D 1379 OULU 2016 D 1379

UNIVERSITY OF OULU P.O. Box 8000 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATIS OULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTA

DMEDICA Reeta-Maria Törmälä Reeta-Maria Törmälä Professor Esa Hohtola University Lecturer Santeri Palviainen ABNORMALITIES – A

Postdoctoral research fellow Sanna Taskila GENETIC APPROACH TO THE UNDERSTANDING OF Professor Olli Vuolteenaho FERTILIZATION FAILURE

University Lecturer Veli-Matti Ulvinen

Director Sinikka Eskelinen

Professor Jari Juga

University Lecturer Anu Soikkeli

Professor Olli Vuolteenaho UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF MEDICINE; Publications Editor Kirsti Nurkkala MEDICAL RESEARCH CENTER OULU; OULU UNIVERSITY HOSPITAL; ISBN 978-952-62-1297-5 (Paperback) NATIONAL GRADUATE SCHOOL OF CLINICAL INVESTIGATION ISBN 978-952-62-1298-2 (PDF) ISSN 0355-3221 (Print) ISSN 1796-2234 (Online)

ACTA UNIVERSITATIS OULUENSIS D Medica 1379

REETA-MARIA TÖRMÄLÄ

HUMAN ZONA PELLUCIDA ABNORMALITIES – A GENETIC APPROACH TO THE UNDERSTANDING OF FERTILIZATION FAILURE

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 Auditorium 4 of Oulu University Hospital, on 23 September 2016, at 12 noon

UNIVERSITY OF OULU, OULU 2016 Copyright © 2016 Acta Univ. Oul. D 1379, 2016

Supervised by Professor Juha Tapanainen Doctor Jouni Lakkakorpi

Reviewed by Professor Sari Mäkelä Docent Virpi Töhönen

Opponent Professor Juha Kere

ISBN 978-952-62-1297-5 (Paperback) ISBN 978-952-62-1298-2 (PDF)

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

Cover Design Raimo Ahonen

JUVENES PRINT TAMPERE 2016 Törmälä, Reeta-Maria, Human zona pellucida abnormalities – a genetic approach to the understanding of fertilization failure. University of Oulu Graduate School; University of Oulu, Faculty of Medicine; Medical Research Center Oulu; Oulu University Hospital; National Graduate School of Clinical Investigation Acta Univ. Oul. D 1379, 2016 University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract Despite the development of assisted reproduction technologies and significant advances in reproductive biology and medicine over the years the cause of infertility remains unexplained in 10–20% of cases. The cause of infertility in these cases may be connected to problems in fertilization or implantation and genetic factors may play a part in this. The zona pellucida (ZP) is an extracellular matrix surrounding the and early-stage . It is important for , fertilization and implantation. In , it is composed of four known ZP glycoproteins that all show varying degrees of structural and functional roles in reproduction. The aim of the present study was to examine the role of zona pellucida genes in cases of total fertilization failure and zona anomalies, and to study their expression in human fetal and ovaries. A total of 34 sequence variations were detected in genes expressing the four human ZP proteins (ZP1–ZP4) among women with fertilization failure and those with varying degrees of zona anomalies in their . Most of the variations were known single nucleotide polymorphisms, while three were novel findings. Women with fertilization failure had a higher mean number of sequence variations in ZP1 and ZP3 when compared with controls. Some of the most frequent zona anomalies may be at least partly explained by sequence variations in ZP1–ZP4 genes. In fetal life, the expression of ZP3 protein and mRNA could already be detected as early as at the 11th week of and it peaked at the 20th week, the time of primordial follicle formation. This suggests that components needed for zona matrix are already present well before the formation of the zona pellucida and may have a role in the development of primordial follicles. Expression of the transcription factor FIGLA (factor in the germline alpha) was increased at around the 20th week of gestation, supporting previous findings of its critical role in the initiation of folliculogenesis and primordial follicle formation. The present study adds to our knowledge on the currently still incomplete picture of formation of the ZP and fertilization in humans. Understanding the genetic background of infertile patients may help us to develop new tools not only to evaluate but also to improve their fertilization potential, and to choose the optimal treatment to achieve .

Keywords: granulosa cell, infertility, oocyte, ovary, total fertilization failure, zona anomaly, zona pellucida, zona pellucida glycoproteins 1–4

Törmälä, Reeta-Maria, Ihmisen alkiokuoren rakennehäiriöt – geneettiset tausta- tekijät osasyynä hedelmättömyyteen? Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Medical Research Center Oulu; Oulun yliopistollinen sairaala; Valtakunnallinen kliininen tutkijakoulu Acta Univ. Oul. D 1379, 2016 Oulun yliopisto, PL 8000, 90014 Oulun yliopisto

Tiivistelmä Diagnostiikan kehityksestä huolimatta hedelmättömyyden syy jää edelleen epäselväksi 10–20 %:ssa tapauksista. Niissä hedelmättömyyden taustalla voivat olla munasolun hedelmöittymiseen ja kohtuun kiinnittymiseen liittyvät ongelmat, jotka voivat osittain johtua geneettisistä syistä. Alkiokuori on munasolua ja varhaista alkiota ympäröivä rakenne, joka osallistuu munarakku- lan kehittymiseen, munasolun hedelmöittymiseen ja alkion tarttumiseen kohdun limakalvolle. Ihmisellä alkiokuori muodostuu neljästä tunnetusta alkiokuoriproteiinista (ZP1–ZP4). Tutkimuk- sessa selvitettiin alkiokuoriproteiineja koodittavien geenien vaikutusta hedelmällisyyteen poti- lailla, joilla koeputkihedelmöitys ei ollut tuottanut yhtään hedelmöittynyttä munasolua (engl. total fertilization failure, TFF) tai joiden munasoluissa havaittiin alkiokuoren rakennemuutoksia (engl. zona anomalies, ZA). Lisäksi selvitettiin alkiokuoriproteiinien ja niiden lähetti-RNA:n esiintymistä sikiöiden ja aikuisten munasarjoissa. TFF- ja ZA-potilaiden alkiokuoriproteiineja koodittavista geeneistä löytyi yhteensä 34 nukleotidimuutosta. Muutoksista kolme oli uusia löydöksiä, mutta suurin osa oli ennalta tunnet- tuja yhden nukleotidin polymorfioita eli geneettisiä monimuotoisuuskohtia. TFF-potilailla havaittiin ZP1- ja ZP3-geeneissä keskimäärin enemmän polymorfioita kuin verrokeilla. Myös osa yleisimmistä alkiokuoren rakennemuutoksista voidaan mahdollisesti selittää ZP1–ZP4-gee- neistä löytyneillä polymorfioilla. Sikiöllä ZP3:n ilmentyminen oli havaittavissa jo 11. raskausviikolla, mutta voimakkainta se oli primordiaalivaiheen munarakkuloiden muodostumisen aikaan 20. raskausviikolla. Tämä voi viitata siihen, että ZP3 saattaa osallistua primordiaalivaiheen munarakkulan kehittymiseen ennen varsinaisen alkiokuoren muodostumista. ZP-geenien säätelytekijän FIGLA:n esiintyminen lisääntyi 20. raskausviikolla, mikä tukee aikaisempia havaintoja FIGLA:n merkityksestä muna- rakkulan kehittymisen aktivaatiossa ja primordiaalivaiheen munarakkuloiden muodostumisessa. Tämä tutkimus tuo lisätietoa alkiokuoren merkityksestä munasolun hedelmöittymisessä ja syventää tietämystämme alkiokuoren muodostumisesta ihmisellä. Hedelmättömyyden taustalla olevien geneettisten tekijöiden tunteminen voi parantaa lapsettomuuspotilaiden hedelmällisyy- den arviointia ja auttaa löytämään heille parhaiten sopivan hoidon.

Asiasanat: alkiokuoren rakennehäiriö, alkiokuori, alkiokuoriproteiini, granuloosasolu, hedelmättömyys, munasarja, munasolu, zona pellucida glykoproteiini 1–4

Acknowledgements

This study was carried out at the Department of and Gynaecology, University of Oulu and at the Clinical Research Center, Oulu University Hospital, during the years 2003-2016. First, I wish to express my deepest and sincere gratitude to my supervisor professor Juha Tapanainen for his positive attitude, trust and endless encouragement during this project. Juha has had a crucial role at every phase of my thesis and I admire how he always found time for it despite his many commitments. I am grateful for his patience during my long periods of parental leave and my move abroad. I wish to express my gratitude to my supervisor Jouni Lakkakorpi, Ph.D., for his guidance throughout this project. I especially admire his writing skills, which have given me a great advantage when writing articles and this thesis. I am grateful to docent Tommi Vaskivuo for his excellent expertise with the project that led to the second article. Professor Hannu Martikainen, Terhi Piltonen, M.D., Ph.D. and docent Laure Morin-Papunen are acknowledged for their encouragement and valuable comments during our research meetings. I wish to thank docent Minna Männikkö, docent Timo Tuuri, Anni Haltia Ph.D., professor Leena Ala-Kokko, Annikki Liakka, M.D., Ph.D. and Sinikka Nuojua- Huttunen M.D., Ph.D., for their valuable collaboration. I warmly thank professor Markku Heikinheimo for kindly inviting me to his innovative research group for one year. Helka Parviainen and the rest of the research group are acknowledged for their support and great company during and after that year. I have had the privilege and joy to work on my thesis in an inspiring and warm- hearted research group. I am grateful to Minna Jääskeläinen, Johanna Puurunen, Sanna Koskela, Kristiina Mäkelä, Mervi Haapsamo, Zdravka Veleva and Outi Uimari for their support and friendship in and out of the lab. I wish to thank professor Sari Mäkelä and docent Virpi Töhönen for their careful review of this thesis. I thank Nicholas Bolton for his excellent linguistic revision of this thesis and the manuscripts over the years. I warmly thank Mirja Ahvensalmi for her skilful assistance in the laboratory. Seija Leskelä is acknowledged for her talented figure editing and her friendly attitude. I also want to thank Risto Bloigu for helping with the statistics. I want to thank my very dear friends and research fellows Anna Hakalahti, Päivi Honkavaara, Irina Nagy and Päivi Fonsén for being there for the ups and

7 downs of the research work and for all the great moments we have had together. I wish to thank Ingrid Huzen for her friendship and encouragement. I want to thank my soul mate Laura Myllymäki for her love and support over the years. I warmly thank my parents Anneli and Pertti for their support and endless willingness to help. I am very grateful to my father who started regularly babysitting Kerttu (and later also Hannes) when she was nine months old so that I could go on with my work. I wish to thank my sister and dear friend Kaisa for her encouragement in work and in life. My brother Juho and his partner Sanna are thanked for the nice moments together. I thank my sister-in-law Nina and her husband Petri for their friendship. I want to thank Panu for his endless love, support and help during my work on this thesis. I am deeply grateful for the adventures in life that we have taken together. I thank Kerttu and Hannes, the treasures of my life, for teaching me how the beauty of life lies in the small things and how to live in the moment. This study was financially supported by the Academy of Finland, the Sigrid Jusélius Foundation, the Finnish Cultural Foundation, the Foundation of the University of Oulu and Oulu University Hospital, who are gratefully acknowledged.

Amstelveen, June 2016 Reeta Törmälä

8 Abbreviations aa amino acid ADAM3 a disintegrin and metalloprotease 3 AMH antimüllerian ART assisted reproduction technology bp base pair cDNA complementary DNA CSGE conformation-sensitive gel electrophoresis DAB diaminobenzidine tetra hydrochloride E12 transcription factor encoded by the E2A gene E-box enhancer box EGF epidermal growth factor EST expressed sequence tag FIGLA factor in the germ line alpha FIVF fertilizers in IVF foxl2 forkhead box L2 Foxo3a forkhead box O3A FSH follicle-stimulating hormone GDF-9 growth differentiation factor 9 ICSI intracytoplasmic injection IHC immunohistochemistry ISH in situ hybridization IVF in vitro fertilization LH MBP-15 bone morphogenic protein 15 mRNA messenger RNA OSP-1 oocyte-specific protein 1 p27 cyclin-dependent kinase inhibitor PCR polymerase chain reaction PTEN phosphatase and tensin homolog rpm rounds per minute SNP single nucleotide polymorphism TFF total fertilization failure TGFα/β transforming growth factor a/β tRNA transfer RNA WPF women with proven

9 ZA zona anomaly ZAP-1 zona pellucida gene activating protein-1 ZP zona pellucida ZP1–4 zona pellucida glycoproteins 1–4

10 List of original publications

This thesis is based on the following articles, which are referred to in the text by their Roman numerals:

I Männikkö M*, Törmälä RM*, Tuuri T, Haltia A, Martikainen H, Ala-Kokko L, Tapanainen JS & Lakkakorpi JT (2005) Association between sequence variations in genes encoding human zona pellucida glycoproteins and fertilization failure in IVF. Hum Reprod 20(6): 1578–85. II Törmälä RM, Jääskeläinen M, Lakkakorpi J, Liakka A, Tapanainen JS & Vaskivuo TE (2008) Zona pellucida components are present in human fetal ovary before follicle formation. Mol Cell Endocrinol 289 (1-2): 10–5. III Pökkylä RM**, Lakkakorpi JT, Nuojua-Huttunen SH & Tapanainen JS (2011) Sequence variations in human ZP genes as potential modifiers of zona pellucida architecture. Fertil Steril 95(8): 2669–72. ***

* = equal contribution

** = Törmälä RM née Pökkylä RM

*** = Some unpublished data of the Study III is presented in the Results and Discussion section.

11

12 Contents

Abstract Tiivistelmä Acknowledgements 7 Abbreviations 9 List of original publications 11 Contents 13 1 Introduction 17 2 Review of the literature 19 2.1 Gonad and development during fetal life ...... 19 2.1.1 FSH-independent follicle development ...... 20 2.1.2 FSH-dependent follicle development ...... 22 2.1.3 ...... 23 2.1.4 Follicular apoptosis ...... 23 2.2 Zona pellucida ...... 25 2.2.1 Ultrastructure of the zona pellucida ...... 26 2.2.2 Structural components of the zona pellucida ...... 27 2.2.3 Genes encoding human ZP proteins ...... 28 2.2.4 Regulation of ZP genes ...... 29 2.2.5 Molecular characteristics of ZP proteins ...... 30 2.2.6 Cell types responsible for ZP protein synthesis ...... 32 2.2.7 Assembly of ZP proteins into zona matrix ...... 33 2.2.8 ZP1–ZP3 mutant mice ...... 34 2.3 Functional characteristics of the zona pellucida ...... 35 2.3.1 Role of the ZP in folliculogenesis ...... 36 2.3.2 reaction, recognition and binding ...... 36 2.3.3 Species specificity ...... 39 2.3.4 Sperm– fusion ...... 40 2.3.5 Block of ...... 41 2.3.6 In vivo hatching prior to implantation ...... 42 2.4 Abnormal ZP structures ...... 43 2.5 Infertility ...... 45 3 Aims of the study 47 4 Subjects and Methods 49 4.1 Subjects and tissue samples ...... 49 4.1.1 Patients with total fertilization failure (TFF) (Study I)...... 49

13 4.1.2 Human ovarian tissues (Study II) ...... 50 4.1.3 Zona anomaly patients (Study III) ...... 50 4.2 Methods ...... 51 4.2.1 Verifying exon–intron boundaries of the human ZP1 gene (Study I) ...... 51 4.2.2 DNA extraction and PCR (Study I and Study III) ...... 51 4.2.3 Conformation-sensitive gel electrophoresis (Study I) ...... 52 4.2.4 Sequencing (Study I and Study III) ...... 53 4.2.5 Allele frequencies (Study I) ...... 53 4.2.6 Statistical analysis (Study I and Study III) ...... 53 4.2.7 Immunohistochemistry (Study II) ...... 53 4.2.8 In situ hybridization (Study II) ...... 54 4.2.9 Image data (Study III) ...... 55 5 Results and Discussion 57 5.1 Sequence variations in genes encoding human zona pellucida glycoproteins, and fertilization failure in IVF (Study I) ...... 57 5.1.1 Verifying exon–intron boundaries of the human ZP1 gene ...... 57 5.1.2 Sequence variations in ZP genes ...... 57 5.1.3 Sequence variations of statistical significance ...... 60 5.1.4 Cumulative effect of sequence variations in the study groups ...... 61 5.2 Zona pellucida components are present in the human fetal ovary before follicle formation (Study II) ...... 61 5.2.1 ZP3 mRNA and protein expression in fetal and adult ovary ...... 62 5.2.2 ZP1 mRNA expression in fetal and adult ovaries ...... 64 5.2.3 FIGLA mRNA expression in fetal and adult ovaries ...... 65 5.3 Sequence variations in human ZP genes as potential modifiers of zona pellucida architecture (Study III) ...... 66 5.3.1 ZP gene-related sequence variations in IVF/ICSI subjects with zona anomalies ...... 66 5.3.2 Zona anomalies and sequence variations ...... 70 5.3.3 Zona anomalies and pregnancy outcome ...... 73 5.4 Methological considerations ...... 74 5.5 Errata ...... 75

