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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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Differentially expressed genes (DEGs) among Day 18 EE tissues.A) Paired comparisons according to SMVar (Table S1). Across all the comparisons performed, 95 statistical occurrences were identified that corresponded to 72 unique DEGs. Multiple occurrences are in bold. Each gene ID is provided as a HUGO term. Among these DEGs, a few had been previously reported: single genes (TUB1A1, B4GALT1) or genes from the CCDC, HSP or TKDP families [17], [19]. B) IPA networks. The DEG list was analyzed with the Ingenuity Pathway Analysis software to identify the top gene networks and the pathways connecting them. SCNT-specific differences (in red) were found in three of four networks. In the IPA database, 4 proteins were located in the extra-cellular space, 8 at the plasma membrane, 20 in the cytoplasm, and 24 in the nucleus, of which 8 were recognised as transcription regulators.
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pone-0038309-g001: Differentially expressed genes (DEGs) among Day 18 EE tissues.A) Paired comparisons according to SMVar (Table S1). Across all the comparisons performed, 95 statistical occurrences were identified that corresponded to 72 unique DEGs. Multiple occurrences are in bold. Each gene ID is provided as a HUGO term. Among these DEGs, a few had been previously reported: single genes (TUB1A1, B4GALT1) or genes from the CCDC, HSP or TKDP families [17], [19]. B) IPA networks. The DEG list was analyzed with the Ingenuity Pathway Analysis software to identify the top gene networks and the pathways connecting them. SCNT-specific differences (in red) were found in three of four networks. In the IPA database, 4 proteins were located in the extra-cellular space, 8 at the plasma membrane, 20 in the cytoplasm, and 24 in the nucleus, of which 8 were recognised as transcription regulators.

Mentions: Consequently, the molecular profile of each Day 18 conceptus (n = 50) was characterized while restricting the high throughput analysis to the extra-embryonic tissues. As each study group was represented by 7–10 elongated conceptuses, sampling was homogeneous enough to proceed to paired comparisons using the array data set. While the control groups were similar, the SCNT groups differed from each other, with the greatest number of gene expression differences occurring between the High and Low groups (19 DEGs, Fig. 1A). AIs, IVPs, and SCNTs had similar numbers of DEGs (18–16; 3–4), with the exception of SCNT Low (10–3). However, AI, IVP, and SCNT Low did not display higher intra-variability than SCNT Med or High (Table S1). As for individual genes, some were found to be pair-specific (SCNM1: AI-IVP pair) whereas others were shared by several pairs (NEAT1 or TXNDC9 for example). Nonetheless, all genes appeared to be connected through in silico networks and pathways (Fig. 1B), suggesting Day 18 SCNT-related alterations were the product of the same differentiation processes having been affected in different ways (especially cellular morphology and cellular development).


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)

Differentially expressed genes (DEGs) among Day 18 EE tissues.A) Paired comparisons according to SMVar (Table S1). Across all the comparisons performed, 95 statistical occurrences were identified that corresponded to 72 unique DEGs. Multiple occurrences are in bold. Each gene ID is provided as a HUGO term. Among these DEGs, a few had been previously reported: single genes (TUB1A1, B4GALT1) or genes from the CCDC, HSP or TKDP families [17], [19]. B) IPA networks. The DEG list was analyzed with the Ingenuity Pathway Analysis software to identify the top gene networks and the pathways connecting them. SCNT-specific differences (in red) were found in three of four networks. In the IPA database, 4 proteins were located in the extra-cellular space, 8 at the plasma membrane, 20 in the cytoplasm, and 24 in the nucleus, of which 8 were recognised as transcription regulators.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038309-g001: Differentially expressed genes (DEGs) among Day 18 EE tissues.A) Paired comparisons according to SMVar (Table S1). Across all the comparisons performed, 95 statistical occurrences were identified that corresponded to 72 unique DEGs. Multiple occurrences are in bold. Each gene ID is provided as a HUGO term. Among these DEGs, a few had been previously reported: single genes (TUB1A1, B4GALT1) or genes from the CCDC, HSP or TKDP families [17], [19]. B) IPA networks. The DEG list was analyzed with the Ingenuity Pathway Analysis software to identify the top gene networks and the pathways connecting them. SCNT-specific differences (in red) were found in three of four networks. In the IPA database, 4 proteins were located in the extra-cellular space, 8 at the plasma membrane, 20 in the cytoplasm, and 24 in the nucleus, of which 8 were recognised as transcription regulators.
Mentions: Consequently, the molecular profile of each Day 18 conceptus (n = 50) was characterized while restricting the high throughput analysis to the extra-embryonic tissues. As each study group was represented by 7–10 elongated conceptuses, sampling was homogeneous enough to proceed to paired comparisons using the array data set. While the control groups were similar, the SCNT groups differed from each other, with the greatest number of gene expression differences occurring between the High and Low groups (19 DEGs, Fig. 1A). AIs, IVPs, and SCNTs had similar numbers of DEGs (18–16; 3–4), with the exception of SCNT Low (10–3). However, AI, IVP, and SCNT Low did not display higher intra-variability than SCNT Med or High (Table S1). As for individual genes, some were found to be pair-specific (SCNM1: AI-IVP pair) whereas others were shared by several pairs (NEAT1 or TXNDC9 for example). Nonetheless, all genes appeared to be connected through in silico networks and pathways (Fig. 1B), suggesting Day 18 SCNT-related alterations were the product of the same differentiation processes having been affected in different ways (especially cellular morphology and cellular development).

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