Establishment of accurate in vitro measurement for testicular toxic te

睾丸毒性试验体外精确测定方法的建立

基本信息

批准号：
07556064
负责人：
KUROHMARU Masamichi
金额：
$ 7.62万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

1. First, we investigated the effect of extracellular matrix on the adhesion, morphology and proliferation of cultured cells. As a result, Laminin was the most effective among the substrate examined. 2. Mixed cell suspensions, prepared by enzymatic digestion of adult rat testicular tissues, were separated into five fractions according to their differences in sedimentation velocity by centrifugal elutriation, a counterflow centrifugation technique. Through these processes, we obtained viable round spermatids. MTT assay was done at 24 hr intervals after plating to examine the cytotoxic effects of cyclosporin A on round spermatids. Cyclosporin A was shown to have little or no effects on metabolic activity of round spermatids in vitro. 3. We examined whether frozen-thawed mouse round spermatids can fertilize oocytes and contribute to normal embryo development. It was demonstrated that mouse round spermatids can be cryopreserved for production of normal offspring. 4. The expression of NGF receptor was recognized in cultured Sertoli cells by RT-PCR method. In the co-culture of spermatids and NGF-added cultured Sertoli cells, DNA synthesis of spermatids was significantly increased. 5. The appearance of immunoreactive follistatin in rat testis was examined by immunohistochemistry using an antiserum raised against synthetic follistatin peptide. Follistatin immunoreactivity was not found in Sertoli and Leydig cells, while it was clearly detected in the cytoplasm and nucleus of late pachytene spermatocytes. Although the reaction in the cytoplasm desappeared after meiosis, it continued intensely in the nucleus from pachytene spermatocytes to round spermatids. 6. Additionaly, the germ cells of birds and reptiles were studied mainly from a morphological viewpoint.

1。首先，我们研究了细胞外基质对培养细胞的粘附，形态和增殖的影响。结果，层粘连蛋白是所检查的底物中最有效的。 2。通过成年大鼠睾丸组织的酶消化制备的混合细胞悬浮液根据其通过离心流离子体离心技术的离心液质量分离为五个部分。通过这些过程，我们获得了可行的圆形精子。 MTT测定在电镀后以24小时的间隔进行，以检查环孢菌素A对圆形精子的细胞毒性作用。环孢菌素A显示出对体外圆形精子的代谢活性几乎没有影响。 3。我们检查了冷冻的小鼠圆形精子是否可以施肥卵母细胞并有助于正常的胚胎发育。已经证明，小鼠圆形精子可以冷冻保存以生产正常后代。 4。通过RT-PCR方法在培养的Sertoli细胞中识别NGF受体的表达。在精子和NGF添加的培养的Sertoli细胞的共同培养中，精子的DNA合成显着增加。 5。使用针对合成卵泡肽的抗血清检查，通过免疫组织化学检查了大鼠睾丸中免疫反应性卵泡素的出现。在Sertoli和Leydig细胞中未发现卵泡蛋白免疫反应性，而在pachytene晚期精子细胞的细胞质和核中清楚地检测到它。尽管细胞质中的细胞质中的反应在减数分裂后呈现，但它在细胞核中一直持续到从粉状的精子细胞到圆形精子。 6。另外，主要从形态学的角度研究了鸟类和爬行动物的生殖细胞。