Cyclic AMP synthesis in Xenopus laevis oocytes inhibition by progesterone

179 Biochimica et Biophysica Acta,582 (1979) 179--184

© Elsevier/North-Holland Biomedical Press

BBA Report BBA 21492 CYCLIC AMP SYNTHESIS IN X E N O P U S L A E V I S OOCYTES INHIBITION BY P R O G E S T E R O N E ODILE MULNER, DENISE HUCHON, CATHERINE THIBIER and RENt~ OZON Laboratoire de Physiologie de la Reproduction, Groupe St6roi'des, Equipe de Recherche associ$e au C.N.R.S. 694, Universit~ Pierre et Marie Curie, 9, Quai Saint Bernard, 75005 Paris (France)

(Received October 2rid, 1978) Key words: Progesterone; Cyclic AMP synthesis; Cholera toxin; (Xenopus oocyte)

Summary [a-32P] ATP was microinjected into X e n o p u s o o c y t e and neosynthesized cyclic AMP was isolated. Cholera toxin inhibited progesterone-induced maturation and stimulated after 3 b of preincubation the amount of neosynthesized cyclic AMP. Progesterone decreased the neosynthesis of cyclic AMP during the first hour following addition of the hormone.

Progesterone induces in vitro meiotic maturation in X e n o p u s laevis oocytes; the action of progesterone involves post-transcriptional events indicated by the failure of actinomycin D or enucleation to affect the induction of maturation by steroids [ 1 ]. Recent evidence favors the view that Ca 2÷ [2 ], cyclic AMP [3] and cyclic AMP-dependent protein kinases [4] play a major role during progesterone-stimulated maturation in amphibian oocytes. Intracellular contents of cyclic AMP were investigated in R a n a pipiens [3, 5] and X. laevis [6,7] oocytes and found to be around 1 pM in both species. The time course for fluctuations in the levels of cyclic AMP during progesteroneinduced maturation shows in R. pipiens a significant decrease of cyclic AMP which occurs within 30 min following progesterone exposure [3, 5]. In contrast, no change in cyclic AMP levels in enzymatically denuded X e n o p u s oocytes was found during the first hours following progesterone treatment [6,7]. However it has been reported in this species [6,8] as well as in R a n a [5] that theophylline and 3-isobutyl-l-methylxanthine which competitively inhibit phosphodiesterase, block progesterone action. Altogether these results suggest that a decrease of intracellular cyclic AMP is a necessary response to the induction stimulus, b u t without distinguishing between activation of a phosphodiesterase and inhibition of an adenylate cyclase. We n o w report in

