kinetic analysis of amino acid pools and protein synthesis in amphibian oocytes and embryos

DEVELOPMENTAL BIOLOGY 66, 172-182 (1978)

Kinetic Analysis of Amino Acid Pools and Protein Synthesis in Amphibian Oocytes and Embryos’-*

ROBERT J. SHIH, CLARE M. O’CONNOR, KIRSTEN KEEM, AND L. DENNIS SMITH Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

Received March 6, 1978; accepted May 4, 1978

Rates of protein synthesis have been measured in Rana pipiens oocytes and embryos and in Xenopus oocytes from the incorporation kinetics of two different concentrations of ammo acid. This method does not require an independent measurement of the amino acid pools, since the pool size can be calculated directly from incorporation data. The effects of the concentration and diffusion of injected amino acid on the calculated values for ammo acid pool size and flow rate are discussed. When the endogenous amino acid pool is appreciably expanded by the injected amino acid, the total amino acid pool in the oocytes or embryos may be considered as the precursor pool for protein synthesis. Under these circumstances, compartment&ion of amino acids does not affect the results, except when lysine is used as tracer. The rates of protein synthesis in ovarian oocytes of Rana pipiens and Xenopus loevis are 18 and 50-54 ng/hr, respectively. In Rana pipiens, the rate increases 70% during maturation and another 50% before the two-cell stage. Finally, the rate approximately doubles between the two-cell and blastula stages.

INTRODUCTION

Several studies have described changes in the total rate of protein synthesis, as well as changes in the rate of synthesis of specific classes of proteins, as a function of developmental stage (review by Davidson, 1976). In most cases, rate calculations have been based on the measured specific activity of the free (extractable) amino acid pool, assuming that it represents the true precursor pool. In support of this view, Regier and Kafatos (1977) reported that rates of protein synthesis in sea urchin eggs and embryos, calculated using the specific activity of the free leucine pool, were within 60-70s of values calculated using the specific activity of leucyl-tRNA. Likewise, rates of protein synthesis in Xenopus laevis oocytes, calculated from histidine incorporation into protein and the specific activity of the free histidine pool, are within about 30% of rates

’ This work was supported by NSF Grant No. GB 39971 and by NIH Grant No. HD 04229.

* Portions of this work were submitted by R.S. as part of a Ph.D. thesis, Purdue University, 1975.

estimated from polysome content (Wood- land, 1974; Davidson, 1976).

In contrast to the above, Ecker (1972) described studies with Rana pipiens oocytes in which both the rate of protein synthesis and the pool size were calculated solely from the kinetics of incorporation of radioactive amino acid into protein. Using [3H]leucine as a precursor, the calculated leucine pool size was reported to be 80 times smaller than the lowest value obtained for the extractable leucine pool (Ecker and Smith, 1968), and the absolute rate of protein synthesis was lower by a factor of 30 than those previously reported (Ecker and Smith, 1968; Smith and Ecker, 1969). Thus, these studies led to the suggestion that the precursor pool “active” in protein synthesis was considerably smaller than the free (extractable) amino acid pool. This in turn has led to questions concerning the feasibility of measuring protein synthetic rates in amphibian oocytes or embryos.

In the present study, we have reinvestigated the question of pool compartmentation in Rana pipiens oocytes and embryos

172 0012-1606/78/0661-0172$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

SHlH ET AL. Protein Synthesis in Frog Embryos 173

and, for comparison, Xenopus laevis oocytes, using essentially the methodology described by Ecker (1972). Specifically, using several amino acids, we have analyzed parameters which might influence the inter- pretation of incorporation data. Based on these studies, we suggest that, under the appropriate experimental conditions, there is no substantial difference between the “active” pool used for protein synthesis and the free amino acid pool, except possibly when lysine is used as the tracer. Taking this into account, we present new data on rates of protein synthesis during oocyte maturation and early development in Rana pipiens.

The increase in protein synthetic rate during maturation reported here is less than that previously reported (Ecker and Smith, 1968; Smith and Ecker, 1969). Fer- tilization results in a further increase in the rate, and by the blastula stage, the rate has increased an additional twofold.

