Sea Urchin Article

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7/28/2019 Sea Urchin Article http://slidepdf.com/reader/full/sea-urchin-article 1/6 Biochem. J. (1988) 252, 257-262 (Printed in Great Britain) Activation of sea urchin eggs by inositol phosphates is independent of external calcium Ian CROSSLEY,* SWANN,* WHITAKER*" *Department of Physiology, University College London, Gower Street, London WCIE 6BT, U.K., an d tDepartment of Physiology and Biophysics, University of Miami Medical School, Miami, FL 33101, U.S.A. We investigated the contribution of external calcium ions to inositol phosphate-induced exocytosis in se a urchin eggs. We show that: (a) inositol phosphates activate eggs of the se a urchin species Lytechinus pictus and Lytechinus variegatus independently of external calcium ions; (b) the magnitude and duration of the inositol phosphate induced calcium changes ar e independent of external calcium; (c) in calcium-free sea- water, increasing the volume of inositol trisphosphate solution injected decreased the extent of egg activation; (d) eggs in calcium-free se a water ar e more easily damaged by microinjection; microinjection of larger volumes increased leakage from eggs pre-loaded with fluorescent dye. We conclude that inositol phosphates do not require external calcium ions to activate se a urchin eggs. This is entirely consistent with their role as internal messengers at fertilization. The increased damage caused to eggs in calcium-free seawater injected with large volumes may allow the EGTA present in the seawater to enter the egg and chelate any calcium released by the inositol phosphates. This may explain the discrepancy between this and earlier reports. INTRODUCTION The transient increase in the cytosolic free calcium ion concentration (Ca2"1) that occurs at fertilization in se a urchin eggs (Steinhardt et al., 1977) is the trigger that initiates development (Whitaker & Steinhardt, 1985). The most obvious sign of egg activation is the elevation of a fertilization envelope. The fertilization envelope rises as a consequence of the exocytosis of cortical secretory granules caused by the increase in Ca2+i (Baker & Whitaker, 1978). Fertilization, parthenogenetic activa- tion and the elevation of the fertilization envelope can occur in calcium-free se a water, which suggests that the increase in Ca2", is due to release of calcium ions from internal stores (Steinhardt & Epel, 1974; Schmidt et al., 1982). It has been suggested that the fertilization calcium transient is the result of the liberation of the calcium- releasing second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. This idea rests on the demonstration of an increased turnover of polyphosphoinositide phospho- lipid (Turner et al., 1984) and the production of inositol trisphosphate at fertilization (Ciapa & Whitaker, 1986). Microinjecting Ins(1,4,5)P3 into se a urchin eggs causes an increase in Ca2"i and the elevation of a fertilization envelope (Whitaker & Irvine, 1984; Swann & Whitaker, 1986). Sea urchin egg homogenates contain an ATP- driven, membrane-bound calcium-sequestering system that is sensitive to Ins(1,4,5)P3 (Clapper & Lee, 1985). It has recently been reported that microinjection of Ins(1,4,5)P3 causes activation of se a urchin eggs (judged by fertilization envelope elevation) only if calcium ions ar e present externally (Slack et al., 1986; Irvine & Moor, 1986). These findings have been taken as an indication that inositol polyphosphates regulate plasma membrane calcium fluxes (Irvine & Moor, 1986). Yet fertilization and development in se a urchins can occur in se a water containing less than 10-8 M-Ca2" (Chambers, 1980), that is, under conditions in which calcium influx cannot occur (Poenie et al., 1985). If Ins(1,4,5)P3 activates eggs only in calcium-containing se a water then it cannot be the crucial internal messenger at fertilization. We undertook this study to determine more precisely the contribution of external calcium ions during InsPJ- induced egg activation. We used microinjection tech- niques to assess the effects of a number of inositol phosphates on cortical granule exocytosis both in the presence and absence of external calcium. We have also assessed the contribution that calcium influx makes to the inositol phosphate-induced calcium transient by measuring Ca2"i using the calcium sensitive fluorescent dye, fura2. We find that all the inositol phosphates tested cause cortical granule exocytosis independently of exter- nal calcium. Measurements of Ca2", indicate that calcium influx makes no significant contribution to the inositol phosphate-induced calcium transient. Our data support the idea that Ins(1,4,5)P3 is the internal messenger that causes the increase in Ca2"1 at fertilization and demon- strate that inositol phosphates do not cause a significant influx of calcium when they activate se a urchin eggs. MATERIALS AND METHODS Obtaining gametes Gametes were obtained by the intracoelomic injection of 0.5 M-KCI. Eggs of the se a urchin species Lytechinus pictus (Pacific Biomarine Laboratories Inc., Venice, CA, U.S.A.) and Lytechinus variegatus (collected off Key Biscayne, FL, U.S.A.) were collected in artificial seawater (ASW; for composition, se e below). The egg jelly was Abbreviations used: InsP3 and InsP4, inositol trisphosphate and Ins(1,2cyc4,5)P3 is inositol 1,2-cyclic 4,5-trisphosphate; ASW, artificial s4 I To whom correspondence should be addressed. tetrakisphosphate respectively; locants ar e given in parentheses, e.g. Vol. 252 257

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B i o c h e m . J . ( 1 9 8 8 ) 2 5 2 , 2 5 7 - 2 6 2 ( P r i n t e d i n G r e a t B r i t a i n )

A c t i v a t i o n o f sea u r c h i n eggs b y i n o s i t o l p h o s p h a t e s i si n d e p e n d e n t o f e x t e r n a l c a l c i u m

Ian CROSSLEY,* SWANN,*

W H I T A K E R * "* D e p a r t m e n t o f P h y s i o l o g y , U n i v e r s i t y C o l l e g e L o n d o n , Gower S t r e e t , London WCIE 6 B T , U . K . , an d t D e p a r t m e n t o f

P h y s i o l o g y a n d B i o p h y s i c s , U n i v e r s i t y o f Miami M e d i c a l S c h o o l , M i a m i , FL 3 3 1 0 1 , U . S . A .

We i n v e s t i g a t e d t h e c o n t r i b u t i o n o f e x t e r n a l c a l c i u m i o n s t o i n o s i t o l p h o s p h a t e - i n d u c e d e x o c y t o s i s i n se a

u r c h i n e g g s . We s h o w t h a t : ( a ) i n o s i t o l p h o s p h a t e s a c t i v a t e eggs o f t h e se a u r c h i n s p e c i e s L y t e c h i n u s p i c t u sa n d L y t e c h i n u s v a r i e g a t u s i n d e p e n d e n t l y o f e x t e r n a l c a l c i u m i o n s ; ( b ) t h e m a g n i t u d e a n d d u r a t i o n o f t h e

i n o s i t o l p h o s p h a t e i n d u c e d c a l c i u m c h a n g e s ar e i n d e p e n d e n t o f e x t e r n a l c a l c i u m ; ( c ) i n c a l c i u m - f r e e sea-

w a t e r , i n c r e a s i n g t h e v o l u m e o f i n o s i t o l t r i s p h o s p h a t e s o l u t i o n i n j e c t e d d e c r e a s e d t h e e x t e n t o f egg

