During the maturation of the oocytes of the frog Xenopus laevis, the rate of protein synthesis shows a twofold increase. Studies of the mechanisms involved in this stimulation have been seriously limited by the lack of an active cell-free translation system. We have now prepared such systems from oocytes, progesterone-matured oocytes and eggs of Xenopus laevis by induction of lysis by centrifugation of whole cells.

The extracts are highly active in incorporation of labelled amino acids and, in the progesterone-matured and egg extracts, a substantial proportion of this is due to reinitiation on endogenous mRNA, as shown by the use of inhibitors. The increased rate of protein synthesis previously observed in intact oocytes following progesterone-induced maturation is reflected in the relative activities of the extracts. The difference in activity is not due to the presence of a dominant inhibitor of translation in the extracts from unstimulated oocytes. Labelling studies with initiator tRNA ([35S]Met-tRNAf) indicate a higher concentration of 43S preinitiation complexes in the extracts from unstimulated oocytes, suggesting an impairment of initiation of translation at or after the mRNA-binding step. Extracts from both oocytes and progesterone-matured oocytes translated endogenous mRNAs to give products ranging over a wide spectrum of molecular weight. However, significant translation of exogenous (globin) mRNA required the presence of reticulocyte postribosomal supernatant, suggesting that one or more factors required for mRNA recruitment is limiting in these extracts.

Xenopus oocytes are arrested in the cell cycle at the stage of meiotic prophase. The process of maturation is triggered in vivo by the hormone progesterone. This hormone is secreted by follicle cells surrounding the oocytes in the ovary, which in turn are stimulated by release of gonadotropins from the pituitary (reviewed by Wasserman et al. 1986). Maturation can also be induced in vitro by treating isolated oocytes with progesterone. The overall process takes several hours and culminates in meiotic division and dissolution of the nuclear envelope. One of the many events associated with maturation is a twofold to threefold increase in the overall rate of protein synthesis (Wasserman et al. 1982), due to recruitment of pre-existing mRNA (Richter et al. 1982). A particular characteristic of the immature Xenopus oocyte is the large proportion of its ribosomes and mRNA molecules not being utilized for protein synthesis (Woodland, 1974; Taylor & Smith, 1985). Upon maturation the proportion of ribosomes engaged in polysomes only rises from about 1 % to 2 % (Woodland, 1974), indicating that a severe restraint on translation exists even in the mature egg. It is widely thought that part of the mRNA in Xenopus oocytes is held in a form unavailable for translation, possibly by the presence of associated inhibitory, or ‘masking’, proteins (Richter, 1987). However, other authors have suggested that, in addition, the rate of protein synthesis in the oocyte may be limited by the activity of one or more other components of the translational apparatus (Laskey et al. 1977; Wasserman et al. 1986; Audet et al. 1987).

Investigation of the components limiting protein synthesis in Xenopus oocytes and the mechanisms promoting the stimulation of translation during maturation has been severely impeded by the lack of suitable cell-free protein-synthesizing systems. In contrast, knowledge of the mechanisms controlling protein synthesis during fertilization of sea urchin eggs has advanced very rapidly over the last 2 years following the development from these cells of cell-free translation systems whose relative activities reflect the stimulation of protein synthesis that occurs in vivo during fertilization (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 in lysates derived from unfertilized eggs is limited by the activity of certain polypeptide initiation factors and that activation of these factors may play a role in the stimulation of translation that occurs in response to fertilization.

This paper describes the preparation and characterization of an active, cell-free protein-synthesizing system from Xenopus oocytes and eggs. Our starting point was the method developed by Lohka and his colleagues for preparing an extract for use in studying pronuclear formation and chromosome condensation in vitro (Lohka & Masui, 1983; Lohka & Mailer, 1985). In this procedure, mature eggs laid in response to the injection of adult Xenopus females with human chorionic gonadotropin were lysed simply by the application of centrifugal force. We have optimized the conditions used to incubate these extracts for the study of protein synthesis, and introduced further modifications to allow us to prepare similar systems from oocytes before and after progesterone-induced maturation in vitro.

