Oviductal protease and trypsin treatment enhance sperm-envelope interaction in Bufo arenarum coelomic eggs

We describe the morphological and biochemical changes in Bufo arenarum coelomic egg envelopes (CE) following passage through the oviduct. In this species, the transformation of the CE into the vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein component. Electrophoretic patterns indicate that a pars recta oviductal protease selectively hydrolyzes in vitro the 84 and the 55 kDa glycoproteins of the CE. During the CE to VE transformation, the relative concentrations of gp48, 42 and 39 kDa also change. In in vitro tests, sperm binding to envelope glycoprotein occurs when they are exposed to VE but not when treated with CE, and VE labeled glycoproteins bind to the head and mid piece of the sperm. The gp39 VE component has 100% identity with internal domains of the sequence deduced from ovarian cDNA for the homologous zona pellucida glycoprotein type C (ZPC) protein precursor in B. arenarum. The effects of trypsin as a substitute for oviductal protease were also examined. Trypsin selectively attacks the 84 and the 55 kDa glycoproteins without hydrolyzing other components and renders coelomic eggs fertilizable in a jelly water preparation. Therefore, trypsin can mimic in vitro the biological action of the oviductal protease. However, it does not wholly mimic the biological action of the oviduct which, in B. arenarum at least, exceeds a mere proteolytic effect. This fact was verified by the lower fertility rates and the abnormal embryo development found when trypsin-treated coelomic eggs were fertilized in vitro.     

The Primary Egg Envelope of the Anuran Lepidobatrachus laevis: Physicochemical and Macromolecular Alterations During Development

The coelomic egg envelope (CE) of the frog Lepidobatrachus laevis has a network of fibrillar bundles which disperse after transit through the oviduct. Following oviposition, the egg vitelline envelope (VE) has an additional amorphous zone on the exterior surface. The fertilization envelope (FE) formed after fertilization, appears to be very similar to the VE. The CEs, VEs, FEs and hatched envelopes (FE_h) were manually isolated. The CE, VE and FE were solubilized at 100° using denaturing conditions, but were only partially solubilized in phosphate buffer, pH 7.0. All envelopes and several purified polypeptides from the VE and FE were analyzed using gel electrophoresis and one-dimensional peptide mapping. Each of the envelopes contained 9 major polypeptides ranging from 118.5 to 22 kD and 8–12 minor polypeptides. Several envelope components were added/removed in the conversions based on the results of experiments in which preparations were incubated with activated egg exudate and crude hatching enzyme; some of these transformations were mimicked by tryptic and chymotryptic digestions. Therefore, serine proteases may be involved in envelope processing in vivo. Lepidobatrachus CE polypeptides and several major components from the VE, FE and FE_h were crossreactive with antibodies against Xenopus VE^*.     

cDNA cloning and sequence analysis of the Xenopus laevis egg envelope glycoprotein gp43

The glycoproteins of the Xenopus laevis egg envelope function in fertilization and development. As the unfertilizable coelomic egg transits the pars recta region of the oviduct, it is converted to a fertilizable egg by limited proteolysis of the envelope glycoprotein gp43 to gp41. This conversion is caused by an oviductally secreted serine active site protease, oviductin. We cloned a cDNA for gp43 from an oocyte cDNA library. The cDNA encoded a 454 amino acid protein homologous to the ZPC family of glycoproteins previously shown to be present in mammalian and fish egg envelopes. Conserved ZPC domains and motifs present in the Xenopus sequence included a signal peptide sequence, an N-linked glycosylation site, and 12 aligned Cys residues. In mammalian and Xenopus sequences, a furin-like (convertase) site and a C-terminal transmembrane domain were present reflecting the biosynthesis of ZPC in these species via the secretory glycoprotein pathway. However, fish envelope glycoproteins lack these sequences since they are synthesized via a different route (in the liver, transported to the ovary, and assembled into the egg envelope surrounding the oocyte). Consensus amino acid residues were identified by sequence comparisons of seven ZPC family members; 19% of the amino acid residues were invariant and 48% of the residues were identical in at least four of the seven sequences. The consensus sequence was used to make structure-fertilization function predictions for this phylogenetically conserved family of glycoproteins.     

