Chronic nitrite exposure impairs testicular energy homeostasis and sperm motility in association with altered hif1a signaling in rare minnow Gobiocypris rarus

Nitrite (NO₂⁻) is a common nitrogenous contaminant in aquatic environments and drinking water, but its mechanisms of action on male reproduction are still not fully understood. In this study, male rare minnow (Gobiocypris rarus) were used as a model to investigate the effects of chronic NO₂⁻ exposure on testicular structure, energy metabolism, and sperm quality, and to elucidate the roles of hypoxia-inducible factor-1 (hif1a) and its associated metabolic genes. Fish were exposed for 21 days to control water or two environmentally relevant NO₂⁻ concentrations (0.5 and 2 mg/L NO₂⁻-N, respectively). Exposure to NO₂⁻ did not significantly affect body length or body weight, but low-dose NO₂⁻ increased testis weight and gonadosomatic index (GSI). At both doses, testicular histology was disrupted, ATP content decreased, and sperm concentration and motility were reduced, suggesting that the elevated GSI more likely reflects pathological changes in the testes rather than enhanced reproductive function. The rare minnow hif1a encoded a protein containing the typical structural domains of Hif1a, was highly conserved with other vertebrates, and was constitutively expressed in the testis. Early during NO₂⁻ exposure (1 week), testicular hif1a mRNA and protein levels were significantly upregulated, before gradually declining with prolonged exposure and falling below control levels by week 3. Immunofluorescence revealed that NO₂⁻ exposure caused disordered Hif1a distribution and patchy accumulation within the seminiferous epithelium of the testis. A series of genes involved in glucose uptake, glycolysis, lactate transport, and mitochondrial function showed time- and dose-dependent expression changes in the testis, and most of them were positively correlated with hif1a expression. These findings indicate that the "early activation followed by suppression" pattern of Hif1a dynamics is closely associated with testicular energy imbalance under NO₂⁻ exposure. In summary, chronic NO₂⁻ exposure impaired spermatogenesis and sperm motility without significantly affecting somatic growth and was associated with altered testicular hif1a/Hif1a dynamics and transcriptional changes in energy metabolism-related genes. These findings provide new evidence for understanding the reproductive toxicity of environmental nitrite in vertebrates.