Quantitative and qualitative analysis of RNA synthesis in stage 6 and stage 4 oocytes of Xenopus laevis

DEVELOPMENTAL

34,

BIOLOGY

Quantitative

106-118

(1973)

and Qualitative

Analysis

of RNA Synthesis

and Stage 4 Oocytes of Xenopus MICHAEL Department

of Biology,

J. LAMARCA,

L. DENNIS

Lawrence Sciences,

University, Appleton, Purdue University, Accepted

SMITH,

April

in Stage 6

laevis 1

AND MARJORIE

C.

STROBEL

Wisconsin, 54911, and Department Lafayette, Indiana 47906

of Biological

21, 1973

RNA synthesis has been studied in oocytes taken from Xenopus laevis females which have not recently ovulated. Such females contain a population of large (stage 6) oocytes which exhibit white equatorial bands and which are considered to represent the terminal stage of oocyte development. Rates of RNA synthesis in these “banded” oocytes were measured by analyzing the kinetics of incorporation of SH-guanosine into acid-precipitable, alkaline-labile material, and changes in precursor pool (GTP) specific activity during incubations. In additional experiments, rates of RNA synthesis were measured after ‘H-GTP was injected directly into stage 6 oocytes. For comparison, rates of RNA synthesis were measured in lampbrush chromosome stage oocytes (stage 4; 0.5-0.6 mm diameter). The results show that, under the in vitro conditions employed, stage 6 oocytes are not metabolically dormant, but synthesize total RNA at a rate at least as great as the stage 4 oocytes. Qualitative studies on newly synthesized RNA in the two oocyte classes have been performed using sucrose density gradient centrifugation and acrylamide gel electrophoresis. Both stage 4 and stage 6 oocytes exhibited similar patterns, and the bulk of the RNA synthesized and accumulated during 12-hr pulses appears to be ribosomal. These observations are discussed in terms of existing concepts concerning synthetic activity in stage 6 oocytes.

Xenopus

stage 4 oocytes by conventional centrifugation and base analysis procedures (Davidson et al., 1964; Mairy and Denis, 1971). In addition, RNA-DNA hybridization experiments leave no doubt that potential “informational RNA” is transcribed in stage 4 oocytes, both from repetitive and nonrepetitive DNA sequences (see Davidson, 1968; Davidson and Hough, 1969). The lampbrush chromosomes contract and apparently becon inactive in RNA synthesis while oocytes still are quite small (Ficq, 1961). Substantial synthesis of DNA-like RNA is observed again only during the period of hormone (gonadotropin) induced ovulation in Xenopus Zaeuis (Brown and Littna 1964b, 1966). On the other hand, considerable sequence homology has been demonstrated between newly synthesized stage 4 RNA, transcribed from repetitive DNA, and the total RNA of large preovulatory (stage 6) oocytes

INTRODUCTION

It is generally accepted that during amphibian oogenesis, oocytes at the maximal lampbrush chromosome stage (stage 4) exhibit the highest levels of RNA synthesis (review by Davidson, 1968). Most, of the RNA synthesized in stage 4 Xenopus laevis oocytes appears to be ribosomal (Davidson et al., 1964; Davidson and Mirsky, 1965; Gall, 1966), and the rapid accumulation of this stable product tends to obscure identification of other RNA classes (Brown and Littna, 1964a; Davidson et al., 1964; Davidson, 1968). However, newly synthesized DNA-like RNA has been detected in 1 Supported by grants from the National Institutes of Health (HD 04229) and the National Science Foundation (GB 8741). One of us (LDS) holds a USPHS research career development award (HD 42549), while the principal investigator was an NSF Science Faculty Fellow in the Department of Biological Sciences, Purdue University, Lafayette, Indiana 47906. 106 Copyright 0 1973 by Academic Press. Inc. All rights of reproduction in any form reserved.

LAMARCA,

SMITH,

AND STROBEL

(review by Davidson, 1968; Davidson and Hough, 1972). Similar comparisons of RNA transcribed from nonrepetitive DNA are not available, but stage 6 oocytes are reported to contain relatively large amounts of nonrepetitive sequence transcripts of high complexity (Davidson and Hough, 1971). These kinds of observations have led to the concept that most of the “informational RNA” needed in early development was synthesized during the lampbrush chromosome stage and conserved (Davidson, 1968; Davidson and Hough, 1972). Synthesis of rRNA appears to be spread over a longer time period. The multiple nucleoli, sites of rRNA synthesis (Brown and Dawid, 1968), continue to incorporate labeled precursors into RNA long after lampbrush chromosomes have contracted (Ficq, 1961; MacGregor, 1967). This synthesis also is considered to decrease substantially as oocytes complete their growth (Brown and Littna, 1964a,b; Brown, 1966). In fact, Crippa et al. (1972) have reported that stage 6 Xenopus oocytes contain an inhibitor of rRNA synthesis. Thus, there has been some question as to whether full-grown Xenopus oocytes synthesize any RNA (Brown and Littna, 1964a, b; Brachet, 1967; Davidson, 1968). In contrast, previous studies with full-grown Rana pipiens oocytes showed readily detectable incorporation of labeled precursor into RNA (Smith and Ecker, 1970). The question of RNA synthesis in the largest ovarian oocytes is of intrinsic interest but, perhaps more importantly, relates directly to interpretation of the several experiments mentioned above. Previous studies indicating synthetic quiescence were based largely on low or undetectable levels of incorporation of radioactive precursors into RNA. However, since precursor pool specific activities were not considered, substantial rates of RNA synthesis cannot be ruled out. In the present investigation, we have measured the actual rates

