Instructor's Objectives
Target Age or Ability Group Audience
Teacher Instructions/Special Precautions
Materials & Equipment Needs
Background [Prior Knowledge or vocabulary necessary to complete activity]
The Student Lab
Method of Evaluation/Assessment
Extension/Reinforcement/Additional Ideas
 

In the first part of this activity, students will use clay to model fertilization and early development  in sea uchins and chordates.  In the second part, students will mix together sperm and ova from live sea urchins to observe, diagram, and record events occuring in fertilization and development over a 1-2 day period including:  cell cleavage, formation of blastula, gastrulation, morphogenesis and organogenesis.  Actual observations will be related to the models constructed earlier.  In an optional section, students examine the effect of sperm concentration and possible pollutants (oil) on rates of fertilization of sea uchin ova. Suggestions are provided for additional student projects on modeling development.

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Classes:  Biology, A.P. Biology, Marine Biology, Anatomy and Physiology, Integrated or Co-ordinated Science
Grade Level:  9-12
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Notes to the Teacher: to top
Part A:  Modeling Steps in Early Development  Make sure you have several pounds of modeling or other clay (obtain from toy or discount stores) for the first part of this activity.  Keep the clay moist.  Contact your art department to see if they have extra clay or know a cheap source.  Clay may be ordered through your own local District warehouse or supply.  Provide each group of 2 students with a piece of clay approximately 4-6 cm in diameter.  For both parts of this activity, groups of 2 students work best.
Click here to see a series of simple sketches representing the major stages of development in the sea urchin.

Part B:  Fertilization and Development of Sea Urchins  Live sea urchins can be obtained on both the East and West Coast or Gulf of Mexico for this investigation by collecting them yourself.  Check your local regulations.  A fishing permit or collecting permit may be required.  Many can be collected at low tide.  Urchins may also be obtained through most Biological supply companies. On the West Coast of the United States, Strongylocentrotus purpuratus, the common intertidal purple urchin, works well during January-June and can usually be easily found in most rocky intertidal habitats. Lytechinus pictus, the white sea urchin, may be used on the West Coast in the Summer and Fall.  It is subtidal.  On the East Coast of the U.S., Strongylocentrotus drobeachiensis, the green sea uchin may be used in the Spring and Arbacia punctulata, the Atlantic purple urchin, may be used in the summer and early Fall. (see Tyler, Developmental Biology:  A guide for Experimental Study, pg 56).
     Inject several sea urchins in the soft tissue between the teste and teeth with approximately 1-2 ml of 0.5M KCl using a 5 cc syringe with a #25-30 needle.  (see caution/disposal suggestions below)  Immediately place the urchins upside down and over small beakers or culture dishes.    Female urchins will release yellow colored eggs or ova. from a pore on the opposite side as the mouth.   Make sure the eggs fall directly into sea water below the urchin.  You may place enough sea water in the container to just touch the aboral surface of the urchin from which the eggs are released.  Collect several small containers of eggs.  Label them clearly.  Rinse the eggs several times with new seawater before storing.  Male urchins will release white sperm.  These can be collected in a dry beaker and stored in the refrigerator for several days.  Once sperm contact seawater, they become active and viable.  Collect several containers of "dry" sperm.  Label them clearly.  Three-five small beakers (100-150ml) each of dry sperm and egg solutions should provide enough gametes for several (2-3 classes).  Standard solutions of sperm should only be made immediately prior to and during the actual student investigation.  To make these solutions, add 1-2 ml of "dry" sperm to every 10ml of sea water.  These sperm cultures are best if used during the first 20 minutes after mixing.  You might want to save several other containers of dry sperm for day 3 of the investigation or for student projects in the optional section. It is very important to keep all glassware in this portion of the activity very clean!!!
    Make sure you have plenty of depression slides, cover slips, and sea water (artificial or natural).  At staggered times and several hours (4-8, if possible) before the student activity, mix up several "cultures" of sperm and ova in culture or small beakers for students to oberve the later developmental stages (2,4,8,16, 32, etc).  Label these cultures with the date and time in which the sperm and ova were mixed so students can monitor developmental stages in reference to time after fertilization.  Store these cultures in the refrigerator if you are working with cold water species.  Provide aeration if possible.

