241 METHYLATION PROFILE OF XIST GENE EXON 1 IN BOVINE SPERMATOGENIC CELLS

Abstract

Mammalian embryos undergo two waves of genome reprogramming, the first in the zygote, when paternal and maternal genomes are demethylated and, shortly thereafter, remethylated, and the second in the primordial germ cells. It is believed that remethylation is reestablished in male germ cells before birth; however, it is not clear when and how this process occurs in the bovine. Thus, this work aimed to verify whether epigenetic reprogramming occurs during spermatogenesis by evaluating the methylation profile of XIST gene exon 1 in different spermatogenic cells. Spermatocytes, elongated spermatids, and spermatozoa from the epididymis head and tail were collected from bovine testes obtained from a slaughterhouse. The DNA was extracted from the collected cells and treated with bisulfite. The treated DNA was amplified through nested PCR, and the product was inserted on a vector and cloned in bacteria. The plasmid DNA was extracted and sequenced. The sequences of the experiment were compared with the sequence of XIST gene from GenBank, and those that presented a bisulfite conversion rate of ≥95% and a homology of ≥97% with the sequence from GenBank were used. To compare the methylation pattern among groups, the total percentage of methylated CpG was calculated in each replicate and each group. The methylation pattern was compared using Kruskal-Wallis and Mann–Whitney tests because the data did not show normality. All data were compared using the Prophet Program, version 5.0 (1996; BBN Systems and Technologies, Cambridge, MA, USA), and are shown as the mean ± SEM. The significance level used was P < 0.05. The spermatocytes, elongated spermatids, and spermatozoa from the epididymis head and tail groups presented 15.70 ± 14.54%, 1.96 ± 1.96%, 74.09 ± 6.78%, and 45.09 ± 20.19% of methylation, respectively. The epididymis head group was more methylated than the spermatocyte and elongated spermatid groups (P < 0.01). When arranged in two groups, one with spermatocytes and elongated spermatids, and the other with epididymidal spermatozoa, and compared, it was observed that the methylation level was different (P < 0.01) between them. These results suggest that epigenetic reprogramming is still occurring during spermatozoa formation.