500 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95631

T H E EFFECT OF F E R T I L I Z A T I O N ON P R O T E I N SYNTHESIS IN T H E

EGG OF T H E SURF CLAM S P I S U L A S O L I D I S S I M A

EUGENE BELL AND RONALD REEDER*

Department of Biology, Institute o/Technology, Cambridge, Mass., and Marine Biological Laboratory, Woods Hole, Mass. (U.S.A.)

(Received January 9th, 1967)

SUMMARY

Mature unfertilized eggs are active metabolically. Fertilization does not initiate protein synthesis: rather it results in a 3-4-fold increase in the rate of protein syn- thesis. The increase in permeability to amino acids which occurs at the time of fer- tilization in the egg of the sea urchin occurs just after meiosis in the clam or about 5o rain after fertilization. After fertilization the rate of protein synthesis increases steadily for at least 3 h. There is at the same time an increase in the number and specific activity of 'polysomes'. I t is suggested that increases in the rate of protein synthesis and in the number of polysomes are controlled by the gradual activation of ribosomes.

INTRODUCTION

The mature unfertilized egg has been thought of as a metabolically inactive cell 1-4. This view has arisen from radioactive isotopic studies which showed that protein synthesis is virtually absent in the mature egg and is initiated by fertiliza- tion. These results were consistent with earlier experiments which reported a marked inciease in 02 consumption at the time of fertilization in some eggs 5.

But most previous experiments have not focused on the permeability change which accompanies fertilization. As a result protein synthesis before fertilization appeared negligible compared with the burst of activity after fertilization when labeled precursor pools increase rapidly.

To s tudy the effect of fertilization on protein synthesis so that changes in permeabili ty did not prejudice results, unfertilized eggs were preloaded with radio- active isotopes, washed and then fertilized in isotope-free medium. Protein synthesis in unfertilized and fertilized eggs was then compared independently of the perme- ability increase which occurs after fertilization.

Our results show that the mature unfertilized clam egg is active metabolically and that fertilization does not initiate protein synthesis but increases the rate of

* Present address: Department of Embryology, Carnegie institution, Baltimore, lVId., (U.S.A.).

Biochim. Biophys. Acta, 142 (1967) 5oo-511

EFFECT OF FERTILIZATION ON PROTEIN SYNTHESIS 501

synthetic activity by a factor of 3-4. The increase in permeabili ty to labeled amino acids occurs in the clam egg at the termination of meiosis 40-5 ° min after fertili- zation rather than at fertilization as it does in the sea urchin egg.

After fertilization the rate of protein synthesis increases steadily until haching. There is a concomitant increase in the number and specific act ivi ty of 'polysomes'. I t is suggested that increases in the rate of protein synthesis and in the number of polysomes are controlled by the gradual activation of ribosomes.

The early development of the clam has been described by ALLEN e and a brief account of procedures for obtaining clam eggs is given by COSTELLO el al. v. MEISEN- HEIMER'S description s of the development of Drisensia, a form which closely resem- bles Spisula is still the most complete, In Spisula, about 15 min after fertilization, the germinal vesicle breaks down and two meiotic divisions follow each other in rapid sequence. After meiosis and the fusion of pronuclei, which occurs 40-5o min after fertilization, cleavage commences. Each cleavage takes approx. 20-25 rain and the first three divisions are relatively synchronous. By five hours a ciliated blastula hatches out of the tough chorion which surrounds the egg. In this paper only develop- ment until the formation of the free-swimming blastula is considered.

MATERIALS AND METHODS

Clams were adult Spisula solidissima which measured about 5-8 inches. Mature eggs were obtained by removing the gonad with surrounding tissue and cutting it open so tha t eggs oozed out. Eggs were collected in sterile sea water and filtered through 4 layers of cheesecloth. They were then allowed to settle by gravity in 250 ml of sterile sea water. After 20 min the sea water and immature eggs remaining in suspension were aspirated off, and the settled eggs resuspended. This process was repeated 3 times to provide a yield of mature eggs. Eggs were then transferred to a I2-ml centrifuge tube and spun in a clinical centrifuge for a time just sufficient to sediment them.

