Abstract
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.
Highlights
Germ cells first arise as primordial germ cells (PGCs) in early embryos [1], and they proliferate and migrate into the genital ridges during embryonic development [2]
Cluster analysis showed that the expression profiles of these miRNAs were classified into three groups, i.e., those highly expressed in somatic tissues or mouse embryonic fibroblasts (MEFs), in spermatogenic cells, and in PGCs and embryonic stem (ES) cells (Fig 1A)
Of the 20 most abundantly expressed miRNAs in PGCs, many miRNAs were highly expressed in ES cells (Fig 1B and S2 Table). miR-741-3p, miR-871-3p, and miR-880-3p were highly expressed in PGCs and spermatogonia, but were rarely expressed in ES cells or somatic tissues
Summary
Germ cells first arise as primordial germ cells (PGCs) in early embryos [1], and they proliferate and migrate into the genital ridges during embryonic development [2]. After arriving at the genital ridges, male germ cells enter G0 mitotic arrest and differentiate into prospermatogonia, which resume proliferation after birth [3, 4]. They further differentiate into spermatogonial stem cells (SSCs), and a subpopulation of SSCs starts the first wave of spermatogenesis [5, 6]. MicroRNAs (miRNAs) are small non-coding RNAs 18–23 nucleotides in length that play a critical role in the regulation of development and differentiation in many organisms and different tissues [8, 9]. MiR-125a modulates self-renewal of hematopoietic stem cells and protects them from apoptosis [10]. miR-1 and miR-206 inhibit cell proliferation and promote myoblast differentiation [11]. miR-29 has multiple activities at different stages of osteoblast differentiation [12]. miRNAs repress gene expression by binding the 30-untranslated region (UTR) of their target mRNAs, thereby decreasing mRNA stability and translational efficiency [13]
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