[SEA URCHIN IVF]
1
Sea Urchin Lab
[In Vitro Fertilization, Infertility, and Fertility Preservation]
Content
In this laboratory investigation, students will harvest gametes from live sea
urchins and conduct in vitro fertilization (IVF) right in the classroom!
Overview
This is a lab designed to lead students to an understanding about fertility
assistance and fertility preservation options. Students will observe haploid
gametes combining to form a diploid zygote, which grows and develops
through cell replication and differentiation. In this lab, students will learn
how to extract sea urchin gametes and conduct in vitro fertilization.
Students will also observe the first few zygotic cell divisions.
Through this investigation, students will answer the following questions:
How does in vitro fertilization work? What do the first few zygotic divisions
look like?
Ultimately, students will understand more about fertility, things that
negatively impact fertility, and ways to preserve fertility. Furthermore,
students will improve their science reasoning and thinking skills.
Approximate Total Time: Four 45 Minute Class Periods
Curriculum Links:
This set of labs links to the larger curriculum through the concepts of gametes,
reproduction, and cell division.
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Section Overview
Part 1: Harvesting gametes and sea urchin IVF
In part 1 of this lab, students will observe fertilization of the sea urchin eggs under the
microscope. They will observe the cell division of the urchin embryos. Students will also
relate these concepts to the fertility preservation methods in oncofertility.
Part 2: Male Factor Infertility
In part 2 of this lab, students will look at several factors that can contribute to male
infertility. Students will conduct an experiment to test for the optimal concentration of
sperm and discuss how sperm sample analysis is used to diagnose male fertility issues.
Finally students will relate their results back to fertility preservation options for male
patients facing cancer treatment.
Core Content Summary
- Utilize the scientific method (developing and testing a hypothesis, collecting and analyzing
data, interpreting results and making predictions based on those results)
- Observe the interaction between gametes
Laboratory Techniques
- In vitro fertilization
- Microscopy
- Basic pipetting
Oncofertility Content
- Harvest and visualize live male and female gametes
- Observe cell division and early stages of embryo development
- Relate experimental findings to fertility preservation methods
Illinois Learning Standards
11.A.4a Formulate hypotheses referencing prior research and knowledge.
11.A.4b Conduct controlled experiments or simulations to test hypotheses.
11.A.4c Collect, organize and analyze data accurately and precisely.
11.A.4e Formulate alternative hypotheses to explain unexpected results. .
11.A.5c Conduct systematic controlled experiments to test the selected hypotheses.
11.B.4b Propose and compare different solution designs to the design problem based upon given
constraints including available tools, materials and time.
11.B.5c Build and test different models or simulations of the design solution using suitable materials, tools
and technology.
11.B.4e Develop and test a prototype or simulation of the solution design using available materials,
instruments and technology.
11.B.4f Evaluate the test results based on established criteria, note sources of error and recommend
improvements.
12.A.4b Describe the structures and organization of cells and tissues that underlie basic life functions
including nutrition, respiration, cellular transport, biosynthesis and reproduction.
13.A.4c Describe how scientific knowledge, explanations and technological designs may change with
new information over time (e.g., the understanding of DNA, the design of computers).
13.A.5d Explain, using a practical example (e.g., cold fusion), why experimental replication and peer
review are essential to scientific claims.
13.B.5b Analyze and describe the processes and effects of scientific and technological breakthroughs.
13.B.5e Assess how scientific and technological progress has affected other fields of study, careers and
job markets and aspects of everyday life.
