Onodera Yohei, Takahashi Kazumasa, Goto Mayumi, Anzai Mibuki, Ono Natsuki, Shirasawa Hiromitsu, Sato Wataru, Miura Hiroshi, Sato Naoki, Sato Akira, Kumazawa Yukiyo, Terada Yukihiro
Department of Obstetrics and Gynecology, Akita University Graduate School of Medicine, Akita, Japan.
PLoS One. 2017 Apr 6;12(4):e0175150. doi: 10.1371/journal.pone.0175150. eCollection 2017.
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.
囊胚孵化时，透明带内外的囊胚部分呈数字“8”的形状，这种孵化被称为“8”形孵化；据报道，这种类型的孵化会影响人类和小鼠胚胎中内细胞团（ICM）的正常呈现。在这里，我们的目的是研究在“8”形孵化过程中影响ICM呈现的因素。我们使用B6D2F1雌性小鼠和ICR雄性小鼠进行体外受精，并使用了捕获的104个囊胚。胚胎在KSOM中于37°C、5%二氧化碳、5%氧气和90%氮气的环境中培养，并使用延时摄影技术对其生长行为进行单独和连续监测。在授精后120小时，对胚胎进行免疫染色并在共聚焦显微镜下检查。我们使用孵化形式来识别“8”形孵化，并将“8”形孵化的囊胚分为两组，一组孵化位点靠近ICM中心，另一组孵化位点远离ICM中心。我们测量了每组的ICM大小和Oct3/4阳性细胞的数量。在95个正在孵化或已孵化的胚胎中，74个是“8”形孵化的囊胚，在这些胚胎中，当孵化位点靠近ICM时，ICM明显比孵化位点远离ICM时更宽（P = 0.0091）。此外，在“8”形孵化的囊胚中，当ICM包含在透明带外的囊胚部分（定义为“囊胚外部”）中时，在这个外部囊胚塌陷后，ICM会附着在与孵化位点相对的外部囊胚的滋养外胚层上。我们的结果表明，在“8”形孵化的囊胚中，孵化位点和外部囊胚的塌陷会影响ICM的形成。因此，对“8”形孵化行为的评估可以产生准确评估胚胎质量的指标。