a c t i v a t i o n ; ( d ) eggs i n c a l c i u m - f r e e se a w a t e r ar e more e a s i l y d a m a g e d b y m i c r o i n j e c t i o n ; m i c r o i n j e c t i o n o f

l a r g e r v o l u m e s i n c r e a s e d l e a k a g e f r o m eggs p r e - l o a d e d w i t h f l u o r e s c e n t d y e . We c o n c l u d e t h a t i n o s i t o lp h o s p h a t e s d o n o t r e q u i r e e x t e r n a l c a l c i u m i o n s t o a c t i v a t e se a u r c h i n e g g s . T h i s i s e n t i r e l y c o n s i s t e n t w i t h

t h e i r r o l e as i n t e r n a l messengers a t f e r t i l i z a t i o n . T h e i n c r e a s e d d a m a g e c a u s e d t o eggs i n c a l c i u m - f r e e

s e a w a t e r i n j e c t e d w i t h l a r g e v o l u m e s may a l l o w t h e EGTA p r e s e n t i n t h e s e a w a t e r t o e n t e r t h e egg a n d

c h e l a t e a n y c a l c i u m r e l e a s e d b y t h e i n o s i t o l p h o s p h a t e s . T h i s may e x p l a i n t h e d i s c r e p a n c y b e t w e e n t h i s a n de a r l i e r r e p o r t s .

INTRODUCTION

T h e t r a n s i e n t i n c r e a s e i n t h e c y t o s o l i c f r e e c a l c i u m i o n

c o n c e n t r a t i o n ( C a 2 " 1 ) t h a t occurs a t f e r t i l i z a t i o n i n se a

u r c h i n eggs ( S t e i n h a r d t e t a l . , 1 9 7 7 ) i s t h e t r i g g e r t h a ti n i t i a t e s d e v e l o p m e n t ( W h i t a k e r & S t e i n h a r d t , 1 9 8 5 ) .T h e most o b v i o u s s i g n o f egg a c t i v a t i o n i s t h e e l e v a t i o no f a f e r t i l i z a t i o n e n v e l o p e . T h e f e r t i l i z a t i o n e n v e l o p e

r i s e s as a consequence o f t h e e x o c y t o s i s o f c o r t i c a ls e c r e t o r y g r a n u l e s c a u s e d b y t h e i n c r e a s e i n C a 2 + i ( B a k e r

& W h i t a k e r , 1 9 7 8 ) . F e r t i l i z a t i o n , p a r t h e n o g e n e t i c a c t i v a -t i o n a n d t h e e l e v a t i o n o f t h e f e r t i l i z a t i o n e n v e l o p e ca n

occur i n c a l c i u m - f r e e se a w a t e r , w h i c h s u g g e s t s t h a t t h ei n c r e a s e i n C a 2 " , i s d u e t o r e l e a s e o f c a l c i u m i o n s f r o mi n t e r n a l s t o r e s ( S t e i n h a r d t & E p e l , 1 9 7 4 ; S c h m i d t e t a l . ,1 9 8 2 ) . I t h a s b e e n s u g g e s t e d t h a t t h e f e r t i l i z a t i o n c a l c i u m

t r a n s i e n t i s t h e r e s u l t o f t h e l i b e r a t i o n o f t h e c a l c i u m -

r e l e a s i n g s e c o n d m e s s e n ge r i n o s i t o l 1 , 4 , 5 - t r i s p h o s p h a t e[ I n s ( 1 , 4 , 5 ) P 3 ] . T h i s i d e a r e s t s on t h e d e m o n s t r a t i o n o f a n

i n c r e a s e d t u r n o v e r o f p o l y p h o s p h o i n o s i t i d e p h o s p h o -l i p i d ( T u r n e r e t a l . , 1 9 8 4 ) a n d t h e p r o d u c t i o n o f i n o s i t o lt r i s p h o s p h a t e a t f e r t i l i z a t i o n ( C i a p a & W h i t a k e r , 1 9 8 6 ) .M i c r o i n j e c t i n g I n s ( 1 , 4 , 5 ) P 3 i n t o se a u r c h i n eggs causes a n

i n c r e a s e i n C a 2 " i a n d t h e e l e v a t i o n o f a f e r t i l i z a t i o ne n v e l o p e ( W h i t a k e r & I r v i n e , 1 9 8 4 ; Swann & W h i t a k e r ,

1 9 8 6 ) . S e a u r c h i n egg h o m o g e n a t e s c o n t a i n an ATP-d r i v e n , m e m b r a n e - b o u n d c a l c i u m - s e q u e s t e r i n g s y s t e mt h a t i s s e n s i t i v e t o I n s ( 1 , 4 , 5 ) P 3 ( C l a p p e r & L e e , 1 9 8 5 ) .

I t h a s r e c e n t l y b e e n r e p o r t e d t h a t m i c r o i n j e c t i o n o f

I n s ( 1 , 4 , 5 ) P 3 causes a c t i v a t i o n o f se a u r c h i n eggs ( j u d g e db y f e r t i l i z a t i o n e n v e l o p e e l e v a t i o n ) o n l y i f c a l c i u m i o n s

ar e p r e s e n t e x t e r n a l l y ( S l a c k e t a l . , 1 9 8 6 ; I r v i n e & M o o r ,1 9 8 6 ) . T h e s e f i n d i n g s h a v e b e e n t a k e n as an i n d i c a t i o nt h a t i n o s i t o l p o l y p h o s p h a t e s r e g u l a t e p l a s m a membranec a l c i u m f l u x e s ( I r v i n e & M o o r , 1 9 8 6 ) . Y e t f e r t i l i z a t i o n

a n d d e v e l o p m e n t i n se a u r c h i n s can occur i n se a w a t e r

c o n t a i n i n g l e s s t h a n 1 0 - 8 M - C a 2 " ( C h a m b e r s , 1 9 8 0 ) , t h a ti s , u n d e r c o n d i t i o n s i n w h i c h c a l c i u m i n f l u x c a n n o t occur

( P o e n i e e t a l . , 1 9 8 5 ) . I f I n s ( 1 , 4 , 5 ) P 3 a c t i v a t e s eggs o n l yi n c a l c i u m - c o n t a i n i n g se a w a t e r t h e n i t c a n n o t b e t h ec r u c i a l i n t e r n a l m e s s e n g e r a t f e r t i l i z a t i o n .We u n d e r t o o k t h i s s t u d y t o d e t e r m i n e more p r e c i s e l y

t h e c o n t r i b u t i o n o f e x t e r n a l c a l c i u m i o n s d u r i n g I n s P J -i n d u c e d e g g a c t i v a t i o n . We u s e d m i c r o i n j e c t i o n t e c h -n i q u e s t o assess t h e e f f e c t s o f a n u m b e r o f i n o s i t o lp h o s p h a t e s on c o r t i c a l g r a n u l e e x o c y t o s i s b o t h i n t h epresence a n d a b s e n c e o f e x t e r n a l c a l c i u m . We h a v e a l s oa s s e s s e d t h e c o n t r i b u t i o n t h a t c a l c i u m i n f l u x m a k e s t o

