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

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.

Live visualisation of electrolytes during mouse embryonic development using electrolyte indicators

“…All animal experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals of Akita University and carried out as previously described with some modifications [ 15 ]. All mice were housed in cages at 23°C with a 12-h light/dark photoperiod and free access to food and water.…”

“…Embryos were cultured under the following conditions: 5% O 2 , 5% CO 2 , 90% N 2 , and a temperature of 37°C. Culture medium was not replaced during the experiments [ 15 ]. Embryos were imaged using the Primo Vision time-lapse monitoring system (Vitrolife) at 5-min intervals from IVF to vitrification, from thawing to experimental use, and from washing after experimental use with the electrolyte indicator for at least 24 h.…”

“…Embryos were stained with the following antibodies: goat polyclonal anti-Oct3/4 (Cat. Sc-8628, Lot #K0615; Santa Cruz Biotechnology, Dallas TX, USA) [ 15 , 19 ], mouse monoclonal anti-Na + /K + -ATPase α-1 (Cat. #05–369, Lot 3170808; C464.6; Merck Millipore, Billerica, MA, USA) [ 15 , 20 ], and Hoechst 33342 (Cat.…”

“…Sc-8628, Lot #K0615; Santa Cruz Biotechnology, Dallas TX, USA) [ 15 , 19 ], mouse monoclonal anti-Na + /K + -ATPase α-1 (Cat. #05–369, Lot 3170808; C464.6; Merck Millipore, Billerica, MA, USA) [ 15 , 20 ], and Hoechst 33342 (Cat. 346–07951, Lot DH039, Dojindo Molecular Technologies, Kumamoto, Japan) [ 15 ].…”

Human frozen-thawed blastocyst morphokinetics observed using time-lapse cinematography reflects the number of trophectoderm cells

“…We performed chromatin, Oct4, and Cdx2 staining as described previously [15]. For Oct4 and Cdx2 staining, embryos were fixed in formaldehyde (3.7%) for 30 min at approximately 26 °C and then washed three times for 45 min in wash buffer-1 (Dulbecco’s phosphate-buffered saline [D-PBS] containing 0.1% bovine serum albumin [BSA]; Sigma—Aldrich, USA).…”