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Differential Utilization of Ribosomes for Protein Synthesis During Oogenesis and Early Embryogenesis of Urechis caupo (Echiura)*
Differentiation
Differentiation (1982) 22: 170-174
0
Springcr-Verlag 1982
Differential Utilization of Ribosomes for Protein Synthesis During Oogenesis and Early Embryogenesis of Urechis caupo (Echiura)" FRANCIS C. DAVIS Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32611, USA
A procedure for the preparation of intact polysomes from immature oocytes and embryos of Urechis caupo is described. Analysis of polysome profiles from immature oocytes at several stages of growth reveals that 7 4 % of the ribosomes are present in polysomes at each stage. This indicates that although immature oocytes utilize only a relatively small fraction of their potential protein synthesizing capacity, the apparent protein synthesizing capacity of the immature oocytes increases in parallel with the accumulation of ribosomes during oogenesis. The polysomes in mature oocytes are barely detectable, with approximately 0.6% of the ribosomes present in polysomes. During early stages of embryogenesis when there is no net increase in the total ribosomes per embryo, ribosomes are transferred into polysomes. The percentage of ribosomes in polysomes increases to 4.1% by the two-cell stage and continues to increase approximately linearly with time of development until 50% of the ribosomes are in polysomes at the trochophore larval stage.
Introduction The mature oocytes of most animals contain relatively large quantities of ribosomes [4]. In the mature oocytes the rate of protein synthesis is typically low or not detectable. Consistent with the low rate of protein synthesis, ribosomes are present as ribosomal subunits and monosomes with few if any present in polysomes [lo, 16, 18, 20, 221. The ribosomes present in the mature oocytes represent a stockpile of machinery for the rapid synthesis of protein which occurs during early stages of embryonic development [lo, 16, 18, 221. Following fertilization, the rate of protein synthesis increases in parallel with the increase in the fraction of ribosomes present in polysomes [14, 16, 18, 221. Although some synthesis of ribosomes occurs during embryogenesis, virtually all of the ribosomes appearing in polysomes during early embryogenesis have been synthesized and accumulated during oogenesis [l, 91. A variety of proteins are present in the mature oocytes which, like the ribosomes, were synthesized and accumulated during oogenesis [4]. Since both ribosomes and proteins are simultaneously accumulated in the growing oocytes, the ribosomes present in the mature oocytes may have functioned in the synthesis of some or all of the proteins accumulated during oogenesis. No information has been reported concerning the extent to which the ribosomes synthesized and accumulated during oogenesis are utilized for the synthesis of proteins simultaneously accumulated in the immature oocytes. Urechis caupo is an ideal system to examine the utilization of ribosomes for protein synthesis during oogenesis. The immature oocytes of Urechis grow as single cells free of accessory cells in the fluid of the coelomic cavity [ll, 171. Procedures are available to isolate immature oocytes free of * Journal Paper No. 3517 from the Florida Agricultural Experimental Station, Gainesville, Florida, USA
contaminating cells and to fractionate the immature oocytes into several stages of growth [6,8,15]. Ribosomes, both RNA and protein, as well as other proteins are synthesized and accumulated in the immature oocytes of Urechis [8, 151. This requires that ribosomes present in the mature oocytes be utilized in the immature oocytes for the synthesis of the proteins accumulated in the growing oocytes. In this paper the fraction of the total ribosomes required for the synthesis of proteins accumulated in the immature oocytes and for protein synthesis in embryos after fertilization is examined. Methods Adult specimen of Urechis caupo were collected from the intertidal mud flats of Bodega Bay, California. The procedures for maintaining the Urechis in the laboratory, collecting mature gametes, and culturing the embryos have been described previously [5, 81. Immature oocytes were isolated free of blood cells and fractionated according to size on 11.0-17.5% Ficoll gradients in Ca2+-freesea water (CaFSW) [6] to which 2.0 mM cyclohexamide was added. Labeling Nascent Polypeptides in Embryos and Immature Oocytes. Gastrula stage embryos grown in Milipore-filtered sea water (FSW)at 17" C were incubated with 5 pCi/ml 3H-leucine (56.6 Ci/mM) for 3 min to label nascent polypeptides. Incorporation was stopped by adding 10 volumes of filtered sea water (4" C) containing 1mM cyclohexamide. Embryos were collected by centrifugation at 2,500 g for 5 min. To label nascent polypeptide in immature oocytes, the total population of immature oocytes was separated from most of the pigmented blood cells as previously described [6]. Immature oocytes were resuspended at 4" C in 5 ml of 22% Ficoll in CaFSW, transferred to a 5 O m l centrifuge tube, 0301-4681/82/0022/0170/$01.OO
F. C. Davis: Polysomes in Urechis Oocytes and Embryos
overlayered with 10 ml of 17% Ficoll in CaFSW followed by 1Oml CaFSW, and centrifuged at 500g for 5min. The immature oocytes were removed from the 17% Ficoll-CaFSW interface, and pelleted by centrifugation at 500g for 1min. The immature oocytes were resuspended in CaFSW, preincubated for 5min at l T C , and labeled with 50pCi/ml 3H-leucine(56.6 Ci/mM) for 3 min. Incorporation was stopped by adding 10 volumes of CaFSW (4" C) containing 2 mM cyclohexamide, and immature oocytes were collected by centrifugation at 2,500 g for 5 min. Polysomes from embryos and immature oocytes were prepared as described below. The susceptibility of the polysomes to ribonuclease (RNase)was determined by incubating the cleared lysate with lOpg/ml RNase A for 30 min at 4OC. The distribution of labeled polypeptidesin sucrosegradients was determined as previously described [6]. Preparation of Polysomes. Polysomes were prepared from oocytes and embryos as previously described [7] except that 1.0mM cyclohexamide was added to the polysome homogenization and sucrose gradient buffers. Approximately 0.1-0.2 ml containing 8-10 Am units of cleared lysate was layered on 4.7ml, 1 7 4 0 % linear sucrose gradients and centrifuged at 50,000 rpm for 45 min in a SW 50.1 rotor (Beckman). The absorbance distribution was monitored at 254nm with an ISCO (Instrument Specialties Co.) density gradient fractionator. The fraction of the ribosomes in polysomes was determined from the area of the absorbance profile in the subunits, monosome, and polysome regions of the gradient. The area of the polysome absorbance profile was corrected for absorbance in the polysome region of the gradient after homogenates were treated with 10 pg/d RNase at 4" C for 30 min prior to centrifugation.
