Purification and partial characterization of a sperm motility‐inhibitory protein of ram cauda epididymal plasma

Abstract

AbstractMammalian sperm remain quiescent but fertile for several weeks in cauda epididymis. Although several sperm quiescent factors of epididymal plasma have been identified in goat, pig and cattle; however, little is known in sheep. In the present study, purification and characterization of a novel sperm quiescent protein of ovine cauda epididymal plasma (CEP) was carried out. The sperm quiescent protein was partially purified by hydroxyapatite gel adsorption chromatography followed by DEAE‐sepharose® anion exchange chromatography. In the latter, the sperm quiescent activity was eluted both in 0.05 and 0.2 M potassium phosphate buffer (pH 7.5) fractions having a predominant protein of about 80 and 70 kDa with 87% and 63% homogeneity, respectively. The proteins were designated as motility‐inhibitory factor of sheep I and II (MIFS‐I and II), respectively. Significant (about 60%) inhibition of sperm motility was observed following treatment of cauda epididymal sperm with 6 and 12 µg/mL of partially purified MIFS‐I and II, respectively. Specific activities of the partially purified MIFS‐I and II were 563 and 261 U/mg of protein, while the fold‐purification of the activity were 5119 and 2373, respectively. Both the proteins were heat‐labile and the activity was completely lost following incubation at 100°C for 5 min. Further, the partially purified MIFS‐I (5 µg/mL) caused significant reduction in in vitro sperm capacitation and slight decline in tyrosine phosphorylated p72 and p52 proteins; however the protein was nontoxic to sperm. Mass spectrometric analysis of MIFS‐I revealed significant identity with human semaphorin 3D. Both dot blot and western blot analysis demonstrated cross‐reactivity of MIFS‐I with polyclonal anti‐human SEMA3D antibody. It was concluded that the MIFS‐I of ovine CEP was putative ovine semaphorin 3D protein having potent sperm quiescent and decapacitating activities and it possibly acts through inhibition of protein tyrosine phosphorylation.