1. Required of students:  Students should be familiar with how to use a dissection and compound microscope.  In addition, students will need to make measurements of cells and other recognizable structures as they make their observations. Students should therefore know how to use a metric ruler slide to estimate cell size under various fields of view.  Students need to understand that 1 millimeter = 1000 micrometers or microns.  Microns are the common unit used to measure cell and cell structures.
2. Preparation time needed:  Obtain clay at least several days before the modeling portion of this activity.  Urchins should be ordered to arrive or collected 1-2 days before the second part of this activity.  Living urchins may be temporarily stored in an insulated cooler with sea water.  Aeration with a simple pump and air stone will help keep the adult urchins alive for a longer time period.
3. Class time needed:  One day (full 45-55 minute period) for part A of this activity.  One-two days for part B.
4. [Hazards/Precatuions]:  Students should wash their hands at the end of each portion of this activity.  Caution and monitor students if they use the syringe to inject the adult urchins with  0.5M KCl.  We recommend that the teacher actually inject the adult urchins to stimulate release of gametes and store the syringe in a secure location.  Needle should be destroyed and/or disposed of properly (consult local policies) when it is no longer useable.
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Before beginning this activity, students should be familiar with the process of mitosis.  Over the course of the activity, cell divisions are frequently referred to;  it is assumed that students understand the details of this process.  Students should also be familiar with the process of fertilization and the events that must occur for fertilization to be possible.  It would be beneficial (though not mandatory) for students to be familiar with the terms used to describe the stages of development and the embryonic tissues that arise during development.
    Part A (Modeling) serves as a nice introduction to Part B(Fertilization and Development of Live Urchins).  Videos or movies on development might be shown as an introduction to this activity or in between Parts A and B.  Appropriate material and diagrams in student textbooks can be used to supplement both parts of this activity and help students better understand the process and sequence of events in development.

This activity helps students answer:

How does a single fertilized egg develop into a multicellular adult?
What do the stages of embryonic development look like?
What are the approximate sizes of a sea urchin embryo in each of the stages of development?
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SEA URCHIN FERTILIZATION AND DEVELOPMENT

Introduction:
    Sea urchins are common seashore animals that can be easily obtained and observed on both the Pacific and Atlantic Coast and in the Gulf of Mexico.  Sea urchins are invertebrates (lacking a backbone) and members of the phylum Echinodermata (Spiny skinned animals) and class Echinoidea.  Urchjns are noted for their spherical shape, pentamerous radial symmetry, endoskeleton of calcareous plates with spines of various lengths protruding from their shell or teste, and a water vascular system with tube feet.  Many sea urchins are herbivores, grazing on seaweeds or kelp.  Sea urchin fertilization and many aspects of its development are similar to many other animals including ourselves and other Chordates.   Therefore sea urchins are commonly used to observe, study, and investigate many aspects of embryological development.
    Sexes are separate in sea uchins. It is difficult to distinguish male and female sea urchins externally.  The eggs and sperm of sea urchins can be easily obtained, however, by injecting the urchins with a solution of 0.5M KCl.  Sperm (white) and Eggs or ova (yellowish) will be secreted to the external environment through an opening opposite the mouth side or oral surface.  Urchins can be turned upside down and the gametes collected in beakers or other small containers.  Solutions of ova and sperm can then be combined to observe fertilization and subsequent development.  Observations are then easily made with a compound microscope.
    The egg or ova are many times larger than sperm and surrounded by a thick jelly coat.  Sperm immediately cluster around the ovum.  Digestive enzymes on the head of the sperm enable it to penetrate the jelly coat.  The head of the sperm also has special protein receptors (species specific) that match the protein receptors on the female ovum.  The sperm nucleus penetrates the outer membrane of the ovum.  Chemical and electrical changes in the membrane of the ovum immediately occur.  These changes lead to the  formation of a special fertilization membrane that eventually block additional sperm from fertilizing the same ovum.  The sperm nucleus (haploid =N) soon fuses with the ovum nucleus (haploid = N) to form a  zygote (2N), the beginnings of a new sea urchin.  The zygote contains a complete number of chromosomes, half from the sperm and half from the ovum.  Cleavage or cell division by mitosis and cytokinesis soon follow.  Two cells are formed, then four, eight, sixteen, thirty two and so forth.  The individual cells in the embryo are termed blastomeres.  Eventually a solid ball of cells or morula is formed. The cells become more closely associated with each other and arrange themselves into a hollow sphere of cells termed a blastula.  All cells at this point in development are still identical. Soon thereafter some dramatic changes in the cells themselves and their organization and arrangement occur.  A  group of cells on one side or pole of the blastula begin moving or migrating inward.  This point on the embryo is termed the blastopore and will actually form the anus of the urchin. Other cells follow and move through the middle of the ball of cells eventually reaching the otherside. This second opening becomes the mouth.  A tube called the archenteron now exists  through the middle of the embryo and forms the lining of the digestive tract.  The embryo is now termed a gastrula and the process of cell differentiation and migration is termed gastrulation.  Three distinct cell layers eventually form:  the endoderm (inner most), mesoderm (middle) and ectoderm (outermost).  Future tissues and organs develop from these three layers. For example, ectoderm forms the outer layers of the animal. In addition, in vertebrates the ectoderm also gives rise to the main organs of the nervous system, the brain and spinal cord.  Mesodermal cells form muscles, skeletal structures, and such organs as the heart, if present.  Endodermal cells form the lining of the digestive tract and associated organs.  The process of organ formation is termed organogenesis.   By the second-third day of development the embryo becomes a pluteus larva.  The larva actively swims in the ocean with tiny cilia as part of the zooplankton, undergoes further development through metamorphosis, and eventually becomes a sea urchin, settling to the bottom.

