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.

The single-cell chromatin landscape in gonadal cell lineage specification.

Gonad development includes sex determination and divergent maturation of the testes and ovaries. Recent advances in measuring gene expression in single cells are providing new insights into this complex process. However, the underlying epigenetic regulatory mechanisms remain unclear. Here, we profiled chromatin accessibility in mouse gonadal cells of both sexes from embryonic day 11.5 to 14.5 using single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq). Our results showed that individual cell types can be inferred by the chromatin landscape, and that cells can be temporally ordered along developmental trajectories. Integrative analysis of transcriptomic and chromatin-accessibility maps identified multiple putative regulatory elements proximal to key gonadal genes Nr5a1, Sox9 and Wt1. We also uncover cell type-specific regulatory factors underlying cell type specification. Overall, our results provide a better understanding of the epigenetic landscape associated with the progressive restriction of cell fates in the gonad.

Is the use of gonad protection protectors necessary during infants chest radiography?

INTRODUCTION AND OBJECTIVES: To compare gonad doses with and without a gonad protector and to optimize the use of gonadal protectors in infants thorax radiography. MATERIALS AND METHODS: Two pediatric anthropomorphic phantoms are used: an X-ray system for KXO-50SS/DRX-3724HD, and a digital radiography system for CALNEO Smart C12, with and without a gonad protector during infants thorax radiography. A real time skin dosimeter is placed on the X-ray system, and a real time skin dosimeter is inserted on the front side of the mammary gland, the front and back sides of the true pelvis level, and on the ovaries and testes. The X-ray system is irradiated 15 times using phantoms with and without a gonad protector. The measured entrance patient doses values of for the real time skin dosimeter are compared for each phantom, with and without the gonad protector. RESULTS: The medium of measured entrance patient doses values for front side dose of the true pelvis level with and without the protector are 10.00 and 5.00 µGy at newborn, and 10.00 and 0.00µGy at one year, respectively. The medium of measured entrance patient doses values for the back side dose of the true pelvis level with and without the protector are 0.00 and 0.00 µGy at both newborn one year, respectively. The measured entrance patient doses cannot be detected in the ovaries and testes with or without the protector. No significant differences are observed in the measured entrance patient doses values for the front and back side doses of the pelvis, ovaries, and testes at newborn and one year, with and without the protector (p>0.05). CONCLUSIONS: No significant difference was observed in gonad dose measurements with and without the gonad protector during infants chest radiography. We believe that gonadal protector wearing is not necessary.

Chronic exposure to gestodene impairs reproductive system in adult female zebrafish (Daniarerio).

Gestodene (GES) is widely used in human therapy and animal husbandry and is frequently detected in aquatic environments. Although GES adversely affects aquatic organisms at trace levels, its effects on the reproductive biology of fish remain inconclusive. In this study, female zebrafish (Danio rerio) were exposed to environmentally relevant levels of GES for the evaluation of the effects of GES on the reproductive system by using endpoints including gene expression, plasma steroid concentrations, histological and morphological analyses, copulatory behavior, and reproductive output. Adult female zebrafish exposed to environmentally relevant concentrations of GES (4.0, 40.2, and 372.7 ng/L) for 60 d demonstrated stagnant ovarian oocyte development, evidenced by an increase in the percentage of perinuclear and atretic oocytes and a decrease in the percentage of late vitellogenic oocytes. GES-exposed females were less attractive to males and had lower copulatory intimacy than females in control. Consequently, spawning (44.3-49.2 %) and egg fertilization rates (27.9-32.0 %) were decreased. The decreased survival of fertilized eggs and hatching rates were accompanied by increased malformations. These negative effects were associated with abnormal transcriptional levels of gonadal steroid hormones, which were regulated by genes (Hsd17ß3, Hsd11ß2, Hsd20ß, Cyp19a1a, and Cyp11b). Overall, our findings suggest that GES impairs the reproductive system of zebrafish, which may threaten population stability.

Exploration of cell-cell interactions and the notch signaling pathway in the gonadal niche of Crassostrea gigas.

