Xenopus Development

LAB 6

EARLY DEVELOPMENT OF XENOPUS

I. Introduction

This week’s lab involves a new model system, Xenopus. This will involve an experimental examination of fertilization and early stages of amphibian development. Normal fertilization involves a number of activities including the initiation of development by activation of the eggs metabolism. However, an egg can develop in the absence of sperm; such a development is known as parthenogenesis. The drone-producing egg of the honey bee is an excellent example of naturally occurring parthenogenetic development. Many other eggs, which do not normally develop parthenogenetically, can be induced to do so by a variety of experimental means.

For example, the eggs of many amphibians may be stimulated to complete the early stages of development in the absence of the nuclear events of normal fertilization if they are inseminated with pretreated sperm, which have been irradiated. Irradiation disrupts the genetic material from the sperm but does not impede mobility or the ability of the sperm to penetrate and stimulate the egg.

Another example is seen in the work of the early experimental embryology experiments demonstrated the artificial activation of an unfertilized egg by pricking it with a sharp needle. This experiment when originally performed utilized dirty instruments. Later investigators could not repeat the results because their instruments were too clean. It turns out that a tiny bit of blood was introduced into the egg cytoplasm during the pricking procedure.

Today’s lab involves a study of parthenogenetically activated frog embryos accompanied by a paralleled study of normal diploid embryos. The success rate for parthenogenetic egg activation is about 6% of the eggs making it through cleavage. Occasionally, a haploid tadpole will be produced, but this is quite rare. Because the success rate is so small, probably only one of two students in a class will have positive results.

We will want to observe the changes in a recently fertilized amphibian egg, especially the organization of the cytoplasm. The distribution of cytoplasm in animal eggs is not a random process. In most species of animals, the newly ovulated egg shows a marked asymmetry. Often, more yolk is accumulated towards one end. Pigmentation may also be seen in parts of the egg surface. This distribution probably reflects the organization of the underlying cytoskeleton. It allows us to discriminate between two poles, animal and vegetal.

The frog egg that we will be studying today is a good example. Pigment of the surface cytoplasm is confined to one half of the egg, the animal hemisphere. The other half is comparatively unpigmented. The nucleus is situated in the pigmented animal hemisphere and the largest yolk platelets in the unpigmented vegetal hemisphere.

This lab utilizes one of the more famous amphibian developmental systems, that of Xenopus laevis. The common name is the South African clawed frog. Its native home is southern Africa, and probably more is known about this vertebrate than any other. It is a favored amphibian because the female ovary contains oocytes of all stages. Injection of gonadotropic hormones at any time of the year will cause egg laying. The frog is easily cultured in aquaria containing dechlorinated water and can subsist well on a diet of uncooked beef liver.

Injection of commercial chorionic gonadotropins into the dorsal lymph sac of female frogs causes egg laying in 12-16 hours. Egg-laying females will be available in the laboratory.

Fertilization can be achieved at a precisely determined time for experimental purposes by sacrificing a male and removing the testis. The isolated testis is homonogized 1ml of 10% Ringers (can also use a 1/3 MMR solution). The suspension is then added to “dry” eggs for fertilization (see detailed procedure below). When sperm is added to eggs, the time is noted as the time of fertilization. After 8 minutes, the eggs are rinsed in 10% Ringers (or a solution of 1/3 MMR) and they stay in the dilute Ringers at 16-20C throughout subsequent development. It is possible for them to develop at room temperature, but they don’t like it, and many may die.

The time scale of developmental events leading to first cleavage is important to you. With 1/2 to 3 minutes of fertilization a wave of exocytosis of cortical granules occurs, leading to the raising and maturing of the fertilization membrane. When the vitelline membrane of the unfertilized egg is changed by fertilization into the raised fertilization membrane, the egg comes free to rotate within it. The egg will rotate so that the yolk-heavy vegetal hemisphere is downward and the pigmented animal hemisphere uppermost in the gravitational field. One sign of a successful fertilization is the change of random orientation of unfertilized eggs to the uniform pigmentation orientation of fertilized ones.

Within 3-5 minutes there is a reversible activation contraction of the cytoplasmic pigment layer in the animal hemisphere into a smaller area around the animal pole. This subsequently relaxes again into the original pigmented area. At about 15 minutes, close examination of the pigmented surface with the dissecting microscope will reveal a densely pigmented spot. This marks the sperm entry point (SEP).

At 40-70 minutes a vague gray crescent may sometimes be seen at the equator between the pigmented and unpigmented hemisphere, although some authors claim there is no gray crescent formation in Xenopus. It forms on the opposite side of the egg from the SEP. This equatorial region is important because it marks the site of the future dorsal lip of the blastopore and the organizing center of the future embryonic body axis. The appearance of the gray crescent (in amphibians that possess one) at about 40-70 minutes indicates that embryonic symmetry is developing through internal cytoplasmic movements.

