DEVELOPMENTAL

BIOLOGY

146,396-405(1991)

The Calcium Content of Cortical Granules and the Loss of Calcium from Sea Urchin Eggs at Fertilization ISABELLE GILLOT,**~BRIGITTECIAPA,~. *Unit& de Biologie tLabwatoire

PATRICKPAYAN,~

AND CHRISTIAN~ARDET*

Cellulaire Marine, URA 671 CNRS, Universite’ de Paris VI, Station Zoologique, 06230 Villefranche-sur-mer, France, and de Physiologie Cellulaire et Comparhe, URA 651 CNRS, Universite’ de Nice, Pare Valrose, 0603.&Nice Cedex, France Accepted April

23, 1991

In many species, fertilization triggers a wave of cortical granule exocytosis in the egg that is the consequence of an increase in intracellular free calcium concentration. We have measured the total calcium content of cortical granules from two species of sea urchins by quantitative X-ray microanalysis and spectrometric measurements. Our results show that cortical granules: (1) contain a high concentration of total calcium (around 30 and 95 mMfor Paracentrotus lividus and Arbacia lixula, respectively), (2) represent a major cortical storage site of calcium in the egg (5 and 11% of total egg calcium for P. lividus and A. lixula, respectively), and (3) exchange part of their accumulated calcium by an ATP dependent mechanism. In addition we have confirmed that at fertilization, sea urchin eggs lose a sizeable amount of their calcium (7% for P. lividus and 15% for A. lixula). The kinetics and magnitude of the loss suggest that some of this calcium could be provided by cortical granules during exocytosis. o 1991 Academic Press, 1~.

is in turn held responsible for the wave of exocytosis and egg activation (Zucker et al., 1978; Schmidt et al, 1982; Most vertebrate and invertebrate eggs contain an Whitaker, 1987). array of cortical secretory vesicles (or cortical granules) Most of the calcium present in unfertilized eggs is tightly adhering to the plasma membrane (Vacquier, bound, but its precise subcellular localization is still un1975,198l; Schuel, 1978,1985; Guraya, 1982; Sarclet and clear (Mazia, 1937; Nakamura and Yasumasu, 1974; disChang, 1987). At fertilization a wave of elevated cytocussed in Gillot et al, 1990). To approach this question, plasmic calcium traverses the egg from the point of some investigators have made use of chlortetracycline sperm entry and triggers a wave of exocytosis of the fluorescence (Ohara and Sato, 1986; Schatten and Hemcortical secretory vesicles (for review see Whitaker and mer, 1979). Others have localized calcium in various egg Steinharclt, 1985; Jaffe, 1988; Gillot et al, 1990). This organelles in the electron microscope after precipitation process has been particularly well studied in sea urchin with fluoride, oxalate, or antimonate combined with eggs where upon exocytosis the enzymes and structural qualitative X-ray microanalysis (Cardasis et al., 1978; proteins contained in thousands of cortical granules Sarclet and Chang, 1985; Poenie and Epel, 1987) or X-ray modify the egg vitelline coat and produce a chemically microanalysis of fixed eggs (Ornberg and Reese, 1981). and mechanically stable fertilization membrane (Veron We have reviewed these approaches and the possible et aZ.,1977; Schuel, 1978; Carol1 and Enclress, 1982; Batmethodological problems associated with them (Nicaise taglia and Shapiro, 1988). The fusion of the cortical granules with the plasma membrane can even be ob- et aZ.,1989). We have recently developed a preparative technique served in isolated or reconstituted cell surface comthat circumvents most of these difficulties. It is based plexes (or cortices) by simply raising the calcium level of on quick-freezing of the eggs, subsequent freeze-substithe surrounding milieu (Vacquier, 1975,198l; Crabb et tution in presence of oxalic acid, and embedding folcd., 1987; Whitaker, 1987; Zimmerberg and Liu, 1988). lowed by quantitative analysis of calcium with X-ray Although there is evidence that calcium influx is inmicroanalysis. The calcium content of individual eggs creased at fertilization, it is commonly thought that inmeasured in this fashion were in complete agreement tracellular stores are the major source of the rise in with spectrophotometric measurements made on egg hocytosolic calcium. This elevated calcium concentration mogenates (Gillot et aZ.,1989). We have now measured the total calcium content of the major subcellular vesicular organelles and combined these observations with isoi To whom correspondence should be addressed. INTRODUCTION

0012-1606/91 $3.00 Copyright All rights

0 1991 by Academic Press, Inc. of reproduction in any form reserved.

