What is known about chemical and mechanical changes during zona hardening of oocytes?

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MSc Chemistry

Track Analytical Sciences

Literature Thesis

What Is Known About Chemical and Mechanical Changes

During Zona Hardening of Oocytes? – A Review

by

Mari-Anne Asseler

UvA #: 12699519, VU #:

2699119

March 2021

12 ECTS-Credits

Period: February 2021 to April 2021

Supervisor/Examiner: Examiner:

Dr. F. Ariese

Dr. B. I. Avci

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Abstract

Mammalian oocytes are surrounded by a transparent layer called the zona pellucida, which consists of either three or four different glycoproteins (ZP1, ZP2, ZP3 and ZP4). The zona pellucida supports the health, nutrition and growth of the oocytes while they prepare to mature. It regulates species-restricted fertilisation of oocytes and helps prevent polyspermy. Polyspermy means that more than one sperm cell fertilises an oocyte. And it protects preimplantation-stage embryos as they move through the fallopian tube on their way to the uterus. Once an oocyte is fertilised, the zona pellucida becomes impenetrable, this process is called zona hardening. There has been a lot of research on the specific events of zona hardening. However, this process is still not fully understood. To understand and to observe zona hardening, different (analytical) techniques are used. This review aims to summarise the different (analytical) techniques to analyse zona hardening. This includes both the chemical and mechanical changes that occur during zona hardening of oocytes. This review first summarises the biochemical processes linked to zona hardening; these include among other things ZP2 cleavage, deglycosylation of ZP3 or other units and zinc sparks. Also, zona hardening without fertilisation is briefly discussed, this can be caused for example due to cryopreservation or oviductal fluids. This is followed by the properties and parameters that describe zona hardening, these include both the chemical and mechanical changes that occur due to zona hardening.

In the second part of this literature review, all the different (analytical) techniques to measure these properties and parameters that describe zona hardening are discussed. The techniques discussed in this review are: mass spectrometry, x-ray crystallography, scanning electron microscopy, transmission electron microscopy, polarised light microscopy, fluorescence microscopy, x-ray fluorescence microscopy, atomic force microscopy, other force measurements (indentation, compression and aspiration), Fourier transform infrared spectroscopy and Raman spectroscopy. In general, zona hardening causes the surface of zona pellucida to become compacter and denser. The β-sheet structures of the glycoproteins increase, which also causes an increase in rigidity. Some specific biological processes can be observed, for instance, zinc sparks. In addition, for bovine and porcine oocytes an increase in disulphide bridges was observed during zona hardening.

Several of the techniques to analyse zona hardening are non-invasive or minimally invasive. This means that it could be used during in vitro fertilisation procedure, and could potentially help to improve the success rate. Especially an aspiration force measurement and Raman measurement showed potential to be used during in vitro fertilisation. However, these techniques first need further research and validation before implementation in clinical use is possible. Additionally, it was recommended to further explore the birefringence parameter with the use of polarised light microscopy, birefringence is an optical parameter as a result of double refraction. Besides, more investigation into the use of Raman spectroscopy to analyse changes in disulphide bridges is recommended, and to investigate the use of multiple non-invasive techniques simultaneously, to help improve the success rate of in vitro fertilisation. Lastly, when analysing oocytes after cryopreservation, it is advised to analyse at least 3 hours post-thaw to avoid measuring a still changing zona pellucida.

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Abbreviations

aa Amino acids

AFM Atomic force microscopy ATR Attenuated total reflection CSFC Consensus furin cleavage site EHP External hydrophobic patch ESI Electrospray ionisation FTIR Fourier transform infrared

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid ICSI Intracytoplasmic sperm injection

IMM Immature

IR Infrared

IVF In vitro fertilisation IVM In vitro matured LC Liquid chromatography

MALDI Matrix-assisted laser desorption ionisation

MII Metaphase II

MPA Micropipette aspiration

MS Mass spectrometry

MTS Micro tactile sensor

OF Oviductal fluid

OVGP1 Oviduct-specific glycoprotein PDMS Polydimethylsiloxane

PLM Polarised light microscopy PTM Posttranslational modification REM Reflection electron microscope SAS1B Sperm Acrosomal SLLP1 Binding

SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis SEM Scanning electron microscopy

SLLP Sperm lysozyme like protein

SP Signal peptide

TEM Transmission electron microscopy TMD Transmembrane like domain XFM X-ray fluorescence microscopy XRC X-ray crystallography

XRF X-ray fluorescence ZP Zona pellucida

ZP-C Zona pellucida C-terminal ZP-N Zona pellucida N-terminal

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1. Introduction

When a mammalian egg cell (oocyte) leaves the ovary during ovulation, the cell has an extracellular coat called zona pellucida (ZP). Zona pellucida consists of glycoproteins and has an important function during fertilisation. It provides sperm recognition, the establishment of the polyspermy block, and some environmental protection.1_{Polyspermy means that more than one sperm cell fertilises an oocyte. When}

one sperm cell penetrates an egg cell (fertilisation) the zona pellucida becomes impenetrable. This process is often referred to as zona hardening. Over the past decades, researchers investigated the different events that occur during zona hardening, this includes both the (bio) chemical and mechanical changes. However, this process is still not fully understood. Various biochemical processes might play a role during zona hardening. To understand these processes and to observe zona hardening researchers utilise different (analytical) techniques. It is important to understand this process since a better understanding of zona hardening and the ability to monitor zona hardening might improve the success rate of in-vitro fertilisation (IVF).

This review aims to summarise the different (analytical) techniques to analyse zona hardening. This includes both the chemical and mechanical changes that occur during zona hardening of oocytes. The first part of this review will discuss the underlying biochemical processes that might influence the zona hardening. The second part will discuss all the (analytical) techniques and their reported results.

This literature thesis will only discuss eutherian mammals. Eutherians are also referred to as placental mammals.2_{Besides eutherians, within the mammals there are two other subclasses, namely}

prototheria and metatheria. Prototherian mammals, also referred to as monotremes, have a very different reproductive system than eutherians since they lay eggs, for example, a platypus.2_{The subclass metatheria,}

also referred to as marsupials, has a different reproductive system as well. They give birth to embryo-like young, the young are very immature and they develop further in their mother’s pouch. For instance, kangaroos and koalas fall under this category. In this review, when referred to mammals or mammalian, it means only the eutherian mammals. Discussed in more details are human, mice, bovine and porcine oocytes.

1.1 Fertilisation an overview

Fertilisation is when a male gamete (sperm cell, also known as spermatozoon), is combined with the female gamete (egg cell/oocyte). Before fertilisation, the oocyte is in the metaphase II (MII) stadium, which is a stadium of meiosis. Meiosis is the process of creating haploid gamete cells.3_{The zona pellucida surrounds}

the oocyte and around the zona pellucida, there is an additional layer called cumulus cells.

