| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1996: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1995: ¥900,000 (Direct Cost: ¥900,000)
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| Research Abstract |
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
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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
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