14 6 Summary and conclusions 77 References 79 Original publications 95

15

16 1 Introduction

Infertility is defined as the inability to conceive after 12 months of regular unprotected . According to the European Society of and (ESHRE, ART Fact Sheet [July 2014]), one in six couples worldwide experience some form of fertility problem at least once in their reproductive lifetime. Despite development of assisted reproduction technologies (ART) such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) and significant advances in reproductive biology and medicine over the years, the cause of infertility still remains unexplained in 10–20% of the cases (ESHRE ART Fact Sheet [July 2014]). Hence, there is still a compelling need for better understanding of the molecular basis of fertilization in order to develop more comprehensive therapies for infertility. The zona pellucida (ZP) is a fibrous extracellular matrix surrounding growing oocytes and early-stage embryos in mammals. It develops between the plasma membrane of the oocyte and the innermost granulosa cells in growing follicles during folliculogenesis (Sinowatz et al. 2001). In humans, the ZP is composed of four ZP glycoproteins, ZP1–ZP4 (Lefievre et al. 2004). Human ZP proteins have been found to be expressed both in the oocyte and the granulosa cells (Gook et al. 2008). A transcription factor known as factor in the germline alpha (FIGLA) is involved in the coordinated expression of all the ZP genes (Liang et al. 1997, Soyal et al. 2000) and its expression increases during mid-gestation at the time of follicle formation (Bayne et al. 2004). ZP is important for successful folliculogenesis, followed by its key roles in taxon-specific fertilization and protection as it passes through the oviduct prior to implantation (Zhao & Dean 2002). Owing to the vital role of the ZP in fertilization, the aim of this study was to investigate whether sequence variations in genes encoding ZP proteins could partly explain unsuccessful IVF treatments and some of the most frequent ZP abnormalities encountered in routine IVF/ICSI. To further deepen our knowledge of the expression of human ZP1 and ZP3 and their transcription factor FIGLA during folliculogenesis, their expression and cellular localization were investigated in fetal and adult ovaries.

17

18 2 Review of the literature

2.1 Gonad and germ cell development during fetal life

In humans, becoming a female or a male is genetically determined at fertilization with the acquisition of an Z or Y from the father. Human preimplantation development starts with the fusion of the egg and sperm procuclei in the and requires embryonic genome activation, and 32 and 129 genes are transcribed during the transition from oocyte to four-cell stage and from four- to eight-cell stage, respectively, during the first 3 days after fertilization (Töhönen et al. 2015). The first signs of gonadal development are seen around the 4th week of gestation, when paired genital ridges become visible (Satoh 1991). At first, human gonads develop as indifferent gonads, which have the potential to develop into either to testes or to ovaries. Primary sex cords are formed at the fifth week of gestation when primordial germ cells (∼100 cells) migrate from the yolk sac to the gonadal ridge (Satoh 1991). During this migratory phase, the primordial germ cells divide mitotically and increase in number (Tam & Snow 1981, Oktem & Urman 2010). When primordial germ cells enter the genital ridges, they lose their motility and begin (Erickson 2001). This involves the differentiation of primordial germ cells into oogonia, which undergo several rounds of mitotic divisions. From the sixth week of gestation the primary sex cords are formed and start to differentiate into testes and ovaries (Satoh 1991, Loffler & Koopman 2002, DeFalco & Capel 2009). Once primordial germ cells arrive at the gonad their number rapidly increases from merely 10 000 at the sixth week to 600 000 at the eight week of gestation (Oktem & Urman 2010). Some oogonia start to develop further into primary oocytes and enter the first stages of at around 11–12th weeks (McGee & Hsueh 2000). The formation of primordial follicles at around the 18th week of gestation (Fulton et al. 2005) appears to protect oocytes from atresia, and oogonia do not persist beyond the 28th week of gestation (Oktem & Urman 2010). Ovarian follicles do not develop in the absence of oocytes, indicating that oocytes participate in follicle formation from the earliest stages (Vanderhyden 2002). A transcription factor FIGLA has been noted to be obligatory for primordial follicle formation and the survival of oocytes, as in FIGLA knockout mice follicle development is disturbed and a massive depletion of oocytes occurs around (Soyal et al. 2000). FIGLA has also been associated with premature ovarian failure in humans (Zhao et al. 2008, Tosh et al. 2014).

19 The total germ cell number peaks at the 18th–20th week of gestation (McGee & Hsueh 2000, Wallace & Kelsey 2010), when about 6–7 million germ cells are present in the ovaries (Baker 1963). Simultaneously, the number of germ cells starts to decline due to the increased rate of apoptosis (Vaskivuo et al. 2001). The reproductive life span of a is determined by the number of primordial follicles in the ovaries and the speed of oocyte demise, and the remaining population of primordial follicles, or ovarian reserve, serves as a resting pool of oocytes available during the female reproductive life span (Tilly & Sinclair 2013). Once the primordial follicle population is established, the follicle is destined to one of three fates: to remain a quiescent member of a follicle reserve for varying lengths of time, to directly, or, during development, undergo atresia (programmed cell via apoptosis), or to be recruited into the growing population of follicles and to mature. Until recently, the general conception has been that there is a finite number of oocytes in the ovary determined in fetal life. However, this concept has been challenged by observations that ovaries of adult mice posses oogonial stem cells (Zou et al. 2009, Pacchiarotti et al. 2010), that can generate fertilization-competent in vivo (Zou et al. 2009; 2011). Further studies have shown that similar cells exist in human ovaries at reproductive age (White et al. 2012). However, these results are challenged by Zhang and co-workers (2015), who propose that these cells are not functional germline stem cells.

2.1.1 FSH-independent follicle development

During the early stages of follicular development, or folliculogenesis, some of the resting primordial follicles are recruited from the resting pool into the growing follicle pool in a process called initial recruitment. This initial activation of primordial follicles occurs throughout life until . When follicles are recruited to the growing phase they first become primary follicles, the granulosa cells around the oocyte become cuboidal and the oocyte increases in size (Motta et al. 1994, Makabe et al. 2006) (Figure 1). Also, the transcription of numerous oocyte-specific genes is initiated during early stages of the primordial to primary follicle transition (Pangas & Rajkovic 2006). An extracellular matrix called the zona pellucida (ZP) is formed between the oocyte and granulosa cells at the primary–secondary follicle stage (Rankin et al. 1996, Makabe et al. 2006, Kierszenbaum 2015). Primary follicles are characterized by frequent ,

20 consequently resulting in an increase of follicular cell number around the oocyte, and several granulosa cell-layers are formed. A secondary follicle is formed when two or more granulosa cell layers surround the oocyte and a layer of somatic thecal cells forms around the granulosa cells. The theca layer can be further divided into theca interna and theca externa. Development up to secondary or pre-antral follicle stage occurs independently of gonadotrophic stimulation (Hirshfield 1985) and relies on paracrine and autocrine regulation by multiple ovarian growth factors (review, Oktem & Urman 2010, Adhikari & Liu 2009).

Fig. 1. Different stages of folliculogenesis. Modified from Edson et al. 2009 with permission from Endocrine Society.

The initial recruitment of primordial follicles is defined by growth of the oocyte to full size (Elvin & Matzuk 1998, Makabe et al. 2006), accompanied by proliferation and differentiation of the surrounding granulosa cells (Adhikari & Liu 2009). The process is well balanced and delicate, since at the same time as primordial follicle recruitment is initiated by factors that activate follicle development, there are factors needed to suppress follicular activation in dormant follicles. The growth and meiotic regulation of the oocyte is dependent on granulosa cells (Elvin & Matzuk 1998, McGee & Hsueh 2000, Vanderhyden 2002), but in turn, oocytes also promote granulosa cell proliferation, differentiation and function (Vanderhyden et al. 1992, Adhikari & Liu 2009). Communication between oocytes and somatic cells is maintained via at least two modes of intercellular communication, gap junctions and paracrine factors (Vanderhyden et al. 2002, Makabe et al. 2006). Throughout folliculogenesis oocyte-derived proteins of transforming growth factor beta (TGFβ) superfamily, such as bone morphogenetic protein 15 (MBP-15) (Elvin et al. 1999, Yan et al. 2001, Gilchrist et al. 2008, Persani et al. 2014) and growth differentiation

21 factor 9 (GDF-9) (McGrath et al. 1995, Hreinsson et al. 2002, Gilchrist et al. 2008, Persani et al. 2014) interact with surrounding somatic cells, which in turn produce their own paracrine factors such as Kit ligand and Kit tyrosine kinase receptor, activins, inhibins and transforming growth factor α (TGFα) and promote oocyte growth, granulosa cell proliferation and thecal cell differentiation (Laitinen et al. 1995, Schmidt et al. 2005, Carlsson et al. 2006, Dumesic et al. 2015, Tuck et al. 2015). Intra-ovarian and intra-oocyte inhibitory factors such as phosphatase and tensin homolog (PTEN), cyclin-dependent kinase inhibitor (p27), forkhead box O3A (Foxo3a), forkhead box L2 (foxl2) and antimüllerian hormone (AMH) stop primordial follicles from being activated and hence a pool of dormant primordial follicles is maintained (review, Adhikari & Liu 2009). Most likely, different combinations of stimulatory, survival and inhibitory local signals interplay to determine the fate of individual resting follicles (Gaytan et al. 2015). In the human ovary, more than 90 days are needed for a primary follicle to reach a secondary follicle stage and another 70 days are needed for development from secondary to early antral stage. After cyclic recruitment, it takes around 15 days for the antral follicle to become a dominant Graafian follicle (Gougeon 2010).

2.1.2 FSH-dependent follicle development

After the secondary stage the follicles become dependent on follicle-stimulating hormone (FSH), and small lacunae arise among granulosa cells, which become filled with a dense follicular fluid that is mainly secreted by granulosa cells (Motta et al. 1994, Makabe et al. 2006). The follicular fluid fills the small lacunae and intercellular spaces to create a single large cavity, the antrum folliculi. This separates granulosa cells into spatially and functionally distinct populations. The newly formed mural granulosa cells in multiple layers line the wall of the follicle and take part in steroidogenesis. Granulosa cells close to the oocyte, called cumulus cells, promote the growth and development of the oocyte. The innermost layers of surrounding cumulus cells are called corona radiata. At this point the oocyte is surrounded by a thick and dense ZP (Motta et al. 1994, Makabe et al. 2006). FSH and luteinizing hormone (LH) coordinate antral follicle development and ovulation. FSH acts via its receptor in the granulosa cell surface membrane to stimulate cell division and formation of glycosaminoglycans, which are essential components of antral fluid (Hillier 1991, Hennet & Combelles 2015).

22 2.1.3 Ovulation

Maturation of a large pre-antral follicle into a pre-ovulatory follicle is dependent upon FSH and LH stimulation. In each , the rise of intercyclic FSH levels recruits a cohort of intermediately mature follicles to enter the initial stages of pre-ovulatory development (Brown 1978, Schipper et al. 1998, Gougeon 2010). Usually only one follicle of the recruited follicles survives until ovulation, while the others become atretic (Gougeon 1982). After the LH surge and immediately before the cumulus-oocyte-complex is released in to the oviduct, meiosis I resumes and the nuclear membrane starts to disintegrate in a process called germinal vesicle breakdown (Matzuk et al. 2002). After the germinal vesicle breaks down the first polar body forms and meiosis is arrested in metaphase II and completed only after sperm penetration. LH-dependent stages of oocyte and cumulus cell maturation are dependent on paracrine signals, such as GDF-9 and BMP-15 (Russell & Robker 2007). Induction of epidermal growth factor (EGF)-like growth factors and activation of EGF receptor signalling are also integral for LH-induced ovulation (Panigone et al. 2008). After ovulation the mural granulosa cells and theca cells remain behind in the ovary and participate in the formation of corpus luteum, while cumulus cells are ovulated with oocytes (Shuezts & Dubin 1981, Gardner et al. 1996, Baerwald et al. 2005). Theca and mural granulosa cells become luteal cells, which are the principal cell type in the corpus luteum. The corpus luteum is responsible for estradiol and progesterone synthesis, to stimulate the uterus and maintain pregnancy.

2.1.4 Follicular apoptosis

Atresia is a coordinated process of follicle degeneration that acts via hormonally controlled apoptosis (Hsueh et al. 1994, Matsuda et al. 2012). During fetal ovarian development, mitotic divisions of germ cells give birth to approximately 6–7 × 106 oocytes (Baker 1971) (Figure 2). However, there is great variability in ovarian reserve (Faddy et al. 1992, Pelosi et al. 2015) as a result of i) markedly different numbers of primordial follicles that normally form during ovarian organogenesis (Erickson 2001) and ii), later on, to a balance of pro- and anti-apoptotic factors (Vaskivuo et al. 2001). The number of germ cells peaks at mid-pregnancy around the time of follicle formation. Shortly thereafter the number of oocytes decreases dramatically, so that at birth, only around 1 million oocytes are left. Atresia occurs at all stages of follicular development but early antral follicles are especially

23 susceptible to it since apoptosis can occur both in somatic and germ cells. Apoptosis occurs in three cell types: oocytes, granulosa cells and luteal cells. In fetal life most of the apoptosis takes place in the oocytes, and in adult life apoptosis mainly occurs in granulosa and luteal cells (Vaskivuo & Tapanainen 2003). Follicular apoptosis continues after birth and by the number of follicles has decreased to roughly 300 000. From these, only about 400 oocytes will be ovulated during a woman´s fertile period and only a few hundred or thousand remain during the last years before menopause (Richardson et al. 1987, Hansen et al. 2008), which occurs around the age of 51 years (Faddy et al. 1992, Hansen et al. 2008).

24 Fig. 2. Life cycle of germ cells. Germ cells first appear as a cluster of ∼100 cells, which migrate, proliferate and colonize the prospective gonads, increasing rapidly from 600 000 at 8 weeks of gestation to 6–7 million at 20 weeks of gestation. Thereafter their number decreases dramatically and at birth, only around 1 million germ cells are left. The number of germ cells decreases towards menopause, leading to only a few hundred or thousand remaining in the last few years before the menopause at around the age of 51. Adapted from Oktem & Urman 2010.

2.2 Zona pellucida

The zona pellucida (ZP) is an extracellular matrix, which forms a viscous border between the plasma membrane of the oocyte and the granulosa cells (Sinowatz et al. 2001). The thickness of the ZP in mature human oocyte is estimated to be approximately 16.18 ± 2μm using light microscopical inspection (Balakier et al. 2012) and it surrounds the oocyte, of around 120μm in diameter (Avella et al. 2013) (Figure 3).

25 Fig. 3. Zona pellucida shown in a fertilized (day one) oocyte. ZP = zona pellucida, O = oocyte, P = polar body, PVS = perivitelline space, PN = .

2.2.1 Ultrastructure of the zona pellucida

The ZP is characterized by its fibrous and porous structure. The filaments are highly ordered and hence have a birefringent structure (which retards the refractive index of polarized light). According to birefringence studies, human ZP can be divided into an inner (9.8 ± 2.1 μm) and an outer (6.1± 1.7μm) birefringent layer separated by a middle (3.7. ± 0.9μm) layer of minimal or negligible birefringence (Pelletier et al. 2004). The thicker and more birefringent inner layer is mainly responsible for the thickness and birefringence variation of the entire zona (Pelletier et al. 2004, Shen et al. 2005). In humans, ZP filaments are straight or curved and are 0.1–0.4 μm in length and 10–14 nm in thickness (Familiari et al. 1992). Human ZP has a different structure in the outer and inner layer, but the structure of the ZP also appears different during the course of folliculogenesis and fertilization. When

26 investigated via scanning electron microscopy (SEM), the outer surface of immature or atretic oocytes contains almost exclusively a tightly meshed network of filaments (Familiari et al. 2006). In turn, the outer surfaces of human ZP in both mature and fertilized oocytes consists of filaments arranged in a multi-layered network that appear compact in the meshes of the network and loose in the holes (Familiari et al. 2006). The pores at the outer surface of the ZP matrix appears wider than those of the inner surface. This spongy porous surface may facilitate sperm penetrability (Familiari et al. 1988; 1992). The inner surface the ZP of unfertilized oocytes is arranged in repetitive structures characterized by numerous short and straight filaments branching with each other, whereas after fertilization this surface is found to contain numerous areas where filaments are fused together to form closely packed structures (Familiari et al 1992; 2006). This condensation of filaments could be related to changes in the inner layer of the ZP during fertilization, such as the zona reaction induced by enzymes released by (Familiari et al. 1992).

