IN VITRO STRESSOR RELATED CHANGES IN OVARIAN

STEROIDOGENESIS IN RAINBOW TROUT (ONCORHYNCHUS MYKISS)

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

SANGJUCTA BARKATAKI

In partial fulfillment of requirements

for the degree of

Master of Science

November, 2007

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While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada ABSTRACT

IN VITRO STRESSOR-RELATED CHANGES IN OVARIAN STEROIDOGENESIS IN RAINBOW TROUT (ONCORHYNCHUS MYKISS)

Advisors: Sangjucta Barkataki Professor J. F. Leatherland University of Guelph, 2007 Professor W. A. King

The study examined the affect of Cortisol on basal and c AMP-stimulated in vitro

steroidogenesis of rainbow trout {Oncorhynchus mykiss) ovarian follicles at different

maturational stages. Basal and cAMP-stimulated 17p-estradiol (E2) and testosterone (T)

production was highest during the mid-vitellogenic (MV) stage, compared with early-

and late-vitellogenic stages. Cortisol suppressed the basal steroid production in the MV

stage, and suppressed cAMP-stimulated steroid production in all three maturational

stages. To determine whether Cortisol exerts its effect on pregnenolone (P5) production, real time RT-PCR was used to measure the expression of genes coding for steroidogenic acute regulatory (StAR) protein, and cytochrome P450scc. There was no significant affect of Cortisol on the expression of either gene. To look for possible actions of Cortisol on steroidogenic enzyme function, the effect of the glucocorticoid on the biotransformation of radiolabeled Ps, and T was examined; Cortisol appeared to suppress the aromatization of androgens to estrogens by a mechanism that is not fully understood. ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my advisor, Dr. John Leatherland for offering the opportunity for me to pursue my goal, as well as for his encouragement, continual support and kindness throughout the course of my study. I would also like to thank my co-advisor Dr. W. A. King and my advisory committee member Dr. M. M.

Vijayan, for their suggestions, support and assistance in the planning and preparation of the thesis.

I would like to thank Dr. J. Raeside for his advice and assistance in interpretation of the HPLC findings.

I am grateful to Dr. N. Aluru for teaching me the techniques in the process of real­ time PCR and Ms. L. Lin for guiding me with RIA technique and many other ways for help during the study. I thank Mr. G. Perry for his help with HPLC aspects of the study;

Ms. H. Christie for her laboratory help, and the staff at Alma Aquaculture Research

Station for providing the fish used in this study. My thank is also extended to Department of Biomedical Sciences administration personal, W. Arthur, S. Cherry, K. Best, F.

Graziorto, and computer assistance, D. Robinson.

My special thank go to my parents, brother and my in-laws for their understanding and encouragement.

Lastly, I would like to express my whole heartedly thank to my husband, Rishi

Barkataki for his unconditional support and encouragement throughout my study.

1 DECLARATION OF WORK PERFORMED

I hereby declare that with the exception of the items indicated below, all the work reported in this thesis was performed by me.

The HPLC steroid separation was performed by Mr. G. Perry, Department of

Biomedical Sciences, University of Guelph. Dr. N. Aluru, Department of Biology,

University of Waterloo, guided me in the completion of the gene expression using quantitative RT-PCR. Immunostaining slide preparation was performed by Lucy Lin,

Department of Biomedical Sciences, University of Guelph. Maintenance of the animals for the study was done by the staff at the Alma Aquaculture Research Station.

Sangjucta (Ria) Barkataki

11 TABLE OF CONTENTS

ACKNOWLEDGEMENTS i

DECLARATION OF WORK PERFORMED ii

TABLE OF CONTENTS iii

LIST OF TABLES vi

LIST OF FIGURES vii

LIST OF ABBREVIATIONS ix

CHAPTER I. LITERATURE REVIEW 1

General introduction 1 Morphology of the ovary, ovulation and spawning in salmonid species 4 Stages of oogenesis in salmonid species 6 Environmental factors regulating ovarian development and maturation in salmonid species 8 Hypothalamus-pituitary gland-ovary (HPO) axis 11 Ovarian steroid hormone metabolism 14 Plasma sex steroid profiles in fish 21 Mode of action of steroid hormones 22 Stress-response in fish 24 General 24 ACTH regulation of Cortisol secretion 26 Cortisol effects on metabolism and the immune system 27 Cortisol and reproduction 27 Mode of action of corticosteroid hormones 29

MAJOR GOALS AND WORKING HYPOTHESIS OF THE STUDY 31

GENERAL EXPERIMENTAL APPROACH 32

CHAPTER 2. EFFECT OF STAGE OF MAURATION OF OVARIAN FOLLICLES ON STEROID HORMONE SECRETION AND RESPONSE TO cAMP STIMULATION 33

Introduction 34 Materials and methods 36 Chemicals 36 Animals used in the study 36

iii Sample collection for hormone measurement and gene expression 37 Measurement of gonadosomatic index (GSI) and follicular mass 38 Radioimmunoassay (RIA) of 17p-estradiol (E2) and testosterone (T) 38 Follicle total RNA extraction 39 Real time RT-PCR measurement of gene expression 39 RNA isolation 39 Primers 40 Relative standard curve 41 Quantification of samples 42 Statistical Analysis 42 Results 43 Discussion 54

CHAPTER 3. EFFECT OF CORTISOL ON OVARIAN STEROIDOGENESIS DURING EARLY, MD3 AND LATE STAGES OF FOLLICLE MATURATION 58

Introduction 59 Materials and methods 61 Chemicals 61 Animals used in the study 62 Sample collection for hormone measurement, gene expression and immunohistochemistry 62 Radioimmunoassay (RIA) of 17P-estradiol (E2) and testosterone (T) and follicle total RNA extraction 63 Histological preparation 63 Real time RT-PCR measurement of StAR and P450scc gene expression 64

RNA isolation and first strand cDNA synthesis 64 Primers 64 Relative standard curve 66 Quantification of samples 66 HPLC 66 Statistical Analysis 68 Results 69 Discussion 85

SUMMARY AND CONCLUSIONS 89

LITERATURE CITED 92

APPENDICES 115

I. Protein phosphorylation inhibition of basal and FS steroidogenesis 115 II. Cortland's Medium 118

iv III. Forskolin preparation 119 IV. Pregnenolone preparation 120 V. Radioimmunoassay [RIA] protocol 121 VI. Total RNA extraction protocol 123 VII. Formaldehyde agrose RNA gel protocol 125 VIII. First-strand cDNA synthesis 127 IX. DNA agrose gel protocol 128 X. Preparation of Cortisol for follicle incubation 131 XI. GR Immunohistochemical staining protocol 132 XII. Sep-Pak Cls column protocol 136 XIII. Effects of varying the levels of Cortisol treatment on 17P-estradiol (E2) productions by rainbow trout ovarian follicles 139 XIV. Effects of ovarian follicles on Cortisol metabolism 142

v LIST OF TABLES

CHAPTER II.

Table 2.1. Representative of the oligonucleotide primers of StAR and CYPscc used in quantitative real-time PCR (qPCR) 41

Table 2.2. Identification of three maturational stages [early-vitellogenesis (EV); mid-vitellogenesis (MV); late-vitellogenesis (LV)] of rainbow trout ovarian follicles based on the correlation of various measured variables... 56

CHAPTER IH.

Table 3.1. Representative of the oligonucleotide primers of StAR, P450scc, GR I, GRII, p-actin and EF-la used in quantitative real-time PCR 65

Table 3.2. Effect of Cortisol on mRNA expression of StAR and CYPscc genes in rainbow trout (Oncorhynchus mykiss) ovarian follicles 74

Table 3.3. Effect of Cortisol on mRNA expression of GR1 and GR2 genes in rainbow trout {Oncorhynchus mykiss) ovarian follicles 75

Table 3.4. Effect of Cortisol on mRNA expression of P-actin and EF-la genes in rainbow trout {Oncorhynchus mykiss) ovarian follicles 76

APPENDICES.

Table XIH.1. Levels of E2 productions by different concentrations [Cortisol lQOOng.ml"1); Cortisol 2 (50 ng.ml"1); Cortisol 3 (10 ng.ml'1)] of Cortisol 139

VI LIST OF FIGURES

CHAPTER I.

Figure 1.1. Pathway of ovarian steroidogenesis 16

CHAPTER II.

Figure 2.1a. Representative data showing the correlation of the date of collection of samples, with gonado-somatic index (GSI) and ovarian follicle size 46

Figure 2.1b. Representative data showing the correlation of gonado-somatic index (GSI) with basal and cAMP-stimulated E2 production 47

Figure 2.1c. Representative data showing the correlation of gonado-somatic index (GSI) and the ratio of FS:basal E2 production 48

Figure 2.2a. Effects of ovarian follicles developmental stages on basal and cAMP-stimulated E2 production 49

Figure 2.2b. Effects of ovarian follicles developmental sages on basal and cAMP-stimulated T production 50

Figure 2.3a. Expression of gene encoding for StAR mRNA expression of the basal and FS-stimulated treatments of all the three developmental stages 52

Figure 2.3b. Expression of gene encoding for CYPscc mRNA expression of the basal and FS-stimulated treatments of all the three developmental stages 53

CHAPTER in.

Figure 3.1. Effect of Cortisol on in vitro basal 17P-estradiol (E2) production by ovarian follicles at early-, mid- and late stages of vitellogenesis 70

Figure 3.2. Effect of Cortisol on in vitro cAMP-stimulated

vii 17p-estradiol (E2) production by ovarian follicles at early-, mid- and late stages of vitellogenesis 72

Figure 3.3. A section through the thecal cell (TC), granulosal cell (GC), zona radiata (ZR) of a rainbow trout (Oncorhynchus mykiss) ovarian follicle at the mid-vitellogenesis stage 78

Figure 3.4. Representative HPLC chromatograph showing the elution time of 18 steroid reference standards with UV absorbance at 254 nm 80

Figure 3.5. Representative HPLC profiles of radiolabeled free steroid hormone metabolites produced by rainbow trout ovarian follicles after in vitro incubation at 8-10°C for 18 h in medium containing [7- H]pregnenolone ([ H]P5) as substrate, either in the absence of Cortisol [A] or the in the presence of Cortisol [B] 81

Figure 3.6. Representative HPLC profiles of radiolabeled free steroid hormone metabolites produced by rainbow trout ovarian follicles after in vitro incubation at 8-10°C for 18 h in the cAMP-stimulated medium containing [7-3H]pregnenolone ([3H]Ps) as substrate, either in the absence of Cortisol [A] or the in the presence of Cortisol [B] 82

Figure 3.7. Representative HPLC profiles of radiolabelled free steroid hormone metabolites produced by rainbow trout ovarian follicles after in vitro incubation at 8-10°C for 18 h in medium containing [1,2,6,7-3H] testosterone ([3H]T) as substrate, either 83

Figure 3.8. Representative HPLC profiles of radiolabelled free steroid hormone metabolites produced by rainbow trout ovarian follicles after in vitro incubation at 8-10°C for 18 h in cAMP-stimulated medium containing [[1,2,6,7-3H] testosterone ([3H]T) as substrate 84

APPENDICES.

Figure 1.1. Effect of cantharidin on basal and cAMP-stimulated steroidogenesis 115

Figure XIV. 1. Representative HPLC profiles of radiolabelled free steroid hormone metabolites produced by rainbow trout ovarian follicles 142

viii LIST OF ABBREVIATIONS

A4 Androstenedione (Androst-4-ene-3, 17-dione)

ACTH Adrenocorticotropic hormone

BSA Bovine serum albumin cAMP Cyclic adenosine monophosphate cDNA Complementary deoxyribonucleic acid

CPM Count per minute

CREB cAMP response element binding

CYPscc Cytochrome P450 side chain cleavage

DHEA Dehydroepiandrosterone

DHP 17a,20p-Dihydroxy-4-pregnen-3-one

E2 17p-Estradiol (Estra-1,3,5(10)-trien-3 a, 17P-diol)

Ei Estrone (Estra-3-ol-1,3,5(10)-trien-17-one)

EV Early-vitellogenic

GH Growth hormone

GnRH Gonadotropin-releasing hormone

GR Glucocorticoid receptor

GSI Gonado-somatic index

GtH Gonadotropic hormone

HPGaxis Hypothalamus-pituitary gland-gonad axis

HPI axis Hypothalamus-pituitary gland-interrenal tissue axis

HREs Hormone response elements

33-HSD 3P-Hydroxysteroid dehydrogenase

ix 170-HSD 17p-Hydroxysteroid dehydrogenase

HSP Heat shocked protein

IGF Insulin-like growth factor

LV Late-vitellogenic

MIH Maturation inducing hormone

MIS Maturation inducing steroid mRNA Messenger ribonucleic acid

MV Mid-vitellogenic

OF Ovarian fluid

P450arom Cytochrome P450 aromatase

P4 Progesterone (Preg-4-ene-3,20-one)

P5 Pregnenolone (Preg-5-ene-3-ol-20-one)

POAH Preoptic anterior hypothalamus qPCR Quantitative RT-PCR

RIA Radioimmunoassay

SEM Standard error of the mean

SER Smooth endoplasmic reticulum

SPE Solid phase extraction

StAR Steroidogenic acute regulatory

T Testosterone

THP 17a,20p,21 -trihydroxy-4-pregnen-3 -one

UV Ultraviolet

VtG Vitellogenin Uxx Unknown steroid metabolite eluting at xx minutes

ZR Zona radiata LITERATURE REVIEW

General introduction

Unlike birds and mammals, in which fertilization is internal, many species offish are oviparous, i.e., they produce gametes that are released into the environment and fertilization is external. There are exceptions; some fish species exhibit internal fertilization associated with ovoviviparity or viviparity, and some species are parthenogenic. For the purpose of this literature review, I will deal mainly with salmonid species in which the oocytes are fertilized outside of the body of the female, and which exhibit either semelparity such as salmon which spawn only once in their life or iteroparity such as trout which spawn in several years. Because gamete fusion and embryo development takes place outside the body cavity, external factors such as temperature, photoperiod, and chemical toxicants, have a major influence on the reproductive cycle.

In salmonid fish, the major phases of ovarian development are growth, maturation and ovulation; these phases are characterized by different metabolic and physiological events taking place inside the ovarian follicles, changes in the rate of production of hormones by the follicles, and the nature of the main hormones produced, particularly the ovarian steroids (Leatherland et al, 2003; Nakamura et al, 2005; Thomas et al, 2006).

In addition, non-ovarian hormones, particularly the gonadotropins, but also hormones such as GH, somatostatin and melatonin are known to exert modulating actions

(Holloway et al, 1999; Leatherland et al, 2004, 2005), although their specific role is not yet well understood. At the time of maturation in lower vertebrates, maturation-inducing

1 hormone (MIH) [17a, 20p-dihydroxy-4-pregnen-3-one (17a, 20p-P, DHP) in salmonid fishes and 17a,20p,21-trihydroxy-4-pregnen-3-one (20P-THP) in sciaenid fishes] acts on the receptors present on the oocyte membrane and induces the activation of maturation- promoting factors in the oocyte cytoplasm (Ishikawa et al, 1977; Godeau et al, 1978;

Masui and Clarke, 1979; Yoshikuni et al, 1993; Thomas et al, 2006). During this period, major morphological changes take place in the oocytes associated with progression of the meiotic cell cycle; the breakdown of the oocyte nuclear envelope, germinal vesicle breakdown (GVBD), which occurs at the prophase/metaphase transition, is usually regarded as the indicator of the progress of oocyte maturation.

The two major steroid hormones involved in ovarian growth and maturation in salmonid fishes are 17p-estradiol (E2) and 17,20P-P (DHP) (Kime, 1993; Goetz and

Garczynski, 1997; Levavi-Sivan et al., 2004); they are produced by the interaction of two cell layers, the thecal and the granulosal cell layers (two-cell type model: Nagahama et al., 1985b). Prior to oocyte maturation in salmonid species, there is a distinct shift in the steroidogenesis from E2 to DHP, probably a consequence of dramatic changes in the expression of genes encoding various steroidogenic enzymes (Nakamura et al, 2005;

Ings and Van Der Kraak, 2006). In addition to its actions on the oocytes, E2 acts on the liver to stimulate the synthesis of vitellogenin (VtG) and zona pellucida (radiata) [ZR] proteins, and on adipose tissue to stimulate lipolysis. Incorporation of VtG and lipid into the oocytes causes the growth of the oocytes (reviewed by Devlin and Nagahama, 2002;

Mosconi et al, 2002; Finn, 2007). Recent studies of ovarian maturation of rainbow trout suggest that a significant hydration of the oocytes occurs during oocyte maturation, and in vitro this can be induced by two steroid hormones - DHP and Cortisol (Milla et al,

2 2006). Once the oocytes have matured, changes in the expression of steroidogenic enzymes result in a decrease in E2 synthesis, and an increase in the synthesis of progestogens; plasma levels of DHP spike, and it is this peak of progestogen that initiates ovulation (Goetz et al, 1987; Kawauchi et al, 1989; Nagahama et al, 1995; Rahman et al, 2001; Senthikumaran et al, 2003). Apart from the role of E2 in ovarian function, in higher vertebrates, the hormone is found to form a complex by interacting with the growth factor in the adult central nervous system (Scharfman and MacLusky, 2006), and it is also involved in Alzheimer's disease (MacLusky, 2004).

Some of the handling techniques that are used in aquaculture practice, endocrine disrupting chemicals, and factors that affect fish in the wild, such as predation, have all been shown to act as stressors in salmonid species, in some instances resulting in an increase in circulating levels of the hormone, Cortisol. In these species, there is evidence of metabolic and physiological effects of stressors possibly acting via the increase in plasma Cortisol levels (Donaldson, 1981; Schreck, 1981; Vijayan and Leatherland, 1990;

Mommsen et al, 1999; Reddy and Leatherland, 2003). Cortisol, which is commonly considered to be a primary stress indicator, is the principle corticosteroid in teleost fishes.

Exposure of fish to stressors has been associated with impaired reproductive function in some species. Whether this is related to altered control of ovarian function via the hypothalamus-pituitary gland axis, or whether Cortisol exerts its effects directly at the level of the ovary, is not known. In this thesis, I focused on the possible actions of

Cortisol on ovarian follicle steroidogenesis in rainbow trout (Oncorhynchus mykiss), examining whether the hormone has an inhibitory or stimulatory action on the follicular production of sex steroid hormones, and if so, evaluating the potential sites of Cortisol

3 action. The following reviews the literature related to the growth and maturation of ovarian follicles in salmonid fishes, and explores the affects of stressors on ovarian maturation.

Morphology of the ovary, ovulation and spawning in salmonid species:

The ovaries in salmonid fishes are located bilaterally along the sides of the abdominal cavity. They are supported by the mesovarian mesentery which attaches the ovary to the body wall. The lamellae of the ovary are rich in blood vessels, ensuring the adequate supply of blood to the maturing ovarian follicles. The blood vessels surrounding the lamellae are supported by septa of connective tissue (de Vlaming, 1972).

The lamellae projecting into the lumen of the ovary are covered by a germinal epithelium

(Van Den Hurk and Peute, 1979).

