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Calcium influx following fertilization of Urechis caupo eggs
DEVELOPMENTAL
BIOLOGY
Calcium
57, 364-374 (1977)
Influx following
N. JOHNSTON’
RANDAL Department
of Biology,
University
Received August
Fertilization
of Victoria,
13,1976;
AND
Victoria,
of Urechis MILES British
caupo
Eggs
PAUL Columbia,
accepted in revised form January
Canada
V8W 2Y2
26,1977
Measurements of %a flux into and out of Urechis eggs indicate that, during the first 10 min alter insemination, the eggs take up 0.24 pmole of Ca/egg. Total egg Ca measured by atomic absorption (AA) spectroscopy increased by 0.23 pmole of Ca/egg (0.56, 0.79, and 0.76 pmole of Ca/egg for unfertilized, lo-min fertilized, and 60-min fertilized eggs, respectively). Thus, the total change in egg Ca is accounted for by the influx even though the rate of efflux, measured as a release of 4sCa from preloaded eggs, increases to twice the unfertilized rate by 15 min. The fertilization influx follows saturation kinetics (K, = 1.3 m&f). It is competitively inhibited by procaine, but is not inhibited by dinitrophenol, mersalyl acid, or ruthenium red. Ten percent of the total Ca influx has occurred by 10 set, and it is, therefore, the most rapid response to fertilization yet known in these eggs. The influx is also observed in eggs partially activated by insemination in pH 7 seawater (SW); the other fertilization responses, except sperm penetration, do not occur in pH 7 SW. Although Ca influx alone is insufficient to activate the eggs, it may be a prerequisite for cytoplasmic activation and development, inducing other secondary responses which are prevented by low external pH. INTRODUCTION
Intracellular Ca is recognized as an important regulator of cellular processes and changes in that it can act as a “trigger” in changing cell behaviour and function. Studies of fertilization of sea urchin eggs have shown an increase in the availibility of intracellular Ca upon activation of the egg (14, 17,331. Uptake of external Ca also occurs in the first few minutes after insemination (1,2, 18). Calcium may initiate the discharge of cortical granules (36) and may be an activating agent for other early changes in sea urchin eggs at fertilization [for example, activation of enzymes such as NAD+ kinase (EC 2.7.1.23) (5) or glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (lo)]. Calcium currents across egg membranes have been demonstrated in tunicate (16, 20) and starfish eggs (15, 30) in response to imposed depolarizations and may also occur at fertilization (30). Also, many kinds ’ Present address: Department of Biological Sciences, Stanford University, Stanford, California 94305.
of eggs can be artificially activated with the Ca ionophore A23187 [sea urchin (33); starfish, limpet, tunicate, toad, hamster (34); surf clam (29); Urechis (2411. Thus, changes in the distribution or availibility of intracellular Ca may be important in the events associated with the activation of animal eggs in general. Since Ca may have an important role in the activation of eggs of the echiuroid worm Urechis caupo, we have investigated the possibility that changes in Ca may occur in these eggs following insemination. Some of the responses to insemination of Urechis eggs are similar to those of sea urchin eggs. Both sea urchin and Urethis eggs release a fertilization acid (24) and elevate a surface coat, although surface coat elevation occurs later and more slowly in Urechis [see Ref. (611. However, in several important respects, the responses of Urechis eggs contrast with those of sea urchin eggs. A cortical reaction like that occurring in sea urchin eggs (extensive cortical granule exocytosis resulting in elevation of the surface coat and 364
Copyright 0 1977 by Academic Press, Inc. All rights
of reproduction
in any form reserved.
ISSN
0012-1606
JOHNSTON AND PAUL
formation of the hyaline layer) does not take place in Urechis eggs (8,241. Thus, in Urechis any “calcium response” would be unrelated to the complicated events of the cortical reaction as it occurs in sea urchins. A second major difference is that Urechis eggs are fertilized as primary oocytes; germinal vesicle breakdown and the meiotic divisions follow insemination (in contrast, sea urchin eggs are fertilized as ova, after meiotic maturation). Also, there is a major change in shape of the Urechis egg as the prominent indentation of the primary oocyte rounds out in the first 4 min after insemination. As in sea urchins, a fertilization acid is released (241, and the respiratory rate increases (221, but NAD+ kinase is apparently not activated.2 A study of a “calcium response” in Urechis eggs may provide insights into the role of Ca in sea urchin and other eggs as well as in Urechis eggs themselves. We have found that a large net influx of Ca, detectable by 10 set, occurs during the first 10 min after insemination of Urechis eggs. This influx begins prior to the other known structural and physiological responses of the egg, including fertilization acid release (the most rapid of these), and can occur in their absence. Therefore, it is not itself induced by any of these changes. So far, we have no clues to the specific role of this large increase in Ca, but our results show that it is a primary event in the activation of Urechis eggs. MATERIALS
AND
METHODS
Handling of worms and gametes. Urethis caupo, obtained from Elk Horn
Slough, Moss Landing, California, were maintained in recirculating seawater (SW) instant ocean aquaria in glass tubes at lo-15°C. Eggs and sperm were obtained by the method of Newby (6) on the day of use. Undiluted semen was stored at 3°C and was diluted with SW just prior to inseminations. Eggs were kept at 16°C. Experiments were performed with 2-4% (v/v) 2 M. Paul, unpublished
observations.
Calcium
Influx
365
egg suspensions. To determine egg concentration, we obtained “packed egg” volumes by centrifuging aliquots of egg suspension in a calibrated Shevky-Stafford tube (Arthur H. Thomas Company, Philadelphia, Pa.) in a Janetzki T5 table centrifuge (Jena Instruments Ltd., Surrey, British Columbia, Canada) at setting “2” (11OOg) for 3 min. A 1.0% egg suspension contained 8.0 x 104eggs/ml by direct count of eggs in dilutions of the egg suspensions. Egg batches in which there was less than 95% normal activation in test inseminations were not used. We refer to the fertilizable primary oocyte and zygote as “unfertilized egg” and “fertilized egg,” respecWe examined morphological tively. changes of eggs activated in low Ca SW by the acetic acid-ethanol procedure (23). General experimental procedure. Eggs were washed into fresh SW just before use, suspended at desired concentrations, and incubated in a water-jacketed Silicladtreated cylindrical glass container at 16°C. During experiments, the egg suspensions were stirred with a magnetic stirring bar. To determine whether they take up Ca from the external medium, we incubated eggs in SW or artificial SW (ASW) containing 45Ca and subsequently measured the radioactivity in the eggs. Isotope was added to egg suspensions at least 5 min prior to sampling; radioactivity was 0.611.17 &i/ml in various experiments except as noted (preloading incubation; experiment, Fig. 3). Eggs were activated either with a freshly prepared sperm suspension or by addition to the egg suspension of an equal volume of a 0.2% (w/v) solution of trypsin in SW or ASW; trypsin is an artificial activator of Urechis eggs (22). Times relative to insemination or activation (T,) are indicated as T, (n = the time in minutes) unless otherwise indicated (i.e., Fig. 2). Sperm penetration was determined by observation of eggs fixed in acetic acidethanol as previously described (23). ASW and SW containing added agents were always adjusted to pH 7 or 8 as appropriate.
366
DEVELOPMENTALBIOLOGY
Determination of radioactivity. Duplicate samples of egg suspensions (0.5 ml) were taken with a Siliclad-treated Pasteur pipet on a Cornwall Pipettor (Becton, Dickinson and Co., Rutherford, N.J.), diluted by addition to 15 ml of chilled SW exactly at the indicated times and washed three times with 15 ml of chilled SW by “hand centrifugation” and aspiration of the supernatant SW to less than 0.1 ml between washings. Washed samples (the volume of eggs plus residual wash medium was approximately 50 ~1) were transferred to 1.5dram plastic-screw-cap glass vials (Fisher Scientific Co., Vancouver, British Columbia, Canada) and were digested overnight in 0.5 ml of Protosol at 55°C. Five milliliters of Omnifluor-toluene scintillation fluid was added to each vial. These small vials were placed within standard 20-ml “glass LSC vials” (New England Nuclear, Dorval, Quebec, Canada) which served as adaptors for counting. Fifty-microliter aliquots of “SW-medium” from egg suspensions were processed similarly to the washed egg samples. All results are corrected for background radioactivity. Calcium influx was calculated with the following formula: counts per minute per sample of eggs X moles of Ca per milliliter of SW medium over eggs per sample x counts per minute per milliliter of SW medium = moles of Ca per egg. For our calculations of the fertilization influx, we have used the net changes in counts per minute from 0 to 10 min after insemination, neglecting any possible contribution from the equilibrium influx of Ca, since we do not know whether it continues during this period. Atomic absorption spectroscopy. Egg samples were each washed four times in ice-cold 0.55 M KCl, dried at 95”C, and digested overnight with 1.0 ml of concentrated HCl. Each sample was then centrifuged in a Janetzki T5 table centrifuge (setting 2) for 10 min, and the supernatant was collected. The remaining pellet was washed with three additional 0.5-ml ali-
VOLUME 57, 1977
quots of HCl, and all of the supernatants were combined in a 5-ml volumetric flask. To each flask was added 0.5 ml of 2% (w/v) EDTA and 0.5 ml of 4% (w/v) SrCl, [these agents minimize interference from other substances in AA determinations of Ca (26)l. Finally, each solution was diluted to 5 ml with doubly distilled water, and the absorbance was determined at 422.7 nm on a Techtron A-A spectrophotometer. Blank samples were prepared in a similar manner, but contained no eggs. Interferences which were not eliminated by this procedure were corrected for, using the method of standard additions (32). Materials. Omnifluor and Protosol were obtained from New England Nuclear, Dorval, Quebec, Canada; trypsin (bovine, pancreatic; lyophilized, 2 x crystallized) from Worthington Biochemical Corp., Freehold, N.J.; procaine, ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis-(@aminoethyl ether)-2\rfl’-tetraacetic acid (EGTA), and mersalyl acid from Sigma Chemical Co., St. Louis, MO.; 2,4dinitrophenol (DNP) from Fisher Scientific Co., Fair Lawn, N.J.; and ruthenium red from K. and K. Laboratories, Plainview, N.Y. Isotope was obtained from Amersham, Oakville, Ontario, Canada (45CaC1,in water, CES.2, Batch 13F) and from New England Nuclear (45CaC1, in water, NEZ-013, Lot S1091). Seawater was collected at a depth of 30 m from the Strait of Georgia, filtered through two layers of Whatman No. 1 filter paper, and stored at 3°C until use. Art&al seawater was prepared from modifications of the MBL Formula (4), with Na+ substitued for Ca2+ in the appropriate molar ratio. RESULTS
Calcium
Influx
Unfertilized Urechis eggs take up Ca at a constant rate which we have observed for 1 hr, this component of “uptake” is shown in Fig. 1 prior to insemination at T,. The rate of Ca uptake during this period is 1.1 f 0.5 fmole/egg *min. We assume that this
JOHNSTON AND PAUL
367 TABLE
0.3
3 k + Q23 sg O.l0, E
Calcium Influx 1
INFLUX OF CALCIUM FOLLOWING ACTIVATION OF URECHIS EGGS Condition
Ca uutake (pmoilegg)
of activation
0.24 (N 0.23 0.25 (N 0.22
pH 8 Sperm Trypsin pH 7 Sperm
* t
I
1
-15
0
15
30
Minutes
FIG. 1. Calcium uptake by Urechis eggs before and after insemination at T,. *%a added to egg suspension in SW at T-,,.
