A novel evolutionary conserved mechanism of RNA stability regulates synexpression of primordial germ cell-specific genes prior to the sex-determination stage in medaka.

Dmrt1 is a highly conserved transcription factor, which is critically involved in regulation of gonad development of vertebrates. In medaka, a duplicate of dmrt1-acting as master sex-determining gene-has a tightly timely and spatially controlled gonadal expression pattern. In addition to transcriptional regulation, a sequence motif in the 3' UTR (D3U-box) mediates transcript stability of dmrt1 mRNAs from medaka and other vertebrates. We show here that in medaka, two RNA-binding proteins with antagonizing properties target this D3U-box, promoting either RNA stabilization in germ cells or degradation in the soma. The D3U-box is also conserved in other germ-cell transcripts, making them responsive to the same RNA binding proteins. The evolutionary conservation of the D3U-box motif within dmrt1 genes of metazoans-together with preserved expression patterns of the targeting RNA binding proteins in subsets of germ cells-suggest that this new mechanism for controlling RNA stability is not restricted to fishes but might also apply to other vertebrates.

Dual role of DMXL2 in olfactory information transmission and the first wave of spermatogenesis.

Gonad differentiation is a crucial step conditioning the future fertility of individuals and most of the master genes involved in this process have been investigated in detail. However, transcriptomic analyses of developing gonads from different animal models have revealed that hundreds of genes present sexually dimorphic expression patterns. DMXL2 was one of these genes and its function in mammalian gonads was unknown. We therefore investigated the phenotypes of total and gonad-specific Dmxl2 knockout mouse lines. The total loss-of-function of Dmxl2 was lethal in neonates, with death occurring within 12 hours of birth. Dmxl2-knockout neonates were weak and did not feed. They also presented defects of olfactory information transmission and severe hypoglycemia, suggesting that their premature death might be due to global neuronal and/or metabolic deficiencies. Dmxl2 expression in the gonads increased after birth, during follicle formation in females and spermatogenesis in males. DMXL2 was detected in both the supporting and germinal cells of both sexes. As Dmxl2 loss-of-function was lethal, only limited investigations of the gonads of Dmxl2 KO pups were possible. They revealed no major defects at birth. The gonadal function of Dmxl2 was then assessed by conditional deletions of the gene in gonadal supporting cells, germinal cells, or both. Conditional Dmxl2 ablation in the gonads did not impair fertility in males or females. By contrast, male mice with Dmxl2 deletions, either throughout the testes or exclusively in germ cells, presented a subtle testicular phenotype during the first wave of spermatogenesis that was clearly detectable at puberty. Indeed, Dmxl2 loss-of-function throughout the testes or in germ cells only, led to sperm counts more than 60% lower than normal and defective seminiferous tubule architecture. Transcriptomic and immunohistochemichal analyses on these abnormal testes revealed a deregulation of Sertoli cell phagocytic activity related to germ cell apoptosis augmentation. In conclusion, we show that Dmxl2 exerts its principal function in the testes at the onset of puberty, although its absence does not compromise male fertility in mice.

The unusual rainbow trout sex determination gene hijacked the canonical vertebrate gonadal differentiation pathway.

Evolutionary novelties require rewiring of transcriptional networks and/or the evolution of new gene functions. Sex determination (SD), one of the most plastic evolutionary processes, requires such novelties. Studies on the evolution of vertebrate SD revealed that new master SD genes are generally recruited from genes involved in the downstream SD regulatory genetic network. Only a single exception to this rule is currently known in vertebrates: the intriguing case of the salmonid master SD gene (sdY), which arose from duplication of an immune-related gene. This exception immediately posed the question of how a gene outside from the classical sex differentiation cascade could acquire its function as a male SD gene. Here we show that SdY became integrated in the classical vertebrate sex differentiation cascade by interacting with the Forkhead box domain of the female-determining transcription factor, Foxl2. In the presence of Foxl2, SdY is translocated to the nucleus where the SdY:Foxl2 complex prevents activation of the aromatase (cyp19a1a) promoter in cooperation with Nr5a1 (Sf1). Hence, by blocking a positive loop of regulation needed for the synthesis of estrogens in the early differentiating gonad, SdY disrupts a preset female differentiation pathway, consequently allowing testicular differentiation to proceed. These results also suggest that the evolution of unusual vertebrate master sex determination genes recruited from outside the classical pathway like sdY is strongly constrained by their ability to interact with the canonical gonadal differentiation pathway.

