245 EFFECTS OF MELATONIN ON THE IN VITRO OOCYTE MATURATION OF PORCINE CUMULUS–OOCYTE COMPLEXES

Abstract

In mammalian species, melatonin is a multifunctional hormone that mediates several circadian and seasonal processes, including reproduction. In addition to its hormonal actions, melatonin has a strong antioxidant effect and acts as a powerful free radical scavenger. Accordingly, the objective of this study was to investigate the local expression of the melatonin-receptor I (Mel-RI) gene and the effect of melatonin as an antioxidant on the in vitro maturation of porcine cumulus–oocyte complexes. Oocytes were obtained from aspiration of slaughterhouse ovaries and then matured in tissue culture medium 199 (TCM199) supplemented with 0.1% polyvinyl alcohol (PVA), 20 ng mL^–1 epidermal growth factor (EGF), and 4 IU mL^–1 pregnant mare serum gonadotropin (PMSG)/hCG. Expression of Mel-RI gene in cumulus cells, granulosa cells, or oocytes was evaluated by RT-PCR (Exp 1). The effect of various concentrations (0, 10, 50, and 100 ng mL^–1) of melatonin on the in vitro maturation in terms of nuclear maturation (polar body extrusion) (Exp 2) and measurement of reactive oxygen species (ROS) by dichlorohydrofluorescein diacetate (Yang et al. 1998 Hum. Reprod. 13, 998–1002) (Exp 3) were investigated. Each experiment was replicated at least four times, and each replication included more than thirty oocytes. For statistical analysis, one-way anova in the Prizm software program (GraphPad, San Diego, CA, USA) was used. The Mel-RI gene was locally expressed in cumulus and granulosa cells but not in oocytes. Melatonin at 10 ng mL^–1 had a beneficial effect on in vitro maturation (84.6%) compared to 0 ng mL^–1 (75.6%), 50 ng mL^–1 (80.9%), and 100 ng mL^–1 (76.5%). Levels of ROS were also significantly decreased by melatonin treatment at 10 (14.07), 50 (14.42), and 100 ng mL^–1 (13.03) of melatonin (P < 0.001) compared to that of the control group (28.21). The presence of melatonin receptor in cumulus cells and granulosa cells indicates a potentially important role for this hormone in regulating porcine ovarian and reproduction function. In conclusion, the present study demonstrated that melatonin had a beneficial effect on in vitro maturation of porcine cumulus–oocyte complexes, probably through binding a receptor and decreased ROS production. Further studies are required to elucidate the relationship between the appearance of melatonin receptor and mechanisms of melatonin function in the embryo.