RAPID COMMUNICATION ZIPGRAM

FERTILIZATION A C I D OF SEA URCHIN EGGS IS NOT A CONSEQUENCE

OF CORTICAL GRANULE EXOCYTOSIS

MILES PAUL (1) JAMES D. JOHNSON ( 2 ) AND D A V I D EPEL ( 2 ) Department of B io logy , U n i v e r s i t y of V i c t o r i a , V i c t o r i a , B.C. V8W 2Y2; U n i v e r s i t y o f C a l i f o r n i a , Scr ipps I n s t i t u t i o n o f Oceanography, La J o l l a , C a l i f o r n i a 92037

ABSTRACT Sea u r c h i n eggs t r e a t e d w i t h 10 mM NH C 1 re lease a " f e r t i l - i z a t i o n a c i d " a1 though c o r t i c a l g ranu le e x o c y t o t i s does n o t take p lace . I f t h e eggs are inseminated f o l l o w i n g ammonia a c t i v a t i o n , t he c o r t i c a l r e a c t i o n occurs and a f e r t i l i z a t i o n membrane e leva tes i n t h e absence o f de tec tab le a c i d re lease. Examination by e l e c t r o n microscopy o f eggs f i x e d between ammonia a c t i v a t i o n and inseminat ion conf i rms t h e presence o f i n t a c t granules. Thus, t h e f e r t i l i z a t i o n a c i d i s n o t caused by t h e re lease o f t h e c o r t i c a l granules. Ammonia t rea tment o f f e r t i l i z e d eggs s t imu la tes f u r t h e r re lease of ac id . The re lease o f a c i d can occur r e - pea ted ly i n ammonia a c t i v a t e d eggs i f they are washed i n t o normal sea- water between successive ammonia t rea tments . Our r e s u l t s suggest t h a t t he re lease o f f e r t i l i z a t i o n a c i d i s r e l a t e d t o some metabo l i c process whichcan be tu rned "on o r o f f " .

One o f t h e e a r l i e s t events f o l l o w i n g f e r t i l i z a t i o n o f sea u r c h i n eggs i s

an e f f l u x o f a c i d ( f e r t i l i z a t i o n a c i d ) i n t o t h e sur round ing seawater (SW).

E a r l y work e l i m i n a t e d the most obvious metabo l ic sources o f a c i d (C02, l a c t i c

ac id , e t c . (Rothsch i ld , ' 5 6 ) ) . Since t h i s a c i d re lease begins a t t he same

t ime t h a t c o r t i c a l g ranu le exocy tos i s begins, a r e l a t i o n t o t h e re lease o f gran-

u l e conten ts i s suggested, and t h e presence o f a c i d mucopolysaccharide i n the

c o r t i c a l granules has supported t h i s view

' 75 ) . However, a c i d re lease cont inues f o r

g ranu le exocy tos i s i s complete. Also, t h e

f o r references, see Paul and Epel ,

severa l minutes a f t e r t he c o r t i c a l

eggs o f Urech is caupo re lease a

f e r t i l i z a t i o n a c i d w i t h no concomitant c o r t i c a l g ranu le exocy tos i s (Paul , '75b) .

These observa t ions have l e d us t o i n v e s t i g a t e o t h e r p o s s i b l e sources o f f e r t i l -

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i z a t i o n ac id , such as e x p o r t from t h e cytoplasm i t s e l f o r inc reased metabo l ic

p roduc t i on o f p ro tons a f t e r f e r t i l i z a t i o n .

Here we present s t r o n g evidence t h a t t he f e r t i l i z a t i o n a c i d i s n o t asso-

c i a t e d w i t h t h e c o r t i c a l g ranu le exocy tos i s b u t i s more d i r e c t l y r e l a t e d t o some

aspect o f metabo l ic a c t i v a t i o n . We were l e d t o t h i s concept through a cont inu-

i n g a n a l y s i s of t he p a r t i a l a c t i v a t i o n o f eggs by ammonia. M i l l i m o l a r amounts

o f ammonia w i l l i n i t i a t e RNA, DNA, and p r o t e i n syn thes is but do n o t i n i t i a t e a

c o r t i c a l r e a c t i o n (S te inha rd t and Mazia, '73; reviewed by Epel e t a l . , ' 74) .

