egg sucking

It is no longer the hot summer it once was. The days so far have been beautiful. The campus is speckled with these trees--half orange and red, the other half still green and yellow, that show a rainbow of autumn colors. Again, its just been gorgeous. The fields, however, are a little bare since the harvest has come and gone. Soon though there will be new crop.

School has been fine; 2 weeks til first exams. Rotation has been going well, concentrating on improving my egg sucking skills. Getting better at identifying them, but still moving slower than I should. A brief explanation into the technical work of egg sucking:

What you are looking for is the germinal vesicle oocytes (Figure 1). This is a clean picture. In reality, you must hunt for these in a brownish pink solution amongst bad oocytes and secondary (larger) oocytes you don't want, and all other cell types out of the ovary that you just mascerated.

Figure 1. Notice that clear circle in the middle of the egg. Within that, is the nucleus. We refer to these eggs as the ones with the 'winkies'.

A good micropipette can make or break you.
1) Pipette is too big-- this will just suck up everything.
2) Pipette is too small--can dent your oocytes and also becomes easily clogged with other unwanted cells.
It will take you longer if you encounter these issues, and oocytes are very fickle, they start degenerating if you don't move quickly. They start looking like this (Figure 2) and even this (Figure 3).

Figure 2. The sides of the oocytes become dented and the nucleus is no longer visible.

Figure 3. The zona pellucida surrounding the oocyte has become thick and dark granules appear inside the oocyte.

The oocytes should not really sit for more than 15 minutes. After collection, you culture them in an incubator overnight, then check how many of them have produced a polar body (Figure 4). Collecting them and putting them in culture inspires the oocytes to undergo meiosis I, to produce the polar body.

Figure 4. Oocyte and its 1st polar body.

Recombination has occurred, but each cell is still diploid and will need one more division to become haploid. Since most of the lab is interested in the recombination events during meiosis I, this is where most people fix their oocytes onto slides. The percentage of ones that did produce a polar body must be 85%, or else you cannot use any of the oocytes. I have yet to fulfill that, but according the Ailene, the lab tech/'egg queen' it can take people weeks-months to get this perfected to get 85%.

On Friday I practiced collecting some 2 cell embryos from pregnant females (Figure 5).

Figure 5. Mouse 2-cell embryos. Have undergone 1 round of mitotic division, polar body still visible.

The embryos are much more stable than the oocytes, and definitely more forgiving when it comes to time. They are also quicker to collect because instead of looking for unfertilized oocytes still in the yucky brownish slurry mess of an ovary, oocytes have already been ovulated out of the ovary, become fertilized and continue their travel down the oviduct/fallopian tube, where you find them. The oviduct which is a much tougher and whiter piece of connective tissue, making it much easier to see and collect the embryos. This is encouraging since my project involves working with embryos (8-cell).

My animals began getting their BPA dosages and I'll be collecting their embryos on Tuesday and Thursday. I may not be able to FISH them because our lab ran out of the X chromosome probe I would need. I may wait for it to come in, or use another probe to look at something else entirely. I'll have to caucus with Pat and the lab on Tuesday to determine the best plan of action. Only 2 more weeks of my lab rotation!