[SEA URCHIN IVF]
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ACT College Readiness Skills
Interpretation of Data
- Understand basic scientific terminology (ID.16-19.b)
- Find basic information in a brief body of text (ID.16-19.c)
- Compare or combine data from a simple data presentation, i.e. order or sum data from a table
(ID.20-23.b)
- Translate information into a table, graph, or diagram (ID.20-23.c)
- Analyze given information when presented with new, simple information (ID.24-27.f)
Scientific Investigations
- Understand the methods and tools used in a moderately complex experiment (SI.20-23.a)
- Understand a simple experimental design (SI.20-23.b)
- Identify a control in an experiment (SI.20-23.c)
- Predict the results of an additional trial or measurement in an experiment (SI.24-27.c)
- Determine the experimental conditions that would produce specified results (SI. 24-27.d)
Evaluation of Models, Inferences, and Experimental Results
- Select a simple hypothesis, prediction, or conclusion that is supported by two or more data
presentations or models (EM.20-23.a)
- Determine whether given information supports or contradicts a simple hypothesis or conclusion,
and why (EM.24-27.b)
- Identify strengths and weaknesses in one or more models (EM.24-27.c)
- Identify similarities and differences between models (EM.24-27.d)
Lab Index
Teacher Lab Guide: Sea Urchin IVF .............................................................................................. 4
Part 1: In vitro Fertilization of Sea Urchin ................................................................................. 5
Section 2: Male Factor Infertility................................................................................................ 8
Background Research Information ............................................................................................. 9
Part 1: In vitro fertilization (IVF) ..................................................................................................11
[Introduction] .............................................................................................................................11
[Protocol] .................................................................................................................................. 13
[Analysis] .................................................................................................................................. 16
[ACT College Readiness Practice] ........................................................................................... 18
Part 2: Male Factor Infertility ....................................................................................................... 20
[Introduction] ............................................................................................................................ 20
[Protocol] .................................................................................................................................. 22
[References] ................................................................................................................................. 24
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Teacher Lab Guide: Sea Urchin IVF
Purchasing live sea urchin IVF kits:
Live Sea Urchin are only shipped on certain days depending on the supplier (i.e.
Carolina Biological only ships live organisms on Mondays or Tuesdays) therefore this
should be taken into account when planning for their arrival.
Supplier
Item Description
Catalog number
Amount
Unit cost*
Carolina Biological
Supply
Sea Urchin
Embryology Kit
162505
1
$125.00
Ward’s Natural Science
Sea Urchin
Embryology Kit
87 V 9030
1
$115.00
Ward’s Natural Science
Sea Urchin
Embryology
Accessory Kit
88 V 9031
1
$42.25
*cost in June 2011
Tips for the arrival of live sea urchin:
Your urchin will arrive shipped in bags within a Styrofoam box. A bag may have leaked
during shipping therefore the urchin will need to be placed into clean saltwater
immediately. It is important to keep the male and female urchins separate. When they
are together in the water, they will release their gametes. Since you cannot determine
the sex of the urchin until spawning, it is best to keep each urchin in a separate
container. E.g. You may keep each one in the individual baggie in which it was shipped,
but just add fresh sea water. Though some kits come with an extra gallon of pre-mixed
seawater more may be needed depending on how long you will need the urchin
between arrival and completion of the lab. Typically, companies ship Atlantic cold-water
urchin so keeping them out of direct sunlight, in a cool part of the room is best.
[SEA URCHIN IVF]
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Part 1: In vitro Fertilization of Sea Urchin
Objective: Students will extract gametes from male and female sea urchin and conduct
in vitro fertilization in order to visualize live cell division.
Materials:
Class Materials:
Male and Female sea urchins
Syringe with 25 gauge or smaller needle
0.55M KCl
2-20μL pipette and 2-200μL pipette tips
1.5ml Epitube
500mL Beakers
Scissors
Per Lab Station:
Light Microscope
8 Glass slides
8 Cover slips (Optional)
4 Transfer pipettes
100mL Sea Water
20mL Graduated cylinder or Syringe
5- 15mL Conical Tubes
Dixie or small plastic cup
Time Requirement Estimates (one or two 40-50 minute lab periods):
 Pre-lab videos, reading, discussion, hypothesis, and procedure design will take
one class period of approximately 50 minutes.

The procedure of this lab (Sea Urchin Embryology Kit IVF) fills a “standard” class
period of approximately 50 minutes but developing embryos can still be
visualized on day two.

“Consulting with the Expert” and discussing results will take 25-50 minutes
depending on how thorough the discussion is.
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Teacher notes:
Injecting Urchins:
Injecting the urchins with potassium chloride will cause them to release their gametes.
The injection procedure is included in the student protocol of the lab. However the
teacher may choose to do the injections as a demo. Extreme safety precautions and
oversight must be used with the syringes of the KCl.