We f i n d t h a t ammonia induces a c i d re lease s i m i l a r t o t h a t f o l l o w i n g normal

inseminat ion . When we f e r t i l i z e d ammonia a c t i v a t e d eggs, a normal c o r t i c a l

granule exocy tos is occurred, b u t t h e r e was no re lease o f a c i d assoc ia ted w i t h

t h i s process.

changes and may have a s i g n i f i c a n t r o l e i n t h e a c t i v a t i o n o f t h e eggs.

The a c i d i s p o s s i b l y a m a n i f e s t a t i o n o f more pro found cy top lasmic

MATERIALS AND METHODS For t h e exper iments descr ibed, we used gametes o f

S t rongy locent ro tus purpuratus ob ta ined near San Diego, C a l i f o r n i a o r Vancouver

I s land , B r i t i s h Columbia. S i m i l a r r e s u l t s were ob ta ined w i t h gametes o f Lyte-

chinus p i c t u s (San Diego) and 5. drobach iens is (Vancouver I s l a n d ) b u t a re n o t

repo r ted i n d e t a i l .

M KC1. The e x t r a -

c e l l u l a r pH o f 2% egg suspensions (17' C) was determined as descr ibed p r e v i o u s l y

(Paul, '75b; Paul and Epel, ' 7 5 ) . The abso lu te amount o f a c i d re leased was

determined a f t e r each experiment by back t i t r a t i o n w i t h s tandard ized NaOH.

Ammonia a c t i v a t i o n o f eggs was by a d d i t i o n o f 2 M NH4C1 i n SW (ad jus ted t o the

pH o f egg suspensions) t o g i v e a f i n a l concen t ra t i on o f 10 mM NH4C1.

examined by l i g h t microscopy w i t h t h e a c e t i c ac id -e thano l procedure (Paul , '75a).

For e l e c t r o n microscopy, eggs were f i x e d and processed as descr ibed by Gould-

Somero and Ho l land ( ' 7 5 ) and examined on a Zeiss EM-9-EM. Other exper imental

Gametes were ob ta ined by i n t racoe lomic i n j e c t i o n o f 0.5

Eggs were d e j e l l i e d by a g i t a t i o n and washing i n f i l t e r e d SW.

Eggs were

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TABLE 1

Amount of acid released from eggs following sperm, ammonia, and successive sperm o r ammoni a treatment.

~~ -

ACID ACID TREATMENT RELEASED WASH TREATMENT RELEASED

# I pmol /ml # 2 umol / m l packed eggs packed eggs

1 sperm 5.1 - - - 2 sperm 7.4 no NH4C1 12.8

3 NH4C1 9.4 no sperm - 4 NH4C1 10.7 Yes sperm 6 .7

5 N H 4 C 1 11.0 yes NH4C1 ( 2 ) 8.0

yes NH4C1 ( 3 ) 7.5

de t a i l s a r e described in the t ex t .

RESULTS AND DISCUSSION The normal k ine t ics of f e r t i l i z a t i o n acid re lease

i s shown in f igure l a .

continues f o r approximately 5 min.

pmol per m l of packed c e l l s (1, tab le 1).

Efflux begins about 30 sec a f t e r sperm addi t ion and

The to t a l amount o f acid released was 5 . 1

"Activation" of the eggs by ammonia r e s u l t s in a par t ia l response which

includes increased protein synthesis , polyadenylation of m R N A , DNA synthesis ,

and chromosome condensation (reviewed by Epel e t a1 . , '74 ) . However, exocyto-

s i s of cor t ica l granules and subsequent events re la ted to i t do not occur.

When ammonia i s added t o unfer t i l i zed eggs, protons a re a l so released ( f i g . l b ;

a l so , f i g . 3a ,b) . The release of ac id , more rapid than with sperm, i s essen-

t i a l l y complete i n 40 sec. Also, the amount of acid released by ammonia, 9.4

pmol per ml packed eggs in t h i s experiment (average, 10.2 f 0.9; N = 5 ) , i s

greater than tha t released a f t e r insemination. Nicotine (1 mM) and procaine

( 5 mM) a lso ac t iva t e protein and DNA synthesis , b u t do not i n i t i a t e detectable

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c o r t i c a l granule exocy tos i s (Johnson and Epel, '75; Vacquier, ' 7 5 ) . We f i n d

t h a t eggs incubated i n these amines re lease a c i d e q u i v a l e n t i n amount t o t h a t

re leased by ammonia.

