Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs.

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.

Interrelationships of vitelline and muscle cells within the vitelline follicles of the blood fluke Aporocotyle simplex (Digenea, Aporocotylidae) and morphological evidence for the modification of vitelline material for eggshell formation.

This study of the fish blood fluke Aporocotyle simplex represents the first detailed transmission electron microscopical (TEM) investigation of the vitellarium of an aporocotylid digenean blood fluke. It revealed some unusual characteristics in the cytoarchitecture of the vitelline follicles and demonstrated modifications of the vitelline granules for eggshell formation. The vitelline follicles consist of vitellocytes at different developmental stages surrounded by sarcoplasmic processes of myocytes which occur throughout each follicle. Sites of intimate contact occur between the vitellocytes and the myocytes. Individual vitelline globules (0.1-0.2 µm in diameter) accumulate in quite small clusters of 10-20 and have a dense, heterogeneous matrix possessing central and peripheral regions with a greater density. Modifications of the vitelline globules take place within the clusters and are first apparent when the vitellocytes reach the lumen of the vitelline duct and vitelline reservoir. Globules within the clusters become confluent, and, when the vitellocytes reach the lumen of the oviduct and proximal ootype, these consolidated clusters contain a shapeless, loosely packed, dense material which is released from the vitellocytes by exocytosis. This investigation has provided morphological evidence for shell formation from modified vitelline globules in the form of a discontinuous, thin layer (~ 0.07 µm in thickness) of electron-dense shell material around the fertilized ovum and associated vitellocytes in the proximal ootype. The eggshell of intra-uterine eggs acquires an additional thin, heterogeneous outer layer, increasing its thickness to ~ 0.1 µm. The cytoarchitecture of the vitellarium, modifications of the vitelline globules within the clusters and the structure of the eggshell of A. simplex may prove to be of value in studies examining relationships between the three distinct lineages of aporocotylid digeneans.

Characterization of structure and protein of vitelline membranes of precocial (ring-necked pheasant, gray partridge) and superaltricial (cockatiel parrot, domestic pigeon) birds.

Of all the known oviparous taxa, female birds lay the most diverse types of eggs that differ in terms of shape, shell pigmentation, and shell structure. The pigmentation of the shell, the weight of the egg, and the composition of the yolk correlate with environmental conditions and the needs of the developing embryos. In this study, we analyzed the structure and protein composition of the vitelline membrane (VM) of ring-necked pheasant, gray partridge, cockatiel parrot, and domestic pigeon eggs. We found that the VM structure is characteristic of each species and varies depending on whether the species is precocial (ring-necked pheasant and gray partridge) or superaltrical (cockatiel parrot and domestic pigeon). We hypothesize that a multilayer structure of VM is necessary to counteract the aging process of the egg. The multilayer structure of VM is only found in species with a large number of eggs in one clutch and is characterized by a long incubation period. An interesting discovery of this study is the three-layered VM of pheasant and partridge eggs. This shows that the formation of individual layers of VM in specific sections of the hen's reproductive system is not confirmed in other species. The number of protein fractions varied between 19 and 23, with a molecular weight ranging from 15 to 250 kDa, depending on the species. The number of proteins identified in the VM of the study birds' eggs is as follows: chicken-14, ring-necked pheasant-7, gray partridge-10, cockatiel parrot-6, and domestic pigeon-23. The highest number of species-specific proteins (21) was detected in the VM of domestic pigeon. This study is the first to present the structure and protein composition in the VM of ring-necked pheasant, gray partridge, cockatiel parrot, and domestic pigeon eggs. In addition, we analyzed the relationship between the hatching specification of birds and the structure of the VM.

Disruption of egg production by triclabendazole-resistant Fasciola hepatica following treatment with a commercial preparation of myrrh (Mirazid).

