Complete male-to-female sex reversal in XY mice lacking the miR-17~92 cluster.

Mammalian sex determination is controlled by antagonistic gene cascades operating in embryonic undifferentiated gonads. The expression of the Y-linked gene SRY is sufficient to trigger the testicular pathway, whereas its absence in XX embryos leads to ovarian differentiation. Yet, the potential involvement of non-coding regulation in this process remains unclear. Here we show that the deletion of a single microRNA cluster, miR-17~92, induces complete primary male-to-female sex reversal in XY mice. Sry expression is delayed in XY knockout gonads, which develop as ovaries. Sertoli cell differentiation is reduced, delayed and unable to sustain testicular development. Pre-supporting cells in mutant gonads undergo a transient state of sex ambiguity which is subsequently resolved towards the ovarian fate. The miR-17~92 predicted target genes are upregulated, affecting the fine regulation of gene networks controlling gonad development. Thus, microRNAs emerge as key components for mammalian sex determination, controlling Sry expression timing and Sertoli cell differentiation.

Cell adhesion and immune response, two main functions altered in the transcriptome of seasonally regressed testes of two mammalian species.

In species with seasonal breeding, male specimens undergo substantial testicular regression during the nonbreeding period of the year. However, the molecular mechanisms that control this biological process are largely unknown. Here, we report a transcriptomic analysis on the Iberian mole, Talpa occidentalis, in which the desquamation of live, nonapoptotic germ cells is the major cellular event responsible for testis regression. By comparing testes at different reproductive states (active, regressing, and inactive), we demonstrate that the molecular pathways controlling the cell adhesion function in the seminiferous epithelium, such as the MAPK, ERK, and TGF-ß signaling, are altered during the regression process. In addition, inactive testes display a global upregulation of genes associated with immune response, indicating a selective loss of the "immune privilege" that normally operates in sexually active testes. Interspecies comparative analyses using analogous data from the Mediterranean pine vole, a rodent species where testis regression is controlled by halting meiosis entry, revealed a common gene expression signature in the regressed testes of these two evolutionary distant species. Our study advances in the knowledge of the molecular mechanisms associated to gonadal seasonal breeding, highlighting the existence of a conserved transcriptional program of testis involution across mammalian clades.

The Biology and Evolution of Fierce Females (Moles and Hyenas).

Talpid moles and spotted hyenas have become the paradigms of anatomical and behavioral female masculinization. Females of many mole species develop ovotestes that produce testosterone, show external genitalia that resemble that of males, and close their vaginal orifice after every estrus, and female spotted hyenas lack an external vaginal orifice and develop a pseudoscrotum and a large pseudopenis through which they urinate, mate, and give birth. We review current knowledge about several significant aspects of the biology and evolution of these females, including (a) their specific study methods; (b) their unique anatomical features, and how these peculiarities influence certain physiological functions; and (c) the role that steroid hormones as well as genetic and environmental factors may have in urogenital system development, aggressive behavior, and social dominance. Nevertheless, both mole and hyena females are exceptionally efficient mothers, so their peculiar genitalia should not call into question their femininity.

Sox9 Is Required for Nail-Bed Differentiation and Digit-Tip Regeneration.

The nail organ is a specialized appendage in which several ectodermal tissues coordinately function to sustain nail growth, a process that is coupled to digit regeneration. In this study, we show that the transcription factor Sox9 is expressed in several cell populations in the mouse digit tip. We found a SOX9+ cell population in the nail bed, and genetic lineage tracing showed that this is a transient cell population differentiated from matrix nail stem cells. In the absence of Sox9, nail matrix stem cells fail to differentiate into epithelial nail-bed cells and proliferate, thus expanding distally and following the corneocyte fate, which results in outlandishly large fingernails. In addition, the tip of the underlying terminal phalanx undergoes bone regression. Sox9-lineage tracing also revealed the existence of a continuous cell supply from a Sox9-expressing population residing in the basal layers to the entire hyponychium epidermis. Furthermore, digit-tip regeneration is compromised in Sox9-knockout mice, revealing an essential role for the gene during this process. These results will contribute to understand the cellular and molecular basis of mammalian nail organ homeostasis and disease and digit-tip regeneration and will help to design new treatment strategies for patients with nail diseases or amputation.

