MicroRNAs-140-5p/140-3p Modulate Leydig Cell Numbers in the Developing Mouse Testis

Abstract

MicroRNAs (miRNAs) have been shown to play key regulatory roles in a range of biological processes, including cell differentiation and development. To identify miRNAs that participate in gonad differentiation, a fundamental and tightly regulated developmental process, we examined miRNA expression profiles at the time of sex determination and during the early fetal differentiation of mouse testes and ovaries using high-throughput sequencing. We identified several miRNAs that were expressed in a sexually dimorphic pattern, including several members of the let-7 family, miR-378, and miR-140-3p. We focused our analysis on the most highly expressed, sexually dimorphic miRNA, miR-140-3p, and found that both miR-140-3p and its more lowly expressed counterpart, the previously annotated guide strand, miR-140-5p, are testis enriched and expressed in testis cords. Analysis of the miR-140-5p/miR-140-3p-null mouse revealed a significant increase in the number of Leydig cells in the developing XY gonad, strongly suggesting an important role for miR-140-5p/miR-140-3p in testis differentiation in mouse.

The role of non-coding RNAs in male sex determination and differentiation

“…The expression of miRNAs in embryonic gonads at the time of sex determination and also at later stages during testis differentiation and in the postnatal testis has been studied extensively in different species. Surprisingly, depending on the experimental setup, there is little overlap between the different studies with respect to the miRNAs that were detected (e.g., Papaioannou et al 2009, Aguilar et al 2010, Rakoczy et al 2013. In addition, deletion of Dicer or Drosha demonstrated only a relatively late role for miRNAs in the postnatal testis (Hayashi et al 2008, Maatouk et al 2008, Papaioannou et al 2009, Huang & Yao 2010, suggesting that miRNAs might not play a role during the early stages of sex differentiation.…”

“…miR-124 was identified in a microarray screen of embryonic mouse gonads as being upregulated in the developing ovary and downregulated in the testis (Real et al 2013), suggesting a role during ovarian differentiation. However, this ovary-enriched expression was not detected by a high-throughput sequencing approach (Rakoczy et al 2013). Nevertheless, inhibition of miR-124 in XX gonadal cells using antagomirs (small, antisense molecules that bind and inhibit miRNAs) resulted in an upregulation of Sox9 expression, indicating that miR-124 is necessary for the repression of Sox9 in the developing ovary (Real et al 2013).…”

“…We have shown recently that the expression of pri-miR-202 is downstream of SOX9 in the developing mouse testis . Both strands, miR-202-3p and miR-202-5p, were initially identified in the embryonic gonad by using highthroughput sequencing of small RNAs from differentiating XY and XX gonads (Rakoczy et al 2013). The sequence of miR-202 is conserved among vertebrates and both strands, miR-202-5p and -3p, are upregulated during testis development not only in mouse but also in chicken embryos.…”

“…Similarly, the miRNA miR-140, expressed in Sertoli cells and shown to be directly upregulated by SOX9 (Nakamura et al 2011, Yang et al 2011, influences Leydig cell differentiation. Loss of miR-140 results in an increase in Leydig cell numbers (Rakoczy et al 2013). However, it is not known which genes are regulated by miR-140 within Sertoli cells that indirectly influence Leydig cell differentiation, although predicted target genes have been shown to play a role in testis development and Leydig cell differentiation and function (Rakoczy et al 2013).…”

“…Loss of miR-140 results in an increase in Leydig cell numbers (Rakoczy et al 2013). However, it is not known which genes are regulated by miR-140 within Sertoli cells that indirectly influence Leydig cell differentiation, although predicted target genes have been shown to play a role in testis development and Leydig cell differentiation and function (Rakoczy et al 2013). DHH produced by Sertoli cells not only controls the differentiation of Leydig cells, but also another testisspecific cell type, so-called peritubular myoid cells; long, flattened cells that surround Sertoli cells at the testis cords and secret, together with Sertoli cells, extracellular matrix proteins, which form a basal lamina between the two cell types (Tung et al 1984).…”