Citation
Sperm increased inositol 1,4,5-trisphospahate mass in Xenopus laevis eggs preinjected with calcium buffers or heparin

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Title:
Sperm increased inositol 1,4,5-trisphospahate mass in Xenopus laevis eggs preinjected with calcium buffers or heparin
Creator:
Roberts, Dawn
Publication Date:
Language:
English
Physical Description:
iv, 75 leaves : illustrations ; 29 cm

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Subjects / Keywords:
Sperm-ovum interactions ( lcsh )
Sperm-ovum interactions ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 71-75).
General Note:
Submitted in partial fulfillment of the requirements for the degree, Master of Arts, Biology.
General Note:
Department of Integrative Biology
Statement of Responsibility:
by Dawn Roberts.

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|University of Colorado Denver
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|Auraria Library
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Resource Identifier:
34015475 ( OCLC )
ocm34015475
Classification:
LD1190.L45 1995m .R63 ( lcc )

Full Text
SPERM INCREASED INOSITOL
1,4,5-TRISPHOSPAHATE MASS IN
XENOPUS LAEVIS EGGS
PREINJECTED WITH CALCIUM BUFFERS
OR HEPARIN
by
Dawn Roberts
B.A., University of Colorado at Boulder, 1991
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Arts
Biology
1995


This thesis for the Master of Arts
degree by
Dawn Roberts
has been approved for the
Department of Biology
by
Corinne Campbell


Roberts, Dawn (M.A., Biology)
Sperm Increased Inositol 1,4,5-Trisphosphate Mass in Xenopus laevis Eggs
Preinjected with Calcium Buffers or Heparin
Thesis directed by Associate Professor Bradley J. Stith.
ABSTRACT
intracellular calcium ([Ca2+]j) is an important second messenger in
fertilization. An increase in [Ca2+],- is necessary for cortical granule exocytosis
and the slow block to polyspermy. However, what triggers this increase in
[Ca ]j is not clearly understood. This study reports a mechanism to explain
the increased [Ca2+]j.
Xenopus eggs were preinjected with the calcium buffer BAPTA, then
fertilized and analyzed for IP3 mass. Despite the presence of BAPTA, inositol
1,4,5-trisphosphate (IP3) levels increased indicating that the phosphoinositide
cascade does not require an increase in [Ca2+]j to produce IP3 upon
fertilization. In fact, IP3 levels in the presence of BAPTA achieved above-
normal levels; thus IP3 metabolism may be inhibited when [Ca2+]j is buffered to
below basal levels. Xenopus eggs were also injected with heparin which
inhibits IP3 action (thus [Ca2+]j release) and the fertilization response. IP3
mass also increased after heparin injection.
iii


Since BAPTA buffers [Ca2+]i, thus inhibiting the polyspermy prevention
mechanism, it could be argued that the BAPTA reduces IP3 release yet an IP3
increase is noted due to the entry of multiple sperm. To determine if this was
indeed the case, IP3 mass was determined for zygotes fertilized under both
polyspermic and monospermic conditions. IP3 levels were similar in both
conditions indicating that a single sperm initiates the increase in [Ca2+]i and
multiple sperm entries do not result in a larger IP3 mass increase.
Based on these data, a pathway for [Ca2+]j increase can be suggested
for Xenopus laevis. Sperm initiate the hydrolysis of PIP2 which breaks down
into IP3. IP3 subsequently is the second messenger that leads to the increase
in [Ca2!.
This abstract accurately represents the content of the candidates thesis. I
recommend its publication.
SignedJ
Bradley J. S
IV


DEDICATION
I wish to dedicate this thesis to my mother, Jin Roberts, for her
unending love and support and to the memory of my father, Willis Roberts.


CONTENTS
CHAPTER
1. INTRODUCTION...................................................1
Fertilization........................ . .....................1
Sperm-Egg Fusion........................................3
Bindin............................................3
Sperm Receptor on the Sea Urchin Egg..............4
Depolarization-The Fast Block to Polyspermy.............6
Intracellular Calcium...................................8
Activation Wave.........................................9
Cortical Granule Exocytosis-The Slow Block To Polyspermy 10
Calcium and Fertilization...................................... 13
Calcium Induced Calcium Release..................7 . 13
IP3 and Calcium......................................... 14
2 +
[Ca ]j Release in Model Systems................................19
Sea Urchin.............................................. 19
Xenopus.................................................22
Other Systems Utilizing IP3.............................26
Experimental Design............................................29
BAPTA...................................................30
Xenoous Model...........................................31
VI


2. MATERIALS AND METHODS .........................................34
Maintenance....................................................34
Priming........................................................34
Fertilization................................................. 35
Sperm Correction Factor........................................35
Injection Parameters...........................................37
Solutions.................................................... 38
BAPTA...................................................38
Dibromo-BAPTA...........................................39
Heparin.................................................39
MaxChelator....................................................40
IP3 Mass Assay.................................................41
Statistical Analysis...........................................42
3. RESULTS ................................................... 43
Fertilization Efficiency.......................................43
IP3 Increase after Fertilization...............................44
BAPTA Concentrations...........................................44
Polyspermy.....................................................52
Heparin........................................................57
VII


4. DISCUSSION......... .'.................................... ... 59
2 +
IP3 Production in the Presence of Ca Chelators...............59
BAPTA and Polyspermy.......................................... 60
A.
Above Normal Induction of IP3 by BAPTA.................... 61
Heparin Injection Prevents Fertilization Events ............. 62
Sea Urchin Egg Activation..................................... 63
G-Proteins............................ ..... ...... 65
Conclusion..................................................... 66
APPENDIX A....................................................... 68
REFERENCES . . . ........................................... 71
viii


ACKNOWLEDGMENTS
I would like to thank Dr. Stith for giving me the opportunity to work
and learn in his lab. Thank you for your guidance and patience in the
creation of this thesis.
I would like to thank Dr. Campbell and Dr. Brockway for their advice
and patience during the writing of this thesis. I have enjoyed learning from
you both.
I would like to thank Tanya Smart for her friendship, support, and
advice. Thanks for being there. I would also like io thank Marc Goalstone
for teaching me the skills needed to work in a lab. Many thanks to the
other members of the lab (Jon Doris, Mozdeh Saffari, Keith Woronoff, Ron
Espinoza, and Tony Ferdensi) for their help and friendship.
And finally, thank you to Dr. Awoniyi for the time to complete this
thesis.
This work was supported by a National Science Foundation grant
awarded to Dr. Stith.
IX


CHAPTER 1
INTRODUCTION
Fertilization is the process by which a sperm and egg fuse, their
chromosomes combine, and a new individual begins to develop (Epel,
1977). One of the first events of fertilization is an increase of intracellular
2 +
calcium ([Ca ]j) which starts at the sperm entry site and traverses as a
24-
wave across the egg. [Ca ]j is thought to be an important ionic signal
responsible for activation of the egg. However, the trigger for increased
2 +
[Ca ]j is not clearly understood. Work described here suggests a
2 +
mechanism for the increase in [Ca ]j at fertilization.
Fertilization
The normal events of fertilization in Xenopus laevis are outlined in
Table 1. First, the sperm and egg fuse. Immediately upon fusion there is a
depolarization of the egg which is the fast block to polyspermy. Up to thirty
seconds later, another, more permanent block to polyspermy occurs. This
2 +
late block is due to cortical granule exocytosis and is [Ca ];. The next
event is the gravitational rotation of the egg which occurs approximately 15
1


minutes after sperm-egg fusion. Intracellular events such as pronuclear
contact and rotation of the yolk free cytoplasm to the dorsal side occur
during the next thirty minutes but are difficult if not impossible to follow
with a dissecting microscope. The next visible event is the return of
pigment granules to the cortex. The last visible event of fertilization is the
appearance of the cleavage furrow which occurs between 80-100 minutes.
Other events such as mitotic prophase, mitotic metaphase and telophase,
and DNA synthesis all occur before the appearance of the cleavage furrow.
However, we were not able to see these events under the microscope
though others have studied and recorded these events (Gerhart and Scharf,
1980).
Since our study concentrates on the early signal transduction
occurring in the egg, I will concentrate mainly on the events immediately
after sperm-egg fusion to cortical granule exocytosis (approximately the first
few minutes of fertilization).
Table 1. Visible events of fertilization through first cleavage in Xenopus
leavis. Adapted from Gerhart and Scharf (1980).
Fertilization Event Time (min)
1. Egg-Sperm Fusion 0
2. Depolarization of egq membrane seconds
3. Activation wave (cortical granule breakdown) 7
4. Activation contraction 12-20
5. Gravitational rotation (perivitelline space fills with liquid) 12-15
6. Pigment granules leave cortex, then return 40-45
7. Cleavaqe furrow appears 100
2


Sperm-Eaa Fusion
In Xenopus. spermatozoa must interact with the egg jelly to acquire
the capacity to fertilize the egg (Wolf and Hedrick, 1971). The egg is
surrounded by a jelly coat, which consists of glycoproteins, hexosamines,
fucosamines and galactosamines. When the sperm and egg jelly coat make
contact, substances in the jelly coat interact with the plasma membrane of
the sperm. At this point, the sperm is considered activated. At the tip of
the sperm head is an acrosome which exocytosis to release enzymes that
dissolve a hole through the jelly coat. Thin filaments, called the acrosomal
process, extend and attach to the vitelline membrane. The plasma
membranes of the two cells fuse and the sperm nuclei is then pulled
forward into the egg (Becker and Deamer, 1991).
Bindin. The fusion between the egg and the sperm in sea urchin
fertilization is partially due to a protein called bindin located on the tip of the
sperm head (Vacquier and Moy, 1977). Xenoous may also utilize bindin
but there are no conclusive studies at this time.
Vacquier and Moy (1977) isolated bindin (or egg receptor located on
sperm) and showed it to be a 30,500 Dalton glycoprotein that agglutinates
unfertilized sea urchin eggs. Even isolated vitelline membranes agglutinate
upon bindin addition. However, no agglutination occurs when the sea
urchin eggs are treated with trypsin (removes sperm receptor) or
3


metaperiodate oxidation (destroys sugar residues and may destroy the
carbohydrate portion of the sperm receptor). These two results suggest
that bindin recognizes a receptor on the vitelline layer of the egg which may
be a sugar portion of a glycoprotein (Vacquier and Moy, 1977).
Sperm Receptor on the Sea Urchin Eqq. Identification of a sperm
receptor on the surface of sea urchin eggs has been hampered due to the
large size of the receptor. Schmell et al. (1977) did one of the earliest
studies on a possible sperm receptor. Fertilization of eggs from the sea
urchin, Arbacia punctulata. were unaffected by the presence of either eggs
or membranes prepared from the eggs of the sea urchin Strongvlocentrotus
purouratus. However, when the membranes from A. ountulata eggs were
added to other intact eggs of A. punctulata. the A. punctulata eggs could
not be fertilized by A. punctulata sperm. This inhibition was due to
membrane associated glycoproteins that competitively bind to sperm,
preventing the sperm from binding and fertilizing the egg.
Next, eggs were treated with trypsin which removes receptor
glycoproteins, therefore these eggs were not fertilizable. The membranes
from eggs were treated with trypsin and added to intact eggs along with
sperm. Unlike the previous experiment, the membranes did not interfere
with the fertilization process. Since the membranes were stripped of their
receptors by the trypsin, sperm were not able to bind to the membranes,
only the intact eggs.
4


