FERTILIZATION PROTOCOL:

FERTILIZATION PROTOCOL:
Brad Stith, Univ of CO. Take a look at Xenopus web sites:
http://www.xenbase.org/., Data (including gene sequences) of Xenopus.
http://worms.zoology.wisc.edu/frogs/welcome.html. This web site shows animations and
videos of the various stages that occur after first cleavage. Take a look at how the braind
and spinal cord form (neurulation).
Time of Fertilization Events in Xenopus laevis (up to first cleavage):
Times can vary due to individual animals, solutions, sperm concentrations and
temperature. The following times points are taken from our data and JC Gerhart (1980;
pages 133-316; in "Biological Regulation and Development," vol II; Plenum Press, NY;
Goldberger RF, editor):
1. Sperm and egg contact: theoretical time zero. However, this is an estimate as it takes
about one minute for the sperm to swim through the egg jelly to reach the egg surface.
(so, the times noted are “insemination times” but sperm and egg contact at a point about 1
min later than insemination).
2. Calcium wave and cortical granule breakdown (exocytosis) ~starts at 3-4 min after
insemination (and the wave takes about 3 min to cross the egg).
3. Gravitational rotation: ~10-15 min after insemination.
4. Cortical contraction (pigment moves toward animal pole--not cortical rotation;
contraction is transient so it can be missed): ~10-20 min.
5. Second polar body extrusion: ~20-25 min. (end of meiosis)
6. DNA synthesis (S phase): ~27 min—getting ready for upcoming mitosis (M phase).
7. Cortical Rotation produces Grey Crescent (latter is very hard to see in Xenopus):
~46-60 min
8. Pronuclei contact each other: ~49 min (sperm and egg nuclei are called pronuclei
because they contain only half the normal number of chromosomes)
9. Mitosis: starting at about 75 min, lasting until about 90 min
10. First Cleavage: ~100 min
--------------------------------------------Done before this lab: 3-4 days before this lab, frogs were “primed” with 100 units of
pregnant mare’s serum gonadotrophin (PMSG), then 12 hours before this lab (time of egg
collection), 850 units of human chorionic gonadotrophin (HCG) were injected.
Next, read through the Appendix…especially pages 11-13
Protocol Steps:
1. Make 100 mls of 10% Modified Barth’s Solution (MBS):
100% MBS:
88 mM NaCl
1 mM KCl
2.4 mM NaHC03 (note this must be added fresh every day; add 0.2 g per 1
1
Liter)
0.8 mM MgS04
0.75 mM CaCl2
10 mM HEPES (pH 7.5)
NOTE THAT 10% BARTHS IS JUST
A 10-FOLD DILUTION OF THE
ABOVE.
2. Obtain a Xenopus laevis male and
excise the 2 testes located along dorsal side (see number 3 in frog
drawing). Wash/clean testes of mesenteric fat with 100%
modified Barth’s Solution (MBS).
3. Cut up both testes up (with small sharp scissors) in 2.4 ml of
100% MBS to create a working sperm suspension. Try to get rid
of major clumps of sperm; make the solution as homogeneous as
you can.
View the sperm using the high powered Swift microscope
that has phase contrast. Phase contrast microscopy has the
advantage of visualizing cell structures in live cells. Typically,
you have to kill the cell and then stain it. But phase contrast
microscopy exploits the differences in thickness and refractive index in different parts of
a cell. As many light waves pass through different parts of the cell, the light waves are
slowed down by different amounts (since light moves as a wave, this means that the light
waves change –or become out of phase- relative to light waves that do not move through
the cell. The phase contrast microscope converts these phase differences into differences
in brightness.
Place a drop of
sperm solution
onto a slide, and
drop a cover slip
over it (place the
cover slip at the
edge of the sperm
solution, then
lower the cover
slip over the
solution).
Remember to
start with the
lowest power,
then gradually
swing over the higher power lenses.
What do the sperm look like?
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4. Eggs are harvested by gently pressing the back and belly of the female frog, and eggs
are “squeezed” out into 100% MBS. These eggs should be healthy looking- eggs are not
in a string, round in shape, uniform in size/shape the liquid clear. The dark animal pole
should not be mottled, but a uniform dark color. Often the first few eggs from a female
are thrown out (poor fertilization). Can you see the thin line in the jelly coat that is the J2
layer? Note that there is a vitelline envelop- it is a matt of protein that lays on the egg
plasma membrane.
