Amphibian oocyte maturation and protein synthesis: Related

Proc. Nati. Acad. Sci. USA Vol. 75, No. 3, pp. 1242-1246, March 1978 Biochemistry Amphibian oocyte maturation and protein synthesis: Related inhibition by cyclic AMP, theophylline, and papaverine (hormone induction/progesterone/cyclic AMP phosphodiesterase/meiosis/translational regulation) RODRIGO BRAVO*, CARLOS OTERO, CATHERINE C. ALLENDE, AND JORGE E. ALLENDEt Departamento de Bioquimica, Facultad de Medicina (Santiago Norte), Universidad de Chile, Casilla 6671, Santiago 7, Chile Communicated by Severo Ochoa, December 27, 1977 ABSTRACT Two inhibitors of cyclic AMP phosphodiesterase (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17), theophylline and papaverine, inhibit the maturation of Xenopus laevis oocytes induced by four different stimuli: human chorionic gonadotropin, progesterone, testosterone, and lanthanum ions. Addition of 1 mm cyclic AMP to the medium delays maturation by approximately 2 hr. Papaverine, theophylline, and cyclic AMP inhibit amino acid incorporation into oocyte proteins by 50% or more but do not inhibit amino acid uptake. The capacity of theophylline to block maturation and protein synthesis is reversed in a parallel fashion by addition of 1-5 mm calcium ion to the medium. Addition of papaverine, theophylline, and cycloheximide to oocytes at different times after hormonal treatment shows that the step sensitive to blockage by the three drugs is coincident and precedes germinal vesicle breakdown by about 1.5 hr. Theophylline and papaverine do not increase endogenous cyclic AMP levels in oocytes but do block the decrease of cyclic AMP levels observed 3 hr after progesterone treatment. Both drugs inhibit oocyte cyclic AMP phosphodiesterase measured in vivo and severely inhibit the stimulus of calcium uptake caused by progesterone and human chorionic gonadotropin. These results suggest that cyclic AMP, theophylline, and papaverine may block oocyte maturation by inhibiting protein synthesis, possibly via a cyclic AMP-dependent protein kinase as shown in reticulocytes [Datta, A., De Haro, C., Sierra, J. & Ochoa, S. (1977) Proc. Nat]. Acad. Sci., USA 74, 1463-1467]. The maturation of full-grown oocytes is a complex process that involves meiotic cell division and prepares the cell for fertilization. Much attention has been devoted to the study of the maturation of amphibian oocytes because these cells can be obtained in large amounts from a single ovary, they are big enough to be microinjected, and they can be induced to mature in vitro. One of the puzzling aspects of the maturation process is that its onset can be triggered by such different stimuli as the hormones human chorionic gonadotropin (hcg), progesterone, and testosterone and by nonphysiological reagents such as ionophore A 23187, organomercurial compounds, and 10 mm lanthanum ions (1-5). Although we are still far from understanding the molecular mechanism that is responsive to these various stimuli, the work of several research groups has provided evidence that de novo protein synthesis, protein phosphorylation, and calcium ions play important roles in the early steps of the maturation process (6-10). Recent evidence has also pointed to the participation of cyclic AMP (camp) in amphibian oocyte maturation induced by progesterone. Speaker and Butcher (11) have reported The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. 1734 solely to indicate this fact. 1242 transient decreases in camp concentration in the first hours after treatment of Rana pipiens oocytes with this hormone, and O'Connor and Smith (12) have shown that theophylline can inhibit Xenopus laevis oocyte maturation triggered by progesterone. In this report we show that theophylline and papaverine, two well-known camp phosphodiesterase (3':5'-cyclic-AMP 5'- nucleotidohydrolase, EC 3.1.4.17) inhibitors, are potent inhibitors of oocyte maturation induced by four different stimuli: hcg, progesterone, testosterone, and lanthanum ions. These drugs, which inhibit endogenous oocyte camp phosphodiesterase (13), do not cause any measurable increase in the overall camp concentration of these cells. However, theophylline or papaverine treatment of oocytes eliminates the increase of Ca2+ uptake induced by progesterone and hcg and results in a very significant inhibition of protein synthesis in these cells. camp itself, added to the oocyte incubation medium, also causes an inhibition of protein synthesis