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Abnormal morphology of the testes of the fyn-deficient mice and analysis of the function of Fyn tyrosine kinase

fyn缺陷小鼠睾丸形态异常及Fyn酪氨酸激酶功能分析

基本信息

批准号：
12670007
负责人：
MAEKAWA Mamiko
金额：
$ 2.05万
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：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670007/
关键词：
Fyn tyrosine kinase testis Sertoli cell spermatogenesis knockout mouse

项目摘要

1. Significant reduction of the testis weight and degenerated germ cells were found at the age of 3 and 4 weeks of fyn-deficient mice. Electron microscopy revealed that fyn-deficient testis showed ultrastructural abnormality in specialized junctional structures of the Sertoli cells, termed the ectoplasmic specializations (ES). Small unusual vesicles were found in the actin filament layer of the ES. Immunohistochemical studies demonstrated that Fyn was concentrated together with actin filaments at the ES. At these sites high level of phosphotyrosine was also immunostained in the wild type testes, while phosphotyrosine immunoreactivity was decreased in the fyn -deficient testes. These findings suggest that Fyn kinase functions at the ectoplasmic specializations of the Sertoli cells in the testis.2. Immunoblot analyzes revealed that Fyn was distributed mostly in the Triton X-100 insoluble cytoskeletal fraction prepared from the wild-type mouse testis, suggesting that Fyn may be associated with cytoskeletal proteins such as actin filaments. A tyrosine phophorylated protein at around 80 kDa (P80) was prominent in the Triton-insoluble fraction from wild-type testis. However, P80 was specifically decreased in the fyn-deficient testes. This finding indicated that P80 might be the target of Fyn kinase, and that P80 may play a role in a Fyn-mediated signal transduction in the mouse testis.3. We found that a mouse Sertoli cell line, TM4, expressed Fyn protein. Tyrosine-phosphorylated proteins were examined in the TM4 cell extracts by two-dimensional immunoblot analysis. When the cells was treated with environmental endocrine disrupters, such as diethylstilbestrol, the level of tyrosine phosphorylation of one protein at approximately 38kDa (P38) was conspicuously increased compared to the control cells. It is possible that Fyn tyrosine kinase may be involved in the phosphorylation of the P38.

1.在3周龄和4周龄时，fyn缺陷小鼠睾丸重量明显减少，生殖细胞退化。电子显微镜显示，fyn缺陷睾丸支持细胞的特化连接结构，称为外质特化（ES）的超微结构异常。在ES的肌动蛋白丝层中发现了不寻常的小泡。免疫组织化学研究表明，Fyn集中在一起，肌动蛋白丝在ES。在这些部位，野生型睾丸中也有高水平的磷酸酪氨酸免疫染色，而fyn缺陷睾丸中磷酸酪氨酸免疫反应性降低。这些结果表明Fyn激酶在睾丸支持细胞的外质特化中起作用.免疫印迹分析显示，Fyn主要分布在Triton X-100不溶性细胞骨架组分制备的野生型小鼠睾丸，表明Fyn可能与细胞骨架蛋白，如肌动蛋白丝。一个酪氨酸磷酸化蛋白在约80 kDa（P80）是突出的Triton不溶性组分从野生型睾丸。然而，P80在fyn缺陷的睾丸中特异性降低。提示P80可能是Fyn激酶的作用靶点，P80可能在Fyn介导的小鼠睾丸信号转导中发挥作用.我们发现小鼠睾丸支持细胞系TM 4表达Fyn蛋白。通过二维免疫印迹分析在TM 4细胞提取物中检查酪氨酸磷酸化蛋白。当细胞被环境内分泌干扰物处理时，如己烯雌酚，与对照细胞相比，一种约38 kDa的蛋白质（P38）的酪氨酸磷酸化水平显著增加。Fyn酪氨酸激酶可能参与P38的磷酸化。