Meiosis progression and donor age affect expression profile of DNA repair genes in bovine oocytes

Several genetic and physiological factors increase the risk of DNA damage in mammalian oocytes. Two critical events are meiosis progression, from maturation to fertilization, due to the extensive chromatin remodelling during genome decondensation and aging which is associated to a progressive oxidative stress. In this work, we studied the transcriptional patterns of three genes, RAD51, APEX-1 and MLH1, involved in DNA repair mechanisms. The analyses were performed by Real-Time quantitative PCR (RT-qPCR) in immature and in vitro matured oocytes collected from 17 ± 3 mo old heifers and 94 ± 20 mo old cows. Batches of 30-50 oocytes for each group (three replicates) were collected from ovarian follicles of slaughtered animals. The oocytes were freed from cumulus cells at the time of follicle removal, or after IVM carried out in M199 supplemented with 10% foetal calf serum, 10 IU LH /ml, 0.1 IU FSH /ml and 1 µg 17β-oestradiol/ml. Total RNA was extracted by Trizol method. The expression of bovine GAPDH gene was used as internal standard, while primers for bovine RAD51, APEX-1 and MLH1 genes were designed from DNA sequences retrieved from GeneBank. Results obtained indicate a clear up-regulation of RAD51, APEX-1 and MLH1 genes after IVM ranging between 2- and 4-fold compared to GV oocytes. However, only RAD51 showed a significant transcript increase between the immature oocytes collected from young and old individuals. This finding candidates RAD51 as gene marker for discriminating bovine immature oocytes in relation to the donor age.