The Notch signaling pathway plays a pivotal role in governing cell fate determinations within the gonadal niche. This study provides an extensive elucidation of the male and female gonadal niches within Crassostrea gigas. Examination via transmission electron microscopy revealed the presence of desmosome-like connection not only between germ cells and niche cells but also among adjacent niche cells within the oyster gonad. Transcriptomic analysis identified several putative Notch pathway components, including CgJAG1, CgNOTCH1, CgSuh, and CgHey1. Phylogenetic analysis indicated a close evolutionary relationship between CgJAG1, CgNOTCH1, and CgHey1 and Notch members present in Drosophila. Expression profiling results indicated a notable abundance of CgHey1 in the gonads, while CgJAG1 and CgNOTCH1 displayed distinct expression patterns associated with sexual dimorphism. In situ hybridization findings corroborated the predominant expression of CgJAG1 in male niche cells, while CgNOTCH1 was expressed in both male and female germ cells, as well as female niche cells. These findings demonstrate the important role of the Notch signaling pathway in the gonadal niche of oysters.

Parthenogenetic Stick Insects Exhibit Signatures of Preservation in the Molecular Architecture of Male Reproduction.

After the loss of a trait, theory predicts that the molecular machinery underlying its phenotypic expression should decay. Yet, empirical evidence is contrasting. Here, we test the hypotheses that (i) the molecular ground plan of a lost trait could persist due to pleiotropic effects on other traits and (ii) that gene co-expression network architecture could constrain individual gene expression. Our testing ground has been the Bacillus stick insect species complex, which contains close relatives that are either bisexual or parthenogenetic. After the identification of genes expressed in male reproductive tissues in a bisexual species, we investigated their gene co-expression network structure in two parthenogenetic species. We found that gene co-expression within the male gonads was partially preserved in parthenogens. Furthermore, parthenogens did not show relaxed selection on genes upregulated in male gonads in the bisexual species. As these genes were mostly expressed in female gonads, this preservation could be driven by pleiotropic interactions and an ongoing role in female reproduction. Connectivity within the network also played a key role, with highly connected-and more pleiotropic-genes within male gonad also having a gonad-biased expression in parthenogens. Our findings provide novel insight into the mechanisms which could underlie the production of rare males in parthenogenetic lineages; more generally, they provide an example of the cryptic persistence of a lost trait molecular architecture, driven by gene pleiotropy on other traits and within their co-expression network.

Gonadal sex and chromosome complement influence the gut microbiome in a mouse model of allergic airway inflammation.

Evidence abounds that gut microbiome components are associated with sex disparities in the immune system. However, it remains unclear whether the observed sex disparity in asthma incidence is associated with sex-dependent differences in immune-modulating gut microbiota, and/or its influence on allergic airway inflammatory processes. Using a mouse model of house dust mite (HDM)-induced allergic inflammation and the four core genotypes (FCGs) model, we have previously reported sex differences in lung inflammatory phenotypes. Here, we investigated associations of gut microbiomes with these phenotypes by challenging FCG mice [mouse with female sex chromosome and male gonad (XXM), mouse with female sex chromosome and female gonad (XXF), mouse with male sex chromosome and male gonad (XYM), and mouse with male sex chromosome and female gonad (XYF); n = 7/group] with HDM (25 µg) or PBS intranasally for 5 wk and collecting fecal samples. We extracted fecal DNA and analyzed the 16S microbiome via Targeted Metagenomic Sequencing. We compared α and ß diversity across genotypes and assessed the Firmicutes/Bacteroidetes (F/B) ratio. When comparing baseline and after exposure for the FCG, we found that the gut F/B ratio was only increased in the XXM genotype. We also found that α diversity was significantly increased in all FCG mice upon HDM challenge, with the highest increase in the XXF, and the lowest in the XXM genotypes. Similarly, ß diversity of the microbial community was also affected by challenge in a gonad- and chromosome-dependent manner. In summary, our results indicated that HDM treatment, gonads, and sex chromosomes significantly influence the gut microbial community composition. We concluded that allergic lung inflammation may be affected by the gut microbiome in a sex-dependent manner involving both hormonal and genetic influences.NEW & NOTEWORTHY Recently, the gut microbiome and its role in chronic respiratory disease have been the subject of extensive research and the establishment of its involvement in immune functions. Using the FCG mouse model, our findings revealed the influence of gonads and sex chromosomes on the microbial community structure before and after exposure to HDM. Our data provide a potential new avenue to better understand mediators of sex disparities associated with allergic airway inflammation.

