DEVELOPMENTAL

BIOLOGY

14%

80-89

(19%)

Repetitive Calcium Transients and the Role of Calcium in Exocytosis and Cell Cycle Activation in the Mouse Egg Department

DOUGLAS

KLINE

of Biological

Sciences, Accepted

AND JOANNE T. KLINE Kent

State

September

University,

Kent,

Ohio 44242

9, 1991

The role of calcium in cortical granule exocytosis and activation of the cell cycle at fertilization was examined in the mouse egg using the calcium chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N:N’tetraacetic acid) and the fluorescent calcium indicator fluo-3. BAPTA and flue-3 were introduced into zona-free mouse eggs by a 30-min incubation with 0.01-50 PM BAPTA acetoxymethyl ester (AM) and/or l-20 ~IM fluo-3 AM prior to in vitro fertilization. Incubation of eggs in a5.0 flBAPTA AM inhibited cortical granule exocytosis in all cases. Introduction of the calcium chelator into the egg blocked second polar body formation at al.0 pM BAPTA AM. Sperm entry occurred in all eggs regardless of the BAPTA AM concentration. Sperm induce a large transient increase in calcium lasting 2.3 + 0.6 min, followed by repetitive transients lasting 0.5 f. 0.1 min and occurring at 3.4 f 1.4-min intervals. Incubation with a5.0 PM BAPTA AM inhibited all calcium transients. Introduction of BAPTA also inhibited calcium transients, exocytosis, and the resumption of meiosis following application of the calcium ionophore A23187 or SrCl,, which activate eggs. These results demonstrate that the calcium increase at fertilization is required for cortical granule exocytosis and resumption of the cell cycle in a mammalian egg. o 19szAcademic PMS, IN. INTRODUCTION

1978; Cuthbertson et a& 1981; Cuthbertson, 1983; Fraser, 1987; Cran et al. 1988; Miyazaki, 1988; Colonna et d, 1989; Marcus, 1990). We tested whether the calcium rise occurring at activation of the mouse egg and the subsequent activation events could be inhibited by introduction of a calcium chelator into the egg before in vitro fertilization or artificial activation. The initial responses of the egg to activation by the sperm include, among others, cortical granule exocytosis and the resumption of meiosis. In this report, we examine calcium changes in the mouse egg and provide supportive evidence that activation of the mammalian egg by sperm requires an increase in intracellular free calcium.

Calcium is thought to be the primary intracellular signal responsible for the initiation of development of the egg following fertilization (Whittingham, 1980; Jaffe, 1983; Whitaker and Steinhardt, 1985). This hypothesis is supported by three experimental observations made in several species: First, an increase in calcium in the egg occurs at fertilization; second, artificially raising intracellular calcium usually initiates egg development; and third, suppressing the natural rise in calcium prevents the initiation of egg development. A rise in calcium during activation of the mammalian egg has been detected with calcium electrodes and with the calcium-dependent luminescent protein, aequorin (Cuthbertson and Cobbold, 1985; Igusa and Miyazaki, 1986; Miyazaki et al, 1986). Artificially increasing intracellular calcium by injection of Ca2’ ion or application of the calcium ionophore A23187 causes parthenogenic activation of mammalian eggs (Steinhardt et ah, 1974; Fulton and Whittingham, 1978; Igusa and Miyazaki, 1983; Ducibella et aZ., 1988). In addition, other agents promoting an increase in intracellular calcium cause egg activation, including ethanol, SrCl,, and inositol trisphosphate (InsP,)l (Whittingham and Siracusa,

MATERIALS

Media and reagents. The media used in these experiments were M16, M2, and in vitro fertilization medium (IVF; Hogan et uL, 1986). The composition of Ml6 was 94.7 mM NaCl, 4.8 mM KCl, 1.7 mM CaCl,, 1.2 mM KH,PO,, 1.2 mM MgSO,, 25.0 mM NaHCO,, 23.3 mM sodium lactate, 0.33 mM sodium pyruvate, 5.6 mM glucose, 100 units/ml penicillin G (potassium salt), 50 pg/ ml streptomycin sulfate, 0.001% phenol red, and 0.4% bovine serum albumin (BSA, type V; Calbiochem, La Jolla, CA). M2 contained 94.7 mM NaCl, 4.8 mM KCl, 1.7 mM CaCl,, 1.2 mM KH,PO,, 1.2 mM MgS04, 4.2 &IM NaHCO,, 23.3 mMsodium lactate, 0.33 mMsodium pyru-

i Abbreviations used: aeetoxymethyl ester, AM; l,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid, BAPTA; bovine serum albumin, BSA; dimethyl sulfoxide, DMSO, fluorescein isothiocyanate, FITC; inositol trisphosphate, InsPa; in vitro fertilization medium, IVF; Len-s culinaria agglutinin, LCA; polyvinyl alcohol, PVA. 0012-1606/92 $3.00 Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