Non-Coding RNAs: lncRNAs, miRNAs, and piRNAs in Sexual Development.

Non-coding RNAs (ncRNAs) are a group of RNAs that do not encode functional proteins, including long non-coding RNAs (lncRNAs), microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and short interfering RNAs (siRNAs). In the last 2 decades an effort has been made to uncover the role of ncRNAs during development and disease, and nowadays it is clear that these molecules have a regulatory function in many of the developmental and physiological processes where they have been studied. In this review, we provide an overview of the role of ncRNAs during gonad determination and development, focusing mainly on mammals, although we also provide information from other species, in particular when there is not much information on the function of particular types of ncRNAs during mammalian sexual development.

Sex Maintenance in Mammals.

The crucial event in mammalian sexual differentiation occurs at the embryonic stage of sex determination, when the bipotential gonads differentiate as either testes or ovaries, according to the sex chromosome constitution of the embryo, XY or XX, respectively. Once differentiated, testes produce sexual hormones that induce the subsequent differentiation of the male reproductive tract. On the other hand, the lack of masculinizing hormones in XX embryos permits the formation of the female reproductive tract. It was long assumed that once the gonad is differentiated, this developmental decision is irreversible. However, several findings in the last decade have shown that this is not the case and that a continuous sex maintenance is needed. Deletion of Foxl2 in the adult ovary lead to ovary-to-testis transdifferentiation and deletion of either Dmrt1 or Sox9/Sox8 in the adult testis induces the opposite process. In both cases, mutant gonads were genetically reprogrammed, showing that both the male program in ovaries and the female program in testes must be actively repressed throughout the individual's life. In addition to these transcription factors, other genes and molecular pathways have also been shown to be involved in this antagonism. The aim of this review is to provide an overview of the genetic basis of sex maintenance once the gonad is already differentiated.

Mediterranean Pine Vole, Microtus duodecimcostatus: A Paradigm of an Opportunistic Breeder.

Most mammalian species of the temperate zones of the Earth reproduce seasonally, existing a non-breeding period in which the gonads of both sexes undergo functional regression. It is widely accepted that photoperiod is the principal environmental cue controlling these seasonal changes, although several exceptions have been described in other mammalian species in which breeding depends on cues such as food or water availability. We studied the circannual reproductive cycle in males of the Mediterranean pine vole, Microtus duodecimcostatus, in the Southeastern Iberian Peninsula. Morphological, hormonal, functional, molecular and transcriptomic analyses were performed. As reported for populations of other species from the same geographic area, male voles captured in wastelands underwent seasonal testis regression in summer whereas, surprisingly, those living either in close poplar plantations or in our animal house reproduced throughout the year, showing that it is the microenvironment of a particular vole subpopulation what determines its reproductive status and that these animals are pure opportunistic, photoperiod-independent breeders. In addition, we show that several molecular pathways, including MAPK, are deregulated and that the testicular "immune privilege" is lost in the inactive testes, providing novel mechanisms linking seasonal testosterone reduction and testis regression.

Divergent Seasonal Reproductive Patterns in Syntopic Populations of Two Murine Species in Southern Spain, Mus spretus and Apodemus sylvaticus.

In most mammals with seasonal reproduction, males undergo testis regression during the non-breeding period. We performed a morphological, hormonal, functional, and molecular study of the testes of sexually inactive males of two species of murine rodents, the wood mouse, Apodemus sylvaticus, and the Algerian mouse, Mus spretus, in syntopic populations of southern Iberian peninsula. Both species reproduce during most of the year, but wood mice stop breeding in the summer whereas Algerian mice do it in winter. Sexually inactive males of A. sylvaticus show complete testis regression with reduced levels of serum testosterone and abnormal distribution of cell-adhesion molecules. Contrarily, inactive males of M. spretus maintain almost normal spermotogenesis despite a significant reduction of androgenic function. The lack of an evident explanation for the divergent seasonal breeding patterns found in southern populations of A. sylvaticus and M. spretus, compared with northern ones, implies that very subtle species/population-specific features and/or non-conspicuous environmental cues probably operate to determine their seasonal breeding pattern. These results also support the notion that multiple models of circannual testis variation are possible for different populations of the same species, showing that the mechanisms controlling seasonal reproduction are in fact very plastic and fast evolving.