396

GILLOTETAL.

Sea Urchin Egg Calcium

Content and Loss at Fertilization

397

(Na+, Cl-, K+, and Ca’+) by a rapid filtration technique (Payan et al., 1981). Two milliliters of egg suspension (5%) was rinsed with an ice-cold Ca’+-free solution containing: 1100 mMglycine, 10 mMTris-HCl, pH 8.2, freed of calcium by passage through Chelex 100 (Bio-Rad Laboratories, Richmond CA). Preparation of samples: Cartices. Eggs were stuck on plastic dishes 9 cm in diameter (Falcon 3003) coated MATERIALS AND METHODS with polylysine. The egg cortices were prepared as previously described by Payan et al. (1986) and Sardet (1984) Biological Material by shearing eggs in a calcium-free buffer adapted from Paracentrotus lividus and Arbacia lixula sea urchins Suprynowicz and Mazia (1985) to mimic the composition were collected from the bay of Villefranche-sur-mer and of the egg cytoplasm (F solution) (K gluconate 250 mM, gametes handled as previously described (Payan et al., N-methyl glucamine 250 mM, Hepes 50 mM, Na,HPO, 5 1981). mM, EGTA 100 PM, pH adjusted to 7.4 with acetic acid). To prepare cortices depleted in cortical granules, corX-Ray Microanalysis tices prepared as above were exposed to 0.005% digitonin in F solution for 3 min, then rapidly rinsed with a Specimen preparation. Eggs were dejellied into a solucalcium free N-methyl glucamine solution (N-methyl tion (EMC) containing 25 mM EGTA, 548 mMNaCJ2.4 glucamine 1100 mM, pH 8, treated with chelex 100). mMNaHCO,, 40 mMTris, pH 8.2 (Detering et al., 1977), After removing the excess milieu, cortices were covered and then washed in sea water. A drop of concentrated with 4 ml of bidistilled water and scraped off. This same eggs suspension was deposited on aluminum foil which 4 ml was used again to cover the second dish, instead of was successively coated with collodion (1% in amyl acebidistilled water, and the operation was repeated five tate) and polylysine (0.5% in EMC), then rinsed with times. The calcium content from five dishes was thus distilled water and dried. The procedures for quickconcentrated in 4 ml for spectrometric analysis. freezing, freeze-substitution, and embedding have been After digitonin treatment, the number of stuck corpreviously described (Gillot et al., 1989). Sections were tices was determined to be equivalent to that of controls stained by uranyl acetate and lead citrate for ultrastrucand results were expressed as femtomoles of calcium/ tural observations. cortex. X-ray microanalysis and quanti&cation. X-ray microMeasurement of total calcium by flame photometry. analysis was performed with a Philips CM 12-TEM anaEggs and cortices collected in bidistilled water were lytical microscope equipped with an energy-dispersive disrupted with a sonicator (W 10 Son&) and calcium device (EDS) Tracer TN 5400 (see Gillot et al., 1989). The content was determined after addition of HCl in a flame unstained sections (150 nm thick) were analyzed during photometer (Eppendorf-Geratebau, Hamburg, FRG). 40 set at 100 kV and with the probe current regulated at The protein contents of egg and cortex samples were approximatively 4 nA. Quantitation was carried out respectively measured by the Lowry and Bradford techwith the BioQ program from Tracer-Northern, using niques. The results were expressed as picomoles of calthe continuum method of Hall (1971). The mass thickcium/egg and femtomoles of calcium/cortex. ness of the section was estimated by taking the integral 45Ca eflux measurements. To study 4SCarelease, corof the specimen spectrum between 4.5 and 6 keV, tices were first loaded for 30 min in F solution containcorrected from a comparable spectrum sector taken in a ing 185 kBq of 45Ca and 100 PM EGTA. CaCl, was added nearby region on the supporting film (recorded during to set the ionized calcium concentration at 0.5 PM. We 200 set). Sections of 150 nm were observed at a magnifistudied the effects of A 23187 and digitonin on 45Caefcation of 7100.During all measurements the probe diamflux by sampling external medium at suitable times (see eter was smaller than that of the analyzed cortical or Payan et al., 1986). yolk granules. For calibration, we used as standard 1% Ca naphtenate graciously given to G. Nicaise by Dr. Ornberg (Bethesda). topic flux measurements and morphometric analysis to assess the calcium content of cortical granules and their role in egg calcium homeostasis. In particular, we discuss the possibility that calcium loss at fertilization originally described by Azarnia and Chambers (1976) could be due in the part to the exocytosis of the calcium rich cortical granules.

Morphometric

Determination

of Calcium Content of Eggs and Cartices

Preparation

of samples: Eggs. Total cellular calcium

content was measured after elimination of external ions

Analysis

of Cortical Granules and Cartices

In order to quantify the total calcium present in cortical granules, we determined the morphometric parameters of cortical granules on isolated cortices. The cor-

398

DEVELOPMENTALBIOLOGYV0~~~~146,1991

tices from eggs of the two sea urchin species were prepared exactly as described above (see Preparation of Samples: Cortices). For some observations (fluorescence, DIC) they were prepared on glass slide rather than on plastic dishes. Dark field (low resolution) and polarized light (high resolution) images of cortices recorded through a newicon camera were digitalized on a AT computer equipped with a Matrox card and the appropriate software (Image I, Universal Imaging). From such images, we measured the number of cortices per surface area and the average diameter of cortices. The average diameter of cortical granules were further estimated with the help of an image analysis program (Imagenia, Biocom). RESULTS

We measured the calcium content of cortical granules in two sea urchin egg species using two complementary strategies: direct measurement of cortical granule content in situ by quantitative X-ray microanalysis, and spectrometric measurement of calcium in cortices originally called cortical granule lawns or layers (Vacquier, 1975,198l) before and after selectively bursting the cortical granules with digitonin. Quantitative in Situ

X-Ray Microanalysis

of Cortical Granules

an anastomosing network of endoplasmic reticulum tubules can be isolated from eggs affixed to a polycationic surface by mechanical shearing with a stream of isotonic calcium-free solution (Vacquier, 1975, 1981; Sardet, 1984; Sardet and Chang, 1987). When exposed to low digitonin concentrations such cortical granule lawns rapidly change their light scattering characteristics as their cortical granules selectively burst (Fig. 3) (Zimmerberg and Liu, 1988). The endoplasmic reticulum network present in isolated cortices (Sardet 1984;Chandler, 1984; Henson et al, 1989) is the major organelle that remains after digitonin treatment. After 2-3 min of exposure to digitonin, the endoplasmic reticulum takes a beaded appearance (Fig. 3~). Because such cortices can still pump calcium in the presence of ATP with twothirds the efficiency of intact cortices (Biyiti et al., 1990), we assume that only cortical granules and some pigment granules are lysed by digitonin treatment. We could thus obtain an estimate of cortical granule calcium content by measuring the calcium content of isolated cortices before and after a 3-min exposure to 40 pib’ digitonin. In P. lividus, 35% of cortical calcium is lost after the opening of cortical granules (control cortices, 64 fmole/cortex; digitonized cortices, 41 fmole/cortex). By measuring the density and the diameter of cortical granules, the average area occupied by a cortex, and the density of stuck cortices per surface area (Table 2), and by the results of X-ray microanalysis to measure the concentration of calcium in cortical granules, we could therefore estimate the calcium content of cortical granules and its contribution to total egg calcium (see discussion).

It is possible to distinguish cortical granules from other vesicular organelles (mitochondria, pigmented vesicles, yolk granules) in unstained sections of rapidly frozen, freeze-substituted, and embedded samples (Fig. 1). We could thus probe individual cortical granules in several eggs from different females and compare their Calcium Exchange in Cortical Granules ionic content to that of yolk granules. In both species It is possible to load 45Cainto the organelles of isoexamined, the calcium content of cortical granules was 9 to 10 times higher than that measured in yolk granules lated cortices in the presence of ATP (Oberdorf et al., 1986; Payan et al., 1986). After we allowed exchange and (Table 1). The values of chlorine, phosphorus, and sulfur concentrations obtained for both organelles showed pumping to occur for 30 min, we could study the natural much less difference. The calcium content of cortical release of 45Caby the cortical organelles (principally granules from the eggs of A. lixula was on average 3 cortical granules and endoplasmic reticulum) and comtimes higher than cortical granules in P. lividus, respec- pare it to that brought about by the bursting of cortical tively about 95 and 30 mM (1 mmole of calcium/kg of granules with digitonin. As illustrated by Fig. 4, about Epon-embedded tissue corresponds to about a concen- one-third of exchangeable calcium was released upon tration of 1 mMaccording to Nicaise et al., 1989). In both digitonin treatment. The remaining calcium could be respecies histogram analysis shows there are large varia- leased by treatment with the calcium ionophore A23187 tions among individual cortical granule (Fig. 2). These (Fig. 4). These experiments indicate that at least part of variations are not related to different eggs within the the calcium sequestered in cortical granules is exchangeable. same batch or to the different egg batches analyzed. Calcium Content oj’Cwtica1 Granules in Isolated Cortices

Calcium Release at Fertilization

A cell surface layer consisting mainly of cortical granules, a few pigment granules, or acidic vesicles and

Using flame spectrophotometry we measured the total calcium content of eggs of P. lividus and A. lixula

GILLOT ET AL.

Sea Urchin

Egg Calcium

Content

and Loss at Fertilization

FIG. 1. Electron micrographs of sections of the cortical zone of an unfixed unfertilized sea urchin egg (Paracentrotus lividus) after quick-freezing, freeze-substitution, and embedding. (a) Stained section: cortical granules which are located just beneath the plasma membrane are characterized by a spherical to elliptical shape, and display typically coiled contents. Yolk platelets which are abundant in the egg and in the vicinity of cortical granules are easily distinguished because of their larger size and their lower electron density. Mitochondria have the same electron density and the same shape as cortical granules, but they are consistently smaller in size and scattered throughout the egg. (b) Unstained section: cortical granules can still be identified. The size of the spot used for X-ray microanalysis is shown. cg, cortical granules; y, yolk granules; m, mitochondria; p, plasma membrane; bar, 0.5 pm.

its contribution to the large loss of calcium detected at fertilization in sea urchin eggs (Azarnia and Chambers, 1976). We used two independent methods to estimate cortical granules calcium content in two sea urchin egg species. They gave values in good agreement.

before and after fertilization (Table 3). We confirmed that total calcium content of unfertilized eggs of P. Zividus was significantly lower than that of A. lixula (Gillot et al., 1989, reviewed in Gillot et al., 1990). Within 4 min (i.e., after all cortical granules have exocytosed), the calcium of eggs of both species had significantly decreased (respectively 7 and 15% in P. lividus and A. lixula, Table 3).