The ammonia t r e a t e d eggs were f i x e d i n a c e t i c ac id -e thano l and examined

by phase microscopy.

had n o t e leva ted t o form a f e r t i l i z a t i o n membrane. We conf i rmed t h e absence

o f c o r t i c a l granule exocy tos i s by e l e c t r o n microscopy ( f i g . 2) and found no

d i f f e r e n c e i n s t r u c t u r e o f these granules i n c o n t r o l and ammonia t r e a t e d eggs.

The c o r t i c a l granules were i n t a c t and t h e v i t e l l i n e l a y e r

When eggs which have been a c t i v a t e d by ammonia a re inseminated (arrowhead,

f i g . l b ) , t he re i s a r a p i d c o r t i c a l g ranu le exocy tos i s r e s u l t i n g i n t h e complete

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 membrane w i t h i n one min (double arrowhead) as

normal ly occurs a t inseminat ion .

t h i s exocy tos is . Thus, i n t h e f i r s t ins tance, t he f e r t i l i z a t i o n a c i d i s r e -

leased w i t h o u t c o r t i c a l g ranu le exocy tos is , and i n t h e second, c o r t i c a l granule

exocy tos is occurs w i t h o u t t he re lease o f f e r t i l i z a t i o n ac id .

e l i m i n a t e the longs tand ing hypothesis t h a t f e r t i l i z a t i o n a c i d i s re leased from

No f e r t i l i z a t i o n a c i d re lease accompanies

These r e s u l t s

FIGURE LEGENDS

1 F e r t i l i z a t i o n a c i d re lease f o l l o w i n g inseminat ion (a ) and ammonia a c t i v a t i o n (b ) a t t ime 0. (Experiments 1 and 3 i n t a b l e 1). Am- monia a c t i v a t i o n i s by a d d i t i o n o f 20 p l o f 2 M NH C1 i n SW a t pH 8. Arrowhead i n d i c a t e s i nsemina t ion o f t h e a m 8 n i a a c t i v a t e d eggs; a t t he double arrowhead, a l l eggs had e leva ted f e r t i l i z a t i o n membranes. C o r t i c a l g ranu le exocy tos i s occurs between t h e t imes i n d i c a t e d by t h e s i n g l e and double arrowheads.

'2 E l e c t r o n micrographs o f u n f e r t i l i z e d ( a ) and ammonia a c t i v a t e d (b ) eggs, showing t h a t ammonia a c t i v a t i o n does n o t i n i t i a t e the exocy- t o s i s o f t h e c o r t i c a l granules. ( X 16,800). F e r t i l i z a t i o n a c i d re lease induced by ammonia ( t ime 0). min the egg suspensions were t i t r a t e d w i t h standard NaOH t o t h e o r i g i n a l pH, and e i t h e r l e f t unwashed (a ) o r washed i n t o f r e s h SW (b ) . The suspensions were inseminated a t 30 min. Ac id re lease recu rs only a f t e r eggs a r e washed i n t o f r e s h SW (b) . 3 and 4, t a b l e 1).

3 A f t e r 5

(Experiments S ing le and double arrowheads as i n f i g u r e 1.

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7.9 T f -z-- 7%-

I n -

8.0 -- 0 5

a b @

I I I I I

I

I I 1 r 30 A 35 40

MINUTES

the cor t ica l granules as ac id ic mucopolysaccharide materials.

When ammonia i s added t o f e r t i l i z e d eggs, there i s an additional acid e f -

f lux.

lowing insemination was as great a s t h a t released by ammonia alone ( 2 , t ab le 1).

However, i n a second experiment with eggs of C. p ic tus , the to t a l acid released

was approximately equal t o t h a t released by unfer t i l i zed eggs only act ivated

by ammonia.

eggs r e su l t s in no fur ther acid re lease .

