Learning Objectives:
- Understand why sea urchins are used in this lab.
- Demonstrate an understanding of basic sea urchin anatomy
- Distinguish mature eggs from immature eggs.
- Distinguish unfertilized eggs from fertilized eggs.
- Demonstrate an understanding of the parts of an oocyte (egg).
- Demonstrate an understanding of the events of fertilization and the effects that interfering with these events.
● Humans and many model genetic organisms have internal fertilization.
● This makes it difficult to study the early events of fertilization in them. It is easier to use organisms with external fertilization to study these early stages.
● Developmental biology has revealed that these events in animals are well conserved and have been shown to be very similar in all animals. Therefore, it is likely that most of these events are similar (with only slight difference).
● Echinoderms are a phylum of marine animals containing over 7,000 species. The name comes from the Greek for "spiny skin".
● Echinoderms include sea stars, sand dollars and sea urchins (Figure 3.1) all of which have external fertilization.
● Sea urchins are easy to acquire through biological supply companies and are easily maintained in a large aquarium.
● Additionally, it is easy to induce echinoderms to spawn.
● Spawning is induced by injecting the sea urchins with 1-2ml of a 0.55M solution of potassium chloride (KCl) through the perisomal membrane.
● KCl induces smooth muscle contraction including in the smooth muscle surrounding their gonads causing them to release their gametes.
Figure 3.1 shows examples of common echinoderms. From left to right; a sea star, a sand dollar and a sea urchin
Sea Urchin Anatomy
• Sea urchins are covered in sharp spines to protect them from predators.
• The lower surface, containing the mouth, is referred to as the oral surface.
• The mouth contains teeth that the sea urchins use to scrape food off rocks and pilings.
• The upper surface is referred to as the aboral surface.
• The aboral surface contains a few openings including the anus, the gonopore, and the madreporite.
• The gonopore is the opening through which the sea urchins release their gametes from their gonads.
• The madreporite is an opening where sea water enters in to their water vascular system. Water enters the Ring canal and is distributed via ampullae (not shown) into individual tube feet.
• The tube feet allow the sea urchin to move and suction tightly to a surface.
Watch the video to see how sea urchin spawning is induced in the lab and how you can distinguish males from females.
First let's become familiar with the sea urchin's eggs.
● When we artificially induce spawning by injecting the sea urchins with KCl most gametes in their gonads will be released whether they are mature or immature.
● You will need to distinguish the immature eggs from the mature eggs since the immature eggs cannot be fertilized.
● You can distinguish an immature sea urchin egg from a mature egg by its appearance.
● An immature egg has a large nucleus called a germinal vesicle. The mature egg has a much smaller nucleus.
● Look at Figure 3.3 to see the difference between an immature egg and a mature egg.
● Unlike mammalian oocytes that are arrested in meiosis II until they are fertilized, the mature sea urchin eggs are ootids that have completed the second meiotic division already.
Parts of the mature unfertilized egg
● Use Figure 3.4 to identify the parts of a mature unfertilized egg.
The mature egg (ootid) has two extracellular coats outside its cell membrane:
1. The vitelline envelope - before fertilization it fits snug up against egg cell membrane and cannot be distinguished.
2. An outer jelly coat– contains chemical sperm attractants (a small polypeptide) that are species -specific.
●The jelly coat is not present in the immature oocyte.
●The chemical attractant diffuses outward from the jelly coat. Sperm swim up its concentration gradient.
●The jelly coat also has a relatively species-specific fructose containing polysaccharide that activates the acrosome reaction in sperm.
● This polysaccharide binds to glycoprotein receptors on head of sperm, causing the acrosomal vesicles within the head of the sperm to fuse with the cell membrane and release enzymes that coat the head of the sperm and eat through the jelly.
● The cortical granules in the outer (cortical) layer of the egg will fuse with the egg cell membrane when fertilization occurs.
● You can distinguish a fertilized egg from an unfertilized egg by the presence of the fertilization envelope around the fertilized egg.
● Notice in Figure 3.5 the fertilized egg appears to have a halo surrounding it.
● You can also distinguish the point of sperm entry by a mark left behind as the sperm enter. There is a cone-shaped elevation in the fertilization envelop called the Fertilization cone. It is formed by a tangle of microfilaments that elongated and wrapped themselves around the sperm.