is an “equilibrium influx” since unfertilized eggs remain fertilizable for prolonged periods (7) and are presumably in an equilibrium condition with respect to Ca content and fluxes. There is a brief phase of rapid association of isotope with the eggs immediately after the addition of isotope to the SW. This initial phase of uptake is revealed by the failure of isotope associated with the eggs to extrapolate to 0 at the time of addition. This apparently represents the equilibration of isotope into a pool of Ca which is rapidly exchanging with the external SW, and may be Ca bound to the egg surface or surface coat [cf., Refs. (3, 28)l. Insemination induces a large uptake of isotope by the eggs (Fig. 1) which is complete by 10 min (16’C). We refer to this as the “Ca influx.” Calculations indicate an uptake of 0.24 + 0.03 pmole of Ca/egg during this period (average rate, 24 fmole/ egg- min). To define the beginning of the increased Ca uptake more accurately, in two experiments we took samples at lo-set intervals after insemination. Of the total uptake, approximately 10% had occurred by 10 set and 25% by 40 sec. Trypsin also initiates an uptake of Ca by the eggs indistinguishable from that induced by insemination (Table 1). This observation eliminates the possibility that the uptake of Ca is an artifact of the activation of the eggs with sperm (for example, due to the penetration of eggs by
Trypsin pH 7 Second influx Time (min)” 60 100 160
+ 0.03 = 8) f 0.12 = 4)
0.11 (N = 2) 0.18 0.22 Percentage control (o/o)
Mersalyl acid (20 mM, T,P DNP (0.25 mM, T-&’ Procaine (0.05 mM; T-,,,I* Procaine (10 mM; T-,,J* Ruthenium red (0.2 @If; T-,,)** c Ruthenium red (4 &f; T_,,)*, c 0 Time after the first partial b Time added. c Concentration determined Luft (1971).
of
93 93 79 0 83 242 (N = 2)
activation. by the method
of
sperm-carrying-bound isotope or due to a possible increased permeability of the egg in the region of the penetration cone). In an effort to distinguish whether the Ca influx might represent a superficial binding of ion (for example, to the surface or surface coat of the egg) or is a true flux into the cell, we attempted to remove &Ca from eggs fertilized in SW containing isotope by washing them in either SW or Cafree SW containing 5 mM EGTA (Fig. 2). Although the radioactivity of the “washing media” increased over 30 min, corresponding to a loss of 8-10% of the original radioactivity of the eggs, the Ca-free SW with EGTA was no more efficient in removing isotope from the eggs than was SW. In another experiment, we transferred eggs which had been inseminated 15 min earlier into SW containing isotope and measured the rate of 45Cainflux between
368
DEVELOPMENTAL BIOLOGY
VOLUME 57. 197'7
I -10
0
10
20
30
MlmJkl
FIG. 2. Release of isotope from fertilized eggs in different washing media. Eggs were fertilized in SW containing “Va. After 15 min, two 5.0-ml samples of the egg suspension were rapidly washed three times in SW and were resuspended to the original volume in either SW (0) or in Ca-free SW containing 5 mM EGTA (A). T, is the time of the final suspension of the eggs in the washing medium. Samples of the eggs in wash media were taken at 5-min intervals and were centrifuged, and the supernatant medium was removed. The radioactivities of triplicate O.l-ml aliquots of each supernatant were determined. The loss of isotope from the eggs is expressed as the percentage of the original radioactivity in the eggs released into the washing medium. In this experiment, the initial uptake of radioactivity was 0.045 cpmlegg (6700 cpm/original 5-ml sample).
20 and 50 min after insemination. These eggs continued to take up Ca at a rate comparable to that of the unfertilized eggs (1.1 fmole/egg *min, unfertilized; 1.O fmole/egg *min, fertilized). Calcium
EffTux
If there were a large increase in Ca efflux accompanying the increased influx just shown, there might be no change in total cellular calcium. To examine this possibility, we determined the efflux of Va from preloaded eggs, both before and after fertilization. Unfertilized eggs released radioactivity at a rate of 0.34 & 0.09% (N = 2) of the total in the eggs per min. Within 1 min after insemination, the rate of efflux begins to increase, doubles within 15 min, and returns to the prefertilization level by 45 min (Fig. 3a, b). Thus, the time courses of the fertilization influx and efflux are different. We “integrated” the function eMux versus time (Fig. 3b) by weighing paper “cutouts” and thus deter-
-iO
-1;
b
1;
3-O
4;
Minuter
FIG. 3. EMux of *Va from preloaded eggs. (a) Release of isotope into-SW from fertilized and unfertilized eggs. 45Ca was added to an egg suspension (20%, v/v) in ASW containing 0.1 mM Ca (30 &i/ ml). After 4 hr (TV,,), the eggs were washed three times in SW. The eggs were washed again after 20 min, resuspended in SW, and divided in half, and one-half was fertilized. At the indicated times, samples of each egg suspension were centrifuged, and the radioactivities of triplicate O.l-ml aliquots of each supernatant were determined. Fertilized (A); unfertilized (0). (b) Rate of release of isotope from preloaded eggs. In a second experiment, the procedure was similar to that in a, but the efflux of isotope was determined over a long period of time. To reduce the effects of random counting errors, the rates of release of isotope were determined using analysis by linear regression of counts per minute for three or four successive time points. To correct for the loss of isotope from the eggs during the experiment, the radioactivity remaining in the eggs at !I’,, was measured, and the et&x was added back to the midpoint of each time interval. The efflux is expressed as the percentage of the isotope in the eggs during each time interval released per minute.
mined that the increased eftlux following fertilization releases 6.4% of the total isotope in the egg. We are unable to calculate the absolute rate of Ca efTlux from the eggs since we do not know the specific activity of the pool (or pools) releasing Ca.
JOHNSTON AND PAUL
Net Movement of Ca: AA Spectroscopy
Mechanism of Calcium Influx In an effort to understand the mechanism of the fertilization influx, we measured the influx in ASW containing varying concentrations of Ca. Fertilization is impaired in low Ca SW (although sperm adhere to eggs in Ca-free SW, the acrosome reaction is defective; neither egg activation nor sperm penetration occur). Thus, in these experiments, we used trypsin as an artificial activator to avoid possible ambiguities in the interpretation of the TABLE
1 pH 2 pH 3 pH 4 pH
8 8 7’ 7’
Influx
369
results due to impaired sperm-egg interaction. The magnitude of the Ca influx varies with external Ca concentration between 0.25 and 2 mM Ca, but is approximately constant from 2 to 20 mM (Fig. 4a, b). To obtain rates of influx at the different Ca concentrations, we used values of Ca influx over the period 1-5 min after activation. The influx exhibits saturation kinetics (Fig. 4c), indicating that binding of Ca is an important step in the permeation. In SW containing less than 2 n&f Ca, egg activation is delayed or inhibited. Eggs in 0.25 mM Ca showed no signs of activation by 25 min. In 0.5 mM Ca, the first stages of chromosome condensation and aster formation were apparent although the germinal vesicle was still intact. In 1 mM Ca, chromosome condensation and aster formation were more advanced, and, in most eggs, the nuclear membrane was broken down. The population of eggs in each case appeared uniform. Therefore, the decreased Ca influx below 2 mM Ca is not likely to be due to a small percentage of eggs taking up Ca in normal amounts while the larger percentage of eggs remain “unactivated”. Surprisingly, the Ca influx always ceases by 10 min, even at low concentrations of external Ca at which the total influx is subnormal (Fig. 4a) and cytoplasmic activation is retarded. Thus, the period of influx may be independent of the intracellular Ca concentration.
We have verified that a net inward flux of Ca occurs at fertilization by measuring total cellular Ca by AA spectroscopy (Table 2). The total cellular calcium increases by 0.23 + 0.05 pmole/egg during the first 10 min after insemination. This value is equivalent to the uptake determined by 45Ca measurements (Fig. 1) and shows that Ca efflux does not significantly affect the accumulation of Ca by the egg. The AA values indicate a decrease in total cellular Ca of 0.03 pmolelegg between 10 and 60 min (Table 2) (this small change is near the limit of resolution by our techniques; the decrease was statistically significant in one experiment and nonsignificant in the other) and are in agreement with the observation that the period of increased calcium efflux extends beyond that of the fertilization influx.
