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

Abstract

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 IC. Initially, a cytotoxicity assay was conducted using tumor and non-tumor cell lines to determine the IC₅₀ of 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 0.3 µg/mL (DXR), or WTA at 0.6 µM or 6.0 µM. DXR or WTA were added to the IVC medium once (₁DXR, ₁WTA0.6, ₁WTA6.0) or three times (₃DXR, ₃WTA0.6, ₃WTA6.0). After the IVC, the ovarian stroma, follicular morphology and development, cell proliferation, senescence, DNA damage, and apoptosis were assessed. The degeneration rate in ₃DXR and WTA6.0 (1x and 3x) was higher (p < 0.05) compared to the DMSO group. ₁DXR and ₃WTA0.6 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 ₃WTA0.6, staining for cleaved caspase-3 was more pronounced (p < 0.05). Additionally, ₃WTA0.6, ₁WTA6.0, and ₃DXR 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.