nov 07 online A.indd - National Association of Biology Teachers

O N L I N E I N Q U I R Y & I N V E S T I G AT I O N
Using Caffeine To Hyperactivate Bull Sperm
T
he introduction of reproduction utilizing only the usual
array of textbook graphs and diagrams can take an otherwise
fascinating subject, particularly to high school and college-age
students, and reduce it to sheer boredom. This exercise gives students the opportunity to see live, swimming mammalian sperm
and to watch how they respond to a trigger that prepares them
for fertilization in the same way that they are prepared to fertilize eggs inside the female. The trigger we use in this experiment
is caffeine, a chemical familiar to students. While it is not the
physiological trigger (which is unknown at present), it produces
the same effect. Although students are taught that cells respond
in many ways to different molecules, they rarely get a chance to
observe these responses or to see how cells might respond to a
chemical that is familiar to them. Here is a chance to actually see a
cell respond to something that a student might drink every day.
This exercise will reinforce students’ understanding of
mammalian reproductive processes by enabling students to see
live sperm. In addition, the use of caffeine to produce a change
in sperm that is normally undergone just before fertilization can
foster an interesting discussion about how caffeine might affect
sperm in the men and women who drink it, including a discussion of how caffeine could get from the digestive tract to the
reproductive tract via the circulatory system. This experiment
includes controls that show the importance of using proper controls in designing experiments.
Background
In different species of mammals, males deposit sperm into
the vagina (e.g., primates and cattle) or directly into the uterus
PAULA JONES is a high school chemistry and biology teacher, Homer Central
School, Homer, NY 13077; e-mail: pjones@homercentral.org. WAYNE
GOTTLIEB is a life science teacher, DeWitt Middle School, Ithaca, NY
14850: e-mail: wgottlieb@yahoo.com. SUSAN S. SUAREZ, Ph.D. is Professor,
Department of Biomedical Sciences, Cornell University, Ithaca, NY 14850;
e-mail: sss7@cornell.edu.
PAULA JONES
W AY N E G O T T L I E B
SUSAN S. SUAREZ
(e.g., pigs). Human sperm are deposited into the vagina right
at the entrance to the cervix, where thousands quickly enter.
This saves the sperm from being killed in the vagina. The pH of
human vaginal fluid is highly acidic, which serves to kill bacteria
and other potentially infectious microbes; however, the acid can
also immobilize sperm that fail to enter the cervix quickly.
Sperm are able to swim through the cervix by following
grooves in the wall. Although the cervix is filled with mucus,
the mucus is very watery during the fertile period of the month,
particularly in the grooves, and sperm can easily swim through
the grooves to reach the uterine cavity.
Sperm that enter the uterus may be helped along towards
the oviduct (fallopian tube) by peristaltic contractions of muscle
in the uterine walls. When sperm reach the entrance to the
oviduct, they must squeeze through the opening to get inside.
Only vigorously motile, well-shaped sperm can make it through.
Sperm may be stored in the oviduct for a few days (or a few
months in hibernating bats) until ovulation occurs and the egg
enters the oviduct.
When a human sperm is first deposited in the vagina, its
flagellum (tail) generates low symmetrical waves that propel it
forward in a straight path. This helps the sperm to pass through
the cervix and uterus, and to enter into the oviduct. When the
time of ovulation nears, the sperm in the oviduct are triggered to
hyperactivate. The waves generated by the flagellum increase in
height, but the beat becomes asymmetrical (reviewed in Suarez
& Pacey, 2006).
If hyperactivated sperm are put on a microscope slide, this
asymmetrical beating causes sperm to swim rapidly in zigzags
or circles (Figure 1)—not the sort of behavior one would expect
from a sperm that is about to fertilize an egg! A microscope slide,
however, is not the same as the inside of the oviduct. In the oviduct, the sperm must swim through a narrow tunnel filled with
a dense mucous secretion and lined by walls of multiple soft
folds. Hyperactivation helps the sperm to move out of pockets
formed by the folds, and to pass through the mucus. The egg
HYPERACTIVATE BULL SPERM
81
itself (oocyte) is embedded in a mass
of cumulus cells and their viscous, elastic secretion (Figure 2). Immediately
around the oocyte is an elastic protein
shell called the zona pellucida. Thus,
sperm must penetrate viscous and elastic substances in order to fertilize an
egg. When artificial viscous and elastic
substances are added to hyperactivated
sperm on a microscope slide, it slows
them down; however, they straighten
out and penetrate it much more effectively than sperm that have not been
hyperactivated (Suarez et al., 1991;
Suarez & Dai, 1992). Also, if eggs are
added to sperm in a dish, only sperm
that are hyperactivated can penetrate
the zona pellucida surrounding each
oocyte (Stauss et al., 1995; Ren et al.,
2001). These experiments indicate that
hyperactivation helps sperm to reach
the egg and fertilize it.
