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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.

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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.

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.

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., (Copyright © 2012 Académie des sciences. Published by Elsevier SAS. All rights reserved.)

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., (Copyright © 2011 S. Karger AG, Basel.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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 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.

The ultrastructure of normal development and JHA-treated egg was examined with the transmission electron microscope. In newly oviposited egg, the chorion consists of exo- and endochorion. Between the chorion and plasma membrane lies vitelline (envelope) membrane, a thin non cellular membrane. The periplasm is free of the yolk spheres with contains vesicles of various sizes and lipid inclusions. The endoplasm is rich in yolk spheres, mitochondria, lipid inclusions, dense vesicles and vacuoles. In 48 h-old egg, cleavage nuclei was seen migrating to the periphery of the egg. The ooplasm around the nuclei contains vesicles, endoplasmic reticulum and few mitochondria. The blastoderm cells contain many organelles and inclusions. Secondary vitellophages and pole cells were observed. In 96 h-old egg, the germ band is formed. It consists of two layers, ectoderm and mesoderm. Amnion and serosa were visible. At (0-1 hour) eggs post-treatment with JHA (Admiral), no real effects was observed except for vacuolation of endoplasm; while at 48 h & 96 h-old post-treatment of eggs, great deterioration were seen which ended by degeneration and lysis of cell components leaving cell debris, vacuolation of nuclei, iregular shape of nuclear membrane and dispersed chromatin material.

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.

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., ((c) 2008 Wiley-Liss, Inc.)

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.

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.

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.

The vitellogenesis of Paraechinophallus japonicus (Yamaguti, 1934), the first pseudophyllidean tapeworm of the family Echinophallidae studied using transmission electron microscope, is described on the basis of ultrastructural observations of specimens from the benthopelagic fish Psenopsis anomala (Temminck et Schlegel, 1844) (Perciformes: Centrolophidae). The process of vitellogenesis in P. japonicus follows the same general pattern observed in other tapeworms. Five stages of vitellocyte development have been distinguished. The first stage corresponds to immature cells containing ribosomes and mitochondria. The second stage of development is characterized by the appearance of granular endoplasmic reticulum and Golgi complexes, formation of shell globules and lipid droplets at the periphery of the cell cytoplasm. Vitellocyte of the third stage presents accumulation of shell globules and lipid droplets. During the fourth stage, shell globule clusters are formed, and lipid droplets and rosettes of alpha-glycogen are accumulated. Mature vitelline cells are characterized by a great number of lipid droplets with glycogen in the centre of the cytoplasm, whereas shell globule clusters are situated more peripherally. The interstitial tissue of vitelline follicles of P. japonicus is syncytial with long cytoplasmic projections extending between vitelline cells. The presence of a large amount of lipid droplets in the vitelline cytoplasm within the eggs of P. japonicus may be related to egg accumulation in the uterine sac.

Yolk sac epitheliocyte differentiation in rat during physiological pregnancy was examined with transmission electron microscopy and morphometry methods. Divergent development of embryonic structures in visceral and parietal layers was noted on day 11. By day 17 of embryonic development visceral layer epitheliocytes were characterized by morphologic features of intensely resorbing cells, while parietal layer cells revealed some signs of active peptide synthesis. Yolk sac epithelium interaction with trophoblastic elements results in the formation of the sinus of Duval, elements of which are involved in selective transport of substances. Polymorphism of yolk sac epithelium is responsible for different trophic functions in rat fetus during gestation.

The present paper aimed to characterize the substance forming the sperm-associated body (SB), to find its producing sites, and to show its functions in the fertilization of chicken. The SB was found both in between the inner and outer layers of vitelline membranes around eggs and in the oviductal infundibulum. Material from which the SB is constructed (SB substance) was isolated from the vitelline membranes. It was a hydrophobic protein with a molecular size of 570 kDa. X-ray microanalysis detected calcium in the aggregates of the SB substance. Immunofluorescence and immunoelectron microscopy showed that the substance was produced in secretory cells in the luminal epithelium of the oviductal infundibulum and was provided to the egg on and in its vitelline membrane. During incubation, the SB substance bound with spermatozoa in the posterior portion of their flagella. Holes and disks were found in the vitelline membranes of fertile eggs at a ratio of 1: 19-24. Over 94% of the holes were accompanied by SB. The presence of SB is necessary for fertile spermatozoa to make holes in the membrane and to enter the fertile egg.

