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

单细胞小鼠胚胎在双细胞阶段表现出阻滞。这种分裂在两细胞阶段之后的失败被称为“两细胞阻滞”。有报道称，通过改变培养基（添加EDTA）或培养体系（共培养）可以克服这种体外发育障碍。最近，我们报道了在输卵管影响下培养的小鼠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细胞期胚胎的形态与单独在培养基中发育的胚胎明显不同（Minami et al., J reprd Fert 96: 753,1992）。这些具有发育能力的4细胞胚胎表现出通常在压实过程中表现出的细胞扁平（过早压实的4细胞），这一过程发生在8细胞阶段。本研究旨在探讨体外输卵管对4细胞胚胎形态的影响，并对E-cadhe、More rin定位和间隙连接的组装进行了研究。单克隆抗体间接免疫荧光法检测e -钙粘蛋白和间隙连接蛋白的信号。在共培养的4细胞胚胎中，e -钙粘蛋白和间隙连接蛋白都定位于细胞粘附区，而单独培养的4细胞胚胎则没有定位。由此可见，输卵管在体外可刺激e -钙粘蛋白的定位和间隙连接蛋白的组装。此外，我们还研究了p34^<cdc2>激酶（细胞周期调节剂）活性与输卵管环境下培养的2细胞胚胎发育能力的关系，因为胚胎在体内停留在输卵管中，在体外发生阻滞的阶段。经输卵管培养的胚胎p34^<cdc2>激酶活性在卵裂后20 h升高，21 h达到峰值，24 h后逐渐降低。而无输卵管培养的胚胎活性直到卵裂后24 h才有所提高。在第一次卵裂后25 h，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%达到4细胞期。虽然2细胞阻断胚胎能够复制DNA，但Hoechst染色显示2细胞阻断胚胎既没有核膜破裂，也没有染色质凝结。从这些数据可以看出，2细胞阻滞胚胎在第二个细胞周期的G_2期被阻滞。此外，用冈田酸处理2细胞阻断胚胎诱导核膜破裂和染色质凝聚的事实表明，在2细胞阻断胚胎中可能存在与减数分裂决定阶段卵母细胞阻滞相同的机制，输卵管环境可能提供体外克服这种G_2阻滞所需的信号。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