Egg Binding Research Articles

Overexpressing sperm surface beta 1,4-galactosyltransferase in transgenic mice affects multiple aspects of sperm-egg interactions.

Sperm surface beta 1,4-galactosyltransferase (GalTase) mediates fertilization in mice by binding to specific O-linked oligosaccharide ligands on the egg coat glycoprotein ZP3. Before binding the egg, sperm GalTase is masked by epididymally derived glycosides that are shed from the sperm surface during capacitation. After binding the egg, sperm-bound oligosaccharides on ZP3 induce the acrosome reaction by receptor aggregation, presumably involving GalTase. In this study, we asked how increasing the levels of sperm surface GalTase would affect sperm-egg interactions using transgenic mice that overexpress GalTase under the control of a heterologous promoter. GalTase expression was elevated in many tissues in adult transgenic animals, including testis. Sperm from transgenic males had approximately six times the wild-type level of surface GalTase protein, which was localized appropriately on the sperm head as revealed by indirect immunofluorescence. As expected, sperm from transgenic mice bound more radiolabeled ZP3 than did wild-type sperm. However, sperm from transgenic animals were relatively unable to bind eggs, as compared to sperm from wild-type animals. The mechanistic basis for the reduced egg-binding ability of transgenic sperm was attributed to alterations in two GalTase-dependent events. First, transgenic sperm that overexpress surface GalTase bound more epididymal glycoside substrates than did sperm from wild-type mice, thus masking GalTase and preventing it from interacting with its zona pellucida ligand. Second, those sperm from transgenic mice that were able to bind the zona pellucida were hypersensitive to ZP3, such that they underwent precocious acrosome reactions and bound to eggs more tenuously than did wild-type sperm. These results demonstrate that sperm-egg binding requires an optimal, rather than maximal, level of surface GalTase expression, since increasing this level decreases sperm reproductive efficiency both before and after egg binding. Although sperm GalTase is required for fertilization by serving as a receptor for the egg zona pellucida, excess surface GalTase is counterproductive to successful sperm-egg binding.

Development of an equine immunocontraceptive agent using sperm plasma membranes

Immunocontraception, and in particular the targeting of antibodies to gamete-specific antigens implicated in sperm–egg binding and fertilisation, offers an attractive approach to the growing global problem of overpopulation. Such an idea is not new; indeed several immunocontraception trials, using animal model systems, have been reported in recent years and a number are reviewed here. However, the results of these studies have been largely disappointing. We believe that two fundamental flaws attribute to the poor success of most of these preliminary immunocontraceptive trials. Firstly, loss of fertility has invariably been used as the assay. This presupposes that immuno-neutralisation of a single, gamete-specific antigen will be sufficient to cause a significant reduction in fertility; however, recent data suggests that such a premise may not be well-founded for a number of reasons. Secondly, and arguably the most important flaw, is the almost universal, but largely inappropriate, use of systemic immunisation as the sole route of antigen delivery. Whilst systemic immunisation regimes may lead to high serum IgG levels, these levels do not correlate with specific antibody levels in the reproductive tract or with contraceptive efficacy. Hence, an alternative antigen delivery approach is required which will induce an effective local immune response in the reproductive tract. Here we discuss the ways in which this might be achieved.

Human sperm–egg binding is inhibited by peptides corresponding to core region of an acrosomal serine protease inhibitor

An antiserum, designated R4 and raised against denatured hamster acrosomes, was shown to localize specifically to the acrosomal region of hamster, rat, mouse, and human spermatozoa, and to inhibit both hamster and human sperm-oocyte binding in vitro. Following screening of a human testis lambda gt11 cDNA expression library with the antiserum R4, a series of cDNA clones were isolated. One (cDNA 134) was selected based on the ability of the beta-galactosidase fusion protein to inhibit human and hamster sperm-zona binding in vitro. The fusion protein was also shown to inhibit the penetration of zona-free hamster oocytes by human spermatozoa. Sequence analysis revealed that cDNA 134 coded for a portion of a serine protease inhibitor (serpin) closely related to plasma Protein C inhibitor. Sequencing of an additional cDNA clone (261) and Northern blot analysis confirmed that a Protein C inhibitor-like mRNA is synthesised in the human testis. Affinity-purified anti-134 antibody specifically localized to the acrosomal region of both hamster and human sperm. Synthetic peptides corresponding to the conserved core region responsible for the interaction of the serpin with its cognate protease also blocked human sperm-zona binding in vitro. The results suggest that this acrosomally located inhibitor plays an important role in the series of binding events that results in human fertilization.

