Localization of a chymotrypsin-like protease to the perivitelline space of Xenopus laevis eggs.

A chymotrypsin-like protease is released from Xenopus laevis eggs at activation and is involved in conversion of the vitelline envelope to the fertilization envelope. To localize this enzyme in unactivated and activated eggs, we used the synthetic peptide substrate succinylalanylalanylprolylphenylalanyl-4-methoxy-2-naphthylamide whose product can be visualized using transmission electron microscopy. Protease product was localized within the perivitelline space of unactivated eggs, appearing as strings of beads. No protease activity was detected in activated eggs, which is consistent with the observation that the protease is released from the egg at activation.     

Factors inducing closure of the micropylar canal in the chum salmon egg

The micropylar canal of the chum salmon egg was almost completely closed following egg activation caused by incubation in a hypotonic salt solution (HSS) for I h. The closure occurred in both inseminated and parthenogenetically activated eggs. Incubation of isolated envelopes from non-activated eggs in HSS or perivitelline fluid (PVF) did not induce any modification in micropylar structure, indicating that normal organization of the egg is essential for inducing closure. To reduce the volume of the perivitelline fluid, the eggs were activated in PVF or HSS containing 8 mM Dextran, Although the envelope showed hardening, closure of the micropyle was not observed in these eggs. The wall of the micropylar canal, however, possessed a slightly rough surface. Following activation in a Ca-free hypotonic salt solution with 10 mM EDTA, hardening of the egg envelope was completely inhibited. Although such eggs possessed an apparent perivitelline space, neither closure of the micropylar canal nor roughening of the canal surface were detected. We conclude that the synergistic action of perivitelline turgor pressure and perivitelline material is responsible for the closure of the micropyle.     

SPERM PENETRATION AND THE FORMATION OF A FERTILIZATION CONE IN THE COMMON CARP EGG*

Sperm penetration and the formation of a fertilization cone in the micropylar canal of the egg of the common carp were examined by electron microscopy. The overwhelming majority of inseminated eggs fixed without immersion in fresh water showed that the first spermatozoon had penetrated into the ooplasm before the cortical reaction had occurred, and in many cases had formed a fertilization cone to plug the micropylar canal. At this stage the sperm head was usually located at the base of the cone, and the tail part did not participate in the formation of the cone. Inseminated eggs fixed soon after immersion in fresh water showed that the elevation of the fertilization membrane and the simultaneous recession of the fertilization cone often permitted the penetration of a few supernumerary spermatozoa into the perivitelline space near the micropylar canal, but polyspermic fertilization was never observed. The mechanism of the block to polyspermy in the egg of the common carp is discussed in connection with the fertilization cone.     

An immunocytochemical localization of the cortical granule lectin in fertilized and unfertilized eggs of xenopus laevis

At fertilization, the vitelline envelope surrounding the egg of Xenopus laevis is modified by the addition of an electron-dense component termed the “F layer.” The F layer functions as a block to polyspermy and as a block to the escape of macromolecules from the perivitelline space, thereby causing an osmotically driven envelope elevation. F-layer formation has been hypothesized to result from interaction between a cortical-granule lectin, released in the cortical reaction, and a jelly-coat ligand. Evidence for this hypothesis was sought by determining the location of the cortical-granule lectin both before and after fertilization, using a specific antibody conjugated to horseradish peroxidase. The cortical-granule lectin was localized only in the cortical granules of the unfertilized egg and was located predominantly in the perivitelline space and the F layer of a fertilized egg. These observations support the hypothesis that the F layer is formed by a cortical-granule-Iectin–jelly layer-ligand interaction.     

