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

## TL;DR

This study explores the role of foxl genes in sex differentiation in Chinese tongue sole, a fish where females grow much faster than males.

## Contribution

The study characterizes three foxl genes and their distinct roles in sex differentiation in Chinese tongue sole.

## Key findings

- foxl2a is more closely associated with female differentiation and maintenance.
- foxl1 and foxl2l are linked to male-related gene expression when knocked down.
- foxl genes are involved in hormone-regulated sex determination pathways.

## Abstract

Chinese tongue sole (Cynoglossus semilaevis) is a cold-water marine fish species in which females grow significantly faster than males. Identifying its key sex-related genes is of great practical significance for improving aquaculture yield. Studies have shown that members of the foxl gene family are closely associated with the hormone-regulated sex determination pathway in C. semilaevis, which provides a fundamental basis for research on sex differentiation and sex maintenance in this species.

Chinese tongue sole (Cynoglossus semilaevis) 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, foxl2 has been characterized in various mammals. Herein, we identified and characterized three foxl genes, foxl1, foxl2a and foxl2l. Three foxl genes exhibited a gonad-biased expression pattern, where foxl2a showed higher expression in ovary than in testis, while foxl1 and foxl2l exhibited higher expression in testis. All foxl genes were detected in testes and ovaries by ISH; foxl1/foxl2l were expressed in oocytes and sperm, and foxl2a in granulosa cells and sperm. Overexpression of foxl 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 foxl1 and foxl2l resulted in upregulation of multiple male-related genes. In contrast, knockdown of foxl2a led to decreased expression of aromatase genes and increased expression of ctnnb1, indicating that foxl2a is more closely associated with female differentiation and maintenance. Our study investigated the functions of the foxl gene family in teleosts and offers valuable insights into their role in sex differentiation and gonadal maintenance in teleost fish.