Results and Mscasaion Conditions for Preparation of Polysomes. Polysomes can be prepared from Urechis embryos using a modification of the procedure described for sea urchin embryos by Hirama and Mano [13]. Gastrula-stage embryos were used to determine the optimal ionic conditions for polysome preparation. Varying the M2+ and K+ concentrations in both homogenization and sucrose gradient buffers indicated that buffers containing 5mM M e and 0.45M k+ were optimal for analysis of polysomes, subunits, and monosomes present in embryos. These concentrations of Mgz+and K+ are similar to the ionic concentrations required to maintain the integrity of polysomes in other marine invertebrate embryos [lo, 13,16,18]. A typical distribution of polysomes, subunits, and monosomes from late gastrula stage embryos is shown in Fig. 1A. The ribosomal subunits and monosomes were identified in the sedimentation profile by extracting RNA from each region of the gradient and characterizing the RNA species by sucrose gradient centrifugation (results not shown). To determine if the ribosomes sedimenting more rapidly than monosomes are polysomes and represent the proportion of ribosomes in polysomes in vivo, the distribution of pulse-labeled protein and RNase sensitivityof the structuresin the polysome region of the gradient were examined. Late gastrula stage embryos were pulsed with 5 pCi/ml 3H-leucine for 3min and the labeled polypeptide distribution in the monosome and polysome regions of the gradient examined (Fig. 1A). The labeled polypeptide either remains at the top of the sucrose gradient or sediments into the polysome region of
171
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Q. 1. Sucrose gradient distribution of ribosomes and 3H-leucine-labeled polypeptides in gastrula stage embryos and immature oocyte plyribosomes before and after FWase digestion. Twenty-four hour embryos (Aand B)were labeled with 5.0 pCilml [3H]leucinefor 3 min, or immature oocytes (C and D) were collected from a discontinuous Ficoll gradient and labeled with 50 pCilml [3H]leucine for 3.5 min. Embryos or oocytes were collected, homogenized, and the postmitochondrial supernate prepared. Samples containing 8-10 Am units were layered on 4.7 ml17-60% sucrose gradients either before (Aand C) or after (Band D) digestionwith 10 pg/mI RNase for 30 min at 4" C. After centrifugation at 288,ooOg for 45 min, the absorbance profie at Am (-) was monitored with a ISCO gradient fractionator, and the distribution of labeled polypeptide (-0--0-) determined by precipitating 0.25ml fractions with 10% CC13COOH. The arrow indicates the position of the monosomes in the sucrose gradients
the gradient. No labeled peak sedimented coincident with the monosomes which would indicate breakdown of polysomes to yield monosomes with nascent peptide attached. Treatment of the homogenate with RNase prior to sucrose gradient centrifugation causes a shift of both the absorbance profile and labeled polypeptide from the polysome region of the gradient to the monosome region (Fig. 1B). This shift in the sedimentation profile of the pulse-labeled protein is consistent with a degradation of the messenger RNA in the polysomes to yield monosomes with nascent peptides attached. Polysomes were prepared from the total population of immature oocytes that had been pulse labeled with 3H-leucine to determine if polysomes remained intact during oocyte isolation and sue fractionation. A cell preparation enriched with immature oocytes was labeled for 3.5 min in vitro with 50 pCi/ml 3H-leucine.The polysome profile from the mixture of immature oocytes is shown in Fig. 1C. A smaller fraction of the ribosomes is present in polysomes than is present in the late gastrula stage embryos (Fig. 1A). Most of the labeled polypeptides are distributed at the top of the gradient and in the polysome region of the gradient. A small peak of label
F. C. Davis: Polysomes in Urechis Oocytes and Embryos
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I
Table 1. Percentage of ribosomes in polysomes in immature oocytes, mature oocytes, and embryosB
Developmental stageb Immature oocytes 10- 40pm 40- 5 5 p 55- 87pm 87- 125 pm Mature oocytes Embryos 2-cell (2 h) 4-cell (2.5 h) 16-cell (3.5 h) (8 h) Blastula Early gastrula (16-17 h) Late gastrula (22-24 h) Trochophore (40h) a
Ribosomes in polysomes"
5.3d 8.5 f 2.9 7.3 f 2.5 8.7 f 1.4 0.6 f 0.6c 4.1 f 0.9 4.9 f 0.6 6.9 f 1.2 8.7 f O . 8 23.3 f 1.9 31.9 f 2 . 4 50.4 f 5.3
(5) (5) (5)
-1
(4) (6) (3) (4)
---___
(4) (5) (4)
(3)
The percentage of ribosomes in polysomes was determined by dividing the area under the absorbance profile of polysomes, by the total area under the absorbance profile of ribosomal subunits, monosomes, and polysomes. The absorbance of the polysome profiles are corrected for absorbance remaining after RNase digestion, and the absorbance profile of ribosomal subunits, monosomes, and polysomes are corrected for absorbance contributed by small molecular weight components. Time in parentheses indicates the time after fertilization Mean f standard deviation. The number of determinations is shown in parentheses. Average of duplicate determinations for a single immature oocyte preparation Determined indirectly by comparing the polysome profiles of sucrose gradients overloaded with 16 A m units of postmitochondrial supernate from either mature oocytes or twocell embryos (Fig. 3, insert). The percentage of ribosomes in polysomes of mature oocytes was calculated by multiplying the percentage of the ribosomes in polysomes at the two-cell stage (4.1%, determined under standard conditions) by the ratio of the area under the polysome profiie of mature oocytes to the area under the polysome profile of the twocell stage
sediments coincident with the monosome peak in the absorbance profile, indicating that some polysome breakdown has occurred during preparation. In three separate labeling experiments, the fraction of the label in the monosome region of the gradient ranged from 4 to 8% of the total label in the monosomes and polysomes. This indicates that minimal degradation of polysomes occurred during the procedures used to fractionate the immature oocytes according to size. The average observed polysome degradation would lower the measured percentage of ribosomes in the polysomes of immature oocytes (Tablel) by about 0.5%. As with the gastrula stage polysomes, treatment of the cleared lysate with RNase caused a shift of both the absorbance profile and labeled polypeptides from the polysome to the monosome region of the sucrose gradients (Fig. 1D). Polysomes in Immature Oocytes at Different Stages of Growth. Polysomes were prepared from immature oocytes that had been fractionated on the basis of differences in size and therefore age [8]. Four immature oocytes fractions were isolated from Ficoll gradients with diameters of 10-40 pm, 40-55 p, 55-87 pm, and 87-125 p. Polysomes were
L -\ ----_--_
10-40
0
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Fig. 2. Distribution of polysomes from immature and mature oocytes. Mature oocytes were collected and washed three times in CaFSW.