Figure 1.
Swimming patterns of pre-hyperactivated (A)
and hyperactivated (B) bull sperm.
Some cases of human male infertility have been linked to
poor hyperactivation of sperm (Chan et al., 2001). Recently, a
mutant strain of mice was developed in which the sperm cannot hyperactivate. The mutant males are completely infertile,
demonstrating that hyperactivation is absolutely required for
fertilization (Carlson et al., 2003).
Sperm hyperactivate when exposed to a trigger that raises
calcium levels in the flagellum (Suarez et al., 1993). The
natural trigger that hyperactivates sperm in the oviducts is still
unknown, but sperm can be hyperactivated by drugs that are
known to increase intracellular calcium. One such drug is caffeine (Ho & Suarez, 2001). Caffeine can raise calcium levels by
opening calcium channels in the plasma membrane of the sperm
flagellum.
For this classroom activity, students will use caffeinated drinks or
even pure caffeine to hyperactivate
bull sperm. Bull sperm behave quite
similarly to human sperm, thus they
serve as an excellent model to use for
these experiments. The main difference between bull sperm and human
sperm is that bull sperm have paddleshaped heads, while human sperm
have conical heads.
This activity would ideally be
undertaken during a unit on reproduction. Students should be familiar
with female reproductive anatomy,
sperm structure, fertilization, and
microscope use. It could also fit nicely
into a unit on cells, where the focus
would be on the effect of caffeine
on cells, or into a unit on human
body systems, where students could consider the connections
between the digestive system, circulatory system, and reproductive system that would allow some of the imbibed caffeine to
reach sperm.
Introducing Students to the
Experiment
Review female anatomy and the process of fertilization with
students. Then, discuss the following concepts and questions.
1. How do sperm get to the egg? What organs do they pass
through? What is it like inside these organs from the
perspective of a sperm?
2. How does a sperm swim? What propels it forward?
3. What happens during fertilization?
Figure 2.
An egg from a rabbit, showing the oocyte, zona pellucida, and cumulus mass containing cells
and viscoelastic matrix. The oocyte is approximately 100 microns in diameter.
A PowerPoint presentation on the
subject of sperm movement through
the female reproductive tract can be
obtained from Suarez (sss7@cornell.
edu).
Overview of
Experiment
Working in pairs, students dilute
bull sperm in a medium made of nonfat dry milk. The students then observe
the sperm under a microscope to see
their linear, progressive swimming
movements. Next, students add coffee to a sample of sperm and see that
it hyperactivates some and kills others, probably because organic chemicals in coffee other than caffeine are
toxic to sperm. As a control, students
treat sperm with decaffeinated coffee.
Students can also treat sperm with a
solution of pure caffeine, which should
hyperactivate nearly all of them without killing any.
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THE AMERICAN BIOLOGY TEACHER, ONLINE PUBLICATION, NOVEMBER/DECEMBER 2007
Materials
• Bull semen (West Hill RD-501, $24.00 at www.west
hillbio.com). Order to arrive the day before the experiment and keep in the refrigerator.
• Carnation Instant Nonfat Dry Milk (we know that
Carnation works; we cannot guarantee that another
brand will)
• Eppendorf tubes (1.5 ml size) or plastic or glass test
tubes
• distilled water
• hot plate or some device for keeping sperm and liquids
at body temperature
• 1 M NaOH for adjusting pH and pH meter or pH paper
for measuring pH
• thermometer for checking temperature of solutions
• plastic pipets
• microscope slides and cover slips (if they are to be reused, clean thoroughly)
• microscope with 40X objective
• caffeinated drinks (coffee, tea, soda) and matching decaffeinated drinks to use as negative controls. Drinks high
in caffeine work best and most coffees contain more
caffeine than most teas. Mountain Dew contains more
caffeine than colas.
