Vitelline Envelope Research Articles

Role of oviductal pars recta extract on interspecific fertilization between anurans.

During their journey through the oviductal pars recta, the vitelline envelope (VE) of Bufo arenarum oocytes encounter structural alterations that make them sensitive to attack by sperm lysin and thus to penetration by sperm cells. The role of pars recta (PR) on the specificity of fertilization between amphibians was analyzed by conditioning Bufo arenarum oocytes with either homologous PR extract (PRE) or Leptodactylus chaquencis PRE. The oocytes were thereafter exposed to sperm lysin preparations from both species. Lysis of the VE only took place when the oocytes were exposed to the homologous PRE. The pattern of protein composition of PRE of these species was strikingly different as shown by Coomassie blue staining of SDS-PAGE. Moreover, antibodies against PR fluid (PRF) of Bufo arenarum produced seven bands of immunoprecipitation in electrophoresed homologous PRE and only one faint band in Leptodactylus chaquencis PRE. Here we show that: (i) the biological activity of PR from Bufo arenarum and Leptodactylus chaquencis over the VE of Bufo arenarum oocytes is species-specific; (ii) this specificity seems to be based in differences in protein structure, which was indicated by the fact that proteins from PRE of Leptodactylus chaquencis and Bufo arenarum were antigenically distinct; (iii) the specificity was solely related to PR activity and not to sperm lysin activity since sperm lysin preparations from both species showed comparable activity.

Crystal structure and subunit dynamics of the abalone sperm lysin dimer: egg envelopes dissociate dimers, the monomer is the active species.

Lysin is a 16-kD acrosomal protein used by abalone spermatozoa to create a hole in the egg vitelline envelope (VE) by a nonenzymatic mechanism. The crystal structure of the lysin monomer is known at 1.9 A resolution. The surface of the molecule reveals two tracks of basic residues running the length of one surface of the molecule and a patch of solvent-exposed hydrophobic residues on the opposite surface. Here we report that lysin dimerizes via interaction of the hydrophobic patches of monomers. Triton X-100 dissociates the dimer. The crystal structure of the dimer is described at 2.75 A resolution. Fluorescence energy transfer experiments show that the dimer has an approximate KD of 1 microM and that monomers exchange rapidly between dimers. Addition of isolated egg VE dissociates dimers, implicating monomers as the active species in the dissolution reaction. This work represents the first step in the elucidation of the mechanism by which lysin enables abalone spermatozoa to create a hole in the egg envelope during fertilization.

Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization.

Abalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after the sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface.

Control of cell volume in oocytes and eggs from Xenopus laevis

The mechanism by which Xenopus laevis oocytes and eggs survive in hypotonic media was investigated by measuring cell volume (Vc) and intracellular pressure (P(ic)) during osmotic swelling. After 2 hr in 10 mOsm buffer, eggs remained intact, while 60.2 +/- 7.0% of oocytes had burst. Water uptake was proportional to buffer osmolarity in oocytes, but not in eggs. We did not see a regulatory volume decrease in the cells, nor was there any evidence for osmolyte efflux. Oocyte P(ic) increased from 0.27 cm H2O (range 0.14-0.61 cm H2O) to 5.92 cm H2O (range 2.52-7.18 cm H2O, P < 0.001) after 70 min in 20 mOsm buffer. Egg P(ic) in 20 mOsm buffer (0.04 cm H2O, range -0.07 to 0.014 cm H2O) was not significantly different from the value in isotonic (200 mOsm) buffer (0.14 cm H2O, range 0-0.16 cm H2O). The data indicate that Vc in oocytes and eggs is not regulated by either plasma membrane ion transport or the mechanical properties of the vitelline envelope. While oocytes appear to behave as passive osmometers, we speculate that the ability of eggs to maintain volume is determined, in part, by the cytoplasm, in particular by its gel-like nature.

Amino acid composition and endocrine control of vitelline envelope proteins in European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata).

The vitelline envelopes of European sea bass and gilthead sea bream are both composed of mainly four proteins with the molecular masses of 90, 52, 48, 45 kDa and 75, 50, 48, 44 kDa, respectively. Each protein has an amino acid composition that is characterized by a high content of proline and glutamic acid and a low content of cysteine, similar to the whole vitelline envelope of both species. The amino acid composition suggests that each protein is distinct but related to the other vitelline envelope proteins. The use of homologous antisera shows that both species have vitelline envelope proteins that are induced by estradiol-17 beta. As males of both species synthesize these proteins after treatment with estradiol-17 beta, the origin is not restricted to the ovaries. Vitellogenin of both European sea bass and gilthead sea bream has the apparent molecular mass of 170 kDa.

