200 EFFECTS OF MELATONIN ON REACTIVE OXYGEN SPECIES GENERATION AND ACQUISITION OF EMBRYONIC DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES MATURED IN VITRO

Abstract

Reactive oxygen species (ROS) are produced under normal culture conditions, but when production increases, it generates a harmful condition called oxidative stress (OS), leading to apoptosis and developmental blocks. Addition of antioxidants as melatonin to culture media has been used to minimize the effects of OS. Our hypothesis was that melatonin could improve oocyte in vitro maturation (IVM) and protect oocytes from ROS under a standard culture condition, thus increasing embryonic development. To test, cumulus-oocyte complexes were matured in TCM-199 with bicarbonate, 0.5 mg mL^–1 of FSH, 100 IU mL^–1 of hCG, and 10% FCS without supplementation (control group) or supplemented with 10^–5 (MT5), 10^–7 (MT7) or 10^–9 (MT9) M melatonin for 22 h at 38.5°C and 5% CO₂ in air. After IVM, a sample of oocytes (control, n = 59; MT5, n = 64; MT7, n = 77; MT9, n = 57) was stained with 1 µg mL^–1 Hoechst 33342 to assess the nuclear maturation, and oocytes were classified as being in the stages of germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase I or telophase I (A/T), or metaphase II (MII). To determine the intracellular ROS levels, other matured oocytes (control, n = 46; MT5, n = 59; MT7, n = 51; MT9, n = 61) were stained with 5 µM CellROX®Green (Molecular Probes, Eugene, OR, USA) and were evaluated immediately under an epifluorescence inverted microscope (excitation 485 nm; emission 520 nm). The images were recorded and further analysed by Q-Capture Pro image software. After subtraction of the background signal intensity from the measured fluorescent signal intensity values, 1 group was chosen as a calibrator (control group), and each treatment value was divided by the mean calibrator value to generate the relative expression level (in arbitrary fluorescence units). Finally, another sample of matured oocytes (control, n = 188; MT5, n = 173; MT7, n = 180; MT9, n = 178) was submitted to IVF (Day = 0), and the presumptive zygotes were cultured in SOF at 38.5°C and 5% CO₂ in air, for up to 7 days. The cleavage rates and embryonic development were evaluated at Days 3 and 7 of IVC, respectively. Data were analysed by ANOVA followed by Tukey’s test (P < 0.05) and are presented as mean ± SEM. There was no effect (P > 0.05) of different concentrations of MT on nuclear status of matured oocytes, as we found no differences in the rates of GV (0% to 5.3% ± 3.4), GVBD (5.4% ± 3.2 to 16.3% ± 5.0), MI (1.7% ± 1.7 to 3.2% ± 3.2), AI/TI (0% to 5.4% ± 3.4), and MII (74.8% ± 2.7 to 87.5% ± 3.7). The cleavage rates did not differ (P > 0.05) among treatments (76.7% ± 4.4 to 83.8% ± 2.7), as well as the embryonic development to the blastocyst stage (31.2% ± 1.9 to 43.7% ± 5.7). The intracellular ROS levels decreased significantly (P < 0.05) in the MT9 group (0.75 ± 0.03) in comparison to Control (1.0 ± 0.06), MT5 (0.97 ± 0.05) and MT7 (0.94 ± 0.05). In conclusion, supplementation with 10^–9 M melatonin during IVM reduced the intracellular ROS levels of oocytes without interfering with the nuclear maturation and the subsequent embryonic development to the blastocyst stage.

Financial support was provided by FAPESP (#2013/07382–6).