oulu 2016 d 1379 university of oulu p.o. box 8000 fi …

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ISBN 978-952-62-1297-5 (Paperback)ISBN 978-952-62-1298-2 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)

U N I V E R S I TAT I S O U L U E N S I S

MEDICA

ACTAD

D 1379

ACTA

Reeta-M

aria Törm

älä

OULU 2016

D 1379

Reeta-Maria Törmälä

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

A C T A U N I V E R S I T A T I S O U L U E N S I SD M e d i c a 1 3 7 9

REETA-MARIA TÖRMÄLÄ


Academic dissertation to be presented with the assent ofthe Doctora l Train ing Committee of Health andBiosciences of the University of Oulu for public defence inAuditorium 4 of Oulu University Hospital , on 23September 2016, at 12 noon

UNIVERSITY OF OULU, OULU 2016

Copyright © 2016Acta Univ. Oul. D 1379, 2016

Supervised byProfessor Juha TapanainenDoctor Jouni Lakkakorpi

Reviewed byProfessor Sari MäkeläDocent Virpi Töhönen

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

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

Cover DesignRaimo Ahonen

JUVENES PRINTTAMPERE 2016

OpponentProfessor Juha Kere

Törmälä, Reeta-Maria, Human zona pellucida abnormalities – a genetic approachto the understanding of fertilization failure. University of Oulu Graduate School; University of Oulu, Faculty of Medicine; MedicalResearch Center Oulu; Oulu University Hospital; National Graduate School of ClinicalInvestigationActa Univ. Oul. D 1379, 2016University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract

Despite the development of assisted reproduction technologies and significant advances inreproductive biology and medicine over the years the cause of infertility remains unexplained in10–20% of cases. The cause of infertility in these cases may be connected to problems infertilization or implantation and genetic factors may play a part in this.

The zona pellucida (ZP) is an extracellular matrix surrounding the oocyte and early-stageembryos. It is important for folliculogenesis, fertilization and implantation. In humans, it iscomposed of four known ZP glycoproteins that all show varying degrees of structural andfunctional roles in reproduction. The aim of the present study was to examine the role of zonapellucida genes in cases of total fertilization failure and zona anomalies, and to study theirexpression in human fetal and adult 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 zonaanomalies in their oocytes. Most of the variations were known single nucleotide polymorphisms,while three were novel findings. Women with fertilization failure had a higher mean number ofsequence variations in ZP1 and ZP3 when compared with controls. Some of the most frequentzona 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 atthe 11th week of gestation 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 theformation 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 ataround the 20th week of gestation, supporting previous findings of its critical role in the initiationof folliculogenesis and primordial follicle formation.

The present study adds to our knowledge on the currently still incomplete picture of formationof the ZP and fertilization in humans. Understanding the genetic background of infertile patientsmay help us to develop new tools not only to evaluate but also to improve their fertilizationpotential, and to choose the optimal treatment to achieve pregnancy.

Keywords: granulosa cell, infertility, oocyte, ovary, total fertilization failure, zonaanomaly, 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 ResearchCenter Oulu; Oulun yliopistollinen sairaala; Valtakunnallinen kliininen tutkijakouluActa Univ. Oul. D 1379, 2016Oulun 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 hedelmöittymiseenja 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:nesiintymistä sikiöiden ja aikuisten munasarjoissa.

TFF- ja ZA-potilaiden alkiokuoriproteiineja koodittavista geeneistä löytyi yhteensä 34nukleotidimuutosta. Muutoksista kolme oli uusia löydöksiä, mutta suurin osa oli ennalta tunnet-tuja yhden nukleotidin polymorfioita eli geneettisiä monimuotoisuuskohtia. TFF-potilaillahavaittiin ZP1- ja ZP3-geeneissä keskimäärin enemmän polymorfioita kuin verrokeilla. Myösosa yleisimmistä alkiokuoren rakennemuutoksista voidaan mahdollisesti selittää ZP1–ZP4-gee-neistä löytyneillä polymorfioilla.