MATERIALS AND METHODS

Animals. Sexually mature Rana pipiens were purchased from dealers in Vermont or Canada. The frogs were kept at 4°C and maintained at this temperature until they were used. Xenopus laevis were obtained from South Africa and maintained in the laboratory as previously described (La- Marca et al., 1973).

Studies on oocytes. Ovaries were removed from either anesthetized or pithed frogs and placed in amphibian Ringer’s solution containing antibiotics (30 pg/ml of penicillin; 50 pg/ml of streptomycin). In some cases the oocytes were transferred to Ringer’s solution containing progesterone (10 pg/ml) for 10 min to induce maturation (Smith et al., 1968). All solutions and glass- ware were routinely sterilized.

Ovulation. Ovulation was induced in gravid Rana females by ip injection of a pituitary suspension (Rugh, 1962). In fall and winter, female frogs were first injected with progesterone (2-4 mg per female) and

kept at 20-21°C for 9-12 hr before injection with pituitary suspension. Fertilization was carried out by pipetting sperm suspensions onto eggs stripped from ovulated females (Rugh, 1962). Fertilized eggs were manually dejellied with watchmakers forceps and dis- secting scissors before injection of isotope.

Measurements of protein synthesis and pool size. Rates of protein synthesis were measured by a modification (Hollinger and Smith, 1976) of the procedure by Ecker (1972). Briefly, parallel groups of oocytes (eggs) were microinjected with two different concentrations of radioactive amino acid. After various incubation times, groups of two or three oocytes were treated with hot (60°C) 0.5 N PCA for 30 min and washed an additional 30 min with distilled water. The acid-extracted oocytes were then cornbusted in a Packard Tri-Carb sample oxidizer, and radioactivity was counted in a Beckman LS 230 scintillation counter. Radioactivity was converted to disintegrations per minute based on exter- nal standardization.

The procedure is based on the following assumptions: (1) expanding the precursor pool does not alter the actual rate of protein synthesis, (2) the injected radioactive amino acid equilibrates with the total precursor pool instantaneously, and (3) amino acid metabolism and (4) protein turnover are small during the course of the experiment (see Shih, 1975).

As shown previously (Ecker, 1972), incorporation of injected precursor into protein can be described by the equation

p* = Lo (1 - e(-f/wu+v,)M),

where LO is the radioactivity (in disintegrations per minute) injected at time = 0, f is the flow rate of a particular amino acid through the precursor pool (picomoles per hour), VO is the size of the nonexpanded precursor pool (picomoles), and Vi is the amount of injected amino acid (picomoles). The initial portion of such a curve approx- imates a straight line whose slope is described by

174 DEVELOPMENTAL BIOLOGY VOLUME 66, 1978

slope = ~LIJ/VO + Vi.

When parallel experiments are conducted using oocytes (eggs) injected with two different amino acid concentrations, two such equations are generated which can be solved simultaneously for the variables f and VO. In practice, LO usually is kept constant, Vi is varied by adding unlabeled amino acid to stock isotope solutions, and only the data points up until the time that about 20% of the injected precursor has been incorporated are fit to a straight line.

For measurements of the total extractable amino acid pool sizes, 350-450 oocytes or embryos were homogenized in cold 5% TCA. The supernatant remaining after cen- trifugation at 10,OOOg for 20 min was brought to a final concentration of 30% TCA by adding cold 60% TCA. After recen- trifugation, the residual TCA in the supernatant was removed with three rinses of excess ether. The supernatants were sub- sequently subjected to pressure dialysis (8 psi of nitrogen) in the presence of 0.01 N HCl for 4-6 hr at 4’C!, neutralized with 0.1 N NaOH, and lyophilized. The amino acid contents were determined using a Beckman 120 C amino acid analyzer. In control experiments, 96% of injected [3H]leucine was recovered using these procedures.

