Berruti G, Borgonovo B
Dipartimento di Biologia, Università di Milano, Italy.
J Cell Sci. 1996 Apr;109 ( Pt 4):851-8. doi: 10.1242/jcs.109.4.851.
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.
sp42是一种最初在射出的公猪精子中发现的42 kDa酪氨酸激酶，是迄今为止唯一从成熟雄性生殖细胞中纯化出来的酪氨酸蛋白激酶。我们已经制备并鉴定了针对公猪精子酶的兔多克隆抗体，该酶已被纯化至同质。抗sp42血清和sp42亲和纯化抗体在蛋白质印迹、免疫沉淀和免疫细胞化学中效果良好，且不抑制sp42的催化活性。免疫印迹分析显示，sp42存在于成熟的公猪附睾（头部、体部和尾部段）精子以及睾丸生精细胞中，从而证实该蛋白在生殖细胞中有效表达，而不是附睾上皮或雄性附属性腺分泌的精子相关蛋白。由于不同的凝集素不能与sp42结合，并且用不同的去糖基化酶处理sp42不会导致sp42分子量降低，即sp42未被糖基化，这一发现得到了进一步加强。当在蛋白质印迹分析中对不同的公猪组织进行免疫筛选时，结果均为sp42阴性。将研究扩展到其他哺乳动物物种（人、小鼠和大鼠）表明，与公猪sp42免疫相关的蛋白质，具有相同的分子量和酪氨酸激酶活性，在生精细胞中表达并保留在成熟精子细胞中。有趣的是，当用sp42抗血清检测多种小鼠和大鼠体细胞组织时，没有组织呈现抗sp42免疫反应性。免疫细胞化学显示，在公猪精子中，sp42局限于尾部中段，而通过对成年大鼠睾丸切片进行免疫组织化学分析，该激酶的出现时间似乎与单倍体精子细胞减数分裂后合成一致。总之，这些结果表明，公猪sp42是一种新的雄性生殖细胞特异性基因产物，其组织表达高度保守并扩展到其他哺乳动物物种，并提示细胞质酪氨酸激酶在单倍体雄性生殖细胞特异性细胞信号网络中可能发挥的作用。
