Acquisition of porcine oocytes

Summary

In vitro maturation of the oocyte includes both nuclear and cytoplasmic maturation. Nucleus maturation consists of the resumption of meiosis by the oocyte, the onset of GVBD, the expulsion of the first polar body, and arrest at mid-phase of the second meiotic division, which is not completed until after fertilization (or activation) and the expulsion of the second polar body. Cytoplasmic maturation includes changes in organelles and changes in the cellular matrix. Cortical granules gradually migrate to the cortical region beneath the plasma membrane after cytoplasmic synthesis. The Golgi complex is initially located around the nucleus and moves to the cortex during maturation. After the cortical granules are fully formed, the Golgi complex disappears in the cortex. Early in development, mitochondria are round or oval, etc., and are mainly distributed in the cortical region. As the oocyte matures, the mitochondria move back around the nucleus to form naïve-type mitochondria. As the oocyte matures, the rough endoplasmic reticulum decreases while the smooth endoplasmic reticulum increases, and the smooth endoplasmic reticulum also becomes scarce in the late stage of maturation. A very important feature of cytoplasmic maturation is the accumulation of a number of stable mRNAs and the translation of proteins at a certain stage in the maturation of the oocyte. During the full maturation of the oocyte, many chemicals are involved in the reaction and interact together in a very complex series of biochemical events. Therefore, a high maturation rate can only be achieved if the oocyte is matured in vitro with a living environment that is very close to that of the oocyte in vivo.

Operation method

Acquisition of porcine oocytes

Materials and Instruments

Equipment:
① Leaky sieve
② Syringe
② Syringe ③ Petri dish
④ CO
2
Incubator
Reagents:
① Material: pig ovary
② Saline, paraffin
③ TL-HEPES
④ Maturation solution
⑤ Hyaluronidase
⑥ Antibiotics

Move

The basic process of obtaining porcine oocytes can be divided into the following steps:
(a) Ovary pickup and delivery
Porcine ovaries collected at the slaughterhouse were transported back to the laboratory in saline within 1 hour; the saline temperature was 32-33 ℃ in summer and 35 ℃ in winter. The temperature of saline was 32-33 ℃ in summer and 35 ℃ in winter. The temperature of the ovaries should be maintained at 35-36 ℃ after transportation to the laboratory. The thermos flasks used should be sterilized before use: boil the flasks for a few minutes in hot water, then pour off the boiling water and refill with tap water containing benzalkonium bromide.
The ovaries should be warmed and gently drained of blood through a sieve, and then rinsed with 37 ℃ saline with double antibiotics while turning to remove the blood.
The cleaned ovaries were divided into 1 or 2 thermos flasks according to the quantity ready for egg extraction. The sieve and the thermos flasks should be cleaned to remove the odor and then dried in an oven at 37 ℃ for the next time.
(ii) Egg extraction
Use a 10 ml syringe with a 10-gauge needle to extract COCs from follicles 2-8 mm in diameter, paying attention to the downward direction of the needle, such as lifting the piston while extracting the needle to suction out the follicular fluid in the follicle. The extracted follicular fluid was placed in a 50 ml sharp-bottomed centrifuge tube at 37 ℃ in a constant temperature water bath, and was extracted as much as possible within 0.5 hours.
(iii) Egg washing
After egg extraction, place the centrifuge tube in a warm oven and let it settle for the first time for 10 minutes, discard the upper layer of follicular fluid and then add TL-HEPES to wash, shake, and let it settle in a warm oven for 3 minutes, discard the upper layer of liquid, repeat the washing for two times, and then add TL-HEPES for the third time, and then pour it into a large domestic flat dish, in order to prepare for picking up the eggs.
Discard the upper layer of follicular fluid: use a 10 ml syringe to aspirate the upper layer quickly, and then aspirate it slowly when it is close to the sediment, in order to prevent the sediment from being drawn.
Prepare a large domestic Petri dish (with pre-scored lines on the bottom about 1 cm apart) with a small domestic Petri dish and add TL-HEPES solution to the Petri dish.
Note: Because of the large amount of TL-HEPES, it is sufficient to pour about 30 ml into the centrifuge tube during washing.
(iv) Egg collection
COCs containing at least 3 layers of intact oocytes are selected at 1x and placed in the petri dish prepared above. Take care to distinguish between dead eggs, naked eggs and eggs with irregular morphology or uneven cytoplasm.
Preparation of mouth and hand pipettes:
1. Wash in tap water with detergent.
2. Place in an ultrasonic shaker at 40 ℃ for 1 hour.
3. Remove and rinse twice with tap water.
4. Soak in distilled water for 1 hour, 3 times.
5. Rinse twice with deionized water.
6. Place in a dry-heat sterilization chamber with tin foil underneath at 180 ℃ for 2 hours.
7. Pack the mouth pipettes into 50-ml imported centrifuge tubes and seal with tape. Seal the mouthpiece with adhesive tape. The hand pipette is wrapped with tin foil.
(v) Maturation culture
The selected COCs were washed with 4 drops of maturation solution and then cultured in 24-well plates with sufficient equilibrium (3-4 hours). 500 μl of maturation culture solution was added to each well, covered with liquid paraffin, and 50 COCs were cultured in each well. The incubation conditions were 38.5 ℃, 5% _CO2, 95% air, saturated humidity. 24-well plates were labeled with the incubation time, number of eggs and incubator.
(VI) Digestion
Preheat the permeabilized enzyme 1 hour in advance.
After 42-44 hours of incubation, about 200 COCs were transferred into a tube of 700 μl of 0.1% hyaluronidase and vortexed for 3 minutes, during which time two small domestic dishes were prepared by adding one more and one less solution, but not less than 2 ml, and pulling the needle of egg collection.
After vortexing, the hyaluronidase was first transferred into the working solution to terminate the digestion, and the oocytes were detected as soon as possible and placed in another small dish containing more working solution. Under the microscope, mature oocytes were selected and the maturation rate was calculated using the release of the first polar body as a criterion for oocyte maturation.
A complete record was kept of all aspects of the maturation culture: time of ovarian transport, ovarian temperature, number of ovarian pairs, number of eggs cultured, incubation time, and number of mature eggs.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/