As a fundamental taxonomic group within vertebrates, fish represent an invaluable resource for investigating the mechanisms underlying sex determination and differentiation owing to their extensive geographical distribution and rich biodiversity. Within this biological cohort, the processes of sex determination and differentiation are intricately governed by both genetic factors and the complex interplay of environmental cues. While variations in external environmental factors, particularly temperature, can exert a modulatory influence on sex differentiation in fish to a limited degree, genetic factors remain the primary determinants of sexual traits. Hermaphroditic fish display three distinct types of sexual transitions: protandry (male to female), protogyny (female-to-male), bidirectional sex change (both directions serially). These fish, characterized by their unique reproductive strategies and sexual plasticity, serve as exemplary natural models for elucidating the mechanisms of sex differentiation and sexual transitions in fish. The present review delves into the histological dynamics during gonadal development across three types of sequential hermaphroditic fish, meticulously delineating the pivotal characteristics at each stage, from the inception of primordial gonads to sexual specialization. Furthermore, it examines the regulatory genes and associated signaling pathways that orchestrate sex determination and differentiation. By systematically synthesizing these research advancements, this paper endeavors to offer a comprehensive and profound insight into the intricate mechanisms governing sex differentiation in sequential hermaphroditic fish.

Hemibagrus guttatus is a commercially valuable freshwater fish in the Pearl River Basin, renowned as the “King of Freshwater Fish.” Due to habitat degradation and overfishing, its wild population has declined sharply, leading to its listing as a National Key Protected Wild Animal of Class II in China. Artificial breeding is therefore crucial for conservation, yet progress is hindered by the lack of clear sexual dimorphism and poor understanding of its sex differentiation mechanism. In this study, we performed high-throughput RNA sequencing (RNA-seq) to compare gonadal transcriptomes of male and female H. guttatus. A total of 3245 differentially expressed genes (DEGs) were identified, including 3122 male-biased and 123 female-biased DEGs, which clustered into three distinct expression patterns. Enrichment analysis revealed that genes associated with the TGF-β (Transforming Growth Factor-beta) and GnRH (Gonadotropin-Releasing Hormone) signaling pathways were significantly enriched in the female gonads, suggesting their potential roles in gonadal differentiation. From the DEG set, we further highlighted five genes with pronounced sex-biased expression: rbm46 (RNA Binding Motif Protein 46) exhibited gonad-specific expression, whereas myc (v-myc avian myelocytomatosis viral oncogene homolog), angptl4 (Angiopoietin-Like 4), sox9 (SRY-Related HMG-Box Gene 9), and fzd2 (Frizzled Class Receptor 2) showed marked expression differences between male and female gonads. These findings provide insights into the molecular mechanisms underlying sex differentiation in H. guttatus, offer potential molecular markers for sex identification, and establish a scientific basis for germplasm conservation and the optimization of breeding techniques.

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.

Monopterus albus is a protogynous hermaphroditic fish that changes from female to male, but the underlying sex change mechanism remains as-yet unknown. In this study, we firstly cloned and characterized the sequence and protein structure of unc-13d of M. albus. We found that the genomic structure of unc-13d was different from other species. Expression was detected in the developing gonad by applying qRT-PCR and in situ hybridization. We found that the expression of unc-13d in the ovotestis was higher than in the ovary and testes. A strong signal of unc-13d was detected in oocytes and granulosa cells in the ovary and spermatogonia and primary spermatocytes in the testes. We found that the promoter methylation of unc-13d was negatively correlated with gene expression in developing gonads, especially at site 114. A dual-luciferase assay was designed and revealed that dmrt1 regulates promoter activity opposite to foxl2. In summary, during sex reversal, DNA methylation affects the binding of the transcription factor dmrt1 and foxl2 in the promoter region through methylation and demethylation interactions to regulate the expression of unc-13d during gonadal development.

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.

Extrachromosomal circular DNA (eccDNA) serves as a vital regulator of gene expression and phenotypic plasticity. The Monopterus albus is a protogynous hermaphroditic fish, and the functional role of eccDNA during its female-to-male sex reversal remains poorly understood. We conducted Circle-seq analysis on gonadal tissues of super-female and male individuals and integrated our findings with previously published gonadal RNA-seq data. A total of 62,574 and 34,562 eccDNAs were, on average, identified in the two groups, and 7881 down-regulated and 2738 up-regulated differentially abundant eccDNAs were screened in male gonads. Three candidate eccDNAs, eccDNA^Dmrt1a, eccDNA^Sox9a1 and eccDNA^ZNF239, were selected for subsequent analysis. Quantitative real-time PCR was applied to detect the overall transcriptional levels of their corresponding host genes, covering transcripts derived from both genomic DNA and eccDNA. Spatiotemporal expression profiling and letrozole induction experiments revealed male-biased expression of Dmrt1a and Sox9a1, while ZNF239 was predominantly expressed in female gonads. Complete sex reversal was observed in female M. albus after 90-day letrozole treatment, accompanied by elevated Dmrt1a/Sox9a1 and reduced ZNF239 transcription. Fluorescence in situ hybridization localized these transcripts in gonadal germ cells and somatic cells across developmental stages. This study verifies the production of eccDNA during gonadal development. The results indicate that Dmrt1a and Sox9a1 may facilitate sex reversal and suggest that ZNF239 is likely involved in ovarian development. This work provides novel clues for deciphering the molecular mechanism underlying sex reversal from the eccDNA perspective.