An in vitro study has been carried out to monitor changes to the female reproductive system in adult triclabendazole (TCBZ)-resistant Fasciola hepatica following treatment with a commercial preparation of myrrh ("Mirazid"). Flukes were immersed for 6 h and 24 h in myrrh extract at a concentration of 200 µg/ml, then processed for histological and transmission electron microscope (TEM) examination of the uterus, Mehlis' gland, ovary and vitellaria. Egg production had become abnormal at 6 h post-treatment (pt), with the uterine lumen being filled with free vitelline cells and masses of shell protein material; few eggs were present. At 24 h pt, no eggs were present. Distinct changes to the ovary and Mehlis' gland were only observed after 24 h incubation in Mirazid. The ovary contained numbers of apoptotic oogonia and oocytes. In the Mehlis' gland, the S1 cells were disorganised and the processes from them were vacuolated, although the disruption was not significant. More severe changes were observed in the vitelline cells and follicles. After 6 h incubation in Mirazid, although the gross organisation of the vitelline follicles appeared to be normal, nuclear changes indicative of the early stages of apoptosis were observed in the stem cells and shell protein production by the mature cells had decreased. At 24 h pt, a distinct shift in cell population was evident, with the follicles containing mainly mature cells and spaces were present between the cells. The shell globule clusters in the mature cells were disorganised. In more severely-affected follicles, cells were seen to be breaking down, with karyolytic nuclei and disintegrating cytoplasm. Overall, the results have shown that exposure to Mirazid treatment had a severe impact on egg production by TCBZ-resistant flukes, an effect that was mediated by disruption of the vitelline cells and of the mechanism co-ordinating egg formation in the ootype.

Xenopus borealis as an alternative source of oocytes for biophysical and pharmacological studies of neuronal ion channels.

For the past 30 years, oocytes from Xenopus laevis have been extensively used to express and characterise ion channels in an easily controlled environment. Here we report the first use of oocytes from the closely related species Xenopus borealis as an alternative expression system for neuronal ion channels. Using the two-electrode voltage-clamp technique, we show that a wide variety of voltage- and ligand-gated ion channels have the same channel properties and pharmacological profiles when expressed in either X. laevis or X. borealis oocytes. Potential advantages of the X. borealis oocytes include a smaller endogenous chloride current and the ability to produce more intense fluorescence signals when studied with voltage-clamp fluorometry. Scanning electron microscopy revealed a difference in vitelline membrane structure between the two species, which may be related to the discrepancy in fluorescence signals observed. We demonstrate that X. borealis oocytes are a viable heterologous system for expression of neuronal ion channels with some potential advantages over X. laevis oocytes for certain applications.

Allantoic, vitelline, and "hybrid" embryonic vestiges of the umbilical cord: an immunohistochemical study.

The embryonic origin of umbilical cord vestiges is well documented; however, their immunophenotype is unknown. This study was conducted to determine whether vitelline and allantoic remnants can be differentiated using immunohistochemical markers. All allantoic remnants were stained with p63 and were negative for CDX2, whereas the vitelline remnants stained with CDX2 and were negative for p63. An unexpected finding was a small number of morphologically ambiguous cases that stained with both markers in a complimentary manner. The term "hybrid" remnant is proposed for these remnants.

Ultrastructure of the vitellarium of Ancyrocephalus paradoxus (Monogenea: Monopisthocotylea), with comments on the nature of the vitellarium in the Monogenea and related platyhelminth groups.

Fine structural features of the vitelline follicles of the monopisthocotylean monogenean Ancyrocephalus paradoxus are revealed and compared with those of other monogenean species. As in other monogeneans, each vitelline follicle of A. paradoxus is composed of a single cell type, i.e. vitellocytes at various stages of development, with no sign of any interstitial cells. There is no special isolation of the vitelline follicles from the surrounding tissue, and both heterologous (between adjacent membranes of the vitelline and surrounding parenchymal cells) and homologous (between adjacent membranes of the vitelline cells) cell junctions (zonulae occludentes) are present. Non-membrane-bound vitelline clusters of A. paradoxus contain 50-100 vitelline globules, moderately electron-dense lipid droplets and glycogen particles present in the mature cell cytoplasm. In a search for phylogenetically informative characters of the fine structure of the vitellarium, the new findings are compared with those known for trematodes, and, to enable this, additional observations were made on the structure of the vitellarium of the aspidogastrean Aspidogaster limacoides. Some new discriminatory traits are revealed in A. limacoides; these include the presence of a single type of cellular component within the vitellarium, sarcoplasmic processes filling the space between and around the vitellocytes and zonulae occludentes between adjacent membranes of vitelline and muscle cells. On the basis of ultrastructural features of the vitellarium, a relationship between the Monogenea and the Aspidogastrea is indicated.