Grey crescent formation occurs through a rotation of the outer cortex with respect to the endoplasm. A contraction of the pigmented endoplasm towards the SEP is superimposed on the more global endoplasmic rotation. The result of these early cytoplasmic movements is that the dorsal/ventral and right/left orientation of the future embryo is being established even before the first cleavage division! First cleavage occurs, at room temperature, about 75-110 minutes after fertilization. The second and the subsequent eight cleavages occur very fast, at 15- to 30-minute intervals in a synchronous fashion.

A. Objectives

1. To become familiar with the harvesting of gametes from amphibians.

2. To observe normal fertilization and embryonic development in amphibians.

3. To attempt to induce artificial parthenogenesis by mechanical manipulation of the egg and by using irradiated sperm (optional).

II. Procedure

A. Pre-lab Injection of Females

Three gravid Xenopus females will be injected by student volunteers with 800 I.U. commercial human chorionic gonadotropin (hCG) the night before the scheduled lab. Note that it takes at least twice as long if gonadotropin from pregnant horse serum is used. Injections are made at an oblique angle just under the skin of the back, using a hypodermic syringe and needle of about 26 gauge. Make a frog burrito with paper towels as described by your instructor. Inject sample, wait 5 seconds then withdraw needle. You will inject just outside the stitch marks with the needle going under the marks. You will initially feel some resistance, which will stop after you have penetrated properly. Below is a good link to this procedure: http://tropicalis.berkeley.edu/home/obtaining_embryos/hcg/hCG.html

All individual male and female frogs should be kept in separate tanks away from any disturbances. At least one female should be placed in a tank with high salt concentration. This should keep any eggs laid overnight viable for the next day’s lab.

B. Harvesting the Eggs

The female frog does not have to be sacrificed to obtain her eggs. The Eggs are removed from the oviducts by a technique called "stripping". In the process of stripping the investigator simulates the role normally played the male frog . You should be fastidious about keeping the females in clean water after ovulation. Water in buckets should be changed every day.

Females should be laying in their containers before attempting egg collection. The idea is to mimic the actions of a male frog and to encourage to lay C not to physically squeeze eggs out of the animal. The action is a gentle touch that has to be learned, the eggs collect in a sac near the cloaca, and gentle lateral and simultaneous vertical pressure should expel them.

If performed gently, the females remain relaxed during the collection, except when vigorously pushing the eggs out. After induction of ovulation, the cloaca becomes red and swollen and is probably rather sensitive, so avoid touching it when picking up the frog. Do not damage the eggs, rough handling and excess heat form microscopes can kill the eggs or early embryos and ruin the experiment.

1. Hold the female frog with her back against the palm of your right hand. Grasp and extend the hind limbs of the female frog with your left hand and place the palm of your right hand over the back of the frog in such a manner that your fingers partially encircle the body just posterior the forelimbs. The tips of your fingers thus come to rest on the ventral surface of the frog.

2. Eggs can be forced from the cloaca by applying initial gentle pressure to the anterior part of the body and then progressively closing the hand toward the cloaca region. She should be held over a cool, dry petri dish (80 mm). If she does not lay, you may need to massage her with more pressure. If the eggs are not to be used immediately, the dish should be covered with a wet kimwipe to keep them from drying out.

Eggs may be laid without movement from the frog, or she may push them out in a couple of vigorous bursts, at which time hold on to her tight (holding her against your shirt really helps), let her settle down and then massage again C the eggs should now be easily laid. Do not continue collection for more than five minutes.

3. Add about 100 eggs into each of three petri dishes. The first two dishes contain either normal freshly prepared sperm suspension or just spring water. They should receive eggs immediately after the sperm suspension is prepared. A third dish contains the irradiated sperm suspensions and should get eggs following the sperm irradiation.

When stripping the eggs squeeze gently and move the female around over the dishes to produce several ribbons or chains of eggs rather than a single heaped mound. To assure complete exposure to sperm, draw sperm suspension into a pasture pipette and squirt the sperm over the eggs. Note the orientation of the black-pigmented animal pole and the creamish-white vegetal pole of the egg.

Mature sperm are present in the testes of male Xenopus virtually all year long (although there is a period of relatively lower spermatogenic activity from late June to mid-September). In common with other vertebrates, the spermatozoa of the frog are a small motile flagellate cell.

1. Pith, decapitate, or anesthetize a male frog and use scissors and forceps to dissect out the pair of testis. The pithed male frog is laid on paper toweling. The abdominal cavity is opened and the testis located. They are 5 x 10 mm, pale ivory structures lying near the posterior dorsal surface of the abdominal cavity in association with fat bodies. They held to the kidneys by means of folds of mesentery. A detailed description is given below:

Use a tissue or sharp forceps to pick up the loose skin on the belly and make a cut with scissors. The skin can now be lifted with forceps. The testes are attached to the fat bodies, and the easiest way to remove them is to make a slit on either side of the dorsal midline, being careful not to cut across the midline where the large abdominal blood vessel is situated. Push aside the liver and pull out the fat body (yellowish, with many fingerlike lobes).

