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

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.


Time-lapse and immunostaining images of a U-shaped-hatching blastocyst that hatched completely, in which an initial “8”-shaped hatching occurred near the ICM.(A) At 90 h 45 min after insemination. Near the ICM (surrounded by arrowheads), an “8”-shaped hatch appears (arrow). (B) At 117 h 57 min after insemination. U-shaped hatching appears (arrow) in this embryo. (C) At 119 h 47 min after insemination. The embryo has hatched completely. (D) Immunostaining at 120 h post-insemination. The result confirmed that the ICM, stained by anti-Oct3/4 (red), is scattered. Scale bar, 20 μm.
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pone.0175150.g006: Time-lapse and immunostaining images of a U-shaped-hatching blastocyst that hatched completely, in which an initial “8”-shaped hatching occurred near the ICM.(A) At 90 h 45 min after insemination. Near the ICM (surrounded by arrowheads), an “8”-shaped hatch appears (arrow). (B) At 117 h 57 min after insemination. U-shaped hatching appears (arrow) in this embryo. (C) At 119 h 47 min after insemination. The embryo has hatched completely. (D) Immunostaining at 120 h post-insemination. The result confirmed that the ICM, stained by anti-Oct3/4 (red), is scattered. Scale bar, 20 μm.

Mentions: Of the 156 embryos retrieved, 120 had attained the blastocyst stage at Day 5, and 104 of the 120 embryos were immunostained and their hatching morphology was evaluated using TLM and immunohistochemistry. Among the 95 hatching blastocysts, “8”-shaped-hatching blastocysts (Fig 1B(a-d), S1 Movie) accounted for 77.9% (74/95) and U-shaped-hatching blastocysts (Fig 1B(e), S2 Movie) for 12.6% (12/95). When we classified the 74 “8”-shaped-hatching blastocysts into the Near group (Figs 1B(a-c) and 5A–5D) and the Far group (Figs 1B(d) and 5E–5H), we found that the Near group accounted for 79.7% (59/74), and that the ICM was included in the outside blastocyst of 71.2% (42/59) of the Near-group embryos (Fig 1B(b and c)). Furthermore, in the TLM performed here, in certain “8”-shaped-hatching blastocysts in which the hatching started near the ICM, the embryos were observed to pass through U-shaped hatching before blastocysts hatched completely (Fig 6).


The location of “ 8 ” -shaped hatching influences inner cell mass formation in mouse blastocysts
Time-lapse and immunostaining images of a U-shaped-hatching blastocyst that hatched completely, in which an initial “8”-shaped hatching occurred near the ICM.(A) At 90 h 45 min after insemination. Near the ICM (surrounded by arrowheads), an “8”-shaped hatch appears (arrow). (B) At 117 h 57 min after insemination. U-shaped hatching appears (arrow) in this embryo. (C) At 119 h 47 min after insemination. The embryo has hatched completely. (D) Immunostaining at 120 h post-insemination. The result confirmed that the ICM, stained by anti-Oct3/4 (red), is scattered. 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.g006: Time-lapse and immunostaining images of a U-shaped-hatching blastocyst that hatched completely, in which an initial “8”-shaped hatching occurred near the ICM.(A) At 90 h 45 min after insemination. Near the ICM (surrounded by arrowheads), an “8”-shaped hatch appears (arrow). (B) At 117 h 57 min after insemination. U-shaped hatching appears (arrow) in this embryo. (C) At 119 h 47 min after insemination. The embryo has hatched completely. (D) Immunostaining at 120 h post-insemination. The result confirmed that the ICM, stained by anti-Oct3/4 (red), is scattered. Scale bar, 20 μm.
Mentions: Of the 156 embryos retrieved, 120 had attained the blastocyst stage at Day 5, and 104 of the 120 embryos were immunostained and their hatching morphology was evaluated using TLM and immunohistochemistry. Among the 95 hatching blastocysts, “8”-shaped-hatching blastocysts (Fig 1B(a-d), S1 Movie) accounted for 77.9% (74/95) and U-shaped-hatching blastocysts (Fig 1B(e), S2 Movie) for 12.6% (12/95). When we classified the 74 “8”-shaped-hatching blastocysts into the Near group (Figs 1B(a-c) and 5A–5D) and the Far group (Figs 1B(d) and 5E–5H), we found that the Near group accounted for 79.7% (59/74), and that the ICM was included in the outside blastocyst of 71.2% (42/59) of the Near-group embryos (Fig 1B(b and c)). Furthermore, in the TLM performed here, in certain “8”-shaped-hatching blastocysts in which the hatching started near the ICM, the embryos were observed to pass through U-shaped hatching before blastocysts hatched completely (Fig 6).

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.