Role of Wnt4 Signaling in Mammalian Sex Determination, Ovariogenesis and Female Sex Duct Differentiation

D 1250 OULU 2014 D 1250

UNIVERSITY OF OULU P.O.BR[ 00 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATIS OULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTA

SERIES EDITORS DMEDICA Renata Prunskaite-Hyyryläinen

ASCIENTIAE RERUM NATURALIUM Renata Prunskaite-Hyyryläinen Professor Esa Hohtola ROLE OF WNT4 SIGNALING BHUMANIORA IN MAMMALIAN SEX University Lecturer Santeri Palviainen CTECHNICA DETERMINATION, Postdoctoral research fellow Sanna Taskila OVARIOGENESIS AND DMEDICA Professor Olli Vuolteenaho FEMALE SEX DUCT ESCIENTIAE RERUM SOCIALIUM DIFFERENTIATION University Lecturer Veli-Matti Ulvinen FSCRIPTA ACADEMICA Director Sinikka Eskelinen GOECONOMICA Professor Jari Juga

EDITOR IN CHIEF Professor Olli Vuolteenaho PUBLICATIONS EDITOR

Publications Editor Kirsti Nurkkala UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF BIOCHEMISTRY AND MOLECULAR MEDICINE; ISBN 978-952-62-0471-0 (Paperback) BIOCENTER OULU; ISBN 978-952-62-0472-7 (PDF) CENTER FOR CELL-MATRIX RESEARCH ISSN 0355-3221 (Print) ISSN 1796-2234 (Online)

ACTA UNIVERSITATIS OULUENSIS D Medica 1250

RENATA PRUNSKAITE-HYYRYLÄINEN

ROLE OF WNT4 SIGNALING IN MAMMALIAN SEX DETERMINATION, OVARIOGENESIS AND FEMALE SEX DUCT DIFFERENTIATION

Academic dissertation to be presented with the assent of the Doctoral Training Committee of Health and Biosciences of the University of Oulu for public defence in the Auditorium of Kastelli Research Centre (Aapistie 1), on 30 May 2014, at 12 noon

Supervised by Professor Seppo J. Vainio

Reviewed by Docent Marja Mikkola Docent Olli Ritvos

Opponent Professor Martin M. Matzuk

ISBN 978-952-62-0471-0 (Paperback) ISBN 978-952-62-0472-7 (PDF)

ISSN 0355-3221 (Printed) ISSN 1796-2234 (Online)

Cover Design Raimo Ahonen

JUVENES PRINT TAMPERE 2014 Prunskaite-Hyyryläinen, Renata, Role of Wnt4 signaling in mammalian sex determination, ovariogenesis and female sex duct differentiation. University of Oulu Graduate School; University of Oulu, Faculty of Biochemistry and Molecular Medicine; University of Oulu, Biocenter Oulu; Center for Cell-Matrix research Acta Univ. Oul. D 1250, 2014 University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract Mammalian female sex development was considered a default developmental pathway. However, the deletion of the Wnt4 gene, a member of the Wnt family of secreted signals, was shown to reverse the sex of XX female mouse embryo and caused exhibition of certain male characteristics. This indicated that the female sexual development cannot be default but depends on active signaling and cell-cell interaction. The aim of the current study was to reveal the functional role of the Wnt4 gene in the control of sex determination, ovariogenesis and female sex duct formation. This study demonstrates that testosterone is produced by the ovary of Wnt4-deficient female embryos. The inhibition of androgen action by an antiandrogen, flutamide, during gestation leads to complete degeneration of the Wolffian ducts in 80% of the Wnt4 mutant females. This suggests that testosterone is the possible mediator of the masculinization phenotype in Wnt4-deficient females. Wnt4 is expressed by ovarian somatic cells, which are vital for the control of female germline development. This work has shown that Wnt4 is the factor maintaining germ cell cysts, cell-cell interaction and early follicular gene expression. In addition, the findings indicate a critical role for Wnt4/5a signaling in meiosis. Our research has proven that Wnt4 has roles during postnatal ovary development as its defective signaling leads to premature ovarian failure associated with diminished Amh levels, defective basement membrane and cell polarization. The Mullerian duct, the anlagen of oviduct, uterus and upper part of vagina, does not form in Wnt4-deficient females. This study indicates that Wnt4 is needed for migration initiation and maintenance during Mullerian duct formation prenatally. During the postnatal uterine differentiation Wnt4 is essential for endometrial gland formation. The present study provides new evidence for Wnt4 function during embryonic and adult female sexual differentiation.

Keywords: anti-Mullerian hormone, cell adhesion, cell polarity, Mullerian duct, ovarian development, premature ovarian failure, sex determination

Prunskaite-Hyyryläinen, Renata, Wnt4-signaloinnin merkitys nisäkkäiden sukupuolen määräytymisessä, munasarjojen kehittymisessä sekä naaraan sukupuolitiehyitten erilaistumisessa. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Biokemian ja molekyylilääketieteen tiedekunta; Biocenter Oulu; Center for Cell-Matrix research Acta Univ. Oul. D 1250, 2014 Oulun yliopisto, PL 8000, 90014 Oulun yliopisto

Tiivistelmä Nisäkkäiden naaraspuolista kehitystä pidettiin aiemmin sukupuolisen erilaistumiskehityksen ole- tusarvona. Signaloivien proteiinien Wnt-perheeseen kuuluvan Wnt4-geenin puutteen todettiin kuitenkin johtavan XX naarasalkion sukupuolen kääntymisen koiraaksi sekä aiheuttavan tiettyjä koiraille ominaisia piirteitä. Tämä osoitti, ettei naaraspuolinen kehitys ole oletusarvo, vaan se riippuu aktiivisesta signaloinnista ja solujen välisestä interaktiosta. Tämän väitöstutkimuksen tarkoitus oli selvittää Wnt4-geenin roolia sukupuolen määräytymi- sessä, munasarjojen kehittymisessä sekä naaraan sukupuolitiehyitten muodostumisessa. Tutkimuksessa osoitettiin, että munasarjat tuottavat testosteronia niillä naaraspuolisilla alki- oilla, joilta puuttuu Wnt4-geeni. 80 prosentilla naaraista, joilla on Wnt4-geenin puute, androgee- nivaikutuksen esto raskauden aikana annettavalla antiandrogeenilla, flutamidilla, estää sukupuolen vaihtumisen fenotyypin. Tämä viittaa siihen, että testosteroni toimii mahdollisena koiraan fenotyypin välittäjänä naarailla, joilta puuttuu Wnt4-geeni. Wnt4 ilmentyy munasarjojen somaat- tisissa soluissa, jotka ovat tärkeitä naaraspuolisen ituradan kehityksen säätelyn kannalta. Väitös- tutkimus osoittaa, että Wnt4 on itusoluryppäitä, solujen välistä interaktiota sekä varhaista follik- keligeeni-ilmentymistä ylläpitävä tekijä. Tulokset osoittavat myös, että Wnt4/5a -signaloinnilla on tärkeä rooli meioosissa. Tutkimus osoittaa lisäksi, että Wnt4 vaikuttaa munasarjojen kehityk- seen myös syntymän jälkeen. Puutteellinen signalointi alentaa Anti-Müllerian hormonin tasoa, heikentää tyvikalvoa ja vähentää solujen polarisaatiota, joka johtaa ennenaikaiseen munasarjojen toiminnan hiipumiseen. Müllerin tiehyet, joista myöhemmin kehittyvät munanjohtimet, kohtu ja vaginan yläosa, jää- vät kokonaan muodostumatta naarailla, joilta puuttuu Wnt4-geeni. Tulokset viittaavat siihen, että Wnt4 on tarpeen alkioaikaiseen Müllerin tiehyen muodostavien solujen liikkeellelähtöön ja yllä- pitoon. Wnt4:llä on myös keskeinen rooli kohturauhasten muodostumisessa sukukypsyyden saa- vuttamisen aikana ja sen jälkeen.

Asiasanat: anti-Müllerian hormoni, munasarjojen kehitys, munasarjojen toiminnan ennenaikainen hiipuminen, Müllerin tiehyt, solujen kiinnittyminen, solujen polaarisuus, sukupuolen määräytyminen

To my family

Happiness is when what you think, what you say, and what you do are in harmony” -Mahatma Gandhi

8 Acknowledgements

This work was carried out at Biocenter Oulu, the Department of Medical Biochemistry and Molecular Biology of the Institute of Biomedicine at the faculty of Medicine, and completed in the newly established Faculty of Biochemistry and Molecular Medicine, University of Oulu. I wish to express my gratitude to Professor Seppo Vainio for giving me the opportunity to discover and explore the fascinating world of developmental biology with its uphills and downhills and for promoting independent and outside-the-box thinking. His passion for science is infectious and I especially value his open-minded thinking and never-ending curiosity. I am grateful to Professor Taina Pihlajaniemi, Professor Johanna Myllyharju, Professor Peppi Karppinen, Professor Kalervo Hiltunen and all the other group leaders in the Faculty for providing an inspiring working environment and excellent working facilities. I would like to thank the members of my PhD follow- up group: PhD Pirkko Huhtala, PhD Ulla Petäjä-Repo, PhD Minna Komu, who has guided my first steps into the field of germ cells; and especially Docent Minna Männikkö for her advice and encouragement. I would like to thank Docent Sinikka Eskelinen for introduction to the imaging techniques and for stimulating discussions. I wish to express my special compliments to Docent Raija Sormunen for guidance into the world of ultrastructures and for open discussions on various aspects of life. I would like to thank PhD Hellevi Peltoketo for inspirational scientific and general discussions. Docent Marja Mikkola and Docent Olli Ritvos are acknowledged for their careful revision and expert comments on this thesis book. Anna Vuolteenaho is acknowledged for the thorough language review. I wish to thank my all co-authors for fruitful collaboration and contributions. I would also like to thank the former and current members of Seppo Vainio’s group for their friendship, discussions and help. I would like to express my gratitude to Hannele Härkman, Johanna Kekolahti-Liias, Paula Haipus, Jaana Kujala, Mervi Matero and Aila Holappa for their excellent technical assistance and above all for being the ‘good fairies’ of the lab, creating a friendly and comfortable working environment. I wish to thank all the members of the previuos Department of Medical Biochemistry and Molecular Biology, the Kieppi-Building and reunited colleagues form the former Department of Biochemistry.

9 I wish to acknowledge Auli Kinnunen, Anne Vainionpää, Teija Luoto, Pertti Vuokila, Seppo Lähdesmäki, Irmeli Nykyri and Hanna-Mari Pesonen for their help and guidance in the practical matters. Antti Viklund owns my special thanks for his patient assistance with computer issues as well as comprehensive discussions on work and matters stretching far beyond it. Special thanks go to my dearest friends and their families: Fumi, Kristina, Ildi, Anna, Irina, Regina, Anastasia, Asta, Eija, Vida, Bernardas, Gražina, Dalia, Viktorija and Neringa - your friendship and support gave me the energy to carry out my work and realize that work is just a part of life; thank you for being there through all the good and bad times. I wish to express my sincere gratitude to my parents-in-law Irja and Rauno, whose warm caring and cheering never failed through all these years. My warmest thanks go to my sister Sigita and her family; knowing that you are there for me, as I am there for you, at all times makes the difference in this world! Esu dėkinga savo dėdei Algimantui, pažadinusiam manyje žingeidumą mokslui bei neįkainuojamą laiką praleistą drauge. Dėkoju savo tėčiui, už skatinimą ir nuolatinį domėjimasį mano veikla. Nuoširdžiai dėkoju savo mamai, už suteiktas pasirinkimo laisves bei visapusišką palaikymą ir meilę. I owe my infinite thanks to my beloved husband, Juha-Petteri - I am fortunate to walk this life hand-in-hand with you; our kids Nora and Lukas - you mean the world to me! This work was supported by Biocenter Oulu, the Sigrid Jusélius Foundation, Oulu University Scholarship Foundation, Orion-Farmos Research Foundation and Jalmari and Rauha Ahokas Foundation.

Oulu, Finland, February 2014 Renata Prunskaitė-Hyyryläinen

10 Abbreviations

Acvr 2/3 Activin A receptor, type 2/3 (ALK2/3) Adamts19 A disintegrin-like and metallopeptidase with thrombospondin type 1 motif, 19 ALK2/3 Activin receptor-like kinase-1 (Acvr 2/3) Amh Anti-Mullerian hormone Amhr1 Anti-Mullerian hormone type 1 receptor Amhr2 Anti-Mullerian hormone type 2 receptor APC Adenomatous polyposis coli Bmp15 Bone morphogenetic protein 15 Bmp4 Bone morphogenetic protein 4 Ca2+ Calcium CamKII Calcium/calmodulin-dependent kinase II Ccnd1 Cyclin D1 Cdh1 Cadherin 1 (E-cadherin) Cdh2 Cadherin 2 (N-cadherin) Celsr Cadherin, EGF LAG seven-pass G-type receptor 3, CK1 Casein kinase 1 CL Corpus luteum Ctnnb1 Catenin (cadherin associated protein), beta 1 (β-catenin) Cx37 Connexin 37 Cx43 Connexin 43 Cyp11a Cytochrome P450 cholesterol side-chain cleavage Cyp17 Cytochrome P450 17 beta-hydroxylase Cyp19a1 Cytochrome P450, family 19, subfamily A, polypeptide 1 Cyp26b1 Cytochrome P450, family 26, subfamily b, polypeptide 1 Daam1 Dishevelled associated activator of morphogenesis 1 Dax1 Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (Nr0b1) Dazl Deleted in azoospermia-like Dg Diego DHEA Dehydroepiandrosterone Dkk Dickkopf homolog Dmc1 DMC1 dosage suppressor of mck1 homolog, meiosis-specific homologous recombination Dpc Days post coitum

11 Dvl Dishevelled ECM Extracelular matrix Emx2 Empty spiracles homeobox 2 ER-β Estrogen receptor beta (NR3A2) Evi Evenness interrupted Fgf9 Fibroblast growth factor 9 Figla Folliculogenesis specific basic helix-loop-helix Fmi Flamingo Fog2 Friend of GATA2 Fos FBJ osteosarcoma oncogene Foxl2 Forkhead transcription factor L2 FSH Follicular stimulating hormone Fst Follistatin Fz Frizzled Gata3 GATA binding protein 3 Gdf9 Growth differentiation factor 9 ɣH2AX ɣ Histone H2A variant Gja1/4 Gap junction protein, alpha 1/4 GnRH Gonadotropin releasing hormone GSK3 Glycogen synthase kinase 3 Hoxa13 Homeobox A13 Hsd17b1 17-beta-hydroxysteroid dehydrogenase 1 Hsd17b3 17-beta-hydroxysteroid dehydrogenase3 HSD3B2 Hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta- isomerase 2 Irx3 Iroquois related homeobox 3 JNK Jun kinase Kit Kit oncogene KitL Kit ligand LEF Lymphoid enhancer factor LH Luteinizing hormone Lhx9 LIM homeobox protein 9 Lim1 LIM homeobox protein 1 LRP5/6 Low-density lipoprotein receptor-related protein 5/6 MD Mullerian duct MET Mesenchymal Epithelial Transition MRKH Mayer-Rokitansky-Kuster-Hauser syndrome

