Aus dem Institut für Reproduktionsmedizin der ...My father, Mr. S. M. Leal, My mother, Ms. E. M. Leal - ... IP (2) Inositol 4, 5 -triphosphate IP (3) Inositol 1, 4, 5 -triphosphate

Aus dem Institut für Reproduktionsmedizin der Tierärztliche Hochschule Hannover und dem Institut für Tierzucht (Mariensee) der Bundesforschungsanstalt für

Landwirtschaft (FAL) Braunschweig __________________________________________________________ BIOCHEMICAL AND STRUCTURAL ALTERATION OF THE PORCINE ZONA PELLUCIDA (ZP) DURING MATURATION

(IN VIVO AND IN VITRO) AND FERTILIZATION (IVF)

INAUGURAL - DISSERTATION Zur Erlangung des Grades einer

Doktorin der Veterinärmedizin (Dr. med. Vet.)

durch die Tierärztliche Hochschule Hannover

Vorgelegt von A. C. Moreira Aus Brasilien

Hannover 2005

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Wissenschaftliche Betreuung: Univ.-Prof. Dr. rer. nat. Dr. med. habil. Edda Töpfer-Petersen

1. Gutachter/in: Univ.-Prof. Dr. rer. nat. Dr. med. habil. Edda Töpfer-Petersen 2. Gutachter: Univ.-Prof. Dr. Wilfried Meyer

Tag der mündlichen Prüfung: 16.11.2005 Angefertigt mit finanzieller Unterstützung: Deutscher Akademischer Austauschdienst – DAAD - Coordenacão CAPES – Brazil – My father, Mr. S. M. Leal, My mother, Ms. E. M. Leal - Deutsche Forschungsgemeinschaft – DFG –

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Dedicated to “Got”

(A.C.M.)

CONTENTS ___________________________________________________________________

CONTENTS

LIST OF ABBREVIATIONS……………………………………………….....9 1 INTRODUCTION…………………………………………………………..13 2 REVIEW OF THE LITERATURE………………………………………...16 2.1 Biology of the Female Gamete……………………………………………..16 2.1.1 Oogenesis………………………………………………………………………........16

2.1.2 Oocyte Growth……………………………………………………………………….16

2.1.3 Folliculogenesis….............................................................................................18

2.2 Maturation of Cumulus-Oocyte Complexes.……………………….........20 2.2.1 In Vitro Maturation (IVM) of Oocytes…………………………….…….................20 2.2.1.1 Role of Cumulus Cells During Maturation………………………………………………..20

2.2.1.2 Maturation Media……………………………………………………………………….......21 2.2.1.2.1 Media Additives………………………………………………………………………………………22

2.2.1.3 Nuclear Maturation…..................................................................................................22

2.2.1.4 Cytoplasmic Maturation…………………………………………………………………….23

2.2.1.5 Distribution of Organelles During Maturation…………………………………………….24 2.2.1.5 1 Cortical Granule Translocation to the Plasma Membrane During Maturation………………...24 2.2.1.5.1 1 Role of Actin Cytoskeleton on Cortical Granule (CG) Translocation During Maturation………………..25

2.3 Biology of the Zona Pellucida (ZP)………………………………………...26 2.3.1 The Zona Pellucida (ZP)…………………………………………………………….26

2.3.2 Morphology, Protein Nomenclature and Genomic Organization of the Zona

Pellucida (ZP)……………………………………………………………………………….27

2.3.3 Carbohydrates of Zona Pellucida (ZP) Glycoproteins and Their Roles in the

Fertilization Process………………………………………………………………………..30

2.3.4 Structural Domains of the Zona Pellucida (ZP) Proteins…………………..........32 2.3.4.1 Position of Cysteine (Cys) on the Zona Pellucida (ZP) Protein Domains…...............32

2.4 Fertilization Cascade (FC)…………………………………………………..34 2.4.1 Sperm Capacitation and the Acrosome Reaction (AR)………………………….34

2.4.2 Sperm Binding to the Oocytes’Zona Pellucida (ZP)……………………………...36

2.4.3 Penetration by Spermatozoa of the Zona Pellucida (ZP)…………....................36

2.4.4 Gamete Interaction: Sperm Fusion and Oocyte Activation……………………...37

CONTENTS ___________________________________________________________________

2.5 Polyspermy Block…………………………………………………………….38 2.6 Cortical Granules (CG)……………………………………………………….39 2.6.1 Cortical Granule Contents and Their Relevance to the Polyspermy Block in

Different Species…….................................................................................................40 2.6.1.1 Cortical Granule Contents and Their Relevance to the Polyspermy Block in Sea

Urchins………………………………………………………………………………………………..40 2.6.1.1.1 Ovoperoxidase……………………………………………………………………………………….41

2.6.1.1.2 Protease………………………………………………………………………………………………42

2.6.1.1.3 Glycosaminoglycans………………………………………………………………………………...42

2.6.1.2 Cortical Granule Contents and Their Relevance to the Polyspermy Block in Mammals

………………………………………………………....................................................................42 2.6.1.2.1 Protease………………………………………………………………………………………………42

2.6.1.2.2 Glucosaminidase…………………………………………………………………………………….44

2.6.1.2.3 Lectin……………………………………………………………………………………………….....44

2.7 Zona Pellucida Hardening……………………………………………..........44

3 MATERIALS AND METHODS…………………………………………...46 3.1 In Vitro Maturation (IVM) of Porcine Oocytes……………………………47 3.1.1 Collection of Ovaries………………………………………………………………...47

3.1.2 Isolation of Cumulus-Oocyte Complexes (COCs)……………………......………47

3.1.3 Classification of Cumulus-Oocyte Complexes (COCs)…..……………………...48

3.1.4 In Vitro Maturation (IVM)……………………………………………………………48

3.1.5 Maturation Rate (IVM)………………………………………………………………49

3.2 In Vivo Maturation (IVM) of Porcine Oocytes…………………………….50 3.2.1 Collection of In Vivo Matured (IVM)Porcine Oocytes…………………………….50

3.3 In Vitro Fertilization (IVF)…………………………………………………....51 3.3.1 Fertilization Rate……………………………………………………………………..51

3.4. Isolation of Intact Zona Pellucida (ZP) from Oocytes of Different Developmental Stages……………………………………………………………52 3.5. Experimental Design………………………………………………………...52 3.6 Pre-Trial I: Selection of Adequate pH Conditions to Indicate Oxidant–Sensitive Thiol Groups…………………………………………………………...54 3.6.1 SDS–PAGE Electrophoresis………………………………………………….........54

CONTENTS ___________________________________________________________________

3.6.1.1 Labelling of Free Thiol Groups……………………………………………………….……54

3.6.1.2 One–Dimensional Gel Electrophoresis (SDS–PAGE)…………………………............55

3.6.1.3 Two–Dimensional Gel Electrophoresis (SDS–PAGE)…………………………............55

3.6.1.4 Protein Blot and Chemiluminescence Analysis ………………………………………...56

3.7 Trial II: Laser Scanning Confocal Microscopy (LSCM)………………...57 3.7.1 Labelling of Free Thiol Groups……...................................................................57

3.7.2 Specificity of the Labelling Procedures……………………………………………57

3.7.3 Labelling of Spermatozoa Bound to the Zona Pellucida (ZP) with

Propidiumiodide (PI)………………………………………………………………………..58

3.7.4. Evaluation and Classifcation of Labelling Patterns of Free Thiol Groups by

Laser Scanning Confocal Microscopy……………………………………………………58 3.7.4.1 Fluorescence Pattern: Intensity Difference (D) Between Inner and Outer Regions of

the Zona Pellucida (ZP)……………………………………………………………………………..59

3.7.4.2 Homogeneity Pattern (H)…………………………………………………………………..60

3.8 Trial III: Scanning Electron Microscopy (SEM)……………………….....61 3.8.1 Labelling of Free Thiol Groups……………………………………………………..61

3.8.2 Classification of Labelling Pattern………………………………………………….62

3.9 Statistical Analysis………………………………………………………….................63

4 RESULTS…………………………………………………………………..64 4.1 Pretrial I: Selection of Adequate pH Conditions to Indicate Oxidant–Sensitive

Thiol Groups………………………………………………………...................................64

4.1.1 Polyacrylamide Gel Electrophoresis (SDS–PAGE)………….............................64 4.1.1.1 One-Dimensional Gel Electrophoresis (SDS–PAGE)…………………………………..64

4.1.1.2 Two-Dimensional Gel Electrophoresis (SDS–PAGE)………………………................65

4.2 Trial II: Laser Scanning Confocal Microscopy (LSCM).………………..67 4.2.1 Specificity of the Labelling Procedures …………………………………………...67

4.2.2 Fluorescence Pattern of the Zona Pellucida (ZP) During In Vitro Maturation

(IVM)…………………………………………………………………………………………68

4.2 3 Fluorescence Pattern of the Zona Pellucida (ZP) During In Vivo Maturation....74

4.2.4 Fluorescence Pattern During In Vitro Fertilization (IVF)…………………………79 4.2.4.1 Fluorescence Pattern of the Zona Pellucida (ZP) from In Vitro Matured (IVM)

Oocytes after Onset of In Vitro Fertilization (IVF)………………………………………………..79

CONTENTS ___________________________________________________________________

4.2.4.2 Fluorescence Pattern of the Zona Pellucida (ZP) from In Vivo Matured Oocytes after

Onset of In Vitro Fertilization (IVF)…………………………………………………………………84

4.2.5 Incubation of Oocytes Matured In Vivo and In Vitro (IVM) but without Exposure

to Spermatozoa……………………………………………………………………………..88

4.2.6 Evaluation of the Fertilization System (IVF)…………….…………………...……89

4.3 Trial III: Scanning Electron Microscopy (SEM)………………………….90 4.4 Sperm Binding to the Zona Pellucida (ZP) under In Vitro Fertilization (IVF) Conditions.............................................................................................96

5 DISCUSSION………………………………………………………………98 5.1 Concluding Remarks……………………………………………………….117

6 ZUSAMMENFASSUNG…………………………………………………118 7 SUMMARY………………………………………………………………..122 8 REFERENCES……………………………………………………………126 9 APPENDIX………………………………………………………………..150 9.1 Composition of Some Media Used in In Vitro Maturation (IVM), In Vitro Fertilization (IVF), and In Vitro Culture (IVC) Experiments.………………150 9.1.1 Physiological Saline Solution Medium……………………………………………150

9.1.2 Dulbecco´s Phosphate Buffered Saline (PBS) Media………………………….150

9.1.3 Maturation Media used for IVM…………………………………………………...151

9.1.4 Fertilization Media used for IVF…………………………………………………..151

9.1.5 Cultivation Media…………………………………………………………………...151

9.2 Composition of Reagents Prepared for Use in Biochemical Experiments………………………………………………………………………153 9.2.1 Acrylamide Solution………………………………………………………………..153

9.2.2 SDS Electrophoresis (LAEMMLI 1970)…………………………………………..153

9.2.3 Loading Buffer ……………………………………………………………………...154

9.2.4 Immobilized pH Gradients (IPG)………………………………………………….154

9.2.5 Blotting to PVDF Membranes……………………………………………………..154

9.2.6 Ponceau Staining…………………………………………………………………..154

9.2.7 TBS/Tween………………………………………………………………………….154

CONTENTS ___________________________________________________________________

9.3 Composition of Reagents Prepared for Use in Biochemical Experiments with Laser Scanning Confocal Microscopy (LSCM) and Scanning Electron Microscopy (SEM)…………………………………….....155 9.3.1 HEPES – Fixation…………………………………………………………………..155

9.3.2 Poly-L-Lysine Coverslips…………………………………………………………..156

9.3.3 Mowiol Mounting Medium pH 6.5…………………………………………………156

9.4 Tables and Figures………………………………………………………….156 9.4.1 Tables………………………………………………………………………………..156

9.4.2 Figures……………………………………………………………………………….158

ACKNOWLEDGMENTS…………………………………………………...163

LIST OF ABBREVIATIONS ___________________________________________________________________

LIST OF ABBREVIATIONS In addition to the commonly used abbreviations for time, weight, volume,

concentration, temperature, and inorganic compounds, the following abbreviations

appear in this dissertation without explanation.

AR Acrosome reaction BIAM Biotin-Conjugated Iodoacetamide – Biotin Iodoacetamide (N-

(Biotinoyl) N´-(Iodoacetyl) Ethylenediamine- Bis-Tris

BisTris N, N-bis (hydroxyethyl)-N,N,N- (Hydroxylmethyl)-Aminomethane

BSA Bovine serum albumin CGs Cortical granules CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanosulfonate COCs Cumulus-oocyte complexes Cys Cysteine residues DIC Differential interference contrast DIP Differential phase contrast DT Dityrosine DTT Dithiothreitol E (2) Estradiol-17ß ECL Enhance chemiluminescence ECM Extracellular matrix EDTA Ethylene-diamine-tetraacetic acid EGF Epidermal growth factor ER Endoplasmic reticulum EtOH Ethanol Exp Experiment FC Fertilization cascade FCS Fetal calf serum FE Fertilization envelope FITC Fluorescein isothiocyanate FITC-PNA Peanut agglutinin labelled with fluorescein isothiocyanate FSH Follicle stimulating hormone GlcNAc N-acetylglucosamine GV Germinal vesicle GVBD Germinal vesicle breakdown hCG Human chorionic gonadotropin HEPES (N-[2-Hydroxyethyl] piperazine-N´-[2-ethanesulfonic acid]) HRP Horseradish peroxidase IAM Iodoacetamide ICM Inner cell mass IEF Isoelectric focusing IP (2) Inositol 4, 5 -triphosphate IP (3) Inositol 1, 4, 5 -triphosphate


IPG–PAGE Immobilized pH gradients of the first-dimension IU International Unit IVM In Vitro Maturation IVF In Vitro Fertilization IVP In Vitro Production LH Luteinizing hormone LSCM Laser scanning confocal microscopy MI Metaphase I MII Metaphase II mBBr Monobromobimane MPN Male pronuclei Mr Molecular weight NBCS Newborn calf serum NCSU North Carolina State University N -Linked NH2 group of the side chain of an asparagine residue O-Linked OH group of the side chain of a serine or threonine residue P Probability PA Plasminogen PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline pKa Acid constant PKC Protein kinase C PMSG Pregnant mare serum gonadotropin pOGP Porcine oviduct-specific glycoprotein pOECs Porcine oviductal epithelial cells PVA Polyvinyl alcohol PVP Polyvinyl pyrrolidone PVDF Polyvinylidene difluoride PVS Perivitelline space pZP Porcine zonae pellucidae ROS Reactive oxygen species RT Room temperature SDS–PAGE Sodium dodecyl sulfate polyacrylamide gels SEM Scanning electron microscopy –SH Free thiol groups (sulfhydryl groups) SeCys Selenocysteine SOF Synthetic oviductal fluid Tab. Table TALP Tyrode, albumin, lactate, pyruvate TCM Tissue culture medium TEM Transmission electron microscopy TEMED N, N, N`N`- tetramethylethylendiamin TRIS Tris (hydroxymethyl)-aminomethan ZP Zona pellucida ZP-free Zona pellucida-free ZR Zona pellucida reaction ZT Zona thinning


1–D

First–dimensional polyacrylamide gel electrophoresis (SDS–PAGE)

2–D

Second–dimensional polyacrylamide gel electrophoresis (SDS–PAGE)

5–IAF 5–iodoacetamidefluorescein

INTRODUCTION ___________________________________________________________________

13

1 INTRODUCTION All vertebrate oocytes are surrounded by an extracellular matrix. In mammals this

matrix is known as the zona pellucida (ZP). One of its functions is to prevent further

sperm penetration (e.g. polyspermy block) caused by the so-called zona block (e.g.

zona reaction), a biochemical alteration of the ZP glycoproteins induced by release of

cortical granule (CGs) exudates (e.g. cortical reaction) during fertilization. After

fertilization in the bovine, oxidation of free cysteine (Cys) thiols between ZP

glycoproteins (ZPA, ZPB, and ZPC) results in the formation of intra– and

intermolecular disulfide bonds (IWAMOTO et al. 1999). The formation of disulfide

bonds is thought to be associated with a crucial alteration of the three–dimensional

structure of the ZP glycoproteins, which may play a strong role in the prevention of

further sperm penetration by the construction of a rigid ZP structure involved in the

so-called hardening of the zona pellucida. In mice, a proteinase released from the

CGs after sperm penetration has been implicated in the conversion of the zona

glycoprotein ZP2 to produce ZP2f, a form that is not capable of binding sperm (BLEIL

et al. 1981, MOLLER and WASSARMAN 1989, MOOS et al. 1994). This proteinase

and other cortical granules enzymes have been implicated in the block to polyspermy

by inhibiting sperm binding to ZP2 (DANDEKAR and TALBOT 1992). The proteinase

described by MOLLER and WASSARMAN (1989) is also observed in the pig

(HEDRICK 1993, HASEGAWA et al. 1994, cited in FUNAHASHI 2003), bovines

(NOGUCHI et al. 1994), and humans (BAUSKIN et al. 1999). However, the exact

correlation of disulfide bond formation and the specific proteolysis of pZP1/ZPA

during fertilization with the hardening of the ZP proteins remain to be determined.

SCHMELL and GULYAS (1980) proposed that mammalian (e.g. murine) CGs contain

another enzyme (e.g. ovoperoxidase) that has been implicated in zona hardening by

cross-linking tyrosine residues of the zona pellucida glycoproteins. In invertebrates

(e.g. sea urchin eggs) enzymatic modification – possibly by the peroxidase present in

the CGs of unfertilized eggs (KLEBANOFF et al. 1979, SOMERS et al. 1989, cited in

WESSEL et al. 2001) – is also responsible for the formation of the fertilization

envelope (FE) proteins by an oxidative process following fertilization (MOTOMURA

INTRODUCTION ___________________________________________________________________

14

1954, MOTOMURA 1957, SHAPIRO 1977, HALL 1978, GULYAS and SCHMELL

1980, SCHMELL and GULYA, 1980, cited in WESSEL et al. 2001 and GREEN

1997). In the pig, the positions of cysteine (Cys) residues in the ZP proteins are

highly conserved (HARRIS et al. 1994, cited in PRASAD et al. 2000) and most of

these residues form disulfide linkages (intramolecular) contributing mainly to the

maintenance of the zona pellucida structure in this species (DUNBAR 1983,

DUNBAR et al. 1994, cited in IWAMOTO et al. 1999). In the present study, the

following types of oocytes were produced and compared with particular reference to

the distribution and reduced state of cysteine thiol residues in the ZP glycoproteins:

immature oocytes (germinal vesicle, Exp. I); in vitro matured (IVM) oocytes for 23 h

(metaphase I, Exp. II); in vitro matured (IVM) oocytes for 46-48 h (metaphase II, Exp.

III); in vivo matured oocytes (metaphase II, Exp. IV); oocytes derived from in vitro

fertilization (IVF) of in vitro matured (IVM) oocytes 0, 3, and 18 h after IVF (Exp. V);

and oocytes derived from IVF of in vivo matured oocytes 0, and 3 h after onset of IVF

(Exp. VI).

The reactivity (i.e. pKa value) of most protein cysteine thiols is approximately 8.5, and

only certain cysteine (Cys) residues exist under physiological pH (≈7.10 to 7.29) as

thiolate anions (Cys-S-) essential for disulfide formation (KIM et al. 2000,

SETHURAMAN et al. 2004). In the biochemical experiments (Pre–Trial I) described

in this thesis, the incorporation of the fluorescence marker in the free cysteine thiols

was tested by two electrophoretic methods (one-dimensional gel electrophoresis, 1–

D SDS–PAGE, and two-dimensional gel electrophoresis, 2–D SDS–PAGE) under

different pH conditions, pH 6.5 and pH 8.5, with a thiol – specific agent, i.e. biotin-

conjugated iodoacetamide, BIAM (KIM et al. 2000), in order to test the pH levels with

the most specific labelling pattern for further morphological evaluation. In the

following morphological study the ZP from different sources was analyzed with

particular reference to the distribution of the free cysteine thiols and the disulfide

status by means of laser scanning confocal microscopy (LSCM, Trial II) and scanning

electron microscopy (SEM, Trial III). For LSCM, the ZP was completely removed and

treated with fluorescent–labelled, i.e. 5-Iodoacetamidefluoresceín, – 5-IAF, at pH 6.5.

For SEM, the ZP was treated with biotin-conjugated iodoacetamide (BIAM) under pH

INTRODUCTION ___________________________________________________________________

15

6.5 and subsequently monitored with streptavidin gold particles. This study also

examined the importance of spermatozoa for the structural changes in the ZP

proteins in order to determine the influence of the fertilization medium (e.g. FertTalp)

on the consumption of free cysteine thiols following fertilization (Exp. VII). Moreover,

this investigation also analyzed the effect of different maturation conditions (in vivo

versus in vitro) on the number of spermatozoa bound to the zona pellucida (ZP) 12 h

after onset of IVF in order to evaluate the functional role of the ovarian

microenvironment in the completion of final ZP maturation.

The initial experiments described in this thesis examined and compared the

distribution of free cysteine thiol residues of the ZP during the initial (germinal

vesicle) to final stages of oocyte maturation (M I [23 h of IVM] and M II [46-48 h of

IVM]) prior to fertilization (IVF) in order to determine if there was a close correlation

between the morphological appearance of the zona and the distribution of free

cysteine thiol residues within the ZP glycoproteins. The porcine oocytes which had

undergone in vivo maturation (MII) were examined in comparison with oocytes

matured in vitro (MII, IVM) as indicators for the physiologic ability of the matured

oocytes to induce biochemical and structural alterations within the ZP following

fertilization (IVF). The bulk of our experiments were aimed at following novel disulfide

bond formation within the ZP of the porcine oocytes of different developmental

stages (in vivo vs. in vitro matured [IVM] oocytes) during in vitro fertilization (0, 3, and

18 h after onset of IVF). The aim was to analyze those changes within the ZP

glycoproteins which may be correlated with the effective establishment of a block to

polyspermy. It is hoped that the results of this examination of the density and

distribution of free cysteine thiol residues of the ZP glycoproteins before maturation

(immature oocytes), after maturation (in vivo vs. in vitro matured [IVM] oocytes), and

following fertilization (IVF) may be an important further contribution towards our

understanding of the mechanisms leading to the block to polyspermy.

REVIEW OF LITERATURE ___________________________________________________________________

16

2 REVIEW OF THE LITERATURE 2.1. Biology of the Female Gamete 2.1.1 Oogenesis The process of oogenesis is initiated early in life, when the primordial germ cells

invade the genital ridge of the embryo (LEIBFRIED-RUTLEDGE et al. 1987).

Towards the end of sexual differentiation, the ovarian germ cells undergo rapid

growth and changes in the distribution of cytoplasmic organelles before being

transformed into oogonia. The oogonia population increases rapidly through

extensive mitotic proliferation for a period before differentiating into primary oocytes.

At this stage the first meiotic division begins (usually before birth in mammals); the

DNA replicates so that each chromosome consists of two sister chromatid, and

crossing-over occurs between non-sister chromatids of these paired chromosomes.

After these events, the cell remains arrested in the prophase of division I of meiosis

as the primary oocyte grows for a period lasting from a few days to many years.

During this long period, the primary oocytes synthesize an extracellular matrix, the –

zona pellucida (ZP) – and cortical granules (CGs). In large non-mammalian oocytes,

these oocytes also accumulate ribosomes, yolk, glycogen, lipid, and the mRNA that

will later direct the synthesis of proteins required for early embryonic growth and the

unfolding of the developmental program.

2.1.2 Oocyte Growth In mammals oocyte growth and development is linked with the acquisition of meiotic

competence called oocyte maturation. Development into mature oocytes does not

occur until sexual maturity, when the oocyte is stimulated by hormones. Under these

hormonal influences, the cell resumes its progress through division I of meiosis


17

(prophase I). The chromosomes recondense, the nuclear envelope breaks down

(GVBD, generally taken to mark the beginning of maturation), and the replicated

homologous chromosomes segregate at anaphase I into two daughter nuclei, each

containing half the original number of chromosomes. To end division I, the cytoplasm

divides asymmetrically to produce two cells that differ greatly in size: one is the small

polar body; and the other is a large secondary oocyte, the precursor of the egg. At

this stage, each of the chromosomes is still composed of two sister chromatids.

These chromatids do not separate until division II of meiosis, when they are

partitioned into separate cells. After this final chromosome separation at anaphase II,

the cytoplasm of the large secondary oocyte again divides asymmetrically to produce

the mature egg or ovum, and a second small polar body, each with a haploid set of

single chromosomes (Fig.1).

Fig. 1: Schematic representation of stages in the developmental transition of oogonia – to

mature eggs (meiosis II) of the female gamete. See text for details.

The ability to complete the transition from metaphase I (MI) to metaphase II (MII) is

achieved in oocytes that have reached their full size (Fig. 2). Oocytes undergo a 500-


18

fold increase in volume, corresponding to an increase in the diameter of the oocyte

from 10–12 µm in a primordial follicle to 70–80 µm in a mature follicle (THIBAULT et

al. 1987, MORENO et al. 2002).

Fig. 2: At ovulation, the arrested secondary oocyte is released from the ovary and undergoes

a rapid maturation step to prepare for fertilization. If fertilization occurs, the oocyte is

stimulated to complete meiosis and begin embryonic development (From LEIBICH 1993,

120x and 300x).

2.1.3 Folliculogenesis Folliculogenesis is described as a process in which the primordial follicle increases in

size 400 to 600 times prior to ovulation and as a rare biological event, because

99.9% of the primordial follicles do not reach ovulation (IRELAND 1987). The

extensive reduction in the number of growing follicles is caused by a wave of atresia

(regression, Fig. 3 A), which is at a maximum between 1 and 4 months after birth.

This occurs first in the oocytes and later in the granulosa cells (MARIANA et al.

1991). DRIANCOURT (1991) classified folliculogenesis into basal and a tonic types

based on the requirements for gonadotropin. Basal folliculogenesis can proceed, at

least partially, independent of gonadotropins support, while tonic folliculogenesis

progresses only under the influence of gonadotropins. After the acquisition of FSH


19

receptors (gonadotropin), the number of follicular granulosa cells increases and they

form concentric layers around the oocytes. Throughout this growth period, the

oocytes remain arrested in prophase of meiosis I (dyctiate stage) for a prolonged

period prior to development into mature oocytes in the preantral developing follicle.

The fully grown follicle thus contains large oocytes surrounded by several layers of

granulosa cells. Many of these cells will stay with the ovulated oocytes, forming the

cumulus oophorus. In addition, during the growth of the follicle, change in the shape

of the granulosa cells forms an antrum (cavity) (Fig. 3 B), which becomes filled with a

complex mixture of proteins, hormones, and other molecules. For oocyte maturation,

follicles need to be at a certain stage of development when the waves of

gonadotropin arise (FSH and LH). After these events, maturing follicles respond to

these hormones by further growth and cellular proliferation. FSH induces the

formation of LH receptors on the granulosa cell, and meiosis continues in response

to LH.

Fig. 3: Schematic representation of development the follicle in female gamete. A: The

reduction in the number of growing follicles is caused by a multiple waves of atresia from

primordial until dominant follicle. B: The population of growing follicles can be classified

according to the size, the number of granulosa cells, the presence or absence of an antrum,

and the number of cell layers as a, primordial follicle, b, secondary follicle, c, d, preantral

follicle, e, early antral follicle, and f, preovulatory Graafian follicle.


20

2.2 Maturation of Cumulus-Oocyte Complexes 2.2.1 In Vitro Maturation (IVM) of Oocytes

2.2.1.1 Role of Cumulus Cells During Maturation The beneficial communication between intact cumulus oophorus and oocyte (gap

junctional communication, GJC) for maturation and/or developmental ability of the

oocyte has been demonstrated in a number of species (mouse, SCHROEDER and

EPPIG 1984; cow, MADISON et al. 1992; pig, LAWRENCE et al. 1978, EPPIG

1982). An intact intercellular communication between the oocyte and its surrounding

cumulus cells is essential for normal oocyte growth and maturation (EPPIG and

SCHROEDER 1986, GIL et al. 2004). This gap junctional communication participates

in signalling meiosis block and resumption, provides energy for oocyte maturation

(SATO et al. 1977), and is required for the oocyte to achieve cytoplasmic maturation

(VANDERHYDEN and ARMSTRONG 1989, VAN SOOM at al. 2002, cited in GIL et

al. 2004) and to prevent precocious hardening of the ZP (De FELICI and SIRACUSA

1982, DOWNS et al. 1986, cited in FATEHI et al. 2002) by preventing premature

migration of cortical granules (CG) underneath the oolemma (GIL et al. 2004).

