Nuclear transfer alters placental gene expression and associated histone modifications of the placental-specific imprinted gene pleckstrin homology-like domain, family A, member 2 (PHLDA2) in cattle.

Abnormal placental development is frequent in nuclear transfer (NT) pregnancies and is likely to be associated with altered epigenetic reprogramming. In the present study, fetal and placental measurements were taken on Day 60 of gestation in cows with pregnancies produced by AI, IVF and NT. Placentas were collected and subjected to histological evaluation, the expression of genes important in trophoblast differentiation and expression of the placental imprinted gene pleckstrin homology-like domain, family A, member 2 (PHLDA2), as well as chromatin immunoprecipitation (ChIP) for histone marks within the promoter of PHLDA2. Fewer binucleated cells were observed in NT cotyledons, followed by IVF and AI cotyledons (P<0.05). Expression of heart and neural crest derivatives expressed 1 (HAND1), placental lactogen (PL), pregnancy-associated glycoprotein 9 (PAG-9) and PHLDA2 was elevated in NT cotyledons compared with AI cotyledons. Expression of PHLDA2 was higher in IVF than AI samples (P<0.05). ChIP revealed an increase in the permissive mark dimethylation of lysine 4 on histone H3 (H3K4me2), surprisingly associated with the silent allele of PHLDA2, and a decrease in the inhibitory mark H3K9me2 in NT samples. Thus, genes critical for placental development were altered in NT placentas, including an imprinted gene. Allele-specific changes in the permissive histone mark in the PHLDA2 promoter indicate misregulation of imprinting in clones. Abnormal trophoblast differentiation could have resulted in lower numbers of binucleated cells following NT. These results suggest that the altered expression of imprinted genes associated with NT are also caused by changes in histone modifications.

Oxygen tension affects histone remodeling of in vitro-produced embryos in a bovine model.

In vitro production of bovine embryos is a biotechnology of great economic impact. Epigenetic processes, such as histone remodeling, control gene expression and are essential for proper embryo development. Given the importance of IVP as a reproductive biotechnology, the role of epigenetic processes during embryo development, and the important correlation between culture conditions and epigenetic patterns, the present study was designed as a 2 × 2 factorial to investigate the influence of varying oxygen tensions (O2; 5% and 20%) and concentrations of fetal bovine serum (0% and 2.5%), during IVC, in the epigenetic remodeling of H3K9me2 (repressive) and H3K4me2 (permissive) in bovine embryos. Bovine oocytes were used for IVP of embryos, cleavage and blastocyst rates were evaluated, and expanded blastocysts were used for evaluation of the histone marks H3K9me2 and H3K4me2. Morulae and expanded blastocysts were also used to evaluate the expression of remodeling enzymes, specific to the aforementioned marks, by real-time polymerase chain reaction. Embryos produced in the presence of fetal bovine serum (2.5%) had a 10% higher rate of blastocyst formation. Global staining for the residues H3K9me2 and H3K4me2 was not affected significantly by the presence of serum. Notwithstanding, the main effect of oxygen tension was significant for both histone marks, with both repressive and permissive marks being higher in embryos cultured at the higher oxygen tension; however, expression of the remodeling enzymes did not differ in morulae or blastocysts in response to the varying oxygen tension. These results suggest that the use of serum during IVC of embryos increases blastocyst rate without affecting the evaluated histone marks and that oxygen tension has an important effect on the histone marks H3K9me2 and H3K4me2 in bovine blastocysts.