Long-term preservation of germ cells and gonadal tissues at ambient temperatures.

Objective: To present an overview of different approaches and recent advances for long-term preservation of germ cells and gonadal tissues at ambient temperatures. Methods: Review of the existing literature. Results: Preserving viable spermatozoa, eggs, embryos, and gonadal tissues for the long term is critical in human fertility treatment and for the management of animal populations (livestock, biomedical models, and wild species). The need and number of banked germplasms are growing very fast in all disciplines, but current storage options at freezing temperatures are often constraining and not always sustainable. Recent research indicates that structures and functions of gametes or gonadal tissues can be preserved for the long term using different strategies based on dehydration and storage at supra-zero temperatures. However, more studies are needed in rehydration and reanimation of germplasms (including proper molecular and cellular evaluations). Conclusions: While a lot of research is still warranted to optimize drying and rehydration conditions for each sample type and each species, alternative preservation methods will change the paradigm in fertility preservation and biobanking. It will transform the way we maintain and manage precious biomaterials for the long term. Lay summary: Living sperm cells, eggs, embryos, and reproductive tissues can be preserved at freezing temperatures for human fertility treatments and used to manage breeding in livestock, laboratory animals, and wild species through assisted reproduction. These cells can be stored in cell banks and demand for them is growing fast. However, current long-term storage options at freezing temperatures are expensive. Instead of using low temperatures, recent research indicates that these cells can be dried and stored above freezing temperatures for an extended amount of time. While a lot of research is still needed to optimize how different samples are dried and rehydrated, alternative methods of preserving cells will make fertility preservation and cell banking easier. It will also transform the way we keep and manage samples for the long term.

Changes in gonadotropin-releasing hormone in the cerebral ganglion of the Manila clam Ruditapes philippinarum during gonadal development.

Gonadotropin-releasing hormone (GnRH) plays an important role in reproduction in both vertebrates and invertebrates; however, little is known about GnRH during gonadal development in bivalves. We developed a time-resolved fluoroimmunoassay (TR-FIA) for Manila clam Ruditapes philippinarum GnRH (rpGnRH) and measured the amount of rpGnRH in the cerebral ganglion (CG) and sex steroid hormones in the hemolymph during gonadal development. The cross-reactivity of the anti-rpGnRH antibody against other forms of GnRH was <0.15%, and the displacement curve obtained for serially diluted CG extracts was parallel to the rpGnRH standard curve, confirming the suitability of the TR-FIA system. Based on histological observation, gonadal development of the clams was classified into early developing (stage 1), late developing (stage 2), ripe (stage 3), and partially spent (stage 4). In female clams, rpGnRH levels in the CG peaked at stage 1, and 17ß-estradiol (E2) levels in the hemolymph peaked at stage 2. The rpGnRH levels in males and hemolymph testosterone levels in both sexes did not differ significantly across stages. Hemolymph E2 levels in males were below the detection limit for the TR-FIA. These results suggest that rpGnRH and E2 secretion in females can activate ovarian development of the Manila clam at the early and late developing stages, respectively.

DNA methylation differences between male and female gonads of the oyster reveal the role of epigenetics in sex determination.

DNA methylation involved in sex determination mechanism by regulating gene expression related to sex determination networks are common in vertebrates. However, the mechanism linking epigenetics in invertebrates and sex determination has remained elusive. Here, methylome of the male and female gonads in the oyster Crassostrea gigas were conducted to explore the role of epigenetics in invertebrate sex determination. Comparative analysis of gonadal DNA methylation of females and males revealed that male gonads displayed a higher level of DNA methylation and a greater number of hypermethylated genes. Luxury genes presented hypomethylation, while housekeeping genes got hypermethylation. Genes in the conserved signaling pathways, rather than the key master genes in the sex determination pathway, were the major targets of substantial DNA methylation modification. The negative correlation of expression and promoter methylation in the diacylglycerol kinase delta gene (Dgkd) - a ubiquitously expressed gene - indicated DNA methylation may fine turn the expression of Dgkd and be involved in the process of sex determination. Dgkd can be used as an epigenetic marker to distinguish male C. gigas based on the different methylation regions in the promoter region. The results suggest that DNA methylation mechanisms played potential functional impacts in the sex determination in oysters, which is helpful to deepen the understanding of sex determination in invertebrate.

Sexual dimorphism in Odontobutis sinensis brain-pituitary-gonad axis and liver highlighted by histological and transcriptomic approach.