Following passage through the oviduct, the coelomic envelope of Discoglossus pictus (amphibia) acquires fertilizability upon reorganization, conversion of gp 42 to gp 40, extensive glycosylation, and formation of a specific layer

This paper describes the morphological and biochemical changes in Discoglossus pictus coelomic oocyte envelope (CE) following passage through the oviduct. As in other anurans, in this species, the transformation of the envelope into vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein. In addition, several features, typical of Discoglossus pictus, were observed. A new layer, VE-D, forms underneath the VE region facing the site of sperm entrance, the dimple. In the VE, arrowhead-like bundles of fibrils are perpendicularly oriented toward the dimple. Ultrastructural observations and staining with UEA-I suggested that VE-D might have a role in supporting sperm penetration into the dimple by orienting VE bundles and exposing sugar residues such as fucose. In 'in vitro' tests, VE binding of sperm occurs only if sperm are exposed to A23187, in agreement with previous data (Campanella et al., 1997: Mol Reprod Dev 47:323-333). Sperm binding occurs all over the VE. Accordingly, extracts of the VE covering the animal or the vegetal hemisphere have the same affinity to lectins (DBA, DSA, GNA, MAA, SBA, SNA, UEA-I, WGA). The CE contains six main glycoproteins. Peptide mapping indicated that during CE transformation into VE, gp 42 shifts to an apparent M(r) of 40 and gp 61 is converted to an apparent M(r) of 63 kDa. Lectin blot analyses showed extensive changes in cross-reactivity of most glycoproteins during the CE-->VE transition. The fact that DBA and UEA-I stain gp 63 rather than gp 61 and that this change is related only to gp 63, suggested that O-glycosylation and terminal fucose might be acquired by gp 63 in preparation of fertilization. Gp 63 has recently been cloned (Vaccaro et al., submitted) and shown to exhibit high homology to Xenopus gp 69/64, a VE sperm ligand (Tian et al., 1997a: J. Cell Biol. 136: 1099-1108; Tian et al., 1997b: Dev Biol 187:143-153), and to ZP2 of mammals.     

Physicochemical characterization of progressive changes in the Xenopus laevis egg envelope following oviductal transport and fertilization

Previous studies have shown that the Xenopus laevis egg envelope exists in three forms with differing ultrastructural, macromolecular, and sperm penetrability properties. The coelomic envelope (CE) is derived from eggs released from the ovary into the body cavity of the female, the vitelline envelope (VE) from eggs which have passed through the oviduct, and the fertilization envelope (FE) from fertilized eggs. In the present study, the physicochemical characteristics of these three envelope types were differentiated. Investigation of envelope solubility, deformability, sulfhydryl reactivity, and hydrophobic dye and ferritin binding capacity demonstrated that profound physicochemical changes occur in envelope conversions CE----VE----FE. The physical strength of the envelopes, as evidenced by deformability studies, ranked FE greater than CE greater than VE. These differences were not accountable by differences in the number of disulfide bonds, although the CE sulfhydryl groups were significantly less accessible than those in the VE or FE. All three envelope forms were hydrophilic in nature, exhibiting little ability to bind 1-anilino-8-naphthalenesulfonic acid. The CE bound greater amounts of ferritin in comparison to the VE and FE, indicating the presence of a basic domain, presumably in the 43-kDa glycoprotein, which is lost upon proteolysis to 41 kDa during the CE----VE conversion. The envelope integrity of all three forms was maintained by both noncovalent and covalent (disulfide) bonds. Measurements of the effect of pH on envelope solubilization indicated the involvement of an ionizable group with pKa of 8.0 in maintaining envelope structure.     

Behaviour of the vitelline envelope in Bufo arenarum oocytes matured in vitro in blockade to polyspermy

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.     

