Extract of Azadirachta indica (neem) leaf induces apoptosis in rat oocytes cultured in vitro

ObjectiveTo determine whether aqueous neem leaf extract (NLE) could induce degeneration of rat oocytes and, if so, whether apoptosis is involved during NLE-induced degeneration of oocytes cultured in vitro.DesignA controlled prospective study.SettingLaboratory research setting at Department of Reproductive Biomedicine of the Institute.Animal(s)Fifty-four sexually immature female rats that were 24–25 days of age.Intervention(s)The immature female rats were injected with 10 IU pregnant mare serum gonadotropin for 48 h followed by 10 IU human chorionic gonadotropin (hCG) for 16 h. After 16 h, the rats were killed and ovulated cumulus oocyte complexes were collected from the oviduct. Cumulus-enclosed as well as denuded oocytes were used in the present study.Main Outcome Measure(s)Rates of shrinkage, membrane leakage, degeneration, assessment of morphological apoptotic changes, bax protein expression, and DNA fragmentation.Result(s)The NLE induced morphologic apoptotic changes such as shrinkage, membrane leakage, and cytoplasmic fragmentation prior to degeneration of oocytes. The NLE-treated oocytes that had morphologic apoptotic features showed overexpression of bax protein and DNA fragmentation as evidenced by terminal deoxynucleotidyl transferase nick-end labeling–positive staining and DNA ladder pattern.Conclusion(s)Neem leaf extract induced apoptosis in rat oocytes prior to degeneration in vitro.