Eggshell ultrastructure in Onychogomphus forcipatus unguiculatus (Vander Linden) (Odonata: Gomphidae)

The eggshell ultrastructure of the exophytic riverine dragonfly Onychogomphus forcipatus unguiculatus (Odonata: Gomphidae) is presented using transmission electron microscopy. A homogeneous vitelline envelope is surrounded by a thick, many-layered endochorion and a relatively thick, homogeneous exochorion. The micropylar projection is covered by a jelly-layer. The structure of the shell is intermediate between known endophytic and exophytic eggshells in the order. A possible evolution of jelly-covered exophytic eggs from endophytic riverine ancestors is discussed.

Extraordinary divergence and positive Darwinian selection in a fusagenic protein coating the acrosomal process of abalone spermatozoa.

During fertilization in marine invertebrates, fusion between sperm and egg cell membranes occurs at the tip of the sperm acrosomal process. In abalone sperm the acrosomal process is coated with an 18-kDa protein. In situ, this protein has no effect on the egg vitelline envelope, but in vitro it is a potent fusagen of liposomes. Thus, the 18-kDa protein may mediate membrane fusion between the gametes, a step in gamete recognition known to restrict heterospecific fertilization in other species. The cDNA and deduced amino acid sequences of the 18-kDa protein were determined for five species of California abalone. The deduced amino acid sequences exhibit extraordinary divergence; the percent identity varies from 27% to 87%. Analysis of nucleotide substitution shows extremely high frequencies of amino acid-altering substitution compared to silent substitution, demonstrating that positive Darwinian selection promotes the divergence of this protein. However, amino acid replacement is conservative with respect to size and polarity of residue. The data support the developing idea that in free-spawning marine invertebrates, the proteins mediating fertilization may be subjected to intense, and as yet unknown, selective forces. The extraordinary divergence of fertilization proteins may be related to the establishment of barriers to heterospecific fertilization.

Long-lasting exocytosis and massive structural reorganisation in the egg periphery during cortical reaction inPlatynereis dumerilii(Annelida, Polychaeta)

The course of the cortical reaction in the Platynereis dumerilii egg is described from live observation and from sectioned fixed material and is found to differ in several aspects from the course of cortical reactions in better-known systems. Cortical granules are unusually numerous. They are discharged by exocytosis during a period of about 25 min following fertilisation (18 degrees C). Most of the surplus membrane material brought to the egg surface by exocytosis is set free into the perivitelline space. Swelling of egg jelly precursor secreted by cortical granule exocytosis may be causal for the detachment of the vitelline envelope from the egg cell surface which, however, remains attached punctately to the vitelline envelope by about 30,000 microvilli. Under the strain of the distending vitelline envelope, the bases of the microvilli move and line up, pulling the cell surface into a network of ridges. The grooves in between the ridges are the sites of exocytoses. Cytochalasin B, generally destabilising actin filaments, induces rupture of the microvilli and exaggerated distension of the vitelline envelope during the cortical reaction. In a final phase of the cortical reaction the vitelline envelope wrinkles and falls back onto the egg cell surface, the microvilli shorten and the egg cell transiently becomes deformed by local contractions. The cortical reaction in the nereid egg is discussed as a process of distortion and reorganisation of the egg cortex and plasmalemma. The abundance of cortical granules accommodating egg jelly precursor in the Platynereis oocyte is attributed to the mode of so-called diffuse oogenesis characteristic of nereids, i.e. of differentiation of oocytes freely suspended in the coelomic fluid. In nereids, egg jelly therefore forms after fertilisation as opposed to ovulation.

Binding of bacterial toxins to glycoproteins in the envelopes of rainbow trout eggs.

The ability of the vitelline and fertilization envelopes of rainbow trout eggs to trap toxins was investigated using cholera enterotoxin B and staphylococcal enterotoxin B in cytochemical or immunocytochemical experiments. Extracts from both envelopes were investigated by immunoblot analysis to identify toxin-binding proteins after SDS-PAGE. Binding studies of cholera enterotoxin B to vitelline envelopes and fertilization envelopes revealed a greater reactive intensity in the former. Treatment with neuraminidase enhanced the reactive intensity (or deposit) in the vitelline envelope and fertilization envelope outermost layers, with more conspicuous reactivity in the former. Cytochemical experiments showed that exogenous ganglioside GM1 considerably enhanced cholera enterotoxin B binding to vitelline and fertilization envelopes. This enhancement was shown by an intense reactivity following the occurrence of new binding sites on the vitelline envelope inner surface and the inner wall of the zona radiata, a simultaneous extreme reduction in the reactivity of the vitelline envelope outermost layer, and a striking increase in reactive products in the fertilization envelope outermost layer. The surface region of the vitelline or fertilization envelope outermost layer was the binding site for staphylococcal enterotoxin B, and neuraminidase treatment caused a considerable reduction of reactive products in these areas. Immunoblot analysis of cholera enterotoxin B- or staphylococcal enterotoxin B-binding substances in extracts from the vitelline envelopes or fertilization envelopes demonstrated that the great majority of the binding substances are glycoproteins. The present results suggest that glycoproteins constituting the vitelline envelope or fertilization envelope may contribute to the protection of the egg itself or the embryo by trapping noxious toxins.

