Efeito do choque térmico durante a maturação in vitro na expressão de micrornas em oócitos e embriões bovinos

Abstract

Thermal stress is one of the main causes for loss of economic productivity in cattle, in Brazil. Regarding reproductive capacity, heat stress affects oocyte quality and embryonic development and results in lower reproductive efficiency. The mechanisms by which the increase in temperature compromises the development of oocytes and embryos are not yet well established; however, the occurrence of transcriptional and translational suggests epigenetic changes to be caused by the elevation of the temperature. MicroRNAs (miRNAs) are an important class of non-coding RNAs classified as essential epigenetic modulators and their relation with thermal stress is not yet established. This study aimed to evaluate the effect of heat shock during in vitro maturation (IVM) on the expression of microRNAs and of genes involved in the processing of miRNAs in bovine oocytes and in embryos derived from these oocytes. Briefly, immature oocytes were randomly distributed in two experimental groups during IVM: 1) Control group (oocytes matured in vitro at 38.8ºC for 24h with 5% CO2), and 2) Thermal shock group (oocytes matured in vitro at 41ºC for 12h with 5% CO2 + 38.8ºC for 12h in 7% CO2), both remained in an atmosphere with 100% humidity. Following IVM, the oocytes were in vitro fertilized (IVF) and then cultured for eight days in CR2aa medium in an incubator with 5% CO2 and at 38.8°C. For miRNA and mRNA analysis, oocytes were collected at the end of the IVM and embryos at the 8-16 cell stage with 72h after the initiation of IVF. Real-time PCR was used for the relative quantification of the following miRNAs: let-7a-5p, -103, -34c, -1246, -19b, and -21-5p, as well as of the mRNAs: Drosha, and Dicer1. We have also evaluated: meiosis progression, embryonic development, and apoptotic index. Thermal shock reduced miR-21-5p (0,51±0,34) expression and increased Drosha (1,43±0,35) in oocytes (p<0.05). There was an increase in the expression of (3,0±2,0), -19b (3,96±2,29) and -1246 (2,29±1,21) in 8-cell stage embryos in the heat shock group (p<0.05). The rates of oocyte maturation, embryonic cleavage and development decreased (p<0.05) and the embryonic apoptotic index increased (p<0.05) in the thermal shock group. We concluded that the thermal shock during IVM promotes changes in the expression of miRNAs and genes involved in their biosynthesis in bovine oocytes. The effects of thermal shock may still be observed due to the alteration of the abundance of microRNAs in embryos derived from stressed oocytes. These alterations may be related to the lower embryonic development, as a result of epigenetic modifications promoted by heat shock.