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.

Estrogen Signaling Inhibits the Expression of anti-Müllerian hormone (amh) and gonadal-soma-derived factor (gsdf) during the Critical Time of Sexual Fate Determination in Zebrafish.

The mechanism of fish gonadal sex differentiation is complex and regulated by multiple factors. It has been widely known that proper steroidogenesis in Leydig cells and sex-related genes in Sertoli cells play important roles in gonadal sex differentiation. In teleosts, the precise interaction of these signals during the sexual fate determination remains elusive, especially their effect on the bi-potential gonad during the critical stage of sexual fate determination. Recently, all-testis phenotypes have been observed in the cyp17a1-deficient zebrafish and common carp, as well as in cyp19a1a-deficient zebrafish. By mating cyp17a1-deficient fish with transgenic zebrafish Tg(piwil1:EGFP-nanos3UTR), germ cells in the gonads were labelled with enhanced green fluorescent protein (EGFP). We classified the cyp17a1-deficient zebrafish and their control siblings into primordial germ cell (PGC)-rich and -less groups according to the fluorescence area of the EGFP labelling. Intriguingly, the EGFP-labelled bi-potential gonads in cyp17a1+/+ fish from the PGC-rich group were significantly larger than those of the cyp17a1-/- fish at 23 days post-fertilization (dpf). Based on the transcriptome analysis, we observed that the cyp17a1-deficient fish of the PGC-rich group displayed a significantly upregulated expression of amh and gsdf compared to that of control fish. Likewise, the upregulated expressions of amh and gsdf were observed in cyp19a1a-deficient fish as examined at 23 dpf. This upregulation of amh and gsdf could be repressed by treatment with an exogenous supplement of estradiol. Moreover, tamoxifen, an effective antagonist of both estrogen receptor α and ß (ERα and Erß), upregulates the expression of amh and gsdf in wild-type (WT) fish. Using the cyp17a1- and cyp19a1a-deficient zebrafish, we provide evidence to show that the upregulated expression of amh and gsdf due to the compromised estrogen signaling probably determines their sexual fate towards testis differentiation. Collectively, our data suggest that estrogen signaling inhibits the expression of amh and gsdf during the critical time of sexual fate determination, which may broaden the scope of sex steroid hormones in regulating gonadal sex differentiation in fish.

The -KTS splice variant of WT1 is essential for ovarian determination in mice.

Sex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pregranulosa cells that govern testis and ovary development, respectively. Although the Y-linked testis-determining gene Sry has been identified, the ovarian-determining factor remains unknown. In this study, we identified -KTS, a major, alternatively spliced isoform of the Wilms tumor suppressor WT1, as a key determinant of female sex determination. Loss of -KTS variants blocked gonadal differentiation in mice, whereas increased expression, as found in Frasier syndrome, induced precocious differentiation of ovaries independently of their genetic sex. In XY embryos, this antagonized Sry expression, resulting in male-to-female sex reversal. Our results identify -KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in gonadal sex differentiation.

How do maternal androgens and estrogens affect sex determination in reptiles with temperature-dependent sex?

Temperature sex determination (TSD) in reptiles has been studied to elucidate the mechanisms by which temperature is transformed into a biological signal that determines the sex of the embryo. Temperature is thought to trigger signals that alter gene expression and hormone metabolism, which will determine the development of female or male gonads. In this review, we focus on collecting and discussing important and recent information on the role of maternal steroid hormones in sex determination in oviparous reptiles such as crocodiles, turtles, and lizards that possess TSD. In particular, we focus on maternal androgens and estrogens deposited in the egg yolk and their metabolites that could also influence the sex of offspring. Finally, we suggest guidelines for future research to help clarify the link between maternal steroid hormones and offspring sex.

Gonadal transcriptomes reveal sex-biased expression genes associated with sex determination and differentiation in red-tail catfish (Hemibagrus wyckioides).

