The Hen's Egg and its Formation

Ruth Bellairs , Mark Osmond , in Atlas of Chick Development (Third Edition), 2014

The Vitelline Membrane (Perivitelline Layer)

The vitelline membrane is the transparent casing that encloses the yolk of the hen's egg and separates it from the albumen. It consists of two major layers, the inner layer, which is laid down in the ovary, and the outer layer, which is secreted in the oviduct. Bellairs et al. (1963) described the fine structure by transmission electron microscopy, and their findings have since been confirmed by others. The fibres of both layers are rich in glycoproteins (reviewed by Wishart and Horrocks, 2000).

The inner layer is about 1–3.5 μm thick, and consists of a meshwork of solid cylindrical fibres that mainly run parallel to the surface of the yolk and vary in thickness from about 0.2 to 0.6 μm ( Plate 2c ). The outer layer varies in thickness from about 0.3 to 9 μm and is composed of a variable number of sublayers lying one above the other. It also contains fibres, but these are thinner than those of the inner layer; strands of the outer fibres are extended out into the chalazae (Text-Figure 1 and see below), where they are spirally arranged and coated with thick albumen. The material between the sublayers of the outer layer of vitelline membrane is probably albumen, and it is likely that there is a penetration of the outer layer by albumen, or that the secretion of the albumen starts to take place before the entire thickness of the outer layer has been laid down.

Each layer of the vitelline membrane consists of proteins, though the amino acid composition differs. The principal components of the outer layer are ovomucin, lysozyme and vitelline membrane outer proteins I and II (Burley and Vadehra, 1989). The ovomucin makes up the fibrous framework on which the outer layer is built, whilst lysozyme forms an electrostatic complex with the ovomucin that provides the bulk and strength.

The principal components of the inner layer are glycoproteins, five having now been identified (Rodler et al., 2011). ZPc is a glycoprotein homologue analogous to ZP3 found in the mammalian zona pallucida, and is present in the inner layer of the chick vitelline membrane (Han et al., 2010). It is thought that the glycoprotein homologous II is concerned with the structural integrity of the inner layer. There is also a carbohydrate-containing fraction (Kido et al. 1976), which includes lectins (Cook et al., 1985). See Wishart and Horrocks (2000) for a fuller discussion of the biochemistry of the vitelline membrane.

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Reproduction and Development

L. Swevers , ... K. Iatrou , in Comprehensive Molecular Insect Science, 2005

1.3.3.8.1 Dipteran insects

1.3.3.8.1.1 Drosophila melanogaster

To date, four Drosophila vitelline membrane protein (VMP) genes have been cloned, VM26A.1, VM26A.2, VM34C, and VM32E (Higgins et al., 1984; Mindrinos et al., 1985; Burke et al., 1987; Popodi et al., 1988; Gigliotti et al., 1989). While VM26A.1, VM34C, and VM26A.2 are expressed continuously from stages 8 to 10, VM32E is considered to be a "late" gene and is expressed only at stage 10. Furthermore, VM34C and VM32E are not expressed in the polar follicular cells, in contrast to VM26A.1 and VM26A.2. However, upon synthesis and secretion, both proteins have been postulated to diffuse towards the poles of the follicles (Andrenacci et al., 2001).

For the VM26A.1 and VM32E genes, analysis of their promoter regions has been carried out in transgenic flies, and cis-regulatory elements for temporal and spatial regulation as well as expression levels have been delineated (Gargiulo et al., 1991; Jin and Petri, 1993; Cavaliere et al., 1997; Andrenacci et al., 2000). However, no trans-acting factors that regulate the expression of the VMP genes have been identified thus far.

Immuno-electron microscopy and confocal immunofluorescence studies have shown that yolk proteins (Yps) and VMPs are co-secreted by the cells of the follicular epithelium (stages 9 and 10 of oogenesis). While the VMPs are incorporated in the incomplete vitelline membrane (VM) matrix, the YPs diffuse through gaps in the vitelline layer to reach the oocyte surface. During stage 10B, the incomplete parts of the VM fuse to form a continuous layer that covers completely the oocyte surface (Margaritis, 1985; Trougakos et al., 2001).

All VMPs become localized in the VM during choriogenesis. However, during the later stages, VM32E (but not the other VMPs) is capable of moving from the VM layer to the endochorion (which is secreted during stages 11–14), indicating its participation in the formation of endochorion structures (Andrenacci et al., 2001). The VMPs also undergo proteolytic processing during choriogenesis (Pascucci et al., 1996), but the functional relevance of this finding remains obscure.

