DEVELOPMENTAL

Changes

BIOLOGY

58,

295-312

in Protein

(1977)

Phosphorylation Accompanying Xenopus laevis Oocytes’

J. MALLER,” Department

of Molecular

Biology,

Maturation

of

M. WV, AND J. C. GERHART University

of California,

Berkeley,

Received September 9, 1976; accepted in revised form February

California

94720

17, 1977

Protein phosphorylation has been measured after injection of [“‘PIphosphate into oocytes of Xenopus laeuis undergoing progesterone-induced meiotic maturation. As oocytes mature, there is a burst of nonyolk protein phosphorylation several hours after progesterone exposure and shortly before germinal vesicle breakdown (GVBD). This burst is not due to changes in the specific activity of the phosphate or ATP pool. Enucleated oocytes exposed to progesterone also experience the burst, indicating the cytoplasmic location of phosphoprotein formation. When an oocyte receives an injection of cytoplasm containing the maturation-promoting factor (MPF), a burst of protein phosphorylation occurs immediately, and GVBD occurs shortly thereafter, even in the presence of cycloheximide. Under a variety of conditions promoting or blocking maturation, oocytes which undergo GVBD are the only ones to have experienced the phosphorylation burst. The results suggest that the protein phosphorylation burst is a necessary step in the mechanism by which MPF promotes GVBD. INTRODUCTION

Prior to maturation, full-grown oocytes of amphibia are uniformly arrested in first meiotic prophase. The prophase arrest is lifted synchronously among oocytes in response to progesterone, and maturation ensues, consisting of meiotic cell division as well as preparation of the cell surface for fertilization (for reviews, see Schuetz, 1974; Smith, 1975). As in mitotic cell division, the nucleus breaks down (referred to as germinal vesicle breakdown, GVBD), chromosomes condense, a spindle forms, and cytokinesis occurs, producing in the case of the oocyte a small polar body and ’ Portions of this work were submitted by J. Maller in partial fulfillment of the requirements for the Ph.D. degree, University of California, Berkeley, California, 1974. * Present address: Department of Biological Chemistry, University of California, Davis, California 95616. 3 Abbreviations used: MPF, maturation-promoting factor; GVBD, germinal vesicle breakdown; MRS, modified Ringer’s saline; MBS, modified Barths’ saline; TCA, trichloroacetic acid; PCA, perchloric acid.

the unfertilized egg. The egg then arrests at second meiotic metaphase. In the conversion of a progesterone-exposed oocyte to an unfertilized egg, several steps have been identified. Progesterone must interact with the oocyte surface, since microinjection of progesterone into the oocyte interior fails to evoke maturation (Masui and Markert, 1971; Smith and Ecker, 1971). Cycloheximide blocks GVBD and other maturation events if applied soon after progesterone, whereas actinomycin D does not inhibit, indicating early steps of maturation depend upon translation but not transcription (Schuetz, 1967; Smith and Ecker, 1969; Merrium, 1972; Baltus et al., 1973). The end of the cycloheximide-sensitive period occurs shortly before GVBD (Wasserman and Masui, 1975) when a cytoplasmic maturation-promoting factor (MPF) appears in the oocyte, as assayed by microinjection of small volumes of cytoplasm into recipient oocytes which then mature without exposure to progesterone (Masui and Markert, 1971; Smith and Ecker, 1969; Merriam, 1972;

295 Copyright 0 19’77by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0012-1606

296

DEVELOPMENTAL BIOLOGY

Schorderet-Slatkine and Drury, 1973; Maller, 1974). Moreover, the matured recipient subsequently serves as a donor for MPF3 activity to the next recipient, and this serial transfer has been carried through 10 oocytes without loss of the activity (Schorderet-Slatkine and Drury, 1973; Reynhout and Smith, 1974). Since only one-twentieth of the cytoplasmic volume is transferred in each injection, progesterone and other substances of the original donor appear to be diluted out during the serial transfers, and MPF appears to have autoamplifying properties. Although MPF is known to promote GVBD in cycloheximide-treated oocytes, there is controversy as to whether autoamplification of MPF occurs in the presence of cycloheximide (Wasserman and Masui, 1975; Drury and Schorderet-Slatkine, 1975). Recently, Wasserman and Masui (1976) reported MPF to be protease sensitive and separable from other cell materials by sucrose gradient centrifugation. Taken together, these findings suggest that MPF may be a cytoplasmic autoactivating enzyme system with a key role in triggering meiotic cell division. We have investigated biochemical events which accompany early steps of maturation in an effort to understand the mechanism of MPF formation and action. The reactions of protein phosphorylation were chosen in particular because: (1) the phosphorylation of certain proteins such as histones attends mitotic cell division (Gurley et al., 1975); (2) protein phosphorylation reactions are known to control enzyme activity changes in hormone-induced physiological states of muscle and other tissues (Krebs, 1972; Rubin and Rosen, 1975); (3) autoactivation reactions are known for the Ca2+-dependent protein kinase of muscle (Delange et al., 1968; Walsh et al., 1971) and may occur for the CAMPdependent protein kinase as well (Rosen and Erlichman, 1975; Rangel-Aldao and Rosen, 1976); and (4) increased protein phosphorylation occurs in amphibian oo-

VOLUME 58, 1977

cytes induced to mature in vivo with gonadotropins (Morrill and Murphy, 1972) and in vitro with progesterone (Maller, 1974; Wallace, 1974). This study describes the invariable association of protein phosphorylation with MPF activity and GVBD. MATERIALS

AND

METHODS

Media. Modified Barths’ saline (MBS) contained millimolar concentrations of the following salts: NaCl, 88; KCl, 1.0; MgSO,, 0.82; Ca(NO,),, 0.33; CaCl,, 0.41; NaHCO,, 2.4; Tris-HCl, pH 7.8, 1.5; and Na-pyruvate, 2.0 (GIBCO). Also included per milliliter were 1 mg of bovine y-globulins (Pentex), 50 pg of streptomycin, and 50 units of penicillin (Pen-Strep, GIBCO). Modified amphibian Ringer’s saline (MRS) contained millimolar concentrations of the following salts: NaCl, 110; KCl, 2.0; MgC&, 1.0; CaCl,, 2.0; NaHCO,, 2.5; with Na-pyruvate, globulins, and antibiotics at the concentrations given above. Xenopus laeuis. Frogs weighing from 150 to 200 g were obtained from the South African Snake Farm, Fish Hoek, South Africa, and were maintained in bath tubs of tap water at 18-22°C. Animals were fed twice weekly to saturation on beef heart and liver, with monthly supplementation of live lCday-old chick embryos. For two months prior to use, frogs were injected every 2 weeks with 100 units of pregnant mare serum gonadotropin (PMSG, obtained from the Rat Pituitary Hormone Distribution Program, NIAMDD) as recommended by Thornton (1971). Ovarian tissue was removed through a slit in the dorsal body wall of frogs anesthetized for 30 min in 0.15% tricaine (ethyl m-aminobenzoate, methane sulfonic acid salt, Calbiochem). Usually one-half of one ovary was cut off, the slit was sutured, and the frog was given a rest of 2 months before use again. The ovary was washed in MBS, cut into pieces approximately 1 cm’, and transferred to fresh MBS. Ovarian tissue was stored at 16°C if not used within 8 hr. Routinely, ovaries were obtained from

MALLER, Wu, AND GERHART

Protein

frogs 7-14 days after their last PMSG injection. Microinjection of [“2Plphosphate. Oocytes (stage VI, 1.25-1.4 mm in diameter) were routinely dissected from their ovarian follicles with watchmaker’s forceps and were stored at room temperature (22 k 2°C) in MRS until use, usually within 2 hr. For incorporation experiments, each oocyte was injected with 30-50 nl of carrier-free [32P]phosphate (ICN) diluted in 20 mM Tris, pH 7.8, to give 0.05-1.0 pCi/ injection, depending upon the level of radioactivity needed in the analysis. Less than 0.5% of the radioactivity was lost into the medium from the oocytes in a 20-hr period. The preparation and calibration of micropipets were as described previously (Maller et al., 1976). Oocytes were induced to mature by incubation in 3 & progesterone (Calbiochem) in MRS. Maturation was scored after 4-8 hr by the presence of a white spot at the animal pole, indicative of GVBD (Merriam, 1971a). In a control experiment, cytological examination of such oocytes confirmed that they had advanced to second meiotic metaphase after progesterone treatment. However, in most experiments reported here, the maturation process was followed only as far as GVBD. Determination of “ZP-labeled phospho,protein. Incorporation was terminated by fixing a group of four oocytes in 3 ml of 7.5% TCA, 1% Na-pyrophosphate at room temperature. After 12 hr, the vials were counted for acid-soluble radioactivity in a Packard Tri-Carb spectrometer with Cerenkov settings. Counting efficiency was 34% of that obtained with Aquasol and 32P settings. This count indicated acid-soluble radioactivity since oocytes retained negligible acid-soluble radioactivity after TCA fixation and since they had converted less than 4% of the total radioactivity to acidinsoluble material. Oocytes were removed, rinsed twice, and homogenized with 1 ml of fresh TCA-pyrophosphate in a Dounce homogenizer. The homogenate was heated for 15 min at 90°C to degrade ribonucleic

Phosphorylation

in Oocytes

297

acids (Schmidt and Thannhauser, 1945) and was filtered through a GF/C filter to collect insoluble material. The filter was washed three times with TCA-pyrophosphate, 5 ml/wash, and once with chloroform-methanol (3:l) to remove phospholipid. Radioactivity on the filter was determined in Aquasol. This material represents the “2P-labeled phosphoprotein reported in all figures. Total protein was determined by the method of Lowry et al. (1951) with bovine serum albumin as standard. The following controls were evaluated. (i) The hot TCA removal of RNA was tested by mixing 3’P-labeled Rous virus RNA (kindly provided by Dr. P. Duesberg) with nonradioactive oocyte extract. Conversion to acid-soluble radioactivity was 99%. (ii) The identity of 32P-labeled phosphoprotein on filters was checked by the solubilization of radioactivity in 2 ml of 0.5 N NaOH for 20 min at 100°C. Over 98% of the counts became TCA soluble. Of these, 89% could be extracted as orthophosphate by the method described below, the same percentage as could be extracted from alkaline-treated [ 3”Plphosphorylase a, prepared by the method of Krebs and Fischer (1962). (iii) Degradation of phosphoprotein in the RNA degradation step was tested by heating radioactive extracts in TCA for prolonged times beyond 15 min. Phosphoprotein radioactivity declined 30% in 1.5 hr, equivalent to 5% in the usual 15 min period. These results are consistent with those of Bylund and Huang (1976) on model phosphopeptides dephosphorylated under mild acid conditions. Acid-insoluble radioactivity in extracts from matured and nonmatured oocytes was fractionated by the above steps and was found to yield respectively: 3.9 and 5.4% as phospholipid (chloroform-methanol extracted); 10.3 and 11.8% as RNA (hot TCA-solubilized); and 85.8 and 83.8% as phosphoprotein. Thus, the data on phosphoprotein radioactivity concern the major portion of acid-insoluble radioactivity.

298

DEVELOPMENTAL BIOLOGY

Determination of orthophosphate. The procedure of Sanui (1974) was used, with the modification that, before the molybdate-sulfuric acid reagent was added, the sample was treated with 8% TCA containing 1.2% sodium silicotungstate in 0.5 N sulfuric acid and the precipitate was removed by centrifugation. Silicotungstate eliminated or counteracted material which prevented the extraction of 132P]phosphate. For determination of orthophosphate pools, 15 oocytes were homogenized at 0°C in 1.2 ml of 5% perchloric acid containing a carefully measured amount of radioactivity from carrier-free [32P]phosphate to serve as a recovery marker. Insoluble material was removed by centrifugation, the supernatant was immediately neutralized with 2 N KOH, and the precipitate was removed by centrifugation. An aliquot of 1.0 ml was used for the Sanui (1974) procedure as described above. Recovery of input radioactivity was approximately 40%. Determination of [3ZP]phosphoserine and [32Plphosphothreonine. Twenty radioactive oocytes (lo5 cpm/oocyte) were homogenized in 2.0 ml of a solution containing 50 mM NaF, 20 mM Tris-HCl, and 2 n&f sodium EDTA, at a final pH of 7.8. The extract was centrifuged at 2000g for 10 min to remove yolk. The supernatant contained 85% of the phosphoprotein radioactivity of the oocyte, when fresh oocytes were extracted. (Frozen oocytes released only 50-70% of their 32P-labeled phosphoprotein into the nonyolk fraction.) To the supernatant was added 0.2 ml of 100% TCA, and the precipitate was collected and treated with 2.0 ml of hot TCA and chloroform-methanol as described previously. The final pellet was dried in a vacuum oven for 15 min at 50°C and was hydrolyzed in 1.0 ml of 6 N HCl in an evacuated tube at 110°C for 1.5 hr. The hydrolyzate was evaporated to dryness, and the residue was dissolved in 2.0 ml of 50 mM HCl. In one case it was chromatographed on a Dowex 50 column as described by Schaffer et al. (1953) with good separation of phos-

VOLUME 58,

1977

phate, phosphoserine, and phosphothreonine, yielding respectively, 49, 38, and 8% of the input radioactivity for matured oocytes. In four other samples, the dissolved hydrolysis residue was passed through a Dowex 1 column as described by Allerton and Perlmann (1965), and the void volume effluent was concentrated and chromatographed on PEI-cellulose thin-layer sheets with 0.5 M Na-formate-0.5 N formic acid, pH 3.4 (Randerath and Randerath, 1964). Phosphate compounds were located with FeCl,-sodium sulfosalicylate spray and heating to 50°C (Wade and Morgan, 1953). Spots were scraped off and counted in Aquasol to determine radioactivity. In duplicate determinations with matured and control oocytes, phosphate (R, 0.401, phosphoserine (R, 0.65), and phosphothreonine (R,O.75) comprised 43.9 f 2.8, 31.9 f 2.1, and 14.2 4 2.3% of input radioactivity, respectively. There was no difference in these values with maturation. We conclude that at least 46% of the radioactivity in the phosphoprotein fraction is in phosphoamino acids; the actual value may be close to 100% since phosphoamino acids in peptides are extensively destroyed under conditions of acid hydrolysis (Bylund and Huang, 1976). Determiantion of ATP specific activity and absolute amount. Fifty oocytes were homogenized with 100 ~1 of 10% perchloric acid at 0°C. After 30 min, the homogenate was centrifuged at 10,OOOgfor 15 min at 4°C. The supernatant was removed and immediately neutralized with 2 N KOH, using thymol blue as indicator. The neutral supernatant was evaporated to a volume of approximately 10 ~1, centrifuged to remove KCIO1, and spotted on a PEI-cellulose thin-layer sheet. The chromatogram was developed in two dimensions with the solvent systems of Hayashi and Hayashi (1972). ATP separated well from the other nucleotides (Rf = 0.1 in the first dimension, 0.3 in the second). Nucleotides were located by uv absorption and autoradiography; the PEI-cellulose was scraped from

MALLER, Wu,

AND

GERHART

Protein

each nucleoside triphosphate spot and was eluted with 1.0 ml of 0.7 M MgCl,. The solution was filtered through a 0.24-pm Millipore filter and was analyzed for uv absorption (for ATP, A,,, 2 0.6) and radioactivity (2 lo4 dpm). In order to determine the relative specific activities at the various phosphate positions of ATP, the eluate from the chromatogram was mixed with a known amount of radioactivity from [14ClATP and was heated for 60 min at 100°C in a sealed tube. The products of partial hydrolysis were separated on PEIcellulose (Randerath and Randerath, 1964), and radioactivity was measured at the AMP, ADP, and ATP positions. The 32P/14Cratios indicated that ADP had 0.5 the 32P specific activity at ATP, whereas AMP had less than 0.01 the 32P specific activity of ATP. When the absolute amount of ATP was determined, the same general procedures were used except that 50 nonradioactive oocytes were homogenized at 0°C with 100 ~1 of 10% perchloric acid containing a carefully measured amount of radioactivity from [32P]ATP (labeled in the y position) or [14ClATP (labeled in the 8 position) to serve as a recovery marker. Overall recovery after the elution from PEI-cellulose was approximately 35%. RESULTS

Time Course of [32P]Phosphate Incorporation in Maturing Oocytes As shown in Fig. 1, there was an initial rapid incorporation of radioactivity into phosphoprotein in oocytes injected with [32Plphosphate and incubated with or without progesterone (phase 1). In this phase, approximately 0.7% of the total intracellular radioactivity was incorporated in a period of 2 hr. In oocytes unexposed to progesterone, incorporation then continued slowly, reaching levels of approximately 1% by 8 hr (phase 2) and 1.4% by 24 hr (see Fig. 5). In contrast, there was an additional burst of protein phosphorylation just prior to GVBD in oocytes exposed

Phosphorylation

in Oocytes

299

FIG. 1. Time course for amount of 32P-labeled phosphoprotein in maturing and nonmaturing oocytes. Oocytes were each injected with approximately 0.05 &i of [““PIphosphate and were incubated in MRS containing: no additions (O), 3 &f progesterone (O), or 100 pg of cycloheximide/ml (A). The cycloheximide-treated oocytes had been injected each with approximately 70 ng of cycloheximide 30 min prior to [*‘PIphosphate injection. At each time point, a group of four oocytes was removed for phosphoprotein analysis as described in Materials and Methods. The amount of :‘2P-labeled phosphoprotein is presented as the percentage of total ?‘P of the oocyte. GVBD refers to germinal vesicle breakdown, scored as oocytes showing a well-defined white spot at the animal pole. The curves are divided into four phases for discussion in the text. The dashed line is a theoretical curve for a synchronous population of oocytes forming ““P-labeled phosphoprotein (PP*) in phases 1 and 2 by two first-order reactions, one with a rate constant (k,) of 0.04 min’ and a maximum level (C,) of 0.5% incorporated :12P, and a second reaction with a 12, of 0.0015 mini and a C, of 0.9% incorporated asP, in the equation: PP* = C,(l e ‘(,‘) + C,(l - em”*‘). Time zero was taken as the time of I:“Plphosphate injection. The solid line is a visual fit to the experimental data of phases 3 and 4.

to progesterone (phase 3), raising the extent of incorporation to approximately 2.5% of the intracellular radioactivity. Subsequently, the rate of net phosphorylation slowed (phase 4) to that of control oocytes in phase 2. As shown by the dashed line in Fig. 1, the incorporation data for phases 1 and 2 can be fit by the sum of two first-order reactions, one proceeding rapidly (tl,2 approximately 17 min) and yielding a maximum incorporation of approximately 0.5% of the total radioactivity and another proceeding slowly (t,,2 approximately 450 min)

300

DEVEWPMENTAL

BIOLOGY

and yielding a maximum incorporation of 0.9% of the total radioactivity. The data could not be fit by a single first-order reaction. The incorporation data of phase 3 will be considered later. In the same experiment, additional oocytes were each injected with approximately 70 ng of cycloheximide and were incubated in MRS containing 100 pg of cycloheximide/ml for 30 min before [32Plphosphate was injected to initiate the labeling period. These oocytes completed phase 1 and phase 2 protein phosphorylation with the same kinetics as oocytes without cycloheximide (Fig. 1). The incorporation of [H”]lysine into protein was depressed 95% in a parallel set of oocytes. Thus, protein synthesis is not necessary for the incorporation of 32P into phosphoproteins of phase 1 and phase 2. In experiments with oocytes from a different female, a detailed study of the phase 3 burst was carried out (Fig. 2), showing its occurrence in the population approximately 30 min prior to GVBD and covering a period of approximately 120 min. The oocyte population was asynchronous as revealed by the fact that GVBD occurred among individual oocytes over a lO.O-min period. This asynchrony of the population prevents the accurate analysis of incorporation kinetics for individual oocytes. However, as indicated by the dashed line in Fig. 2, the data for the phase 3 burst can be fit by a single first-order incorporation reaction (tliz approximately 17 min and maximum incorporation of 1.7% of total radioactivity) initiated 67 min prior to GVBD. The oocytes are assumed to be as asynchronous in the initiation of this phosphorylation reaction as they are for GVBD. Phase 4 incorporation is taken to be equivalent to that of phase 2. The ambiguity about events in the asynchronous population raises the question of whether the phosphorylation burst occurs in all oocytes prior to GVBD. As an answer to this question, Fig. 3 presents an analysis of individual oocytes during the burst

VOLUME 58, 1977

OL Y + progesterone

200

300

MlnUteS (0)

FIG. 2. Detailed time course for the increase of 52P-labeled phosphoprotein during maturation. Oocytes of a different frog were treated as described in Fig. 1, after incubation in the absence (0, A) or presence of (0, A) pfogesterone. Four oocytes were taken for each time point for phosphoprotein determination (0 and 0). GVBD refers to germinal vesicle breakdown, scored within a group of 30 injected oocytes (A and A). The dashed line is a theoretical curve for an asynchronous population of maturing oocytes engaged in a third first-order phosphorylation reaction (see legend of Fig. 1 for reactions 1 and 2) with a k, of 0.04 min-’ and a maximum level (C,) of szP in phosphoprotein equal to 1.7% above the value of the nonmatured oocytes. Each maturing oocyte is assumed to initiate the phosphorylation reaction 67 min prior to its time of GVBD, with a population asynchrony of starting times for phosphorylation equivalent to the asynchrony found for GVBD itself (A). The equation for the theoretical curve is: PP* = 1 fiC,[l - emh8’?)] + Z,, where i ,=, indicates seven groups of oocytes in the population, containing, respectively, 0.07, 0.17, 0.26, 0.26, 0.16, 0.07, and 0.01 as a fraction, fi, of the total oocytes, and differing in starting times (ti) of phosphorylation such that the first group begins at t = 115 min, and each successive group begins at a subsequent 20-min interval. 2, indicates the “2P-labeled phosphoprotein level of the nonmatured control oocytes.

period. At the time when 30% of the population had completed GVBD, 90% of the oocytes had undergone the burst of protein phosphorylation, and among that 90% were all those oocytes which had completed GVBD. In a parallel set, all oocytes completed GVBD within the next 50-min period. Thus, the burst of protein phospho-

MALLER, Wu, AND GERHART

FIG. 3. Protein phosphorylation in individual oocytes. Each oocyte received an injection of approximately 0.1 &i of [32P]phosphate and was incubated in the absence (blank boxes) or presence (crosshatched boxes) of progesterone. After 160 min in progesterone, 30% (9 out of 30) of the oocytes displayed a white spot at the animal pole (GVBD), at which time all oocytes were individually fixed in TCA-pyrophosphate and were analyzed for phosphoprotein as described in Materials and Methods. Oocytes achieving a white spot are indicated by a blank circle in the cross-hatched box. A parallel set of oocytes reached 100% GVBD by 210 min postprogesterone. Note that 90% of the oocytes have increased their incorporation well above the control level at the time of 30% GVBD.

rylation was regularly followed by GVBD, and no oocyte was found to have undergone GVBD in the absence of increased protein phosphorylation. Characterization of “2P-Labeled Materials in Oocytes In a control experiment, oocytes receiving [32P1phosphate by microinjection were matured and fixed in 7.5% TCA-1% Napyrophosphate overnight. Then, the follicle cell layer and vitelline membrane were carefully removed by manual dissection, and their radioactivity was determined. The remainder of the oocyte was processed for “2Plabeled phosphoprotein. The follicle cell-vitelline membrane fraction contained less than 0.4% of the radioactivity found in the phosphoproteins of the remainder of the oocyte, indicating the effectiveness of injection in avoiding labeling of the follicle cells. Figure 4A illustrates the 32P-labeled phosphoprotein after the phosphorylation burst as it was distributed among the major cell fractions separated by the velocity-