Figure 1: Schematic overview of mammalian gametes and the different stages of fertilisation.3

Figure 1 represents the fertilisation process of mammalian gametes. The first contact between the sperm cell and oocyte takes place at the zona pellucida. The sperm recognises the oocyte and multiple

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events cause the activation of acrosomal exocytosis of the sperm. This is called the acrosome reaction; this reaction allows the sperm to penetrate through the zona pellucida. The acrosome-reacted sperm then reaches the egg plasma membrane. It interacts and fuses to this membrane. Following sperm-egg fusion is the exocytosis of the cortical granules, which are membrane-bound organelles that are located along the cortex of the oocyte. Following the release of the cortical granules is a block to prevent polyspermy, these events cause the zona pellucida to become impenetrable. The sperm cell nucleus fuses with the oocyte which enables the fusion of their genetic material and forms a zygote. After fertilisation, the zygote will undergo cell division and will become a blastocyst. This will develop further in an embryo and later in a foetus.

1.2 Scope of this review

This literature review will discuss the different (analytical) techniques to analyse zona hardening. Also, the biochemical processes that are considered to influence zona hardening during fertilisation will be discussed and summarised.

The next chapter will explain the composition, structure and function of zona pellucida, and the biochemical processes associated with zona hardening. This is followed by the properties and parameters describing zona hardening. In chapter 3, the analytical techniques that can analyse the different properties and parameters discussed in chapter 2 will be reviewed and discussed. Chapter 4 will provide an overview of the different (analytical) techniques and their usability, and discuss some reported results in more detail. In addition, the potential use of these techniques to monitor fertilisation and improve the success rate of in-vitro fertilisation will be discussed. The conclusion and further suggestions will close the review.

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2. Zona hardening

This chapter will explain the function, structure and composition of zona pellucida. It will then discuss the biochemical processes that might be responsible for the zona hardening during fertilisation. In addition, zona hardening without fertilisation will be briefly discussed. This is followed by the properties and parameters describing zona hardening, which can be used to analyse and monitor zona hardening.

2.1 Zona pellucida composition, structure and function

As briefly described in chapter 1, the zona pellucida has a crucial role during fertilisation. ZP is a transparent zone surrounding the mammalian eggs. The ZP has several functions with three main stages. First, before the eggs are mature, the zona pellucida supports the health, nutrition and growth of the oocytes as they prepare to mature. Second, it regulates species-restricted fertilisation of eggs by sperm and helps prevent polyspermy. And third, it protects preimplantation-stage embryos as they move through the fallopian tube, on their way to the uterus.1

During oocyte growing, the zona pellucida proteins are synthesised around the egg cell. This happens in the ovary by oocytes and/or their surrounding follicle cells.4_{The synthesised ZP consists of three}

(e.g., mouse) or four (e.g., human) glycoprotein subunits.5_{Namely ZP1, ZP2, ZP3 and ZP4 (zona pellucida}

sperm-binding protein 1-4 is the full name of these abbreviations). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to identify these subunits.4_{These subunits all have a unique}

polypeptide chain, which will be explained in more detail below. ZP1 and ZP4 are not always present in an active form in mammalians, but they can be linked to a pseudogene.6_{A pseudogene is a mutated DNA}

sequence into an inactive form. Researchers categorised mammalian species into three groups: I. Species with a ZP of three ZP subunits with ZP4 pseudogene, at the moment only mouse falls in this category. II. Species with a ZP of three subunits with ZP1 pseudogene, for instance, bovine, dogs and porcine. III. Species with a ZP of four subunits, for instance, humans, rats and hamsters.7_{Sometimes another nomenclature is}

used, especially for porcine and bovine species. The three glycoproteins are called ZPA (ZP2), ZPB (ZP4) and ZPC (ZP3), where ZPA is the largest glycoprotein and ZPC the smallest. The size of the glycoproteins is the basis of this nomenclature.8

Zona pellucida has a different arrangement in different mammal species. Figure 2 represents the arrangement of zona pellucida for a mouse oocyte. The ZP2 and ZP3 form long filaments, and they are covalently cross-linked by ZP1 homodimer. Researchers linked the function of ZP2 and ZP3 to eggshell assembly and regulating sperm binding. The molar ratio between the three glycoproteins in mice oocytes is approximately 1:4:4 (ZP1/ZP2/ZP3).9_{For group III, incorporated in the long filaments is ZP4. ZP4 shares the}

same architecture as ZP1, and the genes are considered paralogous.

Figure 2: Mouse oocyte showing the zona pellucida strands, which are composed of repeating dimers of ZP2 and ZP3 glycoproteins. These strands are cross-linked by ZP1 to form a mesh-like network.10

Mammalians from group II have a different arrangement of the ZP proteins since they lack the ZP1 protein. It is believed that porcine species, for example, have a similar architecture as mouse oocytes, where ZP3 and ZP4 form the long filaments which are cross-linked by ZP2. The estimated molar ratio of porcine ZP is 1:6:6 (ZP2/ZP3/ZP4), which is similar to mice.11_{Bovine species however have a different architecture than}

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Inspecting the unique polypeptide of the different ZP subunits, two important parts can already be identified. Namely the two immunoglobulin‐like domains: the zona pellucida N-terminal (ZP-N) and zona pellucida C-terminal (ZP-C) domain.12_{A linker region connects the two domains.}13_{ZP-N and ZP-C domains}

have been treated in the past as one domain and were called sub-domain or regions of the zona pellucida domain. However, recent research has shown that these are separate domains, and researchers suggested treating them independently or refer to as ZP module.14_{The sequential order is always ZP-N  ZP-C, ZP-N}

can also be present in the absence of ZP-C.15_{ZP-C has been associated as a subunit interaction mediator,}

whereas the ZP-N domain is linked to the polymerisation of the ZP proteins.12,15, 16 _{Figure 3 represents the}

architecture for both the human and mouse zona pellucida and their components.

Figure 3: Scheme of the human and mouse zona pellucida components. Dark grey box is signal peptide, dark green box is ZP-N domain, light green box is ZP-C domain, diamond p is trefoil domain, red box is consensus furin cleavage site, dark yellow box is external hydrophobic patch, brown box is transmembrane like domain, black arrow is Zp2 cleavage site, pink arrow is cross-linking of ZP1, solid inverted tripods experimental carbohydrates, dashed are predicted, blue are O-glycans and black are N-glycans.5

The different components of the polypeptide will be briefly discussed, one by one, from left to right. First, there is a so-called signal peptide (SP) indicated as a dark grey box. This SP is an N-terminal hydrophobic signal peptide to direct it into a secretory pathway.17_{The ZP1, ZP2 and ZP4 can have additional isolated}

copies of the ZP-N domain after the SP. ZP1 and ZP4 have a trefoil (P‐type) domain present before the ZP module starts, indicated with the diamond in the figure. Trefoil domains are thought to be growth factors.17

However, research suggested that interconnection between the different glycoproteins occur via the presence of the trefoil domains in both the ZP1 and ZP4, the specific function of trefoil in the ZP glycoproteins is still unclear.9, 18_{Then the ZP module follows. Attached to the ZP module, all the ZP}

glycoproteins have a consensus furin cleavage site (CFCS), indicated with a red box. The CFCS serves as a processing site for C-terminal cleavage.17_{After the CFCS, there is}_{an external hydrophobic patch (EHP),}

indicated as a dark yellow box. The EHP is involved in the ZP polymerisation.19_{Lastly, on the right there is}

the transmembrane like domain (TMD) located near the C-terminus, TMD is essential for the assembly of the ZP proteins.17

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There are two important positions on the ZP1 and ZP2, paragraph 2.2.1 ZP2 cleavage explains the ZP2 cleavage site, indicated with a black arrow in the figure. The pink arrow indicates the position for the filament cross-linking of the ZP1. The inverted tripods indicate carbohydrates, where the solid inverted

tripods are experimental and the dashed are predicted glycans. The blue-coloured tripods represent O-glycans and black N-glycans. Figure 3 indicates also the number of amino acids (aa) for both the human

and mouse ZP’s.