FOXL2 is a Progesterone Target Gene in the Endometrium of Ruminants.

Forkhead Box L2 (FOXL2) is a member of the FOXL class of transcription factors, which are essential for ovarian differentiation and function. In the endometrium, FOXL2 is also thought to be important in cattle; however, it is not clear how its expression is regulated. The maternal recognition of pregnancy signal in cattle, interferon-Tau, does not regulate FOXL2 expression. Therefore, in the present study, we examined whether the ovarian steroid hormones that orchestrate implantation regulate FOXL2 gene expression in ruminants. In sheep, we confirmed that FOXL2 mRNA and protein was expressed in the endometrium across the oestrous cycle (day 4 to day 15 post-oestrus). Similar to the bovine endometrium, ovine FOXL2 endometrial expression was low during the luteal phase of the oestrous cycle (4 to 12 days post-oestrus) and at implantation (15 days post-oestrus) while mRNA and protein expression significantly increased during the luteolytic phase (day 15 post-oestrus in cycle). In pregnant ewes, inhibition of progesterone production by trilostane during the day 5 to 16 period prevented the rise in progesterone concentrations and led to a significant increase of FOXL2 expression in caruncles compared with the control group (1.4-fold, p < 0.05). Ovariectomized ewes or cows that were supplemented with exogenous progesterone for 12 days or 6 days, respectively, had lower endometrial FOXL2 expression compared with control ovariectomized females (sheep, mRNA, 1.8-fold; protein, 2.4-fold; cattle; mRNA, 2.2-fold; p < 0.05). Exogenous oestradiol treatments for 12 days in sheep or 2 days in cattle did not affect FOXL2 endometrial expression compared with control ovariectomized females, except at the protein level in both endometrial areas in the sheep. Moreover, treating bovine endometrial explants with exogenous progesterone for 48h reduced FOXL2 expression. Using in vitro assays with COS7 cells we also demonstrated that progesterone regulates the FOXL2 promoter activity through the progesterone receptor. Collectively, our findings imply that endometrial FOXL2 is, as a direct target of progesterone, involved in early pregnancy and implantation.

An Assessment of Fixed and Native Chromatin Preparation Methods to Study Histone Post-Translational Modifications at a Whole Genome Scale in Skeletal Muscle Tissue.

BACKGROUND: Genomic loci associated with histone marks are typically analyzed by immunoprecipitation of the chromatin followed by quantitative-PCR (ChIP-qPCR) or high throughput sequencing (ChIP-seq). Chromatin can be either cross-linked (X-ChIP) or used in the native state (N-ChIP). Cross-linking of DNA and proteins helps stabilizing their interactions before analysis. Despite X-ChIP is the most commonly used method, muscle tissue fixation is known to be relatively inefficient. Moreover, no protocol described a simple and reliable preparation of skeletal muscle chromatin of sufficient quality for subsequent high-throughput sequencing. Here we aimed to set-up and compare both chromatin preparation methods for a genome-wide analysis of H3K27me3, a broad-peak histone mark, using chicken P. major muscle tissue. RESULTS: Fixed and unfixed chromatin were prepared from chicken muscle tissues (Pectoralis major). Chromatin fixation, shearing by sonication or digestion and immunoprecipitation performed equivalently. High-quality Illumina reads were obtained (q30 > 93%). The bioinformatic analysis of the data was performed using epic, a tool based on SICER, and MACS2. Forty millions of reads were analyzed for both X-ChIP-seq and N-ChIP-seq experiments. Surprisingly, H3K27me3 X-ChIP-seq analysis led to the identification of only 2000 enriched regions compared to about 15,000 regions identified in the case of N-ChIP-seq. N-ChIP-seq peaks were more consistent between replicates compared to X-ChIP-seq. Higher N-ChIP-seq enrichments were confirmed by ChIP-qPCR at the PAX5 and SOX2 loci known to be enriched for H3K27me3 in myotubes and at the loci of common regions of enrichment identified in this study. CONCLUSIONS: Our findings suggest that the preparation of muscle chromatin for ChIP-seq in cross-linked conditions can compromise the systematic analysis of broad histone marks. Therefore, native chromatin preparation should be preferred to cross-linking when a ChIP experiment has to be performed on skeletal muscle tissue, particularly when a broad source signal is considered.

