Experimental procedure
1. Cloned embryo preparation and embryo culture
The oocytes were cultured in CZB medium containing 5.5 mM glucose. HEPES-CZB solution (HCZB) containing 5.5 mM glucose and 2.5 ug/ml cytochalasin B was used as the working fluid, and the oocyte was removed from the spindle-chromosome complex; the cumulus cells were used as the nuclear donor. . The cumulus cells are repeatedly inhaled and blown out with an injection needle to remove the cell membrane and most of the cytoplasm, and then the nucleus of the naked egg is injected into the oocyte of the spindle-chromosome complex by injection.
The oocytes used in the parthenogenetic embryos are of the same origin as the oocytes used to prepare the nuclear transfer embryos.
Parthenogenetic embryos and nuclear transfer embryos were activated with CZB medium containing no Ca2 but containing 10 mM Sr2 and cytochalasin B. Dimethyl sulfoxide was not used in all steps to avoid uncontrolled effects on cloned embryos. Single cell normal fertilized embryos were obtained by mating of super-arranged (B6D2) F1 females and (B6D2) F1 males.
All embryos were cultured in Whitten's medium (MW). This culture was chosen because fertilized embryos, parthenogenetic embryos, and nuclear transfer embryos prepared using (B6D2) F1 mouse oocytes are more conducive to their embryo development than the two-cell stage in this culture. Other culture fluids that have a better promoting effect on normal embryo development have a blocking effect on cloned embryos and are therefore not used.
2. Analysis of glucose metabolism
Embryos were cultured overnight in WM and used for glucose metabolism analysis in the late single cell or mid-cell phase. We used [1-14C] glucose, [6-14C] glucose, [5-3H] glucose to analyze glucose metabolism. When [1-14C] glucose is used, 14 CO2 is released via the pentose phosphate pathway or the tricarboxylic acid cycle. When [6-14C] glucose is used, 14 CO2 is only released through the tricarboxylic acid cycle. When [5-3H] glucose is used, 3 H2O is released only by the glycolysis pathway. 14CO2 and 3H2O were collected using a device and then quantified by a method. By comparing the metabolic rates of three differently located isotopically labeled glucose, the metabolic rate of different pathways can be determined. In order to prepare a culture solution for measuring metabolism, we freeze an appropriate amount of [1-14C]glucose, [6-14C]glucose, [5-3H]glucose under vacuum, and then dissolve with glucose-free HCZB. The amount of glucose in the solution was increased to 4 mM with cold non-radioactive glucose.
Depending on the radioactivity of the radioactive material, 5-20 embryos were placed in the hollow piston of a 1 ml syringe. 600ul of 0.1M NaOH was placed in the needle to capture the 14 CO2 and 3 H2O released by the embryo. Push the plunger containing the embryo into the syringe and cap the needle. Compared with the Eppendorf hanging drop system, the simple device has no difference in sensitivity and detection accuracy, but we found that the former does not have a large tilt when placing the test substance into the syringe, and the substance is placed. Can be quickly and effectively gathered together. After incubation at 37 ° C for 3 h, the NaOH capture solution was carefully transferred to a luminescent bottle. After 1 h, the amount of radiation of the metabolic species was determined. For each test, there were four blank controls (no embryos), but containing 5 ul of HCZB, incubated under the same conditions to account for the cause of glucose degradation and also for the next calculation.
The efficiency of glucose degradation was calculated and expressed in terms of how many picomoles were degraded per hour per embryo. Each test was repeated at least three times, and the number of embryos used in each test was 3-6.
3. Enzyme and metabolic analysis
Embryos were treated by freeze drying and then used for enzyme and metabolic analysis. Each embryo was extracted under oil and used for enzyme cycle analysis. Calculate the respective ATP content in each embryo. Similarly, the level of glycogen inside each embryo is calculated. The analysis of adenylase and glycogen synthase has been described above. All metabolic calculations are expressed in terms of how many mmole of material per kilogram of wet weight (each embryo weighs 160 ng or 160 pl). Each test is repeated at least three times, and the final analysis number of each embryo must reach 10-20.
4. Number of mitochondrial copies
Quantitative PCR was used to detect the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA), thereby indirectly determining the content of mtDNA. The mtDNA primers used were: 5-CAGGATGAGCCTCAAACTCC-3 (upper primer) and 5-GGTCAGGCTGGCAGAAGTAA-3 (lower primer). The nDNA primers used (18S ribosomal RNA): 5-TCGAGGCCCTGTAATTGGAA-3 (upper primer) and 5-CCCTCCAATGGATCCTCGTT-3 (lower primer). Each individual embryo was placed in a 0.2 ml PCR tube containing 1 ul of 17 uM SDS and 2 ul of 125 ug/ml proteinase K, incubated at 37 ° C overnight, and then at 95 ° C for 15 min to inactivate the protease.
5. Ultrastructure of mitochondria
Five embryos were taken from each treatment group, fixed with a mixture of 2% paraformaldehyde and 0.6% glutaraldehyde, and then fixed with sodium dimethyl citrate containing 1% osmium tetroxide, followed by a series of concentrations. The step-up ethanol was dehydrated and then embedded in PolyBed 812 resin. Ultrathin sections were prepared using a microtome, and the sections were placed on a copper mesh, stained with uranyl acetate and lead citrate, and placed under an Ieol 1200 EX microscope for observation. At least three embryos from each treatment group were used for subjective mitochondrial morphological assessment and were expressed in good, medium, and poor.
6. MitoTracker staining and analysis
In order to be able to see active mitochondria, the embryos were incubated in WM-containing (WM-) or glucose-free WM (WM) for 30 min, both of which contained 500 nM chloromethyl-tetramethyl-rosamine (MitoTracker, molecular probe). MitoTracker is a fluorescent probe specific for active mitochondria. After the above treatment, the sample was washed in the culture solution for 60 minutes, and then fixed with 4% paraformaldehyde for 30 minutes. This staining method was visible and photographed by an embryo microscope (Eclipse 800; Nikon Instruments, Melville, NY). The above microscope is equipped with surface fluorescence and different interference phase difference optical systems. The microscope magnification was X60, focusing on the longest diameter of each embryo; the phase was taken with a Cool Snap camera and the MetaMorph 7.1.0.0 software system. Mitochondrial morphological analysis was performed using a morphological analysis tool in MetaMorph. Manually record the circumference of each embryo (each embryo stage, each treatment group must analyze five embryos), record the relevant pixel intensity values ​​of the perimeter will be recorded by the software, enter these values ​​into Microsoft Excel to calculate each stage , the average of each treatment group.
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