The total thickness of the ZP varies for different mammals since the size of the oocyte also varies. A mouse oocyte has an average diameter of approximately ∼80 µm and the ZP is between 5–7‐µm.20_{A human}

oocyte is approximately ∼120 µm in diameter on average. The zona pellucida thickness varies from 10 to 31 µm, with a mean of 17.5 µm for human oocytes.17_{Bovine and porcine oocytes have similar dimensions as a}

human oocyte, although the oocytes can be larger (+20 µm). The outer surface of mature oocytes has a spongy appearance, composed of wide (pores) and tight (compact) meshes.

2.2 Biochemical processes of zona hardening

Zona hardening means that the zona pellucida becomes impenetrable, this includes the block of polyspermy. Over the past decades, there have been several biochemical processes linked to zona hardening. However, still a lot is unknown. It remains unclear whether one process leads to zona hardening or if several processes are involved. The most important biochemical processes considered responsible for zona hardening are i. ZP2 cleavage, ii. deglycosylation of ZP3 or other ZP subunit, iii. zinc sparks. In the coming paragraphs, this review will discuss these three processes that are believed to be involved in zona hardening. In addition, it will discuss some other processes and zona hardening without fertilisation.

2.2.1 ZP2 cleavage

Already 40 years ago experiments performed by Bleil et al. showed that there is a difference in ZP2 before and after fertilisation.21_{They observed a difference when analysing the ZP2 under reducing}

conditions with SDS-PAGE; under non-reducing conditions they observed no difference. The measured change was that ZP2 from embryos migrated earlier with an apparent molecular weight of 90.000 instead of 120.000 Da. Researchers named this new band ZP2f,since it is the fertilised form. These results suggest a

modification of ZP2 following fertilisation. Because of intramolecular disulphide bonds, this change can only be analysed when the disulphide bonds are broken.

The metalloprotease that mediates proteolytic cleavage of ZP2 is ovastacin.22_{Ovastacin is present}

in cortical granules and is also referred to as SAS1B (Sperm Acrosomal SLLP1 Binding, SLLP is sperm lysozyme like protein).23_{As mentioned in chapter 1, cortical granules release their content after sperm binding.}

However, they also release small amounts of ovastacin before fertilisation, which could cause pre-hardening of the ZP before fertilisation. The serum protein fetuin-B, which is produced in the liver and present in the circulation and follicular fluid, is believed to act as an inhibitor for ovastacin, and thus prevents early cleavage events.24_{The amount of ovastacin released during cortical granule exocytosis is too much for the}

fetuin-B to inhibit and then ZP2 cleavage takes place. ZP2 cleavage affects sperm binding by either directly affecting an interaction site with sperm or changes in the global structure of ZP. The cleavage of ZP2 is not an immediate reaction after fertilisation. Several reports have also indicated that different species have a different time frame for the ZP2 cleavage to occur. This time frame ranges from a few minutes to several hours.22, 25_{Even though ZP2 cleavage can be a slow-acting process, the cleavage of ZP2 provide a definitive}

and irreversible block to polyspermy and is thus an important step in zona hardening.26_{However, it is not}

the only process reported to be responsible for zona hardening.

2.2.2 Deglycosylation of ZP3 or other ZP subunits

All the ZP subunits are highly glycosylated, several studies have suggested the role of ZP glycans in egg-sperm interaction and zona hardening. Over the years the details of this involvement have changed significantly. One aspect to take into consideration is that there might be a difference in glycosylation and function of the glycans between different mammals.5_{To provide a definitive answer, more research is}

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The first suggestion of deglycosylation affecting zona hardening was between 1980 and 1990. Researchers suggested that ZP3 acts as a primary sperm receptor by presenting one or more functionally active O‐glycan chains to sperm. Following fertilisation, a yet‐to‐be‐identified cortical granules glycosidase then modifies these O‐linked carbohydrates to abolish the glycoproteins activity.21_{They suggested that the}

deglycosylation of the O-glycans on the ZP3 leads to the conversion of ZP3 to ZP3f.27 Around 1991, this did

not fit all the results.28_{Other studies suggested that N-glycans on ZP3 instead of O-glycans mediates as a}

sperm receptor.29_{Even without the presence of either O-glycans or N-glycans an oocyte was still fertile,}

which contradicts the previous results that either O- or N-glycans are the sole sperm receptors.30,31_Later

research suggested that specifically O-linked glycans that are attached at residues Ser332 and Ser334 of ZP3 were essential for sperm-binding and thus also the block of sperm binding.32_{However, when these specific}

glycans were not present, the oocyte was still fertile.

Recently, research groups suggested four models that incorporate ZP2 cleavage and deglycosylation of the ZP.6,33_{These models all suggest a different way why sperm recognition can no longer occur after}

fertilisation and thus initiate zona hardening. Figure 4 represents the different models, where model 1 is the glycan model, model 2 is the supramolecular structure model, model 3 is the hybrid model, and last model 4 is the domain-specific model.

Figure 4: Model for sperm binding taken from the book binding protein. Model 1= The Glycan Model, model 2= supramolecular structure model, model 3= hybrid model and model 4 = domain-specific model.34

Model 1, the glycan model, proposed that sperm binds via O-linked glycans attached at residues Ser332 and Ser334 of ZP3. After fertilisation, these residues are deglycosylated and prevent further sperm adhesion. This was the first suggested idea.34_{However, research had contradicted this model. Model 2 is}

the supramolecular structure model. This model is based on the physical structure of the ZP matrix, which was suggested to be critical for sperm binding. Following fertilisation, ZP2 is processed in such a way that it prevents further sperm adhesion.34_{This model does not depend on specific glycans, but on the physical}

structure of the matrix for sperm recognition. Model 3, the hybrid model, incorporates both model 1 and model 2, and proposes that O-linked glycosylation is a critical determinant of sperm recognition. However, these O-glycans are present on residues other than Ser332 and Ser334. The modification of ZP2 that accompanies fertilisation renders these O-glycans inaccessible to sperm.34_{The last model proposed, the}

domain-specific model, suggests that sperm can bind to a variety of N-linked glycans attached to ZP3 or attached to the peptide backbone of the glycoprotein, depending upon its glycosylation status.34

Deglycosylation of these N-glycans makes sperm binding no longer possible.

