The Chinese tongue sole (Cynoglossus semilaevis) is a commercially important mariculture species; however, its fertilization and hatching rates under artificial conditions remain relatively low. Zona pellucida proteins (ZPs), which mediate sperm–egg binding, were previously identified as differentially expressed genes between newly differentiated ovaries and testes in C. semilaevis. In this study, we identified 25 ZPs of C. semilaevis through genomic analysis and classified them into five subfamilies. All genes possessed a conserved ZP domain, characteristic of the gene family from mammals to teleosts. Among them, nine genes were highly expressed in ovary cells, with the expression levels increasing during ovarian development, while another three genes were predominantly expressed in liver cells. Protein–protein interaction analysis predicted that 12 ZPs interacted with key reproductive regulators such as Gdf9, Arid4a, Arid4b, and Rbl, which were involved in steroidogenesis, sperm–egg recognition, and folliculogenesis. Functional analyses using RNA interference revealed that Cszpc7-1 knockdown in ovarian cells led to the downregulation of cyp19a, esr2, bmp15, and adamts-1, while the expression of rbl, gnas, adgrl1, and adgrl2 was upregulated. In contrast, Cszpax1 knockdown resulted in decreased expression of cyp19a, foxl2, arid4a, and zeb1, along with upregulation of arid4b, ogg1, and gdf9. These results suggested that ZP genes might contribute to ovarian homeostasis by regulating steroid hormone synthesis, follicular development, and ovulation. This study contributed to a deeper understanding of the reproductive mechanisms of C. semilaevis and provided evolutionary insights into the functional divergence of the ZP gene family across teleosts.

Chinese tongue sole (Cynoglossus semilaevis) is an important aquaculture species in China. Under high-temperature conditions, genetically female fish can undergo sex reversal and develop into phenotypic males (pseudomale fish). Previous studies have demonstrated that apoptosis might function in sex differentiation. Based on this, we identified and characterized the functions of six caspase genes (caspase1-like, caspase3a, caspase6, caspase8, caspase8-like, caspase9) in Chinese tongue sole. These six caspase genes were expressed in all analyzed tissues of both males and females. They were detected to be expressed in the gonads at various developmental stages, with expression levels peaking between 7 months and 2 years of age. In situ hybridization (ISH) analysis showed that the caspase genes were mainly localized in spermatocytes and oocytes. Promoter activity analysis indicated that with the exception of caspase3a, the remaining five caspase genes exhibited promoter activity and were regulated by transcription factors, including sp1 and gata4. High-temperature stimulation can significantly affect the expression of caspases in the gonads of both male and female fish, with female fish showing a more pronounced response. An siRNA-mediated knockdown experiment revealed that following caspase knockdown, the expression of sex differentiation-related genes, heat shock transcription factors (hsf), and heat shock proteins (hsp) in Chinese tongue sole was significantly altered. Based on these findings, we speculate that caspases play an important role in the sex differentiation process by responding to temperature stimuli.

Chinese tongue sole (Cynoglossus semilaevis), an economically important mariculture species in China, exhibits pronounced sexual dimorphism in growth, underscoring the importance of elucidating sex regulatory mechanisms for aquaculture development. Heterogeneous nuclear ribonucleoprotein K (hnrnpk) critically regulates mammalian reproductive development, yet its role in fish sex regulation remains elusive. Here, we systematically investigated the underlying function and mechanisms of hnrnpk in C. semilaevis through integrated molecular cloning, expression profiling, upstream regulatory analysis, functional assays, and transcriptome sequencing. We found that hnrnpk was highly expressed in the gonad and liver, with female-biased expression during gonadal development. Promoter activity assays revealed that sox2 and c-Jun enhanced hnrnpk transcription, whereas foxl2 and ar suppressed it. Additionally, hnrnpk was directly targeted by miR-460a-5p in C. semilaevis, revealing multi-level transcriptional and post-transcriptional regulation. Functional analyses showed that hnrnpk regulated cyp19a1a in a cell type-dependent and dose-sensitive manner: the expression of cyp19a1a was both upregulated in hnrnpk-knockdown ovarian cells and hnrnpk-overexpression testicular cells. Interestingly, foxl2 was upregulated in hnrnpk-knockdown ovarian cells but suppressed in hnrnpk-overexpression testicular cells, which showed the distinct regulation mechanisms in the different sexual programs. Transcriptomic analyses further revealed that several sex-related genes (sox9a with downregulation, etc.) were significantly regulated, and cell development and cycle pathways were dramatically enriched in functional enrichment analyses. This might indicate that hnrnpk overexpression drives C. semilaevis testis (CSTE) toward feminization reprogramming through sox9 switching and multi-pathway perturbations. Overall, our findings might reveal that hnrnpk, a female-biased gene regulated by miR-460a-5p and transcription factors, influences sex-related gene expression through sox9 switching. This study will offer new insights for C. semilaevis hnrnpk into sex determination and also provide a potential target for monosex breeding in aquaculture.