In this study, sexual dimorphism in Chinese dark sleeper (Odontobutis sinensis) brain-pituitary-gonad axis and liver was highlighted by histological and transcriptomic approach. The results showed that there were two significant differences between males and females. Firstly, males grew larger and faster than females. Transcriptomic analysis and qPCR validation indicated that two key growth genes, insulin-like growth factor (igf) and 25-hydroxyvitamin D-1 alpha hydroxylase (cyp27b), were more highly detected in male liver than that in female liver. Secondly, histological analysis displayed that the liver in males showed an obvious ivory fatty phenomenon with more fat vacuoles and lipid droplet aggregation compared to that in females. Transcriptomic analysis indicated that the transcript level of vitellogenin (vtg) in male liver were significantly lower than that in female liver. After 17ß-estradiol (E2) treatment of primary cultured hepatocytes, the vtg mRNA expression was induced significantly, while dihydrotestosterone (DHT) treatment had little effect on it. Generally, this study will provide some ideas for further exploring the mechanism of sexual dimorphism in Odontobutis sinensis.

Antioxidant capacity differs across social ranks and with ascension in males of a group-living fish.

Animals that live in groups often form hierarchies in which an individual's behaviour and physiology varies based on their social rank. Occasionally, a subordinate can ascend into a dominant position and the ascending individual must make rapid behavioural and physiological adjustments to solidify their dominance. These periods of social transition and instability can be stressful and ascending individuals often incur large metabolic costs that could influence their oxidative status. Most previous investigations examining the link between oxidative status and the social environment have done so under stable social conditions and have evaluated oxidative status in a single tissue. Therefore, evaluations of how oxidative status is regulated across multiple tissues during periods of social flux would greatly enhance our understanding of the relationship between oxidative status and the social environment. Here, we assessed how antioxidant capacity in three tissues (brain, gonad, and muscle) varied among dominant, subordinate, and ascending males of the group-living cichlid fish, Neolamprologus pulcher. Antioxidant capacity in the brain and muscle of ascending males was intermediate to that of dominant (highest levels) and subordinate males (lowest levels) and correlated with differences in social and locomotor behaviours, respectively. Gonad antioxidant capacity was lower in ascending males than in dominant males. However, gonad antioxidant capacity was positively correlated with the size of ascending males' gonads suggesting that ascending males may increase gonad antioxidant capacity as they develop their gonads. Overall, our results highlight the widespread physiological consequences of social ascension and emphasize the importance of tissue-specific measures of oxidative status.

Seasonal reproduction and gonadal function: a focus on humans starting from animal studies.

Photoperiod impacts reproduction in many species of mammals. Mating occurs at specific seasons to achieve reproductive advantages, such as optimization of offspring survival. Light is the main regulator of these changes during the photoperiod. Seasonally breeding mammals detect and transduce light signals through extraocular photoreceptor, regulating downstream melatonin-dependent peripheral circadian events. In rodents, hormonal reduction and gonadal atrophy occur quickly and consensually with short-day periods. It remains unclear whether photoperiod influences human reproduction. Seasonal fluctuations of sex hormones have been described in humans, although they seem to not imply adaptative seasonal pattern in human gonads. This review discusses current knowledge about seasonal changes in the gonadal function of vertebrates, including humans. The photoperiod-dependent regulation of hypothalamic-pituitary-gonadal axis, as well as morphological and functional changes of the gonads is evaluated herein. Endocrine and morphological variations of reproductive functions, in response to photoperiod, are of interest as they may reflect the nature of past population selection for adaptative mechanisms that occurred during evolution.

A new experimental model for the investigation of sequential hermaphroditism.

The stunning sexual transformation commonly triggered by age, size or social context in some fishes is one of the best examples of phenotypic plasticity thus far described. To date our understanding of this process is dominated by studies on a handful of subtropical and tropical teleosts, often in wild settings. Here we have established the protogynous New Zealand spotty wrasse, Notolabrus celidotus, as a temperate model for the experimental investigation of sex change. Captive fish were induced to change sex using aromatase inhibition or manipulation of social groups. Complete female-to-male transition occurred over 60 days in both cases and time-series sampling was used to quantify changes in hormone production, gene expression and gonadal cellular anatomy. Early-stage decreases in plasma 17ß-estradiol (E2) concentrations or gonadal aromatase (cyp19a1a) expression were not detected in spotty wrasse, despite these being commonly associated with the onset of sex change in subtropical and tropical protogynous (female-to-male) hermaphrodites. In contrast, expression of the masculinising factor amh (anti-Müllerian hormone) increased during early sex change, implying a potential role as a proximate trigger for masculinisation. Collectively, these data provide a foundation for the spotty wrasse as a temperate teleost model to study sex change and cell fate in vertebrates.

