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INDUCTION O F M E I O S I S BY INJECTION OF HETEROLOGOUS PROTEIN KIMASE AND

PHOSPHORYLASE KINASE I N XENOPUS LAEVIS OOCYTES (1)

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

ABSTRACT Meios is r e i n i t i a t i o n has been t r i g g e r e d by i n j e c t i o n o f bee f h e a r t p r o t e i n k inase o r r a b b i t phosphorylase k inase i n t o *- opus l a e v i s oocytes. Successful i n j e c t i o n s are fo l l owed by germinal v e s i c l e breakdown, chromosome condensation, fo rmat ion o f a normal m e i o t i c sp ind le , and appearance o f an a m p l i f i a b l e ma tu ra t i on pro- mot ing f a c t o r . Meiosis r e i n i t i a t i o n does n o t occur when t h e enzymes are i n t roduced i n t o the oocytes s imu l taneous ly w i t h EGTA o r a f t e r p re t rea tment w i t h cycloheximide. An t ipa in , an an t i p ro tease which abo l ishes t h e response o f oocytes t o progesterone , does n o t suppress t h e meios is r e i n i t i a t i o n induced by i n j e c t i o n o f p r o t e i n k inase o r phosphoryl ase k inase.

The re lease of t h e prophase b lock d u r i n g oocyte ma tu ra t i on i s assoc ia ted

w i t h i n tense p r o t e i n phosphory la t ion ( M o r r i l l and Murphy, '72; Wallace, '74;

Doree e t a l . , ' 76 ) . I t has been suggested t h a t a new k i n d o f h i s tone k inase

appears i n Xenopus 1 aev i s oocytes t r e a t e d w i t h progesterone (Wi b l e t ,

These f i n d i n g s support the view t h a t appearance o f t h e "matura t ion promot ing

f a c t o r " (MPF) and o f t h e "chromosome condensation f a c t o r " (CCA) migh t be cor -

74) .

r e l a t e d w i t h some new p r o t e i n k inase a c t i v i t y .

W ib le t e t a l . ( ' 7 5 ) ob ta ined germinal v e s i c l e

s a t i o n a f t e r i n j e c t i o n o f heterologous h i s tone

oocytes; they f a i l e d however i n i nduc ing m i o s

l a e v i s oocytes (Wib le t e t a l . , ' 7 5 ) . The o n l y

I n p r e l im ina ry experiments,

reakdown and chromosome conden-

k inase i n t o Ambystoma mexicanum

s r e i n i t i a t i o n us ing Xenopus

p o s i t i v e r e s u l t w i t h Xenopus

l a e v i s oocytes seems t o have been ob ta ined by Ma l le r , who r e p o r t e d t h a t i n j e c -

t i o n o f r a b b i t phosphorylase kinase, b u t n o t CAMP-dependent p r o t e i n k inase,

435

led to the appearance a t the level of the animal pole of the white spot which

i s formed usually a f t e r germinal vesicle breakdown (Maller, ' 74 ) . However t h i s

r e su l t was only obtained using PMSG-pretreated oocytes; in jec t ion of phosphor-

ylase kinase in to untreated oocytes f a i l ed repeatedly. In cont ras t with those

obtained by the other authors, the r e s u l t s reported here show t h a t a well char-

acter ized maturation i s ac tua l ly induced without hormonal pretreatment by the

inject ion in to Xenopus l aev i s oocytes o f e i t h e r rabbi t phosphorylase kinase o r

beef heart CAMP-dependent protein kinase.

MATERIAL AND METHODS Xenopus l aev i s ovarian oocytes ranging from 1.2 t o

1.5 mm were exposed t o collagenase (Worthington, 1 mg/ml, 22' C , 5 h ) in Mer-

riam's medium (Merriam, ' 7 1 ) .

t o HCG (Sigma) indicat ing the absence of functional f o l l i c l e c e l l s , the t a rge t

f o r gonadotropin act ion. A manual micromanipulator Pr ior was used e i t h e r fo r

the d i r ec t inject ion of 50 nl of the enzyme solut ions o r of cytoplasm in to the

oocytes through an Agla microinjection device (Wellcome, London).

introduction of EGTA was performed by passing a constant current of 4 x

A fo r 30 sec through an in t r ace l lu l a r g lass microelectrode f i l l e d with 5 mM

EGTA in buffered sa l ine ; t i p aperture ranged from 0.3 t o 0.5 U. The ind i f -

fe ren t e lectrode was of the Ag-AgC12 type.

on oocytes fixed in Bouin-Hollande, embedded in paraf f in , s e r i a l l y sectioned

8-10 LI thick, and s ta ined with glychemalun-eosin. Beef hear t protein kinase,

3 ' -5 ' CAMP-dependent, rabbi t muscle phosphorylase kinase and phosphorylase 5

were purchased from Sigma, U.S.A. Solutions containing 1 mg/ml of these enzymes

were prepared in Merriam's medium.

against 500 vol o f Merriam's medium before use.

