A sperm factor as the counterpart to the sperm-binding factor of the homologous eggs

A substance was isolated from sperm of the sea urchin, $\text{Hemicentrotus pulcherrimus}$. When unfertilized eggs of homologous species were pre-treated with this substance, they rapidly lost the fertilizability due to loss of the sperm-binding capacity. Such an effect was not exerted upon eggs either in CaMg free sea water or of heterologous species. This substance caused neither iso-agglutination of eggs nor precipitation of jelly coat. When it was pre-incubated with the sperm-binding factor purified from eggs of homologous species, it lost the fertilization-inhibiting effect on eggs. It seems very likely that a complementary relationship exists between the present substance and the sperm-binding factor.     

Partial Purification of the Sperm-binding Factor from the Egg of the Sea Urchin, Anthocidaris Crassispina, Followed by an Immunological Method

Univalent antibody (Fab fragments) against sperm-binding factor inhibits the fertilization of eggs species-specifically. The sperm-binding factor was partially purified from unfertilized eggs of the sea urchin Anthocidaris crassispina by monitoring its neutralizing effect on fertilization inhibiting Fab fragments. It formed two species-specific precipitin lines by the double-immunodiffusion test and gave three main bands of protein with apparent molecular weights of 80,000, 87,000 and 225,000, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate was detected in the first and third of these protein bands.     

Physiological studies on the sperm surface component responsible for sperm-egg bonding in sea urchin fertilization : I. Effect of sperm-binding protein on the fertilizing capacity of sperm

When sea urchin sperm is pretreated with sperm-binding protein prepared from the vitelline membrane of eggs of homologous species, it loses its fertilizing capacity entirely without losing its motility. It is not affected at all by sperm-binding protein from heterologous species. Neither agglutination nor acrosome reaction is evoked by the pretreatment. It is suggested that the sea urchin spermatozoon has on the apical part of its head a component which is complementary to the sperm-binding protein of the egg, and that the observed loss of the fertilizing capacity is caused by antedated interaction of this component with sperm-binding protein added before insemination.     

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.     

Transient recovery from trypsin-caused reduction in fertilizability of sea urchin eggs

Summary The effect of trypsin on the fertilizability of sea urchin eggs was studied withParacentrotus lividus andPseudocentrotus depressus. The main effects were two reductions of fertilizability, with a transient increase intervening. The first decrease was probably caused by degradation of sperm-binding sites at the vitelline sheet and the second by degradation of binding sites on the plasma membrane.     

Immunological evidence that a 305-kilodalton vitelline envelope polypeptide isolated from sea urchin eggs is a sperm receptor

Direct isolation of the sea urchin egg vitelline envelope with intact sperm receptors is difficult because the envelope is firmly attached to the egg plasma membrane. We now report a method for producing an inseminated egg preparation in Strongylocentrotus purpuratus (using soybean trypsin inhibitor [STI] and Ca²⁺, Mg²⁺-free seawater) that contains an elevated vitelline envelope (VE*-STI). The VE*-STI is devoid of cortical granule material, and supernumerary sperm do not detach postinsemination, suggesting that the VE*-STI contains active sperm receptors. VE*-STIs contain a 305-kD polypeptide and additional components that range from 225 to 31 kD, whereas the 305-kD polypeptide was considerably reduced in VE*s. Electrophoresis of sperm receptor hydrolase digests of VE*-STIs showed that the 305-kD polypeptide and several other envelope polypeptides are protease substrates. Univalent Fab fragments against VE*s, VE*-STIs, and 305 and 225-kD polypeptides blocked sperm binding and fertilization in an Fab concentration-dependent manner. The 305 and 225-kD polypeptides were localized in the VE*-STI using indirect immunofluorescence. Enzyme-linked immunosorbent assays showed that the 305 and 225-kD polypeptides share determinants, suggesting that the 225-kD polypeptide may be derived from the 305-kD polypeptide by the proteolysis that occurs at the cell surface during fertilization. Fab fragments against S purpuratus VE*-STI antigens neither bound to nor blocked homologous sperm binding and fertilization of Lytechinus variegatus eggs. Cross fertilizability occurred to the extent of 5% or less between L variegatus and S purpuratus, therefore, we conclude that the 305 kD-polypeptide isolated from S purpuratus is a species-specific vitelline envelope sperm receptor.     