Chinese tongue sole (Cynoglossus semilaevis) is an important marine fish in China. It has sexual dimorphism. The weight and growth rate of female fish are much greater than those of male fish. However, high temperatures can induce sex reversal in genetic female fish (ZW) to phenotypic male fish; thus, identifying the genetic elements involved in temperature perception will provide the molecular basis for sex control. The heat shock transcription factor (hsf) is known as an important component of temperature sensing and mediates the heat shock response in fish such as Danio rerio; however, its function in C. semilaevis is unclear. In this study, five hsf genes (hsf1, hsf2, hsf4, hsf5a, and hsf5b) were identified in tongue sole and found to be expressed in the gonads at different developmental stages, peaking from 7M to 1Y. Gonadal in situ hybridization revealed that hsf gene signals were mainly localized in germ cells, e.g., sperm in the testis and all-stage oocytes in the ovary. Upon high-temperature stimulation, the expression of the hsf gene in the gonads increased gradually with increasing stimulation time, but different hsf genes presented different response patterns. After the RNA interference of hsf in the testis and ovarian cell lines, a series of sex-related genes, such as foxl2 and dmrt1, significantly changed. In vivo RNA interference had an effect on the female gonads and mainly affected neurl3 expression. On the basis of these data, we speculate that hsf responds to temperature stimulation and plays an important role in sex differentiation. This study helps elucidate the relationship between temperature sensing and sex differentiation in C. semilaevis.

Tetramethyl bisphenol A (TMBPA), a novel alternative to Bisphenol A, is widely used as an industrial flame retardant and a raw material for tetramethyl polycarbonate plastics. With the increasing use of TMBPA, its aquatic ecological risks remain unclear. Therefore, this study investigated the developmental toxicity of TMBPA using zebrafish (Danio rerio) as a model, exposing embryos to 0.5, 5, 50, and 200 μg/L TMBPA for 120 h. The results showed that treatment with 5, 50, and 200 μg/L TMBPA decreased the hatching rate of zebrafish embryos at 48 h post-fertilization (hpf), while no significant difference was observed at 72 hpf. Meanwhile, TMBPA exposure at all concentrations showed no significant effect on the survival rate. Furthermore, a high concentration of TMBPA (200 μg/L) significantly reduced the total length and suppressed swimming ability in zebrafish larvae. In addition, gene expression analysis revealed impacts on antioxidant system (cat, gpx, mn-sod, keap1, ucp2, nrf2), hypothalamic–pituitary–thyroid (HPT) axis (ttr, ugt1ab, trβ), cardiac developmental (tbx2b, myl7, bmp4, notch1b, amhc), and the hypothalamic–pituitary–adrenal (HPA) axis (pomca and nr3c1). The results indicated that TMBPA exposure adversely disrupted embryo hatching and larval development of zebrafish, accompanied by altering the expression of functional genes in larvae. These results provide further evidence for the potential environmental hazard posed by TMBPA.

Chinese tongue sole (Cynoglossus semilaevis) is an important mariculture fish in China, and female individuals present a growth advantage. However, genetic females (ZW) can sex reverse to phenotypic males, designated pseudomales. The pseudomale shows abnormal spermatogenesis and produces only Z sperm. Histone is pivotal in spermatogenesis, and post-translational modification could regulate its function. A comparison of testis phosphorylated and ubiquitinated proteins revealed 8 and 12 differentially phosphorylated and ubiquitinated histones in the testes of male and pseudomale Chinese tongue soles, respectively, but there was no difference in the translation level of these proteins. We selected four histone genes, h1.1-like, h1.2-like, h3, and h3.3-like, for further analysis. The expression levels of the h1.1-like, h3, and h3.3-like genes reached their highest levels at 2 years post-hatching (yph), and the expression level of h1.2-like reached its highest level at 1.5 years post-hatching (1.5 yph), indicating that its role began during the late stage of gonadal development. Promoter activity verification revealed that the promoters of the h1.1-like, h1.2-like, h3, and h3.3-like genes were located approximately upstream 2000 bp and six histone-related transcription factor sites were predicted. YY1A, YY1B, C-JUN, and JUNB may have negative regulatory effects on h1.1-like, h1.2-like, h3, and h3.3-like; AR and ETS-2 may have positive regulatory effects on h3 and h3.3-like. The ISH results revealed that h1.1-like, h1.2-like, h3, and h3.3-like mRNAs were located mainly in the sperm cells in the testes and the oocytes at various stages in the ovaries. After siRNA knockdown, the expression of dmrt1 in testis cell lines and the expression of tesk1 and neurl3 in males was downregulated, suggesting that the h1.1-like, h1.2-like, h3, and h3.3-like genes may have a negative regulatory role in spermatogenesis. The regulatory role in female fish remains to be explored. Mass spectrometry analysis revealed that histones have an important role in chromosome remodeling. These results provide a genetic basis for the molecular mechanism of gonadal development and spermatogenesis in Chinese tongue sole.