Investigations on the effects of in vitro exposure of mouse ovaries to withaferin A, a new candidate for chemotherapy.

This study aimed to investigate, in vitro, the toxicity of WTA on ovarian follicles. Initially, a cytotoxicity assay was conducted using tumor and non-tumor cell lines to determine the ICof the WTA and validate its antitumor activity. Mouse ovaries were cultured in vitro (IVC) for 6 days in the presence of 1% dimethyl sulfoxide (DMSO), doxorubicin at 0.3µg/ml (DXR), or WTA at 0.6µM or 6.0µM. DXR or WTA were added to the IVC medium once (DXR, WTA0.6, WTA6.0) or three times (DXR, WTA0.6, WTA6.0). After the IVC, the ovarian stroma, follicular morphology and development, cell proliferation, senescence, DNA damage, and apoptosis were assessed. The degeneration rate in DXR and WTA6.0 (1x and 3x) was higher (p < 0.05) compared to the DMSO group. DXR and WTA0.6 reduced (p < 0.05) the percentage of primordial follicles and increased (p < 0.05) the number of developing follicles compared to the control (CTR) and DMSO groups. An increase (p < 0.05) in lipofuscin granules was observed with DXR and WTA at both concentrations and exposure frequencies compared to the CTR. In the presence of WTA0.6, staining for cleaved caspase-3 was more pronounced (p < 0.05). Additionally, WTA0.6, WTA6.0, and DXR increased (p < 0.05) DNA fragmentation in the stroma compared to the CTR and DMSO groups. We conclude that, like chemotherapy agents used for cancer treatment, WTA induces severe cytotoxic effects on ovarian follicles and stroma, especially at high concentrations and exposure frequencies.