Study On Accessing Parthenogenetic Embryos By Mouse MI Oocytes

current research on parthenogenetic development is often used Mâ…¡oocytes,and The parthenogenetic embryos are usually haploid.Using cytochalasin D(CD)for inhibiting the Release of the first polar body can maintain the genome in 2N.Although for the genomic imprinting,to do this can not make parthenogenetic embryos develop to term,but can provide parthenogenetic embryonic cells a more extensive genetic background.Foreign Researcher reports that the parthenogenetic stem cells matched with the oocyte donor's MHC can be isolated from this Blastocyst.a preliminary study on accessing parthenogenetic embryos by Mâ… oocytes is conducted, which included chosing the methods of activation and Suitable medium for culturing.There are also a comparison on development capability with the parthenogenetic embryo which obtained from Mâ…¡oocytes.In additionn,This experiment adopted somatic cell nuclear transfer technologies to research on access diploid parthenogenetic embryos.1 Effects of different activation on the development of parthenogenetic mouse Mâ… oocytes.5 ways on activation of mouse Mâ… oocytes dealed with CD were investigated.A23187+6-DMAP the cleavage rate and the rate of blastocyst were 69.56%and 28.86%,which significantly higher than that of ethanol,SrCl₂,A23187,and electrical activation.2 Effects of different culture maduim on the development of parthenogenetic mouse Mâ… oocytes. we compared the development of the parthenogenetic embryos cultured in 4 different culture maduim(mCZB,KSOM,M16 and mM16).There got no significant difference in cleavage rate (79.39%,77.54%,77.47%,78.02%,p＞0.05).but percentage of the embryos developing to 4～8-cells are significantly higher in mM16 than the others(68.50%vs.58.63%,48.39%,36.86%, p＜0.01),as well as in rate of blastocyst(43.37%vs.26.85%,16.13%,7.96%,p＜0.01).3 The comparison of the development between Mâ… oocytes and Mâ…¡oocytes.A23187+6-DMAP was used to simulate oocytes and mM16 for culturing.The Mâ… oocytes dealed with CD show a better development capacity than Mâ…¡oocytes after been actived(46.25%vs.34.37%,p＜0.01).But there are no significant difference in activation rate(90.13%vs.87.64%,p＞0.05).4 The effect of different electric field strength on the result of activation with reconstituted oocytes.Mâ… spindle was put together with enucleared Mâ…¡oocyte to form a reconstituted oocytes by spindle transfor.Choosing Fusion madia that contents 0.3 mol/L mannital,0.05 mmol/LCaCl₂,0.05%BSA and 0.5 mmol/L HEPES,fixing electric field duration on 40μs,we use 4 different electric field strength(1.0 KV/cm,1.5 KV/cm,2.0 KV/cm,2.5 KV/cm)to fusion the reconstituted oocytes. When electric field strength are 1.5 KV/cm or 2.0 KV/cm,the fusion rate is significantly higher than that is on 2.5 KV/cm(15.09%,14.92%vs.6.34%,p＜0.05),and very significantly higher than that is on 1.0 KV/cm(15.09%,14.92%vs.4.61%,p＜0.01),but there are no significant difference in fusion rate between 1.5 KV/cm and 2.0 KV/cm.5 The effect of different electric field duration on the result of activation with reconstituted oocytes.Mâ… spindle was put together with enucleared Mâ…¡oocyte to form a reconstituted oocytes by spindle transfor.Choosing Fusion madia that contents 0.3 mol/L mannital,0.05 mmol/LCaCl₂,0.05%BSA and 0.5 mmol/L HEPES,fixing electric field strength on 2.0 KV/cm,we use 3different electric field duration(40μs,80μs,120μs)to fusion the reconstituted oocytes.When electric field duration are 40μs or 80μs fusion rate is high,but there are no significant difference between them.6 The activation and culture of reconstituted oocytes.Choosing Fusion madia that contents 0.3 mol/L mannital,0.05 mmol/LCaCl₂,0.05%BSA and 0.5 mmol/L HEPES,fixing electric field strength on 2.0 KV/cm and electric field duration on 40μ,we got some reconstituted oocytes fused. The activation rate of reconstituted oocytes fused is 86.29%,2-cell rate is 61.78%,4～8-cells rate is 32.88%and the rate of blastocyst is 4.17%.In conclusion:(1)mouse Mâ… oocytes dealed with CD can develop to blastocyst,and it's development capacity is better than Mâ…¡parthenogenetic embryos.(2)A23187+6-DMAP is the best choice for Mâ… oocytes activation and support a highest rate of blastocyst developing among ethanol, SrCl₂,A23187,electrical activation and A23187+6-DMAP.(3)mM16 is the best madium for Mâ… parthenogenetic embryos among mCZB,KSOM,M16 and mM16.(4)When electric field strength is 2.0 KV/cm and electric field duration is 40μs,the reconstituted oocytes got the highest fusion rate. (5)There are few reconstituted oocytes can develop to blastocyst after been activated.(6)Deal the Mâ… mouse oocytes with CD in order to inhibit the Release of the first polar body,and then activate the oocytes can get diploid parthenogenetic embryos.It's not good to access diploid parthenogenetic embryos by reconstituted oocytes because of a low efficiency.