Unraveling the Role of FSHR Gene in Zebrafish (Danio rerio) Sex Differentiation: A Model for Flathead Grey Mullet (Mugil cephalus) Studies

This thesis investigates the functional role of the follicle-stimulating hormone receptor (fshr) gene in sex differentiation, using zebrafish (Danio rerio) as a model to elucidate genetic mechanisms relevant to the flathead grey mullet (Mugil cephalus). Zebrafish were chosen for their genetic tractability and well-established use in reproductive studies. Through CRISPR-Cas9 gene editing, a novel zebrafish fshr mutant line was generated, featuring a nine-amino acid deletion in a conserved region of the fshr gene, mirroring missense variants identified in M. cephalus. This study aimed to examine the impact of fshr mutations on the expression of key genes involved in sex determination and differentiation across two critical tissues—testis and brain—in male zebrafish aged 8 and 13 months. Gene expression was evaluated using quantitative PCR (qPCR) and RNA sequencing (RNAseq) to compare heterozygous (FSHR +/-) and homozygous (FSHR -/-) mutant lines. Results demonstrated alterations in the expression of genes such as cyp11b, vasa, and amh in FSHR -/- males, particularly in the testis, indicating that fshr mutations have profound effects on gonadal development. In addition, RNAseq analysis revealed a broad spectrum of differentially expressed genes, providing further insights into the molecular underpinnings of sex differentiation in zebrafish. These findings suggest that fshr, through its regulation of steroidogenic pathways, may play a pivotal role in controlling male development and could contribute to variations in sex ratios observed in aquaculture settings. This research not only advances our understanding of the genetic factors influencing sex determination in zebrafish but also has broader implications for improving selective breeding strategies and sex control methods in commercially valuable fish species like the flathead grey mullet.