Activity 5: Sea Urchin Fertilization & Cleavage

Question 1

Sea urchin species

Answer 1

Purple urchin
Strongylocentrotus pupuratus
Winter

Banded urchin
Lytechinus pictus
Summer

Sand dollar
Dendraster excentricus
Spring

Question 2

Urchins have reproductive organs that are arranged in five sections in a radial pattern around the mouth. It is possible to cause the ovaries or testes to release the gametes (eggs or sperm) by injecting a solution of ______________.

Answer 2

potassium chloride (KCl)

Question 3

How much KCl to inject:
Inject ________ of KCl per 10mL of urchin volume.

Answer 3

0.1mL (=0.1 cc)

Question 4

KCl activates nerves causing the muscles around the gonads to contract. This induces spawning.

Question 5

Gametes (eggs or sperm) are released through the urchin’s _________, on the upper or ________ side.

The colors of the gametes in males are different than those in females. Their gametes are harvested differently too.

(color) = male
(color) = female (the color depends on species)

Answer 5

gonopores;
“aboral”;
White gametes;
Yellow/pink/orange/dark red/green gametes

Question 6

are a class of echinoderms, marine invertebrates characterized by a spiny endoskeleton and radial symmetry, using a water vascular system for movement and feeding.

Answer 6

Echinoid echinoderms

Question 7

sea urchins and sand dollars are also known as ___________)

Answer 7

irregular urchins

Question 8

Keep them cold (___°C). If they are held all together in a single tank of seawater, 1 sea urchin spawning in the tank will stimulate all the others to spawn.

Answer 8

4–8

Question 9

On the oral side, you will see the hard white mouthparts (“_________”) surrounded by a tough leathery _____________. You will be injecting through this membrane into the perivisceral cavity.

Answer 9

Aristotle’s lantern;
Peristomial membrane

Question 10

For males- Immediately pour off the 1st sperm to get rid of perivisceral fluid, since this will interfere with the sperm’s ability to fertilize. Then allow the animal to shed into the watch glass or petri dish without diluting the sperm. This is called ______.

Answer 10

dry sperm

Question 11

For females- Allow the female to shed eggs into seawater by placing her, inverted, on top of a beaker of seawater. The beaker must be full enough so that seawater touches the aboral side of the animal. As the animal sheds, the eggs will drift down through the seawater and settle in the bottom of the beaker. After shedding is completed, decant off the seawater and add fresh. Do this twice. This washes the eggs of perivisceral fluid which interferes with fertilization.

Question 12

To fertilize the eggs, first make a _________ of 1 drop of dry sperm in 10 ml of seawater. Dry sperm are relatively inactive. Diluted, however, they become very active and quickly use up their energy stores.

Answer 12

standard sperm suspension

Question 13

The seawater for culturing should be sterilized by filtering it through a __________.

Answer 13

0.22-µm porosity filter

Question 14

_________ can be added (2 mg/liter) to retard bacterial growth, but is normally not needed in cultures maintained at cold temperatures.

Answer 14

Streptomycin

Question 15

A ________ is made by nicking the corners of the coverslip against some hard paraffin, so that crumbs of paraffin remain attached at each corner. This will give just enough spacer between the slide and the coverslip to avoid crushing the eggs.

Answer 15

footed coverslip

Question 16

large germinal vesicles = immature

Question 17

In the sea urchin, eggs are mature when they are ________. This means that meiosis is completed and the nucleus is relatively small. An immature egg will be a __________ with a huge nucleus (called a _________).

Answer 17

ootids;
primary oocyte;
germinal vesicle

Question 18

The egg has two extracellular coats: a ________, which before fertilization fits snugly around the egg surface and cannot be distinguished; and an outer layer of jelly. This jelly contains a chemical sperm attractant (a small polypeptide that is species-specific). It is not present in the jelly of an immature egg. From a mature egg’s jelly, this attractant diffuses outward, and
sperm swim up the concentration gradient. The jelly also contains a relatively species-specific fucose-containing polysaccharide that activates the acrosome reaction in the sperm. This polysaccharide binds to _________ on the head of the sperm, causing the ________ within the head to fuse with the cell membrane and release its enzymes.

Answer 18

vitelline envelope;
glycoprotein receptors;
acrosomal vesicle

Question 19

In the process, the acrosomal vesicle becomes inverted and greatly elongated by the assembly of __________. This elongate structure, the ________, is what will fuse with the egg cell surface, and it can be seen under the compound microscope under oil immersion if the contrast is maximized.