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of RNA synthesis in the largest (stage 6) oocytes, and have compared this with rates of synthesis in oocytes at the lampbrush chromosome stage. The results indicate that both synthesize total RNA at comparable rates. We have also compared the qualitative pattern of RNA synthesis in the two oocyte classes, and found no substantial differences. Xenopus

MATERIALS

AND

METHODS

Experimental animals. Large adult Xenopus laevis were obtained directly from

South Africa every 3 months and maintained in tanks with running dechlorinated water at 24°C on a 12-hr light/dark cycle. They were fed ground beef heart enriched with liquid multivitamins (Polyvisol, Mead-Johnson, 30 ml/lb) three times weekly until they were sacrificed by spinal pithing. We used only animals that weighed more than 130 gm, and that were kept less than 4 months under the above conditions. Treatment of oocytes. Ovaries were excised from pithed animals, and rinsed extensively in amphibian Ringer’s solution containing 30 &ml penicillin, 50 &ml streptomycin, and 70 pg/ml gentamycin sulfate. Individual oocytes were manually dissected from their follicles with watchmaker’s forceps and were sized according to diameters, measured with an ocular micrometer in a dissecting microscope. All follicle cells adhering to the oocytes were digested away with pronase as described (Smith and Ecker, 1969), and microscopic analysis confirmed their absence. When large numbers of isolated stage 4 and stage 6 oocytes were required (measurement of total GTP pool sizes or “carriers” for GTP specific activity), ovarian fragments were digested with collagenase (Dumont, 1972); freed oocytes were counted and staged after return to Ringer’s solution. Classification of oocytes. Several methods for classifying oocyte development in Xenopus have been described (see Du-

108

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mont, 1972) and, to avoid ambiguity, it is necessary to define carefully the identification we have used. Oocytes at the maximal lampbrush chromosome stage were taken to be those whose diameters in antibiotic Ringer’s solution measured between 0.5 mm and 0.6 mm, and whose pigmentation was a uniform yellow brown. This corresponds to the stage 4 oocytes of Davidson and Mirsky (1965), a term we have used, but to stage 3 oocytes according to Dumont (1972). Davidson and Mirsky (1965) have considered all oocytes with diameters greater than 1.0 mm as stage 6 oocytes, a term which Dumont (1972) reserves for oocytes containing unpigmented equatorial bands (white bands) and diameters greater than 1.2 mm. These banded oocytes are considered to represent the terminal stage of oocyte development and, by several criteria, are considered to exhibit diminished metabolic activity (Dumont, 1972). For these reasons, we have used only stage 6 white-banded oocytes in the present experiments. Kinetic experiments. In some experiments, a Leitz micromanipulator was used to inject 15 nl of a GTP solution (New England Nuclear, guanosine-S3H(N) triphosphate, tetrasodium salt, 5.28 Gil mmole, dissolved in sterile Ringer’s) into white-banded stage 6 oocytes. Injected oocytes (groups of 5) were extractea overnight in cold (4°C) 0.5 N PCA to remove unincorporated radioactivity. Intact oocytes displayed essentially no loss of PCAprecipitated radioactivity under these conditions. Oocytes were then washed with 5 changes of cold distilled water and 3 changes of cold ethanol (over a period of 4-5 hr), rolled in l-inch squares of cellulose filter paper, and combusted in a Packard Tri-Carb sample oxidizer. Oxidized material was suspended in cocktail D (dioxane, PPO, naphthalene) and counted at 43% efficiency in a Beckman 230 scintillation counter. In most experiments, large numbers of

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34, 1973

stage 6 oocytes and stage 4 oocytes were incubated in a single stender dish containing sterile antibiotic Ringer’s and 100 &i/ml of tritiated guanosine (New England Nuclear, guanosine-8-3H, 6.15 Ci/ mmole). For each time point, 2 groups of 10 stage 6 oocytes and 2 groups of 10 stage 4 oocytes were removed from the dish with a sterile pipette, rinsed quickly in cold Ringer’s and each group was homogenized separately in 10 ml of cold 0.5 N PCA. The precipitate from one group of homogenates of each stage were collected onto Millipore filters (HAWP, 45 ppores), rinsed and dried, oxidized and counted as above. The homogenates of the remaining group of each stage were centrifuged at 10,OOOg for 10 min. An aliquot of the supernatant, neutralized with KOH, was decanted into cocktail D, and counted (at 40% efficiency) as a measure of the uptake of the total acid-soluble material. Pellets were hydrolyzed in 0.3 N KOH for 18 hr at 37”C, then treated with cold PCA to a final concentration of 0.5 N, and remaining particulate matter was collected onto Millipore filters which were processed as above. For each incubation time incorporation into RNA was determined by subtracting the amount of radioactivity remaining after alkali hydrolysis from the PCA-precipitated radioactivity. Nucleoside phosphate determinations. For each determination, 100 stage 4 oocytes and 100 stage 6 oocytes were removed from the incubation dish, rinsed quickly in Ringer’s solution, and homogenized in cold 0.5 N PCA. The PCA-soluble material was freed of precipitate as described by Brown and Littna (1966), and acid-soluble material of 500 “carrier” stage 4 oocytes or of 209 “carrier” stage 6 oocytes was added to the respective PCA supernatants. Nucleoside phosphates in each extract were collected by the method of Brown and Littna (1966), lyophilized, and stored at -70°C. Lyophilized material was later dissolved in 100 ~1 of 50% ethanol, spotted onto commercially prepared polyethyleneimine-cel-

