poly(a)+ rna metabolism during oogenesis in xenopus laevis

DEVELOPMENTAL BIOLOGY 69, 217-236 (1979)

Poly(A)+ RNA Metabolism during Oogenesis in Xenopus /aevis’,*

GREGORY J. DOLECKI” AND L. DENNIS SMITH

Department of Biological Sciences, Purdue Crniversity, West Lafayette, Indiana 47907

Received ,July 17, 1978; accepted in reuised form October 25, 1978

In this study, we have measured the synthesis and turnover of oligo(dT)cellulose-bound RNA [poly(A)’ RNA] in Xenopus laevis oocytes at the maximal lampbrush chromosome stage (stage 3) and at the completion of oocyte growth (stage 6). Oocytes at both stages are shown to be active in the synthesis of poly(A)’ RNA. In stage 6 oocytes, the mean rate of synthesis of stable poly(A)’ RNA is 15% the instantaneous rate of synthesis, while the mean half-life of the unstable component is 1.6 hr. In contrast, the instantaneous rate of synthesis in stage 3 oocytes is about one-third that seen in stage 6, and most of it is devoted to the production of unstable species with an average half-life of 5 hr. Studies on the nuclear versus the cytoplasmic distribution of the newly synthesized poly(A)+ RNA demonstrated that by the end of a 12-hr labeling period for stage 3 oocytes and a 24-hr labeling period for stage 6 oocytes, approximately half of the material was cytoplasmic. This cytoplasmic material had the same electrophoretic mobility as bulk poly(A)’ RNA. Similarly, as with bulk poly(A)’ RNA, little, if any, of the newly synthesized material was found to be polysomal. Also, poly(A) labeling studies indicated that the newly synthesized poly(A)’ RNA was associated with the synthesis of poly(A) of the same length as that appearing on bulk poly(A)’ RNA. Studies on the content of bulk oligo(dT)cellulose-bound RNA indicated that about 86 ng is present in both stage 3 and stage 6 oocytes. The continual synthesis of poly(A)’ RNA throughout oogenesis in the absence of its accumulation led to the conclusion that it must be turning over. These data are discussed in relation to the hypothesis that bulk levels of poly(A)+ RNA are maintained by continually changing rates of synthesis and degradation,

INTRODUCTION

It is clearly established that the full- grown oocytes of many animal species contain a store of maternal mRNAs to be used in postfertilization development. The time of synthesis (and accumulation) of this material, as it relates to specific stages of oogenesis, is less clear. The classical view that most of the maternal mRNA is synthesized during the maximal lampbrush chromosome stage no longer appears feasible, at least in simplest form (review by Davidson, 1976).

Recently, Cabada et al. (1977) have shown that the stored maternal RNA in full-grown oocytes of Xenopus laevis con-

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

’ I’ortions of this work were submitted by GD as part of a Ph.D. thesis to Purdue University, 1977.

’ l’resent address: Kewalo Marine Laboratory. 41 Ahui Street, Honolulu. Hawaii.

tains two distinct size classes of poly(A), designated poly(A)L and poly(A)s, which contain respectively 40-80 (mean, 61) and 15-30 (mean, 20) A residues and are found on different heterogeneous RNA molecules. Only polyp+ RNA binds to oligo (dT)- cellulose, but up to 50% of the poly- (A)s’ RNA can be isolated from the void fraction by binding to PO]YW)- Sepharose. Both classes of poly(A)+ RNA yield identical in vitro protein products.

There appears to be a major shift in poly(A)’ RNA metabolism just prior to the onset of vitellogenesis. Previtellogenic oocytes contain almost exclusively p01y(A)~, which accumulates up to vitellogenesis but remains almost constant from then on (Ca- bada et ccl., 1977). Poly(A)s begins accumulating in early vitellogenic oocytes and continues to do so up to stage 6 (full-grown oocytes).

In previtellogenic oocytes, over 7% of 217

OO12-1606/79/030217-20$02.00/O Copyright C? 1979 by Academic Press. 1~ All rights of reprodurrion in an)- torn1 wserved

218 DEVELOPMENTAL BIOLOGY VOLUME 69,1979

the polyp+ RNA is reported to be on polysomes (Darnbrough and Ford, 1976), while less than 10% is found on polysomes in stage 6 oocytes (Rosbash and Ford, 1974). The poly(A)L+ RNA synthesized by previtellogenic oocytes also is reported to have a half-life of greater than 2 years, and thus apparently would be conserved throughout oogenesis (Ford et al., 1977). In support of this view, there is little new sequence complexity in egg p01y(A)~’ RNA as compared to previtellogenic oocytes (Perlman and Rosbash, 1978). However, these kinds of studies yield information only on the net amounts of poly(A)’ RNA present in oocytes of various stages. As yet, nothing is known about the synthetic or degradative events involved in producing these net amounts.

Our initial hypothesis was that the constant level of poly(A)L+ RNA might indicate a simple steady-state situation, in which there is continual degradation and resynthesis. To test this hypothesis, we have measured the synthesis and turnover of oligo(dT)cellulose-bound RNA in oocytes at the maximal lampbrush chromo- somal stage (stage 3) and in full-grown oocytes (stage 6). These experiments and others designed to determine the electropho- retie mobility of the newly synthesized poly(A)+ RNA, its cellular distribution, and the size of the poly(A) tract associated with it demonstrate that RNA with all of the characteristics of the stored maternal p01y(A)~’ RNA is synthesized both at stage 3 and at stage 6. There are, however, significant differences at the two stages in the rates of synthesis and degradation. These data are discussed in relation to the maintenance of a constant level of polyp+ RNA during oogenesis. We have been unable to detect any newly synthesized poly(A)s+ RNA.

MATERIALS AND METHODS

Experimental animals. Xenopus laevis females were obtained directly from South Africa.

HCG stimulation. Frogs were induced to ovulate with human chorionic gonadotropin as described by Anderson and Smith (1978).

Treatment and classification of oocytes. Oocytes were obtained, handled, and clas- sified as stage 3 or stage 6 as described by Anderson and Smith (1977). Manual dissec- tion of the oocytes does not completely remove all of the follicle cells. However, they contribute insignificantly to the newly synthesized RNA, since the rates of oocyte nuclear RNA synthesis are identical to the rates measured in whole, manually dis- sected oocytes (Anderson and Smith, 1977, 1978).

When large numbers of oocytes (several thousand) were needed, as in experiments to determine poly(A)+ RNA contents, whole ovaries were digested with 0.2% collagenase (Sigma) in 0.1 M phosphate buffer (pH 7.4) for approximately 3 hr at 21°C (Dumont, 1972).

After thorough rinsing, the oocytes were sized by a combination of two methods. First, they were passed upward with Ringer’s through a 2.5cm-diameter chromatography column. As the flow rate was increased, progressively larger oocytes were removed (Woodland and Pestell, 1972). Next, they were sifted through a series of nytex screens of different mesh sizes. Those retained by a screen with a mesh diameter of 1180 pm were taken as stage 6 oocytes. Those which passed through a screen with a mesh diameter of 600 pm but were retained by a screen with a mesh diameter of 350 pm were taken as stage 3 oocytes.

Collagenase treatment was carried out so that it resulted in a loss of the vitelline membrane from some of the oocytes. Hence, follicle cell contamination of these preparations was not a problem.

