Circadian Regulation of Hypothalamic Kiss1 in Neonatal Development, Adulthood and Pregnancy in the Mouse

by

Cassandra Ching-Lin Yap M.Sc.

This thesis is presented for the degree of DOCTOR OF PHILOSOPHY of The University of Western Australia School of Anatomy, Physiology and Human Biology

Submitted: June 2016

ii

Preface

The experimental work presented in this thesis was undertaken in the School of Anatomy, Physiology and Human Biology, The University of Western Australia, under the supervision of Dr. Jeremy T. Smith, Dr. Peter J. Mark and Prof. Brendan J. Waddell, with the financial assistance of an Australian Postgraduate Award, a UWA Safety-Net Top- Up Scholarship and a UWA PhD Completion Scholarship. The experimental work was supported, in part, by an Australian Research Council Discovery Project Grant (Discovery Project 120100521) and the Western Australian Department of Health (Grant number 1062158 2014).

The work described is original and was carried out by myself except where the specific contributions of other persons are acknowledged.

Cassandra Ching-Lin Yap June 2016

iii

iv Abstract

Kisspeptin is the product of the Kiss1 gene; it drives the hypothalamic-pituitary-gonadal (HPG) axis and is crucial for reproduction and pubertal maturation. It is expressed in two discrete populations of hypothalamic neurons, located in the arcuate nucleus (ARC) and the anteroventral periventricular nucleus (AVPV). ARC kisspeptin neurons are negatively regulated by estrogen, suggestive of its role here in negative feedback regulation of the pulsatile secretion of gonadotropin-releasing hormone (GnRH), while AVPV kisspeptin neurons are positively regulated by estrogen, indicating its involvement in the generation of the preovulatory GnRH/luteinizing hormone (LH) surge that occurs in females. The LH surge in female rodents is under tight circadian control and its timing is governed by the central pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus. Kisspeptin neurons in the AVPV receive circadian input through arginine vasopressin (AVP) neurons from the SCN, integrating circadian and estrogen signals, and consequently triggering the LH surge. The overall objective of the studies described herein was to investigate the circadian expression of hypothalamic Kiss1 during neonatal development (pre-pubertal), adulthood (post-pubertal) and pregnancy. Four separate studies were carried out for this thesis.

As AVPV kisspeptin neurons are heavily involved in generating the LH surge, the circadian patterns of kisspeptin neuronal activation and mRNA expression are synchronous with LH levels at proestrus. However, it is not known if this kisspeptin rhythm persists during pregnancy and was therefore investigated in Chapter 5 using real- time PCR. In non-pregnant mice, Kiss1 expression in the AVPV peaked in the evening of proestrus, preceded by an increase Avpr1a mRNA levels at noon and followed by an LH surge. In addition, the circadian variation in Avpr1a and Kiss1 expression seen at proestrus was not observed at any stage of pregnancy despite high E2 concentrations, indicating a disruption of the normal kisspeptin circadian rhythm during pregnancy.

As E2 levels were high during pregnancy and clock gene circadian rhythms remained intact, suppression of the kisspeptin circadian rhythm was potentially due to a disruption of circadian input to the AVPV. Since progesterone (P4) and prolactin (PRL) greatly increase during pregnancy and have direct effects on kisspeptin, Chapter 6 investigated whether either of these hormones is able to affect the evening increase in Kiss1 in E2-

v treated ovariectomised (OVX) mice. Kiss1 and Kiss1r mRNA expression increased in the evening, compared to the morning, in OVX mice that were treated with E2 only. Moreover, time-of-day variation in Bmal1 expression was observed across all treatment groups, suggestive of persistent clock gene rhythmicity in the pregnant state. The study indicates that P4 and PRL may play a role in disrupting circadian input to the AVPV during pregnancy, resulting in a suppression of the circadian kisspeptin rhythm.

Chapter 7 utilised RNA sequencing to examine genes in the anterior hypothalamus that were differentially expressed between proestrus and pregnancy, to elucidate potential mechanisms underlying the suppression of the kisspeptin circadian rhythm during pregnancy. While there was a trend for increased Kiss1 expression in the evening of proestrus similar to Chapter 5, this failed to reach statistical significance, possibly due to the low number of replicates. Several genes involved in haemoglobin production and the suppression of cytokine signalling were elevated during pregnancy compared to proestrus. In addition, although a number of genes were found to exhibit oscillatory expression at proestrus but not during pregnancy, similar to the pattern of Kiss1 expression observed in Chapter 5, their lack of specific association with kisspeptin signalling suggests that they may not be involved in the suppression of the kisspeptin circadian rhythm during pregnancy.

Finally, Chapter 8 in this thesis shifted the focus to Kiss1 expression during the neonatal period and whether the circadian kisspeptin rhythm observed in adult proestrus females is present before puberty. Hypothalamic Kiss1 mRNA expression in neonatal mice at postnatal days (P) 5, 15 and 25 showed no circadian variation. Furthermore, expression of the clock genes Bmal1 and Rev-erbα was not fully rhythmic at P25; Bmal1 demonstrated rhythmicity only in P25 females, while Rev-erbα was rhythmic in P25 males. The results indicate that immature SCN rhythmicity, in combination with low E2 levels, precludes the development of the circadian kisspeptin rhythm before puberty.

Overall, the studies presented in this thesis investigated the circadian patterns of Kiss1 expression in the mouse hypothalamus. In doing so, this thesis provides new insights into kisspeptin and its role in reproduction during the pre-pubertal period, adulthood and in pregnancy.

vi

Acknowledgements

I would first and foremost like to thank my supervisor Jeremy Smith for his guidance, encouragement, patience and understanding throughout this journey. It has been an honour being your first PhD student.

I am very grateful to my co-supervisors Peter Mark and Brendan Waddell; the advice and wisdom they have imparted to me have been invaluable.

A big thank you goes to the ladies of Room 2.36, past and present. You made all of this bearable and I truly appreciate the friendship each and every one of you has extended me, especially Michaela Wharfe, Rachael Crew and Lauren Butchart.

To my parents, I wish to express my heartfelt gratitude for their unwavering support in my doctoral pursuit. I could not have made it this far without their unconditional love and support.

vii

viii Table of Contents

Preface ...... iii

Abstract ...... v

Acknowledgements ...... vii

Table of Contents ...... ix

List of figures ...... xiii

List of tables...... xv

Thesis Format ...... xvii

Abbreviations ...... xix

Publications arising from this and related work ...... xxiii

Presentations ...... xxiii

Chapter 1: Introduction ...... 1

Chapter 2: Literature Review ...... 3 2.1 Reproductive Biology of the Laboratory Mouse ...... 3 2.1.1 Puberty ...... 3 2.1.2 Estrous cycle ...... 3 2.1.3 Ovulation ...... 4 2.1.4 Pregnancy ...... 4 2.1.5 Hormone levels during the estrous cycle and pregnancy ...... 5 2.2 The Hypothalamus ...... 8 2.2.1 Suprachiasmatic nucleus ...... 8 2.2.2 Anteroventral periventricular nucleus ...... 9 2.2.3 Arcuate nucleus ...... 9 2.3 The hypothalamic-pituitary-gonadal (HPG) axis ...... 10 2.4 Kisspeptin ...... 11 2.4.1 General ...... 11 2.4.2 Kisspeptin synthesis ...... 11 2.4.3 Distribution of kisspeptin cells and projections ...... 11 2.4.4 Kisspeptin receptor ...... 14 2.4.5 Kisspeptin action ...... 17 ix 2.5 Kisspeptin and Development ...... 21 2.5.1 Fetal period ...... 21 2.5.2 Neonatal to pre-pubertal/peri-pubertal period ...... 21 2.5.3 Puberty ...... 22 2.6 Circadian Rhythms ...... 23 2.6.1 Master circadian clock and molecular machinery ...... 23 2.6.2 Importance of the SCN as a central circadian pacemaker ...... 24 2.6.3 Functional anatomy of the SCN ...... 24 2.7 Circadian regulation of kisspeptin ...... 27 2.7.1 Circadian regulation of the preovulatory LH surge ...... 27 2.7.2 Pathways from the SCN to GnRH neurons ...... 27 2.7.3 Influence of the circadian system on kisspeptin neurons ...... 28 2.7.4 Circadian rhythms in the developing brain ...... 32 2.8 Kisspeptin and Pregnancy ...... 33 2.8.1 Plasma kisspeptin levels throughout gestation ...... 33 2.8.2 Kisspeptin levels and kisspeptin receptor expression in the placenta ...... 33 2.8.3 Biological actions of kisspeptin in pregnancy ...... 33 2.9 Rfamide-related peptide (RFRP) ...... 36 2.9.1 Distribution ...... 36 2.9.2 Actions ...... 36 2.9.3 RFRP-3 and development ...... 37 2.9.4 Circadian regulation of RFRP-3 ...... 37

Chapter 3: Experimental Objectives ...... 39

Chapter 4: Materials and Methods ...... 43 4.1 Animals ...... 43 4.1.1 Estrous cycle monitoring ...... 43 4.1.2 Mating ...... 43 4.1.3 Litter management ...... 44 4.2 Surgical procedures ...... 44 4.2.1 Ovariectomy ...... 44 4.2.2 Hormone replacement ...... 44 4.3 Tissue and blood sample collection ...... 45 4.4 Quantitative reverse transcription polymerase chain reaction ...... 46 4.4.1 Background ...... 46 4.4.2 Reagents ...... 46 4.4.3 Isolation of the hypothalamus ...... 47 x 4.4.4 RNA sample preparation ...... 49 4.4.5 DNase treatment to remove genomic DNA ...... 49 4.4.6 Reverse transcription ...... 50 4.4.7 Primers ...... 50 4.4.8 Real-time polymerase chain reaction (qPCR) ...... 53 4.4.9 Quantification ...... 53 4.4.10 Melt curve analysis ...... 53 4.5 Plasma hormone analyses...... 57 4.5.1 Background ...... 57 4.5.2 Reagents ...... 57 4.5.3 Protocol for measurement of pituitary hormones ...... 57 4.5.4 Protocol for measurement of steroid and thyroid hormones ...... 58 4.6 RNA-Seq ...... 62 4.6.1 Background ...... 62 4.6.2 cDNA library preparation ...... 62 4.6.3 Sequencing and analysis ...... 63 4.7 Statistical analysis ...... 65

Chapter 5: Diurnal Regulation of Hypothalamic Kisspeptin is

Disrupted During Mouse Pregnancy ...... 67 5.1 Introduction ...... 67 5.2 Materials and methods ...... 69 5.2.1 Animals ...... 69 5.2.2 Tissue collection ...... 69 5.2.3 Hypothalamic gene expression ...... 70 5.2.4 Plasma hormone analyses ...... 73 5.2.5 Statistical analysis ...... 73 5.3 Results ...... 74 5.3.1 Hormone levels ...... 74 5.3.2 Gene expression ...... 76 5.4 Discussion ...... 83

Chapter 6: Effect of Progesterone and Prolactin on the Circadian

Rhythm of Kisspeptin in Ovariectomised E2-Treated Mice ...... 89 6.1 Introduction ...... 89 6.2 Materials and Methods ...... 91 6.2.1 Animals ...... 91 6.2.2 Ovariectomy protocol ...... 91 xi 6.2.3 Tissue collection ...... 91 6.2.4 Hypothalamic gene expression ...... 91 6.2.5 Plasma analyses ...... 93 6.2.6 Statistical analysis ...... 93 6.3 Results ...... 95 6.3.1 Hypothalamic gene expression ...... 95 6.3.2 Plasma hormone levels ...... 95 6.4 Discussion ...... 100

Chapter 7: RNA Sequencing Analysis of the Mouse Anterior

Hypothalamus at Proestrus and During Pregnancy ...... 103 7.1 Introduction ...... 103 7.2 Materials and methods ...... 104 7.2.1 Animals ...... 104 7.2.2 Tissue collection ...... 104 7.2.3 Hypothalamic gene expression ...... 104 7.3 Results ...... 107 7.4 Discussion ...... 122

Chapter 8: Ontogeny of Clock and Kiss1 Gene Expression in the

Prepubertal Mouse Hypothalamus ...... 127 8.1 Introduction ...... 127 8.2 Methods ...... 129 8.2.1 Animals ...... 129 8.2.2 Tissue collection ...... 129 8.2.3 Hypothalamic gene expression ...... 129 8.2.4 Plasma hormone analyses ...... 133 8.2.5 Statistical analysis ...... 133 8.3 Results ...... 135 8.3.1 Gene expression ...... 135 8.3.2 Hormone levels ...... 141 8.4 Discussion ...... 144

Chapter 9: General Discussion ...... 149

Chapter 10: References ...... 161

xii List of figures

Figure 2.1 Relative levels of estradiol (E2) progesterone (P4), prolactin (PRL) and luteinizing hormone (LH) during the a) estrous cycle and b) pregnancy…………………………………………………………... 7

Figure 2.2 Kiss1 mRNA populations in the forebrain of the mouse………...... 13

Figure 2.3 Kisspeptin in the ARC and AVPV are differentially regulated by sex steroids………………………………………………………… 19

Figure 2.4 Key components of the circadian clock molecular machinery…….. 26

Figure 2.5 The synchronous patterns of Kiss1 neuronal activation and LH secretion in mice with constant elevated E2 levels………………… 30

Figure 2.6 Model of the role of AVPV Kiss1 neurons in generating the GnRH/LH surge……………………………………………………. 31

Figure 4.1 Ventral view of the mouse brain…………………………………… 48

Figure 4.2 a) Plot of amplification curve and b) generated standard curve for Sdha mRNA standards……………………………………………... 55

Figure 4.3 Melt curve analysis for Sdha mRNA………………………………. 56

Figure 4.4 Standard curves for a) progesterone and b) prolactin quantitation… 60

Figure 5.1 Plasma hormone concentrations in non-pregnant animals and during pregnancy…………………………………………………... 75

Figure 5.2 Kiss1 diurnal gene expression in non-pregnant animals and during pregnancy. …………………………………………………………. 78

Figure 5.3 Anterior hypothalamus Kiss1r diurnal gene expression in non- pregnant animals and during pregnancy…………………………… 79

Figure 5.4 Avp and Avpr1a diurnal gene expression in the anterior hypothalamus in non-pregnant animals and during pregnancy……. 80

Figure 5.5 Npvf (Rfrp) and Npffr (Rfrpr) diurnal gene expression in non- pregnant animals and during pregnancy…………………………… 81

Figure 5.6 Anterior hypothalamus diurnal clock gene expression in non- pregnant animals…………………………………………………… 82

Figure 5.7 Proposed model indicating the change between proestrus and late pregnancy with the role of AVPV kisspeptin neurons in generating the GnRH/LH surge………………………………………………... 88

xiii Figure 6.1 AVPV Kiss1 mRNA expression and anterior hypothalamic Kiss1r mRNA expression in OVX mice treated with estradiol (E2) only, E2 and progesterone (P4) or E2 and prolactin (PRL)…………………... 96

Figure 6.2 Avp and Avpr1a mRNA expression in the anterior hypothalamus of OVX mice treated with estradiol (E2) only, E2 and progesterone (P4) or E2 and prolactin (PRL)……………………………………... 97

Figure 6.3 Bmal1 and Rev-erbα mRNA expression in the suprachiasmatic nucleus of OVX mice treated with estradiol (E2) only, E2 and progesterone (P4) or E2 and prolactin (PRL)………………………. 98

Figure 6.4 Plasma LH, FSH and PRL concentrations in OVX mice treated with estradiol (E2) only, E2 and progesterone (P4) or E2 and prolactin (PRL)…………………………………………………….. 99

Figure 7.1 Heat map…………………………………………………………… 109

Figure 7.2 Expression of Kiss1, Kiss1r, Avp, Avpr1a, Bmal1, Rev-erbα and Npffr at proestrus and days 10 and 14 of pregnancy……………….. 115

Figure 7.3 Genes that were differentially expressed between proestrus and pregnancy…………………………………………………………… 119

Figure 7.4 Genes that were differentially expressed between ZT1/ZT5 and ZT13 at proestrus and during pregnancy…………………………… 120

Figure 7.5 Genes that were differentially expressed between ZT1/ZT5 and ZT13 at proestrus only……………………………………………… 121

Figure 8.1 Bmal1 and Rev-erbα diurnal gene expression in the anterior hypothalamus of female and male mice at P5, P15 and P25……….. 136

Figure 8.2 Kiss1 and Kiss1r diurnal gene expression in female and male mice at P5, P15 and P25………………………………………………….. 137

Figure 8.3 Avp and Avpr1a diurnal gene expression in the anterior hypothalamus of female and male mice at P5, P15 and P25……….. 139

Figure 8.4 Npvf (RFRP) and Npffr (RFRP receptor) expression in female and male mice at P5, P15 and P25……………………………………… 130

Figure 8.5 Plasma hormone concentrations in female (left panel) and male (right panel) mice at P5, P15 and P25……………………………… 142

Figure 9.1 Proposed model indicating the relative immaturity of the circadian clock and estradiol levels in the pre-pubertal female neonate compared to the adult proestrus female……………………………. 159

xiv List of tables

Table 4.1 PCR conditions...... 52 Table 4.2 CV and R2 values for all pituitary and steroid hormones measured in each experimental chapter...... 61 Table 5.1 Primer sequences and conditions for quantitative PCR...... 72 Table 6.1 Primer sequences and conditions for quantitative PCR ...... 94 Table 8.1 Primer sequences and conditions for quantitative PCR...... 132

xv

xvi Thesis Format

General This thesis is presented in ten chapters, the first four of which are the Introduction, Literature Review, Experimental Objectives and Materials and Methods. The Experimental section is comprised of the next four chapters, each one detailing a major study in the thesis. Within these chapters is presented an Introduction, Methods, Results and Discussion section as it relates to that particular study. The final two chapters provide a General Discussion of the overall findings and a list of all the References cited in this thesis. The Introduction provides an overall perspective of the thesis, including an introduction to the hypotheses and related experimental chapters. The Literature Review and Materials and Methods chapters expand on relevant background that was not included in each of the Results chapters. For the General Discussion, the major findings are reiterated and the potential for further research is considered.

Presentation of Data All of the data is presented using graphical presentations. Figures are generally presented on separate pages, and wherever possible, are placed after the text in which they are cited.

References Published work referred to in the text is cited according to Harvard (UWA Science) format, where author and year of publication are cited in text. If the number of authors exceeds three, only the first is mentioned followed by et al. and the year of publication.

xvii

xviii Abbreviations

°C degrees celsius Actb beta actin ADP AH anterior hypothalamus Alb albumin Amigo2 adhesion molecule with Ig-like domain 2 ANOVA analysis of variance AR androgen receptor ARC arcuate nucleus Arntl aryl hydrocarbon receptor nuclear translocator-like protein 1 AVP arginine vasopressin AVPV anteroventral periventricular nucleus Avpr1a arginine vasopressin receptor 1a B2m beta-2 microglobulin bHLH basic helix-loop-helix BLAST basic local alignment search tool BNST bed nucleus of the stria terminalis bp base pairs C1ra complement component 1, r subcomponent A cDNA complementary deoxyribonucleic acid Ciart circadian associated repressor of transcription Cish cytokine inducible SH2-containing protein Cldn5 claudin 5 CLOCK circadian locomotor output cycles kaput CRY cryptochrome circadian clock DAG diacylglycerol Dbp D site of albumin promoter binding protein Dec1 deleted in esophageal cancer 1 dm dorsomedial DMC dorsomedial nucleus compact part DMD dorsomedial nucleus dorsal part DMDC dimethyl dicarbonate

xix DMH dorsomedial hypothalamus DMN dorsomedial nucleus DNA deoxyribonucleic acid DNase deoxyribonuclease dNTPs dinucleotide triphosphates DYN dynorphin E embryonic day

E2 17-β-estradiol E4bp4 E4 promoter-binding protein 4 EDTA ethylenediaminetetraacetic acid ER estrogen receptor ERE estrogen response element Fgfrl1 fibroblast growth factor receptor-like 1 FSH follicle-stimulating hormone GnRH gonadotropin-releasing hormone GPR G-protein-coupled receptor Hba-a2 haemoglobin alpha, adult chain 2 Hbb-bs haemoglobin, beta adult t chain Hbb-bt haemoglobin, beta adult s chain HCl hydrochloric acid Hif3a hypoxia-inducible factor 3 alpha subunit HPG hypothalamic-pituitary-gonadal Hprt hypoxanthine-guanine phosphoribosyltransferase

IP3 inositol triphosphate Kcnh8 potassium voltage-gated channel subfamily H member 8 Kdr kinase insert domain receptor (vascular endothelial growth factor receptor) Kiss1 kisspeptin Kiss1r Kiss1 receptor KLF10 kruppel-like factor 10 KO knockout KOR kappa-opioid receptor Lap3 leucine aminopeptidase 3 LH luteinising hormone M-MLV moloney murine leukemia virus xx ME median eminence min minute(s) MMPs matrix metalloproteinases mRNA messenger ribonucleic acid n number of samples NKB neurokinin B NPFF1R neuropeptide FF receptor 1 Npvf neuropeptide VF Nr1d1 nuclear receptor subfamily 1 group D member 1 OVX ovariectomised P postnatal day P probability P4 progesterone PAR bZip proline- and acid-rich subfamily of basic region leucine zipper Parpbp poly (ADP-Ribose) polymerase 1 binding protein PAS PER-ARNT-SIM PCR polymerase chain reaction PeN periventricular nucleus PER period circadian clock PH posterior hypothalamus PKC protein kinase C PLCβ phospholipase C beta Plin4 perilipin 4 Pmch pro-melanin concentrating hormone POA preoptic area PR progesterone receptor PRL prolactin PVN paraventricular nucleus q false discovery rate adjusted p-value qRT-PCR quantitative reverse transcriptase polymerase chain reaction r2 coefficient of determination RFRP RFamide-related peptide RNA ribonucleic acid

xxi RNase ribonuclease Rorα RAR-related orphan receptor alpha RP3V rostral periventricular region of the third ventricle RPKM reads per kilobase per one million mapped reads SCN suprachiasmatic nucleus Sdha succinate dehydrogenase subunit A s second(s) SEM standard error of the mean Sgk1 serum/glucocorticoid regulated kinase Slc30a1 solute carrier family 30 (zinc transporter), member 1 TH tyrosine hydroxylase TPRC transient receptor potential canonical Tsc22d3 TSC22 domain family member 3 Ttr transthyretin VMHDM dorsomedial ventromedial hypothalamus VMHVL ventrolateral ventromedial hypothalamus

VPAC2R vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide receptor 2 VIP vasoactive intestinal polypeptide vl ventrolateral Wee1 WEE1 G2 checkpoint kinase x g times gravity Zbtb16 zinc finger and BTB domain containing 16 ZT zeitgeber time

xxii Publications arising from this and related work

Yap CC, Mark PJ, Waddell BJ and Smith JT. (2016) Ontogeny of Clock and Kiss1 Gene Expression in the Prepubertal Mouse Hypothalamus. Reproduction, Fertility and Development: Submitted and currently under review.

Yap CC, Wharfe MD, Mark PJ, Waddell BJ and Smith JT. (2016) Diurnal regulation of hypothalamic kisspeptin is disrupted during mouse pregnancy. Journal of Endocrinology: doi: 10.1530/JOE-16-0086.

Wharfe MD, Mark PJ, Wyrwoll CS, Smith JT, Yap C, Clarke MW and Waddell BJ. (2016) Pregnancy-induced adaptations of the central circadian clock and maternal glucocorticoids. Journal of Endocrinology 228:135-147.

Presentations

Invited presentation: Yap CC, Wharfe MD, Mark PJ, Waddell BJ and Smith JT. (2015) Circadian regulation of hypothalamic kisspeptin during mouse pregnancy. Institute of Metabolic Science, Addenbroke’s Hospital, Cambridge, United Kingdom

Oral presentations: Yap CC, Wharfe MD, Mark PJ, Waddell BJ and Smith JT. (2014) Circadian regulation of Kiss1 and Kiss1r during mouse pregnancy. Endocrine and Reproductive Biology Society of Western Australia Symposium, Perth

Yap CC, Wharfe MD, Mark PJ, Waddell BJ and Smith JT. (2013) Circadian regulation of Kiss1 and Kiss1r during mouse pregnancy. Annual Scientific Meeting of the Australasian Chronobiology Society, Adelaide

Poster presentations: Yap CC, Wharfe MD, Mark PJ, Waddell BJ and Smith JT. (2015) Circadian regulation of Kiss1 and Kiss1r during mouse pregnancy. Science on the Swan Conference, Perth

xxiii Yap CC, Wharfe MD, Mark PJ, Waddell BJ and Smith JT. (2014) Circadian regulation of Kiss1 and Kiss1r during mouse pregnancy. International Congress of Neuroendrinology, Sydney

Yap CC, Wharfe MD, Mark PJ, Waddell BJ and Smith JT. (2014) Circadian regulation of Kiss1 and Kiss1r during mouse pregnancy. Annual Scientific Meeting of the Society for Reproductive Investigation, Florence, Italy

Yap CC, Wharfe MD, Mark PJ, Waddell BJ and Smith JT. (2013) Circadian regulation of Kiss1 and Kiss1r during mouse pregnancy. Annual Scientific Meeting of the Endocrine Society of Australia and the Society for Reproductive Biology, Sydney

xxiv

Chapter 1 Introduction

Kisspeptin is the protein product of the Kiss1 gene. It acts directly on gonadotropin- releasing hormone (GnRH) neurons to stimulate gonadotropin release and hence drives the reproductive axis and fertility. Kisspeptin is abundant in the brain, particularly in the hypothalamus and there are two main populations of kisspeptin neurons – the arcuate nucleus (ARC) and the anteroventral periventricular nucleus (AVPV). These two populations are differentially regulated by sex steroids, indicative of their distinct roles in reproduction. The ARC kisspeptin neurons support the tonic pulsatile release of GnRH whereas AVPV kisspeptin neurons are crucial for the generation of the preovulatory luteinizing hormone (LH) surge in adult proestrus females. In rodents, the precise timing of the LH surge is governed by the suprachismatic nucleus (SCN) of the hypothalamus via the AVPV kisspeptin population, which is itself under circadian control. Previous research has demonstrated that kisspeptin neuronal activation and LH levels show synchronous circadian patterns of expression during proestrus in rodents. It is now believed that the combination of elevated estradiol (E2) levels and a circadian signal is required to activate kisspeptin neurons in the AVPV, thus stimulating GnRH neurons and resulting in the LH surge. The major objective of this thesis was to investigate the circadian expression of hypothalamic Kiss1 during neonatal/pre-pubertal development, and in adulthood and pregnancy. Four separate studies were undertaken (Chapters 5 – 8); the first investigated the circadian Kiss1 rhythm and compared it across the estrous cycle and stages of pregnancy. The second and third studies attempted to identify possible mechanisms underlying the differences in the circadian Kiss1 rhythm between the estrous cycle and pregnancy, through the investigation of hormonal effects and differential gene expression, respectively. The last study assessed the onset of the circadian Kiss1 rhythm during neonatal (pre-pubertal) life.

The circadian kisspeptin rhythm in proestrus female rodents is well documented. However, whether this rhythm persists during pregnancy, despite the absence of an LH surge, is unknown. Moreover, hypothalamic kisspeptin expression in pregnancy remains under-investigated even though circulating kisspeptin levels are known to increase dramatically in humans at this time. Chapter 5 therefore, examined the circadian

1 Chapter 1 expression of hypothalamic kisspeptin in adult female mice at various days of pregnancy and the estrous cycle.

As shown in Chapter 5, the circadian Kiss1 rhythm observed in the AVPV of proestrus females was suppressed during pregnancy in spite of high circulating E2 levels. Chapters 6 and 7 followed up on these findings, with the intention of illuminating potential mechanisms underlying this suppression. Progesterone (P4) and prolactin (PRL) are two hormones essential for the maintenance of pregnancy and are found in elevated concentrations during pregnancy, relative to before pregnancy. Given that both these hormones have demonstrated direct effects on kisspeptin expression and function, P4 and PRL may be involved in blocking the activity of AVPV kisspeptin neurons. Thus, Chapter

6 investigated the effects of P4 and PRL on the circadian rhythm of kisspeptin in ovariectomised (OVX) E2-treated mice. Chapter 7 further attempted to elucidate the mechanisms by which kisspeptin activation is prevented during pregnancy by sequencing the entire transcriptome of the mouse anterior hypothalamus, allowing us to view the overall picture of gene variability within the anterior hypothalamus at proestrus and during pregnancy, and thus identify specific genes that are differentially expressed between these two states.

Kisspeptin is clearly crucial for puberty onset and its expression is significantly increased around the time of puberty, but although kisspeptin has an established role in puberty and reproduction, it is expressed in the hypothalamus considerably earlier. In the ARC, Kiss1 mRNA has been detected as early as embryonic (E) day 13.5, whereas Kiss1 expression in the AVPV emerges at postnatal day (P) 10, around the same time that the circadian rhythms of certain clock genes in the SCN become fully developed. The circadian rhythm of kisspeptin in the AVPV is established by adulthood, but whether the onset of this rhythm occurs prior to puberty is not known. Therefore, Chapter 8 examined the circadian expression of hypothalamic Kiss1 during neonatal development in male and female mice.

Overall, the studies presented in this thesis investigated the circadian patterns of kisspeptin expression in the hypothalamus. In doing so, this thesis provides new insights into how the circadian expression of kisspeptin is regulated during pregnancy and the neonatal period.

2

Chapter 2 Literature Review

2.1 Reproductive Biology of the Laboratory Mouse The laboratory mouse is a very useful model in the field of reproductive study. It has a short ovulatory cycle and reproduces often, and the basic mechanisms that control sexual maturation and reproduction appear to be characteristic of those in humans (Oakley, Clifton & Steiner 2009). The laboratory mouse has an average life span of two to three years, and reaches sexual maturity at seven to eight weeks of age (Danneman, Suckow & Brayton 2012).

2.1.1 Puberty Puberty is characterised by a series of changes culminating in the development of reproductive functions essential for a successful pregnancy in the female, and spermatogenesis in the male. In female mice, the onset of puberty occurs at about 4 weeks of age and the first outward sign is vaginal opening, in response to increasing levels of estrogen (Pritchett & Taft 2006). Between 2 to 10 days after vaginal opening, the first estrus occurs (see section 2.1.2) and this usually signifies full sexual maturity (Pritchett & Taft 2006). In male mice, an external sign of puberty is the separation of the prepuce from the glans penis, termed preputial separation, which occurs around 6 weeks of age (Korenbrot, Huhtaniemi & Weiner 1977). A significant increase in circulating androgen levels occurs 6 – 12 days later (Korenbrot, Huhtaniemi & Weiner 1977)

2.1.2 Estrous cycle The estrous cycle consists of 4 stages (proestrus, estrus, metestrus and diestrus) characterized by various reproductive, physiologic and endocrine events, which typically occur every 4-6 days throughout the reproductive lifetime of the mouse, except during pregnancy or pseudopregnancy (Pritchett & Taft 2006). Beginning in proestrus, estrogen levels rise to reach their peak and trigger a luteinizing hormone (LH) surge that initiates ovulation (Murr, Geschwind & Bradford 1973). Elevated estrogen concentrations in the anabolic phase of the cycle stimulate cell proliferation in the vagina and result in the appearance of nucleated epithelial cells (Pritchett & Taft 2006). Ovulation occurs during

3 Chapter 2 early estrus in the dark phase and the female is receptive to the male at this time (Bingel & Schwartz 1969a). Having reached maximal thickness, if mating does not occur, the vaginal epithelium begins to shed its cornified cells (Pritchett & Taft 2006). Following the LH surge and ovulation, estrogen levels decline in metestrus. This catabolic stage of the estrous cycle sees the shedding of sheets of vaginal epithelium and the presence of leukocytes (Pritchett & Taft 2006). Finally, in diestrus, the quiescent phase of the cycle, estrogen levels remain low and the epithelial cells that built up during proestrus have been completely shed. As the cycle nears proestrus once again, growth in the vaginal epithelium begins anew (Pritchett & Taft 2006).

2.1.3 Ovulation Prolonged exposure to high levels of estrogen trigger a surge of gonadotropin-releasing hormone (GnRH) and in turn, LH early in the afternoon of proestrus (Murr, Geschwind & Bradford 1973). A subsequent cascade of events culminates in ovulation during estrus approximately 12 hours later (Bingel & Schwartz 1969a). The timing of the LH surge and thus, ovulation, is influenced by light-dark cycles and is therefore under circadian control (Pritchett & Taft 2006; See section 2.6).

2.1.4 Pregnancy

Mouse gestation is typically 19-21 days in length (Murray et al. 2010). Progesterone (P4) is critical for the maintenance of pregnancy and it is produced by the ovarian corpora lutea throughout this period. During mating, stimulation of the vagina triggers a neural response that initiates surges of prolactin (PRL) essential for sustaining corpora lutea (Barkley, Geschwind & Bradford 1979; Terkel 1986). Midway through gestation, around embryonic day (E) 11-12, placental lactogens take over from PRL in the role of maintaining pregnancy, through their ability to stimulate corpora luteal production of P4 (Thordarson et al. 1997; Malassine, Frendo & Evain-Brion 2003). Parturition is initiated by the fetus, and though the mechanisms underlying the signals are not fully understood, it is dependent on a decrease in P4 concentrations brought upon by the demise of the corpora lutea (Virgo & Bellward 1974; Pritchett & Taft 2006).

In what is termed post-partum ovulation, mice ovulate approximately 12 to 26 hours after parturition depending on the time of delivery, with the interval shortening the later the delivery occurred during the day (Bingel & Schwartz 1969b). Female mice may mate and

4 Chapter 2 fall pregnant during the post-partum estrus, but if the mouse is lactating then implantation of the pregnancy is delayed between one and seven days (Mantalenakis & Ketchel 1966).

2.1.5 Hormone levels during the estrous cycle and pregnancy

2.1.5.1 Estradiol (E2)

Plasma E2 levels (Figure 2.1) are lowest on the day of estrus then gradually increase over metestrus and diestrus, finally reaching a peak on proestrus, just prior to the LH surge

(Butcher, Collins & Fugo 1974; Smith, Freeman & Neill 1975). In pregnancy, E2 concentrations remain relatively stable during the first half of gestation, then continually increase from mid- to late pregnancy, before declining two days prior to parturition (Barkley, Geschwind & Bradford 1979).

2.1.5.2 Luteinizing hormone (LH) Plasma LH levels (Figure 2.1) remain very low throughout most of the estrous cycle until the afternoon of proestrus, when levels rapidly surge and decline quickly to base line by the early morning of estrus (Butcher, Collins & Fugo 1974; Smith, Freeman & Neill

1975). During the estrous cycle, LH promotes follicular development and enhances E2 secretion from the ovary, while in pregnancy, it stimulates luteal development and P4 synthesis (Pritchett & Taft 2006). During pregnancy, LH concentrations are generally low and begin to increase in the two days leading up to the peaks, which occur on days 3 and 10 of pregnancy. In the second half of pregnancy, LH levels are relatively low and surge again only on the day of parturition (Murr, Bradford & Geschwind 1974).

2.1.5.3 Follicle-stimulating hormone (FSH) Plasma FSH levels are low for most of the estrous cycle and start to rise in the afternoon of proestrus, peaking within a few hours of the LH surge, and return to baseline by noon of estrus (Butcher, Collins & Fugo 1974; Smith, Freeman & Neill 1975). FSH concentrations are high on day 0 of pregnancy and drop thereafter, remaining at low levels during the preimplantation period. After implantation, levels of FSH fluctuate but remain relatively low, before increasing sharply on the day of parturition (Murr, Bradford & Geschwind 1974).