Collecting Urchin Gametes:
After injecting the urchins, observe the gametes that begin to excrete. A male urchin will
release sperm, which is milky white in color. A female urchin will release eggs which can
vary in color, but are typically orange-ish with the species that are recommended here.
Once you know the sex of the urchin, immediately follow with the corresponding
collection procedure described below.
Sperm. After injecting the urchin, place it (mouth side up) on top of an empty beaker.
The sperm must not come into contact with seawater! Thus the sperm that is released is
considered dry inactivated sperm. Seawater will only be added to the sperm to activate
it before initiating fertilization. Activated sperm can only live for approximately 20
minutes. To collect the sperm, cut the tip off of a pipette to create a larger bore. Then
draw up the sperm and place it into a microtubule. The dry inactivated sperm can be
kept for approximately 2-3 days.
Eggs. After injecting the urchin, place it (mouth side up) on top of a beaker filled with
seawater so that the urchin is partially submerged. The eggs will enter the solution and
settle at the bottom. Once all the eggs settle to the bottom of the beaker, wash them 2-3
times with fresh filtered seawater by pouring off the old water and adding more in. After
the washes, let the eggs settle again to the bottom. Remove most of the water and
make a 5% egg solution by adding 5mL of eggs to 100mL of filtered seawater. The eggs
will last a few hours in this solution, but keep them chilled ~13-15oC.
Photo(s) courtesy of OSA San Diego, showing two different methods for harvesting eggs from a female sea urchin.
[SEA URCHIN IVF]
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Be sure to keep the eggs (~15oC) and sperm cold (~4 oC). This can be on ice or in a
refrigerator. (The virtual urchin website is a great resource when to spawning urchins
http://virtualurchin.stanford.edu/fertlab.htm)
After students have harvested and collected their sea urchin gametes, you should now
have at least one 1.5mL epi tube with sperm and 1 beaker with fresh eggs.
Check to make sure the students understand how to identify fertilized eggs so that they
can determine if an egg is fertilized or not for the next section. Teacher should also
make a batch of fertilized eggs to be observed for the next day, this batch of eggs can
sit at room temperature overnight and will be the 24 hour time point.
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Section 2: Male Factor Infertility
Objective: Students will test for the optimal sperm concentration and discuss different
possible causes of infertility in males. Students will then relate this information to fertility
preservation techniques in oncofertility.
Materials:
Group Materials (per lab group):
Light Microscope
4 Glass slides
4 Cover slips (Optional)
6 Transfer pipettes
100mL Sea Water
20mL Graduated cylinder or Syringe
Dixie or small plastic cup
Rubber gloves
Goggles
Class materials
Fertilized sea urchin embryos (saved from previous day)
Sea urchin gametes (saved from previous day)
Time Requirement Estimates (approximately two 40-50 minute lab periods):
 Expert videos, discussion, hypothesis, and procedure design, and classroom lab
procedure will take approximately one class period of 45 minutes.
[SEA URCHIN IVF]
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Background Research Information
This short article highlights the work of Dr. Woodruff and others working to develop
fertility options for women diagnosed with cancer.
 http://www.nibib.nih.gov/HealthEdu/eAdvances/30Sep09
Sea Urchin Embryology. Dr. William H. Heidcamp, Biology Department, Gustavus
Adolphus College.
 http://homepages.gac.edu/~cellab/chpts/chpt13/ex13-1.html
Sea Urchin Development.
 https://www.msu.edu/~gittinsj/seaurchin/development.html
Sea Urchin Embryology
 http://www.stanford.edu/group/Urchin/west.htm
Sea Urchin Animal Maintenance
 http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Urchin/urchin_animals.html
Virtual Urchin Stanford University
 http://virtualurchin.stanford.edu
Basic student-friendly scientific method description
 http://www.sciencebuddies.org/science-fairprojects/project_scientific_method
.shtml
Text description of the scientific method
 http://teacher.pas.rochester.edu/phy_labs/appendixe/appendixe.html
Supplemental Video Clips
The following is a list of short supplemental videos taken from the website
http://www.myoncofertility.org which has a wealth of information that could be
included. These are the most directly related to the lab activities, however many other
portions of the site could be used to add greater depth to the study.
