The mammalian preimplantation embryo is an important object of study in embryology, developmental biology, and animal cell and embryo engineering (1) the study of mammalian reproductive structures (2) the effects of interventions at different developmental stages (3) the diagnosis of genetic diseases.
Operation method
microscopic observation
Principle
The process of sperm-egg union to form a fertilized egg in mice takes place in the juxtaposition of the oviduct. In mice, the first cleavage occurs 18 to 24 hours after fertilization; thereafter, the egg divides every 12 hours or so. According to this law, basically we can deduce the age of the early cleavage embryo in each period, 2-cells for about 24 to 38 hours after fertilization, 4-cells for 38 to 50 hours, 8-cells for 50 to 60 hours, 16-cells for 60 to 72 hours. The mouse embryo begins to densify when the egg cleaves to the 8-cell stage, and the densified cells divide to form the 16-cell mulberry embryo, in which the embryo passes from the oviduct into the uterus. At the 16- to 32-cell stage (66-82 hours) a small blastocyst cavity appears and the blastocyst stage is entered. The blastocyst stage can be divided into four stages: the 16 to 32-cell stage, when the blastocyst cavity is so small that it is not easy to be seen, is the early blastocyst; the late blastocyst when there is a complete blastocyst cavity and it can be clearly differentiated into an inner cell mass; the period in which the blastocyst cavity is further enlarged in size and microscopically resembles a blister, with a relatively small inner cell mass is the expanding blastocyst; and the expanding blastocyst when it hatches out from the zona pellucida, which is not dissolved but is Instead of dissolving the zona pellucida, a part of the zona pellucida is softened by some enzymes in the uterus and the trophoblast, and the blastocyst cells are drilled out from the softened part, and the age of the incubation period is about 96-110 hours. Through the three experimentally arranged periods of embryo collection and observation, several points can be learned about mammals in early embryonic development. The oviduct recovers 2-4 cell embryos, mainly observing the zona pellucida, polar body, and oocyte sphere to understand the mechanism of oocyte cleavage; the oviduct or uterine horn recovers 8 cell embryos to mulberry embryos, observing the process of densification to understand the phenomenon of embryonic densification; the uterus recovers blastocysts, observing the zona pellucida, the intraembryonic cell mass, the trophecyte cells, and the blastocyst cavity, to understand the phenomenon of the embryonic cells' differentiation.
Materials and Instruments
Mouse Move I. Mouse over-exclusion schedule and over-exclusion treatment Caveat 1. When observation is difficult, increase the magnification and carefully observe the cell structure. If the periosteal gap is large, the inner cell mass cells are loose, the cells are of different sizes or in very small clusters, and the cell boundaries are not clear, then it is a degenerated embryo or a metamorphosed embryo. 2. Sometimes some irregular embryos can also be seen. The unfertilized egg has no perivitelline gap, and the zona pellucida is a large cell with more granules or vesicles inside the cell. 3. Animal embryo experiments are experimented in a sterile environment, in which sterile liquid is also a basic requirement, the prepared liquid needs to be observed from time to time to see whether the color changes, whether turbid whether there are colonies produced. Common Problems Morphological quality appraisal of embryos may be based on the following characteristics: 1. the stage of development of the embryo and the morphology of the embryonic cells. 2. the morphology and homogeneity of the embryo, changes in the size of cells within the embryo. 3. the structure and color of the cytoplasm, the presence or absence of vacuoles, cellular debris, and detached cells in the embryo. 4. whether the size of embryo is normal or not. 5. the morphology and integrity of the zona pellucida. The developmental stage of the normal embryo is consistent with the age of the embryo that should be reached at the time of retrieval, the cell structure within the embryo is compact and the embryo is spherical. The boundaries between the cells in the embryo are clearly visible, and the cells are of uniform size and regular arrangement. The transmittance was consistent, neither very bright nor too dark. The cytoplasm contained a number of evenly distributed vesicles, without fine granules or irregularly distributed vacuoles. There are small perivitelline gaps of regular diameter. The zona pellucida is complete, without wrinkles or atrophy. There are no cellular fragments in the embryo. Embryos at the early cleavage stage were clearly distinguishable from each ooglossal sphere, and structures such as individual ooglossal spheres, polar bodies, and zona pellucida could be observed. Densification of 8 cells and mulberry embryo can be seen in the perivitelline gap, and a mass of cells in the zona pellucida. When the angle of the reflector is changed, and the angle of incident light is adjusted appropriately, the blurred boundaries between the cells in the embryo can be seen. For blastocyst stage embryos, mainly observe the structure of intraembryonic cell mass, trophoblast cells, blastocyst cavity, perivitelline space, zona pellucida and other structures. When observing the embryo, it is necessary to pluck the embryo with a foreign body needle and observe it from different sides in order to recognize the number of cells and intra-embryonic structures. For more product details, please visit Aladdin Scientific website.
