Pinto-Correia C, Poccia D L, Chang T, Robl J M
Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst 01003.
Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):7894-8. doi: 10.1073/pnas.91.17.7894.
During fertilization in most mammals, the penetrating sperm organizes an aster of microtubules. We have investigated the mechanisms underlying this function of the sperm by a series of experiments based on microinjection of isolated sperm midpieces into unfertilized oocytes. These midpieces contain antigens recognized by the MPM-2 antibody. These antigens, which are absent from the rest of the tail fraction, correspond to three phosphorylated polypeptides of 77, 81, and 85 kDa. Dephosphorylation with alkaline phosphatase abolishes antigenicity on blots and in whole sperm. Reactivity to the antibody disappears between 1 and 3 hr after calcium stimulation of oocytes, following the decline in H1 kinase activity and coincident with aster formation. In unactivated oocytes, no aster forms and the antigen remains unchanged. MPM-2 treatment of midpieces prior to injection blocks their ability to form asters in oocytes activated by calcium stimulation. The epitope also disappears in 6-methyl-aminopurine-treated oocytes, implying that maintenance of the phosphorylated state requires kinase activity. A result that confirms this view is that sperm midpieces dephosphorylated by alkaline phosphatase can be rephosphorylated after injection into oocytes or by exposure in vitro to a Xenopus oocyte cytoplasmic fraction high in H1 kinase activity. We suggest that the microtubule nucleation activity of sperm midpieces after fertilization is triggered by the calcium-induced decrease in maturation promoting factor, which results in dephosphorylation of specific sperm centrosomal proteins.
在大多数哺乳动物的受精过程中，穿透卵子的精子会组织形成一个微管星体。我们通过一系列实验研究了精子这一功能背后的机制，这些实验基于将分离的精子中段显微注射到未受精的卵母细胞中。这些中段含有可被MPM - 2抗体识别的抗原。这些抗原在尾部其他部分不存在，对应于77、81和85 kDa的三种磷酸化多肽。用碱性磷酸酶去磷酸化会消除印迹和整个精子中的抗原性。在对卵母细胞进行钙刺激后1至3小时，随着H1激酶活性的下降并与星体形成同时发生，对该抗体的反应性消失。在未激活的卵母细胞中，不形成星体且抗原保持不变。在注射前用MPM - 2处理中段会阻断它们在经钙刺激激活的卵母细胞中形成星体的能力。该表位在经6 - 甲基氨基嘌呤处理的卵母细胞中也会消失，这意味着磷酸化状态的维持需要激酶活性。证实这一观点的一个结果是，经碱性磷酸酶去磷酸化的精子中段在注射到卵母细胞中或在体外暴露于富含H1激酶活性的非洲爪蟾卵母细胞胞质组分后可以重新磷酸化。我们认为，受精后精子中段的微管成核活性是由钙诱导的成熟促进因子减少触发的，这导致特定精子中心体蛋白的去磷酸化。
