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

Mitochondria are the richest of organelles in egg cytoplasm, which are the major source of ATP generation for egg, fertilized ovum and embryo. During the early development of embryo, mitochondria undergo a series of changes in morphology and location. During this stage, little change in mitochondria’s function can directly influence the growth and proliferation of preimplantation embryo. If mitochondria respiratory chain get impaired, it would cause ATP reduced, aneuploid generated, and the development potential of ovum suppressed due to the disturbance of the normal fertilization and embryo development.GRIM-19(Genes associated with Retinoid-Interferon-induced Mortality-19) was found to be a part of complex Ⅰ of mitochondrial respiratory chain which catalyzed the first step of electron transfer in the oxidative phosphorylation system (OXPHOS). And it subsequently was demonstrated to be involved in complex Ⅰ-Ⅳ assembly and activity, and maintaining the mitochondrial transmembrane potential (ΔΨ). GRIM-19-/-blastocysts strikingly display abnormal mitochondrial structure, morphology, and cellular distribution. Homologous deletion of GRIM-19causes embryonic lethality at embryonic day9.5. Subsequently, it has been demonstration that GRIM-19was essential to early development of heart. However, there was no report about GRIM-19in preimplantation embryo of mice. So, in this research we studied the function of GRIM-19in preimplantation embryo of mice via detecting the expression of GRIM-19.Methods1. Superovulation and eggs collectionSexual maturity female mice were abdominal injected with10IU each one of human menopausal gonadotropin (HMG), and48h later injected with10IU each one of human chorionic gonadatropin (hCG). One female mice and one male mice as a couple were both placed together for fertilization. Fertilized ovum were collected with G1medium the next day (27h after hCG injection) from uterine tube of females possessing a vaginal plug. Then, embryos were transferred to a four holes of dish to culture with400μl G1medium (which already balanced in incubater) covering mineral oil, at37℃, in a humidified atmosphere of5%CO2in air. And we collected2-cell,4-cell,8-cell, morula and blastocyst embryos at46-48h,55h,65h,75h and84-86h after hCG, respectively.2. Embryonic quality classificationEmbryonic quality classification standard of8-cell embryos:Grade1, blastomere are all homogeneous, transparent and no fragments; Grade2, blastomere are most homogeneous, transparent and no fragments; Grade3, blastomere are all homogeneous, but have a few of fragment existing in egg clearance; Grade4, blastomere are not homogeneous, and have fragments in egg clearance, and darken. Because the number of embryos in some grades are so few and sometimes it’s difficult to classify clearly, we count grade1,2and3as good quality embryos:group A, and grade4as bad quality embryos:group B.3. Western blotWestern blot analysis was performed as described previously (Huang et al.,2007). Briefly, Morphologically normal cells (500embryos/sample) were lysed in20μl RIPA buffer (50mM Tris-HCl, pH7.2,150mM NaCl,1% deoxycholic acid,1%Triton X-100,0.1%SDS,0.25mM EDTA) with1μl phenylmethanesulfonyl fluoride. The lysate was in ultrasound (130watt,20khz,5s), then heated to100℃for5min. For immunoblotting, the proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with phosphate-buffered saline (PBS) containing0.1%Tween20and5%skimmed milk for1h. Then, it was incubated with the rabbit anti-IgG/GRIM-19primary antibodies1:500and the rabbit anti-β-actin primary antibody overnight at4℃, and with the Horse Radish Peroxidase (HRP)-conjugated IgG second antibodies1:5000for1h at normal temperature. Immunoreactivity was detected using the HRP-linked secondary antibody and enhanced chemiluminescence (ECL) detection reagent. All experiments were repeated at least three times. The film scanned and optical densities of the immunoreactive bands were measured using Image J analysis software.4. Real-time RT-PCRFor RNA isolation, embryos were respectively collected at the2-cell stage and cultured to the4-cell、8-cell、morula and blastocyst stage. After removal of Zona Pellucida (ZP) in acidified Tyrode solution (pH2.5), embryos were then placed in2μl NP-40and1μl RRI (Ribonuclease Inhibitor) on ice for up to2h. The RNA was DNase treated and reverse transcribed by using the PrimeScript RT reagent Kit (TaKaRa).For quantitative RT-PCR, the resultant cDNA were performed in triplicate with a Stepone PlusTM Real-Time PCR System (Applied Biosystems), using SYBR green (TaKaRa) within the gene-specific primers GRIM-19: Forward,5’-TCGGGG ACTGTC GGGGTAC-3’, Reverse,5’-AGGGTCCTCCGGTCCTTCT-3’; GAPDH:Forward,5’-GCCTTCCGTGTTCCTACCC-3’, Reverse,5’-CG AAGTCGCAGGAGACAACC-3’, which served as a normalization reference. Each20μl RT-PCR mix included1μl SYBR Premix Ex TagTM,0.2μl PCR F Primer,0.2μl PCR R Primer,2.0μl cDNA,7.6μl H2O. Each qPCR run included negative control reactions without template. The reaction mixtures were subjected to an initial denaturation of95℃for10min, followed by36cycles of95℃for5s,60℃for30s, then subjected to a melting curve analysis. The specificity of each PCR amplification was confirmed by melting point analysis. Real-time RT-PCR with each sample, using the two primer sets, was conducted in triplicate to ensure consistency in cycle threshold (CT) values. The amplification was followed by melt curve analysis and the changes in the CT values were calculated by the equation ΔCT=CT target-CT input where target and input refers to GRIM-19and GAPDH respectively. The fold differences were calculated as per the formula2-Δ(ΔCT).5. MicroinjectionWe collected germ cells of mice as previously described and randomly divided into microinjecting anti-GRIM-19antibody group and blank control group. After that we observed the growing development of germ cells on schedule.Results1. GRIM-19was expressing in every stage of preimplantation embryo of mice. The results of western blotting showed a gradually increase of GRIM-19protein expression from2-cell, until8-cell to peak, and then it decreased progressively.2. The results of real-time PCR showed a gradually increase of GRIM-19mRNA expression from2-cell, until8-cell to peak, and then it decreased progressively.3. In8-cell embryo, mRNA of GRIM-19in good quality group embryo was significantly higher than bad quality group embryo (P<0.05).4. The survival rate of embryo in experimental group is lower than those in the control group3hours after injection. Compared to the control group, the growth and development of the experimental embryos were subjected to different degrees of block after the injection of hCG48hours. And experimental embryos can’t normally develop to blastosphere stage. Conclusion1. GRIM-19was continuously expressing during the development of preimplantation embryo of mice, and its quantity of expression was depended by the development stage. It prompts that GRIM-19might play an important role on embryonic development, and the changes of effect followed with embryo proliferation. Howerer, the mechanism remains further study.2. The expression of GRIM-19mRNA had a positive correlation with embryo quality. It had demonstrated that the expression of GRIM-19mRNA could reflect the potentiality of embryo development. It was worthy of trust that GRIM-19could be used as a indicator of evaluate the quality of early embryo in the future.3. Block of GRIM-19protein would reduce the survival rate of embryos, and block the development of early embryos still further. It showed that GRIM-19was essential for the development of preimplantation embryos.