What is normal female fertility, and how is it affected by cancer treatment?
 http://www.myoncofertility.org/animations/what_normal_female_fertility_and_how
_it_affected_cancer_treatment
What is oncofertiltiy?
 http://www.myoncofertility.org/videos/what_oncofertility
What are some goals of the oncofertility consortium?
 http://www.myoncofertility.org/videos/what_are_some_goals_oncofertility_
consortium
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How can cancer treatment damage fertility in both males and females?
 http://www.myoncofertility.org/videos/how_can_cancer_treatment_damage_
fertility_both_males_and_females
What is the timeline for people with cancer and fertility preservation? The clip describes
male and female fertility preservation.
 http://www.myoncofertility.org/videos/what_timeline_people_cancer_and_fertility_
preservation
Egg & Embryo Banking Video
 http://www.myoncofertility.org/animations/egg_and_embryo_banking_basics
Ovarian Tissue Cryopreservation
 http://www.myoncofertility.org/animations/ovarian_tissue_cryopreservation_basics
[SEA URCHIN IVF]
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Sea Urchin Lab
Part 1: In vitro fertilization (IVF)
[Introduction]
Background Information
Though estimates vary, 10-20% of couples attempting to get pregnant with their first
child experience infertility.
Assisted Reproductive Therapy (or ART) is a field of
medicine that specializes in helping infertile couples conceive children. One way that
infertile couples might conceive is through in vitro fertilization, commonly referred to as
IVF. This method involves joining female gametes (mature eggs) and male gametes
(sperm) outside of the body. Once the egg and sperm join in vitro (outside the body),
then the newly formed embryo can be put back into the woman’s body to develop.
In this series of experiments, you'll begin to explore how IVF works. You will assume
the role of an Assisted Reproductive Therapist, and will extract gametes from sea
urchins. Sea urchins are echinoderms that live in the ocean and feed on algae. They
are round and spiny, with a variety of colors. Sea urchins
in vivo – inside the body
are frequently used as models in developmental biology
because they are simple organisms, and it is easy to
in vitro – outside the body
extract their gametes.
Initial problem to solve: In Biology we use many model organisms to study human
problems. Today you will assume the role of a reproductive scientist that uses sea
urchins as a model to study how to successfully conduct IVF and study early embryonic
development.
The question for you to consider is:
How can you tell if you have successfully fertilized a sea urchin egg using IVF?
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Think about it...
1. Watch the following video about sea urchin fertility in nature:
http://video.nationalgeographic.com/video/player/animals/invertebratesanimals/other-invertebrates/sea_urchin_breeding.html
2. What did you learn about how sea urchins reproduce in nature? Consider how
many gametes are produced by each sex, where the gametes join, and how
many offspring are produced.
3. Why do you think sea urchins reproduce in this way? What are the advantages
of reproducing like this? What are the disadvantages?
4. Why do you think sea urchins are used as models to study IVF?
5. If you wanted to simulate sea urchin reproduction in the laboratory, what are
some of the factors that you will need to consider?
[SEA URCHIN IVF]
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Sea Urchin Lab
Part 1: In vitro Fertilization
[Protocol]
Harvesting and Collecting Female and Male Gametes
1. Identify the sea urchin volume by measuring the amount of salt water displaced
when it is put in a beaker.
Sea Urchin Volume: __________
2. Calculate the amount of 0.55M KCl you will need. Use 0.1 mL per 10mL of
urchin volume.
(0.1mL of 0.55M KCl) x (Sea Urchin Volume/10mL) = ___________
3. Load the syringe and needle with the amount of 0.55M KCl calculated above.
Turn the urchin upside down and inject the KCl solution near the mouth in
between the gonads. Inject at 2 of the 5 injection sites.
Gonads
Bottom view of Urchin
Sites of injection between gonads
Mouth
NOTE: Be as accurate as possible when injecting; too much KCl is lethal to
the urchin!!
Determining Sea Urchin Sex
There is no way to distinguish male sea urchins from female sea urchins until you
extract their gametes. Sea urchin eggs are typically orange, and sea urchin semen is
usually milky white. Therefore, you will determine the sex of your urchin by the type of
gametes it releases after it has been injected with KCl.
