sp42, the boar sperm tyrosine kinase, is a male germ cell-specific product with a highly conserved tissue expression extending to other mammalian species

Abstract

sp42, a tyrosine kinase of 42 kDa originally found in ejaculated boar spermatozoa, is so far the only tyrosine protein kinase to have been purified from mature male germ cells. We have developed and characterized rabbit polyclonal antibodies specifically directed against the boar sperm enzyme, which has been here purified to homogeneity. Anti-sp42 serum and sp42 affinity-purified antibodies work very well in western blot, immunoprecipitation and immunocytochemistry, and do not inhibit sp42 catalytic activity. Immunoblotting analyses reveal the presence of sp42 both in maturing boar epididymal (caput, corpus and cauda segment) spermatozoa and in testicular spermatogenic cells, thus establishing that the protein is effectively expressed in the germ cells and is not a sperm-associated protein secreted by the epididymal epithelium or male accessory glands. This finding is further strengthened by the fact that sp42 is not glycosylated, since different lectins fail to bind to sp42 and treatment of sp42 with different deglycosylation enzymes does not result in a reduction of the molecular mass of sp42. When different boar tissues are immunoscreened in western blot analysis, the results are all sp42-negative. The extension of the study to other mammalian species (human, mouse and rat) demonstrates that proteins immunologically related to boar sp42, which share the same molecular mass and tyrosine kinase activity, are both expressed in spermatogenic cells and maintained in mature sperm cells. Intriguingly, when a wide spectrum of somatic mouse and rat tissues is probed with sp42-antiserum, no tissue presents anti-sp42 immunoreactivity. Immunocytochemistry shows that in boar spermatozoa sp42 is confined to the tail mid-piece, while by immunohistochemistry carried out on sections of adult rat testis the appearance time of the kinase appears to be consistent with a post-meiotic synthesis in haploid spermatids. Altogether, these results demonstrate that boar sp42 is a new male germ cell-specific gene product, with highly conserved tissue expression extended to other mammalian species, and suggest a possible role played by the cytoplasmic tyrosine kinase in the cell signalling network specific to haploid male germ cells.