Wang W H, Abeydeera L R, Han Y M, Prather R S, Day B N
Department of Animal Science, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
Biol Reprod. 1999 Apr;60(4):1020-8. doi: 10.1095/biolreprod60.4.1020.
Porcine embryos produced in vitro have a small number of cells and low viability. The present study was conducted to examine the morphological characteristics and the relationship between actin filament organization and morphology of porcine embryos produced in vitro and in vivo. In vitro-derived embryos were produced by in vitro maturation, in vitro fertilization (IVF), and in vitro development. In vivo-derived embryos were collected from inseminated gilts on Days 2-6 after estrus. In experiment 1, in vitro-derived embryos (</= 8-cell stage) collected 12-48 h after IVF were separately fixed, stained by orcein, and examined under phase contrast microscopy. It was found that 27% of 2-cell, 74% of 3-cell, 51% of 4-cell, and 74% of 5- to 8-cell-stage embryos were abnormal in morphology. Morphological anomalies included fragmentation (no nucleus in one or more than one blastomere) and/or binucleation (two nuclei in one or more than one blastomere). In experiment 2, actin filament distribution of the embryos at 2-cell to blastocyst stages that were produced in vivo and in vitro were stained by rhodamine-phalloidin and examined by confocal microscopy. Actin filaments were distributed in all in vivo-derived embryos at the cell cortex, and at the joints of cells and perinucleus in 2- to 8-cell-stage embryos and in some cells of morulae and blastocysts. Actin filaments were also distributed in the cortex and at the joints of cells of all in vitro-produced embryos. However, only 20% of in vitro-produced embryos at 2- to 8-cell stages had perinuclear actin filaments in all blastomeres. Most in vitro-produced embryos had fewer perinuclear actin filaments or did not have perinuclear actin filaments in some blastomeres. Fragmentation and binucleate blastomeres were not observed in in vivo-derived embryos. In vivo-derived Day 5 (136.5 +/- 60.4 nuclei per blastocyst) and Day 6 (164.5 +/- 51.9 nuclei per blastocyst) blastocysts had significantly (p < 0.001) more cells than in vitro-produced Day 6 blastocysts (37. 3 +/- 11.7 nuclei per blastocyst). In experiment 3, when cytochalasin D, an inhibitor of microfilament polymerization, was included in the culture medium, it prevented 2- to 4-cell-stage embryos from developing to the blastocyst stage. These results indicate that abnormal actin filament distribution is one possible reason for abnormal embryo cleavage and small cell numbers in pig embryos produced in vitro. Culture conditions that mediate normal actin filament distribution may result in an improvement in embryo quality.
体外产生的猪胚胎细胞数量少且活力低。本研究旨在检查体外和体内产生的猪胚胎的形态特征以及肌动蛋白丝组织与形态之间的关系。体外衍生胚胎通过体外成熟、体外受精（IVF）和体外发育产生。体内衍生胚胎在发情后第2 - 6天从授精后备母猪中收集。在实验1中，将IVF后12 - 48小时收集的体外衍生胚胎（≤8细胞期）分别固定，用orcein染色，并在相差显微镜下检查。发现2细胞期胚胎的27%、3细胞期胚胎的74%、4细胞期胚胎的51%以及5 - 8细胞期胚胎的74%形态异常。形态异常包括碎片化（一个或多个卵裂球中无细胞核）和/或双核化（一个或多个卵裂球中有两个细胞核）。在实验2中，用罗丹明 - 鬼笔环肽对体内和体外产生的2细胞至囊胚期胚胎的肌动蛋白丝分布进行染色，并通过共聚焦显微镜检查。肌动蛋白丝分布于所有体内衍生胚胎的细胞皮质，在2 - 8细胞期胚胎的细胞连接处和核周以及桑椹胚和囊胚的一些细胞中也有分布。肌动蛋白丝也分布于所有体外产生胚胎的皮质和细胞连接处。然而，在2 - 8细胞期的体外产生胚胎中，只有20%的胚胎所有卵裂球都有核周肌动蛋白丝。大多数体外产生的胚胎核周肌动蛋白丝较少或在一些卵裂球中没有核周肌动蛋白丝。在体内衍生胚胎中未观察到碎片化和双核化卵裂球。体内衍生的第5天（每个囊胚136.5±60.4个细胞核）和第6天（每个囊胚164.5±51.9个细胞核）囊胚的细胞数量显著（p < 0.001）多于体外产生的第6天囊胚（每个囊胚37.3±11.7个细胞核）。在实验3中，当在培养基中加入微丝聚合抑制剂细胞松弛素D时，它阻止了2 - 4细胞期胚胎发育到囊胚期。这些结果表明，肌动蛋白丝分布异常是体外产生的猪胚胎异常分裂和细胞数量少的一个可能原因。调节正常肌动蛋白丝分布的培养条件可能会改善胚胎质量。
