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ELECTROPHORETIC INTRODUCTION OF CALCIUM I O N S INTO THE CORTEX OF XENOPUS

LAEVIS OOCYTES TRIGGERS M E I O S I S R E I N I T I A T I O N ( 1 )

MARC MOREAU, MARCEL DOREE AND P I E R R E GUERRIER S t a t i o n B io log ique, Roscoff 29211, France

ABSTRACT Meiy$is r e i n i t i a t i o n has been t r i g g e r e d v i a e l e c t r o p h o r e t i c d r i v i n g o f Ca f u n c t i o n a l f o l l i c l e c e l l s by col lagenase t rea tment . be s u b s t i t u t e d f o r Cat'. Cat+ induced matu ra t i on resemtles p roges ter - one induced matura t ion i n s o f a r bo th a re i n h i b i t e d by cycloheximide, g i v e r i s e t o a m p l i f i a b l e MPF i n t h e cytoplasm o f t r e a t e d oocytes and a l l o w chromosome condensation and the fo rmat ion o f a normal m e i o t i c sp ind le .

i n t o the c o r t e x o f Xenopus l a e v i s oocytes f r e e d from Mgtt c o u l d n o t

Me ios is r e i n i t i a t i o n has been ob ta ined i n Xenopus l a e v i s by r a i s i n g t h e tt tt

ex te rna l Ca o r Mg concen t ra t i on above 5 mM i n presence o f t h e ionophore

A 23187 (Wasserman and Masui, ' 75 ) .

b i n a t i o n s of these d i v a l e n t c a t i o n s i n s i d e the oocytes f a i l e d t o t r i g g e r mat-

u r a t i o n (Merriam, '71) . The discrepancy between these two se ts o f r e s u l t s

suggests t h a t ionophore d r i v e s d i v a l e n t c a t i o n s t o a s p e c i f i c t a r g e t cy top lasmic

area which i s n o t r e a d i l y access ib le by c l a s s i c a l m i c r o - i n j e c t i o n techniques.

I n t h i s paper, we r e i n v e s t i g a t e t h i s problem, us ing an e l e c t r i c a l c u r r e n t t o

d r i v e Ca from an i n t r a c e l l u l a r m ic roe lec t rode i n t o var ious cy top lasmic areas

o f Xenopus 1 aev i s oocytes.

MATERIAL AND METHODS

On t h e o t h e r hand, i n j e c t i n g var ious com-

t+

Xenopus l a e v i s ova r ian oocytes rang ing f rom 1.2 t o

1.5 mm were exposed t o col lagenase (Worthington, 1 mg / m l , 22' C, 5h) i n Mer-

r i a m ' s medium (Merriam, '71) .

t o HCG (Sigma), i n d i c a t i n g t h e absence o f f u n c t i o n a l f o l l i c l e c e l l s , t he t a r g e t

f o r gonadotropin a c t i o n .

These oocytes respond t o progesterone b u t n o t

A manual mic romanipu la to r P r i o r was used e i t h e r f o r

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the d i r ec t inject ion o f 50 nl of cytoplasm or of various CaC12 concentrations

in to the oocytes through an Agla microinjection device or for ionophoresis ex-

periments. These were performed by passing a constant current of 4x 10-8A f o r

30 sec t o 1 min through an in t r ace l lu l a r glass microelectrode f i l l e d with 0 ,5

M CaC12, whose t i p aperture ranged from 0 , 3 t o 0,5 p.

was of the Ag-AgC12 type.

the oocytes. Cytological observations were done on oocytes fixed in Bouin-

Hollande, embedded in paraf f in , s e r i a l l y sectioned 8-10 p thick and stained with

glychemal un-eosin.

RESULTS The electrophoret ic driving of Cat' consis tent ly f a i l ed t o induce

The ind i f fe ren t electrode

The same procedure was used to introduce EGTA in to

any cytological change in the oocytes when the t i p of the microelectrode was

inserted deeper than 0.2 mm from the oocyte surface.

maturation spot appeared in about 40% of the oocyte population a t the level of

the animal pole, some 12-15 hours a f t e r ionophoresis, when microelectrode was

introduced into the cortex of e i t h e r the animal o r the vegetal hemisphere, in

the immediate v ic in i ty of the plasma membrane ( t ab le 1).

tha t nei ther K'nor Mg (even 3M) can be subst i tuted fo r Catty although a re la-

t ive ly high concentration o f Mg

for successful calcium appl icat ion.

