CORE LAB

12/03/1998

SUMMARY: This lab is designed to provide students with a laboratory experience with sea urchins in which they will fertilize gametes and observe early developmental stages. In this investigation we will:

1. Induce spawning of gametes by injecting potassium chloride solution.
2. Collect the gametes released.
3. Fertilize the eggs with sperm.
4. Observe development from early cleavage to the pluteus larval stage.

TIMING

The initial lab exercise requires a minimum of one 50-minute period. [Good idea to start on a Monday]
The second day's activities require one 50-minute period to observe cultures, to analyze observations, and to discuss.
The observations will require approximately 15 minutes on subsequent days for about 5 days total.

BACKGROUND

Studies of sea urchins provide information on fertilization and development that apply to all organisms from jellies to humans. These eggs, then, provide a model embryo for understanding development in all forms. It is important to observe early embryonic development because it is during this time that the patterns of development of the organism originate.

Sea urchin gametes are the same size as human gametes and development is very similar through the gastrula stage. Both sea urchins and humans are deuterostomes, meaning that patterns of cleavage are radial and the mouth arises at a site distant from the site of gastrulation. The name deuterostomes means second mouth. In contrast protostomes follow a spiral cleavage pattern and the mouth forms near the blastopore, the origin of gastrulation. See Classification tree. Notice the relationship between humans and sea urchins.

For a visual overview of events in this lab see PATH OF DEVELOPMENT

SETUP

IMPORTANT - to maximize observations the first day, cultures of embryos should be started 1 1/2 and 3 hours before the class period. This will allow viewing of early cleavage stages. If you are teaching multiple periods remember to start cultures each period for subsequent periods to observe. See DETAILED timing and check-off list

MATERIALS

0.5M potassium chloride solution (3.73g of KCL in 100ml of distilled water)
sea water (or Instant Ocean, or artificial sea water)
fertile sea urchins, see also keeping adults
1-5cc syringe
plastic or glass pasteur pipets or eyedroppers
small tubes to store sperm (micro-centrifuge tubes work great) (1-5ml)
beakers a little smaller in diameter than the diameter of female urchins
microscopes (at least 10x and 40x objectives)

PROCEDURE

A. Spawning Animals:

To insure that there is at least one male and one female sea urchin it is best to have approximately 10 sea urchins on hand. (If sea urchins are being shared with multiple classes then save eggs and sperm and inject only one sea urchin for demonstration purposes.)

Inject 0.5 M potassium chloride into the sea urchin.

WARNING - ONLY TEACHERS should handle the syringe with the potassium chloride. Potassium chloride itself is not particularly hazardous, being the main ingredient in substitute salt formulations, but injection into a vein or artery can cause an electrical imbalance in the heart or in the brain resulting in death.

>>> see animation

Note how the needle goes over the top of the teeth to inject KCl into the body cavity and not into the mouth..

Side View /\ and Top View \/

Inject about 0.1-0.2 ml per inch of urchin width in each side of the urchin. A total of two injections are made. It is best to use as small a needle as possible as a larger needle leaves a larger wound, subjecting the urchin to infection. (#25 - #30).
Gently shake the urchin for a few seconds to mix the KCl solution inside the sea urchin. (shaking too hard can kill the urchin by ripping apart the delicate internal membranes).

Males: see animation. sperm are a milky white color

After injection, place the male urchin mouth side down onto a small dish, like a petri dish. (NOT UNDER WATER).
In a minute or two you will start to see the white sperm appearing on the surface of the urchin.
Collect the sperm "dry" in a pipet or eyedropper and place it into a small test tube. [Seawater "activates" the sperm reducing their life span from days to minutes! Keep concentrated until just before use.]
After you have collected all that you can, place the tube into a refrigerator (not freezer) or an ice bucket with ice. Sperm will keep at 4 C for 2-5 days.

Females: see animation eggs are pale yellow to orange to dark maroon, depending on species.

After injection place the female urchin mouth side up onto a beaker FULL OF SEAWATER. Eggs will not shed unless in contact with the seawater. Beaker should be slightly smaller than the diameter of the sea urchin.
The eggs will be shed into the seawater and collected at the bottom of the beaker. This can take from 10-30 minutes to finish shedding the eggs, although, within a few minutes you will have enough eggs to begin work.
It is best to keep the eggs and the urchins at the same temperature. (This is species specific.)

WHEN THINGS GO WRONG AND YOU GET EITHER [NO EGGS OR NO SPERM!]

B. Fertilization:

Appropriate sperm dilution is essential for successful fertilization. Too low a sperm concentration results in fewer eggs being fertilized (safer than too high). Too high a sperm concentration will result in polyspermy (more than one sperm per egg) and abnormal development of the embryo. There are a number of built in mechanisms to help prevent polyspermy, but they are not full proof and at very high sperm concentrations they can be overwhelmed.

see animations Just right sperm, Too many sperm, Too few sperm

Sperm Dilution:

A sperm dilution series is a useful way of showing just how few sperm are needed to fertilize eggs. For comparison purposes, it is important to use the same sperm concentration for each fertilization. Use a pasteur pipet to make the initial dilution . Mark a line part way up the narrow end of the pipet. see illustration below

Always use this quantity of sperm as the starting point for dilutions. Add "one notch" of sperm (2mm?) to 100ml of sea water (adjust this volume up or down if necessary to get proper fertilization levels without polyspermy). This is referred to as the STOCK SPERM SUSPENSION. A single drop of this suspension will be enough to fertilize 5 ml of a dilute egg suspension (1-2%). A single drop can be used to demo fertilization under a microscope. Although this is too many sperm for proper development, the embryos are unlikely to develop under a microscope for a variety of reasons (heat from lamp will make them too warm, the slide will dry out, etc.). This does, however, allow the student to witness, first hand, the raising of the fertilization membrane.

Eggs:

Pour the egg suspension gathered from spawning into a 100 ml graduated cylinder
Fill to 100ml with seawater, and let the eggs settle. Without centrifugation the eggs will swell (egg jelly swells) to about 2x their actual size. 4ml of eggs at the bottom of a 100ml cylinder is really a 2% egg concentration.
Dilute the 2% eggs 1:20 (10 ml of 2% eggs into 180 ml of seawater) to a 0.1% egg concentration. This concentration is better for long term storage of unfertilized eggs (a few hours to one day without antibiotics) or development (3-5 days).
Alternatively suspend the eggs in a large flask no greater than 1 cm in depth to insure adequate exchange of gases. (concentrations as high as 1% can be used in this way to make it easier for students to find the eggs in early stages of development)

Eggs can be re-concentrated to allow for easier viewing by letting them settle in a beaker or test tube and pouring off the excess seawater.

To fertilize:

Have the students place a drop of egg suspension on a glass depression slide under their microscope (a less that 1% egg concentration will allow students to see individual eggs).
Let them see and focus on the eggs.
Next, while the student focuses on the eggs, add a small drop of stock sperm suspension to the drop of eggs and add a cover slip. Quickly, let the students focus on the eggs and watch the fertilization membranes rise.

see animations:

Normal Development 94K

A Time Lapse Video from Sea Studios of early events, 241K

C. Development:

Keep dilute cultures of embryos, at less than 1% concentration, in sea water no more than 1 cm deep or in slowly stirring cultures.

Timing:

This is different for each species and is HIGHLY temperature dependent (at warmer temperatures under a microscope, division is faster). examples:

species	1st division	2 nd division	blastula	gastrula	pluteus
L. pictus 18C	90'	2.5 hrs	24 hrs	2 days	5 days
S. purp 12C	120'	3 hrs	24 hrs	2 days	5 days

What you usually have to do is set cultures up at different times.
See STORAGE for suggestions on how to store gametes for long periods of time.
See DETAILS for timing suggestions on how to set up cultures.

OBSERVATIONS

This lesson is often done when mitosis and meiosis are discussed. Students will definitely be able to observe the first division, if they are present when it occurs, but may not see "centering", "streak" and "metaphase".

Students should keep careful records of when each of the recognizable development steps occurs. Detailed drawings are very helpful. If you are lucky enough to have two species or two different temperature environments, students can compare stages and rates of development.

See URCHIN animation (55K)

IMPLICATIONS

What would happen if the sperm could not get into the egg?
How do organisms develop from the size of a sperm and egg to an adult? What directs these changes?
Why is there such a difference in the size of the egg and the sperm?
Water currents in the ocean are much stronger than any sperm. How do sperm and egg find each other? [Remind students that water currents can bring together as well as separate and not all eggs will be fertilized, that is why so many are produced.]
Why do sea urchins have external fertilization? [Discuss the life style of the developing sea urchin embryo in the water column compared to a very different ecological niche of the adults on the ocean floor.] What are the adaptive advantages of external fertilization? What are the adaptive advantages of internal fertilization?
What would happen if one cell was removed or damaged at the 2 cell, 4 cell, 8 cell stage?
What would happen if the two cells were separated after the first cleavage and allowed to develop on their own?
Why do cells with multiple chromosomes need the complex steps of mitosis to divide in comparison to the steps of binary fission in bacteria?

EVALUATION

Active participation. This is a group assignment. Make sure everyone is allowed to and does participate.
Lab reports - layout, tables, drawings, etc.
Answers to IMPLICATION questions.

NEW - self evaluated "Check Your Understanding"

S.U.E. - CONTENTS