The Studies On Expression Of GRIM-19in Preimplantation Embryo Of Mice And Its Location

PurposesGRIM-19is one of the members of the family GRIMs, which belongs to IFN/RA induced cell death regulators. GRIM-19is involved in the process of regulation in proliferation, apoptosis of cells and its expression decreased or mutation can lead to abnormal proliferation and malignant transformation of cells.During the early development of embryo, the number of mitochondria, the activity and location undergo a series of changes. During this period, little changes of mitochondria’s function may be directly influence the proliferation and growth of preimplantation embryo. As the basic functional unit of mitochondrial complex I, GRIM-19is of vital importance in the process of mitochondrial I in breath, so its deletion and abnormal expression will lead to abnormal embryonic development. But it shows that there is no research which reported the expression and location of GRIM-19in preimplantation embryos so far. During the early development of embryo, the action and mechanism of GRIM-19is still not clear too. So, in this research we studied the function of GRIM-19in preimplantation embryo of mice via detecting the expression and distribution of GRIM-19, which provide a new idea to further explore the developmental potential and quality evaluation method of embryo.Methods1The collection of superovulation and eggsIn this experiment, ten of sexual maturity female mice were cultured carefully. Each of them was abdominally injected with10IU human menopausal gonadotropin (HMG) on the first day, and after48h each of them was abdominal injected with10IU human chorionic gonadatropin (HCG). One female mice and one male mice were both placed together in one cage for fertilization. Fertilized ovums were collected the next day (24h after HCG injection) from uterine tube of females possessing a vaginal plug. After treated with HGG females were killed and we removed the oocytes. After that, the oocytes and embryos were transferred to a dish to culture with400μl G1medium (which already balanced in in37℃cubator) covering with mineral oil, at37℃, in a humidified atmosphere of6%CO2in air. And we collected2-cell,4-cell,8-cell, morula and blastocyst embryos after2days every10h after HCG, respectively. At the same time, female mice treated with HCG were sacrificed and removed the mature oocytes.2Detection of GRIM-19expression in mice embryos by immunofluorescenceThe embryo cells were collected and washed2-3times with PBS. Adding freshly prepared4%paraformaldehyde cells were fixed15min at room temperature and washed3-4times with PBS. They were placed in room temperature for8min in PBS, then closed1hours containing10%of goat serum PBS. Cells were washed2times by PBS, then adding to100μl antibody (1:100dilution) putted into the wet box at room temperature incubation1H or4℃overnight. Cells were washed3times by PBS and added to200μL2antibody (1:100dilution) at room temperature incubation1H. We take DAPI and covered the film with DAPI, then washed3times by PBS.15μl resistant quencher was added to slide treated in poly lazy ammonia acid, then covered with cover glass and coated with Nail Polish. We observed the expression and location of GRIM-19protein in each stage of mice embryos by Zeiss confocal laser scanning microscopy with543nm wavelength of the exciting light.3Analysis of ΔΨm in blastocystAdded JC-1working liquid according to1:1to place in incubator37℃with6%of carbon dioxide, The blastocysts were stained30min. After washed several times the blastocysts were observed under fluorescence microscope respectively in green fluorescence and red fluorescence and obtained images. We measured the same blastocysts of red and green fluorescence intensity values and then obtained their ratios. After repeated many times, we compared and analysised the ΔΨm of different phases of blastocysts.ResultsUsing nuclear staining and immunohistochemistry, the expression profile of GRIM-19gene was observed in oocytes,2cells,4cells,8cells, morula under confocal laser microscopy, expression in the blastocysts. It was proved that GRIM-19were expressed in mice preimplantation embryos for each phase and mainly located in the cytoplasm, nucleus almost no expression. At the same time changes of ΔΨm was detected of blastocysts. Compared with the expression of GRIM-19, we found that both of them showed increasing tread.Conclusion1GRIM-19was continuously expressing during the development of preimplantation embryo of mice. The changes of ΔΨm of mouse blastocysts and the expression of GRIM-19were coincident. It prompts that GRIM-19might play an important role on embryonic development. However, the mechanism remains further study.2GRIM-19was mainly distributed in the cytoplasm, the nucleus almost no expression, which suggested that the GRIM-19may regulate other death associated proteins in the cytoplasm. Specific functions in the nucleus need to be further confirmed.