5 Chapter 2

2.1.5.4 Progesterone (P4)

In the estrous cycle, P4 secreted from the corpus luteum (Figure 2.1) increases slowly from metestrus to reach a peak in the morning of diestrus before dropping sharply. Values remain low until the afternoon of proestrus, when progesterone levels rapidly increase, coinciding with the LH surge (Butcher, Collins & Fugo 1974; Smith, Freeman & Neill

1975). The drop off in P4 levels on the morning of diestrus indicates the regression of the corpus luteum. However, if a cervical stimulus is applied or in the event of pregnancy, the corpus luteum continues to secrete P4, in which case P4 levels continue to rise through day 2 of pregnancy (or pseudopregnancy), instead of decreasing (Butcher, Collins & Fugo 1974; Smith, Freeman & Neill 1975).

In early pregnancy, plasma P4 levels rise steadily until day 10, when a significant decrease in P4 secretion occurs. This decline in circulating P4 correlates with the cessation of the biphasic PRL surges (see section 2.1.5.5). Thereafter, plasma P4 continues to rise until very late in pregnancy, when levels then decline precipitously to trigger parturition (Murr et al. 1974; Virgo & Bellward 1974; Barkley, Geschwind & Bradford 1979; Waddell, Bruce & Dharmarajan 1989).

2.1.5.5 Prolactin (PRL) PRL concentrations (Figure 2.1) are low through metestrus and diestrus, and increase rapidly on the afternoon of proestrus, around the time of the LH surge. Levels then decline to baseline by the morning of estrus (Butcher, Collins & Fugo 1974; Smith, Freeman & Neill 1975). A second PRL peak may occur sometime between the late morning and afternoon of estrus (Taya & Igarashi 1973; Butcher, Collins & Fugo 1974). For the first half of pregnancy, PRL is responsible for the maintenance of the corpus luteum and its continued P4 secretion in response to cervical stimulation (Malassine, Frendo & Evain-Brion 2003). In early pregnancy, plasma PRL levels are high and exhibit a biphasic pattern of secretion, with one nocturnal and one diurnal surge. These biphasic surges are terminated after day 8 of pregnancy, corresponding with the initiation of placental lactogen secretion, which takes over the luteotrophic functions of PRL (Smith & Neill 1976). PRL concentrations are low throughout mid- to late pregnancy, then surge immediately before parturition, and decrease once again on the day of parturition (Morishige, Pepe & Rothchild 1973; Murr, Bradford & Geschwind 1974). Post-partum PRL concentrations remain low until a sharp increase occurs on day 4 of lactation, and high levels are maintained until day 10. Subsequently, PRL levels fall and remain low until the end of lactation (Taya & Sasamoto 1981). 6 Chapter 2

Figure 2.1 Relative levels of estradiol (E2), progesterone (P4), prolactin (PRL) and luteinizing hormone (LH) during the A) estrous cycle and B) pregnancy. Adapted from Butcher, Collins & Fugo 1974; Smith, Freeman & Neill 1975; Barkley, Geschwind & Bradford 1979 and Murr, Bradford & Geschwind 1974.

7 Chapter 2 2.2 The Hypothalamus Most organisms have the need to maintain homeostasis in an ever-changing and challenging external environment. Thus, one of the main functions of the hypothalamus is to regulate the body’s internal environment in response to the external environment. This is a complex process involving the successful integration of endocrine, visceral and somatomotor control mechanisms, systems to which the hypothalamus provides an essential interface (Simerly, 2004).

The hypothalamus is situated below the thalamus at the base of the forebrain, and is connected to the pituitary gland by a network of blood vessels termed the hypophyseal portal system, which allows endocrine communication between the two (Bear, Connors, & Paradiso, 2006). The ability of the hypothalamus to integrate information from the forebrain, brainstem and spinal cord, gives it a large degree of control over whole body physiology (Purves et al., 2007).

The hypothalamus is generally divided into functional zones: periventricular, medial and lateral (Crosby & Woodburne, 1940). The periventricular zone houses neurons known as parvocellular neurosecretory cells, which release hypophysiotropic hormones that act on the anterior pituitary and regulate the secretion of hormones (Bear et al., 2006; Simerly, 2004). Rather than extending their axons directly into the pituitary, these hypothalamic neurons secrete hormones into the hypophyseal portal system (Bear et al., 2006; Purves et al., 2007).

Of the many areas in the hypothalamus, the periventricular zone will be the focus of this literature review, with three nuclei of particular interest. The subsequent sections will provide information specifically relating to these nuclei.

2.2.1 Suprachiasmatic nucleus A group of small, densely packed neurons constitutes the suprachiasmatic nucleus (SCN), which receives direct retinal innervation and has been shown to be vital to the regulation of circadian rhythms in mammals (Rusak & Zucker, 1979; Simerly, 2004). See section 2.6.3 for more detail on the structure and function of the SCN.

8 Chapter 2 2.2.2 Anteroventral periventricular nucleus The anteroventral periventricular nucleus (AVPV) consists of an oval cluster of densely packed neurons, which has been identified as having a central role in controlling gonadotropin secretion (Simerly, 2004). The AVPV receives numerous inputs, particularly from within the periventricular zone and other hypothalamic nuclei, as well as from the amygdala, hippocampus and brainstem nuclei. The strongest projections from the AVPV are to other nuclei in the periventricular zone and the hypothalamus (Simerly, 2004).

2.2.3 Arcuate nucleus The arcuate nucleus (ARC) is situated caudally to the AVPV in the mediobasal hypothalamus, along the base of the third ventricle. The majority of neurons in the ARC contain hypophysiotropic hormones that are secreted from terminals in the median eminence and travel to the anterior pituitary via the hypophyseal portal system (Simerly, 2004), and as such plays a large role in homeostatic neuroendocrine mechanisms. Inputs to and outputs from the ARC are mostly confined to the periventricular zone, particularly the AVPV.

9 Chapter 2 2.3 The hypothalamic-pituitary-gonadal (HPG) axis

The HPG axis regulates reproduction and fertility, and key to this system are hypothalamic GnRH, as well as the pituitary gonadotropins LH and FSH, the release of which is stimulated by the pulsatile secretion of GnRH (Roa et al. 2009). In the ovary, FSH, as its name suggests, stimulates follicular maturation and the production of estrogen through aromatisation of androgens, while LH facilitates this process by stimulating androgen production in thecal cells (Themmen & Huhtaniemi 2013). LH also triggers ovulation and subsequently aids in the differentiation and maintenance of the corpus luteum. In the testis, the role of FSH is still relatively unclear but it appears to act directly on Sertoli cells to govern spermatogenesis, while LH stimulates androgen production in Leydig cells (Themmen & Huhtaniemi 2013).

The secretion of GnRH itself is governed by gonadal steroids, which exert mostly negative but also positive feedback effects (Naftolin et al. 2007; Herbison 2008). In both sexes, the gonadal steroids produced in response to GnRH/LH secretion primarily inhibit GnRH release, thus forming the negative feedback loop that prevails for much of the ovulatory cycle (Naftolin et al. 2007). In females, however, there is a small window just prior to ovulation, during which rising E2 levels stimulate GnRH secretion and generate the preovulatory LH surge that triggers ovulation, thus forming a positive feedback loop (Herbison 2008).

The specific neuronal pathways that govern the negative and positive feedback effects of estrogen remained elusive for decades, particularly because GnRH neurons were not thought to express sex steroid receptors, implying that any action of sex steroids on GnRH neurons was mediated by an intermediary neuronal population situated in the hypothalamus (Herbison & Pape 2001; Wintermantel et al. 2006). That neuronal population has now been identified as kisspeptin-containing neurons (discussed in the following section). Some studies have shown that GnRH neurons express estrogen receptor (ER) β (Hrabovszky et al. 2001, 2007; Kalló et al. 2001), although this particular subtype does not appear to have much of a role in estrogen feedback (Wintermantel et al. 2006; Radovick, Levine & Wolfe 2012).

10 Chapter 2 2.4 Kisspeptin 2.4.1 General KISS1 was initially discovered as a human metastasis-suppressor gene in 1996 (Lee et al. 1996). In 1999, the then orphan G-protein coupled receptor GPR54 was identified in the rat (Lee et al. 1999), and is now known as Kiss1r due to its role as a kisspeptin receptor. The human homolog of GPR54, KISS1R (previously known as AXOR12), was discovered shortly thereafter, along with the link between KISS1R and its ligand KISS1 (Kotani et al. 2001; Muir et al. 2001; Ohtaki et al. 2001). Then in 2003, two research groups made independent and almost simultaneous discoveries that loss-of-function mutations in the human kisspeptin receptor were linked to idiopathic hypogonadotropic hypogonadism and impaired pubertal maturation (de Roux et al. 2003; Seminara et al. 2003). Studies of mice bearing targeted disruptions of Kiss1r exhibited reproductive developmental abnormalities very similar to those of their human counterparts, supporting kisspeptin-KISS1R signaling as being vital for normal reproduction (Funes et al. 2003; Seminara et al. 2003).

2.4.2 Kisspeptin synthesis The KISS1 gene, located on the long arm of chromosome 1, encodes a 145-amino acid pro-peptide, from which shorter kisspeptin proteins are cleaved (West et al. 1998; Kotani et al. 2001). The longest, active kisspeptin product in humans is a 54-amino acid protein is known as kisspeptin-54, and along with the shorter peptides kisspeptin-10, -13, and - 14, together they are termed kisspeptins (Oakley, Clifton & Steiner 2009). All kisspeptins bind to KISS1R with equal affinity and efficacy, owing to the conserved sequence of 10 amino acids at the C terminal-end, and although they are all biologically active, the precise in vivo relevance of the shorter peptides is as yet unknown (West et al. 1998; Kotani et al. 2001; Muir et al. 2001). The shorter peptides are thought to be the products of kisspeptin-54 cleavage (Kotani et al. 2001).

2.4.3 Distribution of kisspeptin cells and projections Kisspeptin expression has been localised in the brain, in particular to the hypothalamus and pituitary, and expression is also particularly abundant in the placenta (Muir et al. 2001). In the adult rodent brain, the ARC and AVPV are the two hypothalamic areas in which kisspeptin-responsive neurons are most abundant (Gottsch et al. 2004; Smith et al. 2005a; 11 Chapter 2 see Figure 2.2). These findings were supported and expanded upon in a later study by Clarkson and colleagues (2009), who mapped the distribution of kisspeptin- immunoreactive cell bodies and fibres in the adult mouse brain. Three populations of kisspeptin-immunoreactive cell bodies were identified, the largest being located in the continuum of cells comprising the AVPV and periventricular preoptic nucleus, collectively termed the rostral periventricular region of the third ventricle or RP3V (Clarkson et al. 2009b; Hanchate et al. 2012). The second most abundant population was located in the ARC (Clarkson et al. 2009b; Hanchate et al. 2012), and the third was relatively less densely distributed throughout the dorsomedial nucleus (DMN) and posterior hypothalamus (Clarkson et al. 2009b), possibly as continuations of the ARC population. Kisspeptin immunoreactive fibres were concentrated within the hypothalamus, particularly surrounding kisspeptin cell bodies in the AVPV and periventricular preoptic nuclei in the RP3V, as well as in the ARC, from which a dense collective of fibres extended (Clarkson et al. 2009b; Kalló et al. 2012). Kisspeptin fibres were also found in the supraoptic, paraventricular, periventricular and dorsomedial nuclei, in the internal zone of the median eminence, the ventral aspect of the lateral septum and the preoptic area (Clarkson et al. 2009b). Outside of the hypothalamus, kisspeptin immunoreactive fibres were relatively scarce, detected only in the bed nucleus of the stria terminalis (BNST), anterior portion of the paraventricular thalamus, medial amygdala, periaqueductal grey, and around the locus coeruleus (Clarkson et al. 2009b). Studies in rats show that the distribution of kisspeptin cell bodies and fibres in the adult rat brain mirrors that of the mouse (Desroziers et al. 2010; Xu et al. 2012).

In the sheep, Kiss1/kisspeptin cells are present in large clusters in the ARC, POA and dorsomedial hypothalamus (DMH) (Estrada et al. 2006; Franceschini et al. 2006; Smith et al. 2007). Kisspeptin fibre distribution is relatively scattered but high densities are found in the ARC, POA, DMH and the median eminence (Franceschini et al. 2006). Kisspeptin distribution in the human brain has also been characterised and groups of kisspeptin neurons have been identified that are analogous to the ARC and AVPV populations in the rodent. The largest population of kisspeptin cells resides in the infundibular area and this probably corresponds to the ARC in rodents (Rometo et al. 2007; Hrabovszky et al. 2010). A smaller group of cells in the rostral periventricular area likely corresponds to the rodent AVPV (Hrabovszky et al. 2010). Immunoreactive fibres were most abundant around the third ventricle, including the periventricular nucleus (PeN), paraventricular nucleus (PVN), infundibulum and DMH (Hrabovszky et al. 2010).

12 Chapter 2

Figure 2.2 Kiss1 mRNA populations in the forebrain of the mouse. Kiss1 mRNA- expressing cells, as reflected by red dots in the top schematic and white clusters of silver grains in the bottom photomicrographs, in the A) anteroventral periventricular nucleus (AVPV) and B) arcuate nucleus (ARC). Adapted from Smith et al. 2005a and Smith, Clifton & Steiner 2006.

13 Chapter 2 2.4.4 Kisspeptin receptor Kiss1r is part of the G protein-coupled receptor (GPCR) superfamily (Kotani et al., 2001; Muir et al., 2001), which can be divided into three main classes: the rhodopsin-, secretin- and metabotropic glutamate receptor-like families (Marchese et al. 1999). The kisspeptin receptor, which is part of the rhodopsin family, has a secondary structure consisting of seven transmembrane domains, shared by all members of the GPCR superfamily.

2.4.4.1 Distribution and development In the mouse hypothalamus, Kiss1r is usually found on GnRH neurons, and double-label in situ hybridisation experiments reveal that the percentage of GnRH neurons expressing Kiss1r ranges from 77% to >90% (Irwig et al. 2004; Han et al. 2005). GnRH neuron sensitivity to kisspeptin increases gradually over development; from 27% of GnRH neurons responding to kisspeptin stimulation at P8 – P19, to 44% at P26 – P33, and finally to 90% in adult male mice (Han et al. 2005).

2.4.4.2 Signal transduction The binding of kisspeptin to its receptor stimulates G protein-activated phospholipase C

β (PLCβ), leading to the generation of inositol triphosphate (IP3) and diacylglycerol (DAG), second messenger molecules that signal the release of intracellular Ca2+ and activation of calcium-dependent protein kinase C (PKC), respectively (Stafford et al. 2002; Constantin et al. 2009). These second messengers are also thought to mediate the activation of transient receptor potential canonical (TPRC)-like channels and the inhibition of inwardly rectifying potassium channels, allowing kisspeptin to stimulate GnRH secretion (Stafford et al. 2002; Constantin et al. 2009).

2.4.4.3 Mutation of kisspeptin and kisspeptin receptor genes in rodents Observations from studies of genetically engineered mice bearing loss-of-function mutations in the Kiss1r gene bear remarkable similarities to those from human studies; both human subjects and their rodent counterparts demonstrate a failure to progress to puberty, low circulating sex steroids, reduced pituitary gonadotropin levels, underdeveloped gonads and infertility (Funes et al. 2003; Seminara et al. 2003). All four independently developed strains of Kiss1r knockout (KO) mice show largely the same phenotype with slight variations.

One of the first Kiss1r KO strains was generated by Paradigm Therapeutics in 2003, with a deletion of 702 base pairs in total from exons 1 and 2, and the entire intron 1 of the 14 Chapter 2 Kiss1r gene (Seminara et al. 2003). In the same year, a second strain was designed by Schering-Plough Research Institute that was missing a portion of exon 2 (Funes et al. 2003). Characterisation of another two strains of Kiss1r KO mice occurred in 2007. One strain, developed by the Harvard Reproductive Endocrine Sciences Center, is missing the entire exon 2 (Lapatto et al. 2007). The other strain, developed by Omeros Corp., was generated by inserting a retrovirus into the Kiss1r gene, thereby preventing its transcription without any deletions in the coding sequence (Dungan et al. 2007; Kauffman et al. 2007b; Dror, Franks & Kauffman 2013).

Although all Kiss1r KO strains of mice displayed the same characteristics such as absent pubertal maturation, low levels of sex steroids and complete infertility, female mice from the viral targeting lineage reportedly responded to E2 treatment with GnRH neuronal activation and an LH surge (Dungan et al. 2007). In contrast, female mice of the Paradigm Therapeutics strain were unable to show activation in GnRH neurons or mount an LH surge in response to sex steroid replacement protocols (Clarkson et al. 2008). Since the only females that responded to E2 treatment were from a strain generated by retroviral insertion without any deletions in the Kiss1r coding sequence, it is possible that residual gene expression could account for the persistence of the LH surge in these animals (Clarkson et al. 2008). However, a more recent study by Dror et al. (2013) subjected Kiss1r KO mice from this same strain to four different LH surge-inducing hormonal paradigms, and found that they were unable to produce LH surges in response to any single one. The authors suggest that the discrepancy between their study and that of Steiner and colleagues (2007) could be due to the latter’s flawed qualification of a surge. In the Dungan study, mean LH surge levels were relatively low, and furthermore, GnRH neuronal activation appeared to be unusually high in the morning, with values much closer to evening levels than is typical (Dungan et al. 2007; Dror, Franks & Kauffman 2013).

A new strain of Kiss1r KO mice was created by Lexicon Pharmaceuticals in 2012 using the Cre-LoxP technique, where one animal bearing LoxP sites flanking the Kiss1r gene, is crossed with a second animal expressing the Cre recombinase enzyme, thus yielding a mouse strain in which the Kiss1r gene has been excised from all cells (Gavériaux-Ruff & Kieffer 2007; García-Galiano et al. 2012). Tena-Sempere and colleagues (2012) reported that this strain exhibited a severe hypogonadotropic hypogonadism, and that administration of glutamate receptor agonists and galanin-like peptide, but not a

15 Chapter 2 neurokinin B agonist, were able to elicit an LH response independently of kisspeptin signalling. Taking into account all the evidence to date, it would seem that kisspeptin- Kiss1r signalling is indeed mandatory for positive feedback induction of the LH surge and ovulation in mice.

The exact cellular location of of kisspeptin-Kiss1r signalling has also been determined using two different genetically modified mouse models. The first, a GnRH neuron- specific Kiss1r KO mouse, was infertile, failed to go through puberty and was unable to respond to kisspeptin stimulation (Kirilov et al. 2013). The second model selectively re- introduced Kiss1r to GnRH neurons in global Kiss1r KO mice, resulting in the restoration of kisspeptin-Kiss1r signalling, as well as fertility in these animals (Kirilov et al. 2013). The combination of these two phenotypes provides convincing evidence that kisspeptin- Kiss1r signalling specifically at GnRH neurons is both necessary and sufficient for fertility and reproduction in mice.

Mice lacking the Kiss1r ligand, kisspeptin, have also been studied; the year 2007 saw the development of two such strains. The strain generated by the Harvard Reproductive Endocrine Sciences Center contained a deletion of exon 1 from the Kiss1 gene (Lapatto et al. 2007), while the strain generated by Paradigm Therapeutics contained deletions in exons 1 and 2 (d’Anglemont de Tassigny et al. 2007). Both strains exhibited a phenotype that paralleled the Kiss1r KO mice, whereby Kiss1 null mice failed to reach sexual maturation and were infertile (d’Anglemont de Tassigny et al. 2007; Lapatto et al. 2007). However, direct comparison of the Kiss1 and Kiss1r KO models revealed that the kisspeptin-deficient mice suffered from a less severe reproductive phenotype that appeared to be intermediate between wild-type and receptor-deficient animals (Lapatto et al. 2007). This phenotypic variability was particularly evident in Kiss1 KO females; about half of the mice had small vaginal openings and small ovaries akin to Kiss1r null animals, whereas the other half had larger vaginal openings and larger ovaries that more closely resembled their wild-type counterparts (Lapatto et al. 2007). Thus, although it appears that kisspeptin-Kiss1r signalling is necessary for full reproductive function, the relatively moderate phenotype of Kiss1 KO mice suggests that either a peptide other than kisspeptin may interact with Kiss1r, or that the receptor may exhibit some constitutive activity (Lapatto et al. 2007; Chan et al. 2009).

16 Chapter 2 2.4.5 Kisspeptin action Numerous studies in rodents have shown that kisspeptin directly stimulates GnRH neurons to enhance GnRH secretion (Han et al. 2005), leading to subsequent gonadotropin release, particularly LH (Gottsch et al. 2004; Irwig et al. 2004; Kauffman et al. 2007b). Moreover, blocking GnRH signalling with an antagonist or antibody abolishes kisspeptin-induced LH secretion in female rodents (Gottsch et al. 2004; Kinoshita et al. 2005), indicating that kisspeptin does not stimulate gonadotropin release via direct action on the pituitary, and this was subsequently confirmed in sheep (Smith et al. 2008). Altogether these findings indicate that kisspeptin plays an important role in generating LH secretion through its action on GnRH neurons.

2.4.5.1 Kisspeptin in the AVPV is involved in the preovulatory GnRH/LH surge

The AVPV is involved in driving E2-mediated positive feedback induction of the LH surge, because this region not only communicates with GnRH neurons, but lesions to this area result in acyclicity and an absence of E2-induced surges (Wiegand et al. 1980; Herbison 2008). Multiple lines of evidence now support the AVPV kisspeptin population as being the conduit for positive feedback signals to reach GnRH neurons. First, Kiss1 expression in the AVPV decreases in ovariectomised (OVX) animals, but increases in response to E2 treatment (Smith et al. 2005a, 2006). Second, while GnRH neurons lack ERα, the subtype that mediates positive feedback, virtually all Kiss1 neurons in the AVPV express ERα (Smith et al. 2005a, 2006; Adachi et al. 2007). Third, Kiss1 neurons in the AVPV are transcriptionally activated at the time of the LH surge in proestrus females or OVX females treated with E2, but not in diestrus or OVX females (Smith et al. 2006; Adachi et al. 2007; Robertson et al. 2009). Fourth, male rodents do not exhibit LH surges (Buhl, Norman & Resko 1978) and have correspondingly smaller numbers of AVPV Kiss1 neurons than females (Clarkson & Herbison 2006; Kauffman et al. 2007a), suggesting that the purpose of the larger AVPV Kiss1 population in females may be to generate the LH surge (see Figure 2.3).

Interestingly, elevated levels of E2 accompanied by an increase in Kiss1 expression are not necessarily sufficient to generate an LH surge; OVX animals given E2 treatment showed high AVPV Kiss1 levels but not circulating LH in the morning (Smith et al.

2005a). At proestrus, although E2 is at constantly elevated levels, AVPV Kiss1 expression increases significantly from the morning to the evening (Smith et al. 2006). Furthermore,

OVX + E2 treated rodents show a preovulatory-like release of LH in the evening. This 17 Chapter 2 indicates that other inputs, specifically, circadian signals, also regulate Kiss1 expression in the AVPV (see section 2.6).

2.4.5.2 Kisspeptin in the ARC as the GnRH pulse generator The ARC has been previously linked to negative feedback of sex steroids (Soper & Weick 1980; Simonian, Spratt & Herbison 1999), and the kisspeptin population in this region is likely responsible for the tonic release of GnRH. Like kisspeptin neurons in the AVPV, ARC kisspeptin neurons also express ERα, along with androgen receptor (AR) (Smith et al. 2005a, 2006). In contrast to AVPV Kiss1 expression, however, OVX females demonstrate increased Kiss1 expression in the ARC, in association with elevated LH secretion, that is reversed with E2 replacement (Smith et al. 2005a, 2005b). Likewise in males, castration causes an increase in Kiss1 expression that is inhibited by testosterone (Smith et al. 2005b). It has also been proposed that neurokinin B (NKB) and dynorphin (DYN), which are coexpressed in virtually all ARC Kiss1-expressing neurons, act autosynaptically to coordinate the pulsatile discharge of kisspeptin from the ARC (Navarro et al. 2009). Therefore, kisspeptin neurons in the ARC are well-equipped to receive estrogen negative feedback signals and regulate the activity of GnRH neurons to maintain tonic secretion of GnRH and LH (see Figure 2.3).

18 Chapter 2

Figure 2.3 Kisspeptin in the ARC and AVPV are differentially regulated by sex steroids. Kisspeptin from the ARC and AVPV stimulate GnRH release, causing LH and FSH release from the pituitary, which in turn, induces gonadal secretion of sex steroids. The sex steroids then feed back onto kisspeptin neurons, negatively regulating Kiss1 expression in the ARC, while positively regulating its expression in the AVPV. From Dungan, Clifton & Steiner 2006.

19 Chapter 2 2.4.5.3 Differential regulation of Kiss1 in the ARC and AVPV

The mechanism underlying the differential regulation of Kiss1 by E2 in different brain regions has not been identified, but a few possibilities have been raised.

Phenotypic differences between the two kisspeptin populations could account for their differing responses to E2. As previously mentioned, nearly all ARC kisspeptin neurons also express NKB and DYN and their respective receptors, while AVPV neurons do not (Navarro et al. 2009). Conversely, in mice, 80% of kisspeptin neurons in the AVPV coexpress tyrosine hydroxylase (TH), but no TH expression is observed in the ARC (Semaan et al. 2010). However in the rat, only 5-30% of Kiss1 neurons in the AVPV coexpress TH, depending on E2 levels, and TH neurons and kisspeptin neurons are evidently distinct populations (Kauffman et al. 2007a).

Another potential explanation for the differential responses could lie in different estrogen signalling pathways. It is known that ERα signalling can result in an array of cellular effects, dependent on the particular pathway activated. In the classical pathway, E2 binds to estrogen response elements (EREs) in the gene promoter region of target genes (eg. Kiss1) to alter transcription; this is known as an ERE-dependent mechanism (Björnström & Sjöberg 2005). The non-classical signalling pathway is thus termed due to the involvement of ERE-independent mechanisms, such as protein-protein interactions at heterologous response elements (Björnström & Sjöberg 2005). Genetically modified mice have been developed which allow analysis of ERE-dependent and -independent signalling pathways in vivo. Studies utilizing these mice demonstrate that classical pathways are required for positive feedback regulation of Kiss1 in the AVPV, while non- classical pathways mediate negative feedback in the ARC (Glidewell-Kenney et al. 2007; Gottsch et al. 2009).

There is also limited evidence to suggest that epigenetic regulation of the Kiss1 gene by

E2 underpins its differential effects on the ARC and AVPV. High levels of histone acetylation were present on the Kiss1 promoter region in the AVPV; conversely in the ARC, histones were strongly de-acetylated on the Kiss1 promoter region (Tomikawa et al. 2012).

20 Chapter 2 2.5 Kisspeptin and Development Kisspeptin is considered to be a critical component of puberty onset, becoming highly activated around this time. Despite its function in activating puberty, kisspeptin is also expressed in the brain well before this important developmental stage.

2.5.1 Fetal period Kisspeptin mRNA is detectable in the fetal mouse brain as early as E13.5, but only in the ARC (Fiorini & Jasoni 2010), and this increases substantially by E17 (Knoll et al. 2013). Recent work has revealed that functional neural circuits between ARC kisspeptin neurons and Kiss1r-expressing GnRH neurons are established before birth (Kumar et al. 2014), although the purpose of potential kisspeptin signalling at this time is unknown.

2.5.2 Neonatal to pre-pubertal/peri-pubertal period Kiss1 mRNA is present in the mouse ARC at birth and expression does not change within the first 20 hours of life (Poling et al. 2012). Although Kiss1 levels in ARC are not different between male and female rodents in adulthood, data suggests there is a sexual dimorphism that exists at birth and early postnatal life that converges closer to puberty (Cao & Patisaul 2011; Takumi, Iijima & Ozawa 2011; Poling et al. 2012); Kiss1 expression in female rats decreases around 3 weeks of age to more closely match that of the male (Cao & Patisaul 2011; Takumi, Iijima & Ozawa 2011). Similarly, Semaan and Kauffman (2015) found that in the female mouse ARC, total Kiss1 mRNA is higher at postnatal day (P) 15 than P20-25.

The ontogeny of kisspeptin expression differs quite significantly between the AVPV and the ARC. While Kiss1 expression in the ARC is higher in early postnatal life than closer to puberty, Kiss1 in the AVPV exhibits the opposite pattern. Kiss1-expressing neurons have been detected in the AVPV in mice as early as P10; sex differences are not apparent at this age but emerge a few days later at P12, with twice the number of Kiss1 cells in females compared to males (Semaan et al. 2010). Kiss1 levels then increase steadily from P15 to past puberty (Semaan & Kauffman 2015). Kisspeptin protein is first detectable at P15 (Clarkson et al. 2009a), and kisspeptin cell bodies and fibres, as well as kisspeptin fibre appositions to GnRH neurons, are present at P25 (Clarkson & Herbison 2006). The number of kisspeptin cells rapidly increases thereafter, with female mice exhibiting 10- fold greater expression than males in adulthood (Clarkson & Herbison 2006).

21 Chapter 2

2.5.3 Puberty Kiss1 mRNA expression in the entire rat hypothalamus is significantly elevated around the time of puberty in both sexes (Navarro et al. 2004). In female rats at P26, 3-4 days before vaginal opening, Kiss1 mRNA expression in the ARC and AVPV increased more than four-fold compared to P21. Kisspeptin immunoreactivity also increased considerably from P26 to P31, after vaginal opening (Takase et al. 2009). Both ARC and AVPV Kiss1 mRNA levels in male rats were significantly higher at P45 than at P15, and higher at P45 than at P60 in the AVPV alone (Bentsen et al. 2010). For females, the developmental increase in AVPV kisspeptin neurons, in particular, appears to be contingent upon the action of E2, as female mice with pre-pubertal OVX see a 70% reduction in AVPV kisspeptin neurons at P30, compared to intact mice (Clarkson et al. 2009a).

While it is generally accepted that kisspeptin plays a crucial role in pubertal maturation, the observations from an experiment by Mayer and Boehm (2011) contradict this widely- held belief. They created a strain of mice that co-expressed diphtheria toxin with the Kiss1 gene, which resulted in the elimination of all Kiss1-expressing cells. Unexpectedly, the lack of Kiss1 neurons from early in development did not affect reproductive function. In fact, mutant females showed the same timing of vaginal opening as control females, regular estrous cycles, normal conception rates and they delivered typical-size litters; the only exception was a significant reduction in ovarian mass (Mayer & Boehm 2011). A complementary strain of mutant mice, featuring the loss of all cells expressing the kisspeptin receptor, exhibited a remarkably similar phenotype to mice lacking Kiss1 neurons (Mayer & Boehm 2011). These surprising results suggest that either kisspeptin signalling is expendable for puberty or that a small number of Kiss1- and Kiss1r- expressing cells are sufficient for driving the HPG axis and fertility (Popa et al. 2013). The latter is a possibility due to a very small percentage of Kiss1- and Kiss1r-expressing cells remaining after ablation with diphtheria toxin (Mayer & Boehm 2011). However, inducing the loss of Kiss1 neurons just before the onset of puberty at P20 causes acyclicity and infertility (Mayer & Boehm 2011). Thus potentially, elimination of kisspeptin cells in embryonic life initiates the development of compensatory reproductive neural circuits, and that this is possible only before puberty (Mayer & Boehm 2011).

22 Chapter 2 2.6 Circadian Rhythms Most biological processes, from gene expression to overt behavior, occur in circadian rhythms that are governed by circadian clocks, which ensure that these processes are coordinated to the 24-hour day (Reppert & Weaver 2001). The SCN is entrained by a range of periodic environmental cues, but the light-dark cycle is the most powerful and ubiquitous among them (Reppert & Weaver 2001). Circadian clocks are self-sustaining and endogenous, meaning that even if the organism is deprived of cues from the external world, these circadian rhythms can persist for weeks and beyond (Hastings, O’Neill & Maywood 2007). As mentioned briefly, the SCN of the hypothalamus is the seat of the master circadian clock responsible for the generation of circadian rhythms, and governs peripheral clocks present in nearly all tissues including skeletal muscle, liver and lungs (Yamazaki et al. 2000).

2.6.1 Master circadian clock and molecular machinery The molecular machinery driving circadian clocks comprises interacting positive and negative transcriptional-translational feedback loops, of which there is a core circadian oscillator and additional accessory loops (Figure 2.4).

In the negative-feedback loop, transcription of the Period (Per1-3) and Cryptochrome (Cry1-2) genes are driven by the transcription factors CLOCK and BMAL1, which form a heterodimer (Reppert & Weaver 2001). Both CLOCK and BMAL1 contain basic helix- loop-helix (bHLH) motifs that bind DNA, and PER-ARNT-SIM (PAS) domains that facilitate protein-protein interactions (Hastings, O’Neill & Maywood 2007). The CLOCK/ BMAL1 heterodimer activates transcription from the Per and Cry loci. The translated PER and CRY proteins form complexes in the cytoplasm and translocate to the nucleus, where they inhibit the transcriptional activity of the CLOCK/ BMAL1 heterodimer, thereby negatively regulating their own production (Reppert & Weaver 2001). While CRY proteins dominate the negative feedback loop, PER proteins are more involved in positive transcriptional regulation. Simultaneously, in the positive-feedback loop, PER2 augments the transcription of Bmal1. This regeneration of BMAL1 promotes CLOCK:BMAL1 dimerisation, restarting Per/Cry transcription, and thus allows the clock cycle to continue (Reppert & Weaver 2001). This describes the core circadian loop.

23 Chapter 2 Accessory loops exist that augment and stabilize the core oscillatory loop. One such pathway involves the orphan nuclear receptor proteins Rev-erbα and Rorα (Hastings, O’Neill & Maywood 2007). While each of their transcription is driven by the CLOCK/ BMAL1 heterodimer, Rev-erbα represses Bmal1 expression while Rorα positively regulates it. The end result is that Bmal1 levels are low during the day but high at night, therefore oscillating in anti-phase with Per/Cry (Preitner et al. 2002; Sato et al. 2004).

Downstream of the core loop are a range of clock-controlled genes, including transcriptional regulators such as D site of albumin promoter binding protein (Dbp), deleted in esophageal cancer 1 (Dec1) and E4 promoter-binding protein 4 (E4bp4), which control a whole host of circadian-regulated genes involved in numerous physiological processes (Asher & Schibler 2011; Waddell et al. 2012; see Figure 2.4).

2.6.2 Importance of the SCN as a central circadian pacemaker Multiple lines of evidence have established the importance of the SCN as the master circadian pacemaker. First, lesions to the SCN in rodent models result in the disruption of physiological and behavioural circadian rhythms (Moore & Eichler 1972; Stephan & Zucker 1972; Janssen et al. 1994). Second, rat SCN explants cultured for weeks in vitro are able to maintain rhythmicity in both electrical activity and vasopressin release, demonstrating the self-sustaining nature of this circadian oscillator (Earnest & Sladek 1986; Bos & Mirmiran 1990; Murakami et al. 1991). Moreover, the transplantation of these cells into the brains of SCN lesioned rodents can restore circadian rhythms (Ralph, Joyner & Lehman 1993). Lastly, the SCN receives direct light input from melanopsin- expressing photoreceptors in the retina, allowing light to synchronise circadian rhythms (Moore, Speh & Card 1995; Hattar et al. 2006). Disruption to the normal light schedule impacts rhythmicity in the SCN and many other peripheral clocks (Yamazaki et al. 2000; Fonken et al. 2010; Varcoe et al. 2011).