The mole genome reveals regulatory rearrangements associated with adaptive intersexuality.

Linking genomic variation to phenotypical traits remains a major challenge in evolutionary genetics. In this study, we use phylogenomic strategies to investigate a distinctive trait among mammals: the development of masculinizing ovotestes in female moles. By combining a chromosome-scale genome assembly of the Iberian mole, Talpa occidentalis, with transcriptomic, epigenetic, and chromatin interaction datasets, we identify rearrangements altering the regulatory landscape of genes with distinct gonadal expression patterns. These include a tandem triplication involving CYP17A1, a gene controlling androgen synthesis, and an intrachromosomal inversion involving the pro-testicular growth factor gene FGF9, which is heterochronically expressed in mole ovotestes. Transgenic mice with a knock-in mole CYP17A1 enhancer or overexpressing FGF9 showed phenotypes recapitulating mole sexual features. Our results highlight how integrative genomic approaches can reveal the phenotypic impact of noncoding sequence changes.

Intronic variation of the SOHLH2 gene confers risk to male reproductive impairment.

OBJECTIVE: To evaluate whether SOHLH2 intronic variation contributes to the genetic predisposition to male infertility traits, including severe oligospermia (SO) and different nonobstructive azoospermia (NOA) clinical phenotypes. DESIGN: Genetic association study. SETTING: Not applicable. PATIENT(S): Five hundred five cases (455 infertile patients diagnosed with NOA and 50 with SO) and 1,050 healthy controls from Spain and Portugal. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Genomic DNA extraction from peripheral blood mononuclear cells, genotyping of the SOHLH2 polymorphisms rs1328626 and rs6563386 using the TaqMan allelic discrimination technology, case-control association analyses using logistic regression models, and exploration of functional annotations in publicly available databases. RESULT(S): Evidence of association was observed for both rs6563386 with SO and rs1328626 with unsuccessful sperm retrieval after testicular sperm extraction (TESE-) in the context of NOA. A dominant effect of the minor alleles was suggested in both associations, either when the subset of patients with the manifestation were compared against the control group (rs6563386/SO: P=.021, odds ratio [OR] = 0.51; rs1328626/TESE-: P=.066, OR = 1.46) or against the group of patients without the manifestation (rs6563386/SO: P=.014, OR = 0.46; rs1328626/TESE-: P=.012, OR = 2.43). The haplotype tests suggested a combined effect of both polymorphisms. In silico analyses evidenced that this effect could be due to alteration of the isoform population. CONCLUSION(S): Our data suggest that intronic variation of SOHLH2 is associated with spermatogenic failure. The genetic effect is likely caused by different haplotypes of rs6563386 and rs1328626, which may predispose to SO or TESE- depending on the specific allelic combination.

Deficiency of the onco-miRNA cluster, miR-106b∼25, causes oligozoospermia and the cooperative action of miR-106b∼25 and miR-17∼92 is required to maintain male fertility.

The identification of new genes involved in sexual development and gonadal function as potential candidates causing male infertility is important for both diagnostic and therapeutic purposes. Deficiency of the onco-miRNA cluster miR-17∼92 has been shown to disrupt spermatogenesis, whereas mutations in its paralog cluster, miR-106b∼25, that is expressed in the same cells, were reported to have no effect on testis development and function. The aim of this work is to determine the role of these two miRNA clusters in spermatogenesis and male fertility. For this, we analyzed miR-106b∼25 and miR-17∼92 single and double mouse mutants and compared them to control mice. We found that miR-106b∼25 knock out testes show reduced size, oligozoospermia and altered spermatogenesis. Transcriptomic analysis showed that multiple molecular pathways are deregulated in these mutant testes. Nevertheless, mutant males conserved normal fertility even when early spermatogenesis and other functions were disrupted. In contrast, miR-17∼92+/-; miR-106b∼25-/- double mutants showed severely disrupted testicular histology and significantly reduced fertility. Our results indicate that miR-106b∼25 and miR-17∼92 ensure accurate gene expression levels in the adult testis, keeping them within the required thresholds. They play a crucial role in testis homeostasis and are required to maintain male fertility. Hence, we have identified new candidate genetic factors to be screened in the molecular diagnosis of human males with reproductive disorders. Finally, considering the well-known oncogenic nature of these two clusters and the fact that patients with reduced fertility are more prone to testicular cancer, our results might also help to elucidate the molecular mechanisms linking both pathologies.