Preparation of egg extracts

Extracts from unfertilized Xenopus laevis eggs were prepared by a modification of the method described by Lohka & Mailer (1985). Mature wild female frogs (Xenopus Ltd, UK) were stimulated to lay eggs by injection of 650 i.u. human chorionic gonadotrophin (Chorulon, Intervet Laboratories) into their dorsal lymph sacs, 16 hours before the eggs were required. Eggs were collected at room temperature into saline tap water (110mm-NaCl in tap water) to prevent activation, and dejellied in 5mm-dithiothreitol, 20mm-Tris-HCl, pH8·5, in saline tap water. Following the removal of their jelly coats, the eggs were rinsed twice in saline tap water and then twice in ice-cold extraction buffer: 20mm-Hepes-KOH, pH 7·5, 125mm-KCl, 2mm-MgC12, 2mm-2-mercaptoethanol, 3 μg ml-1 leupeptin. The eggs were transferred to 5 ml centrifuge tubes and allowed to sediment under gravity. As much excess buffer as possible was removed before the eggs were centrifuged at 10 000g for 10min at 4°C using an 8×5 ml swing-out rotor in an MSE 18 centrifuge. The procedure resulted in a stratified extract containing a large pellet of yolk platelets, a particulate soluble phase of variable colour and texture and a lipid pellicle. For most of the experiments described, the material between the lipid pellicle and the yolk platelet pellet was removed and kept on ice until required. For the experiments investigating the effect of cytochalasin B (Sigma), the collected material was treated with the chemical at a final concentration of 50 μg ml-1 and centrifuged again at 10000g for 10 min at 4 °C. The supernatant was then removed and kept on ice until required.

Preparation of extracts from oocytes and progesterone-matured oocytes

Adult lab-bred female frogs were stimulated by injection of 50 i.u. follicle-stimulating hormone (Folligon, Intervet Laboratories) into their dorsal lymph sacs, 5 to 7 days in advance of the experiment. Each animal was then killed and the ovary removed into Barth’s saline (88· 0mm-NaCl, 1·0mm-KCl, 2·4mm-NaHCO3, 10 mm-Tris-HCl (pH7·6), 0·30mm-Ca(NO3)2.4H2O, 0·41 mm-CaCl2.6H2O, 0·82mm-MgSO4. 7H2O). The lobes of the ovary were dissected into clumps of 30 to 50 oocytes and washed thoroughly in Barth’s saline. Approximately half of the clumps were treated with progesterone (Sigma) from a stock solution of 1 mg ml-1 in 50 % ethanol to a final concentration of 2 μg ml-1 in Barth’s saline for 15 min. The clumps of oocytes were then treated with 250 μg ml-1 collagenase (EC 3.4.24.3, Boehringer, Clostridium hystolyticum) in 50% high-salt Barth’s saline (115 mm-NaCl), 50 % 0·1 m-sodium phosphate buffer as follows. Keeping the progesterone-treated oocytes separate from the others, the clumps of oocytes were transferred to 50 ml conical centrifuge tubes and the tubes filled three-quarters full with the collagenase solution. The tubes were then placed horizontally in a shaking water bath at 23 °C and shaken at 120 revs min-1. The length of time taken to liberate the oocytes varied from one ovary to another. Free oocytes were collected up to 3 hours after the start of the treatment and rinsed three times in Barth’s saline (or high-salt Barth’s saline for progesterone-treated batches to prevent activation after maturation). All the oocytes were manually sorted and selected for undamaged stage VI oocytes (as defined by Dumont, 1972), or for appearance of the white spot to indicate maturation. The cells were then rinsed in extraction buffer and the extracts prepared exactly as described for eggs except that, for most experiments, human placental ribonuclease inhibitor (RNA-sin, Boehringer) and soy-bean trypsin inhibitor (Sigma) were added to the prepared extracts at concentrations of 267unitsml-1 and 0·5 mg ml-1, respectively (Lopo et al. 1988).

Amino acid incorporation assays with Xenopus extracts

Standard assays contained 0·6 volumes of extract and 0·4 volumes of a reaction mix with the following components at final concentrations in the assays: 35 mm-creatine phosphate, 250 μg ml-1 creatine phosphokinase (EC 2.7.3.2), 0·5mm-spermidine, and 40 μCiml-1 L-[35S]methionine (800–1500Ci mmol-1, Amersham International). Incubations were carried out at 21 °C. For assay of amino acid incorporation, samples were removed from the assay tube and spotted onto 2·1 cm circles of Whatman No. 1 filter paper. After air drying for 10 seconds, the filters were dropped into 10% trichloroacetic acid containing 2 mm unlabelled methionine and processed as described by Pain et al. (1980).

Calculation of the rate of protein synthesis in extracts

For each extract, a series of incorporation assays was carried out as described above with the addition of unlabelled methionine at a range of final concentrations between 10 and 100 μM. A graph was then constructed in which the concentration of cold methionine added was plotted against the reciprocal of incorporation of [35S]methionine into protein. The negative intercept on the ordinate gives the concentration of methionine contributed by the extract itself to the assay. Knowledge of this value permits the calculation of the specific radioactivity of the precursor pool of [35S]methionine in the assay (assuming 100% purity of the radioactive material), which in turn allows the actual protein synthesis rate to be calculated from the incorporation data. For the purpose of calculating protein synthesis rates as pmol of methionine incorporated, the efficiency of estimation of radioactivity of [35S]protein on the filters was taken as 50%.