Protein

Phosphorylation

in Oocytes

301

density centrifugation procedure of Jared et al. (1973). From a comparison of protein and radioactivity in different regions of the gradient, it was found that yolk comprised 65% of the cell protein, yet contained only 3% of the “‘P-labeled phosphoprotein. The mitochondrial band comprised 11% of the protein and 6% of the radioactivity, whereas the supernatant protein comprised 24% of total protein and 91% of the radioactivity. A similar distribution was found for oocytes not exposed to progesterone (data not shown). Thus, almost 97% of the 32P-labeled phosphoprotein even material, is nonyolk-associated though yolk contains high levels of phosphoprotein as phosvitin and lipovitellin subunits (Wallace et al., 1972; Bergink and Wallace, 1974). Our measurements on whole oocytes indicate approximately 120 nmole of protein-bound phosphate/oocyte, in agreement with the data of Colman and Gadian (1976). From homogenates of oocytes that had undergone the phosphorylation burst, supernatant protein was prepared by centrifugation at 1O”g for 10 min to remove yolk platelets. Analysis of the supernatant protein by SDS-gel electrophoresis (Fig. 4B) revealed that most of the radioactivity was in the high molecular weight regions of the gel, and a peak in the region of 55 x lo3 molecular weight. This material was not RNA or DNA since it released radioactive orthophosphate when heated in alkali; and, as described in the Methods and Materials section, RNA accounted for only 10% of the total acid-insoluble radioactivity. Nonradioactive orthophosphate was also measured after alkaline treatment and found in large amounts in the molecular weight range of 17,000 to 25,000 Daltons, slightly smaller than expected for yolk platelet phosvitin. Summation of the area under the curve in Fig. 4B, as well as direct measurements, of total phosphoprotein in separately prepared supernatant fractions, gave values of lo-20 nmole of protein-bound phosphateloocyte. These

302

DEVELOPMENTAL

1.0 (a)

Yolk

.---r

t

0

5

so

BIOLOGY

(b)

10 33,000 i

'7'~oo

15 55,000 1

18

0

x E 8

10 .’ 0 z

VOLUME

58, 1977

predominant phosphoproteins released 65 75% of the orthophosphate present in the gel but only 10% of the radioactivity. Clearly, they are not significant targets of phosphorylation during progesteroneinduced maturation in uitro. Oocytes labeled with [32P]phosphate in the absence of progesterone yielded a similar gel profile of radioactive phosphoproteins (data not shown). Due to the large amount of nonradioactive phosphoprotein in oocytes, we were unable to determine whether there is an increase of total phosphoprotein during the phosphorylation burst. Pools of Phosphate and ATP during uration

OP

I 5 BOTTOM

IO

15 Fraction

20 number

25

IO 30 TOP

FIG. 4. Analysis of 32P-labeled phosphoproteins in matured oocytes. Oocytes were injected with approximately 0.15 &i of [32Plphosphate and were incubated in the presence of 3 @f progesterone in MRS for 12 hr to obtain 100% maturation. Panel A: sucrose gradient centrifugation. Twenty oocytes (4.2 mg of protein) were extracted in 0.5 ml of PVPsucrose and were centrifuged in a 20-60% sucrose gradient, following the procedure of Jared et al. (1973). After 2 hr at 34,000 rpm, yolk and mitochondrial bands were visible. Drops were collected from the bottom, 0.28 ml/fraction. A 50-yl aliquot was analyzed for protein by the method of Lowry et al. (1951). The remainder of each fraction was brought to 1.0 ml with 10% TCA-1% sodium pyrophosphate, and the precipitate was collected on GF/C filters which were processed for 32P-labeled phosphoprotein as described in Materials and Methods. Values on the graph indicate the amount of protein or radioactivity per fraction. Panel B: SDS-polyacrylamide gel electrophoresis. Fifteen oocytes were extracted in 20 ~1 of a solution of 10 mM Tris-HCl, 2.5 mM EDTA, 15% sucrose, pH 7.8. The homogenate was centrifuged 10 min at 30,000 rpm at 4°C. The supernatant (20 ~1) was electrophoresed for 2 hr at 8 mA/gel by the procedure of Laemmli (1970) on a polyacrylamide gel (0.8 x 12 cm) prepared from 12% acrylamide and 0.32% Bis. Included with the sample were the fluorescamine-labeled marker proteins (Handschin and Ritschard, 1976), E. coli aspartate transcarbamylase catalytic subunit and regulatory subunit, as well as hog brain microtubule protein, at 33, 17, and 55 x lo3 Daltons, respectively, as indicated

Mat-

As shown in Table 1, the absolute amount and specific activity of orthophosphate and the four nucleoside triphosphates varied by less than 13% during maturation. These small changes of precursor pools cannot account for the 180% increase in phosphoprotein radioactivity during maturation, and, therefore, the increase must be due to an increased amount of incorporated phosphate. Our values for the total quantity of nucleoside triphosphates per oocyte (3.6-3.9 nrnole/oocyte) are somewhat higher than those obtained by Woodland and Pestell (1972) without the use of recovery markers and are in good agreement with those reported by Colman and Gadian (1976) from 31P nuclear-magnetic resonance studies. In Table 1 is reported the distribution of above the graph. Their movement during and after electrophoresis was visualized with black light. The gel was fixed for 10 hr in 15% methanol-lo% acetic acid and for 10 hr in 25% isopropanol to remove SDS and soluble radioactivity and was then stained with Stains-All to visualize phosphoproteins (Cutting and Roth, 1973). A strong blue band indicative of phosphoprotein appeared in the position at 17 to 25 x lo3 Daltons. The gel was frozen and sliced into 4mm pieces. Each piece was heated for 20 min at 100°C in 2 ml of 0.5 N NaOH. The supernatant was analyzed for phosphate and radioactivity (Sanui, 1974). Values on the graph indicate butyl acetateextractable phosphate and radioactivity liberated from phosphoprotein.

MALLER,

Protein

WV, AND GERHART

TABLE NUCLEOSIDE

TRIPHOSPHATES

Absolute amount (nmole/oocyte)

Pi

ATP

UTP

CTP

GTP

Other Total

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 2 4 1 2

-

-

3.lC 2.5’

2.1’ 2.4e 1.6” 1.4 0.63 0.30 -

l.Ob LO* l.Ob

-

1.86 1.7”

-

1.1 1.3

-

0.47 0.65

-

0.22 0.29

-

(2.0)’ 1.8 1.9 1.7 1.9 2.0 1.6 1.5 -

6.7 6.4

IN XENOPUS OOCYTEP

Exchangeable residues (nmole/oocyte)