MicroRNAs May Play an Important Role in Sexual Reversal Process of Chinese Soft-Shelled Turtle, Pelodiscus sinensis.

The Chinese soft-shelled (Pelodiscus sinensis) turtle exhibits obvious sex dimorphism, which leads to the higher economic and nutritional value of male individuals. Exogenous hormones can cause the transformation from male to female phenotype during gonadal differentiation. However, the molecular mechanism related to the sexual reversal process is unclear. In this study, we compared the difference between the small RNAs of male, female, and pseudo-female turtles by small RNA-seq to understand the sexual reversal process of Chinese soft-shelled turtles. A certain dose of estrogen can cause the transformation of Chinese soft-shelled turtles from male to female, which are called pseudo-female individuals. The result of small RNA-seq has revealed that the characteristics of pseudo-females are very similar to females, but are strikingly different from males. The number of the microRNAs (miRNAs) of male individuals was significantly less than the number of female individuals or pseudo-female individuals, while the expression level of miRNAs of male individuals were significantly higher than the other two types. Furthermore, we found 533 differentially expressed miRNAs, including 173 up-regulated miRNAs and 360 down-regulated miRNAs, in the process of transformation from male to female phenotype. Cluster analysis of the total 602 differential miRNAs among females, males, and pseudo-females showed that miRNAs played a crucial role during the sexual differentiation. Among these differential miRNAs, we found 12 miRNAs related to gonadal development and verified their expression by qPCR. The TR-qPCR results confirmed the differential expression of 6 of the 12 miRNAs: miR-26a-5p, miR-212-5p, miR-202-5p, miR-301a, miR-181b-3p and miR-96-5p were involved in sexual reversal process, which was consistent with the results of omics. Using these six miRNAs and some of their target genes, we constructed a network diagram related to gonadal development. We suggest that these miRNAs may play an important role in the process of effective sex reversal, which would contribute to the breeding of all male strains of Chinese soft-shelled turtles.

Serum anti-Müllerian hormone as a marker of ovarian reserve after cancer treatment and/or hematopoietic stem cell transplantation in childhood: proposal for a systematic approach to gonadal assessment.

OBJECTIVE: Female patients treated with alkylating agents in childhood are at risk for ovarian impairment. We aimed at describing the pattern of residual ovarian function in a cohort of survivors of hematological malignancies and/or hematopoietic stem cell transplantation (HSCT) and assessing the relationship between cyclophosphamide equivalent dose (CED) and anti-Müllerian hormone (AMH). DESIGN AND METHODS: Gonadal health was clinically and biochemically assessed in 124 post-menarchal survivors who underwent treatment for pediatric hematological malignancies and/or HSCT between 1992 and 2019. RESULTS: Overt 'premature ovarian insufficiency' (POI) was detected in 72.1 and 3.7% of transplanted and non-transplanted patients, respectively; milder 'diminished ovarian reserve' (DOR) in 16.3 and 22.2%. In non-transplanted patients, increasing CED values were associated with lower AMH-SDS (P = 0.04), with the threshold of 7200 g/m2 being the best discriminator between DOR/POI and normal ovarian function (AUC: 0.75 on ROC analysis) and with an observed decrease of 0.14 AMH-SDS for each CED increase of 1 g/m2. In addition, age at diagnosis ≥10 years played a detrimental role on ovarian reserve (P = 0.003). In the HSCT group, irradiation was associated with a statistically significant reduction in AMH-SDS (P = 0.04). CONCLUSIONS: In non-transplanted patients, CED ≥ 7200 mg/m2 was associated with a DOR, while younger age at diagnosis played a protective role on ovarian reserve. As a result of the data collected, we propose a systematic algorithm to assess iatrogenic gonadal impairment in young female patients exposed to chemo-radiotherapy in childhood for hematological disorders.

DMRT1 gene disruption alone induces incomplete gonad feminization in chicken.

