Expression and function of small non-coding RNAs in the mouse testis
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Spermatogenesis is complex and fascinating process of differentiation. It starts with the renewal of the rare spermatogonial stem cells, followed by the proliferation of differentiated spermatogonia. The latter cells form primary spermatocytes, which enter a prolonged meiotic prophase to effect pairing of homologous chromosomes and genetic recombination. With the two reduction divisions and random segregation of chromosomes, spermatocytes yield haploid spermatids. Then, during spermiogenesis, the spermatids form the mature spermatozoa through a remarkable sequence of differentiation that includes chromatin condensation, nucleus reorganization, acrosome formation, and tail assembling. Given the complexity of spermatogenesis, the normal development of male germ cells requires the accurate regulation of gene expression. Small non-coding RNAs including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and endogenous small interfering RNAs (endo-siRNAs) can regulate gene expression especially at the post-transcriptional level. It is intriguing to study whether these small RNAs have function in spermatogenesis. Using mouse as a research model, we found that all three types of small RNAs are abundantly expressed in testis. In late spermatocytes, when meiotic sex chromosome inactivation (MSCI) takes place and the transcription of all protein-coding genes on the X and Y chromosomes ceases, our data showed that the miRNA genes on X chromosomes are still actively transcribed, suggesting that these miRNAs may function in MSCI or gene regulation in post-meiotic stages. During spermiogenesis, the most featured post-transcriptional regulation is that spermatid-specific mRNAs are sequestrated in ribonucleoproteins (RNPs) of early spermatids until their translation in polyribosomes (polysomes) of late spermatids. We showed that miRNAs are involved in this post-transcriptional regulation by repressing mRNA translation in RNPs and activating mRNA translation in polysomes. We showed that piRNAs are abundantly expressed during spermatogenesis, especially in pachytene spermatocytes and round spermatids. Our data also revealed that hundreds or even thousands of piRNAs from a single piRNA gene cluster can be produced from one single long transcript, which provides essential preliminary data for the future study of piRNA biogenesis. We first indentified endo-siRNAs in the mouse testis, and bioinformatic analysis showed that these endo-siRNAs are likely derived from naturally occurring long double-stranded RNAs (dsRNAs). In addition to their roles as post-transcriptional regulators, endo-siRNAs may function as epigenetic regulators at the transcriptional level.Overall, we showed that all three types of small RNAs are abundantly expressed in male germ cells and they are involved in gene regulation during spermatogenesis. Our work will benefit the deeper understanding of spermatogenesis and male fertility.