2.6.3 Functional anatomy of the SCN The SCN is anatomically and functionally divided into two parts with differing architecture and connections; the ventrolateral (vl) core and the dorsomedial (dm) shell (Abrahamson & Moore 2001). The core receives direct light input through the retinohypothalamic tract and communicates this information to the shell (Abrahamson & Moore, 2001). The SCN as a whole sends projections to numerous other areas within the

24 Chapter 2 hypothalamus, including the preoptic area (POA), PeN and PVN (Abrahamson & Moore 2001; Deurveilher & Semba 2005), which enable the regulation of the circadian rhythms of gonadotropins and metabolic hormones, among others (Hastings, O’Neill & Maywood 2007). Several neuropeptides are responsible for disseminating information both within and from the SCN; vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP) are two that have been implicated in the regulation of circadian rhythms (Khan & Kauffman 2012). The SCN core synthesises and releases VIP, while the shell expresses AVP (Card et al. 1988). VIP appears to be essential for the communication of light information within the SCN, as well as circadian information to downstream tissues (Vosko et al. 2007). Indeed, the importance of VIP-signalling is demonstrated by the disrupted circadian rhythms or total arrhythmicity observed in VIP null mice (Colwell et al. 2003). Unlike VIP, AVP does not seem to be necessary for endogenous rhythm generation (Groblewski, Nunez & Gold 1981), although it exhibits circadian rhythmicity both in vitro and in vivo (Reppert et al. 1981; Murakami et al. 1991). However, AVP may be able to evoke excitatory responses in SCN neurons (Liou & Albers 1989; Mihai et al. 1994). Additionally, AVP-containing projections from the SCN extend to various regions of the hypothalamus including the PVN, POA, AVPV and DMN (Dai et al. 1998; Abrahamson & Moore 2001), supporting the role of AVP as a critical output signal of the SCN, responsible for regulating circadian rhythms such as that of corticosterone (Kalsbeek et al. 1992, 1996).

Both VIP and AVP have also been implicated in reproductive function. Blocking VIP signalling with a VIP antiserum significantly delayed and attenuated the proestrus LH surge in female rats (Van der Beek, Swarts & Wiegant 1999). Moreover, VIP null female mice present some degree of infertility; they exhibit irregular estrous cycles and produce half the number of offspring as their wild-type counterparts (Loh et al. 2014). Similarly, administering an AVP receptor antagonist results in the diminution of the proestrus LH surge (Funabashi et al. 1999), and female Brattleboro rats (incapable of synthesising vasopressin) are sub-fertile with abnormal estrous cycles (Boer, Boer & Swaab 1981).

25 Chapter 2

Figure 2.4 Key components of the circadian clock molecular machinery. The core loop comprises transcription of the Period (Per1, Per2, Per3) and Cryptochrome (Cry1, Cry2) genes driven by the transcription factors CLOCK and BMAL1, and subsequent auto-inhibition of Per and Cry. Accessory loops stabilise the core loop through positive and negative regulation by the nuclear orphans receptors Rorα and Rev-erbα, respectively. Together, the core and accessory loops drive the transcription of target genes via clock-controlled genes, such as E4 promoter-binding protein 4 (E4bp4), basic helix-loop-helix (bHLH), proline- and acid-rich subfamily of basic region leucine zipper (PAR bZip) and Kruppel-like factor 10 (KLF10). From Waddell et al. 2012.

26 Chapter 2 2.7 Circadian regulation of kisspeptin 2.7.1 Circadian regulation of the preovulatory LH surge There is now overwhelming evidence that the timing of the preovulatory LH surge in female rodents is tightly controlled by the master circadian clock in the SCN. The first of this evidence was observed over sixty years ago, when a series of seminal experiments showed that the LH surge occurs consistently in the afternoon of proestrus, and that administration of dibenamine, atropine or neumbutal during proestrus blocks the LH surge for approximately 24 hours (Everett, Sawyer & Markee 1949; Everett & Sawyer 1950). These data highlighted the involvement of circadian input in the events leading up to the LH surge. Second, ovariectomised (OVX) rats implanted with E2 capsules, to induce constant levels of elevated E2, exhibit a daily LH surge just prior to the dark phase (Legan & Karsch 1975; Legan, Coon & Karsch 1975), further supporting circadian involvement in the surge. Third, investigation of the temporal patterns of LH levels and c-fos expression in SCN and GnRH neurons demonstrate that SCN neurons are activated immediately before GnRH neurons and the LH surge (Tsukahara 2006). Finally, lesions of the SCN, as well as mutations in the Clock gene, result in the disruption of the LH surge and ovulation (Brown-Grant & Raisman 1977; Wiegand et al. 1980; Miller et al. 2004).

2.7.2 Pathways from the SCN to GnRH neurons Although evidence of circadian control of the LH surge is robust, indications that GnRH neurons receive circadian information through direct connections from the SCN is comparatively weak. GnRH neurons that express the VIP receptor VPAC2R are directly targeted by VIP neurons from the SCN core (Van der Beek et al. 1997; Smith, Jennes & Wise 2000). It is therefore possible that VIP provides the circadian input to initiate the GnRH/LH surge and some data exists to support this notion; VIP-innervated GnRH neurons are activated at the time of the LH surge, and antagonising VIP signalling in the SCN delays and diminishes the surge (Van der Beek et al. 1994; Harney et al. 1996).

Further evidence points to an indirect pathway linking the SCN to GnRH neurons, and involves AVP-expressing neurons from the SCN shell projecting to the AVPV, which in turn projects to GnRH neurons (Leak & Moore 2001; Kriegsfeld et al. 2004; Herbison 2008). Firstly, the AVP receptor V1a is expressed in the AVPV and estrogen has a stimulatory effect on its expression (Funabashi et al. 2000; Kalamatianos et al. 2004). 27 Chapter 2

Secondly, vasopressin is capable of producing an LH surge in SCN-lesioned, OVX, E2- treated rats, as well as in 50% of Clock mutant mice, both of which characteristically do not display LH surges (Palm et al. 1999; Miller et al. 2006). Thirdly, the prevention of, or lack of AVP signalling as seen in Brattleboro rats, results in attenuation of the LH surge and sub-fertility, respectively (Boer, Boer & Swaab 1981; Funabashi et al. 1999). Lastly, desynchronisation of the dm and vl regions of the SCN in rats demonstrates that the dmSCN, rather than the vlSCN, exhibits a rhythm that is always in phase with the LH surge, indicating that the circadian signals timing the LH surge arise from the dmSCN and accordingly, are transmitted via AVP-ergic neurons (Smarr, Morris & de la Iglesia 2012).

2.7.3 Influence of the circadian system on kisspeptin neurons With the bulk of the evidence pointing towards AVP-ergic cells providing indirect circadian input to GnRH neurons, kisspeptin neurons in the AVPV are the most obvious link between them, due to the fact that they are indispensable for the LH surge, as well as the for connectivity of the AVPV with the SCN and GnRH neurons (Herbison 2008).

The most convincing evidence is that the circadian patterns of kisspeptin mRNA expression and neuronal activation have been shown to coincide with the pattern of LH secretion (Robertson et al. 2009). To demonstrate this synchrony, female mice were first entrained to a 14 : 10 h light/dark cycle, OVX and given E2 replacement, and then transferred into constant darkness for two days before being killed. These mice showed an LH surge in the subjective late afternoon/early evening (defined as circadian time (CT) 11 and 12, where CT12 denotes the onset of locomotor activity), co-incident with peaks in both Kiss1 mRNA expression and cfos/Kiss1 co-expression occurring at the same time (Robertson et al. 2009; see Figure 2.5). A later study replicated these findings in rats; Kiss1 mRNA expression in the AVPV peaked at the time of the LH surge, but not if the SCN was lesioned (Smarr, Morris & de la Iglesia 2012). Taken together, these data strongly suggest that the timing of the LH surge is controlled by the circadian activation of AVPV Kiss1 neurons.

There is also robust evidence to support the theory that kisspeptin neurons in the AVPV receive circadian information through AVP neurons from the SCN. Two studies in female mice and hamsters report similar observations, with both showing AVP-ergic fibres (but not VIP fibres) from the SCN in apposition with kisspeptin neurons in the AVPV (Vida et al. 2010; Williams 3rd et al. 2011). Significantly, AVP immunoreactive fibre 28 Chapter 2 appositions on kisspeptin neurons were abolished when the hamsters received SCN lesions (Williams 3rd et al. 2011). Moreover, the AVP receptor V1a has been detected in

AVPV Kiss1 neurons (Williams 3rd et al. 2011), and shows E2-dependent circadian expression in the AVPV (Smarr, Gile & de la Iglesia 2013), further supporting the theory that the circadian pattern of kisspeptin neurons in the AVPV is regulated by the SCN via AVP signalling.

Interestingly, the study by Robertson et al (2009) also showed that mice that were OVX but not treated with E2 did not display the same circadian patterns of LH, Kiss1 gene expression or neuronal activation that were seen in OVX + E2 mice. This implies that the activation of AVPV kisspeptin neurons in the late afternoon of proestrus is dependent on high levels of E2. This data has been corroborated by Smarr et al (2013), although the circadian expression of Kiss1 in this study using rats was blunted rather than eliminated in the absence of E2. In another level of regulation, GnRH neurons appear to include a gating mechanism of control by exhibiting time-dependent sensitivity to kisspeptin stimulation. Williams et al. (2011) showed that kisspeptin administration was able to activate GnRH neurons much more effectively in the afternoon than in the morning, thus restricting the LH surge to the late afternoon. GnRH cells demonstrate circadian rhythms (Zhao & Kriegsfeld 2009; Hickok & Tischkau 2010), and accordingly may modulate sensitivity to kisspeptin stimulation throughout the day, either by changing kisspeptin receptor expression or inhibitory tone (Williams 3rd et al. 2011).

The current model of how the AVPV generates the LH surge in an adult non-pregnant female rodent is that the AVPV acts as an integration centre for circadian signals from the SCN and estrogenic signals, and the combination of these two inputs leads to the release of kisspeptin, that ultimately gives rise to the LH surge (Smarr, Gile & de la Iglesia 2013; see Figure 2.6). However, it is not known how the feedback organisation of kisspeptin expression, or if the circadian expression of kisspeptin in the AVPV or ARC, is altered during pregnancy.

29 Chapter 2

Figure 2.5 The synchronous patterns of Kiss1 neuronal activation and LH

secretion in mice with constant elevated E2 levels. A) Photomicrographs of Kiss1 mRNA (visualised with Vector Red substrate) and c-fos co-expression (represented by silver grains) in the AVPV. B) Percentage of Kiss1 neurons in the AVPV coexpressing c-fos, indicating neuronal activation, peaking at CT11 – CT12. C) Serum LH levels peaking at CT11 – CT12. Adapted from Robertson et al. 2009.

30 Chapter 2

Figure 2.6 Model of the role of AVPV Kiss1 neurons in generating the GnRH/LH surge. Kiss1 neurons in the AVPV receive circadian information from the SCN via vasopressin receptors V1a, and sense circulating estradiol levels via estrogen receptors (ER), located on these neurons. Kiss1 neurons are activated in the event that estrogenic and circadian signals are both high, releasing kisspeptin and activating GnRH neurons via kisspeptin receptors GPR54, which in turn drives the release of GnRH, and subsequently LH. Adapted from Smith et al. 2006.

31 Chapter 2 2.7.4 Circadian rhythms in the developing brain Development of the rodent SCN is a gradual process and much of it occurs postnatally, compared to prenatally in humans, non-human primates or sheep (Serón-Ferré et al. 2012). In the rat, formation of the SCN takes place from E14 through to E17, but synaptogenesis is largely a postnatal event. Late in gestation, relatively few synapses are formed, but this is followed by a rapid increase in synaptic number between P2 and P10 (Moore & Bernstein 1989). By P10, synaptic density is equivalent to that of an adult, although total synaptic number continues to grow until early adulthood (Moore & Bernstein 1989).

Rhythms in glucose utilisation (determined by 2-deoxyglucose uptake), Avp mRNA and neuronal firing rate are detectable in the SCN even as early as the late embryonic stage (Reppert & Schwartz 1984; Reppert & Uhl 1987; Shibata & Moore 1987). Despite the appearance of these intrinsic rhythms, it is unclear whether they are truly driven by the clock gene machinery of the fetal SCN, or simply a response to cyclic maternal signals (Sumová et al. 2008). A functional central clock requires the core clock genes to be expressed in a rhythmic fashion, and current evidence suggests that the fetal SCN may not be capable of generating endogenous rhythms. Studies in rats and mice show that although clock genes are expressed during fetal life, coordinated oscillatory expression does not develop until postnatally, and rhythmicity of some clock genes (namely the Pers and Crys) appear to be established by P10 (Shimomura et al. 2001; Sladek et al. 2004; Kováciková et al. 2006; Ansari et al. 2009).

While kisspeptin expression and clock gene rhythms in the neonatal mouse brain have been studied, little is known about the development of the circadian rhythms of kisspeptin. The rhythmicity in kisspeptin expression at proestrus is thought to develop around time of puberty, coincident with LH surge development, but the precise period of onset is unknown.

32 Chapter 2 2.8 Kisspeptin and Pregnancy 2.8.1 Plasma kisspeptin levels throughout gestation In humans, circulating kisspeptin levels are profoundly increased throughout pregnancy and evidence indicates a placental origin for the peptide (Horikoshi et al. 2003), although the role of high levels of plasma kisspeptin during pregnancy is still unclear. A study in pregnant women demonstrated that kisspeptin levels rose 900-fold during the first trimester and this further increased to 7000-fold by the third trimester, but returned to normal levels within 5 days of delivery (Horikoshi et al. 2003). Plasma kisspeptin levels during rodent pregnancy have not been studied.

2.8.2 Kisspeptin levels and kisspeptin receptor expression in the placenta The spatial expression of kisspeptin and KISS1R is similar in both the human and rodent placenta. Kiss1 mRNA is expressed in human extravillous trophoblasts and in rodent trophoblast giant cells, both of which are responsible for early invasion of the maternal decidua (Bilban et al. 2004; Terao et al. 2004).

Human placental trophoblasts express higher levels of Kiss1 (Bilban et al. 2004) and Kiss1r (Janneau et al. 2002; Bilban et al. 2004) in the first trimester than at term. A study in rat placenta showed that Kiss1 expression was highest at E12.5 and gradually decreased to undetectable levels by E18.5 (Terao et al. 2004). These data seemingly show that placental kisspeptin expression contrasts with circulating kisspeptin levels, and is maximally expressed at a time that coincides with peak trophoblast invasion, suggesting that kisspeptin may be involved in restraining this process. In contrast, more recent studies in rodents have found that placental Kiss1 and Kiss1r increase over gestation (Mark et al. 2013; Herreboudt et al. 2015), consistent with the slowing of placental growth particularly towards term.

2.8.3 Biological actions of kisspeptin in pregnancy The previously discussed findings on placental kisspeptin expression, combined with in vitro evidence that kisspeptin inhibits trophoblast migration and invasion (Bilban et al. 2004; Roseweir, Katz & Millar 2012; Francis et al. 2014), while increasing trophoblast adhesion (Taylor et al. 2014), indicate that kisspeptin likely plays a role in the implantation process through regulation of trophoblast invasion. 33 Chapter 2 However, the evidence from studies investigating pregnancies associated with poor placentation is conflicting. Higher Kiss1 mRNA and protein levels have been found in placental tissue from women with pre-eclampsia (a serious placental disorder associated with poor trophoblast invasion into maternal spiral arteries) compared to women with normal pregnancies (Qiao et al. 2005; Vazquez-Alaniz et al. 2011; Zhang et al. 2011). Conversely, low circulating kisspeptin levels in early to mid-pregnancy have also been found in women who delivered growth restricted neonates and/or suffered pre-eclampsia (Smets et al. 2008; Armstrong et al. 2009; Logie et al. 2012); this would seem to contradict the proposed role of kisspeptin in inhibiting invasion, since these conditions are associated with reduced invasive capacity. There are a few theories to explain the contradictory findings; it is possible that low placental kisspeptin expression is indicative of low invasive capacity (Smets et al. 2008), or that the development of less invasive placentas precedes the low production of kisspeptin. All currently available data regarding altered kisspeptin levels in pre-eclampsia is correlative, and so it cannot be determined whether altered kisspeptin levels are a cause of the disease, or merely a consequence.

Nevertheless, there is experimental evidence to suggest that kisspeptin is directly involved in the processes of implantation and placentation. Matrix metalloproteinases (MMPs) are tissue remodelling factors that govern cell behaviour, such as cell division, migration and morphogenesis (Mott & Werb 2004). Kisspeptin has been shown to inhibit the expression and activity of these critical factors in vitro (Yan, Wang & Boyd 2001; Yoshioka et al. 2008; Francis et al. 2014). Kisspeptin may also have a role in angiogenesis, which is essential for establishing the placenta, as it has been shown to mediate vasoconstriction in coronary arteries (Mead et al. 2007).

Moreover, Kiss1 null female mice show a failure to implant despite gonadotropin and E2 treatment, which restored ovulation and fertilization, indicating that kisspeptin signalling is required for a successful pregnancy (Calder et al. 2014). Further study confirmed that kisspeptin signalling in the uterus plays a role in regulating embryo implantation in the mouse (Fayazi et al. 2015). Intriguingly, a recent study characterising Kiss1 mutant placentas showed that neither placental structure nor function was affected by the lack of Kiss1 expression (Herreboudt et al. 2015), contradicting numerous findings that kisspeptin regulates placental trophoblast invasion, and hence is important for placental development (Bilban et al. 2004; Roseweir, Katz & Millar 2012; Francis et al. 2014;

34 Chapter 2 Taylor et al. 2014). However, the authors acknowledge the possibility that since the mothers were heterozygous for Kiss1, maternal kisspeptin could have had an effect on placental development (Herreboudt et al. 2015).

Another potential role for kisspeptin in pregnancy involves the stimulation of oxytocin secretion, the levels of which rise throughout gestation (Otsuki 1983), and are important for parturition and lactation (Russell 2003). It has been shown that intravenous administration of kisspeptin-10 increases both plasma oxytocin levels and the firing rate of oxytocin neurons in female rats (Kotani 2001, Scott and Brown 2011). Thus, high circulating kisspeptin levels during pregnancy may serve to activate the oxytocin system at this time (Scott and Brown 2013).

35 Chapter 2 2.9 Rfamide-related peptide (RFRP) RFamide-related peptides (RFRPs) belong to the family of arginine-phenylalanine-amide (RFamide) peptides long known for their neuroendocrine functions; indeed, kisspeptin is also a member of this family. RFRP-3, the mammalian orthologue to avian gonadrotropin-inhibitory hormone, in particular, has been shown to have significant effects on the neuroendocrine control of reproduction and is proposed as a key inhibitor of the HPG axis (Kriegsfeld 2006). The actions of RFRP-3 are mediated through its receptor NPFF1R, also known as GPR147 or RFRPR (Yoshida et al. 2003).

2.9.1 Distribution In rodents, RFRP-3 is exclusively expressed in the dorsal-medial nucleus (DMN) of the hypothalamus (Kriegsfeld 2006; Smith et al. 2010), and its fibres project to a number of areas in the brain, including the ARC, PVN, lateral hypothalamus, and the POA, where they contact GnRH neurons (Ukena & Tsutsui 2001; Gibson et al. 2008).

2.9.2 Actions Central and peripheral administration of RFRP-3 suppresses LH secretion and inhibits GnRH neuronal activity in rodents (Kriegsfeld et al. 2006; Anderson et al. 2009; Ducret, Anderson & Herbison 2009). Due to its inhibitory effects on GnRH neurons and direct connections with the SCN, RFRP-3 is proposed to regulate the negative feedback effects of estrogen that dominate most of the ovulatory cycle (Kriegsfeld 2006; Gibson et al. 2008; Ducret, Anderson & Herbison 2009; Williams 3rd & Kriegsfeld 2012).

RFRP-3 may also have a part to play in relaying and/or “permitting” E2-positive feedback to GnRH neurons. Using Syrian hamsters, Gibson et al. (2008) demonstrated that both the number of RFRP cells and RFRP activity were greatest during diestrus and reduced on the day of proestrus, returning to diestrus levels after the surge. Interestingly, this pattern of activity and expression of RFRP is reciprocal to that observed for kisspeptin, but both require the presence of estrogen, suggesting that there exists a fine balance between these two systems operating throughout the ovulatory cycle.

Although the findings of Gibson et al (2008) suggest that RFRP-3 is inhibited by elevated estrogen, studies looking into the effects of E2 on RFRP in female rodents have yielded remarkably inconsistent results, ranging from decreased Rfrp levels (Molnár et al. 2011;

36 Chapter 2 Poling et al. 2012), to increased Rfrp levels (Iwasa et al. 2012), to having no effect at all (Quennell et al. 2010).

A recent study by Tena-Sempere and colleagues (2014) using a RFRPR KO mouse model supports the role of RFRP-3 as an inhibitor of gonadotropic function in rodents, but also demonstrates the expendable role of RFRP-3 in puberty and fertility. The removal of inhibitory signalling resulted in higher LH levels in pubertal males but did not affect pubertal timing. Fertility was also unaffected, in fact, RFRPR null mice had an increased litter size compared to wild type animals (León et al. 2014).

2.9.3 RFRP-3 and development Unlike kisspeptin expression in the AVPV, Rfrp expression in adult mice is not sexually dimorphic (Poling et al. 2012). The lack of difference in Rfrp expression between the sexes potentially downplays the purported role that RFRP-3 plays in the sexually dimorphic LH surge in female rodents (Gibson et al. 2008). In both sexes, there is a substantial decline in the number of Rfrp-expressing cells between birth and P20, and a smaller decrease between P20 and adulthood (Poling et al. 2012; Semaan & Kauffman 2015).

Only a small subset of GnRH neurons (15%) express Rfrpr suggesting that RFRP-3 may either exert its effects directly on GnRH neurons via Rfrpr, or indirectly via other GnRH- regulating pathways (Poling et al. 2012). However, this low co-expression of Rfrpr in GnRH neurons is inconsistent with data from Ubuka et al. (2012) in male Siberian hamsters who observed a much higher co-expression figure of 80%. Kauffman and colleagues (2012) also discovered that Rfrp-expressing cells are split into two subpopulations; a small group expressing very high levels of Rfrp mRNA (high- expressing), and a larger group with approximately 3-fold lower Rfrp mRNA levels (low- expressing). These two populations are present in similar numbers in both sexes, but exhibit different developmental patterns; high-expressing cells increase in number from birth to P20 and decrease substantially in adulthood, whereas the number of low- expressing cells decreases steadily from birth through to adulthood (Poling et al. 2012).

2.9.4 Circadian regulation of RFRP-3

SCN projections to RFRP-3 neurons in the DMN, along with the E2-dependent temporal regulation of RFRP-3 neurons (Gibson et al. 2008), suggests that RFRP-3 may also be 37 Chapter 2 involved in the circadian regulation of the LH surge. How the SCN communicates circadian information to RFRP-3 neurons is still somewhat unclear, but a recent study proposes the involvement of VIP-signalling (Russo et al. 2015). Immunofluorescence showed that VIP inhibits RFRP-3 neuronal activity in a time-dependent manner, with Fos expression in RFRP-3 neurons being reduced in response to afternoon but not morning injections (Russo et al. 2015). Moreover, RFRP-3 cells were observed to express PER1 in a rhythmic fashion, potentially providing a mechanism by which these cells coordinate their responsiveness to VIP signaling from the SCN (Russo et al. 2015). Both VIP and AVP fibres from the SCN contacted RFRP cells, however the proportion of cells receiving these projections was small (10% and 15%, respectively), and only a small minority of RFRP-3 neurons were found to express VIP receptors (Russo et al. 2015). Altogether, the results suggest that the RFRP-3 system does not receive circadian information directly from the SCN; rather, there likely exists an intermediary system that is stimulated by VIP-signalling, which in turn inhibits RFRP-3 neurons (Russo et al. 2015).

38

Chapter 3 Experimental Objectives

The overall aim of the experiments presented in this thesis was to investigate the circadian expression of hypothalamic Kiss1 and other key neuroendocrine genes, from neonatal life, through to pre-puberty and adulthood, as well as during pregnancy. As detailed in the previous chapter, kisspeptin is essential for pubertal maturation, fertility and reproduction. Although hypothalamic kisspeptin expression has been studied during the fetal, neonatal and pubertal periods, the circadian variation in kisspeptin levels, and in particular the onset of the circadian kisspeptin rhythm that is seen in adult proestrus females, has yet to be examined. It is known that in females kisspeptin exhibits a circadian-controlled increase at proestrus around the time of the LH surge, but whether this pattern of expression is preserved at all during pregnancy is unknown. Therefore, using the mouse as an experimental model, the circadian expression of Kiss1 in the hypothalamus was investigated during various developmental milestones, in adulthood and during pregnancy. For this thesis, four separate studies were undertaken and the specific experimental objectives of each are outlined below.

Study 1 (Chapter 5): Diurnal regulation of hypothalamic kisspeptin is disrupted during mouse pregnancy The objective of this study was to examine circadian Kiss1 expression in the hypothalamus in pregnant mice and compare it to that in non-pregnant mice. Expression of Kiss1 and Kiss1r, the clock genes Bmal1 and Rev-erbα, the carrier of circadian information Avp and its receptor Avpr1a, as well as the proposed GnRH inhibitor Rfrp and its receptor Rfrpr, was determined in the hypothalamus by real-time quantitative RT- PCR. Plasma pituitary and steroid hormone concentrations were also measured using multiplex assays. These analyses, performed in four-hour intervals, enabled the observation of a circadian profile of Kiss1 mRNA, and of the various signalling pathways that ultimately lead to the LH surge. In addition, this study allowed the comparison of the Kiss1 circadian profile between proestrus and pregnancy. We hypothesised that the normal circadian rhythm of hypothalamic Kiss1 is altered during pregnancy, leading to the suppression of gonadotropin secretion and ovulation in the pregnant state.

39 Chapter 3

Study 2 (Chapter 6): Effect of progesterone and prolactin on the circadian rhythm of kisspeptin in ovariectomised E2-treated mice Study 1 (Chapter 5) showed that during pregnancy, the circadian pattern of Kiss1 expression that is seen in a proestrus female is abolished, in spite of elevated E2 levels and intact circadian clock gene rhythms. P4 and PRL are two hormones that have direct actions on kisspeptin neurons in the AVPV, and greatly increase in concentration during pregnancy, therefore potentially suppressing the circadian-controlled increase in Kiss1 expression at this time. This study investigated the effects of P4 and PRL on Kiss1 expression in isolation by administering ovariectomised E2-treated mice, which experience a daily LH surge, either P4 or PRL, to mimic the concentrations of these hormones in pregnancy. Kiss1, Kiss1r, Bmal1, Rev-erbα, Avp, Avpr1a, Rfrp and Rfrpr mRNA expression was determined once again by real-time quantitative RT-PCR.

Analysis of the circadian expression of hypothalamic Kiss1 in the presence of elevated P4 or PRL concentrations enabled the assessment of whether either (or both) of these hormones is responsible for the suppression of kisspeptin activation during pregnancy.

We hypothesised that mice receiving E2 only treatment will exhibit an evening increase in Kiss1 mRNA expression, which is abolished by additional treatment with P4 and PRL.

Study 3 (Chapter 7): RNA Sequencing analysis of the mouse anterior hypothalamus at proestrus and during pregnancy Study 1 (Chapter 5) showed that the peak in Kiss1 mRNA expression that occurs in the late afternoon of proestrus is suppressed during pregnancy. Elevated E2 levels in pregnancy suggest that disruption of the circadian signal to AVPV kisspeptin neurons is a likely cause. The alteration may lie within the AVPV and it is possible that the rhythmicity of other genes in this region show differential expression patterns between proestrus and pregnancy, similar to Kiss1. This study sequenced the entire transcriptome of the mouse anterior hypothalamus and identified differentially expressed genes between proestrus and pregnancy, with the aim of shedding light on the mechanism underlying the suppression of the kisspeptin circadian rhythm during pregnancy.

Study 4 (Chapter 8): Ontogeny of clock and Kiss1 gene expression in the neonatal (pre-pubertal) mouse hypothalamus Kisspeptin expression has been studied during fetal and neonatal development, but it is not yet known precisely when the onset of the kisspeptin circadian rhythm occurs, but it has been assumed to take place after puberty. Oscillatory expression of clock genes does 40 Chapter 3 not develop until after birth and rhythmicity of some clock genes is not established until P10. The objective of this study was to examine circadian Kiss1 expression during neonatal/pre-pubertal life and investigate the onset of the kisspeptin circadian rhythm that is observed in the adult proestrus female. Analyses of clock gene expression, Kiss1, Kiss1r, Avp, Avpr1a, Rfrp and Rfrpr and plasma levels of pituitary and steroid hormones, allowed us to determine whether a kisspeptin circadian rhythm is present or even possible before puberty onset. We hypothesised that an adult-like kisspeptin circadian rhythm would not be present in female neonatal or pre-pubertal mice.

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Chapter 4 Materials and Methods

4.1 Animals Nulliparous C57BL/6J mice (6 weeks old) were supplied by the Animal Resources Centre (Murdoch, Western Australia, Australia) and housed at the Preclinical Animal Facility, The University of Western Australia. Mice were maintained in an environmentally controlled room (mean temperature 21°C, average humidity 55%) under a 12-hour light, 12-hour dark cycle (lights on 0700 – 1900 h) with ad libitum access to rat and mouse cubes (Specialty feeds, Glen Forrest, Australia) and acidified tap water (0.008% HCl; pH 2.5-3). Mice were housed four to five per cage prior to mating, and then housed individually upon during mating and confirmation of pregnancy.

4.1.1 Estrous cycle monitoring Female mice were subjected to a daily vaginal smear to determine estrous cycle stage and monitored for three full cycles. Vaginal smears were collected through vaginal lavage using a pipette with 5 µL of saline, and were mounted on glass slides. The slides were placed under a light microscope with 10X and 20X objective lens to identify cellular morphology and indicate the estrous cycle stage. The proestrus stage vaginal smear is predominated by nucleated epithelial cells, estrus is characterised by cornified epithelial cells with no visible nucleus, metestrus usually contains a mix of leucocytes and cornified or nucleated epithelial cells, while diestrus consists primarily of leucocytes (Pritchett & Taft 2006).

4.1.2 Mating A subset of female mice in experiments 1 and 3, and all female mice in experiment 4 were mated. Female mice in proestrus were removed from their home cage and placed with a male overnight. Pregnancy was confirmed by observation of a vaginal plug the following morning, which was designated day 1 of pregnancy.

43 Chapter 4 4.1.3 Litter management Female mice in experiment 4 carried their pregnancies to term and delivered. Parturition in C57Bl/6J mice typically occurs on the morning of day 19, designated postnatal day 1. Within 12 h of birth, all offspring were sexed by examination of external genital morphology. Pups remained with their mother until they were killed for tissue collection.

4.2 Surgical procedures 4.2.1 Ovariectomy Mice were bilaterally ovariectomised (OVX) under isoflurane anaesthesia (induction 5%, maintenance 2.5%) in oxygen (1.5 L/min). The mouse’s dorsal surface was shaved and cleaned before making a 1.5 cm wide incision in the skin approximately halfway between the caudal rib and the top of the pelvis. The skin was blunt dissected away from the muscle wall and the ovary visualised through the muscle. The ovary was exteriorised through a small incision made in the muscle wall and the proximal uterine horn was clamped. The ovary was then severed and the ligated stump placed back within the peritoneal cavity. The muscle incision was sutured with 4-0 vicryl suture (Ethicon, Somerville, New Jersey, USA) and the procedure was repeated on the opposite side. The skin incision was closed with sterile wound clips. A subcutaneous injection of buprenorphine (1 mg/kg) was administered immediately after surgery. Mice were allowed to recover on a heat pad before being returned to their cages.

4.2.2 Hormone replacement Micro-osmotic pumps (Alzet, Cupertino, California, USA; Model# 1002) containing ovine PRL (Sigma-Aldrich, St Louis, Missouri, USA; Cat# L6520) were prepared according to the manufacturer’s instructions. Each pump contained 400 µg of PRL dissolved in 100 µL of saline. The solution was drawn up into a 1 mL syringe, which was then attached to a blunt-tipped 27-gauge filling tube (included with the micro-osmotic pumps). With the flow moderator removed, the filling tube was inserted into the pump and the syringe depressed slowly, in order to prevent the formation of air bubbles. Once the pump was full, the filling tube was removed and the flow moderator was fully inserted into the body of the pump to seal it.

At the time of OVX, mice were implanted with a 0.1 mg E2 pellet (Innovative Research of America, Sarasota, Florida, USA; Cat# NE-121), either alone, or in combination with

44 Chapter 4 a 25 mg P4 pellet (Innovative Research of America, Sarasota, Florida, USA; Cat# P-131) or a PRL pump. At the dorsal incision, the skin was blunt dissected away from the body and using a pair of forceps, the P4 pellet or PRL pump was inserted subcutaneously just below the shoulder blades. The E2 pellet was inserted subcutaneously posterior to the initial skin incision. Mice were given one week to recover from surgery before being killed for tissue collection.

4.3 Tissue and blood sample collection By convention in chronobiology, lights off at 1900 h was defined as Zeitgeber time (ZT) 12, with sampling times described as relative to ZT12. Collections were carried out at 0800 (ZT1), 1200 (ZT5), 1600 (ZT9), 2000 (ZT13), 2400 (ZT17) and 0400 (ZT21), or at 0800 (ZT1) and 1800 (ZT11), under isoflurane anaesthesia (induction 5%, maintenance 2.5%) in oxygen (1.5 L/min). A red light was used to facilitate collection of tissues in the dark phase.

In experiments 1 and 3, whole brain and blood sample collections were made at 4-hour intervals commencing at 0800 h on proestrus/diestrus of the cycle or days 6, 10, 14 or 18 of pregnancy (term = 19 days). In experiment 2, whole brain and blood sample collections were made at 0800 h and 1800 h one week after OVX surgery. A blood sample was obtained through cardiac puncture using a 26-gauge needle connected to a 1 mL syringe, and collected in a tube containing EDTA (100 µL per mL of blood). Plasma was obtained following centrifugation of the blood sample and stored at -20°C until required. Following blood sample collection, the mouse was decapitated and the whole brain removed from the skull. Whole brains were frozen by being placed on aluminium foil over dry ice immediately following removal, then stored at -80°C for later use.

In experiment 4, whole brain and blood sample collections were made at 4-hour intervals commencing at 0800 h from one male pup and one female pup per litter at postnatal days five (P5), fifteen (P15) and twenty-five (P25). A trunk blood sample was obtained following decapitation and collected in a tube containing EDTA (100 µL per mL of blood). The whole brain was removed from the skull and frozen by being placed on aluminium foil over dry ice immediately following removal, then stored at -80°C for later use.

45 Chapter 4 4.4 Quantitative reverse transcription polymerase chain reaction 4.4.1 Background Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a highly sensitive and reliable method for mRNA quantitation. RNA is first extracted from a tissue homogenate, and then reverse transcribed to generate complementary DNA (cDNA), which is relatively stable compared to RNA. Amplification of a target gene from the cDNA template is recorded via fluorescence measurement, where the degree of fluorescence is relative to the amount of PCR product. The fluorescent dye utilised in the experiments presented is SYBR green, which fluoresces only when bound to the minor groove of double stranded DNA. During the denaturing phase of the PCR cycle, all DNA becomes single stranded and hence there is little fluorescence. In the annealing phase, PCR primers hybridise to target sequences, resulting in small amounts of double stranded DNA. Finally, after the elongation phase, the primers are fully extended such that all DNA is double stranded and maximum fluorescence is achieved. The cycle at which fluorescence output exceeds background levels is termed the quantification cycle (Cq), which is inversely proportional to the starting concentration of the RNA transcript.

4.4.2 Reagents Reagents used in the extraction of RNA from hypothalamic tissue included Qiazol Lysis Reagent (Qiagen, Hilden, Germany; Cat# 79306) and 4-bromoanisole (BAN) (Molecular Research Center, Cincinnati, Ohio, USA; Cat# BN191).