Total immature oocytes were collected from the surface of a cell pellet formed by centrifuging the coelomic cavity contents in a hand centrifuge for 1min. The immature oocytes were suspended in 21% Ficoll in CaFSW containing 2.0mM cyclohexamide, layered under 11.0-17.5% Ficoll gradients in CaFSW containing 2.0 mM cyclohexamide, and centrifuged at 5OOg for 1Omin. Gradient fractions containing immature oocytes in sue ranges of 10-40 pm, 40-55 pm, 55-87pn1, and 87-125pn were pooled. The ptmitochondrial supernates were prepared and the sucrose gradient distribution of ribosomes and polysomes analyzed as described in Fig. 1. The polysome region of the absorbance profie is shown before (-) and after (---) RNase digestion
prepared from each immature oocyte stage and fractionated on sucrose gradients. Polysome profiles from each immature oocyte stage are shown in Fig. 2. A similar fraction of the ribosomesis present in polys6mes at each stage. Repeated analyses of the polysome profiles of the immature oocytes indicate that the 1 0 - 4 0 p , 4 0 - 5 5 p , 5 5 - 8 7 p , and 87-125 p immature oocytes have 5.3,8.5,7.3, and 8.7% of their ribosomes in polysomes, respectively (Table 1). The values for the percentage of ribosomes in polysomes in each of the immature oocyte stages do not differ significantly during growth of the immature oocytes. As the immature o w e s grow, the ribosomal RNA content increases and hence the total ribosome content increases [8], The fraction of ribosomes present in polysomes is approximately constant which indicates that the rate of protein synthesis increases during growth of the immature oocytes. As the immature oocytes grow from 10-40 pm, to 40-55 pm, to 55-87 p,and to 87-125 p;the average RNA content per cell is 0.7,5.2,11.0, and 32.0 pg per cell respectively [8]. Since the total RNA is directly proportional to the ribosome content of each oocyte sue class, the increasing ribosome content and constant fraction of ribosomes in polysomes suggests that the rate of protein synthesis increases two-fold from the 40-55 pm immature o w e s to the 5 5 - 8 7 p immature oocytes and three-fold from the 55-87 p immature oocytes to the 87-125 pm immature oocytes. No estimate of a change in the rate of protein synthesis from the 10-40 pimmature oocytes has been made due to the possible errors in the measurements of both the fraction of ribosomes in polysomes (Table 1) and the total RNA content of this size class of oocytes [8]. The
F. C. Davis: Polysomes in Urechis Oocytes and Embryos
173
protein to RNA ratio in the immature oocytes increases as the oocytes grow [8, 151. At present, no information is available about the turnover of RNA and protein during oogenesis in Urechis. If it is assumed that the rate of protein turnover is constant, the apparent increase in the rate of protein synthesis is consistent with what is known about the accumulation of RNA and protein during oogenesis [8, 151. The rate of synthesis and accumulation of RNA in the 40-55 pm immature oocyte is approximately equal to that in the 55-87 pm immature oocytes; however, the observed increase in the protein to RNA ratio suggests that an increase in the rate of protein synthesis must have occurred between the two stages. The rate of RNA synthesis is seen to increase about 4- to 5-fold in the 87-125 pm immature oocytes and the protein to RNA ratio continues to increase. Consequently a further increase in the rate of protein synthesis (i.e., total number of ribosomes utilized to synthesis the proteins) is expected between 55-87 pm and 87-125 pm stages. Polysomes in Mature Oocytes. The mature oocytes continue to synthesizeproteins prior to fertilization [12].To determine the fraction of the ribosomes utilized to form the newly synthesued proteins in the mature oocytes, polysomes were prepared from mature oocytes. The absorbance profile of the distribution of ribosomes in subunits, monosomes, and polysomes indicates that only a small fraction of the ribosomes are in polysomes (Figs. 2 and 3). Comparison of the mature oocyte absorbance profiles with the absorbance profiles from the two-cell stage before and after RNase digestion of the cleared lysate (Fig. 3, insert) indicates that about 0.6% of the ribosomes are in polysomes (Table 1). The mature oocytes have an average RNA content of 89 pg per cell, approximately a 4-fold increase from the average RNA content of the 85-125 pm immature oocytes. The fraction of the ribosomes in polysomes in mature oocytes is approximately 15-fold less than in the 87-125 pm immature oocytes. The increase in the total number of ribosomes in the mature oocytes coupled with the decrease in the fraction of the ribosomes in polysomes, indicate that there is a decrease in the total number of ribosomes in polysomes in the mature oocyte compared to the 87-125 pm immature oocyte. As a consequence of the decrease of ribosomes in polysomes, the rate of protein synthesis in the mature oocytes appears to be approximately one fifth of that in the late stage immature oocytes. The mature oocytes may be held in the storage organ for months before spawning [3] and since there is no evidence of protein accumulationin the mature oocytes during storage, the continued protein synthesis appears to be the level of synthesis required to replace the proteins lost due to turnover. Polysomes in Developing Embryos. Polysomes were prepared from mature oocytes and embryos at different stages of development, from the two-cell stage at 2 h postfertilizationto the trochophore larval stage at 40 h postfertilization. Polysome profiles from each stage of development are shown in Fig. 3. The fraction of the ribosomes in polysomes increases throughout early development. The increase in the fraction of ribosomes in polysomes begins soon after fertilization and by the two-cell stage has increased to approximately4% from the 0.6% that was present in the mature oocyte (Table 1). The increase in the fraction of the ribosomes in polysomesis nearly linear with time of development through 40 h postfertilization (Table 1).