AND METHODS

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KLINEANDKLINE

Calcium and Mcwe Egg Activation

vate, 5.6 mMglucose, 20.9 mMHepes, 100 units/ml penicillin G (potassium salt), 50 pg/ml streptomycin sulfate, and 0.001% phenol red. BSA was replaced by 0.1 YO polyvinyl alcohol (PVA). PVA was used to prevent damage to eggs during washing and transfer by pipet. The composition of IVF was 99.3 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl,, 0.5 mM MgCl,, 0.36 mM NaHzPO,, 25.0 mM NaHCO,, 25.0 mM sodium lactate, 0.5 mM sodium pyruvate, 5.6 mM glucose, 100 units/ml penicillin G (potassium salt), 50 pg/ml streptomycin sulfate, 0.001% phenol red, and 3% BSA. Ca2+, Mg2+-free IVF consisted of IVF without CaCl, or MgCl,, with NaCl increased to 102.8 mM; BSA was replaced by 0.1% PVA. Similar Ca2+, M $+-free media have been used in previous studies of artificial activation of mammalian eggs (Steinhardt et c& 1974; Ducibella et al, 1988; Marcus, 1990). Media were made with cell culture reagents and tissue culture grade water. All chemicals, except where noted, were obtained from Sigma Chemical Co. (St. Louis, MO). The pH of each standard medium was 7.4. The pH of IVF with reduced BSA or without BSA was adjusted by addition of HCl. All media were filtered through 0.2-pm Acrodisc filters (Gelman Sciences, Ann Arbor, MI) and equilibrated at 37°C in an atmosphere of 5% C02, 95% air. Preparation of gametes.Eggs were obtained from 8- to 12-week-old NSA (CF-1) female mice (Harlan SpragueDawley, Indianapolis, IN). Superovulation was induced by injection of 10 IU of pregnant mare’s serum gonadotropin followed 48 hr later by injection of 10 IU human chorionic gonadotropin. Eggs were removed from the ampulla of the oviduct 13-14 hr later and collected in Ml6 medium under light mineral oil (Fisher Scientific, Pit&burg, PA). The cumulus cells were removed by treatment (approximately 5 min) with 0.3 mg/ml hyaluronidase (type IV-S). Only normal, mature eggs, with a fully formed first polar body, were used in these experiments. Egg chromatin was visualized by loading with 1.0 pg/ml Hoechst 33342, a DNA-specific fluorochrome, for 15 min. Preloading the eggs with Hoechst also allowed detection of sperm-egg fusion following fertilization (see Conover and Gwatkin, 1988). The zonae pellucidae were removed by a brief treatment (approximately 1 min) with 10 pg/ml Ly-chymotrypsin (Type II). Near the end of the treatment, the eggs were drawn into and expelled from a small bore pipet, which aided in the removal of the zona pellucida. Using this procedure, the zona pellucida was removed from more than 90% of the eggs. Following each treatment, the eggs were thoroughly washed in Ml6 medium. Eggs were cultured in 35-mm Petri dishes in 200-ccl drops of Ml6 under mineral oil and incubated in a humidified atmosphere of 5% CO2 and 95% air at 37°C.

