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

对生殖细胞分化过程的完整理解需要对生殖细胞特异性分子的分子功能和时空表达进行阐明。最近，我们发现了一个细胞表面标记蛋白，命名为TEX101，这是男性和女性生殖细胞所特有的。生殖细胞中TEX101表达的开关与配子发生动力学密切相关。本研究采用抗tex101抗体免疫沉淀法分离睾丸蛋白，并采用液相色谱/串联质谱法对蛋白进行鉴定。在鉴定的三个蛋白（膜联蛋白2，ly6k和cellubrevin）中，TEX101和cellubrevin之间的生化关联通过免疫沉淀- western blotting实验得到证实。使用纤维素蛋白特异性抗体的免疫组织化学研究结果表明，该分子在某些生殖细胞（如精母细胞和早期精子细胞）中含量丰富，而在支持细胞、精原细胞、精子和晚期精子细胞中含量可忽略不计。大多数细胞内的纤维素蛋白似乎与细胞内的TEX101并列，而膜相关的纤维素蛋白似乎停靠在细胞质侧的TEX101阳性质膜附近。在质膜的外表面从未观察到这种密切的联系。从这些结果我们得出结论，依赖纤维素蛋白的膜运输参与了TEX101到男性生殖细胞表面的转运。此外，这些发现提示了TEX101可能的分子功能，从而有助于增加我们对精子发生基本机制的理解。

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.