0.05
-.----___
0 Top
--. Trochophore
Bottom
Fii. 3. Distribution of polysomes from mature oocytes and embryos.
Mature oocytes were fertilized, washed by settling three-times from filtered sea water, and embryos collected at the stages indicated. Cyclohexamide was added to 2.0mM, the cultures chilled, and embryos collected by centrifuging at 5,OOO g for 2 min. The postmitochondrial supernates were prepared and the sucrose gradient distribution of ribosomes and polysomes analyzed as described in Fig. 1. The polysomesregion of the absorbanceprofile is shown before (-) and after (- - -) RNase digestion. The insert shows greater d e w of the polysome profilesfrom sucrose gradients on which 16 Am units of postmitochondrial supernate from mature oocytes and the two-cellstage were layered
The redistribution of ribosomes into polysomes is very similar to the increase in the fraction of ribosomes present during early development of other animals [lo, 16,18,21,22]. In Urechis the total RNA content of the mature oocytes and embryos through the trochophore larval stage is constant [19]. Since the ribosomal RNA comprises the bulk of the RNA in the embryos, a constant RNA content indicates that the number of ribosomes per embryo is essentially constant through early development. The increase in the fraction of the ribosomes in polysomes should reflect an increase in the rate of protein synthesis per embryo [14,16,18,221. If it is assumed that the efficiency of translation by ribosomes in polysomes is equal at each stage, the increase in the polysome content of embryos indicates that the rate of protein synthesis increases more than 50-fold from the mature oocyte to the trochophore larval stage. However, if the rate of polypeptide chain elongation increases after fertilization as has been observed in the sea urchin [2],the increase in the rate of protein synthesis could be even greater. Acknowledgemen&. I would like to thank Dr. J. W. Brookbank and
Dr. L. 0. Ingram for their helpful and critical review of this manuscript. This work was supported in part by U. S. Public Health Service Grant RRo7021.
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References 1. Brown DD, Littna E (1964) RNA synthesis during the develop ment of XenopuF laevis, the South African clawed toad. J Mol Biol 8: 669 2. Danilchik MV, Hille MB (1981) Sea urchin eggs and embryo ribosomes: Differences in translational activities in a cell-free system. Develop Biol 84: 291 3. Das NK (1976) Cytochemical and biochemical analysis of development of Urechis oocytes. Am Zoo1 16: 345 4. Davidson EH (1976) Gene activity in early development. Academic Press, New York 5. Davis FC (1975) Unique sequence DNA transcripts present in mature oocytes of Urechis caupo. Biochim Biophys Acta 390: 33 6. Davis FC, Davis RW (1978) Polyadenylationof RNA in immature
oocytes and early cleavage of Urechb cuupo. Develop Biol 66: 86 7. Davis FC, Mullersman RW (1981) Processing of the ribonucleic acid in the large ribosomal subunits of Urechis cuupo. Biochemistry 20: 3554 8. Davis FC, Wilt FH (1972) RNA synthesis during oogenesis in the echiuroid worm Urechis caupo. Develop Biol 27: 1 9. Emmerson CP, Humphreys T (1970) Regulation of DNA-like RNA and the apparent activation of ribosomal RNA synthesis in sea urchin embryos: Quantitative measurements of newly synthesized RNA. Develop Biol 23: 86 10. Firtel RA, Monroy A (1970) Polysomes and RNA synthesis during early development of the surf clam Spisda solidissima. Develop Biol 21: 87 11. Gould M (1%7) Echiuroid worms: Urechis. In: Wilt FH, Wessels N (eds) Methods in developmental biology. Crowell-Collier,New York, p 163
12. Gould MC (1%9) A comparison of RNA and protein synthesis in
fertilized and unfertilized eggs of Urechis cuupo. Develop Biol 19: 482 13. Huama M, Mano Y (1973) Polysomes of the sea urchin embryo: An improved method for extraction of integrated polysomes. Develop Growth Differentiation 15: 269 14. Humphreys T (1%9) Efficiency of translation of messenger-RNA 15.