High-throughput sequencing analyses of XX genital ridges lacking FOXL2 reveal DMRT1 up-regulation before SOX9 expression during the sex-reversal process in goats.

FOXL2 loss of function in goats leads to the early transdifferentiation of ovaries into testes, then to the full sex reversal of XX homozygous mutants. By contrast, Foxl2 loss of function in mice induces an arrest of follicle formation after birth, followed by complete female sterility. In order to understand the molecular role of FOXL2 during ovarian differentiation in the goat species, putative FOXL2 target genes were determined at the earliest stage of gonadal sex-specific differentiation by comparing the mRNA profiles of XX gonads expressing the FOXL2 protein or not. Of these 163 deregulated genes, around two-thirds corresponded to testicular genes that were up-regulated when FOXL2 was absent, and only 19 represented female-associated genes, down-regulated in the absence of FOXL2. FOXL2 should therefore be viewed as an antitestis gene rather than as a female-promoting gene. In particular, the key testis-determining gene DMRT1 was found to be up-regulated ahead of SOX9, thus suggesting in goats that SOX9 primary up-regulation may require DMRT1. Overall, our results equated to FOXL2 being an antitestis gene, allowing us to propose an alternative model for the sex-determination process in goats that differs slightly from that demonstrated in mice.

DMRT1 is a testis-determining gene in rabbits and is also essential for female fertility.

DMRT1 is the testis-determining factor in several species of vertebrates, but its involvement in mammalian testes differentiation, where SRY is the testis-determining gene, remains ambiguous. So far, DMRT1 loss-of-function has been described in two mammalian species and induces different phenotypes: Disorders of Sex Development (46, XY DSD) in men and male infertility in mice. We thus abolished DMRT1 expression by CRISPR/Cas9 in a third species of mammal, the rabbit. First, we observed that gonads from XY DMRT1-/- rabbit fetuses differentiated like ovaries, highlighting that DMRT1 is involved in testis determination. In addition to SRY, DMRT1 is required in the supporting cells to increase the expression of the SOX9 gene, which heads the testicular genetic cascade. Second, we highlighted another function of DMRT1 in the germline since XX and XY DMRT1-/- ovaries did not undergo meiosis and folliculogenesis. XX DMRT1-/- adult females were sterile, showing that DMRT1 is also crucial for female fertility. To conclude, these phenotypes indicate an evolutionary continuum between non-mammalian vertebrates such as birds and non-rodent mammals. Furthermore, our data support the potential involvement of DMRT1 mutations in different human pathologies, such as 46, XY DSD as well as male and female infertility.

Animals that reproduce sexually have organs called gonads, the ovaries and testes, which produce eggs and sperm. These organs, which are different in males and females, originate from the same cells during the development of the embryo. As a general rule, the chromosomal sex of an embryo, which gets determined at fertilization, leads to the activation and repression of specific genes. This in turn, controls whether the cells that will form the gonads will differentiate to develop testes or ovaries. Disruption of the key genes involved in the differentiation of the gonads can lead to fertility problems, and in some cases, it can cause the gonads to develop in the 'opposite' direction, resulting in a sex reversal. Identifying these genes is therefore essential to know how to maintain or restore fertility. DMRT1 is a gene that drives the differentiation of gonadal cells into the testicular pathway in several species of animals with backbones, including species of fish, frogs and birds. However, its role in mammals ­ where testis differentiation is driven by a different gene called SRY ­ is not well understood. Indeed, when DMRT1 is disrupted in male humans it leads to disorders of sex development, while disrupting this gene in male mice causes infertility. To obtain more information about the roles of DMRT1 in mammalian species, Dujardin et al. disrupted the gene in a third species of mammal: the rabbit. Dujardin et al. observed that chromosomally-male rabbits lacking DMRT1 developed ovaries instead of testes, showing that in rabbits, both SRY and DMRT1 are both required to produce testes. Additionally, this effect is similar to what is seen in humans, suggesting that rabbits may be a better model for human gonadal differentiation than mice are. Additionally, Dujardin et al. were also able to show that in female rabbits, lack of DMRT1 led to infertility, an effect that had not been previously described in other species. The results of Dujardin et al. may lead to better models for gonadal development in humans, involving DMRT1 in the differentiation of testes. Interestingly, they also suggest the possibility that mutations in this gene may be responsible for some cases of infertility in women. Overall, these findings indicate that DMRT1 is a key fertility gene.