Cat+ microinjection, performed a t the same cor t ica l s i t e s , gave no posi t ive

resu l t s .

On the contrary, a typical

We found moreover tt

tt (10-20 mM) was required i n the external medium

On the other hand, we ver i f ied tha t manual

+t When successfully Ca t rea ted oocytes, bearing an animal white spot, were

fixed 3 hours l a t e r by standing 10 min in boiling medium and sectioned, they

appeared deprived o f germinal ves ic le . These observations have been confirmed

on 11 oocytes which were processed fo r cytological s tudies . In 6 of them, we

were even able t o f i n d a typical meiotic spindle w i t h highly condensed chromo-

somes. In one case, t h i s spindle was found migrating towards the animal pole

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TABLE 1

% o f me ios is r e i n i t i a t i o n observed a f t e r o r Cat+ ionophores is (performed i n presence o f 20 mM ex te rna l MgC1,). A d i f f e r e n t female was used f o r each exper iment. oocytes appears i n b rackets .

M progesterone a p p l i c a t i o n

The number o f t r e a t e d

Expt. Cont ro ls :

No. Calcium ionophores is ( 4 ~ 1 0 - ~ A f o r 3 sec) :

Pro es terone endoplasm * + e t h i d i u m b r . + cyc lohex imide 10- B M o r c o r t e x 50 w / m l 10 d m l

1 82 (45) 2 37 (40) 3 75 (40) 4 87 (45) 5 67 (40) 6 88 (40) 7 37 (40 ) 8 80 (40)

O ( 15)*52 (25) - 0 ( 20 ) *40 ( 12 ) - O ( 18)*40( 30) -

44125) 481 25) 42(12) 30( 10) 25 (12) 34(23) 40( 30) - - -

TABLE 2

E f f e c t o f a p rev ious i onophore t i c i n t r o d u c t i o n o f EGTA (5mM, 4 ~ 1 0 - ~ A f o r 30 sec) on the % o f me ios is r e i n i t i a t i o n ob ta ined i n a t y p i c a l exper iment, us ing oocytes f rom t h e same female.

I n i t i a t o r Cont ro l oocytes EGTA Treated

Progesterone I O - ~ M 86 (45) ~ H M P S ~ o - ~ M 84 (45) Ionophore A 23187 10-5M 65 (20) Cat' ionophores is 42 (30)

( f i g . lA), wh i le , i n the o t h e r 5 oocytes, i t was a l ready anchored t o t h e c o r t e x

( f i g . l B ) . Moreover, cytoplasm taken f rom Ca s t i m u l a t e d oocytes a t t he t ime

o f appearence o f t h e ma tu ra t i on d o t was found t o induce germinal v e s i c l e break-

down (GVBD), some 4 h l a t e r , when i n j e c t e d ( 5 0 n l ) i n t o un t rea ted r e c i p i e n t

oocytes.

tt

The matu ra t i on promot ing f a c t o r (MPF) produced i n these c o n d i t i o n s

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t ru ly possesses amp1 i f i ca t ion capacity since se r i a l t r ans fe r of cytoplasm taken

from a l o t of 100 Ca

through 3 cycles of t r ans fe r (56 of 75 a t 1 s t cycle ; 10 o f 14 a t the 2nd cycle

and 3 out of 4 a t 3rd cyc le) .

organomercur ia 1 dependent maturations, the cal c i um- induced maturation s t i 1 1

occurs in presence of 50 ug/ml ethidium bromide and i s inhibi ted by 5 o r 10

ug/ml cycloheximide ( t ab le 1 ) . On the other hand, we found t h a t maturation

never occurred when the oocytes have been previously loaded ( in jec t ion o r ion-

ophoresis) using a 5mM buffered EGTA so lu t ion , j u s t as does t h i s treatment when

oocytes a re stimulated w i t h progesterone, ionophore o r pHMPS ( t ab le 2 ) .