2.2.2 Structural components of the zona pellucida

In mammals, ZP filaments are composed, depending on the species, of either three or four glycoproteins designated zona pellucida glycoprotein -1 (ZP1), -2 (ZP2), - 3 (ZP3) and -4 (ZP4) (Table 1).

Table 1. ZP proteins in different mammalian species.

ZP1, ZP2, ZP3 (ZP4)1 ZP2, ZP3, ZP4 (ZP1)1 ZP1, ZP2, ZP3, ZP4 Mouse Pig (Goudet et al. 2008) Human (Lefievre et al. 2004) (Bleil & Wassarman 1980b, Cow (Noguchi et al. 1994) Rat (Hoodbhoy et al. 2005) Goudet et al. 2008) Dog (Goudet et al. 2008) Hamster (Izquierdo-Rico et al. 2009) Bonnet monkey (Ganguly et al. 2008) Rabbit (Stetson et al. 2012) Cat (Stetson et al. 2015 1 pseudogene

ZP2 and ZP3 genes are common to all mammals studied so far, while ZP1 and ZP4 are, depending on the mammalian species, present either as active or as pseudogenes. A pseudogene is regarded as a gene that has evolved by generating a stop codon and/or insertion/deletion disrupting the reading frame and resulting in the loss of their protein-coding ability. It is interesting to note that different

27 organisms construct the ZP with various combinations of ZP proteins, which may have implications for species-specific fertilization (Claw & Swanson 2012). According to mouse studies, ZP1 is structurally a dimer composed of identical polypeptides, which are held together by intermolecular disulphide bonds, where as ZP2 and ZP3 appear as monomers (Wassarman & Litscher 2012).

2.2.3 Genes encoding human ZP proteins

Human ZP1, ZP2 and ZP3 show 32–93%, 57–96% and 67–95% identity in their nucleotide sequences compared with other mammals (McLeskey et al. 1998). It is believed that ZP proteins have evolved from a common ancestral gene via gene duplication (Spargo & Hope 2003) and pseudogenization (Claw & Swanson 2012). Goudet and co-workers (2008) determined phylogenetically that ZP1 and ZP4 are the most recent duplications to occur. ZP1 and ZP4 are paralogous and supposedly have arisen from duplication of a common ancestral gene (Goudet et al. 2008). In humans, ZP3 is not a single-copy gene – the chromosome region 7q11.23 where ZP3 is located contains a polymorphic locus that may give rise to a non-functional polypeptide, POM-ZP3 (van Duin et al. 1993). The 3’ end of the POM-ZP3 transcript is 99% identical to that of ZP3 and it appears to have arisen from duplication of the last four exons (exon 5–8) of ZP3 (Kipersztok et al. 1995). The locations, numbers of protein-coding exons and lengths of the human ZP genes are represented in Table 2.

Table 2. The locations, numbers of exons and lengths of human ZP genes.

Gene Chromosome No of exons Length (aa) References

ZP1 11 12 638 Hughes & Barratt 1999

ZP2 16 19 745 Liang & Dean 1993

ZP3 7 8 424 Chamberlin & Dean 1990

ZP4 1 12 540 Hughes & Barrat 1999, Lefievre et al. 2004

28 2.2.4 Regulation of ZP genes

According to mouse studies, ZP genes are expressed in coordination during the growth phase of (Epifano et al. 1995). As the ribonucleic acid (RNA) accumulation profiles of ZP1, ZP2 and ZP3 transcripts maintain the same 1:4:4 ratios throughout oocyte growth (Epifano et al. 1995), it suggests that ZP genes are regulated, in part, by identical transcription factors binding to the promoter regions of all three mouse ZP genes (Liang et al. 1997). When comparing the sequence data, ZP genes have been found to share TATA boxes, a type of promoter DNA 5´- TATAA-3´sequence or similar indicating where genetic code can be read and decoded, approximately 25–35 base pairs (bp) upstream of their transcription start sites. The 5´flanking regions of ZP2 and ZP3 in mice and humans also contain five conserved elements (I, IIA, IIB, III and IV), and the element IV, containing the motif CANNTG (DNA sequence where N can be any nucleotide), known as the E- box (Enhancer box), located approximately 200 bp downstream of the transcription start site, plays a critical role in directing the ZP expression in oocytes (Millar et al. 1991). E-box sequences act as protein-binding sites regulating gene expression. An oocyte-specific protein–DNA complex (also called zona pellucida gene activating protein-1 [ZAP-1]) has been found to bind specifically to element IV of the mouse ZP2 and ZP3 promoter regions, suggesting that it may serve as a transcription factor regulating coordinated ZP gene expression (Millar et al. 1991; 1993). The timing of the developmental appearance of this complex correlates approximately with the onset of transcription of mouse ZP2 at around the time of entry of the oocytes into the dictyate (resting) stage of oogenesis (at meiotic prophase I) (Millar et al. 1993). DNA binding activity similar to that of ZAP-1 is present in human ovarian extracts (Liang & Dean 1993, Millar et al. 1993). An oocyte-specific protein (OSP-1), binds the sequence 5´-TGATAA-3 within the first 100 bp of the promoter region of mouse ZP3 and is suggested to be a transcription factor regulating mouse ZP3 expression (Schickler et al. 1992). A transcription factor FIGLA is involved in the coordinated expression of ZP genes in mice (Liang et al. 1997, Soyal et al. 2000) and together with transcription factor encoded by the E2A gene (E12) it forms a heterodimer that binds to an E- box in the promoter regions of murine ZP genes (Liang et al. 1997). According to a study by Bayne and co-workers (2004), with human material, both FIGLA and E12 are required to form a complex on a ZP2 E-box, and the binding site requires an intact E-box. However, additional transcription factors are most likely also required for activation of the ZP genes. Since the ZP plays such an important role

29 in fertilization and early embryonic survival, it is likely that redundant strategies have evolved to ensure coordinated, oocyte-specific activation of the ZP genes (Liang et al. 1997).

2.2.5 Molecular characteristics of ZP proteins

A considerable amount of homology is observed between ZP proteins among the species. In humans, 20–47% identity is seen in amino acids between the proteins (Gupta et al. 2007). ZP1 and ZP4 share the highest identity (47%) and ZP2 and ZP3 the lowest (20%). Human ZP glycoproteins have a signal peptide at the N-terminus (Figure 4), which directs them into the secretory pathway (Gupta et al. 2007) and is cleaved off the mature protein. ZP glycoproteins have an approximately 260 amino acids- long ZP domain, defined by eight or ten (ZP3 and ZP1, ZP2 and ZP4, respectively) conserved cysteine residues. The ZP domain has been associated with the polymerization of the ZP proteins, formation of filaments and assembly of the ZP matrix (Bork & Sander 1992, Jovine et al. 2002). The ZP domain consists of an N- terminal subdomain and a C-terminal subdomain connected by a by a short linker. The linker region is suggested to be important for N-/C-terminal subdomain rearrangements during polymerization (Han et al. 2010). N-terminal subdomain is thought to constitute a basic building block of ZP filaments (Monne et al. 2008), and C-terminal subdomain may mediate interaction with other ZP proteins (Kanai et al. 2008). Additional copies of N-terminal subdomain are found within N- terminal extensions of ZP1, ZP2 and ZP4, which may play a role in species-specific gamete binding (Callebaut et al. 2007). Internal to the C-terminal subdomain, there is a hydrophobic peptide termed the internal hydrophobic patch and it is essential for incorporation of mouse ZP3 into the ZP (Jovine et al. 2004). ZP1 and ZP4 have a 42 aa-long cysteine rich trefoil/P domain prior to the ZP domain (Gupta et al. 2007), whose function is proposed to be involved in ZP assembly, maintaining its ultrastructure (McLeskey et al. 1998). In ZP3, a putative sperm-binding site is located downstream of the C-terminal subdomain in a region encoded by exon 7 (ZP3 subdomain) (Litscher et al. 2009).

30 Fig. 4. Schematic representation of mammalian ZP proteins. Red arrow: site of signal peptidase. The ZP domain consists of N-terminal (ZP-N) and C-terminal (ZP-C) subdomains connected by a linker region. ZP1, ZP2 and ZP4 contain additional copies of ZP-N in N-terminal extensions. ZP1 and ZP4 contain a trefoil domain prior to the ZP domain (circle marked T). Green box: internal hydrophobic patch (IHP). Box with stripes: subdomain unique to ZP3. Red box: consensus furin cleavage site (CFCS)/dibasic motif. Blue box: external hydrophobic patch (EHP). Black box: transmembrane domain (TMD). Green arrow: site cleaved by ovastacin, a cortical granule enzyme released after fertilization. Adapted from Yonesawa 2014.

A consensus furin cleavage site is located downstream of the ZP domain, and is believed to separate the mature protein from a C-terminal propeptide and to release the ZP proteins into the extracellular space (Jovine et al. 2007). However, consensus furin cleavage site is not present in human ZP4 (Zhao et al. 2003), but instead a dibasic motif is located in the same region of all mammalian ZP proteins, suggesting that alternative cleavage site(s) beside consensus furin cleavage site are available (Zhao et al. 2003). The presence of consensus furin cleavage site/other cleavage sites upstream of the transmembrane domain suggests that precursors of ZP are translated as transmembrane proteins, which then become secretory proteins, following cleavage around the consensus furin cleavage site (Yonezawa 2014). The C-terminal propeptide consists of a hydrophobic peptide termed the external hydrophobic patch, a transmembrane domain and a short cytoplasmic tail (Monne & Jovine 2011). The external hydrophobic patch, external to the ZP domain, is paired with the internal hydrophobic patch and is essential for incorporating ZP3

31 into the ZP (Jovine et al. 2004). ZP proteins must contain external hydrophobic patch (but not necessarily transmembrane domain) to be secreted and both external hydrophobic patch and transmembrane domain must be cut off in order to incorporate the nascent ZP proteins into the ZP (Jovine et al. 2004). In addition, internal and external hydrophobic patches are proposed to interact during transportation, ensuring an inactive conformation of individual zona proteins, which prevents intracellular polymerization (Jovine et al. 2004, Han et al. 2010). It is further suggested that external hydrophobic patch blocks premature protein polymerization during intracellular trafficking by acting as a “molecular glue” that keeps the ZP module in a conformation that is essential for secretion but not compatible with formation of ZP matrix (Han et al. 2010). Transmembrane domain is believed to anchor the glycoproteins in secretory vesicles and the plasma membrane (Jovine et al. 2007). The short cytoplasmic tail may be involved in preventing intracellular interactions among ZP precursor proteins when trafficking through the cell to the plasma membrane, and after secretion, it is needed for incorporation of nascent proteins into the ZP (Jimenez-Movilla & Dean 2011). Human ZP glycoproteins are highly and heterogeneously glycosylated and the carbohydrate content of ZP proteins is estimated to represent 15–54% of the mass of the proteins (Yonezawa 2014). Human ZP has exposed mannosyl, N- acetylglucosaminyl and beta-galactosyl residues (Jimenez-Movilla et al. 2004, Gupta 2015). It has been suggested that human ZP2, ZP3 and ZP4 might have predominantly N-linked glycosylation (Chiu et al. 2008b). A sialyl-Lewisx sequence present in N- and O-linked glycans of human ZP has been shown to play an important role in sperm–egg binding (Pang et al. 2011).

2.2.6 Cell types responsible for ZP protein synthesis

The location of ZP protein synthesis in mammals has been controversial for a long time. However, it seems likely that both oocyte and granulosa cells contribute to the production of ZP proteins (Lee & Dunbar 1993, Sinowatz et al. 1995; 2001, Kölle et al. 1996, Grootenhuis et al. 1996, Lee 2000, Bogner et al. 2004, Xie et al. 2010). In humans, immunohistochemical studies have revealed that ZP1–ZP3 proteins are expressed within adult human primordial follicles both in oocytes and granulosa cells, and that their expression increases with development (Gook et al. 2008).

32 2.2.7 Assembly of ZP proteins into zona matrix

Jimenez-Movilla and Dean (2011) have hypothesized that a putative transmembrane protease recognizes the intracellular cytoplasmic tails of the ZP glycoproteins at the plasma membrane of the oocyte and releases the extracellular domain (ectodomain) from the C-terminal propeptide around the area of the consensus furin cleavage site. They further postulated that as CTs of both ZP2 and ZP3 differ from each other, this would imply that cytoplasmic tails could account for the recognition of ZP2 and ZP3 at the plasma membrane to ensure correct stoichiometry of the ZP. Membrane-anchoring of ZP proteins may play an important role in ZP assembly by orienting the ZP3 precursor in such a manner that it can properly interact with other subunits upon cleavage (Han et al. 2010). Cleavage alters the conformation of the ectodomains, permitting oligomerization of the ZP proteins and formation of the ZP (Jimenez-Movilla & Dean 2011). The conformational change is believed to be due to cleavage of external hydrophobic patch, which leaves the ectodomain retaining only internal hydrophobic patch, which in turn could then interact with other zona ectodomains and form the ZP (Jovine et al. 2004). In mice, the filamentous structure of the zona matrix is formed by multiple ZP2/ZP3 heterodimers that are joined together in a head-to-tail fashion (Bleil & Wassarman 1980b, Greve & Wassarman 1985, Wassarman & Mortillo 1991). Homodimers of ZP1 (Bleil & Wassarman 1980b, Greve & Wassarman 1985) stabilize this structure by cross-linking ZP2/ZP3 multimeres together to create a 3D matrix. In humans, ZP filaments are believed to be composed of ZP2, ZP3 and ZP4, which are then linked together with ZP1 (Familiari et al. 2006, Huang et al. 2014). The correct architecture of the ZP relies greatly on correct stoichiometry of secreted mature ZP proteins. Mouse ZP2 and ZP3 seem to be present in the ZP in equimolar amounts and they are both significantly more abundant than ZP1 (ratio of 1:4:4) (Epifano et al. 1995). In humans, ZP4 levels are equivalent to those of ZP2 and ZP3, whereas ZP1 is a minor component (Lefievre et al. 2004). The secreted nascent ZP proteins are believed to be deposited in the innermost layer of the ZP, and more precisely, it is hypothesized that they can only be incorporated into growing ends of the ZP filaments (Qi et al. 2002). Formation of ZP matrix is developmentally regulated, since it occurs during distinct stages of oogenesis (Dunbar & O´Rand 2013). ZP matrix is not present in

33 the resting primordial follicles, but begins to appear in growing oocytes. However, human ZP3 mRNA (Huntriss et al. 2002) and protein (Gook et al. 2008) can already be detected at primordial follicle stage. Based on the ultra structural studies of ZP in mammals, the ZP is not formed as a complete and continuous extracellular membrane around the oocyte. Instead, it seems to appear as discrete and discontinuous portions, which later fuse together (Chiquoine 1960). The first traces of growing ZP are seen under the microscope when amorphous intercellular material is gathered in small intercellular clefts between the oocyte and follicle cells at the primary follicle stage (Chiquoine 1960).

