# Comparative RNA-Seq Analysis of Differentially Expressed Genes in the Testis and Ovary of Mudskipper, Boleophthalmus pectinirostris

## TL;DR

This study identifies genes involved in sex determination in mudskipper fish, revealing key genes like amh and their roles in reproductive development.

## Contribution

The first transcriptome analysis of sex-biased gene expression in Boleophthalmus pectinirostris, revealing amh isoforms and sex-specific gene networks.

## Key findings

- 17,214 differentially expressed genes identified in testis and ovary of B. pectinirostris.
- Key genes like dmrt1, amh, and bmp15 are associated with sex determination and reproductive pathways.
- amh gene has two isoforms, both expressed in ovaries and testes, with a deletion in the second exon.

## Abstract

Gobioidei fishes are key inhabitants of mangrove mudflats and intertidal zones, representing an important lineage in the transition from marine to terrestrial environments and occupying crucial ecological niches. They serve as essential food sources for many commercially important fishes, and some species, such as Boleophthalmus pectinirostris, also serve as a direct protein resources for humans. Gobioidei exhibit remarkable diversity and form one of the largest teleost groups, with over 2000 described species. However, morphological similarities among certain genera complicate accurate species and sex identification, underscoring the need for molecular tools. Here, we present the first transcriptome analysis of sex-biased gene expression in B. pectinirostris, an economically important species. We identified amh, a key gene in sex determination and differentiation, with two protein-coding isoforms. This study lays a foundation for understanding sex determination and differentiation in B. pectinirostris and advances insight into reproductive developmental mechanisms.

Sex determination and differentiation in teleosts are governed by complex genetic regulatory networks that include evolutionarily conserved mechanisms. In this study, we investigated Boleophthalmus pectinirostris, a Gobiidae species lacking heterogametic sex chromosomes, using comparative gonadal transcriptome analysis to identify sex differentially expressed genes (DEGs). RNA sequencing of ovarian and testicular tissues identified 17,214 DEGs, including 14,302 upregulated in males and 2912 upregulated in females. These DEGs were primarily associated with male (e.g., dmrt1, amh, amhr2) or female (e.g., bmp15, gdf9, rspo1) sex determination and differentiation, steroidogenesis (e.g., hsd17b1, hsd3b1, cyp17a1), and meiosis (e.g., cyp26b1, aldh1a2, piwil2). Functional enrichment analysis revealed that male upregulated DEGs were involved in spermatogenesis pathways such as calcium signaling, while female upregulated DEGs were associated with oogenesis pathways including oocyte meiosis and progesterone-mediated oocyte maturation. Conserved regulators, notably dmrt1 and amh, were predicted to act as key hubs in protein–protein interaction networks, being primarily associated with reproductive processes and sex differentiation in B. pectinirostris. The amh gene produces two alternatively spliced isoforms that differ by a partial deletion in the second exon, both expressed in ovaries and testes. Collectively, this study provides the first comprehensive molecular framework of sex determination and differentiation in Gobiidae species, offering critical insights into the regulatory mechanisms of B. pectinirostris reproductive development.