The VMPs are characterized by a conserved hydrophobic domain of 38 aa, the VM domain (Scherer et al., 1988) which is necessary for their assembly in the VM. Alternatively, the domain responsible for the localization of the VM32E protein to the endochorion during choriogenesis resides at the C-terminus of the protein (Andrenacci et al., 2001).

In the female sterile mutation fs(2)QJ42, the follicles fail to accumulate VM26A.2, and this results in the formation of an altered VM, onto which the endochorion layer collapses during stage 14 (Savant and Waring, 1989). Thus, the preformation of a properly assembled VM is a prerequisite to the formation of a stable chorion structure (Pascucci et al., 1996).

Another genetic locus implicated in vitelline membrane and chorion formation in Drosophila is the defective chorion-1 (dec-1) locus (Waring et al., 1990). The dec-1 gene encodes multiple protein products that are generated by alternative splicing and protein processing. Three proproteins, fc177, fc125, and fc106, are produced that share a common N-terminus but are distinguished by differential C-termini. Following their secretion, the proproteins undergo distinct extracellular maturation pathways: fc106 is processed during stage 10B into s80, which undergoes an additional cleavage that generates s60 during stage 14; fc125 is initially cleaved to a 125   kDa protein at stage 10A and subsequently to 110 and 95   kDa proteins during stage 10B; and fc177 undergoes a cleavage to a 125   kDa and a second one that yields an 85   kDa protein during stages 12 and 13, respectively (Noguerón and Waring, 1995; Noguerón et al., 2000). The DEC-1 proteins are synthesized concomitantly with the VMPs and accumulate extracellularly between the follicular epithelium and the oocyte. The VM functions both as the site where the proteolytic cleavage products are generated and as a reservoir for the release of the different cleavage products to the oocyte and distinct regions of the chorion, where they presumably exert unique functions (Noguerón et al., 2000). Mutants of dec-1 fail to organize the endochorion, which, as is the case of the fs(2)QJ42 mutation, collapses into the VM during late choriogenesis (Waring et al., 1990).

Recently, the distinct functions of the three DEC-1 proproteins were also dissected genetically. Using specific dec-1 mutations, in conjunction with introduced dec-1 transgenes, it was shown that gross morphological abnormalities occur only in the absence of fc177, although all three proproteins are essential for female fertility. It was proposed that fc177 acts as a scaffolding protein necessary for the erection of the chorion structure (Mauzy-Melitz and Waring, 2003).

The VM represents also a storing site for secreted follicular epithelium cell products involved in embryonic pattern formation (see Chapter 1.2). Extracellular proteins that signal the patterning of the embryo are anchored in the VM and participate actively in the VM assembly. The Nudel protease, for example, a large modular protein with a trypsin-like serine protease domain, functions both in the generation of the extracellular signal that determines the dorsoventral axis of the embryo, and the cross-linking of the vitelline membrane (LeMosy et al., 1999; LeMosy and Hashimoto, 2000). Similarly, the fs(1)Nasrat and fs(1)polehole genes are not only required for the generation of the ligand for the local activation of the Torso (TOR) receptor at the termini of embryos, but also have structural roles in the biogenesis of the VM (Cernilogar et al., 2001).

1.3.3.8.1.2 Aedes aegypti

Three genes encoding VMPs have been cloned in A. aegypti, 15a-1, 15a-2, and 15a-3 (Lin et al., 1993; Edwards et al., 1998). All are characterized by a short 46 aa sequence that bears similarities with the VM domain of the Drosophila VMPs. The expression patterns of the vitelline membrane proteins during oogenesis overlap those of the YPs. In situ hybridization has also shown that the different VMP genes are expressed in spatially distinct domains of the follicular epithelium: 15a-1 and 15a-3 are expressed in the middle and the posterior regions of the follicle, while 15a-2 is expressed over the entire surface (Edwards et al., 1998). Finally, a moderate stimulation of expression levels of the VMPs could be achieved by addition of high concentrations 20E (Lin et al., 1993; Edwards et al., 1998).