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4. Gently shake the sea urchin for a few seconds to distribute the injected KCl and
place the urchin with its mouth down on a petri dish or beaker. Gametes will be
released from the urchin’s gonopore (opposite side of the mouth) after a few
minutes.
What is the sex of your urchin? ____________
Follow the directions below for “collecting eggs” or “collecting sperm”
based on whether your sea urchin is a female or a male.
Collecting Eggs
5. Place the sea urchin with its mouth side up on
top of a beaker filled with seawater so that the
urchin is partially submerged. The eggs will enter
the solution and settle at the bottom.
6. Once all the eggs settle to the bottom of the
beaker, wash them 2-3 times with fresh filtered
seawater by pouring off the old water and adding
more in.
7. After the washes, let the eggs settle again to the bottom.
8. Remove most of the water and make a 5% egg solution by adding 5mL of
eggs to 100mL of filtered seawater.
9. Place the tube in cool water or in refrigerator (a directed by your teacher). The
eggs will last a few hours in this solution, but keep them chilled at about 1315oC.
Collecting Sperm
5. Cut the end of a pipette tip with scissors to allow for a larger diameter.
6. Using a 2-20L pipette, collect as much sperm as you can from the sea
urchin and place it into a 1.5mL tube.
7. Place the tube on ice or in refrigerator (a directed by your teacher). The
sperm can last for one week if kept cold (~4oC).
NOTE: Do not mix the sperm with any sea water at this step! (Once the sperm
are activated with seawater they will only survive for approximately one hour.)
[SEA URCHIN IVF]
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Fertilization and Early Embryonic Development
Each lab station receives a small sample of 5% egg solution and inactivated sperm solution.
1. Sperm Activation: pipette 10L of sperm into 10mL of filtered seawater. Label this
Dilution A, mix the tube gently and keep it cold (~4oC).
What is the ratio of sperm to seawater in Dilution A? _________
NOTE: Dilution A solution should be used within 20 minutes; if kept longer,
make again fresh.
2. Label a glass slide “A” and pipette a few drops of the 5% Egg solution onto the
center of the glass slide.
3. Place the slide onto a light microscope and focus on the eggs to see how they
look unfertilized.
4. Pipette a drop of the diluted sperm to the eggs on the microscope.
5. Observe what happens. You can distinguish fertilized eggs by the presence of a
halo, which should form within 1 minute of the sperm addition.
Note the time at which slide A is fertilized: ______________
6. Draw a picture of the egg before and after fertilization. Label the halo that is
present.
Before Fertilization
After Fertilization
7. Remove slide from microscope and set aside for use the following day.
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Sea Urchin Lab
Part 1: In vitro Fertilization
[Analysis]
1. Based on your observations, how is it possible to determine if you have
successfully fertilized a sea urchin egg using IVF?
2. Minutes after fertilization is complete a protective coat called the fertilization
envelope develops around the newly fertilized egg. Why do you think the
protective envelope forms?
EGG BEFORE FERTILIZATION
“EGG” AFTER FERTILIZATION
Little
dots are
sperm
Photo courtesy of the Hamdoun Lab displays the appearance of the egg before and after fertilization.
3. In human IVF clinics, eggs and sperm are also combined in vitro, similarly to the
sea urchin IVF you conducted. After fertilization, the embryo must be implanted
into the mother. Why might determining successful fertilization be useful when
trying to help infertile couples conceive?
[SEA URCHIN IVF]
17
4. The lab process we did today is known as IVF or In vitro Fertilization. “In vitro”
literally translates to “within the glass” while “in vivo” means “within the living.”
Why do you think people sometimes decide to have doctors perform In Vitro
Fertilization?
5. What do you suppose might be some risks associated with going through the
process of IVF?
6. What might be some of the controversial or moral/ethical debates that can come
up with this process?
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Sea Urchin Lab
Part 1: In vitro Fertilization
[ACT College Readiness Practice]
Experiment 1
In the first experiment students timed how long it took for
individual sperm cells to swim 10 mm at different
temperatures and recorded their data in table 1.
Table 1
Temp. of
water
bath
(°C)
Time for
sperm to
swim
10mm
(sec)
Number
of eggs
fertilized
5
22
20
10
15
32
15
8
31
20
5
40
25
9
25
Experiment 2
Next, students placed 50 Sea Urchin eggs and 1000 Sea
Urchin sperm into each of the five different containers and
recorded the number of Sea Urchin eggs that were
fertilized and recorded their data in table 1.
