Parthenogenetic Activation And Homogeneous And Heterogeneous Embryo Reconstruction In Mouse

(The college of Animal Science and Veterinary Medicine, Northwest Science and Technology University of Agriculture and Forestry, Yangling, Shaanxi, R.R.China, 712100)In order to define the best electroactivation condition of oocytes and discuss possibility of heterogeneous cloned embryos reconstruction and interaction between donor nucleus and recipent cytoplasm, the experiments were designed to research effects of different factors on electroactivation and parthenogenesis of mouse oocytes and inject a single type fresh cumulus cell nucleus of mouse, rat, bovine into enucleated mouse MII oocytes to reconstruct homogeneous and heterogeneous embryos. Reconstructed embryos were electroactivated and cultured in vitro. Following is the results:The mouse oocytes of different age (time between HCG injected and mouse killed) of 13h, 15h, 17h, 19h were electroactivated. If other conditions were same, following the age lengthened, the rates of electroactivation and parthenogenesis increased. When oocytes age is 17 hours, the rates of electroactivation and parthenogenesis reach highest (73.1% and 34.2). When the age of oocytes is more than 17 hours, the rates of electroactivation and parthenogenesis no longer increase, the rate of cleavage begin to increase. So the mouse MII oocytes of 17 hours age are easiest to electroactivated.The electroactivation parameters of field strength, pulse duration, pulse times have important influence to rates of electroactivation and parthenogenesis of oocytes. The research shows that the parameters of field strength 1.0kv/cm, pulse duration 30μs, two times pulse interval 1 minute are best in electroactivation of mouse MII oocytes. Ca2+ Mg2+are necessary elements in solution of oocytes electroactivation. When Sr2+ (10mmol/L) was added into electroactivation solution contained Ca2+ (0.1mmol/L), Mg2+ (0.1mmol/L), it could induce intracellular Ca2+ to release. So the electroactivated rate of mouse MII oocytes could be elevated further and reached 78.6%. But the developmental rate of parthenogenetic embryos was not influenced in vitro. CHX (Cycloheximide) contained in cultured solution can inhibit protein synthesis of oocyte and make MPF in low level. So it can increase electroactivated rate (83.3%) of oocytes and morulae/blastocysts rate (57.5%) of parthenogenetic embryos in vitro.Hoechst 33342 was used to stain mouse MII oocytes for examination whether nucleus was enucleated. Enucleated mouse MII oocytes were each injected with a single type cumulus nucleus of mouse, rat, bovine and left for 3 hours in CZB solution before electroactivated. Electroactivated solution contained Ca2+ ,Mg2 ,Sr2+. Reconstructed embryos were cultured in solution contained CHX for 20 hours, then moved to solution free CHX to culture in vitro. The rates of 2-cell and blastocyst of homogeneous embryos are 45.1% and 9.8% respectively. The rates of 2-cell of heterogeneous embryos reconstructed with enucleated mouse MII oocytes and rat or bovine cumulus nucleus are 43.5% and 44.2% respectively. The 2-cell rates of homogeneous and heterogeneous embryos are no significant difference (p>0.05). The development of heterogeneous embryos can not beyond 2-cell stage for possible reasons of maternal-zygotic transition failure and nuclear-cytoplasmic incompatibility.