Zebrafish serve as a valuable model organism for studying germ cell biology and reproductive processes. The AB strain of zebrafish is proposed to exhibit a polygenic sex determination system, where most males initially develop juvenile ovaries before committing to male fate. In species with chromosomal sex determination, gonadal somatic cells are recognized as key determinants of germ cell fate. Notably, the loss of germ cells in zebrafish leads to masculinization, implying that germ cells harbor an intrinsic feminization signal. However, the specific signal triggering oogenesis in zebrafish remains unclear. In the present study, we identified foxl2l as an oocyte progenitor-specific gene essential for initiating oogenesis in germ cells. Results showed that foxl2l-knockout zebrafish bypassed the juvenile ovary stage and exclusively developed into fertile males. Further analysis revealed that loss of foxl2l hindered the initiation of oocyte-specific meiosis and prevented entry into oogenesis, leading to premature spermatogenesis during early gonadal development. Furthermore, while mutation of the pro-male gene dmrt1 led to fertile female differentiation, simultaneous disruption of foxl2l in dmrt1 mutants completely blocked oogenesis, with a large proportion of germ cells arrested as germline stem cells, highlighting the crucial role of foxl2l in oogenesis. Overall, this study highlights the unique function of foxl2l as a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.
斑马鱼是研究生殖生物学的重要模型。AB品系的斑马鱼拥有多基因性别决定系统，大多数雄性的性腺最初会发育为类卵巢，然后再逆转为精巢。在具有染色体性别决定系统的物种中，性腺体细胞在决定生殖细胞命运中起关键作用。有趣的是，一系列研究发现生殖细胞的丧失会导致斑马鱼雄性化，这意味着生殖细胞自身存在向雌性发育的能力。然而，何种信号在斑马鱼中起始卵子发生仍不清楚。在该研究中，我们鉴定出一个卵母细胞前体特异性表达的基因 foxl2l，它是生殖细胞进入卵子发生的不可或缺的信号。我们发现， foxl2l敲除的斑马鱼的性腺发育不经历类卵巢阶段，而直接发育为可育的雄性。进一步的研究揭示， foxl2l的丧失阻碍了卵母细胞中减数分裂的启动，并阻止了卵子发生的起始，从而导致在性腺发育早期提前开启精子发生。此外，我们发现尽管促雄基因 dmrt1的突变会导致斑马鱼大部分发育为雌性，在 dmrt1突变体中同时敲除 foxl2l会导致卵子发生完全阻断，大量生殖细胞细胞停留在未分化状态，进一步证明了 foxl2l在卵子发生中的重要作用。综上，这项研究揭示了 foxl2l在斑马鱼生殖细胞向卵母细胞分化过程中，发挥“看门人”的独特作用。.

In reptiles, such as the red-eared slider turtle ( Trachemys scripta elegans), gonadal sex determination is highly dependent on the environmental temperature during embryonic stages. This complex process, which leads to differentiation into either testes or ovaries, is governed by the finely tuned expression of upstream genes, notably the testis-promoting gene Dmrt1 and the ovary-promoting gene Foxl2. Recent studies have identified epigenetic regulation as a crucial factor in testis development, with the H3K27me3 demethylase KDM6B being essential for Dmrt1 expression in T. s. elegans. However, whether KDM6B alone can induce testicular differentiation remains unclear. In this study, we found that overexpression of Kdm6b in T. s. elegans embryos induced the male development pathway, accompanied by a rapid increase in the gonadal expression of Dmrt1 at 31°C, a temperature typically resulting in female development. Notably, this sex reversal could be entirely rescued by Dmrt1 knockdown. These findings demonstrate that Kdm6b is sufficient for commitment to the male pathway, underscoring its role as a critical epigenetic regulator in the sex determination of the red-eared slider turtle.
许多爬行动物（如红耳龟）的性别取决于胚胎发育的环境温度。该性别决定过程涉及一系列上游基因如促睾丸分化的 Dmrt1及促卵巢分化的 Foxl2的精细调控。作者前期研究表明，组蛋白去甲基化酶KDM6B的表达是直接激活 Dmrt1转录的必要条件。然而，KDM6B是否能单独诱导睾丸分化尚不清楚。在该研究中，我们发现在产雌温度下对红耳龟胚胎进行 Kdm6b过表达会迅速上调性腺中 Dmrt1的表达，并诱导性腺分化为睾丸。此外，敲低 Dmrt1能够阻断 Kdm6b过表达导致的雌向雄性逆转过程，性腺最终仍发育成卵巢。实验结果表明 Kdm6b通过上调 Dmrt1使性腺发育成睾丸。因此，KDM6B是红耳龟性别决定过程中的关键表观遗传调控因子。.