Condition of insemination
Calcium
2
AA SPECTROSCOPYVALUES OF CALCIUM IN URECHIS EGGS Calcium present Ca increase Percentage CT,-T,,; pmol) of increase (pmole/eggP (To-T,,) T 60 TO TlO 0.56kO.01 0.57kO.05 0.84kO.03 0.70+0.13
0.79~0.00 0.71*?0.02 1.11?0.03 0.94+0.05
0.76t0.01 0.68b~0.01 -
0.23 0.14 0.23 0.24
41 24 32 35
Perce$age decrease CT,,-Tad 4.2 3.8 -
a T,, unfertilized; T,, and T,,, 10 and 60 min after fertilization, respectively. Values are the averages of four determinations. b Adjusted upward to correct for only 70% activation in this experiment. The Ca influx may have been subnormal. c Activation of the egg does not occur.
370
VOLUME 57, 1977
DEVELOPMENTALBIOLOGY
-10
0
IO
20
5
hkvter
Ca2+
10
IS
Concentration,
20
mM
FIG. 4. Effects on Ca influx of varying external calcium concentrations. 45Cawas added to egg suspensions in ASW (0.2520 mM Cal at T-,,. The eggs were activated with trypsin. (a) Calcium uptake at varying Ca concentrations. For clarity, data from experiments with 5 and 20 mM Ca SW are omitted from the figure; the uptake at these concentrations was similar to that at 2 and 10 r&f (see Fig. 4b). (b) The magnitude of the fertilization influx of Ca (O-10 min) plotted against Ca concentration. (c) Average rate of Ca influx between 1 and 5 min plotted against Ca concentration.
We tested the effects on the Ca influx of substances which have been shown to affect the movement of Ca across membranes (Table 1). Mersalyl acid, which inhibits Ca flux in trout gill (131, and DNP, an uncoupler of oxidative phosphorylation, had no effect on Ca influx. Both agents have other rapid effects on the eggs (DNP increased the rate of acid release by the eggs, detected as a decrease in pH of the external SW and possibly due to respiratory CO,; mersalyl acid activates unfertilized eggs). Ruthenium red, an inhibitor of the Ca-H anti-port in mitochondria at a concentration of 0.27 fl (271, had no effect at 0.2 a, but increased the fertilization influx of Ca two- to threefold over controls at 4 fi. The reason for this is obscure to us; however, ruthenium red clearly does not inhibit the fertilization influx. The local anesthetic procaine, at a concentration of 10 m&f, blocks both the fertilization influx of Ca (Fig. 5) and cytoplasmic activation, but blocks neither sperm attachment nor formation of the sperm penetration cone. The inhibition of activation by procaine is reversible; after transfer to SW, these eggs can be activated. Procaine does not inhibit the uptake of Ca by unfertilized eggs (Fig. 5). To investigate further the effects of pro-
0.38 Y % +
0.2-
c 5E
O.l-
8 0
-io
b
i0
20
Minutes
FIG. 5. Inhibition of the fertilization influx of calcium by procaine. Eggs were suspended in either SW (0) or SW containing 10 mM procaine (Al at T-*5. 45Ca was added at T-,,, and the eggs were inseminated at T,. The percentages of eggs containing sperm nuclei were 100% (n = 100) in the control and 41% (N = 100) in eggs inseminated in procaine SW.
Caine on the Ca influx, we measured the influx at external Ca concentrations between 2 and 20 n&f in the presence of 0.05 mM procaine. This was the highest procaine concentration which permitted egg activation at the lowest Ca concentration (2 mM) with this batch of eggs (we observed some variability in the responses of different egg batches to procaine). An Eadie-Hofstee (9) analysis of the results (Fig. 6) indicated that procaine is a competitive inhibitor of Ca influx, presumably compet-
JOHNSTON
AND
PAUL
Calcium
Influx
ing for specific Ca binding sites at the egg membrane. This result suggests that procaine specifically inhibits the Ca influx and only indirectly blocks activation of the other responses. The parameters of the Ca influx are (from Fig. 6), in the absence of procaine, K, = 1.3 n&f, V,,, = 0.04 pmole of Ca/egg*min and, in the presence of 0.05 mM procaine, K, = 5.1 m&f, V,,, = 0.04 pmole of Ca/egg* min. The binding constant for procaine is calculated as Ki = 0.02 mM.
4
Calcium
Rote
Influx
in
Partially
Activated
Is the Ca influx dependent upon egg activation? To answer this question we used partially activated eggs. [We restrict the term “activation” (cytoplasmic activation) for any response of eggs which includes germinal vesicle breakdown. Any response which does not include germinal vesicle breakdown we term “partial activation.” When eggs are incubated for 1 hr in SW adjusted to pH 7 and then inseminated, the known physiological and morphological responses of the egg are absent, with the single exception of the localized events associated with sperm penetration (“sperm partial activation”). Treatment of eggs with trypsin in pH 7 SW results in loss of indentation and surface coat elevation (“trypsin partial activation”)].3 Surprisingly, we found an apparently normal influx of calcium into these eggs following insemination or treatment with trypsin in pH 7 SW (Fig. 7, Table 1). AA spectroscopy measurements also showed that, in the partially activated eggs, there is an increase in total Ca similar to that following a normal activation in pH 8 SW (Table 2). We also examined the possibility that the Ca influx might recur upon a second insemination. Eggs were inseminated in pH 7 SW and then reinseminated at various times following the first insemination (60-160 min in different experiments). When the second insemination followed 3 M. Paul, manuscript in preparation.
14
lb of Cd+
Influx
/
[Co’+]
FIG. 6. Eadie-Hofstee analysis of procaine inhibition of the fertilization influx of Ca. Eggs were in ASW (2-20 mkf Ca) containing 0.06 mkf procaine and %a (A). The rates of influx between 1 and 5 min are presented with the results of the experiment without procaine (0) shown in Fig. 4c (l-20 mkf Ca). Ordinate: picomoles of Ca x 102/egg.min; abscissa: picomoles of Calegg *min. mole of Ca. Intersection of the plots at the ordinate suggests competitive inhibition by procaine.
so
40
Ii0
do
Minu,*r
FIG. 7. Influx of Ca in pH 7 SW. After eggs had been in pH 7 SW for 2 hr, 45Ca was added (T-,,). Thirty minutes after insemination, eggs were washed three times and were resuspended to the original concentration in pH 7 SW. Isotope was added again at Tlz5, and the eggs were reinseminated at T,,,.
the first by less than 2 hr, an influx of Ca was induced, but the amount was variable and always less than that of the first influx (Table 1). When reinsemination was delayed for more than 2 hr, the amount of Ca taken up by the eggs was similar to that associated with the first insemination (Fig. 7, Table 1). Thus, under these conditions, the influx can be reactivated after a refractory period.
372
DEVELOPMENTAL BIOLOGY TABLE
3
EFFECT ON EGG ACTIVATION OF TRANSFERRING EGGS TO Ca-FREE SEAWATER AT INTERVALS AFTER ACTIVATION” Time of transfer (min)
Percentage of eggs activated*
0.5 1 1.5 2 3 5 10
1.7c 1.7c 36.2c 85.2 95.4 98.7 99.5
a Trypsin was added to eggs in seawater (SW), and, at the indicated times, O.l-ml aliquots were transferred to 1.0 ml of Ca-free SW containing both trypsin and 10 mM EGTA. * Polar body formation. The number of eggs counted in each sample was greater than 120. c Polar body formation was delayed in those eggs which activated.
Is the Influx of Calcium at Fertilization Necessary for Cytoplasmic Activation?
To explore this possibility, we initiated activation of eggs with trypsin in SW and then, at intervals after treatment, transferred the eggs to Ca-free SW containing EGTA and trypsin. Egg activation depended on the time of transfer; 85% of the eggs activated “normally” when they were transferred at 2 min (Table 3). In the normal egg response, the Ca influx is 40% complete by 2 min after insemination (see Fig. 1). DISCUSSION
Our results show that a large and rapid influx of Ca, increasing the total egg Ca by approximately 40%, is one of the first responses to insemination of Urechis eggs. The “washing experiments” suggest that the Ca is not simply bound at the egg surface (for example due to changes in surface coat materials), and the possibility that it might be Ca Yrapped” in the perivitelline space which increases in volume during this time is eliminated by the observation that the “uptake” occurs in partially activated eggs with no accompanying morphological changes.
