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 FERTILIZATION

10/8/97

| TIMING | BACKGROUND | MATERIALS | PROCEDURE | MATH | IMPLICATIONS | EVALUATION |

TIMING

• If done in combination with the GAMETES lab, one 45-50 minute period will be needed for both labs

BACKGROUND:

This is how our own lives began. The early events in sea urchins are very similar to that in humans and other animals. This lesson is often described as "THE best lab of the year."

>>> Do show the students just how little sperm is necessary to fertilize an entire beaker of eggs. (barely visible amount on the end of a glass pipet)

URCHIN JOKE:

Why does it take 200 million sperm to fertilize a human egg?

NONE OF THEM ARE WILLING TO STOP AND ASK DIRECTIONS.

MATERIALS:

• Urchin Kit
• Microscopes

PROCEDURE:

See GAMETES lab for spawning directions.

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: See SPERM DILUTION lesson.

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). 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:

1. Pour the egg suspension gathered from spawning into a 100 ml graduated cylinder
2. Fill to 100ml with sea water, 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.
3. Dilute the 2% eggs 1:20 (10 ml of 2% eggs into 180 ml of sea water) 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).
4. 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 sea water.

1. 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).
2. Let them see and focus on the eggs.
3. 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:

Acrosome reaction 86K

Early fertilization 118K

An important event at fertilization is the fusion (67K) of the cortical granules with the plasma membrane. This is what raises the fertilization membrane from the surface of the egg, helping to prevent further sperm entry.

Normal Development 94K

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

This lesson is often combined with the DEVELOPMENT lesson. If you do this you may want to start cultures of embryos at different times BEFORE class starts to let the students see the different developmental stages.

MATH:

If you do a sperm dilution series there will be some math, but largely this is an observation lab.

IMPLICATION:

1. 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.]
2. In humans there are no water currents and the volume of the female reproductive tract is relatively limited. Then, why does the male have so many sperm?
3. 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. Besides, would you want to give live birth to a sea urchin! ouch!]
4. Then why don't the parents take care of the young? [In fact the adults do in a way. After the embryo has grown large enough and metamorphosed into a young urchin they hide in the spines of the adults, living off of the scraps of food produced from the messy eating of the adults]
5. Because the urchin life involves two or more ecological niches, they are more susceptible to predation and exposure to environmental toxins. What environmental conditions, predators and toxins might they be exposed to at each stage?

EVALUATION:

• Active participation - walk around to each lab group and see that everyone is seeing the eggs and fertilization. Often they do not have the microscopes adjusted properly and are looking at air bubbles, or too light an image without enough contrast, etc. Some microscopes can be adjusted to give a partial dark field image. This is great for viewing eggs and sperm. see Introduction to the Microscope.
• Lab Reports - check for detail on drawings. reflections on size, number of sperm seen, timing of events, etc. Look for evidence of normal as well as abnormal development, such as polyspermic eggs with the membrane coming up in several locations at once.
• Evidence of Independent Thinking, such as - Did they try an experiment with varying sperm concentrations? Did they question if eggs would be affected by exposure to heat from the microscope light?
• Answers to IMPLICATION questions. Can be part of their lab reports or done as an oral activity with the entire class.

S.U.E. - CONTENTS