| The success of cloning animals is the great improvement for biological research. The technology of nuclear transfer is very useful in many researches, such as the gametogenesis, cell differentiation and genetic expression and regulation, et al. Cloned offspring have been produced by somatic cell nuclear transfer (SCNT) in many mammalian species. The golden hamster is an excellent model animal for many research fields. It represents an attractive species for studies ranging from reproductive physiology, oncology, genetics and virology. In order to establish animal models for human diseases using golden hamsters by somatic cell neclear transfer, this study was conducted.The activation conditions of golden hamster oocytes were examined and optimized. Golden hamster oocytes of different ages were tested with three treatments: (1) an electrical pulse ranging from 10V/mm to 600V/mm; (2) incubation for 2h to 6h in 2mM of 6-DMAP; (3) combination of electrical and 6-DMAP stimulus. Our results demonstrated that oocytes exhibited an intensity-dependent increase in cleavage from 10V/mm to 500V/mm, but younger oocytes (13.5h post hCG) could not bear pulse higher than 500V/mm; the highest cleavage rates (46%) of 15h-post-hCG oocytes were observed with 4h 6-DMAP treatment, but 6-DMAP had difficulty to induce cleavage of 17h and 19h post-hCG oocytes. Under combined treatment, the cleavage of oocytes rates reached to 96.49%.Ageing and anti-ageing of golden hamster oocytes were studied. Oocytes were collected under three different conditions: (1) oocyte direct recovery from the oviduct of hormonally treated donor at different age; (2) oocyte recovery from the oviduct of hormonally treated donor (15h post hCG) followed by 5h, 10h, 20h in vitro culture with or without cumulus cells; (3) 15h-post-hCG oocytes treated with TSA and caffeine for 5h to 20h. Then the spontaneous parthenogenetic activation, cortical granules and developmental potential of oocytes were investigated. Our results demonstrated In vivo matured oocytes were ageing with the age, and spontaneous parthenogenetic activation reached to 36.96% in oocytes of 35h post hCG; cumulus cells had little effect on ageing of in vitro-cultured oocytes; TSA and caffeine could delay the ageing process, the developmental potential of oocytes in different ages was higher than that of untreated oocytes; cortical granules stained were decreased with age.Changes in the reciprocal position of the first polar body (FPB) and chromosome set of MII oocytes of golden hamsters were examined. The oocytes were collected under three different conditions: oocytes of in vivo; oocytes of in vitro culture; oocytes of in vitro maturation; oocytes treated with colchicine and/or cytochalasin B. Then denuded oocytes were stained with Hoechst 33342 and propidium iodide and evaluated under an inverted fluorescence microscope and a confocal laser scanning microscope. Our results demonstrated that 82.10% FPBs of 13.5h-post-hCG oocytes were in the zone of 0o-30o, the change in FPBs position increased with age in in vivo-matured oocytes; cumulus cells could protect the FPBs of in vitro-cultured oocytes from degeneration, while they did not significantly affect the repositioning of FPBs; In vitro-matured oocytes aged slower, still 77.94% FPBs were in the zone of 0o-30o when they were cultured for 18 h; The oocytes could be induced cytoplasmic protrusions in colchicine, while the cytochalasin B almost had no effect on FPBs repositioning.Concentrations of colchicine and demecolcine for inducing cytoplasmic protrusion (containing chromosomes) and assisting enucleation of golden hamster oocytes were examined. Denuded oocytes of different ages were treated with different concentration of colchicine and demecolcine. Then cytoplasmic protrusions of oocytes were removed with a micromanipulation pipette. Our results demonstrated that: after 13.5-18h post hCG injection, approximately 90% of oocytes treated with 10μg/ml of colchicine for 1h had formed cytoplasmic protrusions, and some 13.5h-post-hCG oocytes could be induced enucleation (23.25%) directly as the chromosomes eliminated with the second polar body; when treated with 0.4μg/ml of demecolcine for 1h, the cytoplasmic protrusion rate of oocytes reached nearly 100%; when the cytoplasmic protrusions induced by colchicine or demecolcine were removed, assisted enucleation rates were over 80%, much more successful than 31.53% of blind enucleation.Fetuses fibroblasts and cumulus cells of golden harmsters were collected as nuclear donor cells in nuclear transfer. The 10-days fetuses were collected and digested to prepare golden harmster fibroblasts. Cumulus cells were collected when the cumulus–oocyte complexes being treated with hyaluronidase. The oocytes were enucleated and electrofused with donor cells. Then the fusion efficiency was examined 30min after treatment. Our results demonstrated that 22.14% fibroblasts and 16.59% cumulus cells were fused with enucleated oocytes.In conclusion, in this study conditions of oocytes activation and cleavage had been optimized; spontaneous parthenogenetic activation, cortical granules, developmental potential and reciprocal position of the FPB were observed in oocytes ageing process; colchicine and demecolcine were used in assisting enucleation of hamster oocytes; hamster cloned embryos were constructed by cell fusion. In one word, all of these experiments were useful to establish animal model for human diseases using golden hamsters. |
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