Sikiöllä ZP3:n ilmentyminen oli havaittavissa jo 11. raskausviikolla, mutta voimakkainta seoli primordiaalivaiheen munarakkuloiden muodostumisen aikaan 20. raskausviikolla. Tämä voiviitata siihen, että ZP3 saattaa osallistua primordiaalivaiheen munarakkulan kehittymiseen ennenvarsinaisen alkiokuoren muodostumista. ZP-geenien säätelytekijän FIGLA:n esiintyminenlisääntyi 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 jasyventää tietämystämme alkiokuoren muodostumisesta ihmisellä. Hedelmättömyyden taustallaolevien 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

7

Acknowledgements

This study was carried out at the Department of Obstetrics 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

8

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

9

Abbreviations

aa amino acid

ADAM3 a disintegrin and metalloprotease 3

AMH antimüllerian hormone

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 sperm injection

IHC immunohistochemistry

ISH in situ hybridization

IVF in vitro fertilization

LH luteinizing hormone

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 fertility

10

ZA zona anomaly

ZAP-1 zona pellucida gene activating protein-1

ZP zona pellucida

ZP1–4 zona pellucida glycoproteins 1–4

11

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.

13

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 germ cell development during fetal life ................................ 19 2.1.1 FSH-independent follicle development ........................................ 20 2.1.2 FSH-dependent follicle development ........................................... 22 2.1.3 Ovulation ...................................................................................... 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 Acrosome reaction, gamete recognition and binding ................... 36 2.3.3 Species specificity ........................................................................ 39 2.3.4 Sperm–egg fusion ......................................................................... 40 2.3.5 Block of polyspermy .................................................................... 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

14

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

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6 Summary and conclusions 77 References 79 Original publications 95

17

1 Introduction

Infertility is defined as the inability to conceive after 12 months of regular

unprotected sexual intercourse. According to the European Society of Human

Reproduction and Embryology (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 embryo 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.

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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 chromosome from the father. Human

preimplantation development starts with the fusion of the egg and sperm procuclei

in the zygote 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 gametogenesis (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 meiosis 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 birth

(Soyal et al. 2000). FIGLA has also been associated with premature ovarian failure

in humans (Zhao et al. 2008, Tosh et al. 2014).

20

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 woman 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 death 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

eggs 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 menopause. 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 mitosis,

21

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

22

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).

23

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 menstrual cycle, 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

24

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 puberty 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).

25

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).

26

Fig. 3. Zona pellucida shown in a fertilized (day one) oocyte. ZP = zona pellucida, O =

oocyte, P = polar body, PVS = perivitelline space, PN = pronucleus.

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

27

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 cortical

reaction (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

(Bleil & Wassarman 1980b,

Goudet et al. 2008)

Pig (Goudet et al. 2008)

Cow (Noguchi et al. 1994)

Dog (Goudet et al. 2008)

Human (Lefievre et al. 2004)

Rat (Hoodbhoy et al. 2005)

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

28

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

29

2.2.4 Regulation of ZP genes

According to mouse studies, ZP genes are expressed in coordination during the

growth phase of oogenesis (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

30

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).

31

Fig. 4. Schematic representation of mammalian ZP proteins. Red arrow: cleavage 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

32

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).

33

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

34

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

35

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 acrosome reaction in zona-bound

spermatozoa and post-fertilization blockage of polyspermy (Yanagimachi 1994,

36

Gupta 2015). In early cleavage-stage embryos the ZP has been suggested to have a

shaping function, as it holds dividing blastomeres 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,

37

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).

38

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

ZP

protein

Binds to Effect Site of interest References

ZP1 Capacitated sperm, acrosome-

intact sperm: mainly acrosomal

cap, but also equatorial segment

AR ZP domain Ganguly et al. 2010a, b

ZP2 Acrosome-reacted sperm:

equatorial segment, acrosomal

cap, mid-piece

Sperm

binding

N-terminal domain,

aa residues 51–149

Chakravarty et al. 2008, Chiu

et al. 2008b, Avella et al.

