The overall goals of this experimental approach are to, first, morphologically and biochemically characterize horse oocytes and, second, to compare gametes from the same animal that were processed in vitro or in vivo. This experimental approach can help answer key questions in oocyte biology and fertility in monovulatory species. It might also improve the safety of gamete manipulation procedures.
This approach was developed to overcome the restricted sample availability of oocytes, especially from monovulatory species. Demonstrating the OPU procedures will be Professor Stefan Deleuze of the University of Liege, Cecile Douet, the lab manager of my lab, and Fabrice Reigner, the manager of the equine unit at the INRA Centre Val de Loire. On a daily basis, assess the diameter of the ovarian follicles in a cohort of adult mares using ultrasound.
Search for a large follicle with a diameter greater than 33 millimeters. Once found, prepare to inject 1, 500 units of human chorionic gonadotropin to induce the maturation of the oocyte into a dominant follicle. Next, locate the jugular furrow, and swab the injection site with alcohol.
Then, to make the vein rise, apply some force two or three centimeters below the site of injection. Insert the needle almost parallel to the neck, aspirate slightly to ensure that the needle is in the vein, and inject the hormone. Prepare an ultrasound probe for vaginal access.
Then, connect the needle to a suction system. 35 hours after hCG injection, set up the instrumentation for OPU, and sedate the mare. Good analgesia is extremely important for the animal's and the operator's safety.
An animal that does not feel pain or discomfort will allow manipulation of the ovary, making punctures and flushes easier to perform. Once the needle probe is inserted, one operator must manipulate the ovary to face the probe while the other operator manipulates the needle. Locate the dominant follicle in the echographic image.
Then, use the needle to pierce the vaginal wall and the wall of the dominant follicle. Once the follicle space is accessed, aspirate the follicular fluid. Rotate the needle to scrape the follicular wall and attach the cumulus-oocyte complex.
Then, pour the fluid into a large petri dish. And under a stereoscope, search for the COC. Meanwhile, flush out the follicle with warm Dulbecco's modified PBS containing heparin.
It may take several flushes to collect the COC. After the COC is found, change the needle to a double-lumen needle and collect the immature COCs from all of the follicles that are between five and 25 millimeters in diameter. These will be used for in vitro maturation.
Once all of the follicles from one ovary are aspirated, repeat the same procedures to collect the immature COCs from the other ovary. Now, choose between the COCs. Keep only COCs with several complete layers of cumulus cells and a brown, finely-granulated ooplasm.
Discard the others. At the end of the procedure, inject the mare with 15, 000 units of benzyl-penicillin to prevent infection. Then, house the mare in a quiet, clean stable for at least two hours while she recovers.
Before the OPU starts, warm a bottle of supplemented HEPES-buffered TCM-199 to 37 degrees Celsius. Next, prepare the IVM medium. Dispense 500 microliters of supplemented sodium bicarbonate-buffered TCM-199 into the wells of a four-well dish.
Then, incubate the dish at 38.5 degrees Celsius for at least two hours. Transfer the recovered immature COCs to a 3.5-centimeter dish filled with two milliliters of the warmed HEPES TCM-199. Move the COCs through different regions of the dish to gently wash away surrounding debris.
Now, transfer the COCs to the IVM medium, and incubate them for 28 hours at 38.5 degrees Celsius. To perform a chromosome count on mature COCs, incubate them in 100-micromolar monastrol for an hour at 38.5 degrees Celsius. Next, remove the cumulus cells by treating the COCs with 0.5%hyaluronidase in HEPES-buffered TCM-199.
Monitor this reaction on a heated stage set to 38 degrees Celsius. Within about five minutes, remove all of the cells. Use gentle pipetting to remove the last few stubborn cells.
Next, remove the zona pellucida using 0.2%pronase in HEPES-buffered TCM-199. Monitor this reaction on a heated stage as well. It should take about five minutes to complete.
Then, fix the oocytes in 4%paraformaldehyde in DPBS for 15 minutes at 38.5 degrees Celsius, followed by 45 more minutes of fixation at four degrees Celsius. After the fixation, wash the oocytes by sequentially transferring them between three wells of 0.1%polyvinyl alcohol in DPBS. Use about half a milliliter of solution per well.
Then, transfer the oocytes to a well filled with 0.3%Triton X-100 in PBS, and let them incubate for 10 minutes at room temperature. To block non-specific signals, bathe the cells in DPBS with 1%BSA and 10%normal donkey serum. Incubate the cells for at least 30 minutes at room temperature.
After 30 minutes, apply the primary antibody, and then incubate the cells at four degrees Celsius overnight. The following day, wash the oocytes, and apply the secondary antibody for an hour at room temperature, shielded from light exposure. After applying the secondary, wash the oocytes again.
Then, before mounting, attach double-sided tape strips to slides to serve as coverslip supports. Now, mount the oocytes three to four per slide in a drop of non-hardening, antifade mounting medium supplemented with 20 micromolar of YO-PRO-1. Allow the oocytes to settle for about 10 minutes, and then apply the coverslips.
Out of 32 COCs retrieved by OPU from dominant follicles, 28 were in metaphase II.Using in vitro maturation, 37 of 44 COCs collected from non-dominant follicles were matured to metaphase II.The chromosomes of both groups were then compared. The in vitro-matured oocytes were often affected by aneuploidy, mostly hyperploidy. This was not observed in the in vivo-matured oocytes.
Next, the pole-to-pole spindle length and the diameter of the spindle at its maximum were both measured. The in vitro-matured oocytes were significantly longer and wider than the in vivo-matured oocytes. In the same samples, the global acetylation level of H4K16 was measured.
The results were as expected. Acetylation of H4K16 was significantly lower in in vitro-matured oocytes than in in vivo-matured oocytes. A well-trained team can process five mares in a morning of OPU with an 80%retrieval rate for the preovulatory oocyte and 50 to 60%retrieval of the immature oocytes.
During the OPU, always monitor the ess and well-being of the animal, and be ready to provide adequate veterinary care if needed. The experimental approach can be applied to oocytes of other species as well and is especially applicable where samples availability is an issue, such as with humans. Based on our results, studies must be undertaken to better understand the culture conditions needed for proper chromosome segregation and epigenetic changes.
The overarching goal is to improve in vitro maturation efficiency for both veterinary medicine and human fertility treatment.