t h e i n o s i t o l p h o s p h a t e - i n d u c e d c a l c i u m t r a n s i e n t b ym e a s u r i n g C a 2 " i u s i n g t h e c a l c i u m s e n s i t i v e f l u o r e s c e n td y e , f u r a 2 . We f i n d t h a t a l l t h e i n o s i t o l p h o s p h a t e s t e s t e dcause c o r t i c a l g r a n u l e e x o c y t o s i s i n d e p e n d e n t l y o f e x t e r -

n a l c a l c i u m . M e a s u r e m e n t s o f C a 2 " , i n d i c a t e t h a t c a l c i u mi n f l u x m a k e s no s i g n i f i c a n t c o n t r i b u t i o n t o t h e i n o s i t o lp h o s p h a t e - i n d u c e d c a l c i u m t r a n s i e n t . Our d a t a s u p p o r t

t h e i d e a t h a t I n s ( 1 , 4 , 5 ) P 3 i s t h e i n t e r n a l m e s s e n ge r t h a tcauses t h e i n c r e a s e i n C a 2 " 1 a t f e r t i l i z a t i o n a n d d e m o n -s t r a t e t h a t i n o s i t o l p h o s p h a t e s d o n o t cause a s i g n i f i c a n ti n f l u x o f c a l c i u m w h e n t h e y a c t i v a t e se a u r c h i n eggs.

MATERIALS AND METHODS

O b t a i n i n g g a m e t e s

G a m e t e s we re o b t a i n e d b y t h e i n t r a c o e l o m i c i n j e c t i o no f 0 . 5 M - K C I . E g g s o f t h e se a u r c h i n s p e c i e s L y t e c h i n u sp i c t u s ( P a c i f i c B i o m a r i n e L a b o r a t o r i e s I n c . , V e n i c e , C A ,U . S . A . ) a n d L y t e c h i n u s v a r i e g a t u s ( c o l l e c t e d o f f K e yB i s c a y n e , F L , U . S . A . ) w e r e c o l l e c t e d i n a r t i f i c i a l s e a w a t e r

(ASW; f o r c o m p o s i t i o n , se e b e l o w ) . T h e egg j e l l y was

A b b r e v i a t i o n s u s e d : I n s P 3 a n d I n s P 4 , i n o s i t o l t r i s p h o s p h a t e a n d

I n s ( 1 , 2 c y c 4 , 5 ) P 3 i s i n o s i t o l 1 , 2 - c y c l i c 4 , 5 - t r i s p h o s p h a t e ; ASW, a r t i f i c i a l s 4

I To whom c o r r e s p o n d e n c e s h o u l d b e a d d r e s s e d .

t e t r a k i s p h o s p h a t e r e s p e c t i v e l y ; l o c a n t s ar e g i v e n i n p a r e n t h e s e s , e . g .

V o l . 2 5 2

2 5 7

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I . C r o s s l e y a n d o t h e r s

r e m o v e d b y t r i p l e p a s s a g e t h r o u g h 1 0 0 ,m N i t e x m e s h

a n d t h e e g g s w e r e w a s h e d t w i c e w i t h , t h e n r e s u s p e n d e d

i n , e i t h e r A S W o r c a l c i u m - f r e e ASW. F o r m i c r o i n j e c t i o nt h e e g g s w e r e a t t a c h e d t o c o v e r s l i p s p r e t r e a t e d w i t h

p o l y ( L - l y s i n e ) ( 0 . 0 2 m g / m l ) . S p e r m w a s c o l l e c t e d i n a

m i n i m a l v o l u m e o f ASW a n d k e p t a t 4 ° C u n t i l u s e .T h e v i a b i l i t y o f e g g s f r o m e a c h f e m a l e w a s a s s e s s e d a t

t h e e n d o f a s e r i e s o f e x p e r i m e n t s o n e g g s f r o m t h a t

f e m a l e . A s a m p l e o f e g g s w a s f e r t i l i z e d a n d d e v e l o p m e n tw a s o b s e r v e d . F o r a l l d a t a r e p o r t e d h e r e > 9 0 0 o f t h es a m p l e e l e v a t e d f e r t i l i z a t i o n e n v e l o p e s a n d > 8 0 0 %d e v e l o p e d t o a t l e a s t t h e f o u r - c e l l s t a g e .

A l l e x p e r i m e n t s w e r e c o n d u c t e d a t 1 6 ' C .

C o m p o s i t i o n o f a r t i f i c i a l s e a w a t e r

ASW: 4 3 5 m M - N a C l , 4 0 m M - M g C l 2 , 1 5 m M - M g S O 4 ,1 1 m M - C a C l 2 , 1 0 m M - K C l , 2 . 5 mM-NaHCO3, 1 mM-EDTA, pH 8 . 0 .

C a l c i u m - f r e e ASW: 4 4 5 m M - N a C l , 5 0 m M - M g C l 2 , 1 0m M - K C l , 2 . 5 mM-NaHCO3, 2 mM-EGTA, 1 mM-EDTA,pH 8 . 0 .

C a l c i u m - f r e e ASW w a s c a l c u l a t e d t o h a v e a f r e ec a l c i u m

i o nc o n c e n t r a t i o n o f l e s s t h a n 1 0 0 nM b y u s i n g

t h e b i n d i n g c o n s t a n t s o f M a r t e l l & S m i t h ( 1 9 7 4 ) . T h i s

w a s c o n f i r m e d b y u s i n g a c a l c i u m - s e n s i t i v e e l e c t r o d e( W o r l d P r e c i s i o n I n s t r u m e n t s , New H a v e n , C T , U S A ) .

M i c r o i n j e c t i o n t e c h n i q u e s

M i c r o p i p e t t e s w e r e p u l l e d f r o m g l a s s c a p i l l a r y t u b e

( 1 . 5 mm i . d . ; C l a r k e E l e c t r o m e d i c a l , P a n g b o u r n e , U . K . )

u s i n g a P a l m e r B i o s c i e n c e M i c r o e l e c t r o d e P u l l e r ( S h e e r -n e s s , U . K . ) . T h e t i p r e s i s t a n c e w a s t y p i c a l l y 5 - 1 0 M oh mw h e n f i l l e d w i t h 3 M - K C I a n d t h e d i a m e t e r w a s s u c h t h a ta 1 0 - 5 0 ms p u l s e o f 4 5 0 k P a p r e s s u r e e j e c t e d a v o l u m e o f1 p l ( S w a n n & W h i t a k e r , 1 9 8 6 ) . P u l s e s w e r e d e l i v e r e d t ot h e p i p e t t e a t a f r e q u e n c y o f 1 Hz d u r i n g p e r i o d s o f

i m m e r s i o n i n s e a w a t e r t o p r e v e n t c o n t a m i n a t i o n o f t h e

p i p e t t e c o n t e n t s . T h e m i c r o p i p e t t e w a s h e l d i n am i c r o m a n i p u l a t o r ( P r i o r I n s t r u m e n t s , C a m b r i d g e ,

U . K . ) m o u n t e d o n t o t h e s t a g e o f a L e i t z D i a v e r t

m i c r o s c o p e .M i c r o i n j e c t i o n s w e r e p e r f o r m e d b y u s i n g b r i g h t f i e l d

m i c r o s c o p y w i t h a 4 0 x , 0 . 6 5 NA a c h r o m a t o b j e c t i v e .T h e i n j e c t e d v o l u m e w a s e s t i m a t e d b y m e a s u r i n g t h ec y t o p l a s m i c d i s p l ac e m e n t o f t h e i n j e c t e d f l u i d w i t h a n

e y e p i e c e g r a t i c u l e . F o r e x p e r i m e n t s t o d e t e r m i n e t h ed a m a g e d o n e t o e g g s b y m i c r o i n j e c t i o n a l o w p r e s s u r ei n j e c t i o n p r o c e e d u r e b a s e d o n t h a t d e s c r i b e d b y T u r n e r

e t a l . ( 1 9 8 6 ) w a s u s e d .

