Vitelline Envelope Research Articles

Immunochemical detection of the major vitelline envelope proteins in the plasma and oocytes of the maturing female rainbow trout, Oncorhynchus mykiss.

The major vitelline envelope proteins were detected in the plasma of female rainbow trout maturing under natural conditions by using the Western blot technique. Females were sampled every month from July until ovulation in January. The amount of vitelline envelope proteins in plasma increased markedly as the gonads increased in size from 0.4 to about 15% of the total body weight. The plasma level of oestradiol-17 beta largely followed the alterations in the amount of vitelline envelope proteins, indicating the endocrine control of vitelline envelope protein synthesis. In addition, plasma vitellogenin changed in a manner that resembled the changes in the amount of plasma vitelline envelope proteins. The appearance and growth of the vitelline envelope during oocyte development was demonstrated using immunohistochemical methods. The vitelline envelopes from oocytes at different stages of development were immunoreactive with the antibodies directed against the major vitelline envelope proteins. No immunoreactivity could be observed in the ooplasm or in the surrounding follicular cells, which indicated that the major vitelline envelope proteins were of extraovarian origin. The present study further supports the hypothesis that the major protein constituents of the vitelline envelope in teleosts are under the endocrine control of oestradiol-17 beta and that the site of synthesis is outside the ovary.

Evaluation of corneal epithelial cell replication after alkali injury by immunocytochemistry for proliferating cell nuclear antigen

The extracellular matrix of Xenopus laevis eggs was analyzed at fixed intervals after prick-activation using quick-freeze, deep-etch, rotary-shadow electron microscopy. This technique revealed that the modifications of the matrix seen at fertilization do not occur simultaneously, but that instead there is an orderly progression of alterations at activation. The first modification, conversion of the vitelline envelope (VE) to the altered vitelline envelope (VE∗), occurs within 2 to 3 min after activation. Intermediate stages of the VE to VE∗ transformation can be visualized traveling around the egg in a wave-like fashion. Upon completion of the wave, the loosely woven outer surface of the VE, believed to be the prefertilization layer, remains unaltered. Subsequent formation of the fertilization (F) layer at this VE-jelly interface occurs between 4 and 8 min postactivation. Finally, between 10 and 15 min postactivation, the smooth (S) layer forms on the tips of the microvilli and surrounds the entire egg.

Post-fertilization changes in Discoglossus pictus (Anura) eggs result in the formation of a capsular chamber where the egg rotates.

Discoglossus pictus is one of the few anurans with an egg where a capsular chamber forms as a consequence of fertilization; the egg with its vitelline envelope rotates in this chamber according to gravity. We investigated the formation of the capsular chamber through various experimental cytochemical and ultrastructural approaches, and found that it is the product of plug liquefaction. The plug is a lens-shaped jelly coat typical of Discoglossus, and covering only part of the egg animal half. About 15 min after fertilization, granular material coming from the egg enters the plug, which gradually dissolves and, once liquefied, reorganizes itself around the entire egg, thus forming the chamber. This process goes through stages of rearrangement of the 25-A- and 250-A-thick filaments which constitute the plug matrix. The material entering the plug derives from the exocytosis of two vacuole types, with electron transparent and granular PAS-positive contents. Liquefaction of the plug correlates with the reduction of disulfide bonds present in its matrix. Furthermore, in vitro tests showed that the substances released from the egg are active in selectively dissolving only the plug, and lose activity upon boiling.

Enzymatic basis for protection of fish embryos by the fertilization envelope

The mechanism by which the fertilization envelope (FE) is able to protect the embryo of fish until hatching is almost unknown, except for its function as a physical barrier. FE extract from activated or fertilized eggs of the fish Salmo gairdneri was demonstrated to contain enzyme activities using an agar plate enzyme assay. The enzymes apparently active were carboxymethylcellulase (cellulase; EC 3.2.1.4), laminaranase (endo-1,3(4)-beta-glucanase; EC 3.2.1.6), carboxymethylchitinase (chitinase; EC 3.2.1.14), xylanase (endo-1,4-beta-xylanase; EC 3.2.1.8), mannanase (mannan 1,2-(1,3)-alpha-mannosidase; EC 3.2.1.77), dextranase (EC 3.2.1.11), a protease and lysozyme (EC 3.2.1.17). The FE extract exerted an antifungal or fungicidal action on the fungus Saprolegnia parasitica, whereas an extract from the vitelline envelopes (VE) has no apparent enzyme activity nor antifungal or fungicidal action. Enzymes acquired by the FE through the cortical reaction may have an important defensive role, protecting the embryo against invaders or pathogens.

