Incomplete epigenetic reprogramming is one of the major factors affecting the development of embryos cloned by somatic cell nuclear transfer (SCNT). Histone 3 lysine 9 (H3K9) trimethylation has been identified as a key barrier to efficient reprogramming by SCNT. The aim of this study was to explore a method of downregulating H3K9me3 levels in donor cells by using histone lysine demethylase (KDM) protein. When sheep fetal fibroblast cells were treated with recombinant human KDM4D protein (rhKDM4D), the levels of H3K9 trimethylation and dimethylation were both significantly decreased. After SCNT, rhKDM4D-treated donor cells supported significantly higher percentage of cloned embryos developing into blastocysts as compared to non-treated control cells. Moreover, the blastocyst quality was also improved by rhKDM4D treatment of donor cells, as assessed by the total cell number in blastocysts and the expression of developmental genes including SOX2, NANOG and CDX2. These results indicate that treatment of donor cells with recombinant KDM4D protein can downregulate the levels of H3K9 trimethylation and dimethylation and improve the developmental competence of SCNT embryos. This strategy may be convenient to be used in KDM4-assisted SCNT procedure for improving the efficiency of cloning.
BackgroundThe high mortality of pre-weanling piglets is a dominant challenge which severely restricts the development of pig industry. A number of factors including nutrients imbalance and temperature variation during postnatal period of piglets have been reported to closely associated with the high mortality of postnatal piglets. This study aims to find out the relationship between fat deposition and survival of newborn piglets.ResultsThere were no differences in organ coefficient and bone density between the surviving and dead piglets (P > 0.05). The body weight and the fat deposition in the dead piglets were lower than the live individuals (P < 0.05). Consistently, the average sizes of white adipocytes in back and abdominal adipose tissues of dead piglets were smaller than the survivals (P < 0.05). The protein expression levels of adipocyte differentiation markers PPARγ and C/EBPα in the back and abdominal adipose tissues were lower in dead piglets compared to live piglets. The mRNA expressions of thermogenic markers PGC1α and PRDM16 in adipose tissues were decreased in the dead piglets (P < 0.05). The microarray of back fat samples from the surviving and dead piglets were conducted; two down-regulated genes namely AAMDC and CASTOR1 were identified from the dead piglets. According to quantitative real-time PCR (RT-PCR) analysis, the mRNA expression of AAMDC decreased, whereas CASTOR1 expression elevated in the dead piglets compared to the surviving piglets (P < 0.05).ConclusionsThe fat deposition and adipocyte differentiation in the dead piglets are insufficient compared to the surviving piglets, which may attenuate the thermogenic ability of white adipose tissue (WAT). Our data indicate that fat deposition in newborn piglets is vital to their survival.
Vitamin C is not only an antioxidant but also a regulator of epigenetic modifications that can enhance the activity of the ten‐eleven translocation (TET) family dioxygenases and promote the oxidation of 5‐methylcytosine (5mC) to 5‐hydroxymethylcytosine (5hmC). Here, we investigated the effects of vitamin C in regulating DNA methylation in sheep somatic cells or embryos in an effort to improve the cloned embryo development. Vitamin C treatment of sheep foetal fibroblast cells significantly increased the 5hmC levels but did not affect the 5mC levels in cells. After nuclear transfer, vitamin C‐treated donor cells could not support a higher blastocyst development rate than non‐treated cells. Although combination of serum starvation and vitamin C treatment could induce significant 5mC decrease in donor cells, it failed to promote the development of resultant cloned embryos. When cloned embryos were directly treated with vitamin C, the pre‐implantation development of embryos and the 5hmC levels in blastocysts were significantly improved. This beneficial role of vitamin C on embryo development was also observed in fertilized embryos. Our results suggest that vitamin C treatment of the embryos, but not the donor cells, can improve the development of cloned sheep embryos.
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