Ultra-low temperature sectioning. Ultra- low-temperature sectioning was performed by a modification of Horowitz’s (1974) procedure. At various times after injection with tritiated amino acids, oocytes were blotted with filter paper and transferred into a sample holder containing embedding medium (95% O.C.T. compound, A. H. Thomas Co., and 5% propylene glycol). The sample holder was then immersed into Freon which was cooled to -160°C with liquid nitrogen. The total time needed from initial blotting to final freezing of the oocyte was 45-65 sec. Sectioning was carried out at -50°C on a Sorvall ultramicrotome equipped with a cryostat chamber. Serial sections (60 pm thick) were collected individually, wrapped with paper, oxidized, and counted.

All labeled amino acids were obtained in 0.01 N HCl and neutralized with 1 M Tris, except tryptophan, which was suspended in 50% ethanol. To prepare labeled amino acid solutions with low specific activity, 5 ~1 of unlabeled amino acid solutions (2.5 to 36 mg/ml) were added to 200 ~1 of stock isotope solution. The low specific activity [3H]tryptophan was prepared by adding solid L-tryptophan to the stock solution.

Amino acids were obtained as follows: r,-[4,5-3H]leucine, 1 mCi/ml, 5 Ci/mmole, or 41 Ci/mmole; L-[3-3H]arginine, 1 mCi/ ml, 24.2 Ci/mmole; L-[3H]phenylalanine (G), 1 mCi/ml, 6.15 Ci/mmole; ~-[2,3-~H]- proline, 1 mCi/ml, 11.14 Ci/mmole were purchased from New England Nuclear Corp., Boston, Mass. L-[4,5-3H]Lysine, 0.5 mCi/ml, 38 Ci/mmole was purchased from Schwartz/Mann., Bioresearch Inc., Orange- burg, N. Y.

RESULTS

Determination of Protein Synthetic Rates

Figure 1 shows typical incorporation kinetics obtained after injection of two-cell Rana embryos with [3H]leucine of two different specific activities. The two groups of embryos were injected with the same amount of radioactivity, but as expected, the embryos injected with the larger amount of leucine (136 as compared to 52 pmoles) incorporate isotope into protein

TIME Iminutes)

FIG. 1. The incorporation kinetics of [‘Hlleucine in R. pipiem two-cell embryos. Embryos were injected with 13 nl of amino acid solutions with specific activities of 0.07 Ci/mmole (open circles) and 0.2 Ci/mmole (closed circles).

SHIH ET AL. Protein Synthesis in Frog Embryos 175

more slowly. In this experiment, the calculated value for VO is 76 pmoles, while the value for f is 36 pmoles hr-‘. Both values are substantially larger than the values reported by Ecker (1972) for Rana oocytes. The major distinction between Ecker’s experiments and our experiments, outside of the developmental stage used, is that Ecker injected much less leucine into the oocytee than we did.

Table 1 shows other calculated values of Vo and f for Rana pi’iens oocytes and two- cell embryos and for Xenopus laevis oocytes. In these experiments the oocytes or embryos were injected with several different concentrations of leucine. In all cases, it is apparent that the calculated pool size increases as the amount of injected leucine

TABLE 1

EFFECT OF THE CONCENTRATION OF INJECTED LEUCINE ON THE KINETIC ANALYSIS OF THE AMINO

ACID POOL SIZE ( VO) AND FLOW RATE ( f) Picomoles of in-

jected amino VO f acid’ ___-

High Low concen- concen-

(pmoles) c$yd$

tration tration tine hr-1)

R. pipiens oo- 150 50 13 19.2 cytes 11 5.3 17.4

2 0.9 16.8 50 11 3.7 16.2

2 0.6 15.0 11 2 1.0 12.0

R. pipiens two- 150 50 19.3 40.0 ceil embryos 11 12.4 37.0

2 9.0 37.6 50 11 10.6 35.0

2 7.8 33.4 11 2 6.2 28.0

X. laevis oocytes 234 117 42 40.7 59 35 39.9 20 44 41.3

2 12 36.4 117 59 30 37.8

20 45 20.7 2 11 16.4

59 20 55 48.8 2 9 28.9

20 2 3 12.8

o In aII cases, oocytes and embryos were injected with 26,000 dpm (13 nl) of [3H]leucine of varying specific activities.

increases. A similar result is apparent in comparing the different values for flow rate (fl, although the relative magnitude of the change is considerably less than in the case of pool size. Furthermore, as noted partic- ularly in the study with Xenopus oocytes, expanding the pool beyond a certain point causes no additional changes in the calculated values of either for Vo. In these experiments, the injected amino acid would have expanded the extractable pool at most two- to fivefold.