t l stimulated oocytes gave 75% of e f fec t ive maturation

Alike the progesterone, chemical ionophore o r

DISCUSSION There i s l i t t l e doubt t ha t divalent cations play a key role

in the hormonal mwtianism of meiosis r e i n i t i a t i o n in Xenopus laevis oocytes:

progesterone dependent maturation requires the presence of divalent cat ions

in the external medium (Merriam, ' 71 ) ; moreover, progesterone action can be mim-

icked by driving divalent cat ions inside the oocytes, using the ionophore A

23187 (Wasserman and Masui, ' 75) .

using Ca45, tha t pCMB-induced maturation (Brachet e t a l . , '75) i s coupled with

a net increase of Ca

inf l uence of progesterone (Marot e t a1 . , ' 76 ) .

I n addition, i t has been shown recent ly ,

+t inf lux, although t h i s does not seem to occur under the

The present study confirms t h a t true maturation including amplifiable MPF

production can be t r iggered, without pr ior treatment by progesterone o r organo-

mercurials, by the d i r ec t action of Ca on the oocyte i t s e l f . I t appears in-

deed tha t the eff icacy of e l ec t r i ca l ionophoresis, a1 ike chemical ionophoresis

(Wasserman and Masui, ' 7 6 ) does not depend on the release of progesterone by

the f o l l i c l e c e l l s which pe r s i s t a f t e r collagenase treatment:

not functional since they do not respond to HCG by producing progesterone, in

opposition t o untreated f o l l i c l e c e l l s . Moreover, calcium induced maturation

tt

these c e l l s are

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F I G U R E L E G E N D S

l A , 1B f i r s t matura t ion sp ind les observed i n s e c t i o n prepared from Cat+ s t i m u l a t e d oocytes which were f i x e d th ree hours a f t e r t he appearance of t h e ma tu ra t i on dot.

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i s n o t i n h i b i t e d by e th id ium bromide, a drug which u s u a l l y suppresses t h e pro-

duc t i on o f progesterone by normal f o l l i c l e c e l l s ( t a b l e 1 ) . Thus, t h e de lay

we observed f o r GVBD i s l i k e l y t o depend on an a d d i t i o n a l s tep necessary f o r

t h e g e n e r a l i z a t i o n o f t he Ca dependent process which, i n our experiments, has

been evoked i n a r a t h e r r e s t r i c t e d cytoplasmic area and n o t t o the t ime r e q u i r e d

f o r an eventual p roduc t i on and re lease o f progesterone by remain ing f o l l i c l e

c e l l s .

dependent matura t ion was n o t due t o some s e n s i b i l i z a t i o n by t h e col lagenase

treatment i t s e l f s ince oocytes w i t h i n t a c t f o l l i c l e c e l l s a1 so resume matu ra t i on

under Ca s t i m u l a t i o n a f t e r t h e same delay.

Our r e s u l t s make two more p o i n t s c l e a r .

tt

I n a d d i t i o n , we checked t h a t t h e a b i l i t y o f t he oocytes t o undergo Cat+

tt

The f i r s t p o i n t i s t h a t v a r i a t i o n

o f Cat' r a t h e r than Mgtt concen t ra t i on p lays a key r o l e i n me ios is r e i n i t i a t i o n

s ince Mg

since i n t r a c e l l u l a r EGTA t r u l y i n h i b i t s progesterone as w e l l as organomercur ia ls,

ionophore A 23187 and Cat' induced matu ra t i on ( t a b l e 2 ) .

a t i o n t r i g g e r e d by r a i s i n g t h e e x t e r n a l Mg

ophore A 23187 (Wasserman and Masui, ' 75 ) can be t e n t a t i v e l y accounted f o r by

assuming a Mg

c o r t i c a l s i t e s .

tt cannot be s u b s t i t u t e d f o r Cat' i n o u r i onophore t i c technique and

Thus, me ios is r e i n i t i - ++

concen t ra t i on i n presence o f i on -

t+ tt enhanced re lease o f Ca from t h e plasma membrane o r f rom some

+t The second p o i n t i s t h a t t he t a r g e t area f o r Ca ions i s t h e c o r t i c a l

compartment o f t he oocyte, perhaps t h e c e l l membrane i t s e l f which was l o c a l l y

clamped i n ou r exper iments d u r i n g Ca ions i n t r o d u c t i o n . Th is migh t e x p l a i n

why e l e c t r i c a l Ca ionophoresis, which a f f e c t s e l e c t r i c a l membrane p r o p e r t i e s

i s e f f i c i e n t i n t r i g g e r i n g ma tu ra t i on whereas manual i n j e c t i o n o f Ca

even when performed a t t he same topograph ica l s i t e , remains w i t h o u t e f f e c t .

