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

精子发生分为四类。精原细胞经过反复的有丝分裂来创造自己的池。(2)这些细胞致力于分裂，随后与初级精母细胞一样。(3)初级精母细胞进行两次减数分裂，形成次级精母细胞和精子细胞。(4)精子细胞变成精子。在本工作中，我们通过几种方式分析了青冈稻的体外生精过程。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。为了了解体外分化的精子是否具有生育能力，对掺入BrdU的精母细胞的分化过程进行了观察。培养3d后，免疫细胞化学检测掺入BrdU的精子。通过人工授精…可以非常直接地检测体外分化的精子的可育性另一方面，据报道，许多激素对精子发生有作用。本实验研究了人绒毛膜促性腺激素(HCG)、11-酮睾酮、睾酮和17-β雌二醇对体外培养的睾丸碎片分化的影响。加入人绒毛膜促性腺激素、11-酮-睾酮和雌二醇-17β后，精原细胞囊泡数量增加，精子发生被激活。最后，我们克隆了青海鱼精蛋白的cDNA，以阐明精子发生的分子机制。在lambda gtll中构建的青竹睾丸c DNA文库有2.78×10^6个独立重组子。用抗青海鱼精蛋白的多克隆抗血清进行免疫筛选，获得多个阳性克隆。测序结果表明，其中一个阳性克隆MP-1编码精氨酸簇，这是鱼精蛋白的特征。MP-1的氨基酸序列与其他鱼精蛋白有显著的同源性，与蓝鳍金枪鱼、金枪鱼、Thynnin Z1的同源性为71%。用MP-1cDNA探针进行Northern杂交表明，MP-1mRNA仅存在于睾丸中，并可检测到两条带。一种是1400b，另一种是400b。互补RNA探针的原位杂交实验正在进行中。较少

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