It is difficult to explain the observations described above if we assume, as reported by Ecker and Smith (1968), that in all cases the injected amino acid equilibrates instantaneously with the precursor pool. On the other hand, if only a portion of the precursor pool became labeled during the incorporation period used in the rate calculations, and if this proportion is different with the two amino acid concentrations, then these calculations would be seriously af- fected.

Diffusion of Isotope in Oocytes and Em- bryos

To estimate the time required for at least some radioactive amino acid to occupy all portions of the cytoplasm, we have measured radioactivity in serial sections cut through oocytes (embryos) at various times after injection of amino acid into the center of the oocytes (embryo). The results provide an estimate of the time course of diffusion in only one dimension (perpendicular to the plane of sectioning), although it should be emphasized that within each section radioactivity is not distributed homo- geneously. In addition, since sections cut from the center of the oocyte would contain more radioactivity than sections cut closer to the periphery, the results have been expressed as disintegrations per minute per unit volume for each section.

An example of the diffusion of C3H]leu- tine (0.1 Ci/mmole) in a two-cell embryo is shown in Fig. 2. Essentially the same results were obtained with oocytes. In this partic-


SECTION NO.

FIG. 2. Ultra-low-temperature sectioning of R. p&piens two-cell embryos at various times after injection of [3H]leucine. The embryos were injected with 13 nl of leucine (0.1 Ci/mmole), frozen at various times after injection, and sectioned parallel to the animal-vegetal axis. Embryos were frozen at 42 set (0), 5 min (O), 15 min (A), and 45 min (A).

ular case, sections were cut parallel to the animal-vegetal axis, although identical results were obtained when the sections were cut perpendicular to the axis. At the earliest time (42 set) after injection, the major portion of the radioactivity is restricted to a “sphere” about 600 pm in diameter and extending to an absolute maximum of 960 pm. In this particular embryo, the injected isotope occupies about 30% of the embryo volume. With time, the diameter of the sphere expands until by 15 min radioactivity is distributed throughout the embryo.

The results of several experiments describing the distribution of injected [3H]leu- tine and [3H]lysine at several different specific activities in two-cell embryos and oocytes are summarized in Table 2. At any of the specific activities, injected lysine appears to be distributed throughout the oocyte within about 15 min. In addition, less than half the injected isotope has been incorporated into protein at this time. Similar results were obtained with the lowest specific activity of leucine (0.1 Ci/mmole), but as the specific activity of the injected leu- tine is increased, the movement of injected isotope becomes progressively reduced. Since the injected isotope is also incorporated into protein more rapidly as the spe-

cific activity is increased, the restriction of the apparent diffusion of injected leucine most probably is due to its incorporation into more slowly diffusing proteins. In the extreme case (41 Ci/mmole), the initial slope describing isotope incorporation into protein would be based on kinetics obtained within the first 3 min after injection, during which considerably less than 20% of the precursor pool (two-cell embryos) was labeled.

Comparison of Calculated and Extracta- ble Pools

Based on the above considerations, the size of the kinetically active pool (V,) for several amino acids was calculated from incorporation kinetics under conditions in which the incorporation of label into protein was approximately linear for at least one hour. The results are summarized in Table 3. For comparison, the size of the free (extractable in cold acid) pools of the same amino acids also are shown. It is clear that there is considerable variability in the measured pool sizes. The material used for the chemical and kinetic analysis was obtained from different frogs, although the donor frogs were obtained from dealers at the same time. This may reduce some of the

&III ET AL. Protein Synthesis in Frog Embryos

TABLE 2

DIFFUSION OF ISOTOPES IN Rana pipiens OOCYTES AND EGGS”