YANG and LU (1990) reported that only 48.8% of the denuded oocytes cleaved in

vitro progressed to the later stages, in contrast to 82% of the cumulus intact cells and

none of the denuded oocytes. Similarly, other workers found that the naked oocytes

had lower frequencies of maturation and fertilization in vitro (SHIOYA et al. 1988,

KIM and PARK 1990, YAMAUCHI and NAGAI 1999, FATEHI et al. 2002, VAN

SOOM et al. 2002).


21

2.2.1.2 Maturation Media Many different media including North Carolina State University medium 37 (NCSU–

37, PETTERS and WELLS 1993), North Carolina State University medium 23

(NCSU–23, PETTERS and WELLS 1993, HYUN et al. 2003), TCM 199 (RHO et al.

2001, HYUN et al. 2003), and Whitten´s medium (FUNAHASHI et al. 1994, KA et al.

1997) have been used for in vitro maturation (IVM) of porcine oocytes. HYUN et al.

(2003) compared two oocyte maturation media (NCSU–23 and TCM199) and

obtained similar results with both. However, in a similar study, MAO et al. (2002)

found that the choice of maturation media influenced the differentiation rate of

subsequent embryo development. Further, they observed that the maturation media

NCSU–23 resulted in higher follicular growth rate and antrum formation than that

obtained with TCM199.

In most of the maturation media pH regulation is achieved with HCO3/CO2, but it has

been demonstrated that supplementation with HEPES achieves greater stability of

the pH. The pH of the commonly used media varies between 7.1 and 7.4 (WRIGHT

and O´FALLON 1987). Most workers have used media with pH adjusted to 7.4.

SHEA et al. (1976) matured the oocytes in media with the pH ranging from 6.70 to

7.59 and reported that in the bovine the highest maturation rate (69%) was obtained

at pH 7.00 to 7.29.

The osmolarity of the commonly used maturation media varies between 276 and 316

mOs (WRIGHT and O´FALLON 1987). While THIBAULT (1977) suggested an

osmolarity of 290 mOs for the maturation medium, SATO et al. (1977) preferred

media with high osmolarity of 316-355 mOs.

The importance of water quality in defined in vitro culture media has also been

reported in a study on cattle (BAVISTER et al. 1992). KEEFER et al. (1991) indicated

that the quality of water has an influence on the success of embryo culture in vitro. In


22

their study, none of the oocytes progressed to the blastocyst stage when cultured in

media prepared from purchased deionized distilled water.

2.2.1.2.1 Media Additives Sera, gonadotropic hormones, and granulosa cells (HENG et al. 2004) are the

common additives used in the maturation media to improve the in vitro maturation

conditions. In addition, low-molecular weight thiols such as cysteamine, (for reviews,

see YOSHIDA et al. 1993b, GRUPEN et al. 1995), amino acids (KA et al. 1997),

follicular fluid (YOSHIDA et al. 1992, KIM et al. 1997), oviductal epithelial cells (BUHI

et al. 1993, KOUBA et al. 2000, BUHI 2002, McCAULEY et al. 2003), and a number

of growth factors have also been used to improve the in vitro development of

oocytes.

2.2.1.3 Nuclear Maturation

The prophase I immature oocyte displays a nucleus, and the germinal vesicle, which

appears spherical in shape, and contains a single, large eccentrically located

nucleolus. The germinal vesicle moves from a central position in the ooplasm to a

more peripheral situation at the time of the LH surge and just prior to the germinal

vesicle breakdown (GVBD) in the first step of meiosis resumption. The metaphase I

(MI) stage is determined by default in an oocyte displaying no polar body and no

germinal vesicle (MANDELBAUM 2000). Generally, the nucleus of the oocyte is

mature when the first polar body is extruded and the oocyte is arrested in metaphase

II (MII). The occurrence of defects in the meiotic spindle may be one of the reasons

for failure of the normal termination of subsequent development (CHEN et al. 2004).

In mammals, VOGEL and SPIELMANN (1992) found that chromosomal aberrations

are largely confined to the female pronucleus, indicating developmental compromise

prior to fertilization.


23

2.2.1.4 Cytoplasmic Maturation Several investigators have demonstrated that the cytoplasmic development of

matured oocytes seems to be essential to confer the capability for normal embryonic

development in oocytes matured and fertilized in vitro. In the pig, the failure of most

in vitro matured (IVM) and fertilized oocytes is attributable to abnormalities of

fertilization, including polyspermy and asynchrony of male and female pronuclear

formation (for reviews, see HUNTER 1990, cited in GRUPEN et al. 1995).

Asynchronous pronuclear formation is characterized by a delay in male pronuclear

development following the normal formation of the female pronucleus. The synthesis

and accumulation of male pronuclear growth factor(s), by which the oocyte acquires

the ability to decondense sperm heads, is thought to occur as a result of complete

cytoplasmic maturation during oocyte maturation (IVM) and fertilization (IVF) in vitro

(for review, see DING et al. 1992, cited in GRUPEN et al. 1995).

In pigs, a major problem of the polyspermic penetration with IVM and IVF systems

seems to be delayed and/or reduced ability to protect the oocytes against multiple

sperm penetration (RATH 1992), which is characterized by a incompetent maturation

oocyte system. Oocytes that have completely reached MII develop the full ability to

prevent further sperm entry, thus, ensuring monospermic fertilization (for reviews,

see SCHMELL et al. 1983, HOODBHOY and TALBOT 1994, YANAGIMACHI 1994,

cited in WANG et al. 1998). Under physiologic conditions, almost all of the oocytes

fertilized are monospermic (> 95%) (HUNTER 1972), and both nuclear and

cytoplasmic maturation is believed to assist the mechanisms whereby pig oocytes

establish a functional block to polyspermy. MANDELBAUM (2000) has proposed that

the migration of CGs, which is thought to occur during oocyte maturation (WANG et

al. 1998), is an essential mechanism crucial for the establishment of an efficient

defence against polyspermy during fertilization.


24

2.2.1.5 Distribution of Organelles During Maturation 2.2.1.5.1 Cortical Granule Translocation to the Plasma Membrane During Maturation Cortical granules (CGs) are membrane-bound secretory organelles derived from the

Golgi apparatus. They are usually unique to oocytes and originate during the onset of

follicular growth in nearly all animals (HOODBHOY and TALBOT 1994). Cortical

granules accumulate and are randomly distributed within the cytoplasm during

oogenesis in most animals. Cortical granule translocation is an early indicator of

meiotic resumption, or may even precede the stimulus for meiosis in the oocyte. At

some points during maturation, the granules (CGs) translocate to below the oolemma

and anchor to the plasma membrane. In bovine oocytes, meiotic maturation and CG

translocation are inseparable processes (WESSEL et al. 2002). Comparison of CG

translocation in both in vivo and in vitro matured (IVM) oocytes shows that the

majority of oocytes matured in vitro exhibit delays in cortical granule migration

compared to oocytes matured in vivo (WESSEL et al. 2001). It is hypothesized that

CG translocation is in some way coordinated with nuclear and cytoplasmic

maturation by the regulatory mechanism responsible for cortical granule progression

(for reviews, see WESSEL et al. 2001, CHEN et al. 2004). In porcine oocytes, the

progression through germinal vesicle breakdown (GVBD) initiates the mass migration

of all cortical granules (CGs) towards the oolemma. Therefore it is hypothesized that

CG translocation is also coordinated with meiotic maturation (Fig. 4 A and Fig. 4 B,

see also 2.2.1.4 Cytoplasmic Maturation). In bovine oocytes, the key to this process

may be associated with the influence of the cumulus cells (WESSEL et al. 2001).


25

Fig. 4: Cortical granule migration. A: Cortical granules have already translocated to the

cortex at MII (BERG and WESSEL 1997). B: Cortical granules are usually poised at the

cytoplasmic face of the plasma membrane (WESSEL et al. 2001). C: Cortical granules are

attached or docked to the plasma membrane of the unfertilized eggs in sea urchin eggs.

2.2.1.5.1.1 Role of Actin Cytoskeleton on Cortical Granule (CG) Translocation During Maturation

There is strong evidence that actin microfilaments are critical in cortical granule (CG)

translocation (WESSEL et al. 2001). Cortical granules are associated with a dense

cortical microfilament. Just after germinal vesicle breakdown (GVBD), CGs attach to

microfilaments and translocate to the cell surface (from about 0.5 µm to 1.0 µm from

the surface of the cell at metaphase II). By this time, approximately 40% of the CGs

have already translocated to the periphery of the oocyte (BERG and WESSEL 1997).

Treatment with cytochalasin B to disrupt microfilaments resulted in a clustering of CG

and a failure to orient towards the oocyte surface (i.e. below the oolemma), which

strongly indicated that the process of CGs translocation is dependent on actin

rearrangement during maturation (SANTELLA et al. 1999, cited in WESSEL et al.

2001).


26

2.3 Biology of the Zona Pellucida (ZP) 2.3.1 The Zona Pellucida (ZP) Mammalian oocytes are surrounded by a transparent extracellular matrix which is

composed of a complex glycoprotein matrix known as the zona pellucida (ZP). The

developmental expression and extracellular assembly of this unique matrix occurs

during the early stages of ovarian folliculogenesis following the recruitment of the

oocyte from meiotic arrest (PRASAD et al. 2000). The ZP proteins have been shown

to be produced by granulosa cells as well as by the oocyte and are thought to be

important in granulosa cell differentiation and folliculogenesis. In the mouse, the

growing oocyte is the only source of zona glycoproteins, whereas in domestic

animals, these proteins are expressed in both oocyte and granulosa cell

differentiation (PRASAD et. al. 2000, SINOWATZ et al. 2001).The different ZP

proteins are synthesized, secreted, and organized in a specific temporal sequence

(PRASAD et al. 2000).

In addition to the potential roles of these matrix proteins in granulosa cell

differentiation, they are intimately involved in several critical stages of fertilization (for

review, see PRASAD et al. 2000). This includes: (i) attachment and binding of

capacitated sperm to the ZP, (ii) induction of the sperm acrosome reaction, (iii)

subsequent sperm penetration of the ZP matrix, and (iv) fertilization process-induced

modifications of the ZP which result in the prevention of polyspermy (SKINNER and

DUNBAR 1986, TIMMONS and DUNBAR 1988, WASHENIK et al. 1989, TIMMONS

et al. 1990, WASSARMAN 1990, DUNBAR et al. 1991, GUPTA et al. 1996,

SKINNER et al. 1996). The molecular basis of sperm–ZP interaction has been shown

by intensive studies in the mouse model. However, it is becoming increasingly

apparent that the molecular mechanisms of sperm–ZP interaction are distinct among

non-rodent species (PRASAD et al. 2000).


27

2.3.2 Morphology, Protein Nomenclature and Genomic Organization of the Zona Pellucida (ZP) The zona matrix first appears as an amorphous material deposited in the space

between the oocyte and the surrounding cumulus cells. The single most distinctive

morphological feature of the ZP of different mammalian species is its relative

thickness (PRASAD et al. 2000). This matrix varies in thickness from 1 to 2 µm in the

invertebrate, 5 µm in the mouse, to between 13 and 16 µm in the human and pig

(DUNBAR and WOLGEMUTH 1984, DUNBAR et al. 1991, cited in PRASAD et al.

2000). By electron microscopy the zona pellucida (ZP) of mature pig oocytes appears

to consist of a dense filamentous meshwork, which is less compact on the inner and

outer faces. The uneven surface of the ZP is made of unordered and stretched fibrilis

with surrounding deep funnels which are openings of the radial canaliculi (for review,

see FLÉCHON et al. 2004). The topography of the ZP surface may contribute to the

initial interplay between male and female gametes. FLÉCHON et al. (2004) found

that the ZP of the pig oocyte has bundles of fibrils distributed in concentric layers

(except in the innermost and outer parts), which can be visualized by cytochemical

techniques. Morphological alteration of the zona pellucida (ZP) of ovulated and in

vitro matured oocytes (IVM) was demonstrated by studies of FUNAHASHI et al.

(2000). The thickness of intact ZP was larger in ovulated oocytes than in oocytes

after in vitro maturation (IVM). FUNAHASHI et al. (2000) demonstrated via scanning

electron microscopy (SEM) the presence of a homogeneous fine network of filaments

from ovulated oocytes, whereas a much more compacted structure was present in

IVM oocytes. The zona pellucida (ZP) proteins of most mammalian species studied to

date are classified into three major glycoprotein families, ZP1, ZP2, and ZP3, which

exhibit extreme heterogenity in molecular weight and charge due to posttranslational

modifications including glycosylation and sulfation (for reviews, see DUNBAR et al.

1980, DUNBAR et al. 1981, WASSARMAN 1988, DUNBAR et al. 1991, DUNBAR et

al. 1994, cited in PRASAD et al. 2000). Much of the confusion surrounding the

identification of different ZP proteins has been due to the various nomenclatures that

have been applied to describe the complex nature of the heterogeneity of the ZP


28

proteins (PRASAD et al. 2000). The characteristics of representative ZP proteins

from different species and their nomenclature are listed in Table 1. Because of the

molecular heterogeneity, various approaches have been used to identify individual

glycoproteins. Determination of the primary amino acid sequence of ZP proteins

deduced from cDNA cloned from a number of species has allowed the detailed

characterization of glycoprotein families (for reviews see, PRASAD et al. 2000, BOJA

et al. 2003).

Each of the three protein families from different mammalian species have been

shown to have between 50% and 98% homology at the nucleic acid level (PRASAD

et al. 2000). The ZP glycoproteins are classified in the order of the molecular mass of

the genes from high (ZPA) to low (ZPC) (HARRIS et al. 1994), whereby the

glycoproteins families are ZPA (ZP2 in mouse), ZPB (ZPA in mouse) and ZPC (ZP3

in mouse). The characteristics of the ZP proteins from different species and their

nomenclature are also given inTable 1.

Tab. 1: Characteristics of representative members of the zona pellucida glycoprotein

families (modified from DUNBAR et al. 2001).

ZP

protein

family ọ

Species Former ZP

protein

nomenclature

No. of amino

acid.

residues a

Mr

(kDa)º

Relative

M (kDa)

No. of N-

glycosylation

sites

ZP1 Mouse ZP1 623 70 200 6

Rabbit 55 kDa 540 57 85-95 6

ZPB Pig ZP3α 536 57 55 5

ZP2 Mouse ZP2 713 80 120 7

Rabbit 75 kDa/ZP2 684 75 100-130 7

ZPA Pig ZP1/ZP2 716** 80 70-100 7

ZP3 Mouse ZP3 424 46 83 6

Rabbit 45 kDa 419 43 68-120 5

ZPC Pig ZP3β 421 44 78-120 4

ọ Based on mouse ZP nomenclature. a. Comparison at the amino acid level.

◦ Deduced from cDNA sequence (PRASAD et al. 2000).


29

Despited the variation in the molecular weights of individual native ZP proteins, there

is considerable conservation in the number and position of cysteine residues as well

as potential N-linked glycosylation sites within each family (PRASAD et al. 2000).

The genomic structures of each protein family also exhibit similar intron/extron

organization (for reviews, see EPIFANO et al. 1995, McLESLEY et al. 1998, cited in

PRASAD et al. 2000). However, of the three ZP families, the ZP1/ZPB family seem to

be less conserved both in structure and function (PRASAD et al. 2000). The mouse

ZP1, which is the orthologue of the rabbit, pig, and human ZPB family proteins, is

only 39% identical to the human ZPB at the amino acid level, while rabbit and pig

ZPB are 70% identical to human ZPB (PRASAD et al. 2000). In addition, mouse ZP1

is 623 amino acids long and contains a stretch of sequence in the N-terminal region

that is absent in the ZPB of other species (EPIFANO et al. 1995, PRASAD et al.

1997, cited in PRASAD et al. 2000).

The three glycoproteins of mouse zona pellucida (ZP1, ZP2, and ZP3) form long

filaments between ZP2 and ZP3 that are crosslinked by ZP1 (WASSARMAN et al.

2004), as is shown in Figure 5.


30

Fig. 5: Schematic representation of the mouse ZP glycoproteins ZP1, ZP2, and ZP3. The

ZP2 and ZP3 glycoproteins form long filaments and are crosslinked by ZP1 glycoproteins

(PRASAD et al. 2000, WASSARMAN et al. 2004).

In the pig, the three glycoproteins ZPA, ZPB, and ZPC form hetero-oligomeric

complexes which can contribute toward the assembly of the zona matrix (YUREWICZ

et al. 1998). The ZPB and ZPC glycoproteins cannot be separated by gel filtration

unless the acidic N-acetyllactosamine regions of their carbohydrate chains are

removed (KUDO et al. 1998). The partially deglycosylated ZPB proteins retain

ligands important for the sperm-binding activity (KUDO et al. 1998).

2.3.3 Carbohydrates of Zona Pellucida (ZP) Glycoproteins and Their Roles in the Fertilization Process It has long been observed that there are two stages of sperm interaction with the

zona pellucida (ZP) (HARTMAN 1983, cited in PRASAD et al. 2000) that occur after


31

the process of sperm capacitation, sperm attachment, and sperm binding (for review,

see FRASER 1995, cited in PRASAD et al. 2000). It is becoming increasingly

apparent that multiple molecules on a single ZP protein (carbohydrate, protein, other

post-translational modifications including sulfation, or combinations of all these) as

well as multiple ZP proteins may interact with sperm membrane proteins (PRASAD et

al. 2000). The initial stage of sperm-egg interaction, referred to as “attachment”, most

likely occurs through interactions with carbohydrate moieties or other post-translation

modification (GWATKIN 1978, cited in PRASAD et al. 2000). It is likely that the

subsequent “tight binding” events may be the result of sperm interaction with the

peptide core of the protein (PRASAD et al. 2000). The carbohydrate moieties are

thought to greatly influence cell-cell interactions within the developing follicle as well

as those occurring between the fully developed zona pellucida (ZP) and the fertilizing

spermatozoon (for reviews, see BLEIL and WASSARMAN 1983, MORI et al. 1991,

NAGDAS et al. 1994, cited in PRASAD et al. 2000). In the mouse, the ZP ligand,

primarily responsible for sperm-zona interactions, has been found to be a class of O-

linked oligosaccharides that are covalently linked to ZP3 (FLORMAN and

WASSARMAN 1985, BLEIL and WASSARMAN 1988, LITSCHER et al. 1995, cited in

PRASAD et al. 2000). However, another study has reported that it is the N-

acetylglucosaminyl residues and not the α-galactose residues on mZP3 that have

sperm receptor function and bind to a galactosyltransferase on mouse spermatozoa

(MILLER et al. 1992, cited in PRASAD et al. 2000). In the pig, both O-linked and N-

linked carbohydrate structures of the ZP have been shown to be involved in sperm–

binding to the ZP (for reviews, see MORI et al. 1991, NOGUCHI and NAKANO 1992,

KUDO et al. 1998, cited in PRASAD et al. 2000). Many studies have demonstrated

the presence of fucosylated N-linked glycans carrying lactosaminoglycans repeats

(for reviews, see YUREWICZ et al. 1987, MORI et al. 1991, NOGUCHI and NAKANO

1992, 1993, NOGUCHI et al. 1992, cited in PRASAD et al. 2000). In addition, it has

been demonstrated in the pig that it is the N-linked carbohydrates and not the O-

linked carbohydrates that mediate sperm-zona interaction (NOGUCHI et al. 1992,

cited in PRASAD et al. 2000).


32

Sugar-mapping analysis of the carbohydrate chains of the ZP glycoproteins has

shown that the triantennary and tetraantennary chains are localized mainly in the N-

terminal region at Asn220 of pig ZPB, and diantennary chains were shown to be

present in three other potential residues at Asn203, Asn220, and Asn333 (KUDO et

al. 1997). These results suggest that the carbohydrate chains linked to Asn220 of pig

ZPB possess the ligands important for sperm-zona interaction (for reviews, see

YUREWICZ et al. 1993, NAKANO et al. 1996, KUDO et al. 1998, YONEZAWA et al.

1997, PRASAD et al. 2000).

2.3.4 Structural Domains of the Zona Pellucida (ZP) Proteins Several structural domains have now been identified in the zona pellucida (ZP)

glycoproteins on the basis of the amino acid sequences. Some of these domains are

common to all three ZP protein families (PRASAD et al. 2000). A domain referred to

as the ZP module has been identified in all three of the ZP families on the basis of

pattern-based sequence analysis (for review, see PRASAD et al. 2000).

The ZP domain has been found to be responsible for the polymerization process of

the glycoproteins into filaments forming the supramolecular structure of the ZP matrix

(JOVINE et al. 2002). All three glycoproteins are characterized by the presence of an

N-terminal hydrophobic signal sequence, a furin processing site, and a C-terminal

hydrophobic transmembrane domain (BORK and SANDER 1992, McLESKEY et al.

1998, cited in PRASAD et al. 2000). The functions of the furin cleavage site and

transmembrane domain have not yet been defined. However, YUREWICZ et al.

(1993) have suggested that proteolytic processing of an integral membrane precursor

may be an intermediary step in the secretion of ZP proteins (PRASAD et al. 2000).

2.3.4.1 Positions of Cysteine (Cys) in the Zona Pellucida (ZP) Protein Domains The zona pellucida (ZP) domain of pig ZPC contains eight cysteine (Cys) residues,

whereas the ZP domains of ZPA and ZPB each have ten Cys residues, eight of


33

which correspond to those in the ZP domain of ZPC (Fig. 6). The positions of the Cys

residues in the ZP domains are well conserved (for reviews, see PRASAD et al.

2000, YONEZAWA and NAKANO 2003), suggesting similarity in the three-

dimensional structure of all three ZP protein families. When at least two cysteine

residues are oxidized to cystine (L-cystine), this results in a disulfide bond. The

formation of disulfide linkages suggests their importance for the correct folding of the

ZP domain (for reviews, see PRASAD et al. 2000, YONEZAWA and NAKANO 2003).

The ZP domains contain different clusters of disulfide bonds which could be

constituted of two subdomains (YONEZAWA and NAKANO 2003). The intriguing

possibility that these subdomains play different functional roles in sperm binding,

polyspermic penetration block, and/or zona formation remains to be investigated. In

the pig, it has been reported that ZPB is contaminated with a trace of ZPC, and that

ZPB that is completely free of ZPC loses sperm ligand activity (YUREWICZ et al.

1998). Triantennary and tetraantennary chains of both ZPB and ZPC might then be

involved in sperm binding (PRASAD et al. 2000, NAKANO and YONEZAWA 2001).

Fig. 6: Schematic presentation of Cys residues in porcine ZPB and ZPC. The circles denote

groups of Cys (C) residues in porcine ZPB and ZPC that form possible disulfide bonds with

each other. There is conservation in the number and position of the cysteine residues within

each ZP glycoprotein (YONEZAWA and NAKANO 2003).


34

Fig. 7: Overview of the oxidation of the two cysteine (Cys) residues to cystine (L-cystine).

2.4 Fertilization Cascade (FC) 2.4.1 Sperm Capacitation and the Acrosome Reaction The capacitation of mammalian spermatozoa involves a spectrum of molecular

changes in sperm membranes which ultimately modify ion channels in the

plasmalemma (i.e. oolemma) of mature spermatozoa (for reviews, see AHUJA 1984,

LANGLAIS and ROBERTS 1985, YANAGIMACHI 1988, PETRUNKINA et al. 2001,

TARDIF et al. 2001). In turn, this modification of the membrane permits the

transmembrane flux of Ca2+ ions (for reviews, see BEDFORD 1991, cited in

FLESCH and GADELLA 2000) that seems to be a key to the initiation of the two

functional end points of capacitation, hyperactivation and the acrosome reaction

(AR). Although, similar in the time of onset, these two events are independent of

each other and certainly involve different region–specific membrane changes in the

spermatozoa (BEDFORD 1991). Other molecular events that coincide with


35

mammalian capacitation are also probably associated with the phosphorylation of

sperm proteins by cAMP-dependent protein kinase A (PKA), although the enzymes

(kinases and phosphatases) and protein substrates implicated in these process have

not yet been fully characterized (FLESCH and GADELLA 2000, TARDIF et al. 2001).

Capacitation is a conditioning phase that naturally takes place within the female

genital tract (BEDFORD 1991, ZANEVELD et al. 1991). In pigs, the caudal region of

the oviductal isthmus appears to be particularly efficient in exerting effects on the

sperm cell that are responsible for the modulation and initiation of sperm capacitation

(HUNTER et al. 1998) believed to be necessary for triggering the acrosome reaction

(TOEPFER-PETERSEN et al. 2002) and for allowing sperm-egg fusion to occur

(YANAGIMACHI 1977). The acrosome reaction (AR) is an exocytotic event requiring

the fusion of two membranes; the (inner) plasma membrane, and the (outer) sperm

membrane - to cause the release of acrosomal components. Like other membrane

fusion events, Ca2+ has been proposed as assisting in the coalescence of the two

membranes involved (BEDFORD 1991). The influx of Ca2+ during the AR has been

shown to cause a rise in cAMP concentrations within sperm. Several other molecules

have been implicated as important regulators of the AR such as prostaglandins,

bicarbonate ions (HYNE 1984), phospholipids (FLEMING and YANAGIMACHI 1984),

and factors from follicular fluid (HYNE and GARBERS 1981).

It is generally accepted that the AR of sperm follows its binding to the zona pellucida

(ZP). Once bound to the zona pellucida, the acrosome-reacted sperm is allowed to

penetrate to the ZP and finally to fertilize the egg (HUANG et al. 1981, BEDFORD

1991, TOEPFER-PETERSEN et al. 2000, 2002). WU et al. (2004) proposed that

there are alternative mechanistic pathways for the acrosome reaction during

fertilization which differs from the widely accepted sperm–ZP receptor models.


36

2.4.2 Sperm Binding to the Oocyte Zona Pellucida (ZP) Initial studies using rodent species suggested that sperm attachment to the zona is

species-specific. These studies prompted the search for specific sperm receptors on

the surface of the zona pellucida (ZP). Such receptors have been described in

analogous structures of the vitelline envelope from invertebrate species (e.g. sea

urchin; for review, see RUIZ-BRAVO and LENNARZ 1989). Although it is apparent

that there is a critical need for the specificity of species recognition between gametes

in environments in which external fertilization occurs, the need may be less critical in

mammalian species, in which physiological and behavioral barriers exist to prevent

gametes from contacting each other. For instance, the species specificity of sperm–

zona interaction has been described for some mammals (e.g. mouse, hamster, pig),

but less specificity is exhibited by sperm of other mammalian species (SWENSON

and DUNBAR 1982). The sperm–egg binding is best understood in the mouse,

because the acrosome is intact when the murine sperm initially encounter the zona

pellucida glycoproteins of the oocyte (ZP1, ZP2, and ZP3). In this species, initial

sperm receptor activity has been associated with the ZP3 glycoprotein (for reviews,

see WASSARMAN and LITSCHER 1995, McLESKEY et al. 1998, cited in PRASAD

et al. 2000), which is known to contain the initial sperm–binding site and acrosome–

reaction–inducing activity and subsequently to bind to ZP2 following the acrosome

reaction (BLEIL and WASSARMAN 1986). In the pig, the ZP1 (ZPB) glycoproteins

appear to be involved in the interaction with the sperm (for review, see SACCO et al.

1989, in PRASAD et al. 2000). More recently, YUREWICZ et al. (1998) demonstrated

that boar sperm binds to hetero-complexes of ZP1 and ZP3 (cited in PRASAD et al.

2000).

2.4.3 Penetration by Spermatozoa of the Zona Pellucida (ZP) Many studies have also been carried out to analyze the enzymes associated with

sperm function in the female reproductive tract prior to and during fertilization (for


37

review, see ZANEVELD and DE JONGE 1991, cited in PRASAD et al. 2000), for

which sperm must penetrate the zona pellucida (ZP) during the fertilization process

without disrupting the ZP matrix, which is important for the support and protection of

the embryo. Sperm penetration of the ZP has long been thought to be facilitated by

limited proteolysis of proteins of the ZP glycoprotein matrix (STAMBAUGH 1978,

cited in PRASAD et al. 2000). Extensive studies provide evidence for the role of the

trypsin-like acrosomal proteinase acrosin (for reviews, see TOPFER-PETERSEN and

HENSCHEN 1987, 1988) in the mediation of penetration of the ZP during fertilization.