2.2.8 ZP1–ZP3 mutant mice

Knockout mouse technology has provided an interesting perspective to study the significance of each ZP protein in ZP assembly. Mutant mice without either active ZP1, ZP2 or ZP3 genes (ZP1–3 null mice) were produced using homologous recombination and insertional mutagenesis in embryonic stem cells (Rankin et al. 1996; 1999; 2001, Wassarman et al. 1997). Mice homozygous for an insertional mutation in the ZP3 (mZP3-/-) (ZP3 null) produced zonaless oocytes despite of the synthesis of ZP1 and ZP2, and were infertile (Rankin et al. 1996). Although oocyte growth and follicle development proceeded in ZP3-/- females, there seemed to be fewer antral follicles present than in wild-type females. In addition, the extent of follicle cell–oocyte interaction was severely disturbed. Interestingly, mice with a single ZP3 allele (ZP+/-) synthesized less ZP3 proteins than wild-type animals, resulting in a thinner ZP with reduced levels of both ZP3 and ZP2. Despite this, these mice seemed to be as fertile as their wild-type counterparts (Wassarman et al. 1997). In ZP2-deficient mice, a very thin ZP has been observed in the follicles until antral stage, but no ZP was observed later in folliculogenesis or in ovulated eggs (Rankin et al. 2001). The abnormal ZP did not affect initial folliculogenesis, but there was a significant decrease in the number of antral-stage follicles compared with wild-type mice (Rankin et al. 2001). The absence of a ZP seemed to preclude the formation of a normal oophorus-cumulus complex and very few eggs were present in the oviducts of null females, and the respective mice were infertile (Rankin et al. 2001, Wassarman & Litscher 2012). Mice lacking ZP1 produced a somewhat thinner ZP consisting of ZP2 and ZP3 in roughly equimolar amounts (Rankin et al. 1999). However, the matrix was

34 structurally flawed (loosely organized and distorted) and about 10% of the growing follicles had anomalies, such as granulosa cells in the perivitelline space. Wassarman and Litscher (2012) speculated that the presence of mouse ZP2 and ZP3 might support formation of heterodimers, which, in turn, would allow assembly into long fibrils. When ZP1 is absent, the fibrils are insufficiently packed together creating an unusually porous matrix, permitting even follicular cells to enter the perivitelline space (Wassarman & Litscher 2012). ZP surrounding oocytes from ZP1-/- females was fragile when compared with the ZP of wild-type oocytes. The poorly organized ZP permitted fertilization, but ZP1-null females had decreased fertility because of precocious hatching of early embryos from the structurally compromised zona matrix (Rankin et al. 1999). Taken together, these studies suggest that two ZP glycoproteins are sufficient to produce a ZP matrix if one of them is ZP3. Functionally, both nascent mouse ZP2 and ZP3 must be present for proper assembly of the ZP around the oocyte in order to support normal fertilization (Wassarman & Litscher 2012). In humans, a frame shift mutation causing a premature stop codon in the zona domain region of ZP1 results in a truncated ZP1 protein and subsequently in oocytes completely lacking a ZP (Huang et al. 2014). The authors hypothesize that retention of the first half of the zona domain and simultaneous absence of both cytoplasmic tail and transmembrane domain in this particular mutant ZP1 not only promotes interaction with other ZP proteins but also leads to sequestration of the other ZP proteins. This leads to imbedding the traffic through the oocyte, thus preventing the formation of the ZP and finally resulting in sterility.

2.3 Functional characteristics of the zona pellucida

Unlike the egg coats found in lower species, mammalian ZP is generally less flexible and more difficult to penetrate, enabling it to serve as a substantial physical barrier for the protection of the oocyte (Clark 2010). During oocyte maturation and folliculogenesis (Zhao & Dean 2002), the ZP maintains oocyte–granulosa cell interactions (Eppig 1991) via intercellular communications to facilitate oocyte growth (Zhao & Dean 2002). After ovulation, ZP allows oocyte a free passage through the oviduct and prevents its early aggregation and implantation on the tubal wall (Modlinski 1970). Crucial functions of the ZP are the provision of species- specificity at fertilization, induction of the in zona-bound spermatozoa and post-fertilization blockage of polyspermy (Yanagimachi 1994,

35 Gupta 2015). In early cleavage-stage embryos the ZP has been suggested to have a shaping function, as it holds dividing closely together (Edwards 1964) and maintains the integrity of the inner cell mass (Trounson & Moore 1974). This ensures maximum contact between blastomeres, which, in turn, is a prerequisite for compaction (Dunbar 1989). The ZP is also a key barrier protecting the early embryo against microorganisms, viruses and immune cells as it passes down the oviduct (Loret de Mola et al. 1997).

2.3.1 Role of the ZP in folliculogenesis

During folliculogenesis, expression of ZP proteins is needed to provide extracellular matrix for granulosa cell attachment (Rankin et al. 1996). The authors postulated that in mice the lack of ZP3 glycoprotein synthesis has deleterious effects on folliculogenesis, as corona radiata cells of such mice were consistently non-uniformly arranged. In addition, the cumulus oophorus was poorly organized, when compared with that of wild-type animals. A similar phenomenon was also observed in mice lacking ZP2 (Rankin et al. 2001). Since ZP is a highly viscous extracellular matrix, it possibly also serves to stabilize gap junctions that form between the oocyte and the corona radiata cells (Wassarman & Litscher 2012). These intercellular connections through the ZP are believed to be crucial for transporting small molecules, which facilitates oocyte growth and maintains it in meiotic arrest (Zhao & Dean 2002). Interestingly, when folliculogenesis of mice lacking functional ZP1, ZP2 or ZP3 was studied (Rankin et al. 2001), it turned out that these mice had normal amounts of primary and secondary follicles but a reduced amount of antral follicles. This clearly suggests that disruption of the ZP complicates the ability of follicles to continue their development to antral stage. It remained unclear, however, whether the observed follicular loss of antral follicles was a direct effect of an abnormal (ZP1-null) or missing (ZP2- and ZP3-null) ZP, or an indirect effect supposedly mediated by altered somatic-germ cell interactions (Rankin et al. 2001).

2.3.2 Acrosome reaction, gamete recognition and binding

The general concept is that sperm must be acrosome-intact when interacting with the ZP, as sperm that have undergone an AR before contact with the ZP are not able to fertilize the egg/oocyte (Bleil & Wassarman 1983, Saling et al. 1979). Indeed,

36 acrosome-reacted sperm have lost the anterior plasma membrane, which, in turn, carries molecules needed for interacting with ZP proteins (Fraser 2010). It has also been thought that the AR occurs at the surface of the ZP (Jin et al. 2011), as ZP1, ZP3 and ZP4 have been found to bind to capacitated acrosome-intact human spermatozoa and induce an acrosome reaction (Caballero–Campo et al. 2006, Chakravarty et al. 2008, Chiu et al. 2008a, b; Ganguly et al. 2010a, b). However, studies now suggest that cumulus cells might be mainly responsible for inducing the acrosome reaction in vivo (Jin et al. 2011) and most sperm reaching the ZP are in fact already acrosome-reacted (Sun et al. 2011). In addition, Inoue and co- workers (2011) have shown that acrosome-reacted sperm are able to penetrate the ZP and subsequently fertilize the egg. Studies with mice (Bleil & Wassarman 1980a, Bleil & Wassarman 1983, Bleil et al. 1988, Greve and Wasserman 1985) have suggested that ZP3 functions as a primary sperm receptor responsible for binding to capacitated spermatozoa and subsequent induction of the acrosome reaction. In turn, ZP2 has been regarded as a secondary sperm receptor that mediates the binding of acrosome-reacted spermatozoa to the ZP. The role of ZP1 in sperm binding does not seem obvious, but it is believed to play a structural role in maintaining the filamentous structure of the zona matrix by covalently cross-linking these filaments together. In humans, all ZP proteins except ZP2 bind to capacitated acrosome-intact human spermatozoa and induce an acrosome reaction (for review see, for example Gupta et al. 2012) (Table 3). It appears that ZP2 is the zona ligand to which human sperm binds, since human sperm bind to humanized mouse ZP with human ZP2, but not with human ZP1, ZP3 or ZP4 (Yauger et al. 2011, Baibakov et al. 2012). At the molecular level, it is assumed that sperm attach to an N-terminal domain (aa 51–149) of ZP2 prior to penetration and gamete fusion (Baibakov et al. 2012, Avella et al. 2014).

37 Table 3. Human ZP proteins in sperm binding and inducing acrosome reaction (AR).

ZP Binds to Effect Site of interest References protein ZP1 Capacitated sperm, acrosome- AR ZP domain Ganguly et al. 2010a, b intact sperm: mainly acrosomal cap, but also equatorial segment

ZP2 Acrosome-reacted sperm: Sperm N-terminal domain, Chakravarty et al. 2008, Chiu equatorial segment, acrosomal binding aa residues 51–149 et al. 2008b, Avella et al. cap, mid-piece 2014, Baibakov et al. 2012

ZP3 Capacitated, acrosome-intact AR, C-terminal domain, Chakravarty et al. 2008, Chiu sperm: mainly equatorial region Sperm N-linked et al. 2008a, b, but also acrosomal cap and mid- binding? glycosylation Caballero-Campo et al. piece 2006, Bansal et al. 2009

ZP4 Capacitated, acrosome-intact AR N-linked Chakravarty et al. 2008, sperm: acrosomal cap, equatorial glycosylation Caballero-Campo et al. region 2006, Chiu et al. 2008a,b

More specifically, recognition between sperm and ZP is suggested to be organized as a complex plug: different sperm proteins associated in a large complex recognize sperm binding moieties on ZP glycoproteins (Petit et al. 2014). Such a complex could prevent interspecies breeding, but absence of one to these proteins could not definitely halt recognition (Petit et al. 2014). At a molecular level, multiple ligands on spermatozoa such as β-1-4- galactosyltranseferase, ZP glycoprotein 3 receptor, zonadhesin, SED1 and disintegrin and metalloprotease 3 (ADAM3) are proposed as potential candidates for mediating the actual binding of the two (review by Gupta 2014, Avella et al. 2013). According to the literature, there seems to be some controversy as regards how gamete binding actually occurs at a molecular level. According to the `glycan model´, initial species-restricted binding between mammalian gametes is believed to be mediated by distinct O-glycans present in ZP3 protein (Florman & Wassarman 1985), which are deglycosylated after fertilization to prevent further sperm binding. This sperm-binding region is found in the C-terminal region of the mature ZP3 (Bansal et al. 2009), which is encoded by exon 7 of the mouse ZP3 gene (Wassarman & Litcher 2008, Kinloch et al. 1995). Chen and co-workers (1998) reported that O-glycans at two distinct serine residues (i.e. Ser332 and Ser334) of murine ZP3 were essential for mediating mouse sperm–ZP binding. However,

38 results obtained by Boja and co-workers (2003) indicate that these two serine residues are not occupied by O-linked oligosaccharide side chains. The `supramolecular complex model´ suggests that binding of the two gametes does not solely rely on carbohydrate moieties, but instead is best described by a distinct supramolecular structure of the ZP (Rankin et al. 2003). Cleavage of the ZP2 after fertilization is supposed to render the supramolecular complex non-permissive for subsequent sperm binding (Rankin et al. 2003). The third model, known as the `hybrid model´ combines some key elements from both these models above, suggesting that sperm binds to a different O-linked site (i.e. other than Ser332 and Ser334) (Visconti & Florman 2010). Access to this glycan moiety is dependent on the proteolytic cleavage state of ZP2. A putative alternative for such an O-linked glycan moiety might be Thr155, as its surrounding residues are highly conserved in ZP3 (Visconti & Florman 2010). The `domain-specific model´ suggests that besides carbohydrate moieties, the protein backbone of ZP glycoproteins is also responsible for constituting the required three-dimensional sperm-binding site (Clark & Dell 2006, Clark 2010). This site is proposed to be located at the C- terminal region of mouse ZP3 (Clark 2011). Here, due to variation in glycosylation between different ZP3 proteins of a single oocyte, some of these molecules carry glycans that sterically hinder access to peptide sequences, and binding is proposed to be mediated via lectin-like interaction. In other ZP3 molecules, glycosylation sites are unoccupied, and the peptide sequences that mediate sperm binding will be accessible, and protein–protein interactions predominate (Clark 2011). What is clear from these models is that sperm most likely engage in multiple binding events with a variety of ligands in the ZP (Redgrove et al. 2012).

2.3.3 Species specificity

Sperm–egg interaction is a highly species-specific event. However, a considerable amount of interspecies cross-reactivity may exist in mammalian sperm-egg interaction (Bedford 1977). While mouse sperm are promiscuous in their egg recognition (Bedford 1977), human spermatozoa display a high degree of specificity as they bind only to oocytes of higher primates (Homo sapiens, Gorilla gorilla and Hylobates lar (gibbon)) (Bedford 1977, Lanzendorf et al. 1992). However, when the ZP is completely removed, human spermatozoa are able to penetrate into mature ova of species as diverse as the hamster (Yanagimachi et al. 1976).

39 The ZP has been believed to be the key structure maintaining species specificity at the time of fertilization. In search of the ZP protein(s) responsible for taxon specificity at fertilization, Rankin and co-workers (1998, 2003) showed that when murine ZP2 and ZP3 were replaced with human equivalents, murine sperm was able to bind these `humanized´ oocytes, while human sperm was not. A similar phenomenon was observed when human ZP4 was expressed in transgenic mice (Yauger et al. 2011), suggesting that neither human ZP4 nor human ZP2/ZP3 are sufficient for taxon-specific sperm recognition in humans. Later on, however, Baibakov and co-workers (2012) showed that human spermatozoa indeed bind to human ZP2 rescue and human ZP2 transgenic mouse eggs and not to corresponding ZP1, ZP3 or ZP4 eggs, and that gamete recognition was located at the N-terminus of ZP2. Alternatively, it has been hypothesised that taxon-specific sperm–egg binding may rely on taxon-specific glycosylation of ZP proteins (Primakoff & Myles 2002, Talbot et al. 2003). This hypothesis is supported by the findings that human ZP possesses a unique carbohydrate composition that is quite different that of other mammalian species (Jimenez-Movilla et al. 2004). It has been found out that the region immediately downstream of ZP domain at the C-terminus of ZP3, possesses a high level of sequence divergence between mammals when compared with the rest of the ZP3 glycoprotein (Wassarman & Litscher 1995). This divergence is suggested to be a result of rapid evolution in this region, indicating that this region is under positive Darwinian selection (Swanson et al. 2001). Since this region has also been suggested to be the primary binding site for the (Rosiere & Wassarman 1992, Kinloch et al. 1995, Li et al. 2007) and induction of the acrosome reaction (Bansal et al. 2009), it has been proposed that sequence divergence in this region may result in species-specificity of sperm–ZP binding (Williams et al. 2003; 2006).

2.3.4 Sperm–egg fusion

After penetrating the ZP, gamete fusion continues with merging of the sperm plasma membrane overlying the equatorial segment with the area of egg plasma membrane covered in microvilli (Bedford et al. 1979, McLeskey et al. 1998). Targeted deletion studies have revealed four proteins, CD9 and Juno on oocytes and Izumo1 and Spaca6 on spermatozoa, to be necessary in sperm–egg fusion (Inoue et al. 2013, Bianchi et al. 2014). In mice lacking CD9 or Juno and Izumo1

40 or Spaca6, membrane fusion is almost completely (CD9-deficient mice) or completely (Juno- or Izumo1- or Spaca6-deficient mice) impaired (Kaji et al. 2000, Le Naour et al. 2000, Miyado et al. 2000, Inoue et al. 2005, Bianchi et al. 2014, Lorenzetti et al. 2014). It has been further shown that Juno on the egg plasma membrane (Bianchi et al. 2014) is the receptor for Izumo1 located at the surface of acrosome-reacted sperm (Inoue et al. 2005). To date, this is the only known cell receptor pair essential for gamete recognition identified (Bianchi et al. 2014).

