Efeito da melatonina na maturação in vitro sobre a expressão gênica e a qualidade de oócitos bovinos sob choque térmico

Abstract

The objective of this study was to evaluate the effect of melatonin addition to the in vitro maturation (IVM) medium of bovine oocytes subjected to heat shock on quality and expression of specific genes. Ovarian follicles from 3 to 8 mm of diameter, were aspirated from ovaries obtained in a slaughterhouse. Oocytes were matured under heat shock in IVM medium with melatonin, in the following concentrations: 0; 10 12 ; 10 -9 ; 10 -6 e 10 -3 M. In the positive control, oocytes were matured in the absence of melatonin (NS) on conventional condition. Three to six batteries were performed for each analysis. The randomized block design was utilized, being the blocks formed by the batteries. Maturation and apoptosis rate, reactive oxygen species (ROS) and mitochondrial activity were analyzed by generalized linear models through the GLIMIMIX procedure (SAS ® ). To the gene expression analysis, the treatment NS was used as a calibrator and the primer efficiency was calculated through the program LinRegPCR, with the results being analyzed by the REST ® software. Maturation rate did not differ between oocytes matured under heat shock with different melatonin concentration and did not differ between oocytes from treatments 0 M of melatonin under heat shock and 0 M of melatonin under conventional conditions. The concentrations 10 -3 and 10 -9 M of melatonin reduced the maturation rate in comparison to NS. Melatonin did not influence the apoptosis rate. Apoptosis rate from NS group was lower than observed on group 0 M, but it did not differ from the rate observed in the treatment 10 -3 . ROS production was lower in the treatments 10 -9 , 10 -6 , and 10 -3 M and greater in the treatments 0 and 10 -12 . Mitochondrial activity of oocytes matured at 10 -12 , 10 -9 , and 10 -6 M of melatonin did not differ from the one observed in oocytes of treatment NS. Oocytes from group 0 M presented greater mitochondrial activity compared to oocytes from group NS. There was subexpression of genes c-MOS in the treatments 10 -9 and 10 -12 M of melatonin and of MAPK in the treatment 10 -12 M. The treatments 10 -3 ; 10 -6 and 10 -9 M of melatonin presented subexpression of the gene GDF-9. Genes ZAR1 and HSP40 were not affected by the melatonin addition to the IVM medium or by the heat shock. Concluding, melatonin is able to protect oocytes from the damage caused by heat shock, being the concentration 10 -6 the most efficient.