This study has assessed the effect of zinc on human sperm motility and the acrosome reaction in vitro. Progressively motile human sperm were selected by swim-up and by glass bead columns and then incubated in a medium in which capacitation happened in an asynchronous way. Different doses of zinc (1, 10, 100 and 1000 microM) were added for periods of 2, 4 or 6 h. Other samples were incubated with zinc (1000 microM), and after 1 h incubation, the zinc was removed. Aliquots of each culture were used to evaluate progressive motility and the acrosome reaction using a triple-stain technique. Sperm motility was reduced when the amount of zinc added was greater than or equal to 100 microM, and these doses also caused a significant reduction in the % of sperm undergoing the acrosome reaction. After removal of zinc and further incubation in zinc-free medium for 1 h, an increase in the percentage of motile and acrosome-reacted sperm was observed. However, the increase in acrosome reaction did not reach the values observed in controls. Results suggest that extracellular zinc acts as an inhibitor of human sperm motility and the acrosome reaction (and/or capacitation and the acrosome reaction). This inhibitory effect is reversible and occurs in a dose-dependent fashion. The probable mechanisms involved are discussed.

Phospholipase A2 (PLA2), its localization on human sperm and its involvement in sperm-egg interaction, was investigated. Sperm-egg interaction was examined using an in vitro assay of the interaction between human sperm and zona-free or zona-intact hamster egg. PLA2- specific antibodies and/or lysophosphatidylcholine (LPC) were added to the coincubation medium. PLA2 was localized on the anterior tip of the human sperm head by an immunogold silver staining method in light microscopy (IGSS) and TEM. PLA2-specific antibodies inhibited human sperm-zona-free oocyte fusion significantly. LPC treatment allows interspecies fertilization of zona-intact hamster oocytes. PLA2 plays an important role in membrane-fusion events. This statement is supported by the fact that PLA2 is localized in the region where an exocytotic event, such as the acrosome reaction, occurs in the spermatozoon. PLA2-specific antibodies inhibited sperm-egg fusion, but not sperm-oolemma adhesion. LPC may stimulate the fertilizing ability of spermatozoa and induce changes on the zona pellucida and on the oolemma promoting in sperm-egg fusion. Based on these findings, it is suggested that sperm PLA2 and one of its modulators, the LPC, may contribute to membrane-fusion events in mammalian fertilization.

Two-cell mouse embryos cultured in vitro in the presence of either purified rabbit uteroglobin (UG) or recombinant human UG developed and proliferated faster than controls cultured in the absence of this protein. Both the percentage of embryos developing to the blastocyst stage and the number of cells per embryo were increased. Treatment with UG for 3 hr was enough to trigger this response. The effect of UG was blocked by genistein, an inhibitor of tyrosine protein kinases, suggesting the involvement of these kinases in the stimulation of the embryo by UG. To further support this suggestion, embryos were metabolically labeled in vitro with [ 32 P] and the phosphorylated proteins were immunoprecipitated with anti-phosphotyrosine. Analysis of the immunoprecipitates by SDS-PAGE showed that UG induced the phosphorylation of several proteins of M r between 200 and 37 kDa. This induction was observed after 1 hr of stimulation with UG and further increased after 3 hr of treatment. Since UG is synthesized and secreted in the uterus and the oviduct, these results suggest a physiological role of this protein in the correct development of the embryo in vivo.

Phospholipase A2 and lysophospholipids have been implicated in the mammalian sperm acrosome reaction. In this study we further investigated the role of this enzyme and lysophospholipids on the acrosome reaction of hamster spermatozoa. Hamster epididymal spermatozoa were incubated under capacitation and acrosome reaction-inducing conditions. After 3.0 and 3.5 h, the spermatozoa were treated with different doses of lysophosphatidylcholine for 12 min. Then the percentage of motility, hyperactivation, and acrosome reaction was evaluated by light microscopy. Lysophosphatidylcholine, 10 micrograms/ml, was the highest acrosome reaction-inducing dose without an effect on sperm motility. Lysophosphatidylcholine induced the acrosome reaction only when added to spermatozoa capacitated for a minimum of 2 h. This effect was apparent after 1 min of its addition and reached a plateau after 5 min. Lysophosphatidylethanolamine and lysophosphatidylinositol were also effective in inducing the acrosome reaction. Lysophosphatidylserine did not have any effect on the reaction, but caused an increase in sperm hyperactivation. Sperm treated with the phospholipase A2 inhibitors quinacrine dihydrochloride and p-bromophenacyl-bromide showed an inhibition of the spontaneous occurrence of the acrosome reaction. These inhibitors, however, did not block the acrosome reaction induced by lysophosphatidylcholine. The time course of the lysophosphatidylcholine-induced acrosome reaction was the same whether control or inhibitor treated spermatozoa were used. These results suggest that the membrane events of the acrosome reaction initiate with the activation of the phospholipase A2, thus producing the fusogen agents necessary for this exocytotic event.