2.3.5 Block of polyspermy

Different organisms have evolved distinct mechanisms to prevent polyspermy. In mammals, the female seems to have been designed for stringent sperm selection, as only a few hundred sperm out of hundreds of millions released actually reach the egg (Bianchi & Wright 2014). Accordingly, multiple extracellular layers around mammalian eggs may have evolved as an egg’s defence strategy against polyspermy (Claw & Swanson 2012). In addition, oviductal fluid seems to cause pre-fertilization modifications in ZP (Kolbe & Holtzt 2005, Coy et al. 2008a, b), such as binding of the oviduct-specific glycoprotein OVGP1 (Coy et al. 2008a). This renders the ZP more resistant to protease digestion and sperm penetration (Coy & Aviles 2010). Block of polyspermy has been found to occur primarily at two levels in the mammalian egg: the egg plasma membrane and the ZP. The mechanisms of plasma membrane block are largely unknown, while ZP block or `zona reaction´ involving cortical granules is extensively documented (Gardner & Evans 2006). After fertilization, cortical granules, i.e. regulatory secretory organelles located at the cortex of an unfertilized oocyte, migrate to the plasma membrane of the oocyte and are then subjected to exocytosis. Their contents are released into the perivitelline space in an event known as a `cortical reaction´, which is believed to remove ZP carbohydrates involved in sperm–ZP binding (Miller et al. 1993) and cleave ZP glycoproteins, finally causing `zona hardening´ (Coy & Aviles 2010). Based on mouse studies, two different models have emerged to explain the ZP block of polyspermy. According to the `ZP2 cleavage model´, ovastacin, an oocyte- specific metalloendoprotease released from cortical granules (Quesada et al. 2004), cleaves ZP2 at the N-terminal domain (Gahlay et al. 2010, Burgart et al. 2012) and renders the ZP non-permissive to sperm binding (Burkart et al. 2012). After this initial cleavage, a further cleavage of ZP2 occurs at the N-terminal domain

41 destroying the sperm-binding domain (Baibakov et al. 2012, Burkart et al. 2012). These studies suggest that uncleaved ZP2 maintains the zona matrix in a state, which is permissive to sperm, as cleavage of ZP2 alters the matrix so that it blocks sperm binding (Gahlay et al. 2010). The second model, the `ZP3 glycan-release model´, suggests that release of glycosidase after cortical granule exocytosis leads to removal of O-glycans from ZP3 at locations Ser332 and Ser334, and this would make sperm unable to bind to the ZP after fertilization (Avella et al. 2013). However, this is in contrast to findings by Boja and co-workers (2003), who reported that both the serine residues are unoccupied in mouse ZP3 (Boja et al. 2003). Interestingly, the results of a recent study by Bianchi and co-workers (2014) suggest an entirely new mechanism for block of polyspermy. They found that Juno, a sperm receptor located at the egg plasma membrane, becomes undetectable there within 30–40 min after fertilization. This is interesting, since it is the time-frame in which the plasma membrane block to polyspermy is established (Gardner & Evans 2006). Juno was shown to disappear from the plasma membrane and become redistributed within vesicles in the perivitelline space, and the authors hypothesize that these Juno containing vesicles may bind and rapidly neutralise subsequent incoming sperm, thereby reducing the possibility of polyspermy (Bianchi et al. 2014).

2.3.6 In vivo hatching prior to implantation

For nearly a decade, time-lapse imaging has allowed embryologists to follow the development of early embryos. This fascinating technique has revealed that the human , like those in other mammals, undergoes a series of expansions and contractions before hatching, i.e. lysis of the ZP. When the diameter of the blastocyst increases, ZP thickness decreases dramatically and becomes almost invisible. It is feasible to believe that this thinning requires distinct structural properties of the ZP. Simultaneous action of lysins proteases (e.g. trypsin), which are synthesized by the blastocyst and/or the uterus (Gordon & Dapunt 1993, Schiewe et al. 1995, Hammadeh et al. 2011), finally causes rupture of the ZP in such a manner that hatching of the blastocyst is possible. The embryo flows into the uterine cavity, where it implants into the endometrium (Kutlu et al. 2010). Lack of hatching could lead to implantation failure (De Vos & Steirteghem 2000).

42 Interestingly, a specific arginine residue (at aa location R167) in ZP3 has been identified as a target cleavage site of hatching enzymes in fish (medaka, Oryzias latipes) (Yasumasu et al. 2010). This residue has also been found in chick ZP3 (at location R142), which is important for homodimeric arrangements in the ZP domain required for secretion (Han et al. 2010), suggesting that enzymes responsible for hatching could solubilize egg-coat filaments by disrupting the stability of ZP dimers through this residue (Han et al. 2010). As this cleavage site R↓T(R130) is conserved in human ZP3 as well, a similar mechanism may be involved in human embryo hatching and implantation (Han et al. 2010).

2.4 Abnormal ZP structures

The development of assisted reproduction technologies (ART) has helped in revealing detailed structural features of the human oocyte, including those of the ZP. It should be emphasized, however, that overall oocyte quality is regarded as the key limiting factor in female fertility, reflecting its intrinsic developmental potential (e.g. Gilchrist et al. 2008). Focusing on human ZP, irregularities in shape, thickness or composition may impair its optimal function and lead to reduced sperm binding and fertility (Ebner et al. 2008). However, ZP dysmorphisms do not necessarily predict an unsuccessful pregnancy outcome. According to a case report by Paz and co-workers (2004), oocytes with ZPs of irregular thickness and abnormal shape can be associated with successful outcome if ICSI is used. Similarly, Esfandiari (2005) reported of a successful pregnancy after ICSI, using oocytes with severe ZP (thick irregular zona, `cucumber shaped´ oocyte) and cytoplasm abnormalities. ZP thickness is not constant, as it increases progressively during follicle maturation but becomes thinner prior to hatching (Dirnfeld et al. 2003, Pelletier et al. 2004). It has been noted that zona thickness and zona thickness variation progressively decreases with a woman´s age as well (Gabrielsen et al. 2000, Sun et al. 2005, Valeri et al. 2011). Interestingly, the results of previous studies have suggested that zona thickness variation might be beneficial as regards IVF , presumably because of increased competency to hatch prior to implantation (Palmstierna et al. 1998, Sun et al. 2005). Furthermore, Bertrand and co-workers (1995) have observed that oocytes with thinner ZPs are associated with higher fertilization rates.

43 In contrast, a thick ZP (≥22 μm) has been associated with poor fertilization in IVF (Bertrand et al. 1995). One of the known key reasons for this is smoking, which has been found to increase ZP thickness of oocytes and embryos (Shiloh et al. 2004). Theoretically, a thick ZP may have a negative impact on blastocyst expansion, which presumably has an effect on pregnancy outcome. There is no clear evidence in the literature, however, to support this theory unequivocally. Despite this, assisted hatching (AH) has been developed as a supplementary technique to support both IVF and ICSI treatments, and is used by some IVF clinics worldwide. This technique is assumed to improve implantation rates among patients with poor prognosis or those having embryos with a thick ZP. Assisted hatching involves either thinning or making a hole in the zona matrix using either weak acid, laser technology or a glass micro-needle. Anomalies such as an oval or irregular ZP, a dark ZP or an enlarged perivitelline space have been associated with diminished pregnancy and implantation rates in IVF (Sauerbrun-Cutler et al. 2015). Indeed, a spherical shape is believed to ensure an ideal milieu to obtain maximal contacts between blastomeres, which is prerequisite for differentiation of the inner cell mass and its further development into the . Remaining blastomeres surrounding the inner cell mass will give rise to trophoblast and later on, the formation of a major proportion of the placenta. It is reasonable to believe that embryos derived from ovoid oocytes may have a reduced ability to express optimal cell associations (Sauerbrun-Cutler et al. 2015). In addition, an ovoid ZP may favour generation of an atypical cleavage pattern, which may result in delayed compaction and blastocyst formation (Ebner et al. 2008). Similarly, oocytes with either a large perivitelline space (Rienzi et al. 2008) or a dark zona (Shi et al. 2014) have been associated with a decreased fertilization rate, a low rate of good-quality embryos and adverse pregnancy outcome in IVF/ICSI. Zona splitting has been associated with non-conception in ICSI (Shen et al. 2005) and it is likely that zona splitting and perivitelline space size influence the maintenance of normal preimplantation development (Ebner et al. 2008). Zona splitting may be caused by temporarily interrupted patterning or secretion of ZP proteins during the formation of the zona matrix or by rupturing caused by mechanical stress at retrieval or separation from cumulus cells (Shen et al. 2005). Studies on knockout mice have suggested that the origins of ZP thickness and abnormalities may also be genetic, involving mutation(s) in the genes encoding ZP glycoproteins (Rankin 1999; 2001). To date, there are only a few studies in the

44 literature concerning a putative link between distinct genetic factors and human zona anomalies. Margalit and co-workers (2012) found eight sequence variations in ZP1–ZP4 among women with abnormally shaped ZPs. However, none of them could explain the observed dysmorphisms. Despite of their ZP abnormalities, the oocytes of these patients showed normal ability to bind sperm, suggesting that infertility in these women may be explained by factors other than those in sperm– oocyte interaction or in early developmental processes (Margalit et al. 2012). In another study, performed by Ferre and co-workers (2014), ZP1–ZP4 genes were studied among women with oocyte lysis and fragile ZPs. They found five sequence variations, but none of them seemed to be associated with oocyte lysis. They postulated that this apparently rare phenomenon was a result of some other factors, perhaps ovarian stimulation used in IVF (Ferre et al. 2014).

2.5 Infertility

Increased age of the female partner is one of the most common explanations for female infertility today. In Finland the average age of a woman giving birth to her first was 28.6 years in 2014 (http://www.stat.fi/til/sysyvasy/index.html) and women aged 35–39 years received the majority of IVF and ICSI treatments (36,7%) in 2013 (https://www.thl.fi/fi/tilastot/), which corresponds well with the latest statistics from other Nordic countries and Europe in general (Kupka et al. 2014). Oocyte aging is one of the most important factors involved in the failure of fertilization in ART. Aged oocytes are characterized by a thinner ZP and reduced total and cytoplasmic diameters (Valeri et al. 2011). In ICSI the role of ZP proteins in sperm binding, the ZP-induced acrosome reaction and penetration through the ZP are bypassed. Despite development in ART and significant advances in reproductive biology and medicine over the years, the cause of infertility still remains unexplained for 10–20% of patients. In these cases, infertility may be connected to genetic factors such as mutations in the genes encoding ZP proteins (Huang et al. 2014).

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46 3 Aims of the study

The structure and functions of the ZP have been studied extensively over the past few decades. For ethical reasons and because of limited access to human oocytes, most of these studies have been carried out by using animal models. Studies on human ZP and human fertilization are essential for understanding infertility and the mechanisms underlying it. Owing to the vital role of the ZP in fertilization, the detailed aims of the study were:

1. To explore whether putative sequence variations in genes encoding human ZP glycoproteins (ZP1–ZP4) could explain unsuccessful IVF treatment in a subset of infertile women suffering from total fertilization failure. 2. To study the expression and localization of ZP1 mRNA and ZP3 mRNA/protein and their transcription factor FIGLA mRNA during folliculogenesis in fetal and adult human ovaries. 3. To investigate whether some of the most frequent abnormalities in ZPs encountered in routine IVF/ICSI could be explained by sequence variations in genes encoding human ZP glycoproteins.

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48 4 Subjects and Methods

4.1 Subjects and tissue samples

All the studies were evaluated and approved by the Ethics Committee of Northern Ostrobothnia Hospital District. An informed consent document was obtained from all participants. A permit to study human autopsy tissue was obtained from the National Authority for Medico-legal Affairs.

4.1.1 Patients with total fertilization failure (TFF) (Study I)

Eighteen Finnish infertile couples from Oulu University Hospital and the Family Federation of Finland (Oulu, Helsinki and Turku Clinics) were studied. After routine IVF, none of their oocytes were fertilized after overnight incubation (following ), but total fertilization failure was avoided with these patients when ICSI was performed in following cycles. A figure of four or more oocytes produced in a single IVF cycle was set as an inclusion criterion to avoid fertilization failure by chance. The age of the women in this group varied between 25 and 36 years (mean 30 years). All the men revealed normal sperm parameters (Kruger strict criteria; Kruger et al. 1986; 1988) either in their native sperm samples and/or after standard sperm washing. Two distinct control groups were set for the study. `Fertilizers in IVF´ (i.e. the FIVF group) had to have had at least one fertilized oocyte in IVF (the mean proportion of fertilized oocytes in IVF cycles studied was 61%, range 21–91%). Twenty-three infertile Finnish couples from Oulu University Hospital and the Family Federation of Finland (Oulu, Helsinki and Turku Clinics) constituted this group, and similarly to the TFF group, women of this group had to have had at least four oocytes in a single IVF cycle and their partners had to reveal normal sperm parameters. The age of the women in the FIVF group was 25–45 years (mean 34 years). The second control group consisted of sixty-eight Finnish women who had given birth to at least of one healthy offspring after a spontaneous pregnancy. This group was termed `women with proven fertility´(WPF) and no ART was used.

49 4.1.2 Human ovarian tissues (Study II)

A total of 18 ovarian samples were obtained from 10 (fetal age 11–21 weeks) after spontaneous or therapeutic abortions and from six fetuses (fetal age 23–37 weeks) after intrauterine fetal death followed by spontaneous or induced delivery or Caesarean section. In addition, ovarian samples from two neonates (fetal ages 23 and 31 weeks) who died because of perinatal asphyxia or infection within eight hours after birth were studied. All fetuses and neonates had normal karyotypes and samples with visible autolysis were excluded from the study. Adult ovarian samples were obtained from seven patients undergoing oophorectomy as a result of . Ovarian autopsy tissue samples were collected from autopsies performed at the Department of Pathology, Oulu University Hospital. The samples were fixed in 4% phosphate-buffered neutral formaldehyde for 24h. After dehydration they were embedded in paraffin and histological sections (4 μm) were cut and processed for immunohistochemistry and in situ hybridization.

4.1.3 Zona anomaly patients (Study III)

Thirty-one study subjects of Finnish origin were selected among volunteers undergoing IVF or ICSI treatment at the Department of Obstetrics and Gynaecology, Oulu University Hospital, and Väestöliitto Fertility Clinic, Oulu in 2003–2007. As inclusion criteria, the patients in the zona anomaly (ZA) group had to produce four or more oocytes in a single IVF/ICSI cycle and to have at least one type of zona anomaly (i.e. split zona, oval-shaped zona, thick zona or thin zona) as confirmed by visual observation through an inverted microscope during routine IVF laboratory investigations. The age of the study subjects varied from 25 to 41 years (mean 32 years) and BMI ranged from 19 to 38 kg/m2 (mean 24 kg/m2). Twenty-two of the 31 patients were non-smokers, while five women smoked on a daily basis (3–17 cigarettes/day). Regarding the remaining four subjects, no information on smoking was available. Image data was collected for each oocyte on day one following ovum pick-up. Morphological analyses were performed by a single embryologist with several years of experience in IVF. Collection of suitable subjects for a control group was challenging, as only a marginal number of infertile women were able to produce morphologically sound oocytes with no indication of zona anomalies. Therefore, despite the missing

50 oocyte image data, sixty-eight women with proven fertility (WPF) from Study I were investigated as reference material.

4.2 Methods

4.2.1 Verifying exon–intron boundaries of the human ZP1 gene (Study I)

The exon–intron boundaries of human ZP1 were determined by amplifying the full- length complementary DNA (cDNA), using a Human Ovary Marathon Ready cDNA kit (Clontech, Palo Alto, USA) and an ExpandTM Long Template PCR system (Roche, Mannheim, Germany). In the first PCR cycle a specific primer for ZP1 cDNA based on the predicted sequence and the AP1 primer from the Marathon Ready kit were used. Nested PCR was performed with ZP1 cDNA-specific primers. The PCR reactions were carried out in a volume of 50 µl containing 5 µl of human ovary cDNA (Clontech), 5 pmol of each primer, 1.75 mM MgCl2, 0.2 mM deoxynucleotides and 3.5 U Expand Long Enzyme mix (Roche). The conditions, after initial denaturation at 94 °C for 2 min, were 25 cycles of 30 s at 94 °C, 30 s at 60 °C and 2 min at 68 °C, followed by final extension at 68 °C for 4 min. Nested PCR was performed under identical conditions with the exception that 5 µl of the first PCR product was used as a template. The PCR products were purified from 1.2% agarose gel and sequenced (ABI PRISMTM 377 sequencer). The obtained cDNA sequence was compared with the genomic sequence to determine the exon– intron boundaries of the human ZP1 gene.