Ultrastructural study of vitellogenesis and oogenesis of Metadena depressa (Stossich, 1883) Linton, 1910 (Digenea, Cryptogonimidae), intestinal parasite of Dentex dentex (Pisces, Teleostei).

The ultrastructural organization of the female reproductive system of Metadena depressa, digenean intestinal parasite of Sparidae (Dentex dentex), was investigated by electron microscopy. The vitellogenesis is divided into four stages: stage I, vitellocytes have a cytoplasm mainly filled with ribosomes and few mitochondria; stage II, beginning of the synthetic activity; stage III, active shell globule clusters synthesis; stage IV, mature vitellocytes are filled with shell globule clusters and generally contain several large lipid droplets. Glycogen granules are grouped at the periphery of the cell. The three stages of the oogenesis process take place in the ovary: stage I, oogonia are undifferentiated small cells located at the periphery of the organ; stage II, primary oocytes possess a higher nucleo-cytoplasmic ratio and a nucleus with a nucleolus and synaptonemal complexes indicating the zygotene-pachytene stage of the first meiotic division; stage III, mature oocytes are located in the proximal region of the organ and possess a cytoplasmic chromatoid body and cortical granules in a monolayer close to the periphery of the cell.

Assembly of the inner perivitelline layer, a homolog of the mammalian zona pellucida: an immunohistochemical and ultrastructural study.

The avian inner perivitelline layer (IPVL), a homologous structure to the mammalian zona pellucida, is deposited between the granulosa cells and the oocyte cell membrane during folliculogenesis. The glycoprotein meshwork of the IPVL forms a 3-dimensional matrix and possesses important functions in the fertilization process: it contributes to the binding of avian spermatozoa to the oocyte and induces acrosomal exocytosis. In contrast to the zona pellucida of mammals, the IPVL does not prevent the physiological polyspermy found in birds. Previous studies have shown that in the Japanese quail (Coturnix japonica) at least 5 glycoproteins are constituents of the IPVL (ZP1, ZP2, ZP3, ZP4, and ZPD). In this study, we investigated the spatiotemporal assembly pattern of the IPVL during folliculogenesis using immunohistochemical and ultrastructural methods. The obtained results clearly show that these glycoproteins are incorporated into the IPVL at distinct points during follicular development, supporting the hypothesis that ZP2 and ZP4 form a type of prematrix into which ZP1, ZP3, and ZPD are integrated at a later stage of development.

Egg structure of Zorotypus caudelli Karny (Insecta, Zoraptera, Zorotypidae).

The structural features of eggs of Zorotypus caudelli Karny are described in detail. The egg is elliptic with long and short diameters of 0.6 and 0.3 mm respectively, and creamy white. The egg shows a honeycomb pattern on its surface, without any specialized structures for hatching such as an operculum or a hatching line. The fringe formed by a fibrillar substance secreted after the completion of the chorion encircles the lateral surface. The egg layer is composed of an exochorion, an endochorion, and a vitelline envelope. The exochorion and endochorion are electron-dense and homogeneous in structure. The exochorion shows a perforation of numerous branching aeropyles. The exo- and endochorion are connected by numerous small columnar structures derived from the latter. The vitelline envelope is very thin and more electron-dense than the chorion. A pair of micropyles is present at the equator on the dorsal side of the egg. Originating at the micropyle, the micropylar canal runs through the chorion obliquely. The structural features of the eggs of Zoraptera were compared with those of other polyneopteran and paraneopteran orders.

Micropores in the vitteline layer of the eggs of the dragonfly Oligoaeshna pryeri: a preliminary observation from the viewpoint of oxygen uptake.