The bulk of the ovary comprises ovarian follicles, and each follicle contains the oocyte, with its associated zona pellucida or zona radiata (ZR), surrounded by layers of steroid hormone secreting cells, the thecal and granulosal layers. The oocytes are derived from primordial germ cells (PGCs) that migrated through the gut mesentery and the gonadal ridges of the mesonephros of the early embryo to reach the primitive ovary

(Byskov and Hoyer, 1994). During the first year of gonadal maturation in salmonid fishes (sometimes referred to as puberty), the PGCs begin to differentiate into oogonia which undergo a species-specific number of mitotic divisions until they enter meiosis and become oocytes. On average, a mature Pacific salmon, such as coho salmon

(Oncorhynchus kisutch) has 2000 - 3500 oocytes; the number for rainbow trout is approximately 1500-2000.

4 The developing follicles take up ZR proteins, VtG and lipid from the maternal blood and these are incorporated into the growing oocytes by means of transport proteins; oxygen also moves by diffusion from maternal blood into the oocytes. Other factors, such as steroid, thyroid and pituitary hormones, and lipophilic contaminants, are also transferred to the oocytes from the maternal circulation (Scott et al, 1980a; Supriya et al, 2005).

During the late stages of ovarian maturation, when steroid hormone synthesis has largely changed from estrogen to progestogen as the major product (Nakamura et al,

2005), the first polar body is formed, and the second meiotic division is arrested at metaphase until fertilization. Ovulation involves the release of the oocyte and its surrounding ZR (now defined as an egg) into the body cavity of the female, where they may be retained for several days before spawning takes place; a fluid matrix, called ovarian fluid (OF), comprising exudates of the ovary and secretion of tissues in the body cavity (Lahnsteiner et al, 1995; Lahnsteiner, 2002) maintains the plasticity of the ZR, and in some species the OF released with the eggs at spawning is the medium in which the spermatozoa are motile for purposes of fertilization of the oocyte. Ovulation is usually associated with the maturation of the ovulatory "vent" which acts as the conduit for release of the eggs. Any delay in the spawning or artificial removal of the eggs from the body cavity results in detrimental changes in the viability of the gametes, and if spawning is delayed for an extended period, the eggs are usually reabsorbed. Normal {i.e., non- induced) spawning, including the final maturation and opening of the vent requires appropriate external stimuli, often including pheromonal stimulation from the male, and possibly also visual and tactile stimuli (Whitehead, 1978; Duston and Bromage, 1986).

5 Penetration of the spermatozoa occurs through a single pore in the ZR, the micropyle; penetration of the head of the spermatozoa into the oocyte induces the completion of meiotic division and the extrusion of a polar body. Penetration of the spermatozoa through the micropyle must occur rapidly, because the ZR, on contact with water, absorbs water and begins to swell, and at this time the micropyle portal is closed.

This process is commonly called "water hardening", and results in the formation of a relatively impermeable and protective acellular membrane within which the embryo will develop until hatching.

Stages of oogenesis in salmonid species:

Oogenesis is the process by which primordial germ cells (PGCs) become ova that are ready to be fertilized. In salmonid species, oogenesis is a complete cycle of egg development which is initiated with the recruitment of oogonia and terminates at ovulation. A complex series of events occur before ovulation and subsequent fertilization in salmonid fishes as well as most other teleostean families, namely (1) the formation of

PGC's; (2) the transformation of PGCs into oogonia (differentiation); (3) the transformation of oogonia into oocytes (onset of meiosis); (4) the growth of oocytes; (5) the maturation of the oocytes; and (6) ovulation (Wallace and Selman, 1981; Breton et ai, 1998; Celius and Walther, 1998).

The total number of oocytes present in the adult ovary is determined by the number of PGC's formed in the yolk sac epithelium of the embryo. As stated earlier, these differentiated oogonia undergo a species-specific number of mitotic divisions until they enter meiosis and become oocytes (Patino and Redding, 2000; Strussmann and

6 Nakamura 2002). Oocyte growth, discussed in the context of the whole ovarian follicle, can be classified into previtellogenic and vitellogenic stages. The basic structure of the ovarian follicle is established when granulosal cells and their associate basement membrane entirely surround the oocyte at its late pachytene or early diplotene stage of chromosomal development. Granulosal cells form a monolayer around the oocyte and the connective tissues surrounding the granulosal layer form a heterogeneous second layer called thecal cell layer which is added over the granulosal cells. A non-cellular protein layer, the ZR, forms between the granulosal cells and the oocyte of each ovarian follicle.

This follicular structure remains unchanged throughout the growth stage (Wallace and

Selman 1990; Patino and Takashima 1995). During the vitellogenesis stage, lipid and

VtG, are transferred from maternal blood into the oocyte (Mommsen and Walsh 1988).

At this stage, the growth of the oocyte reaches its maximum, and this is associated with the peak production of E2 (Kawaga et al, 1982; Idler and Ng, 1983; Nagahama et al,

1994). The oocyte maturation phase follows the vitellogenesis phase, during which the oocyte prepares for ovulation (Goetz, 1983; Van Der Kraak and Donaldson, 1986).

In fish, oocyte growth and maturation is primarily mediated by the action of two pituitary gonadotropins (GtH), GTH I and GtH II (Fontaine and Burzawa-Gerard, 1978;

Fontaine, 1980); these are the homologues of mammalian follicle stimulating hormone

(FSH) and luteinizing hormone (LH), respectively (Suzuki et al, 1988). During the oocyte maturation phase there is an increase in GtH II secretion; the hormone binds to its receptor on the granulosal cells and stimulates the production of DHP (Levavi-

Zermonsky and Yaron, 1986; Kawauchi et al, 1989), otherwise known as MIH

(Nagahama et al, 1983, 1994; Khan et al, 1999). In salmonid species, GtH II is the

7 predominant gonadotropin in the plasma and pituitary gland during final oocyte maturation stage (Dickhoff and Swanson, 1990; Swanson, 1991; Prat et al, 1996). The

GtH actions do not appear to directly affect oogenesis, but they are mediated through

steroid hormones produced in the ganulosal and thecal cells. DHP acts on transmembrane receptors on the oocytes and acts to re-initiate meiosis (Patifio et al, 2001). During this

stage there is a distinct steroidogenic shift from E2 to DHP synthesis (Senthikumaran et al, 2003) reflected in changes in the plasma level of these steroids (Goetz et al, 1987;

Nagahama et al, 1995). In the Japanese huchen (Hucho perryi) and the common carp

(Cyprinus carpio) there is evidence that suggests E2 and DHP significantly promote DNA synthesis in the ovarian germ cells, however E2 acts directly on oogonial proliferation while DHP acts directly on the initiation of the first meiotic division of oogenesis

(Higashino et al, 2002; Miura et al, 2007). The final stage is ovulation, which involves the expulsion of the oocyte from its follicle, is promoted by DHP (Pinter and Thomas,

1999).

Environmental factors regulating ovarian development and maturation in salmonid species:

The production of viable gametes is essential for the survival of any given species, and the quality of the gametes plays a critical role in determining the developmental potential of the embryo. The quality of the oocyte is established throughout the period of vitellogenesis when the oocytes grow and during the final stages of oocyte maturation. The use of different reproductive technologies, such as the isolation of oocytes for in vitro fertilization and development of the embryo, affect the quality of

8 the oocyte, as does exposure of the animal at key developmental stages to stressors. Even though there may be no apparent affect of the treatments on the meiotic process and nuclear maturation, or on the successful completion of the early developmental process, other functions may be affected.

The regulation of ovarian development and maturation in salmonid fishes depends on a range of biotic and abiotic factors, including environmental conditions of photoperiod and temperature, nutritional factors, which influence the endocrine regulation of gonadal development. Some are discussed below.

The reproductive cycle of many salmonid species is dependent on temperature and photoperiod. The modulation of reproductive development in rainbow trout is dependent on the rhythmic processes of photosensitivity, and circadian and circannual rhythms that regulate the timing and entrainment of this cycle (Duston and Bromage,

1986). For most strains of autumn-spawning rainbow trout, photoperiod is thought to be the main factor involved in determining the time of onset of gonadal development, since gonadal maturation can be induced artificially by photoperiod manipulation (Whitehead,

1978). For autumn-spawning stocks of trout, a gradually increasing photophase, followed by decreasing photophase appears to be the principal environmental factor in regulating reproductive cycles and gametogenesis (Breton etal, 1977; Whitehead, 1978). However, for some spring-spawning genetic strains of rainbow trout, the photoperiod regulation of the time of onset of ovarian (and testicular) development is reversed. For some species temperature is also a factor, although it does not appear to be limiting in trout reproduction (Breton ef al, 1977; Billard etal, 1978).

9 Elevated water temperatures impair reproduction in trout; and trout embryos need cool, well oxygenated water to develop properly. Interestingly, environmental water temperature is one of the factors in sex determination in some species of fish, with high temperatures increasing the male:female ratio (Patifio et al, 1996; Blaquez et al, 1998;

Baroiller, 1999). Recently it has been reported that in Atlantic salmon (Salmo salar) high temperature spikes can affect reproductive success as strongly as more prolonged exposures, and also that the critical period of reproductive sensitivity to elevated temperature is late February and early March (King et al, 2007). There is also an effect of elevated temperatures on gonadal steroid production, vitellogenesis and egg quality in the same species (King et al, 2003). In addition, factors such as conspecific interactions, ambient pH, and xenobiotic chemicals in the environment are known to determine sex differentiation or reproductive function in fish (Maradonna and Carnevali, 2007; Ritchie et al., 2007). In rainbow trout, environmental temperature has an effect on ovulation, egg fertility, plasma levels sex hormones and in vitro ovarian steroidogenesis (Pankhurst et al, 1996, Suzuki et al, 1997). In cold water fishes, e.g., Arctic charr {Salvelinus alpinus}, increasing temperature results in delay in time of ovulation as well as lower rate of viability of eggs (Gillet, 1991). In male rainbow trout, cooler water temperature encouraged the production of spermatogonia and spermatocytes, and warmer temperatures stimulate the formation of spermatids and spermatozoa; this might be a

GtH-regulated transition since the magnitude of the rise in plasma GtH concentrations was greater at higher temperature than in lower water temperature (Breton et al, 1977).

Environmental factors other than photoperiod and water temperature, such as the availability of suitable diets, are also known to affect reproductive success. In fish,

10 nutrition is known to have profound effect on gonadal growth and fecundity (Watanabe,

1989). In captive female broodstock, a restricted diet during early stages of ovarian development delays the first maturation age of the animal; restricted food availability during oocyte differentiation reduces the number of eggs produced, while a reduction of food levels during the last phases of oogenesis has only a small effect on egg size, composition and hatchability (Luquet and Watanabe, 1986).

Other factors play roles in ovarian maturation and spawning; for example, suitable spawning habitats are needed for spawning of many fish species, and if these are not available, the gametes may be retained and reabsorbed; similar disturbances in water flow

(stream discharge) adversely affect natural reproduction in a trout population (Banks and

Bettoli, 2000; Orth et al, 2001; Pender and Kwak, 2002).

Hypothalamus-pituitary gland-ovary (HPO) axis:

As in all other vertebrates, the HPO axis in teleost fishes is very significant in the regulation of the rate of steroid hormone synthesis. It is well established that the hormones of the hypothalamus regulate the activity of the gonadotrophs in the pituitary gland (Yaron et al, 2003; Ando and Urano, 2005), and that GtH I and GtH II play a major role in regulating gonadal activity (Peter, 1970, 1973). Different concentrations of the circulating steroids are involved in the maintenance of the feedback regulation of GtH secretion and the optimization of HPO axis regulatory feedback (Ying, 1988; Khan et al,

1999; Rousseau et al, 2002).

11 In most fish species studied to date the hypothalamic hormones, gonadotropin- releasing hormone (GnRH) and dopamine (L-DOPA) play key roles in regulating the

HPO axis activity, although other hypothalamic factors may also be involved. The secretion rate of both GnRH and L-DOPA is influenced by changes in the environmental stimuli (photoperiod and water temperature) that regulate to onset of reproductive development (Kime, 1993; Grober etal., 1995).

GnRH is a decapeptide hormone, synthesized and release by the hypothalamus and is responsible for the release of GtH I and GtH II from the anterior pituitary. L-

DOPA is a phenethylamine which acts both as a neurotransmitter and a neurohormone.

As a member of the catecholamine family, it acts as a precursor for the neurotransmitters, norepinephrine and epinephrine. GnRH, acts on the pituitary gonadotrophs via a seven- transmembrane receptor type belonging to the G protein-coupled receptor family, and initiates several intracellular signalling pathways (Robinson et ah, 2001; Pati and Habibi,

2002). The release of GtH from the pituitary gland is stimulated by GnRH, and inhibited by dopamine (Szabo et al, 1991; reviewed by Trudeau et al, 2000; Vacher et. ah, 2002).

In T-primed immature rainbow trout, GtH release is also stimulated by the glutamate agonist, N-methyl-D, L-aspartate (NMA) (Flett etah, 1996), suggesting the involvement of other secretagogues in the modulation of GtH secretion.

During the period of ovarian development in rainbow trout, there is differential production and regulation of GtH I and GtH II by the pituitary gland (Naito et ah, 1991).

The plasma GtH profiles in salmonid fishes suggest that GtH I and GtH II are secreted in gradually increasing amounts to stimulate gonadal recrudescence; a sharp increase the levels of GtH II occurs at the time of spermiation or ovulation (Billard et ah, 1977;

12 Fostier et ah, 1978). In fish, GtH synthesis and release by pituitary gonadotrophs is partly influenced by the gonadal glycoprotein hormones, activin, inhibin, and follistatin

(reviewed by Ge, 2000). In female teleost fishes, the early developmental stage of the ovarian follicle appears to be independent of GtH; during the vitellogenic stage the oocytes are under the influence of GtH I (Breton et ah, 1998), whereas in the later stage, during the final maturation of oocytes, GtH II promotes the synthesis of DHP (Nagahama etah, 1994; Breton ef ah, 1998).

In teleost species, GtHs induce oocyte maturation through the synthesis of C21 steroids (Jalabert, 1976). At puberty, the ovary begins to secrete steroid hormones for the first time in response to stimulation by GtH I at first, and then by GtH II (Miller and

Strauss, 1999). Receptors for GtH I and GtH II have been found both in thecal and granulosal cells of coho salmon (Yan et ah, 1992). Other pituitary hormones in addition to GtH I and II, and hormones from non-pituitary sources, such as the thyroid hormones, and Cortisol may also regulate gonadal function. In addition, sex steroids and perhaps non-steroid factors exert both positive and negative feedback control on the hypothalamus, the pituitary gland and the gonad itself (Mendez et. ah, 2005).

In addition to its role in the regulation of GtH synthesis and release, GnRH and

GnRH receptors are also produced in the ovary and testis offish (Baker, 2001; Adams et ah, 2002). GnRH may act as a paracrine factor controlling some aspects of gonadal development and function and coordinates reproduction with the environment (Grober et ah, 1995; Sherwood etah, 1997; Adams etah, 2002).

13 Ovarian steroid hormone metabolism:

Steroid hormones are derivatives of cholesterol; the basic structure of all steroid hormones is the cyclopentanoperhydrophenanthrene ring. These hormones are produced by steroidogenic tissues of adrenal and gonadal origin and are released into the blood stream; in the gonads the steroids also pass directly either into the oocytes, or into the seminiferous lobules. As discussed above, the major "end-product" steroids of the salmonid ovary are E2 and T, although several intermediary steroids may also appear in plasma; the production of the main endpoint steroids is the focus of this review.

Steroidogenesis is the production of biologically active steroid hormones

(androgens, estrogens and progestogens) from pregnenolone (P5). The pathway of ovarian steroidogenesis is shown in Figure 1.1. Steroid hormone excretion is brought about by the biologically active steroid hormones being transformed into more water soluble steroids that can be readily excreted. The synthesis of steroid hormones involves a sequence of reactions catalyzed by monooxygenases associated with the cytochrome P450 (CYP) family. Some of these reactions occur in the inner compartment of the mitochondria of steroid secreting cells, and others occur in the cytoplasm in association with the smooth endoplasmic reticulum. As steroids are formed, they leave the steroid secreting cells, probably by diffusion, and enter the extracellular fluid in the ovary; some of these find their way into the main circulatory system, and some enter the oocytes.

As described previously, during the period of development of the ovarian follicle in teleost fishes, the thecal and granulosal cell layers form around the immature oocytes

(Wallace and Selman, 1990); the production of E2 by the granulosal cells stimulates the

14 synthesis of the lipophosphoprotein, VtG by the liver and the mobilization of lipid from various tissues. The incorporation of VtG and lipid into the ooplasm (together with the cortisol-induced hydration discussed earlier) leads to an increase in the size of the oocyte

(reviewed by Devlin and Nagahama, 2002; Mosconi et al, 2002). During the vitellogenic stages, GtH I acts on the thecal cells stimulating them to produce androgens as the end- point steroids; the androgens pass into the granulosal cells which contain P450 aromatase

la (P450arom-la); this enzyme catalyzes the conversion of androgen into predominantly

E2 (Goetz et al, 1987; Kime, 1993; Nagahama et al, 1995; Goetz and Garczynski, 1997).

After the oocyte maturation phase is complete, during the post-vitellogenic stage, leading to ovulation, there is a switch in steroid synthesis to progestogens, primarily DHP in salmonid species (Nagahama and Aldachi, 1985a; Canario and Scott, 1990; Kime,

1993; Goetz and Garczynski, 1997). DHP, acting via membrane receptors on the oocyte, stimulates the germinal vesicle breakdown and final oocyte maturation (Goetz and

Garczynski, 1997), hence its alternate name of MIS (Nagahama and Aldachi, 1985a).

15 P450sc 30-HSD N /Cholesterol » Pregnenolone [P5] > Progesterone [P4]

17a-hydroxylase 17a-hydroxylase

3P-HSD a 17 -OHP5 17a-OHP4

17,20-lyase 17,20-lyase I 3P-HSD I DHEA ^ Androstenedione[A« Estrone P450arom Ei]

17P-HSD 17P-HSD P45J)trom 1 Testosterone [T] —^. Estradiol [E2]

Thecal Cell Granulosa! Cell

Figure 1.1. Pathway of ovarian steroidogenesis by the thecal and granulosal cells of ovarian follicle. The arrows point to the steroid hormones produced, and the captions beside the arrows are the enzymes required at each step.

Abbreviations: P450scc, cytochrome P450 side chain cleavage; 3a-HSD, 3a- hydroxysteroid dehydrogenase; DHEA, dehydroepiandrosterone; 170-HSD, 170- hydroxysteroid dehydrogenase.

16 The site of production of gonadal steroid hormones by the thecal and granulosal cells is the smooth endoplasmic reticulum, where many of the steroidogenic enzymes are located; however, a key enzyme, cytochrome P450 side chain cleavage (CYPscc), is found only in the mitochondria. The rate-regulating step in steroidogenesis is the movement of cholesterol into the inner compartment of the mitochondria. Since cholesterol is hydrophobic, the hydrophilic inter-membrane space of the mitochondrion acts as a barrier to the movement of cholesterol into the mitochondrion. Trophic hormones such as ACTH and GtH induce adrenal and gonadal steroidogenesis via cAMP-regulated pathways that stimulates P5 synthesis by facilitating the delivery of cholesterol into the mitochondria. Two proteins are known to be involved in cholesterol transport into the mitochondria, steroidogenic acute regulatory (StAR) protein, and peripheral-type benzodiazepine receptor (PBR) protein; insertion of the StAR protein into the outer mitochondrial membrane allows cholesterol to move into the inner chamber of the mitochondria (Strauss et al, 1999; Hauet et al, 2002; Liu et al, 2006); details of the relationship between StAR protein and PBR protein are still not fully known. Most information pertaining to the roles of StAR and PBR proteins has been determined by studies of the mammalian adrenal cortex, but the pattern appears to be similar in ovarian steroidogenic cells and tissues.