The ultrastructure of the follicular wall in primordial, previtellogenic and vitellogenic follicles of the sexually immature ostrich is described in the present study. The follicular wall consists of a zona radiata, granulosa cell layer, basal lamina and thecal layer. Cytoplasmic processes from the plasma membranes of the granulosa cell layer and the ovocyte form the zona radiata in previtellogenic and vitellogenic follicles. The granulosa cell layer transforms from simple cuboidal epithelium in primordial follicles to simple columnar or pseudostratified columnar epithelium in previtellogenic and vitellogenic follicles. Transosomes were observed along the apical and lateral plasma membranes of granulosa cells. The thecal layer in previtellogenic and vitellogenic follicles consists of interna and externa components. The fibroblasts in the theca externa contain microfilaments, which are thought to be actin filaments. The study revealed ultrastructural features, which are associated with the transportation of yolk precursors and nutrients into the ovoplasm. In addition, the study indicates that, although the cells in the theca externa contain microfilaments, they do not possess the ultrastructural characteristics of smooth muscle cells.

Oocytes from virtually all organisms are surrounded by at least one coat. This specialized extracellular matrix, called the zona pellucida (ZP) in mammals and the vitelline envelope (VE) in nonmammals, has a structural function and plays essential roles in oogenesis, fertilization, and early development. During the last 15 years, compelling evidence has accumulated that all ZP/VE subunits polymerize using a conserved sequence, the ZP domain, so that the basic structural features of egg coat matrices have been maintained through evolution. Moreover, ZP domains have been identified in many other polymeric extracellular proteins from eukaryotes. This review compares the ultrastructure and molecular composition of egg coats from mollusc to human, suggests a common mechanism for assembly of ZP/VE proteins, and discusses alternative models of how these could be arranged within filaments.

During activation of amphibian eggs, cortical granule exocytosis causes elaborate ultrastructural changes in the vitelline envelope. These changes involve modifications in the structure of the vitelline envelope and formation of a fertilization envelope (FE) that can no longer be penetrated by sperm. In Bufo arenarum, as the egg traverses the oviduct, the vitelline envelope is altered by a trypsin-like protease secreted by the oviduct, which induces an increased susceptibility of the vitelline envelope to sperm lysins. Full-grown oocytes of B. arenarum, matured in vitro by progesterone, are polyspermic, although cortical granule exocytosis seems to occur within a normal chronological sequence. These oocytes can be fertilized with or without trypsin treatment, suggesting that the vitelline envelope is totally sperm-permeable. Vitelline envelopes without trypsin treatment cannot retain either gp90 or gp96. This suggests that these glycoproteins are involved in the block to polyspermy and that trypsin treatment of matured in vitro oocytes before insemination is necessary to enable vitelline envelopes to block polyspermy. The loss of the binding capacity in vitelline envelopes isolated from B. arenarum oocytes matured in vitro with trypsin treatment and activated by electric shock suggests that previous trypsin treatment is a necessary step for sperm block to occur. When in vitro matured oocytes were incubated with the product of cortical granules obtained from in vitro matured oocytes (vCGP), vitelline envelopes with trypsin treatment were able to block sperm entry. These oocytes exhibited the characteristic signs of activation. These results support the idea that B. arenarum oocytes can be activated by external stimuli and suggest the presence of unknown oocyte surface receptors linked to the activation machinery in response to fertilization. Electrophoretic profiles obtained by SDS-PAGE of solubilized vitelline envelopes from oocytes matured in vitro revealed the conversion of gp40 (in vitro matured oocytes, without trypsin treatment) to gp38 (ascribable to trypsin activity or cortical granule product activity, CGP) and the conversion of gp70 to gp68 (ascribable to trypsin activity plus CGP activity). Taking into account that only the vitelline envelopes of in vitro matured oocytes with trypsin treatment and activated can block sperm entry, we may suggest that the conversion of gp70 to gp68 is related to the changes associated with sperm binding.