LAMARCA,

SMITH,

AND STROBEL

lulose (PEI) thin layers on plastic sheets (Brinkman Instruments, Westbury, New York) and the nucleoside phosphates were separated by the 2-dimensional chromatography method (KH,PO, ; Formateborate) of Cashel et al. (1969). Spots were visualized in UV light, marked, cut out, and eluted in 0.5 ml, 0.1 N HCI for 4 hr at room temperature. The OD,,, of each eluent was measured against eluents of equal blank areas from the same chromatogram on a Zeiss spectrophotometer, and the radioactivity determined by placing an aliquot in cocktail D. Specific activities for each time point were computed after the optical densities were corrected for the contribution of the carrier oocytes. The identity of the spots on the chromatograms was established from migration rates, by the UV absorption spectra of the eluents, and by comigration with known standard nucleoside phosphates. In addition, standard radioactive nucleoside phosphates were added to sample extracts and chromatographed, and the dried chromatograms were sprayed with Omnispray (New England Nuclear) and exposed to X-ray film which was developed after 4 days. This procedure provided good separation of GTP and dGTP (and ATP and dATP) from other nucleoside phosphates, but the diphosphates of guanosine (GDP and dGDP) comigrated with CTP and dCTP, respectively. Since mild acid hydrolysis (Wilt, 1969) of RNA revealed no conversion of guanosine to cytidine derivatives, radioactivity in the combined spots (GDP-CTP and dGDP-dCTP was considered as representing only GDP and dGDP. There was minimal conversion of guanosine to adenosine derivatives, with ATP accounting for less than 2% of the acid-soluble radioactivity. The small amount of radioactivity entering RNA from the ATP pool (substantially larger than GTP, Woodland and Pestell, 1972) was ignored in our calculations. Total GTP pools were extracted as above from 1000 stage 6 oocytes and 2000 stage 4

RNA

Synthesis

in Xenopus

Oocytes

109

oocytes. The amount of GTP was calculated from the extinction coefficient at ODz6,,, corrected for the efficiency of recovery by adding known amounts of 3H GTP prior to homogenization. Qualitative analysis of RNA. RNA was extracted using the cold phenol procedure of Brown and Littna (1964a), modified to include reextraction of the interface with pH 5.0 acetate buffer; RNA was precipitated overnight with ethanol at -20°C. Recovery of the RNA was consistently between 87 and 88%, based on a comparison of counts in precipitated RNA with counts incorporated into total RNA in kinetic experiments, quantity of RNA in extracts (OD,,,) and known RNA content of oocytes, and recovery of radioactive bacterial RNA added prior to homogenization. Sucrose gradients (5 to 20%) prepared according to Brown and Littna (1964a) were centrifuged at 25,000 rpm for 16-17 hr (4°C) in a Spinco LZ-65B ultracentrifuge with an SW 27 rotor. Fractions were collected from the top, and absorbance at 254 nm was monitored continuously with an Isco UV analyzer. RNA in each tube was precipitated with cold 5% TCA with carrier yeast RNA, collected onto Millipore filters, and counted (18% efficiency) in toluenebased scintillation fluid. Autoradiography. Individual oocytes injected with 3H-GTP were fixed in Carnoy’s fixative (alcohol:chloroform:glacial acetic acid, 6:3:1) after 2-hr incubations. Sections, cut at 8 pm, were extracted with cold 10% TCA for 1 hr, dipped in 0.04% PPO in toluene according to Przybylski (1969), and then processed as described previously (Ecker and Smith, 1971b). RESULTS

Quantitation

of RNA Synthesis

Initial attempts to measure rates of RNA synthesis involved direct injection of 3HGTP into individual stage 6 white-banded oocytes. Figure 1 shows that incorporation into acid-precipitable material occurs in a

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DEVELOPMENTAL

BIOLOGY

VOLUME

34,

1973 TABLE GTP

Animal 1800

-

E 1600

-

.

Stage

POOL 6

1 .%ZES= Stage

4

1

189.9

51.1

3.7

2 3

343.2 237.5

82.9 94.1

4.1 2.5

“Determined from 1000 stage 6 oocytes and 2000 stage 4 oocytes from each animal. Values are expressed in picomoles per oocyte.

2

3 Hours

4

5

FIG. 1. Incorporation of 3H-GTP into precipitable material by oocytes injected with cpm SH-GTP. Each point represents the amount incorporated by 5 oocytes from which subtracted the mean amount “incorporated” oocytes in 0 time. The line was fit to data by least squares analysis.