Acute stimulation of steroidogenesis by the cAMP pathway occurs through the phosphorylation of pre-existing StAR protein and the rapid synthesis of new StAR protein. The enzyme, adenylyl cyclase, converts ATP into cAMP leading to the activation of protein phosphatase which activates StAR protein (Sewer and Waterman,

2002a, 2003; Donghui et al, 2007). Increases in the insertion of StAR protein into the

17 outer mitochondrial membrane increases the flow of cholesterol into the mitochondria, thus increasing substrate availability to whatever amount of cytochrome P450scc

(CYPscc) is present. Activation of the steroidogenic cell by its pituitary tropic hormone stimulates the insertion of StAR protein into the outer membrane of the mitochondria

(Vahouny et ah, 1978). StAR protein is rapidly deactivated (Liu et ah, 2006), and thus the amount of cholesterol transport into the mitochondria is dependent on the cAMP- regulated StAR protein insertion process. In mammals, the role of cAMP-dependent phosphorylation in the process of steroidogenesis is fairly well established (Lin et ah,

2001; Sewer and Waterman, 2002b; Ozbay et ah, 2006); a preliminary study in our laboratory has shown that basal and cAMP-stimulated steroidogenesis is inhibited in the presence of the phosphorylation inhibitor, cantharidin (Appendix I), suggesting that phosphorylation is part of the process in the regulation of steroidogenesis by rainbow trout ovarian follicles.

It appears that cAMP partly regulates the synthesis of StAR and other possible protein candidates through its interaction with, and the phosphorylation of, a member of cAMP response element binding (CREB) protein family (Manna et ah, 2002). There is a rapid expression of the gene encoding StAR protein during the acute phase of GtH stimulation in gonads, and ACTH stimulation in adrenal cortical cells. This phenomenon is presumed to be regulated by the increase in intracellular cAMP, and occurs in association with the members of the cAMP response element binding (CREB) protein family; CREB is known to interact with the cAMP response element promoter of target genes (Manna et ah, 2002).

18 In addition to the role of cAMP as the secondary intracellular messenger in gonadal steroidogenic cells, other cytoplasmic signaling pathways are also involved. Of these, calcium-dependent signal transduction pathways have been shown to play essential roles in some species (Pace and Thomas, 2005; Benninghoff and Thomas, 2006).

Within the inner compartment of the mitochondrion (Figure 1.1), cholesterol is cleaved by CYPscc (Hall, 1986) to produce P5 (Stocco, 2000; Aluru et al, 2005). As it is produced, P5 diffuses into the cytoplasm and, in the presence of the SER enzymes 30- hydroxysteroid dehydrogenase (3P-HSD) and A5-A4 isomerase (Hall, 1986), it is converted to progesterone (P4); P5 and P4 can then potentially be converted to 17a-OHPs and 17a-OHP4, respectively by 17a-hydroxylase; 17a-OHPs and 17a-OHP4, in turn, can be converted into dehydroepiandrosterone (DHEA) and androstenedione (A4), respectively by the activity of the enzyme 17,20-lyase (17,20-desmolase). In the presence of the enzyme, 17p-hydroxysteroid dehydrogenase (17P-HSD), T can be derived from A4 in the thecal cells. Some of the T formed in the thecal cells enters the granulosal cells and is converted into E2 in the presence of enzyme P450arom-la (Hall, 1986); some T also enters the circulation.

As discussed previously, hormones other than GtH are also known to affect ovarian steroidogenesis, but the biological significance of their roles is still not well understood. For example, steroidogenesis, in vitro, by rainbow trout ovarian follicles was suppressed by the glutamate agonist, N-methyl-D, L-aspartate (NMA), L-glutamic acid

(GA) and somatostatin (SRIF), even when exogenous cAMP was administered or endogenous synthesis of cAMP was enhanced (Leatherland et al, 2004, 2005).

Similarly, melatonin at concentration (1 x 10"2) and (1 x 10"4) M was found to have

19 inhibitory effect on the basal steroid production by rainbow trout ovarian follicles at the peak-vitellogenic and preovulatory stages (Leatherland and Lin, 2001). In addition, IGF-

I increases GtH I, and GnRH-stimulated GtH I release from the pituitary gonadotrops of coho salmon (Baker et al., 2001, 2007), and Campbell et al. (2006) proposed that the

IGF-I-induced increases in plasma levels of GtHs may be a mechanism by which pubertal development is promoted by somatic growth and nutrition. There is a positive correlation between plasma IGF-I, E2, and pituitary GtH I during growth and these factors may be an important link by which body growth influences the rate of oocyte development in fish.

The thyroid hormones may also play an important role during early stage of ovarian development and vitellogenesis in teleost species (Supriya etal, 2005), possibly acting to potentiate GtH-stimulated gonadal steroid hormone secretion by ovarian follicles (Cyr andEales, 1988; Cyr etal, 1988; Flette/a/., 1989).

In vertebrates, the regulation of steroid hormone homeostasis involves both the synthesis of steroid hormones as well as the regulation of steroid hormone metabolism and excretion. In order to avoid steroidogenic tissues becoming overexposed to steroid hormones, it is essential for the tissues to regulate their steroid environment. Thecal and granulosal cells in addition to being the major steroid production sites can also inactivate their products by forming sulphated and glucuronidated steroids (conjugated steroids).

These sulphate or glucuronide conjugates are more water soluble than the "free" steroid making them more readily excreted, and their binding affinity to the specific steroid receptors markedly decreases (Norman and Litwack, 1997).

20 Plasma sex steroid profiles in fish:

Earlier studies on steroidogenesis in salmonid fishes emphasized the "classical" teleost steroid hormones, particularly E2, DHP and T in females and T, 11- ketotestosterone (11KT) and DHP in males. There are low levels of plasma sex steroids in immature female fish and sexual maturation is associated with the increasing levels of plasma sex steroids. As discussed previously, although the main circulating sex steroid hormone in female fishes is E2, T is also present in the plasma in significant levels since it is the immediate precursor in E2 biosynthesis. In female salmonid fish, there is an increase in the plasma levels of E2 and T during the vitellogenesis phase and DHP levels increase rapidly at the time of ovulation (Goetz et ah, 1987; Nagahama et ah, 1995).

Conversely, in male salmonid fish, androgens, such as T and 11KT are secreted during early spermatogenesis, and DHP, which is involved in spermiation, is secreted when the spermatozoa mature (Planas et ah, 1993). However, measurement of only these hormones can be misleading, since blood steroid hormone levels are frequently low, and a range of hormones other than the "classical" steroids are present in the blood (Canario and Scott, 1987; Kime, 1993). Steroid hormones such as 11-deoxycorticosterone and 11- deoxycortisol (Colombo and Belvedere, 1976), epipregnanolone (3a-hydroxy-5P- pregnan-20-one) and pregnanediol (5P-pregnan-3a-20a-diol) (Ungar, 1977), and P4 have also been found in the blood of some species (e.g., winter flounder, Pseudopleuronectes americanus [Campbell et ah, 1976)]; however, their biological significance (if any) has yet to be established. Similarly, it is not yet known whether T has physiological functions in female fish (Kime, 1979; Sandor, 1979); however, in rainbow trout, plasma levels of 11-KT, the primary androgen in males, is undetectable or extremely low during

21 most of the reproductive cycle (Simpson and Wright, 1977) suggesting that it probably plays no significant role in ovarian development.

Mode of action of steroid hormones:

The best known mode of action of steroid hormones is their interaction with receptors that act as transcription factors that regulate the expression of specific genes

(McDonnell et al, 1993; Baker, 2001; Thomas et al, 2006). However, transmembrane steroid receptors have now also been identified in the gonads of some fish species; some of the roles of these types of receptors in ovarian function in fishes are beginning to be identified (Hawkins and Thomas, 2004; Thomas et al, 2006; Tokumoto etal, 2006).

As regards the "nuclear" receptors, steroid hormones pass from the interstitial fluid into the target cells and bind to specific steroid hormone receptor proteins that are either present in the cytoplasm or in the nucleus of the target cell, depending on the receptor. These receptors are members of the nuclear receptor family that include a group of homologous structured receptors that binds to non-steroid ligands such as thyroid hormones, vitamin D, and vitamin A. Of the two types of nuclear hormone receptors, namely "type I" and "type II", type I receptors include the androgen (AR), estrogen (ER), progesterone (PR), glucocorticoid (GR) and mineralocorticoid receptors

(MR). Type II nuclear receptors include the thyroid hormone (T3R), vitamin A (VAR), vitamin D (VDR), trans-retinoic acid (RAR), and 9-cis retinoic acid (RXRs) receptors.

The receptors for specific ligands can have different isoforms (variations) which have different DNA hormone response element (HRE) specificities, and different ligand affinities.

22 Only the type I receptors will be considered here; they are thought to be normally present in the cytoplasm of target cells. Free steroid enters the cell; interacts with its specific receptor to form an activated receptor-ligand complex. As the ligand binds to its receptor, heat shock protein (HSP) which is associated with the non-liganded form of receptor (Kawarsky and King, 2001; Matwee et ah, 2001) disassociates from the receptor, and the receptor-ligand complex then translocates via pores in the nuclear membrane to the nucleus. The activated receptor complex then either dimerizes, or binds sequentially to unique nucleotide sequences, the HREs, in the promoter region of specific genes. This ligand-receptor-HRE complex, then acts as a transcription factor for specific genes. The transcription factor may act to stimulate or inhibit the transcription of the gene (depending on the sequence of the HRE), thus regulating the synthesis of specific mRNA.

As indicated above, in addition to the nuclear receptors for steroid hormones, plasma membranes steroid receptors are found in some target cells (Luconi et al, 2004).

Far less is known about the roles of these cell membrane steroid receptors, but ligand activation of these receptors activates several intracellular signal transduction pathways.

In fish gonads, in which these steroid membrane receptors were first reported, there is evidence to suggest the presence of several G-protein associated steroid membrane receptors, including progestin receptor-a (mPRa), an estrogen receptor (mER), and an androgen receptor (mAR) (Thomas et ah, 2004, 2006). In fish, the mPRa receptor has been reported to induce oocyte maturation, increase sperm motility and exert rapid actions in the preoptic anterior hypothalamus (POAH) to down-regulate GnRH secretion

(Thomas et ah, 2004). Two other membrane steroid receptors, namely P4 membrane

23 receptor component 1 (PGMRC1) and membrane progestin receptor-P (mPRp), have been identified in rainbow trout, and appear to play a significant role in induction of oocyte maturation (Mourot et al, 2006; Nutu et al, 2006). Another cell membrane steroid receptor specific for DHP (DHPR) found on the oocyte membrane might also be involved in the regulation of oocyte growth and maturation in teleost fishes (Thomas et al, 2001).

Stress-response in fish:

General:

The term "stress" is used to describe a situation in which an organism is exposed to an unfavorable or unfamiliar environmental condition resulting in some alteration in normal physical and physiological functioning. The factor causing the stress is called a stressor; the physiological response of the organism is the stress-response. Stress can be described as a wide range of strong external stimuli, both physiological and psychological that can act on physiological response of the body. In fish, as in other vertebrates, the stress response can be separated into three phase, the so-called primary, secondary and tertiary responses. The primary response is rapid, and is associated with the release of the catecholamine hormone, epinephrine, and the epinephrine-stimulated elevation of blood glucose, changes in the cardiovascular system, and ionoregulatory changes (Zhang et al, 1992; Gamperl and Boutilier, 1994). The secondary response occurs within minutes to hours, and involves the elevation of plasma Cortisol concentrations, associated with cortisol-driven metabolic changes, and suppression of

24 immune responses. The tertiary responses are the longer term responses to chronic stress, such as increased infection (related to a suppressed immune system), reduced growth, or impaired reproductive function (Pickering and Pottinger, 1987; Vijayan and Leatherland,

1988; Reddy and Leatherland, 1998). The tertiary responses may, themselves, be related to the episodes of hypercortisolism (Renaud and Moon, 1980; Laidley and Leatherland,

1988; Vijayan and Leatherland, 1990; Ding et al, 1994; Gamperl et al, 1994; Vijayan et al, 1993, 1994, 1996, 1997; McCormick, 1995; Mommsenef a/., 1999).

In teleostean species, the major corticosteroids identified in blood plasma are

Cortisol and cortisone, but Cortisol is found in higher concentrations compared to cortisone (Donaldson and Fagerlund, 1972; Idler and Truscott, 1977). Serum Cortisol levels are significantly higher in stressed fish compared to unstressed rainbow trout

(Laidley and Leatherland, 1988; Kubilay and Ulukoy, 2002; Laiz-Carrion et al, 2002) and tilapia {Oreochromis mossambicus) (Foo and Lam, 1993a). In the freshwater fish

Notopterus notopterus, serum Cortisol levels are markably higher during the breeding phase as compared to the non-breeding phase (Shankar and Kulkarni, 2007), suggesting either a role for Cortisol in reproduction, or indicative of stressor-related responses during the breeding phase. Chronically elevated plasma Cortisol levels can have significant consequences; for example, a recent study on rainbow trout suggests that Cortisol implantation resulted in impaired memory (Barreto et al, 2006). As discussed in the later section, the elevated Cortisol during reproduction may also be related to the fact that fish go off feeding during that period, and the Cortisol response may be compensatory, inducing gluconeogenesis to ensure the production of carbohydrates needed to maintain central nervous system function.

25 ACTH regulation of Cortisol secretion:

Cortisol acts both as a glucocorticoid and mineralocorticoid in fish, and its secretion is regulated by the hypothalamus-pituitary gland-interrenal tissue (HPI) axis.

ACTH, released from the anterior pituitary gland is the main secretagogue for the regulation of Cortisol secretion (Mommsen et ah, 1999; Hagen et ah, 2006). The signaling pathways leading to acute ACTH-stimulated Cortisol secretion by the interrenal tissue of rainbow trout is stimulated by cAMP-dependent protein kinase (PKA) and inhibited by protein kinase C (PKC) (Liu et ah, 1992; Lacroix and Hontela, 2001).

Exposure offish to various stressors induces an elevation in the plasma Cortisol levels via the secretion of ACTH release and subsequent activation of the ACTH receptors on the interrenal cells; plasma Cortisol levels are commonly used to measure the stress response in fishes (Donaldson, 1981; Mazeaud and Mazeaud, 1981; McDonald and Milligan,

1992; Rotllant et ah, 2001; Tort et ah, 2001). The range of situations that result in changes in Cortisol secretion or the response to ACTH stimulation in fishes is broad, and includes dietary deficiencies of essential fatty acids (Ganga et ah, 2006), treatment with

P-naphthoflavone (Wilson et al., 1998), and other contaminants (Brown, 1993), and various handling stressors such as netting and handling (Laidley and Leatherland, 1988), and crowding (Rotllant et ah, 2000, 2001).

In salmonid species, an acute stressor-induced increase in circulating Cortisol levels is rapid, appearing within 10 minutes and being maintained for approximately 4 hours (Sumpter et ah, 1986; Davis and Parker, 1986; Laidley and Leatherland, 1988;

McDonald and Milligan, 1992). The degree and duration of the Cortisol elevation depends

26 on both the social and reproductive status of an individual fish, as does the reproductive capacity of individual fish (Cameron, 1997; Fox et al, 1997); the response of the reproductive axis to stress depends on the type of stressor, the magnitude and duration of the exposure to the stressor, the perception of the stress by the individual, the social status of the individual, the concurrent level of aggressive behavior displayed by the individual, seasonal cues, and the prior level of activity within the reproductive axis. This will be discussed further in a later section of this chapter.

Cortisol effects on metabolism and the immune system:

Cortisol, binding to its receptor, activates a number of enzymes that control many aspects of the intermediary metabolism offish (Kime, 1978; Truscott, 1979; Vijayan and

Leatherland, 1988, 1989; Vijayan et al, 1993, 1994, 1996, 1997; Leatherland, 1993;

Laiz-carrion et al, 2003, 2004; McDonald and Wood, 2004). In addition, immunosuppressive effects of Cortisol have been well documented in fish (Maule et al,

1987; Woo et al, 1987, Wiik et al, 1989); indeed the highly elevated plasma levels of

Cortisol have been associated with the post-spawning mass die-off of Pacific salmon. In this phenomenon it is proposed that the hormone causes tissue degradation, suppresses the immune system and impairs various homeostatic mechanisms (Dickhoff, 1989; Stein-

Behren and Sapolsky, 1992).

Cortisol and reproduction:

The reports of the actions of Cortisol on reproductive function in fishes are contradictory. On the one hand, Cortisol appears to have actions on the normal function

27 of the gonads of fishes, but other studies have found impaired reproductive function in fish that are in hypercortisolic states. Examples of the disparate responses to Cortisol in fishes are presented below.

The expression of hepatocyte nuclear factor (HNF-3P) was affected in vitro by both E2 and Cortisol which suggests that glucocorticoids may play a regulatory role during oocyte maturation (Huang etal, 2007). In addition, Cortisol was found to increase hepatic transcription of the VtG gene in tilapia (Ding et al, 1994) suggesting a role in enhancing yolk protein production. In addition, Cortisol administration appeared to increase the ovarian GSI, and the hepato-somatic index during the non breeding phase, possibly providing energy that is available for use during the reproductively active phase

(Shankar and Kulkarni, 2006). Conversely, Cortisol has been reported to interfere with the reproductive function in immature and maturing rainbow trout (Chakraborti et al, 1987).

The hormone appears to decrease hepatic estradiol binding capacity (Pottinger and

Pickering, 1990), suppress plasma VtG levels and inhibit VtG synthesis (Carragher et al,

1989), which may explain reported reductions in the growth of oocytes during the vitellogenic phase. In addition, in cortisol-implanted trout, the ovaries were small, plasma E2, T and VtG levels were low, as was the pituitary GtH content (Carragher et al,

1989; Pottinger et al, 1996); similarly, in cortisol-implanted tilapia, there was retardation of ovarian growth and depressed serum E2 and T levels (Foo and Lam, 1993 b). Further, in rainbow trout, acute stress during reproductive development resulted in a significant delay in ovulation, and reduced egg size (Campbell et al, 1992, 1994). Moreover, stressing fish or treating them with Cortisol was reported to cause ovarian failure by reducing ovarian development, reduced egg size, causing extensive follicular destruction,

28 reducing ovarian steroid hormone production (Armstrong, 1986; Janz et al, 1997; Hoyer,

2005), affect certain reproductive performance parameters, such as the timing of ovulation and relative fecundity (Campbell et al, 1992, 1994; Eriksen et al, 2006; King et al, 2007), and inhibiting spermatogenesis (Shankar and Kulkarni, 2000). These findings suggest that chronic elevation of plasma Cortisol can influence a wide range of reproductive parameters in tilapia, and some salmonid species.

