Development 106, 1-9 (1989) ]Printed in Great Britain © The Company of Biologists Limited 1989

Preparation and characterization of cell-free protein synthesis systems from

oocytes and eggs of Xenopus laevis

TINA D. PATRICK, CLARE E. LEWER and VIRGINIA M. PAIN

Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK

Summary

During the maturation of the oocytes of the frog Xenopuslaevis, the rate of protein synthesis shows a twofoldincrease. Studies of the mechanisms involved in thisstimulation have been seriously limited by the lack of anactive cell-free translation system. We have now pre-pared such systems from oocytes, progesterone-maturedoocytes and eggs of Xenopus laevis by induction of lysisby centrifugation of whole cells.

The extracts are highly active in incorporation oflabelled amino acids and, in the progesterone-maturedand egg extracts, a substantial proportion of this is dueto reinitiation on endogenous mRNA, as shown by theuse of inhibitors. The increased rate of protein synthesispreviously observed in intact oocytes following pro-gesterone-induced maturation is reflected in the relativeactivities of the extracts. The difference in activity is notdue to the presence of a dominant inhibitor of translation

in the extracts from unstimulated oocytes. Labellingstudies with initiator tRNA ([35S]Met-tRNAf) indicate ahigher concentration of 43S preinitiation complexes inthe extracts from unstimulated oocytes, suggesting animpairment of initiation of translation at or after themRNA-binding step. Extracts from both oocytes andprogesterone-matured oocytes translated endogenousmRNAs to give products ranging over a wide spectrumof molecular weight. However, significant translation ofexogenous (globin) mRNA required the presence ofreticulocyte postribosomal supernatant, suggesting thatone or more factors required for mRNA recruitment islimiting in these extracts.

Key words: Xenopus laevis, translation, maturation, cell-free extracts, initiation of translation, oocytes.

Introduction

Xenopus oocytes are arrested in the cell cycle at thestage of meiotic prophase. The process of maturation istriggered in vivo by the hormone progesterone. Thishormone is secreted by follicle cells surrounding theoocytes in the ovary, which in turn are stimulated byrelease of gonadotropins from the pituitary (reviewedby Wasserman et al. 1986). Maturation can also beinduced in vitro by treating isolated oocytes withprogesterone. The overall process takes several hoursand culminates in meiotic division and dissolution of thenuclear envelope. One of the many events associatedwith maturation is a twofold to threefold increase in theoverall rate of protein synthesis (Wasserman et al.1982), due to recruitment of pre-existing mRNA(Richter et al. 1982). A particular characteristic of theimmature Xenopus oocyte is the large proportion of itsribosomes and mRNA molecules not being utilized forprotein synthesis (Woodland, 1974; Taylor & Smith,1985). Upon maturation the proportion of ribosomesengaged in polysomes only rises from about 1 % to 2 %(Woodland, 1974), indicating that a severe restraint ontranslation exists even in the mature egg. It is widely

thought that part of the mRNA in Xenopus oocytes isheld in a form unavailable for translation, possibly bythe presence of associated inhibitory, or 'masking',proteins (Richter, 1987). However, other authors havesuggested that, in addition, the rate of protein synthesisin the oocyte may be limited by the activity of one ormore other components of the translational apparatus(Laskey et al. 1977; Wasserman et al. 1986; Audet et al.1987).

Investigation of the components limiting proteinsynthesis in Xenopus oocytes and the mechanismspromoting the stimulation of translation during matu-ration has been severely impeded by the lack of suitablecell-free protein-synthesizing systems. In contrast,knowledge of the mechanisms controlling protein syn-thesis during fertilization of sea urchin eggs has ad-vanced very rapidly over the last 2 years following thedevelopment from these cells of cell-free translationsystems whose relative activities reflect the stimulationof protein synthesis that occurs in vivo during fertiliz-ation (Winkler et al. 1985; Lopo & Hershey, 1985;Hansen et al. 1987; Colin et al. 1987; Lopo et al. 1988).This work suggests that the rate of protein synthesis inlysates derived from unfertilized eggs is limited by the

T. D. Patrick, C. E. Lewer and V. M. Pain

activity of certain polypeptide initiation factors and thatactivation of these factors may play a role in thestimulation of translation that occurs in response tofertilization.

This paper describes the preparation and characteriz-ation of an active, cell-free protein-synthesizing systemfrom Xenopus oocytes and eggs. Our starting point wasthe method developed by Lohka and his colleagues forpreparing an extract for use in studying pronuclearformation and chromosome condensation in vitro(Lohka & Masui, 1983; Lohka & Mailer, 1985). In thisprocedure, mature eggs laid in response to the injectionof adult Xenopus females with human chorionic gona-dotropin were lysed simply by the application of cen-trifugal force. We have optimized the conditions used toincubate these extracts for the study of protein syn-thesis, and introduced further modifications to allow usto prepare similar systems from oocytes before andafter progesterone-induced maturation in vitro.

Materials and methods

Preparation of egg extractsExtracts from unfertilized Xenopus laevis eggs were preparedby a modification of the method described by Lohka & Mailer(1985). Mature wild female frogs (Xenopus Ltd, UK) werestimulated to lay eggs by injection of 650 i.u. human chorionicgonadotrophin (Chorulon, Intervet Laboratories) into theirdorsal lymph sacs, 16 hours before the eggs were required.Eggs were collected at room temperature into saline tap water(llOmM-NaCl in tap water) to prevent activation, and dejel-lied in 5mM-dithiothreitol, 20mM-Tris-HCl, pH8-5, in salinetap water. Following the removal of their jelly coats, the eggswere rinsed twice in saline tap water and then twice in ice-coldextraction buffer: 20mM-Hepes-KOH, pH7-5, 125mM-KCl,2mM-MgCl2, 2mM-2-mercaptoethanol, 3/igml"1 leupeptin.The eggs were transferred to 5 ml centrifuge tubes andallowed to sediment under gravity. As much excess buffer aspossible was removed before the eggs were centrifuged at10000g for lOmin at 4°C using an 8x5 ml swing-out rotor inan MSE 18 centrifuge. The procedure resulted in a stratifiedextract containing a large pellet of yolk platelets, a particulatesoluble phase of variable colour and texture and a lipidpellicle. For most of the experiments described, the materialbetween the lipid pellicle and the yolk platelet pellet wasremoved and kept on ice until required. For the experimentsinvestigating the effect of cytochalasin B (Sigma), the col-lected material was treated with the chemical at a finalconcentration of SO^gmP1 and centrifuged again at lOOOOgfor lOmin at 4°C. The supernatant was then removed andkept on ice until required.

Preparation of extracts from oocytes and progesterone-matured oocytesAdult lab-bred female frogs were stimulated by injection of50 i.u. follicle-stimulating hormone (Folligon, Intervet Lab-oratories) into their dorsal lymph sacs, 5 to 7 days in advanceof the experiment. Each animal was then killed and the ovaryremoved into Barth's saline (88-0mM-NaCl, 1-OrtiM-KCl,2-4mM-NaHCO3, 10 mM-Tris-HCl (pH7-6), 0-30mM-Ca(NO3)2.4H2O, 0-41mM-CaCl2 .6H2O, 0-82mM-MgSO4.7H2O). The lobes of the ovary were dissected into clumps of30 to 50 oocytes and washed thoroughly in Barth's saline.Approximately half of the clumps were treated with pro-

gesterone (Sigma) from a stock solution of 1 mg ml * in 50 %ethanol to a final concentration of 2/igml"1 in Barth's salinefor 15min. The clumps of oocytes were then treated with250/igml"1 collagenase (EC 3.4.24.3, Boehringer, Clostri-dium hystolyticum) in 50% high-salt Barth's saline (115 mM-NaCl), 50 % 0-1 M-sodium phosphate buffer as follows. Keep-ing the progesterone-treated oocytes separate from theothers, the clumps of oocytes were transferred to 50 ml conicalcentrifuge tubes and the tubes filled three-quarters full withthe collagenase solution. The tubes were then placed horizon-tally in a shaking water bath at 23 °C and shaken at 120 revsmin~x. The length of time taken to liberate the oocytes variedfrom one ovary to another. Free oocytes were collected up to3 hours after the start of the treatment and rinsed three timesin Barth's saline (or high-salt Barth's saline for progesterone-treated batches to prevent activation after maturation). Allthe oocytes were manually sorted and selected for undamagedstage VI oocytes (as defined by Dumont, 1972), or forappearance of the white spot to indicate maturation. The cellswere then rinsed in extraction buffer and the extracts pre-pared exactly as described for eggs except that, for mostexperiments, human placental ribonuclease inhibitor (RNA-sin, Boehringer) and soy-bean trypsin inhibitor (Sigma) wereadded to the prepared extracts at concentrations of267unitsml~1 and O-Smgml"1, respectively (Lopo et al.1988).

Amino acid incorporation assays with XenopusextractsStandard assays contained 0-6 volumes of extract and 0-4volumes of a reaction mix with the following components atfinal concentrations in the assays: 35 mM-creatine phosphate,250/igml"1 creatine phosphokinase (EC 2.7.3.2), 0-5mM-spermidine, and 40/iCiml"1 L-[35S]methionine (800-1500Ciminor1, Amersham International). Incubations were carriedout at 21 °C. For assay of amino acid incorporation, sampleswere removed from the assay tube and spotted onto 2-1 cmcircles of Whatman No. 1 filter paper. After air drying for 10seconds, the filters were dropped into 10% trichloroaceticacid containing 2 MM unlabelled methionine and processed asdescribed by Pain et al. (1980).

Calculation of the rate of protein synthesis in extractsFor each extract, a series of incorporation assays was carriedout as described above with the addition of unlabelledmethionine at a range of final concentrations between 10 and100 HM. A graph was then constructed in which the concen-tration of cold methionine added was plotted against thereciprocal of incorporation of [35S]methionine into protein.The negative intercept on the ordinate gives the concentrationof methionine contributed by the extract itself to the assay.Knowledge of this value permits the calculation of the specificradioactivity of the precursor pool of [35S]methionine in theassay (assuming 100% purity of the radioactive material),which in turn allows the actual protein synthesis rate to becalculated from the incorporation data. For the purpose ofcalculating protein synthesis rates as pmol of methionineincorporated, the efficiency of estimation of radioactivity of[35S]protein on the filters was taken as 50%.

Estimation of RNA content in extracts20 ji\ samples of extract were assayed by a procedure based onthat of Munro & Fleck (1969). Samples were precipitated onice with 3 ml of 2% (v/v) perchloric acid (HC1O4) andcentrifuged at 3000revsmin"1 for lOmin at 4°C in a SorvallRT 6000 centrifuge. The pellets were washed twice more with2 % HC1O4 and the RNA was then hydrolysed for 60 min with

Xenopus cell-free protein synthesis systems 3

0-3M-NaOH at 37°C. Protein was reprecipitated with 0-8 mlof 20% HC1O4 on ice for 30min. After centrifugation, theprecipitate was retained for protein determination by themethod of Lowry et al. (1951). The supernatants wereremoved and absorbances at 232 and 260 run read in quartzcuvettes in an SP-500 Pye Unicam spectrophotometer againsta blank of 2 ml 0-3M-NaOH and 0-8ml 20% HC1O4. RNAcontent was then calculated from the following equation(Ashford & Pain, 1986): [(3-llxA2<»)-(0-58xA232)]x10-53x2-8x50 = ug RNA per ml extract.