The Divergence of Species-Specific Abalone Sperm Lysins is Promoted by Positive Darwinian Selection.

Recognition by sperm lysin of the egg vitelline envelope may be one component in determining the species-specificity of fertilization in abalones. The amino acid sequences of lysin proteins of seven California abalone species were deduced from the cDNA sequences. This is the first extensive comparison of a gamete recognition protein from congeneric species. Each prelysin has a highly conserved signal peptide of 18 amino acids, followed by a mature sequence of 136-138 residues. Of 136 aligned positions, 68 have the same amino acid in all seven sequences. The % identity relative to the red abalone lysin sequence is: white 90%, flat 83%, pinto 82%, pink 78%, black 71%, and green 65%. Hydropathy plots and a distance tree of the seven lysins show that red, white, and flat lysins are more closely related to each other than to the lysins of the other four species. A hypervariable, species-specific, domain exists in all sequences between positions 2-12. Amino acid replacements between any two lysins are mostly nonconservative. Analysis of the cDNA sequences shows the number of nonsynonymous substitutions (amino acid altering) exceeds the number of synonymous substitutions (silent) in 20 of the 21 pairwise comparisons of the seven sequences, indicating that positive Darwinian selection must promote the divergence of lysin sequences.

Ultrastructural rearrangements of the vitelline envelope during egg development inEyprepocnemis plorans(Charp.) (Orthoptera, Acrididae)

Abstract The vitelline envelope of Eyprepocnemis plorans eggs undergoes rearrangement after ovulation. This modification involves the gradual condensation of the original structure, which separates from the oolemma, and the elaboration of an underlying granular layer. Both these layers reach their definitive arrangement at an advanced stage of embryogenesis, probably fulfilling a similar functional requirement for egg water‐exchange. A study of parthenogenetic eggs was also undertaken to evaluate the role played by the male gamete in these modifications. The results show that while vitelline envelope rearrangements are independent of fertilization, this seems to be indispensable for the genesis of the granular layer.

Oestradiol-17β induces the major vitelline envelope proteins in both sexes in teleosts

During growth of the ovarian follicle, the teleost oocyte becomes surrounded by an acellular coat, the vitelline envelope. The nature, origin and number of the vitelline envelope proteins in fish appear to vary with species. In this work, polyclonal antibodies directed against vitelline envelope proteins from rainbow trout, brown trout and turbot were used to show that oestradiol-17 beta induces the major vitelline envelope proteins in juveniles, both males and females, from different species. The fact that males can synthesize vitelline envelope constituents shows that the origin of these proteins is not confined to the ovary. The vitelline envelope of rainbow trout eggs consists of three major proteins, designated alpha (60 kDa), beta (55 kDa) and gamma (50 kDa). The amino acid composition of each of the three proteins indicated that the three proteins are alike and the suggestion that these proteins represent a separate class of structural proteins is sustained.

The eggs of Forficula auricularia L. (Dermaptera, Forficulidae): ultrastructure and resistance to low and high temperatures

The morphology, ultrastructure, and resistance to low and high temperatures of the fertilized egg of Forficula auricularia were studied both before and after water absorption. The increase in volume resulting from water absorption did not modify the ultrastructure of the envelopes. The chorion was found to be made of five distinct layers and of a vitelline envelope under which a serosal cuticle was secreted by the embryo. Before hydration, the supercooling point of the egg was −27 °C; after hydration, it was −22 °C. Hydration did not alter the resistance of the egg to heat. It withstood a thermal rise to 36 °C without apparent damage. Morphological and physiological changes due to water absorption did not result in a modification of the viability of the eggs when they were exposed to temperature differences.

Enzyme activities and antifungal action of fertilization envelope extract from fish eggs

AbstractExtracts from fertilization envelopes (FE) of activated eggs of two species of fish, Plecoglossus altivelis and Tribolodon hakonensis, have been demonstrated to contain carboxymethylcellulase, carboxymethylchitinase, β‐1,3‐glucanase (or laminaranase), lichenase, xylanase, dextranase, mannase, protease, and lysozyme activities by an agar plate method. Vitelline envelope (VE) extracts in contrast had no enzyme activities. The FE extracts exerted an antifungal or fungicidal action on the fungus Saprolegnia parasitica which was isolated from the body surface of a carp with mycosis. Inhibition of hyphal growth from the mycelial pieces was time‐dependent. The VE extracts had no antifungal action. FE extracts treated with pronase exerted no antifungal or fungicidal action on S. parasitica, but trypsinized FE extracts still retained their antifungal or fungicidal effect. These results suggest that the FE may be able to protect the embryo through enzymatic activity, which is acquired after the process of cortical reaction, and may help to degrade the cell walls of potential pathogens.