Labeling of oocytes. With respect to [3H]GTP (Schwarz-Mann; [8-3H]guanosine triphosphate, tetrasodium salt; 15 Ci/ mmole), 1 mCi was desiccated to dryness and resuspended in 35 ~1 of Ringer’s solution. Stage 6 oocytes were injected with 20

DOLECKI AND SMITH Poly(A)+ RNA Mrtnholism in X. inelu 219

nl containing approximately 50 pmoles of [“H]GTP (-1.5 x 10” dpm). Stage 3 oocytes were injected with 15 nl containing approximately 38 pmoles of [“H]GTP (-1.1 X 10” b-d.

With respect to [,‘H]ATP (New England Nuclear; [2,8-“Hladenosine triphosphate, tetrasodium salt; 32 Ci/mmole), 1 mCi was desiccated to dryness and resuspended in 35 ~1 of Ringer’s solution. Stage 6 oocytes were injected with 20 nl containing approximately 25 pmoles of [“H]ATP (-1.5 X 10” dpm). Stage 3 oocytes were injected with 15 nl containing approximately 19 pmoles of [“H]ATP (-1.1 x 10”).

With respect to [“Hlleucine (Schwarz- Mann; L-[4,5-“Hlleucine; 61 Ci/mmole), 300 ~1 of isotope in 0.01 N HCl was neutralized by the addition of 5 ~1 of 1.0 M Tris. Stage 6 oocytes were injected with 20 nl containing approximately 2 pmoles of [“Hlleucine (-2.4 x 10” dpm).

Cellulnr fractionation. For most experiments, oocytes were fractionated into nuclei (germinal vesicles) and cytoplasms as described by Anderson and Smith (1977). Cytoplasmic recovery was monitored by comparing the amount of RNA extracted from this fraction to that extracted from total oocytes, the amount of RNA being measured by OD,,;,,. The assumption here is that the vast majority of the OD,,jo ex- tractable from oocytes is cytoplasmic RNA. Nuclear recovery was monitored by comparing the amount of 40 S ribosomal RNA precursor extracted from this fraction to that extracted from total oocytes. Recovery for both fractions was approximately 84%. RNA extraction efficiency was monitored by including a “‘C-labeled bacterial RNA marker at the time of homogenation.

To prepare enucleated oocytes for polysome studies, the method of Ford and Gur- don (1977) was used. The oocytes were not allowed to heal but were, instead, immedi- ately homogenized.

Polysome preparation. All solutions, except sucrose and detergent solutions, were autoclaved prior to use. Glassware and

utensils were made RNase free by baking overnight at 220°C. Ultracentrifuge tubes and plastic centrifuge tubes were soaked in 27.5% HzO, for 30 min and then rinsed wit,h sterile distilled water.

Oocytes were homogenized in 1 ml ot basic buffer plus detergent and centrifuged as described by Woodland (1974), except that Brij was the detergent used. The clear solution between the pellet and the lipid skin was divided into two 0.5-ml portions. Fifty microliters of 0.35 M EDTA (pH 7.4) was added to one, and both were then incubated on ice for 5 min (Humphreys, 1971). The EDTA-treated half was layered on a 12-ml linear gradient from 15 to 304 sucrose (w/w) over a 0.5-ml 60’; sucrose (w/w) cushion. The sucrose was dissolved in 0.3 M KCl, 0.01 M EDTA, 0.02 M Tris-HCl (pH 7.4). The untreated half was layered on an identical gradient, only the sucrose was dissolved in basic buffer. Gra- dients were spun at 37,000 rpm in the SW 41 rotor of a Beckman ultracentrifuge for :1 hr at 4°C. Gradients were scanned at 254 nm and fractionated using an Isco gradient fractionator.

To look at the RNA present on the gradient, O.6-ml fractions were collected and pooled into two groups: those containing material heavier than 80 S and those containing material 80 S and lighter. The RNA was then extracted as described below after the addition of p-aminosalicylic acid, SDS, and polyvinyl sulfate to the appropriate concentrations. EDTA was added to the fractions from the gradients run in basic buffer.

GTP pool specific activity. For kinetic experiments, the specific activity of the GTP pool was measured as described by Dolecki et al. (1976), with the following exceptions. Oocytes were homogenized in 1 ml of 10% TCA and the homogenizer was subsequently rinsed with an additional 0.5 ml of 10% TCA for a combined total of 1.5 ml of extract. The charcoal was eluted in 1% NH:, in 50% ethanol. This t.reatment causes an 119 exchange of the ‘H label,


which was taken into account. Finally, after for 1 min, chilled to 4”C, made 0.5 M in the luciferase reaction, 10 ml of Mono- NaCl, and rebound to the column as above. phase-40 (Packard Instruments) was added The column was again washed with binding as the scintillation cocktail. buffer at 4°C until no more RNA came off.

RNA extraction. RNA was extracted The bound RNA was finally eluted with from Xenopus oocytes by a modification of elution buffer at room temperature. This the phenol-cresol procedure of Kirby (1965; last eluate was called the bound fraction. See Anderson and Smith, 1977). All of the other column effluent was pooled

Poly(A)+ RNA isolation. Poly(A)+ RNA to give the unbound fraction. In all cases, was isolated by oligo(dT)cellulose chroma- recovery of oocyte poly(A)+ RNA was mon- tography. The oligo(dT)cellulose was a itored by adding 14C-labeled poly(A)’ RNA mixture of two different types. One was from Blastocladiella emmersonii (gift of S. purchased from Collaborative Research (T- A. Johnson) at the initial homogenization 2), and the other was identically prepared step. by Mr. S. A. Johnson in the laboratory of Several controls were run to determine Dr. P. T. Gilham. This mixture provided a the validity of oligo(dT)cellulose fraction- high binding capacity and good flow char- ation of poly(A)’ and poly(A)- RNA. Pos- acteristics. For the purpose of isolating bulk sible nonspecific sticking of ribosomal RNA poly(A)+ RNA present in different-stage (Bantle et al., 1976) was tested by electro- oocytes, columns were made in 12-ml plas- phoresing oligo(dT)cellulose-bound RNA tic syringes with a 3-ml bed of oligo- on 2.2% polyacrylamide gels (Fig. 1). Radio- (dT)cellulose. These columns had a binding active ribosomal RNA was not a visible capacity of 40 ODZeO units of poly(A) contaminant. (Sigma). In all other experiments, columns Contamination of the poly(A)+ RNA were made in 6-ml plastic syringes with a preparations by heterodisperse poly(A)- 0.6-ml bed of oligo(dT)cellulose. These col- RNA was tested by hybridizing [“HI- umns had a binding capacity of 8 ODXW hnRNA from sea urchin blastulas (gift of units of poly(A) (Sigma). Binding and elu- D. Lowensteiner) with poly(U) to mask the tion buffers were autoclaved prior to use. poly(A) stretches and then isolating the Glassware was made RNAase free by bak- oligo(dT)cellulose-bound material. In three ing overnight at 220°C. replicates, only 0.045, 0.040, and 0.050% of