Stressor-related affects on reproductive function in mammals is well established

(Tilbrook et al, 2000, 2002; Reeder and Kramer, 2005), but the situation in fish remains controversial. Pankhurst et al. (1995, 1996) emphasize that the effects of Cortisol on ovarian function in fishes are elusive; studies are just as likely to show stimulatory, inhibitory or no affects. It may be that the stress response in fish is polymorphic; the same stressor may have different level of severity depending on the species of fish, the stage of maturity of the fish, the duration of exposure to the stressor and the nature of the stressor (Donaldson, 1981; Van Der Kraak et al, 1997; Ruane et al, 1999; McQuillan et al, 2003).

Mode of action of corticosteroid hormones:

Corticosteroids bind to their tissue-specific receptors (CRs) and act as transcription factors by binding to specific HREs. Unlike mammals, which have separate glucocorticoid (GR) and mineralocorticoid receptors (MR), fish appear to have only the

GR (Ducouret et al, 1995), although there may be two isoforms of this receptor (Bury et al, 2003; Greenwood et al, 2003). Rainbow trout GR (rtGR) exerts a transcriptional interference on the expression of the estrogen receptor (rtER) which may account for

29 negative effects of stress or Cortisol on vitellogenesis (Lethimonier et al, 2000). Unlike other vertebrates, the overlap in the processes controlled by CRs may be different in fish, as the members of this taxon are thought to synthesize only glucocorticoids, whereas mammals express both GR and MR (Bury et al, 2003; Prunet et al, 2006). The presence of different subtypes of CRs in fish suggests a more complicated corticosteroid signaling in this taxon (Ducouret etal, 1995; Greenwood etal, 2003).

30 RATIONALE AND WORKING HYPOTHESIS OF THE STUDY

The primary aim of this study was to examine changes in the endocrine physiology of the maturing ovary of rainbow trout during the reproductive season, and determine if Cortisol has an affect on the secretion of the primary end point sex steroids,

E2 and T. The overriding null hypothesis for the series of studies is that Cortisol has no affect on ovarian steroidogenesis in rainbow trout. To test the general hypothesis of this study, specific objectives were developed as follows:

1. To determine whether there is an affect of Cortisol on basal and cAMP-stimulated ovarian follicle steroidogenesis, in vitro, by measuring the production of the major end point ovarian steroids, E2 and T.

2. To determine whether any actions of Cortisol are related to the stage of maturation of the follicle.

3. To determine whether any actions of Cortisol may operate via known GR proteins in rainbow trout ovarian follicles, using immunohistochemistry techniques.

4. To determine whether Cortisol exerts any affects on the profiles of the steroid hormone metabolites those are synthesized by the thecal and granulosal cells from P5 and

T substrate in vitro.

5. To determine, if there is evidence of a Cortisol affect on steroidogenesis, and if so determine the position (or positions) in the ovarian steroidogenic pathway at which

Cortisol exerts its action(s).

6. To determine whether Cortisol is metabolized by the ovarian follicle.

31 GENERAL EXPERIMENTAL APPROACH

Many studies of the stress response of fishes have demonstrated an acute and sometimes chronic increase in the secretion of Cortisol in response to stressors, and even when plasma levels of Cortisol have returned to pre-stressor levels, physiological responses suggest that target tissues continue to be stimulated by Cortisol. Studies have also shown that increased plasma Cortisol levels in sexually mature adult female salmonid fish elevate Cortisol levels in the oocytes (Donaldson, 1981; Davis and Parker, 1986;

McDonald and Milligan, 1992; Tagawa et al, 2000; Eriksen et al, 2006, 2007; Garcia-

Lopez, 2007). To achieve the objectives of the study, which is essentially to examine stressor-related changes in ovarian steroidogenesis in rainbow trout, I examined the effect of elevating the level of exposure of ovarian follicles to Cortisol, in vitro, as a way to examine direct actions of the hormone, rather than indirect actions of Cortisol that might operate via the HPG axis. For this study I used rainbow trout ovarian follicles of different maturational stages as my working sample, using some without cAMP stimulation to provide measurements of basal steroidogenic rates, and some incubated in the presence of the adenylate cyclase activator, Forskolin (FS), to increase intracellular cAMP levels in the thecal and granulosal cells; Cortisol was added to the medium of untreated and FS-treated follicles to assess the effect of the hormone on the two levels of steroidogenic activity. In addition, follicles were harvested for extraction of RNA and assessment of the expression of key steroidogenesis-related genes, and GR genes, using

"real time" (quantitative) RT-PCR (qPCR) to determine if the effects of Cortisol (if any) could be explained on the basis of changes in gene expression.

32 CHAPTER II

EFFECT OF THE STAGE OF MATURATION OF OVARIAN FOLLICLES ON

STEROID HORMONE SECRETION AND RESPONSE TO cAMP-

STIMULATION

33 INTRODUCTION

As discussed in the previous chapter, a major focus of the thesis is the possible

effect of stressors on the function of the ovarian follicles in rainbow trout (Oncorhynchus mykiss) at different developmental stages. In order to undertake such a study it is necessary to have a sound understanding of the normal developmental patterns of the ovary of the specific species. Consequently, this chapter focuses on developing a better understanding of the normal physiology of the ovarian follicles during gonadal recrudescence. Particular focus is placed on identifying the physiological responses that can be used to identify the stage of development of the ovarian follicles. Categorization of the stage of development is predominantly important for species such as rainbow trout because ovarian maturation within a stock is not synchronous and it is not always possible to identify maturational stages based solely on morphological characteristics of the oocytes.

To classify the different stage of development of ovarian follicles in rainbow trout, follicles were collected from a total of 33 fish from the month of August to the end of November over a two year period; the follicles were incubated in vitro, to determine basal steroidogenic activity by measuring E2 and T production, the major steroid hormones produced during ovarian steroidogenesis in salmonid species (Kime, 1993;

Lowartz et al., 2003; Petkam, 2003). In addition, to determine the basal expression of specific steroidogenesis-related genes, specifically StAR and cytochrome P450scc

(CYPscc), the mRNA abundance of the specific genes were obtained from qPCR studies.

In addition to incubation of the basal treatment group, follicles from each fish were also

34 incubated in the presence of the adenylate cyclase activator, FS, to determine the

sensitivity of follicles to cAMP-stimulation at different developmental stages.

Information such as fish total body mass, total ovary mass, age of fish, ovarian follicular size, and date of collection of follicles were recorded from each fish. The GSI was calculated from the body and ovary mass of each fish. The data of ovarian follicle

steroid production were analysed relative to all the above mentioned parameters. Also, the ratio of cAMP-stimulated to basal E2 production (FSE2:E2) was also considered for comparison with all of the other factors (above) to look for possible correlations that could be used as indicators of the stage of ovarian follicle maturation for further studies of the effect of Cortisol on ovarian steroidogenesis in rainbow trout.

35 MATERIALS AND METHODS

The protocols for the use of all animals for this study were reviewed and approved by the University of Guelph Animal Care Committee. Moreover a mandatory animal care course has been taken before handling animals for experimental purposes.

All the samples for the study were collected from Alma Research Station, University of

Guelph, ON, Canada.

Chemicals:

Non-radioactive pregnenolone (P5), T and E2, Medium 199 (Ml99), bovine serum albumin (BSA), L-glutamine, P-D-glucose, bovine P-glucuronidase, and normal rabbit serum were purchased from Sigma (Sigma-Aldrich Canada Ltd, Oakville, ON, Canada).

Forskolin, extracted from Coleus forskohlii, was purchased from Sigma (Sigma-Aldrich

Canada Ltd, Oakville, ON, Canada).

Animals used in the study:

A total of 33 female rainbow trout from the stock maintained at the Alma

Research Station (ARS) were used for the study. The fish were held in constantly aerated and running well water in outside holding aquaria; they were fed daily to satiety with a commercial trout diet, and examined weekly by the ARS Staff to assess non-invasively their reproductive state; random fish were captured from holding tanks and the abdomen was gently palpated to determine whether ovulation had occurred; pre-ovulatory fish were killed by a blow to the head; the ovaries were removed and placed in ice-cold

36 Cortland's medium (supplemented with 0.1% glucose, 0.1% bovine serum albumin, and

0.1% streptomycin sulphate at pH 7.55; Appendix II) for transfer to the laboratory at the

University of Guelph.

Sample collection for measurement of steroid hormone production and gene expression:

In the Guelph laboratory, the in vitro incubation procedures prior to incubation were carried out on ice-chilled trays. Individual follicles were gently separated from the ovary using forceps, and placed in 24-well cell culture plates (Fisher Scientific); 10 follicles were randomly assigned to each well and each well contained 1 ml of fresh

Cortland's medium. After 30-40 minutes, the medium was aspirated and replaced with 1 ml of fresh treatment medium. Two treatment groups (12 replicates) were used, as follows: Basal (control) and FS-treated (cAMP- stimulated) (Appendix III). All incubations were carried out at 8-10C for 18 h, unless otherwise specified; the medium was stored at -20°C until hormone assays could be performed.

Additional follicles were collected and placed in RNAlater. They were kept overnight at 8-IOC and then frozen at -20C for later gene expression studies. A piece of ovary was taken from each animal used in the study in the second collection season, and was placed in 10% formalin for measurement of the size of the follicles and for histological preparations.

37 Measurement of gonado-somatic index (GSI) and follicle mass:

The body mass and ovary mass for each fish were used to calculate the GSI using the formula:

GSI = (ovary mass/body mass) x 100.

The mean ovarian follicle mass of each fish was measured by weighing several fragments of the formalin-fixed ovary tissue, and then counting the total number of follicles in each fragment.

Radioimmunoassays (RIAs) for 17f}-estradiol (£2) and testosterone (T) measurements:

Tritium (3H)-based RIAs were used to measure the concentration of E2 and T in the incubation medium. The general procedure of a competitive RIA is as described by

McMaster et al. (1995). Specifically, a fixed concentration of radiolabeled steroid

(approximately 10,000 counts) was incubated with polyclonal antibody against E2

(1:7,500) or T (1:5,000) in the presence of the sample (straight media). The mixture was incubated at 4°C for 18 h, and then the bound and unbound radiolabeled steroid was separated by adding activated charcoal to the mixture followed by centrifugation (Sorvall

RT6000B, Dupont) at 2,700 x g for 20 minutes. The supernatant with bound titrated steroid was decanted and mixed with 4 ml of scintillation cocktail and the radioactive content was measured using a P-counter, Tracor Analytic Delta 300 (NCS

Instrumentation Inc., Mississauga, ON). The steroid concentration in the unknown samples was determined by comparing with a standard curve. The assay methods used

38 were those described in Petkam et al. (2005); details of the assays are described in

Appendix V.

Follicle total RNA extraction:

A RNeasy Lipid Tissue Mini kit (Qiagen Sciences, Marryland, USA) was used for the extraction of total RNA from ovarian follicles. The frozen follicles (stored at -

20 C) were quickly thawed in lysis buffer (3-4 follicles.mr1), and then homogenized following the protocol described in Appendix VI. The total RNA was measured at 260 nm in a UV spectrophotometer, and calculated as total RNA content per follicle. Each assay of total follicle RNA was performed in triplicate.

Real time RT-PCR (qPCR) measurement of StAR and CYPscc gene expression:

RNA isolation:

Total RNA was extracted from the ovarian follicles of fish of different stages by using the RNeasy® Lipid Tissue Mini kit (Qiagen Sciences, Marryland, USA) following the manufacturer's protocol, and quantified sprectrophotometrically at 260 nm by means of a Qenequat spectrophotometer (Biochrom, UK). The procedure for the total RNA extraction is presented in Appendix VI. The integrity of the RNA was examined using a

1.2 % formaldehyde agrose RNA gel with ethidium bromide. (Appendix VII). The total

RNA for each sample was reverse-transcribed to cDNA using the First Strand cDNA synthesis kit (MBI Fermantas, USA), using procedures recommended by the supplier

(Appendix VIII). For cDNA synthesis, the total RNA was diluted to O.lug.ul"1 in RNase

39 free water. Briefly, total RNA was heat denatured (70°C) and cooled on ice. The sample was used in a 20 ul reverse transcriptase reaction that included 0.5 ug of oligo d(T) primers [1 mM of each dNTP], 20 U of ribonuclease inhibitors, and 40 U of M-MuLV reverse transcriptase. Incubation was for 1 h at 37°C, stopped by heating at 70°C for 10 min.

Primer design and validation:

Dr. MM. Vijayan, Department of Biology, University of Waterloo, ON,

Canada, kindly provided the oligonucleotide primers for StAR and CYPscc used in the study. The primers were designed using rainbow trout StAR protein and CYPscc cDNA sequences (GenBank accession nos: AB047032 and S57305, respectively) to amplify

-101 bp for StAR, and for P450scc ~140bp product in qPCR; the annealing temperature was 60°C for all the genes used in the qPCR procedures. The primer sequences are shown in Table 2.1.

TABLE 2.1. Representative of the oligonucleotide primers used in quantitative real-time

PCR (qPCR).

Gene Primers

StAR Forward 5' -TGGGGAAGGTGTTTAAGCTG-3'

Reverse 5'-AGGGTTCCAGTCTCCCATCT-3'

P450ssc Forward 5'-GCTTCATCCAGTTGCAGTCA-3'

Reverse 5' -C AGGTCTGGGG AAC AC ATCT-3'

40 Preparation of standards and generation of standard curves:

The target sequence of StAR and CYPscc were amplified using the following

PCR conditions: target 500 pg.ul"1 (as we were only able to quantify the total RNA) and cycle: 95°C - 3 min; 40 cycles: denature 95°C - 30 sec, anneal 60°C - 30 sec, extend

72°C - 30 sec; 1 cycle: 72°C - 10 min, followed by a 4°C hold. The amplified products were ligated into pGEM-Teasy plasmid vector system and cloned into E.Coli competent cells. Plasmids with insert sequences were isolated using plasma purification kit (Sigma) and quantified using a spectrophotometer. The plasmids were sequenced to confirm the target gene identity. These plasmid + insert stocks were used to generate standard curves for qPCR.

To generate standard curves, plasmid + insert stocks were serially diluted (108 to

101) to attain varying copy number of inserts sequences. The qPCR was carried using iCycler (BIORAD) and the PCR conditions include, 2 min at 94°C followed by 40 cycles of 15 sec at 95°C and 30 sec at 60°C. The PCR reaction consisted of 1 ul of cDNA, 4 pM of each primer and SYBR Green mix (50 U.ml"1 of iTaq DNA poltmerase, 40 mM of

Tris-HCl (pH 8.4), 100 mM of KCL, 6 mM of MgCl2, 0.4 mM of each dNTP component

(dATP, dGTP, dCTP and dTTP), SYB Green I, 20 uM of flouresein, and stabilizers,

BIORAD) in total volume of 25 ul. At the end, PCR products were subjected to melt curve analysis to confirm the presence of single amplicon. Amplification plots were analysed using iQ optical system software (version 3.0A) and base line substracted threshold cycles (Ct) were obtained for all the samples in the dilution series. Ct values were ploted against the starting quantity of plasmid template to generate the standard

41 curve. The efficiency of the standard curve was calculated using the formula, (10" d/siopey 1Q0 and it ranged from 97.100o/o.

Quantification of samples:

PCR reactions contained 1 ul of cDNA, 4 pM of each primer and SYBR Green

super mix (BIORAD) in a total volume of 25 ul. qPCR protocol and melting temperatures are similar to that described above. The control reactions, with no template

(water; negative control) and no amplification control (RNA) were also run to check for background and genomic contamination respectively. The copy numbers in each sample were calculated using the standard curve (above). All the qPCR reactions were carried out in triplicate (3 x 25 ul) and master mixes were prepared at each step to reduce pipetting errors.

Statistical Analysis:

All statistical analyses was performed with SigmaStat version 3.00 (Copyright©

1992-2003 SPSS. Inc., Chicago, IL, USA) or SigmaPlot version 8.02 (Copyright© 1986-

2001 SPSS Inc., Chicago, IL, USA). The data were transformed, wherever necessary, for homogeneity of variance. Mean Basal (control, logio) E2 and T production values and

GSI of each trial (fish) were compared using Student's t-test. The data of the concentration of T and E2 in the medium were separately subjected to one-way ANOVA.

Where F value indicated significant differences (P<0.05), Tukey's W test was used to test the differences between means. A probability level of P<0.05 was considered to be significant.

42 RESULTS

Correlation of measured variables (GSI, mean mass of ovarian follicles, date of

sample collection, £2 and T production by the follicles):

Correlation analysis was performed for all of the measured parameters (date of

collection of samples, GSI, follicle size, basal E2 production, the ratio of cAMP-

stimulated:basal E2 production) obtained from 33 fishes during 2 reproductive seasons

(August-December) to examine for correlations that might be indicators of the maturational stage of the ovarian follicles. Respective correlation of the date of collection, GSI and follicle size (Figure 2.1a); GSI, basal and FS-stimulated E2 production (Figure 2.1b); and GSI and the ratio of cAMP-stimulated:basal E2 production

(Figure 2. lc) are shown in this section.

There was a significantly (P<0.01) positive linear correlation between the date of collection, and GSI and follicle size; as the reproductive season progressed there was an increase in GSI as well as in follicle mass (Figure 2.1a).

The data of steroid hormone production showed no significant linear correlation, but scatterplots suggested a marked seasonal pattern, with low basal and cAMP- stimulated ovarian E2 production by follicles harvested from fish sampled in late August to early September, higher basal and cAMP-stimulated E2 production by follicles offish sampled from mid-September to mid-October, and lower production again by follicles of fish sampled in late October onwards (Figure 2.1b). The pattern was suggestive of a differentiation of steroid hormone production into an early-, mid- and late-vitellogenesis

43 stage that was strongly correlated with the reproductive season, and with GSI. Correlation analysis showed a significant (P<0.01) inverse correlation between GSI and cAMP- stimulated:basal E2 production ratio (Figure 2. lc).

When the steroid hormone production data were grouped together to reflect the early-, mid- and late-stages, the statistical analysis supported the assumptions based on seasonal progression and GSI. Basal E2 and T production values by the mid-vitellogenic stage were significantly (P<0.001) higher than those of early- and late-vitellogenic stages.

Although basal E2 production values of early- and late-vitellogenic stages were not statistically significant from one another (Figure 2.2a), for basal T production, the early- stage follicle production was significantly (P<0.05) lower than that of late-vitellogenic stages (Figure 2.2b). The mean basal E2 and T production by follicles at the EV stage is

0.32 ± 0.03 ng.follicle'1 and 0.009 ± 0.001 ng.follicle'1, respectively. The productions of basal E2 (Figure 2.2a) and T (Figure 2.2b) by follicles at early-, mid-, and late vitellogenic stages are shown as a percent of the mean basal EV values.

The FS-stimulated E2 and T productions by the early-, mid- and late- vitellogenesis stages of the ovarian follicles are shown in Figures 2.2a and 2.2b, respectively. The production of E2 and T by the FS-treated groups was significantly

(PO.01 and P<0.001) higher than that of basal treated group for all the maturational stages. In addition, with an increase in the maturation stage of the ovarian follicles the stimulatory affect of FS was reduced (Figure 2.2a); however, the mean cAMP-stimulated

E2 production for the early-stage follicles was very low (1.05 ± 0.10). The increase in T secretion in response to FS was significantly (P<0.001) highest in the mid-vitellogenic stage follicles, and relatively low in the early- and late-vitellogenic stages (Figure 2.2b).

