Melatonin in in vitro maturation and their effect on the expression of antioxidant and apoptosis related genes of murine cumulus-oocyte complexes

Melatonin (MLT) is a hormone synthesized primarily in the pineal gland and participates in the control of the circadian cycle and reproductive seasonality in some animal species. In addition to these functions, MLT also acts as a powerful antioxidant and antiapoptotic factor. The aim of this study was to assess the effect of the addition of MLT during in vitro maturation (IVM) on nuclear maturation and expression of genes related to antioxidant and apoptotic activity in murine cumulus cells (CC) and oocytes (OO). Female F1 hybrids (n=20; C57BL/6 x CBA) were subject to intraperitoneal injection of eCG (5IU/0.1 mL/animal) and after 48h, the cumulus-oocyte complexes (COCs) were collected. COCs (n=25 per group/treatment) were randomly selected and in vitro matured with MLT (10-9, 10-6 and 10-3 M) or 0.5 µg/mL FSH (control) for 17h in an incubator at 37oC and 5% CO2 in air. Only MLT and not its association with FSH was used in order to evaluate their individual action on IVM and gene expression. Maturation rate was assessed according to the presence of the first polar body under an inverted microscope. Gene expression was evaluated by real-time quantitative PCR (4 replicates) for BAX and BCL2L1 (apoptosis) and GPX1, SOD1 and SOD2 (antioxidant) in CC and OO. As endogenous control, the geometric mean of H2AFZ and HPRT1 genes was used. Statistical analyses were performed by ANOVA followed by Tukey’s test (4 replicates) with 5% significance level. No differences were detected (P>0.05) between groups matured with MLT at different concentrations (56.0%, 56/100; 53.7%, 51/95; and 48.9%, 43/88; for 10-9, 10-6 and 10-3 M MLT, respectively) compared with the control group (57.3%, 55/96; FSH). The expression of GPX1 and SOD1 in CC was increased by MLT at 10-9 and 10-6 M (P=0.0006 e 0.0045, respectively). For OO, the group treated with 10-6 M MLT showed increased expression (P=0.0208) of the BAX pro-apoptotic gene. For the other genes, there was no difference between treatments (P>0.05). In conclusion, under the conditions studied, MLT was unable to improve the IVM rate, but alone was as efficient as FSH in promoting maturation of murine oocytes, indicating its potential effect on stimulating meiosis. In the real-time quantitative PCR analysis, the group treated with melatonin at 10-9 M presented increased expression of an antioxidant enzyme, suggesting the ability to enhance antioxidant action in CC.