# The Function of Heat Shock Transcription Factors in Sex Differentiation in Cynoglossus semilaevis

## TL;DR

This study explores how heat shock transcription factors (hsf) influence sex differentiation in Chinese tongue sole fish, revealing their role in temperature-induced sex reversal.

## Contribution

The study identifies and characterizes five hsf genes in Cynoglossus semilaevis and their potential role in temperature-mediated sex differentiation.

## Key findings

- Five hsf genes were found to be expressed in gonads, with signals concentrated in germ cells.
- High-temperature stimulation increased hsf gene expression in gonads, with varied response patterns among genes.
- RNA interference of hsf affected sex-related genes and neurl3 expression in female gonads.

## Abstract

As an important economic fish, Cynoglossus semilaevis has sexual dimorphism, and the growth difference between males and females is great. Studies have shown that the genotypic females of Cynoglossus semilaevis can be transformed into pseudomales at high temperatures. Therefore, we studied the heat shock transcription factor hsf gene. In this study, we located five hsf genes of Cynoglossus semilaevis in gonad tissue and found that they were mainly concentrated in sperm cells and oocytes. Under high-temperature stimulation, with the increase in stimulation time, the expression of the hsf gene in the gonad gradually increased, but different hsf genes showed different reaction patterns. In vivo siRNA interference had effects on female gonads, mainly affecting the expression of neurl3. According to these data, we speculate that hsf not only responds to temperature stimuli, but also plays an important role in gender differentiation. This study is helpful to clarify the relationship between the temperature perception and sex differentiation of Cynoglossus semilaevis.

Chinese tongue sole (Cynoglossus semilaevis) is an important marine fish in China. It has sexual dimorphism. The weight and growth rate of female fish are much greater than those of male fish. However, high temperatures can induce sex reversal in genetic female fish (ZW) to phenotypic male fish; thus, identifying the genetic elements involved in temperature perception will provide the molecular basis for sex control. The heat shock transcription factor (hsf) is known as an important component of temperature sensing and mediates the heat shock response in fish such as Danio rerio; however, its function in C. semilaevis is unclear. In this study, five hsf genes (hsf1, hsf2, hsf4, hsf5a, and hsf5b) were identified in tongue sole and found to be expressed in the gonads at different developmental stages, peaking from 7M to 1Y. Gonadal in situ hybridization revealed that hsf gene signals were mainly localized in germ cells, e.g., sperm in the testis and all-stage oocytes in the ovary. Upon high-temperature stimulation, the expression of the hsf gene in the gonads increased gradually with increasing stimulation time, but different hsf genes presented different response patterns. After the RNA interference of hsf in the testis and ovarian cell lines, a series of sex-related genes, such as foxl2 and dmrt1, significantly changed. In vivo RNA interference had an effect on the female gonads and mainly affected neurl3 expression. On the basis of these data, we speculate that hsf responds to temperature stimulation and plays an important role in sex differentiation. This study helps elucidate the relationship between temperature sensing and sex differentiation in C. semilaevis.