71 MELATONIN IMPROVES PORCINE IN VITRO MATURATION VIA SONIC HEDGEHOG SIGNALLING

Abstract

Melatonin (N-acetyl-5-methoxytryptamine) is the hormone synthesised from the mammalian pineal gland, which has an antioxidant property and regulates physiological processes such as cellular metabolism. It is well known that melatonin affects in vitro maturation of oocytes and embryonic development in many species. However, limited information is available on the underlying beneficial effects of melatonin. Sonic Hedgehog (Shh) signalling is important for follicular development, oocyte maturation, and embryo development. To elucidate the relationship between melatonin and Shh signalling, we designed an experiment with the following three groups: (1) control, (2) melatonin, and (3) melatonin with cyclopamine (smoothened inhibitor) during porcine in vitro maturation. Porcine ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory at 28 to 32°C. The contents of follicles 3 to 6 mm in diameter were recovered by aspiration with an 18 G needle. Cumulus–oocyte complexes were pooled and cultured in TCM-199 medium for 44 h. The aim of this study was to evaluate the effects of melatonin (10⁻⁹ M) with or without cyclopamine (2 μM) on cumulus cell expansion (a total of 432 cumulus–oocyte complexes were used in 3 replicates), embryo development after parthenogenetic activation (a total of 432 oocytes were used in 4 replicates). Moreover, we detected gene expression related to cumulus expansion, oocyte maturation, and hedgehog signalling in cumulus cells and oocyte. Results indicated that melatonin treatment significantly increased cumulus expansion index (3.75 ± 0.02 v. 3.51 ± 0.03 and 3.59 ± 0.05, respectively; P < 0.05) and blastocyst formation rates (30.4 ± 2.4 v. 21.9 ± 2.2 and 20.0 ± 2.2, respectively; P < 0.05) compared with control and melatonin with cyclopamine. In addition, the expression of cumulus expansion-related genes (Ptgs1, Ptgs2, Has2, Ptx-3, and Tnfaip6) and hedgehog signalling-related genes (Shh, Pthc1, Smo, and Gli-1) in cumulus cells were up-regulated in melatonin treatment compared with control and melatonin with cyclopamine. Similarly, the expressions of oocyte maturation-related genes (GDF9 and BMP15) in porcine oocytes were up-regulated in melatonin treatment compared with control and melatonin with cyclopamine. In conclusion, Shh signalling mediated melatonin to improve porcine cumulus cell expansion, oocyte maturation, and subsequent embryo development. Further studies are needed to evaluate the effect of melatonin on protein levels of Shh signalling. Statistical analyses were performed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA). All data were tested for normality and homoscedasticity and then subjected to one-way ANOVA, followed by Duncan’s multiple range test (when the variances were assumed to be equal) or Dunnet’s T3 test (when the variances were assumed to be unequal) to determine differences among experimental groups. All results are expressed as means ± SEM; P-values < 0.05 were considered to be statistically significant.

This study was supported by Ministry of Trade, Industry and Energy (#10048948), Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries (#311011–05–4-SB010, #114059–03–2-SB010), National Research Foundation (2016M3A9B6903410), China Scholarship Council (CSC, No. 2015–3022), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.