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Electrophysiology and Microinjection

D. Bertrand , ... M. Ballivet , in Methods in Neurosciences, 1991

Removal of Vitelline Membrane

Defolliculated oocytes are surrounded by a fibrous vitelline membrane that acts as a protective coat. For patch-clamp recording, this membrane must be removed to expose the oocyte plasma membrane containing the ionic channels. The easiest way to remove the vitelline membrane is to peel it off mechanically with forceps. Normally, the vitelline membrane adheres closely to the plasma membrane, but the space between the two membranes will enlarge if the oocyte is placed in a hypertonic solution (450–500 mOsm) for 10 to 20 min. With the aid of a dissecting microscope (80 ×), it is possible to strip away the vitelline membrane with sharpened forceps without damaging the plasma membrane; usually the vitelline membrane comes off in a complete shell. As hypertonic solutions, we have used either a potassium aspartate solution (pH 7.3) (in m M): potassium aspartate, 200; KCl, 20; MgCl2, 0.5; HEPES, 10; or OR2 plus CaCl2, 1; MgCl2, 1; potassium aspartate, 100; or simply 2 × OR2. We have found little difference in the ability to strip off the vitelline membrane when one solution is used over the other.

It has been reported that the vitelline membrane can be digested off with proteases (21); however, the membrane does not come off completely, and therefore, "clean" areas need to be selected prior to recording. Also, there is a danger that the protease may alter the ionic channels in the plasma membrane.

Once stripped, the oocytes are extremely delicate and can be easily ruptured mechanically, or by surface tension. To minimize manipulation of stripped oocytes, we remove the vitelline membrane after the oocyte is transferred into the recording chamber which contains extracellular media used for recording (see below). Our recording chambers are 35-mm plastic Petri dishes (1008; Falcon, Oxnard, CA).

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Innate defenses of the avian egg

Sophie Réhault-Godbert , ... Joel Gautron , in Avian Immunology (Third Edition), 2022

Perivitelline layer (vitelline membrane)

The perivitelline layer, also known as the vitelline membrane, is a thin protein layer covering and encapsulating the egg yolk nutrients destined for the developing embryo. In addition to its role in fertilization and in early embryonic development, the perivitelline layer also participates in the protection of both the embryo and the yolk nutrients during the early incubation. Indeed, this biological barrier protects embryonic cells against the alkalinity of egg white, and it limits the diffusion of egg yolk nutrients to the egg white, and the penetration of bacteria from the egg white to the egg yolk.

In chicken eggs, the perivitelline layer is almost entirely composed of proteins. About 140 different proteins were identified by mass spectrometry [63]. The perivitelline layer consists of two superposed layers, the inner and the outer perivitelline layers, in contact with the egg yolk contents and the egg white, respectively, which are separated by a very thin granular "continuous membrane" [64]. The chicken internal perivitelline layer is initially produced in the ovarian follicle before ovulation and consists of a three-dimensional network of fibers [64,65] (Fig. 13.3). At the molecular level, the filamentous structures are mainly composed of Zona Pellucida proteins ZP1–ZP3 complexes assembled into helical fibrils, which further assemble into bundles to form the ZP1–ZP3 matrix. Besides, Zona Pellucida D protein (ZPD) aggregates and binds to ZP1–ZP3 matrix to form the inner perivitelline layer matrix [66]. ZP proteins are presumably involved in sperm binding and the acrosome reaction. No direct innate immune functions were reported for chicken ZP proteins to date, although ZPD might have some immune roles by homology with the human protein uromodulin, which is involved in antibacterial protection and immune regulation within the human urinary tract [66]. The outer perivitelline layer is synthesized in the proximal part of oviduct (infundibulum) following ovulation and covers the inner perivitelline layer. It consists of a variable number of sublayers superposed on each other and containing long and straight fibrils [64] (Fig. 13.3). The major proteins composing chicken outer perivitelline layer are ovomucin, lysozyme, vitelline membrane outer layer proteins 1 and 2 (VMO1 and VMO2, now referred to as avian β-defensin-11, AvBD11) [67,68]. Although the perivitelline layer protein pattern may vary depending on avian species [69], the innate immune properties of these major compounds (see Section 13.2.2) strongly support the antimicrobial function of outer perivitelline layer. It has been hypothesized that lysozyme contributes to perivitelline layer structure via electrostatic interactions with ovomucin [67,70]. As demonstrated in quail, VMO2/AvBD11 might have a structural role in outer perivitelline layer, in mediating the binding of outer perivitelline layer with inner perivitelline layer, possibly via an association with ZP1 and ZP3 proteins [71]. The maintenance of outer perivitelline layer integrity is crucial to prevent or limit bacterial penetration from the egg white into the yolk. However, outer perivitelline layer integrity can be challenged by several parameters, especially the conditions of egg storage prior to incubation (e.g., high temperature, long duration).

Figure 13.3. Micrographs of chicken egg perivitelline layer in cross-section by transmission electron microscopy (A), and of outer and inner surfaces by scanning electron microscopy (B and C, respectively). OPL, outer perivitelline membrane; IPL, inner perivitelline membrane; CM; continuous membrane.