180 X. laevis that both cholera toxin and Escherichia coli enterotoxin (which exert their effects in vertebrate cells through activation of adenylate cyclase) irreversibly block progesterone-induced o o c y t e maturation and that in ovo cyclic AMP synthesis decreases during the first hour after hormonal stimulation. X. laevis females were obtained from South Africa. Ovaries were removed surgically after immersion anaesthesia in I g/1 M.S. 222 (Sandoz). The ovaries were transferred in medium A : 88 mM NaC1/0.33 mM Ca(NO3)2/1 mM KC1/ 0.41 mM CaClz/0.82 mM MgSO4/2 mM Tris, pH 7.4 [9]. Stage VI oocytes [10] were collected after collagenase (1 mg/ml) digestion at 30°C for 6 h [11]. Crude extract of E. coli enterotoxin (from Doctor Alouf, Institut Pasteur, Paris) blocks progesterone-induced maturation. Commercially purified cholera toxin (Schwarz/Mann) also inhibits maturation. As low as 0.5 ng of toxin per ml of medium A inhibited, more or less, progesterone-induced maturation depending on the females; in all tested females a dose of 50 ng/ml of toxin caused a 100% irreversible inhibition. A 5-min preincubation in cholera toxin was sufficient to completely abolish subsequent maturation by progesterone. Fig. 1 shows that cholera toxin inhibits maturation only when added during the first 2 h following exposure to progesterone. Thus only the early steps of maturation are sensitive to inhibition by toxins. Preliminary experiments showed that neosynthesized cyclic AMP could be isolated after microinjection into o o c y t e of 40--50 nl of [~-32P]ATP (10 Ci/ mmol; The Radiochemical Center, Amersham) in pit 7 MES buffer (2-[Nmorpholino]ethanesulfonic acid). In a typical experiment 25 oocytes were each microinjected with 2.4-106 cpm of [a-32P]ATP and then incubated for 3 h in medium A in the presence of I mM 3-isobutyl-l-methylxanthine. At the end of the incubation period 25 400 cpm were recovered as cyclic AMP following extraction and a two-step column chromatography purification procedure [12]. Authenticity of cyclic AMP was established by incubating eluates of the alumina column with beef heart phosphodiesterase (Sigma). Less than 4% of the radioactivity was then recovered in the cyclic AMP fraction after a second passage over the columns. It is not possible to estimate the true amount of cyclic AMP formed, since the specific activity of ATP at the enzyme level cannot be measured under our experimental conditions. However, the endogenous pool of ATP in X. laevis o o c y t e has been measured and was found to be approx. 610 p m o l / o o c y t e [13]. In these conditions the amount of cyclic AMP formed can be expressed in p m o l / o o c y t e per h and is 0.14 (S.D. 0.05, 11 determinations). Since it is difficult to microinject constant amounts of precursor [a-32P]ATP, it was important to show that the amount of cyclic AMP formed increased linearly with the amount of microinjected precursor (Fig. 2). It is generally assumed that 3-isobutyl-l-methylxanthine inhibits cyclic AMP phosphodiesterase in isolated cells. The amount of de novo synthesized radioactive cyclic AMP from [ ~ Y P ] A T P has been estimated in Xenopus o o c y t e in the presence or absence of 3-isobutyl-l-methylxanthine. When 3-isobutyl-l-methylxanthine was added to the incubation medium at the time of [ a Y P ] ATP microinjection, no, or only slight, stimulation (depending on the female tested) of the amount of cyclic AMP recovered after a 60-min

181

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o~32-P--A TP m/cro,njected

(x lOdcpm)

Fig. 1. I n h i b i t i o n o f p r o g e s t e r o n e - i n d u c e d m a t u r a t i o n b y c h o l e r a t o x i n . E n z y m i c a l l y d e f o U i c u l a t e d o o c y t e s w e r e e x p o s e d t o 10 pM p r o g e s t e r o n e for 5 m i n u t e s in m e d i u m A a n d t h e n t r a n s f e r r e d in an h o r m o n e - f r e e m e d i u m . A t v a r i o u s t i m e s t h e r e a f t e r 30 o o c y t e s w e r e t a k e n a n d i n c u b a t e d in 1 m l of m e d i u m A c o n t a i n i n g 50 ng o f c h o l e r a t o x i n ( S c h w a r z / M a n n ) . M a t u r a t i o n was s c o r e d b y o b s e r v i n g t h e f r e q u e n c y of g e r m i n a l vesicle b r e a k d o w n ( G V B D ) e v i d e n t f r o m t h e f o r m a t i o n o f a w h i t e s p o t s u r r o u n d e d b y p i g m e n t o n t h e a n i m a l p o l e o f t h e o o c y t e . G V B D was t h e n a s c e r t a i n e d b y t h e a b s e n c e of t h e g e r m i n a l vesicle u n d e r d i s s e c t i o n of t h e b o i l e d o o c y t e s . C o n t r o l o o c y t e s , t h a t h a v e n o t b e e n treated by cholera toxin, show 93% GVBD, 6 h after progesterone treatment. Fig. 2. R a d i o a c t i v e cyclic AMP r e c o v e r y in five o o c y t e s as a f u n c t i o n o f t h e a m o u n t of [~-3~P]ATP m i c r o i n j e c t e d . Five o o c y t e s w e r e m i c r o i n j e c t e d w i t h 50 nl e a c h of MES b u f f e r c o n t a i n i n g v a r i o u s a m o u n t s o f [c~-32p] A T P ( 1 . 0 - 1 0 6 t o 8 . 0 . 1 0 ~ c p m / 5 o o c y t e s ) . T h e i n j e c t i o n o f five o o c y t e s t a k e s no m o r e t h a n 5 rain. T h e m i c r o i n j e c t e d o o e y t e s w e r e t h e n i n c u b a t e d f o r 60 m i n in m e d i u m A c o n t a i n i n g 3 - i s o b u t y l - l - m e t h y l x a n t h i n e (1 m M ) . T h e r e a c t i o n was s t o p p e d b y h o m o g e n i z i n g t h e o o c y t e s in 500 pl o f 2% SDS, 1 m M cyclic AMP, 2.5 m M A T P , 1 0 0 m M Tris-HCl ( p H 7.5) a n d 3 0 0 0 c p m of cyclic [ 3 H ] A M P as i n t e r n a l r e c o v e r y s t a n d a r d . P u r i f i c a t i o n o f cyclic AMP was a c c o m p l i s h e d b y sequential c o l u m n c h r o m a t o g r a p h y over Dowex AG 1 X 8 (H + form) and neutral alumina according to S a l o m o n et al. [ 1 2 ] .