Micro analysis of the egg shell of Adela metallica (Poda) (Lepidoptera : Adelidae) by energy-filtering transmission electron microscopy (EFTEM)

The egg shell of the incurvarioid moth Adela metallica (Lepidoptera : Adelidae) was studied by conventional (cTEM) and energy-filtering transmission electron microscopy (EFTEM). The shell of the laid egg consists of 3 envelopes. The vitelline envelope is 0.1–0.2μm thick and homogeneous, thus exhibiting the non-exoporian character state. The single-layered chorion, which is covered by a fibrogranular mucous layer, is 0.5–0.9μm thick and homogeneous, thus exhibiting the non-ditrysian character state. The chorion is highly electron-lucent. Neither cTEM nor EFTEM revealed any sub-structural details. However, electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS), revealing the elemental composition of the egg shell, indicate that the chorion and vitelline envelope are proteinaceous and hence, similar to the egg shells of other lepidopteran species. The presence of high sulphur signals associated with the vitelline envelope and the thin basal lamella of the chorion indicates that these components may be stabilized via sulphur-bridges.

Isolation and Characterization of Ovochymase, a Chymotrypsin-like Protease Released during Xenopus laevis Egg Activation

A chymotrypsin-like protease contained in the perivitelline space of unactivated Xenopus eggs is released during egg activation and appears to participate in vitelline envelope conversion. This 30-kDa protease, which we have termed ovochymase, was isolated from the exudate of activated eggs using a soy bean trypsin inhibitor-agarose affinity column. The column eluant contained only two proteins, the 30-kDa ovochymase plus a 78-kDa chymotrypsin-like proteolytic activity, The 78-kDa protease was not usually observed in fresh egg exudate samples and thus was activated during the purification process and may represent the proposed precursor of the 30-kDa protease. The 30- and 78-kDa proteases were separated by gel filtration HPLC or by SDS-PAGE. The N-terminal amino acid sequence of SDS-PAGE-isolated ovochymase was determined to be VVGGQQAAPR. This conserved amino acid sequence, plus active site specific inhibition and substrate specificity studies, places ovochymase in the serine protease I family of enzymes. A two-dimensional protease activity gel revealed that ovochymase is present as several isozymes with a wide range of pI's.

Eggshell fine structure of Bradysia aprica (Winnertz) (Diptera : Sciaridae)

The eggshell fine structure of the dark-winged fungus-gnat Bradysia aprica (Winnertz) (Diptera : Sciaridae) was investigated by scanning and transmission electron microscopy. At the anterior pole of the ovoid egg is a single micropyle, centrally located in a well-defined micropylar area. The latter is covered by many long drumstick-like chorionic processes that are longer and more numerous than those of the rest of the egg surface. Cross-sections of the eggshell show 3 concentric envelopes: the vitelline envelope, wax layer and chorion. The chorion consists of 3 components with different morphological features: the inner, intermediate and outer chorion. The latter 2 layers, involved in the organization of the drumstick-like processes, have homogeneous features, whereas the former is crystalline and resembles the innermost chorionic layer of other Diptera.

