172 Effect of Melatonin on Meiosis Progression and Lipid Content in Bovine Oocytes Matured In Vitro

Abstract

Melatonin (MLT) may have positive effects on oocyte nuclear maturation and may influence their lipid metabolism. The aim of this study was to assess the effect of MLT on nuclear maturation and lipid content inbovine oocytes during in vitro maturation (IVM). Cumulus-oocyte complexes (30-35/group) were submitted to IVM in TCM-199 supplemented with 3 mg mL⁻¹ BSA (negative control; NC) or with added hormones: 1 µg mL⁻¹ FSH (positive control; PC) or MLT (10⁻¹¹, 10⁻⁹ and 10⁻⁷ M), to evaluate the individual action of each treatment on meiosis resumption (metaphase I, MI) at 9 h and progression to metaphase II (MII) at 24 h IVM. Lipid content in oocytes was also assessed. Oocytes were denuded and stained with Hoechst 33342 and Nile Red, and evaluated by epifluorescence microscopy to determine the nuclear maturation stage and lipid content, respectively. The fluorescence intensity (FI) was measured by ImageJ software (National Institutes of Health, Bethesda, MD, USA). Data were analysed by ANOVA followed by Tukey’s test to compare effects of treatments on meiosis resumption and lipid contents at 9 h and on meiosis progression and lipid contents at 24 h; and t-test to compare the same treatment at 9 and 24 h IVM regarding lipid contents (4 replicates/treatment). Significance level was 5% (GraphPrism software; GraphPad Inc., San Diego, CA, USA). At 9 h IVM, meiosis resumption rate (MI) increased (P < 0.05) for MLT 10⁻⁹ M (32.6 ± 14.8%, 45/138) and FSH groups (33.9 ± 18.1%, 38/112) compared with NC (4.2 ± 2.7%, 5/120). The other treatments did not differ in relation to NC (20.5 ± 12.5%, 27/132 and 14.9 ± 9.0%, 20/134 to MLT 10⁻¹¹ and 10⁻⁷ M, respectively). Regarding lipid contents at 9 h, MLT 10⁻¹¹ M showed higher lipid content (8.43 ± 7.61FI; P < 0.0001) compared with other treatments [MLT 10⁻⁹ M (5.59 ± 4.13), MLT 10⁻⁷ M (4.87 ± 3.21), FSH (5.34 ± 3.66), and NC (4.27 ± 3.28)]. Maturation rate (MII) at 24 h IVM was highest in FSH group (70.1 ± 10.2%, 94/134). No differences were observed (P > 0.05) between groups matured in different concentrations of MLT (65.9 ± 4.2%, 83/126; 62.2 ± 0.6%, 56/90 and 53.5 ± 10.0%, 61/114 for MLT 10⁻¹¹, 10⁻⁹ and 10⁻⁷ M, respectively) compared with NC (86.3 ± 1.2%, 88/102; P < 0.05). Similarly, lipid content at 24 h IVM was higher (P < 0.05) in FSH group (10.72 ± 4.71 FI) compared with NC (7.98 ± 3.45 FI) or MLT treatments with 10⁻¹¹ M (8.90 ± 3.91), 10⁻⁹ M (7.85 ± 3.56), and 10⁻⁷ M (8.29 ± 3.87). However, MLT 10⁻⁷ M, FSH, and NC groups had higher (P < 0.0001) lipid content at 24 than at 9 h. In conclusion, MLT 10⁻⁹ M alone was able to stimulate meiosis resumption (9 h) at a rate similar to FSH, but was insufficient to stimulate progression to MII at 24 h, indicating that it may affect nuclear maturation only during the initial steps of meiosis. Lipid contents were increased during IVM but for lower MLT concentrations, this increase was not observed. Whether these observations have any impact on oocyte quality remains to be determined. Further studies are necessary to improve our knowledge of the role of MLT on lipid metabolism and by which mechanism it may affect meiosis resumption in bovine oocytes.

This research was funded by FAPESP (HF- 2016/24884-3, scholarship; CLVL, financial support 2015/20379-0).