Table 2
Experiment 3
In order to determine the ideal salinity (concentration of salt) for
Sea Urchin egg fertilization, a controlled experiment was
conducted. 50 Sea Urchin eggs and 1000 sperm were placed
into five different aquariums and fertilization rates were
observed (temperature of the water was maintained at 25 °C
for each aquarium). The data for experiment 3 was recorded in
data table 2.
Salinity
(g/L)
Number of
eggs
fertilized
15
15
25
20
35
23
45
19
65
10
1) At which of the following temperatures would you expect Sea Urchin eggs to be
fertilized the quickest? (ID.16-19.d)
a. 5
b. 15
c. 20
d. 25
[SEA URCHIN IVF]
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2) According to experiments 1, 2, and 3 what is the optimal (best) temperature and
salinity for successful Sea Urchin egg fertilization? (ID.20-23.a)
a. 35° and 20 g/L
b. 20° and 25 g/L
c. 5° and 65 g/L
d. 20° and 35g/L
3) In experiments 2 and 3, the students used a microscope to make it easier to
determine: (SI.20-23.a)
a. The salinity of the water
b. The number of sperm and egg cells
c. The temperature of the water
d. The density of the water
4) If a student conducted another trial of experiment 1 in which the temperature of
the water was 3 °C, which of the following would be the most likely time for the
sperm to swim 10mm? (ID.24-27.c)
a. 22
b. 17
c. 35
d. 25
5) According to table 1 and table 2, if experiment 3 was repeated with a
temperature of 20 °C what would happen to the number of successful
fertilizations? (ID.24-27.a)
a. It would increase
b. There would be no change
c. It would decrease
d. First decrease and then increase
6) A student conducted experiment 1 and stated that water at 18° C produced the
fastest swimming sperm. Does the data trend support his statement?
(EM.24-27.b)
a. Yes, because that was the optimal salinity level for fertilization.
b. No, because temperature has no effect on swimming rates.
c. No, because temperatures near 18° C produced the slowest swimming
rates.
d. Yes, because temperatures near 18° C produced the fastest swimming
rate
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Sea Urchin Lab
Part 2: Male Factor Infertility
[Introduction]
Background Information
Infertility may be caused a variety of different things and both males and females can be
infertile. One form of infertility is called male factor infertility. The most common cause
of male factor infertility is a disorder involving the sperm. These include problems with
the production and maturation of sperm.
For example, sperm may be immature,
abnormally shaped, or unable to move properly. These factors can affect a sperm’s
motility, or ability to move/swim.
Alternatively, normal sperm may be produced in
abnormally low numbers or not at all.
This is often called “low sperm count.”
(http://www.umm.edu/urology-info/infertil.htm)
If the sperm count or motility is extremely low, this is usually assumed to be the cause of
infertility for a couple. What ultimately matters is not how many sperm there are, or how
fast they can swim, but whether they can fertilize the female partner's eggs. This is
really a biochemical issue working at the molecular level. Looking at the sperm under
the microscope is not necessarily an accurate way to assess their ability to fertilize
eggs. However, a semen analysis is still the best test available to assess male factor
infertility at this time. (http://www.advancedfertility.com/sperm.htm)
In this series of experiments, you will explore male factor infertility. You will continue
your role as an Assisted Reproductive Therapist, and will conduct a series of
experiments to determine how low sperm count has an impact on fertilization.
Initial problem to solve: As a reproductive scientist you
must determine what concentration of sperm constitutes
optimum fertility probability.
The question for you to consider is:
How do you determine if someone has male factor infertility?
Motility – the ability to move
spontaneously and actively
Molecular – the atoms or
compounds that compose a
substance
Biochemical – the chemical
processes of living things
[SEA URCHIN IVF]
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Think about it...
Sea Urchins reproduce by a method called “broadcast spawning.” This means that they
release their gametes into the water, as seen in the video (“Think About It…” Part 1). In
order for fertilization to occur, a sperm and an egg must come into contact, then fuse to
combine their genetic material. A sperm and egg coming into contact in the ocean
happens purely by chance.