To date, the acrosomal enzyme acrosin has been shown to have both ZP- and

fucose-binding properties, which may facilitate sperm binding to and penetration of

the ZP during fertilization (TOEPFER-PETERSEN and HENSCHEN 1987). However,

other investigators have questioned the role of this enzyme in sperm penetration.

These controversies can be attributed to the considerable species variation in the

morphological, physicochemical, and biochemical properties of the ZP (BROWN

1982). It is also likely that the specific enzymes of spermatozoa of different species

play a complex role in successful penetration of the zona pellucida (ZP) matrix.

2.4.4 Gamete Interaction: Sperm Fusion and Oocyte Activation After a sperm has successfully penetrated the zona pellucida (ZP) and encountered

the plasma membrane of the egg, interaction between the two gametes leads to the

fusion of their respective membranes. One of the earliest events of oocyte activation

is an increase in the level of intracellular Ca2+. In mammals, the levels of intracellular

Ca2+ waves oscillate repeatedly for a period of up to several hours after sperm-

oocyte fusion (for review, see MIYAZAKI et al. 1993, cited in WILLIAMS 2002). While

lower species do not generate multiple Ca2+ waves (WILLIAMS 2002), these are

critical for successful oocyte activation in mammals (for reviews, see OZIL 1990,

KLINE and KLINE 1992). Elevations in intracellular Ca2+ trigger exocytosis, – an

enzymatic process of the cortical granules (CG) –, which contain enzymes that are

thus released into the perivitelline space (PVS), where they are responsible for


38

modification of the ZP to prevent additional sperm from binding to and penetrating

the ZP (BLEIL and WASSARMAN 1980).

Spatial and temporal characteristics of Ca2+ oscillations regulate many different

cellular responses, including cell proliferation, gene transcription, and vesicle

secretion (BERRIDGE et al. 1998). A recent study confirmed that an abnormal

frequency of Ca2+ oscillations induces abnormal patterns of oocyte activation and

can induce cell death in aged oocytes (GORDO et al. 2000). A critical component of

oocyte activation is the resumption of meiosis II, which occurs during the time of the

sperm chromatin decondensation; this resumption marks the re-entry of the oocyte

into the cell cycle, which is controlled by a balance of the activities of kinases and

phosphatases such as maturation promoting factor (MPF) and mitogen-activated

protein (MAP) kinase that modulate the activity of cellular proteins. Morphological

changes during meiotic resumption include rotation of the metaphase spindle, entry

into anaphase, and emission of the second polar body. At this point, the DNA

originating from the female gamete becomes haploid (WILLIAMS 2002). Several

hours after sperm-oocyte fusion, the Ca2+ oscillations cease (JONES et al. 1995),

the male and female pronuclei form, and DNA synthesis begins. Under the control of

a microtubular network, the two pronuclei migrate to the center of the newly formed

unicellular zygote.

2.5 Polyspermy Block Following fertilization, the fusion of the cortical granule (CG) membranes with the

oolemma induces CG exocytosis (cortical reaction), and the CG exudates contains

enzymes that modify the zona pellucida (ZP) matrix, leading to an event known as

the zona reaction. The cortical reaction is regulated by a wave of free cytoplasmic

Ca2+ following the fusion of sperm and egg that causes rapid exocytosis of the

cortical granules (CG)

In the pigs, immediately after fusion of spermatozoa with the oolemma, exocytosis of

the CG contents into the perivitelline space (PVS) leads the ZP to become refractory


39

to both, binding of free-swimming sperm, and penetration by sperm that had partially

entered the extracellular coat (ZP) prior to fertilization. In most mammals, both the

CG reaction and zona reaction prevent further sperm entry and ensure monospermic

fertilization at the level of the ZP and/or plasma membrane (SCHMELL et al. 1983,

HOODBHOY and TALBOT 1994, YANAGIMACHI 1994, cited in WANG et al. 1998).

In the mouse, there are two important mechanisms that participate in blocking

polyspermy: 1) ZP3/ZPC is inactivated by a CG glycosidase which removes the

terminal monosaccharide (e.g. O-linked oligosaccharides) responsible for gamete

recognition and adhesion, so that sperm no longer can bind to the ZP3/ZPC protein

(BLEIL and WASSARMAN 1988, HOODBHOY and TALBOT 1994, NIXON et al.

2001) (also described in 2.6.1.2.2 Glucosaminidase); and 2) at the same time,

ZP2/ZPA (pZP1) undergoes limited proteolytic cleavage, which is catalyzed by a CG

protease (FUNAHASHI 2003) causing a structural rearrangement of the ZP that

promotes filament-filament interactions, and thereby makes the ZP more insoluble

(also described in 2.7 Zona Pellucida Hardening). In addition, proteolysis of ZP2/ZPA

may preclude maintenance of the binding of acrosome-reacted sperm to the ZP by

inactivating ZP2/ZPA (pZP1) as a secondary sperm receptor in order to block

polyspermy.

Under physiologic conditions, almost all fertilized oocytes (> 95%) are monospermic

(HUNTER 1972, cited in WANG et al. 1998). When in vitro matured (IVM) oocytes

are inseminated in vitro, the polyspermic rate is particularly high (NIWA 1993, cited in

WANG et al. 1998). It is possible that in vivo maturation and the oviduct

microenvironment assist in the completion of the mechanisms whereby pig oocytes

establish a functional block to polyspermy.

2.6 Cortical Granules (CG) The contents of the cortical granules (CGs) are central to the block to polyspermy

(WESSEL et al. 2001) in all mammals, most vertebrates, and many invertebrates.

This is accomplished by modification of the extracellular environment of the oocyte.


40

Most contents of the CGs are species-specific, e.g. ovoperoxidase, protease,

glucanase, and proteoliaisin in sea urchins; lectin, and glucosamindase in

amphibians; and protease, glycoconjugates, glucosaminidase, and lectin in mammals

(for review, see WESSEL et al. 2001). Nevertherless, the general process is

important to blocking polyspermy in all species (SCHMELL et al. 1983, WESSEL et

al. 2001, VELILLA et al. 2004). In sea urchin eggs, there is evidence that the CG

contents (protease, ovoperoxidase) modify the oocyte cell surface to prevent sperm

entry by construction of the fertilization envelope (FE). The role of the CG contents in

blocking polyspermy is conserved throughout evolution, since similar events modify

the extracellular matrix, the ZP. Inhibitors of the exocytosis mechanism (CONNER

and WESSEL 1998, cited in WESSEL et al. 2001) have been used to show clearly

that blocking of the cortical reaction and the zona reaction leads to polyspermy.

2.6.1 Cortical Granule Contents and Their Relevance to the Polyspermy Block Function in Different Species

2.6.1.1 Cortical Granule Contents and their Relevance to the Polyspermy Block in Sea Urchins Upon fertilization of the sea urchin eggs, cortical granules (CG) release their

contents, which modify the vitelline layer to form a fertilization envelope (FE). The FE

coupled with the plasma membrane generates a physical block to polyspermy.

Several diverse types of molecules responsible for FE are synthesized and stored in

the CG from sea urchin eggs. These include enzymes such as an ovoperoxidase

(described in 2.6.1.1.1 Ovoperoxidase), a protease (described in 2.6.1.1.2 Protease),

glycosidase, structural proteins such as SFE9, proteoliasin, and SFE1 (for review,

see WESSEL et al. 2001), glycosaminoglycans (described in 2.6.1.1.3

Glycosaminoglycans), and perivitelline molecules (e.g. glucanase and hyaline) (for

review, see WESSEL et al. 2001). Of the estimated twelve CG proteins, all except

two are unique to oocytes and CGs (WESSEL et al. 2001).


41

2.6.1.1.1 Ovoperoxidase The cortical granule-derived enzyme ovoperoxidase is targeted to the fertilization

envelope (FE) in a reaction that requires extracellularly generated hydrogen peroxide

(H2O2) (HEINECKE and SHAPIRO 1990, cited in WESSEL et al. 2001, WONG et al.

2004) (Fig. 8). Within minutes of fertilization, the FE hardens and becomes resistant

(mechanically and enzymatically) as a result of an oxidative process (for review, see

MOTOMURA 1957, cited in WESSEL et al. 2001). FORDER and SHAPIRO (1977)

and HALL (1978) proposed that ovoperoxidase activity is responsible for FE

hardening via catalysis of covalent dityrosine bonds between proteins in the FE

(WESSEL et al. 2001). Some experimental data support this: (i) inhibitors of

peroxidase activity inhibit hardening of the fertilization envelope (FORDER and

SHAPIRO 1977, cited in WESSEL et al. 2001), and (ii) purified ovoperoxidase

catalyzes the oxidation of tyrosine in solution, whereas an FE formed following

inhibition of ovoperoxidase activity has no dityrosine (WONG et al. 2004).

Fig. 8: The essential substrate involved with cell signalling is hydrogen peroxide (H2O2).

There is a temporal correlation between the peak productions of hydrogen peroxide

(H2O2) and ovoperoxidase activity; both have evolved into a tightly regulated system

which limits the potentially lethal oxidant effects without compromising FE integrity

(WONG et al. 2004).


42

2.6.1.1.2 Protease

The protease in sea urchin eggs is a trypsin-like serine protease that is active at

fertilization (HALEY and WESSEL 1999). Two protease activities from the cortical

granule (CG) exudates seem to be involved in blocking polyspermy: (i) cleaving of

supernumerary sperm receptors, and (ii) cleaving of connections between the

vitelline layer and the plasma membrane, thereby allowing them to separate during

fertilization (vitelline delaminase activity) (WESSEL et al. 2001).

2.6.1.1.3 Glycosaminoglycans Researchers speculate that, like molecules, the glycosaminoglycans (GAG) have

several functions, both before and after fertilization (WESSEL et al. 2001). GAGs

may participate in the biosynthesis of cortical granules (CGs), serving to condense

and to concentrate the contents, and in forming aggregates of proteins destined for

the cortical granule (for review, see SCHUEL 1985, in WESSEL et al. 2001).

While the CAGs are in the CG, they may also function to inhibit enzymatic activities

(WESSEL et al. 2001). Following fertilization, rapid hydration of the

glycosaminoglycans (GAGs) in the cortical granules (CGs) may serve to propel

exocytosis of the contents (CHANDLER et al. 1989, in WESSEL et al. 2001) and to

lift the vitelline layer of the plasma membrane.

2.6.1.2 Cortical Granule Contents and Their Relevance to the Polyspermy Block in Mammals 2.6.1.2.1 Protease Proteins from cortical granules (CG) including proteases have been characterized in

many species biochemically (SCHUEL 1985, cited in WESSEL et al. 2001) and by


43

molecular isolation of cDNAs (sea urchin: LAIDLAW and WESSEL 1994, cited in

WESSEL et al. 2001; mouse: MILLER et al. 1993; human: BAUSKIN et al. 1999; pig:

FUNAHASHI 2003).

A cortical granule protease cleaves human glycoprotein ZP2 (ZPA) in the amino

terminal domain to a 60-73 kDa, denoted ZP2p, which remains linked to the

proteolysed fragments by intra-molecular disulfide bonds (-S-S- bonds) (BAUSKIN et

al. 1999). FUNAHASHI (2003) demonstrated in pigs that, after the cortical reaction,

pZP1 (ZPA) is divided into pZP2 and pZP4 (ZPA to ZPAf) by reduction of the -S-S-

bonds (Fig. 9). In the mouse, MOLLER and WASSARMAN (1989) showed that the

involvement of a specific protease resulted in the conversion of 60% of ZP2 to ZP2f.

Fig. 9: Porcine zona pellucida is divided into pZP2 (ZP2) and pZP4 (ZP2f) by reduction of -S-

S- bonds (marked in red).


44

2.6.1.2.2 Glucosaminidase

Following fertilization, glycoprotein ZP3 is modified by the removal of terminal sugars,

which releases bound sperm and prevents further binding of additional sperm

(WESSEL et al. 2001). Competitive inhibitors of enzyme activity during fertilization

cause a loss of the zona block to polyspermy (MILLER et al. 1993). The model

proposed from these results is that the N-acetylglucosaminidase cleaves a terminal

N-acetylglucosamine from glycoprotein ZP3, which would otherwise serve as a

substrate for binding by a sperm cell surface galactosyltransferase (WESSEL et al.

2001).

2.6.1.2.3 Lectin The binding of lectin released from the cortical granules (CG) triggers modifications in

the gel coat that forms the interface to the vitelline envelope (Xenopus laevis eggs;

for review, see GREVE and HEDRICK 1978), and therefore is an additional factor in

the block to polyspermy (HEDRICK, MCB abstract). In the mouse, recent reports

suggested that a galactose-specific lectin may be very similar to the lectin in

amphibians.

2.7 Zona Pellucida (ZP) Hardening Zonae pellucidae (ZP) from fertilized eggs become hardened relative to those of

unfertilized eggs. Investigations in bovines indicate that cysteine (Cys) residues in

the ZP are oxidized to cystine (L-cystine) during fertilization (IVF) (IWAMOTO et al.

1999). It is postulated that the formation of intra- and intermolecular disulfide bonds

modifies the body of the ZP to prevent further sperm penetration through the

construction of a rigid ZP structure, responsible for zona pellucida hardening (De

FELICI and SIRACUSA 1982, KURASAWA et al. 1989, DUCIBELLA et al. 1990,

GREEN 1997, IWAMOTO et al. 1999).


45

Specific cleavage of the glycoproteins ZP2 (mice: MOLLER and WASSARMAN 1989;

pig: FUNAHASHI 2003) by a protease from exocytosis of the cortical granules (CGs)

during fertilization, results in the formation of ZP2f (pig, pZP2 and pZP4), which

remains linked by intramolecular disulfide bonds (-S-S- bonds) (BAUSKIN et al.

1999) (described in 2.6.2.1 Protease). It is suggested that this event resulting to the

construction of a more rigid ZP structure, which is responsible for hardening of the

zona pellucida.

Numerous investigations (MOTOMURA 1957, cited in WESSEL et al. 2001, ZHANG

et al. 1991, HOODBHOY and TALBOT 1994, GREEN 1997, DELLÁQUILA et al.

1999, IWAMOTO et al. 1999, CHOI et al. 2001, ALBERTS et al. 2002, LANDIM-

ALVARENGA et al. 2002, LEE et al. 2003, SUN 2003) suggest that the hardening of

the zona pellucida by inhibition of sperm binding to ZP glycoproteins is the main

factor contributing to the block to polyspermy.

In sea urchin eggs, zona hardening may result from an oxidative process by catalysis

of covalent di-tyrosine bonds, which produces hardening by linking fertilization

envelope (FE) proteins (FORDER and HALL 1978, in WESSEL et al. 2001, GREEN

1997) (described in 2.6.1.1.1 Ovoperoxidase). The FE hardens and becomes

resistant to both mechanical and enzymatic modification (WESSEL et al. 2001,

WONG et al. 2004). There is accumulating evidence that the hardening in sea

urchins prevents further sperm penetration by linking fertilization envelope

(DELLÁQUILA et al. 1999).

MATERIALS AND METHODS ___________________________________________________________________

46

3 MATERIALS AND METHODS Laser scanning confocal microscopy (LSCM) and scanning electron microscopy

(SEM) were used to investigate the distribution pattern of free thiol groups within the

zona pellucida (ZP) of oocytes of different development stages. The following flow

chart shows the experimental design of this study (Fig. 10).

Fig. 10: Experimental design: Porcine ZP from seven different developmental stages were

labelled with 5-Iodoacetamidefluorescein (5-IAF) and biotin-conjugated iodoacetamide

(BIAM) and identified by laser scanning confocal microscopy (LSCM) and scanning electron

microscopy (SEM).


47

3.1 In Vitro Maturation (IVM) of Porcine Oocytes 3.1.1 Collection of Ovaries Porcine ovaries were obtained from prepubertal gilts at a local slaughterhouse and

transported in pre-warmed (38.5 °C) physiological saline solution (as described in

Table 11, Appendix) to the laboratory within 2-3 h. Upon arrival in the laboratory, the

ovaries were washed with room temperature (RT) physiological saline solution before

aspiration of the cumulus-oocyte complexes (COCs). Collection of the oocytes was

completed within 2 h of the arrival of the ovaries at the laboratory. Oocytes at the

germinal vesicle (GV) stage were used either for isolation of ZP or for in vitro

maturation (IVM) and/or in vitro fertilization (IVF).

3.1.2 Isolation of Cumulus-Oocyte Complexes (COCs) Cumulus-oocyte complexes (COCs) were aspirated from follicles approximately 2-5

mm in diameter showing a translucent surface, fine vascularization, and clear

follicular fluid. This was accomplished with a short bevelled 18-gauge needle

(Microlance 3, Becton Dickinson GmbH, Heidelberg, Germany), attached to a

disposable 50-ml collection tube (Greiner, Frickenhausen, Germany) connected to

vacuum pump (Model VMAR–5100, William A. Cook Australia Ltd., Queensland,

Australia). The vacuum was set to a flow rate of 20 ml/min. The follicular aspirates

were pooled in collection tubes and allowed to sediment for 10-15 min after

collection. The sediment (2-3 ml) was then removed and resuspended with an equal

amount of a warm (38.5 °C) Dulbecco´s phosphate buffered saline (DPBS) (Cat. D-

6650, SIGMA-ALDRICH Chemie GmbH, Taufkirchen, Germany, as described in

Table 12, Appendix) supplemented with 1% heat-inactivated newborn calf serum

(NBCS, Cat. 16010-159, Gibco / Invitrogen GmbH, Karlsruhe, Germany); PBS + 1%

newborn calf serum (NBCS; as described in Table 12, Appendix,). This suspension

was allowed to stand for 15 min. The sediment (2-3 ml) was removed again and

mixed with an equal amount of PBS + 1% NBCS in 10-ml plastic dishes (35-mm


48

plastic Petri dishes, Cat. 627102, Greiner BIO-ONE GmbH, Frickenhausen,

Germany) before viewing under a stereomicroscope at between 20 and 50x

magnification (Model SZ112, Olympus Optical Co. Europa GmbH, Hamburg,

Germany).

3.1.3 Classification of Cumulus-Oocyte Complexes (COCs) Depending on the requirements of a particular experiment, cumulus-oocyte

complexes (COCs) were classified into four categories based upon the character of

their cellular investments (as for cattle by LEIBFRIED and FIRST 1979):

Category I: Oocytes surrounded by a compact, complete, cumulus oophorus (cells)

with more than three layers.

Category II: Oocytes enclosed by a complete, compact, but fewer than three layers

or with only partial cumulus investment.

Category III: Oocytes surrounded only by corona radiata cells.

Category IV: Denuded oocytes without any cellular investments.

Regardless of the category, no oocytes with degenerated cytoplasm or surrounded

by expanded cumulus cells were used for the experiments.

3.1.4 In Vitro Maturation (IVM) For in vitro maturation (IVM) experiments, only high quality COCs of categories I and

II were selected and transferred to the holding medium (PBS + 1% NBCS, as

described in Table 12, Appendix). These oocytes were washed twice in 2 ml holding

medium, according to the requirements of the individual experiment as outlined in the

experimental design later in this section.

Groups of 50 COCs were the washed three times in oocyte maturation media (as

described in Table 13, Appendix), and placed dropwise into 500 µl media in five-well

embryo culture dishes (Cat. 19021005, MINITÜB Abfüll– und Labortechnik GmbH,


49

Tiefenbach, Germany). The oocyte maturation medium was supplemented with

hormones (as described in Table 13, Appendix) and oocytes were matured in this

medium for 23–24 h at 39 °C and in 5% CO2 at 100% humidity in an incubator

(Nuaire type NU 2700 E, Zapf Instruments, Sarstedt, Germany). After this hormone

treatment, the COCs were washed in oocyte maturation medium without hormonal

supplements (as described in Table 13, Appendix) and transferred dropwise into 500

µl drops of the same medium without hormones for another 22 to 23 h of culture.

Following maturation, the oocytes were vortex agitated for 1-2 min for removal of

cumulus cells. Denuded oocytes were selected for in vitro fertilization or prepared for

isolation of ZP (see 3.4. Isolation of Intact Zona Pellucida [ZP] from Oocytes of

Different Developmental Stages).

3.1.5 Maturation Rate (IVM) At the end of the in vitro maturation phase, an aliquot of oocytes was assessed

subjectively for cumulus cell expansion under a stereomicroscope (Model SZ122,

Olympus Optical Co. Europa GmbH, Hamburg, Germany) at 20 to 50x magnification

and classified into one of five categories:

Category 0: no expansion.

Category 1: very slight expansion observed only in the outer most layers.

Category 2: slight expansion observed in 2-3 layers from the periphery.

Category 3: moderate expansion, observed in about 50% of the cumulus cell layers.

Category 4: full expansion except in the corona radiata layer.

Only oocytes of categories 3 and 4 were used for further experiments.


50

3.2 In Vivo Maturation of Porcine Oocytes 3.2.1 Collection of In Vivo Matured Porcine Oocytes In vivo matured oocytes were collected from superovulated gilts. Prepubertal 5.5-

month-old gilts (German Landrace) received a single intramuscular injection of 1500

IU pregnant mare serum gonadotropin (PMSG) (Intergonan, Vemie Veterinär Chemie

GmbH, Kempten, Germany) followed by 500 IU human chorionic gonadotropin (hCG)

(Ekluton, Vemie Veterinär Chemie GmbH, Kempten, Germany) 72 h later. Gilts were

slaughtered 37 to 38 h after hCG injectiom and the whole genital tract was removed

immediately after the blood was drained from the carcass. The genital tract was

transported within 5 min to the laboratory in a thermos flask filled with warm

physiological saline solution (38 °C). The ovaries were removed from the genital

tract.

Follicles were evaluated by size and quality. Only intact 6- to 12-mm follicles with a

translucent surface, fine vascularization, and clear follicular fluid were flushed 2 to 3

times with flushing medium (PBS + 1% NBCS, as described in Table 12, Appendix)

with a short bevelled 18-gauge needle (Microlance 3, Becton Dickinson GmbH,

Heidelberg, Germany). The aspirated follicular fluid was placed into pre-warmed five-

well embryo culture dishes (Cat. 19021005, MINITÜB Abfüll- und Labortechnik

GmbH, Tiefenbach, Germany) and maintained as individual minidrops to simplify

oocyte identification and transfer into the washing medium (PBS + 1% NBCS, as

described in Table 12, Appendix). The COCs were repeatedly washed and evaluated

under a stereomicroscope at 50x magnification, with emphasis on cumulus

expansion and integrity of the cytoplasm and plasma membrane (i.e. oolemma).

Oocytes potentially at MII stages were washed, denuded and pre-equilibrated in Fert-

TALP (as described in Table 13, Appendix) for at least 2 to 5 hours in humidified air

(39 °C, 5% CO2). Alternatively, the oocytes were prepared for isolation of the zona

pellucida (ZP) (see 3.4. Isolation of Intact Zona Pellucida [ZP] from Oocytes of

Different Developmental Stages).


51

3.3 In Vitro Fertilization (IVF) In vitro and in vivo matured oocytes were washed three times in in vitro fertilization

medium (FertTalp), and groups of 50 oocytes were placed in 90 µl of FertTalp. The

medium was pre-equilibrated under paraffin oil for 2-3 h at 39 °C in 5% CO2.

Cryopreserved epididymal semen (0.25-ml straw) was thawed in 10 ml Androhep

(MINITÜB Abfüll- und Labortechnik GmbH, Tiefenbach, Germany), then in

Dulbecco´s phosphate buffered saline (DPBS) (Cat. D-6650, SIGMA-ALDRICH

Chemie GmbH, Taufkirchen, Germany) supplemented with 0.1% (w/v) bovine serum

albumin (BSA) (Cat. A-9647, SIGMA-ALDRICH Chemie GmbH, Taufkirchen,

Germany). Spermatozoa were washed once by centrifugation at 820 x g for 3 min.

The supernatant was discharged, and the pellet was resuspended in 430 µl FertTalp,

sperm concentration was determined as described above, and 10 µl of the final

semen suspension was added to the fertilization drop to yield a final concentration of

3 X 105 spermatozoa/ml. Oocytes were co-incubated with spermatozoa for 1, 2, 3, 6,

and 18 hours at 39 °C in a humified atmosphere with 5% CO2. Alternatively, oocytes

of both groups were incubated under the same IVF conditions without sperm for 12-

18 hours.

3.3.1 Fertilization Rate (IVF)

For monitoring of cleavage of the blastocyst, IVF was performed for 18 h and the

presumptive zygotes were washed three times in NCSU23 (as described in Table 13,

Appendix) containing 0.4% (w/v) BSA (Cat. A-9647, SIGMA-ALDRICH Chemie

GmbH, Tiefenbach, Germany). Groups of 30- to 35 embryos were cultivated for 7

days in 500 µl NCSU23 at 39 °C in 5% CO2. The blastocysts were then labelled with

a solution containing 10 µm/ml propidium iodide (Cat. P-4170, SIGMA-ALDRICH

Chemie GmbH, Taufkirchen, Germany) for 5 min at RT, and the relative spatial

distribution of DNA was evaluated by laser scanning confocal microscopy (LSCM)

(Microscope Model LSM 510, Carl Zeiss Jena GmbH, Jena, Germany).


52

3.4 Isolation of Intact Zona Pellucida (ZP) from Oocytes of Different Developmental Stages The following different developmental stages were distinguished: oocytes in the

germinal vesicle (GV) stage (Exp. I), those derived from 23 h of in vitro maturation

GV (IVM) (MI; Exp. II) and from 46 to 48 h of IVM (MII; Exp. III), in vivo matured

oocytes (MII; Exp. IV); and oocytes sampled for 0, 1, 2, 3, 6, 12, and 18 h after in

vitro fertilization (IVF; Exp. V and Exp. VI). All were washed three times in PBS + 2%

polyvinyl alcohol (PVA) (Cat. P-8136, SIGMA-ALDRICH Chemie GmbH, Taufkirchen,

Germany; as described in Table 12, Appendix). Isolated ZP were retrieved after

removal of the ooplasm by pipetting the oocyte suspensions up and down under a

stereomicroscope through a glass pipette with a tip diameter smaller than that of the

oocytes. Isolated ZP were washed by passage through three drops of PBS + 2%

PVA and immediately prepared for experiments.

3.5 Experimental Design The presence of free thiol groups of the ZP from different developmental stages was

morphologically studied by LSCM (Trial II) and SEM (Trial III). Changes in the content

of free thiol groups correlates with the formation of new disulfide bonds and can be

used as a marker for biochemical alteration of the ZP. Free thiol groups were

detected by labelling with iodacetamide derivatives. For LSCM, the ZP were treated

with fluorescence–labelled iodoacetamide (5-Iodoacetamidofluorescein. 5-IAF)

(Cat.I3, Molecular Probes Europe, Leiden, The Netherlands), whereas biotinylated

iodoacetamide (Biotin Iodoacetamide (N- (Biotinoyl) N´-(Iodoacetyl) Ethylenediamine

(biotin–conjugated iodoacetamide, BIAM) (B-1591; Molecular Probes Europe,

Leiden, The Netherlands) was used for SEM, for which BIAM was identified with a

streptavidin gold secondary label.


53

Figure 11 shows the experimental design for BIAM labelling of free thiol groups prior

to scanning electron microscopy (SEM, Trial III) or electrophoresis (SDS-PAGE, 1- or

2-D), while 5-IAF was used for laser scanning confocal microscopy (LSCM, Trial II).

Fig.11: Overview of the experimental design.

The zonae pellucidae (ZP) were investigated in seven different experiments on

oocytes:

• Experiment I: Immature oocytes (GV).

• Experiment II: Oocytes matured in vitro (IVM) for 23 h (MI).

• Experiment III: Oocytes matured in vitro (IVM) for 46-48 h (MII).

• Experiment IV: Oocytes matured in vivo (MII).

• Experiment V: Oocytes derived from IVF of in vitro matured (IVM) oocytes, 0,

3, and 18 h after onset of IVF.

• Experiment VI: Oocytes derived from IVF of in vivo matured oocytes 0, and 3 h

after onset of IVF.

• Experiment VII: In vivo and in vitro matured (IVM) oocytes cultured under IVF

conditions but without exposure to spermatozoa for between 12 and 18 hours.


