SU9516, A New CDK Inhibitor, Induce Tetraploid Blastocyst In Porcine Parthenogenetic Activation Embryo

Tetraploid embryos have been widely used in animal reproduction including production mice from embryonic stem cells (ESCs) and tetraploid:diploid (4n:2n) chimeras for rescuing extraembryonic defects. There are many methods to produce tetraploid embryos by different principles. But the different producing methods have different degrees of defects. So we need to develop new methods for the preparation of tetraploid. The objective of this study was to use SU9516, a CDK inhibitor, on inducing tetraploid blastocyst in porcine PA embryo in high efficiently. and in vitro development of pig parthenogenetic activation (PA) embryos by inhibit MPF. The SU9516 can induce tetraploid blastocyst by inhibiting the second polar body and preventing the duplication of centrosome. SU9516 is a new CDK inhibitor that an important part of MPF. So SU9516 can inhibit the expression level of MPF in the process of development of oocytes, and make the oocytes come across MIl retardation.1. SU9516 can product procine PA tetraploid blastocyst.Experiment 1:Followed porcine embryos were addressed with electrical, porcine PA embryos were cultivated in IVC medium including different concentration SU9516. The result show that Porcine PA embryos treated with 10 μM SU9516 for 4h had a greater rate of blastocyst formation compared with CB or embryos treated with 1 μM,5 μM or 100 μM SU9516 (47.5% vs. 26.2%,28.9%,40.6%, and 25.8%, P< 0.05). It examined the in vitro developmental competence of pig PA embryos incubated in 10 μM SU9516 for various intervals after electrical activation. Result indicates that PA embryos treated for 4 h had higher rates of blastocyst formation than those treated for 2,6 or 8 h (47.5%vs.39.0%,33.7%, and 39.1%, P< 0.05).Experiment 2:The situation of second polar body expel from porcine PA embryos under the action of 10 μM SU9516 or 5ug/ml CB after 2h of electrical activation were observed. The result shows that 10 μM SU9516 group or 5ug/ml group can notably inhibit the second polar discharge compared with untreated group (14.0%,13.8% vs.78.8% P< 0.05). In order to determine the number of sets of chromosomes. Blastocysts induced from 10μM SU9516 group or 5ug/ml CB group were karyotype analysis. The result showed that SU9516 group notably high CB group in inducing tetraploid blastocyst(55.8% vs.14.0 P< 0.05).Experiment 3:In order to further confirm the number of sets of in different treatment PA blastocyst groups. Using Hoechest 33342 to stain the cell number in the different treatment groups showed that the mean cell number in SU9516 group blastocysts was significantly lower than that of CB group and 6-DMAP group (34.4±17.5b vs.46.4±10.5a,45.1±10.6a)2. SU9516 can improve the development of porcine PA embryo in vitro though decreasing the expression of MPFExperiment 1:PA embryos treated in 10 μM SU9516 for 4h to observe the level of MPF. The result of MPF level were significantly lower in PA embryos induced by SU9516 than CB group (103.2 pg/ml vs.131 pg/ml). We can concluded that SU9516 can improve the development of pig PA embryos by deduce the activity of MPF.Conclusion:In this study demonstrates that SU9516 can induce tetraploid blastocyst in high efficiency and significantly influence the in vitro developmental competence of porcine PA embryos by deduce the level of MPF at 4h for 10μM SU9516.