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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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Validated gene expression differences.The gene expression differences are presented according to their decreasing rank (or adjusted P-value) within the SMVar output lists. In A: differences between SCNT and AI, in B: differences between SCNT and IVP, in C: differences among SCNT. The y-axis of the each histogram corresponds to the relative expression values of each DEG in EE tissues (AI, IVP and SCNT High, Med, Low). Array data are in grey, QPCR data in black.
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pone-0038309-g002: Validated gene expression differences.The gene expression differences are presented according to their decreasing rank (or adjusted P-value) within the SMVar output lists. In A: differences between SCNT and AI, in B: differences between SCNT and IVP, in C: differences among SCNT. The y-axis of the each histogram corresponds to the relative expression values of each DEG in EE tissues (AI, IVP and SCNT High, Med, Low). Array data are in grey, QPCR data in black.

Mentions: Instead of focusing on a single gene network, we chose 4 networks (presented in Fig. 1B) and selected 14 genes based on their rank (or decreasing adjusted P-values) and their implication in: i) cellular morphology or development (ALG5, APLP2, CCNE1, FN1, LHFPL2, MYO6, PLIN2, WNT5B) and ii) alterations in chromatin modification or DNA repair (ASF1A, BIN3, DFFA, EED, EIF2S3X, MSH3). Validated expression differences are provided in Fig. 2; they mainly included differences between SCNT and control groups and within SCNTs (SCNT-AI: 6; SCNT-IVP: 4; SCNT Low-Med: 1). In addition, most of the differences resulted from weaker expression in SCNTs than in controls (8/9), except for WNT5B, which was more strongly expressed in SCNT High than in IVP. Non-validated DEGs were related to differences within SCNTs (Low-High: BIN3, CCNE1) or between SCNTs and controls (AI-SCNT Med: LHFPL2; AI-SCNT High: MSH3), except for FN1. To determine the cellular location of the validated transcripts in SCNTs as well as in controls, several in situ hybridisations were performed; three in particular revealed an interesting labelling pattern on the panel of tissue sections (APLP2, FN1, PLIN2). APLP2 and FN1 appeared to be restricted to endodermal cells and PLIN2 to trophoblast cells (Fig. 3A, 3C). Using 2–3 conceptuses per group, we further determined that the cellular location of these genes was unchanged in SCNTs and IVPs (Fig. 3A), even in cases of delayed elongation (5 tubular shapes out of 9). Unfortunately, because the shortest conceptuses were extremely limited in size (<3 cm), gene cellular location could not be evaluated for abnormal elongations.


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)

Validated gene expression differences.The gene expression differences are presented according to their decreasing rank (or adjusted P-value) within the SMVar output lists. In A: differences between SCNT and AI, in B: differences between SCNT and IVP, in C: differences among SCNT. The y-axis of the each histogram corresponds to the relative expression values of each DEG in EE tissues (AI, IVP and SCNT High, Med, Low). Array data are in grey, QPCR data in black.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038309-g002: Validated gene expression differences.The gene expression differences are presented according to their decreasing rank (or adjusted P-value) within the SMVar output lists. In A: differences between SCNT and AI, in B: differences between SCNT and IVP, in C: differences among SCNT. The y-axis of the each histogram corresponds to the relative expression values of each DEG in EE tissues (AI, IVP and SCNT High, Med, Low). Array data are in grey, QPCR data in black.
Mentions: Instead of focusing on a single gene network, we chose 4 networks (presented in Fig. 1B) and selected 14 genes based on their rank (or decreasing adjusted P-values) and their implication in: i) cellular morphology or development (ALG5, APLP2, CCNE1, FN1, LHFPL2, MYO6, PLIN2, WNT5B) and ii) alterations in chromatin modification or DNA repair (ASF1A, BIN3, DFFA, EED, EIF2S3X, MSH3). Validated expression differences are provided in Fig. 2; they mainly included differences between SCNT and control groups and within SCNTs (SCNT-AI: 6; SCNT-IVP: 4; SCNT Low-Med: 1). In addition, most of the differences resulted from weaker expression in SCNTs than in controls (8/9), except for WNT5B, which was more strongly expressed in SCNT High than in IVP. Non-validated DEGs were related to differences within SCNTs (Low-High: BIN3, CCNE1) or between SCNTs and controls (AI-SCNT Med: LHFPL2; AI-SCNT High: MSH3), except for FN1. To determine the cellular location of the validated transcripts in SCNTs as well as in controls, several in situ hybridisations were performed; three in particular revealed an interesting labelling pattern on the panel of tissue sections (APLP2, FN1, PLIN2). APLP2 and FN1 appeared to be restricted to endodermal cells and PLIN2 to trophoblast cells (Fig. 3A, 3C). Using 2–3 conceptuses per group, we further determined that the cellular location of these genes was unchanged in SCNTs and IVPs (Fig. 3A), even in cases of delayed elongation (5 tubular shapes out of 9). Unfortunately, because the shortest conceptuses were extremely limited in size (<3 cm), gene cellular location could not be evaluated for abnormal elongations.

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