In vitro analysis of spermatogenesis in the medaka, Oryzias latipes.

精子发生福尔斯分为四类。精原细胞经过反复的有丝分裂形成一个自身的细胞池。(2)这些细胞致力于减数分裂，随后作为初级精母细胞。(3)初级精母细胞进行两次减数分裂，产生次级精母细胞和精子细胞。(4)本研究以青鳉（Oryzias latipes）为材料，通过几种不同的方式，对青鳉精子发生过程进行了分析。超微结构观察表明，从初级精母细胞到精子的生精过程正常进行。为了判断体外分化的精子是否具有生育能力，我们观察了BrdU掺入的精母细胞的分化过程。培养后第3天，用免疫细胞化学方法检测BrdU掺入精子。通过人工授精，可以非常直接地观察体外分化的精子的生育力。 关于我们 另一方面，有报道称许多激素对精子发生有效。我们研究了几种激素，人绒毛膜促性腺激素（hCG），11-酮睾酮，睾酮和雌二醇-17 β对体外培养的睾丸碎片的分化的影响。当加入hCG、11-酮睾酮和17 β-雌二醇时，精原囊肿数量增加，精子发生被激活。最后，我们克隆了青鳉鱼精蛋白的cDNA，以阐明精子发生的分子机制。在λ gt 11中构建的青鳉精巢cDNA文库有2.78 × 10^6个独立重组子。用青鳉鱼精蛋白多克隆抗血清进行免疫筛选，获得了大量阳性克隆。测序结果表明，其中一个阳性克隆MP-1编码精蛋白特征性精氨酸簇。MP-1蛋白的氨基酸序列与其它鱼类鱼精蛋白的氨基酸序列有很大的同源性，与蓝鳍金枪鱼、Thunnus thynnus、Thynnin Z1的氨基酸序列同源性为71%。用MP-1cDNA探针进行的DNA杂交表明，MP-1 mRNA仅存在于睾丸中，并有两条可检测的条带。一种是1400 b，另一种是400 b。互补RNA探针原位杂交的实验正在进行中。少

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 … More n.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