12 MRKHBL Mayer-Rokitansky-Küser-Hauser-Biason-Lauber syndrome Myc Myelocytomatosis oncogene Nobox Newborn ovary homeobox Nr0b1 Nuclear receptor subfamily 0, group B, member 1 (Dax1) NR5A1 Nuclear receptor subfamily 5group A member 1 (Sf1) Pax2 Paired-box gene 2 Pax8 Paired-box gene 8 PCOS Polycystic ovary syndrome PCP Planar cell polarity PGC Primordial germ cells Pk Prickle PKC Protein kinase C Pod1 Transcription factor 21 (Tcf21) POF Premature ovarian failure Pou5f1 POU domain, class 5, transcription factor 1 (Oct4) Prickle1 Prickle homolog 1 RA Retinoic acid Rac RAS-related C3 botulinum substrate 1 RALDH Retinal dehydrogenase Rec8 REC8 homolog RhoA Ras homolog gene family, member A Rspo1 R-spondin1 Runx2 Runt related transcription factor 2 RYK Receptor tyrosine kinase Sf1 Steroidogenic factor 1 (Nr5a1) Shh Sonic hedgehog Sox9 SRY (sex determining region Y)-box 9 Spo11 SPO11 meiotic protein covalently bound to DSB homolog Star Steroidogenic acute regulatory gene Stbm Strabismus Stra8 Stimulated by retinoic acid 8 Sycp3 Synaptonemal complex protein 3 TCF T Cell factor Tcf21 Transcription factor 21 (Pod1) TGF Transcription growth factor Vangl1/2 Vang-like 1/2 WD Wolffian duct

13 Wls Wntless Wnt4 Wingless-related MMTV integration site 4 Wnt5a Wingless-related MMTV integration site 5A Wnt7a Wingless-related MMTV integration site 7A Wnt8 Wingless-related MMTV integration site 8 Wnt9b Wingless-related MMTV integration site 9b Wt1 Wilms tumor 1 homolog Xlr X-linked lymphocyte-regulated β-TrCP β-transduction repeat containing protein

14 List of original articles

Present thesis is based on the articles listed below; they are referred in the text by the specified Roman numbers:

I Heikkila M*, Prunskaite R*, Naillat F, Itaranta P, Vuoristo J, Leppaluoto J, Peltoketo H & Vainio S (2005) The partial female to male sex reversal in Wnt4-deficient females involves induced expression of testosterone biosynthetic genes and testosterone production, and depends on androgen action. Endocrinology 146(9): 4016–4023. II Naillat F*, Prunskaite-Hyyryläinen R*, Pietilä I, Sormunen R, Jokela T, Shan J & Vainio SJ (2010) Wnt4/5a signalling coordinates cell adhesion and entry into meiosis during presumptive ovarian follicle development. Hum Mol Genet 19(8): 1539–1550. III Prunskaite-Hyyryläinen R, Shan J, Railo A, Heinonen KM, Miinalainen I, Yan W, Shen B, Perreault C & Vainio SJ (2013) Wnt4, a pleiotropic signal for controlling cell polarity, basement membrane integrity and Anti-Mullerian hormone expression during oocyte maturation in the female follicle. FASEB J. IV Prunskaite-Hyyryläinen R, Skovorodkin I, Shan J & Vainio SJ (2014) Wnt4+ Progenitor Cells Construct the Mullerian Duct and its Hypomorphic Allele Demonstrates a Key Role in Uterus Development. Manuscript. (*) Equal contribution

16 Contents

Abstract Tiivistelmä Acknowledgements 9 Abbreviations 11 List of original articles 15 Contents 17 1 Introduction 21 2 Review of the literature 23 2.1 Wnt gene family ...... 23 2.1.1 The canonical Wnt/beta catenin signaling pathway ...... 24 2.1.2 Non-canonical: planar cell polarity and calcium pathways ...... 25 2.2 Mammalian sex determination ...... 27 2.2.1 Bipotential gonads ...... 27 2.2.2 Female sex determination ...... 28 2.2.3 Meiosis ...... 31 2.3 Adult ovary ...... 33 2.3.1 Ovarian Folliculogenesis ...... 33 2.3.2 Extracellular matrix and cell-cell junctions in the developing follicle ...... 36 2.3.3 Molecular basis of ovarian development and function ...... 37 2.4 Steroidogenesis ...... 41 2.5 Female reproductive tract ...... 42 2.5.1 Mullerian duct development ...... 43 2.5.2 Uterus differentiation ...... 44 2.5.3 Genetic control of female reproductive tract development ...... 45 2.6 Wnt4 functions in reproductive system ...... 48 2.6.1 Disorders of WNT4 function in female reproductive system ...... 49 3 Aims of the study 51 4 Materials and methods 53 5 Results 55 5.1 The Partial Female to Male Sex Reversal in Wnt4 knock-out Females Implicates Induced Expression of Testosterone (I) ...... 55 5.1.1 Testosterone was detected in the plasma and gonads of Wnt4-deficient females ...... 55

17 5.1.2 Wnt4-deficient ovaries have changed expression of the hormone biosynthesis genes ...... 55 5.1.3 Blocking of the androgen action causes the degeneration of the Wolffian ducts in Wnt4 knock-out females but has no influence on endothelial cell organization ...... 56 5.2 Wnt4/5a signaling controls cell-cell adhesion and meiosis initiation during ovarian folliculogenesis (II) ...... 57 5.2.1 Wnt4 is expressed in somatic cells and plays an important role in maintenance of cell-cell interaction in the developing ovary ...... 57 5.2.2 Wnt4 signaling plays a role in follicle development and meiosis initiation ...... 58 5.3 Wnt4 as a pleiotropic signal for controlling multiple aspects of follicle maturation in the adult female ovary (III) ...... 59 5.3.1 Wnt4 deficiency by Amhr2+/Cre in the ovarian somatic cells compromises folliculogenesis and female fertility ...... 59 5.3.2 Postnatal inactivation of Wnt4 function with TetO-Cre;RosartTA/+ leads to premature ovarian failure ...... 60 5.3.3 Wnt4mCherry, a novel hypomorphic mouse model, develops premature ovarian failure ...... 60 5.3.4 Wnt4 signaling contributes to ovarian development by coordinating cell polarity within the follicle ...... 61 5.3.5 Wnt4 signaling in the control of Amh expression ...... 62 5.4 Wnt4+ progenitor cells construct the Mullerian duct and Wnt4 hypomorphic allele demonstrates a key role in uterus development (IV) ...... 63 5.4.1 Mullerian duct growth initiation and further extension requires Wnt4 signal ...... 63 5.4.2 Imbalanced expression of Wnt4 gene leads to MD defects early in embryogenesis and uterine gland agenesis with hydrouterus condition in adult mice ...... 65 6 Discussion 67 6.1 Female to male sex reversal in Wnt4-deficient ovary involves ectopic testosterone synthesis and anti-androgen administration prevents Wolffian duct formation (I) ...... 67 6.2 Wnt4/5a signaling controls cell-cell adhesion and meiosis initiation during ovarian folliculogenesis (II) ...... 68

18 6.2.1 Wnt4 plays a role in assembly of female ovarian follicles ...... 68 6.2.2 Wnt4 signaling regulates the development of the follicular complex and meiosis ...... 68 6.3 Wnt4 signal is involved in controlling multiple aspects of follicle maturation in the adult female ovary (III) ...... 69 6.4 Wnt4 signal is needed for Mullerian Duct cell migration and Wnt4 hypomorphic allele demonstrates a key role in Uterus Development (IV) ...... 71 7 Summary and conclusions 73 References 75 Original articles 91

20 1 Introduction

Sex determination, the process determining the sex of a developing embryo, has been one of the great questions of humankind since ancient times. A great number of folk tales and scientific explanations for it were discussed over the centuries. By now we know that mammalian sex is determined genetically; yet in many other species it can be influenced by environmental factors. The development of mammalian gonads and sex ducts involves highly unique processes. In response to presence or absence of Sry signals from the male Y chromosome the bipotential gonad will differentiate either to testes, in XY organisms, or ovaries, in XX individuals. The anlagen of the male - Wolffian and female - Mullerian duct both form in males and females during embryogenesis. After the initiation of Sry expression testicular hormone production leads to regression of the Mullerian duct (MD) and facilitates the Wolffian duct (WD) differentiation to epididymis, vas deferens and seminal vesicles in male. In females, the absence of the testicular hormones causes the regression of WD whereas MD develops into oviduct, uterus and upper part of the vagina (Brennan & Capel 2004, Edson et al. 2009, Wilhelm et al. 2007). Due to the fact that the Sry gene was sufficient to induce male sex development it was previously considered that its absence would lead to female pathway differentiation by default (Bernard & Harley 2007). The deletion of the Wnt4 gene, a member of the Wnt family of secreted signals, however has demonstrated partial female-to-male sex reversal of XX embryos (Vainio et al. 1999). The MD did not form and the WD persisted along with the changes in the ovary in the Wnt4-deficient females. This descovery has brought a new understanding in the sex determination field and assigned novel functions for the Wnt4 gene. However, the mechanism of Wnt4 function remained largely unknown, especially in the adult ovariogenesis and sex duct development, partially due to early postnatal lethality of Wnt4-deficient mice. The urge for gaining a better understanding of reproductive organ formation and function is increasing with the growing rate of couples who cannot conceive. Around one in six couples can have difficulty producing offspring. Infertility can be caused by a variety of disorders and lifestyle aspects. In women, it can commonly occur due to premature ovarian failure (POF), polycystic ovary syndrome, endometriosis, congenital reproductive tract and other anomalies. Unfortunately, fertility problems cannot be explained in around a third of the cases.

21 The current study addresses the functional role of the Wnt4 gene in the control of sex determination, ovariogenesis and female sex duct formation. This work shows that the sex reversal phenotype caused by Wnt4 deficiency occurs partially due to ectopic testosterone biosynthesis. Study demonstrates that Wnt4 signaling is needed for fine-tuned gene signaling and appropriate cell-cell interaction, early follicular gene expression and meiosis initiation in embryonic female gonads. In postnatal ovaries Wnt4 controls folliculogenesis in part with Amh, whereas Wnt4-deficient signaling leads to POF. This work also demonstrates that Wnt4 serves as a migration initiation factor during MD duct growth and is furthermore essential for the endometrial gland formation in the adult mammals. This work helps to gain a better understanding of the pathological conditions causing female infertility and could provide useful information when looking for means to prevent it.

22 2 Review of the literature

2.1 Wnt gene family

Wnts (Wingless) are conserved in all metazoan animals. Mammals have 19 Wnt ligands, which are cysteine-rich proteins of approximately 350–400 amino acids and contain an N-terminal signal peptide for secretion (MacDonald et al. 2009, Clevers 2006, Miller et al. 1999). Wnts are hydrophobic proteins, but they can still travel over long distances to induce cell-type specific responses, partially due to extracellular carriers such as exosomes (Clevers & Nusse 2012, Gross & Boutros 2013). Mouse Wnt3a protein was the first to be purified and biochemically characterized. Thirty years after Wnts were initially described another Wnt (Wnt8) was purified in complex with its receptor Frizzled8- cysteine – rich – domain and the crystal structure of entire complex Wnt8-Fz8-CRD was solved (Bienz & He 2012, Janda et al. 2012). Wnts mediate numerous processes of invertebrate and vertebrate development as well as tissue homeostasis of adult organisms due to their influences on cell proliferation, differentiation, and migration (Barker & Clevers 2006, Nusse 2005). A few conserved genes, namely, Wntless (Wls) (Banziger et al. 2006) and evenness interrupted (Evi) (Bartscherer et al. 2006), showed to be essential for Wnt secretion. In the absence of these genes Wnts are retained in the cells of origin and are not secreted out. Wls is also important for the generation of a Wnt gradient (Coudreuse et al. 2006). Some studies showed that Wnts serve as gradient morphogens and would be able to act in a long- or short-range manner (Zecca et al. 1996). Recent studies in drosophila have challenged Wnt’s long- range signaling properties. In the given model the Wnt secretion was ablated and caused only minor phenotype in the affected individuals. Due to this it was concluded that Wnts act as auto- and paracrine signals (Alexandre et al. 2014). Wnt family members have been assigned into different types based on their ability to induce transformation of the mouse epithelial cell line (C57MG). One of those highly transforming group contains Wnt1, Wnt3, Wnt3a and Wnt7a. The intermediately transforming and non-transforming group comprises Wnt2, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7b and Wnt11 (Kikuchi et al. 2012, MacDonald et al. 2009).

23 Currently, there are three different pathways known to be activated upon Wnt receptor activation: the canonical Wnt/β-catenin cascade, the noncanonical planar cell polarity (PCP) pathway, and the Wnt/Ca2+ pathway. Despite this classification, Wnt ligands probably excite complex, non-linear networks that share many downstream effectors (Anastas & Moon 2013).

2.1.1 The canonical Wnt/beta catenin signaling pathway

The β-catenin-dependent or canonical Wnt signaling pathway centers on β- catenin protein. In the absence of Wnt, cytoplasmic β-catenin is degraded by the action of the Axin complex, composed of scaffolding protein Axin, the tumor suppressor adenomatous polyposis coli (APC), casein kinas 1 (CK1) and glycogen synthase 3 (GSK3). CK1 and GSK3 phosphorylate the amino terminal region of β-catenin. This allows for ubiquitin ligases β-transduction repeat- containing protein (β-TrCP) to recognize the β-catenin leading to subsequent β- catenin ubiquitination and proteosomal degradation. As a result of β-catenin inactivation, the nuclear T cell factor/lymphoid enhancer factor (TCF/LEF) will not be transcribed and so the Wnt target genes will remain inactive (Anastas & Moon 2013, Barker & Clevers 2006, Clevers 2006, Logan & Nusse 2004, MacDonald et al. 2009). Once Wnt binds to its receptor Frizzled (Fz), the seven-pass transmembrane receptor that cooperates with the low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), the Wnt/β-catenin pathway becomes activated. The Wnt-Fz- LRP5/6 complex activates the scaffolding protein Dishevelled (Dvl), causing LRP6 phosphorylation and Axin complex interaction with the receptors. As a result, β-catenin phosphorylation is inhibited and β-catenin is stabilized. It accumulates and enters the nucleus where it forms complexes with the TCF/LEF and activates multiple target genes such as Axin2, Cycline D1, Runx2, Bmp4, Fgf9, Dickkopf, Myc and others (Anastas & Moon 2013, Logan & Nusse 2004, MacDonald et al. 2009, Nusse 2005). The secreted Wnt agonists R-spondins (1– 4) bind to leucine-rich repeat G-protein-coupled receptors (Lgr4–6) and act as enhancers of canonical Wnt signals (Barker et al. 2013, de Lau et al. 2014). The Wnt/β-catenin signaling pathways are illustrated in Figure 1 in the case of Wnt absence (A) and presence (B).