Evaluation of Male Fertility-Associated Loci in a European Population of Patients with Severe Spermatogenic Impairment.

Infertility is a growing concern in developed societies. Two extreme phenotypes of male infertility are non-obstructive azoospermia (NOA) and severe oligospermia (SO), which are characterized by severe spermatogenic failure (SpF). We designed a genetic association study comprising 725 Iberian infertile men as a consequence of SpF and 1058 unaffected controls to evaluate whether five single-nucleotide polymorphisms (SNPs), previously associated with reduced fertility in Hutterites, are also involved in the genetic susceptibility to idiopathic SpF and specific clinical entities. A significant difference in the allele frequencies of USP8-rs7174015 was observed under the recessive model between the NOA group and both the control group (p = 0.0226, OR = 1.33) and the SO group (p = 0.0048, OR = 1.78). Other genetic associations for EPSTI1-rs12870438 and PSAT1-rs7867029 with SO and between TUSC1-rs10966811 and testicular sperm extraction (TESE) success in the context of NOA were observed. In silico analysis of functional annotations demonstrated cis-eQTL effects of such SNPs likely due to the modification of binding motif sites for relevant transcription factors of the spermatogenic process. The findings reported here shed light on the molecular mechanisms leading to severe phenotypes of idiopathic male infertility, and may help to better understand the contribution of the common genetic variation to the development of these conditions.

Germ cell desquamation-based testis regression in a seasonal breeder, the Egyptian long-eared hedgehog, Hemiechinus auritus.

Testes of seasonally breeding species experience a severe functional regression before the non-breeding period, which implies a substantial mass reduction due to massive germ-cell depletion. Two alternative mechanisms of seasonal germ-cell depletion have been described in mammals, apoptosis and desquamation (sloughing), but their prevalence has not been determined yet due to reduced number of species studied. We performed a morphological, hormonal, and molecular study of the mechanism of seasonal testicular regression in males of the Egyptian long eared-hedgehog (Hemiechinus auritus). Our results show that live, non-apoptotic, germ cells are massively depleted by desquamation during the testis regression process. This is concomitant with both decreased levels of serum testosterone and irregular distribution of the cell-adhesion molecules in the seminiferous epithelium. The inactive testes maintain some meiotic activity as meiosis onset is not halted and spermatocytes die by apoptosis at the pachytene stage. Our data support the notion that apoptosis is not the major testis regression effector in mammals. Instead, desquamation appears to be a common mechanism in this class.

Sertoli cell-specific ablation of miR-17-92 cluster significantly alters whole testis transcriptome without apparent phenotypic effects.

MicroRNAs are frequently organized into polycistronic clusters whose transcription is controlled by a single promoter. The miR-17-92 cluster is expressed in most embryonic and postnatal organs. It is a potent oncogene associated to several types of cancer and it is involved in several important developmental processes. In the testis, expression of the miR-17-92 cluster in the germ cells is necessary to maintain normal spermatogenesis. This cluster is also expressed in Sertoli cells (the somatic cells of the seminiferous tubules), which require miRNAs for correct cell development and survival. To study the possible role of miR-17-92 in Sertoli cell development and function and, in order to overcome the postnatal lethality of miR-17-92-/ mice, we conditionally deleted it in embryonic Sertoli cells shortly after the sex determination stage using an Amh-Cre allele. Mutant mice developed apparently normal testes and were fertile, but their testis transcriptomes contained hundreds of moderately deregulated genes, indicating that testis homeostasis is tightly controlled in mammals and that miR-17-92 expression in Sertoli cells contribute to maintain normal gene expression levels, but is unnecessary for testis development and function. Our results show that significant deregulation of hundreds of genes might have no functional consequences.

