16 Effect of cell surface N-linked oligosaccharide chains on the compaction of preimplantation mouse embryos

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1 16 Effect of cell surface N-linked oligosaccharide chains on the compaction of preimplantation mouse embryos H.Hayashi, N.Minami, M.Yamada and K.Utsumi Department of Animal science, College of Agriculture, Kyoto University, Kitashirakawa, sakyoku, Kyoto , Japan 1. Delayed compaction of 8-cell embryo by N-glycanase treatment N-glycanase is an enzyme which cleaves N-linked oligosaccharide chain of an asparagine residue of the protein. The effect of N-glycanase treatment of 8-cell embryos on the compaction and the subsequent development to blastocysts was studied in two ways. a) early 8-cell embryos were treated with N-glycanase either in modified-whitten's medium (m-w.m.)(condition 1) or in Ca2+ Mg2+ free phosphate buffered saline (PBS(-))(Condition 2) at 37 Ž for 1 h, and then the embryos were cultured in normal medium (m-w.m.). b) compacted 8-cell embryos were experimentally decompacted by culturing in PBS(-) at 37 Ž for 1 h and then, they were treated with N-glycanase in PBS(-) for 1 h. After the treatment, embryos were further cultured in normal m-w.m. (Condition 3) or in m-w.m. containing N-glycanase (Condition 4). In each control experiment, embryos were subjected to identical procedures except that N-glycanase was omitted. In some experiments, the heat inactivated enzyme was used as an additional control [1,2]. The time taken for compaction of 50% of embryos under Condition 1 was almost the same as that of untreated control embryos, and the time was 3 h. However the time taken for compaction of 50% of embryos under Condition 2 was 4.7 h, compared with 3.3 h for control embryos that were incubated in PBS(-) for 1 h and then were cultured in m-w.m. continuously. On the other hand, in the case of experimentally decompacted embryos by PBS(-), after the treatment with N-glycanase in PBS(-) for 1 h, the time taken for recompaction of 50% of embryos under Condition 3 was 2.5 h, compared with 0.8 h for untreated control embryos and with 1.3 h for another control embryos treated with heat inactivated enzyme. The recompaction of 50% of embryos treated under Condition 4 was even further delayed, and the time taken for it was 4.3 h. A delay in compaction was observed both in early 8-cell and in decompacted 8-cell embryos treated with N-glycanase

2 only in PBS(-). It is reported that cell surface oligosaccharide are recognized by endogenous lectins of adherent cells in Ca2+ dependent manner [3], and as a reason of this result, it is thought that by the deletion of Ca2+ in PBS, the binding of oligosaccharide and endogenous lectins was inhibited and so, the enzyme in PBS(-) worked effectively. This result indicates that N-linked oligosaccharide chains cleaved by the enzyme are involved in compaction and recompaction process, and that the N-linked oligosaccharide have already been expressed in early 8-cell embryos. And it is inferred that the completion of compaction and recompaction in embryos which were treated with N-glycanase followed the reappearance of cell surface carbohydrateṣ By 96 h after hcg, the percentages of blastocysts developed after treatment under Condition 1, 2, 3 and 4 were 63.2%, 70.8%, 90.1% and 64.6%, respectively. More than 85% of embryos treated under Condition 1,2 and 3 could developed to morphologically normal expanded blastocysts until 120 h after hcg. The rate, however, was reduced to 7.3% when the embryos were cultured under Condition 4. The blastulation rates of embryos treated under Condition 1 and 4 were only a little lower than those of embryos treated under Condition 2 and 3. However, the percentage of embryos developed to expanded blastocysts after the treatment under Condition 4 were much lower than those of embryos treated under any other conditions. This result indicates that N glycanase treatment inhibits the normal development to expanded blastocysts, and this may be due to the removal or suppression of reappearance of cell surface N-linked oligosaccharide chains in blastocysts by continuous enzyme treatment. But to define the picture of how N-glycanase affects the development to expanded blastocysts, further investigations are required. 2. Changes in total amount of cell surface oligosaccharide in decompacted 8-cell embryos after N-glycanase treatment. Fluorescein isothiocyanate (FITC) conjugated lectins were used to examine the dissolution of N linked oligosaccharide by N-glycanase and the reappearance of cell surface oligosaccharide in 8 cell embryos after treatment with N-glycanase [4,5]. Embryos treated under Condition 3,4 were stained with Concanavalin A (Con A), Wheat germ agglutinin (WGA) or Dolichos biflorus agglutinin (DBA) at 0 h and 5 h after treatment with N-glycanase. The primary carbohydrate specificity of Con A, WGA and DBA are mannose, N-acetylglucosamine and N-acetylgalactosamine, respectively, and it is known