Estimation of RNA content in extracts

20 μl samples of extract were assayed by a procedure based on that of Munro & Fleck (1969). Samples were precipitated on ice with 3 ml of 2% (v/v) perchloric acid (HC1O4) and centrifuged at 3000 revs min-1 for 10 min at 4°C in a Sorvall RT 6000 centrifuge. The pellets were washed twice more with 2 % HClO4 and the RNA was then hydrolysed for 60 min with 0·3M-NaOH at 37°C. Protein was reprecipitated with 0·8 ml of 20% HC1O4 on ice for 30 min. After centrifugation, the precipitate was retained for protein determination by the method of Lowry et al. (1951). The supernatants were removed and absorbances at 232 and 260 nm read in quartz cuvettes in an SP-500 Pye Unicam spectrophotometer against a blank of 2 ml 0·3M-NaGH and 0·8ml 20% HC1O4. RNA content was then calculated from the following equation (Ashford & Pain, 1986): [(3·11×A260)—(0·58× A232)] × 10·53 × 2·8× 50 = μg RNA per ml extract.

Labelling of 43S preinitiation complexes k

Incubations (2min) were carried out as described above, but with [35S]methionine replaced with [35S]Met-tRNAf (approximately 5×106 cts min-1 ml-1), prepared as described by Clemens et al. (1974). Emetine was added at 1 mm. Reactions were terminated by the addition of an equal volume of gradient buffer (25mm-sodium cacodylate, pH 6·6, 80mm-KC1, 2 mm-magnesium acetate) containing 2% (v/v) neutralized formaldehyde. The mixtures were layered onto 12ml linear gradients of 20–40 % sucrose in the same buffer and centrifuged for 16·5 h at 92000 g in a Beckman SW 40 rotor. The gradients were analysed at 254 nm in an ISCO Model UA-5 recording spectrophotometer and divided into 20 fractions, which were processed as described by Pain et al. (1980).

Preparation of reticulocyte components

Procedures described by Clemens (1984) were used for the following: preparation and incubation of rabbit reticulocyte lysates, preparation and incubation of the messenger-dependent lysate system and isolation of total reticulocyte RNA by extraction of untreated lysate with sodium dodecyl sulphate, phenol and chloroform. For preparation of reticulocyte S100, reticulocyte lysate was supplemented with 15μM-haemin, diluted with 2 volumes of buffer (20 mm-Mops-KOH, pH 7·6, 0·1 M-KC1, 0·1 mm-EDTA, 1 mm-dithiothreitol, 10 % glycerol) and supplemented with MgCl2 and GTP at a final concentration of 2mm each. The mixture was then centrifuged at 100000 g for 4 h at 4 °C in a Beckman 50 Ti rotor to generate the G-100 supernatant.

Analysis of translation products by polyacrylamide gel electrophoresis

Protein synthesis assays (25 μl) were carried out as described above except that [35S]methionine was added at a concentration of 400 μCiml-1. Incubation was for 1h. For analysis, 50 μl of 100mm-Tris, pH 6·8, was added to each assay, then the samples were centrifuged at 11500g for 10 min in an MSE Microcentaur microcentrifuge in a cold room. The supernatants were dialysed against 100mm-Tris, pH6·8 for 4h at 4°C. Concentrated electrophoresis sample buffer was then added to give the following concentrations: 1 % sodium dodecyl sulphate, 40mm-Tris-HCl, pH6·8, 5% glycerol, 0·002% bromophenol blue. Samples were boiled for 4 min, and 40 μl aliquots were applied to polyacrylamide slab gels containing 10% acrylamide/0·1 % bisacrylamide/0·1 % sodium dodecyl sulphate and subjected to electrophoresis as described by Laemmli (1970) with the modifications of Anderson et al. (1973). Autoradiographs were prepared by exposure of Amersham Hyperfilm Betamax film to the gels for 6 days.

Preliminary characterization of the cell-free system from eggs

The initial experiments were performed using egg extracts prepared exactly as described by Lohka & Mailer (1985). This involved treatment of the extracts prepared as described in Materials and methods with 50 μg ml-1 cytochalasin B and centrifuging the mixture again at 10000g for 10 min. However, omission of the treatment with cytochalasin B and the second centrifugation had no effect on the incorporation of [35S] methionine into protein. In all the experiments described in this paper, the procedure described in Materials and methods, omitting these treatments, was used.