24 Fig. 1. Wnt/β-catenin signaling pathway, adapted from MacDonald et al. 2009.

2.1.2 Non-canonical: planar cell polarity and calcium pathways

The non-canonical or planar cell polarity (PCP) pathway mediates cell polarity and cell movements during gastrulation, neural tube closure, orientation of hair bundle in inner ear sensory cells, cilia motility in the embryonic node and other processes (Belotti et al. 2012, Chien et al. 2009, Habas & Dawid 2005, Simons & Mlodzik 2008). In vertebrates the PCP pathway is mediated by noncanonical Wnts, such as Wnt5a and Wnt11, through recruitment of principal components: Fzd, Dishevelled (Dvl), cadherin, EGF LAG seven-pass G-type receptor 3, flamingo homolog (Celsr), Vang-like1/2 (Vangl 1/2), and prickle homolog 1 (Prickle) (Habas & Dawid 2005, Wang 2009). Noncanonical-Wnt (Wnt5a, Wnt11) binds 7-transmembrane Fzd receptor and activates the recruitment of cytoplasmic scaffold protein Dvl to the plasma membrane. At the level of Dvl, two independent and parallel pathways lead to the activation of the small GTPases Rho and Rac (Dale et al. 2009, Habas & Dawid

25 2005, Kikuchi et al. 2012). Activation of Rho requires the homology protein Daam1 that binds to the PDZ domain of Dvl gene, leads to the activation of the Rho-associated kinase ROCK, and mediates cytoskeletal re-organization and cell movement. Rac activation is independent of Daam1, requires the DEP domain of Dvl, and stimulates Jun kinase (JNK) activity inducing JNK stress response pathway (Chien et al. 2009, Habas & Dawid 2005, Kikuchi et al. 2012, Schlessinger et al. 2009). PCP signaling is regulated at multiple levels. Seven-transmembrane protocadherin Celsr regulates the asymmetrical localization of Fzd and four- transmembrane protein Vangl, contributing to the maintenance of planar cell polarity. The cytoplasmic tail of Vangl binds and recruits Prickle to plasma membrane, where Prickle binds Dvl and antagonizes Dvl enrolment by Fzd, therefore inhibiting PCP signaling. (Dale et al. 2009, Wang 2009). The noncanonical, PCP Wnt signaling pathway is illustrated in Figure 2, A. Calcium (Ca2+) is an important factor in non-canonical Wnt signaling pathway. This was demonstrated by injection of Wnt5a or Wnt11 mRNA into 1- cell zebra fish embryos which caused remarkable increase of Ca2+ levels in the blastocyst. However, injections of Wnt8, the canonical Wnt, which activates the β-catenin, did not have impact of Ca2+ levels (Kohn & Moon 2005). Wnt/Ca2+ is mediated through G-proteins leading to an increase of Ca2+ levels, which in turn activates the Ca2+-dependent enzymes protein kinase C (PKC) and calcium/calmodulin-dependent kinase II (CamK2) (Habas & Dawid 2005, Kohn & Moon 2005). The non-canonical, Ca2+-dependent Wnt signaling pathway is illustrated in Figure 2, B. In addition, Wnt proteins activate distinct signaling branches by binding to the tyrosine kinase-like orphan receptor 1/2 (Ror 1/2) and the receptor tyrosine kinase (RYK) to activate JNK (Kikuchi et al. 2012, Kohn & Moon 2005).

26 Fig. 2. Non-canonical Wnt signaling pathways: planar cell polarity (PCP) (A) and Ca2+ dependent (B). Adapted from Habas and Dawid (Habas & Dawid 2005).

2.2 Mammalian sex determination

2.2.1 Bipotential gonads

Sex determination in mammals is divided into primary or chromosomal and secondary or hormonal. The presence of the second X or Y chromosome determines if the embryo will be genetically female (XX) or male (XY). The primary sex is chromosomal and is generally considered as not influenced by environment (Vaiman & Pailhoux 2000). Interestingly, controversial evidence from evolutionary biology and information on molecular mechanisms suggests that mammalian mothers could have a role in influencing the sex of the offspring (reviewed in (Grant & Chamley 2010). However, this is an emerging field that requires further investigation.

27 The gonads are paired organs deriving from the intermediate mesoderm; they form in close proximity with the developing kidney. Both ovaries and testes derive from a common precursor, the bipotential or indifferent gonad. At that time they do not have male or female features, around 10.5–11 dpc in mouse and 4–7 weeks in human (Gilbert 2010). Later, gonads develop sexually dimorphic phenotypes under the influence of differentially expressed genes leading to the specific hormone secretion. Loss of function mutations in numerous transcription factor genes such as Empty spiracles homeobox 2 (Emx2), LIM homeobox protein 9 (Lhx9), Sf1, Wt1 and Pod1 causes degeneration of gonads prior to sex determination, indicating that these genes are needed for the development and specification of gonadal primordium (Brennan & Capel 2004, Camerino et al. 2006, Ross & Capel 2005, Ungewitter & Yao 2013). The genital ridge epithelium forms the somatic cell part of the gonads which is later populated by primordial germ cells (PGC). The PGC originate in the base of the allantois at the posterior end of the primitive streak around day 6–6.5 dpc. By 7 dpc they form a population of about 45 cells committed for PGC linage and situated in the area of the developing hindgut. Around 9.5 dpc PGC start entering the genital ridge. During migration PGCs continually divide and by 11.5 dpc most of the PGC have arrived to the gonad and comprise a population of about 3,000 cells (Adams & McLaren 2002, Ewen & Koopman 2010, McLaren 2003, Molyneaux et al. 2001, Wilhelm et al. 2007). PGC make clusters called germ cell nests or cysts; they are defined as a group of cells that originates from one founder cell. These cells are connected by intercellular bridges. This is an important step for further sperm and egg formation (Pepling & Spradling 1998, Pepling et al. 1999). At around 11.5 dpc sex determination takes place in the mouse, and under the action of a cascade of genes triggered by the presence or absence of a second X or Y chromosome, the bipotential gonad develops into ovary or testes.

2.2.2 Female sex determination

In the male the presence of the Y chromosome prompts testes development and androgen synthesis by Leydig cells. This induces the male genital tract differentiation from the Wolffian duct, namely vas deferens, epididymis and seminal vesicles. In the absence of Y chromosome the female sex determination factors start acting, leading to ovary and female genital tract development: the

28 Mullerian duct, the precursor of the oviduct, uterus, cervix and upper part of the vagina (Kim & Capel 2006, Vaiman & Pailhoux 2000). During the bipotential gonad phase, a number of genes important for further sexually dimorphic organ development are expressed: Wnt4, Sf1, Sox9, Lhx9, Fgf9, Wt1 and Gata4. The genes important for female development continue to be expressed in the ovary beyond bipotential faze, whereas the ones defining male sex will be suppressed. Nuclear receptor subfamily 0, group B, member 1 (Nr0b1) also known as Dax1, a nuclear receptor, is expressed in adult testes and adrenal glands. It was shown as a testis antagonist in humans (Zanaria et al. 1995) and to act as a male pathway inhibitor in mice (Swain et al. 1998). However, Dax1 inactivation in mice does not cause phenotype in female mice, indicating that it is not required for the ovary development (Yu et al. 1998). The Wnt4 gene is initially expressed in both sexes in the developing mesonephros, but around 11.5 dpc it becomes suppressed in males but continues to be expressed in females (Vainio et al. 1999). Wnt4 knock-out mice studies have shown that the loss of function leads to female-to-male sex reversal phenotype (Vainio et al. 1999) and male-specific vasculature formation (Jeays-Ward et al. 2003, Yao et al. 2004). The expression of downstream genes follistatin and Bmp2 is abolished (Kim & Capel 2006). Wnt4 does not only promote the female pathway, the male pathway since certain male-specific genes, namely Fgf9 and Sox9, are de-repressed in the Wnt4-/- XX gonads. (Kim et al. 2006). Foxl2 is a basic helix-loop-helix transcription factor. Its expression starts in somatic cells of the XX gonads at 12.5 dpc and continues into adult life. Foxl2 is essential for granulosa cell development and is present in the pre-granulosa cells (Ottolenghi et al. 2005, Schmidt et al. 2004). Inactivation of Foxl2 leads to female-to-male sex reversal phenotype. Ovaries of XX mice form testis tubules and spermatogonia (Ottolenghi et al. 2007). Foxl2 gene is not required for the initial differentiation of follicle cells or for the assembly of ovarian follicles, but Foxl2 mutant follicle cells acquire male characteristics as they start expressing male-specific genes after birth (Loffler et al. 2003, Ottolenghi et al. 2005, Uda et al. 2004).The forced expression of Foxl2 impairs testis tubule differentiation in XY transgenic mice, pointing to anti-testes role for Foxl2 where it would have a synergistic role together with the Wnt4 (Ottolenghi et al. 2007). The R-spondin1 (Rspo1) belongs to small family of growth factors. It is expressed in the bipotential gonad in both sexes but by 12.5 dpc there is a strong increase in Rspo1 expression in XX gonads (Parma et al. 2006). Mutations in

29 RSPO1 gene in XX patients cause female-to-male sex reversal (Parma et al. 2006). Rspo1 knock-out mice have demonstrated gonad masculinization phenotype. Additionally, it was revealed that β-catenin activation is controlled by Rspo1 and is required for female somatic cell differentiation and germ cell commitment to meiosis. The germ cells of Rspo1 knock-out mice fail to enter meiosis typical of XX individuals. The Wnt4 gene is not activated and as a result of this Rspo1 XX ovaries develop male type of vasculature (Chassot et al. 2008a, Chassot et al. 2008b, Tomizuka et al. 2008). Catenin beta 1 (Ctnnb1/ β-catenin), a downstream target of canonical Wnt signaling, is present in the XX and XY gonads, but it appears to be essential only for the ovary development. The ovaries lacking β-catenin, as revealed by conditional deletion by SF1Cre, have morphological defects, develop testes- specific coelomic vessel, androgen-producing cells and loose ovarian germ cells (Liu et al. 2009). Ectopic expression and stabilization of the β-catenin in the somatic cells of XY gonads leads to male-to-female sex reversal phenotype (Maatouk et al. 2008). β-catenin is also identified as a pro-ovarian and anti-testis signaling molecule (Maatouk & Capel 2008, Maatouk et al. 2008). The sex determination and the genes involved are illustrated in Figure 3.

30 Fig. 3. Sex determination. During mouse embryogenesis, bipotential gonads differentiate from the genital ridges by 10.5 dpc. In somatic cells of XY genital ridges, Sry gene expression peaks at 11.5 dpc. Later, Sox9 expression precludes differentiation of Sertoli cells. Subsequently Fgf9, Amh and other male-specific gene expression is initiated. In the absence of Sry, Rspo1, Wnt4, Foxl2, Fst and other genes (see text) are expressed and induce ovarian development.

2.2.3 Meiosis

Defined sex causes a differential onset of meiosis, the proper control of which is required for subsequent fertility. Meiosis is comprised of two rounds of cell division, meiosis I and meiosis II. Each of these includes several stages: progress through the prophase, metaphase, anaphase and telophase. The timing and regulation of meiosis differs significantly between the sexes. In mammalian males it is first initiated at puberty and continues through adult life (Hunt & Hassold 2002, Morelli & Cohen 2005). The female germ cells enter first meiotic division during embryogenesis around 13.5 dpc in mice (McLaren & Southee 1997). Germ cells enter meiosis in the rostro-caudal wave during development of the mouse ovary (Bullejos & Koopman 2004). They progress through leptotene, zygotene, pachytene and get arrested in the diplotene stages of prophase I. Oocytes continue meiosis only after female sexual maturation when ovulation starts. After ovulation of mature follicle, its oocyte complete the first meiotic division and get arrested

31 again, whereas the second meiotic division is completed only after fertilization (McLaren & Southee 1997, McLaren 2003, Morelli & Cohen 2005). Retinoic acid (RA) plays an important role in meiotic induction in fetal XX and postnatal XY germ cells. RA is derived from the retinol (vitamin A) in the mesonephros (as well as numerous other tissues). It is reversibly converted to retinal, and retinal is later irreversibly oxidized to retinoic acid by retinal dehydrogenase (RALDH) (Spiller et al. 2013). Cytochrome P450, family 26, subfamily b, polypeptide 1 (Cyp26b1) the RA degradation enzyme is expressed in embryonic male gonads, thus preventing germ cells from entering into meiosis (Bowles et al. 2006, Menke & Page 2002, Saga 2008). In agreement Cyp26- deficient XY germ cells express meiotic markers (Bowles et al. 2006, Bowles et al. 2010). In females RA stimulates Stra8 (Stimulated by retinoic acid 8) gene expression, which is expressed by ovarian germ cells shortly before initiation of prophase I (Bowles et al. 2006, Menke et al. 2003). Male and female germ cells lacking Stra8 fail to initiate meiosis (Anderson et al. 2008, Baltus et al. 2006). The expression pattern of pre-meiotic markers Rec8 and Scp3 becomes distorted. Other meiotic markers ɣH2AX, which labels double strand DNA breaks, as well as Dmc1 and Spo11, genes required for double strand DNA break repair, are not expressed in the Stra8 deficient germ cells (Baltus et al. 2006). The germ cells respond to RA by entering meiosis but other cells do not. This could be explained by the fact that germ cells undergo genome-wide de-methylation around 12.5 dpc (Maatouk et al. 2006). Additionally, they lose repressive histone modifications and are susceptible to active histone modification. All this makes germ cell chromatin to be in an ‘open’ state (Hajkova et al. 2002, Spiller et al. 2012). Several studies have demonstrated that very low levels of endogenous RA are sufficient to induce meiosis in in vitro conditions (reviewed in (Spiller et al. 2012). After entering meiosis primordial germ cells are called oocytes (Pepling & Spradling 1998). Later studies of Stra8-deficient mice, which do not initiate meiosis, have shown that they still form oocyte like-cells and express oocyte differentiation markers. This has led to the conclusion that oocyte differentiation and meiosis are genetically distinct processes (Dokshin et al. 2013). The meiosis process and the genes involved are illustrated in Figure 4.

32 Fig. 4. Meiosis. Retinoic acid (RA) is secreted by mesonephros (ME). The RA signal reaches female gonads and triggers Stra8 expression, which induces Dazl & Sycp3 and initiates meiosis around 13.5 dpc. In embryonic testes somatic Leydig cells secret the Cyp26b1 enzyme, which degrades RA; due to this Stra8 transcription is not stimulated and embryonic male germ cells do not enter meiosis. Adapted from (Saga 2008).

2.3 Adult ovary

2.3.1 Ovarian Folliculogenesis

In the embryonic ovaries, as described above, oocytes are in clusters called germ line cysts. They are surrounded by somatic cells which are the presumptive granulosa cells (Pepling & Spradling 1998, Pepling et al. 1999, Tingen et al. 2009). The granulosa cells acquire squamous shape, encircle the oocyte and encapsulate all structure by basement membrane layer, forming a structure called the primordial follicle (Da Silva-Buttkus et al. 2008, Edson et al. 2009, Pepling et al. 1999). In mice the primordial follicles form during the first few days after birth. They comprise the resting pool of oocytes from which follicles are recruited for maturation through the mammalian female reproductive age (McGee & Hsueh 2000). Adolescence ovarian folliculogenesis comprise three major steps: the initial follicular recruitment, the cyclic recruitment and the ovulation followed by corpus luteum formation (Edson et al. 2009, McGee & Hsueh 2000, Thomas & Vanderhyden 2006).