Aoyagi, University of Tokyo.

These oocytes responded t o progesterone, b u t not

Ionophoretic

Cytological observations were done

Protein kinase was dialyzed f o r 8 h a t 4' C

Antipain was a g i f t of Doctor

RESULTS Three t o f ive hours a f t e r in jec t ion of 50 nl of s a l ine containing

436

TABLE 1

P r o t e i n k inase- induced matu ra t i on

% GVBD * (number of oocytes counted i n b racke ts ) 50 n l i n j e c t i o n s ex te rna l appl i c a t i o n Separate

experiments 50 ng p r o t e i n k inase - lom6 M (1 female i n f o r each) Merr iam's medium M e r r i am's medi um progesterone

-CAMP + CAMP 10m6M

65 44 76 44 71 47

- 50 (20)

50 (11) 100 ( 9 )

40 (20 ) -

40 (20) - 30 (20) - 65 (80) -

0 (20) -

0 (47) -

0 125) 88 (63) 67 (143) 80 (72) 67 (33)

100 (31) 55 (28) 88 (75)

0 (80) 83 (45)

0 (48)

0 (30) 66 (53)

* ** ***

**** EGTA was in t roduced i n t o t h e oocytes j u s t be fo re enzyme i n j e c -

assumed from t h e appearance o f a w h i t e spo t a t t h e animal p o l e 10 pg/ml cycloheximide were added i n t h e medium 30 min be fo re s t a r t i n g t h e experiment. 1 mg/ml a n t i p a i n was added i n t h e medium 1 h be fo re s t a r t i n g the experiment.

t i o n , accord ing t o the i o n o p h o r e t i c procedure descr ibed under "met hods I ' .

50 ng o f b e e f h e a r t p r o t e i n k inase o r o f r a b b i t phosphorylase k inase i n t o f u l l -

grown immature oocytes o f Xenopus l a e v i s , t h e animal hemisphere pigment accumu-

l a t e d as a dark brown r i n g near the animal po le c i r cumscr ib ing a l i g h t l y pigment-

ed area. The same pigment d i s t r i b u t i o n occur red i n c o n t r o l oocytes some 2 t o 3

hrs a f t e r t rea tment M progesterone. Oocytes i n j e c t e d w i t h 50 n l o f s a l i n e

437

TABLE 2

P h o s p ho ry 1 as e k i n as e - i n d u ce d ma t 11 r a t i on

Separate 50 ng/50 nl inject ion of External application experiments p hos p ho ry 1 a se k i n ase I O - ~ M progesterone

(1 female compound added** % GVBD* % GVBD* f o r each)

I None I1 do I11 do IV do

None cycl oheximide

v1[; None EGTA

None an t i pain

46 (13) 100 (15) 25 ( 1 2 )

88 ( 6 ) 62 (21)

25 (12) 0 (11)

0 (30) 100 (20) 100 (11)

89 (27) 89 (32) 60 (51) 97 (46) 80 (23)

80 (81)

98 (80)

0 (12)

0 (35)

0 (15)

* assumed from the appearance of a white spot a t the animal pole. Number of oocytes counted in brackets. cycloheximide and ant ipain were added in the medium as indicated in table 1. EGTA was introduced in to the oocytes j u s t before enzyme inject ion (ionophoretic procedure : see "methods").

k*

alone, with 50 rl of heat-denatured enzyme so lu t ions , or with 50 nl of phoshor-

ylase a solution did not undergo any change. The above experiments were per-

formed using 9 (protein kinase) and 7 females (phosphorylase kinase) purchased

from two d i f f e ren t breeding centers . These females did not receive HCG nor

PMSG inject ions before the experiments. Tables 1 and 2 show t h a t the r e su l t s

are f a i r l y reproducible. In many experiments percentages of posi t ive response

were as high a f t e r enzyme in jec t ion as a f t e r

When successful ly injected oocytes bearing a white spot a t the aninal pole

M progesterone treatment.

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

438

FIGURE LEGENDS

1 a, 1 b f i r s t maturation spindle observed in sections prepared from protein kinase (1 a ) or phosphorylase kinase (1 b ) stimulated oocytes which were fixed three hours a f t e r the appearance of the maturation dot.

