Research On Parthenogenetic Development Of Mouse Recombinant Oocyte Which Was Removed Sperm Nucleus After Engraft PB1

In 2007,Kitai Kim,etc.used the cytochalasin D to deal with the first meiotic metaphase(MI) mouse oocytes in order to prevent the extrusion of the first polar body(PB1),after mature cultivation and parthenogenetic activation,diploidy is maintained,and the result that pES(pES line)can be isolated which similar to those embryonic stem using for therapeutic cloning.With the detection of this line by the pES oocyte donor shows that the gene locus of the major histocompatibility complex (MHC) are exactly the same,this method can be expect to substitute the somatic cell cloning embryonic stem cells for Histiooytic replacement therapy.The somatic cells from the sick animals are differentiated cells implied the mutant gene,not only generate a low rate of cloned blastocysts but also the ES cells which contains mutant gene have quality problem.Nevertheless,this new parthenogenetic embryonic stem cells with heterozygous diploid karyotype derived from the germ cells which still maintain telomerase activation and not yet via the individual differentiation. Theoretically,this ES cells does not exist the mutant gene,so this pES lines have a broader prospects for medical applications.In the above-mentioned study we find that the ratio of the diploid parthenogenetic blastocysts is low,the reason may ascribe to the maturity of the immature MI period oocyte,drug non-physiologic poisoning effect to the oocyte,mature cultivation times in vitro after preventing the extrusion of the first polar body and the activating method,all this problems need to further in-depth study.However,from the investigation above-mentioned we demonstrated that in the condition of preventing the extrusion of the first polar body,PB1 has the same function as the oocyte chromosome,assume that in a short time during the extrusion of the PB1, transfer the PB1 to the newborn MII oocyte via nuclear transplantation technology,and then the reconstruction oocytes were activated by sperms.Moreover,because of the male pronucleus is larger than the female pronucleus,when we analyze the development of male pronucleus and the female pronucleus,to observe the condition of the reconstruction oocytes and the development of the parthenogenetic embryo which has engrafted the PB1 and enucleated the male pronucleus.This experimental design depleted the cleavage inhibition which the chemical materials effect on the immature oocytes and the unphysiologic action of the parthenogenetic activation.In this experiment,we use the natural developmental stage oocyte which can be controled, implantation technique,and male/female pronucleus which can be discrimination to observe the merogenesis of the nonage oocytes,using the sperm activation,according to the reproduction rules in order to obtain diploid karyotype and to simplify the generation of parthenoblastular,aiming to explore the new pathway for mouse therapeutic embryonic stem cell.This reaserch base on the experiment above,first,investigate in the precisely time of when the first polar body was extrude,and an easy method to obtain a devil of highly activity PB1;By enucleation,polar body transplantation and adosculation technique to observe the development of the reconstructed embryo;Adosculation on the basis of engrafting the PB1 to enucleate oocyte,when the male/female pronucleus was formed,remove the male pronucleus to investgate the development andmerogensis of the Heterozygous diploid oocytes.In this study,nuclear transplantation, adosculation,oocyte matural cultivation,Parthenogenetic activation and karyotype analysis technique were used to explore the ways to generate a new type of Heterozygous diploid parthenogenetic embryos and to increase the efficiency.Test are summarized as follows:1 Research on the time of PB1 discharges by manual controlIn this experiment,pregnant mare serum gonadotrophin(PMSG)and human chorionic gonadotrophin(hCG)were used to stimulate super ovulation of Kunming of 6-8 weeks old.Ten hours after injection of hCG.Cumulus ooeyte complexes(COCs)were collected from each group of mice every 1 hours.The number of ooeyte and the shape ofthe first polar body(PB1)were checked,and morphological classes of PB1 were counted.We carry on the graduation of PB1 according to this experiment self-restraint a set of polocyte graduation chart(Figure 3-1),The aim of this experiment is to determine best acquisition just discharged time of ovocyte's PB1.The result showed that 12,13,14 honrs after hCG treatment the PbI had the higher rate, and the 12h is the best rigor of all groups(60.87%) ofⅠandⅡgrade account the number of PB1),moreover,the difference of this three time is not remarkable (P＞0.05);12h(except 13h and 14h) compares the even difference with other each group to be remarkable(P＜0.05);15h and 16h difference not remarkable speaking ofⅠ,Ⅱgrade of PB1 rate(P＜0.05).Therefore,the ooeyte of 12h after hCG injectionto just discharge the PB1 to enter the oviduct from the ovary,we should choose this time's ovocyte and ovocyte's PB1 as the experimental subject when carries on the polocyte reorganization experiment.2 Research on collecting high-activity PB1 effectivelyIn this experiment.we advanced the process of eliminating transparent belt on the ovocyte of including the PB1 by catenin enzyme of different concentration(1.5%,1%,0.5%) and