33 Initial follicular recruitment is characterized by ongoing activation of primordial follicles which takes place throughout entire female reproductive life until the menopause. This is a gonadotropin-independent stage (Edson et al. 2009, McGee & Hsueh 2000). The gonadotropin-independent cyclic recruitment starts after puberty once follicles are in early antral stage (in rodents) as a result of increased levels of circulating Follicular stimulating hormone (FSH) (McGee & Hsueh 2000). The beginning of the primary follicle development is associated with oocyte growth and division of the granulosa cells. At the same time granulosa cells undergo intense morphological transformation from flattened to cuboidal (Da Silva-Buttkus et al. 2008, Fortune 2003). The shift from the primordial to primary stage can be lengthy and follicles with admixed population of flattened and cuboidal granulosa cells can be observed in vivo (Fortune 2003, Myers et al. 2004). At the primary stage stromal cells are recruited to form the theca-cell layer by the factors secreted from activated primary follicles. In the secondary follicles the flattened thecal cells envelop the basement membrane, which encapsulates the granulosa-oocyte complex (Young & McNeilly 2010). The theca cells layer is comprised of an outer part called theca externa and an inner par, theca interna. The theca cells are important for follicular growth; they provide the androgens needed for estrogen production in granulosa cells of the growing follicle and the follicle’s own vascular network develops within the surrounding thecal cells (Young & McNeilly 2010). Primary follicles have been shown to be growing; however, due to the difficulties involved in distinguishing between non-growing and growing follicles, primordial and primary follicles have often been considered as a contiguous group (McGee & Hsueh 2000, Myers et al. 2004). The secondary stage begins when the development of a second layer of granulosa cells takes place. Gradually up to about six or seven layers of granulosa cells surround the oocyte. Once the follicle reaches the species-specific size, it forms a fluid-filled space called antral cavity (Edson et al. 2009, Myers et al. 2004, Thomas & Vanderhyden 2006). Pre-antral follicle growth depends on endocrine and paracrine factors but is gonadotropin-independent, whereas antral- stage follicles become gonadotropin-dependent to sustain further growth (Edson et al. 2009). The pituitary gland-derived FSH is important in granulosa cell apoptosis and follicular atresia prevention. The formed antrium divides granulosa cells to two populations. The one situated along the outer wall of developing follicle is important for steroidogene production and ovulation, whereas the other,

34 cumulus cell population, surrounds the oocyte and maintains its growth (Diaz et al. 2007). The atresia rates increase throughout folliculogenesis, reaching their peak at the antral stage; despite this only a few selected follicles survive and reach the preovulatory stage (Young & McNeilly 2010). The FSH becomes suppressed whereas Luteinizing hormone (LH) levels increase and cause oocyte meiotic re- initiation followed by follicle rapture, ovulation and corpus luteum (CL) formation (Edson et al. 2009). The egg release is cyclic in females. Humans and most of the other primates have menstrual cycle, whereas other mammals have estrous cycle both of them refers to the changes that occur in the uterus. The major difference between these two is the fate of the uterine lining if pregnancy does not happen. In menstrual cycles, the endometrium is shed from the uterus through the cervix and vaginal bleeding called menstruation. In estrous cycles, the endometrium is reabsorbed by the uterus, and no extensive bleeding occurs. The human female menstrual cycle lasts about 24–32 days; the mouse estrous cycle 4–5 days. Parallel to menstrual cycle there is ovarian cycle. It starts with follicular phase during which several ovarian follicles initiate growth. In humans only one and in mice few will continue to enlarge and mature. The follicular phase ends with the ovulation and then the ovulatory phase starts. At that time follicle and the adjacent ovary wall rapture and oocyte is being released. The luteal phase begins and remaining follicular cells transform into corpus luteum which secrets estrogen and progesterone (Summarized from (Campbell 1996, Johanson & Everitt 2001). During the follicular phase of the ovarian cycle, the pituitary secrets small amounts of FSH and LH in response to gonadotropin-releasing hormone (GnRH) from hypothalamus. The FSH stimulates follicle growth and in response follicle cells secret estrogen. This small increase in estrogen inhibits the secretion of the pituitary hormones, keeping FSH and LH levels rather low. However, high estrogen level stimulates gonadotropin secretion and as a result increased LH concentration leads to ovulation. Estrogen and progesterone levels decline near the end of luteal phase. The pituitary gland begins again to secret FSH to stimulate the growth of new follicles in the ovary, so the next ovarian cycle gets initiated (Summarized from (Campbell 1996, Johanson & Everitt 2001). The folliculogenesis is schematically illustrated in Figure 5.

35 Fig. 5. Mammalian folliculogenesis. Primordial follicles form in 1- to 2-day-old mice, granulosa cells making more layers leading to secondary follicle formation which later develops to pre-antral follicle. This follicle growth does not require gonadotropins secreted by pituitary gland. Further follicle growth requires gonadotropins FSH and LH to progress through the antral, preovulatory, ovulation stages and CL formation. Basement membrane - BM; Granulosa cell - GC; Thecal cell - TH.

2.3.2 Extracellular matrix and cell-cell junctions in the developing follicle

The extracellular matrix (ECM) provides structural support to the follicle. It sustains cellular organization and provides biochemical signals that promote follicle development and maturation (Irving-Rodgers et al. 2006, Rodgers et al. 2003). There are many different types of extracellular matrices in different follicle compartments (Berkholtz et al. 2006b, Irving-Rodgers & Rodgers 2006). Before the growth initiation of the follicle basement membrane, specific sheet of the ECM, contains all six α chains of type IV collagen, whereas later on only two remain expressed, α1 and α2. Laminins, perlecans and nidogens are other components of follicular basement membrane (Berkholtz et al. 2006a, Nakano et al. 2007, Rodgers et al. 2003). The follicular basement membrane separates the granulosa cells from the surrounding stroma starting from the primordial stage onwards. The basement membrane is secreted by granulosa cells and the thecal cells get organized on its opposite side (Berkholtz et al. 2006b, Irving-Rodgers & Rodgers 2006, Young & McNeilly 2010). In healthy follicles the basement membrane excludes capillaries, blood cells and nerve processes from invading the granulosa cell layer until ovulation. During the ovulation the follicular basement

36 membrane becomes degraded, while in case of atretic follicle it can be penetrated by cells from the thecal layer and gradually disassembles (Irving-Rodgers & Rodgers 2006). Several defects in proper ECM organization have been reported to cause failure in folliculogenesis. In Foxl2 knock-out mice basement membrane was not synthesized properly, leading to failure in primordial follicle formation (Uda et al. 2004). ADAMTS1 deficiency was associated with ECM remodeling during folliculogenesis and lymphangiogenesis (Brown et al. 2006). The adult estrogen - receptor – beta (ER-β) null females were subfertile and had reduced numbers of growing follicles along with increased expression of ECM genes (Zalewski et al. 2012). Gap junctions are composed of connexons, a collection of intercellular membrane channels that allow adjacent cells to share small molecules. The oocyte-granulosa cells are connected by Connexin37 (Cx) (Kidder & Mhawi 2002, Kidder & Vanderhyden 2010). The Cx37 is encoded by the Gja4 gene. The Gja4 null mice are infertile due to impaired folliculogenesis (Simon et al. 1997). The Cx43 is encoded by the Gja1 gene; the knock-out mice of this gene have disrupted ovaries as oocyte and follicle growth has been slowed down. This indicates that Cx43 is essential for connection between the granulosa cells to sustain their proliferation (Ackert et al. 2001, Kidder & Vanderhyden 2010). E-cadherin/Cdh1 (epithelial cadherin) is a transmembrane molecule that links nearby cells via hemophilic interactions. The intracellular tail of E-cadherin binds β-catenin, which binds ɑ-catenin to form a functional adherence junction (Young et al. 2003). E-cadherin plays an important role in PGC migration and homing. It was also suggested that it might function as a modulator of PGC development due to its differential expression in female and male PGC (Di Carlo & De Felici 2000). Interestingly, E-cadherin is not expressed in the granulosa cells of large preantral and antral follicles, but is present in theca cells. This could be due to partial epithelial to mesenchymal transition that granulosa cells are undergoing (Childs & McNeilly 2012, Mora et al. 2012). In contrast, expression of N- cadherin/Cdh2 (neural cadherin) is preserved in granulosa cells at all stages of follicle development (Machell & Farookhi 2003).

2.3.3 Molecular basis of ovarian development and function

Anti-Mullerian hormone (Amh), also known as Mullerian inhibiting substance, is a glycoprotein, a member of the transforming growth factor-beta gene (TGF-β) family which mediates Mullerian duct regression during male sexual

37 differentiation. It is secreted by testes Sertoli cells in male (Behringer et al. 1994, Kobayashi et al. 2011). In females, Amh is produced by granulosa cells of ovarian early developing follicles until menopause. The highest Amh expression is observed in small antral follicles, but not in the late antral follicles. Amh production gradually declines as follicles grow. This is an important requirement for dominant follicle selection and progression to ovulation. Amh controls the primordia follicle recruitment since as long as follicles express Amh they are insensitive to FSH due to inhibition of aromatase and LH receptor expression (di Clemente et al. 1994, Durlinger et al. 2001, Pellatt et al. 2010). Amh knock-out mice have an increased amount of growing follicles at young age, which leads to early exhaustion of follicle pool, seen as reduction of the amount of primordial follicles followed by premature ovarian failure (Durlinger et al. 1999, Durlinger et al. 2002). The serum levels of Amh/AMH have been shown to correlate with small antral follicle number. For this reason it is clinically used as a biomarker to estimate the ovarian reserve (Kevenaar et al. 2008, Piltonen et al. 2005, Visser et al. 2012). Amh transduces signal by binding to its receptor Amhr2, which is expressed in the ovarian granulosa cell, oviduct and uterus (Mishina et al. 1996), whereas Amhr1 serves as a receptor to Activin A, type 2/3 (ALK2/3) (Orvis et al. 2008). Amh is an important factor for predicting the growing follicle numbers; this is why it is of great interest to find out what are the genes and factors that regulate its expression. Testosterone was shown to be as one of the potential factors, as prenatal testosterone treatment in sheep has led to reduced amounts of the Amh protein expression in granulosa cells of preantral follicles postnatally (Veiga-Lopez et al. 2012). Kit (kit oncogene) tyrosine kinase receptor is referred to as c-kit and Kit ligand (KitL), it is also known as steel factor or stem cell factor (SCF). The Kit is expressed on the membrane of mouse PGSs, whereas KitL is expressed on the membrane of somatic cells (Pesce et al. 1997). Interestingly, in humans KIT is expressed both in the oocyte and granulosa cells (Carlsson et al. 2006). Both Kit and KitL are important during early folliculogenesis. In postnatal ovaries Kit is expressed by oocytes and KitL in granulosa cells at all stages of folliculogenesis (Driancourt et al. 2000, Motro et al. 1991). Kit and KitL mediate follicular growth from the primordial pool to follicular fluid and preovulatory follicle formation. Mutations in these genes lead to defect in PGC growth and differentiation (Parrott & Skinner 1999, Yoshida et al. 1997). Growth Differentiation Factor 9 (Gdf9), a member of TGFβ superfamily, is localized exclusively to the oocytes at all stages of the follicular growth except

38 for primordial follicles (Dube et al. 1998, Jaatinen et al. 1999, Laitinen et al. 1998, McGrath et al. 1995). The follicles of Gdf9 knock-out mice do not progress beyond primary stage (Dong et al. 1996). Studies suggest that GDF9 might function as an autocrine factor to regulate oocyte maturation/function and as a paracrine factor controlling granulosa cell differentiation and propagation (Elvin et al. 1999a, Elvin et al. 1999b). Bone morphogenetic protein 15 (Bmp15) also known as Gdf9b, belongs to TGFβ superfamily ligand. Bmp15 and Gdf9 are highly homologous oocyte- secreted growth factors and are known to control folliculogenesis and ovulation through direct effects on granulosa cells in the developing follicles (Dube et al. 1998, Fortune 2003, Jaatinen et al. 1999). Bmp15 knock-out mice are subfertile and have reduced rates of ovulation and fertilization (Yan et al. 2001). Interestingly, while mice are subfertile, sheep with the inactivating mutations of the BMP15 gene are infertile (Galloway et al. 2000, Galloway et al. 2002). In humans, natural BMP15 mutations cause increased ovulation rate and infertility phenotypes in a dosage-sensitive manner (Galloway et al. 2000). The phenotype difference between species, shown by BMP15, emphasizes the importance of employing multiple study models for gaining a better understanding of gene function. Taken together, in mammals with a low ovulation rate phenotype, both oocyte-derived GDF9 and BMP15 proteins are critical for appropriate follicular development in early and later stages of growth. In addition, it was proposed that both BMP15 and GDF9 could be involved in POF (Laissue et al. 2006). Gata binding protein-4 (Gata-4) encodes a member of the GATA family of zinc-finger transcription factors. GATA4 is a granulosa cell factor employed in inhibin-alpha activation by the TGFβ pathway (Anttonen et al. 2006). Studies of adult Gata4 heterozygous mice showed that they had smaller ovaries, which had released fewer oocytes, and had reduced production of estrogen. Gata4 conditional knock-out mice had impaired fertility and cystic ovaries (Kyronlahti et al. 2011). Gata4 is a key transcriptional regulator of ovarian somatic cell function in both fetal and adult mice. It exhibits its function with the Friend of GATA2 (Fog2) (Manuylov et al. 2008). Assembly of functioning testis and ovary requires a Gata4-Fog2 transcriptional complex as revealed by studies of Gata4 conditional ablation and of Gata4/Fog2 double knock-out mice (Manuylov et al. 2008). Foxl2 is a transcriptional factor specifically expressed in XX gonads is important not only during embryonic ovary growth, but is needed throughout

39 mouse ovary development (Garcia-Ortiz et al. 2009, Ottolenghi et al. 2005). In animal models, Foxl2 is required for maintenance, and possibly induction, of female sex determination independently of other critical genes, e.g., Rspo1 (Ottolenghi et al. 2005). Post-natal Foxl2 is expressed in granulosa cells of small and medium ovarian follicles and for this reason it is used as a marker for granulosa cell differentiation. Foxl2 is essential for follicle maturation, in particular for transition from squamous to cuboidal granulosa cells, as no primary follicles are formed in Foxl2-deficient ovaries (Kuo et al. 2012, Schmidt et al. 2004, Uda et al. 2004). Foxl2 functions as a repressor of the steroidogenic acute regulatory gene (Star). The Star regulates the transcription of aromatase, P450scc, and cyclin D2, which are the key genes involved in granulosa cell proliferation, differentiation, and steroidogenesis (Yang et al. 2010). Foxl2 mutation is associated with POF in women with blepharophimosis-ptosis-epicanthus inversus syndrome, type I. Follistatin (Fst) is an activin-binding glycoprotein. It is differentially expressed in the developing testes and ovaries. Fst knock-out mice develop female-to-male sex reversal phenotype and ovaries have a male type of vasculature. It was shown to act downstream of Wnt4 (Yao et al. 2004). Fst specifically inhibits follicle-stimulating hormone release. Fst knock-out mice die within hours after birth due to multiple disorders such as retarded growth, skeletal defects and respiratory failure (Matzuk et al. 1995). Foxl2 and Bmp2 act cooperatively to regulate Fst gene expression during ovarian development (Kashimada et al. 2011). It is expressed in the cytoplasm of granulosa and luteal cells but not in thecal cells (Singh & Adams 1998). Granulosa cell-specific inactivation of Fst in mice caused ovarian defects and infertility (Jorgez et al. 2004). FST has also been reported as a candidate gene for polycystic ovary syndrome (Jones et al. 2007). Newborn ovary homeobox (Nobox) is a homeobox transcription factor that is necessary throughout the folliculogenesis for expression of several key oocyte- specific genes, including Gdf9 and Pou5f1 (Oct4) (Choi & Rajkovic 2006). Nobox knock-out mice form primordial follicles but folliculogenesis does not progress any further (Rajkovic et al. 2004). They develop premature ovarian failure due to defective germ cell cyst breakdown (Lechowska et al. 2011). R-spondin 2 is an oocyte expressed gene which has capastiy to indorse granulosa cell proliferation via the canonical Wnt signaling pathway. R-sondin 2 functions in cloase synergy with the granulosa cell derived Wnts and stimulates β- catenin pathway which in response stimulates primary to secondary and antral

40 follicle development (Cheng et al. 2013). These recent discovery is worthwhile deeper research. A number of other genes are important in ovarian development and maturation but only ones that were relevant to this work were discussed herein.

