Inhibition of RNA synthesis during Scriptaid exposure enhances gene reprogramming in SCNT embryos

in Reproduction
Correspondence should be addressed to V Bordignon; Email: vilceu.bordignon@mcgill.ca
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Insufficient epigenetic reprogramming is incompatible with normal development of embryos produced by somatic cell nuclear transfer (SCNT), but treatment with histone deacetylases inhibitors (HDACi) enhances development of SCNT embryos. However, the mechanisms underpinning HDACi benefits in SCNT embryos remain largely uncharacterized. We hypothesized that, in addition to enhancing reprogramming, HDACi treatment may promote expression of genes not required for early development of SCNT embryos. To test this hypothesis, RNA synthesis was inhibited by treating bovine SCNT embryos with 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DBR), which were concomitantly treated or not with Scriptaid (Scrip; an HDACi). Development to the blastocyst stage was significantly increased by treatment with Scrip alone (26.6%) or associated with DRB (28.6%) compared to Control (17.9%). The total number of nuclei was significantly improved only in embryos that were treated with both Scrip + DRB. Nuclear decondensation after SCNT was significantly increased by DRB treatment either alone or associated with Scrip. The relative mRNA expression, evaluated during the embryo genome activation (EGA) transition, revealed that some KDMs (KDM1A, KDM3A, KDM4C and KDM6A) and DNMT1 where prematurely expressed in Scrip-treated embryos. However, treatment with Scrip + DRB inhibited early mRNA expression of those genes, as well as several other KDMs (KDM4A, KDM4B, KDM5A, KDM5B, KDM5C and KDM7A) compared to embryos treated with Scrip alone. These findings revealed that HDACi improved development in SCNT embryos compared to Control, but altered the expression of genes involved in epigenetic regulation and did not improve embryo quality. Inhibition of RNA synthesis during HDACi treatment enhanced nuclear chromatin decondensation, modulated gene expression and improved SCNT embryo quality.

Downloadable materials

  • Table S1. List of primers and accession number of analyzed genes.
  • Figure S1. RNA synthesis detection (A) and nuclear area (B) in control and DRB treated (12 h) fibroblast cells.

 

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    Number of nuclei in SCNT-derived embryos from Control, DRB, Scrip and DRB + Scrip-treated embryos. (A) Total number of nuclei on D7 blastocysts and ICM/total number of nuclei ratio. (B) Representative pictures of CDX2 immunofluorescence staining for differential cell count in D7 blastocysts. Results are presented as means ± s.e.m., and P < 0.05 was considered statistically significant.

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    Nuclear area at 12 hpa in 1-cell SCNT embryos from different experimental groups (A) and representative pictures showing nuclear swelling (B). Results are presented as means ± s.e.m., and P < 0.05 was considered statistically significant.

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    Relative mRNA expression of H3K4 lysine demethylases on days 3, 4 and 5 of development in SCNT embryos from Control (black bars), Scrip (gray bars) and DRB + Scrip (white bars) treatments. Results are presented as means ± s.e.m., and P < 0.05 was considered statistically significant. Different letters indicate statistical significance in the same treatment between days, and asterisks indicate statistical significance between treatments in the same day.

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    Relative mRNA expression of H3K9 lysine demethylases on days 3, 4 and 5 of development in SCNT embryos from Control (black bars), Scrip (gray bars) and DRB + Scrip (white bars) treatments. Results are presented as means ± s.e.m., and P < 0.05 was considered statistically significant. Different letters indicate statistical significance in the same treatment between days, and asterisks indicate statistical significance between treatments in the same day.

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    Relative mRNA expression of H3K27 lysine demethylases on days 3, 4 and 5 of development in SCNT embryos from Control (black bars), Scrip (gray bars) and DRB + Scrip (white bars) treatments. Results are presented as means ± s.e.m., and P < 0.05 was considered statistically significant. Different letters indicate statistical significance in the same treatment between days, and asterisks indicate statistical significance between treatments in the same day.

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    Relative mRNA expression of DNMT1 and XIST on days 3, 4 and 5 of development in SCNT embryos from Control (black bars), Scrip (gray bars) and DRB + Scrip (white bars) treatments. Results are presented as means ± s.e.m., and P < 0.05 was considered statistically significant. Different letters indicate statistical significance in the same treatment between days, and asterisks indicate statistical significance between treatments in the same day.

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