To begin, place the experimental female mouse on a working platform. Intraperitoneally, administer 7.5 international units of pregnant mare serum gonadotropin around 8:00 PM on the first day. After 48 hours, intraperitoneally inject 7.5 international units of human chorionic gonadotropin.
Divide a cell culture dish lid into upper and lower halves. In the upper half, place four to six droplets of five microliter each of PVP for sperm placement. Add 8 to 10 droplets of five microliter each of M2 medium in the lower half for the ICSI procedure.
Cover the PVP and M2 medium droplets with approximately three milliliters of mineral oil. After isolating epididymal cauda from the euthanized male mouse, gently blot it on sterilized gauze. Cut open the cauda epididymis and gently squeeze, using forceps to release the sperm, collecting them in pre-equilibrated human tubal fluid tubes.
Rinse the cauda epididymis in the pre-warmed human tubal fluid medium. Place the tube uncovered at 37 degrees Celsius and 5%carbon dioxide incubator for sperm capacitation. On the fourth day after injecting human chorionic gonadotropin, place the euthanized female mouse under a dissecting microscope.
Using a 25-gauge needle, tear open the ampulla of the oviduct to release numerous cumulus oocyte complexes. After capacitation, sonicate 100 microliters of the sperm for five seconds to separate the heads from the tails. Fill the micromanipulation needle with the operating fluid, ensuring the filled portion measures 0.5 centimeters inside the needle.
Install the needle on the right arm of the micromanipulator and secure it by tightening the holding cap. Then install a flat-tipped micromanipulation holding pipette on the left arm of the micromanipulator. Position the injection needle and the holding pipette in the microscope's field of view.
Under a polarization microscope, determine the position of the metaphase spindle within the oocyte to ensure that the spindle apparatus is not on the injection side. Upon contact of the injection needle with the zona pellucida, activate Piezo pulses with an intensity of five and frequency of one, creating a perforation in the zona pellucida and allowing the injection needle to pass through. Using Piezo pulses of intensity one and frequency one, create a small pore in the plasma membrane, ensuring the sperm head is injected into the ooplasm.
Using atraumatic forceps, carefully exteriorize the ovaries, oviducts, and uterus from the anesthetized pseudo-pregnant female mouse. Make a small upward-angled incision using the tip of a syringe needle on the uterine wall below the uterotubal junction, avoiding blood vessels. Gently insert the oviduct two to three millimeters through the small hole.
Using the ICSI method, a fertilization rate of 89.57%was achieved in mice, with 87.38%of zygotes advancing to the 2-cell embryo stage within 24 hours post ICSI. The birth rate of live pups post embryo transfer was observed at 42.5%There was no significant fluctuation in random blood glucose levels between mice born naturally and ICSI mice. However, male ICSI mice consistently exhibited elevated fasting blood glucose levels, suggesting impaired glucose homeostasis.
In glucose tolerance tests, significant differences in blood glucose levels were noted between male ICSI and control mice, while no difference was observed in female mice. In body weight tracking, both male and female ICSI mice exhibited an overweight phenotype compared to controls.