54

3.6 Pre-Trial I: Selection of Adequate pH Conditions to Indicate Oxidant–Sensitive Thiol Groups The pKa of the thiol side chain of the cysteine residues has been determined to be ≈

8.3. However, free thiol groups are highly sensitive to the microenvironment within a

protein. Oxidant-sensitive cysteine residues with low pKa can be selectively modified

at pH 6.5 with labelled iodoacetamide and monitored by chemiluminescence using

streptavidin blot analysis (according CHOI et al. 2001).

In order to test the pH levels with the most specific labelling pattern for further

morphological evaluation (LSCM, Trial II, and SEM, Trial III) the reaction of this thiol-

reactive stain with zona pellucida (ZP) glycoproteins ZPA, ZPB, and ZPC, was

evaluated in the Pre-Trial by electrophoretic methods (one-dimensional gel

electrophoresis, –1-D SDS-PAGE, –and by two-dimensional gel electrophoresis, –2-

D SDS-PAGE) under different pH conditions, –pH 6.5 and pH 8.5, – with a thiol-

specific agent, i.e. biotin-conjugated iodoacetamide, –BIAM, (KIM et al. 2000).

3.6.1 SDS–PAGE Electrophoresis 3.6.1.1 Labelling of Free Thiol Groups Zona pellucida of germinal vesicle (GV) oocytes (from approximately 3000 ovaries)

were dissolved for 2 h in water at 72 °C. The samples were freed from insoluble

compounds by centrifugation at 15000 x g for 10 min at RT. The dissolved ZPs were

immediately lyophilized in SpeedVac Plus (Model Sc 110 A, Savant, Instrument, Inc.,

Farmington, NY, USA). Aliquots (equivalent to between 75 and 100 zonae

pellucidae) were then redissolved either in 10 µl 50 mM BIS-TRIS (Cat. 103252,

Merck, Darmstadt, Germany), pH 6.5, or in 20 mM TRIS-HCl (Cat. 77-86-1,

AppliChen GmbH, Darmstadt, Germany), pH 8.5. The labelling procedure was

performed according KIAM et al. (2000). Briefly, BIAM was dissolved in dimethyl


55

sulfoxide (DMSO) (Cat. D-2650, SIGMA-ALDRICH Chemie GmbH, Taufkirchen,

Germany) and added to the ZP solutions to a final concentration of 0.025 mM BIAM

and < 0.1% DMSO. The suspension was incubated at 25 °C for 30 min in the dark.

Bubbling with nitrogen gas at low flow rate for 60 sec eliminated oxygen from the

buffer. The labelling reaction was terminated by addition of iodoacetamide (Cat. I-

1149, SIGMA-ALDRICH Chemie GmbH, Taufkirchen, Germany) to a final

concentration of 20 mM. The reaction mixture was centrifuged 2000 xg for 30 min at

25 °C and the supernatant was subjected to sodium dodecyl sulfate-polyacrylamide

gels (SDS-PAGE) as described in the Appendix. (9.2. Composition of Reagents

Prepared for Use in Biochemical Experiments).

3.6.1.2 One–Dimensional Gel Electrophoresis (SDS–PAGE) Aliquots of ZP (equivalent to between 75 and 100 zonae pellucidae/lane) were

separated on 10% (w/v) polyacrylamide gels using a discontinuous buffer system

(see Appendix, 9.2 Composition of Reagents Prepared for Use in Biochemical

Experiments) as described by LAEMMLI (1970). Electrophoresis was carried out at

100 V for 30 min and then at 200 V for 1 h until the tracking dye reached the bottom

of the gel. The molecular weight of the purified protein was estimated using the

proteins of known protein molecular mass as standard (Low Molecular Weight [LMW]

Standard, Cat. RP N 2280, Pharmacia LKB, Uppsala, Sweden).

3.6.1.3 Two–Dimensional Gel Electrophoresis (SDS–PAGE) For 2–D SDS–PAGE, aliquots of approximately 150 zonae pellucidae/gel were mixed

overnight at RT (20 °C) with rehydration buffer (as described in the Appendix, 9.2.4

Immobilized pH Gradients. IPG), 0.5% IPG buffer (Cat. 17-6000-87, Amersham

Biosciences Europe GmbH, Freiburg, Germany) and applied to a 7-cm long

ImmobilineR DryStrips gel (Cat. 80-6416-87, Amersham Biosciences Europe GmbH,

Freiburg, Germany) at pH 3 to10 in an IPGphor isoeletric focusing system (Cat. 80-

6505-03, Amersham Biosciences Europe GmbH, Freiburg, Germany). The focusing


56

protocol was: (1) 50 µA per strip at 20 °C; (2) re-hydration for 15 h; (3) 150 V for 1 h

(step and hold); (4) 500 V for 1 h (step and hold); (5) 1000 V for 1 h (step and hold);

(6) 4000 V to 17500 V (step and hold). After electrofocusing, the strips were

equilibrated for 20 min in 0.05 M TRIS-HCl, pH 6.8 containing 2% SDS, 6 M urea,

30% (w/v) glycerol, and 2% (w/v) SDS, and subjected to 10% SDS-PAGE as

described by LAEMMLI (1970). All IEF strips were checked for complete elution with

Coomassie Blue staining (Coomassie Brillant Blue) (Cat.161-04000, Bio-Rad

Laboratories GmbH, Munich, Germany) as described by GÖRG et al. (1998). The

molecular weight of the purified protein was estimated using the marker proteins of

known molecular mass as standard (Low Molecular Weight [LMW] Standard) (Cat.

RP N 2280, Pharmacia LKB, Uppsala, Sweden).

3.6.1.4 Protein Blot and Chemiluminescence Analysis Free thiol groups modified with BIAM were detected with enhanced

chemiluminescence. Protein blot analyses were performed immediately after SDS-

PAGE electrophoresis. The gels were removed and then electrotransferred to PVDF

membranes (Cat. 1722026, Boeringer Mannheim, Mannheim, Germany) for 2 h at RT

using semidry blotting equipment (Biometra, Göttingen, Germany). Thereafter they

were blocked overnight with 1% Tween 20 in TBS (as described in Appendix,

9.2.7.TBS/Tween). The next day, the membranes were washed in the same buffer

and incubated with peroxidase-conjugated streptavidin (Cat. 016-030-084, Dianova,

Hamburg, Germany) in a dilution of 1:10000, for 30 min at RT, and detected with

enhanced chemiluminescence (Super Signal West Pico, Pierce über KMF

Laborchemie, St. Augustin-Buisdorf, Germany) according to the manufacturer’s

recommendations.


57

3.7 Trial II: Laser Scanning Confocal Microscopy (LSCM) 3.7.1 Labelling of Free Thiol Groups Intact ZP of all groups were labelled with 5-iodoacetamidefluoresceín (5-IAF) (Cat. I3,

Molecular Probes Europe BV, Leiden, The Netherlands) with a stock solution of 200

mM 5-IAF in dimethylformamide (DMF) (Cat. 14073-2, SIGMA-ALDRICH Chemie

GmbH, Taufkirchen, Germany) stored at – 20 °C. The stock solution was added to

100 mM BIS-TRIS (Cat. 103252, Merck, Darmstadt, Germany) at pH 6.5 to give a

final concentration of 0.2 mM. Five microliters of this solution were added to between

10 and 25 porcine ZP in 50 µl BIS-TRIS at pH 6.5, resulting in a final concentration of

0.02 mM. The suspension was incubated at 25 °C for 30 min in the dark. ZP’-s

incorporating 5-IAF were washed twice in PBS + 2% PVA to remove excess

fluorescent stain and were mounted on coverslips coated with poly-L-lysine agarose

(Cat. P-6893, SIGMA-ALDRICH Chemie GmbH, Taufkirchen, Germany) coated

cover slips with Mowiol (Cat. 4-88, Hoechst, Frankfurt, Germany, as described in the

Appendix, 9.3.3. Mowiol Mounting Medium) in 0.2 M BIS-TRIS at pH 6.5 for 30 min at

RT and subsequently monitored by a LSCM (Microscope model LSM 510, Carl Zeiss

Jena GmbH, Jena, Germany) equipped with an argon laser. Fluorescence intensity

was quantified at the same confocal microscope settings by calculating the mean

pixel intensity for each stained ZP using gain 680-640 and viewed in differential

interference contrast (DIC) 530-640 at an excitation wavelength of 488 nm with a

barrier filter at 550 nm. Composite images of the ZP were created using between 5

and 25 sections. Composite images and/or optical sections were recorded using the

standard software of the Zeiss confocal microscope and saved as TIFF files.

3.7.2 Specificity of the Labelling Procedures Intact ZP was incubated with iodoacetamide (Cat. I-1149, SIGMA-ALDRICH Chemie

GmbH, Taufkirchen, Germany) with a stock solution of 92.5 mg iodoacetamide in

1000 µl double-distilled water, and 0.5 µl of this solution were added to 11 µl BIS-


58

TRIS at pH 6.5 (Cat. 103252, Merck, Darmstadt, Germany) to give a final

concentration of 20 mM. The suspension was incubated at 25 °C for 30 min in the

dark. ZP were then washed three times with PBS + 2% PVA (as described in Table

12, Appendix) to remove excess iodoacetamide and were subsequently labelled with

5-iodoacetamidofluoresceín (5-IAF) (Cat. I3, Molecular Probes Europe, The

Netherlands) as described in details in 3.7.1. Labelling of Free Thiol Groups.

3.7.3 Labelling of Spermatozoa Bound to the Zona Pellucida (ZP) with Propidiumiodide (PI) Twelve h after onset of in vitro fertilization (IVF) intact oocytes matured in vivo and in

vitro (IVM) were washed two times to remove loosely bound spermatozoa in PBS +

2% PVA (as described in Table 12, Appendix) and were then labelled with propidium

iodide (PI) (Cat. P-4170, SIGMA-ALDRICH Chemie GmbH, Taufkirchen, Germany).

The stock solution of 0.5 mg/ml PI in double-distilled water was stored at –28 °C in

the dark. One microliter of this solution was added to a drop containing 50 oocytes.

The suspension was incubated at 25 °C for 5 min in the dark. Oocytes were then

washed twice in PBS + 2% PVA to remove excess fluorescent stain and were

mounted on coverslips coated with poly-L-lysine agarose (Cat. P-6893, SIGMA-

ALDRICH Chemie GmbH, Taufkirchen, Germany). The oocytes were subsequently

viewed by LSCM (Microscope model LSM 510, Carl Zeiss Jena GmbH, Jena,

Germany) with the differential phase contrast (DPC), and the background was

examined with differential interference contrast (DIC) as an internal control.

3.7.4 Evaluation and Classification of Labelling Patterns of Free Thiol Groups by Laser Scanning Confocal Microscopy The labelling patterns of free thiol groups of the ZP glycoproteins were classified

according to the intensity difference (D) between the inner (I) and outer surfaces (A)

(D = I - A) and by the homogeneity pattern (H) of the distribution of free thiol groups.


59

An absolute measure of the intensity was recorded by LSCM with the differential

phase contrast (DPC).

3.7.4.1 Fluorescence Pattern: Intensity Difference (D) Between Inner and Outer Regions of the Zona Pellucida (ZP) The fluorescence intensity difference (D) was calculated as the difference between

the intensity of the inner (I) and outer (A) surfaces of the ZP. The intensity differences

were classified as small (D = 0, Fig. 12 A), medium (D = 1, Fig. 12 B) and strong (D =

2, Fig. 12 C), as shown in Figure 12. Alternatively, the total fluorescence intensity (I)

was subjectively classified as none (I = 0, Fig. 13 A), low (I = 1, Fig. 13 B), medium (I

= 2, Fig. 13 C), or strong (I = 3, Fig. 13 D), as shown in Figure 13.

Fig.12: Confocal microscope image of the fluorescence intensity difference (D) pattern

between the distribution across the inner and outer regions of the ZP: a, no difference (D =

0), b, medium difference (D = 1), c, strong difference (D = 2).


60

Fig. 13: Confocal microscope image of the distribution of the fluorescence intensity (I)

pattern across the ZP: a, strong intensity (I = 3), b, medium intensity (I = 2), c, low intensity

(I=1), d, no fluorescence (I = 0).

3.7.4.2 Homogeneity (H) Pattern The homogeneity of the fluorescence signal (H) was classified in four categories:

minor homogeneity (H = 0, Fig.14 A), low homogeneity (H = 1, Fig.14 B), medium

homogeneity (H = 2, Fig. 14 C), and high homogeneity (H = 3, Fig.14 D), as

documented in Figure 14.

Fig. 14: Confocal microscope images of homogeneity (H) labelling pattern distributed across

the ZP: a, minor homogeneity (H = 0), b, low homogeneity (H = 1), c, medium homogeneity

(H = 2), d, high homogeneity (H = 3).


61

3.8 Trial III: Scanning Electron Microscopy (SEM) 3.8.1 Labelling of Free Thiol Groups The labelling procedure was performed on:

1. Isolated porcine zonae pellucidae (ZP) and

2. Intact porcine oocytes

About 10 to 25 ZP and/or oocytes were resuspended in 10 µl of a 50 mM BIS-TRIS

(Cat.103252, Merck, Darmstadt, Germany) solution containing 0.025 mmol BIAM

(Cat. B-1591, Molecular Probes Europe, Leiden, The Netherlands) and < 0.1%

dimethyl sulfoxide (DMSO) (Cat. D-2650, SIGMA-ALDRICH Chemie GmbH,

Taufkirchen, Germany). The suspension was incubated at 25 °C for 30 min in the

dark. The labelling reaction was terminated by the addition of unlabelled

iodoacetamide (Cat. I-1149, SIGMA-ALDRICH Chemie GmbH, Taufkirchen,

Germany) at a final concentration of 20 mM. After incorporation of BIAM, ZP and

oocytes were washed twice in PBS + 2 % PVA (Table 12) to remove all superfluous

BIAM, and were then stored a fixative solution (as described in the Appendix, 9.3.1

HEPES-Fixation). The following procedures were performed in cooperation with Dr.

P. Schwartz (Department of Anatomy, Georg- August- University, Goettingen,

Germany).

Before labelling, samples were washed twice in PBS for 10 min followed by

incubation at RT for 30 min in streptavidin-gold (streptavidin–coated 20nm gold

particles, Cat. EM.STP 20, BBInternational Ltd, Cardiff, England) in PBS at

concentrations of 1:100, 1:500, and 1:1000. After two washing steps (10 min each) in

PBS, samples were transferred to poly-L-lysine-coated cover slips (Poly-L-Lysine,

Cat-P-6893, SIGMA-ALDRICH Chemie GmbH, Taufkirchen, Germany). Cover slips

were rinsed and then placed for 10 minutes in a fixative solution containing 0.1%

glutaraldehyde 25% (Cat. 820603, Merck, Darmstadt, Germany). After subsequent

washing for 10 min in PBS, the samples were incubated for 20 to 30 min with silver

enhancing solution (Silver Enhancing Kit-Light Microscopy, Cat. SEKL15,


62

BBInternational Ltd, Cardiff, England, UK). The preparations were then dried in a

critical point dryer (Model Polaron, Waterford, England, UK) followed by mounting on

aluminium grids. Specimens were coated with gold/palladium in a sputter coater

(Model SC510 sputter coater, Fisons Instruments, Vertriebs-GmbH, Mainz-Kastel,

Germany) and examined with a Zeiss DS 960 SEM (Microscope model DSM 960,

Zeiss, Oberkochen, Germany).

3.8.2 Classification of Labelling Pattern For semi-quantitative classification, the labelling pattern was analyzed on the basis of

the density of gold particles, as shown in Figure 15.

Streptavidin gold particles bound to BIAM (biotin-conjugated iodoacetamide)

appeared in clusters which were classified into four “features” (F) groups based on

the distribution of gold particle aggregated at the outer oocyte surface, as shown in

Figure 15: (i) F = 0, no features (Fig. 15 A), (ii) F = 1, few features (Fig.15 B), (iii) F =

2, medium number of features (Fig.15 C), and (iv) F = 3, completed distribution of

features (Fig.15 D).


63

Fig. 15: Classification of free thiol group distribution (F) by scanning electron microscopy

(SEM) (x 1000; x 900; x 2000, and x 2000). A: no gold particles (F = 0), B: few gold particles

(F = 1), C: moderate number of gold particles (F = 2), D: complete distribution of gold

particles (F = 3).

3.9 Statistical Analysis All data were analyzed with the SAS statistical package. In Trial II, porcine ZP

(LSCM) analyzed by LSCM were compared in various groups according to the length

of in vitro maturation (IVM): (i) immature (GV) vs. oocytes matured 23 h in vitro

matured (MI) (IVM), (ii) oocytes matured 23 h in vitro matured (MI) (IVM) vs. oocytes

matured 46-48 h in vitro matured (MII) (IVM), (iii) MII from in vivo matured oocytes vs.

in vitro matured (IVM) oocytes (MII), (iv) oocytes matured in vitro (IVM) for 0, 3, and

18 h after onset of IVF, (v) in vivo matured oocytes 3 h after the onset of IVF vs. in

vitro matured oocytes 3 h after the onset of IVF, for each treatment.

In Trial III (SEM), porcine ZP were analyzed from (i) immature oocytes, (ii) MII stages

after IVM, and from (iii) oocytes matured in vitro (IVM) derived 0, 3, and 18 h after the

onset of IVF. All data are given as mean ± SEM. Data were considered to be

significantly different at P < 0.05 and very significantly different when P < 0.0001.

RESULTS ___________________________________________________________________

64

4 RESULTS 4.1 Pretrial I: Selection of Adequate pH Conditions to Indicate Oxidant–Sensitive Thiol Groups A labelling procedure with biotin-conjugated iodoacetamide (BIAM) at both pH 6.5

and pH 8.5 was used in this study to estimate the optimum pH for the identification of

the thiol side chains of cysteine residues with low and high pKa. For electrophoretic

analyses (1–D and 2–D SDS-PAGE) dissolved ZP glycoproteins were coupled with

BIAM and monitored by blot analysis with HRP–conjugated streptavidin.

4.1.1 Polyacrylamide Gel Electrophoresis (SDS–PAGE) 4.1.1.1 One–dimensional Gel Electrophoresis (SDS–PAGE) Proteins containing active cysteine residues in the ZP of immature oocytes (GV) were

identified by 1–D SDS–PAGE. Labelling with BIAM at pH 8.5 resulted in the oxidation

of virtually all free cysteine residues of the glycoproteins ZPA, ZPB, and ZPC. The

ZPA glycoprotein showed strong staining and appeared as a broad band between 80

and 120 kDa (Fig. 16 B). The glycoproteins ZPB/ZPC were not separated and

appeared as a broad, strongly labelled band between 55 and 60 kDa (Fig. 16 B).

In contrast, staining with BIAM at pH 6.5 resulted in selective labelling within the ZPA

glycoprotein (Fig. 16 A), while no labelling of glycoproteins ZPB/ZPC was present

(Fig. 16 A).

RESULTS ___________________________________________________________________

65

Fig. 16: Analyses of BIAM-labelled ZP glycoproteins by means of 1–D SDS–PAGE at pH 6.5

and 8.5. Between 75 and 100 zonae pellucidae were dissolved from the germinal vesicle

(GV) stage and labelled with BIAM at pH 6.5 and pH 8.5. Equal portions of each mixture

were detected by chemiluminescent blot analysis with HRP–conjugated streptavidin. (A):

Selective labelling of the ZPA glycoprotein was demonstrated at pH 6.5. No labelling of

glycoproteins ZPB/ZPC was present. (B): Biotinylation (BIAM) at pH 8.5 resulted in the

labelling of virtually all free cysteine of the glycoproteins ZPA, ZPB, and ZPC.

4.1.1.2 Two–dimensional Gel Electrophoresis (SDS–PAGE) Because of the heterogeneity of the glycans, ZP glycoproteins separated in two–

dimensional gel electrophoresis over a broad pI range. In order to analyze the

occurrence of free thiol groups within ZP glyco-isoforms, 2–D SDS–PAGES were

RESULTS ___________________________________________________________________

66

performed on ZP glycoproteins labelled with biotin-conjugated iodoacetamide (BIAM)

at pH 8.5 and pH 6.5 (Fig. 17). Biotinylation of the free thiol groups at pH 8.5 showed

a dominant labelling of the ZPA glycoproteins at between pH 4 and 6, whereas the

labelling of ZPB/ZPC glycoproteins was strong over a wide pH range of between 3

and 6.5 (Fig.17 B). As already demonstrated by 1–D SDS–PAGE under pH 6.5

conditions, biotinylated BIAM led to selective labelling of the ZPA glycoproteins. The

glycoprotein ZPA appeared as a single protein band in the pH range of between 4

and 6 (Fig.17 A).

Fig. 17: Zona pellucida (ZP) from immature (GV) porcine oocytes was labelled with BIAM at

pH 6.5 and pH 8.5 and separated by 2–D SDS–PAGE. Approximately 150 zonae pellucidae

were dissolved and labelled with BIAM at pH 6.5 (A) and at pH 8.5 (B). SDS–PAGE was

carried outer under non–reducing conditions in 10% acrylamide separating gel with 4%

stacking gel, transferred to PVDF membrane, and visualized with streptavidin-peroxidase

and chemiluminescence.

RESULTS ___________________________________________________________________

67

When considering these electrophoretic results, it must be recalled that ZPA

glycoproteins contain oxidant-sensitive Cys residues with a low pKa, which may be

predominantly involved in the formation of the three–dimensional architecture and

structural changes of the ZP. Therefore, we used labelling conditions at pH 6.5 for

further morphological studies with laser scanning confocal microscopy (LSCM) and

scanning electron microscopy (SEM).

4.2 Trial II: Laser Scanning Confocal Microscopy (LSCM) The distribution of free thiol groups within the ZP of oocytes of different

developmental stages were studied by laser scanning confocal microscopy (LSCM)

using 5-iodoacetamidefluoresceín (5-IAF) at pH 6.5. The fluorescence patterns were

subjectively classified according to homogeneity (H), intensity difference (D), and

overall intensity (I) of the fluorescence pattern as outlined in Material and Methods.

4.2.1 Specificity of the Labelling Procedures The specificity of the interaction was controlled by reacting the free thiol groups with

iodoacetamide prior to the fluorescence labelling procedure with 5-IAF. When

cysteine residues are blocked, the fluorescent stain cannot incorporate into the intact

zona pellucida (ZP). As shown in Figure 18, no fluorescent stain was verified at the

ZP matrix, indicating that the fluorescent dye did not bind unspecifically to the ZP.

RESULTS ___________________________________________________________________

68

Fig. 18: Specificity of fluorescent labelling with 5-IAF to the ZP matrix. The fluorescent dye

(5-IAF) did not react with free thiol groups (B). The image (A) was evaluated by differential

interference contrast (DIC) as one of our controls for the non–reaction of the free thiol groups

with fluorescent stain (5-IAF) as analyzed by differential phase contrast (DPC, B).

4.2.2 Fluorescence Pattern of the Zona Pellucida (ZP) during In Vitro Maturation (IVM) Immature oocytes (GV) and oocytes matured in vitro (IVM) for 23 h (MI) and for 46 to

48 h after IVM (MII) were observed according to the homogeneity (H) and intensity

difference (D) patterns of free thiol groups within the intact ZP.

(i) Homogeneity (H) Pattern The data obtained for the homogeneity (H) pattern in the ZP from oocytes in the

germinal vesicle (GV) stage and after in vitro maturation (23 h and 46-48 h of IVM, MI

and MII) are presented in Table 2.1 (Exp. I, Exp.II, and Exp. III).

RESULTS ___________________________________________________________________

69

Tab. 2.1: Free thiol group distribution according to homogeneity (H) pattern classified

as (0), (1), (2), or (3) at GV stage, 23 h (MI) and at 46 to 48 h (MII) after in vitro

maturation (IVM).

Developmental

status of the

oocyte/

Homogeneity

pattern

Homogeneity

(0)

% (n)*

Homogeneity

(1)

% (n)*

Homogeneity

(2)

% (n)*

Homogeneity

(3)

% (n)*

GV

26.1 (6)

47.8 (11) 17.4 (4) 8.7 (2)

MI (23 h of

IVM)

4.4 (2) 35.6 (16) 51.1 (23) 8.9 (4)

MII (46-48 h of

IVM)

0.0 (0) 5.3 (5) 36.8 (35) 57.9 (55)

*The number (n) of ZP examined is shown in parentheses. The dominant population from germinal vesicle (GV) stages showed a minor (H = 0,

26.1%) and low (H = 1, 47.8%) homogeneity pattern distributed across the ZP. The

dominant homogeneity pattern among the oocytes matured in vitro (IVM) for 23 h

(MI) was between low (H = 1, 35.6%) and medium (H = 2, 51.1%), while that of the

oocytes matured in vitro (IVM) for 46 to 48 h (MII) ranged from medium (H = 2;

36.8%) and high (H = 3, 57.9%).

The results obtained from probability values (P) are given in Table 2.2.

RESULTS ___________________________________________________________________

70

Tab. 2.2: Probability values (P) for the comparison of the homogeneity (H) pattern

distribution at different ZP stages (GV, MI, and MII) of in vitro maturation (IVM) (Chi-

Square Test).

Developmental

status of the

oocyte/

Homogeneity

pattern

Homogeneity

(0)

Homogeneity

(1)

Homogeneity

(2)

Homogeneity

(3)

GV vs. MI

(23 h of IVM)

0.008 0.32 0.007 0.97

MI vs. MII

(46 h of IVM)

0.03 0.0001 0.10 0.0001

After IVM (46-48 h, MII), there was a statistically significant shift to greater

homogeneity (3) (P < 0.0001).

Figure 19 shows the dominant homogeneity (H) patterns within the ZP from GV, 23

and 46-48 h after in vitro maturation (IVM).

RESULTS ___________________________________________________________________

71

Fig. 19: Confocal microscope images of homogeneity (H) pattern showed the distribution of

free thiol groups in the ZP surface from oocytes at the germinal vesicle (GV) stage, at 23

(MI), and at 46-48 h after IVM (MII). (a) H = 0 from GV, (b) H = 1 from GV, (c) H = 2 from

oocytes matured in vitro (IVM) for 23 h (MI), and (d) H = 3 from oocytes matured in vitro

(IVM) for 46-48 h (MII). Images were evaluated by differential phase contrast (DPC).

RESULTS ___________________________________________________________________

72

(i) Intensity Pattern (Intensity Difference) The data obtained for fluorescence of intensity difference (D) between the inner (I)

and the outer (A) surface of the ZP from oocytes in the germinal vesicle (GV) stage

and after IVM (23 and 46-48 h; MI and MII, respectively) is presented in Table 3.1

(Exp. I, Exp. II, and Exp. III).

Tab. 3.1: Free thiol group distribution according to intensity difference (D) pattern

classified as (0) (1) or (2) at GV stage, 23 h (MI) and at 46-48 h (MII) after in vitro

maturation (IVM).

Developmental status of the oocyte/ Difference pattern

Difference (0) % (n)*



Immature Oocytes (GV I)

0.0 (0) 39.1 (9) 60.9 (14)

MI (23 h of IVM)

2.2 (1) 19.6 (9) 78.2 (36)

MII (46-48 h of IVM)

0.0 (0) 68.5 (76) 31.5 (35)

*The number (n) of ZP examined is shown in parentheses.

There were strong differences in the dominant populations from germinal vesicle

(GV) stage, and from metaphase I (23 h after IVM) between the inner and outer

regions of the ZP (D = 2, 60.9% and 78.2%, respectively), while there were moderate

differences in the dominant populations from metaphase II oocytes (MII, 46-48 h after

IVM) between the inner and the outer regions of the ZP (D = 1, 68.5%). These results

indicated that the fluorescence intensity within the outer zona surface increases

during maturation in comparison with the inner zona surface.

The probability values (P) at different stages of in vitro maturation (IVM) are also

included in Table 3.2.

RESULTS ___________________________________________________________________

73

Tab. 3.2: Probability values (P) for the comparison of the intensity difference (D)

pattern distribution at different ZP stages (GV, MI, and MII) of in vitro maturation (46-

48 h of IVM) (Chi-Square Test).

Developmental status of the oocyte/ Difference pattern

Difference (0)

Difference (1)

Difference (2)

GV vs. MI (23 h of IVM)

0.47 0.08 0.12

MI vs. MII (46 h of IVM)

0.11 0.0001 0.0001

There was no statistically signficant difference in distribution between GV stage and

MI oocytes at medium and strong intensities (D = 1, P < 0.08, and D = 2, P < 0.12,

respectively). On the other hand, there was a highly statistically significant difference

between MI (23 h of IVM) and MII (46-48 h of IVM) with respect to the distribution in

both dominant categories (medium, D = 1, and strong, D = 2), P < 0.0001. Figure 20

demonstrates exemplarily the intensity difference patterns (D) classified as (0), (1),

and (2) of the ZP from different stages (GV, MI, and MII of onset of IVM).

