Study On The Interspecific Nuclear Transfer Of Przewalski’s Gazelle And Bovine

Przewalski’s gazelle (Procapra przewalskii), also known as Platts antelope, is one of the most endangered species which is unique to China in the world; it is Artiodactyla, Bovidae, antelope subfamily, gazelle genus, has historically distributed in Inner Mongolia, Gansu, Ningxia and Qinghai. It has the similar shape and size to the Tibetan gazelle, and has been regarded as a subspecies of the Tibetan gazelle, really an independent species. Studies of gazelle-bovine interspecific cloning r are important both in the trials for protection of the endangered species, and might be a model in the researches of nuclear-cytoplast interaction. In this study, the Przewalski’s gazelle adult cells were used as as nuclear donors, and bovine oocytes as recipient cytoplasts to investigate the feasibility of interspecies cloning between Przewalski’s gazelle and bovine. We collected a piece of about0.5cm2ear tissue from a healthy adult Przewalski’s gazelle from the Qinghai Wild Zoo. After in vitro culture, the fibroblast cells were obtained. We did a systematic study of the cells including the cell growth, growth curve, karyotype analysis, cell cycle, and the optimal transfection conditions. We collected the bovine ovaries from slaughterhouse, obtained the cumulus-oocyte complexes. After in vitro culture, we obtained the matured oocytes. We used histone deacetylase inhibitors for treatment of embryos, reverse nuclear transfer protocols to improve the in vitro development. We have the results as follows:The Przewalski’s gazelle ear fibroblast cells are in typical spindle, triangle or multangular. The recovery and vitality of the frozen cells have no obvious changes. When the cell confluences reached to50-60%,70-80%,95-100%the proportions of cells at G0/G1stages were77.03%,77.51%and92.25%, respectively. The proportion of cells at G0/G1stages was with no difference between cells at95-100%confluence and cells treated with0.5%serum starving. Therefore, cells at95-100%confluence instead of serum starvation were used as nuclear donors during nuclear transfer. Chromosomal analysis of the cultured cells showed that the number of chromosomes is60. Cells less than8passages maintained a normal chromosome diploidy. With the increase of passages cell diploid composition gradually decreased. In this study, five experiments were designed to examine the developmental potential of interspecific gazelle-cattle reconstructed embryos.(1) The gazelle-bovine reconstructed embryos were treated with a deacetylase inhibitor valproic acid (VPA) for different time and at different concentrations. The result showed that VPA at0.5mM treated the cloned embryos for24h significantly increased the8-16-cell stage embryo development when compared to the control (61.9%vs33.8%). However, the morula (1.2%Vs1.6%, P>0.05) and blastocyst (0.8%vs1.6%, P>0.05) development did not improve.(2) When the Oct-4-eGFP confected cells were used as donors and the cloned embryos were treated with0.5mM VPA for24h, The gazelle-bovine cloned embryos were with similar cleavage (78.4%vs71.4%),8-16stage (62.1%vs58.5%), morula (1.3%vs1.9%) and blastocyst (0.7%vs1.3%) development to the control.(3) When TSA at30nM was used to treat the gazelle-bovine embryos the blastocyst development was significantly higher than the control group (3.6%/3.0%vs0.8%/0.9%, P<0.05). Three gazelle-bovine blastocysts obtained which all expressed Oct-4-eGFP.(4) In order to improve the gazelle-bovine cloned embryo development a modified reverse nuclear transfer protocol was used to reconstruct the embryos. The results suggested that there were no significant differences in The cleavage (61.1%vs63.5%) and8-16-stage embryo development,(45.6%vs49.0%, P>0.05) of the gazelle-bovine clones were with no differences to the control.(5) To examine if incomplete reprogramming of donor cells occurred in the gazelle-bovine cloned embryos microarray analyses of8-to16-cell interspecific and bovin-bovine cloned embryos at same stage, the donor cells were conducted. The results indicated that643genes associated with nuclear reprogramming were activated in gazelle-cattle embryos, while1527genes were activated in bovine-bovine clones.There were1010genes which were special to the gazelle cells did not silence and still active in the cloned embryos. These results suggested that gazelle donor cells have not been reprogrammed during the cloned embryo development. The maternal RNA degradation level in gazelle-cattle cloned embryos was less than the bovine-bovine same stage clones.In conclusion, the present study showed that the Przewalski’s gazelle somatic cells could support the gazelle-cattle cloned embryo development to blastocyst stage although with extreme low efficiency. Treatment of gazelle-cattle reconstructed embryos with VPA did not improve blastocyst development. Treatment of embryos with TSA increased gazelle-cattle cloned embryo development. Oct4, the reprogramming factor confected cells, and reverse nuclear transfer protocol did not improve embryo development. Microarray analyses of the gazelle-cattle embryos suggested that many nuclear reprogramming associated genes have not been activated and obviously incomplete reprogramming occurred.