2014, Baibakov et al. 2012

ZP3 Capacitated, acrosome-intact

sperm: mainly equatorial region

but also acrosomal cap and mid-

piece

AR,

Sperm

binding?

C-terminal domain,

N-linked

glycosylation

Chakravarty et al. 2008, Chiu

et al. 2008a, b,

Caballero-Campo et al.

2006, Bansal et al. 2009

ZP4 Capacitated, acrosome-intact

sperm: acrosomal cap, equatorial

region

AR N-linked

glycosylation

Chakravarty et al. 2008,

Caballero-Campo et al.

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 gametes (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,

39

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).

40

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 spermatozoon (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

41

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 reproductive system 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

42

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 blastocyst, 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).

43

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

pregnancies, 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.

44

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 fetus. 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

45

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 child 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).

47

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.

49

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 insemination), 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.

50

4.1.2 Human ovarian tissues (Study II)

A total of 18 ovarian samples were obtained from 10 fetuses (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 endometriosis.

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

51

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

52

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).

53

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

54

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

55

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.

56

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

Study Subjects & Material Methods Main results

I human DNA samples

from women with total

fertilization failure,

proven fertility and

fertilizers in IVF

DNA extraction

PCR

CSGE

sequencing

Women in the TFF group had a higher mean

number of sequence variations in ZP1 and

ZP3 when compared with the FIVF and WPF

groups.

II human fetal ovaries

human adult ovaries

ZP1: ISH

ZP3: IHC and ISH

FIGLA: ISH

The expression of ZP3 and FIGLA mRNA in

fetal ovaries increases at the time of follicle

formation, suggesting that these factors may

have a role in the development of PFs before

ZP formation in human.

III human DNA samples

from women with

zona anomalies

and proven fertility

DNA extraction

PCR

sequencing

Some of the most frequent zona anomalies

may be at least partly explained by sequence

variations in genes expressing the four

human ZP proteins.

57

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

58

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.

59

Ta

ble

5. O

bs

erv

ed

seq

ue

nc

e v

ari

ati

on

s i

n Z

P1

, Z

P2

, Z

P3

an

d Z

P4.

Ge

ne

S

eq

ue

nce

va

ria

tion

P

osi

tion

T

FF

1 n

(%

) F

IVF

2 n

(%

) W

PF

3 n

(%

) p4

S

NP

ZP1

c.2

77

G>

A,

p.V

93

I e

2

1 (

6)

0 (

0)

2 (

3)

0.5

52

rs

14

50

67

88

3

c.

473T

>C

, p.I158T

5

e 3

7

(3

9)

1 (

4)

18

(2

6)

0.0

26

rs

489172

c.

858C

>T

, p.F

286

5

e 5

8

(4

4)

8 (

35

) 2

8 (

41

) 0

.80

2

rs1

08

97

12

2

c.

1573

−81G

>A

i 9

10 (

56)

7 (

30)

22 (

32)

0.1

58

rs

2074418

ZP2

c.528+

60T

>C

5

i 6

4 (

22

) 5

(2

2)

18

(2

6)

0.8

69

rs

22

42

55

1

c.

747T

>C

, p.P

249

5

e 8

7

(3

9)

4 (

17

) 2

2 (

32

) 0

.27

5

rs2

07

55

26

c.

1830+

25G

>A

i 1

5

1 (

6)

5 (

22)

5 (

7)

0.1

10

rs

16971219

c.

20

95

+7

7T

>G

i 1

8

1 (

6)

0

(0

) 0

(0

) 0

.07

8

-

ZP3

c.1

−87

T>

G

5´U

TR

6

(3

3)

3 (

13

) 6

(9

) 0.0

27

rs

132332187

c.

91

G>

A,

p.G

31

R

e 1

5

(2

8)

7 (

30

) 2

0 (

29

) 0

.98

3

rs2

28

64

28

c.