Fig. 1A shows the time course of incorporation of [35S]methionine into protein by the egg extract. It also shows a dramatic inhibition of incorporation resulting from a single pass of a loose-fitting pestle in a Dounce homogenizer. Another treatment highly deleterious to protein synthetic activity was snap-freezing of the extract in liquid nitrogen (Fig. IB). Addition of glycerol (10% v/v) did not protect the samples from loss of activity upon freezing (not shown). Freshly prepared cytoplasmic extracts were therefore used for all the experiments reported in this paper.

Fig. 1.

(A) Time course of incorporation of [35S]methionine into protein using egg extracts prepared normally as described in Materials and methods (•), or with the inclusion of a single pass of a loose-fitting pestle in a Dounce homogenizer before centrifugation (◼). Incubations were performed as described in Materials and methods. 10 μl samples were removed from the incubation mixture at the times shown. RNA concentrations in both extracts were measured as described in Materials and methods and found to be identical. (B) Effects of freezing and the inhibitor edeine on incorporation of [35S]methionine in egg extracts. Each point represents the removal of 10 μl of the incubation mixture from the assay tube at the time indicated. The extracts were either used fresh in the assay (•) or frozen before incubation by rapid immersion in liquid nitrogen. Small aliquots of extract of less than 200 μl were frozen in this manner and then thawed before use (◼). Edeine was added to the assay tubes before incubation at a final concentration of 1 μM in fresh (◻) and frozen (◼) extracts.

Fig. 1.

(A) Time course of incorporation of [35S]methionine into protein using egg extracts prepared normally as described in Materials and methods (•), or with the inclusion of a single pass of a loose-fitting pestle in a Dounce homogenizer before centrifugation (◼). Incubations were performed as described in Materials and methods. 10 μl samples were removed from the incubation mixture at the times shown. RNA concentrations in both extracts were measured as described in Materials and methods and found to be identical. (B) Effects of freezing and the inhibitor edeine on incorporation of [35S]methionine in egg extracts. Each point represents the removal of 10 μl of the incubation mixture from the assay tube at the time indicated. The extracts were either used fresh in the assay (•) or frozen before incubation by rapid immersion in liquid nitrogen. Small aliquots of extract of less than 200 μl were frozen in this manner and then thawed before use (◼). Edeine was added to the assay tubes before incubation at a final concentration of 1 μM in fresh (◻) and frozen (◼) extracts.

To investigate whether initiation of protein synthesis on endogenous mRNA was occurring in vitro, incorporation assays were performed in the presence of specific inhibitors of initiation such as edeine (Fig. IB), or 7-methylguanosine 5’ triphosphate (7-methyl GTP) (Fig. 2). Fig. IB shows that a significant proportion of the incorporation during the incubation is edeine-sensitive, suggesting that initiation of protein synthesis is occurring. To estimate how long the capacity for initiation is maintained, the inhibitors were added at various times after the start of incubation, adjusting the amounts added to ensure the same final concentration if samples for time points had been removed before inhibitor addition. In these experiments, inhibitors were added up to 40 min into the assay, and the results shown for 7-methyl GTP in Fig. 2 suggest that initiation continues at least up until then. Incorporation ceased approximately 10 min after the addition of the inhibitor, suggesting that a round of initiation occurs every 10 min.

Fig. 2.

The effect of adding the inhibitor 7-methyl GTP to an egg extract at various times after the start of the incorporation assays. Incubations were carried out as described in Materials and methods, with 10 μl samples being removed at the times shown. 7-methyl GTP was added to a final concentration of 500 μM to incubation tubes at 0 min (◼), 20min (◼), and 40 min (◻). A control incubation with no inhibitor added is also shown (•).

Fig. 2.

The effect of adding the inhibitor 7-methyl GTP to an egg extract at various times after the start of the incorporation assays. Incubations were carried out as described in Materials and methods, with 10 μl samples being removed at the times shown. 7-methyl GTP was added to a final concentration of 500 μM to incubation tubes at 0 min (◼), 20min (◼), and 40 min (◻). A control incubation with no inhibitor added is also shown (•).

Optimum conditions for incorporation assays

Studies were carried out with Xenopus egg extracts to determine the optimal conditions for translation. Although there were minor differences between individual extracts, addition of extra potassium or magnesium ions over and above those already contributed by the extract rarely resulted in significant enhancement of the rate of incorporation. Addition of magnesium and potassium ions by more than 2mm and 50 mm, respectively, were invariably inhibitory (results not shown). The effect of addition of spermidine (buffered to pH7·5) has also been investigated. The.polyamine was added at various concentrations between 0 and 3mm and the optimum found to be approximately 0·5 mm. This resulted in a stimulation of approximately 50%.