Labelling of 43S preinitiation complexesIncubations (2min) were carried out as described above, butwith [35S]methionine replaced with [35S]Met-tRNAf (ap-proximately 5X106 ctsmin"1 ml"1), prepared as described byClemens et al. (1974). Emetine was added at lmin. Reactionswere terminated by the addition of an equal volume ofgradient buffer (25mM-sodium cacodylate, pH6-6, 80 min-KC1, 2 mM-magnesium acetate) containing 2% (v/v) neutral-ized formaldehyde. The mixtures were layered onto 12mllinear gradients of 20-40 % sucrose in the same buffer andcentrifuged for 16-5 h at 92 000 g in a Beckman SW 40 rotor.The gradients were analysed at 254 nm in an ISCO ModelUA-5 recording spectrophotometer and divided into 20 frac-tions, which were processed as described by Pain et al. (1980).

Preparation of reticulocyte componentsProcedures described by Clemens (1984) were used for thefollowing: preparation and incubation of rabbit reticulocytelysates, preparation and incubation of the messenger-depen-dent lysate system and isolation of total reticulocyte RNA byextraction of untreated lysate with sodium dodecyl sulphate,phenol and chloroform. For preparation of reticulocyte S100,reticulocyte lysate was supplemented with 15/*M-haemin,diluted with 2 volumes of buffer (20 mM-Mops-KOH, pH7-6,0-1 M-KCI, 0-1 mM-EDTA, 1 mM-dithiothreitol, 10 % glycerol)and supplemented with MgCl2 and GTP at a final concen-tration of 2mM each. The mixture was then centrifuged at100 000 g for 4 h at 4°C in a Beckman 50 Ti rotor to generatethe G-100 supernatant.

Analysis of translation products by polyacrylamide gelelectrophoresisProtein synthesis assays (25 jA) were carried out as describedabove except that [ S]methionine was added at a concen-tration of 400^Ciml"1. Incubation was for lh. For analysis,50jul of lOOmM-Tris, pH6-8, was added to each assay, thenthe samples were centrifuged at 11500 g for 10 min in an MSEMicrocentaur microcentrifuge in a cold room. The super-natants were dialysed against lOOmM-Tris, pH6-8 for 4h at4°C. Concentrated electrophoresis sample buffer was thenadded to give the following concentrations: 1 % sodiumdodecyl sulphate, 40mM-Tris-HCl, pH6-8, 5% glycerol,0-002% bromophenol blue. Samples were boiled for 4 min,and 40^1 aliquots were applied to polyacrylamide slab gelscontaining 10% acrylamide/0-1% bisacrylamide/0-1%sodium dodecyl sulphate and subjected to electrophoresis asdescribed by Laemmli (1970) with the modifications of Ander-son et al. (1973). Autoradiographs were prepared by exposureof Amersham Hyperfilm Betamax film to the gels for 6 days.

Results

Preliminary characterization of the cell-free systemfrom eggsThe initial experiments were performed using egg

extracts prepared exactly as described by Lohka &Mailer (1985). This involved treatment of the extractsprepared as described in Materials and methods with50/igml"1 cytochalasin B and centrifuging the mixtureagain at 10 000g for 10 min. However, omission of thetreatment with cytochalasin B and the second centrifu-gation had no effect on the incorporation of [35S]meth-ionine into protein. In all the experiments described inthis paper, the procedure described in Materials andmethods, omitting these treatments, was used.

Fig. 1A shows the time course of incorporation of[35S]methionine into protein by the egg extract. It alsoshows a dramatic inhibition of incorporation resultingfrom a single pass of a loose-fitting pestle in a Douncehomogenizer. Another treatment highly deleterious toprotein synthetic activity was snap-freezing of theextract in liquid nitrogen (Fig. IB). Addition ofglycerol (10% v/v) did not protect the samples fromloss of activity upon freezing (not shown). Freshlyprepared cytoplasmic extracts were therefore used forall the experiments reported in this paper.

To investigate whether initiation of protein synthesison endogenous mRNA was occurring in vitro, incorpor-ation assays were performed in the presence of specificinhibitors of initiation such as edeine (Fig. IB), or 7-methylguanosine 5' triphosphate (7-methyl GTP)(Fig. 2). Fig. IB shows that a significant proportion ofthe incorporation during the incubation is edeine-sensitive, suggesting that initiation of protein synthesisis occurring. To estimate how long the capacity forinitiation is maintained, the inhibitors were added atvarious times after the start of incubation, adjusting theamounts added to ensure the same final concentration ifsamples for time points had been removed beforeinhibitor addition. In these experiments, inhibitorswere added up to 40 min into the assay, and the resultsshown for 7-methyl GTP in Fig. 2 suggest that initiationcontinues at least up until then. Incorporation ceasedapproximately 10 min after the addition of the inhibitor,suggesting that a round of initiation occurs every10 min.

Optimum conditions for incorporation assaysStudies were carried out with Xenopus egg extracts todetermine the optimal conditions for translation.Although there were minor differences between indi-vidual extracts, addition of extra potassium or mag-nesium ions over and above those already contributedby the extract rarely resulted in significant enhancementof the rate of incorporation. Addition of magnesiumand potassium ions by more than 2 HIM and 50 mM,respectively, were invariably inhibitory (results notshown). The effect of addition of spermidine (bufferedto pH7-5) has also been investigated. The.polyaminewas added at various concentrations between 0 and3mM and the optimum found to be approximately0-5 HIM. This resulted in a stimulation of approximately50%.