Morphology, composition and fertilization of carp eggs: a review

This paper is a review of the literature on eggs of carp ( Cyprinus carpio L.) and some related species and includes data taken from the former USSR and eastern European countries. Reported data relate to fecundity, egg morphology and composition, and fertilization. Information is also available on the ovarian fluid composition with characterization of proteins and lipids. The fecundity of common carp is very high, ranging from 100 000 to 300 000 eggs · kg −1 body weight per oogenetic cycle, with one cycle/year in the case of females reared in an outside natural pond environment. The diameter and weight of the eggs are in the range of 1.24–1.42 mm and 0.86–1.41 mg, respectively. The ooplasm includes large amounts of yolk and various organelles (endoplasmic reticulum, Golgi apparatus, mitochondria), cortical granules and alveolae. The main characteristics of cyprinid ovarian fluid are an osmotic pressure of about 300 mOsm, a Mg 2+ concentration of 2.58 mM and a high ovarian fluid pH of 9. Energy in the form of ATP which is necessary for egg metabolism originates from glycolytic and oxidative reactions. The egg has a relatively thick vitelline envelope (VE) or zona radiata which is reorganized into the fertilization envelope (FE) after fertilization. The micropyle located at the animal pole is a funnel-shaped structure leading spermatozoa to the ooplasmic surface on which a fertilization cone develops after fertilization. A site of sperm attachment is identified on the plasma membrane at the level of the internal aperture of the micropylar canal. Major differences are observed between the two outermost VE and FE layers, as revealed by electron microscopy, enzyme or carbohydrate cytochemistry, and imunohistochemistry. FE extracts have strong bactericidal and fungicidal effects.

Induction of Vitelline Envelope Proteins by Estradiol-17β in 10 Teleost Species

Induction of vitelline envelope proteins by estradiol-17β was investigated in 14 teleost species from five systematic groups to assess whether estradiol-17β controls the synthesis of vitelline envelope proteins in teleost fish. Vitelline envelope proteins were detected in plasma from fish treated with estradiol-17β using three different antisera directed against vitelline envelope proteins from three teleosts. Induction of vitelline envelope proteins was demonstrated in 10 species. In 6 of these species males were available and used to demonstrate that the synthesis of vitelline envelope proteins is not restricted to the ovaries. The immunoreactivity of the proteins varied considerably within and between species. It is suggested that the synthesis of vitelline envelope proteins is controlled by estradiol-17β, in the majority of teleost species.

Ultrastructure of the eggshell of Aeshna Juncea (L.) (Odonata : Aeshnidae)

The eggshell of Aeshna juncea L. (Odonata : Aeshnidae) is described using scanning and transmission electron microscopy. The vitelline envelope is homogenous. The endochorion is many-layered, the thickness of the sub-layers increasing from the inside and out. About 50 or more thin strata are arranged in a crystalline-like fashion, 0.17 μm thick, facing the vitelline envelope. This structure corresponds to the innermost chorionic layer (ICL) reported from Drosophila melanogaster. About 70 thin, bilaminar crystalline-like strata associated with a more electron-dense material, appear between the endochorionic sub-layers near the anterior end of the egg. The electron-dense exochorion is perforated with thin canals opening in small pores on the surface. The filamentous pre-chorion substance forming the exochorion, previously reported from Aeshna, is believed to form a paracrystalline region with a spherulite-like configuration comprising the inner half of the layer. At the anterior end of the egg, the endo- and the exochorion are anchored to each other in a regular manner. The micropylar chutes are 5 μm wide. Corresponding passages in the vitelline envelope are believed to close after fertilization. A standardized nomenclature for endophytic dragonfly eggs is proposed.

Formation of the vitelline envelope precedes the active uptake of vitellogenin during oocyte development in the rainbow trout, Oncorhynchus mykiss.

In order to study the initial formation of the vitelline envelope and the appearance of vitellogenin in oocytes of rainbow trout, females were sampled monthly from 19 to 5 mo before ovulation. Immunohistochemistry revealed that the formation of the vitelline envelope starts when the oocytes reach a diameter of about 450 microns. Oocytes of this size were first found in females sampled a year before ovulation at the time when plasma levels of estradiol-17 beta increased from 0.2 to 0.6 ng/ml. An antiserum directed against vitellogenin crossreacted with small vesicles (around 2 microns) present just inside the oolemma, when the oocytes reached a diameter of 600 microns. This was interpreted as an active uptake of vitellogenin. Oocytes of this size were first found in females sampled 9 mo before ovulation at the time when estradiol-17 beta levels increased from 0.6 to 1.0 ng/ml and the gonadal somatic index was doubled. Oocytes with a diameter of 600 microns had an immunoreactive vitelline envelope with a thickness of about 3 microns. It is apparent that the initial formation of the vitelline envelope starts before the active uptake of vitellogenin and that the low previtellogenic plasma levels of estradiol-17 beta observed in females are of physiological significance.

Pre-fusion events of sperm-oocyte interaction in the marine shrimp, Sicyonia ingentis

The non-motile sperm of Sicyonia ingentis, mixed with eggs by a spawning female, undergo a primary binding to the vitelline envelope (VE) of the oocyte. Once bound to the VE, sperm undergo exocytosis of the acrosomal vesicle, penetrate the VE, and secondarily bind to a surface coat that is closely associated with the oolemma. Unreacted sperm preincubated with solubilized VE components exhibit diminished binding to VEs in a concentration dependent manner. The ligand responsible for this binding is a carbohydrate moiety in the VE. The ligand preferentially binds to the anterior tip of unreacted sperm, as demonstrated with anti-VE polyclonal antibodies. Acrosome intact sperm will not bind to surface coats; however, acrosome reacted sperm do bind to surface coats via an externalized acrosomal granule.