Relative specific radioactivity* Nonmatured

-

-

6.3

-

303

in Oocytes

1

AND ORTHOPHOSPHATE

Matured

Nonmatured

Phosphorylation

Matured

1.1 1.0 (2.0)? 1.8 2.0 1 1.6 -

1

2.0 1.4 -

1

Distribution of radioactivity (S total 32P in each compound) Expected

2.7

28

3.2

33

2.2

1.1

23

11

0.40 9.6

4 -

Nonmatured

Matured

-

-

33 35 37 36 17 15 7.2 7.2 5.1 4.7 0.Y 2.0 -

35 38 15 7.0

5.3 0.4 -

* Oocytes were extracted with PCA and were analyzed as described in Materials and Methods. Each experiment involved oocytes of a single frog. Absolute amounts were determined from an extract of 50 nonradioactive oocytes to which was added a radioactive recovery marker for Pi or ATP. The recovery of the other nucleotides was assumed to be equal to that of ATP. Relative specific activity was calculated from the specific activity data of the four nucleotides normalized to the specific activity found for orthophosphate in nonmatured oocytes. For each determination of specific activity, 50 oocytes were allowed to equilibrate injected radioactivity for 6 hr prior to extraction. Exchangeable residues comprise the amount of phosphate residues equilibrating with injected [32P1phosphate within 6 hr in each compound, expressed as nanomoles per oocyte. The values are averages calculated by multiplying the average absolute amount of each compound by its average relative specific activity. The total of the exchangeable residues indicates the total phosphate residues of acid-soluble metabolites able to exchange with 13*Plphosphate, in nanomoles per oocyte. The distribution of radioactivity was determined on an extract of five oocytes equilibrated with injected [32P]phosphate for 60 min. The two-dimensional PEI thin-layer chromatogram was autoradiographed for 4 days to locate all compounds with greater than 0.2% the applied radioactivity. The expected distribution was calculated as the percentage of total exchangeable residues in each compound. b Measured specific activities for phosphate: 9.2, 3.1, and 9.9 dpmlpmole for Expts 1, 3, and 4, respectively. ’ Values calculated from recoveries measured for each sample: approximately 40% recovery of the input marker lS2Plphosphate in the butyl acetate phase. At least 50% of the phosphate measured in this fraction results from the acid and molybdate catalyzed breakdown of creatine phosphate (Lee, Wu, and Gerhart, unpublished results). ’ Values calculated from recoveries measured for each sample: approximately 50% recovery of the input marker [‘QATP or [SZP]ATP in the MgSO, eluate from the chromatogram. ’ In this single determination, only ATP specific activity was measured in nonmatured and matured oocytes. The specific activity of the nonmatured sample was set to 2.0 for normalization purposes. The actual specific activity of this sample was 0.47 dpm/pmole. ’ This radioactive material remained largely at the origin.

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VOLUME 58, 1977

radioactivity among acid-soluble com[32Plphosphate in oocytes, these sites (0.27 pounds 60 min after [32P]phosphate injecnmole) are still a very small percentage of tion. This distribution was obtained the total phosphorylated sites in the oowithin 8 min after injection and was cyte. largely complete even at 1 min. Thus, As discussed earlier (Fig. 4A), at least equilibration of injected [32Plphosphate 85% of the radioactive phosphoprotein of with nucleoside triphosphates occurred matured oocytes is located in the nonyolky rapidly as compared to the period of time supernatant obtained when extracts are over which incorporation of 32P into phos- centrifuged at 104g. This supernatant fracphoprotein was measured. There was no tion was found to contain approximately experimental advantage achieved by in40 pg of proteinloocyte. Therefore, 0.23 jecting [y-32PlATP into oocytes as a specific nmole of incorporated phosphate (85% of donor of phosphoprotein radioactivity; it 0.27 nmole) is contained in 40 pug of prowas found that the y-phosphate equilitein, or 1 phosphate incorporated/l.7 x lo5 brated rapidly with the other nucleotides Daltons of nonyolk protein. and with orthosphosphate, so that, within Labeling and Turnover of 2 min after injection, the distribution was Long-Term Phosphoproteins identical to that of injected [32Plphosphate. From the data of Figs. 1 and 2, it is not These results will be presented in detail clear whether the level of 32P-labeled phoselsewhere (Lee, Wu and Gerhart, manuphoprotein in maturing oocytes is ever script in preparation). produced by nonmaturing oocytes, or As presented in Table 1, our measured whether the nonmaturing oocytes are value for the absolute amount and the relative specific activity of each nucleoside merely approaching that same level more triphosphate and of orthophosphate can be slowly. This is a question as to whether involves changes in rates or multiplied together, and the products can maturation be summed to calculate the total amount levels of phosphoprotein formation or changes of both. As shown in Fig. 5, ooof phosphate residues with which injected [32P]phosphate or [y-32PlATP rapidly ex- cytes were allowed to incubate in the abchanges. This amount will be essential for sence of progesterone for a 24-hr period by which time their phosphorylation level calculations of the phosphate incorporated had gradually approached 1.4% of the total into phosphoprotein during the labeling 0.13 radioactivity, i.e., approximately experiments. As shown in Table 1, there nmole of incorporated phosphate (curve 1). are approximately 9.6 nmole of exchangeable phosphate residues per oocyte. These After 17 hr, oocytes were removed and residues contain almost 100% of the in- treated with progesterone to mature. The phosphorylation burst occurred to the exjected 32P, since 4% or less of the total radioactivity enters acid-insoluble mate- tent of a further 1.8% increase, i.e., 0.17 rial. Therefore, when 2.8% of the total ra- nmole of incorporated phosphate (curve 3). dioactivity and total exchangeable phos- This increase was identical to the 1.8% increase observed 17 hr earlier when fresh phate becomes incorporated into phosphooocytes from the same female were treated protein after maturation (Fig. 2), approxiwith progesterone (curve 2). Thus, there mately 0.27 nmole of phosphate would have been incorporated. As described ear- appears to be a class of phosphoprotein lier (Fig. 4), there are very large pools of sites (0.17 nmole in size) unique to maturphosphoprotein in the yolk platelets and ing oocytes. This result is consistent with cytosol, amounting to approximately 120 either the appearance of new phosphoprotein species during maturation or with nmole of protein-bound phosphate/oocyte. quantitative changes in phosphoproteins Thus, while many sites are labeled with

MALLER, WV, AND GERHART

‘0 t” K h?(Shours 2o 3o 5 befofeG”BD3%oftel GVBD I. 0 0) ,., FIG. 5. Long-term incorporation of [32Plphosphate into phosphoproteins. Oocytes were prepared as described in Fig. 1. A fraction (N = 28) of the population was exposed to progesterone immediately (0; curve 2); another fraction (N = 32) was exposed 20 hr later (0, curve 3); and the remainder (N = 48) was unexposed (0, curve 1). In a parallel experiment, nonradioactive oocytes (N = 52) were incubated in progesterone until after GVBD and then were injected with [32Plphosphate (A, curve 4). Four oocytes were taken for each sample point.

present before maturation. In the same experiment, it was possible to examine the basis for the plateau in the amount of radioactive phosphoprotein reached after GVBD in progesteronetreated oocytes (phase 4 of Fig. 1). The plateau could represent either a steady state or an exhaustion of substrates or enzymes. To distinguish between these possibilities, nonradioactive oocytes were allowed to achieve GVBD and were then injected with [32Plphosphate. As shown in Fig. 5 (curve 41, [32Plphosphate enters phosphoprotein rapidly and extensively with kinetics similar to those of oocytes containing radioactivity during their phosphorylation burst. This result clearly shows that the phase 4 plateau is a steady state, with active phosphatases and kinases engaged in phosphoprotein turnover. In a comparable experiment (data not shown), nonradioactive oocytes have been allowed to incubate in MRS without progesterone for 8 or 20 hr to become equivalent to phase 2 oocytes, as defined in Fig. 1. Then these oocytes were injected with

Protein

Phosphorylation

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305

[32P]phosphate and were found to form radioactive phosphoprotein with kinetics equivalent to those shown in Fig. 1 for phase 1 and 2. Thus, we conclude the plateau of phase 2 (approximately 0.14 nmole of incorporated phosphate) represents a steady state, with phosphoprotein engaging in rapid turnover of the phosphate groups. The phosphorylation burst at maturation can be interpreted as a switching by the oocyte from a steady state of one level to that of a higher level. Phosphorylation Oocytes

of Proteins in Enucleated

Since MPF production and maturation of the oocyte surface are stimulated by progesterone in the absence of the nucleus (Smith and Ecker, 1969; Masui and Markert, 1971; Schorderet-Slatkine and Drury, 19731, it was desirable to examine the effects of enucleation on protein phosphorylation. Oocytes were dissected from the ovary, injected with 13”P]phosphate, and, after 30 min, punctured gently with a tungsten needle in the animal pole, through which the germinal vesicle was extruded by light pressure exerted on the cell. Only oocytes were used from which the nucleus was removed intact. Figure 6 shows that these enucleated progesteronetreated oocytes also underwent a burst of phosphorylation, reaching the same level of incorporation as nucleated controls. This result indicates that the maturationdependent protein phosphorylation utilizes cytoplasmic enzymes and substrates. In a separate experiment with radioactive nonmatured nucleated oocytes, the germinal vesicle was found to contain 30% of the ““P-labeled phosphoprotein of the oocyte after 6 hr of labeling. (The germinal vesicle was dissected from TCA-fixed oocytes and was processed for phosphoprotein.) To reconcile this result with the observation of a complete phosphorylation burst in enucleated oocytes, we can cite evidence that cytoplasmic proteins move to the nucleus (Merriam, 1969; Feldherr,

