Ruggeri Elena, DeLuca Keith F, Galli Cesare, Lazzari Giovanna, DeLuca Jennifer G, Stokes Joanne E, Carnevale Elaine M
1Department of Biomedical Sciences,Colorado State University,1693 Campus Delivery,Fort Collins,CO 80523,USA.
3Department of Biochemistry and Molecular Biology,Colorado State University,1870 Campus Delivery,Fort Collins,CO 80523,USA.
Microsc Microanal. 2017 Dec;23(6):1197-1206. doi: 10.1017/S1431927617012740. Epub 2017 Dec 6.
Confocal microscopy was used to image stages of equine zygote development, at timed intervals, after intracytoplasmic sperm injection (ICSI) of oocytes that were matured in vivo or in vitro. After fixation for 4, 6, 8, 12, or 16 h after ICSI, zygotes were incubated with α/β tubulin antibodies and human anticentromere antibody (CREST/ACA), washed, incubated in secondary antibodies, conjugated to either Alexa 488 or Alexa 647, and incubated with 561-Phalloidin and Hoechst 33258. An Olympus IX81 spinning disk confocal microscope was used for imaging. Data were analyzed using χ 2 and Fisher's exact tests. Minor differences in developmental phases were observed for oocytes matured in vivo or in vitro. Oocytes formed pronuclei earlier when matured in vivo (67% at 6 h and 80% at 8 h) than in vitro (13% at 6 and 8 h); 80% of oocytes matured in vitro formed pronuclei by 12 h. More (p=0.04) zygotes had atypical phenotypes, indicative of a failure of normal zygote development, when oocyte maturation occurred in vitro versus in vivo (30 and 11%, respectively). Some potential zygotes from oocytes matured in vivo had normal phenotypes, although development appeared to be delayed or arrested. Confocal microscopy provided a feasible method to assess equine zygote development using limited samples.
共聚焦显微镜用于对体内或体外成熟卵母细胞经胞浆内单精子注射（ICSI）后，在设定的时间间隔内马受精卵发育的各个阶段进行成像。在ICSI后4、6、8、12或16小时固定后，将受精卵与α/β微管蛋白抗体和人抗着丝粒抗体（CREST/ACA）孵育，洗涤，再与偶联有Alexa 488或Alexa 647的二抗孵育，并与561-鬼笔环肽和Hoechst 33258孵育。使用奥林巴斯IX81旋转盘共聚焦显微镜进行成像。数据采用χ2检验和Fisher精确检验进行分析。观察到体内或体外成熟的卵母细胞在发育阶段存在微小差异。体内成熟的卵母细胞比体外成熟的卵母细胞更早形成原核（体内成熟6小时时为67%，8小时时为80%；体外成熟6小时和8小时时为13%）；体外成熟的卵母细胞80%在12小时时形成原核。与体内成熟相比，体外成熟卵母细胞来源的受精卵出现非典型表型（分别为30%和11%），表明正常受精卵发育失败的情况更多（p = 0.04）。一些体内成熟卵母细胞来源的潜在受精卵具有正常表型，尽管发育似乎延迟或停滞。共聚焦显微镜为使用有限样本评估马受精卵发育提供了一种可行的方法。
