Studies on the cell adhesion and cell to cell communication during development and differentiation in mammalian preimplantation embryos

单细胞小鼠胚胎在两细胞阶段表现出障碍。这种不能分裂出两细胞阶段的现象被称为“两细胞块”。有报道称，在体外，通过对培养液的某些修改(添加EDTA)或培养体系(共培养)可以克服这一发育障碍。最近，我们报道了在输卵管的影响下培养的小鼠1-细胞胚胎在体外可以正常发育到囊胚期。此外，在共培养中发育的4-细胞期胚胎的形态明显不同于在单独的培养液中发育的胚胎(Minami等人，J Reprod Fert 96：753,1992)。这些具有发育能力的4-细胞胚胎表现出细胞扁平化(过早致密的4-细胞)，这通常在紧凑化过程中表现出来，这一过程发生在8-细胞阶段。本研究的目的是评价输卵管对早熟致密4-细胞胚胎形态的影响，其中E-CADHE…检查了更多的环定位和缝隙连接组装。用单抗间接免疫荧光法检测E-钙粘蛋白和缝隙连接蛋白信号。在共培养的4-细胞胚胎中，E-钙粘附素和缝隙连接蛋白都定位于细胞粘附区，而单独培养的4-细胞胚胎不显示定位。提示输卵管在体外可促进E-钙粘蛋白的定位和缝隙连接蛋白的组装。此外，我们还研究了p34^&lt；cdc2&gt；激酶(细胞周期调节因子)活性与体外培养的2-细胞胚胎发育能力的关系，因为胚胎在体内处于输卵管内，而体外受阻发生在这个阶段。与输卵管共同培养的胚胎，其p34^-lt；cdc2&gt；激酶活性在卵裂后20h开始升高，21h达到高峰，之后逐渐下降。然而，无输卵管培养的胚胎在卵裂后24小时才出现活性增加。直到第一次卵裂后25h，有输卵管培养的胚胎有50%达到4-细胞期，而无输卵管培养的胚胎只有7.6%能达到4-细胞期。虽然2-细胞阻断的胚胎能够复制DNA，但Hoechst染色显示，2-细胞阻断的胚胎既没有核膜的破裂，也没有染色质的凝聚。从这些数据可以看出，2-细胞受阻胚胎被停滞在第二个细胞周期的G_2期。此外，冈田酸处理2-细胞受阻胚胎导致核膜破裂和染色质凝聚的事实表明，在2-细胞受阻胚胎中可能存在与减数分裂分裂中期受阻卵母细胞相同的机制，输卵管环境可能提供了在体外克服这种G2受阻所需的信号。较少

One-cell mouse embryos exhibit a block at the two-cell stage. This falure to cleave beyond the two-cell stages has been termed the 'two-cell block'. It has been reported that some modification of culture medium (addition of EDTA) or culture system (co-culture) can overcome this developmental block in vitro. Recently, we reported that mouse 1-cell embryos cultured under the influence of oviducts can normally develop to the blastocyst stage in vitro. In addition, the morphology of the 4-cell stage embryos developed in co-culture is apparently different from that of embryos developed in culture medium alone (Minami et al., J Reprod Fert 96 : 753,1992). These developmentally competent 4-cell embryos exhibit cell flattening (premature compacted 4-cell) that is normally exhibited during compaction, a process which takes place at the 8-cell stage. The objective of this study was to assess the in vitro effects of oviduct on the morphology of premature compacted 4-cell embryos, in which E-cadhe … More rin localization and gap junction assembly were examined. The signals of E-cadherin and gap junction protein were detected by indirect immunofluorescence assay using monoclonal antibodies. Both E-cadherin and gap junction protein were localized to the cell adhesion region in the 4-cell embryos developed in co-culture, although the 4-cell embryos developed in culture alone do not exhibit localization. From the data, it is suggested that the oviduct can stimulate the localization of E-cadherin and the assembly of gap junction protein in vitro. In addition, we examined the relationship between p34^<cdc2> kinase (cell cycle regulator) activity and developmental competence of 2-cell embryos cultured under oviductal enviromment, because embryos stays in oviduct in vivo at which stage the block occurs in vitro. The p34^<cdc2> kinase activity of embryos cultured with oviduct increased at 20 h and reached the peak at 21 h, and then gradually decreased by 24 h after cleavage. However, the activity of embryos cultured without oviduct did not increase until 24 h after cleavage. Until 25 h after first cleavage, 50% of embryos cultured with oviduct reached to the 4-cell stage although only 7.6% of embryos cultured without oviduct were able to reach the 4-cell stage. Although 2-cell blocked embryos were able to duplicate DNA,Hoechst staining revealed that the 2-cell blocked embryos showed neither the breakdown of nuclear envelope nor the chromatin condensation. From these data, it is demonstrated that the 2-cell blocked embryos are arrested at G_2 phase of the second cell cycle. Furthermore, the fact that treatment of 2-cell blocked embryos with okadaic acid induce nuclear envelope breakdown and chromatin condensation indicates that common mechanisms as in the oocyte arrested at dictyate stage of meiosis may exist in the 2-cell blocked embryos, and oviductal environment may supply the signals required to overcome this G_2 arrest in vitro. Less