BACKGROUND: Chinese medaka (Oryzias sinensis) is widely distributed in freshwater rivers in China. Similar to the medaka (Oryzias latipes), Chinese medaka has the characteristics of small size, rapid reproductive cycle, and strong adaptability, which makes it suitable as a model organism for studies in basic biology and environmental toxicology. Chinese medaka exhibits distinct sexual dimorphism. However, due to the lack of complete genomic information, the regulation of sex determination and differentiation-related genes in Chinese medaka remains unclear.
METHODS: Chinese medaka dmrt1 (Osdmrt1) was cloned by PCR, and transgenic individuals of medaka [Tg(CMV:Osdmrt1)] overexpressing Osdmrt1 were generated to investigate the role of Osdmrt1 in sex determination. Western blot was used to validate the integration of the Osdmrt1 into the medaka genome. Tissue sectioning and HE staining were used to identify Tg(CMV:Osdmrt1) physiological gender and phenotype. qRT-PCR was used to analyze the expression of gonad-specific genes.
RESULTS: Osdmrt1 was cloned and identified, and it shared similar evolutionary relationships with medaka dmrt1. Tg(CMV:Osdmrt1) exhibited partial sex reversal from female to male in the F2 generation, with genetically female individuals developing testes and producing functional sperm. Additionally, the secondary sexual characteristics of the transgenic females also changed to males.
CONCLUSIONS: The Chinese medaka dmrt1 gene could convert females to males in medaka.
CONCLUSIONS: These results not only elucidate the function of Chinese medaka dmrt1, but also accumulate knowledge for studying the function of economically important fish genes in model fish by transgenic technology.

The testis-specific double sex and mab-3-related transcription factor 1 (DMRT1) has long been recognized as a crucial player in sex determination across vertebrates, and its essential role in gonadal development and the regulation of spermatogenesis is well established. Here, we report the cloning of the key spermatogenesis-related DMRT1 cDNA, named Tc-DMRT1, from the gonads of Tridacna crocea (T. crocea), with a molecular weight of 41.93 kDa and an isoelectric point of 7.83 (pI). Our hypothesis is that DMRT1 machinery governs spermatogenesis and regulates gonadogenesis. RNAi-mediated Tc-DMRT1 knockdown revealed its critical role in hindering spermatogenesis and reducing expression levels in boring giant clams. A histological analysis showed structural changes, with normal sperm cell counts in the control group (ds-EGFP) but significantly lower concentrations of sperm cells in the experimental group (ds-DMRT1). DMRT1 transcripts during embryogenesis exhibited a significantly high expression pattern (p < 0.05) during the early zygote stage, and whole-embryo in-situ hybridization confirmed its expression pattern throughout embryogenesis. A qRT-PCR analysis of various reproductive stages revealed an abundant expression of Tc-DMRT1 in the gonads during the male reproductive stage. In-situ hybridization showed tissue-specific expression of DMRT1, with a positive signal detected in male-stage gonadal tissues comprising sperm cells, while no signal was detected in other stages. Our study findings provide an initial understanding of the DMRT1 molecular machinery controlling spermatogenesis and its specificity in male-stage gonads of the key bivalve species, Tridacna crocea, and suggest that DMRT1 predominantly functions as a key regulator of spermatogenesis in giant clams.

Disorders of sexual development (DSDs) encompass a group of congenital conditions associated with atypical development of internal and external genital structures. Among those with DSDs are 46,XX males, whose condition mainly arises due to the translocation of SRY onto an X chromosome or an autosome. In the few SRY-negative 46,XX males, overexpression of other pro-testis genes or failure of pro-ovarian/anti-testis genes may be involved, even if a non-negligible number of cases remain unexplained. A three-year-old boy with an SRY-negative 46,XX karyotype showed a normal male phenotype and normal prepubertal values for testicular hormones. A heterozygous de novo in tandem duplication of 50,221 bp, which encompassed exons 2 and 3 of the Doublesex and Mab-3-related transcription factor 1 (DMRT1) gene, was detected using MPLA, CGH-array analysis, and Sanger sequencing. Both breakpoints were in the intronic regions, and this duplication did not stop or shift the coding frame. Additional pathogenic or uncertain variants were not found in a known pro-testis/anti-ovary gene cascade using a custom NGS panel and whole genome sequencing. The duplication may have allowed DMRT1 to escape the transcriptional repression that normally occurs in 46,XX fetal gonads and thus permitted the testicular determination cascade to switch on. So far, no case of SRY-negative 46,XX DSD with alterations in DMRT1 has been described.

Exosomes are endosome-derived extracellular vesicles about 100 nm in diameter. They are emerging as promising delivery platforms due to their advantages in biocompatibility and engineerability. However, research into and applications for engineered exosomes are still limited to a few areas of medicine in mammals. Here, we expanded the scope of their applications to sex-determining gene studies in early vertebrates. An integrated strategy for constructing the exosome-based delivery system was developed for efficient regulation of dmrt1, which is one of the most widely used sex-determining genes in metazoans. By combining classical methods in molecular biology and the latest technology in bioinformatics, isomiR-124a was identified as a dmrt1 inhibitor and was loaded into exosomes and a testis-targeting peptide was used to modify exosomal surface for efficient delivery. Results showed that isomiR-124a was efficiently delivered to the testes by engineered exosomes and revealed that dmrt1 played important roles in maintaining the regular structure and function of testis in juvenile fish. This is the first de novo development of an exosome-based delivery system applied in the study of sex-determining gene, which indicates an attractive prospect for the future applications of engineered exosomes in exploring more extensive biological conundrums.

Bacterial or viral infections, such as Brucella, mumps virus, herpes simplex virus, and Zika virus, destroy immune homeostasis of the testes, leading to spermatogenesis disorder and infertility. Of note, recent research shows that SARS-CoV-2 can infect male gonads and destroy Sertoli and Leydig cells, leading to male reproductive dysfunction. Due to the many side effects associated with antibiotic therapy, finding alternative treatments for inflammatory injury remains critical. Here, we found that Dmrt1 plays an important role in regulating testicular immune homeostasis. Knockdown of Dmrt1 in male mice inhibited spermatogenesis with a broad inflammatory response in seminiferous tubules and led to the loss of spermatogenic epithelial cells. Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) revealed that Dmrt1 positively regulated the expression of Spry1, an inhibitory protein of the receptor tyrosine kinase (RTK) signaling pathway. Furthermore, immunoprecipitation-mass spectrometry (IP-MS) and co-immunoprecipitation (Co-IP) analysis indicated that SPRY1 binds to nuclear factor kappa B1 (NF-κB1) to prevent nuclear translocation of p65, inhibit activation of NF-κB signaling, prevent excessive inflammatory reaction in the testis, and protect the integrity of the blood-testis barrier. In view of this newly identified Dmrt1- Spry1-NF-κB axis mechanism in the regulation of testicular immune homeostasis, our study opens new avenues for the prevention and treatment of male reproductive diseases in humans and livestock.
细菌或病毒感染会破坏睾丸的免疫稳态，如布鲁氏菌、腮腺炎病毒、单纯疱疹病毒和寨卡病毒，都会导致精子发生障碍和不育。最新研究表明，SARS-CoV-2可感染男性性腺，破坏支持细胞和间质细胞，并导致男性生殖功能障碍。由于抗生素治疗的局限性，迫切需要探寻生殖系统炎症的替代治疗方法。在该研究中，我们发现 Dmrt1在调节睾丸免疫稳态中发挥着重要作用。 Dmrt1缺陷小鼠的生精能力降低，曲细精小管中存在广泛的炎症反应，导致生精上皮细胞的脱落和丢失。ChIP-seq和RNA-seq分析表明， Dmrt1直接调节许多与炎症途径相关的基因且正向调控 Spry1的表达。在用脂多糖（LPS）刺激后，睾丸中 Dmrt1或 Spry1的敲除都导致更严重的炎症反应、BTB损伤和NF-κB信号的激活。IP-MS和Co-IP结果表明，SPRY1蛋白与NF-κB1结合，从而抑制NF-κB通路的激活，防止睾丸过度炎症反应，并保护血-睾屏障的完整性。该研究发现了Dmrt1-Spry1-NF-κB轴在调节睾丸免疫稳态中的新机制，为预防、治疗人类和家畜的生殖系统疾病开辟了新的途径。.

Aromatase (encoded by Cyp19a1) in the ovarian follicular cells catalyzes the production of estradiol from testosterone, which plays important roles in the ovarian development of vertebrates. In the present study, the interaction of Dmrt1, Foxl2, and Nr5a1a on the regulation of cyp19a1a transcription in ovarian follicles was examined in a teleost, the ricefield eel Monopterus albus. The expression of dmrt1a, foxl2, and nr5a1a was detected in ovarian follicular cells together with cyp19a1a at the mRNA and/or protein levels. Sequence analysis identified one conserved Foxo binding site in the proximal promoter region of ricefield eel cyp19a1a. Transient transfection assay showed that Foxl2 may bind to the conserved Foxo site to activate cyp19a1a transcription and act synergistically with Nr5a1a. Mutation of either the conserved Nr5a1 site or Foxo site abolished or significantly decreased the synergistic effects of Nr5a1a and Foxl2 on cyp19a1a transcription. The sequence between Region III and I-box of Nr5a1a was critical to this synergistic effect. Dmrt1a modulated the Foxl2- and Nr5a1a-induced activation of cyp19a1a transcription and their synergistic effects in a biphasic manner, with inhibitory roles observed at lower doses (10-50 ng) but release of the inhibition or even potentiating effects observed at higher doses (100-200 ng). Collectively, data of the present study suggest that the interaction of Dmrt1a, Foxl2, and Nr5a1a in the ovarian follicular cells may facilitate the adequate expression of cyp19a1a and the production of estradiol, and contribute to the development and maturation of ovarian follicles in ricefield eels and other vertebrates as well.

Primary sex determination is the developmental process that results in the sexual differentiation of the gonads. Vertebrate sex determination is generally considered to follow the model based on the mammalian system, where a sex-specific master regulatory gene activates one of the two different gene networks that underlie testis and ovary differentiation.
It is now known that, while many of the molecular components of these pathways are conserved across different vertebrates, a wide variety of different trigger factors are utilized to initiate primary sex determination. In birds, the male is the homogametic sex (ZZ), and significant differences exist between the avian system of sex determination and that of mammals. For example, DMRT1, FOXL2, and estrogen are key factors in gonadogenesis in birds, but none are essential for primary sex determination in mammals.
Gonadal sex determination in birds is thought to depend on a dosage-based mechanism involving expression of the Z-linked DMRT1 gene, and it may be that this \"mechanism\" is simply an extension of the cell autonomous sex identity associated with avian tissues, with no sex-specific trigger required.

During gonadal sex determination, the supporting cell lineage differentiates into Sertoli cells in males and pre-granulosa cells in females. Recently, single cell RNA-seq data have indicated that chicken steroidogenic cells are derived from differentiated supporting cells. This differentiation process is achieved by a sequential upregulation of steroidogenic genes and downregulation of supporting cell markers. The exact mechanism regulating this differentiation process remains unknown. We have identified TOX3 as a previously unreported transcription factor expressed in embryonic Sertoli cells of the chicken testis. TOX3 knockdown in males resulted in increased CYP17A1-positive Leydig cells. TOX3 overexpression in male and female gonads resulted in a significant decline in CYP17A1-positive steroidogenic cells. In ovo knockdown of the testis determinant DMRT1 in male gonads resulted in a downregulation of TOX3 expression. Conversely, DMRT1 overexpression caused an increase in TOX3 expression. Taken together, these data indicate that DMRT1-mediated regulation of TOX3 modulates expansion of the steroidogenic lineage, either directly, via cell lineage allocation, or indirectly, via signaling from the supporting to steroidogenic cell populations.