81

Sperm were obtained from the caudae epididymides and vas deferens of 14- to 16-week-old male ND4 SwissWebster mice (Harlan Sprague-Dawley). Sperm, at 2-5 x lo6 sperm/ml, were incubated l-2 hr in IVF at 37°C in a humidified atmosphere of 5% CO, and 95% air. Under similar conditions, sperm were capacitated and about 35 to 40% underwent a spontaneous acrosome reaction (Fraser and Herod, 1990). Fertilization and artificial activation. Eggs were inseminated in a 200-~1 drop of IVF, under oil. The final sperm concentration was 2-5 x 10’ sperm/ml. Eggs were artificially activated by treatment with the Ca2+ ionophore A23187 or SrCl, in Ca2’, Mg2+-free IVF. For ionophore activation, eggs were treated with 5.0 pM A23187 for 2 min in Ca2+, Mg2+-free IVF, then washed and incubated in Ca2’, M g2+-free IVF for 30 min. For activation with SrCl,, eggs were incubated with 4.6 or 9.2 mMSrC1, for 30 to 40 min in Ca2+, Mg2+-free IVF. Introduction of the calcium chelator. Zona-free eggs were loaded with BAPTA before fertilization or artificial activation by incubation in 0.01 to 50.0 pM of the cell permeant acetoxymethyl ester of BAPTA (Molecular Probes, Eugene, OR) for 30 min at 37°C in M2 medium. To enhance loading, the solution also contained 1 pi/ml of a 25% w/w solution of the dispersing agent Pluronic F-127 (Molecular Probes) in anhydrous dimethyl sulfoxide (DMSO, Aldrich Chemical Co., Milwaukee, WI; final DMSO ~2 pi/ml). Cells accumulate millimolar levels of polycarboxylate chelators such as BAPTA in the presence of micromolar extracellular concentrations of the acetoxymethyl ester (Tsien et al, 1982). In some cases, the loading medium also contained 2.5 mM probenecid (see below). Addition of probenecid had no adverse effect on the egg nor did it alter experimental results. Control eggs were incubated without BAPTA AM in media with an equivalent volume of the solvent DMSO (final DMSO ~2 pi/ml). Labeling the cortical granule em&ate. Thirty to 40 min after fertilization or artificial activation, unfixed eggs were washed in Ml6 and incubated for 15 min in 10 pg/ ml fluorescein isothiocyanate (FITC)-conjugated Lens culinaris agglutinin (LCA; E-Y Laboratories, San Mateo, CA). Before use, the FITC-LCA solution was centrifuged 5 min at 8OOOg to remove particulates. The eggs were thoroughly washed (five washes, 5 min each) before examination by fluorescence microscopy approximately 2 hr after fertilization or activation. LCA has been shown to label the cortical granule contents in mouse (Ducibella et a& 1988) and hamster eggs (Cherr et a& 1988). The cortical granule exudate remains on the surface of the activated egg after fertilization (Cherr et aZ., 1988, Lee et aL, 1988). The resumption of meiosis in fertilized or activated eggs was determined by noting if the eggs had progressed from the metaphase II stage

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DEVELOPMENTALBIOLOGY vOLUME149,1992

and formed a second polar body when the eggs were examined for cortical granule exocytosis. Visualization of intracellular calcium changes. After removing the zona pellucida, eggs were loaded with the fluorescent Ca2’ indicator fluo-3 (Minta et ah, 1989). Fluo-3 was introduced into the egg by incubation for 30 min at 3’7°C with cell permeant fluo-3 AM (Molecular Probes). The loading medium (M2) contained 1,5, or 20 PM fluo-3 AM (from a 1 mlM stock in anhydrous DMSO), 1 pi/ml Pluronic, and 2.5 mM probenecid to inhibit intracellular compartmentalization and excretion of the indicator during loading (Di Virgilio et al, 1988). The medium in which fluorescence measurements were made contained 2.5 mM probenecid. Basal fluorescence of unfertilized eggs loaded with fluo-3 remained unchanged for over 1 hr, indicating little indicator loss. Fluo-3 and BAPTA were coloaded by simultaneous incubation with fluo-3 AM and BAPTA AM (final DMSO: 2-22 pi/ml). Eggs loaded with fluo-3 or coloaded with fluo-3 and BAPTA were washed in IVF containing 0.4% BSA and then placed on a Petri dish in 60 ~1 of IVF containing no BSA under mineral oil. BSA was omitted from the medium so the eggs would more readily adhere to the dish. Some BSA was introduced with the sperm suspension during in vitro fertilization, giving a final BSA concentration of about 0.4%. The center of the Petri dish was treated with 3.5 pg/cm2 Cell-Tak (Collaborative Research, Bedford, MA) before use so the eggs would adhere to the dish prior to the addition of lo-15 ~1 sperm suspension. In activation experiments, eggs loaded with fluo-3, or fluo-3 and BAPTA were washed in Ca2+, Mg2+free IVF, then transferred to Ca2+, Mg2+-free IVF without PVA on a dish treated with Cell-Tak. PVA was omitted from the medium so the eggs would more readily adhere to the dish. Eggs were activated with the nonfluorescent Ca2+ ionophore 4-bromo A23187 (5.0 PUM; Molecular Probes) or by 4.6 or 9.2 mM SrCl, in Ca2+, Mg2+-free IVF (no PVA). In experiments examining the Ca2+ changes caused by 4-bromo A23187, the ionophore was present in the recording dish throughout the experiment (in studies of exocytosis, the eggs were transferred into Ca2+, Mg2+-free IVF without ionophore after 2 min). Fluorescence was measured on a Zeiss Axiovert microscope equipped with a 50 W mercury arc bulb for epifluorescence. The eggs were illuminated with blue light through an exciter filter (450-490 nm) and emission fluorescence was obtained through an FT 510 dichroic beamsplitter and 530 nm emission filter. Hoechst was excited with uv light (exciter filter 365 nm, dichroic 395, emission filter 420 nm). Light from the mercury arc bulb was reduced in intensity by neutral density filters. A low-light level SIT camera (Hamamatsu Photonic Sys-