2.4 Steroidogenesis

The hypothalamus secrets GnRH and it stimulates pituitary gland to produce LH and FSH. Steroidogenesis in most mammals occur via the ‘two-cell, two- gonadotropin’ model. This means that androgens are synthesized from cholesterol in LH-stimulated theca cells, followed by conversion to estrogens in FSH- exposed granulosa cells. The LH receptors and the enzyme CYP17, which converts pregnenolone and progesterone to dehydroepiandrosterone (DHEA) and androstenedione, respectively, are expressed primarily in theca cells, while FSH receptors and aromatase (CYP19), which converts androgens to estrogens, are expressed mainly in granulosa cells (Jamnongjit & Hammes 2006). The circulating estrogens in mammalian females are mostly produced by the granulosa cells of the growing ovarian follicles from the androgens secreted by the thecal cells. The mitochondria of ovarian thecal cells produce androgens from circulating cholesterol. This process is initiated by LH which activates the cAMP. Cholesterol is subsequently converted to pregnenolone in the reaction catalyzed by cytochrome P450 cholesterol side-chain cleavage (Cyp11A1). The further conversion to androstenedione is controlled by cytochrome P450 17 beta- hydroxylase (Cyp17) and 3-beta-hydroxysteroid dehydrogenase type 2 (HSD3B2). Most of the androstenedione from thecal cells enters granulosa cells, but some of it is converted to testosterone by 17-beta-hydroxysteroid dehydrogenase type 1 (HSD17B1). Secreted androgens defuse via basement membrane and enter granulosa cells where the steroidogenesis is regulated by FSH action. Androstenedione is converted to estrone in the reaction controlled by cytochrome P450 aromatase (CYP19A1) and later to estradiol by HSD17B1 (Reviewed in (Craig et al. 2011, Lapointe & Boerboom 2011, Miller & Auchus 2011, Vanderhyden 2002, Young & McNeilly 2010)). The steroidogenesis control in the growing follicles is illustrated in Figure 6.

41 Fig. 6. Steroidogenesis control in the growing follicles. LH stimulates theca cells; this initiates the cholesterol conversion to pregnenolone, followed by formation of progesterone, dehydroepiandrosterone (DHEA), androstenedione and testosterone. Androstenedione and testosterone defuse to granulosa cells via basement membrane; the signals from FSH initiate androgen conversion to estrogens: estrone and estradiol. GnRH – Gonadotropin Releasing Hormone, LH - luteinizing hormone, LHR - luteinizing hormone receptor, FSH - follicular stimulating hormone, FSHR - follicular stimulating hormone receptor, DHEA - dehydroepiandrosteron. Modified from (Lapointe & Boerboom 2011).

2.5 Female reproductive tract

The female reproductive system consists of the ovaries and the reproductive tract, which includes the oviduct, uterus and vagina.

42 2.5.1 Mullerian duct development

The internal mammalian sex organs are established during embryogenesis from the male Wolffian ducts, also referred to as mesonephros, and the female Mullerian duct, also called the paramesonephros. Before the sex differentiation both ducts persist in males and females. Later, as the sex-specific genes initiate the cascade of differential gene expression, the MD degrades in males due to the action of Amh, and the WD differentiates to epididymis, vas difference and seminal vesicles. In XX individuals, on the contrary, the WD degenerates, due to the low levels of androgens, whereas the MD persists and predisposes formation of the oviduct, the uterus and the upper part of vagina (Guioli et al. 2007, Kobayashi et al. 2011, Orvis & Behringer 2007). The origination of the anlagen of the MD was actively discussed in the field since it was previously thought that MD originates from WD (Guioli et al. 2007). However, later studies have demonstrated that the MD originates from placode- like thickening of the coelomic epithelium that extends caudally at 11.75 dpc, and at 13.5 dpc it reaches cloaca and its full length in mice (Guioli et al. 2007, Jacob et al. 1999, Kobayashi et al. 2011). However, WD is necessary for MD formation and growth as WD provides the mechanical guidance cues and promotes the caudal elongation of the MD via signals such as the Wnt9b (Carroll et al. 2005). Interestingly, there is no known case where MD would have formed in the absence of WD. The MD represents the only duct in the mammalian body whose formation depends on another duct, thus providing a unique biological model system (Orvis & Behringer 2007). The MD is composed of three cellular components: a) the epithelial cells forming the inner part of the MD epithelium, b) the mesenchymal cells surrounding the tube and c) the coelomic epithelial cells that mark the outer borders of the MD and are called Mullerian coelomic epithelium. The apical end of the MD is a luminal epithelial tube surrounded by several layers of mesenchymal cells, whereas the proximal tip of the MD is mesenchymal and in close contact with the WD (Guioli et al. 2007). The MD is considered to be composed of the mesoepithelial cells since the epithelial cell polarity markers (such as E- cadherin) appear relatively late during the development (Orvis & Behringer 2007). Mullerian duct development is illustrated in Figure 7.

43 Fig. 7. Mullerian duct development. The Mullerian duct forms at around 11.75 dpc at the apical side of the mesonephros laterally to the Wolffian duct. The cells start migrating proximally to extend the duct which reaches the cloaca by 13.5 dpc. Adapted from (Kobayashi & Behringer 2003).

2.5.2 Uterus differentiation

The MD-generated oviduct, uterus and the upper part of the vagina become matured only after birth in females. The differential tissue identity is initially determined in the mesenchyme, which sends differentiation signals to the epithelium. Subsequently the oviduct develops from the apical part of the MD, the uterus from the intermediate fragment and the upper part of the vagina from the proximal part of MD (Kurita et al. 2001). The mouse uterus consists of undifferentiated mesenchyme, a simple luminal epithelium, and does not have endometrial glands at birth (Hayashi et al. 2011). After birth during postnatal days (P) 0–3 the uterine mesenchyme differentiates into endometrial stromal cells, the inner (circular) and outer (longitudinal) myometrium cell types. At around P6 luminal epithelium folds, this represents the prospective endometrial glands (Hayashi et al. 2011). During the following days (by P12) the glands grow from luminal epithelium into endometrial stroma. At the same time myometrium longitudinal smooth muscle cells organize into bundles, providing the contraction capacity for the uterus (Hayashi et al. 2011). The established uterine wall in the adult consists of two compartments, the endometrium and the myometrium (Hu et al. 2004). The endometrium is the inner mucosal lining of the uterus, derived from the inner layer of ductal mesenchyme

44 (Hu et al. 2004, Spencer et al. 2012). Histologically, the mouse endometrium consists of few components: two types of epithelial cells, the luminal epithelium and the glandular epithelium, which are surrounded by stroma, blood vessels and immune cells. The myometrium is the smooth muscle part composed of inner and outer layers. The inner circular layer develops from the intermediate layer of MD mesenchymal cells, and an outer longitudinal layer, derived from subperimetrial mesenchyme (Hu et al. 2004). The endometrial glands provide nutrients, growth factors and cytokines to promote pregnancy. (Hayashi et al. 2011, Hempstock et al. 2004). The myometrium smooth muscles provide the basis for the contraction capacity for the uterus. The failure in any of these can lead to infertility (Hempstock et al. 2004). The uterus development is illustrated in Figure 8.

Fig. 8. Uterus development. The uterus differentiates from the Mullerian duct during embryogenesis. After birth around P 0–3 the uterine mesenchyme differentiates into myometrium and endometrial cell types with the uterine lumen in the center. At around P6 the luminal epithelium folds, forming endometrial glands. Mullerian duct - MD, Wolffian duct - WD, Myometrium - Myo, Endometrium E, Endometrial glands - eG, Lumen - L.

2.5.3 Genetic control of female reproductive tract development

Gene targeting experiments indicated that MD development is regulated by the transcription factors Paired-box gene 2 (Pax2), Paired-box gene 8 (Pax8), LIM homeobox protein 1 (Lim1), empty spiracles homeobox 2 (Emx2), homeobox A13 (Hoxa13) and the signaling molecules Wnt4, Wnt5a, Wnt7a and Wnt9b (Carroll et

45 al. 2005, Grote et al. 2008, Kobayashi & Behringer 2003, Kobayashi et al. 2004, Miller & Sassoon 1998, Simpson 1999, Taylor & Fei 2005, Torres et al. 1995, Vainio et al. 1999). The Pax2 knock-out mice do initially form WD and MD, but they both degenerate later: WD at 12.5 dpc, while MDs are only present at the uppermost levels of the genital ridge. By 16.5 dpc the part of the MD initially formed degenerates, too, in both sexes (Torres et al. 1995). The Pax8 gene is expressed in developing WD and MD as is the Pax2 gene (Bouchard et al. 2002). The Pax8 null mice are infertile as they lack a functional uterus, which consists only of degenerated myometrium. In addition, the vaginal opening is missing in these mice (Mittag et al. 2007). Therefore Pax8 is needed for uterine development rather than its formation. Lim1 is expressed in the WD and MD. It is needed for formation of the MD. The Lim1 null mice die around 10 dpc. Studies on Lim1 chimera mice have shown that it is required for MD epithelium formation (Kobayashi & Behringer 2003). The conditional Lim1 deletion by Pax2Cre has led to truncated MD (Pedersen et al. 2005). Emx2 is needed for the formation of both ducts. The WD in Emx2 knock-out mice degenerates and MD does not form at all (Miyamoto et al. 1997). Hoxa13 expression is present in WD and MD. The deletion of the gene leads to multiple defects in MD derivatives (Warot et al. 1997). Wnt4 is found in the mesenchyme around the MD at 12 dpc and is not expressed in the WD (Vainio et al. 1999). Wnt4 is essential for the formation of the MD since Wnt4 knock-out mice fail to develop MD. WD develops normally in Wnt4 -/- male mice and strikingly it persists in XX females (Vainio et al. 1999). Conditional Wnt4 deletion by PRCre in mice has revealed Wnt4 function in adult mouse uterus gland and luminal epithelium formation. The mice were sub-fertile and had defects in embryo implantation and decidualization (Franco et al. 2011). The primordial germ cell entry to the gonads is altered in Wnt5a -/- mice leading to decreased numbers of germ cells (Chawengsaksophak et al. 2012). Wnt5a is expressed in the mesenchyme of the uterus, cervix and vagina of the adult mouse (Miller et al. 1998). In Wnt5a deficiency, the uterus does not develop endometrial glands. Additionally, the cervix and vagina do not form properly (Mericskay et al. 2004). Wnt7a is specifically expressed in the epithelium of the MD from 12.5 dpc. Wnt7a expression persists in the MD-derived organs. Wnt7a knock-out mice have defective MD development and both male and female mice are infertile (Parr &

46 McMahon 1998). The uterus of the adult mice lacks the secretory glands, which derive from the MD epithelium, and stromal layer, which differentiates from MD mesoderm (Miller & Sassoon 1998). Wnt7a is needed for postmenstrual endometrial regeneration, where it should be downregulated prior to embryo implantation in the primate endometrium (Fan et al. 2012). The conditional Wnt7a deletion by PgrCre has led to disrupted endometrial gland development, uterine gene expression and defective implantation (Dunlap et al. 2011). Wnt9b is expressed by WD epithelium and the loss of this gene results in incomplete development of the MD. The MD is capable of initiating apical- proximal elongation, but extension is ceased at 1/3 of its original length as caudal elongation does not progress. It is considered that Wnt9b is needed for the second phase of MD elongation (Carroll et al. 2005). Vangl2 is mutated in loop-tail mutant mice, which have defects in PCP signaling pathway. They display deficiencies in multiple tissues including the female reproductive tract, homozygote mice die shortly after birth and heterozygote mice are subfertile. The mice have defects in the fusion of uterine horns at the level of the cervix, and oviduct coiling and uterine horns are shortened. The uterine epithelium displays altered cell polarity, associated with changes in cytoskeletal actin localization (Vandenberg & Sassoon 2009). The genes discussed in this chapter and the phenotypes caused by their deficiency are summarized in Table 1.

47 Table 1. List of genes needed for the development of the Mullerian duct and Mullerian duct-derived organs: oviduct, uterus and upper part of vagina.

Gene name Phenotype Reference Pax2 MD degenerate (Torres et al. 1995) Lim1 MD degenerate (Kobayashi et al. 2004, Pedersen et al. 2005) Emx2 MD does not form (Miyamoto et al. 1997) Wnt4 MD does not form (Vainio et al. 1999) Wnt5a Defects in MD differentiation (Mericskay et al. 2004) Wnt7a Defective MD differentiation (Parr & McMahon 1998)(Miller & Sassoon 1998) Wnt9b Defect in MD extension (Carroll et al. 2005) Hoxa13 Defects in MD differentiation (Warot et al. 1997) Vangl2 Defective MD differentiation (Vandenberg & Sassoon 2009)

2.6 Wnt4 functions in reproductive system

Wnt4 function is important for the development of multiple organs: kidney (Stark et al. 1994, Vivante et al. 2013), mammary gland (Brisken et al. 2000), adrenal gland (Heikkila et al. 2002), brain (Miyakoshi et al. 2009, Potok et al. 2008) testes (Jordan et al. 2003, Yu et al. 2006), epididymis (Deshpande et al. 2009), ovary, Mullerian duct and its derivatives (Franco et al. 2011, Hayashi et al. 2011, Vainio et al. 1999). Wnt4 is expressed during the development of ovaries in many species in the animal kingdom: fish - rainbow trout (Oncorhynchus mykiss) (Nicol et al. 2012), protandrous black porgy (Acanthopagrus schlegeli) (Wu & Chang 2009), avian - chicken (Gallus gallus) (Lim et al. 2013), marsupials - tammar wallaby (Macropus eugenii) (Yu et al. 2006), rodents - mouse (Mus musculus) (Vainio et al. 1999), rat (Rattus norvegicus) (Richards et al. 2002) and primates - human (Biason-Lauber et al. 2004, Jaaskelainen et al. 2010, Mandel et al. 2008). The Wnt4 gene is highly conserved across species (Yu et al. 2006). Studies of homozygous Wnt4 knock-out mice revealed that female genitalia were masculinized (Vainio et al. 1999). The MD was absent and the WD had continued to develop. Wnt4 is initially required in both sexes for the formation of the Mullerian duct. Wnt4 suppress the development of Leydig cells in the emerging ovary. Wnt4-mutant female ovaries have less than 10% of the germ cells left at birth and have enhanced germ cell apoptosis at 13,5–16,5 dpc (Jaaskelainen et al. 2010, Vainio et al. 1999). Wnt4 knock-out ovaries develop a male-specific coelomic blood vasculature (Jeays-Ward et al. 2003).

48 Studies have shown that Wnt4 is needed in adult ovaries for follicle maturation. The microarray of follicular granulosa cells with Wnt4 gain-of- function demonstrated that this leads to enhanced expression of the genes involved in steroid biosynthesis (Boyer et al. 2010). In humans up-regulation of WNT4 signaling leads to male-to-female sex reversal (Jordan et al. 2001). Therefore, Wnt4 is suggested to acts as an anti-male factor by disrupting recruitment of β-catenin at or around steroidogenic factor 1 (Sf1) binding sites which exist in multiple steroidogenic genes (Jordan et al. 2003).

