The Insulin/IGF System in Mammalian Sexual Development and Reproduction.

Persistent research over the past few decades has clearly established that the insulin-like family of growth factors, which is composed of insulin and insulin-like growth factors 1 (IGF1) and 2 (IGF2), plays essential roles in sexual development and reproduction of both males and females. Within the male and female reproductive organs, ligands of the family act in an autocrine/paracrine manner, in order to guide different aspects of gonadogenesis, sex determination, sex-specific development or reproductive performance. Although our knowledge has greatly improved over the last years, there are still several facets that remain to be deciphered. In this review, we first briefly outline the principles of sexual development and insulin/IGF signaling, and then present our current knowledge, both in rodents and humans, about the involvement of insulin/IGFs in sexual development and reproductive functions. We conclude by highlighting some interesting remarks and delineating certain unanswered questions that need to be addressed in future studies.

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.

miR-373 is regulated by TGFß 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 TGFß 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 TGFß signaling, we validated the Nodal inhibitor Lefty. Our work suggests a crucial role for the interplay between miRNAs and signaling pathways in ESCs.

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.

Sertoli cell Dicer is essential for spermatogenesis in mice.

Spermatogenesis requires intact, fully competent Sertoli cells. Here, we investigate the functions of Dicer, an RNaseIII endonuclease required for microRNA and small interfering RNA biogenesis, in mouse Sertoli cell function. We show that selective ablation of Dicer in Sertoli cells leads to infertility due to complete absence of spermatozoa and progressive testicular degeneration. The first morphological alterations appear already at postnatal day 5 and correlate with a severe impairment of the prepubertal spermatogenic wave, due to defective Sertoli cell maturation and incapacity to properly support meiosis and spermiogenesis. Importantly, we find several key genes known to be essential for Sertoli cell function to be significantly down-regulated in neonatal testes lacking Dicer in Sertoli cells. Overall, our results reveal novel essential roles played by the Dicer-dependent pathway in mammalian reproductive function, and thus pave the way for new insights into human infertility.

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.

Genetic programs that regulate testicular and ovarian development.

The gonadal primordium is the only tissue in mammals that has two divergent developmental fates leading ultimately to the formation of either a testis or an ovary. The goal of this review is to summarize the major characteristics of the male and female transcriptional programs triggered in the developing mouse gonads during the critical time window of sex determination. Expression profiling studies reveal that both male and female genetic programs are initiated as early as embryonic day (E) 11.5. By E13.5, more than 1000 genes are overexpressed either in developing ovaries or testes. A large fraction of these have so far no known roles during gonadal differentiation, yet interestingly some of their human orthologues map to chromosomal loci associated with sexual disorders. Identifying the functional roles for these candidate genes will improve our understanding of sex determination and provide new insights into the causes of gonadal dysgenesis and reproductive disorders.

An essential role for insulin and IGF1 receptors in regulating sertoli cell proliferation, testis size, and FSH action in mice.

Testis size and sperm production are directly correlated to the total number of adult Sertoli cells (SCs). Although the establishment of an adequate number of SCs is crucial for future male fertility, the identification and characterization of the factors regulating SC survival, proliferation, and maturation remain incomplete. To investigate whether the IGF system is required for germ cell (GC) and SC development and function, we inactivated the insulin receptor (Insr), the IGF1 receptor (Igf1r), or both receptors specifically in the GC lineage or in SCs. Whereas ablation of insulin/IGF signaling appears dispensable for GCs and spermatogenesis, adult testes of mice lacking both Insr and Igf1r in SCs (SC-Insr;Igf1r) displayed a 75% reduction in testis size and daily sperm production as a result of a reduced proliferation rate of immature SCs during the late fetal and early neonatal testicular period. In addition, in vivo analyses revealed that FSH requires the insulin/IGF signaling pathway to mediate its proliferative effects on immature SCs. Collectively, these results emphasize the essential role played by growth factors of the insulin family in regulating the final number of SCs, testis size, and daily sperm output. They also indicate that the insulin/IGF signaling pathway is required for FSH-mediated SC proliferation.

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.

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.

microRNAs in the testis: building up male fertility.

Spermatogenesis is a strictly regulated process, at both the transcriptional and the posttranscriptional level, which allows continuous gamete production throughout adulthood. A novel mechanism of posttranscriptional control mediated by microRNAs (miRNAs) has lately emerged as an important regulator of spermatogenesis. miRNAs are endogenous, small, noncoding RNAs produced through a multistep enzymatic process, which involves the action of Dicer, an RNaseIII endonuclease. Here, we first present a short overview of classic posttranscriptional control during spermatogenesis, and then concentrate on recent findings that have unraveled the important role of miRNAs in male reproductive function. Particular focus is given to the in vivo role of miRNAs that has been demonstrated through the generation of Sertoli cell-specific or germ cell-specific Dicer knockouts, as well as the potential application of these findings in the treatment of human male infertility and the development of male contraceptives. It is anticipated that unraveling miRNA functions in the testis will further our understanding of the regulatory mechanisms of mammalian spermatogenesis.