Reproductive toxic effects of chronic exposure to bisphenol A and its analogues in marine medaka (Oryzias melastigma).

As awareness of BPA's health risks has increased, many countries and regions have implemented strict controls on its use. Consequently, bisphenol analogues like BPF and BPAF are being increasingly used as substitutes. However, these compounds are also becoming increasingly prevalent in the environment due to production, use and disposal processes. The oceans act as a repository for various pollutants, and recent studies have revealed the extensive presence of bisphenols (BPs, including BPA, BPF, BPAF, etc.) in the marine environment, posing numerous health hazards to marine wildlife. Nevertheless, the reproductive toxicity of these chemicals on marine fish is not comprehensively comprehended yet. Thus, the histological features of the gonads and the gene expression profiles of HPG (Hypothalamic-Pituitary-Gonadal) axis-related genes in marine medaka (Oryzias melastigma) were studied after exposure to single and combined BPs for 70 days. The effects of each exposure group on spawning, embryo fertilization, and hatching in marine medaka were also assessed. Furthermore, the impacts of each exposure group on the genes related to methylation in the F2 and F3 generations were consistently investigated. BPs exposure was found to cause follicular atresia, irregular oocytes, and empty follicles in the ovary; but no significant lesions in the testis were observed. The expression of several HPG axis genes, including cyp19b, 17ßhsd, 3ßhsd, and fshr, resulted in significant changes compared to the control group. The quantity of eggs laid and fertilization rate decreased in all groups treated with BPs, with the BPAF-treated group showing a notable reduction in the number of eggs laid. Additionally, the hatching rate showed a more significant decline in the BPF-treated group. The analysis of methylated genes in the offspring of bisphenol-treated groups revealed significant changes in the expression of genes including amh, dnmt1, dnmt3ab, mbd2, and mecp2, indicating a potential transgenerational impact of bisphenols on phenotype through epigenetic modifications. Overall, the potential detrimental impact of bisphenol on the reproduction of marine medaka emphasizes the need for caution in considering the use of BPAF and BPF as substitutes.

Valorisation of sea urchin (Paracentrotus lividus) gonads through canning.

Fresh sea urchin (Paracentrotus lividus) gonads are a delicacy with short seasonal availability, very often heterogeneous in size and intrinsic characteristics. This study aimed to valorise this resource through the preparation of canned products (with/without Porphyra spp.) and evaluate their physicochemical and sensory quality (3-12 months). Canning contributed to a decrease in protein, K and most carotenoids contents; and a concentration of lipids, ash, Na and Se levels. A simulated 12-month ageing led to decrease the protein and ß-carotene contents; and the Na and lutein levels concentration. The macroalgae addition resulted in an orange, darker and less soft product, with higher carbohydrates, Na, Se and carotenoids contents. A 25 g-dose contributes to significant daily intakes of protein (8-9%), EPA+DHA (47-53%), I (35-62%) and Se (30-47%). The products were commercially stable/sterile and had good sensory acceptance. Overall, canning constitutes a strategy to provide a nutritionally balanced product available all year-round.

The expression profiles of cyp19a1, sf-1, esrs and gths in the brain-pituitary during gonadal sex differentiation in juvenile Japanese eels.

Eels are gonochoristic species whose gonadal differentiation initiates at the yellow eel stage and is influenced by environmental factors. We revealed some sex-related genes were sex dimorphically expressed in gonads during gonadal sex differentiation of Japanese eel (Anguilla japonica); however, the expression of sex-related genes in the brain-pituitary during gonadal sex differentiation in eels is still unclear. This study aimed to investigate the sex-related gene expressions in the brain-pituitary and tried to clarify their roles in the brain and gonads during gonadal sex differentiation. Based on our previous histological study, the control eels developed as males, and estradiol-17ß (E2) was used for feminization. Our results showed that during testicular differentiation, the brain cyp19a1 transcripts and aromatase proteins were increased significantly; moreover, the cyp19a1, sf-1, foxl2s, and esrs (except gperb) transcripts in the midbrain/pituitary also were increased significantly. Forebrain gnrh1 transcripts increased slightly during gonadal differentiation of both sexes, but the gnrhr1b and gnrhr2 transcripts in the midbrain/pituitary were stable during gonadal differentiation. The expression levels of gths and gh in the midbrain/pituitary were significantly increased during testicular differentiation and were much higher in males than in E2-feminized females. These results implied that endogenous estrogens might play essential roles in the brain/pituitary during testicular differentiation, sf-1, foxl2s, and esrs may have roles in cyp19a1 regulation in the midbrain/pituitary of Japanese eels. For the GnRH-GTH axis, gths, especially fshb, may be regulated by esrs and involved in regulating testicular differentiation and development in Japanese eels.