2.6.1 Disorders of WNT4 function in female reproductive system

WNT4 in combination with three other genes, DAX1, FOXL2 and RSPO1, is considered as a factor involved in the ovarian development; however none of them has been proven to be the ovarian determining factor. WNT4 signaling disturbance is one of causes involved in pathological conditions generally called disorders of sex development (Kousta et al. 2010). SERKAL syndrome is an autosomal recessive disorder in XX females caused by loss of function in the WNT4 gene. The disorder is linked to a mutation in the Wnt4 gene, an intraexonic homozygous C to T transition at cDNA position 341. This leads to an alanine to valine residue substitution at amino acid position 114 (Mandel et al. 2008). The human phenotype closely resembles the Wnt4-/- mouse (Vainio et al. 1999) phenotype causing female-to-male sex reversal, along with renal, adrenal, and lung dysgenesis (Mandel et al. 2008). Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome is characterized by primary amenorrhea and infertility. The patients have congenital aplasia of the uterus and the upper part of the vagina. They have 46, XX karyotype and normal secondary sexual characters. The MRKH syndrome is considered to be caused by defective development of the Mullerian ducts between the fifth and sixth weeks of gestation (Pizzo et al. 2013). Biason-Lauber and colleagues have found mutation in the WNT4 gene in a patient diagnosed with MRKH syndrome. The patient lacked a uterus and had an excess of androgens, which was associated with the defects in the WNT4 gene located on chromosome 1 (Biason-Lauber et al. 2004, Biason-Lauber et al. 2007). Later analysis of young women with primary amenorrhea and lack of Mullerian ducts has identified a L12P mutation within exon 1 of the WNT4 gene. These findings have shown that WNT4 could be the factor behind the development of Mullerian ducts and would control biosynthesis of the ovarian androgens also in

49 humans (Philibert et al. 2008). By now there were reported four WNT4 mutation associated with Mullerian duct development and androgen biosynthesis abnormalities (Philibert et al. 2011). Only the cases of MRKH syndrome associated with Mullerian duct defects and hyperandrogenism proved to be worth screening for the mutations in WNT4 gene, whereas many other MRKH syndrome cases were not associated with defects in the WNT4 gene (Chang et al. 2012, Drummond et al. 2008, Ravel et al. 2009). The WNT4 gene plays an important role in the development of the female phenotype with or without renal anomalies, which is close to, but is considered to be different from MRKH syndrome. In order to distinguish classical MRKH from syndrome caused by disorders in WNT4 singling it was suggested to be called MRKH-Biason-Lauber syndrome (Philibert et al. 2011). Recently one more mutation in the WNT4 gene was found and published. It leads to a renal phenotype; no sex organogenesis related defects were reported (Vivante et al. 2013). Polycystic ovary syndrome (PCOS) is characterized by chronic anovulation with either oligoamenorrhea or amenorrhea and hyperandrogenism. This is a relatively frequent condition in women of reproductive age, but the etiology of PCOS is unknown, however. It has been suggested that genomic variants in genes related to the regulation of androgen biosynthesis and function could be involved in genetic predisposition to PCOS. The direct sequencing of the WNT4 gene in 25 women with PCOS demonstrated no mutant alleles in studied patients (Canto et al. 2006). Based on these studies WNT4 is not considered to be a candidate gene for PCOS.

50 3 Aims of the study

The goal of this research was to reveal the functional role of the Wnt4 gene in the control of sex determination, ovariogenesis and female sex duct formation. The specific aims of this study were as follows: 1. Wnt4 knock-out mice undergo female-to-male sex reversal; the study aimed to find out the mechanism behind the sex reversal phenotype caused by Wnt4 deficiency. 2. The germ cell development was altered in the mice lacking Wnt4 signaling. The study assessed the reasons for the loss of the germ cells and studied early folliculogenesis in the Wnt4 knock-out females. 3. The study aimed to reveal Wnt4 function in postnatal gonads by using Wnt4 conditional deletion and novel hypomorphic Wnt4 mCheery mice models. 4. This work characterized Wnt4 roles in the Mullerian duct formation and in postnatal uterine development and function.

52 4 Materials and methods

Table 2.

Method Original publication Antibody blocking assay IV Beta-galactosidase assay II, IV Bioinformatics III Cell culture III-IV Cell migration assay (Incu Cyte) VI Estrous circle follow-up III Flutamide-treatment I Follicular count III Hematoxylin-Eosin assay I-IV Immunohistochemistry I-IV Laser micro dissection (PALM) III Optical Projection Tomography (OPT) VI Organ culture I, II, IV Polymerase chain reaction (PCR) I-IV Real-time quantitative polymerase chain reaction (qPCR) II-IV Section in situ hybridization II Statistics I-IV Time lapse imaging VI Transgenic mouse lines I-IV Transmission electron microscopy (TEM) II, III Western Blotting II Whole mount in situ hybridization I-IV Wnt reporter assay III

54 5 Results

5.1 The Partial Female to Male Sex Reversal in Wnt4 knock-out Females Implicates Induced Expression of Testosterone (I)

A previous study (Vainio et al. 1999) has shown that the Wnt4-deficient female developed Wolffian ducts instead of Mullerian ducts. This raised the possibility that androgens, namely testosterone, could be expressed in Wnt4 knock-out females and would maintain the Wolffian duct persistence.

5.1.1 Testosterone was detected in the plasma and gonads of Wnt4- deficient females

Hormonal measurement has revealed testosterone presence in the plasma samples of the wild-type and Wnt4-deficient males and females, whereas no testosterone was detected from wild-type females. In order to detect the testosterone origin we have checked if the adrenal gland was the source of testosterone. No testosterone was detected in wild-type or experimental group of male and female adrenal glands. Nevertheless, consistent with the results in plasma, testosterone production was identified in wild-type and Wnt4 deficient testes and also in the Wnt4-deficient ovary, whereas no synthesis was detected in the wild-type newborn ovary. This led to the conclusion that testosterone is synthesized by the Wnt4-deficient ovary. Taken together, testosterone could be considered as the main signal for the partial male-to-female sex reversal phenotype in Wnt4- deficient females. Testosterone is produced via precursors such as androstenedione and DHEA; thus we have measured their amounts in the gonads. The concentration of androstenedione was not detectable, but that of DHEA was elevated in Wnt4- deficient females compared with wild-type females. No significant differences were observed between wild-type and Wnt4-deficient males.

5.1.2 Wnt4-deficient ovaries have changed expression of the hormone biosynthesis genes

Microarray analysis was used to reveal the genetic changes that lead to the synthesis of testosterone in the embryonic ovaries of Wnt4-deficient females. The

55 results indicate that Cyp11a, Cyp11b2, Cyp17, Cyp19, Cyp21a, Hsd3b1, Hsd3b6, Hsd17b1, and Hsd17b3 gene expression was induced in the ovaries of Wnt4- deficient mice in comparison to the wild type. The in situ hybridization analysis confirmed the ectopic expression of the Cyp17 and Hsd17b1 in the Wnt4 knock- out ovaries whereas it has remained unchanged in the wild-type and Wnt4 deficient males.

5.1.3 Blocking of the androgen action causes the degeneration of the Wolffian ducts in Wnt4 knock-out females but has no influence on endothelial cell organization

We have used the antiandrogen flutamide to determine whether testosterone is in charge of maintaining the Wolffian duct in Wnt4-deficient females. Flutamide is a non-steroidal anti-androgen drug, which competes with testosterone and dihydrotestosterone for binding to androgen receptors. The pregnant females were given flutamide injections of 100mg/kg every 24h until the pups were born. This caused testes size reduction in newborn males in comparison to placebo, those recived corn oil injections. The obvious phenotype was seen in the Wnt4-deficient female since no sex ducts were formed whereas untreated females had Wolffian duct-derived cords. Flutamide did not have any effect on the expression of the Cyp17 and Hsd17b1 genes in either sex or genotype, as judged by in situ hybridization analysis. The increased testosterone levels have prompted interest to analyze whether they would have any influence on external genitalia differentiation since the ano- genital region development is controlled by testosterone (Mahendroo et al. 2001). The ano-genital distance is longer in males than in females. The ano-genital distances of newborn Wnt4-deficient and wild-type males that had received daily injections of flutamide were significantly reduced compared with the corresponding placebo males. However, this was not observed in the wild-type or in the Wnt4-deficient females when compared with placebo controls. It was previously reported that Wnt4-deficient ovaries develop testes-specific coelomic blood vessel (Jeays-Ward et al. 2003). We have raised the question of whether the vessel formation is due to ectopic testosterone function or to Wnt4 signal ablation. The experiments showed that administration of flutamide did not change the sex-specific organization of endothelial cells in either sex or genotype studied, thus suggesting that those androgens cause the WD persistence in XX Wnt4 knock-out but do not regulate coelomic vessel formation. Taken together,

56 this study differentiated the features of the sex reversal phenotype that are caused by enhanced androgen function and those that are caused by Wnt4 deficiency.

5.2 Wnt4/5a signaling controls cell-cell adhesion and meiosis initiation during ovarian folliculogenesis (II)

Wnt4 was identified as a critical signal that promotes female development, since characteristic female features are impaired in its absence and are replaced by some male ones (Heikkila et al. 2005, Jeays-Ward et al. 2003, Vainio et al. 1999, Yao et al. 2004). As we have reported earlier, Wnt4-deficient ovary ectopically expresses several genes of the testosterone biosynthesis pathway (Heikkila et al. 2005). Given the fact that signals from somatic cells are crucial for germ cell differentiation (Adams & McLaren 2002) and they were seemingly affected in the case of Wnt4 deficiency we set to analyze Wnt4 function in the developing ovary.

5.2.1 Wnt4 is expressed in somatic cells and plays an important role in maintenance of cell-cell interaction in the developing ovary

The lineage tracing experiments using Wnt4EGFPCre;Rosa26LacZ mice showed only somatic cells of the ovary to be positive for beta-galactosidase staining. This led to the conclusion that Wnt4 regulates female development as a somatic signal since it was not detected in the female germ cells at any time point studied. Immunostainings revealed that female gonads at 12.5 dpc and 14.5 dpc had numerous PGC both in wild-type and Wnt4-deficient mice. However, Wnt4 knock-out germ cells failed to accumulate to the cortical part of the ovary, which was even more evident at 16.5 dpc. At the same time, wild-type germ cells were associated in the clusters comprised of several cells. The Wnt4 knock-out germ cell cysts consisted of one to a few germ cell assemblies. Ultrastructure analysis pointed out that the germ cells in the Wnt4-deficient ovary were separated from each other, with gaps visible in the germ-somatic cell complexes at 13.5 dpc. This led us to analyze the cell junction components in developing oocytes and somatic cells. Transmission electron microscopy revealed that the adherence junctions were poorly differentiated in Wnt4-deficient ovary, whereas they were already detectable in the endothelial cells of the emerging blood vessels of the same ovary. Additionally, protein levels of the gap junction protein Cx43 (Gittens & Kidder 2005, Gittens et al. 2005) and RNA levels of Iroquois3 (Irx3) were downregulated in the Wnt4-deficient ovary. Based on these results we concluded

57 that Wnt4 signaling is critical for the maintenance of cell-cell interaction in the developing ovary.

5.2.2 Wnt4 signaling plays a role in follicle development and meiosis initiation

Further analysis of the genes involved in folliculogenesis by in situ hybridization, qRTPCR or microarray revealed that a great number of female-specific genes were downregulated in Wnt4 deficient ovaries. Among those were: Adamts19 ovary specific gene; the granulosa cell-derived Kitl involved in growth control of the oocyte; the transcription factor Folliculogenesis-specific basic helix-loop- helix (Figla) which regulates the zona pellucida (Zp1–3) gene expression; Nobox gene, essential for folliculogenesis as it controls oocyte specific gene expression as well as expression of Gdf9 and Bmp15. These results have shown that Wnt4 signaling is critical for the maintenance of the developing ovarian follicles. Immunostainings showed that about 80% of wild-type germ cells had initiated meiosis at 14.5 dpc, whereas only about 20% of the Wnt4-deficient germ cells had expressed meiosis markers by that time. Further analysis of meiosis- specific genes showed that the meiosis entry regulating gene Stra8 was downregulated in Wnt4 knock-out ovaries in comparison to the wild type. The Cyp26b1 gene encodes an enzyme, which prevents male gem cells from entering meiosis by degrading RA in the embryonic testis (Bowles et al. 2006, Koubova et al. 2006). Cyp26b1 was not detected in the wild-type ovaries from 12.5 dpc onwards since they initiate meiosis. Interestingly, the Cyp26b1 gene was ectopically expressed in the Wnt4-deficient mesonephros and ovary at 12.5 dpc. This suggests that Wnt4 signaling contributes to the initiation of meiosis in female germ cells and changes meiosis-specific gene expression towards a male pattern. Wnt5a expression was upregulated in the Wnt4-deficient ovary in comparison to the wild type and localized under the germinal epithelium at 12.5 dpc and 14.5 dpc in female gonads. The ovary of the Wnt5a-deficient embryo expressed reduced amount of Stra8 and ɣH2AX. Interestingly, the germ cells of Wnt4/Wnt5a double knock-out mice did not express meiosis markers Stra8, Xlr or ɣH2AX at 14.5 dpc. Due to this and the fact that about 20% of the germ cells in the Wnt4-deficient ovary still had the capacity to initiate meiosis, we hypothesize that Wnt4 may function in concert with other Wnt factors to regulate meiosis.

58 5.3 Wnt4 as a pleiotropic signal for controlling multiple aspects of follicle maturation in the adult female ovary (III)

Work from our laboratory and others have shown that Wnt4 is an important signal for many aspects of female sexual development. However, the studies of later Wnt4 function in adult ovaries are limited, mainly due to early lethality of Wnt4 knock-out mice. An in situ hybridization analysis and fate mapping cells of the Wnt4 origin with the help of the mT/mG:Wnt4Cre dual-reporter crossing revealed that Wnt4 RNA and the Wnt4 linage cells were found among the follicular, granulosa, theca, and interstitial cells. These suggested a possible role for Wnt4 in ovarian folliculogenesis. In this study we have addressed whether Wnt4 signal is involved in the development and maturation of the ovary after initial promotion. We took advantage of Wnt4 floxed, anti-Mullerian hormone receptor 2 Cre (Amhr2Cre), doxycycline-inducible tetO-Cre ROSArtTA/- (tetO-Cre ROSArtTA/-) knock-in and novel Wnt4 monomeric cherry (Wnt4mCherry; Wnt4mCh/mCh) hypomorphic allele inheriting mouse models.

5.3.1 Wnt4 deficiency by Amhr2+/Cre in the ovarian somatic cells compromises folliculogenesis and female fertility

The crosses of Amhr2Cre with Wnt4flox/flox (Wnt4f/f) mice led to the Wnt4 deletion in the follicles where the reporter gene was expressed. However, the efficiency of Amhr2Cre-mediated recombination varied. Wnt4 expression was reduced by around 74% leaving about 26% of gene function intact as judged by the real-time qPCR. The Wnt4f/f;Amhr2+/Cre crosses produced 3.28±1.8 (n=7) pups, whereas the wild-type females generated 8.2±1.3 (n=7; P≤0,001) pups, making the litter size about 60% smaller in comparison to the wild-type controls. The estrous cycle time was prolonged in Wnt4f/f;Amhr2+/Cre. Despite this no significant changes were detected in the amounts of serum FSH, LH or progesterone levels. As the Wnt4 deletion rate varied, so did the phenotype. At 4 months of age, the most extreme Wnt4f/f;Amhr2+/Cre females had ovaries that were considerably reduced in size (2/21 females). The ovarian shape was changed from round to oval, they contained numerous zona pellucida remnants, and such ovaries produced neither antral follicles nor CLs.

59 The rest of the analyzed Wnt4f/f;Amhr2+/Cre ovaries (19/21) had unchanged follicle counts. However, they had a higher frequency of polyovular follicles and cleaved caspase-3 positive cells.