16. 17. 18. 19. 20. 21. 22.
before and after fertilization in sea urchin. Develop Biol 20: 435 Miller JH, Epel D (1973) Studies of oogenesis in Urechiscuupo 11. Accumulation during oogenesis of carbohydrate, RNA, microtubule protein, and soluble, mitochondrial and lysosornal enzymes. Develop Biol 32: 331 Mirkes PE (1972) Polysomes and protein synthesis during development of Ilyanassa obsoletu. Exptl Cell Res 74: 503 Newby WW (1940) The embryology of the echiuroid worm, Urechis caupo. Mem Am Phil Soc 16: 1 Rinaldi AM, Monroy A (1%9) Polyribosome formation and RNA synthesis in the early postfertilization stages of the sea urchin egg. Develop Biol 19: 73 Schwartz MC (1970) Nucleic acid metabolism in oocytes and embryos of Urechis cuupo. Develop Biol 23: 241 Smith DL (1975) Molecular events during oocyte maturation. In: Weber R (ed) The biochemistry of animal development, Vol. 111. Academic Press, New York, p 1 Spieth J, Whiteley AH (1981) Polysome formation and Poly(A)antaining RNA in embryos of the sand dollar, Dendraster excentricus. Wilhelm Roux’s Arch 190: 111 Woodland HR (1974) Changes in the polysome content of developing Xenopus laevb embryos. Develop Biol 40:90
Received February 1982Accepted in revised form June 1982
Differentiation (1982) 22: 170-174
0
Springcr-Verlag 1982
Differential Utilization of Ribosomes for Protein Synthesis During Oogenesis and Early Embryogenesis of Urechis caupo (Echiura)" FRANCIS C. DAVIS Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32611, USA
A procedure for the preparation of intact polysomes from immature oocytes and embryos of Urechis caupo is described. Analysis of polysome profiles from immature oocytes at several stages of growth reveals that 7 4 % of the ribosomes are present in polysomes at each stage. This indicates that although immature oocytes utilize only a relatively small fraction of their potential protein synthesizing capacity, the apparent protein synthesizing capacity of the immature oocytes increases in parallel with the accumulation of ribosomes during oogenesis. The polysomes in mature oocytes are barely detectable, with approximately 0.6% of the ribosomes present in polysomes. During early stages of embryogenesis when there is no net increase in the total ribosomes per embryo, ribosomes are transferred into polysomes. The percentage of ribosomes in polysomes increases to 4.1% by the two-cell stage and continues to increase approximately linearly with time of development until 50% of the ribosomes are in polysomes at the trochophore larval stage.
Introduction The mature oocytes of most animals contain relatively large quantities of ribosomes [4]. In the mature oocytes the rate of protein synthesis is typically low or not detectable. Consistent with the low rate of protein synthesis, ribosomes are present as ribosomal subunits and monosomes with few if any present in polysomes [lo, 16, 18, 20, 221. The ribosomes present in the mature oocytes represent a stockpile of machinery for the rapid synthesis of protein which occurs during early stages of embryonic development [lo, 16, 18, 221. Following fertilization, the rate of protein synthesis increases in parallel with the increase in the fraction of ribosomes present in polysomes [14, 16, 18, 221. Although some synthesis of ribosomes occurs during embryogenesis, virtually all of the ribosomes appearing in polysomes during early embryogenesis have been synthesized and accumulated during oogenesis [l, 91. A variety of proteins are present in the mature oocytes which, like the ribosomes, were synthesized and accumulated during oogenesis [4]. Since both ribosomes and proteins are simultaneously accumulated in the growing oocytes, the ribosomes present in the mature oocytes may have functioned in the synthesis of some or all of the proteins accumulated during oogenesis. No information has been reported concerning the extent to which the ribosomes synthesized and accumulated during oogenesis are utilized for the synthesis of proteins simultaneously accumulated in the immature oocytes. Urechis caupo is an ideal system to examine the utilization of ribosomes for protein synthesis during oogenesis. The immature oocytes of Urechis grow as single cells free of accessory cells in the fluid of the coelomic cavity [ll, 171. Procedures are available to isolate immature oocytes free of * Journal Paper No. 3517 from the Florida Agricultural Experimental Station, Gainesville, Florida, USA
contaminating cells and to fractionate the immature oocytes into several stages of growth [6,8,15]. Ribosomes, both RNA and protein, as well as other proteins are synthesized and accumulated in the immature oocytes of Urechis [8, 151. This requires that ribosomes present in the mature oocytes be utilized in the immature oocytes for the synthesis of the proteins accumulated in the growing oocytes. In this paper the fraction of the total ribosomes required for the synthesis of proteins accumulated in the immature oocytes and for protein synthesis in embryos after fertilization is examined. Methods Adult specimen of Urechis caupo were collected from the intertidal mud flats of Bodega Bay, California. The procedures for maintaining the Urechis in the laboratory, collecting mature gametes, and culturing the embryos have been described previously [5, 81. Immature oocytes were isolated free of blood cells and fractionated according to size on 11.0-17.5% Ficoll gradients in Ca2+-freesea water (CaFSW) [6] to which 2.0 mM cyclohexamide was added. Labeling Nascent Polypeptides in Embryos and Immature Oocytes. Gastrula stage embryos grown in Milipore-filtered sea water (FSW)at 17" C were incubated with 5 pCi/ml 3H-leucine (56.6 Ci/mM) for 3 min to label nascent polypeptides. Incorporation was stopped by adding 10 volumes of filtered sea water (4" C) containing 1mM cyclohexamide. Embryos were collected by centrifugation at 2,500 g for 5 min. To label nascent polypeptide in immature oocytes, the total population of immature oocytes was separated from most of the pigmented blood cells as previously described [6]. Immature oocytes were resuspended at 4" C in 5 ml of 22% Ficoll in CaFSW, transferred to a 5 O m l centrifuge tube, 0301-4681/82/0022/0170/$01.OO