Addition of an amino acid mixture containing 19amino acids to the system at a final concentration of200 UU each did not affect translation. Addition of

T. D. Patrick, C. E. Lewer and V. M. Pain

20 40Time (min)

Fig. 1. (A) Time course of incorporation of [35S]methionineinto protein using egg extracts prepared normally asdescribed in Materials and methods ( • ) , or with theinclusion of a single pass of a loose-fitting pestle in aDounce homogenizer before centrifugation ( • ) .Incubations were performed as described in Materials andmethods. 10/A samples were removed from the incubationmixture at the times shown. RNA concentrations in bothextracts were measured as described in Materials andmethods and found to be identical. (B) Effects of freezingand the inhibitor edeine on incorporation of[35S]methionine in egg extracts. Each point represents theremoval of 10 /d of the incubation mixture from the assaytube at the time indicated. The extracts were either usedfresh in the assay (#) or frozen before incubation by rapidimmersion in liquid nitrogen. Small aliquots of extract ofless than 200 fi\ were frozen in this manner and then thawedbefore use ( • ) . Edeine was added to the assay tubes beforeincubation at a final concentration of 1 /UM in fresh ( • ) andfrozen ( • ) extracts.

exogenous ATP together with equimolar magnesium tothe Xenopus egg system was found to be inhibitory.This suggests that the presence of 35 mM-creatine phos-

20 40Time (mm)

Fig. 2. The effect of adding the inhibitor 7-methyl GTP toan egg extract at various times after the start of theincorporation assays. Incubations were carried out asdescribed in Materials and methods, with 10 fi\ samplesbeing removed at the times shown. 7-methyl GTP wasadded to a final concentration of 500 ^M to incubation tubesat Omin ( • ) , 20min ( • ) , and 40 min ( • ) . A controlincubation with no inhibitor added is also shown (• ) .

phate and 250 jUg mP1 creatine phosphokinase togetherwith the endogenous ATP pool generates sufficientATP to maintain translation during the incubation.Assays were also carried out to examine the effect ofextract concentration. Optimal activity was obtainedwhen the extract constituted 60 % of the assay volume.

Effect of progesterone-induced maturation on proteinsynthesis in extracts from oocytesFig. 3A shows a time course of incorporation intoprotein in extracts prepared from unstimulated oocytesand those induced to undergo maturation in vitro bytreatment with progesterone. The endogenous concen-trations of unlabelled methionine and of RNA in theextracts were estimated as described in Materials andmethods, and the results are presented as pmol meth-ionine incorporated per mg RNA. This compensatedfor variations between extracts in ribosome concen-tration and in the endogenous methionine pool size.

The results in Fig. 3A show a twofold higher rate ofmethionine incorporation in the extract derived fromprogesterone-matured oocytes. At an early stage in theincubation, at least part of this difference must reflectthe greater amount of 'run-off incorporation thatwould be expected because of the greater polysomecontent of progesterone-matured oocytes (Woodland,1974). However, studies with the initiation inhibitor,edeine, indicate that the system from progesterone-matured oocytes is also more efficient at reinitiation oftranslation on endogenous mRNA than that from the

Xenopus cell-free protein synthesis systems

20 30Time (min)

10 20 30Time (min)

45

Fig. 3. (A) Effect of progesterone maturation ontranslational activity of oocyte extracts. Extractswere made from oocytes (•) and progesterone-matured oocytes (•) and incubations andcalculations at the rate of protein synthesis wereperformed as described in Materials and methods.Edeine was added at a final concentration of 1 )m tothe oocyte system (•) or the progesterone-maturedoocyte system (O). (B) The edeine-sensitiveincorporation by the oocyte (•) and progesterone-matured oocyte (O) extracts.

unstimulated oocytes (Fig. 3A). This is illustrated bythe data in Fig. 3B, showing the edeine-sensitive (i.e.initiation-dependent) incorporation of the two types ofextract. This ability of cell-free systems from progester-one-matured oocytes to sustain edeine-sensitive proteinsynthesis at later stages in the incubation is dependenton the presence of placental ribonuclease inhibitor andsoy-bean trypsin inhibitor to inhibit ribonuclease andprotease activities in the extracts.

In a series of experiments comparing extracts fromoocytes from different frogs we have found that,although progesterone-induced maturation always re-sults in an increase in the protein synthetic activity ofthe extracts, both the rates of protein synthesis ob-tained and the degree of stimulation show considerablevariation. The mean ± S.E.M. of the initial rates ofincorporation in cell-free systems from 6 animals was1-07 ±0-20 and 1-54 ± 0-22 nmol methionine mg"1

RNA h"1 for extracts from oocytes and progesterone-matured oocytes, respectively, a stimulation of 44%(P = 0-003, by paired Mest). The main difference be-tween the approximately 20% of our experiments inwhich a low degree of stimulation is seen and those likethat shown in Fig. 3 is that, in the former, the systemsfrom the unstimulated oocytes tend to show kineticsand edeine sensitivity more similar to those from theprogesterone-matured oocytes. This could be due to asomewhat elevated level of gonadotropins in some ofthe donor frogs.

Approximate comparisons can be made between the

rates of protein synthesis in our oocyte extracts andrelated values in the literature. Assuming that totaloocyte protein contains 2% methionine by weight, theinitial rate of protein synthesis in the oocyte extract isapproximately 7-5 ng protein h~x /xg"1 RNA. This com-pares well with the rate of 4-7 ng protein h"1 ^g"1 RNAdetermined for intact oocytes by Taylor & Smith (1985).In the units of Blow & Laskey (1988), the mean rate ofprotein synthesis in our oocyte extracts is 23 ng pro-tein h"1 ml"1 extract, which is of the same order as thatstated by these workers to occur in similarly derivedextracts from Xenopus eggs.

Labelling of 43S preinitiation complexes with initiatortRNA in extracts from oocytes and progesterone-induced oocytesThe experiments with edeine indicated that the cell-freesystems from progesterone-matured oocytes were moreactive in the reinitiation of translation on endogenousmRNA. To identify more closely the stage in theinitiation pathway that might be regulated by matu-ration, we investigated the labelling of native 40Sribosomal subunits with initiator tRNA ([35S]Met-tRNAf) (Darnborough et al. 1973; Austin et al. 1982).The results are shown in Fig. 4. As previously observedin similar analyses of native ribosomal subunits fromcultured cells (Hirsch et al. 1973), the 40S subunit peakexhibited heterogeneity, presumably due to the exist-ence of multiple species containing different amounts ofassociated protein (Hirsch et al. 1973). In these gradi-

oX

2-

A 1

1\40s

it

\ •'"•

\ ' \

\ ' \ \V ' /\ ' ' /

\ i \

5 10 15Fraction no.

