Sugimura Satoshi, Akai Tomonori, Hashiyada Yutaka, Aikawa Yoshio, Ohtake Masaki, Matsuda Hideo, Kobayashi Shuji, Kobayashi Eiji, Konishi Kazuyuki, Imai Kei
National Livestock Breeding Center, Fukushima 961-8511, Japan.
J Reprod Dev. 2013;59(2):115-22. doi: 10.1262/jrd.2012-113. Epub 2012 Nov 15.
To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 μl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density.
为了在体外发育过程中对胚胎进行个体识别，我们之前开发了一种多孔微滴培养皿（WOW培养皿），它包含25个微孔。在此，我们研究了胚胎密度（每单位体积培养基中的胚胎数量）对在WOW培养皿中培养的牛体外受精胚胎的体外发育和基因表达的影响。使用传统微滴培养和WOW培养两种方式，将5、15和25枚牛胚胎在125 μl培养基中培养168小时。对第7天的囊胚进行细胞数量分析以及10个基因（CDX2、IFN-tau、PLAC8、NANOG、OCT4、SOX2、AKR1B1、ATP5A1、GLUT1和IGF2R）表达的检测。在微滴培养中，每滴培养5枚胚胎时形成的>4细胞期分裂胚胎和囊胚的比例显著低于每滴培养15枚或25枚胚胎时，但在WOW培养中并非如此。在微滴培养和WOW培养中，各实验组之间的发育动力学和囊胚细胞数量均无差异。每滴培养25枚胚胎时培养的胚胎中IFN-tau的表达显著高于每滴培养15枚胚胎时培养的胚胎以及人工授精（AI）来源的囊胚。此外，25枚胚胎组中IGF2R的表达显著低于5枚胚胎组以及AI来源的囊胚。在WOW培养中，这些基因的表达不受胚胎密度影响，且与AI来源的囊胚相似。这些结果表明，与传统微滴培养相比，微孔系统中胚胎的体外发育以及IFN-tau和IGF2R的表达可能对胚胎密度不敏感。
