To investigate the effect of epigenetic modifications on sex determination and differentiation in northern pike (Esox lucius), we employed Whole-Genome Bisulfite Sequencing (WGBS) to analyze the DNA methylation patterns in gonadal tissues of females, males, and neomales. First, we obtained high-quality sequencing data, including a total of 410.16 Gb of raw reads and 361.48 Gb of clean reads, with an 86% unique mapping rate, and a bisulfite conversion efficiency of 99.6%. Subsequently, comparative analysis revealed that 66,581 differentially methylated CG regions (i.e., DNA regions with a high frequency of CG dinucleotides), 1215 differentially methylated CHG regions (i.e., DNA regions where CG is followed by another nucleotide), and 3185 differentially methylated CHH regions (i.e., regions where cytosine is methylated in a CHH sequence, with ‘H’ representing A, T, or C) were identified among the three groups. Furthermore, we identified four key differentially methylated candidate genes (Rspo1, hsd11b2, CYP27A1 and smad3) associated with sex determination and differentiation processes in E. lucius. Finally, by integrating GO and KEGG enrichment analyses, we explored the role of epigenetic modification regulatory networks in the sex determination and differentiation of E. lucius and identified multiple metabolic pathways related to sex determination and differentiation processes (Notch signaling pathway, Wnt signaling pathway and Ovarian steroidogenesis). This study thereby lays a foundation for subsequent functional verification.

Fish represent the most species-rich group within the phylum Chordata, possessing exceptional nutritional and ornamental value. Global aquaculture, particularly finfish farming, is experiencing rapid expansion worldwide, and fish serve as crucial model organisms for vertebrate developmental biology and functional genomics research. However, traditional breeding methods are plagued by limitations such as low precision and lengthy breeding cycles. Currently, gene editing technologies represented by the CRISPR/Cas system, base editing, and prime editing have provided revolutionary tools for dissecting gene function, modeling human diseases, targeted trait improvement, and ecological adaptation studies. This review describes the evolutionary history of gene editing technology, compares gene delivery strategies in fish embryos, and highlights landmark applications in key areas, including gene function research, aquaculture breeding, ornamental fish coloration regulation, and human disease model construction. Finally, we propose that innovation should be pursued while ensuring biosafety and regulatory compliance, to promote the transformation of fish gene editing toward large-scale and safe application.

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.

Northern pike (Esox lucius) exhibits significant sexual growth dimorphism, yet the molecular basis of this sex-biased trait remains largely unexplored. In this study, we performed an integrative analysis of whole-genome DNA methylation (WGBS) and whole-transcriptome (RNA-seq) data from female and male gonadal tissues of E. lucius. Promoter methylation exhibited sexually dimorphic associations with gene expression: in females, promoter hypermethylation showed a positive correlation with elevated expression, whereas no such association was observed in males; gene body methylation was not correlated with expression in either sex. Integrative analysis identified a total of 508 methylation-associated differentially expressed genes (mDEGs), comprising 377 Hyper-Down (hypermethylated with downregulated expression) and 131 Hypo-Up (hypomethylated with upregulated expression) genes, from which 13 candidate genes were prioritized. Pathway enrichment analyses implicated TGF-β signaling, Wnt signaling, and progesterone-mediated oocyte maturation pathways, suggesting crosstalk between sex differentiation and metabolic regulation/signal transduction. Collectively, this study provides an exploratory perspective on the epigenetic landscape associated with sex differentiation in E. lucius. Our results reveal associations between promoter methylation and the expression of core sex-related genes, including dmrt1 (male-biased) and rspo1 (female-biased), which may be relevant to sex differentiation. The potential link between these epigenetic patterns and sexual growth dimorphism remains speculative and requires direct validation using growth-related phenotypic data and tissues.