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Analysis of the interaction between cytoskeleton and Fyn tyrosine kinase during spermatogenesis in the mouse

小鼠精子发生过程中细胞骨架与Fyn酪氨酸激酶相互作用的分析

基本信息

批准号：
10670007
负责人：
MAEKAWA Mamiko
金额：
$ 2.18万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670007/
关键词：
Fyn tyrosine kinase testis Setoli cell cytoskeleton アクチンフィラメント

项目摘要

1. Localization of Fyn tyrosine kinase in the mouse testisImmunohistochemical studies revealed that Fyn tyrosine kinase was localized in the Sertoli cells in the mouse testis, and not in the spermatogenic cells.2. Colocalization of Fyn and actin filaments in the ectoplasmic specializationFyn was localized abundantly in the ectoplasmic specialization of the Sertoli cells, and Fyn was colocalized with actin filaments in the specializations.3. Western blot analysesTriton-soluble and insoluble fractions were prepared from the mouse testis, and the samples were applied for SDS-PAGE, transferred to the membrane, and immunostained with anti-Fyn antibody. The results indicated that Fyn protein was detected mostly in the Triton-insoluble, cytoskeletal fraction. These data strougly suggest that Fyn kinase is associated with cytoskeletal proteins. Using anti-phosphotyrosine antibody, a tyrosine-phosphorylated protein at 80 kDa(p80) was abundantly detedted in the Triton -insoluble cytoskeletal fractions of the wild-type testis. In contrast, p80 was significantly reduced in the cytoskeletal fraction of the fyn -/- testes. This finding indicated that p80 might be the target of Fyn kinase.4. Abnormalities in the fyn -/- testis during postnatal developmentAdultfyn -/- male mice are fertile. However, we found that the weight of fyn -/- immature testes at the age of 3 to 4 weeks significantly decreased compared to that of wild-type testes. The diameter of the seminiferous tubules were also decreased, and necrotic germ cells were frequently found in the fyn -/- testes. Furthermore, uncommon ultrastructures of ectoplasmic specializations were also detected in the fyn -/- testes. These findings showed that Fyn Kinase is involved in the survival and/or differentiation of the germ cells at the specific stages via Sertoli cells.

1.Fyn酪氨酸激酶在小鼠睾丸中的定位免疫组织化学研究表明，Fyn酪氨酸激酶定位于小鼠睾丸的Sertoli细胞，而不是生精细胞。Fyn和肌动蛋白细丝在胞外特化中的共定位Fyn在Sertoli细胞的胞质特化中大量定位，Fyn与肌动蛋白细丝在胞质特化中共定位。免疫印迹分析从小鼠睾丸制备Triton可溶性和不溶性部分，并进行SDS-PAGE，转移到细胞膜上，用抗Fyn抗体进行免疫染色。结果表明，Fyn蛋白主要存在于不溶于Triton的细胞骨架部分。这些数据有力地表明Fyn激酶与细胞骨架蛋白有关。利用抗磷酸化酪氨酸抗体，在野生型睾丸不溶于Triton的细胞骨架部分中大量检测到80 kDa(P80)的酪氨酸磷酸化蛋白。相反，p80在fyn-/-睾丸的细胞骨架部分显著减少。这一发现表明p80可能是Fyn激动酶的靶点。Fyn-/-雄性小鼠在出生后发育过程中的异常。然而，我们发现，与野生型相比，3-4周龄的fyn/-未成熟睾丸的重量显著减轻。曲细精管直径缩小，睾丸内常可见坏死性生殖细胞。此外，在FYN-/-精巢中还检测到罕见的外质特化的超微结构。这些发现表明，Fyn Kinase通过Sertoli细胞参与生殖细胞在特定阶段的存活和/或分化。