAT and in the olfactory system. The reduced granule density of mast cells suggest already engaged inflammatory processes or perhaps weakened inflammatory potential.

Three weeks into the HF diet, subjects showed modest increases in body weight and significant increases in WAT weight and percentage. This outcome is consistent with reports of mice fed a HF diet for three days showing increased body weight and WAT weight and insulin resistance (Lee et al., 2011b). The current study also found that the size of adipose cells was larger in the HF group than in controls and that these cells expanded due to enhanced TG storage.

219 LD accumulations in the liver were analysed because, along with WAT, the liver has a considerable capacity for storage of lipids (Walther and Farese Jr, 2012) and because a HF diet delays uptake and/or slows turnover of dietary fat in the liver (Janssens et al., 2015). The results presented here show rats on a short-term HF diet had hepatic steatosis, steatohepatitis, and steatofibrosis, and had high levels of lipid content in the liver as well as glucose intolerance and systemic insulin resistance.

It is known that when WAT generates excessive fatty acids, the liver produces more glucose in an unregulated fashion, involving β-insulocytes. This situation can evolve into type 2 diabetes, which may require high doses of exogenous insulin (Saltiel, 2000; van der Heijden et al., 2015). Hyperinsulinemia also causes elevated levels of TG, lower levels of HDL, increased vascular resistance, changes in steroid hormone levels, attenuation of peripheral blood flow, and weight gain (Saltiel, 2000).

It is not clear what types of inflammatory cells are involved and what role they have in inflammation that promotes metabolic dysregulation. A number of studies have demonstrated increased accumulation of WAT macrophages and lymphocytes in obese humans and rodents (Weisberg et al., 2003; Xu et al., 2003). WAT from obese subjects contained more macrophages than those from lean donors. These inflammatory macrophages furnish cytokines, growth factors, chemokines, and proteases in WAT and it has been suggested that macrophage-induced pro-inflammatory signalling is a key mediator of obesity-induced tissue inflammation (Fantuzzi, 2005; Wu et al., 2007). Lee et al. (2011b) showed a significant elevation of inflammatory gene expression in WAT following three days of a HF diet. It has been shown that T cells accumulate in WAT and may be involved in inflammation and obesity (Wu et al., 2007; Rausch et al., 2008). T lymphocytes interacted with macrophages to regulate the inflammatory cascade. There is the potential for mast cells to contribute to obesity-induced WAT inflammation because mast cells are potent inducers of inflammation through degranulation and release of their immunoreactive mediators (Liu et al., 2009; Zhang and Shi, 2012; Chmelař et al., 2016). One study which stained mast cells with tryptase monoclonal antibodies found that WAT from obese humans and mice contained more mast cells than lean counterparts (Liu et al., 2009). Adoptive transfer experiments of cytokine-deficient mast cells demonstrated that mast cells contributed to diet-induced obesity by producing the cytokines IL-6 and INF-γ (Zhang and Shi, 2012).

Insulin resistance may be caused by other pro-inflammatory mediators, such as inducible nitric oxide synthases (iNOS), monocyte chemoattractant protein (MCP)-1, and IL-1 (Amrani et al., 1996; Fried et al., 1998; Hrnciar et al., 1999). Further, kidneys of diabetic patients with nephropathy were found to have increased density of mast cells

220 (Rüger et al., 1996; Okon and Stachura, 2007). In cases of renal injury, mast cell infiltration is frequent (Jones et al., 2003). The mast cells degranulation and release of mediators, such as TGF-β, chymase, tryptase, cathepsin G, and rennin may contribute to renal complications in diabetes (Sharma and Ziyadeh, 1993; Kondo et al., 2001; Lindstedt et al., 2001).

The current study analysed TB-stained mast cells in the tongue, liver, WAT, and the OE of the olfactory system in control and HF groups. These results show mast cell degranulation. Granules generally remained clustered around individual mast cells. Many mast cells retained only a few granules rendering these cells almost invisible. The HF group had reduced mast cell density (numbers) in the tongue, liver, and WAT, which may be due to mast cell degranulation in these tissues.

The fact that significantly less mast cells were observed in the tongue suggests that they may have degranulated and released mediators that may affect taste perception. As discussed in Chapter 1, the tongue plays a key role in taste perception. Taste buds, which are located predominantly on the tongue and soft palate, initiate gustatory signalling. Taste receptors (TRs), together with ORs and somatosensory receptors, can recognise the chemicals that comprise food. TRs relay sensory signals to the brain, which processes the stimuli and interprets them as “flavour” (Rolls, 2005; Veldhuizen et al., 2009; Small, 2012). The processing of taste information is essential for mediating food preference and, as a result, weight maintenance (Dotson et al., 2012). Taste function can be modulated by endocrine and paracrine mechanisms. Hormones, such as leptin, oxytocin, insulin and neuropeptides, such as vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), bind to TRs on taste chemoreceptor cells (TRCs) and affect taste, which in turn influences food consumption (Loper et al., 2015). For example, leptin reduces taste sensitivity to sweetness (Kawai et al., 2000; Di Marzo et al., 2001; Shigemura et al., 2004; Nakamura et al., 2008). NPY may modulate the bitter taste (Herness and Zhao, 2009). VIP modulates the sweet, bitter and sour tastes (Martin et al., 2010). Mast cell granules contain many biologically-active mediators, including NPY and VIP, as discussed in Section 1.4.2, substances which were found to modulate taste sensitivity. It is possible that some crosstalk between mast cells and TRCs are takes place and mast cells in the tongue may represent the first stage in modulating gustatory sensory perception.

Mast cell granules also contain acidic mucopolysaccharides and glycosaminoglycans, such as histamine and heparin (Metcalfe et al., 1997; Moon et al., 2014). Histamine and heparin are known to induce vasodilation, vascular permeability, and angiogenesis. Further, there are significant changes in the brain histamine system

221 in diseases such as multiple sclerosis, Alzheimer’s disease, and Down syndrome (Onodera et al., 1994; Langlais et al., 2002; Haas and Panula, 2003). In these cases, there were clear changes in the number or morphology of histaminergic neurons. It was suggested that histamine may participate in these processes by contributing to changes in vascular function, blood-brain barrier (BBB) permeability, and/or immune activity. Although histamine is not stored in neurons outside of the CNS, mast cell derived histamine can modify peripheral sensory nerve function (Siegel et al., 2006). In line with these findings, the present study showed that a HF diet promoted inflammation, angiogenesis, and caused vascular permeability, such as erythrocyte clustering in blood vessels and erythrocyte infiltration in the parenchyma of liver and WAT. Additionally, this study revealed that the total area of the portal vein increased in livers of the HF group, indicating induced vasodilation in the liver.

A relationship between the number of TB(+) mast cells and area of the portal veins in the liver of control and HF groups of Sprague-Dawley male rats was documented. The results showed that mast cells were located primarily around the portal triad in both groups. A strong correlation between the number of TB(+) mast cells and area of the portal veins in the control group was observed. The number of mast cells significantly increased in proportion to the area of the portal veins. These results are consistent with those of Chan et al. (2001) who found a strong correlation between mast cell numbers and the area of portal veins in the liver in two strains of female rats— Australian albino Wistar and Brown Norway rats—using Alcain blue/safranin staining to detect mast cells. This study expanded existing knowledge on the correlation between mast cells and area portal veins by examining livers of rats fed a HF diet. A significant correlation was found between the number of TB(+) mast cells and area of the portal veins in the HF group. The number of mast cells increased in proportion to the area of the portal veins.

Energy balance and olfaction are intimately linked (Palouzier-Paulignan et al., 2012). The sense of smell is fundamental to eating behaviours including food detection and consumption. The addition of certain odorants to a diet profoundly influences the amount of food rats consume (Le Magnen, 1956, 2001), and loss of smell in bulbectomised rats leads to long-term compulsive overeating (hyperphagia) (Seguy and Perret, 2005; Palouzier-Paulignan et al., 2012). Conversely, nutritional status regulates olfaction (Chaput and Holley, 1976; Riera and Dillin, 2016). For example, fasted rats have a better sense of smell than satiated ones (Albrecht et al., 2009; Tong et al., 2011; Cameron et al., 2012). The relationship between olfaction and food intake is mediated by peptide hormones transported by blood. For example, ghrelin, secreted from the

222 stomach, signals hunger, whereas insulin from the pancreas and leptin from the adipose tissue mainly signal satiety. These peptides prompt the food-intake regulatory centres of the brain to produce orexigenic peptides (e.g., orexin) or anorexigenic peptides (leptin and insulin) to initiate or to stop food intake (Schwartz et al., 2000; Plum et al., 2005; Stanley et al., 2005; Arora, 2006). Intracerebroventricular injection of orexin or leptin has been shown to increase or decrease olfactory sensitivity, respectively (Julliard et al., 2007).

Receptors for metabolic hormones and peptides, such as insulin, NPY, ghrelin, leptin, orexins, and cholecystokinin are expressed in the OE and OB (Caillol et al., 2003; Baly et al., 2007; Lacroix et al., 2008). Because metabolic receptors are located in the areas related to an olfactory signal transduction pathway, it is possible that communication takes place between these receptors and the olfactory system. Orexin, insulin, and leptin receptors are found in OSNs and other cells in the OE (Getchell et al., 2006; Baly et al., 2007; Gorojankina et al., 2007; Lacroix et al., 2008). Elevated levels of insulin and leptin decreased olfactory sensitivity of the OE by altering the spontaneous and odorant induced activity of OSNs in rats (Savigner et al., 2009). Metabolic disorders modify the expression of olfactory signalling components, including ORs, Golf, and AC3 proteins (Jeffery and Reid, 1975; Joo et al., 2010).

It has been suggested that the OB serves as a sensor of internal chemistry as modified by metabolic activity (Tucker et al., 2013). The OB contains concentrations of insulin that are as high as those in the hypothalamus, and is, therefore, a major site of insulin activity in the brain (Baskin et al., 1983; Hill et al., 1986). The OB is also the site with the highest transport rate of insulin across the BBB (Banks et al., 1999). Mice with olfactory bulbectomy showed increased sensitivity to peripherally induced insulin, lower body weights and reduced blood sugar levels (Perassi et al., 1975). These results show that the OB may be affected by metabolic status (Riera and Dillin, 2016). However, more data are required to confirm this association. Surgical removal of the OB causes variations in ablation and healing, which can impact body weight and appetite. These results in behaviours can be misinterpreted (Primeaux et al., 2007; Oral et al., 2013; Riera and Dillin, 2016). Thus, non-surgical, non-invasive methods, such as performed in the current study, are preferable to investigate how metabolic status affects the OB.

The current study showed that overconsumption of fat, even for a short period, affected the OE and OB. It revealed that the intensity of OMP expression was significantly reduced in mOSNs in the OE of the HF group. Intensity of OMP expression was also significantly reduced in each individual rat of this group. This circumstance suggests that fat has a harmful effect on mOSNs and that it can modify expression of

223 ORs. Equal numbers of cell nuclei were observed in both groups. These combined findings are consistent with those of Thiebaud et al. (2014) who reported that OE thickness was not modified by a six-month HF diet and suggested that this diet was not of sufficient duration to affect OE thickness in mice (Thiebaud et al., 2014). The authors also observed, however, a marked loss of OMP-expressing mOSNs with their axonal projections in animals with long-term HF diets, suggesting that the loss of OSNs and associated circuitry was linked to changes in proliferation of neuronal cells and their apoptotic cycles. They suggested that a long-term HF diet may affect the kinetics of regeneration in the OE by increasing the apoptosis of the neuronal population (Thiebaud et al., 2014).

Studies have examined mitral cells of the OB with respect to nutritional status (Fadool et al., 2000; Hardy et al., 2005; Fadool et al., 2011; Tucker et al., 2013). For example, chronic exposure to dietary fat impaired the action potential firing frequency and interspike intervals and reduced EOG amplitude in the mitral cells of the OB (Fadool et al., 2011). However, little research has been conducted on other neuronal cells of the OB. A six-month HF diet in mice reduced the density of OSNs expressing M72 and depleted their axonal projections to the M72 glomerulus. Additionally, it reduced the size of the M72 lateral glomerulus, causing changes to associated circuitry but the number of glomeruli remained unchanged. HF-challenged mice showed significantly less OE response to odour ligand M72, acetophenone, as compared to controls using EOG recordings. This reduction in EOG amplitude was proportional to the number of activated OSNs and may be linked to the loss of OSNs in the OE or to the multiple changes that fat caused in different levels of the olfactory system. A HF diet modulated olfactory-based behaviour in mice. For example, mice fed a HF diet exhibited reduced olfactory learning and olfactory discrimination as well as behavioural and cognitive inflexibility (Thiebaud et al., 2014).

The current study expanded on previous research by investigating CB and TH expression in the neurons of the GL of the OB. The results showed that a short-term HF diet affected the CB(+) JG cells. There was a significant reduction in the mean density of CB(+) JG cells in the GL of the OB. CB(+) JG cells showed variable CB expression— strong, medium, and weak. This finding is consistent with those by Philpot et al. (1997), who suggested that the variation in intensity of staining may indicate divergences in cell functions. The current study also showed that a short-term HF diet affected the TH(+) JG cells of the OB. It found that there was a reduction in the mean density of TH(+) JG cells. TH(+) JG cells showed variable TH expression—strong, medium, and weak expression—similar to that of CB(+) cells.

224 The current study demonstrated that a short-term HF diet caused a reduction of OMP expression in mOSNs in the OE and reduced the density of CB(+) and TH(+) JG cells in the GL of the OB. Overconsumption of fat, even for a short period, affected the neuroarchitecture of the olfactory system and altered chemoreception.

A HF diet had a profound effect on mast cells in the OE. TB(+) mast cells were observed only in the lamina propria of the OE in both control and HF groups. Mast cells in the HF group showed evidence of degranulation. The finding, that pro-inflammatory cells were present in the OE is consistent with observations by Thiebaud et al. (2014). These authors found that a long-term HF diet activated microglia and increased the number of pro-inflammatory macrophages in the OE of mice (Thiebaud et al., 2014). The current study’s finding that mast cells degranulated in the OE of the HF group suggests that mast cells might contribute to inflammation and sensory dysregulation in the OE that would affect eating and smell.

6.6 Conclusion In summary, a short-term, HF diet produced adverse effects on blood, tongue, liver, WAT, mast cells, and the olfactory system. It caused a reduction of OMP expression in mOSNs in the OE and reduced the density of CB(+) and TH(+) JG cells in the GL of the OB. It also caused degranulation of mast cells in the lamina propria of the OE, similar to degranulation of mast cells in the tongue, liver, and WAT. This degranulation suggests that mast cells also may evoke local inflammation in the OE and contribute to HF diet-induced inflammation and metabolic dysregulation. The extent to which mast cells contribute to this dysregulation requires further investigation. The results also demonstrate that a HF diet changes the neuroarchitecture of the olfactory system, thus affecting chemoreception. These changes may affect energy balance and body weight by altering metabolism and feeding-related behaviours. Disruptions in chemoreception can change olfactory identification, odorant detection threshold, discrimination, and hedonic value of food and may have lasting effects on health. Any future studies on metabolism and diet should include a study of olfaction.

225 Conclusions and Future Directions Olfactory receptors (ORs), olfactory marker protein (OMP), olfactory specific G protein (Golf), and adenylyl cyclase III (AC3) all serve roles in the olfactory system as mediators of the sense of smell (chemoreception) (Buck and Axel, 1991). These molecules have also been identified in some non-olfactory tissues (Kang et al., 2015).

My thesis confirmed the distribution of OMP, Golf, and AC3 in the olfactory system so that I could explore chemoreception in non-olfactory cells. I paid special attention to mobile cells that might use chemoreception as guidance cues to navigate around the body.

The results showed that OMP, Golf, and AC3 are co-expressed in certain cells of non-olfactory systems (reproductive and immune), including spermatozoa, mast cells (of tongue, liver, and white adipose tissue), and Leydig cells. They also showed that OMP is expressed in these cell types in three mammalian species: humans, rats, and mice. These results demonstrate similarities in OMP expression in different mammals, with some species-specificity. These observations demonstrated that OMP is expressed in specific cells that share the functions of chemoreception and mobility. OMP was co- expressed with an OR in control, activated, and hyper-activated human spermatozoa. The functional state of the spermatozoa affected co-expression locations and it was observed that OMP had a strong affiliation with ORs. Thus, OMP expression is a reliable indicator of OR-mediated chemoreception in non-olfactory tissues.

OR-mediated chemoreception occurs in mammals in cells of non-olfactory tissues that share common features. However, some species-specificity in expression of ORs is evident. Overconsumption of fat disrupted the immune system and affected the neuroarchitecture of the olfactory system, which, in turn, affected chemoreception. Disruptions in chemoreception can change olfactory identification, odorant detection threshold, discrimination, and hedonic value of food and may have lasting effects on health. It also caused degranulation of mast cells in tongue, liver, white adipose tissue and olfactory epithelium that may evoke local inflammation and contribute to HF diet- induced inflammation and metabolic dysregulation.

It will be important to characterise the functional roles of ectopic ORs to determine the specific ligand(s) that activate the olfactory signalling pathway in non-olfactory systems. It will also be useful to investigate OMP expression in mast cells and spermatozoa in subjects with obesity, given that OMP intensity is affected by fat.

G protein-coupled receptors (GPCRs) constitute a large and diverse that mediates the physiological responses to light, hormones, neurotransmitters, chemotactic peptides and growth factors. Of these, ORs are the largest and most heterogeneous group. Although all members of the OR group serve a chemical detection

226 function, the structural similarity between members is not conserved. Moreover, mammalian ORs may serve additional functions such as axon guidance in the olfactory system, chemotaxis of spermatozoa, and migration of mast cells and embryonic Leydig cells. It remains to be determined what other functions might be served by ORs in different systems. This functional and structural diversity is not surprising when one considers that there are no essential differences between chemical binding of neurotransmitters, hormones, or odorants. Whether they are expressed at spermatozoa, mast cells or in sensory cells, chemoreceptors serve the function of chemical detection of a ligand in surrounding environment. This wide range of chemoreception demonstrates nature’s economy of function for biological systems.

Close examination of the OR gene family across species suggests multiple evolutionary time points at which new OR sequences appear. ORs in nematodes show little similarity to ORs in flies, which in turn show little similarity to vertebrate ORs. It is thought that the OR multigene family arose from an ancestral gene or gene cluster by a series of duplications followed by adaptive and neutral mutations (Lancet and Ben-Arie, 1993). Comparisons of OR gene sequences in informative species showed that fish-like OR repertoires change little between species. In contrast, mammalian-like OR repertoires dramatically expand in diversity and size during evolution (Freitag et al., 1998). This situation suggests that these two broad subgroups of OR genes might be subjected to different evolutionary processes, possibly owing to differences in ecological niches to adjust to new environmental requirements (Dryer, 2000). For any new environmental challenge, the organism must respond with adequate phenotypic plasticity that adapts to new conditions with appropriate acquired capabilities (Fordyce, 2006). ORs may thus be adaptable to new and unpredictable chemical combinations in its environment. The dual roles of ORs and their diverse forms across species is a crucial evolutionary feature that adapts to selection pressure and maximises survival.

227 References Abaffy T, Malhotra A, Luetje CW (2007) The molecular basis for ligand specificity in a mouse olfactory receptor a network of functionally important residues. Journal of Biological Chemistry 282:1216-1224. Abe K, Takano H (1989) Early degeneration of the epithelial cells in the initial segment of the epididymal duct in mice after efferent duct cutting. Archives of histology and cytology 52:299-310. Abou-Haïla A, Fain-Maurel MA (1984) Regional differences of the proximal part of mouse epididymis: morphological and histochemical characterization. The Anatomical Record 209:197-208. Abraham-Peskir JV, Chantler E, Uggerhøj E, Fedder J (2002) Response of midpiece vesicles on human sperm to osmotic stress. Human Reproduction 17:375-382. Åbrink M, Grujic M, Pejler G (2004) Serglycin is essential for maturation of mast cell secretory granule. Journal of Biological Chemistry 279:40897-40905. Abu-Abid S, Szold A, Klausner J (2002) Obesity and cancer. Journal of medicine 33:73-86. Ache BW, Young JM (2005) Olfaction: diverse species, conserved principles. Neuron 48:417- 430. Adamali HI, Hermo L (1996) Apical and narrow cells are distinct cell types differing in their structure, distribution, and functions in the adult rat epididymis. Journal of andrology 17:208-222. Adipietro KA, Mainland JD, Matsunami H (2012) Functional evolution of mammalian odorant receptors. PLoS genetics 8:e1002821. Agarwal S, Choudhury M, Banerjee A (1987) Mast cells and idiopathic male infertility. International journal of fertility 32:283-286. Agasandyan KV (1990) Microvillar cells in swine olfactory epithelium. Journal of evolutionary biochemistry and physiology (USA). Ahmed MH, Sabry SM, Zaki SM, El-Sadik AO (2009) Histological, immunohistochemical and ultrastructural study of the epididymis in the adult albino rat. Australian J Basic Appl Sci 3:2278-2289. Aitken RJ, Koopman P, Lewis SE (2004) Seeds of concern. Nature 432:48-52. Akaishi S, Ogawa R, Hyakusoku H (2008) Keloid and hypertrophic scar: neurogenic inflammation hypotheses. Medical hypotheses 71:32-38. Akiyama T, Tachibana I, Shirohara H, Watanabe N, Otsuki M (1996) High-fat hypercaloric diet induces obesity, glucose intolerance and hyperlipidemia in normal adult male Wistar rat. Diabetes research and clinical practice 31:27-35. Albrecht J, Schreder T, Kieemann A, Schopf V, Kopietz R, Anzinger A, Demmel M, Linn J, Kettenmann B, Wiesmann M (2009) Olfactory detection thresholds and pleasantness of a food-related and a non-food odour in hunger and satiety. Rhinology 47:160. Albrecht M, Frungieri MB, Gonzalez-Calvar S, Meineke V, Köhn FM, Mayerhofer A (2005) Evidence for a histaminergic system in the human testis. Fertility and sterility 83:1060- 1063. Aloe L, Levi-Montalcini R (1977) Mast cells increase in tissues of neonatal rats injected with the nerve growth factor. Brain research 133:358-366. Altini M, Coleman H, Doglioni C, Favia G, Maiorano E (2000) Calretinin expression in ameloblastomas. Histopathology 37:27-32. Altman J (1969) Autoradiographic and histological studies of postnatal neurogenesis. III. Dating the time of production and onset of differentiation of cerebellar microneurons in rats. Journal of Comparative Neurology 136:269-293. Alvarez-Buylla A, García-Verdugo JM, Tramontin AD (2001) A unified hypothesis on the lineage of neural stem cells. Nature Reviews Neuroscience 2:287. Amann R, Seidel G, Mortimer R (2000) Fertilizing potential in vitro of semen from young beef bulls containing a high or low percentage of sperm with a proximal droplet. Theriogenology 54:1499-1515. Amann RP (1989) Structure and function of the normal testis and epididymis. International Journal of Toxicology 8:457-471. Amrani A, Jafarian-Tehrani M, Mormede P, Durant S, Pleau J, Haour F, Dardenne M, Homo- Delarche F (1996) Interleukin-1 effect on glycemia in the non-obese diabetic mouse at the pre-diabetic stage. Journal of endocrinology 148:139-148.

228 Angelova P, Davidoff M (1989) Immunocytochemical demonstration of substance P in hamster Leydig cells during ontogenesis. Zeitschrift fur mikroskopisch-anatomische Forschung 103:560-566. Angelova P, Davidoff M, Kanchev L (1991) Substance P-induced inhibition of Leydig cell steroidogenesis in primary culture. Andrologia 23:325-327. Anton F, Morales C, Aguilar R, Bellido C, Aguilar E, Gaytan F (1998) A comparative study of mast cells and eosinophil leukocytes in the mammalian testis. Journal of Veterinary Medicine Series A 45:209-218. Apa D, Cayan S, Polat A, Akbay E (2002) Mast cells and fibrosis on testicular biopsies in male infertility. Archives of andrology 48:337-344. Arai R, Winsky L, Arai M, Jacobowitz DM (1991) Immunohistochemical localization of calretinin in the rat hindbrain. Journal of Comparative Neurology 310:21-44. Araneda RC, Kini AD, Firestein S (2000) The molecular receptive range of an odorant receptor. Nature neuroscience 3:1248-1255. Armon L, Eisenbach M (2011) Behavioral mechanism during human sperm chemotaxis: involvement of hyperactivation. PloS one 6:e28359. Armon L, Ben-Ami I, Ron-El R, Eisenbach M (2014) Human oocyte-derived sperm chemoattractant is a hydrophobic molecule associated with a carrier protein. Fertility and sterility 102:885-890. Arora S (2006) Role of neuropeptides in appetite regulation and obesity–a review. Neuropeptides 40:375-401. Arrang J-M, Garbarg M, Schwartz J-C (1983) Auto-inhibition of brain histamine release mediated by a novel class (H3) of histamine receptor. Nature 302:832-837. Arya M, Vanha-Perttula T (1986) Comparison of lectin-staining pattern in testis and epididymis of gerbil, guinea pig, mouse, and nutria. American journal of anatomy 175:449-469. Asai H, Kasai H, Matsuda Y, Yamazaki N, Nagawa F, Sakano H, Tsuboi A (1996) Genomic structure and transcription of a murine odorant receptor gene: differential initiation of transcription in the olfactory and testicular cells. Biochemical and biophysical research communications 221:240-247. Asarian L, Yousefzadeh E, Silverman A-J, Silver R (2002) Stimuli from conspecifics influence brain mast cell population in male rats. Hormones and behavior 42:1. Ashmole I, Bradding P (2013) Ion channels regulating mast cell biology. Clinical & Experimental Allergy 43:491-502. Astic L, Pellier-Monnin V, Saucier D, Charrier C, Mehlen P (2002) Expression of netrin-1 and netrin-1 receptor, DCC, in the rat olfactory nerve pathway during development and axonal regeneration. Neuroscience 109:643-656. Au E, Roskams AJ (2003) Olfactory ensheathing cells of the lamina propria in vivo and in vitro. Glia 41:224-236. Augusto D, Leteurtre E, De La Taille A, Gosselin B, Leroy X (2002) Calretinin: a valuable marker of normal and neoplastic Leydig cells of the testis. Applied Immunohistochemistry & Molecular Morphology 10:159-162. Aumüller G, Wilhelm B, Seitz J (1999) Apocrine secretion—fact or artifact? Annals of Anatomy- Anatomischer Anzeiger 181:437-446. Aumüller G, Renneberg H, Schiemann P-J, Wilhelm B, Seitz J, Konrad L, Wennemuth G (1997) The role of apocrine released proteins in the post-testicular regulation of human sperm function. In: The Fate of the Male Germ Cell, pp 193-219: Springer. Aungst J, Heyward P, Puche A, Karnup S, Hayar A, Szabo G, Shipley M (2003) Centre–surround inhibition among olfactory bulb glomeruli. Nature 426:623. Babcock DF (2003) Smelling the roses? Science 299:1993-1994. Bakalska M, Atanassova N, Angelova P, Koeva I, Nikolov B, Davidoff M (2001) Degeneration and restoration of spermatogenesis in relation to the changes in Leydig cell population following ethane dimethanesulfonate treatment in adult rats. Endocrine regulations 35:209-216. Bakalyar HA, Reed RR (1990) Identification of a specialized adenylyl cyclase that may mediate odorant detection. Science 250:1403-1406. Baker H, Grillo M, Margolis FL (1989) Biochemical and immunocytochemical charaterization of olfactory marker protein in the rodent central nervous system. Journal of Comparative Neurology 285:246-261. Baker H, Kawano T, Margolis F, Joh T (1983) Transneuronal regulation of tyrosine hydroxylase expression in olfactory bulb of mouse and rat. Journal of Neuroscience 3:69-78.

