Date Published: February 1, 2019
Publisher: Public Library of Science
Author(s): Hiromitsu Ota, Yumi Ito-Matsuoka, Yasuhisa Matsui, Jean-Pierre Rouault.
MicroRNAs (miRNAs) play a critical role in multiple aspects of biology. Dicer, an RNase III endonuclease, is essential for the biogenesis of miRNAs, and the germ cell-specific Dicer1 knockout mouse shows severe defects in gametogenesis. How miRNAs regulate germ cell development is still not fully understood. In this study, we identified germ cell-specific miRNAs (miR-741-3p, miR-871-3p, miR-880-3p) by analyzing published RNA-seq data of mouse. These miRNA genes are contiguously located on the X chromosome near other miRNA genes. We named them X chromosome-linked miRNAs (XmiRs). To elucidate the functions of XmiRs, we generated knockout mice of these miRNA genes using the CRISPR/Cas9-mediated genome editing system. Although no histological abnormalities were observed in testes of F0 mice in which each miRNA gene was disrupted, a deletion covering miR-871 and miR-880 or covering all XmiRs (ΔXmiRs) resulted in arrested spermatogenesis in meiosis in a few seminiferous tubules, indicating their redundant functions in spermatogenesis. Among candidate targets of XmiRs, we found increased expression of a gene encoding a WNT receptor, FZD4, in ΔXmiRs testis compared with that in wildtype testis. miR-871-3p and miR-880-3p repressed the expression of Fzd4 via the 3′-untranslated region of its mRNA. In addition, downstream genes of the WNT/β-catenin pathway were upregulated in ΔXmiRs testis. We also found that miR-871, miR-880, and Fzd4 were expressed in spermatogonia, spermatocytes and spermatids, and overexpression of miR-871 and miR-880 in germ stem cells in culture repressed their increase in number and Fzd4 expression. Previous studies indicated that the WNT/β-catenin pathway enhances and represses proliferation and differentiation of spermatogonia, respectively, and our results consistently showed that stable β-catenin enhanced GSC number. In addition, stable β-catenin partially rescued reduced GSC number by overexpression of miR-871 and miR-880. The results together suggest that miR-871 and miR-880 cooperatively regulate the WNT/β-catenin pathway during testicular germ cell development.
Germ cells first arise as primordial germ cells (PGCs) in early embryos , and they proliferate and migrate into the genital ridges during embryonic development . After arriving at the genital ridges, male germ cells enter G0 mitotic arrest and differentiate into prospermatogonia, which resume proliferation after birth [3, 4]. Subsequently, they further differentiate into spermatogonial stem cells (SSCs), and a subpopulation of SSCs starts the first wave of spermatogenesis [5, 6]. Spermatogenesis is a highly complex differentiation process, during which gene expression is highly orchestrated and strictly regulated .
In this study, we identified miR-741-3p, miR-871-3p, and miR-880-3p as being highly and preferentially expressed in germ cells. The genes for these three miRNAs are clustered on the X chromosome. In mice, 28.1% of miRNA genes form clusters on various chromosomes . miRNA gene clusters may arise by de novo formation of miRNA-like hairpin structures in existing primary miRNA transcript units or by tandem duplication of a single miRNA gene . XmiRs and additional miRNA gene clusters nearby may also be generated by similar molecular mechanisms.