8•0-15

6

4-

2

B\ 40s

\1

1 f\ ' \\ ' ' 1

V ' ' /\ M.V\ ? ^*

5 10 15Fraction no.

0-15

Ec

• 3 -

auocCO

X)

bsor

<

Fig. 4. Labelling of 43S preinitiationcomplexes with [35S]Met-tRNAf in cell-freesystems from oocytes (A) and progesterone-matured oocytes (B). Incubations, densitygradient analysis and processing of fractionswere as described in Materials and methods.Sedimentation was from left to right, and theposition of native, 40S ribosomal subunits(40-43S) is indicated. The major peak risingat the bottom of the gradients is that of 80Smonomeric ribosomes. Absorbance at254 nm, • • [35S] radioactivityprecipitable by cetyltrimethylammoniumbromide.

6 T. D. Patrick, C. E. Lewer and V. M. Pain

ents from Xenopus oocyte extracts, the peak of native60S subunits is not visible, due to its being swamped bythe enormous peak of 80S monomeric ribosomes,which, as previously demonstrated by Woodland (1974)make up more than 95 % of the ribosomal particles inthese cells. Our data on the labelling of the subunitswith initiator tRNA demonstrate a greater number of([35S]Met-tRNAf .40S subunit) initiation complexes inthe extracts from unstimulated oocytes than in thosefrom progesterone-matured oocytes. This suggests thatthe utilization of these complexes in subsequent steps ofinitiation is less efficient in these extracts.

Investigation of possible inhibitory activity of extractsA possible model for explaining the difference inprotein synthetic activity between unstimulated andprogesterone-matured oocytes is that the former maycontain an inhibitor of translation. This has been testedin two experiments: first, the effects of adding oocyteand progesterone-matured oocyte extracts were testedin a reticulocyte lysate translation system. The resultsare shown in Fig. 5A and indicate that neither extractcontains a dominant inhibitor of the reticulocyte lysatesystem. The second experiment involved adding variousamounts of oocyte extract or the extraction buffer to afixed amount of progesterone-matured oocyte extractand measuring the rate of incorporation in each incu-bation. Fig. 5B shows the result of this experiment, andagain suggests that a dominant inhibitor of translation isnot present in the oocyte extract.

Analysis of translation products of endogenous andexogenous mRNA in Xenopus oocyte cell-free systemsTracks 1 and 2 of Fig. 6 show the labelled products oftranslation of endogenous mRNA in the cell-free sys-tems from unstimulated and progesterone-maturedoocytes. It can be seen that both systems translatedproducts encompassing a wide range of molecularweights, similar to that seen in the intact cells. Thissuggests that premature termination is not a majorproblem in these extracts, as it is in some other cell-freetranslation systems (Kerr et al. 1972). Comparison oftracks 1 and 2 indicates some characteristic changes inthe pattern of products resulting from progesterone-induced maturation, but this is outside the scope of thepresent study. More detailed analysis, involving two-dimensional gel electrophoresis, will be required toexamine changes in individual translation products, ashas been carried out with intact cells (Ballantine et al.1979).

The remaining tracks in Fig. 6 show the effect ofadding exogenous mRNA to the extracts. In thesestudies, we used total reticulocyte RNA as a source ofglobin mRNA. Addition of increasing amounts of thisRNA to the oocyte cell-free system (tracks. 3 and 5-8)resulted in very little, if any, production of globin. Thiswas also the case in the extract from progesterone-matured oocytes (track 4), where the picture is slightlycomplicated by the greater translation of an endogen-ous product with similar migration characteristics (seetrack 2). Tracks 9-13, however, show a dramatic

15 30Oocyte extract or buffer added

% final volume

Fig. 5. Investigation of potential inhibitors in oocyte andprogesterone-matured oocyte extracts. All extracts wereprepared as described in Materials and methods. (A) Theeffect of adding 7 [A of oocyte ( • ) or progesterone-maturedoocyte ( • ) extracts to a reticulocyte lysate system at a finalvolume of 50^1. Control reticulocyte lysate containingextraction buffer is also shown (•) . 6/il samples wereremoved from the incubation mix at the times shown.(B) The effect of adding an increasing percentage of eitheroocyte extract (•) or extraction buffer (•) to a fixedamount (60%) of progesterone-matured oocyte extract.Incubations were carried out as described in Materials andmethods at a final volume of 50/il for 30min. Theincubation tubes were then plunged into iced water and10 (A samples removed from each tube and spotted ontofilters in quadruplicate. Filters were then processed andcounted as described in Materials and methods.

potentiating effect on globin mRNA translation ofadding a small quantity of postribosomal supernatant(S-100) prepared from rabbit reticulocyte lysate. Thiswas not due to the presence of globin mRNA in thereticulocyte S-100 itself (track 14). The effect of reticu-locyte S-100 appeared to be specific for the translationof the exogenous mRNA, since overall amino acidincorporation into protein was not affected. Similar

Xenopus cell-free protein synthesis systems

4 2 - • * <

3 1 -

21-

14-

Fig. 6. Translation products fromcell-free systems fromunstimulated and progesterone-matured oocytes. Incubationconditions and processingprocedures were as described inMaterials and methods. Thetracks show the products of thefollowing incubations: endogenoustranslation products of cell-freesystems from unstimulated (track1) and progesterone-matured(track 2) oocytes; track 3: oocyteextract + 1-4 /ig total reticulocyteRNA; track 4: progesterone-matured extract + 1-4 figreticulocyte RNA; tracks 5-8:oocyte extract + 0-35 ng, 0-70 fig,1-05 fig, 1-75 fig reticulocyte RNA;tracks 9-14: oocyte extract + 3 jAreticulocyte S-100 + 0 - 35 fig,0-70 fig, hO5 fig, 1-40 fig, 1-75 figand Ofig reticulocyte RNA; track15: globin standard, prepared bytranslating 1-4 fig reticulocyteRNA in a 12 jA messenger-dependent lysate system anddiluting directly (1:1) intoelectrophoresis sample buffer. 5 fAwas applied to the gel. TotalRNA, S-100 and the messenger-dependent lysate system wasprepared from rabbit reticulocytelysates as described in Materialsand methods.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

results were obtained when reticulocyte S-100 wasadded to extracts from progesterone-matured oocytes(not shown).