Participation of the 39-kDa glycoprotein (gp39) of the vitelline envelope of Bufo arenarum eggs in sperm-egg interaction

The acquisition of egg fertilizability in Bufo arenarum takes place during the oviductal transit and during this process the extracellular coelomic envelope (CE) of the eggs is converted into the vitelline envelope (VE). It has been stated that one of the necessary events leading to a fertilizable state is the proteolytic cleavage of CE glycoproteins in the oviductal pars recta by oviductin, a serine protease. Consequently, there is a marked increase in the relative quantity of glycoproteins with 39 (gp39) and 42 kDa (gp42) in the VE. In the present study, sperm-VE binding assays using heat-solubilized biotin-conjugated VE glycoproteins revealed that both gp39 and gp42 have sperm binding capacity. According to this result, our study was focused on gp39, a glycoprotein that we have previously reported as a homologue of mammalian ZPC. For this purpose, rabbit polyclonal antibodies against gp39 were generated at our laboratory. The specificity of the antibodies was confirmed with western blot of VE glycoproteins separated on SDS-PAGE. Immunohistochemical and immunoelectron studies showed gp39 distributed throughout the width of the VE. In addition, immunofluorescence assays probed that gp39 bound to the sperm head. Finally, as an approach to elucidate the possible involvement of gp39 in fertilization, inhibition assays showed that pretreatment of eggs with antibodies against gp39 generated a significant decrease in the fertilization rate. Therefore, our findings suggest that gp39, which is modified by oviductal action, participates as a VE glycoprotein ligand for sperm in Bufo arenarum fertilization.     

Radioiodination studies of the envelopes from Xenopus laevis eggs

To investigate the molecular basis of the observed morphological and biological characteristics of coelomic egg envelopes (CE), vitelline envelopes (VE), and fertilization envelopes (FE) of Xenopus laevis eggs, envelopes were radioiodinated under a variety of conditions: in situ, isolated and intact, or solubilized. The distribution of 125I in envelope components was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each envelope type displayed unique profiles when iodinated in the intact state. A major constituent of VE, the 41,500 molecular weight component, was not labeled in the intact state, although the corresponding component of CE was heavily labeled. After dissociation of the envelope by guanidine-HCl or sodium dodecyl sulfate, all of the components could be radioiodinated. However, when the envelopes (VE and FE) were dissolved by heating and subsequently radioiodinated by lactoperoxidase, the resulting radioactivity profile was similar to that of the intact envelopes, suggesting that in the heat-dissolved envelope, the individual components retain similar structural relations as in the intact envelope. Quantitative but not qualitative differences were found between the inner and outer aspects of VE and FE. The significance of these findings is discussed in relation to what is known about the morphological, biological, and molecular properties of the envelopes.     

Immunohistochemical localisation of gp69/64 molecules in Xenopus egg envelopes in relation to their sperm binding activity

A monoclonal antibody (6964M) was generated against the envelope component gp69/64 of Xenopus laevis eggs. On indirect immunofluorescence using this antibody, the positive reaction was seen on the surface of both vitelline envelope (VE) and coelomic envelope (CE). On immunoelectron microscopy, gp69/64 was preferentially distributed on the thick bundles forming the edge of the tunnel openings on CE, and this distribution pattern was fundamentally inherited by VE. Counting the number of immunogold particles indicated that VE has about twice as many particles as CE, with a 3-4 times higher density at the animal pole than vegetal pole. The number of sperm bound to CE was small, being approximately one-twentieth of the number of sperm bound to VE. An extremely small number of sperm (< 2 per animal hemisphere) was found to bind to VE* of activated eggs as a background. The sperm binding to CE was inhibited by pretreatment of the envelopes with 6964M or in the presence of purified gp69/64 from VE on insemination, confirming that sperm binding is mediated by gp69/64 exposed on the CE surface. In spite of at most a 2-fold increase in the amount of exposed gp69/64, the sperm binding increased about 20-fold upon CE-to-VE conversion, suggesting that the increase in the amount of exposed gp69/64 is itself insufficient to explain the increase in the number of bound sperm.     