Purpose:  In part A, students will use clay to model the events that occur in fertilization and early development.   In Part B, students will fertilize sea urchin eggs and observe, investigate, and measure developmental events and processes.

Equipment:  Modeling clay, adult sea urchins, egg solutions, pipettes, dry sperm, sperm solutions, microscope (depression) slide, coverslips, metric ruler slide for measurement, compound microscope, sea water, cultures of urchin eggs fertilized at different times.

Procedure:

A.    Modeling Fertilization and Early Development in Sea Urchins (Day One)

1.    Take your lump of clay and roll it into a ball approximately 6 cm in diameter.  Set aside a small portion of clay for the next step.  The ball you have just created will represent a sea urchin egg.

2.    Using your knowledge of cell sizes, use some of the remaining clay to create a sea urchin sperm cell of appropriate size.

3.    The first step in sea urchin reproduction is fertilization.  In this event, the egg and sperm fuse to form a zygote.  Using your clay cells, model this event.

4.    Following fertilization, the single-celled zygote must develop into a multicellular adult.  This development begins with a process called cleavage.  In cleavage, the fertilized egg goes through several divisions without increasing in size. Using the piece of string provided, take your fertilized egg through its first division.

5.    Repeat step four three times;  your teacher will demonstrate where the plane of division is.    How many cells are now present?

6.    At this stage, the embryo is called a morula.  It is a solid ball of cells, equal in size to the original fertilized egg.  In the next set of divisions, the embryo will change from a solid to a hollow ball of cells.  During these divisions, the embryo will also begin to increase in size.  The cells of the morula will move outward to form the hollow sphere called the blastula.

7.    As best you can, convert your model from the morula stage to the blastula stage.  This will require that you remove tissue from the interior of your embryo and add tissue to the outer layer.  Obviously, the process by which blastula formation occurs cannot be accurately replicated in this exercise;  however, we can create something similar to the final product.

8.    So far, our embryo has maintained a spherical shape as it has increased its total number of cells.  In the next stage of development, the overall shape of the embryo begins to change.  To mimic this next stage, you should place your finger on the surface and gently press inward.  The pore you are creating is called a blastopore.  Continue to push in at the blastopore until a long tube is formed.  This process is called gastrulation.  The tube you have created is called the archenteron; ultimately this tube will become the gut of the animal.

9.    The embryo at this stage is called  a gastrula.  Up until this point, all of the cells in the embryo have been identical.  Once gastrulation begins, cell differentation takes place.  When this process occurs, distinct cell layers begin to form.  The embryo also begins to lose its spherical shape.  To model this in your clay embryo, you should roll the embryo between your hands so it elongates.

10.    Lay the embryo flat on your desk.  The surface of the embryo facing up is referred to as the dorsal surface.  Place your finger on the anterior end of the dorsal surface.  (The anterior end is the end where ultimately the head will form.  In the sea urchin, the anterior end of the embryo is opposite the end of the blastopore).  Press gently at the this point to make an indentation in the embryo.

11.    The indentation you have made is called the neural plate;  the edges of the indentation are the neural crests. In Vertebrates, the edges of the neural crests fold together to form a neural tube that eventually gives rise to the brain and spinal cord.  Simulate this folding with your clay model.  This process is termed neurulation.

B.  Fertilization and Development of Living Sea Urchins (Days 2-3)

1.  Obtain a microscope (depression) slide and compound microscope.

2.  Add a drop of "egg solution" to your slide.  Observe the egg cells or ova under low magnification of your microscope.  Draw and label several ova on the data sheet provided by your teacher or on your own paper.  Using your metric ruler slide, measure the field of view under low magnification in millimeters, then microns (1mm = 1000 microns).  Record this value on your data or observation sheet. Use it to determine the sizes (diameter) of several egg cells.  Record the size (diameter) of the ova on your data sheet. Rinse and clean your slide.

3.   Add a drop of "sperm solution" to your slide.  Observe the sperm under low, then medium, and if needed high magnification.  Draw and label several sperm on your data sheet or your own paper.  Again, using your ruler slide, measure the field view in millimeters and microns.  Use this value to help you estimate the size of sperm. Record the size (length) of the sperm on your data sheet.  Rinse and clean your slide.