44 There was no significant difference between E2 production by early- and mid-vitellogenic follicles that were incubated with FS, but both were significantly (P<0.05) higher than for the late-stage follicles (Figure 2.2a). Conversely, cAMP-stimulated T production between early- and mid-vitellogenic follicle stages, and between mid- and late- vitellogenic follicle stages were statistically (P<0.001) different from one another, with highest cAMP-stimulated T output in the mid-vitellogenic follicles,and the lowest in the early- and late-vitellogenic stages (Figure 2.2b). Basal and cAMP-stimulated E2 production in each of the maturational stages was significantly different from one another

(PO.001, <0.01 and <0.01, respectively). Likewise, the difference between basal and cAMP-stimulated T production in all the three stages was statistically significant

(P<0.001).

45 Follicle GSI " Size (g)

' Equation: Equation: y= (4.9 0.821+(0.19 0.04)x ' y = (0.025 0.007)+(0.0009 0.0003)x

Date of Collection Date of Collection

14 r 0.06

12 A L 0.05 A A 10

A ii A A 0-04 Follicle GSI • A« Size(g) A A A 0.03 A A

0.02

0.01 Aug Sept I Oct Nov Date of Collection

Figure 2.1a. Linear correlation of the date of collection of samples, with gonadosomatic index (GSI) and ovarian follicles size. Symbols: clear triangles = GSI and solid circles = follicle size (g). Correlation analysis shows there is a significant (P<0.01) positive correlation between date of collection and GSI as well as date of collection and follicle size.

46 1.6 - 3.0

A 1.4 - 2.5 /^S • "i 12- a 2.0 •JS • • *"! 10- A e A s^* A 8 • • J °- " • % •a 0.6 - £ • • A A ,.i •• n. A A • • • t «s 0.4 - A 4 * 0.5 is? W • • A A a o.2- A A • • t to 03 • I 0.0 0.0-

' " I i 1 1 6 8 10 12 14 GSI

Figure 2.1b. Correlation of gonadosomatic index (GSI) with basal and cAMP-stimulated E2 productions. Arbitary lines have been added to separate the GSI values into three different range; 0-4, 4.1-9.9, and 10-14. Clear triangle represents basal E2 production and solid circle represents FS E2. The vertical lines indicate arbitary points of separation of the collection into three stages based on GSI.

47 4.0 -i

3.5 A Equation: y = (2.8 ± 0.36) + (-0.15 ± 0.04)x £ 30 a 1 2.5 13 O i- a. c* 2.0 W 13 I 1.5

1.0 H '• •

0.5 —i 2 4 6 8 10 12 14 GSI

Figure 2.1c. Linear correlation of gonadosomatic index (GSI) and the ratio of cAMP- stimulated:basal E2 production. The line is a significant (P<0.01) inverse correlation between the variables.

48 a]

400 -i x X ** AAA ^M BASAL E2 •1 FSE2 B G O 300 A • mm •mm u 3 e m. a.t* 2O0-\ & y

I 100 H ca

EARLY MID LATE

Vitellogenic Stages

Figure 2.2a: Effects of ovarian follicles developmental stages on in vitro basal and cAMP-stimulated E2 production by rainbow trout {Oncorhynchus mykiss) ovarian follicles. All data are shown as mean ± SEM (n=6) and expressed as a % of the basal E2 production (0.32 ± 0.25 ng.follicle"1). ***, **, statistically significant (PO.001) and (P<0.01) compared to control group at the same maturational stage; capital letters indicate differences between basal groups; lower case letters indicate differences between cAMP-stimulated groups and the differences are based on real numbers, not on percentage value. Bars with different letters are significantly different (P<0.001) from one another. The basal E2 production of the mid-vitellogenic stage follicles was significantly (P<0.001) higher than that of the early- and late-vitellogenic stages.

49 b]

6OOOO1 AAA ^•BASALT | mm FST 50000- L

Co 40000-

S 30000- B X

AAA

(T ) product i ' •

| **A ^|

•>:-M Testosteron e 1 c A n , I 1 EARLY MID LATE

Vitellogenic Stages

Figure 2.2b. Effects of ovarian follicles developmental stages on in vitro basal and cAMP-stimulated T production by rainbow trout (Oncorhynchus mykiss) ovarian follicles. All the data are shown as mean ± SEM (n=6) and expressed as a percent of the EV basal T production (0.009 ± O.OOlng.follicle"1). ***, statistically significant (PO.001) compared to control groups at the same maturational stage; capital letters indicate differences between basal groups; lower case letters indicate differences between cAMP- stimulated groups and the differences are based on real numbers, not based on percentage value. Bars with different letters are significantly different (P<0.001) from one another. The basal T production of mid-vitellogenic stage follicles was significantly (P<0.001) higher than that of the early- and late-vitellogenic stages.

50 StAR and CYPscc gene expression during basal and cAMP-stimulated steroidogenesis by ovarian follicles:

The gene expression of StAR (Figure 2.3a) and cytochrome P450scc (CYPscc)

(Figure 2.3b), by all the three vitellogenic stages (early-, mid-, and late-vitellogenesis) are shown as mRNA copy numbers for each treatment group. There were no statistically significant differences in the accumulation of mRNA encoding for StAR protein among the three maturational stages, although there was a trend for the mRNA expression to be higher in the MV stage basal group compared with the other two, and low in the early- and late-vitellogenic stages (Figure 2.3a). A similar (not significant) pattern was seen with the StAR mRNA expression in the FS treatment groups of all the maturational stages in rainbow trout follicles (Figure 2.3a).

The basal and cAMP-stimulated expression of the gene encoding for CYPscc was significantly (P<0.001) higher in the mid-vitellogenesis stage compare with the early- and late-vitellogenesis stages (Figure 2.3b).

51 a]

1.4e+5 ^H BASAL ill FS 1.2e+5

u •g 1.0e+5 s s c £ 8.0e+4- o g 6.0e*4- s

^ 4.0e+4

2.0e+4 A 0.0 i EARLY MID LATE Vitellogenic Stages

Figure 2.3a: Accumulation of mRNA encoding for StAR protein following basal and cAMP-stimulated treatments of early-, mid- and late-vitellogenesis stage ovarian follicles. No statistical significant difference between any groups. Copy numbers of StAR were obtained from the standard curve generated using plasmid stock.

52 b]

^H BASAL B 1e+5 -i V3%>M% FS

T 8e+4 v .o

£ 6e+4 o B

4e+4

0. U 2e+4 A x

EARLY MID LATE

Vitellogenic Stages

Figure 2.3b: Accumulation of mRNA encoding for CYPscc protein following basal and cAMP-stimulated treatments of early-, mid- and late-vitellogenesis stage ovarian follicles. Capital letters indicate differences between basal groups; lower case letters indicate differences between cAMP-stimulated groups. Bars with different letters are significantly different (P<0.001) from one another. Copy numbers of CYPscc were obtained from the standard curve generated using plasmid stock.

53 DISCUSSION

The results of this study demonstrated a correlation between the date of collection of samples and follicle mass and GSI (Figure 2.1a). With the progression of the season from mid-August until the end of November there was an increase in GSI and follicle sizes of the collected samples. This is consistent with other earlier studies of the same stock of rainbow trout (Holloway et al., 1999, 2000) that showed an increase in GSI and follicle size with the seasonal progression of ovarian developmental period in trout. The progressive increase in GSI was associated with developments in the maturational stage of the ovarian follicles as indicated by the level of basal and cAMP-stimulated E2 production. These response are low in fish that have low GSI; they tend to peak during the mid-GSI range, and decrease when GSI values are high (Figure 2.1b). This association is supportive of previous studies in salmonid fishes (Scott et al., 1980b;

Down et al, 1990; Nakamura et al., 2005). Further, with an increase in GSI, the ratio of cAMP-stimulated:basal E2 production tends to decrease, and there is a negative correlation between the two variables (Figure 2. lc). It would seem, therefore, that these associations are useful indicators of the seasonal pattern of reproduction in female rainbow trout, and I used these correlations to identify trout at early-, mid- and late- vitellogenic stages for further examination of the effects of Cortisol on steroidogenesis at these stages. A synthesis of the various variables I used to determine the maturational stage is shown in Table 2.2.

The data of basal and cAMP E2 and T production by follicles that were determined to be in the different maturational stages provided support for the

54 determinations. The data suggest that steriodogenesis is significantly higher in the mid- vitellogenic stage than in the early-vitellogenic stage (Figure 2.2a, b) and support earlier reports by Scott et al. (1980a, b). The low E2 production during the early-vitellogenic stage is probably because the steroidogenic pathway is not fully activated during that period, probably because the HPG axis is not fully functional at that stage. There was also a low E2 production by the late-vitellogenic stage follicles (Figure 2.2a, b) is likely because of the steroidogenic shift from E2 to DHP production as the follicles approach ovulation. Previous studies in other species show that E2 levels increase dramatically and

T levels increase modestly during vitellogenesis, while DHP levels increase dramatically just prior to ovulation and decrease after ovulation (Taylor et al., 2004).

55 Table 2.2. Criteria I used to identify early-vitellogenesis, mid-vitellogenesis, and late- vitellogenesis stages of rainbow trout {Oncorhynchus mykiss) ovarian follicles.

STAGES MEASURED VARIABLES

Low GSI Low basal E2 production Low basal T production Early-Vitellogenesis Early season collection of samples Smallest oocytes High cAMP-stimulated:basal E2 output ratio

Medium GSI High basal E2 production High basal T production Mid-Vitellogenesis Mid season colloction of samples Medium size oocytes Medium cAMP-stimulated:basal E2 output ratio

High GSI No/very low basal E2 production Low basal T production Late-Vitellogenesi s Late season collection of samples Largest oocytes High cAMP-stimulated:basal E2 output ratio

56 The cAMP-stimulated steroid hormone production data showed an increased rate

of steroidogenesis (compared with basal values) by follicles at all three maturational

stages (Figure 2.2a, b); however, cAMP-stimulated E2 production was most marked in

follicles at the mid-vitellogenesis stage (Figure 2.2a), which also supports the hypothesis

of lower steroidogenic activity in early stage follicles

The expression of mRNA encoding for P450scc (CYPscc) protein was highest in

the mid-vitellogenic stage compared to early- and late-vitellogenic stages; a similar

pattern, albeit not significant, was suggested for the expression of the StAR gene. These

findings are in close agreement with the steroid hormone production data. The absence of

a significant difference in the accumulation of mRNA encoding StAR protein between the maturational stages may be because StAR protein is continuously produced in the cytoplasm of a cell and the amount of this protein used for cholesterol transfer is

substituted immediately because the synthesis of protein may not be directly related to the accumulation of mRNA. Thus, significant changes in gene expression may not be evident. In contrast, CYPscc expression was highest in both basal and cAMP-stimulated groups in the mid-vitellogenic stage compared to early- and late-vitellogenic stages suggesting that expression of the gene encoding for the cholesterol converting enzyme does vary with developmental stage in rainbow trout, as it appears to do in zebrafish (Ing and Van Der Kraak, 2006).

In conclusion, based on the measured variables identified, representative fish from three ovarian follicles developmental stages were identified and used for further assessment of the effect of Cortisol on steroid production by rainbow trout ovarian follicles (see Chapter in).

57 CHAPTER in

EFFECT OF CORTISOL ON OVARIAN STEROIDOGENESIS DURING

EARLY-, MD3- AND LATE STAGES OF FOLLICLE MATURATION INTRODUCTION

This chapter focuses on the stress hormone, Cortisol, and its possible affects on the function of ovarian follicles in rainbow trout at different stages of ovarian

development. In Chapter II, the ovarian follicles collected over the two years of study were grouped into three developmental stages, early- , mid- and late-vitellogenic, based on the criteria of basal and forskolin (FS)-treated 17p~estradiol (E2) production, gonado-

stomatic index (GSI), follicle size and date in the season when the ovaries were harvested. Follicles at these three stages were incubated either with or without Cortisol in the medium to determine if the glucocorticoid exerts an affect on steroidogenesis; the assumption made is that Cortisol, which is released as part of the stress response in vertebrates, may be responsible for some of the reported affects of stress on reproduction in vertebrates.

It is well established that many different forms of stressors can inhibit reproductive processes in higher vertebrates, and a reasonable amount is known about the mechanisms underlying the phenomenon in mammals in particular (Moberg, 1991;

Tilbrook et al, 2000, 2002; Moore and Jessop, 2003; Maeda and Tsukamura, 2006). In fish, however, much less is known about the effect of stressors (or the stress response) on reproduction, and as discussed in Chapter I, the response appears to vary markedly among species. In the study reported here, the effects of Cortisol on in vitro steroidogenesis in rainbow trout (Oncorhynchus mykiss) ovarian follicles were examined.

Both basal and cAMP-stimulated steroidogenesis were measured; the adenylate cyclase activator, FS was used to increase cytoplasmic cAMP levels (Hylka et al, 1989; Leung

59 and Steele; 1992; Srivastava and Van der Kraak; 1994); the end point steroids E2 and T

were used as measures of overall steroidogenesis. In addition, qPCR technology was

used to determine if Cortisol affects the expression of genes encoding for the

steroidogenesis-related proteins, steroidogenic acute regulatory (StAR) protein and

cytochrome P450side chain cleavage protein (CYPscc). Also, the expression of genes

encoding for glucocorticoid receptor (GR) proteins (GR 1, and GR 2), was examined.

Expression of the reference genes, P-actin and EF-la were also measured as an indicator

of the possible seasonal changes in overall gene expression (i.e., non-steroidogenesis-

related genes). To look for evidence of GR presence in the follicles,

immunohistochemistry was applied to sections of ovary at different developmental

stages. High performance liquid chromatography (HPLC) was used to separate steroids in the incubation medium to determine whether Cortisol changes the metabolites produced

from radiolabeled substrates (pregnenolone [P5]) and T), and similar methods were used to determine if ovarian follicles of rainbow trout metabolize Cortisol in the medium (see

Appendix XIV).

60 MATERIALS AND METHODS

All the animals used for this research study were reviewed and approved by the

University of Guelph Animal Care Committee. Moreover a mandatory animal care course

has been taken before handling animals for experimental purposes. All the samples for

the study were collected from Alma Research Station, University of Guelph, ON,

Canada. A total of 33 animals were collected and parameters were recorded for each fish

as reported in Chapter II. Based on the correlation analysis the fish were divided into 3

maturational stages. For Chapter III, 2 fish were considered for early-, 5 for mid- and 4

for late-vitellogenic stages. Animals at the peak of basal E2 production were choosen as

representatives of the mid-vitellogenic stage. For details see Appendix XV.

Chemicals:

The sources of many of the chemicals used for this part of the study were reported

3 3 in Chapter II. In addition, radiolabeled, [7- H(N)]pregnenolone ([ H]P5; specific activity

250 pCi per mmol) and [1,2,6,7-3H] testosterone ([3H]T) were purchased from New

England Nuclear (Mandel Scientific Co, Ltd, Guelph, ON, Canada). Radiolabeled

[l,2,6,7-3H]cortisol was purchased from Amersham Pharmacia Biotech, Quebec, Canada; the material used in this study was kindly donated by Dr. J. Raeside. Non-radioactive T

and E2, Medium 199 (Ml99), bovine serum albumin, L-glutamine, P-D-glucose, bovine

P-glucuronidase, and normal rabbit serum were purchased from Sigma (Sigma-Aldrich

Canada Ltd, Oakville, ON, Canada). FS was purchased from Sigma (Sigma-Aldrich

Canada Ltd, Oakville, ON, Canada). Cortisol [hydrocortisone (Up, 17a, 21-

61 trihydroxypregn-4-ene-3, 20-dione)] was purchased from Sigma Chemicals Co. (St.

Louis, MO, USA).

Animals used in the study:

The fish used in the study and the methods used to process the animals were as

described in Chapter n.

Sample collection for hormone measurement, gene expression and

immunohistochemistry:

The method used for sample collection was same as those described in Chapter II.

Eight treatment groups (12 replicates) were used for the study of Cortisol effects on

steroidogenesis, as follows: Basal (control); Basal + Cortisol 1 (100 ng.ml"1); Basal +

Cortisol 2 (50 ng.ml"1); Basal + Cortisol 3 (10 ng.ml"1); cAMP-stimulated [by the addition of FS to the medium] (Appendix III); FS + Cortisol 1 (100 ng.ml'1); FS +

Cortisol 2 (SOng.ml"1); FS + Cortisol 3 (10 ng.ml"1). The preparation of the Cortisol solutions is described in Appendix X. Based on preliminary studies of the steroid production by the follicles; the low Cortisol concentration was used for the purposes of this section of the thesis. The data for the higher applications of Cortisol are presented in

Appendix XIII, as is the rationale for choosing to use the lower Cortisol concentration data for further analysis.

Some incubations were carried out in the presence of tritium-labeled P5, T and

Cortisol; the medium from these preparations was subjected to high performance liquid

62 chromatography (HPLC) to separate the steroid metabolites formed from the precursor

radiolabeled steroids (see below for further details).

Following incubation, some follicles were collected and placed in RNAlater for

later gene expression studies. Also, a piece of ovary was taken from each animal used in the study, and placed in 10% formalin for immunohistochemical examination.

Radioimmunoassay (RIA) of £2 and T and follicle total RNA extraction:

Tritium (3H)-based RIAs were used to measure the concentration of E2 and T in the

incubation medium, and follicle total RNA extraction using protocols described in

Chapter II.

Histological preparations:

Pieces of formalin-fixed ovary sections were processed by a Rivic 1530 paraffin tissue processor, which dehydrated the samples in a graded series of ethanol and perfused them with paraffin at 58°C. The embedded follicles were sectioned at 5 to 7 urn using a

Spencer 820 microtome. Sections were mounted on glass slides and allowed to dry for 48 hours at 40 C before immunostaining, or staining with hematoxylin and eosin for routine histological examination.

