Department of Public Health and Cellular Biology, University of Rome Tor Vergata, Rome, Italy.
PLoS One. 2010 Feb 4;5(2):e9064. doi: 10.1371/journal.pone.0009064.
A critical step of spermatogenesis is the entry of mitotic spermatogonia into meiosis. Progresses on these topics are hampered by the lack of an in vitro culture system allowing mouse spermatogonia differentiation and entry into meiosis. Previous studies have shown that mouse pachytene spermatocytes cultured in simulated microgravity (SM) undergo a spontaneous meiotic progression. Here we report that mouse mitotic spermatogonia cultured under SM with a rotary cell culture system (RCCS) enter into meiosis in the absence of any added exogenous factor or contact with somatic cells. We found that isolated Kit-positive spermatogonia under the RCCS condition enter into the prophase of the first meiotic division (leptotene stage), as monitored by chromosomal organization of the synaptonemal complex 3 protein (Scp3) and up-regulation of several pro-meiotic genes. SM was found to activate the phosphatidyl inositol 3 kinase (PI3K) pathway and to induce in Kit-positive spermatogonia the last round of DNA replication, typical of the preleptotene stage. A PI3K inhibitor abolished Scp3 induction and meiotic entry stimulated by RCCS conditions. A positive effect of SM on germ cell differentiation was also observed in undifferentiated (Kit-negative) spermatogonia, in which RCCS conditions stimulate the expression of Kit and Stra8. In conclusion, SM is an artificial environmental condition which promotes postnatal male germ cell differentiation and might provide a tool to study the molecular mechanisms underlying the switch from mitosis to meiosis in mammals.
精子发生的一个关键步骤是有丝分裂精原细胞进入减数分裂。由于缺乏允许小鼠精原细胞分化并进入减数分裂的体外培养系统，这些研究进展受到阻碍。先前的研究表明，在模拟微重力（SM）下培养的小鼠粗线期精母细胞会自发进入减数分裂。在这里，我们报告说，在没有任何外加外源因子或与体细胞接触的情况下，使用旋转细胞培养系统（RCCS）在 SM 下培养的小鼠有丝分裂精原细胞进入减数分裂。我们发现，在 RCCS 条件下分离的 Kit 阳性精原细胞进入第一次减数分裂的前期（细线期），如联会复合体 3 蛋白（Scp3）的染色体组织和几个前减数分裂基因的上调所监测到的。SM 被发现激活磷脂酰肌醇 3 激酶（PI3K）途径，并在 Kit 阳性精原细胞中诱导最后一轮 DNA 复制，这是典型的早细线期。PI3K 抑制剂消除了 RCCS 条件诱导的 Scp3 诱导和减数分裂进入。SM 对未分化（Kit 阴性）精原细胞中生殖细胞分化也有积极影响，其中 RCCS 条件刺激 Kit 和 Stra8 的表达。总之，SM 是一种人工环境条件，可促进出生后雄性生殖细胞的分化，并且可能为研究哺乳动物有丝分裂向减数分裂转变的分子机制提供一种工具。
