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

1. 在3和4周龄时，发现睾丸重量明显减少，生殖细胞退化。电镜显示，fynn缺陷睾丸的支持细胞特化连接结构出现超微结构异常，称为外质特化（ES）。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与肌动蛋白丝在ES集中。在野生型睾丸中，高水平的磷酸酪氨酸也被免疫染色，而在fyn缺乏的睾丸中，磷酸酪氨酸的免疫反应性降低。这些发现表明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主要分布在野生型小鼠睾丸制备的Triton X-100不溶性细胞骨架中，提示Fyn可能与肌动蛋白丝等细胞骨架蛋白有关。在野生型睾丸的tryon不溶性部分中，有一个约80 kDa的酪氨酸磷酸化蛋白（P80）。然而，P80在fyn缺乏的睾丸中特异性降低。这一发现提示P80可能是Fyn激酶的靶点，P80可能在Fyn介导的小鼠睾丸信号转导中发挥作用。我们发现小鼠支持细胞系TM4表达Fyn蛋白。二维免疫印迹法检测TM4细胞提取物中酪氨酸磷酸化蛋白。当细胞被环境内分泌干扰物（如己烯雌酚）处理时，与对照细胞相比，一种蛋白约38kDa （P38）的酪氨酸磷酸化水平显著增加。Fyn酪氨酸激酶可能参与了P38的磷酸化。

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.