 ©Plateforme IBiSA de Microscopie Electronique, University and CHRU of Tours, France, S. Georgeault.

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Use of Whole Embryo Cultures in In Vitro Teratogenicity Testing

BEAT SCHMID , ... PAVEL KUCERA , in In Vitro Methods in Pharmaceutical Research, 1997

2 Culture conditions

After preincubation for 20 h the eggs opened and the vitelline membrane with the attached blastoderm is excised from the yolk and transferred to an incubation chamber moulded from a transparent silicone elastomere.

The incubation medium is obtained by mixing one part of the thin albumen collected from the eggs during the explanation with one part of Tyrode solution. The compounds to be tested are dissolved in the medium or suspended in gelatin at the desired concentration. Constant volumes of culture medium are poured over the preparation, the chamber is closed, incubated at 37.5°C, and the embryo is allowed to develop for a further 3 days. The incubation medium is not changed during the incubation period. On average, 11 embryos are used per substance and concentration.

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Techniques

Ruth Bellairs , Mark Osmond , in Atlas of Chick Development (Third Edition), 2014

Albumen–agar technique

The purpose of this modification is to provide a semi-solid base beneath the vitelline membrane that is helpful when carrying out microsurgery. The addition of agar to the albumen, however, raises the risk of bacterial infection, so that strict sterility should be maintained. A few drops of Indian ink may be incorporated if a dark background is required. Chapman et al. (2001) recommend substituting a disc of filter paper for the glass ring so that the vitelline membrane adheres to the paper. This adaptation is not suitable for embryos younger than stage 3 because of a high death rate and microcephaly (Voiculescu et al., 2008). More recently, El-Ghali et al. (2010) reported better results by replacing the filter paper with rings made from nitrocellulose or PVDF membranes.

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Snake Protease of Drosophila

Robert DeLotto , in Handbook of Proteolytic Enzymes (Third Edition), 2013

Biological Aspects

The current view of the dorsal–ventral signaling pathway suggests that an asymmetric cue is laid down in the vitelline membrane or perivitelline space of the egg during oogenesis [10]. After fertilization, a protease cascade is initiated in the perivitelline space of the embryo, resulting in the sequential activation of three serine protease proenzymes, Gastrulation defective, Snake and Easter. The result of this cascade is the ventrally restricted processing of Spaetzle, resulting in a graded distribution of ligand with a local maximum on the ventral side of the embryo [11]. The protease cascade has been reconstituted by co-expression studies in using baculovirus and in transfected Drosophila Schneider cells [3,4].

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Role of Oxidative Stress in the Etiology of Male Infertility and the Potential Therapeutic Value of Antioxidants

R. John Aitken , ... Joel R. Drevet , in Oxidants, Antioxidants and Impact of the Oxidative Status in Male Reproduction, 2019

Sperm–Oocyte Fusion

The ability of human spermatozoa to acrosome react and generate a fusogenic equatorial segment capable of initiating fusion with the vitelline membrane of the oocyte is also notoriously sensitive to oxidative stress [25]. The underlying mechanisms presumably involve the induction of lipid peroxidation in the sperm plasma membrane and a consequential loss of membrane fluidity, fusability, and function. Whether proteins that are intimately involved in sperm–oocyte fusion such as IZUMO (which must become translocated to the equatorial segment following the acrosome reaction in order to interact with its receptor on the egg, JUNO) [35] are vulnerable to oxidative attack or alkylation by lipid aldehydes generated as a consequence of oxidative stress has not yet been investigated.

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Reproduction and Development

B. Loppin , T.L. Karr , in Comprehensive Molecular Insect Science, 2005

1.6.4.1.1 casanova

In animals, sperm-egg recognition usually relies on the interaction of carbohydrate residues on the surface of the egg's vitelline membrane with complementary molecules on the plasma membrane of the sperm. In Drosophila, biochemical analysis have identified two distinct glycosidases at the surface of male gametes: β-N-acetylglucosaminidase and α-d-mannosidase (Cattaneo et al., 1997, 2002; Pasini et al., 1999; Perotti et al., 2001). casanova (csn) is a Drosophila male sterile mutant with a unique phenotype: casanova mutant males produce motile sperm that are stored in females but that fail to enter wild-type eggs (Perotti et al., 2001). Ultrastructural analysis of casanova sperm revealed that β-N-acetylglucosaminidase is absent from the spermatozoa membrane overlying the acrosome, where it is normally present. Although the csn gene has not been identified yet and probably does not encode this enzyme, this study suggests that a molecular sperm-egg recognition system (involving at least β-N-acetylglucosaminidase and its substrate β-N-acetylglucosamine) exists in insects, like in higher animals.

