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黏附、形态和增殖的影响。结果表明，在所考察的底物中，层粘连蛋白是最有效的。2.采用逆流离心法，将成年大鼠睾丸组织酶消化法制备的混合细胞悬液按沉降速度的不同分成五个组分。通过这些过程，我们获得了有活力的圆形精子细胞。培养24小时后，用四甲基偶氮唑盐比色法检测环孢素A对圆形精子细胞的细胞毒作用。环孢菌素A在体外对圆形精子细胞的代谢活性几乎没有影响。3.我们检测了冷冻解冻的小鼠圆形精子细胞能否使卵母细胞受精并促进胚胎的正常发育。结果表明，小鼠圆形精子细胞可以超低温保存以产生正常后代。4.用RT-PCR方法检测到培养的Sertoli细胞表达NGF受体。在精子细胞与NGF共同培养的支持细胞中，精子细胞的DNA合成显著增加。5.用合成的卵泡抑素多肽抗血清进行免疫组织化学染色，观察卵泡抑素在大鼠睾丸中的免疫反应。支持细胞和间质细胞中未见卵泡抑素免疫反应，粗线期晚期精母细胞胞浆和胞核中可见明显的卵泡抑素免疫反应。虽然减数分裂后细胞质中的反应消失，但从粗线型精母细胞到圆形精细胞，核内的反应仍很强烈。6.此外，对鸟类和爬行动物生殖细胞的研究主要从形态角度进行。

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.