Glycoproteins from Bufo arenarum vitelline envelope with fertility-impairing effect on homologous spermatozoa

When spermatozoa from Bufo arenarum are incubated with molecules extracted from the vitelline envelopes of homologous oocytes, they lose their fertilizing capacity. Those molecules are glycoproteins, and the elimination of mannoside residues from them results in activity loss, while digestion of the proteic moiety did not alter their biological effect. Sepharose-concanavalin A columns were used to purify the glycoproteins, since the active fraction binds to the column. The fertility-impairing effect observed does not seem to be mediated by an acrosome reaction-inducing effect.

Gamete interactions during in vitro fertilization of lobster ( Homarus americanus) Oocytes

Preovulatory lobster oocytes were mechanically removed from follicles and fertilized in vitro with sperm from the vas deferens of males or the seminal receptacle of females. Oocytes were fixed for ultrastructural analysis at various times after insemination. Stages in gamete interaction beginning with sperm binding to envelope 1A and extending through nuclear envelope breakdown of the fertilizing sperm were examined microscopically. Numerous sperm bind to envelope 1A of mature oocytes. Only properly oriented sperm undergoing normal acrosome reactions penetrate envelopes 1A and 1B (together these form the vitelline envelope). During the acrosome reaction, the inner acrosomal material comes in direct contact with envelope 1B and does not appear to release a lysin for penetration of envelopes 1A and 1B. The acrosomal membrane overlying the acrosomal filament appears to be the first membrane of the sperm to contact the oolemma and is probably the initial site of gamete membrane fusion. Following fusion, egg cytoplasm flows around the sperm nucleus forming a small fertilization cone. The acrosomal contents are left outside the oocyte in the pervitelline space. Once gamete membrane fusion has occurred, the nuclear envelope fragments, and by 120 min after insemination, it has completely disappeared around some sperm nuclei. As sperm are incorporated into the ooplasm, the microtubules associated with the membrane/microtubule complex of the nucleus depolymerize, and the membranes of this complex are less frequently observed. After gamete membrane fusion, the acrosomal filament is contiguous with oocyte cytoplasm, and it remains structurally unchanged by 120 min after insemination. It is probable that the oocyte releases some high density cortical granules within the first 120 min of insemination; however, there was no evidence for the release of low density vesicles, ring vesicles, and moderately dense vesicles during this time.

The interaction of boar sperm proacrosin with its natural substrate, the zona pellucida, and with polysulfated polysaccharides

Boar sperm acrosin is an acrosomal protease with trypsin-like specificity, and it functions in fertilization by assisting sperm passage through the zona pellucida by limited hydrolysis of this extracellular matrix. In addition to a proteolytic active site domain, acrosin binds the zona pellucida at a separate binding domain that is lost during proacrosin autolysis. In this study, we quantitate the binding of proacrosin to the physiological substrate for acrosin, the zona pellucida, and to a non-substrate, the polysulfated polysaccharide fucoidan. Binding was analogous to sea urchin sperm bindin that binds egg jelly fucan and the vitelline envelope of sea urchin eggs. Proacrosin was found to bind to fucoidan and to the zona pellucida with binding affinities similar to bindin interaction with egg jelly fucan. These interactions were competitively inhibited by similar relative molecular mass polysulfated polymers. Since bindin and proacrosin have distinctly different amino acid sequences, their interaction with acidic sulfate esters demonstrates an example of convergent evolution wherein different macromolecules localized in analogous sperm compartments have the same biological function. From cDNA sequence analysis of proacrosin, this binding may be mediated through a consensus sequence for binding sulfated glycoconjugates. Proacrosin binding to the zona pellucida may serve as both a recognition or primary sperm receptor, as well as maintaining the sperm on the zona pellucida once the acrosome reaction has occurred.

In vitro fertilization of lobster oocytes

AbstractThe feasibility of fertilizing preovulatory lobster oocytes was examined in vitro under various experimental conditions. Large (1.6 mm diameter) and small (0.6 mm diameter) oocytes were compared in fertilization trials. Small oocytes were surrounded by thin envelope thought to correspond to envelope 1A of mature oocytes. Large oocytes were surrounded by a fully formed vitelline envelope comprised of two distinct sublayers, 1A and 1B. Although sperm bound very effectively to the coat surrounding small oocytes, none penetrated the coat or fertilized the oocytes. Large diameter oocytes that were removed from follicles by dissection were fertilized by sperm from the proximal vas deferens of males and by sperm from the seminal receptacle of females. Fertilized oocytes showed a high degree of polyspermy. Higher numbers of sperm bound to, penetrated, and fertilized large diameter oocytes when inseminations were done in a saline solution (2.5LSH) than when done in artificial sea water (ASW). Sperm failed to bind to large diameter oocytes that were induced to ovulate in vitro by collagenase treatment. Contaminating enzymes may have destroyed the sperm receptor in envelope 1A of collagenase treated oocytes. Our in vitro fertilization method will allow the process of fertilization to be studied experimentally in lobsters, and it may be applicable to other decapods.

