Identification and Functional Analysis of tgfb2b Gene in Ovarian Development of Chinese Tongue Sole (Cynoglossus semilaevis)

Transforming growth factor β (TGF-β) superfamily members are critical in teleost sex determination and differentiation. <i>Tgfb2b</i> is an important TGF-β ligand gene exhibiting dominant expression in the ovary of Chinese tongue sole (<i>Cynoglossus semilaevis</i>), 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. <i>In situ</i> 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 <i>tgfb2b</i> promoter. <i>In vitro</i> knockdown of <i>tgfb2b</i> in <i>C. semilaevis</i> ovarian cells led to downregulation of its downstream genes (e.g., <i>smad1</i> and <i>smad2</i>) as well as other sex-related genes (e.g., <i>foxl2</i> and <i>esr2b</i>). Moreover, multi-omics analysis indicated that, in <i>C. semilaevis</i> gonads, a miRNA named novel-m0083-3p showed an opposite expression pattern with <i>tgfb2b</i> and might have a binding site with the gene. By dual-luciferase assay, <i>tgfb2b</i> was validated to be directly targeted and suppressed by the miRNA. These results demonstrate that <i>tgfb2b</i> plays a significant role in ovarian differentiation and development. Further functional and molecular studies on the interplay between <i>tgfb2b</i> and the <i>foxl2-cyp19a-esr</i> axis will help elucidate the regulatory network underlying sex development in teleost.