BACKGROUND: Red-tail catfish (Hemibagrus wyckioides) is an important commercially farmed catfish in southern China. Males of red-tail catfish grow faster than females, suggesting that all-male catfish will produce more significant economic benefits in aquaculture practice. However, little research has been reported on sex determination and gonadal development in red-tail catfish. RESULTS: In this study, we performed the first transcriptomic analysis of male and female gonads at four developmental stages at 10, 18, 30, and 48 days post hatching (dph) using RNA-seq technology. A total of 23,588 genes were screened in 24 sequenced samples, of which 28, 213, 636, and 1381 differentially expressed genes (DEGs) were detected at four developmental stages, respectively. Seven candidate genes of sex determination and differentiation were further identified. Real-time quantitative PCR (RT-qPCR) further confirmed that anti-Mullerian hormone (amh), growth differentiation factor 6a (gdf6a), testis-specific gene antigen 10 (tsga10), and cytochrome P450 family 17 subfamily A (cyp17a) were highly expressed mainly in the male, while cytochrome P450 family 19 subfamily A polypeptide 1b (cyp19a1b), forkhead box L2 (foxl2), and hydroxysteroid 17-beta dehydrogenase 1 (hsd17b1) were highly expressed in the female. The KEGG pathway enrichment data showed that these identified DEGs were mainly involved in steroid hormone biosynthesis and TGF-ß signaling pathways. CONCLUSIONS: Based on RNA-seq data of gonads at the early developmental stages, seven DEGs shared by the four developmental stages were identified, among which amh and gdf6a may be the male-biased expression genes, while foxl2, cyp19a1b and hsd17b1 may be the female-biased expression genes in red-tail catfish. Our study will provide crucial genetic information for the research on sex control in red-tail catfish, as well as for exploring the evolutionary processes of sex determination mechanisms in fish.

Loss- and gain-of-function analyses reveal the essential role of Cyp19a1 in ovarian determination of the red-eared slider turtle.

Estrogen signaling exerts a decisive role in female sex determination and differentiation in chicken and fish. Aromatase encoded by Cyp19a1 is the key enzyme that catalyzes the conversion of androgen to estrogen. Correlative analyses implicate the potential involvement of aromatase in reptilian sexual development, however, the direct genetic evidence is lacking. Herein, we found that Cyp19a1 exhibited temperature-dependent sexually dimorphic expression, and located in the medullary somatic cells in early female embryos of the red-eared slider turtle (Trachemys scripta elegans), before the gonad is distinct. To determine the functional role of Cyp19a1 in turtle ovarian determination, we established loss- and gain-of-function models through in ovo lentivirus-mediated genetic manipulation. At female-producing temperature, inhibition of aromatase or knockdown of Cyp19a1 in turtle embryos resulted in female-to-male sex reversal, with the formation of a testis-like structure and a male distribution pattern of germ cells, as well as ectopic expression of male-specific markers (SOX9 and AMH) and disappearance of ovarian regulator FOXL2. On the contrary, overexpression of Cyp19a1 at male-producing temperature led to male-to-female sex reversal. In conclusion, our results suggest that Cyp19a1 is both necessary and sufficient for ovarian determination in the red-eared slider turtle, establishing causality and a direct genetic link between aromatase and reptilian sex determination and differentiation.

Role and function of the Hintw in early sex differentiation in chicken (Gallus gallus) embryo.

Mono-Sex culturing is an important methodology for intensive livestock and poultry production. Here, Hintw was identified as a potential key gene in sex-determination process in chickens via RNA-seq. Then we developed an effective method to interfere or overexpress Hintw in chicken embryos through the intravascular injection. QRT-PCR, ELISA and H&E staining were used to detect the effects of Hintw on gonadal development of chicken embryos. Results showed that Hintw exhibited a female-biased expression pattern in the early stage of PGCs (primordial germ cells) in embryonic gonads. The qRT-PCR analysis showed that Foxl2, Cyp19a1 in females were upregulated under the overexpression of Hintw, while Sox9 and Dmrt1 were downregulated Hintw. Overexpression of Hintw can promote the development of gonadal cortex, while interference with Hintw show the opposite result. Additionally, we found that overexpression of the Hintw in male chicken embryos could inhibit androgen levels and increase estrogen levels. On the other hand, interfering with Hintw in female chicken embryos decreased estrogen levels and increased androgen levels. In conclusion, this work sets the basis for the understanding of the molecular regulatory network for the sex-determination process in chicken embryos as well as providing the theoretical basis for mono-sex culturing of poultry.