Discussion

Cell-free systems for use in studies of protein synthesishave been prepared from many eukaryotic sources,including rabbit reticulocytes (Pelham & Jackson, 1976;Jackson & Hunt, 1983), Ehrlich ascites tumour cells(Henshaw & Panniers, 1983), mouse and rat liver(Eisenstein & Harper, 1984; Morley & Jackson, 1985),L cells (Skup & Millward, 1980) and yeast (Gasior et al.1979). With the single exception of the rabbit reticulo-cyte lysate, most of these extracts synthesize protein ata low rate relative to that in the intact cells and tend toreinitiate on endogenous mRNA inefficiently. Theyhave, however, been used successfully to identify mech-anisms regulating the rate of translation in mammaliancells by a variety of physiological treatments (Pain et al.1980; Austin et al. 1986; Panniers et al. 1985). Animportant prerequisite in such studies is that the rela-tive translational activities of the extracts preparedfrom cells in two different physiological states reflectthe differences in the rate of protein synthesis seen inthe intact cells from which the extracts are derived.

Recently, several laboratories have reported interest-ing results from work with cell-free translation systemsderived from sea urchin eggs and zygotes (Winkler et al.1985; Lopo & Hershey, 1985; Colin et al. 1987; Hansenet al. 1987; Lopo et al. 1988). The use of these extractshas provided much valuable information on the mech-anisms contributing to the 20-fold stimulation of proteinthat occurs in sea urchin eggs during fertilization. First,the roles of individual initiation factors in mediating theresponse to fertilization have been examined by addingexogenous purified factors to the cell-free systems. Inthis way, Winkler et al. (1985) and Colin et al. (1987)showed cell-free systems from unfertilized eggs ofLytechinus pictus to be stimulated by addition of theinitiator-tRNA-binding protein, eIF-2, or the guanine-nucleotide-exchange factor, termed GEF or eIF-2B,that modulates its activity (see review by Pain (1986)).Lopo et al. (1988), using extracts from unfertilized eggsof Strongylocentrotus purpuratus, found that the moststimulatory initiation factor was the cap-binding com-plex, eIF-4F, and this is consistent with the observationsof Hansen et al. (1987) and Huang et al. (1987) thatunfertilized eggs from this species contain a potentinhibitor of protein synthesis that appears to antagonizeeIF-4F activity. A second important, and closely re-lated, area of investigation regarding the stimulation ofprotein synthesis in early development concerns the

8 T. D. Patrick, C. E. Lewer and V. M. Pain

mechanisms regulating the recruitment of the cellularmRNA for translation. Two aspects of this are the stateof the mRNA itself (e.g. whether it is sequestered byassociated 'masking' proteins) and the ability of thecellular translation system to recruit previously untrans-lated mRNA. The work of Colin et al. (1987) on thetranslation of exogenous mRNA and polyribosomalmRNA by L. pictus extracts in the presence andabsence of different combinations of added initiationfactors illustrates the versatility offered by the use ofcell-free systems to studies addressing the question ofmRNA translatability in early development.

We have now prepared extracts from Xenopusoocytes and eggs that reflect the stimulation of trans-lational activity seen in the intact cells (Richter et al.1982). Our experiments show (Fig. 3) that the ability ofthe extracts to reinitiate translation on exogenousmRNA is greater following progesterone-inducedmaturation. The measurements of labelling of native40S ribosomal subunits with [35S]Met-tRNAf in thesecell-free systems (Fig. 4) show a greater accumulationof 43S preinitiation complexes in cell-free systems fromunstimulated oocytes, suggesting that progesterone-induced maturation may increase the efficiency ofutilization of these complexes by accelerating one of thesubsequent steps in the initiation pathway. An obviouscandidate for such regulation would be the very nextstep, the binding of the preinitiation complex tomRNA. This could be limited by the availability oftranslatable mRNA, by the activity of one or more ofthe initiation factors already known to be involved inthis complicated process, or by the activity of as yetunknown factor(s) involved in recruitment of mRNAsnot already being translated (see below). Our resultsalso indicate (Fig. 5) that the difference in activitybetween extracts from oocytes and progesterone-matured oocytes is not due to the presence of adominant translational inhibitor in the former. Thissuggests that the mechanisms regulating protein syn-thesis in early development in Xenopus laevis differfrom that seen in the sea urchin S. purpuratus byHansen et al. (1987) and Huang et al. (1987). Furtherwork on the roles of individual initiation factors is nowin progress. This is of particular interest in the light ofthe recent observation that microinjection of the puri-fied initiation factor, eIF-4A, into intact Xenopusoocytes stimulates overall protein synthesis to an extentsimilar to that resulting from progesterone-inducedmaturation (Audet et al. 1987).