Using cell culture systems from the Persian Gulf Arabian yellowfin sea bream, Acanthopagrus arabicus, to assess the effects of dexamethasone on gonad and brain aromatase activity and steroid production.

Dexamethasone (DEX) is a synthetic glucocorticoid widely used as pharmaceutical and usually exists in effluents with varying degrees of concentrations. In this study, cultivated Brain, ovary and testis cells from Arabian Sea bream, Acanthopagrus arabicus, were treated by DEX at concentrations of 0, 0.3, 3.0, 30.0 and 300.0 µg/ml for 48 h. The aromatase activity and steroid (17-ß-estradiol (E2), progesterone (P) and testosterone (T)) production by cells were measured at 12, 24 and 48 h of the experiment. The results showed that the sensitivity of cultivated ovarian, testicular and brain cells to DEX increased dose dependently. DEX was potent inhibitor of aromatase activity at specially 30.0 and 300.0 µg/ml in the cultivated ovarian and testicular cells at different sampling time. On the other hand, DEX was found to stimulate the aromatase activity of fish brain. DEX also decreased E2, P and T production by cultivated ovarian and testicular cells during the experiment. While, DEX caused an increase in the production of E2 and P by brain cells, which seems logical considering the stimulating effect of this drug on brain aromatase activity. In conclusion, results highlight that DEX is able to change the activity of aromatase, and disrupt the biosynthesis of estrogens and thus affect reproduction in fish.

Gonads under stress: A systematic review and meta-analysis on the effects of acute psychosocial stress on gonadal steroids secretion in humans.

Animal research has shown that the hypothalamus-pituitary-gonadal (HPG) axis is inhibited by (chronic and/or severe) stress, which can lead to impaired fertility and reproductive functioning, presumably caused by the inhibition of gonadal steroid secretion and in interactions with glucocorticoids. However, what has not been clarified is how acute psychosocial stress modulates gonadal steroid secretion in humans. Here we summarize the experimental research on the acute effects of stress on the secretion of gonadal steroids in humans. A systematic literature search revealed 21 studies (with N=881 individuals) measuring testosterone, progesterone or estradiol in response to a standardized acute laboratory stressor in healthy humans. Both our literature review and quantitative meta-analysis suggest that in humans, acute stress stimulates rather than inhibits HPG axis activity, although there is a considerable heterogeneity in the reported methods and results. Increased gonadal steroids in response to acute stress contrasts with many animal studies reporting the opposite pattern, at least regarding severe and/or chronic stressors. We discuss methodological issues and challenges for future research and hope to stimulate experimental studies within this area. A better understanding of these mechanisms is needed, and may have important implications for health and disease, as well as the modulation of various behaviors by acute stressors.

Biological patterns of reproduction of the Brazilian sardine Sardinella brasiliensis in the purse seine fishery of Southwest Atlantic Ocean: A long-term assessment.

Recent estimates of the size at first maturity (L50) of Sardinella brasiliensis showed contradictory results with a decreasing in the fish stock biomass encompassed by increasing values of L50. The methodological approach used hereby allowed to separate sardines classified in the virginal maturity stage from those categorized in the recovery stage, and ready for one next spawning event. This study evaluated the hypothesis of the existence of separated stocks experiencing distinct environmental conditions and fishing pressures which may have altered L50 estimates using a robust dataset based on biological samples collected along the entire species distribution area in the southeast-south Brazilian coast [Rio de Janeiro (RJ), São Paulo (SP), Paraná (PR), Santa Catarina (SC) and Rio Grande do Sul (RS)] between 2000 and 2018. A reclassification of the gonadal maturity stages provided a more realistic estimate of L50. Combining biological, reproductive, fishing data and the mean temperature of the catch (MTC), the leave-one-out classification correctly re-assigned individuals with an overall accuracy of 85% [100% (RJ), 45% (SP), 99% (PR), 99% (SC) and 82% (RS)]. The connectivity between the local populations of S. brasiliensis off RJ (23°S) and the southern populations is limited, contrasting to spatial structured semi-discrete population-units found between SP and RS (24°S-30°S). The northern extreme population-unit (RJ, 22°S-23°S) showed an expressive reduction of L50, and a negative correlation was detected between the increasing MTC values and the abundance of early maturing individuals and recruits of the species. Stock specific L50 estimates seemed to act as indicators of long term environmental fluctuations.

