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: (i) meiosis progression, from maturation to fertilization, due to extensive chromatin remodelling during genome decondensation; and (ii) aging, which is associated with a progress...

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Bibliographic Details
Published in:Zygote (Cambridge) 2015-02, Vol.23 (1), p.11-18
Main Authors: Bilotto, S., Boni, R., Russo, G.L., Lioi, M.B.
Format: Article
Language:English
Subjects:
Age Factors
Animals
Cattle
Deoxyribonucleic acid
DNA
DNA Repair - genetics
DNA-(Apurinic or Apyrimidinic Site) Lyase - genetics
Female
Gene Expression Profiling
In Vitro Oocyte Maturation Techniques
Meiosis
Nuclear Proteins - genetics
Oocytes - physiology
Oxidative stress
Rad51 Recombinase - genetics
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Summary:Several genetic and physiological factors increase the risk of DNA damage in mammalian oocytes. Two critical events are: (i) meiosis progression, from maturation to fertilization, due to extensive chromatin remodelling during genome decondensation; and (ii) aging, which is associated with 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-month-old heifers and 94 ± 20-month-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 in vitro maturation (IVM) carried out in M199 supplemented with 10% fetal calf serum, 10 IU luteinising hormone (LH)/ml, 0.1 IU follicle-stimulating hormone (FSH)/ml and 1 μg 17β-oestradiol/ml. Total RNA was extracted by Trizol method. The expression of bovine GAPDH gene was used as the internal standard, while primers for bovine RAD51, APEX-1 and MLH1 genes were designed from DNA sequences retrieved from GenBank. Results obtained indicate a clear up-regulation of RAD51, APEX-1 and MLH1 genes after IVM, ranging between two- and four-fold compared with germinal vesicle (GV) oocytes. However, only RAD51 showed a significant transcript increase between the immature oocytes collected from young or old individuals. This finding highlights RAD51 as a candidate gene marker for discriminating bovine immature oocytes in relation to the donor age.
ISSN:0967-1994
1469-8730
DOI:10.1017/S0967199413000154