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

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(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.