# Differential Expression Characteristics of Two Isoforms nr5a2f and nr5a2m in Gonadal Differentiation of Chinese Giant Salamanders, Andrias davidianus

## TL;DR

This study explores how two versions of the Nr5a2 gene affect gonadal development in Chinese giant salamanders, showing distinct roles in male and female sex differentiation.

## Contribution

The study identifies and characterizes two Nr5a2 isoforms with sex-specific expression patterns in Chinese giant salamanders.

## Key findings

- Nr5a2f is highly expressed in ovaries and decreases during sex reversal, while Nr5a2m is more active in testes.
- Inhibiting Nr5a2f reduces female-specific genes and increases male-specific genes in salamander gonads.
- In situ hybridization shows Nr5a2f starts in female gonads and Nr5a2m in male gonads during early development.

## Abstract

The aim of this research was to characterize the structure, expression and function of Nr5a2f and Nr5a2m, which was generated by alternative splicing of the Nr5a2 gene. The expression profile suggested that Nr5a2f and Nr5a2m had different expression profiles in the gonads of females and males. Nr5a2f expressed significantly high in the ovary, while Nr5a2f in the testis. Furthermore, when Nr5a2f expression was inhibited, we observed that female-based gene expression was decreased, while male-based gene expression was increased.

Nr5a2 (nuclear receptor subfamily 5, group a, member 2) is involved in gonad development and sex hormone synthesis. In this study, the full length of Nr5a2f and Nr5a2m were obtained by Nr5a2 variable splicing from Andrias davidianus, and the tissue distribution was detected. We identified Nr5a2f of 2455 bp and Nr5a2m of 2150 bp length, encoding 479 and 325 amino, respectively. We first characterized Nr5a2f and Nr5a2m gene expression in developing gonads. Results showed that Nr5a2f had significantly high expression in the ovary and little expression in other tissues, during the sex differentiation and sex reversal, Nr5a2f expression was gradually decreased in the ovary and the expression in the testis was significantly lower than in the ovary from 1 year to 6 year old. Significantly high expression was observed in the ovary and reversal ovary, while low expression was in the testis and reversal testis. While Nr5a2m expression exhibited the opposite profile, high expression was observed in the brain and testis. During sex differentiation and sex reversal, high expression was shown in the testis and low expression in the ovary from one year to six years old and significantly higher expression emerged in testis and reversal testis than in ovary and reversal ovary. In situ hybridization, results showed that Nr5a2f began to express in female undifferentiated gonads and the expression level increased from 48 dpf to 91, while Nr5a2m was expressed in male undifferentiated gonads. Three RNA interference sites were designed and we detected that site 293 exhibited a significant inhibitory effect in ovary cells. After Nr5a2f expression was inhibited by site 293, we observed that female-based gene Nr5a2f, foxl2 and cyp19 expression were decreased, while the male-based gene dmrt1 and cyp17 expression was increased. These results suggested that Nr5a2f and Nr5a2m exhibited different expression patterns in the process of sex differentiation, which provided a foundation for further functional characterizations.