• If using ground coffee: filter paper & funnel
• Pure caffeine can be purchased from Flinn Scientific
(www.flinnsci.com), catalog number C0344.
Methods
1. Milk medium for sperm can be prepared one day ahead
and stored in a refrigerator.
Dissolve 9.58 grams of Carnation Instant Nonfat Dry
Milk in distilled water and bring volume up to 100 ml.
Increase the pH to 8.3 by adding 1 M NaOH drop-wise.
2. Coffee and other caffeinated drinks can be prepared one
day ahead and stored in refrigerator.
Coffee: Dissolve 1 tsp Folgers Instant Coffee to 30 ml of
milk medium. Add 1 M NaOH drop-wise to pH 8.3. Do
the same with decaffeinated coffee as a negative control.
The higher the caffeine content, the better the results. A
strong ground coffee, such as expresso, can be prepared
by placing 1 tbsp ground coffee in a filter paper in a
funnel. Pour boiling milk solution on the coffee. Collect
filtrate and re-filter through the coffee a second time.
Readjust pH to 8.3. For a negative control this can be
done with a decaffeinated version of the coffee.
Tea: Bring 30 ml of milk medium to a boil. Remove from
heat and immediately add a bag of strong black tea.
Allow it to brew for five minutes. Remove tea bag and
readjust to pH 8.3. For a negative control this can be
done with a decaffeinated version of the tea.
Soda: Allow 30 ml of cola, Mountain Dew, or other caffeinated soda to go flat. Add 1 M NaOH to bring pH to
8.3.
3. Make a 40 mM solution of pure caffeine by dissolving
0.077 grams caffeine in 10 ml of milk medium.
4. Just before adding to sperm, warm milk and test solutions to 37˚ C (98.6˚ F). Also warm all tubes, pipets,
slides, and coverslips.
5. To prepare sperm, add about 0.1 ml of the semen to 0.9
ml of pre-warmed milk medium in a tube. The remaining semen should be kept cooled for use later in the day
or the next day. Once the sperm have been diluted in
the warm milk medium for the experiment, they should
be kept at 37˚ C in an incubator, on a hot plate, or in a
warm water bath.
6. Stir up the sperm very gently by flicking the bottom of
the tube. Using a plastic pipet, place a small drop of the
sperm suspension onto a microscope slide. Gently cover
with a coverslip.
7. Place the slide on the microscope stage and focus on
the sperm using the 40X objective. The sperm are 70
microns long, so they are barely visible under a 10X
objective. If the microscope is equipped with a substage
diaphragm in addition to the field diaphragm, the sperm
will be easier to see if the diaphragm is closed down to
increase depth of field.
8. Describe the swimming patterns of the sperm and estimate by eye the percentage of sperm that are motile.
9. Place a small drop of warm coffee solution on a clean
slide. Immediately add a small drop of sperm suspension into the drop of coffee solution and top with a
coverslip.
10. Describe the swimming patterns of the sperm and estimate by eye the percentage of sperm that are motile.
11. Repeat Steps 9-10 using decaffeinated coffee. Compare
the effect on sperm with that of caffeinated coffee.
12. If time permits, the effects of tea or caffeinated soft drinks
can be compared with those of coffee (Are they as effective as coffee? Do they kill more or fewer sperm?). Repeat
Steps 9-10 and use decaffeinated controls if available.
Different groups of students could test different drinks.
13. If pure caffeine is to be used, which is highly recommended, repeat Steps 9-10 using the solution of caffeine
in milk medium. For the negative control, use milk
medium without caffeine.
Precautions
1. Sperm are delicate. It is important that they are kept
warm for the duration of the experiment and handled
gently. Those that will be used at a later time must be
kept cool, according to the instructions provided with
the package.
2. Whenever transferring sperm, students should pipet
smoothly and slowly to avoid killing them.
3. Sperm are diluted in milk rather than in water in order
to approximate the fluids encountered by sperm in the
female reproductive tract. Adding plain water to sperm
will kill them immediately.
4. Any container used for sperm, medium, or test substances should be very clean, rinsed free of any soap residue
using tap water, given a thorough final rinse in distilled
water, and then completely air dried before use.