6

PCA45,500 mean was by 5 linear

linear fashion. We believe that this incorporation represents synthesis of RNA since, in other experiments, such radioactive material is sensitive to RNase and hydrolyzable by KOH at 37°C. Provided the specific activity (SA) of the GTP pool is known, the actual rate of incorporation (R) can be calculated from the observed incorporation (0 according to the equation.

dlidt

= R x SA

(1)

In order to obtain the GTP specific activity, we first measured the total GTP pool size in oocytes from several females. Table 1 lists these results, both for banded stage 6 oocytes as well as those of stage 4 oocytes of the same animals. These data are consistent with the observations of Woodland and Pestell (1972), who found that GTP pools in “1arge”Xenopus oocytes and unfertilized eggs ranged from 150 to 470 pmoles. We have assumed an “average” endogenous pool size of 256.9 4 78.5 pmoles and under these conditions, the injected GTP (about 10 pmoles in Fig. 1) would expand the endogenous pool by only about 4%. Furthermore, during the course

of the experiments, less than 4% of the pool radioactivity was incorporated into RNA, and less than 5% of the injected label leaked from the oocytes during incubation. Thus, we have considered specific activity of the GTP pool, estimated from the ratio of injected radioactivity and the “average” pool size, to remain essentially constant during the course of the experiments. Under these conditions, the value of R in Fig. 1 calculated from the slope of a line fit to the data and the estimated specific activity, was. 1.46 f 0.45 pmoles of GTP incorporated.into RNA per hour per oocyte (Table 2). An additional experiment with oocytes from a second female provided an estimate of 1.07 f 0.33 pmoles/hr per oocyte. Because of the large variation in GTP pool size in oocytes from different females, and the difficulty in injecting stage 4 oocytes for comparison, all additional experiments were conducted with oocytes which were incubated continuously in aHguanosine. Figure 2 indicates that the rate of uptake of radioactivity into total acidsoluble material, as well as appearance of radioactivity in phosphorylated derivatives of guanosine, was essentially linear with time both for stage 6 and stage 4 oocytes. Consequently, equilibrium conditions were not obtained during the course of our experiments, and the precursor pools cannot be saturated with exogenous precursors. It should be noted that even with stage 4 oocytes, the amount of radioactivity in triphosphates at the end of 15 hr

SMITH,

LAMARCA,

RNA

AND STROBEL

TABLE RATES

Type

Experimentb 1 2

{

3 4 5

Kinetics injected

OF INCORPORATION

Synthesis

1

111

Oocytes

2 OF GTP

analysis’

INTO RNA”

Stage4oocytes

microGTP

Kinetics incorp. guanosine from medium i

6 7

in Xenopus

RNA separated on sucrose gradients

Stage

6 oocytes

-

1.46 zt 0.456 1.07 * 0.33

1.37 f 0.12 0.78 f 0.03 -

1.72 zt 0.14 1.58 zt 0.08 1.51 * 0.07

0.8 f 0.1 1.5 f 0.1

1.2 l 0.1 1.4 * 0.2

a Values are expressed as picomoles of GTP incorporated per hour per oocyte. b Each experiment was performed with oocytes from different females. c See text for further description. d Uncertainty intervals are * one standard deviation (0) which were calculated in experiments 1 and 2 from (r of the slopes of the lines fit to the incorporation data (Fig. l), the 0 of the average amount of 8H-GTP injected, and the 0 of the “average” GTP pool size (Table 1) (Radioactivity for both the experimental points and injected isotope was counted at the same efficiency, eliminating the necessity for correction to dpm.) In experiments 3-5 the uncertainty interval was calculated from the 0 of the constant (R/z/2) of the parabolas fit to the incorporation data (Fig. 3B), and the g of the slopes of the lines (k) fit to the data showing increase in GTP specific activities (Fig. 3A). In experiments 6 and 7, it was calculated from c of the slopes of the lines (k) fit to the data showing increase in GTP specific activities. In all experiments, lines or curves were fit to the data by least squares analysis. 60

70-

r STAGE 4

STAGE 6

60-

2c IC

.

LA 5

HOllrS

IO

15

HOUTS

FIG. 2. Uptake of PCA-soluble material by stage 4 oocytes and stage 6 oocytes incubated in 100 &i/ml 3H-guanosine. Total acid soluble material (0) represents average of 10 oocytes. Each nucleoside phosphate determination is the average of 100 oocytes corrected for recovery. 0, GTP plus dGTP; A, GDP plus dGDP. Lines were fit to the data by linear least squares analysis.

woulti correspond to expansion of the GTP pool by no more than 2%, and substantially less for stage 6 oocytes. The proportion of total acid-soluble radi-

oactivity found in GTP and dGTP at any given time during the incubations (calculated from slopes in Fig. 2) averages from 27% (stage 4) to 37% (stage 6). In both

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DEVELOPMENTALBIOLOGY

cases, the triphosphates account for about 85% of the total radioactivity in the phosphorylated derivatives of guanosine (data for monophosphates not shown in Fig. 2). Similar results have been reported by Woodland (1969) in experiments in which 3H-uridine and adenosine were injected directly into Xenopus oocytes. The remaining acid-soluble radioactivity has not been identified in our experiments, but we presume that it represents intracellular nucleoside. The possible existence of a “pool” of intracellular nucleoside (or base) might indicate phosphorylation as a limiting step in the incorporation of label into RNA. Nevertheless, we suggest that the incorporation of exogenous nucleosides into both the nucleotide pools and RNA is limited largely by the rate of entry of exogenous precursors into oocytes. In this connection, we have observed as much as a lo-fold variation in the rate of uptake into total acid-soluble material among oocytes of the same size class from different females. These kinds of observations emphasize the difficulties involved in attempting to estimate rates of RNA synthesis from incorporation data alone. Since the size of the endogenous GTP pool in stage 4 oocytes is approximately one-third of that in stage 6 oocytes (Table l), and the rate of uptake of radioactive guanosine is at least 3-fold greater in the smaller oocytes (Fig. 2), specific activities of GTP pools in stage 4 oocytes should be considerably greater. Direct measurements of GTP specific activity confirm this (Fig. 3A). The linear rate of increase of GTP pool specific activity in stage 4 oocytes shown in the figure is about 6 times greater than in white-banded oocytes. In several additional experiments, we observed rates of increase of GTP pool specific activities in stage 4 oocytes ranging from 3 to 6 times that in stage 6 oocytes from the same animals. The linear increase with time in GTP specific activity can be represented by the expression,