Reagents used in DNase treatment and reverse transcription were all purchased from Promega (Madison, Wisconsin, USA) and included RQ1 RNase-free DNase (Cat# M6101), RQ1 RNase-Free DNase 10x Buffer, RQ1 DNase Stop Solution, random primers (Cat# C1181), 10mM dNTP mix (Cat# U1511), Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase RNase H Minus Point Mutant (Cat# M3683) and M-MLV Reverse Transcriptase 5X reaction buffer.

Real-time PCR reagents included iQ SYBR Green Supermix (Bio-Rad, Hercules, California, USA; Cat# 170-8880) and Rotor-Gene SYBR Green PCR Master Mix (Qiagen, Hilden, Germany; Cat# 204074).

46 Chapter 4

4.4.3 Isolation of the hypothalamus The hypothalamus was isolated from the whole brain and dissected into anterior and posterior portions according to the method described by Quennell et al. (2011). Hypothalami were dissected along the following boundaries: laterally at the peri- hypothalamic sulci, 2 mm either side of the third ventricle to the anterior border at the optic chiasm and the posterior border of the mammillary bodies, and the level of the anterior commissure/thalamus dorsally. Hypothalami were then bisected in the coronal plane immediately anterior to the pituitary stalk (Figure 4.1). The anterior portion contained the following major nuclei: AVPV, preoptic area, PeN, striohypothalamic nucleus, SCN, retrochiasmatic area, PVN and anterior hypothalamic area. The posterior portion contained the following major nuclei: ARC, VMH, DMH, dorsal hypothalamic area, premamillary nucleus. Hypothalamic samples were immediately placed in 2 mL microfuge tubes and stored at -80°C until extraction.

47 Chapter 4

Optic chiasm

Mammillary bodies

Figure 4.1 Ventral view of the mouse brain. The hypothalamus was dissected out according to the boundaries of the black square. Anterior and posterior hypothalamic portions were divided along the dotted line.

48 Chapter 4

4.4.4 RNA sample preparation Samples of anterior and posterior hypothalami were homogenised using a micro rotary tool (Dremel, Racine, Wisconsin, USA) in 750 µL of ice-cold Qiazol Lysis Reagent. Samples were kept on dry ice until homogenisation to prevent RNA degradation. The homogeniser was cleaned with RNase Zap and rinsed in dimethyl dicarbonate (DMDC) water to remove any contaminating RNases, both before homogenisation and between samples. The homogenate was supplemented with 37.5 µL of 4-bromoanisole (BAN), thoroughly mixed and centrifuged at 12,000 x g for 15 min at 4°C to initiate phase separation. All centrifugations were performed in a refrigerated centrifuge (Eppendorf, Hamburg, Germany). Following centrifugation, the aqueous phase (375 µL) was transferred into a fresh tube and mixed with 375 µL of isopropanol to precipitate the RNA. Samples were inverted 3 times, stored at room temperature for 10 min and then centrifuged at 12,000 x g for 5 min at 4°C. The supernatant was discarded and the RNA pellet was washed by adding 750 µL of 75% ethanol to the tube and subsequently centrifuging at 7,500 x g for 5 min at 4°C. The ethanol was discarded and the washing process repeated. After removal of the ethanol wash, the RNA pellet was air-dried for 5 min. The RNA was resuspended in 50 µL of RNase-free water, placed on ice for 5 min and thoroughly vortexed.

RNA concentration and purity was determined using the Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA) by measuring absorbance at 260 and 280 nm and stored at -80°C until DNase treatment and reverse transcription.

4.4.5 DNase treatment to remove genomic DNA RNA samples were treated with RQ1 RNase-free DNase to remove any genomic DNA present. Samples were made up to a total volume of 10 µL with 1 µg of RNA, 1 µL of RQ1 RNase-Free DNase 10x Buffer, 2 µL of RQ1 RNase-Free DNase and nuclease-free water, and incubated at 37°C for 30 min. RQ1 DNase Stop Solution (1 µL) was added to each sample to terminate the reaction, and samples were incubated at 65°C for 10 min to deactivate the DNase.

49 Chapter 4 4.4.6 Reverse transcription Following DNase treatment, 11 µL of RNA (1 µg) was combined with 2.5 µL of DEPC water and 0.5 µL of random primers for a sample volume of 14 µL. Samples were heated at 70°C for 5 min and then chilled on ice for 5 min. To achieve a final volume of 25 µL, 1 µL Moloney Murine Leukemia Virus Reverse Transcriptase, RNase H Minus, Point Mutant (M-MLV RT (H-)),5 µL of M-MLV Reverse Transcriptase 5X reaction buffer, 1.3 µL of 10mM dNTP mix, and 3.7 µL of DEPC water were added. Samples were placed in a thermal cycler and incubated at 25°C for 10 min, 55°C for 50 min and 70°C for 15 min.

The resultant cDNAs were purified using the Ultraclean PCR Cleanup kit (MoBio Laboratories, Carlsbad, Calfornia, USA; Cat# 12500-250), according to the manufacturer’s instructions. SpinBind (125 µL) was added to each cDNA sample (25 µL) and mixed well by pipetting. The mixture was transferred to a spin column and centrifuged for 30 s at 10,000 x g in order to retain the DNA in the membrane while filtering out leftover components from reverse transcription. The liquid flow-through was discarded and 300 µL of SpinClean Buffer added to the Spin Filter unit. The filter was centrifuged first for 30 s, and again for 60 s, at 10,000 x g to allow the SpinClean buffer to pass through and remove traces of unwanted contaminants from the DNA. The spin basket containing cDNA was transferred to a clean 2 mL collection tube and 50 µL of sterile water was added directly onto the centre of the membrane. After 1 minute, the spin filter unit was centrifuged for 60 s at 10,000 x g to elute the purified cDNA, which was stored at -20°C.

4.4.7 Primers Primer pairs for Kiss1, Kiss1r, Arntl which encodes for Bmal1, and Nr1d1 which encodes for Rev-erbα, and the reference genes hypoxanthine-guanine phosphoribosyltransferase (Hprt), succinate dehydrogenase subunit A (Sdha), beta actin (Actb) and beta-2 microglobulin (B2m) were designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast; Rozen & Skaletsky 2000) and synthesised by GeneWorks (Thebarton, South Australia, Australia). All primer pairs were positioned to span an intron to ensure no product was amplified from genomic DNA.

Primer specificity was first determined by melt curve analysis (see section 4.4.10) to demonstrate amplification of a single PCR product. Products were then separated by gel 50 Chapter 4 electrophoresis on a 2% agarose gel containing ethidium bromide for 45 min at 110 V (in 1x TAE buffer). Single bands of the expected size for each primer pair were visualised under UV light and excised for gel extraction using QIAEX II gel extraction kit (Qiagen, Hilden, Germany). After extraction, the DNA products were sequenced to confirm specificity using Big Dye Terminator version 3.1 (Applied Biosystems, Foster City, California, USA). The generated primer sequences are shown in Table 4.1.

Primer pairs for Arginine vasopressin (Avp), Avp receptor 1a (Avpr1a), neuropeptide VF precursor (Npvf) and neuropeptide FF receptor (Npffr) were purchased as QuantiTect Primer Assays (Qiagen, Hilden Germany; Cat# QT00249389, QT00113169, QT00278551, QT001169196).

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Table 4.1 PCR conditions. Primer sequences, annealing temperatures, amplicon sizes used for quantitative PCR.

Gene Primer sequence Size Annealing (bp) temp (°C) Kiss1 F: 5’-CTCTGTGTCGCCACCTATGG-3’ 126 60 R: 5’-AGGCTTGCTCTCTGCATACC-3’ Kiss1r F: 5’-TGCTGGGAGACTTCATGTGC-3’ 102 60 R: 5’-CATACCAGCGGTCCACACTC-3’ Bmal1 F: 5’-CGTGCTAAGGATGGCTGTTC-3’ 166 60 R: 5’-CTTCCCTCGGTCACATCCTA-3’ Rev-erbα F: 5’-ATTGCCCAGGGGGCGAGAGA-3’ 292 60 R: 5’-GCCAAAAGAGCGGGCAGGGT-3’ Hprt F: 5’-GCAGTACAGCCCCAAAATGG-3’ 80 60 R: 5’-AGTCTGGCCTGTATCCAACAC-3’ Sdha F: 5’-ATGGAAAATGGGGAGTGCCG-3’ 123 60 R: 5’-ACAGCTGAAGTAGGTTCGGC-3’ Actb F: 5’-TCCACACCCGCCACCAG-3’ 197 62 R: 5’-GGCCTCGTCACCCACATAG-3’ B2m F: 5’-TGACCGGCCTGTATGCTATC-3’ 128 60 R: 5’-GATTTCAATGTGAGGCGGGTG-3’ Avp 93 60

Avpr1a 133 60

Npvf 93 60

Npffr 76 60

52 Chapter 4

4.4.8 Real-time polymerase chain reaction (qPCR) Preliminary qPCR runs were conducted for each gene to evaluate the viability of the cDNA being amplified, the efficiency of primers and to optimise PCR conditions. Quantitative PCR was performed in 10 µL reaction volumes on the Rotorgene 6000 (Corbett Life Science, Concorde, New South Wales, Australia).

For GeneWorks primers, each 10 µL reaction volume was prepared with 5 µL of iQ SYBR

Green Supermix, 1 µL of forward primer, 1 µL of reverse primer, 2 µL of ddH2O and 1

µL cDNA or ddH2O (no template control). The PCR cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 1 s, annealing at a temperature specified in Table 4.1 for 15 s, and extension at 72°C for 5 s. The fluorescence was monitored and recorded after the completion of the extension phase of each cycle.

For Qiagen primers, each 10 µL reaction volume was prepared with 5 µL of Rotor-Gene SYBR Green PCR Master Mix, 2 µL of QuantiTect Primer Assay, 2 µL of RNase-free water and 1 µL cDNA or ddH2O (no template control). The PCR conditions were as follows: initial activation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 5 s and combined annealing/extension at 60°C for 10 s. The fluorescence was monitored after the completion of the annealing/extension phase of each cycle.

4.4.9 Quantification Standard curves for each product were generated from gel-extracted (QIAquick Gel Extraction Kit, Qiagen) PCR products using 10-fold serial dilutions and the Rotorgene 6000 software. All samples were standardised against Hprt, Sdha and Actb or B2m using the GeNorm algorithim (Vandesompele et al. 2002).

4.4.10 Melt curve analysis The properties of SYBR Green dye dictate that it will bind to any double stranded DNA, including primer dimers and contaminating cDNA, therefore a melt curve analysis was performed to confirm amplification of a single and specific product. Since cDNA of equal size and composition will denature at the same temperature, heating the product generated from qPCR from 72 to 99°C, while continuously measuring

53 Chapter 4 fluorescence produces ‘melting peaks’ at the temperature at which the DNA transcripts denature. Thus, the identity of a PCR product can be confirmed by a single melting peak at the correct temperature, while multiple peaks are indicative of non-specific amplification.

54 Chapter 4

Figure 4.2 a) Plot of amplification curves and b) generated standard curve for Sdha mRNA standards. Amplification profiles of 6 standards (10-fold serial dilutions) are shown against the number of PCR cycles. The threshold line indicates the point at which all samples fall within the log-linear phase of amplification and is used to determine the Cq for each sample. Amplification profiles were used to generate the standard curve.

55 Chapter 4

Figure 4.3 Melt curve analysis for Sdha mRNA. Fluorescence is plotted against temperature to show a single peak representing the denaturation of a single amplified product for each sample.

56 Chapter 4

4.5 Plasma hormone analyses 4.5.1 Background Plasma levels of FSH, LH and PRL were measured using the Milliplex Map Mouse Pituitary Magnetic Bead kit (Merck Millipore, Billerica, Massachusetts, USA; Cat# MPTMAG-49K).

Plasma levels of E2 and P4 were measured using the Milliplex Map multi species Steroid/Thyroid Hormone panel kit (Merck Millipore, Billerica, Massachusetts, USA; Cat # STTHMAG-21K)

This assay utilises magnetic beads known as microspheres, colour-coded with fluorescent dyes to give numerous distinct colours, each of which is coated with an antibody that captures a specific analyte. A detection antibody along with a reporter molecule completes the reaction on each microsphere. The microspheres are then passed through two lasers, the first excites the fluorescent dyes on the colour-coded microspheres and the second excites the phycoerythrin on the reporter molecule. Finally, each specific analyte is identified and quantified by digital-signal processors. The unique fluorescence signature of each microsphere thus allows multiple analytes to be quantified in a single assay.

4.5.2 Reagents All reagents specific to each protocol were included in the kit, with the exception of MAGPIX drive fluid (Merck Millipore, Billerica, Massachusetts, USA; Cat# MPXDF- 4PK).

4.5.3 Protocol for measurement of pituitary hormones All reagents were allowed to warm to room temperature prior to use. Working standards were generated through 4-fold serial dilutions of the original reconstituted standard in assay buffer. Assay buffer (200 µL) was added to each well of a 96-well plate, which was then sealed and mixed on a plate shaker for 10 min at room temperature. After the assay buffer was decanted, 25 µL of matrix solution was added to standard and quality control wells, while 25 µL of assay buffer was added to sample wells. Each standard, quality control and plasma sample (25 µL) was aliquoted in single to the appropriate wells. Finally, 25 µL of antibody-immobilised bead mixture was added to each well, while shaking the bead bottle intermittently to avoid settling. The plate was sealed, covered 57 Chapter 4 with aluminium foil and incubated with agitation on a plate shaker for 16 – 18 hours (overnight) at 4°C. The following day, well contents were removed and the plate was washed twice with 200 µL of wash buffer per well. The plate was placed on a hand-held magnet when decanting well contents to retain the magnetic beads. Detection antibodies (50 µL) were added into each well and the plate was sealed, covered with aluminium foil and incubated with agitation on a plate shaker for 60 min at room temperature. The fluorescent reporter molecule streptavidin-phycoerythrin (50 µL) was added to each well and the plate was sealed, covered with aluminium foil and incubated with agitation on a plate shaker for 30 min at room temperature. The well contents were removed and the plate was washed twice with 200 µL of wash buffer per well. Drive fluid (100 µL) was added to all wells, the plate was sealed and the beads were resuspended on a plate shaker for 5 min. The plate was then run on a MAGPIX system (Luminex Corporation, Austin, Texas, USA) with xPONENT software to analyse the median fluorescent intensity data and obtain analyte concentrations.

4.5.4 Protocol for measurement of steroid and thyroid hormones Prior to the assay, plasma samples were extracted by adding 75 µL of acetonitrile to 50 µL of sample, vortexing for 5 s and incubating for 10 min at room temperature. Samples were centrifuged at 17,000 x g for 5 min and 100 µL of the supernatant was transferred into fresh microfuge tubes. The samples were dried in a CentriVap Benchtop Vacuum Concentrator (Labconco, Kansas City Missouri, USA) at 37 °C and reconstituted with 40 µL of assay buffer for subsequent assay.

All reagents were allowed to warm to room temperature prior to use. Working standards were generated through 3-fold serial dilutions of the original reconstituted standard in assay buffer. Assay buffer (200 µL) was added to each well of a 96-well plate, which was then sealed and mixed on a plate shaker for 10 min at room temperature. After the assay buffer was decanted, another 25 µL of assay buffer was added to all wells and 25 µL of standard, quality control, or plasma sample was aliquoted in single to the appropriate wells. Steroid/thyroid HRP conjugate (25 µL) and antibody-immobilized beads (25 µL) were added to each well, while shaking the bead bottle intermittently to avoid settlings. The plate was sealed, covered with aluminium foil and incubated with agitation on a plate shaker for 16 – 20 hours (overnight) at 4°C. Well contents were removed and the plate was washed four times with 200 µL of wash buffer in each well. The plate was placed on a hand-held magnet when decanting well contents to retain the magnetic beads. Detection 58 Chapter 4 antibodies (25 µL) were added into each well and the plate was sealed, covered with foiled and incubated with agitation on a plate shaker for 60 min at room temperature. The fluorescent reporter molecule streptavidin-phycoerythrin (25 µL) was added to each well and the plate was sealed, covered with aluminium foil and incubated with agitation on a plate shaker for 30 min at room temperature. The well contents were removed and the plate was washed twice with 200 µL of wash buffer per well. Drive fluid (100 µL) was added to all wells and the beads were resuspended on a plate shaker for 5 min. The plate was then run on a MAGPIX system (Luminex Corporation, Austin, Texas, USA) with xPONENT software to analyse the median fluorescent intensity data and obtain analyte concentrations.

59 Chapter 4

Figure 4.4 Standard curves for a) progesterone and b) prolactin quantitation. Values for standards are shown where fluorescence is plotted against concentration.

60 Chapter 4

Table 4.2 CV and R2 values for all pituitary and steroid hormones measured in each experimental chapter.

LH FSH PRL E2 P4

Chapter 5 CV(%) 2.41 2.12 0.91 6.26 3.22 R2 0.992 0.999 1 0.942 0.985 Chapter 6 CV(%) 1.74 2.38 0.86 R2 0.999 0.998 1 Chapter 8 CV(%) 2.59 0.431 3.00 0.11 0.102 R2 0.999 0.997 0.997 1 0.998

61 Chapter 4 4.6 RNA-Seq 4.6.1 Background RNA-Seq (RNA sequencing) is a high-throughput DNA sequencing method that allows the profiling of an entire transcriptome, including mRNAs, non-coding RNAs and small RNAs, in a quantitative manner. Extracted RNA from a tissue sample is converted to a library of cDNA fragments, and adapters – containing an index or barcode allowing identification of the sample – are attached to one or both ends. The cDNA is then loaded onto a flow cell and placed in a sequencer to obtain short sequences of 30-400 bp from one end (single-end sequencing) or both ends (paired-end sequencing). During sequencing, DNA strands are held in place by oligonucleotides that coat the bottom of the flow cell and bend over to attach to the adjacent oligonucleotide, forming a ‘bridge’. Polymerases then synthesise complementary strands and the double stranded DNA is denatured to separate the two strands. This process is known as bridge amplification and it occurs repeatedly to ensure that all the DNA strands in one area are from a single source (clonal amplification). Once bridge amplification is complete, all the reverse strands are washed off, leaving only forward strands. Primers attach and add fluorescent-tagged nucleotides to the DNA strand, allowing the sequencer to identify and record which base was added through its unique fluorescence. This process is known as sequence by synthesis. The resulting sequence reads are aligned to a reference genome or transcriptome in order to discover the true location of each read, giving transcript information and allowing transcript abundance to be determined through the number of reads aligned (the number of reads generated from a transcript is directly proportional to its relative abundance). RNA-Seq was performed by the Institute of Metabolic Science, University of Cambridge, UK.

4.6.2 cDNA library preparation RNA was extracted from anterior hypothalamic samples as previously described (see section 4.4.4). cDNA libraries were prepared using the Illumina TruSeq Stranded mRNA Library Preparation Kit (Illumina, San Diego, California, USA; Cat# RS-122-2101) according to manufacturer’s instructions. 1 µg of RNA from each of 48 samples was purified using poly-T oligo attached magnetic beads. Following purification, the RNA was fragmented using divalent cations under elevated temperature, and then primed with random

62 Chapter 4 hexamers for cDNA synthesis. The cleaved RNA fragments were reverse transcribed into first strand cDNA using reverse transcriptase and random primers, while second strand cDNA was synthesised using DNA Polymerase I and RNase H. A single ‘A’ nucleotide was added to the 3’ ends of the blunt fragments to prevent them from ligating to one another. Multiple indexing adapters with a corresponding single ‘T’ nucleotide on the 3’ end were ligated to the ends of the ds cDNA, and the products were enriched using PCR. Ten µL of each sample library was normalised 10 nM using Tris-HCl and two pooled libraries (24 samples each) were made using 10 µL of each normalised sample library.

4.6.3 Sequencing and analysis Single-end sequencing was performed in a HiSeq 2500 (Illumina, San Diego, California USA) for 50 cycles.

The first step in the analysis process involved filtering and trimming the sequence reads. The quality of sequence reads was assessed by viewing FASTQ files generated by the sequencer, which contain the nucleotide sequence and corresponding base call quality. Reads that were determined to have a high error rate were trimmed or discarded.

All subsequent stages of analysis were carried out within the R programming language and software environment (https://www.r-project.org) using various software packages. Sequence reads were mapped to the mouse genome (NCBI m38 build), including intron/exon annotations (both downloaded from the UCSC Genome Bioinformatics Site), using a tool called TopHat (https://ccb.jhu.edu/software/tophat/index.shtml; Trapnell et al. 2012).

Cufflinks (http://cole-trapnell-lab.github.io/cufflinks/) was used to assemble the aligned reads into individual transcripts, and normalise raw read counts by sequencing coverage and transcript length, as more reads per transcript are observed for longer genes (Trapnell et al. 2012). This normalisation procedure is known as RPKM (reads per kilobase per one million mapped reads), adjusting read counts for gene length and library size.

The resulting BAM file containing the aligned sequence reads was fed to Cuffdiff (included in Cufflinks) to calculate expression levels and the statistical significance of

63 Chapter 4 observed changes, thus identifying differentially expressed genes across all samples (Trapnell et al. 2012). A heat map of all differentially expressed genes was generated using GeneSpring software (Agilent Technologies, Santa Clara, California, USA). Genes that were differentially expressed between specific days or time points were identified and statistically analysed using a more powerful tool called EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html; Robinson, McCarthy & Smyth 2010).

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4.7 Statistical analysis All data are expressed as mean ± SEM, with each animal representing an n of one. Statistical analyses on hypothalamic gene expression and plasma hormone levels were conducted using GraphPad Prism 6 (GraphPad Software, La Jolla, California, USA). Clock gene rhythmicity was assessed by cosinor analysis using a non-linear regression model (Genstat Version 9, VSN International Ltd, Hemel Hempstead, UK). Statistical analyses on RNA Seq data were conducted using EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html)

In experiment 1, variation in hypothalamic gene expression and plasma LH levels was assessed by two-way ANOVA, with variation attributed to day of pregnancy/estrous cycle and time-of-day. Variation in plasma hormone levels other than LH was assessed by one-way ANOVA, with variation attributed to day of pregnancy/estrous cycle. In experiment 2, variation in hypothalamic gene expression and plasma hormone levels was assessed by two-way ANOVA, with variation attributed to hormone replacement treatment and time-of-day. In experiment 3, variation in hypothalamic gene expression was assessed by two-way ANOVA, with variation attributed to day of pregnancy/estrous cycle and time-of-day, one-way ANOVA, with variation attributed to day of pregnancy/estrous cycle or t-test, with variation attributed to time-of-day. Differences were considered significant at q < 0.05 (where q is the false discovery rate (FDR)-adjusted P-value). In experiment 4, variation in hypothalamic gene expression and plasma hormone levels was assessed by two-way ANOVA, with variation attributed to postnatal age and time-of-day. For all ANOVAs, when significant interaction between independent variables was observed, separate comparisons were conducted by one-way ANOVA or unpaired t-test as appropriate. For all ANOVAs, where the F test reached statistical significance (P < 0.05), subsequent post hoc analyses were performed using Tukey’s tests to correct for multiple comparisons.

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Chapter 5 Diurnal Regulation of Hypothalamic Kisspeptin is Disrupted During Mouse Pregnancy

5.1 Introduction Kisspeptin neurons are the afferent population that is critical for stimulation of GnRH neurons, to drive the hypothalamic-pituitary gonadal axis. Gonadal sex steroids exert feedback actions onto GnRH neurons via kisspeptin regulation, with kisspeptin populations in the AVPV and ARC being involved in positive and negative feedback control of E2, respectively (Gottsch et al. 2004; Smith et al. 2005a, 2005b). Neurons expressing RFamide-related peptides such as RFRP-3, produced from a precursor peptide encoded by the neuropeptide VF precursor (Npvf) gene and the actions of which are conducted through the neuropeptide FF receptor (NPFFR) (Clarke et al. 2009), are found in the dorsomedial nucleus (DMN). RFRP-3 neurons are proposed inhibitors of GnRH secretion that are thought to modulate the negative feedback effects of estrogen across most of the ovulatory cycle (Kriegsfeld et al. 2006; Ducret, Anderson & Herbison 2009; Williams 3rd & Kriegsfeld 2012). For a short period just prior to ovulation, estrogen feedback to the reproductive axis switches from negative to positive and this causes a surge of GnRH and thus LH secretion from the pituitary (Karsch et al. 1997; Levine 1997). This switch is thought to be governed, in part, by the interplay between kisspeptin and RFRP-3.

The pre-ovulatory LH surge that occurs in female rodents is a product of interactions between circadian inputs and estrogenic signals. Specifically, high concentrations of E2 and a circadian signal predicting the onset of darkness are both essential for triggering the increase in kisspeptin levels and the subsequent LH surge (Levine 1997; Christian & Moenter 2010; Williams 3rd et al. 2011). In female rodents, the LH surge is precisely timed to occur in the late afternoon of proestrus, being tightly controlled by the master circadian oscillator located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Kisspeptin also appears to be under circadian control, as evidenced by studies showing that increases in kisspeptin expression in the AVPV of the hypothalamus are synchronous with the LH surge

67 Chapter 5 during proestrus in female rats (Smith et al. 2006), mice (Robertson et al. 2009) and hamsters (Williams 3rd et al. 2011). Furthermore, studies in hamsters have shown that RFRP-3 cells are indirectly modulated via SCN vasoactive intestinal peptide (VIP)-ergic neurons (Russo et al. 2015), and act directly on GnRH neurons to inhibit their activity (Gibson et al. 2008). Data also suggests that RFRP-3 exhibits reduced expression during proestrus, and thus lowers GnRH inhibition and positively drives the GnRH/LH surge and ovulation (Gibson et al. 2008). Other studies showing that estrogen lowers RFRP-3 mRNA levels also suggest that RFRP-3 may also play a role in estrogen positive feedback (Molnár et al. 2011; Poling et al. 2012)

AVP neurons are responsible for circadian input to the AVPV and originate from the dorsomedial SCN to contact kisspeptin neurons expressing the AVP receptor subtype V1a (Leak & Moore 2001; Williams 3rd et al. 2011). As the master pacemaker, the SCN exerts tight circadian control over many biological processes through endogenous rhythms generated by positive and negative feedback gene transcription and translation loops of clock genes including Clock, Bmal1, Per1-3, Cry 1-2 and Rev-erbα (Reppert & Weaver 2001). These clock genes are also expressed in numerous peripheral tissues (Boden et al. 2010). We have recently shown that the expression of core clock genes in the SCN changes significantly across gestation in the mouse (Wharfe et al. 2016), and Wharfe et al. (2011) has also previously demonstrated that clock gene rhythms in the rat liver are altered by pregnancy (Wharfe, Mark & Waddell 2011). Additionally, despite the high E2 levels during pregnancy, the hypothalamic-pituitary-gonadal (HPG) axis is dormant and ovulation does not occur, suggesting the shutdown of kisspeptin signalling, reduced GnRH neuron or gonadotrope sensitivity, or a combination of each. We therefore speculate that disruption of normal hypothalamic circadian rhythms, including those of kisspeptin in the AVPV, occurs in the pregnant state and suppresses brain mechanisms controlling ovulation. In the present study, we sought to characterize the hypothalamic expression of the kisspeptin signaling system in the mouse during pregnancy, and to investigate whether this expression exhibits a circadian pattern, as is observed in proestrus females.

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5.2 Materials and methods 5.2.1 Animals Nulliparous C57Bl/6J mice (6 weeks old) were supplied by the Animal Resources Centre (Murdoch, Australia). Mice were maintained in an environmentally controlled room under a 12-hour light, 12-hour dark cycle (lights on from 0700 – 1900 h) with ad libitum access to food and water. By convention in chronobiology, lights off at 1900 h was defined as Zeitgeber time (ZT) 12, with sampling times described as relative to ZT12. Female mice were subjected to a daily vaginal smear to determine estrous cycle stage and monitored for three full cycles. A sub-group of female mice were mated overnight, and pregnancy was confirmed by observation of a vaginal plug the following morning, which was designated day 1 of pregnancy. Whole brain and blood sample collections were made at 4-hour intervals commencing at 0800 h on diestrus II/proestrus of the cycle or days 6, 10, 14 or 18 of pregnancy (term = 19 days). Diestrus and proestrus were chosen as days of the cycle as hormone levels and gene expression typically show the most variation between those two days. The days of pregnancy chosen represent developmental milestones: day 6 is post-implantation, day 10 is the embryonic period, day 14 represents early fetal life and day 18 late fetal life. We have previously shown 6 time-points (4-hour intervals) is adequate for the determination of circadian regulation of genes (Wharfe et al. 2016). All procedures involving animals were conducted with the approval of the Animal Ethics Committee of The University of Western Australia.

5.2.2 Tissue collection Whole brains were collected from mice under isoflurane anaesthesia at 0800 (ZT1), 1200 (ZT5), 1600 (ZT9), 2000 (ZT13), 2400 (ZT17) or 0400 (ZT21) in order to obtain a reasonable circadian profile of gene expression and plasma hormone levels. A red light (>600 nm wavelength) was used to facilitate collection of tissues in the dark phase. Whole brains were frozen on dry ice immediately following removal. A blood sample was obtained from each mouse under anaesthesia through a cardiac puncture and collected in a tube containing EDTA (100 µL per mL of blood). Plasma was obtained following centrifugation of the blood sample and stored at -20°C until required.

69 Chapter 5 5.2.3 Hypothalamic gene expression

5.2.3.1 RNA extraction Hypothalami were dissected from whole brain samples and bisected into anterior (containing the AVPV and SCN) and posterior (containing the ARC) portions as described in (Quennell et al. (2011). Total RNA was extracted from anterior and posterior hypothalami using Qiazol (Qiagen) according to the manufacturer’s instructions. The RNA pellet was dissolved in 50 µL of RNase-free water, placed on ice for 5 min and thoroughly vortexed. RNA was quantitated using the Nanodrop ND-1000 spectrophotometer (Thermo Scientific) at 260 nm and stored at -80 °C until required.

5.2.3.2 Reverse transcription RNA samples were treated with RQ1 RNase-free DNase (Promega, cat# M6101) to remove any genomic DNA present. Reactions were made up to a total volume of 10 µL with 1 µg of RNA, 1 µL of RQ1 RNase-Free DNase 10x Buffer, 2 µL of RQ1 RNase- Free DNase and nuclease-free water, and incubated at 37°C for 30 min. RQ1 DNase Stop Solution (1 µL) was added to each sample to terminate the reaction, and samples were incubated at 65°C for 10 min to deactivate the DNase. Total RNA (1 µg) was reverse transcribed to cDNA with random primers (Promega, cat# C1181) using Moloney Murine Leukemia Virus Reverse Transcriptase, RNase H Minus, Point Mutant (M-MLV RT (H-)) (Promega, cat# M3683). The resultant cDNAs were purified using the Ultraclean PCR Cleanup kit (MoBio Industries, cat# 12500-250), according to the manufacturer’s instructions.

5.2.3.3 Real-time PCR Analyses of mRNA levels for total Kiss1, Kiss1 receptor (Kiss1r), arginine vasopressin (Avp), arginine vasopressin receptor 1a (Avpr1a), neuropeptide VF precursor (Npvf) and neuropeptide FF receptor (Npffr) transcripts were performed by quantitative RT-PCR on the Rotorgene 6000 (Corbett Life Science, New South Wales, Australia) using iQ SYBR Green Supermix (Bio-Rad, cat# 170-8880). Primers (Table 5.1) for total Kiss1, Kiss1r, Bmal1, Rev-erbα and the reference genes hypoxanthine-guanine phosphoribosyltransferase (Hprt), succinate dehydrogenase subunit A (Sdha) and beta actin (Actb) were designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov). Each of the selected primer pairs was positioned to span an intron to ensure no product was amplified from genomic DNA. The resulting amplicons were sequenced to confirm

70 Chapter 5 specificity. Standard curves for each product were generated from gel-extracted (QIAquick Gel Extraction Kit, Qiagen) PCR products using 10-fold serial dilutions and the Rotorgene 6000 software. The PCR cycling conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 1 s, annealing at 60 °C for 15 s and extension at 72 °C for 5 s. Melt curve analysis was performed to confirm amplification specificity for each gene. Primers for Avp, Avpr1a, Npvf and Npffr were purchased as QuantiTect Primer Assays (Qiagen, cat# QT00249389, QT00113169, QT00278551, QT001169196). The PCR conditions were as follows: initial activation 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 5 s and combined annealing/extension at 60 °C for 10 s. Melt curve analysis was performed to confirm amplification specificity. All target genes were standardized against housekeeping genes Hprt, Sdha and Actb using the GeNorm algorithim (Vandesompele et al. 2002). No differences were seen in these genes across days or time-points (data not shown).

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Table 5.1 Primer sequences and conditions for quantitative PCR.

Gene Sequence Size Annealing (bp) temp (°C) Kiss1 F: 5’-CTCTGTGTCGCCACCTATGG-3’ 126 60 R: 5’-AGGCTTGCTCTCTGCATACC-3’ Kiss1r F: 5’-TGCTGGGAGACTTCATGTGC-3’ 102 60 R: 5’-CATACCAGCGGTCCACACTC-3’ Bmal1 F: 5’-CGTGCTAAGGATGGCTGTTC-3’ 166 60 R: 5’-CTTCCCTCGGTCACATCCTA-3’ Rev-erbα F: 5’-ATTGCCCAGGGGGCGAGAGA-3’ 292 60 R: 5’-GCCAAAAGAGCGGGCAGGGT-3’ Hprt F: 5’-GCAGTACAGCCCCAAAATGG-3’ 80 60 R: 5’-AGTCTGGCCTGTATCCAACAC-3’ Sdha F: 5’-ATGGAAAATGGGGAGTGCCG-3’ 123 60 R: 5’-ACAGCTGAAGTAGGTTCGGC-3’ Actb F: 5’-TCCACACCCGCCACCAG-3’ 197 62 R: 5’-GGCCTCGTCACCCACATAG-3’

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5.2.4 Plasma hormone analyses

5.2.4.1 Pituitary hormones Plasma levels of FSH, LH and PRL were measured in a 10 µl sample using the MILLIPLEX Map Mouse Pituitary Magnetic Bead kit (Merck Millipore, cat# MPTMAG- 49K) according to the manufacturer’s instructions. The plate was run on a MAGPIX system (Luminex Corporation) with xPONENT software to analyse the median fluorescent intensity data and obtain analyte concentrations. The lower limits of detection for this assay are as follows: FSH - 9.5 pg/mL, LH - 1.9 pg/mL, PRL - 46.2 pg/mL.

5.2.4.2 Steroid hormones Prior to assay, plasma samples (50 µL) were extracted in 75 µL of acetonitrile, vortexed for 5 s and incubated for 10 min at room temperature. Samples were centrifuged at 17,000 x g for 5 min and the supernatant was transferred into separate tubes. The samples were dried by CentriVap Benchtop Vacuum Concentrator (Labconco, Kansas City Missouri, USA) at 37 °C and reconstituted with 40 µL of assay buffer for subsequent assay.

Plasma levels of E2 and P4 were measured in 25 µl of extracted sample using the MILLIPLEX Map multi species Steroid/Thyroid Hormone panel kit (Merck Millipore, cat # STTHMAG-21K) according to the manufacturer’s instructions. The plate was run on a MAGPIX system (Luminex Corporation) with xPONENT software to analyse the median fluorescent intensity data and obtain analyte concentrations. The lower limits of detection for this assay are as follows: E2 - 16 pg/mL, P4 - 90 pg/mL.