229 Balakumar P, Singh AP, Ganti SS, Krishan P, Ramasamy S, Singh M (2008) Resident cardiac mast cells: are they the major culprit in the pathogenesis of cardiac hypertrophy? Basic & clinical pharmacology & toxicology 102:5-9. Baldi E, Luconi M, Muratori M, Marchiani S, Tamburrino L, Forti G (2009) Nongenomic activation of spermatozoa by steroid hormones: facts and fictions. Molecular and cellular endocrinology 308:39-46. Baldisseri DM, Margolis JW, Weber DJ, Koo JH, Margolis FL (2002) Olfactory marker protein (OMP) exhibits a β-clam fold in solution: implications for target peptide interaction and olfactory signal transduction. Journal of molecular biology 319:823-837. Baltanás F, Weruaga E, Airado C, Valero J, Recio J, Díaz D, Alonso J (2007) Chemical heterogeneity of the periglomerular neurons in the olfactory bulb. A review. European Journal of Anatomy 11:123. Baly C, Aioun J, Badonnel K, Lacroix M-C, Durieux D, Schlegel C, Salesse R, Caillol M (2007) Leptin and its receptors are present in the rat olfactory mucosa and modulated by the nutritional status. Brain research 1129:130-141. Banek L, Hittmair A, Pezerović-Panijan R, Goluža T, Schulze W (1999) Mast cells in testicular biopsies of infertile men with ‘mixed atrophy’of seminiferous tubules. Andrologia 31:203- 210. Banks WA, Kastin AJ, Pan W (1999) Uptake and degradation of blood-borne insulin by the olfactory bulb. Peptides 20:373-378. Bannister LH, Dodson HC (1992) Endocytic pathways in the olfactory and vomeronasal epithelia of the mouse: ultrastructure and uptake of tracers. Microscopy research and technique 23:128-141. Banuelos-Cabrera I, Valle-Dorado MG, Aldana BI, Orozco-Suárez SA, Rocha L (2014) Role of histaminergic system in blood–brain barrier dysfunction associated with neurological disorders. Archives of medical research 45:677-686. Barber P, Dahl D (1987) Glial fibrillary acidic protein (GFAP)-like immunoreactivity in normal and transected rat olfactory nerve. Experimental brain research 65:681-685. Bardin C, Cheng CY, Musto N, Gunsalus G (1988) The sertoli cell. The physiology of reproduction 1:933-974. Barnea G, O'Donnell S, Mancia F, Sun X, Nemes A, Mendelsohn M, Axel R (2004) Odorant receptors on axon termini in the brain. Science 304:1468-1468. Bartolomei JC, Greer CA (2000) Olfactory ensheathing cells: bridging the gap in spinal cord injury. Neurosurgery 47:1057-1069. Baskin D, JR P, Guest K, Dorsa D (1983) Regional concentrations of insulin in the rat brain. Endocrinology 112:898-903. Batbayar B, Somogyi J, Zelles T, Fehér E (2003) Immunohistochemical analysis of substance P containing nerve fibres and their contacts with mast cells in the diabetic rat's tongue. Acta Biologica Hungarica 54:275-284. Batbayar B, Zelles T, Vér Á, Fehér E (2004) Plasticity of the different neuropeptide-containing nerve fibres in the tongue of the diabetic rat. Journal of the Peripheral Nervous System 9:215-223. Baxendale RW, Fraser LR (2003a) Evidence for multiple distinctly localized adenylyl cyclase isoforms in mammalian spermatozoa. Molecular reproduction and development 66:181- 189. Baxendale RW, Fraser LR (2003b) Immunolocalization of multiple Gα subunits in mammalian spermatozoa and additional evidence for Gαs. Molecular reproduction and development 65:104-113. Bédard A, Parent A (2004) Evidence of newly generated neurons in the human olfactory bulb. Developmental brain research 151:159-168. Bedford J (1975) Maturation transport and fate of spermatozoa in the epididymis. Beets M (1970) The molecular parameters of olfactory response. Pharmacological reviews 22:1- 34. Bellgrau D, Gold D, Selawry H, Moore J, Franzusoff A, Duke RC (1995) A role for CD95 ligand in preventing graft rejection. Nature 377:630. Belluscio L, Gold GH, Nemes A, Axel R (1998) Mice deficient in G olf are anosmic. Neuron 20:69- 81. Belluzzi O, Benedusi M, Ackman J, LoTurco JJ (2003) Electrophysiological differentiation of new neurons in the olfactory bulb. Journal of Neuroscience 23:10411-10418. Benoist C, Mathis D (2002) Mast cells in autoimmune disease. Nature 420:875.

230 Benton L, Shan L-X, Hardy MP (1995) Differentiation of adult Leydig cells. The Journal of steroid biochemistry and molecular biology 53:61-68. Bertschy S, Genton CY, Gotzos V (1997) Selective immunocytochemical localisation of calretinin in the human ovary. Histochemistry and cell biology 109:59-66. Bhandawat V, Reisert J, Yau K-W (2005) Elementary response of olfactory receptor neurons to odorants. Science 308:1931-1934. Bhushan S, Tchatalbachev S, Klug J, Fijak M, Pineau C, Chakraborty T, Meinhardt A (2008) Uropathogenic Escherichia coli block MyD88-dependent and activate MyD88- independent signaling pathways in rat testicular cells. The Journal of Immunology 180:5537-5547. Bienenstock J, Stead R, Marshall J (1993) Mast cells and the nervous system. Lung biology in health and disease 62:687-698. Bischoff S, Sellge G, Lorentz A, Sebald W, Raab R, Manns M (1999) IL-4 enhances proliferation and mediator release in mature human mast cells. Proceedings of the National Academy of Sciences 96:8080-8085. Bishop GA, Berbari NF, Lewis J, Mykytyn K (2007) Type III adenylyl cyclase localizes to primary cilia throughout the adult mouse brain. Journal of Comparative Neurology 505:562-571. Blache P, Gros L, Salazar G, Bataille D (1998) Cloning and tissue distribution of a new rat olfactory receptor-like (OL2). Biochemical and biophysical research communications 242:669-672. Black J, Duncan W, Durant CJ, Ganellin CR, Parsons E (1972) Definition and antagonism of histamine H2-receptors. Nature 236:385-390. Blank U, Rivera J (2004) The ins and outs of IgE-dependent mast-cell exocytosis. Trends in immunology 25:266-273. Blank U, Benhamou M (2013) Deciphering new molecular mechanisms of mast cell activation. Frontiers in immunology 4:100. Bleil JD, Wassarman PM (1983) Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein. Developmental biology 95:317-324. Böhmer M, Van Q, Weyand I, Hagen V, Beyermann M, Matsumoto M, Hoshi M, Hildebrand E, Kaupp UB (2005) Ca2+ spikes in the flagellum control chemotactic behavior of sperm. The EMBO journal 24:2741-2752. Bolteus AJ, Bordey A (2004) GABA release and uptake regulate neuronal precursor migration in the postnatal subventricular zone. Journal of Neuroscience 24:7623-7631. Bouchard C (2009) Childhood obesity: are genetic differences involved? The American journal of clinical nutrition 89:1494S-1501S. Bovetti S, Bovolin P, Perroteau I, Puche AC (2007a) Subventricular zone-derived neuroblast migration to the olfactory bulb is modulated by matrix remodelling. European Journal of Neuroscience 25:2021-2033. Bovetti S, Hsieh Y-C, Bovolin P, Perroteau I, Kazunori T, Puche AC (2007b) Blood vessels form a scaffold for neuroblast migration in the adult olfactory bulb. Journal of Neuroscience 27:5976-5980. Bozza T, Feinstein P, Zheng C, Mombaerts P (2002) Odorant receptor expression defines functional units in the mouse olfactory system. The Journal of neuroscience 22:3033- 3043. Braga T, Grujic M, Lukinius A, Hellman L, Åbrink M, Pejler G (2007) Serglycin proteoglycan is required for secretory granule integrity in mucosal mast cells. Biochemical Journal 403:49-57. Braun T, Voland P, Kunz L, Prinz C, Gratzl M (2007) Enterochromaffin cells of the human gut: sensors for spices and odorants. Gastroenterology 132:1890-1901. Breer H, Fleischer J, Strotmann J (2006) The sense of smell: multiple olfactory subsystems. Cell Mol Life Sci 63:1465-1475. Breipohl W, Laugwitz H, Bornfeld N (1974) Topological relations between the dendrites of olfactory sensory cells and sustentacular cells in different vertebrates. An ultrastructural study. Journal of anatomy 117:89. Breitbart H (2003) Signaling pathways in sperm capacitation and acrosome reaction. Cellular and molecular biology-Paris-Wegmann 49:321-328. Bremner W, Millar M, Sharpe R, Saunders P (1994a) Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens. Endocrinology 135:1227-1234.

231 Bremner WJ, Millar MR, Sharpe RM, Saunders P (1994b) Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens. Endocrinology 135:1227-1234. Brenker C, Goodwin N, Weyand I, Kashikar ND, Naruse M, Krähling M, Müller A, Kaupp UB, Strünker T (2012) The CatSper channel: a polymodal chemosensor in human sperm. The EMBO journal 31:1654-1665. Brenker C, Zhou Y, Müller A, Echeverry FA, Trötschel C, Poetsch A, Xia X-M, Bönigk W, Lingle CJ, Kaupp UB (2014) The Ca2+-activated K+ current of human sperm is mediated by Slo3. Elife 3:e01438. Brennan J, Tilmann C, Capel B (2003) Pdgfr-α mediates testis cord organization and fetal Leydig cell development in the XY gonad. Genes & development 17:800-810. Brenner T, Soffer D, Shalit M, Levi-Schaffer F (1994) Mast cells in experimental allergic encephalomyelitis: characterization, distribution in the CNS and in vitro activation by basic protein and neuropeptides. Journal of the neurological sciences 122:210- 213. Breslin PA (2013) An evolutionary perspective on food and human taste. Current Biology 23:R409-R418. Brinon J, Alonso J, Arévalo R, Garcia-Ojeda E, Lara J, Aijón J (1992) Calbindin D-28k-positive neurons in the rat olfactory bulb. Cell and tissue research 269:289-297. Brookes S, Steele P, Costa M (1991) Calretinin immunoreactivity in cholinergic motor neurones, interneurones and vasomotor neurones in the guinea-pig small intestine. Cell and tissue research 263:471-481. Brooks D (1981) Secretion of proteins and glycoproteins by the rat epididymis: regional differences, androgen-dependence, and effects of protease inhibitors, procaine, and tunicamycin. Biology of reproduction 25:1099-1117. Brooks D, Higgins S (1980) Characterization and androgen-dependence of proteins associated with luminal fluid and spermatozoa in the rat epididymis. Journal of reproduction and fertility 59:363-375. Brosnan CF, Claudio L, Tansey FA, Martiney J (1990) Mechanisms of autoimmune neuropathies. Annals of Neurology: Official Journal of the American Neurological Association and the Child Neurology Society 27:S75-S79. Brown D, Montesano R (1981) Membrane specialization in the rat epididymis. II. The clear cell. The Anatomical Record 201:477-483. Brown D, Breton S (1996) Mitochondria-rich, proton-secreting epithelial cells. The Journal of experimental biology 199:2345-2358. Brown KL, Hancock RE (2006) Cationic host defense (antimicrobial) peptides. Current opinion in immunology 18:24-30. Brown MA, Tanzola MB, Robbie-Ryan M (2002) Mechanisms underlying mast cell influence on EAE disease course. Molecular immunology 38:1373-1378. Brown-Woodman P, Sale D, White I (1976) The glycerylphosphorylcholine content of the rat epididymis after injecting alpha-chlorohydrin and ligating the vasa efferentia. Acta Europaea fertilitatis 7:155-162. Brunet LJ, Gold GH, Ngai J (1996) General anosmia caused by a targeted disruption of the mouse olfactory cyclic nucleotide–gated cation channel. Neuron 17:681-693. Buck L, Axel R (1991) A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 65:175-187. Buck LB (1996) Information coding in the vertebrate olfactory system. Annual review of neuroscience 19:517-544. Buck LB (2000) The molecular architecture of odor and sensing in mammals. Cell 100:611-618. Budanova E, Bystrova M (2010) Immunohistochemical detection of olfactory marker protein in tissues with ectopic expression of olfactory receptor genes. Biochemistry (Moscow) Supplement Series A: Membrane and Cell Biology 4:120-123. Buehr M (1997) The primordial germ cells of mammals: some current perspectives. Experimental cell research 232:194-207. Buehr M, Gu S, McLaren A (1993) Mesonephric contribution to testis differentiation in the fetal mouse. Development 117:273-281. Bugajski A, Chłap Z, Bugajski J (1994) Effect of isolation stress on brain mast cells and brain histamine levels in rats. Agents and Actions 41:C75-C76.

232 Buiakova O, Baker H, Scott J, Farbman A, Kream R, Grillo M, Franzen L, Richman M, Davis L, Abbondanzo S (1996) Olfactory marker protein (OMP) gene deletion causes altered physiological activity of olfactory sensory neurons. Proceedings of the National Academy of Sciences 93:9858-9863. Buiakova OI, Krishna NR, Getchell TV, Margolis FL (1994) Human and rodent OMP genes: conservation of structural and regulatory motifs and cellular localization. Genomics 20:452-462. Burgos MH (1964) Uptake of colloidal particles by cells of the caput epididymidis. The Anatomical Record 148:517-525. Burkett BN, Schulte BA, Spicer SS (1987) Histochemical evaluation of glycoconjugates in the male reproductive tract with lectin-horseradish peroxidase conjugates: I. Staining of principal cells and spermatozoa in the mouse. American journal of anatomy 178:11-22. Burnett LA, Anderson DM, Rawls A, Bieber AL, Chandler DE (2011) Mouse sperm exhibit chemotaxis to allurin, a truncated member of the cysteine-rich secretory protein family. Developmental biology 360:318-328. Caballero J, Frenette G, Sullivan R (2010) Post testicular sperm maturational changes in the bull: important role of the epididymosomes and prostasomes. Veterinary medicine international 2011. Caballero-Campo P, Buffone MG, Benencia F, Conejo-García JR, Rinaudo PF, Gerton GL (2014) A role for the chemokine receptor CCR6 in mammalian sperm motility and chemotaxis. Journal of cellular physiology 229:68-78. Caflisch C (1992) Acidification of testicular and epididymal fluids in the rat after surgically-induced varicocele. International journal of andrology 15:238-245. Caflisch CR, DuBose T (1990) Direct evaluation of acidification by rat testis and epididymis: role of carbonic anhydrase. American Journal of Physiology-Endocrinology And Metabolism 258:E143-E150. Caggiano M, Kauer JS, Hunter DD (1994) Globose basal cells are neuronal progenitors in the olfactory epithelium: a lineage analysis using a replication-incompetent retrovirus. Neuron 13:339-352. Cai D, Yuan M, Frantz DF, Melendez PA, Hansen L, Lee J, Shoelson SE (2005) Local and systemic insulin resistance resulting from hepatic activation of IKK-β and NF-κB. Nature medicine 11:183. Caillol M, Aı̈oun J, Baly C, Persuy M-A, Salesse R (2003) Localization of orexins and their receptors in the rat olfactory system: possible modulation of olfactory perception by a neuropeptide synthetized centrally or locally. Brain research 960:48-61. Cali JJ, Zwaagstra JC, Mons N, Cooper D, Krupinski J (1994) Type VIII adenylyl cyclase. A Ca2+/calmodulin-stimulated enzyme expressed in discrete regions of rat brain. Journal of Biological Chemistry 269:12190-12195. Callahan CA, Thomas JB (1994) Tau-beta-galactosidase, an axon-targeted fusion protein. Proceedings of the National Academy of Sciences 91:5972-5976. Calof AL, Chikaraishi DM (1989) Analysis of neurogenesis in a mammalian neuroepithelium: proliferation and differentiation of an olfactory neuron precursor in vitro. Neuron 3:115- 127. Calof AL, Lander AD, Chikaraishi DM (1991) Regulation of neurogenesis and neuronal differentiation in primary and immortalized cells from mouse olfactory epithelium. In: Regeneration of vertebrate sensory receptor cells, pp 249-276: Wiley Chichester. Calof AL, Bonnin A, Crocker C, Kawauchi S, Murray RC, Shou J, Wu HH (2002) Progenitor cells of the olfactory receptor neuron lineage. Microscopy research and technique 58:176-188. Cameron JD, Goldfield GS, Doucet É (2012) Fasting for 24 h improves nasal chemosensory performance and food palatability in a related manner. Appetite 58:978-981. Cani PD, Bibiloni R, Knauf C, Waget A, Neyrinck AM, Delzenne NM, Burcelin R (2008) Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet– induced obesity and diabetes in mice. Diabetes 57:1470-1481. Cani PD, Amar J, Iglesias MA, Poggi M, Knauf C, Bastelica D, Neyrinck AM, Fava F, Tuohy KM, Chabo C (2007) Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes 56:1761-1772. Carleton A, Petreanu LT, Lansford R, Alvarez-Buylla A, Lledo P-M (2003) Becoming a new neuron in the adult olfactory bulb. Nature neuroscience 6:507. Carr VM, Farbman A, Colletti L, Morgan J (1991) Identification of a new non-neuronal cell type in rat olfactory epithelium. Neuroscience 45:433-449.

233 Carr VM, Walters E, Margolis FL, Farbman AI (1998) An enhanced olfactory marker protein immunoreactivity in individual olfactory receptor neurons following olfactory bulbectomy may be related to increased neurogenesis. Journal of neurobiology 34:377-390. Carter LA, MacDonald JL, Roskams AJ (2004) Olfactory horizontal basal cells demonstrate a conserved multipotent progenitor phenotype. Journal of Neuroscience 24:5670-5683. Castells M (1999) Mast cells: molecular and cell biology. Internet J Asthma Allergy Immunol 1:1- 20. Caughey GH (2007) Mast cell tryptases and chymases in inflammation and host defense. Immunological reviews 217:141-154. Çelik A, Fuss SH, Korsching S (2002) Selective targeting of zebrafish olfactory receptor neurons by the endogenous OMP promoter. European Journal of Neuroscience 15:798-806. Celio M (1990) Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35:375-475. Chabot B, Stephenson DA, Chapman VM, Besmer P, Bernstein A (1988) The proto-oncogene c- kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus. Nature 335:88-89. Chan A, Cooley MA, Collins AM (2001) Mast cells in the rat liver are phenotypically heterogeneous and exhibit features of immaturity. Immunology and cell biology 79:35. Chaput M, Holley A (1976) Olfactory bulb responsiveness to food odour during stomach distension in the rat. Chemical senses 2:189-201. Chauhan SP, Sheth N, Suhagia B (2015) Effect of fruits of Opuntia elatior Mill on mast cell degranulation. Journal of pharmacy & bioallied sciences 7:156. Chen S, Lane AP, Bock R, Leinders-Zufall T, Zufall F (2000) Blocking adenylyl cyclase inhibits olfactory generator currents induced by “IP3-odors”. Journal of neurophysiology 84:575- 580. Chen X, Fang H, Schwob JE (2004) Multipotency of purified, transplanted globose basal cells in olfactory epithelium. Journal of Comparative Neurology 469:457-474. Chess A, Simon I, Cedar H, Axel R (1994) Allelic inactivation regulates olfactory receptor gene expression. Cell 78:823-834. Chevrot M, Passilly-Degrace P, Ancel D, Bernard A, Enderli G, Gomes M, Robin I, Issanchou S, Vergès B, Nicklaus S (2014) Obesity interferes with the orosensory detection of long- chain fatty acids in humans. The American journal of clinical nutrition 99:975-983. Chmelař J, Chatzigeorgiou A, Chung K-J, Prucnal M, Voehringer D, Roers A, Chavakis T (2016) no role for Mast cells in Obesity-related Metabolic Dysregulation. Frontiers in immunology 7. Choi E-J, Xia Z, Storm DR (1992) Stimulation of the type III olfactory adenylyl cyclase by calcium and calmodulin. Biochemistry 31:6492-6498. Chyczewski L, Debek W, Chyczewska E, Debek K, Bankowski E (1996) Morphology of lung mast cells in rats treated with bleomycin. Experimental and Toxicologic Pathology 48:515-517. Claman HN (1985) Mast cells, T cells and abnormal fibrosis. Immunology Today 6:192-195. Clapham DE, Neer EJ (1993) New roles for G-protein beta gamma-dimers in transmembrane signalling. Nature 365:403. Cleland K (1957) The structure and fuction of the epididymis. 1. The histology of the rat epididymis. Australian Journal of Zoology 5:223-246. Clermont Y, Flannery J (1970) Mitotic activity in the epithelium of the epididymis in young and old adult rats. Biology of reproduction 3:283-292. Clulow J, Jones R, Hansen L, Man S (1998) Fluid and electrolyte reabsorption in the ductuli efferentes testis. Journal of reproduction and fertility-Supplement-:1-14. Cohen-Dayag A, Tur-Kaspa I, Dor J, Mashiach S, Eisenbach M (1995) Sperm capacitation in humans is transient and correlates with chemotactic responsiveness to follicular factors. Proceedings of the National Academy of Sciences 92:11039-11043. Cohen-Dayag A, Ralt D, Tur-Kaspa I, Manor M, Makler A, Dor J, Mashiach S, Eisenbach M (1994) Sequential acquisition of chemotactic responsiveness by human spermatozoa. Biology of reproduction 50:786-790. Collington SJ, Williams TJ, Weller CL (2011) Mechanisms underlying the localisation of mast cells in tissues. Trends in immunology 32:478-485. Combes AN, Wilhelm D, Davidson T, Dejana E, Harley V, Sinclair A, Koopman P (2009) Endothelial cell migration directs testis cord formation. Developmental biology 326:112- 120.

234 Connell CJ (1976) A scanning electron microscope study of the interstitial tissue of the canine testis. The Anatomical Record 185:389-401. Cooper DM, Mons N, Karpen JW (1995) Adenylyl cyclases and the interaction between calcium and cAMP signalling. Nature 374:421. Cooper G, Overstreet J, Katz D (1979) The motility of rabbit spermatozoa recovered from the female reproductive tract. Molecular Reproduction and Development 2:35-42. Cooper T (1986) Function of the epididymis and its secretory products (Part III). The Epididymis, Sperm Maturation and Fertilization Springer Verlag, Berlin:117-230. Cooper T (2005) Cytoplasmic droplets: the good, the bad or just confusing? Human reproduction 20:9-11. Cooper T (2006) Sperm cytoplasmic droplets and ART. Embryo Talk 1:129-136. Cooper T (2007) The human epididymis, sperm maturation and storage. ANIR-ANHP 9:18-24. Cooper TG (2011) The epididymis, cytoplasmic droplets and male fertility. Asian journal of andrology 13:130. Cooper TG, Yeung CH (2003) Acquisition of volume regulatory response of sperm upon maturation in the epididymis and the role of the cytoplasmic droplet. Microscopy research and technique 61:28-38. Cooper TG, Yeung C-H, Fetic S, Sobhani A, Nieschlag E (2004) Cytoplasmic droplets are normal structures of human sperm but are not well preserved by routine procedures for assessing sperm morphology. Human reproduction 19:2283-2288. Coren S (2003) Sensation and perception: Wiley Online Library. Cornwall GA (2009) New insights into epididymal biology and function. Human reproduction update 15:213-227. Cosentino M, Cockett A (1986) Structure and function of the epididymis. Urological research 14:229-240. Cosson MP (1990) Sperm chemotaxis. Controls of sperm motility: biological and clinical aspects:104-135. Costello S, Michelangeli F, Nash K, Lefievre L, Morris J, Machado-Oliveira G, Barratt C, Kirkman- Brown J, Publicover S (2009) Ca2+-stores in sperm: their identities and functions. Reproduction 138:425-437. Coward K, Wells D (2013) Textbook of clinical embryology: Cambridge University Press. Crabo B (1964) Studies on the composition of epididymal content in bulls and boars. Acta Veterinaria Scandinavica 22:SUPPL 5: 1-94. Cudicini C, Lejeune H, Gomez E, Bosmans En, Ballet Fo, Saez J, Jégou B (1997) Human Leydig cells and Sertoli cells are producers of interleukins-1 and-6. The Journal of Clinical Endocrinology & Metabolism 82:1426-1433. Cunha A, Vitković L (1992) Transforming growth factor-beta 1 (TGF-β1) expression and regulation in rat cortical astrocytes. Journal of neuroimmunology 36:157-169. Cunningham A, Ryugo DK, Sharp A, Reed RR, Snyder SH, Ronnett GV (1993) Neuronal inositol 1, 4, 5-trisphosphate receptor localized to the plasma membrane of olfactory cilia. Neuroscience 57:339-352. Curtis MA, Kam M, Nannmark U, Anderson MF, Axell MZ, Wikkelso C, Holtås S, van Roon-Mom WM, Björk-Eriksson T, Nordborg C (2007) Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension. science 315:1243-1249. D'agostino P, Milano S, Barbera C, Di Bella G, La Rosa M, Ferlazzo V, Farruggio R, Miceli D, Miele M, Castagnetta L (1999) Sex hormones modulate inflammatory mediators produced by macrophages a. Annals of the New York Academy of Sciences 876:426- 429. D’mello AXP, Sylvester TV, Ramya V, Britto FP, Shetty PK, Jasphin S (2016) Metachromasia and Metachromatic Dyes: A review. International journal of advance health sciences 2:12-17. Dacheux J, Dacheux F, Paquignon M (1989) Changes in sperm surface membrane and luminal protein fluid content during epididymal transit in the boar. Biology of reproduction 40:635- 651. Dacheux J-L, Dacheux F (2002) Protein secretion in the epididymis. The Epididymis: From Molecules to Clinical Practice New York: Kluwer Academic/Plenum Publishers:151-168. Dacheux JL, Gatti JL, Dacheux F (2003) Contribution of epididymal secretory proteins for spermatozoa maturation. Microscopy research and technique 61:7-17. Dachman WD, Bedarida G, Blaschke TF, Hoffman BB (1994) Histamine-induced venodilation in human beings involves both H 1 and H 2 receptor subtypes. Journal of allergy and clinical immunology 93:606-614.