Based on the research available, it cannot be ruled out that ZP protein glycosylation plays a role in the interaction between sperm and oocyte and, consequently, the block to polyspermy and zona hardening. The interactions at the molecular level and whether ZP3 is the only sperm receptor remains unclear.6

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2.2.3 Zinc sparks

More recently, research suggested zinc sparks play a role during zona hardening.35,36_{A few minutes}

after a sperm has penetrated the zona pellucida, the oocyte releases about 12 billion zinc atoms in the first 90 minutes of activation.35_{Zinc is present in the cortical granules, where a portion of the zinc is bound to}

ovastacin, the rest of the zinc is believed to reside in the interstices between ovastacin molecules.3_Studies

have shown that increasing the zinc concentration partially or directly inhibits sperm penetration through the zona pellucida.3_{Research proposed two possibilities of zinc interactions with zona pellucida. The first}

possibility is that zinc perturbs the zona structure density, and as a result, the zona may no longer be able to support sperm binding. Que et al. suggested this idea; they reported that ZP3 could have the ability to act as zinc-binding moieties. Upon fertilisation, zinc-mediated crosslinking occurs, which leads to changes in the ZP structure.35_{Alternatively, zinc may prevent sperm penetration by affecting sperm motility of the bound}

and/or penetrating sperm during interaction with the egg.36_{Researchers suggested that these zinc sparks}

decrease sperm penetration before the cortical granule’s reaction initiates, and thus during the time to cleave ZP2. This event of zinc spark has been represented by Tokuhiro and Dean for mouse oocytes, see Figure 5.36

Figure 5: Zona block to polyspermy proposed by Tokuhiro and Dean for mouse oocytes. A glycan-independent sperm binding to the N terminus of ZP2 in the zona pellucida surrounding eggs. Following fertilisation, sperm penetration of the zona is transiently blocked by zinc exocytose from egg cortical granules. This provides a temporal window for ZP2 cleavage by ovastacin (lower graph) that prevents sperm binding and ensures monospermy.36

The model represented in Figure 5 also suggests that there are no glycans needed for sperm recognition, only the ZP2 N terminus is necessary for sperm recognition. Directly after fertilisation, the zinc spark occurs and provides a block for sperm penetration before ZP2 cleavage.

2.2.4 Other factors associated with ZP hardening

Besides the three mentioned processes, research reported many other processes that were linked to zona hardening as well. One of these other processes has to do with the Juno receptors. Juno receptors are located on the oolemma. Bianchi et al. identified Juno as the egg receptors for the sperm protein Izumo.37_{Izumo is a protein displayed on the surface of acrosome-reacted sperm. Bianchi has shown that}

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fertilisation suggested a mechanism for the membrane block to polyspermy. This block is quick compared to ZP2 cleavage. Research had shown that female mice lacking this Juno receptor are infertile.37_Later

research showed that humans with a certain sequence variant of Juno-encoding gene also had been related to unexplained fertilisation failure.38_{They suggested Juno receptors prevent polyspermy by regulating}

sperm-oolemma interactions. They play a crucial role in the block of polyspermy, which is part of zona hardening.

Several studies reported cross-linking during zona hardening. They suggested lectins released upon cortical granules exocytosis can potentially cross-link zona pellucida proteins non-covalently. Lectins had already been identified as a major compound of vertebrate cortical granule (e.g. fish and frogs). And there their function is to block polyspermy.39_{Some research indicated that lectins might play a role in mouse}

oocyte zona hardening. Incubated oocytes with lectins showed they inhibit fertilisation by blocking sperm binding in mouse, hamster, pig and rabbit.28_{More than a dozen different lectins have been used to}

cytochemically demonstrate granules in unfertilised oocyte and/or to localise cortical granule exudates following fertilisation.40

Research showed an increase in disulphide bonds after fertilisation in bovine and porcine oocytes ZP. 4142_{The biochemical process behind the increased disulphide bonds remains unsolved. In addition, this}

event has not been reported for other mammalian species yet.

The mammalian cortical granules contain between four and fourteen different proteins. However, the exact number is unknown.40_{Some of these proteins are hydrolase, proteinase and ovoperoxidase.}

Studies suggest that these enzymes also play a role in preventing the binding and penetration of sperm after fertilisation by modifying the ZP glycoproteins. However, the mechanistic details are unknown.35

To conclude these biochemical processes that occur during fertilisation, it is likely that zona hardening does not depend on one event, but rather on multiple events, which can also happen independently. Several studies have indicated evidence of different binding sides. It remains unclear whether fertilisation depends on carbohydrate-carbohydrate interactions, carbohydrate-protein interactions, protein-protein interactions, or some combination of all three mechanisms.43_{An advantage of}

these multiple/complex adhesion systems is that they enhance the opportunities of sperm to bind to the oocytes and maximise the change of fertilisation. However, this also complicates the events of zona hardening, and several processes remain to be discovered.

2.3 Zona hardening without fertilisation

Several researchers have reported zona hardening without fertilisation. One event that is frequently reported is the cryopreservation-induced zona hardening.44, 45_{Cryopreservation is the process of preserving}

cells at an extremely low temperature, there are two types of cryopreservation, namely slow freezing and flash freezing. Flash freezing is also referred to as vitrification and happens in a matter of minutes compared to hours for slow cooling.45_{Cryopreservation has several challenges and undesirable effects on IVF. One of}

them is the decreased ability of sperm penetration, this may be because of a structural change in the zona pellucida which results in zona hardening. Studies demonstrated that vitrification alters the ZP glycoprotein matrix. These alterations might occur because of premature cortical granule exocytosis. This effect has been reported in both bovine and human oocytes.46_{To overcome this problem, research proposed}

intracytoplasmic sperm injection (ICSI) as an elective route to achieve fertilisation in IVF. In ICSI a single sperm cell is directly injected into the cytoplasm of an oocyte.43_{Also, research showed that a calcium-free}

vitrification medium reduced the zona hardening during vitrification.47

Next to cryopreservation-induced zona hardening, studies reported zona hardening pre-fertilisation by oviductal modulators for different mammalian species. Oviductal fluid (OF) induces this specific zona hardening.48_{Researchers reported this effect first for bovine and porcine; later research has shown that this}

effect also occurs in mouse, rat, hamster, rabbit, sheep and goat oocytes, however, it does not occur in humans.49_{These oviductal modulators bind to the zona pellucida and affect its biological function of}

unfertilised oocytes.48_{At least two components of the OF have been linked to affecting the zona pellucida,}

these components are oviduct-specific glycoprotein (OVGP1) and heparin-like glycosaminoglycans.50_The

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if these two components of the OF are the only responsible factors. In rat oocyte, for example, pre-zona hardening by oviductal fluid also occurs even though it does not contain OVGP1.50

In addition, which was already mentioned under 2.2.1 ZP2 cleavage, fetuin-B is an important plasma protein to avoid fertilisation hardening. Genetic ablation of fetuin-B in mouse oocytes caused pre-fertilisation hardening, and studies performed with oocytes without the fetuin-B were not fertile.51, 52_{It is}

therefore crucial for IVF to keep the oocytes in a special medium before fertilisation, which contains fetuin-B, but also serum and other compounds, to improve the fertilisation rates. Other medium characteristics such as redox potential, osmolality and pH may affect zona hardening.53

2.4. Properties and parameters describing zona hardening

Over the past decades, researchers linked many different biochemical processes to zona hardening, which are all discussed in the previous paragraphs. These biochemical processes cause several parameters and properties of zona pellucida to change. These changes can be analysed and monitored to describe zona hardening. First, it is important to understand the chemical composition of zona pellucida.