VOLUME 34, 1973

SA = kt

(2)

Substituting this in Eq. (l), followed by integration and evaluation at t = 0 gives the expression,

I = (Rk/2)t2

(3)

where k is the rate of increase in specific activity. Figure 3B shows that the parabola described by the equation fits reasonably well to the kinetics of incorporation of 3H-guanosine into RNA, both for stage 4 and stage 6 oocytes. Using the incorporation data, R (the actual rate of incorporation) was calculated from the incorporation constant (Rk/2) of the curve which best fits the data, and the slope (k) of the line fit to the specific activity data. The results of 3 experiments, performed with oocytes from 3 different females, are shown in Table 2. As indicated, the calculated rates of incorporation in stage 6 oocytes are comparable to those obtained by injecting precursor directly into oocytes, and are at least as great as the rates of incorporation in stage 4 oocytes. In a final group of experiments, actual rates of incorporation were determined from quantitative analysis of RNA separated on sucrose gradients. In these experiments, specific activities of GTP were determined for oocytes incubated for 1, 6, and 12 hr in SH-guanosine. At the end of 12 hr, RNA was extracted from oocytes and the radioactivity in acid-precipitated fractions from sucrose gradients (next section) was totaled, corrected for recovery of RNA, and counting efficiency. Assuming the actual rate of incorporation remains constant over the incubation period, the value obtained at the single incorporation point was evaluated in Eq. (3) at t = 12 hr, with k derived from the slope of the line fitting the three specific activity points. The results of 2 separate experiments also are shown in Table 2. Measurements of the actual rate of incorporation calculated from the kinetic data (Fig. 3) probably are the most accu-

LAMARCA,

SMITH,

AND STROBEL

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Synthesis

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Oocytes

113

FIG. 3. (A) Specific activities of oocytes incubated in 100 &i/ml 3H-guanosine; ---, stage 6 oocytes-each point calculated from the GTP extracted from 100 incubated oocytes and 206 “carrier” oocytes; -, stage 4 oocytes-each point calculated from the GTP extracted from 100 incubated oocytes and 500 “carrier” oocytes. The lines were fit to the data by linear least squares analysis. (B) Incorporation of radioactivity into PCAprecipitable, alkali-soluble material by stage 6 oocytes (--) and stage 4 oocytes (---) incubated in 100 &i/ml in 3H-guanosine. Each point represents the mean value for 10 oocytes. Curves are parabolas of the type I = (Bk/2)tz, which were fit to the data by least squares analysis.

rate, but measurements from all three experimental approaches are remarkably consistent. The results show clearly that, under our experimental conditions, stage 6 oocytes incorporate GTP into RNA at least at the rate it occurs in lampbrush stage oocytes. These experiments do not indicate the synthesis of metabolically unstable RNA, but it is doubtful if anything but substantial synthesis of such a class could be identified because of the low specific activity of the precursor pool at short incubation times. Hence, absolute rates of RNA synthesis cannot be calculated. Nevertheless, accepting our estimated rates of incorporation, and assuming newly synthesized RNA has a ribosome-like base composition (next section), then for every picomole of GTP incorporated into RNA, oocytes would synthesize approximately 1500 pg of total RNA. Using this conversion, stage 4 oocytes would synthesize and accumulate 1.1-2.1 ng of RNA per oocyte

per hour, while the value for stage 6 oocytes would be 1.6 to 2.4 ng of RNA per oocyte per hour.

Qualitative

Aspects of RNA Synthesis

The RNA extracted from both stage 6 and stage 4 oocytes after a 12-hr incubation with 3H-guanosine exhibited similar sedimentation behavior on sucrose gradients (Fig. 4). In both cases the bulk (unlabeled) RNA sedimented as two major and one minor peaks, which correspond to 28 S and 18 S ribosomal RNA and 4 S RNA. The radioactive RNA in both groups of oocytes sediments with a peak in the 4 S region, a peak coincident with the 18 S RNA, and a third peak slightly heavier (by one fraction) than the 28 S RNA. This slight displacement has been observed frequently in our experiments, and has been reported by others in Xenopus oocytes (Mairy and Denis, 1971) and in RNA extracted from the nuclei of Z’riturus oocytes (Gall, 1966).

114

DEVELOPMENTAL

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1973

STAGE

6

1500 .80

.60

300 CPM

0.D aa4 .40

200

.20

too

0

30

25

20

15

IO

5

30 FRACTIONS

25

20

15

IO

5

0

FIG. 4. Sedimentation patterns of RNA synthesized by 200 stage 6 oocytes and 200 stage 4 oocytes that had been incubated for 12 hr in 100 &i/ml *H-rmanosine. -, OD,,,; O---O, radioactivity in acid-precipitated RNA.