I n t h i s respect, i t i s wor th emphasizing t h a t a l l p r e v i o u s l y known e f f i c i e n t

tt

tt

tt ions,

t reatments, i n c l u d i n g chemical ionophoresis, a c t u a l l y mod i fy t h e e l e c t r i c a l

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p r o p e r t i e s o f t h e plasma membrane (Moreau e t a1 . , '76 ) .

F i n a l l y , a reasonable hypothes is t o account f o r p h y s i o l o g i c a l ma tu ra t i on +t would be t h a t progesterone induces a small inc rease o f Ca

i n t h e c o r t e x o f t h e oocytes, p o s s i b l y by i n c r e a s i n g the Ca

the c e l l membrane.

r e s u l t s of Marot e t a l . ( '76) , s ince we found t h a t t h e amount o f Ca ions,

which was s u f f i c i e n t t o t r i g g e r me ios is r e i n i t i a t i o n i n o u r experiments, cou ld

n o t be de tec ted us ing c l a s s i c a l i s o t o p i c methods.

Cat+ s t i m u l a t e d process necessary f o r r e l e a s i n g t h e oocyte f rom meios is i n h i -

b i t i o n remains l a r g e l y a ma t te r o f specu la t ion , i t i s c l e a r t h a t i t c o n s t i t u t e s

a very e a r l y event occurr ing p r i o r t o syn thes is , unmasking o r l i b e r a t i o n o f MPF.

i ons c o n c e n t r a t i o n

i n f l u x across tt

This i s n o t u n l i k e l y , even when one considers t h e nega t i ve +t

While t h e na tu re o f t h a t

LITERATURE C I T E D

Brachet, J . , E. Ba l tus , A. de Schutter-Pays, J. Hanocq-Quert ier , E . Hubert and G. S t e i n e r t 1975 I n d u c t i o n o f ma tu ra t i on (me ios i s ) i n Xenopus l a e v i s oocytes by t h r e e organomercur ia ls. Acad. Sc i . USA. 72: 1574-1578.

Proc. Nat:

Marot, J . , R. B e l l 6 and R. Ozon ck l ' ovocy te de Xenopus l a e v i s . i o n s calc ium. C.R. Acad. Sc. Par is , 282: 1301-1304.

Merriam, R. W. 1971 Progesterone-induced matu ra t i ona l events i n oocytes o f Xenopus l a e v i s . ca lc ium and magnesium. Exp. C e l l . Res., 68: 75-80.

Moreau, M. , P. Guer r i e r and M. Dore'e 1976 M o d i f i c a t i o n s pr6coces des p r o p r i e t g s e ' l ec t r i ques de l a membrane plasmique des ovocytes de Xenopus l a e v i s au c o w s de l a r e i n i t i a t i o n me'iot ique i n d u i t e p a r l a progest&one, l e parachloromercuribenzoate (pCMB) ou l ' i o n o p h o r e A 23187.C.R. Acad. Sc. Pa r i s , 282: 1309-1312.

Wasserman, W.J., and Y. Masui 1975 I n i t i a t i o n o f m e i o t i c matura- t i o n i n Xenopus l a e v i s oocytes by t h e combinat ion o f d i v a l e n t ca t i ons and ionophore A 23187. J . Exp. Zool., 193: 369-375.

1976 Recherches sur l a ma tu ra t i on Arguments en faveur du r 6 l e des

I. cont inuous necess i t y f o r d i f f u s i b l e

REFERENCES

1 Th is work was supported by C.N.R.S. ATP 1890 and D.G.R.S.T. ACC A 659-1340.

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