235 Dal Col JA, Matsuo T, Storm DR, Rodriguez I (2007) Adenylyl cyclase-dependent axonal targeting in the olfactory system. Development 134:2481-2489. Dalton RP, Lyons DB, Lomvardas S (2013) Co-opting the unfolded protein response to elicit olfactory receptor feedback. Cell 155:321-332. Danciger E, Mettling C, Vidal M, Morris R, Margolis F (1989) Olfactory marker protein gene: its structure and olfactory neuron-specific expression in transgenic mice. Proceedings of the National Academy of Sciences 86:8565-8569. Darszon A, Nishigaki T, Beltran C, Treviño CL (2011) Calcium channels in the development, maturation, and function of spermatozoa. Physiological Reviews 91:1305-1355. Davidoff M, Schulze W, Middendorff R, Holstein A-F (1993) The Leydig cell of the human testis— a new member of the diffuse neuroendocrine system. Cell and tissue research 271:429- 439. Davidoff MS, Middendorff R, Holstein AF (1996) Dual nature of Leydig cells of the human testis. Biomedical Reviews 6:11-41. Davidoff MS, Middendorff R, Mayer B, Holstein AF (1995) Nitric oxide synthase (NOS-I) in Leydig cells of the human testis. Archives of histology and cytology 58:17-30. Davidoff MS, Middendorff R, Mueller D, Holstein AF (2009) Introduction. In: The Neuroendocrine Leydig Cells and their Stem Cell Progenitors, the Pericytes, pp 1-2: Springer. Davidoff MS, Middendorff R, Köfüncü E, Müller D, Ježek D, Holstein A-F (2002) Leydig cells of the human testis possess astrocyte and oligodendrocyte marker molecules. Acta histochemica 104:39-49. Dawson R, Mann T, White I (1957) Glycerylphosphorylcholine and phosphorylcholine in semen, and their relation to choline. Biochemical Journal 65:627. Dawson TM, Arriza JL, Jaworsky DE, Borisy FF, Attramadal H, Lefkowitz RJ, Ronnett GV (1993) Beta-adrenergic receptor kinase-2 and beta-arrestin-2 as mediators of odorant-induced desensitization. Science 259:825-830. de La Cruz O, Blekhman R, Zhang X, Nicolae D, Firestein S (2009) A signature of evolutionary constraint on a subset of ectopically expressed olfactory receptor genes. Mol. Biol. Evol 26 3:491-494. De S, Chen H, Pace J, Hunt J, Terranova P, Enders G (1993) Expression of tumor necrosis factor- alpha in mouse spermatogenic cells. Endocrinology 133:389-396. Defer N, Best-Belpomme M, Hanoune J (2000) Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase. American Journal of Physiology-Renal Physiology 279:F400-F416. Defer N, Marinx O, Poyard M, Lienard MO, Jégou B, Hanoune J (1998) The olfactory adenylyl cyclase type 3 is expressed in male germ cells. FEBS letters 424:216-220. DeHamer MK, Guevara JL, Hannon K, Olwin BB, Calof AL (1994) Genesis of olfactory receptor neurons in vitro: regulation of progenitor cell divisions by fibroblast growth factors. Neuron 13:1083-1097. Dejucq N, Dugast I, Ruffault A, Van der Meide P, Jegou B (1995) Interferon-alpha and-gamma expression in the rat testis. Endocrinology 136:4925-4931. Dejucq N, Lienard M-O, Guillaume E, Dorval I, Jégou B (1998) Expression of interferons-α and-γ in testicular interstitial tissue and spermatogonia of the rat. Endocrinology 139:3081- 3087. Delahunt B, Eble J, Srigley J, Thornton A (2000) Paratesticular adenomatoid tumor: assessment of calretinin immunoexpression and cell proliferation indices. Journal of urologic pathology 12:105-116. Demott R, Suarez SS (1992) Hyperactivated sperm progress in the mouse oviduct. Biology of Reproduction 46:779-785. Dhallan RS, Yau K-W, Schrader KA, Reed RR (1990) Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons. Nature 347:184. Di Marzo V, Goparaju SK, Wang L, Liu J, Bátkai S, Járai Z, Fezza F, Miura GI, Palmiter RD, Sugiura T (2001) Leptin-regulated endocannabinoids are involved in maintaining food intake. Nature 410:822. Diamond G, Legarda D, Ryan LK (2000) The innate immune response of the respiratory epithelium. Immunological reviews 173:27-38. Diemer T, Hales D, Weidner W (2003) Immune–endocrine interactions and Leydig cell function: the role of cytokines. Andrologia 35:55-63. Dietsch G, Hinrichs D (1989) The role of mast cells in the elicitation of experimental allergic encephalomyelitis. The Journal of Immunology 142:1476-1481.

236 Dimitriadou V, Rouleau A, Tuong MT, Newlands G, Miller H, Luffau G, Schwartz J-C, Garbarg M (1997) Functional relationships between sensory nerve fibers and mast cells of dura mater in normal and inflammatory conditions. Neuroscience 77:829-839. Doetsch F, Alvarez-Buylla A (1996) Network of tangential pathways for neuronal migration in adult mammalian brain. Proceedings of the National Academy of Sciences 93:14895-14900. Doetsch F, Garcı́a-Verdugo JM, Alvarez-Buylla A (1997) Cellular composition and three- dimensional organization of the subventricular germinal zone in the adult mammalian brain. Journal of Neuroscience 17:5046-5061. Dong H, Zhang X, Qian Y (2014) Mast cells and neuroinflammation. Medical science monitor basic research 20:200. Dong X, Han S-k, Zylka MJ, Simon MI, Anderson DJ (2001) A diverse family of GPCRs expressed in specific subsets of nociceptive sensory neurons. Cell 106:619-632. Dotson CD, Colbert CL, Garcea M, Smith JC, Spector AC (2012) The consequences of gustatory deafferentation on body mass and feeding patterns in the rat. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 303:R611-R623. Doucette R (1991) PNS-CNS transitional zone of the first cranial nerve. Journal of Comparative Neurology 312:451-466. Doucette R (1993) Glial cells in the nerve fiber layer of the main olfactory bulb of embryonic and adult mammals. Microscopy research and technique 24:113-130. Downes G, Gautam N (1999) The G protein subunit gene families. Genomics 62:544-552. Draber P, Halova I, Polakovicova I, Kawakami T (2016) Signal transduction and chemotaxis in mast cells. European journal of pharmacology 778:11-23. Dráberová E, Dráber P, Viklický V (1986) Cellular distribution of a protein related to neuronal microtubule-associated protein MAP-2 in Leydig cells. Cell biology international reports 10:881-890. Dreyer WJ (1998) The area code hypothesis revisited: olfactory receptors and other related transmembrane receptors may function as the last digits in a cell surface code for assembling embryos. Proceedings of the National Academy of Sciences 95:9072-9077. Droller MJ, Roth TF (1966) An electron microscope study of yolk formation during oogenesis in Lebistes reticulatus guppyi. The Journal of cell biology 28:209-232. Dropp JJ (1972) Mast cells in the central nervous system of several rodents. The Anatomical Record 174:227-237. Dropp JJ (1976) Mast cells in mammalian brain. Cells Tissues Organs 94:1-21. Drutel G, Arrang J-M, Diaz J, Wisnewsky C, Schwartz K, Schwartz J-C (1994) Cloning of OL1, a putative olfactory receptor and its expression in the developing rat heart. Receptors & channels 3:33-40. Dryer L (2000) Evolution of odorant receptors. BioEssays 22:803-810. Du Plessis SS, Cabler S, McAlister DA, Sabanegh E, Agarwal A (2010) The effect of obesity on sperm disorders and male infertility. Nature Reviews Urology 7:153-161. Dubacq C, Jamet S, Trembleau A (2009) Evidence for developmentally regulated local translation of odorant receptor mRNAs in the axons of olfactory sensory neurons. The Journal of Neuroscience 29:10184-10190. Durzyński Ł, Gaudin J-C, Myga M, Szydłowski J, Goździcka-Józefiak A, Haertlé T (2005) Olfactory-like receptor cDNAs are present in human lingual cDNA libraries. Biochemical and biophysical research communications 333:264-272. Dvorak AM (2005a) Ultrastructural studies of human and mast cells. Journal of Histochemistry & Cytochemistry 53:1043-1070. Dvorak AM (2005b) Piecemeal degranulation of basophils and mast cells is effected by vesicular transport of stored secretory granule contents. In: Ultrastructure of Mast Cells and Basophils, pp 135-184: Karger Publishers. Dvorak AM, Kissell S (1991) Granule changes of human skin mast cells characteristic of piecemeal degranulation and associated with recovery during wound healing in situ. Journal of leukocyte biology 49:197-210. Dvorak AM, Morgan ES (1997) Diamine oxidase-gold enzyme-affinity ultrastructural demonstration that human gut mucosal mast cells secrete histamine by piecemeal degranulation in vivo. Journal of allergy and clinical immunology 99:812-820. Dvorak AM, Massey W, Warner J, Kissell S, Kagey-Sobotka A, Lichtenstein LM (1991) IgE- mediated anaphylactic degranulation of isolated human skin mast cells. Blood 77:569- 578.

237 Dvorak AM, Tepper R, Weller P, Morgan E, Estrella P, Monahan-Earley R, Galli S (1994) Piecemeal degranulation of mast cells in the inflammatory eyelid lesions of interleukin-4 transgenic mice. Evidence of mast cell histamine release in vivo by diamine oxidase-gold enzyme-affinity ultrastructural cytochemistry. Blood 83:3600-3612. Dvorak AM, McLeod R, Onderdonk A, Monahan-Earley RA, Cullen J, Antonioli DA, Morgan E, Blair J, Estrella P, Cisneros R (1992) Ultrastructural evidence for piecemeal and anaphylactic degranulation of human gut mucosal mast cells in vivo. International archives of allergy and immunology 99:74-83. Dym M (1983) The male reproductive system. In: Histology, pp 1000-1053: Springer. Dyson A, Orgebin-Crist M-C (1973) Effect of hypophysectomy, castration and androgen replacement upon the fertilizing ability of rat epididymal spermatozoa. Endocrinology 93:391-402. Ebeigbe AB, Talabi OO (2014) Vascular effects of histamine. Nigerian Journal of Physiological Sciences 29:07–10. Edmonds RH, Nagy F (1973) Crystalline inclusion bodies in the epididymis of the nine-banded armadillo. Journal of ultrastructure research 42:82-86. Ehmcke J, Schlatt S (2006) A revised model for spermatogonial expansion in man: lessons from non-human primates. Reproduction 132:673-680. Ehrlich ME, Grillo M, Joh TH, Margolis FL, Baker H (1990) Transneuronal regulation of neuronal specific gene expression in the mouse olfactory bulb. Molecular Brain Research 7:115- 122. Eickhoff R, Wilhelm B, Renneberg H, Wennemuth G, Bacher M, Linder D, Bucala R, Seitz J, Meinhardt A (2001) Purification and characterization of macrophage migration inhibitory factor as a secretory protein from rat epididymis: evidences for alternative release and transfer to spermatozoa. Molecular Medicine 7:27. Eisenbach M, Tur-Kaspa I (1999) Do human eggs attract spermatozoa? BioEssays 21:203-210. Eisenbach M, Giojalas LC (2006) Sperm guidance in mammals—an unpaved road to the egg. Nature reviews Molecular cell biology 7:276-285. Elsaesser R, Montani G, Tirindelli R, Paysan J (2005) Phosphatidyl-inositide signalling proteins in a novel class of sensory cells in the mammalian olfactory epithelium. European Journal of Neuroscience 21:2692-2700. Enerback L (1981) The gut mucosal mast cell. Monogr Allergy 17:222-232. Enerbäck L (1965) Mast cells in rat gastrointestinal mucosa. I. Effects of fixation. Acta pathologica et microbiologica Scandinavica 66:289-302. Eng DL, Kocsis JD (1987) Activity-dependent changes in extracellular potassium and excitability in turtle olfactory nerve. Journal of neurophysiology 57:740-754. Ennis M, Hayar A (2008) The Senses: A Comprehensive Reference, Volume 4: Olfaction and Taste. Ennis M, Zhou F-M, Ciombor KJ, Aroniadou-Anderjaska V, Hayar A, Borrelli E, Zimmer LA, Margolis F, Shipley MT (2001) Dopamine D2 receptor–mediated presynaptic inhibition of olfactory nerve terminals. Journal of Neurophysiology 86:2986-2997. Epstein JB, Scully C, Spinelli J (1992) Toluidine blue and Lugol's iodine application in the assessment of oral malignant disease and lesions at risk of malignancy. Journal of oral pathology & medicine 21:160-163. Epstein JB, Oakley C, Millner A, Emerton S, van der Meij E, Le N (1997) The utility of toluidine blue application as a diagnostic aid in patients previously treated for upper oropharyngeal carcinoma. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 83:537-547. Erpenbach KH (1991) Systemic treatment with Interferon-α2b: an effective method to prevent sterility after bilateral mumps orchitis. The Journal of urology 146:54-56. Esposito P, Gheorghe D, Kandere K, Pang X, Connolly R, Jacobson S, Theoharides TC (2001) Acute stress increases permeability of the blood–brain-barrier through activation of brain mast cells. Brain research 888:117-127. Ezeh P, Wellis D, Scott J (1993) Organization of inhibition in the rat olfactory bulb external plexiform layer. Journal of neurophysiology 70:263-274. Fabro G, Rovasio RA, Civalero S, Frenkel A, Caplan SR, Eisenbach M, Giojalas LC (2002) Chemotaxis of capacitated rabbit spermatozoa to follicular fluid revealed by a novel directionality-based assay. Biology of Reproduction 67:1565-1571. Fabry Z, Raine CS, Hart MN (1994) Nervous tissue as an immune compartment: the dialect of the immune response in the CNS. Immunology today 15:218-X.

238 Fadool D, Tucker K, Phillips J, Simmen J (2000) Brain insulin receptor causes activity-dependent current suppression in the olfactory bulb through multiple phosphorylation of Kv1. 3. Journal of neurophysiology 83:2332-2348. Fadool DA, Tucker K, Pedarzani P (2011) Mitral cells of the olfactory bulb perform metabolic sensing and are disrupted by obesity at the level of the Kv1. 3 ion channel. PloS one 6:e24921. Fantuzzi G (2005) Adipose tissue, adipokines, and inflammation. Journal of Allergy and Clinical Immunology 115:911-919. Farbman AI (1992) Cell biology of olfaction: Cambridge University Press. Farbman AI, Margolis FL (1980) Olfactory marker protein during ontogeny: immunohistochemical localization. Developmental biology 74:205-215. Farbman AI, Buchholz JA (1996) Transforming growth factor-α and other growth factors stimulate cell division in olfactory epithelium in vitro. Journal of neurobiology 30:267-280. Farbman AI, Buchholz JA, Walters E, Margolis FL (1998) Does olfactory marker protein participate in olfactory neurogenesis? Annals of the New York Academy of Sciences 855:248-251. Faria MJS, Simões ZLP, Lunardi LO, Hartfelder K (2003) Apoptosis process in mouse Leydig cells during postnatal development. Microscopy and Microanalysis 9:68-73. Farooqi IS, O’Rahilly S (2007) Genetic factors in human obesity. Obesity Reviews 8:37-40. Fawcett DW (1975) Ultrastructure and function of the Sertoli cell. Handbook of physiology. Fawcett DW, Porter KR (1954) A study of the fine structure of ciliated epithelia. Journal of Morphology 94:221-281. Fawcett DW, Neaves WB, Flores MN (1973) Comparative observations on intertubular lymphatics and the organization of the interstitial tissue of the mammalian testis. Biology of Reproduction 9:500-532. Fehr J, Meyer D, Widmayer P, Borth HC, Ackermann F, Wilhelm B, Gudermann T, Boekhoff I (2007) Expression of the G-protein α-subunit gustducin in mammalian spermatozoa. Journal of Comparative Physiology A 193:21-34. Feinstein PG, Schrader KA, Bakalyar HA, Tang W-J, Krupinski J, Gilman AG, Reed RR (1991) Molecular cloning and characterization of a Ca2+/calmodulin-insensitive adenylyl cyclase from rat brain. Proceedings of the National Academy of Sciences 88:10173-10177. Feldmesser E, Olender T, Khen M, Yanai I, Ophir R, Lancet D (2006) Widespread ectopic expression of olfactory receptor genes. BMC genomics 7:121. Fernández-Real JM, Broch M, Richart C, Vendrell J, López-Bermejo A, Ricart W (2003) CD14 monocyte receptor, involved in the inflammatory cascade, and insulin sensitivity. The Journal of Clinical Endocrinology & Metabolism 88:1780-1784. Feuchter F, Tabet A, Green M (1988) Maturation antigen of the mouse sperm flagellum. I. Analysis of its secretion, association with sperm, and function. Developmental Dynamics 181:67-76. Fijak M, Meinhardt A (2006) The testis in immune privilege. Immunological reviews 213:66-81. Fijak M, Schneider E, Klug J, Bhushan S, Hackstein H, Schuler G, Wygrecka M, Gromoll J, Meinhardt A (2011) Testosterone replacement effectively inhibits the development of experimental autoimmune orchitis in rats: evidence for a direct role of testosterone on regulatory expansion. The Journal of Immunology 186:5162-5172. Firestein S, Darrow B, Shepherd GM (1991) Activation of the sensory current in salamander olfactory receptor neurons depends on a G protein-mediated cAMP . Neuron 6:825-835. Flegel C, Manteniotis S, Osthold S, Hatt H, Gisselmann G (2013) Expression profile of ectopic olfactory receptors determined by deep sequencing. PLoS One 8:e55368. Flegel C, Vogel F, Hofreuter A, Schreiner BS, Osthold S, Veitinger S, Becker C, Brockmeyer NH, Muschol M, Wennemuth G (2015) Characterization of the olfactory receptors expressed in human spermatozoa. Frontiers in molecular biosciences 2. Fleischer J, Schwarzenbacher K, Besser S, Hass N, Breer H (2006) Olfactory receptors and signalling elements in the Grueneberg ganglion. Journal of neurochemistry 98:543-554. Flickinger CJ (1979) Synthesis, transport and secretion of protein in the initial segment of the mouse epididymis as studied by electron microscope radioautography. Biology of reproduction 20:1015-1030. Flickinger CJ (1981) Regional differences in synthesis, intracellular transport, and secretion of protein in the mouse epididymis. Biology of reproduction 25:871-883.

239 Flickinger CJ (1985) Radioautographic analysis of the secretory pathway for glycoproteins in principal cells of the mouse epididymis exposed to [3H] fucose. Biology of reproduction 32:377-389. Flickinger CJ, Wilson KM, Gray HD (1984) The secretory pathway in the mouse epididymis as shown by electron microscope radioautography of principal cells exposed to monensin. The Anatomical Record 210:435-448. Florenzano F, Bentivoglio M (2000) Degranulation, density, and distribution of mast cells in the rat thalamus: a light and electron microscopic study in basal conditions and after intracerebroventricular administration of nerve growth factor. Journal of Comparative Neurology 424:651-669. Florman HM, Storey BT (1982) Mouse gamete interactions: the zona pellucida is the site of the acrosome reaction leading to fertilization in vitro. Developmental biology 91:121-130. Florman HM, Ducibella T (2006) Fertilization in mammals. Knobil and Neill’s physiology of reproduction 1:149-196. Fordyce JA (2006) The evolutionary consequences of ecological interactions mediated through phenotypic plasticity. Journal of Experimental Biology 209:2377-2383. Foreman JC (1993) Non-immunological stimuli of mast cells and leucocytes. Immunopharmacology of Mast Cells and Basophils 57. Fornes M, Barbieri A, Cavicchia J (1995) Morphological and enzymatic study of membrane-bound vesicles from the lumen of the rat epididymis. Andrologia 27:1-5. Foster SR, Roura E, Thomas WG (2014) Extrasensory perception: odorant and taste receptors beyond the nose and mouth. Pharmacology & therapeutics 142:41-61. Fox SI (1996) Human Physiology. Wm. C. Brown Publishers USA. Francis F, Koulakoff A, Boucher D, Chafey P, Schaar B, Vinet M-C, Friocourt G, McDonnell N, Reiner O, Kahn A (1999) Doublecortin is a developmentally regulated, microtubule- associated protein expressed in migrating and differentiating neurons. Neuron 23:247- 256. Fraser LR (1977) Motility patterns in mouse spermatozoa before and after capacitation. Journal of Experimental Zoology Part A: Ecological Genetics and Physiology 202:439-444. Freitag J, Ludwig G, Andreini I, Rössler P, Breer H (1998) Olfactory receptors in aquatic and terrestrial vertebrates. Journal of Comparative Physiology A 183:635-650. Frenette G, Sullivan R (2001) Prostasome-like particles are involved in the transfer of P25b from the bovine epididymal fluid to the sperm surface. Molecular reproduction and development 59:115-121. Frenette G, Girouard J, Sullivan R (2006) Comparison between epididymosomes collected in the intraluminal compartment of the bovine caput and cauda epididymidis. Biology of reproduction 75:885-890. Frenette G, Légaré C, Saez F, Sullivan R (2005) Macrophage migration inhibitory factor in the human epididymis and semen. Molecular human reproduction 11:575-582. Fried SK, Bunkin DA, Greenberg AS (1998) Omental and subcutaneous adipose tissues of obese subjects release interleukin-6: depot difference and regulation by . The Journal of Clinical Endocrinology & Metabolism 83:847-850. Friedrich BM, Jülicher F (2007) Chemotaxis of sperm cells. Proceedings of the National Academy of Sciences 104:13256-13261. Friend D (1977) The organization of the spermatozoal membrane. In: Cambridge University Press, Cambridge. Friend DS, Farquhar MG (1967) Functions of coated vesicles during protein absorption in the rat vas deferens. The Journal of cell biology 35:357-376. Frisch D (1967) Ultrastructure of mouse olfactory mucosa. Developmental Dynamics 121:87-119. Frungieri MB, Weidinger S, Meineke V, Köhn FM, Mayerhofer A (2002) Proliferative action of mast-cell tryptase is mediated by PAR2, COX2, prostaglandins, and PPARγ: possible relevance to human fibrotic disorders. Proceedings of the National Academy of Sciences 99:15072-15077. Fuchs T, Glusman G, Horn-Saban S, Lancet D, Pilpel Y (2001) The human olfactory subgenome: from sequence to structure and evolution. Human genetics 108:1-13. Fujishima H, Mejia ROS, Bingham CO, Lam BK, Sapirstein A, Bonventre JV, Austen KF, Arm JP (1999) Cytosolic phospholipase A2 is essential for both the immediate and the delayed phases of generation in mouse bone marrow-derived mast cells. Proceedings of the National Academy of Sciences 96:4803-4807.

240 Fukuda N, Touhara K (2006) Developmental expression patterns of testicular olfactory receptor genes during mouse spermatogenesis. Genes to Cells 11:71-81. Fukuda N, Yomogida K, Okabe M, Touhara K (2004) Functional characterization of a mouse testicular olfactory receptor and its role in chemosensing and in regulation of sperm motility. Journal of cell science 117:5835-5845. Fuss SH, Omura M, Mombaerts P (2005) The Grueneberg ganglion of the mouse projects axons to glomeruli in the olfactory bulb. European Journal of Neuroscience 22:2649-2654. Gage F, Kempermann G, Song H (2008) Adult neurogenesis: CSHL Press. Gaillard I, Rouquier S, Pin JP, Mollard P, Richard S, Barnabé C, Demaille J, Giorgi D (2002) A single olfactory receptor specifically binds a set of odorant molecules. European Journal of Neuroscience 15:409-418. Galindo MM, Voigt N, Stein J, van Lengerich J, Raguse J-D, Hofmann T, Meyerhof W, Behrens M (2011) G protein–coupled receptors in human fat taste perception. Chemical senses:bjr069. Galizia CG, Menzel R (2000) Odour perception in honeybees: coding information in glomerular patterns. Current opinion in neurobiology 10:504-510. Gall CM, Hendry SH, Seroogy KB, Jones EG, Haycock JW (1987) Evidence for coexistence of GABA and dopamine in neurons of the rat olfactory bulb. Journal of Comparative Neurology 266:307-318. Galli S (1987) New approaches for the analysis of mast cell maturation, heterogeneity, and function. In: Federation proceedings, pp 1906-1914. Galli SJ, Tsai M (2008) Mast cells: versatile regulators of inflammation, tissue remodeling, host defense and homeostasis. Journal of dermatological science 49:7-19. Galli SJ, Grimbaldeston M, Tsai M (2008) Immunomodulatory mast cells: negative, as well as positive, regulators of innate and acquired immunity. Nature reviews Immunology 8:478. Gao B, Gilman AG (1991) Cloning and expression of a widely distributed (type IV) adenylyl cyclase. Proceedings of the National Academy of Sciences 88:10178-10182. Garcia-Esparcia P, Schlüter A, Carmona M, Moreno J, Ansoleaga B, Torrejón-Escribano B, Gustincich S, Pujol A, Ferrer I (2013) Functional genomics reveals dysregulation of cortical olfactory receptors in Parkinson disease: novel putative chemoreceptors in the human brain. Journal of Neuropathology & Experimental Neurology 72:524-539. Garcia-Segura L, Baetens D, Roth J, Norman A, Orci L (1984) Immunohistochemical mapping of calcium-binding protein immunoreactivity in the rat central nervous system. Brain research 296:75-86. Garty NB, Galiani D, Aharonheim A, Ho Y, Phillips DM, Dekel N, Salomon Y (1988) G-proteins in mammalian gametes: an immunocytochemical study. Journal of cell science 91:21-31. Gat U, Nekrasova E, Lancet D, Natochin M (1994) Olfactory receptor proteins. The FEBS Journal 225:1157-1168. Gatti J-L, Métayer S, Belghazi M, Dacheux F, Dacheux J-L (2005) Identification, proteomic profiling, and origin of ram epididymal fluid exosome-like vesicles. Biology of reproduction 72:1452-1465. Gatti J-L, Métayer S, Moudjou M, Andréoletti O, Lantier F, Dacheux J-L, Sarradin P (2002) Prion protein is secreted in soluble forms in the epididymal fluid and proteolytically processed and transported in seminal plasma. Biology of reproduction 67:393-400. Gaudin J-C, Breuils L, Haertle T (2001) New GPCRs from a human lingual cDNA library. Chemical senses 26:1157-1166. Gaudin J-C, Breuils L, Haertlé T (2006) Mouse orthologs of human olfactory-like receptors expressed in the tongue. Gene 381:42-48. Gautier-Courteille C, Salanova M, Conti M (1998) The olfactory adenylyl cyclase III is expressed in rat germ cells during spermiogenesis 1. Endocrinology 139:2588-2599. Gaytan F, Carrera G, Pinilla L, Aguilar R, Bellido C (1989) Mast cells in the testis, epididymis and accessory glands of the rat: effects of neonatal steroid treatment. Journal of andrology 10:351-358. Gaytan F, Bellido C, Aceitero J, Aguilar E, Sanchez-Criado JE (1990a) Leydig cell involvement in the paracrine regulation of mast cells in the testicular interstitium of the rat. Biology of reproduction 43:665-671. Gaytan F, Aceitero J, Bellido C, Pinilla L, Aguilar R, Aguilar E (1990b) Are eosinophil leucocytes involved in the oestrogenic response of the postnatal rat epididymis? International journal of andrology 13:500-507.

