Genome-Wide Identification and Expression Analysis of Zona Pellucida (ZP) Gene Family in Cynoglossus semilaevis

The Chinese tongue sole (<i>Cynoglossus semilaevis</i>) 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 <i>C. semilaevis</i>. In this study, we identified 25 ZPs of <i>C. semilaevis</i> 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 <i>Cszpc7-1</i> knockdown in ovarian cells led to the downregulation of <i>cyp19a</i>, <i>esr2</i>, <i>bmp15</i>, and <i>adamts-1</i>, while the expression of <i>rbl</i>, <i>gnas</i>, <i>adgrl1</i>, and <i>adgrl2</i> was upregulated. In contrast, <i>Cszpax1</i> knockdown resulted in decreased expression of <i>cyp19a</i>, <i>foxl2</i>, <i>arid4a</i>, and <i>zeb1</i>, along with upregulation of <i>arid4b</i>, <i>ogg1</i>, and <i>gdf9</i>. 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 <i>C. semilaevis</i> and provided evolutionary insights into the functional divergence of the ZP gene family across teleosts.