Each ml of eggs to be fertilized was suspended in IOO ml of sterile sea water containing o.17 mg/ml penicillin and o.i mg/ml streptomycin. One ml of sperm sus- pension made by diluting o.I ml dry sperm with IO ml sea water was added to each IOO ml of egg suspension.

After 5 min the eggs were sedimented in 4o-ml tubes in a clinical centrifuge and resuspended in sterile sea water in the presence of antibiotics.

The amounts and time of addition of the radioactive isotope which was generally aaC-labeled algal protein hydrolysate or 14C-labeled reconstituted yeast protein hy- drolysate, are specified with results.

At various intervals aliquots of eggs were removed and their specific act ivi ty determined. Each aliquot was spun lightly in a clinical centrifuge; the supernatant was removed and I ml of cold 2 M NaOH was added. After IO h each aliquot was precipitated with an equal volume of 30 % trichloroacetic acid; the precipitate was collected on a millipore filter and washed twice with 5 ml of 5 °fo trichloroacetic acid. Precipitation with hot trichloroacetic acid did not alter noticeably the number of radioactive counts due to acid-precipitable material.

Filters were cemented to planchettes, air dried, and counted in a low back- ground gas-flow counter. To determine the total radioactive pool, aliquots of eggs

Biochim. Biophys. Acta, 142 (1967) 5oo-511

502 E. BELL, R. REEDER

withdrawn from sea water with radioactive isotope were layered into a mixture of sucrose (o.81 M), isotonic with sea water and sea water mixed one to one, in a I2-ml centrifuge tube. Eggs were pelleted and washed again with 2 ml of sucrose-sea water without resuspending. After discarding the supernatant I ml of i M NaOH was added and after 12 h the sample was transferred to a scintillation vial containing 0.5 ml of i M NH4OH; after IO min 15 ml of Bray 's solution was added and the radio- act ivi ty counted.

The method for extracting and displaying cytoplasmic polysomes is described elsewhere 9. Deoxycholate (0.05 ml io ~o deoxycholate/ml of extract) had no effect on the yield of material having an ultraviolet absorption at 2max 260 m/~.

Autoradiographs were made as follows: eggs were labeled, then washed and fixed on a microscope slide with either 4 % glutaraldehyde in sea water or 5 O//o tri- chloroacetic acid for 12 h. They were then washed with distilled water and allowed to dry. The material was boiled on the slide in 5 ~o trichloroacetic acid for 2 addi- tional hours to extract the acid-soluble radioactivity. Slides were rinsed in distilled water several times and dried. They were dipped in Kodak NTB emulsion and stored in the cold until they were developed.

Eggs collected by settling under gravi ty contained acid-insoluble radioactive isotope and were relatively uniformly radioactive. Rarely an egg was observed which was somewhat more radioactive than the others, but no eggs were seen which did not give evidence of incorporation into acid-insoluble material. An aliquot of the eggs used for autoradiography was fertilized and the incidence of development was greater than 95 %, indicating that virtually all eggs, if not all, were mature.

RESULTS

Amino acid incorporation in un/ertilized and/ertilized eggs. Three types of experiments are presented: in the first, unfertilized eggs were

exposed continuously to radioactive isotope, then fertilized in the presence of radio- active isotope; in the second, unfertilized eggs were pulse-labeled and in the third, unfertilized eggs were labeled, washed and fertilized in sea water without radioactive isotope.

(i) Unfertilized eggs were incubated in sterile sea water in the presence of antibiotics and I0/zC/m114C-labeled algal protein hydrolysate (New England Nuclear), and I-ml aliquots were withdrawn at equal intervals for determination of total and acid-insoluble incorporation and protein by the method of Lowry. In Fig. i it is seen tha t incorporation into acid-insoluble material occurred at a uniform rate throughout the experiment. Radioactivity in the acid-soluble pool can be estimated by the dif- ference between total counts/min and acid-insoluble counts/min. Radioactivity in the acid-soluble pool increased rapidly for the first 2 h, then reached equilibrium and remained constant for the remainder of the experiment.

Incorporation of precursor into acid-insoluble radioactivity in fertilized eggs exposed to radioactivity isotope continuously is shown in Fig. 2. For the first 50 min of development fertilized eggs incorporate amino acids into acid-insoluble material at the same rate as unfertilized controls. When meiosis is completed and the pro- nuclei become visible the rate of incorporation into acid-insoluble material increases sharply.

