Regulation Of Testis-specific MiRNA-469 By GRTH/DDX25 Controls Male Germ Cells Development

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), present in Leydig and germ cells (meiotic spermatocytes and round spermatids), regulated developmentally by androgen at the transcriptional level. GRTH knock-out male mice are sterile, with azoospermia caused by a complete arrest of spermiogenesis at step 8/9 of round spermatids and failure to elongate. The CB was condensed and greatly reduced in size in the round spermatids of GRTH-null mice. Accumulated evidence indicate that members of the small interfering RNA (siRNA) and microRNA (miRNA) pathway, which include Dicer to process si-, mi-RNA precursors to mature small RNAs, the effector complex RNA induced silencing complex (RISC) and MVH, another germ cell helicase are present in the CB. Preservation of the expression of relevant mRNAs with failure of protein expression in the GRTH-null mice indicate that GRTH is also involved in posttranscriptional events.To elucidate the miRNAs and pathway(s) related to GRTH regulation in male germ cells, their differential expression profile was first determined in purified round spermatids of WT and GRTH^-/- mice by high-throughput microRNA array and validated by RT-PCR. A total of 119 miRNAs including the ubiquitous let-7 family, testis preferentially (miR-470/miR-34c) and testis exclusively expressed (miR-469) were up-regulated in GRTH^-/- mice.Further in vitro studies in NIH3T3 cells showed that miR-469 inhibits the expression of luciferase constructs containing the coding and 3'UTR of transition protein 2 (TP2) and protamine 2 (Prm2) genes, which are chromatin remodelers crucial in the elongation of round spermatids were absent in the GRTH^-/-. Unlike the commonly defined 3'UTR as miRNA targeting region, deletion and mutagenesis analyses demonstrated that miR-469 with perfect seed sequence to coding regions of TP2 (376-382nt) and Prm2 (271-277; 292-298nt) repressed protein expression but did not cause RNA degradation. This is consistent with the preservation of TP2 and Prm2 mRNA but failure of their protein expression in the GRTH^-/- mice. Unlike the earlier proposed miRNA regulation associated with CB in WT, the regulatory functions of up-regulated miRNAs in the GRTH^-/- mice may occur at cytoplasmic sites other than the CB, whose morphological and functional identity is maintained by GRTH.The significant increases in the primary-miRNAs of let-7d/g, -34c, -469 and -470 found in the GRTH^-/- mice compared to wild type. The expression of Drosha/DGCR8 microprocessor complex, required for the generation of precursor pre-miRNA, was significantly increased at both mRNA and protein level in the absence of GRTH (GRTH KO versus WT).Thus, GRTH has a critical role in the negative regulation of a subset of miRNAs biogenesis to maintain homeostasis of mature miRNA synthesis which could control the temporal progression of spermatogenesis. The finding of an inhibitory action of GRTH associated with miRNA biogenesis has provided insights into a novel molecular control mechanism of this helicase in the regulation of male germ cell development.