Experiments on collection and structural observation of oocytes from mouse oviducts
Experiments on collection and structural observation of oocytes from mouse oviducts
Mouse oviduct oocyte collection and structural observation can be applied to recognize the structural composition of female reproductive organs and their locations in mice, and to recognize reproductive organs such as ovaries, oviducts, and uterus.
Operation method
microscopic observation
Principle
Mammalian oogenesis takes place within the follicle of the ovary. During the oocyte maturation stage of development, the primary oocyte undergoes germinal vesicle breakdown (GVBD) and completes the first maturation division (meiosis, characterized by homologous chromosome segregation) and expels the first polar body. The onset of GVBD and the expulsion of the first polar body are generally considered to be the hallmarks of nuclear maturation of the oocyte. Subsequently, the secondary oocyte undergoes a second maturation division (generally mitotic, characterized by chromosome segregation) and arrests at the midpoint of the second maturation division. During this period, final maturation of the cytoplasm is accomplished. At this point, the follicle matures and the oocyte in the middle of the second maturation division is expelled with the follicular fluid, along with the surrounding oocytes. The expelled oocyte passes through the funnel of the fallopian tube into the fallopian tube and runs to the inflated portion of the fallopian tube (jugular), the site of fertilization. The oocyte matures fully only after fertilization, completing the late and terminal stages of division, followed by the formation of a fertilized syncytium and the release of a small second polar body.
Materials and Instruments
Mouse Move I. Dispensing and preparation of hormones Common Problems Oocytes are usually spherical. Mouse oocytes are small, about 70-90 um in diameter, and consist of a zona pellucida surrounding the cytoplasm. The zona pellucida is surrounded by a radial crown of radially arranged cells (oocytes) with cytoplasmic protrusions.The first polar body is discharged as a sign of maturation of the oocyte at stage MII, and the first polar body of the mouse oocyte is large and easy to recognize. For more product details, please visit Aladdin Scientific website.
Saline Fluid Embryo Recovery Solution
Microscope Scrubbing paper Constant temperature water bath Water-separated constant temperature incubator Instrument tray Surgical scissors Surgical forceps Ophthalmic scissors Ophthalmic forceps Ophthalmic foreign body needles Surface dishes Oocyte straws Oocyte test cups Alcohol cotton balls Straws Syringe Needles Needle cases Alcohol lamps Syringes Needles Gestational equine serum gonadotropin Human chorionic gonadotropin Alcohol cotton balls Tweezers Electronic balances Medication spoons Penicillin vials Gelatin tape Marker pens
Hormone manufacturers produce reproductive hormones for superovulation, such as pregnant horse serum gonadotropin and human chorionic gonadotropin. When used for superovulation in mice, they are packaged in large doses and are not usually used up at once. And due to the chemical properties of these hormones, once diluted, the biological activity in solution will quickly decline and inactivate, and large doses of diluted can not be kept for long even if not used. Therefore, commercially available packages should be divided before use. When dispensing, you need to weigh the net weight of the hormones in a package and convert the number of hormone units per unit weight according to the net weight and the hormone units labeled on the package. Then, according to the number of units of hormone needed for each super-excretion, weigh the hormone with a precision electronic balance, divide the hormone, put it into sterilized dry penicillin vials, seal each small package with adhesive tape, and mark the date, weight, and number of units of hormone with a marker pen, and put it into the refrigerator at 4 ℃ to be used. Before use, take out a small package, according to each mouse intraperitoneal injection of 0.2 mL (or 0.5 mL) of the solution volume, with a solvent (generally use sterilized saline) for dilution.
II. Mouse overdischarge treatment schedule and overdischarge treatment
On the day of the experiment (day 0), from 3:00 to 5:00 p.m., young female mice with light pink coloration of the vulva were selected, which were close to the onset of oestrus, and the effect of superovulation treatment was better. After catching the mice, 8 to 10 units of PMSG were injected intraperitoneally. Forty-eight to 50 hours after the injection of PMSG, each superovulated mouse was then given an intraperitoneal injection of 8 to 10 units of hCG. 15 to 17 hours after the injection of hCG, the oocyte complex was recovered from the juxtapetal region of the oviduct.
III. Collection of mouse oocytes and structural observation
Experimental mice were executed by the cervical dislocation method, and the abdomen was sterilized with 70% alcohol cotton balls. A small incision was cut in the middle of the lower abdomen (Fig. 4-2A), and the upper and lower skin of the incision was grasped by both hands or with forceps and pulled toward the head and tail, respectively, until the abdomen was fully exposed (Fig. 4-2B). The peritoneum is incised and the viscera are turned upward, i.e., exposing the ovaries and fallopian tubes on both sides (Fig. 4-2C). The uterus-tubal junction was clamped with ophthalmic forceps, and the tubal tunic was cut with sharp scissors (Fig. 4-3A), after which the connecting structures between the ovaries and fallopian tubes were cut first (Fig. 4-3B), and then the uterus-tubal junction was cut, i.e., the tubal portion was obtained, and the tubal portion was transferred to a surface dish containing PBS.
The fat on the oviduct is removed as much as possible with ophthalmic scissors and rinsed out so that blood or fat globules do not mix with the fluid to prevent egg detection. Since the mouse oviduct is very thin, it is not easy to rinse the lumen directly. Place the surface dish on a solid microscope carrier stage and locate the inflated portion of the oviduct (juxtamedulla) at 20 or 40x (Fig. 4-4). Hold the oviduct in place with an ophthalmic foreign body needle and tear open the juxtamedulla with another foreign body needle, and the oviductal-oocyte mass will normally move out spontaneously. If the beginner is not able to judge the inflated part of the fallopian tube, the whole fallopian tube can be torn longitudinally with a foreign body needle. At this point, the oocyte is surrounded by the oocyte mound cells, and multiple eggs are aggregated into a cumulus cloud. When the oocyte mass is transferred to hyaluronidase-containing fluid, the oocytes disperse into individual cells. The oocytes were examined with a thin pipette into an egg examination cup, washed three times with PBS, and the morphologic structure of the oocytes was observed under a solid microscope.
Oocytes are usually spherical. Mouse oocytes are small, about 70-90 um in diameter, and consist of a zona pellucida surrounding the cytoplasm. The zona pellucida is surrounded by a radial crown of radially arranged cells (oocytes) with cytoplasmic protrusions.The first polar body is discharged as a sign of maturation of the oocyte at stage MII, and the first polar body of the mouse oocyte is large and easy to recognize. 