Fig. 20: Confocal microscope images showing the intensity difference (D) patterns of

fluorescence labelling in the inner (I) and outer (A) regions of the ZP. (A), strong intensity

difference (D = 2, from the germinal vesicle [GV] stage), (B), medium intensity difference (D

= 1, MI from 23 h of IVM), and (C), medium intensity difference (D = 1, MII from 46-48 h of

IVM). Images were evaluated by differential phase contrast (DPC).

RESULTS ___________________________________________________________________

74

4.2 3 Fluorescence Pattern of the Zona Pellucida (ZP) During In Vivo Maturation The ZP from oocytes matured in vivo (MII) were found to be significantly thinner than

those obtained after in vitro maturation (46-48 h after onset of IVM) (P < 0.0001). It

was not possible to differentiate between the outer (A) and inner (I) layers (Fig. 21).

Therefore, the fluorescence labelling of the ZP from in vivo and in vitro matured (IVM)

oocytes was calculated as the overall intensity (I) (Exp.III vs. Exp. IV).

Fig. 21: The results reveal the fine structure of the ZP among porcine oocytes after in vivo

and in vitro maturation (IVM). (A), image by reverse microscopy of the thickness of the ZP

from in vivo matured oocytes, evaluated at between 2.5 µm to 11.8 µm (X = 7.6, SD = 2.8),

(B) image by interference contrast of the thickness of the ZP from in vitro matured (IVM)

oocytes between 10.3 µm to 15 µm (X = 12.0, SD = 1.4).

(i) Homogeneity (H) Pattern The data obtained for the homogeneity (H) pattern in the ZP from in vivo and in vitro

matured (IVM) oocytes (MII) are presented in Table 4.1 (Exp. III and Exp. IV).

RESULTS ___________________________________________________________________

75

Tab. 4.1: Free thiol group distribution according to homogeneity (H) pattern classified

as (1), (2), or (3) at MII from in vivo and in vitro matured oocytes (46-48 h of IVM).

Developmental

status of the oocyte

/Homogeneity

pattern

Homogeneity (1)

% (n)*

Homogeneity (2)

% (n)*

Homogeneity (3)

% (n)*

MII (In- vivo matured

oocytes)

28.6 (8) 53.6 (15) 17.8 (5)

MII (46-48 h of IVM) 5.3 (5) 36.8 (35) 57.9 (55) *The number (n) of ZP examined is showed in parentheses.

The homogeneity pattern distributed across the ZP in the dominant population from

in vivo matured (MII) oocytes was low (H = 1, 28.6%) or medium (H = 2, 53.6%),

while that of the dominant population of oocytes matured in vitro (IVM) for 46-48 h

(MII) was medium (H = 2, 36.8%) to high (H = 3, 57.9%).

The results obtained from probability values (P) are given in Table 4.2.


distribution at in vivo and in vitro maturation (46-48 h of IVM) (Chi-Square Test).

Developmental status of the oocyte /Homogeneity pattern

Homogeneity (1)

Homogeneity (2)

Homogeneity (3)

MII vs. MII

(In vivo maturation

vs. 46-48 h of IVM)

0.0004 0.11 0.0002

The homogeneity patterns of the ZP from in vivo and in vitro matured (IVM) oocytes

(46-48 h of IVM) differed significantly (P < 0.0001) and were correlated with the

distribution of free thiol groups across the ZP surface.

RESULTS ___________________________________________________________________

76

(i) Intensity Pattern (I) The data obtained for overall intensity (I) pattern classified as (0), (1), (2), or (3) in the

ZP from in vivo and in vitro matured oocytes (IVM) was presented in Table 5.1 (Exp.

III and Exp. IV).

Tab. 5.1: Free thiol group distribution according to intensity (I) labelling pattern

classified as (1), (2), or (3) in the ZP from in vivo and in vitro matured (46-48 h of

IVM) oocytes.

Developmental

status of the

oocyte/ Intensity

pattern

Intensity (1)

% (n)*

Intensity (2)

% (n)*

Intensity (3)

% (n)*

MII (In vivo

maturation)

3.5 (1) 37.9 (11) 68.6 (17)

MII (46 -48 h of

IVM)

0.0 (0) 4.5 (5) 95.5 (106)

*The number (n) of ZP examined is shown in parentheses.

The intensity patterns in the dominant populations from in vivo matured oocytes (MII)

were medium (I = 2, 37.9%) or strong (I = 3, 68.6%), but those of the dominant

population from in vitro matured (IVM) oocytes (MII, 46-48 h of IVM) was always

strong (I = 3, 95.5%).

Table 5.2 shows the probability values (P) for in vivo and in vitro maturation (IVM).

RESULTS ___________________________________________________________________

77

Tab. 5.2: Probability values (P) for the comparison of the intensity (I) pattern

distribution for in vivo and in vitro maturation (IVM) (Chi-Square Test).

Developmental

status of the oocyte/

Intensity pattern

Intensity

(1)

Intensity

(2)

Intensity

(3)

MII vs. MII

(In vivo vs. 46-48 h

of IVM)

0.05 0.0001 0.0001

The intensity pattern of the ZP from oocytes matured in vivo and in vitro (IVM)

differed significantly (P < 0.0001). Figure 22 indicates exemplarily the difference in

the overall intensity (I) and the homogeneity (H) pattern between in vivo and IVM

oocytes (46-48 h of IVM).

RESULTS ___________________________________________________________________

78

Fig. 22: Confocal microscope image showing the overall intensity (I) and homogeneity (H)

patterns in the ZP from oocytes matured in vivo and in vitro (IVM). (A), metaphase II after in

vitro maturation of oocytes (IVM) (I = 3, H = 3), (B), (C), and (D), metaphase II after in vivo

maturation of oocytes (I = 3, H = 1), (I = 3, H = 2), (I = 3, H = 3), respectively. Images were

evaluated by differential phase contrast (DPC).

There are clear differences in morphology, overall intensity (I), and homogeneity (H)

fluorescence pattern of the distribution of free thiol groups in the ZP from in vivo and

in vitro matured (IVM) oocytes. Most in vivo matured oocytes showed intensive

RESULTS ___________________________________________________________________

79

labelling across the whole ZP, while the intensity fluorescence of IVM oocytes was

concentrated in the inner layers (i.e. ad-oolemma) of the zona pellucida (ZP).

4.2.4 Fluorescence Pattern During In Vitro Fertilization (IVF) ZP from in vivo (MII) and in vitro matured (IVM) oocytes (46-48 h of IVM, MII) 0, 3,

and 18 h after onset of in vitro fertilization (IVF) were classified according to their

homogeneity (H) and overall intensity (I) fluorescence patterns.

4.2.4.1 Fluorescence Pattern of the Zona Pellucida (ZP) from In Vitro Matured (IVM) Oocytes after Onset of In Vitro Fertilization (IVF)

(i) Homogeneity (H) Pattern The data obtained for the homogeneity (H) patterns of ZP from in vitro matured

oocytes (MII) after IVF were classified only at 0 and 3 h after IVF (Tables 6.1 and

6.2). Evaluation of the homogeneity pattern 18 h after IVF was not possible because

of the decreased fluorescence labelling. Therefore, the fluorescence labelling of the

ZP from in vitro matured (IVM) oocytes 18 h after onset of IVF was calculated only as

the overall intensity (I) and is presented in Tables 7.1 and 7.2.

Table 6.1 shows homogeneity (H) patterns classified as (0), (1), (2), and (3) of ZP

from in vitro matured (IVM) oocytes 0 and 3 h after onset of in vitro fertilization (IVF)

(Exp. V).

RESULTS ___________________________________________________________________

80

Tab. 6.1: Homogeneity (H) patterns (0-3) of the distribution of free thiol groups in

porcine ZP derived from IVM oocytes (46-48 h of IVM) 0 and 3 h after onset of IVF.

Developmental

status of the

oocyte

/Homogeneity

pattern

Homogeneity

(0)

% (n)*

Homogeneity

(1)

% (n)*

Homogeneity

(2)

% (n)*

Homogeneity

(3)

% (n)*

0 h after IVF 0.0 (0) 5.3 (5) 36.8 (35) 57.9 (55)

3 h after IVF 0.0 (0) 3.3 (3) 15.4 (14) 81.3 (74) *The number (n) of ZP examined is shown in parentheses. At 0 h after IVF the homogeneity patterns distributed across the ZP in the dominant

population were medium (H = 2, 36.8%) and high (H = 3, 57.9%). At 3 h after onset

of IVF there were two dominant oocyte populations (H = 2, 15.4% and H = 3, 81.3%).

There was no difference in the fluorescence of these oocytes (0 and 3 h after IVF)

from that of IVM oocytes (46-48 h of IVM). (For comparison see also Table 2.1 and

Fig. 19).

The probability values (P) are shown in Table 6.2 for the homogeneity (H) pattern (0-

3) 0 and 3 h after onset of IVF.

Tab. 6.2: Probability values (P) for the comparison of the distribution of the

homogeneity (H) pattern at 0 and 3 h after onset of IVF (Chi-Square Test).

Developmental

status of the

oocyte

/Homogeneity

pattern

Homogeneity

(1)

Homogeneity

(2)

Homogeneity

(3)

MII vs. 3 h IVF

(46-48 h of IVM) 0.50 0.009 0.005

RESULTS ___________________________________________________________________

81

There was a significant difference in the homogeneity pattern of the ZP derived 0 h

and 3 h after onset of IVF, with P < 0.009 (H = 2) and P < 0.005 (H = 3).

(i) Intensity Pattern (I) Table 7.1 presents the intensity (I) fluorescence labelling classified as (1), (2) and (3)

of free thiol groups distributed across the ZP from in vitro matured (IVM) oocytes 0, 3,

and 18 h after onset of IVF (Exp. V).

Tab.7.1: Intensity (I) fluorescence patterns (1-3) of the distribution of free thiol groups

in porcine ZP from in vitro matured (IVM) oocytes 0, 3, and 18 h after onset of IVF.

(Exp. V).

Developmental

status of the

oocyte/ Intensity

pattern

Intensity (0)

% (n)*

Intensity (1)

% (n)*

Intensity (2)

% (n)*

Intensity (3)

% (n)*

0 h after IVF 0.0 (0) 0.0 (0) 4.5 (5) 95.5 (106)

3 h after IVF 0.0 (0) 2.3 (4) 27.1 (46) 70.6 (120)

18 h after IVF 60.0 (72) 26.7 (32) 8.3 (10) 5.0 (6) *The number (n) of ZP examined is shown in parentheses. Immediately (0 h) after IVF, the intensity fluorescence pattern of the dominant

population distributed across the ZP was strong from (I = 3, 95.5%). Three h after

onset of IVF there were two dominant oocyte populations with intensity fluorescence

patterns (I = 2, 27.1%, and I = 3, 70.6%). Eighteen h after IVF, the intensity

fluorescence patterns in the dominant oocyte population were either completely

absent (i.e. zero intensity: I = 0, 60.0%) or low (I = 1, 26.7%).

Table 7.2 shows the probability values (P) of intensity (I) patterns for the overall

distribution of free thiol groups at different times after onset of IVF (3 and 18 h, Exp.

V).

RESULTS ___________________________________________________________________

82

Tab. 7.2: Probability values (P) for the comparison of the distribution of the intensity

(I) pattern at 0, 3, and 18 h after onset of IVF (Chi-Square Test).

Developmental status of the oocyte /Intensity pattern

Intensity

(0)

Intensity

(1)

Intensity

(2)

Intensity

(3)

3 h vs. 18 h IVF 0.0001 0.0001 0.0001 0.0001

There was a highly significant difference in the intensity (I) pattern (0-3) of fertilized

ZP 3 h and 18 h after onset of IVF in all categories (0-3) at P < 0.0001.

Figure 23 shows the intensity (I) patterns for IVM oocytes 0 and 3 h after onset of IVF

(Exp. V).

RESULTS ___________________________________________________________________

83

Fig. 23: Confocal microscope images of the fluorescence intensity (I) patterns in the ZP from

IVM oocytes 0, and 3 h after onset of IVF. (a), (b), and (c), the intensity patterns of IVM

oocytes at metaphase II 0 h after IVF were medium (I = 2) or strong (I = 3); (d), (e), and (f), the fluorescence intensity pattern of in vitro matured (IVM) oocytes 3 h after onset of IVF was

greater (strong, I = 3). Images were evaluated by differential phase contrast (DPC).

Figure 24 shows the intensity (I) fluorescence pattern of the distribution of free thiol

groups from IVM oocytes 18 h after onset of IVF.

RESULTS ___________________________________________________________________

84

Fig. 24: Confocal microscope images of the fluorescence intensity (I) pattern in the ZP from

IVM oocytes 18 h after onset of IVF. (A), (B), (C), (D), (E), and (F): There was no

fluorescence labelling (I = 0) in IVM oocytes incubated for 18 h after IVF. Images were

evaluated by differential phase contrast (DPC).

4.2.4.2 Fluorescence Pattern of the Zona Pellucida (ZP) from In Vivo Matured Oocytes after Onset of In Vitro Fertilization (IVF) In vivo matured oocytes (MII) collected from superovulated gilts were fertilized in vitro

(IVF) and classified according to the overall intensity (I) pattern 0 and 3 h after onset

of in vitro fertilization (IVF) (Exp. VI). Three h after onset of IVF, the ZP of oocytes

matured in vivo was compared with that of those matured in vitro (IVM) (46-48 h of

IVM, MII) (Exp. V vs. Exp. VI).

RESULTS ___________________________________________________________________

85

(i) Intensity Pattern (I) Table 8.1 compares the intensity (I) patterns classified as (0), (1), (2), and (3)

between ZP from in vitro matured (IVM) oocytes (Exp. V) and ZP from in vivo

matured (MII) oocytes 3 h after onset of IVF system (Exp. V vs. Exp. VI).

Tab. 8.1: Free thiol group distribution according to the intensity (I) of the fluorescence

pattern (0-3) from oocytes matured in vivo and in vitro (IVM) 3 h after onset of IVF.

Developmental

status of the

oocyte /Intensity

pattern

Intensity (0)

% (n)*

Intensity (1)

% (n)*

Intensity (2)

% (n)*

Intensity (3)

% (n)*

In vivo matured

oocytes

67.6 (71) 26.7 (28) 5.7 (6) 0.0 (0)

In vitro matured

(IVM) oocytes

0.0 (0) 2.3 (4) 27.1 (46)

70.6 (120)

*The number (n) of ZP examined is shown in parentheses

The dominant population of in vivo matured oocytes 3 h after onset of IVF showed

absent (no) fluorescence labelling (I = 0, 67.6%), while at the same time after in vitro

maturation (IVM) (3 h after IVF) 70.6% of the oocytes demonstrated strong intensity

pattern (I = 3).

Table 8.2 shows probabilities values (P) of intensity labelling pattern (0-3) of in vivo

and IVM oocytes 3 h after IVF.

RESULTS ___________________________________________________________________

86

Tab. 8.2: Probability values (P) for the comparison of the intensity (I) pattern

distribution of oocytes matured in vivo and in vitro (IVM) 3 h after onset of IVF (Chi-

SquareTest).

Developmental

status of the

oocyte

/Intensity

pattern

Intensity

(0)

Intensity

(1)

Intensity

(2)

Intensity

(3)

3 h of IVF after

in vivo vs. IVM

0.0001 0.0001 0.0001 0.0001

The intensity (I) pattern of fertilized ZP (3 h after onset of IVF) derived from in vivo

and in vitro matured (IVM) oocytes demonstrated high significantly different in all

categories (0-3) at P < 0.0001.

Figure 25 shows the intensity patterns for in vivo and IVM oocytes 3 h after onset of

IVF.

RESULTS ___________________________________________________________________

87


oocytes matured in vivo and in vitro (IVM) 3 h after onset of IVF. (A), (B), and (C), oocytes

matured in vitro (IVM) 3 h after IVF exhibited a high fluorescence pattern (I = 3), whereas

oocytes matured in vivo (D), (E), and (F) demonstrated no (zero) intensity fluorescence

labelling (I = 0) 3 h after IVF. The images (D1), (E1), and (F1) were evaluated by differential

interference contrast (DIC) as one of our controls for the zero-intensity fluorescence images

(I = 0) from oocytes matured in vivo (3 h after IVF). Compare with images (D), (E), and (F)

which were analyzed by differential phase contrast (DPC).

RESULTS ___________________________________________________________________

88

4.2.5 Incubation of Oocytes Matured In Vivo and In Vitro (IVM) but without Exposure to Spermatozoa In order to determine the influence of the medium on the consumption of free thiol

groups, oocytes matured in vivo and in vitro (46-48 h of IVM) were incubated with

FertTalp medium but without exposure to spermatozoa for 18 hours (Exp. VII; Fig.

26).

Fig. 26: Confocal microscope images of the fluorescence intensity (I) pattern from oocytes

matured in vivo and in vitro (IVM) (46-48 h of IVM) and incubated in FertTalp medium for 18

h without exposure to spermatozoa. Oocytes matured in vivo (A) and in vitro (B) (IVM)

exhibited high fluorescence labelling at the ZP. Images were recorded by differential phase

contrast (DPC).

These results demonstrated that there was no change in intensity (I) of the

fluorescence labelling in the ZP from both sources, indicating that both in vivo and in

vitro matured (IVM) oocytes develop the ability to form disulfide bonds only after

penetration of spermatozoa, confirming the results of experiment V (4.2.4.1

Fluorescence Pattern of the Zona Pellucida [ZP] from In Vitro Matured [IVM] Oocytes

after Onset of In Vitro Fertilization [IVF]) and experiment VI (4.2.4.2 Fluorescence

RESULTS ___________________________________________________________________

89

Pattern of the Zona Pellucida [ZP] from In Vivo Matured Oocytes after Onset of In

Vitro Fertilization [IVF]).

4.2.6 Evaluation of the Fertilization System (IVF) In vitro fertilization (IVF) was performed for 18 h and the presumptive zygotes were

cultured in vitro (IVC) for 7 days for monitoring of cleavage of the blastocyst as one of

our controls for the IVF system. The relative spatial distribution of DNA was

evaluated by laser scanning confocal microscopy (LSCM) and analyzed for the

determination of the cleavage and development of the morula/blastocyst stage.

Figure 27 shows the relative spatial distribution of nuclei (DNA) in an early blastocyst

(day 7) labelled with propidium iodide (PI) evaluated by laser scanning confocal

microscopy (LSCM).

Fig. 27: Laser scanning confocal microscope image (LSCM) from 7- day-old blastocyst with

relative spatial distributions of DNA. Images were recorded by differential phase contrast

(DPC).

RESULTS ___________________________________________________________________

90

These data demonstrated that oocytes matured in vivo and in vitro (IVM) progressed

to the morula/–blastocyst stage when cultured in our routine IVF/IVC system (84%

vs. 60%).

4.3 Trial III: Scanning Electron Microscopy (SEM) Detailed information was obtained on free thiol group distribution of the ZP surface

under different conditions, by analyzing oocytes at different developmental stages

(GV I, MII after 46-48 h of IVM, oocytes derived from IVM 3 and 18 h after onset of

IVF) according to the BIAM method outlined in Pre-trial I experiments. The reaction

was visualized with streptavidin gold and silver enhancement and documented by

scanning electron microscopy (SEM). The gold particles appeared as spherical

vesicles surrounded by a well-defined form, which was subjectively classified in four

features categories: no gold particles (0), few gold particles (1), medium (i.e.

moderate) number of gold particles (2) and complete coverage of the outer surface of

the ZP with gold particles (3) as demonstrated in Table 9.1.

Tab. 9.1: Density (F) distribution of gold particles of the ZP from immature (GV), M II

stages (46-48 h of IVM) oocytes, and IVM oocytes 3 and 18 h after onset of IVF.

Developmental status of the oocyte /Features

Features (0) % (n)*

Features (1) % (n)*

Features (2) % (n)*

Features (3) % (n)*

Immature oocytes (GV)

20.7 (6) 6.9 (2) 41.4 (12) 31.0 (9)

Metaphase II (46-48 h of IVM)

0.0 (0) 0.0 (0) 13.6 (3) 86.4 (19)

3 h after IVF 0.0 (0) 13.8 (4) 10.3 (3) 75.9 (22)

18 h after IVF 70.7 (29) 26.8 (11) 2.5 (1) 0.0 (0)

*The number (n) of oocytes examined is shown in parentheses. There was a significant increase in the distribution of free thiol groups in the outer

surface of the ZP derived from immature (GV) and in vitro matured (IVM) oocytes

during maturation phase, resulting in almost complete accumulation of gold particles

RESULTS ___________________________________________________________________

91

(F = 3, 86.4%). These patterns did not change substantially until 3 h after IVF, but no

oocytes with complete features (F = 3, 0.00%) were present 18 h after onset of IVF.

While none of these oocytes were without gold particles on the surface of the ZP 3 h

after IVF (I = 0, 0.00%), this was the case in the dominant population 18 h after onset

of IVF, when 70.7% had no features (I = 0). These results are similar to our findings

from the experiments using LSCM (Exp. IV, 3 and 18 h after IVF), in which IVM

oocytes showed the absence (no) of intensity fluorescence on the ZP surface 18 h

after IVF. (See also Tables 7.1, 7.2 and Fig. 24).

Table 9.2 contains the probability values (P) for the density distribution of gold

particles on the ZP from oocytes at different developmental stages (immature, in vitro

matured [IVM], and IVM oocytes 3 and 18 h after IVF).

Tab.9.2: Probability values (P) for the comparison of the density (F) distribution of

gold particles on the ZP from immature (GV) oocytes, in vitro matured (IVM) oocytes,

and oocytes derived from IVM 3 and 18 h after onset of IVF (Chi-SquareTest).

Developmental status of the oocyte /Features

Features (0)

Features (1)

Features (2)

Features (3)

GV vs. MII (46-48 h of IVM)

0.02 0.20 0.03 0.0001

MII vs. 3 h IVF (46-48 h of IVM)

-- 0.07 0.71 0.34

MII vs. 18 h IVF (46-48 h of IVM)

0.0001 0.0075 0.082 0.0001

After IVM (46-48 h, MII), there was a statistically significant shift to complete features

(F = 3) at P < 0.0001. There is a highly significant difference between no features (F

= 0) and complete features (F = 3) in MII and 18 h after onset of IVF (P < 0.0001).

Figures 28 and 29 show medium (moderate) numbers clusters of gold particles (F =

2) from immature oocytes (GV) and complete clusters of gold particles (F = 3) from in

vitro matured (IVM) oocytes (46-48 h of IVM). Figure 30 shows oocytes matured in

RESULTS ___________________________________________________________________

92

vitro (IVM) 3 h after onset of IVF (complete cluster, F = 3), while Figure 31 shows

oocytes matured in vitro (IVM) 18 h after onset of IVF (no features, F = 0).

Fig. 28: Scanning electron microscope (SEM) image from an immature (GV) oocyte. The

figure shows the distribution of free thiol groups within the ZP oocytes from immature porcine

oocytes. In general, immature oocytes had free thiol groups distributed in medium-sized

clusters of gold particles (medium, F = 2) over the outer ZP surface. Oocytes were

photographed on the SEM at 2000x magnification.

RESULTS ___________________________________________________________________

93

Fig. 29: Scanning electron microscope (SEM) image from an oocyte matured in vitro (IVM)

(MII, 46-48 h of IVM) with complete clusters of gold particles (F = 3) at the periphery of the

outer surface of the ZP, which forms a monolayer beneath the plasma membrane. Oocytes

were photographed on the SEM at 3000x magnification.

RESULTS ___________________________________________________________________

94

Fig. 30: Scanning electron microscope (SEM) image of an IVM oocyte 3 h after onset of IVF

showing the presence of complete clusters of gold particles (F = 3) at the outer surface of the

ZP. Oocytes were photographed on the SEM at 1000x.

RESULTS ___________________________________________________________________

95

Fig. 31: Scanning electron microscopy (SEM) image of an IVM oocyte 18 h after onset of IVF

showing the absence of gold particles (no features, F = 0) on the outer surface of the intact

oocyte. Oocytes were photographed on the SEM at 1000x.

RESULTS ___________________________________________________________________

96

4.4 Sperm Binding to the Zona Pellucida (ZP) under In Vitro Fertilization (IVF) Conditions The major objective of the current study was to determine whether any of the factors

in the oviduct specifically influenced the number of sperm binding to the ZP under our

in vitro fertilization conditions. Table 10 shows the minimum and maximum numbers

of spermatozoa bound to the zona pellucida (ZP) from ovulated and in vitro matured

(IVM) oocytes 12 h after onset of IVF.

Tab.10: Sperm binding to the ZP from oocytes matured in vivo and in vitro matured

(IVM) 12 h after onset of IVF.

Developmental

status of the

oocyte

Time after

onset of

sperm

exposure to

oocyte (h)

Number of

Oocytes

Mean

(%)

Std.Dev.

(%)

Minimum

Maximum

MII (In vivo) 12 36 4.19 (a) 2.15 1 10

MII (In vitro) 12 45 21.04 (b) 9.94 6 53 Different letters (a, b) indicates values significantly different (P<0.0001).

Twelve h after IVF, there was a significant reduction in the number of sperm binding

to the ZP (4.19%) in oocytes matured in vivo, while a high number of spermatozoa

were bound to the ZP (21.04%) 12 h after IVF in oocytes matured in vitro (IVM). The

difference in the number of sperm binding to in vivo and in vitro matured (IVM) ZP 12

h after onset of IVF is statistically significant at P < 0.0001.

Figure 32 compares the number of spermatozoa bound to the zona pellucida (ZP)

from oocytes matured in vivo and in vitro (IVM) 12 h after onset of IVF.

RESULTS ___________________________________________________________________

97

Fig. 32: Fluorescence images from oocytes matured in vivo (A) and in vitro (B) (46-48 h of

IVM) 12 h after onset of IVF. There were significantly fewer spermatozoa bound to the ZP

during the fertilization process in oocytes matured in vivo (A) than in those matured 46-48 h

in vitro (IVM) (B).

DISCUSSION ___________________________________________________________________________

98

5 DISCUSSION

Oocytes are surrounded by a glycoprotein envelope known as the zona pellucida

(ZP) glycoproteins (LINDSAY and HEDRICK 2004). This envelope serves as the site

of specific interactions with sperm and also induces the sperm acrosome reaction

(AR). The zona envelopes of porcine oocytes contain at least three major

glycoproteins, which are designated ZPA, ZPB, and ZPC and belong to a family of

structurally and functionally conserved molecules which upon fertilization are

modified by the release of oocyte cortical granule (CG) factors to prevent further

sperm interactions (i.e penetration). Studies by IWAMOTO et al. (1999) showed in

bovines that the oxidation of free cysteine (Cys) thiol residues between ZP

glycoproteins following fertilization is involved in the prevention of further sperm

penetration (i.e. the polyspermy block).

The first goal of this study was to analyze the distribution of the oxidant-sensitive

cysteine (Cys) residues in the ZP glycoproteins ZPA, ZPB, and ZPC by

electrophoretic methods. For this purpose we used different pH levels (pH 6.5 and

pH 8.5) in order to obtain the most specific labelling pattern for the further

morphologic evaluation by laser scanning confocal microscopy (LSCM) (Trial II) and

scanning electron microscopy (SEM) (Trial III) of the ZP from different sources

(immatured vs. in vitro matured [IVM] oocytes, in vivo vs. in vitro matured [IVM]

oocytes, and in vivo and IVM oocytes sampled at different times after the onset of

IVF). Our protocol for biochemical analysis was designed from tests with immature

oocytes derived from a population of growing follicles which appeared to have

achieved maturation competence.