306

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1975). Furthermore, the phosphoproteins formed during the progesterone-induced burst appear not to be located in the nucleus, since radioactive oocytes taken in the burst period but before GVBD reveal, on dissection, an increase of radioactivity in the cytoplasm but not in the nucleus (data not shown). It is also apparent from Fig. 6 that enucleated oocytes exposed to progesterone undergo the phosphorylation burst earlier than nucleated oocytes, and that, even in the absence of progesterone, enucleated oocytes undergo at least a partial burst. The significance of this phosphorylation in enucleated control oocytes is not clear at present. Such oocytes undergo the viscosity decrease characteristic of maturation (Merriam, 1971b). Whether other events of maturation occur is presently under investigation. Protein plasmic

Phosphorylation Transfers

following

Cyto-

One of the most interesting features of the oocyte maturation system is the production of the cytoplasmic maturation-pro-

FIG. 6. Protein phosphorylation by enucleated oocytes. Oocytes were injected with approximately 0.05 +i of YPlphosphate and, after 30 min, were enucleated by squeezing the germinal vesicle through a small puncture at the animal pole. After 30-90 min of healing in MRS, the oocytes were (A) or were not (A) exposed to progesterone. Nucleated oocytes were sampled in parallel after progesterone exposure (0) or nonexposure (0). Three oocytes were taken for each sample point and were analyzed for phosphoprotein as described in Materials and Methods.

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58, 1977

moting factor (MPF) and its assay by microinjection. To determine what effects MPF had on protein phosphorylation, oocytes were injected with [32P]phosphate and were incubated in the absence of progesterone until protein phosphorylation had entered phase 2. Volumes of cytoplasm were withdrawn from mature nonradioactive oocytes and were injected into these radioactive recipients, which were sampled at intervals for the extent of protein phosphorylation. As shown in Fig. 7, protein phosphorylation accelerated immediately after injection of donor cytoplasm in volumes of 17 or 70 nl, approximately 1.5 and 6.0% of the oocyte volume, respectively. The rapid increase in 32P-labeled phosphoprotein proceeded with a first-order reaction half-time of approximately 20 min, in good agreement with the rate assumed for maturing oocytes in an asynchronous population responding to progesterone (Fig. 2). As shown in Fig. 7, progesterone-exposed oocytes only began protein phosphorylation after a lOO-min delay. In a similar fashion, MPF-injected oocytes underwent GVBD within loo-140 min after injection, while progesterone-induced GVBD required 200-260 min, an effect previously reported by Reynhout and Smith (1974). Thus, both GVBD and protein phosphorylation appear to be MPFstimulated reactions. In the same experiment, radioactive oocytes were incubated for 30 min in MRS containing 100 kg of cycloheximide/ml and were then injected with either 17 or 70 nl of MPF. Although protein synthesis was inhibited by 95% in terms of 13Hllysine incorprotein phosphorylation inporation, creased immediately and followed the same kinetics, as shown in Fig. 7. This result indicates that the phosphorylation burst does not depend on newly synthesized protein and occurs after the cycloheximide-sensitive steps. Furthermore, the results indicate that the phase 4 plateau level of 32P-labeled phosphoprotein is approached in the presence of cycloheximide

MALLER, Wu, AND GERHART

Protein

FIG. 7. 32P-Labeled phosphoprotein formation in oocytes receiving an injection of cytoplasm from matured oocytes (MPF). Oocytes were injected with approximately 0.05 &i of [32P]phosphate, and, at the times indicated, some were (0) or were not (0) exposed to progesterone, and others were each injected with 17 (0 and +) or 70 nl (0 and n ) of cytoplasm from a matured oocyte (GVBD at least 1 hr before use as a donor). Three oocytes were taken for each time point. Note that progesterone-treated oocytes show a delay of 100 min before their major increase of 32P-labeled phosphoprotein, whereas the cytoplasmic transfer recipients increase their 32Plabeled phosphoprotein without delays. The symbols + and n indicate recipients which had been injected with 70 ng of cycloheximide 30 min prior to their cytoplasm injection and, thereafter, were incubated in MRS containing 100 pg of cycloheximide/ml.

and, therefore, that turnover of the phosphate groups is cycloheximide-insensitive. Attempts to Dissociate Protein Phosphorylation and Germinal Vesicle Breakdown In experiments described thus far, maturation induced by progesterone or by MPF was always accompanied by phosphorylation. The following four conditions were tested to determine if protein phosphorylation could be experimentally separated from maturation. First, a frog was induced to lay eggs by injection of 500 unit of human chorionic gonadotropin (Antuitrin S), and, 48 hr later (24 hr after egg laying ceased), the ovary was removed surgically. Large oocytes (1.2-1.3 mm in diameter) were dissected manually from their follicles, injected with [32P]phosphate, and exposed to progesterone. Mat-

Phosphorylation

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307

uration was very rapid, with 50% of the oocytes completing GVBD within 100 min after progesterone exposure. The burst of protein phosphorylation occurred shortly before GVBD (data not shown) and involved an increase of 0.17 nmole of incorporated phosphate, similar to the data of Figs. 1 and 2. Thus, gonadotropin stimulation of the frog did not by itself stimulate phosphorylation in nonmaturing oocytes, in contrast to the effect of gonadotropins on protein synthesis and thymidine kinase activity (Woodland, 1969; Adamson and Woodland, 1977). In a second experimental approach, large oocytes from an unstimulated frog were injected with [32Plphosphate and were exposed in groups to increasing concentrations of progesterone, from 0.005 to 1 @g/ml in order to find a range where less than 100% of the oocytes would mature. At 0.02 pg/ml, maturation occurred in 50% of the oocytes within 8 hr. Oocytes which had matured showed a high level of proteinbound [32P]phosphate (0.29 nmole of phosphate incorporated), whereas the remaining oocytes showed the basal level of incorporation (0.11 nmole). Thus, exposure to progesterone stimulated incorporation only when maturation resulted. In a third type of experiment, radioactive oocytes were exposed simultaneously to progesterone and cycloheximide (100 pg/ml), and GVBD failed to occur in accordance with previous reports (Wasserman and Masui, 1975; Drury and Schorderet-Slatkine, 1975). Under these conditions, 32P-labeled phosphoprotein formation was not stimulated, either. However, as mentioned earlier, when cycloheximide-blocked radioactive oocytes were injected with MPF (17 or 70 nl), the phosphorylation burst began immediately, and the oocytes underwent GVBD. Thus, the phosphorylation event occurs after the cycloheximide block, as does GVBD. As a fourth test, small oocytes (0.7-0.9 mm in diameter) were injected with 132Plphosphate and were exposed continuously to 1 pg/ml of progesterone. Such

308 oocytes failed not display These results of GVBD and rylation.