Addition of an amino acid mixture containing 19 amino acids to the system at a final concentration of 200 μM each did not affect translation. Addition of exogenous ATP together with equimolar magnesium to the Xenopus egg system was found to be inhibitory. This suggests that the presence of 35 mm-creatine phosphate and 250 μg ml-1 creatine phosphokinase together with the endogenous ATP pool generates sufficient ATP to maintain translation during the incubation. Assays were also carried out to examine the effect of extract concentration. Optimal activity was obtained when the extract constituted 60 % of the assay volume.

Effect of progesterone-induced maturation on protein synthesis in extracts from oocytes

Fig. 3A shows a time course of incorporation into protein in extracts prepared from unstimulated oocytes and those induced to undergo maturation in vitro by treatment with progesterone. The endogenous concentrations of unlabelled methionine and of RNA in the extracts were estimated as described in Materials and methods, and the results are presented as pmol methionine incorporated per mg RNA. This compensated for variations between extracts in ribosome concentration and in the endogenous methionine pool size.

Fig. 3.

(A) Effect of progesterone maturation on translational activity of oocyte extracts. Extracts were made from oocytes (◼) and progesterone-matured oocytes (•) and incubations and calculations at the rate of protein synthesis were performed as described in Materials and methods. Edeine was added at a final concentration of 1 μM to the oocyte system (◻) or the progesterone-matured oocyte system (○). (B) The edeine-sensitive incorporation by the oocyte (◼) and progesterone-matured oocyte (○) extracts.

Fig. 3.

(A) Effect of progesterone maturation on translational activity of oocyte extracts. Extracts were made from oocytes (◼) and progesterone-matured oocytes (•) and incubations and calculations at the rate of protein synthesis were performed as described in Materials and methods. Edeine was added at a final concentration of 1 μM to the oocyte system (◻) or the progesterone-matured oocyte system (○). (B) The edeine-sensitive incorporation by the oocyte (◼) and progesterone-matured oocyte (○) extracts.

The results in Fig. 3A show a twofold higher rate of methionine incorporation in the extract derived from progesterone-matured oocytes. At an early stage in the incubation, at least part of this difference must reflect the greater amount of ‘run-off ’incorporation that would be expected because of the greater polysome content 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 of translation on endogenous mRNA than that from the unstimulated oocytes (Fig. 3A). This is illustrated by the data in Fig. 3B, showing the edeine-sensitive (i.e. initiation-dependent) incorporation of the two types of extract. This ability of cell-free systems from progesterone-matured oocytes to sustain edeine-sensitive protein synthesis at later stages in the incubation is dependent on the presence of placental ribonuclease inhibitor and soy-bean trypsin inhibitor to inhibit ribonuclease and protease activities in the extracts. .

In a series of experiments comparing extracts from oocytes from different frogs we have found that, although progesterone-induced maturation always results in an increase in the protein synthetic activity of the extracts, both the rates of protein synthesis obtained and the degree of stimulation show considerable variation. The mean ± S.E.M. of the initial rates of incorporation in cell-free systems from 6 animals was 1·07 ±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 t-test). The main difference between the approximately 20% of our experiments in which a low degree of stimulation is seen and those like that shown in Fig. 3 is that, in the former, the systems from the unstimulated oocytes tend to show kinetics and edeine sensitivity more similar to those from the progesterone-matured oocytes. This could be due to a somewhat elevated level of gonadotropins in some of the donor frogs.

Approximate comparisons can be made between the rates of protein synthesis in our oocyte extracts and related values in the literature. Assuming that total oocyte protein contains 2% methionine by weight, the initial rate of protein synthesis in the oocyte extract is approximately 7·5 ng protein h-1pg-1 RNA. This compares well with the rate of 4·7 μg protein h-1pg-1 RNA determined for intact oocytes by Taylor & Smith (1985). In the units of Blow & Laskey (1988), the mean rate of protein synthesis in our oocyte extracts is 23 μg protein h-1 ml-1 extract, which is of the same order as that stated by these workers to occur in similarly derived extracts from Xenopus eggs.

Labelling of 43S preinitiation complexes with initiator tRNA in extracts from oocytes and progesterone-induced oocytes

The experiments with edeine indicated that the cell-free systems from progesterone-matured oocytes were more active in the reinitiation of translation on endogenous mRNA. To identify more closely the stage in the initiation pathway that might be regulated by maturation, we investigated the labelling of native 40S ribosomal subunits with initiator tRNA ([35S]Met-tRNAf) (Darnborough et al. 1973; Austin et al. 1982). The results are shown in Fig. 4. As previously observed in similar analyses of native ribosomal subunits from cultured cells (Hirsch et al. 1973), the 40S subunit peak exhibited heterogeneity, presumably due to the existence of multiple species containing different amounts of associated protein (Hirsch et al. 1973). In these gradients from Xenopus oocyte extracts, the peak of native 60S subunits is not visible, due to its being swamped by the enormous peak of 80S monomeric ribosomes, which, as previously demonstrated by Woodland (1974) make up more than 95 % of the ribosomal particles in these cells. Our data on the labelling of the subunits with initiator tRNA demonstrate a greater number of ([35S]Met-tRNAf .40S subunit) initiation complexes in the extracts from unstimulated oocytes than in those from progesterone-matured oocytes. This suggests that the utilization of these complexes in subsequent steps of initiation is less efficient in these extracts.

