The first time I sanded a hole in a chicken egg, peeled back the shell, and looked at the embryo, I was amazed. I could watch the blood being pumped in and out of the heart, and once I looked through the dissecting microscope, I was even able to see the individual red blood cells flowing through the vessels. Now, having worked in the Reproductive Physiology Division for a year opening numerous eggshells for injections, I still take a moment to look in wonder at this spectacle.
I work as a research associate studying avian reproduction. We are currently working on a model system to aid in preserving endangered birds by transferring gonadal stem cells (self-renewing cells that are located in the gonads and produce sperm or eggs) into chicken embryos. The cells that I inject, or transfer, are adult gonadal stem cells collected from male quails. I inject quail cells to develop the method, because it is easier to get quail testes than testes from exotic birds.
Let me give you a little background in the development of birds as it relates to our project. Their gonadal stem cells circulate throughout the bloodstream before they migrate to the gonad, where they mature and produce either sperm or eggs. Because we inject the adult male quail gonadal stem cells into early stage chicken embryos, they will also migrate to the gonadal ridge. When this rooster reaches maturity, he will produce both chicken sperm and quail sperm derived from the injected cells. The goal is to be able to use this technique to collect and inject gonadal stem cells from valuable exotic birds that have died of natural causes, so that even after death the sperm of that valuable bird can still be used for artificial insemination.
Let me take you through a typical day of embryo injections. The first thing that needs to be looked after are the needles. As you can imagine, the blood vessels of a chicken embryo three days after it is laid are very tiny. Our needles are smaller than a strand of hair at the sharpened tip. It takes a lot of time and patience to learn how to grind these glass capillary tubes (small, hollow glass tubes) into a fine, sharp point. The needles are mounted onto a micromanipulator apparatus that holds the needle steady during the injections. I load the needle with quail gonadal stem cells that are stained a fluorescent red so that we know which cells we injected and which are the chicken embryo’s own gonadal stem cells.
I sand a hole about the size of a quarter into the shell of a chicken embryo using your everyday belt sander. Gently, I peel off the shell to expose the embryo. Next, I very carefully inject the cells into one of the blood vessels while looking through the microscope. I seal the egg up using melted parafilm (a kind of scientist’s cling wrap) and a hot glue gun (minus the hot glue) and put the embryo back into the warm incubator.
Did I mention that with the exception of needle sharpening, all of this is done in the dark? The fluorescent-stained cells will lose their color if exposed to long periods of light. To keep this from happening, we use only one light directed away from where we are injecting, and the injected eggs are kept in a darkened incubator. At the end of our experiments, we identify the injected cells within the gonads of the chicken embryo by looking for red fluorescent cells using a microscope or a laser cell counter. This tells us whether the quail gonadal stem cells migrated to the gonads as predicted.
Our next step will be to use gonadal stem cells from exotic birds to assess whether they will migrate in the same way as the quail gonadal stem cells.
Mandi Roe is a research associate for the San Diego Zoo Institute for Conservation Research.