Biochim. Biophys. Acta, 142 (1967) 5oo-511

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EFFECT OF FERTILIZATION ON PROTEIN SYNTHESIS 503

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The acid-soluble pools in fertilized and unfertilized eggs are similar. At the end of meiosis a sharp increase is observed in fertilized eggs.

(2) Pulse labeling: Similar results were observed when equal aliquots of eggs were pulse-labeled with 5 #C/ml x4C-labeled algal protein hydrolysate for 5-rain in- tervals during the 5 h between fertilization and hatching (Fig. 3). In this experiment only acid-insoluble radioactivity was measured. I t can be seen that fertilized and un- fertilized eggs incorporate at the same rate until then meiosis is completed; the rate

Biochim. Biophys. Acta, 142 (1967) 5oo-511

504 E. BELL, R. REEDER

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of incorporation in the fertilized eggs increases sharply. Eventually the rate begirJs to level off.

Correctly interpreted these experiments reflect an increase in cell permeability at the end of meiosis, signaled by the increased rate of amino acid incorporation into acid-soluble and acid-insoluble material.

(3) Prelabeling: To look at the effect of fertilization on protein synthesis apart from changes in permeability the following experiment was devised.

Two batches of eggs were placed in sea water containing radioactive amino acids for 2 h. One batch was then fertilized; both were washed 3 times and resuspended in plain sea water without isotope. Total radioactivity and acid-insoluble radioac- t ivi ty were determined at intervals. Figs. 4 a and b show the results of 2 experiments. Total radioactivity in the eggs did not change materially for the duration of the experiment after the eggs were resuspended in plain sea water. This implies that little, if any, radioactivity leaked from the cells into the surrounding medium. The only change appeared to be a transfer of radioactivity from the acid-soluble to the acid-insoluble fraction of the eggs. The rate of incorporation into acid-insoluble material remained constant throughout the experiment in the unfertilized eggs. In the fertilized eggs, however, the rate of incorporation in the acid-insoluble fraction increased immediately after fertilization and then gradually tapered off. No change at all was seen in the rate of incorporation at the end of meiosis. These results must be interpreted with caution since the pool of radioactive amino acids available for incorporation is steadily decreasing throughout the experiment. Likewise, the non- radioactive pool size is unknown and may vary for different aminio acds. I t seems certain, however, that there is a significant increase in the real rate of protein syn- thesis immediately after fertilization. The apparent increase in protein synthetic rate seen in the first two experiments at the end of meiosis can be at tr ibuted solely to an increase in permeabili ty to labeled amino acids.

Biochim. Biophys. Acta, 142 (1967) 5oo-511

E F F E C T OF F E R T I L I Z A T I O N ON P R O T E I N S Y N T H E S I S

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F i g . 4- T w o e x p e r i m e n t s s h o w i n g i n c o r p o r a t i o n o f p r e c u r s o r i n t o t o t a l a n d a c i d - i n s o l u b l e r a d i o - a c t i v i t y a f t e r e g g s a r e p r e l a b e l e d a n d w a s h e d , a. I n c o r p o r a t i o n is s h o w n b e f o r e a n d a f t e r f e r t i l i - z a t i o n , b . O n l y a f t e r f e r t i l i z a t i o n . I n (b) r a d i o a c t i v i t y is e x p r e s s e d p e r m l o f e g g s u s p e n s i o n a s d e s c r i b e d i n MATERIALS AND METHODS. I n (a) a s m a l l d e c r e a s e in t o t a l i n c o r p o r a t i o n ( G - C ) ) is o b s e r v e d . I n (b) t o t a l i n c o r p o r a t i o n i n u n f e r t i l i z e d e g g s ( ~ , - A ) a n d i n f e r t i l i z e d e g g s ( & - L X ) r e m a i n s c o n s t a n t a f t e r r e m o v a l o f e g g s f r o m r a d i o a c t i v e i s o t o p e .

