Sette C, Bevilacqua A, Bianchini A, Mangia F, Geremia R, Rossi P
Dipartimento di Sanitá Pubblica e Biologia Cellulare, Sezione di Anatomia, Università di Roma Tor Vergata, Rome, Italy.
Development. 1997 Jun;124(11):2267-74. doi: 10.1242/dev.124.11.2267.
A truncated form of the c-kit tyrosine kinase receptor, corresponding to the phosphotransferase portion of the cytoplasmic catalytic domain and the carboxyterminus (tr-kit), is accumulated during late mouse spermiogenesis. Here we report that tr-kit is specifically localized in the residual sperm cytoplasm, with maximal accumulation in the midpiece of the flagellum, suggesting that it can enter the egg during fertilization. Microinjection of extracts from COS cells expressing a recombinant tr-kit protein into metaphase II-arrested mouse oocytes caused complete oocyte activation, including cortical granule exocytosis, completion of the 2nd meiotic division, formation of a parthenogenetic pronucleus and progression through cleavage stages. No activation above background levels was obtained with extracts from mock-transfected COS cells. Similar results were obtained by microinjection of in vitro synthesized tr-kit mRNA into metaphase II-arrested oocytes. Tr-kit-induced parthenogenetic egg activation was completely inhibited by oocyte preincubation with the Ca2(+)-chelating agent BAPTA-AM or with a specific inhibitor of phospholipase C activity. Tr-kit-induced egg activation was associated with a decrease in activity of mitogen-activated protein kinase, an essential component of the cytostatic factor. These results candidate tr-kit as a putative sperm factor required for triggering activation of mouse eggs at fertilization.
一种截短形式的c-kit酪氨酸激酶受体，对应于细胞质催化结构域的磷酸转移酶部分和羧基末端（tr-kit），在小鼠精子发生后期积累。我们在此报告，tr-kit特异性定位于残留的精子细胞质中，在鞭毛的中段积累最多，这表明它在受精过程中可以进入卵子。将表达重组tr-kit蛋白的COS细胞提取物显微注射到处于减数分裂中期II期阻滞的小鼠卵母细胞中，可导致卵母细胞完全激活，包括皮质颗粒胞吐、完成第二次减数分裂、形成孤雌生殖原核并通过分裂阶段。用mock转染的COS细胞提取物未获得高于背景水平的激活。通过将体外合成的tr-kit mRNA显微注射到处于减数分裂中期II期阻滞的卵母细胞中也获得了类似结果。用Ca2(+)-螯合剂BAPTA-AM或磷脂酶C活性的特异性抑制剂对卵母细胞进行预孵育，可完全抑制tr-kit诱导的孤雌生殖卵激活。tr-kit诱导的卵激活与有丝分裂原激活蛋白激酶活性降低有关，有丝分裂原激活蛋白激酶是细胞静止因子的重要组成部分。这些结果使tr-kit成为受精时触发小鼠卵激活所需的一种假定精子因子。