and delays the onset of hormone-induced maturation. Several characteristics of the effect of theophylline, papaverine, and camp in blocking oocyte maturation seem to indicate that this effect is due to their inhibition of cellular protein synthesis. MATERIALS AND METHODS X. laevis were obtained from South African Snake Farm, Fish Hoek, South Africa. Full-grown oocytes [stage VI (14)] were obtained and selected manually as described (13). Isolated oocytes were maintained in a modified amphibian saline (63 mm NaCl/l mm KC1/0.5 mm CaCl2/1 mm MgCl2/20 mm Tris-HCl, ph 7.4/penicillin at 10,g/ml/streptomycin at 10,qg/ml. Microinjections were performed essentially as described by Gurdon (15). Maturation was induced by exposure of the stage VI oocytes to hormone or lanthanum and scored by measuring germinal vesicle breakdown (GVBD) by microscopic inspection and manual removal of the germinal vesicle (16) or by dissecting oocytes fixed in 5% trichloroacetic acid. Oocytes were classified as mature if nuclear breakdown was complete. camp levels were determined by the camp-binding protein method of Gilman (17) as described for X. laevis (13). camp phosphodiesterase activity was measured in duplicate groups of five oocytes in vivo by microinjection of each oocyte with a known concentration of [3H]cAMP followed, at different time Abbreviations: camp, cyclic AMP; GVBD, germinal vesicle breakdown; hcg, human chorionic gonadotropin. * Present address: Department of Biochemistry, University of Oxford, Oxford, England. t To whom reprint requests should be addressed.

100B- 0.01 0.1 1.0 10 0.001 0.01 0.1 1.0 Theophylline, mm Papaverine, mm FIG. 1. Inhibition of maturation by theophylline (A) and papaverine (B). Oocytes were incubated for 18 hr at 220 in the presence of different concentrations of inhibitors with hcg at 60 units/ml (-), 1 AM progesterone (0), 1 ;zm testosterone (A), or 10 mm lanthanum nitrate (o). Each point represents 100 oocytes scored for GVBD. periods, by measurement of the 5'-[3H]AMP formed, by thinlayer chromatography as described (13). The assay for amino acid uptake into intact oocytes has been published (18). Essentially, triplicate groups of five oocytes each were incubated in radioactive amino acid and were washed extensively with amphibian saline and squashed on glass fiber filters for assay of radioactivity in a liquid scintillation counter Ṗrotein synthesis determinations were carried out with triplicate groups of five intact oocytes; incorporation of 20,tM labeled amino acid, added to the external medium, into oocyte acid-precipitable material was measured (18). Progesterone, testosterone, theophylline, and papaverine were from Sigma Chemical Co. hcg was purchased from Ayerst Laboratory, Santiago. Cycloheximide was from Calbiochem. [s5s]methionine (specific activity, 11 Ci/mmol), [3H]alanine (specific activity, 154 mci/mmol), [3H]theophylline (specific activity, 27 Ci/mmol), and 45CaCl2 (specific activity, 17.6 mci/mg) were from New England Nuclear. 0 E 100 _ 80 - T60- o 40 Biochemistry: 200 Bravo et al. 2 3 4 5 6 7 18 Time, hr FIG. 2. Effect of camp on maturation of X. laevis oocytes. Groups of 35 oocytes were incubated in the presence of 1,M progesterone and examined at the times given for GVBD (0). Other groups of 35 oocytes were preincubated for 2 hr with 1 mm camp and then with 1,uM progesterone, and GVBD was scored at the times indicated (-). B Table 1. Proc. Natl. Acad. Sci. USA 75 (1978) 1243 Hydrolysis of [3H]cAMP microinjected into oocytes in the presence of hormone and inhibitors 5'-[3H]AMP formed, pmol Additions 10 min 60 min None 260 1300 Theophylline, 1 mm 114 600 Papaverine, 0.1 mm 102 540 None 180 1480 Progesterone, 0.1 gm 220 1340 Full-grown oocytes were microinjected with 2 nmol of [3H]cAMP, (150,000 cpm per oocyte), incubated in amphibian saline, and, at the times given, processed for 5'-[3H]AMP formation by thin-layer chromatography (13). Duplicate groups of five oocytes each were analyzed. Progesterone, theophylline, and papaverine were added to oocytes 3 hr before the microinjection. RESULTS The results shown in Fig. 1 demonstrate that theophylline and papaverine, well-known inhibitors of camp phosphodiesterases, are potent inhibitors of X. laevis oocyte maturation as assayed by the breakdown of the giant nucleus or germinal vesicle (GVBD). At 1 mm, theophylline completely inhibited the maturation of oocytes triggered by either hcg, progesterone, testosterone, or