In dragonfly eggs, oxygen diffusing in, and carbon dioxide diffusing out, encounter barriers in the shell.According to Tullett and Board, in avian eggs the most important of these barriers results from the geometry of the pores through the shells. As in birds, dragonfly egg shells consist of three layers: the exochorion, endochorion and the innermost vitelline membrane. Trueman has described pores and fine anchorlike structures in the endochorion but the vitelline membrane does not seem to have been studied. In the present work we have used scanning electron microscopy to examine the vitelline membrane in hatching eggs of Oligoaeschna pryeri. We have assumed that the numerous openings seen on the micrographs are pores through the membrane.Results are expressed as means ± SD. The pore diameter, pore area and number per µm2 of the vitelline membrane were 74.7 ± 61.3 nm, 4380 ± 3555 nm2 and 4.16 ±1.3 pores/µm2 (4.16 x108 pores/cm2), respectively. The total pore area was calculated to be 18,222 nm2/ µm(2). In avian egg shells pore density depends on the weight of the egg. Results given by Tullett and Board suggest that an egg weighing 1 g may have a pore density of 300 pores/cm2, which is much lower than the present result for dragonflies. It seems likely that the difference reflects the fact that in Oligoaeshna pryeri the eggs are immersed in water.

Formation and structure of egg envelopes in Russian sturgeon Acipenser gueldenstaedtii (Acipenseriformes: Acipenseridae).

The covering of the eggs in Russian sturgeon Acipenser gueldenstaedtii consists of three envelopes (the vitelline envelope, chorion and extrachorion) and is equipped with multiple micropyles. The most proximal to the oocyte is the vitelline envelope that consists of four layers of filamentous and trabecular material. The structural components of this envelope are synthesized by the oocyte (primary envelope). The chorion encloses the vitelline envelope. The extrachorion covers the external surface of the egg. Examination of the arrangement of layers that comprise the egg envelopes together with the ultrastructure of follicular cells revealed that the chorion and extrachorion are secondary envelopes. They are secreted by follicular cells and are built of homogeneous material. During formation of egg envelopes, the follicular cells gradually diversify into three morphologically different populations: 1) cells covering the animal oocyte region (cuboid), (2) main body cells (cylindrical) and (3) micropylar cells. The apical surfaces of follicular cells from the first two populations form processes that remain connected with the oocyte plasma membrane by means of gap junctions. Micropylar cells are located at the animal region of the oocyte. Their apical parts bear projections that form a barrier to the deposition of materials for egg envelopes, resulting in the formation of the micropylar canal.

Comparative study on histological structures of the vitelline membrane of hen and duck egg observed by cryo-scanning electron microscopy.

The histological structures of the vitelline membranes (VM) of hen and duck eggs were observed by cryo-scanning electron microscopy (cryo-SEM), and the chemical characteristics were also compared. The outer layer surface (OLS) of duck egg VM showed networks constructed by fibrils and sheets (0.1-5.2 microm in width), and that of hen egg presented networks formed only by sheets (2-6 microm in width). Thicker fibrils (0.5-1.5 microm in width) with different arrangement were observed on the inner layer surface (ILS) of duck egg VM as compared to those (0.3-0.7 microm in width) of hen egg VM. Upon separation, the outer surface of the outer layer (OSOL) and the inner surface of the inner layer (ISIL) of hen and duck egg VMs were quite similar to fresh VM except that the OSOL of duck egg VM showed networks constructed only by sheets. Thin fibrils interlaced above a bumpy or flat structure were observed at the exposed surface of the outer layer (ESOL) of hen and duck egg VMs. The exposed surfaces of inner layers (ESIL) of hen and duck egg VMs showed similar structures of fibrils, which joined, branched, and ran in straight lines for long distances up to 30 microm; however, the widths of the fibrils shown in ESOL and ESIL of duck egg VM were 0.1 and 0.7-1.4 microm, respectively, and were greater than those (<0.1 and 0.5-0.8 microm) of hen egg VM. The continuous membranes of both hen and duck egg VMs were still attached to the outer layers when separated. The content of protein, the major component of VM, was higher in duck egg VM (88.6%) than in hen egg VM (81.6%). Four and six major SDS-soluble protein patterns with distinct localization were observed in hen and duck egg VMs, respectively. Overall, the different histological structures of hen and duck egg VMs were suggested to be majorly attributable to the diverse protein components.

