| The technique of interspecies somatic cell nuclear transfer, in which interspecies cloned embryos can be reconstructed by using domestic animals oocytes as nuclear recipient and endangered animal or human somatic cell as nuclear donor, can afford more opportunities in endangered animals rescue and human organ transplantation by obtaining embryonic stem cell or cloned offspring from interspecies cloned embryos which were cultured in vitro or in vivo. But the application of this technique was limited by its extremely low efficiency which maybe can be attributed to donor nucleus not fully reprogrammed by xenogenic recipient cytoplasmy. Moreover, two types of mitochondria, which were derived respectively from nuclear donor and nuclear recipient, were observed in many interspecies cloned embryos and offspring. But the factors which affected mitochondrial heteroplasmy were still unclear. The zygotic gene activation (ZGA) is the key affair during the early embryogenesis. After ZGA, many kinds of glycoprotein antibodies can be produced by zygotic genes and transported to the surface of blastomere. Therefore, analyzing the antibody on the surface of cloned embryos can be used to estimate when the ZGA happened and whether the donor nucleus were reprogrammed by the recipient cytoplasmy.In present study, intra- and inerspecies cloned embryos were constructed using in vitro matured sheep oocytes as nuclear recipient and sheep- (SFFs) or goat fetal fibroblasts (GFFs) as nuclear donor. The difference in developmental ability and developmental speed betweem intra- and interspecies cloned embryos were investigated by cultured them in vitro. Moreover, at various developmental stage (1-, 2-, 4-, 8-, 16-cell, morula and blastocyst) of interspecies cloned embryos, the percentage of two types of mtDNA and mtRNA was examined using real-time PCR and real-time reverse transcription-PCR (real-time RT-PCR). Last, at various developmental stage (2-, 4-, 8-, morula) of intra- and interspecies cloned embryos, the species-specific antibodies on the surface of cloned embryos were examined using immunocytochemistry.After 22～24 h of cultured in OM medium in vitro, the rate of matured sheep oocyte (with apparent first polar body), which were collected from slaughterhouse-derived ovaries, was 72.8%, and the rate of high qualitative oocytes (with good shape and can be used for nuclear transfer) was 70.3%. The result of development in vitro showed that the rate of intra- blastocyst (16.0%) was higher than that of interspecies blastocyst (7.4%), and this difference was mainly due to lower developmental ability (p<0.05) of intra- than that of interspecies cloned embryos during 8- to 16-cell stage and 16-cell to morula. Moreover, during 2-cell to 8-cell stage, the developmental speeds of two types of cloned embryos were same; however, during 8-cell to blastocyst stage, the developmental speed of inter- was 12～24 h slower than that of intraspecies cloned embryos.The results of examining two types of mitochondria in interspecies cloned embryos showed that at all developmental stage, the mtDNA from both GFF and sheep oocyte can coexisted in interspecies cloned embryos; in the process of 1-cell to morula stage, the copy number of two kinds of mtDNA was stable relatively; However, in the process of morula to blastocyst stage, the copy number of GFF-derived mtDNA decreased significantly from 5.6×103±0.8×103 to 6.1×102±1.5×102 (p<0.01), while the sheep oocyte-derived increased significantly from 3.2×105±1.3×105 to 5.4×106±0.4×106 (p<0.01). This contrary change in copy number resulted in the ratio of GFF-derived mtDNA to those sheep oocyte-derived decreased significantly from 2.0%±1.0% to 0.012%±0.004% (p<0.01).The results of examining two types of ND-1 mRNA in interspecies cloned embryos showed that the copy number of goat-specific ND-1 mRNA decreased from 1-cell stage (2.0×103±0.4×103) to 2-cell stage (4.8×102±1.2×102), and could not be detected from 4-cell stage onward to blastocyst stage. However, the copy number of sheep-specific ND-1 mRNA was roughly constant from 1-cell stage (6.9×103±0.6×103) to 8-cell stage(7.6×103±0.9×103), increased gradually at 16-cell stage (2.8×104±0.7×104) and sharply at morula stage (2.4×105±0.4×105) and blastocyst stage (8.5×106±1.4×106). This resulted in declined ratio of goat-specific ND-1 mRNA copy number to sheep-specific ND-1 mRNA copy number during the embryogenesis of goat-sheep cloned embryos. According the results above, we strongly argued a mechanism, that is donor-cell-derived mitochondria were degraded for the depression of bioenergetic functions, and then selectively eliminated during the embryogenesis of interspecies cloned embryos.The results of examing the antibody on the surface of intra- and interspecies cloned embryos showed that at the stage of 2-, 4-, 8-cell of intraspecies cloned embryos, a small amount of sheep-specific antibody can be detected on the surface of blastomere, and its quantity increased significantly at the stage of morual. This indicated that the ZGA was beginning in the process of 8-cell to morula of intraspecies cloned embryos. At the stages of 2-, 4-cell of interspecies cloned embryos, a small amount of sheep-specific antibody also can be detected on the surface of blastomere; however, no goat-specific antibody can be detected. At the stages of 8-cell and morula of interspecies cloned embryos, sheep-specific antibody can hardly be detected on the surface of blastomere; whereas goat-specific antibody increased significantly. This result indicated that the ZGA was also beginning in the process of 8-cell to morula of goat-sheep interspecies cloned embryos, and sheep oocytes have the ability to reprogram the goat somatic cell nucleus.
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