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

青鳉、Oryzias latipes 精子发生的体外分析。

基本信息

批准号：
03804058
负责人：
ONITAKE Kazuo
金额：
$ 1.34万
依托单位：
Yamagata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

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

精子发生分为四类。精原细胞经过反复的有丝分裂形成一个自身的细胞池。这些细胞进行减数分裂，随后成为初级精母细胞。(3)初级精母细胞随后进行两次减数分裂，产生次级精母细胞和精母细胞。(4)精子变为精子。在本研究中，我们通过几种方式分析了米达科植物的体外生精过程。超微结构观察显示，从初级精母细胞到精子的生精过程正常进行。为了确定体外分化的精子是否具有可育性，我们对brdu掺入的精母细胞的分化过程进行了检测。培养3天后，免疫细胞化学检测brdu掺入精子。通过人工授精，可以非常直接地测定体外分化精子的生育能力。另一方面，有报道称许多激素对精子的发生是有效的。研究了人绒毛膜促性腺激素（hCG）、11-酮睾酮、睾酮和雌二醇-17 β对体外培养睾丸碎片分化的影响。添加hCG、11-酮睾酮和雌二醇-17 β后，精子原囊数量增加，精子发生被激活。最后，我们克隆了medaka鱼精蛋白的cDNA，以阐明精子发生的分子机制。用lambda gtll构建的睾丸medaka cDNA文库有2.78 × 10^6个独立的重组。用多克隆抗血清对medaka鱼精蛋白进行免疫筛选，获得了许多阳性克隆。结果表明，其中一个阳性克隆被命名为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类鱼精蛋白具有显著的同源性；与蓝鳍金枪鱼、Thunnus thynnus、Thynnin Z1的氨基酸序列一致性达71%。用MP-1 cDNA探针进行北杂交，发现MP-1 mRNA只存在于睾丸中，有两条可检测到的条带。一个是1400b，另一个是400b。互补RNA探针原位杂交实验正在进行中。少