Paternal neonatal sevoflurane exposure induces intergenerational anxiety via epididymal DNMT2-dependent sperm transfer RNA-derived small RNA remodelling in rats
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Updated: Sep 18, 2026
Stable Isotope In-Vivo Labeling for Mass-Spectrometry Identification of Paternal Metabolites Transferred from Sperm to Oocyte During Fertilization
Published on: June 17, 2025
Xiaoyu Hu1, Qingsheng Meng2, Sai Chen3
1Department of Anesthesiology, Pain and Perioperative Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
Background:
Perioperative exposures such as general anaesthesia can induce long-term neurodevelopmental changes with potential intergenerational effects, but the paternal epigenetic mechanisms remain unclear. We investigated whether neonatal sevoflurane exposure in male rats drives intergenerational anxiety through DNA methyltransferase 2 (DNMT2)-dependent remodelling of sperm transfer RNA-derived small RNAs (tsRNAs).
Methods:
Anxiety-like behaviours and hypothalamic-pituitary-adrenal (HPA) axis function were assessed in F0 males (sevoflurane-exposed or control generation), their offspring (F1), and grand-offspring (F2). Glucocorticoid receptor (GR)-mediated DNMT2 regulation was verified using chromatin immunoprecipitation quantitative polymerase chain reaction and primary cell assays. Sperm tsRNA profiles and 5-methylcytosine (m5C) methylation were analysed using small RNA sequencing and liquid chromatography-tandem mass spectrometry. Epididymal exosomes were isolated, and their role in tsRNA transfer was evaluated. Pharmacological and genetic interventions (GR blockade, DNMT2 knockdown, exosome inhibition) were used for validation of the pathway.
Results:
Paternal neonatal sevoflurane exposure induced anxiety-like behaviours in F0 males (open-field test, P=0.041; elevated plus maze, P=0.005) and F1 (not F2) offspring. Sevoflurane-induced HPA axis sensitisation, sustained corticosterone elevation (P=0.004), and GR-mediated DNMT2 upregulation in the cauda epididymis (P=0.014) of F0 males, with increased sperm m5C methylation (P=0.019) and aberrant tsRNA profiles. Epididymal exosomes mediated tsRNA transfer; DNMT2 knockdown or exosome blockade prevented tsRNA remodelling and intergenerational anxiety. Similar alleviating effects were observed after paternal treatment with the DNMT inhibitor decitabine.
Conclusions:
Neonatal sevoflurane exposure triggers a paternal GR-DNMT2 pathway in the epididymis, reprogramming sperm tsRNA profiles and driving intergenerational anxiety in a rat model. Epididymal DNMT2 is a key epigenetic mediator of anaesthesia-induced inheritance, and the alleviating effects of decitabine suggest additional mediating mechanisms.