Immunohistochemistry was carried out using an antibody raised in rabbit against rainbow trout glucocorticoid receptor (GR) protein, according to the established protocols

(Boone and Vijayan, 2002; Sathiyaa and Vijayan, 2003). The antibody does not discriminate between the two isoforms of the receptor. The avidin-biotin complex (ABC,

Vectastain Laboratories, Inc., Burlingame, CA) protocol was used as described

63 previously (Farbridge and Leatherland, 1991), using diaminobenzidine as the enzyme

substrate. Details of the protocol are described in Appendix XI.

qPCR measurement of StAR, P450scc, GR 1, GR 2, p-actin and EF-la gene

expression:

RNA isolation:

The total RNA extraction procedures used were as described in Chapter H

Primer design and validation:

Dr. M.M. Vijayan, Department of Biology, University of Waterloo, ON, Canada, kindly provided the oligonucleotide primers for StAR, CYPscc, GR 1, GR 2, P-actin and

EF-la used in the study; the primers were designed using rainbow trout StAR, P450scc,

GR1, GR2, p-actin, and EF-la c-DNA sequences (GenBank accession nos: AB047032,

S57305, AJ622902, AJ566190, AF157514, and AF498320, respectively). These were

-101 bp for StAR, -140 bp for P450scc, -105 bp for p-actin, -115 bp for GR1, -147 bp for GR2, and -95 bp for EF-la. The amplifyng temperature was 56°C for EF-la, and

o

60 C for all the other genes used in the qPCR procedure. The primer sequences are shown in Table 3.1.

64 Table 3.1. Sequences of the oligonucleotide primers used for the qPCR procedure

Gene Primers

StAR Forward 5'-TGGGGAAGGTGTTTAAGCTG-3'

Reverse 5'-AGGGTTCCAGTCTCCCATCT-3'

P450ssc Forward 5'-GCTTCATCCAGTTGCAGTCA-3'

Reverse 5'-CAGGTCTGGGGAACACATCT-3'

P-actin Forward 5 '-TGTCCCTGTATGCCTCTGGT-3'

Reverse 5'-AAGTCCAGACGGAGGATGG-3'

GR 1 Forward 5' -TTCCAAGTCCACCACATCAA-3'

Reverse 5' -GGAGAGCTCCATCTGAGTCG-3'

GR 2 Forward 5' -GGGGTGATCAAACAGGAGAA-3'

Reverse 5' -CTCACCCCACAGATGGAGAT-3'

EF-1 a Forward 5' - CATTGACAAGAGAACCATTGA-3'

Reverse 5' - CCTTCAGCTTGTCCAGCAC-3'

Preparation of standards and generation of standard curves:

The target sequence of StAR, CYPscc, GR1, GR2, P-actin and EF-la were amplified using the following PCR conditions: target 500 pg.ul"1 (as we were only able to quantify the total RNA) and cycle: 95°C - 3 min; 40 cycles: denature 95°C - 30 sec, anneal 60°C - 30 sec, extend 72°C - 30 sec; 1 cycle: 72°C - 10 min, followed by a 4°C hold. The amplified products were ligated into pGEM-Teasy plasmid vector system and

65 cloned into E.Coli competent cells. Plasmids with insert sequences were isolated using plasma purification kit (Sigma) and quantified using a spectrophotometer. The plasmids were sequenced to confirm the target gene identity. These plasmid + insert stocks were used to generate standard curves for qPCR.

To generate standard curves, plasmid + insert stocks were serially diluted (108 to

101) to attain varying copy number of inserts sequences. The qPCR was carried using iCycler (BIORAD) and the PCR conditions include, 2 min at 94°C followed by 40 cycles

of 15 sec at 95°C and 30 sec at 60°C. The PCR reaction consisted of 1 ul of cDNA, 4 pM

of each primer and SYBR Green mix (50 U.ml"1 of iTaq DNA poltmerase, 40 mM of

Tris-HCl (pH 8.4), 100 mM of KCL, 6 mM of MgCl2, 0.4 mM of each dNTP component

(dATP, dGTP, dCTP and dTTP), SYB Green I, 20 uM of flouresein, and stabilizers,

BIORAD) in total volume of 25 ul. At the end, PCR products were subjected to melt curve analysis to confirm the presence of single amplicon. Amplification plots were analysed using iQ optical system software (version 3.0A) and base line substracted threshold cycles (Ct) were obtained for all the samples in the dilution series. Ct values were ploted against the starting quantity of plasmid template to generate the standard curve. The efficiency of the standard curve was calculated using the formula,

(10-(i/siopey 10Q and it ranged from 97_100o/o

Quantification of samples:

PCR reatctions contained 1 ul of cDNA, 4 pM of each primer and SYBR Green super mix (BIORAD) in a total volume of 25 ul. qPCR protocol and melting temperatures are similar to that described above. The control reactions, with no template

(water; negative control) and no amplification control (RNA) were also run to check for

66 background and genomic contamination respectively. The copy numbers in each sample were calculated using the standard curve (above). All the qPCR reactions were carried

out in triplicate (3 x 25 ul) and master mixed were prepared at each step to reduce

pipetting errors.

HPLC:

Some incubations were conducted with [3H]Ps or [3H]T added to each well in 10

ul of ethanol to make a final concentration of 0.025 nM (18.5 Kbq); in separate trials, the

effect of this level of ethanol in the incubation was examined, and there were found to be no affects on the level of steroidogenesis. Following incubation, the medium was

aspirated from each well, and stored frozen at -20°C before further processing for HPLC

separation of the steroids.

The medium was thawed and initially subjected to solid phase extraction (SPE) as

described previously (Khan et al., 1997). The medium was perfused through a Sep-Pak

Cig column which had been primed with 5 ml of methanol and 5 ml of distilled water

(Appendix XII). Each column was then washed with 5 ml of water, and allowed to drain.

The radioactivity of aliquots (100 ul) of the initial incubation medium, the first wash and the second wash was measured. The columns were washed with 5 ml of hexane, and the free steroid fraction was eluted using 5 ml of diethyl ether; the conjugated steroid fraction was than eluted using 5 ml of methanol. The ether and methanol fractions were dried separately under nitrogen at 45 C. The ether extract was reconstituted in 100 ul of acetonitrile containing a mixture of unlabeled steroid standards, and 20 ul of this mixture was injected into the HPLC column. The methanol extract was processed sequentially by acid solvolysis and enzyme hydrolysis to convert sulphate and glucuronide conjugates,

67 respectively, into free steroids for separate separation and identification; these methods

have been described previously (Khan et ah, 1997).

The HPLC system was equipped with a Waters Model 2695 Alliance Separation

Module and Waters 996 Photodiode Array Detector. The Empower 2 Software was used to create an acetonitrile, water and methanol binary gradients on a Nova-Pak Cig (150 mm x 3.9 mm, 4um) column. The gradient was a multistep mobile phase consisting of

27.5% acetonitrile in water from 0 to 5 min, increasing to 36% at 9.5 min, remaining at

36% from 9.5 to 21.0 min, increasing to 60% at 27 min, and then running from 85 to

100% between 27 and 35 min; the flow rate was 0.6 ml.min"1.

Detection was made by extracting chromatograms at 200, 254 and 280 nm from the photodiode array detector. Radioactive counts were obtained by mixing the radioactive elute with 4 ml of scintillation cocktail and the radioactive contented was measured using a P-counter.

Statistical Analysis:

All statistical analyses was performed with SigmaStat version 3.00 (Copyright©

1992-2003 SPSS. Inc., Chicago, IL, USA) and SigmaPlot version 8.02 (Copyright©

1986-2001 SPSS Inc., Chicago, IL, USA) and data were expressed as mean ± SEM. The data were transformed (logarithmic), wherever necessary, for homogeneity of variance.

The basal (control) E2 and T production data and GSI data were analysed using Student's t-test. The RIA data and gene expression data were also subjected to one-way analysis of variance (ANOVA) to test for treatment effects. Where F value indicated significant

68 differences, Tukey's W test was used to test the differences between means. A probability level of P < 0.05 was considered significant. RESULT

Effect of Cortisol on basal E2 production:

Cortisol significantly (P<0.001) reduced basal E2 production by the mid- vitellogenic stage, but there was no suppressive effect of Cortisol in the early- or late- vitellogenic stages (Figure 3.1); indeed, in the early-vitellogenic stages, in which basal E2

production was low, Cortisol significantly (P<0.001) enhanced E2 production by the follicles (Figure 3.1). The mean basal E2 production of the control groups were 0.32 ±

0.03, 0.78 ± 0.06, and 0.29 ± 0.03 ng.follicle"1 for the early-, mid- and late-vitellogenic

stages, respectively. The mean basal E2 production of the cortisol-treated groups was 0.74

± 0.1, 0.55 ± 0.04, and 0.31 ± 0.05 ng.follicle"1 for the early-, mid- and late-vitellogenic

stages, respectively; these values were all significantly (P<0.01) different from one

another (Figure 3.1).

70 BASAL 180 COR 3 AAA 160

w 140

a 120 s B o a, 100 AAA " 80 H

60 A

I 40 H

20 H

EARLY MID LATE Vitellogenic Stages

Figure 3.1: Effect of Cortisol on in vitro basal 17p-estradiol (E2) production by rainbow trout ovarian follicles at early-, mid- and late stages of vitellogenesis; the data are shown as mean ± SEM (n = 6) and presented as a percent of the control value for each maturational stage. The mean (± SEM) E2 production for the control groups of the early-, mid- and late-vitellogenic stages are 0.32 ± 0.25, 0.78 ± 0.06, and 0.29 ± 0.03 ng.follicle" l, respectively. ***, significantly (PO.001) different compared to the control group at the same maturational stage. The mean basal E2 production of the cortisol-treated groups was 0.74 ± 0.1, 0.55 ± 0.04, and 0.31 ± 0.05 ng.follicle"1 for the early-, mid-, and late- vitellogenic stages, respectively; capital letters indicate differences between cortisol- treated groups, lower case letters indicate differences between basal control groups and the difference are based on real numbers. Bars with different letters are significantly different (P<0.01) from one another.

71 Effect of Cortisol on cAMP-stimulated E2 production:

Cortisol reduced E2 production by ovarian follicles significantly at early-

(P<0.001), mid- (P<0.01), and late-vitellogenic stages (P<0.001), compared with the control FS-treated follicles (Figure 3.2). The mean FS-treated E2 production of the control groups for the early-, mid- and late-vitellogenic stages was 1.06 ± 0.14, 1.05 ±

0.05, and 0.42 ± 0.07 ng.follicle"1, respectively.

The mean cAMP-stimulated E2 production of the cortisol-treated groups was 0.67 ± 0.03,

0.9 ± 0.4, and 0.2 ± 0.03 ng.follicle"1 for the early-, mid- and late-vitellogenic stages,, respectively; these values were all significantly (P<0.001) different from one another

(Figure 3.2).

72 nam FS 120 B FS COR 3

B 100 ? ** ^^ e • mmo A -4-> 80 ISs © C u a fS 60 AAA s ^O^ •a 40 -4-* V) T CO. 1-H 20

EARLY MID LATE

Vitellogenic Stages

Figure 3.2: Effect of Cortisol on in vitro cAMP-stimulated 17P-estradiol (E2) production by rainbow trout ovarian follicles at early-, mid- and late stages of vitellogenesis; the data are shown as mean ± SEM (n = 6) and presented as a percent of the control value for each maturational stage. The mean (± SEM) E2 production for the control groups of the early-, mid- and late-vitellogenic stages are 1.061 ± 0.1, 1.05 ± 0.05, and 0.42 ± 0.07 ng.follicle"1, respectively. ***, **, significantly (PO.001, <0.01, respectively) different compared the control group at the same maturational stage. The mean cAMP-stimulated E2 production of the cortisol-treated groups was 0.67 ± 0.03, 0.9 ± 0.4, and 0.2 ± 0.03 ng.follicle"1 for the EV, MV, and LV stages, respectively; capital letters indicate differences between cortisol-treated groups, lower case letters indicate differences between FS-treated control groups and the difference are based on real numbers. Bars with different letters are significantly different (P<0.001) from one another.

73 qPCR analysis:

The gene expression of StAR and CYPscc; GR 1 and GR 2; 0-actin and EF-la, by all the three vitellogenic stages are shown as copy number of each treatment group

(Tables 3.2, 3.3, and 3.4, respectively).

As shown in Table 3.4, there were no significant changes in the expression of 0- actin and EF-la genes associated with the stage of maturation and there were no significant affects of Cortisol on the expression of these genes by follicles undergoing either basal or cAMP-stimulated steroidogenesis.

The expression rates of the two GR genes (GR1 and GR2) and the two steroidogenesis-related genes (StAR and CYPscc), as measured by qPCR, by follicles undergoing either basal or cAMP-stimulated steroidogenesis were not significantly affected by the presence of Cortisol in the medium (Tables 3.2 and 3.3).

74 Table 3.2: Effect of Cortisol on the accumulation of mRNA encoding for StAR and P450scc proteins in rainbow trout (Oncorhynchus mykiss) ovarian follicles. The data represent the number (mean ± SEM; n = 6) of mRNA copies for each treatment x 10'2. Means with similar superscript letters in the same row are not significantly different from one another; all significant differences were P<0.001.

Genes Treatments Early Mid Late

StAR Basal 120 ± 63 580 ±230 260+90

Basal+cortisol 70 + 46 250+100 240 ±160

FS 430+160 900 ± 330 550 ± 270

FS+cortisol 460 ± 200 830 + 210 310+140

P450scc Basal 60±10a 750+180 b 450 +70 b

Basal+cortisol 61 + 15 580+100 220 ± 60

FS 61±17a 680 +200 b 360 +60 b

FS+cortisol 63+4 890 ± 200 300 ± 50

75 Table 3.3: Effect of Cortisol on the accumulation of mRNA encoding for glucocorticoid receptors, GR1 and GR2 in rainbow trout (Oncorhynchus mykiss) ovarian follicles. The data represent the number (mean ± SEM; n = 6) of mRNA copies for each treatment.

Genes Treatments Early Mid Late

GRI Basal 200+100 1300+1200 150+100

Basal+cortisol 80 + 20 150 + 80 120 ± 70

FS 70 + 30 130 + 50 130 + 50

FS+cortisol 100 + 30 200+100 200 ±100

GRH Basal 600 ±400 600 ±140 310 + 60

Basal+cortisol 300+100 630+150 900 ± 500

FS 200 ± 70 570 ±140 250 ± 70

FS+cortisol 300+100 710+110 180 + 70

76 Table 3.4: Effect of Cortisol on the accumulation of mRNA encoding for P-actin and EF- la in rainbow trout (Oncorhynchus mykiss) ovarian follicles. The data represent the number (mean ± SEM; n = 6) of mRNA copies for each treatment x 10"2 for p-actin and 10-5forEF-la.

Genes Treatments Early Mid Late

P-Actin Basal 60 + 34 41 + 15 37+17

Basal+cortisol 46+16 50+ 18 60 + 30

FS 32 + 9 95 + 39 50 + 26

FS+cortisol 46+ 18 75 + 31 55 + 24

EF-la Basal 62 + 38 200+150 80 + 31

Basal+cortisol 50 + 32 90 + 44 61+40

FS 47 + 21 220 ± 84 61+33

FS+cortisol 75 + 40 180 + 75 37 + 20

77 Detection of GR protein in thecal and granulosal cells by immunohistochemistry:

Relative to the negative control, both the thecal and granulosal cells appear to be intensely immunostained (Figure 3.3A). There also appears to be some immunostaining through the body of the zona radiata (ZR) in the anti-GR section, probably of granulosal cell cytoplasm passing through pores in the ZR. There were no apparent differences in the extent of immunostaining present in the follicles at the three vitellogenic stages. The images shown in Figure 3.3 are of tissues of a mid-vitellogenic stage ovary.

78 Figure 3.3. A section through the thecal cell (TC), granulosal cell (GC), zona radiata (ZR) of a rainbow trout (Oncorhynchus mykiss) ovarian follicle at the mid-vitellogenesis stage.

Figure 3.3A was immunostained with anti-rainbow trout GR antibody; Figure 3.3B is an adjacent section treated identically to that shown in Figure 3.3A except GR antibody was not included in the primary antibody incubation. Effects of Cortisol on steroid profiles of metabolites formed from radiolabeled

steroid substrates:

The elution profile of the 18 steroid hormone authentic reference standards (at

absorbance 254nm is shown in Figure 3.4. The radiolabeled precursors P5, and T were

used in separate incubation series; both basal and cAMP-stimulated steroidogenesis was

examined.

The free steroid metabolite profiles following both basal and FS-treated group

incubated with P5 (Figure 3.5a, b and Figure 3.6a, b) suggest that Cortisol tends to

suppress E2 production compared to control. Similarly, with radiolabeled T in the

incubation, Cortisol appeared to markedly suppress E2 production in both basal and

cAMP-stimulated treatments; moreover, labelled steroids that co-eluted with

norandrostenedione (norA4) and nortestosterone (norT) were also present in the Cortisol treated groups (Figure 3.7a, b and Figure 3.8a, b).

In the conjugated steroid metabolite profiles following both basal and cAMP-

stimulated treatments incubated with radiolabeled P5, Cortisol tended to suppress E2 production in sulphate conjugate; whereas there was no marked affect of Cortisol on the glucuronide conjugate profile. On the other hand, the profiles from follicles incubated with radiolabeled T, exhibited no clear difference in sulphated E2 production in both basal and cAMP-stimulated groups; conversely, in the steroid glucuronide conjugate profiles there appeared to be a higher production of T glucuronide in the cAMP-

stimulated Cortisol group compared to control, but little evidence of an affect of Cortisol for the basal steroidogenesis treatment groups.

80 0.010 -,

0.008

0.000

Elution time (min)

1. Aldosterone 2. Cortisol 3. Cortisone 4. lipOHT 5. 11KT 6. Corticosterone 7. lipOHA 8. 11KA 9. 11DOC 10. 19NOR-T 11. E2 12. 17,20P 13. 19NOR-A 14. T 15. 110OHP 16. El 17. A4 18. 17QOHP

Figure 3.4. Representative HPLC chromatograph showing the elution time of 18 steroid reference standards with UV absorbance at 254 nm. Abbreviations: lipOHT, lip-hydroxytestosterone; 11KT, 11-ketotestosterone; lipOHA, lip-hydroxyandrostenedione; 11KA, 11-androstenetrione; 11DOC, 11-deoxycortisol; 19NOR-T, 19-nortestosterone;E2, 17p-estradiol; 17,20P-dihydroxy-4-pregnen-3-one; 19NOR-A, 19-norandrostenedione; T, testosterone; lipOHP, lip-hydroxyprogesterone; Ei, estrone; A4, androstenedione; 17aOHP, 17a-hydroxyprogesterone.

81 A] ^ norA norT \ s

hi

norA E2 ±1

E,

^ 10 15 E lution tlm e (m in)

Figure 3.5: Representative HPLC profiles of radiolabeled free steroid hormone metabolites produced by rainbow trout (Onchorhynchus mykiss) ovarian follicles after in vitro incubation at 8-10°C for 18 h in medium containing [7-3H]pregnenolone ([3H]Ps) as substrate, either in the presence [B] or absence of Cortisol [A]; the free steroids are shown in A and B; the sulphated-conjugates are shown in Al and A2; the glucuronide- conjugates are shown in A2 and B2; CPM, counts per minute.

82 A] 10000 -• norA

E ^^\ 1

B] Bl] norA 11POHA 11KA NM V

^L

Figure 3.6: Representative HPLC profiles of radiolabelled free steroid hormone metabolites produced by rainbow trout {Onchorhynchus mykiss) ovarian follicles after in vitro incubation at 8-10°C for 18 h in cAMP-stimulated medium containing [7- H]pregnenolone ([ H]Ps) as substrate, either in the presence [B] or absence of Cortisol [A]; the free steroids are shown in A and B; the sulphated-conjugates are shown in Al and A2; the glucuronide-conjugates are shown in A2 and B2; CPM, counts per minute.

83 Al]

norT V/

[!•«•)• aa*

/

10 15 20 Elutlon time (mln) IkitfeBiBaM»h>

B] Bl]

norT E, norA

W H EMIM *•*«•*)

E2 /

10 15 EMhiaatteM Elution time (min)

Figure 3.7: Representative HPLC profiles of radiolabelled free steroid hormone metabolites produced by rainbow trout (Onchorhynchus mykiss) ovarian follicles after in vitro incubation at 8-10°C for 18 h in medium containing [1,2,6,7-3H] testosterone ([3H]T) as substrate, either in the presence [B] or absence of Cortisol [A]; the free steroids are shown in A and B; the sulphated-conjugates are shown in Al and A2; the glucuronide-conjugates are shown in A2 and B2; CPM, counts per minute.

