Dicer1 Depletion in Male Germ Cells Leads to Infertility Due to Cumulative Meiotic and Spermiogenic Defects

Background: Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice.Principal Findings: We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes.Conclusions/Significance: Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis

A brief history of sex determination

A fundamental biological question that has puzzled, but also fascinated mankind since antiquity is the one pertaining to the differences between sexes. Ancient cultures and mythologies poetically intended to explain the origin of the two sexes; philosophy offered insightful albeit occasionally paradoxical perceptions about men and women; and society as a whole put forward numerous intuitive observations about the traits that distinguish the two sexes. However, it was only through meticulous scientific research that began in the 16th century, and gradual technical improvements that followed over the next centuries, that the study of sex determination bore fruit. Here, we present a brief history of sex determination studies from ancient times until today, by selectively interviewing some of the milestones in the field. We complete our review by outlining some yet unanswered questions and proposing future experimental directions

miR-373 is regulated by TGFβ signaling and promotes mesendoderm differentiation in human Embryonic Stem Cells

MicroRNAs (miRNAs) belonging to the evolutionary conserved miR-302 family play important functions in Embryonic Stem Cells (ESCs). The expression of some members, such as the human miR-302 and mouse miR-290 clusters, is regulated by ESC core transcription factors. However, whether miRNAs act downstream of signaling pathways involved in human ESC pluripotency remains unknown. The maintenance of pluripotency in hESCs is under the control of the TGFβ pathway. Here, we show that inhibition of the Activin/Nodal branch of this pathway affects the expression of a subset of miRNAs in hESCs. Among them, we found miR-373, a member of the miR-302 family. Proper levels of miR-373 are crucial for the maintenance of hESC pluripotency, since its overexpression leads to differentiation towards the mesendodermal lineage. Among miR-373 predicted targets, involved in TGFβ signaling, we validated the Nodal inhibitor Lefty. Our work suggests a crucial role for the interplay between miRNAs and signaling pathways in ESCs. © 2014 Elsevier Inc

Insulin and IGF1 receptors are essential for XX and XY gonadal differentiation and adrenal development in mice

Mouse sex determination provides an attractive model to study how regulatory genetic networks and signaling pathways control cell specification and cell fate decisions. This study characterizes in detail the essential role played by the insulin receptor (INSR) and the IGF type I receptor (IGF1R) in adrenogenital development and primary sex determination. Constitutive ablation of insulin/IGF signaling pathway led to reduced proliferation rate of somatic progenitor cells in both XX and XY gonads prior to sex determination together with the downregulation of hundreds of genes associated with the adrenal, testicular, and ovarian genetic programs. These findings indicate that prior to sex determination somatic progenitors in Insr;Igf1r mutant gonads are not lineage primed and thus incapable of upregulating/repressing the male and female genetic programs required for cell fate restriction. In consequence, embryos lacking functional insulin/IGF signaling exhibit (i) complete agenesis of the adrenal cortex, (ii) embryonic XY gonadal sex reversal, with a delay of Sry upregulation and the subsequent failure of the testicular genetic program, and (iii) a delay in ovarian differentiation so that Insr;Igf1r mutant gonads, irrespective of genetic sex, remained in an extended undifferentiated state, before the ovarian differentiation program ultimately is initiated at around E16.5

Dicer is required for haploid male germ cell differentiation in mice.

BACKGROUND: The RNase III endonuclease Dicer is an important regulator of gene expression that processes microRNAs (miRNAs) and small interfering RNAs (siRNAs). The best-characterized function of miRNAs is gene repression at the post-transcriptional level through the pairing with mRNAs of protein-encoding genes. Small RNAs can also act at the transcriptional level by controlling the epigenetic status of chromatin. Dicer and other mediators of small RNA pathways are present in mouse male germ cells, and several miRNAs and endogenous siRNAs are expressed in the testis, suggesting that Dicer-dependent small RNAs are involved in the control of the precisely timed and highly organised process of spermatogenesis. PRINCIPAL FINDINGS: Being interested in the Dicer-mediated functions during spermatogenesis, we have analysed here a male germ cell-specific Dicer1 knockout mouse model, in which the deletion of Dicer1 takes place during early postnatal development in spermatogonia. We found that Dicer1 knockout testes were reduced in size and spermatogenesis within the seminiferous tubules was disrupted. Dicer1 knockout epididymides contained very low number of mature sperm with pronounced morphological abnormalities. Spermatogonial differentiation appeared unaffected. However, the number of haploid cells was decreased in knockout testes, and an increased number of apoptotic spermatocytes was observed. The most prominent defects were found during late haploid differentiation, and Dicer was demonstrated to be critical for the normal organization of chromatin and nuclear shaping of elongating spermatids. CONCLUSIONS/SIGNIFICANCE: We demonstrate that Dicer and Dicer-dependent small RNAs are imperative regulators of haploid spermatid differentiation and essential for male fertility