1. If sea urchin eggs and sperm only come into contact by chance, what are some
ways to increase the likelihood that they will come into contact?
2. In human reproduction, sperm are deposited into the female’s reproductive tract.
A sperm and egg that come into contact still happens by chance, but the human
system has been designed to increase the chances that this will occur. How is
sea urchin reproduction similar to human reproduction? How is it different?
Compare and contrast the two in the space provided, below.
3. Consider male fertility preservation options for patients diagnosed with cancer.
How might you think oncofertility relates to this part of the lab?
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Sea Urchin Lab
Part 2: Male Factor Infertility
[Protocol]
Setting up a Serial Dilution
1. Sperm Activation: pipette 10L of sperm into 10mL of filtered seawater Label this
Dilution A, mix the tube gently and keep it cold (~4oC).
NOTE: Dilution A solution should be used within 20 minutes; if kept longer, make
again fresh.
2. Label 4 other tubes with B, C, D, and E. Fill each tube with 8ml of artificial or
filtered seawater using a graduated cylinder or syringe.
3. Using a transfer pipette place 2mL of Dilution A into
tube marked B; mix well and place on ice.
4. Using a transfer pipette place 2mL of Dilution B
into tube marked C; mix well and place on ice.
NOTE: Mix each dilution
well by using the transfer
pipette to mix up and down
before making the next
solution and to keep them
on ice at all times.
5. Using a transfer pipette place 2mL of Dilution C into tube marked D; mix well
and place on ice.
6. Using a transfer pipette place 2mL of Dilution D into tube marked E; mix well
and place on ice.
Dilution
A
B
C
D
E
Procedure
10L sperm + 10mL of seawater
2mL Dilution A + 8mL of seawater
2mL Dilution B + 8mL of seawater
2mL Dilution C + 8mL of seawater
2mL Dilution D + 8mL of seawater
Ratio of Sperm to Seawater
1:1000
1:5000
1:25000
1:125000
1:625000
7. Label 5 glass slides with one letter, A-E, with a marker.
8. Starting with slide A, place a few drops of the 5% Egg solution onto the center,
and observe the eggs under the light microscope just as in Part 1.
[SEA URCHIN IVF]
23
9. Add a drop of Dilution A to the eggs on Slide A; wait 2 minutes then look for
fertilized eggs. Draw what you see in Table 2. Note the number of fertilized eggs
for Dilution A, and record in the Table 2.
10. Repeat for dilutions B, C, D, and E.
Table 2. Male Factor Infertility Observations
Dilution
Draw Diagram
(after 2 minutes)
A
B
C
D
E
Number of
Fertilized Eggs
24
[NUBIO]
[References]
Reference(s): John Yamauchi1 and Dominic Dirksen2 1Dept. of Pharmacology, Skaggs School of
Pharmacy and Pharmaceutical Sciences, Univ. of California-San Diego, La Jolla, CA, 92037-0650
2
Steele Canyon High School, 12440 Campo Road, Spring Valley, CA 91978
Sea Urchin Embryology. Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus
College. http://homepages.gac.edu/~cellab/chpts/chpt13/ex13-1.html
Photo(s) courtesy of OSA San Diego. Photo shows Dr. Hamdoun demonstrates proper technique when
stimulating sea urchins to release gametes.
Reference(s): John Yamauchi1 and Dominic Dirksen2 1Dept. of Pharmacology, Skaggs School of
Pharmacy and Pharmaceutical Sciences, Univ. of California-San Diego, La Jolla, CA, 92037-0650
2Steele Canyon High School, 12440 Campo Road, Spring Valley, CA 91978
Sea Urchin Embryology. Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus
College. http://homepages.gac.edu/~cellab/chpts/chpt13/ex13-1.html
Sea Urchin Development. https://www.msu.edu/~gittinsj/seaurchin/development.html
Sea Urchin Embryology http://www.stanford.edu/group/Urchin/west.htm
Sea
Urchin
Animal
Maintenance
http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Urchin/urchin_animals.html
Igelsrud, Don. Sea Urchins. The American Biology Teacher, Vol. 49, No. 7 (Oct., 1987), pp.446-450