incubation could be detected. However when the oocytes were incubated for at least 2 or 3 h in the presence of 3-isobutyl-l-methylxanthine, a significant increase in the a m o u n t of isolated cyclic AMP was found (Fig. 3). On the other hand, it has been previously shown that theophylline, another methylxanthine, always blocks the degradation of microinjected free cyclic AMP [6]. Cyclic AMP phosphodiesterase activities of Xenopus oocytes were also assayed in vitro; the intracellular distribution of the activities show that about 50% of the total activity is in the soluble fraction and the remaining is detectable in the membrane fraction. Both activities are totally inhibited by 1 mM 3-isobutyl-l-methylxanthine. In the oocyte, newly synthesized cyclic AMP may remain in a free form or may be b o u n d to the regulatory (R) subunit of cyclic AMP-dependent protein kinase. Only free cyclic AMP is accessible to degradation by phosphodiesterase. An explanation of the lag time necessary to observe an increase of radioactive cyclic AMP induced by 3-isobutyl-1methylxanthine could be that newly synthesized cyclic AMP is readily b o u n d

182 c2~

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E

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100 /ncubahon hrne (hours) Fig. 3. E f f e c t o f 3 - i s o b u t y l - l - m e t h y l x a n t h i n e o n t h e f o r m a t i o n o f c y c l i c A M P . O o c y t e s w e r e prei n c u b a t e d f o r 6 0 m i n in m e d i u m A in t h e p r e s e n c e o r in t h e a b s e n c e o f 1 m M 3 - i s o b u t y l - l - m e t h y l x a n t h i n e . D u p l i c a t e s a m p l e s o f f i v e o o c y t e s e a c h w e r e m i c r o i n j e e t e d w i t h [ao32p] A T P a n d f u r t h e r i n c u b a t e d f o r v a r i o u s t i m e s in m e d i u m A: • . . . . • in t h e p r e s e n c e o f 1 m M 3 - i s o b u t y l - l - m e t h y l x a n t h i n e ; ×--× in t h e a b s e n c e o f 3 - i s o b u t y l - l - m e t h y l x a n t h i n e . O o c y t e s w e r e h o m o g e n i z e d a n d c y c l i c A M P i s o l a t e d (see Fig. 2). T h e r e s u l t s a r e e x p r e s s e d as t h e a m o u n t o f c y c l i c A M P r e c o v e r e d v e r s u s t h e amount of [aJ2P]ATP microinjected.