Probable asymmetry in the organization of components of the hamster zona pellucida

The distribution of the sperm-, lectin- and antibody-binding components was examined on isolated fragments of the hamster zona pellucida. In all three studies, binding was much stronger on the outer than on the inner surface of the zona pellucida. Fab antibody-binding sites were different from those for various lectins since there appeared to be no competition for binding. Multiple components are obviously exposed on the surfaces of the zona pellucida because the sperm receptor sites are also inaccessible to Fab fragments. We conclude that although the components may be present throughout the zona they are asymmetrically arranged in relation to both surfaces. Microdisc PAGE of zonae from ovulated eggs revealed three major components of molecular weight 240 000, 150 000 and 80 000. These are probably glycoproteins and thus resemble the glycoprotein composition of the mouse zona.     

Purification of the sperm-binding factor from the egg of the sea urchin,Hemicentrotus pulcherrimus

The substance which seems to be responsible for the sperm-binding at fertilization was successfully purified from unfertilized eggs of the sea urchin, $\text{Hemicentrotus pulcherrimus}$. It completely cancelled the fertilizing capacity only of homologous sperm without reducing their motility. The antiserum against this substance made only homologous eggs incapable of binding sperm. The methods employed for purification were (1) extraction by urea, (2) fractionation by calcium acetate, (3) salting-out by ammonium sulfate, (4) gel filtration and (5) ion-exchange chromatography. This substance was electrophoresed on cellulose-acetate strip as a single band which was stained with Amido Black, and could not be split by 6 M guanidine hydrochloride.     

Use of heterologous sperm for the dispermic induction of androgenesis in barbs

Dispermic activation of genome‐inactivated eggs of the grey tiger barb Puntius tetrazona , facilitated by 2·5% polyethylene glycol (PEG)‐incubated sperm of the golden rosy barb Puntius conchonius , resulted in the generation of interspecific androgenetic clones of the golden rosy barb. A 10 min incubation of the golden rosy barb sperm in increasing concentrations (1·0, 1·5, 2·0, 2·5 and 3·0%) reduced the frequency of motile sperm (to 70%), motility duration (110–50 s) and fertilizability (to 80%) of the sperm; however, the frequency of sperm with double head size increased. At 3% PEG, motility pattern of the sperm completely changed from 'zig‐zag' to 'circular'. Incubation in 2·5% PEG facilitated the dispermic entry and production of diploid androgenetic female and male progenies at the ratio of 0·27 : 0·73. Polymerase chain reaction (PCR) analysis using Tc1 transposon specific primers confirmed the purity of paternal inheritance of P. conchonius through the surrogate eggs of P. tetrazona . Survival and breeding, body colour, diploidy (karyotyping and erythrocyte measurements) and 0·27F : 0·73M sex ratio of the androgenotes provided evidence for the successful induction of dispermic androgenesis. Despite increased heterozygosity and reduced 'damage cost' on restoration of diploidy, survival of dispermic androgenotes induced in heterologous eggs was lower (1·7%) than those reported for androgenetic golden rosy barbs induced using homologous sperm (14·0%) and heterologous eggs (7·0%), i.e . tiger barb eggs.     

Antibody to a sperm surface glycoprotein inhibits the egg jelly-induced acrosome reaction of sea urchin sperm

When the surface of sea urchin (Strongylocentrotus purpuratus) sperm is radioiodinated, 75% of the protein-incorporated radioactivity is associated with two glycoproteins of M_r 84,000 (84K) 64,000 (64K) (Lopo and Vacquier 1980). Antibodies were prepared against these two components by separating a Triton X-100 extract of sperm on SDS-polyacrylamide gels, cutting out the band containing the glycoprotein and injecting the homogenized gel into rabbits. Both anti-84K and anti-64K sera agglutinate sperm. Light and EM immunoperoxidase localization show both antigens are distributed over the entire sperm surface. By the immunoperoxidase technique there is some degree of cross-reactivity of both antisera with sperm of other Strongylocentrotus species, but not with those of other genera. Living sperm incubated with anti-84K Fab fragments are completely inhibited from undergoing the egg jelly-induced acrosome reaction and fertilizing eggs. Anti-64K Fab fragments have no effect on the ability of the sperm to undergo the acrosome reaction or fertilize eggs. Sperm incubated in anti-84K or anti-64K Fab fragments undergo the acrosome reaction in response to the Ca²⁺ ionophore A23187, or when the extracellular pH is increased to 9.2 with NH₄OH, indicating that the inhibition of the egg jelly-induced acrosome reaction results from the binding of the anti-84K Fab to an external molecule involved in the initiation or propagation of the acrosome reaction. The 84K glycoprotein is the first sperm surface component identified that might have a role in the induction of the acrosome reaction.     

Inhibition of in-vitro fertilization of intact and denuded hamster eggs by univalent anti-sperm antibodies.

Univalent (Fab) rabbit anti-hamster sperm antibodies added to an in-vitro fertilization system did not interfere with the sperm acrosome reaction or motility, but inhibited cumulus dispersion by the spermatozoa, sperm binding to and passage through the zona pellucida as well as sperm-egg fusion. Addition of the Fab preparations to the capacitated spermatozoa at various times before or up to 40-45 min after the sperm-egg mixing prevented penetration of spermatozoa through the zona pellucida. Detachment of the spermatozoa already bound as well as those partly inside the zona pellucida was achieved by a late addition of antibodies. In experiments with zona-free hamster eggs, addition of the Fab antibodies to the spermatozoa 10 min to 5 h before the introduction of unfertilized eggs reduced the rate of adhesion and fertilization to very low levels. These antibodies were not absorbed on hamster ovary, liver or kidney and had no direct effect on the fertilizability of zona-intact or zona-free eggs.     

Immunization of female rabbits with heat-solubilized bovine zonae: Production of anti-zona antibody and inhibition of fertility

Bovine zonae were solubilized by heating to 80°C in buffered saline. On injection of female rabbits with this solution, zona-specific antisera were produced that could be assayed on bovine eggs in vitro by immunofluorescence, coating, and inhibition of sperm-binding. When the latter was complete, the rabbits were found to be infertile. Immunofluorescence assays showed that the rabbit anti-bovine zona serum reacted as strongly with rabbit and rhesus monkey zonae as with homologous zonae. Strong cross-reaction was also observed with marmoset and dog zonae, but with hamster zonae the reaction was relatively weak.     

