Institute of Human Genetics, University of Goettingen, Goettingen 37073, Germany.
Biol Cell. 2012 Nov;104(11):677-92. doi: 10.1111/boc.201200014. Epub 2012 Sep 24.
Recently, it became apparent that microRNAs (miRNAs) can regulate gene expression post-transcriptionally. Despite the advances in identifying the testis-expressed miRNAs and their role in spermatogenesis, only few data are available showing the spatiotemporal expression of miRNAs during this process.
To understand how different miRNAs can regulate germ cells differentiation, we generated a transgenic mouse model and purified pure populations of premeiotic (PrM) cells and primary spermatocytes (meiotic cells). We also established spermatogonial stem cell (SSC) culture using relatively simple and robust culture conditions. Comparison of global miRNA expression in these germ cell populations revealed 17 SSC-, 11 PrM- and 13 meiotic-specific miRNAs. We identified nine miRNAs as specific for both SSC and PrM cells and another nine miRNAs as specific for PrM and meiotic cells. Additionally, 45 miRNAs were identified as commonly expressed in all three cell types. Several of PrM- and meiotic-specific miRNAs were identified as exclusively/preferentially expressed in testis. We were able to identify the relevant target genes for many of these miRNAs. The luciferase reporter assays with SSC (miR-221)-, PrM (miR-203)- and meiotic (miR-34b-5p)-specific miRNAs and 3'-untranslated region constructs of their targets, c-Kit, Rbm44 and Cdk6, respectively, showed an approximately 30%-40% decrease in reporter activity. Moreover, we observed a reduced expression of endogenous proteins, c-Kit and Cdk6, when the testis-derived cell lines, GC-1 and GC-4, were transfected with miRNA mimics for miR-221 and miR-34b-5p, respectively.
Taken together, we established the miRNA signature of SSC, PrM and meiotic cells and show evidence for their functional relevance during the process of spermatogenesis by target prediction and validation. Through our observations, we propose a working model in which the stage-specific miRNAs such as miR-221, -203 and -34b-5p coordinate the regulation of spermatogenesis.
最近，人们发现微小 RNA（miRNA）可以在后转录水平上调节基因表达。尽管在鉴定睾丸表达的 miRNA 及其在精子发生中的作用方面取得了进展，但仅有少数数据显示 miRNA 在这一过程中的时空表达。
为了了解不同 miRNA 如何调节生殖细胞的分化，我们生成了一种转基因小鼠模型，并纯化了原始生殖细胞（PrM）细胞和初级精母细胞（有丝分裂细胞）的纯群体。我们还使用相对简单和强大的培养条件建立了精原干细胞（SSC）培养。对这些生殖细胞群体中的全局 miRNA 表达进行比较，发现 17 个 SSC、11 个 PrM 和 13 个减数分裂特异性 miRNA。我们确定了 9 个 miRNA 是 SSC 和 PrM 细胞特异性的，另外 9 个 miRNA 是 PrM 和减数分裂细胞特异性的。此外，45 个 miRNA 被鉴定为在所有三种细胞类型中共同表达。一些 PrM 和减数分裂特异性 miRNA 被鉴定为仅在睾丸中特异性表达或优先表达。我们能够鉴定出许多这些 miRNA 的相关靶基因。使用 SSC（miR-221）-、PrM（miR-203）-和减数分裂（miR-34b-5p）-特异性 miRNA 的荧光素酶报告基因检测和它们各自靶基因的 3'-非翻译区构建体，c-Kit、Rbm44 和 Cdk6，报告基因活性分别降低约 30%-40%。此外，当用 miRNA 模拟物分别转染睾丸衍生细胞系 GC-1 和 GC-4 时，观察到内源性蛋白 c-Kit 和 Cdk6 的表达减少。
总之，我们建立了 SSC、PrM 和减数分裂细胞的 miRNA 特征，并通过靶基因预测和验证显示了它们在精子发生过程中的功能相关性。通过我们的观察，我们提出了一个工作模型，其中阶段特异性 miRNA，如 miR-221、-203 和 -34b-5p，协调精子发生的调节。