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Reproductive and developmental toxicity in fishes

Helmut Segner , in Reproductive and Developmental Toxicology, 2011

Fertilization and egg/sperm quality

The egg is bounded by a series of membranes, with an outer, relatively tough one (chorion), and the vitelline membrane around the yolk. It must contain all the nutrients required by the developing embryo from fertilization until start of exogenous feeding. The egg yolk has to support both fueling of embryonic development as well as building up of the tissues. The yolk material has been deposited in the growing oocyte by the mother, with liver-derived vitellogenin being the major precursor ( Hiramatsu et al., 2005). The ovulated eggs contain water (between 60 and 90%, depending on whether the eggs are buoyant or demersal), proteins and amino acids, lipids and lipoproteins, micronutrients such as vitamins, as well as hormones such as estradiol or thyroid hormones. Deficiencies in egg composition can have adverse effects on development, as it is exemplified by the M74 syndrome. This syndrome, which leads to high mortalities among early life stages of Baltic salmon, is associated with low thiamine levels in salmon eggs and yolk sac fry (Börjeson and Norrgren, 1997).

Mature sperm cells, in order to be able to fertilize the eggs, have to acquire motility. In salmonids, the spermataozoa in the lumen of the seminiferous tubulus are not yet mobile. They acquire the potential for motility only during migration down the sperm ducts, and this is caused by an increase of semen pH (Morisawa, 1994). Sperm motility is then actually realized upon release into the aquatic environment, and the trigger for this are extracellular ionic and osmotic changes (Morisawa, 1994). To sustain flagellar movement, the spermatozoa face a huge energy demand. In species with external fertilization, this is usually provided from endogenous substrates, while species with internal fertilization appear to utilize also exogenous substrates (Ingermann, 2008). Survival time of the sperm cells in the water is generally very short and rarely exceeds 2 minutes (Kime, 1998). In contrast to mammals, fish sperm fertilizes the egg not by penetrating the oocyte wall but fertilization takes place through a funnel-like micropyle located at the animal pole of the egg. After the egg is activated by the sperm, the micropyle closes thereby preventing polyspermy. Furthermore, the egg envelope or chorion undergoes hardening and separates from the vitelline membrane leading to the formation of the perivitelline space. This includes also protection against pathogens, as the egg shell contains anti-microbial factors (Modig et al., 2007).

The vast majority of teleost species have external fertilization; however, a few species such as guppy, Poecilia reticulata, have internal fertilization. In most species with internal fertilization, the fertilized eggs are retained and undergo development in the maternal body. Hatching either precedes or coincides with parturition, i.e. the females give birth to free-living young fish.

Egg and sperm quality may be defined as the ability to fertilize or to be fertilized, respectively. The quality is determined by the intrinsic properties of the gametes themselves as they have been provided by the parents. Numerous factors such as broodstock nutrition or stress can influence egg and sperm quality (Bobe and Labbé, 2010). The question what makes a good egg or sperm quality and how this can be assessed has been debated for a long time but has yet to be answered. Egg size, weight and morphology can provide estimates on the developmental potential of eggs, but the parameters show huge variation and the relation with egg quality is not simple and straightforward (Kjorsvik et al., 1990). In fractional spawners, the quality of eggs produced in different batches over a spawning season may vary considerably, even if the husbandry conditions are apparently equivalent over the whole period (Kjesbu et al., 1996). Much attention has been given on the role of maternal nutrition on egg quality; however, the evidence that diet can directly affect egg quality is limited. On the sperm side, motility, together with morphology, appear to be useful parameters, but it still can be difficult to correlate sperm quality parameters to the fertilization rate obtained with the same sperm (Bobe and Labbé, 2010). One of the more meaningful quality parameters which reflect both egg and sperm is fertilization success. There have been also numerous attempts to use molecular and biochemical parameters to estimate egg and sperm quality. For instance, high expression of apoptosis-related markers, or high cathepsin enzymatic activities, may be indicative of eggs with low viability (e.g., Carnevali, 2007). Also maternal mRNA transcripts have been evaluated for their utility as egg/sperm quality indicators (Aegerter et al., 2005). Transcriptomic and proteomic profiling of eggs and sperm may be able to identify sets of quality-indicating genes or protein sets; however, the analysis gets complicated due to the rapid temporal changes of gene and protein expression in developing embryos (Ziv et al., 2008).

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