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Analysis of the mechanism underlying meiosis initiation using in vitro culture system.

使用体外培养系统分析减数分裂启动的机制。

基本信息

批准号：
05454654
负责人：
ABE Shinich
金额：
$ 3.97万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454654/
关键词：
spermatogenesis meiosis initiation spermatogonia Sertoli cells FSH Annexin V JAK 1 Activin A 第一精母細胞 cDNA activin A cDNAクローニング JAK1 アネキシン

项目摘要

(1)Stimulation of spermatogonial proliferation by sertolicells ; FSH stimulated [^3H] thymidine incorporation into spermatogonia in aggrtegation culture of spermatogonia and Sertolicells, whereas FSH failed in stimulation in culture of spermatogonia alone. Since spermatogonia in this culture system died rapidly, dissociated testicular cells were centrifuged, the pellet was embedded in collagen matrix and cultured ; FSH stimulated differentiation into primary spermatocytes. These results indicate that FSH stimulates proliferation and differentiation of spermatogonia into primary spermatocytes via Sertoli cells. (2) Isolation of annexin cDNA clone : By immunodifferential screening of expression cDNA libraries, annexin cDNA was isolated ; its gene and protein expression increased at the initiation of meiosis. (3) Screening of growth factors for spermatogonia : By reverse transcription-polymerase chain reaction (RT-PCR) of spermatogonial RNA using tyrosine kinase domain, newt JAK 1 cDNA clone was isolated. In situ hybridization (ISH) showed that JAK 1 mRNA was expressed in spermatogonia alone. (4) Expression of newt activin/inhibin bA subunit mRNA : Newt activin/inhibin bA and bB subunit cDNAs were isolated by RT-PCR.ISH showed that only bA subunit mRNA was expressed in Sertoli cells alone. In organ cultute of newt testes, FSH stimulated expression of bA subunit mRNA.(5) Stimulation of spermatogonial proliferation by human activin A : In organ culture of newt testes human activin A remarkably stimulated spermatogonial proliferation. These results indicate that FSH activates activin A gene expression in Sertoli cells and activin A stimulates spermatogonial proliferation.

（1）支持细胞刺激精原细胞增殖：在精原细胞和支持细胞的聚集培养中，FSH刺激[^3H]胸苷掺入精原细胞，而在单独培养的精原细胞中，FSH不能刺激。由于该培养体系中的精原细胞死亡迅速，将解离的睾丸细胞离心，将沉淀包埋在胶原基质中并培养; FSH刺激分化为初级精母细胞。这些结果表明FSH通过支持细胞刺激精原细胞增殖和分化为初级精母细胞。(2)膜联蛋白cDNA克隆的分离：通过表达cDNA文库的免疫差异筛选，分离出膜联蛋白cDNA;其基因和蛋白表达在减数分裂起始时增加。(3)精原细胞生长因子的筛选：采用逆转录-聚合酶链反应（RT-PCR）技术，以精原细胞酪氨酸激酶结构域为模板，克隆蝾螈JAK 1cDNA克隆。原位杂交（ISH）显示JAK 1 mRNA仅在精原细胞中表达. (4)蝾螈激活素/bA亚基mRNA的表达：用RT-PCR方法分离蝾螈激活素/bA亚基和bB亚基的cDNA，原位杂交结果表明，在支持细胞中只有bA亚基mRNA表达。在蝾螈睾丸器官培养中，FSH刺激bA亚基mRNA的表达。(5)人激活素A对精原细胞增殖的刺激：在蝾螈睾丸器官培养中，人激活素A显著刺激精原细胞增殖。这些结果表明，FSH激活激活素A基因表达的支持细胞和激活素A刺激精原细胞增殖。