Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells.

The insulin family of growth factors (insulin, IGF1, and IGF2) are critical in sex determination, adrenal differentiation, and testicular function. Notably, the IGF system has been reported to mediate the proliferation of steroidogenic cells. However, the precise role and contribution of the membrane receptors mediating those effects, namely, insulin receptor (INSR) and type-I insulin-like growth factor receptor (IGF1R), have not, to our knowledge, been investigated. We show here that specific deletion of both Insr and Igf1r in steroidogenic cells in mice leads to severe alterations of adrenocortical and testicular development. Double-mutant mice display drastic size reduction of both adrenocortex and testes, with impaired corticosterone, testosterone, and sperm production. Detailed developmental analysis of the testes revealed that fetal Leydig cell (LC) function is normal, but there is a failure of adult LC maturation and steroidogenic function associated with accumulation of progenitor LCs (PLCs). Cell-lineage tracing revealed PLC enrichment is secondary to Insr and Igf1r deletion in differentiated adult LCs, suggesting a feedback mechanism between cells at different steps of differentiation. Taken together, these data reveal the cell-autonomous and nonautonomous roles of the IGF system for proper development and maintenance of steroidogenic lineages.-Neirijnck, Y., Calvel, P., Kilcoyne, K. R., Kühne, F., Stévant, I., Griffeth, R. J., Pitetti, J.-L., Andric, S. A., Hu, M.-C., Pralong, F., Smith, L. B., Nef, S. Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells.

Sertoli cells control peritubular myoid cell fate and support adult Leydig cell development in the prepubertal testis.

Sertoli cells (SCs) regulate testicular fate in the differentiating gonad and are the main regulators of spermatogenesis in the adult testis; however, their role during the intervening period of testis development, in particular during adult Leydig cell (ALC) differentiation and function, remains largely unknown. To examine SC function during fetal and prepubertal development we generated two transgenic mouse models that permit controlled, cell-specific ablation of SCs in pre- and postnatal life. Results show that SCs are required: (1) to maintain the differentiated phenotype of peritubular myoid cells (PTMCs) in prepubertal life; (2) to maintain the ALC progenitor population in the postnatal testis; and (3) for development of normal ALC numbers. Furthermore, our data show that fetal LCs function independently from SC, germ cell or PTMC support in the prepubertal testis. Together, these findings reveal that SCs remain essential regulators of testis development long after the period of sex determination. These findings have significant implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.

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.

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.

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.

Sertoli Cell Number Defines and Predicts Germ and Leydig Cell Population Sizes in the Adult Mouse Testis.

Sertoli cells regulate differentiation and development of the testis and are essential for maintaining adult testis function. To model the effects of dysregulating Sertoli cell number during development or aging, we have used acute diphtheria toxin-mediated cell ablation to reduce Sertoli cell population size. Results show that the size of the Sertoli cell population that forms during development determines the number of germ cells and Leydig cells that will be present in the adult testis. Similarly, the number of germ cells and Leydig cells that can be maintained in the adult depends directly on the size of the adult Sertoli cell population. Finally, we have used linear modeling to generate predictive models of testis cell composition during development and in the adult based on the size of the Sertoli cell population. This study shows that at all ages the size of the Sertoli cell population is predictive of resulting testicular cell composition. A reduction in Sertoli cell number/proliferation at any age will therefore lead to a proportional decrease in germ cell and Leydig cell numbers, with likely consequential effects on fertility and health.

Research resource: the dynamic transcriptional profile of sertoli cells during the progression of spermatogenesis.

Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1ß), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility.

DNA methylation profiling of the fibrinogen gene landscape in human cells and during mouse and zebrafish development.

The fibrinogen genes FGA, FGB and FGG show coordinated expression in hepatocytes. Understanding the underlying transcriptional regulation may elucidate how their tissue-specific expression is maintained and explain the high variability in fibrinogen blood levels. DNA methylation of CpG-poor gene promoters is dynamic with low methylation correlating with tissue-specific gene expression but its direct effect on gene regulation as well as implications of non-promoter CpG methylation are not clear. Here we compared methylation of CpG sites throughout the fibrinogen gene cluster in human cells and mouse and zebrafish tissues. We observed low DNA methylation of the CpG-poor fibrinogen promoters and of additional regulatory elements (the liver enhancers CNC12 and PFE2) in fibrinogen-expressing samples. In a gene reporter assay, CpG-methylation in the FGA promoter reduced promoter activity, suggesting a repressive function for DNA methylation in the fibrinogen locus. In mouse and zebrafish livers we measured reductions in DNA methylation around fibrinogen genes during development that were preceded by increased fibrinogen expression and tri-methylation of Histone3 lysine4 (H3K4me3) in fibrinogen promoters. Our data support a model where changes in hepatic transcription factor expression and histone modification provide the switch for increased fibrinogen gene expression in the developing liver which is followed by reduction of CpG methylation.

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.

FSH-stimulated PTEN activity accounts for the lack of FSH mitogenic effect in prepubertal rat Sertoli cells.

Follicle-stimulating hormone (FSH) controls the proliferation and differentiation of Sertoli cells of the testis. FSH binds a G protein-coupled receptor (GPCR) to stimulate downstream effectors of the phosphoinositide-3 kinase (PI3K)-dependent pathway, without enhancing PI3K activity. To clarify this paradox, we explored the activity of phosphatase and tensin homolog deleted in chromosome 10 (PTEN), the PI3K major regulator, in primary cultures of rat Sertoli cells. We show that, within minutes, FSH increases PTEN neo-synthesis, requiring the proteasomal degradation of an unidentified intermediate, as well as PTEN enzymatic activity. Importantly, introducing an antisense cDNA of PTEN into differentiating Sertoli cells restores FSH-dependent cell proliferation. In conclusion, these results provide a new mechanism of PTEN regulation, which could serve to block entry into S phase of Sertoli cells, while they are proceeding through differentiation in prepubertal animals.

Dcp1-bodies in mouse oocytes.

Processing bodies (P-bodies) are cytoplasmic granules involved in the storage and degradation of mRNAs. In somatic cells, their formation involves miRNA-mediated mRNA silencing. Many P-body protein components are also found in germ cell granules, such as in mammalian spermatocytes. In fully grown mammalian oocytes, where changes in gene expression depend entirely on translational control, RNA granules have not as yet been characterized. Here we show the presence of P-body-like foci in mouse oocytes, as revealed by the presence of Dcp1a and the colocalization of RNA-associated protein 55 (RAP55) and the DEAD box RNA helicase Rck/p54, two proteins associated with P-bodies and translational control. These P-body-like structures have been called Dcp1-bodies and in meiotically arrested primary oocytes, two types can be distinguished based on their size. They also have different protein partners and sensitivities to the depletion of endogenous siRNA/miRNA and translational inhibitors. However, both type progressively disappear during in vitro meiotic maturation and are virtually absent in metaphase II-arrested secondary oocytes. Moreover, this disassembly of hDcp1a-bodies is concomitant with the posttranslational modification of EGFP-hDcp1a.

Gene expression during sex determination reveals a robust female genetic program at the onset of ovarian development.

The primary event in mammalian sexual development is the differentiation of the bipotential gonads into either testes or ovaries. Our understanding of the molecular pathways specifying gonadal differentiation is still incomplete. To identify the initial molecular changes accompanying gonadal differentiation in mice, we have performed a large-scale transcriptional analysis of XX and XY Sf1-positive gonadal cells during sex determination. In both male and female genital ridges, a robust genetic program is initiated pre-dating the first morphological changes of the differentiating gonads. Between E10.5 and E13.5, 2306 genes were expressed in a sex-specific manner in the somatic compartment of the gonads; 1223 were overexpressed in XX embryos and 1083 in XY embryos. Although sexually dimorphic genes were scattered throughout the mouse genome, we identified chromosomal regions hosting clusters of genes displaying similar expression profiles. The cyclin-dependent kinase inhibitors Cdkn1a and Cdkn1c are overexpressed in XX gonads at E11.5 and E12.5, suggesting that the increased proliferation of XY gonads relative to XX gonads may result from the overexpression of cell cycle inhibitors in the developing ovaries. These studies define the major characteristics of testicular and ovarian transcriptional programs and reveal the richness of signaling processes in differentiation of the bipotential gonads into testes and ovaries.