3 that N-linked oligosaccharide chains contain many of those carbohydrates. The staining intensity with each lectin tended to vary between experiments even when the staining procedures were identical. On the average, there were a little difference in the level of binding of each lectin between control and treatment groups under Condition 3 stained immediately after enzyme treatment,. The fluorescence intensity with Con A, WGA. or DBA on enzyme treated embryos was lower than that of control embryos stained immediately after enzyme treatment. However, the intensity at 5 h after enzyme treatment under Condition 3 was similar to those of control embryos stained immediately after decompaction. In addition, there was little difference in the fluorescence intensity with each lectin between embryos treated under Condition 4 and Condition 3 when the embryos were stained at 5 h after treatment with the enzyme. This result suggests that the reappearance of cell surface oligosaccharide parallels recompaction, and the oligosaccharide chains that contain carbohydrates recognized by Con A, WGA and DBA are involved extremely in compaction and recompaction process. 3. Changes in distribution of E-cadherin in decompacted 8-cell embryos after N-glycanase treatment. It is known that E-cadherin molecules is distributed uniformly over the blastomeres of 2- and 4-cell embryos and become restricted to the cell contact regions between adjacent cells during compaction of 8-cell embryos. It is also reported that E-cadherin molecules and carbohydrates are involved in the compaction process in mouse embryos [6,7]. The localization of E- cadherin molecules is essential for compaction process and the compaction completes successfully after E-cadherin molecules moves to the adhesion region between the blastomeres of 8-cell embryos. We examined whether the distribution of E-cadherin molecules in 8-cell embryos is affected by N-glycanase treatment using monoclonal antibody for E-cadherin (ECCD-2). The distribution of E-cadherin molecules in decompacted embryos after treatment with the enzyme (Condition 3 and 4) was not different from that of untreated control embryos. From the result, it is suggested that the delay in compaction by enzyme treatment was not due to the redistribution of E-cadherin molecules but due to the cleavage of cell surface oligosaccharide chains of decompacted 8-cell embryos. In conclusion, it is obvious that the removal of N-linked oligosaccharide chains from the surface of blastomere have some effects on the compaction of 8-cell embryos. In addition, it is

4 supposed that the oligosaccharide chains of E-cadherin molecules are also involves in coupling of E-cadherin molecules during compaction. If it is true, the lack of oligosaccharide chains of E-cadherin inhibits the binding of E-cadherin molecules so that compaction is delayed. However, the role of cell surface oligosaccharide for the compaction remains to be investigated. Acknowledgement The authors would like to thank Dr. M. Takeichi (Faculty of Science, Kyoto University) for the generous gift of ECCD-2 antibody. References [1]S.Rastan, S.J.Thorpe, P.Brown, H.C.Gool and T.Feizi (1985): Cell interaction in preimplantation embryos: evidence for involvement of saccharides of the poly-n-acetyllactosamine series. J. Embryol. exp. Morph. 87: [2]H.A.H.Surani, S.J.Kimber and A.H.Handyside (1981): Synthesis and role cell surface glycoproteins in preimplantation mouse development. Exp. Cell Res. 133: [3]M.Tiemeyer, S.J.Swiedler, M.Isihara, M.Moreland, H.Schweingruber, P.Hirtzer and B.K.Brandley (1991): Carbohydrate ligands for endothelial leukocyte adhesion molecule 1. Proc. Natl. Acad. Sci. USA. 88: [4]G.B.Dealtry, M.R.Curry and M.H.Sellens (1987): Fucosylated glycoconjugates appear on mouse embryos during blastocyst formation. J. Exp. Zool. 243: [5]S.J.Kimber and J.M.Bird (1985) Cell surface changes in preimplantation mouse embryos during compaction investigated using FITC conjugated lectins after proteolytic enzyme treatment. Roux's Arch. Dev. Biol. 194: [6]G.K.Winkel, J.E.Ferguson, M.Takeichi and R.Nuccitelli (1990): Activation of protein kinase C triggers premature compaction in the four cell stage mouse embryo. Dev. Biol. 138:1-15.

5 [7]Y.Shirayoshi, T.S.Okada and M.Takeichi (1983): The calcium-dependent cell-cell adhesion system regulates inner cell mass formation and cell surface polarization in early mouse development. Cell 35: