Differential effects of alcohol-derived serum metabolites and direct ethanol exposure on mouse preantral follicle development.

Acute alcohol exposure is known to impair female reproductive function; however, it remains unclear whether the biological effects of circulating metabolites generated after alcohol ingestion on follicular development differ from those of direct ethanol exposure. Therefore, we compared the effects of serum from mice with acute alcohol exposure and ethanol-supplemented medium on the in vitro development of mouse preantral follicles and explored the underlying mechanisms of these effects. Female Kunming (KM) mice were administered 56% (volume/volume, v/v) ethanol intragastrically to establish an acute alcohol exposure model, followed by serum collection. Preantral follicles were cultured in three systems: foetal bovine serum (FBS) control, serum from alcohol-exposed mice, and medium supplemented with ethanol at an equivalent concentration. Follicular morphology, steroid hormone secretion, gene expression, oxidative stress, apoptosis, oocyte maturation, and subsequent embryonic developmental competence were evaluated. Acute alcohol exposure altered serum composition, and this alteration was characterised by increased ethyl linoleate and reduced cholesterol levels. Compared with the FBS control, follicles cultured with serum from mice exposed to alcohol exhibited enlarged antral cavities and cumulus expansion, whichwas accompanied by meiotic delay at the germinal vesicle (GV) and mid-metaphase I (mid-MI) stages. Alcohol-exposed serum increased follicle-stimulating hormone levels and upregulated forkhead box L2 (Foxl2) expression, thereby promoting oestradiol synthesis while suppressing progesterone secretion during follicular development. In contrast, direct ethanol exposure impaired follicular development by decreasing mitochondrial membrane potential and inducing apoptosis. Although oxidative stress marker levels were elevated in serum from alcohol-exposed mice, oocytes maintained normal reactive oxygen species levels through enhanced antioxidant defence, including increased expression of superoxide dismutase 1 (Sod1), glutathione peroxidase 1 (Gpx1), translocase of inner mitochondrial membrane 23 (Tim23) and polyribonucleotide nucleotidyltransferase 1 (Pnpt1) in oocytes. Notably, oocytes derived from follicles cultured with alcohol-exposed serum exhibited improved embryonic developmental competence after in vitro fertilisation. Thus, serum from mice with acute alcohol exposure exerts biological effects distinct from those of direct ethanol exposure during preantral follicle development. Alcohol-derived serum metabolites partially mitigate ethanol-induced reproductive toxicity through endocrine modulation and activation of antioxidant defence pathways, providing new insights into the complex effects of alcohol metabolism on female reproductive function.