terns, Oak Brook, IL) and a time-lapse video recorder were used to record Ca2’ changes. Some measurements were made with a photon counting photomultiplier tube (Photon Technology International, South Brunswick, New Jersey). A warm stage, with laminar flow of 5% C02, 95% air, (Micro Devices, Newtown, PA) was used to maintain a temperature of 37°C and to prevent pH changes in the medium, which was kept under mineral oil. RESULTS

Introduction of BAPTA inhibited cortical granule exocytosis. Loading mouse eggs with BAPTA by incubation for 30 min with 25.0 PLLMBAPTA AM prior to the addition of sperm inhibited cortical granule exocytosis in all cases (Fig. 1E and 2A). We also tested if exocytosis induced by artificial activators could be inhibited by prior incubation in BAPTA AM. Cortical granule exocytosis occurred in all cells treated with 5.0 PM A23187 in Ca2’, Mg2+-free IVF but exocytosis was completely suppressed by pretreating the eggs with 25.0 PM BAPTA AM (Fig. 3A). Addition of 4.6 m&f SrCl, in Ca2+, Mg2+free IVF caused cortical granule exocytosis in 71% of control eggs and 9.2 mM SrCl, caused exocytosis in 93% of eggs treated. Cortical granule exocytosis induced by 4.6 mM SrCl, was completely inhibited by incubating eggs in al.0 p&l BAPTA AM, and activation by 9.2 mM SrCl, was usually blocked by incubation in al.0 PM BAPTA AM and always inhibited by >,lO.O PM BAPTA AM (Fig. 3B). Introduction of BAPTA inhibited the resumption of meiosis. Incubation of eggs in al.0 PMBAPTA AM prior to in vitro fertilization blocked the resumption of meiosis; the eggs remained at the metaphase II stage (an apparent metaphase spindle was observed with Hoechst) and second polar body formation did not occur (Fig. 1D and 2B). With prior incubation in 1.0 PLM BAPTA AM, the addition of the calcium ionophore, A23187, caused the resumption of meiosis in only 16% of the eggs (n = 52), while incubation in a2.0 pil4 BAPTA AM prior to addition of A23187 inhibited meiosis in all cases (n = 43). Incubation in ~0.5 1MBAPTA AM prior to addition of ionophore did not inhibit the resumption of meiosis (n = 45). Meiosis resumed and second polar bodies formed in 42% of control eggs activated with 4.6 mMSrC1, (n = 25) and in 88% of eggs activated with 9.2 miUSrC1, (n = 30). Following incubation in 0.01 or 0.1 PLM BAPTA AM, 6070% of the eggs examined formed second polar bodies after activation with 4.6 or 9.2 mM SrCl, (n = 67). The resumption of meiosis was suppressed for both concentrations of SrCl, by preincubation in al.0 p&l BAPTA AM (n = 103).

KLINE Am KLINE

83

Calcium and Mouse Egg Activation

1OOpm FIG. 1. Inhibition of cortical granule exocytosis and second polar body formation by chelation of intracellular calcium. Images were recorded on videotape and photographed from the video monitor. (A-C) Control eggs not treated with BAPTA formed second polar bodies (A, the first polar bodies dissociated during removal of the zona pellucida). These eggs underwent exocytosis as indicated by cortical granule exudate identified by FITC-LCA (B) and were penetrated by one or more sperm indicated by Hoechst staining (C). (D-F) Experimental eggs were incubated in 5.0 PMBAPTA AM, washed, and inseminated. Treated eggs did not form second polar bodies (D), did not undergo cortical granule exocytosis as indicated by the absence of material stained by FITC-LCA (E), but were penetrated by one or more sperm as shown by Hoechst staining (F).

Calcium oscill4xtions following fertilization were sup pressed or abolished by introduction of BAPTA. Follow-

ing addition of sperm to eggs, we detected regular, transient increases in intracellular Ca” in fertilized eggs (Fig. 4A). Sperm induce a large, initial transient increase in Ca2+ lasting 2.3 + 0.6 min, followed by periodic transients lasting 0.5 + 0.1 min and occurring at 3.4 f 1.4-min intervals (n = 93). We typically made a baseline measurement of Ca2’ prior to adding sperm, then continued for 30-60 min following sperm addition. Transients occurred for over 1 hr in most eggs and, when examined at 2 hr, Ca2’ oscillations were still present. The number of sperm that fused with each egg was determined in a group of eggs in which Ca2’ measurements were made. The number of fused sperm per egg was 1.7 f 0.7 (SD, n = 69). Up to 24 Ca2’ transients in 1 hr were seen in monospermic eggs. The frequency of oscillations was not related to the number of sperm entries.