Cortical Granules Calcium

DISCUSSION

Contain a Hi,yh Concentration

of

It has been repeatedly reported that sea urchin cortical granules contain calcium precipitates after qualitative histochemical localization (Cardasis et al., 1978;

The main purpose of the present work was to analyze the calcium content of cortical granules and appreciate TABLE

1

ELEMENTALCONCENTRATIONSINCORTICALGRANULESANDYOLKGRANULESOFTWOSPECIESOFSEA URCHIN EGGS Concentration Species Pn mcentrofl~s livid1rs A rbacirr lixulrr

Organelle CG YG CG

YG

71 31 14 32 13

Calcium 32 4.6 97 10.3

i k + t

3.8 1.56* 9.5 3.08**

(mmol/kg Chlorine

370 192 536 503

* * * k

19.0 35.3 33.3 127.3

of Epon-embedded Phosphorus 34 19.8 34 29.5

* -t * k

4.1 4.16 5.0 5.6

tissue) Sulphur 64 19.7 159 60

k f f +

4.6 3.20 15.1 9.9

ATote. CG, cortical granules; YG, yolk granules. 16,number of granules analyzed. Concentrations were calculated from X-ray spectra obtained from the two different organelles according to Materials and Methods and are expressed as the mean * SEM. P. statistical significance of calcium content calculated by Student’s t test; calcium content of yolk granules is significantly different from 0 (*P cc 0.002 **P < 0.001). Cortical granules contains significantly more calcium than the yolk granule: P < 0.001 for both Paraceutrotxs and Arbncia. Nine eggs were analyzed for CG in the two species. For yolk granules, 11 eggs were analyzed in Parncenfrotus li~~idus and 7 eggs in Arbtccio Li.ruln.

400

DEVELOPMENTALBIOLOGY

VOLUME146,1991

granules and that these values differed considerably in the two species examined (see Table 1). We do not know at present if the fairly large scatter in the values of individual cortical granules represents real variations in the maturity of granules or reflects normal variations in their internal content. Cortical granules are secretory vesicles found in many other eggs and comparable to other storage granules in secretory cells, including acinar cells of exocrine pancreas (Clemente and Meldolesi, 1975), blood platelets (Sat0 et al, 1975), anterior pituitary (Cramer et ah, 1976), and presynaptic terminals (Politoff et ah, 1974). Quantitative estimates have also been made of calcium in several types of secretory granules (see review by Nemere, 1990). Most of these secretory granules contain large amount of calcium. In this respect, it is noteworthy that cortical granules in polychaete oocytes (Emmanuelson and Odselius, 1985) and pigment granules in amphibians oocytes (Andeuccetti et al., 1984) and sea urchin eggs (Poenie and Epel, 1987) are also known to contain calcium. The high calcium concentration in the millimolar range found in cortical granules of sea urchin eggs is 20 40 60 80 100 120 140 160 180 0 comparable in concentration to that measured in sarcomoles/kg of Upon-embedded tissue plasmic reticulum of muscle (Somlyo et ah, 1981), secreFIG. 2. Distribution histograms of calcium concentration (mmole/ tory granules of chromaffin cells (Ornberg et al., 1988), kg of Epon-embedded tissue) of cortical granules from unfertilized sea and egg endoplasmic reticulum (Gualtieri and Sardet, urchin eggs of Paracentrotus lividus (A) and Arbacia lixulu (B) deter1989), where high-capacity calcium-binding proteins mined by X-ray microanalysis. The area probed was systematically in the central part of the organelles. The histogram scale is 10 units per have been characterized (Oberdorf et al., 1988;Henson et class in abscissa. Means of the data set are 32 f 3.8 and 97 f 9.5 al., 1989; Sardet et al., in preparation). It is therefore (tSEM) respectively but scatter and possible plurimodal distribution likely that the cortical granules also contains calciumin Arbacia is apparent. In insert, energy dispersive spectrum obtained binding macromolecules. These may be in part strucafter probing one cortical granule of an unfertilized Arbaciu eggs. The tural proteins precursor of the fertilization membrane copper signal comes from the grid, where the section is deposited. P, phosphorus; S, sulphur; Cl, chlorine; Ca, Calcium; Cu, copper. densely packed in the granules such as the calcium precipitable protein hyalin (Stephens and Kane, 1970) or enzymatic components that have been shown to bind Ornberg and Reese, 1981; Sardet and Chang, 1985; calcium (Battaglia and Shapiro, 1988). Poenie and Epel, 1987). For quantitative estimate of this calcium, we used a method based on rapid freezing and Cortical Granules and Fertilization freeze-substitution (Gillot et al., 1989). Its application to At least part of the calcium present in cortical granmeasuring cortical granules and yolk granules ionic content showed that individual cortical granules con- ules is exchangeable and our experiments with isolated tain a much higher amount of calcium than do yolk cortices suggest that an ATP-dependent calcium pump

