Establishment of accurate in vitro measurement for testicular toxic te

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。结果表明，层粘连蛋白是在所检测的底物中最有效的。2. 以成年大鼠睾丸组织为原料，采用酶切法制备混合细胞悬浮液，根据其沉降速度的差异，采用离心洗脱（逆流离心）技术将细胞悬浮液分离成5份。通过这些过程，我们获得了可存活的圆形精子。每隔24小时用MTT法检测环孢素A对圆形精子的细胞毒作用。环孢素A对圆形精子体外代谢活性的影响很小或没有影响。3. 我们研究了冷冻解冻的小鼠圆形精子是否能使卵母细胞受精并促进正常胚胎发育。结果表明，小鼠圆形精子可以冷冻保存以产生正常后代。4. RT-PCR法检测培养的支持细胞中NGF受体的表达。在精子细胞与添加ngf的培养的支持细胞共培养时，精子细胞的DNA合成显著增加。5. 采用合成卵泡素肽抗血清免疫组化方法观察大鼠睾丸中卵泡素的免疫反应性。卵泡抑素免疫反应性在支持细胞和间质细胞中未发现，而在厚线期晚期精母细胞的细胞质和细胞核中明显检测到。虽然减数分裂后细胞质中的反应消失，但从粗粒精母细胞到圆形精母细胞，细胞核中的反应持续强烈。6. 另外，主要从形态学角度对鸟类和爬行动物的生殖细胞进行了研究。

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.