In both of the latter cases, this labeled RNA was interpreted as being the 30 S intermediate of 28 S ribosomal RNA. Finally, a peak of radioactivity with a calculated sedimentation of 37 S is consistently seen in stage 6 and stage 4 oocytes. The migration of RNA from stage 6 oocytes on polyacrylamide gels (data not shown) corresponds closely to the pattern obtained from sucrose gradients. We have assumed that labeled RNA sedimenting in the 26 S and 18 S regions of sucrose gradients includes predominantly ribosomal RNA. Support for this comes from autoradiographic studies (Fig. 5) showing an accumulation of grains over the nucleoli of stage 6 oocytes injected with 3H-GTP. Similarly, the labeled 37 S peak, also reported by Crippa and TocchiniValentini (1971) in Xenopus oocytes, might be considered analogous to the 40 S rRNA precursor identified by several others (Gall, 1966; Landesman and Gross, 1969; Loening et al., 1969). Further support for this comes from our observations (not shown) that the slowest-moving band of radioactivity seen on polyacrylamide gels (Loening, 1968) has a calculated molecular weight of 2.5 x lo6 daltons. This agrees well with the reported value for the Xenopus ribosomal precursor (Loening et al., 1969). Thus, the qualitative experi-

ments described above are consistent with the conclusion that the bulk of the RNA synthesized and accumulated over a 12-hr period is ribosomal RNA, both in stage 6 and stage 4 oocytes. DISCUSSION

At least two considerations suggest the need for caution in interpreting quantitative comparisons between stage 6 and stage 4 oocytes. First, the possibility always exists that removal of oocytes from their ovarian environment, coupled with pronase treatment and incubation in saline media may, in itself, alter the nature of synthetic events (Smith, 1972). For example, Ringer’s solution appears to stimulate the rate of protein synthesis in Rana pipiens oocytes compared to that seen in lower ionic strength media (Ecker and Smith, 1971a). Reciprocally, there are reports that long-term incubation of oocytes in Ringer’s solution (greater than 12-24 hr) can be deleterious (Gall, 1966; Rogers, 1968; Wallace and Jared, 1969). In our experiments, oocytes were sometimes maintained in Ringer’s solution for up to 24 hr (beginning with dissection of oocytes from their follicles). In either case, we suggest that possible environmental effects on synthetic activity would affect both oocyte size classes equally.

LAMARCA,

SMITH,

AND STROBEL

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Synthesis

in Xenopus

Oocytes

115

FIG. 5. Autoradiograph of a section from a stage 6 oocyte injected with 25,000 cpm of aH-GTP 2 hr before fixation. The section was stained with lithium carmine and developed after 4 days’ exposure. Grains are apparent over the germinal vesicle and are particularly concentrated over the multiple nucleoli. The photograph was taken by double exposure with reflected and transmitted light, using a Zeiss Universal microscope. Magnification approximately x 616.

116

DEVELOPMENTAL

BIOLOGY

There also are indications that the level of synthetic activity in different-sized oocytes is dependent on the physiological status of donor females. While whitebanded oocytes are reported to incorporate minimal radioactivity, oocytes remaining in the ovary after the induction of ovulation do exhibit detectable incorporation (Davidson et al., 1964; Brown, 1966). Davidson (1968) mentions that HCG (used to induce ovulation) stimulates ribosomal RNA synthesis in stage 6 oocytes to a rate about 15% of that seen in lampbrush stage oocytes. Recently, Mairy and Denis (1971) reported that RNA synthesis in oocytes taken from females recently induced to ovulate was preferentially reduced in small oocytes (500-700 /*rn in diameter) and elevated in large oocytes (1.0-1.2 mm in diameter). In females not ovulated for several months, small oocytes exhibited the highest levels of incorporation. On the other hand, differential hormonal effects on permeability to exogenous precursors also could account for these observations. Gonadotropins are known to stimulate pinocytosis in Xenopus oocytes, and this surface response is retained for several weeks after a single injection of females (Wallace et al., 1970). All our experiments have been performed with oocytes taken from “nonstimulated” females since, according to Dumont (1972), only females not having recently ovulated contain a population of banded oocytes. Thus, it is unlikely that the high rates of RNA synthesis we have measured in stage 6 oocytes result from hormonal stimulation analogous to that suggested above. However, our studies do show that stage 6 oocytes exhibit greatly reduced incorporation of precursors into RNA, compared to smaller oocytes. Correlated with this, stage 6 oocytes exhibit greatly reduced uptake of exogenous precursors, compared to smaller oocytes (Fig. 2), and contain substantially larger precursor nools (Table These two factors alone

VOLUME

34, 1973

can account for low or undetectable levels of incorporation into RNA over a given time period, particularly if availability of the radioactive precursor is restricted enough to result in slow increases in pool specific activity. Such conditions could apply when isotope is injected into intact females (Brown and Littna, 1964a,b; Brown, 1966), and may explain why significant levels of incorporation into the RNA of stage 6 oocytes have not been detected in most previous studies. Thus, a conservative interpretation of our data is that oocytes are capable of exhibiting at least a constant rate of RNA synthesis between stage 4 and stage 6. We suggest that the same situation normally occurs during oogenesis in viva. Without additional data, one cannot rule out the possibility that the largest oocytes synthesize RNA more rapidly than do stage 4 oocytes (Table 2). Davis and Wilt (1972) have reported that the rate of RNA synthesis during the final one-third of oogenesis in Urechis caupo oocytes actually is 4- to &fold greater than at earlier times. Our calculated values for the actual rate of incorporation are based on the assumption that the total acid soluble GTP pool is used as a source of nucleotides for RNA synthesis, i.e., precursor pools are not compartmentalized. However, in a series of studies on tissue culture cells, Plagemann (1971a,b; 1972) has reported that most exogenous nucleosides enter and expand a precursor pool not used directly for RNA synthesis. Likewise, Ecker (1972) has reported that the amino acid pools “active” in protein synthesis in Rana pipiem oocytes are considerably smaller than the amount of extractable free amino acid. Similar studies have not yet been done concerning the nucleotide pools in Xenopus. Thus, our values are estimates at best, and are useful primarily in comparisons between stage 6 and stage 4 oocytes. Nevertheless, if one accepts a rate of RNA svnthesis in stage 4 oocvtes of l-2 ng per