FOXL2 is regulated during the bovine estrous cycle and its expression in the endometrium is independent of conceptus-derived interferon tau.

FOXL2, a winged-helix/forkhead domain transcription factor, is a key gene involved in the differentiation and biological functions of the ovary. In a recent transcriptomic analysis, we found that FOXL2 expression in bovine caruncular endometrium was different from that in intercaruncular endometrium. In order to gain new insights into FOXL2 in this tissue, we determined the expression of this transcription factor during the estrous cycle and the establishment of pregnancy in cattle. The endometrial expression of FOXL2 did not vary during maternal recognition of pregnancy (Days 16-20). Using an in vivo bovine model and primary cell cultures, we showed that FOXL2 was not an interferon-tau target gene. Both FOXL2 transcript and protein were expressed from Day 5 to Day 20 of the estrous cycle, and their levels showed a significant increase during the luteolytic phase. A 2-day progesterone supplementation in heifers led to a clear down-regulation of FOXL2 protein levels, suggesting the negative impact of progesterone on FOXL2 expression. Immunohistochemistry data revealed the localization of FOXL2 in endometrial stromal and glandular cells. FOXL2 subcellular distribution was shown to be nuclear in endometrial samples collected during the luteolytic period, while it was not detected in nuclei during the luteal phase and at implantation. Collectively, our findings provide the first evidence that FOXL2 is involved in the regulation of endometrial tissue physiology.

Absence of Testicular Estrogen Leads to Defects in Spermatogenesis and Increased Semen Abnormalities in Male Rabbits.

Estrogens are steroid hormones produced by the aromatization of androgens by the aromatase enzyme, encoded by the CYP19A1 gene. Although generally referred to as "female sex hormones", estrogen is also produced in the adult testes of many mammals, including humans. To better understand the function of estrogens in the male, we used the rabbit model which is an important biomedical model. First, the expression of CYP19A1 transcripts was localized mainly in meiotic germ cells. Thus, testicular estrogen appears to be produced inside the seminiferous tubules. Next, the cells expressing ESR1 and ESR2 were identified, showing that estrogens could exert their function on post-meiotic germ cells in the tubules and play a role during sperm maturation, since ESR1 and ESR2 were detected in the cauda epididymis. Then, CRISPR/Cas9 CYP19A1-/- genetically modified rabbits were analyzed. CYP19A1-/- males showed decreased fertility with lower sperm count associated with hypo-spermatogenesis and lower spermatid number. Germ/sperm cell DNA methylation was unchanged, while sperm parameters were affected as CYP19A1-/- males exhibited reduced sperm motility associated with increased flagellar defects. In conclusion, testicular estrogens could be involved in the spermatocyte-spermatid transition in the testis, and in the acquisition of sperm motility in the epididymis.

Fetal Estrogens are not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits.

AROMATASE is encoded by the CYP19A1 gene and is the cytochrome enzyme responsible for estrogen synthesis in vertebrates. In most mammals, a peak of CYP19A1 gene expression occurs in the fetal XX gonad when sexual differentiation is initiated. To elucidate the role of this peak, we produced 3 lines of TALEN genetically edited CYP19A1 knockout (KO) rabbits that were devoid of any estradiol production. All the KO XX rabbits developed as females with aberrantly small ovaries in adulthood, an almost empty reserve of primordial follicles, and very few large antrum follicles. Ovulation never occurred. Our histological, immunohistological, and transcriptomic analyses showed that the estradiol surge in the XX fetal rabbit gonad is not essential to its determination as an ovary, or for meiosis. However, it is mandatory for the high proliferation and differentiation of both somatic and germ cells, and consequently for establishment of the ovarian reserve.

[Sex differentiation: state of the art and future prospects]. / La différenciation du sexe - Acquis et perspectives.

Our knowledge on sex differentiation in mammals has considerably progressed during the last decennials, beginning with the discovery of the testis-determining factor. Here, the morphogenetic processes involved in the early gonadic switch will be presented, together with the major genes involved in testis and ovary formation. Existing differences between the widely used mouse model and other mammals, such as human and goat, will be highlighted.