to R and therefore inaccessible to cyclic AMP phosphodiesterase in the shortterm incubation period. On the other hand, 3-isobutyl-l-methylxanthine had been found to inhibit protein synthesis in Xenopus o o c y t e (between 10 to 40%; unpublished results). This confirms the reported effect of theophylline by Bravo et al. [14]. However, experiments with cycloheximide have shown that maturation is still possible when protein synthesis is inhibited by 50% [15]. The role of cyclic AMP in this inhibition is far from clear, since cholera toxin does not block but rather increases total protein synthesis in o o c y t e (unpublished results). Whatever is the exact mechanism of action of 3-isobutyl-l-methylxanthine in the living oocyte, the presence of 3-isobutyl-l-methylxanthine in the incubation medium allowed estimation of the synthesis of radioactive cyclic AMP (both free and bound) with minimal action of phosphodiesterase. All further experiments were then performed after microinjections of [a.32p] ATP (from 0.5.106 to 2.106 cpm per oocyte) into five oocytes isolated from the same female and preincubated for 60 min in 1 mM 3-isobutyl-l-methylxanthine, followed by an incubation period of 60 min in the presence of 3-isobutyl-l-methylxanthine. When oocytes were preincubated in cholera toxin for 2 h and then microinjected with [a-32p]ATP, the amotmt of cyclic AMP isolated was increased three times (Fig. 4). A lag of 2 h was necessary before the stimulation of adenylate cyclase was apparent. The action of progesterone was investigated and we found that the steroid hormone always caused a significant decrease (between 30 and 60%) in the amount of newly synthesized cyclic AMP during the 60-min incubation period. This decrease was obtained either in the presence of 1 mM 3-isobutyl1-methylxanthine or in the absence of 3-isobutyl-l-methylxanthine (Table I). Furthermore, it was shown that progesterone is capable to decrease the formation of cyclic AMP, even in the presence of cholera toxin (Fig. 4); it

183

TABLE I E F F E C T O F P R O G E S T E R O N E ON C Y C L I C AMP S Y N T H E S I S I N X E N O P U S

OOCYTE

D u p l i c a t e g r o u p s o f five o o c y t e s e a c h w e r e m i c r o i n j e c t e d w i t h [~.32p] A T P a n d i n c u b a t e d f o r 6 0 rain in m e d i u m A. I pM p r o g e s t e r o n e w a s a d d e d t o t h e i n c u b a t i o n m e d i u m at t h e t i m e o f t h e m i e r o i n j e c t i o n o f A T P . W h e n 1 m M 3 - i s o b u t y l - l - m e t h y l x a n t h i n e w a s p r e s e n t , t h e d r u g w a s a d d e d 60 rain b e f o r e t h e microinjection of ATP and the addition of progesterone. At the end of the incubation period oocytes w e r e h o m o g e n i z e d a n d cyclic AMP isolated (see Fig. 2). E x p e r i m e n t s r e p o r t e d w e r e o b t a i n e d f r o m 11 d i f f e r e n t f e m a l e s . T h e r e s u l t s are e x p r e s s e d as ( c p m c y c l i c A M P i s o l a t e d / c p m A T P m i c r o i n j e c t e d ) × 106.

Witb 3 - i s o b u t y l - l - m e t h y l x a n t h i n e

Female:

