Effect of Phosphatidylcholine on Acrosome Breakdown and Fertilizing Capacity of Amphibian Spermatozoa: (phosphatidylcholine vesicles/amphibian fertilization/acrosome breakdown).

The effect of vesicles of purified egg yolk phosphatidylcholine on the fertilizing capacity and acrosome breakdown of amphibian spermatozoa was studied. When Bufo arenarum spermatozoa were incubated with either small unilamellar vesicles (prepared by sonication) or with large unilamellar vesicles (prepared by reverse-phase evaporation) a decrease in the fertilizing capacity of spermatozoa was found. At the same phosphatidylcholine concentration, large unilamellar vesicles were more inhibitory than small unilamellar vesicles. The inhibition was dependent upon the phospholipid concentration and the length of the incubation period. Small unilamellar vesicles did not modify the time course of acrosome breakdown in Leptodactylus chaquensis, while large unilamellar vesicles markedly accelerated the rate of acrosome breakdown. In both biossays, the charge of the vesicles (made either positive or negative by the addition of 5% stearylamine or 5% phosphatidic acid) did not influence their biological effect. Multilamellar vesicles did not alter the fertilizing capacity nor the acrosome breakdown. We conclude that the size and the structure of the vesicles are important parameters in determining the inhibitory capacity of phosphatidyl choline on amphibian fertilization.

Lectin binding patterns and carbohydrate mediation of sperm binding to llama oviductal cells in vitro.

Sperm binding to oviductal epithelium would be involved in sperm reservoir formation in the utero tubal junction (UTJ). Although in other mammals sperm-oviduct interaction has been proved to be mediated by carbohydrate-recognition mechanisms, the factors implicated in the sperm adhesion to oviductal epithelium of llama are still unknown. In order to assess the role of carbohydrates present in the mucosa surface, we examined the distribution of glycoconjugates in the llama oviduct by confocal lectin-histochemistry. Mannosyl, glucosyl, N-acetylglucosaminyl, galactosyl, N-acetylgalactosaminyl and sialic acid residues were detected in the oviductal mucose glycocalyx. By incubation of UTJ oviductal explants with LCA, DBA, UEA-1 or PNA lectin previous to co-culture with sperm, we observed a significant decrease in sperm binding only with LCA lectin. In the mucosa surface there were numerous d-glucosyl and D-manosyl residues, which were spotted by this lectin. Probably, this fact promotes the whole covering of the oviduct luminal surface by the sugar-lectin complex, preventing sperm access and adhesion of further residues. However, sperm incubation with mannose or glucose does not significantly prevent binding, which means that glucose and mannose would not be involved in a specific sperm-oviduct interaction. On the other hand, we observed a high reduction in sperm binding to UTJ explants with N-acetylgalactosamine and galactose (p<0.001). Coincidentally, binding sites for N-acetylgalactosamine-PAA-FITC conjugate were observed on the whole surface of the sperm, supporting the concept that llama sperm have lectin-like molecules in their surface, as is the case in other mammals. Probably, these lectin-like molecules, by means of N-acetylgalactosamine and galactose recognition, could link the sperm to the oviductal mucosa with the purpose of forming storing sites in the UTJ. Our results support the idea that more than one carbohydrate could participate in sperm reservoir formation in the llama UTJ oviductal segment.

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.