FIG. 3. The unfertilized sea urchin egg cortex in light microscopy. (a) In dark field microscopy, a field of cortices (Paracentrotus Zividus) isolated by shearing eggs stuck onto a polylysine-coated coverslip (moderate shear). Cortical granules cause the light to scatter intensely on each cortex. Bar, 50 Km. (b) The same field of cortices after digitonin treatment (0.005% during 3 min): cortical granules have disappeared, leading to a decrease in light scattering. The pigment granules band remains clearly visible (see Sardet and Chang, 1985). Bar, 50 pm. (c, d) Phase-contrast microscopy of a piece of stuck cortex (Puracentrotus lividus), before digitonin treatment (c) and after digitonin treatment. Most cortical vesicles have disappeared (d). Bar, 5 Km. (e, f) Same field of cortex in light microscopy using a blue filter in which the vesicles forming the pigment granule band are clearly visible, before (e) or after digitonin treatment (f). Most of the pigmented vesicles have disappeared. Bar, 5 pm. (g, h) Endoplasmic reticulum of a single cortex (Arbucia lizulu) visualized with DioC,3 (1 ~Mduring 1 min). (g) control; (h) after digitonin treatment (3 min); bar, 10 pm.

GILLOTETAL.

Sea Urchin

Egg Calcium

Cvntwt

und Loss at Fertiliznticm

401

402

DEVELOPMENTAL BIOLOGY

TABLE 2 MORPHOMETRIC CHARACTERISTICS AND CALCIUM CONTENT OF CORTICAL GRANULES AND STUCK DOWN CORTICES Species Cortical granules Diameter (pm) Density (number of cortical granules/fim’) Calcium content in cortical granules of a single egga (X-ray microanalysis) Cortex Surface (km’) Density x lo5 (number of cortices/fim’) Calcium contained in one full isolated cortex (flame photometry)* Calcium contained in cortical granules of a full cortexC

Paraeentrotus

lividus

Arbacia

VOLUME 146, 1991 loo%-.

A corm01

lixula

60 % 0.55 f 0.049 (,n = 127)

0.66 f 0.01 (n = 234)

1.37 f 0.048 (n = 4)

0.94 f 0.06 (n = 8)

95 fmole

279 fmole

7363 * 177 (n = 65)

5394 + 67 (n = 70)

8.7 + 0.32 (n = 5)

13.2 f 0.34 (n = 4)

243 fmole

n.d.

87 fmole

n.d.

Note. Values are given as means f SEM; n, number of measurements. nd.: not determined. ’ Calculated from the calcium concentration in cortical granules (Table l), cortical granule diameter, density in present table, and egg diameter (Gillot et al., 1989). *The area of the full cortex is 3.8 times the average stuck down cortex area calculated from the ratios of whole egg area and average stuck down cortex area (in present table). ’ The calcium content is deduced from flame photometry measurements made before and after digitonin treatment (respectively 64 and 41 fmole per cortex).

