FERTILIZATION, EMBRYOLOGY AND DEVELOPMENT Introduction This lab will be a cursory study of fertilization, embryology and early development.

Knowledge of stages and an appreciation of the trend of developmental processes will provide a reasonable comprehension of the subject. There are marked similarities in the development of all embryos; thus, this study will include sea urchins, fish, and humans. A logical account of development must begin with a consideration of the process which initiates that development, fertilization. Fertilization is the union of sperm and egg, bringing together haploid sets of chromosomes from two organisms of the same species to form a single diploid cell which is the beginning of a new organism. Sea urchins provide a good model in which to observe these early stages. Objectives A. B. C. D. Inject fertile sea urchins to induce gamete release. Observe fertilization and the formation of the fertilization membrane. Classify sea urchin gametes according to species by observing fertilization results. Describe cleavage and the early sea urchin development including the germ layers and structures derived from each. E. Compare human development with that of other animals.

Part I. Donating to the Sperm/Egg Bank. Sea urchin eggs can be fertilized only by sperm from sea urchins of the same species. Today in lab you will be given 1 sea urchin, which you will stimulate to release gametes. Using those gametes you will sort gametes from unknown sea urchins according to the species. Procedure. A. Procurement of Gametes (see illustrations on the next page) 1. In preparation for gamete collection, have a microscope, a slide, and a clean petri dish in place. Eggs are released with a jelly-like fluid and so have some protection. But the sperm are tricky. As soon as they hit water they frantically begin to swim in search of an egg, and will quickly use up their limited energy supply. Activated sperm are viable for only about 40 minutes. They can survive longer under "dry" conditions, undiluted, covered, and kept at 10C. 2. Obtain a living sea urchin from the tank and place it on a disposable petri dish with the oral opening up. You will recognize the oral opening by the presence of 5 triangular teeth that surround it. Fill a syringe with 2.5cc of the 0.55M KCl solution. Inject the sea urchin through several points in the perioral membrane. The test of the sea urchin is hard, but the area around the oral opening is soft. Make sure that the hypodermic needle penetrates well into the body cavity. 3. Watch the "underside" of the sea urchin. Within a few minutes the gametes should be released. Note: KCl will damage the gametes. Blot the underside of the sea urchin often to remove any dripping KCl. As soon as gametes appear, transfer the urchin to a dry dish. a. If grainy, amber-brown fluid is released, it probably contains eggs. Microscopic observation is the only way to confirm sex. Make a slide. Eggs are easily visible under the 4x objective. b. If milky, white or yellow-white fluid is released, its probably sperm. Make a slide. Sperm can be seen under the 10x objective and very low light (close down the iris diaphragm) as tiny, vibrating specks. The 40x objective gives and even better view; use a coverslip. 1

B. Preparing and submitting the sample. 1. Once you have identified your gametes, prepare your sea urchin for a visit to the Fertility Clinic. It should be resting comfortably in a clean dish. 2. Fill out a submission slip and transport the mother and eggs to the Donor Facility, or the father and sperm to the Sperm Bank in the back room.

GAMETE PROCUREMENT

KCl

mouth

gonads

eggs
anus

gonadopore

sperm

Keep the eggs away from KCl. The coelomic fluid will hydrate them for a short time.

Keep sperm "dry" and covered.

ALLOW 20 MINUTES FOR PROCESSING EGG/SPERM DONOR AND RECIPIENT REQUESTS.

PART II. In Vitro Fertilization, the Creation of Test Tube Babies.

Procedure. A. Return to the Clinic window and withdraw the gametes that have been assigned to your table. Note the small quantity, about 4 drops in each vial. You will use 2-4 drops from each. B. Decide which sperm and eggs to combine first. One of these should be of a known sex and species.

C. Prepare a slide and watch for fertilization. 1. Select a washed egg preparation and record the identifying information in Table A. 2. Place two drops of this eggs/seawater mixture on a depression slide and do not add a coverslip. Tip: Eggs are heavy so will fall to the bottom of the tube. Draw your drop from the bottom of the tube, but dont use all of the eggs. You may need more for another fertilization. Observe the eggs with the bright field microscope using the 4x, and then the 10x objective. The eggs are round and usually amber, brown or black in color. You may see a clear jelly coat surrounding each egg. Close down the iris diaphragm for the next part. 3. Select sperm for fertilization and record the identifying information in Table A. Using a clean pipette, withdraw the tiniest possible drop of sperm. So that you may better control the fertilization process, add the drop to the depression slide just outside the drop of eggs.

