Mitosis: In Vivo and In Vitro Microscopy

Eileen Fong, Jin-Hong Kim, Alexander Lin

Mitosis in Sea Urchin Embryos

Mitosis: In Vivo and In Vitro Microscopy - Final Project

.: Introduction

The goal of this lab was to visualize mitosis in vivo by employing time-lapse microscopy of the fertilization and subsequent nuclear division of the embryo using a high resolution camera. The following types of sea urchins were utilized in the experiments:

Lytechinus variegatus (green sea urchin)
Lytechinus variegatus are handsomely pink and white, with an explosion of short spines emanating from a fat round body. Eggs are remarkably clear and easy to study, particularly for demonstrating mitotic spindles. Huge volumes of eggs can be produced from a single large specimen, hence it is also prized by seafood loving gourmets. Specimens are fertile May through September.

Image of: Lytechinus variegatus (green sea urchin)

Arbacia punctulata (purple-spined sea urchin)
This urchin bristles long, sharp, formidable looking spines as it rapidly moves about the aquarium. Their powerful teeth scrape away algae, and chew into sponges. Although they add action and beauty to the salt water aquarium, they have been classically used in embryology. Specimens are fertile mid-January through April.

Strongylocentrotus purpuratus (purple sea urchin)
Purple sea urchins are found on the pacific coastline from Alaska to Cedros Island, Mexico. The purple sea urchin thrives amid strong wave action and areas with churning aerated water. They must be kept in cold conditions. This urchin has adapted the ability to burrow itself into the substrate, often times rock. It uses its five bony teeth in concert with its spines to slowly gauge and scrape away at the substrate. The result is a depression in the substrate into which the rest of the urchin can settle with a firm hold. This is a unique feature that can sometimes prove deadly. When S. purpuratus is young, it may begin to scrape into the substrate. As it grows, the urchin may find that it has trapped itself for life. January, February, and March are the primary reproductive months.

Sea urchin

.: Methods & Results

Slide Preparation and Rehydration

Based on studies from Week 1 & 2, one of the biggest issues we faced with time-lapse microscopy was dehydration of the medium that the cells were in due to heat and light from the microscopy, both necessary to visualize the processes. To prevent dehydration we employed two suggestions:

1. Provide a slide well using molten wax to increase the volume of artificial sea water between the slide cover and the slide as shown below:

2. Then monitor the slide every 30 minutes to determine the degree of dehydration. If the slide begins to dehydratre, use a very small pipette tip and draw out approximately 10 uL of artificial sea water and slow push it under the slide. Let diffusion even out the dried portions of the slide. Be careful not to expel the sea water too quickly on to the slide otherwise the image will shift due to the force of the flow.

Strongylocentrotus purpuratus (purple sea urchin)

Efforts were made to obtain purple sea urchin as they are abundant along the California coastline and they are in the ideal time of fertility. A quick road trip was made to the Palos Verdes Peninsula, just south of San Pedro. At the tip of the peninsula, White Point Beach is home to some of the most diverse tidepools on the West Coast. Hundreds of purple sea urchins could be found there (see Picture Gallery). It is important to arrive at the tide pools at low tide (check tide tables!) Approximately 40 sea urchins were collected, transported at sea water temperature, and transferred to the Keck 040 urchin tanks. Unfortunately there was no way to control the temperature in the tanks. All of the sea urchins perished before they could be utilized for experimentation. Furthermore, fertilization and embryology would have to occur under cold water conditions, making this a difficult

Arbacia punctulata (purple-spined sea urchin)

Although the the purple-spined sea urchin were vey hearty, fertilizing them proved to be very difficult. Several attempts were made to fertilize the species but all attempts were either unsuccessful or abortive. Either the eggs would not fertilize at all or they would begin mitosis but would stop at some time in the process. In the movie shown below, on the left, you can see that the cell begins to go through telophase but at some point it no longer proceeds and eventually fuses back into one cell again. A similar deformity occurs in the movie below, right, where during telophase, the process is "corrupted" as can be seen from the shape of the inner membrane of the cell.

Deform.avi (2.7MB) Deform2.avi (4.3 MB)

Lytechinus variegatus (green sea urchin)

Fortunately, the green sea urchin was much more amenable to fertilization. The only difficulty with this part of the study was that 90% of the sea urchins shipped from Carolina Bio were dead on arrival (see image to the left). Only two of the twenty sea urchins survived the journey of which we were very fortunate to have one female and one male. The fertilization was successful and the images provide an excellent view of mitosis. The nucleus can be identified and followed. Unfortunately at the very end of the images, dehydration does take place but not before exceeding well over 6 cell cycles.

Success.avi (9MB)