O b s e r v a t i o n o f t h e e g g c o r t i c a l g r a n u l e s

Weu s e d

c o r t i c a lg r a n u l e

e x o c y t o s i s a sa m o r p h o l o g i c a l

m a r k e r t o i n d i c a t e t h a t a n e l e v a t i o n o f C a 2 + 1 h a do c c u r r e d . H o w e v e r , a s w e l l a s l o o k i n g a t f e r t i l i z a t i o ne n v e l o p e e l e v a t i o n a s a n i n d i r e c t a s s a y o f e x o c y t o s i s , weo b s e r v e d t h e c o r t i c a l g r a n u l e s t h e m s e l v e s . T h e c o r t i c a lg r a n u l e s w e r e o b s e r v e d b y u s i n g d i f f e r e n t i a l i n t e r f e r e n c ec o n t r a s t m i c r o s c o p y w i t h a L e i t z 1 0 0 x , 1 . 2 NA o i l -i m m e r s i o n o b j e c t i v e .

F l u o r e s c e n c e m e a s u r e m e n t s o f C a 2 + ; a n d o f d y e e f f l u x

C a 2 + w a s m e a s u r e d b y u s i n g t h e c a l c i u m - s e n s i t i v ef l u o r e s c e n t d y e f u r a 2 . T h e f u r a 2 ( p e n t a p o t a s s i u m s a l t ;M o l e c u l a r P r o b e s , J u n c t i o n C i t y , O R , U . S . A . ; 1 0 mM i n0 . 5 M - K C I / 2 0 m M - P i p e s , pH 6 . 7 ) w a s i n t r o d u c e d i n t o t h ee g g s b y m i c r o i n j e c t i o n t o g i v e a c y t o p l a s m i c c o n c e n t r a -

t i o n o f a b o u t 1 0 0 / I M . T h e f l u o r e s c e n c e f r o m s i n g l e e g g s

w a s m e a s u r e d b y t h e m e t h o d s d e s c r i b e d b y S w a n n &W h i t a k e r ( 1 9 8 6 ) . C a 2 " i w a s c a l c u l a t e d b y u s i n g t h em e t h o d s a n d b i n d i n g c o n s t a n t s o f P o e n i e e t a l . ( 1 9 8 5 ) .On o c c a s i o n , e g g s w e r e l o a d e d w i t h t h e f l u o r e s c e n t d y ef l u o r e s c e i n ( 2 0 1 t M - f l u o r e s c e i n d i a c e t a t e f o r 3 0 m i n ) a n d

t h e l e a k a g e o f t h e d y e f o l l o w i n g i n j e c t i o n w a s m e a s u r e d

u s i n g a N i k o n O p t i p h o t e p i f l u o r e s c e n c e m i c r o s c o p e

( e x c i t a t i o n 4 5 0 - 4 9 0 n m , e m i s s i o n 5 1 0 n m ) a n d a S I Tc a m e r a a t t a c h e d t o a v i d e o r e c o r d e r a n d v i d e o a n a l y s e r( C o l o r a d o V i d e o I n c . , B o u l d e r , C O , U . S . A . ) .

I n o s i t o l p h o s p h a t e s

T h e i n o s i t o l p h o s p h a t e s w e r e a g e n e r o u s g i f t f r o m D r .

R . F . I r v i n e o f t h e I n s t i t u t e o f A n i m a l P h y s i o l o g y ,B a b r a h a m , U . K . T h e y w e r e d i s s o l v e d i n a n i n j e c t i o nv e h i c l e c o n t a i n i n g 5 0 0 m M - K C l , 2 0 m M - P i p e s a n d 0 . 1mM-EGTA, pH 6 . 7 .

RESULTS

I n o s i t o l p h o s p h a t e s a n d c o r t i c a l g r a n u l e e x o c y t o s i s

We m i c r o i n j e c t e d I n s ( 1 , 4 , 5 ) P 3 , i n o s i t o l 2 , 4 , 5 - t r i s p h o s -p h a t e [ I n s ( 2 , 4 , 5 ) P 3 ] , i n o s i t o l 1 , 2 - c y c l i c 4 , 5 - t r i s p h o s p h a t e[ I n s ( 1 , 2 c y c 4 , 5 ) P 3 ] , i n o s i t o l 1 , 3 , 4 - t r i s p h o s p h a t e[ I n s ( 1 , 3 , 4 ) P 3 ] a n d i n o s i t o l 1 , 3 , 4 , 5 - t e t r a k i s p h o s p h a t e[ I n s ( 1 , 3 , 4 , 5 ) P 4 ] t o t e s t t h e i r e f f e c t s o n c o r t i c a l g r a n u l e

e x o c y t o s i s . I n s ( 1 , 4 , 5 ) P 3 i n d u c e d t h e e x o c y t o s i s o f c o r t i c a lg r a n u l e s a n d t h e r e s u l t a n t e l e v a t i o n o f a f e r t i l i z a t i o ne n v e l o p e . F i g . 1 s h o w s t w o e g g s , o n e m i c r o i n j e c t e d i na r t i f i c i a l s e a w a t e r (ASW) a n d o n e i n c a l c i u m - f r e e ASW.B o t h e g g s h a v e e l e v a t e d a c o m p l e t e f e r t i l i z a t i o n e n v e l o p e .H o w e v e r , t h e f e r t i l i z a t i o n e n v e l o p e i n c a l c i u m - f r e e ASWi s much l e s s r e f r a c t i l e a n d m o r e d i f f i c u l t t o s e e . F o r t h i sr e a s o n , we d e c i d e d t o o b s e r v e t h e c o r t i c a l g r a n u l e sd i r e c t l y w h e n d e t e r m i n i n g t h e e f f e c t s o f i n o s i t o l p h o s -p h a t e s .