Vitelline envelope gps 63 and 75 specifically bind sperm in "in vitro" assays in Discoglossus pictus

In Xenopus, conflicting data related to sperm-vitelline envelope (VE) binding suggest that further experiments should be performed to study the role of VE glycoproteins in sperm binding. In this article, we studied the VE of Discoglossus pictus, where gp63, the product of the Dp ZP2 gene, has high molecular identity to Xenopus gp69/64 and to mouse ZP2 and only A23187-treated sperm bind to VE. Sperm bind to VE all over the egg, yet a sperm tuft was found only in the animal half of the egg, where the dimple, the site of fertilization, is located and an intense immunostain was detected in VE by an antiserum directed against gp69/64. The same antiserum inhibited sperm binding to VE. Sperm binding to beads coated with gp63, gp40, or gp75 was in the range of 62-70% for gp63-beads, 67-75% for 75 beads, and about 20% for BSA beads and gp40-coated beads. Soluble purified gp63 and gp75 competitively inhibited binding of sperm to gp63-coated beads. Similarly, the same glycoproteins inhibited sperm binding to gp75-coated beads. SDS-polyacrylamide gels (PAGE) of FE and comparison of VE and FE peptide maps showed that gp63 undergoes a minor shift to about 62 kDa in FE. In sperm binding assays with beads coated with FEs gp62, there was no binding. Following fertilization, in the region of the dimple, an F-layer is formed as well as an alteration of the VE structure. Lectin blots of the FE showed that the FE and in particular gp62 acquires a stronger affinity to Maackia amurensis agglutinin (MAA) with respect to VEs gp63. These results indicate that gps 63 and 75 are the sperm binding glycoproteins of D. pictus VE, where major post-fertilization changes occur as in other anuran species.     

The coelomic envelope to vitelline envelope conversion in eggs of Xenopus laevis

An amphibian egg recovered from the body cavity is enclosed by a coelomic egg envelope. Upon transport down the oviduct, the envelope is converted to the vitelline envelope. The coelomic and vitelline envelopes are distinct in terms of sperm penetrability, ultrastructural morphology, and radioiodination profiles. In this study, the macromolecular compositions of these two envelopes were determined. Isolated envelopes were compared by one- and two-dimensional gel electrophoresis, peptide mapping, and radiolabeling. A protein with a molecular weight of 57,000 (57K) was present in the vitelline envelope but was absent in the coelomic envelope. A glycoprotein with a molecular weight of 43K in the coelomic envelope was converted to a component with a molecular weight of 41K in the vitelline envelope. The 43K-molecular weight component of the coelomic envelopes could be radioiodinated by lactoperoxidase but no labeling of the 41K-molecular weight component occurred in the vitelline envelope. Peptide mapping using limited proteolysis established that the 43K-molecular weight component of the coelomic envelope was a precursor to the 41K-molecular weight component of the vitelline envelope. These molecular alterations may underlie the ultrastructural and physiological changes occurring in these envelopes.     

Alteration of structure and penetrability of the vitelline envelope after passage of eggs from coelom to oviduct in Xenopus laevis.

The vitelline envelope (VE) that surrounds an egg released from the ovary into the coelom of Xenopus laevis differs markedly, in structure and penetrability, from the VE surrounding an oviposited egg. In a coelomic egg, the filaments that form the VE are arranged in distinct fascicles or bundles. The exterior surface of the VE is irregular in contour and is permeated by channels. In an oviposited egg, the filaments are evenly dispersed and lack a fasciculated arrangement; the exterior surface is smooth and no channels are present. The fascicular arrangement of fibrils in the coelomic VE is maintained only at neutral pH, and is not visibly altered by the cortical reaction. VEs from coelomic eggs retain their fasciculated morphology after isolation from the egg. In an in vitro test system, sperm penetrated VEs isolated from oviposited eggs, but failed to penetrate VEs isolated from coelomic eggs. The structural transformation of the VE from the coelomic type to the oviposited type occurs in the first 1-cm segment of the oviduct, prior to addition of jelly to the egg. Neither intact jelly, solubilized jelly, nor jelly extracts were capable of altering the structural organization of coelomic VEs, suggesting that the structural transformation of the VE is effected by some oviducal factor other than jelly.     