Sox9 and Sox8 protect the adult testis from male-to-female genetic reprogramming and complete degeneration.

The new concept of mammalian sex maintenance establishes that particular key genes must remain active in the differentiated gonads to avoid genetic sex reprogramming, as described in adult ovaries after Foxl2 ablation. Dmrt1 plays a similar role in postnatal testes, but the mechanism of adult testis maintenance remains mostly unknown. Sox9 and Sox8 are required for postnatal male fertility, but their role in the adult testis has not been investigated. Here we show that after ablation of Sox9 in Sertoli cells of adult, fertile Sox8(-/-) mice, testis-to-ovary genetic reprogramming occurs and Sertoli cells transdifferentiate into granulosa-like cells. The process of testis regression culminates in complete degeneration of the seminiferous tubules, which become acellular, empty spaces among the extant Leydig cells. DMRT1 protein only remains in non-mutant cells, showing that SOX9/8 maintain Dmrt1 expression in the adult testis. Also, Sox9/8 warrant testis integrity by controlling the expression of structural proteins and protecting Sertoli cells from early apoptosis. Concluding, this study shows that, in addition to its crucial role in testis development, Sox9, together with Sox8 and coordinately with Dmrt1, also controls adult testis maintenance.

Circannual Testis Changes in Seasonally Breeding Mammals.

In the non-equatorial zones of the Earth, species concentrate their reproductive effort in the more favorable season. A consequence of seasonal breeding is seasonal testis regression, which implies the depletion of the germinative epithelium, permeation of the blood-testis barrier, and reduced androgenic function. This process has been studied in a number of vertebrates, but the mechanisms controlling it are not yet well understood. Apoptosis was assumed for years to be an important effector of seasonal germ cell depletion in all vertebrates, including mammals, but an alternative mechanism has recently been reported in the Iberian mole as well as in the large hairy armadillo. It is based on the desquamation of meiotic and post-meiotic germ cells as a consequence of altered Sertoli-germ cell adhesion molecule expression and distribution. Desquamated cells are either discarded alive through the epididymis, as in the mole, or subsequently die by apoptosis, as in the armadillo. Also, recent findings on the reproductive cycle of the greater white-toothed shrew at the meridional limits of its distribution area have revealed that the mechanisms controlling seasonal breeding are in fact far more plastic and versatile than initially suspected. Perhaps these higher adaptive capacities place mammals in a better position to face the ongoing climate change.

The testis of greater white-toothed shrew Crocidura russula in Southern European populations: a case of adaptive lack of seasonal involution?

Males of all seasonal breeding mammals undergo circannual periods of testis involution resulting in almost complete ablation of the germinative epithelium. We performed a morphometric, histological, hormonal, and gene-expression study of the testes from winter and summer males of the greater white-toothed shrew, Crocidura russula, in populations of the southeastern Iberian Peninsula. Unexpectedly, we found no significant differences between the two study groups. Surprisingly, female data confirmed a non-breeding period in the summer, evidencing that males retain full testis function even when most females are not receptive. This situation, which has not been described before, does not occur in northern populations of the same species where, in addition, the reproductive cycle is inverted with respect to those in the south, as the non-breeding period occurs in winter instead in summer. Considering that the non-reproductive period shortens at lower latitude locations, we hypothesize that in southern populations the non-breeding period is short enough to make testis regression inefficient in terms of energy savings, because: (1) testes of C. russula are very small, a condition derived from their monogamy that implies low investment in spermatogenesis; and (2) the spermatogenic cycle of this species is slow and long. The inverted seasonal breeding cycle and the lack of seasonal testis regression described here are new adaptive processes that deserve further research, and provide evidence that the genetic and hormonal mechanisms controlling reproduction timing in mammals are more plastic and versatile than initially suspected.

