Characterization of Forkhead Box Transcription Factor (foxl) in Sex Differentiation of Chinese Tongue Sole (Cynoglossus semilaevis)

Chinese tongue sole (<i>Cynoglossus semilaevis</i>) is an important mariculture product in northern China, exhibiting significant sexual dimorphism: females grow 2-4 times faster than males and ultimately attain much greater body weights. As a well-known transcription factor crucial for regulating sex differentiation, <i>foxl2</i> has been characterized in various mammals. Herein, we identified and characterized three <i>foxl</i> genes, <i>foxl1</i>, <i>foxl2a</i> and <i>foxl2l</i>. Three <i>foxl</i> genes exhibited a gonad-biased expression pattern, where <i>foxl2a</i> showed higher expression in ovary than in testis, while <i>foxl1</i> and <i>foxl2l</i> exhibited higher expression in testis. All <i>foxl</i> genes were detected in testes and ovaries by ISH; <i>foxl1</i>/<i>foxl2l</i> were expressed in oocytes and sperm, and <i>foxl2a</i> in granulosa cells and sperm. Overexpression of <i>foxl</i> in testicular cells led to KEGG enrichment in DNA repair, MAPK, FOXO and progesterone-mediated oocyte maturation pathways. In tongue sole testicular cell line, knockdown of <i>foxl1</i> and <i>foxl2l</i> resulted in upregulation of multiple male-related genes. In contrast, knockdown of <i>foxl2a</i> led to decreased expression of aromatase genes and increased expression of <i>ctnnb1</i>, indicating that <i>foxl2a</i> is more closely associated with female differentiation and maintenance. Our study investigated the functions of the <i>foxl</i> gene family in teleosts and offers valuable insights into their role in sex differentiation and gonadal maintenance in teleost fish.