Understanding the mechanical properties of zona pellucida and the mechanical property differences of ZP before, during and after fertilisation can help understand the process of zona hardening. Research groups used mechanical properties more frequently than chemical properties to measure zona hardening. An oocyte is like most cells often described as a viscoelastic material. Since it can exhibit both liquid-like and solid-like mechanical properties.53_{Frequently analysed mechanical parameters during zona hardening are}

viscoelastic properties, especially the stiffness which is expressed in the Young’s modulus. Viscoelastic properties can be analysed by applying a force to the oocyte. Researchers achieved this with the use of atomic force microscopy, but also with several other techniques.

Besides mechanical properties, also quite some research has been conducted on morphological parameters of zona pellucida. For instance, the thickness of the zona pellucida but also the irregularities of the surface, for example, the porosity and density of the glycoproteins. Research groups analysed most morphological parameters with the use of microscopy. Additionally, an interesting optical property is birefringence, which can describe zona hardening. Next to morphological parameters also the architectural characteristics of the zona pellucida are important to understand, for example, the three-dimensional structure of the glycoproteins and their different domains. Changes in zona pellucida architecture are accompanied by changes in the secondary structures of the zona pellucida proteins. During zona hardening, there is an increase in β-sheet structures content. Researchers analysed the secondary structures of proteins with the use of vibrational spectroscopy. An increase in β-sheet formation also causes an increase in rigidity.54

For a long time, studies described the hardness of zona pellucida according to enzymatic digestion time. Additionally, some biochemical processes have been monitored, for example, the zinc sparks, OVGP1 and ovastacin. Also, the disulphide bridges could be analysed before and after fertilisation.

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3. Techniques

This chapter discusses several (analytical) techniques that can analyse and monitor one or several properties and parameters mentioned in paragraph 2.4.

3.1 Mass spectrometry

Studies have been performed on the composition of the zona pellucida proteins in mammals. The results of these studies have already been described in chapter 2. A technique that has contributed to these studies is mass spectrometry (MS). MS is a technique used to measure the mass-to-charge ratio of ions. MS is also the main technique used, in combination with a variety of separation methods, in proteomics. There are two approaches of proteomics, namely top-down and bottom-up.55_{In top-down proteomics proteins}

are analysed intact. And by bottom-up proteomics peptides are analysed by proteolytic digestion.56_Both

approaches provide insight into the amino acid sequence of the zona pellucida glycoproteins and posttranslational modifications (PTM), such as glycosylation.

The variety of separation methods that can be used are SDS-PAGE, liquid chromatography (LC), two dimensional LC, two dimensional SDS-PAGE or a combination of SDS-PAGE and LC. As ionisation method, either electrospray ionisation (ESI) or matrix-assisted laser desorption ionisation (MALDI) are commonly used for the analysis of zona pellucida. Also, tandem MS is frequently used in combination with one of the separation methods. Many combinations have been used successfully.57, 58, 59_{All these different methods}

will not be discussed in detail. MS provides information about the composition of zona pellucida and can also provide information on changes in composition. Unfortunately, it is a destructive analysis, and it requires a time-consuming sample preparation and separation method.

3.2 X-ray crystallography

X-ray crystallography (XRC) is a technique that uses x-radiation to determine the three-dimensional structure of proteins. A protein needs to be crystallised prior to analysis. The specific structure of the crystal causes the beam of x-rays to diffract in a specific matter, these scattered beams form a diffraction pattern. Rotating the crystallised protein causes the angle and intensities of these diffracted beams to change, and a three-dimensional image of the electron density can be observed. Based on the electron density, the positions of the atoms and their chemical bonds within the crystal can be determined, which results in a three-dimensional molecular structure of the protein.60

Several studies have used XRC successfully for the analysis of zona pellucida. These studies have focused specifically on measuring the different structures of ZP-N and ZP-C domains on ZP2 and ZP3. Through these studies it is known that these domains have an immunoglobulin‐like structure.12,13, 61_There

has been little research so far in the structures of ZP1 or ZP4. This is because the crystallisation of mammalian ZP proteins is very difficult since they are highly glycosylated, and glycosylation hinders protein crystallisation.6_{The crystallisation of proteins is mainly a trial-and-error procedure in which the protein is}

slowly precipitated from its solution. First, the protein that needs to be analysed should be expressed and purified. With the use of MS several aspects can be checked for the crystallised proteins, for example, the expression of recombinant protein, the purity and heavy-atom derivatives.60

It is not possible to use XRC and measure a whole oocyte since the proteins need to be crystallised. This crystallisation process is very complex and time-consuming. When a protein crystal is formed successfully, it provides three-dimensional structure information. XRC does not directly provide information about zona hardening, but indirectly it can provide useful information about the three-dimensional structures of the glycoproteins to better understand the different zona pellucida proteins.

3.3 Enzymatic digestion

The hardness of the zona pellucida can be defined as its resistance to enzymatic digestion. This resistance can be quantified in an index for ZP hardness. This index represents the duration of time to complete digestion. To monitor the digestion, researchers often used a simple microscopy at x400 (or x200) magnification. As soon as the ZP is no longer visible, the digestion time is reached. The digestion is performed

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on a heated plate, to maintain the physiological temperature. Most often α-chymotrypsin is used for this enzymatic digestion. Other solutions have been used as well, for example, a pronase solution or protease solution.62,63_{Over the years, different concentrations of α-chymotrypsin have been utilised, ranging from}

0.2 mg/ml till 3 mg/ml α-chymotrypsin.64, 65_{What should be noted is that researchers used higher}

concentrations when digesting 10 to 20 oocytes simultaneously. One major disadvantage of this analysis is that it is destructive. Several researchers have indicated that the time to digest the zona pellucida increases after fertilisation, which was expected.

Unfortunately, enzymatic activity and environmental factors, such as pH and temperature could influence the digestion time, this limits the precision of this analysis.66_{This analysis has not been used}

frequently over the past decade since other non-destructive alternatives have become available.

3.4 Microscopy

There are many types of microscopes developed over the years. The main principle of microscopy is to view samples and objects that cannot be seen with the unaided eye. Microscopes can be separated into different groups. This review uses one classification, which is based on the interactions with the sample to generate an image. These groups are I. optical microscopy, where light is used II. electron microscopy, where electrons are used III. x-ray microscopy, where x-ray photons are used. And the last group IV.

scanning probe microscopy, where specific probes are used, for instance, in atomic force microscopy. The

microscopy techniques that will be discussed per group, from group I. both scanning and transmission electron microscopy. From group II. polarised and fluorescence microscopy. From group III. x-ray fluorescence microscopy and from the last group only atomic force spectroscopy. These different microscopy techniques will be discussed one by one and their contribution to analyse zona hardening will be discussed.