The RNA was dissolved in elution buffer the input material bound to the column. (0.01 M Tris-HCl, pH 7.0) and heat dena- The other two problems associated with tured at 60°C for 1 min to reduce aggrega- oligo(dT)cellulose chromatography are in- tion and, hence, rRNA contamination. The efficient binding and irreversible binding. sample was then chilled to 4°C in an ice- 14C-Labeled poly(A)+ RNA from Blastocla- water bath and NaCl was added to 0.5 M. diella was chosen to monitor these two After addition of the NaCl, the RNA was phenomenon since it has a size distribution passed four times in succession over the (data not shown) and a poly(A) length (60 column, which previously had been equili- residues; S. A. Johnson, personal commu- brated with binding buffer (0.5 M NaCl, nication) similar to that of the poly(A)+ 0.01 M Tris-HCl, pH 7.2) at 4°C. The col- RNA from Xenopus oocytes (Cabada et al., umn was then washed thoroughly with ice- 1977). cold binding buffer until no more RNA Initially, the oligo(dT)cellulose columns came off. were regenerated after each use with 0.1 N

The bound RNA was eluted with elution KOH (Aviv and Leder, 1972). Under these buffer at room temperature and the column conditions, irreversible binding of the 14C- was reequilibrated with binding buffer at labeled marker seemed to be a severe prob- 4°C. The eluate was heat denatured at 60°C lem; the bound plus the unbound fractions

DoLECKI AND SMITH Poly(A)’ RNA Metabolism in X. laecis

oocyte RNA and the recovery marker was taken into account in all calculations.

221

2 4 6 0

MIGRATION km) FIG. 1. Newly synthesized oligo(dT)cellulose-

bound RNA. Stage 6 oocytes were injected with [‘H]GTP and incubated in Eppig and Dumont’s medium (Eppig and Dumont, 1976) for 24 hr. Poly(A)’ RNA was isolated by oligo(dT)cellulose chromatography. Electrophoresis was on aqueous 2.2% polyacrylamide gels. (M) ‘H-Labeled oligo(dT)cellulose- bound RNA; (A-A) ‘%-labeled stage 6 oocyte RNA added to sample prior to electrophoresis.

from duplicate ohgo(dT)cellulose fraction- ations totaled on1.y 84 and 83% of the input radioactivity. Prel?mpting the column with Escherichia coli tRNA (Bantle et al., 1976) did not prevent the problem. It was then decided to omit the KOH regeneration step. After several additional uses of these un- regenerated columns, irreversible binding decreased to 0%

Inefficient binding of poly(A)+ RNA to oligo(dT)cellulose was less of a problem than irreversible binding. The “‘C-labeled Blastocladiella marker bound with 81 and 83% efficiencies in duplicate experiments. Xenopus oocyte poly(A)+ RNA bound with 90, 95, and 100%~ efficiencies, as determined by “H-labeled poly(U) hybridization (Do- lecki et al., 1977) with three separate preparations of stage 6 oocyte RNA. The slight difference in binding efficiency between the

Polyacrylamide gel electrophoresis. Poly(A) preparations produced by nuclease digestion were run at 20°C through 10% polyacrylamide gels (0.5 X 9.5 cm; Loening, 1967) at. 7 mA per gel in E buffer (Loening, 1969). After scanning at 260 nm, the gels were frozen to lengths of 9.5 cm (scanning length), sliced, and counted. The tolu- ene-PPO cocktail used for these gels was 6% NCS (Nuclear Chicago Solubilizer).

Other RNA preparations were sized on 2.2%’ polyacrylamide gels as described by Anderson and Smith (1977). RNA was layered onto the gels in 0.1 M NaCl. 0.005 1M EDTA, 0.5%’ SDS, 15% sucrose (pH 7.4) (McKnight and Schimke, 1974). To dena- ture the RNA prior to electrophoresis, the RNA was dissolved in the above buffer, heated to 60°C for 1 min, quickly cooled to 4°C in an ice-water bath, and then layered onto the gel (S. A. ,Johnson, personal com- munication).

Nuclease digestion. RNA extracted from oocytes labeled with [“H]ATP was digested at 37°C for 30 min with a combination of nucleases: 10 units of T1 RNAase (Sigma)/ ml and 5 pg of pancreatic RNAase (Sigma)/ ml (Wilt, 1973). The basic digestion buffer was 0.3 M NaCl, 0.001 M EDTA. 0.01 M Tris-HCl (pH 7.4).

After digestion, the samples were made 0.5% in SDS and the RNA was extracted as described above, with the following exceptions. Phenol saturated with digestion buffer was used instead of the phenol-cresol mixture, and no p-aminosalicylic acid or polyvinyl sulfate was added. After extraction, carrier RNA was added and the samples were ethanol precipitated twice and not passed over Sephadex.

Treatment of Millipore filters. Millipore filters were cornbusted in a Packard Tri- Carb oxidizer to produce “HZ0 and “CO,. This machine effectively separates the two isotopes with a greater than 95q recovery of each. The scintillation cocktails used foi the “HZ0 and “CO, were Monophase 40


and Permafluor V (Packard Instruments), from the following relationship: Kd = ln2/ respectively. Samples were counted in a t1/2. Beckman LS-230 scintillation counter.

Rate measurements and quantitation of RESULTS

data. For measuring rates of RNA synthe- Accumulation of Poly(A)+ RNA during Oo- sis, groups of 7-30 oocytes were injected genesis

with [“HIGTP and incubated for various lengths of time in Eppig and Dumont’s

Determinations of the amount of poly(A)

medium (Eppig and Dumont, 1976). They per oocyte at different stages of oogenesis,

were then rinsed in Ringer’s solution, frozen based on “H-labeled poly( U) binding, led to

on dry ice, and stored at -70°C. 14C-La- the conclusion that poly(A)’ RNA accu-

beled Blastocladiella poly(A)+ RNA was mulates up to early vitellogenesis and then

added to monitor recovery and RNA was remains constant (Rosbash and Ford, 1974;

extracted. The RNA was then passed over Cabada et al., 1977). We attempted to con-

oligo(dT)cellulose. Radioactivity in the firm this observation’directly by extracting

bound fraction was plotted as poly(A)+ RNA from large numbers of stage 3 and

RNA and radioactivity in the bound plus stage 6 oocytes and measuring the ODzso of

the unbound fractions was plotted as total the oligo(dT)cellulose-bound fraction. To-

RNA, after converting to picomoles of GTP tal RNA contents for stage 3 and stage 6 oocytes averaged, respectively, 2.2 and 5.6

incorporated using precursor pool specific pg. These values are in good agreement activity data. with those of Cabada et al. (1977). Table 1

The following equation was used to de- shows the poly(A)+ RNA contents of both scribe the synthesis of stable RNA: stage 3 and stage 6 oocytes. The values are

dRNA,/dt = KS:,, essentially identical. The 86-ng content

where KS is the synthetic constant (pmoles measured in the current study agrees very

of GTP/hr), t is time in hours, and RNA, is well with the 84-ng content of polyp’ RNA which can be calculated from the data

picomoles of GTP incorporated into stable RNA.

of Cabada et al. (1977) assuming an average

The following equation was used to de- molecular weight of 7.5 x lo” for polyp’

scribe the synthesis of unstable RNA: RNA and a poly(A) length of 45 (also measured in the current study; see p. 229. The

dRNA,,/dt = K,, - Kd(RNA,,), rRNA contamination in these experiments

where KU, is the synthetic constant (pmoles averaged less than 5% (Dolecki, 1977).

of GTP/hr), t is time in hours, Kd is the TABLE 1

first-order degradation constant (hr-‘), and OLIco(dT)CELLuLosE-BOUND RNA CONTENTS OF

RNA,, is picomoles of GTP incorporated STACE~ANDSTAGE~OOCYTES~

into unstable RNA. Expt Oocyte stage Poly(A)+ RNA

These equations can be combined and W

manipulated into the following form to de- 1 III 71

scribe the accumulation of total RNA: 1 VI 75 2 III 83

RNAT = KSt + (K,,/KJ(l - epKdt), where 2 VI -

K,,/Kd is the steady-state level of unstable 3 III 108

RNA (pmoles of GTP) and RNAT = RNA, 3 VI 94

+ RNA,,. The data were fit to this last Average III 87

equation by a least-squares method using Average VI 85

the SPSS 21 computer program to deter- ” The data were calculated from the percentage of

mine the kinetic constants (Nei et al., 1970). the total ODZ~ which bound to oligo(dT)cellulose after correcting for recovery. ‘%-Labeled BlastocladielZa

Degradation half-lives ( t1,2) were calculated emmersonii poly (A)’ RNA was the recovery marker.