43

1+

11

5G

>T

i 2

1

(6

) 0

(0

) 0

(0

) 0

.07

8

-

c.

536-1

7C

>A

5

i 3

3 (

17

) 0

(0

) 1

(1

) 0.0

06

rs

6966715

c.

54

7G

>A

, p

.A1

83

T

e 4

0

(0

) 1

(4

) 2

(3

) 0

.69

2

rs7

46

76

08

2

c.

714

−22

T>

C5

i 4

7 (

39

) 6

(2

6)

23

(3

4)

0.6

70

rs

39

72

76

8

c.

714

−51

C>

T5

i 4

0 (

0)

1 (

4)

2 (

3)

0.6

92

rs

14

61

47

90

2

c.

89

4G

>A

, p

.K2

98

e

6

9 (

50

) 0

(0

) 1

6 (

24

) 0.0

08

rs

73363163

ZP4

c.1

8C

>T

, p

.C6

e

1

1 (

6)

4 (

17

) 2

(3

) 0

.05

0

rs3

59

05

27

8

c.

401

−12

3A

>G

5

i 3

11

(6

1)

13

(5

7)

39

(5

7)

0.2

63

rs

22

36

59

5

c.

1495+

47A

>G

5

i 11

4

(2

2)

6

(2

6)

17

(2

5)

0.9

58

rs

22

75

69

4

c.

1623+

132A

>G

3´U

TR

8 (

42)

9 (

39

)

25

(3

7)

0.8

36

rs

27

94

81

3

1 to

tal n

um

be

r o

f w

om

en

in t

he

TF

F g

rou

p =

18

, 2 t

ota

l num

ber

in t

he F

IVF

gro

up =

23,

3 tota

l num

ber

in the W

PF

gro

up =

68,

4 p

-va

lue

s fo

r ch

i-sq

ua

red

te

st f

or

trend. S

tatis

tically

sig

nifi

cant diff

ere

nce

show

n in

bold

. e =

exo

n,

i = in

tro

n.

5 T

ypogra

phic

err

ors

in the c

orr

esp

ondin

g table

in the o

rigin

al a

rtic

le h

ave

been

corr

ect

ed in

this

ta

ble

.

60

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.

61

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

62

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.

63

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)

64

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

65

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

66

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.

67

Ta

ble

7. O

bs

erv

ed

seq

ue

nc

e v

ari

ati

on

s i

n Z

P1

, Z

P2

, Z

P3 a

nd

ZP

4.

Gene

S

equence

variatio

n

Posi

tion

Z

A1

n (

%)

W

PF

2 n

(%

) p3

S

NP

ZP1

c.2

77

G>

A,

p.V

93

I e

2

1 (

3%

) 2

(3

%)

1

rs1

45

06

78

83

c.

47

3 T

>C

, p

.I1

58

T

e 3

1

1 (

36

%)

18

(2

6%

) 0

.47

54

rs

48

91

72

c.

85

8C

>T

, p

.F2

86

e

5

12

(3

9%

) 2

8 (

41

%)

1

rs1

08

97

12

2

c.

1573

−81

G>

A

i 9

19

(6

1%

) 2

2 (

32

%)

0.0

08

6

rs2074418

ZP2

c.1

−73

G>

T

5´U

TR

2

5 (

81

%)

47

(6

9%

) 0

.33

09

rs

20

75

52

1

c.

10

7G

>T

, p

.G3

6V

e

2

1

1 (

35

%)

37

(5

4%

) 0

.08

84

rs

20

75

52

0

c.

11

6T

>G

, p

.I3

9R

e

2

12

(3

9%

) 0

(0

%)

0.0

00

1

-

c.

528+

60T

>C

i 6

8 (

26%

) 18 (

26%

) 1

rs2242551

c.

74

7T

>C

, p

.P2

49

e

8

19

(6

1%

) 2

7 (

39

%)

0.0

53

3

rs2

07

55

26

c.