4.2.2 DNA extraction and PCR (Study I and Study III)

EDTA-treated blood samples were collected, frozen and stored at −20 °C for genomic DNA extraction by salt-extraction. First, the samples were half-defrosted in water bath at 37 °C and placed immediately on ice. A lysis buffer (saccharose, 1

M Tris pH 7.5, 1 M MgCl2) was added to maintain a stable pH and the samples were then incubated on ice and centrifuged (2800 rpm for 15 min) in 4 °C. The supernatant was discarded and another lysis buffer (5 M NaCl, 250 mM EDTA, 1 M Tris pH 8.0), 20% SDS and proteinase K (10 mg/ml) was added to break open the cells and to digest the contaminating proteins. After overnight incubation at 37 °C, 5 M NaCl was added to stabilize the double helical structure of DNA and to

51 neutralize the negative charge present in DNA. After centrifugation (3000 rpm for 15 min) at 4 °C, the supernatant was transferred to a clean tube and absolute ethanol was added to precipitate the DNA. DNA was collected, dried and dissolved in TE- buffer (1 M Tris pH 7.5, 250 mM EDTA pH 7.5). The DNA concentration was measured spectrophotometrically. The extracted genomic DNA was used to study potential mutations in ZP genes (ZP1–ZP4). The sequences corresponding to 12 exons of ZP1 (Genebank accession number AC004126), 19 exons of ZP2 (NT_010393), 5 out of 8 exons of ZP3 (NT_007933), 12 exons of ZP4 (NT_004836) and exon-flanking sequences were amplified by PCR. The sizes of the PCR products varied between 200 and 500 bp. The PCR-reaction mixture (25 µl) contained 20 ng of genomic DNA, 5 to 10 pmol of each primer, 1.5 mM MgCl2, 0.2 mM deoxynucleotides and 0.6 U AmpliTaq Gold DNA polymerase (Applied Biosystems Roche, Brenchburg NJ, USA) or Biotools DNA polymerase (B&M Labs, S.A., c/Valle de Tobalina, Madrid, Spain). Initial denaturing at 95 °C for 10 min was used to fully denature the target DNA. Thereafter, each of the 35 cycles consisted of the following steps: 30 s denaturation at 95 °C, 30 s annealing at 54 to 67 °C and 30 s extension at 72 °C, followed by final extension at 72 °C for 8 min to finish elongation of the PCR product. A 3µl aliquot of the PCR product was run in 1.2% agarose gel to evaluate the quantity and quality of the PCR product.

4.2.3 Conformation-sensitive gel electrophoresis (Study I)

Conformation-sensitive gel electrophoresis (CSGE) (Körkkö et al. 1998) was used for mutation screening of genomic DNA extracted from EDTA-treated blood samples. In this assay, conformational changes caused by single-base mismatches in the double-stranded DNA lead to different migration patterns of heteroduplexes and homoduplexes in the electrophoretic gel. After a standard PCR procedure, the PCR products were denatured at 95 °C for 5 min, followed by annealing at 68 °C for 30 min in order to generate heteroduplexes and homoduplexes for CSGE. Approximately 20 ng of the PCR product was used for heteroduplex analysis by CSGE. Samples were loaded in 12% CSGE gel (10% polyacrylamide-1,4- Bis(acraloyl)piperazine, 10% ethyleneglycol, 15% formamide, 0.5 × TTE (44.4 mM Tris, 14.25 mM taurine, 0.1 mM EDTA), 10% ammoniumpersulfate, TEMED) and run at 20 w for 9.5 h. Gels were stained with SYBR Gold nucleic acid gel stain (Molecular Probes, Eugene, Oregon, USA).

52 4.2.4 Sequencing (Study I and Study III)

PCR products were sequenced using a DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech, Buckinghamshire, England) and an ABI PRISMTM 377 sequencer to detect sequence variations. The genotypes of the homozygotes were determined either by digestion or sequencing. The nomenclature of sequence variations is based on instructions by den Dunnen and Antonarakis (2001).

4.2.5 Allele frequencies (Study I)

Allele frequencies were determined for three sequence variations found in the ZP3, c.1−87T>G, c.536−17C>A and c.894G>A, p.K298 that were found as heterozygotes more often in TFF subjects than in the other two groups. Digestion with a restriction enzyme was performed on c.1−87T>G with PvuII and on c.894G>A, p.K298 with StuI. Sequencing by means of the ABI PRISMTM 377 sequencer was used for c.536−17C>A.

4.2.6 Statistical analysis (Study I and Study III)

Fisher’s exact test was used to analyse the statistical significance of the observed allele and genotype frequencies. Calculations were performed by means of GraphPad Calculations Software, 2002–2005 (GraphPad Calculations Software, San Diego, CA).

4.2.7 Immunohistochemistry (Study II)

ZP3 protein expression in fetal and adult ovarian tissue was studied by immunohistochemistry. Histological sections of 4 µm thickness were deparaffinised in xylene and rehydrated in a graduated series of alcohol solutions. For adult tissue, sodium citrate treatment in a microwave oven was conducted to enhance tissue permeability. Incubation in 3% hydrogen peroxide in methanol was used to block endogenous peroxidase activity. Normal rabbit serum was used to prevent non-specific staining. Polyclonal ZP3 antibody obtained from Aviva Systems Biology (San Diego, Ca, USA) was used as a primary antibody at 1:200 and the incubation time was 2 h at RT. For adult tissue, fetal calf serum was added to the primary antibody (final concentration 5%) in order to block non-specific

53 binding, and the tissue incubated overnight at 4 °C. For negative controls, phosphate-buffered saline was used instead of primary antibody. Tissues were washed in phosphate-buffered saline and incubated in rabbit secondary antibody. Visualization of the bound antibody was carried out by using an avidin–biotin immunoperoxidase system Vectastain Elite ABC Kits (Vector Laboratories, Burlingame, CA, USA). Diaminobenzidine tetra hydrochloride (DAB, DakoCytomation Ltd., Ely, UK) was used for staining the bound antibody and haematoxylin to counterstain the tissues.

4.2.8 In situ hybridization (Study II)

In situ hybridization is a method used to visualize mRNA expression in a tissue, thus providing anatomical localization. Probes for ZP1, ZP3 and FIGLA in situ hybridization were prepared from Expressed Sequence Tag (EST) clones (image clone 1837179 for ZP1, 5724267 for ZP3 and 5744748 for FIGLA) from MRC gene service (Cambridge, UK). Plasmid DNA preparations were made using PhoenIXTM Maxiprep Kits (Qbiogene, Morgan Irvine, CA, USA) according to the instructions of the manufacturer. Templates were linearized by digestion with suitable restriction enzymes (ZP1 sense and antisense RsaI, ZP3 sense XmaI, ZP3 antisense SalI, FIGLA sense XbaI, FIGLA antisense EcoRI). DNA was purified according to the instructions in the QIAquick® PCR Purification Kit (QIAGEN, Hilden, Germany). Antisense and sense probes were transcribed from the linearized templates by T7, SP6 or T3 RNA polymerases (Promega Corporation, Madison, WI, USA) and labelled with 35S-UTP (GE Healthcare, UK). The probes were precipitated in a tenth volume of 3 M sodium acetate, pH 5.5 and a 2.5 volume of absolute ethanol overnight at −20 °C. There after the probes were centrifuged (14000 rpm for 10 min), washed in 70% EtOH and air-dried. 20 μl of 1 M DTT was added to the probe and 180 μl of hybridization solution containing a ¼ volume of 50% dextran sulphate, a 1/40th volume of 50× Denhardt´s solution and 5 M NaCl, 1 M Tris-HCl pH 8.0, 0.5 M EDTA pH 8.0, formamide and Depc-H2O. The activity of the probes was measured in a liquid scintillation counter and thereafter the probes were stored at −20 °C. For hybridization, deparaffinised ovary samples were treated with proteinase K (10 mg/ml) to improve the access of the probe to the sample mRNA. To prevent non-specific labelling, the tissues were incubated in 0.1 M triethanolamine pH 8.0 and triethanolamine pH 8.0 with acetic anhydride. The

54 tissues were washed in SSC buffer and dehydrated gradually in an ascending series of alcohol solutions. A 1/20th volume of tRNA (10 mg/ml) was added to the probes, which were then heated at 80 °C for 2 minutes. Sense and antisense RNA probes (45 µl) were applied to the tissue sections (1.5–2 million counts per minute/slide), which were then sealed with a plastic coverslip and hybridized in a humidified chamber at 56 °C for 16 hours. The sections were digested with RNAse A (0.02 mg/ml) in NaCl, Tris-HCl, EDTA and Depc H2O. The slides were washed in a decreasing series of SSC buffer, and dried in graded series of alcohol solutions. Finally, the slides were dried in a vacuum. To visualize the bound probes, the slides were coated with photographic emulsion (Kodak NTB, Kodak, Rochester, NY, USA) diluted 1:1 with 1% glycerol. The slides were exposed for 11 to 25 days and developed using Kodak Professional D-19 developer (Kodak, Rochester, NY, USA) for 3.5 min and fixed using Kodak Professional fixer (Kodak, Rochester, NY, USA) for 2 min. The slides were lightly counterstained with haematoxylin. All samples were evaluated by using light and dark-field microscopy. The mRNA expression levels were semi-quantified by scoring the samples according to degrees of intensity from +/- (lowest) to +++ (highest).

4.2.9 Image data (Study III)

The FertiMorph system (IHMedical, Denmark), assembled around a Nikon inverted microscope, was used to collect 3D image data (Z-stack program) from fertilized oocytes at pronuclear stage on day 1. Immature or degenerated/ruptured oocytes were omitted from the analyses. Depending on oocyte diameter, 25 to 35 optical sections at sequential focal planes or depths were recorded using a step size of 2.5 μm (duration required to acquire the stack images was 6–8 seconds). By analysing these saved images rather than the oocyte itself, we were able to collect all the necessary information without exposing the oocytes for prolonged times outside the incubator. Morphological parameters included analyses of zona structure (zona splitting), shape (ovality) and variation in thickness (thin or thick). Oocytes were regarded as oval when the roundness index, i.e. zona length divided by width (Richter et al. 2001, Ebner et al. 2008), was 1.20 or higher. Likewise, they were regarded as thin or thick when the respective measurements were either less than 13 μm (Garside et al. 1997) or more than 20 μm.

55 Table 4. Summary of material, methods and main results of the studies.

Study Subjects & Material Methods Main results

I human DNA samples DNA extraction Women in the TFF group had a higher mean from women with total PCR number of sequence variations in ZP1 and fertilization failure, CSGE ZP3 when compared with the FIVF and WPF proven fertility and sequencing groups. fertilizers in IVF

II human fetal ovaries ZP1: ISH The expression of ZP3 and FIGLA mRNA in human adult ovaries ZP3: IHC and ISH fetal ovaries increases at the time of follicle FIGLA: ISH formation, suggesting that these factors may have a role in the development of PFs before ZP formation in human.

III human DNA samples DNA extraction Some of the most frequent zona anomalies from women with PCR may be at least partly explained by sequence zona anomalies sequencing variations in genes expressing the four and proven fertility human ZP proteins.

56 5 Results and Discussion

5.1 Sequence variations in genes encoding human zona pellucida glycoproteins, and fertilization failure in IVF (Study I)

Owing to the vital role of the ZP in fertilization, intensive research has been carried out to resolve its structural and functional characteristics. Studies in mice have shown that silencing of individual ZP genes has serious effects on ZP architecture and fertility (Rankin et al. 1996; 1999; 2001, Wassarman et al. 1997). Only a little is known about how alterations in ZP genes affect the ZP matrix and fertilization in humans. One of the primary aims of Study I was to determine whether a single sequence variation in ZP1–ZP4 either alone or together with others could reduce fertilization capability among a subpopulation of infertile couples.

5.1.1 Verifying exon–intron boundaries of the human ZP1 gene

Predicted cDNA for ZP1 was verified by amplifying full-length cDNA from a human ovary cDNA library (Clontech) and exon–intron boundaries were determined by comparing the obtained cDNA with the genomic sequence (AC004126). Four PCR products were obtained, one containing ZP1 exons 1–11 (1629 bp), the second, exons 1, 2 and 4–11 (1332 bp), and intron 8 (69 bp), the third, exons 1 and 2 (318 bp) and the fourth, exons 10–12 (320 bp). Based on these results, the ZP1 cDNA from human ovary was found to be 1952 bp in size and to contain 12 exons, thus confirming the predicted ZP1 cDNA (Hughes & Barratt 1999). Exon 3 was found to be missing from the second PCR product. This causes a premature stop-codon in exon 4, theoretically resulting in a truncated protein of only 109 amino acids.

5.1.2 Sequence variations in ZP genes

Eighteen study subjects (TFF group) and two control groups (FIVF, n = 23 and WPF, n = 68) were studied for mutations in the four ZP genes. Altogether, 20 sequence variations were detected in ZP genes: four in both ZP1 and ZP2, eight in ZP3 and four in ZP4 (Table 5). Most of them are known single nucleotide polymorphisms (SNPs). As oocyte–sperm interaction has been proposed to involve

57 multiple binding sites and events (Castle 2002, Redgrove et al. 2012, Avella et al. 2013), perhaps to prevent complete fertilization failure arising from a putative single de novo nucleotide change, it was not surprising to discover that no single point mutation observed solely explained fertilization failure in IVF among women in the TFF group.

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Table 5. Observed sequence variations in ZP1, ZP2, ZP3 and ZP4.

Gene Sequence variation TFF 1 n (%) FIVF 2 n (%) WPF 3 n (%) p4 SNP

ZP1 c.277G>A, p.V93I e 2 1 (6) 0 (0) 2 (3) 0.552 rs145067883 c.473T>C, p.I158T 5 e 3 7 (39) 1 (4) 18 (26) 0.026 rs489172 c.858C>T, p.F2865 e 5 8 (44) 8 (35) 28 (41) 0.802 rs10897122 c.1573−81G>A i 9 10 (56) 7 (30) 22 (32) 0.158 rs2074418

ZP2 c.528+60T>C 5 i 6 4 (22) 5 (22) 18 (26) 0.869 rs2242551 c.747T>C, p.P249 5 e 8 7 (39) 4 (17) 22 (32) 0.275 rs2075526 c.1830+25G>A i 15 1 (6) 5 (22) 5 (7) 0.110 rs16971219 c.2095+77T>G i 18 1 (6) 0 (0) 0 (0) 0.078 -

− ZP3 c.1 87T>G 5´UTR 6 (33) 3 (13) 6 (9) 0.027 rs132332187 c.91G>A, p.G31R e 1 5 (28) 7 (30) 20 (29) 0.983 rs2286428 c.431+115G>T i 2 1 (6) 0 (0) 0 (0) 0.078 - c.536-17C>A5 i 3 3 (17) 0 (0) 1 (1) 0.006 rs6966715 c.547G>A, p.A183T e 4 0 (0) 1 (4) 2 (3) 0.692 rs74676082 c.714−22T>C 5 i 4 7 (39) 6 (26) 23 (34) 0.670 rs3972768 c.714−51C>T 5 i 4 0 (0) 1 (4) 2 (3) 0.692 rs146147902 c.894G>A, p.K298 e 6 9 (50) 0 (0) 16 (24) 0.008 rs73363163

ZP4 c.18C>T, p.C6 e 1 1 (6) 4 (17) 2 (3) 0.050 rs35905278 c.401−123A>G 5 i 3 11 (61) 13 (57) 39 (57) 0.263 rs2236595 c.1495+47A>G5 i 11 4 (22) 6 (26) 17 (25) 0.958 rs2275694 c.1623+132A>G 3´UTR 8 (42) 9 (39) 25 (37) 0.836 rs2794813 1 total number of women in the TFF group = 18, 2 total number in the FIVF group = 23, 3 total number in the WPF group = 68, 4 p-values for chi-squared test for trend. Statistically significant difference shown in bold. e = exon, i = intron5 .Typographic errors in the corresponding table in the original article have been 59 corrected in this table.

Although no statistically significant difference between the studied groups was found, the sequence variation c.91G>A, p.G31R in ZP3 is interesting since it removes the last G residue in the LWLL - - G amino acid sequence, which, in turn, has been found to be one of the structures binding to the equatorial segment and the post-acrosomal sheath of human spermatozoa (Eidne et al. 2000), suggesting that this sequence is of importance in at least sperm–oocyte recognition and fusion. Interestingly, according to studies by Swanson and co-workers (2001; 2002), the LWLL - - G sequence is likely to be under positive Darwinian selection and therefore important for speciation. As loss of the LWLL - - G sequence was also observed in both control groups, we may only conclude that this sequence variation cannot solely explain the complete fertilization failure in the TFF group, suggesting that additional factors are involved.

