Sea urchins

Lytechinus variegatus from North Carolina
Develop normally at room temperature, large, optically clear.
Keep them in ambient temperature running sea water tank.

To collect gametes:

Materials:
0.5 M KCl
50 ml glass beaker
1 ml disposable syringe, 24 gauge needle (can use many times)
0.5 ml eppendorf tubes
pasteur pipets
box 12 x 75 mm test tubes

Each urchin has 5 gonads. To save urchins, spawn gametes from one gonad at a time:
Locate the madrepore on the top of the animal, then find the gonopore clockwise to it.
Turn the animal over so that the gonad corresponding to the gonopore is slanted downwards.
Inject 0.15 to 0.2 ml of 0.5 M KCl between aristotle's lantern and the test in the region of the gonad.
Hold animal in same position for about 30 - 40 sec
Turn over in a way that the KCl does not run over other gonads, and check whether the animal is male (white), female (yellow/orange) or not gravid (nothing).

If male, collect the sperm "dry".
Use a pasteur pipet to transfer sperm to an eppendorf tube.
Transfer tube to ice bucket or refrigerator.
The sperm are good for at least a day, maybe 3 days.

If female, transfer the eggs by pasteur pipet to a test tube.
(can add a bit of sea water to the top of the animal to make transfer easier)
Once you have collected the eggs, you may want to wash them one or two times by removing the sea water and replacing with fresh sea water.
Keep eggs at room temperature or somewhat cooler (20 C), not on ice.
Eggs are good for 4 hrs, possibly 12 hrs, maybe even overnight, but best not to.

Keep track of males and females, and which gonopore has been injected.

Alternatively, if there are many animals, or there is use for many eggs, the whole animal can be spawned by injecting 1 ml 0.5 M KCl.

Fertilization

Materials:
1 ml eppendorf tube
microscope slide
Microscope with 10-16x objective

In a test tube:
Put a drop of eggs into 1 ml sea water in a test tube.
Make a 1:1000 sperm suspension by adding 1 ul sperm to 1 ml sea water.
(You can get approximately 1 ul sperm by taking up 1 mm in the tip of a pasteur pipet)
Add one or two drops sperm suspension to the eggs
(sea urchin sperm lose ability to fertilize within a few minutes after diluting into sea water, so use them within that time)
Wait 3-5 minutes before transferring fertilized eggs to a slide, as transfer may shear off fertilization envelope.
Look at eggs in a microscope.
With good animals, you should get 99% fertilization.

Exercises:
devise a chamber which allows you to add sperm while watching the eggs in the microscope
Try to watch and see the fertilizing sperm - it suddenly straightens out and becomes still
Watch for the fertilization cone, which develops in the first 2-3 minutes then fades away.
Watch for the appearance of the sperm aster (about 5 min-10 min)
Watch sperm aster migration, egg pronucleus migration, and nuclear fusion.
Try to see when the two poles separate
Determine the timing of nuclear envelope breakdown and cleavage

Some materials
Double stick tape
silicon grease
22 x 22 mm cover slips

Reading material:
Kume and Dan, Invertebrate Embryology (chapter 1 sections on sea urchins)
Gilbert, Developmental Biology, to see cleavage patterns for sea urchins
Maienschein, Transforming Traditions, about cleavage patterns

Starfish

Asterina miniata from California.
Large, easy to inject.
Keep in cooled sea water (12-18 C)

To collect gametes

Materials:
MBL Ca free sea water (CFSW)
Ice bucket
2 50 ml glass beakers
sample corer
small forceps
microscope
microscope slides, pasteur pipets

Locate the madrepore for orientation in describing which arm is being used
Use small sample corer to make hole in starfish along side of arm, near the body.
Reach in with small forceps and pull out orange ovary parts.
In a ripe animal, the ovary is very large; you don't need all of it.
Transfer ovary parts to beaker with CFSW on ice
The CFSW causes the follicle cells around the oocytes to come off
After about 5 min, swirl the beaker, and transfer the free oocytes to another beaker on ice, leaving the larger parts in the original beaker
Monitor the follicle cells on the ooyctes. When about 1/4 of the follicle cell layer is left on the oocytes, transfer them to room temperature sea water.
Wash corer and forceps in water, as the sea water corrodes them.

Keep the oocytes at 20C or cooler.
They are good for a day's work, and can be cultured for about 3 days, but they go downhill

To collect sperm:
Use same procedure for making hole in starfish arm
Pull out a small piece of testes and transfer it dry to an eppendorf tube
Keep on ice or in refrigerator
Good for 1 day.

Maturation and fertilization:
Fully grown starfish oocytes are 170-180 microns diameter. Smaller ones may not undergo maturation
1-methyl adenine at 1 µM causes maturation of fully grown oocytes (not of smaller ones)
20-30 min after 1-MA, the germinal vesicle breaks down.
Normal fertilization occurs after ~10-15 minutes after germinal vesicle breakdown and before the 1st meiotic division. There is no meiotic block during maturation (in contrast to frog).
Use similar techniques for starfish fertilization as with sea urchins
The fertilization envelope in starfish is slower to come up and is lower.
The fertilized egg must complete the 1st and 2nd meiotic divisions before the sperm nucleus can do anything, so the first cleavage of starfish eggs is late, sometimes 2 hrs after fertilization.