Vitrification of the inner perivitelline layer of chicken eggs for use in the sperm-egg interaction assay.

The sperm-egg interaction assay is a good predictor of the fertilizing potential of rooster semen; the ability of chicken sperm to interact with the egg can be assessed by counting the number of holes in the inner perivitelline layer (IPVL) of a freshly laid egg. Although isolated IPVL can be stored for up to 24h, preservation of IPVL for prolonged intervals in liquid nitrogen would facilitate the sperm-egg interaction assay. The objective of this study was to adapt the technique of vitrifying swine oocytes for use with the IPVL. Our hypothesis was that vitrification would not alter the ability of the membrane to bind sperm; therefore, there would be no difference between vitrified and fresh IVPL in the number of hydrolysis holes made by sperm. Our hypothesis was supported; there were no differences in the mean+/-SEM number of holes made by the same sample of sperm in vitrified and in fresh membranes (146.0+/-17.7 holes/mm(2) IPVL and 159.5+/-17.7 holes/mm(2) IPVL, respectively, P>0.05; n=123 IVPLs tested). Furthermore, 80% of frozen-thawed membranes were recovered intact. Because vitrification did not significantly change the ability of membranes to bind sperm, vitrified membranes can be safely used for the sperm-egg interaction assay. Vitrified IVPL would ensure availability for sperm evaluation and facilitate wide distribution of IPVL, enabling assays to be conducted even in the absence of facilities or expertise to prepare membranes.

Organization of the vitelline envelope in ovarian follicles of Torpedo marmorata Risso, 1810 (Elasmobranchii: Torpediniformes).

In Torpedo marmorata, the vitelline envelope (VE), an extracellular envelope surrounding the growing oocyte, consists of fibrils and amorphous materials that are deposited in the perivitelline space starting from the initial steps of oocyte growth. SDS-PAGE analysis of the isolated and purified VE reveals that it consists of different glycoproteins. Furthermore, our investigations showed that the 120 and 66 kDa glycoproteins are positive to an antibody directed against gp69/64 of the Xenopus laevis VE and are synthesized under the control of 17beta-estradiol in the liver, that, together follicle cells and the oocyte, is the biosynthetic site of VE components.

The study of vitelline gland of Haploporus lateralis (Digenea: Trematoda).

The study of vitelline glands of digenean Haploporus lateralis, indicates that the glands consist of vitelocytes in various stages of maturation. The cytoplasmic process of nurse cells which surrounded the vitelline lobes, intrude between vitelocytes and surround them. Immature vitelline cells are undifferentiated, they possess small amount of cytoplasm and large nucleus. The synthesis of shell globules begin in the maturing vitelline cells. Increasing numbers of vitellocytes reach develop of mature vitelline cells. The cytoplasm of mature vitelline cells is filled with shell globules. Mature vitelline cells usually found in the center of vitelline lobe. They release into the vitelline duct and vitelline reservoir. The vitelline reservoir and duct are lined with a syncytial epithelium. Basement membrane is present between nurse cells and vitelline cells.

Ultrastructure of the eggshell and its formation in Planipapillus mundus (Onychophora: Peripatopsidae).

Although the majority of onychophorans are viviparous or ovoviviparous, oviparity has been described in a number of species found exclusively in Australia and New Zealand. Light microscopy and scanning and transmission electron microscopy were used to examine developing eggs and the reproductive tract of the oviparous Planipapillus mundus. Deposited eggs and fully developed eggs dissected from the terminal end of the uteri have an outer thick, slightly opaque chorion, and an inner thin, transparent vitelline membrane. The chorion comprises an outermost extrachorion, sculptured with domes equally spaced over the surface; a middle exochorion, with pores occurring in a pattern of distribution equivalent to that of the domes of the extrachorion above; and an innermost, thick endochorion consisting of a spongelike reticulum of cavities comparable to the respiratory network found in insect eggs. The vitelline membrane lies beneath the chorion, from which it is separated by a fluid-filled space. The vitelline membrane tightly invests the developing egg. Examination of oocytes in the ovary and developing eggs at various stages of passage through the uterus indicate that the majority of chorion deposition occurs in the midregion of the uterus, where vast networks of endoplasmic reticulum are present in the columnar epithelium. The vitelline membrane, however, is believed to begin its development as a primary egg membrane, surrounding the developing oocytes in the ovary. The vitelline membrane is transformed after fertilization, presumably by secretions from the anterior region of the uterus; hence, it should be more accurately referred to as a fertilization membrane. Aspects of the reproductive biology of P. mundus are also included.