The present study describes the holes in the inner vitelline membrane of fertile eggs of the quail Coturnix japonica, which remain after the spermatozoa pass through. It was shown that the light-microscopically observable 'holes' correspond mostly to electron-microscopically defined 'disks', and, to a lesser extent (about 5%), real holes. Immunofluorescent staining of the vitelline membranes with an antiquail ZPC antiserum was used to discriminate the holes from the disks light-microscopically. Over 96% of holes were accompanied by calcium-coated sperm-associated bodies, indicating a close relationship between the two. There was no preferential localization of the disks, holes or sperm-associated bodies in the vitelline membrane around the egg. The sperm-associated bodies bound with the spermatozoa at the posterior end of sperm flagella. Incubation of the inner vitelline membranes, isolated from the largest follicles, with spermatozoa resulted in production only of the disks, whereas the holes (about 9%) were produced when the sperm-associated bodies were added to the system. It was suggested that the sperm-associated bodies assist fertile spermatozoa in binding to the inner vitelline membrane, making holes in the membrane and passing through them in fertile eggs.

Oogenesis was examined in nine species of Antarctic fish to verify the existence of morphological peculiarities. The analyses were carried out on specimens belonging to three different families of Notothenioids (Nototheniidae, Channichthyidae and Bathydraconidae), all captured in the Ross Sea, in front of the Italian Station of Terra Nova Bay. Following dissection, the ovaries were processed and examined at the light and electron microscopes to determine the oocyte gross and fine morphology. The attention, in particular, was focused on the presence of cytoplasmic round bodies and on the organization of the cortical alveoli and the vitelline envelope. Results reveal significant specie-specific differences that could be partly correlated to the phylogenetic radiation but not to the peculiar environmental conditions being essentially comparable to those observed among temperate species.

Synbranchus marmoratus is a protogynous diandric teleost fish widely distributed throughout South America. The aim of this work was to study the ultrastructure of the vitelline envelope and the relationship among oocyte and their follicular cells during oogenesis. During perinucleolar stage, the oocyte and the follicular cells form microvillar processes that project into the perivitelline space. The oocyte secretes a dense and amorphous material, which appears as the first evidence of the vitelline envelope (VE) development. The VE passes from a double to a multilayered structure during oocyte growth. In mature oocytes, the VE reach a mean thickness of 11 microm, having up to 30 layers. Oocyte microvilli are thinner than the follicular ones and were seen in contact with the follicular plasmalema, however we could not find any contact between the follicular microvilli and the oolemma. Before ovulation, microvillar processes retract and the pore canals seem to collapse. An outer electron dense layer occludes the superficial pore and forms a continuous layer. No jelly or adhesive coatings were seen at least in ovulated eggs sampled from ovarian lumen. Follicular cell and oocyte cytological characteristics do not differ from those described in other teleosts species., (Copyright 2003 Elsevier Science Ltd.)

Purpose: To study the effect of the presence of coarse granules in the perivitelline space (PVS) of oocytes on embryonic development, and on implantation and pregnancy rates in IVF., Methods: The study population included 24 patients treated during the period 1995-2000. The majority or all of their oocytes exhibited repeatedly coarse granulation in the PVS. Clinical and laboratory cycle characteristics of their 65 IVF-ICSI cycles and the resulting implantation and pregnancy rates were compared to a matched control group of 65 IVF- ICSI cycles without granulation in the PVS., Results: A total of 623 oocytes were retrieved, 418 oocytes fertilized, and 246 embryos were transferred in the study group. No difference was detected between the study and control group with regard to patients' clinical data, IVF cycle characteristics, mean number of oocytes retrieved and fertilized, and mean number of embryos transferred. Only seven pregnancies were achieved in the study group, leading to pregnancy and implantation rates of 10.7 and 5.7%, respectively. Pregnancy and implantation rates were significantly higher in the control group of matched IVF-ICSI cycles without granulation in the PVS (32.5 and 11.5%, respectively)., Conclusions: The presence of coarse granules in the PVS correlates with low implantation and pregnancy rates in IVF-ICSI cycles and might be regarded as a distinct entity, part of the yet poorly defined condition of "egg factor infertility."

