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Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer.

Degrelle SA, Jaffrezic F, Campion E, Lê Cao KA, Le Bourhis D, Richard C, Rodde N, Fleurot R, Everts RE, Lecardonnel J, Heyman Y, Vignon X, Yang X, Tian XC, Lewin HA, Renard JP, Hue I - PLoS ONE (2012)

Bottom Line: SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular "uncoupling".Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls.When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1198 Biologie du Développement et Reproduction, Jouy-en-Josas, France.

ABSTRACT
Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular "uncoupling". Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters re-differentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way.

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KLF4 and ACVR2A discriminate among normal and abnormal SCNTs.A) Box plots of KLF4 and ACVR2A expression in normal and abnormal SCNTs. B) PCR results from normal and abnormal EE tissues, pooled as indicated in Table S1. C) Expression of KLF4 and ACVR2A in the fibroblasts (5538, 7711 and 0029) used to generate SCNT High, Med, and Low conceptuses. Triangles correspond to cell passages temporally proximate to biopsy (p2-p3), squares to the passages closer to nuclear transfer (p7–p8).
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pone-0038309-g007: KLF4 and ACVR2A discriminate among normal and abnormal SCNTs.A) Box plots of KLF4 and ACVR2A expression in normal and abnormal SCNTs. B) PCR results from normal and abnormal EE tissues, pooled as indicated in Table S1. C) Expression of KLF4 and ACVR2A in the fibroblasts (5538, 7711 and 0029) used to generate SCNT High, Med, and Low conceptuses. Triangles correspond to cell passages temporally proximate to biopsy (p2-p3), squares to the passages closer to nuclear transfer (p7–p8).

Mentions: Over the totality of the SCNT conceptuses, half were normal and half were abnormal. To be able to discriminate between them and preserve embryonic tissues for other analyses, we searched for extra-embryonic genes that could distinguish between normal and abnormal SCNTs. Using three classification methods and taking into account the genes commonly identified by these methods, two genes were identified that could serve this function: KLF4 and ACVR2A (Table S1; Fig. 7A). Both were more strongly expressed in abnormal versus normal SCNT extra-embryonic tissues. Because the SCNT High, Med and Low groups all contained normal and abnormal conceptuses, differences in expression of these two genes were unlikely to stem from differences in somatic origin. Instead, they could have indicated poor post-SCNT differentiation. Moreover, these genes displayed different expression patterns in somatic cells (Fig. 7C); KLF4 was highly expressed in the 5538 fibroblasts (as compared to the others: 7711, 0029). It thus seemed that KLF4 was downregulated in normal Day 18 conceptuses from the SCNT High group but upregulated in abnormal conceptuses from SCNT Med or Low groups (as compared to its initial levels in the corresponding fibroblasts). Conversely, ACVR2A seemed upregulated in abnormal conceptuses (as compared to its level in different somatic cells; Fig. 7A, 7C).


Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer.

Degrelle SA, Jaffrezic F, Campion E, Lê Cao KA, Le Bourhis D, Richard C, Rodde N, Fleurot R, Everts RE, Lecardonnel J, Heyman Y, Vignon X, Yang X, Tian XC, Lewin HA, Renard JP, Hue I - PLoS ONE (2012)

KLF4 and ACVR2A discriminate among normal and abnormal SCNTs.A) Box plots of KLF4 and ACVR2A expression in normal and abnormal SCNTs. B) PCR results from normal and abnormal EE tissues, pooled as indicated in Table S1. C) Expression of KLF4 and ACVR2A in the fibroblasts (5538, 7711 and 0029) used to generate SCNT High, Med, and Low conceptuses. Triangles correspond to cell passages temporally proximate to biopsy (p2-p3), squares to the passages closer to nuclear transfer (p7–p8).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368877&req=5

pone-0038309-g007: KLF4 and ACVR2A discriminate among normal and abnormal SCNTs.A) Box plots of KLF4 and ACVR2A expression in normal and abnormal SCNTs. B) PCR results from normal and abnormal EE tissues, pooled as indicated in Table S1. C) Expression of KLF4 and ACVR2A in the fibroblasts (5538, 7711 and 0029) used to generate SCNT High, Med, and Low conceptuses. Triangles correspond to cell passages temporally proximate to biopsy (p2-p3), squares to the passages closer to nuclear transfer (p7–p8).
Mentions: Over the totality of the SCNT conceptuses, half were normal and half were abnormal. To be able to discriminate between them and preserve embryonic tissues for other analyses, we searched for extra-embryonic genes that could distinguish between normal and abnormal SCNTs. Using three classification methods and taking into account the genes commonly identified by these methods, two genes were identified that could serve this function: KLF4 and ACVR2A (Table S1; Fig. 7A). Both were more strongly expressed in abnormal versus normal SCNT extra-embryonic tissues. Because the SCNT High, Med and Low groups all contained normal and abnormal conceptuses, differences in expression of these two genes were unlikely to stem from differences in somatic origin. Instead, they could have indicated poor post-SCNT differentiation. Moreover, these genes displayed different expression patterns in somatic cells (Fig. 7C); KLF4 was highly expressed in the 5538 fibroblasts (as compared to the others: 7711, 0029). It thus seemed that KLF4 was downregulated in normal Day 18 conceptuses from the SCNT High group but upregulated in abnormal conceptuses from SCNT Med or Low groups (as compared to its initial levels in the corresponding fibroblasts). Conversely, ACVR2A seemed upregulated in abnormal conceptuses (as compared to its level in different somatic cells; Fig. 7A, 7C).

Bottom Line: SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular "uncoupling".Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls.When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1198 Biologie du Développement et Reproduction, Jouy-en-Josas, France.

ABSTRACT
Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular "uncoupling". Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters re-differentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way.

Show MeSH
Related in: MedlinePlus