Breakdown of the cortical alveoli of medaka eggs at the time of fertilization,with a particular reference to the possible role of spherical bodies in the alveoli

Summary The process of cortical change upon fertilization of eggs of the teleostean fish,Oryzias latipes was investigated. A cortical alveolus (CA) contains colloidal material, a spherical body, and often a membranous structure. Upon insemination, breakdown of the cortical alveoli and elevation of the chorion began around the animal pole and ended at the vegetal pole. It was found that the spherical body was extruded with the colloidal material from the CA: the spherical body swelled after the opening of an aperture and was extruded into the perivitelline space through a large aperture. The empty CA shrank and disappeared completely as a result of the transformation of its envelope to numerous microvilli. The spherical body isolated or in the perivitelline space could be digested quickly by proteolytic enzymes. When spherical bodies in the perivitelline space of a fertilized egg were digested enzymatically, the vitellus came into direct contact with the chorion. The present study seems to show that swollen spherical bodies derived from CA play a role in maintaining a certain distance between the chorion and the vitellus after fertilization.     

Immunocytochemical localization of the 35-kDa sea urchin egg trypsin-like protease and its effects upon the egg surface

Trypsin-like protease in sea urchin eggs is thought to reside in cortical granules since it is secreted at fertilization and has been isolated with cortical granule fractions from unfertilized eggs. A 35-kDa serine protease has been purified from Strongylocentrotus purpuratus eggs by soybean trypsin inhibitor-affinity chromatography. For this report the protease was localized by immunocytochemistry before and after fertilization, and its potential biological activity was examined by application of the isolated enzyme to the unfertilized egg surface. The protease was localized on sections by immunofluorescence and immunoelectron microscopy, and was found to reside in the spiral lamellae of S. purpuratus cortical granules and in the electron-dense stellate core of Arbacia punctulata granules. At fertilization the enzyme is secreted into the perivitelline space and accumulates only very briefly between the hyaline layer and the nascent fertilization envelope. Shortly thereafter the enzyme is lost from the perivitelline space and immunological reactivity is no longer associated with the egg surface. The 35-kDa cortical granule protease has vitelline delaminase activity but does not appear to destroy vitelline envelope sperm receptors as judged by the fertility of protease-treated eggs.     

A cortical granule-specific enzyme,B-1,3-glucanase,in sea urchin eggs

The ultrastructural localization of B-1,3-glucanase in three species of sea urchin eggs was determined using a monospecific antibody in an electronmicroscopic immunogold procedure. In all three species, Lytechinus variegatus, Strongylocentrotus purpuratus, and Arbacia punctulata, B-1,3-glucanase was localized specifically to the cortical granules. No other organelle within the egg contained significant label. During the fertilization reaction, B-1,3-glucanase was released from cortical granules into the perivitelline space and became associated with the hyaline layer. No significant label was found in association with the fertilization envelope.     

The extracellular matrix of Xenopus laevis eggs: a quick-freeze, deep- etch analysis of its modification at fertilization

Eggs of the amphibian, Xenopus laevis, were quick-frozen, deep-etched, and rotary-shadowed. The structure of the extracellular matrix surrounding these eggs, including the perivitelline space and the vitelline envelope (VE), was visualized in platinum replicas by electron microscopy. The perivitelline space contains an elaborate filamentous glycocalyx which connects microvillar tips to the plasma membrane, to adjacent microvilli, and to the overlying VE. The VE is comprised of two layers, the innermost of which is a thin network of horizontal fibrils lying on the tips of the microvilli. The outermost is a thicker layer of large, cable-like fibers which twist and turn throughout the envelope. Upon fertilization, three dramatic modifications of the matrix occur. A thin sheet of smooth material, termed the smooth layer, is deposited on the tips of the microvilli and separates the egg from the overlying envelopes. The VE above is transformed from a thick band of cable-like fibers to concentric fibrous sheets, the altered VE. Finally, an ornate band of particles, corresponding to the fertilization layer in previous studies, is deposited at the altered VE/jelly interface. The altered VE and the fertilization layer comprise the fertilization envelope, which effects the structural block to polyspermy.     