Answer 19

actin microfilaments;
acrosomal process

Question 20

The sperm first binds to the vitelline envelope using a species-specific cell surface protein called ______, which binds to a _________ protein on the vitelline envelope. The sperm then fuses with the egg cell membrane, and in so doing causes a brief influx of ________. This influx raises the resting membrane potential of the egg from ______ to _________.

Answer 20

bindin;
bindin-receptor;
sodium ions;
–70 mV;
above 0 mV

Question 21

Sperm cannot fuse with an egg whose membrane potential is above about _______, so this change in membrane potential effectively prevents any additional sperm from fusing. This is called the _________. It takes only one-tenth of a second to occur, but is not permanent, lasting only about a minute. Since the fast block depends on the availability of sodium in the medium, you can circumvent it by keeping the sodium concentrations in the surrounding seawater artificially low.

Answer 21

–10 mV;
fast block to polyspermy

Question 22

Binding of the sperm with the egg cell membrane also sets up a second block to polyspermy, the _______. This block takes a minute to occur, and it is permanent. You will see it as a lifting of the vitelline envelope away from the egg cell surface. This membrane then toughens (involving a chemical process much like tanning leather) and is now called the ________. Sperm cannot penetrate this tough layer.

Answer 22

slow block;
fertilization envelope

Question 23

The point of sperm entry can be identified by a cone-shaped elevation called the ________. It represents a tangle of microvilli that have elongated and wrapped themselves around the sperm.

Answer 23

fertilization cone

Question 24

The biochemical events that are causing the lifting of the vitelline envelope are initiated through the ________, which is set off by the binding of the sperm to the egg cell membrane.

Answer 24

phosphatidylinositol bisphosphate (PIP) cycle

Question 25

Once the PIP cycle is activated, there is a sudden spike in _______ levels within the egg due to the release of ------- from the ___________. This spike in ------ levels causes hundreds of vesicles (the __________) housed in the cortical cytoplasm of the egg to fuse with the egg cell membrane and to empty their contents into the _______ between the egg cell membrane and the vitelline envelope. The released contents of the cortical granules swell, lifting the vitelline envelope away from the egg cell surface, and tan the envelope, making it tough and impenetrable by other sperm.

Answer 25

calcium; smooth endoplasmic reticulum; cortical granules; perivitelline space

Question 26

The PIP cycle also causes a rise in internal pH by activating a _________. Sodium ions are pumped into the egg while hydrogen ions are pumped out. The resulting rise in internal pH activates the egg, causing protein synthesis to start and the egg to begin its development.

Answer 26

sodium-hydrogen ion pump

Question 27

Sequence for the development of fertilized sea urchin eggs.

Answer 27

1. Formation of fertilization envelope 2. First cleavage 3. Second cleavage 4. Third cleavage 5. Blastula 6. Hatching 7. Gastrula 8. Pluteus = 1-2 days

Question 28

In sea urchin, cleavage is _______ (the entire egg cleaves) and _____ (the cleavage planes are parallel or at right angles to the animal vegetal pole).

Answer 28

holoblastic; radial

Question 29

At the 16-cell stage, a small group of _______ are cleaved at the vegetal pole. These are the _________ and will be the first to show gastrulation movements.

Answer 29

micromeres; primary mesenchyme cells

Question 30

At 5–6 hours, the embryo is at the _____ stage, and by 7–8 hours, the embryo has hatched out of its fertilization envelope and is spinning around the dish using its cilia for locomotion. This is called a ________.

Answer 30

blastula; hatched blastula

Question 31

________ begins at about the hatched blastula stage. Primary mesenchyme cells first migrate into the blastocoel and then form a necklace of cells that will secrete the skeletal supports for the larva, the spicules.

Answer 31

Gastrulation

Question 32

spicules are first _______, and they eventually branch to have several arms.

Answer 32

tripartite rods

Question 33

Gastrulation continues by invagination of the vegetal plate to form the __________ (meaning “ancient gut”).

Answer 33

archenteron

Question 34

The forming gut will elongate, capped by a loose collection of cells, the ______.

Answer 34

secondary mesenchyme

Question 35

The secondary mesenchyme aid in elongating the gut with their _______, pulling the archenteron toward the far wall of the blastocoel and guiding it to its final destination. They later disperse to form mesodermal organs.

Answer 35

contractile filopodia

Question 36

As the gut is developing, the embryo goes through a _______ stage (between 18–20 hours), looking like an exquisite rotating jewel, and finally becomes a _______ (at about 22–24 hours).

Answer 36

prism larval; pluteus (echinopluteus) larva

Question 37

______ is an ornate organism, projecting long, delicate arms supported by branched spicules.

Answer 37

pluteus

Question 38

The timing of sea urchin development will vary considerably with temperature.