Digitoxin-induced apoptosis in ovarian granulosa cells disrupts follicular development and impairs reproductive performance

BACKGROUND: Digitoxin, a cardiac glycoside from Digitalis spp., is clinically employed for heart failure and atrial fibrillation, and yet its reproductive toxicity is not well understood. We have therefore assessed digitoxin′s influence on follicular development and reproductive outcomes in both cell-culture models and animal models. METHODS: The in vitro culture of porcine follicles was employed to assess gene expression levels related to proliferation and apoptosis, whereas an in vivo mouse model was utilized to evaluate hormone levels (via ELISA), ovarian morphology (H&E staining), apoptosis (TUNEL assay), and reproductive performance. RESULTS: Transcriptomic analysis revealed that, in the digitoxin-treated group, 1,577 genes were upregulated, whereas 4,359 genes were downregulated compared with the control group. KEGG enrichment analysis demonstrated significant involvement of these differentially expressed genes in pathways associated with amino acid metabolism, steroid biosynthesis, and oocyte maturation. Additionally, GO enrichment analysis underscored their role in regulating cellular functions and metabolic processes. GSEA further indicated that digitoxin influences pathways related to DNA function, meiotic progression, and steroid biosynthesis. Moreover, treatment with 100 nM digitoxin for 48 h significantly reduced granulosa cell (GC) viability to ~ 15%, decreased proliferation to 3.02% (p < 0.05), and increased apoptosis to 34.9% (p < 0.05). Digitoxin treatment also downregulated PCNA, CDK4, and MCL1, while upregulating CASP3, CASP7, CASP8, and CASP9. In vivo, digitoxin administration resulted in lower serum levels of estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) (p < 0.001), an increase in antral follicle counts, a decrease in corpus luteum numbers (p < 0.05), a delayed onset of first estrus, and a reduction in litter size (p < 0.05). CONCLUSION: Our study elucidates the multifaceted effects of digitoxin on GC function and female reproductive health, highlighting significant implications for fertility and reproductive toxicity. Whereas digitoxin is beneficial in treating cardiovascular diseases and certain tumors, the careful selection and monitoring of therapeutic doses are crucial, particularly for women of reproductive age requiring prolonged treatment.