Fertilization‐Induced changes in the vitelline envelope of echinoderm and amphibian eggs: Self‐assembly of an extracellular matrix

The surface of the unfertilized sea urchin egg is covered by the vitelline layer (VL), a fibrous extracellular matrix that contains receptors for sperm. At fertilization, cortical granule exocytosis releases enzymes and structural proteins that cause the VL to elevate and become remodelled into the mechanically and chemically tough fertilization envelope. This envelope prevents further penetration of sperm and protects the embryo during early development. A thicker, more complex vitelline envelope surrounds the Xenopus laevis egg. This fibrous coat is also restructured at fertilization to produce an impenetrable barrier to sperm. The biochemical steps that occur during self-assembly of these fertilization envelopes are reviewed, and the ultrastructural changes that occur, as seen in platinum replicas of quick-frozen, deep-etched, and rotary-shadowed eggs, are illustrated.

Control of follicular epithelium development and vitelline envelope formation in the mosquito; role of juvenile hormone and 20-hydroxyecdysone

Using microsurgical manipulations, hormone applications, and transmission electron microscopy we have investigated the regulation of differentiation of the follicular epithelium and formation of the vitelline envelope (VE) in primary follicles in the ovary of the mosquito, Aedes aegypti. During the first 3 days after eclosion, the primary follicle grows, and cells of the follicular epithelium differentiate, their content of mitochondria, rough endoplasmic reticulum, and Golgi complexes increases significantly. Growth and differentiation of the follicular epithelium appear to be under the control of juvenile hormone (JH), because they are blocked by removal of corpora allata in newly closed adult females and can be restored by either implantation of corpora allata or application of JH III. In insects, including mosquitoes, VE is the first layer of the eggshell to be deposited. It is formed from the secretory products of the follicle cells and its deposition coincides with yolk accumulation by developing oocytes. Only follicle cells adjacent to the oocyte deposit VE. In decapitated females, given a blood meal by enema and injected with picogram doses of 20-hydroxyecdysone (20-HE), follicle cells synthesize the VE precursors and deposit morphologically normal VE, in contrast to saline injected controls which deposit no VE. We conclude that 20-HE, as well as factors originating from the blood meal and the oocyte, are required for the normal formation of VE in the mosquito follicles.

Egg envelopes in diplopods, a comparative ultrastructural study

By means of electron microscopy two types of egg envelope have been described in representatives of two diplopod subclasses, the Chilognatha and the Pselaphognatha. The vitelline envelope appears on the oocyte surface in early previtellogenesis and persists till ovulation. In its thin and filamentous structure it resembles basement membranes. During vitellogenesis electron dense material is deposited on filamentous scaffolding which fills the space between the oolemma and the vitelline envelope. As a result, the thick and spongy or filamentous chorion is formed. In the present study it has been shown that regardless of the type of oogenesis (solitary—the Chilognatha, or follicular—the Pselaphognatha) both envelopes in diplopods are produced by the oocyte itself, and although completely different in structure and time of appearance, they must be both considered as primary.

Ultrastructural and histochemical studies of the egg capsules of Perla marginata (Panzer, 1799) and Dinocras cephalotes (Curtis, 1827) (Plecoptera : Perlidae)

Eggshells of stone flies P. marginata and D. cephalotes (Plecoptera : Perlidae), inhabiting mountain streams, were examined using scanning and transmission electron microscopes, a phase-contrast light microscope and histochemical methods to detect proteins, lipids and polysaccharides. The eggshells of the species investigated consist of a vitelline envelope, chorion and gelatinous sheet decorated on its outer surface with mushroom-like structures. An anchoring structure (attachment disc) is situated on the posterior pole of the egg. The structure and function of the attachment disc, as well as the possible taxonomic applications, are discussed. The morphology and histochemical composition of all these elements of the shell clearly demonstrate good adaptation to land and aquatic habitats; the chorion consists of 2 layers, the internal layer being finely perforated by numerous aeropyles. The external layer, with fewer, regularly placed aeropyles, protects the egg interior against dehydration in the land habitat. The gelatinous sheet seems to provide additional protection. Mushroom-like structures, situated on its surface, correspond with the positions of aeropylar openings. These and other interrelations between chorion structure and function are discussed.

