| Traditionally, Somatic nuclear transfer in mammal is completed by micromanipulation. But micromanipulation is evidently damaging to cytoplast cytoskelton, causing the lost of some important factors involved in programming of donor karyoplast, reducing the volume of cytoplasm. Alternatively, induced enucleation is carried out with agents disturbing the meiosis or inhibiting protein synthesis of oocyte during polar body emites. Consequently all chromatin are extruded as the form of polar body. Here, we used mouse oocytes enucleated by chemical induction as recipients in mouse somatic cloning. Recovery of microtubule organizing centers in enucleated oocytes and condensation of chromosome and organization of spindle in reconstructed embryos were analysed.A non-invasive method of enucleation mouse oocytes has been developed. Firstly, the progression of mouse oocytes in meitosis I was ascertained by immunofluoresence technique. Consequently Cumulus-oocytes complexes were in the stage of germinal vesicle when isolated from the antral follicles. Within 2h isolation from the follicle 100% of oocytes underwent germinal vesicle breakdown and both the nucleolus and the nuclear envelope disappeared. After culturing for 6 h, prometaphase occurred in 90% oocytes. By 8 h of in vitro maturation 33.3% oocytes were at the stage of MI. Anaphase I is most apparent at approximate 12h following isolation with number of 71.4%. At the same time, telophase I occurred in 28.6% of oocytes. After 15h of maturation. 98% of oocytes released the first polar body and thus the first meiosis of mouse oocytes ended. Secondly, Strong chromosome to chromosome binding was induced by culturing pro-MI oocytes in demecolcine supplemented medium. Subsequent expulsion of entirely chromosome complex during polar body extrusion was facilitated by exposing the demecolcine treated oocytes to a combination of cytoheximide and demecolcine. This simple two-step chemical enucleation procedure yields fully enucleated mouse oocytes in 85.6% of cumulus-oocytes complexes and 56% of natural oocytes, significantly different (p < 0.05). Since the enucleation rate of cumulus-oodles complexes is equivalent to that by micromanipulation, induced enucleation will greatly improve the whole efficiency in mouse somatic nulear transfer.The preimplantation embryos of several mammalian species undergo blocks in development in vitro. The stage when the block occurs has been correlated to the time the embryonic genome takes over control of development from the maternal genome, which occurs in mouse at the 2-cell stage. The embryos of BALB/CxICR Fl mouse show 2-cell block in vitro and the effects of several culture media on overcoming the 2-cell block in embryos development were studied. M16(Sigma) , in which 96.2% of 1-cell embryos developed to 4-cell stage, were shown to be effective in overcoming the 2-cell block. The modified M16 added with essential amino acid (1%), non-essential amino acid (1%) and glucose significantly improved the developmental rate such as morula rate> blastocyst rate and inhatching rate after the 4-cell stage embryos were removed into the medium. We conclude that the amino acid and glucose may promote the lately development of mouse embryos. Birth of mouse offspring after transferring of embryos in vitro cultured further demonstrated the availability of theculture system of mouse embryos.Since the success of Dolly, the first cloned sheep with the adult somatic cells as karyoplast donor, new approaches have been developed for nuclear transfer technology. Here we describe a handmade cloning method which combines the chemical induced enuleation and zona-free technology in embryo culture. Enuleated oocytes were derived by exposing the oocytes to demecolcine and cytoheximide supplemented mdium sequently and its chromosome was depleted to the first polar body. Then the zona and polar body of oocytes treated with drug were removed by transferring into the M2 containing 0.5% protease. The mouse fetal fibroblast cells were glued to the oocytes memb...
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