VOLUME 57, 1977
Although we do not know the specific role of the Ca influx, changes in intracellular Ca might regulate processes mediated by microtubules or microfilaments, properties of intracellular membranes, enzyme activities, or other cellular events. It is not required for rounding of the egg or surface coat elevation, since both occur when eggs are treated with trypsin in Ca-free SW. It is apparently not involved in fertilization acid formation by a Ca-H exchange mechanism, similar to the Na-H exchange in sea urchin eggs (111, since acid release is normal in Ca-free SW when eggs are activated with Ca ionophore (24), and the Ca influx occurs when eggs are inseminated in pH 7 SW although fertilization acid is not released. The calcium influx may correspond to the transmembrane Ca currents which have been described in tunicate (16, 20) and starfish eggs (15, 30). The Ca influx follows saturation kinetics, K, = 1.3 mM. Thus, binding of Ca is an important step in its permeation. Energy for the translocation of Ca could be provided by a Ca gradient between the SW and the inside of the egg. Although we do not know the concentration of free Ca in Urechis eggs, it is likely to be much lower than that of the external SW (approximately 11 n&0, since the free Ca concentration in most cells when known is found to be in the micromolar range [for examples, see Ref. (3111.The free Ca concentration in unfertilized sea urchin eggs has been estimated as 0.1 mM (17). We are led to think that the Ca influx has a primary role in the activation of Urechis eggs. First, the beginning of the influx is rapid. By 10 see, 10% of the total influx is already complete. The earliest other response known is fertilization acid release which begins at 10 set (24). Second, the Ca influx occurs in the partially activated eggs in the absence of the other known physiological and morphological responses (fertilization acid, surface coat elevation, change in shape and volume, respiratory increase, germinal vesicle
JOHNSTON AND PAUL
breakdown); it cannot itself be dependent on any of them. Third, cytoplasmic activation of eggs treated with trypsin in low Ca SW is delayed or absent, in extent, correlated with the reduced Ca influx. Fourth, results of the experiment in which eggs were transferred to Ca-free SW at various times after addition of trypsin suggest that at least a part of the Ca influx is necessary for initiation of subsequent events (although this experiment does not exclude the possibility of a Ca-sensitive event independent of the influx but necessary for egg activation). Finally, procaine blocks both the fertilization influx of Ca and “cytoplasmic activation.” Procaine has been shown to affect the mobilities of surface receptors on cells (25) possibily through its effect on cytoskeletal elements associated with the plasma membranes. Activation of Urechis eggs might be inhibited by such an effect. However, procaine is also known to displace Ca from membranes [(21) also see Ref. (19) for other effects of procaine]. Our results suggest that procaine competes for Ca binding sites associated with the Ca permeation. Thus, the primary effect of procaine is apparently to directly block Ca influx, whereas its affect on activation is secondary and indirect. That Urechis eggs can be activated in Ca-free SW with the Ca ionophore A23187 (24) appears inconsistent with the idea that influx of external Ca is necessary for egg activation. However, the ionophore may release bound intracellular Ca from egg stores [e.g., see Ref. (34)l; or, it may activate eggs abnormally in some other way, bypassing the Ca influx. The Ca response by itself it not sufficient to cause the cytoplasmic activation of the eggs since this activation and subsequent development do not proceed at pH 7 (but Ca influx does); pH 7 inseminated eggs do not activate when returned to pH 8 SW unless reinseminated. Low pH is not generally inhibitory but appears specific to the early period, since eggs transferred to pH 7 SW 3 min after insemination con-
Calcium
Influx
373
tinue to develop (35). The Ca response is the most rapid activation response of Urechis eggs known so far. It is apparently necessary for the activation of most of the other egg responses and for further development; therefore, it must induce a secondary response (or several) which does not proceed at pH 7. We feel that this aspect of the activation response is worthy of further investigation. We are grateful to M. Gould-Somero, D. H. Paul, and J. T. Buckley for helpful discussions of this work. We thank them and also L. Holland, J. H. Miller, and V. D. Vacquier for critically reading the manuscript, and T. K. Davies and L. B. Humphrey for assistance in the atomic absorption spectroscopy measurements. Some of this work was done in partial fulfillment for a Bachelor of Science Honours Degree by R. N. J. Supported by a Faculty Research Grant from the University of Victoria and a grant from the National Research Council of Canada (A6946) to M.P. REFERENCES 1. AZARNIA, R., and CHAMBERS, E. L. (1976). The role of divalent cations in activation of the sea urchin egg. 1. Effect of fertilization on divalent cation content. J. Exp. 2001.198,65-78. 2. AZARNIA, R., and CHAMBERS, E. L. (1970). Effect of fertilization on the calcium and magnesium content of the eggs of A&a& punctulafa. Biol. Bull. 139, 413-414. 3. BORLE, A. B. (1970). Kinetic analyses of calcium movements in cell cultures. III. Effects of calcium and parathyroid hormone in kidney cells. J. Gen. Physiol. 55, 163-186. 4. CAVANAUGH, G. M. (ed.) (1956). “Marine Biological Laboratory: Formulae and Methods V,” p. 55. Marine Biological Laboratory, Woods Hoie, Mass. 5. EPEL, D., PRESMAN, B. C., ELSAESSER, S., and WEAVER, A. M. (1969). The program of structural and metabolic changes following fertilization of sea urchin eggs. In “The Cell Cycle” (G. M. Padilla, G. L. Whitson, and I. L. Cameron, eds.), p. 294. Academic Press, New York. 6. GOULD, M. (1967). Echiuroid worms: Urechis. In “Methods in Developmental Biology” (F. Wilt and N. Wessells, eds.), p. 165. Thomas Y. Crowell, New York. 7. GOULD, M. (1969). A comparison of RNA and protein synthesis in fertilized and unfertilized eggs of Urechis caupo. Develop. Bid. 19, 482497. 8. GOULD-SOMERO, M., and HOLLAND, L. (1975).
DEVELOPMENTAL BIOLOGY
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9. 10.
11.
12.
13.
14.
15.
Fine structural investigation of the insemination response in Urechis caupo. Develop. Biol. 46. 358-369. HOFSTEE, B. H. J. (1959). Non-inverted versus inverted plots in enzyme kinetics. Nature (London) 184, 1296-1298. ISONO, N., TSUSAKA, A., and NAKANO, E. (1963). Studies on glucose-gphosphate dehydrogenase in sea urchin eggs. I. J. Fat. Sci. Univ. Tokyo Sect. 4 lo,?55-66. JOHNSON, J. D., EPEL, D., and PAUL, M. (1976). Na+-H+ exchange is required for activation of sea urchin eggs after fertilization. Nature (London) 262, 261-264. LUFT, J. H. (1971). Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anat. Rec. 171, 347-368. MA, S. W. Y., SHAMI, Y., MESSER, H. H., and COPP,D. H. (1974). Properties of Ca2+-ATPase from the gill of rainbow trout (Salmo gairdneri). Biochim. Biophys. Acta 345, 243-251. MAZIA, D. (1937). The release of calcium in Arback eggs on fertilization. J. Cell. Comp. Physiol. 10, 291-304. MIYAZAKI, S., OHMORI, H., and SASAKI, S. (1975). Action potential and non-linear current-voltage relation in starfish oocytes. J. Physiol.
246, 37-54.
16. MIYAZAKI, S., TAKAHASHI, K., and TSUDA, K. (1972). Calcium and sodium contributions to regenerative responses in the embryonic excitable cell membrane. Science 176, 14411443. 17. NAKAMURA, M., and YASUMASU, I. (1974). Mechanism for increase in intracellular concentration of free calcium in fertilized sea urchin egg. A method for estimating intracellular concentration of free calcium. J. Gen. Physiol.
63, 374-388.
18. NAKAZAWA, T., ASAMI, K., SHOGER, R., FUJIWARA, A., and YA~UMASU, I. (1970). Ca*+ uptake, H+ ejection and respiration in sea urchin eggs on fertilization. Exp. Cell Res. 63, 143-146. 19. NICOLSON, G. L., SMITH, J. R., and POBTE, G. (1976). Mprphology and membrane-associated cytosketetal organization in BALB/3T3 cells. J. Cell Biol.
63, 395-402.
20. OKAMOTO, H., TAKAHASHI, K., and YOSHII, M. (1976). Two components of the calcium current in the egg cell membrane of the tunicate. J. Physiol.
255, 527-561.
21. PAPAHADJOPOULOS, D. (1972). Studies on the mechanism of action of local anesthbtics with phospholipid model membranes. Biochim. Biophys. Acta 265, 169-186.
VOLUME 57. 1977
22. PAUL, M. (1970). Fertilization-associated changes in the eggs of Strongylocentrotus purand Urechis caupo. Ph.D. Thesis, puratus Stanford University, Stanford, Calif. 23. PAUL, M. (1975a). The polyspermy block in eggs of Urechis caupo. Evidence for a “rapid” block. Exp. Cell Res. 90, 137-142. 24. PAUL, M. (1975b). Release of acid and changes in light scattering properties following fertilization of Urechis caupo eggs. Develop. Biol. 43, 299-312.
25. POSTE, G., PAPAHADJOPOULOS, D., and NICOLSON, G. L. (1975). Local anesthetics affect transmembrane cytoskeletal control of mobility and distribution of cell surface receptors. Proc. Nat. Acad. Sci. USA 72, 4430-4434.
26. RAMAKRISHNA, T. V., ROBINSON, J. W., and WEST, P. W. (1966). The determination of calcium and magnesium by atomic absorption spectroscopy. Anal. Chim. Acta 36, 57-64. 27. REED, K. C., and BYGRAVE, F. L. (1974). The inhibition of mitochondrial calcium transport by lanthanides and ruthenium red. B&hem. J. 140, 143-155. 28. ROBINSON,K. R., and JAFFE, L. F. (1973). Ion movements in a developing fucoid egg. Develop. Biol. 35, 349-361. 29. SCHUETZ, A. W. (1975). Induction of nuclear breakdown and meiosis in Spisula solidissima oocytes by calcium ionphore. J. Exp. 2001. 191, 433-440. 30. SHEN, S., and STEINHARDT, R. A. (1976). An electrophysiological study of the membrane properties of the immature and mature oocyte of the batstar, Patiria miniatu. Develop. Biol. 48, 148-162. 31. SIMKISS, K. (1974). Calcium translocation by cells. Endeavour 33, 119-123. 32. SKOOG,D. A., and WEST, D. M. (1971). “Principles of Instrumental Analysis,” pp. 299-300. Holt, Rinehart and Winston, New York. 33. STEINHARDT, R. A., and EPEL, D. (1974). Activation of sea urchin eggs by a calcium ionophore. Proc. Nat. Acad. Sci. USA 71, 19151919. 34. STEINHARDT, R. A., EPEL, D., CARROLL, E. J., and YANAGIMACHI, R. (1974). Is calcium ionophore a universal activator for unfertilized eggs? Nature (London) 252, 41-43. 35. TYLER, A., and J. SCHULTZ. (1932). Inhibition and reversal of fertilization in eggs of the echinoid (sic) worm, Urechis caupo. J. Exp. Zool. 63, 509-531. 36. VACQUIER, V. D. (1975). The isolation of intact cortical granules from sea urchin eggs: Calcium ions trigger granule discharge. Develop. Biol.