Identification of a novel C1q family member in color crucian carp (Carassius auratus) ovary

Potential roles of C1q/tumor necrosis factor (TNF) superfamily proteins have been observed in vertebrate oogenesis and oocyte maturation, but no ovary-specific member has been identified so far. In this study, we have cloned and identified a novel member of C1q family with a C1q domain in the C-terminal from fully grown oocyte cDNA library of color crucian carp and demonstrated that the gene might be specifically expressed in ovary and therefore designated as Carassius auratus ovary-specific C1q-like factor, CaOC1q-like factor. It encodes a 213 amino acid protein with a 17 amino acid signal peptide. There is only one protein band of about 24.5 kDa in the extracts from phase I to phase IV oocytes, but two positive protein bands are detected in the extracts of mature eggs and fertilized eggs. Furthermore, the mobility shift of the smaller target protein band cannot be eliminated by phosphatase treatment, but the larger protein band increases its mobility on the gel after phosphatase treatment, suggesting that the larger protein might be a phosphorylated form. Immunofluorescence localization indicates that the CaOC1q-like proteins localize in cytoplasm, cytoplasm membrane and egg envelope of the oocytes at cortical granule stage and vitellogenesis stage, whereas they were compressed to cytoplasm margin in ovulated mature eggs and discharged into perivitelline space between cytoplasm membrane and egg envelope after egg fertilization. Further studies on distribution and translocation mechanism of the CaOC1q-like factor will be benefit to elucidate the unique function in oogenesis, oocyte maturation and egg fertilization.     

A CYTOLOGICAL STUDY OF ARTIFICIAL PARTHENOGENESIS IN THE SEA URCHIN ARBACIA PUNCTULATA

Eggs of the sea urchin Arbacia punctulata were artificially activated with hypertonic seawater. The artificially activated eggs undergo the cortical reaction which is not distinguished by a wavelike progression as in the case of inseminated eggs. The cortical granules are released at random loci at the surface of the egg and result in spaces separated by large cytoplasmic projections. Unreacted cortical granules and ribosomes are found within the matrix comprising the large cytoplasmic projections. No "fertilization cone" is formed. The subsequent release of additional cortical granules results in the formation of a continuous perivitelline space, 15 min following activation. 85 min postactivation, an organization of annulate lamellae, endoplasmic reticulum of the smooth variety, and microtubules around a centriole is observed prior to nuclear division. Before the breakdown of the nuclear envelope a streak stage is formed. The streak is composed of a central core of annulate lamellae and is encompassed by endoplasmic reticulum and vesicular components. Condensation of chromatin is followed by the establishment of the mitotic apparatus. Centrioles were not found in the mature egg; however, they are present after activation prior to the first nuclear division, in the four-cell embryo, multicellular embryo, and at blastula. Artificially activated eggs have been observed to develop to the pluteus stage in more than 50% of the eggs treated.     

Microgravity and hypergravity effects on fertilization of the salamander Pleurodeles waltl (urodele amphibian)

Effects of microgravity (microG) on fertilization were studied in the urodele amphibian Pleurodeles waltl on board the MIR space station. Genetic and cytomorphologic analyses ruled out parthenogenesis or gynogenesis and proved that fertilization did occur in microG. Actual fertilization was demonstrated by the analysis of the distribution of peptidase-1 genes, a polymorphic sex-linked enzyme, in progenies obtained in microG. Further evidence of fertilization was provided by the presence of spermatozoa in the perivitelline space and in the fertilization layer of the microG eggs and by the presence of a female pronucleus and male pronuclei in the egg cytoplasm. Experiments in microG and in 1.4G, 2G, and 3G hypergravity showed for the first time that, compared to eggs in 1G, several characteristics of the fertilization process including the cortical reaction and the microvillus transformations were altered depending on the gravitational force applied to the eggs. Microvillus elevation, the most evident feature, was reduced on microG-eggs and amplified on eggs submitted to 2G and 3G. No lethal consequences of these alterations on the early development of microG-eggs were observed.     