439

appeared deprived of germinal ves ic le . 20 oocytes successful ly injected with

protein kinase were processed f o r cytological s tud ies , which confirmed the

absence of a germinal ves ic le . In 14 oocytes we were even able t o find a typ-

ical meiotic spindle already anchored t o the cortex ( f i g . 1 a ) . Typical meiotic

spindles were a l so observed in 4 o u t of 4 oocytes injected with phosphorylase

kinase ( f i g . 1 b ) .

ed oocytes a t the time of appearance of the maturation dot was found to induce

germinal vesicle breakdown some 4 hours l a t e r when injected (50 n l ) in to un-

t reated rec ip ien t oocytes. MPF produced in these conditions t ru ly possesses

amplification capacity since se r i a l t r ans fe r of cytoplasm taken from a l o t of

20 phosphorylase kinase-stimulated oocytes gave 100% to 60% of e f fec t ive matu-

ra t ion t h r o u g h 3 cycles of t r ans fe r (20 of 20 a t 1 s t cycle, 10 of 10 a t 2nd

cycle, and 3 of 5 a t 3rd cyc le) .

using protein-kinase st imulated oocytes as cytoplasm donors.

o r phosphorylase kinase dependent maturation d i d not occur i n presence of 10

ug/ml cycloheximide o r a f t e r an ionophoretic loading of the oocytes w i t h EGTA,

treatments which have been shown t o suppress the progesterone , organomercurials,

o r Ca induced maturation (Wasserman and Masui, '75; Moreau e t a l . , ' 7 6 ) .

On the contrary, protein kinase and phosphoryl ase kinase t r iggered maturations

a re not affected by an t ipa in , a low molecular weight ant iprotease which actual-

l y suppresses progesterone o r ionophore A 23187 induced maturations (Guerrier

e t a l . , ' 7 6 ) .

n o t necessary fo r inducing maturation, a1 though this s l igh t ly enhanced the

eff ic iency of the treatment.

Moreover, cytoplasm taken from phosphorylase kinase in j ec t -

Essent ia l ly ident ical r e s u l t s were obtained

The protein kinase

++

Finally we found tha t addition of CAMP t o protein kinase was

DISCUSSION The above experiments demonstrate t h a t the release of Xenopus

laevis oocytes from meiosis inhibi t ion can be induced by inject ion of beef

heart CAMP-dependent protein kinase and rabbi t phosphoryl ase kinase, and t h a t

440

germinal v e s i c l e breakdown then occurs several hours e a r l i e r than w i t h proges-

te rone. These r e s u l t s conf i rm t h a t t he phosphory la t ion o f some p r o t e i n s p l a y s

a key r o l e i n the sequence o f events l e a d i n g t o me ios is r e i n i t i a t i o n .

While CAMP-dependent p r o t e i n k inase has 1 i t t l e s p e c i f i c i t y , phosphorylase

k inase a c t i o n seems t o be r e s t r i c t e d t o phosphorylase b and several muscle

p r o t e i n s d i r e c t l y concerned i n t h e c o n t r a c t i l e process (Rubin and Rosen, ' 7 5 ) .

Since phosphorylase 5 i n j e c t i o n proved t o be i n e f f i c i e n t i n i n d u c i n g meios is

r e i n i t i a t i o n , a reasonable hypothes is would be t h a t some c o n t r a c t i l e p r o t e i n ,

p o s s i b l y l o c a l i z e d i n t h e c o r t e x o f t he oocytes, may be t h e t a r g e t f o r phosphor-

y l a s e k inase a c t i o n i n Xenopus l a e v i s . O f course, t h e hypothes is t h a t phosphor-

y l a s e k inase a c t u a l l y p lays a key r o l e i n t h e normal ma tu ra t i on process ( i . e . ,

t he process t r i g g e r e d by progesterone) has n o t y e t rece ived any suppor t and i t

remains p r e s e n t l y l a r g e l y specu la t i ve .

e s i s s ince bo th a c t i v a t i o n and a c t i v i t y o f t h i s enzyme are s t r o n g l y dependent

on Ca (Brostrom e t a1 . , ' 7 1 ) , and t h i s i s a l s o a main c h a r a c t e r i s t i c o f t h e

progesterone- induced matu ra t i on (Merriam, ' 7 1 ) . Moreover ma tu ra t i on can be

t r i g g e r e d by d r i v i n g Ca i o n s i n t o Xenopus l a e v i s oocytes, p rov ided t h a t these

ions are i n t roduced i n t o t h e c o r t i c a l reg ion (Moreau e t a l . , ' 7 6 ) , where st imu-

l a t i o n o f p r o t e i n phosphory la t ion has been shown t o occur e s s e n t i a l l y a f t e r

hormonal t rea tment ( M o r r i l l and Murphy, ' 7 2 ) . F i n a l l y i onophore t i c i n t r o d u c t i o n

o f EGTA abo l ishes bo th t h e progesterone and the phosphorylase k inase dependent

matura t ion .