DOLECKI AND SMITH Poly(A)’ RNA Metabolism in X. laevis 223

Synthesis of Poly(A)+ RNA A molar accumulation curve for radioac-

tive poly(A)+ RNA in stage 6 oocytes injected with [“HIGTP is presented in Fig. 2. Also included in Fig. 2 is the change in GTP pool specific activity during the course of the experiment; a decline of 13% was observed. As previously reported for total RNA synthesis (Anderson and Smith, 1977; Dolecki, 1977), the kinetics are biphasic, indicating the synthesis of both stable and unstable RNA. A computer analysis of the data yields a rate of incorporation into unstable RNA of 0.18 pmole hr-‘. The mean half-life of this component is calculated to be 2.8 hr. The rate of incorporation into stable RNA is 0.032 pmole hr-‘, or 15% of the instantaneous rate of incorporation (0.212 pmole hr-‘).

Since the bulk amount of oligo(dT)- cellulose-bound RNA per oocyte remains essentially constant from early vitellogenesis on, the “stable” component described above must ultimately be unstable, perhaps having a half-life only slightly longer than the duration of the experiments. To inves- tigate this possibility, we attempted to

carry out experiments for longer periods of time. It soon became apparent, however, that continued incubation of oocytes in ci- tro posed a problem. Figure 3 shows the effects of extended culture periods on the molar accumulation of radioactive poly(A)’ RNA in stage 6 oocytes. Injected oocytes were maintained in Eppig and Dumont’s medium for 72 hr, with daily replacement of the medium. On each successive day, a kinetic experiment spanning a 24-hr period was performed. It is clear from Fig. 3 that the most dramatic effect was on the rate of stable accumulation; by the third day in culture, it had become nonexistent. In an- other such study, the molar accumulation curve for poly(A)+ RNA not only plateaued by 14 hr of incorporation, as shown in Fig. 3C, but then began to decline so that after 24 hr of incorporation, it had reached a level which was only 10% of the plateau value. The various kinetic constants appear in Table 2. Although the change in the rate of stable accumulation is most pronounced, all of the constants are affected. Similar effects on the molar accumulation of total radioactive RNA were observed during the

16 r . 14-

i? 0.6-

d

z a 04- . p------

4 8 12 16 20 24

TIME (hr)

4 8 12

TIME (hr)

i 16 20 24

FIG. 2. Molar accumulation curve for poly(A)’ RNA from stage 6 oocytes. Groups of 7-30 stage 6 oocytes were injected with [‘H]GTP and incubated in Eppig and Dumont’s medium (Eppig and Dumont. 1976). The extracted RNA was fractionated by oligo(dT)cellulose chromatography and the radioactivity in the bound fraction was used to produce the curve. The inset shows the GTP pool specific activity during the course of the study.

224 DEVELOPMENTAL BIOLOGY VOLUME 69.1979

experiment; the instantaneous rate of total RNA synthesis declined by a factor of 2.5 and the rate of stable accumulation by a factor of 4 during the 72-hr period.

Elaborate control studies such as the one just described were not performed on stage 3 oocytes, due to the greater difficulty in-

20

16

12

OS

04

r g 2.0

c 1.6

2.0 c

16 C 12

/.

1 I I I / I

4 8 12 16 20 24

TIME (hr)

FIG. 3. The effect of in vitro culture on the molar accumulation of poly(A)’ RNA in stage 6 oocytes. Oocytes were maintained in Eppig and Dumont’s medium (Eppig and Dumont, 1976) for 72 hr, with daily replacement of the medium. On each successive day, groups of 7-30 oocytes were injected with [“H]GTP to perform a kinetic experiment spanning a 24-hr period. The extracted RNA was fractionated by oligo (dT)cellulose chromatography and the radioactivity in the bound fraction was used to produce the curves. (A) Day 1, (B) Day 2, (C) Day 3.

volved in handling these smaller oocytes. For convenience and uniformity, then, measurements of only the instantaneous rates of synthesis of total RNA and poly(A)+ RNA were made to monitor the effects of in vitro culture on both stage 3 and stage 6 oocytes. There was a good deal of variation from experiment to experiment. The most frequently observed phenomenon was a general decline in the rates of synthesis with time in culture, although the relationship was not. always linear. However, some groups of oocytes exhibited increases in the rates of RNA synthesis, while others maintained constant rates for at least 5 days. Several attempts were made to prevent this aberrant behavior, but none was successful. Hence, it was decided to restrict the stage 6 studies to a 24-hr period and the stage 3 studies to a 12-hr period. This would produce, on the average, a decline in the instantaneous rate of RNA synthesis of approximately 12%, which is less than the error involved in the measurement of this parameter. As a safeguard, the instantaneous rates of both poly(A)+ RNA and total RNA synthesis were measured at the end of each kinetic study. In cases where these varied by more than 12% from the values measured at the beginning of the study, the experiment was discarded.

A summary of the data on poly(A) RNA kinetics in stage 3 and stage 6 oocytes is presented in Table 3. Also included in Table 3 is a single experiment on stage 4 oocytes. In stage 6 oocytes, the mean rate of synthesis of unstable poly(A)’ RNA is 0.25 pmole hr-‘, while the mean half-life of this component is 1.6 hr. The mean rate of synthesis

TABLE 2

THE EFFECTS OF in Vitro CULTURE ON THE KINETIC PARAMETERS FOR PoLY(A)+ RNA SYNTHESIS“

Day KS (stable) Ku, (unstable) K,r (total) Kus/Kd t1/2 KS/KY x (pmoles

GTP/hr/oo- G’#~l$~o- G$:?i$to- (h:“, (pmoles (hr) 100

GTP) We) We)

1 0.047 0.400 0.447 0.690 0.580 1.00 11 2 0.029 0.500 0.529 0.610 0.820 1.13 5 3 0.000 0.220 0.220 0.340 0.647 2.03 0

U The data were calculated from the curves in Fig. 3.