● As the sea urchins release their gametes eggs are collected in a beaker of sea water by inverting the female sea urchin over the beaker and letting the eggs sink to the bottom of the beaker of sea water.
● Sperm are collected by inverting the male sea urchin over a dry watch glass or petri dish lid.
● It is important to keep the sperm free of seawater until you are ready to use them. "Dry sperm" can last hours to days and still be capable of fertilizing an egg once placed in seawater.
● When you are ready to use the sperm a standard sperm solution is made by adding 1 drop of dry sperm to 10 ml of seawater.
● This activates the sperm, and they will now start swimming. This will use up their energy supply in about 20 minutes.
● If your standard sperm solution is more than 20 minutes old it will likely be unable to fertilize the eggs.
● A drop of fresh sperm solution is added to a drop of mature eggs the sperm will detect the chemical attractant produced by the jelly coat and swing toward them.
Observe the following event of fertilization in Figure 3.6.
1. The acrosome releases enzymes to digest the jelly coat.
2. Actin filaments bind to receptors in the vitelline envelope.
3. The sperm and egg cell membranes fuse and become depolarized by an influx of sodium from the seawater preventing additional sperm from fusing with the egg cell membrane.
4. The sperm nucleus enters and fuses with the egg nucleus.
5. The vitelline envelope swells to form the fertilization envelope.
● Sperm first bind to a receptor on the vitelline envelope using a species- specific cell surface protein called Bindin.
● The sperm then fuses with the egg cell membrane. The fusing of the two membranes sets off a series of events including an influx of sodium from the surrounding seawater, a spike in calcium ion concentration in the eggs cytoplasm and an increase in the pH of the egg cytoplasm. All of these events are key to fertilization occurring properly. Let's look and see what each causes and how they are involved in fertilization
Influx of sodium ions from surrounding seawater
● The sodium influx increases the resting membrane potential of the egg from -70mV to over 0mV.
● Sperm cannot fuse with an eggs with a membrane potential above -10mV.
this change in the membrane potential is called the fast block to polyspermy and effectively prevents additional sperm from fusing with the egg cell membrane.
● It is important that one and only one sperm fuses with the egg. If two or more sperm fuse the resulting zygote will be have additional copies of each chromosome and will not be viable.
● The fast block to polyspermy occurs in 1/10 of a second after the sperm and egg, membrane fuse (that really is fast). ● This change in the resting membrane potential only lasts about 1 minute.
Calcium ion spike
● Calcium ions are released from the smooth endoplasmic reticulum (sER) inside the egg.
● The spike in calcium ion concentration caused hundreds of Cortical granules (vesicles within the outer cortex of the egg) to fuse with the egg cell membrane and empty their contents into the perivitelline space (space between the egg cell membrane and the vitelline envelope).
● This caused the vitelline envelope to lift off of the egg surface.
● Substances with in the granules also cause the vitelline envelope to toughen.
● The vitelline envelope is now referred to as the fertilization envelope.
● Sperm cannot penetrate this toughen fertilization envelope. This is refereed to ass the slow block to polyspermy. It takes about 1 minute to occur and is permanent.
Increase in internal pH
● Binding of the sperm to the egg cell membrane activates a sodium ion pump.
● Sodium is pumped into the egg while hydrogen ions (H+) are pumped out. This causes an increase in the internal pH of the egg.
● The rise in internal pH activates the egg causing protein synthesis to start and the egg to begin to develop.
● We can learn a great deal about normal fertilization by interfering with it and seeing what happens.
● Many events of early fertilization rely on the jelly coat.
● We know this by observing what happens when the jelly coat is missing, as in an immature egg, or if it is experimentally removed.
● The jelly coat is easily removed by placing the eggs in seawater that has been adjusted to a pH of 5.
● The sodium in the seawater also plays a vital role in the early steps of fertilization. We know this because we can create sodium free seawater and observe what happens.
● Calcium ions also play a crucial role in the events of early fertilization.
● We can experimentally increase the calcium ion concentration in an unfertilized egg by adding a calcium ionophore, A23187.
● This ionophore transports calcium ions across the egg cell membrane mimicking the calcium spike that normally occurs when the sperm and egg cell membranes fuse.