The Thai Journal of Veterinary Medicine


Our study investigated proteins in bovine spermatozoa using 2-DE and LC MS/MS and found a markedly different expression in mature bovine proacrosin binding protein (sp32), which is the focus of the present study. Spermatozoa extract from nine fertile Brahman bulls was separated using 2-DE followed by Coomassie brilliant blue staining. From over 600 protein spots, a particular protein spot with two different patterns was detected at 32 kDa. The high relative protein content of spot Y1 (32 kDa pI 5.3, pattern A) was found in six bulls whereas the remaining three bulls expressed two small spots Y1 and Y2 (32 kDa pI 5.3 and 31.5 kDa pI 5.5, respectively; pattern B). Identification of spots Y1 and Y2 both in pattern A and B, by using LC MS/MS, revealed the predicted protein (derived from a genomic sequence)—the proacrosin binding protein, sp32 (62.3 kDa and pI 5.1) found in Bos taurus. The amino acid sequence coverage of these spots corresponded to the carboxyl-terminal half of sp32. Immunoblotting and immunocy to chemistry with the anti-phosphotyrosine antibody were performed to determine whether the spot Y2 was a tyrosine phosphorylated form (p32) of sp32 as reported on Sus scrofa. The results obtained did not confirm that Y2 was a p32 form of sp32. In conclusion, we applied proteomic approaches to characterize bovine spermatozoa proteins and reported a mature bovine sp32, with a relative molecular mass of ~32 kDa. The markedly different expressions of sp32 were investigated for any post-translational modification, specifically phosphorylation; however, phosphotyrosine protein (p32) could not be confirmed. Further characterization and functional analysis are needed.

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