HYPERACTIVATE BULL SPERM
83
5. Make sure that students are able to make wet mounts
and focus a microscope using 40X objectives well before
the start of the experiment. If they take more than a few
minutes trying to find sperm because they are unfamiliar
with using the microscope, the sperm may slow down
or die.
Realistically, very little caffeine from a cup of coffee would make
it to the sperm in the reproductive tract. The other organic chemicals in the drinks that kill sperm are unlikely to kill sperm in the
body, because most will be broken down by digestive enzymes.
Caffeine is a small molecule that is not quickly destroyed by
digestive enzymes.
6. If pure caffeine is used, note that it can be toxic to people
if ingested in amounts greater than a few hundred milligrams.
During the class discussion or in the lab write-up, students
could be given the opportunity to design their own experiment.
If students have seen other swimming microorganisms under
the microscope in the past, they could compare the anatomy and
behavior of those organisms to the swimming of sperm.
Results
Prior to treatment with coffee or decaffeinated coffee, the
sperm should be seen swimming in straight lines (Figure 1).
After treatment with coffee, about half of the sperm die, due to
other organic chemicals in the coffee. About half of the sperm
that remain motile should be hyperactivating, which means they
will swim rapidly in circles or show a zigzag movement pattern.
While these patterns may not seem useful for getting to the egg,
it should be pointed out that hyperactivated sperm can penetrate
thick elastic substances around the egg. Other caffeinated and
decaffeinated drinks will produce similar effects and the strength
of the hyperactivation response will depend on the amount of
caffeine present in the drink.
Treatment of sperm with decaffeinated drinks serves as
a negative control to demonstrate that it is the caffeine in the
drinks that causes hyperactivation, while the other ingredients
kill sperm. The same percentage of sperm should be killed as
those treated with caffeinated drink; however, the sperm remaining motile will continue swimming in straight lines.
Acknowledgment
Development of the classroom activity was supported by a
“Research Experiences for Teachers” supplement to NSF Grant
MCB-0421855.
References
Carlson, A.E., Westenbroek, R.E., Quill, T., Ren D., Clapham, D.E.,
Hille, B., Garbers, D.L. & Babcock, D.F. (2003). CatSper 1
required for evoked Ca2+ entry and control of flagellar function
in sperm. Proceedings of the National Academy of Sciences USA,
100, 14864-14868.
Chan, P.J., Corselli, J.U., Patton, W.C., Jacobson, J.D., Chan, S.R. &
King, A. (2001). A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free oocytes. Fertility and Sterility, 75, 186-192.
Treatment with pure caffeine will hyperactivate most of
the sperm without killing any of them. A movie of fully hyperactivated bull sperm can be seen online as a supplemental file
to a published scientific paper by Ignotz and Suarez (2005).
The Web address is: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_
uids=15878888&query_hl=1&itool=pubmed_docsum.
Ho, H-C. & Suarez, S.S. (2001). An inositol 1,4,5-trisphosphate
receptor-gated intracellular Ca2+ store is involved in regulating sperm hyperactivated motility. Biology of Reproduction, 65,
1606-1615.
Discussion
Ren, D., Navarro, B., Perez, G., Jackson, A.C., Hsu, S., Shi, Q., Tilly,
J.L. & Clapham, D.E. (2001). A sperm ion channel required for
sperm motility and male fertility. Nature, 413, 603-609.
In class, the following questions could be discussed:
1. How did you describe the swimming patterns of the
sperm?
2. How did the swimming patterns change when caffeine
was added?
3. What were the experimental controls? What was the
purpose of the controls? Were these the best possible
controls?
4. What could be the purpose of these different swimming
patterns?
5. If a woman drank a strong cup of coffee while sperm
were in her oviduct, what route would the caffeine take
to reach the sperm?
6. What could happen if a man drank a strong cup of coffee?
During the discussion, students should be reminded that
caffeine is a drug that simulates whatever it is that actually
hyperactivates sperm during the natural fertilization process.
The natural trigger for hyperactivation is yet to be discovered.
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Ignotz, G.G. & Suarez, S.S. (2005) Calcium/calmodulin and calmodulin kinase II stimulate hyperactivation in demembranated
bovine sperm. Biology of Reproduction, 73, 519-526.