I n e x p e r i m e n t s t o d e t e r m i n e t h e s e n s i t i v i t y o f e g g s t oi n o s i t o l p h o s p h a t e s w e i n j e c t e d a c o n s t a n t v o l u m e o f

i n o s i t o l p h o s p h a t e ( 1 . 5 p l , 0 . 3 0 e g g v o l u m e ) a n d v a r i e dt h e p i p e t t e c o n c e n t r a t i o n . T h e r e a s o n s f o r t h i s w e r et w o f o l d . F i r s t , t h e s e n s i t i v i t y o f t h e e g g s t o i n o s i t o lp h o s p h a t e s may b e a f u n c t i o n o f t h e c o n c e n t r a t i o n

i n j e c t e d a s w e l l a s t h e t o t a l d o s e . S e c o n d , w e w e r ec o n c e r n e d a b o u t p o s s i b l e d a m a g e t o e g g s c a u s e d b ym i c r o i n j e c t i n g l a r g e r v o l u m e s , p a r t i c u l a r l y i n c a l c i u m -

f r e e ASW ( s e e b e l o w ) . T h e r e s u l t s o f m i c r o i n j e c t i n gv a r i o u s i n o s i t o l p h o s p h a t e s i n t o L y t e c h i n u s p i c t u s e g g s

a r e s h o w n i n F i g . 2 . I n ASW t h e m o s t a c t i v e i n o s i t o lp h o s p h a t e t e s t e d w a s I n s ( 1 , 4 , 5 ) P 3 : h a l f - m a x i m a l a c t i v a -t i o n o c c u r s a t a n I n s ( 1 , 4 , 5 ) P 3 c o n c e n t r a t i o n o f 2 . 5 / M ,s i m i l a r t o t h e c o n c e n t r a t i o n d e t e r m i n e d i n e a r l i e r w o r k( W h i t a k e r & I r v i n e , 1 9 8 4 ; T u r n e r e t a l . , 1 9 8 6 ) . O t h e r

i n o s i t o l p h o s p h a t e s a l s o c a u s e d c o r t i c a l g r a n u l e e x o -

c y t o s i s . I n s ( 1 I , 2 c y c 4 , 5 ) P 3 , I n s ( 2 , 4 , 5 ) P 3 a n d I n s ( 1 , 3 , 4 , 5 ) P 4a c t i v a t e d e g g s a t h a l f - m a x i m a l c o n c e n t r a t i o n s o f 1 9 , 5 6

a n d 3 9 0 / M r e s p e c t i v e l y . We a l s o t e s t e d I n s ( 1 , 3 , 4 ) P 3 . I ts h o w e d l i t t l e a c t i v i t y : a - p i p e t t e c o n c e n t ra ti o n o f 1 m Mc a u s e d o n l y 2 0 0 o f t h e e g g s i n j e c t e d t o u n d e r g o c o m p l e t ec o r t i c a l g r a n u l e e x o c y t o s i s . M i c r o i n j e c t i o n o f v e h i c l ea l o n e c a u s e d n o s i g n o f e x o c y t o s i s ( n = 2 8 ) .

F i g . 2 a l s o s h o w s t h a t r e m o v i n g t h e e x t e r n a l c a l c i u m

d id n o t a f f e c t t h e s e n s i t i v i t y o f c o r t i c a l g r a n u l e e x o c y t o s i st o a n y o f t h e i n o s i t o l p h o s p h a t e s t e s t e d . T h i s r e s u l t w a s

v e r y d i f f e r e n t f r o m t h e p r e v i o u s r e p o r t s ( S l a c k e t a l . ,1 9 8 6 ; I r v i n e & M o o r , 1 9 8 6 ) o f a n e x t e r n a l c a l c i u m

1 9 8 8

2 5 8

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I n o s i t o l p h o s p h a t e s a n d c a l c i u m i n f l u x i n s e a u r c h i n e g g s

. z - s.:

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l e c l l . S t : . . ..s .___.._ I I > . s a : . . . . . . . . . . . . : ,; . < s P . . ; : : : s . . . . . .

u s: £e: . , , . :: .: , : : : : : :- _ a l t : x . > = . . . . }. : : : . . . . . . . . . : : . . . : . : : . , . ,I N , ; , s . : . . . : . . . . : . ; < > . :. . . . . . . l g f , . : : a : : . : . : . ;

il F s 5 > > t.. . . , s . : : : . :. . . l S o x e E . . . . .: F L ' : . : : : ' : :

.5 : ? t e f ' . : : ' . : . : : . :| b 9 i f D ' : . : : . : .s , a : : : : : . . . . . . . . . : : :. : : : :X . . . . . . . . . X . : S . - : : : : : :E l a t s , ; ; . . . . . . . . . . . , s .: : } . : :

R C t ; r : : : , .i_ f s s 5 . : : : : . : . . { ! S

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l l rl l i i : i s ' : ' . .:rS S i X . # S g > ' . : ' l . :^;

l | g X ' . a 8 : . ' " '| ? s ? S s s e s p i t rXs#@l E f . , r , . M , . . . . . . . . . . , ? W & , . ? .i s. : : w . s w 6 : ^ :| , , , . 5 : , . . . . . . . . . .r . S e . . . . . . . . . .a ? : . . . : . w ? s : e x t .. e A g D S s - . B s . . . ;

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. . . . . : 2 0 p r l l

F i g . 1 . T h e I n s P 3 - i n d u c e d e l e v a t i o n o f a f e r t i l i z a t i o n e n v e l o p e i n n o r m a l a n d c a l c i u m - f r e e ASW

Tw o e g g s m i c r o i n j e c t e d w i t h I n s ( 1 , 4 , 5 ) P 3 ( 1 . 5 p l , 1 0 # u M p i p e t t e c o n c e n t r a t i o n ) . On t h e l e f t , a n e g g i n o r d i n a r y ASW; o n t h e r i g h t ,a n e g g i n c a l c i u m - f r e e ASW. B o t h h a v e e l e v a t e d f e r t i l i z a t i o n e n v e l o p e s , b u t t h e e n v e l o p e o f t h e e g g i n c a l c i u m - f r e e ASW i s muchl e s s d i s t i n c t

I n s ( 1 , 4 , 5 ) P 3 I n s ( 1 , 2 c y c 4 , 5 ) P 3 n s ( 2 , 4 , 5 ) P 3 n s ( 1 , 3 , 4 , 5 ) P 4

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P i p e t t e c o n c en t r at i on o f i n o s i t o l p h o s p h a t e ( p M )

1 0 0 0

F i g . 2 . I n o s i t o l p h o s p h a t e - i n d u c e d c o r t i c a l g r a n u l e e x o c y t o s i s

E g g s w e r e m i c r o i n j e c t e d w i t h a c o n s t a n t v o l u m e o f i n o s i t o l p h o s p h a t e ( 1 . 5 p l ) a n d t h e p i p e t t e c o n c e n t r a t i o n w a s v a r i e d . T h e e g g s

w e r e s c o r e d f o r c o r t i c a l g r a n u l e e x o c y t o s i s b y d i r e c t o b s e r v a t i o n o f t h e c o r t i c a l g r a n u l e s b y u s i n g d i f f e r e n t i a l i n t e r f e r e n c ec o n t r a s t m i c r o s c o p y . C l o s e d s y m b o l s r e f e r t o i n j e c t i o n s p e r f o r m e d i n n o r m a l ASW. O p e n s y m b o l s r e f e r t o i n j e c t i o n s p e r f o r m e di n c a l c i u m - f r e e ASW. E a c h p o i n t o n t h e g r a p h r e p r e s e n t s t h e i n j e c t i o n o f b e t w e e n 1 0 a n d 1 5 e g g s .

