IDENTIFICATION OF TESTICULAR PROTEINS ASSOCIATED WITH THE GERM CELL-SPECIMC PROTEIN, TEX101 : CELLUBREVIN IS INVOLVED IN TEX101-TRAFFICKING TO THE GERM CELL SURFACE DURING SPERMATOGENESIS IN MICE

要全面了解生殖细胞的分化过程，需要阐明生殖细胞特异性分子的分子功能和时空表达。最近，我们发现了一种细胞表面标记蛋白，命名为TEX 101，这是男性和女性生殖细胞所特有的。生殖细胞中TEX 101表达的开/关转换与配子发生的动力学密切相关。在本研究中，我们分离睾丸蛋白的免疫沉淀与抗TEX 101抗体和鉴定的蛋白质，使用液相色谱/串联质谱。三种蛋白质（膜联蛋白2，ly 6 k和cellubrevin），TEX 101和cellubrevin之间的生物化学协会被证实免疫沉淀-蛋白质印迹实验。免疫组化研究结果表明，使用cellubrevin特异性抗体的分子是丰富的某些生殖细胞，如精母细胞和早期精子细胞，而可以忽略不计的量被发现在支持细胞，精原细胞，精子，和晚期精子细胞。大多数细胞内的cellubrevin似乎是并列与细胞内的TEX 101，和膜相关cellubrevin似乎是停靠在附近的TEX 101阳性质膜的细胞质侧。在质膜的外表面上从未观察到这种密切的联系。从这些结果中，我们得出结论，cellubrevin依赖的膜贩运参与运输从头TEX 101的表面的男性生殖细胞。此外，这些发现表明了TEX 101可能的分子功能，因此将有助于增加我们对精子发生基本机制的了解。

A complete understanding of the germ cell differentiation process requires the elucidation of the molecular functions and spatiotemporal expression of germ cell-specific molecules. Recently, we identified a cell-surface marker protein, designated TEX101, that is unique to male and female germ cells. On/off switching of TEX101 expression in germ cells is closely linked to the kinetics of gametogenesis. In the present study, we isolated testicular proteins by immunoprecipitation with anti-TEX101 antibody and identified the proteins using liquid chromatography/tandem mass spectrometry. Of three proteins identified (annexin 2, ly6k and cellubrevin), a biochemical association between TEX101 and cellubrevin was confirmed by immunoprecipitation-Western blotting experiments. Results of immunohistochemical studies using a cellubrevin-specific antibody indicated that the molecule is abundant on certain germ cells, such as spermatocytes and early-stage spermatids, whereas negligible amounts are found in Sertoli cells, spermatogonia, spermatozoa, and late-stage spermatids. Most of the intracellular cellubrevin appeared to be juxtaposed with intracellular TEX101, and membrane-associated cellubrevin appeared to be docked near TEX101-positive plasma membranes on the cytoplasmic side. This close association was never observed on the outer surface of the plasma membrane. From these results we conclude that cellubrevin-dependent membrane trafficking is involved in transport of de novo TEX101 to the surface of male germ cells. In addition, these findings suggest possible molecular functions for TEX101, and will thus help to increase our understanding of the fundamental mechanisms of spermatogenesis.