Detection of antigalactosyltransferase antibodies in plasma from patients with antisperm antibodies

Sperm-associated galactosyltransferase has been implicated as a macromolecule involved in sperm to egg binding interactions during fertilization. An analysis of human plasma from 18 patients with antibodies to sperm antigens detected antigalactosyltransferase antibodies when compared with 10 control patients. These plasma antibodies were all able to immunoprecipitate enzyme activity from a preparation of human milk galactosyltransferase but varied in their ability to detect the protein by Western blot analyses. These results confirm the antigenic presence of galactosyltransferase in human sperm.

Starfish acrosomal acid phosphatase: a cytochemical and biochemical study

The acrosome reaction of spermatozoa from the starfish Marthasterias glacialis was induced with the ionophore A23187. Reacted cells were then processed for acid phosphatase ultrastructural cytochemistry, but significant enzyme activity was not detected. However, when the supernates from suspensions of ionophore-treated sperm were assayed for acid phosphatase, a net enzyme activity was observed. Supernatant proteins were run in starch gel electrophoresis and fluorescent zymograms revealed a single band of acid phosphatase. SDS-PAGE of proteins eluted from the active spots of starch gels showed one major band of about 63 kDa. The results obtained support the hypothesis that the acid phosphatase whose activity has been detected only at the time of binding of sperm and egg originates from the sperm acrosome.

Starfish acrosomal acid phosphatase: a cytochemical and biochemical study

The acrosome reaction of spermatozoa from the starfish Marthasterias glacialis was induced with the ionophore A23187. Reacted cells were then processed for acid phosphatase ultrastructural cytochemistry, but significant enzyme activity was not detected. However, when the supernates from suspensions of ionophore-treated sperm were assayed for acid phosphatase, a net enzyme activity was observed. Supernatant proteins were run in starch gel electrophoresis and fluorescent zymograms revealed a single band of acid phosphatase. SDS-PAGE of proteins eluted from the active spots of starch gels showed one major band of about 63 kDa. The results obtained support the hypothesis that the acid phosphatase whose activity has been detected only at the time of binding of sperm and egg originates from the sperm acrosome.

Electron microscopic study of the in vivo zona pellucida binding ability of epididymal ram spermatozoa

AbstractIn the ram, spermatozoa develop the ability to initiate pregnancy only after reaching the body of the epididymis. To determine the zona pellucida binding ability of ram spermatozoa collected from different levels of the epididymis, sufficient numbers of motile sperm cells of different epididymal origin were inseminated surgically below the uterotubal junction of ewes at the time of ovulation. Intratubal ova were recovered 24 hr later, and those having spermatozoa attached to the zona were examined by transmission electron microscopy to assess the characteristics of the bound spermatozoa. Data indicate that the ability of the capacitated spermatozoa to adhere to the zona pellucida depends on sperm egg binding sites that develop on the acrosomal membranes from the apex to equatorial segment during epididymal transit.

Galactosyltransferase activities on mouse sperm bearing multiple tlethal and tviable haplotypes of the T/t-complex.

SUMMARYSegregation-distortingt-sperm show a specific increase inN-acetylglucosamine: galactosyltransferase activity over wild-type (+/+) due to a deficiency of a wild-type galactosyltransferase inhibitor (Shur & Bennett, 1979). Eight other enzymic activities are indistinguishable between +- andt-sperm suspensions. In this study, three additional points are analysed. First, galactosyltransferases are assayed on sperm homozygous for a semilethal haplotype (tW2/tW2), relative to heterozygous (+ /tw2) and wild-type (+/+) controls.tW2/tW2assays circumvent the +-sperm inhibition oft-sperm galactosyltransferases that occurs in heterozygous + /t-assays and show thatt-sperm are actually four times as active as wild-type. Second, sperm which are compound heterozygotes for two complementing lethalt-haplotypes (tlx/tly), have nearly twice the theoretical enzyme level oftlx/tlysperm. Thus, in either homozygous (tW2/tW2) or double heterozygous (tlx/tly) formt-haplotypes act synergistically on sperm galactosyltransferase activity.Third, and most interesting, sperm bearing either recombinant, viablet-haplotypes (+/tv,tvx/tvy), or one of three dominantT/t-complex mutations, were assayed to determine which portions of theT/t-complex are responsible for elevated galactosyltransferase activity. Results show that sperm bearing recombinant, non-segregation-distorting, viabletv-haplotypes no longer show elevated transferase activity. Therefore, the elevatedt1-sperm galactosyltransferase activity strictly correlates with the increased transmission frequency oft1-sperm. These studies strengthen further the hypothesis that sperm surface galactosyltransferases are involved in egg binding during fertilization, and thatt1-sperm segregation-distortion results, at least in part, from increased galactosyltransferase activity.

Egg Binding in Broiler Chickens Fed Heptachlor

Egg binding was observed at 25 to 29 weeks of age in broiler chickens fed .1 or .3 ppm of heptachlor in the ration during the first 8 weeks of life. Eggshell thickness was not affected, but heavier and misshapen eggs were laid by chickens fed heptachlor.