288 INFLUENCE OF MELATONIN ON IN VITRO MATURATION OF BOVINE OOCYTES

Abstract

The present study evaluated the effects of melatonin (MEL) during IVM of bovine cumulus-oocyte complexes (COC). The COC were cultured in droplets (25–30/100 µL) under mineral oil at 38.5°C and 5% CO₂ in air. Medium (TCM199 + 0.1% polyvinyl alcohol, 0.25 mM sodium pyruvate, and 25 µg mL^–1 of gentamycin) was supplemented with FSH (0.5 µg mL^–1), MEL (10^–9 and 10^–6 M), or no hormones (control). In experiment 1, oocytes were assessed for nuclear maturation rates (6, 12, 18, and 24 h of IVM). In experiment 2, relative abundance of antioxidant enzymes copper, zinc superoxide dismutases (CuZnSOD), manganese superoxide dismutases (MnSOD), and glutathione peroxidase (GPx) was evaluated in oocytes and cumulus cells (0 and 24 h) by real time PCR. The immature group was the reference and endogenous controls were actin β and glyceraldehyde 3-phosphate dehydrogenase. In experiment 3, nuclear fragmentation in cumulus was assessed by TUNEL and flow cytometry (24 h). In experiment 4, embryo development after in vitro fertilization and culture was analysed (cleavage rates D2, blastocysts Day 8, and hatching Day 10). The control group was matured in complete IVM medium (10% FCS, 0.5 µg mL^–1 of FSH, 50 µg mL^–1 of LH, and 1 µg mL^–1 of oestradiol). The MEL and FSH groups were the same. Data (3–4 replicates) were analysed by Chi-square (experiment 1; GraphPad Prism) or ANOVA and Tukey test (SAS, 1995) and 5% significance. At 6 h of IVM, all oocytes were at germinal vesicle stage. At 12 h, hormone groups had similar metaphase I rates (71–81%, P > 0.05) and were superior to control (54%, P < 0.05). At 18 h, metaphase II (MII) rates were similar (57–74%, P > 0.05). After 24 h, MEL (10^–9 and 10^–6 M) was similar to control (51, 67, and 69% MII, respectively, P > 0.05) and FSH had the highest MII rates (90%, P > 0.05). The 10^–6 M MEL was similar to FSH (P > 0.05). Antioxidant enzymes were unaffected in oocytes (P > 0.05%). The CuZnSOD transcripts increased in cumulus after IVM with 10^–6 M MEL compared to immature cells (3.5 and 1.0, respectively, P < 0.05). Control, FSH, and 10^–9 M MEL did not differ (2.1–2.5, P > 0.05) or increased relative to immature cells (P < 0.05), but were inferior to 10^–6 M MEL (P < 0.05). The MnSOD relative abundance was similar for immature, control, and 10^–9 M MEL (1.0–3.0, P > 0.05). The 10^–6 M MEL increased MnSOD relative to immature cells (3.0 and 1.0, respectively, P < 0.05), but was similar to control and 10^–9 M MEL (P > 0.05). The FSH showed the highest levels (9.1, P < 0.05); GPx4 transcripts were not affected (P > 0.05). Nuclear fragmentation in cumulus was not influenced (33.4–41.5/10 000 cells; P > 0.05). Embryo development rates were similar for all groups (cleavage: 82–87%, blastocysts: 49–54, hatching: 91–96%, P > 0.05). In conclusion, MEL during IVM stimulates meiosis resumption at rates similar to FSH and embryo development similar to FSH and complete IVM medium. The MEL increased CuZnSOD expression in cumulus, but no parallel effect was observed on nuclear fragmentation.