r e q u i r e m e n t f o r I n s ( 1 , 4 , 5 ) P 3 - i n d u c e d e x o c y t o s i s . Tod e t e r m i n e w h e t h e r a s p e c i e s d i f f e r e n c e m i g h t a c c o u n t f o rt h i s d i s c r e p a n c y , we r e p e a t e d o u r e x p e r i m e n t s u s i n g e g g sf r o m L . v a r i e g a t u s , t h e s p e c i e s u s e d i n t h e f u l l e r o f t h et w o r e p o r t s . We o b t a i n e d v e r y s i m i l a r r e s u l t s i n L .

v a r i e g a t u s a n d L . p i c t u s ( T a b l e 1 ) .We a l s o a t t e m p t e d t o r e p r o d u c e t h e r e p o r t e d s y n e r g i s m

b e t w e e n I n s ( 1 , 3 , 4 , 5 ) P 4 a n d I n s ( 2 , 4 , 5 ) P 3 ( I r v i n e & M o o r ,1 9 8 6 ) . We c o - i n j e c t e d s u b m a x i m a l d o s e s : 1 0 / , M -

I n s ( 1 , 3 , 4 , 5 ) P 4 w i t h e i t h e r 1 0 / t M - I n s ( 2 , 4 , 5 ) P 3 o r 1 / t M -I n s ( 1 , 4 , 5 ) P 3 . We d i d n o t o b s e r v e a n y s y n e r g i s t i c e f f e c t s .

D y e l e a k a g e f r o m m i c r o i n j e c t e d e g g s

O u r o b s er v at i o n s w e r e s o c l e a r l y d i f f e r e n t f r o m t h o s er e p o r t e d b y I r v i n e & M oor ( 1 9 8 6 ) t h a t we madee x p e r i m e n t s i n w h i c h we a t t e m p t e d t o r e p r o d u c e t h e i rm e t h o d s a s c l o s e l y a s we c o u l d . F o r t h e s e e x p e r i m e n t s w ea t t e m p t e d t o u s e t h e i r l o w - p r e s s u r e m i c r o i n j e c t i o n

V o l . 2 5 2

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1 . C r o s s l c y a n d o t h e r s

T a b l e 1 . I n o s i t o l p h o s p h a t e - i n d u c e d c o r t i c a l g r a n u l e e x o c y t o s i s i n e g g s o f t h e s e a u r c h i n s p e c i e s L . v a r i e g a t u s

O n l y c o m p l e t e f e r t i l i z a t i o n e n v e l o p e e l e v a t i o n w a s s c o r e d . N . D . , n o t d e t e r m i n e d .

S c o r e

V o l u m e P i p e t t e N o r m a l C a l c i u m -( p I ) c o n c e n t r a t i o n ASW f r e e ASW

I n s ( l , 4 , 5 ) P 3I n s ( 1 , 3 , 4 , 5 ) P 4

I n s ( 2 , 4 , 5 ) P 3

I n s ( l , 2 c y c 4 , 5 ) P I

0 . 5 1 0 I U M0 . 5 1 0 0 I t M0 . 5 1 mM0 . 5 1 0 , U M5 1 0 #M0 . 5 l O / I M

0 . 5 1 0 0 #M

m e t h o d a n d t h e i r l a r g e r i n j e c t i o n v o l u m e s ( 4 e g g

v o l u m e ) . We f o u n d t h a t l a r g e r i n j e c t i o n v o l u m e s (> 2eg g v o l u m e ) c a u s e d more v i s i b l e d a m a g e t o eggs,

p a r t i c u l a r l y i n c a l c i u m - f r e e s e a w a t e r . We d i d n o t f i n d

l o wpressure

i n j e c t i o n t o b e a s a t i s f a c t o r y m e t h o d f o ri n t r o d u c i n g l a r g e v o l u m e s o f s o l u t i o n i n t o t h e egg. Tot e s t t h e i d e a t h a t l a r g e i n j e c t i o n v o l u m e s may d a m a g eeggs we us e h i g h pressure t o m i c r o i n j e c t eggs i n c a l c i u m -f r e e ASW w i t h e i t h e r 1 . 5 p l or 2 0 p l o f 10 U t M -I n s ( 1 , 4 , 5 ) P 3 . I n t h e s e e x p e r i m e n t s , t h e i n j e c t i o n o f 1 . 5 p l( 0 O . 3 0 0 egg v o l u m e ) o f 10 / M - I n s ( l , 4 , 5 ) P 3 c a u s e d 8 5 0 %( n = 3 ) eggs t o u n d e r g o c o m p l e t e c o r t i c a l g r a n u l ee x o c y t o s i s i n c a l c i u m - f r e e ASW, w h i l e t h e i n j e c t i o n o f2 0 p l ( 4 egg v o l u m e ) o f 1 0 / t M - I n s ( 1 , 4 , 5 ) P 3 c a u s e d

3 8 . 6 0 % ( n = 8 ) o f t h e eggs i n j e c t e d t o u n d e r g o c o m p l e t ec o r t i c a l g r a n u l e e x o c y t o s i s . T h u s m i c r o i n j e c t i n g l a r g e rv o l u m e s o f t h e same c o n c e n t r a t i o n o f I n s ( 1 , 4 , 5 ) P 3 c a u s e d

s i g n i f i c a n t l y l e s s a c t i v a t i o n ( P < 0 . 0 0 5 ) .We made a n e s t i m a t e o f t h e l e a k i n e s s o f eggs a f t e r

m i c r o i n j e c t i o n b y p r e - l o a d i n g t h e eggs w i t h a f l u o r e s c e n td y e a n d m e a s u r i n g t h e r a t e o f l e a k a g e o f t h e d y e f r o m t h eeggs a f t e r l a r g e a n d s m a l l v o l u m e m i c r o i n j e c t i o n s u s i n gh i g h pressure p u l s e s ( s e e t h e M a t e r i a l s a n d m e t h o d ss e c t i o n ) . We f o u n d t h a t d y e l e a k a g e was n o t d e t e c t a b l ea f t e r m i c r o i n j e c t i o n i n n o r m a l ASW w i t h i n j e c t e dv o l u m e s o f up t o 1 2 p l ( 2 . 4 % egg v o l u m e ) . I n c a l c i u m -f r e e ASW, l a r g e v o l u m e i n j e c t i o n s ( 1 2 p l , 2 . 4 0 % eg g

v o l u m e ) c a u s e d more r a p i d d y e l e a k a g e t h a n s m a l lv o l u m e i n j e c t i o n s ( 1 . 5 p l , 0 . 3% gg v o l u m e ) : t h e f o r m e rc a u s e d a l o s s o f 14+ 1 . 3 % ( m e a n + S . E . M . , n =3 ) o f t h ed y e 2 m i n a f t e r i n j e c t i o n , t h e l a t t e r o n l y 2 . 7 + 2 . 6 6 %( n = ) . T h i s d i f f e r e n c e i s s i g n i f i c a n t ( P < 0 . 0 2 , o n e t a i l e dt - t e s t ) .