lanthanum ions. Papaverine at 'Ao the concentration was able to block oocyte maturation induced by these agents. Oocyte maturation can also be inhibited by addition of 1 mm camp to the external medium. As shown in Fig. 2, oocytes treated with camp and then with progesterone matured fully when scored after 18 hr of hormonal treatment, but the kinetics of GVBD lagged with respect to the control cells by approximately 2 hr. The permeability of camp into X. laevis oocytes is very low, which explains the high concentration of the cyclic nucleotide required to obtain the effect. Unfortunately, this difficulty cannot be resolved by microinjecting camp into these cells because the high endogenous camp phosphodiesterase activity quickly reestablishes the normal intracellular levels (13). It is significant that the concentrations of theophylline and papaverine required to block oocyte maturation are the concentrations that are effective in inhibiting camp phosphodiesterase from several tissues (19). Previous in vvo studies of camp phosphodiesterase of X. laevis oocytes assayed by the degradation of microinjected camp showed that coinjection of theophylline significantly inhibits the camp phosphodiesterase of the living cell (13). Table 1 demonstrates that the in vivo activity of the enzyme is also inhibited by >50% when 1 mm theophylline or 0.1 mm papaverine is added to the external medium. It also shows that the levels of camp phosphodiesterase assayed in this manner do not change significantly 3 hr after maturation has been induced by treatment of the oocytes with progesterone. X. kaevis oocytes have an endogenous camp concentration of 1-4,uM (13). Surprisingly, analysis of camp content of oocytes treated with theophylline or papaverine failed to show any significant increment. These analyses were carried out at hourly intervals for 6 hr after exposure of oocytes to the drug, in the presence or absence of a maturation-inducing hormone. These results confirm those of Speaker and Butcher (11) and of O'Connor and Smith (12) who likewise did not observe changes in camp after theophylline treatment of amphibian oocytes.

1244 Biochemistry: Bravo et al. Proc. Nati. Acad. Scz. USA 75 (1978) t W 0- a> 0014- CL 12- 'O60 -~ ~ ~ U60 C > =o I 0~~~~~~~~~~~~~~ :E40 - o 40 20 20 1 2 3 4 5 6 Time after progesterono, hr FIG. 3. camp content of oocytes during maturation. Duplicate groups of five oocytes were incubated in the presence of 1 MM progesterone (0) or 1,MM progesterone and 1 mm theophylline (0) and were assayed for camp at the times given (7, 13). Batches of oocytes that showed a well-synchronized response to progesterone induction of maturation underwent a significant but transient decrease in camp levels between 2 and 4 hr after hormone treatment (Fig. 3). The recovery of the levels coincided approximately with GVBD in these cells. Treatment of these oocytes with 1 mm theophylline in the external medium eliminated the change in camp levels caused by the hormone. A similar decrease in camp levels after progesterone treatment has been observed with R. pipiens oocytes (11) but was not detected with X. laevtis cells (12). The effect of theophylline, papaverine, and camp on oocyte protein synthesis was studied. From the data presented in Table 2 it is clear that, at the concentration that causes inhibition of maturation, these compounds have a strong inhibitory effect on amino acid incorporation into proteins but do not significantly affect the uptake of the amino acid into the cells. Hydrolysis of the exogenous camp with camp phosphodiesterase resulted in the elimination of inhibitory effect of this com- Table 2. Inhibition of oocyte protein synthesis by theophylline, papaverine, and camp Amino acid Amino acid incorporation uptake, into protein Inhibitor cmp/oocyte cpm/oocyte % inhibition None 217,720 21,825 Theophylline, 0.1 mm 207,263 11,557 47 Theophylline, 1 mm 221,409 8,670 60 None 11,893 1,668 Theophylline, 1 mm 11,656 982 41 Papaverine, 0.1 mm 9,892 752 55 Papaverine, 1.0 mm 10,919 466 74 camp, 1 mm 11,584 1,013 40 camp(1 mm) + PDE* 10,671 1,487 11 PDE 11,579 1,671 0 Triplicate groups of five oocytes were used for measurement of amino acid uptake and protein synthesis, incubating in 50 1A of amphibian saline solution containing 20 MAM [35S~methionine, specific activity 11 Ci/mmol (Exp. 1) or 20MuM [14C]alanine, 154 mci/mmol, for 2 hr at 220. Assays were as described in Materials and Methods. Inhibitors when