Identification and involvement of DAX1 gene in spermatogenesis of boring giant clam Tridacna crocea.

DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenital critical region on X chromosome gene 1), a key sex determinant in various species, plays a vital role in gonad differentiation and development and controls spermatogenesis. However, the identity and function of DAX1 are still unclear in bivalves. In the present study, we identified a DAX1 (designed as Tc-DAX1) gene from the boring giant clam Tridacna crocea, a tropical marine bivalve. The full length of Tc-DAX1 was 1877 bp, encoding 462 amino acids, with a Molecular weight of 51.81 kDa and a theoretical Isoelectric point of 5.87 (pI). Multiple sequence alignments and phylogenetic analysis indicated a putative ligand binding domain (LBD) conserved regions clustered with molluscans DAX1 homologs. The tissue distributions in different reproductive stages revealed a dimorphic pattern, with the highest expression trend in the male reproductive stage, indicating its role in spermatogenesis. The DAX1 expression data from embryonic stages shows its highest expression profile (P < 0.05) in the zygote stage, followed by decreasing trends in the larvae stages (P > 0.05). The localization of DAX1 transcripts has also been confirmed by whole mount in situ hybridization, showing high positive signals in the fertilized egg, 2, and 4-cell stage, and gastrula. Moreover, RNAi knockdown of the Tc-DAX1 transcripts shows a significantly lower expression profile in the ds-DAX1 group compared to the ds-EGFP group. Subsequent histological analysis of gonads revealed that spermatogenesis was affected in a ds-DAX1 group compared to the ds-EGFP group. All these results indicate that Tc-DAX1 is involved in the spermatogenesis and early embryonic development of T. crocea, providing valuable information for the breeding and aquaculture of giant clams.

Edible Sea urchins Echinus esculentus from Norwegian waters- Effect of season on nutritional quality and chemical contaminants.

This study aimed to characterize Echinus esculentus gonads in terms of biometric parameters and nutritional quality at two sites in Mid-Norway at four different seasons. The chemical contamination of the gonads was also investigated for the first time through the evaluation of 28 macro- and trace elements and 32 components from the emerging and persistent group per- and polyfluoroalkyl substances (PFAS). The spawning period was determined in summer, given that the gonad index was the lowest in this season for both sites. Protein concentrations were constant (8%-10%). However, lipid contents (1%-3%) were noticed to be higher in gonads during autumn and winter. The gonads had high contents of PUFA mainly EPA and DHA, followed by SFA, and MUFA year around for both locations. E. esculentus gonads constitute a good source of fatty acids, macro, and trace elements. This species could also be a bioindicator for the monitoring of marine environments.

The effect of gonadal hormones on the gene expression of brain-pituitary in protandrous black porgy, Acanthopagrus schlegelii.

In black porgy (Acanthopagrus schlegelii), the brain-pituitary-testis (Gnrh-Gths-Dmrt1) axis plays a vital role in male fate determination and maintenance, and then inhibiting female development in further (puberty). However, the feedback of gonadal hormones on regulating brain signaling remains unclear. In this study, we conducted short-term sex steroid treatment and surgery of gonadectomy to evaluate the feedback regulation between the gonads and the brain. The qPCR results show that male phase had the highest gths transcripts; treatment with estradiol-17ß (E2) or 17α-methyltestosterone (MT) resulted in the increased pituitary lhb transcripts. After surgery, apart from gnrh1, there is no difference in brain signaling genes between gonadectomy and sham fish. In the diencephalon/mesencephalon transcriptome, de novo assembly generated 283,528 unigenes; however, only 443 (0.16%) genes showed differentially expressed between sham and gonadectomy fish. In the present study, we found that exogenous sex steroids affect the gths transcription; this feedback control is related to the gonadal stage. Furthermore, gonadectomy may not affect gene expression of brain signaling (Gnrh-Gths axis). Our results support the communication between ovotestis and brain signaling (Gnrh-Gths-testicular Dmrt1) for the male fate.