177

Amino acid Oocyte Two-cell

Time after Volume oc- Averaged P/z Time after injection

(min) cupied b? (min) injection isotope (min)

Leucine 0.1 Ci/mmol

6 Ci/mmole

41 Ci/mmole

Lysine 0.55 Ci/mmole

5.5 Ci/mmole

38 Ci/mmole

1 26.0 30f 13 0.7 29.6 5 78.7 5 79.4

15 100.0 15 100.0 45 100.0 45 100.0

0.83 20.0 5.8 + 3.1 0.7 28.0 5 39.0 5 43.0

10 89.0 - - 1 37.0 3 3 19.0 5 47.0 - -

0.83 38.0 110 1.1 15 100.0 5 30 100.0 15

1 26.0 110 0.7 15 100.0 15

1 16.0 - 8 100.0 - 2

Volume occupied b? isotope

30.0 88.5

100.0 16.4 89.0 -

50.0

Averaged tM’ (min)

43 + 18

12 f 8.2

3

138

138

-

n The ultra-low-temperature sectionings were carried out as described in Materials and Methods. Thirteen nanoliters of amino acid solutions were injected into oocytes or two-cell embryos.

* These values were expressed as the percentage of the total oocyte or egg volume which was occupied by the isotope.

c The incorporation half times (t%) were derived from the incorporation kinetics of each isotope. t% represents the time at which half the injected radioactivity is in acid-precipitable material.

variability in the amino acid pool size which normally occurs both between females and with the season of the year (see Shih, 1975). Nevertheless, the data show clearly that, with the exception of lysine, there is no substantial difference between the pool size calculated from the incorporation kinetics and that determined by amino acid analysis of the acid-extractable pool. In some cases, the calculated pool is actually larger than the free pool. In those cases in which the calculated pool is smaller, the differences are no more than 50%.

The data shown in Table 3 for lysine were obtained by injecting oocytes and embryos with C3H]lysine at specific activities of 8.0 and 0.8 Ci/mmole. As shown in Table 2, incorporation of [3H]lysine at these levels is relatively slow, and diffusion of the isotope should not be a problem. Nevertheless,

the data support the view that the lysine pool “active” in protein synthesis is considerably smaller than the free pool. To inves- tigate this further, two-cell embryos were injected with varying amounts of [3H]lysine. The data, summarized in Table 4, fall into two groups. The three combinations using the highest amounts of lysine gener- ate both pool sizes ( Vo) and flow rates (f) approximately twice those obtained with the combinations using lower amounts. We should point out that due to the extremely large lysine pool (2315 pmoles) in embryos, even the largest amount of injected lysine expands the free pool only 25%. In other experiments (not shown), injecting an additional 500 pmoles of lysine did not appreciably alter the calculated values for VO or f. Based on such data, we suggest that the lysine pool “active” in protein synthesis

DEVELOPMENTAL BIOLOGY VOLUME 66, 1978 178

TABLE 3 COMPARISON OF Srx AMINO ACID POOL SIZES

DETERMINED KINETICALLY OR BY ACID EXTRACTION OF Rana pipiens OOCYTES, EGGS, AND EMBRYOS

AIllillO VO (pmoles)” V (pmole& acid

OvariaD oocytes

Fist meta- ph* oocytes

Two-cell embryos

Leucine

Leucine

Lysine Leucine

Lysine Arginine Phenyl-

alanine Proline Trypto-

ph=

55 +- 27 (3)

15 f 7 (3)

7 (1) 39 f 14 (5)

7.1+- 2 (2) 43 f 8 (3) 63 +- 23 (3)

41+ 7 (2) 21 f 8 (3)

65 + 11 (3)

22 + 4 (2)

1900 f 424 (2) 34 * 5 (2)

2315 k 417 (2) 48 f 32 (2) 34 f 20 (2)

64 f 20 (2) 20 (1)

’ Pool sixes were calculated from the incorporation kinetics of two concentrations of amino acid. The C3H]leucine had specific activities of 0.07 and 0.2 Ci/mmole. Thirteen nanoliters of amino acid solutions were injected.

b Pools were extracted from oocytes and embryos with cold 5% TCA (Materials and Methods).

