Phelps B M, Koppel D E, Primakoff P, Myles D G
Department of Physiology, University of Connecticut Health Center, Farmington 06030.
J Cell Biol. 1990 Nov;111(5 Pt 1):1839-47. doi: 10.1083/jcb.111.5.1839.
On terminally differentiated sperm cells, surface proteins are segregated into distinct surface domains that include the anterior and posterior head domains. We have analyzed the formation of the anterior and posterior head domains of guinea pig sperm in terms of both the timing of protein localization and the mechanism(s) responsible. On testicular sperm, the surface proteins PH-20, PH-30 and AH-50 were found to be present on the whole cell (PH-20) or whole head surface (PH-30, AH-50). On sperm that have completed differentiation (cauda epididymal sperm), PH-20 and PH-30 proteins were restricted to the posterior head domain and AH-50 was restricted to the anterior head domain. Thus these proteins become restricted in their distribution late in sperm differentiation, after sperm leave the testis. We discovered that the differentiation process that localizes these proteins can be mimicked in vitro by treating testicular sperm with trypsin. After testicular sperm were treated with 20 micrograms/ml trypsin for 5 min at room temperature, PH-20, PH-30, and AH-50 were found localized to the same domains to which they are restricted during in vivo differentiation. The in vitro trypsin-induced localization of PH-20 to the posterior head mimicked the in vivo differentiation process quantitatively as well as qualitatively. The quantitative analysis showed the process of PH-20 localization involves the migration of surface PH-20 from other regions to the posterior head domain. Immunoprecipitation experiments confirmed that there is protease action in vivo on the sperm surface during the late stages of sperm differentiation. Both the PH-20 and PH-30 proteins were shown to be proteolytically cleaved late in sperm differentiation. These findings strongly implicate proteolysis of surface molecules as an initial step in the mechanism of formation of sperm head surface domains.
在终末分化的精子细胞上，表面蛋白被分隔到不同的表面区域，包括头部的前部和后部区域。我们从蛋白质定位的时间以及相关机制方面，分析了豚鼠精子头部前部和后部区域的形成。在睾丸精子上，发现表面蛋白PH - 20、PH - 30和AH - 50存在于整个细胞（PH - 20）或整个头部表面（PH - 30、AH - 50）。在完成分化的精子（附睾尾部精子）上，PH - 20和PH - 30蛋白局限于头部后部区域，而AH - 50局限于头部前部区域。因此，这些蛋白在精子分化后期、离开睾丸后，其分布受到限制。我们发现，通过用胰蛋白酶处理睾丸精子，可在体外模拟这些蛋白定位的分化过程。在室温下用20微克/毫升胰蛋白酶处理睾丸精子5分钟后，发现PH - 20、PH - 30和AH - 50定位于它们在体内分化过程中所局限的相同区域。体外胰蛋白酶诱导的PH - 20定位于头部后部，在数量和质量上都模拟了体内分化过程。定量分析表明，PH - 20定位过程涉及表面PH - 20从其他区域迁移到头部后部区域。免疫沉淀实验证实，在精子分化后期，体内精子表面存在蛋白酶作用。PH - 20和PH - 30蛋白在精子分化后期都被证明发生了蛋白水解切割。这些发现强烈表明，表面分子的蛋白水解是精子头部表面区域形成机制的初始步骤。
