Altered imprinted gene expression and methylation patterns in mid-gestation aborted cloned porcine fetuses and placentas

Abstract

Purpose To determine the expression patterns of imprinted genes and their methylation status in aborted cloned porcine fetuses and placentas. Methods RNA and DNA were prepared from fetuses and placentas that were produced by SCNT and controls from artificial insemination. The expression of 18 imprinted genes was determined by quantitative real-time PCR (q-PCR).Bisulfite sequencing PCR (BSP) was conducted to determine the methylation status of PRE-1 short interspersed repetitive element (SINE), satellite DNA and H19 differentially methylated region 3 (DMR3). Results The weight, imprinted gene expression and genomewide DNA methylation patterns were compared between the mid-gestation aborted and normal control samples. The results showed hypermethylation of PRE-1 and satellite sequences, the aberrant expression of imprinted genes, and the hypomethylation of H19 DMR3 occurred in mid-gestation aborted fetuses and placentas. Conclusions Cloned pigs generated by somatic cell nuclear transfer (SCNT) showed a greater ratio of early abortion during mid-gestation than did normal controls because of the incomplete epigenetic reprogramming of the donor cells. Altered expression of imprinted genes and the hypermethylation profile of the repetitive regions (PRE-1 and satellite DNA) may be associated with defective development and early abortion of cloned pigs, emphasizing the importance of epigenetics during pregnancy and implications thereof for patient-specific embryonic stem cells for human therapeutic cloning and improvement of human assisted reproduction.

Faithful expression of imprinted genes in donor cells of SCNT cloned pigs

“…Our results showed that these genes were hemi‐methylated (∼50%) in PEF and C‐PEF; however, they were either completely methylated or unmethylated in P‐PEF and CP‐PEF by BSP and COBRA analysis. Previous reports have observed altered methylation patterns of imprinted genes, especially the demethylation of imprinted H19 , IGF2 and XIST in mid‐gestation aborted or short‐lived cloned animals while not in the healthy and full‐term development cloned animals [27–29], which demonstrated better understanding of the aberrant methylation in cloned animals may provide insight into improving the efficiency of SCNT and the subsequent health of cloned animals [30]. Although we didn't specifically distinguish between the paternal and maternal alleles using single nucleotide polymorphisms (SNPs), it has been shown that the DMRs of H19 , NNAT and MEST were either fully methylated or completely unmethylated in parental specific alleles [31].…”

Distribution of 5-methylcytosine and 5-hydroxymethylcytosine in bovine fetal tissue of the placenta

“…Our study is one of the first to evaluate the levels of 5-mC and 5-hmC in such extraembryonic tissues. The correct pattern of DNA methylation in placenta is probably related to normal pregnancy and healthy newborn animals (Zhang et al 2014). The literature has shown that almost all cloned bovine animals show a hypertrophied funiculus umbilicalis, which is strictly related to high mortality rates (Wells et al 1999).…”

DNA methylation patterns and gene expression associated with litter size in Berkshire pig placenta

“…These proteins were overexpressed in LLG, and the authors suggested that their expression plays a crucial role in placental efficiency. Another study compared the expression and genome-wide DNA methylation patterns of 18 imprinted genes between mid-gestational, aborted, cloned porcine fetuses and placentas [ 24 ]. However, no studies have investigated methylation in placental tissue in relation to litter size in pigs.…”