Stauss, C.R., Votta, T.J. & Suarez, S.S. (1995). Sperm motility hyperactivation facilitates penetration of the hamster zona pellucida.
Biology of Reproduction, 53, 1280-1285.
Suarez, S.S. & Dai, X.B. (1992). Hyperactivation enhances mouse
sperm capacity for penetrating viscoelastic media. Biology of
Reproduction, 46, 686-691.
Suarez, S.S. & Pacey, A.A. (2006). Sperm transport in the female
reproductive tract. Human Reproduction Update, 12, 23-37.
Suarez, S.S., Varosi, S.M. & Dai, X. (1993). Intracellular calcium
increases with hyperactivation in intact, moving hamster sperm
and oscillates with the flagellar beat cycle. Proceedings of the
National Academy of Sciences USA, 90, 4660-4664.
Suarez, S.S., Katz, D.F., Owen, D.H., Andrew, J.B. & Powell, R.L.
(1991). Evidence for the function of hyperactivated motility in
mammalian sperm. Biology of Reproduction, 44, 375-381.
THE AMERICAN BIOLOGY TEACHER, ONLINE PUBLICATION, NOVEMBER/DECEMBER 2007
APPENDIX. Handout
Give Your Bull Sperm a Pick-Me-Up!
Introduction
In this lab you and a partner will examine bull sperm and see
how caffeine can affect it. What does caffeine do to you when
you ingest it? What might it do to sperm? Before reading the lab,
answer the following questions:
1. Describe the path taken by sperm through the mammalian female reproductive tract to reach the egg (including
humans). What organs do the sperm pass through?
6. Quickly examine the sperm using the 40X objective on
your microscope. Note your observations in the table
below.
7. Repeat Steps 5 and 6 using decaffeinated coffee. Compare
the effect on sperm with that of caffeinated coffee and
write your observations in the table.
8. Repeat Steps 5 and 6 using other caffeinated and decaffeinated drinks or a solution of pure caffeine.
______________________________________________
______________________________________________
Treatment
______________________________________________
2. What do you think will happen when caffeine is added
to sperm?
Swimming patterns
of moving sperm
milk medium alone
______________________________________________
coffee
______________________________________________
decaffeinated coffee
3. How could caffeine get from the digestive system into the
reproductive system?
Approximate
percentage of
sperm that are
moving
caffeine
______________________________________________
Discussion
______________________________________________
After competing the experiment, answer the following questions
______________________________________________
Materials
bull semen
hot plate, incubator, or warm water bath
thermometer
plastic tubes
plastic pipets
slides and cover slips
microscope with 40X objective
milk medium for sperm
caffeinated coffee
decaffeinated coffee
Procedure
1. Warm milk and all test solutions to 37˚ C (98.6˚ F).
2. Add 0.1 ml semen to 0.9 ml of pre-warmed milk medium
in a tube. The sperm will work best if it is kept warm, treated
gently, and protected from light as much as possible. Perform
all tests as soon as possible after warming sperm. Return the
unused portion to the refrigerator without allowing it to
warm up.
3. Using a plastic pipet, place a small drop of the sperm
suspension onto a microscope slide. Gently cover with a
coverslip.
4. Quickly place the slide under a microscope and try to
find sperm using the 40X objective. If you have trouble
finding sperm, go to the edge of the coverslip and focus
on the glass edge of the coverslip. This will put you at the
right focal length for seeing sperm. Use the table below
to record your observations.
5. Place a small drop of warm coffee-in-milk solution on a
slide and immediately add a small drop of sperm suspension into this drop. Gently cover with a coverslip.
1. What effect did coffee have on sperm? How do you know
it was the caffeine in the coffee that had this effect?
______________________________________________
______________________________________________
______________________________________________
2. What was the control in this experiment?
______________________________________________
3. Was it an appropriate control? If not, what would be a
better control?
______________________________________________
______________________________________________
4. What ingredients in the drinks might have killed
sperm?
______________________________________________
______________________________________________
5. Why was the sperm diluted in milk? What might happen
if it was diluted in water?
______________________________________________
______________________________________________
6. Describe an experiment that you would do to find out if
drinking caffeine can really affect sperm movement in an
animal.
______________________________________________
______________________________________________
______________________________________________
______________________________________________
______________________________________________
______________________________________________
EVOLUTIONARY TREES
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