present were added 2 hr before the amino acid. * PDE, phosphodiesterase 1 2 3 4 5 6 Time after hcg, hr FIG. 4. Effect of time of addition of theophylline and papaverine on oocyte maturation. Groups of 100 oocytes were incubated with hcg at 60 units/ml, and, at the times shown, 1 mm theophylline (0), 0.1 mm papaverine (0), or 0.01 mm cycloheximide (A&) was added. Percentage inhibition of GVBD was estimated after 18 hr. Oocytes (100 per point) from the same batch were also scored for the time of GVBD in the absence of inhibitors (o). pound. Protein synthesis in oocytes treated with hormones was inhibited by these drugs to a similar extent (not shown). An experiment was performed in which theophylline, papaverine, and cycloheximide were added to the external medium of oocytes at different times after hormone treatment. This experiment was designed to detect possible differences in the action of the camp phosphodiesterase inhibitors and the classical protein synthesis inhibitor (Fig. 4). From the results obtained, it is clear that the step that is sensitive to theophylline and papaverine occurs at about the same time as the step sensitive to cycloheximide and precedes GVBD by 1-2 hr. Similar a rm 80 (D 0 ~ 0-0.c 40 9II I[- )k 20 _- I I _ I I I o3- o I 1 2 3 Ca2+. mm I 4 5 50 40 C.. c s.2 C 30 Q >.0._C 2000 20 8 L FIG.. 5. Effect of Ca2+ on the inhibition of oocyte GVBD and protein synthesis by theophylline and papaverine. Groups of 100 oocytes were incubated in amphibian saline with 1MgM progesterone and varying amounts of calcium chloride as shown and with added 1 mm theophylline (0), or 0.1 mm papaverine (o). GVBD was scored at 18 hr. Protein synthesis was measured by incubating duplicate groups of 10 oocytes in 20 MAM [35S]methionine (specific activity, 11 Ci/mmol) for 2 hr with added calcium chloride and theophylline (0) or papaverine (-). -I 10

Biochemistry: Bravo et al. results have been obtained with other inducers of matusration such as testosterone and progesterone, with some disp met in the time scale (not shown). The effect of theophylline and papaverine on oocyte maturation and on protein synthesis run parallel courses in two more aspects. The inhibition of both phenomena caused by theophylline can be reversed by the removal of the drug from the external medium. The inhibition obtained with papaverine is not affected by repeated washing of the cells and a 22-hr incubation in the absence of the drug. The data from several laboratories have established the involvement of Ca2+ in the maturation process (4, 10). An increase in the concentration of Ca2+ in the external medium from 0.5 mm to 5 mm eliminated almost completely the inhibition of oocyte maturation and of oocyte protein synthesis caused by 1 mm theophylline (Fig. 5). The addition of Ca2+ did not affect the inhibition caused by papaverine. Using radioactive theophylline it has been possible to measure the uptake of this drug into oocytes and to study its release from the cells when they are placed in a drug-free medium. [3H]- Theophylline was readily taken up by the oocytes, and the presence of 5 mm Ca2+ did not significantly inhibit its transport into the cell. Also, [3H]theophylline was rapidly released from oocytes that were placed in a drug-free medium: after 2 hr, only 40% of the compound was found inside the oocyte. These findings explain the reversibility of the inhibition caused by this drug. In order to explore further the relationship between theophylline and papaverine inhibition and Ca2, the effect of the inhibitors on the uptake of 45Ca2+ by oocytes was studied. The results (not shown) demonstrated that these compounds did not affect the basal uptake of 45Ca2+ but that their presence eliminated the 2.5-fold stimulation caused by treatment of the oocytes with progesterone. With hcg, the stimulation of 45Ca2+ uptake was 5.5-fold but again in this case the addition of papaverine significantly inhibited its transport. Recent work in this laboratory has established that hcg, but not progesterone, significantly stimulates the uptake of several amino acids and nucleosides into X. laevis oocytes (20). DISCUSSION The study of the inhibition of oocyte maturation by theophylline, papaverine, and camp has revealed some interesting facts about the nature of this process. Despite the diversity of substances that can trigger its onset, there seem to be specific steps that are required for maturation to occur. One of these steps is sensitive