43, 62-74.
BIOLOGY
Calcium
57, 364-374 (1977)
Influx following
N. JOHNSTON’
RANDAL Department
of Biology,
University
Received August
Fertilization
of Victoria,
13,1976;
AND
Victoria,
of Urechis MILES British
caupo
Eggs
PAUL Columbia,
accepted in revised form January
Canada
V8W 2Y2
26,1977
Measurements of %a flux into and out of Urechis eggs indicate that, during the first 10 min alter insemination, the eggs take up 0.24 pmole of Ca/egg. Total egg Ca measured by atomic absorption (AA) spectroscopy increased by 0.23 pmole of Ca/egg (0.56, 0.79, and 0.76 pmole of Ca/egg for unfertilized, lo-min fertilized, and 60-min fertilized eggs, respectively). Thus, the total change in egg Ca is accounted for by the influx even though the rate of efflux, measured as a release of 4sCa from preloaded eggs, increases to twice the unfertilized rate by 15 min. The fertilization influx follows saturation kinetics (K, = 1.3 m&f). It is competitively inhibited by procaine, but is not inhibited by dinitrophenol, mersalyl acid, or ruthenium red. Ten percent of the total Ca influx has occurred by 10 set, and it is, therefore, the most rapid response to fertilization yet known in these eggs. The influx is also observed in eggs partially activated by insemination in pH 7 seawater (SW); the other fertilization responses, except sperm penetration, do not occur in pH 7 SW. Although Ca influx alone is insufficient to activate the eggs, it may be a prerequisite for cytoplasmic activation and development, inducing other secondary responses which are prevented by low external pH. INTRODUCTION
Intracellular Ca is recognized as an important regulator of cellular processes and changes in that it can act as a “trigger” in changing cell behaviour and function. Studies of fertilization of sea urchin eggs have shown an increase in the availibility of intracellular Ca upon activation of the egg (14, 17,331. Uptake of external Ca also occurs in the first few minutes after insemination (1,2, 18). Calcium may initiate the discharge of cortical granules (36) and may be an activating agent for other early changes in sea urchin eggs at fertilization [for example, activation of enzymes such as NAD+ kinase (EC 2.7.1.23) (5) or glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (lo)]. Calcium currents across egg membranes have been demonstrated in tunicate (16, 20) and starfish eggs (15, 30) in response to imposed depolarizations and may also occur at fertilization (30). Also, many kinds ’ Present address: Department of Biological Sciences, Stanford University, Stanford, California 94305.
of eggs can be artificially activated with the Ca ionophore A23187 [sea urchin (33); starfish, limpet, tunicate, toad, hamster (34); surf clam (29); Urechis (2411. Thus, changes in the distribution or availibility of intracellular Ca may be important in the events associated with the activation of animal eggs in general. Since Ca may have an important role in the activation of eggs of the echiuroid worm Urechis caupo, we have investigated the possibility that changes in Ca may occur in these eggs following insemination. Some of the responses to insemination of Urechis eggs are similar to those of sea urchin eggs. Both sea urchin and Urethis eggs release a fertilization acid (24) and elevate a surface coat, although surface coat elevation occurs later and more slowly in Urechis [see Ref. (611. However, in several important respects, the responses of Urechis eggs contrast with those of sea urchin eggs. A cortical reaction like that occurring in sea urchin eggs (extensive cortical granule exocytosis resulting in elevation of the surface coat and 364
Copyright 0 1977 by Academic Press, Inc. All rights
of reproduction
in any form reserved.
ISSN
0012-1606
JOHNSTON AND PAUL
formation of the hyaline layer) does not take place in Urechis eggs (8,241. Thus, in Urechis any “calcium response” would be unrelated to the complicated events of the cortical reaction as it occurs in sea urchins. A second major difference is that Urechis eggs are fertilized as primary oocytes; germinal vesicle breakdown and the meiotic divisions follow insemination (in contrast, sea urchin eggs are fertilized as ova, after meiotic maturation). Also, there is a major change in shape of the Urechis egg as the prominent indentation of the primary oocyte rounds out in the first 4 min after insemination. As in sea urchins, a fertilization acid is released (241, and the respiratory rate increases (221, but NAD+ kinase is apparently not activated.2 A study of a “calcium response” in Urechis eggs may provide insights into the role of Ca in sea urchin and other eggs as well as in Urechis eggs themselves. We have found that a large net influx of Ca, detectable by 10 set, occurs during the first 10 min after insemination of Urechis eggs. This influx begins prior to the other known structural and physiological responses of the egg, including fertilization acid release (the most rapid of these), and can occur in their absence. Therefore, it is not itself induced by any of these changes. So far, we have no clues to the specific role of this large increase in Ca, but our results show that it is a primary event in the activation of Urechis eggs. MATERIALS
AND
METHODS
Handling of worms and gametes. Urethis caupo, obtained from Elk Horn
Slough, Moss Landing, California, were maintained in recirculating seawater (SW) instant ocean aquaria in glass tubes at lo-15°C. Eggs and sperm were obtained by the method of Newby (6) on the day of use. Undiluted semen was stored at 3°C and was diluted with SW just prior to inseminations. Eggs were kept at 16°C. Experiments were performed with 2-4% (v/v) 2 M. Paul, unpublished
observations.
Calcium
Influx
365
egg suspensions. To determine egg concentration, we obtained “packed egg” volumes by centrifuging aliquots of egg suspension in a calibrated Shevky-Stafford tube (Arthur H. Thomas Company, Philadelphia, Pa.) in a Janetzki T5 table centrifuge (Jena Instruments Ltd., Surrey, British Columbia, Canada) at setting “2” (11OOg) for 3 min. A 1.0% egg suspension contained 8.0 x 104eggs/ml by direct count of eggs in dilutions of the egg suspensions. Egg batches in which there was less than 95% normal activation in test inseminations were not used. We refer to the fertilizable primary oocyte and zygote as “unfertilized egg” and “fertilized egg,” respecWe examined morphological tively. changes of eggs activated in low Ca SW by the acetic acid-ethanol procedure (23). General experimental procedure. Eggs were washed into fresh SW just before use, suspended at desired concentrations, and incubated in a water-jacketed Silicladtreated cylindrical glass container at 16°C. During experiments, the egg suspensions were stirred with a magnetic stirring bar. To determine whether they take up Ca from the external medium, we incubated eggs in SW or artificial SW (ASW) containing 45Ca and subsequently measured the radioactivity in the eggs. Isotope was added to egg suspensions at least 5 min prior to sampling; radioactivity was 0.611.17 &i/ml in various experiments except as noted (preloading incubation; experiment, Fig. 3). Eggs were activated either with a freshly prepared sperm suspension or by addition to the egg suspension of an equal volume of a 0.2% (w/v) solution of trypsin in SW or ASW; trypsin is an artificial activator of Urechis eggs (22). Times relative to insemination or activation (T,) are indicated as T, (n = the time in minutes) unless otherwise indicated (i.e., Fig. 2). Sperm penetration was determined by observation of eggs fixed in acetic acidethanol as previously described (23). ASW and SW containing added agents were always adjusted to pH 7 or 8 as appropriate.
366
DEVELOPMENTALBIOLOGY
Determination of radioactivity. Duplicate samples of egg suspensions (0.5 ml) were taken with a Siliclad-treated Pasteur pipet on a Cornwall Pipettor (Becton, Dickinson and Co., Rutherford, N.J.), diluted by addition to 15 ml of chilled SW exactly at the indicated times and washed three times with 15 ml of chilled SW by “hand centrifugation” and aspiration of the supernatant SW to less than 0.1 ml between washings. Washed samples (the volume of eggs plus residual wash medium was approximately 50 ~1) were transferred to 1.5dram plastic-screw-cap glass vials (Fisher Scientific Co., Vancouver, British Columbia, Canada) and were digested overnight in 0.5 ml of Protosol at 55°C. Five milliliters of Omnifluor-toluene scintillation fluid was added to each vial. These small vials were placed within standard 20-ml “glass LSC vials” (New England Nuclear, Dorval, Quebec, Canada) which served as adaptors for counting. Fifty-microliter aliquots of “SW-medium” from egg suspensions were processed similarly to the washed egg samples. All results are corrected for background radioactivity. Calcium influx was calculated with the following formula: counts per minute per sample of eggs X moles of Ca per milliliter of SW medium over eggs per sample x counts per minute per milliliter of SW medium = moles of Ca per egg. For our calculations of the fertilization influx, we have used the net changes in counts per minute from 0 to 10 min after insemination, neglecting any possible contribution from the equilibrium influx of Ca, since we do not know whether it continues during this period. Atomic absorption spectroscopy. Egg samples were each washed four times in ice-cold 0.55 M KCl, dried at 95”C, and digested overnight with 1.0 ml of concentrated HCl. Each sample was then centrifuged in a Janetzki T5 table centrifuge (setting 2) for 10 min, and the supernatant was collected. The remaining pellet was washed with three additional 0.5-ml ali-
VOLUME 57, 1977
quots of HCl, and all of the supernatants were combined in a 5-ml volumetric flask. To each flask was added 0.5 ml of 2% (w/v) EDTA and 0.5 ml of 4% (w/v) SrCl, [these agents minimize interference from other substances in AA determinations of Ca (26)l. Finally, each solution was diluted to 5 ml with doubly distilled water, and the absorbance was determined at 422.7 nm on a Techtron A-A spectrophotometer. Blank samples were prepared in a similar manner, but contained no eggs. Interferences which were not eliminated by this procedure were corrected for, using the method of standard additions (32). Materials. Omnifluor and Protosol were obtained from New England Nuclear, Dorval, Quebec, Canada; trypsin (bovine, pancreatic; lyophilized, 2 x crystallized) from Worthington Biochemical Corp., Freehold, N.J.; procaine, ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis-(@aminoethyl ether)-2\rfl’-tetraacetic acid (EGTA), and mersalyl acid from Sigma Chemical Co., St. Louis, MO.; 2,4dinitrophenol (DNP) from Fisher Scientific Co., Fair Lawn, N.J.; and ruthenium red from K. and K. Laboratories, Plainview, N.Y. Isotope was obtained from Amersham, Oakville, Ontario, Canada (45CaC1,in water, CES.2, Batch 13F) and from New England Nuclear (45CaC1, in water, NEZ-013, Lot S1091). Seawater was collected at a depth of 30 m from the Strait of Georgia, filtered through two layers of Whatman No. 1 filter paper, and stored at 3°C until use. Art&al seawater was prepared from modifications of the MBL Formula (4), with Na+ substitued for Ca2+ in the appropriate molar ratio. RESULTS
Calcium
Influx
Unfertilized Urechis eggs take up Ca at a constant rate which we have observed for 1 hr, this component of “uptake” is shown in Fig. 1 prior to insemination at T,. The rate of Ca uptake during this period is 1.1 f 0.5 fmole/egg *min. We assume that this
JOHNSTON AND PAUL
367 TABLE
0.3
3 k + Q23 sg O.l0, E
Calcium Influx 1
INFLUX OF CALCIUM FOLLOWING ACTIVATION OF URECHIS EGGS Condition
Ca uutake (pmoilegg)
of activation
0.24 (N 0.23 0.25 (N 0.22
pH 8 Sperm Trypsin pH 7 Sperm
* t
I
1
-15
0
15
30
Minutes
FIG. 1. Calcium uptake by Urechis eggs before and after insemination at T,. *%a added to egg suspension in SW at T-,,.
