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. 2015 Feb;17(1):28-40.
doi: 10.1089/cell.2014.0075. Epub 2014 Dec 30.

Oxamflatin treatment enhances cloned porcine embryo development and nuclear reprogramming

Jiude Mao  1 , Ming-Tao Zhao, Kristin M Whitworth, Lee D Spate, Eric M Walters, Chad O'Gorman, Kiho Lee, Melissa S Samuel, Clifton N Murphy, Kevin Wells, Rocio M Rivera, Randall S Prather
Affiliations

Oxamflatin treatment enhances cloned porcine embryo development and nuclear reprogramming

Jiude Mao et al. Cell Reprogram. 2015 Feb.

Abstract

Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low cloning efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro-fertilized (IVF) embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the cloned embryos, indicating these genes were successfully reprogrammed. However, compared to the lack of methylation of XIST in day-7 IVF embryos, a higher methylation level in day-7 cloned embryos was observed, implying that X chromosomes were activated in day-7 IVF blastocysts, but were not fully activated in cloned embryos, i.e., reprogramming of XIST was delayed. A time-course analysis of XIST DNA methylation on day-13, -15, -17, and -19 in vivo embryos revealed that XIST methylation initiated at about day 13 and was not completed by day 19. The methylation of the XIST gene in day-19 control cloned embryos was delayed again when compared to in vivo embryos. However, methylation of XIST in Oxamflatin-treated embryos was comparable with in vivo embryos, which further demonstrated that Oxamflatin could accelerate the delayed reprogramming of XIST gene and thus might improve cloning efficiency.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Percentage of embryos cleaved at 28 h (A) and blastocysts formed at day 7 (B) postactivation in the zero control and 150, 300, and 500 nM Oxamflatin groups. Embryos were treated with various concentrations of Oxamflatin for 16 h and then washed and cultured in PZM3 medium until day 7. Means with different letters (a, b) differ significantly (p<0.05).
<b>FIG. 2.</b>
FIG. 2.
Percentage of cleavage at 28 h postactivation, blastocysts formed (%) at day 7, and total cell number of blastocysts in the Oxamflatin and Scriptaid groups. Embryos were treated with 150 nM Oxamflatin or 500 nM Scriptaid for 16 h, then were washed and cultured in PZM3 until day 7.
<b>FIG. 3.</b>
FIG. 3.
Methylation status of POU5F1 locus in the donor cells and day-7 SCNT embryos. A dramatic decrease in methylation from donor cells to day-7 embryos; Oxamflatin treatment further decreased its methylation (p<0.05). (Solid-filled circles) Methylated cytosine; (open circles) unmethylated cytosine in each CpG site; (gray circles) mutated and/or single nucleotide polymorphism (SNP) variation at certain CpG sites. Each row of circles represents an individual clone that contains the inserted amplicon. The top diagram in each figure following schematically denotes the genomic location of the target DNA methylation region. ATG, start codon. Note, the same legend applies to Figures 4–7. The top diagrams that indicate the location of interest genes studied were the same promoter region of POU5F1 and NANOG and centromeric repeat elements gene as reported by Zhao et al. (2013).
<b>FIG. 4.</b>
FIG. 4.
Methylation status of NANOG locus in the donor cells and day-7 SCNT embryos. A dramatic decrease in methylation from donor cells to day-7 embryos was observed. Oxamflatin treatment did not have any effect on NANOG methylation.
<b>FIG. 5.</b>
FIG. 5.
Methylation status of centromeric repeat elements in the donor cells and day-7 SCNT embryos. There was a decrease in methylation from donor cells to day-7 embryos. Oxamflatin treatment further decreased methylation from 27.3±3.1% of controls to 18.2±3.2% of the Oxamflatin-treated group (p<0.05).
<b>FIG. 6.</b>
FIG. 6.
Methylation status of the XIST locus in the donor cells and day-7 SCNT and IVF embryos. There was a decrease in the XIST DNA methylation level from donor cells to day-7 embryos. Oxamflatin treatment did not have an effect on XIST methylation. A high level of methylation in the XIST gene indicated that reprogramming of XIST was not completed in SCNT embryos, as compared to no methylation of IVF embryos.
<b>FIG. 7.</b>
FIG. 7.
Association between XIST gene expression and its methylation level in day-7 SCNT embryos. Each dot represents one pooled blastocyst sample. (Triangle) Oxamflatin-treated group; (diamond) control group.
<b>FIG. 8.</b>
FIG. 8.
XIST DNA methylation in day-13, -15, -17, and -19 in vivo (A) and day-19 SCNT embryos (B). The overall percentage of methylated CpGs out of total CpG sites was not different between different days of in vivo embryos (A) or between Oxamflatin and control SCNT embryos (B).
<b>FIG. 9.</b>
FIG. 9.
Validation of XIST methylation by COBRA. A group of selected samples were used to confirm bisulfite sequencing data. Even band intensity (50/50) for both the cut methylated allele and the uncut unmethylated allele in the donor cells and day-19 embryonic membrane was measured, whereas day 13 had almost no cut band. COBRA was performed using the restriction enzyme BstUI, which cuts the methylated allele at 121 bp between CpG 7 and 8 (amplicon size 191 bp).
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