Intermolecular cross-linking of vitelline envelope polypeptides predominates in the hardened sea urchin fertilization envelope

At fertilization, the sea urchin egg vitelline envelope (VE) elevates, and a subset of released cortical granule proteins, paracrystalline protein fraction (PCF), associates with the VE to form the fertilization envelope (FE). Cortical granule peroxidase cross-links FE polypeptides by phenolic coupling of tyrosyl residues. We have used an immunological approach to determine which polypeptides are linked together in the hardened FE of Strongylocentrotus purpuratus. Soluble polypeptides were extracted from hardened FEs, and antibodies were prepared in rabbits against the insoluble envelope matrix (FE ghost). Whole immune serum and purified IgGs each reacted with FE ghosts when using an enzyme-linked immunosorbent assay. VEs isolated by means of three published procedures cross-reacted with the immune serum and purified IgGs. Soluble FE polypeptides also cross-reacted with whole immune serum and IgGs owing to the presence of VE polypeptides. Hyalin, a protein not found in FEs, and PCF did not cross-react with antiserum against FE ghosts. To determine which VE polypeptides were cross-linked in the hardened FE, VE polypeptides were immunoblotted by using antiserum against FE ghosts. Most of the VE polypeptides that ranged from 68,000 to 283,000 molecular weight cross-reacted with the antibody.     

Oviductin, the Xenopus laevis oviductal protease that processes egg envelope glycoprotein gp43, increases sperm binding to envelopes, and is translated as part of an unusual mosaic protein composed of two protease and several CUB domains

The glycoprotein envelope surrounding the Xenopus laevis egg is converted from an unfertilizable to a fertilizable form during transit through the pars recta portion of the oviduct. Envelope conversion involves the pars recta protease oviductin, which selectively hydrolyzes envelope glycoprotein gp43 to gp41. Oviductin cDNA was cloned, and sequence analysis revealed that the protease is translated as the N terminus of an unusual mosaic protein. In addition to the oviductin protease domain, a protease domain with low identity to oviductin was present, possessing an apparent nonfunctional catalytic site. Three CUB domains were also present, which are related to the mammalian spermadhesin molecules implicated in mediating sperm-envelope interactions. We propose that during post-translational proteolytic processing of the mosaic oviductin glycoprotein, the processed N-terminal protease domain is released coupled to two C-terminal CUB domains and constitutes the enzymatically active protease molecule. In functional studies, isolated coelomic egg envelopes treated with oviductin purified from the oviduct showed a dramatic increase in sperm binding. This observation established that oviductin alone was the oviductal factor responsible for converting the egg envelope to a sperm-penetrable form, via an increase in sperm binding. Trypsin mimicked oviductin's effect on envelope hydrolysis and sperm binding, demonstrating that gp43 processing is the only requirement for envelope conversion.     

The Synthesis and Localization of Envelope Glycoproteins in Oocytes of Xenopus laevis using Immunocytochemical Methods

The site of origin and the mode of differentiation of the coelomic envelope (CE) in growing oocytes of Xenopus laevis were studied using the rabbit antiserum raised against the isolated envelope from oviposited eggs. The antiserum preabsorbed with egg extracts reacted with most components of CE glycoproteins detectable by SDS-PAGE, and stained specifically the CE of full-grown (st. VI) oocytes using indirect immunofluorescence methods. Transmission electron microscopy employing IgG-conjugated colloidal gold demonstrated that the CE antigens were distributed throughout the whole cytoplasm of st. I oocytes, and began to be deposited around the oocyte surface at late st. I. During st. II to VI the density of CE antigens in the oocyte cytoplasm decreased markedly, while the deposition of extracellular CE antigens increased gradually in association with the formation of a fibrillar network. The CE antigens were observed in and around the highly extended oocyte microvilli during st. II to IV, but were never found in follicle cells at any stages of oocyte growth. On western blot analyses, the extracellular CE components appeared first at st. II, and increased both in amount and number of bands during st. III - V to attain a typical electrophoretic profiles of well-developed CE. The cytosols of growing oocytes, however, possessed several antigenic components which were distinct from those of extracellular CE, suggesting the occurrence of intracellular precursor molecules for CE. The CEs of st. IV oocytes defolliculated and cultured in [³H] leucine contained certain CE components that expressed the radiolabel on fluorography. These results indicate that in Xenopus laevis the oocyte is directly involved in the synthesis and assembly, as well as secretion of CE with least contribution by the follicle cells.     