241 Gaytan F, Aceitero J, Lucena C, Aguilar E, Pinilla L, Garnelo P, Bellido C (1992) Simultaneous proliferation and differentiation of mast cells and Leydig cells in the rat testis. Journal of andrology 13:387-397. Ge R, Shan L, Hardy M (1996) Pubertal development of Leydig cells. The Leydig Cell 1:159-174. Ge R, Chen G, Hardy MP (2009) The role of the Leydig cell in spermatogenic function. In: Molecular Mechanisms in Spermatogenesis, pp 255-269: Springer. Ge R, Dong Q, Sottas C, Hardy M (2005) Development of androgen biosynthetic capacity in an enriched fraction of stem Leydig cells. In: Biology of reproduction, pp 104-104: Soc study reproduction 1603 Monroe st, Madison, WI 53711-2021 USA. Ge R-S, Hardy MP (1998) Variation in the end products of androgen biosynthesis and metabolism during postnatal differentiation of rat Leydig cells. Endocrinology 139:3787-3795. Gebhardt T, Sellge G, Lorentz A, Raab R, Manns MP, Bischoff SC (2002) Cultured human intestinal mast cells express functional IL-3 receptors and respond to IL-3 by enhancing growth and IgE receptor-dependent mediator release. European journal of immunology 32:2308-2316. Gerdprasert O, O'Bryan M, Nikolic-Paterson D, Sebire K, De Kretser D, Hedger M (2002a) Expression of monocyte chemoattractant protein-1 and macrophage colony-stimulating factor in normal and inflamed rat testis. MHR: Basic science of reproductive medicine 8:518-524. Gerdprasert O, O'Bryan MK, Muir JA, Caldwell AM, Schlatt S, de Kretser DM, Hedger MP (2002b) The response of testicular leukocytes to lipopolysaccharide-induced inflammation: further evidence for heterogeneity of the testicular macrophage population. Cell and tissue research 308:277-285. Gerton GL (2002) Function of the sperm acrosome. In, pp 265-302: Academic Press, San Diego. Gesteland R (1986) Speculations on receptor cells as analyzers and filters. Cellular and Molecular Life Sciences 42:287-291. Getchell TV, Kwong K, Saunders CP, Stromberg AJ, Getchell ML (2006) Leptin regulates olfactory-mediated behavior in ob/ob mice. Physiology & behavior 87:848-856. Ghosh S, Sinha-Hikim A, Russell L (1991) Further observations of stage-specific effects seen after short-term hypophysectomy in the rat. Tissue and Cell 23:613-630. Gibson S, Miller H (1986) Mast cell subsets in the rat distinguished immunohistochemically by their content of serine proteinases. Immunology 58:101. Gierer A (1998) Possible involvement of gradients in guidance of receptor cell axons towards their target position on the olfactory bulb. European Journal of Neuroscience 10:388-391. Gilad Y, Man O, Glusman G (2005) A comparison of the human and chimpanzee olfactory receptor gene repertoires. Genome research 15:224-230. Gilfillan AM, Tkaczyk C (2006) Integrated signalling pathways for mast-cell activation. Nature reviews Immunology 6:218. Gilman AG (1995) G proteins and regulation of adenylyl cyclase. Bioscience reports 15:65-97. Giojalas L (1998) Mouse spermatozoa modify their motility parameters and chemotactic response to factors from the oocyte microenvironment. international journal of andrology 21:201- 206. Giorgi F, Maggio R, Bruni LE (2011) Are olfactory receptors really olfactive? Biosemiotics 4:331- 347. Gispen W, Verhaagen J, Oestreicher A, Margolis F (1989) The expression of the growth- associated protein B-50/GAP43 in the olfactory system of neonatal and adult rats. Journal of Neuroscience 9:683-691. Glass CK, Olefsky JM (2012) Inflammation and in the etiology of insulin resistance. Cell metabolism 15:635-645. Glassner M, Jones J, Kligman I, Woolkalis MJ, Gerton GL, Kopf GS (1991) Immunocytochemical and biochemical characterization of guanine nucleotide-binding regulatory proteins in mammalian spermatozoa. Developmental biology 146:438-450. Gleeson JG, Lin PT, Flanagan LA, Walsh CA (1999) Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons. Neuron 23:257-271. Glusman G, Yanai I, Rubin I, Lancet D (2001) The complete human olfactory subgenome. Genome research 11:685-702. Gold GH (1999) Controversial issues in vertebrate olfactory transduction. Annual Review of Physiology 61:857-871.

242 Goldstein BJ, Schwob JE (1996) Analysis of the globose basal cell compartment in rat olfactory epithelium using GBC-1, a new monoclonal antibody against globose basal cells. Journal of Neuroscience 16:4005-4016. Goldstein BJ, Fang H, Youngentob SL, Schwob JE (1998) Transplantation of multipotent progenitors from the adult olfactory epithelium. Neuroreport 9:1611-1617. Gomariz R, Arranz A, Juarranz Y, Gutierrez-Canas I, Garcia-Gomez M, Leceta J, Martínez C (2007) Regulation of TLR expression, a new perspective for the role of VIP in immunity. Peptides 28:1825-1832. Gonçalves I, Hubbard PC, Tomás J, Quintela T, Tavares G, Caria S, Barreiros D, Santos CR (2016) ‘Smelling’the cerebrospinal fluid: olfactory signaling molecules are expressed in and mediate chemosensory signaling from the choroid plexus. FEBS Journal 283:1748- 1766. Gordon MK, Mumm JS, Davis RA, Holcomb JD, Calof AL (1995) Dynamics of MASH1 expression in vitro and in vivo suggest a non-stem cell site of MASH1 action in the olfactory receptor neuron lineage. Molecular and Cellular Neuroscience 6. Gornstein RA, Lapp CA, Bustos-Valdes SM, Zamorano P (1999) Androgens modulate interleukin- 6 production by gingival fibroblasts in vitro. Journal of Periodontology 70:604-609. Gorojankina T, Grébert D, Salesse R, Tanfin Z, Caillol M (2007) Study of orexins signal transduction pathways in rat olfactory mucosa and in olfactory sensory neurons-derived cell line Odora: multiple orexin signalling pathways. Regulatory peptides 141:73-85. Gorski JP, Hugli TE, Müller-Eberhard HJ (1979) C4a: the third anaphylatoxin of the human . Proceedings of the National Academy of Sciences 76:5299-5302. Goto T, Salpekar A, Monk M (2001) Expression of a testis-specific member of the olfactory receptor gene family in human primordial germ cells. Molecular human reproduction 7:553-558. Gow R, O'Bryan M, Canny B, Ooi G, Hedger M (2001) Differential effects of dexamethasone treatment on lipopolysaccharide-induced testicular inflammation and reproductive hormone inhibition in adult rats. Journal of endocrinology 168:193-201. Goyal HO (1985) Morphology of the bovine epididymis. Developmental Dynamics 172:155-172. Goyal HO, Williams CS (1991) Regional differences in the morphology of the goat epididymis: a light microscopic and ultrastructural study. Developmental Dynamics 190:349-369. Grant J (1958) The passage of trypan blue through the epididymis and its uptake by this organ. In: Proc Soc Stud Fertil, p 95. Graziadei GM, Stanley R, Graziadei P (1980) The olfactory marker protein in the olfactory system of the mouse during development. Neuroscience 5:1239-1252. Graziadei P (1973) Cell dynamics in the olfactory mucosa. Tissue and Cell 5:113-131. Graziadei P, Graziadei GM (1979) Neurogenesis and neuron regeneration in the olfactory system of mammals. I. Morphological aspects of differentiation and structural organization of the olfactory sensory neurons. Journal of neurocytology 8:1-18. Graziadei P, Graziadei GM (1985) Neurogenesis and plasticity of the olfactory sensory neurons. Annals of the New York Academy of Sciences 457:127-142. Gregory M, Dufresne J, Hermo L, Cyr DG (2001) Claudin-1 is not restricted to tight junctions in the rat epididymis** This work was supported by the Toxic Substances Research Initiative (to DC and LH) and the Medical Research Council of Canada (to LH). Endocrinology 142:854-863. Griffin CA, Kafadar KA, Pavlath GK (2009) MOR23 promotes muscle regeneration and regulates cell adhesion and migration. Developmental cell 17:649-661. Grillo M, Margolis F (1990) Use of reverse transcriptase polymerase chain reaction to monitor expression of intronless genes. Biotechniques 9:262, 264, 266-268. Griswold MD (1998) The central role of Sertoli cells in spermatogenesis. In: Seminars in cell & developmental biology, pp 411-416: Elsevier. Griswold S, Behringer R (2009) Fetal Leydig cell origin and development. Sexual Development 3:1-15. Groneberg DA, Welker P, Fischer TC, Dinh QT, Grützkau A, Peiser C, Wahn U, Henz BM, Fischer A (2003) Down-regulation of vasoactive intestinal polypeptide receptor expression in atopic dermatitis. Journal of allergy and clinical immunology 111:1099-1105. Gross KJ, Pothoulakis C (2007) Role of neuropeptides in inflammatory bowel disease. Inflammatory bowel diseases 13:918-932. Gruber BL, Marchese MJ, Kew RR (1994) Transforming growth factor-beta 1 mediates mast cell chemotaxis. The Journal of Immunology 152:5860-5867.

243 Gu HF (2010) AC3: a novel gene plays a role in the regulation of body weight. The Open Diabetes Journal 3. Guidobaldi HA, Teves ME, Uñates DR, Giojalas LC (2012) Sperm transport and retention at the fertilization site is orchestrated by a chemical guidance and oviduct movement. Reproduction 143:587-596. Guo Z, Packard A, Krolewski RC, Harris MT, Manglapus GL, Schwob JE (2010) Expression of pax6 and sox2 in adult olfactory epithelium. Journal of Comparative Neurology 518:4395- 4418. Gurish MF, Boyce JA (2006) Mast cells: ontogeny, homing, and recruitment of a unique innate effector cell. Journal of Allergy and Clinical Immunology 117:1285-1291. Gutièrrez-Mecinas M, Crespo C, Blasco-Ibáñez JM, Gracia-Llanes FJ, Marqués-Marí AI, Martínez-Guijarro FJ (2005) Characterization of somatostatin-and cholecystokinin- immunoreactive periglomerular cells in the rat olfactory bulb. Journal of Comparative Neurology 489:467-479. Gwathmey TM, Ignotz GG, Suarez SS (2003) PDC-109 (BSP-A1/A2) Promotes Bull Sperm Binding to Oviductal Epithelium In Vitro and May Be Involved in Forming the Oviductal Sperm Reservoir 1. Biology of reproduction 69:809-815. Gwathmey TM, Ignotz GG, Mueller JL, Manjunath P, Suarez SS (2006) Bovine seminal plasma proteins PDC-109, BSP-A3, and BSP-30-kDa share functional roles in storing sperm in the oviduct 1. Biology of reproduction 75:501-507. Haas H, Panula P (2003) The role of histamine and the tuberomamillary nucleus in the nervous system. Nature reviews Neuroscience 4:121. Hack I, Bancila M, Loulier K, Carroll P, Cremer H (2002) Reelin is a detachment signal in tangential chain-migration during postnatal neurogenesis. Nature neuroscience 5:939. Hadley K, Orlandi RR, Fong KJ (2004) Basic anatomy and physiology of olfaction and taste. Otolaryngologic Clinics of North America 37:1115-1126. Haider SG (2004) Cell biology of Leydig cells in the testis. International review of cytology 233:181-241. Halasz N, Hökfelt T, Ljungdahl A, Johansson O, Goldstein M (1976) Dopamine neurons in the olfactory bulb. Advances in biochemical psychopharmacology 16:169-177. Halász N, Johansson O, Hökfelt T, Ljungdahl Å, Goldstein M (1981) Immunohistochemical identification of two types of dopamine neuron in the rat olfactory bulb as seen by serial sectioning. Journal of neurocytology 10:251-259. Hales DB (2007) Regulation of Leydig Cell Function as it Pertains to the Inflammatory Response. In: The Leydig Cell in Health and Disease, pp 305-321: Springer. Halova I, Draberova L, Draber P (2012) Mast cell chemotaxis–chemoattractants and signaling pathways. Frontiers in immunology 3. Hamilton DW, Jones AL, Fawcett DW (1969) Cholesterol biosynthesis in the mouse epididymis and ductus deferens: a biochemical and morphological study. Biology of reproduction 1:167-184. Hammerstedt RH (1981) Monitoring the metabolic rate of germ cells and sperm. In: Reproductive Processes and Contraception, pp 353-391: Springer. Hanchate NK, Kondoh K, Lu Z, Kuang D, Ye X, Qiu X, Pachter L, Trapnell C, Buck LB (2015) Single-cell transcriptomics reveals receptor transformations during olfactory neurogenesis. Science 350:1251-1255. Hanoune J, Defer N (2001) Regulation and role of adenylyl cyclase isoforms. Annual review of pharmacology and toxicology 41:145-174. Hardy AB, Aïoun J, Baly C, Julliard KA, Caillol M, Salesse R, Duchamp-Viret P (2005) Orexin A modulates mitral cell activity in the rat olfactory bulb: patch-clamp study on slices and immunocytochemical localization of orexin receptors. Endocrinology 146:4042-4053. Hartman BK, Margolis FL (1975) Immunofluorescence localization of the olfactory marker protein. Brain research 96:176-180. Hartmann K, Henz BM, Krüger-Krasagakes S, Köhl J, Burger R, Guhl S, Haase I, Lippert U, Zuberbier T (1997) C3a and C5a stimulate chemotaxis of human mast cells. Blood 89:2863-2870. Harvima I, Naukkarinen A, Paukkonen K, Harvima R, Aalto M-L, Schwartz L, Horsmanheimo M (1993) Mast cell tryptase and chymase in developing and mature psoriatic lesions. Archives of dermatological research 285:184-192.

244 Hashimoto J, Nagai T, Takaba H, Yamamoto M, Miyake K (1988) Increased mast cells in the limiting membrane of seminiferous tubules in testes of patients with idiopathic infertility. Urologia internationalis 43:129-132. Head JR, Billingham RE (1985) Immune privilege in the testis. II. Evaluation of potential local factors. Transplantation 40:269-275. Head JR, Neaves WB, Billingham RE (1983) Immune privilege in the testis. I. Basic parameters of allograft survival. Transplantation 36:423-431. Hedger M, Meinhardt A (2000) Local regulation of T cell numbers and -inhibiting activity in the interstitial tissue of the adult rat testis. Journal of reproductive immunology 48:69-80. Hedger M, Klug J, Fröhlich S, Müller R, Meinhardt A (2005) Regulatory cytokine expression and interstitial fluid formation in the normal and inflamed rat testis are under Leydig cell control. Journal of andrology 26:379-386. Hedger MP (2002) Macrophages and the immune responsiveness of the testis. Journal of reproductive immunology 57:19-34. Hedger MP (2011) Toll-like receptors and signalling in spermatogenesis and testicular responses to inflammation—a perspective. Journal of reproductive immunology 88:130-141. Heindel JJ, Treinen KA (1989) Physiology of the male reproductive system: endocrine, paracrine and autocrine regulation. Toxicologic pathology 17:411-445. Hellevuo K, Yoshimura M, Mons N, Hoffman PL, Cooper D, Tabakoff B (1995) The characterization of a novel human adenylyl cyclase which is present in brain and other tissues. Journal of Biological Chemistry 270:11581-11589. Hempstead J, Morgan J (1983) Monoclonal antibodies to the rat olfactory sustentacular cell. Brain research 288:289-295. Hendriksen E, van Bergeijk D, Oosting RS, Redegeld FA (2017) Mast cells in neuroinflammation and brain disorders. Neuroscience & Biobehavioral Reviews 79:119-133. Hermo L, Robaire B (2002) Epididymal cell types and their functions. In: The epididymis: from molecules to clinical practice, pp 81-102: Springer. Hermo L, Dworkin J, Oko R (1988) Role of epithelial clear cells of the rat epididymis in the disposal of the contents of cytoplasmic droplets detached from spermatozoa. Developmental Dynamics 183:107-124. Herness S, Zhao F-l (2009) The neuropeptides CCK and NPY and the changing view of cell-to- cell communication in the taste bud. Physiology & behavior 97:581-591. Hevér H, Altdorfer K, Zelles T, Batbayar B, Fehér E (2013) Changes in the innervation of the taste buds in diabetic rats. Orvosi hetilap 154:443-448. Hibi H, Kato K, Mitsui K, Taki T, Yamada Y, Honda N, Fukatsu H, Yamamoto M (2002) Treatment of oligoasthenozoospermia with tranilast, a mast cell blocker, after long-term administration. Archives of andrology 48:451-459. Higashijima T, Burnier J, Ross E (1990) Regulation of Gi and Go by mastoparan, related amphiphilic peptides, and hydrophobic amines. Mechanism and structural determinants of activity. Journal of Biological Chemistry 265:14176-14186. Higashijima T, Uzu S, Nakajima T, Ross EM (1988) Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G proteins). Journal of Biological Chemistry 263:6491-6494. Hildebrandt MA, Hoffmann C, Sherrill–Mix SA, Keilbaugh SA, Hamady M, Chen YY, Knight R, Ahima RS, Bushman F, Wu GD (2009) High-fat diet determines the composition of the murine gut microbiome independently of obesity. Gastroenterology 137:1716-1724. e1712. Hill J, Lesniak M, Pert C, Roth J (1986) Autoradiographic localization of insulin receptors in rat brain: prominence in olfactory and limbic areas. Neuroscience 17:1127-1138. Hinsch KD, Schwerdel C, Habermann B, Schill WB, Müller-Schlösser F, Hinsch E (1995) Identification of heterotrimeric G proteins in human sperm tail membranes. Molecular reproduction and development 40:345-354. Ho H-C, Suarez SS (2001) Hyperactivation of mammalian spermatozoa: function and regulation. Reproduction 122:519-526. Ho K, Wolff CA, Suarez SS (2009) CatSper-null mutant spermatozoa are unable to ascend beyond the oviductal reservoir. Reproduction, Fertility and Development 21:345-350. Hoffer AP (1976) The ultrastructure of the ductus deferens in man. Biology of reproduction 14:425-443.

245 Hoffer AP, Hamilton DW, Fawcett DW (1973) The ultrastructure of the principal cells and intraepithelial leucocytes in the initial segment of the rat epididymis. The Anatomical Record 175:169-201. Hofstra CL, Desai PJ, Thurmond RL, Fung-Leung W-P (2003) Histamine H4 receptor mediates chemotaxis and calcium mobilization of mast cells. Journal of Pharmacology and Experimental Therapeutics 305:1212-1221. Holbrook EH, Szumowski KEM, Schwob JE (1995) An immunochemical, ultrastructural, and developmental characterization of the horizontal basal cells of rat olfactory epithelium. Journal of Comparative Neurology 363:129-146. Holstein AF, Maekawa M, Nagano T, Davidoff MS (1996) Myofibroblasts in the lamina propria of human seminiferous tubules are dynamic structures of heterogeneous phenotype. Archives of histology and cytology 59:109-125. Hong S-H, Goh S-H, Lee SJ, Hwang J-A, Lee J, Choi I-J, Seo H, Park J-H, Suzuki H, Yamamoto E (2013) Upregulation of adenylate cyclase 3 (ADCY3) increases the tumorigenic potential of cells by activating the CREB pathway. Oncotarget 4:1791. Horstmann E (1962) Elektronenmikroskopie des menschlichen Nebenhodenepithels. Zeitschrift für Zellforschung und Mikroskopische Anatomie 57:692-718. Hotamisligil GS (2006) Inflammation and metabolic disorders. Nature 444:860. Hrnciar J, Gabor D, Hrnciarova M, Okapcova J, Szentiványi M, Kurray P (1999) Relation between cytokines (TNF-alpha, IL-1 and 6) and homocysteine in android obesity and the phenomenon of insulin resistance syndromes. Vnitrni lekarstvi 45:11-16. Hsu C-L, Neilsen CV, Bryce PJ (2010) IL-33 is produced by mast cells and regulates IgE- dependent inflammation. PloS one 5:e11944. Hu H (1999) Chemorepulsion of neuronal migration by Slit2 in the developing mammalian forebrain. Neuron 23:703-711. Hu H, Tomasiewicz H, Magnuson T, Rutishauser U (1996) The role of polysialic acid in migration of olfactory bulb interneuron precursors in the subventricular zone. Neuron 16:735-743. Huard JM, Schwob JE (1995) Cell cycle of globose basal cells in rat olfactory epithelium. Developmental dynamics 203:17-26. Huard JM, Youngentob SL, Goldstein BJ, Luskin MB, Schwob JE (1998) Adult olfactory epithelium contains multipotent progenitors that give rise to neurons and non-neural cells. Journal of Comparative Neurology 400:469-486. Hudgins PM, Weiss GB (1968) Differential effects of calcium removal upon vascular smooth muscle contraction induced by norepinephrine, histamine and potassium. Journal of Pharmacology and Experimental Therapeutics 159:91-97. Hugli TE, Müller-Eberhard HJ (1978) Anaphylatoxins: C3a and C5a. Advances in immunology 26:1-53. Huhtaniemi I, Toppari J (1995) Endocrine, paracrine and autocrine regulation of testicular steroidogenesis. In: Tissue Renin-Angiotensin Systems, pp 33-54: Springer. Humar RA, Ojha P, Anand Kumar T (1990) Ultrastructure of paracriny/steroidogenesis in principal cells of the marmoset epididymis. Archives of andrology 24:167-175. Huntley J, Gooden C, Newlands G, Mackellar A, Lammas D, Wakelin D, Tuohy M, Woodbury R, Miller H (1990) Distribution of intestinal mast cell proteinase in blood and tissues of normal and Trichinella-intected mice. Parasite immunology 12:85-95. Hussein MR, Abou-Deif ES, Bedaiwy MA, Said TM, Mustafa MG, Nada E, Ezat A, Agarwal A (2005) Phenotypic characterization of the immune and mast cell infiltrates in the human testis shows normal and abnormal spermatogenesis. Fertility and sterility 83:1447-1453. Ibrahim MZ, Reder AT, Lawand R, Takash W, Sallouh-Khatib S (1996) The mast cells of the multiple sclerosis brain. Journal of neuroimmunology 70:131-138. Ichikawa H, Jacobowitz DM, Winsky L, Helke CJ (1991) Calretinin-immunoreactivity in vagal and glossopharyngeal sensory neurons of the rat: distribution and coexistence with putative transmitter agents. Brain research 557:316-321. Ignotz GG, Cho MY, Suarez SS (2007) Annexins Are Candidate Oviductal Receptors for Bovine Sperm Surface Proteins and Thus May Serve to Hold Bovine Sperm in the Oviductal Reservoir 1. Biology of reproduction 77:906-913. Ikeda R (1906) Über das Epithel im Nebenhoden des Menschen. Anat Anz 29:76. Iosub R, Klug J, Fijak M, Schneider E, Fröhlich S, Blumbach K, Wennemuth G, Sommerhoff C, Steinhoff M, Meinhardt A (2006) Development of testicular inflammation in the rat involves activation of proteinase-activated receptor-2. The Journal of pathology 208:686- 698.

246 Ishii T, Serizawa S, Kohda A, Nakatani H, Shiroishi T, Okumura K, Iwakura Y, Nagawa F, Tsuboi A, Sakano H (2001) Monoallelic expresion of the odourant receptor gene and axonal projection of olfactory sensory neurones. Genes to Cells 6:71-78. Ishijima S (2011) Dynamics of flagellar force generated by a hyperactivated spermatozoon. Reproduction 142:409-415. Isnard-Bagnis C, Da Silva N, Beaulieu V, Alan S, Brown D, Breton S (2003) Detection of ClC-3 and ClC-5 in epididymal epithelium: immunofluorescence and RT-PCR after LCM. American Journal of Physiology-Cell Physiology 284:C220-C232. Isobe T, Minoura H, Tanaka K, Shibahara T, Hayashi N, Toyoda N (2002) The effect of RANTES on human sperm chemotaxis. Human Reproduction 17:1441-1446. Itakura S, Ohno K, Ueki T, Sato K, Kanayama N (2006) Expression of Golf in the rat placenta: Possible implication in olfactory receptor transduction. Placenta 27:103-108. Itoh M (2017) Testicular Autoimmunity: A Cause of Male Infertility: Springer. Ivic L, Pyrski MM, Margolis JW, Richards LJ, Firestein S, Margolis FL (2000) Adenoviral vector- mediated rescue of the OMP-null phenotype in vivo. Nature neuroscience 3:1113. Iwai N, Zhou Z, Roop DR, Behringer RR (2008) Horizontal basal cells are multipotent progenitors in normal and injured adult olfactory epithelium. Stem cells 26:1298-1306. Jackson NE, Wang HW, Tedla N, McNeil HP, Geczy CL, Collins A, Grimm MC, Hampartzoumian T, Hunt JE (2005) IL-15 induces mast cell migration via a pertussis toxin-sensitive receptor. European journal of immunology 35:2376-2385. Jacobowitz DM, Winsky L (1991) Immunocytochemical localization of calretinin in the forebrain of the rat. Journal of comparative neurology 304:198-218. Jacobson MF (2002) Nutrition, physical activity, and obesity. The Lancet 360:1250. Jaiswal BS, Eisenbach M (2002) Capacitation. Fertilization:57-117. Jaiswal BS, Cohen-Dayag A, Tur-Kaspa I, Eisenbach M (1998) Sperm capacitation is, after all, a prerequisite for both partial and complete acrosome reaction. FEBS letters 427:309-313. James PS, Hennessy C, Berge T, Jones R (2004) Compartmentalisation of the sperm plasma membrane: a FRAP, FLIP and SPFI analysis of putative diffusion barriers on the sperm head. Journal of cell science 117:6485-6495. Jang W, Youngentob SL, Schwob JE (2003) Globose basal cells are required for reconstitution of olfactory epithelium after methyl bromide lesion. Journal of Comparative Neurology 460:123-140. Jang W, Kim KP, Schwob JE (2007) Nonintegrin laminin receptor precursor protein is expressed on olfactory stem and progenitor cells. Journal of Comparative Neurology 502:367-381. Jankovski A, Sotelo C (1996) Subventricular zone-olfactory bulb migratory pathway in the adult mouse: Cellular composition and specificity as determined by heterochronic and heterotopic transplantation. Journal of Comparative Neurology 371:376-396. Janssens S, Heemskerk MM, Van Den Berg SA, van Riel NA, Nicolay K, van Dijk KW, Prompers JJ (2015) Effects of low-stearate palm oil and high-stearate lard high-fat diets on rat liver lipid metabolism and glucose tolerance. Nutrition & metabolism 12:57. Jeffery P, Reid L (1975) New observations of rat airway epithelium: a quantitative and electron microscopic study. Journal of anatomy 120:295. Jégou B (1992) 3 The sertoli cell. Baillière's clinical endocrinology and metabolism 6:273-311. Jégou B, Pineau C (1995) Current aspects of autocrine and paracrine regulation of spermatogenesis. In: Tissue Renin-Angiotensin Systems, pp 67-86: Springer. Jenkins AD, Lechene CP, Howards SS (1980) Concentrations of seven elements in the intraluminal fluids of the rat seminiferous tubules, rete testis, and epididymis. Biology of Reproduction 23:981-987. Jensen BM, Beaven MA, Iwaki S, Metcalfe DD, Gilfillan AM (2008) Concurrent inhibition of Kit- and FcϵRI-mediated signaling: coordinated suppression of mast cell activation. Journal of Pharmacology and Experimental Therapeutics 324:128-138. Jensen LJ, Stuart-Tilley AK, Peters LL, Lux SE, Alper SL, Breton S (1999) Immunolocalization of AE2 anion exchanger in rat and mouse epididymis. Biology of reproduction 61:973-980. Jia Z, Yuan Y, Shi Q (1997) The transducing pathway of Ca2+ influx during progesterone-initiated acrosome reaction of guinea pig sperm. Sheng li xue bao:[Acta physiologica Sinica] 49:349-353. Johnson D, Krenger W (1992) Interactions of mast cells with the nervous system—recent advances. Neurochemical research 17:939-951.