DOLECKI APED SMITH Poly(A)’ RNA Metabolism in X. laeris 225

TABLE :I

KINETIC PARAMETERS FOR PoLY(A)’ RNA SYNTHESIS IN STACE 3 ANI) STA(:E 6 Oocu~r~?;

Oocyte stage Expt KS (stable) K,,, (unsta- (pmoles ble)

GTP/hr/ (pmoles oocyte) GTP/hr/

oocyte)

VI 1 0.047 0.400 VI 2 0.032 0.180 VI 3 0.055 0.170 VI Average 0.045 0.250 III 1 0.000 0.061 III 2 0.030 0.098 III 3 0.000 0.070 III Average 0.010 0.076 III 1 0.000 0.102

Stimulated P III 2 0.025 0.053

Stimulated P IV 1 0.032 0.148

for the stable component is 0.045 pmol hr-I, or 15% of the instantaneous rate of synthesis (0.295 pmole hr-‘). In contrast, the instantaneous rate of synthesis in stage 3 oocytes is about one-third that seen in stage 6 oocytes, and most of it is devoted to the production of unstable species with an average half-life of 5 hr. Unlike the situation with respect to heterogeneous RNA synthesis (Anderson and Smith, 1978), these values are essentially unchanged in oocytes taken from females recently injected with human chorionic gonadotropin,

Identification of a stable component in stage 3 oocytes is more difficult, due to the restricted time span over which the experiments were conducted, coupled with the longer half-life of the unstable component. A best fit of the data by computer indicates the presence of a stable component in oocytes from two of the five females (one nonstimulated and one hcG stimulated). The single experiment on stage 4 oocytes reveals a rate of unstable poly(A)’ RNA synthesis intermediate between that in stage 3 oocytes and that in stage 6 oocytes. Stable poly(A)+ RNA in this case accumulated at almost the same rate as in stage 6 oocytes, but represented a slightly higher percentage (18%) of the instantaneous synthetic rate.

K.1 (total) K,i KJKd ti 2 K./K, (pmoles (hr ‘) (pmoles (hr) x 100

GTP/hr/ GTP) oocyte)

0.447 0.690 0.580 1 .oo 11 0.212 0.250 0.720 2.77 lC5 0.225 0.670 0.254 1.04 ‘5 0.295 0.540 0.518 1.60 17 0.061 0.202 0.302 3.43 0 0.128 0.260 0.376 2.67 21 0.070 0.078 0.897 8.92 0 0.086 0.180 0.525 5.01 8 0.102 0.114 0.895 Ii.08 0

0.078 0.460 0.115 1.50 32

0.180 0.193 0.770 3.69 18

Intracellular Location of Newly Synthe- sized Poly(A) RNA Previous kinetic studies by Anderson and

Smith (1977, 1978) have shown that unstable heterogeneous RNA is restricted to the nucleus, while stable heterogeneous RNA accumulates in the cytoplasm. Presumably, since poly(A)’ RNA represents a subset of total heterogeneous RNA, the unstable and stable components would exhibit nuclear and cytoplasmic localization, respectively. For technical reasons, it was judged im- practical to attempt detailed kinetic studies on the accumulation of newly synthesized nuclear and cytoplasmic poly(A).’ RNA. In- stead, nuclei and cytoplasms were isolated at only two time points, one at the beginning (2 hr for stage 3 oocytes and 3 hr foi stage 6 oocytes) and one at the end (12 hr for stage 3 oocytes and 24 hr for stage 6 oocytes) of a kinetic study, and the RNA was fractionated on oligo(dT)cellulose.

Figure 4 shows the electrophoretic profiles of radioactive poly(A)’ RNA from the nuclear and cytoplasmic fractions of both stage 3 and stage 6 oocytes at the latest time points. In each case, the RNA was heat denatured twice to eliminate aggre- gates during the oligo(dT)fractionation (see Materials and Methods). The newly synthesized nuclear and cytoplasmic poly(A) ’


N 6

b

I I I I I I I I III 2 4 6 8 2 4 6 8

MIGRATION km)

FIG. 4. The electrophoretic mobility of newly synthesized nuclear and cytoplasmic poly(A)+ RNA. Groups of 30 oocytes were injected with [3H]GTP and incubated in Eppig and Dumont’s medium (Eppig and Dumont, 1976) for 12 hr (stage 3) or for 24 hr (stage 6). Nuclei were manually removed in the acetate buffer system described in Materials and Methods. The extracted RNA was fractionated by oligo(dT)cellulose chromatography. Electrophoresis was on aqueous 2.2% polyacrylamide gels. (A) Stage 3 nuclei, (B) stage 3 cytoplasms, (C) stage 6 nuclei, (D) stage 6 cytoplasms.

RNAs have similar heterodisperse profiles, with the nuclear material being, on the average, slightly larger than the cytoplasmic material. This is analogous to observations on heterodisperse RNA in sea urchin embryos (Kung, 1974). The size distribution of the newly synthesized cytoplasmic poly(A)+ RNA closely parallels that of bulk poly(A)+ RNA (Rosbash and Ford, 1974; Dolecki, 1977), of which at least 80% is cytoplasmic (Rosbash and Ford, 1974).

There is also a great deal of similarity between the two preparations from the two different stages, with one exception. There is a distinct peak of radioactivity coincident with the 40 S rRNA precursor in the electrophoretic profile of the nuclear preparation from stage 6 oocytes which is not apparent in that from stage 3 oocytes. Since stage 3 oocytes synthesize 40 S rRNA precursor at rates equivalent to those in stage 6 oocytes (Anderson and Smith, 1977, 1978), this material would not appear to be a rRNA contaminant. Similarly, radioactivity in this region of the gel always represented about 13% of the radioactivity in newly synthesized poly(A)+ RNA from the total oocyte, regardless of the time of incorporation. Thus, its molar accumulation is biphasic, as is that of total poly(A)+ RNA, and not first order, as is that of 40 S pre- rRNA (Anderson and Smith, 1977).

Data on the percentage of poly(A)+ RNA and total RNA which is nuclear and that which is cytoplasmic at the two time points are summarized in Table 4. The data on total RNA are in good agreement with those of Anderson and Smith (1977, 1978). Newly synthesized RNA is predominantly nuclear at the early time points. As time proceeds, cytoplasmic accumulation occurs. In sharp contrast, the nuclear:cytoplasmic distribution of poly(A)+ RNA at both time points is close to l:l, in both stage 3 and stage 6 oocytes. The final column in Table 4 shows that the newly synthesized cytoplasmic poly(A)+ RNA is a greater proportion of the total newly synthesized cytoplasmic RNA at early times than at later times. This is consistent with the observations of Anderson and Smith (1977, 1978) which showed that heterogeneous RNA begins to accumulate in the cytoplasm several hours earlier than does rRNA.

Poly(A)’ RNA in Polysomes

As described above, a significant proportion of the newly synthesized poly(A)+ RNA is cytoplasmic after 12 hr of labeling at stage 3 or after 24 hr of labeling at stage

DOLECKI AND SMITH Poly(A)’ RNA Metabolism in X. Iaeris 227

6. To determine whether this material is in somal structures by EDTA treatment was polysomes and hence potentially active in first tested. A homogenate of stage 6 oo- protein synthesis, or whether it represents cytes labeled with [“Hlleucine was divided mRNA stored in the form of free RNPs for in two: One half was treated with EDTA, use after fertilization, oocyte homogenates and the other served as a control. Each was were run on sucrose gradients and the pol- then run on a linear 15-30% sucrose gra- ysome and supernatant fractions were an- dient as described in Materials and Meth- alyzed for the presence of newly synthe- ods. sized poly(A)’ RNA. Figure 5A is an OD,,ic, scan of the control

The efficiency of dissociation of poly- gradient. The two peaks of material repre-

TABLE 4

INTRACELLULAR LOCATION OF NEWLY SYNTHESIZED RNA”

Oocyte Incuba- 7 Total RNA B Total RNA R Poly(A)+ % PolyfA)’ “, Nuclear RNA ‘; Cvtopl;tsmk stage tion time in nucleus in cytoplasm RNA in nu- RNA in cy- which is pol,~lA)~ RNA which is

(hrl cleus toplasm RNA polviA) ’ RNA

VI 3 80 (78-82) 21 (18-22) 50 (37-71) 50 (29-63 I 6.1 (4.5-8.8) 23 ( IiXil) VI 24 26 (21-31) 74 (69-79) 39 (36-42) 61 (58-64) 9.0 (6.8-10) 4.; (4.0-rr.2) III 2 90 (90-91) IO (9-10) 53 (46-65) 47 (35-54) 2.2 i I .9-2.8) licil4-21) III 12 56 (52-63) 44 (37-48) 62 (58-68) 38 (32-42) 5.5 (4.5-6.4) 4.3 (3.5~5.0)

” Groups of 30 oocytes were injected with [‘H]GTP and incubated in Eppig and Dumont’s medium (Eppig and Dumont, 1976) for the appropriate times. Nuclei were manually removed in the acetate huffer system described in Materials and Methods. The extracted RNA was fractionated hy oligo(dT)cellulose chromatography. The radioactivity in the hound plus the unbound fractions was summed for total RNA. The values listed are averages of three experimental determinations. The numbers in parentheses depict the ranges 01’ values obtained.

A

!,I SUPERNATANT POLYSOMES

TOP BOTTOM TOP BOTTOM

FIG. 5. Ultraviolet absorbance profiles of poiysome gradients. A group of 60 oocytes was injected with [:‘H]leucine and incubated in Ringer’s for I5 min. The homogenate was split in two. One half served as a control (A). The other half was treated with EDTA to release nascent peptides (B). Centrifugation was for 3 hr at 37,000 rpm in an SW 41 rotor.

B

228 DEVELOPMENTAL BIOLOGY VOLUME 69.1979

sent the KCl-dissociated ribosome subunits (Baierlein and Infante, 1974). Stage 6 oocytes have too little polysomal material to be detected by this method (Woodland, 1974). Figure 5B is an ODatiO scan of the EDTA-treated half of the homogenate. The subunits exhibited the abnormally low sed-

4

3

l-u

b x

B

2

I m

1

I-

I 1 1

8 16 24 32

FRACTION FIG. 6. Incorporation of [JH]leucine into nascent

peptides. Fractions of 0.4 ml were collected from the gradients shown in Fig. 5. One milliliter of 1 N PCA was added to each fraction, and they were heated to 60°C for 15 min. They were then chilled to 4°C and carrier RNA was added. Precipitates were collected on Millipore filters and counted after combustion in a Packard Tri-Carb oxidizer. (U) Control; (A---A) EDTA-released half.

imentation rates characteristic of EDTA- derivatized ribosomes (Tashiro and Sieke- vitz, 1965; Baierlein and Infante, 1974).

Fractions of 0.4 ml were collected from each of these gradients, and the [“Hlleucine incorporated into nascent peptides and pro- teins was determined as described in Ma- terials and Methods. The data are presented in Fig. 6. The radioactivity in fractions 16-32 of the EDTA-treated preparation is greatly diminished compared to that in the identical fractions of the control gradient. Due to the brief nature of the labeling period, this disappearance of radioactivity was assumed to be indicative of EDTA release of nascent peptides. The treatment was calculated to be at least 81% effective. From these data, it was concluded that polysomes were being isolated intact and that the EDTA treatment was producing the appropriate effect.

Data on the polysome versus the supernatant distribution of newly synthesized RNA in stage 3 and stage 6 oocytes is presented in Table 5. Oocytes were injected with [3H]GTP and incubated for either 12 hr (stage 3) or 24 hr (stage 6). In control gradients, 7686% of the total newly synthesized RNA was in the supernatant fraction; this material is predominantly ribosome subunits and preribosome particles. However, only 53-64s of the newly synthesized poly(A)+ RNA was in the supernatant fraction. EDTA treatment resulted in the expected shift of material from the polysome to the supernatant region of the gradient, so that 89-92s of the total newly synthesized RNA and 63-77s of the newly

TABLE 5

POLYSOME vs SUPERNATANT DISTRIBUTION OF NEWLY SYNTHESIZED RNA”

Oocyte +EDTA %, Total RNA in 8 Total RNA in 9 Total RNA % Poly(A)+ %, Poly(Aj+ % Poly(Aj+ stage polysome re- supernatant re- which is EDTA RNA in poly- RNA in super- RNA which is

gion gion sensitive some region nant region EDTA sensitive

6 -EDTA 16 [17,14] 84 [83, 861 (84) 37 [37, 361 (42) 64 [63, 641 (58) 6 +EDTA 9 [8, 101 (7) 91 [92, 901 (93) 7 61 (9) 26 129, 231 (34) 74 [71, 771 (66) 11 [8, 131 (8) 3 -EDTA 20 [ 16, 241 80 [84, 761 43 [47, 391 57 [53,61] 3 +EDTA 9 1% 111 91 [92,89] 11 [8, 131 33 [37, 281 68 [63, 721 IO [lo, 111

0 The appropriate fractions from gradients such as those shown in Fig. 5 were pooled and the RNA was extracted. The extracted RNA was fractionated by oligo(dT)cellulose chromatography. The radioactivity in the bound plus the unbound fractions was summed for total RNA. The values listed are averages of two experimental determinations; the individual determinations are given in brackets. The data in parentheses are on enucleated oocytes.

DOLECKI AND SMITH Poly(A)’ RNA Metnholism in K InetG 229

synthesized poly(A)’ RNA were then in the supernatant fraction. There remained, however, a substantial proportion of EDTA-resistant poly(A)’ RNA in the polysome region. Only about 27% of the poly(A)+ RNA in the polysome region or 10% of the total poly(A)+ RNA in the control gradients was EDTA sensitive. Pre- sumably, the poly(A)+ RNA not released by EDTA from the polysome region represents large RNP particles.

When one takes into consideration the percentage of the newly synthesized poly(A)+ RNA which was found to be nuclear (61%; see Table 4) and the effective- ness of the EDTA release (81%,), it can be calculated that an average of 22% of the newly synthesized cytoplasmic poly(A)’ RNA should be on the polysomes in stage 6 oocytes after 24 hr of labeling; the comparable number for stage 3 oocytes after 12 hr of labeling should be 38%. As part of the first experiment listed in Table 5 on stage 6 oocytes, an equal number of oocytes was enucleated as described in Materials and Methods prior to polysome versus supernatant fractionation. The results on the distribution of the RNA in these gradients are given in parentheses in Table 5; they are hardly different from those obtained with whole oocytes. The proportion of the newly synthesized cytoplasmic poly(A)+ RNA which is EDTA sensitive (8%/0.81 = 9.9%) is identical to the proportion of newly synthesized whole oocyte poly(A)+ RNA which is EDTA sensitive, and does not approximate the 16%) figure predicted from the whole oocyte data (8%/0.81/0.61 = 16%). This indicates that the resolution of the system does not permit a high degree of confidence in the measured proportion of EDTA-sensitive poly(A)’ RNA. The ac- tual measured value (-10%) is so small that the error associated with it and with the calculations based on it may be large. It would, however, seem safe to conclude that the bulk of the newly synthesized poly(A)’ RNA is not polysomal after 12 hr of labeling at stage III or after 24 hr of labeling at stage

VI. It may represent maternal mRNA stored in the form of an RNP particle, as described by Rosbash and Ford (1974).

The Length of the Newly Synthesized Pal-y(A) Tract

In order to determine whether the synthesis of oligo(dT)cellulose-bound poly(A)’ RNA was associated with the concomitant synthesis of the poly(A) characteristic of the stored maternal mRNA, the following experiments were performed. Oocytes were labeled with [“H]ATP, and the oligo- (dT)cellulose-bound and -unbound RNA was isolated. After digestion with a combination of T1 and pancreatic RNAses, the resistant material was analyzed by polyacrylamide gel electrophoresis as described in Materials and Methods.