84 norT

10 15 20 Elution time (mln)

Figure 3.8: Representative HPLC profiles of radiolabeled free steroid hormone metabolites produced by rainbow trout (Onchorhynchus mykiss) ovarian follicles after in vitro incubation at 8-10°C for 18 h in cAMP-stimulated medium containing [1,2,6,7-3H] testosterone ([3H]T) as substrate, either in the presence [B] or absence of Cortisol [A]; the free steroids are shown in A and B; the sulphated-conjugates are shown in Al and A2; the glucuronide-conjugates are shown in A2 and B2; CPM, counts per minute.

85 DISCUSSION

Basal E2 production by rainbow trout (Oncorhynchus mykiss) ovarian follicles was significantly (P<0.001) suppressed by Cortisol in the mid-vitellogenic stage follicles compared to the controls, but did not affect steroidogenesis in early- and late-vitellogenic

stages; indeed there was a significant increase in E2 production in cortisol-treated early

stage follicles (Figure 3.1). The reason for the suppressive effect of Cortisol on the mid- vitellogenic stage and not on the early-vitellogenic stage may be because the

steroidogenic pathway is not fully activated; thus, E2 production was already at a very low level and frequent suppression may not have been measurable. Similarly, the absence of a significant suppresive action of Cortisol on E2 production by the late-vitellogenic

stage follicles is because of the steroidogenic shift from E2 to DHP production. In contrast, in the FS-treated groups, E2 production was significantly suppressed in all three maturational stages when Cortisol was included in the incubation medium (Figure 3.2).

The above findings suggest that when E2 production is low, the suppressive action of

Cortisol is not evident using the criteria of steroid hormone production, but it is evident wherever E2 production rates are elevated.

The expression of the genes encoding for StAR and CYPscc proteins was examined to determine if any of the measured actions of Cortisol where mediated by changes in cholesterol transfer to the mitochondria of the thecal/granulosal cells or the formation of P5. The absence of an affect of Cortisol on the expression of two steroidogenesis-linked genes (StAR and CYPscc) (Table 3.2) suggests that the glucocorticoid may not exert its affect on the regulation of StAR protein recruitment into

86 mitochondria, or the conversion of cholesterol to P5 within the mitochondria. However, it must be emphasized that the study only considered mRNA transcript numbers, and did not measure protein production.

The expression of the two housekeeping genes (P-actin and EF-la) was measured to determine if there were changes in expression rates of genes not related to

steroidogenesis during ovarian maturation of rainbow trout of the kind reported for zebrafish {Dania rerio) (Ings and Van Der Kraak, 2006); and if so, whether Cortisol affected the expression. The results of the quantitative real-time PCR studies (Table 3.4)

suggest that there is no significant effect of Cortisol on the respective genes, and nor is there a change in the expression of these genes related to the stage of follicle maturation.

The absence of maturational changes in this study using rainbow trout study contrasts with the findings in zebrafish by Ings and Van Der Kraak (2006). The differences are probably because of the different gonadal maturation events in the two species; trout exhibit a single annual spawning event, whereas zebrafish exhibit multiple spawnings within a year. Moreover, the ranges of stages of follicular maturation in the two studies were quite different; a much broader spectrum of maturation was considered in the zebrafish report.

The expression of the two glucocorticoid receptor (GR) genes was measured to determine if any of the observed effects of Cortisol were exerted via the GR receptors

(GR1 and GR2), with the possibility that gene expression would be suppressed in conditions of elevated glucocorticoid (as shown by Aluru et al., 2005 for interrenal tissue). Interestingly, the genes encoding for the glucorticoid receptors, GR 1, GR 2 were also unaffected by the addition of Cortisol to the incubation medium. This suggests that

87 the glucocorticoid-related decreases in E2 production are not brought about via ligand- receptor activated pathways.

The immunohistological study showed that there were no differences in anti-GR immunostaining among the three different developmental stages. Similarly, there were no differences in the expression of the GR genes by follicles at different stages. These findings suggest that genes are expressed and that GR protein is present throughout gonadal recrudescence, regardless of the steroidogenic levels.

The tissues that were used for immunohistological staining had not been incubated in the presence of Cortisol, and therefore, in the present study, there is no measure of an affect of Cortisol on GR protein content; however, the gene expression results suggest that Cortisol does not affect the rate of GR gene expression. Taken together, these findings suggest that the E2 suppressive action of Cortisol does not operate via pathways that involve a cortisol-GR activated pathway.

Taken together, the findings described above suggest that Cortisol has no apparent affect on the expression of key steroidogenesis-related genes. I was interested, therefore, to see if the glucocorticoid would affect any of the steroidogenic enzyme systems directly; the approach chosen was to incubate ovarian follicles with radiolabeled P5, or

T, to determine if the range of steroid metabolites produced was affected by the presence of Cortisol in the medium. There was evidence of suppressive effect of Cortisol on the E2 production when follicles incubated with radiolabeled P5 for both basal and cAMP- stimulated treatments (Figure 3.5a, b) and (Figure 3.6a, b), respectively. In the basal treatment group of free steroid set (Figure 3.5a, b), E2 production was reduced and there were higher fractions of androgens (norT and 11 P-hydroxyandrostenedione [lipOHA])

88 in the Cortisol treated group compared to control. Similar suppressive effect of Cortisol was seen in both steroid-sulphates and steroid-glucuronides. In the free steroid group of cAMP-stimulated treatments, Cortisol appeared to inhibit E2 production in association with the appearance of more metabolites of androgen (1 lpOHA and 11KT), whereas both the conjugate sets showed similar suppressive effect of Cortisol on E2 production. In both basal and cAMP-stimulated groups P5 was metabolized into more androgen products in

Cortisol treated group, suggesting that there might be an inhibition of the enzyme

P450arom, which catalyzes the conversion of androgens to estrogens (Kamat et al.,

1998). This would explain the suppression of E2 production by Cortisol, and the hypothesis is supported by the marked affect of Cortisol on the metabolism of radiolabeled T. E2 production was suppressed by Cortisol in both basal and cAMP- stimulated follicles (Figures 3.7a, b, Figure 3.8a, b). These results imply that P450arom might be affected directly by Cortisol. There is evidence of direct effect of Cortisol on stimulation of androgen production by thecal cells from porcine ovarian follicles

(Raeside and Xun, 1986). Studies with mammals also suggest that Cortisol exerts a selective inhibition of FSH-induction of aromatase activity in granulosal cells (Hsueh and

Erickson, 1978; Hsueh et al, 1984; DanisovA et al., 1987). The suppression of E2 production by Cortisol in the present study is not operating via a GtH stimulation pathway, but it does impact'on cAMP-stimulated steroidogenesis. The absence of any affects on the expression of key steroidogenic genes, and the results of the steroid metabolite profile studies suggest that the enzyme (P450arom) involved in the conversion of T to E2 in the rainbow trout follicles) might be suppressed directly by Cortisol.

89 SUMMARY, CONCLUSIONS AND FUTURE DIRECTIONS

This study identified physiological characteristics that allowed the identification of different vitellogenic stages of ovarian development in rainbow trout (Oncorhynchus mykiss) (early-, mid-, and late-vitellogenesis). Basal E2 and T production was highest in the mid-vitellogenic stage compared to early- and late-vitellogenic stages. Following

Forskolin (FS) treatment, and the subsequent increase in intracellular levels of cyclic

AMP (cAMP), increased steroid production by follicles at all three stages of maturation, suggesting that the steroidogenic enzymes were in place, and that the activation of steroidogenic acute regulatory protein (StAR) protein has occurred presumably resulting in an increased delivery of cholesterol into the inner mitochondrial compartment; however, there were no significant maturational stage-related differences in the gene expression of StAR protein; there was, however, a change in the expression of the cytochrome P450scc (CYPscc) gene from low values in the early-vitellogenic stage follicles (both basal and FS-treated) to significantly higher values in the later stages.

The findings of this study showed a direct effect of Cortisol on steroidogenesis by rainbow trout ovarian follicles. Exposure to Cortisol in the medium for 18 h resulted in decreased production of E2 and T by ovarian follicles, especially by those follicles at mid-vitellogenic stage of ovarian development. However, there was no effect of Cortisol on the expression of genes that encode for StAR, and CYPscc, key steroidogenesis- related genes, suggesting that Cortisol is not exerting its effect on the respective genes in the steroidogenic pathway. Similarly, Cortisol had no affect on the expression of genes that encode for GR protein, nor were there differences in the expression of these genes, or in the amount of GR protein (based on immunohistochemistry) in thecal and granulosal

90 cells of follicles at early-, mid- and late-vitellogenic stages; taken together, these finding

suggest that Cortisol may not exert its inhibitory affect on steroidogenesis E2 and T via

the cortisol-receptor pathway. Studies examining the metabolites formed from the in

vitro conversion of radiolabeled P5 and T suggested that the suppressive action of

Cortisol on E2 production occurs at the late stage of steroidogenesis, namely the

aromatization of androgens into estrogens; this conclusion was supported by the

observation of high levels of androgens, including norT, nor A, T, lipOHA and 11KT, in the incubation medium. These findings suggest that Cortisol exerts its steroidogenic

suppressive effects by direct inhibition of the function of the enzyme P450arom which

converts androgens into estrogens in granulosal cells; however, the mechanism of action

of Cortisol on in vitro ovarian steroidogenesis in rainbow trout has to be explored further.

Taken together the findings in chapter II and III, I was able to achieve most of my

study objectives demonstrating that Cortisol inhibits basal and cAMP-stimulated ovarian follicle steroidogenesis, and that the actions of Cortisol are related to the stage of maturation of the follicle. The working hypothesis that formed the basis of part of the

study was that Cortisol exerted its effects on steroidogenesis via modulation of StAR activation, thus inhibiting cholesterol transfer into the inner mitochondrial membrane.

The results of the gene expression work tended to counter that hypothesis, as did the results of the biotransformation of radio labelled P5 and T by ovarian follicles, particularly the appearance of different species of androgens in the medium following incubation of follicles with radiolabeled T, may suggest a direct action of Cortisol on aromatase

activity in the granulosal cells. Further work is needed to test that hypothesis further.

Future work might include:

91 1. Additional steroid biotransformation trials using other radiolabeled substrates, with

particular interest in the range of steroid conjugates that are formed.

2. Additional biotransformation trials using nonlabelled T or A4 as a substrate and

examining the affect of Cortisol at different levels estrogen production using RIA, and

using HPLC separation of steroids.

3. Comparison of the effects of Cortisol with those of commercially available aromatase

inhibitors.

4. Comparison of the effects of adrenal steroids other than Cortisol on ovarian follicle

steroidogenesis.

5. Additional trials for mRNA expression of genes encoding for the enzymes P450arom

and 17P-HSD, enzymes that are involved in the later part of the ovarian steroidogenic

pathway.

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115 APPENDIX I

PROTEIN PHOSPHORYLATION INHIBITION OF BASAL & FS STEROIDOGENESIS

120 n

*** *** ** 100 H

I 80 A i 1 «°

40 02.

20 A

MID LATE

BASAL Vitdlogenic Stages CANTHARIDIN FS 1 FS-CANTHARIDIN

Figure 1.1: Effects of cantharidin (protein phosphorylation inhibitor) on basal and cAMP-stimulated (forskolin) steroidogenesis. The mean [ng.follicle"1] E2 production (±SEM) values of the mid-vitellogenesis stage was 0.43±0.02 and 0.66±0.043 for the basal and cAMP-stimulated treatment groups, respectively (P<0.05). The mean [ng.follicle"1] E2 production (±SEM) values of the late-vitellogenesis stage was 0.12±0.004 and 0.12±0.002 for the basal and cAMP-stimulated treatment groups, respectively. The asterisks (*, **, ***) indicate significant differences (PO.05, 0.01, 0.001, respectively) between control and cantheridin-treated follicles.

116 Experimental method:

Ovarian follicles were incubated in vitro in the presence or absence of the protein phosphorylation inhibitor, cantheridin (concentration 1 uM) to determine if cAMP steroidogenesis involves phosphorylation events. Cantheridin used in the study was kindly donated by Dr. G. Pyle. The incubation methods and procedures used were as described in Chapter II. Trout at mid- and late-vitellogenesis were used in the study. The cantheridan was kindly donated by Dr Glen Pyle.

Results:

Cantheridin significantly suppressed E2 production by ovarian follicles at mid- and late-vitellogenic stages (Figure 1).

Discussion:

The findings suggest that cAMP-stimulated steroidogenesis in trout ovarian follicles involves protein phosphorylation events; the effect on basal steroidogenesis may suggest that cAMP-stimulated processes were in affect in the follicles, even without forskolin activation of adenylate cyclase.

Tropic hormones (ACTH and gonadotropins) acutely stimulate adrenal and gonadal steroidogenesis by activating cAMP-dependent protein kinase A (PKA) signaling pathway and hence subsequent induction of steroidogenic acute regulatory

(StAR) protein expression. In mammals, cAMP is known to regulate the process of steroidogenesis through its interaction with, and the phosphorylation of; a member of cAMP response element binding (CREB) protein family. There is evidence that

117 phosphokinase A acts at the post-translational level to regulate StAR expression and

suggest that phosphorylation of StAR by PKA contributes to protein stability (Clark et al., 2001). In fish so far, to the best of my knowledge, there has been no evidence presented of the role of protein phosphatase in cAMP-dependent steroidogenesis. To determine that similar to that of mammals, protein phosphorylation is an intermediate step in the cAMP-dependent steroidogenesis we have considered two stages of rainbow trout ovarian developmental stages (mid- and late-vitellogenic stage) and each group were treated with a known protein phosphorylation inhibitor, cantharidin and compared with control and cAMP-stimulated (forskolin).

118 APPENDIX H

CORTLAND'S MEDIUM

Stock solution A (1L):

Chemicals

NaCl 72.5 g

CaCl2.2H20 2.3 g

KC1 3.8 g

NaH2P04H20 4.1 g

MgCl2.6H20 2.03 g

MgS047H20 2-3 g

Stock solution B (1L):

Chemicals

NaHC03 10 g

Cortland's working solution (2L):

Stock solution A 200 ml

Stock solution B 200 ml

Glucose 2g

BSA 2g

Streptomycin sulphate 0.2 g

Make to 2 liters with dd H20, adjusting pH to 7.55, kept in fridge for use.

119 APPENDIX HI

FORSKOLIN PREPARATION

1. Forskolin (10-5 M; 10 uM):

Newly ordering, dissolve 10 mg in 200 ul DMSO; gives 122 mM Forskolin,

Aliquot in lOx 12 ul, 2 x 40ul, and kept in -20°C for future use.

Make 1:11.2 dilutions of 122 mM Forskolin, [10 ul of 122 mM Forskolin + 112 ul of

DMSO; give 10 mM stock].

Add 1 ul of 10 mM Foskolin stock to 1 ml of incubation (Cortland's) medium to give 10

uM.

Make 120 ul of 10 mM Foskolin stock for each incubation.

120 APPENDIX IV

PREGNENOLONE (P5) PREPARATION

1. Pregnenolone [FW 316.5] (5 uM):

Dissolve 0.016 gm of Pregnenolone and dissolve in 10 ml of absolute alcohol, to make 5

uM stocks.

Aliquot 10 x 1 ml and kept at -20° C.

Add 1 ul of 5 mM stock to 1 ml of incubation medium (Cortland's) medium to prepare a

5 uM solution.

121 APPENDIX V

RADIOIMMUMO ASSAY (RIA) PROTOCOL

Testosterone / Estradiol RIA

Volume (ul) of Hi of Cone. stock sof. ul of ul of ul of ul of total ng.ml'1 (1 ng.mr1) phosgel H3 Antibody Charcoal volume

Total X 700 500 X X 1200

NSB X 200 500 X 500 1200 0 0 100 500 100 500 1200 0.1 10 100 500 100 500 1200 0.25 25 100 500 100 500 1200 0.5 50 100 500 100 500 1200 1 100 100 500 100 500 1200 2.5 250 100 500 100 500 1200 5 500 100 500 100 500 1200 10 1000 100 500 100 500 1200

* The concentration of the working solution of Testosterone/Estradiol STD is 1 ng.ml"

(1 pg.mr1).

Pipet 10, 25, 50, 100, 250, 500, 1000 ul of the working solution to the set of std. tubes

to cover a range of 0, 0.1, 0.25, 0.5, 1, 2.5, 5, 10 (ng.ml"1).

• The cpm for tracer of 500 ul should be about 10000.

• ~80 ul 3H-estradiol/60ml of phosgel

• ~70 ul 3H-testosterone/70 ml of phosgel

122 1. Dry standard under N2 at 35°C. Reconstitute each std. with 100 ul of phosgel.

2. Pipet 100 ul of each sample in duplicate on ice.

3. Set up tubes for Total, NSB and interassay sample with each assay.

4. Add 500 ul of tracer and then 100 ul of antiserum to each tube.

5. Vortex and incubate in fridge overnight.

6. Add 500 ul of charcoal/dextran mixture to each tube except tubes of Total contents.

Charcoal 0.5 gm

Dextran 0.05 gm

Phosgel 100 ml

7. Vortex and centrifuge at 3000 rpm for 20 min.

8. Decant and collect the supernatant in scintillation vial, add 4.5 ml of scintillation

cocktail, vortex and count.

Phosgel buffer (2 liter)

Na2HP04 11.5 gm

NaH2P04.H20 2.56 gm

Gelatin 2 gm

Thimerosol 0.2 gm

H20 make to 2 1

Heat H20 to 45-50°C. Dissolve gelatin in warm H20. Add phosphates; then thimerosol.

Adjust pH to 7.6. Store at 4°C.

123 APPENDIX VI

TOTAL RNA EXTRACTION PROTOCOL

1. Take 2 ml centrifuge tubes (n=3-4). Work on ice.

2. 1 ml or 800 ul QIAZOL lysis reagent.

3. Homogenize (30-45 sec).

4. Leave at room temperature for 5 min.

5. 200 ul chloroform.

6. Vortex/Shake 15 sec.

7. Leave at room temperature for 2-3 min

8. Centrifuge at 12, 000 x G for 15 min (4°C)

9. Transfer to 1.5 ml tube.

10. Add 700 ul ethanol (70%) in the 1.5 ml tube [Do not centrifuge].

11. Put add 700 ul of the mixture to the RNase mini column.

12. Spin for 8000 x G for 20 sec.

13. Repeat with 700 ul of mixture in the column. 8000 x G for 20 sec.

14. RW1 buffer 350 ul to the column, spin 8000 x G for 20 sec.

15. 10 ul DNase + 70 ul RDD buffer = 80ul/sample, add in the column.

16. Leave for 10 mins.

17. RW1 buffer 350 ul add to the column, spin 8000 x G for 20 sec.

18. Take new collection tube.

124 19. RPE buffer 500 ul, spin at 8000 x G for 20 sec.

20. Repeat RPE buffer 500 ul, spin at 12000 x G for 2 min.

21. Take out the column, place the column in 1.5 ml centrifuge tube.

22. Put 25 ul RNase free H20 to the column

23. Leave for 10 mins at room temperature.

24. Spin at 8000 x G for 1 min.

25. Repeat 20 ul RNase free H20 to the column, sit for 10 min on ice.

26. Centrifuge at 8000 x G for 1 min.

Check the quality in spectrophotometer. Store total RNA at -80°C for future use.