Fig. 4.

Labelling of 43S preinitiation complexes with [35S]Met-tRNAf in cell-free systems from oocytes (A) and progesterone-matured oocytes (B). Incubations, density gradient analysis and processing of fractions were as described in Materials and methods. Sedimentation was from left to right, and the position of native, 40S ribosomal subunits (40–43S) is indicated. The major peak rising at the bottom of the gradients is that of 80S monomeric ribosomes. — Absorbance at 254 nm, • – – – • [35S] radioactivity precipitable by cetyltrimethylammonium bromide.

Fig. 4.

Labelling of 43S preinitiation complexes with [35S]Met-tRNAf in cell-free systems from oocytes (A) and progesterone-matured oocytes (B). Incubations, density gradient analysis and processing of fractions were as described in Materials and methods. Sedimentation was from left to right, and the position of native, 40S ribosomal subunits (40–43S) is indicated. The major peak rising at the bottom of the gradients is that of 80S monomeric ribosomes. — Absorbance at 254 nm, • – – – • [35S] radioactivity precipitable by cetyltrimethylammonium bromide.

Investigation of possible inhibitory activity of extracts

A possible model for explaining the difference in protein synthetic activity between unstimulated and progesterone-matured oocytes is that the former may contain an inhibitor of translation. This has been tested in two experiments: first, the effects of adding oocyte and progesterone-matured oocyte extracts were tested in a reticulocyte lysate translation system. The results are shown in Fig. 5A and indicate that neither extract contains a dominant inhibitor of the reticulocyte lysate system. The second experiment involved adding various amounts of oocyte extract or the extraction buffer to a fixed amount of progesterone-matured oocyte extract and measuring the rate of incorporation in each incubation. Fig. 5B shows the result of this experiment, and again suggests that a dominant inhibitor of translation is not present in the oocyte extract.

Fig. 5.

Investigation of potential inhibitors in oocyte and progesterone-matured oocyte extracts. All extracts were prepared as described in Materials and methods. (A) The effect of adding 7 μl of oocyte (◻) or progesterone-matured oocyte (◼) extracts to a reticulocyte lysate system at a final volume of 50 μl. Control reticulocyte lysate containing extraction buffer is also shown (•). 6 μl samples were removed from the incubation mix at the times shown.(B) The effect of adding an increasing percentage of either oocyte extract (◼) or extraction buffer (•) to a fixed amount (60%) of progesterone-matured oocyte extract. Incubations were carried out as described in Materials and methods at a final volume of 50 μl for 30 min. The incubation tubes were then plunged into iced water and 10 μl samples removed from each tube and spotted onto filters in quadruplicate. Filters were then processed and counted as described in Materials and methods.

Fig. 5.

Investigation of potential inhibitors in oocyte and progesterone-matured oocyte extracts. All extracts were prepared as described in Materials and methods. (A) The effect of adding 7 μl of oocyte (◻) or progesterone-matured oocyte (◼) extracts to a reticulocyte lysate system at a final volume of 50 μl. Control reticulocyte lysate containing extraction buffer is also shown (•). 6 μl samples were removed from the incubation mix at the times shown.(B) The effect of adding an increasing percentage of either oocyte extract (◼) or extraction buffer (•) to a fixed amount (60%) of progesterone-matured oocyte extract. Incubations were carried out as described in Materials and methods at a final volume of 50 μl for 30 min. The incubation tubes were then plunged into iced water and 10 μl samples removed from each tube and spotted onto filters in quadruplicate. Filters were then processed and counted as described in Materials and methods.

Analysis of translation products of endogenous and exogenous mRNA in Xenopus oocyte cell-free systems

Tracks 1 and 2 of Fig. 6 show the labelled products of translation of endogenous mRNA in the cell-free systems from unstimulated and progesterone-matured oocytes. It can be seen that both systems translated products encompassing a wide range of molecular weights, similar to that seen in the intact cells. This suggests that premature termination is not a major problem in these extracts, as it is in some other cell-free translation systems (Kerr et al. 1972). Comparison of tracks 1 and 2 indicates some characteristic changes in the pattern of products resulting from progesterone-induced maturation, but this is outside the scope of the present study. More detailed analysis, involving twodimensional gel electrophoresis, will be required to examine changes in individual translation products, as has been carried out with intact cells (Ballantine et al. 1979).