This sperm receptor was also shown to bind to the lectin Concavalin
A. Lectins bind carbohydrates. Concavalin A-treated eggs were
unfertilizable. Based on the trypsin and Concavalin A data, Schmell et al.
(1977) surmised that the sperm receptor on the sea urchin egg was a
glycoprotein.
Rossignol et al. (1984) were able to further characterize this sperm
receptor using iodinated membranes. The iodinated sperm receptor binds to
bindin and this allowed partial purification of the sperm receptor. Besides
protein, the sperm receptor is made up of neutral sugars, amino sugars,
uronic acid, and sulfate. Hexosamine, sulfate, and uronic acid indicate that
there are glycosaminoglycan-like chains present while glucosamine and
mannose suggest a N-linked oligosaccharide. These purified sperm
receptors were then added to activated sperm. Since the receptors bound
to the sperm, the sperm were unable to subsequently fertilize intact eggs.
Further characterization was difficult due to the insolubility and/or the high
molecular weight of the sperm receptor (Rossignol et al., 1984).
In 1990, the extracellular fragment of the sperm receptor was
elucidated by utilizing lysylendoproteinase C (Lys C). A 70 kDa proteolytic
fragment was formed when the receptor was treated with Lys C. When Lys
C treated eggs were exposed to acrosome reacted sperm, fertilization was
inhibited in a specific manner. This 70 kDa glycoprotein was also able to
5


inhibit fertilization. It also bound to acrosome-reacted sperm or bindin (Foltz
and Lennarz, 1990).
In 1993, Ohlendieck et al. found that the sea urchin egg has
1.25 x 10 sperm receptor molecules on its surface. The receptor has an
apparent molecular weight of 350 kDa and is a highly glycoslylated
transmembrane protein containing approximately 70% carbohydrates and
30% protein. The carbohydrate component is in oligosaccharide chains,
sulfated, and N and 0 linked chains. Both the polypeptide backbone and
carbohydrate chains are involved in the binding process and the isolated
receptor is able to retain its biological activity. The receptor inhibits
fertilization in a species-specific manner and the receptor binds to acrosome
reacted sperm (Ohlendieck et al., 1993).
Depolarization of the Egg Membrane-The Fast Block to Polvspermv
Once the sperm and egg membranes merge, a rapid depolarization
of the egg membrane occurs, preventing the entry of multiple sperm. This
depolarization is transient in nature and is known as the fast block to
polyspermy, the fertilization potential, or the electrical block to polyspermy
(Webb and Nuccitelli, 1985). In Xenoous. the egg depolarizes due to the
opening of chloride channels. The egg has a negative membrane potential
and since intracellular concentration of chloride is higher than external
chloride, there is a net efflux of chloride ions and the egg becomes more
6


24-
positive. The trigger that opens the chloride channels is elevated [Ca ]j
(Webb and Nuccitelli, 1985).
Webb and Nuccitelli (1985) experimented with the external chloride
concentration and what affect it had on the membrane potential of the
Xenopus egg. As the external chloride concentration increased, the
fertilization potential decreased. However, basal membrane potentials were
unaffected by external chloride concentrations. These results indicated that
the fertilization potential was associated with a net efflux of chloride ions.
Increasing the extracellular concentration of other halides had a similar
effect. Bromide or iodide entered the egg the easiest and counteracted the
depolarization effect of any chloride efflux. The presence of bromide and
iodide in the fertilization medium resulted in polyspermic zygotes (multiple
sperm entry sites) and multiple first cleavage furrows. As demonstrated by
the appearance of the fertilization envelope and normal egg rotation, the
fertilization potential was not required for the cortical reaction (cortical
granule exocytosis) (Webb and Nuccitelli, 1985).
Webb and Nuccitelli (1985) determined that the initial potential of an
unfertilized Xenopus egg was -33.1 mV. Insertion of a electrode caused a
slight depolarization but not activation of the eggs. After fertilization, the
membrane potential increased to +3.0 +_ 4.1 mV for approximately 15
minutes. Repolarization consisted of an initial slow phase followed by a
shorter, faster phase. Cleavages were accompanied by a hyperpolarization
7


which began 6 minutes after the furrow formed (Webb and Nuccitelli,
1985).
In contrast to Webb and Nuccitelli, Grey et al. (1982) found that
unfertilized eggs had a membrane potential of -19 mV with an increase to
+ 8 mV upon fertilization. They also concluded that the fertilization
potential was essential for the prevention of polyspermy in Xenopus. In
vitro studies showed that 20 mM Nal solutions caused hyperpolarizations
and prevented the fertilization depolarization. After fertilization in 20 mM
Nal, approximately 14 sperm entry sites were observed and 50-90% of the
eggs showed polyspermy. Thus, a depolarization prevents polyspermy.
Intracellular Calcium
One of the first cytosolic events after fertilization is an increase in
intracellular calcium. Steinhardt et al. (1977) noticed an intracellular
2 +
calcium increase in sea urchin eggs after fertilization. Free [Ca ];
increased from 2.5 uM to 4.5 uM 45-60 seconds after activation and this
2 +
increase lasted 2-3 minutes. The [Ca ]j increase occurred even in a zero
2+ "2 +
Ca external medium, indicating that external Ca does not play a
2+ 2 +
significant role in the increase of [Ca ]j. The zero Ca external medium
contained ethylene glycol-bis (amino ethyl ether) N,N,N',N'-tetraacetic acid
2 +
(EGTA), a Ca chelator.
8


The activation current was measured and graphed against the
amount of aequorin injected. They discovered that the two have a second
2 +
power relationship. From this data they determined that 2.5 -4.5 uM Ca
2 +
was released during fertilization. Next, cortices were isolated and Ca
was added based on the preceding calibrations. Using scanning electron
microscopy (SEM), they watched for the discharge of cortical granules
2 +
which occurred at 2.5 -4.5 uM Ca
Based on these two experiments, Steinhardt et al. (1977) determined
2+ 2 +
that Ca release is localized in the cortical layer and the highest Ca
levels are confined to the subsurface of the egg.
Activation Wave
Takeichi and Kubota (1984) were able to characterize the activation
wave in the Xenopus egg by using time lapse cinematography and time
lapse video tape recording. The eggs were prick activated. A light area
appeared near the pricking point and expanded as a circular light zone. The
egg first lightened, then the lightness traveled as a wave at the rate of 9
um/second from the area of sperm contact to the opposite side of the egg.
During this light wave, cortical granule exocytosis occurred as well as
elongation of microvilli.
9


A dark wave began about 60 seconds after the light wave. During
the dark wave, the microvilli shortened and became more globular
(Takeichi and Kubota, 1984).
2 +
Kline and Nuccitelli (1985) theorized that the [Ca ]j increase may
actually occur as a wave in Xenopus and cause the activation wave. A ring
shaped wave of inward current went across the egg, from the site of
activation to the opposite side to sperm entry. The wave reflected the
propagated opening of the calcium sensitive chloride channels responsible
for the fertilization potential. Other morphological changes included
exocytosis and the activation contraction (the movement of the pigmented
region toward the animal pole). The activation current was followed by
exocytosis and two or more contraction waves. The inward current was
primarily due to a chloride ion efflux.
Cortical Granule Exocvtosis-The Slow Block to Polvspermv
The fast block due to membrane depolarization occurs within a few
seconds after insemination and is transient in nature. A second, permanent
block to polyspermy is initiated in 2-3 minutes after insemination. This
block is chemical in nature and is called the slow block to polyspermy. Both
the early and late blocks are steps to prevent more sperm from entering the
egg. The second, late block is due to cortical granule exocytosis.
In the unfertilized egg, the vitelline membrane and plasma membrane
are approximately 1 um thick. The perivitelline space has little electron
10


dense material. Upon sperm egg fusion, the fertilization envelope forms and
consist of the vitelline membrane plus a layer of electron dense material,
which comes from cortical granule exocytosis. The fertilization envelope
forms on the outer surface of the vitelline envelope (Grey et al., 1974).
The cortical layer of the unfertilized egg is located just inside the
plasma membrane and contains small cortical granules (Grey et al., 1974).
These granules are approximately 1.0 um in diameter and contain enzymes.
These vesicles fuse with the egg plasma membrane after sperm-egg fusion
2 +
and an increase in [Ca ]j. The enzymes contained in the granules are
released as the granules exocytose and have important functions in the
fertilization scheme. One of the enzymes from the cortical granule destroys
the sperm receptors on the egg. Another enzyme breaks down the proteins
that connect the egg and the vitelline membrane.
The enzymes released into the perivitelline space cause a shift in the
osmolarity. The osmolarity difference causes water to enter the space and
the vitelline membrane separates slightly from the egg plasma membrane.
This is sometimes referred to as membrane lift-off (Grey et al., 1974).
The vitelline membrane also hardens. Both events prevent more sperm from
entering the egg. This event is known as cortical granule exocytosis.
Electron microscopy show that the cortical granules ruptured about three
minutes after sperm addition (Grey et al., 1974).


In another study. Grey et al. (1976) showed that the fertilization
envelope may block polyspermy. The fertilization envelope and vitelline
envelope were isolated from Xenopus eggs. These envelopes were then
exposed to a sperm solution, fixed, and analyzed using light microscopy.
The sperm penetrated the vitelline envelope; however, the sperm did not
penetrate the fertilization envelope. . ...
2 +
Zucker and Steinhardt (1978) determined that Ca is necessary
for cortical exocytosis in fertilized sea urchin eggs by microinjecting EGTA
or EDTA into the egg. EGTA prevented cortical granule exocytosis and
formation of the fertilization membrane. In EDTA injected eggs the
fertilization membrane did develop but was slower than normal to develop.
Since EGTA and EDTA are calcium chelators, this suggested that calcium is
necessary for early events of fertilization (Kline, 1988). In our study,
2 +
BAPTA also functions to inhibit the increase in Ca and thus cortical
2 +
granule exocytosis. If the increase in [Ca ]j is prevented by chelation of
2 +
Ca the events associated with exocytosis should not occur. The slow
block is inhibited and eggs would exhibit polyspermy.
12


Calcium and Fertilization
Calcium and other messenger waves are for communication in and
out of the cell. Calcium responses often spread spatially as waves that
expand local excitation within cells (Meyer, 1991). These messenger
waves are usually slow and the length of concentration gradient at the
wave front can vary. The wave front is defined as the product of the rise
time and velocity. The velocity of the calcium wave is approximately 8-100
um/second, while the wave front can be 7-50 urn. The forward
propagation of calcium waves may be driven by a sequence of calcium
diffusion and calcium amplification steps (CICR) or IP3- induced release
(Meyer, 1991).
Calcium Induced Calcium Release
There are two current theories as to the cause of this increase in
calcium. One theory is the calcium induced calcium release (CICR) theory.
This calcium release is mediated by calcium channels in the endoplasmic
reticulum (ER) that are sensitive to agonists like cyclic adenosine phosphate-
2+ 2 +
ribose (cADPR), to [Ca ]j, and to the amount of Ca in the lumen of the
ER (Galione et al., 1993). The ryanodine receptor (RyR) is the receptor that
triggers the release from the ER. The RyR appears to be regulated by
cADPR (Galione et al., 1991) as well as caffeine and ryanodine. In sea
13



2 +
urchin eggs, [Ca ]j release appears to involve a combination of IP3 (the
second method) ahd ryanodine receptors (Galione et al., 1993).
IP3 and Calcium
The second theory, reviewed by Berridge and Irvine (1984), is that
the phosphatidyl inositol (PI) turnover is the primary signal for intracellular
calcium increase. Phosphatidylinositol 4,5-bisphosphate (PIP2) is broken
- - 2 +
down to inositol 1,4,5, trisphosphate (IP3) which in turn releases Ca
from intracellular stores. The biochemical cycles concerned with the role of
the inositol lipids in signal transduction are seen in figure 1. The lipid and
inositol phosphate cycles ultimately unite to synthesize phosphatidylinositol,
which is fed into the phosphatidyl inositol\inositol 4,5-bisphosphate futile
cycles.
Phosphatidylinositol 4,5-bisphosphate (PIP2) is one of the inositol
lipids and is located in the inner leaflet of the plasma membrane. PIP2 is
formed by phosphorylation of phosphatidylinositol (PI) at the 4 and 5
positions of its inositol head group. PIP2 can convert back into PI by
phospho-monoesterases. These phosphomonoesterases constitute two
futile cycles known as the phosphatidylinositol\PIP2 cycles. Phosphates are
constantly being added to and removed from the 4 and 5 positions of the
inositol head group.
14


i- pip Cell j
I kinase : PIP PIP2 plasma | membrane !
X \
PLC
>
DAG IP3 |

PKC Ca
Lipid cycle
PIP phospho-
monoesterase

PIP2 phospho-
monesterase
Figure
Phosphoincsi'i
1984.
cascade, adapted from 3erridge and Irvine,
15


This futile cycle is rigidly controlled since it may be metabolically expensive.
PIP2 Is diverted from this futile cycle when a,receptor is occupied by an
agonist. PIP2 is made accessible to a phosphodiesterase to be cleaved into
sn-1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) (Berridge
and Irvine, 1984).
The phosphodiesterase that cleaves PIP2 is called phospholipase C
(PLC). Agonists may induce a conformational change in the receptor, which
in turn activates PLC. PLC may be controlled by coupling receptors to the
enzyme through a GTP-binding protein, as in the mechanism used to link
receptors to adenylate cyclase (Berridge and Irvine, 1984). There is
evidence that a G-protein initiates the breakdown of PIP2. When
nonhydrolyzable analogues of guanine nucleotides were added to ceil types,
IP3 and DAG production were stimulated (Berridge and Irvine, 1984). A
second method of activation of PLC is through tyrosine phosphorylation by a
receptor tyrosine kinase.
The lipid cycle begins with DAG kinase, which converts DAG to
phosphatidic acid (PA). PA reacts with CTP to form CDP-diacylglycerol
which reacts with inositol to reform phosphatidylinositol.
IP3 is degraded by removal of the 5- position phosphate by inositol
trisphosphatase. This results in inositol-1,4 bisphosphate (IP2). Inositol
bisphosphatase hydrolyzes IP2 to inositol 1- phosphate (IP1). IP1 is then
converted to free inositol via an inositol 1 phosphatase. This last step is
16


markedly inhibited by lithium which may block the inositol phosphate cycle
and reduce the supply of the inositol lipids needed by the receptor
mechanism (Berridge and Irvine, 1984).
2 +
IP3 releases Ca while DAG stimulates protein kinase C. In the
short term, these second messengers may contribute to such biological
functions as contraction, secretion and metabolism. In the long term
aspect, IP3 and DAG may have a function in growth and information
storage (Berridge, 1987).
IP3-induced calcium release has been shown in several various cell
types. For example, when insulinoma cells are injected with IP3, there is a
rapid release of calcium followed by a slower re-uptake to an original
2 +
equilibrium value. It is believed that IP3 releases Ca from stores located
in the endoplasmic reticulum (ER). The mitochondria were ruled as a
possible calcium store when inhibition of mitochondrial function by removing
metabolic substrates or addition of inhibitors had no effect on the ability of
IP3 to release calcium from an intracellular pool.
Use of PIP2 antibodies has led to further support for the role of the PI
cascade during fertilization (Matuoka et al., 1988). PIP2 antibodies reduce
IP3 production at fertilization by masking the site on PIP2 for PLC. To
produce the antibody, mice are injected with PIP2 from bovine spinal cord
and then hybridoma cells are cultured accordingly. Clones then secrete an
17


lgG2b class antibody which has been labeled kt3g-. This antibody was
showed to bind to PIP2 and no other lipids when tested by enzyme linked
immunosorbant assay (Matuoka et al., 1988). The PIP2 antibody was been
shown to decrease PIP2 hydrolysis by 92.4% in NIH 3T3 cells (Matuoka et
al., 1988).
Another example of partial PIP2 antibody effectiveness was
demonstrated by Han et al. (1992). Blastomeres injected with the PIP2 or
antibody or heparin inhibits further embryological development. The PIP2
antibody also inhibited the further cell proliferation when injected into
carcinoma cells (Matuoka et al., 1988).
Using this PIP2 antibody, Larabell and Nuccitelli (1992) were able to
2 +
show that the antibody reduces the sperm induced [Ca ]j in Xenopus
eggs. Any eggs exhibiting artificial activation were due to PIP2 antibody
injection were removed. However, the PIP2 antibody injected eggs had a
only 50% reduction in cortical intracellular calcium increase. This agreed
with findings that the IP3 receptor is found on both the perinuclear envelope
of the ER and in the cortical layer. With this finding, Larabell and Nuccitelli
theorized that PIP2 hydrolysis and IP3 induced calcium release travel around
the egg in a wave. In the meantime, cycles of PIP2 turnover produce the
fluctuations of calcium at the peak of the wave. DAG activates PKC which
may lead to the down regulation of PIP2 hydrolysis and phosphorylation of
the IP3 receptor (Ferris et al., 1991).
18


2 +
fCa1j Release in Model Systems
Sea Urchin
As mentioned earlier, Lee et al. (1993) have shown that two
2 +
different receptors are apparently necessary for Ca release in the
fertilization of sea urchin eggs. Lee and his colleges investigated the effects
2 +
of heparin on the IP3 induced Ca mobilization. Heparin completely
2 +
blocked IP3 induced Ca release; however, heparin had no effect on
2 +
cADPR induced Ca release. The 8-amino derivative of cADPR is an
2 +
effective antagonist for cADPR induced Ca release. Addition of 8-amino-
cADPR (8-NH2) inhibited cADPR induced calcium release but IP3 was still
able to induce calcium release in the presence of 8-NH2. Lee and his
2 +
colleagues showed that there are two independent Ca release systems in
a sea urchin egg, and each can be blocked by its own specific antagonist.
Many believe that it is this calcium increase, CICR, that triggers the
phosphatidyl inositol cascade which eventually leads to the release of more
calcium (Lee et al., 1993).
Lee's ideas are supported by the earlier findings of Whitaker and
Irvine (1984) and Ciapa and Whitaker (1986). Whitaker and Irvine (1984)
showed that IP3 injected into sea urchin eggs activated the eggs in a similar
19


2 +
manner as sperm induced egg activation. A cytoplasmic [Ca ]j
concentration greater than 1 uM stimulates cortical granule exocytosis and
the fertilization envelope appears. IP3 injected into eggs, at concentrations
of 2 x10"6 pmol and 1.6 x 10 6 pmol, elevated the fertilization envelope in
half of the eggs. Inositol bisphophates (IP2) were less effective, requiring
one hundred fold higher concentrations to achieve similar results. From
these data, Whitaker and Irvine suggested that 10 nM IP3 stimulated
2 +
cortical granule exocytosis and IP3 may act indirectly via elevated [Ca ]f.
2 +
This amount is much lower than that required for IP3 to release Ca in
permeabilized cells but there is a similar rank order for IP3 and IP2
efficiency.
2 +
Whitaker and Irvine (1984) also showed that IP3 released Ca
locally. After injection of less IP3, the fertilization envelope only rose over
the region of the plasma membrane close to the tip of micropipette.
Injection of higher amounts of IP3 induced a wave of exocytosis that began
at the tip of the pipette and reached the antipode 25 seconds later. The
transit time was constant over a 5 fold range of IP3 concentrations and was
equivalent to the transit time at fertilization. This was consistent with the
2 +
notion that IP3 induces a propagated wave of release Ca similar to that
which may occur at fertilization. Whitaker and Irvine concluded that IP3
20


sets in motion the ionic changes which constitute egg activation. IP3-
2+2 +
stimulated Ca and Ca stimulated IP3 production may cooperate to
2 +
produce a wave of Ca release which propagated through the sea urchin
eQ9 cytoplasm. However, our results for Xenopus disagree with this finding
(see results).
In sea urchin eggs, Ciapa and Whitaker (1986 ) reported an early
increase in IP3 turnover as the calcium wave traversed the egg. Sea Urchin
3 3
eggs were incubated in sea water with myo-2- H-inositol or H-arachidonic
3
acid and eggs retained 5% of the H-inositol, 35% of this in polyphospho-
3
inositide phospholipids. The eggs retained 20% of the H-arachidonic acid
with 5% remaining in the polyphosphoinositide phospholipids and 75%
remaining in other lipids. After fertilization, ice cold TCA was added to the
homogenates. The organic phase was analyzed using chromatography and
the aqueous layer was placed on a Dowex column which separated
polyphosphoinositides into IP3 and IP. Their results showed an increase in
3
H-inositol labeled IP3 within 10 seconds after fertilization. This increase
3
remained elevated for 30 minutes. Similarly, H-arachidonic acid-labeled
DAG increased within 10 seconds. Later, IP3 increased again as the calcium
declined and pH increased. This discrepancy does not lend credence to this
methodology. Ciapa and Whitaker (1986) noted that the calcium transient
21


began 7 seconds after fertilization. Their data suggested that a calcium
induced hydrolysis of PIP2 occurred at fertilization. Our data for Xenopus
fertilization disagrees with this conclusion for sea urchins.
32 3
Kamel et al. (1985) pulse-labeled inositol with P04 and H in sea
urchin eggs. There was a 50% increase in isotope Incorporation into PIP2
within 1 minute after insemination. A decrease in phosphatidyl inositol (PI)
3
indicated the phosphorylation of PI to form PIP2. H-labeled IP3 increased 5
fold in 10 minutes post insemination. The burst of PIP2, IP3 and the
2 +
transient increase in [Ca ]j suggested that PIP2 and IP3 were associated
2 +
with [Ca ]j mobilization (Kamel et al., 1985) but it was unknown whether
the increase caused the IP3 increase.
The temporal correlation between the early burst of PIP2 and IP3
2 +
formation and the increase in Ca suggest that P1P2 conversion into IP3
may be associated with calcium mobilization within the egg (Kamel et al.,
1985).
Xenopus
2 +
The mechanism of the fertilization [Ca ]j increase has not been
determined in Xenopus. There have been many studies that indicate IP3 is
an important component in Xenopus fertilization. Busa et al. (1985)
2 +
induced a transient [Ca ]j and activation increase by injecting Xenopus
22


2 +
eggs with IP3. This [Ca ]j increase was very similar to that 2.2 minutes
2 +
after the sperm-induced fertilization. [Ca ]j levels returned to basal
conditions in the next twelve minutes after IP3 injection or sperm addition.
Electrodes were placed in both the animal and vegetal hemispheres. Sperm
2 +
binding always occurred in the animal hemisphere. [Ca ]j was first
detected in the animal hemisphere 3.2 minutes after the onset of the
fertilization potential and in the vegetal hemisphere 2 minutes later. The
2 +
propagation rate of [Ca ]j wave was similar between fertilized eggs and
eggs injected with IP3.
Further evidence for a role for IP3 comes from the studies of Stith et
al. (1993). Changes in IP3 mass were monitored from fertilization through
first cleavage. IP3 levels increased 45 seconds after sperm addition and
peaked at 5 minutes. Levels returned to near basal by 10 minutes and
remained here until first cleavage. At this time, approximately 90 minutes,
IP3 mass increased; however, the increase was not as great as the initial
increase. An increase in IP3 mass was also associated with the onset of
sperm motility but the acrosome reaction exhibited a decrease in IP3 mass
(Stith et al. 1993).
Le Peuch et al. (1985) showed that PIP2 decreased from
approximately 1 nmol/egg to less than 0.5 nmol/egg after prick activation.
These were then prick activated and measured for the amount of 32-P04
23


incorporated into PIP and PIP2. Upon activation, PIP2 content dropped
significantly while PIP levels remained relatively constant.
Using Xenopus oocytes, DeLisle and Welsh (1992) determined that
2 +
IP3 is required for the propagation of [Ca ]j waves. 1P3 injected into
2 +
oocytes released [Ca ]j from discrete intracellular sites. Each of these
2+ 24-
sites released [Ca ]j periodically and then [Ca ]j propagated away.
Wave fronts did not decrease in intensity as they swept through the
cytoplasm and wave fronts inhibited each other when they collided. DeLisle
24-
and Welsh theorized that IP3 released [Ca ]j from a localized IP3-sensitive
24- 24-
store. [Ca ]j then diffused to an adjacent IP3 insensitive [Ca ]j store and
2-t-
more [Ca ]j was released via CICR and so on. To test this hypothesis,
24-
CaCl2 was injected into oocytes leading to an increase in [Ca ]; but no
2-i-
waves. Injection of [Ca ]j first then IP3 however did produce waves.
24- 24-
lonomycin is a Ca ionophore while thapsigargin inhibits Ca uptake by
24-
paralyzing the ATP-ase necessary for the Ca pump. When these two
24-
agents were injected (in Ca -free media) into oocytes, a localized increase
2-t-
in [Ca ]j persisted or slowly enlarged. No wave was apparent.
24-
Ryanodine or caffeine produced no increase in [Ca ]j and no waves.
24


Based on these data, the waves are not due to CICR or IP3- insensitive
2 +
[Ca ]j stores and are probably not due to a positive feedback mechanism
2 +
whereby [Ca ]j stimulates the production of endogenous IP3. If this had
2 +
been the case, [Ca ]j injection should have initiated the wave. An
2 +
alternative theory is that the [Ca ]j wave was due to IP3-sensitive stores.
2 +
This was tested using heparin. Heparin abolished[Ca ]j waves generated
with IP3S3 (a poorly metabolized IP3 derivative). When IP3S3 then CaCl2
2+ 2 +
were injected, [Ca ]j continuous waves were observed. [Ca ]j
2 +
stimulation of IP3- induced [Ca ]-, release contributes to the propagation of
2 +
[Ca ]j waves in Xenopus. The mechanism is similar to CICR but uses
2 +
IP3-sensitive [Ca ]f stores. Thus, IP3 must be an important factor in the
2 +
propagation of [Ca ]; waves in oocytes (DeLisle and Welsh, 1992).
Further support for the priority of the IP3 system comes from the
presence of an IP3 receptor in the Xenopus oocyte. No ryanodine receptor
has yet been found. The IP3 receptor (IP3R) has been characterized from
Xenopus oocytes by Parys et al. (1992). Oocytes and eggs from Xenopus
2 +
were homogenized and centrifuged to form a microsome pellet. [Ca ]j
2 +
stores accumulated Ca in an ATP dependent manner. Oligomycin and
25


2 +
anitmycin had no effect indicating that the [Ca ]j stores were not
2 +
mitochondria. Addition of IP3 led to [Ca ]j release from vesicles so the
microsomes must have a functional IP3R. The IP3R is composed of four
monomers each being 256 kDa. Binding between IP3 and its receptor is
highly specific. This new IP3 receptor is a new shorter form of the
mammalian brain type. A ryanodine receptor was not located, suggesting
that IP3 is a more likely candidate for calcium release in the Xenopus model.
Other Systems Utilizing IP3
Streb et al. (1983) were also able to show that IP3 induces calcium
release from nonmitochondrial stores in pancreatic acinar cells. The cells
were first permeablized by washing them in a calcium-free then high
2 +
potassium solution. Changes in [Ca ] in the incubation medium were
then measured. Intracellular organelles of the leaky cells were shown to
take up excess calcium from the medium until the cell reached a steady
2 +
state. IP3 was then added which resulted in an increase in Ca in the
incubation medium. This calcium was then taken up by the cells. In ghost
2 +
red blood cells, IP3 addition resulted in only a small Ca increase. In cells
preincubated with A23187 to deplete intracellular calcium stores, IP3
released similar small amounts of calcium. These data suggested that the
increase in calcium following IP3 addition to the cells is indeed due to
calcium release from vesicle cellular stores. Inositol 1-phosphate (IP),
26


inositol 1,4-bisphosphate (IP2) and cyclic IMP could not release calcium,
suggesting that calcium release may depend on the 5' phosphate on the
inositol ring. Using the mitochondrial inhibitors antimycin A and oligomycin,
IP3 was still able to release calcium. In cells treated with IP3 then
2 +
carbachol (which also induces [Ca ]j release in cells), carbachol induced
2 +
[Ca ]j release was inhibited. In cells treated with carbachol first, then IP3,
2+ 2 +
[Ca ]j release decreased by one-third. The sum of the [Ca ]j released by
carbachol and IP3 was constant. Thus, when saturating concentrations of
- 2 +
IP3 are present, carbachol can no longer release [Ca ]j. This suggests that
both carbachol and IP3 may act on the same pool. In turn, carbachol action
may be mediated by IP3. These data together suggest that IP3 functions as
an internal calcium mobilizer in pancreatic acinar cells (Streb et al., 1983).
Hill et al. (1987) examined the effect of heparin on IP3-stimulated
2 +
release of [Ca ]j in permeabilized neoplastic rat liver epithelial cells. A
2 +
concentration of 0.5 uM IP3 released [Ca ]j to 200-300 uM and the
maximum levels were reached in 15 seconds. Sequestration into
2 +
intracellular storage sites occurred during the next-2-3 minutes until [Ca ]j
reached resting levels. When heparin, 40 ug/mL, was injected first, the
cells did not respond to IP3 addition. Inositol 2,4,5 trisphosphate and IP2
27


actions were also blocked by heparin. When cells were chilled to 4C,
Ca passively leaked from intracellular storage pools. At 30C, Ca
2 +
ATPase pumps reduced Ca to starting levels. Heparin had no effect on
this uptake.
Hill et al. concluded that heparin blocks the IP3 receptor and heparin
has become an important tool for studying the IP3 pathway. Channels for
2+ 2 +
passive Ca release and ATP-dependeiit uptake of Ca on the ER are
unaffected by heparin at concentrations that completely inhibit IP3 action.
3
Heparin displaces H-IP3 from membrane binding sites but does not affect
on IP3-phosphokinase or phosphatase activity.
Hill et al. showed that heparin (5.0 uM) inhibits IP3 induced calcium
2 +
release in permeabilized rat liver cells. Ca release reached maximum
2 +
levels within 15 seconds, then Ca was sequestered during the next 2-3
2 +
minutes and this lowered the Ca back to resting levels. When these
same cells were pretreated with 40 ug/mL of heparin for 10 minutes, the
2+
cells did not show an increase in [Ca ]j with IP3 addition. Hill et al.
(1987) concluded that heparin blocks IP3 binding to membrane receptor
sites. Chopra et al. (1989) suggested that heparin competes for a common
IP3 binding site. They discovered that low molecular weight heparin (10
ug/mL) reduced the IP3 response yet showed no stimulation or release of
28


calcium. De-N-sulfated heparin and high molecular weight heparin showed
only a very small inhibition of response.
Experimental Design
The hypothesis of this experiment is that IP3, not CICR, induces the
2 +
[Ca ]j increase associated with fertilization.
Other researches have used BAPTA and heparin successfully in their
experiments. Kline (1988) injected BAPTA into Xenopus eggs to determine
2 +
the effect of prevention of the increase in [Ca ]j, Bement and Capco
(1990) showed that BAPTA injection inhibits cortical breakdown in Xenopus
eggs. Grandin and Charbonneau (1992) used BAPTA to determine the
2 +
effect of [Ca ]; on intracellular pH. Speksnijder et al. (1989) determined
that dibromo-BAPTA was the most potent BAPTA derivative in suppressing
gradients in fucoid eggs. Han et al. (1992) also used dibromo-BAPTA to
prevent cytokinesis in Xenopus blastomeres.
2 +
To determine if the [Ca ]j wave associated with fertilization is
2 +
initiated by calcium or IP3, the [Ca ]j buffer, BAPTA or dibromo-BAPTA,
will be injected into Xenopus laevis eggs. These eggs are then fertilized and
2 +
measured for IP3 content. If elevated [Ca ]j is necessary for PIP2
hydrolysis, IP3 levels should remain low. However, if IP3 release occurs
2 +
before any [Ca ]j release, BAPTA will not have any affect on the sperm-
29


2 +
induced IP3 increase. Heparin, a blocker of IP3-induced [Ca ]j release,
will be injected to determine its effect on sperm-induced increase in IP3
levels.
Heparin has also been used to examine the PI cascade. Heparin was
2 +
shown to inhibit the sperm-induced [Ca ]j release in Xenopus by Nuccitelli
et al. (1993) and Galione et al. (1993). Han et al. (1992) also used heparin
to prevent cytokinesis in Xenopus blastomeres. Heparin has been shown to
induce polyspermy in Xenopus zygotes. In this experiment, heparin will be
used to induce polyspermy to determine the effects of multiple sperm entry
on IP3 levels.
BAPTA
The calcium buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-
2 +
tetraacetic acid (BAPTA) binds Ca without proton release. In addition, it
2 +
binds calcium faster than EGTA (which releases a proton when Ca is
bound). The proton may acidify the cytoplasm. By injecting BAPTA into an
2+ 2 +
egg, the sperm induced [Ca ]; rise is inhibited and [Ca ]j dependent
fertilization events are inhibited (Kline, 1988). For example, eggs
preinjected with BAPTA to a final concentration of 0.7 mM, the fertilization
potential did not occur.
Injection of BAPTA also inhibited exocytosis, the cortical
contractions, elevation of the fertilization envelope and gravitational
30


rotation. Some of these zygotes were analyzed histologically and the
preinjected zygotes still contained cortical granules in the membrane
whereas uninjected eggs lacked these granules. Finally, Kline showed that
although multiple sperm may enter a BAPTA injected egg, chromatin
decondensation and pronuclear formation are inhibited. Kline surmised that
2+ 2 +
a sperm-induced increase in Ca acts to trigger a sequence of Ca
dependent events associated with activation of the egg.
Xenopus model
The use of the African clawed frog, Xenopus laevis. offers many
advantages in laboratory experiments. The animal is easily maintained since
Xenopus are carnivorous frogs and can survive on an inexpensive diet of
cubed beef heart. The frogs also require little maintenance since they are
totally aquatic. Female Xenopus are able to produce oocytes or eggs year-
round The cells are approximately 1.3 mm (see figure 2) and can be seen
with the naked eye. Since they are large, the cells are easy to microinject
with desired solutions. In vitro fertilization is also easy to accomplish with
Xenopus eggs and sperm. Fertilization can be confirmed by gravitational
rotation and cleavage (figures 3 and 4). Gravitational rotation occurs when
the animal pole rotates to point upward. This occurs approximately 15
minutes after insemination. Mitotic cleavage occurs 90 minutes after
insemination. This was observed by Gerhart and Scharf (1980) and our
data confirm their findings.
31


Figure 2 Author's adaptation of the Xenopus egg.
32


Figure 3 Author's adaptation of gravitational rotation in Xenoous zygotes
looking directly down oh the egg
Figure 4 Author's adaptation of mitotic cleavage in Xenopus zygotes
looking directly down on the egg
>5
33


CHAPTER 2
MATERIALS AND METHODS
Maintenance
Xenopus laevis frogs (Xenopus One, Ann Arbor, Ml) were fed with a
diet of 1.5 -2.0 g of cubed beef heart every other day. The water in which
the frogs lived was replaced every other day with fresh, oxygenated water.
A twenty-four hour clock was used to set a 12 hour light and 12 hour dark
cycle. Ambient temperature was 23 Celsius.
Priming
Females injected with human chorionic gonadotropin (HCG) can
produce viable eggs as needed. The hormone is injected in the dorsal
lymph sac and the frogs can be recycled every 6 months. Selected female
frogs were primed one to three days in advance of each experiment with
100 I.U. of pregnant mare serum gonadotropin (PMSG) (Sigma Chemical
Company, ST. Louis, MO). These females were then reprimed with 850
I.U. of HCG (Sigma Chemical Company, St. Louis, MO) approximately 12
hours before use.
34


Fertilization
Fresh eggs were milked from the female and placed in 100%
Modified Bath's Solution (MBS) (see appendix). A.male frog was sacrificed
and the testes removed and placed in 100% MBS. Twenty eggs were
placed in a small glass petri dish. Excess 100% MBS was removed with a
kimwipe and 300 uL of 10% MBS was added to the eggs. The sperm
solution was prepared by mincing one-quarter of a testis in 300 uL of 100%
MBS. 50 uL of this sperm solution was added to the eggs. Fertilization
was confirmed by gravitational rotation (occurs at approximately 15-20
minutes) and the appearance of cleavage furrows (occurs at approximately
90-100 minutes) (figures 3 and 4). To induce polyspermy, eggs were
placed in 300 uL of 20 mM Nal or 67% MBS.
Sperm Correction Factor
Sperm that may still be attached to the zygote need to be taken into
account since these non fertilizing sperm also contain IP3. A correction
factor was determined (Stith et al., 1993). The difference in IP3 at time
zero (eggs alone) and time points collected at 8 and 15 seconds after sperm
35


addition was taken as IP3 in added sperm since sperm-egg contact occurs
well after 15 seconds after sperm addition. Fifteen eggs had 567 + 79
fmol of IP3 while the average zygote had 1452 +_ 372 fmol of IP3 at the 8
and 15 second points. The difference of 885 fmol represents the IP3
associated with sperm. Based on 1 amol per sperm that has contacted egg
jelly (Stith et al., 1993), this would amount to approximately 1.9 million
sperm still on the outside of the zygote.
This correction factor assumes that sperm IP3 does not change
dramatically from 8-15 seconds to 15 minutes after insemination. To
examine this assumption, approximately 1.9 million sperm were added to
one egg jelly and the IP3 value per spermatozoon was determined as a
function of time. The IP3 mass in one spermatozoon was 1.12 _+ 0.02
amole at 5 minutes after sperm addition to egg jelly. At 10 minutes, the
IP3 value was 0.945 _+ 0.05 amol/spermatozoon and at 15 minutes, it was
0.917 _+ 0.04 amole/spermatozoon. With 1 amole of IP3/spermatozoan,
885 fmol IP3 in nonfertilizing sperm, and the removal { washing ) of excess
sperm solution, about 885,000 sperm remained with the 15 zygotes. Since
1.9 million sperm were initially added to the zygotes, 53% of the sperm
were removed when 85% of the external medium was removed.
36


The final method of correction for excess sperm IP3 involved fertilization
with different amounts of sperm and using the difference in IP3 as the amount
of nonfertilizing sperm. BAPTA injected eggs were fertilized with high and low
amounts of sperm and total IP3 mass quantified five minutes after
insemination. The two values of IP3 were 5940 and 3446 fmol/sperm + 15
zygotes. The difference between the two IP3 values was divided by the
difference in the number of total sperm in the IP3 samples (3,057,440 and
201,2760 sperm), the correction value was found to be 0.9 amole of IP3 per
spermatozoon. This value was not significantly different from that obtained
with sperm in the presence of uninjected eggs, about 1 amole/spermatozoon
(Stith et al 1993).
Injection Parameters
A Pico-pump PV830 (World Precision Instruments, Inc., Sarasota, FL)
was used to inject solutions. The size of the droplet to be injected
depended on the concentration of the solution, the concentration desired in
the egg, and the volume of the egg. The formula II x (d)3/6 is used to
determine the size of the droplet. In this formula, ud equals the diameter of the
droplet which is measured under the microscope with a micrometer. For
BAPTA experiments, eggs were shed dry into a petri dish and injected with
33.5 nL of BAPTA:CaCI2 (see later section for specific calculations).
37


The eggs were then flooded with a 10 mM chlorobutanol/ 100% MBS
24-
solution. Chlorobutanol has the ability to prevent elevated [Ca ]j due to
microinjection (Webb and Nuccitelli, 1985). After two minutes, the eggs
were rinsed twice and stored in 100% MBS for twenty minutes. This
allowed for the diffusion of BAPTA throughout the egg. The eggs were
then placed in a small petri dish and the excess 100% MBS was removed.
Fertilization was then performed by sperm addition.
For heparin injection, eggs were first placed into a 10 mM
cholorbutanol/100% MBS solution, then injected with 33.5 nL of various
stock concentrations of heparin. The eggs were rinsed and stored in 100%
MBS. The heparin was allowed to diffuse through the egg for 1 hour before
fertilization.
Solutions
BAPTA
The BAPTA concentration needed to buffer [Ca2+]j levels was based
on three studies using Xenopus laevis eggs. Kline (1988) estimated that
approximately ImM BAPTA effectively blocks the increase in [Ca2+]j,
assuming a 560 nL egg volume. Based on the data of Grandin and
Charbonneau (1992), these authors found 3-6 mM BAPTA an effective
concentration. This calculation was based upon an injection of 35 nL of 50-
38


100 mM BAPTA into an egg volume of 560 nL. Finally, Bement and Capco
(1990) suggested using a 5.4 mM BAPTA stock concentration. The
injection volume was 8 nL of 360 to 388 mM BAPTA stock solution. Kline
(1988) also established that BAPTA to Ca ratios from 1:1 to 10:1 were all
equally effective. In other words, 2:1 BAPTA:Ca is just as effective as a
solution of 6:1 BAPTA:Ca. Using the Maxchelator program (see later
2 +
section) it was possible to estimate the amount of free [Ca ]j in the
24-
presence of the buffer. The solution of 1.56:1 BAPTA :Ca buffered [Ca ]j
to approximately 345 nM; 2:1 BAPTA:Ca to 194 nM; and 6:1 BAPTA:Ca to
39 nM.
Dibromo-BAPTA
Dibromo-BAPTA can diffuse faster than BAPTA, therefore dibromo-
2 +
BAPTA can inhibit [Ca ]j gradients better. Dibromo-BAPTA solutions were
similar to the concentrations of the BAPTA:Ca solutions. The final actual
stock solution was made up to a concentration of 388 mM dibromoBAPTA
to 65 mM total CaCI2.
Heparin
Three different solutions of heparin (low molecular weight, 3000
kDa) (Sigma Chemical Co, St. Louis, MO) were prepared. The high
39


concentration was 13 mg/mL; the medium solution was 3.0 mg/mL; and the
low solution was 0.6 mg/mL- The solution of de-n-sulfated heparin
(denso) was prepared to a concentration of 13 mg/mL.
MaxChelator
MaxChelator (MAXC) is a computer program designed by Chris
2 +
Patton of the Hopkins Marine station that calculates free [Ca ]j in the
presence of chelators. MAXC also provides the total metal concentrations
2 +
that are needed to achieve the free Ca wanted. Finally, the amount of
chelator to add to achieve a certain free metal concentration can be
calculated. The information can be adjusted to accommodate different
environments by entering the temperature, pH, and ionic strength into the
program. In our study, the constants were 23 C ambient temperature, an
intracellular pH of 7.3, and ionic strength of 0.25 Osm. The final estimated
intracellular buffer concentrations were used to estimate the buffered


IP3 Mass Assay
IP3 mass was measured with a receptor (isolated from bovine brain
extract) competitive ligand binding assay (New England Nuclear,
Wilmington, DE). More specifically, IP3 mass was determined by measuring
the decrease in cpm as cold IP3 from the eggs or zygotes displaced tritiated
IP3 bound to IP3 receptors.
Each treatment consisted of five groups of twenty eggs or zygotes.
At designated time points, each group was transferred to a 1.5 mL
eppendorf v-vial and homogenized in 300 uL of 25% trichloroacetic acid
(TCA). Each vial was centrifuged for 1 minute at 15,000 x g and 5 C.
The supernatant was transferred to a new v-vial and 300 uL of
freon:tri-n-octylamine (Sigma Chemical Co., St. Louis, MO) (3:1, v/v) was
added. Vials were vortexed for 20 seconds and centrifuged for 20 minutes
at 15,000 x g and 5 C. This organic step extracted TCA, since the acid
would displace tritiated IP3 from the receptor. An aqueous and organic
layer formed. The aqueous contained the IP3 and was removed and placed
in a 500 uL v-vial. This usually yielded about 200-250 uL of sample. A
100 uL aliquot was placed in reaction vessels. 400 uL of working
3
receptor tracer solution (IP3 receptor bound to H-IP3) was added to the
reaction vessels containing 100 uL sample, IP3 standards (New England
41


Nuclear) (48nM; 9.6nM; 4.8 nM; or 2.4 nM), water, or blanking solution.
The blanking solution, which contained a high concentration (12nM) of
inositol 1,2,3,4,5,6-hexaphosphate (IP6), displaced all 3H-IP3 from its
receptors. This resulted in a cpm value that represented background,
nonspecific trapping of tritiated IP3 in the receptor pellet (obtained later).
Samples, standards, water, and blanking solution were incubated for
an hour at 5 C. The reaction vessels were then centrifuged for 20 minutes
at 15,000 x g at 5 C. The supernatant was removed and 50 uL of 0.15 M
NaOH was added to each vessel. Vessel were vortexed for ten minutes at
room temperature. Each vessel was placed into a scintillation vial with 7
mL of aqueous scintillation fluid (Budget Solve and Biosafe 2, Research
Products, Inc., Mt. Prospect, IL) and counted in the scintillation counter.
Statistical Analysis
Standard errors bars are shown in the figures while the mean +_
standard deviation are given in the text. Each experiment had four to six
determinations per treatment group and each determination was made with
15 eggs. A two tailed, pooled student's t test was used for tests of
significance.
42


CHAPTER 3
' - RESULTS
Fertilization Efficiency
First, the in vitro concentration of ions for optimal fertilization was
determined. Twenty eggs were placed in a small, tilted glass petri dish in
300 uL of varying percentages of MBS. Fertilization was quantified by time
and percentage of zygotes undergoing gravitational rotation and cleavage.
Eggs fertilized in 20% MBS had 84.5% rotations in 16-18 minutes; eggs
fertilized in 10% MBS had 91.7% rotations in approximately 15 minutes;
while eggs fertilized in 5% MBS had 80% rotations in approximately 15
minutes. Eggs were followed through cleavage which usually occurred 80-
100 minutes after insemination. Eggs fertilized in 10% and 20% MBS had
similar cleavage times, however 5% MBS fertilized eggs had a lower
percentage of cleavages occurring over a longer period of time. Thus we
adopted a procedure where twenty eggs were placed in 300 uL of 10%
MBS and fertilized with a 50 uL sperm solution. Fertilization is optimal in a
dilute saline medium (Wolf and Hedrick, 1971). The sperm solution, made
in 100% MBS, was added to eggs in a dilute ,10% MBS medium. With this
protocol, rotations were typically 100%.
43


IP3 Increase After Fertilization
A measurement of the time course of the IP3 increase at fertilization
is shown in figure 5. The sperm and egg data from part A (figure 5) were
corrected by the amount of IP3 in sperm (885 fmole) and the result was
divided by 15 to calculate IP3 mass per zygote. IP3 levels in eggs were 40
fmol/zygote within eight seconds. After fertilization, the IP3 peak occurred
at approximately 6-7 minutes and reached a peak value of 211 +.47
fmol/zygote (three determinations). After approximately 10 minutes, IP3
mass decreased to approximately control levels. A wave of contraction
occurs at approximately the same time as peak IP3 values: the contraction
was observed using a videocamera attached to a microscope. The
contraction wave traveled across the egg starting at about 3-5 min and
ending at about 5-7 min.
BAPTA Concentrations
A stock solution of 400 mM BAPTA and a second solution of 1.8 M
CaCI2 were prepared. The two solutions were added to each other in
different ratios. For a 2:1 BAPTA:CaCI2 solution, 196.4 uL of 400 mM
BAPTA was combined with 19.64 uL of 1.8 M CaCI2. The final
2 +
concentration of free [Ca ]j were 363 mM BAPTA and 163 mM CaCI2,
respectively. Eight nanoliters, which was calculated to be a 0.25 mm drop,
44


IP3 (f MOL/ZYGOTE) IP3 (f MOL/15 ZYGOTES AND
ASSOCIATED SPERM)
Figure 5 Time course of IP3 mass after insemination. (A) Uncorrected
values show that the addition of sperm was associated with
an increase in IP3 mass. Time zero is IP3 in 15 eggs before
sperm addition and time of insemination. (B) IP3 mass per
zygote increased from 37.8 .+ 5.3 (five determinations) to
211 +_ 47 fmole/zygote. insert is the first two points after
sperm addition at 8 and 1 5 seconds after insemination. Insert
has the same units as the large axes. Asterisks denote
numbers significantly greater than unfertilized egg (P<0.003).
45


of this solution was injected into each of 12-25 eggs. BAPTA did not
prevent the fertilization IP3 increase Injection did not artificially activate
eggs as unfertilized, injected eggs did not exhibit gravitational rotation for
up to 2 hours. In addition, when injected with approximately 5.5 mM
BAPTA, the IP3 mass in nonfertilized eggs was not significantly different
from that found in eggs that had not been injected with BAPTA (figure 6
compares egg alone with BAPTA alone). In the presence different BAPTA
solutions (i.e., 6:1 and 2:1) there was no significant difference in IP3 mass
2 +
so the data were pooled. Since artificial [Ca ]; elevation can increase IP3,
2 +
this suggests that injection and subsequent Ca influx did not activate the
egg-
Eggs injected with higher concentrations of BAPTA had a
significantly higher increase in IP3 mass than those in the control
fertilization (figure 7). Eggs preinjected with BAPTA, then fertilized, had a
greater than normal increase in IP3 after fertilization. There was no
significant difference in IP3 production when BAPTA injected eggs were
allowed to incubate for 20 minutes (774 +_157 fmole of IP3/zygote), 40
minutes (825 __ 292 fmole of IP3/zygote) and 90 minutes (805 + 300
fmole of !P3/zygote) before sperm addition.
46


IP3 (f MOL/CELL)
600
500
400
300
2 00
100
0
T__
(18)
*
(13)
(5)
(13)
ILI < LU <
o z h- h- z l
o o OC CL O Q. +
LU _J LU < _J <
< LL CQ < CQ
Figure 6 BAPTA preinjection did not prevent fertilization IP3 increase.
EGG ALONE refers to control conditions while FERTIL refers
to normal fertilization parameters. BAPTA ALONE refers to
BAPTA only injected eggs. BAPTA + FERTIL refers to BAPTA
injected eggs that have been fertilized. Asterisks denote groups
that are significantly higher than egg alone (P>0.001).
47
FERTIL


IP3 (f MOL/CELL)
Figure 7 A concentration of BAPTA:Ca was unable to prevent fertilization
events and did not alter the normal fertilization IP3 increase.
BAPTA Alone are injected to a final estimated intracellular
concentration of 5.1 mM BAPTA.
48


A time course compared IP3 mass in BAPTA injected eggs to IP3
mass changes in noninjected eggs. IP3 levels in BAPTA injected eggs were
greater than in control fertilization (see figures 5 and 8). In BAPTA injected
eggs, the maximum IP3 value is higher and peaks a few minutes later than
the IP3 produced after insemination of uninjected eggs. Even though
multiple sperm enter the BAPTA injected egg, IP3 levels decrease rapidly
after about 8 minutes.
An above normal IP3 production was noted in the BAPTA injected
eggs upon insemination. We measured the fertilization IP3 increase with a
BAPTA solution that would buffer calcium to a higher concentration of 345
nM instead of 39 nM. The 1.56:1 BAPTA:Ca solution should buffer to a
2 +
[Ca ]j to approximately 200 to 400 nM which is closer to the basal,
unstimulated level (as seen by Busa and Nuccitelli (1985) and Nuccitelli et
al. (1993)). The 1.56:1 solution did not produce an above average
production of IP3 (figure 9); however, it was just as potent as the 2:1 or
6:1 BAPTA:Ca solutions in that all solutions completely prevented the
contraction wave, gravitational rotation, and cleavage. This is in agreement
with Kline (1988). Thus, the above average production of IP3 in the higher
BAPTA concentrations is an artifact of buffering calcium to levels much
lower than basal.
49


IP3(-f MOL/CELL)
Figure 8 Time course of the IP3 increase after insemination of BAPTA
injected eggs.
50


IP3(f MOL/CELL)
Figure 9
- +
CD
6:1 BAPTA versus 1.56:1 BAPTA injected eggs. No induction
of an above average IP3 increase occurs in the 1.56:1 BAPTA
injected eggs.
51
1.56:1 BAPTA
+ FERTIL


Speksnijder et al. (1989) found that 5,5' dibromo BAPTA (dibromo-
BAPTA) was the most potent of the various BAPTA derivatives in
2 +
suppressing [Ca Ij gradients in the fucus egg. Dibromo-BAPTA, in our
experiments, did not inhibit the fertilization IP3 increase. The final
2 +
concentration of dibromo-BAPTA was 5.5 mM and should buffer [Ca ]j to
approximately 547 nM. In three experiments, normal fertilization produced
an increase to 280 +. 196 fmol/zygote, whereas dibromo-BAPTA injected
cells showed an IP3 increase to 289 +_ 241 fmole/zygote. The large
standard deviations are due to a combination of the results of three
experiments; within each experiment, the standard deviations were about
15% of the average and there was no a significant difference between the
IP3 peaks after fertilization of uninjected or dibromo-BAPTA injected eggs.
Polyspermy
These experiments were conducted to examine whether multiple
sperm entries could explain why BAPTA injected eggs show full IP3
increase. That is many sperm enter a BAPTA injected egg since there is no
activation potential and early block to polyspermy. And, even though
BAPTA reduces IP3, a normal measure is not all due to many sperm entries.
Eggs were fertilized using high tonicity MBS (67%) or 10% MBS with 20
52


mM Nal. Though these eggs are polyspermic, IP3 levels were no greater
than for monospermic fertilized eggs (see figure 10).
The use of high and low concentrations of sperm added to BAPTA
injected zygotes may be expected to produce different levels of polyspermy,
yet similar IP3 production was noted. The amount of IP3 in excess sperm
(3,057,440 x 1 amole/spermatozoon = 3057 fmole) was subtracted from
the IP3 in all sperm and 15 zygotes (5940 fmole) and divided by 15 and the
result is 193 fmole/zygote. Fertilizing with 15-fold lower sperm
concentration, a similar peak was calculated (216 fmole/zygote).
Increasing extracellular chloride or iodide induced multiple sperm
entries as seen by Grey et al. (1982). Under these conditions, IP3 levels do
not increase to a value greater than that obtained after a single sperm entry
(figures 11 and 12).
Polyspermy was confirmed by observing multiple and atypical
cleavage furrow formation. Sperm entries were determined by recording
the number of dark sperm entry sites, as described by Grey et al. (1982). In
0 mM Nal, fertilization produced only one sperm entry site per zygote, in 10
mM Nal 3-5 sperm entry sites per zygote was noted, in 20 mM Nal 10 or
more sperm entry sites per zygote were noted (Grey et al., 1982). DNA
dyes were not used since they often leak out of the cell and fluoresce all
53


IP3 (fMOL/ZYGOTE)
G g NORMAL 67% MBS 20mM Nal
OO (10% MBS)
LU _l
<
IP3 AT 5 MIN AFTER FERTILIZATION
Figure 10 IP3 levels in normal (10%) MBS, 67% MBS and MBS + 20
mM Nal. The latter two solutions induce polyspermy,
however, polyspermic eggs did not exhibit a greater increase
in IP3 levels than monospermic eggs.
54


IP3 (I MOL/CELL)
O o
lu _j
<
N a I CONC. '(mM)
Figure 11 Varying the concentration of Nal and presumably the number
of sperm entries, did not vary the level of IP3 in polyspermic
eggs versus monospermic eggs. There were three
experiments with up to 11 determinations per treatment.
55


IP3 (fMOL/ZYGOTE)
300 -
200 -
100 -
0
Figure 12
0 w NORMAL 67% MBS 20mM Nal
oo (10% MBS)
<
IP3 AT 5 MIN AFTER FERTILIZATION
Heparin and de-N-sulfated heparin and IP3 levels after
insemination.
56


attached sperm. Also the large, opaque Xenopus egg makes observation
with DNA dyes difficult. :
Heparin
Heparin inhibits IP3 action by blocking the IP3 receptor. Three
different stock concentrations (13 mg/mL, 3 mg/mL, 0.6 mg/mL) were
injected in Xenoous eggs. Final intracellular concentrations were estimated
to be 9 ug/mL do), 44 ug/mL (med), and 190 ug/mL (high). Heparin injected
eggs were incubated for an hour before sperm was added. De-N-sulfated
heparin (denso) was also injected into eggs to a final intracellular
concentration of 190 ug/mL. De-N-sulfated heparin has been shown to be
unable to bind to the IP3 receptor and block IP3 action. These eggs were
fertilized after an hour incubation. Among the injected, fertilized eggs, none
showed a significant difference in the amount of IP3 produced as compared
to uninjected, fertilized eggs (figure 13). All groups had four to five
determinations with 15 eggs per determination.
High heparin injection blocked gravitational rotation and cleavage
De-N-Sulfated heparin did not affect the IP3 increase, nor did it block
gravitational rotation and cleavage.
57


APPROXIMATE IP3 MASS
LD
f
o
o
>
N
\
_J
O
5
LO
I-
<
ESTIMATED SPERM ENTRIES
Figure 13 IP3 mass is independent of the number of sperm entry under
controlled polyspermic conditions.
58


CHAPTER 4
DISCUSSION
2 +
Upon fertilization, an initial increase in [Ca ]; is believed to occur at
2 +
the sperm binding site. This [Ca ]; increase then travels (as a wave) the
length of the egg and this propagation occurs without external calcium.
Consequently, intracellular stores must be responsible for the increase.
2 +
There is an abundant amount of information concerning the Ca wave
since it can be artificially induced and thus easily studied. However, what
24-
causes the initial [Ca ]j increase remains a mystery.
2 +
IP3 Production in the Presence of Ca Chelators or Heparin
We have shown that BAPTA, dibromo-BAPTA, or heparin preinjection
does not lower the IP3 increase noted after Xenopus fertilization. This
suggests that sperm increase PIP2 breakdown to produce IP3 before there
2 +
is a large increase in [Ca ];. Xenopus eggs have 50-75 fmoles of IP3/cell
under normal conditions. After fertilization, IP3 levels jump to 300
fmoles/cell. If the initial fertilization PIP2 breakdown is dependent upon an
2 +
increase in [Ca ]j, IP3 levels should remain low in eggs preinjected with
59


BAPTA. This was not the case as seen in figure 5. BAPTA-injected eggs
had IP3 levels that were actually higher in IP3 mass than in fertilized eggs
not injected with BAPTA. This result is explained in detail later.
Similar results were found using heparin and dibromo-BAPTA in that
the sperm induced IP3 increase was never inhibited fully. From these data,
2 +
we conclude that PIP2 breakdown must occur prior to [Ca ]j increase in
the fertilization of Xenopus eggs.
Studies done by Nuccitelli et al. (1993) support the initial PI cascade
theory. When the PIP2 antibody was injected into Xenopus eggs, both the
2 +
[Ca ]j and wave were inhibited. Inhibitors to CICR, procaine or ruthenium
red, were also injected but calcium release was not inhibited. Similarly,
CICR stimulators, caffeine and ryanodine, did not induce any rise in
calcium.
The PIP2 antibody appears to be highly variable depending on cell
type. If the antibody can be optimized for each cell type concerned, the
antibody may become an effective tool in analyzing PIP2 hydrolysis and
thus IP3 production.
BAPTA and Polvspermv
Polyspermy leads to the improper allotment of genetic material and
abnormalities may result (Kline, 1991). To guard against polyspermy, an
increase in intracellular calcium upon fertilization leads to cortical granule
60


exocytosis. When these granules undergo exocytosis, a number of
morphological changes occur in the egg which form a permanent block to
multiple sperm entries. BAPTA was shown to induce polyspermy in
Xenopus eggs by buffering intracellular calcium and thus inhibiting the block
to polyspermy (Kline, 1988). Histological analysis revealed multiple sperm
entry sites in BAPTA-injected eggs. Thus, it could be argued that the IP3
increase in BAPTA-injected eggs is due to multiple sperm entering the egg.
However, our fertilization experiments done under polyspermic conditions
did not support this theory. Fertilization done under polyspermic conditions,
in 20 mM Nal or high tonicity (67% MBS), did not show a greater than
normal increase in IP3 than when 10% MBS (monospermic) was used.
Egg fertilized under polyspermic conditions had IP3 levels around 250
fmole/zygote which is comparable to the 280 fmole/zygote IP3 levels found
under monospermic conditions. Also, approximate IP3 mass remained
constant despite the number of sperm entry (see figure 13). Thus we can
assume the IP3 mass at various concentrations of BAPTA (figure 7) is due
to the initial sperm and not to subsequent sperm entry, as seen in various
concentrations of Nal (figure 13).
Above Normal Induction of IP3 bv BAPTA
2 +
The BAPTA solutions prevented the wave of [Ca ]j without
2 +
decreasing IP3 production. This suggests that the [Ca ]j wave does not
61


produce IP3 in a positive feedback loop as previously suggested. Whitaker
and Swann (1993) suggested that there is a positive feedback from
calcium-stimulated IP3 release (Berridge, 1987).
A key to this loop is the dephosphorylation of IP3 by IP3-5'
phosphatase which terminates the second messenger action of IP3. IP3 is
metabolized to IP2 which is not capable of releasing calcium (Berridge,
1987). Inhibition of 1,4,5-trisphosphatase by 2,3-diphosphoglyceric acid
could play a regulatory role in insulin-secreting beta cell (Berridge, 1987). It
is also believed that the DAG/C kinase pathway may act to stimulate the
hydrolysis of IP3 via phosphorylation reactions concerning IP3-5
phosphatase (Berridge, 1987).
Eggs preinjected with higher amounts of BAPTA:CaCI2 (6:1 or 2:1)
showed higher increases in IP3 upon fertilization than uninjected eggs. This
may be explained by the fact that the degradation mechanisms for IP3 may
be calcium dependent.
Heparin Injection Prevents Fertilization Events.
In this study, heparin did not inhibit IP3 production after fertilization
2 +
but did prevent the [Ca ]j dependent events of fertilization. This suggests
2 +
that IP3 is necessary to initiate fertilization and occurs before the [Ca ];
increase. Nuccitelli et al. (1993) demonstrated that Xenoous eggs injected
2 +
with heparin did not exhibit the normal increase in [Ca ]j as seen in
62


uninjected eggs upon fertilization.. These data lend support to our findings
that IP3 is required for calcium release in fertilization of Xenopus.
Sea Urchin Eqq Activation.
Our results in Xenopus may be compared with other systems. In the
sea urchin, it appears that the calcium increase is due to CICR and IP3.
Whitaker and Irvine (1984) isolated plasma membranes and associated
cortical granules in an EGTA-containing medium. These media were then
exposed to various amounts of free calcium solutions buffered with EGTA.
After separation via TLC, the lipids were identified- by comparison with
known standards. Sea urchin eggs were incubated in the presence of P-
orthophosphate which incorporated predominantly into PIP2. There was
little PIP2 breakdown in the presence of excess EGTA. This provided some
evidence that it is calcium-stimulated breakdown of PIP2 to IP3 that leads
to the release of calcium. PIP2 breakdown in the plasma membrane was
2 +
stimulated by [Ca ], concentrations of 3-5 uM. This also the
concentration at which cortical exocytosis occurs in the sea urchin egg
(Whitaker and Baker, 1983). However, injection of IP3 also was able to
2 +
stimulate Ca release indicating that both CICR and IP3 induced calcium
release may work together to increase intracellular calcium release in sea
urchin eggs.
63


2 +
Support for Ca -stimulated PIP2 breakdown was found when Lee
2 +
et al. (1993) discovered that dual receptors led to the increase in Ca in
2 +
sea urchin eggs. Heparin was shown to block IP3 induced Ca release but
2 +
not Ca induced release. Cyclic-ADPR has been shown to be a potent
calcium mobilizer in sea urchin eggs. Cyclic-ADPR was shown desensitize
microsomes to subsequent action of caffeine or ryanodine. Thus it is
possible that the calcium mechanism activated by cADPR is
pharmacologically similar to CICR (Lee, 1993). The antagonist of cADPR,
8-amino-cADPR (8NH2), was able to block CICR but not IP3 induced
calcium release. Inhibition of either IP3 or CICR was not sufficient to
prevent the sperm induced calcium release indicating there is more than one
mechanism involved in intracellular calcium release in sea urchin egg.
Buck et al. (1994) showed that the calcium release in sea urchin egg
can be induced by ryanodine and cyclic ADP-ribose. Both IP3 and ryanodine
receptors have been identified in sea urchin eggs. Though IP3 is the
endogenous regulator of the IP3 receptor, it is not clear what the
endogenous regulator of the ryanodine receptor is. It may be cyclic ADP-
ribose (a metabolite NAD) (Buck et al. 1994).
24-
In amphibians and hamsters, [Ca ]j release is not due to CICR but
IP3 induced calcium release. In our study, it appears as if IP3 is the
64


2 +
mechanism for [Ca ]j increase upon fertilization. BAPTA and dibromo-
BAPTA did not lower IP3 levels suggesting calcium is not necessary for PIP2
breakdown. If calcium had been required, IP3 levels would not have
increased (as seen in figures 5 through 9).
G-Proteins
Sperm may activate the PI cascade through tyrosine kinase activity
(Ciapa et al., 1992). or G-proteins. In one hypothesis, the sperm binds to
an egg receptor protein on the membrane. This binding stimulates the G-
protein which activates phospholipase C (PLC). This activated enzyme then
hydrolyzes PIP2 into IP3 and DAG (Kline, 1991). This theory has some
support in that Schmell et al. (1977) and Rossignol et al. (1984) have
described sperm receptor proteins in sea urchin egg. This receptor is an
approximately 350 kDa heavily glycosylated protein. In addition to the sea
urchin receptor, G-proteins have been isolated in the sea urchin egg, lending
further support to the sperm activated G-protein theory (Kline, 1991). It will
be interesting to see what mechanism induces PIP2 breakdown to IP3 and
subsequent increase in intracellular calcium in response to insemination.
Gillmen (1984) characterized some of the G-proteins possibly
involved in the activation of PLC. G-proteins are receptors for hormones,
neurotransmitters, etc., that interact with ligands at the cell surface. They
either inhibit or stimulate adenylate cyclase. Stimulatory receptors (Gs) are
beta-adrenergic and respond to acetylcholine and gonadotropins. Inhibitory
65


(Gj) receptors are alpha-adrenergic and include muscarinic agonists and
opiods. Gs is oligometric with two subunits of 45 kDa and 35 kDa. Gj is
also oligometric but with 41 kDa and 35 kDa subunits. G; and Gs share the
same subunit. Gomperts (1983) showed that GTP analogues cause mast
cells to undergo exocytotic secretion in response to extracellular calcium
addition. GTP releases more calcium than IP3 but this release can be
blocked by GDP (Berridge, 1987).
Chueh et al. (1987) showed that both GTP and IP3 release calcium
in smooth muscle cell lines. However, GTP was shown to release up to
70% more calcium than IP3. Also, IP3 depleted calcium stores upon
subsequent addition of IP3. GTP did not display this phenomenon upon
subsequent treatments. This suggests that GTP and IP3 may release
calcium from the some pool but GTP may work elsewhere as well.
Lechleiter and Clapham (1992) also showed that GTP-gamma-S induced
calcium release in Xenopus oocytes. In opposition, PLC may be stimulated
directly by the sperm receptor.
Conclusion
These data suggest that sperm increase IP3 via PIP2 breakdown to
2 +
induce the Ca release needed to initiate fertilization. We also determined
2 +
that IP3 production occurs at [Ca ]j levels below basal and basal levels of
2 +
Ca are needed to allow for rapid IP3 metabolism. The level of IP3
66


initiation depends on only one sperm, the first one to fertilize. Subsequent
sperm entry does not induce a higher level of IP3 mass than a single sperm
entry.
The next important step is to determine the first messenger system,
specifically the receptor, that induces PIP2 breakdown and the opening of
chloride ion channels.
2 +
Determining the [Ca ]j mechanism that may play a part in the
2 +
metabolism of IP3 may also be an interesting aspect of [Ca ]j research.
67


APPENDIX A
CHEMICALS, SOLUTIONS AND EQUIPMENT
Chemicals
BAPTA
CaCI2 2HzO
Ca(N03) 4H20
Chlorobutanol
Freon
HEPES
HCG
IP3 Standards
[3H] IP3
KCI
MgS04'7H20
NaCI
NaHC03
NaOH
(Calbiochem, LaJolla, CA)
(Baker)
(Sigma)
(Sigma)
(Sigma) 1,1,2-trichloro-1,2,2-trifluroethane
(R.P.I.)
(Sigma)
(New England Nuclear)
(New England Nuclear) [3H] IP3 kit,
cat. # NEK-064
(Baker)
(Sigma)
(Baker)
68


PMSG
- (Sigma)
Scintillation Fluor (IP# Assay) - (R.P.I.) Budget Solve and BioSafe II
TCA - (Sigma) Trichloroacetic Acid
Trioctylamine - (Sigma)
Solutions
BAPTA - 400 mM stock 50 mg BAPTA 196.4 uLdH20
BAPTA:CaCL2 - 2:1; 363 mM BAPTA :163 mM CaCI2 19.6 uL 1.8 M CaCI2 196.4 uL 400 mM BAPTA
CaCI2 - 1.8 M 0.2 mg CaCI2 2H20 756 uL dH20
Chlorobutanol - 1 M stock solution 18.65 g chlorobutanol 100 ml. alcohol - 10 mM chlorobutanol in MBS 1 ml. 1 M chlorobutanol 99 mL 100% MBS
Freon : Trioctylamine -3:1 (v/v)
HCG - Human Chorionic Gonadotropin 2500 U/mL
69


Modified Barth's Solution
Modified Barth's Solution - 88 mM NaCI 1.0 mM KCI 2.4 mM NaHCOg 10 mM HEPES pH 7.5 0.82 mM MgS04'7H20 0.33 mM Ca(N03) 4H20 0.41 mM CaCI2 2H20
PMSG - Pregnant Mare Serum Gonadotropin 116.67 U/mL
TCA - 25% solution, 5 g in 20 ml dH20
Working Receptor/Tracer - Dilute Receptor/Tracer 15 fold with Assay Buffer (NEN)
Centrifuge EauiDment Clinical Microfuge
Pneumatic Pico Pump - World Precision Instruments, Inc. PV 830 Sarasota, FL
Scintillation counter - LKB-Wallace, Pharmacia Gaithersburg, MD
70


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