|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1993 : ¥200,000 (Direct Cost : ¥200,000)
Fiscal Year 1992 : ¥300,000 (Direct Cost : ¥300,000)
Fiscal Year 1991 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Spermatogenesis falls into four categories. Speramtogonial cells undergo repeated mitotic division to createa pool of itself. (2) These cells committed to miosis, as subsequently as primary spermatocytes. (3) Primary spermatocytes carried out two meiotic divisions follw, giving rise to secondary spermatocytes and then spermatids. (4) Spermatid becomes spermatozoa.
In the present work, we have analyzed the spermatogenic process in vitro by the several manners in the medaka, Oryzias latipes. In the ultrastructural observations, it was revealed that the spermatogenic process from the primary spermatocyte to the sperm progessed normally. In order to very whether the sperm differentiated in vitro is fertile or not, the differentiating process of BrdU-incorporated spermatocytes was examined. At 3 days after cultivation, BrdU-incorporated sperm was detedcted immunocytochemically. It became possible to very directly the fertility of the sperm differentiated in vitro by the artifical inseminatio
On the other hand, it has reported that many hormones were effective on the spermatogenesis. We investigated the effect of the several hormones, human chorionic gonadotropin (hCG), 11-ketotestosterone, testosterone and estradiol-17 beta on the differentiation of the testis fragments cultured in vitro. When hCG, 11-ketotestosterone and estradiol-17beta were added, a number of spermatogonial cysts increased and the spermatogenesis was activated.
Finally, we cloned cDNA of the medaka protamine in order to elucidate the molecular mechanisms of spermatogenesis. The medaka testis cDNA library construced in lambda gtll had 2.78 X 10^6 independent recombinants. A numberof positive clones were obtained by immunoscreening with polyclonal antiserum against the medaka protamine. The results of these sequences showed that one of these positive clones named MP-1 was found to encode arginine clusters, characteristic of protamine. The amino acid sequence of MP-1 revealed a remarkable extent of homology with other fish protamine ; 71% identity of the amino acid sequence with that of Bluefine tuna, Thunnus thynnus, Thynnin Z1. Nothern hybridization using MP-1 cDNA probe showed that MP-1 mRNA is exclusively present in testis and there were two bands detectable. One is a 1400b, and the other is a 400b.
Experiments by in situ hybridization of complementary RNA probe are now progressing. Less