is an “equilibrium influx” since unfertilized eggs remain fertilizable for prolonged periods (7) and are presumably in an equilibrium condition with respect to Ca content and fluxes. There is a brief phase of rapid association of isotope with the eggs immediately after the addition of isotope to the SW. This initial phase of uptake is revealed by the failure of isotope associated with the eggs to extrapolate to 0 at the time of addition. This apparently represents the equilibration of isotope into a pool of Ca which is rapidly exchanging with the external SW, and may be Ca bound to the egg surface or surface coat [cf., Refs. (3, 28)l. Insemination induces a large uptake of isotope by the eggs (Fig. 1) which is complete by 10 min (16’C). We refer to this as the “Ca influx.” Calculations indicate an uptake of 0.24 + 0.03 pmole of Ca/egg during this period (average rate, 24 fmole/ egg- min). To define the beginning of the increased Ca uptake more accurately, in two experiments we took samples at lo-set intervals after insemination. Of the total uptake, approximately 10% had occurred by 10 set and 25% by 40 sec. Trypsin also initiates an uptake of Ca by the eggs indistinguishable from that induced by insemination (Table 1). This observation eliminates the possibility that the uptake of Ca is an artifact of the activation of the eggs with sperm (for example, due to the penetration of eggs by
Trypsin pH 7 Second influx Time (min)” 60 100 160
+ 0.03 = 8) f 0.12 = 4)
0.11 (N = 2) 0.18 0.22 Percentage control (o/o)
Mersalyl acid (20 mM, T,P DNP (0.25 mM, T-&’ Procaine (0.05 mM; T-,,,I* Procaine (10 mM; T-,,J* Ruthenium red (0.2 @If; T-,,)** c Ruthenium red (4 &f; T_,,)*, c 0 Time after the first partial b Time added. c Concentration determined Luft (1971).
of
93 93 79 0 83 242 (N = 2)
activation. by the method
of
sperm-carrying-bound isotope or due to a possible increased permeability of the egg in the region of the penetration cone). In an effort to distinguish whether the Ca influx might represent a superficial binding of ion (for example, to the surface or surface coat of the egg) or is a true flux into the cell, we attempted to remove &Ca from eggs fertilized in SW containing isotope by washing them in either SW or Cafree SW containing 5 mM EGTA (Fig. 2). Although the radioactivity of the “washing media” increased over 30 min, corresponding to a loss of 8-10% of the original radioactivity of the eggs, the Ca-free SW with EGTA was no more efficient in removing isotope from the eggs than was SW. In another experiment, we transferred eggs which had been inseminated 15 min earlier into SW containing isotope and measured the rate of 45Cainflux between
368
DEVELOPMENTAL BIOLOGY
VOLUME 57. 197'7
I -10
0
10
20
30
MlmJkl
FIG. 2. Release of isotope from fertilized eggs in different washing media. Eggs were fertilized in SW containing “Va. After 15 min, two 5.0-ml samples of the egg suspension were rapidly washed three times in SW and were resuspended to the original volume in either SW (0) or in Ca-free SW containing 5 mM EGTA (A). T, is the time of the final suspension of the eggs in the washing medium. Samples of the eggs in wash media were taken at 5-min intervals and were centrifuged, and the supernatant medium was removed. The radioactivities of triplicate O.l-ml aliquots of each supernatant were determined. The loss of isotope from the eggs is expressed as the percentage of the original radioactivity in the eggs released into the washing medium. In this experiment, the initial uptake of radioactivity was 0.045 cpmlegg (6700 cpm/original 5-ml sample).
20 and 50 min after insemination. These eggs continued to take up Ca at a rate comparable to that of the unfertilized eggs (1.1 fmole/egg *min, unfertilized; 1.O fmole/egg *min, fertilized). Calcium
EffTux
If there were a large increase in Ca efflux accompanying the increased influx just shown, there might be no change in total cellular calcium. To examine this possibility, we determined the efflux of Va from preloaded eggs, both before and after fertilization. Unfertilized eggs released radioactivity at a rate of 0.34 & 0.09% (N = 2) of the total in the eggs per min. Within 1 min after insemination, the rate of efflux begins to increase, doubles within 15 min, and returns to the prefertilization level by 45 min (Fig. 3a, b). Thus, the time courses of the fertilization influx and efflux are different. We “integrated” the function eMux versus time (Fig. 3b) by weighing paper “cutouts” and thus deter-
-iO
-1;
b
1;
3-O
4;
Minuter
FIG. 3. EMux of *Va from preloaded eggs. (a) Release of isotope into-SW from fertilized and unfertilized eggs. 45Ca was added to an egg suspension (20%, v/v) in ASW containing 0.1 mM Ca (30 &i/ ml). After 4 hr (TV,,), the eggs were washed three times in SW. The eggs were washed again after 20 min, resuspended in SW, and divided in half, and one-half was fertilized. At the indicated times, samples of each egg suspension were centrifuged, and the radioactivities of triplicate O.l-ml aliquots of each supernatant were determined. Fertilized (A); unfertilized (0). (b) Rate of release of isotope from preloaded eggs. In a second experiment, the procedure was similar to that in a, but the efflux of isotope was determined over a long period of time. To reduce the effects of random counting errors, the rates of release of isotope were determined using analysis by linear regression of counts per minute for three or four successive time points. To correct for the loss of isotope from the eggs during the experiment, the radioactivity remaining in the eggs at !I’,, was measured, and the et&x was added back to the midpoint of each time interval. The efflux is expressed as the percentage of the isotope in the eggs during each time interval released per minute.
mined that the increased eftlux following fertilization releases 6.4% of the total isotope in the egg. We are unable to calculate the absolute rate of Ca efTlux from the eggs since we do not know the specific activity of the pool (or pools) releasing Ca.
JOHNSTON AND PAUL
Net Movement of Ca: AA Spectroscopy
Mechanism of Calcium Influx In an effort to understand the mechanism of the fertilization influx, we measured the influx in ASW containing varying concentrations of Ca. Fertilization is impaired in low Ca SW (although sperm adhere to eggs in Ca-free SW, the acrosome reaction is defective; neither egg activation nor sperm penetration occur). Thus, in these experiments, we used trypsin as an artificial activator to avoid possible ambiguities in the interpretation of the TABLE
1 pH 2 pH 3 pH 4 pH
8 8 7’ 7’
Influx
369
results due to impaired sperm-egg interaction. The magnitude of the Ca influx varies with external Ca concentration between 0.25 and 2 mM Ca, but is approximately constant from 2 to 20 mM (Fig. 4a, b). To obtain rates of influx at the different Ca concentrations, we used values of Ca influx over the period 1-5 min after activation. The influx exhibits saturation kinetics (Fig. 4c), indicating that binding of Ca is an important step in the permeation. In SW containing less than 2 n&f Ca, egg activation is delayed or inhibited. Eggs in 0.25 mM Ca showed no signs of activation by 25 min. In 0.5 mM Ca, the first stages of chromosome condensation and aster formation were apparent although the germinal vesicle was still intact. In 1 mM Ca, chromosome condensation and aster formation were more advanced, and, in most eggs, the nuclear membrane was broken down. The population of eggs in each case appeared uniform. Therefore, the decreased Ca influx below 2 mM Ca is not likely to be due to a small percentage of eggs taking up Ca in normal amounts while the larger percentage of eggs remain “unactivated”. Surprisingly, the Ca influx always ceases by 10 min, even at low concentrations of external Ca at which the total influx is subnormal (Fig. 4a) and cytoplasmic activation is retarded. Thus, the period of influx may be independent of the intracellular Ca concentration.
We have verified that a net inward flux of Ca occurs at fertilization by measuring total cellular Ca by AA spectroscopy (Table 2). The total cellular calcium increases by 0.23 + 0.05 pmole/egg during the first 10 min after insemination. This value is equivalent to the uptake determined by 45Ca measurements (Fig. 1) and shows that Ca efflux does not significantly affect the accumulation of Ca by the egg. The AA values indicate a decrease in total cellular Ca of 0.03 pmolelegg between 10 and 60 min (Table 2) (this small change is near the limit of resolution by our techniques; the decrease was statistically significant in one experiment and nonsignificant in the other) and are in agreement with the observation that the period of increased calcium efflux extends beyond that of the fertilization influx.