5.2.5 Statistical analysis Statistical analyses were conducted using GraphPad Prism 6 (GraphPad Software). Data are expressed as the mean ± SEM, with an n of 5-7 per ZT on each day measured. Differences were considered significant when P < 0.05. One-way ANOVAs were used to analyse plasma E2, P4, PRL and FSH levels. Two-way ANOVAs were used to analyse gene expression data and plasma LH levels, with day and time as factors; followed by Tukey’s post hoc tests where appropriate. One-way ANOVAs with time as a factor were conducted within each day, where significant interaction effects were observed in two- way ANOVA analyses.

73 Chapter 5 5.3 Results 5.3.1 Hormone levels There was a significant effect of day on plasma LH concentrations (P < 0.001), which was highest at proestrus. Within proestrus LH increased around two-fold between ZT5 and ZT17 (P < 0.05), and no diurnal variation was observed at diestrus or day 18 of pregnancy (Figure 5.1A). Plasma FSH levels showed no difference between any of the days measured (Figure 5.1B).

Plasma E2 levels increased significantly during pregnancy, almost doubling from diestrus to day 18 of pregnancy (P < 0.05) (Figure 5.1C). Similarly, plasma P4 levels rose dramatically during pregnancy, increasing 45-fold by day 6 (P < 0.01) and 170-fold by day 18 (P < 0.0001; Figure 5.1D). Plasma PRL concentrations peaked at day 6 of pregnancy, increasing nearly two-fold compared to proestrus (P < 0.0001) and falling to pre-pregnancy levels by day 18 (P < 0.0001; Fig 5.1E).

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Figure 1.

A B # 1000 1000 * 800 800

600 600

400 400

200 200 Plasma LH levels (pg/ml) levels LH Plasma Plasma FSH levels (pg/ml)

0 0

ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Diestrus Proestrus Day 18 Diestrus Proestrus Day 18 C D E

c 250 60 60 * c 200 40 b 40 150 ab

100 a 20 20 b 50 a a 0 0 0 Plasma estradiol (pg/ml) Plasma P4 levels (ng/ml) Die Pro Day 6 Day 18 Die Pro Day 6 Day 18 Plasma prolactin (ng/ml) Die Pro Day 6 Day 18

Figure 5.1 Plasma hormone concentrations in non-pregnant animals and during pregnancy. A) LH levels peaked at ZT17 on the day of proestrus. Main effect of day, F (2, 96) = 9.205, P < 0.001; main effect of time-of-day, F (5, 96) = 0.5542, P > 0.05; interaction between day and time-of-day, F (10, 96) = 1.171, P > 0.05. B) FSH levels were unchanged in non-pregnant animals and pregnant mice. Main effect of day, F (2, 44) = 0.2955, P > 0.05; main effect of time-of-day, F (5, 44) = 1.163, P > 0.05; interaction between day and time-of-day, F (10, 44) =

0.4451, P > 0.05. C) E2 levels were significantly increased at day 18 of pregnancy compared to day 16 proestrus (Pro) and diestrus (Die). Main effect of day, F (3,

76) = 6.409, P < 0.001. D) P4 levels were significantly increased at day 6 of pregnancy and further increased at day 18 of pregnancy. Main effect of day, F (3, 76) = 86.58, P < 0.0001. E) PRL levels peaked at day 6 of pregnancy. Main effect of day, F (3, 76) = 17.30, P < 0.0001. Data are means ± SEM, n = 4-7 per group. Grey panel indicates lights off from zeitgeber time (ZT)12 to ZT0. Two-way

75 Chapter 5 5.3.2 Gene expression Kiss1 expression in the anterior hypothalamus, representing the AVPV population, differed significantly between days and peaked at day 10 of pregnancy, increasing two- fold compared to diestrus (P < 0.001). As for within-day effects, on the day of proestrus, Kiss1 increased more than two-fold from ZT9 to ZT13 (P < 0.05) and returned to baseline by ZT17 (Figure 5.2A). No time-of-day variation was observed during diestrus or any day of pregnancy (Figure 5.2A). In the posterior hypothalamus, representing the ARC population of Kiss1 neurons, no change was noted between days. Within days, Kiss1 levels increased over four-fold from ZT5 to ZT9 (P < 0.05) and returned to baseline by ZT13 on day 18 of pregnancy, while diurnal variation was not observed on any other day of pregnancy, diestrus or proestrus (Figure 5.2B). Kiss1r expression in the anterior hypothalamus was different among days (P < 0.0001; Figure 5.3), with elevated mRNA levels at day 14 of pregnancy, having nearly doubled compared to diestrus (P < 0.0001), but showed no diurnal variation on any day of the cycle or pregnancy.

Avp expression in the anterior hypothalamus changed between days as pregnancy progressed (P < 0.0001); levels peaked in the late stages, with an increase of 2.4-fold from day 10 to day 14 of pregnancy (P < 0.0001) which was sustained at day 18 (P < 0.01). However, no diurnal variation was observed at any stage (Figure 5.4A). There was an effect of day on Avpr1a mRNA levels in the anterior hypothalamus (P < 0.0001); Avpr1a was high throughout diestrus/proestrus and the early stages of pregnancy, then decreased by 55% from day 10 to day 14 and 18 of pregnancy (P < 0.0001; Figure 5.4B). As for within-day effects, Avpr1a levels in the anterior hypothalamus increased nearly two-fold from ZT1 to ZT5 during proestrus (P < 0.05) and 1.6-fold between ZT5 and ZT13 on day 10 of pregnancy (P < 0.05; Figure 5.4B).

Npvf expression in the posterior hypothalamus, representing the DMH population of RFRP-3 neurons, was different among days (P < 0.05), with mRNA levels declining after day 10 of pregnancy to 65% by day 18 (P < 0.05). However, diurnal variation was not observed at any stage (Figure 5.5A). Npffr mRNA levels in the anterior hypothalamus varied across pregnancy (P < 0.01); Npffr was high throughout diestrus/proestrus and the early stages of pregnancy, then fell by 55-65% at day 14 through to day 18 of pregnancy (P < 0.01). As for time-of-day effects, Npffr levels increased 3.5-fold from ZT1 to ZT13

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(P < 0.01) during proestrus, but there was no diurnal variation evident at diestrus or during pregnancy (Figure 5.5B).

Bmal1 and Rev-erbα exhibited strong time-of-day effects on both diestrus and proestrus (P < 0.05 for both genes on both days) (Figure 5.6). They also demonstrated an anti-phase relationship, with Rev-erbα peaking in the afternoon between ZT5 and ZT9, and Bmal1 peaking in the early hours of the morning between ZT21 and ZT1. Bmal1 and Rev-erbα and other central circadian clock genes also exhibited robust diurnal rhythms during pregnancy, which appeared unchanged despite an apparent upward shift in overall expression from mid to late gestation (Wharfe et al. 2016).

The reference genes Hprt, Sdha and B2m showed no difference in expression between any of the days measured or across time points (data not shown).

77 Figure 2. Chapter 5

A 15 #

* 10 mRNA mRNA Kiss1

5 AVPV (Relative expression) (Relative

0

ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Diestrus Proestrus Day 6 Day 10 Day 14 Day 18 B

15 *

mRNA 10 Kiss1

5 ARC (Relative expression) (Relative

0

ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Diestrus Proestrus Day 6 Day 10 Day 14 Day 18

Figure 5.2 Kiss1 diurnal gene expression in non-pregnant animals and during pregnancy. A) Kiss1 levels in the AVPV were significantly increased at ZT13 on the day of proestrus. This effect was not seen at diestrus or during pregnancy. Main effect of day, F (5, 179) = 3.579, P < 0.01; main effect of time-of- day, F (5, 179) = 1.144, P > 0.05; interaction between day and time-of-day, F (25, 179) = 1.355, P > 0.05. B) Diurnal variation in Kiss1 seen at day 18 of pregnancy in the ARC. Main effect of day, F (5, 178) = 2.135, P > 0.05; main effect of time-of- day, F (5, 178) = 2.182, P > 0.05; interaction between day and time-of-day, F (25, 178) = 2.646, P = 0.0001. Data are means ± SEM, n = 5-7 per group. Grey panel indicates lights off from zeitgeber time (ZT)12 to ZT0. Two-way ANOVA, *, P < 0.05 within day effect. #, P < 0.001 between day effect compared to diestrus.

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Figure 3.

20 #

15 mRNA 10 Kiss1r

AH 5 (Relative expression) (Relative

0

ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Diestrus Proestrus Day 6 Day 10 Day 14 Day 18

Figure 5.3 Anterior hypothalamus Kiss1r diurnal gene expression in non- pregnant animals and during pregnancy. No diurnal variation was observed at any stage. Main effect of day, F (5, 189) = 6.784, P < 0.0001; main effect of time- of-day, F (5, 189) = 0.2065, P > 0.05; interaction between day and time-of-day, F (25, 189) = 0.3836, P > 0.05. Data are means ± SEM, n = 5-7 per group. Grey panel indicates lights off from zeitgeber time (ZT)12 to ZT0. Two-way ANOVA, #, P < 0.0001 between day effect compared to diestrus and proestrus.

79 FigureChapter 4. 5

A 20 ## #

15

mRNA 10 Avp AH 5 (Relative expression) (Relative

0

ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Diestrus Proestrus Day 6 Day 10 Day 14 Day 18 B 15 * * 10 mRNA ## ## Avpr1a 5 AH (Relative expression) (Relative

0

ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Diestrus Proestrus Day 6 Day 10 Day 14 Day 18

Figure 5.4 Avp and Avpr1a diurnal gene expression in the anterior hypothalamus in non-pregnant animals and during pregnancy. A) Avp mRNA levels did not exhibit diurnal variation at any stage. Main effect of day, F (5, 186) = 21.52, P < 0.0001; main effect of time-of-day, F (5, 186) = 0.6809, P > 0.05; interaction between day and time-of-day, F (25, 186) = 0.6568, P > 0.05. B) Avpr1a expression increased at ZT5 on the day of proestrus, and showed some diurnal variation at day 10 of pregnancy. Main effect of day, F (5, 188) = 38.38, P < 0.0001; main effect of time-of-day, F (5, 188) = 1.665, P > 0.05; interaction between day and time-of-day, F (25, 188) = 2.099, P < 0.01. Data are means ± SEM, n = 5-7 per group. Grey panel indicates lights off from zeitgeber time (ZT)12 to ZT0. Two-way ANOVA, *, P < 0.05 within day effect compared to ZT1, 9, 13, and 21. #, P < 0.01, ##, P < 0.0001 between day effect compared to diestrus, proestus day 6 and 10.

80 Chapter 5 Figure 5.

A 10

# 8

6 mRNA

Npvf 4 PH PH

(Relative expression) (Relative 2

0

ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Diestrus Proestrus Day 6 Day 10 Day 14 Day 18 B 10 * 8 ##

6 ## mRNA

Npffr 4 AH

(Relative expression) (Relative 2

0

ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT1 ZT5 ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Diestrus Proestrus Day 6 Day 10 Day 14 Day 18

Figure 5.5 Npvf (Rfrp) and Npffr (Rfrpr) diurnal gene expression in non- pregnant animals and during pregnancy. A) Npvf expression in posterior hypothalamus showed no diurnal variation at any stage. Main effect of day, F (5, 178) = 3.076, P < 0.05; main effect of time-of-day, F (5, 178) = 1.989, P > 0.05; interaction between day and time-of-day, F (25, 178) = 0.7820, P > 0.05. B) Npffr expression in the anterior hypothalamus increased from ZT1 to ZT13 on the day of proestrus. Main effect of day, F (5, 182) = 9.147, P < 0.0001; main effect of time-of-day, F (5, 182) = 1.215, P > 0.05; interaction between day and time-of- day, F (25, 182) = 1.031, P > 0.05. Data are means ± SEM, n = 5-7 per group. Grey panel indicates lights off from zeitgeber time (ZT)12 to ZT0. Two-way ANOVA, *, P < 0.01 within day effect. #, P < 0.05 between day effect compared to day 6, 14 and 18, ##, P < 0.01 between day effect compared to diestrus, proestus day 6

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Figure 6.

A B 20 15 * * * * * * * * 15

mRNA 10 α mRNA 10

Bmal1 5

5 Rev-erb AH AH (Relative expression) (Relative (Relative expression) (Relative 0 0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Diestrus Proestrus Diestrus Proestrus

Figure 5.6 Anterior hypothalamus diurnal clock gene expression in non- pregnant animals. A) Bmal1 expression showed a robust diurnal rhythm on diestrus and proestrus. Main effect of day, F (5, 194) = 59.72, P < 0.0001; main effect of time-of-day, F (5, 194) = 43.40, P < 0.0001; interaction between day and time-of-day, F (25, 194) = 2.569, P < 0.001. B) Rev-erbα showed a robust diurnal rhythm on diestrus and proestrus Main effect of day, F (5, 194) = 40.04, P < 0.0001; main effect of time-of-day, F (5, 194) = 42.60, P < 0.0001; interaction between day and time-of-day, F (25, 194) = 2.170, P < 0.01. Expected anti-phase relationship between Bmal1 and Rev-erbα was observed. Data are means ± SEM, n = 5-7 per group. Grey panel indicates lights off from zeitgeber time (ZT)12 to ZT0. Two-way ANOVA, *, P < 0.05 within day effect.

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5.4 Discussion The present findings demonstrate an alteration in the diurnal regulation of AVPV Kiss1 and Avpr1a expression during pregnancy in the mouse, that is associated with the blockade of the pre-ovulatory LH surge, in spite of elevated E2 concentrations and a functional master circadian clock. Kiss1 gene expression in the AVPV peaked at ZT13 during proestrus, four hours before the LH peak at ZT17, which is consistent with that reported in the literature, although the peak in Kiss1 mRNA levels has previously been found to be roughly coincident with the LH surge (Smith et al. 2006; Robertson et al. 2009).

We also found that AVP receptor Avpr1a increased in expression in the AVPV at ZT5 during proestrus, prior to the rise in Kiss1 expression and the LH surge. This is consistent with work by de la Iglesia and colleagues in the rat, showing a peak in Avpr1a gene expression four hours before that of Kiss1 (Smarr, Gile & de la Iglesia 2013). It is possible that this temporal arrangement of events may reflect the transmission of circadian information from the SCN to kisspeptin neurons in the AVPV, although the eight-hour gap observed in the current study decreases the likelihood of a direct connection between the two events. While its receptor showed an increase in expression at proestrus, Avp itself did not mirror this increase at any time on the day of proestrus. However, previous data have shown that Avp gene expression in the mouse SCN follows a circadian pattern, being highest at the end of the day and lowest at the end of the night, although the sex of the mice was not specified (Dardente et al. 2004). This pattern of Avp expression would be consistent with the sequence of events observed in the present study, which comprise the increase in Avpr1a and Kiss1 expression and subsequent LH surge. It is possible that in the current study the diurnal rhythm of Avp mRNA specifically within the SCN has been masked; numerous parvocellular and magnocellular neurosecretory cells of the paraventricular and supraoptic nuclei located in the anterior hypothalamus express AVP as well (Alves et al. 1998; Nomura et al. 2002). In situ hybridization is needed to determine Avp mRNA levels specifically in the SCN.

The transcription factor albumin D-site binding protein (Dbp) in the AVPV has been hypothesised to be the conduit through which AVP drives the circadian rhythm of Kiss1; Dbp is capable of triggering Kiss1 transcription and exhibits elevated levels in the afternoon of proestrus, similar to Kiss1 expression (Xu et al. 2011). Data from Kriegsfeld

83 Chapter 5 and colleagues (2011) suggests that the initiation of the LH surge by AVP is secondarily gated by GnRH neurons, whereby kisspeptin neurons are indiscriminately activated by AVP but GnRH neurons are selectively responsive to kisspeptin stimulation, depending on the time of day (Williams 3rd et al. 2011). Although AVP stimulation of kisspeptin is already under strict circadian control, this mechanism of control further ensures that the LH surge is limited to the late afternoon of proestrus. In a further layer of circadian control, AVPV kisspeptin neurons appear to possess a circadian oscillator independent of the master pacemaker in the SCN. Recent work by Chassard et al. (2015) demonstrates a daily PER1 rhythm within AVPV kisspeptin cells that is E2-sensitive and phase delayed compared to that in the SCN, suggesting that there may exist yet another level of circadian gating of the LH surge.

In the current study, both Kiss1 and Avpr1a mRNA in the anterior hypothalamus exhibited a peak in expression during proestrus that was absent at diestrus. These estrous cycle differences in expression profiles do not appear to be driven by clock genes as the rhythms of Bmal1 and Rev-erbα were unchanged between diestrus and proestrus, suggesting that the likely major stimulus for the increase in Kiss1 and Avpr1a expression at proestrus is rising E2 levels (Robertson et al. 2009; Williams 3rd et al. 2011; Smarr, Gile & de la Iglesia 2013). Unlike Kiss1 expression in the AVPV, Kiss1 expression in the ARC was not different between proestrus and diestrus, exhibiting no diurnal variation on either day. The different expression patterns reflect the distinct roles of the two neuronal populations, with the kisspeptin neurons in the ARC responsible for the tonic control of gonadotropin secretion, as opposed to generating the preovulatory LH surge (Smith et al. 2005b; Beale et al. 2014).

Although the suppression of diurnal regulation of Kiss1 and Avpr1a expression in the AVPV during pregnancy compared to proestrus is consistent with our hypothesis, the mechanisms underlying this suppression are unclear and require further investigation.

Despite the rise in E2 levels throughout pregnancy, established in the literature and confirmed by our data, the surge in kisspeptin and LH does not occur at any stage of pregnancy. As both the LH surge and AVPV Kiss1 levels require the combination of elevated E2 and a circadian signal (Robertson et al. 2009), the lack of an LH surge in the face of peak E2 concentrations would indicate a disruption of the circadian signal. Because diurnal rhythmicity of all the core clock genes in the SCN appears to be intact during pregnancy (Wharfe et al. 2016), the master pacemaker in the SCN is therefore

84 Chapter 5 likely to be functioning normally and thus any changes in circadian input to the AVPV are likely to occur downstream of the SCN. One possibility is that there is an alteration in AVP receptor expression on kisspeptin neurons or in downstream signal transduction, leading to a loss in circadian information. Alternatively, it could be that other hormone(s) that experience an upsurge during pregnancy are overriding the estrogenic and circadian signals that give rise to the increase in kisspeptin in the AVPV.

Potential candidate hormones are P4 and PRL, both of which are elevated in the plasma during pregnancy in the present study and exhibit expression patterns that are in agreement with previously reported data from both the rat and mouse. Plasma PRL concentrations are high in early pregnancy with a biphasic pattern of secretion, characterized by one nocturnal and one diurnal surge, which cease after day 8 of pregnancy (Smith & Neill 1976). This coincides with the beginning of an increase in placental lactogen secretion (which peaks at day 11) (Smith & Neill 1976), and there is evidence to suggest that the latter causes the former (Tonkowicz & Voogt 1983). PRL levels fall and plateau in mid- and late pregnancy, then surge again immediately before parturition (Morishige, Pepe & Rothchild 1973; Murr, Bradford & Geschwind 1974).

Plasma P4 exhibits almost the opposite pattern to PRL, rising continually until very late in pregnancy, when levels fall precipitously to signal the onset of parturition (Murr et al. 1974; Barkley, Geschwind & Bradford 1979; Waddell, Bruce & Dharmarajan 1989). Our data failed to capture the sudden decrease in P4 levels prior to parturition, however, it is likely that this would have occurred at a later time had the pregnancies been allowed to progress further. In addition, recent work has shown that AVPV kisspeptin neurons require progesterone receptor (PR) signalling to display normal c-fos induction and to mount an LH surge in response to elevated E2 (Stephens et al. 2015). However, it does not appear to be gonadal P4 but rather E2-induced local synthesis of neuro-progesterone in the hypothalamus that is critical for the LH surge (Micevych & Sinchak 2011). Furthermore, kisspeptin expression in the ARC and AVPV is inhibited by high PRL levels, resulting in the suppression of LH secretion (Araujo-Lopes et al. 2014; Brown,

Herbison & Grattan 2014). The substantial effects of P4 and PRL on kisspeptin, as well as the important role that these hormones play during gestation, likely implicate them in the disruption of circadian signaling that results in the suppression of kisspeptin activation in pregnancy.

85 Chapter 5 Although no diurnal regulation of AVPV Kiss1 mRNA expression during pregnancy was noted in our study, we did reveal a number of changes in gene expression during pregnancy. AVPV Kiss1 expression was elevated at day 10 of pregnancy compared to diestrus, in line with the findings of Roa et al. (2006) showing elevated Kiss1 mRNA in the whole hypothalamus during pregnancy, although the authors suggest that this is not likely attributable to high levels of E2 and P4. Moreover, at day 18 we saw a within day increase in ARC Kiss1, despite relatively high E2 levels and no observable increase in plasma LH levels. We also saw an increase in Kiss1r at day 18. Avp mRNA expression increased at day 14 and 18 of pregnancy and within day effects in Avpr1a were seen at day 10. We can only speculate what these changes reflect, but an attractive idea may be that changes relate to parturition or the phenomenon of postpartum ovulation in the mouse. Clearly this requires further study.

Although the present study demonstrated a four-hour time lag between the peak in Kiss1 mRNA levels and that of plasma LH levels, Robertson et al. (2009) have previously shown these two events occur in synchrony. This discrepancy in timing may be due in part to the shorter time intervals between collections in the Robertson (2009) study; one- to two-hour intervals around the time of the LH surge, compared to four-hour intervals in the current study, which may have failed to capture the true peak of LH concentrations as well as Kiss1 mRNA levels. Another contributing factor could be the large variation in plasma LH concentrations in the current study at each time-point. Samples were collected across a one-hour period, within 30 min of the designated time, thus variations in short- lived peaks of LH levels would contribute to variation in the four-hourly measures.

Lastly, we observed no difference in Npvf expression between diestrus and proestrus. This is in contrast to some of the known literature, which indicates that RFRP expression is lowest at proestrus and maximal at diestrus (Gibson et al. 2008), consistent with its role as a GnRH inhibitor and in the negative feedback effects of E2 present for the majority of the female mouse ovulatory cycle (Kriegsfeld et al. 2006; Gibson et al. 2008; Ducret, Anderson & Herbison 2009). The disparity between our data and previous research may be explained by the differing methods used to measure RFRP levels; Gibson and colleagues (2008) used immunohistochemical procedures to determine the number of RFRP-expressing cells. It is likely in this case that Npvf message levels are not an accurate reflection of RFRP protein levels, thus further studies using either western blots or immunohistochemistry are needed to confirm our results. Moreover, our anterior

86 Chapter 5 hypothalamus Npffr data would seem to show the inverse of the expected trend, with expression at proestrus increased around the time of the LH surge, suggesting that RFRP may even follow a similar pattern to kisspeptin activation. However, it is also important to note that there is currently no consensus in the literature on the regulation of RFRP neurons by estradiol. Estradiol treatment has been shown to have markedly different effects on RFRP levels in female rodents; in some instances decreasing (Molnár et al. 2011; Poling et al. 2012), increasing (Iwasa et al. 2012) or having no effect (Quennell et al. 2010).

The current findings reveal a spike in kisspeptin expression during proestrus that is concurrent with the pre-ovulatory LH surge and preceded by an increase in AVP receptor expression, which may indicate the transmission of a circadian signal from the SCN. Pregnancy onset abolishes these temporal patterns and the subsequent LH surge. While RFRP-3 may inhibit GnRH and/or gonadotropin secretion, we find that neither the peptide nor its receptor exhibits a lower level of expression during proestrus around the time of the LH surge; furthermore, there was no evidence of diurnal variation in RFRP-3 prior to or during pregnancy. Our data are the first to show that the diurnal variation in hypothalamic kisspeptin expression seen at proestrus is abolished during pregnancy, in spite of high estradiol levels. We speculate that this is due to a disruption of the circadian signal, possibly along the SCN-AVPV pathway or at a hormonal level.

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Figure 7.

Proestrus PM SCN AVPV mPOA Circadian Clock Kisspeptin Neuron GnRH Neuron

V1a Kiss1 GnRH Kisspeptin

ER GPR54

Estradiol Late Pregnancy SCN AVPV mPOA Circadian Clock Kisspeptin Neuron GnRH Neuron

V1a Kisspeptin GnRH

ER GPR54

Estradiol Progesterone ? Prolactin

Figure 5.7 Proposed model indicating the change between proestrus and late pregnancy with the role of AVPV kisspeptin neurons in generating the GnRH/LH surge. At proestrus, kisspeptin neurons in the AVPV receive circadian information from the SCN via vasopressin receptors (V1a), and sense circulating estradiol via estrogen receptors (ER). Kisspeptin neurons are activated in the event that estrogenic and circadian signals are both high, activating GnRH neurons. In late pregnancy, despite high estrogen and an operant clock there is a disruption of kisspeptin output possibly due to reduction in V1a and high progesterone and/or prolactin. Figure adapted from (Smith et al. 2006).

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Chapter 6 Effect of Progesterone and Prolactin on the Circadian Rhythm of Kisspeptin in Ovariectomised E2-Treated Mice

6.1 Introduction The pre-ovulatory GnRH/LH surge that occurs in female rodents requires the combination of high E2 concentrations and a circadian signal predicting the onset of darkness. The subsequent spike in kisspeptin levels in the AVPV of the hypothalamus ultimately triggers the surge in LH (Levine 1997; Christian & Moenter 2010; Williams 3rd et al. 2011). The master pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus orchestrates the precise timing of the LH surge along with the synchronous activation of kisspeptin in the late afternoon of proestrus (Smith et al. 2006; Robertson et al. 2009; Williams 3rd et al. 2011).

We have previously shown that the circadian rhythm of kisspeptin observed during proestrus is absent during pregnancy and the LH surge does not occur, in spite of the high

E2 levels at this time (see Chapter 5). As such, it would appear that circadian input from the SCN to kisspeptin neurons in the AVPV is disrupted during pregnancy. Given that the circadian rhythms of core clock genes in the SCN persist throughout pregnancy (Wharfe et al. 2016), the absence of the kisspeptin rhythm during pregnancy is not likely due to a loss of circadian input. Besides, any disruption of clock gene rhythms is likely to have a detrimental effect on pregnancy or the health of the fetus, as evidenced by the considerably lower rates of pregnancy success in mice exposed to altered photoperiods (Summa, Vitaterna & Turek 2012). Rather, it is possible that certain hormones, other than

E2, that change during pregnancy override the signals that give rise to the kisspeptin spike and subsequent LH surge.

P4 and PRL are two such hormones. High levels of P4 are essential for implantation and the maintenance of pregnancy (Bole-Feysot et al. 1998); plasma P4 rises continually until late in gestation, then falls precipitously just prior to parturition (Murr et al. 1974; Barkley, Geschwind & Bradford 1979; Waddell, Bruce & Dharmarajan 1989). PRL possesses luteotrophic functions, among many others, and is indirectly involved in P4 production through its effects on the corpus luteum (Bole-Feysot et al. 1998). Plasma

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PRL concentrations during pregnancy exhibit almost the opposite pattern to that of P4; levels are high in early pregnancy and exhibit a biphasic pattern, consisting of one nocturnal and one diurnal surge, that ceases after day 8 of pregnancy (Smith & Neill 1976). In mid- and late pregnancy PRL levels decline and plateau, but surge again closer to parturition (Morishige, Pepe & Rothchild 1973; Murr et al. 1974).

P4 signalling in AVPV kisspeptin neurons is critical for kisspeptin neuronal activation, and thus normal LH surges and fertility in female rodents (Stephens et al. 2015). High PRL levels have been shown to inhibit kisspeptin expression in both the ARC and AVPV, thereby suppressing LH release (Araujo-Lopes et al. 2014; Brown, Herbison & Grattan

2014). The direct effects that both P4 and PRL have on kisspeptin, along with the essential roles that these hormones have during gestation, likely implicate them in the suppression of kisspeptin activation during pregnancy.

In the present study we sought to investigate the effect that P4 and PRL have on the circadian rhythm of kisspeptin in pregnant mice. In order to isolate the effects of each hormone, we performed these investigations on ovariectomised mice receiving E2 replacement.

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6.2 Materials and Methods 6.2.1 Animals Nulliparous C57BL/6J mice (6 weeks old) were supplied by the Animal Resources Centre (Murdoch, Australia). Mice were maintained in an environmentally controlled room under a 12-h light, 12-h dark cycle (lights on from 0700 – 1900 h) with ad libitum access to food and water. Lights on at 0700 h was defined as Zeitgeber time (ZT)-zero, with sampling times described as relative to ZT-zero. All procedures involving animals were conducted with the approval of the Animal Ethics Committee of The University of Western Australia.

6.2.2 Ovariectomy protocol Mice were bilaterally ovariectomised (OVX) under isoflurane anaesthesia as described in

Robertson et al. (2009). At the time of OVX, mice were implanted with a 0.1 mg E2 pellet

(Innovative Research of America, cat# NE-121), either alone (E2), or in conjunction with a 25 mg P4 pellet (E2 + P4; Innovative Research of America, cat# P-131) or a micro- osmotic pump (Alzet, model# 1002) containing ovine PRL (E2 + PRL; Sigma cat# L6520; 400 µg in 100 µl saline = 1 mg/kg body weight based on Sapsford et al. 2012). Mice were given one week to recover from surgery before being killed for tissue collection.

6.2.3 Tissue collection Whole brains and blood samples were collected from mice at 0800 (ZT1) or 1800 (ZT11) under isoflurane anaesthesia. Whole brains were frozen on dry ice immediately following removal. A trunk blood sample was obtained following decapitation and collected in a tube containing EDTA (100 µL per mL of blood). Plasma was obtained following centrifugation of the blood sample and stored at -20°C until required.

6.2.4 Hypothalamic gene expression

6.2.4.1 RNA extraction Hypothalami were dissected from whole brain samples and bisected into anterior (containing the AVPV) and posterior (containing the ARC) portions as described in Quennell et al. (2011). Total RNA was extracted from anterior and posterior hypothalami using Qiazol (Qiagen,) according to the manufacturer’s instructions. The RNA was dissolved in 40 µL of RNase-free water, placed on ice for 5 min and thoroughly vortexed. 91 Chapter 6 RNA was quantitated using the Nanodrop ND-1000 spectrophotometer (Thermo Scientific) at 260 nm and stored at -80 °C until required.

6.2.4.2 Reverse transcription RNA samples were treated with RQ1 RNase-free DNase (Promega, cat# M6101) to remove any DNA present. Samples were made up to a total volume of 10 µL with 1 µg of RNA, 1 µL of RQ1 RNase-Free DNase 10x Buffer, 2 µL of RQ1 RNase-Free DNase and nuclease-free water, and incubated at 37°C for 30 min. RQ1 DNase Stop Solution (1 µL) was added to each sample to terminate the reaction, and samples were incubated at 65°C for 10 min to deactivate the DNase.

Total RNA (1 µg) was reverse transcribed to cDNA with random primers (Promega, cat# C1181) using Moloney Murine Leukemia Virus Reverse Transcriptase, RNase H Minus, Point Mutant (M-MLV RT (H-)) (Promega, cat# M3683). The resultant cDNAs were purified using the Ultraclean PCR Cleanup kit (MoBio Industries, cat# 12500-250), according to the manufacturer’s instructions.

6.2.4.3 Real-time PCR Analyses of mRNA levels for total Kiss1, Kiss1 receptor (Kiss1r), arginine vasopressin (Avp), arginine vasopressin receptor 1a (Avpr1a), neuropeptide VF precursor (Npvf), neuropeptide FF receptor (Npffr), Arntl which encodes for Bmal1, and Nr1d1 which encodes for Rev-erbα were performed by quantitative RT-PCR on the Rotorgene 6000 (Corbett Industries). Primers (Table 6.1) for total Kiss1, Kiss1r, Bmal1, Rev-erbα and the reference genes hypoxanthine-guanine phosphoribosyltransferase (Hprt), succinate dehydrogenase subunit A (Sdha) and beta-2 microglobulin (B2m) were designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov). Each of the selected primer pairs was positioned to span an intron to ensure no product was amplified from genomic DNA. The resulting amplicons were sequenced to confirm specificity. Standard curves for each product were generated from gel-extracted (QIAquick Gel Extraction Kit, Qiagen) PCR products using 10-fold serial dilutions and the Rotorgene 6000 software. The PCR cycling conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 1 s, annealing at 60 °C for 15 s and extension at 72 °C for 5 s. Melt curve analysis was performed to confirm amplification specificity for each gene.

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Primers for Avp and Avpr1a were purchased as QuantiTect Primer Assays (Qiagen, cat# QT00249389, QT00113169, QT00278551, QT001169196). The PCR conditions were as follows: initial activation 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 5 s and combined annealing/extension at 60 °C for 10 s. Melt curve analysis was performed to confirm amplification specificity.

All samples were standardized against Hprt, Sdha and B2m using the GeNorm algorithim (Vandesompele et al. 2002).

6.2.5 Plasma analyses

6.2.5.1 Pituitary hormones Plasma levels of FSH, LH and PRL were measured in a 10 µl sample using the Milliplex Map Mouse Pituitary Magnetic Bead kit (Milliplex, cat# MPTMAG-49K) according to the manufacturer’s instructions. The plate was run on a MAGPIX system (Luminex Corporation) with xPONENT software to analyse the median fluorescent intensity data and obtain analyte concentrations.

6.2.6 Statistical analysis Statistical analyses were conducted using GraphPad Prism 6 (GraphPad Software). Data are expressed as the mean ± SEM, with an n of 6-8 per ZT in each treatment group. Differences were considered significant when P < 0.05. Two-way ANOVAs were used to analyse all data with treatment and circadian time as factors, followed by Fisher’s LSD post hoc tests where appropriate. Unpaired t-tests within treatment groups, and one-way ANOVAs between treatment groups were conducted where significant interaction effects were observed in two-way ANOVA analyses.

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Table 6.1 Primer sequences and conditions for quantitative PCR

Gene Sequence Size Annealing (bp) temp (°C) Kiss1 F: 5’-CTCTGTGTCGCCACCTATGG-3’ 126 60 R: 5’-AGGCTTGCTCTCTGCATACC-3’ Kiss1r F: 5’-TGCTGGGAGACTTCATGTGC-3’ 102 60 R: 5’-CATACCAGCGGTCCACACTC-3’ Bmal1 F: 5’-CGTGCTAAGGATGGCTGTTC-3’ 166 60 R: 5’-CTTCCCTCGGTCACATCCTA-3’ Rev-erbα F: 5’-ATTGCCCAGGGGGCGAGAGA-3’ 292 60 R: 5’-GCCAAAAGAGCGGGCAGGGT-3’ Hprt F: 5’-GCAGTACAGCCCCAAAATGG-3’ 80 60 R: 5’-AGTCTGGCCTGTATCCAACAC-3’ Sdha F: 5’-ATGGAAAATGGGGAGTGCCG-3’ 123 60 R: 5’-ACAGCTGAAGTAGGTTCGGC-3’ B2m F: 5’-TGACCGGCCTGTATGCTATC-3’ 128 60 R: 5’-GATTTCAATGTGAGGCGGGTG-3’

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6.3 Results 6.3.1 Hypothalamic gene expression Kiss1 mRNA expression in the AVPV varied with treatment (P < 0.01) and there was a significant Treatment x Time-of-day interaction. Subsequent t-tests revealed that Kiss1 levels were higher at ZT11 than ZT1 for the OVX mice receiving only E2 but not P4 or PRL (P < 0.05; Figure 6.1A). Kiss1r mRNA expression in the anterior hypothalamus showed a significant Treatment x Time-of-day interaction and was subsequently found to be higher at ZT11 than at ZT1 only in the E2 group (P < 0.05; Figure 6.1B). A significant Treatment x Time-of-day interaction was seen in Avp mRNA expression in the anterior hypothalamus and levels were higher at ZT 11 compared to ZT1 only in mice treated with

E2 + P4 (P < 0.01; Figure 6.2A). Both Avpr1a (Figure 6.2B) and Rev-erbα (Figure 6.3B) mRNA expression were not affected by treatment or time-of-day, although Rev-erbα displayed a trend for greater expression at ZT11 in E2 only mice. There was a significant time-of-day effect on Bmal1 expression in the SCN (P < 0.001; Figure 6.3A).

