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Improved development of somatic cell cloned mouse embryos by vitamin C and latrunculin A.

Mallol A, Santaló J, Ibáñez E - PLoS ONE (2015)

Bottom Line: The use of LatA alone significantly improved in vitro development, but not full-term development.On the other hand, the simultaneous treatment of cloned embryos with VitC and the histone deacetylase inhibitor psammaplin A (PsA), in combination with the use of LatA, resulted in cloning efficiencies equivalent to those of VitC or PsA treatments alone, and the effects on pluripotency and nuclear reprogramming markers were less evident than when only the PsA treatment was applied.These results suggest that although both epigenetic modifiers improve cloning efficiencies, possibly through different mechanisms, they do not show an additive effect when combined.

View Article: PubMed Central - PubMed

Affiliation: Departament de Biologia Cellular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra, Spain.

ABSTRACT
Impaired development of embryos produced by somatic cell nuclear transfer (SCNT) is mostly associated with faulty reprogramming of the somatic nucleus to a totipotent state and can be improved by treatment with epigenetic modifiers. Here we report that addition of 100 μM vitamin C (VitC) to embryo culture medium for at least 16 h post-activation significantly increases mouse blastocyst formation and, when combined with the use of latrunculin A (LatA) during micromanipulation and activation procedures, also development to term. In spite of this, no significant effects on pluripotency (OCT4 and NANOG) or nuclear reprogramming markers (H3K14 acetylation, H3K9 methylation and DNA methylation and hydroxymethylation) could be detected. The use of LatA alone significantly improved in vitro development, but not full-term development. On the other hand, the simultaneous treatment of cloned embryos with VitC and the histone deacetylase inhibitor psammaplin A (PsA), in combination with the use of LatA, resulted in cloning efficiencies equivalent to those of VitC or PsA treatments alone, and the effects on pluripotency and nuclear reprogramming markers were less evident than when only the PsA treatment was applied. These results suggest that although both epigenetic modifiers improve cloning efficiencies, possibly through different mechanisms, they do not show an additive effect when combined. Improvement of SCNT efficiency is essential for its applications in reproductive and therapeutic cloning, and identification of molecules which increase this efficiency should facilitate studies on the mechanism of nuclear reprogramming and acquisition of totipotency.

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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.
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pone.0120033.g001: 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.

Mentions: ICSI and cloned embryos were fixed at different developmental stages and immunostained for H3K14ac, H3K9me2, 5meC and 5hmeC marks. With regards to histone acetylation, we observed that H3K14 was highly acetylated in the cumulus cell nucleus 10 min after injection, but its acetylation markedly decreased 1 h after nuclear transfer into the enucleated oocyte (Fig. 1A). By the end of the treatment with the epigenetic modifiers (16 h post-activation), the VitC group displayed similar levels of H3K14ac as the non-treated group, both significantly lower than those of the ICSI group (Fig. 1). VitC-PsA-treated embryos were significantly more acetylated than embryos treated with VitC alone, at levels equivalent to those of PsA-treated cloned embryos and ICSI embryos. At the 2-cell stage (24 h post-activation), no significant differences were detected between treated and non-treated cloned embryos or among the three treated groups. In spite of this, only the three groups of treated embryos showed equivalent levels of H3K14ac to the control ICSI group. Finally, at the blastocyst stage (120 h post-activation), there were no differences between cloned and ICSI embryos, except for PsA-treated embryos which showed the highest levels of H3K14ac, even when compared with ICSI controls.


Improved development of somatic cell cloned mouse embryos by vitamin C and latrunculin A.

Mallol A, Santaló J, Ibáñez E - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352067&req=5

pone.0120033.g001: 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.
Mentions: ICSI and cloned embryos were fixed at different developmental stages and immunostained for H3K14ac, H3K9me2, 5meC and 5hmeC marks. With regards to histone acetylation, we observed that H3K14 was highly acetylated in the cumulus cell nucleus 10 min after injection, but its acetylation markedly decreased 1 h after nuclear transfer into the enucleated oocyte (Fig. 1A). By the end of the treatment with the epigenetic modifiers (16 h post-activation), the VitC group displayed similar levels of H3K14ac as the non-treated group, both significantly lower than those of the ICSI group (Fig. 1). VitC-PsA-treated embryos were significantly more acetylated than embryos treated with VitC alone, at levels equivalent to those of PsA-treated cloned embryos and ICSI embryos. At the 2-cell stage (24 h post-activation), no significant differences were detected between treated and non-treated cloned embryos or among the three treated groups. In spite of this, only the three groups of treated embryos showed equivalent levels of H3K14ac to the control ICSI group. Finally, at the blastocyst stage (120 h post-activation), there were no differences between cloned and ICSI embryos, except for PsA-treated embryos which showed the highest levels of H3K14ac, even when compared with ICSI controls.

Bottom Line: The use of LatA alone significantly improved in vitro development, but not full-term development.On the other hand, the simultaneous treatment of cloned embryos with VitC and the histone deacetylase inhibitor psammaplin A (PsA), in combination with the use of LatA, resulted in cloning efficiencies equivalent to those of VitC or PsA treatments alone, and the effects on pluripotency and nuclear reprogramming markers were less evident than when only the PsA treatment was applied.These results suggest that although both epigenetic modifiers improve cloning efficiencies, possibly through different mechanisms, they do not show an additive effect when combined.

View Article: PubMed Central - PubMed

Affiliation: Departament de Biologia Cellular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra, Spain.

ABSTRACT
Impaired development of embryos produced by somatic cell nuclear transfer (SCNT) is mostly associated with faulty reprogramming of the somatic nucleus to a totipotent state and can be improved by treatment with epigenetic modifiers. Here we report that addition of 100 μM vitamin C (VitC) to embryo culture medium for at least 16 h post-activation significantly increases mouse blastocyst formation and, when combined with the use of latrunculin A (LatA) during micromanipulation and activation procedures, also development to term. In spite of this, no significant effects on pluripotency (OCT4 and NANOG) or nuclear reprogramming markers (H3K14 acetylation, H3K9 methylation and DNA methylation and hydroxymethylation) could be detected. The use of LatA alone significantly improved in vitro development, but not full-term development. On the other hand, the simultaneous treatment of cloned embryos with VitC and the histone deacetylase inhibitor psammaplin A (PsA), in combination with the use of LatA, resulted in cloning efficiencies equivalent to those of VitC or PsA treatments alone, and the effects on pluripotency and nuclear reprogramming markers were less evident than when only the PsA treatment was applied. These results suggest that although both epigenetic modifiers improve cloning efficiencies, possibly through different mechanisms, they do not show an additive effect when combined. Improvement of SCNT efficiency is essential for its applications in reproductive and therapeutic cloning, and identification of molecules which increase this efficiency should facilitate studies on the mechanism of nuclear reprogramming and acquisition of totipotency.

Show MeSH