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

## TL;DR

This study identifies the tgfb2b gene's role in ovarian development in Chinese tongue sole fish and explores its regulation and interactions with other genes and miRNA.

## Contribution

The study reveals the functional role of tgfb2b in ovarian development and its regulation by C/EBPα, c-Jun, and a specific miRNA in Chinese tongue sole.

## Key findings

- tgfb2b expression increases during ovarian development and is localized in oocytes but not in testis.
- C/EBPα and c-Jun repress tgfb2b promoter activity, while its knockdown reduces downstream and sex-related gene expression.
- A miRNA (novel-m0083-3p) inversely regulates tgfb2b and directly targets it, as confirmed by dual-luciferase assays.

## Abstract

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