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. 2021 May 27;11:11167. doi: 10.1038/s41598-021-90653-1

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Discordance in developmental ability for isolated single sister blastomeres of the same parental embryos. (a) 2CB and 4CB pseudoblastocysts (96 hpi) derived from twin or quadruplet blastomeres, respectively, of parental embryos were subjected to immunofluorescence staining with anti-OCT4 and anti-CDX2 for determination of the segregation rate for the ICM and TE lineages. The proportion of pseudoblastocysts in which the ICM and TE were segregated out of all pseudoblastocysts derived from each twin or quadruplet set of blastomeres is represented by x. Parental embryos were classified into three categories on the basis of this proportion. The P value was determined with the two-sided Fisher’s exact test. n indicates the number of twin or quadruplet blastomere sets (parental embryos). All data were obtained from three or four experiments performed on different days for each blastomere stage and were then analyzed. (b) 2CB and 4CB pseudoblastocysts that had been cultured in vitro up to 96 hpi were then cultured further for determination of their survival rate at 120 hpi (judged on the basis of a maintained cavity). The proportion of pseudoblastocysts that survived during this extended culture period out of all pseudoblastocysts derived from each twin or quadruplet set of blastomeres is represented by x. Parental embryos were classified into three categories on the basis of this proportion. **P < 0.001, determined as in (a). n indicates the number of twin or quadruplet blastomere sets (parental embryos). All data were obtained from three or four experiments performed on different days for each blastomere stage and were then analyzed. (c,d) 2CB and 4CB pseudoblastocysts cultured in vitro up to 120 hpi in (b) were subjected to immunofluorescence staining with anti-NANOG and anti-GATA4 for evaluation of EPI and PrE formation. The phenotypes of pseudoblastocysts with regard to formation of EPI and PrE were classified into four categories as shown in Fig. 5e. The proportion of pseudoblastocysts that showed mutually exclusive scattered or sorted signals for NANOG and GATA4 (c) or sorted signals for these proteins (d) in the ICM out of all pseudoblastocysts derived from each twin or quadruplet set of blastomeres is represented by x. Parental embryos were classified into three categories on the basis of these proportions. *P < 0.01, determined as in (a). n indicates the number of twin or quadruplet blastomere sets (parental embryos).