6.3.2 Plasma hormone levels Overall, LH was affected by both time-of-day (P < 0.05) and treatment (P < 0.001), but post-hoc analysis showed no difference in LH levels between ZT1 and ZT13 within any of the treatment groups (Figure 6.4A). There was an effect of both time-of-day (P < 0.05) and treatment (P < 0.0001) on FSH concentrations and levels were lower at ZT1 compared to ZT11 only in the E2 group (P < 0.01; Figure 6.4B). There was a treatment effect (P < 0.0001) as well as a significant Treatment x Time-of-day interaction for PRL concentrations. Subsequent t-tests showed that PRL levels were higher at ZT1 compared to ZT11 only in mice receiving E2 alone (P < 0.05; Figure 6.4C). One-way ANOVAs revealed that at ZT1, PRL levels were highest in the E2 + P4 group (P < 0.01), while at

ZT11, PRL levels were higher in the E2 + P4 group compared to the E2 + PRL group only (P < 0.001; Figure 6.4C).

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A 15 E2 * E2+P4 E2+PRL 10 mRNA Kiss1 5 AVPV AVPV (Relative expression) (Relative 0

ZT1 ZT1 ZT1 B ZT11 ZT11 ZT11 15 *

10 mRNA

Kiss1r 5 AH (Relative expression) (Relative 0

ZT1 ZT1 ZT1 ZT11 ZT11 ZT11

Figure 6.1 AVPV Kiss1 mRNA expression and anterior hypothalamic

Kiss1r mRNA expression in OVX mice treated with estradiol (E2) only, E2 and progesterone (P4) or E2 and prolactin (PRL). A) Kiss1 expression was higher at ZT11 than at ZT1 within the E2 group only. Main effect of treatment, F (2, 29) = 6.288, P < 0.01; main effect of time-of-day, F (1, 29) = 0.5431, P > 0.05; interaction between treatment and time-of-day, F (2, 29) = 4.254, P < 0.05. B)

Kiss1r expression was higher at ZT11 than ZT1 only in mice treated with E2 alone. Main effect of treatment, F (2, 31) = 0.4035, P > 0.05; main effect of time- of-day, F (1, 31) = 0.8138, P > 0.05; interaction between treatment and time-of- day, F (2, 31) = 5.108, P < 0.05. Data are means ± SEM, n = 6-8 per group. *, P < 0.05 time-of-day effect.

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A 10 E2 8 E2+P4 * E2+PRL 6 mRNA

4 Avp Avp

AH 2 (Relative expression) (Relative 0

ZT1 ZT1 ZT1 ZT11 ZT11 ZT11 B 10

8

mRNA 6

4 Avpr1a 2 AH (Relative expression) (Relative 0

ZT1 ZT1 ZT1 ZT11 ZT11 ZT11

Figure 6.2 Avp and Avpr1a mRNA expression in the anterior hypothalamus of OVX mice treated with estradiol (E2) only, E2 and progesterone (P4) or E2 and prolactin (PRL). A) Avp expression was higher at ZT11 than ZT1 only in mice treated with E2 + P4. Main effect of treatment, F (2, 31) = 1.559, P > 0.05; main effect of time-of-day, F (1, 31) = 1.197, P > 0.05; interaction between treatment and time-of-day, F (2, 31) = 5.407, P < 0.01. B) Avpr1a expression was not affected by treatment or time-of-day. Main effect of treatment, F (2, 31) = 0.1018, P > 0.05; main effect of time-of- day, F (1, 31) = 0.002815, P > 0.05; interaction between treatment and time- of-day, F (2, 31) = 1.980, P > 0.05. Data are means ± SEM, n = 6-8 per group. *, P < 0.01 time-of-day effect.

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A 20 E2 E2+P4 15 E2+PRL mRNA * * 10 * Bmal1 5 SCN (Relative expression) (Relative 0

ZT1 ZT1 ZT1 B ZT11 ZT11 ZT11 20

15 mRNA α 10

Rev-erb 5 (Relative expression) (Relative SCN 0

ZT1 ZT1 ZT1 ZT11 ZT11 ZT11

Figure 6.3 Bmal1 and Rev-erbα mRNA expression in the suprachiasmatic nucleus of OVX mice treated with estradiol (E2) only, E2

and progesterone (P4) or E2 and prolactin (PRL). A) There was a significant time-of-day effect on Bmal1 expression. Main effect of treatment, F (2, 31) = 1.566, P > 0.05; main effect of time-of-day, F (1, 31) = 14.23, P < 0.001; interaction between treatment and time-of-day, F (2, 31) = 0.2646, P > 0.05. B) Rev-erbα expression was not affected by treatment or time-of-day. Main effect of treatment, F (2, 30) = 0.2027, P > 0.05; main effect of time-of-day, F (1, 30) = 2.136, P > 0.05; interaction between treatment and time-of-day, F (2, 30) = 1.053, P > 0.05. Data are means ± SEM, n = 6-8 per group. *, P < 0.05 time-of- day effect.

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A 200 E2 E2+P4 150 E2+PRL

100

50

PlasmaLH levels (pg/ml) 0

ZT1 ZT1 ZT1 B ZT11 ZT11 ZT11 1000

800 * 600

400

200

0 PlasmaFSH levels (pg/ml)

ZT1 ZT1 ZT1 C ZT11 ZT11 ZT11 25000 ## #

20000 ##

15000 *

10000

5000

0

Plasmaprolactin levels (pg/ml) ZT1 ZT1 ZT1 ZT11 ZT11 ZT11

Figure 6.4 Plasma LH, FSH and PRL concentrations in OVX mice treated with estradiol (E2) only, E2 and progesterone (P4) or E2 and prolactin (PRL). A) Luteinizing hormone levels were significantly affected by treatment and time-of-day. Main effect of treatment, F (2, 41) = 9.525, P < 0.001; main effect of time-of-day, F (1, 41) = 5.834, P < 0.05; interaction between treatment and time-of-day, F (2, 41) = 0.2682, P > 0.05. B) Follicle-stimulating hormone levels were lower at ZT11 compared to ZT1 only in E2-treated mice. Main effect of treatment, F (2, 41) = 21.30, P < 0.0001; main effect of time-of-day, F (1, 41) = 6.451, P < 0.05; interaction between treatment and time-of-day, F (2, 41) = 1.318, P > 0.05. C) PRL levels were higher at ZT11 compared to ZT1 only in

E2-treated mice. Main effect of treatment, F (2, 41) = 16.99, P < 0.0001; main effect of time-of-day, F (1, 41) = 0.3085, P > 0.05; interaction between treatment and time-of-day, F (2, 41) = 3.728, P < 0.05. Data are means ± SEM, n = 6-8 per group. *, P < 0.05 time-of-day effect. #, P < 0.01, ##, P < 0.001 treatment effect.

99 Chapter 6 6.4 Discussion Chapter 5 shows that the peak in AVPV Kiss1 gene expression observed in the late afternoon of proestrus is abolished during pregnancy despite elevated levels of E2 and a functional circadian clock. P4 and PRL are two hormones potentially involved in the suppression of the circadian kisspeptin rhythm during pregnancy. The findings of the present study show that the increase in AVPV Kiss1 expression in OVX mice in the presence of elevated E2 levels is abolished with P4 and PRL treatment.

Kiss1 mRNA levels in the AVPV were increased at ZT11 compared to ZT1 in OVX mice treated only with E2, as expected. This is analogous to the increase in Kiss1 expression seen in the late afternoon of proestrus in intact cycling females (Chapter 5; Smith et al.

2006; Adachi et al. 2007). Female mice receiving P4 or PRL in addition to E2 did not demonstrate the same pattern of Kiss1 mRNA expression, suggesting that these hormones play a role in preventing kisspeptin activation even in the presence of elevated E2 concentrations and an active circadian signal. The exact mechanism is unclear but it is possible that P4 and PRL exert their effects directly on kisspeptin neurons, as kisspeptin neurons in the AVPV express both P4 and PRL receptors (Clarkson et al. 2008; Kokay,

Petersen & Grattan 2011). In line with the effects of P4 and PRL on Kiss1, Kiss1r expression was also increased in the anterior hypothalamus at ZT11 in mice receiving E2 only, but not in those treated with P4 or PRL.

The expression of the clock gene Bmal1 was strongly affected by the time-of-day, suggesting that SCN rhythmicity is intact in all treatment groups and this is consistent with our previous data demonstrating robust clock gene rhythms throughout pregnancy (Wharfe et al. 2016). Thus, any potential suppression of the circadian kisspeptin rhythm by P4 and PRL must occur downstream of SCN gene transcription. In contrast, Rev-erbα expression was not affected by the time-of-day. However, it is important to note that a substantial limitation exists in relation to the interpretation of this clock gene expression data; the rhythmicity of clock genes is difficult to determine through an ANOVA based on two time points alone. Circadian rhythmicity is typically assessed by a cosinor analysis, which involves fitting a cosine curve to a number of time points representative of a 24-h period, and calculating the strength of the fit. ANOVA aside, Bmal1 expression appears to decrease from ZT1 to ZT11, while Rev-erbα expression showed a trend for an increase from ZT1 to ZT11, which is consistent with their known antiphase relationship (Preitner et al. 2002; Wharfe et al. 2016), and confirms our observations from Chapter 5. 100 Chapter 6

Although Kiss1 mRNA expression was increased one hour before lights off (ZT11) as compared to the morning time point (ZT1), this was not accompanied by the expected surge in LH levels. One possibility is that because the rise in kisspeptin expression can precede the LH surge (Chapter 5; Chassard et al. 2015), the increase in circulating LH may have occurred at a later time. This theory may bear some weight given that the most recent studies in E2-treated OVX mice report LH surge induction at the time of lights off (Dungan et al. 2007; Dror, Franks & Kauffman 2013). However, Robertson et al. (2009) show that LH levels are significantly increased in E2-treated mice one hour before lights off, which informed the sampling time of the current study. Although the mice in their study were housed in a 14-h light, 10-h dark cycle (Robertson et al. 2009), as opposed to the 12-h light, 12-h dark cycle in the present study, it is unlikely that the different light cycle would have affected the timing of the LH surge, given that it is the onset of darkness which signals the rise in the LH, but it may be a possibility. It is also important to consider that studies in E2-treated animals do not measure LH at short enough intervals around the time of lights off and hence do not capture the potential spread of the LH surge. By sampling hourly, Miller et al. (2004) showed that individual mice surged at different times between ZT10 and ZT17, with the average peak amplitude occurring 1-2 hours after lights off. This was however, in intact cycling mice, which are subject to natural variation in the timing of rising E2 levels unlike OVX E2-treated mice.

FSH levels were significantly lower at ZT11 compared to ZT1 in mice treated with E2 alone, which may be attributed to the negative feedback effects of estrogen on gonadotropins that operate prior to the switch to positive feedback and the subsequent LH surge. In line with this, a study in women showed that FSH levels declined in response to

E2 infusion until the occurrence of the LH and FSH surge (Liu & Yen 1983).

PRL levels were increased between ZT1 and ZT11 in mice treated with E2 alone, consistent with literature showing that PRL starts to increase in the late afternoon of proestrus, and peaks at the time of the LH surge (Butcher, Collins & Fugo 1974; Smith, Freeman & Neill 1975). Unexpectedly, overall PRL levels were higher in mice treated with P4 than in those treated with E2 alone or with PRL. This is consistent with previous studies in rats and guinea pigs demonstrating that treatment with E2 and P4 increases circulating PRL levels compared to treatment with E2 alone (Caligaris, Astrada &

Taleisnik 1974; Bethea et al. 1995). This P4-stimulated release of endogenous PRL appears to have resulted in levels of PRL that are greater than those in PRL-supplemented 101 Chapter 6 mice. The dose of PRL used in the current study (1 mg/kg body weight per day; Sapsford et al. 2012) may have only been effective in producing circulating PRL concentrations at the low end of the range normally observed in pregnancy; the literature shows that PRL levels during the second half of rodent pregnancy (which are relatively low compared to the first half) vary from 10 ng/mL (Amenomori, Chen & Meites 1970; Morishige, Pepe & Rothchild 1973) to 100 ng/mL (Murr et al. 1974). However, it is important to note that differences in sample preparation and assay technique (radioimmunoassay vs Luminex assay in the present study) may mean that PRL concentrations between the current and previous studies are not necessarily directly comparable. Nonetheless, we are confident that our PRL treatment regime achieved the desired effect. Moreover, the observation that

P4 treatment resulted in elevated PRL levels suggests that the suppressive effect of P4 on Kiss1 expression may, in fact, be indirectly caused by the increase in PRL levels, rather than P4 itself, although this requires further investigation.

Avpr1a expression in the anterior hypothalamus was not increased at ZT11 in the E2 only group (or any of the other treatment groups), consistent with data from Chapter 5 and that of Smarr and colleagues (2013), showing that the rise in Avpr1a expression occurs between 4 to 8 hours before the increase in Kiss1 expression. Avp expression was not different between ZT1 and ZT11 in mice treated with E2 only, which again is consistent with the findings in Chapter 5. In contrast, Avp levels were higher at ZT11 in the E2 + P4 group. P4 administration has been shown to elevate AVP levels in the pituitary gland of female rats and this is thought to involve noradrenergic inhibition in the paraventricular nucleus (Crowley et al. 1978). However, whether this mechanism underlies the P4- induced increase in Avp mRNA expression in the current study is unknown, as the effect of P4 on hypothalamic AVP has not been previously studied.

In conclusion, we show that the rise in AVPV Kiss1 expression that precedes the LH surge in E2-treated mice, does not occur in the presence of elevated levels of P4 or PRL.

This indicates that P4 and PRL may therefore be responsible for the suppression of the circadian kisspeptin rhythm during pregnancy, potentially acting directly on their respective receptors located on kisspeptin neurons.

102

Chapter 7 RNA Sequencing Analysis of the Mouse Anterior Hypothalamus at Proestrus and During Pregnancy

7.1 Introduction The kisspeptin neuronal population in the AVPV is crucial to the preovulatory LH surge that occurs in female rodents. The central circadian clock, located in the suprachiasmatic nucleus (SCN) of the hypothalamus, governs the precise timing of the LH surge via the AVPV kisspeptin neurons (Smarr, Morris & de la Iglesia 2012). Studies have shown that in the late afternoon of proestrus, the circadian pattern of increased AVPV Kiss1 expression and LH concentrations are synchronous (Smith et al. 2006; Robertson et al.

2009; Williams 3rd et al. 2011). High circulating E2 levels, in combination with information from the SCN indicating the time-of-day, are required to trigger the LH surge (Levine 1997; Christian & Moenter 2010; Williams 3rd et al. 2011). The AVPV integrates the estrogenic and circadian signals, resulting in kisspeptin activation and the subsequent rise in LH levels (Smarr, Gile & de la Iglesia 2013).

As shown in Chapter 5, the evening (ZT13) increase in Kiss1 expression observed in proestrus females was abolished during pregnancy, along with the LH surge, in spite of elevated circulating E2 levels, indicating that the circadian signal may be disrupted in pregnancy. Given that clock gene rhythmicity in the SCN remains relatively robust during pregnancy (Wharfe et al. 2016), it is likely that potential alterations responsible lie downstream within, but not restricted to, the AVPV. It is possible that the rhythmicity of other genes in the anterior hypothalamus show a similar circadian pattern at proestrus that is disrupted during pregnancy.

In the present study, in order to shed light on genes that could be involved in the suppression of the kisspeptin circadian rhythm in pregnancy, we utilised RNA sequencing in the anterior hypothalamus to uncover the genes that are expressed differently between proestrus and pregnancy. RNA sequencing is a next generation sequencing technique that allows high throughput sequencing and enables characterisation of the entire transcriptome.

103 Chapter 7 7.2 Materials and methods 7.2.1 Animals Anterior hypothalami samples used in this study were previously collected in the study reported in Chapter 5. Briefly, nulliparous C57Bl/6J mice (6 weeks old) were supplied by the Animal Resources Centre (Murdoch, Western Australia, Australia). Mice were maintained in an environmentally controlled room under a 12-h light, 12-h dark cycle (lights on from 0700 – 1900 h) with ad libitum access to food and water. By convention in chronobiology, lights off at 1900 h was defined as Zeitgeber time (ZT) 12, with sampling times described as relative to ZT12. Female mice were subjected to a daily vaginal smear to determine estrous cycle stage and monitored for three full cycles. A sub- group of female mice were mated overnight, and pregnancy was confirmed by observation of a vaginal plug the following morning, which was designated day 1 of pregnancy. Whole brain and blood sample collections were made at 4-hour intervals commencing at 0800 h on proestrus of the cycle, or days 10 or 14 of pregnancy (term = 19 days). All procedures involving animals were conducted with the approval of the Animal Ethics Committee of The University of Western Australia.

7.2.2 Tissue collection Whole brains were collected from mice under isoflurane anaesthesia at 0800 (ZT1), 1200 (ZT5), 1600 (ZT9), 2000 (ZT13), 2400 (ZT17) or 0400 (ZT21) in order to obtain a reasonable circadian profile of gene expression (n = 3 at proestrus and day 10 of pregnancy, n = 2 at day 14 of pregnancy). A red light (>600 nm wavelength) was used to facilitate collection of tissues in the dark phase. Whole brains were frozen by being placed on dry ice immediately following removal.

7.2.3 Hypothalamic gene expression

7.2.3.1 RNA extraction Anterior hypothalami (containing the AVPV and SCN) were dissected from whole brain samples as described in (Quennell et al. (2011). Total RNA was extracted from anterior hypothalami using Qiazol (Qiagen, Hilden, Germany; Cat# 79306) according to the manufacturer’s instructions. The RNA pellet was dissolved in 50 µL of RNase-free water, placed on ice for 5 min and thoroughly vortexed. RNA was quantitated using the Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA) at 260 nm and stored at -80 °C until required. 104 Chapter 7

7.2.3.2 Reverse transcription RNA samples were treated with RQ1 RNase-free DNase (Promega, Madison, Wisconsin, USA; cat# M6101) to remove any genomic DNA present. Reactions were made up to a total volume of 10 µL with 1 µg of RNA, 1 µL of RQ1 RNase-Free DNase 10x Buffer, 2 µL of RQ1 RNase-Free DNase and nuclease-free water, and incubated at 37°C for 30 min. 1 µL of RQ1 DNase Stop Solution was added to each sample to terminate the reaction, and samples were incubated at 65°C for 10 min to deactivate the DNase. Total RNA (1 µg) was reverse transcribed to cDNA with random primers (Promega, cat# C1181) using Moloney Murine Leukemia Virus Reverse Transcriptase, RNase H Minus, Point Mutant (M-MLV RT (H-)) (Promega, Madison, Wisconsin, USA; cat# M3683). The resultant cDNAs were purified using the Ultraclean PCR Cleanup kit (MoBio Laboratories, Carlsbad, Calfornia, USA, cat# 12500-250), according to the manufacturer’s instructions.

7.2.3.3 RNA sequencing RNA sequencing was performed by the Institute of Metabolic Science, University of Cambridge, UK. cDNA libraries were prepared using the Illumina TruSeq Stranded mRNA Library Preparation Kit (Illumina, San Diego, California, USA; Cat# RS-122- 2101) according to manufacturer’s instructions, using 1 µg RNA. Single-end sequencing was performed in an Illumina HiSeq 2500 (Illumina, San Diego, California, USA) for 50 cycles.

7.2.3.4 Analysis Analysis was carried out within the R programming language and software environment (https://www.r-project.org) using various software packages as previously detailed in Trapnell et al. (2012). Sequence reads were mapped to the mouse genome (NCBI m38 build), including intron/exon annotations (both downloaded from the UCSC Genome Bioinformatics Site), using TopHat (https://ccb.jhu.edu/software/tophat/index.shtml; Trapnell et al. 2012). Cufflinks (http://cole-trapnell-lab.github.io/cufflinks/) was used to assemble the aligned reads into individual transcripts, and to normalise raw read counts by gene length and library size (Trapnell et al. 2012). A heat map of differentially expressed genes was generated using GeneSpring software (Agilent Technologies, Santa Clara, California, USA).

105 Chapter 7 Statistical analysis to determine which genes were differentially expressed between days or time points was conducted using EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html; Robinson, McCarthy & Smyth 2010). Differences were considered significant at q < 0.05 (where q is the false discovery rate (FDR)-adjusted P-value). In addition, Graphpad Prism was used to conduct Two-way ANOVAs to determine and confirm specific differences in target genes of the effect of day or time-of-day on the expression of Kiss1, Kiss1r, Avp, Avpr1a, Npffr, Bmal1 and Rev-erbα, followed by Tukey’s post-hoc tests where appropriate. Differences were considered significant at P < 0.05.

106 Chapter 7

7.3 Results Figure 7.1 shows a heat map featuring all genes that were differentially expressed by ≥ 2-fold.

Genes of interest (Figure 7.2) Kiss1 expression was significantly affected by day (P < 0.0001), and exhibited no time- of-day variation at any stage, although there looked to be a trend for a peak in Kiss1 expression at ZT13 of proestrus (~1.5-fold increase compared to ZT1, ZT5 and ZT9). Kiss1r and Avpr1a expression was not affected by either day or time-of-day. There was an effect of day on the expression of Avp and Npffr (P < 0.01), and neither showed time- of-day variation at any stage. Both Bmal1 and Rev-erbα expression was significantly affected by time-of-day (P < 0.0001) and appeared to exhibit an antiphase relationship. Bmal1 showed time-of-day variation at proestrus and on day 10 of pregnancy but not day 14, while Rev-erbα showed time-of-day variation at all stages.

Genes that were differentially expressed between proestrus and pregnancy (Figure 7.3) Expression of haemoglobin, beta adult s chain (Hbb-bs), haemoglobin, beta adult t chain (Hbb-bt), haemoglobin alpha, adult chain 2 (Hba-a2), complement component 1, r subcomponent A (C1ra), suppressor of cytokine signalling 2 (Socs2), cytokine inducible SH2-containing protein (Cish), adhesion molecule with Ig-like domain 2 (Amigo2) and potassium voltage-gated channel subfamily H member 8 (Kcnh8) was significantly higher at days 10 and 14 of pregnancy compared to proestrus (q < 0.05). Expression of albumin (Alb) was higher at only day 10 of pregnancy compared to proestrus (q < 0.05).

Genes that were differentially expressed between ZT1/ZT5 and ZT13 at proestrus and during pregnancy (Figure 7.4) Expression of perilipin 4 (Plin4), which coats intracellular lipid storage droplets, hypoxia- inducible factor 3 alpha subunit (Hif3a), the transcription factors zinc finger and BTB domain containing 16 (Zbtb16) and TSC22 domain family member 3 (Tsc22d3), serum/glucocorticoid regulated kinase 1 (Sgk1), circadian associated repressor of transcription (Ciart), and period circadian clock 3 (Per3) was significantly higher at ZT13 compared to ZT1/ZT5 at proestrus and at days 10 and 14 of pregnancy (q < 0.05).

107 Chapter 7 Expression of integral membrane protein claudin 5 (Cldn5) was significantly lower at ZT13 compared to ZT1/ZT5 at proestrus and at days 10 and 14 of pregnancy (q < 0.05).

Genes that were differentially expressed between ZT1/ZT5 and ZT13 at proestrus only (Figure 7.5) Expression of kinase insert domain receptor (Kdr), which is a receptor for vascular endothelial growth factor (VEGF), leucine aminopeptidase 3 (Lap3), which is involved in the processing of intracellular proteins and fibroblast growth factor receptor-like 1 (Fgfrl1) was significantly lower at ZT13 compared to ZT1/ZT5 at proestrus (q < 0.05), but not during day 10 or day 14 of pregnancy. WEE1 G2 checkpoint kinase (Wee1), which acts as a negative regulator of entry into mitosis, and period circadian clock 2 (Per2) was significantly higher at ZT13 compared to ZT1/ZT5 at proestrus (q < 0.05), but not during day 10 or day 14 of pregnancy.

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115 Chapter 7 2-fold in 2-fold ≥ Figure 7.1 Heat map of all genes that are differentially expressed by by expresseddifferentially are that genes all of map Heat Figure 7.1 the anterior of mice at proestrus, hypothalamus 14 of pregnancy. 10 and at days and Numbers in square brackets at the top of each column indicate the day of estrous cycle/ estrous of day the indicate column each of top the at brackets square in Numbers pregnancy (first number) and the time-of-day in ZT (second number).

116 Chapter 7

A Kiss1 B Kiss1r 2.5 4

2.0 ### 3 ### 1.5 2 1.0 1 0.5 Counts per million Counts per million

0.0 0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Proestrus Day 10 Day 14 Proestrus Day 10 Day 14 C Avp D Avpr1a 8 # ## 3

6 2

4

1 2 Counts per million Counts per million

0 0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Proestrus Day 10 Day 14 Proestrus Day 10 Day 14 E Bmal1 F Rev-erba 5.5 7.0 * * * * * * 5.0 6.5

4.5 6.0 Counts per million Counts per million4.0 5.5 0.5 0.5 0.0 0.0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Proestrus Day 10 Day 14 Proestrus Day 10 Day 14 G Npffr 2.5 # # 2.0

1.5

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0.5 Counts per million

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ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Proestrus Day 10 Day 14

117 Chapter 7

Figure 7.2 Expression of Kiss1, Kiss1r, Avp, Avpr1a, Bmal1, Rev-erbα and Npffr at proestrus and days 10 and 14 of pregnancy. A) Kiss1 expression was lower during pregnancy than at proestrus. No time-of-day variation was observed at any stage. Main effect of day, F (2, 30) = 13.96, P < 0.0001; main effect of time-of-day, F (5, 30) = 0.6029, P > 0.05; interaction between day and time-of-day, F (10, 30) = 0.4837, P > 0.05. B) Kiss1r expression was not affected by day or time-of-day. Main effect of day, F (2, 30) = 0.4436, P > 0.05; main effect of time-of-day, F (5, 30) = 1.893, P > 0.05; interaction between day and time-of-day, F (10, 30) = 0.5142, P > 0.05. C) Avp expression was higher during pregnancy than at proestrus. Main effect of day, F (2, 30) = 6.331, P < 0.01; main effect of time-of-day, F (5, 30) = 0.8700, P > 0.05; interaction between day and time-of-day, F (10, 30) = 1.122, P > 0.05. D) Avpr1a expression was not affected by day or time-of-day. Main effect of day, F (2, 30) = 3.169, P > 0.05; main effect of time-of-day, F (5, 30) = 1.903, P > 0.05; interaction between day and time-of- day, F (10, 30) = 1.825, P > 0.05. E) There was a time-of-day effect on Bmal1 expression. Main effect of day, F (2, 30) = 2.950, P > 0.05; main effect of time-of-day, F (5, 30) = 12.75, P < 0.0001; interaction between day and time-of-day, F (10, 30) = 1.109, P > 0.05. F) There was a time-of-day effect on Rev-erbα expression. Main effect of day, F (2, 30) = 0.2058, P > 0.05; main effect of time-of-day, F (5, 30) = 24.70, P < 0.0001; interaction between day and time-of-day, F (10, 30) = 1.314, P > 0.05. G) Npffr expression was higher during pregnancy than at proestrus. Main effect of day, F (2, 30) = 5.693, P < 0.01; main effect of time-of-day, F (5, 30) = 1.854, P > 0.05; interaction between day and time-of-day, F (10, 30) = 0.6399, P > 0.05. Data are means ± SEM, n = 2-3 per group. Two-way ANOVA, #, P < 0.05, ##, P < 0.01, ###, P < 0.001 between- day effect compared to proestrus. *indicates the peak of the circadian rhythms of Bmal1 and Rev-erbα.

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0 0 0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Proestrus Day 10 Day 14 Proestrus Day 10 Day 14 Proestrus Day 10 Day 14 D C1ra E Socs2 F Cish 3 5 # 5 # # # # # 4 4 2 3 3

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ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Proestrus Day 10 Day 14 Proestrus Day 10 Day 14 Proestrus Day 10 Day 14

Figure 7.3 Genes that were differentially expressed between proestrus and pregnancy. Expression of A) haemoglobin, beta adult s chain (Hbb-bs), B) haemoglobin, beta adult t chain (Hbb-bt), C) haemoglobin alpha, adult chain 2 (Hba- a2), D) complement component 1, r subcomponent A (C1ra), E) suppressor of cytokine signalling 2 (Socs2), F) cytokine inducible SH2-containing protein (Cish), G) adhesion molecule with Ig-like domain 2 (Amigo2) and H) potassium voltage-gated channel subfamily H member 8 (Kcnh8) was higher during pregnancy than at proestrus. I) Expression of albumin (Alb) was higher at day 10 of pregnancy compared to proestrus and day 14 of pregnancy. Data are means ± SEM, n = 2-3 per group. #, q < 0.05 between-day effect compared to proestrus.

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3.5 4.0 5.5 3.0 3.5 Counts per million Counts per million Counts per million2.5 5.0 3.0 0.5 0.5 0.5 0.0 0.0 0.0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Proestrus Day 10 Day 14 Proestrus Day 10 Day 14 Proestrus Day 10 Day 14

Figure 7.4 Genes that were differentially expressed between ZT1/ZT5 and ZT13 at proestrus and during pregnancy. A) perilipin 4 (Plin4) expression was higher at ZT13 compared to ZT1 and ZT5 at proestrus and during pregnancy. Expression of B) hypoxia-inducible factor 3 alpha subunit (Hif3a), C) zinc finger and BTB domain containing 16 (Zbtb16) and D) TSC22 domain family member 3 (Tsc22d3) was higher at ZT13 compared to ZT1 and ZT5 at proestrus and day 10 of pregnancy, and higher at ZT13 compared to ZT1 at day 14 of pregnancy. E) serum/glucocorticoid regulated kinase 1 (Sgk1) expression was higher at ZT13 compared to ZT1 at proestrus, and higher at ZT13 compared to ZT1 and ZT5 during pregnancy. F) circadian associated repressor of transcription (Ciart) expression was higher at ZT13 compared to ZT1 at proestrus and day 10 of pregnancy, and higher at ZT13 compared to ZT1 and ZT5 at day 14 of pregnancy. G) period circadian clock 3 (Per3) expression was higher at ZT13 compared to ZT1 and ZT5 at proestrus, and higher at ZT13 compared to ZT1 during pregnancy. H) claudin 5 (Cldn5) expression was lower at ZT13 compared to ZT1 and ZT5 at proestrus, and lower at ZT13 compared to ZT1 during pregnancy. Data are means ± SEM, n = 2-3 per group. *, P < 0.05 between time-points.

120 Chapter 7

A Kdr B Lap3 5.0 * 6.5 4.5 * * 6.0 4.0

3.5 5.5 3.0 Counts per million Counts per million 2.5 5.0 0.5 0.5 0.0 0.0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Proestrus Day 10 Day 14 Proestrus Day 10 Day 14 C Fgfrl1 D Wee1 4.5 4.0 * * 4.0 3.5

3.5 3.0 Counts per million 3.0 Counts per million2.5 0.5 0.5 0.0 0.0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Proestrus Day 10 Day 14 Proestrus Day 10 Day 14 E Per2 6.5 * 6.0

5.5

5.0

Counts per million4.5 0.5 0.0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 Proestrus Day 10 Day 14

Figure 7.5 Genes that were differentially expressed between ZT1/ZT5 and ZT13 at proestrus only. A) Kdr expression was lower at ZT13 compared to ZT1 and ZT5 at proestrus. B) Lap3 expression was lower at ZT13 compared to ZT1 at proestrus. C) Fgfrl1 expression was lower at ZT13 compared to ZT5 at proestrus. Expression of D) Wee1 and E) Per2 was higher at ZT13 compared to ZT5 at proestrus. Data are means ± SEM, n = 2-3 per group. *, P < 0.05 between time- points.

121 Chapter 7 7.4 Discussion Our findings show a number of genes in the anterior hypothalamus are differentially expressed between the proestrus and pregnancy, and certain genes have also altered their circadian expression in response to pregnancy. Although there appeared to be a trend for an increase in Kiss1 expression in the evening of proestrus, there was no statistically significant difference found between ZT13 and other time points. This is most likely due to the biological variation present and the low number of replicates available for RNA Seq. Across days, the expression of Kiss1 at proestrus was higher than at days 10 and 14 of pregnancy, in line with the findings of Grattan and colleagues who show that AVPV Kiss1 mRNA at proestrus is higher compared to days 6 and 18 of pregnancy (Liu et al. 2014). The present findings, however, differ from Chapter 5, where Kiss1 expression throughout the day during proestrus was lower than at day 10, but not day 14 of pregnancy. Once again, this discrepancy may be attributable to the low number of replicates used for RNA Seq. Alternatively, although the reference genes used in qRT- PCR showed no statistically significant differences between days of estrous cycle or pregnancy (or across time-points), a trend for lower reference gene expression on day 10 of pregnancy may have translated into significantly higher Kiss1 mRNA expression upon standardisation. Similarly, RNA Seq revealed no peak in Avpr1a expression at proestrus, otherwise seen with qRT-PCR in Chapter 5. Furthermore, Npffr expression was higher during pregnancy compared to proestrus, which again contradicts the findings of Chapter 5, showing that Npffr expression was relatively low at day 14 and 18 of pregnancy. The increased expression of Npffr during pregnancy may reflect the purported role of RFRP- 3 as an inhibitor of the reproductive axis (Gibson et al. 2008; Ducret, Anderson & Herbison 2009). Nevertheless, Avp expression in the present study was higher during pregnancy compared to proestrus, consistent with Chapter 5. Avp mRNA levels in the whole hypothalamus have also been found to be nearly double in pregnant rats near term compared to their non-pregnant counterparts (Xu et al. 1996), although this increase is most likely restricted to the magnocellular neurons of the supraoptic and paraventricular nuclei of the hypothalamus and related to body fluid homeostasis during pregnancy (Xu et al. 1996), rather than circadian control of kisspeptin. Bmal1 and Reverba expression showed rhythmic variation at proestrus and during pregnancy, as well as an antiphase relationship, consistent with data from Chapter 5 and Wharfe et al. (2016). Even though the samples used to conduct RNA-Seq in the present study were the same as those used for qPCR in Chapter 5, the expression of many target genes was inconsistent. The

122 Chapter 7 differences in the two techniques may account for much of the discrepancy, as could the relatively small sample size for RNA-Seq compared to qPCR.