LAMARCA, SMITH, AND STROBEL

hour, and assumes the RNA to be stable, oocytes would accumulate about 2 pg of total RNA in 45-90 days. This is the approximate time period separating stage 4 and stage 6 oocytes (Davidson, 1968), and this amount of RNA added to that already present at stage 4 (Davidson and Mirsky, 1965) would result essentially in the level measured in stage 6 oocytes (about 4 fig; Brown and Littna, 1966). Examination of the sedimentation pattern of RNA labeled in stage 4 and stage 6 oocytes reveals no differences, and we have interpreted these patterns, coupled with autoradiography, to mean that the bulk of the RNA synthesized and accumulated in both oocyte size classes is ribosomal RNA. However, accumulation of radioactive rRNA over the 12-hr incubation period could obscure label in other RNA species, and our extraction procedure is not ideally suited for the recovery of DNA-like RNA (see Perry et al., 1972). Additional studies are needed to determine what proportion, if any, of the “informational RNA” present in stage 6 oocytes (Davidson and Hough, 1971; Hough and Davidson, 1972) is actually synthesized by stage 6 oocytes. We wish to acknowledge the advice and assistance given us by Dr. R. E. Ecker of the Argonne National Laboratories, by Dr. A. I. Aronson and U. Clever of Purdue University, and by Dr. D. M. Cook of Lawrence University. We appreciate also the valuable technical assistance provided by Mr. T. Hollinger, Mrs. M. Williams, and Mrs. K. Keem. REFERENCES BRACHET,J. (1967). Biochemical changes during fertilization and early embryonic development. Cell Differentiation, Ciba Found. Symp., pp. 39-64. BROWN, D. D. (1966). The nucleolus and synthesis of ribosomal RNA during oogenesis and embryogenesis of Xenopus laevis. Nat. Cancer Inst. Monogr. 23, 297-309. BROWN, D. D., and DAWID, I. D. (1968). Specific gene amplification in oocytes. Science 160, 272-280. BROWN, D. D., and LITTNA, E. (1964a). RNA synthesis during the development of Xenopus laevis, the South African clawed toad. J. Mol. Biol. 8669-687. BROWN, D. D., and LIT~NA, E. (1964b). Variations in the synthesis of stable RNA’s during oogenesis and

RNA

Synthesis

in Xenopus

development

of Xenopus

117

Oocytes laevis.

J. Mol.

Biol.

8,

688-695.

BROWN, D. D., and LEA, E. (1966). Synthesis and accumulation of DNA-like RNA during embryogenesis of Xenopus laevis. J. Mol. Biol. 20, 81-94.

CASHEL, M., LAZZARINI, R. A., and KALBACHER, B. (1969). An improved method for thin-layer chromatography of nucleotide mixtures containing 32Plabeled Orthophosphate. J. Chromatogr. 40, 103-109. CRIPPA, M., and TOCCHINI-VALENTINI, G. P. (1971). Synthesis of amplified DNA that codes for ribosomal RNA. Proc. Nut. Acad. Sci. U.S. 68, 2769-2776.

CRIPPA, M ., TOCCHINI-VALENTINI, G. P., and ANDRONICO,F. (1972). Regulation of ribosomal RNA synthesis during oogenesis of Xenopus laevis. In “Oogenesis” (J. D. Biggers and A. W. Schuetz, eds.), pp. 193-214. Univ. Park Press, Baltimore, Maryland. DAVIDSON, E. H. (1968). “Gene Activity in Early Development.” Academic Press, New York. DAVIDSON, E. H. and HOUGH, B. R. (1969). High sequence diversity in the RNA synthesized at the lampbrush stage of oogenesis. Proc. N&Z. Acad. Sci.

U.S.

63, 342-349.

DAVIDSON, E. H., and HOUGH, B. R. (1971). Genetic information in oocyte RNA. J. Mol. Biol. 56, 491406.

