Immunolocalization of bovine sperm protease BSp120 by light and electron microscopy during capacitation and the acrosome reaction: Its role in in vitro fertilization

Abstract

Mammalian fertilization involves various steps in which the participation of specific enzymes has been demonstrated by numerous studies. Acrosin is one of the most widely acrosomal protease in mammalian spermatozoa studied, including bovine; however, other proteases have also been described. A new trypsin-like serine protease named bovine serine protease of 120 kDa (BSp120) and its pre-cursor BSp66 (66 kDa) were identified in bovine spermatozoa. Cytological and ultrastructural immunolocalization studies on BSp120 were performed in live and fixed cells. Immunoflorescence assays with specific polyclonal antibodies revealed localization of BSp120 on the sperm head, with a signal homogeneously distributed over the acrosome resembling a horseshoe. After the acrosome reaction, sperm showed a patchy pattern in the acrosomal cap. Immune electron microscopy analysis indicated that BSp120 is located over the head plasma membrane of capacitated spermatozoa and acrosome reacting spermatozoa. To assess BSp120 function in sperm-oocyte interaction, in vitro fertilization studies were conducted. Oocytes were incubated with spermatozoa pre-treated with anti-BSp120, anti-guinea pig acrosin, and anti-BSp120 plus anti-guinea pig acrosin. Pre-treatment of bovine spermatozoa with antibodies towards each protein did not significantly modify fertilization rates. However, when both anti-acrosin and anti-BSp120 antibodies were simultaneously added, there was a significant decrease in the fertilization rate, suggesting that both enzymes may be required for fertilization. Altogether, the results from the present study described the localization of BSp120 over the acrosome of bovine sperm, and suggest its involvement in fertilization.

Serine protease activity, bovine sperm protease, 66 kDa (BSp66), is present in hamster sperm and is involved in sperm–zona interaction

“…AR induction depicted a different exposure of BSp66 in hamster live spermatozoa, showing that BSp66 was located not only over the external membranes but also within the acrosomal content, as described for acrosin (Honda et al 2002). These results are consistent with our previous findings in bovine spermatozoa (Cesari et al 2004b).…”

“…The capacitation period involves surface changes in several species (Visconti et al 1999), some of them associated with the unmasking of surface receptors such as the progesterone receptor (Sirivaidyapong et al 2001, Pietrobon et al 2003. In agreement with these findings, our previous results with live bovine spermatozoa showed that capacitation induction promoted the exposure of BSp66 onto the sperm surface, and a redistribution during the AR (Cesari et al 2004b). As BSp66 codifying genes were not yet identified, we ignored this protein if it was expressed in the spermatogonial cell or if it was superficially acquired during the journey across the male duct (Boue et al 1992).…”

“…The fact that blocking antibodies against sperm surface antigens did not completely impair fertilization was also observed in our previous assays with bovine sperm (Cesari et al 2004b) and by other authors (Ramalho-Santos et al 2000). Although the physiological interpretation of IVF results in the presence of antibodies against specific proteins is controversial, the role of sperm and oocyte surface proteins has been determined by this experimental approach (Hasegawa et al 2000, Peknicova et al 2001.…”

“…2). The location of the protein homologous to BSp66 in hamster is coincident with that observed in bovine sperm (Cesari et al 2004b). No signal was observed in the negative control ( Fig.…”

“…In acrosome reacting spermatozoa the signal towards BSp66 was observed surrounding the exocytotic vesicles (Fig. 3B) suggesting that BSp66 is located onto the PM or the outer acrosomal membrane (OAM), as described for bovine spermatozoa (Cesari et al 2004b). In some cases, the acrosomal content showed gold labelling ( Fig.…”