Once we had the ovary tissue in the lab, we sliced and then minced it to release any oocytes (eggs). Our goal was to extract the oocytes so we could mature them, fertilize them, and if all went well, cryopreserve the resulting embryos in our Frozen Zoo®. We found only three eggs that were big enough to develop in vitro with current technology. Because ovaries also contain small, undeveloped oocytes, we always cryopreserve the tissue so our frozen samples can be utilized when the appropriate technology is developed in the future.
Working with oocytes is rather complicated for us because oocytes extracted from tissue have not yet matured (i.e., their chromatin [DNA of the cell nucleus] is not yet at the right stage for fertilization). We must put them in culture to mature them, a process called in vitro maturation (IVM). The oocytes of different species require different culture conditions, which presents another challenge because we work with a wide assortment of species and for most no IVM protocol has been established. We had never had the opportunity to work with okapi eggs before, and so for these three eggs we used a model protocol that was developed for domestic cattle. Okapis, of course, are not in the bovid (cattle) family (they are most closely related to giraffes), but since there is no giraffe IVM protocol, we started with a protocol we know works for other hoofed mammals.
After 24 hours in culture, bovine eggs are ready to be fertilized, but would the okapi eggs be ready? If we had a large number of eggs, we could stain a few to see if the chromatin had matured, but with only three, staining wasn’t an option, so we decided to use the same timing required for cattle oocytes. There are two basic ways we can go about fertilizing an oocyte in vitro. If the sperm are relatively robust, fertilizing the eggs is quite easy: we just add a drop to the eggs in a Petri dish. Okapi sperm, however, are very sensitive to the freeze–thaw process, and no one has found an effective way to cryopreserve them. We knew if we thawed a vial, the sperm would be in poor condition and would not be able to break through the oocyte’s membrane. In these cases, we must fertilize by intracytoplasmic sperm injection (ICSI), in which sperm is actually injected into the egg.
In theory, ICSI is a simple procedure, but anyone who has ever attempted it can tell you that actually performing ICSI is no easy task. It involves tremendous hand–eye coordination and a certain amount of finesse. The people who perform ICSI at human fertility clinics practice daily for several months before they are allowed to do the procedure for a patient. Needless to say, with all the other projects we have going on here, we don’t get that much practice. But we do know people who are willing to volunteer their expertise in this area, and so we made some phone calls. None of the people who helped us in the past were available, but one of them asked a coworker and he was happy to help. And one full day after the eggs had been placed in culture, Bill from the San Diego Fertility Center came to our lab and injected the eggs with the thawed sperm. As we’d expected, the sperm was not very good, but no worse than the sperm Bill often sees at his job.
After the eggs were fertilized, we moved them into a medium that is good for embryo development and we waited. Would the eggs cleave? If they had matured properly and the sperm had successfully fertilized them, we would see some cell division after 36 hours. However, there was no cell division. Feeling rather disappointed, we took the eggs out of the culture and stained them to look for pronuclei (the DNA of the sperm or of the egg before the two have united). We did not find evidence that DNA from the sperm had decondensed after being injected into the eggs. We did, however, see the chromatin of the eggs, and we learned that we had fertilized the eggs a little too early. The eggs had been in the process of maturing but weren’t quite ready for the sperm.
From this we learned two important things: (1) okapi eggs take longer to mature in vitro than cow eggs and (2) the media in the bovine protocol were indeed appropriate choices for beginning work with okapi eggs. Now the next time we get ovaries from an okapi, we’ll be one step further in knowing what to do. I hope the rest of our okapis will stay healthy, and this event won’t repeat itself for a long, long time.
Dianne Van Dien is a research technician in the San Diego Zoo’s Reproductive Physiology Lab.
Read her previous post, Freezing and Thawing: Not So Easy