A study of goat SRY protein expression suggests putative new roles for this gene in the developing testis of a species with long-lasting SRY expression.

The testis-determining gene SRY is not well-conserved among mammals, and particularly between mouse and other mammals. To evaluate SRY function in a nonrodent species, we produced an antibody against goat SRY and used it to investigate the expression pattern of SRY throughout goat testicular development. By contrast with the mouse, SRY is primarily expressed in most cells of XY genital-ridges and not solely in pre-Sertoli cells. Between cord formation and prepuberty, SRY remains expressed in both Sertoli and germinal cells. During adulthood, SRY expression declines and then disappears from meiotic germ cells, only remaining present at low levels in some spermatogonia. Unlike the germinal lineage, SRY continues to be highly expressed in adult Sertoli cells with a typical nuclear staining. Our data indicate that in goat, the role of SRY may not be limited to testis determination and could have other functions in testicular maintenance and hence male fertility.

PR-SET7 and SUV4-20H regulate H4 lysine-20 methylation at imprinting control regions in the mouse.

Imprinted genes are important in development and their allelic expression is mediated by imprinting control regions (ICRs). On their DNA-methylated allele, ICRs are marked by trimethylation at H3 Lys 9 (H3K9me3) and H4 Lys 20 (H4K20me3), similar to pericentric heterochromatin. Here, we investigate which histone methyltransferases control this methylation of histone at ICRs. We found that inactivation of SUV4-20H leads to the loss of H4K20me3 and increased levels of its substrate, H4K20me1. H4K20me1 is controlled by PR-SET7 and is detected on both parental alleles. The disruption of SUV4-20H or PR-SET7 does not affect methylation of DNA at ICRs but influences precipitation of H3K9me3, which is suggestive of a trans-histone change. Unlike at pericentric heterochromatin, however, H3K9me3 at ICRs does not depend on SUV39H. Our data show not only new similarities but also differences between ICRs and heterochromatin, both of which show constitutive maintenance of methylation of DNA in somatic cells.

Goat RSPO1 over-expression rescues sex-reversal in Rspo1-knockout XX mice but does not perturb testis differentiation in XY or sex-reversed XX mice.

RSPO1 is a newly discovered gene involved in sex differentiation. Two goat BAC clones encompassing the RSPO1 gene (gRSPO1) were injected into mouse oocytes and several transgenic lines derived. Both clones induced gRSPO1 over-expression in various tissues, including male and female gonads, with no obvious phenotype and normal sex-ratios. Introgression of the gRSPO1 transgene into a mouse RSPO1 knockout genotype resulted in the rescue of the fertility and the disappearance of the masculinized gonadic features of the females, demonstrating the functionality of the goat protein in a mouse context. On the contrary, over-expression of gRSPO1 within a mSRY or a gSRY-XX genotypes did not interfere with the SRY-induced male phenotype.

RUNX1 maintains the identity of the fetal ovary through an interplay with FOXL2.

Sex determination of the gonads begins with fate specification of gonadal supporting cells into either ovarian pre-granulosa cells or testicular Sertoli cells. This fate specification hinges on a balance of transcriptional control. Here we report that expression of the transcription factor RUNX1 is enriched in the fetal ovary in rainbow trout, turtle, mouse, goat, and human. In the mouse, RUNX1 marks the supporting cell lineage and becomes pre-granulosa cell-specific as the gonads differentiate. RUNX1 plays complementary/redundant roles with FOXL2 to maintain fetal granulosa cell identity and combined loss of RUNX1 and FOXL2 results in masculinization of fetal ovaries. At the chromatin level, RUNX1 occupancy overlaps partially with FOXL2 occupancy in the fetal ovary, suggesting that RUNX1 and FOXL2 target common sets of genes. These findings identify RUNX1, with an ovary-biased expression pattern conserved across species, as a regulator in securing the identity of ovarian-supporting cells and the ovary.

R-spondin1 and FOXL2 act into two distinct cellular types during goat ovarian differentiation.

