Embryos that are produced in vitro frequently present epigenetic modifications. However, maternal supplementation with folic acid (FA) may improve oocyte maturation and embryo development, preventing epigenetic errors in the offspring. We sought to evaluate the influence of FA supplementation during in vitro maturation of grade I (GI) and grade III (GIII) bovine oocytes on embryo production rate and the expression of IGF2 and KCNQ1OT1 genes. The oocytes were matured in vitro with different concentrations of FA (0, 10, 30 and 100 μM), followed by in vitro fertilization and embryo culture. On the seventh day (D7) of culture, embryo production was evaluated and gene expression was measured using real-time qPCR. Supplementation with 10 μM of FA did not affect embryo production for GI and GIII oocytes. Moderate supplementation (30 μM) seemed to be a positive influence, increasing embryo production for GIII (P = 0.012), while the highest dose (100 μM) reduced embryo production (P = 0.010) for GI, and IGF2 expression was not detected. In GIII, only embryos whose oocyte maturation was not supplemented with FA demonstrated detected IGF2 expression. The lowest concentration of FA (10 μM) reduced KCNQ1OT1 expression (P = 0.05) on embryos from GIII oocytes. Different FA concentrations induced different effects on bovine embryo production and gene expression that was related to oocyte quality. Despite the epigenetic effects of FA, supplementation seems to be a promising factor to improve bovine embryo production if used carefully, as concentration is an important factor, especially in oocytes with impaired quality.

Intrauterine growth restriction (IUGR) due to uteroplacental insufficiency results in a placenta that is unable to provide adequate nutrients and oxygen to the fetus. These growth-restricted babies have an increased risk of hypertension and chronic kidney disease later in life. In rats, both male and female growth-restricted offspring have nephron deficits but only males develop kidney dysfunction and high blood pressure. In addition, there is transgenerational transmission of nephron deficits and hypertension risk. Therefore, epigenetic mechanisms may explain the sex-specific programming and multigenerational transmission of IUGR-related phenotypes. Expression of DNA methyltransferases (Dnmt1and Dnmt3a) and imprinted genes (Peg3, Snrpn, Kcnq1, and Cdkn1c) were investigated in kidney tissues of sham and IUGR rats in F1 (embryonic day 20 (E20) and postnatal day 1 (PN1)) and F2 (6 and 12 months of age, paternal and maternal lines) generations (n = 6–13/group). In comparison to sham offspring, F1 IUGR rats had a 19% decrease in Dnmt3a expression at E20 (P < 0.05), with decreased Cdkn1c (19%, P < 0.05) and increased Kcnq1 (1.6-fold, P < 0.01) at PN1. There was a sex-specific difference in Cdkn1c and Snrpn expression at E20, with 29% and 34% higher expression in IUGR males compared to females, respectively (P < 0.05). Peg3 sex-specific expression was lost in the F2 IUGR offspring, only in the maternal line. These findings suggest that epigenetic mechanisms may be altered in renal embryonic and/or fetal development in growth-restricted offspring, which could alter kidney function, predisposing these offspring to kidney disease later in life.

Androgenetic embryonic stem (AgES) cells offer a possible tool for patient-specific pluripotent stem cells that will benefit genomic imprinting studies and clinic applications. However, the difficulty in producing androgenetic embryos and the unbalanced expression of imprinted genes make the therapeutic applicability of AgES cells uncertain. In this study, we produced androgenetic embryos by injecting two sperm into an enucleated metaphase II (MII) oocyte. By this method, 88.48% of oocytes survived after injection, and 20.24% of these developed to the blastocyst stage. We successfully generated AgES cell lines from the androgenetic embryos and assayed the expression of imprinted genes in the cell lines. We found that the morphological characteristics of AgES cells were similar to that of fertilized embryonic stem cells (fES), such as expression of key pluripotent markers, and generation of cell derivatives representing all three germ layers following in vivo and in vitro differentiation. Furthermore, activation of paternal imprinted genes was detected, H19, ASC12 and Tss3 in AgES cell activation levels were lower while other examined genes showed no significant difference to that of fES cells. Interestingly, among examined maternal imprinted genes, only Mest and Igf2 were significantly increased, while levels of other detected genes were no different to that of fES cells. These results demonstrated that activation of some paternal imprinted genes, as well as recovery of maternal imprinted genes, was present in AgES cells. We differentiated AgES cells into a beating embryoid body in vitro, and discovered that the AgES cells did not show significant higher efficiency in myocardial differentiation potential.