OPS玻璃化冷冻对小鼠四倍体胚胎体外发育的影响
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摘要
为探究开放式拉长细管(OPS)玻璃化冷冻对四倍体胚胎发育的影响,本实验利用2-细胞胚胎电融合法制备四倍体胚胎,再对四倍体胚胎进行OPS玻璃化冷冻,分别观察记录二倍体胚胎、四倍体胚胎以及冷冻解冻后四倍体胚胎的发育情况。结果表明:2-细胞胚胎电融合效率为96.1%;二倍体胚胎组与电融合后四倍体胚胎组的囊胚率和孵化囊胚率差异不显著;冷冻解冻后四倍体胚胎的囊胚率(100%)与四倍体新鲜组(93.3%)差异不显著,其孵化囊胚率(72.3%)较新鲜组(64.9%)显著增高(P<0.05);四倍体冷冻解冻组的囊胚细胞数(31.96)与新鲜组(32.54)无显著差异;冷冻解冻后的四倍体早期囊胚进行体外培养时其发育速度比对照组更快。可见,冷冻对小鼠四倍体胚胎的囊胚率和囊胚细胞数均无显著影响,但孵化囊胚率显著提高,且OPS玻璃化冷冻后使四倍体胚胎的发育速度更快。
In order to explore the effect of open lengthened tubule(OPS) vitrification on development of tetraploid embryos,tetraploid embryos were prepared by 2-cell embryo electrofusion method, and then the tetraploid embryos were OPS vitrification. The development of tetraploid embryos after freezing and thawing, fresh diploid embryos and fresh tetraploid embryos was observed and recorded. Results showed that the efficiency of 2-cell embryo electrofusion was 96.1%. There was no significant difference in blastocyst rate and incubation blastocyst rate between diploid embryo group and tetraploid embryo group after electrofusion. After freezing and thawing, the blastocyst rate of tetraploid embryos(100%) was not significantly different from that of the tetraploid fresh group(93.3%), and the hatching blastocyst rate(72.3%) was significantly higher than that of the fresh group(64.9%)(P<0.05). There was no significant difference in the number of cyst cells between the tetraploid group(31.96) and the fresh group(32.54). The frozen thawing tetraploid early blastocysts developed faster than control group when cultured in vitro. It can be seen that freezing had no significant effect on the blastocyst rate and the number of blastocyst cells of mouse tetraploid embryos in mice, but the incubation blastocyst rate was significantly increased, and the OPS vitrification cryopreservation accelerated the development of tetraploid embryos.
In order to explore the effect of open lengthened tubule(OPS) vitrification on development of tetraploid embryos,tetraploid embryos were prepared by 2-cell embryo electrofusion method, and then the tetraploid embryos were OPS vitrification. The development of tetraploid embryos after freezing and thawing, fresh diploid embryos and fresh tetraploid embryos was observed and recorded. Results showed that the efficiency of 2-cell embryo electrofusion was 96.1%. There was no significant difference in blastocyst rate and incubation blastocyst rate between diploid embryo group and tetraploid embryo group after electrofusion. After freezing and thawing, the blastocyst rate of tetraploid embryos(100%) was not significantly different from that of the tetraploid fresh group(93.3%), and the hatching blastocyst rate(72.3%) was significantly higher than that of the fresh group(64.9%)(P<0.05). There was no significant difference in the number of cyst cells between the tetraploid group(31.96) and the fresh group(32.54). The frozen thawing tetraploid early blastocysts developed faster than control group when cultured in vitro. It can be seen that freezing had no significant effect on the blastocyst rate and the number of blastocyst cells of mouse tetraploid embryos in mice, but the incubation blastocyst rate was significantly increased, and the OPS vitrification cryopreservation accelerated the development of tetraploid embryos.
引文
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[2]Snow M H.The immediate postimplantation development of tetraploid mouse blastocysts[J].J Embryol Exp Morphol,1976,35(1):81-86.
[3]Misra R P,Bronson S K,Xiao Q,et al.Generation of singlecopy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation[J].BMC Biotechnol,2001,1(1):12.
[4]Li H,Zhao C,Xu J,et al.Rapid generation of gene-targeted EPS-derived mouse models through tetraploid complementation[J].Protein Cell,2019,10(1):20-30.
[5]Amano T,Kato Y,Tsunoda Y.Comparison of heat-treated and tetraploid blastocysts for the production of completely ES-cellderived mice[J].Zygote,2001,9(2):153-157.
[6]Zhou D,Ren J X,Ryan T M,et al.Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts[J].Nucleic Acids Res,2004,32(16):e128.
[7]Pralong D,Lim M L,Vassiliev I,et al.Tetraploid embryonic stem cells contribute to the inner cell mass of mouse blastocysts[J].Cloning Stem Cells,2005,7(4):272-278.
[8]Thomas R,Marks D H,Chin Y,et al.Whole chromosome loss and associated breakage-fusion-bridge cycles transform mouse tetraploid cells[J].EMBO J,2017,37(2):201-218.
[9]Snow M H.Tetraploid mouse embryos produced by cytochalasin B during cleavage[J].Nature,1973,244(5417):513-515.
[10]Eglitis M A.Formation of tetraploid mouse blastocysts following blastomere fusion with polyethylene glycol[J].J Exp Zool,2010,213(2):309-313.
[11]Machado Júnior J,Urio M,Aguiar L H D,et al.Mice embryos cryopreservation:vitrification or ultra-rapid freezing?[J].Acta Sci Vet,2010,38(4):357-362.
[12]Raz A,Amer-Alshiek J,Goren-Margalit M,et al.Donation of surplus frozen pre-embryos to research in Israel:underlying motivations[J].Isr J Health Policy Res,2016,5(1):25.
[13]Martino N A,Dell Aquila M E,Cardone R A,et al.Vitrification preserves chromatin integrity,bioenergy potential and oxidative parameters in mouse embryos[J].Reprod Biol Endocrinol,2013,11(1):1-11.
[14]Oikonomou Z,Chatzimeletiou K,Sioga A,et al.Effects of vitrification on blastomere viability and cytoskeletal integrity in mouse embryos[J].Zygote,2017,25(1):75-84.