The arrows in Fig. 7 indicate the electrophoretic mobility of poly(A) standards of g&58,27, and 14 residues, as well as that of Xenopus 4 S and 5 S RNA. When the mobilities of the standards are plotted against the log of their molecular weights, a straight line is produced. The slope of the curve becomes very steep for poly(A) of less than 14 residues (Cabada et cd., 1977). Due to the peculiar migration of poly(A) during gel electrophoresis (Burness et ctl., 1975), the Xenopus 4 S and 5 S RNA species exhibit mobilities comparable to 40 and 63 A residues, respectively, although their ac- tual nucleotide lengths are nearly twice as great.

The data from the digestion experiments are presented in Fig. 7. Digests of the oligo(dT)cellulose-unbound material contain almost exclusively short poly(A) tracts, which migrate faster than the dye marker. Material equivalent to polyp,. migrating in the 4-5 S region of the gels, is confined to the oligo(dT)cellulose-bound fractions. These fractions also contain some short poly(A) tracts and some longer poly(A) migrating more slowly than poly(A)r.. The or- igin of the latter material is unknown; perhaps it is the nuclear precursor (Sheiness et al., 1975) to poly(A)l. and l~)ly(A)~.


14

4 T I . :

cc ‘9

3

X

52

8

vi= 1

18

18

8-

f P-T

6 'CJ

2 4 6 8

MIGRATION km 1 Fro 7. Electrophoretic mobility of newly synthesized poly(A) from Xenopus oocytes. Groups of 60 oocytes

were injected with L3H]ATP and incubated in Eppig and Dumont’s medium (Eppig and Dumont, 1976). The extracted RNA was fractionated by oligo(dT)cellulose chromatography and then digested with T1 and pancreatic RNAses. Electrophoresis was on aqueous 10% polyacrylamide gels. Poly(A) standards of 95, 58, 27, and 14 residues were obtained from Miles Laboratories. An additional 14-residue standard was obtained from the laboratory of Dr. P. T. Gilham. (A) Stage 3, 2 hr; (B) stage 3, 12 hr; (C) stage 6, 3 hr; (D) stage 6, 24 hr. (@---O) Bound fraction; (A-A) unbound fraction.

These electrophoretic patterns are in ex- cellent agreement with those obtained by Cabada et al. (1977) using “H-labeled poly(U) hybridization, in that the mobilities of the various Xenopus poly(A) species are the same with respect to the 4 S and 5 S RNA markers. However, there is some dis- agreement as to the absolute size of these

species. Cabada et al. (1977) assign the 4 S and 5 S RNA markers lengths of 58 and 82 A residues, respectively, whereas in the present study, they are assigned lengths of 40 and 63 A residues, respectively. Hence, Fig. 7 indicates that polyp is between 58 and 28 residues. Poly(A)s is of some unde- termined length less than 14 residues. The

DOLECKI AND SMITH Poly(A)’ RNA Metabolism m X. laeris 231

c

c . ’

_ I

‘, i

I 4

I

h fi i2'

I I I I

2 4 6 8

MIGRATION (cm) FIG. 7. C-D.

most rapidly moving component of poly(A)s has the same electrophoretic mobility as a mixture of 2’,3’-AMP.

In order to obtain a more accurate esti- mate of the length of the “poly(A),,” stage VI oocytes were injected with [“H]ATP and incubated for 24 hr. The RNA was extracted and processed as just described. The poly(A)s was then eluted from the polyacrylamide gels. Eluates were lyophilized and fractionated on a Bio-Gel P-4 column. The results are shown in Fig. 8. The poly(A)s from the oligo(dT)cellulose-

unbound fraction is quite small, between 2 and 6 nucleotides in length. The p01y(A)~ from the bound fraction also contains material of the size just mentioned, in addition to some even shorter material (l-2 nucleotides in length). We suggest that in both cases, the poly(A)s largely represents nonspecific digestion products not completely removed from the nuclease digests by ethanol precipitation. Thus, we are unable to detect any newly synthesized poly(A) sequences analogous to the p01y(A)~ reported by Cabada et al. (1977).


87654 3 21

20 30 40

Fraction FIG. 8. Bio-Gel P-4 sizing of poly(A)s. The experiment presented in Fig. 7D was repeated and the poly(A)s

was eluted from the polyacrylamide gels overnight with NET buffer (0.1 M NaCl, 0.001 M EDTA, 0.05 &f Tris-HCl, pH 7.4). The eluates were lyophilized and passed over a Bio-Gel P-3 column equilibrated with NET buffer. Fractions of 1 ml were collected and counted. The column was calibrated with oligo(A) markers prepared according to Asteriadis et al. (1976). (W) B ound fraction; (A-A) unbound fraction.

DISCUSSION

The results of this study show clearly that oocytes continually synthesize a class of RNA which, by virtue of its binding to oligo(dT)cellulose, is equivalent to the polyp+ RNA previously reported. At all stages examined, the bulk of the newly synthesized poly(A)+ RNA is unstable, with a half-life ranging from one to a few hours. Based on the data in Table 3, it represents an insignificant fraction of the stored poly(A)+ RNA; its steady-state level is only about 0.8 ng, both at stage III an8 at stage VI. Since less than 10% of the newly synthesized poly(A) RNA appears to be on polysomes, it is unlikely that it represents the unstable mRNA seen in other systems. Most likely, the unstable poly(A)+ RNA represents nuclear material. Although its half-life is somewhat longer than that of hnRNA in other systems (Brandhorst and Humphreys, 1972; Brandhorst and Mc- Conkey, 1974), Anderson and Smith (1977)

report an hnRNA population with a half- life of several hours in stage VI Xenopus oocytes.

The data on poly(A)’ RNA synthesis in stage VI oocytes clearly show the presence of a stable component during the term of the experiments. Identification of a stable component in stage 3 oocytes has been more difficult, due to the restricted time span over which the experiments were conducted, coupled with the longer half-life of the unstable component. However, a stable component was indicated in stage III oocytes from two of the five females examined, as well as in the single experiment on stage IV oocytes. In these cases, stable poly(A)+ RNA accumulated at almost the same rate as in stage VI oocytes (Table 3).