participates in the accumulation of calcium in cortical granules. This has been already suggested by Schatten and Hemmer (1979). It is possible that after exocytosis such a calcium pump is incorporated in the plasma membrane and participates in the maintenance of a calcium efflux (Steinhardt and Epel, 1974; Walter et aZ., 1989), thus contributing to restore a low cytosolic calcium concentration, a role usually mainly attributed to intracellular organelles (see Nemere, 1990). It is difficult at Jresent to know if cortical granules calcium participates in the intracellular release of calcium observed at fertilization. This role is generally attributed to the endoplasmic reticulum although there is no definite experimental evidence for it (see F’sen and Reynolds, 1985; Ohara and Sato, 1986; Poenie and Epel, 1987; and Gillot et ah, 1990 for experimental evidence and discussions). The fact that egg activation and development can proceed without cortical granules exocytosis (Schmidt and Epel, 1983) and that calcium release

5

0

10 Time

15

(min.)

FIG. 4. Loss of 45Ca from isolated egg cortices (Paracentrotus lividus). Cortices were preloaded with 45Caduring 30 min in the presence of ATP, then the isotope was removed. After a period of 5 min, digitonin was added (arrow 1) to a final concentration of (0.005%) (a). Calcium ionophore A23187 (50 &I was added (arrow 1) to a similar preparation immediately (B) or 5 min after digitonin (arrow 2) (w). A control experiment showing the usual course of 45Ca efflux is also shown (A). Results are expressed as a percentage of the total 46Ca originally accumulated during the pumping period.

and wave propagation are still observed in eggs that do not have cortical granules (ascidians for example, see Speknisjder et al, 1989, 1990) argues against a preeminent role for cortical granules in intracellular calcium release. The Loss of Calcium at Fertilization Cortical Granules Exocytosis

and Its Relaticm to

Our quantitative measurements of cortical granules content by X-ray microanalysis in two species of sea urchin eggs show that there is a good correspondence between what is effectively lost by the egg after cortical granule exocytosis following fertilization and the calTABLE 3 VARIATION OF TOTAL CALCIUM CONTENT OF SEA URCHIN EGGS AT FERTILIZATION Paracentrotus

Unfertilized Postfertilization (4 min) Difference Percentage of decrease

lividus

Arbacia

lixula

1.78 t 0.130 1.66 2 0.164 A = 0.12 f 0.052 6.9%

2.50 AZ0.433 1.81 + 0.447 A = 0.44 + 0.164 14.7%

P < 0.05

P < 0.02

Note. Egg calcium content was determined by flame photometry, and expressed in pmoles of calcium/egg. Values are the mean of 20 experiments from eight different females in unfertilized and fertilized eggs for 4 min. Paired data comparison gave P values.

GILLOT

ET AL.

The Cortex of the Egg is a Calcium-Rich

TABLE 4 DETERMINATION OF RELATIVE CALCIUM CONTENT IN CORTICAL GRANULES AND EGGS

Paracentrotus Parameters a Egg diameter * Egg surface ’ Egg volume ’ Unitary cortical granules volume e Total number of cortical granules per egg fTotal volume represented by cortical granules Ratio cortical granules volume/egg volume *Calcium content of a single egg h Calcium content in cortical granules of a single egg Ratio cortical granules calcium/egg calcium W/7) ’ Measured decrease in egg calcium

Units

lividus

Arbacia lixula

pm l.rm’ pm3

94 27,744 434,672

73 16,732 203,585

pm3

0.077

0.150

38,120

15,728

2989

2359

5%

0.6

1.2

pmole

1.78

2.5

fmole

95

279

5%

5.3

11.1

a

6.9

14.7

pm3

403

Sea Urchin Egg CalciwL Content and Loss at Fertilization

a Egg diameter from Gillot et al., 1989. *sc Calculation of egg surface and egg volume assume the egg to be a sphere. ’ Calculation of cortical granule volume was done by assimilating cortical granule to a sphere of 0.55 pm and 0.66 pm of diameter respectively in Parucentrotus lividus and Arbacia lixula (see Table 2). ‘Total number of cortical granule in one egg was obtained by the product of the density of cortical granule measured in cortices (Table 2) and the surface of one egg. fTotal volume of cortical granule in one egg is given by multiplying the number of cortical granules by their unitary volume. B Calcium content in one egg was obtained by spectrometric measurement (see Table 3). h Calcium content in cortical granule of a single egg is the product of intravesicular calcium (microanalysis) and the total volume of cortical granule in one egg (see Table 2). ’ From Table 3.

