| In the process of nuclear transfer, karyoplast and cytoplast are two important components of reconstructed embryos. Especially cytoplast, it's key to the reprogramming of karyogene and the development of embryos. At present, a lot of researchers are used second metaphase (MII) oocytes as cytoplasm. But this method request aspirating lots of cytoplasm, and also request oocytes under UV irradiation to examine enucleated whether or not complete. So it's very harmful to oocytes and the development of reconstructed embryos. Recently some people take use of activated telophase of the second meiotic division, by aspirating the second polar body and surrounding cytoplasm to enucleate nucleus (TII enucleation). This method request aspirating little cytoplasm and didn't need under UV irradiation, so its' little harmful to oocytes.Several labs have made studies on TII enucleation, but about effects of oocytes TII enucleation on pig somatic cell nuclear transfer and the development of reconstructed embryos. Meanwhile, the domestic report related to this hasn't been observed. This paper main studied and compared the device and effect of oocytes TII enucleation on pigs. On this fundamental also optimizations the procedure of use oocytes TII enucleation to product pig somatic cell nuclear transfer embryos. Provide some experiences and dates on generalization this technique product pig somatic cell nuclear transfer embryos.On the basis of nuclear transfer technique which have existed, this essay studied several factors (the procedure of oocytes preactivation, the action time of nucleus with cytoplasm after nuclear transfer and pretreatment of donor cells) that effected the development of reconstructed embryos which oocytes TII enucleation on pig somatic cell nuclear transfer take experiments.1. Selected material for pig oocytes TII preactivation: this experiment use the rate of second polar body extrusion as index, three material with different concentration were selected, exerted on maturation oocytes of pig then observe and statistic the result of preactivation. After the preactivation of ethanol with 5%,7% and 10% ,the rate of second polar body extrusion were from 20.83%(25/120) and 25.00%(30/120) to 22.50%(27/120);use concentration with 4μg/mL,6μg/mL, 8μg/mL and 10μg/mL of cycloheximide preactivation pig oocytes, after activation the rate of second polar body extrusion were 16.67% (20/120), 19.17% (23/120), 25.83% (31/120) and 20.83% (25/120); concentration with 5μmol/mL,10μmol/mL,15μmol/mL and 20μmol/mL of Calcium ionophore A23187 (CaA) preactivation pig oocytes, after activation the rate of second polar body extrusion were 54.17% (65/120),58.33% (70/120),64.17% (77/120) and 57.50%;the group of 15μmol/mL were highest, The differences of each group were not significant (p>0.05). Compared among the three materials of above, we can discover that the group of CaA with 15μmol/mL preactivation pig oocytes were highest, and the differences were very significant(P<0.01).2. The effect of different recipient cytoplasts to pig somatic cell nuclear transfer. This experiment use the cleavage of reconstructed embryo and the development rate of embryos with more than 4 cells as index, adopt oocytes which MII enucleation and TII enucleation as recipient cytoplasts, used cumulus cells that by serum starved as donor cells.Compared the effects of two differences recipient cytoplasts to pig somatic cell nuclear transfer. results demonstrated that: the cleavage rate and proportion of embryos with more than 4 cells of oocytes in MII enucleation group were both higher than those in TII enucleation group (31.67% vs 24.17%,14.17% vs 10.83%), the difference between them was significant (p<0.05).3. The effects of action time which nucleus with cytoplasm to nuclear transfer. After the reconstruction of nuclear transfer embryos was fusioned, donor cells were incubated in the oocyte cytoplasm with TII enucleation for 0h, 1h, 2h, 3h, 4h or 5h before, then activated with 1.3kv/cm, 80us and single pulse, use the cleavage of reconstructed embryo and the development rate of embryos with more than 4 cells as index, Results demonstrated that: nucleus and cytoplasm incubated for 4h group was higher than other groups, the cleavage rate was 35.83% (45/120), the cleavage rate was significant higher than other groups (22.50% 25.83%,26.67%,28.83%,30.83%, p<0.05). The embryos with more than 4 cells rate compared with 3h group difference was not significant (12.50% vs 12.50%, p>0.05). But compared with other groups the difference among them was significant (12.50% vs 4.17%,9.17%,10.00%, 7.50%,p<0.05).The results indicated that use TII enucleation oocytes as recipient cytoplasts, nucleus with cytoplasm incubated 4h then undertakes supplementary activation could elevate the development of reconstructed embryos from pig somatic cell nuclear transfer.4. The effects of different pretreatments of donor cell to nuclear transfer. This experiment adopted serum starved cells as control group, use 7% ethanol or Calcium ionophore A23187 (5μmol/mL)pretreated donor cells, after the reconstruction of nuclear transfer embryos was fusioned, nucleus with cytoplasm incubated 4h before activated. Results demonstrated that: use Calcium ionophore pretreated group the cleavage rate (37.50% vs 30.83) and embryos with more than 4 cells rate(15.83% vs 10.83)all higher than control group, the difference between them was significant (p<0.05). The cleavage rate also little higher than ethanol pretreated group (37.50% vs 34.16%, p>0.05), but embryos with more than 4 cells rate significant higher than ethanol pretreated group (15.83% vs 9.17%, p<0.05). The results indicated that, use TII enucleation oocytes as recipient cytoplasts and Calcium ionophore pretreated donor cells, then nucleus with cytoplasm incubated 4h before activated. It's profited to the development of reconstructed embryos.In conclusion, while use TII enucleation oocytes as recipient cytoplasts, the most suitable procedure is: use 15μmol/mL Calcium ionophore treated oocytes for 5min, aspirated second polar body and surrounding cytoplasm, use 5μmol/mL Calcium ionophore pretreated donor cells and subzonal injected to pig oocyte which use TII enucleationed, adopt parameter as 1.0kv/cm, 80us and single pulse fusioned mathod injected donor cells to reconstructed embryos, then nucleus with cytoplasm incubated 4h, finally use parameter as 1.3kv/cm, 80us and single pulse activated reconstructed embryos .could significantly elevate the development of reconstructed embryos for pig somatic cell nuclear transfer, simplified the operational procedure of nuclear transfer.
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