247 Johnson D, Seeldrayers PA, Weiner HL (1988) The role of mast cells in demyelination. 1. Myelin proteins are degraded by mast cell proteases and myelin basic protein and P2 can stimulate mast cell degranulation. Brain research 444:195-198. Johnson EW, Eller PM, Jafek BW (1993) An immuno-electron microscopic comparison of olfactory marker protein localization in the supranuclear regions of the rat olfactory epithelium and vomeronasal organ neuroepithelium. Acta oto-laryngologica 113:766-771. Jones DT, Reed RR (1989) G (olf): an olfactory neuron specific-G protein involved in odorant signal transduction. Science 244:790-796. Jones R (1989) Membrane remodelling during sperm maturation in the epididymis. Oxford reviews of reproductive biology 11:285-337. Jones SE, Kelly DJ, Cox AJ, Zhang Y, Gow RM, Gilbert RE (2003) Mast cell infiltration and chemokine expression in progressive renal disease. Kidney international 64:906-913. Joo JI, Kim DH, Choi J-W, Yun JW (2010) Proteomic analysis for antiobesity potential of capsaicin on white adipose tissue in rats fed with a high fat diet. Journal of proteome research 9:2977-2987. Jourdan F (1975) Ultrastructure of the olfactory epithelium of the rat: polymorphism of the receptors. Comptes rendus hebdomadaires des séances de l'Académie des sciences Série D: Sciences naturelles 280:443. Jourdan KB, Mason NA, Long L, Philips PG, Wilkins MR, Morrell NW (2001) Characterization of adenylyl cyclase isoforms in rat peripheral pulmonary arteries. American Journal of Physiology-Lung Cellular and Molecular Physiology 280:L1359-L1369. Julliard A, Chaput M, Apelbaum A, Aime P, Mahfouz M, Duchamp-Viret P (2007) Changes in rat olfactory detection performance induced by orexin and leptin mimicking fasting and satiation. Behavioural brain research 183:123-129. Jungnickel MK, Marrero H, Birnbaumer L, Lémos JR, Florman HM (2001) Trp2 regulates entry of Ca 2+ into mouse sperm triggered by egg ZP3. Nature Cell Biology 3:499. Kadowaki T, Hara K, Yamauchi T, Terauchi Y, Tobe K, Nagai R (2003) Molecular mechanism of insulin resistance and obesity. Experimental Biology and Medicine 228:1111-1117. Kägi U, Chafouleas JG, Norman AW, Heizmann CW (1988) Developmental appearance of the Ca 2+-binding proteins parvalbumin, calbindin D-28K, S-100 proteins and calmodulin during testicular development in the rat. Cell and tissue research 252:359-365. Kajiya K, Inaki K, Tanaka M, Haga T, Kataoka H, Touhara K (2001) Molecular bases of odor discrimination: reconstitution of olfactory receptors that recognize overlapping sets of odorants. Journal of Neuroscience 21:6018-6025. Kang N, Koo J (2012) Olfactory receptors in non-chemosensory tissues. BMB reports 45:612- 622. Kang N, Kim H, Jae Y, Lee N, Ku CR, Margolis F, Lee EJ, Bahk YY, Kim M-S, Koo J (2015) Olfactory marker protein expression is an indicator of olfactory receptor-associated events in non-olfactory tissues. PloS one 10:e0116097. Karnik NS, Newman S, Kopf GS, Gerton GL (1992) Developmental expression of G protein α subunits in mouse spermatogenic cells: Evidence that Gαi is associated with the developing acrosome. Developmental biology 152:393-402. Kasturi SS, Tannir J, Brannigan RE (2008) The metabolic syndrome and male infertility. Journal of andrology 29:251-259. Kaupp UB, Kashikar ND, Weyand I (2008) Mechanisms of sperm chemotaxis. Annu Rev Physiol 70:93-117. Kawai K, Sugimoto K, Nakashima K, Miura H, Ninomiya Y (2000) Leptin as a modulator of sweet taste sensitivities in mice. Proceedings of the National Academy of Sciences 97:11044- 11049. Kawai T, Akira S (2006) TLR signaling. Cell death and differentiation 13:816. Keegan AD, Paul WE (1992) Multichain immune recognition receptors: similarities in structure and signaling pathways. Immunology today 13:63-68. Keller A, Margolis F (1975) Immunological studies of the rat olfactory marker protein. Journal of neurochemistry 24:1101-1106. Keller A, Vosshall LB (2008) Better smelling through genetics: mammalian odor perception. Current opinion in neurobiology 18:364-369. Kelsall MA (1966) Aging on mast cells and plasmacytes in the brain of hamsters. The Anatomical Record 154:727-739. Kempuraj D, Papadopoulou NG, Lytinas M, Huang M, Kandere-Grzybowska K, Madhappan B, Boucher W, Christodoulou S, Athanassiou A, Theoharides TC (2004) Corticotropin-

248 releasing hormone and its structurally related urocortin are synthesized and secreted by human mast cells. Endocrinology 145:43-48. Kerr DS, Von Dannecker LEC, Davalos M, Michaloski JS, Malnic B (2008) Ric-8B interacts with Gαolf and Gγ13 and co-localizes with Gαolf, Gβ1 and Gγ13 in the cilia of olfactory sensory neurons. Molecular and Cellular Neuroscience 38:341-348. Kerr J, De Kretser D (2006) Functional morphology of the testis. Endocrinology 5:3089-3120. Kerr J, Millar M, Maddocks S, Sharpe R (1993) Stage-dependent changes in spermatogenesis and Sertoli cells in relation to the onset of spermatogenic failure following withdrawal of testosterone. The Anatomical Record 235:547-559. Kerr J, Loveland K, O’bryan M, De Kretser D (2006) Cytology of the testis and intrinsic control mechanisms. Knobil and Neill’s physiology of reproduction 1:827-947. Khalil M, Ronda J, Weintraub M, Jain K, Silver R, Silverman A-J (2007) Brain mast cell relationship to neurovasculature during development. Brain research 1171:18-29. Kierszenbaum AL, Laura L (2007) Digestive glands. In “Histology and Cell Biology. ”. In: Elsevier, PA. Kirchhoff C (1998) Molecular characterization of epididymal proteins. Reviews of reproduction 3:86-95. Kishi K, Peng JY, Kakuta S, Murakami K, Kuroda M, Yokota S, Hayakawa S, Kuge T, Asayama T (1990) Migration of bipolar subependymal cells, precursors of the granule cells of the rat olfactory bulb, with reference to the arrangement of the radial glial fibers. Archives of histology and cytology 53:219-226. Kispélyi B, Lohinai Z, Altdorfer K, Fehér E (2014) Neuropeptide analysis of oral mucosa in diabetic rats. Neuroimmunomodulation 21:213-220. Kiyokage E, Pan Y-Z, Shao Z, Kobayashi K, Szabo G, Yanagawa Y, Obata K, Okano H, Toida K, Puche AC (2010) Molecular identity of periglomerular and short axon cells. Journal of Neuroscience 30:1185-1196. Kleene SJ (1994) Inhibition of olfactory cyclic nucleotide-activated current by calmodulin antagonists. British journal of pharmacology 111:469-472. Köberle M, Kaesler S, Kempf W, Wölbing F, Biedermann T (2012) Tetraspanins in mast cells. Frontiers in Immunology 3:106. Kohno S, Munoz J, Williams T, Teuscher C, Bernard C, Tung K (1983) Immunopathology of murine experimental allergic orchitis. The Journal of Immunology 130:2675-2682. Kondo S, Kagami S, Kido H, Strutz F, Muller GA, Kuroda Y (2001) Role of mast cell tryptase in renal interstitial fibrosis. Journal of the American Society of Nephrology 12:1668-1676. Koo JH, Gill S, Pannell LK, Menco BPM, Margolis JW, Margolis FL (2004) The interaction of Bex and OMP reveals a dimer of OMP with a short half-life. Journal of neurochemistry 90:102- 116. Kopf GS (1991) The mammalian sperm acrosome reaction and the acrosome reaction. Elements of mammalian fertilization:153-203. Kopf GS (2002) Signal transduction mechanisms regulating sperm acrosomal exocytosis-6. Kosaka K, Kosaka T (2007) Chemical properties of type 1 and type 2 periglomerular cells in the mouse olfactory bulb are different from those in the rat olfactory bulb. Brain research 1167:42-55. Kosaka K, Toida K, Aika Y, Kosaka T (1998) How simple is the organization of the olfactory glomerulus?: the heterogeneity of so-called periglomerular cells. Neuroscience research 30:101-110. Kosaka K, Aika Y, Toida K, Heizmann CW, Hunziker W, Jacobowitz DM, Nagatsu I, Streit P, Visser TJ, Kosaka T (1995) Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb. Neuroscience research 23:73-88. Kosaka T, Kosaka K (2005) Structural organization of the glomerulus in the main olfactory bulb. Chemical senses 30:i107-i108. Kosaka T, Kosaka K (2011) “Interneurons” in the olfactory bulb revisited. Neuroscience research 69:93-99. Koster N, Norman A, Richtand N, Nickell W, Puche A, Pixley S, Shipley M (1999) Olfactory receptor neurons express D2 dopamine receptors. Journal of Comparative Neurology 411:666-673. Kratskin IL, Belluzzi O (2003) Anatomy and neurochemistry of the olfactory bulb. In: Handbook of olfaction and gustation: CRC Press. Krautwurst D, Yau K-W, Reed RR (1998) Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell 95:917-926.

249 Krieger J, Mameli M, Breer H (1997) Elements of the olfactory signaling pathways in insect antennae. Invertebrate neuroscience 3:137-144. Krishnaswamy G, Chi DS (2006) Mast cells: methods and protocols: Springer Science & Business Media. Krystel-Whittemore M, Dileepan KN, Wood JG (2016) Mast cell: a multi-functional master cell. Frontiers in immunology 6:620. Ku JH, Kim Y, Jeon Y, Lee N (1999) The preventive effect of systemic treatment with interferon- alpha2B for infertility from mumps orchitis. BJU international 84:839-842. Kudrycki K, Stein-Izsak C, Behn C, Grillo M, Akeson R, Margolis F (1993) Olf-1-: characterization of an olfactory neuron-specific promoter motif. Molecular and cellular biology 13:3002-3014. Kuehn HS, Gilfillan AM (2007) G protein-coupled receptors and the modification of FcɛRI- mediated mast cell activation. Immunology letters 113:59-69. Kulka M, Sheen CH, Tancowny BP, Grammer LC, Schleimer RP (2008) Neuropeptides activate human mast cell degranulation and chemokine production. Immunology 123:398-410. Kuster CE, Hess RA, Althouse GC (2004) Immunofluorescence reveals ubiquitination of retained distal cytoplasmic droplets on ejaculated porcine spermatozoa. Journal of andrology 25:340-347. Kwon HJ, Koo JH, Zufall F, Leinders-Zufall T, Margolis FL (2009) Ca2+ Extrusion by NCX Is Compromised in Olfactory Sensory Neurons of OMP−/− Mice. PLoS One 4:e4260. Kwon RY, Temiyasathit S, Tummala P, Quah CC, Jacobs CR (2010) Primary cilium-dependent mechanosensing is mediated by adenylyl cyclase 6 and cyclic AMP in bone cells. The FASEB journal 24:2859-2868. Lacroix MC, Badonnel K, Meunier N, Tan F, Poupon C, Durieux D, Monnerie R, Baly C, Congar P, Salesse R (2008) Expression of insulin system in the olfactory epithelium: first approaches to its role and regulation. Journal of neuroendocrinology 20:1176-1190. Lagunoff D, Martin T, Read G (1983) Agents that release histamine from mast cells. Annual review of pharmacology and toxicology 23:331-351. Lambracht-Hall M, Dimitriadou V, Theoharides TC (1990) Migration of mast cells in the developing rat brain. Developmental Brain Research 56:151-159. Lancet D, Ben-Arie N (1993) Olfactory receptors. Current Biology 3:668-674. Langlais PJ, McRee RC, Nalwalk JA, Hough LB (2002) Depletion of brain histamine produces regionally selective protection against thiamine deficiency-induced lesions in the rat. Metabolic brain disease 17:199-210. Le Magnen J (1956) Effets sur la prise alimentaire du rat blanc des administrations postprandiales d'insuline et le mécanisme des appétits caloriques. Journal de Physiologie 48:789-802. Le Magnen J (2001) My scientific life: 40 years at the College de France. Neuroscience & Biobehavioral Reviews 25:375-394. Le Tortorec A, Denis H, Satie A-P, Patard J-J, Ruffault A, Jégou B, Dejucq-Rainsford N (2008) Antiviral responses of human Leydig cells to mumps virus infection or poly I: C stimulation. Human reproduction 23:2095-2103. Le Trong H, Neurath H, Woodbury RG (1987) Substrate specificity of the chymotrypsin-like protease in secretory granules isolated from rat mast cells. Proceedings of the National Academy of Sciences 84:364-367. Leal-Berumen I, Conlon P, Marshall JS (1994) IL-6 production by rat peritoneal mast cells is not necessarily preceded by histamine release and can be induced by bacterial lipopolysaccharide. The Journal of Immunology 152:5468-5476. Lee AC, He J, Ma M (2011a) Olfactory marker protein is critical for functional maturation of olfactory sensory neurons and development of mother preference. Journal of Neuroscience 31:2974-2982. Lee CH, Park D, Wu D, Rhee S, Simon MI (1992) Members of the Gq alpha subunit gene family activate phospholipase C beta isozymes. Journal of Biological Chemistry 267:16044- 16047. Lee YS, Li P, Huh JY, Hwang IJ, Lu M, Kim JI, Ham M, Talukdar S, Chen A, Lu WJ (2011b) Inflammation is necessary for long-term but not short-term high-fat diet–induced insulin resistance. Diabetes 60:2474-2483. Légaré C, Bérubé B, Boué F, Lefièvre L, Morales CR, El-Alfy M, Sullivan R (1999) Hamster sperm antigen P26h is a phosphatidylinositol-anchored protein. Molecular reproduction and development 52:225-233.

250 Lejeune H, Chuzel F, Thomas T, Avallet O, Habert R, Durand P, Saez J (1996) Paracrine regulation of Leydig cells. In: Annales d'endocrinologie, pp 55-63. Lemanske RF, Kaliner MA (1983) Late phase allergic reactions. International journal of dermatology 22:401-409. Leung CT, Coulombe PA, Reed RR (2007) Contribution of olfactory neural stem cells to tissue maintenance and regeneration. Nature neuroscience 10:720-726. Leurs R, Smit M, Timmerman H (1995) Molecular pharmacological aspects of histamine receptors. Pharmacology & therapeutics 66:413-463. Leveteau J, MacLeod P (1966) Olfactory discrimination in the rabbit olfactory glomerulus. Science 153:175-176. Levine N, Kelly H (1978) Measurement of pH in the rat epididymis in vivo. Journal of reproduction and fertility 52:333-335. Li J, Ishii T, Feinstein P, Mombaerts P (2004) Odorant receptor gene choice is reset by nuclear transfer from mouse olfactory sensory neurons. Nature 428:393. Li N, Wang T, Han D (2012) Structural, cellular and molecular aspects of immune privilege in the testis. Frontiers in immunology 3:152. Li Z, Danis V, Brooks P (1993) Effect of gonadal steroids on the production of IL-1 and IL-6 by blood mononuclear cells in vitro. Clinical and experimental rheumatology 11:157-162. Liang Y, Li Z, Liang S, Li Y, Yang L, Lu M, Gu H, Xia N (2016) Hepatic adenylate cyclase 3 is upregulated by Liraglutide and subsequently plays a protective role in insulin resistance and obesity. Nutrition & diabetes 6:e191. Liew FY, Pitman NI, McInnes IB (2010) Disease-associated functions of IL-33: the new kid in the IL-1 family. Nature reviews Immunology 10:103. Lindstedt KA, Wang Y, Shiota N, Saarinen J, Hyytiäinen M, Kokkonen JO, Keski-Oja J, Kovanen PT (2001) Activation of paracrine TGF-β1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase. The FASEB Journal 15:1377- 1388. Lishko PV, Botchkina IL, Kirichok Y (2011) Progesterone activates the principal Ca2+ channel of human sperm. Nature 471:387-391. Lishko PV, Botchkina IL, Fedorenko A, Kirichok Y (2010) Acid extrusion from human spermatozoa is mediated by flagellar voltage-gated proton channel. Cell 140:327-337. Lishko PV, Kirichok Y, Ren D, Navarro B, Chung J-J, Clapham DE (2012) The control of male fertility by spermatozoan ion channels. Annual review of physiology 74:453-475. Litscher ES, Williams Z, Wassarman PM (2009) Zona pellucida glycoprotein ZP3 and fertilization in mammals. Molecular reproduction and development 76:933-941. Liu G, Rao Y (2003) Neuronal migration from the forebrain to the olfactory bulb requires a new attractant persistent in the olfactory bulb. Journal of Neuroscience 23:6651-6659. Liu J, Divoux A, Sun J, Zhang J, Clément K, Glickman JN, Sukhova GK, Wolters PJ, Du J, Gorgun CZ (2009) Genetic deficiency and pharmacological stabilization of mast cells reduce diet- induced obesity and diabetes in mice. Nature medicine 15:940-945. Liu WL, Shipley MT (1994) Intrabulbar associational system in the rat olfactory bulb comprises cholecystokinin-containing tufted cells that synapse onto the dendrites of GABAergic granule cells. Journal of Comparative Neurology 346:541-558. Livera G, Xie F, Garcia M, Jaiswal B, Chen J, Law E, Storm D, Conti M (2005) Inactivation of the mouse adenylyl cyclase 3 gene disrupts male fertility and spermatozoon function. Molecular endocrinology 19:1277-1290. Lledo P-M, Merkle FT, Alvarez-Buylla A (2008) Origin and function of olfactory bulb interneuron diversity. Trends in neurosciences 31:392-400. Lo KC, Lei Z, Rao CV, Beck J, Lamb DJ (2004) De novo testosterone production in luteinizing hormone receptor knockout mice after transplantation of leydig stem cells. Endocrinology 145:4011-4015. Lois C, Alvarez-Buylla A (1994) Long-distance neuronal migration in the adult mammalian brain. Science 264:1145-1148. Lois C, Garcia-Verdugo J-M, Alvarez-Buylla A (1996) Chain migration of neuronal precursors. Science 271:978-981. Loper HB, La Sala M, Dotson C, Steinle N (2015) Taste perception, associated hormonal modulation, and nutrient intake. Nutrition reviews 73:83-91. Lording D, De Kretser D (1972) Comparative ultrastructural and histochemical studies of the interstitial cells of the rat testis during fetal and postnatal development. Journal of Reproduction and Fertility 29:261-269.

251 Lovercamp K, Safranski T, Fischer K, Manandhar G, Sutovsky M, Herring W, Sutovsky P (2007) High resolution light microscopic evaluation of boar semen quality sperm cytoplasmic droplet retention in relationship with boar fertility parameters. Archives of andrology 53:219-228. Lowe G, Gold GH (1993) Contribution of the ciliary cyclic nucleotide-gated conductance to olfactory transduction in the salamander. The Journal of Physiology 462:175. Lowman MA, Rees PH, Benyon RC, Church MK (1988) Human mast cell heterogeneity: histamine release from mast cells dispersed from skin, lung, adenoids, tonsils, and colon in response to IgE-dependent and nonimmunologic stimuli. Journal of Allergy and Clinical Immunology 81:590-597. Luger T, Lotti T (1998) Neuropeptides: role in inflammatory skin diseases. Journal of the European Academy of Dermatology and Venereology 10:207-211. Luskin MB (1993) Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone. Neuron 11:173-189. Luskin MB, Boone MS (1994) Rate and pattern of migration of lineally-related olfactory bulb interneurons generated postnatally in the subventricular zone of the rat. Chemical Senses 19:695-714. Lyman M, Lloyd DG, Ji X, Vizcaychipi MP, Ma D (2014) Neuroinflammation: the role and consequences. Neuroscience research 79:1-12. Ma M, Grosmaitre X, Iwema CL, Baker H, Greer CA, Shepherd GM (2003) Olfactory signal transduction in the mouse septal organ. Journal of Neuroscience 23:317-324. Macrides F, Schneider SP (1982) Laminar organization of mitral and tufted cells in the main olfactory bulb of the adult hamster. Journal of Comparative Neurology 208:419-430. Maddocks S, Setchell B (1988) The rejection of thyroid allografts in the ovine testis. Immunology & Cell Biology 66:1-8. Majeed S (1994) Mast cell distribution in rats. Arzneimittel-forschung 44:370-374. Malnic B, Hirono J, Sato T, Buck LB (1999) Combinatorial receptor codes for odors. Cell 96:713- 723. Maneely R (1959) Epididymal structure and function: a historical and critical review. Acta Zoologica 40:1-21. Manglapus GL, Youngentob SL, Schwob JE (2004) Expression patterns of basic helix-loop-helix transcription factors define subsets of olfactory progenitor cells. Journal of Comparative Neurology 479:216-233. Mania-Farnell B, Farbman AI (1990) Immunohistochemical localization of guanine nucleotide- binding proteins in rat olfactory epithelium during development. Developmental Brain Research 51:103-112. Manova K, Bachvarova R, Huang E, Sanchez S, Pronovost S, Velazquez E, McGuire B, Besmer P (1992) C-kit receptor and ligand expression in postnatal development of the mouse cerebellum suggests a function for c-kit in inhibitory interneurons. Journal of Neuroscience 12:4663-4676. Margolis F (1980) A marker protein for the olfactory chemoreceptor neuron. Proteins of the nervous system 2:59-84. Margolis FL (1972) A brain protein unique to the olfactory bulb. Proceedings of the National Academy of Sciences 69:1221-1224. Margolis FL, Tarnoff JF (1973) Site of biosynthesis of the mouse brain olfactory bulb protein. Journal of Biological Chemistry 248:451-455. Marquis NR, Fritz IB (1965) The distribution of carnitine, acetylcarnitine, and carnitine acetyltransferase in rat tissues. Journal of Biological Chemistry 240:2193-2196. Marshall JS, Gauldie J, Nielsen L, Bienenstock J (1993) Leukemia inhibitory factor production by rat mast cells. European journal of immunology 23:2116-2120. Martan J, Risley PL (1963) The epididymis of mated and unmated rats. Journal of Morphology 113:1-15. Martin B, Shin Y-K, White CM, Ji S, Kim W, Carlson OD, Napora JK, Chadwick W, Chapter M, Waschek JA (2010) Vasoactive intestinal peptide null mice demonstrate enhanced sweet taste preference, dysglycemia and reduced taste bud leptin receptor expression. Diabetes. Martínez-García F, Regadera J, Cobo P, Palacios J, Paniagua R, Nistal M (1995) The apical mitochondria-rich cells of the mammalian epididymis. Andrologia 27:195-206.