Taken from Hedrick and Hardy, Methods in Cell Biology vol 36; Ed by BK Kay, HB Peng.
5. Just before use (adding sperm and egg), eggs are put into 10% MBS. To do this, take
the group of eggs, remove excess 100% MBS, then add 10% MBS. Repeat- remove 10%
MBS excess, then add more 10% MBS. The final volume of solution should be 1 ml—
you will have to tilt the small 15 ml Petri dish to make sure that all the eggs are covered
by 10% MBS.
IT IS CRUCIAL THAT NEITHER EGGS OR SPERM REMAIN IN LOW
TONICITY 10% BARTH'S SOLUTION FOR MORE THAN A FEW MINUTES
BEFORE FERTILIZATION (sperm are motile for no more than 15 min in low tonicity
medium, and egg jelly becomes hydrated and very hard -if allowed to sit in low tonicity
medium and this prevents or slows fertilization/sperm penetration).
6. Get ready for fertilization; you will be recording the number of eggs that are vegetal
(light color) side up. Some 10-20 min after fertilization, the dark side will rotate upward
if there was successful fertilization. You want to record the percentage of light colored
vegetal eggs that rotate so that the dark side is up- and the time that the cells rotated. Set
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up a camera to record the information but remember that you often flood the cells after 10
min and this will mean that you have to reselect vegetal side up zygotes.
7. FERTILIZATION: 167 µl of working sperm suspension is added to 1 ml of (up to
300-400) eggs in 10% MBS. The sperm become activated when the tonicity of the
solution is reduced from 100% to about 10% (can you calculate the final concentration of
MBS? What percentage after you add a bit of 100% and 10% together?).
18. Typically, at 6-10 min after insemination, you flood the Petri dish with 10% MBS
(this dilutes the sperm; for fertilization we wanted a specific HIGH concentration of
sperm but now we can simply flood the Petri dish since it is easier than tilting the dish).
If you flood the Petri dish too early, you will inhibit fertilization.
19. Also, count the dark sperm entry spots; there should be only one per zygote. Watch
cells as they undergo first cleavage at about 100 min postinsemination.
19. We also want to induce polyspermy (more than one dark sperm entry site- see
photo). When more than one sperm enters, there will be too much DNA present and
zygote will eventually die. To prevent this, the cell uses the fast block to polyspermy (a
depolarization caused by the first sperm to enter; once depolarized, the sperm receptors –
located in the plasma membrane- no longer bind sperm), and a slow block to polyspermy
(due to cortical granule exocytosis that induces elevation of the fertilization envelopewhich pushes away other sperm- and releases dregradative enzymes that destroy the
sperm receptors). We will inhibit the fast block to polyspermy by the use of NaI (see
PowerPoint slides for reason why…). To do this:
a. Repeat the fertilization protocol but utilize 66% MBS
instead of 10% MBS; higher chloride concentration
outside the cell presumably inhibits efflux of
chloride…preventing the depolarization..
b. 20 mM NaI added to the 10% MBS (may have to try
30 mM NaI) that contains the eggs….then fertilize with
sperm.
Count the dark sperm entry spots.
------------------------------------------------------------------------We will take video of the fertilization response: you should seen that the zygote rotates
due to gravity (dark animal pole moves upward, heavier light vegetal pole moves down)
after the calcium wave and the wave of cortical granule exocytosis pass across the egg.
The calcium wave typically starts at about 3 min and lasts until about 6 min after
insemination. Gravitational rotation typically begins after this but all process can slow
down- you would want about 100% of the zygotes undergoing gravitational rotation by
about 20 min after insemination. Similar procedures are used to optimize human in vitro
fertilization.
You can purchase a C mount (C for camera) for microscopes (as shown in photo). Then,
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you attach a video or still camera to the C
mount.
C Mount
Our video setup:
Video
Camera
(high
resolution
But blk &
white only
and no sound)
We pass the video feed through a Time Stamp (a device that is like a stop watch; it puts
the time on the video picture) to produce videos such as those found on my web site (see:
http://carbon.cudenver.edu/~bstith/waverot.htm).
You combine the video camera, time stamp, video recorder and monitor as follows:
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Appendix:
(Early Development of Xenopus laevis; A laboratory manual, but Hazel L Sive, Robert Grainger, and RH
Harland; Cold Spring Harbor Laboratory Press, 1998)
6
7
8
9
10
11
12
sequent batch because egg quality can vary from one laying
to the next. Good quality eggs should produce fertilization
efficiencies near 100%.
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