From the Epel lab depel@leland.stanford.edu

The deterioration of oocytes after ovulation results from spoilage by contaminating bacteria. The evidence for this is that a variety of antibiotics that suppress bacterial growth retards deterioration. Also, there is a direct correlation of the presence of bacteria with egg deterioration, indicating that deterioration is actually egg spoilage.

These bacteria come from many sources. Indeed, our microbiological study indicates many different species of bacteria can be associated with spoilage, suggesting that the "infection" is by opportunistic bacteria (mostly Vibrios). Their initial growth is most likely on nutrients provided by a few lysed eggs, and the increased titer of bacteria then allows further spoilage of the eggs from the secretion of digestive enzymes the bacteria. The bulk of the bacteria probably arise from the KCl injection procedure to induce spawning, in which the emptying of the digestive tract deposits bacteria amongst the eggs. Bacteria are also present in the seawater and on the urchin surface and these also contribute to spoilage.

Using the techniques described below, it is possible to ovulate and store eggs for at least one week with excellent subsequent development. Strongylocentrotus purpuratus is the more difficult species, and one week storage is typical. Lytechinus pictus is easier to store, and we have maintained eggs for as long as three weeks (fertilization rate around 70-80% by this time).

We have not developed any new procedures for sperm storage. As recognized for many years, collecting the sperm "dry" (with as little dilution as possible), and storing them in the refrigerator at 4-5 ^OC, provides sperm that are viable for long periods of time (we find that such cold temperatures are detrimental to eggs, as noted below).

Given the correlation of spoilage with bacteria, the objective for long-term storage is to minimize bacterial growth as much as possible. Although it is impossible to be completely sterile with eggs prepared with current procedures, minimization of the initial bacteria titer leads to longer storage and normality of development. Our experience has been that improved practices leading to sterility has led to longer storage. It is also important to minimize egg lysis, since any cell breakage provides material which residual bacteria will utilize and grow.

1.Sterile seawater. Use bacteria-filtered seawater. We use the 0.2 micron disposable Nalgene filters and collect the seawater into sterilized glassware.

DO NOT USE AUTOCLAVED SEAWATER. Something happens during autoclaving that results in the clumping of the eggs during storage.

2. Preparation of antibiotic solution. We have tested a large number of antibiotics and find that a combination of two sulfa drugs that interfere with bacterial folic acid metabolism provides the best control of bacteria and subsequent normal development.

The drugs are a mixture of two antibiotics, sulfamethoxazole (final concentration at 200 ugms/ml and trimethoprim(final concentration of 10 ugms/ml). These concentrations are almost at the limits of solubility in seawater and getting them into solution is time-consuming. Continuous stirring and periodic adjustment of the pH is required until they are dissolved (~2 hours). At the end of the dissolution procedure, the antibiotic solution is passed through the Nalgene bacterial filter.

Most of our procedures have been developed using KCl-induced spawning (we have also used an electric shock method as described later). The urchin surface is first washed in sterile seawater and then injected in the peristomal membrane with 0.5 M KCl. After the first gametes are shed, the urchin is washed again with sterile seawater, removing the first exudate of eggs, which is discarded. This washing is critical, as the bulk of fecal exudate occurs shortly after shedding commences.

Lytechinus pictus: With L. pictus, the eggs are collected by inverting the urchin over a sterile beaker containing sterile seawater. Do not let the urchins shed for more than 5-10 minutes (longer times increase the probability of infection). Any fecal pellets are removed with a sterile Pasteur pipette. The eggs are then washed three times by aspiration and resuspension with sterile seawater. It is not advisable to wash the eggs too many times, as extensive washing increases the probability of egg lysis.

Strongylocentrotus purpuratus: Storage of S. purpuratus is more difficult than L. pictus . Eggs can be stored for one week, but longer storage periods are problematic. The eggs are less hardy, contamination is more frequent, and at times we have had problems with protozoan contamination.

The ovulation procedure that works best for S. purpuratus is either "dry spawning" or electric shock/dry spawning. For dry spawning, the urchins are injected with KCl. Upon shedding of eggs, the aboral surface is quickly washed with sterile seawater and then the animals are allowed to shed with the aboral surface UP (not down into a beaker of seawater). Keeping the animal in the up position minimizes fecal deposition.