Chimeric fab fragments of antibodies to recombinant Ebola virus glycoprotein

Previously, we have determined the nucleotide and amino acid sequences of the variable domains of three mouse monoclonal antibodies specific to the individual epitopes of the Ebola virus glycoprotein: GPE118 (IgG), GPE325 (IgM) and GPE534 (IgG) [1]. In the present paper, chimeric Fab fragments of Fab118, Fab325, and Fab534 antibodies were obtained based on the variable domains of murine antibodies by attaching CH1 and CL constant regions of human kappa-IgG1 to them. The recombinant chimeric Fab fragments were synthesized in the heterologous expression system Escherichia coli, isolated and purified using metal chelate affinity chromatography. The immunochemical properties of the obtained Fab fragments were studied by immunoblotting techniques as well as indirect and competitive ELISA using recombinant Ebola virus proteins: EBOV rGPdTM (recombinant glycoprotein of Ebola hemorrhagic fever virus without the transmembrane domain), NP (nucleoprotein) and VP40 (structural protein). The identity of recombinant chimeric Fab fragments, as well as their specificity to the recombinant glycoprotein of Ebola hemorrhagic fever virus (EBOV GP) was proved. The results of indirect ELISA evidence the absence of immunological cross-reactivity to NP and VP40 proteins of Ebola virus. The dissociation constants of the antigen-antibody complex K _d equal to 5.0, 1.0 and 1.0 nM for Fab118, Fab325 and Fab534, respectively, were determined; they indicate high affinity of the obtained experimental samples to EBOV GP. The epitope specificity of Fab fragments was studied using a panel of commercial neutralizing antibodies. It was found that all studied antibodies to EBOV GP are targeted to different epitopes, while the epitopes of the recombinant chimeric Fab fragments and original murine monoclonal antibodies (mAbs) coincide. All the obtained and studied mAbs to EBOV GP are specific to epitopes that coincide or overlap the epitopes of three commercial neutralizing mAbs to Ebola virus: epitopes Fab118 and Fab325 overlap the epitope of the known commercial mAb h13F6; Fab325 epitope also overlaps mAb c6D8 epitope; Fab534 epitope is located near mAb KZ52 conformational epitope, in the formation of which amino acid residues of GP1 and GP2 domains of EBOV GP are involved.     

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.     

Protein D is differentially expressed and regulated in the rat epididymis.

We report the use of a sensitive and specific enyzme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) to study the expression of protein D, a major androgen-regulated sperm-binding glycoprotein at the protein and mRNA level in different anatomical regions of the rat epididymis. The concentration of protein D in the caput, corpus and cauda region of the epididymis was 10.2 +/- 0.67, 7.3 +/- 0.61 and 22.8 +/- 1.34 ng/micrograms total protein, respectively. The total RNA extracted from the caput, corpus and cauda regions of the rat epididymis was amplified by PCR with oligonucleotide primers specific for the 5' and 3' portion of protein D cDNA. Compared to the caput and cauda region, a significant reduction (greater than 82 +/- 3%) in the expression of protein D mRNA levels was observed for corpus epididymal RNA. This data demonstrates regional differences in the concentration of protein D and suggests that protein D expression may be regulated at the level of mRNA within the corpus epididymidis.     

Identification and preliminary characterization of a sperm-binding protein in normal human semen

In the seminal plasma of normal men a protein, immunologically related to a major protein (RSV-IV) secreted from the rat seminal vesicle epithelium, was detected by competition with RSV-IV in a specific radioimmunoassay using a rabbit anti RSV-IV antiserum. The protein was partly (80%) purified by column chromatography; characterization by gel electrophoresis indicated that the protein is slightly basic and has a molecular weight of 140 000. The protein was present on the surface of human spermatozoa as well as in the ejaculates of azoospermic men and it is believed to be a sperm-binding protein.     

Aggregation of beta-1,4-galactosyltransferase on mouse sperm induces the acrosome reaction

beta-1,4-Galactosyltransferase (GalTase) is present on the surface of mouse sperm, where it functions during fertilization by binding to oligosaccharide residues in the egg zona pellucida. The specific oligosaccharide substrates for sperm GalTase reside on the glycoprotein ZP3, which possesses both sperm-binding and acrosome reaction-inducing activity. A variety of reagents that perturb sperm GalTase activity inhibit sperm binding to the zona pellucida, including UDP-galactose, N-acetylglucosamine, alpha-lactalbumin, and anti-GalTase Fab fragments. However, none of these reagents are able to cross-link GalTase within the membrane nor are they able to induce the acrosome reaction. On the other hand, intact anti-GalTase IgG blocks sperm-zona binding as well as induces the acrosome reaction. Anti-GalTase IgG induces the acrosome reaction by aggregating GalTase on the sperm plasma membrane, as shown by the inability of anti-Gal-Tase Fab fragments to induce the acrosome reaction unless cross-linked with goat anti-rabbit IgG. These data suggest that zona pellucida oligosaccharides induce the acrosome reaction by clustering GalTase on the sperm surface.     