3.4.1 Electron microscopy

Electron microscopy uses electrons as a source of illumination to generate an image. There are several types of electron microscopy. The most common types are scanning electron microscope (SEM) and transmission electron microscope (TEM). The wavelength of an electron is much smaller than that of photons, which results in a higher resolution than optical microscopy.67_{Both SEM and TEM have been}

frequently used for the analysis of zona pellucida. Both will be explained in the following paragraphs.

3.4.1A Scanning electron microscopy

Scanning electron microscopy produces images of a sample by scanning the surface with a focused beam of electrons. The electrons that bounce back from the surface are used to generate an image. SEM can analyse zona pellucida in three ways. Either by analysing the whole oocyte intact or by stripping the zona pellucida from the oolemma or by analysing only a cross-section of the ZP. Prior to analysis, the oocyte undergoes a sample preparation procedure for chemical fixation and dehydration. Different sample preparation procedures have been used over the past decades, but the main step in the sample preparation procedure was dehydration of the oocytes after they had been washed with different solutions (buffers). After the dehydration, they were dried at a critical point and placed in a specimen holder and then coated with a heavy metal. Dehydration of the oocytes was mostly performed with ethanol.67_{Sample preparation}

of SEM is time-consuming and invasive for the oocytes, thus living cell imaging is not possible.

With SEM, the outer surface structure of the zona pellucida can be analysed. Also the pore size has been measured with the use of SEM and sperm binding patterns could be analysed. Magerkurth et al. showed that there were four different surface structures to be distinguished in human ZP, these four different surface structures are represented in Figure 6 on the next page.68_{Types A and B for the more}

porous and types C and D for the compact and smooth surfaces. Type C and D were more common for fertilised oocytes, and type A and B were more common for unfertilised oocytes. These results were also obtained by other research groups on other mammal species. They distinguished also a more porous structure for unfertilised oocytes vs a more compact and smooth structure for fertilised oocytes.69, 70, 71

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Figure 6: SEM micrographs of four different types of surface strucutres of human zona pellucida, types to distinguish were type A, B, C and D.68

However, it should be noted that these analyses might not reflect the in-vivo state because of dehydration during sample preparation. Several papers also assumed that there was a correlation between the type of surface morphology and the stage of maturity.72, 73_{However, there are also some papers that}

did not find any correlation between the zona pellucida surface and maturity of the oocytes.73_{Maybe there}

was no correlation found due to the fact that the oocytes undergo different sample preparation procedures. During sample preparation, fixation and/or dehydration can cause differences since many variable conditions are used. When cells are hydrated in their native state, hydrophilic surfaces of macromolecules are free to interact with water. When water is removed, these surfaces rearrange and may attract each other, forming aggregates. The only way to find out is to use a different technique that can analyse the ZP in a hydrated environment. Such an analysis might also confirm if the found structure represents the in-vivo state.73_{Another disadvantage of SEM is that the signal obtained comes from the heavy metals and not from}

the zona pellucida itself. The signal depends on how far the stain penetrates, and differences in density can occur.

Recently a solution to overcome some disadvantages has been proposed to use on oocytes, namely cryo-electron microscopy (cryo-EM). In Cryo-EM the sample state is as close to reality as possible. It is in a fully hydrated state, free of chemical fixatives or stains.67_{In cryo-EM the oocyte is rapidly vitrified, the}

analyses take place of frozen-hydrated specimens at a temperature so low that water does not evaporate. In order to achieve uniform vitrification, oocytes are frozen under high pressure.73_{Cryo-EM shows promising}

results but has not been utilised much yet. However, it can analyse an oocyte in its near-native state, without the use of chemical fixatives or stains which is a main advantage compared to regular SEM. The contrast of the image with cryo-EM is lower than regular SEM, since the contrast is from natural biological material and not from heavy metal stains, this results in a limited resolution.74

3.4.1B Transmission electron microscopy

Transmission electron microscopy is different from SEM, since the beam of electrons is transmitted through the sample. The detector is located on the opposite side of the sample than the illumination source.67_{Since the electrons need to penetrate through the sample in order to generate an image, TEM has}

a thickness limitation. 1 µm is often used as the maximum thickness for the sample. Therefore, to analyse zona pellucida, a section from the ZP needs to be cut. This can be done with a diamond knife on an ultramicrotome. TEM is often used besides SEM to determine the internal structure instead of the surface.75

It can also be used to determine filament dimensions. For human oocytes, Familiari et al. determined that the length of the filaments is between 0.1-0.4 μm with a thickness of 10-14 nm.72_{The major disadvantage}

of TEM is that it requires a very thin sample, which also makes the analysis invasive.

3.4.2 Optical microscopy

In optical microscopy, light ranging from the ultraviolet to the near-infrared region is used as an emission source. There are many different optical microscopes developed over the years. Two types of optical microscopy provide useful information in measuring and monitoring zona hardening. These are polarised light microscopy and fluorescence microscopy. They will both be discussed in more detail in the following paragraphs.

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3.4.2A Polarised light microscopy

Normally light is unpolarised, this means that the plane of the electric field vector is randomly distributed. Polarised light can be produced by passing unpolarised light through an optical filter, this optical filter is also known as a polariser. There are different types of polarised light. For example, plane or linearly polarised light, then the electric field vector is fixed. But also circular polarisation, then the plane of the electric field vector rotates about its propagation direction.76_{The type of polarised light used for the coming}

polarised light microscopy (PLM) experiments is circular polarised light. One interesting parameter to analyse with PLM is birefringence. Birefringence is an optical property, it is also called double refraction.77

Birefringence is the decomposition of a ray of light into two rays when it passes through a certain material with birefringence properties.77

There are two birefringent structures in oocytes: the spindle and the inner layer of the zona pellucida.45

Meiotic spindle is formed within the oocyte in the final stages of maturation, but will not be further discussed. The matrix of zona pellucida is composed of filaments organised in layers in differing orientation.78_{PLM analyses the density (measured as retardance, unit is nm) and thickness of individual}

zona layers. The inner layers of ZP have a higher degree of birefringence than the outer layers. Additionally, within the zona pellucida, the retardance and thickness are variable.62_{This variation is also shown in Figure}

7, here two different mature human oocytes are analysed. Where one has a very low birefringence and the other a high degree but considerable variations around one individual oocyte are observed as well.

Figure 7: Mature human oocytes showing different degrees of ZP inner layer birefringence, left has a low degree of birefringence and right has a high degree of birefringence. The light blue dot around 4 and 5 o’clock is the meiotic spindle, 400x magnification. 79

The analysis is performed in a Petri dish on a heating plate, to maintain the physiological temperature. The oocytes are placed in a handling medium which is covered with mineral oil to prevent medium evaporation. Most frequently used as handling medium is G-MOPS or a HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid). Maintaining the temperature and using a specific handling medium allows this technique to be non-invasive and allows living cell imaging.