Condition of insemination
Calcium
2
AA SPECTROSCOPYVALUES OF CALCIUM IN URECHIS EGGS Calcium present Ca increase Percentage CT,-T,,; pmol) of increase (pmole/eggP (To-T,,) T 60 TO TlO 0.56kO.01 0.57kO.05 0.84kO.03 0.70+0.13
0.79~0.00 0.71*?0.02 1.11?0.03 0.94+0.05
0.76t0.01 0.68b~0.01 -
0.23 0.14 0.23 0.24
41 24 32 35
Perce$age decrease CT,,-Tad 4.2 3.8 -
a T,, unfertilized; T,, and T,,, 10 and 60 min after fertilization, respectively. Values are the averages of four determinations. b Adjusted upward to correct for only 70% activation in this experiment. The Ca influx may have been subnormal. c Activation of the egg does not occur.
370
VOLUME 57, 1977
DEVELOPMENTALBIOLOGY
-10
0
IO
20
5
hkvter
Ca2+
10
IS
Concentration,
20
mM
FIG. 4. Effects on Ca influx of varying external calcium concentrations. 45Cawas added to egg suspensions in ASW (0.2520 mM Cal at T-,,. The eggs were activated with trypsin. (a) Calcium uptake at varying Ca concentrations. For clarity, data from experiments with 5 and 20 mM Ca SW are omitted from the figure; the uptake at these concentrations was similar to that at 2 and 10 r&f (see Fig. 4b). (b) The magnitude of the fertilization influx of Ca (O-10 min) plotted against Ca concentration. (c) Average rate of Ca influx between 1 and 5 min plotted against Ca concentration.
We tested the effects on the Ca influx of substances which have been shown to affect the movement of Ca across membranes (Table 1). Mersalyl acid, which inhibits Ca flux in trout gill (131, and DNP, an uncoupler of oxidative phosphorylation, had no effect on Ca influx. Both agents have other rapid effects on the eggs (DNP increased the rate of acid release by the eggs, detected as a decrease in pH of the external SW and possibly due to respiratory CO,; mersalyl acid activates unfertilized eggs). Ruthenium red, an inhibitor of the Ca-H anti-port in mitochondria at a concentration of 0.27 fl (271, had no effect at 0.2 a, but increased the fertilization influx of Ca two- to threefold over controls at 4 fi. The reason for this is obscure to us; however, ruthenium red clearly does not inhibit the fertilization influx. The local anesthetic procaine, at a concentration of 10 m&f, blocks both the fertilization influx of Ca (Fig. 5) and cytoplasmic activation, but blocks neither sperm attachment nor formation of the sperm penetration cone. The inhibition of activation by procaine is reversible; after transfer to SW, these eggs can be activated. Procaine does not inhibit the uptake of Ca by unfertilized eggs (Fig. 5). To investigate further the effects of pro-
0.38 Y % +
0.2-
c 5E
O.l-
8 0
-io
b
i0
20
Minutes
FIG. 5. Inhibition of the fertilization influx of calcium by procaine. Eggs were suspended in either SW (0) or SW containing 10 mM procaine (Al at T-*5. 45Ca was added at T-,,, and the eggs were inseminated at T,. The percentages of eggs containing sperm nuclei were 100% (n = 100) in the control and 41% (N = 100) in eggs inseminated in procaine SW.
Caine on the Ca influx, we measured the influx at external Ca concentrations between 2 and 20 n&f in the presence of 0.05 mM procaine. This was the highest procaine concentration which permitted egg activation at the lowest Ca concentration (2 mM) with this batch of eggs (we observed some variability in the responses of different egg batches to procaine). An Eadie-Hofstee (9) analysis of the results (Fig. 6) indicated that procaine is a competitive inhibitor of Ca influx, presumably compet-
JOHNSTON
AND
PAUL
Calcium
Influx
ing for specific Ca binding sites at the egg membrane. This result suggests that procaine specifically inhibits the Ca influx and only indirectly blocks activation of the other responses. The parameters of the Ca influx are (from Fig. 6), in the absence of procaine, K, = 1.3 n&f, V,,, = 0.04 pmole of Ca/egg*min and, in the presence of 0.05 mM procaine, K, = 5.1 m&f, V,,, = 0.04 pmole of Ca/egg* min. The binding constant for procaine is calculated as Ki = 0.02 mM.
4
Calcium
Rote
Influx
in
Partially
Activated
Is the Ca influx dependent upon egg activation? To answer this question we used partially activated eggs. [We restrict the term “activation” (cytoplasmic activation) for any response of eggs which includes germinal vesicle breakdown. Any response which does not include germinal vesicle breakdown we term “partial activation.” When eggs are incubated for 1 hr in SW adjusted to pH 7 and then inseminated, the known physiological and morphological responses of the egg are absent, with the single exception of the localized events associated with sperm penetration (“sperm partial activation”). Treatment of eggs with trypsin in pH 7 SW results in loss of indentation and surface coat elevation (“trypsin partial activation”)].3 Surprisingly, we found an apparently normal influx of calcium into these eggs following insemination or treatment with trypsin in pH 7 SW (Fig. 7, Table 1). AA spectroscopy measurements also showed that, in the partially activated eggs, there is an increase in total Ca similar to that following a normal activation in pH 8 SW (Table 2). We also examined the possibility that the Ca influx might recur upon a second insemination. Eggs were inseminated in pH 7 SW and then reinseminated at various times following the first insemination (60-160 min in different experiments). When the second insemination followed 3 M. Paul, manuscript in preparation.
14
lb of Cd+
Influx
/
[Co’+]
FIG. 6. Eadie-Hofstee analysis of procaine inhibition of the fertilization influx of Ca. Eggs were in ASW (2-20 mkf Ca) containing 0.06 mkf procaine and %a (A). The rates of influx between 1 and 5 min are presented with the results of the experiment without procaine (0) shown in Fig. 4c (l-20 mkf Ca). Ordinate: picomoles of Ca x 102/egg.min; abscissa: picomoles of Calegg *min. mole of Ca. Intersection of the plots at the ordinate suggests competitive inhibition by procaine.
so
40
Ii0
do
Minu,*r
FIG. 7. Influx of Ca in pH 7 SW. After eggs had been in pH 7 SW for 2 hr, 45Ca was added (T-,,). Thirty minutes after insemination, eggs were washed three times and were resuspended to the original concentration in pH 7 SW. Isotope was added again at Tlz5, and the eggs were reinseminated at T,,,.
the first by less than 2 hr, an influx of Ca was induced, but the amount was variable and always less than that of the first influx (Table 1). When reinsemination was delayed for more than 2 hr, the amount of Ca taken up by the eggs was similar to that associated with the first insemination (Fig. 7, Table 1). Thus, under these conditions, the influx can be reactivated after a refractory period.
372
DEVELOPMENTAL BIOLOGY TABLE
3
EFFECT ON EGG ACTIVATION OF TRANSFERRING EGGS TO Ca-FREE SEAWATER AT INTERVALS AFTER ACTIVATION” Time of transfer (min)
Percentage of eggs activated*
0.5 1 1.5 2 3 5 10
1.7c 1.7c 36.2c 85.2 95.4 98.7 99.5
a Trypsin was added to eggs in seawater (SW), and, at the indicated times, O.l-ml aliquots were transferred to 1.0 ml of Ca-free SW containing both trypsin and 10 mM EGTA. * Polar body formation. The number of eggs counted in each sample was greater than 120. c Polar body formation was delayed in those eggs which activated.
Is the Influx of Calcium at Fertilization Necessary for Cytoplasmic Activation?
To explore this possibility, we initiated activation of eggs with trypsin in SW and then, at intervals after treatment, transferred the eggs to Ca-free SW containing EGTA and trypsin. Egg activation depended on the time of transfer; 85% of the eggs activated “normally” when they were transferred at 2 min (Table 3). In the normal egg response, the Ca influx is 40% complete by 2 min after insemination (see Fig. 1). DISCUSSION
Our results show that a large and rapid influx of Ca, increasing the total egg Ca by approximately 40%, is one of the first responses to insemination of Urechis eggs. The “washing experiments” suggest that the Ca is not simply bound at the egg surface (for example due to changes in surface coat materials), and the possibility that it might be Ca Yrapped” in the perivitelline space which increases in volume during this time is eliminated by the observation that the “uptake” occurs in partially activated eggs with no accompanying morphological changes.
