Rhinogobio nasutus, an endangered fish species endemic to the upper and middle reaches of the Yellow River in China, lacks essential genomic information on gonadal development, hindering research into its reproductive biology. To address this, mRNA-seq and miRNA-seq datasets derived from adult testis (n = 3) and ovary (n = 3) were integrated to characterize sex-biased expression profiles and potential regulatory mechanisms. A total of 34,813 genes and 68,623 transcripts were detected, and 16,105 differentially expressed genes (DEGs) were identified between testis and ovary, including 9365 testis-biased and 6740 ovary-biased genes. Small-RNA profiling identified 51 differentially expressed miRNAs (DEMs: 31 testis-biased; 20 ovary-biased). The sex-biased mRNA profiles highlighted conserved candidate genes associated with germ-cell maintenance, somatic regulation, ovarian differentiation, and oocyte maturation, including vasa, piwi, dmrt1, amh, cyp19a1a, zar1, zar1l, and rbpms2. Integrated miRNA–mRNA analysis further predicted potential interactions involving key sex-related genes, suggesting that DEMs may contribute to post-transcriptional regulation during gonadal differentiation. Functional enrichment (GO and KEGG analyses) highlighted pathways associated with gonadal differentiation, germline maintenance, and signal transduction pathways. qRT-PCR validation of nine mRNAs and nine miRNAs showed expression patterns consistent with the sequencing results. Collectively, these results provide an integrated mRNA and miRNA resource for R. nasutus gonads and identify candidate genes and miRNAs for future studies on sex-biased gonadal development, reproductive regulation, and artificial propagation.

Rhinogobio nasutus, a freshwater fish belonging to the family Cyprinidae, is primarily distributed in the upper and middle reaches of the Yellow River. However, overfishing and habitat degradation have resulted in a marked decline in its wild populations, and the species was listed as a Class II nationally protected aquatic wildlife species in China in 2021. Spermatogonial stem cells (SSCs) provide a valuable resource for fish germplasm conservation and reproductive biotechnology. In this study, we successfully established a long-term cultured germ-cell population with spermatogonial stem cell-like characteristics derived from R. nasutus (RnSSCs). These cells exhibited a morphology consistent with an undifferentiated spermatogonial phenotype and showed a diploid chromosome number of 2n = 50. Alkaline phosphatase (AP) staining showed strong positive activity, while RT-PCR detected the expression of germ-cell- and stem-cell-associated markers, with only weak expression of the somatic-cell-associated marker. Immunofluorescence staining revealed strong signals for both proliferating cell nuclear antigen (PCNA) and the germ cell-specific protein Vasa. After more than four months of in vitro culture, the cells continued to proliferate and retained expression of germ-cell- and stem-cell-associated markers. Following transplantation into zebrafish blastulae, fluorescently labelled cells were detected in the gonadal region of a small number of recipient embryos. Following hormonal induction, some cells developed a sperm-like morphology, accompanied by the expression of the meiotic-associated markers rec8 and dmc1. The establishment of the RnSSC line provides a renewable cellular resource for studying spermatogenesis and germ cell transplantation in R. nasutus. These findings provide a useful cellular resource for studying the reproductive biology of R. nasutus.

To explore the mechanisms underlying toxicity effects induced by acute ammonia nitrogen stress on juvenile Silurus lanzhouensis, individuals with an average body weight of (2.64±0.68) g were selected as experimental subjects for ammonia nitrogen stress experiments. A 96h acute toxicity test was conducted to determine the median lethal concentration (LC₅₀) and safe concentration (SC). Based on these results, a control group (0) and an experimental group (14 mg/L) were established for a 48h acute ammonia nitrogen stress experiment, followed by a 48h post-stress recovery period. The activities of antioxidant enzymes, pathological damage, and expression changes of immune-related genes in gill and liver tissues were analyzed. Results indicated that the LC₅₀ values of ammonia nitrogen for juvenile Silurus lanzhouensis at 24h, 48h, 72h, and 96h were 18.69, 16.61, 15.39, and 14.27 mg/L, respectively, with a safe concentration of 3.94 mg/L. Under ammonia nitrogen stress, the activity of Na⁺/K⁺-ATPase in gill tissue exhibited a trend of initially decreasing, subsequently increasing, and then decreasing again. Antioxidant enzyme activities (SOD, CAT, T-AOC) and lipid peroxidation products (MDA) reached their peak levels at 12h of stress exposure and then significantly declined. Pathological damage in gill and liver tissues progressively worsened over time, characterized by fusion of gill lamellae, vacuolation, epithelial cell death, liver cell necrosis, and expansion of blood sinusoids. This damage remained irreversible even after 48h of recovery. Expression levels of the HSP90 gene in gill and liver tissues peaked at 12h of stress exposure and subsequently decreased, while IL-1 gene expression reached its maximum at 48h of stress exposure and did not fully recover to control group levels post-recovery. These findings suggest that acute ammonia nitrogen stress poses a significant threat to the health of juvenile Silurus lanzhouensis by disrupting the antioxidant system, inducing irreversible tissue structural damage, and causing abnormal expression of immune-related genes. This study provides a theoretical basis for water quality management in healthy breeding practices for Silurus lanzhouensis and contributes to understanding environmental toxicity tolerance and response mechanisms.