BACKGROUND: Up to now, two loci have been involved in XX sex-reversal in mammals following loss-of-function mutations, PIS (Polled Intersex Syndrome) in goats and R-spondin1 (RSPO1) in humans. Here, we analyze the possible interaction between these two factors during goat gonad development. Furthermore, since functional redundancy between different R-spondins may influence gonad development, we also studied the expression patterns of RSPO2, 3 and 4. RESULTS: Similarly to the mouse, RSPO1 shows a sex-dimorphic expression pattern during goat gonad development with higher levels in the ovaries. Interestingly, the PIS mutation does not seem to influence its level of expression. Moreover, using an RSPO1 specific antibody, the RSPO1 protein was localized in the cortical area of early differentiating ovaries (36 and 40 dpc). This cortical area contains the majority of germ cell that are surrounded by FOXL2 negative somatic cells. At latter stages (50 and 60 dpc) RSPO1 protein remains specifically localized on the germ cell membranes. Interestingly, a time-specific relocation of RSPO1 on the germ cell membrane was noticed, moving from a uniform distribution at 40 dpc to a punctuated staining before and during meiosis (50 and 60 dpc respectively). Interestingly, also RSPO2 and RSPO4 show a sex-dimorphic expression pattern with higher levels in the ovaries. Although RSPO4 was found to be faintly and belatedly expressed, the expression of RSPO2 increases at the crucial 36 dpc stage, as does that of FOXL2. Importantly, RSPO2 expression appears dramatically decreased in XX PIS-/- gonads at all three tested stages (36, 40 and 50 dpc). CONCLUSION: During goat ovarian development, the pattern of expression of RSPO1 is in agreement with its possible anti-testis function but is not influenced by the PIS mutation. Moreover, our data suggest that RSPO1 may be associated with germ cell development and meiosis. Interestingly, another RSPO gene, RSPO2 shows a sex-dimorphic pattern of expression that is dramatically influenced by the PIS mutation.

Epigenetic stability of embryonic stem cells and developmental potential.

Recent studies highlight the tremendous potential of human embryonic stem (ES) cells and their derivatives as therapeutic tools for degenerative diseases. However, derivation and culture of ES cells can induce epigenetic alterations, which can have long lasting effects on gene expression and phenotype. Research on human and mouse stem cells indicates that developmental, cancer-related genes, and genes regulated by genomic imprinting are particularly susceptible to changes in DNA methylation. Together with the occurrence of genetic alterations, epigenetic instability needs to be monitored when considering human stem cells for therapeutic and technological purposes. Here, we discuss the maintenance of epigenetic information in cultured stem cells and embryos and how this influences their developmental potential.

Goat SRY induces testis development in XX transgenic mice.

The testis-determining gene SRY is not well-conserved among mammals, particularly between mouse and other mammals, both in terms of protein structure and of expression regulation. To evaluate SRY phylogenic conservation in regards to its function, we expressed the goat gene (gSRY) in XX transgenic mouse gonads. Here, we show that gSRY induces testis formation, despite a goat expression profile. Our results demonstrate that sex-reversal can be induced in XX-mice by a non-mouse SRY thus suggesting a conserved molecular mechanism of action of this testis-determining gene across mammalian species.

FOXL2 activates P450 aromatase gene transcription: towards a better characterization of the early steps of mammalian ovarian development.

Previous studies have equated FOXL2 as a crucial actor in the ovarian differentiation process in different vertebrate species. Its transcriptional extinction in the polled intersex syndrome (PIS) leads primarily to a drastic decrease of aromatase (CYP19) expression in the first steps of goat ovarian development. In this study, we provide a better characterization of early ovarian development in goat, and we provide experimental evidence demonstrating that FOXL2 represents a direct transcriptional activator of the CYP19 gene through its ovarian-specific promoter 2. Moreover, the ovarian location of FOXL2 and CYP19 proteins, together with their expression profiles in the female gonads, stress the involvement of FOXL2 co-factor(s) for regulating CYP19 transcription. Expressional analyses show that activin-betaA can be considered as a strong candidate for being one of these FOXL2 co-factors. Finally, we discuss evidence for a role of activin and estrogens in somatic and germinal cell proliferation occurring before germ cell meiosis. This period, of 20 days in goat, seems to have no equivalent in mouse. This species-specific difference could explain the phenotype discrepancy observed between XX goat PIS(-/-) and XX mouse Foxl2(-/-).