The Chinese tongue sole (Cynoglossus semilaevis) is a marine flatfish of significant economic value, characterized by pronounced female-biased sexual size dimorphism (SSD). Sexual differences of cell number and gene expression within the PIT-1 lineage of the pituitary gland may be crucial for interpreting the female-biased SSD of C. semilaevis. Among hormones secreted by PIT-1 cell lineage, growth hormone (gh), prolactin (prl), prolactin 2 (prl2), and somatolactin (sl) comprise a gene family within the extensive superfamily of class-1 helical cytokines. To better understand the function of the gh/prl/sl in teleost SSD, we firstly identified five genes of the gh/prl/sl family (gh, sl, prl, prl2a, and prl2b) and their receptors (ghra, ghrb, prlra, prlrb, and prlr-like) from C. semilaevis at the genome-wide level. Phylogenetic analyses revealed that the gh/prl/sl family and their receptors were each clustered into five distinct groups. More microsatellites were revealed in the intron 2 of gh gene of female rather than the male and pseudo-male individuals, which is positively correlated with its sexual expression pattern. Interaction network prediction indicated that gh, prl, and sl may collectively contribute to individual growth and development. A FRET experiment showed that ghra can act as a receptor for sl. Additionally, the transcripts of the gh/prl/sl family and their receptors exhibited varying abundances in the pituitary, brain, gonad, and liver of both female and male C. semilaevis, with most ligands showing the highest abundance in the female pituitary. Furthermore, gh and sl were found to be maternally expressed. The knock-down of gh, prl, and sl in the pituitary cells could lead to the expression change of igf1, c-fos, and sos2. This study provided a foundation for further functional characterization of the gh/prl/sl gene family, contributing to a deeper understanding of the growth and reproductive mechanisms in C. semilaevis.

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.