Immunocytochemical Localization and Biochemical Characterization of Two Seminal Plasma Proteins That Protect Ram Spermatozoa Against Cold Shock

Abstract

We have already shown that seminal plasma proteins in the ram can repair cold-shock sperm membrane damage and that the addition of seminal plasma proteins before cold-shock treatment prevents sperm membrane injury. In this study, we prove that 2 protein bands of approximately 14 (P14) and 20 (P20) kd isolated from seminal plasma are responsible for this protective effect. In vitro capacitation (CA) and acrosome reaction (AR) modified the content of both proteins on the sperm surface. P20 release began at the beginning of CA, and the induction of the AR accounted for an additional release of both proteins; not more than 35% of these proteins remained on acrosome-reacted sperm. Cytochemical analysis detected that there are several binding sites for P14 and P20 on the sperm surface and that membrane alterations induced by CA and the AR accounted for the loss and redistribution of both proteins to the equatorial and postequatorial regions. The P14-sequenced fragment showed a high homology with several seminal plasma proteins of different species and contained the FN 2 domain like bovine PDC-109. However, the sequence of the P20 fragment was not homologous with any reported protein. By immunochemical analysis, we obtained evidence that P14 is phosphorylated in serine and threonine residues and that P20 is a glycosylated protein. These results suggest that both proteins are involved in sperm CA and gamete interaction, first by stabilizing the sperm membrane and then by participating in CA in the female reproductive tract. The protective effect of P14 and P20 could be related to their decapacitating role.