Characterization and immunolocalization of HBP, FA-1 and TIMP-2 like proteins in cattle bull semen: HBP modification in in vitro capacitated spermatozoa

Characterization and localization of HBP, FA-1 and TIMP-2 like proteins in spermatozoa and seminal plasma of cattle bulls was carried out by immunoblotting and immunofluoresence. Anti-HBP, anti-TIMP-2 and anti-FA-1 reacted with 55, 48, 45, 42, 35, 30, 24, 18, 16 kDa; 65, 45, 24, 16 kDa and 55, 48, 16 kDa sperm proteins on immunoblots. Immunofluoresence indicated that HBP/TIMP-2 are localized mainly on acrosomal cap, whereas, FA-1 predominantly on post acrosomal cap. Among the bulls, positive for 60, 45, 16 kDa FA-1 like proteins in sperm extracts and 11/16 kDa in SP; 7, 8, 6 and 7 bulls also showed higher rate of in vitro AR. Number of bulls positive for 65, 24 kDa-TIMP-2 and with higher rate of AR was more as compared to other anti-TIMP-2 reactive sperm/SP proteins. Therefore, 60, 45 and 16 kDA-FA-1, 65 and 24 kDa-TIMP-2 like proteins may serve as indicators of higher rate of in vitro AR vis a vis fertility of cattle bulls. Immunoblotting of cpapacitatd and un-capacitated spermatozoa with anti-HBP suggested that removal of 55, 48, 45, 40, 37 and 30 kDa HBP from in vitro acrosome reacted cattle bull spermatozoa allowed heparin to mediate in vitro AR and an increase in intensity of 110, 90 kDa and exposure of 65, 60, 26 and 16 kDa HBP after AR may be important for binding of sperm to ovum, penetration and fertilization.