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

哺乳动物植入前胚胎发育和分化过程中细胞粘附和细胞间通讯的研究

• 批准号: 07660378
• 负责人: MINAMI Naojiro
• 金额: $ 1.09万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660378/
• 关键词: Mouse embryo; Development; Oviduct; E-cadherin; Gap Junction; Protein kinase C; p34^<cdc2> kinase; マウス初期発生; ギャプ結合; p34cdc2キナーゼ; mouse embryo; oviduct; E-cadherin; gap junction; protein kinase C

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

单细胞小鼠胚胎在两细胞阶段表现出阻滞。这种在两细胞阶段以后不能裂解的现象被称为“两细胞块”。据报道，对培养基（添加EDTA）或培养系统（共培养）进行一些修改可以克服这种体外发育障碍。最近，我们报道了小鼠1-细胞胚胎在输卵管的影响下，可以在体外正常发育到囊胚期。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培养基中发育的胚胎的形态（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（过早压实的4-细胞），这通常在压实过程中表现出，这一过程发生在8-细胞阶段。本研究的目的是评估输卵管在体外对早期致密4-细胞胚胎形态的影响，其中E-cadhe ...更多信息 检测Rin定位和间隙连接组装。用单克隆抗体间接免疫荧光法检测E-cadherin和缝隙连接蛋白的信号。E-cadherin和缝隙连接蛋白均定位于共培养中发育的4-细胞胚胎的细胞粘附区域，尽管单独培养中发育的4-细胞胚胎不表现出定位。提示输卵管在体外可刺激E-cadherin的定位和缝隙连接蛋白的组装。此外，我们还研究了<cdc2>在输卵管环境下培养的2-细胞胚胎的p34激酶（细胞周期调节因子）活性与发育能力之间的关系，因为胚胎在体内停留在输卵管中，而在体外发生阻塞。与<cdc2>输卵管一起培养的胚胎p34^激酶活性在卵裂后20 h升高，21 h达到高峰，24 h后逐渐下降。而无输卵管培养的胚胎，卵裂后24 h才有活性增加。第一次卵裂后25 h，有输卵管的胚胎有50%达到4-细胞期，而无输卵管的胚胎只有7.6%达到4-细胞期。Hoechst染色显示，2-细胞阻断的胚胎既没有核膜破裂，也没有染色质凝聚。结果表明，2-细胞阻滞胚胎阻滞于第二细胞周期的G_2期。此外，冈田酸处理2-细胞阻滞胚胎引起核膜破裂和染色质浓缩的事实表明，在2-细胞阻滞胚胎中可能存在与卵母细胞减数分裂停滞在网状期相同的机制，输卵管环境可能提供克服这种G_2停滞所需的信号。少

项目成果

N.Minami et al.: "Relationship between p34^<CDC2> kinase activity and developmental competence of 2-cell mouse embryos cultured under oviductal environment." Biol. Reprod.(in press).

N.Minami 等人：“p34^<CDC2> 激酶活性与输卵管环境下培养的 2 细胞小鼠胚胎发育能力之间的关系。”

N.Minami et al.: "Relationship between p34^<cdc2> kinase activity and developmental competence of 2-cell mouse embryos cultured under oviductal environment." Biol.Reprod.(in press).

N.Minami 等人：“p34^<cdc2> 激酶活性与输卵管环境下培养的 2 细胞小鼠胚胎发育能力之间的关系。”

Naojiro Minami: "Early embryonic development under oviductal influence in vitro" Anim. Reprod. Sci.(in press). (1996)

Naojiro Minami：“体外输卵管影响下的早期胚胎发育”动画。

N.Minami et al.: "E-cadherin localization and gap junction assembly in the mouse embryos are accelerate by the oviduct in vitro." Biol. Reprod.54. 170 (1996)

N.Minami 等人：“体外输卵管加速了小鼠胚胎中 E-钙粘蛋白的定位和间隙连接组装。”

N.Minami, A.Takahashi, M.Yamada and K.Utsumi: "E-cadherin localization and gap junction assembly in the mouse embryos are accelerate by the oviduct in vitro." Biol.Reprod.54. 170 (1996)

N.Minami、A.Takahashi、M.Yamada 和 K.Utsumi：“体外输卵管加速了小鼠胚胎中 E-钙粘蛋白的定位和间隙连接组装。”