Biochim. Biophys. Acta, 142 (1967) 5 o o - 5 1 1

500 E. BELL, R. REEDER

Distribution o/ absorbance and radioactivity in cytoplasmic extracts o/un/ertilized eggs, I-h zygotes and 3-h blastulae

A cytoplasmic extract of unfertilized eggs labeled for 2 h with IO ffC/ml 14C- labeled reconstituted yeast protein hydrolysate (Schwartz) was divided into 2 parts. One part was treated with 4 fig of ribonuelease for IO rain in the cold and each was layered on a 15-3 ° % sucrose gradient. Gradients were spun at 25 ooo rev./min for 3 h in an S.W. 25 rotor on a Spinco ultracentrifuge. Figs. 5a and b show the distri- bution of material with ultraviolet absorption at 2max 260 mff and of radioactivity in the gradients. Only about 30 o~,,o of the counts associated with material heavier than ribosomal particles are moved into the region of single ribosomes after treat- ment with ribonuelease. It can be seen in the control gradient that radioactivity is also associated with single ribosomes and the percent increase in radioactivity in the region of single ribosomes is small (16 %) after treatment with ribonuclease. In other extracts treated with ribonuclease the number of counts moved from the region of particles which were heavier than ribosomal particles, into the peak of single ribosomes was less than in the example illustrated above.

Cytoplasmic extracts were prepared from I-h zygotes and 3-h blastulae after labeling for 5 min with IO ffC 14C-labeled algal protein hydrolysate. After displaying

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Biochim. Biophys. Acta, 142 (1967) 500 511

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EFFECT OF FERTILIZATION ON PROTEIN SYNTHESIS 507

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the extracts in sucrose gradients the specific activity of material sedimenting more rapidly than ribosomes was compared. Material from extracts of 3-h blastulae had an average specific activity of 1.12, nearly twice as great as that from I-h zygote which had a specific activity of 0.68.

By 3 h after fertilization ribonuclease had a pronounced effect on the A260 m~, of a cytoplasmic extract (Figs. 6a and b) whereas the effect was very much smaller when cytoplasmic extracts from the unfertilized egg were treated. Also 80 % of the radioactivity which sediments in the region of the gradient where material is heavier than ribosomal particles is moved into the zone of single ribosomes by ribonuclease, compared with 30 % or less in unfertilized eggs.

Still the absorbance which remains after treatment of the cytoplasm from unfertilized eggs with low concentrations of ribonuclease is due at least in part to aggregates of ribosomes. These aggregates taken from a region of a sucrose gradient in which particles between 17o S and 200 S sediment have been visualized in the electron microscope (Fig. 7). Material from the same region was sedimented, resus- pended in sodium dodecyl sulfate buffer and displaced in a 5-20 % sodium dodecyl

Biochim. Biophys. Acta, I42 (I967) 5oo-511

5 0 8 E. BELL, R. REEDER

sulfate sucrose gradient 1° and yielded I8-S and 28-S RNA. The aggregates could be non-functional polysomes whose messenger RNA is not accessible to attack by en- zyme or non-specific aggregates of ribosomes.

DISCUSSION

Oogenesis may be considered to consist of two distinct phases: (i) the period of synthesis, accumulation, and storage of products which will be used after ferti- l ization and (2) a period of readiness when the egg is fully developed but awaits sperm entry. During the second period the clam egg is fairly impermeable to pre- cursors such as radioactive amino acids but, it is not quiescent. Our results clearly show incorporation of radioactive amino acids into acid-insoluble material in the mature unfertilized clam egg. This incorporation occurs in batches of eggs which subsequently yield close to zoo % fertilization and development. Autoradiographs of labeled unfertilized eggs show that virtually all eggs are labeled. This makes it unlikely that the incorporation is due to contamination by immature oocytes. The incorporation cannot be due to bacterial contamination for two reasons: (I) eggs were washed and allowed to develop in sterile, antibiotic-containing sea water, and (2) unfertilized eggs which were preloaded with radioactive amino acids, washed, and placed in plain sea water continue to incorporate radioactivity from endogenous

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Biochim. Biophys. Acta, 142 (1967) 5oo-511

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pools for at least 6 h. In the unlikely event that bacteria were still stuck to the outside of these washed eggs, the bacteria would quickly exhaust their own endo- genous pools of radioactivity. We conclude, therefore, that mature unfertilized clam eggs do synthesize protein. The apparent rate change in protein synthesis observed when meiosis is completed can be explained by the change in membrane permeability which occurs at that time. A true rate change of the order of 3-4-fold occurs at the time of fertilization. It is possible that continued synthetic activity is necessary to maintain the unfertilized egg in a condition suitable for fertilization.

