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.

Induction of kidney-related gene programs through co-option of SALL1 in mole ovotestes.

Changes in gene expression represent an important source of phenotypic innovation. Yet how such changes emerge and impact the evolution of traits remains elusive. Here, we explore the molecular mechanisms associated with the development of masculinizing ovotestes in female moles. By performing integrative analyses of epigenetic and transcriptional data in mole and mouse, we identified the co-option of SALL1 expression in mole ovotestes formation. Chromosome conformation capture analyses highlight a striking conservation of the 3D organization at the SALL1 locus, but an evolutionary divergence of enhancer activity. Interspecies reporter assays support the capability of mole-specific enhancers to activate transcription in urogenital tissues. Through overexpression experiments in transgenic mice, we further demonstrate the capability of SALL1 to induce kidney-related gene programs, which are a signature of mole ovotestes. Our results highlight the co-option of gene expression, through changes in enhancer activity, as a plausible mechanism for the evolution of traits.

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.

In vivo dissection of a clustered-CTCF domain boundary reveals developmental principles of regulatory insulation.

Vertebrate genomes organize into topologically associating domains, delimited by boundaries that insulate regulatory elements from nontarget genes. However, how boundary function is established is not well understood. Here, we combine genome-wide analyses and transgenic mouse assays to dissect the regulatory logic of clustered-CCCTC-binding factor (CTCF) boundaries in vivo, interrogating their function at multiple levels: chromatin interactions, transcription and phenotypes. Individual CTCF binding site (CBS) deletions revealed that the characteristics of specific sites can outweigh other factors such as CBS number and orientation. Combined deletions demonstrated that CBSs cooperate redundantly and provide boundary robustness. We show that divergent CBS signatures are not strictly required for effective insulation and that chromatin loops formed by nonconvergently oriented sites could be mediated by a loop interference mechanism. Further, we observe that insulation strength constitutes a quantitative modulator of gene expression and phenotypes. Our results highlight the modular nature of boundaries and their control over developmental processes.

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.

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.

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.

A microRNA (mmu-miR-124) prevents Sox9 expression in developing mouse ovarian cells.

In mammals, sex differentiation depends on gonad development, which is controlled by two groups of sex-determining genes that promote one gonadal sex and antagonize the opposite one. SOX9 plays a key role during testis development in all studied vertebrates, whereas it is kept inactive in the XX gonad at the critical time of sex determination, otherwise, ovary-to-testis gonadal sex reversal occurs. However, molecular mechanisms underlying repression of Sox9 at the beginning of ovarian development, as well as other important aspects of gonad organogenesis, remain largely unknown. Because there is indirect evidence that micro-RNAs (miRNA) are necessary for testicular function, the possible involvement of miRNAs in mammalian sex determination deserved further research. Using microarray technology, we have identified 22 miRNAs showing sex-specific expression in the developing gonads during the critical period of sex determination. Bioinformatics analyses led to the identification of miR-124 as the candidate gene for ovarian development. We knocked down or overexpressed miR-124 in primary gonadal cell cultures and observed that miR-124 is sufficient to induce the repression of both SOX9 translation and transcription in ovarian cells. Our results provide the first evidence of the involvement of a miRNA in the regulation of the gene controlling gonad development and sex determination. The miRNA microarray data reported here will help promote further research in this field, to unravel the role of other miRNAs in the genetic control of mammalian sex determination.

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.

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.

Role of apoptosis and cell proliferation in the testicular dynamics of seasonal breeding mammals: a study in the Iberian mole, Talpa occidentalis.

Apoptosis and cell proliferation are two important cellular processes known to be involved in the normal functioning of the testis in nonseasonally breeding mammals, but there is some controversy concerning their roles in the gonads of males from seasonally breeding species. We have studied the processes of apoptosis and cell proliferation in the testes of males of the Iberian mole (Talpa occidentalis), a species showing a strict seasonal reproduction pattern. Both males and females are sexually active during the winter and completely inactive in the summer, with two transitional periods, in the autumn and the spring. Adult males from these four reproductive stages were captured, and their testes were immunohistochemically studied for the presence of apoptotic and proliferation molecular markers as well for other testicular and meiotic cell-specific markers. We found that apoptosis varies in a season-dependent manner in the testes of male moles, affecting mainly late zygotene and pachytene cells during the period of sexual inactivity, but it does not differentially affect the number of Sertoli cells. More interestingly, apoptosis is not responsible for the massive germ-cell depletion occurring during mole testis regression. In addition, a wave of spermatogonial cell proliferation appears to restore the number of spermatogonia lost during the period of testis inactivity. According to current knowledge, data from moles indicate that mammals do not form a homogeneous group regarding the mechanisms by which the cell-content dynamics are regulated in the testes of males from seasonally breeding species.

SOX9 is not required for the cellular events of testicular organogenesis in XX mole ovotestes.

Mammalian sex determination is the genetic process that commits the undifferentiated bipotential gonads to develop as either testes or ovaries. The differentiation of SOX9-expressing Sertoli cells is assumed to be necessary to initiate testis development. Insectivorous moles of the genus Talpa represent a unique case of generalized true hermaphroditism, as XX female moles constitutively develop two ovotestes instead of normal ovaries. In this work, we have investigated the expression patterns of a number of genes known to play key roles in gonad organogenesis, throughout the entire process of ovotestis development in female moles. Molecular and morphological evidence are provided that these ovotestes contain primary medullary testis-like cords, Leydig cells, peritubular myoid cells, and a testis-specific vasculature, but no Sertoli cells. Our results show for the first time that SOX9 is not required for the formation of the primary testis cords, but it is necessary for the maintenance and subsequent development of these cords. In addition, the expression pattern of WNT4 in male and female moles indicates that this gene inhibits Leydig cell differentiation and, contrary to the proposed scenario in the mouse, it is not required for the colonization and survival of primordial germ cells. According to our data, mole ovotestes result from a process of PDGFRalpha-mediated mesonephric cell migration, which occurs simultaneously in both sexes. The fact that FST remains inactive during the critical stages of female gonad development, explains the lack of migration inhibition, and may be a consequence of improper WNT4 signalling in the mole.