TABLE 4 EFFECT OF THE CONCENTRATION OF INJECTED

LYSINE ON THE KINETIC ANALYSIS OF THE AMINO ACID POOL SIZE (V) AND FLOW RATE (f) IN Rana

phiens TWO-CELL EMBRYOS Picomoles of lysine in- V f

jetted” (pmoles) (pmoles lysine

High con- Low concen- h-1) centration tration

523 52.3 380 25 5.2 289 23 0.52 283 23

52.3 5.2 141 11 0.52 143 11

5.2 0.52 159 13

“In ah cases, embryos were injected with 55,200 dpm (25 nI) of [3H]lysine of varying specific activities.

averages about 300 pmoles per embryo. The location and potential availability of the remaining “free” lysine is not known.

Protein Synthetic Rates in Oocytes and Early Embryos

Table 5 summarizes the data obtained from several experiments in which rates of protein synthesis were measured after the injection of [3H]leucine (0.07 and 0.2 Ci/mmole) into R. pipiens oocytes and two- cell embryos. The actual rates of protein synthesis were calculated from the amino acid flow rate (f) by assuming that proteins contain 10% leucine by weight. The results show that during maturation (first metaphase), the mean rate of synthesis has al- most doubled over that seen in ovarian oocytes. In the two experiments involving second metaphase (mature) oocytes, the rate may increase even further. However, it should be pointed out that these latter oocytes can be activated in response to the injection pipet and may thus be more equiv- alent to fertilized eggs (Smith et al., 1968). Clearly, the rate of synthesis is about 75% higher in two-cell embryos than in first meiotic metaphase oocytes. This result is not in agreement with earlier studies (Ecker

TABLE 5 RATES OF PROTEIN SYNTHESIS IN Rana pipiens

OOCYTES, EGGS, AND EARLY EMBRYOS=

Frog Rates of protein synthesis?

Ovarian First Second Two- oocytes meiotic meiotic cell-

meta- meta- phase phase

stage embryos

oocytes oocytes 1 16.0 29.0 - - 2 24.0 25.0 49.0 44.0 3 22.0 31.0 36.0 57.0 4 16.0 47.0 - - 5 12.0 - - - 6 - 25.0 - 43.0 7 - - - 53.0 8 - - - 51.0 9 - - - 59.0

Mean f SE 18 + 5 31 f 9 43 A 9 51 f 7 Ratio (1) (1.7) (2.3) (2.8)

D AU pools were calculated from the incorporation kinetics of [3H]leucine (0.07 and 0.2 Ci/mmole) into protein (Materials and Methods).

bThe rates are expressed as nanograms per hour per oocyte (or egg), assuming newly synthesized proteins are 10% leucine by weight.

.%IIH ET AL. Protein Synthesis in Frog Embryos 179

and Smith, 1968; Smith and Ecker, 1969) which reported that maximal rates of synthesis were achieved at about first meiotic metaphase and were maintained constant throughout the remainder of maturation as well as through activation or fertilization.

Injection of Precursor into Blastulae

Figure 3 shows an example of the incorporation kinetics obtained when two different concentrations of [3H]leucine were injected into the blastocoel of Rana stage 8 blastulas (Rugh, 1962). In contrast to the situation with oocytes or two-cell embryos, these kinetics show a definite lag of several minutes before incorporation becomes approximately linear. We interpret this to rep- resent the time required for the injected isotope to diffuse from the blastocoel into the surrounding cells. Calculations of both

. k 06 - c

TIME (minutes)

FIG. 3. The incorporation kinetics of [3H]leucine in R. pipiens blastulas. Embryos were injected with 23 nl (51,400 dpm) of lysine solutions with specific activities of 0.06 Ci/mmole (closed circles) and 0.2 Ci/mmole (open circles).

f and Vo were based on the linear portion of the incorporation curves, assuming that, as with oocytes or two-cell embryos, the isotope has diffused into all the blastula cells early during the long linear portion of the incorporation curve. To the extent that this assumption is not valid, we will have un- derestimated the actual synthetic rates.