F. C. Davis: Polysomes in Urechis Oocytes and Embryos
overlayered with 10 ml of 17% Ficoll in CaFSW followed by 1Oml CaFSW, and centrifuged at 500g for 5min. The immature oocytes were removed from the 17% Ficoll-CaFSW interface, and pelleted by centrifugation at 500g for 1min. The immature oocytes were resuspended in CaFSW, preincubated for 5min at l T C , and labeled with 50pCi/ml 3H-leucine(56.6 Ci/mM) for 3 min. Incorporation was stopped by adding 10 volumes of CaFSW (4" C) containing 2 mM cyclohexamide, and immature oocytes were collected by centrifugation at 2,500 g for 5 min. Polysomes from embryos and immature oocytes were prepared as described below. The susceptibility of the polysomes to ribonuclease (RNase)was determined by incubating the cleared lysate with lOpg/ml RNase A for 30 min at 4OC. The distribution of labeled polypeptidesin sucrosegradients was determined as previously described [6]. Preparation of Polysomes. Polysomes were prepared from oocytes and embryos as previously described [7] except that 1.0mM cyclohexamide was added to the polysome homogenization and sucrose gradient buffers. Approximately 0.1-0.2 ml containing 8-10 Am units of cleared lysate was layered on 4.7ml, 1 7 4 0 % linear sucrose gradients and centrifuged at 50,000 rpm for 45 min in a SW 50.1 rotor (Beckman). The absorbance distribution was monitored at 254nm with an ISCO (Instrument Specialties Co.) density gradient fractionator. The fraction of the ribosomes in polysomes was determined from the area of the absorbance profile in the subunits, monosome, and polysome regions of the gradient. The area of the polysome absorbance profile was corrected for absorbance in the polysome region of the gradient after homogenates were treated with 10 pg/d RNase at 4" C for 30 min prior to centrifugation.
Results and Mscasaion Conditions for Preparation of Polysomes. Polysomes can be prepared from Urechis embryos using a modification of the procedure described for sea urchin embryos by Hirama and Mano [13]. Gastrula-stage embryos were used to determine the optimal ionic conditions for polysome preparation. Varying the M2+ and K+ concentrations in both homogenization and sucrose gradient buffers indicated that buffers containing 5mM M e and 0.45M k+ were optimal for analysis of polysomes, subunits, and monosomes present in embryos. These concentrations of Mgz+and K+ are similar to the ionic concentrations required to maintain the integrity of polysomes in other marine invertebrate embryos [lo, 13,16,18]. A typical distribution of polysomes, subunits, and monosomes from late gastrula stage embryos is shown in Fig. 1A. The ribosomal subunits and monosomes were identified in the sedimentation profile by extracting RNA from each region of the gradient and characterizing the RNA species by sucrose gradient centrifugation (results not shown). To determine if the ribosomes sedimenting more rapidly than monosomes are polysomes and represent the proportion of ribosomes in polysomes in vivo, the distribution of pulse-labeled protein and RNase sensitivityof the structuresin the polysome region of the gradient were examined. Late gastrula stage embryos were pulsed with 5 pCi/ml 3H-leucine for 3min and the labeled polypeptide distribution in the monosome and polysome regions of the gradient examined (Fig. 1A). The labeled polypeptide either remains at the top of the sucrose gradient or sediments into the polysome region of
171
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15
Bottom Fraction Number
Q. 1. Sucrose gradient distribution of ribosomes and 3H-leucine-labeled polypeptides in gastrula stage embryos and immature oocyte plyribosomes before and after FWase digestion. Twenty-four hour embryos (Aand B)were labeled with 5.0 pCilml [3H]leucinefor 3 min, or immature oocytes (C and D) were collected from a discontinuous Ficoll gradient and labeled with 50 pCilml [3H]leucine for 3.5 min. Embryos or oocytes were collected, homogenized, and the postmitochondrial supernate prepared. Samples containing 8-10 Am units were layered on 4.7 ml17-60% sucrose gradients either before (Aand C) or after (Band D) digestionwith 10 pg/mI RNase for 30 min at 4" C. After centrifugation at 288,ooOg for 45 min, the absorbance profie at Am (-) was monitored with a ISCO gradient fractionator, and the distribution of labeled polypeptide (-0--0-) determined by precipitating 0.25ml fractions with 10% CC13COOH. The arrow indicates the position of the monosomes in the sucrose gradients
the gradient. No labeled peak sedimented coincident with the monosomes which would indicate breakdown of polysomes to yield monosomes with nascent peptide attached. Treatment of the homogenate with RNase prior to sucrose gradient centrifugation causes a shift of both the absorbance profile and labeled polypeptide from the polysome region of the gradient to the monosome region (Fig. 1B). This shift in the sedimentation profile of the pulse-labeled protein is consistent with a degradation of the messenger RNA in the polysomes to yield monosomes with nascent peptides attached. Polysomes were prepared from the total population of immature oocytes that had been pulse labeled with 3H-leucine to determine if polysomes remained intact during oocyte isolation and sue fractionation. A cell preparation enriched with immature oocytes was labeled for 3.5 min in vitro with 50 pCi/ml 3H-leucine.The polysome profile from the mixture of immature oocytes is shown in Fig. 1C. A smaller fraction of the ribosomes is present in polysomes than is present in the late gastrula stage embryos (Fig. 1A). Most of the labeled polypeptides are distributed at the top of the gradient and in the polysome region of the gradient. A small peak of label
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I
Table 1. Percentage of ribosomes in polysomes in immature oocytes, mature oocytes, and embryosB
Developmental stageb Immature oocytes 10- 40pm 40- 5 5 p 55- 87pm 87- 125 pm Mature oocytes Embryos 2-cell (2 h) 4-cell (2.5 h) 16-cell (3.5 h) (8 h) Blastula Early gastrula (16-17 h) Late gastrula (22-24 h) Trochophore (40h) a
