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

(1)浆膜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，卵泡刺激素刺激[^3H]胸腺嘧啶并入精原细胞，而单独培养精原细胞时，卵泡刺激素没有刺激作用。由于该培养体系中精原细胞死亡快，故离心分离睾丸细胞，包埋于胶原基质中培养；卵泡刺激素刺激分化为原代精母细胞。这些结果表明，卵泡刺激素通过支持细胞刺激精原细胞增殖并向原代精母细胞分化。(2)膜联蛋白cDNA克隆的分离：通过免疫差异筛选表达cDNA文库，分离膜联蛋白cDNA；其基因和蛋白表达在减数分裂开始时增加。(3)精原细胞生长因子筛选：利用酪氨酸激酶结构域对精原细胞RNA进行逆转录聚合酶链反应（RT-PCR），分离得到蝾螈JAK 1 cDNA克隆。原位杂交（ISH）结果显示，JAK - 1 mRNA仅在精原细胞中表达。(4)蝾螈激活素/抑制素bA亚基mRNA表达：RT-PCR分离蝾螈激活素/抑制素bA和bB亚基cdna。ISH结果显示，仅bA亚基mRNA在支持细胞中表达。在蝾螈睾丸器官培养中，卵泡刺激素刺激bA亚基mRNA的表达。(5)人激活素A对精原细胞增殖的刺激作用：在蝾螈睾丸器官培养中，人激活素A对精原细胞增殖有显著的刺激作用。这些结果表明，卵泡刺激素激活了支持细胞中激活素A基因的表达，激活素A促进了精原细胞的增殖。

(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.