time.Then the PB1 was observed Trypan hlue staining.The aim of this experiment is to elect efficacious device of separation of PB1 from mouse oocytes.The result showed that 1%catenin enzyme and affecting 5 minutes on oocytes obtained high-activity PB1(80.00%),which rate to be higher than other each group to achieve, and difference remarkable(P＜0.05).Therefore,we can be able to obtain many high-activity PB1 when take PB1 as the material of tests by 1%catenin enzyme and affecting 5 minutes.3 research on Fertilization in vitro of the mouse oocytes recombined by first polar bodiesCollecting PbⅠof oocytes was in 12h after superovulation,then the best PB1 was obtained by 1%pronase treatment in 5 min,and recombined another oocytes whose nuclei was enucleated by micromanipulation,and the recombined oocytes were fertilized and cultured in vitro in order to observe its ability of furtherDevelopment and established foundation after the following experiment.(1) research on fertility In vitro for different time during the maturation culture culture In vitro of the mouse oocytes recombined by PB1PbⅠwere injected into the oocyte whose nuclei was enucleated and the recombined oocytes were fertilized and cultured in vitro in order to observe its ability of further development and choose the best culture stage.the results show that 2-cells rate was the highest(16.67%) after culturing 3h in vivo.oocytes recombined between 3h and 4h was similar(P＞0.05),but was not similar among others.Apart from 3h and 4h was similar(P＞0.05),the others were not similar(P＜0.05).In conclusion,the highest 2-cells rate was obained(16.67%) by culturing 3h of oocytes recombined in vivo(2)research on on emergence time of female pronucleus and male pronucleus after Fertilization in vitro of the mouse oocytes recombinedThe recombined oocytes by 3h in vitro culture was added to sperm drops in 6h,then transfered and cultured.Observing fertilized egg each 2h and Recording condition in different pronucleus stages.The results show that female pronucleus and male pronucleus rates were the most hightest by 6h culure(22.58%),which was not similar than others(P＜0.05).In conclusion,the highest female pronucleus andmale pronucleus were obtained by 6h cultured that was the optim period for operating pronucleus by micromanipulation(3) research on pronucleus embryos culture in vivo63 pronucleus embryos was obtained and cultured in vivo from(1),(2) culture media:①②③Observing the development and results of pronucleus embryos in order to explore culture condions and optimum.The results show that Only mM16 could support embryos development after 4cell.(4.54%) it was not similarthan others (P＜0.05).M16 and KSOM could not overcome embryo development retardation after 2-cell stage.In conclusion,MM16 media is the most appropriate for in vitro culture of early embryos of Kunming mice.4 research on the development of the heterozygous diploid oocyte witch contain the PB1 with sperm nucleus removedfluorescence staining to the oocytes contain PB1 which were collected from the oviducts of adult mice 12h after injection of human chorionic gonadotropin,which was given 48 h after pregnant mare serum gonadotropin injection.Putting the discharged PB1 into the place where properly next to the proocyte nuclei by micromanipulation, incubation for 6h after adosculation,and then transfer to the embryo culture medium in order to observe the formation of the female and male pronucleus and the expulsion of the PB2.Enucleating the male pronucleus from the embryo and then culture the parthenogenetic embryo in vitro for research.This experiment aimed at investigating whether the diploid formulated by female pronucleus activated by sperm and the female pronucleus of its own can support the parthenogenetic embryo growth.The results showed that:MII stage oocyte via artificial engraft PB1 can finish the second meiosis and discharge the second polar body after sperm activation,manual removal of male pronucleus can form reconstruction diploid karyotype parthenogenetic embryos,the rate of 2-cell was 2.32%.further cultivante the 2-cell,this embryo can pass through the 4-cell and continue develop.Conclusion:(1) the oocyte with PB1 obtain from the oviducts of adult mice 12h after injection of human chorionic gonadotropin,which was given 48h after pregnant marc serum gonadotropin injection.At that time PB1 was just discharge by oocyte,the development process of the polar body's genomic is the most coincidence with the development of oocyte cytoplasm,this period is considered to be the very time for the polar body recombination.(2) It was found that the embryo had higher gymnoblast rate after 0.5%protease treated,so we can separate single highly activity polar body. (3)When we recombinate the genome of newly born polar body to the newly born MII stage enucleate oocyte and then adosculation,the recombinate oocyte was fertilized after culture in vitro for 3h can gain a higher fertility rate(16.67%),this data shows that the genome of the PB1 can support the embryonic development,provid theory and technique directions for the parthenogenetic development of the sperm nucleus removed oocyte.(4) MII stage oocyte via artificial engraft PB1 can finish the second meiosis and discharge the second polar body after sperm activation,manual removal of male pronucleus can form reconstruction diploid karyotype parthenogenetic embryos.