Calcium transients were not observed in eggs simultaneously loaded with l-20 ~iI!ffluo-3 AM and 5,10, or 100 &II BAPTA AM and then inseminated (n = 71; Fig. 4B). Sperm entry did occur and all eggs examined contained one or more fused sperm. A single Ca” transient occurred in 90% of eggs incubated in 1 piI!BAPTA AM and then inseminated (n = 21; Fig. 4C); no change in Ca2’ was seen in the other eggs. Two to ten Ca2’ transients occurred in eggs incubated in 0.1 pM BAPTA AM (n = 24; Fig. 4D). Eggs incubated in 0.01 pM BAPTA and then (n = 24; Fig. 4E) had transients similar to inseminated those in control eggs. Calcium changes induced b artificial activation were suppressed by BAPTA. Addition of the nonfluorescent Ca2’ ionophore, 4-bromo A23187 (5.0 PM), caused a sin-

gle transient increase in Ca2+ in a Ca2+, Mg2+-free medium (n = 25; Fig. 5A). The single Ca2’ transient was similar in amplitude and duration to the first sperm-

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DEVELOPMENTALBIOLOGY

VOLUME 149,1992

A 100 .u, 80 g 5 60

60

W g

40

40

20

20

70

0

BWTA

b&4 @,I

BN’TA

AM [+4I

FIG. 2. Suppression of cortical granule exoeytosis (A) and second polar body formation (B) by chelating intracellular calcium. Eggs were incubated in 0.01-50 &f BAPTA AM, washed, and inseminated. After approximately 2 hr the presence of second polar bodies was noted, and eggs having undergone exoeytosis were identified by FITC-LCA labeling of the cortical granule exudate. Fractions indicate the fraction of eggs in which exoeytosis or second polar body formation occurred in each condition. BAPTA AM concentrations are plotted on a log scale. No exocytosis or second polar body formation occurred in control eggs which were not inseminated (0191). Inseminated eggs without prior BAPTA treatment underwent cortical granule exocytosis and formed second polar bodies (93193).

induced Ca’+ transient. Following the initial increase induced by ionophore, the relative Ca’+ concentration remained somewhat higher than the level before the addition of ionophore. Prior incubation in 5.0 pM BAPTA prevented the Ca” transient normally induced by 4bromo A23187 (n = 19; Fig. 5B). The Ca” transient induced by the Ca2+ ionophore was not suppressed by prior incubation in 1.0 FM BAPTA AM (n = 9) or by 0.1 PM BAPTA AM (n = 8). Application of 4.6 or 9.2 mM &Cl, in Ca2’, Mg’+-free IVF initiated Ca2+ transients in 25/25 and 26/26 eggs (Fig. 6A). No apparent difference in magnitude, duration, or frequency of transients was observed following treatment with 4.6 or 9.2 mM &Cl,. The transients induced by ,%-Cl, in the Ca2+, Mg2+-free medium were lower in relative amplitude, somewhat longer in duration, and occurred less frequently than those induced by -413/13

u)

5 +4

A23187

sperm in normal Ca2+ -containing medium. Fluo-3 will bind other divalent cations in addition to Ca2+, so the change in fluorescence is probably due to both an increase in intracellular Ca2’ triggered by Sr2+ and an increase in intracellular S?+ as it substitutes for Ca2’. The relative fluorescence intensities can not be compared directly because of the difference in media and because the change in fluo-3 fluorescence on binding the divalent cation, S?‘, has not been examined. Ca2+ transients are initiated by SrCl, in IVF containing Ca2’, but much higher concentrations of SrCl, are required; 12 of 14 eggs produced Ca2+ transients after the addition of 20 or 40 mM SrCl, in IVF with Ca2+, while 10 mil4 SrCI, initiated transients in only 1 of 7 eggs. The Ca2’ transients caused by 4.6 or 9.2 mM SrCl, in Ca2’, Mg2’-free IVF were inhibited by prior incubation in 5 or 10 PM BAPTA AM (n = 23; Fig. 6B). Ca2’ tran‘.O..

17117

4.6 =2

TM

-o-

92 =2

fwi

80

z 0

5

60

w2

40

EnPTA

AA4 [+.I!