DEVELOPMENTAL BIOLOGY

to undergo GVBD and did the phosphorylation burst. reveal the close association the burst of protein phosphoDISCUSSION

Our data demonstrate that, in progesterone-treated oocytes, the amount of [32Plphosphate in phosphoproteins increases approximately 2.5fold at a time shortly before GVBD. The radioactive phosphoproteins of the oocyte are not associated with yolk platelets nor with the predominant soluble phosphoprotein extracted in yolk-free supernatants from oocytes. The level of incorporation of [32P]phosphate represents approximately 0.28 nmole of incorporated phosphate after maturation, equivalent to one phosphate incorporated per 1.7 x lo5 Daltons of soluble protein (see calculations in Results). There are other metabolic changes known to occur in the period shortly before GVBD, and the question arises as to which of these have a cause or effect relationship to the protein phosphorylation burst. In Rana oocytes, the rate of protein synthesis increases many fold (Ecker and Smith, 1968; Smith and Ecker 1969); in Xenopus oocytes, protein synthesis increases only slightly although histone synthesis accelerates greatly (Adamson and Woodland, 1977; Pennequin et al., 1975); thymidine conversion to dTTP increases at least lofold (Woodland, 19691; and oxygen consumption goes up 50% (Brachet et al., 1975). However, protein synthesis and thymidine conversion are also extensively stimulated in nonmaturing oocytes taken from HCG-injected frogs (Woodland, 1969; Adamson and Woodland, 1977)) indicating their dissociability from maturation events such as GVBD. Even the maturation events of microvillus retraction and preparation of the cortex for activation may be separable from GVBD, as shown in starfish oocytes (Schuetz, 1975). In contrast, the phosphorylation burst still oc-

VOLUME 58, 1977

curs in oocytes taken from HCG-treated frogs and matured in vitro with progesterone, indicating its close association with GVBD. This association is further documented by the absence of the burst in oocytes failing to mature due to cycloheximide blockage, immaturity, or insufficient progesterone. One other metabolic change invariably associated with maturation is MPF formation, and it is noteworthy that both the phosphorylation burst and MPF formation occur shortly before GVBD, and both may occur in cycloheximide-treated oocytes injected with small amounts of MPF (Wasserman and Masui, 1975; Figs. 3 and 71, i.e., after the cycloheximide-sensitive steps of maturation. In addition, neither MPF formation nor protein phosphorylation requires nuclear participation, as demonstrated by their occurrence in enucleated oocytes (Fig. 5; Masui and Markert, 1971; Maller, 1974; Reynhout and Smith, 1974). These results suggest that protein phosphorylation is an essential step in the formation of MPF and its action in effecting GVBD and other metabolic changes during maturation. However, we cannot exclude at present that GVBD and the protein phosphorylation burst are separate responses to progesterone or MPF, initiated independently and in parallel. In discussing the phosphorylation reactions, it is simplest to begin with nonmaturing oocytes, which also incorporate [32P]phosphate into phosphoproteins, though to a lesser extent: approximately 1.0% after 4 hr (when most maturation experiments are done) and 1.4% of the total intracellular radioactivity after 24 hr when a plateau value is reached (phases 1 and 2 of Fig. 1). Since the oocyte contains approximately 9.6 nmole of rapidly equilibrating phosphate groups in nucleotides (in the p and y positions) and in free phosphate, we estimate that the phase 2 plateau incorporation value is equivalent to approximately 0.13 nmole of protein-bound phosphate. Since this plateau value is approached with the same kinetics whether

MALLER, Wu, AND GERHART

the oocytes are injected with radioactivity immediately after removal from the ovary, or after a delay of a day, the plateau must represent a steady state maintained by turnover of phosphoproteins. The turnover probably involves only the phosphate groups of the phosphoproteins since cycloheximide does not perturb the kinetics, indicating that newly synthesized protein is not required for phosphoprotein formation. Thus, the steady state of phase 2 is probably due to the balanced activity of protein kinases and phosphoprotein phosphatases. The phosphorylation reactions must involve cytoplasmically localized enzymes and substrates since [32P]phosphate incorporation proceeds in enucleated oocytes. There appear to be at least two turnover reactions distinguished by the kinetics of phases 1 and 2 (see legend of Fig. 1): one reaction incorporating approximately 0.05 nmole of phosphateloocyte and occurring rapidly (tliz of 15-20 min for the approach to its steady state) and a second reaction incorporating 0.08 nmole of phosphate slowly (tliz of 400-500 min to approach its steady state). In the presence of progesterone, a third phosphorylation reaction begins before GVBD (phase 3 of Fig. 1). Of the three reactions, this one leads to the greatest incorporation: approximately 0.17 nmole/ oocyte. As outlined in the legend of Fig. 2, the reaction probably proceeds rapidly ( tliz of 15-20 min for the approach to its steady state) and may begin as much as 67 min prior to GVBD, although accurate kinetic analysis is precluded by the asynchronous response of the oocytes to progesterone. This reaction also involves rapid turnover of phosphate groups on protein, since it is not perturbed by cycloheximide, and occurs with cytoplasmic enzymes and substrates, as demonstrated in enucleated oocytes. Since this last reaction has been inseparable from GVBD, it would be informative to identify what aspect of phosphoprotein metabolism changes to initiate the increase of [“*PIphosphate incorporation.

Protein

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309

in Oocytes

For the purpose of discussion, it will be assumed that, just before the incorporation accelerates, the oocyte is in phase 1 or 2 and turns over phosphoprotein by a single reaction, rather than by two or more reactions. Then the partial reactions for 32P-labeled phosphoprotein formation in phase 1 and 2 can be written as follows: ATP*

+ P

kin %PP* + ADP and PP* + H,O

k

Out ,P + pi*.

The equation for the rate of change of amount of radioactive phosphoprotein in the oocyte will be: dPP* = ki,(ATP*)(P) - k,,,(PP*), dt PP* = PP ss*(l - e-“‘)

or

in the integrated form, where PP* is the amount of radioactive phosphoprotein per oocyte; PP,*, the amount at steady state of phase 2; P, the amount of protein substrate for phosphorylation; ATP*, the amount of radioactive ATP; Pi*, the radioactive orthophosphate; ki,, the rate constant of transfer of phosphate from ATP* to protein P; kout, the rate constant for dephosphorylation of PP*; and A, the apparent rate constant (ki,[ATP*] + k,,,,) for the approach to steady state. These rate constants are measures of protein kinase activity (kin) and phosphoprotein phosphatase activity (k,,,). If the amount of PP* is to increase rapidly in phase 3 incorporation shortly before GVBD, one or several terms of the equation must change to establish the increase. We can eliminate ATP” as a possibility since the amount and specific activity of ATP do not change during maturation. Unfortunately, kin, k,,,, and P cannot be evaluated from the kinetic measurements, although we can divide their possible changes into two categories: the first, in which enzyme activities change (i.e., the rate constants) while the pool of exchangeable phosphoproteins (i.e., P + PP*) re-

310

DEVELOPMENTAL BIOLOGY

mains constant during maturation; and second, its opposite in which the pool of changes exchangeable phosphoproteins while enzyme activities remain constant. In the first category, the increase in radioactive phosphoprotein would be caused by an increase of the activity of one or more protein kinases (i.e., increase of kin) or a decrease in the activity of one or more phosphoprotein phosphatases (i.e., decrease of K,,,). Since the phosphorylation burst occurs in cycloheximide-treated oocytes injected with small volumes of MPF, the change of enzyme activity would not involve newly synthesized enzymes? but rather modification of pre-existing ones. The kinetic analysis does not distinguish changes of ki, or K,,,. However, it may be noted that Wiblet (1974) has reported a new kinase activity after maturation. In the second category, the phosphoprotein pool (P + PP*) would increase its size before GVBD, whereas the kinases and phosphatases would be unchanged. The newly available protein substrates might be new kinds of proteins or increased amounts of the kinds previously available. The cycloheximide results suggest newly synthesized proteins are not involved, leaving unmasking as the main possibility . Since we have no measure of the total exchangeable pool (P + PP*) but only of PP*, we cannot distinguish changes of enzyme activity from changes of substrate level. Furthermore, these two possibilities have been treated independently for discussion purposes, while it is plausible that the pool as well as the rate constants may change simultaneously. This would be the case if a new kinase were to appear with a specificity for proteins not previously phosphorylated, or if a new phosphatase were to appear and remove nonradioactive phosphate from a phosphoprotein not previously involved in turnover, thereby exposing it to [32P]phosphate incorporation. However, gel electrophoresis thus far reveals few, if any, major new phosphopro-