Most insect eggshells are ornately sculptured; that of the Karner Blue butterfly, Lycaeides melissa samuelis, exhibits a series of interwoven ridges and depressions. Scanning electron microscopic views of the shell show that the patterning resides in the outer chorion, while the inner vitelline membrane is relatively flat and featureless. We here describe the morphology of the egg and introduce a physical technique, use of a Dynamitron accelerator, to identify and localize elements in the eggshell. Most elements present are represented in the chorion, but sulfur appears restricted to the vitelline membrane. The micropyle is particularly rich in calcium and, in unhatched eggs, phosphorus as well., (Copyright 2002 Wiley-Liss, Inc.)

The investigation of ovaries and the formation of egg envelopes of the stonefly Leuctra autumnalis was carried out with light and transmission electron microscopes. The ovary of the studied species is paired and consists of several dozen panoistic ovarioles opening individually to the oviduct. The process of egg capsule formation already begins in previtellogenesis. At this time the follicular cells secrete precursors of the vitelline envelope. Analysis of the presented data suggests that the oocyte itself also takes part in the formation of the vitelline envelope during late vitellogenesis. Simultaneously, the follicular cells produce precursors of further layers of the egg capsule, i.e. two-layered chorion and extrachorion, consisting of two gelatinous layers and a flocculent one. The completely developed capsule contains channels, probably micropylar ones.

A study was made of the localization and assembly of the VM32E protein, a putative vitelline membrane component of the Drosophila eggshell. The results highlight some unique features of this protein compared with the other proteins of the same gene family. At the time of its synthesis (stage 10), the VM32E protein is not detectable in polar follicle cells. However, it is able to move in the extracellular space around the oocyte and, by stage 11 is uniformly distributed in the vitelline membrane. During the terminal stages of oogenesis the VM32E protein is partially released from the vitelline membrane and becomes localized in the endochorion layer also. By analyzing transgenic flies carrying variously truncated VM32E proteins, we could identify the protein domains required for the proper assembly of the VM32E protein in the eggshell. The highly conserved vitelline membrane domain is implicated in the early interactions with other components and is required for cross-linking VM32E protein in the vitelline membrane. The terminal carboxylic domain is necessary for localization to the endochorion layer. Protein with the C-end domain deleted is localized solely to the vitelline membrane and cross-linked only in laid eggs, as occurs for the other vitelline membrane proteins.

Oocyte maturation is a key issue of current animal biotechnology. This study was designed to examine the morphodynamics of the cumulus-oocyte association during oocyte maturation. Porcine cumulus-oocyte complexes were recovered from slaughterhouse ovaries; matured in vitro for 0, 24, 36, and 44 h; and evaluated by scanning electron microscopy either combined or not combined with the osmium-dimethyl sulfoxide-osmium maceration (ODO) method. The cytoskeleton distribution was also observed by fluorescence staining. Prior to maturation culture (0 h), the spherical cumulus cells were tightly clustered around the oocyte, with narrow intercellular spaces. They showed active secretion at 36 h and were fully expanded at 44 h of culture. The ODO methods revealed that the cumulus cells projected numerous long and thin transzonal projections at 0 h, but these were largely disconnected at 44 h. The outer surface of the zona pellucida showed a meshwork surface regardless of time of incubation, whereas the inner surface changed from a fine fibrous surface to a spongy surface that was coated with mucin. The vitelline surface changed from a sparse distribution of short microvilli (MV) to a dense distribution of well-developed MV. Fluorescence staining showed that the cumulus cell projections consisted mainly of microfilaments, which were abundant at the germinal vesicle and metaphase-I (M-I) stages (0-24 h) but which were decreased in number at the M-II stage (36-44 h). We conclude that the cumulus-oocyte transzonal projections became disconnected between the M-I and M-II stages as a result of cumulus expansion. The cumulus-cumulus communications, however, remained intact at these stages, although the biological functions of these communications were not clear.