EiPPTA.%4@lj

FIG. 3. Suppression of cortical granule exocytosis by introduction of BAPTA into eggs that would normally be activated by the calcium ionophore, A23187, or by SrCI,. Eggs were incubated in 0.01-50 nhfBAPTA AM, washed, and treated with 5.0 fiA23187 (A) and 4.6 or 9.2 mM SrCl, (B). Eggs having undergone exocytosis were identified by FITC-LCA labeling of the cortical granule exudate. Fractions indicate the fraction of eggs responding in each condition. No exocytosis occurred in eggs loaded with BAPTA but not treated with ionophore or SrCl, (O/45). Control eggs were not loaded with BAPTA. All (82182) control eggs treated with 5.0 &f A23187 underwent cortical granule exocytosis, 71% (42/59) underwent cortical granule exocytosis following addition of 4.6 mM SrCl,, and 93% (37/40) with addition of 9.2 m&f SrCl,.

KLINE AND KLINE

85

Calcium and Mouse Egg Activation 2.0 r

1.0 @A BAPTA

AM

0.01

0 L

pv

BAPTA

5.0

+I

BAPTA

AM

0.1

@A BAPTA

AM

AM

1

FIG. 4. Intracellular calcium changes following fertilization are suppressed or inhibited by introduction of BAPTA into the eggs before in vitro fertilization. Eggs were loaded with fluo-3 or fluo-3 and BAPTA, washed, and inseminated. Recordings of fluo-3 fluorescence were made from single eggs with a photon counting photomultiplier tube. The excitation or emission spectra for fluo-3 do not change on binding Ca*’ so fluo-3 cannot be used as a ratio indicator to indicate steady-state Ca” levels. Furthermore, Ca2+ does not rise uniformly in the egg but spreads as a wave of increased Ca*‘, so Caa’ activities will vary in different parts of the egg. Therefore, we show fluorescence intensity (106 counts/set) rather than converting to calcium activities. Data was acquired using Photoscan software (Photon Technology International) at a collection rate of 4 pointskec. (A) Control egg loaded with 1.0 ~Mfluo-3 AM only. (B-E) Eggs loaded with 1.0 /.&fluo-3 AM and 5.0 PMBAPTA AM (B), 1.0 pM BAPTA AM (C), 0.1 pM BAPTA AM (D), and 0.01 @M BAPTA AM (E).

sients were induced by 4.6 or 9.2 mM SrCl, in 14 of 15 eggs previously incubated in 1 PM BAPTA AM, although these transients were lower in magnitude than those in control eggs. Ca2+ transients caused by SrCl, after prior incubation in 0.1 piV BAPTA AM were similar to control eggs (n = 14). DISCUSSION

We have tested the hypothesis that an increase in intracellular calcium is required for cortical granule exo-

cytosis and second polar body formation in a mammalian egg. Our results and those of others have shown that a change in intracellular calcium occurs at fertilization in mouse and hamster eggs and that artificially increasing intracellular calcium activates cortical granule exocytosis and cell cycle activation. The results of this study support the hypothesis that a calcium rise is necessary for these activation events. Introduction of the calcium chelator, BAPTA, into the egg at concentrations that inhibit calcium transients

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DEVELOPMENTAL BIOLOGY

-!----2

1.0

-

0.5

- 4-B

P

4-B

A23187

9

A23187

t 0 1

0 L

1

FIG. 5. Intracellular calcium changes following addition of 4-bromo A23187. (A) Control egg activated with 5.0 &f4-bromo A23187 in Cal+, Mga+-free IVF. (B) Addition of 5.0 rM4-bromo A23187 in Ca2’, Mg 2+-free IVF after prior incubation in 5.0 gbf BAPTA AM.

calcium-dependent step in exocytosis is not known. Calcium may act through a calcium-sensitive protein kinase or phosphatase (see Whalley et cd, 1991) or may directly or indirectly affect some other protein involved in exocytosis (see Jackson and Modern, 1990). Some evidence from studies of mouse and frog suggests that the calcium change may be related to activation of protein kinase C, which might regulate or modulate exocytosis (Endo et al, 198’7; Colonna et aL, 1989; Bement and Capco, 1990). Introduction of BAPTA into the mouse egg by incubation in al.0 PM BAPTA AM prior to fertilization prevented resumption of the cell cycle and formation of the second polar body. Prevention of the calcium rise at fertilization also inhibits the resumption of meiosis in the frog egg (Kline, 1988). The calcium signal at fertilization could act on one or more proteins that control the cell cycle. The most likely target is cytostatic factor (CSF), a calcium-sensitive factor that stabilizes the activity of maturation promoting factor (MPF) in the unfertilized egg and which disappears after fertilization. The results for mouse and frog eggs, which show that the resumption of meiosis is inhibited by preventing the calcium rise at fertilization, are consistent with the calcium-

