80 DNA methylation of insulin-like growth factor 2 (igf2) gene in day 14 in vitro-produced bovine embryos of different sizes.

Abstract

In mammals, a correct DNA methylation reprogramming and the maintenance of genomic imprinting after fertilization are essential for embryo development and pregnancy. One important imprinted gene, related to embryo development and placentation, is the insulin-like growth factor 2 (IGF2) gene. Therefore, embryos with different sizes could show differences in the methylation pattern of IGF2 gene. The aim of this study was to evaluate the methylation pattern of the differentially methylated region (DMR) located within exon 10 of the IGF2 gene, of in vitro-produced Nellore bovine embryos that were different in size on day D14 of development. The embryos were produced from oocytes obtained by follicular aspiration of slaughter house ovaries. On D7 after in vitro fertilization only grade I blastocysts were selected and, in groups of 10 embryos, were transferred non-surgically to the uteri of previously synchronized recipients with similar conditions. Seven days after being transferred, embryos were collected (Day 14 of development) and measured using Motic Images Plus 2.0 program (Motic, Richmond, BC, Canada). Embryos >45mm were considered large (L) and those <25mm were considered small (S). After being measured, a portion of each trophoblast layer was biopsied and used to determine the methylation status of the IGF2 gene by bisulfite sequencing. The methylation pattern was evaluated on individual embryos considered as separate replicates. At least 5 to 8 clones were evaluated per embryo and the sequences were analysed with the BiQAnalyser software (Max-Planck-Institut für Informatik, Saarbrücken, Germany), using the GenBank sequence NM_174087.3 as reference. The methylation pattern of the different groups was compared using Kruskal-Wallis test (P<0.05). No differences in DNA methylation were found between S (26.7±8.3%, n=37 clones, 5 embryos) and L (34.8±2.9%, n=20 clones, 4 embryos) embryos. It is already known that the region studied is hypermethylated in sperm and hypomethylated in oocytes and, in some somatic cell types, it is expected to be around 50% methylated, being an imprinted region. Although we found a lower percentage of methylation than that expected for an imprinted region, this pattern may be the physiological pattern for trophoblast cells. This is the first report describing the methylation pattern of this region of the IGF2 gene in Day 14 bovine embryos of different sizes. It can be concluded that the methylation pattern of the intragenic DMR on exon 10 of IGF2 gene of in vitro-produced embryos on Day 14 of development is not affected by embryo size.

Full text

DNA methylation of IGF2 gene in D14 in vitro produced bovine embryos with
different sizes
Carvalho JO1; Franco MM2, Machado GM2; Dode MAN2.
1University of São Paulo - Piracicaba, SP - Brazil; 2Embrapa Genetic Resources and
Biotechnology- Brasília, DF - Brazil.
In mammals, a correct DNA methylation reprogramming and the maintenance of
genomic imprinting after fertilization are essential for embryo development and
pregnancy. One important imprinted gene, related with embryo development and
placentation, is the IGF2 gene. Many studies have associated changes in the IGF2
methylation pattern with assisted reproductive technology. The aim of this study was to
evaluate the methylation pattern of the differentially methylated region (DMR) located
into the exon 10 of the IGF2 gene, of in vitro produced bovine embryos that were
different in size on day D14 of development. The embryos were produced from oocytes
obtained by follicular aspiration of slaughterhouse ovaries. On D7 after in vitro
fertilization only grade I blastocysts were selected and, in number of 10 embryos, were
transferred non-surgically to the uteruses of previously synchronized recipients, with
similar conditions. Seven days after being transferred, embryos were collected (Day 14
of development – D14) and measured using Motic Images Plus 2.0 program. Embryos
greater than 45 mm were considered large (L) and smaller than 25 mm were considered
small (S). After being measured, a portion of each trophoblast layer was biopsied and
used to determine the methylation status of the IGF2 gene, using bisulfite sequencing.
The methylation pattern was performed individually, and each embryo was considered
as a replicate. At least 5-8 clones were evaluated per embryo and the sequences were
analyzed using the BiQAnalyser software, using the Genbank sequence NM_174087.3
as reference. The methylation pattern of the different groups was compared using
Kruskal-Wallis test (P<0.05). No differences in DNA methylation were found between
S (26.7±8.3%, n=37 clones) and L (34.8±2.9%, n=20 clones) embryos. As already
known the region studied is hypermethylated in sperm and hypomethylated in oocytes,
and in some somatic cell types it is expected to be around 50% methylated, considering
an imprinted region. Although we found a lower percentage of methylation than that
expected for an imprinted region, this pattern may be the physiological pattern for
trophoblast cells. This is the first report describing the methylation pattern of this region
of the IGF2 gene on Day 14 bovine embryos with different sizes. It can be concluded
that the methylation pattern of the intragenic DMR on exon 10 of IGF2 gene of in vitro
produced embryos on D14 of development was not affected by embryo size.
keyword:epigenetic, mutilation, DNA
Financial support: CNPq, FAP-DF