VOLUME 56, 1977

teins in the molecular weight range of lo150 x 103. These results may indicate that quantitative rather than qualitative changes of phosphoproteins account for the incorporation burst prior to GVBD. In a continued examination of this point, we are comparing proteins of still higher molecular weight for possible changes of phosphorylation at maturation. It is anticipated that characterization of the phosphorylation reactions at maturation will provide insight into the formation of MPF and into its mechanism of action in promoting GVBD. To this end, we are using model protein substrates in microinjection assays in vivo to monitor changes of kinase or phosphatase activity, and we are examining these activities in MPF preparations. We thank Dr. Peter Condliffe of the NIAMDD Rat Pituitary Distribution Program for a generous gift of PMSG, and John Compton and Larry Honig for advice concerning the analysis of kinetic data. This research was supported by United States Public Health Service Grant No. GM19363. J. Maller was an NIH predoctoral trainee (GM 01369) during the tenure of this work. REFERENCES ADAMSON, E. D., and WOODLAND, H. R. (1977). Changes in the rate of histone synthesis during oocyte maturation and very early development of Xenopus laevis. Develop. Biol., in press. ALLERTON, S. E., and PERLMANN, G. E. (19651. Chemical characterization of the phosphoprotein phosvitin. J. Biol. Chem. 240, 3892-3898. BALTUS, E., BRACHET, J., HANOCQ-QUERTIER, J., and HUBERT, E. (1973). Cytochemical and biochemical studies on progesterone-induced maturation in amphibian oocytes. I. Ribonucleic acid and protein synthesis (effects of inhibitors and of a “maturation promoting factor”). Differentiation 1, 127-143. BERGINK, E. W., and WALLACE, R. A. (1974). Precursor-product relationship between amphibian vitellogenin and the yolk proteins, lipovitellin and phosvitin. J. Biol. Chem. 249, 2897-2903. BRACHET, J., PAYS-DE SCHUTTER, A., and HUBERT, E. (1975). Studies on maturation inxenopus laevis oocytes. III. Energy production and requirements for protein synthesis. Differentiation 3, 3-14. BYLUND, D. B., and HUANG, T. S. (19761. Decomposition of phosphoserine and phosphothreonine

MALLER, Wu, AND GERHART during acid hydrolysis. Anal. Biochem. 73, 477485. COLMAN, A., and GADIAN, D. G. (1976). 3’P-Nuclearmagnetic-resonance studies on the developing embryos ofxenopus laeuis. Eur. J. Biochem. 61,387396. CUTTING, J. A., and ROTH, T. F. (1973). Staining of phosphoproteins on acrylamide gel electropherograms. Anal. Biochem. 54, 386-394. DELANGE, R., KEMP, R., RILEY, W., COOPER, R., and KREBS, E. (1968). Activation of skeletal muscle phosphorylase kinase by adenosine triphosphate and adenosine 3’5’-monophosphate. J. Biol. Chem. 243, 2200-2208. DETTLAFF, T. A., NIKITINA, LA.A., and STROEVA, 0. G. (1964). The role of the germinal vesicle in oocyte maturation in anurans as revealed by the removal and transplantation of nuclei. J. Embryol. Exp. Morphol. 12, 851-873. DRURY, D. C., and SCHORDERET-SLATKINE, S. (1975). Effects of cycloheximide on the “autocatalytic” nature of the maturation-promoting factor (MPF) in oocytes of Xenopus laeuis. Cell 4, 269-274. ECKER, R. E., and SMITH, L. D. (1968). Protein synthesis in amphibian oocytes and early embryos. Develop. Biol. 18, 232-249. FELDHERR, C. M. (1975). The uptake of endogenous proteins by oocyte nuclei. Exp. Cell Res. 93, 411419. GURLEY, L. R., WALTERS, R. A., and TOBEY, R. A. (1975). Sequential phosphorylation of histone subfractions in the Chinese hamster cell cycle. J. Biol. Chem. 250, 3936-3944. HANDSCHIN, U. E., and RITSCHARD, W. J. (1976). Spectrophotometric determination of fluorophor, protein, and fluorophor/protein ratios in fluorescamine and MDPF fluorescent antibody conjugates. Anal. Biochem. 71, 143-155. HAYASHI, M. N., and HAYASHI, M. (1972). Isolation of 0X174 specific messenger ribonucleic acids in uiuo and identification of their 5’ terminal nucleotides. J. Viral. 9, 207-215. JARED, D. W., DUMONT, J. N., and WALLACE, R. A. (1973). Distribution of incorporated and synthesized protein among cell fractions of Xenopus oocytes. Develop. Biol. 35, 19-28. KREBS, E. G. (1972). Protein kinases. In “Current Topics in Cellular Regulation” (B. L. Horecker and E. R. Stadtman, eds.), Vol. 5, pp. 99-134. Academic Press, New York. KREBS, E. G., and FISCHER, E. H. (1962). Phosphorylase b kinase from rabbit skeletal muscle. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 5, pp. 373-376. Academic Press, New York. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and

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RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265 275. MALLER, J. (1974). Studies on the mechanism of cytoplasmic control of meiotic maturation of Xenopus oocytes. Ph.D. Thesis, University of California, Berkeley, Calif. MALLER, J., POCCIA, D., NISHIOKA, D., KIDD, P., GERHART, J., and HARTMAN, H. (1976). Spindle formation and cleavage in Xenopus eggs injected with centriole-containing fractions from sperm. Exp. Cell Res. 99, 285-294. MASUI, Y., and MARKERT, C. L. (1971). Cytoplasmic control of nuclear behavior during meiotic maturation offrog oocytes. J. Exp. 2001. 177,129-146. MERRIAM, R. W. (1969). Movement of cytoplasmic proteins into nuclei induced to enlarge and initiate DNA or RNA synthesis. J. Cell Sci. 5,333-349. MERRIAM, R. W. (1971a). Progesterone-induced maturational events in oocytes of Xenopus laeuis. I. Continuous necessity for diffusable calcium and magnesium. Exp. Cell Res. 68, 75-80. MERRIAM, R. W. (1971b). Progesterone-induced maturational events in oocytes of Xenopus Zaeuis. II. Changes in intracellular calcium and magnesium distribution at germinal vesicle breakdown. Exp. Cell Res. 68, 81-87. MERRIAM, R. W. (1972). On the mechanism of action in gonadotropic stimulation of oocyte maturation in Xenopus laevis. J. Exp. Zool. 180, 421-426. MORRILL, G. A., and MURPHY, J. B. (1972). Role for protein phosphorylation in meiosis and in early cleavage phase of amphibian embryonic development. Nature (London) 238, 282-284. PENNEQUIN, P., SCHORDERET-SLATKINE, S., DRURY, K. C., and BAULIEU, E-E. (1975). Decreased uptake of 13Hl leucine during progesterone-induced maturation of Xenopus laeuis oocytes. FEBS Lett. 51, 156-160. RANDERATH, E., and RANDERATH, K. (1964). Resolution of complex nucleotide mixtures by two-dimensional anion-exchange thin-layer chromatography. J. Chromatogr. 16, 126-129. RANGEL-ALDAO, R., and ROSEN, 0. M. (1976). Dissociation and reassociation of phosphorylated and non-phosphorylated forms of the adenosine 3’5’monophosphate-dependent protein kinase from bovine cardiac muscle. J. Biol. Chem. 251, 33753380. REYNHOUT, J. K., and SMITH, L. D. (1974). Studies on the appearance and nature of a maturationinducing factor in the cytoplasm of amphibian oocytes exposed to progesterone. Deuelop. Biol. 38, 394-400. ROSEN, 0. M., and ERLICHMAN, J. (1975). Reversible autophosphorylation of a cyclic 3’-5’ AMP-dependent protein kinase from bovine cardiac muscle. J. Biol. Chem. 250, 7788-7794. RUBIN, S. C., and ROSEN, 0. M. .(1975). Protein

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