Stable and persistent male-like behavior during male-to-female sex change in the common clownfish Amphiprion ocellaris.

Many fish species exhibit natural sex change as part of their life, providing unique opportunities to study sexually-differentiated social behaviors and their plasticity. Past research has shown that behavioral sex change in the female-to-male (protogynous) direction occurs rapidly and well before gonadal sex change. However, little is known about the timecourse of behavioral sex change in male-to-female (protandrous) sex-changing species, limiting our ability to compare patterns of behavioral sex change across species and identify conserved or divergent underlying mechanisms. Using the protandrous sex changing anemonefish Amphiprion ocellaris, we assessed behavior (aggression and parental care) and hormones (estradiol and 11-ketotestosterone) in fish over six months of sex change, and compared those fish against their non-changing partners as well as control males and females. Contrary to expectations, we found that sex-changing fish displayed behavior that was persistently male-like, and that their behavior did not become progressively female-like as sex change progressed. Hormones shifted to an intermediate profile between males and females and remained stable until gonads changed. These results support a new perspective that the timecourse for protandrous sex change in anemonefish is completely distinct from other well-established models, such that behavioral sex change does not occur until after gonadal sex change is complete, and that sex-changing fish have a stable and unique behavioral and hormonal phenotype that is distinct from a male-typical or female-typical phenotype. The results also identify aspects of sex change that may fundamentally differ between protandrous and protogynous modes, motivating further research into these remarkable examples of phenotypic plasticity.

Temperature fluctuations and estrone sulfate affect gene expression via different mechanisms to promote female development in a species with temperature-dependent sex determination.

Variation in developmental conditions can affect a variety of embryonic processes and shape a number of phenotypic characteristics that can affect offspring throughout their lives. This is particularly true of oviparous species where development typically occurs outside of the female, and studies have shown that traits such as survival and behavior can be altered by both temperature and exposure to steroid hormones during development. In species with temperature-dependent sex determination (TSD), the fate of gonadal development can be affected by temperature and by maternal estrogens present in the egg at oviposition, and there is evidence that these factors can affect gene expression patterns. Here, we explored how thermal fluctuations and exposure to an estrogen metabolite, estrone sulfate, affect the expression of several genes known to be involved in sexual differentiation: Kdm6b, Dmrt1, Sox9, FoxL2 and Cyp19A1. We found that most of the genes responded to both temperature and estrone sulfate exposure, but that the responses to these factors were not identical, in that estrone sulfate effects occur downstream of temperature effects. Our findings demonstrate that conjugated hormones such as estrone sulfate are capable of influencing temperature-dependent pathways to potentially alter how embryos respond to temperature, and highlight the importance of studying the interaction of maternal hormone and temperature effects.

Isolation and Characterization of Germline Stem Cells in Protogynous Hermaphroditic Monopterus albus.

Germline stem cells (GSCs) are a group of unique adult stem cells in gonads that act as important transmitters for genetic information. Donor GSCs have been used to produce offspring by transplantation in fisheries. In this study, we successfully isolated and enriched GSCs from the ovary, ovotestis, and testis of Monopterus albus, one of the most important breeding freshwater fishes in China. Transcriptome comparison assay suggests that a distinct molecular signature exists in each type of GSC, and that different signaling activities are required for the maintenance of distinct GSCs. Functional analysis shows that fGSCs can successfully colonize and contribute to the germline cell lineage of a host zebrafish gonad after transplantation. Finally, we describe a simple feeder-free method for the isolation and enrichment of GSCs that can contribute to the germline cell lineage of zebrafish embryos and generate the germline chimeras after transplantation.