Prior studies on cryopreserving embryos of several non-drosophilid flies established that two Drosophila melanogaster embryo cryopreservation protocols were not directly suitable for use with these species. This paper describes our work on developing a protocol for cryopreservation of embryos of the housefly, Musca domestica. Significant progress was made when permeabilization of the vitelline membrane was optimized, a vitrification solution containing ethylene glycol, polyethylene glycol, and trehalose was formulated, and when cooling and recovery of the cryopreservation protocol included a step which passed the embryos through liquid nitrogen vapor. More than 70% of housefly embryos withstand treatments of dechorionation, permeabilization, loading with cryoprotectant, and dehydration in vitrification solution, but the cooling, warming, and poststorage rearing steps still cause a considerable reduction in survival. About 53% of the vitrified M. domestica embryos hatched into larvae. Relative to the percentage of the control adult emergence, about 13% of the embryos stored in liquid nitrogen developed into fertile adults. Hatching of the F(1) progeny of adults having been cryopreserved as embryos was similar to control levels., (Copyright 2000 Academic Press.)

Oocyte activation is a critical component of the current animal cloning scheme. This study was designed to examine surface characteristics of bovine oocytes by scanning electron microscopy (SEM) after activation by calcium ionophore A23187 (A23187) and electric pulse combined with cycloheximide (CHX) or 6-dimethylaminopurine (6-DMAP) treatments. In vitro matured (IVM) oocytes were activated then harvested at 0 to 19 hours after the onset of treatments for SEM processing. The zona pellucida (ZP) of untreated IVM oocytes exhibited an open mesh structure. The ZP surface showed little changes after A23187 alone, but dramatically changed to a less porous surface 3 hours after combined treatments with CHX or 6-DMAP. The vitelline membrane of IVM oocytes was covered with well-developed microvilli (MV). The MV became shorter (0.83 vs. 1.35 microm, p < 0.01) 8 hours after A23187 treatment alone. The vitelline membrane was altered in all oocytes examined 3 hours after incubation with A23187 plus CHX or 6-DMAP. A 1.5-fold increase in the diameter of MV in the CHX group and a higher incidence of large cytoplasmic protrusions (more than 1 microm width) in the 6-DMAP group were observed. After removal of inhibitors, the surface morphologies of the ZP and vitelline membrane were returned nearly to those of untreated IVM oocytes in both groups. The present study clearly showed that surface characteristics of the bovine oocyte were more profoundly changed by a combination of agents for parthenogenetic stimulation, and that the ultrastructural effects were reversible.

The present study analyses, by transmission electron microscopy, vitellogenesis in two anuran amphibian families: Leptodactilidae (Ceratophrys cranwelli) and Bufonidae (Bufo arenarum). These differ in the type of stimulus that sets off their reproductive period, pluvial changes being the trigger in C. cranwelli and temperature increase in B. arenarum. We found that vitellogenesis follows an endocytic pathway that involves membranous structures (coated pits, coated vesicles, endosomes and multivesicular bodies). This process results in a fully grown yolk platelet of similar structure in both species. Despite the above similarity, a distinctive feature in B. arenarum was that the multivesicular bodies exhibited condensed proteins together with lipid droplets, the latter remaining as such even in the primordial yolk platelet. In C. cranwelli, however, lipids droplets were only found attached to the primordial yolk platelet. The coexistence of lipid droplets together with proteins in the nascent precursor yolk platelets observed in B. arenarum is similar to that found in B. marinus. This fact might constitute a characteristic feature of the Bufonidae family.

