40 Administration of 0.1 μM melatonin during in vitro maturation of bovine oocytes regulates autophagy levels in produced embryos

Abstract

Melatonin, the antioxidant pineal hormone, is a strong regulator for various cellular processes essential for reproduction. Although the protective role of 0.1 µM melatonin against the toxicity of different anti-developmental compounds has been elucidated in numerous studies, its effect on the autophagy level in in vitro-produced blastocysts has not been entirely clarified. In this study, oocytes were incubated for 24 h in the presence and absence of melatonin, administered during IVM, to investigate the effect of 0.1 µM melatonin on the developmental competence of bovine oocytes and pre-implantation embryos, autophagy, and quality of embryos. The developmental potential of embryos were basically the stages from oocytes fertilization to blastocyst production. Gene expression levels were evaluated in matured oocytes, whereas blastocysts were used for immunofluorescence experiments. The differences between treated and control groups were analysed using Student’s t-test (GraphPad Prism version 6; GraphPad Inc.), where P-values <0.05 were considered significant. Results showed that oocyte maturation, Day-4 total cleavage, and Day-8 blastocyst development rates were not significantly improved (melatonin: 72 ± 2 vs. control: 69 ± 2 for cleavage rate, and melatonin: 33 ± 1 vs. control: 31 ± 2 for control for Day-8 blastocyst; P > 0.05), whereas the level of reactive oxygen species (ROS) was reduced (P < 0.05) with addition of melatonin. Using RT-qPCR, cumulus cells-related (HAS2) and apoptosis-related (Bcl2 and SOD2) genes were upregulated, whereas BAX was downregulated in melatonin-treated oocytes. Using immunofluorescence, apoptosis (caspase-3) and autophagy (Beclin-1 and LC3) markers were underexpressed, whereas the PI3K survival protein (P < 0.05) and matrix metalloproteinases (MMP-2 and MMP-9; P > 0.05) were overexpressed, in Day-8 embryos of melatonin-treatment. Additionally, the total number of cells per blastocysts, inspected via nuclei-based 4′,6-diamidino-2-phenylindole (DAPI) staining was higher in the melatonin-treated group (P < 0.05). Taken together, our study demonstrates that 0.1 µM melatonin treatment during IVM does not interfere with developmental competence, but improves the quality of IVF-produced embryos by lowering the incidence of autophagy.