Protective Effect of Cimicifuga racemosa (L.) Nutt Extract on Oocyte and Follicle Toxicity Induced by Doxorubicin during In Vitro Culture of Mice Ovaries

SIMPLE SUMMARY: Doxorubicin (DOXO) is a chemotherapeutic drug that promotes the loss of follicular reserve through atresia and overactivation of primordial follicles and, consequently, decrease fertility in female patients. Gonadotoxic protective agents that act independently of chemotherapy drug have great value for human medicine. Cimicifuga racemosa (L.) Nutt extract (CIMI) improves antioxidant status and has the potential to prevent formation of reactive oxygen species (ROS) in the ovary. However, there are no reports about the potential of CIMI to protect oocytes and follicles against damages caused by DOXO in ovarian tissues cultured in vitro. This study evaluated the potential of CIMI extract to reduce the deleterious effects of DOXO in oocytes, follicles and stromal cells in mice ovaries cultured in vitro. The results show that DOXO reduces the percentage of normal follicles and the density of stromal cells in cultured ovaries, but these harmful effects were blocked by CIMI. Higher staining intensity for caspase-3 was seen in ovaries cultured in control medium alone or with DOXO when compared with those cultured with CIMI alone or both CIMI and DOXO. Furthermore, ovaries cultured with CIMI had higher levels of mRNA for antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT). ABSTRACT: This study evaluated the potential of Cimicifuga racemosa (L.) Nutt extract (CIMI) to reduce the deleterious effects of doxorubicin (DOXO) in oocytes, follicles and stromal cells in mice ovaries cultured in vitro. In experiment 1, mice ovaries were cultured in DMEM(+) alone or supplemented with 5, 50 or 500 ng/mL CIMI, while in experiment 2, mice ovaries were cultured in DMEM(+) alone or supplemented with 5 ng/mL CIMI (better concentration), 0.3 μg/mL DOXO or both. Thereafter, the ovaries were processed for histological (morphology, growth, activation, extracellular matrix configuration and stromal cell density), immunohistochemical (caspase-3) analyses. Follicle viability was evaluated by fluorescence microscopy (ethidium homodimer-1 and calcein) while real-time PCR was performed to analyses the levels of (mRNA for SOD, CAT and nuclear factor erythroid 2–related factor 2 (NRF2) analyses. The results showed that DOXO reduces the percentage of normal follicles and the density of stromal cells in cultured ovaries, but these harmful effects were blocked by CIMI. The DOXO reduced the percentage of primordial follicles, while the presence of CIMI alone did not influence percentage of primordial follicles. A higher staining for caspase-3 was seen in ovaries cultured in control medium alone or with DOXO when compared with those cultured with CIMI alone or both CIMI and DOXO. In addition, follicles from ovaries cultured with both CIMI and DOXO were stained by calcein, while those follicles cultured with only DOXO were stained with ethidium homodimer-1. Furthermore, ovaries cultured with CIMI or both CIMI and DOXO had higher levels of mRNA for SOD and CAT, respectively, than those cultured with only DOXO. In conclusion, the extract of CIMI protects the ovaries against deleterious effects of DOXO on follicular survival and ovarian stromal cells.