Fig. 6.

Translation products from cell-free systems from unstimulated and progesterone-matured oocytes. Incubation conditions and processing procedures were as described in Materials and methods. The tracks show the products of the following incubations: endogenous translation products of cell-free systems from unstimulated (track 1) and progesterone-matured (track 2) oocytes; track 3: oocyte extract + 1·4μg total reticulocyte RNA; track 4: progesterone-matured extract + 1·4 μg reticulocyte RNA; tracks 5–8: oocyte extract + 0·35μg, 0·70 μg, 1·05μg, 1·75μg reticulocyte RNA; tracks 9–14: oocyte extract + 3 μl reticulocyte S-100 + 0·35 μg, 0·70 μg, 1·05μg, 1·40 μg, 1·75 μg and 0 μg reticulocyte RNA; track 15: globin standard, prepared by translating 1·4μg reticulocyte RNA in a 12 μl messenger-dependent lysate system and diluting directly (1:1) into electrophoresis sample buffer. 5 μl was applied to the gel. Total RNA, S-100 and the messenger-dependent lysate system was prepared from rabbit reticulocyte lysates as described in Materials and methods.

Fig. 6.

Translation products from cell-free systems from unstimulated and progesterone-matured oocytes. Incubation conditions and processing procedures were as described in Materials and methods. The tracks show the products of the following incubations: endogenous translation products of cell-free systems from unstimulated (track 1) and progesterone-matured (track 2) oocytes; track 3: oocyte extract + 1·4μg total reticulocyte RNA; track 4: progesterone-matured extract + 1·4 μg reticulocyte RNA; tracks 5–8: oocyte extract + 0·35μg, 0·70 μg, 1·05μg, 1·75μg reticulocyte RNA; tracks 9–14: oocyte extract + 3 μl reticulocyte S-100 + 0·35 μg, 0·70 μg, 1·05μg, 1·40 μg, 1·75 μg and 0 μg reticulocyte RNA; track 15: globin standard, prepared by translating 1·4μg reticulocyte RNA in a 12 μl messenger-dependent lysate system and diluting directly (1:1) into electrophoresis sample buffer. 5 μl was applied to the gel. Total RNA, S-100 and the messenger-dependent lysate system was prepared from rabbit reticulocyte lysates as described in Materials and methods.

The remaining tracks in Fig. 6 show the effect of adding exogenous mRNA to the extracts. In these studies, we used total reticulocyte RNA as a source of globin mRNA. Addition of increasing amounts of this RNA to the oocyte cell-free system (tracks. 3 and 5–8) resulted in very little, if any, production of globin. This was also the case in the extract from progesterone-matured oocytes (track 4), where the picture is slightly complicated by the greater translation of an endogenous product with similar migration characteristics (see track 2). Tracks 9–13, however, show a dramatic potentiating effect on globin mRNA translation of adding a small quantity of postribosomal supernatant (S-100) prepared from rabbit reticulocyte lysate. This was not due to the presence of globin mRNA in the reticulocyte S-100 itself (track 14). The effect of reticulocyte S-100 appeared to be specific for the translation of the exogenous mRNA, since overall amino acid incorporation into protein was not affected. Similar results were obtained when reticulocyte S-100 was added to extracts from progesterone-matured oocytes (not shown).

Cell-free systems for use in studies of protein synthesis have 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 veast (Gasior et al. 1979). With the single exception of the rabbit reticulocyte lysate, most of these extracts synthesize protein at a low rate relative to that in the intact cells and tend to reinitiate on endogenous mRNA inefficiently. They have, however, been used successfully to identify mechanisms regulating the rate of translation in mammalian cells by a variety of physiological treatments (Pain et al. 1980; Austin et al. 1986; Panniers et al. 1985). An important prerequisite in such studies is that the relative translational activities of the extracts prepared from cells in two different physiological states reflect the differences in the rate of protein synthesis seen in the intact cells from which the extracts are derived.