New insights into the all-testis differentiation in zebrafish with compromised endogenous androgen and estrogen synthesis.

The regulatory mechanism of gonadal sex differentiation, which is complex and regulated by multiple factors, remains poorly understood in teleosts. Recently, we have shown that compromised androgen and estrogen synthesis with increased progestin leads to all-male differentiation with proper testis development and spermatogenesis in cytochrome P450 17a1 (cyp17a1)-/- zebrafish. In the present study, the phenotypes of female-biased sex ratio were positively correlated with higher Fanconi anemia complementation group L (fancl) expression in the gonads of doublesex and mab-3 related transcription factor 1 (dmrt1)-/- and cyp17a1-/-;dmrt1-/- fish. The additional depletion of fancl in cyp17a1-/-;dmrt1-/- zebrafish reversed the gonadal sex differentiation from all-ovary to all-testis (in cyp17a1-/-;dmrt1-/-;fancl-/- fish). Luciferase assay revealed a synergistic inhibitory effect of Dmrt1 and androgen signaling on fancl transcription. Furthermore, an interaction between Fancl and the apoptotic factor Tumour protein p53 (Tp53) was found in vitro. The interaction between Fancl and Tp53 was observed via the WD repeat domain (WDR) and C-terminal domain (CTD) of Fancl and the DNA binding domain (DBD) of Tp53, leading to the K48-linked polyubiquitination degradation of Tp53 activated by the ubiquitin ligase, Fancl. Our results show that testis fate in cyp17a1-/- fish is determined by Dmrt1, which is thought to stabilize Tp53 by inhibiting fancl transcription during the critical stage of sexual fate determination in zebrafish.

Expression of IZUMO1 and JUNO in the gonads of domestic cats (Felis catus).

Because of the time-consuming nature of surgical neutering and the rapid rate of reproduction among domestic cats, it is crucial to investigate alternative, nonsurgical methods of contraception for this species. Sperm protein IZUMO1 and its oocyte receptor JUNO have been proposed as potential targets for nonsurgical contraceptives. This study aimed to demonstrate (1) the protein coding sequence of feline IZUMO1 and JUNO, (2) gene expression in specific organs by measuring mRNA levels in different visceral tissues, and (3) the expression of IZUMO1 and JUNO during sperm maturation and folliculogenesis, respectively. Amplification for sequencing of feline IZUMO1 and JUNO was performed using the RT-PCR method. Levels of gene expression in different tissues were evaluated using real-time PCR. In situ hybridization was performed to localize JUNO mRNA in ovarian tissues. The complete coding sequences of IZUMO1 and JUNO were obtained and analyzed. A comparison between protein orthologs demonstrated the conservation of IZUMO1 and JUNO in Felidae. The real-time PCR results from various visceral organs indicated that IZUMO1 was significantly higher in the testis than in other organs, whereas JUNO was significantly higher in the ovary than in other organs. Expression of IZUMO1 was found to be higher in the testes than in the caput, corpus, and cauda of epididymides. In situ hybridization revealed that JUNO mRNA was in the ooplasm and nucleus of the primordial, primary, secondary, and antral follicles. Importantly, this was the first study to demonstrate the IZUMO1 and JUNO genes in the testis and ovary of cats. The results are useful for future research related to these genes and for developing contraceptives against these targets.

Reprograming skin fibroblasts into Sertoli cells: a patient-specific tool to understand effects of genetic variants on gonadal development.