cium contained in the cortical granules after the first 4 min when exocytosis is complete (Table 4). It is therefore tempting to hypothesize that calcium loss at fertilization representing 7 to 15% of total egg calcium is mainly due to the release of cortical granule content during exocytosis. However, we cannot exclude that a part of the calcium release at fertilization is due to cytoplasmic calcium that has risen to micromolar concentrations at fertilization (reviewed in Gillot et ah, 1990). Further experiments on the exact kinetics of the loss of calcium with a vibrating calcium electrode will be necessary (Kiihtreiber and Jaffe, 1990).

Zone

We could estimate that calcium in P. lividus retained in the isolated cortex and contained in cortical granules, the endoplamic reticulum network and pigmented vesicles amounted to about 14% of the egg calcium although on a protein basis this layer only accounts for 1% of total egg protein. From morphometric measurements (Table 2) we calculated that the surface of a single egg is equivalent to the area occupied by 3.8 stuck down isolated cortices; one egg contains 1.78 pmole of calcium while the corresponding cortical area retains 64 X 3.8 = 0.243 pmole of calcium). Our estimates indicate that a third of the calcium is contained in cortical granules, the remaining part must be mainly sequestered in the endoplasmic reticulum network (Chandler, 1984; Sardet, 1984; Oberdorf et al., 1986; Payan et ah, 1986; Henson et al., 1989) which has been shown to contain a high-capacity calcium-binding protein (Oberdorf et al, 1988; Henson et al., 1989) and calcium antimonate deposits (Sardet and Chang, 1985; Poenie and Epel, 1987). In addition in P. lividus some of this cortical calcium must be contained in pigmented vesicles (Sardet and Chang, 1985). We thank Ghislain Nicaise for his comments about the manuscript and his support and advice with the X-ray microanalysis experiments performed in the “Centre Commun de Microscopic Appliquee”; Claudine Gleyzal for her expertise with this technique; and Christian Rouviere for his help with image analysis. We wish to thank the CEA (Department of Biology) for facilitating the purchase of radioactive products.

REFERENCES ANDREUCCETTI, P., DENIS-DONINI, S., BURRINI, A. G., and CAMPANELLA, C. (1984). Calcium ultrastructural localization in Xenopus Zaevis eggs following activation by pricking or by calcium ionophore A 23187. J. Exp. Zool. 229,295-308. AZARNIA, R., and CHAMBERS, E. L. (1976). The role of divalent cations in activation of the sea urchin egg. I. Effect of fertilization on divalent cation content. J. Exp. Zool. 198, 65-77. BAT~AGLIA, D. E., and SHAPIRO, B. M. (1988). Hierarchies of protein cross-linking in the extracellular matrix: Involvement of an egg surface transglutaminase in early stages of fertilization envelope assembly. J. Cell Biol. 107, 2447-2454. BIYITI, L., PESANDO, D., PUISEUX-DAO, S., GIRARD, J.-P., and PAYAN, P. (1990). Effect of antibacterial plant flavanones on the intracellular calcium compartment involved in the first cleavage of sea urchin eggs. Toxicon. 28,275-283. CARDASIS, C. A., SCHUEL, H., and HERMAN, L. (1978). Ultrastructural localization of calcium in unfertilized sea-urchin eggs. J. Cell Sci. 77, 101-115. CAROLL, E. J., and ENDRESS, A. G. (1982). Sea urchin fertilization envelope: Uncoupling of cortical granule exocytosis from envelope assembly and isolation of an envelope intermediate from Strong& centrotus pwpuratus embryo. Dev. Biol. 94,252-258. CHANDLER, D. E. (1984). Exocytosis in v&o: Ultrastructure of the

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