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.

Transforming growth factor β (TGF-β) superfamily members are critical in teleost sex determination and differentiation. Tgfb2b is an important TGF-β ligand gene exhibiting dominant expression in the ovary of Chinese tongue sole (Cynoglossus semilaevis), yet its function in sex regulation remains unclear. In the present study, the gene expression pattern, transcriptional regulation, and knockdown effect were examined. Its expression persisted and showed a gradual increase throughout ovarian development from 3 months to 1.5 years post-hatching. In situ hybridization (ISH) revealed that the gene was distributed across oocytes at stages I–III, while scarcely detectable in the testis. The transcriptional factors CCAAT/enhancer binding protein α (C/EBPα) and Jun proto-oncogene AP-1 transcription factor subunit (c-Jun) could repress the activity of tgfb2b promoter. In vitro knockdown of tgfb2b in C. semilaevis ovarian cells led to downregulation of its downstream genes (e.g., smad1 and smad2) as well as other sex-related genes (e.g., foxl2 and esr2b). Moreover, multi-omics analysis indicated that, in C. semilaevis gonads, a miRNA named novel-m0083-3p showed an opposite expression pattern with tgfb2b and might have a binding site with the gene. By dual-luciferase assay, tgfb2b was validated to be directly targeted and suppressed by the miRNA. These results demonstrate that tgfb2b plays a significant role in ovarian differentiation and development. Further functional and molecular studies on the interplay between tgfb2b and the foxl2–cyp19a–esr axis will help elucidate the regulatory network underlying sex development in teleost.