4.  Place1-2 drops of "egg solution" on your microscope depression slide.  Carefully add 1-2 drops of "sperm solution" on top of it.  Add a coverlsip.  Immediately begin observing what happens under low and then medium magnification on your microscope.  Observe the mixture for the next 10-20 minutes.  Repeat the process again, but on the second attempt, add the sperm form the side of the coverslip to the egg solution instead of directly mixing the two suspensions together.  Draw two separate sketches.  The first sketch should illustrate sperm as they cluster around an ovum.  Count and record the number of sperm around each ovum you sketch. Label your sketch. Continue looking  for ova in which a special membrane has moved off the surface of the egg to form a fertilization membrane blocking additional sperm from fertilizing the same ovum. A gap should be noticeable between this outer membrane and the inner membrane of the egg itself.  This indicates the ovum has been fertilized. Diagram a fertilized ovum illustrating the fertilization membrane for your second sketch.

5.  Rinse and clean your slide.  Using a pipette, obtain several drops from the culture labeled developing embryos.  Your teacher may have several cultures with embryos developing at different time periods after fertilization (2, 4, 6, 12, 18, 24 hrs, etc).  Place several drops on your slide.  Observe under low, then medium magnification.  Draw and label various stages of development on your data sheet or on your own paper.  Your diagrams should illustrate the 2,4, 8, 16 cell stage, blastula (hollow ball of cells), gastrula with archenteron (primitive gut), and pluteus larva.  Using your ruler slide and measurements of the field of view taken earlier, estimate the size of invidual cells (blastomeres) when visible as well as any other recognizable structures. Also record the time in minutes and/or hours at which you view various stages in the developmental process.  Assist your teacher, if directed, by mixing up several cultures of eggs and sperm for observing the later stages of development on another day.  Make sure all glassware used for these new cultures is very clean and dry before you begin.

Observations:  For each diagram you make, you should label the magnification at which the sketch was taken, time in minutes or hours after fertilization if appropriate, and size of cells (blastomeres) or other recognizable structures. Also identify and label appropriate structures such as sperm flagella, egg membrane, fertilization membrane, archenteron, etc.
 
Student Data Table:  Sea Urchin Fertilization and Development
(construct a table similar to the one below, leave plenty of space for your observations and diagrams)
 

Conclusions:  Write a 1-2 page paper describing the development of the sea urchin from fertilization through the pluteus larva.  Describe the processes that occurred and that you observed including:  fertilization, cell division or cleavage, cell differentiation, gastrulation, morphogenesis (development of shape and structure), and organogensis. How many minutes or hours after fertilzation did each of these major events occur?  How did cell size and embryo size change during development?

2.    The modeling activity included in the student activity assumes that the embryo is undergoing holoblastic radial cleavage, and it does not account for the presence of the yolk.  If you want students to model an embryo in which there is a significant amount of yolk, consider the following model:

Again, students will use clay to represent the yolk-free portion of the egg;  the clay will be placed on an orange or a grapefruit, which will represent the remainder of the egg.  The clay should be made into the shape of a round loaf of bread and placed on the top of the fruit.

In the first five divisions, the cleavage plane is from top to bottom.  The first cleavage will produce two adjacent cells, equal in size.  The second cleavage produces four cells, also equal in size.  The third cleavage produces two rows of four cells.  This cleavage is slightly unequal, so the cells in the middle are slightly larger than those on the end.  The fourth cleavage will produce four rows of four cells.  The fifth cleavage will produce four rows of eight cells.  Through each of these cleavages, the embryo is not gaining cytoplasmic material;  therefore, the cells are occupying the same amount of space as the orginal blastodisc did.

If you wish to continue through subsequent cleavages, you should then add layers of cells.  In the sixth cleavage, you will see layers of cells, each containing 32 cells.  In further cleavages, cells will continue to pile up until the fruit is somewhat top-heavy.

(This modeling exercise was presented by Don Cronkite at the 1997 Woodrow Wilson Biology Summer Institute and is from an earlier activity entitled Visualizing Early Development Patterns in Eggs which was initially presented at the Neurobiology Institute, 1996.  A copy of this activity can be obtained by requesting it from Don Cronkite.  His email address is:  cronkite@hope.edu)

2.  Fertilized Medaka or Zebra fish eggs can be easily obtained from adult fish and the stages of development monitored over a one-two week period.  Later stages of development such as neurulation, morphogenesis, and organogenesis are clearly observable.  In addition, students willl then be able to compare development in an invertebrate versus a vertebrate animal.  See the activity in this module entitled:  UTILIZING THE JAPANESE MEDAKA TO INVESTIGATE EMBRYONIC DEVELOPMENT:  Student Activitie.

3.  Have students design their own experiments to investigate the effects of sperm concentration or environmental factors such as pH, salinity, or possible pollutants (i.e. oil) on fertilization success.  The number of eggs fertilized can be counted and compared to unfertilized ova because of the distinct presence of the fertilization membrane. Many ideas for student designed investigations are provided at the Stanford Sea Urchin Education Site listed below in the References section.
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