5.3.2 Postnatal inactivation of Wnt4 function with TetO-Cre;RosartTA/+ leads to premature ovarian failure

Given the restricted efficiency of Amhr2+/Cre, we took advantage of the tetracycline-inducible TetO-Cre;RosartTA/+ where Wnt4 was globally deleted at birth. The TetO-Cre;RosartTA/+ deletion efficiency varied between 80 and 99%. We have noted that the reduction in ovarian size and follicle count correlated with the efficiency of Wnt4 gene inactivation. When the Wnt4 gene was deleted with 95– 99% efficiency (n=2/5), the ovaries were composed of noticeably reduced primary and antral follicle counts and the total number of follicles was reduced by approximately half. Strikingly, when Wnt4 inactivation efficiency was less than 90% (n=3), the follicle count remained normal in comparison to controls. These results provided evidence that a minute amount of Wnt4 is sufficient to obtain a normal ovary in vivo, whereas a more significant Wnt4 deficiency leads to POF.

5.3.3 Wnt4mCherry, a novel hypomorphic mouse model, develops premature ovarian failure

Wnt4mCherry is a novel hypomorphic mouse model that has residual Wnt4 signaling. The mCherry fluorescent protein was fused with Wnt4 cDNA, the construct was targeted at exon 1 of the Wnt4 gene in embryonic stem cells, and transgenic mice were generated. We made crosses between Wnt4mCh/+ and Wnt4mCh/+ to generate Wnt4mCh/mCh mice. It has appeared that the phenotype of Wnt4mCh/mCh resembled Wnt4-/- due to defective function of the fusion protein. Interestingly, some of the newborn mice have survived to adulthood and provided a tool to study Wnt4 function postnatal. The Wnt4mCh/mCh females failed to generate any offspring. The rudimentary ovaries were either reduced or larger, which was attributable to the formation of intraovarian cysts. Wnt4mCh/mCh females had polyovular follicles. The well-arrayed organization of the granulosa cells in the follicle was altered and they had a theca cell layer that was only 1 cell thick whereas multiple theca cell layers were seen

60 in the controls. The ovaries had abundant zona pellucida remnants not observed in the controls. Due to Wnt4 hypomorphism, the primordial follicle counts at all stages of differentiation were drastically reduced and the total follicle count was diminished by 90%. Additionally, to MD derivatives the WD has persisted in Wnt4mCh/mCh females, most probably due to ectopic testosterone synthesis. The analysis of the genes previously implicated in POF development, namely Foxl2, Star and Fos, has shown reduced Star and Fos expression in all studied mouse models, but not the Foxl2. The observed phenotypes and the changed gene expression pattern has led to the conclusion that failure in appropriate Wnt4 signaling leads to POF.

5.3.4 Wnt4 signaling contributes to ovarian development by coordinating cell polarity within the follicle

In wild-type follicles, the BM encapsulates the granulosa and theca cells, situated on the opposite sides of the BM. Unusually, in some of the Wnt4mCh/mCh ovaries, ectopic laminin and type IV collagen positive BM-like sheets had accumulated within the granulosa cell layers. Unlike in control follicles, the granulosa cells of Wnt4mCh/mCh mice could not form well-arrayed, ring-like assemblies within the follicles. Moreover, the pattern of expression of the adherent junction components Cdh2 (N-cadherin), Ctnnb1 and the gap junction protein Cx43 had changed significantly. The analysis of the ultrastructure of the BM showed a clearly distinguished BM in the newborn normal follicle but the BM of the ovarian primordial follicle of the Wnt4mCh/mCh females was loose, fragmented and had loops not noted in the controls. Immunostaining of a wild-type newborn ovary with laminin highlighted the BM around the emerging primordial follicles assemblies, whereas Wnt4 knock- out ovaries had robust laminin expression around the partially sex-reversed ovaries, reminiscent of the normal testis. The primordial follicles did not form in the ovaries of the Wnt4 knock-out females. Therefore, Wnt4 function seems to be pleiotropic and contributes to follicle development by coordinating granulosa cell organization, polarization marker expression and BM assembly.

61 5.3.5 Wnt4 signaling in the control of Amh expression

Real-time qPCR and in situ hybridization showed that expression of Amh was reduced in all the analyzed Wnt4 mouse models, both in the cases when RNA was extracted from whole ovary and also in laser capture dissected granulosa cells of the secondary follicles. Our previous studies shown that Wnt4 deficiency leads to ectopic testosterone synthesis in genotypic females (Heikkila et al. 2005, Vainio et al. 1999); due to this, a report that in utero exposure to testosterone reduces ovarian Amh expression raised the hypothesis that the two might be related (Veiga-Lopez et al. 2012). We turned to test the possible interactions between Wnt4, testosterone and Amh signaling in the KK1 murine granulosa cell line model. The KK1 cells were transfected with Wnt4 or Wnt4mCherry expressing vectors supplemented with or without the synthetic androgen methyltrienolone/R1881 in order to mimic the testosterone effect. After 24 hrs cells were harvested and extracted RNA was used for qRT-PCR for the gene expression analysis. The non-transfected KK1 cells served as control and all the gene expression was normalized to housekeeping gene Gapdh. It became evident that, Wnt4 induced Amh expression alone and even more in synergy with R1881. The Wnt4mCherry had also increased Amh expression; however the effect was weaker pointing to the hypomorphic function of the fusion protein. Interestingly, the R1881 alone did not affect Amh expression. Next we used the MetaCore database to describe potential mediators between AMH/AMHR2 and WNT4 signaling. The transcription factors GATA and SOX/SRY were highlighted as putative mediators; however, no direct interactions were described before. We conclude that Wnt4 signaling is involved in the control of oocyte development, partially by affecting the expression of Amh and testosterone. The results indicated that Wnt4 signaling controls ovarian function, not only during embryogenesis, but also after birth and in adulthood. Wnt4-mediated control of follicle maturation involves Amh. Besides this, Wnt4 coordinates granulosa cell polarity and accumulation of BM components within the developing follicle, providing a putative molecular mechanism by which impaired Wnt4 signaling in the ovarian somatic cells contributes to POF.

62 5.4 Wnt4+ progenitor cells construct the Mullerian duct and Wnt4 hypomorphic allele demonstrates a key role in uterus development (IV)

The internal mammalian sex organs are established during embryogenesis from the female Mullerian (MD) and the male Wolffian ducts (WD), the indifferent gonad and the integrated germ cells. Of these the MD, also called the paramesonephros, is the primordia of the oviduct, the uterus and the upper part of the vagina. Gene targeting experiments indicated that MD development is regulated by the various factors Pax2, Pax8, Lim1, Emx2, Hoxa13, Wnt4, Wnt7a and Wnt9b (Carroll et al. 2005, Grote et al. 2008, Kobayashi & Behringer 2003, Kobayashi et al. 2004, Miller & Sassoon 1998, Simpson 1999, Taylor & Fei 2005, Torres et al. 1995, Vainio et al. 1999). Of these the Wnt4 gene encodes the key signal for MD development since in its absence the duct fails to develop and only the very anterior Wnt7a positive primordia is specified, but other than that the role of Wnt4 in MD development remains poorly understood (Vainio et al. 1999).

5.4.1 Mullerian duct growth initiation and further extension requires Wnt4 signal

Wnt4 is expressed by the mesenchyme cells of the growing MD (Vainio et al. 1999). The WD forms normally in the Wnt4 knock-out mice, however MD does not form despite the presence of MD precursor cell population in the anterior part of the mesonephros (Vainio et al. 1999). This would suggest that Wnt4 is not required for the MD placode-like structure formation but is needed for subsequent MD formation. Based on this we raised the hypothesis that Wnt4 could be the factor behind the mobility of the MD-forming cells. To check this out we used Wnt4EGFPCre:R26YFPflox/+ and double reporter mT/mG:Wnt4Cre mice lines to trace the migration of the Wnt4 linage cells by time lapse imaging. The time lapse movies of the genital ridges derived from Wnt4EGFPCre:R26YFPflox/+ show strong YFP expression in the mesenchyme, coelomic epithelium region and mesonephric tubules but not in the Wolffian duct. During cultivation starting after 11.5 dpc a large group of YFP positive cells started rapid migration from the area at the apical side situated between WD on one side and ceolomic epithelium on the other side. The duct extended and

63 reached its full length by 13.5 dpc. This experiment showed that the cells of Wnt4 origin were abundantly present among the MD-forming cells. However, the resolution of the whole mount image did not enable us to distinguish single cells sufficiently well. Next we used mT/mG:Wnt4Cre and confocal imaging with several Z sections, which provided high resolution and single cell visibility as well as cell tracing possibility. We witnessed the placode-type structure forming at the most apical lateral side of WD at around 11.5 dpc. Some of the cells in the placode-like assembly were GFP positive, indicating the origin from Wnt4 linage. Strong GFP expression was seen in coelomic epithelium along the WD and mesonephric tubules but never in the WD itself. After a placode-like cell group had formed, one of the GFP positive cells made a long extension and started to protrude between WD and coelomic epithelium. This elongated cell became the MD tip, and the rest of the cells followed it. The cells in the stream were both Wnt4GFP positive and negative. Noteworthy is the observation that the coelomic epithelium cells were ‘turning on’ Wnt4 expression, becoming GFP positive from GFP negative cells, as the MD elongated. This led us to the speculation that the Wnt4 signal in the coelomic epithelium could serve as a landmark for the migrating MD cells. The observations described above raised the question of whether Wnt4 is needed only for the preceding cell differentiation and migration initiation, or also for MD extension. We used Wnt4 antibodies to block the Wnt4 activity in the gonadal-mesenchyme explants in ex vivo organ culture conditions. The results showed that in the control group the MD was fully grown (10/10). In the Wnt4 antibody supplemented specimen the MD did not extend or its growth was stopped without reaching the end of the Wolffian duct (8/10). Thus we concluded that Wnt4 is needed not only for the initiation of migration but also for the extension of the MD. We set to analyze if Wnt4 has an effect on cell migration only in the context of MD growth and endothelial cell migration (Jeays-Ward et al. 2003) or whether it has a broader effect. We took advantage of the embryonic fibroblast NIH3T3 cell line, which does not express endogenous Wnt4 (Kispert et al. 1998) served as control, and Wnt4-NIH3T3 cells that stably express Wnt4. After wound making the cells were grown in 10% serum and serum free conditions for 24h. The time lapse image analysis showed that in Wnt4-NIH3T3 cells assay wound closed faster in comparison to control NIH3T3 cells both in the serum-supplemented and serum-free conditions. The relative value of 50% wound closure in serum-

64 supplemented conditions was 4 hrs for the NIH3T3-Wnt4 cells and 8 hrs for the controls. An even more noticeable difference in wound closure was seen in serum-free conditions. In NIH3T3-Wnt4 cells this process took 15 hrs, whereas in controls it took 64 hrs. This data suggests that Wnt4 might be a factor that enhances and controls the migration of various cell types.

5.4.2 Imbalanced expression of Wnt4 gene leads to Mullerian duct defects early in embryogenesis and uterine gland agenesis with hydrouterus condition in adult mice

The hypomorphic Wnt4mCherry/mCherry (Wnt4mCh/mCh) mice with only a fraction of the Wnt4 activity provided a tool for the analysis of the Wnt4 functions in the developing MD and in its derivatives both prenatally and postnatally. Most of the Wnt4mCh/mCh mice had formed MD, but its growth was altered. The duct was narrow, contained less epithelial cells per cross section, and was situated closer to the WD; stromal cells were loosely assembled and eosinophilic. The grooves of the MD ridge commonly forming in the wild-type did not form in the Wnt4mCh/mCh. Interestingly, the expression pattern of other Wnts involved in MD differentiation was unchanged, namely Wnt5a and Wnt7a. The immunostainings of Wnt4mCh/mCh mice showed no signs of endometrial gland formation, whereas control mice had emerging uterine glands at the age of P21. Some of the Wnt4mCh/mCh survived to adulthood, but they often developed hydrouterus. This led to disturbed uterine architecture. The uterine glands did not form, the myometrium was undifferentiated and the epithelium of the uterine lumen got detached and formed the debris in the liquid-filled uterus. These observations led to the conclusion that a rather minor Wnt4 signal is sufficient to initiate MD growth, but apparently it has to be more prominent for the formation of the fully functional MD capable of differentiating into oviduct, uterus and upper part of the vagina. The lack of Wnt4 function precludes endometrial gland formation, whereas the vaginal malformation causes hydrouterus condition leading to defects in reproduction.

66 6 Discussion

6.1 Female to male sex reversal in Wnt4-deficient ovary involves ectopic testosterone synthesis and anti-androgen administration prevents Wolffian duct formation (I)

The analysis of Wnt4-deficient females revealed partial masculinization, because they had Wolffian ducts with structures resembling an epididymis and the embryonic ovary expressed some genes encoding enzymes involved in the testosterone synthesis pathway. Our study showed that testosterone was certainly synthesized by the gonads of the Wnt4-deficient females. Testosterone was also detected in the plasma and at the same time the DHEA levels were increased in the gonad extract of the newborn mice. It was previously reported (Heikkila et al. 2002) that Wnt4 is needed for adrenal gland development, and our current results demonstrated that testosterone was not detected in adrenal glands either sex or genotype of the studied mice. This indicated that embryonic adrenal glands do not contribute to the testosterone increase in the Wnt4-/- mice. The current study demonstrated that the pathway leading to the production of testosterone was induced in Wnt4-deficient ovary precluding the androgen production. Wnt4-deficient females produced testosterone, but it was not converted to significant amounts of DHT. This could be the reason why the ano-genital distance was not changed in response to flutamide, even though it was reduced by antiandrogen treatment in the wild-type and Wnt4-deficient males. The flutamide administration was sufficient to prevent Wolffian duct differentiation in Wnt4-deficient females. This indicated that duct persistence phenotype was due to ectopic testosterone secretion. In this work we raised the hypothesis that testosterone could play a role in the organization of the male type of coelomic vessel in the developing testis and Wnt4-deficient ovary. Further experiments ruled out this possibility since flutamide injections did not lead to a change in the organization of the male- specific coelomic blood vessel in wild-type male or Wnt4-deficient female. This excludes the role of androgen action in the process of coelomic blood vessel formation.

67 6.2 Wnt4/5a signaling controls cell-cell adhesion and meiosis initiation during ovarian folliculogenesis (II)

The current work provided evidence that Wnt4 serves as a critical somatic cell component controlling the development of the female germ cell line, involving germ line cyst break down, cell-cell interaction, oocyte specific gene expression and meiosis control.

6.2.1 Wnt4 plays a role in assembly of female ovarian follicles

Wild-type female germ cells localize in the cortex of the developing ovary. The germ cells of Wnt4 deficient embryos largely fail to reach the ovarian cortical region. In addition, germ line cysts reduce prematurely to single cells or to assemblies of a few germ cells. Transmission electron microscopy micrographs have shown gaps between cells, pointing to cell-cell adhesion defects. Cell junction marker studies have shown that Cx43 and Irx3 were reduced and adherence junction formation was affected in the case of Wnt4 deficiency. The protein levels of the E-cadherin and β-catenin were elevated and their expression pattern was changed from a female type to a male type when Wnt4 function was impaired in line with the reported sex-specific distribution of E-cadherin (Di Carlo & De Felici 2000, Liu et al. 2009). The data support the conclusion that Wnt4 function is important for the maintenance of female germline cysts and interaction between somatic - somatic and somatic - germ cells.