Loss of Raptor induces Sertoli cells into an undifferentiated state in mice.

In mammals, testis development is triggered by the expression of the sex-determining Y-chromosome gene SRY to commit the Sertoli cell (SC) fate at gonadal sex determination in the fetus. Several genes have been identified to be required to promote the testis pathway following SRY activation (i.e., SRY box 9 (SOX9)) in an embryo; however, it largely remains unknown about the genes and the mechanisms involved in stabilizing the testis pathway after birth and throughout adulthood. Herein, we report postnatal males with SC-specific deletion of Raptor demonstrated the absence of SC unique identity and adversely acquired granulosa cell-like characteristics, along with loss of tubular architecture and scattered distribution of SCs and germ cells. Subsequent genome-wide analysis by RNA sequencing revealed a profound decrease in the transcripts of testis genes (i.e., Sox9, Sox8, and anti-Mullerian hormone (Amh)) and, conversely, an increase in ovary genes (i.e., LIM/Homeobox gene 9 (Lhx9), Forkhead box L2 (Foxl2) and Follistatin (Fst)); these changes were further confirmed by immunofluorescence and quantitative reverse-transcription polymerase chain reaction. Importantly, co-immunofluorescence demonstrated that Raptor deficiency induced SCs dedifferentiation into a progenitor state; the Raptor-mutant gonads showed some ovarian somatic cell features, accompanied by enhanced female steroidogenesis and elevated estrogen levels, yet the zona pellucida 3 (ZP3)-positive terminally feminized oocytes were not observed. In vitro experiments with primary SCs suggested that Raptor is likely involved in the fibroblast growth factor 9 (FGF9)-induced formation of cell junctions among SCs. Our results established that Raptor is required to maintain SC identity, stabilize the male pathway, and promote testis development.

Methamphetamine can induce alteration of histopathology and sex determination gene expression through the oxidative stress pathway in the testes of human post-mortem.

Methamphetamine is a recreational drug that can be taken ingestion orally, injected, smoked or snorted. Methamphetamine abuse may lead to male infertility. The purpose of this study was to evaluate the long-term effects of methamphetamine abuse on the sex reprogramming of human post-mortem testis. Testes were collected from the autopsies of methamphetamine users (n = 10) and healthy males (reference group) (n = 10). They were then taken for stereological studies and RNA extraction to evaluate the expressions of PCNA, DMRT1, SOX8, c-Kit, TNF-α, IL6 and FOXL2 genes. In addition, Reactive Oxygen Species (ROS) level and Glutathione Disulfide (GSH) were assessed. Autopsied testicular samples of methamphetamine revealed a significant reduction in stereological parameters and histopathological findings, suggesting methamphetamine as a practical approach to prevention strategies in reproductive medicine that can disrupt spermatogenesis. Moreover, the results indicated the expressions of the genes involved in testis function and male-to-female genetic reprogramming (PCNA, DMRT1, SOX8, c-Kit, TNF-α, IL6 and FOXL2) (16) as well as in increasing inflammation (TNF-α and IL-6). The results also showed a high level of ROS and a decrease in GSH activity. The results of SOX9 immunohistochemistry indicated a significant decrease in the expression of SOX9 as well as in the number of Sertoli cells in the methamphetamine group. Overall, the results suggested that methamphetamine abuse caused spermatogenesis disruption and genetic reprogramming, probably through oxidative stress and changes in the expression of sex-determining genes.

Sex-specific splicing of Z- and W-borne nr5a1 alleles suggests sex determination is controlled by chromosome conformation.