press t h e progesterone o r the c a l c i urn-dependent matura t ion , suggest ing t h a t some

pro tease m igh t be i nvo l ved i n t h i s process. It i s perhaps i n t e r e s t i n g i n t h i s

connect ion t o n o t i c e t h a t phosphorylase k inase may be a c t i v a t e d ove r 100 f o l d

by Ca" ions , CAMP-dependent phosphory la t ion , and by p r o t e a s i c d i g e s t i o n o f a

po l ypep t ide arm o f t he enzyme (Cohen, ' 7 4 ) .

-

It i s however a ve ry a t t r a c t i v e hypoth-

tt

tt

We have shown on t h e o t h e r hand t h a t s p e c i f i c an t i p ro teases sup-

441

LITERATURE CITED

Brostrom, C.O., F.L. Hunkeler and E.G. Krebs +I971 s k e l e t a l muscle phosphorylase k inase by Ca . J. B i o l . Chem.,

Cohen, P. 1974 The r o l e o f phosphorylase k inase i n the nervous and hormonal c o n t r o l o f g l ycogeno lys i s i n muscle. Biochem. SOC. Symp. ,

Doree, M., M. Moreau and P. G u e r r i e r 1976 The r o l e o f 1-methylade- n i n e i n Echinoderms. I n : Proceedings o f t h e Vth I n t e r n a t i o n a l Congress o f Endocr inology. V.H.T. James, ed. Excerpta Medica, Amsterdam.

Guer r ie r , P., M. Moreau and M. Doree 1976 I n h i b i t i o n de l a r 6 i n i - t i a t i o n de l a mei'ose des ovocytes de Xenopus l a e v i s p a r t r o i s an t i p ro teases microbiennes, 1 ' a n t i p a i n e , l a c h y m s t a t i n e e t l a l eupep t ine . C.R. Acad. Sc. Pa r i s , i n press.

Ma l l e r , J.L. 1974 Stud ies on t h e mechanism o f cy top lasmic c o n t r o l o f m e i o t i c ma tu ra t i on o f Xenopus l a e v i s oocytes. Ph.D. D isse r ta - t i o n , Uni v e r s i t y o f Cal i f o r n i a, Berke ley .

Merriam, R.W. 1971 Progesterone- induced matu ra t i ona l events i n 00- cy tes o f Xenopus l a e v i s . I. cont inuous n e c e s s i t y f o r d i f f u s i b l e ca lc ium and magnesium. Exp t l C e l l Res., 68: 75-80.

Moreau, M., M. Doree and P. G u e r r i e r 1976 E l e c t r o p h o r e t i c i n t r o d u c - t i o n o f ca l c ium i o n s i n t o the c o r t e x o f Xenopus l a e v i s oocytes t r i g g e r s me ios i s r e i n i t i a t i o n . J. Exp. Zool. , submi t ted f o r p u b l i c a t i o n .

M o r r i l l , G.A., and J.B. Murphy 1972 Role f o r p r o t e i n phosphory la t i on i n me ios is and i n the e a r l y c leavage phase o f amphibian embryonic development. Nature, 238: 282-284.

Rubin, C.S., and O.R. Rosen 1975 P r o t e i n phosphory la t i on . Ann. Rev. Biochem. , 44: 831-887.

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Wasserman, W.J. , and Y. Masui 1975 E f f e c t s o f cyc lohex imide on a cy top lasmic f a c t o r i n i t i a t i n g m e i o t i c ma tu ra t i on i n Xenopus oocytes. E x p t l . C e l l Res. , 91: 381-388.

Wib le t , M. 1974 P r o t e i n k inase a c t i v i t i e s d u r i n g ma tu ra t i on i n Xenopus 1 aev i s oocytes. B i ochem. B i ophys. Res . Comm. , 60 : 991-998.

Wib le t , M., E. B a l t u s and J.B. Brachet 1975 Meiose: r61e d 'une h i stone k inase dans 1 a condensat ion des chromosomes d ' oocytes ova-

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REFERENCES

1 This work was supported b y C.N.R.S. ATP 1890 and D.G.R.S.T. ACC 659-1340.

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