The potentiating effect of the reticulocyte S-100preparation on the translation of exogenous globinmRNA in these extracts raises the question of theidentity of the factor(s) present in the reticulocyteextract but absent or limiting in the Xenopus cell-freesystems. Reticulocyte S-100 has been shown to stimu-late the translational activity of cell-free extracts frommouse liver (Morley et al. 1985), from sea urchin eggs(Lopo et al. 1988) and from cultured mammalian cellssubjected to heat shock or treatment with hypertonicmedia (Lane, 1988). However, the stimulatory activityhas proved to be very labile during attempts to purify

and characterize it (Morley et al. 1985; Lane, 1988). Inthe cell-free systems described here, the pronouncedstimulation of globin synthesis occurred in the absenceof any effect on overall protein synthesis, suggestingthat the stimulatory factor(s) is needed more for therecruitment of newly added mRNA than for continuedreinitiation on to endogenous polysomal RNA alreadybeing translated. This distinction seems a reasonablepossibility in the light of the recent studies of Nelson &Winkler (1987), suggesting differences in mechanismbetween initiation and reinitiation. It is also consistentwith the data of Asselbergs et al. (1979), who studiedthe translation of exogenous mRNAs (including globinmRNA) microinjected into intact Xenopus oocytes.These messages only become maximally recruited intopolysomes after about 6h and, as this happened, theirtranslation became progressively less susceptible tocompetition from a second species of mRNA, given in asecond microinjection. Additionally, there may be vari-ations in behaviour between different exogenousmRNAs in our cell-free systems, since the oocyteextract was able to translate SP6 transcripts of theinfluenza virus HA mRNA without the addition ofreticulocyte S-100 (T. Patrick & A. Colman, unpub-lished). Clearly the translation of a number of exogen-ous mRNAs relative to that of endogenous XenopusmRNAs, and the influence of added factors on this,requires further study.

We would like to thank Drs Chris Ford, Chris Hutchison,Mike Clemens, Alan Colman, John Kay, Julian Blow and IanJeffrey for invaluable discussions, and also to Chris Ford forcriticism of the manuscript. We are also extremely grateful toMrs Eileen Willis for typing the manuscript.

References

ANDERSON, C. W., BAUM, P. R. & GESTELAND, R. F. (1973).Processing of adenovirus 2-induced proteins. J. Virol. 12,241-252.

ASHFORD, A. J. & PAIN, V. M. (1986). Effect of diabetes on therates of synthesis in rat muscle and liver in vivo. J. biol. Chem.261, 4059-4065.

ASSELBERGS, F. A. M., VAN VENROOU, W. J. & BLOEMENDAL, H.

(1979). Messenger RNA competition in living Xenopus oocytes.Eur. J. Biochem. 94, 249-254.

AUDET, R. G., GOODCHILD, J. & RICHTER, J. D. (1987). Eukaryotic

initiation factor 4A stimulates translation in microinjectedXenopus oocytes. Devi Biol. 121, 58-68.

AUSTIN, S. A., PAIN, V. M., LEWIS, J. A. & CLEMENS, M. J.(1982). Investigation of the role of uncharged tRNA in theregulation of polypeptide chain initiation by amino acidstarvation in cultured mammalian cells: a reappraisal. Eur. J.Biochem. 122, 519-526.

AUSTIN, S. A., POLLARD, J. W., JAGUS, R. & CLEMENS, M. J.

(1986). Regulation of polypeptide chain initiation and activity ofinitiation factor eIF-2 in Chinese-hamster ovary cell mutantscontaining temperature-sensitive aminoacyl-tRNA synthetases.Eur. J. Biochem. 157, 39-47.

BALLANTINE, J. E. M., WOODLAND, H. R. & STURGESS, E. A.

(1979). Changes in protein synthesis during the development ofXenopus laevis. J. Embryol. exp. Morph. 51, 137-153.

BLOW, J. J. & LASKEY, R. A. (1988). A role for the nuclearenvelope in controlling DNA replication within the cell cycle.Nature, Lond. 332, 546-548.

CLEMENS, M. J. (1984). In Transcription and Translation: a

Xenopus cell-free protein synthesis systems

Practical Approach (ed. B. D. Hames and S. J. Higgins), pp.231-270. Oxford and Washington: IRL Press.

CLEMENS, M. J., HENSHAW, E. C , RAHAMIMOFF, H. & LONDON, I.M. (1974). Met-tRNAf binding to 40S ribosomal subunits: a sitefor the regulation of initiation of protein synthesis by hemin.Proc. nam. Acad. Sci. U.S.A. 71, 2946-2950.

COLIN, A. M., BROWN, B. D., DHOLAKIA, J. N., WOODLEY, C. L.,

WAHBA, A. J. & HILLE, M. B. (1987). Evidence for simultaneousderepression of messenger RNA and the guanine nucleotideexchange factor in fertilized sea urchin eggs. Devi Biol. 123,354-363.

DARNBOROUGH, C , LEGON, S., HUNT, T. & JACKSON, R. J. (1973).

Initiation of protein synthesis: evidence for mRNA-independentbinding of Met-tRNAf to the 40S ribosomal subunit. J. molec.Biol. 76, 379-403.

DUMONT, J. N. (1972). Oogenesis in Xenopus laevis (Daudin). 1.Stages of oocyte development in laboratory maintained animals.J. Morph. 136, 153-180.

EISENSTEIN, R. S. & HARPER, A. E. (1984). Characterization of aprotein synthesis system from rat liver. Translation ofendogenous and exogenous messenger RNA. J. biol. Chem. 259,9922-9928.

GASIOR, E., HERRERA, F., SADNIK, I., MCLAUGHLIN, C. S. &MOLDAVE, K. (1979). The preparation and characterization of acell-free system from Saccharomyces cerevisiae that translates anatural messenger ribonucleic acid. J. biol. Chem. 254,3965-3969.

HANSEN, L. J., HUANG, W.-I. & JAGUS, R. (1987). Inhibitor oftranslational initiation in sea urchin eggs prevents mRNAutilization. J. biol. Chem. 262, 6114-6120.

HENSHAW, E. C. & PANNIERS, R. (1983). Translational systemsprepared from the Ehrlich ascites tumour cell. MethodsEnzymol. 101, 616-629.