Purification, physicochemical characterization, and immunohistochemical localization of a major 11.7 S glycoprotein from the jelly coats of the anuran Lepidobatrachus laevis

Embryos of the frog Lepidobatrachus laevis are encased by a fertilization envelope and two jelly layers, termed J1 (innermost) and J2 (outermost). From preparations of total jelly solubilized from cleavage-stage embryos by a solution of alkaline beta-mercaptoethanol we have purified one jelly coat glycoprotein to homogeneity via FPLC gel permeation chromatography on Superose 6H. The purified glycoprotein was 94% protein and 6% carbohydrate, had an s0(20),w of 11.7 S, with a molecular weight of 245,000 measured by sedimentation equilibrium and 263,000 by gel permeation chromatography. SDS-PAGE revealed that the glycoprotein is composed of a single subunit near 29,700 molecular weight; thus we propose that eight of these subunits comprise the native molecule. Amino acid analysis of the glycoprotein indicated a high content of Glx + Asx (32.4 mole%), a low content of basic amino acids (Arg + Lys = 12.2 mole%), and a single cysteine residue per subunit. The N-terminal amino acid was threonine and the sequence of the first twenty amino acids was determined. Monospecific antisera to the glycoprotein were prepared in rabbits and were used to immunohistochemically localize the glycoprotein throughout the matrix of both jelly layers. Antiserum against the glycoprotein had virtually no effect on the fertilizability of jellied eggs in vitro; thus we hypothesize that the glycoprotein fulfills a structural role in both jelly layers.     

Induction of the acrosome reaction on the surface of de-jellied sea urchin eggs

The vitelline coat of sea urchin eggs was disrupted by DTT and trypsin after removal of the jelly layer. Thereafter the percentage of acrosome reaction was determined and the fertilization rate was estimated, employing the treated eggs. Electron microscopical investigation of these eggs showed that the vitelline coat was disrupted but no morphological difference was observed between eggs treated with DTT and those treated with trypsin. However, the fertilizability of the eggs was markedly decreased by the treatment with trypsin. In contrast, DTT treatment did not affect the fertilizability of the eggs, indicating that some surface substance(s) necessary for fertilization which were not eliminated by DTT were digested by trypsin. At the same time, the percentage of acrosome reaction of supernumerary spermatozoa in the presence of variously treated eggs was estimated as an index of the acrosome reaction-inducing activity of the egg surface. The acrosome reaction of spermatozoa actually occurred at the surface of de-jellied and DTT-treated eggs. However, the eggs treated with trypsin lost the capacity to induce the acrosome reaction. The surface substance which induces the acrosome reaction and renders the eggs fertile was removed by trypsin and found in the supernatant fraction. The necessity of an acrosome reaction for fertilization was demonstrated by the fact that the low fertilizability of trypsin-treated eggs was brought back to the control level by insemination with spermatozoa previously treated with egg water to evoke the reaction of the acrosomes.     

Adhesion-blocking antibodies prepared against gp150 react with gp80 of Dictyostelium

A membrane glycoprotein of 150000 D, gp150, has been implicated in the mechanism of cell-cell adhesion which arises during development of Dictyostelium discoideum. This conclusion was founded on the observation that monovalent Fab′ fragments prepared from an antiserum raised against partially purified gp150 are able to block cell-cell adhesion. We show that this serum contains antibodies to a distinct membrane glycoprotein, gp80, previously implicated in cell-cell adhesion. Reaction of Fab′ to this surface molecule can account for the adhesion-blocking activity in the antiserum to gp150. Moreover, binding of gp80 neutralized Fab′ to gp150 does not block adhesion. If gp150 carries other determinants which bind adhesion-blocking Fab′, these determinants must also be present on gp80. Thus, it is not clear that gp150 is directly involved in cell-cell adhesion of Dictyostelium.