When adult cat fleas, Ctenocephalides felis (Bouché), were fed concentrations of < or = 0.08 ppm lufenuron in cattle blood, egg hatch did not differ significantly from the controls. However, as the concentration of lufenuron in blood increased from 0.125 to 1.0 ppm egg hatch decreased to 64 and 2%, respectively. Most of these eggs contained fully developed larvae. Microscopic examination of unhatched larvae, revealed that the cuticle epidermal cells, chorion, and vitelline membrane all were affected by lufenuron treatment. Larvae often produced 2 separate cuticles in response to treatment. The 1st cuticle consisted of an indistinct layer of epicuticle and a procuticle composed of randomly deposited chitin microfibrils. After the 1st layer of procuticle separated from the epidermal cells, a 2nd layer of procuticle was deposited. It was not possible to determine whether the egg tooth was functional during larval hatch. The surface of the egg tooth appeared normal, but the cuticle may have had structural abnormalities similar to those seen in other areas of the exoskeleton. Structural defects appeared to be due to the cytotoxic effects of lufenuron. The epidermal cells of treated larvae showed evidence of disintegration (i.e., the nuclei and mitochondria appeared to be degenerating and the amount of endoplasmic reticulum and other cytoplasmic organelles was decreased). The chorion of lufenuron-treated larvae consisted of an outer layer, middle and inner layers that were thinner and less electron dense than those of controls, and lacked the innermost chorionic layer found in the control larvae. The vitelline membrane also was thinner than that of the controls. Larval hatching was prevented by ruptures in the cuticle, which opened during eclosion resulting in the loss of hemolymph and desiccation of the larva. Evidently, tearing of the cuticle was caused by abnormal formation of the procuticle that was not strong enough to withstand the cuticular expansion and muscular movement of the larva within the egg shell.

Electrical activation of oocytes is a key component of the current mammalian cloning regimen. The present study examined the surface ultrastructure alterations of bovine oocytes at different ages following electrical pulse (EP) activation. In Experiment 1, bovine oocytes 22, 30 and 40 h after in vitro maturation were activated by EP. Significantly higher pronuclear formation (57 or 59%) was obtained for the 30- or 40-h oocytes than for the 22-h oocytes (10%). More oocytes (58%) in the 40-h group activated spontaneously than in the 22- or 30-h groups. In Experiment 2, oocytes were cultured for 1, 2, 4 and 8 h after activation and then evaluated by scanning electron microscopy (SEM). The zona pelucida (ZP) meshwork was completely abolished within 1 h after EP in the majority of the oocytes in the 22- and 30-h groups, but no obvious changes were observed in the 40-h group. From 2 h onward, the ZP meshwork was gradually restored in most treated oocytes. It was also noted that the microvilli pattern on the vitelline surface was altered dramatically after EP treatment, with the proportion affected increasing with oocyte age. The present study extends the previous findings that older oocytes are more readily activated and that the age-dependent surface ultrastructural alterations correlate very well with the age dependent activation of oocytes.

Aim: To observe the ultrastructural changes in the body wall and the vitelline cells of Paragonimus westermani in vitro and in vivo before and after triclabendazole treatment., Methods: The worms were obtained from in vitro and in vivo tests. All of the samples were processed by conventional techniques, and observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM)., Results: The external plasma membrane and matrix were cracked or disappeared after the treatment. The necrosis of the muscular layer differed. The cell membranes of cortex and vitelline cells were damaged. Nuclear membrane was damaged partially, heterochromatin solidified and condensed to brim and dissolved. The Golgi complex disappeared, endoplasmic reticulum expanded, mitochodria denatured and dissolved. The damage was more serious in vivo than in vitro., Conclusion: Triclabendazole is remarkablely effective against Paragonimus westermani by damaging the body wall and vitelline cells, mainly affecting the nuclei, membrane structures and microtubular system.