Thiol groups have long been recognized as the site of formation of disulfide bonds

(i.e. S-S bonds) between two oxidized Cys residues (L-cystine) within a polypeptide

chain. There is a strong suggestion that disulfide bond formation is responsible for

the increase in the stability of proteins through an alteration of the global

conformation (SHAPIRO 1991, BARFORD 2004). The acid constant (pKa) of the thiol

DISCUSSION ___________________________________________________________________________

99

side chain of the cysteine (Cys) residues has been determined to be ≈ pKa 8.3;

however, free thiol groups are highly sensitive to the microenvironment within a

protein, and thus, to the function of proteins and glycoproteins. Oxidant-sensitive

cysteine (Cys) residues with low pKa can be selectively labelled at pH 6.5 with

iodoacetamide. In this study, the reaction of a thiol-reactive stain with zona pellucida

(ZP) glycoproteins was evaluated by electrophoretic methods (one–dimensional gel

electrophoresis, 1–D SDS–PAGE, and two–dimensional gel electrophoresis, 2–D

SDS-–PAGE) under different pH conditions (pH 6.5 and pH 8.5). After protein

blotting, the formerly free thiol groups, now bound to biotin–conjugated

iodoacetamide (BIAM), were visualized by chemiluminescence using streptavidin-

peroxidase (according to KIM et al. 2000). Although, all electrophoretic methods (1–D

SDS–PAGE and 2–D SDS–PAGE) responded at pH 6.5 with a selective labelling of

the glycoprotein ZPA, there was no labelling of the glycoproteins ZPB/ZPC. These

results are also in contrast to those obtained with BIAM stained at pH 8.5 (1–D SDS–

PAGE and 2–D SDS–PAGE). In that experiment, virtually all free cysteine (Cys)

residues at the glycoproteins ZPA, ZPB, and ZPC responded to the labelling

procedure. In 1–D SDS–PAGE, ZPA appears as a strong band between 80 and 120

kDa, and the glycoproteins ZPB/ZPC appear as a band between 55 and 60 kDa

which could be not separated. In 2–D SDS–PAGE after biotinylation of the free thiol

groups at pH 8.5, the ZPA glycoprotein showed a dominant labelling between pH 4

and 6, whereas ZPB/ZPC glycoproteins demonstrated strong labelling in a wide pH

range between 3 and 6.5. All these electrophoretic results indicated that the ZPA

glycoprotein contains oxidant-sensitive Cys residues with low pKa which may be

predominantly involved in the formation of the three–dimensional architecture and

structural changes in the ZP after fertilization (IVF). Two important factors which

could potentially account for these observations are the sensitivity of Cys residues

and the effect of pH during the fertilization process (IVF). First, most protein Cys

residues have a pKa of 8.5, and only a few proteins seem to possess oxidant-

sensitive Cys residues (low pKa) (KIM et al. 2000). Second, there is a dramatic

difference in specificity of the oxidation of Cys residues on ZP glycoproteins (ZPA,

ZPB, and ZPC) between pH 6.5 and 8.5. Studies in our laboratory demonstrated that

DISCUSSION ___________________________________________________________________________

100

only the glycoprotein ZPA contains oxidant-sensitive cysteine (Cys) residues. The pH

of the IVF media (e.g. FertTalp) used in our study (pH 7.0 ≈ 7.2) ensures that

oxidant-sensitive cysteine residues (pKa 6.5) of the ZP glycoproteins can identified as

markers for the oxidative process following fertilization. This suggests that the novel

intra- intermolecular disulfide bond formation affects the global ZP conformation, thus

altering the structural stability of the protein (BALASUBRAMANIAN and KANWAR

2002) and improving most of their biological activities, ultimately resulting in the

hardening of the zona pellucida. It is postulated that zona hardening may have three

post-fertilization zona functions, namely, the zona block to polyspermic fertilization

(SCHMELL et al. 1983, cited in DROBNIS et al. 1988, SCHROEDER et al. 1990);

protection of the developing embryo (GWATKIN 1977, cited in DROBINS 1988); and

transport of the early embryo to the uterus (ORTIZ et al. 1986).

The number and position of the Cys residues within each ZP glycoprotein are well

conserved, suggesting that disulfide bonds (S-S) are important for correct folding of

the ZP (YONEZAWA and NAKANO 2003). In the pig, the mature polypeptide ZPA

contains 20 cysteine residues. Comparison with ZP glycoproteins of other species

revealed that at least 10 cysteine residues may be present in the reduced state

(YUREWICZ et al. 1993, TAYA et al. 1995) at positions Cys 85, Cys 103, Cys 371,

Cys 463, Cys 402, Cys 423, Cys 613, Cys 618, Cys 628, and Cys 632 residues. It

has been reported that the mature polypeptide ZPB contains 16 Cys residues

(HARRIS et al. 1994) and no free thiol groups (NAKANO and YONEZAWA;

unpublished results, cited in NAKANO and YONEZAWA 2000). The N-terminal six

Cys residues (Cys 144, Cys 155, Cys 165, Cys 170, Cys 171, and Cys 180) possibly

form disulfide bonds in the same combinations as the trefoil factor (HARRIS et al.

1994, YONEZAWA and NAKANO 2003). The following four Cys residues at positions

Cys 190, Cys 224, Cys 243, and Cys 286 are part of the first disulfide bond fragment

of the ZP domain, while the C-terminal six residues (Cys 368, Cys 389, Cys 442, Cys

447, Cys 455, and Cys 459) form disulfide bonds separately (HARRIS et al. 1994). It

has been reported that mature polypeptide ZPC has no free cysteine thiol residues

(YONEZAWA and NAKANO 2003). This indicates that all cysteine (Cys) residues are

DISCUSSION ___________________________________________________________________________

101

involved in disulfide bond formation among the four Cys residues at positions Cys 45,

Cys 77, Cys 98, and Cys 139 (YONEZAWA and NAKANO 2003) and among the

eight Cys residues at positions Cys 216, Cys 238, Cys 281, Cys 299, Cys 318, Cys

320, Cys 321, and Cys 326 (YONEZAWA and NAKANO 2003).

Since all cysteine residues in porcine ZPB and ZPC are involved in disulfide bond

formation, this indicates that only a small proportion of Cys residues of the

glycoprotein ZPA are susceptible to the oxidation process following fertilization.

Those oxidant-sensitive Cys residues are most probably found among the eight Cys

residues at positions Cys 52, Cys 135, Cys 152, Cys 260, Cys 267, Cys 328, Cys

357, Cys 359, and among the two Cys residues in domains proteins at positions Cys

543 and Cys 564. The positions of Cys residues in the ZP glycoproteins are highly

conserved (HARRIS et al. 1994), and most of these residues form disulfide linkages

(BOJA et al. 2003, YONEZAWA and NAKANO 2003). Our data clearly indicate that

the glycoprotein ZPA contains low pKa Cys residues and that a small proportion of

these residues appear to be susceptible to oxidation at pH 6.5. The oxidation of

these residues in the ZPA glycoprotein, which can be detected by labelling with BIAM

at pH 6.5, could be important for the construction of the rigid zona pellucida (ZP)

structure (e.g. zona pellucida hardening) responsible for the block to polyspermy.

Although there are various reports on the role of Cys residues on the formation of

disulfide bonds (ZHANG et al. 1991, HARRIS et al. 1994, IWAMOTO et al. 1999),

this study is the first to evaluate selective oxidant-sensitive Cys residues in the ZP

glycoproteins under different pH conditions (pH 6.5 and pH 8.5). IWAMOTO et al.

(1999) demonstrated in bovine zona pellucida glycoproteins that the oxidation of

cysteine (Cys) residues to cystine (L-cystine) during fertilization (IVF) and their

formation of novel intra- and intermolecular disulfide linkages may be one reason for

the construction of a rigid zona pellucida structure (i.e. zona pellucida hardening),

which is thought to contribute to the polyspermy block. Similar results have been

reported by ZHANG et al. (1991) for rat ZP glycoproteins.

DISCUSSION ___________________________________________________________________________

102

Maturation of the Zona Pellucida (ZP) In Vitro Maturation (IVM) Previous studies have demonstrated that the biological properties of the ZP are

modified during folliculogenesis (DUNBAR and RAYNOR 1980, DUNBAR et al. 1980,

DUNBAR 1983, DIETL 1989, DUNBAR et al. 1994, EPIFANO et al. 1995,

WASSARMAN 1998, PRASAD et al. 2000, DUNBAR et al. 2001, SINOWATZ et al.

2001, and FLÉCHON et al. 2003). Those biological changes occurring during oocyte

maturation have been collectively referred to as zona maturation (AVILÉS et al.

2000). It has been demonstrated in several species that the carbohydrate residues

are heterogeneously redistributed in the ZP during maturation. The zona maturation

process includes an increase in resistance of the ZP glycoproteins to proteases; in

the penetrability of sperm through the ZP; and in the sperm binding ability of the ZP

(CROZET and DUMONT 1984, SHALGI et al. 1989, RUFAS and SHALGI 1990, and

OEHNINGGER et al. 1991, cited in AVILÉS et al. 2000), however, the biochemical

basis of these changes remains unknown. The present study examined the binding

sites of free cysteine (Cys) thiols distributed throughout the ZP of immature oocytes

(germinal vesicle stages) and those matured in vitro (IVM). The analysis of the

fluorescence pattern of the ZP structure under both oocytes conditions (immature

and matured in vitro) revealed significant differences in the location, density, and

distribution of the free cysteine thiols sites (P < 0.0001). In the immature ZP, there

was relatively heavy (i.e. strong) labelling of free cysteine thiols sites, which are

dispersed heterogeneously throughout its entire (inner and outer) body, but slightly

more concentrated in the interior (inner) than at the exterior (outer) surface of the ZP.

Once oocyte growth has begun (MI vs. MII), free cysteine thiol sites are then

homogeneously rearranged between the inner and the outer surface of the zona

network, and remain in that form when oocytes undergo full meiotic development.

Most binding sites of free cysteine thiols observed in this study were detected within

the inner zona, rather than the outer zona (P < 0.0001). The functional significance of

this regional difference is unknown, but the concentration of the free thiol groups at

DISCUSSION ___________________________________________________________________________

103

the inner surface could be essential to the reaction with the CG exudates, which are

released by the cortical reaction into the perivitelline space (PVS), inducing the zona

reaction. Further studies are necessary for the analysis of the impact of the variability

of this difference in concentration on the fertilization process and on the individual

incidence of oocytes to block polyspermy.

AVILÉS et al. (2000) demonstrated in the mouse that the secretion of the ZP

glycoproteins was always restricted to the inner region of the zona matrix. It is also

known that the only source of all three glycoproteins in this species is the growing

oocyte in the ovary (WASSARMAN 1988), where these proteins are synthesized and

secreted (WASSARMAN 1988, cited in PRASAD et al. 2000). In other domestic

animals, including pigs, these proteins are expressed during follicular development in

both, the oocyte and the granulosa cells (PRASAD et. al. 2000, SINOWATZ et al.

2001). The ZP increases in thickness as the oocyte increases in diameter.

At this stage, the primordial follicle cell contains no ZP, and the oocyte is surrounded

only by a thin layer of granulosa cells. As the follicle develops, the surrounding

granulosa cells multiply and establish extensive processes towards the oocyte. The

ZP is then built in the cleft between cumulus cells and corona radiata. Initially the ZP

is assembled as isolated islands of material which presumably coalesce as they

enlarge (DIETL 1989, cited in GREEN 1997). The oocyte expands during the

synthesis of ZP and continues to expand after ZP protein expression has stopped. It

appears that the oocyte stretches the emerging ZP after the individual islands of ZP

material have coalesced, and continues to deposit more material underneath the by

then continuous layer of glycoproteins (GREEN 1997). There is evidence that the

mature ZP is more closely packed on its inner surface (DIETL 1989, cited in: GREEN

1997), and this may reflect the secretion of ZP proteins into a mechanically

compressed space under a stretched ZP (GREEN 1997). The oocyte is presumably

the spherical mould upon which the ZP is built, which is the source of the remarkably

uniform spherical shape of the ZP.

DISCUSSION ___________________________________________________________________________

104

The individual native ZP proteins exhibit considerable variations in post-translation

modification (e.g. processing, glycosylation, and disulfide bond formation). Our

results suggest that there are clear differences in the number and position of free

cysteine thiols distributed in the zona from immature oocytes (GV) and metaphase II

(MII) oocytes. One possible reason for these observations could be that additional

layers of ZP glycoproteins are deposited underneath the ZP during maturation.

Despite considerable speculation about the origins of the ZP, at least in the mouse, it

has now been established unequivocally that all three zona pellucida proteins are

synthesized by the oocyte in a co-ordinate manner (EPIFANO et al. 1995, cited in

GREEN 1997), while in other domestic species, including the pig, the ZP

glycoproteins are expressed in both the oocyte and the granulosa cells (PRASAD et

al. 2000). Antibodies to porcine ZP glycoproteins have been used to detect a similar

localization pattern of ZP glycoproteins in the oocyte and granulosa cells of growing

and matured porcine follicles (TOEPFER-PETERSEN, umpublished observations).

KAUFMANN et al. (1989) observed that murine ZP glycoproteins are constructed of

different layers reflecting real differences in the location, density, and distribution of

glycoproteins formed during the final stages of oocyte maturation prior to and

immediately following GVBD. It is possible that the addition of more glycoproteins

(ZPA, ZPB, and ZPC) underneath the zona matrix leads to a great increase in the

amount of free cysteine residues, which would account for our results.

Finally, we found that free cysteine thiol binding sites were homogeneously

distributed within the ZP during the in vitro maturation (IVM) process, but, most of

these binding sites were concentrated at the inner area of the ZP, suggesting that

newly synthesized ZP glycoproteins derived from the oocyte are associated with

continuous layers up to the final stage of folliculogenesis. The bulk of free cysteine

thiols sites would constitute the inner region of the ZP and could therefore not be

involved in the sperm-egg recognition phenomena, but may play a role in the

initiation of the ZP hardening as one result of the fertilization process (e.g. cortical

DISCUSSION ___________________________________________________________________________

105

reaction). A layer of modified ZP adjacent to its inner surface may be then required to

block polyspermy.

In Vivo Maturation

The goal of these experiments was to determine whether there are differences in the

distribution of the free thiol groups (Cys-SH) between oocytes matured in vivo and in

vitro (IVM) and to examine and compare those differences as indicators of the

physiological ability of the oocyte to induce biochemical and structural alteration of

the ZP following fertilization. Our results demonstrated that the distribution of

fluorescence of oocytes matured in vivo was uniform (i.e. homogeneous) across the

whole structure of the ZP without regional differences, while there was a

concentration of fluorescence intensity in in vitro matured (IVM) ZP at the outer and

the inner (i.e. ad-oolemma) layers of the ZP.

As in vivo matured ZP was shown to be significantly thinner than that of in vitro

matured oocytes (46-48 h after onset of IVM), it was therefore not possible to a

differentiate between outer (A) and inner (I) layers, despite our confocal microscopy

analysis. Perhaps the most surprising finding is that the oocytes matured in vivo had

significantly thinner zonae pellucidae (ZP) than those matured in vitro (IVM). These

results are in contrast to those of other studies, which report significantly thicker zona

of oocytes matured in vivo than in those matured in vitro (IVM) (WANG et al. 1998).

Our findings also demonstrate that there was less rearrangement of the distribution

of the free thiol groups in oocytes matured in vivo than in those matured in vitro (46-

48 h of IVM). In the former, the homogeneity pattern was low (H = 1, 28.6%) to

medium (H = 2, 53.6%), while it was medium (H = 2, 36.8%) to high (H = 3, 57.9%) in

the latter (IVM). Despite these differences, there was at least some rearrangement of

the free thiol groups across the ZP in all mature oocytes, both in vivo and in vitro, in

comparison with immature oocytes (germinal vesicle stage).

DISCUSSION ___________________________________________________________________________

106

Our observations also clearly indicate that there are differences in the morphology of

the ZP under both in vivo and in vitro conditions. It is probable that the maturation of

the oocytes is not identical under physiologic conditions to that produced in the in

vitro maturation (IVM) system, in which differences in many dynamic events alter the

ultrastructure of the ZP.

The maturation associated with zona pellucida morphology may not be fully achieved

in the oocytes matured in vitro (IVM). Our results suggest that there are differences in

the distribution of free cysteine thiol residues in the ZP glycoproteins between

oocytes matured in vivo and in vitro (IVM).

Morphological studies have reported that many of these changes in the ZP were

initiated within the ovary during the final stages of oocyte maturation (FOWLER and

GRAINGE 1985, FOWLER et al. 1986, cited in KAUFMAN et al. 1989). It has been

known for more than fifty years that mammalian oocytes removed from antral follicles

undergo spontaneous nuclear maturation in vitro when cultured in a simple medium.

Under in vitro experimental conditions, meiotic resumption of mammalian oocytes is

induced in two different phases: first, the phase of hormone-induced maturation by

gonadotropic hormones (e.g. PMSG and hCG), in which PMSG/hCG stimulation

produces changes in the structure and composition (e.g. ultrastructural and

histochemical) of the zona pellucida (KAUFMAN et al. 1989); and second, the final

phase without hormonal induction and without cumulus cell influence (i.e. the final

stages of oocyte maturation), in which detachment of the cumulus cells from the

oocyte surface produces deep defects in the external layer of the ZP (KAUFMAN et

al. 1989). At this point, the concentric layers of the zona pellucida are completed.

It is of considerable significance that the female reproductive tract (e.g. ovary,

oviduct) plays an important role in the fine structure of the final zona modifications

during oocyte maturation by transportating materials via intercellular coupling

between the oocyte and corona cells (i.e. gap junctional communication, GJC), which

DISCUSSION ___________________________________________________________________________

107

is more active under in vivo conditions (MOOR et al. 1990, cited in FUNAHASHI et al.

2003). KAUFMAN et al. (1989) observed that the ultrastructural appearance of the

ZP is correlated with stages of the follicle maturation during physiologic oocyte

development. In those same morphological studies, the zona pellucida appeared to

be composed of five concentric layers in some oocytes in which the germinal vesicle

breakdown (GVBD) had not yet occurred. In the majority of oocytes of preovulatory

follicles, in which GVBD has occurred, the concentric layers of the ZP become less

evident (KAUFMAN et al. 1989).

These layers of the ZP are then penetrated by long cumulus cells processes (i.e.

denticular processes), by which the cells assume a characteristic shape and are

anchored to the ZP surface (KAUFMAN et al. 1989). At this time, the microvilli at the

oocyte surface are very prominent. By the time of the first polar body extrusion (i.e.

metaphase II), the cumulus cells have elongated and become progressively

detached from each other. At this point, the microvilli on the oocyte surface are still

prominent, and the ZP surface is no longer anchored to the cumulus cells

presumably owing to abrogation of typical cumulus cell functions.

There are many reports evaluating the difference between in vivo and in vitro

matured oocytes (WANG et al. 1997b, 1998, MOOR and DAI 2001, VAN BLERKOM

and DAVIS 2001, SUTTON et al. 2003). However, the present study is the first to

evaluate and compare the distribution of the free cysteine thiol residues between in

vivo and in vitro matured (IVM) ZP glycoproteins. It is likely that the changes we have

observed in the intensity and distribution of free cysteine thiol residues within ZP

glycoproteins (e.g. in vivo and in vitro matured oocytes) have a significant correlation

with the ability of oocytes to perform the block to polyspermy following fertilization

through induction of global conformation changes in the zona pellucida glycoproteins.

However, further studies are clearly needed before the biological significance of

these changes can be fully assessed.

DISCUSSION ___________________________________________________________________________

108

Disulfide Bond Formation between Zona Pellucida Glycoproteins and Their

Relevance to the Polyspermy Block

Another aim of the current study was to examine and compare the distribution of the

free cysteine thiol residues (Cys-SH) available on the zona pellucida (ZP) surface

under in vivo and in vitro conditions throughout the biochemical and structural

alterations of the ZP following fertilization (IVF). The results obtained in the present

experiments show that there are differences in the times when the alterations of the

ZP occur following fertilization. No fluorescence intensity was detected 18 h after

fertilization (IVF) in ZP matured in vitro (IVM). In contrast, fluorescence disappeared

already within 3 h after IVF in ZP matured in vivo, suggesting that there is a delayed

or incomplete reaction of cortical granule (CG) exudates (e.g. cortical reaction) in IVM

oocytes, which may be one of the major reasons for this delay (i.e. 18 h after IVF). In

our IVF system, both in vivo and in vitro matured (IVM) oocytes were incubated with

the same number of spermatozoa. It has been reported that exposing porcine

oocytes to excess numbers of spermatozoa is one of several major causes of the

high incidence of polyspermic penetration in porcine IVF system (FUNAHASHI 2003).

Regulation of the number of spermatozoa binding to the zona pellucida (ZP) just after

insemination, i.e. reduction of free sperm receptors on the porcine ZP, should reduce

the chances of simultaneous sperm penetration in porcine IVF systems (FUNAHASHI

2003). In our IVF system, both in vivo and in vitro matured (IVM) oocytes were

exposed to the same number of spermatozoa (3 x 105 spermatozoa/ml), and the

oocytes appeared not to be affected by the difference in the changes in the ZP over

time observed between the two sources of oocytes (in vivo vs. in vitro maturation

[IVM]). In addition, a recent study demonstrated that ZP in the pig occurs over a

gradient of sperm-binding sites which decreases from the outside to the inside zona

layer (FAZELI et al. 1997, cited in FUNAHASHI 2003), whereas only capacitated

acrosome-intact and/or partially acrosome-reacted spermatozoa contributed to the

successful zona pellucida reaction by CG components. Consequently, the incidence

of monospermic penetration may increase (FUNAHASHI 2003).

DISCUSSION ___________________________________________________________________________

109

It has been reported that the cortical granules (CGs) fuse with the overlying oolemma

just after sperm penetration, when they release their material into the perivitelline

space (PVS), thereby altering the properties of the ZP (BLEIL et al. 1981, MOLLER

and WASSARMAN 1989). This event is crucial to the functional block to polyspermy

(IWAMOTO et al. 1999), which is achieved by the production of a rigid zona pellucida

(ZP) refractory to sperm penetration, and comprises the process known as zona

pellucida hardening (MOLLER and WASSARMAN 1989, DANDEKAR and TALBOT

1992, HATANAKA et al. 1992, IWAMOTO et al. 1999).

Analysis of oocytes before and after fertilization in mouse zona pellucida (ZP)

revealed that proteolysis of glycoprotein ZP2 following oocyte activation triggers the

events responsible for the zona reaction (MOLLER and WASSARMAN 1989) and

which are required for the formation of the zona block to polyspermic binding

(MILLER et al. 1993) and for hardening of the zona pellucida. The proteolytic

cleavage of the ZP produces two subunits (e.g. glycopeptides), ZP2f forms, which

remain attached by intramolecular disulfide bonds (MOLLER and WASSARMAN

1989).

It is probable that there are one or more different processes that are important for the

modification of the architecture of the ZP after sperm penetration. In boar

spermatozoa, proacrosine has been considered to be a second ligand molecule that

binds acrosome–reacted cells tightly to the matrix of the ZP (FUNAHASHI 2003).

However, other studies revealed that acrosomal supernatants from acrosome–

reacted spermatozoa did not induce limited proteolysis of ZP2 (BAUSKIN et al.

1999). Furthermore, a protease released from the oocyte (CG) in the pig induces

proteolysis of ZPA (pZP1) following the cortical reaction (FUNAHASHI 2003). Porcine

ZPA (pZP1, 90 kDa) glycoprotein is specifically cleaved at the amino acid terminus

between Ala 168 and Asp 169 residues resulting in pZP2 (65 kDa) and pZP4 (20

kDa) subunits which remain attached by disulfide (S-S) bonds (HEIDRICK et al.

1987, HATANAKA et al. 1992, HEDRICK 1993, HASEGAWA et al. 1994, cited in

IWAMOTO et al. 1999, FUNAHASHI 2003). The amount of intact ZPA (pZP1) after

DISCUSSION ___________________________________________________________________________

110

specific cleavage decreases substantially (FUNAHASHI 2003). However, accessory

spermatozoa continue to penetrate into the outer zona layer of the fertilized oocytes

(FUNAHASHI 2003), but are not able to complete the penetration through the inner

zona layer. These data suggest that the cleavage of ZPA (pZP2 to pZP4) is limited to

the inner zona layer. Studies by TATEMOTO and TERADA (1999) corroborate the

suggestion that the zona reaction appears in the pig to be completed only within the

inner zona layer of fertilized oocytes. In Xenopus laevis eggs, another step of the

block to polyspermy involves a lectin-like mechanism (LINDSAY and HEDRICK

2004). In this species, a galactose-specific lectin forms a dense layer to block sperm

contact with the egg envelope (LINDSAY and HEDRICK 2004). This cortical granule

lectin mechanism has also been identified in mammals (PEAVY and HEDRICK 2000,

WESSEL et al. 2001). In addition, an N-acetylglucosaminidase from the cortical

granules (CGs) removes the carbohydrate sperm binding sites from the zona glycans

(WESSEL et al. 2001). In the mouse, three important mechanisms are involved in the

zona block to polyspermy after sperm–egg fusion. One is the inactivation of the

ZP3/ZPC glycoproteins by a CG glycosidase, which removes the terminal

monosaccharide (i.e. O-linked oligosaccharides) responsible for gamete recognition

and adhesion, after which sperm can no longer bind to the ZP3/ZPC. Another is the

limited proteolytic cleavage of the ZP2 (ZPA) glycoproteins as described in another

species. The third is the cross-linkage formation between tyrosine residues of the

zona pellucida glycoproteins. SCHMELL and GULYAS (1980) proposed that

mammalian CGs contain another enzyme (e.g. ovoperoxidase) that has been

implicated in zona hardening by cross-linking tyrosine residues of the zona pellucida

glycoproteins. In invertebrates (e.g. sea urchins eggs) the enzymatic modification

which is possibly also performed by the peroxidase present in the CGs of unfertilized

eggs (KLEBANOFF et al. 1979, SOMERS et al. 1989, cited in WESSEL et al. 2001)

is responsible for the formation of the fertilization envelope (FE) proteins by an

oxidative process following fertilization (MOTOMURA 1954, MOTOMURA 1957,

SHAPIRO 1977, HALL 1978, GULYAS and SCHMELL 1980, SCHMELL and

GULYA, 1980, cited in WESSEL et al. 2001, GREEN 1997). Consequently it appears

that the cortical granule material (i.e. cortical reaction process) in several species is

DISCUSSION ___________________________________________________________________________

111

responsible for the rearrangement of the three–dimensional structure of the ZP,

which mainly promotes filament–filament interactions, thereby making the ZP more

insoluble (i.e. zona hardening), and thus establishing a functional block to

polyspermy.

We have shown that the loss of free cysteine thiols during fertilization could be a

result of the formation of predominantly novel intramolecular disulfide bonds of the

ZPA glycoprotein. It is postulated that the formation of disulfide linkages, together

with alteration of the three–dimensional architecture of the ZP during fertilization,

mainly induces the construction of a more rigid structure (i.e. zona hardening), thus

contributing to the establishment of a functional block to polyspermy. Our results

demonstrate that the oxidation of free thiol groups and the subsequent formation of

disulfide bonds after sperm penetration were more efficient in oocytes matured in

vivo (3 h after onset of IVF) than in those matured in vitro (IVM) (18 h after onset of

IVF). One possible reason for this could be differences in the distribution and

rearrangement of the free cysteine thiol sites associated with the zona derived from

both sources (in vivo and in vitro). It is possible that in vivo conditions (i.e. the ovarian

microenvironment) assist or enhance differences in the density and distribution of

Cys-SH sites on the ZP glycoproteins during the final stages of oocyte maturation,

unlike those of ZP glycoproteins from oocytes matured in vitro (IVM). In all animals

evaluated, oocytes that have reached metaphase II (MII) develop the full ability to

undergo the cortical reaction (DUCIBELLA 1996, WANG et al. 1997c, WANG et al.

1998). BUHI (2002) reported that mammalian oviductal microenvironment also plays

an important role in establishing essential morphological, biochemical, and

physiological changes during the final maturation of the oocytes. During late follicular

growth, the oviduct, under specific hormonal control, produces proteins, e.g. oviduct-

secretory glycoproteins (OGP) associated with the ZP and PVS of the oocytes and

embryos. In most species, including pigs, it has been recognized that among the

influences of the OGP on the oocyte are increased sperm penetration, increased

fertilization rates, and decreased polyspermy.

DISCUSSION ___________________________________________________________________________

112

Under physiological conditions, almost all fertilized oocytes (< 95%) are

monospermic (HUNTER 1972). However, when IVM oocytes are inseminated in vitro,

the polyspermic rate is especially high (NIWA 1993). Mammalian cortical granules

(CG) migrate to the cortex area of the ooplasm during maturation, and, thus

demonstrated that the timing of this migration and the reorganization of

microfilaments to the oocyte cortex area in IVM oocytes (KIM et al. 1996, cited in

FUNAHASHI 2003) are similar to those of oocytes matured in vivo (YOSHIDA et al.

