[Abstract unavailable

Oocytes are sequestered in primordial follicles before birth and remain quiescent in the ovary, often for decades, until recruited into the growing pool throughout the reproductive years. Therefore, activation of follicle growth is a major biological checkpoint that controls female reproductive potential. However, we are only just beginning to elucidate the cellular mechanisms required for either maintenance of the quiescent primordial follicle pool or initiation of follicle growth. Understanding the intracellular signalling systems that control oocyte maintenance and activation has significant implications for improving female reproductive productivity and longevity in mammals, and has application in domestic animal husbandry, feral animal population control and infertility in women.

BACKGROUND Achieving the correct spatial and temporal expression of germ-cell-specific genes is fundamental to the production of viable healthy spermatozoa. Notably, post-transcriptional gene regulation resulting in the repression of protein translation is central to many embryonic processes, and is particularly active during spermatogenesis. In this review, we discuss microRNA (miRNA) regulation of target gene expression in relation to mammalian spermatogenesis, the establishment of testicular germ cell tumours (TGCT) and the potential use of miRNA manipulation for cancer therapy and fertility regulation. METHODS Journal databases such as PubMed were searched using key words, including miRNA, testis, spermatogenesis, germ cell, testicular cancer and cancer. RESULTS In the past decade, the deployment of small non-coding RNA molecules, including miRNA, by the cell, has been recognized as among the most important mechanisms of fine-tuning translational regulation in differentiating cell types. For key regulators of male gametogenesis, high levels of gene expression do not always correspond to elevated levels of protein expression. Cumulatively this indicates that enhancement and repression of post-transcriptional regulatory mechanisms are essential to the success of spermatogenesis. There is also growing evidence that this form of regulation contributes to the aetiology of both TGCT and spermatocytic tumours. CONCLUSIONS miRNA plays an essential role in regulation of genes during the process of spermatogenesis. Disruption of this regulation has the ability to contribute to the neoplastic development of germ cell tumours. However, targeted knockdown of specific miRNA molecules has the potential to form both anti-oncogenic reagents and underpin the basis for novel contraceptive technologies.

Key Points Exosome complex components are endogenous suppressors of erythroid cell maturation. GATA-1 and Foxo3 transcriptionally repress exosome complex components, thus abrogating the erythroid maturation blockade.

Since the highly conserved exosome complex mediates the degradation and processing of multiple classes of RNAs, it almost certainly controls diverse biological processes. How this post-transcriptional RNA-regulatory machine impacts cell fate decisions and differentiation is poorly understood. Previously, we demonstrated that exosome complex subunits confer an erythroid maturation barricade, and the erythroid transcription factor GATA-1 dismantles the barricade by transcriptionally repressing the cognate genes. While dissecting requirements for the maturation barricade in Mus musculus, we discovered that the exosome complex is a vital determinant of a developmental signaling transition that dictates proliferation/amplification versus differentiation. Exosome complex integrity in erythroid precursor cells ensures Kit receptor tyrosine kinase expression and stem cell factor/Kit signaling, while preventing responsiveness to erythropoietin-instigated signals that promote differentiation. Functioning as a gatekeeper of this developmental signaling transition, the exosome complex controls the massive production of erythroid cells that ensures organismal survival in homeostatic and stress contexts.DOI: http://dx.doi.org/10.7554/eLife.17877.001

The last 100 years have seen a concerning decline in male reproductive health associated with decreased sperm production, sperm function and male fertility. Concomitantly, the incidence of defects in reproductive development, such as undescended testes, hypospadias and testicular cancer has increased. Indeed testicular cancer is now recognised as the most common malignancy in young men. Such cancers develop from the pre-invasive lesion Carcinoma in Situ (CIS), a dysfunctional precursor germ cell or gonocyte which has failed to successfully differentiate into a spermatogonium. It is therefore essential to understand the cellular transition from gonocytes to spermatogonia, in order to gain a better understanding of the aetiology of testicular germ cell tumours. MicroRNA (miRNA) are important regulators of gene expression in differentiation and development and thus highly likely to play a role in the differentiation of gonocytes. In this study we have examined the miRNA profiles of highly enriched populations of gonocytes and spermatogonia, using microarray technology. We identified seven differentially expressed miRNAs between gonocytes and spermatogonia (down-regulated: miR-293, 291a-5p, 290-5p and 294*, up-regulated: miR-136, 743a and 463*). Target prediction software identified many potential targets of several differentially expressed miRNA implicated in germ cell development, including members of the PTEN, and Wnt signalling pathways. These targets converge on the key downstream cell cycle regulator Cyclin D1, indicating that a unique combination of male germ cell miRNAs coordinate the differentiation and maintenance of pluripotency in germ cells.

SUMMARY Hematopoietic development requires the transcription factor GATA-2, and GATA-2 mutations cause diverse pathologies including leukemia. GATA-2-regulated enhancers regulate Gata2 expression in hematopoietic stem/progenitor cells and control hematopoiesis. The +9.5 kb enhancer activates transcription in endothelium and hematopoietic stem cells (HSCs), and its deletion abrogates HSC generation. The −77 kb enhancer activates transcription in myeloid progenitors, and its deletion impairs differentiation. Since +9.5−/− embryos are HSC-deficient, it was unclear whether the +9.5 functions in progenitors or if GATA-2 expression in progenitors solely requires −77. We further dissected the mechanisms using −77;+9.5 compound heterozygous (CH) mice. The embryonic lethal CH mutation depleted megakaryocyte-erythrocyte progenitors (MEPs). While the +9.5 suffices for HSC generation, the −77 and +9.5 must reside on one allele to induce MEPs. The −77 generated Burst Forming Unit-Erythroid through induction of GATA-1 and other GATA-2 targets. The enhancer circuits controlled signaling pathways that orchestrate a GATA factor-dependent blood development program.