also inhibits exocytosis and second polar body formation. BAPTA AM at concentrations 35.0 PIM blocks calcium transients and egg activation in all cases. When eggs were loadedewith BAPTA in 1.0 pikf BAPTA AM and then fertilized, no change in calcium occurred in 10% of the eggs and a single calcium transient occurred in 90% of the eggs. In parallel experiments, exocytosis occurred in only 30% of the eggs incubated in 1.0 pM BAPTA AM prior to in vitro fertilization. This suggests that the magnitude or duration of the single calcium rise in eggs incubated in 1.0 ~4f BAPTA AM may not always have been large enough to cause exocytosis. To determine if the single calcium transient represents an increase of enough calcium to cause exocytosis, it would be necessary to use a calcium indicator with an excitation wavelength different from that of the fluorescent molecule used to detect exocytosis and make quantitative measurements of calcium and exocytosis in the same egg. Inhibition of cortical granule exocytosis by introduction of calcium buffers or chelators in the egg before fertilization has also been demonstrated in sea urchin and frog eggs (Zucker and Steinhardt, 19’78; Hamaguchi and Hiramoto, 1981; Kline, 1988). The identity of the 2.0

A

2.0

1

B

1 5.0

1.5

h4

BAPTA

AM

1 I

FIG. 6. Intracellular calcium changes following addition of &Cl,. (A) Control eggs activated with 4.6 mM SrCl, in Ca2+, Mg”+-free IVF. (B) Addition of 4.6 mM SrCl, in Ca2+, Mg2+-f ree IVF after prior incubation in 5.0 PM BAPTA AM.

KLINEANDKLINE

Calcium

dependent loss (following sperm-egg fusion) of cytoplasmic factors that maintain meiotic arrest (see Masui and Shibuya, 1987). Repeated, transient increases in intracellular calcium during fertilization were first reported in mouse eggs injected with the calcium-dependent luminescent protein, aequorin. Cuthbertson et al. (1981) observed a series of small calcium transients preceding a single large rise in calcium that occurred loo-150 min after adding sperm. They referred to the large increase as the “activating calcium rise.” However, this early work examined only three eggs and sperm entry could not be confirmed. We have never observed such a pattern of calcium transients and suspect that the small transients may have been part of the sperm-induced response, but that the larger single transient may have occurred because the egg had become damaged by the end of the experiment. A later paper (Cuthbertson and Cobbold, 1985) on mouse egg activation reported data on calcium transients from four fertilized eggs previously injected with aequorin. In these experiments, periodic calcium transients were reported, but not the later, large increase described in the earlier paper. Compared with our results, a much longer delay between sperm addition and the first calcium transient was reported (more than 45 min). This difference may have been due to differences in the procedures used for capacitating sperm and the number of acrosome-reacted sperm able to fertilize the zona-free eggs. Cuthbertson and Cobbold (1985) also reported somewhat longer durations for each calcium transient and a longer interval between transients (10 min between the first and second transients and 20 min between later transients). The differences between the data reported in the earlier two papers on calcium transients in mouse eggs and the data presented in this paper could be because the measurements were made in different strains of mice, but we also found the same pattern and kinetics of calcium transients in eggs from another strain of mice (C57BL/6J; unpublished data). The disparities could result from differences in the preparation of media and gametes or in the methods used to examine calcium changes. The aequorin used in the earlier work was probably a mixture of many isoaequorins and the response to changes in calcium is nonlinear for such mixtures. Heterogeneous mixtures are also toxic to a number of cells and it would be interesting to reexamine the calcium changes with aequorin using the specific semisynthetic or recombinant aequorins that have been shown to be nontoxic (Shimomura et aZ., 1990). Cuthbertson et al. (1981) also observed an intracellular calcium increase in mouse eggs incubated in calcium-free ,medium containing 1 mM Sr’+; oscillations

