Caracterização molecular dos componentes do sistema angiotensina-(1-7) durante a divergência folicular e expressão de genes de reparo da fita dupla de DNA em embriões bovinos

Abstract

The first study characterized the expression of MAS receptor and key enzymes for Ang-(1-7) production, such as, ACE2, NEP and PEP during follicular development. Furthermore, the regulation of local Ang1-7 system was evaluated after the intrafollicular injection of fulvestrant (an estradiolreceptor inhibitor) in the dominant follicle. Cows were ovariectomized when the size between the largest (F1) and the second largest follicle (F2) was not statistically different (Day 2), slightly different (Day 3), or markedly different (Day 4). The mRNA abundance of genes encoding MAS receptor, ACE2, NEP and PEP was evaluated in the follicular cells from F1 and F2. The mRNA expression of MAS receptor was upregulated in the granulosa cells of F2 after the establishment of follicular deviation (Day 4), while PEP mRNA increased during (Day 3) and after (Day 4) the deviation process. However, the mRNA expression of ACE2 was upregulated in the granulosa cells of F1 during and after the deviation process. The mRNA expression of NEP was not regulated in F1 and F2. The MAS receptor was immunolocated in the granulosa and theca cells of F1 and F2 during follicular deviation. Moreover, MAS receptor gene expression increased when the F1 was treated with the estrogen receptor-antagonist in vivo. In conclusion, the expression profile of MAS receptor, ACE2, NEP and PEP in dominant and subordinate follicles indicated that Ang-(1-7) play a role in the regulation of the follicular dominance in cattle. A second study was performed to investigate the expression of genes that control homologous recombination (HR; 53BP1, ATM, RAD50, RAD51, RAD52, BRCA1, BRCA2 and NBS1), and non-homologous end-joining (NHEJ; KU70, KU80 and DNAPK), DNArepair pathways in bovine embryos with high, intermediate or low developmental competence. We also evaluated whether bovine embryos can respond to DNA double-stranded breaks (DSBs) induced by ultraviolet (UV) irradiation by regulating the expression of genes involved in the HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro fertilization and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation (EGA). All studied genes were expressed before, during and after the EGA period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before EGA in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-fertilization in bovine embryos with DSBs induced by UV irradiation. In conclusion, important genes controlling HR and NHEJ repair pathways are expressed in bovine embryos before, during or after EGA. Lower developmental competence seems to be associated with a higher mRNA expression of 53BP1 and RAD52. Bovine embryos can response to UV-induced DSBs after the EGA but HR and NHEJ repair pathways seem to be particularly regulated at the blastocyst stage.