Skip to content

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:

Freshly spawned eggs of Strongylocentrotus purpuratus. 4 November 2015 © Allison J. Gong
Freshly spawned eggs of Strongylocentrotus purpuratus.
4 November 2015
© Allison J. Gong

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:

Two-cell embryo of Strongylocentrotus purpuratus, approx. two hours post-fertilization. 4 November 2015 © Allison J. Gong
Two-cell embryo of the sea urchin Strongylocentrotus purpuratus, approx. two hours post-fertilization.
4 November 2015
© Allison J. Gong

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.

16-cell embryo of the sea urchin Strongylocentrotus purpuratus. 4 November 2015 © Allison J. Gong
16-cell embryo of the sea urchin Strongylocentrotus purpuratus, approx. five hours post-fertilization.
4 November 2015
© Allison J. Gong

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

We are finally heading into the time of the year that our local intertidal sea urchin, Strongylocentrotus purpuratus, spawns. Usually I would wait until December or January to try to spawn urchins in the lab, but next week my students will be dissecting urchins in lab and I thought I might as well evaluate gonad development in the animals that are going to be sacrificed anyway. In early December I'm going to loan several urchins to a colleague who will be spawning them to show the earliest stages of development to students in one of the lower-division classes at the end of the semester. If I have any luck today, I'll be able to: (1) start my own cultures of urchin larvae so that I can show the later larval stages to students in my upper-division class; and (2) let my colleague know how likely it is that the urchins I loan to her will be spawnable.

4 November 2015 © Allison J. Gong
4 November 2015
© Allison J. Gong

I know, it ain't as romantic as the Ritz-Carlton but this is where I hope to make the sea urchins have sex. We have our victims lucky individuals in their "live only" tub, two beakers for eggs, two sperm dishes on ice, a box of glass pipets, a bottle of magic juice, and a syringe with needle to get the magic juice into the animals. Ready to go!

What is the magic juice, you ask? It's a solution of KCl in filtered seawater. I'm not sure exactly how it works, but here's what I think happens. We use a solution of MgCl2, a similar salt, to narcotize animals before dissecting them. Sea urchins sitting in a bath of  MgCl2 isotonic with seawater get sleepy pretty quickly, becoming entirely nonresponsive after about 30 minutes. I suspect that KCl has a similar effect. We inject KCl into the main body cavity of the urchin (I call this "shooting them up") and I think it relaxes the muscles surrounding the gonopores. If the gonads are ripe, then gametes are released as the gonopores open. If gonads are immature, then nothing happens.

A sea urchin is a well-armored beast. Its endoskeleton, or test, is a solid structure composed of calcareous ossicles that are perforated only where tube feet extend. Getting a needle through the test without damaging the animal is pretty much impossible, so we go through the peristomial membrane instead. This membrane surrounds the mouth on the oral (bottom) side of the urchin. It's the only way to get into an urchin without breaking the test.

The urchins don't seem to like being injected with KCl--they wave their tube feet and spines all around and generally appear somewhat agitated--but they don't suffer any lasting effects.

If the urchins are ripe, they should start spawning shortly after being injected with KCl. Sometimes the response is immediate, with urchins pouring out gametes through all five gonopores at an astounding rate. Today it was much slower. It took about 5 minutes for the first female to spawn:

Spawning female sea urchin (Strongylocentrotus purpuratus). 4 November 2015 © Allison J. Gong
Spawning female sea urchin (Strongylocentrotus purpuratus).
4 November 2015
© Allison J. Gong

That little blotch of pale orange is is the mass of eggs that she is spawning. At this point you can pipet off the eggs into a beaker of filtered seawater, but I decided to go the less-invasive route and simply invert the spawning animal onto a beaker filled with water and let the eggs drop to the bottom as they flowed out of her.

The only difficulty with this method is that the animal doesn't like being upside down and immediately tries to right herself. I kept having to remove her from the beaker and replace her in the orientation we wanted. I designated this urchin as F1. She gave us a decent number of eggs. A second, smaller female (F2) spawned just a few eggs but we kept them all.

Sperm get a different treatment. I had only one male spawn this morning and he wasn't exactly a gusher. I pipetted off the concentrated sperm into a cold dish on ice, and didn't dilute the sperm until the eggs were ready for fertilization.

UP NEXT: Fertilization and subsequent events.

%d bloggers like this: