Vol.
172,
No.
October
2, 1990
30,
BIOCHEMICAL
AND
BIOPHYSICAL
1990
RESEARCH
COMMUNICATIONS Pages
High Concentration of Inositol 1,4,5trisphosphate Kuni H. Iwasa*, Gerald Ehrenstein*, Louis J. DeFelice**,
932-938
in Sea Urchin Sperm and James T. Russell***
*Laboratory of Biophysics, NINDS, National Institutes of Health, Maryland 20892 **Department ***Laboratory
of Anatomy, Emory University, Atlanta, Georgia 30332
of Developmental Neurobiology, NICHD,
National Institutes of Health, Bethesda,
Maryland 20892 Received
September
13,
1990
We measured inositol 1,4,5-trisphosphate (InsP3) content of sea urchin gametes by using a specific protein binding assay, and found that a spermatozooncontains 4x10-19to 1x10-18moles of InsPgbefore the acrosomereaction. Since the acrosomereaction haspreviously beenshown to increasethe InsP3content of spermseveralfold, our measurementindicates that a spermatozoon contains at least 2x10-18 molesof InsP3at fertilization, corresponding to a concentration in the spermatozoonof about 1 mM. The thresholdfor activation of eggsby injection of InsPl dissolved in a much larger volume of solution hasbeenfound to be about 3~10-1smoles,correspondingto a concentration in the injectate of 1 PM. This suggeststhat seaurchin spermmay contain enough InsPsto activate eggs.With an electroporation method, we also showedthat spermextract actson eggsonly from inside, consistentwith a primary messengerrole for InsP3. r Ll3ilAcKkmri PreSs,Inc. How spermactivate eggsat fertilization has beenelusive despiteintensive efforts for many yearsi-4. It has been shown that injection of InsPq triggers the activation of eggss-7.It hasalso beenshown that a solublecomponentof spermtriggers activationl. On the basisof theseresults, Whitaker and Irvine5 suggestedthat InsP3 could be the primary messengerthat triggers activation. However, since it has not been determined how much InsPj a spermatozooncarries, it has not beenclear whether InsPgis the primary messengeror whether, asin other cell@, its role is limited to that of a secondmessengerproducedwithin the target celld-6.In order to test the possibility that InsP3 is the primary messenger,we determined how much InsPg a sea urchin spermatozoon carries.We alsoaddressthe questionof how generalthis putative mechanismmight be.
Materials
and Methods
gametes
Gameteswere obtained from Lytechinus variegatus collected in Florida. The seaurchins were openedand eggswere collected with a spatula.They were filtered through a tissuepaper to remove grossdebris. Egg jelly wasremoved by washing with artificial seawater(four times with 10 volumes). Eggs were testedfor their viability before use.If more than 90 5%of the eggstested formed fertilization membrane, the batch of the eggswas usedfor the experiments. Sperm was collected ‘dry’, i. e. the gonadswere washedwith Ca-free seawater and thick spermwhich comes out of the gonadswas collected with a pipette. The number density of the eggsin the suspension was determined with a microscopewith a low power objective (x5). The number density of the spermwas determinedwith a microscopewith a x25 objective. This measurementwas facilitated 0006.29 I X190 $1.50 Copyright 0 1990 by Academic Press. inc. All rights of reproduction in arg form resented.
932
Vol.
172,
No.
2, 1990
by formaldehyde water9.
BIOCHEMICAL
AND
BIOPHYSICAL
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fixation and drying after dilution (40,000-fold
COMMUNICATIONS
for dry sperm) with distilled
InsP3 extraction A 5 ml gamete suspension was prepared for quantifying InsP3. An egg suspension was washed with artificial sea water to remove jelly. A spermatozoa suspension was obtained from either purified sperm obtained by centrifugation in a Ficoll density gradient10 or from whole dry sperm. 5 ml of an aqueous solution of 15 % trichloroacetic acid was added and incubated for 20 minutes on ice. The suspension was then centrifuged at 4000 g for 15 minutes, and the pellet was discarded. Trichloroacetic acid was extracted from the supernatant with diethyl ether (five repetitive extractions with 4 volumes each). The aqueous solution was then adjusted to pH 7.5 with sodium bicarbonate. determination of InsP? content Tritium-labeled InsP3 and InsP3-binding protein were obtained from Amersham (Arlington Heights, IL). The assay has been described by Palmer et al.11 100 ~1 samples were added to a 100 ~1 buffer which contained 4 mM EDTA, 4mg/ml bovine serum albumin, and 0.1 M Tris HCI adjusted to pH 9.0, and then a 100 yl solution of tritium labeled-InsP3 (about 2.5 kBq/ml) was added. Then a 100 ~1 suspension of InsP3 binding protein from bovine adrenal gland was added, and the mixture was centrifuged (4000g for 20 min.) to remove unbound labeled InsP3. The pellet was counted for labeled complex to obtain a relative concentration of InsP3. The above procedure was also performed with known samples of InsPg and the results plotted to obtain a calibration curve (Cf. Fig. 1). Absolute concentrations were obtained by comparing the amount of labelled complex in each sample with this calibration curve. ion exchaneer chromatoeraDhv Mono-Q column (Pharmacia-LKB) was used to fractionate the sperm extract. The low ionic-strength buffer contained 10 mM KC1 and 10 mM potassium phosphate and the high ionicstrength buffer was 1 M KC1 and 10 mM potassium phosphate. Both of the buffers were adjusted to pH. 6.3 with KOH. The column was calibrated with tritium labeled inositol phosphates. electronoration of em Eggs were placed in a chamber equipped with two platinum electrodes (0.02 mm thick, 2 mm wide) with a separation of 8 mm. The chamber had a thin #1 glass slide bottom was topped with a smaller cover glass. These two glass plates were held by strips of Vaseline at two sides. The spacing of the glass plates was about 0.2 mm, and the spacing between two strips of Vaseline about 3 mm. (The resistance between the two electrodes was about 200 Ohms.) The chamber was mounted on a microscope for observation. The eggs were bathed in glycine medium12 (600 mM glycine, 420 mM potassium gluconate, 100 n-&l NaCl, 20 mM MgC12, and 20 mM K-Pipes at pH 7.8 and with osmolality adjusted with mannitol to 1000 mOs/kg) with sperm extract which contained 0.5 PM InsP3. When this medium is introduced internally, it makes the cortical granules of eggs more sensitive to calcium elevation than a medium containing high KC112. To the medium, Indo-l (Molecular Probes, Eugene, OR) was added to a final concentration of 10 PM. Tndo-l worked both as a chelator to lower the free calcium concentration and as a calcium indicator. Nominally Ca-free medium showed 200 nM free Ca2+ concentration. We applied an electrical field by discharging a 1 PF capacitor. The amplitude was regulated by a dc supply (up to 300 V) used to charge the capacitor and the time constant is controlled by a parallel resistor (5 to 100 Ohms). An example is given in Figure 2.
Results InsP,
and Discussion In order to determine InsP3 concentration, we measured the specific binding of labeled to InsPg-binding protein as a function of concentration of cold InsP3 (Fig. 1). The
calibration curve is plotted against the InsP, content in moles, and the sample curve is plotted against the dilution factor in percentage. consistency check on our measurements.
These curves are of the same shape, providing By comparing 933
a
the two curves, we obtained the
Vol.
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No.
2, 1990
BIOCHEMICAL
AND
concentration
dilution
BIOPHYSICAL
RESEARCH
of the standard
factor
of sample
COMMUNICATIONS
(nM)
(%)
Figure 1. InsPg concentration determination by competitive binding to protein. The relative amount of protein-bound labeled InsPs is plotted against the InsPs concentration for the standard. Unfilled circles represent InsPs standard, and filled circles sample. Error bars show standard deviation. The standard is plotted against InsP3 concentration. For the sample, it is plotted against the dilution factor in percent, 100 being undiluted. An estimate of the concentration is obtained at 60 % binding. A 0.5 % dilution of the sample is equivalent to 30 nM InsP3. Thus the sample concentration was 6 PM. This figure represents one of the experiments to determine InsPs content of spermatozoa before acrosome reaction.
concentration
of InsP3 in the sample, 6pM.
of the gametes in the starting suspension, I). In the table the number
Combining we obtained
of experiments
microscopic
examination
undergone
the acrosomal
that the whole,
for whole,
dry sperm and for purified
based on relative after the acrosomal
reaction
reaction
and Garbersls
are listed. We verified
reaction
increases
InsP3, using
column.
Measurement
binding
had not the same
the InsP3 content of sperm was
spermatozoa
incubated
of absolute
assay was difficult
with labeled
concentrations
of InsP3
because of the presence of
our measured value of the absolute concentration increase caused by the acrosome reaction
to obtain the absolute concentration
of InsP3 before the reported
by Domino
after the acrosome reaction.
Table I. Measured InsPs in sea urchin gametes
dry spermatozoa purified spermatozoa -7223
by
sperm, ie 4x 1O- ly - 1x10-18 moles InsP3 per spermatozoon.
by our protein
with the relative
(Table
dry sperm samples used in our measurements
of labeled
ion exchange
egg jelly. We have combined acrosome
the lnsP3 content in single gametes
reaction 10. As shown in Table I, results were approximately
measurements
and an HPLC
with the number density
(n) and standard deviations
The recent report13 that the acrosomal inositol
this concentration
concentration of sample (PM)
density of gametes (number/ml)
6+1 0.05+0.01 0.08f0.02
(1.5+0.1)~10~~ (4+1.3)x10’ (3-+0.2)x104
per gamete (moles)
InsP3
(4f0.8)~10-‘~ (lf0.4)~10-‘~ (3+1)x10-”
average InsPj concentration (FM) 2OOk40 500+200 6f2
n (4) (2) (2)
To calculate average concentrations, we assumed the following volumes for gametes: 2 pm3 (2x10-l5 1) for spermatozoa and 5~10~ pm3 (SX~O-‘~ I) for eggs. 934
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Table II shows that the concentration of InsP3 in spermis three ordersof magnitudehigher than the injectate and that the total amountof InsP3in spermis comparableto the thresholdvalues that activate eggs by injection. If the total amount of InsP3 brought into an egg is the only important factor in activating the egg, the amountof InsPgin a spermatozoonis near the threshold. However, there is evidence that a higher concentrationof InsP3increasesthe efficiency. The total quantity of InsP3 required to activate an egg, about 3~10.~~moless*7is insignificant compared with 3x10-l5 moles already presentin an egg (Cf. Table I). Thus, the parameterthat determines the threshold cannot be the total amount of InsP3 present, but must depend on InsP3 concentrationl4. The effectiveness of sperm InsP3 in activating an egg is also dependenton the way it is transferred to the egg. It would be lessefficient if the transfer takes place slowly. It has been calculated that the time required for spermInsPj to diffuse into the egg is much shorter than the time from the fusion of the plasmamembranesto the onsetof Ca2+ elevationr5. This indicatesthat the transfer rate is high enough to allow most of the sperm InsP? to be effective. Since the spermatozoon carries virtually all of this into the egg during fertilization and since InsP3 concentration seemsto be an important factor in activation, it is likely that the amount of InsP3 presentin a singlespermatozoonis enoughto causeactivation. Evidence that activation occurs from insidethe egg haspreviously beenreportedl. On the other hand, a recent report4 suggestedthat activation is triggered externally. In order to directly addressthe questionasto whether activation by spermoccursinside or outsidethe egg, we placed eggsin a mediumcontaining spermextract, and then permeabilizedthe eggsto allow this medium to enter the cytoplasm. According to fluorescencemeasurement,the free calcium concentration outsidethe egg was 200 nM. No fertilization membraneswere formed around the eggs. Then an exponentially decaying electrical pulsethat correspondsto an initial field strengthof 300 V/cm and time constant 20 ps was applied, and fertilization membraneswere formed (Fig. 2). The electric field makes small holes in the egg membrane, allowing small molecules into the cell16. We observedmore than 20 eggsin one experiment, and we repeatedthe samecondition at leasttwice. Control medium did not contain InsP3or the sperm extract. Only when the medium contained either the sperm extract or standardInsP3 at concentrations above about 100 nM, fertilization membranes were formed. This result shows that activation in our experiments is triggered internally. Although a previous worktt hasshownthat the binding protein we usedis highly specific to InsP3,it cannot be ruled out that someother componentin the extract interactedwith the protein. Table II. Comparison of InsP3 in acrosome-reacted spermatozoon with threshold dose of InsP3 in injection experiments amount(moles) InsP3 in acrosome-reacted spermatozoon threshold dose of InsP3 in injection experiment
concentration@M)
2x10-‘8
1.ooo*
4x10-‘*
1
reference present paper and ref. 13 ref. 5, 7
*This is a round number corresponding to either 5-fold increase of dry spermatozoa value or twofold increase of purified spermatozoa value on sperm activation. 935
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Figure 2. Fertilization membrane raised by electroporation of the egg in the glycine medium containing sperm extract. The free calcium ion concentration is 200 nM. The time course of the electric field strength is exponential with the peak strength 300 V/cm and the relaxation time 20 us. The picture was taken 15 s after the field application. Bar represents 50 urn.
For corroborating that the effective component is InsP3, we fractionated the extract with an anion exchanger column (mono-Q, Pharmacia-LKB)
and tested the eluents for interactions
with the
protein (Fig. 3). The column was first calibrated with tritium labeled Ins (1,4) P2, Ins (1,3,4) P3, and Ins (1,3,4,5) Pd. In Figure 3, broken lines represent elution patterns of these labeled inositol phosphates. The radio-binding method shown in Fig. 1 was used to characterize the effluent of the sample run. In the figure, apparent InsP3 concentration is used to quantify the effluents. Figure 3 shows that the component of the extract that interacts with InsPj-binding protein matches exactly with the sharp peak of InsP3. We also tested the efficiency of these effluents in activating eggs and found that it also agrees with the peak. Thus the component has the elution pattern of InsP3 in FPLC ion exchanger chromatography, strong interaction with InsPj-binding protein, and a high efficiency in activating eggs. These observations are consistent with the hypothesis that the effective component in the extract is indeed InsP3. The InsPg concentration in eggs, which is very much lower than its concentration in sperm (Cf. Table I), does not appear to be unusual compared to other tissues. For example, the InsP3 concentration in brain tissue has been reported to be similar to the 6 PM value we found for 936
Vol. 172, No. 2, 1990
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AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 3. Elutionpatternof spermextractin FPLCanionexchangerchromatography. A pre-packed mono-Q column(Pharmacia-LKB)wasused.Theionic strengthof the eluentis reuresented bv a solidthin line. The scaleof the ionic strengthis shownon thi right. Calibrationswith tritiuk labeledIns (1,4) P2,Ins (1,3,4) P3, andIns (1,3,4,5)P4 are shownin brokenlinesin an arbitrary scale. ApparentInsP3concentrations of thefractionatedsamplearedetermined by a radiobindingassay, asdescribedin Fig. 1, andplotted with a thick solidline. The scalefor InsP3concentrationis shownon theleft.
eggs17,1s.This concentration is higher than the threshold concentrationsof InsP3in the injection experimentsand in our electroporationexperiments. A likely explanationof this result is that InsP3 is not uniformly distributed within the egg, and that its concentrationnear the plasmamembraneis lower than average. While we cannot rule out the possibility that other transmitters are also involved in the fertilization process,our resultsindicate that InsP3may function asthe primary messenger.It is of interest whether or not spermatozoa in other animals have high concentration of TnsP3.Our preliminary result showsthat a goat spermatozooncontains3~10-‘~mole InsP3,an amount similar to that of a seaurchin spermatozoon(seeTable I). This suggeststhat the role of InsP3 as the primary messengerin the activation of eggsmay be quite general. The high concentration of this putative primary messengerin the spermatozoon is reminiscent of the high concentration of transmitter present in secretory vesicles19.20,but this proposed mechanismof communication during fertilization has two unusualfeatures: the molecule proposedas the primary messengeris one that is often usedin other cells asa secondmessengerand it is transmitteddirectly to the inside of the target cell. These features would provide several advantages.One advantage is that the activation processwould be simplified by the elimination of several steps. Another advantageis that carrying the messengerdirectly into the egg avoids dilution into the external medium, In addition, the joint entry into the egg of the primary messengerand the genetic componentof the spermatozoonavoidsthe counterproductiveformation of fertilization membranesin eggsthat might receive the former without the latter. Joint entry of messengerand geneticmaterial alsoinsuresthe synchronization of the variousprocessesinitiated by their entry. References 1 Dale, B. DeFelice, L. J. and Ehrenstein,G. (1985) Experientia 41, 1068-1070. 2 Gould, M. and Stephano, J. L. (1987) Science235, 1654-1656. 937
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3 Longo, F. J., Lynn, J. W., McCulloh, D. H., and Chambers, E. L. (1986) Dev. Biol. 118, 155-166. 4 Kline, D., Simoncini, L., Mandel, G., Maue, R. A., Kado, R. T. and Jaffe, L. A. (1988) Science 241, 464-467. 5 Whitaker, M. and Irvine, R. F. (1984) Nature 312, 638-639 6 Turner, P. R., Jaffe, L. A., and Fain, J. J. Cell Biol. 102, 70-76 (1986) 7 Crossley, I., Swann, K., Chambers, E., and Whitaker, M. (1988) Biochem. J. 252, 257262 Berridge,M. J., and Irvine, R. F. (1984) Nature 312, 315 - 321 Brokaw, C. J. (1986) In “Methods in Cell Biology” (T. E. Schroeder, Ed.) Vol. 27, pp. 4156. Academic Press, San Diego, CA. 10 Vacquier, V. D. (1986) In “Methods in Cell Biology” (T. E. Schroeder, Ed.) Vol. 27, pp. 1540. Academic Press, San Diego, CA. 11 Palmer, S., Hughes, K. T., Lee, D. Y., and Wakelam, M. J. 0. (1989) Cellular Signalling 1, 147-156. 12 Sasaki, H. (1984) Dev. Biol. 101, 125-135. 13 Domino S. E. and Garbers, D. L. (1988) J. Biol. Chem. 263, 690-695 14 Ehrenstein G. and FitzHugh, R. (1986) In “Ionic Channels in Cells and Model Systems,” (R. Latorre, Ed.), pp. 421-430, Plenum, New York. 15 Whitaker, M., Swann, K., and Crossley, I. (1989) In “Mechanisms of Egg Fertilization” (R. Nuccitelli, G. N. Cherr, and W. H. Clark, Ed.), pp. 157-171. Plenum, New York, NY. 16 Zimmermann, U. and Urnovitz, H. B. (1987) Methods in Enzymol. 151, 195 - 221 17 Chaliss, R. A., Batty, I. H., and Nahorski, S. R. (1988) Biochem. Biophys. Res. Commun. 157, 684-69 1 18 Bredt, D. S., Mourey. R. J., and Snyder, S. H. (1989) Biochem. Biophys. Res. Commun. 159, 976-982 19 Winkler H. and Westhead, E. W. (1980) Neurosci. 5, 1803-1823 20 Russell, J. T. (1987) Current Topics in Membrane and Transport 31, 277-312.
938
172,
No.
October
2, 1990
30,
BIOCHEMICAL
AND
BIOPHYSICAL
1990
RESEARCH
COMMUNICATIONS Pages
High Concentration of Inositol 1,4,5trisphosphate Kuni H. Iwasa*, Gerald Ehrenstein*, Louis J. DeFelice**,
932-938
in Sea Urchin Sperm and James T. Russell***
*Laboratory of Biophysics, NINDS, National Institutes of Health, Maryland 20892 **Department ***Laboratory
of Anatomy, Emory University, Atlanta, Georgia 30332
of Developmental Neurobiology, NICHD,
National Institutes of Health, Bethesda,
Maryland 20892 Received
September
13,
1990
We measured inositol 1,4,5-trisphosphate (InsP3) content of sea urchin gametes by using a specific protein binding assay, and found that a spermatozooncontains 4x10-19to 1x10-18moles of InsPgbefore the acrosomereaction. Since the acrosomereaction haspreviously beenshown to increasethe InsP3content of spermseveralfold, our measurementindicates that a spermatozoon contains at least 2x10-18 molesof InsP3at fertilization, corresponding to a concentration in the spermatozoonof about 1 mM. The thresholdfor activation of eggsby injection of InsPl dissolved in a much larger volume of solution hasbeenfound to be about 3~10-1smoles,correspondingto a concentration in the injectate of 1 PM. This suggeststhat seaurchin spermmay contain enough InsPsto activate eggs.With an electroporation method, we also showedthat spermextract actson eggsonly from inside, consistentwith a primary messengerrole for InsP3. r Ll3ilAcKkmri PreSs,Inc. How spermactivate eggsat fertilization has beenelusive despiteintensive efforts for many yearsi-4. It has been shown that injection of InsPq triggers the activation of eggss-7.It hasalso beenshown that a solublecomponentof spermtriggers activationl. On the basisof theseresults, Whitaker and Irvine5 suggestedthat InsP3 could be the primary messengerthat triggers activation. However, since it has not been determined how much InsPj a spermatozooncarries, it has not beenclear whether InsPgis the primary messengeror whether, asin other cell@, its role is limited to that of a secondmessengerproducedwithin the target celld-6.In order to test the possibility that InsP3 is the primary messenger,we determined how much InsPg a sea urchin spermatozoon carries.We alsoaddressthe questionof how generalthis putative mechanismmight be.
Materials
and Methods
gametes
Gameteswere obtained from Lytechinus variegatus collected in Florida. The seaurchins were openedand eggswere collected with a spatula.They were filtered through a tissuepaper to remove grossdebris. Egg jelly wasremoved by washing with artificial seawater(four times with 10 volumes). Eggs were testedfor their viability before use.If more than 90 5%of the eggstested formed fertilization membrane, the batch of the eggswas usedfor the experiments. Sperm was collected ‘dry’, i. e. the gonadswere washedwith Ca-free seawater and thick spermwhich comes out of the gonadswas collected with a pipette. The number density of the eggsin the suspension was determined with a microscopewith a low power objective (x5). The number density of the spermwas determinedwith a microscopewith a x25 objective. This measurementwas facilitated 0006.29 I X190 $1.50 Copyright 0 1990 by Academic Press. inc. All rights of reproduction in arg form resented.
932
Vol.
172,
No.
2, 1990
by formaldehyde water9.
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
fixation and drying after dilution (40,000-fold
COMMUNICATIONS
for dry sperm) with distilled
InsP3 extraction A 5 ml gamete suspension was prepared for quantifying InsP3. An egg suspension was washed with artificial sea water to remove jelly. A spermatozoa suspension was obtained from either purified sperm obtained by centrifugation in a Ficoll density gradient10 or from whole dry sperm. 5 ml of an aqueous solution of 15 % trichloroacetic acid was added and incubated for 20 minutes on ice. The suspension was then centrifuged at 4000 g for 15 minutes, and the pellet was discarded. Trichloroacetic acid was extracted from the supernatant with diethyl ether (five repetitive extractions with 4 volumes each). The aqueous solution was then adjusted to pH 7.5 with sodium bicarbonate. determination of InsP? content Tritium-labeled InsP3 and InsP3-binding protein were obtained from Amersham (Arlington Heights, IL). The assay has been described by Palmer et al.11 100 ~1 samples were added to a 100 ~1 buffer which contained 4 mM EDTA, 4mg/ml bovine serum albumin, and 0.1 M Tris HCI adjusted to pH 9.0, and then a 100 yl solution of tritium labeled-InsP3 (about 2.5 kBq/ml) was added. Then a 100 ~1 suspension of InsP3 binding protein from bovine adrenal gland was added, and the mixture was centrifuged (4000g for 20 min.) to remove unbound labeled InsP3. The pellet was counted for labeled complex to obtain a relative concentration of InsP3. The above procedure was also performed with known samples of InsPg and the results plotted to obtain a calibration curve (Cf. Fig. 1). Absolute concentrations were obtained by comparing the amount of labelled complex in each sample with this calibration curve. ion exchaneer chromatoeraDhv Mono-Q column (Pharmacia-LKB) was used to fractionate the sperm extract. The low ionic-strength buffer contained 10 mM KC1 and 10 mM potassium phosphate and the high ionicstrength buffer was 1 M KC1 and 10 mM potassium phosphate. Both of the buffers were adjusted to pH. 6.3 with KOH. The column was calibrated with tritium labeled inositol phosphates. electronoration of em Eggs were placed in a chamber equipped with two platinum electrodes (0.02 mm thick, 2 mm wide) with a separation of 8 mm. The chamber had a thin #1 glass slide bottom was topped with a smaller cover glass. These two glass plates were held by strips of Vaseline at two sides. The spacing of the glass plates was about 0.2 mm, and the spacing between two strips of Vaseline about 3 mm. (The resistance between the two electrodes was about 200 Ohms.) The chamber was mounted on a microscope for observation. The eggs were bathed in glycine medium12 (600 mM glycine, 420 mM potassium gluconate, 100 n-&l NaCl, 20 mM MgC12, and 20 mM K-Pipes at pH 7.8 and with osmolality adjusted with mannitol to 1000 mOs/kg) with sperm extract which contained 0.5 PM InsP3. When this medium is introduced internally, it makes the cortical granules of eggs more sensitive to calcium elevation than a medium containing high KC112. To the medium, Indo-l (Molecular Probes, Eugene, OR) was added to a final concentration of 10 PM. Tndo-l worked both as a chelator to lower the free calcium concentration and as a calcium indicator. Nominally Ca-free medium showed 200 nM free Ca2+ concentration. We applied an electrical field by discharging a 1 PF capacitor. The amplitude was regulated by a dc supply (up to 300 V) used to charge the capacitor and the time constant is controlled by a parallel resistor (5 to 100 Ohms). An example is given in Figure 2.
Results InsP,
and Discussion In order to determine InsP3 concentration, we measured the specific binding of labeled to InsPg-binding protein as a function of concentration of cold InsP3 (Fig. 1). The
calibration curve is plotted against the InsP, content in moles, and the sample curve is plotted against the dilution factor in percentage. consistency check on our measurements.
These curves are of the same shape, providing By comparing 933
a
the two curves, we obtained the
Vol.
172,
No.
2, 1990
BIOCHEMICAL
AND
concentration
dilution
BIOPHYSICAL
RESEARCH
of the standard
factor
of sample
COMMUNICATIONS
(nM)
(%)
Figure 1. InsPg concentration determination by competitive binding to protein. The relative amount of protein-bound labeled InsPs is plotted against the InsPs concentration for the standard. Unfilled circles represent InsPs standard, and filled circles sample. Error bars show standard deviation. The standard is plotted against InsP3 concentration. For the sample, it is plotted against the dilution factor in percent, 100 being undiluted. An estimate of the concentration is obtained at 60 % binding. A 0.5 % dilution of the sample is equivalent to 30 nM InsP3. Thus the sample concentration was 6 PM. This figure represents one of the experiments to determine InsPs content of spermatozoa before acrosome reaction.
concentration
of InsP3 in the sample, 6pM.
of the gametes in the starting suspension, I). In the table the number
Combining we obtained
of experiments
microscopic
examination
undergone
the acrosomal
that the whole,
for whole,
dry sperm and for purified
based on relative after the acrosomal
reaction
reaction
and Garbersls
are listed. We verified
reaction
increases
InsP3, using
column.
Measurement
binding
had not the same
the InsP3 content of sperm was
spermatozoa
incubated
of absolute
assay was difficult
with labeled
concentrations
of InsP3
because of the presence of
our measured value of the absolute concentration increase caused by the acrosome reaction
to obtain the absolute concentration
of InsP3 before the reported
by Domino
after the acrosome reaction.
Table I. Measured InsPs in sea urchin gametes
dry spermatozoa purified spermatozoa -7223
by
sperm, ie 4x 1O- ly - 1x10-18 moles InsP3 per spermatozoon.
by our protein
with the relative
(Table
dry sperm samples used in our measurements
of labeled
ion exchange
egg jelly. We have combined acrosome
the lnsP3 content in single gametes
reaction 10. As shown in Table I, results were approximately
measurements
and an HPLC
with the number density
(n) and standard deviations
The recent report13 that the acrosomal inositol
this concentration
concentration of sample (PM)
density of gametes (number/ml)
6+1 0.05+0.01 0.08f0.02
(1.5+0.1)~10~~ (4+1.3)x10’ (3-+0.2)x104
per gamete (moles)
InsP3
(4f0.8)~10-‘~ (lf0.4)~10-‘~ (3+1)x10-”
average InsPj concentration (FM) 2OOk40 500+200 6f2
n (4) (2) (2)
To calculate average concentrations, we assumed the following volumes for gametes: 2 pm3 (2x10-l5 1) for spermatozoa and 5~10~ pm3 (SX~O-‘~ I) for eggs. 934
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Table II shows that the concentration of InsP3 in spermis three ordersof magnitudehigher than the injectate and that the total amountof InsP3in spermis comparableto the thresholdvalues that activate eggs by injection. If the total amount of InsP3 brought into an egg is the only important factor in activating the egg, the amountof InsPgin a spermatozoonis near the threshold. However, there is evidence that a higher concentrationof InsP3increasesthe efficiency. The total quantity of InsP3 required to activate an egg, about 3~10.~~moless*7is insignificant compared with 3x10-l5 moles already presentin an egg (Cf. Table I). Thus, the parameterthat determines the threshold cannot be the total amount of InsP3 present, but must depend on InsP3 concentrationl4. The effectiveness of sperm InsP3 in activating an egg is also dependenton the way it is transferred to the egg. It would be lessefficient if the transfer takes place slowly. It has been calculated that the time required for spermInsPj to diffuse into the egg is much shorter than the time from the fusion of the plasmamembranesto the onsetof Ca2+ elevationr5. This indicatesthat the transfer rate is high enough to allow most of the sperm InsP? to be effective. Since the spermatozoon carries virtually all of this into the egg during fertilization and since InsP3 concentration seemsto be an important factor in activation, it is likely that the amount of InsP3 presentin a singlespermatozoonis enoughto causeactivation. Evidence that activation occurs from insidethe egg haspreviously beenreportedl. On the other hand, a recent report4 suggestedthat activation is triggered externally. In order to directly addressthe questionasto whether activation by spermoccursinside or outsidethe egg, we placed eggsin a mediumcontaining spermextract, and then permeabilizedthe eggsto allow this medium to enter the cytoplasm. According to fluorescencemeasurement,the free calcium concentration outsidethe egg was 200 nM. No fertilization membraneswere formed around the eggs. Then an exponentially decaying electrical pulsethat correspondsto an initial field strengthof 300 V/cm and time constant 20 ps was applied, and fertilization membraneswere formed (Fig. 2). The electric field makes small holes in the egg membrane, allowing small molecules into the cell16. We observedmore than 20 eggsin one experiment, and we repeatedthe samecondition at leasttwice. Control medium did not contain InsP3or the sperm extract. Only when the medium contained either the sperm extract or standardInsP3 at concentrations above about 100 nM, fertilization membranes were formed. This result shows that activation in our experiments is triggered internally. Although a previous worktt hasshownthat the binding protein we usedis highly specific to InsP3,it cannot be ruled out that someother componentin the extract interactedwith the protein. Table II. Comparison of InsP3 in acrosome-reacted spermatozoon with threshold dose of InsP3 in injection experiments amount(moles) InsP3 in acrosome-reacted spermatozoon threshold dose of InsP3 in injection experiment
concentration@M)
2x10-‘8
1.ooo*
4x10-‘*
1
reference present paper and ref. 13 ref. 5, 7
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Figure 2. Fertilization membrane raised by electroporation of the egg in the glycine medium containing sperm extract. The free calcium ion concentration is 200 nM. The time course of the electric field strength is exponential with the peak strength 300 V/cm and the relaxation time 20 us. The picture was taken 15 s after the field application. Bar represents 50 urn.
For corroborating that the effective component is InsP3, we fractionated the extract with an anion exchanger column (mono-Q, Pharmacia-LKB)
and tested the eluents for interactions
with the
protein (Fig. 3). The column was first calibrated with tritium labeled Ins (1,4) P2, Ins (1,3,4) P3, and Ins (1,3,4,5) Pd. In Figure 3, broken lines represent elution patterns of these labeled inositol phosphates. The radio-binding method shown in Fig. 1 was used to characterize the effluent of the sample run. In the figure, apparent InsP3 concentration is used to quantify the effluents. Figure 3 shows that the component of the extract that interacts with InsPj-binding protein matches exactly with the sharp peak of InsP3. We also tested the efficiency of these effluents in activating eggs and found that it also agrees with the peak. Thus the component has the elution pattern of InsP3 in FPLC ion exchanger chromatography, strong interaction with InsPj-binding protein, and a high efficiency in activating eggs. These observations are consistent with the hypothesis that the effective component in the extract is indeed InsP3. The InsPg concentration in eggs, which is very much lower than its concentration in sperm (Cf. Table I), does not appear to be unusual compared to other tissues. For example, the InsP3 concentration in brain tissue has been reported to be similar to the 6 PM value we found for 936
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Figure 3. Elutionpatternof spermextractin FPLCanionexchangerchromatography. A pre-packed mono-Q column(Pharmacia-LKB)wasused.Theionic strengthof the eluentis reuresented bv a solidthin line. The scaleof the ionic strengthis shownon thi right. Calibrationswith tritiuk labeledIns (1,4) P2,Ins (1,3,4) P3, andIns (1,3,4,5)P4 are shownin brokenlinesin an arbitrary scale. ApparentInsP3concentrations of thefractionatedsamplearedetermined by a radiobindingassay, asdescribedin Fig. 1, andplotted with a thick solidline. The scalefor InsP3concentrationis shownon theleft.
eggs17,1s.This concentration is higher than the threshold concentrationsof InsP3in the injection experimentsand in our electroporationexperiments. A likely explanationof this result is that InsP3 is not uniformly distributed within the egg, and that its concentrationnear the plasmamembraneis lower than average. While we cannot rule out the possibility that other transmitters are also involved in the fertilization process,our resultsindicate that InsP3may function asthe primary messenger.It is of interest whether or not spermatozoa in other animals have high concentration of TnsP3.Our preliminary result showsthat a goat spermatozooncontains3~10-‘~mole InsP3,an amount similar to that of a seaurchin spermatozoon(seeTable I). This suggeststhat the role of InsP3 as the primary messengerin the activation of eggsmay be quite general. The high concentration of this putative primary messengerin the spermatozoon is reminiscent of the high concentration of transmitter present in secretory vesicles19.20,but this proposed mechanismof communication during fertilization has two unusualfeatures: the molecule proposedas the primary messengeris one that is often usedin other cells asa secondmessengerand it is transmitteddirectly to the inside of the target cell. These features would provide several advantages.One advantage is that the activation processwould be simplified by the elimination of several steps. Another advantageis that carrying the messengerdirectly into the egg avoids dilution into the external medium, In addition, the joint entry into the egg of the primary messengerand the genetic componentof the spermatozoonavoidsthe counterproductiveformation of fertilization membranesin eggsthat might receive the former without the latter. Joint entry of messengerand geneticmaterial alsoinsuresthe synchronization of the variousprocessesinitiated by their entry. References 1 Dale, B. DeFelice, L. J. and Ehrenstein,G. (1985) Experientia 41, 1068-1070. 2 Gould, M. and Stephano, J. L. (1987) Science235, 1654-1656. 937
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3 Longo, F. J., Lynn, J. W., McCulloh, D. H., and Chambers, E. L. (1986) Dev. Biol. 118, 155-166. 4 Kline, D., Simoncini, L., Mandel, G., Maue, R. A., Kado, R. T. and Jaffe, L. A. (1988) Science 241, 464-467. 5 Whitaker, M. and Irvine, R. F. (1984) Nature 312, 638-639 6 Turner, P. R., Jaffe, L. A., and Fain, J. J. Cell Biol. 102, 70-76 (1986) 7 Crossley, I., Swann, K., Chambers, E., and Whitaker, M. (1988) Biochem. J. 252, 257262 Berridge,M. J., and Irvine, R. F. (1984) Nature 312, 315 - 321 Brokaw, C. J. (1986) In “Methods in Cell Biology” (T. E. Schroeder, Ed.) Vol. 27, pp. 4156. Academic Press, San Diego, CA. 10 Vacquier, V. D. (1986) In “Methods in Cell Biology” (T. E. Schroeder, Ed.) Vol. 27, pp. 1540. Academic Press, San Diego, CA. 11 Palmer, S., Hughes, K. T., Lee, D. Y., and Wakelam, M. J. 0. (1989) Cellular Signalling 1, 147-156. 12 Sasaki, H. (1984) Dev. Biol. 101, 125-135. 13 Domino S. E. and Garbers, D. L. (1988) J. Biol. Chem. 263, 690-695 14 Ehrenstein G. and FitzHugh, R. (1986) In “Ionic Channels in Cells and Model Systems,” (R. Latorre, Ed.), pp. 421-430, Plenum, New York. 15 Whitaker, M., Swann, K., and Crossley, I. (1989) In “Mechanisms of Egg Fertilization” (R. Nuccitelli, G. N. Cherr, and W. H. Clark, Ed.), pp. 157-171. Plenum, New York, NY. 16 Zimmermann, U. and Urnovitz, H. B. (1987) Methods in Enzymol. 151, 195 - 221 17 Chaliss, R. A., Batty, I. H., and Nahorski, S. R. (1988) Biochem. Biophys. Res. Commun. 157, 684-69 1 18 Bredt, D. S., Mourey. R. J., and Snyder, S. H. (1989) Biochem. Biophys. Res. Commun. 159, 976-982 19 Winkler H. and Westhead, E. W. (1980) Neurosci. 5, 1803-1823 20 Russell, J. T. (1987) Current Topics in Membrane and Transport 31, 277-312.
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