125 APPENDIX VH

FORMALDEHYDE AGROSE RNA GEL PROTOCOL

1. Clean up equipment

• Wash electrophoresis tanks, tubes and so on with 0.5% SDS, rinse in

water, dried with 100% ethanol

• Fill with 3% hydrogen peroxide, 10 min at room temperature, rinse

thoroughly with RNase-free water, let dry at room temperature

2. 1.2% Formaldehyde agrose (FA) gel (50 ml)

• 0.6 g Agrose

5 ml lOx FA gel buffer, and add RNase-free water to 50 ml (mark on the

flask), microwave to melt agrose, cool to 65°C in a water bath

• Add 0.9 ml of 37% formaldehyde and 0.5 ul of ethidium bromide (EtBr,

10 mg.ml"1) and RNase-free water to the mark because of evaporation,

mix and pour the gel solution support, place comb in position.

• Equilibrate in lx FA gel running buffer up to 30 min.

• Add 1 volume of 5x RNA loading buffer to 4 volume of RNA sample,

65°C heat for 5 min, chill on ice, and load on to the equilibrated FA gel.

3. Run gel at 70 V for 40-60 min depending on the gel size

126 Solutions lOx FA gel buffer

200 mM 3-[N-morpholino]propanesulfonic acid (MOPS) (free acid)

50 mM sodium acetate

lOmMEDTA

Make with dd water to 1,000 ml, adjust pH to 7.0 with NaOH, add 1 ml DEPG-A

in 37°C over night, then autoclave, lx FA gel buffer

100 ml lOx FA gel buffer

20 ml 37% formaldehyde

880 ml RNase-free H20

5x RNA loading buffer

1.6 ul saturated bromophenol blue solution

8 ul 500 mM EDTA (pH 8.0)

72 ul 37% (12.3M) formaldehyde

200 ^il 100% glycerol

308.4 ul formamide

400 ul 1 Ox FA gel buffer

Add RNase-free water to 1 ml

127 APPENDIX VHI

FIRST-STRAND cDNA SYNTHESIS

The first strand cDNA was synthesized from 1 ug of total RNA using cDNA synthesis kit

(MBI Fermantas)

First Step: Dilute RNA to 1 ug per 10 JJ.1 (in RNase free water).

Add 1 ul of Oligo dT, pulse it and heat at 70° C for 5 min and 4°C for 10

min (in thermocycler).

Second Step: Pulse the tube

Add 4 ul of 5x reaction buffer, 2 ul dNTP and 1 ul of RNase inhibitor.

Heat at 37°C for 5 min.

Third Step: Add 2 ul MMuLV (reverse transcriptase) and incubate at 37°C for 60 min

and 70°C for 10 min.

128 APPENDIX IX

DNA AGROSE GEL PROTOCOL

1. 2% agarose gel:

(A) 0.5gagrose(25ml) (B) 0.6 g agrose (30 ml) (C) 0.9 g agrose (45 ml)

24.5 ml dd water 29.4 ml dd water 44.1 ml dd water

0.5ml50xTAE 0.6 ml 50xTAE 0.9ml50xTAE

2. Cover flask with watch glass or small Petri dish

3. Mark level of liquid in case of evaporation

4. Boil in microwave and swirl occasionally, until no agrose crystals left

5. Add 1.0 or 1.5 ul of 10 mg.ml"1 EtBr (25ml) and gently swirl flask to mix

6. Swirl occasionally and cool to about 60°C (flask is hot but touchable)

7. Level and tape ends of gel tray

8. Slowly pour gel solution into one corner of tray, avoid production of bubbles which

can be removed with pipette

9. Place comb in position and level for 30-60 min or until gel has solidified

10. Carefully remove comb and just cover with running buffer

11. Gently press a piece of Parafilm into the well of an Eppendorf rack

12. Pipette 5-10 ul PCR product and 3 ul of loading dye into Parafilm well and mix

13. Pipette 10 fj.1 (0.1 ug) of DNA ladder into a well also

129 14. Carefully load sample into the wells in the gel load from right to left and label as

ladder, and samples

15. Run at 100 V briefly and then decrease into 50-75 V run until dye front is about 3A on

the gel (about 45 min), which depends on what kind of dye used

16. Turn off power pack and use UV light to view gel and take photograph

Solutions:

1. 50 x TAE buffer:

242gofTrisbase

57.1 ml of Glacial Actetic acid

100 ml of 0.5 M EDTA (pH 8.0)

Add ddH20 up to 1000 ml, store in the fridge

2. lx TAE buffer with 0.5 ug.ml'1 EtBr (running buffer)

20 ml 50x TAE

50 ul 10 mg.ml"1 EtBr

Bring to 1000 ml with dd water

3. 25 bp DNA ladder

1 ug.ml"1 ladder — 20 ul

130 TE buffer — 167 ul

4. 6x bromophdnel blue loading buffer — 33 ul

Total 220 ul of 1 ug. 10"1 25 bp DNA ladder

5. TE buffer:

1.2 g of Tris base (pH 7.5), or 10 mM Tris base

2 ml of 0.5 M EDTA (ImM EDTA)

Add ddH20 upto 1000 ml, store in the fridge

6. 0.5 M EDTA with pH 8.0

18.6gofNa2EDTA.2H20

80mlofddH2O

2 g of NaOH pellets

Adjust the pH to 8.0 with NaOH pellets

Bring the volume up to 100 ml with ddH20

Bring the volume up to 100 ml with ddH20

Autoclave

131 APPENDIX X

PREPARATION OF CORTISOL FOR FOLLICLE INCUBATION

1. Dissolve 2 mg of Cortisol in 2 ml of absolute alcohol, then make a 1:9 dilution to

give 0.1 mg.ml"1 (Cortisol 1).

Add lul of Cortisol 1 to 1 ml of incubation medium gives incubation

concentration of 100 ng.ml"1; a 1:1 dilution of Cortisol 1 gives 0.05 mg.ml"1

(Cortisol 2); add 1 ul of Cortisol 2 to 1 ml of incubation medium gives

incubation concentration of 50 ng.ml"1; a 1:9 dilution of Cortisol 1 gives 0.01

mg.ml-1 (Cortisol 3); 1 ul of Cortisol 3 to 1 ml of incubation medium gives

incubation concentration of 10 ng.ml"1.

132 APPENDIX XI

GR IMMUNOfflSTOCHEMICAL STAINING PROTOCOL

(Vectastain ABC method)

1 Xylene 10 min 2 Xylene 10 min 3 100% Ethanol (used) 10 min 4 100% Ethanol (new) 10 min 5 90% Ethanol 5 min 6 70% Ethanol 5 min 7 Running tap water 10 min 8 DD water rinse 5 min

9 1% H202 in methanol 15 min 10 Running tap water 10 min 11 DD water rinse 5 min 12 5% normal goat serum 40 min 13 Blot away excess to dry 14 Primary antibody (GR AB 1:500) 60 min 3 changes of 15 PBS wash min Biotinylated secondary antibody (goat anti-rabbit 16 IgG) 60 min 3 changes of 17 PBS wash min 18 Vectastain ABC 60 min 3 changes of 19 PBS wash min 20 Peroxidase substrate 2-7 min 21 Running tap water 10 min 22 DD water rinse 5 min 23 70% Ethanol 3 min 24 90% Ethanol 3 min 25 100% Ethanol 3 min 26 100% Ethanol 3 min 27 Xylene 3 min 28 Xylene 3 min 29 Mount with DPX vfotes:

133 1. Remove mercuric chloride crystals with sample fixed in SBH solution

2. 3% Sodium thiosulphate bleaches out coloring produced by iodine

3. Quenching of endogenous peroxide if necessary - do not store solution for more

than 2 weeks

4. Normal serum should be from the same species that the secondary serum is raised

in: 500 ul/10 ml buffer (approximately 50 \il per section)

5. Dilution to be determined by trial and error

Polyclonal:

a. 1 part PBS with 1% BSA

b. 1 part rainbow trout liver extract - 5 g liver in 200 ml PBS with 1%

BSA, homogenize, centrifuge 3000 rpm for 20 min, collect supernatant

To guard against errors, first make 10-fold dilution and aliquot and then

dilute aliquots to desired dilution for staining.

Prepare 20 to 30 ml of antibody put in a pinch of rat liver powder.

Incubate at 4°C for 6 h, centrifuge at 3000 rpm for 15 min and collect

supernatant.

Divide into 1 ml aliquot and store in -80°C

To use antibody for staining, thaw aliquot centrifuge at 3000 rpm 15 min

or less, collect supernatant, do not re-use once thawed.

6. PBS (pH 7.5)

Solution a: 1.4 g NaH2P04 in 100 ml dd water

134 Solution b: 1.4 g Na2HP04 in 100 ml dd water

15.9 ml A

84.5 ml B

8.5gNaCl

made up to 1000 ml with dd water

7. Secondary antisera is biotinylated and supplied by Vector laboratories

50 ul/10 ml buffer

Do not use azide after this step as it inhibits peroxidase

8. Vectastain ABC reagent

Add exactly 2 drops of reagent A to 10 ml buffer in mixing bottle

provided

Add exactly 2 drops of reagent B to above mixture

Mix immediately and allow standing for 30 minutes

Prepare during incubation of secondary antibody, do not use azide

9. Peroxide substrate

Mix together an equal volume of

A. Hydrogen peroxide 0.02% (made in distilled water from 30%

stock), 10 ul/50 ml

B. Diaminobenzidine tetrahydrochloride (DAB) 0.1% (1 mg/ml)

(made in 0.1 M Tris buffer pH 7.2)

1 ml Stock DAB

4 ml Tris buffer

135 0.1 MTris buffer (pH 7.2):

a: concentrated HC1 8.3 ml made up to 1000 ml (IN)

b: Tris 1.212 g made up to 100 ml (0.1M)

c: 25.0 ml solution b, add solution a until pH 7.2 (approx. 25 ml)

Made up to 100 ml

StockDAB(5mg/ml):

0.1 g in 20 ml Tris buffer, stored in 1 ml aliquots at -20°C

The hydrogen peroxide solution should be prepared fresh daily from concentrated stock.

Peroxide substrate is unstable in the presence of the hydrogen peroxide in light so prepare just before using.

DAB is a carcinogenic substance; take care in handling and disposing of all solutions.

136 APPENDIX Xn

SEP-PAK Cig COLUMN

Ci8, 3cc Sep-Pak cartridges Waters # 20805

10ml syringes with Luer lock (reservoir)

16x100mm glass tubes Fisher 14-961-29

B-glucuronidase, Type B-3

From Bovine liver Sigma G-0376

Methanol Caledon 6701-7-40

Hexane Caledon 5601-7-40

Diethyl Ether Caledon 4700-1-10

Ethyl Acetate Fisher E195-4

Trifluoroacetic Acid (TFA) Fisher 04902-100

137 Prime Sep-Pak cartridge methanol (5ml) followed by H20 (5ml) sample (eg lml plasma in 5ml H2O)

XI free (unconjugated) steroids Sep-Pak wash with H2O 5 ml let ether stand minimum lOmins for water to displace water with hexane 5 ml come out elute with diethyl ether 5 ml •=> decant ether into another tube leaving behind elute with methanol 5 ml water drop evaporate under N2, 35-40°C -a sample ready for analysis conjugated steroids evaporate methanol incubate in ethyl acetate/TFA (100:1, v:v; 5mls/50ul) 18h(o/n)at45°C evaporate and redissolve in H2O 5ml hydrolyzed sulfate conjugated steroids a ^ Sep-Pak (primed) wash with H20 5 ml displace water with hexane 5ml elute with diethyl ether 5 ml elute with methanol 5 ml

138 £ glucuronide conjugated steroids evaporate methanol incubate in .5M sodium acetate pH5 (500ul) and p-glucuronidase lOOU/ul (20ul) 18h(o/n)at37°C add water 5ml

Sep-Pak (primed) [^N Hydro,yzed glucoronide conjugated wash with H20 5ml * steroids displace water with hexane 5ml elute with diethyl ether 5 ml elute with methanol 5ml £ other conjugates

139 APPENDIX Xm

EFFECTS OF VARYING THE LEVELS OF CORTISOL TREATMENT ON 17p-

ESTRADIOL (E2) PRODUCTION BY RAINBOW TROUT OVARIAN FOLLICLES

Table XIII. 1: Levels of E2 productions by different concentrations [Cortisol

lQOOng.ml"1); Cortisol 2 (50 ng.mr1); Cortisol 3 (10 ng.ml"1)] of Cortisol in the three maturational stages [EV- early-vitellogenic; MV- mid-vitellogenic; LV- late-vitellogenic stages] of ovarian follicles of rainbow trout. All values represent mean ± SEM (n=6).

Stage of vitellogenesis Cortisol Early mean E2 Mid mean E2 Late mean E2 concentration production production (ng.mr1) production (ng-mf1) (ng.mr1) (ng.mr1) 0 0.32 ±0.03 0.78 ± 0.06 0.29 ± 0.03 10 0.69 ±0.01 0.55 ±0.04 0.31 ±0.05 50 0.64 ± 0.09 0.52 ±0.04 0.26 ± 0.04 100 0.34 ±0.03 0.64 ± 0.05 0.26 ± 0.03

Experimental method:

Ovarian follicles were incubated in vitro in the presence of Cortisol at one of the three concentrations (100, 50 or 10 ng.mr1) to determine if there is a dose-related effect of Cortisol on ovarian steroidogenesis, specifically the production of E2; the study was

140 carried out on follicles at early-, mid-, and late-vitellogenesis stages. The incubation

methods and procedures used were as described in Chapter III.

Result:

For the early stage follicles, the high Cortisol treatment significantly (P<0.05)

suppressed E2 production relative to the other two treatment groups (Table XIII. 1). A

similar pattern was seen for the late stage follicles, with both the 50 and lOOng.ml"1

significantly (P<0.05) suppressing E2 production relative to the 10 ng.ml-1 treatement

group. In contrast, for the mid-stage follicles, E2 production by the follicles exposed to the high Cortisol treatment was significantly (P<0.05) higher than for the other two treatment groups (Table XIII. 1) in the level of E2 production by follicles exposed to 100

ng.ml"1 of Cortisol compared with animals exposed to 10 ng.ml'1 of the hormone; while

Cortisol 2 and 3 are statistically non-significant in these stages. However in the late stage

there is statistical significance between Cortisol 2 and Cortisol 3. The level of E2 in

Cortisol 1 (100 ng ml"1) is variable in all the three stages, whereas Cortisol 3 (10 ng ml'1)

has a stable response.

Discussion:

From Table XIII. 1 we can see that, for the mid-vitellogenesis stage which had the highest basal E2 output, there appeared to be an attenuation of the inhibitory response to

Cortisol with increasing Cortisol concentrations; the highest levels of the 100 ng.ml-1 group may be eliciting a down-regulation of glucocorticoid receptors (GRs). Also there are possibilities that other pathways (as yet unknown) might be undergoing some

141 compensatory down-regulation. In the early- and late-vitellogenesis stages, where basal

E2 output is lower combared to mid-vitellogenesis basal E2 production, the pattern of

response to the Cortisol (measured as E2 production) is different. This may be possibly because of the low level of steroidogenesis leading to attenuation of basal E2 output.

However, here too, there was evidence of an altered effect of increased level of Cortisol

exposure.

On the basis of these findings I decided to consider only the actions of Cortisol at the lower concentration level (10 ng.ml"1) for purposes to examine the effect of Cortisol

on basal and FS-stimulated ovarian steroidogenesis in rainbow trout (Chapter HI).

142 APPENDIX XIV

EFFECTS OF OVARIAN FOLLICLES ON CORTISOL METABOLISM

A] B]

Corticosterone 2. Cortisol 3. Cortisone 4. lipOHT 5. 11KT 6. Corticosterone 7.110OHA 0.G06

0.006 H

0.004 4000 <

Q000 8 10 Uution time (nin)

C] Dl Cortisol Cortisol

Corticosterone Corticosterone

8 10 12 EiutJon Unif (min) Edition time (mla)

Figure XIV. 1: Representative HPLC profiles of radiolabeled free steroid hormone metabolites produced by rainbow trout ovarian follicles after in vitro incubation at 8- 10°C for 18 h in medium containing [l,2,6,7-3H]cortisol as substrate. A] reference standard; B] follicles were incubated in the presence of non-radiolabelled Cortisol; in C] follicles were incubated in the presence of forskolin and non-radiolabelled Cortisol; and in D] the medium containing radiolabeled Cortisol was incubated without follicles. The concentration of non-radiolabelled Cortisol is 100 ng.ml"1.

143 Experimental method:

Ovarian follicles were incubated in vitro in the presence of radiolabelled [1,2,6,7-

H]cortisol to determine whether ovarian follicles can metabolise Cortisol; the study was carried out on ovarian follicles at vitellogenesis stages. The incubation methods and procedures used were as described in Chapter III. One of the incubations was carried out with radiolabelled Cortisol in the medium without follicles, to examine the quality of the labeled Cortisol.

Result and Discussion:

Figure XIV. 1 shows that several corticosteroids were expressed in the medium with and without ovarian follicles, suggesting that the Cortisol was not pure Cortisol.

There were no marked difference in the profiles of steroids incubated in the presence of follicles and radiolabelled steroid alone suggesting that, at least in this study, Cortisol was not metabolized in significant quantities by the follicles during the 18 h incubation.

144 APPENDIX XV

Date of Collection Sample # GSI Basal E2 Aug 16 '05' 2005-5 4.74 0.16 Aug 18 '05' 2005-6 8.31 0.16 Aug 18'05' 2005-7 6.55 0.2 Sep 08 '05' 2005-8 3.27 0.31 Sep 08 '05' 2005-9 8.13 0.05 Sep 08 '05' 2005-10 8.23 0.37 Sep 12 '06' 2006-1$ 2.4 0.24 Sep 12 '06' 2006-2$ 6.3 0.41 Sep 14 '05' 2005-1® 12.22 0.34 Sep 14 '05' 2005-2 5.83 0.14 Sep 14 '05' 2005-11 3.05 0.14 Sep 19 '06 2006-3® 6.4 0.41 Sep 21 '05' 2005-3® 5.47 0.46 Sep21'05' 2005-4 4.47 0.2 Sep 21 '05' 2005-12 8.5 0.3 Sep 26 '06' 2006-4* 6.9 0.43 Sep 29 '05' 2005-13 4.33 0.35 Sep 29 '05' 2005-14 7.89 0.52 Oct 03 '06' 2006 - 5® 9.4 1.45 Oct 03 '06' 2006 - 6® 8.5 0.51 Oct 03 '06' 2006 - 7 11.3 0 Oct 10'06' 2006 - 8* 13.4 0.44 Oct 10 '06' 2006 - 9® 12.1 0.73 Oct 17 '06' 2006-10* 10.3 0.27 Oct 17 '06' 2006-11® 9.148 0.85 Oct 24 '06' 2006 - 12* 10.84 0.15 Oct 24 '06' 2006 - 13 6.8352 Oct31'06' 2006 - 14 10.827 Oct3r06' 2006-15 9.96 Nov 07 '06 2006 - 16 10.56 Nov 07 '06' 2006 - 17 9.76 Nov 07 '06' 2006 - 18 11.92 Nov 22 '06' 2006 - 19 11.22 Symbols:

$ - Early-vitellogenic stage @ - Mid-vitellogenic stage * - Late-vitellogenic

145