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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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Gastrulation patterns.A) Definition of gastrulation classes. Normal Brachyury patterns are shown in N1 (a) and D (b) embryonic discs. Abnormal Brachyury patterns (U-shaped and broadened labelling) are shown in Ab1 (c) embryonic discs. These are whole-mount in situ hybridisations with an anti-sense Brachyury DIG-labelled riboprobe performed on embryonic discs from two SCNT High (a, b) and two SCNT Low conceptuses (c, right and left panels). Scale bar: 100 µm. B) Overview of all conceptuses.
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pone-0038309-g004: Gastrulation patterns.A) Definition of gastrulation classes. Normal Brachyury patterns are shown in N1 (a) and D (b) embryonic discs. Abnormal Brachyury patterns (U-shaped and broadened labelling) are shown in Ab1 (c) embryonic discs. These are whole-mount in situ hybridisations with an anti-sense Brachyury DIG-labelled riboprobe performed on embryonic discs from two SCNT High (a, b) and two SCNT Low conceptuses (c, right and left panels). Scale bar: 100 µm. B) Overview of all conceptuses.

Mentions: Relying on morphological signs of gastrulation and the expression pattern of an early mesoderm marker, the Brachyury gene, this staging method describes D18 patterns in AI conceptuses as being at stage 4 or 3 (referred to as normal: N1 and N2, respectively; Fig. 4). Accordingly, these stages were the most frequently observed and their presence best predicted successful development to term in AI, IVP and SCNT High groups (Table 1B). When fewer N1 and N2 stages were observed, more unusual patterns were recorded including late and abnormal development. In SCNT Med and Low conceptuses, a few embryonic discs were delayed but normal, i.e. they appeared morphologically younger than those in controls (AI), and corresponded to stage 2 [24]. They were thus recorded as delayed (D). Since the D pattern occurred with equal frequency in all groups except for AI, this delay in embryonic development was not typical of post-SCNT differentiation. Conversely, several other discs from SCNT Med and Low groups were atypical, i.e. not previously observed in controls [23], [24], [31], [38]. Depending on the severity of their phenotype, they were called Ab1 or Ab2. In the mild phenotype (Ab1), discs were small or folded, with atypical U-shaped or broadened Brachyury labelling (Fig. 4A). In contrast, the severe phenotype (Ab2) displayed no disc, or at least none that was morphologically recognisable or molecularly detectable using the Pou5f1 gene as a molecular marker for the epiblast [23]. The highest numbers of Ab1 and Ab2 patterns were found in those conceptuses that had the lowest success rates at Day 21 and at term, namely those in the SCNT Med and Low groups (Table 1).


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)

Gastrulation patterns.A) Definition of gastrulation classes. Normal Brachyury patterns are shown in N1 (a) and D (b) embryonic discs. Abnormal Brachyury patterns (U-shaped and broadened labelling) are shown in Ab1 (c) embryonic discs. These are whole-mount in situ hybridisations with an anti-sense Brachyury DIG-labelled riboprobe performed on embryonic discs from two SCNT High (a, b) and two SCNT Low conceptuses (c, right and left panels). Scale bar: 100 µm. B) Overview of all conceptuses.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038309-g004: Gastrulation patterns.A) Definition of gastrulation classes. Normal Brachyury patterns are shown in N1 (a) and D (b) embryonic discs. Abnormal Brachyury patterns (U-shaped and broadened labelling) are shown in Ab1 (c) embryonic discs. These are whole-mount in situ hybridisations with an anti-sense Brachyury DIG-labelled riboprobe performed on embryonic discs from two SCNT High (a, b) and two SCNT Low conceptuses (c, right and left panels). Scale bar: 100 µm. B) Overview of all conceptuses.
Mentions: Relying on morphological signs of gastrulation and the expression pattern of an early mesoderm marker, the Brachyury gene, this staging method describes D18 patterns in AI conceptuses as being at stage 4 or 3 (referred to as normal: N1 and N2, respectively; Fig. 4). Accordingly, these stages were the most frequently observed and their presence best predicted successful development to term in AI, IVP and SCNT High groups (Table 1B). When fewer N1 and N2 stages were observed, more unusual patterns were recorded including late and abnormal development. In SCNT Med and Low conceptuses, a few embryonic discs were delayed but normal, i.e. they appeared morphologically younger than those in controls (AI), and corresponded to stage 2 [24]. They were thus recorded as delayed (D). Since the D pattern occurred with equal frequency in all groups except for AI, this delay in embryonic development was not typical of post-SCNT differentiation. Conversely, several other discs from SCNT Med and Low groups were atypical, i.e. not previously observed in controls [23], [24], [31], [38]. Depending on the severity of their phenotype, they were called Ab1 or Ab2. In the mild phenotype (Ab1), discs were small or folded, with atypical U-shaped or broadened Brachyury labelling (Fig. 4A). In contrast, the severe phenotype (Ab2) displayed no disc, or at least none that was morphologically recognisable or molecularly detectable using the Pou5f1 gene as a molecular marker for the epiblast [23]. The highest numbers of Ab1 and Ab2 patterns were found in those conceptuses that had the lowest success rates at Day 21 and at term, namely those in the SCNT Med and Low groups (Table 1).

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