Without 3-isobutyl1-methylxanthine

1

2

3

4

5

6

7

8

9

10

11

Control

233 236

173 137

289 295

166 120

179 102

379 326

138 139

175 156

130 119

165 196

94 137

Progesterone

93 103

67 105

135 103

60 61

105 130

171 114

74 122

103 113

80 82

111 103

85 63

was n o t possible to totally reverse the action of the toxin by progesterone. It is known that the activation of the adenyl cyclase by cholera toxin is irreversible, and we have shown that the inhibition of maturation by cholera toxin is also irreversible. These results suggest that progesterone and cholera toxin act at different sites to control, in Xenopus oocyte, the synthesis of cyclic AMP. It was shown that the decrease of the synthesis of cyclic AMP is specific to maturing steroids. In contrast, non-maturing steroids, as estradiol-17~, do not modify cyclic AMP synthesis. Furthermore there is a good correlation between the percentage of maturation, the concentration of progesterone and the a m o u n t of radioactive cyclic AMP formed during the first hour following addition of progesterone (Fig. 5). It is interesting to note that low doses of progesterone which do n o t induce maturation, change the rate of cyclic AMP formation; this could be related to the facilitation of o o c y t e maturation by subthreshold doses of progesterone [16]. The results presented in Fig. 5 suggest that maturation is initiated only when the cyclic AMP concentration reaches a threshold level. Our experiments show that it is possible to study cyclic AMP synthesis in a living cell, the Xenopus oocyte. We report for the first time that a steroid hormone, progesterone, which initiates o o c y t e maturation, is capable of decreasing the steady state concentration of cyclic AMP. In fact the a m o u n t of radioactive cyclic AMP isolated after microinjection of [~.32p] ATP, represents a steady state concentration of de novo synthesized cyclic AMP, which depends both on the rate of synthesis and on the rate of disappearance of the cyclic nucleotide. Since our experiments are conducted in the presence of an inhibitor of phosphodiesterase, one possibility could be that progesterone acts on adenylate cyclase activity. Testing this hypothesis would require to distinguish between a direct inhibition of the enzyme or a change in the specific activity of the precursor ATP pool at the enzymatic site. This work was supported b y the D~l~gation G~n~rale ~ la Recherche Scientifique et Technique (D.G.R.S.T.).

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Fig. 4. A c t i o n o f c h o l e r a t o x i n on cyclic AMP f o r m a t i o n in X e n o p u s oocyte. Oocytes were microi n j e c t e d w i t h [c~-32P]ATP a n d t h e cyclic AMP i s o l a t e d (see Fig. 2) a f t e r 60 rain i n c u b a t i o n . All experim e n t s w e r e p e r f o r m e d in t h e p r e s e n c e of 1 m M 3 - i s o b u t y l - l - m e t h y l x a n t h i n e . C, C o n t r o l o o c y t e s . T0, c h o l e r a t o x i n ( 5 0 n g / m l ) was a d d e d at t h e t i m e o f m i c r o i n j e c t i o n . T2, o o c y t e s w e r e p r e i n c u b a t e d for 2 h in c h o l e r a t o x i n b e f o r e m i c r o i n j e c t i o n . Pg, 1 pM p r o g e s t e r o n e was a d d e d a t t h e t i m e o f m i c r o i n j e c t i o n . Pg + T2, o o c y t e s w e r e p r e i n c u b a t e d f o r 2 h in c h o l e r a t o x i n t h e n p r o g e s t e r o n e was a d d e d at t h e t i m e of m i c r o i n j e c t i o n . Fig. 5. Cyclic A M P f o r m a t i o n as a f u n c t i o n of t h e p r o g e s t e r o n e c o n c e n t r a t i o n in i n c u b a t i o n m e d i u m . D u p l i c a t e g r o u p s o f five o o c y t e s each w e r e m i c r o i n j e c t e d w i t h [~-S2P]ATP a n d i n c u b a t e d for 60 rain in m e d i u m A c o n t a i n i n g 1 m M 3 - i s o b u t y l - l - m e t h y l x a n t h i n e a n d v a r i o u s c o n c e n t r a t i o n s of p r o g e s t e r o n e (0.01 p M - - 1 0 / ~ M ) or e s t r a d i o l - 1 7 ~ (10 pM). A t t h e e n d o f t h e i n c u b a t i o n p e r i o d o o c y t e s w e r e h o m o g e n i z e d a n d cyclic AMP i s o l a t e d (see Fig. 2). T h e results are e x p r e s s e d as t h e a m o u n t of cyclic AMP r e c o v e r e d versus t h e a m o u n t o f [~-S2p]ATP m i c r o i n j e c t e d . Parallel e x p e r i m e n t s w e r e c o n d u c t e d w i t h 50 n o n - m i c r o i n j e e t e d o o c y t e s i n c u b a t e d in p r e s e n c e of t h e s a m e a m o u n t of p r o g e s t e r o n e or estradiol but w i t h o u t 3 - i s o b u t y l - l - m e t h y l x a n t h i n e , until m a t u r a t i o n ; the n u m b e r above each bar represents the percentage of GVBD.