5.1.3 Sequence variations of statistical significance

A statistically significant difference (p < 0.05) between the studied groups was found in four of the found sequence variations, one in ZP1 (c.473T>C, p.I158T) and three in ZP3 (c.1−87T>G, c.536-17C>A, c.894G>A, p.K298). Most interestingly, the sequence variation c.1−87T>G in ZP3, was found to be more common in the TFF group than in the WPF group and it is located in the regulatory region of ZP3. This promoter region contains five conserved elements termed I, IIA, IIB, II and IV and it is suggested that element IV is both necessary and sufficient for transcription from the ZP3 promoter (Millar et al. 1991). The sequence variation c.1−87T>G was found to be located among the element IIA, changing T into G. Since this sequence variation is located among a putative conserved element and was more frequently found in the TFF group than in the control groups, there is a minor possibility that element IIA also has a role in controlling human ZP3 transcription. In theory, this sequence variation may reduce the rate of transcription of the ZP3 gene and subsequently restrict the expression of ZP3 protein, which could result in the formation of a thinner ZP. Indeed, when only single ZP3 allele is present, it results in 50% reduction of the respective gene product in vivo and, subsequently, in the formation of oocytes with 50% reduction in their zona thickness (Wassarman et al. 1997). Here, thinning of the ZP seemed to have no significant effect on mouse reproduction, as litter sizes were comparable with those of wild-type mice.

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The second statistically significant variation in ZP3, c.536-17C>A, was found to be more common in the TFF group than in the WPF group. Considering that it is located within the intronic region of the gene, its effect is supposedly not very significant. The third sequence variation, c.894G>A, p.K298, is located in the ZP domain, and was found significantly more frequently in the TFF group than in the FIVF and WPF groups. Because it does not alter the amino acid sequence, its effect on the respective zona protein is most likely insignificant. The sequence variation in ZP1 (c.473T>C, p.I158T) changes isoleucine to threonine. However, this sequence variation was observed to be significantly less frequent in the FIVF than in TFF and WPF groups. Concerning this variation, and a few other ones (discussed in section 5.5. Errata), there are unfortunately typos in the text and in Table III of the original article, and the correct data is shown in the Table 5.

5.1.4 Cumulative effect of sequence variations in the study groups

To give a general view as to whether some of the zona proteins possess a higher number of sequence variations in the TFF group than in the two reference groups, putatively resulting in complete fertilization failure, the mean number of cumulative sequence variations per person in each group was calculated. This calculation, however, may give us only a rough estimate, but it was interesting to find that ZP3 and ZP1 contained on average the highest number of sequence variations (Table V of the original article). The results further revealed that women in the TFF group had a roughly 1.5-fold greater mean number of sequence variations in ZP3 and ZP1 than women in the FIVF and WPF groups. It is tempting to speculate, that these results might suggest a more significant role for ZP1 in human fertilization (Ganguly et al. 2010 a, b), even though the cumulative effect of the four sequence variations found in ZP1 is difficult to predict.

5.2 Zona pellucida components are present in the human fetal ovary before follicle formation (Study II)

The ZP is not present in resting primordial follicles, but appears later in growing oocytes. During human fetal life, ZP3 transcripts can be detected in oocytes from primordial-stage follicles up to blastocyst-stage embryos (Huntriss et al. 2002). ZP3 protein can be detected in the oocytes and granulosa cells of primordial

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follicles up to antral follicles in human ovaries from study subjects aged 14–40 years (Gook et al. 2008). The aim of Study II was to investigate the expression and localization of ZP1 mRNA and ZP3 mRNA/protein and their key regulator FIGLA mRNA in human fetal and adult ovaries and to elucidate their roles in human ovarian development and folliculogenesis.

5.2.1 ZP3 mRNA and protein expression in fetal and adult ovary

Eighteen ovary samples from fetuses of age 11th to 37th week of gestation were studied to reveal the time and location of ZP3 mRNA expression. ZP3 mRNA could already be detected at 11th week of gestation and its expression increased towards mid-pregnancy, peaking at around the 20th week, the time of follicle formation (Table 6) (Figure 1 (a) of the original article). The expression ZP3 mRNA remained high until around the 32nd week of gestation, after which it diminished, reaching a similar level of expression as observed at 11th week. Common understanding is that the ZP is formed around the oocyte in growing follicles at primary-secondary follicle stage (Kierszenbaum 2015). The expression of ZP genes before the formation of primordial follicles suggests that crucial ZP components are already present at early stages of ovarian development.

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Table 6. Expression of ZP1, ZP3 and FIGLA mRNA.

Age (weeks + days) ZP1 ZP3 FIGLA 11+3 +/− + +

12 +/− + +

13 +/− na +

15 +/− + +

16 +/− ++ +

17+5 +/− ++ ++

18+1 +/− na na 19+1 + +++ +

20+6 +/− na + 21+6 na +++ ++

23+1 +/− +++ ++ 25+6 + na ++

27 + +++ ++

31+2 na +++ ++

32+3 + ++ ++

33+4 + ++ ++

36+2 na na ++

37+6 + + na na = specimen not available

The localization of mammalian ZP proteins in the ovary has been controversial and in humans, ZP1–ZP3 have been found both in oocytes and the granulosa cells in study subjects aged 14–44 years (Gook et al. 2008). In our study, after around the 20th week of gestation all oocytes clearly expressed ZP3 mRNA, but before that the location was difficult to determine. Due to the high level of expression in the oocyte, it was difficult to exclude possible expression in the granulosa cells as well. However, it was clear that the oocytes showed greater mRNA expression compared with granulosa cells. At mid-gestation, a dense spherical staining of ZP3 mRNA was seen around some of the oocytes, suggesting that the ZP may be formed already at the time of follicle formation. In adult ovaries, ZP3 mRNA was mainly localized to oocytes (from primordial to antral follicles) (Figure 3 (A–D) of the original article), but expression in granulosa cells can not be excluded. In a study by Huntriss and co-workers (2002)

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ZP3 transcripts were detected in oocytes from primordial-stage follicles up to blastocyst-stage embryos, but not in granulosa cells. Similar to mRNA, ZP3 protein was already detected at the 11th week of gestation but its location was difficult to determine (Figure 2 of the original article). At the 17th week of gestation the expression in the oocytes was high and it was difficult to exclude ZP3 staining in the granulosa cells. Similar to mRNA, the strongest expression of ZP3 protein was seen between 20th and 30th week of gestation. At this stage and thereafter, the most intense staining was localized in follicles, and especially in oocytes, and only minimal staining was observed in the stroma. In adult ovaries (Figure 3 (E–H) of the original article), ZP3 protein expression was localized to the oocytes of primordial and secondary follicles, but only negligible staining was observed in the antral follicles. In a study by Gook and co-workers (2008), human ZP3 protein was detected in the oocytes and granulosa cells of primordial follicles up to antral follicles and the expression increased with development in study subjects aged 14–40 years. In the study by Gook and co-workers (2008), ZP1 and ZP3 proteins were consistently present in ∼90% of the primordial oocytes of 18 study subjects between the ages 14–44 years, suggesting that this initial synthesis of ZP proteins does not occur after primordial follicles are recruited into the growing follicular pool, but takes place in the quiescent primordial follicles constituting the ovarian reserve. Since our results demonstrated that ZP3 protein is present in the primordial follicles already in fetal life, it indeed seems that ZP proteins can be retained in the primordial follicles throughout reproductive life, as hypothesized by Gook and co- workers (2008).

5.2.2 ZP1 mRNA expression in fetal and adult ovaries

Only minimal or negligible ZP1 mRNA expression was detected in fetal ovaries between 11th and 25th week of gestation (Table 6) (Figure 1 (b) of the original article). Thereafter, expression was slightly increased and it remained constant until the 37th week of gestation. Owing to a generally low expression level of ZP1 mRNA it was difficult to determine its location, as similar level of expression was detected both in the oocyte and granulosa cells. These results suggest that ZP1 may not have a major impact on follicle development in early fetal life in humans. However, the generally low expression rate of ZP1 mRNA is in agreement with the results reported by Lefievre and co-workers (2004), suggesting that ZP1 is less abundant

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than ZP2–ZP4 in humans (Lefievre et al. 2004). Similarly, in mice ZP1 has been found to be the least abundant of the ZP proteins, accounting for only 9% of the total ZP protein content (Green 1997). Only negligible expression of ZP1 mRNA was detected in adult ovaries (Figure 3 (I–L) of the original article). Previously, ZP1 protein has been detected in the oocytes of adult human ovaries from primordial stage onwards (Eberspaecher et al. 2001) and expression increases towards the antral follicle stage (Gook et al. 2008).

5.2.3 FIGLA mRNA expression in fetal and adult ovaries

FIGLA, a germ cell-specific transcription factor, is involved in the coordinated expression of ZP genes during murine oogenesis (Liang et al. 1997). In our study, FIGLA mRNA expression could be detected from the 11th week of gestation but it was relatively low until the 20th week (Table 6) (Figure 18 (c) of the original article). Thereafter, its expression rose and it remained moderately high until the 36th week of gestation. Bayne and co-workers (2004) found a 40-fold increase in the FIGLA transcript expression in human fetal ovaries between 14th and 19th week of gestation. Studies on FIGLA-deficient mice have demonstrated a critical role of FIGLA in the initiation of folliculogenesis and primordial follicle formation (Soyal et al. 2000). Our results support these findings, as the expression of FIGLA is clearly increased at the time of primordial follicle formation. In addition, the ZP3 mRNA expression increased together with FIGLA mRNA expression in early fetal life, suggesting that ZP3 may have a role in the development of primordial follicles before formation of the ZP in humans. In our study, FIGLA mRNA expression could be localized to oocytes, especially after the 20th week of gestation, but the expression in granulosa cells as well can not be excluded, especially during early fetal life. This is in concordance with the previous studies (Huntriss et al. 2001, Bayne et al. 2004). The importance of FIGLA in the formation of ovarian follicles is emphasized by observations in FIGLA-deficient mice, which neither form primordial follicles and nor express ZP genes (Soyal et al. 2000). Furthermore, FIGLA gene mutations have been associated in primary ovarian insufficiency in humans (Zhao et al. 2008, Tosh et al. 2015). In adult ovaries (Figure 3 (M–P) of the original article), moderate FIGLA mRNA expression could be located in primordial follicles but not in antral follicles, a fact which supports earlier observations that FIGLA is detected in ovarian

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follicles from primordial stage to secondary stage follicles and metaphase II oocytes (Huntriss et al. 2002).

5.3 Sequence variations in human ZP genes as potential modifiers of zona pellucida architecture (Study III)

The aim of the Study III was to investigate whether sequence variations in genes encoding ZP glycoproteins (ZP1–ZP4) could explain the most frequently encountered abnormalities in routine IVF/ICSI.

5.3.1 ZP gene-related sequence variations in IVF/ICSI subjects with zona anomalies

Thirty-one study subjects with zona anomalies (ZA) and 68 women with proven fertility (WPF) were investigated in this study. All four known human ZP genes, ZP1–4, were investigated for sequence variations within exonic and promoter (250 bp upstream from the translation-initiation site) regions in the ZA group, and the results were compared with those obtained from women with proven fertility (WPF group). A total of 31 sequence variations were detected: four in ZP1, seven in ZP2, nine in ZP3 and eleven in ZP4 (Table 7). The majority of these variations have already been described, either in Study I or they are known as single nucleotide polymorphisms (SNPs) (http://www.ncbi.nlm.nih.gov/SNP/). Their frequencies corresponded well with those in some representative reference populations, as presented in the International HapMap project (http://hapmap.ncbi.nlm.nih.gov/). Only the first four exons of ZP3 were studied, as the existence of a very similar POM-ZP3 interferes with interpretation of the results concerning the last four exons. Unfortunately, there are two typographic errors in Table 1 of the original article. The sequence variation c.91G>A, p.G30R in ZP3 should be c.91G>A, p.G31R and the sequence variation c.313−75G>T should be c.313−65G>T. These are corrected in Table 7.

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Table 7. Observed sequence variations in ZP1, ZP2, ZP3 and ZP4.

Gene Sequence variation Position ZA 1 n (%) WPF 2 n (%) p3 SNP ZP1 c.277G>A, p.V93I e 2 1 (3%) 2 (3%) 1 rs145067883 c.473 T>C, p.I158T e 3 11 (36%) 18 (26%) 0.4754 rs489172 c.858C>T, p.F286 e 5 12 (39%) 28 (41%) 1 rs10897122 c.1573 −81G>A i 9 19 (61%) 22 (32%) 0.0086 rs2074418

ZP2 −73G>Tc.1 5´UTR 25 (81%) 47 (69%) 0.3309 rs2075521 c.107G>T, p.G36V e 2 11 (35%) 37 (54%) 0.0884 rs2075520 c.116T>G, p.I39R e 2 12 (39%) 0 (0%) 0.0001 - c.528+60T>C i 6 8 (26%) 18 (26%) 1 rs2242551 c.747T>C, p.P249 e 8 19 (61%) 27 (39%) 0.0533 rs2075526

c.973−37C>T i 9 1 (3%) 0 (0%) 0.4133 rs75227459 c.1830+25G>A i 15 5 (16%) 5 (7%) 0.2786 rs16971219

ZP3 c.1−87T>G 5´ UTR 10 (32%) 6 (9%) 0.0065 rs13232187 c.91G>A, p.G31R4 e 1 8 (26%) 20 (29%) 0.8123 rs2286428 c.313+65G>T4 i 1 23 (74%) 42 (62%) 0.261 rs6465128 c.431+115G>T i 2 1 (3%) 0 (0%) 0.3131 - c.536−143G>C i 3 8 (26%) 26 (38%) 0.261 rs6966715 c.547G>A, p.A183T e 4 3 (9%) 2 (3%) 0.1755 rs74676082 c.662C>G, p.P221R e 4 1 (3%) 0 (0%) 0.3131 rs139729790 c.714−22T>C i 4 20 (65%) 23 (34%) 0.0081 rs3972768 c.714−51C>T i 4 3 (9%) 2 (3%) 0.1755 rs146147902

ZP4 c.1−429A>G 5´UTR 2 (6%) 5 (7%) 1 rs75647222 c.18C>T, p.C6 e 1 2 (6%) 2 (3%) 0.5873 rs35905278 c.176−48G>A i 1 16 (52%) 33 (49%) 0.8305 rs538499 67

68 68 Gene Sequence variation Position ZA 1 n (%) WPF 2 n (%) p3 SNP c.401 −123A>G i 3 23 (74%) 39 (57%) 0.1227 rs2236595 c.554 −40G>A i 4 2 (6%) 1 (1,5%) 0.2303 rs114491365 c.774A>G, p.E258 e 6 2 (6%) 1 (1,5%) 0.2303 rs520720 c.839A>G, p.S277G e 6 2 (6%) 1 (1,5%) 0.2303 rs36017138 c.839+28A>G i 6 2 (6%) 1 (1,5%) 0.2303 rs510549 c.1160+31A>G i 8 2 (6%) 1 (1,5%) 0.2303 rs1209519 c.1495+47A>G i 11 14 45%) 17 (25%) 0.0616 rs2275694 c.1623+132A>G 3´UTR 21 (68%) 25 (37%) 0.005 rs2794813 1 total number of women in the ZA group = 31, 2 total number in the WPF group = 68, 3 p-values for chi-squared test for trend. Statistically significant difference shown in bold. e = exon, i = intron.4 Typographic errors in the corresponding table in the original article have been corrected in this table.

When comparing the ZA and WPF groups, five sequence variations were found to be statistically significantly more common in the former than in the latter group. One of them is located in ZP1 (c.1573−81G>A), one in ZP2 (c.116T>G, p.I39R), two in ZP3 (c.1−87T>G and c.714−22T>C) and one in ZP4 (c.1623+132A>G). Our primary interest was focused on c.116T>G, p.I39R in ZP2, as none of our reference women (WPF group) carried this variation and because it was a novel finding (i.e. is not a known SNP). Interestingly, this together with another variation, i.e. c.107G>T, p.G36V, are both located at a putative signal peptide cleavage site of ZP2. In theory, loss of this consensus sequence might result in decreased amounts of soluble ZP2 protein. According to the results of studies on knockout mice, a complete loss of ZP2 may result in a thinner ZP (Rankin et al. 2001). However, we observed no increase in the existence of thinner zona matrices in connection with sequence variation c.116T>G, p.I39R, suggesting that its impact on zona matrix thickness is minimal. Two sequence variations in ZP3, c.1−87T>G and c.714−22T>C (both also found in Study I) were statistically more frequent in the ZA group than in the WPF group. The former of these two variations is located at the promoter region, among a conserved element (IIA) (Millar et al. 1991). Based on its location, this variation may theoretically reduce the rate of transcription and subsequently restrict the expression of ZP3 protein. As a consequence, this may result in the formation of thinner zonas. Accordingly, when only single ZP3 allele is present, a 50% reduction of respective gene product is observed in vivo, together with the formation of oocytes with 50% reduction in zona thickness (Wassarman et al 1997). Interestingly, this sequence variation was associated with increasing numbers of thin zonas in the present study (Table 8). Sequence variations in ZP1 (c.1573−81G>A), ZP3 (c.714−22T>C) and in ZP4 (c.1623+132A>G) were found to be located in non-coding regions. All of them had already been found in Study I. The sequence variation c.1573−81G>A is located in intron 9 and is surrounded by exons encoding part of the ZP domain. The sequence variation c.714−22T>C is found downstream within intron 4 and is surrounded by exons that encode part of the ZP domain. The sequence variation c.1623+132A>G is located near the 3’ end of the respective mRNA, i.e. between the translation stop codon and the poly-A tail. Due to the locations of these variations, their impact on zona anomalies is most likely negligible. The sequence variation c.662C>G, p.P221R in ZP3 was identified only in one patient, but its location is interesting, as according to Monné and Jovine (2011), it

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affects a residue located next to C-terminal subdomain β-strands E´ and F´, suggesting that it might destabilize dimer formation or impair the ability of strand F to take part in polymerization following external hydrophobic patch ejection. The significance of this variant remains to be established using larger sample sets.