During the 5-h period after fertilization the rate of synthesis continues to increase as the rate curve in Fig. 3 shows. The increase in the rate of protein syn- thesis which occurs between fertilization and hatching may be related to the number of functional polysomes which also increases.

Two ways in which the number of functional polysomes can increase are through an increase in the number of ribosomes available for protein synthesis and through an increase in the amount or availability of messenger RNA. At the moment the former appears to be the more plausible. In work which will be reported elsewhere we present data which indicate that the rate of protein synthesis increases in the absence of RNA synthesis. This has also been observed in the sea urchin. We have

Biochim. Biophys. Acta, 142 (1967) 5oo - 5 I I

510 E. BELL, R. REEDER

Fig. 7- Electronmicrograph of material sedimenting more rapidly than single ribosomes (Fraction 21, Fig. 5b). Stained with saturated uranyl acetate on carbon-filmed grid. Magnification 12o ooo ×.

reported that most ribosomes in the unfertilized clam egg are larger than adult ribosomes and appear to be non-functionaP 1. Non-functional ribosomes have been observed in the unfertilized egg of the sea urchin 12 and in metaphase HeLa cells 13. As late as 3 h after fertilization many ribosomes in developing blastulae are still of the large variety (E. BELL, unpublished results). Study of the kinetics of activation of ribosomes during early sea urchin development as well as during early clam devel- opment in relation to the increasing rate of protein synthesis will be of special interest.

We would like to suggest that a principal factor involved in control of the rate of protein synthesis during early development may be the gradual activation of ribosomes. The actual rate change of protein synthesis observed at the time of fer- tilization may be due largely to other factors 1~.

ACKNOWLEDGEMENTS

This work was supported by Grant GB 614 from the National Science Foun- dation.

Dr. REEDER was the recipient of an N.I.H. fellowship while a portion of this research was in progress.

We acknowledge the collaboration of Dr. A. BELL of Syracuse University and the Marine Biological Laboratory who did the electron microscopy.

We are grateful to Prof. A. MONROY for having read the manuscript.

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EFFECT OF FERTILIZATION ON PROTEIN SYNTHESIS : 511

R E F E R E N C E S

I T. HULTIN, Exptl. Cell Res., 3 (1952) 494. 2 E. NAKANO AND A. MONROY, Exptl. Cell Res., 14 (1958) 236. 3 P. R. GRoss, L. I. MALKIN AND W. MOYER, Proc. Natl. Acad. Sci. U.S., 51 (1964) 4o7 . 4 D. W. STAFFORD, W. H. SOFER AND R. M. IVERSON, Proc. Natl. Acad. Sci. U.S., 52 (1964) 313 • 5 0 . WARBURG, Z. Physiol. Chem., 66 (191o) 305 . 6 R. D. ALLEN, Biol. Bull., lO 5 (1953) 213. 7 D. P. COSTELLO, M. E. DAVIDSON, A. EGGEKS, M. H. FOX AND C. HENLEY, Methods /or ob-

taining and handling marine eggs and embryos, Marine Biological Labora to r i e s , 1957, p. 113. 8 J. MEISENHEIMER, Z. Wiss. Zool. Abt. A., 69 (19Ol) i. 9 T. HUMPHREYS, S. PENMAN AND E. BELL, Biochem. Biophys. Res. Commz*n., 17 (1964) 618.

io ~T. GILBERT, J. Mol. Biol., 6 (1963) 389 . I I E. BELL, 2rid Decennial Con]. o/the Tissl*e Culture Association, in the press. 12 A. MONROY, R. MAGGIO AND A. M. RINALDI, Proc. Natl. Acad. Sci. U.S., 54 (1965) lO7. 13 J. M. SALB AND P. 1. MARCUS, Proc. Natl. Acad. Sci. U.S., 54 (1965) 1353. 14 F. RoY MACKINTOSH AND E. BELL, Science, in the press.

Biochim. Biophys. Acta, I42 (I967) 5oo 511