Table 6 summarizes the values for f and VO calculated from several experiments in which sibling two-cell embryos and blastulas were injected with two concentrations of [3H]leucine, [3H]arginine, or [3H]lysine. The calculated value for f in blastulas is always higher than that in two-cell embryos, although the magnitude of the increase varies from 40% when lysine is used as tracer to 170% when arginine is used. This may reflect a difference in the classes of proteins being synthesized at the two stages.

DISCUSSION

As pointed out by others, the most direct estimate of absolute protein synthetic rates involves the measurement of aminoacyl- tRNA or peptidyl-tRNA specific activities (Airhart et al., 1974; Regier and Kafatos, 1977; Ilan and Singer, 1975). This has not yet been possible with amphibian embryos due to both the small amounts of the req- uisite materials (polysomes, acylated tRNA) and the necessity of microinjecting isotope into individual embryos. Hence, more indirect procedures have been used.

The major assumption on which these latter procedures are based is that injected

TABLE 6

AMINO ACID POOL SIZES AND FLOW RATES IN Rana pipiens TWO-CELL EMBRYOS AND BLASTULAS”

Amino acid V (pmoles/embryo) f (pmoles/hr/embryo)

Two-cell Blastula Two-cell Blastula

Leucine (2) 18.6 f 2.6 37.1 f 14.2 27.8 1- 3.1 46.3 -+ 9.8 Arginine (4) 113 + 17 258 -t 115 23.2 + 1.1 62.2 + 24.1 Lvsine (31 252 f 116 263 k 39 48.3 f 18 69+ 16

a Pool sixes and flow rates were calculated from the incorporation kinetics of two concentrations of amino acids. Embryos were injected with 20-30 nl of [3H]leuine (0.06 and 0.2 Ci/mmole), [3H]arginine (0.2 and 0.7 Ci/mmole or 0.2 and 21 Ci/mmole), or [3H]lysine (0.2 and 43 Ci/mmole). Numbers in paentheses refer to the number of experiments. Mean values and standard errors for V and fare listed in the table.


amino acid equilibrates essentially instantaneously with the precursor pool (Ecker and Smith, 1968). The present work shows, however, that amino acid diffusion can seriously affect rate determinations. Among other solutes whose diffusion has been stud- ied in Rana pipiens oocytes, sucrose (MW 341) was found to have a diffusion coefficient of about 2 X low6 cm2/sec, or about one-third its diffusivity in water (Horowitz, 1972).

If we assume that the diffusion coefficient for leucine (MW 131) in cytoplasm is similar, then from the equations described by Horowitz (1972), we estimate that about 6 min would be required for radioactive leu- tine near the oocyte periphery to reach 10% the level at the edge of an injection sphere 600 pm in diameter. This assumes that cytoplasmic diffusion is not effectively slowed by incorporation of the diffusing radioactive precursor into protein (see Ho- rowitz et al., 1970). As seen in Table 2, the minimum time that we have observed for injected radioactive amino acids to distrib- ute to all portions of the oocyte (embryo) is about 15 min. This time was obtained under conditions in which isotope was injected approximately into the center of oocytes. Injection at eccentric locations pre- sumably would result in even longer times before all portions of the precursor pool became labeled to some degree.

Since in calculations of the protein synthetic rate, total oocyte (embryo) proteins are extracted and counted, differences in incorporation resulting from a nonhomo- geneous distribution of isotope are averaged out; diffusional equilibrium is not required provided that all portions of the precursor pool contain some radioactive amino acid. The data in Table 2 show that when high specific activity [3H]leucine is injected, this condition is not satisfied during the time period over which the kinetics of incorporation are measured. Thus, under such conditions, we can reproduce the result reported by Ecker (1972) that the “active” leucine pool is in the range of 2-6

pmoles per oocyte. In contrast, under conditions in which incorporation of precursor into protein is linear for an appreciably longer time than that required for diffusion, the calculated pool size approaches that of the free pool (Table 3). We cannot conclude from this data that amino acid pools are not compartmentalized in amphibian oocytes (embryos). In fact, we already have pointed out that restricted diffusion cannot account for the results obtained with [3H]- lysine (Table 3). We do suggest that, if compartmentalization is a general phenom- enon, it is circumvented when the precursor pools are sufficiently expanded. This situation would not be significantly different from that described in studies with mam- malian cells (Airhart et al., 1974).