Closure of the micropyle during embryonic development of some pelagic fish eggs

The fine structure of the egg envelope and micropyle was studied in unfertilized and developing eggs of the flounder Paralichthys olivaceus (Temminck & Schlegel), the Alaska pollack Theragra chalcogramma (Pallas), the Japanese tilefish Branchiostegus japonicus (Houttuyn) and the porgy Pagrus major (Temminck & Schlegel). The outer envelope surface of the unfertilized egg was wrinkled, while the inner surface was folded. The micropyle of the unfertilized egg consisted of a shallow vestibule and a distinct canal. The micropylar region of the inner surface of the envelope had a conical- or bowl-shaped protrusion. In developing eggs, the thickness of the envelope decreased and showed smooth outer and inner surfaces which indicated that it had been stretched tangentially at the time of the perivitelline space formation. The lumen of the micropylar canal was invariably occupied with envelope material. We postulate that the blockage of the micropylar canal is a result of the stretching of the envelope. The closure of the micropyle inhibits sperm and external pathogens from penetrating into the perivitelline space and seems to be involved in both the permanent prevention of polyspermy and the protection of the developing embryo from bacterial infection.     

Formation and structure of the fertilization envelope in Xenopus laevis

This paper reports the morphological events that occur when the vitelline envelope (VE) of an unfertilized egg of Xenopus laevis is transformed into the fertilization envelope (FE) surrounding the zygote. The VE is about 1 μm thick and is composed of an interlacing network of small filaments. The FE is constructed from the VE plus an electron-dense layer (fertilization layer), about 2–6 μm thick, on the outer surface of the VE, i.e., at the interface between the VE and the innermost jelly-coat layer. The fertilization layer is a stable component of the FE and is not removed by mercaptan solutions used to dejelly eggs. The events of FE formation were observed in the light and electron microscopes after dejellied eggs were activated by pricking. The FE is established when material from the cortical granules is extruded into the perivitelline space. The cortical granule material passes through the VE as the envelope lifts away from the egg surface. Some cortical granule material deposits in the interstices of the VE, but most of it forms the fertilization layer on the outer surface of the envelope. The cortical reaction is completed about 8–9 min after addition of sperm when eggs are fertilized in vitro.     

Hierarchies of protein cross-linking in the extracellular matrix: involvement of an egg surface transglutaminase in early stages of fertilization envelope assembly

The involvement of transglutaminase activity in fertilization envelope (FE) formation was investigated using eggs from the sea urchin, Strongylocentrotus purpuratus. Eggs fertilized in the presence of the transglutaminase inhibitors, putrescine and cadaverine, had disorganized and expanded FEs with inhibition of the characteristic I-T transition. The permeability of the FE was increased by these agents, as revealed by the loss of proteins from the perivitelline space and the appearance of ovoperoxidase activity in supernates from putrescine- treated eggs. [3H]putrescine was incorporated into the FE during fertilization in a reaction catalyzed by an egg surface transglutaminase that could also use dimethylcasein as a substrate in vitelline layer-denuded eggs. Egg secretory products alone had no transglutaminase activity. The cell surface transglutaminase activity was transient and maximal within 4 min of activation. The enzyme was Ca2+ dependent and was inhibited by Zn2+. We conclude that sea urchin egg surface transglutaminase catalyzes an early step in a hierarchy of cross-linking events during FE assembly, one that occurs before ovoperoxidase-mediated dityrosine formation (Foerder, C. A., and B. M. Shapiro. 1977. Proc. Natl. Acad. Sci. USA. 74:4214-4218). Thus it provides a graphic example of the physiological function of a cell surface transglutaminase.     

Incorporation of ZP1 into perivitelline membrane after in vivo treatment with exogenous ZP1 in Japanese quail (Coturnix japonica)