Pogona vitticeps has female heterogamety (ZZ/ZW), but the master sex-determining gene is unknown, as it is for all reptiles. We show that nr5a1 (Nuclear Receptor Subfamily 5 Group A Member 1), a gene that is essential in mammalian sex determination, has alleles on the Z and W chromosomes (Z-nr5a1 and W-nr5a1), which are both expressed and can recombine. Three transcript isoforms of Z-nr5a1 were detected in gonads of adult ZZ males, two of which encode a functional protein. However, ZW females produced 16 isoforms, most of which contained premature stop codons. The array of transcripts produced by the W-borne allele (W-nr5a1) is likely to produce truncated polypeptides that contain a structurally normal DNA-binding domain and could act as a competitive inhibitor to the full-length intact protein. We hypothesize that an altered configuration of the W chromosome affects the conformation of the primary transcript generating inhibitory W-borne isoforms that suppress testis determination. Under this hypothesis, the genetic sex determination (GSD) system of P. vitticeps is a W-borne dominant female-determining gene that may be controlled epigenetically.

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.

Identification and expression pattern of the sex determination gene fruitless-like in Cherax quadricarinatus.

The fruitless (fru) gene has an important function in the courtship behavior and sex determination pathway of Drosophila melanogaster; however, the fru gene has never been reported in shrimps. In this study, the fruitless-like gene was identified in Cherax quadricarinatus (Cqfru) and is reported here for the first time. A sequence analysis revealed a conserved BTB domain in Cqfru which is the same as fru in D. melanogaster. An analysis of the expression level of Cqfru showed that it was highly expressed in the gastrula stage during embryonic development. Furthermore, in situ hybridization and expression distribution in tissues showed that its sexually dimorphic expression may be focused on the hepatopancreas, brains, and gonads. The gonads, brains, and hepatopancreas of males had a higher expression level of Cqfru than those of females; however, the expression level of the abdominal ganglion was found to be higher in females than in males in this study. The results of an RNA interference treatment showed that a knockdown of Cqfru reduced the expression of the insulin-like androgenic gland hormone (IAG) and tumor necrosis factor (TNF). The characteristic fru gene in shrimps is reported here for the first time, with the results providing basic information for research into the sex-determination mechanism in C. quadricarinatus.

Estrogenic signaling and sociosexual behavior in wild sex-changing bluehead wrasses, Thalassoma bifasciatum.

Estrogenic signaling is an important focus in studies of gonadal and brain sexual differentiation in fishes and vertebrates generally. This study examined variation in estrogenic signaling (1) across three sexual phenotypes (female, female-mimic initial phase [IP] male, and terminal phase [TP] male), (2) during socially-controlled female-to-male sex change, and (3) during tidally-driven spawning cycles in the protogynous bluehead wrasse (Thalassoma bifasciatum). We analyzed relative abundances of messenger RNAs (mRNAs) for the brain form of aromatase (cyp19a1b) and the three nuclear estrogen receptors (ER) (ERα, ERßa, and ERßb) by qPCR. Consistent with previous reports, forebrain/midbrain cyp19a1b was highest in females, significantly lower in TP males, and lowest in IP males. By contrast, ERα and ERßb mRNA abundances were highest in TP males and increased during sex change. ERßa mRNA did not vary significantly. Across the tidally-driven spawning cycle, cyp19a1b abundances were higher in females than TP males. Interestingly, cyp19a1b levels were higher in TP males close (~1 h) to the daily spawning period when sexual and aggressive behaviors rise than males far from spawning (~10-12 h). Together with earlier findings, our results suggest alterations in neural estrogen signaling are key regulators of socially-controlled sex change and sexual phenotype differences. Additionally, these patterns suggest TP male-typical sociosexual behaviors may depend on intermediate rather than low estrogenic signaling. We discuss these results and the possibility that an inverted-U shaped relationship between neural estrogen and male-typical behaviors is more common than presently appreciated.

Successful Spermatogonial Stem Cells Transplantation within Pleuronectiformes: First Breakthrough at inter-family Level in Marine Fish.