Compared with the well-described XY sex determination system in mammals, the avian ZW sex determination system is poorly understood. Knockdown and overexpression studies identified doublesex and mab-3-related transcription factor 1 (DMRT1) as the testis-determining gene in chicken. However, the detailed effects of DMRT1 gene disruption from embryonic to adult development are not clear. Herein, we have generated DMRT1-disrupted chickens using the clustered regularly interspaced short palindromic repeats-associated protein 9 system, followed by an analysis of physiological, hormonal, and molecular changes in the genome-modified chickens. In the early stages of male chicken development, disruption of DMRT1 induced gonad feminization with extensive physiological and molecular changes; however, functional feminine reproductivity could not be implemented with disturbed hormone synthesis. Subsequent RNA-sequencing analysis of the DMRT1-disrupted chicken gonads revealed gene networks, including several novel genes linearly and non-linearly associated with DMRT1, which are involved in gonad feminization. By comparing the gonads of wild type with the genome-modified chickens, a set of genes were identified that is involved in the ZW sex determination system independent of DMRT1. Our results extend beyond the Z-dosage hypothesis to provide further information about the avian ZW sex determination system and epigenetic effects of gonad feminization.

Genetic homogeneity, lack of larvae recruitment, and clonality in absence of females across western Mediterranean populations of the starfish Coscinasterias tenuispina.

We here analysed the populations' genetic structure of Coscinasterias tenuispina, an Atlantic-Mediterranean fissiparous starfish, focusing on the western Mediterranean, to investigate: the distribution and prevalence of genetic variants, the relative importance of asexual reproduction, connectivity across the Atlantic-Mediterranean transition, and the potential recent colonisation of the Mediterranean Sea. Individuals from 11 Atlantic-Mediterranean populations of a previous study added to 172 new samples from five new W Mediterranean sites. Individuals were genotyped at 12 microsatellite loci and their gonads histologically analysed for sex determination. Additionally, four populations were genotyped at two-time points. Results demonstrated genetic homogeneity and low clonal richness within the W Mediterranean, due to the dominance of a superclone, but large genetic divergence with adjacent areas. The lack of new genotypes recruitment over time, and the absence of females, confirmed that W Mediterranean populations were exclusively maintained by fission and reinforced the idea of its recent colonization. The existence of different environmental conditions among basins and/or density-depend processes could explain this lack of recruitment from distant areas. The positive correlation between clonal richness and heterozygote excess suggests that most genetic diversity is retained within individuals in the form of heterozygosity in clonal populations, which might increase their resilience.

The embryonic ontogeny of the gonadal somatic cells in mice and monkeys.

In the early fetal stage, the gonads are bipotent and only later become the ovary or testis, depending on the genetic sex. Despite many studies examining how sex determination occurs from biopotential gonads, the spatial and temporal organization of bipotential gonads and their progenitors is poorly understood. Here, using lineage tracing in mice, we find that the gonads originate from a T+ primitive streak through WT1+ posterior intermediate mesoderm and appear to share origins anteriorly with the adrenal glands and posteriorly with the metanephric mesenchyme. Comparative single-cell transcriptomic analyses in mouse and cynomolgus monkey embryos reveal the convergence of the lineage trajectory and genetic programs accompanying the specification of biopotential gonadal progenitor cells. This process involves sustained expression of epithelial genes and upregulation of mesenchymal genes, thereby conferring an epithelial-mesenchymal hybrid state. Our study provides key resources for understanding early gonadogenesis in mice and primates.

Concerted morphogenesis of genital ridges and nephric ducts in the mouse captured through whole-embryo imaging.

During development of the mouse urogenital complex, the gonads undergo changes in three-dimensional structure, body position and spatial relationship with the mesonephric ducts, kidneys and adrenals. The complexity of genital ridge development obscures potential connections between morphogenesis and gonadal sex determination. To characterize the morphogenic processes implicated in regulating gonad shape and fate, we used whole-embryo tissue clearing and light sheet microscopy to assemble a time course of gonad development in native form and context. Analysis revealed that gonad morphology is determined through anterior-to-posterior patterns as well as increased rates of growth, rotation and separation in the central domain that may contribute to regionalization of the gonad. We report a close alignment of gonad and mesonephric duct movements as well as delayed duct development in a gonad dysgenesis mutant, which together support a mechanical dependency linking gonad and mesonephric duct morphogenesis.

Sex dimorphism in European sea bass (Dicentrarchus labrax L.): New insights into sex-related growth patterns during very early life stages.

