251 METHYLATION PATTERN OF A DIFFERENTIALLY METHYLATED REGION LOCATED IN THE LAST EXON OF THE Igf2 GENE IN OOCYTES FROM NELLORE COWS

Abstract

Assisted reproductive technologies are essential in modern cattle breeding. The relevance of in vitro embryo production (IVP) has been increasing around the world, particularly in Brazil. Acknowledging the positive effect of IVP regarding the utilization of female reproductive potential, its productivity can be improved. Methylation pattern of DNA in oocytes is a key factor for the improvement of IVP efficiency because it is related to oocyte competence. The objective of this study was to evaluate the methylation pattern of a differentially methylated region (DMR) located in the exon 10 of the imprint gene IGF2 in immature and in vitro-maturated oocytes from different follicle sizes. Small follicles (1-3 mm in diameter) and large follicles (≥8.1 mm in diameter) were dissected from slaughterhouse ovaries, and the COC were removed. Immature and in vitro-maturated oocytes, presenting the first polar body, were denuded and frozen in PBS at -80°C until DNA extraction. After extraction, DNA was treated with sodium bisulfite using EZ DNA Methylation™ Kit (Zymo Research, Orange, CA, USA). Bisulfite-treated DNA was amplified in a 2-round PCR strategy (nested-PCR), the amplicons purified with Geneclean® III Kit (MP Biomedicals, Irvine, CA, USA), cloned using pGEM®-T Easy Vector System (Promega, Madison, WI, USA), and sequenced. The number of follicles and in vitro maturation data were analyzed with chi-square test, and the methylation status of 27 CpG islands was analyzed with Student (normal distribution) or Mann-Whitney tests in the Prophet software (BBN Systems and Technologies, Cambridge, MA, USA). Cumulus-oocyte complexes from ≥8.1-mm follicles presented greater percentages of viable oocytes (40.5%) and nuclear maturation (60.6%) (P ≤ 0.05). Immature oocytes from 1- to 3-mm follicles were less methylated (33.33%) than those from ≥8.1-mm follicles (83.69%). After in vitro maturation, oocytes from ≥8.1-mm follicles were less methylated (18.51%) than the immature oocytes from ≥8.1-mm follicles (83.69%) and 1- to 3-mm maturated oocytes (49.62%). The less methylated pattern in the oocytes from ≥8.1-mm maturated follicles can be associated with greater competency of those oocytes, because this was the expected pattern. The lower level of methylation in the immature oocytes group (1- to 3-mm follicles) can be associated with the incomplete establishment of imprinting reprogramming pattern. Finally, we concluded that the methylation pattern of DMR, in the last exon of the IGF2 gene, can be used as a molecular marker for epigenetic reprogramming status in oocytes, helping the development of new IVP protocols. Supported by Embrapa Genetic Resources and Biotechnology and CAPES.