Recently, several laboratories have reported interesting results from work with cell-free translation systems derived from sea urchin eggs and zygotes (Winkler et al. 1985; Lopo & Hershey, 1985; Colin et al. 1987; Hansen et al. 1987; Lopo et al. 1988). The use of these extracts has provided much valuable information on the mechanisms contributing to the 20-fold stimulation of protein that occurs in sea urchin eggs during fertilization. First, the roles of individual initiation factors in mediating the response to fertilization have been examined by adding exogenous purified factors to the cell-free systems. In this way, Winkler et al. (1985) and Colin et al. (1987) showed cell-free systems from unfertilized eggs of Lytechinus pictus to be stimulated by addition of the initiator-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 eggs of Strongylocentrotus purpuratus, found that the most stimulatory initiation factor was the cap-binding complex, eIF-4F, and this is consistent with the observations of Hansen et al. (1987) and Huang et al. (1987) that unfertilized eggs from this species contain a potent inhibitor of protein synthesis that appears to antagonize eIF-4F activity. A second important, and closely related, area of investigation regarding the stimulation of protein synthesis in early development concerns the mechanisms regulating the recruitment of the cellular mRNA for translation. Two aspects of this are the state of the mRNA itself (e.g. whether it is sequestered by associated ‘masking’ proteins) and the ability of the cellular translation system to recruit previously untranslated mRNA. The work of Colin et al. (1987) on the translation of exogenous mRNA and polyribosomal mRNA by L. pictus extracts in the presence and absence of different combinations of added initiation factors illustrates the versatility offered by the use of cell-free systems to studies addressing the question of mRNA translatability in early development.

We have now prepared extracts from Xenopus oocytes and eggs that reflect the stimulation of translational activity seen in the intact cells (Richter et al. 1982). Our experiments show (Fig. 3) that the ability of the extracts to reinitiate translation on exogenous mRNA is greater following progesterone-induced maturation. The measurements of labelling of native 40S ribosomal subunits with [35S]Met-tRNAf in these cell-free systems (Fig. 4) show a greater accumulation of 43S preinitiation complexes in cell-free systems from unstimulated oocytes, suggesting that progesterone-induced maturation may increase the efficiency of utilization of these complexes by accelerating one of the subsequent steps in the initiation pathway. An obvious candidate for such regulation would be the very next step, the binding of the preinitiation complex to mRNA. This could be limited by the availability of translatable mRNA, by the activity of one or more of the initiation factors already known to be involved in this complicated process, or by the activity of as yet unknown factor(s) involved in recruitment of mRNAs not already being translated (see below). Our results also indicate (Fig. 5) that the difference in activity between extracts from oocytes and progesterone-matured oocytes is not due to the presence of a dominant translational inhibitor in the former. This suggests that the mechanisms regulating protein synthesis in early development in Xenopus laevis differ from that seen in the sea urchin 5. purpuratus by Hansen et al. (1987) and Huang et al. (1987). Further work on the roles of individual initiation factors is now in progress. This is of particular interest in the fight of the recent observation that microinjection of the purified initiation factor, eIF-4A, into intact Xenopus oocytes stimulates overall protein synthesis to an extent similar to that resulting from progesterone-induced maturation (Audet et al. 1987).

The potentiating effect of the reticulocyte S-100 preparation on the translation of exogenous globin mRNA in these extracts raises the question of the identity of the factor(s) present in the reticulocyte extract but absent or limiting in the Xenopus cell-free systems. Reticulocyte S-100 has been shown to stimulate the translational activity of cell-free extracts from mouse liver (Morley et al. 1985), from sea urchin eggs (Lopo et al. 1988) and from cultured mammalian cells subjected to heat shock or treatment with hypertonic media (Lane, 1988). However, the stimulatory activity has proved to be very labile during attempts to purify and characterize it (Morley et al. 1985; Lane, 1988). In the cell-free systems described here, the pronounced stimulation of globin synthesis occurred in the absence of any effect on overall protein synthesis, suggesting that the stimulatory factor(s) is needed more for the recruitment of newly added mRNA than for continued reinitiation on to endogenous polysomal RNA already being translated. This distinction seems a reasonable possibility in the light of the recent studies of Nelson & Winkler (1987), suggesting differences in mechanism between initiation and reinitiation. It is also consistent with the data of Asselbergs et al. (1979), who studied the translation of exogenous mRNAs (including globin mRNA) microinjected into intact Xenopus oocytes. These messages only become maximally recruited into polysomes after about 6h and, as this happened, their translation became progressively less susceptible to competition from a second species of mRNA, given in a second microinjection. Additionally, there may be variations in behaviour between different exogenous mRNAs in our cell-free systems, since the oocyte extract was able to translate SP6 transcripts of the influenza virus HA mRNA without the addition of reticulocyte S-100 (T. Patrick & A. Colman, unpublished). Clearly the translation of a number of exogenous mRNAs relative to that of endogenous Xenopus mRNAs, 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 Ian Jeffrey for invaluable discussions, and also to Chris Ford for criticism of the manuscript. We are also extremely grateful to Mrs Eileen Willis for typing the manuscript.

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