In birds, the egg envelope surrounding the oocyte prior to ovulation is called the perivitelline membrane and it plays important roles in fertilization. In a previous study we demonstrated that one of the components of the perivitelline membrane, ZP3, which is secreted from the ovarian granulosa cells, specifically interacts with ZP1, another constituent that is synthesized in the liver of Japanese quail. In the present study, we investigated whether ZP1 injected exogenously into the blood possesses the ability to reconstruct the perivitelline membrane of Japanese quail. When ZP1 purified from the serum of laying quail was injected into other female birds, the signal of this exogenous ZP1 was detected in the perivitelline membrane. In addition, we revealed, by means of ligand blot analysis, that serum ZP1 interacts with both ZP1 and ZP3 of the perivitelline membrane. By contrast, when ZP1 derived from the perivitelline membrane was administered, it failed to become incorporated into the perivitelline membrane. Interestingly, serum ZP1 recovered from other Galliformes, including chicken and guinea fowl, could be incorporated into the quail perivitelline membrane, but the degree of interaction between quail ZP3 and ZP1 of the vitelline membrane of laid eggs from chicken and guinea fowl appeared to be weak. These results demonstrate that exogenous ZP1 purified from the serum, but not ZP1 from the perivitelline membrane, can become incorporated into the perivitelline membrane upon injection into other types of female birds. To our knowledge, this is the first demonstration that the egg envelope component, when exogenously administered to animals, can reconstruct the egg envelope in vivo.     

Egg coats of the rock shrimp Rhynchocinetes typus: ultrastructural characterization and their function during fertilization

The aim of the present work was to characterize structurally and ultrastructurally the egg coats of the rock shrimp, Rhynchocinetes typus, and to describe their functional roles during fertilization. Oocytes fixed directly from the ovary, have a total diameter of 549 microm and are covered by a 10- microm-thick transparent envelope. Electron microscope sections (dehydrated) of the egg envelope revealed an electron-dense external coat of 0.4 microm covered by filamentous processes, and a granular inner coat of 4- microm thickness. Oocytes placed for 5 min in seawater had a significantly larger diameter (573 microm), because of the increase in the thickness of the egg coats (32 microm) and the formation of a 16- microm perivitelline space. The diameter of the egg proper was reduced by the same extent as the size of the perivitelline space. All these changes were associated to the loss of the egg fertilizability. SDS-PAGE of isolated and solubilized egg coats with 20% beta-mercaptoethanol or 25 mM dithiothreitol (DTT) showed bands between 58 and 105 kDa and between 44 and 103 kDa, respectively. During normal fertilization, the sperm undergoes a drastic change in shape after first contact with the egg. We observed a similar change when solubilized egg coats were placed with vas deferens sperms. When the solubilized egg coat proteins were ultrafiltrated with a membrane of 10,000 MWCO (pore size) and then assayed for their effect on fertilization, an inhibitory effect of 30%, 41%, and 59% was found when oocytes were incubated with spermatozoa pre-treated with 30, 60, and 120 microg/ml of proteins solubilized with beta-mercaptoethanol. A similar inhibitory effect was found when egg coat proteins solubilized with 25 mM DTT were used. Our results suggest that, in the shrimp, the egg coats play an active role during the morphological changes of the sperm during their passage through them.     

Electron microscopic study of the cortical reaction of an ophiuroid echinoderm.

The egg coats of an ophiuroid echinoderm (Ophiopholis aculeata) are described by electron microscopy before and after fertilization. The unfertilized egg is closely invested by a vitelline coat about 40 A thick, and the peripheral cytoplasm is crowded with cortical granules five or six deep. During the cortical reaction, which rapidly follows insemination, exocytosis of cortical granules takes place. Some of the cortical granule material is evidently added to the vitelline coat to form a composite structure, the fertilization envelope, which is made up of a 400 A thick middle layer separating inner and outer dense layers, each about 50 A thick. The elevation of the fertilization envelope from the egg surface creates a perivitelline space in which the hyaline layer soon forms. The hyaline layer is about 2 micron thick, finely granular, and apparently derived from cortical granule material. The extracellular layers of the early developmental stages of ophiuroids and echinoids are quite similar in comparison to those of asteroids; this finding helps support Hyman's argument that the ophiuroids are more closely related to the echinoids than to the asteroids.     