1993a, cited in FUNAHASHI 2003). Although the morphology and migration of CGs

appear to be similar in IVM and in vivo (e.g. ovulated) porcine oocytes (FUNAHASHI

2003), differences in cortical granule (CG) contents may account for increased

incidence of polyspermy. In addition, it is possible that in oocytes matured in vitro the

CG material (i.e. contents) released into the perivitelline space (PVS) results in the

prevention of sperm penetration into the ZP, but not at the plasma membrane (i.e.

oolemma) level. This is an important event necessary for the complete establishment

of the membrane block to polyspermy, and thus, could in part be explained as

evidence for the high incidence of polyspermic penetration in IVF system when the

porcine oocyte undergoes maturation in vitro (IVM).

WANG et al. (1997b) demonstrated that cortical granule (CG) exocytosis is

accompanied by nuclear activation, suggesting that both nuclear and cytoplasmic

factors are responsible for the ability of oocytes to release their contents. Therefore,

differences in the cytoplasmic maturation of in vivo and in vitro matured (IVM)

oocytes could also be correlated with the ability of oocytes to block polyspermy, but

this correlation has not been investigated (WANG et al. 1998).

Elevations in the intracellular calcium (Ca2+) cause exocytosis of CGs (WILLIAMS

2002). It has been demonstrated in oocytes that the endoplasmic reticulum (ER) is

the source of free calcium (Ca2+) necessary for exocytosis. Just prior to fertilization,

the extracellular stimulus (e.g. sperm induction) elevates the cytoplasmic calcium

(Ca2+) levels mediated by signals from a series of protein interaction events

(WESSEL et al. 2001), resulting in the inositol triphosphate (IP3) system, which is

acquired only during the progression of nuclear maturation (FUJIWARA et al. 1993).

DISCUSSION ___________________________________________________________________________

113

This is known to be a prerequisite for mammalian oocyte activation and exocytosis of

CGs (MIYAZAKI et al. 1993, WILLIAMS 2002) requires for modification of the ZP to

prevent additional sperm from binding to and penetrating the ZP (BLEIL and

WASSARMAN 1980). In our findings, the major difference in the course of the

biochemical and structural alterations of the ZP over time between in vivo and IVM

oocytes (3 h vs. 18 h after IVF) could be explained as the result of suboptimal in vitro

maturation system (in vitro vs. in vivo system), in which insufficient and/or delayed

wave of free Ca2+ produced from the cytoplasmic endoplasmic reticulum (ER)

caused a delayed or incomplete mechanism of the cortical granule exocytosis. It may

be possible that the completion of nuclear maturation is not necessary for the

completion of many processes (i.e. aspects) of cytoplasmic maturation.

The crucial ability of the oocyte to influence Ca2+ signalling in order to block

polyspermic penetration at the correct time is acquired through coordination of

various processes involved in the complete maturation phase. In some species (e.g.

sea urchin eggs), only a single wave of free cytoplasmic calcium (Ca2+) is

necessary, and leads to exocytosis. However, in other species (e.g. mice), multiple

and/or transient calcium waves are usually necessary to trigger exocytosis.

Considering that, the single calcium (Ca2+) spikes (i.e. waves) last only a few

minutes (WESSEL et al. 2001) and continue for at least 3 h post-fertilization (SUN

and NAGAI 2003), this may not coincide in time with the alterations of the in vitro

matured (IVM) porcine oocytes (18 h after onset of IVF). It is thus possible that our

IVM system does not support the full developmental potential required for

synchronous nuclear and cytoplasmic maturation. In contrast, studies with in vivo

matured cells showed they possess equal ability to complete disulfide bond formation

within the ZP glycoproteins at an exact time (i.e. 3–5 hours after sperm penetration)

as the physiologic mechanism that prevents or reduces simultaneous sperm

penetration (e.g. establishment of the polyspermy block).

In fact, there are more well-defined physiological factors regulating many other

events which allow the oocytes to resume and complete meiosis (MII) during normal

DISCUSSION ___________________________________________________________________________

114

oocyte maturation (i.e. intracellular glutathione [GSH] content, histone kinase activity

[MAP], and maturation promoting factor [MPF]). However, it would be very difficult to

establish the exact time during oocyte growth and nuclear maturation, when some of

these processes of cytoplasmic maturation occur unless the specific factors

associated with that process are identified (EPPIG et al. 1996). Nevertheless, some

processes of cytoplasmic maturation appear to be coordinated with, and in some

cases might depend upon, the completion of nuclear maturation. However, the exact

definition of cytoplasmic maturation includes processes or events that may not

coincide with nuclear maturation (EPPIG et al. 1996). For example, the conditions

necessary for the changes in the surface structure (i.e. architecture) of the ZP during

the final stages of oocyte maturation (e.g. syntheses of glycoproteins and

carbohydrate modifications) seem to depend in part on post-translational

modifications of proteins present in the cytoplasm of the oocyte. Moreover, in certain

circumstances, the synthesis and secretion of ZP glycoproteins is broadly defined as

the cytoplasmic maturation (EPPIG et al. 1996). For normal fertilization and embryo

development to occur in an in vitro maturation system, it is necessary to eliminate

any sources of failure in the maturation process itself. The completion of nuclear

maturation in vitro does not necessarily ensure completion of normal cytoplasmic

maturation. Some of the reasons why oocytes do not develop to the blastocyst stage

are related to incomplete or abnormal processes of cytoplasmic maturation (EPPIG

et al. 1996). For example, the dramatic difference between in vivo and in vitro

matured (IVM) oocytes described in our results shows that an in vitro maturation

system that seemingly promotes the completion of nuclear maturation is not

necessarily sufficient to cause completion of all the events needed for the completion

of cytoplasmic maturation. In vitro matured oocytes required a period of 18 h after

insemination before they had acquired the ability to form disulfide bonds. In contrast,

the ability of in vivo matured oocytes to form disulfide bonds is acquired 3 h after

sperm penetration. In many laboratories, failure to select suitable oocytes for routine

IVM and suboptimal IVM culture (ABEYDEERA 2002) result in inadequate nuclear

and cytoplasmic maturation, both of which are necessary for developmental

DISCUSSION ___________________________________________________________________________

115

competence. In some cases it is therefore crucial to have a better understanding of

all the steps involved in the process of in vitro production.

It is as yet unknown how the specific cleavage of ZPA glycoprotein is related to the

sequence of disulfide bond formation following fertilization demonstrated in our

studies. LINDSAY and HEDRICK (2004) showed in Xenopus laevis eggs that

proteolytic ZPA cleavage at a single site is the single biochemical event responsible

for the conformational changes of the ZP that is necessary for hardening of the

envelope. The modifications of the ZP2 (ZPA) following fertilization appear to be the

result of the protease exuded by the CGs, which is released during the cortical

reaction approximately 5 to 8 min after fertilization. In IVF systems, evidence is

present that sperm penetration of porcine oocytes begins at approximately 3 h after

insemination (SUN et al. 2003). However, our results indicate that the alterations

(biochemical and structural) over time of the ZP derived from both sources (in vivo

and in vitro matured zona) did not coincide temporally with the cortical reaction and

sperm penetration. A possible explanation for the apparent difference in timing

between the loss of free cysteine thiol residues within the ZP glycoproteins and the

two events after fertilization (e.g. sperm penetration and cortical reaction) may also

be that more biochemical events are involved in addition to the proteolytic cleavage

of the ZPA glycoprotein and disulfide bond formation. The specific cleavage of ZP2

glycoprotein by a proteinase released from the cortical granules (CGs) induces the

global conformation changes in the ZP matrix, thereby facilitating the formation of

disulfide bonds and orientating two Cys residues in a suitable location (e.g. three–

dimensional) where they can interact with each other along the extended polypeptide

chains (ZP). However, the results of in vitro incubations support the assumption that

the oxidation of proteins in -SH groups to disulfide bond formation also occurs

independently of the environment, presumably by spontaneous oxidation of thiol

groups for disulfide bonding.

Our observations provide strong evidence that the mechanism by which the zona

pellucida (ZP) is completely modified chiefly involves the ZPA glycoprotein. By

DISCUSSION ___________________________________________________________________________

116

biochemical analysis (one– and two–dimensional gel electrophoresis, SDS–PAGE) of

the oxidant-sensitive Cys residues, we demonstrated that, under physiologic

conditions (≈pH 6.5), only the ZPA glycoprotein contains oxidant-sensitive Cys

residues with low pKa, which possibly are necessary for the formation of the three–

dimensional architecture and structural changes in the ZP after fertilization. The two

ZP glycoprotein subunits (pZP2 to pZP4) were found to be linked with each other by

disulfide bonds. These results suggest that disulfide bond formation during

fertilization occurs predominantly by intramolecular linkages. Despite the fact that the

ZPB and ZPC glycoproteins were shown also to contain free Cys residues at pH 8.5

(1–D SDS–PAGE and 2–D SDS–PAGE); however, they did not contain those

oxidant-sensitive Cys residues with low pKa that are readily susceptible to oxidation

(i.e. disulfide bonds), which were shown to be present only in the ZPA glycoprotein.

Although there are numerous studies evaluating the differences of in vivo and in vitro

matured ZP before and after fertilization (DUCIBELLA et al. 1995, HATANAKA et al.

1992, and WANG et al. 1997c, 1998), this study is the first to quantify and to

compare the distribution of free cysteine thiol residues on the in vivo and in vitro

matured ZP and to follow the temporal course of novel intra- intermolecular disulfide

bond formation after fertilization associated with biochemical and structural

alterations of the ZP glycoproteins (ZPA, ZPB, and ZPC). It is possible that the

formation of disulfide bonds triggered by the proteolysis of pZP1 (ZPA) to pZP2 and

pZP4 (ZPA to ZPAf) following fertilization and the global conformational changes of

the three–dimensional structure of the zona pellucida induce the hardening of the

zona pellucida which is responsible for an effective block to polyspermy.

DISCUSSION ___________________________________________________________________________

117

5.1 Concluding Remarks This study provides for the first time an efficient biochemical marker for the

identification of oxidant-sensitive cysteine residues in porcine ZP glycoproteins. In

general, the morphological difference in the distribution of the free cysteine thiol

residues in the ZP architecture when compared to immature (germinal vesicle) and in

vitro matured (IVM) oocytes provides insights into how all three ZP glycoproteins are

formed and synthesized. These comparative studies of in vivo and in vitro matured

(IVM) oocytes suggest that factors within the ovary are also necessary to enable the

follicle to grow and the oocyte to achieve its fully ability to make/undergo

developmental alterations (e.g. biochemical and structural) of the ZP during the final

stages of oocyte maturation. Morphological studies of the ZP from in vivo and in vitro

matured (IVM) oocytes at different times after IVF provide an approach to better

understanding of the major problem of polyspermy during IVF. Oxidation of free thiol

groups and the subsequent formation of disulfide bonds after sperm penetration were

efficient in in vivo matured oocytes (3 h after IVF) in comparison to in vitro matured

(IVM) oocytes (18 h after IVF). This may be a possible reason for the high

polyspermic penetration in in vitro matured (IVM) oocytes derived from IVF. Apart

from the implications of our work for this research program, it is possible that the

formation of disulfide bonds triggered by ZP glycoproteins following fertilization and

the probably global conformational changes in the three–dimensional structure of the

ZP are responsible for the hardening of the zona pellucida (ZP). However, the exact

mechanism of this process clearly needs to be elucidated before it will be possible to

make a full assessment of the biological significance of this correlation between

biochemical and structural alterations of the ZP glycoproteins and the formation of

disulfide bonds for the polyspermy block.

ZUSAMMENFASSUNG ___________________________________________________________________

118

6 ZUSAMMENFASSUNG

Moreira, Ana Claudia Biochemische und strukturelle Veränderungen an der porcinen Zona pellucida (ZP) während der Reifung (In Vivo und In Vitro) und In-Vitro Befruchtung (IVF)

Die Zona pellucida (ZP) der Oozyten spielt bei der Befruchtung eine zentrale Rolle.

Matrixoberfläche und dreidimensionale Architektur der ZP unterliegen dabei einer

Reihe von Reifungs-und Transformationsprozessen. Hier steht insbesondere das

sog. Zona hardening im Vordergrund, das für den Polyspermieblock essentiell ist.

Aufgabe der vorliegenden Arbeit war es, die Zona pellucida (ZP) während der

Reifung und der In vitro Befruchtung (IVF) von porcine Oozyten näher zu

charakterisieren, mit besonderem Blick auf die biochemischen und strukturellen

Veränderungen der Zona pellucida (ZP). Dabei sollte insbesondere die Bildung und

die Verteilung von den Disulfidbrücken analysiert und deren Rolle für die Etablierung

der Polyspermieblock untersucht werden.

Für das Erreichen dieser Zielsetzung wurde dieses Projekt in sieben Abschnitte

gegliedert, wobei in jeder eine (1) der folgenden Gruppen untersucht wurde: (i)

Experiment I: Immature Oozyten (germinal vesicle), (ii) Experiment II: In Vitro

gereiften (IVM) Oozyten von 23 Stunden (MI), (iii) Experiment III: In vitro gereiften

(IVM) Oozyten von 46-48 Stunden (MII), (iv) Experiment IV: In vivo gereiften Oozyten

von präpuberalen Saureren (MII), (v) Experiment V: In vitro gereiften (IVM) Oozyten

(46-48 Stunden, MII) nach 0, 3, und 18 Stunden In vitro Befruchtung (IVF), (vi)

Experiment VI: In vivo gereiften Oozyten (MII) nach 0, und 3 Stunden In vitro

Befruchtung (IVF), (vii) Experiment VII: In vitro und In vivo gereiften Oozyten nach 18

Stunden In vitro Spermienfreien FertTalp Medium.

In der vorliegenden biochemischen Untersuchung („Pretrial I“) wurden die drei

Glycoproteine der pZP mittels eindimensionaler (1–D SDS–PAGE) und


119

zweidimensionaler Polyacrylamidgelelektrophorese (2–D SDS–PAGE) aufgetrennt.

Es folgte eine Charakterisierung der freien Thiolgruppen der Cysteine innerhalb der

Glykoproteine der ZP (ZPA, ZPB, und ZPC) von ungereiften porcine Oozyten (GV).

Die ZP wurde mit BIAM (biotin- conjugated iodoacetamide) unter mehreren pH-

Bedingungen (pH 8,5 und pH 6,5) umgesetzt (KIM et al. 2000), über SDS–PAGE (1–

D SDS–PAGE und 2–D SDS–PAGE) separiert und die biotinilierten Cystein

Aminosäuren selektiv nachgewiesen.

In der vorliegenden Strukturanalyse („Trial II“ und „Trial III“) wurde die ZP durch

konfokale Mikroskopie (LSCM, „Trial II“) und Rasterelektronenmikroskopie (SEM,

„Trial III“) morphologisch untersucht. Für LSCM wurden die ZP Glykoproteine mit 5-

Iodoacetamidefluorescein (5-IAF) unter pH 6,5 behandelt. Es wurde eine

multikomparative und semi-quantitative Analyse von dem Muster der

Fluoreszenzintensität (I), der Fluoreszenzhomogenität (H) und Muster der Differenz

(D) der Fluoreszenzintensitäten der inneren (I) und äußeren (A) Oberfläche der ZP

durchgeführt. Für die SEM wurden die ZP Glykoproteine mit BIAM unter pH 6,5

behandelt und mittels Streptavidin- Goldenpartikel die Verteilung der Thiolgruppen

innerhalb der äußeren Oberfläche der ZP nachgewiesen.

Die Ergebnisse können wie folgt zusammengefasst werden:

1. In der 1–D SDS–PAGE zeigte die Reaktion des ZP Glykoproteins mit BIAM unter

pH–Wert von 8,5 die Oxidation aller virtually freien Thiolgruppen Glycoproteins

ZPA, ZPB und ZPC. Im Gegensatz dazu konnte unter pH-Bedingung von 6,5 eine

selektive Markierung von ZPA beobachtet werden.

2. In der 2–D SDS–PAGE zeigte ZPA Glykoprotein eine dominante Markierung im

pI-Bereich 4–6 mit BIAM unter pH–Wert von 8,5, ZPB/ZPC Glykoproteins eine

starke Markierung in einer breiten Markierung von pI-Bereich 3–6,5. Die Reaktion

mit BIAM unter pH 6,5 führte zur selektiven Markierung der ZPA Glycoprotein.

ZPA erschien als ein Single-Protein-Band.


120

3. Im Experiment I (LSCM) zeigte die Analyse von ungereiften ZP (GV) zwei

dominante Populationen von Fluoreszenzhomogenitätmustern (H): (a) niedrige (H

= 1, 47,8%), and (b) keine Homogenität (H = 0, 26,1%).

4. 51,1% von der ZP (LSCM) von Metaphase I Eizellen (MI, 23 Stunden IVM)

besitzen mittlere (H = 2) Fluoreszenzhomogenitätmuster (H). Statistische

signifikante Differenzen zwischen GV und MI wurden gefunden (P < 0,007).

5. Im Experiment III 57,9% von der ZP von In vitro gereiften (IVM) Metaphase II

Eizellen (46–48 Stunden, IVM) führen zu einer erhöhten Fluoreszenzhomogenität

(H = 3). Statistische signifikante Differenzen zwischen MI und MII wurden

gefunden (P < 0,0001).

6. Experiment IV: In vivo Reifung von Oozyten führt zur Etablierung einer niedrigen

(H = 1, 23,6%) bis mittleren (H = 2, 53,6%) Fluoreszenzhomogenität.

Währendessen zeichnen sich 57,9% der ZP von In vitro gereiften (IVM) Oozyten

durch das Fluoreszenzhomogenitätmuster H = 3 aus. Signifikante Unterschiede

zwischen In vivo und In vitro gereiften (IVM) Oozyten wurden gefunden (P <

0,0001).

7. Experiment V: 95,5% von der ZP von IVM Eizellen nach 0 Stunden des IVF

Prozesses führen zu einem hohen Fluoreszenzintensitätenmuster (I = 3) und

70,6% von der ZP von IVM Eizellen nach 3 Stunden des IVF Prozesses besitzen

ein Fluoreszenzintensitätenmuster (I = 3). Im gegensatz dazu zeigen 60,0% die

ZP von IVM nach 18 Stunden des IVF Prozesses keine

Fluoreszenzintensitätenmuster (I = 0). Signifikante Unterschiede zwischen 3

Stunden und 18 Stunden wurden gefunden (P < 0,0001).

8. Experiment VI: 67,6% von der ZP von In vivo gereiften Oozyten nach 3 Stunden

des IVF Prozesses zeigen keine Fluoreszenzintensitätenmuster (I = 0).

Signifikante Unterschiede zwischen In vivo und In vitro gereiften Oozyten nach 3

Stunden des IVF Prozesses wurden gefunden (P < 0,0001).

9. Experiment VII: In vivo und IVM Oozyten, die in einem spermienfreien FertTalp

Medium für 18 Stunden inkubiert waren, zeigten ein hohes

Fluoreszenzintensitätenmuster (I = 2; I = 3). Das lässt vermuten, dass sowohl In


121

vivo als auch In vitro gereifte Oozyten nun nach der Penetration zur Bildung

neuer Disulfidbrücken (i.e. Verbrauch von freien Thiolgruppen) befähigt sind.

10. SEM: Die Verteilung der Thiolgruppen innerhalb der äußeren Oberfläche der ZP

zeigten signifikante Unterschiede zwischen ungereiften (GV) und In vitro gereiften

(IVM) Eizellen (MII) (P < 0,0001).

11. Bei Benutzung von SEM auf dem IVF Prozess der Oozyten zeigten nach 18

Stunden (IVF) 70,7% keine (Null) Streptavidin Goldpartikel an der ZP Oberfläche.

Signifikante Unterschiede mit dem Muster der ZP von MII wurden gefunden (P <

0,0001).

12. Basierend auf den vorgelegten Ergebnissen der Arbeitsgruppe ist zu sagen, dass

der Verlust von Thiolgruppen während des Befruchtungsprozesses (IVF) eine

mögliche Begründung für die Modifikation von ZP-Struktur sein könnte, welche

als Barriere zur polyspermischen Eindringung dient. In In vitro gereiften (IVM)

Oozyten wächst die strukturelle Alteration der ZP relativ spät (18 Stunden nach

IVF) verglichen mit in In vivo gereiften Oozyten (3 Stunden nach IVF). Dies zeigt

an, dass die verzögerte Bildung von neuen Disulfidbrücken (i.e. Verbrauch von

freien Thiolgruppen) von In vitro gereiften (IVM) Oozyten, wenn sie verglichen

werden mit In vivo gereiften Oozyten (nach 3 Stunden IVF), eine mögliche

Erklärung für den hohen Anteil polyspermie Befruchtung nach IVM und IVF ist.

Jedoch müssen zu dieser Thematik weitere Studien angefertigt werden. Es

könnte weiterhin noch hinterfragt werden, welche Rolle nach unterschiedlichem

Maturation-Prozess die Zona pellucida (ZP) während der In vivo und In vitro

Reifung (IVM) für das Zona hardening durch die Bildung von Disulfidbrücken

spielt.

SUMMARY ___________________________________________________________________

122

7 SUMMARY

Moreira, Ana Claudia

Biochemical and Structural Alteration of the Porcine Zona Pellucida (ZP) during (In Vivo and In Vitro) Maturation and Fertilization (IVF)

All vertebrate oocytes are surrounded by an extracellular matrix. In mammals this

matrix is known as the zona pellucida (ZP). One of its functions is to prevent

polyspermy by - the so-called zona block - which is a biochemical and structural

alteration of the ZP glycoproteins induced by release of cortical granule (CG)

contents. The goal of the study was to examine and compare the distribution of free

cysteine thiol residues of the ZP glycoproteins. Seven different groups of oocytes

were used for the investigation: (i) immature oocytes (germinal vesicle, Exp. I); (ii)

oocytes matured in vitro (IVM) for 23 h (metaphase I, Exp. II); (iii) oocytes matured in

vitro (IVM) for 46- to 48 h (metaphase II, Exp. III); (iv) oocytes matured in vivo

(metaphase II, Exp. IV); (v) oocytes derived from IVF of in vitro matured (IVM)

oocytes, 0, 3, and 18 h after IVF (Exp. V); (vi) oocytes derived from IVF of in vivo

matured oocytes 0, and 3 h after onset of IVF (Exp. VI); and (vii) oocytes derived

from in vivo and in vitro maturation (IVM) cultured under IVF conditions, but without

exposure to spermatozoa.

The biochemical aim (Pre-Trial I) of this study was to analyze the distribution of the

oxidant-sensitive cysteine (Cys) residues in the ZP glycoproteins ZPA, ZPB, and ZPC

by electrophoretic methods. For this purpose different pH levels (pH 6.5 and 8.5)

were used in order to optimize the labelling technique for the further morphologic

evaluation by laser scanning confocal microscopy (LSCM) (Trial II) and scanning

electron microscopy (SEM) (Trial III) of ZP from the different sources described

above. For LSCM the ZP was treated with direct fluorescent labelled 5-

iodoacetamidefluoresceín (5-IAF at pH 6.5). For SEM the ZP was treated with biotin-

conjugated iodoacetamide (BIAM) at below pH 6.5 and subsequently monitored with

SUMMARY ___________________________________________________________________

123

streptavidin gold particles. Multi-comparative and semi-quantitative statistical analysis

was applied to the intensity fluorescence pattern (I), homogeneity pattern (H), and

the difference (D) between inner (I) and outer (A) surface of the ZP of all stages. The

following results were obtained:

1. One dimensional (1–D SDS–PAGE) BIAM (pH 8.5) revealed oxidation of virtually

all free cysteine thiols of the glycoproteins ZPA, ZPB, and ZPC. In contrast, BIAM

at pH 6.5 led to selective labelling within ZPA glycoprotein, while no labelling of

glycoproteins ZPB and ZPC was present.

2. Two dimensional (2–D SDS–PAGE) BIAM (pH 8.5) revealed dominant labelling of

the ZPA glycoprotein between pH 4– and 6, whereas there was strong labelling of

ZPB and ZPC glycoproteins over a wide pH range from pH 3 to– 6.5. In contrast,

BIAM at pH 6.5 led to selective labelling of the ZPA glycoproteins, while there was

no staining of ZPB and ZPC glycoproteins.

3. The staining homogeneity of the ZP from the germinal vesicle (GV) stages was

minor (H = 0, 26.1%) and low (H = 1, 47.8%). The staining homogeneity of the

metaphase I (MI) oocytes was low (H = 1, 35.6%) to medium (H = 2, 51.1%),

while that of metaphase II oocytes (MII) was medium (H = 2, 36.8%) to high (H =

3, 57.9%). After IVM (46–48 h, MII) there was a statistically significant shift to

increased homogeneity (3) at P < 0.0001.

4. There were strong differences between the inner and outer region of the ZP from

the germinal vesicle (GV) stages and metaphase I (23 h of IVM) (D = 2, 60.9%

and 78.2%, respectively), while there were moderate (i.e. medium) differences

between the inner and the outer regions of the ZP from MII (46–48 h of IVM) (D =

1, 68.5%). There was a statistically significant difference (D) pattern in the

distribution of labelling in the ZP (P < 0.0001) between MI (23 h of IVM) and MII

(46–48 h).

5. The homogeneity of oocytes matured in vivo (MII) was low (H = 1, 28.6%) and

medium (H = 2, 53.6%), while that of IVM (MII) was medium (H = 2, 36.8%) to

high (H = 3, 57.9%). There was a highly significant difference in the homogeneity

of staining of the ZP (P < 0.0001) between in vivo and IVM (46–48 h) oocytes.

SUMMARY ___________________________________________________________________

124

6. The staining intensity of oocytes matured in vivo (MII) was medium (I = 2, 37.9%)

and strong (I = 3, 68.6%), while that of oocytes matured in vitro (IVM) was always

strong (I = 3, 95.5%). There was a significant difference in the staining intensity

distribution of the ZP (P < 0.0001) between IVM oocytes and those matured in

vivo.

7. There was a strong fluorescence distributed across the ZP 0 and 3 h after IVF (I =

3, 95.5%, and I = 3, 70.6%, respectively). On the other hand, the fluorescence

intensity of IVM oocytes was zero 18 h after onset of IVF (I = 0, 60.0%). There

was a highly signicant difference in fluorescence intensity (I) classified as (1), (2),

(3) of fertilized ZP 3 and 18 h after onset of IVF at P < 0.0001.

8. No fluorescence labelling occurred in oocytes matured in vivo 3 h after onset of

IVF (I = 0, 67.6%), while there was strong fluorescent staining in 70.6% of the

oocytes matured in vitro (IVM) (3 h after IVF) (I = 3). There was a significant

difference (P < 0.0001) in intensity of fluorescence staining between fertilized ZP

(3 h after onset of IVF) derived from in vivo and of IVM oocytes.

9. After incubation in FertTalp medium for 18 h without exposure to spermatozoa,

both in vivo and in vitro matured (IVM) oocytes exhibited high fluorescence (I = 3,

and I = 2, respectively), which suggests that both in vivo and in vitro matured

(IVM) oocytes develop the ability to formation disulfide bonds only after

penetration of spermatozoa.

10. SEM examination revealed that there was strong labelling with gold particles in

the outer surface of the ZP (F = 3) from immature (GV) and IVM (MII) oocytes.

These patterns apparently did not change substantially until 3 h after IVF, but 18

h after IVF there were no gold particles on the ZP surface (F = 0, 70.7%), and this

is highly significant at P < 0.0001.

In conclusion, the present results indicate that loss of free thiol groups during

fertilization (IVF) could be the results of the formation of new intramolecular disulfide

bonds of the ZPA glycoprotein, which may be crucial to the tertiary structure of the

ZP, which prevents further sperm penetration. In porcine IVM, the alterations (e.g.

biochemical and structural) of the ZP occurs relatively late (18 h after IVF) compared

SUMMARY ___________________________________________________________________

125

with oocytes matured in vivo (3 h after IVF). This may be a reason for the high

polyspermic penetration in in vitro matured (IVM) oocytes derived from IVF. Further, it

is possible that the formation of disulfide bonds triggered by ZP glycoproteins

following fertilization and the global conformational changes of the three–dimensional

structure of the ZP induce the hardening of the zona pellucida. However, further

studies are clearly needed before the significance of this correlation can be fully

assessed.

