278 METHYLATION STATUS IN THE INTRAGENIC DIFFERENTIALLY METHYLATED REGION OF THE IGF2 LOCUS IN UNSORTED AND SEX-SORTED SPERM

Abstract

Methylation is the main coordinator of epigenetic inheritance between generations, influencing the regulation of gene expression. Therefore, changes of its pattern in any cell can cause important alterations affecting its function. The methylation pattern of imprinted genes has been reported to be altered by numerous environmental factors such as nutrition, diseases, and drugs. Sexing by flow cytometry exposes the sperm cells to various procedures that can affect sperm quality and could induce methylation changes, resulting in lower fertilization when compared with conventional sperm. Although many studies have related changes in methylation pattern to in vitro culture of oocytes and embryos, no reports have evaluated these changes in sperm caused by the sexing process. The IGF2 imprinted gene, which is predominantly expressed by the paternal allele with the maternal one silenced, is expected to have its intragenic differentially methylated region (DMR) highly methylated in the sperm. Therefore, it became an excellent candidate region to be used for evaluating changes in methylation status of the sperm. The objective of this study was to evaluate the influence of sexing by flow cytometry on the methylation pattern of the DMR located in exon 10 of the IGF2 gene. Frozen–thawed unsorted and sex-sorted sperm samples from 4 Nellore bulls were used (5–10 years old). Each ejaculate was separated into 3 fractions: nonsexed (NS), sexed for X-sperm (SX), and sexed for Y-sperm (SY). Then, 1 straw of each treatment/bull was thawed, placed in 40:70 Percoll gradient (GE Bioscience^®, Uppsala, Sweden) and centrifuged at 700 × g for 45 min to separate the somatic cells from the sperm. Sperm pellets were then used for DNA extraction. After that, DNA was treated with sodium bisulfite using the EZ DNA methylation kit (Zymo Research^®, Orange, CA, USA). Bisulfite-treated DNA was amplified in 2-round PCR strategy (nested-PCR) and the amplicons were purified using GenClean III kit (MP Biomedical^®, Solon, OH, USA). The purified amplicons were cloned into the pGEM-T easy vector system (Promega^®, Madison, WI, USA) and transformed into Escherichia coli cells (XL-1 Blue). The resulting individual clones were sequenced, using a dideoxy fluorescence terminator system (ABI 3130xl, Applied Biosystems, Foster City, CA, USA). The experiment was performed in 3 replicates per bull. Sequences were analysed using the BiQ Analyzer software with a GenBank sequence (X53553) as a reference. At least 60 clones were sequenced per group. The methylation status of the 28 CpG sites was compared among the groups using analysis of variance and Tukey test in the Prophet software. No differences in DNA methylation were found between NS (97.5 ± 1.7%), SX (95.8 ± 1.8%), and SY (97.3 ± 0.2%) (P = 0.36). Moreover, 100% of analysed sequences in all groups were hypermethylated. Therefore, we can conclude that the process of sexing by flow cytometry does not change the methylation pattern in the intragenic DMR located in exon 10 of the IGF2 gene.