973

−37

C>

T

i 9

1 (

3%

) 0 (

0%

) 0.4

133

rs

75227459

c.

1830+

25G

>A

i 1

5

5 (

16%

) 5 (

7%

) 0.2

786

rs

16971219

ZP3

c.1

−87

T>

G

5´

UT

R

10

(3

2%

) 6

(9

%)

0.0

06

5

rs13232187

c.

91G

>A

, p.G

31R

4

e 1

8

(2

6%

) 2

0 (

29

%)

0.8

12

3

rs2

28

64

28

c.

313+

65

G>

T4

i 1

23

(7

4%

) 4

2 (

62

%)

0.2

61

rs

64

65

12

8

c.

431+

11

5G

>T

i 2

1 (

3%

) 0 (

0%

) 0.3

131

-

c.

536

−14

3G

>C

i 3

8

(2

6%

) 2

6 (

38

%)

0.2

61

rs

69

66

71

5

c.

54

7G

>A

, p

.A1

83

T

e 4

3

(9

%)

2 (

3%

) 0

.17

55

rs

74

67

60

82

c.

662C

>G

, p.P

221

R

e 4

1 (

3%

) 0 (

0%

) 0.3

131

rs

139729790

c.

714

−22

T>

C

i 4

20

(6

5%

) 2

3 (

34

%)

0.0

08

1

rs3972768

c.

714

−51

C>

T

i 4

3 (

9%

) 2 (

3%

) 0.1

755

rs

146147902

ZP4

c.1

−42

9A

>G

5

´UT

R

2 (

6%

) 5

(7

%)

1

rs7

56

47

22

2

c.

18C

>T

, p.C

6

e 1

2 (

6%

) 2 (

3%

) 0.5

873

rs

35905278

c.

176

−48

G>

A

i 1

16

(5

2%

) 3

3 (

49

%)

0.8

30

5

rs5

38

49

9

68

Gene

S

equence

variatio

n

Posi

tion

Z

A1

n (

%)

W

PF

2 n

(%

) p3

S

NP

c.

401

−12

3A

>G

i 3

2

3 (

74

%)

39

(5

7%

) 0

.12

27

rs

22

36

59

5

c.

554

−40G

>A

i 4

2 (

6%

) 1 (

1,5

%)

0.2

303

rs

114491365

c.

77

4A

>G

, p

.E2

58

e

6

2 (

6%

) 1

(1

,5%

) 0

.23

03

rs

52

07

20

c.

83

9A

>G

, p

.S2

77

G

e 6

2

(6

%)

1 (

1,5

%)

0.2

30

3

rs3

60

17

13

8

c.

839+

28A

>G

i 6

2 (

6%

) 1 (

1,5

%)

0.2

303

rs

510549

c.

1160+

31A

>G

i 8

2 (

6%

) 1 (

1,5

%)

0.2

303

rs

1209519

c.

14

95

+4

7A

>G

i 1

1

14

45

%)

17

(2

5%

) 0

.06

16

rs

22

75

69

4

c.

16

23

+1

32

A>

G

3´U

TR

2

1 (

68

%)

25

(3

7%

) 0.0

05

rs

2794813

1 to

tal n

um

ber

of w

om

en in

the Z

A g

roup =

31,

2 to

tal n

um

ber

in the W

PF

gro

up =

68,

3 p

-valu

es

for

chi-sq

uare

d t

est

for

trend. S

tatis

tically

sig

nifi

cant

diff

ere

nce

show

n in

bold

. e =

exo

n, i =

intr

on.

4 T

ypogra

phic

err

ors

in the c

orr

esp

on

din

g table

in the o

rigin

al a

rtic

le h

ave

been c

orr

ect

ed in

this

table

.

69

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

70

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).

71

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.

72

Table 8. Sequence variations and zona anomalies.

Gene Sequence variation Zona splitting Oval shaped

zona

Thin zona Thick 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

73

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

pregnancy rate (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.

74

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.

75

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.

77

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

78

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.

79

References

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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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