VOLUME 57, 1977
Although we do not know the specific role of the Ca influx, changes in intracellular Ca might regulate processes mediated by microtubules or microfilaments, properties of intracellular membranes, enzyme activities, or other cellular events. It is not required for rounding of the egg or surface coat elevation, since both occur when eggs are treated with trypsin in Ca-free SW. It is apparently not involved in fertilization acid formation by a Ca-H exchange mechanism, similar to the Na-H exchange in sea urchin eggs (111, since acid release is normal in Ca-free SW when eggs are activated with Ca ionophore (24), and the Ca influx occurs when eggs are inseminated in pH 7 SW although fertilization acid is not released. The calcium influx may correspond to the transmembrane Ca currents which have been described in tunicate (16, 20) and starfish eggs (15, 30). The Ca influx follows saturation kinetics, K, = 1.3 mM. Thus, binding of Ca is an important step in its permeation. Energy for the translocation of Ca could be provided by a Ca gradient between the SW and the inside of the egg. Although we do not know the concentration of free Ca in Urechis eggs, it is likely to be much lower than that of the external SW (approximately 11 n&0, since the free Ca concentration in most cells when known is found to be in the micromolar range [for examples, see Ref. (3111.The free Ca concentration in unfertilized sea urchin eggs has been estimated as 0.1 mM (17). We are led to think that the Ca influx has a primary role in the activation of Urechis eggs. First, the beginning of the influx is rapid. By 10 see, 10% of the total influx is already complete. The earliest other response known is fertilization acid release which begins at 10 set (24). Second, the Ca influx occurs in the partially activated eggs in the absence of the other known physiological and morphological responses (fertilization acid, surface coat elevation, change in shape and volume, respiratory increase, germinal vesicle
JOHNSTON AND PAUL
breakdown); it cannot itself be dependent on any of them. Third, cytoplasmic activation of eggs treated with trypsin in low Ca SW is delayed or absent, in extent, correlated with the reduced Ca influx. Fourth, results of the experiment in which eggs were transferred to Ca-free SW at various times after addition of trypsin suggest that at least a part of the Ca influx is necessary for initiation of subsequent events (although this experiment does not exclude the possibility of a Ca-sensitive event independent of the influx but necessary for egg activation). Finally, procaine blocks both the fertilization influx of Ca and “cytoplasmic activation.” Procaine has been shown to affect the mobilities of surface receptors on cells (25) possibily through its effect on cytoskeletal elements associated with the plasma membranes. Activation of Urechis eggs might be inhibited by such an effect. However, procaine is also known to displace Ca from membranes [(21) also see Ref. (19) for other effects of procaine]. Our results suggest that procaine competes for Ca binding sites associated with the Ca permeation. Thus, the primary effect of procaine is apparently to directly block Ca influx, whereas its affect on activation is secondary and indirect. That Urechis eggs can be activated in Ca-free SW with the Ca ionophore A23187 (24) appears inconsistent with the idea that influx of external Ca is necessary for egg activation. However, the ionophore may release bound intracellular Ca from egg stores [e.g., see Ref. (34)l; or, it may activate eggs abnormally in some other way, bypassing the Ca influx. The Ca response by itself it not sufficient to cause the cytoplasmic activation of the eggs since this activation and subsequent development do not proceed at pH 7 (but Ca influx does); pH 7 inseminated eggs do not activate when returned to pH 8 SW unless reinseminated. Low pH is not generally inhibitory but appears specific to the early period, since eggs transferred to pH 7 SW 3 min after insemination con-
Calcium
Influx
373
tinue to develop (35). The Ca response is the most rapid activation response of Urechis eggs known so far. It is apparently necessary for the activation of most of the other egg responses and for further development; therefore, it must induce a secondary response (or several) which does not proceed at pH 7. We feel that this aspect of the activation response is worthy of further investigation. We are grateful to M. Gould-Somero, D. H. Paul, and J. T. Buckley for helpful discussions of this work. We thank them and also L. Holland, J. H. Miller, and V. D. Vacquier for critically reading the manuscript, and T. K. Davies and L. B. Humphrey for assistance in the atomic absorption spectroscopy measurements. Some of this work was done in partial fulfillment for a Bachelor of Science Honours Degree by R. N. J. Supported by a Faculty Research Grant from the University of Victoria and a grant from the National Research Council of Canada (A6946) to M.P. REFERENCES 1. AZARNIA, R., and CHAMBERS, E. L. (1976). The role of divalent cations in activation of the sea urchin egg. 1. Effect of fertilization on divalent cation content. J. Exp. 2001.198,65-78. 2. AZARNIA, R., and CHAMBERS, E. L. (1970). Effect of fertilization on the calcium and magnesium content of the eggs of A&a& punctulafa. Biol. Bull. 139, 413-414. 3. BORLE, A. B. (1970). Kinetic analyses of calcium movements in cell cultures. III. Effects of calcium and parathyroid hormone in kidney cells. J. Gen. Physiol. 55, 163-186. 4. CAVANAUGH, G. M. (ed.) (1956). “Marine Biological Laboratory: Formulae and Methods V,” p. 55. Marine Biological Laboratory, Woods Hoie, Mass. 5. EPEL, D., PRESMAN, B. C., ELSAESSER, S., and WEAVER, A. M. (1969). The program of structural and metabolic changes following fertilization of sea urchin eggs. In “The Cell Cycle” (G. M. Padilla, G. L. Whitson, and I. L. Cameron, eds.), p. 294. Academic Press, New York. 6. GOULD, M. (1967). Echiuroid worms: Urechis. In “Methods in Developmental Biology” (F. Wilt and N. Wessells, eds.), p. 165. Thomas Y. Crowell, New York. 7. GOULD, M. (1969). A comparison of RNA and protein synthesis in fertilized and unfertilized eggs of Urechis caupo. Develop. Bid. 19, 482497. 8. GOULD-SOMERO, M., and HOLLAND, L. (1975).
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9. 10.
11.
12.
13.
14.
15.
Fine structural investigation of the insemination response in Urechis caupo. Develop. Biol. 46. 358-369. HOFSTEE, B. H. J. (1959). Non-inverted versus inverted plots in enzyme kinetics. Nature (London) 184, 1296-1298. ISONO, N., TSUSAKA, A., and NAKANO, E. (1963). Studies on glucose-gphosphate dehydrogenase in sea urchin eggs. I. J. Fat. Sci. Univ. Tokyo Sect. 4 lo,?55-66. JOHNSON, J. D., EPEL, D., and PAUL, M. (1976). Na+-H+ exchange is required for activation of sea urchin eggs after fertilization. Nature (London) 262, 261-264. LUFT, J. H. (1971). Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anat. Rec. 171, 347-368. MA, S. W. Y., SHAMI, Y., MESSER, H. H., and COPP,D. H. (1974). Properties of Ca2+-ATPase from the gill of rainbow trout (Salmo gairdneri). Biochim. Biophys. Acta 345, 243-251. MAZIA, D. (1937). The release of calcium in Arback eggs on fertilization. J. Cell. Comp. Physiol. 10, 291-304. MIYAZAKI, S., OHMORI, H., and SASAKI, S. (1975). Action potential and non-linear current-voltage relation in starfish oocytes. J. Physiol.
246, 37-54.
16. MIYAZAKI, S., TAKAHASHI, K., and TSUDA, K. (1972). Calcium and sodium contributions to regenerative responses in the embryonic excitable cell membrane. Science 176, 14411443. 17. NAKAMURA, M., and YASUMASU, I. (1974). Mechanism for increase in intracellular concentration of free calcium in fertilized sea urchin egg. A method for estimating intracellular concentration of free calcium. J. Gen. Physiol.
63, 374-388.
18. NAKAZAWA, T., ASAMI, K., SHOGER, R., FUJIWARA, A., and YA~UMASU, I. (1970). Ca*+ uptake, H+ ejection and respiration in sea urchin eggs on fertilization. Exp. Cell Res. 63, 143-146. 19. NICOLSON, G. L., SMITH, J. R., and POBTE, G. (1976). Mprphology and membrane-associated cytosketetal organization in BALB/3T3 cells. J. Cell Biol.
63, 395-402.
20. OKAMOTO, H., TAKAHASHI, K., and YOSHII, M. (1976). Two components of the calcium current in the egg cell membrane of the tunicate. J. Physiol.
255, 527-561.
21. PAPAHADJOPOULOS, D. (1972). Studies on the mechanism of action of local anesthbtics with phospholipid model membranes. Biochim. Biophys. Acta 265, 169-186.
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22. PAUL, M. (1970). Fertilization-associated changes in the eggs of Strongylocentrotus purand Urechis caupo. Ph.D. Thesis, puratus Stanford University, Stanford, Calif. 23. PAUL, M. (1975a). The polyspermy block in eggs of Urechis caupo. Evidence for a “rapid” block. Exp. Cell Res. 90, 137-142. 24. PAUL, M. (1975b). Release of acid and changes in light scattering properties following fertilization of Urechis caupo eggs. Develop. Biol. 43, 299-312.
25. POSTE, G., PAPAHADJOPOULOS, D., and NICOLSON, G. L. (1975). Local anesthetics affect transmembrane cytoskeletal control of mobility and distribution of cell surface receptors. Proc. Nat. Acad. Sci. USA 72, 4430-4434.
26. RAMAKRISHNA, T. V., ROBINSON, J. W., and WEST, P. W. (1966). The determination of calcium and magnesium by atomic absorption spectroscopy. Anal. Chim. Acta 36, 57-64. 27. REED, K. C., and BYGRAVE, F. L. (1974). The inhibition of mitochondrial calcium transport by lanthanides and ruthenium red. B&hem. J. 140, 143-155. 28. ROBINSON,K. R., and JAFFE, L. F. (1973). Ion movements in a developing fucoid egg. Develop. Biol. 35, 349-361. 29. SCHUETZ, A. W. (1975). Induction of nuclear breakdown and meiosis in Spisula solidissima oocytes by calcium ionphore. J. Exp. 2001. 191, 433-440. 30. SHEN, S., and STEINHARDT, R. A. (1976). An electrophysiological study of the membrane properties of the immature and mature oocyte of the batstar, Patiria miniatu. Develop. Biol. 48, 148-162. 31. SIMKISS, K. (1974). Calcium translocation by cells. Endeavour 33, 119-123. 32. SKOOG,D. A., and WEST, D. M. (1971). “Principles of Instrumental Analysis,” pp. 299-300. Holt, Rinehart and Winston, New York. 33. STEINHARDT, R. A., and EPEL, D. (1974). Activation of sea urchin eggs by a calcium ionophore. Proc. Nat. Acad. Sci. USA 71, 19151919. 34. STEINHARDT, R. A., EPEL, D., CARROLL, E. J., and YANAGIMACHI, R. (1974). Is calcium ionophore a universal activator for unfertilized eggs? Nature (London) 252, 41-43. 35. TYLER, A., and J. SCHULTZ. (1932). Inhibition and reversal of fertilization in eggs of the echinoid (sic) worm, Urechis caupo. J. Exp. Zool. 63, 509-531. 36. VACQUIER, V. D. (1975). The isolation of intact cortical granules from sea urchin eggs: Calcium ions trigger granule discharge. Develop. Biol.
43, 62-74.