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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.

No MeSH data available.


Time-lapse and immunostaining images of “8”-shaped hatching.(A) At 119 h 30 min post-insemination. The outside blastocyst is expanded. (B) At 119 h 35 min post-insemination. The outside blastocyst has collapsed (arrow). (C) At 119 h 59 min post-insemination. The outside blastocyst has re-expanded. (D) Immunostaining at 120 h post-insemination, immediately after the time point shown in C. A merged image of Oct3/4 (red), Cdx2 (green), and Hoechst (blue) staining is shown. Oct3/4-positive cells (arrowhead) were attached to the TE of the outside blastocyst opposite the hatching site. Scale bar, 20 μm.
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pone.0175150.g008: Time-lapse and immunostaining images of “8”-shaped hatching.(A) At 119 h 30 min post-insemination. The outside blastocyst is expanded. (B) At 119 h 35 min post-insemination. The outside blastocyst has collapsed (arrow). (C) At 119 h 59 min post-insemination. The outside blastocyst has re-expanded. (D) Immunostaining at 120 h post-insemination, immediately after the time point shown in C. A merged image of Oct3/4 (red), Cdx2 (green), and Hoechst (blue) staining is shown. Oct3/4-positive cells (arrowhead) were attached to the TE of the outside blastocyst opposite the hatching site. Scale bar, 20 μm.

Mentions: The “8”-shaped-hatching blastocysts and U-shaped-hatching blastocysts showed no significant differences in ICM size (width: 75.2 ± 3.4 μm versus 71.2 ± 6.4 μm, P = 0.5617; Fig 7A) and the number of Oct3/4-positive cells (20 ± 1 versus 21 ± 2, P = 0.4573; Fig 7B). In our examination of the effect of hatching position, we found that the ICM was significantly wider in the Near group than in the Far group (83.1 ± 5.2 μm versus 66.3 ± 2.9 μm, P = 0.0091; Fig 7C). However, the Near and Far groups showed no significant differences in the number of Oct3/4-positive cells (21 ± 2 versus 18 ± 2, P = 0.2782; Fig 7D). Furthermore, after sorting based on the rate of occurrence of outside collapse, no significant difference was observed in ICM width (77.5 ± 3.5 μm versus 62.7 ± 9.4 μm, P = 0.1124; Fig 7E) or cell number (20 ± 1 versus 15 ± 3, P = 0.1254; Fig 7F), and the outside-collapse count was not significantly correlated with ICM size (P = 0.4639). Lastly, in two blastocysts among embryos that included the ICM in the outside blastocyst (as depicted in Fig 1B(b)), in the blastocysts that exhibited outside collapse (Fig 8), immunostaining revealed that Oct3/4-positive cells adhered to the TE of the outside blastocyst, opposite the hatching site (Fig 8D, S3 Movie).


The location of “ 8 ” -shaped hatching influences inner cell mass formation in mouse blastocysts
Time-lapse and immunostaining images of “8”-shaped hatching.(A) At 119 h 30 min post-insemination. The outside blastocyst is expanded. (B) At 119 h 35 min post-insemination. The outside blastocyst has collapsed (arrow). (C) At 119 h 59 min post-insemination. The outside blastocyst has re-expanded. (D) Immunostaining at 120 h post-insemination, immediately after the time point shown in C. A merged image of Oct3/4 (red), Cdx2 (green), and Hoechst (blue) staining is shown. Oct3/4-positive cells (arrowhead) were attached to the TE of the outside blastocyst opposite the hatching site. Scale bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0175150.g008: Time-lapse and immunostaining images of “8”-shaped hatching.(A) At 119 h 30 min post-insemination. The outside blastocyst is expanded. (B) At 119 h 35 min post-insemination. The outside blastocyst has collapsed (arrow). (C) At 119 h 59 min post-insemination. The outside blastocyst has re-expanded. (D) Immunostaining at 120 h post-insemination, immediately after the time point shown in C. A merged image of Oct3/4 (red), Cdx2 (green), and Hoechst (blue) staining is shown. Oct3/4-positive cells (arrowhead) were attached to the TE of the outside blastocyst opposite the hatching site. Scale bar, 20 μm.
Mentions: The “8”-shaped-hatching blastocysts and U-shaped-hatching blastocysts showed no significant differences in ICM size (width: 75.2 ± 3.4 μm versus 71.2 ± 6.4 μm, P = 0.5617; Fig 7A) and the number of Oct3/4-positive cells (20 ± 1 versus 21 ± 2, P = 0.4573; Fig 7B). In our examination of the effect of hatching position, we found that the ICM was significantly wider in the Near group than in the Far group (83.1 ± 5.2 μm versus 66.3 ± 2.9 μm, P = 0.0091; Fig 7C). However, the Near and Far groups showed no significant differences in the number of Oct3/4-positive cells (21 ± 2 versus 18 ± 2, P = 0.2782; Fig 7D). Furthermore, after sorting based on the rate of occurrence of outside collapse, no significant difference was observed in ICM width (77.5 ± 3.5 μm versus 62.7 ± 9.4 μm, P = 0.1124; Fig 7E) or cell number (20 ± 1 versus 15 ± 3, P = 0.1254; Fig 7F), and the outside-collapse count was not significantly correlated with ICM size (P = 0.4639). Lastly, in two blastocysts among embryos that included the ICM in the outside blastocyst (as depicted in Fig 1B(b)), in the blastocysts that exhibited outside collapse (Fig 8), immunostaining revealed that Oct3/4-positive cells adhered to the TE of the outside blastocyst, opposite the hatching site (Fig 8D, S3 Movie).

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.