Induction of calcium-dependent, localized cortical granule breakdown in sea-urchin eggs by voltage pulsation

A technique that employs a high-voltage pulses to produce pores in cell membranes (Kinosita and Tsong (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1923) has been used to investigate the role of Ca2+ in the early events of activation of sea-urchin eggs. Exposure of eggs to a voltage pulse of 1 kV/cm for 100 microseconds resulted in localized exocytosis of the contents of cortical granules and development of a partial fertilization envelope. This effect was triggered by entrance of Ca2+ through the voltage-induced pores. In a medium containing 100 microM Ca2+ and 45Ca2+ tracer, the voltage-treated eggs admitted 3.6 +/- 0.3 fmol Ca2+/egg within a few seconds. Untreated eggs took up only 1.0 +/- 0.2 fmol/egg after minutes of incubation. Furthermore, depletion of Ca2+ or the presence of EGTA in the external medium prevented elevation of the fertilization envelope by the voltage pulsation. Delay in Ca2+ addition after the voltage pulsation reduced the fraction of eggs that developed partial fertilization envelope. Loss of essential cytoplasmic components during the delay period is judged unlikely, since these eggs were viable, could form partial fertilization envelopes if re-pulsed in the presence of Ca2+, and could develop to normal blastula stage embryos upon fertilization with sperm. Thus, we interpret this effect as due to a resealing of pores; the half-life of pores being 20 s. The elevation of partial fertilization envelopes occurred only at the loci facing the anode, and multiple pulses with mixing resulted in the formation of multiple fertilization envelopes. These envelopes were stable for up to several hours; further propagation (wave spreading) was not observed. The above results indicate that a primary reaction in the sequence of steps in fertilization envelope formation involves Ca2+ to trigger cortical granule breakdown and formation of the fertilization envelope.     

Fate of the sea urchin egg receptor for sperm following fertilization

The sea urchin egg receptor for sperm is a 350 kDa glycoprotein containing a large extracellular domain that contains the sperm binding site, a transmembrane domain and a short COOH- terminal intracellular domain. During oogenesis, the receptor protein is first detected in Golgi-associated vesicles and cortical granules. Not until the egg is mature does the receptor appear on the cell surface; at this stage the intact receptor is found in approximately equal quantities on the egg cell surface and in cortical granules. As a potentially unique type of receptor, we were interested in its fate following fertilization. Several techniques have revealed that, following sperm binding, the amount of receptor markedly decreases. Using western blot analysis as well as direct measurement of the receptor protein, it was found that the membrane-bound form of the receptor rapidly disappeared following sperm binding to the egg, with only 3% of the receptor remaining after 30 s. Analysis by immupoelectron microscopy revealed that 30 s after sperm binding, 30% of the initial level of receptor was present. This remaining 30% was found mostly within the perivitelline space formed by the raised fertilization envelope. The disparity between these two sets of results (i.e. 3 vs 30%) is most likely accounted for by the exocytosis of receptor molecules from cortical granules; this fraction of the receptor would have been lost during isolation of the membrane-bound form of the receptor. Thus, unlike other cell surface receptors, the sea urchin egg receptor for sperm is not endocytosed and recycled following ligand binding. Rather, it disappears, presumably as a result of proteolysis. Transiently, the cortical granule form of the receptor is found released into the perivitelline space where it may bind to sperm and thereby prevent polyspermy. Despite the apparent secretion of this form of the receptor, experiments with antibodies to the extracellular and intracellular domains indicate that the receptors in cortical granules and in the plasmic membrane are similar, if not identical.     

The effect of light exposure on buoyancy of halibut eggs

Effects of light-exposure on eggs of the Atlantic halibut ( Hippoglossus hippoglossus ) were investigated. Egg buoyancy, yolk sac osmolality and perivitelline space (PVS) in light and dark-exposed eggs were followed during 3–12 days after fertilization. In light-exposed eggs, the density increased to a maximum at day 6 while the density in dark exposed eggs significantly decreased between day 4 and 10. There was no significant difference between treatments at day 12. The pattern of yolk osmolality reflected these changes in density. Embryonic volume, calculated from estimates of total volume and PVS, was found to decrease rapidly at days 3–4 after fertilization in the light exposed group, whereas the control group during the same period showed no change. After this period, the embryonic volume showed a parallel decrease in both groups. The increased egg density is caused by both the loss of water from the embryonic compartment and by increased yolk osmolality. These results are discussed in relation to changes in vertical distribution in both natural and culture systems.