To obtain the eggs, we suck the eggs directly from the surface with a sterile Pasteur pipette with a large opening (Fisher large volume Pasteur pipet/ cat # 13-678-8). A problem with this procedure is the increased probability of egg lysis. To avoid this, first wet the pipette with sterile seawater, and then suck up the eggs as gently as possible, avoiding rapid inspiration and potential lysis of the eggs. The eggs are gently added to sterile seawater and then the washing procedure as above. This seems to minimize infection and has given consistent good results with S. purpuratus.

The other approach is to use electric shock, which works when the urchins are at the height of the season and quite gravid. The electrostimulation apparatus for S. purpuratus is composed of two stainless steel electrodes (we use weighing spatulas) that are 8 cm apart. They are mounted on a lucite sheet that fits over an one liter beaker. The apparatus, which utlizes 120 V AC current, must be properly grounded and protected from the user so that no electric shock can occur. The leads to the electrodes are attached to a variac -type controlled voltage transformer.

The urchin is placed aboral side up into sterile sea water, which does not quite cover the urchin, at the bottom of a beaker. The electrodes are placed into the water and 30-50 volts are applied until the urchin begins to shed. The urchin is then removed from the beaker, washed with sterile seawater, and then allowed to release eggs unto the aboral surface as described above for the dry spawning procedure. Note with this electrical shock procedure, that the animals continue to release gametes after cessation of the electrical stimulation.

Following the third wash, the eggs are placed into 60 ml Corning tissue culture treated flasks (cat# 430168) for long-term storage. We find that the eggs store best in a 0.1to 0.2% suspension. Our usual procedure is to adjust the concentration of the last wash to 0.2-0.4% (volume/volume) and add 30 mls of this to 30 mls of SW-antibiotic solution into the culture flask. The bottles are laid down on the wide side, protected from light and stored at 15C in a constant temperature room. [Refrigerator temperatures (4-5C) are not good for storage; the eggs deteriorate]. Agitation or shaking does not appear to be necessary (there is some agitation from the fan in the constant temperature room, and this potential source of agitation appears to be adequate)

As noted above, we have been able to obtain excellent fertilization and development with S. purpuratus and L. pictus eggs stored for one week and continued support with L. pictus of fertilization and development for up to three weeks. Carla Falugi of Genoa University has also used earlier versions of this method with urchin eggs in Italy. She has transported the eggs by train to a collaborator in Germany and has reported excellent results.

Fertilization and cleavage are no different from controls. Later embryonic development is also very good; the one difference we have seen is as that development of the hindgut is affected with eggs as they are stored for longer and longer times. With L. pictus, after 2-3 weeks of storage, the hind gut the hindgut is more tube-like and deflated in a significant percentage of cases (as opposed to being large and inflated).

A major problem in obtaining sterile eggs is that standard KCl-spawning also stimulates emptying of the intestinal tract with associated bacteria and protozoa. As noted, this is minimized by having the animals spawn dry, with the gonoducts in the upright position. A possibility which we have not tried is to open up the sea urchin and remove the gonads into sterile seawater. Perhaps with careful dissection (to avoid puncturing the intestine) and addition of KCl to the isolated gonads, one might get good emptying of the ovaries under more sterile conditions.

Another possibility would be to actually seal the anal opening. We have considered using plasticine or epoxy cement but have not tried this. This approach might also minimize contamination.

One aspect of our study is the finding of variation in egg quality in terms of storing the eggs for long periods of time. The first factor is seasonality; at the end of the season, egg quality is bad and most females ovulate eggs that are fragile and cannot be stored for more than a few days.

The other factor is some sort of intrinsic variation in quality that is routinely seen amongst different females, even at the height of the season. We routinely find that only about 50% of the females have eggs that can be stored for long periods of time. Although most batches of eggs yield good development immediately after spawning, only about 50% of the different females provide batches of eggs that can be stored for longer than one week. We do not know the source of this variation.

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