We have shown by means of conventional electron microscopy that the eggshell of Drosophila virilis at the main body of the laid egg consists of the vitelline membrane and the multilayered chorion, which includes the wax layer, the innermost chorionic layer, the endochorion, and the exochorion, while several specialized regions of the eggshell are seen across the anterior-posterior axis of the egg. Biochemical analysis revealed the existence of six quantitatively enriched chorion proteins. Among them, Dvs38 and Dvs36 are synthesized when the innermost chorionic layer and the endochorion are assembled. Immunogold electron microscopy has shown that these two proteins are incorporated in the morphologically complete vitelline membrane apparently through an intercalation process and represent structural components of the endochorion in all the specialized regions of the eggshell. Additionally, by cytochemical means, the enzyme eggshell peroxidase, which is synthesized in parallel with Dvs38 and Dvs36, has been identified as a structural component of the innermost chorionic layer and the endochorion. These findings suggest a complex protein-protein recognition pattern during the formation of the eggshell since the cosecretion of its components (i.e., Dvs38, Dvs36 chorion proteins and eggshell peroxidase) does not recommend their colocalization in the eggshell sublayers and the timing of their synthesis is not related to their final position on the eggshell (i.e., the identification of Dvs38 and Dvs36 chorion proteins as vitelline membrane components)., (Copyright 1998 Academic Press.)

A mechanism for eggshell production in Schistosoma mansoni has been proposed (Wells & Cordingley, 1991), and suggests that the release of eggshell protein globules from the vitelline cells occurs under alkaline conditions within the ootype followed by their subsequent fusion to form the eggshell. Fusion and tanning of these components produces eggshell which autofluoresces. The present study was carried out to determine whether a similar process operates in Fasciola hepatica. A number of drug treatments were used to disrupt key steps in the maturation of vitelline cells. Treatment with the calcium ionophore lasalocid (1 x 10(-5) M) led to the premature release of eggshell globules from the vitelline cells but not their fusion. Incubation in monensin (1 x 10(-6)M), a sodium ionophore and ammonium chloride (NH4Cl) (5 x 10(-2) M), a weak base, resulted in the premature fusion of eggshell protein globules within the vitelline cells and premature tanning of the eggshell protein material. The copper-containing enzyme, phenol oxidase, is thought to be involved in the tanning process during the production of eggs. Diethyldithiocarbamate, (DDC, 1 x 10(-3) M) is a phenol oxidase inhibitor and treatment with this compound, in combination treatments with monensin and NH4Cl, prevented fusion of the vitelline cell globules and tanning of the shell protein material. The results of the study suggest that the mechanism for eggshell formation in F. hepatica is similar to that proposed for S. mansoni and may be common to other trematodes as well.

1. The frequency distribution of points of sperm hydrolysis (or holes) produced per unit area of the inner perivitelline layer was examined in samples of approximately 60 laid eggs, taken on the same day from each of 19 flocks of broiler breeder hens. 2. The holes counted in samples of perivitelline layer from eggs varied from 0 to greater than 100; lower numbers being found in eggs from flocks with lower fertility. 3. The median frequency of holes in the inner perivitelline layer was strongly correlated (r = 0.92) with the median frequency of spermatozoa found trapped in the corresponding outer perivitelline layer. 4. The median frequency of holes in the inner perivitelline layer and of spermatozoa in the outer perivitelline layer were both strongly correlated (r = 0.80 and 0.77, respectively) with flock fertility. 5. It is suggested that counting 'holes' in the inner perivitelline layer of laid eggs is a more convenient method for assessing breeding efficiency and predicting flock fertility than counting spermatozoa trapped in the outer perivitelline layer.

A series of in vitro assays of sperm-egg binding were developed to identify potentially subfertile roosters. Initial assays used either segments of intact hen's egg perivitelline membrane (PVM) placed on a microscope slide or a heat-solubilized extract of PVM (HS-PVM) dried within a flat-bottomed microwell plate, with bound sperm detected with a DNA-specific stain and epifluorescence microscopy. An automated assay was developed using prestained sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetate-HS-PVM and enumeration of bound sperm with a fluorometric microwell plate reader. Four populations of chickens differing in fertility were evaluated with the following results: 1) the correlation across lines between in vitro sperm binding and fertility was 0.83 (N = 40; p < 0.0001); 2) correlations with other seminal parameters were low; and 3) the relationship between sperm binding and fertility was not linear, but a threshold plot allowed identification of males with low binding and low fertility. Motile sperm from roosters, turkeys, bulls, humans, mice, rams, and stallions bound in a dose responsive manner. Features of binding were revealed by both scanning and transmission electron microscopy. Use of this assay to cull males whose semen appears normal by traditional modes of analysis but differs in the obligatory trait of sperm-egg binding could be of value to avoid expensive progeny testing.