Structural changes of the zona pellucida during fertilization and embryo development.

The zona pellucida (ZP) is a unique extracellular coat surrounding the maturing oocyte, during ovulation, fertilization, and early embryo development. It is formed by three/four glycoproteins. Ultrastructural data obtained with transmission (TEM) and scanning electron microscopy (SEM) were compared with molecular data on the glycoproteins network from ovulation to blastocyst formation. Molecular models are quite different to the morphology obtained with TEM, which shows a microfibrillar architecture, or with SEM, which shows a spongy or smooth surface. The saponin-ruthenium red-osmium tetroxide-thiocarbohydrazide technique allows to show the ZP real microfilamentous structure and the related functional changes. These results support an ultrastructural supramolecular model, more similar and comparable to molecular models related with the glycoprotein network. A detailed mapping of single mammalian ZP proteins and their relationship within the supramolecular architecture of the zona matrix would clearly supply insights into the molecular basis of sperm-egg recognition. Differences in ZP glycoproteins among mammals do not affect structural morphology; further studies are needed to clarify the relationships between ultrastructural and molecular organizations.

Egg envelope synthesis and chorion modification after oviposition in the dragonfly Libellula depressa (Odonata, Libellulidae).

Libellula depressa (Odonata, Libellulidae) is an exophytic dragonfly ovidepositing eggs in clutches on the surface of floating plants and algae. The present work investigates, at ultrastructural level, the gradual differentiation of the egg envelopes and the chorionic changes after egg deposition in water. The ovary of the mature female of L. depressa is composed of numerous strings of panoistic ovarioles, where the eggshell formation takes place gradually throughout the activity of the follicle cells. The present data show that the egg envelopes are constituted of a very thick electrondense vitelline envelope, a thin endochorion and an extremely thick exochorion composed of a fibrillar matrix resting on a thin electrondense layer. After deposition in water, L. depressa eggs, initially white and almost transparent, gradually become brown spots in a semitransparent jelly coat, rich of incorporated debris. The jelly coat enveloping the eggs of L. depressa derives exclusively from the exochorion, constituted of a fibrillar matrix, which swell at contact with water. The jelly-like coat performs an adhesive function and presumably a protective role during egg segmentation and ensuing larval hatching.

Analysis of vitelline envelope synthesis and composition during early oocyte development in gilthead seabream (Sparus aurata).

The oocyte vitelline envelope (VE) of gilthead seabream is composed of four known zona pellucida (ZP) proteins, ZPBa, ZPBb, ZPC, and ZPX. We have previously shown that the gilthead seabream ZP proteins are differentially transcribed in liver and ovary, with the expression in liver being under estrogenic control. However, although mRNA was found in both liver and ovary, only low ZPBa protein levels were detected in liver and plasma. Using isoform-specific ZP antibodies we show that ZPBa and ZPX translation products are present in the cytosol of stage I and II oocytes. In addition, the zpBa and zpX mRNAs were detected in early developing oocytes. During oocyte growth (vitellogenesis), the VE increased in thickness (>10 microm), and we show that the four ZP isoforms are present in different regions of the VE. ZPX was detected closest to the oocyte plasma membrane while the intermediate region was composed of ZPBa, ZPBb, and ZPC. At the outer layer, only ZPC was detected. When oocytes reach the fully grown stage they resume meiosis and hydration. As the oocyte expands, thinning to 4 microm, the VE acquire a striped and compact appearance at the electron microscopy level. This study provides further evidence for the oocyte origin of some ZP proteins in the gilthead seabream and suggests that the ZP proteins are differentially distributed within the VE.