Iwayama et al. researched with the use of polarised light microscopy if a non-invasive birefringence

parameter could be used instead of the enzymatic digestion to determine the hardness of the ZP.62_They

proposed to use retardance and thickness as an indicator for ZP hardness. Their outcome was that there was a positive correlation between retardance and thickness compared to the time required for full enzymatic digestion. However, Gu et al. investigated the birefringence of the ZP in human embryos before and after cryopreservation.66_{They reported that there was no significant difference in retardance and}

thickness of the zona pellucida structure between the two groups. Thus there is no significant change in the zona pellucida birefringence of human embryos before and after cryopreservation. Montag et al. performed a similar experiment on human unfertilised oocytes. 45, 80_{They reported a significant change in birefringence}

directly after cryopreservation, however, 3.5 hours after thawing it returned close to its initial value before freezing. They concluded that the time of the measurement after thawing is crucial and affects the results.

There has not been any report of measuring the birefringence during fertilisation to monitor the zona hardening. Pelletier et al. did research differences in retardance and thickness between human immature, mature oocytes and 3 days old embryos after ICSI. They reported that the embryos had a

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significantly thinner zona pellucida than both immature and mature oocytes.81_{The ZP of embryos was}

thinner primarily owing to thinning of the outer ZP layer.

Birefringence has often been performed on human, mouse and hamster oocytes. Only in the past decade there has been an increase in studies that use PLM for the analysis of farm animals such as bovine and porcine species.82, 83, 84, 85 _{Despite that the technique has considerable variations when analysing one}

oocyte, it might be able to measure differences in thickness and retardance during zona hardening. However, it remains to be discovered if this change is significant to be measured. A major advantage of this technique is that it does not require special equipment or instruments that are technically difficult to perform. It is also a very fast analysis.

3.4.2B Fluorescence microscopy

In fluorescence spectroscopy, a photon is absorbed by a molecule which is then excited to a higher energy state. Then the excited molecule relaxes to a lower energy state by emission of a photon. The release of a fluorescence photon causes the molecule to return to its ground state. Fluorescence is only possible when the energy of the photon is equal or greater than the difference between the ground state (S0) and

the first excited state (S1) of a molecule. Fluorescence microscopy uses fluorescence to generate an image.

This means that the object of interest needs to have fluorescence properties or needs to be labelled with a fluorescence label. Two frequently used geometries of fluorescence microscopy are epifluorescence wide field and confocal fluorescence microscopy. In epifluorescence wide field the excitation of the fluorophore and the detection of the fluorescence is done through the same light path, which causes emission fluorescence from both inside and outside the focal plane. This means that also out-of-focus fluorescence is measured, this contributes to detail obscurity and limits the depth that can be imaged.86_{In confocal}

fluorescence, both the excitation and emission optics are focused on one spot. Out-of-focus light is blocked, which makes confocal microscopy to have a higher spatial resolution in depth.

To measure zona pellucida with fluorescence microscopy, fluorescent probes are needed. Table 1 summarises some fluorescence probes that have been used. As an illumination source, a laser has been used with the wavelength stated by λ excitation in the table. A general rule for biological sample is that probes with long-wavelength excitation are less harmful to living cells and organisms compared to short-wavelength. When the oocytes needed to be preserved, the analysis was performed on a heated plate and in a specific handling medium, to maintain physiological conditions. Similar to PLM, a mineral oil was used to prevent medium evaporation.

Table 1: Overview of fluorescent probes with excitation and emission wavelengths(λ).

Fluorescent probe What is measured λ excitation λ emission FluoZin-3 AM Zinc 488 nm 515 nm

ZincBY-1 Zinc 520 nm 543 nm

Fura-2-AM Calcium 405 nm 510 nm

Fluo-4-AM Calcium 488 nm 525 nm

Monobromobimane Thiols 405 nm 483 nm

Intracellular labile zinc can be analysed with the use of fluoZin-3 AM probe or ZincBY-1. They have both been used successfully to measure the zinc in oocytes.36,87,88,89,90_{Intracellular labile zinc means that}

only the zinc that is loosely bound or free will be analysed. To measure zinc that is tightly bound to proteins or membrane as well, x-ray fluorescence microscopy needs to be used, which will be explained in the next paragraph. Next to zinc, also calcium can be interesting to analyse, since many biological processes are calcium-dependent, for example, the release of the cortical granules. For calcium two different probes have been used: Fura-2-AM and Fluo-4-AM.87,89_{Measuring calcium and zinc can be performed on living oocytes,}

which makes it also possible to monitor these before, during and after fertilisation.

Monobromobimane has been used by Iwamoto et al. to measure the presence of free thiol groups in bovine oocytes.41_{They showed that there was a significant difference between fertilised oocytes and}

unfertilised oocytes; the unfertilised oocytes had a significantly greater coupling to this probe than fertilised oocytes. This indicates, together with other experiments, an increase in disulphide bridges during zona

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hardening. Iwamoto et al. isolated the zona pellucida, by removing the ooplasm from the oocyte, prior to analyses, which makes this specific analysis invasive.

Besides the probes mentioned in Table 1, also more specific dyes have been used for immunofluorescence microscopy. In immunofluorescence, antibodies are used to detect specific proteins within a sample. There are two types of immunofluorescence, namely direct and indirect. In the direct approach, the fluorescence dye molecules are chemically conjugated to the antibodies. In the indirect approach first a primary antibody is bound to the target and then a secondary antibody, which is chemically conjugated, is used for detection. With the use of immunostaining, the different glycoproteins can be stained separately, to observe the distribution of the different glycoproteins. This, however, does not provide information about zona hardening. Next to the glycoproteins also other proteins have been analysed with immunofluorescence microscopy, for example, ovastacin. Ovastacin has been measured indirect in mice oocytes, to determine the cellular distribution.65_{Since ovastacin mediates ZP2 cleavage, this is an}

important parameter for zona hardening. Next to ovastacin also OVGP1 has been analysed with immunofluorescence in porcine oocytes. Since OVGP1 has been linked to pre-fertilisation zona hardening, analysing this protein can also provide some insight in zona hardening.63_{Many more immunofluorescence}

microscopy experiments have been performed on oocytes. A disadvantage of immunofluorescence microscopy is that it depends on the quality and concentration of the antibodies used. Also, most immunofluorescence microscopy experiments fixated the oocyte prior to staining, which makes living cell imaging not possible.

3.4.3 X-ray fluorescence microscopy

X-ray fluorescence microscopy (XFM) is a combination of x-ray fluorescence (XRF) with microscopy. It can be used to both quantify and visualise the metal content of biological samples, since the number of XRF photons are directly related to the quantity of an element. With XFM the distribution of elements can be mapped at micro- and nanoscale, to determine the subcellular distribution in cells.91_{Elements can be}

distinguished by the characteristic x-ray transitions associated with their core electron energy levels. With XFM, the metal contents have been analysed either in intact oocytes or in isolated zona pellucida.90_{For most}

XFM experiments they fixated and or dehydrated the oocyte prior to the analysis, also; some studies embedded the oocyte in resins after sample preparation. Embedding makes preserving the sample for long periods of time possible. The sample preparation of XFM makes the analysis invasive. Mostly zinc is analysed with the use of XFM in oocytes but also iron and copper.35,89, 90, 91_{However, zinc is the most interesting for}

analysing zona hardening. The zinc content is also an order of magnitude higher in oocytes than Fe and Cu. XFM can determine the total concentration of zinc present in the oocyte compared to only labile zinc with fluorescence microscopy. Advantages of XFM are that it has a low background, high sensitivity, selective excitation and no dyes are needed. A disadvantage is that x-ray radiation can alter and/or change an oocyte negatively.92