As a promising biotechnology, fish germ cell transplantation shows potentials in conservation germplasm resource, propagation of elite species, and generation of transgenic individuals. In this study, we successfully transplanted the Japanese flounder (P. olivaceus), summer flounder (P. dentatus), and turbot (S. maximus) spermatogonia into triploid Japanese flounder larvae, and achieved high transplantation efficiency of 100%, 75-95% and 33-50% by fluorescence tracking and molecular analysis, respectively. Eventually, donor-derived spermatozoa produced offspring by artificial insemination. We only found male and intersex chimeras in inter-family transplantations, while male and female chimeras in both intra-species and intra-genus transplantations. Moreover, the intersex chimeras could mature and produce turbot functional spermatozoa. We firstly realized inter-family transplantation in marine fish species. These results demonstrated successful spermatogonial stem cells transplantation within Pleuronectiformes, suggesting the germ cells migration, incorporation and maturation within order were conserved across a wide range of teleost species.

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.

Cyp17a1 is Required for Female Sex Determination and Male Fertility by Regulating Sex Steroid Biosynthesis in Fish.

In teleost fish, sex steroids are involved in sex determination, sex differentiation, and fertility. Cyp17a1 (Cytochrome P450 family 17 subfamily A member 1) is thought to play essential roles in fish steroidogenesis. Therefore, to further understand its roles in steroidogenesis, sex determination, and fertility in fish, we constructed a cyp17a1 gene mutant in Nile tilapia (Oreochromis niloticus). In XX fish, mutation of the cyp17a1 gene led to a female-to-male sex reversal with a significant decline in 17ß-estradiol (E2) and testosterone (T) production, and ectopic expression of male-biased markers (Dmrt1 and Gsdf) in gonads from the critical window of sex determination. Sex reversal was successfully rescued via T or E2 administration, and ovarian characteristics were maintained after termination of E2 supplementation in the absence of endogenous estrogen production in cyp17a1-/- XX fish. Likewise, deficiencies in T and 11-ketotestosterone (11-KT) production in both cyp17a1-/- XX sex-reversed males and cyp17a1-/- XY mutants resulted in meiotic initiation delays, vas deferens obstruction and sterility due to excessive apoptosis and abnormal mitochondrial morphology. However, 11-KT treatment successfully rescued the dysspermia to produce normal sperm in cyp17a1-/- male fish. Significant increases in gonadotropic hormone (gth) and gth receptors in cyp17a1-/- mutants may excessively upregulate steroidogenic gene expression in Leydig cells through a feedback loop. Taken together, our findings demonstrate that Cyp17a1 is indispensable for E2 production, which is fundamental for female sex determination and differentiation in XX tilapia. Additionally, Cyp17a1 is essential for T and 11-KT production, which further promotes spermatogenesis and fertility in XY males.

Autosomal sex-associated co-methylated regions predict biological sex from DNA methylation.

Sex is a modulator of health that has been historically overlooked in biomedical research. Recognizing this knowledge gap, funding agencies now mandate the inclusion of sex as a biological variable with the goal of stimulating efforts to illuminate the molecular underpinnings of sex biases in health and disease. DNA methylation (DNAm) is a strong molecular candidate for mediating such sex biases; however, a robust and well characterized annotation of sex differences in DNAm is yet to emerge. Beginning with a large (n = 3795) dataset of DNAm profiles from normative adult whole blood samples, we identified, validated and characterized autosomal sex-associated co-methylated genomic regions (sCMRs). Strikingly, sCMRs showed consistent sex differences in DNAm over the life course and a subset were also consistent across cell, tissue and cancer types. sCMRs included sites with known sex differences in DNAm and links to health conditions with sex biased effects. The robustness of sCMRs enabled the generation of an autosomal DNAm-based predictor of sex with 96% accuracy. Testing this tool on blood DNAm profiles from individuals with sex chromosome aneuploidies (Klinefelter [47,XXY], Turner [45,X] and 47,XXX syndrome) revealed an intimate relationship between sex chromosomes and sex-biased autosomal DNAm.