Alternately, expose most of the belly, then similarly remove the muscle layer to expose the viscera. The testes each lie at the base of the fat bodies C they are whitish and covered with capillaries. Using scissors or forceps, free each testis from the fat body and surrounding connective tissue. If you are unsure whether you have the testis, crush a little on a microscope slide and view with phase contrast. The sperm are easy to see with a fine helical shape.

2. After removing the testes, rinse with 1/3 MMR, then place in 1 X MMR on ice. Next, cut a small portion (2-3 mm²) of the testis and place in 2ml of chilled 1/3 MMR) in a tissue homogonizer and break up the tissue of the testis to make a sperm suspension. Some authors suggest cutting the testis and rubbing them over the eggs, but we will not use that procedure today.

3. Transfer 1 ml of the sperm suspension to a Petri dish for irradiation. The sperm suspension should be spread thinly over the bottom of the dish. Label the dish “experimental” and irradiate as described under number (1) below. Here is a good link: http://www.xlaevis.com/why.html

4. The sperm "slurry" is next mixed with the eggs. Make sure the eggs are dispersed in the dish. A pair of forceps can be used to break up clumps of eggs and spread them out. The eggs are very tough and will not break easily at this point. After 8 minutes, wash the eggs free of testis debris in 1/3 MMR or 10% amphibian Ringers. The second testis should be placed in a chilled Petri dish with full strength (1X Ringers), where it should remain viable for a week if kept chilled.

5. Place a drop of suspension of a glass slide and examine under high-dry. Observe the shape of the spermatozoa and check for motility.

6. The first sign of fertilization is a contraction of the pigment so that the unpigmented territory is now maybe 2/3 to 3/4 of the egg. After about 30 minutes the fertilized eggs will rotate within the vitelline membrane so that the animal hemisphere faces up. Always perform test fertilizations on a few eggs soon after laying, so that you can assess the quality of the eggs. If the first fertilization is poor, perform another, on eggs laid at a different time to those used in the first test since the quality of eggs may vary during the day. Fertilization efficiencies range from 100% on down, with "good" frogs typically giving greater than 80% fertilization.

Testis keep adequately in 1x MMR. Antibiotics such as pen/strep or gentamycin (1μl per ml) may be added and then store the testes on ice or at 4^oC. They are good for a couple of days, when kept intact (wholly or partially) after which sperm viability drops. Note that both sperm and testis should be kept chilled as much as possible.

Nuclear damage is one of the demonstrable effects of ultraviolet (UV) irradiation of the sperm cell. In particular, the energy of UV radiation induces abnormal bonding of the pyrimidine bases of nucleic acids. Thus, chromatin material of the sperm cell is disrupted by UV radiation without diminishing the capacity of the sperm to enter the egg. A phenomenon peculiar to UV exposure is that of photoreactivation in which the effect of the UV radiation is perceptibly lessened by the presence of intense visible light (like overhead illumination). Therefore it is advisable to irradiate in a dimly lit room.

1. Position the ultraviolet lamp 15 inches above the table top. Place the uncovered "experimental" petri dish beneath the lamp and expose the sperm suspension to the rays of the lamp for 10 minutes. Occasionally swirl the sperm suspension gently to ensure equal exposure of all sperm to the rays. Do not expose your skin to UV radiation unnecessarily and do not look directly at the lamp. You are now ready to inseminate your eggs.

You will need some capillary tubes coated with heparin to inhibit clotting, and filled with frog blood. You will strip the female of eggs and place them in a finger bowl. These should sit covered for about 30 minutes so as to allow the CO₂ to escape but not the water. Therefore this part of the experiment should be done at the beginning of the period.

Next get five of these eggs and place them on a microscope slide or in a Petri plate. Pipette a small amount of blood onto the eggs, then gently, but firmly, prick each egg with a sharp point of a glass or tungsten needle. The puncture should be applied within the animal hemisphere but not in its exact center where the second maturation spindle is likely to be located. When finished puncturing all of the eggs, transfer them to a petri dish, cover them with spring water, and follow their development over the next few days. Compare your results with the normally inseminated controls.

1. Johnson, L.G., and Volpe P.E. 1973. Patterns and Experiments in Developmental Biology. WM. C. Brown Co. Dubuque, Iowa.

2. Biroc, S.L. 1986. Developmental Biology, a Laboratory Course with Readings. Macmillian Pub. Co. New York.

3. Gerhart, J., M. Danilchik, J. Roberts, B. Rowning, and J.-P. Vincent, 1986, Primary and secondary polarity of the amphibian oocyte and egg. In: J. G. Gall, ed., Gametogenesis and the Early Embryo, Alan R. Liss, New York, pp. 305-320.

4. Scharf, S. R., J.-P. Vincent, and J. C. Gerhart, 1984, Axis determination in the Xenopus egg. In: E. H. Davidson and R. A. Firtel, eds., Molecular Biology of Development, Alan R. Liss, New York, pp. 51-74.