to theophylline and papaverine because these drugs are able to block maturation induced by four different stimuli. The presence of theophylline and papaverine in the external medium inhibits the oocyte camp phosphodiesterase measured in vivo but does not increase the overall camp content of the cell. This paradoxical result might be explained by the fact that these giant cells have compartmentalized camp. Evidence for the existence of a high camp compartment in the oocyte nucleus has been presented (13). In other systems, it has been observed that significant amounts of camp are bound to membranes and that hormone treatment causes a redistribution of this nucleotide (21). The involvement of Ca2+ and cyclic nucleotides in hormone action seems to be a rather general phenomenon but the details of the mutual relationship between these two cellular signals remains obscure. The discovery of a calcium-binding protein Proc. Natl. Acad. Sci. USA 75 (1978) 1245 that activates camp phosphodiesterase (22) may explain the Ca2rMedfated reversal of the inhibitory effect of theophylline because the ion and the drug have opposite effects on the activity of this enzyme. The most promising observation presented above is the effect of theophylline and papaverine on oocyte protein synthesis and the close correlation between this effect and the capacity of these drugs to block oocyte maturation. No evidence has been obtained as to the mechanism through which these compounds could inhibit protein synthesis. Experiments in our laboratory, however, show that they do not inhibit poly(u)-directed poly(phe) synthesis in an in vitro system derived from X. laevis oocytes (0. Orellana, personal communication). The fact that incubation of the oocytes in camp also inhibits the incorporation of amino acids into proteins and delays the onset of maturation, however, favors the idea that these drugs might cause localized increments in camp and that this increment inhibits protein synthesis. In this light it is tempting to relate our findings to the recent observation of Datta et al. (23) on the existence of a camp- -dependent protein kinase that activates a camp-independent protein kinase that in turn inhibits reticulocyte protein synthesis. These authors have postulated that a similar mechanism might mediate some of the regulatory effects of camp levels on the metabolism of many cell types. As an attempt to fit this idea with the data available about the regulation of oocyte maturation, the following working hypothesis can be envisaged: 1. Oocyte maturation requires the synthesis of a specific protein or proteins that may be the maturation-promoting factor described by several authors (1, 4, 7, 24). 2. The mrna coding for this protein is present in an extranuclear compartment of full-grown oocytes but is not translated in the unstimulated oocyte because of the inhibition of the translation machinery. 3. The inhibition of the translation components available to the maturation-factor mrna is due to phosphorylation of key proteins such as initiation factors or ribosomal proteins catalyzed by protein kinase(s). 4. The activity of the inhibitory protein kinases and of possible counteracting protein phosphatases is controlled by the intracompartment levels of camp. Calcium ions may regulate these enzyme activities directly or through localized effects on camp levels. 5. Any factor that decreases protein phosphorylation or activates protein dephosphorylation will therefore trigger maturation. This hypothesis incorporates the proposal of Maller and Krebs (25) for the role of protein phosphorylation in oocyte maturation but, in addition, includes the relationship between camp and protein synthesis postulated by Datta et al. (23) and suggested by our results. Strong support for the idea that such a mechanism for translational regulation may be operative in X. Iaevis oocytes is afforded by experiments in which microinjection of the catalytic subunit of camp-dependent protein kinase from beef heart resulted in a strong inhibition of oocyte protein synthesis (S. Ochoa and S. Pestka, personal communication). This research was supported by Grant 770-0309 from the Ford Foundation, by United Nations Development Program/United Nations Educational, Scientific and Cultural Organizations Project RLA 76/006, by the Organization of American States Program in Biochemistry, and by the University of Chile.

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