6.2.2 Wnt4 signaling regulates the development of the follicular complex and meiosis

A number of genes involved in folliculogenesis such as Figla, Nobox, Gdf9 and Bmp15 presented reduced expression in Wnt4-deficient ovary. This suggested that Wnt4 plays a role in controlling the folliculogenes. The meiosis-specific gene expression was reduced in the Wnt4-deficient ovaries along with the above-mentioned genes. Meiosis was shown to be initiated by RA signaling, which triggers the expression of a transcription factor, Stra8 (Anderson et al. 2008). During embryogenesis, meiosis is inhibited in the male by the enzyme Cyp26b1, which degrades RA (Anderson et al. 2008, Trautmann et al.

68 2008). Due to Wnt4 deletion the Stra8 expression was reduced whereas Cyp26b1 became up-regulated, mimicking the male phenotype to some extent. In support of the Wnt4 role in meiosis, we found that factors such as Spo11, Rec8, Msh5 and Syp3 were down-regulated in the Wnt4-deficient ovary. This may lead to the subsequent failure of meiosis in association wit Wnt4 deficiency. As an outcome of the defective meiosis initiation, only about 20% of the Wnt4 deficient germ cells expressed meiosis-specific markers. Additionally, the E-cadherin expression remained high in the Wnt4-deficient ovaries while being reduced in the wild-type ovaries as a sign of meiosis initiation (Di Carlo & De Felici 2000). The masculinization phenotype was enhanced by the formation of spermatogonia-like cells within a sex cord-like structures, resembling seminiferous tubules. Correspondingly, the Wnt4/5a double-mutant had germ cells, but they did not express Stra8 or other meiotic markers. Based on these findings we concluded that Wnt4 regulates meiosis in synergy with Wnt5a. It is worth noting that a later study from another laboratory suggested an alternative interpretation for the increased Wnt5a expression in the Wnt4 knock- out ovary, attributing it solely to the masculinization phenotype and excluding Wnt5a role in meiosis (Chawengsaksophak et al. 2012). Taken together, the data suggest that Wnt4 signaling promotes the expression of ovary-specific genes and endorses initiation of meiosis by influencing the RA pathway.

6.3 Wnt4 signal is involved in controlling multiple aspects of follicle maturation in the adult female ovary (III)

This study indicates closely that Wnt4 signaling is essential not only for mammalian female sex determination but also later in the organogenesis of the ovary and in folliculogenesis. The impaired Wnt4 signaling destabilized the estrous cycle and led to reduction in litter size by 60% in Wnt4f/f;Amhr2+/Cre mice and infertility in Wnt4mCh/mCh mice. We have previously reported defects in germ cell cyst formation in the Wnt4 knock-out mice (Naillat et al. 2010, Vainio et al. 1999). The adult mice that had impaired prenatal Wnt4 signaling were more prone to develop polyovular follicles, which could correlate with the disturbances in the early selection of individual oocytes from the cyst (Chen et al. 2007, Chen et al. 2009, Iguchi et al. 1990, Tingen et al. 2009). Thus, reduced Wnt4 function may promote formation

69 of the polyovular follicle, based on its early signaling roles in the embryonic ovary. The main phenotypic consequence of the conditional Wnt4 knock-out and the Wnt4mCherry-knock-in allele mouse models was POF. It represented a severe condition in which female reproduction failed prematurely. We considered Wnt4 to be a critical contributing factor in the development of POF due to its signaling importance both during the prenatal and postnatal steps of folliculogenesis. Wnt4 involvement in POF was reinforced by the reduced expression of the Star and Fst genes, which have been implicated in the development of POF in knock-out models (Jorgez et al. 2004, Kaku et al. 2008). Moreover, the BM and some cell polarity control factors were deregulated when Wnt4 function was altered, namely intrafollicular accumulation of laminin and type IV collagen in BM-like sheets were noted. Additionally, abnormal Wnt4 functional ablation led to disturbed expression of adherent junction proteins: Cdh2 and Ctnnb1, and a gap junction component, Cx43. This indicated compromised follicular cell polarity caused by the loss of Wnt4 function. The importance of the BM in the development of POF was highlighted in the Foxl2 knock-out, where the follicular BM is affected as well (Uda et al. 2004). Taken together, an important function of Wnt4 is to serve as a cell polarity signal to coordinate folliculogenesis. Our experiments demonstrated that Wnt4 deficiency greatly reduced the number of follicles within the ovary. We speculated that this condition involved enhanced follicular atresia and premature recruitment of primordial follicles caused by a decrease in Amh expression. Consistently with this working hypothesis, we found that Wnt4 and Amh gene expression correlated during follicle development and expression of the Amh gene was reduced in the analyzed mouse models. A gain in Wnt4 function also induced Amh expression in the cell line model in vitro. It was documented that a decline in the amount of Amh made the follicle more sensitive to pituitary gland-derived FSH. Since Wnt4-deficient female embryos also express testosterone ectopically, testosterone could be considered as a signal that coordinates Wnt4 and Amh expression in the developing follicle. In line with this suggestion, the synthetic androgen R1881 increased Amh expression in synergy with Wnt4, but not alone, whereas Wnt4 signal alone increased Amh expression in KK1 cells. Therefore, androgens synthetized by follicles may couple Amh and Wnt4 signaling to coordinate oocyte maturation in the follicle. The phenotypes noted in the Wnt4mCherry hypomorph are reminiscent of those in human Mayer-Rokitansky-Küser-Hauser-Biason-Lauber (MRKHBL)

70 syndrome, since the described mutations in the Wnt4 gene lie behind the development of this syndrome (Biason-Lauber et al. 2004, Biason-Lauber et al. 2007, Biason-Lauber & Konrad 2008, Philibert et al. 2011). These points present the suitability of the Wnt4mCherry transgenic mouse lines generated in this study as models for addressing in detail the mechanisms of human MRKHBL syndrome. These results suggested that Wnt4 signaling controls ovarian function, not only prenatally, but also postnatally and in adulthood. Wnt4-mediated regulation of follicle maturation involves Amh. Furthermore, Wnt4 coordinates granulosa cell polarity and accumulation of BM components within the developing follicle, providing a presumptive molecular mechanism by which impaired Wnt4 signaling in the ovarian somatic cells contributes to POF.

6.4 Wnt4 signal is needed for Mullerian Duct cell migration and Wnt4 hypomorphic allele demonstrates a key role in Uterus Development (IV)

This work provides evidence that Wnt4 is a factor required for MD cell migration needed for ductal growth and for endometrial gland formation in the adult mouse uterus. The origination of the anlagen of the MD is still actively discussed in the field. The first model, not supported today, suggested that Wolffian duct derived cells would contribute to MD growth. WD-derived cell lineage tracing experiments have shown that WD derived cells do not contribute to MD growth (Guioli et al. 2007). However, WD is needed for MD elongation as a physical guide and signal provider, as illustrated by Wnt9b knock-out mice where MD stops extending despite WD presence (Carroll et al. 2005). The second, and the most widely supported theory, suggests that the MD originates from placode-like thickening of the coelomic epithelium that extends caudally (Guioli et al. 2007, Jacob et al. 1999, Kobayashi & Behringer 2003, Kobayashi et al. 2011). Our work supports the second model. Additonaly, in our work we did not witness invagination of coelomic epithelium rather it was cell migration. It is of high interest to analyze if the MD formation could follow the pattern of ectodermal appendage or kidney development, in respect of MET, where Wnt4 was shown to play a role (Biggs & Mikkola 2014, Stark et al. 1994). This remains to be analyzed, whereas the current results show that Wnt4 expression is needed for the tip cell differentiation and initiation of the MD-specific cell migration. These cells

71 are guided not only by the Wolffian duct but also by the signals coming from nearby coelomic epithelium, based on the observation that coelomic epithelium cells initiate Wnt4 expression as the MD growth progresses. The migrating cell stream is composed of a mixed cell population as not all cells are Wnt4 positive, providing evidence that multiple cell types are needed for appropriate MD growth and differentiation. Antibody blocking assays have proven to be a useful tool in protein function studies (Oberlender & Tuan 1994). The Wnt4 antibody blocked the MD elongation in genital ridge cultures. This served as proof that Wnt4 is required not only for MD growth initiation, but continuously to maintain MD elongation. The cell culture wound healing assays with NIH3T3 and NIH Wnt4–3T3 cells indicate that Wnt4 endorses cell migration of a variety of cell types. Wnt4 involvement in cell migration coordination was shown in the context of endothelial and steroidogenic cell migration to form sex-specific vasculature (Jeays-Ward et al. 2003). Our earlier experiments with anti-androgen flutamide have shown that the androgen action blockage could not rescue the MD formation (Heikkila et al. 2005) attributing the MD growth control to Wnt4 signaling. There is a certain minimal amount of Wnt4 signal sufficient to initiate MD forming cell migration since Wnt4mCh/mCh hypomorphic mice are capable of forming MD. However, the lack of proper Wnt4 function leads to defects in MD differentiation but does not have influence on the topological expression of the Wnt5a and Wnt7a genes. The postnatal analysis of Wnt4mCh/mCh uterus revealed that uterine glands did not form, the myometrium was disturbed and the uterus was smaller at P21 whereas it became dilated due to hydrouterus formation. Fewer uterine glands and a defective decidualization process were reported in the mice with conditional Wnt4 deletion by PR-Cre (Franco et al. 2011). Additionally, our previous studies showed prolonged estrous cycle in the Wnt4Amhr2Cre mice (Prunskaite- Hyyrylainen et al. 2014). In summary, Wnt4 is a crucial factor behind MD-forming cell migration initiation and maintenance during embryogenesis. In the adult mouse it is needed for proper myometrium layering, luminal and endometrial gland formation. Taken together, we show that balanced Wnt4 signaling is needed continuously through embryogenesis, adolescence and adult life to ensure proper formation and function of the female sex ducts. A deeper understanding of Wnt4 function in female sex duct development would help to explain pathophysiological mechanisms leading to endometriosis and/or infertility.

72 7 Summary and conclusions

This study was prompted by the finding that Wnt4 deficiency leads to female-to- male sex reversal phenotype (Vainio et al. 1999). The present work provides a detailed analysis of the Wnt4 gene function in the female reproductive system. Herein is showed that testosterone is partially responsible for the sex reversal phenotype and the testosterone inhibitor flutamide can prevent WD persistence in the Wnt4 deficient female. The analysis of embryonic ovaries demonstrated that Wnt4 is an essential signal needed for ovarian-specific gene expression, meiosis initiation and cell-cell interaction establishment and maintenance. This demonstrated that the changed signaling pattern in the female granulosa cells is changing the gem cell environment and lead to male specific gene expression. The conditional deletion of Wnt4 in the ovarian granulosa cells (Wnt4;Amhr2Cre), global Wnt4 deletion (Wnt4f/f;TetO-Cre;RosartTA/+) and Wnt4mCherry hypomorphic mice have made it possible to analyze Wnt4 function in adult ovaries. These have shown that rather a minute amount of Wnt4 is enough to maintain ovarian function. We demonstrated that Wnt4 signaling deficiency leads to premature ovarian failure, sub- or infertility due to altered folliculogenesis since follicle-specific gene expression becomes deregulated and follicular basement membrane and cell polarization becomes altered. This study demonstrated that Wnt4 signal is needed for the differentiation of the MD-tip cell, initiation and maintenance of the MD cell migration. Additionally, the Wnt4mCherry hypomorphic mice revealed that Wnt4 is needed for the epithelisation process and is essential for the formation of the endometrial glands in the adult mouse uterus. Further studies should address the Wnt4 downstream genes in the developing ovary, MD and its derivatives as well as possible epigenetic control. It remains to be answered what signaling pathway Wnt4 uses for transducing its signal at different developmental stages and if there are any additional players or/effectors involved. It would be of interest to analyses how Wnt4 expression in the gonads correlates with the Wnt4 expression in the pituitary gland during the embryogenesis and adult female life, and is there any interdependence between these two. The knowledge gained during this work provides a better understanding of the complexity of female sex organogenesis and the pathogenesis of infertility.

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90 Original articles

I Heikkila M, Prunskaite R, Naillat F, Itaranta P, Vuoristo J, Leppaluoto J, Peltoketo H & Vainio S (2005) The partial female to male sex reversal in Wnt4-deficient females involves induced expression of testosterone biosynthetic genes and testosterone production, and depends on androgen action. Endocrinology 146(9): 4016–4023. II Naillat F, Prunskaite-Hyyryläinen R, Pietilä I, Sormunen R, Jokela T, Shan J & Vainio SJ (2010) Wnt4/5a signalling coordinates cell adhesion and entry into meiosis during presumptive ovarian follicle development. Hum Mol Genet 19(8): 1539–1550. III Prunskaite-Hyyryläinen R, Shan J, Railo A, Heinonen KM, Miinalainen I, Yan W, Shen B, Perreault C & Vainio SJ (2013) Wnt4, a pleiotropic signal for controlling cell polarity, basement membrane integrity and Anti-Mullerian hormone expression during oocyte maturation in the female follicle. FASEB J. IV Prunskaite-Hyyryläinen R, Skovorodkin I, Shan J & Vainio SJ (2014) Wnt4+ Progenitor Cells Construct the Mullerian Duct and its Hypomorphic Allele Demonstrates a Key Role in Uterus Development. Manuscript. Reprinted with the permission from Bioscientifica (I), Oxford University Press (II), and FASEB Journal (III).

Original publications are not included in the electronic version of the dissertation.

92 ACTA UNIVERSITATIS OULUENSIS SERIES D MEDICA

1232. Vatjus, Ritva (2014) Kohti suhdekeskeisyyttä lääkärin ja potilaan kohtaamisessa : laadullinen tutkimus potilas-lääkärisuhteen hahmottumisesta yleislääkäreiden koulutuksessa 1233. Mankinen, Katariina (2014) Neuropsychological performance and functional MRI findings in children with non-lesional temporal lobe epilepsy 1234. Karvonen, Henna (2014) Ultrastructural and functional characterization of myofibroblasts in lung diseases 1235. Sipola, Seija (2014) Colectomy in an ICU patient population : clinical and histological evaluation 1236. Lipponen, Kaija (2014) Potilasohjauksen toimintaedellytykset 1237. Jansson, Miia (2014) The effectiveness of education on critical care nurses' knowledge and skills in adhering to guidelines to prevent ventilator-associated pneumonia 1238. Turunen, Sanna (2014) Protein-bound citrulline and homocitrulline in rheumatoid arthritis : confounding features arising from structural homology 1239. Kuorilehto, Ritva (2014) Moniasiantuntijuus sosiaali- ja terveydenhuollon perhetyössä : monitahoarviointi Q-metodologialla 1240. Sova, Henri (2014) Oxidative stress in breast and gynaecological carcinogenesis 1241. Tiirinki, Hanna (2014) Näkyvien ja piilotettujen merkitysten rajapinnoilla : terveyskeskukseen liittyvät kulttuurimallit asiakkaan näkökulmasta 1242. Syrjänen, Riikka (2014) TIM family molecules in hematopoiesis 1243. Kauppila, Joonas (2014) Toll-like receptor 9 in alimentary tract cancers 1244. Honkavuori-Toivola, Maria (2014) The prognostic role of matrix metalloproteinase-2 and -9 and their tissue inhibitor-1 and -2 in endometrial carcinoma 1245. Pienimäki, Tuula (2014) Factors, complications and health-related quality of life associated with diabetes mellitus developed after midlife in men 1246. Kallio, Miki (2014) Muuttuuko lääketieteen opiskelijoiden käsitys terveydestä peruskoulutuksen aikana : kuusivuotinen seurantatutkimus 1247. Haanpää, Maria (2014) Hereditary predisposition to breast cancer – with a focus on AATF, MRG15, PALB2, and three Fanconi anaemia genes 1249. Nagy, Irina I. (2014) Wnt-11 signaling roles during heart and kidney development

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