GATA-2 levels must be stringently regulated to ensure normal hematopoiesis, and human GATA-2 mutations cause hematologic disorders. GATA-2-regulated enhancers differentially control Gata2 expression in hematopoietic stem/progenitor cells and are essential for hematopoiesis and embryonic development. Mechanisms underlying how the enhancers control Gata2 expression and GATA-2 instigated genetic networks in a cell-specific manner are not completely understood. Targeted deletion of an intronic Gata2 enhancer 9.5 kb downstream of the transcription start site (+9.5) abrogates HSC genesis in the aorta-gonad-mesonephros (AGM) region (Gao et al., JEM, 2013). By contrast, the -77 kb enhancer (-77) activates transcription in myeloid progenitors, and its deletion impairs progenitor differentiation (Johnson et al., Science Advances, 2015). To dissect relationships between the enhancers, we developed a compound heterozygous (CH) mouse model bearing +9.5 and -77 enhancer mutations on different Gata2 alleles. While the CH embryos were alive at E13.5, nearly all died by E14.5 (p = 3.58 x 10-5). Flow cytometric analyses and embryo confocal imaging demonstrated that CH embryos have modestly reduced HSC numbers in the fetal liver (2.9-fold) and the AGM (41%, p = 7.8 x 10-5), which was comparable to +9.5+/- embryos. Thus, -77 does not genetically interact with +9.5 to control HSC emergence. Flow cytometric analysis revealed that Lin-Sca1-Kit+ myelo-erythroid progenitors were 6.6-fold lower in CH vs. WT embryos (p = 1.8 x 10-11), with the difference involving disproportionate losses of GMP (8.6-fold; p = 3.7 x 10-6) and MEP (379-fold; p = 3.2 x 10-9). By contrast, +9.5+/- fetal livers had 2-fold fewer myeloid progenitors, which involved similar reductions of CMP (2.1-fold; p = 1 x 10-6), GMP (2.6-fold; p = 0.0007) and MEP (1.9-fold; p = 0.002). Consistent with the myelo-erythroid progenitor reductions and MEP depletion, CH fetal livers lacked BFU-E (p < 0.001) and CFU-GEMM (p < 0.001) in a colony assay. These results illustrate a genetic interaction between +9.5 and -77 in progenitors, but not HSCs, and a new paradigm in which both enhancers must reside on a single allele to generate MEPs. As erythroid precursor cells express GATA-2, we tested whether the -77 deletion impairs erythroid maturation due to a reduction in myelo-erythroid progenitors or due to a cell-autonomous requirement of the enhancer in erythroid precursors. -77-/- E14.5 fetal livers were pale and smaller than WT counterparts, and -77-/- fetal liver cellularity was reduced 7.2-fold (5.3 x 10-4). When liver size was taken into account, there was little difference in the number of E14.5 R1 cells in -77-/- liver vs. WT littermates (p = 0.31). However, -77-/- R2-R5 cells declined sharply (R2, 8.2-fold, p = 0.004; R3, 14-fold, p < 10-5; R4, 9.7-fold, p = 0.002; R5, 14-fold, p = 0.087). The mutant R1 cells were defective in forming BFU-Es and CFU-Es. Analysis of transcriptomes of purified 77-/- and WT R1 cells from E14.5 fetal livers revealed 2805 and 2519 upregulated and downregulated (p < 0.05) genes, respectively, in -77-/- R1 cells. The -77 enhancer conferred GATA-2 expression, which strongly upregulated GATA-1 and therefore a large GATA-1 target gene cohort. A comparison of WT and -77-/- R1 cell transcriptomes with those of early (Tgbfr3low) and late (Tgbfr3high) BFU-Es (Gao et al., Blood, 2016) revealed a -77-/- R1 signature that correlated with the early BFU-E signature (r = 0.73, p < 10-4) and negatively correlated with the late BFU-E signature (r = -0.42, p = 4 x 10-4) differing from WT cells. In addition to GATA-1 target gene alterations, 253 of the -77-activated genes were not GATA-1-regulated in the G1E-ER-GATA-1 system. These genes included Ryk, which encodes a non-canonical Wnt receptor, and had not been studied in erythroid cells. Two Ryk shRNAs significantly decreased BFU-Es and CFU-GMs in lineage-depleted fetal liver cells. Ongoing studies are integrating Ryk function into signaling circuits that control erythroid maturation and analyzing other -77-regulated targets predicted to constitute new regulators of erythroid cell maturation/function. Thus, loss of the -77 enhancer creates multi-faceted defects in erythroid precursors, involving deficiencies of constituents of signaling and transcriptional circuitry required to enable and drive erythroid maturation. Figure Figure. Disclosures No relevant conflicts of interest to declare.