In one experiment the amount of acid released by ammonia addition fo l -

A second additionof ammonia to e i t h e r unfer t i l i zed o r f e r t i l i z e d

When ammonia i s removed, however, the capacity of the eggs t o re lease

acid i s "regenerated".

complete, then washed the eggs and l e f t them in f resh SW fo r 20-30 m i n . When

we added sperm ( f i g . 3b; 4, t ab le 1) o r ammonia (5 , t ab le 1) t o these eggs,

additional acid was released, in cont ras t t o the behavior of ammonia t rea ted

eggs which were not washed before addition of sperm ( f i g . 3a; 3, t ab le 1) o r

more ammonia.

following an i n i t i a l ammonia ac t iva t ion (5, t ab le 1 ) . The repeated acid re-

lease following annnonia ac t iva t ion i s dependent on the incubation of eggs i n

fresh SW, suggesting tha t acid may accumulate in the eggs when ammonia i s re-

moved.

of sperm or ammonia.

We incubated eggs i n ammonia unt i l acid release was

We have stimulated eggs t o re lease acid a second and th i rd time

This acid i s then avai lable f o r re lease upon the subsequent addition

These r e su l t s , summarized i n t ab le 1, eliminate cor t ica l granule exocy-

t o s i s as a source of the acid and are most consis tent w i t h a metabolic source

of the " f e r t i l i z a t i o n acid". Acid release i s st imulated by compounds such a s

ammonia, nicot ine, and procaine which ac t iva t e metabolism of the eggs, and the

a b i l i t y of the eggs t o re lease acid can be regenerated. Although the s igni f -

icance o f the acid e f f lux i s s t i l l unclear, the amount o f acid i s qu i t e large

(equivalent t o more than 5 mM on the basis of ce l l water) . The exportation

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of the acid could r e su l t i n a substant ia l change in cytoplasmic pH, which in

t u r n would have an important ro l e in the cytoplasmic ac t iva t ion of the eggs.

We a re presently invest igat ing t h i s poss ib i l i ty .

ACKNOWLEDGMENTS We thank E . Baker and R. N . Johnston fo r technical as-

s i s tance . Aided by grants from the National Research Council of Canada and

the National Science Foundation, and by a National In s t i t u t e s of Health

Training grant.

LITERATURE CITED

Epel, D . , R . S te inhardt , T. Humphreys and D. Mazia 1974 An analysis of the par t ia l metabolic derepression of sea urchin eggs by am- monia: the existence of independent pathways. Develop. Biol . ,

Gould-Somero, M . , and L . Holland 1975 Oocyte d i f f e ren t i a t ion in Urechis caupo (Echiura): a f ine s t ruc tura l study. J . Morph.,

Johnson, J . D . , and D. Epel 1975 Relationship between release of surface proteins and metabolic act ivat ion o f sea urchin eggs a t f e r t i l i z a t i o n . Proc. Nat. Acad. Sci . USA, 72: 4474-4478.

Paul, M. 1975a The polyspermy block in eggs of Urechis caupo: evidence f o r a "rapid block". Exptl. Cell Res., 90: 137-142.

Paul, M. 1975b Release of acid and changes in l igh t -sca t te r ing propert ies following f e r t i l i z a t i o n of Urechis caupo eggs. Develop. Biol . , 43: 299-312.

Paul, M. and D. Epel 1975 Formation of f e r t i l i z a t i o n acid by sea urchin eggs does not require spec i f i c cat ions. Exptl. Cell Res.,

Rothschild, Iord 1956 Fe r t i l i za t ion . Hol t , Rinehart and Winston, New York.

Steinhardt , R. A . and D. Mazia and membrane potent ia l in unfer t i l i zed sea urchin eggs a f t e r ex- posure t o NH40H. Nature (London), 241: 400-401.

Vacquier, V . D. 1975 The i so la t ion of i n t ac t cor t ica l granules from sea urchin eggs: calcium ion t r iggers granule discharge. Develop. Biol . , 43: 62-74.

40: 245-255.

147: 475-506.

94: 1-6.

1973 Development of Kt- conductance

REFERENCES

1 Department of Bio'logy, University o f Victor ia , Victor ia , B .C . V8W 2Y2. 2 University o f California , Scripps Ins t i t u t ion of Oceanography,

La Jo l l a , California 92037.

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