DAVIDSON,E. H., and HOUGH, B. R. (1972). Utilization of genetic information during oogenesis. In “Oogenesis” (J. D. Biggers and A. W. Schuetz, eds.), pp. 129-139. University Park Press. Baltimore, Maryland. DAVIDSON, E. H., and MIRSKY, A. E. (1965). Gene activity in oogenesis. Genetic control of differentiation. Brookhoven Symp. Biol. 18, 77-98. DAVIDSON, E. H., CRIPPA, M., KRAMER, F. R., and MIRSKY, A. E. (1964). Genomic function during the lampbrush chromosome stage of amphibian oogenesis. Proc. Nat. Acad. Sci. U.S. 56, 856-863. DAVIS, F. C., and WILT, F. H. (1972). RNA synthesis during oogenesis in the echiuroid worm Urechis caupo. Develop. Biol. 27, l-12. DUMONT, J. N. (1972). Oogenesis in Xenopus laevis (Daudin) I. Stages of oocyte development in laboratory maintained animals. J. Morphol. 136, 153-180. ECKER, R. E. (1972). The regulation of protein synthesis in anucleate frog oocytes. In: Biology and Radiobiology of Anucleate Systems. I. Bacteria and Animal Cells (S. Bonotto, R. Goutier, R. Kirchmann, and J. R. Maisin, eds.), pp. 165-179. Academic Press, New York. ECKER, R. E., and SMITH, L. D. (1971a). Influence of exogenous ions on the events of maturation in Rana pipiens. J. Cell Physiol. 77, 61-70. ECKER, R. E., and SMITH, L.D. (1971b). The nature

118

DEVELOPMENTAL

BIOLOGY

and fate of Rana pipiens proteins synthesized during maturation and early cleavage. Develop. Biol. 24, 559-576. FICQ, A. (1961). Metabolisme de l’oogenese chez les Amphibiens. Germ Cells and the Earliest Stages of Development. Symp. Int. Inst. Embryol., Pallanza, Italy. GALL, J. G. (1966). Nuclear RNA of the salamander oocyte. Nat. Cancer Inst. Monogr. 23, 475-488. HOUGH, B. R., and DAVIDSON, E. H. (1972). Studies on the repetitive sequence transcripts of Xenopus oocytes. J. Mol. Biol. 70, 491-509. LANDESMAN, R., and GROSS, P. R. (1969). Patterns of macromolecular synthesis during development of Xenopus laeuis. II. Identification of the 405 precursor to ribosomal RNA. Develop. Biol. 19, 244-260. LOENING, U. E. (1968). In “Chromatographic and Electrophoretic Techniques” (I. Smith, ed.), Vol. 2, p. 437. Heinemann, London. LOENING, U. E., JONES, K. W., and BIRNSTIEL, M. L. (1969). Properties of the ribosomal RNA precursor in Xenopus laeuis: Comparison to the precursor in mammals and plants. J. Mol. Biol. 45, 353-366. MACGREGOR, H. C. (1967). Pattern of incorporation of [8H]uridine into RNA of amphibian oocyte nucleoli. J. Cell Sci. 2, 145-150. MAIRY, and DENIS, H. (1971). Recherches biochimiques sur I’oogenese 1. Synthese et accumulation du RNA pendant l’oogenese du crapaud sudafricain Xenopus laevis. Develop. Biol. 24,143-165. PERRY, R. P., LATORRE, J., KELLEY, D. E., and GREENBERG, J. R. (1972). On the lability of poly(A) sequences during extraction of messenger RNA from polyribosomes. Biochim. Biophys. Acta 262, 220-226. PLAGEMANN, P. G. W. (1971a). Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. I. Kinetics of incorporation of nucleosides into nucleotide pools and pool sizes during growth cycle. J. Cell Physiol. 77, 213-240. PLAGEMANN, P. G. W. (1971b). Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. II. Independent nucleotide pools for nucleic

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34, 1973

acid synthesis. J. Cell Physiol. 77, 241-258. PLAGEMANN, P. G. W. (1972). Nucleotide pools in Novikoff rat hepatoma cells growing in suspension culture. III. Effects of nucleosides in medium on levels of nucleotides in separate nucleotide pools for nuclear and cytoplasmic RNA synthesis. J. Cell Biol. 52, 131-146. PRZYBYLSKI, R. J. (1969). Scintillation radioautography: A new technique designed to augment silver grain number in radioautographs. J. Cell Biol. 43, 108a. ROGERS, M. E. (1968). Ribonucleoprotein particles in the amphibian oocyte nucleus. Possible intermediates in ribosome synthesis. J. Cell Biol. 36,421-432. SMITH, L. D. (1972). Protein synthesis during oocyte maturation. In “Oogenesis” (J. D. Biggers and A. W. Schuetz, eds.), pp. 227-239. University Park Press. Baltimore, Maryland. SMITH, L. D., and ECKER, R. E. (1969). Role of the oocyte nucleus in physiological maturation in Rana pipiens. Develop. Biol. 19, 281-309. SMITH, L. D., and ECKER, R. E. (1970). Regulatory processes in the maturation and early cleavage of amphibian eggs. Curr. Top. Develop. Biol. 5, l-38. WALLACE, R. A., and JARED, D. W. (1969). Studies on amphibian yolk VIII, The estrogen-induced synthesis of a serum lipophosphoprotein and its selective uptake by the ovary and transformation into yolk platelet proteins in Xenopus laevis. Develop. Biol. 19, 498-526. WALLACE, R. A., JARED, D. W., and NELSON, B. L. (1970). Protein incorporation by isolated amphibian oocytes. J. Exp. Zool. 175, 259-270. WIT, F. H. (1969). An artifact in the alkaline hydrolysis of RNA labeled with guanosine-8-SH Anal. Biochem. 27, 1. 186189. WOODLAND, H. R. (1969). The phosphorylation of thymidine by oocytes and eggs of Xenopus laevis Daudin. Biochim Biophys. ACTA 186, l-12. WOODLAND, H. R., and PESTELL, R. Q. W., (1972). Determination of the nucleoside triphosphate contents of eggs and oocytes of Xenopus laevis. Biothem. J. 127,597-605.