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

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酪氨酸激酶在小鼠睾丸中定位于Sertoli细胞，而在生精细胞中不存在. Fyn与肌动蛋白丝在支持细胞胞质特化中的共定位Fyn在支持细胞胞质特化中大量定位，Fyn与肌动蛋白丝在支持细胞胞质特化中共定位. Western印迹分析从小鼠睾丸中制备Triton可溶性和不可溶性组分，并将样品用于SDS-PAGE，转移到膜上，并用抗Fyn抗体免疫染色。结果表明，Fyn蛋白主要在Triton不溶性的细胞骨架组分中检测到。这些数据充分表明Fyn激酶与细胞骨架蛋白相关。利用抗磷酸酪氨酸抗体，在野生型睾丸的Triton不溶性细胞骨架组分中大量检测到80 kDa的酪氨酸磷酸化蛋白（p80）。与此相反，p80显着减少的fyn -/-睾丸的细胞骨架部分。这一发现表明p80可能是Fyn激酶的作用靶点.出生后发育过程中fyn -/-睾丸中的脱落成年fyn-/-雄性小鼠具有生育能力。然而，我们发现，与野生型睾丸相比，fyn -/-未成熟睾丸在3至4周龄时的重量显著降低。精小管直径减小，睾丸生殖细胞坏死。此外，不常见的超微结构的内质网专业化也检测到在fyn -/-睾丸。这些发现表明Fyn激酶通过支持细胞参与特定阶段生殖细胞的存活和/或分化。

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.