Place eggs on a depression slide

Place a drop of sperm on the flat portion of the slide. While focused on the eggs, push the drop of sperm into the eggs with a dissecting needle.

View at 40-100x

4. While looking through the microscope at the eggs, unite them with the sperm by gently drawing the sperm drop into the egg drop with a dissection needle. 5. Within seconds, you should notice the sperm swimming around the eggs. compared to eggs. Look for the following events of fertilization: Sperm are very, very tiny

EVENTS OF FERTILIZATION (see illustrations below) 0 seconds 30-40 seconds 35-50 seconds Penetration of ovum by head and neck of sperm (tail drops off) Breakdown of cortical granules - the surface will look smoother Elevation of fertilization membrane at point of sperm entrance. The fertilization membrane will prevent multiple fertilization. Multiple fertilization produces gross defects in the embryo. Hyaline layer formed (translucent "glass-like") between egg and fertilization membrane

2 minutes

Unfertilized egg

Fertilization

Fertilized egg with fertilization membrane

6. If fertilization occurs, you know that the sperm and egg were from the same species. If no fertilization occurs, they were from different species. Record your results in Table A. 7. You may make subsequent withdrawals from the Fertility Clinic to test your unknown. Repeat the fertilization process as many times as needed, using appropriate combinations, to determine the species.

Table A Sample # Example: AO3215

M/F

Experiment

Result (No fertilization )

Species

AO3215 +

Pencil male

AO3215 is not of the Pencil species

PART III.

Observing Early Development.

Procedure. A. Observing early development of a fertilized egg. Following fertilization, the egg undergoes many mitotic cell divisions called cleavage. During this time, the number of cells increases but the embryo does not change in size. Eventually there is a tiny hollow ball of cells called a blastula. The folding in of the blastula is called gastrulation and produces a gastrula. When the gastrula is formed all 3 tissue layers are established.

Cleavage (dividing with no growth) 2 cell stage Continued cleavage Blastula Gastrulation (folding in) Gastrula

The gastrula possesses the 3 primary germ layers from which all tissues are derived: 1. ectoderm skin, nervous tissue 2. mesoderm organs, bone, blood, muscle tissue 3. endoderm gut, some digestive organs

B. Observe the embryonic developmental stages on display that were prepared by your lab instructor. Compare each to the developmental stages chart and determine the stage of each preparation. Embryo A ______________ Embryo B _______________ Embryo C ______________

Part IV. Human Development a. On what day of the menstrual cycle (the first day of the menstrual period is day 1) does a fertilized egg begin implantation? _____ Complete implantation?____ b. A woman may suspect that she is pregnant when her menstrual period is 1 week late. By this time, has the embryos brain begun to form? Yes / No c. Observe the developmental photographs of a human fetus. On what day does the fetuss heart first begin to beat? ________ weeks

d. At what age are the beginnings of webbed fingers and toes first seen?

e. At what age can a fetus first suck its thumb? __________________

f.

Which vitamin can cause deformations in the kidneys and gonads when taken in excess by the mother during the first 5 months of pregnancy? ___________

g. What fetal organs can be affected when a woman takes birth control pills during pregnancy? _______________

Conclusion 1. Sequence the events of early embryological development, by numbering the following steps 1 - 9. ____first cleavage, _____gastrulation, ____unfertilized egg, ____fertilization, _____blastula, ____ fertilization membrane forms, 2. _____ 2-cell stage, _____ 8-cell stage, ____gastrula,

List the 3 primary germ layers, and some of the tissues/organs fromed from each. a. b. c.

3.

What is the function of the fertilization membrane?

4.

What might happen if two or more sperm entered the egg? (Hint: Can you imagine a human with 69 chromosomes?)

5.

What species of sea urchin was your unknown sample? #_________ - Species _______________________