Detection Of Spindle Morphology And Associated Proteins Of Human Somatic Cell Nuclear Transfer Embryos

Background:Nuclear Mitotic Apparatus Protein (NuMA),Kinesin spindle kinesin (Eg5) andγ-tubulin play important role in the human embryonic fibroblasts cell multiplioation, oocytes maturation, preimplantation development of human somatic cell nuclear transfer (SCNT) cloned embryos. NuMA is one of macromolecular nucleoprotein that molecular weight is 236-240kDa. NuMA can initiate spindle microtubule assembly and tether spindle microtubules to their poles. Eg5 is one of the members of Kinesin family who mediates transportation and location of NuMA in spindle poles, and guides to gather tubulin by distal end of NuMA.γ-tubulin is a representative protein in pericentriolar material with the function of initiating formation of microtubule crystal nucleus and regulating the spindle assembly. It has been proved that both the absence and over-expression of above proteins can lead to abnormal spindle morphology, and further lead to abnormal cell division. In the study of SCNT in nonhuman primates, Simerly found that the SCNT embryos had the absence of NuMA, distribution irregularly of kinesin EG5 as well as abnormal spindle morphology. So he considered that the failure of non-human primate SCNT experiment was mainly caused by the loss of NuMA and Eg5 kinesin during the process of oocyte enucleation. But the subsequent studies showed that the most of the SCNT embryos in monkey had normal distribution of NuMA. Up to now, it has not been reported that how the spindle and associated proteins distribute in hunman oocytes at different developmental stages and in human SCNT embryos.Objective:To search for the distribution of spindle and its associated proteins in human embryonic fibroblasts, human oocytes at various maturation stages and human somatic cell nuclear transfer embryos. And to explore whether the abnormal expression of spindle and associated proteins during the enucleation of human oocytes can cause the low efficiency of human somatic cell nuclear transfer. According to the studies, we expect to help to solve the technical obstacles of human somatic cell nuclear transfer and accelerate the applications of human therapeutic cloning.Methods:We observed the distribution of the spindle and its associated proteins in human embryonic fibroblasts, human oocytes at various maturation stages and human nuclear transfer embryos by indirect immunofluorescence staining under a normal fluorescence microscope or laser confocal microscopy.Germinal vesicle (Gv), the human oocytes of MI derived from the discarded oocytes after the patients undergoing infertility treatment in the ICSI programs at the CITIC-XIANGYA Reproductive & Genetic Hospital; Most of the MⅡoocytes were in vitro maturation cultured by the abandoned oocytes from the patients who undergoed infertility treatment in the ICSI programs, others were donated by women with polycystic ovary (PCOS) who undergoed the treatment of puncture. Human embryonic fibroblast cells were derived from the National Engineering and Research Center of Human Stem Cells. We used the human embryonic fibroblast cells as donor cells and removed the nuclear of human MⅡoocytes under the polarized light microscope. The donor cells were injected to cell cytoplasm of the enucleated MⅡoocytes by using an injection pipette [inner diameter (I.D.) 8-10μm]. The human somatic cells nuclear transfer embryos were cultured by G1.5/G2.5 sequentially way after in vitro chemical activation (A23187 combined 6-DMAP).Results:1. During the interphase of human embryonic fibroblasts and the Gv stage of human oocytes, NuMA was detected in the GV bubble except the nucleolus,α-tubulin was distributed in the cytoplasm in a characteristic reticular shape. During both the meiotic MI and MⅡstages of human oocytes and the metaphase of human embryonic fibroblasts cells, NuMA appeared in the spindle poles.2. According to immunofluorescence staining, Eg5 was distributed in karyoplasms andγ-tubulin was apeared in the nuclear membrane around presenting an aggregation of point-like distribution during the mitotic interphase of human embryonic fibroblast. Eg5 and γ-tubulin were concentrated in the spindle poles during metaphase, Eg5 also appeared in the cytoplasm and the central of spindle.3. We didn't detect NuMA andα-tubulin in enucleated oocytes cytoplasm. However, the removed cytoplast contained normal bipolar spindle poles with associated NuMA.2 hours After injection of the donor cell (before the activation of nuclear transfer embryos), premature chromosome condensation (PCC) appeared in donor cell nuclei with a temporary spindle-like structure, and NuMA was accumulated at the spindle poles. At the pronuclear formation period, NuMA was detected in the karyoplasms except of the nucleolus; whileα-tubulin was appeared in the cytoplasm. At the first time mitosis metaphase of human nuclear transfer embryos, NuMA was mainly distributed in the spindle poles. NuMA was detected in interphase nucleus of human embryos developmentally blocked.Conclusion:NuMA was distributed in the nuclear of human embryonic fibroblasts at interphase stage and human oocytes at Gv stage, as well as in the spindle poles of human embryonic fibroblasts at metaphase stage and oocytes at MI and MⅡstages. At different developmental stages of human SCNT embryos, NuMA can be detected with different distribution. At interphase stage, NuMA was distributed in the karyoplasms,α-tubulin distributed in the cytoplasm. At metaphase stage, NuMA was accumulated at spindle poles, normal spindle and NuMA expression were found in human SCNT embryos at different developmental stages. Our results show that donor cell nuclei contain NuMA that contributed to the SCNT embryos and maintenance of normal spindle morphology. Accroding to the above experiments, it is almost imposssible that the loss of NuMA in the process of nuclear transfer which caused the nuclear transfer embryos poor development.