Identification of live germ-cell desquamation as a major mechanism of seasonal testis regression in mammals: a study in the Iberian mole (Talpa occidentalis).

In males of seasonally breeding species, testes undergo a severe involution at the end of the breeding season, with a major volume decrease due to massive germ-cell depletion associated with photoperiod-dependent reduced levels of testosterone and gonadotropins. Although it has been repeatedly suggested that apoptosis is the principal effector of testicular regression in vertebrates, recent studies do not support this hypothesis in some mammals. The purpose of our work is to discover alternative mechanisms of testis regression in these species. In this paper, we have performed a morphological, hormonal, ultrastructural, molecular, and functional study of the mechanism of testicular regression and the role that cell junctions play in the cell-content dynamics of the testis of the Iberian mole, Talpa occidentalis, throughout the seasonal breeding cycle. Desquamation of live, nonapoptotic germ cells has been identified here as a new mechanism for seasonal testis involution in mammals, indicating that testis regression is regulated by modulating the expression and distribution of the cell-adhesion molecules in the seminiferous epithelium. During this process, which is mediated by low intratesticular testosterone levels, Sertoli cells lose their nursing and supporting function, as well as the impermeability of the blood-testis barrier. Our results contradict the current paradigm that apoptosis is the major testis regression effector in vertebrates, as it is clearly not true in all mammals. The new testis regression mechanism described here for the mole could then be generalized to other mammalian species. Available data from some previously studied mammals should be reevaluated.

Genes promoting and disturbing testis development.

Mammals have an XX/XY sex chromosomal sex determination system in which males represent the heterogametic sex. The Y-linked gene, SRY, determines sex by inducing the undifferentiated, bipotential gonads to differentiate as testes, which produce androgens and promote in this way the development of a male phenotype. Thus, in mammals, sex determination can be equated to testis determination, which involves several important cell processes, including Sertoli cell differentiation, mesonephric cell migration, testis cord formation, testis-specific vascularization, and myoid and Leydig cell differentiation. Many genes are currently known to be involved in testis development. Some of them, including SF1, WT1, GATA4 and FOG2, are necessary for the formation of the bipotential, undifferentiated gonad but also have important roles in testis differentiation. Others can be considered testis-promoting, differentaition and/or maintenance genes: these include SRY, SOX9, FGF9, PTGDS, SOX8, SOX3, NR0B1, PDGFRa, DMRT1, AMH, NGF, NTF3 and NGFR as the most important examples. Finally, there is a smaller group of genes which are involved in ovarian development and which can cause aberrant testis development if mutated, including RSPO1, WNT4, CTNNB1, FST, BMP2 and FOXL2. In this paper, we review our current knowledge on the function, spatio-temporal expression pattern and mutant sexual phenotypes associated with these genes, and discuss the various roles they play in gonad development.

Pattern and density of vascularization in mammalian testes, ovaries, and ovotestes.

According to the classical paradigm, the vasculature of the embryonic testis is more dense and complex than that of the ovary, but recent studies based on whole-mount detection of Caveolin-1 (CAV1) as an endothelial cell marker, have suggested that the level of ovarian vascularization is higher than previously assumed. However, this new hypothesis has been neither tested using alternative methodology nor investigated in other mammalian species. In this paper, we have studied the vascularization process in the gonads of males and females of two mammalian species, the mouse (Mus musculus) and the Iberian mole (Talpa occidentalis). Our results show that the pattern of testis vascularization is very well conserved among mammals, including both pre- and postnatal stages of development and, at least in the mole, it is conserved irrespectively of whether the testicular tissue is XY or XX. We have shown that CAV1 is present not only in endothelial cells but also in prefollicular oocytes and in an ovarian population of somatic cortical cells. These data clearly establish that: (1) according to the classical hypothesis, the degree of vascularization of the developing ovary is lower than that of the testis, (2) ovarian vascularization is also evolutionarily conserved as it occurs similarly both in moles and in mice, and (3) that the degree of vascular development of the mammalian ovary is age-dependent increasing significatively at puberty. The expression of CAV1 in the ovary of most animal taxa, from nematodes to mammals, strongly suggests a role for this gene in the female meiosis.