M e a s u r e m e n t o f C a 2 + i a f t e r m i c r o i n j e c t i o n o f i n o s i t o lp h o s p h a t e s

Our o b s e r v a t i o n s t h a t i n o s i t o l p h o s p h a t e - i n d u c e dc o r t i c a l g r a n u l e e x o c y t o s i s i s i n d e p e n d e n t o f e x t e r n a l

c a l c i u m i on s d i d n o t e x c l u d e t h e p o s s i b i l i t y t h a t i n o s i t o lp h o s p h a t e s cause a c a l c i u m i n f l u x t h a t a f f e c t s t h e

d u r a t i o n a n d m a g n i t u d e o f t h e i n o s i t o l p h o s p h a t e -i n d u c e d i n c r e a s e i n C a 2 + i , s i n c e e x o c y t o s i s i s n o t a s t r i c t l yq u a n t i t a t i v e m e a s u r e o f C a 2 ' + . We t h e r e f o r e measured

c h a n g e s i n C a 2 " , f o l l o w i n g t h e i n j e c t i o n o f I n s ( 1 , 4 , 5 ) P 3a n d I n s ( 1 , 3 , 4 , 5 ) P 4 b y u s i n g t h e c a l c i u m - s e n s i t i v e f l u o r e s -

c e n t d y e f u r a 2 . The r e s u l t s o f t h e s e e x p e r i m e n t s ar e

shown i n F i g . 3 ( a ) . We f o u n d t h a t t h e i n j e c t i o n o f

I n s ( 1 , 4 , 5 ) P 3 ( 1 . 5 p l o f 1 0 , g M ) i n ASW c a u s e d a t r a n s i e n t

i n c r e a s e i n C a 2 " , w i t h a p e a k o f 2 /M a n d a d u r a t i o no f 3 m i n . R e m o v i n g t h e e x t e r n a l c a l c i u m i o n s

a f f e c t e d n e i t h e r t h e m a g n i t u d e n or d u r a t i o n o f t h ec a l c i u m response. We o b t a i n e d s i m i l a r r e s u l t s when

I n s ( l , 3 , 4 , 5 ) P 4 ( 1 p l o f I m M) was m i c r o i n j e c t e d ( F i g . 3 ( b ) ) .

DISCUSSION AND CONCLUSION

We h a v e i n v e s t i g a t e d t h e p a r t h e n o g e n e t i c a c t i v a t i o no f se a u r c h i n eggs b y i n o s i t o l p h o s p h a t e s a n d t h e r e q u i r e -ments f o r e x t e r n a l c a l c i u m . I n s ( 1 , 4 , 5 ) P 3 was t h e most

a c t i v e i n o s i t o l p h o s p h a t e t e s t e d , c a u s i n g c o m p l e t ec o r t i c a l g r a n u l e e x o c y t o s i s a t a h a l f - m a x i m a l concen-

t r a t i o n o f 2 . 5 /!M, a v a l u e i n g o o d a g r e e m e n t w i t h e a r l i e rf i n d i n g s ( W h i t a k e r & I r v i n e , 1 9 8 4 ; T u r n e r e t a l . , 1 9 8 6 ) .We h a v e c o n f i r m e d I r v i n e & M o o r ' s ( 1 9 8 6 ) o b s e r v a t i o n

t h a t I n s ( 1 , 3 , 4 , 5 ) P 4 h a s l i t t l e c a l c i u m - r e l e a s i n g a c t i v i t y i nse a u r c h i n e g g s . T h e e f f e c t s we o b s e r v e d may b ea c c o u n t e d f o r b y t h e c o n t a m i n a t i o n o f o ur s a m p l e o f

I n s ( 1 , 3 , 4 , 5 ) P 4 w i t h I n s ( 1 , 4 , 5 ) P 3 . I n c o n t r a s t we f o u n d t h a tI n s ( 2 , 4 , 5 ) P 3 h a d a n a c t i v i t y t wo o r d e r s o f m a g n i t u d eg r e a t e r t h a n t h a t p r e v i o u s l y r e p o r t e d ( I r v i n e & M o o r ,1 9 8 6 ) . Our o b s e r v a t i o n s p o i n t t o a s i m i l a r s e n s i t i v i t y f o rI n s ( 2 , 4 , 5 ) P 3 i n se a u r c h i n eggs as i n o t h e r t i s s u e s ( I r v i n ee t a l . , 1 9 8 4 ) . Our f i n d i n g t h a t t h e two m a i n m e t a b o l i t e so f I n s ( l , 4 , 5 ) P 3 , I n s ( l , 3 , 4 , 5 ) P 4 a n d I n s ( l , 3 , 4 ) P 3 ( B e r r i d g e ,1 9 8 7 ) , h a v e l i t t l e c a l c i u m - m o b i l i z i n g a c t i v i t y i n un -

f e r t i l i z e d se a u r c h i n eggs argues a g a i n s t t h e i r h a v i n g a

s i g n i f i c a n t r o l e as i n t e r n a l messengers c o n t r o l l i n g Ca2+a t f e r t i l i z a t i o n .We f i n d t h a t i n o s i t o l p h o s p h a t e - i n d u c e d c o r t i c a l

g r a n u l e e x o c y t o s i s occurs i n d e p e n d e n t l y o f e x t r a c e l l u l a r -c a l c i u m i o n s . M o r e o v e r , we f i n d t h a t t h e i n o s i t o l

p h o s p h a t e - i n d u c e d c a l c i u m t r a n s i e n t i s n o t m e a s u r a b l ya f f e c t e d b y t h e r e m o v a l o f e x t e r n a l c a l c i u m . T h e s e d a t a

i n d i c a t e t h a t i n o s i t o l p h o s p h a t e s d o n o t i n c r e a s e c a l c i u mi n f l u x i n se a u r c h i n eggs i n a way t h a t s i g n i f i c a n t l y a f f e c t s

C a 2 + . Our f i n d i n g s ar e e n t i r e l y c o n s i s t e n t w i t h t he i d e a

t h a t I n s ( 1 , 4 , 5 ) P 3 i s t h e c r u c i a l i n t e r n a l messenger a t

f e r t i l i z a t i o n , w h e r e i t b r i n g s a b o u t r e l e a s e o f c a l c i u m

f r o m an i n t e r n a l s t o r e ( W h i t a k e r & I r v i n e , 1 9 8 4 ; C l a p p e r& L e e , 1 9 8 5 ; Swann & W h i t a k e r , 1 9 8 6 ) . T h e s e r e s u l t s

a n d , i n d e e d , p r e v i o u s work ( C h a m b e r s , 1 9 8 0 ; S c h m i d t e t

a l . , 1 9 8 2 ) make s c h e m e s i n w h i c h c a l c i u m i n f l u x p l a y s a

s i g n i f i c a n t p a r t a t f e r t i l i z a t i o n ( I r v i n e & M o o r , 1 9 8 6 ;M i c h e l l , 1 9 8 6 ; H o u s l a y , 1 9 8 7 ) h i g h l y u n l i k e l y . Ou ro b s e r v a t i o n s c l e a r l y d i f f e r from e a r l i e r f i n d i n g s ( S l a c k e t

a l . , 1 9 8 6 ; I r v i n e & M o o r , 1 9 8 6 ) . On e p o s s i b l e e x p l a n a t i o n

1 9 8 8

2 0 / 2 00 / 1 09 / 1 14 / 1 0

1 0 / 1 00 / 1 0

1 0 / 1 1

2 0 / 2 0N . D .1 2 / 1 9N . D .N . D .N . D .N . D .