Loss of Dicer in Sertoli Cells Has a Major Impact on the Testicular Proteome of Mice

Sertoli cells (SCs) are the central, essential coordinators of spermatogenesis, without which germ cell development cannot occur. We previously showed that Dicer, an RNaseIII endonuclease required for microRNA (miRNA) biogenesis, is absolutely essential for Sertoli cells to mature, survive, and ultimately sustain germ cell development. Here, using isotope-coded protein labeling, a technique for protein relative quantification by mass spectrometry, we investigated the impact of Sertoli cell-Dicer and subsequent miRNA loss on the testicular proteome. We found that, a large proportion of proteins (50 out of 130) are up-regulated by more that 1.3-fold in testes lacking Sertoli cell-Dicer, yet that this protein up-regulation is mild, never exceeding a 2-fold change, and is not preceeded by alterations of the corresponding mRNAs. Of note, the expression levels of six proteins of interest were further validated using the Absolute Quantification (AQUA) peptide technology. Furthermore, through 3′UTR luciferase assays we identified one up-regulated protein, SOD-1, a Cu/Zn superoxide dismutase whose overexpression has been linked to enhanced cell death through apoptosis, as a likely direct target of three Sertoli cell-expressed miRNAs, miR-125a-3p, miR-872 and miR-24. Altogether, our study, which is one of the few in vivo analyses of miRNA effects on protein output, suggests that, at least in our system, miRNAs play a significant role in translation control

Absence of <i>Insr</i> and <i>Igf1r</i> significantly reduces proliferation of somatic cell progenitors prior to sex determination.

<p>Photomicrographs of control and dko embryos at E10.5 (A, scale bar 2.5 mm), E11.5 (B, scale bar 4 mm) and E12.5 (C, scale bar 5 mm). (D) Body weights of control and dko animals at E10.5 (n≥9), E11.5 (n≥22) and E12.5 (n≥11). (E) Scanning electron microscopy pictures of control and dko genital ridges at E11.5 (scale bar 200 µm). A pink overlay marks the genital ridges. (F) FACS analysis was used to determine the number of SF1⁺ somatic progenitor cells in genital ridges of control and dko animals at E10.5 (n≥9), E11.5 (n≥12), E12.5 (n≥13) and E13.5 (n≥6). (G) Double IF using the gonadal progenitor marker GATA4 (red) along with proliferating marker Ki67 (green) in control and dko genital ridges at E11.5. Quantification of GATA4⁺/Ki67⁺ cells in XX and XY genital ridges of control and dko animals at E10.5 (H) and E11.5 (I) revealed a significant reduction in the proliferation rates of somatic gonadal precursors at E10.5 and E11.5 in dko genital ridges. Values are expressed as means ± SEM, *p<0.05, **p<0.01, ***p<0.001 vs control. Scale bar: 100 µm.</p

Absence of <i>Insr</i> and <i>Igf1r</i> negatively affects <i>SF1</i> gene expression and causes adrenal agenesis.

<p>Whole mount <i>in situ</i> hybridization on E10.5–E13.5 genital ridges (A), double IF with anti-SF1 (green) and anti-WT1 (red) at E11.5 (B) and qRT-PCR on genital ridges/mesonephroi at E11.5 and 12.5 (C) revealed that <i>Sf-1</i> is expressed at reduced levels both in XX and XY dko embryos during sex determination. Haematoxylin and eosin staining (D,H,L) as well as immunostaining for the adrenocortical markers SF1 (E,I,M) and 3βHSD (F,J,N) and chromaffin cell marker tyrosine hydroxylase, TH (G,K,O) from control (D–G) and dko (H–O) XY embryos at E16.5 revealed that adrenal glands are either massively reduced in size (H–K, 3 out of 27 embryos) or absent (L–O, 24 out of 27 embryos) in dko mutants. Fluorescence photomicrographs of XY genital ridges at E10.5 (P,T), E11.5 (Q,U), E12.5 (R,V) and E13.5 (S,W) from control (P–S) and dko (T–W) embryos expressing the <i>Sf1;eGFP</i> transgene. Note the absence of adrenal development (arrowhead) and gonadal primordium differentiation into testis (arrows) in dko animals. REL, relative expression levels. Values are expressed as means ± SEM, p*<0.05, ***p<0.001 vs control. Scale bar: 100 µm.</p