Monoclonal antibodies were raised against zona-free mouse oocytes by intrasplenic immunizations. Antibodies, which detected antigen epitopes in unfertilized but not in fertilized mouse eggs (fertilization-specific antibodies) and which in addition recognized epitopes in unfertilized human oocytes (mouse-human cross-reacting antibodies) were used to visualize the distribution of their corresponding antigens by confocal laser fluoresence scanning microscopy (CLFSM). We found that among 26 antibodies tested, two ones showed a patch pattern. Since a patchy distribution of cell membrane molecules indicates the presence of integral proteins with ability to transduce signals to the cytoplasm, we suggest that the method here applied will select for those oolemma molecules that could be involved in the fusion of sperm cells.

The vitelline coat (VC) glycoproteins of the Unio elongatulus egg, purified as previously described (Focarelli and Rosati, 1993: Mol Reprod Dev 35:44-51) and indicated as gp220 and gp180 by virtue of their apparent molecular weights in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The analysis confirmed the purity of our preparations and the mass of gp180, but gave a mass of 273,000 for gp220. Intact VCs and purified VC components were then visualized in stereo images of platinum replicas produced by the quick-freeze, deep-etch, and rotary shadowing techniques: gp180 revealed a c-like shape and gp273 a rosette-like shape. The intact VCs were found to consist of two layers, the internal one clearly fibrous and the external one compact. Since purified preparations of gp180 spontaneously formed fibrils of similar width to those present in the inner VC layer, this layer presumably consists mainly of this component. The prevalence of gp273 in the outer layer is also suggested and discussed.

The biochemical and ultrastructural changes in the envelope of the Xenopus laevis egg that occur during oviposition and fertilization have been thoroughly studied (Hedrick, J. L., and Nishihara, D. M., Methods Cell Biol. 36, 231-247, 1991; Larabell, C. A., and Chandler, D. E., J. Electron Microsc. Tech. 17, 294-318, 1991). However, the biological significance of these changes with respect to gamete interaction has been unclear. In the current study, it was found that changes in the envelope are directly responsible for regulating sperm-egg adhesion, an initial step of fertilization. As a result of these transformations, sperm bind only to unfertilized oviposited eggs, not to oocytes or coelomic eggs. In addition, they do not bind to fertilized eggs. The molecular and cellular basis of the regulation of the sperm binding process was investigated in the context of our recent findings that two structurally related envelope glycoproteins, gp69/64, serve as sperm receptors during fertilization (Tian, J.-D., Gong, H., Thomsen, G. H., and Lennarz, W. J., J. Cell Biol. 136, 1099-1108, 1997). Although the purified gp69/64 glycoproteins isolated from the oocyte or coelomic egg envelopes exhibited sperm binding activity, when these proteins are part of the intact oocyte or coelomic egg envelopes, they are not accessible to either anti-gp69/64 antibodies or to sperm. During the conversion from the coelomic to the vitelline envelope, the gp69/64 sperm receptors become exposed on the surface, an event that correlates with proteolytic cleavage of gp43 and accompanying ultrastructural alterations in the envelope. Conversely, after fertilization, when the vitelline envelope of the egg is converted to the fertilization envelope of the zygote, limited proteolytic cleavage of the sperm receptor results in loss of sperm binding activity. In addition, formation of a fertilization layer on top of the structurally altered VE adds another physical block to sperm binding. These results provide new insights into structure-function relationships between envelope components of the anuran egg, and provide further evidence supporting the key role of gp69/64 as sperm receptors during X. laevis fertilization.