2 6 0

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F i g . 3 . C h a n g e s i n C a 2 + ; i n d u c e d b y m i c r o i n j e c t i o n o f I n s ( 1 , 4 , 5 ) P 3 a n d 1 n s ( 1 , 3 , 4 . ) 5 ) P 4

( a ) C a 2 + . was m e a s u r e d w i t h t h e c a l c i u m - s e n s i t i v e d y e f u r a 2 f o l l o w i n g t h e m i c r o i n j e c t i o n o f I n s ( 1 , 4 , 5 ) P 3 a t t =0 ( 1 . 5 p l , 1 0 p mp i p e t t e c o n c e n t r a t i o n ) . C l o s e d s y m b o l s ( A , * , * ) r e p r e s e n t i n j e c t i o n s i n o r d i n a r y ASW a n d o p e n s y m b o l s ( E , F 2 , O ) r e p r e s e n t

i n j e c t i o n s p e r f o r m e d i n c a l c i u m - f r e e ASW. T h r e e e x p e r i m e n t s were p e r f o r m e d i n e a c hc a s e

a n d e a c h i n j e c t i o n c a u s e d t h ee l e v a t i o n o f a c o m p l e t e f e r t i l i z a t i o n e n v e l o p e . T h e r e i s no o b v i o u s d i f f e r e n c e i n t h e e x t e n t a n d d u ra ti o n o f t h e I n s P 3 - i n d u c e dc a l c i u m t r a n s i e n t i n n o r m a l a n d c a l c i u m - f r e e ASW. ( b ) C a 2 + i f o l l o w i n g t h e m i c r o i n j e c t i o n o f I n s ( 1 , 3 , 4 , 5 ) P 4 a t t = O ( 1 . 5 p l ,I mm p i p e t t e c o n c e n t r a t i o n ) . As i n ( a ) , c l o s e d s y m b o l s r e p r e s e n t i n j e c t i o n s p e r f o r m e d i n o r d i n a r y ASW a n d o p e n s y m b o l si n j e c t i o n s p e r f o r m e d i n c a l c i u m - f r e e ASW. T h r e e e x p e r i m e n t s were p e r f o r m e d i n e a c h c a s e a n d e a c h i n j e c t i o n c a u s e d t h ee l e v a t i o n o f a c o m p l e t e f e r t i l i z a t i o n e n v e l o p e .

f o r t h i s d i f f e r e n c e may l i e i n t h e d a m a g e t h a t t h e r e l e a s e d b y t h e i n o s i t o l p h o s p h a t e s . We c h o s e t o

m i c r o i n j e c t i o n o f l a r g e v o l u m e s c a u s e s t o e g g s i n c a l c i u m - m i c r o i n j e c t s m a l l v o l u m e s t o a v o i d t h i s p r o b l e m .f r e e ASW. I t i s p o s s i b l e t h a t t h e l e a k i n e s s t h a t we We h a v e no e x p l a n a t i o n f o r our f a i l u r e t o o b s e r v e t h em e a s u r e d u s i n g f l u o r e s c e n t d y e s i s s u f f i c i e n t t o a l l o w t h e s y n e r g i s m b e t w e e n I n s ( 1 , 3 , 4 , 5 ) P 4 a n d I n s ( 2 , 4 , 5 ) P 3 p r e v i -EGTA p r e s e n t i n t h e c a l c i u m - f r e e ASW t o e n t e r t h e e g g s o u s l y r e p o r t e d ( I r v i n e & M o o r , 1 9 8 6 ) , t h o u g h i t a p p e a r sd u r i n g m i c r o i n j e c t i o n a n d c h e l a t e a n y c a l c i u m i o n s t h a t o t h e r s h a v e h a d s i m i l a r d i f f i c u l t i e s ( I r v i n e & M o o r ,

V o l . 2 5 2

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2 6 2 I . C r o s s l e y a n d o t h e r s

1 9 8 7 ; I r v i n e e t a l . , 1 9 8 8 ) . I r v i n e & Moor ( 1 9 8 7 ) h a v es u g g e s t e d t h a t t h e i r i n a b i l i t y t o r e p r o d u c e t h i s c r u c i a lo b s e r v a t i o n s t e m s f r o m a s e a s o n a l v a r i a t i o n i n t h er e s p o n s e o f L . v a r i e g a t u s e g g s t o i n o s i t o l p h o s p h a t e s . Wef i n d n o s u c h v a r i a t i o n i n t h e r e s p o n s e s o f L . p i c t u s e g g s .I t h as b e e n s u g g e s t e d t h a t w h e t h e r o r n o t a c e l l r e q u i r e se x t e r n a l a s w e l l a s i n t e r n a l c a l c i u m f o r a r e s p o n s e

d e p e n d s c r u c i a l l y o n t h e s i z e o f t h e i n t e r n a l s t o r e r e l a t i v et o t h e c y t o s o l i c f r e e c a l c i u m c o n c e n t r a t i o n c h a n g e

r e q u i r e d t o p r o d u c e t h e r e s p o n s e . T h i s p o s t u l a t e i m p l i e st h a t t h e s y n e r g i s m b e t w e e n I n s ( 2 , 4 , 5 ) P 3 a n d I n s ( 1 , 3 , 4 , 5 ) P 4m i g h t b e o b s e r v e d o n l y i n e g g s w i t h a d e p l e t e d i n t r a -c e l l u l a r c a l c i u m s t o r e . T h i s d o e s n o t s e e m t o u s a

s a t i s f a c t o r y e x p l a n a t i o n , s i n c e e g g s t h a t m a n i f e s t e d t h es y n e r g i s m h a d a n o r m a l c e l l u l a r r e s p o n s e t o I n s P 3 . W e l l -

b e h a v e d L . v a r i e g a t u s e g g s h a v e a r o b u s t i n t r a c e l l u l a rs t o r e t h a t c a n n o t b e d e p l e t e d b y l o n g i n c u b a t i o n s i nc a l c i u m - f r e e s e a w a t e r ( A z a r n i a & C h a m b e r s , 1 9 7 6 ) : t h ep o s t u l a t e i s t h e r e f o r e d i f f i c u l t t o t e s t .

T h i s w o r k w a s s u p p o rt e d b y g r a n t s f r o m t h e W e l l c o m eT r u s t , a n d t h e B r o w n e F u n d o f t h e R o y a l S o c i e t y . I . C . h o l d s

a W e l l c o m e R e s e a r c h S t u d e n t s h i p a n d K . S . i s a n SERCs c h o l a r . We t h a n k D r . R . F . I r v i n e f o r p r o v i d i n g t h e i n o s i t o lp h o s p h a t e s , a n d D . M i d d l e m a s s f o r h i s h e l p i n p r e p a r i n g t h ef i g u r e s .

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R e c e i v e d 1 6 N o v e m b e r 1 9 8 7 / 8 J a n u a r y 1 9 8 8 ; a c c e p t e d 2 6 F e b r u a r y 1 9 8 8

1 9 8 8