Saline Pregnant horse serum gonadotropin Human chorionic gonadotropin Embryo retrieval operating solution
Electronic balance Medicine spoon Penicillin vial Tape Marker Syringe Needle Alcohol cotton ball Tweezers Microscope Scrubbing paper Constant temperature water bath Insulated constant temperature incubator Instrument tray Alcohol lamp Surgical scissors Surgical forceps Ophthalmic forceps Ophthalmic scissors Ophthalmic forceps Ophthalmic foreign body needles Surface dish Petri dish Flat dish Embryo pipette Ovitrap cup Alcohol cotton ball Straws Straws Syring Needles Needle case
Note: If the number of experimental subjects is large and the number of mice required is large, in order to avoid a shortage of mating males, multiple batches of experimental mice can be selected for cross-over mating, and the experiments will be conducted in a cross-over manner accordingly.
After hCG injection, put 1 or 2 treated female mice into a cage, and later put 1 male mouse into the same cage overnight. Before 7:00 to 8:00 a.m. on the 3rd day, the negative bolus of the mated female mice was observed and recorded. The dosage of gonadotropins for superovulation should be appropriate, too little when the number of ovulated eggs is low, and too much when the number of ovulated eggs is too high and the degradation of embryos after fertilization is increased, which in turn reduces the number of usable embryos and may lower the quality of usable embryos. Due to the difference in hormone activity between hormone manufacturers or different batch numbers produced by the same manufacturer, and due to the difference in the responsiveness of mice to superovulatory hormones depending on the strain, it is necessary to conduct preparatory tests on hormones from a certain manufacturer or a certain batch number to determine the appropriate dosage prior to the official experiments.
Embryo collection
1. Tubal embryo collection
Oviduct collection and separation. Since the mouse oviduct is very thin, it is not easy to directly flush the lumen to collect embryos. A surface dish containing the oviduct and egg flushing fluid is placed on a solid microscope carrier stage, and under 20 or 40x magnification, the oviduct is held in place with an ophthalmic foreign body needle, and the oviduct is torn open longitudinally with another foreign body needle, and the entire oviduct is lacerated in this way, and the embryo will be free into the fluid.
2. Uterine horn embryo retrieval
Expose the uterine horns on both sides as described in experiment III. Hold the cervix of the uterus with ophthalmic forceps (Fig. 4-5A) and cut the cervix with ophthalmic scissors (Fig. 4-5B). The uterus is gently lifted with forceps and the uterine tunica is cut with ophthalmic scissors up to the tip of the uterus, and the uterine horns are taken by cutting between the fallopian tubes and the ovaries (Fig. 4-5C). In a table glass dish containing egg wash, the appendages such as the tethered membrane on the uterine horn are removed with ophthalmic scissors and rinsed out, and the embryo is recovered by the rinsing method.
For embryo flushing, the uterine horn was placed into a flat dish, the uterine tube junction was cut longitudinally with ophthalmic scissors, respectively, and the uterine cavity was flushed with a syringe containing egg-flushing fluid inserted with a needle through the cervical opening, so that the embryo was flushed out of the uterus with the flow of the fluid (Fig. 4-6A), with about 1.0 mL of fluid used on each side. Another method is to cut from the base of the uterine horn and flush the embryo out of the uterine cavity by inserting a needle through the head of the uterine tube junction and toward the cervix (Figure 4-6B).
Morphological observation and identification of embryos and embryos
The embryonic fluid is left to stand for a few minutes and then the embryo is detected. When detecting the embryo, put the embryo-containing vessel under the solid microscope, first adjust the focus under low magnification (15-20 times), after seeing the embryo, use the egg detection needle to remove the foreign matter such as the reproductive tract detached cells in the recycling liquid, and then move the embryo with the egg suction tube into the egg detection cup containing freshly washed out eggs, and then carry out purification treatment after all the embryos are detected. Embryo decontamination was performed by transferring the detected embryos into fresh egg wash for 2-3 times to remove contaminants adhering to the embryos. The liquid is changed each time during washing, and when transferring the embryos by suction of the egg tubes, it is important to minimize suctioning into the liquid in the previous container to prevent carrying contaminants into the fresh liquid. The operation process should be light and rapid to avoid damaging the embryos.
After the embryos were purified and processed, they were put into the egg inspection cup containing fresh egg flushing liquid for observation under 60-80x microscope, and morphological quality appraisal was carried out to distinguish between normal and abnormal embryos.