252 Mason HA, Ito S, Corfas G (2001) Extracellular signals that regulate the tangential migration of olfactory bulb neuronal precursors: inducers, inhibitors, and repellents. Journal of Neuroscience 21:7654-7663. Mason KE, Shaver SL (1952) Some functions of the caput epididymis. Annals of the New York Academy of Sciences 55:585-593. Matafome P, Santos-Silva D, Sena C, Seiça R (2013) Common mechanisms of dysfunctional adipose tissue and obesity-related cancers. Diabetes/metabolism research and reviews 29:285-295. Matarazzo V, Clot-Faybesse O, Marcet B, Guiraudie-Capraz G, Atanasova B, Devauchelle G, Cerutti M, Etiévant P, Ronin C (2005) Functional characterization of two human olfactory receptors expressed in the baculovirus Sf9 insect cell system. Chemical senses 30. Matsson L (1992) Mast cell heterogeneity in various oral mucosal sites in the rat. Archives of oral biology 37:445-450. Matsuki S, Sasagawa I, Suzuki Y, Yazawa H, Tateno T, Hashimoto T, Nakada T, Saito H, Hiroi M (2000) The use of ebastine, a mast cell blocker, for treatment of oligozoospermia. Archives of andrology 44:129-132. Matsuoka M, Osada T, Yoshida-Matsuoka J, Ikai A, Ichikawa M, Norita M, Costanzo RM (2002) A comparative immunocytochemical study of development and regeneration of chemosensory neurons in the rat vomeronasal system. Brain research 946:52-63. Mayerhofer A (2007) Neuronal Signaling Molecules and Leydig Cells. In: The Leydig Cell in Health and Disease, pp 291-304: Springer. Mayerhofer A, Seidl K, Lahr G, Bitter-Suermann D, Christoph A, Barthels D, Wille W, Gratzl M (1992) Leydig cells express neural cell adhesion molecules in vivo and in vitro. Biology of reproduction 47:656-664. Mayorga LS, Tomes CN, Belmonte SA (2007) Acrosomal exocytosis, a special type of regulated secretion. IUBMB life 59:286-292. McCurdy J, Lin T-J, Marshall JS (2001) Toll-like receptor 4-mediated activation of murine mast cells. Journal of leukocyte biology 70:977-984. Mehlem A, Hagberg CE, Muhl L, Eriksson U, Falkevall A (2013) Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. Nature protocols 8:1149- 1154. Meineke V, Frungieri MB, Jessberger B, Vogt H-J, Mayerhofer A (2000) Human testicular mast cells contain tryptase: increased mast cell number and altered distribution in the testes of infertile men. Fertility and sterility 74:239-244. Meinhardt A, Hedger MP (2011) Immunological, paracrine and endocrine aspects of testicular immune privilege. Molecular and cellular endocrinology 335:60-68. Meinhardt A, Bacher M, McFarlane JR, Metz CN, Seitz J, Hedger MP, De Kretser D, Bucala R (1996) Macrophage migration inhibitory factor production by Leydig cells: evidence for a role in the regulation of testicular function. Endocrinology 137:5090-5095. Meinhardt A, Bacher M, Metz C, Bucala R, Wreford N, Lan H, Atkins R, Hedger M (1998) Local regulation of macrophage subsets in the adult rat testis: examination of the roles of the seminiferous tubules, testosterone, and macrophage-migration inhibitory factor. Biology of reproduction 59:371-378. Meininger CJ, Yano H, Rottapel R, Bernstein A, Zsebo KM, Zetter B (1992) The c-kit receptor ligand functions as a mast cell chemoattractant. Blood 79:958-963. Meizel S (1984) The importance of hydrolytic enzymes to an exocytotic event, the mammalian sperm acrosome reaction. Biological Reviews 59:125-157. Meizel S (2004) The sperm, a neuron with a tail:‘neuronal’receptors in mammalian sperm. Biological Reviews 79:713-732. Melaine N, Liénard M-O, Guillaume E, Ruffault A, Dejucq-Rainsford N, Jégou B (2003) Production of the antiviral proteins 2′ 5′ oligoadenylate synthetase, PKR and Mx in interstitial cells and spermatogonia. Journal of reproductive immunology 59:53-60. Menco B (1995) Freeze-Fracture, Deep-Etch, and Freeze-Substitution Studies of Olfactory Epithelia, With Special Emphasis on Immunocytochemical Variables. Microscopy research and technique 32:337-356. Menco BP, Farbman AI (1985) Genesis of cilia and microvilli of rat nasal epithelia during pre-natal development. I. Olfactory epithelium, qualitative studies. Journal of cell science 78:283- 310. Menco BP, Tekula FD, Farbman AI, Danho W (1994) Developmental expression of G-proteins and adenylyl cyclase in peripheral olfactory systems. Light microscopic and freeze-

253 substitution electron microscopic immunocytochemistry. Journal of neurocytology 23:708-727. Menco BPM (1989) Electron-microscopic demonstration of olfactory-marker protein with protein G-gold in freeze-substituted, Lowicryl K11M-embedded rat olfactory-receptor cells. Cell and tissue research 256:275-281. Menco BPM (1992) Lectins bind differentially to cilia and microvilli of major and minor cell populations in olfactory and nasal respiratory epithelia. Microscopy research and technique 23:181-199. Menco BPM (1994) Ultrastructural aspects of olfactory transduction and perireceptor events. In: Seminars in cell biology, pp 11-24: Elsevier. Menco BPM (1997) Ultrastructural aspects of olfactory signaling. Chemical senses 22:295-311. Menco BPM, Jackson J (1997) Cells resembling hair cells in developing rat olfactory and nasal respiratory epithelia. Tissue and Cell 29:707-713. Menco BPM, Morrison EE (2003) Morphology of the mammalian olfactory epithelium: form, fine structure, function, and pathology. Neurological disease and therapy 57:17-50. Menco BPM, Cunningham AM, Qasba P, Levy N, Reed RR (1997) Putative odour receptors localize in cilia of olfactory receptor cells in rat and mouse: a freeze-substitution ultrastructural study. Journal of neurocytology 26:691-706. Merlet F, Weinstein LS, Goldsmith PK, Rarick T, Hall JL, Bisson J-P, de Mazancourt P (1999) Identification and localization of G protein subunits in human spermatozoa. Molecular human reproduction 5:38-45. Metcalfe DD, Boyce JA (2006) Mast cell biology in evolution. Journal of allergy and clinical immunology 117:1227-1229. Metcalfe DD, Baram D, Mekori YA (1997) Mast cells. Physiological reviews 77:1033-1079. Metz M, Maurer M (2007) Mast cells–key effector cells in immune responses. Trends in immunology 28:234-241. Michaloudi H, Grivas I, Batzios C, Chiotelli M, Papadopoulos GC (2003) Parallel development of blood vessels and mast cells in the lateral geniculate nuclei. Developmental brain research 140:269-276. Michetti F, Lauriola L, Rende M, Stolfi VM, Battaglia F, Cocchia D (1985) S-100 protein in the testis. Cell and tissue research 240:137-142. Milho R, Frederico B, Efstathiou S, Stevenson PG (2012) A heparan-dependent herpesvirus targets the olfactory neuroepithelium for host entry. PLoS pathogens 8:e1002986. Miller H, Huntley J, Newlands G, Mackellar A, Irvine J, Haig D, MacDonald A, Lammas A, Wakelin D, Woodbury R (1989) Mast cell granule proteases in mouse and rat: a guide to mast cell heterogeneity and activation in the gastrointestinal tract. Mast cell and basophil differentiation and function in health and disease Raven Press, New York:81-91. Miller M, Andringa A, Evans J, Hastings L (1995) Microvillar cells of the olfactory epithelium: morphology and regeneration following exposure to toxic compounds. Brain research 669:1-9. Mombaerts P (1996) Targeting olfaction. Current opinion in neurobiology 6:481-486. Mombaerts P (2004) Odorant receptor gene choice in olfactory sensory neurons: the one receptor–one neuron hypothesis revisited. Current opinion in neurobiology 14:31-36. Mombaerts P, Wang F, Dulac C, Chao SK, Nemes A, Mendelsohn M, Edmondson J, Axel R (1996) Visualizing an olfactory sensory map. Cell 87:675-686. Mons N, Yoshimura M, Cooper DM (1993) Discrete expression of Ca2+/calmodulin-sensitive and Ca2+-insensitive adenylyl cyclases in the rat brain. Synapse 14:51-59. Monti-Graziadei G, Margolis F, Harding J, Graziadei P (1977) Immunocytochemistry of the olfactory marker protein. Journal of Histochemistry & Cytochemistry 25:1311-1316. Moon TC, Befus AD, Kulka M (2014) Mast cell mediators: their differential release and the secretory pathways involved. Frontiers in immunology 5. Moore H, Bedford J (1979a) Short-term effects of androgen withdrawal on the structure of different epithelial cells in the rat epididymis. The Anatomical Record 193:293-311. Moore H, Bedford J (1979b) The differential absorptive activity of epithelial cells of the rat epididymus before and after castration. The Anatomical Record 193:313-327. Morales A, Cavicchia J (1991) Release of cytoplasmic apical protrusions from principal cells of the cat epididymis, an electron microscopic study. Tissue and Cell 23:505-513. Morales C, Clermont Y (1993) Structural changes of the Sertoli cell during the cycle of the seminiferous epithelium. The Sertoli Cell, Russell LD, Griswold MD (eds) pp:306-329.

254 Moran DT, Rowley JC, Jafek BW (1982a) Electron microscopy of human olfactory epithelium reveals a new cell type: the microvillar cell. Brain research 253:39-46. Moran DT, Rowley III JC, Jafek BW, Lovell MA (1982b) The fine structure of the olfactory mucosa in man. Journal of neurocytology 11:721-746. Moreno RD, Ramalho-Santos J, Chan EK, Wessel GM, Schatten G (2000a) The Golgi apparatus segregates from the lysosomal/acrosomal vesicle during rhesus spermiogenesis: structural alterations. Developmental biology 219:334-349. Moreno RD, Ramalho-Santos Jo, Sutovsky P, Chan EK, Schatten G (2000b) Vesicular traffic and Golgi apparatus dynamics during mammalian spermatogenesis: implications for acrosome architecture. Biology of Reproduction 63:89-98. Mori K, Nagao H, Yoshihara Y (1999) The olfactory bulb: coding and processing of odor molecule information. Science 286:711-715. Moriyama M, Sato T, Inoue H, Fukuyama S, Teranishi H, Kangawa K, Kano T, Yoshimura A, Kojima M (2005) The neuropeptide neuromedin U promotes inflammation by direct activation of mast cells. Journal of Experimental Medicine 202:217-224. Morrison EE, Costanzo RM (1990) Morphology of the human olfactory epithelium. Journal of Comparative Neurology 297:1-13. Mortimer D, Leslie E, Kelly R, Templeton A (1982) Morphological selection of human spermatozoa in vivo and in vitro. Journal of Reproduction and Fertility 64:391-399. Moulton D, Fink R (1972) Cell proliferation and migration in the olfactory epithelium. Olfaction and taste 4:20-26. Mousli M, Bueb J-L, Bronner C, Rouot B, Landry Y (1990a) G protein activation: a receptor- independent mode of action for cationic amphiphilic neuropeptides and venom peptides. Trends in pharmacological sciences 11:358-362. Mousli M, Bronner C, Landry Y, Bockaert J, Rouot B (1990b) Direct activation of GTP-binding regulatory proteins (G-proteins) by substance P and compound 48/80. FEBS letters 259:260-262. Mousli M, Bronner C, Bockaert J, Rouot B, Landry Y (1990c) Interaction of substance P, compound 4880 and mastoparan with the α-subunit C-terminus of G protein. Immunology letters 25:355-357. Murase S-i, Horwitz AF (2002) Deleted in colorectal carcinoma and differentially expressed integrins mediate the directional migration of neural precursors in the rostral migratory stream. Journal of Neuroscience 22:3568-3579. Nacher J, Rosell DR, McEwen BS (2000) Widespread expression of rat collapsin response- mediated protein 4 in the telencephalon and other areas of the adult rat central nervous system. Journal of Comparative Neurology 424:628-639. Naessen R (1971) The “receptor surface” of the olfactory organ (epithelium) of man and guinea pig: A descriptive and experimental study. Acta oto-laryngologica 71:335-348. Nagao H, Yoshihara Y, Mitsui S, Fujisawa H, Mori K (2000) Two mirror-image sensory maps with domain organization in the mouse main olfactory bulb. Neuroreport 11:3023-3027. Nagayama S, Homma R, Imamura F (2014) Neuronal organization of olfactory bulb circuits. Frontiers in neural circuits 8:98. Nakagawa Y, Nagasawa M, Yamada S, Hara A, Mogami H, Nikolaev VO, Lohse MJ, Shigemura N, Ninomiya Y, Kojima I (2009) Sweet taste receptor expressed in pancreatic β-cells activates the calcium and cyclic AMP signaling systems and stimulates insulin secretion. PloS one 4:e5106. Nakamura Y, Sanematsu K, Ohta R, Shirosaki S, Koyano K, Nonaka K, Shigemura N, Ninomiya Y (2008) Diurnal variation of human sweet taste recognition thresholds is correlated with plasma leptin levels. Diabetes 57:2661-2665. Nara K, Saraiva LR, Ye X, Buck LB (2011) A large-scale analysis of odor coding in the olfactory epithelium. Journal of Neuroscience 31:9179-9191. Nautiyal KM, Liu C, Dong X, Silver R (2011) Blood-borne donor mast cell precursors migrate to mast cell-rich brain regions in the adult mouse. Journal of neuroimmunology 240:142- 146. Nautiyal KM, Dailey CA, Jahn JL, Rodriquez E, Son NH, Sweedler JV, Silver R (2012) Serotonin of mast cell origin contributes to hippocampal function. European Journal of Neuroscience 36:2347-2359. Navarro B, Miki K, Clapham DE (2011) ATP-activated P2X2 current in mouse spermatozoa. Proceedings of the National Academy of Sciences 108:14342-14347.

255 Navarro M, Valencia J, Vazquez C, Cozar E, Villanueva C (1998) Crude mare follicular fluid exerts chemotactic effects on stallion spermatozoa. Reproduction in Domestic Animals 33:321- 324. Neary MT, Batterham RL (2009) Gut hormones: implications for the treatment of obesity. Pharmacology & therapeutics 124:44-56. Neer EJ (1995) Heterotrimeric C proteins: organizers of transmembrane signals. Cell 80:249-257. Nef S, Parada LF (1999) Cryptorchidism in mice mutant for Insl3. Nature genetics 22:295. Nelissen S, Lemmens E, Geurts N, Kramer P, Maurer M, Hendriks J, Hendrix S (2013) The role of mast cells in neuroinflammation. Acta neuropathologica 125:637-650. Neuhaus EM, Mashukova A, Barbour J, Wolters D, Hatt H (2006) Novel function of β-arrestin2 in the nucleus of mature spermatozoa. Journal of cell science 119:3047-3056. Neuhaus EM, Zhang W, Gelis L, Deng Y, Noldus J, Hatt H (2009) Activation of an olfactory receptor inhibits proliferation of prostate cancer cells. Journal of Biological Chemistry 284:16218-16225. Neutra M, Leblond C (1966) Radioautographic comparison of the uptake of galactose-H3 and glucose-H3 in the Golgi region of various cells secreting glycoproteins or mucopolysaccharides. The Journal of cell biology 30:137-150. Newlands G, Huntley J, Miller H (1984) Concomitant detection of mucosal mast cells and eosinophils in the intestines of normal and Nippostrongylus-immune rats. Histochemistry 81:585-589. Ng KL, Li J-D, Cheng MY, Leslie FM, Lee AG, Zhou Q-Y (2005) Dependence of olfactory bulb neurogenesis on prokineticin 2 signaling. Science 308:1923-1927. Ngai J, Chess A, Dowling MM, Necles N, Macagno ER, Axel R (1993) Coding of olfactory information: topography of odorant receptor expression in the catfish olfactory epithelium. Cell 72:667-680. Nicander L (1965) An electron microscopical study of absorbing cells in the posterior caput epididymidis of rabbits. Cell and Tissue Research 66:829-847. Niemi M, Sharpe R, Brown W (1986) Macrophages in the interstitial tissue of the rat testis. Cell and tissue research 243:337-344. Nigrovic PA, Lee DM (2004) Mast cells in inflammatory arthritis. Arthritis Res Ther 7:1. Niissalo S, Hietanen J, Malmström M, Hukkanen M, Polak J, Konttinen YT (2000) Disorder- specific changes in innervation in oral lichen planus and lichenoid reactions. Journal of oral pathology & medicine 29:361-369. Nilsson G, Metcalfe DD (2000) Mast cells and basophils. In: Haematopoietic and Lymphoid Cell Culture, pp 165-179: Cambridge University Press Cambridge. Nilsson G, Butterfield JH, Nilsson K, Siegbahn A (1994) Stem cell factor is a chemotactic factor for human mast cells. The Journal of Immunology 153:3717-3723. Nilsson G, Hjertson M, Andersson M, Greiff L, Svensson C, Nilsson K, Siegbahn A (1998) Demonstration of mast-cell chemotactic activity in nasal lavage fluid: characterization of one chemotaxin as c-kit ligand, stem cell factor. Allergy 53:874-879. Nishida K, Yamasaki S, Ito Y, Kabu K, Hattori K, Tezuka T, Nishizumi H, Kitamura D, Goitsuka R, Geha RS (2005) FcεRI-mediated mast cell degranulation requires calcium- independent microtubule-dependent translocation of granules to the plasma membrane. J Cell Biol 170:115-126. Nishimura M, Naito S (2005) Tissue-specific mRNA expression profiles of human toll-like receptors and related genes. Biological and Pharmaceutical Bulletin 28:886-892. Nistal M, Santamaria L, Paniagua R (1984) Mast cells in the human testis and epididymis from birth to adulthood. Cells Tissues Organs 119:155-160. Nonogaki T, Noda Y, Narimoto K, Shiotani M, Mori T, Matsuda T, Yoshida O (1992) Localization of CuZn-superoxide dismutase in the human male genital organs. Human Reproduction 7:81-85. Nordlind K, Azmitia EC, Slominski A (2008) The skin as a mirror of the soul: exploring the possible roles of serotonin. Experimental dermatology 17:301-311. Norrby K (2002) Mast cells and angiogenesis. Apmis 110:355-371. O'Toole CM, Arnoult C, Darszon A, Steinhardt RA, Florman HM (2000) Ca2+ entry through store- operated channels in mouse sperm is initiated by egg ZP3 and drives the acrosome reaction. Molecular Biology of the Cell 11:1571-1584. O’Bryan MK, Schlatt S, Phillips DJ, de Kretser DM, Hedger MP (2000a) Bacterial lipopolysaccharide-induced inflammation compromises testicular function at multiple levels in vivo. Endocrinology 141:238-246.

256 O’Bryan MK, Schlatt S, Gerdprasert O, Phillips DJ, de Kretser DM, Hedger MP (2000b) Inducible nitric oxide synthase in the rat testis: evidence for potential roles in both normal function and inflammation-mediated infertility. Biology of reproduction 63:1285-1293. O’Donnell L, McLachlan Rl, Wreford NG, de Kretser DM (1996) Testosterone withdrawal promotes stage-specific detachment of round spermatids from the rat seminiferous epithelium. Biology of Reproduction 55:895-901. O’Shaughnessy P, Fleming L, Jackson G, Hochgeschwender U, Reed P, Baker P (2003) Adrenocorticotropic hormone directly stimulates testosterone production by the fetal and neonatal mouse testis. Endocrinology 144:3279-3284. Oboti L, Peretto P, De Marchis S, Fasolo A (2011) From chemical neuroanatomy to an understanding of the olfactory system. European journal of histochemistry: EJH 55. Ogawa M, Miyata T, Nakajimat K, Yagyu K, Seike M, Ikenaka K, Yamamoto H, Mikoshibat K (1995) The reeler gene-associated antigen on Cajal-Retzius neurons is a crucial molecule for laminar organization of cortical neurons. Neuron 14:899-912. Ohkubo T, Shibata M, Inoue M, Kaya H, Takahashi H (1994) Autoregulation of histamine release via the histamine H3 receptor on mast cells in the rat skin. Archives internationales de pharmacodynamie et de therapie 328:307-314. Ohno I, Ohyama M, Hanamure Y, Ogawa K (1981) Comparative anatomy of olfactory epithelium. Biomedical research-Tokyo 2:455-458. Okano Y, Takagi H, Tohmatsu T, Nakashima S, Kuroda Y, Saito K, Nozawa Y (1985) A wasp venom mastoparan-induced polyphosphoinositide breakdown in rat peritoneal mast cells. FEBS letters 188:363-366. Okayama Y, Kawakami T (2006) Development, migration, and survival of mast cells. Immunologic research 34:97-115. Oko R, Hermo L, Chan P, Fazel A, Bergeron J (1993) The cytoplasmic droplet of rat epididymal spermatozoa contains saccular elements with Golgi characteristics. The Journal of cell biology 123:809-821. Okon K, Stachura J (2007) Increased mast cell density in renal interstitium is correlated with relative interstitial volume, serum creatinine and urea especially in diabetic nephropathy but also in primary glomerulonephritis. Pol J Pathol 58:193-197. Oliveira R, Tomasi L, Rovasio R, Giojalas L (1999) Increased velocity and induction of chemotactic response in mouse spermatozoa by follicular and oviductal fluids. Journal of reproduction and fertility 115:23-27. Onodera K, Yamatodani A, Watanabe T, Wadas H (1994) Neuropharmacology of the histaminergic neuron system in the brain and its relationship with behavioral disorders. Progress in neurobiology 42:685-702. Ophir D, Lancet D (1988) Expression of intermediate filaments and desmoplakin in vertebrate olfactory mucosa. The Anatomical Record 221:754-760. Oral E, Aydin M, Aydin N, Ozcan H, Hacimuftuoglu A, Sipal S, Demirci E (2013) How olfaction disorders can cause depression? The role of habenular degeneration. Neuroscience 240:63-69. Oren-Benaroya R, Orvieto R, Gakamsky A, Pinchasov M, Eisenbach M (2008) The sperm chemoattractant secreted from human cumulus cells is progesterone. Human reproduction 23:2339-2345. Orgebin-Crist M (1967) Sperm maturation in rabbit epididymis. Orgebin-Crist M, Danzo B, Davies J (1975) Endocrine control of the development and maintenance of sperm fertilizing ability in the epididymis. Orgebin-Crist MC, Fournier-Delpech S (1982) Sperm-Egg Interaction. Journal of Andrology 3:429-433. Oschatz C, Maas C, Lecher B, Jansen T, Björkqvist J, Tradler T, Sedlmeier R, Burfeind P, Cichon S, Hammerschmidt S (2011) Mast cells increase vascular permeability by heparin- initiated bradykinin formation in vivo. Immunity 34:258-268. Ottaviano G, Zuccarello D, Menegazzo M, Perilli L, Marioni G, Frigo A, Staffieri A, Foresta C (2013) Human olfactory sensitivity for bourgeonal and male infertility: a preliminary investigation. European Archives of Oto-Rhino-Laryngology 270:3079-3086. Overman EL, Rivier JE, Moeser AJ (2012) CRF induces intestinal epithelial barrier injury via the release of mast cell proteases and TNF-α. PLoS One 7:e39935. Owens T, Renno T, Taupin V, Krakowski M (1994) Inflammatory cytokines in the brain: does the CNS shape immune responses? Immunology today 15:566-571.

257 Pacey A, Davies N, Warren M, Barratt C, Cooke L (1995) Hyperactivation may assist human spermatozoa to detach from intimate association with the endosalpinx. Human Reproduction 10:2603-2609. Page ST, Plymate SR, Bremner WJ, Matsumoto AM, Hess DL, Lin DW, Amory JK, Nelson PS, Wu JD (2006) Effect of medical castration on CD4+ CD25+ T cells, CD8+ T cell IFN-γ expression, and NK cells: a physiological role for testosterone and/or its metabolites. American Journal of Physiology-Endocrinology and Metabolism 290:E856-E863. Palacios J, Regadera J, Nistal M, Paniagua R (1991) Apical mitochondria-rich cells in the human epididymis: An ultrastructural, enzymohistochemical, and immunohistochemical study. The Anatomical Record 231:82-88. Palacios J, Regadera J, Paniagua R, Gamallo C, Nistal M (1993) Immunohistochemistry of the human ductus epididymis. The Anatomical Record 235:560-566. Palladino M, Johnson T, Gupta R, Chapman J, Ojha P (2007) Members of the Toll-like receptor family of innate immunity pattern-recognition receptors are abundant in the male rat reproductive tract. Biology of reproduction 76:958-964. Palouzier-Paulignan B, Lacroix M-C, Aimé P, Baly C, Caillol M, Congar P, Julliard AK, Tucker K, Fadool DA (2012) Olfaction under metabolic influences. Chemical senses 37:769-797. Panzanelli P, Fritschy J, Yanagawa Y, Obata K, Sassoè-Pognetto M (2007) GABAergic phenotype of periglomerular cells in the rodent olfactory bulb. Journal of Comparative Neurology 502:990-1002. Parmentier M (1990) Structure of the human cDNAs and genes coding for calbindin D28K and calretinin. In: Calcium binding proteins in normal and transformed cells, pp 27-34: Springer. Parmentier M, Libert F, Schurmans S, Schiffmann S, Lefort A, Eggerickx D, Ledent C, Mollereau C, Gerard C, Perret J (1992) Expression of members of the putative olfactory receptor gene family in mammalian germ cells. Nature 355:453-455. Parrish-Aungst S, Shipley M, Erdelyi F, Szabo G, Puche A (2007) Quantitative analysis of neuronal diversity in the mouse olfactory bulb. Journal of Comparative Neurology 501:825-836. Parvinen M (1982) Regulation of the Seminiferous Epithelium*. Endocrine Reviews 3:404-417. Pastor-Soler N, Beaulieu V, Litvin TN, Da Silva N, Chen Y, Brown D, Buck J, Levin LR, Breton S (2003) Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH- dependent V-ATPase recycling. Journal of Biological Chemistry 278:49523-49529. Păunescu TG, Shum WW, Huynh C, Lechner L, Goetze B, Brown D, Breton S (2014) High- resolution helium ion microscopy of epididymal epithelial cells and their interaction with spermatozoa. MHR: Basic science of reproductive medicine 20:929-937. Pearce F, Ali H, Barrett K, Befus A, Bienenstock J, Brostoff J, Ennis M, Flint K, Hudspith B, Johnson N (1985) Functional characteristics of mucosal and connective tissue mast cells of man, the rat and other animals. International Archives of Allergy and Immunology 77:274-276. Perassi NI, Peralta M, Loyber I (1975) Removal of olfactory bulbs in rats of different ages: repercussion on some metabolic constants. Archives internationales de physiologie et de biochimie 83:855-862. Peretto P, Merighi A, Fasolo A, Bonfanti L (1997) Glial tubes in the rostral migratory stream of the adult rat. Brain research bulletin 42:9-21. Pérez CV, Theas MS, Jacobo PV, Jarazo-Dietrich S, Guazzone VA, Lustig L (2013) Dual role of immune cells in the testis: Protective or pathogenic for germ cells? Spermatogenesis 3:e23870. Persinger MA (1977) Mast cells in the brain: possibilities for physiological psychology. Physiological Psychology 5:166-176. Persinger MA (1980) Handling factors not body marking influence thalamic mast cell numbers in the preweaned albino rat. Behav Neural Biol 30:448-459. Persinger MA (1981) Developmental alterations in mast cell numbers and distributions within the thalamus of the albino rat. Developmental neuroscience 4:220-224. Peterlin Z, Li Y, Sun G, Shah R, Firestein S, Ryan K (2008) The importance of odorant conformation to the binding and activation of a representative olfactory receptor. Chemistry & biology 15:1317-1327. Petreanu L, Alvarez-Buylla A (2002) Maturation and death of adult-born olfactory bulb granule neurons: role of olfaction. Journal of Neuroscience 22:6106-6113.