HILLE, M. B. (1974). Inhibitor of protein synthesis isolated fromribosomes of unfertilised eggs and embryos of sea urchins.Nature, Lond. 249, 556-558.

HIRSCH, C. A., Cox, M. A., VAN VENROOIJ, W. J. & HENSHAW, E.C. (1973). The ribosome cycle in mammalian protein synthesis:association of the native smaller ribosomal subunit with proteinfactors. J. biol. Chem. 248, 4377-4385.

HUANG, W.-I., HANSEN, L. J., MERRICK, W. C. & JAGUS, R.(1987). Inhibitor of eukaryotic initiation factor 4F activity inunfertilised sea urchin eggs. Proc. natn. Acad. Sci. U.S.A. 84,6359-6363.

JACKSON, R. J. & HUNT, T. (1983). Preparation and use ofnuclease-treated rabbit reticulocyte lysates for the translation ofeukaryotic messenger RNA. Methods Enzymol. 96, 50-74.

KERR, I. M., BROWN, R. E. & TOVELL, D. R. (1972).

Characterisation of the polypeptides formed in response toencephalomyocarditis virus ribonucleic acid in a cell-free systemfrom mouse ascites tumour cells. J. Virol. 10, 73-81.

LAEMMLI, U. K. (1970). Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature, Lond. 221,680-685.

LANE, R. M. (1988). The control of protein synthesis in stressedmammalian cells. Ph.D. Thesis, University of Cambridge.

LASKEY, R. A., MILLS, A. D., GURDON, J. B. & PARTINGTON, C.

A. (1977). Protein synthesis in oocytes of Xenopus laevis is notregulated by the supply of messenger RNA. Cell 11, 345-351.

LOHKA, M. J. & MALLER, J. L. (1985). Induction of nuclearenvelope breakdown, chromosome condensation, and spindleformation in cell-free extracts. J. Cell Biol. 101, 518-523.

LOHKA, M. J. & MASUI, Y. (1983). Formation in vitro of spermpronuclei and mitotic chromosomes induced by amphibianooplasmic contents. Science 220, 719-721.

LOPO, A. & HERSHEY, J. W. B. (1985). Translational control duringearly embryogenesis in sea urchins. Fed. Proc. 44, 1801.

LOPO, A. C , MACMILLAN, S. & HERSHEY, J. W. B. (1988).Translational control in early sea urchin embryogenesis: initiationfactor eIF-4F stimulates protein synthesis in lysates fromunfertilized eggs of Strongylocentrotus purpuratus. Biochemistry27, 351-357.

LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J.(1951). Protein measurement with the Folin phenol reagent. J.biol. Chem. 193, 265-275.

MORLEY, S. J. & JACKSON, R. J. (1985). Preparation and propertiesof an improved cell-free protein synthesis system frommammalian liver. Biochim. Biophys. Ada 825, 45-56.

MORLEY, S. J., BUHL, W.-J. & JACKSON, R. J. (1985). A rabbitreticulocyte factor which stimulates protein synthesis in severalmammalian cell-free systems. Biochim. Biophys. Acta 825,57-69.

MUNRO, H. N. & FLECK, A. (1969). In Mammalian ProteinMetabolism, vol. 3 (ed. H. N. Munro), Ch. 30, pp. 424-526.New York: Academic Press.

NELSON, E. M. & WINKLER, M. (1987). Regulation of mRNA entryinto polysomes. J. biol. Chem. 262, 11 501-11 506.

PAIN, V. M. (1986). Initiation of protein synthesis in mammaliancells. Biochem. J. 235, 625-637.

PAIN, V. M., LEWIS, J. A., Huvos, P., HENSHAW, E. C. &CLEMENS, M. J. (1980). The effects of amino acid starvation onregulation of polypeptide chain initiation in Ehrlich ascitestumour cells. J. biol. Chem. 255, 1486-1491.

PANNIERS, R., STEWART, E. B., MERRICK, W. C. & HENSHAW, E.C. (1985). Mechanism of inhibition of polypeptide chaininitiation in heat shocked Ehrlich cells involves reduction ofeukaryotic initiation factor 4F activity. J. biol. Chem. 260,9648-9653.

PELHAM, H. R. B. & JACKSON, R. J. (1976). An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J.Biochem. 67, 247-256.

RICHTER, J. D. (1987). Molecular mechanisms of translationalcontrol during the early development of Xenopus laevis. InTranslational Regulation of Gene Expression (ed. J. Ilan), pp.111-139. New York: Plenum Press.

RICHTER, J. D. , WASSERMAN, W. J. & SMITH, L. D. (1982). Themechanism for increased protein synthesis during Xenopusoocyte maturation. Devi Biol. 89, 159-167.

SKUP, D. & MILLWARD, S. (1980). Highly efficient translation ofmessenger RNA in cell-free extracts prepared from L-cells.Nucleic Acid Res. 4, 3581-3587.

TAYLOR, M. A. & SMITH, L. D. (1985). Quantitative changes inprotein synthesis during oogenesis in Xenopus laevis. Devi Biol.110, 230-237.

WASSERMAN, W. J., PENNA, M. J. & HOULE, J. G. (1986). Theregulation of Xenopus laevis oocyte maturation. InGametogenesis and the Early Embryo (ed. J. G. Gall), pp.111-130. New York: A. R. Liss Inc.

WASSERMAN, W. J., RICHTER, J. D. & SMITH, L. D. (1982). Proteinsynthesis during maturation-promoting factor and progesterone-induced maturation in Xenopus oocytes. Devi Biol. 89, 152-158.

WINKLER, M. W., NELSON, E. M., LASHBROOK, C. & HERSHEY, J.W. B. (1985). Multiple levels of regulation of protein synthesis atfertilization in sea urchin eggs. Devi Biol. 107, 290-300.

WOODLAND, H. (1974). Changes in the polysome content ofdeveloping Xenopus laevis embryos. Devi Biol. 40, 90-101.

{Accepted 18 January 1989)