Table 1.
Effect of different durations of 100 μM VitC treatment on the in vitro development and quality of cloned mouse embryos.
Table 2.
Effect of different epigenetic modifier treatments on the in vitro development of cloned mouse embryos.
Fig 1.
Psammaplin A, but not vitamin C, increases H3K14 acethylation at 16 h post-activation.
ICSI embryos and cloned embryos non-treated (NT) and treated with 100 μM vitamin C (VitC), 10 μM psammaplin A (PsA) or the combination of both (VitC-PsA) during 16 h were immunostained for H3K14ac and DNA detection. A) Representative images of DNA and H3K14ac staining. Scale bar = 20 μm. B) Average intensity of H3K14ac/DNA signal ratio (+SEM) relative to ICSI embryos.
Fig 2.
Psammaplin A, but not vitamin C, reduces H3K9 dimethylation at 72 h post-activation.
ICSI embryos and cloned embryos non-treated (NT) and treated with 100 μM vitamin C (VitC), 10 μM psammaplin A (PsA) or the combination of both (VitC-PsA) during 16 h were immunostained for H3K9me2 and DNA detection. A) Representative images of DNA and H3K9me2 staining. Scale bar = 20 μm. B) Average intensity of H3K9me2/DNA signal ratio (+SEM) relative to ICSI embryos.
Fig 3.
Psammaplin A, but not vitamin C, reduces DNA methylation at 72 h post-activation.
ICSI embryos and cloned embryos non-treated (NT) and treated with 100 μM vitamin C (VitC), 10 μM psammaplin A (PsA) or the combination of both (VitC-PsA) during 16 h were immunostained for 5meC and DNA detection. A) Representative images of DNA and 5meC staining. Scale bar = 20 μm. B) Average intensity of 5meC/DNA signal ratio (+SEM) relative to ICSI embryos.
Fig 4.
Vitamin C does not alter 5hmeC/5meC ratio.
ICSI embryos and cloned embryos non-treated (NT) and treated with 100 μM vitamin C (VitC), 10 μM psammaplin A (PsA) or the combination of both (VitC-PsA) during 16 h were immunostained for 5meC and 5hmeC detection. A) Representative images of 5meC and 5hmeC staining. Scale bar = 20 μm. B) Average intensity of 5hmeC/5meC signal ratio (+SEM) relative to ICSI embryos.
Fig 5.
Psammaplin A, but not vitamin C, increases NANOG levels.
ICSI blastocysts and cloned blastocysts non-treated (NT) and treated with 100 μM vitamin C (VitC), 10 μM psammaplin A (PsA) or the combination of both (VitC-PsA) during 16 h were immunostained for NANOG and DNA detection. A) Representative images of DNA and NANOG staining. Scale bar = 20 μm. B) Mean percentage of blastocyst cells (+SEM) positive for NANOG staining. C) Average intensity of NANOG signal (+SEM) relative to ICSI embryos.
Fig 6.
Psammaplin A alone or combined with vitamin C increases OCT4 but not CDX2 levels.
ICSI blastocysts and cloned blastocysts non-treated (NT) and treated with 100 μM vitamin C (VitC), 10 μM psammaplin A (PsA) or the combination of both (VitC-PsA) during 16 h were immunostained for OCT4, CDX2 and DNA detection. A) Representative images of DNA, OCT4 and CDX2 staining. Scale bar = 20 μm. B) Mean percentage of blastocyst cells (+SEM) positive for OCT4 or CDX2 staining. C) Average intensity of OCT4 and CDX2 signals (+SEM) relative to ICSI embryos.
Fig 7.
Vitamin C increases reduced glutathione content.
Cloned embryos non-treated (NT) and treated with 100 μM vitamin C (VitC), 10 μM psammaplin A (PsA) or the combination of both (VitC-PsA) during 16 h were stained for reduced glutathione content detection at 16 h post-activation. A) Representative images of monochlorobimane (MCB) staining. Scale bar = 20 μm. B) Average intensity of MCB signal (+SEM) relative to non-treated embryos.
Table 3.
Comparative full-term development and body and placenta weights of fertilized and cloned mouse embryos.