258 Philpot BD, Lim JH, Brunjes PC (1997) Activity-dependent regulation of calcium-binding proteins in the developing rat olfactory bulb. Journal of Comparative Neurology 387:12-26. Pi-Sunyer X (2009) The medical risks of obesity. Postgraduate medicine 121:21-33. Pierce KL, Premont RT, Lefkowitz RJ (2002) Seven-transmembrane receptors. Nature Reviews Molecular Cell Biology 3:639-650. Pietrement C, Sun-Wada G, Da Silva N, McKee M, Marshansky V, Brown D, Futai M, Breton S (2006) Distinct expression patterns of different subunit isoforms of the V-ATPase in the rat epididymis. Biology of reproduction 74:185-194. Pifferi S, Boccaccio A, Menini A (2006) Cyclic nucleotide-gated ion channels in sensory transduction. FEBS letters 580:2853-2859. Pilpel Y, Lancet D (1999) The variable and conserved interfaces of modeled olfactory receptor proteins. Protein Science 8:969-977. Pinart E, Bonet S, Briz M, Sancho S, Garcı́a N, Badia E (2001) Cytology of the interstitial tissue in scrotal and abdominal testes of post-puberal boars. Tissue and Cell 33:8-24. Pinching A, Powell T (1971a) The neuropil of the periglomerular region of the olfactory bulb. Journal of cell science 9:379-409. Pinching A, Powell T (1971b) The neuron types of the glomerular layer of the olfactory bulb. Journal of cell science 9:305-345. Pinching A, Powell T (1972a) A study of terminal degeneration in the olfactory bulb of the rat. Journal of cell science 10:585-619. Pinching A, Powell T (1972b) Experimental studies on the axons intrinsic to the glomerular layer of the olfactory bulb. Journal of cell science 10:637-655. Pixley SK (1992) The olfactory nerve contains two populations of glia, identified both in vivo and in vitro. Glia 5:269-284. Pixley SK, Farbman AI, Menco BPM (1997) Monoclonal antibody marker for olfactory sustentacular cell microvilli. The Anatomical Record 248:307-321. Plum L, Schubert M, Brüning JC (2005) The role of insulin receptor signaling in the brain. Trends in Endocrinology & Metabolism 16:59-65. Pluznick JL, Zou D-J, Zhang X, Yan Q, Rodriguez-Gil DJ, Eisner C, Wells E, Greer CA, Wang T, Firestein S (2009) Functional expression of the olfactory signaling system in the kidney. Proceedings of the National Academy of Sciences 106:2059-2064. Pluznick JL, Protzko RJ, Gevorgyan H, Peterlin Z, Sipos A, Han J, Brunet I, Wan L-X, Rey F, Wang T (2013) Olfactory receptor responding to gut microbiota-derived signals plays a role in renin secretion and blood pressure regulation. Proceedings of the National Academy of Sciences 110:4410-4415. Pohl V, Van Rampelbergh J, Mellaert S, Parmentier M, Pochet R (1992) Calretinin in rat ovary: an in situ hybridization and immunohistochemical study. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology 1160:87-94. Polyzoidis S, Koletsa T, Panagiotidou S, Ashkan K, Theoharides TC (2015) Mast cells in meningiomas and brain inflammation. Journal of neuroinflammation 12:170. Polyzonis B, Kafandaris P, Gigis P, Demetriou T (1979) An electron microscopic study of human olfactory mucosa. Journal of anatomy 128:77. Poo C, Isaacson JS (2009) Odor representations in olfactory cortex:“sparse” coding, global inhibition, and oscillations. Neuron 62:850-861. Popovic N, McLeod D, Borski A (1973) Ultrastructure of the human vas deferens. Investigative urology 10:266-277. Portela-Gomes GM, Grimelius L, Johansson H, Efendic S, Wester K, Abdel-Halim SM (2002) Increased expression of adenylyl cyclase isoforms in the adrenal gland of diabetic Goto- Kakizaki rat. Applied Immunohistochemistry & Molecular Morphology 10:387-392. Preininger AM, Hamm HE (2004) G protein signaling: insights from new structures. structure 7:8. Price J, Powell T (1970a) The mitral and short axon cells of the olfactory bulb. Journal of Cell Science 7:631-651. Price J, Powell T (1970b) The morphology of the granule cells of the olfactory bulb. Journal of cell science 7:91-123. Primeaux SD, Barnes MJ, Bray GA (2007) Olfactory bulbectomy increases food intake and hypothalamic neuropeptide Y in obesity-prone but not obesity-resistant rats. Behavioural brain research 180:190-196. Primiani N, Gregory M, Dufresne J, Smith CE, Liu YL, Bartles JR, Cyr DG, Hermo L (2007) Microvillar size and espin expression in principal cells of the adult rat epididymis are regulated by androgens. Journal of andrology 28:659-669.

259 Prince F (2001) The triphasic nature of Leydig cell development in humans, and comments on nomenclature. Journal of Endocrinology 168:213-216. Prince FP (2007) The human Leydig cell. In: The Leydig Cell in Health and Disease, pp 71-89: Springer. Probst WC, Snyder LA, Schuster DI, Brosius J, Sealfon SC (1992) Sequence alignment of the G- protein coupled receptor superfamily. DNA and cell biology 11:1-20. Pudney J (1996) Comparative cytology of the Leydig cell. The Leydig cell Cache River, Vienna 101:98-142. Pundir P, Kulka M (2010) The role of G protein-coupled receptors in mast cell activation by antimicrobial peptides: is there a connection? Immunology and cell biology 88:632. Puopolo M, Bean BP, Raviola E (2005) Spontaneous activity of isolated dopaminergic periglomerular cells of the main olfactory bulb. Journal of neurophysiology 94:3618-3627. Purcell W, Atterwill C (1995) Mast cells in neuroimmune function: neurotoxicological and neuropharmacological perspectives. Neurochemical research 20:521-532. Puri N, Kruhlak MJ, Whiteheart SW, Roche PA (2003) Mast cell degranulation requires N- ethylmaleimide-sensitive factor-mediated SNARE disassembly. The Journal of Immunology 171:5345-5352. Qiu L, LeBel RP, Storm DR, Chen X (2016) Type 3 adenylyl cyclase: a key enzyme mediating the cAMP signaling in neuronal cilia. International journal of physiology, pathophysiology and pharmacology 8:95. Rajalakshmi M, Prasad M (1969) Changes in sialic acid in the testis and epididymis of the rat during the onset of puberty. Journal of Endocrinology 44:379-385. Rajangam K, Behanna HA, Hui MJ, Han X, Hulvat JF, Lomasney JW, Stupp SI (2006) Heparin binding nanostructures to promote growth of blood vessels. Nano letters 6:2086-2090. Rakusan K, Sarkar K, Turek Z, Wicker P (1990) Mast cells in the rat heart during normal growth and in cardiac hypertrophy. Circulation research 66:511-516. Ralt D, Goldenberg M, Fetterolf P, Thompson D, Dor J, Mashiach S, Garbers DL, Eisenbach M (1991) Sperm attraction to a follicular factor (s) correlates with human egg fertilizability. Proceedings of the National Academy of Sciences 88:2840-2844. Ralt D, Manor M, Cohen-Dayag A, Tur-Kaspa I, Ben-Shlomo I, Makler A, Yuli I, Dor J, Blumberg S, Mashiach S (1994) Chemotaxis and chemokinesis of human spermatozoa to follicular factors. Biology of reproduction 50:774-785. Ramirez-Zacarias J, Castro-Munozledo F, Kuri-Harcuch W (1992) Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Histochemistry 97:493-497. Ramos EJ, Xu Y, Romanova I, Middleton F, Chen C, Quinn R, Inui A, Das U, Meguid MM (2003) Is obesity an inflammatory disease? Surgery 134:329-335. Ramos-Nino ME (2013) The role of chronic inflammation in obesity-associated cancers. ISRN oncology 2013. Rangachari P (1998) The fate of released histamine: reception, response and termination. The Yale journal of biology and medicine 71:173. Rausch M, Weisberg S, Vardhana P, Tortoriello D (2008) Obesity in C57BL/6J mice is characterized by adipose tissue hypoxia and cytotoxic T-cell infiltration. International journal of obesity 32:451. Reichel H (1921) Die Saisonfunktion des Nebenhodens vom Maulwurf. Anat Anz 54:129-149. Reid AC, Silver RB, Levi R (2007) Renin: at the heart of the mast cell. Immunological reviews 217:123-140. Reisert J, Yau KW, Margolis FL (2007) Olfactory marker protein modulates the cAMP kinetics of the odour-induced response in cilia of mouse olfactory receptor neurons. The Journal of physiology 585:731-740. Rejraji H, Sion B, Prensier G, Carreras M, Motta C, Frenoux J-M, Vericel E, Grizard G, Vernet P, Drevet JR (2006) Lipid remodeling of murine epididymosomes and spermatozoa during epididymal maturation. Biology of reproduction 74:1104-1113. Résibois A, Rogers J (1992) Calretinin in rat brain: an immunohistochemical study. Neuroscience 46:101-134. Ressler KJ, Sullivan SL, Buck LB (1993) A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell 73:597-609. Ressler KJ, Sullivan SL, Buck LB (1994a) Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell 79:1245- 1255.

260 Ressler KJ, Sullivan SL, Buck LB (1994b) A molecular dissection of spatial patterning in the olfactory system. Current opinion in neurobiology 4:588-596. Rettew JA, Huet-Hudson YM, Marriott I (2008) Testosterone reduces macrophage expression in the mouse of toll-like receptor 4, a trigger for inflammation and innate immunity. Biology of reproduction 78:432-437. Riar S, Setty B, Kar AB (1973) Studies on the physiology and biochemistry of mammalian epididymis: biochemical composition of epididymis. A comparative study. Fertility and sterility 24:355-363. Ribatti D (2015) The crucial role of mast cells in blood–brain barrier alterations. Experimental cell research 338:119-125. Riccioli A, Starace D, Galli R, Fuso A, Scarpa S, Palombi F, De Cesaris P, Ziparo E, Filippini A (2006) Sertoli cells initiate testicular innate immune responses through TLR activation. The Journal of Immunology 177:7122-7130. Rich KA, De Kretser D (1977) Effect of differing degrees of destruction of the rat seminiferous epithelium on levels of serum follicle stimulating hormone and androgen binding protein. Endocrinology 101:959-968. Rich KA, Kerr JB, de Kretser DM (1979) Evidence for Leydig cell dysfunction in rats with seminiferous tubule damage. Molecular and cellular endocrinology 13:123-135. Riera CE, Dillin A (2016) Emerging role of sensory perception in aging and metabolism. Trends in Endocrinology & Metabolism 27:294-303. Robaire B, Hermo L (1988) Efferent ducts, epididymis, and vas deferens: structure, functions, and their regulation. The physiology of reproduction 1:999-1080. Robaire B, Viger RS (1995) Regulation of epididymal epithelial cell functions. Biology of Reproduction 52:226-236. Robaire B, Hinton B, Orgebin-Crist M-C (2002) The Epididymis: from molecules to clinical practice: a comprehensive survey of the efferent ducts, the epididymis and the vas deferens: Springer Science & Business Media. Robas N, Mead E, Fidock M (2003) MrgX2 is a high potency cortistatin receptor expressed in dorsal root ganglion. Journal of Biological Chemistry 278:44400-44404. Rodriguez-Martinez H, Ekstedt E, Einarsson S (1990) Acidification of epididymal fluid in the boar. International journal of andrology 13:238-243. Rogers K, Dasgupta P, Gubler U, Grillo M, Khew-Goodall Y, Margolis F (1987) Molecular cloning and sequencing of a cDNA for olfactory marker protein. Proceedings of the National Academy of Sciences 84:1704-1708. Rolls ET (2005) Taste, olfactory, and food texture processing in the brain, and the control of food intake. Physiology & behavior 85:45-56. Roosen-Runge EC, Anderson D (1959) The development of the interstitial cells in the testis of the albino rat. Cells Tissues Organs 37:125-137. Roskams A, Cai X, Ronnett GV (1998) Expression of neuron-specific beta-III tubulin during olfactory neurogenesis in the embryonic and adult rat. Neuroscience 83:191-200. Rössler P, Mezler M, Breer H (1998) Two olfactory marker proteins in Xenopus laevis. Journal of Comparative Neurology 395:273-280. Roth TF, Porter KR (1964) Yolk protein uptake in the oocyte of the mosquito Aedes aegypti. L. The Journal of cell biology 20:313-332. Royer B, Varadaradjalou S, Saas P, Guillosson J, Kantelip J, Arock M (2001) Inhibition of IgE- induced activation of human mast cells by IL-10. Clinical & Experimental Allergy 31:694- 704. Rüger B, Hasan Q, Greenhill N, Davis P, Dunbar P, Neale T (1996) Mast cells and type VIII collagen in human diabetic nephropathy. Diabetologia 39:1215-1222. Russell L (1993a) Form, dimensions, and cytology of mammalian Sertoli cells. The Sertoli Cell 1:1-37. Russell L (1993b) Morphological and functional evidence for Sertoli-germ cell relationships. The Sertoli Cell. Russell L (1996) Mammalian Leydig cell structure. In: The Leydig Cell, pp 43-96: Cache River Press, Vienna, IL. Russell L, Peterson R, Freund M (1979) Direct evidence for formation of hybrid vesicles by fusion of plasma and outer acrosomal membranes during the acrosome reaction in boar spermatozoa. Journal of Experimental Zoology Part A: Ecological Genetics and Physiology 208:41-55.

261 Russell LD, Clermont Y (1977) Degeneration of germ cells in normal, hypophysectomized and hormone treated hypophysectomized rats. The Anatomical Record 187:347-365. Russell LD, Ren HP, Hikim IS, Schulze W, Hikim APS (1990) A comparative study in twelve mammalian species of volume densities, volumes, and numerical densities of selected testis components, emphasizing those related to the Sertoli cell. Developmental Dynamics 188:21-30. Rüther U, Stilz S, Röhl E, Nunnensiek C, Rassweiler J, Dörr U, Jipp P (1995) Successful Interferone-α (2) a Therapy for a Patient with Acute Mumps Orchitis. European urology 27:174-176. S Melendrez C, Meizel S, Berger T (1994) Comparison of the ability of progesterone and heat solubilized porcine zona pellucida to initiate the porcine sperm acrosome reaction in vitro. Molecular Reproduction and Development 39:433-438. Saez J, Lejeune H (1996) Regulation of Leydig cell functions by hormones and growth factors other than LH and IGF-1. The leydig cell 1:383-406. Saez JM (1994) Leydig cells: endocrine, paracrine, and autocrine regulation. Endocrine reviews 15:574-626. Safina F, Tanaka S, Inagaki M, Tsuboi K, Sugimoto Y, Ichikawa A (2002) Expression of L-histidine decarboxylase in mouse male germ cells. Journal of Biological Chemistry 277:14211- 14215. Saghatelyan A, De Chevigny A, Schachner M, Lledo P-M (2004) Tenascin-R mediates activity- dependent recruitment of neuroblasts in the adult mouse forebrain. Nature neuroscience 7:347. Saito H, Chi Q, Zhuang H, Matsunami H, Mainland JD (2009) Odor coding by a Mammalian receptor repertoire. Science signaling 2:ra9. Saling PM (1982) Development of the ability to bind to zonae pellucidae during epididymal maturation: reversible immobilization of mouse spermatozoa by lanthanum. Biology of reproduction 26:429-436. Saltiel AR (2000) Series introduction: the molecular and physiological basis of insulin resistance: emerging implications for metabolic and cardiovascular diseases. Journal of Clinical Investigation 106:163. Sanz G, Schlegel C, Pernollet J-C, Briand L (2005) Comparison of odorant specificity of two human olfactory receptors from different phylogenetic classes and evidence for antagonism. Chemical senses 30:69-80. Sarin S, Malhotra V, Gupta SS, Karol A, Gaur S, Anand B (1987) Significance of eosinophil and mast cell counts in rectal mucosa in ulcerative colitis. Digestive diseases and sciences 32:363-367. Savigner A, Duchamp-Viret P, Grosmaitre X, Chaput M, Garcia S, Ma M, Palouzier-Paulignan B (2009) Modulation of spontaneous and odorant-evoked activity of rat olfactory sensory neurons by two anorectic peptides, insulin and leptin. Journal of neurophysiology 101:2898-2906. Sawada M, Kaneko N, Inada H, Wake H, Kato Y, Yanagawa Y, Kobayashi K, Nemoto T, Nabekura J, Sawamoto K (2011) Sensory input regulates spatial and subtype-specific patterns of neuronal turnover in the adult olfactory bulb. Journal of Neuroscience 31:11587-11596. Schild D, Restrepo D (1998) Transduction mechanisms in vertebrate olfactory receptor cells. Physiological reviews 78:429-466. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B (2012) Fiji: an open-source platform for biological-image analysis. Nature methods 9:676-682. Schlatt S, Arslan M, Weinbauer GF, Behre HM, Nieschlag E (1995) Endocrine control of testicular somatic and premeiotic germ cell development in the immature testis of the primate Macaca mulatta. European journal of endocrinology 133:235-247. Schmahl J, Eicher EM, Washburn LL, Capel B (2000) Sry induces cell proliferation in the mouse gonad. Development 127:65-73. Schoenfeld TA, Marchand JE, Macrides F (1985) Topographic organization of tufted cell axonal projections in the hamster main olfactory bulb: an intrabulbar associational system. Journal of Comparative Neurology 235:503-518. Schulze W, Rehder U (1984) Organization and morphogenesis of the human seminiferous epithelium. Cell and tissue research 237:395-407.

262 Schulze W, Davidoff MS, Holstein A-F (1987) Are Leydig cells of neural origin? Substance P-like immunoreactivity in human testicular tissue. Acta endocrinologica 115:373-377. Schwartz MW, Woods SC, Porte Jr D, Seeley RJ, Baskin DG (2000) Central nervous system control of food intake. Nature 404:661. Schwob JE (2002) Neural regeneration and the peripheral olfactory system. The Anatomical Record 269:33-49. Schwob JE, Youngentob SL, Mezza RC (1995) Reconstitution of the rat olfactory epithelium after methyl bromide-induced lesion. Journal of Comparative Neurology 359:15-37. Schwob JE, Huard JM, Luskin MB, Youngentob SL (1994) Retroviral lineage studies of the rat olfactory epithelium. Chemical senses 19:671-682. Secor VH, Secor WE, Gutekunst C-A, Brown MA (2000) Mast cells are essential for early onset and severe disease in a murine model of multiple sclerosis. Journal of Experimental Medicine 191:813-822. Sedar A (1966) Transport of exogenous peroxidase across the epididymal epithelium. In: International Conference for Electron Microscopy, Kyoto, Japan, p 591. Seeldrayers PA, Yasui D, Weiner HL, Johnson D (1989) Treatment of experimental allergic neuritis with nedocromil sodium. Journal of neuroimmunology 25:221-226. Seguy M, Perret M (2005) Changes in olfactory inputs modify the energy balance response to short days in male gray mouse lemurs. Physiology & behavior 84:23-31. Seifert K (1970) Die Ultrastruktur des Riechepithels beim Makrosmatiker: eine elektronenmikroskopische Untersuchung; 4 Tabellen: Thieme. Serizawa S, Miyamichi K, Nakatani H, Suzuki M, Saito M, Yoshihara Y, Sakano H (2003) Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse. Science 302:2088-2094. Serrano H, Canchola E, García-Suárez MD (2001) Sperm-attracting activity in follicular fluid associated to an 8.6-kDa protein. Biochemical and biophysical research communications 283:782-784. Serre V, Robaire B (1998) Segment-specific morphological changes in aging Brown Norway rat epididymis. Biology of reproduction 58:497-513. Setchell BP, Granholm T, Ritzén EM (1995) Failure of thyroid allografts to function in the testes of cynomolgous monkeys. Journal of reproductive immunology 28:75-80. Shah AS, Ben-Shahar Y, Moninger TO, Kline JN, Welsh MJ (2009) Motile cilia of human airway epithelia are chemosensory. Science 325:1131-1134. Shan L, Hardy MP (1992) Developmental changes in levels of luteinizing hormone receptor and androgen receptor in rat Leydig cells. Endocrinology 131:1107-1114. Shan L, Phillips DM, Bardin CW, Hardy MP (1993) Differential regulation of steroidogenic enzymes during differentiation optimizes testosterone production by adult rat Leydig cells. Endocrinology 133:2277-2283. Shang T, Zhang X, Wang T, Sun B, Deng T, Han D (2011) Toll-like receptor-initiated testicular innate immune responses in mouse Leydig cells. Endocrinology 152:2827-2836. Sharma K, Ziyadeh F (1993) The transforming growth factor-beta system and the kidney. In: Seminars in nephrology, p 116. Sharpe RM (1990) Intratesticular control of steroidogenesis. Clinical endocrinology 33:787-807. Sheedfar F, Biase SD, Koonen D, Vinciguerra M (2013) Liver diseases and aging: friends or foes? Aging cell 12:950-954. Shepherd GM (1988) Neurobiology: Oxford University Press. Shepherd GM (1994) Discrimination of molecular signals by the olfactory receptor neuron. Neuron 13:771-790. Shigemura N, Ohta R, Kusakabe Y, Miura H, Hino A, Koyano K, Nakashima K, Ninomiya Y (2004) Leptin modulates behavioral responses to sweet substances by influencing peripheral taste structures. Endocrinology 145:839-847. Shoelson SE, Lee J, Goldfine AB (2006) Inflammation and insulin resistance. Journal of Clinical Investigation 116:1793. Shukla SA, Veerappan R, Whittimore JS, Miller LE, Youngberg GA (2005) Mast cell ultrastructure and staining in tissue. Mast Cells: Methods and Protocols:63-76. Shykind BM, Rohani SC, O'Donnell S, Nemes A, Mendelsohn M, Sun Y, Axel R, Barnea G (2004) Gene switching and the stability of odorant receptor gene choice. Cell 117:801-815. Siegel G, Albers R, Brady S, Price D (2006) Basic Neurochemistry: Molecular, Cellular, and Medical Aspects. (with CD-ROM). American journal of neuroradiology 27:465.

263 Silver R, Ramos C, Silverman AJ (1992) Sexual behavior triggers the appearance of non- neuronal cells containing gonadotropin-releasing hormone-like immunoreactivity. Journal of neuroendocrinology 4:207-210. Silver R, Silverman A-J, Vitković L, Lederhendler II (1996) Mast cells in the brain: evidence and functional significance. Trends in neurosciences 19:25-31. Silverman A-J, Sutherland AK, Wilhelm M, Silver R (2000) Mast cells migrate from blood to brain. Journal of Neuroscience 20:401-408. Simpson K, Sweazey R (2006) Olfaction and taste. Fundamental neuroscience for basic and clinical applications Churchill Livingstone, Philadelphia:366-378. Sinclair ML, Wang XY, Mattia M, Conti M, Buck J, Wolgemuth DJ, Levin LR (2000) Specific expression of soluble adenylyl cyclase in male germ cells. Molecular reproduction and development 56:6-11. Sipilä P, Cooper TG, Yeung C-H, Mustonen M, Penttinen J, Drevet Jl, Huhtaniemi I, Poutanen M (2002) Epididymal dysfunction initiated by the expression of simian virus 40 T-antigen leads to angulated sperm flagella and infertility in transgenic mice. Molecular Endocrinology 16:2603-2617. Skaper S, Facci L, Giusti P (2014) Neuroinflammation, microglia and mast cells in the pathophysiology of neurocognitive disorders: a review. CNS & Neurological Disorders- Drug Targets (Formerly Current Drug Targets-CNS & Neurological Disorders) 13:1654- 1666. Skinner MK (1991) Cell-cell interactions in the testis. Endocrine reviews 12:45-77. Slominski AT (2006) Proopiomelanocortin signaling system is operating in mast cells. Journal of Investigative Dermatology 126:1934-1936. Small DM (2012) Flavor is in the brain. Physiology & behavior 107:540-552. Smith PC, Firestein S, Hunt JF (2002) The crystal structure of the olfactory marker protein at 2.3 Å resolution. Journal of molecular biology 319:807-821. Söder O, Sultana T, Jonsson C, Wahlgren A, Petersen C, Holst M (2000) The interleukin-1 system in the testis. Andrologia 32:52-55. Spehr M, Schwane K, Heilmann S, Gisselmann G, Hummel T, Hatt H (2004a) Dual capacity of a human olfactory receptor. Current Biology 14:R832-R833. Spehr M, Gisselmann G, Poplawski A, Riffell JA, Wetzel CH, Zimmer RK, Hatt H (2003) Identification of a testicular odorant receptor mediating human sperm chemotaxis. Science 299:2054-2058. Spehr M, Schwane K, Riffell JA, Barbour J, Zimmer RK, Neuhaus EM, Hatt H (2004b) Particulate adenylate cyclase plays a key role in human sperm olfactory receptor-mediated chemotaxis. Journal of Biological Chemistry 279:40194-40203. Spiteri-Grech J, Nieschlag E (1993) Paracrine factors relevant to the regulation of spermatogenesis-a review. Journal of reproduction and fertility 98:1-14. Stanley S, Wynne K, McGowan B, Bloom S (2005) Hormonal regulation of food intake. Physiological reviews 85:1131-1158. Starace D, Galli R, Paone A, Cesaris PD, Filippini A, Ziparo E, Riccioli A (2008) Toll-like receptor 3 activation induces antiviral immune responses in mouse sertoli cells. Biology of reproduction 79:766-775. Statter MB, Foglia RP, Parks DE, Donahoe PK (1988) Fetal and postnatal testis shows immunoprivilege as donor tissue. The Journal of urology 139:204-210. Stensmyr MC, Giordano E, Balloi A, Angioy A-M, Hansson BS (2003) Novel natural ligands for Drosophila olfactory receptor neurones. Journal of Experimental Biology 206:715-724. Stéphan J-P, Syed V, Jégou B (1997) Regulation of Sertoli cell IL-1 and IL-6 production in vitro. Molecular and cellular endocrinology 134:109-118. Stevens RL, Austen KF (1989) Recent advances in the cellular and molecular biology of mast cells. Immunology today 10:381-386. Strader CD, Fong TM, Tota MR, Underwood D, Dixon RA (1994) Structure and function of G protein-coupled receptors. Annual review of biochemistry 63:101-132. Strauss KI, Isaacs KR, Ha QN, Jacobowitz DM (1994) Calretinin is expressed in the Leydig cells of rat testis. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression 1219:435-440. Strobel S, Miller H, Ferguson A (1981) Human intestinal mucosal mast cells: evaluation of fixation and staining techniques. Journal of clinical pathology 34:851-858.

264 Strotmann J, Levai O, Fleischer J, Schwarzenbacher K, Breer H (2004) Olfactory receptor proteins in axonal processes of chemosensory neurons. Journal of Neuroscience 24:7754-7761. Strotmann J, Conzelmann S, Beck A, Feinstein P, Breer H, Mombaerts P (2000) Local permutations in the glomerular array of the mouse olfactory bulb. Journal of Neuroscience 20:6927-6938. Strünker T, Alvarez L, Kaupp U (2015) At the physical limit—chemosensation in sperm. Current opinion in neurobiology 34:110-116. Strünker T, Goodwin N, Brenker C, Kashikar ND, Weyand I, Seifert R, Kaupp UB (2011) The CatSper channel mediates progesterone-induced Ca2+ influx in human sperm. Nature 471:382-386. Suarez SS, Varosi SM, Dai X (1993) Intracellular calcium increases with hyperactivation in intact, moving hamster sperm and oscillates with the flagellar beat cycle. Proceedings of the National Academy of Sciences 90:4660-4664. Sullivan R, Frenette G, Girouard J (2007) Epididymosomes are involved in the acquisition of new sperm proteins during epididymal transit. Asian journal of andrology 9:483-491. Sullivan RM, Wilson DA (2003) Molecular biology of early olfactory memory. Learning & Memory 10:1-4. Sullivan SL, Adamson MC, Ressler KJ, Kozak CA, Buck LB (1996) The chromosomal distribution of mouse odorant receptor genes. Proceedings of the National Academy of Sciences 93:884-888. Sun B, Qi N, Shang T, Wu H, Deng T, Han D (2010) Sertoli cell-initiated testicular innate immune response through toll-like receptor-3 activation is negatively regulated by Tyro3, Axl, and mer receptors. Endocrinology 151:2886-2897. Sun F, Giojalas LC, Rovasio RA, Tur-Kaspa I, Sanchez R, Eisenbach M (2003) Lack of species- specificity in mammalian sperm chemotaxis. Developmental biology 255:423-427. Sun F, Bahat A, Gakamsky A, Girsh E, Katz N, Giojalas LC, Tur-Kaspa I, Eisenbach M (2005) Human sperm chemotaxis: both the oocyte and its surrounding cumulus cells secrete sperm chemoattractants. Human Reproduction 20:761-767. Sunahara RK, Dessauer CW, Gilman AG (1996) Complexity and diversity of mammalian adenylyl cyclases. Annual review of pharmacology and toxicology 36:461-480. Sutovsky P (2003) Ubiquitin-dependent proteolysis in mammalian spermatogenesis, fertilization, and sperm quality control: Killing three birds with one stone. Microscopy research and technique 61:88-102. Suzuki Y, Schafer J, Farbman AI (1995) Phagocytic cells in the rat olfactory epithelium after bulbectomy. Experimental neurology 136:225-233. Suzuki Y, Takeda M, Farbman AI (1996) Supporting cells as phagocytes in the olfactory epithelium after bulbectomy. Journal of Comparative Neurology 376:509-517. Sweet MJ, Hume DA (1996) Endotoxin signal transduction in macrophages. Journal of leukocyte biology 60:8-26. Sydor W, Teitelbaum Z, Blacher R, Sun S, Benz W, Margolis F (1986) Amino acid sequence of a unique neuronal protein: rat olfactory marker protein. Archives of biochemistry and biophysics 249:351-362. Tähkä K (1986) Current aspects of Leydig cell function and its regulation. Journal of reproduction and fertility 78:367-380. Takamiya K, Yamamoto A, Furukawa K, Zhao J, Fukumoto S, Yamashiro S, Okada M, Haraguchi M, Shin M, Kishikawa M (1998) Complex gangliosides are essential in spermatogenesis of mice: possible roles in the transport of testosterone. Proceedings of the National Academy of Sciences 95:12147-12152. Talero E, Sanchez-Fidalgo S, Calvo J, Motilva V (2007) Chronic administration of galanin attenuates the TNBS-induced colitis in rats. Regulatory peptides 141:96-104. Tan L, Li Q, Xie XS (2015) Olfactory sensory neurons transiently express multiple olfactory receptors during development. Molecular Systems Biology 11:844. Tatemoto K, Nozaki Y, Tsuda R, Konno S, Tomura K, Furuno M, Ogasawara H, Edamura K, Takagi H, Iwamura H (2006) Immunoglobulin E-independent activation of mast cell is mediated by Mrg receptors. Biochemical and biophysical research communications 349:1322-1328. Tatsura H, Nagao H, Tamada A, Sasaki S, Kohri K, Mori K (2001) Developing germ cells in mouse testis express pheromone receptors. FEBS letters 488:139-144.