and Mouse

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were apparently not detected. We found that calcium transients induced in CaBf, Mg2+-free IVF required less &Cl, than needed in a calcium-containing medium. This may be why Whittingham and Siracusa (1978) and Fraser (1987) found that SrCl, was more effective in activating mouse eggs in a calcium-free medium rather than a calcium-containing medium. It was suggested that Srz’ activates eggs by displacing bound calcium in the egg (Whittingham and Siracusa, 1978). Srz’ probably initiates calcium release from intracellular stores in the mouse egg. In muscle cells, Sr2+ is handled much like Ca2+; Sr2+ can be taken up by the sarcoplasmic reticulum, can compete with calcium for uptake by the ATPdependent transport system of the sarcoplasmic reticulum, and can cause calcium release from isolated sarcoplasmic reticulum (Mermier and Hasselbach, 1976; reviewed in Endo, 1985). The calcium transients we detect in fertilized mouse eggs resemble those observed in the hamster egg with both aequorin and calcium-selective electrodes. Like the mouse, repetitive, transient increases in calcium in the hamster egg continue for more than 100 min after the first transient. However, in hamster, the first transient is usually the same duration as the remaining transients, all transients are short (15-25 set), and the interval between transients is less, particularly in polyspermic eggs where it is reduced to 40-50 set (Igusa and Miyazaki, 1986; Miyazaki et ok, 1986). In hamster, the frequency and number of calcium oscillations depends on the number of attached sperm and presumably on the number of fused sperm (Miyazaki and Igusa, 1981). We found no relationship between the number of sperm fused with the mouse egg and the frequency or number of calcium oscillations. In the hamster, the calcium transients stop if extracellular calcium is removed from the medium, and the frequency of transients is reduced by adding cobalt chloride to inhibit calcium entry (Igusa and Miyazaki, 1983). Our preliminary results suggest that the calcium transients in the mouse egg are also dependent on influx of extracellular calcium. Addition of 5 or 10 mM external BAPTA after the first or second transient in normal IVF limited further calcium transients; only one or two transients occurred in the following 20- to 30-min interval (n = 5). These results suggest that the repetitive transient increases in intracellular calcium in the mouse egg are dependent on calcium influx. Further experimentation will be necessary to determine what portion of the first sperm-induced calcium transient is due to release from intracellular stores and what amount, if any, is dependent on calcium influx. These results suggest that an increase in intracellular calcium is an obligatory step in the activation of the mouse egg by sperm or by artificial activating agents;

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however, it will be important to examine the steps that precede and follow the calcium rise to determine how sperm initiate the change in calcium and determine how subsequent activation events are regulated by calcium. Certainly, calcium must be a trigger for a number of parallel pathways involved in the organization of the early events of development. There is evidence to suggest that the rise in calcium in eggs, like some other cells, is initiated by activation of a guanine-nucleotide binding protein, subsequent production of InsP3, and release of calcium from intracellular stores (reviewed in Turner and Jaffe, 1989; Jaffe, 1990; Miyazaki, 1990; Kline et al., 1991). An alternative hypothesis is that sperm may introduce a factor into the egg after spermegg fusion that directly initiates calcium release (reviewed in Swann, 1990; Swann and Whitaker, 1990). Among the species so far studied, only the eggs of mammals (mouse and hamster) and ascidians (Speksnijder et ah, 1990) produce oscillatory increases in intracellular calcium following fertilization. The role of these calcium oscillations is not known. The oscillations, and the elevated calcium levels they produce, could influence the meiotic or pronuclear events following fertilization or effect some other aspect of early embryonic development. Since the calcium transients in mouse eggs appear to be dependent on extracellular calcium, events could be examined by inhibiting all but the first transient and examining later development. Our preliminary observations indicate that the second and later calcium transients are initiated in the hemisphere opposite the second metaphase spindle and proceed as a wave of increased free calcium traveling toward the hemisphere containing the egg chromatin. Such waves were not seen in hamster (Miyazaki et ab, 1986) but were noted in an ascidian egg. It has been suggested that calcium waves in the ascidian egg, since they are attenuated in the animal hemisphere, would expose the vegetal hemisphere to a greater concentration of calcium and may influence early developmental events (Speksnijder et ah, 1990). The mouse egg would be a good mammalian egg in which to examine this further. Agonist-induced calcium oscillations occur in a number of mammalian cell types (see Berridge and Galione, 1988; Petersen and Wakui, 1990) and models have been proposed to account for them, involving both InsP,-induced calcium release and calcium-induced calcium release (Parker and Ivorra, 1990; Berridge, 1991; DuPont et al, 1991). Miyazaki has suggested that calcium oscillations in the hamster egg may be induced by a sustained elevated level of InsP, and periodic calcium-induced calcium release (Miyazaki, 1991). This hypothesis could be tested in mouse by utilizing agents that inhibit or potentiate calcium release from InsP,-sensitive or InsP,insensitive calcium stores.

VOLUME I49,1992

This work was supported by a grant from the National Institute Child Health and Human Development (HD 26032) to D. Kline.

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