The European sea bass (Dicentrarchus labrax) exhibits female-biased sexual size dimorphism (SSD) early in development. New tagging techniques provide the opportunity to monitor individual sex-related growth during the post-larval and juvenile stages. We produced an experimental population through artificial fertilization and followed a rearing-temperature protocol (~16°C from hatching to 112 days post-hatching, dph; ~20°C from 117 to 358 dph) targeting a roughly balanced sex ratio. The fish were tagged with microchips between 61 and 96 dph in five tagging trials of 50 fish each; individual standard length (SL) was recorded through repeated biometric measurements performed between 83 to 110 dph via image analyses. Body weight (BW) was modelled using the traits measured on the digital pictures (i.e. area, perimeter and volume). At 117 dph, the fish were tagged with microtags and regularly measured for SL and BW until 335 dph. The experiment ended at 358 dph with the sexing of the fish. The sex-ratio at the end of the experiment was significantly in favor of the females (65.6% vs. 34.4%). The females were significantly longer and heavier than the males from 103 dph (~30 mm SL, ~0.44 g BW) to 165 dph, but the modeling of the growth curves suggests that differences in size already existed at 83 dph. A significant difference in the daily growth coefficient (DGC) was observed only between 96 and 103 dph, suggesting a physiological or biological change occurring during this period. The female-biased SSD pattern in European sea bass is thus strongly influenced by very early growth differences between sexes, as already shown in previous studies, and in any case long before gonadal sex differentiation has been started, and thus probably before sex has been determined. This leads to the hypothesis that early growth might be a cause rather than a consequence of sex differentiation in sea bass.

Mechanisms of temperature modulation in mammalian seasonal timing.

Global warming is predicted to have major effects on the annual time windows during which species may successfully reproduce. At the organismal level, climatic shifts engage with the control mechanism for reproductive seasonality. In mammals, laboratory studies on neuroendocrine mechanism emphasize photoperiod as a predictive cue, but this is based on a restricted group of species. In contrast, field-oriented comparative analyses demonstrate that proximate bioenergetic effects on the reproductive axis are a major determinant of seasonal reproductive timing. The interaction between proximate energetic and predictive photoperiodic cues is neglected. Here, we focused on photoperiodic modulation of postnatal reproductive development in common voles (Microtus arvalis), a herbivorous species in which a plastic timing of breeding is well documented. We demonstrate that temperature-dependent modulation of photoperiodic responses manifest in the thyrotrophin-sensitive tanycytes of the mediobasal hypothalamus. Here, the photoperiod-dependent expression of type 2 deiodinase expression, associated with the summer phenotype was enhanced by 21°C, whereas the photoperiod-dependent expression of type 3 deiodinase expression, associated with the winter phenotype, was enhanced by 10°C in spring voles. Increased levels of testosterone were found at 21°C, whereas somatic and gonadal growth were oppositely affected by temperature. The magnitude of these temperature effects was similar in voles photoperiodical programmed for accelerated maturation (ie, born early in the breeding season) and in voles photoperiodical programmed for delayed maturation (ie, born late in the breeding season). The melatonin-sensitive pars tuberalis was relatively insensitive to temperature. These data define a mechanistic hierarchy for the integration of predictive temporal cues and proximate thermo-energetic effects in mammalian reproduction.

Involvement of glycolysis activation in flatfish sexual size dimorphism: Insights from transcriptomic analyses of Platichthys stellatus and Cynoglossus semilaevis.

The starry flounder (Platichthys stellatus), a flatfish cultured at the margins of the North Pacific, displays an obvious female-biased growth advantage, similar to many other fish species. To reveal the molecular mechanism underlying sexual size dimorphism, a comparative transcriptomic analysis of the somatotropic and reproductive axes was conducted. In total, 156, 67, 3434, and 378 differentially expressed genes (DEGs) between female and male samples were obtained in the brain, liver, gonad, and muscle tissues (q < 0.05). These DEGs were significantly enriched for various GO terms, including ion channel activity, protein binding, lipid transporter activity, and glycolytic process. The significantly enriched KEGG pathways included insulin secretion, axon guidance, and glycolysis/gluconeogenesis. In a detailed analysis of DEGs in these significantly enriched pathways, 35 genes showed higher expression levels in female muscle tissues than in male muscle tissues. A protein-protein interaction network further revealed specific interactions involving the glycolysis related-protein enolase (ENO), triosephosphate isomerase (TPI), Bisphosphoglycerate mutase (BPGM), fructose-bisphosphate aldolase (ALDO), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Interestingly, the role of glycolysis/gluconeogenesis was supported by an analysis of common DEGs between P. stellatus and Chinese tongue sole (Cynoglossus semilaevis). These results indicate that the activation of glycolysis in female muscle tissues contributes to flatfish sexual size dimorphism.