265 Tatti R, Bhaukaurally K, Gschwend O, Seal RP, Edwards RH, Rodriguez I, Carleton A (2014) A population of glomerular glutamatergic neurons controls sensory information transfer in the mouse olfactory bulb. Nature communications 5:3791. Teerds KJ, Dorrington JH (1993) Localization of transforming growth factor β1 and β2 during testicular development in the rat. Biology of Reproduction 48:40-45. Teves ME, Barbano F, Guidobaldi HA, Sanchez R, Miska W, Giojalas LC (2006) Progesterone at the picomolar range is a chemoattractant for mammalian spermatozoa. Fertility and sterility 86:745-749. Teves ME, Guidobaldi HA, Uñates DR, Sanchez R, Miska W, Publicover SJ, Garcia AAM, Giojalas LC (2009) Molecular mechanism for human sperm chemotaxis mediated by progesterone. PloS one 4:e8211. Theas M, Rival C, Jarazo-Dietrich S, Jacobo P, Guazzone V, Lustig L (2008) Tumour necrosis factor-α released by testicular macrophages induces apoptosis of germ cells in autoimmune orchitis. Human reproduction 23:1865-1872. Theoharides T (1990) Mast cells: the immune gate to the brain. Life sciences 46:607-617. Theoharides T (1996) The mast cell: a neuroimmunoendocrine master player. International journal of tissue reactions 18:1-21. Theoharides T, Spanos C, Pang X, Alferes L, Ligris K, Letourneau R, Rozniecki J, Webster E, Chrousos G (1995) Stress-induced intracranial mast cell degranulation: a corticotropin- releasing hormone-mediated effect. Endocrinology 136:5745-5750. Theoharides TC, Kalogeromitros D (2006) The critical role of mast cells in allergy and inflammation. Annals of the New York Academy of Sciences 1088:78-99. Theoharides TC, Bondy PK, Tsakalos ND, Askenase PW (1982) Differential release of serotonin and histamine from mast cells. Nature 297:229-231. Thiebaud N, Johnson MC, Butler JL, Bell GA, Ferguson KL, Fadool AR, Fadool JC, Gale AM, Gale DS, Fadool DA (2014) Hyperlipidemic diet causes loss of olfactory sensory neurons, reduces olfactory discrimination, and disrupts odor-reversal learning. Journal of Neuroscience 34:6970-6984. Thomas MB, Haines SL, Akeson RA (1996) Chemoreceptors expressed in taste, olfactory and male reproductive tissues. Gene 178:1-5. Thundathil J, Palasz A, Barth A, Mapletoft R (2001) The use of in vitro fertilization techniques to investigate the fertilizing ability of bovine sperm with proximal cytoplasmic droplets. Animal reproduction science 65:181-192. Tian H, Ma M (2008) Activity plays a role in eliminating olfactory sensory neurons expressing multiple odorant receptors in the mouse septal organ. Molecular and Cellular Neuroscience 38:484-488. Tietjen I, Rihel JM, Cao Y, Koentges G, Zakhary L, Dulac C (2003) Single-cell transcriptional analysis of neuronal progenitors. Neuron 38:161-175. Tilg H, Moschen AR (2008) Insulin resistance, inflammation, and non-alcoholic fatty liver disease. Trends in Endocrinology & Metabolism 19:371-379. Tilly B, Tertoolen L, Lambrechts A, Remorie R, De Laat S, Moolenaar W (1990) Histamine-H1- receptor-mediated phosphoinositide hydrolysis, Ca2+ signalling and membrane-potential oscillations in human HeLa carcinoma cells. Biochemical Journal 266:235-243. Tobin VA, Hashimoto H, Wacker DW, Takayanagi Y, Langnaese K, Caquineau C, Noack J, Landgraf R, Onaka T, Leng G (2010) An intrinsic vasopressin system in the olfactory bulb is involved in social recognition. Nature 464:413. Toida K, Kosaka K, Aika Y, Kosaka T (2000) Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb—IV. Intraglomerular synapses of tyrosine hydroxylase-immunoreactive neurons. Neuroscience 101:11-17. Tong J, Mannea E, Aimé P, Pfluger PT, Yi C-X, Castaneda TR, Davis HW, Ren X, Pixley S, Benoit S (2011) Ghrelin enhances olfactory sensitivity and exploratory sniffing in rodents and humans. Journal of Neuroscience 31:5841-5846. Tore F, Tuncel N (2009) Mast cells: target and source of neuropeptides. Current pharmaceutical design 15:3433-3445. Touhara K, Sengoku S, Inaki K, Tsuboi A, Hirono J, Sato T, Sakano H, Haga T (1999) Functional identification and reconstitution of an odorant receptor in single olfactory neurons. Proceedings of the National Academy of Sciences 96:4040-4045. Trasler JM, Hermo L, Robaire B (1988) Morphological changes in the testis and epididymis of rats treated with cyclophosphamide: a quantitative approach. Biology of reproduction 38:463-479.

266 Tucker K, Cho S, Thiebaud N, Henderson MX, Fadool DA (2013) Glucose sensitivity of mouse olfactory bulb neurons is conveyed by a voltage-gated potassium channel. The Journal of physiology 591:2541-2561. Turner T (1979) On the epididymis and its function. Investigative urology 16:311-321. Turner T (1991) Spermatozoa are exposed to a complex microenvironment as they traverse the epididymis. Annals of the New York Academy of Sciences 637:364-383. Turner T, Giles R (1982) Sperm motility-inhibiting factor in rat epididymis. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 242:R199-R203. Turner T, Reich G (1985) Cauda epididymidal sperm motility: a comparison among five species. Biology of reproduction 32:120-128. Usselman MC, Cone RA (1983) Rat sperm are mechanically immobilized in the caudal epididymis by “immobilin,” a high molecular weight glycoprotein. Biology of reproduction 29:1241- 1253. Usselman MC, Cone RA, Rossignol DP (1985) Rat cauda epididymal fluid is a mucus. Journal of andrology 6:315-320. Vaidehi N, Floriano WB, Trabanino R, Hall SE, Freddolino P, Choi EJ, Zamanakos G, Goddard WA (2002) Prediction of structure and function of G protein-coupled receptors. Proceedings of the National Academy of Sciences 99:12622-12627. Val P, Jeays-Ward K, Swain A (2006) Identification of a novel population of adrenal-like cells in the mammalian testis. Developmental biology 299:250-256. van der Heijden RA, Sheedfar F, Morrison MC, Hommelberg PP, Kor D, Kloosterhuis NJ, Gruben N, Youssef SA, de Bruin A, Hofker MH (2015) High-fat diet induced obesity primes inflammation in adipose tissue prior to liver in C57BL/6j mice. Aging (Albany NY) 7:256. Vanderbeld B, Kelly GM (2000) New thoughts on the role of the βγ subunit in G protein signal transduction. Biochemistry and Cell Biology 78:537-550. Vanderhaeghen P, Schurmans S, Vassart G, Parmentier M (1993) Olfactory receptors are displayed on dog mature sperm cells. Journal of Cell Biology 123:1441-1452. Vanderhaeghen P, Schurmans S, Vassart G, Parmentier M (1997) Specific repertoire of olfactory receptor genes in the male germ cells of several mammalian species. Genomics 39:239- 246. Vassar R, Chao SK, Sitcheran R, Nun JM, Vosshall LB, Axel R (1994) Topographic organization of sensory projections to the olfactory bulb. Cell 79:981-991. Veitinger T, Riffell JR, Veitinger S, Nascimento JM, Triller A, Chandsawangbhuwana C, Schwane K, Geerts A, Wunder F, Berns MW (2011) Chemosensory Ca2+ dynamics correlate with diverse behavioral phenotypes in human sperm. Journal of Biological Chemistry 286:17311-17325. Veldhuizen MG, Shepard TG, Wang M-F, Marks LE (2009) Coactivation of gustatory and olfactory signals in flavor perception. Chemical senses 35:121-133. Vendrely E (1981) Histology of the epididymis in the human adult. Prog Reprod Biol 8:21-23. Veri JP, Hermo L, Robaire B (1993) Immunocytochemical localization of the Yf subunit of glutathione S-transferase P shows regional variation in the staining of epithelial cells of the testis, efferent ducts, and epididymis of the male rat. Journal of andrology 14:23-44. Vernon RB, Sage H (1989) The calcium-binding protein SPARC is secreted by Leydig and Sertoli cells of the adult mouse testis. Biology of reproduction 40:1329-1340. Versteeg DH, Van der Gugten J, De Jong W (1976) Regional concentrations of noradrenaline and dopamine in rat brain. Brain research 113:563-574. Vig M, Kinet J-P (2009) in immune cells. Nature immunology 10:21-27. Visconti PE (2009) Understanding the molecular basis of sperm capacitation through kinase design. Proceedings of the National Academy of Sciences 106:667-668. Vliagoftis H, Befus AD (2005) Rapidly changing perspectives about mast cells at mucosal surfaces. Immunological reviews 206:190-203. Vollrath M, Altmannsberger M, Weber K, Osborn M (1985) An ultrastructural and immunohistological study of the rat olfactory epithelium: unique properties of olfactory sensory cells. Differentiation 29:243-253. von Köckritz-Blickwede M, Goldmann O, Thulin P, Heinemann K, Norrby-Teglund A, Rohde M, Medina E (2008) Phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation. Blood 111:3070-3080. Vornberger W, Prins G, Musto NA, Suarez-Quian CA (1994) Androgen receptor distribution in rat testis: new implications for androgen regulation of spermatogenesis. Endocrinology 134:2307-2316.

267 Vosseller K, Stella G, Yee N, Besmer P (1997) c-kit receptor signaling through its phosphatidylinositide-3'-kinase-binding site and protein kinase C: role in mast cell enhancement of degranulation, adhesion, and membrane ruffling. Molecular Biology of the Cell 8:909-922. Vosshall LB (2004) Olfaction: attracting both sperm and the nose. Current biology 14:R918-R920. Waberski D, Weitze K, Gleumes T, Schwarz M, Willmen T, Petzoldt R (1994) Effect of time of insemination relative to ovulation on fertility with liquid and frozen boar semen. Theriogenology 42:831-840. Walensky LD, Snyder SH (1995) Inositol 1, 4, 5-trisphosphate receptors selectively localized to the acrosomes of mammalian sperm. The Journal of cell biology 130:857-869. Walensky LD, Roskams A, Lefkowitz R, Snyder S, Ronnett G (1995) Odorant receptors and desensitization proteins colocalize in mammalian sperm. Molecular Medicine 1:130. Walensky LD, Ruat M, Bakin RE, Blackshaw S, Ronnett GV, Snyder SH (1998) Two novel odorant receptor families expressed in spermatids undergo 5′-splicing. Journal of Biological Chemistry 273:9378-9387. Walls AF, He S, Teran LM, Buckley MG, Jung K-S, Holgate ST, Shute JK, Cairns JA (1995) Granulocyte recruitment by human mast cell tryptase. International archives of allergy and immunology 107:372-373. Walther TC, Farese Jr RV (2012) Lipid droplets and cellular lipid metabolism. Annual review of biochemistry 81:687-714. Wang C, Zhuang Y, Zhang Y, Luo Z, Gao N, Li P, Pan H, Cai L, Ma Y (2012) Toll-like receptor 3 agonist complexed with cationic liposome augments vaccine-elicited antitumor immunity by enhancing TLR3–IRF3 signaling and type I interferons in dendritic cells. Vaccine 30:4790-4799. Wang F, Nemes A, Mendelsohn M, Axel R (1998) Odorant receptors govern the formation of a precise topographic map. Cell 93:47-60. Wang MM, Reed RR (1993) Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast. Nature 364:121. Wang MM, Tsai R, Schrader KA, Reed RR (1993) Genes encoding components of the olfactory signal transduction cascade contain a DNA binding site that may direct neuronal expression. Molecular and cellular biology 13:5805-5813. Wang Z, Li V, Chan GC, Phan T, Nudelman AS, Xia Z, Storm DR (2009) Adult type 3 adenylyl cyclase–deficient mice are obese. PloS one 4:e6979. Ward GE, Brokaw CJ, Garbers DL, Vacquier VD (1985) Chemotaxis of Arbacia punctulata spermatozoa to resact, a peptide from the egg jelly layer. The Journal of cell biology 101:2324-2329. Watson PA, Krupinski J, Kempinski AM, Frankenfield CD (1994) Molecular cloning and characterization of the type VII isoform of mammalian adenylyl cyclase expressed widely in mouse tissues and in S49 mouse lymphoma cells. Journal of Biological Chemistry 269:28893-28898. Weber M, Pehl U, Breer H, Strotmann J (2002) Olfactory receptor expressed in ganglia of the autonomic nervous system. Journal of neuroscience research 68:176-184. Weiler E, Benali A (2005) Olfactory epithelia differentially express neuronal markers. Journal of neurocytology 34:217-240. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante Jr AW (2003) Obesity is associated with macrophage accumulation in adipose tissue. Journal of clinical investigation 112:1796. Wendling F, Shreeve M, McLeod D, Axelrad A (1985) A self-renewing, bipotential erythroid/mast cell progenitor in continuous cultures of normal murine bone marrow. Journal of cellular physiology 125:10-18. Wensley CH, Stone DM, Baker H, Kauer JS, Margolis F, Chikaraishi D (1995) Olfactory marker protein mRNA is found in axons of olfactory receptor neurons. Journal of Neuroscience 15:4827-4837. Wernersson S, Pejler G (2014) Mast cell secretory granules: armed for battle. Nature Reviews Immunology 14:478. Wershil B, Mekori Y, Galli S (1989) The contribution of mast cells to immunological responses with IgE-and/or T cell-mediated components. Mast Cell and Basophil Differentiation and Function in Health and Disease 229. West DB, York B (1998) Dietary fat, genetic predisposition, and obesity: lessons from animal models. The American journal of clinical nutrition 67:505S-512S.

268 Wetzel CH, Oles M, Wellerdieck C, Kuczkowiak M, Gisselmann G, Hatt H (1999) Specificity and Sensitivity of a Human Olfactory Receptor Functionally Expressed in Human Embryonic Kidney 293 Cells andXenopus Laevis Oocytes. Journal of Neuroscience 19:7426-7433. Weyand I, Godde M, Frings S, Weiner J, Müller F, Altenhofen W, Hatt H, Kaupp UB (1994) Cloning and functional expression of a cyclic-nucleotide-gated channel from mammalian sperm. Nature 368:859. Whitman MC, Greer CA (2007) Adult-generated neurons exhibit diverse developmental fates. Developmental neurobiology 67:1079-1093. WHO (2010) WHO laboratory manual for the examination and processing of human semen: World health organization. Wichterle H, García-Verdugo JM, Alvarez-Buylla A (1997) Direct evidence for homotypic, glia- independent neuronal migration. Neuron 18:779-791. Wickman K, Clapham DE (1995) Ion channel regulation by G proteins. Physiological reviews 75:865-885. Wiesner B, Weiner J, Middendorff R, Hagen V, Kaupp UB, Weyand I (1998) Cyclic nucleotide- gated channels on the flagellum control Ca2+ entry into sperm. The Journal of cell biology 142:473-484. Wilborn C, Beckham J, Campbell B, Harvey T, Galbreath M, La Bounty P, Nassar E, Wismann J, Kreider R (2005) Obesity: prevalence, theories, medical consequences, management, and research directions. Journal of the International Society of Sports Nutrition 2:4. Wilhelm M, King B, Silverman A-J, Silver R (2000) Gonadal steroids regulate the number and activational state of mast cells in the medial habenula. Endocrinology 141:1178-1186. Williams KC, Dooley NP, Ulvestad E, Waage A, Blain M, Yong V, Antel J (1995) Antigen presentation by human fetal astrocytes with the cooperative effect of microglia or the microglial-derived cytokine IL-1. Journal of Neuroscience 15:1869-1878. Wilson D, Leon M (1988) Spatial patterns of olfactory bulb single-unit responses to learned olfactory cues in young rats. Journal of Neurophysiology 59:1770-1782. Wilson EO (1963) Pheromones. Scientific American 208:100-115. Winsky L, Nakata H, Martin BM, Jacobowitz DM (1989) Isolation, partial amino acid sequence, and immunohistochemical localization of a brain-specific calcium-binding protein. Proceedings of the National Academy of Sciences 86:10139-10143. Winzell MS, Ahrén B (2004) The High-Fat Diet–Fed Mouse. Diabetes 53:S215-S219. Wong ST, Baker LP, Trinh K, Hetman M, Suzuki LA, Storm DR, Bornfeldt KE (2001) Adenylyl cyclase 3 mediates prostaglandin E2-induced growth inhibition in arterial smooth muscle cells. Journal of Biological Chemistry 276:34206-34212. Wong ST, Trinh K, Hacker B, Chan GC, Lowe G, Gaggar A, Xia Z, Gold GH, Storm DR (2000) Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice. Neuron 27:487-497. Wong V, Russell LD (1983) Three-dimensional reconstruction of a rat stage V Sertoli cell: I. Methods, basic configuration, and dimensions. Developmental Dynamics 167:143-161. Woolf CJ, Safieh-Garabedian B, Ma Q-P, Crilly P, Winter J (1994) Nerve growth factor contributes to the generation of inflammatory sensory hypersensitivity. Neuroscience 62:327-331. Woolley DE (2003) The mast cell in inflammatory arthritis. New England Journal of Medicine 348:1709-1711. Wright NT, Margolis JW, Margolis FL, Weber DJ (2005) Refinement of the solution structure of rat olfactory marker protein (OMP). Journal of biomolecular NMR 33:63-68. Wu H, Wang H, Xiong W, Chen S, Tang H, Han D (2008) Expression patterns and functions of toll-like receptors in mouse sertoli cells. Endocrinology 149:4402-4412. Wu H, Ghosh S, Dai Perrard X, Feng L, Garcia GE, Perrard JL, Sweeney JF, Peterson LE, Chan L, Smith CW (2007) T-cell accumulation and regulated on activation, normal T cell expressed and secreted upregulation in adipose tissue in obesity. Circulation 115:1029- 1038. Wu H, Shi L, Wang Q, Cheng L, Zhao X, Chen Q, Jiang Q, Feng M, Li Q, Han D (2016) Mumps virus-induced innate immune responses in mouse Sertoli and Leydig cells. Scientific reports 6:19507. Wu W, Wong K, Chen J-h, Jiang Z-h, Dupuis S, Wu JY, Rao Y (1999) Directional guidance of neuronal migration in the olfactory system by the protein Slit. Nature 400:331. Xanthos DN, Sandkühler J (2014) Neurogenic neuroinflammation: inflammatory CNS reactions in response to neuronal activity. Nature Reviews Neuroscience 15:43.

269 Xiong Y, Hales DB (1993) The role of tumor necrosis factor-alpha in the regulation of mouse Leydig cell steroidogenesis. Endocrinology 132:2438-2444. Xu H, Barnes GT, Yang Q, Tan G, Yang D, Chou CJ, Sole J, Nichols A, Ross JS, Tartaglia LA (2003) Chronic inflammation in fat plays a crucial role in the development of obesity- related insulin resistance. Journal of clinical investigation 112:1821. Yamagishi M, Hasegawa S, Nakano Y, Takahashi S, Iwanaga T (1989) Immunohistochemical analysis of the olfactory mucosa by use of antibodies to brain proteins and cytokeratin. Annals of Otology, Rhinology & Laryngology 98:384-388. Yamamoto M, Hibi H, Miyake K (1995) New treatment of idiopathic severe oligozoospermia with mast cell blocker: results of a single-blind study. Fertility and sterility 64:1221-1223. Yanagimachi R (1972) Fertilization of guinea pig eggs in vitro. The Anatomical Record 174:9-19. Yanagimachi R (1994) Mammalian fertilization. The physiology of reproduction:189-317. Yanagimachi R, Kamiguchi Y, Mikamo K, Suzuki F, Yanagimachi H (1985) Maturation of spermatozoa in the epididymis of the Chinese hamster. Developmental Dynamics 172:317-330. Yang M-F, Chien C-L, Lu K-S (1999) Morphological, immunohistochemical and quantitative studies of murine brain mast cells after mating. Brain research 846:30-39. Yeung C, Anapolski M, Depenbusch M, Zitzmann M, Cooper T (2003) Human sperm volume regulation. Response to physiological changes in osmolality, channel blockers and potential sperm osmolytes. Human reproduction 18:1029-1036. Yeung C-H, Sonnenberg-Riethmacher E, Cooper TG (1999) Infertile spermatozoa of c-ros tyrosine kinase receptor knockout mice show flagellar angulation and maturational defects in cell volume regulatory mechanisms. Biology of Reproduction 61:1062-1069. Young JM, Friedman C, Williams EM, Ross JA, Tonnes-Priddy L, Trask BJ (2002) Different evolutionary processes shaped the mouse and human olfactory receptor gene families. Human molecular genetics 11:535-546. Youngentob SL, Margolis FL (1999) OMP gene deletion causes an elevation in behavioral threshold sensitivity. Neuroreport 10:15-19. Youngentob SL, Margolis FL, Youngentob LM (2001) OMP gene deletion results in an alteration in odorant quality perception. Behavioral neuroscience 115:626. Youngentob SL, Kent PF, Margolis FL (2003) OMP gene deletion results in an alteration in odorant-induced mucosal activity patterns. Journal of neurophysiology 90:3864-3873. Yu TT, McIntyre JC, Bose SC, Hardin D, Owen MC, McClintock TS (2005) Differentially expressed transcripts from phenotypically identified olfactory sensory neurons. Journal of Comparative Neurology 483:251-262. Yuan TT-T, Toy P, McClary JA, Lin RJ, Miyamoto NG, Kretschmer PJ (2001) Cloning and genetic characterization of an evolutionarily conserved human olfactory receptor that is differentially expressed across species. Gene 278:41-51. Yudin AI, Gottlieb W, Meizel S (1988) Ultrastructural studies of the early events of the human sperm acrosome reaction as initiated by human follicular fluid. Molecular Reproduction and Development 20:11-24. Zamir N, Rivenkreitman R, Manor M, Makler A, Blumberg S, Ralt D, Eisenbach M (1993) Atrial natriuretic peptide attracts human spermatozoa in vitro. Biochemical and biophysical research communications 197:116-122. Zhang C, Finger TE, Restrepo D (2000) Mature olfactory receptor neurons express connexin 43. Journal of Comparative Neurology 426:1-12. Zhang J, Shi G-P (2012) Mast cells and metabolic syndrome. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1822:14-20. Zhang J, Yuan Y, Liu Q, Yang D, Liu M, Shen L, Zhou Y, Wang Z (2017) Differentially expressed genes in the testicular tissues of adenylyl cyclase 3 knockout mice. Gene 602:33-42. Zhang X, Firestein S (2002) The olfactory receptor gene superfamily of the mouse. Nature neuroscience 5:124-133. Zhang X, Bedigian AV, Wang W, Eggert US (2012) G protein-coupled receptors participate in cytokinesis. Cytoskeleton 69:810-818. Zhang X, Rogers M, Tian H, Zhang X, Zou D-J, Liu J, Ma M, Shepherd GM, Firestein SJ (2004) High-throughput microarray detection of olfactory receptor gene expression in the mouse. Proceedings of the National Academy of Sciences of the United States of America 101:14168-14173. Zhao H, Ivic L, Otaki JM, Hashimoto M, Mikoshiba K, Firestein S (1998) Functional expression of a mammalian odorant receptor. Science 279:237-242.

270 Zhao S, Zhu W, Xue S, Han D (2014) Testicular defense systems: immune privilege and innate immunity. Cellular & molecular immunology 11:428. Zhou Z, Zheng Y, Steenstra R, Hickey W, Teuscher C (1989) Actively-induced experimental allergic orchitis (EAO) in Lewis/NCR rats: sequential histo-and immunopathologic analysis. Autoimmunity 3:125-134. Zhu L-J, Hardy MP, Inigo IV, Huhtaniemi I, Bardin CW, Moo-Young AJ (2000) Effects of androgen on androgen receptor expression in rat testicular and epididymal cells: a quantitative immunohistochemical study. Biology of reproduction 63:368-376. Zhuang X, Machuca H, Silver R (1993) Changes in brain mast cells during development in doves. In: Soc. Neurosci. Abstr, p 18. Zhuang X, Silverman AJ, Silver R (1999) Distribution and local differentiation of mast cells in the parenchyma of the forebrain. Journal of Comparative Neurology 408:477-488. Zierau O, Zenclussen AC, Jensen F (2012) Role of female sex hormones, estradiol and progesterone, in mast cell behavior. Frontiers in immunology 3:169. Zou D-J, Chesler AT, Le Pichon CE, Kuznetsov A, Pei X, Hwang EL, Firestein S (2007) Absence of adenylyl cyclase 3 perturbs peripheral olfactory projections in mice. Journal of Neuroscience 27:6675-6683. Zozulya S, Echeverri F, Nguyen T (2001) The human olfactory receptor repertoire. Genome biology 2:research0018. 0011.

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