3.4.4 Atomic force microscopy

Atomic force microscopy (AFM) is also referred to as scanning force microscopy. The basic set-up of AFM is a sharp needle tip attached to a cantilever, this tip can move across the surface of the material that is being analysed. On the cantilever, a laser is reflected towards a quadrant photodiode. Differences in the direction of these reflections can be translated into information such as topographic imaging. AFM can be used for different analyses: it can provide insight into the morphology by measuring topographic imaging, but also mechanical characterisation by force measurements can be conducted.93_{Especially the}

determination of mechanical properties is interesting to understand zona hardening. The basic principle for AFM force measurements is to indent the probe into the sample and record the applied force, which is proportional to the cantilever deflection, and the distance travelled by the probe in a force-distance curve. From such force-distance curves the apparent stiffness can be calculated, and the Young’s modulus. To extract the mechanical properties from the AFM data, different models have been proposed. Some models that have been used are: modified biomembrane point-load model, modified Hertz model, half-space model, layered model and shell model.93, 94_{These mathematical models will not be discussed.}

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Papi et al. analysed bovine oocytes during maturation and fertilisation with AFM.73, 95_{They separated}

the zona pellucida by removing the ooplasm from the oocyte. They analysed both the ZP with the outer surface upside, but also with the inner surface upside. Prior to analysis, the zona pellucida was immobilised. They found that the stiffness decreased during maturation and after fertilisation increased. The differences between the inner and outer surfaces were minor.73,95_{Giolo et al. analysed human oocytes and the effect}

of cryopreservation with AFM. 96_{Human oocytes were evaluated immediately after retrieval with the use of}

AFM and then vitrified and stored in nitrogen. Afterwards, the same oocytes were thawed and analysed again. Their results indicated that healthy oocytes preserve their morphological features after cryopreservation and maintain their stiffness. However, oocytes with dysmorphic characteristics (irregular ZP or cell rupture or cytoplasmic anomalies) showed significant variations in their mechanical response.96

A disadvantage of AFM is that the analysis is time-consuming. Also, any movement of the sample must be avoided during an AFM measurement, which means that the zona pellucida must be properly immobilised to the substrate. However, cell immobilisation could influence the mechanical response and should be a boundary condition in the theoretical models.97

3.5 Other force measurement methods

Other devices have been developed besides AFM to measure the viscoelastic properties of cells. These techniques can be categorised into three groups: indentation, compression or aspiration. Several examples are shown in Figure 8. Within these three classes, different types of devices have been developed. One thing all these devices have in common is that a microscope is used to control the technique, since it provides information about the position of the oocyte compared to the pipette or sensor. These techniques are non-invasive/minimally invasive under the right circumstances (e.g. temperature, measuring medium and minimal stress). Since there are quite a lot of different devices developed over the years, only a few will be discussed in the following paragraphs.

Figure 8: Different devices used to probe oocyte mechanical properties, based on three main approaches: indentation, compression and aspiration.53

3.5.1 Indentation

AFM also falls under the different indentation methods, but next to AFM both micropipette and micro tactile sensors will be discussed.

3.5.1A Micropipette indentation

The second indentation method to be discussed next to AFM is the micropipette indentation. Micropipette indentation has been developed by Liu et al. 98_{and is also shown in Figure 8 A top left. It shows}

an oocyte being pushed against a group of flexible posts. This method has also been referred to as vision-based force measurement technique.98_{This group has developed a holding device from}

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poly(dimethylsiloxane) (PDMS). On this device, every oocyte is contained in its own microwell and is surrounded by flexible posts. A microneedle is then used to apply an indentation force on the oocyte. And the oocyte will be compressed against the posts, which will also deform. Then based on the deformation of the posts, the mechanical properties of the oocytes can be calculated.

The study performed by Lui et al. used this set-up to measure the differences between young and aged mice oocytes. The results obtained showed that aged oocytes were significantly softer but more viscous than young oocytes. This study has not measured zona hardening, but the set-up could be used to provide insight into the different elastic properties during zona hardening. However, there are also many other alternatives that can be used and do not require this unique measuring platform.

3.5.1B Micro tactile sensors

Thirdly by Murayama et al.99_{a micro tactile sensor (MTS) has been developed. This is also shown in}

Figure 8 A bottom left. This micro tactile sensor consists of a cylindrical ceramic probe. This probe is composed of piezoelectric transducers. Based on the slope of the change in the probe’s resonance frequency upon indentation of the ZP, the Young’s modulus had been calculated. The studies by Murayama et al. performed elasticity measurements during oocyte maturation, fertilisation, and early embryo development of bovine oocytes. They found that the Young’s modulus increased after fertilisation, from 8.26 kPa to 22.3 kPa. They also reported that the zona gradually softens again after fertilisation and becomes softer than the mature egg by the 8-cell stage.99

3.5.2 Compression

Two compression devices that have been developed over the years were developed by Wacogne et

al.100_{and Abadie et al..}101_{The first compression device was developed by Wacogne et al.}100_{, which is}

represented in Figure 8 B bottom. Wacogne et al. developed a device to measure oocyte stiffness, a micro-beam that acts as a force sensor during oocyte compression. Based on the deformation of the micro-beam, the stiffness can be calculated. This study analysed the mechanical properties of human oocyte; the oocytes used were not suited for ICSI technique.

The second device was developed by Abadie et al. 101_{, which is represented in Figure 8 B top. In this}

set-up, the oocyte was compressed between a micropipette and the edge of a floating platform. Here magnetic springs are attached to the platform. Compression lengths of these springs were used to calculate the force applied to the oocyte. This report used human oocytes and monitored differences during the maturation process. The report did not mention a Young’s modulus, but they reported stiffness values of oocytes.101

3.5.3 Aspiration

Decades ago, the stiffness of oocytes was already measured with the use of micropipette aspiration (MPA). The principle of this technique is still used frequently nowadays. The elastic modulus of cells is determined with MPA by drawing the zona pellucida into a micropipette. The zona hardness is estimated from the deformation of the zona pellucida. This deformation has been tracked microscopically via image analysis, and more recently an optical fibre inside the micropipette has been used to track the deformation based on phase variations. Many researchers have used this technique to analyse viscoelastic properties of oocytes. Khalilian et al. which is represented in Figure 8 C bottom, analysed both human and mouse oocytes before and after fertilisation.102, 103_{They investigated if there was a change in Young’s modulus of the ZP}

during fertilisation. They also tested three different theoretical models (half-space model, layered model and shell model).102, 103_{The results from the three theoretical models were different, but they all indicated}

that there was an increase in hardening of the zona pellucida following fertilisation.

Yanez et al. which is represented in Figure 8 C top, used micropipette aspiration in combination with

gene expression.104_{They analysed both human and mouse oocytes a few hours after fertilisation, and}

investigated if there was a correlation between embryo viability and mechanical properties. Their results suggested that the potency of an embryo was largely determined by the quality and maturation of the