MOLECULAR REPRODUCTION AND DEVELOPMENT 69:411–418 (2004) Immunolocalization of Bovine Sperm Protease BSp120 by Light and Electron Microscopy During Capacitation and the Acrosome Reaction: Its Role in In Vitro Fertilization ANDREINA CESARI, 1 * JORGE J. SA ´ NCHEZ, 1 JUAN C. BIANCOTTI, 2 MO ´ NICA H. VAZQUEZ-LEVIN, 2 GERMA ´ N KAISER, 3 GUSTAVO A. PALMA, 3 RICARDO ALBERIO, 3 AMANDA E. VINCENTI, 4 AND MIGUEL W. FORNE ´ S 4 1 Instituto de Investigaciones Biolo´gicas, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata. Mar del Plata, Buenos Aires, Argentina 2 Instituto de Biologı´a y Medicina Experimental. CONICET-UBA Vuelta de Obligado, Buenos Aires, Argentina 3 Laboratorio de Biotecnologı´a de la Reproduccio´n, INTA, Balcarce, Provincia de Buenos Aires, Argentina 4 Instituto deHistologı´a yEmbriologı´a ‘‘Dr. Mario H.Burgos’’, Facultad de Ciencias Me´dicas UNCU-CONICET, Mendoza, Argentina 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 spermato- zoa. Cytological and ultrastructural immunolocaliza- tion 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 micro- scopy analysis indicated that BSp120 is located over the head plasma membrane of capacitated spermato- zoa 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. Mol. Reprod. Dev. 69: 411–418, 2004. ß 2004 Wiley-Liss, Inc. Key Words: serine protease; membrane sperm pro- tein; mammalian fertilization INTRODUCTION Mammalian fertilization is a process that involves several steps, including the sperm acrosome reaction (AR) and zona pellucida (ZP) penetration. During penetration, mechanical and enzymatic tools are appar- ently necessary (Yanagimachi, 1994). In this regard, the presence of serine–proteases have been described, and their specific inhibitors have been found to decrease the rates of fertilization (Zaneveld et al., 1971; Fraser, 1982; Llanos et al., 1993). Trypsin-like proteases have been involved in membrane fusion processes and in signaling events during fertilization (Tesarik, 1995; Dell et al., 1999; Haden et al., 2000; Yoshitani et al., 2001; Klinefelter et al., 2002). In mammalian spermatozoa, the most widely studied serine protease is acrosin, which is located within the acrosomal vesicle (Urch, 1991). ß 2004 WILEY-LISS, INC. Andreina Cesari, affiliated to Universidad Nacional de Mar del Plata; Jorge J. Sa ´nchez, Juan C. Biancotti, Mo ´nica H. Vazquez-Levin, Amanda E. Vincenti, and Miguel W. Forne ´s, affiliated to CONICET, Germa ´ n Kaiser, Gustavo A. Palma, and Ricardo Alberio, affiliated to INTA. Grant sponsor: Universidad Nacional de Mar del Plata; Grant sponsor: CIUDA; Grant sponsor: SECYT; Grant sponsor: FIRCA; Grant sponsor: CONICET (to RDC); Grant number: PIP2842; Grant sponsor: CONICET (to MHVL); Grant number: PIP4404; Grant sponsor: PLACIRH/ PROGRESAR (fellowship). *Correspondence to: Andreina Cesari, Instituto de Investigaciones Biolo ´gicas, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, CC:1245, Zip Code: 7600, Mar del Plata, Argentina. E-mail: acesari@mdp.edu.ar Received 25 March 2004; Accepted 18 May 2004 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.20100