REFERENCES ___________________________________________________________________

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APPENDIX ___________________________________________________________________

150

9 APPENDIX 9.1 Composition of Some Media Used in In Vitro Maturation (IVM), In

Vitro Fertilization (IVF), and In Vitro Culture (IVC) Experiments 9.1.1 Physiological Saline Solution Medium Table 11 summarizes the contents of the components used here Tab. 11: Physiological saline solution medium Component 0.9% NaCl Na Cl 9 g Penicillin 0.06 g Streptomicin sulfat 0.131 g 9.1.2 Dulbecco’s Phosphate Buffered Saline (PBS) Media Table 12 summarizes the contents of the components used.

APPENDIX ___________________________________________________________________

151

Tab. 12: Composition of dulbecco´s phosphate buffered saline (PBS) used in various

ZP experiments.

Component PBS* PBS + 2% PVA PBS + 1% NBCS

Bovine serum albumin (BSA)

1mg/ml 1mg/ml 1mg/ml

Streptomycin-sulphate 50 µg/ml 50 µg/ml 50 µg/ml

Penicillin G 60 µg/ml 60 µg/ml 60 µg/ml

KCL 2.38 mM 2.38 mM 2.38 mM

NaCL 130.0 mM 130.0 mM 130.0 mM

KH2PO4 1.47 mM 1.47 mM 1.47 mM

NaH2PO4 8.10 mM 8.10 mM 8.10 mM

MgSO4 x 7 H2O 0.92 mM 0.92 mM 0.92 mM

CaCL2 x 2 H20 0.71 mM 0.71 mM 0.71 mM

PVA** NBCS***

0.02g/ml 0.01ml/ml

*Medium based on Dulbecco´s Phosphate Buffered Saline without energy substrate, ** PVA

(Polyvinypyrrolidone), *** NBCS (newborn calf serum)

9.1.3 Maturation Media Used for IVM Table 13 summarize the contents of the maturation media used, NCSU-37 (North

Carolina State University) free BSA according to PETTERS and WELLS (1993).

9.1.4 Fertilization Media Used for IVF The composition of Fert-TALP media used for IVF is summarized in Table 13. 9.1.5 Cultivation Media

Table 13 summarizes the contents of the applied media, NCSU-23 (North Carolina

State University) with BSA according to PETTERS and WELLS (1993).

APPENDIX ___________________________________________________________________

152

Tab.13: Composition of media used in IVM, IVF, and IVC experiments Component TCM199 NCSU-37

(PETTERS u. WELLS 1993)

Fert-TALP (PARRISH u. FIRST 1993)

NCSU-23 (PETTERS u. WELLS 1993)

BSA Fraction V *0.4% *0.4% CaCL2 x 2 H20 1.7 mM 1.7 mM Coffein 2.0 mM Ca-Lactat x 5 H20 2.5 g/l Cystein *0.6 mM db-cAMP **1.0 mM EGF *10 ng/ml FCS 10% FSH *1.0 µg/ml Gentamycin 0.5 g/l Glucose 5.6 mM 5.0 mM 5.6 mM Glutamin *1.0 mM *1.0 mM hCG **10 IE/ml Hepes Hypotaurin *5.0 mM Immature Foll. Fl. *10 % *10 % Insulin * 5.0 mg/l 0.1 g/l Kanamycin KCL 4.8 mM 3.2 mM 4.8 mM KH2PO4 1.2 mM 1.2 mM Mercaptoethanol * 50 µM MgCL2 x 6 H2O 0.5 mM MgSO4 x 7 H2O 1.2 mM 1.2 mM Na-pyruvat *1.0 mM NaCL 108.7 mM 114 mM 108.7 mM NaH2PO4 0.34 mM NaHCO3 25.1 mM 25.1 mM 25.1 mM 25.1 mM Na-Lactat 10 mM Penicillin G 100 IE/l 100 IE/l Phenolrot 0.001% 0.001% PMSG **10 IE/ml PVA 1.0 g/l Sorbitol 12 mM Streptomycin 50 mg/l 50 mg/l Taurin *7.0 mM

APPENDIX ___________________________________________________________________

153

9.2 Composition of Reagents Prepared for Use in Biochemical Experiments 9.2.1 Acrylamide Solution Unless indicated otherwise, all manufacturers are located in Germany. 10% Dense Solution: 30% Acrylamide solution (Roth, Karlsruhe) 3.3 ml

1.5 M Tris/HCl pH 8.8 2.5 ml

10 ml - 10% SDS Ultrapure (Roth, Karlsruhe) 100 µl

Distilled water 4.0 µl

TEMED (Serva, Heidelberg) 4.0 µl

40% Ammonium persulfate (Serva, Heidelberg) 100 µl

4.5% Light Solution: 30% Acrylamide solutionl (Roth, Karlsruhe) 0.5 ml

1.0 M Tris/HCL pH 6, 8 0.38 ml

3 ml - 10% SDS Ultrapure (Roth, Karlsruhe) 30 µl

Distilled water 2.1 ml

TEMED (Serva, Heidelberg) 3 µl

40% Ammonium persulfate (Serva, Heidelberg) 30 µl

9.2.2 SDS Electrophoresis (LAEMMLI 1970): 50 mM Tris pH 6.8, 2% SDS (w/v), 10 % glycerol, bromophenol blue

APPENDIX ___________________________________________________________________

154

9.2.3 Loading Buffer 25 mM Tris, 192 mM glycin, 0.1 % SDS (w/v) 9.2.4 Immobilized pH Gradients (IPG) IPG strip rehydration: 9 M urea, 2% CHAPS, 0.5 % IPG buffer,

IPG strip equilibration: (a) 6 M urea, 2% SDS (w/v), 2.5 ml 0.5 M Tris/ HCL buffer

pH 6.8, 30 % glycerol (w/v)

Equilibration solution I: (a) + 65 mM DTT (Dithiothreitol, AppliChem, Darmstadt)

Equilibration solution II: (a) + 0.24 M iodoacetamide (Sigma Aldrich Chemie

GmbH, Taufkirchen)

9.2.5 Blotting to PVDF Membranes 0.039 M glycine, 0.048 M Tris, 0.0375% SDS (w/v), 20% methanol (v/v) 9.2.6 Ponceau Staining 0.5% Ponceau red (Roth, Karlsruhe) to 1% acetic acid. 9.2.7 TBS/ Tween TBS + 1% Tween (v/v)

APPENDIX ___________________________________________________________________

155

9.3 Composition of Reagents Prepared for Use in Biochemical Experiments with Laser Scanning Confocal Microscopy (LSCM) and Scanning Electron Microscopy (SEM) 9.3.1 HEPES- Fixation Stock solution:

2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethane sulfonic acid sodium salt MG: 260.28g/l,

0.3 M Hepes = 78.084g + 800 ml distilled water.

The final pH adjusted to pH 7.35.

The final volume was adjusted to 1000 ml with distilled water.

The final concentration was adjusted to ~ 750 mOsm.

1000 ml Solution Fixation: 500 ml 0.3 M Hepes + 60 ml 25% glutaraldehyde (Merck) + 60 ml 25%

paraformaldehyde (PFA) (Merck)

The volume was adjusted to 900 ml with distilled water.

The final pH adjusted to 7.35.

The final volume was adjusted to 1000 ml with distilled water.

The final concentration was adjusted to 800-900 mOsm/l

25% Paraformaldehyd (PFA): 15 g PFA + 50 ml distilled water allowed to stand at 65 °C, 1 N NaOH was added to

solution until to clear. The volume was adjusted to 60 ml distilled water. The solution

was aliquoted into sterile plastic tubes (each 10 ml) and stored at -20 °C.

Before use, the medium was thawed at RT. Repeated freeze - thawing was avoided.

APPENDIX ___________________________________________________________________

156

9.3.2 Poly-L-Lysine Coverslips 0.1% (w/v) solution of poly-L-lysine (Sigma, Deisenhofen, Germany) 0.1% (w/v) was

made by dissolving 2 mg/ml distilled water.

9.3.3 Mowiol Mounting Medium pH 6.5 2.4 g Mowiol (Hoechst, Cat. 4-88),

6.0 g Glycerol AR (Merck, Cat. 4094),

6 ml H2O,

0.2 M Bis-Tris pH 6.5.

Clarification by centrifugation at 4000-5000 rpm for 20 min, and aliquots taken.

This remained stable for 12 months at -20°C.

Once thawed at RT, it was stable for one month at RT.

9.4 Tables and Figures 9.4.1 Tables Tab. 1: Characteristics of representative members of the zona pellucida glycoprotein families

(modified from DUNBAR et al. 2001). ----------------------------------------------------------------------28

Tab. 2.1: Free thiol group distribution according to homogeneity (H) pattern classified as (0),

(1), (2), or (3) at GV stage, 23 h (MI) and at 46-48 h (MII) after in vitro maturation (IVM). ---69


distribution at different ZP stages (GV, MI, and MII) of in vitro maturation (46-48 h of IVM)

(Chi-Square Test). ----------------------------------------------------------------------------------------------70

Tab. 3.1: Free thiol group distribution according to intensity difference (D) pattern classified

as (0) (1) or (2) at GV stage, 23 h (MI) and at 46-48 h (MII) after in vitro maturation (IVM). -72

Tab. 3.2: Probability values (P) for the comparison of the intensity difference (D) pattern

distribution at different ZP stages (GV, MI, and MII) of in vitro maturation (46-48 h of IVM)

(Chi-Square Test). ----------------------------------------------------------------------------------------------73

APPENDIX ___________________________________________________________________

157

Tab. 4.1: Free thiol group distribution according to homogeneity (H) pattern classified as (1),

(2), or (3) at MII from in vivo and in vitro (IVM) matured (46-48 h of IVM) oocytes. ------------75


distribution at in vivo and in vitro maturation (46-48 h of IVM) (Chi-Square Test). -------------75

Tab. 5.1: Free thiol group distribution according to the intensity (I) labelling pattern classified

as (1), (2), or (3) in the ZP from in vivo and in vitro matured oocytes (46-48 h of IVM). ------76

Tab. 5.2: Probability values (P) for the comparison of the intensity (I) pattern distribution for

in vivo and in vitro maturation (IVM) (Chi-Square Test). ----------------------------------------------77

Tab. 6.1: Homogeneity (H) pattern (0-3) of the distribution of free thiol groups in porcine ZP

derived from IVM oocytes (46-48 h of IVM) 0 and 3 h after onset of IVF. -------------------------80

Tab. 6.2: Probability values (P) for the comparison of the distribution of the homogeneity (H)

pattern distribution at 0 and 3 h after onset of IVF (Chi-Square Test). ----------------------------80

Tab.7.1: Intensity (I) fluorescence patterns (1-3) of the distribution of free thiol groups in

porcine ZP from in vitro matured (IVM) oocytes 0, 3, and 18 h after onset of IVF. -------------81

Tab. 7.2: Probability values (P) for the comparison of the distribution of the intensity (I)

pattern distribution at 0 h, 3 h, and 18 h after onset of IVF (Chi-Square Test). -----------------82

Tab. 8.1: Free thiol group distribution according to the intensity (I) of the fluorescence pattern

(0-3) from oocytes matured in vivo and in vitro (IVM) 3 h after onset of IVF. --------------------85

Tab. 8.2: Probability values (P) for the comparison of the intensity (I) pattern distribution of

oocytes matured in vivo and in vitro (IVM) 3 h after onset of IVF (Chi-SquareTest). ----------86

Tab. 9.1: Density (F) distribution of gold particles of the ZP from immature oocytes (GV), MII

stage (46-48 h of IVM) oocytes, and IVM oocytes 3 and 18 h after onset of IVF. --------------90

Tab.9.2: Probability values (P) for the comparison of the density (F) distribution of gold

particles on the ZP from immature (GV) oocytes, in vitro matured (IVM) oocytes, and oocytes

derived from IVM 3 and 18 h after onset of IVF (Chi-Square Test). --------------------------------90

Tab.10: Sperm binding to the ZP from oocytes matured in vivo and in vitro matured (IVM) 12

h after onset of IVF. ---------------------------------------------------------------------------------------------96

Tab. 11: Physiological saline solution medium. --------------------------------------------------------150 Tab. 12: Composition of Dulbecco´s phosphate buffered saline (PBS) used in various ZP

experiments. ----------------------------------------------------------------------------------------------------151

Tab.13: Composition of media used in IVM, IVF, and IVC experiments. ------------------------152

APPENDIX ___________________________________________________________________

158

9.4.2 Figures Fig. 1: Schematic representation of stages in the developmental transition of oogonia – to

mature eggs (meiosis II) of the female gamete. See text for details. -------------------------------17

Fig. 2: At ovulation, the arrested secondary oocyte is released from the ovary and undergoes

a rapid maturation step to prepare for fertilization. If fertilization occurs, the oocyte is

stimulated to complete meiosis and begin embryonic development (From LEIBICH 1993,

120x and 300x). --------------------------------------------------------------------------------------------------18

Fig. 3: Schematic representation of development the follicle in female gamete. A: The

reduction in the number of growing follicles is caused by a multiple waves of atresia from

primordial until dominant follicle. B: The population of growing follicles can be classified

according to the size, the number of granulosa cells, the presence or absence of an antrum,

and the number of cell layers as, a, primordial follicle, b, secondary follicle, c, d, preantral

follicle, e, early antral follicle, and f, preovulatory Graafian follicle. ---------------------------------19

Fig. 4: Cortical granule migration. A: Cortical granules have already translocated to the

cortex at MII (BERG and WESSEL 1997). B: Cortical granules are usually poised at the

cytoplasmic face of the plasma membrane (WESSEL et al. 2001). C: Cortical granules are

attached or docked to the plasma membrane of the unfertilized eggs in sea urchin eggs. --25

Fig. 5: Schematic representation of the mouse ZP glycoproteins ZP1, ZP2, and ZP3. The

ZP2 and ZP3 glycoproteins form long filaments and are crosslinked by ZP1 glycoproteins

(PRASAD et al. 2000, WASSARMAN et al. 2004). -----------------------------------------------------30

Fig. 6: Schematic presentation of Cys residues in porcine ZPB and ZPC. The circles denote

groups of Cys (C) residues in porcine ZPB and ZPC that form possible disulfide bonds with

each other. There is conservation in the number and position of the cysteine residues within

each ZP glycoprotein (YONEZAWA and NAKANO 2003). -------------------------------------------33

Fig. 7: Overview of the oxidation of the two cysteine (Cys or C) residues to cystine (L-

cystine). ------------------------------------------------------------------------------------------------------------34

Fig. 8: The essential substrate involved with cell signalling is hydrogen peroxide (H2O2). ---41

Fig. 9: Porcine zona pellucida is divided into pZP2 (ZP2) and pZP4 (ZP2f) by reduction of -S-

S- bonds (marked in red). -------------------------------------------------------------------------------------43

Fig. 10: Experimental design: Porcine ZP from seven different developmental stages were

labelled with 5- Iodoacetamidefluorescein (5-IAF) and biotin-conjugated iodoacetamide

(BIAM) and identified by laser scanning confocal microscopy (LSCM) and scanning electron

microscopy (SEM). ----------------------------------------------------------------------------------------------46

APPENDIX ___________________________________________________________________

159

Fig.11: Overview of the experimental design. -----------------------------------------------------------53

Fig.12: Confocal microscope image of the fluorescence intensity difference (D) pattern

between the distribution across the inner and outer regions of the ZP: a, no difference (D =

0), b, medium difference (D = 1), c, strong difference (D = 2). --------------------------------------59

Fig. 13: Confocal microscope image of the distribution of the fluorescence intensity (I)

pattern across the ZP: a, strong intensity (I = 3), b, medium intensity (I = 2), c, low intensity (I

= 2), d, no fluorescence (I = 0). ------------------------------------------------------------------------------60

Fig. 14: Confocal microscope images of homogeneity (H) labelling pattern distributed across

of the ZP: a, minor homogeneity (H = 0), b, low homogeneity (H = 1), c, medium

homogeneity (H = 2), d, high homogeneity (H = 3). ----------------------------------------------------60

Fig. 15: Classification of free thiol group distribution (F) by scanning electron microscopy

(SEM) (x 1000; x 900; x 2000, and x 2000). A: no gold particles (F = 0), B: few gold particles

(F = 1), C: moderate number of gold particles (F = 2), D: complete distribution of gold

particles (F = 3). -------------------------------------------------------------------------------------------------63

Fig. 16: Analyses of BIAM–labelled ZP glycoproteins by means of 1-D SDS-PAGE at pH 6.5

and 8.5. Between 75 and 100 zonae pellucidae were dissolved from the germinal vesicle

(GV) stage and labelled with BIAM at pH 6.5 and to pH 8.5. Equal portions of each mixture

were detected by chemiluminescent blot analysis with HRP- conjugated streptavidin. (A):

Selective labelling of the ZPA glycoprotein was demonstrated at pH 6.5. No labelling of

glycoproteins ZPB/ZPC was present. (B): Biotinylation (BIAM) at pH 8.5 resulted in labelling

of virtually all free cysteine of the glycoproteins ZPA, ZPB, and ZPC. -----------------------------65

Fig. 17: Zona pellucida (ZP) from immature (GV) porcine oocytes were labelled with BIAM at

pH 6.5 and pH 8.5 and separated by 2-D SDS-PAGE. Approximately 150 zonae pellucidae

were dissolved and labelled with BIAM at pH 6.5 (A) and at pH 8.5 (B). SDS-PAGE was

carried outer under no–reducing conditions in 10% acrylamide separating gel with 4%

stacking gel, transferred to PVDF membrane, and visualized with streptavidin-peroxidase

and chemiluminescence. --------------------------------------------------------------------------------------66

Fig. 18: Specificity of fluorescent labelling with 5-IAF to the ZP matrix. The fluorescent dye

(5-IAF) did not react with free thiol groups (B). The image (A) was evaluated by differential

interference contrast (DIC) as one of our controls for the no reaction of the free thiol groups

with fluorescent stain (5-IAF) analyzed by differential phase contrast (DPC, B). ---------------68

APPENDIX ___________________________________________________________________

160

Fig. 19: Confocal microscope images of homogeneity (H) pattern showed the distribution of

free thiol groups in the ZP surface from oocytes at the germinal vesicle (GV) stage, at 23 h

(MI), and at 46-48 h after IVM (MII). (a) H = 0 from GV; (b) H = 1 from GV; (c) H = 2 from

oocytes matured in vitro for 23 h (MI), and (d) H = 3 from oocytes matured in vitro (IVM) for

46-48 h (MII). Images were evaluated by differential phase contrast (DPC). --------------------71

Fig. 20: Confocal microscope images showing the intensity difference (D) patterns of

fluorescence labelling in the inner (I) and the outer (A) regions of the ZP. (A), strong intensity

difference, (D = 2 from germinal vesicle [GV] stage), (B), medium intensity difference (D = 1

MI from 23 h of IVM), and (C) medium intensity difference (D = 1, MII from 46-48 h of IVM).

Images were evaluated by differential phase contrast (DPC). ---------------------------------------73

Fig. 21: The results reveal the fine structure of the ZP among porcine oocytes after in vivo

and in vitro maturation (IVM). (A), image by reverse microscopy of the thickness of the ZP

from in vivo matured oocytes, evaluated at 2.5 µm to 11.8 µm (X = 7.6; SD = 2.8); (B), image

by interference contrast of the thickness of the ZP from in vitro matured (IVM) oocytes,

between 10.3 µm to 15 µm (X = 12.0; SD = 1.4). -------------------------------------------------------74

Fig. 22: Confocal microscope image showing the overall intensity (I) and homogeneity (H)

pattern in the ZP from oocytes matured in vivo and in vitro (IVM). (A), metaphase II after in

vitro matured (IVM) oocytes (I = 3, H = 3), (B), (C), and (D), metaphase II after in vivo

matured oocytes (I = 3, H = 1), (I = 3, H = 2), (I = 3, H = 3), respectively. Images were

evaluated by differential phase contrast (DPC). ---------------------------------------------------------78

Fig. 23: Confocal microscope images of the fluorescence intensity (I) patterns in the ZP from

IVM oocytes 0 h, and 3 h after onset of IVF. (a), (b), and (c), the intensity patterns of IVM

oocytes at metaphase II 0 h after IVF were medium (I = 2) or strong (I = 3): (d), (e), and (f), the fluorescence intensity pattern of in vitro matured (IVM) oocytes 3 h after onset of IVF was

greater (strong, I = 3). Images were evaluated by differential phase contrast (DPC). ---------83


IVM oocytes 18 h after onset of IVF. (A), (B), (C), (D), (E), and (F): There was no

fluorescence labelling (I = 0) in IVM oocytes incubated for 18 h after IVF. Images were

evaluated by differential phase contrast (DPC). ---------------------------------------------------------84

APPENDIX ___________________________________________________________________

161


oocytes matured in vivo and in vitro (IVM) 3 h after onset of IVF. (A), (B), and (C): oocytes

matured in vitro (IVM) 3 h after IVF exhibited a high fluorescence pattern (I = 3), whereas

oocytes matured in vivo (D), (E), and (F) demonstrated no (zero) intensity fluorescence

labelling (I = 0) 3 h after IVF. The images (D1), (E1), and (F1) were evaluated by differential

interference contrast (DIC) as one of our controls for the zero-intensity fluorescence images

(I = 0) from oocytes matured in vivo (3 h after IVF). Compare with images (D), (E), and (F)

which were analyzed by differential phase contrast (DPC). ------------------------------------------87

Fig. 26: Confocal microscope images of the fluorescence intensity (I) pattern from oocytes

matured in vivo and in vitro (IVM) (46-48 h of IVM) and incubated in FertTalp medium for 18

h without exposure to spermatozoa. Oocytes matured in vivo (A) and in vitro (B) (IVM)

exhibited high fluorescence labelling at the ZP. Images were recorded by differential phase

contrast (DPC). --------------------------------------------------------------------------------------------------88

Fig. 27: Laser scanning confocal microscope image (LSCM) from 7- day-old blastocyst with

relative spatial distributions of DNA. Images were recorded by differential phase contrast

(DPC). --------------------------------------------------------------------------------------------------------------89

Fig. 28: Scanning electron microscope (SEM) image from an immature (GV) oocyte. The

figure shows the distribution of free thiol groups within the ZP oocytes from immature porcine

oocytes. In general, immature oocytes had free thiol groups distributed in medium-sized

clusters of gold particles (medium, F = 2) over the outer ZP surface. Oocytes were

photographed on the SEM at 2000x magnification. ----------------------------------------------------92

Fig. 29: Scanning electron microscope (SEM) image from an oocyte matured in vitro (IVM)

(MII, 46-48 h of IVM) with complete clusters of gold particles (F = 3) at the periphery of the

outer surface of the ZP, which forms a monolayer beneath the plasma membrane. Oocytes

were photographed on the SEM at 3000x magnification. ---------------------------------------------93


showed the presence of the complete clusters of gold particles (F = 3) at the outer surface of

the ZP. Each oocyte was photographed on the SEM at 1000 x. ------------------------------------94


showing the absence of gold particles (no features, F = 0) on the outer surface of the intact

oocyte (ZP). Oocyte was photographed on the SEM at 1000 x. ------------------------------------95

APPENDIX ___________________________________________________________________

162

Fig. 32: Fluorescence images from oocytes matured in vivo (A) and in vitro (B) (46-48 h of

IVM) 12 h after onset of IVF. There were significantly fewer spermatozoa bound to the ZP

during the fertilization process in oocytes matured in vivo (A) than in those matured 46-48 h

in vitro (IVM) (B). ------------------------------------------------------------------------------------------------97

ACKNOWLEDGMENTS ___________________________________________________________________

163

ACKNOWLEDGMENTS I would like to thank Univ.-Prof. Dr. rer. nat. Dr. med. habil. Edda Toepfer-Petersen,

Institute for Reproductive Medicine, University of Veterinary Medicine, Hannover,

Foundation, for her valuable academic help and for her support during my studies in

Germany.

I thank Prof. Dr. D. Rath, Institute for Animal Science, Mariensee (FAL), – for allowing

me to work in this institute.

I thank Dr. M. Eklasi-Hundrieser, Ms. C. Hettel, Ms. C. Kochel, and Ms. E. Podhajsky,

Institute for Reproductive Medicine, University of Veterinary Medicine, Hannover,

Foundation, and Ms. A. Frenzel and Ms. P. Westermann, Institute for Animal

Science, Mariensee (FAL), for their help in the laboratory.

I thank Prof. Dr. H. Y. Naim, Director of the Department of Physiological Chemistry,

University of Veterinary Medicine, Hannover, Foundation, Prof. Dr. H. Niemann,

Institute of Animal Science, Mariensee (FAL), Prof. Dr. D. Rath, Institute of Animal

Science, Mariensee (FAL), and Prof. Dr. E. Toepfer-Petersen, Institute for

Reproductive Medicine, University of Veterinary Medicine, Hannover, Foundation, for

allowing me to work in Germany in cooperation with German Academic Exchange

Service (DAAD).

I am very grateful to Dr. A. Petrunkina, Institute for Reproductive Medicine, University

of Veterinary Medicine, Hannover, Foundation, for her valuable suggestions and for

the computing of data.

My thanks are also due to Dr. J. W. Carnwath, Institute of Animal Science, Mariensee

(FAL), and Ph. D J. McAlister-Hermann, English Editorial Office (EEO), University of

Veterinary Medicine, Hannover, Foundation for the critical reading of this thesis and

for valuable suggestions. Thank you for everything!

ACKNOWLEDGMENTS ___________________________________________________________________

164

I am very grateful to Ms. H. Schäfer and to Dr. C. Gebert for their special

contributions during my life in Germany.

German Research Council (DFG), German Academic Exchange Service (DAAD),

and CAPES – Brazil are gratefully acknowledge for the financial support.

Finally, special thanks to my father, Mr. S. M. Leal, my mother, Ms. E. M. Leal, and

my both sister, Ms. S. M. Frediani and Ms. I. C. Moreira, for financial support during

my studies in Germany and for their understanding during my long absence from

home – Brazil during my study.

„Die Wissenschaft scheint mich in höchster Art und stärkster Weise die Art der

großen Wahrheit zu lehren.

Lasse Dich vor diesem Faktum nieder und sei vorbereitet, alles vorgefassten

Meinung fallenzulassen, Folge demütig der Spur, wohin auch immer und zu welchen

Abgründen Dich die Natur führt wird oder Du wirst nichts lernen“ (A.C. Moreira,

1975-)

„In dem Wissen wirft man eine gelöste Frage mindestens drei neue auf“. (A. Einstein,

1879-1955)

ERKLÄRUNG ___________________________________________________________________

165

Erklärung Hiermit erkläre ich, dass ich die Dissertation (Biochemical And Structural Alteration

Of The Porcine Zona Pellucida [ZP] During [In Vivo And In Vitro] Maturation And

Fertilization [IVF]) selbständig verfasst habe. Bei der Anfertigung der Dissertation

wurden die folgenden Hilfen Dritter in Anspruch genommen:

Die Durchführung der Rasterelektronischen Mikroskopie (scanning electron

microscopy [SEM]) wurde in Kooperation mit Herrn Dr. P. Schwartz, Zentrum

Anatomie, Georg -August - Universität Göttingen, Germany, durchgeführt.

Ich habe keine entgeltliche Hilfe von Vermittlungs- bzw. Beratungsdiensten

(Promotionsberater oder anderer Personen) in Anspruch genommen. Niemand hat

von mir unmittelbar oder mittelbar entgeltliche Leistungen für Arbeiten erhalten, die

im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen.

Ich habe die Dissertation an folgenden Institutionen angefertigt:

1. Institut für Reproduktionsmedizin in der Stiftung Tierärztliche Hochschule Hannover, Germany

2. Institut für Tierzucht (Mariensee) der Bundesforschungsanstalt für Landwirtschaft (FAL) Braunschweig, Germany;

3. Institut für Physiologische Chemie in der Stiftung Tierärztliche Hochschule Hannover, Germany;

4. Zentrum Anatomie, Georg - August - Universität Göttingen, Germany. Die Dissertation wurde bisher nicht für eine Prüfung oder Promotion oder für einen

ähnlichen Zweck zur Beurteilung eingereicht.

Ich versichere, dass ich die vorstehenden Angaben nach bestem Wissen vollständig

und der Wahrheit entsprechend gemacht habe.

Hannover, den 01 September 2005_______________________________________ (A. C. Moreira, Dr. Med. Vet.)

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