BACKGROUND: Disorders/differences of sex development (DSD) are congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. With overlapping phenotypes and multiple genes involved, poor diagnostic yields are achieved for many of these conditions. The current DSD diagnostic regimen can be augmented by investigating transcriptome/proteome in vivo, but it is hampered by the unavailability of affected gonadal tissue at the relevant developmental stage. We try to mitigate this limitation by reprogramming readily available skin tissue-derived dermal fibroblasts into Sertoli cells (SC), which could then be deployed for different diagnostic strategies. SCs form the target cell type of choice because they act like an organizing center of embryonic gonadal development and many DSD arise when these developmental processes go awry. METHODS: We employed a computational predictive algorithm for cell conversions called Mogrify to predict the transcription factors (TFs) required for direct reprogramming of human dermal fibroblasts into SCs. We established trans-differentiation culture conditions where stable transgenic expression of these TFs was achieved in 46, XY adult dermal fibroblasts using lentiviral vectors. The resulting Sertoli like cells (SLCs) were validated for SC phenotype using several approaches. RESULTS: SLCs exhibited Sertoli-like morphological and cellular properties as revealed by morphometry and xCelligence cell behavior assays. They also showed Sertoli-specific expression of molecular markers such as SOX9, PTGDS, BMP4, or DMRT1 as revealed by IF imaging, RNAseq and qPCR. The SLC transcriptome shared about two thirds of its differentially expressed genes with a human adult SC transcriptome and expressed markers typical of embryonic SCs. Notably, SLCs lacked expression of most markers of other gonadal cell types such as Leydig, germ, peritubular myoid or granulosa cells. CONCLUSIONS: The trans-differentiation method was applied to a variety of commercially available 46, XY fibroblasts derived from patients with DSD and to a 46, XX cell line. The DSD SLCs displayed altered levels of trans-differentiation in comparison to normal 46, XY-derived SLCs, thus showcasing the robustness of this new trans-differentiation model. Future applications could include using the SLCs to improve definitive diagnosis of DSD in patients with variants of unknown significance.

Individuals with disorders/differences of sex development (DSD) frequently do not get a specific genetic diagnostic. A limitation in the field is that the relevant cell types that would be needed to study the molecular events occurring at the time of onset of many DSD are found in the embryonic gonad. This, of course, is not accessible for research or diagnostic purposes. We set out to develop a method for directly transforming more accessible cells, from adult skin, into the cells known to organize the male gonad in the embryo, Sertoli cells. A combination of unique transcription factors was stably integrated into skin fibroblasts, and culture under appropriate conditions allowed differentiation into Sertoli-like cells (SLC), but not other gonadal cell types. The SLCs recapitulated known patterns of gene expression, shape, and behavior of Sertoli cells. The method was also tested on commercially available fibroblasts from a variety of DSD genetic backgrounds. The resulting cells exhibited condition-specific behavior (gene expression, adhesion to substrate, division rate…). This method provides a new tool to study molecular events occurring at the time of onset of DSD in the embryonic gonad, and the impact of patient-specific mutations on those. It could allow identification of new developmental pathways (and, thus, new candidate genes for DSD), as well as a provide models to validate the impact of variants of unknown significance, or to test approaches to correct the genetic anomaly in patient cells.

Dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Sex is determined by multiple factors derived from somatic and germ cells in vertebrates. We have identified amhy, dmrt1, gsdf as male and foxl2, foxl3, cyp19a1a as female sex determination pathway genes in Nile tilapia. However, the relationship among these genes is largely unclear. Here, we found that the gonads of dmrt1;cyp19a1a double mutants developed as ovaries or underdeveloped testes with no germ cells irrespective of their genetic sex. In addition, the gonads of dmrt1;cyp19a1a;cyp19a1b triple mutants still developed as ovaries. The gonads of foxl3;cyp19a1a double mutants developed as testes, while the gonads of dmrt1;cyp19a1a;foxl3 triple mutants eventually developed as ovaries. In contrast, the gonads of amhy;cyp19a1a, gsdf;cyp19a1a, amhy;foxl2, gsdf;foxl2 double and amhy;cyp19a1a;cyp19a1b, gsdf;cyp19a1a;cyp19a1b triple mutants developed as testes with spermatogenesis via up-regulation of dmrt1 in both somatic and germ cells. The gonads of amhy;foxl3 and gsdf;foxl3 double mutants developed as ovaries but with germ cells in spermatogenesis due to up-regulation of dmrt1. Taking the respective ovary and underdeveloped testis of dmrt1;foxl3 and dmrt1;foxl2 double mutants reported previously into consideration, we demonstrated that once dmrt1 mutated, the gonad could not be rescued to functional testis by mutating any female pathway gene. The sex reversal caused by mutation of male pathway genes other than dmrt1, including its upstream amhy and downstream gsdf, could be rescued by mutating female pathway gene. Overall, our data suggested that dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.