5.3.2 Zona anomalies and sequence variations

The mean age of the women in the zona anomaly (ZA) group was 32 years (25 to 41 years), which represented well those women attending to infertility treatments in general. It has been previously noted that both ZP thickness and its variation decline with a woman´s age (Sun et al. 2005). The mean zona thickness variation value in women less than 30 years has been reported to be significantly greater than in women older than 35 years (Gabrielsen et al. 2000). Smoking may have a negative impact on zona architecture, as thick zonas have been observed more often in smokers than in non-smokers (Shiloh et al. 2004). Most of the subjects (22 out of 31) in our study were non-smokers, and smoking did not seem to have any significant effect on ZP thickness, as thick zonas were not observed in any of the smokers (five subjects) or in those whose smoking behaviour was unknown (four subjects). Morphological features found in the oocytes were first categorized into four groups (i.e. zona splitting, oval, thin and thick) (Figure 5), all being supposedly more or less disadvantageous as regards sperm recognition, binding and penetration through the zona matrix as well as in vivo hatching. To study whether any of the found sequence variations are involved in modifying zona architecture, sequence variations were compared with the morphological data (Table 8).

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Fig. 5. Representative images of zona anomalies. A: zona splitting, B: oval zona, B: thin zona, D: thick zona. The arrows indicate the location of each zona anomaly.

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Table 8. Sequence variations and zona anomalies.

Gene Sequence variation Zona splitting Oval shaped Thin zona Thick zona zona − + − + − + − + ZP1 c.1573−81G>A 18% 4%

ZP2 c.107G>T, p.G36V 45% 23% c.116T>G, p.I39R 15% 27% 45% 25% c.528+60T>C 34% 47% c.1830+25G>A 33% 58%

ZP3 c.1−87T>G 23% 12% 30% 53% c.91G>A, p.G31R 40% 29% c.313−65G>T 27% 41% c.536−143G>C 40% 29%

ZP4 c.401−123A>G 17% 7% Note: Mean frequencies of zona anomalies, either in the absence (-) or presence (+) of sequence variations in the ZA group, are shown only if the difference exceeds 10%.

Based on the results, three notable observations emerged. First, none of the found sequence variations here were clearly associated with an increased number of thick zona matrices. Thus, mechanisms which ultimately result in formation of thick zonas must be sought elsewhere. This is no doubt a crucial question to resolve, as thick zona is considered to be a potential candidate as regards poor fertilization (Bertrand et al. 1995, Loret de Mola et al. 1997). In contrast, several variations were found in association with oocytes with either decreased (c.107G>T, p.G36V and c.116T>G, p.I39R in ZP2 and c.91G>A, p.G31R and c.536−143G>C in ZP3) or increased (c.528+60T>C and c.1830+25G>A in ZP2 and c.1−87T>G and c.313−75G>T in ZP3) numbers of thin zonas. Since the sequence variations associated with increased numbers of thin zonas are located in non-coding sequences, their impact on zona thinning is difficult to interpret. We may speculate, however, that certain variations may act together in such a manner that they disturb the production of respective mRNAs. This, in turn, may lead to decreased protein synthesis and subsequently thin zona formation. Thirdly, the majority of the sequence variations were located within either the ZP2 or ZP3. Since ZP2 and ZP3 proteins are two of the most abundant components of ZP matrix, it is not surprising that the majority of the sequence variations were located in ZP2 and ZP3. On the

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other hand, since ZP1 is considered to be important for the structure of ZP matrix by crosslinking the ZP filaments composed of ZP2 and ZP3, one would have expected to find many sequence variations in ZP1. However, considering that in ZP1-deficient mice (Rankin et al. 1999) only 10% of the oocytes had distinct zona anomalies, it is reasonable to believe that the sequence variations described here may have only a marginal effect on zona morphology. Interestingly, Huang and co- workers (2014) reported a frame shift deletion in ZP1, which was found to lead to a truncated form of ZP1 and oocytes completely devoid of ZP. The authors propose that the truncated ZP1 causes intracellular interaction and accumulation of the other ZP proteins and thus prevents formation of the ZP. In the literature, there are only a few studies on causal relationships between sequence variations in ZP genes and zona anomalies. Margalit and co-workers (2012) studied the ZP1–ZP4 genes of three patients with abnormal ZPs. Altogether, eight variations were found, all of which were regarded as known SNPs. Three of the eight variations turned out to be the same as described here, i.e. c.473T>C, p.I158T in ZP1 and c.107G>T, p.G36V and c.747T>C, p.P249 in ZP2, but none of these sequence variations were found to be significantly more common in the ZA group in our study. Neither were these variations associated with an oval zona. However, c.107G>T, p.G36V was associated with decreased numbers of thin zonas. Ferre and co-workers (2014) identified five sequence variations in ZP1–ZP4 in three patients with oocyte lysis (including the disruption of the ZP) in IVF, none of which, however, could explain the observed lysis of the oocytes. Interestingly, four of them (c.858C>T, p.F286 in ZP1, c.1−73G>T and c.747T>C, p.P249 in ZP2 and c.91G>A, p.G31R in ZP3) were also found in the present work and c.747T>C, p.P249 in ZP2 also in the study by Margalit and co-workers (2012).

5.3.3 Zona anomalies and pregnancy outcome

Although the main emphasis here was to focus on putative associations between given zona anomalies and variations in genes encoding the four known human ZP proteins, we further studied the possible impact of zona anomalies on pregnancy outcome. These results were not included in the original article (Paper III). The (IVF, n =13 and ICSI, n =18) in the study group was 26.3%, which was somewhat lower than the overall pregnancy rate (31.2%) in the two clinics during the study period (2003–2007). The implantation and birth rates were 25.8 % vs. 27.4% and 19.4% vs. 22.6%, respectively.

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A thin zona was clearly the most typical anomaly in transferred embryos in the ZA group. It was present in 18 out of 40 embryos transferred (i.e. 20 single- and 10 double-embryo transfers), from which four (22.2%) resulted in live birth. Eight of the transferred embryos (out of 40) showed oval-shaped zona matrices. Perhaps surprisingly, all studied zona anomalies, except thick zona, were relatively frequent among embryos that progressed to term. The results also suggest that oval-shaped embryos are not necessarily destined to pregnancy failure, as biochemical pregnancy was achieved with four and live birth with two oval embryos (out of 10 pregnancies). Others have also reported successful pregnancies if ICSI is used in connection with oocytes with abnormal ZPs (Esfandiari 2005, Paz et al. 2004).

5.4 Methological considerations

There are some factors that may affect the results described here. Collecting a large representative study group was challenging, as only a limited amount of patients fulfilled the strict inclusion criteria set for the present study. Hence, the study groups remained relatively small in Study I and Study III. As DNA extraction and PCR are sensitive to contamination, all procedures were carried out accurately, using aseptic working. A sample without DNA was included in every PCR set as a negative control to evaluate the quality of the set of PCRs carried out. The human fetal and adult ovary samples were collected from autopsies routinely performed at the Department of Pathology, Oulu University Hospital. During collection these samples were carefully processed to minimize the damage and protein degradation that may occur before sample fixation. Due to the low number of fetal autopsies conducted yearly, the sample size is understandably low. Consequently, these samples are highly valuable and a rare opportunity to study human fetal ovaries. Immunohistochemical studies on these samples were performed by avoiding contamination in all steps and by minimizing non-specific binding of antibody by using normal blocking serum. The specificity of the staining was assured by using negative controls. However, despite carefully planned protocols and use of methods diminishing non-specific antibody binding, some non-specific staining may exist, which has to be taken into consideration when interpreting the results of the immunohistochemical studies. Probes for the in situ hybridization were carefully designed for reliable and specific binding to target mRNA. A sense control was used for every sample. The tissue samples were carefully handled to avoid contamination and ribonuclease (RNAse)-free conditions were assured at every step of the protocol.

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5.5 Errata

Unfortunately, there are some typographic errors in the text and in Table III of Paper I. These mistakes do not change the type of the sequence variation but merely are unfortunate errors concerning the location of the sequence variation in nucleotide or amino acid sequences or nucleotides changed. The sequence variation c.471T>G, p.I158T in ZP1 is at location c.473 and not at c.471 and it changes T into C and not to G. Thus the correct form of the sequence variation is c.473T>C, p.I158T. The amino acid number of the sequence variation c.858C>T, p.F268 in ZP1 should be 286, and thus the correct form of the sequence variation is c.858C>T, p.F286. Sequence variation c.528+60C>T in ZP2 should be c.528+60T>C. Sequence variation c.747T>C, p. P229 in ZP2 should be c.747T>C, p.P249. Sequence variation c.535−17C>A in ZP3 should be c.536−17C>A. Sequence variation c.741−22C>T in ZP3 should be c.714−22T>C. Sequence variation c.741−51C>T in ZP3 should be c.714−51C>T. Sequence variation c.401−122A>G in ZP4 should be c.401−123A>G. Sequence variation c.1391+47A>G in ZP4 should be c.1495+47A>G. All these are now corrected in Table 5. Unfortunately, there are also two typographic errors in Table 1 of Paper III. The sequence variation c.91G>A, p.G30R in ZP3 should be c.91G>A, p.G31R and the sequence variation c.313−75G>T should be c.313−65G>T. These are now corrected in Table 7.

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6 Summary and conclusions

Altogether, 34 sequence variations were detected in ZP1–ZP4 among women suffering from total fertilization failure in IVF and various zona anomalies investigated in Study I and Study III. It was not surprising that no single point mutation solely explained total fertilization failure, as oocyte–sperm interaction is thought to involve multiple binding sites and events at a molecular level. Most of the sequence variations found were known single nucleotide polymorphisms (SNPs) and only three of them represented novel findings not known as SNPs. The results in Study I revealed that ZP3 and ZP1 contained on average the highest number of sequence variations. It was also interesting to discover that women in the TFF group had an approximately 1.5-fold greater mean number of sequence variations in ZP3 and ZP1 than those in the FIVF and WPF groups. It is tempting to speculate that these results might suggest a significant role for ZP1 in human fertilization (Ganguly et al. 2010 a, b), even though the cumulative effect of the four sequence variations found in ZP1 is difficult to predict. Sequence variation c.1−87T>G in ZP3 is interesting, since it is located among a conserved sequence, element IIA in the promoter region of ZP3. Due to its specific location, this sequence variation may reduce the transcription rate of ZP3 and subsequently restrict the expression of ZP3 protein. This may lead to the formation of thinner zonas, therefore potentially decreasing fertility. Interestingly, this sequence variation was found to be associated with an increased number of thin zonas, and furthermore, it was found to be significantly more common in the study groups compared with the controls in both Study I and Study III. Sequence variation c.116T>G, p.I39R, was found to be significantly more common in the ZA group compared with the control group and to be located at a putative signal peptide cleavage site of ZP2. In theory, loss of this consensus sequence might result in decreased amounts of soluble ZP2, disrupting the arrangement of ZP2-ZP3-ZP4 polymers and ultimately the zona itself. According to results of studies on knockout mice, a complete loss of ZP2 results at an early stage of folliculogenesis in a thinner ZP, but it is finally lost from ovulated eggs. However, we did not observe an increase in the number of thinner zona matrices associated with sequence variation c.116T>G, p.I39R, suggesting that its impact on zona thickness is minimal. In fetal ovaries, the expression of ZP3 mRNA and protein could already be detected as early as the 11th week of gestation and it peaked at the 20th week, the time of primordial follicle formation. Therefore, the crucial component needed for

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zona matrix is already present well before the formation of the ZP. This suggests that ZP3 may have a role in the development of primordial follicles before the formation of the ZP in humans. Expression of transcription factor FIGLA mRNA was also increased at around the 20th week of gestation, supporting previous findings of its critical role in the initiation of folliculogenesis and primordial follicle formation. The present results add new information on the currently still incomplete picture of human fertilization and hopefully will enable better understanding of the existence of various zona anomalies and whether, for example, a gradual increase of certain sequence variations will reduce the recognition and/or binding capacity of the two gametes in such a manner that ultimately results in total fertilization failure. Understanding the structure–function relationships more precisely in the two human gametes, together with the corresponding genetic data may enable future development of new diagnostic tools, e.g. microchip technology. This, in turn, would help in selection of the most suitable infertility treatment for each infertile couple.

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Original publications

I Männikkö M, Törmälä RM, Tuuri T, Haltia A, Martikainen H, Ala-Kokko L, Tapanainen JS & Lakkakorpi JT (2005) Association between sequence variations in genes encoding human zona pellucida glycoproteins and fertilization failure in IVF. Hum Reprod 20(6): 1578–85. II Törmälä RM, Jääskeläinen M, Lakkakorpi J, Liakka A, Tapanainen JS & Vaskivuo TE (2008) Zona pellucida components are present in human fetal ovary before follicle formation. Mol Cell Endocrinol 289 (1-2): 10–5. III Pökkylä RM, Lakkakorpi JT, Nuojua-Huttunen SH & Tapanainen JS (2011) Sequence variations in human ZP genes as potential modifiers of zona pellucida architecture. Fertil Steril 95(8): 2669–72. Reprinted with permission from Oxford University Press (I) and Elsevier (II and III).

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

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96 ACTA UNIVERSITATIS OULUENSIS SERIES D MEDICA

1364. Heikkilä, Vesa-Pekka (2016) New techniques and methods for decreasing healthy tissue dose in prostate radiotherapy, with special reference to rectal doses 1365. Aro, Jani (2016) Novel load-inducible factors in cardiac hypertrophy 1366. Myllymäki, Mikko (2016) Hypoxia-inducible factor prolyl 4-hydroxylase-2 in Tibetan high-altitude adaptation, extramedullary erythropoiesis and skeletal muscle ischemia 1367. Rubino, Antonino S. (2016) Efficacy of the Perceval sutureless aortic valve bioprosthesis in the treatment of aortic valve stenosis 1368. Krökki, Olga (2016) Multiple sclerosis in Northern Finland : epidemiological characteristics and comorbidities 1369. Mosorin, Matti-Aleksi (2016) Prognostic impact of preoperative and postoperative critical conditions on the outcome of coronary artery bypass surgery 1370. Pelkonen, Sari (2016) Frozen embryo transfer : early pregnancy, perinatal outcomes, and health of singleton children 1371. Pohjanen, Vesa-Matti (2016) Toll-like receptor 4 and interleukin 6 gene polymorphisms in Helicobacter pylori related diseases 1372. Hekkala, Anne (2016) Ketoacidosis at diagnosis of type 1 diabetes in children under 15 years of age 1373. Kujanpää, Tero (2016) Generalized anxiety disorder and health care utilization 1374. Hintsala, Hanna-Riikka (2016) Oxidative stress and cell adhesion in skin cancer 1375. Lehtonen, Ville (2016) Dental and otologic problems in cleft lip and palate patients from Northern Finland : cleft associated problems 1376. Koivukangas, Jenni (2016) Brain white matter structure, body mass index and physical activity in individuals at risk for psychosis : The Northern Finland Birth Cohort 1986 Study 1377. Väyrynen, Sara (2016) Histological and molecular features of serrated colorectal adenocarcinoma and its precursor lesions 1378. Kujanpää, Kirsi (2016) Mechanisms behind stem cell therapy in acute myocardial infarction

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