Study On Bovine Parthenogenetic And Cloning Polyploid Embryos

The efficiency of producing animals through somatic cell nuclear transfer (SCNT) in mammalian is still low. Even when fetuses develop to term, the phenomena such as large offspring and abnormal placenta development is often present, which affect animal cloning efficiengy. Polyploid cells can give rise to extraembryonic tissues. Transfer of aggregated embryos of polyploid and cloned embryos can improve animal producing efficiency. Therefore, obtaining polyploid embryos is very important for further study on developmental mechanism of cloned animals. In this research, techniques such as immunfluorescent staining, drug treatment, parthenogenetic activation, reverse nuclear transfer were employed to study the methods of getting parthenogenetic and cloned polyploid embryos and to analyze the effect of bovine oocyte nuclear on somatic cell nuclear reprogramming. This research will provide experimental basis for perfection of SCNT technique.1. Nuclear maturation process and dynamic changes of cytoskeleton and chromosome of oocytes from bovine, goat and ovine during in vitro maturationIn this experiment, the bovine, goat and sheep oocytes matured in vitro for different time were collected, respectively. The nuclear maturation process and dynamic changes of cytoskeleton and chromosome were analyzed by immunofluorescent staining. The nuclear maturation process of bovine and goat oocytes exhibited similar characteristics, with GVBD initiate 4h after beginning of maturation.50% -60% of oocytes arrived at MⅠat 12h. Peak of AⅠ/TⅠappeared at 16h (42% ), then most of the oocytes begin to expel polar body and enter MⅡNuclear maturation process of sheep oocytes was slower than bovine and goat. The peak of AⅠ/TⅠappeared at 20h after IVM. The dynamic changes of cytoskeleton and chromosome of oocytes from three domestic animals were similar, but more irregular spindle structure tend to occur in sheep oocytes.2. Effects of cytochalasin B on in vitro maturation of bovine oocytes and formation of diploid chromosome oocytesThis study was designed to investigate the effects of cytochalasin B (CB) on bovine oocyte maturation, and to induce the formation of diploid oocytes. The results showed that:(1) Incubation of oocytes in CB at≥2.0μg/mL concentrations for 24 h significantly decreased oocyte maturation and the matured oocytes'haploid composition. Over 50% of the CB-treated oocytes did not expel PB1 (non-PB1), and 50-65% of the non-PB1 oocytes contained 2n (60) chromosomes. (2) Pretreatment of oocytes with CB at concentrations of 7.5 and 15μg/mL for 6-8h did not affect the maturation rates compared to the control oocytes (about 80% vs. 83% ), but prolonged pretreating time to 10h significantly decreased the oocyte maturation(60%). (3) Oocytes were cultured for the first 10h and then transferred to 3.0-7.5μg/mL CB-containing medium and cultured to 24h (post-treatment). The results showed that 58.5% -92.8% of the oocytes could not expel PB1. (4) Prolonging the post-treatment of oocytes in CB at 7.5μg/mL to 30h,36h, and 48h, respectively resulted in significantly decreased AI/TI oocytes to 1.9-6.4% compared to 24h treatment group (15.3% ) and increased the number of oocytes containing big spindles comprised of 60 chromosomes.These results indicated that CB disfigures spindle structure, inhibits PB1 extrusion, decreases oocyte maturation, and results in formation of diploid (2n or 4c) oocytes.3. Effect of Cytochalasin B treatment of bovine oocytes on tetraploid parthenogenetic embryo developmentThis study was designed to induce the formation of tetraploid embryos. The oocytes were matured in CB-containing media at different concentrations for 24h, or pretreated with CB for 6-10h then into normal culture media up to 24h, or pre-culture in normal media for 10h then post-treatment of oocytes with CB to 24h. The results showed that:(1) The parthenogenetic blastocysts (25-28% ) derived from the non-PB 1 oocytes of posttreatment group was significantly higher than that from whole period treatment (5% -15% ), pretreatment (7% -16% ), and the control oocytes (12-16% ). (2) Cytogenetic analysis of the embryos derived from CB-treated non-PB 1 oocytes resulted in 74% of the one-cell stage embryos being 4n=120 chromosomes,82% of two-cell stage embryos contained 4n chromosomes in each blastomere, and 75% of the blastocysts were tetraploidy (4n=120). (3) The stopped uncleaved one-cell embryos showed an amazing phenomenon of over 15% of them containing extra chromosomes, which suggested multiple DNA duplication occurred within 40 h after activation.In conclusion, CB treatment results in formation of diploid (2n or 4c) oocytes. The diploid oocytes resulted in a higher development of tetraploid embryos.4. Effects of the presence of oocyte nuclei on the donor cell nuclear remodelingand polyploid embryo developmentIn this experiment, the non-enucleated oocytes were used as recipient to reconstruct embryos and examined the effects of the presence of oocyte nuclei on the donor cell nuclear remodeling, including premature chromosome condensation (PCC) and DNA configuration, and subsequent embryo development. The results showed that:(1) the presence of oocyte MⅡspindles was more likely to induce donor cell PCC. (2) The positional relationship between the fused donor cell and the oocyte metaphase spindle had an effect on oocyte PB2 extrusion. When the fused donor cell was widely separated from the MⅡspindle,94.4% of the reconstructed oocytes expelled a PB2. When the donor cell was fused adjacently to the MⅡspindle, almost all of the reconstructed oocytes did not expel the PB2; the majority (67.9% ) formed a very large M-phase spindle in which the oocyte and the donor cell chromosomes merged. (3) After activation, the oocyte and donor nuclei exhibited a variety of pronuclear patterns and asynchronous development. (4) The embryos reconstituted with nonenucleated oocytes resulted in a similar cleavage rate as observed in the control embryos reconstituted with enucleated oocytes. Blastocyst developmental rates were no different between nonenucleated and enucleated cloned embryos; however, the development rates from early to hatching blastocysts significantly decreased in the nonenucleation group compared to enucleation controls (0 vs.23.1% ; 27.5 vs.67.8%), regardless with either cumulus cells or fibroblasts as donor cells. (5) All nonenucleated oocyte-derived blastocysts contained mixed polyploidy with a variety of compositions that included 2n/4n, 2n/6n,2n/8n, and 2n/4n/8n; The blastocyst cell number in the nonnucleated groups (about 60 cells) was significantly lower than the enucleated control groups (around 90 cells). (6) The in vitro developmental data of embryos produced by two reverse nuclear transfer protocols showed that development of the RNT-1h group was much higher than the RNT-3h group (47.2 vs.20% , p<0.01). The enucleation 3h after nuclear transfer induced abnormal DNA configuration.We conclude that oocyte MⅡspindles induce donor cell PCC, the developmental capacity of cloned embryos reconstituted with nonenucleated oocytes is inferior to those with enucleated oocytes, and that all such derived blastocysts are polyploidy.