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[Abstract]:objective
Mitochondria are the most abundant organelles in the cytoplasm of eggs. The ATP produced by mitochondria is the main energy source of eggs, fertilized eggs and embryos. If the mitochondrial respiratory chain is damaged, ATP production will decrease, aneuploidy will occur, and normal fertilization and embryonic development will be interfered with, affecting the development potential of the egg.
Gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) is a part of mitochondrial respiratory chain complex I. Its assembly of mitochondrial respiratory chain complex I-IV, charge transfer of mitochondrial respiratory chain and maintenance of electrochemical potential energy are also involved. Necessary, the lack of GRIM-19 leads to abnormal mitochondrial structure, distribution, and function. Studies have shown that the death of mouse embryos lacking GRIM-19 at 9.5 days suggests that GRIM-19 plays a crucial role in embryonic development, and GRIM-19 also plays an important role in the early development of the heart. The expression of GRIM-19 in mouse preimplantation embryos was detected to observe the dynamic expression of GRIM-19 in preimplantation embryos and to explore the effect of GRIM-19 on the growth and development of early embryos.
Method
1, superovulation and embryo acquisition in mice.
Female mature Kunming mice were intraperitoneally injected with HMG10IU/mice, 48 hours later, intraperitoneally injected with hCG10IU/mice, and sexually mature male Kunming mice were caged in a ratio of 1:1 overnight. The next morning, female rats were examined for pudendal suppository, and the female rats were killed by the method of pudendal suppository positive (27 hours after hCG). Bilateral fallopian tubes were taken out under the solid microscope, fertilized eggs were transferred into four-hole dishes. A 400 ml pre-balanced G1 medium was added into the hole and covered with mineral oil. The embryos were cultured in the incubator at 37 C and 5% CO2. The 2-cell, 4-cell, 8-cell, mulberry and blastocyst stage embryos were taken at 46-48 h, 55 h, 65 h, 75 h, 84-86 h after injection of hCG.
2, embryo grading
8-Cell embryo grading standard: 1 grade, large blastomere, homogeneous transparent, no fragments; 2 grade, blastomere incomplete large, homogeneous transparent, no fragments; 3 grade, blastomere large, homogeneous but a few fragments exist in the egg space; 4 grade, blastomere is not large, there are fragments, granular non-uniform black and so on. The 1,2,3 grade embryos were classified as the excellent embryo group (group A), and the 4 group was the non embryo group (group B).
3, Western blot
The collected embryos (500 embryos) were washed with PBS for 1-2 times, then added with 20 ml protein extract buffer, and ultrasonic 20 times (130 watt, 20 khz, 5 s). SDS sample buffer was added, boiled at 100 C for 5 min, SDS-PAGE was separated and the protein was transferred to PVDF membrane, then sealed with 5% milk powder, and then mixed with rabbit anti-mouse IgG/GRIM-19 antibody (1. Horseradish peroxidase (HRP) conjugated sheep anti-rabbit IgG as a second antibody (1:5000), incubated at room temperature for 1 h. ECL chemiluminescence method was used to develop the color. The film was exposed in darkroom. The light density values of each band were obtained by image J software, and the content was expressed by the ratio of target gene to internal reference beta-actin.
4, Real-time PCR
I. Configuration of embryo lysate and reverse transcription fluid (50 ml system, prepared on ice) 2 UL NP-40, 1 u l RRI (Ribonuclease Inhibitor), 10 U L 5 *Prime ScriptTM Buffer (for Real Time), 2.5 u l Oligo DT Primer (50 u M)*1,5 u l Random 6mers (100 u M), 22 u l Rnase Freed H2O.
Ii. Embryos obtained by the above-mentioned method were washed with PBS after removal of zona pellucida by Tyrode's acid. Twenty blastomeres were sucked by micropipette under stereoscopy. The volume of inhalation was controlled within 5 ml. The blastomeres were added to RNA-free PCR tubes containing cell lysate, and the blank control tubes were added with the same amount of acellular PBS. 2H, fully lysate cells.
I I I. Reverse transcription was performed by adding 3 ml Prime ScriptTM RT Enzyme Mix I into the PCR tubes. The reaction conditions were as follows: the first step, 37 20 min, the second step, 85 5 sec, the third step, 12 forever, the fourth part, stop.
IV. Real Time PCR (20 ull system) 10O_ L SYBR Premix Ex TagTM, 0.2_ L PCR F Primer, 0.2_ L PCR R Primer, 2.0_ L cDNA template, 7.6_ L 3 DW sterilization. Reaction conditions: 95 30sec, Holding stage; 95 5sec, 60.0 30sec, 40cycles; 95.0 15sec, 60.0 sec, 1 min, 90 15sec, Melt curve stage.
5, microinjection antibody.
Mice were randomly divided into two groups, group a (injection antibody group) was injected with anti-GRIM.19 antibody into the cytoplasm of fertilized eggs, group B (blank control group) was injected with the same amount of G1 culture medium into the cytoplasm of fertilized eggs to observe the development of fertilized eggs.
Result
1. GRIM-19 protein was expressed in mouse embryos at different stages before implantation. Its protein level gradually increased from 2-cell stage to 8-cell stage, reached the peak, then decreased, and reached the lowest level at blastocyst stage.
2. The transcription level of GRIM-19 mRNA increased gradually from 2-cell stage to 8-cell stage, reached a peak, then decreased to the lowest level in blastocyst stage.
The level of GRIM-19mRNA in 3,8- embryos was significantly higher than that in the non embryo group (P0.05).
4. The survival rate of embryos in the microinjection group was lower than that in the control group. Compared with the control group, the growth and development of embryos in the experimental group were blocked to some extent, and the embryos could not develop to blastocyst stage normally.
conclusion
1. GRIM-19 was continuously expressed in mouse preimplantation embryos. The expression of GRIM-19 changed with the development of mouse preimplantation embryos, suggesting that GRIM-19 plays a certain role in early embryonic development and changes with the process of embryo proliferation. The specific mechanism needs further study.
2. The expression of GRIM-19 was positively correlated with embryo grading, indicating that the expression of GRIM-19 was correlated with embryo quality and developmental potential, suggesting that GRIM-19 could be used as a potential indicator for evaluating the developmental potential of early embryos.
3. The blockade of GRIM-19 protein can affect the survival rate of embryos and block the development of mouse early embryos, suggesting that GRIM-19 plays an important role in the early development of embryos.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R321-33

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