小鼠精原细胞体外分化为精子细胞的研究

目的探讨在辅以外源生殖激素混合培养的小鼠睾丸细胞中,精原细胞向精子细胞的转化。方法采用7~8 d龄小鼠睾丸组织,以组合酶消化法制备睾丸细胞,在含重组卵泡刺激素(r-FSH)和睾酮的培养基中进行体外培养;定期观察细胞的生长和形态变化;用分子生物学和流式细胞技术对生长各阶段细胞进行分析。结果培养5 d即可观察到形态与大小类似于圆形精子细胞,7 d后可见带有鞭毛与变形的精子细胞;逆转录聚合酶链反应(RT-PCR)结果显示,培养前细胞的睾丸特异蛋白激酶(TESK1)与鱼精蛋白2(Prm2)mRNA表达阴性,培养9与17 d细胞TESK1与Prm2 mRNA表达均阳性;DNA倍体分析显示,培养5 d细胞有单倍体峰出现,并随培养天数增加而增加。结论在体外混合培养的小鼠睾丸细胞中加入外源生殖激素可以使精原细胞转化为精子细胞。

Study on in vitro differentiation of mouse spermatogonia into spermatids

Objective: To investigate the in vitro differentiation of spermatogonia into spermatids in an exogenous hormone supplemented culture system.Methods: Mouse testicular cells at 7~8 d of age were obtained using combined enzyme digestion method and cultured in an exogenous hormone supplemented culture system with r-FSH and testosterone.The cell development was observed by periodic observation of the cell growth and morphological appearance.And the stage-specific markers,TESK1 and Prm2 for spermatogonia differentiation were determined and DNA ploid analysis was performed using flow cytometry.Results: Round spermatids-like cells were found at day 5 of culture.Round spermatids with flagella and elongating spermatids were observed from the day 7 of culture.Cell suspension before the culture showed TESK1 and Prm2 mRNA negative,while cultured cells exhibited positive expression of TESK1 from day 5 and Prm2 from day 9.DNA ploid analysis showed the consistent results with the histological observation.Haploid peaks appeared at day 5 of culture and the proportion of haploid peak increased with time.Conclusion: Mouse spermatogonia could differentiate into spermatids in the experimental culture system where immature mouse testicular cells were incubated with supplementation of exogenous hormones.