References 1 S m i t h , L.D. ( 1 9 7 5 ) in T h e B i o c h e m i s t r y o f A n i m a l D e v e l o p m e n t (Weber, R., ed.), Vol. 3, pp. 1 - - 4 6 , A c a d e m i c Press, N e w Y o r k 2 W a s s e r m a n , W.J. a n d Masui, Y. ( 1 9 7 5 ) J. Exp. Zool. 193, 3 6 9 - - 3 7 5 3 S p e a k e r , M.C. a n d B u t c h e r , F.R. ( 1 9 7 7 ) N a t u r e 267, 8 4 8 - - 8 4 9 4 Mallet, J.L. a n d K r e b s , E.G. ( 1 9 7 7 ) J. Biol. C h e m . 2 5 2 , 1 7 1 2 - - 1 7 1 8 5 Morrill, G . A . , S c h a t z , F., K o s t e l l o w , A.B0 a n d Poupko~ J.M. ( 1 9 7 7 ) D i f f e r e n t i a t i o n 8, 9 7 - - 1 0 4 6 O ' C o n n o r , C.M. a n d S m i t h , L.D. ( 1 9 7 6 ) Develop. Biol. 52, 3 1 8 - - 3 2 2 7 Pays de S c h u t t e r , A., K r a m , R., H u b e r t , E. a n d B r a c h e t , J. ( 1 9 7 5 ) Exp. Cell Res. 96, 7 - - 1 4 8 O z o n , R., Bell6, R., H u c h o n , D. a n d Marot, J. ( 1 9 7 8 ) J. A m . Biol. A n i t a . B i o c h i m . Biophys. 18, 483--491 9 T b i b i e r - F o u c h e t , C., Mulner, O. a n d O z o n , R. ( 1 9 7 6 ) Biol. R e p r o d . 14, 3 1 7 - - 3 2 6 10 D u m o n t , J.N. ( 1 9 7 2 ) J. M o r p h o l . 136, 1 5 3 - - 1 7 9 11 S e h o r d e r e t - S l a t k i n e , S. a n d D r u r y , K.C. ( 1 9 7 3 ) Cell Diff. 2, 2 4 7 - - 2 5 4 12 S a l o m o n , Y., L o n d o s , C. a n d R o d b e l l , M. ( 1 9 7 4 ) Anal. B i o c h e m . 58, 5 4 1 - - 5 4 8 13 W o o d l a n d , H.R. a n d PesteU, R.Q.W. ( 1 9 7 2 ) B i o c h e m . J. 127, 5 9 7 - - 6 0 5 14 Bravo, R., O t e r o , C., A l l e n d e , C.C. a n d A l l e n d e , J.E. ( 1 9 7 8 ) Proc. Natl. A c a d . Sci. U.S. 75, 1 2 4 2 - 1246 15 B r a c h e t , J., Baltus, E., De S c h u t t e r , A., H a n o c q , F., H a n o c q - Q u e r t i e r , J., H u b e r t , E., IacobeUi, S. a n d S t e i n e r t , G. ( 1 9 7 4 ) Mol. Cell. B i o c h e m . 3, 1 8 9 - - 2 0 5 16 M a r o t , J., Bell~, R. a n d O z o n , R. ( 1 9 7 7 ) D e v e l o p . Biol. 59, 9 1 - - 9 5