Ribosomes in polysomes"
5.3d 8.5 f 2.9 7.3 f 2.5 8.7 f 1.4 0.6 f 0.6c 4.1 f 0.9 4.9 f 0.6 6.9 f 1.2 8.7 f O . 8 23.3 f 1.9 31.9 f 2 . 4 50.4 f 5.3
(5) (5) (5)
-1
(4) (6) (3) (4)
---___
(4) (5) (4)
(3)
The percentage of ribosomes in polysomes was determined by dividing the area under the absorbance profile of polysomes, by the total area under the absorbance profile of ribosomal subunits, monosomes, and polysomes. The absorbance of the polysome profiles are corrected for absorbance remaining after RNase digestion, and the absorbance profile of ribosomal subunits, monosomes, and polysomes are corrected for absorbance contributed by small molecular weight components. Time in parentheses indicates the time after fertilization Mean f standard deviation. The number of determinations is shown in parentheses. Average of duplicate determinations for a single immature oocyte preparation Determined indirectly by comparing the polysome profiles of sucrose gradients overloaded with 16 A m units of postmitochondrial supernate from either mature oocytes or twocell embryos (Fig. 3, insert). The percentage of ribosomes in polysomes of mature oocytes was calculated by multiplying the percentage of the ribosomes in polysomes at the two-cell stage (4.1%, determined under standard conditions) by the ratio of the area under the polysome profiie of mature oocytes to the area under the polysome profile of the twocell stage
sediments coincident with the monosome peak in the absorbance profile, indicating that some polysome breakdown has occurred during preparation. In three separate labeling experiments, the fraction of the label in the monosome region of the gradient ranged from 4 to 8% of the total label in the monosomes and polysomes. This indicates that minimal degradation of polysomes occurred during the procedures used to fractionate the immature oocytes according to size. The average observed polysome degradation would lower the measured percentage of ribosomes in the polysomes of immature oocytes (Tablel) by about 0.5%. As with the gastrula stage polysomes, treatment of the cleared lysate with RNase caused a shift of both the absorbance profile and labeled polypeptides from the polysome to the monosome region of the sucrose gradients (Fig. 1D). Polysomes in Immature Oocytes at Different Stages of Growth. Polysomes were prepared from immature oocytes that had been fractionated on the basis of differences in size and therefore age [8]. Four immature oocytes fractions were isolated from Ficoll gradients with diameters of 10-40 pm, 40-55 p, 55-87 pm, and 87-125 p. Polysomes were
L -\ ----_--_
10-40
0
TOP
J
-----------________.
Bottom
Fig. 2. Distribution of polysomes from immature and mature oocytes. Mature oocytes were collected and washed three times in CaFSW.
Total immature oocytes were collected from the surface of a cell pellet formed by centrifuging the coelomic cavity contents in a hand centrifuge for 1min. The immature oocytes were suspended in 21% Ficoll in CaFSW containing 2.0mM cyclohexamide, layered under 11.0-17.5% Ficoll gradients in CaFSW containing 2.0 mM cyclohexamide, and centrifuged at 5OOg for 1Omin. Gradient fractions containing immature oocytes in sue ranges of 10-40 pm, 40-55 pm, 55-87pn1, and 87-125pn were pooled. The ptmitochondrial supernates were prepared and the sucrose gradient distribution of ribosomes and polysomes analyzed as described in Fig. 1. The polysome region of the absorbance profie is shown before (-) and after (---) RNase digestion
prepared from each immature oocyte stage and fractionated on sucrose gradients. Polysome profiles from each immature oocyte stage are shown in Fig. 2. A similar fraction of the ribosomesis present in polys6mes at each stage. Repeated analyses of the polysome profiles of the immature oocytes indicate that the 1 0 - 4 0 p , 4 0 - 5 5 p , 5 5 - 8 7 p , and 87-125 p immature oocytes have 5.3,8.5,7.3, and 8.7% of their ribosomes in polysomes, respectively (Table 1). The values for the percentage of ribosomes in polysomes in each of the immature oocyte stages do not differ significantly during growth of the immature oocytes. As the immature o w e s grow, the ribosomal RNA content increases and hence the total ribosome content increases [8], The fraction of ribosomes present in polysomes is approximately constant which indicates that the rate of protein synthesis increases during growth of the immature oocytes. As the immature oocytes grow from 10-40 pm, to 40-55 pm, to 55-87 p,and to 87-125 p;the average RNA content per cell is 0.7,5.2,11.0, and 32.0 pg per cell respectively [8]. Since the total RNA is directly proportional to the ribosome content of each oocyte sue class, the increasing ribosome content and constant fraction of ribosomes in polysomes suggests that the rate of protein synthesis increases two-fold from the 40-55 pm immature o w e s to the 5 5 - 8 7 p immature oocytes and three-fold from the 55-87 p immature oocytes to the 87-125 pm immature oocytes. No estimate of a change in the rate of protein synthesis from the 10-40 pimmature oocytes has been made due to the possible errors in the measurements of both the fraction of ribosomes in polysomes (Table 1) and the total RNA content of this size class of oocytes [8]. The
