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The location of “ 8 ” -shaped hatching influences inner cell mass formation in mouse blastocysts

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ABSTRACT

The hatching of a blastocyst where the blastocyst portions on the inside and the outside of the zona pellucida feature a figure-of-eight shape is termed “8”-shaped hatching; this type of hatching has been reported to affect the proper presentation of the inner cell mass (ICM) in both human and mouse embryos. Here, our aim was to investigate the factors that affect ICM presentation during “8”-shaped hatching. We performed IVF by using B6D2F1 female mice and ICR male mice, and used the 104 captured blastocysts. Embryos were maintained in KSOM at 37°C in a 5% CO2, 5% O2, and 90% N2 environment, and their growth behavior was monitored individually and continuously using time-lapse cinematography. At 120 h after insemination, embryos were immunostained and examined under a confocal microscope. We used the hatching form to identify “8”-shaped hatching, and we classified the “8”-shaped-hatching blastocysts into two groups, one in which the hatching site was near the ICM center, and the other in which the hatching site was far from the ICM center. We measured each group for ICM size and the number of Oct3/4-positive cells. Of the 95 hatching or hatched embryos, 74 were “8”-shaped-hatching blastocysts, and in these embryos, the ICM was significantly wider when the hatching site was near the ICM than when the hatching site was far from the ICM (P = 0.0091). Moreover, in the “8”-shaped-hatching blastocysts in which the ICM was included in the blastocyst portion outside the zona pellucida―the portion defined as the “outside blastocyst”―after the collapse of this outside blastocyst, the ICM adhered to the trophectoderm of the outside blastocyst, opposite the hatching site. Our results indicate that in “8”-shaped-hatching blastocysts, the hatching site and the collapse of outside blastocyst affect ICM formation. Thus, the assessment of “8”-shaped hatching behaviors could yield indices for accurately evaluating embryo quality.

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ICM size and cell number at 120 h post-insemination, sorted according to type of developmental behavior of blastocysts: “8”-shaped hatching and U-shaped hatching, hatching position relative to the ICM, and occurrence of outside collapse.(A, B) Comparison by hatching mode. (C, D) Comparison by hatching position. The ICM was significantly wider in the Near group than in the Far group (*P = 0.0091, Welch’s t test). (E, F) Comparison based on occurrence of outside collapse.
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pone.0175150.g007: ICM size and cell number at 120 h post-insemination, sorted according to type of developmental behavior of blastocysts: “8”-shaped hatching and U-shaped hatching, hatching position relative to the ICM, and occurrence of outside collapse.(A, B) Comparison by hatching mode. (C, D) Comparison by hatching position. The ICM was significantly wider in the Near group than in the Far group (*P = 0.0091, Welch’s t test). (E, F) Comparison based on occurrence of outside collapse.

Mentions: Cdx2 (green) and Oct3/4 (red) were stained with specific antibodies, and nuclei were stained with Hoechst (blue). In the case of “8”-shaped hatching, the figure shows embryos in the Near group (A–D) and Far group (E–H). Whereas the ICM tended to be morphologically compact in the Far group (G), it was scattered in the Near group (C; see Fig 7C).


The location of “ 8 ” -shaped hatching influences inner cell mass formation in mouse blastocysts
ICM size and cell number at 120 h post-insemination, sorted according to type of developmental behavior of blastocysts: “8”-shaped hatching and U-shaped hatching, hatching position relative to the ICM, and occurrence of outside collapse.(A, B) Comparison by hatching mode. (C, D) Comparison by hatching position. The ICM was significantly wider in the Near group than in the Far group (*P = 0.0091, Welch’s t test). (E, F) Comparison based on occurrence of outside collapse.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383253&req=5

pone.0175150.g007: ICM size and cell number at 120 h post-insemination, sorted according to type of developmental behavior of blastocysts: “8”-shaped hatching and U-shaped hatching, hatching position relative to the ICM, and occurrence of outside collapse.(A, B) Comparison by hatching mode. (C, D) Comparison by hatching position. The ICM was significantly wider in the Near group than in the Far group (*P = 0.0091, Welch’s t test). (E, F) Comparison based on occurrence of outside collapse.
Mentions: Cdx2 (green) and Oct3/4 (red) were stained with specific antibodies, and nuclei were stained with Hoechst (blue). In the case of “8”-shaped hatching, the figure shows embryos in the Near group (A–D) and Far group (E–H). Whereas the ICM tended to be morphologically compact in the Far group (G), it was scattered in the Near group (C; see Fig 7C).

View Article: PubMed Central - PubMed

ABSTRACT

The hatching of a blastocyst where the blastocyst portions on the inside and the outside of the zona pellucida feature a figure-of-eight shape is termed “8”-shaped hatching; this type of hatching has been reported to affect the proper presentation of the inner cell mass (ICM) in both human and mouse embryos. Here, our aim was to investigate the factors that affect ICM presentation during “8”-shaped hatching. We performed IVF by using B6D2F1 female mice and ICR male mice, and used the 104 captured blastocysts. Embryos were maintained in KSOM at 37°C in a 5% CO2, 5% O2, and 90% N2 environment, and their growth behavior was monitored individually and continuously using time-lapse cinematography. At 120 h after insemination, embryos were immunostained and examined under a confocal microscope. We used the hatching form to identify “8”-shaped hatching, and we classified the “8”-shaped-hatching blastocysts into two groups, one in which the hatching site was near the ICM center, and the other in which the hatching site was far from the ICM center. We measured each group for ICM size and the number of Oct3/4-positive cells. Of the 95 hatching or hatched embryos, 74 were “8”-shaped-hatching blastocysts, and in these embryos, the ICM was significantly wider when the hatching site was near the ICM than when the hatching site was far from the ICM (P = 0.0091). Moreover, in the “8”-shaped-hatching blastocysts in which the ICM was included in the blastocyst portion outside the zona pellucida―the portion defined as the “outside blastocyst”―after the collapse of this outside blastocyst, the ICM adhered to the trophectoderm of the outside blastocyst, opposite the hatching site. Our results indicate that in “8”-shaped-hatching blastocysts, the hatching site and the collapse of outside blastocyst affect ICM formation. Thus, the assessment of “8”-shaped hatching behaviors could yield indices for accurately evaluating embryo quality.

No MeSH data available.


Related in: MedlinePlus