Abstract
BackgroundMicroRNAs (miRNAs) are critical regulators of transcriptional and post-transcriptional gene silencing, which are involved in multiple developmental processes in many organisms. Apart from miRNAs, mouse germ cells express another type of small RNA, piwi-interacting RNAs (piRNAs). Although it has been clear that piRNAs play a role in repression of retrotransposons during spermatogenesis, the function of miRNA in mouse germ cells has been unclear.Methodology/Principal FindingsIn this study, we first revealed the expression pattern of miRNAs by using a real-time PCR-based 220-plex miRNA expression profiling method. During development of germ cells, miR-17-92 cluster, which is thought to promote cell cycling, and the ES cell-specific cluster encoding miR-290 to -295 (miR-290-295 cluster) were highly expressed in primordial germ cells (PGCs) and spermatogonia. A set of miRNAs was developmentally regulated. We next analysed function of miRNA biogenesis in germ cell development by using conditional Dicer-knockout mice in which Dicer gene was deleted specifically in the germ cells. Dicer-deleted PGCs and spermatogonia exhibited poor proliferation. Retrotransposon activity was unexpectedly suppressed in Dicer-deleted PGCs, but not affected in the spermatogonia. In Dicer-deleted testis, spermatogenesis was retarded at an early stage when proliferation and/or early differentiation. Additionally, we analysed spermatogenesis in conditional Argonaute2-deficient mice. In contrast to Dicer-deficient testis, spermatogenesis in Argonaute2-deficient testis was indistinguishable from that in wild type.Conclusion/SignificanceThese results illustrate that miRNAs are important for the proliferation of PGCs and spermatogonia, but dispensable for the repression of retrotransposons in developing germ cells. Consistently, miRNAs promoting cell cycling are highly expressed in PGCs and spermatogonia. Furthermore, based on normal spermatogenesis in Argonaute2-deficient testis, the critical function of Dicer in spermatogenesis is independent of Argonaute2.
Highlights
MicroRNAs ranging between 18–23 bp are critical regulators of transcriptional and post-transcriptional gene silencing in many organisms
To identify miRNAs that are expressed during primordial germ cells (PGCs) development, we performed real-time PCR analyses using primers containing stem-loop structure by which miRNAs can be representatively amplified from a small number of cells, and from single cells [27,28]
Amongst the miRNAs detected in the samples, miRNAs belonging to miR-17-92 cluster was highly expressed in PGCs
Summary
MicroRNAs (miRNA) ranging between 18–23 bp are critical regulators of transcriptional and post-transcriptional gene silencing in many organisms. Only a single Dicer gene has so far been identified, and its disruption leads to defects in miRNA generation as well as in RNA interference-mediated gene silencing [6,7]. Dicer-mediated miRNA biogenesis is required for multiple developmental processes, and not just in the initial differentiation of the early embryo [8] and embryonic stem (ES) cells [6,7]. MicroRNAs (miRNAs) are critical regulators of transcriptional and post-transcriptional gene silencing, which are involved in multiple developmental processes in many organisms. It has been clear that piRNAs play a role in repression of retrotransposons during spermatogenesis, the function of miRNA in mouse germ cells has been unclear
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