Recent estimates of oocyte growth rates (Keem, Smith, Wallace, and Wolf, submitted for publication) from the end of stage III (0.6-mm diameter) to stage VI (1.2-mm diameter) indicate that the total time re-

DOLECKI AND SMITH Poly(A)’ RNA Metuholism in X. IcrerG 233

quired to progress through this size increase is 16-24 weeks in unstimulated females and 9-12 weeks in hcG-stimulated females. Even at the lowest rate of stable poly(A)+ RNA synthesis in stage 3 oocytes (the average for all stage III experiments), the 16- to 24-week period in unstimulated females would result in a 40- to 60-ng increase in total poly(A)’ RNA content between stages III and VI. However, the experiments on bulk amounts of oligo(dT)cellulose-bound RNA in stage III and stage VI oocytes indicate essentially no change between the two stages. Thus, the component we have identified as stable is not accumulating over the long term.

Two possibilities can be considered. One is that the “stable” newly synthesized poly(A)+ RNA represents a subset of the total poly(A)+ RNA which, in contrast to the bulk, is continually turning over. For example, assuming minimal half-lives for the stable poly(A)’ RNA equal to the duration of the longest experiments (12 hr for stage III oocytes and 24 hr for stage VI oocytes), steady-state levels of 0.26 and 2.3 ng are predicted for this material at stage III and at stage VI, respectively. In other systems (Spradling et al., 1975; Puckett et al., 1975; Lengyel and Penman, 1977), polysomal poly(A)’ RNA populations of different stabilities have been described. How- ever, although the 0.26- and 2.3-ng steady- state levels calculated above are in the range of the 2-ng content of polysomal mRNA present in stage VI oocytes (Wood- land, 1974), less than 10% of the newly synthesized poly(A)’ RNA is found on the polysomes either at stage III or at stage VI. The vast majority of the bulk poly(A)+ RNA is also nonpolysomal in oocytes of both of these stages. Of course, the possibility of there being two populations of nonpolysomal poly(A)’ RNA with different stabilities cannot be eliminated.

At this point, it might be worth consid- ering the contribution of newly synthesized mitochondrial RNA to the poly(A)’ RNA investigated in the current study. The ex-

istence of mitochondrial poly(A)’ RNA in other cell types has been demonstrat,ed (Ojala and Attardi, 1974; Hirsch and Pen- man, 1973). In stage VI oocytes which have been incubated for 24 hr after injection with [“HIGTP, 30% of the newly synthesized mitochondrial RNA binds to oligo(dT)cellulose (Dolecki and Webb, unpublished data). If one assumes that 30% of the rate of mitochondrial RNA synthesis measured by Webb et aE. (1975) (0.3 x 0.53 pg/min, oocyte) is devoted to the production of stable poly(A)+ RNA, then it can be calculated that only 14% of the rate of stable poly(A)+ RNA accumulation in stage VI oocytes could be the result of mitochondrial synthesis. This assumption may not be valid for stage III oocytes, since the rate of mitochondrial RNA synthesis in these oo-- cytes is not known. However, assuming the rate in stage III oocytes is at least equiva.. lent to that in stage VI oocytes, then in those cases in which stage III oocytes show an accumulation of stable poly(A)’ RNA, only 23% of this accumulation could be the result of mitochondrial synthesis.

The second possibility is that the entire stockpile of maternal poly(A) + RNA is continually turning over, i.e., is at steady state, and the stable newly synthesized poly(A) ’ RNA replaces that which is lost by degradation. In this case, it is clear that the concept of a simple steady-stage situation, in which the bulk level of poly(A) + RNA is maintained by constant rates of synthesis and degradation, is not applicable. Alter- natively, we suggest a somewhat more com- plex steady-state concept, in which a constant amount of material is maintained h> continually changing rates of synthesis and degradation. An example of such a situation is provided by the unstable poly(A) + RNA. The steady-state level of this material is approximately the same at stage III and stage VI, yet its rates of synthesis and degradation are greater at stage VI than at stage III. Following this same reasoning, and using the 86-ng content of poly(A)’ RNA measured in the current study as a.

234 DEVELOPMENTAL BIOLOGY VOLUME 69, 1979

steady-state plateau the average rates of accumulation, we calculate that a half-life of 38 days for the stable poly(A)+ RNA in stage VI oocytes would be required to main- tain the steady-state value. The corre- sponding half-life value for the stable component in stage III oocytes, when present, would average 63 days.

According to Dumont (1972)) 45% of the stage II-VI oocyte population is represented by stage II oocytes. This observation suggests that stage II oocytes represent a “reserve” from which groups are selected to undergo further oogenesis. The recent data of Keem et al. (unpublished) on oocyte growth rates further suggest that, as oocytes leave the stage II population, they move through stage III and the early por- tion of stage IV relatively slowly. Thereaf- ter, the rate of growth accelerates until oocytes reach stage V. At that time, growth again slows down as the ovary accumulates a population of stage VI oocytes. These may remain in the ovary for some time before becoming atretic or being ovulated (Dumont, 1972).

Taking into consideration the data obtained in the current study, and the variable residence time of oocytes at any given stage, the following situation is proposed to explain the metabolism of poly(A)+ RNA during oogenesis in Xenopus laevis. Stage I oocytes (or perhaps premeiotic oogonia) begin synthesizing polyp+ RNA. By the start of stage II, they have accumulated all that is needed for early embryogenesis. They then enter a dormant period, awaiting an as yet unknown signal to continue with further oogenesis. During this period, the p01y(A)~+ RNA may be completely stable, and its synthesis may be greatly reduced. As groups of oocytes are selected to begin the lengthy process of vitellogenesis, ~01~ WL+ RNA synthesis is reactivated. During this period, poly(A)t,+ RNA degradation also comes into play, perhaps for the first time in oogenesis. As the months pass, there is an increasing probability that the oocyte contains a population of maternal

mRNA which has somehow become dam- aged. This increasing probability is coun- teracted by continually increasing the rates of degradation and resynthesis, while always maintaining the same steady-state level.

The situation just described would imply that polyp’ RNA sequences synthesized beyond stage II are identical to those al- ready present in previtellogenic oocytes. In fact, there is little new sequence complexity in egg poly(A)+ RNA as compared to previtellogenic stages (Perlman and Rosbash, 1978). Similarly, based on the observation that sea urchin embryos synthesize and turn over mRNAs which belong to the same sequence set as do mature oocyte mRNAs, Hough-Evans et al. (1977) have proposed a similar situation for the oocyte. Perhaps, as they suggest, the whole period from early oogenesis until well into embryogenesis should be thought of as one in which a certain set of mRNAs presumably required for early morphogenesis is continually synthesized and turned over, while their protein products slowly accumulate (Hough- Evans et al., 1977).

The situation described above would also explain why the polyp+ RNA synthesized by previtellogenic oocytes appears to have a half-life in vivo in excess of 2 years. In those experiments, immature frogs whose ovaries contained only previtello- genie oocytes were injected with [“Hluri- dine and [“Hlguanosine. The radioactivity in oligo(dT)cellulose-bound RNA extracted from whole ovaries was then measured as the animals matured. As suggested by Ford et al. (1977), perhaps only a fraction of the oocytes labeled during previtellogenesis proceeds into vitellogenesis and becomes stage VI oocytes during these experiments. If the polyp’ RNA is stable in those oocytes which do not proceed and degraded in those that do, then the apparent stability (half-life > 2 years) of the labeled RNA would, at least in part, be the result of asynchronous oogenesis.

The authors wish to thank Ms. Kirsten Keem for

DOLECKI AND SMITH Poly(A)’ RNA Metabolism cn X. laevis 235

technical assistance and Kerry Bloom and Dr. David Anderson for many helpful discussions.

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