RNA Seq revealed a number of genes that were differently expressed between proestrus and pregnancy were all upregulated. The most commonly upregulated genes coded for haemoglobin subunits, which is consistent with the requirement for more haemoglobin due to blood volume expansion during pregnancy (Longo 1983), although this is unusual and may not be physiologically relevant given the hypothalamus is not considered a site of haemoglobin production. Expression of Alb, the gene which encodes for the main protein in blood plasma, albumin, was highly elevated at day 10 of pregnancy compared to proestrus, but returned to low levels at day 14 of pregnancy. In pregnant women, albumin concentrations fall as pregnancy progresses and this is thought to be due to the increased plasma volume in relation to albumin levels (Maher et al. 1993). However, a study in mice has shown that providing blastocysts with access to albumin during a preimplantation culture resulted in increased fetal growth (Kaye & Gardner 1999). As fetal growth is influenced by maternal protein nutrition, it is possible that despite decreased maternal circulating levels of albumin, the production of albumin is increased, particularly during the first half of mouse pregnancy, and transferred into the fetal circulation to facilitate fetal growth. The expression of C1ra, part of the complement system, which itself is a crucial component of innate immunity, was increased during pregnancy. Although increased C1ra gene expression in the mouse placenta has been found in spontaneous pregnancy loss (Parks et al. 2011), and a deficiency in the inhibitor of the C1 complex (of which C1ra is a component) is associated with preeclampsia in human pregnancy (Derzsy et al. 2010), moderate increases in components of the complement cascade have also been found during normal pregnancies (Derzsy et al. 2010). In the brain, complement activation is usually associated with pathological events such as traumatic brain injury (Stahel, Morganti-Kossmann & Kossmann 1998), or neurological conditions like Alzheimer’s disease (Mukherjee & Pasinetti 2000) and epilepsy (Aronica et al. 2007), but it is possible that the complement system is also activated in the hypothalamus as a normal part of pregnancy. Socs2 and Cish are both part of the SOCS family of proteins which act to negatively regulate the JAK-STAT pathway that is mediated by cytokines; thus the upregulation of SOCS proteins during pregnancy is important to control the effects of cytokine signalling (Fitzgerald et al. 2009). Expression of Socs1, Socs 3 and Cish is elevated in the rat ARC in late pregnancy, stimulated by the high circulating levels of PRL at this time (Anderson et al. 2006). SOCS 123 Chapter 7 proteins may also be a potential mediator of the leptin resistance typical of pregnancy; hypothalamic Socs3 mRNA expression increases in response to leptin treatment, and is increased in a mouse model of leptin-resistant obesity (Bjørbaek et al. 1998). Among the genes that were differentially expressed between ZT1 and ZT13 at proestrus and during pregnancy, many are part of the clock gene machinery that regulates the circadian expression of downstream targets: Per3 and Ciart. Ciart, also known as Gm129 or Chrono, is a more recently discovered regulator of the core circadian loop. It acts as a transcriptional repressor of CLOCK-BMAL1 activity, much like the Per1-3 and Cryptochrome (Cry1-2) genes, and thus exhibits strong oscillatory expression in the SCN as well as many peripheral tissues (Annayev et al. 2014; Goriki et al. 2014). Consistent with its role as a negative regulator of Bmal1, the circadian rhythms of Ciart and Bmal1 were in antiphase in the present study.

There were several genes that were differentially expressed between ZT1 and ZT13 or ZT5 and ZT13 at proestrus but not during pregnancy, exhibiting a similar pattern to Kiss1 expression (in Chapter 5). It is possible, therefore, that their time-of-day variation may be related to, or associated with, suppression of the kisspeptin circadian rhythm during pregnancy. Expression of Kdr or the VEGF receptor has been shown to exhibit oscillatory expression in the heart, with expression reaching a trough at the start of the dark phase, and peaking at the start of the light phase (Zhang et al. 2014). Rhythmicity in the heart appears to be phase-delayed compared to the SCN, as Kdr expression in the present study was found to be be lowest 3 hours before lights off. Lap3 is a member of the leucine aminopeptidase enzyme family and is therefore expressed in many tissues throughout the body. Circadian oscillatory expression has not been widely investigated except for in digestive and renal contexts. In the small intestine, the phase of the Lap rhythm appeared to be opposite to that exhibited by the SCN in the current study, being closely related to food intake (Saito et al. 1975). Lap rhythmicity in the kidney (peak at around 6pm), as measured in the urine, showed a similar phase to that in the small intestine (Cal et al. 1987). The rhythm of Lap in the small intestine and kidney thus appears to be phase- delayed compared to that of the SCN, which peaks at around 8 am. Fgfrl1 has not been extensively studied, but one study in Fgfrl1 knockout mice demonstrated that it is essential for early kidney development, and its absence resulted in death within minutes of birth (Gerber et al. 2009). It also promotes cell-cell fusion, a process that is fundamental to embryonic development (Trueb, Amann & Gerber 2013). However, its oscillatory expression has not been studied. Wee1 is a key cell cycle regulator that is a

124 Chapter 7 known direct target of the core clock genes, as circadian control is crucial for the regulation of cell cycle progression (Matsuo et al. 2003). Like Per3 and Ciart, Per2 is an essential component of the core circadian loop and disruption of this gene has been found to have adverse effects on the circadian clock and fertility; Per2 mutant mice eventually become arrhythmic in complete darkness (Zheng et al. 1999), and middle-aged female mutants exhibit lower reproductive success, akin to aged wild-type mice, suggestive of accelerated aging (Pilorz & Steinlechner 2008).

Expression of Kdr, Lap3, Fgfrl1, Wee1 and Per2 showed time-of-day variation at proestrus but not during pregnancy, thus exhibiting circadian patterns that appear to correlate with that of Kiss1 expression in Chapter 5. However, given the numerous important functions that these genes perform in a wide range of tissues, it seems implausible that their rhythmicity in the SCN is suppressed during pregnancy. While it is possible that oscillatory expression of these genes may persist in the peripheral tissues where their actions are critical, despite a loss of rhythmicity in the SCN, it is high unlikely given that the circadian rhythms of peripheral clocks are typically dependent on the master clock in the SCN. Per2, in particular, is a clock gene, and clock gene rhythmicity has previously been shown to be preserved in the hypothalamus during pregnancy (Wharfe et al. 2016). Moreover, other clock genes such as Per3 and Ciart demonstrated robust circadian rhythmicity during pregnancy in the current study. In addition, none of these genes are associated with Kiss1, with the exception of Kdr. Kdr and Kiss1 are both found in the placenta, responsible for trophoblast invasion and angiogenesis, respectively, and are thus important for ensuring successful placentation (Matjila et al. 2013). However, the functions of kisspeptin in the placenta are distinct from its role in the hypothalamus as a driver of gonadotropin secretion.

In summary, although there was a trend for an increase in Kiss1 expression in the evening of proestrus, a low number of replicates may have prevented this from reaching statistical significance, and is inconsistent with data from Chapter 5. The study also identified numerous genes that are differentially expressed between proestrus and pregnancy. While several genes demonstrated time-of-day variation at proestrus but not during pregnancy, and thus appeared to exhibit an expression pattern similar to that previously seen in Kiss1 (Chapter 5), they may not be involved in the suppression of the kisspeptin circadian rhythm during pregnancy. Therefore, the present study was unable to conclusively

125 Chapter 7 identify the genes that are responsible for suppressing kisspeptin circadian rhythmicity in pregnancy.

126

Chapter 8 Ontogeny of Clock and Kiss1 Gene Expression in the Prepubertal Mouse Hypothalamus

8.1 Introduction The Kiss1/Kiss1r signaling system is widely considered to be the gatekeeper for puberty onset, permitting the activation of GnRH neurons and subsequent release of gonadotropins, a key event in the initiation of puberty (Seminara & Kaiser 2005). The importance of kisspeptin signaling in puberty onset was first observed in Kiss1r-deficient humans and mice, which exhibit hypogonadotropic hypogonadism (Seminara et al. 2003). Subsequent work in the rat showed that Kiss1 mRNA expression in the hypothalamus is significantly elevated around the time of puberty in both sexes (Navarro et al. 2004). The two main hypothalamic kisspeptin populations, the ARC and the AVPV, are positively and negatively regulated by E2, respectively (Gottsch et al. 2004; Smith et al. 2005a, 2005b). This differential regulation reflects their distinct feedback functions; the ARC kisspeptin neurons are responsible for the tonic/pulsatile release of GnRH, while the AVPV is involved in the generation of the pre-ovulatory LH surge in post-pubertal females (Smith et al. 2005b; Beale et al. 2014).

After the onset of puberty in females, the timing of the LH surge is strictly controlled by circadian input in the presence of high circulating E2 (Legan & Karsch 1975; Legan, Coon & Karsch 1975). The central circadian clock, the suprachiasmatic nucleus (SCN), regulates many physiological and behavioural rhythms generated by transcriptional- translational feedback loops of clock genes including Clock, Bmal1, Per1-3, Cry 1-2 and Rev-erbα (Reppert & Weaver 2001). The core clock genes in the SCN are present during fetal life in the rodent, but oscillatory expression develops gradually only after birth and the rhythmicity of some of the clock genes appears to be established by around postnatal day (P) 10 (Shimomura et al. 2001; Sladek et al. 2004; Kováciková et al. 2006; Ansari et al. 2009).

The circadian controlled LH surge in proestrus females is synchronous with the rise in AVPV kisspeptin expression in rats (Smith et al. 2006), mice (Robertson et al. 2009) and

127 Chapter 8 hamsters (Williams 3rd et al. 2011). It is the proposed mediator between the master circadian pacemaker of the SCN and the activation of GnRH neurons, ensuring that the timing of the GnRH/LH surge is strictly controlled (Robertson et al. 2009). The particular circadian rhythm of AVPV kisspeptin expression exhibited during proestrus is not seen in diestrus, linking this pattern of activity to the LH surge and ovulation (Chapter 5; Smith et al. 2006; Adachi et al. 2007). The onset of this circadian rhythm in Kiss1 is generally thought to occur after puberty, but it is not known whether any circadian variability in kisspeptin exists prior to this.

We know that kisspeptin is expressed in the brain early in development, even though its established function is in reproduction and puberty onset. Kiss1 mRNA has been identified in the mouse AVPV as early as P10, with sex differences (females > males) emerging a few days later at P12 (Semaan et al. 2010). Furthermore, levels increase steadily from P15 to post puberty (Semaan & Kauffman 2015). Kisspeptin cell bodies and fibers, as well as kisspeptin fiber appositions to GnRH neurons, are present in the mouse AVPV at P25 and rapidly increase in numbers through to adulthood, with females exhibiting 10-fold greater kisspeptin expression than males (Clarkson & Herbison 2006). The sexual dimorphism of the AVPV kisspeptin population is well-established, owing to the region’s involvement in the generation of the pre-ovulatory LH surge in adult females (Gottsch et al. 2004). In the ARC, Kiss1 mRNA is detectable in the fetal mouse brain as early as E13.5 (Fiorini & Jasoni 2010; Knoll et al. 2013). Functional neural circuits between ARC kisspeptin neurons and GnRH neurons are also fully established in utero (Kumar et al. 2014), although the purpose of Kiss1/Kiss1r signaling during this time is as yet unknown. While Kiss1 levels in the ARC are not different between male and female rodents in adulthood, sexual dimorphism exists at birth (female > male) and diminishes closer to puberty, with female ARC Kiss1 expression close to that of the male at P20 (Cao & Patisaul 2011; Poling et al. 2012).

While kisspeptin expression and clock gene rhythms in the mouse brain have been studied extensively in adults, much less is known about their development in the neonate, and little is known about the ontogeny of the circadian peak in Kiss1 mRNA expression within the AVPV that occurs in the evening of proestrus. Thus we aimed to investigate whether the onset of the circadian regulation of kisspeptin expression occurs prior to puberty. In addition, we investigated expression of clock genes, and that of the AVP system, an intermediary between the SCN and AVPV kisspeptin neurons (Williams 3rd et al. 2011),

128 Chapter 8 to identify possible mediators of a kisspeptin peak prior to puberty. Finally, due to its proposed role in inhibiting GnRH and LH secretion (Kriegsfeld 2006), the expression of RFRP was also examined.

8.2 Methods 8.2.1 Animals Nulliparous C57BL/6J mice (6 weeks old) were supplied by the Animal Resources Centre (Murdoch, Australia). Mice were maintained in an environmentally controlled room under a 12-h light, 12-h dark cycle (lights on from 0700 – 1900 h) with ad libitum access to food and water. Lights off at 1900 h was defined as Zeitgeber time (ZT)-12, with sampling times described as relative to ZT-0. Female mice were mated and the day of birth was designated as postnatal day 0. Whole brain and blood sample collections were made at 4-h intervals commencing at ZT1 (0800 h) at postnatal day (P) 5, 15 and 25. All procedures involving animals were conducted with the approval of the Animal Ethics Committee of The University of Western Australia.

8.2.2 Tissue collection Whole brains were collected from one male pup and one female pup per litter at 0800 (ZT1), 1200 (ZT5), 1600 (ZT9), 2000 (ZT13), 2400 (ZT17) or 0400 (ZT21) at each postnatal age. A red light (>600 nm wavelength, outside of the visible range for mice) was used to facilitate collection of tissues in the dark phase. Whole brains were frozen on dry ice immediately following removal. A trunk blood sample was obtained following decapitation and collected in a tube containing EDTA (100 µL per mL of blood). Plasma was obtained following centrifugation of the blood sample and stored at -20°C until required.

8.2.3Hypothalamic gene expression

8.2.3.1RNA extraction Hypothalami were dissected from whole brain samples and bisected into anterior (containing the AVPV and SCN) and posterior (containing the ARC) portions as described in Quennell et al. (2011). Total RNA was extracted from anterior and posterior hypothalami using Qiazol Lysis Reagent (Qiagen, Hilden, Germany; Cat# 79306) according to the manufacturer’s instructions. The RNA was dissolved in 40 µL of RNase-

129 Chapter 8 free water, placed on ice for 5 min and thoroughly vortexed. RNA was quantitated using the Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA) at 260 nm and stored at -80 °C until required.

8.2.3.2 Reverse transcription RNA samples were treated with RQ1 RNase-free DNase (Promega, Waltham, Massachusetts, USA; cat# M6101) to remove any genomic DNA present. Briefly, samples were made up to a total volume of 10 µL with 1 µg of RNA, 1 µL of RQ1 RNase- Free DNase 10x Buffer, 2 µL of RQ1 RNase-Free DNase and nuclease-free water, and incubated at 37°C for 30 min. RQ1 DNase Stop Solution (1 µL) was added to each sample to terminate the reaction, and samples were incubated at 65°C for 10 min to deactivate the DNase. Total RNA (1 µg) was reverse transcribed to cDNA with random primers (Promega, Waltham, Massachusetts, USA; cat# C1181) using Moloney Murine Leukemia Virus Reverse Transcriptase, RNase H Minus, Point Mutant (Promega, Waltham, Massachusetts, USA; cat# M3683). The resultant cDNAs were purified using the Ultraclean PCR Cleanup kit (MoBio Laboratories, Carlsbad, Calfornia, USA; Cat# 12500-250), according to the manufacturer’s instructions.

8.2.3.3 Quantitative real-time RT-PCR Analyses of mRNA levels for total Kiss1, kisspeptin receptor (Kiss1r), arginine vasopressin (Avp), arginine vasopressin receptor 1a (Avpr1a), RFRP (neuropeptide VF precursor, Npvf), RFRP receptor (neuropeptide FF receptor, Npffr), Bmal1 (also known as Arntl), and Rev-erbα (also known as Nr1d1) were performed by quantitative RT-PCR on the Rotorgene 6000 (Corbett Life Science, New South Wales, Australia). Bmal1 and Rev-erbα were selected as they showed the most robust rhythms out of all the clock genes (Wharfe 2016). Primers (Table 8.1) for total Kiss1, Kiss1r, Bmal1, Rev-erbα and the reference genes hypoxanthine-guanine phosphoribosyltransferase (Hprt), succinate dehydrogenase subunit A (Sdha) and beta-2 microglobulin (B2m) were designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov). Each of the selected primer pairs was positioned to span an intron to ensure no product was amplified from genomic DNA. The resulting amplicons were sequenced to confirm specificity. Standard curves for each product were generated from gel-extracted (QIAquick Gel Extraction Kit, Qiagen) PCR products using 10-fold serial dilutions and the Rotorgene 6000 software. The PCR cycling 130 Chapter 8 conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 1 s, annealing temperature (Table 1) for 15 s and extension at 72 °C for 5 s. Melt curve analysis was performed to confirm amplification specificity for each gene.

Primers for Avp, Avpr1a, Npvf and Npffr were purchased as QuantiTect Primer Assays (Qiagen, cat# QT00249389, QT00113169, QT00278551 and QT001169196, respectively). The PCR conditions were as follows: initial activation 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 5 s and combined annealing/extension at 60 °C for 10 s. Melt curve analysis was performed to confirm amplification specificity.

All samples were standardized against Hprt, Sdha and B2m using the GeNorm algorithim (Vandesompele et al. 2002).

131 Chapter 8

Table 8.1 Primer sequences and conditions for quantitative PCR.

Gene Sequence Size Annealing (bp) temp (°C) Kiss1 F: 5’-CTCTGTGTCGCCACCTATGG-3’ 126 60 R: 5’-AGGCTTGCTCTCTGCATACC-3’ Kiss1r F: 5’-TGCTGGGAGACTTCATGTGC-3’ 102 60 R: 5’-CATACCAGCGGTCCACACTC-3’ Bmal1 F: 5’-CGTGCTAAGGATGGCTGTTC-3’ 166 60 R: 5’-CTTCCCTCGGTCACATCCTA-3’ Rev-erbα F: 5’-ATTGCCCAGGGGGCGAGAGA-3’ 292 60 R: 5’-GCCAAAAGAGCGGGCAGGGT-3’ Hprt F: 5’-GCAGTACAGCCCCAAAATGG-3’ 80 60 R: 5’-AGTCTGGCCTGTATCCAACAC-3’ Sdha F: 5’-ATGGAAAATGGGGAGTGCCG-3’ 123 60 R: 5’-ACAGCTGAAGTAGGTTCGGC-3’ B2m F: 5’-TGACCGGCCTGTATGCTATC-3’ 19 7 60 R: 5’-GATTTCAATGTGAGGCGGGTG-3’

132 Chapter 8 8.2.4 Plasma hormone analyses Hormone levels were measured in plasma samples collected at ZT1 and ZT13 only.

8.2.4.1 Pituitary hormones Plasma levels of FSH, LH and PRL were measured in a 10 µl sample using the MILLIPLEX Map Mouse Pituitary Magnetic Bead kit (Merck Millipore, Billerica, Massachusetts, USA; Cat# MPTMAG-49K) according to the manufacturer’s instructions. The plate was run on a MAGPIX system (Luminex Corporation, Austin, Texas, USA) with xPONENT software to analyse the median fluorescent intensity data and obtain analyte concentrations. The lower limits of detection for this assay are as follows: FSH - 9.5 pg/mL, LH - 1.9 pg/mL, PRL - 46.2 pg/mL.

8.2.4.2 Steroid and thyroid hormones Prior to assay, plasma samples (50 µL) were extracted in 75 µL of acetonitrile, vortexed for 5 s and incubated for 10 min at room temperature. Samples were centrifuged at 17,000 x g for 5 min and the supernatant was transferred into separate tubes. The samples were dried by CentriVap Benchtop Vacuum Concentrator (Labconco, Kansas City Missouri, USA) at 37 °C and reconstituted with 40 µL of assay buffer for subsequent assay.

Plasma levels of E2 and P4 were measured using the MILLIPLEX Map multi species Steroid/Thyroid Hormone panel kit (Merck Millipore, Cat # STTHMAG-21K) according to the manufacturer’s instructions. The plate was run on a MAGPIX system (Luminex Corporation) with xPONENT software to analyse the median fluorescent intensity data and obtain analyte concentrations. The lower limits of detection for this assay are as follows: E2 - 0.02 ng/mL, P4 - 0.09 ng/mL.

8.2.5 Statistical analysis Statistical analyses were conducted using GraphPad Prism 6 (GraphPad Software, La Jolla, California, USA). Data are expressed as the mean ± SEM, with an n of 6-8 per ZT on each postnatal day. Differences were considered significant when P < 0.05. Two-way ANOVAs were used to analyse all data with age and time as factors; followed by Tukey’s post hoc tests where appropriate. Where significant interaction effects were observed in two-way ANOVA analyses, unpaired t-tests or one-way ANOVAS with time as a factor were conducted within each day. When variances were unequal (Kiss1 expression in the anterior and posterior hypothalamus, female Npffr expression) data were log-transformed 133 Chapter 8 prior to analysis. Clock gene rhythmicity was assessed by cosinor analysis using a non- linear regression model (Genstat Version 9, VSN International Ltd, Hemel Hempstead, UK).

134 Chapter 8 8.3 Results 8.3.1 Gene expression There was an age effect on both Bmal1 and Rev-erbα expression in the anterior hypothalamus in female and male neonates (P<0.0001; Figure 8.1), with overall expression of Bmal1 lower at P15 than P5 (Figure 8.1A and B) while overall Rev-erbα expression was higher at P15 than P5 (Figure 8.1C and D). Cosinor analysis revealed Bmal1 expression to be rhythmic in females only at P25 (r2 = 0.131; P < 0.05, Figure 8.1A) but not rhythmic in males at any age (Figure 8.1B). In females, Rev-erbα expression demonstrated no rhythmicity at any age (Figure 8.1C), while in males, Rev-erbα expression was rhythmic only at P25 (r2 = 0.123; P < 0.05; Figure 8.1D).

Kiss1 expression in both the AVPV and ARC increased with age in both sexes (P<0.0001), but no significant time-of-day variation was seen (Figure 8.2A-D). In females, Kiss1r expression in the anterior hypothalamus was lower at P25 than P15 (P < 0.001), but no time-of-day variation was seen at any age (Figure 8.2E), while in males, Kiss1r expression was not affected by either age or time-of-day (Figure 8.2F).

Female Avp expression in the anterior hypothalamus was significantly affected by age, with P25 expression levels higher than those at P5 (P < 0.05), but no time-of-day variation was seen at any age (Figure 8.3A), while male Avp expression in the anterior hypothalamus were not affected by either age or time-of-day (Figure 8.3B). Avpr1a expression in the anterior hypothalamus decreased from P5 to P15 and again to P25 in both sexes (P < 0.0001; Figure 8.3C and 8.3D). No time of day variation was seen.

Male and female Npvf expression in the posterior hypothalamus was significantly affected by age, with female expression lower at P25 compared to P5 and P15 (P < 0.01) and male expression lower at P25 (P < 0.0001) and P15 (P < 0.05) compared to P5. No time-of- day variation was seen at any age (Figure 8.4A and B). Male Npffr expression in the anterior hypothalamus was higher at P25 compared to P5 and P15 (P < 0.0001), but no time-of-day variation was seen at any age (Figure 8.4D), while female Npffr expression was not affected by either age or time-of-day (Figure 8.4C).

135 Chapter 8

A B 250 350

300 200 ## ##

mRNA 250 ## ## mRNA 150 Bmal1 Bmal1 100 100

50 50 (Relative expression) (Relative expression) Male AH Female AH

0 0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 C P5 P15 P25 D P5 P15 P25 600 500 ## ## 400 mRNA mRNA

α #

400 α 300 Rev-erb

Rev-erb 200 200

100 (Relative expression) (Relative expression) Male AH Female AH 0 0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 P5 P15 P25 P5 P15 P25

Figure 8.1 Bmal1 and Rev-erbα diurnal gene expression in the anterior hypothalamus of female and male mice at P5, P15 and P25. A) Female Bmal1 expression was rhythmic at P25 but not at P5 or P15 (note: broken y-axis scale). Main effect of age, F (2, 99) = 177.4, P < 0.0001; main effect of time-of-day, F (5, 99) = 0.7773, P > 0.05; interaction between age and time-of-day, F (10, 99) = 1.073, P > 0.05. B) Male Bmal1 expression was not rhythmic at any age. Main effect of age, F (2, 110) = 55.41, P < 0.0001; main effect of time-of-day, F (5, 110) = 2.095, P > 0.05; interaction between age and time-of-day, F (10, 110) = 0.9839, P > 0.05. C) Female Rev-erbα expression was not rhythmic at any age. Main effect of age, F (2, 98) = 56.13, P < 0.0001; main effect of time-of-day, F (5, 98) = 0.4616, P > 0.05; interaction between age and time-of-day, F (10, 98) = 1.484, P > 0.05. D) Male Rev-erbα expression was rhythmic at P25 but not at P5 or P15. Main effect of age, F (2, 111) = 13.76, P < 0.0001; main effect of time-of-day, F (5, 111) = 0.9258, P > 0.05; interaction between age and time-of-day, F (10, 111) = 1.590 P > 0.05. Data are means ± SEM, n = 6-8 per group. Solid line indicates rhythmicity according to significant cosinor fit; dotted line indicates non-rhythmicity. Grey panel indicates lights off from zeitgeber time (ZT)12 to ZT0. Two-way ANOVA, #, P < 0.001, ##, P < 0.0001 age effect compared to P5.

136 Chapter 8

A B 250 200 ### ###

200 150 mRNA mRNA 150 ##

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AVPV Kiss1 # 50 50 (Relative expression) (Relative expression) Male AVPV Female 0 0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 C P5 P15 P25 D P5 P15 P25 400 400 ### ###

300 300 mRNA mRNA

Kiss1 200 ## 200 Kiss1

### 100 100 (Relative expression) (Relative expression) Male ARC Female ARC 0 0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 E P5 P15 P25 F P5 P15 P25 300 300

## mRNA

200 mRNA 200 Kiss1r Kiss1r

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0 0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 P5 P15 P25 P5 P15 P25

137 Chapter 8

Figure 8.2 Kiss1 and Kiss1r diurnal gene expression in female and male mice at P5, P15 and P25. A) Female Kiss1 levels in the AVPV increased with age, but did not show any time-of-day variation at any age. Main effect of age, F (2, 88) = 53.29, P < 0.0001; main effect of time-of-day, F (5, 88) = 0.6797, P > 0.05; interaction between age and time-of-day, F (10, 88) = 1.025, P > 0.05. B) Male Kiss1 levels in the AVPV increased with age, but did not show any time-of-day variation. Main effect of age, F (2, 94) = 97.31, P < 0.0001; main effect of time-of-day, F (5, 94) = 0.9992, P > 0.05; interaction between age and time-of-day, F (10, 94) = 0.7496, P > 0.05. C) Female Kiss1 levels in the ARC increased with age but did not show any time-of-day variation. Main effect of age, F (2, 101) = 77.51, P < 0.0001; main effect of time-of- day, F (5, 101) = 0.5020, P > 0.05; interaction between age and time-of-day, F (10, 101) = 0.8526, P > 0.05. D) Male Kiss1 levels in the ARC increased with age, but did not show any time-of-day variation. Main effect of age, F (2, 98) = 148.9, P < 0.0001; main effect of time-of-day, F (5, 98) = 2.683, P < 0.05; interaction between age and time-of-day, F (10, 98) = 1.500, P > 0.05. E) Female Kiss1r levels in the anterior hypothalamus were lower at P25 compared to P5 and P15, but did not show time-of- day variation. Main effect of age, F (2, 100) = 9.723, P < 0.0001; main effect of time- of-day, F (5, 100) = 1.510, P > 0.05; interaction between age and time-of-day, F (10, 100) = 0.8617, P > 0.05. F) Male Kiss1r levels in the anterior hypothalamus were not affected by age or time-of-day. Main effect of age, F (2, 111) = 1.428, P > 0.05; main effect of time-of-day, F (5, 111) = 1.023, P > 0.05; interaction between age and time- of-day, F (10, 111) = 0.5179, P > 0.05. Data are means ± SEM, n = 6-8 per group. Grey panel indicates lights off from zeitgeber time (ZT)12 to ZT0. #, P < 0.05, ##, P < 0.01, ###, P < 0.0001 age effect compared to P5 (Two-way ANOVA).

138 Chapter 8

A B 400 300 #

300

mRNA 200 mRNA

Avp 200 Avp

100 100 Male AH (Relative expression) (Relative expression) Female AH

0 0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 P5 P15 P25 P5 P15 P25 C D 300 250 ## 200

mRNA ##

200 mRNA 150 Avpr1a Avpr1a 100 ## 100 ##

50 (Relative expression) (Relative expression) Male AH Female AH 0 0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 P5 P15 P25 P5 P15 P25

Figure 8.3 Avp and Avpr1a diurnal gene expression in the anterior hypothalamus of female and male mice at P5, P15 and P25. A) Female Avp levels were higher at P25 compared to P5, but did not show time-of-day variation at any age. Main effect of age, F (2, 101) = 8.226, P < 0.001; main effect of time-of-day, F (5, 101) = 1.418, P > 0.05; interaction between age and time-of-day, F (10, 101) = 0.4833, P > 0.05. B) Male Avp levels were not affected by age or time-of-day. Main effect of age, F (2, 114) = 2.095, P > 0.05; main effect of time-of-day, F (5, 114) = 0.9510, P > 0.05; interaction between age and time-of-day, F (10, 114) = 0.8200, P > 0.05. C) Female Avpr1a levels decreased with age, but did not show any time-of-day variation. Main effect of age, F (2, 96) = 145.4, P < 0.0001; main effect of time-of- day, F (5, 96) = 1.824, P > 0.05; interaction between age and time-of-day, F (10, 96) = 1.452, P > 0.05. D) Male Avpr1a levels decreased with age, but did not show any time-of-day variation. Main effect of age, F (2, 114) = 161.2, P < 0.0001; main effect of time-of-day, F (5, 114) = 1.483, P > 0.05; interaction between age and time-of-day, F (10, 114) = 1.375, P > 0.05. Data are means ± SEM, n = 6-8 per group. Grey panel indicates lights off from zeitgeber time (ZT)12 to ZT0. Two-way ANOVA, #, P < 0.001, ##, P < 0.0001 age effect compared to P5.

139 Chapter 8

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# 150 150

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Npvf 100 100 Npvf

50 50 Male PH (Relative expression) (Relative expression) Female PH

0 0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 P5 P15 P25 P5 P15 P25 C D 250 200

### 200 150 mRNA

150 mRNA

Npffr Npffr 100 Npffr 100

50 50 Male AH (Relative expression) (Relative expression) Female AH

0 0

ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT1ZT5ZT9 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 ZT13ZT17ZT21 P5 P15 P25 P5 P15 P25

Figure 8.4 Npvf (RFRP) and Npffr (RFRP receptor) expression in female and male mice at P5, P15 and P25. A) Female Npvf expression in the posterior hypothalamus was lower at P25 compared to P5 and P15, but showed no time-of-day variation at any age. Main effect of age, F (2, 102) = 7.231, P < 0.01; main effect of time-of-day, F (5, 102) = 1.115, P > 0.05; interaction between age and time-of-day, F (10, 102) = 0.6259, P > 0.05. B) Male Npvf expression in the posterior hypothalamus decreased with age, but did not show any time-of-day variation. Main effect of age, F (2, 110) = 18.50, P < 0.0001; main effect of time-of-day, F (5, 110) = 1.701, P > 0.05; interaction between age and time-of-day, F (10, 110) = 0.6346, P > 0.05. C) Female Npffr expression in the anterior hypothalamus was not affected by age or time-of-day. Main effect of age, F (2, 99) = 3.057, P > 0.05; main effect of time-of-day, F (5, 99) = 2.177, P > 0.05; interaction between age and time-of-day, F (10, 99) = 0.5936, P > 0.05. D) Male Npffr expression in the anterior hypothalamus was higher at P25 compared to P5 and P15, but showed no time-of-day variation at any age. Main effect of age, F (2, 109) = 26.04, P < 0.0001; main effect of time-of-day, F (5, 109) = 0.7640, P > 0.05; interaction between age and time-of-day, F (10, 109) = 0.5135, P > 0.05. Data are means ± SEM, n = 6-8 per group. Grey panel indicates lights off from zeitgeber time (ZT)12 to ZT0. Two-way ANOVA, #, P < 0.05, ##, P < 0.01, ###, P < 0.0001 age effect compared to P5.

140 Chapter 8 8.3.2 Hormone levels There was a significant effect of age on LH and FSH concentrations (P < 0.0001) in both sexes, but levels were not different between ZT1 and ZT13 at any age (Figure 8.5A-D). In females, LH and FSH concentrations were highest at P15 compared to P5 and P25 (P < 0.0001; Figure 8.5A and C), while LH levels at P25 were lower than those at P5 (P = 0.0001). In males, LH and FSH levels increased from P5 to P15 (P < 0.001) and again to P25 (P < 0.0001; Figure 8.5B and D).

There was a significant effect of age on E2 concentrations in males (P < 0.0001) and females (P < 0.01); levels were highest at P5 and decreased to below detection at P15 and

P25 (P < 0.0001 in males, P < 0.05 in females). E2 levels were similar between ZT1 and ZT13 at all ages in both sexes (Figure 8.5E and F).

There was a significant effect of both time-of-day and age on P4 concentrations in female neonates (P < 0.05); levels at P25 were higher at ZT13 compared to ZT1 (P < 0.05), but no different at P5 or P15 (Figure 8.5G). In males, P4 levels were not affected by either age or time (Figure 8.5H).

141 Chapter 8

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1500 ## 1000

1000 ## 500 500 PlasmaLH levels (pg/ml) PlasmaLH levels (pg/ml) 0 0

ZT1 ZT1 ZT1 ZT1 ZT1 ZT1 ZT13 ZT13 ZT13 ZT13 ZT13 ZT13 P5 P15 P25 P5 P15 P25

C D ### 6000 ### 5000

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0 0 PlasmaFSH levels (pg/ml) PlasmaFSH levels (pg/ml)

ZT1 ZT1 ZT1 ZT1 ZT1 ZT1 ZT13 ZT13 ZT13 ZT13 ZT13 ZT13 P5 P15 P25 P5 P15 P25 E F

0.15 0.15

0.10 0.10

0.05 0.05 # # ### ###

0.00 0.00 Plasmaestradiol levels (ng/ml) ZT1 ZT1 ZT1 Plasmaestradiol levels (ng/ml) ZT1 ZT1 ZT1 ZT13 ZT13 ZT13 ZT13 ZT13 ZT13 P5 P15 P25 P5 P15 P25 G H

0.3 0.25

* 0.20 0.2 0.15

0.10 0.1 0.05

0.0 0.00

ZT1 ZT1 ZT1 ZT1 ZT1 ZT1 ZT13 ZT13 ZT13 ZT13 ZT13 ZT13 Plasmaprogesterone levels (ng/ml) Plasmaprogesterone levels (ng/ml) P5 P15 P25 P5 P15 P25

142 Chapter 8

Figure 8.5 Plasma hormone concentrations in female (left panel) and male (right panel) mice at P5, P15 and P25. A) Female LH levels were lower at P25 compared to P5 and P15, and higher at P15 compared to P5, but similar between ZT1 and ZT13 at all ages. Main effect of age, F (2, 35) = 51.70, P < 0.0001; main effect of time-of-day, F (1, 35) = 0.6763, P > 0.05; interaction between age and time-of-day, F (2, 35) = 1.368, P > 0.05. B) Male LH increased with age, but were similar between ZT1 and ZT13. Main effect of age, F (2, 36) = 52.81, P < 0.0001; main effect of time- of-day, F (1, 36) = 0.1033, P > 0.05; interaction between age and time-of-day, F (2, 36) = 0.7939, P > 0.05. C) Female FSH was highest at P15, and similar between ZT1 and ZT13 at all ages. Main effect of age, F (2, 35) = 37.83, P < 0.0001; main effect of time- of-day, F (1, 35) = 0.07269, P > 0.05; interaction between age and time-of-day, F (2, 35) = 0.1441, P > 0.05. D) Male FSH increased with age. Main effect of age, F (2, 36) = 94.45, P < 0.0001; main effect of time-of-day, F (1, 36) = 0.6682, P > 0.05; interaction between age and time-of-day, F (2, 36) = 0.8000, P > 0.05. E-F) Female and male E2 was below detectability at P15 and P25 compared to P5, but not different between ZT1 and ZT13 at any age. Female: Main effect of age, F (2, 35) = 6.578, P < 0.01; main effect of time-of-day, F (1, 35) = 1.446, P > 0.05; interaction between age and time-of- day, F (2, 35) = 1.365, P > 0.05. Male: Main effect of age, F (2, 36) = 47.74, P < 0.0001; main effect of time-of-day, F (1, 36) = 1.332, P > 0.05; interaction between age and time-of-day, F (2, 36) = 1.332, P > 0.05. G) Female P4 levels were higher at ZT13 compared to ZT1 at P25, but not different between ages. Main effect of age, F (2, 35) = 3.788, P < 0.05; main effect of time-of-day, F (1, 35) = 6.233, P < 0.05; interaction between age and time-of-day, F (2, 35) = 1.852, P > 0.05. H) Male P4 levels were not affected by age or time-of-day. Main effect of age, F (2, 36) = 0.3847, P > 0.05; main effect of time-of-day, F (1, 36) = 1.304, P > 0.05; interaction between age and time-of- day, F (2, 36) = 1.700, P > 0.05. Data are means ± SEM, n = 6-8 per group. Two-way ANOVA, #, P < 0.05, ##, P < 0.001, ###, P < 0.0001 age effect compared to P5. *, P < 0.05 time of day effect compared to ZT1.