Circulating miR-30b levels increase during male puberty.

OBJECTIVE: The role of miRNA as endocrine regulators is emerging, and microRNA mir-30b has been reported to repress Mkrn3. However, the expression of miR-30b during male puberty has not been studied. DESIGN AND METHODS: Circulating relative miR-30b expression was assessed in sera of 26 boys with constitutional delay of growth and puberty (CDGP), treated with low-dose testosterone (T) (n =11) or aromatase inhibitor letrozole (Lz) (n =15) for 6 months and followed up to 12 months (NCT01797718). The associations between the relative expression of miR-30b and hormonal markers of puberty were evaluated. RESULTS: During the 12 months of the study, circulating miR-30b expression increased 2.4 ± 2.5 (s.d.) fold (P = 0.008) in all boys, but this change did not correlate with corresponding changes in LH, testosterone, inhibin B, FSH, or testicular volume (P = 0.25-0.96). Lz-induced activation of the hypothalamic-pituitary-gonadal (HPG) axis was associated with more variable miR-30b responses at 3 months (P < 0.05), whereas those treated with T exhibited significant changes in relative miR-30b levels in the course the study (P < 0.01-0.05). CONCLUSIONS: Circulating miR-30b expression in boys with CDGP increases in the course of puberty, and appears to be related to the activity of the HPG axis.

Broadening the role of osteocalcin in the hypothalamic-pituitary-gonadal axis.

Bone is emerging as a versatile endocrine organ and its interactions with apparently unrelated organs are being more widely recognized. Osteocalcin (OCN), a polypeptide hormone secreted by osteoblasts, has been found to exert multiple endocrine functions through its metabolically active form, uncarboxylated OCN (uOCN). Mounting evidence has shown that following its binding to G-protein coupled receptor 6a (Gprc6a) in the peripheral tissues, uOCN acts on pancreatic ß cells to increase insulin secretion, and on muscle and white adipose tissue to promote glucose and lipid metabolism. More strikingly, researchers have found a surprising role of uOCN in testicular function to facilitating testosterone biosynthesis and regulating male fertility via a pancreas-bone-gonadal axis. However, the detailed functional mechanisms of uOCN on the hypothalamic-pituitary-gonadal axis or the pancreas-bone-gonadal axis are not fully understood. Besides highlighting the regulatory mechanisms of uOCN in the hypothalamus and pituitary, we also discuss its role in male as well as female fertility and its potential clinical implications in some reproductive endocrine diseases and pubertal developmental disorders.

The conditional deletion of steroidogenic factor 1 (Nr5a1) in Sox9-Cre mice compromises testis differentiation.

Steroidogenic factor 1 (NR5A1) is essential for gonadal development. To study the importance of NR5A1 during early gonadal sex differentiation, we generated Sox9-Cre-Nr5a1 conditional knockout (cKO) mice: Sox9-Cre;Nr5a1flox/flox and Sox9-Cre;Nr5a1flox/- mice. Double-immunostaining for NR5A1 and AMH revealed silenced NR5A1 in Sertoli cells and reduced AMH+ cells in the gonads of XY Sox9-Cre-Nr5a1 cKO mice between embryonic days 12.5 (E12.5) and E14.5. Double-immunostaining for SOX9 and FOXL2 further indicated an early block in Sertoli cells and ectopic granulosa cell differentiation. The number of cells expressing the Leydig cell marker 3ßHSD obviously reduced in the gonads of XY Sox9-Cre;Nr5a1flox/- but not Sox9-Cre;Nr5a1flox/flox mice at E15.5. The presence of STRA8+ cells indicated that germ cells entered meiosis in the gonads of XY Sox9-Cre-Nr5a1 cKO mice. The results of qRT-PCR revealed remarkably reduced and elevated levels of testis and ovary markers, respectively, in the gonads of XY Sox9-Cre-Nr5a1 cKO mice at E12.5‒E13.5. These data suggested that the loss of Nr5a1 abrogates the testicular pathway and induces the ectopic ovarian pathway, resulting in postnatal partial/complete male-to-female gonadal sex reversal. Our findings provide evidence for the critical role of NR5A1 in murine gonadal sex determination in vivo.