Deaminated neuraminic acid-rich glycoprotein of rainbow trout egg vitelline envelope. Occurrence of a novel alpha-2,8-linked oligo(deaminated neuraminic acid) structure in O-linked glycan chains.

Deaminated neuraminic acid-rich glycoprotein (KDN-gp), first found and isolated from the vitelline envelope of rainbow trout eggs (Inoue, S., Kanamori, A., Kitajima, K., and Inoue, Y. (1988) Biochem. Biophys. Res. Commun. 153, 172-176), has been found to contain a number of O-linked glycan. Oligosaccharides were released by alkaline borohydride treatment of KDN-gp. Following fractionation by DEAE-Sephadex A-25 and thin-layer chromatography, a series of acidic oligosaccharides were obtained and analyzed for their chemical structures. The structure is based on composition analysis, methylation analysis, alkali-catalyzed "peeling" reactions, periodate oxidation, 400-MHz one- and two-dimensional 1H NMR spectroscopy, and molecular secondary ion mass spectrometry. The O-linked oligosaccharides isolated from KDN-gp have been shown to contain a common core trisaccharide Gal beta 1-3GalNAc alpha 1-3GalNAc in which the terminal Gal residue is blocked by a single residue of deaminated neuraminic acid (KDN) and the proximal GalNAc residue is substituted by alpha-2,8-linked oligo(KDN) chains. Structures of KDN-oligosaccharide chains in the glycoprotein are novel and expressed by the following general formula, where n = 0-5, for which data are available. [formula: see text]

Ultrastructural aspects of oogenesis and oocyte growth in fish and amphibians

Oogenesis, the early events of primary oocyte growth (meiotic arrest, synapsis, ribosomal gene duplication), and folliculogenesis can be seen to particular advantage in the germinal ridge of the syngnathan ovary. After budding off the germinal ridge (a compartment of the luminal epithelium), nascent follicles then enter into a linear array of developing follicles within which temporal and stage-specific events can be correlated with spatial distribution. Prominent features of the later phase of primary oocyte growth include intense transcriptional activity and the formation and subsequent dispersal of the Balbiani vitelline body (mitochondrial cloud) concomitant with an increase in cytoplasmic organelles and volume. Further oocyte growth is characterized by a period of cortical alveolus (in teleosts) or cortical granule (in anurans) formation, in which Golgi elements play a predominant role, and finally vitellogenesis. The latter process, which is responsible for the preponderance of oocyte growth, includes the hepatic synthesis and secretion of vitellogenin (VTG), the uptake of VTG from the bloodstream into the oocyte by receptor-mediated endocytosis, and the transport of VTG via endosomes and multivesicular bodies to forming yolk platelets. In the process, VTG is proteolytically cleaved into the yolk proteins, which assume either a monoclinic (in cyclostomes) or orthorhombic (in teleosts and amphibians) crystalline array. Other structures associated with the growing oocyte are also briefly discussed, including nuage, the vitelline envelope, intercellular junctions between the oocyte and overlying follicle cells, pigment, intramitochondrial crystals in ranidae, and annulate lamellae.

Ultrastructure of sperm, spermiogenesis, and sperm‐egg interactions in selected invertebrates and lower vertebrates which use external fertilization

This review discusses the ultrastructure of sperm with reference to their development, the surface morphology of the egg, and the processes of sperm binding and penetration during fertilization. These topics are treated for selected invertebrates and lower vertebrates which live in aquatic environments and fertilize their eggs externally. Specifically, sperm eggs from cnidarians, echinoderms, decapod crustaceans, ascidians, lampreys, bony fishes, and amphibians are discussed. Sperm from the majority of these groups exhibit the classical head-midregion-tail configuration characteristic of primitive sperm. Specific variations within this general morphology have been described. The notable exceptions to the primitive-sperm paradigm are the sperm of decapod crustaceans and amphibians. Eggs from all of the animals considered are covered by complex vitelline envelopes except those of cnidarians. In general, the ultrastructural analysis of these egg envelopes shows that they are composed of fibrous subunits. Sperm bind to the vitelline envelope and then penetrate through it to fertilize the egg in all groups reviewed except fishes. In sperm ultrastructure which occur during penetration of the egg envelopes in both flagellated and non-flagellated sperm. These changes, which involve membrane fusion and reorganization as well as movement of membranous organelles, aid the sperm in reaching the actual site of gamete fusion.