The data in Table 2 show that sufficient pool expansion occurs in Rana pipiens oocytes only when at least 46 pmoles of leu- tine (corresponding to a specific activity of 0.1 Ci/mmole) is injected into the oocytes. Under these conditions, the rate of leucine incorporation (Table 1) is 19.2 pmoles hr-’ in oocytes and 40 pmoles m-’ in two-cell embryos. The more extensive data for Xenopus oocytes show that values for f ranging from 21 to 48 pmoles l-n-’ were obtained when 20 pmoles (0.6 C/mmole) minimally was injected in combination with greater amounts; with 50 pmoles (0.2 C/mmole) the range was only 38 to 41 pmoles hr-‘. These values correspond to 28-63 and 50-54 ng of protein l-K’, respectively, assuming that newly synthesized proteins are 10% leucine by weight. For comparison, one can estimate that oocytes are synthesizing protein at rates of 17-48 ng hr-’ using the polysome content of Xen- opus oocytes (Woodland, 1974) and assuming certain translation rate parameters (Woodland, 1974; Davidson, 1976). Clearly, these values obtained by the two independent means are essentially the same.

The major point of the preceeding discussion is that calculation of protein synthetic rates from two sets of incorporation kinetics can provide a convenient method

SHIH ET AL. Protein Synthesis in Frog Embryos 181

to obtain reasonably accurate values. How- ever, this method requires a high degree of experimental precision since two variables (Lo and Vi) are determined independently for each incorporation curve. Because the two equations describing the incorporation kinetics are then solved simultaneously, any errors in measurement can be rapidly propagated. Thus, we suggest that for general use it may be simpler to calculate rates based on a single set of incorporation kinetics after sufficient pool expansion, cou- pled with measurements of the extractable pool size. Alternatively, Regier and Kafa-. tos, (1971, 1977) have described another isotopic method for measuring directly the total pool specific activity.

Considering the problems raised in the previous pages, we have reinvestigated the nature of rate changes in protein synthesis during oocyte maturation and early development of Rana pipiens. Our data (Table 6) show that the rate of protein synthesis during maturation increases less than twofold. This result is substantially different from the initial studies of Ecker and Smith (1968) and Smith and Ecker (1969) who reported increases of 20- to 30-fold in ma- turing oocytes. Likewise, the data are some- what different than those reported by Ecker (1972), showing minimum increases of 3- to lo-fold during maturation. On the other hand, O’Connor and Smith (1976) reported that the rate of protein synthesis in Xenopus oocytes also approximately doubled during maturation, consistent with an approximate doubling of polysome content during this time (Woodland, 1974).

In Xenopus, the polysome content has increased an additional twofold by shortly after fertilization and, by the blastula stage, again has increased an additional threefold. Our data for Rana (Table 6) also show a 50% increase in the rate of protein synthesis as a result of fertilization (two-cell embryos compared to mature oocytes). Neverthe- less, the conclusion that fertilization (or artificial activation) has no discernible effect on protein synthetic rates (Ecker and

Smith, 1968) no longer appears valid. Esti- mates of the rate of protein synthesis in Rana blastulae are about twice those of two-cell-stage embryos (Table 6), which is less than the increase predicted from the polysome data in Xenopus. On the other hand, the blastula stage in Xenopus is reached within about 6-8 hr after fertilization (2O”C), while in Rana (l@C), 16-21 hr usually is required. Thus, based on the changes indicated, both embryos would ac- cumulate similar amounts of protein during this developmental period.

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kinetic analysis of amino acid pools and protein synthesis in amphibian oocytes and embryos

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