Isolation, Partial Characterization, Induction, and the Occurrence in Plasma of the Major Vitelline Envelope Proteins in the Atlantic Halibut (Hippoglossus hippoglossus) during Sexual Maturation

The vitelline envelope of Atlantic halibut (Hippoglossus hippoglossus) is composed mainly of two major and two minor proteins. Estradiol-17ß induces the two major vitelline envelope proteins in halibut of both sexes. These proteins were also found in plasma of vitellogenic females. The origin of the two major vitelline envelope proteins is not restricted to the ovary, as male halibut synthesize these proteins after treatment with estradiol-17ß. Individual female halibut were followed and plasma sampled from May to March. Plasma levels of estradiol-17ß increased in October, peaked in early February, and declined until the last ovulation in March. In October, the two major vitelline envelope proteins were first detected in plasma and the amount remained elevated until early March. Prior to the last ovulation, the amount decreased. Plasma vitellogenin varied in a similar way. The results provide strong evidence that the physiological regulation of the synthesis of the major vitelline envelope proteins in teleosts is controlled by the female sex steroid estradiol-17ß.

The species-specificity and structure of abalone sperm lysin

Abalone sperm lysin is a 16 kDa protein that creates a hole in the egg vitelline envelope (VE) to allow the sperm to fuse with the egg. Purified lysin exhibits quantitative species-specificity in the dissolution of isolated VE. The molecular basis for this specificity has been studied by sequencing lysin cDNA and by solving the lysin crystal structure. In the deduced amino acid sequences of lysins of seven species of California abalones 50% of the positions are invariant. The most highly variable and strictly species-specific region is the amino-terminal domain of residues 2-12. The crystal structure of lysin reveals a highly α-helical protein with a novel fold. Two tracks of basic amino acids run the length of the molecule. A hydrophobic patch of 11 residues lies on the opposite surface from the basic tracks. The species-specific domain of positions 2-12 extends away from the helical core. Mapping the species-variable positions onto the lysin structure indicates regions which could be involved in species-specific molecular recognition.

Fertilization of the lamprey (Lampetra japonica) eggs: Implication of the presence of fast and permanent blocks against polyspermy

AbstractWe have carried out a morphological examination to analyze the mechanism of monospermic fertilization in the lamprey, Lampetra japonica. Upon insemination, many sperm passed through a “tuft” of jelly that is found only on the animal pole area of the egg and underwent the acrosome reaction after they came into contact with the outer surface of the two‐layered vitelline envelope. The acrosomal filaments were admitted into the envelope and their tips touched the ooplasmic surface. The Hoechst dye transfer method revealed that a single sperm showed intense fluorescence before its nucleus penetrated the vitelline envelope; this indicated the actual fusion of the gametes. No other sperm with acrosomal filaments fluoresced, however. These findings indicate that a fast block against polyspermy is established at the plasma membrane level and prevents additional sperm‐egg fusion.The separation of the ooplasmic surface from the vitelline envelope (perivitelline space formation) was initiated beneath the margin of the animal pole region and progressed toward the animal and vegetal poles of the egg. At this stage, the head of a fertilizing sperm passed through the thickness of the vitelline envelope. The nuclei of supernumerary sperm were unable to enter the inner layer of the vitelline envelope. During the early phase of the perivitelline space formation at the animal pole region, many thin threads and one, or a small number, of the thick projection(s) of the ooplasm connected the envelope with the vitellus. A fertilizing sperm invariably moved along one of the thin threads and was embedded in the ooplasm at the animal pole region. Cytochalasin B inhibited sperm from passing through the inner layer of the vitelline envelope. When ionophore‐activated eggs were inseminated, the tips of the acrosomal filaments were prevented from touching the ooplasmic surface by the intervening presence of the perivitelline space. None of these sperm passed through the vitelline envelope. These findings suggest that the fusion of the acrosomal filament with the egg plasma membrane is a prerequisite for the passage of sperm through the inner layer of the vitelline envelope. Furthermore, actin‐mediated ooplasmic activity is probably involved in the passage. We conclude that the formation of the perivitelline space at the animal pole region contributes to the establishment of a permanent block against polyspermy in the lamprey egg. © 1994 Wiley‐Liss, Inc.