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protein to RNA ratio in the immature oocytes increases as the oocytes grow [8, 151. At present, no information is available about the turnover of RNA and protein during oogenesis in Urechis. If it is assumed that the rate of protein turnover is constant, the apparent increase in the rate of protein synthesis is consistent with what is known about the accumulation of RNA and protein during oogenesis [8, 151. The rate of synthesis and accumulation of RNA in the 40-55 pm immature oocyte is approximately equal to that in the 55-87 pm immature oocytes; however, the observed increase in the protein to RNA ratio suggests that an increase in the rate of protein synthesis must have occurred between the two stages. The rate of RNA synthesis is seen to increase about 4- to 5-fold in the 87-125 pm immature oocytes and the protein to RNA ratio continues to increase. Consequently a further increase in the rate of protein synthesis (i.e., total number of ribosomes utilized to synthesis the proteins) is expected between 55-87 pm and 87-125 pm stages. Polysomes in Mature Oocytes. The mature oocytes continue to synthesizeproteins prior to fertilization [12].To determine the fraction of the ribosomes utilized to form the newly synthesued proteins in the mature oocytes, polysomes were prepared from mature oocytes. The absorbance profile of the distribution of ribosomes in subunits, monosomes, and polysomes indicates that only a small fraction of the ribosomes are in polysomes (Figs. 2 and 3). Comparison of the mature oocyte absorbance profiles with the absorbance profiles from the two-cell stage before and after RNase digestion of the cleared lysate (Fig. 3, insert) indicates that about 0.6% of the ribosomes are in polysomes (Table 1). The mature oocytes have an average RNA content of 89 pg per cell, approximately a 4-fold increase from the average RNA content of the 85-125 pm immature oocytes. The fraction of the ribosomes in polysomes in mature oocytes is approximately 15-fold less than in the 87-125 pm immature oocytes. The increase in the total number of ribosomes in the mature oocytes coupled with the decrease in the fraction of the ribosomes in polysomes, indicate that there is a decrease in the total number of ribosomes in polysomes in the mature oocyte compared to the 87-125 pm immature oocyte. As a consequence of the decrease of ribosomes in polysomes, the rate of protein synthesis in the mature oocytes appears to be approximately one fifth of that in the late stage immature oocytes. The mature oocytes may be held in the storage organ for months before spawning [3] and since there is no evidence of protein accumulationin the mature oocytes during storage, the continued protein synthesis appears to be the level of synthesis required to replace the proteins lost due to turnover. Polysomes in Developing Embryos. Polysomes were prepared from mature oocytes and embryos at different stages of development, from the two-cell stage at 2 h postfertilizationto the trochophore larval stage at 40 h postfertilization. Polysome profiles from each stage of development are shown in Fig. 3. The fraction of the ribosomes in polysomes increases throughout early development. The increase in the fraction of ribosomes in polysomes begins soon after fertilization and by the two-cell stage has increased to approximately4% from the 0.6% that was present in the mature oocyte (Table 1). The increase in the fraction of the ribosomes in polysomesis nearly linear with time of development through 40 h postfertilization (Table 1).
0.05
-.----___
0 Top
--. Trochophore
Bottom
Fii. 3. Distribution of polysomes from mature oocytes and embryos.
Mature oocytes were fertilized, washed by settling three-times from filtered sea water, and embryos collected at the stages indicated. Cyclohexamide was added to 2.0mM, the cultures chilled, and embryos collected by centrifuging at 5,OOO g for 2 min. The postmitochondrial supernates were prepared and the sucrose gradient distribution of ribosomes and polysomes analyzed as described in Fig. 1. The polysomesregion of the absorbanceprofile is shown before (-) and after (- - -) RNase digestion. The insert shows greater d e w of the polysome profilesfrom sucrose gradients on which 16 Am units of postmitochondrial supernate from mature oocytes and the two-cellstage were layered
The redistribution of ribosomes into polysomes is very similar to the increase in the fraction of ribosomes present during early development of other animals [lo, 16,18,21,22]. In Urechis the total RNA content of the mature oocytes and embryos through the trochophore larval stage is constant [19]. Since the ribosomal RNA comprises the bulk of the RNA in the embryos, a constant RNA content indicates that the number of ribosomes per embryo is essentially constant through early development. The increase in the fraction of the ribosomes in polysomes should reflect an increase in the rate of protein synthesis per embryo [14,16,18,221. If it is assumed that the efficiency of translation by ribosomes in polysomes is equal at each stage, the increase in the polysome content of embryos indicates that the rate of protein synthesis increases more than 50-fold from the mature oocyte to the trochophore larval stage. However, if the rate of polypeptide chain elongation increases after fertilization as has been observed in the sea urchin [2],the increase in the rate of protein synthesis could be even greater. Acknowledgemen&. I would like to thank Dr. J. W. Brookbank and
Dr. L. 0. Ingram for their helpful and critical review of this manuscript. This work was supported in part by U. S. Public Health Service Grant RRo7021.
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before and after fertilization in sea urchin. Develop Biol 20: 435 Miller JH, Epel D (1973) Studies of oogenesis in Urechiscuupo 11. Accumulation during oogenesis of carbohydrate, RNA, microtubule protein, and soluble, mitochondrial and lysosornal enzymes. Develop Biol 32: 331 Mirkes PE (1972) Polysomes and protein synthesis during development of Ilyanassa obsoletu. Exptl Cell Res 74: 503 Newby WW (1940) The embryology of the echiuroid worm, Urechis caupo. Mem Am Phil Soc 16: 1 Rinaldi AM, Monroy A (1%9) Polyribosome formation and RNA synthesis in the early postfertilization stages of the sea urchin egg. Develop Biol 19: 73 Schwartz MC (1970) Nucleic acid metabolism in oocytes and embryos of Urechis cuupo. Develop Biol 23: 241 Smith DL (1975) Molecular events during oocyte maturation. In: Weber R (ed) The biochemistry of animal development, Vol. 111. Academic Press, New York, p 1 Spieth J, Whiteley AH (1981) Polysome formation and Poly(A)antaining RNA in embryos of the sand dollar, Dendraster excentricus. Wilhelm Roux’s Arch 190: 111 Woodland HR (1974) Changes in the polysome content of developing Xenopus laevb embryos. Develop Biol 40:90
Received February 1982Accepted in revised form June 1982