Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation

Protein tyrosine phosphorylation is an important intracellular event accomp anying the in-vitro capacitation of mouse, bovine and human spermatozoa. He re, we demonstrate that bovine serum albumin (BSA) and NaHCO3 are required for protein tyrosine phosphorylation in ejaculated human spermatozoa. The a bsence of protein tyrosine phosphorylation in media minus these two constit uents could be recovered by addition to the media of cAMP analogues and/or phosphodiesterase inhibitors. Since BSA is postulated to modulate capacitat ion by removal of cholesterol from the sperm plasma membrane, we determined whether cholesterol release leads to changes in protein tyrosine phosphory lation. Incubation of spermatozoa in media containing BSA resulted in the r elease of significant amounts of cholesterol when compared with media devoi d of BSA. Preloading BSA with cholesterol-SO4 inhibited protein tyrosine ph osphorylation, as well as capacitation, and this inhibitory effect was over come by the addition of dibutyryl cAMP plus isobutylmethylxanthine (IBMX). The functional significance of BSA-mediated cholesterol release, protein ty rosine phosphorylation and capacitation was confirmed by examining the effe cts of the cholesterol-binding heptasaccharides, methyl-acyclodextrin or OH -propyt-beta-cyclodextrin. Both cyclodextrins caused cholesterol efflux fro m the spermatozoa, increased protein tyrosine phosphorylation, and stimulat ed capacitation. Therefore, cholesterol release is associated with the acti vation of a signal transduction pathway involving protein kinase A and tyro sine kinase second messenger systems, and resulting in protein tyrosine pho sphorylation and capacitation.