Having obtained decent-ish amounts of gametes from sea urchins, the next step is to get eggs and sperm together. The first thing I did was examine the spawned eggs to make sure they were round and all the same size. Lumpy eggs or a variety of sizes of eggs indicates that they are probably not fertilizable. These eggs from F1 looked just about perfect:
Note that the eggs are all similarly sized (80 µm in diameter) and round. These look good to go.
The next step is to dilute the sperm in filtered seawater and introduce a small amount to the eggs. The sperm need to be diluted because, believe it or not, in this case too much of a good thing is bad. There's a phenomenon called "polyspermy" which is pretty much exactly what it sounds like: an egg being penetrated by more than one sperm. Polyspermy leads to wonky development down the road, and while it probably rarely happens in the field, where sperm would be diluted immediately upon being spawned, it definitely does occur in the lab. However, eggs are smart and have evolved a couple of mechanisms to prevent polyspermy.
The fast block to polyspermy occurs within a few seconds of the fusion of the sperm and egg plasma membranes. As the sperm nucleus begins to enter the cytoplasm of the egg, Na+ ion channels in the egg membrane open and cause a depolarization of the egg membrane; this depolarization makes the egg impenetrable to other sperm. However, the egg membrane cannot remain depolarized indefinitely, so after about a minute the slow block to polyspermy takes effect.
The slow block is the rising of the egg's vitelline layer above the surface of the egg, creating what we call the fertilization membrane. This envelope acts as a physical barrier against additional sperm. The really cool thing about studying fertilization in sea urchins is that you can watch it happen in real time. I mean, how often do you get to observe the formation of a brand new life at the moment that is is being formed?
In this video there are 2.5 eggs in the field of view. Concentrate on the two whole eggs. The one on the top has already been fertilized, which you know because you can see the fertilization membrane surrounding it. You can also see a lot of sperm zooming around. Keep an eye on the lower of the whole eggs; can you see the rising of its fertilization membrane?
Of the two female urchins that spawned for me this morning, F2 had only a few eggs to give but her fertilization rate was 100%. F1, on the other hand, spawned a lot of eggs but only about 50% of them were fertilized. I have no explanation for this. Sometimes (quite a lot of times, actually) things simply don't work.
That said, at our local ambient temperature the first cleavage division occurs about two hours post-fertilization. That's when I saw this:
A few hours later the embryos had progressed to what I think is the 16-cell stage. At this point it starts getting difficult to distinguish the different cells without focusing up and down through the embryo. But if you know what you're looking at, the three-dimensional structure does make some sense. In the embryo below I can talk myself into seeing two rings of eight cells each, one ring lying on top of the other.
If the embryo is at the 16-cell stage, then it has undergone four cleavage divisions. The early divisions of an embryo are called "cleavages" because the cells divide in half to form equal-sized daughter cells. In other words, the cell cleaves. During cleavage the embryo doesn't grow, which means that the average cell size necessarily decreases. Cleavage divisions will continue for a total of about 24 hours, resulting in a stage called a blastula.
UP NEXT (hopefully): hatching and swimming