143 Chapter 8 8.4 Discussion The present findings demonstrate that clock gene rhythmicity in the anterior hypothalamus is not fully mature at P5 and P15 and is only partially established by P25. Consequently, the onset of the peak in AVPV Kiss1 expression in females, which stimulates ovulation at proestrus, does not occur until later, presumably at puberty.

The oscillatory expression of Bmal1 and Rev-erbα in the anterior hypothalamus (containing the SCN) was variable depending on age and sex; it was absent at P5 and P15, but exhibited sex differences at P25. These data suggest that clock gene rhythmicity in the SCN is not fully established even at P25 and this is consistent with data showing that while oscillatory expression in the Per and Cry genes is observed relatively early in postnatal life, oscillatory expression in Bmal1 and Clock develops later (Sladek et al. 2004; Ansari et al. 2009).

While kisspeptin is present in the hypothalamus from early in development and kisspeptin circuits are reportedly fully functional (Semaan et al. 2010; Knoll et al. 2013; Kumar et al. 2014), its biological role at this time is still unknown. We demonstrate here that the diurnal/circadian pattern of AVPV Kiss1 that we and others have observed in proestrus females does not occur at P25 or prior to that. This is not entirely surprising given that the Kiss1 surge requires high estradiol levels during proestrus, and circulating estradiol levels remained undetectable between P15 and P25. Moreover, clock gene rhythmicity does not appear to be fully established at this time, and this unique rhythm of Kiss1 is dependent upon circadian inputs. Thus, LH levels showed no sign of diurnal variation or a surge and in fact, circulating LH levels decreased between P15 and P25. Ovariectomised adult female mice exposed to a hormonal paradigm that produces constantly elevated estradiol levels demonstrate a daily LH surge (Robertson et al. 2009; Dror, Franks & Kauffman 2013); this raises the possibility that treating female neonates with estradiol may be sufficient to induce a similar response even before puberty, although the immaturity of SCN clock gene rhythmicity may prevent this.

Although no statistically significant time-of-day variation was noted in Kiss1 mRNA expression in our study, we did see a trend for a peak in Kiss1 expression in both the AVPV and ARC of females at P15. The immaturity of clock gene rhythmicity at this age would likely preclude a circadian pattern of Kiss1 expression comparable to that observed in post-pubertal females at proestrus. Nonetheless, it is possible that other clock genes 144 Chapter 8 besides Bmal1 and Rev-erbα exhibit a greater degree of rhythmicity by P15 and these are driving the apparent peak in Kiss1 mRNA levels seen in the current study. This could also be associated with the relatively high circulating LH concentrations in P15 females, although no time-of-day patterns were seen in LH at P25. Here we noted greater variability in the data set and this may be indicative of a premature organisation of kisspeptin at this time.

In both sexes, Kiss1 expression in the AVPV increased with age, consistent with the known role of kisspeptin in puberty onset (Clarkson & Herbison 2006; Semaan & Kauffman 2015). Kiss1 expression in the ARC also increased with age in both sexes and this is somewhat in agreement with the study by Clarkson & Herbison (2006) which showed that the number of kisspeptin cell bodies significantly increased between P10 and P25 in females, but not in males. In contrast, a recent study found that although the number of Kiss1 neurons in the ARC in females did not change between P15 and P25, total Kiss1 expression decreased (Semaan & Kauffman 2015). However, Semaan and Kauffman (2015) designated day of birth as P1, as opposed to P0 for our current study, therefore making our P25 equivalent to Semaan and Kauffman’s P26, and by P26 Kiss1 mRNA increased such that it was no longer different from that at P15 (Semaan & Kauffman 2015).

We (in Chapter 5) and others have previously observed an increase in AVP receptor Avpr1a expression in the anterior hypothalamus in adult females in the evening of proestrus (Smarr, Gile & de la Iglesia 2013). Like that for Kiss1, this pattern is both time- and estradiol-dependent (Smarr, Gile & de la Iglesia 2013), and so the low estradiol levels at P5 and P25 likely prevent the peak in Avpr1a expression at this time. Interestingly, overall Avpr1a expression decreased markedly between P5 and P25 in both sexes, suggestive of reduced overall sensitivity to AVP.

Kiss1r expression in the anterior hypothalamus in females was lower at P25 than at P15 or P5. This is contrary to the findings of Semaan and Kauffman (2015) that Kiss1r mRNA levels did not change at any age measured between P15 and adulthood. The discrepancy may be due to the different techniques used; Semaan and Kauffman used in situ hybridisation, allowing more specific targeting of GnRH neurons than the quantitative PCR in the present study, which would have measured total Kiss1r expression in the anterior hypothalamus, including Kiss1r that is potentially expressed outside of GnRH

145 Chapter 8 neurons. Our current observations are also in contrast to another study which reported that kisspeptin receptor expression increases from P5 to P20 (Herbison et al. 2010), however unlike the present work, that study used immunostaining and measurements were not restricted to the anterior hypothalamus.

Rfrp expression decreased in both males and females as they developed, consistent with data reported by Semaan and Kauffman (2015) showing that Rfrp mRNA in the dorsomedial nucleus (DMN) decreased from P15 across all ages until adulthood. Given that RFRP-3 has been proposed to inhibit the reproductive axis (Gibson et al. 2008; Ducret, Anderson & Herbison 2009), a decrease in Rfrp expression from P5 through to P25 would fit with a lifting of inhibition on the reproductive axis in the lead up to puberty. Rfrpr expression was increased in P25 males, but it is unclear if this would counteract the concomitant decrease in Rfrp expression.

Studies have shown that the AVPV itself exhibits circadian rhythms, evidenced by circadian expression of the clock genes Per1 and Bmal1, which is phase-delayed relative to the SCN (Smarr, Gile & de la Iglesia 2013; Chassard et al. 2015). Thus, another possibility is that because clock gene expression in the current study was measured in the entire anterior hypothalamus, the data obtained represents the combination of two separate, but related, rhythms emerging from the SCN and the AVPV (which is phase delayed compared to the SCN by 4 – 8 hours depending on estrous cycle stage), resulting in a masking or blunting of the SCN rhythmicity. However, this possibility is unlikely given that Bmal1 expression in females and Rev-erbα expression in males showed rhythmicity at P25.

In the present study, male and female neonates often exhibited similar patterns of gene expression and hormone levels, however, sexual dimorphism was evident in gonadotropin levels. In females, LH and FSH levels showed similar patterns, rising from P5 to P15 and decreasing substantially at P25. Data from C57BL/6Cr mice are consistent with the current work; LH generally increases from early neonatal life and peaks around day 19, dropping sharply thereafter (Michael, Kaplan & Macmillan 1980). FSH concentrations in the present study were also in agreement with previous reports showing a transient spike in FSH at P10 that declines over several days (Stiff, Bronson & Stetson 1974; Michael, Kaplan & Macmillan 1980). In contrast, both LH and FSH concentrations increased with age in males in the current study, in line with observations from a study

146 Chapter 8 on C57BL/10 and DBA/1 mice, which also showed a general increase in age in LH and FSH levels (Selmanoff, Goldman & Ginsburg 1977).

The high estradiol levels at P5 compared to P15 and P25 in both sexes may reflect the process of sexual differentiation that occurs within the first week of birth (Montano, Welshons & Saal 1995). Similar low-to-undetectable levels of estradiol were observed in

P15 and P25 female mice (Mayer et al. 2010). P4 levels in female neonates increased steadily from P5 to P25, consistent with data in rats (Dohler & Wuttke 1974).

Interestingly, P25 females show a time-of-day difference in P4 concentrations, possibly due to impending pubertal onset, as adult females also show higher levels of P4 at ZT13 compared to ZT1 (Wharfe et al. 2016). However, given that the levels of estradiol and P4 at all time points are near the lower level of detection, it is possible that these statistically significant differences may not be of any physiological relevance.

In conclusion, we show that the AVPV kisspeptin circadian rhythm that occurs in proestrus females in the lead up to the preovulatory LH surge is not developed by 25 days of age in the neonatal mouse. This is likely due in part to SCN clock gene rhythmicity not being fully established at this stage, and therefore the circadian input that is essential for the kisspeptin surge is absent. The kisspeptin surge also requires elevated estradiol levels, which were clearly absent at P25. We speculate that kisspeptin rhythmicity in the AVPV develops only with puberty onset, corresponding with the large increases in kisspeptin expression around this time, the associated rise in gonadotropin secretion and the commencement of estrous cyclicity.

147

148

Chapter 9 General Discussion

The overall objective of the studies described in this thesis was to investigate the circadian regulation of hypothalamic Kiss1 mRNA expression during neonatal/pre-pubertal development, adulthood and pregnancy, in a mouse model. Four separate studies were undertaken to achieve this objective, and the findings of each have been presented and discussed in detail within their respective chapters. In this concluding chapter, the major findings are summarised, integrated and their biological significance addressed.

The first study (Chapter 5) characterised circadian hypothalamic Kiss1 expression in adult female mice during the estrous cycle and pregnancy. The key finding that emerged from this study was that pregnancy abolished the rise in Kiss1 and AVP receptor mRNA expression observed at proestrus in the AVPV, despite high circulating E2 levels and a functional clock in the SCN. Our findings demonstrated a peak in Kiss1 expression in the AVPV one hour after the onset of the dark phase, which is consistent with the literature (Smith et al. 2006; Robertson et al. 2009). Interestingly, the LH “surge” was observed 4 hours later, as opposed to occurring simultaneously, as seen in previous studies. The discrepancy may be explained by our sampling intervals being 4 hours apart, while Robertson et al. (2009) sampled every hour around the time of the expected surge. The relatively short-lived peaks in LH levels, typical of the mouse LH surge, may have occurred shortly after the ZT13 collection time point and resulted in the true peak of LH concentrations being missed. Indeed, Chassard et al. (2015) observed a 2-hour difference between the increase in Kiss1 levels and the LH surge, suggesting that the two events may not necessarily be concurrent. Moreover, although the LH surge typically occurs around the onset of darkness, mice do exhibit natural variations in its timing (Miller et al. 2004; as described below).

In the ARC, an increased variability in Kiss1 expression was observed towards the end of pregnancy. As this observation is restricted to the latter half of pregnancy, the variability may be either related to the preparation for parturition, or the phenomenon of post-partum ovulation, where female rodents typically ovulate within 24 hours of parturition (Mantalenakis & Ketchel 1966; Bingel & Schwartz 1969b). As in cyclic ovulation, post-partum ovulation requires a preceding rise in E2 and gonadotropin surge, 149 Chapter 9 the timing of which is dependent on the time of delivery (Hoffmann & Schwartz 1965; Ying et al. 1973; Connor & Davis 1980), and tends to be more variable relative to the cyclic rodent surge (Bingel & Schwartz 1969b). This time-dependence indicates a circadian input to the timing of the post-partum LH surge and ovulation, akin to cyclic ovulation. The involvement of circadian signalling may therefore also explain the increased variability in Avp expression towards the end of pregnancy. However, given that the post-partum LH surge and ovulation are most likely mediated by the AVPV, which also mediates estrogen positive feedback, the increased variability in Kiss1 expression in the ARC may not be linked to post-partum ovulation. Another possible reason for the increased Kiss1 variability, or potentially a variable increase in activity, could be the stimulation of oxytocin secretion, in preparation for parturition and lactation (Russell, Leng & Douglas 2003). Peripheral administration of kisspeptin-10 has been shown to increase circulating oxytocin levels (Kotani et al. 2001), as well as oxytocin neuronal firing rate in both non-pregnant and pregnant rats (Scott & Brown 2011, 2013). In contrast, central administration of kisspeptin fails to elicit an oxytocin response in non- pregnant rats, initially suggesting that kisspeptin in the peripheral circulation may not cross the blood brain barrier to act on oxytocin neurons (Scott & Brown 2013). This is supported by studies in the non-pregnant rat showing that the supraoptic nucleus contains few kisspeptin fibres (Desroziers et al. 2010), and weak expression of Kiss1r (Higo et al. 2016). Intriguingly, oxytocin neurons do respond to centrally administered kisspeptin during pregnancy, although this response differs from that elicited by peripheral kisspeptin (Scott & Brown 2013). In late pregnancy, while oxytocin neurons exhibit a robust increase in firing rate in response to centrally administered kisspeptin, the duration of this increase varies between neurons, unlike when kisspeptin is administered peripherally (Scott & Brown 2013). Therefore, the development of central kisspeptin excitation of oxytocin neurons in late pregnancy may be related to increased Kiss1 variability in the ARC at this time.

Prior to the rise in Kiss1 mRNA expression in the AVPV, levels of the AVP receptor Avpr1a also increased, similar to observations in the rat using in situ hybridisation, where Avpr1a expression peaked at the time of lights off and this was followed by a peak in Kiss1 expression four hours later (Smarr, Gile & de la Iglesia 2013). However, given that the interval between the Avpr1a and Kiss1 expression peaks was 8 hours (see Chapter 5), compared to 4 hours in the study by Smarr et al. (2013), the temporal arrangement may not necessarily reflect the transmission of direct circadian information from the SCN to

150 Chapter 9 AVPV kisspeptin neurons, as might be expected. Measuring the circadian expression of Avp mRNA specifically in the SCN (via in situ hybridisation) may have been helpful in further elucidating the temporal link between Avp and Kiss1, especially considering the recent suggestion that circadian variation in Avpr1a is not necessarily reflective of changes in AVP signalling in AVPV kisspeptin neurons (Piet et al. 2015). In that study, the authors observed that the responsiveness of AVPV kisspeptin neurons to AVP is similar regardless of time-of-day or estrous cycle stage (Piet et al. 2015), in agreement with a previous report by Williams 3rd et al. (2011). However, Avp levels in this thesis reflect the entire anterior hypothalamus, consequently encompassing numerous AVP- expressing magnocellular neurons of the paraventricular and supraoptic nuclei (Alves et al. 1998; Nomura et al. 2002), in addition to the AVP neurons projecting from the SCN to the AVPV. These particular AVP neurons are not involved in circadian signalling, but rather, regulate blood osmolality (Dunn et al. 1973). Accordingly, any time-of-day variation in Avp expression related to circadian signalling in the present study could have been masked by the non-rhythmic magnocellular neuronal population, which would potentially explain the lack of diurnal variation in Avp expression in the current study. In situ hybridisation would be an improvement over qPCR by allowing visualisation of Avp mRNA specifically in the SCN may reveal a rhythmic expression of Avp, as previously observed (Dardente et al. 2004). Here, Avp mRNA expression in the SCN of adult mice kept in constant darkness was found to be strongly rhythmic, peaking in the late subjective day (Dardente et al. 2004).

Given the purported role of RFRP-3 as an inhibitor of GnRH and/or gonadotropin secretion (Kriegsfeld et al. 2006; Ducret, Anderson & Herbison 2009), it has been proposed to modulate the negative feedback effects of estrogen that operate for most of the ovulatory cycle (Kriegsfeld 2006; Gibson et al. 2008; Ducret, Anderson & Herbison 2009; Williams 3rd & Kriegsfeld 2012). Gibson et al. (2008) have previously shown that the number of cells expressing RFRP is lowest at proestrus and maximal at diestrus, indicating that the withdrawal of RFRP-3 may also be important for positive estrogen feedback. Taken together, these findings suggest that RFRP-3 may be involved in regulating negative estrogen feedback and allowing positive feedback; an outcome not consistent with the observed lack of difference in Rfrp expression between diestrus, proestrus and pregnancy in the current study. Notably, the literature regarding the regulation of hypothalamic RFRP-3 by E2 in rodents is highly contradictory; with E2 treatment having been shown to decrease (Molnár et al. 2011; Poling et al. 2012), increase

151 Chapter 9 (Iwasa et al. 2012) or have no effect (Quennell et al. 2010) on RFRP levels. Furthermore, the specific role of RFRP-3 in the female-only LH surge is doubtful, given that Rfrp expression is not sexually dimorphic (Poling et al. 2012). Clearly, the field of RFRP biology is still subject to considerable debate. Based on the current study, our findings do not point to a clear role for Rfrp in the regulation of GnRH secretion.

The peak in Kiss1 expression was restricted to proestrus and not observed at diestrus, most likely due to the relatively low levels of E2 in the latter stage, especially considering clock gene expression between the two stages was not different. As high levels of E2 are a potent trigger for the positive feedback rise in Kiss1 and the GnRH/LH surge (Robertson et al. 2009; Williams 3rd et al. 2011; Smarr, Gile & de la Iglesia 2013), how these responses are abolished during pregnancy, in spite of elevated E2 levels, is unclear. The underlying mechanisms of this pregnancy-induced suppression of the Kiss1 peak in expression were subsequently investigated in Chapters 6 and 7.

The current model of kisspeptin involvement in the LH surge posits that kisspeptin neurons in the AVPV act as an integration centre, receiving both estrogenic and circadian signals. The kisspeptin neurons are activated only when the daily AVP signal from the

SCN coincides with elevated E2 levels, as in proestrus. This has the downstream effect of activating GnRH neurons and eventually results in the LH surge. Adding to this model, we propose that during pregnancy, despite elevated E2 levels and an intact circadian clock, circadian input to AVPV kisspeptin neurons is disrupted, possibly by the actions of other hormones elevated during pregnancy, resulting in a suppression of kisspeptin output. Two likely candidates for this are P4 and PRL, and these were chosen for further study.

Given that triggering the LH surge requires the combination of estrogenic and circadian signals, the findings initially indicated that circadian input to the AVPV was disrupted or deficient in pregnancy. However, the persistence of robust clock gene rhythmicity into pregnancy indicated that the SCN itself was still functional (Wharfe et al. 2016), therefore pointing to the possibility that the circadian signal was being overridden by rising levels of hormones during pregnancy. P4 and PRL are two such hormones that play essential roles in the maintenance of pregnancy. P4 has long been known to be a potent inhibitor of gonadotropin secretion, as detailed below (Goodman & Karsch 1980; Skinner et al.

1998; Bashour & Wray 2012). Alternatively, P4 signalling in kisspeptin neurons in the

152 Chapter 9 AVPV is also necessary for neuronal activation, and hence, LH surge induction (Stephens et al. 2015). Moreover, both kisspeptin expression and LH release are inhibited by high levels of PRL in rodents (Araujo-Lopes et al. 2014; Brown, Herbison & Grattan 2014). The importance of these hormones in pregnancy, and their direct effects on kisspeptin and the subsequent LH surge, suggest that they could be involved in the suppression of the E2-induced increase in Kiss1 mRNA expression during pregnancy. Thus the effects of P4 and PRL on the circadian expression of Kiss1 mRNA in the AVPV was examined in Chapter 6.

In Chapter 6 we determined that OVX mice treated with E2 were able to replicate the increase in Kiss1 mRNA expression in the evening, as seen in proestrus females in Chapter 5, and consistent with observations from previous studies (Smith et al. 2006;

Adachi et al. 2007). However, additional treatment with P4 or PRL abolished this pattern of Kiss1 expression, without affecting the oscillatory expression of the clock gene Bmal1, indicating that these hormones are involved in the suppression of the kisspeptin circadian rhythm. The underlying mechanisms of how P4 and PRL achieve this are still unclear.

Kisspeptin neurons express both P4 and PRL receptors (Clarkson et al. 2008; Kokay, Petersen & Grattan 2011) and it is likely that these hormones exert their effects through direct action on kisspeptin neurons (Brown, Herbison & Grattan 2014; Stephens et al.

2015). P4 is known to have inhibitory effects on GnRH secretion (Goodman & Karsch 1980; Skinner et al. 1998; Bashour & Wray 2012), although the exact mechanism is not completely clear as the classic nuclear PR has not been detected in GnRH neurons

(Skinner, Caraty & Allingham 2001). It is therefore possible that P4 exerts its actions on GnRH secretion via kisspeptin neurons, which express PR. It has been shown in mice that PRL suppresses GnRH secretion during lactation by reducing Kiss1 mRNA and protein levels in the AVPV and ARC (Liu et al. 2014). Furthermore, brain slice recordings revealed that GnRH neurons in lactating mice were responsive to exogenous kisspeptin, but not kisspeptin input from the AVPV, suggesting that AVPV kisspeptin neurons were unable to release adequate endogenous kisspeptin to stimulate GnRH neurons (Liu et al. 2014). It is possible that PRL acts in a similar fashion to prevent the kisspeptin increase and subsequent LH surge during pregnancy.

Although it was expected that mice receiving PRL treatment would exhibit the highest plasma levels of PRL, surprisingly, mice in the E2 + P4 group had higher PRL levels compared to mice treated with E2 + PRL and mice treated with E2 alone. Somewhat

153 Chapter 9 consistent with this, previous studies have found that giving rats and guinea pigs E2 in combination with P4 results in increased PRL concentrations compared to E2 administration alone (Caligaris, Astrada & Taleisnik 1974; Bethea et al. 1995). As for mice treated with PRL in Chapter 6, the dose used (1 mg/kg body weight per day; based on Sapsford et al. 2012) was unable to produce an increase in circulating PRL levels that was greater than the P4-stimulated release of endogenous PRL. Indeed, PRL levels in the PRL-treated mice of Chapter 6 appear to be at the low end of the range seen in rodent pregnancy (Amenomori, Chen & Meites 1970; Morishige, Pepe & Rothchild 1973), although differences in assay technique and other factors may prevent direct comparison between our study and others. An interesting implication arising from this unexpected observation is that the suppression of the Kiss1 increase in P4-treated mice may be due to the P4-stimulated increase in PRL, rather than P4 itself. Support for this theory may come from a study in ewes showing that, although P4 strongly inhibits GnRH/LH secretion (Goodman & Karsch 1980; Skinner et al. 1998; Bashour & Wray 2012), this suppressive action may not be directly mediated by kisspeptin, as P4 treatment had a relatively small effect on Kiss1 expression (Smith et al. 2007). While kisspeptin itself may not be directly involved in the negative feedback actions of P4, there is evidence that dynorphin (DYN) co-expressed in ARC kisspeptin/neurokinin B/dynorphin (KNDy) neurons plays this role, albeit from studies in ewes. Firstly, virtually all KNDy neurons express PR (Foradori et al. 2002); secondly, administering a DYN receptor (kappa-opioid receptor; KOR) antagonist resulted in an increased LH pulse frequency in luteal phase ewes (roughly equivalent to the metestrus and diestrus phases in the rodent; Goodman et al. 2004); and finally, the expression of preprodynorphin mRNA decreased with OVX (Foradori et al.

2005). These data strongly suggest that P4 acts via DYN to inhibit GnRH secretion. However, the evidence is less clear in rats compared to ewes. In support of an inhibitory role for DYN, administration of a KOR antagonist increased LH pulse amplitude and frequency in rats (Gallo 1990; Zhen & Gallo 1992), and conversely, a KOR agonist decreased serum LH levels in a mouse model (Navarro et al. 2009). In contrast, E2 suppressed, rather than stimulated, Dyn mRNA levels, and Dyn/KOR knock-out mice exhibited reduced LH levels (Navarro et al. 2009). Thus, the role of DYN as a mediator of P4 negative feedback in rodents remains uncertain. Additionally, more recent evidence suggests DYN (in conjunction with NKB) acts autosynaptically on KNDy neurons to regulate kisspeptin secretion, and hence GnRH release (Navarro et al. 2009; Goodman et al. 2013; Ruka, Burger & Moenter 2013). Another possible mechanism for the inhibitory action of P4 on GnRH secretion, independent of kisspeptin, is via direct effects on GnRH

154 Chapter 9 neurons. Although GnRH neurons do not express the classic nuclear PR isoforms, a study has shown that P4 can act directly on GnRH neurons by binding to the non-classical PR isoform, progesterone receptor membrane component 1 (PgRMC1; Bashour & Wray

2012). Thus, while it is well established that P4 inhibits GnRH secretion, it is still unclear if P4 acts directly on GnRH or via kisspeptin. Accordingly, the mechanism of Kiss1 suppression by P4 and PRL, needs further investigation.

Although AVPV kisspeptin neurons are thought to integrate estrogenic and circadian signals to ensure appropriate timing of the LH surge, these cells may not be the only gating locus. Evidence suggests that GnRH neurons themselves possess a gating mechanism that mediates the timing of the LH surge and thus ovulation; GnRH neuronal activation in response to kisspeptin stimulation is not indiscriminate, but time-dependent, responsive only in the afternoon and not the morning (Williams 3rd et al. 2011). Moreover, the degree of GnRH neuronal activity in response to kisspeptin stimulation is not static, but enhanced by the presence of E2 (Williams 3rd et al. 2011). The potential mechanism underlying the changing sensitivity of GnRH to kisspeptin may be either circadian variation in kisspeptin receptor expression, or a high inhibitory tone in the morning (Williams 3rd et al. 2011). These findings, that GnRH neurons exhibit time- and estrogen- dependent responses to kisspeptin stimulation, highlight the importance of GnRH neurons as a gating locus in mediating ovulation, in addition to AVPV kisspeptin neurons.

Chapter 7 involved sequencing the entire transcriptome of the mouse anterior hypothalamus. The aim was to identify differentially expressed genes between proestrus and pregnancy, in the hope of identifying those which could potentially be involved in suppressing the kisspeptin circadian rhythm. The RNA Seq data showed that Kiss1 expression at proestrus was not significantly increased at ZT13 compared to other time points, contrary to previous observations in Chapter 5 and other studies (Smith et al. 2006; Adachi et al. 2007). While there was a trend for a peak in Kiss1 expression at ZT13, the low number of replicates afforded to RNA Seq, and biological variation likely prevented a statistically significant result. With a limit of 48 samples in total to be sequenced, we were restricted to a maximum of 3 samples per time point and stage of estrous cycle/pregnancy. This may also account for the lack of increase in Avpr1a expression at proestrus. On the other hand, the clock genes Bmal1 and Reverba demonstrated a robust rhythmic expression at proestrus and during pregnancy, as well as an antiphase

155 Chapter 9 relationship, which is consistent with previous studies (Wharfe et al. 2016; Yap et al. 2016).

From Chapter 7, genes found to be differently expressed between proestrus and pregnancy were all upregulated throughout pregnancy, with the exception of albumin, which was increased only at day 10 of pregnancy. The upregulation of genes coding for haemoglobin subunits is in line with blood volume expansion during pregnancy (Longo 1983), while increases in the SOCS family of proteins is crucial for modulating the potentially harmful effects of cytokine signalling (Fitzgerald et al. 2009). Per3 and Ciart were among the genes found to exhibit robust oscillatory expression at proestrus that persisted at days 10 and 14 of pregnancy. Both Per3 and Ciart are transcriptional repressors of CLOCK-BMAL1 activity, and hence are important components of the clock gene machinery that drives the circadian expression of downstream targets (Reppert & Weaver 2001; Annayev et al. 2014; Goriki et al. 2014). The role of Per3 and Ciart as negative regulators of Bmal1 transcription is confirmed by their antiphase relationship with Bmal1 as seen in Chapter 7. The sustained rhythmicity of Per3 and Ciart between proestrus and pregnancy is also consistent with a previous study showing robust clock gene rhythmicity in the mouse SCN throughout pregnancy (Wharfe et al. 2016).

A handful of genes, including the VEGF receptor Kdr and the clock gene Per2, were found to be differentially expressed between the morning and evening exclusively at proestrus, and not at day 10 or 14 of pregnancy. While the expression pattern of these genes appears to be similar to that of Kiss1, it was unexpected that their rhythmicity was disrupted in pregnancy. As a clock gene and an essential component of the core circadian loop, the rhythmicity of Per2 is most likely preserved during pregnancy, given the persistent circadian rhythmicity of clock genes in pregnant mice as previously mentioned (Wharfe et al. 2016). The lack of circadian variation of Per2 at days 10 and 14 of pregnancy may therefore be attributed again to the low replicate number used. In addition, the clock genes Per3 and Ciart have been found to exhibit oscillatory expression at proestrus and during pregnancy in the current study. Further confirmation of these results by qRT-PCR is needed, as is always the case with RNA Seq experiments.

Although the kisspeptin circadian rhythm is well established in adult female rodents, the timing of onset is still not known. As the kisspeptin rhythm is vital for the LH surge and hence reproduction, it is possible that the rhythm appears in conjunction with puberty.

156 Chapter 9 However, given that kisspeptin is expressed in the hypothalamus long before puberty (Clarkson et al. 2009a; Semaan et al. 2010; Poling et al. 2012), kisspeptin circuits are reported to be functional in utero (Kumar et al. 2014), and some clock gene circadian rhythms are well developed fairly early in neonatal life (Sladek et al. 2004; Ansari et al. 2009), it is possible that the kisspeptin rhythm may begin prior to puberty. This was investigated in Chapter 8.

Our findings revealed that the kisspeptin circadian rhythm does not develop before postnatal (P) day 25. Clock genes in the anterior hypothalamus (which likely represent the SCN), as represented by Bmal1 and Rev-erbα, did not exhibit fully developed oscillatory expression by P25. Bmal1 and Rev-erbα were selected as representatives of clock gene rhythmicity in our studies based on their robust oscillatory expression profiles, as observed by Wharfe et al. (2016). However, previous studies have shown that oscillatory expression of Bmal1 and Clock develops later than that of the Per and Cry genes, which appear to become rhythmic around P10 (Sladek et al. 2004; Ansari et al. 2009). Regardless, the full complement of core clock genes was not completely “mature” by 25 days of age. As the kisspeptin circadian rhythm is dependent on a circadian signal, the lack of complete clock gene rhythmicity in the SCN during neonatal development, precludes circadian expression of kisspeptin in the AVPV at this time. Moreover, E2 concentrations were extremely low at P15 and P25, and as such would be insufficient to stimulate a surge in Kiss1 mRNA levels.

Whether treating female (or even male) neonatal/pre-pubertal mice with elevated levels of E2 would induce a proestrus-like kisspeptin rhythm (as it does in ovariectomised adult females) is a potential avenue for further research. Success is doubtful however, given the immaturity of the SCN clock gene rhythmicity in the neonatal period. The combination of deficient SCN rhythmicity and low E2 levels during neonatal life strongly suggest that the kisspeptin circadian rhythm is unable to develop before puberty. Even if neonatal E2 treatment could stimulate a late afternoon/evening increase in kisspeptin expression, this may not necessarily translate to a surge in GnRH/LH, given that GnRH neurons possess a gating mechanism of control that is independent of kisspeptin neurons, demonstrated by the time-dependent sensitivity of GnRH neurons to kisspeptin stimulation (Williams 3rd et al. 2011), as described above. Moreover, premature exposure to excessive exogenous estrogens during neonatal life can have adverse effects on both male and female rodents. Male rats treated with estrogens during the early neonatal period 157 Chapter 9 have reduced testosterone levels, poor spermatogenesis and decreased fertility in adulthood (Atanassova et al. 1999; Goyal et al. 2003). Female rats receiving estrogen treatments neonatally exhibit a “defeminisation” of the AVPV kisspeptin population and lose their ability to mount an E2-induced LH surge (Kauffman et al. 2007a; Homma et al.

2009); thus, treating neonates with E2 could potentially prevent a stimulation of an adult- like kisspeptin rhythm.

It is possible that Bmal1 and Rev-erbα expression specifically in the SCN was rhythmic prior to puberty but oscillatory expression was masked, due to a different rhythm in the AVPV, given that both nuclei were present in our sample of the anterior hypothalamus. Recent studies have shown that the AVPV possesses a subordinate circadian clock that is tied to the central clock in the SCN (Smarr, Gile & de la Iglesia 2013), but phase-delayed (Chassard et al. 2015). Because clock gene expression was measured in the entire anterior hypothalamus (containing the SCN and the AVPV), and due to potential phase differences between the two oscillators, it is conceivable that clear circadian clock gene expression was masked by the overlapping rhythms. Nonetheless, this scenario is made less likely by previous observations of clear and robust circadian rhythms in clock gene expression, measured in the entire anterior hypothalamus of adult female mice (Wharfe et al. 2016; Yap et al. 2016).

In Chapter 8, Rfrp expression was also found to decrease with age, from P5 to P25; given its GnRH-inhibiting effects, the high levels of Rfrp could be a contributing factor to the lack of kisspeptin rhythmicity during the neonatal period. Although only 12% of AVPV Kiss1 neurons express RFRP-3 receptors (Poling et al. 2013), it is still possible that Rfrp could have an effect on kisspeptin expression by acting directly on kisspeptin neurons in the AVPV. The decrease in Rfrp from the early neonatal to pre-pubertal period may therefore represent a gradual removal of inhibition on the reproductive axis as puberty approaches (Semaan & Kauffman 2015), strengthening the theory that the kisspeptin circadian rhythm does not develop until after puberty. Conversely, it is also possible that full pubertal maturation is contingent upon the development of the kisspeptin circadian rhythm. We propose that the combination of an immature central circadian oscillator and low E2 levels during neonatal life, with a possible input from Rfrp expressing neurons, prevents time-of-day variation in Kiss1 expression in the AVPV, such as that seen in the adult female rodent at proestrus (Figure 9.1).

158 Chapter 9

Figure 9.1 Proposed model indicating the relative immaturity of the circadian clock and estradiol levels in the pre-pubertal female neonate compared to the adult proestrus female. In the neonatal period, incomplete clock gene rhythmicity in the SCN in combination with low estradiol levels, and a potential suppressive action of high RFRP- 3 expression, prevent the circadian activation of Kiss1 neurons. In the evening of proestrus, Kiss1 neurons in the AVPV receive circadian information from the SCN via vasopressin receptors (Avpr1a or V1a), and sense circulating estradiol levels via estrogen receptors (ER), located on these neurons. Kiss1 neurons are activated in the event that estrogenic and circadian signals are both high, releasing kisspeptin and activating GnRH neurons via kisspeptin receptors GPR54, which in turn drives the release of GnRH. Adapted from Smith et al. 2006.

159 Chapter 9 One of the major limitations with interpreting the results of the studies within this thesis is that gene expression, as measured by quantitative PCR, while meaningful, does not give an indication of physiological function. An increase or decrease gene expression may not necessarily translate to a change in peptide activity, or even an increase in peptide levels, although it has shown that Kiss1 mRNA levels in the female mouse correlate with kisspeptin-immunoreactivity (Overgaard et al. 2013). Moreover, kisspeptin synthesised within the cell requires packaging and exocytosis before being able to exert its intended function. Furthermore, with regard to receptor expression, an additional step of receptor insertion into the cell membrane is required for a functional outcome.

Overall, the studies in this thesis investigated diurnal changes in kisspeptin expression in the hypothalamus during adulthood and pregnancy, as well as during neonatal development. The kisspeptin circadian rhythm that is observed in proestrus females and is responsible for the pre-ovulatory GnRH/LH surge, is suppressed during pregnancy.

This suppression is due, in part, to the influence of P4 and PRL, hormones that are found in elevated concentrations during pregnancy. The kisspeptin circadian rhythm does not appear to begin before puberty, probably because clock gene rhythmicity is not fully established at this time, although further studies are required to confirm exactly when it does start. It is likely that oscillatory kisspeptin expression does not occur until puberty onset, when gonadotropin levels are elevated and estrous cyclicity is established.

160 Chapter 10

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