Identification of the ZPC oligosaccharide ligand involved in sperm binding and the glycan structures of Xenopus laevis vitelline envelope glycoproteins

The Xenopus laevis egg vitelline envelope is composed of five glycoproteins (ZPA, ZPB, ZPC, ZPD, and ZPX). As shown previously, ZPC is the primary ligand for sperm binding to the egg envelope, and this binding involves the oligosaccharide moieties of the glycoprotein (Biol. Reprod., 62:766-774, 2000). To understand the molecular mechanism of sperm-egg envelope binding, we characterized the N-linked glycans of the vitelline envelope (VE) glycoproteins. The N-linked glycans of the VE were composed predominantly of a heterogeneous mixture of high-mannose (5-9) and neutral, complex oligosaccharides primarily derived from ZPC (the dominant glycoprotein). However, the ZPA N-linked glycans were composed of acidic-complex and high-mannose oligosaccharides, ZPX had only high-mannose oligosaccharides, and ZPB lacked N-linked oligosaccharides. The consensus sequence for N-linked glycosylation at the evolutionarily conserved residue N113 of the ZPC protein sequence was glycosylated solely with high-mannose oligosaccharides. This conserved glycosylation site may be of importance to the three-dimensional structure of the ZPC glycoproteins. One of the complex oligosaccharides of ZPC possessed terminal beta-N-acetyl-glucosamine residues. The same ZPC oligosaccharide species isolated from the activated egg envelopes lacked terminal beta-N-acetyl-glucosamine residues. We previously showed that the cortical granules contain beta-N-acetyl-glucosaminidase (J. Exp. Zool., 235:335-340, 1985). We propose that an alteration in the oligosaccharide structure of ZPC by glucosaminidase released from the cortical granule reaction is responsible for the loss of sperm binding ligand activity at fertilization.     

Morphological Modifications of Oocyte Vitelline Envelope from Bufo arenarum during Different Functional States

The submicroscopic morphology of the vitelline envelope of Bufo arenarum's oocyte change significantly during the maturation and fertilization processes. The morphological changes are related to physiological activity in vivo and can be triggered in vitro by experimental procedures. It is our scope to present the ultrastructure differences of fascicular components of the vitelline envelope in coelomic, "pars recta" conditioned, oviductal, oviposited and fertilized oocytes. Our experimental results indicate that artificial "pars recta" treatment of coelomic oocytes arrange the fascicular components as those of oviposited oocyte, although differences still remain indicating that additional maturation processes take place while the egg pass througth the oviduct. Fertilized or activated oocytes which are refractary to sperm penetration, change the vitelline envelope fascicular components orientation giving a submicroscopical image quite different to those of none fertilized oocytes. These ultrastructural changes define in a clear cut manner the functional states of Bufo arenarum's oocyte.     

Analyses of Oviductal Pars Recta-Induced Fertilizability of Coelomic Eggs in Xenopus laevis

The acquisition of fertilizability in coelomic eggs of Xenopus laevis has been shown to be correlated with the physical, biochemical, and ultrastructural alterations of the egg envelope [coelomic envelope (CE)] induced during the passage of eggs through the pars recta portion of the oviduct. However, no direct evidence that the pars recta renders eggs fertilizable has yet been presented. In this study, we show that coelomic eggs are highly fertilizable when they are incubated with continuous shaking for 4 h at 15 degrees C in pars recta extract (PRE) derived from females prestimulated by pregnant mare serum gonadotropin. The PRE from pituitary-stimulated Bufo japonicus was as potent as homologous PRE in rendering Xenopus eggs fertilizable. Incubation of coelomic eggs in PRE for 30 min induced a dramatic increase in the rates of sperm binding to the envelope to a level equivalent to that exhibited by the envelope from uterine eggs (VEs). The CE-to-VE ultrastructural conversion and a 43k-to-41k hydrolysis of the envelope glycoprotein component started 5 min after, and were completed by 15 min after, the start of incubation in PRE and were accompanied by an exposure of a new N-terminal sequence typical to gp41. Thus, the biochemical and ultrastructural conversions and the sperm-binding activity of the envelope induced by PREs, although being prerequisite, were not sufficient to render coelomic eggs fully accessible to fertilizing sperm.