小鼠致密化前胚胎卵裂球体外培养体系的研究

目的探索改善小鼠致密化前胚胎卵裂球体外发育能力。方法用酶消化与机械法结合分离小鼠2-、4-、8-细胞胚胎卵裂球为单个卵裂球及2/4、4/8多卵裂球胚胎,并将其装入空透明带后于兽用人绒毛膜促性腺激素(hCG)注射后138 h,观察透明带包裹、胚胎密度及胰岛素(Ins)、葡萄糖(Glu)添加对各卵裂球、囊胚形成率及囊胚细胞数目的影响。结果透明带包裹提高了2-细胞胚胎卵裂球来源的单腔囊胚的发育率。25/50μl胚胎密度及培养基添加Ins、Glu能显著提高2-细胞胚胎卵裂球囊胚发育率和囊胚细胞数;且对4-和8-细胞胚胎卵裂球发育具有同样的促进作用。结论采用透明带包裹、卵裂球密集培养、培养基添加Ins、Glu的方法成功建立了一种维持致密化前胚胎卵裂球体外高效发育的培养体系。其中,透明带在卵裂球发育中具维持囊胚正常形态作用,避免了多腔囊胚的形成;卵裂球密集培养和培养基添加Ins、Glu均提高囊胚形成率和(或)囊胚细胞数目。该体系有效改善了致密化前胚胎卵裂球体外发育质量。

Study on in Vitro culture of blastomeres derived from mouse precompacted embryos

Objective: To improve the in vitro development of the blastomere(s) derived from mouse precompacted embryo.Methods: 1/2 blastomere(single blastomere from 2-cell embryos),1/4,2/4 blastomere(s)(single or two blastomere(s) from 4-cell embryos),and 1/8,4/8 blastomeres(single or four blastomere(s) from 8-cell embryos) were obtained by enzyme digestion and mechanical methods and cultured in CZB medium(or supplemented with insulin and glucose) at several densities following inserted into an empty zona pellucida.The blastocyst formation rate and the cell number per blastocyst were examined at 138 hours after hCG injection.Results: The rate of blastocyst with double or several blastocoelic cavities from 1/2 blastomeres with zona pellucida was lower than that without zona pellucida.Increasing the embryo density,from 5/50 μl to 25/50 μl,significantly increased the cell number per blastocyst from 1/2 blastomeres.Addition of insulin and glucose to CZB medium caused a significant increase in the cell number per blastocyst from 1/2 blastomeres.The same results were also observed when 1/4,1/8,2/4,4/8 blastomeres were cultured with an empty pellucida in CZB supplemented with insulin and glucose at a density of 25/50 μl.Conclusions: An effective approach was established for the in vitro development of blastomeres from mouse precompacted embryos,in which zona pellucida had the effect of maintaining morphology of embryo during the development in vitro.Higher embryo density and addition of insulin and glucose to CZB medium can improve the development capacity of blastomeres derived from mouse precompacted embryos.