DEVELOPMENTAL BIOLOGY 39, 191-197 (1974)

RNA Metabolism in Previtellogenic Oocytes of Xenopus laevis

CHRISTIAN THOMAS 1

Laboratoire de Cytologie et Embryologie mol~culaires, Universit~ libre de Bruxelles, Rhode-Saint-Gen~se, Belgium

Accepted March 25, 1974

RNA synthesis was analyzed in previtellogenic oocytes of about 120 #m diameter of Xenopus laevis. Follicle cells were removed, and germinal vesicles and cytoplasm were separated by micromanipulation. RNA was analyzed by gel filtration on Sephadex G-100 and by electrophore- sis in agarose gels. The previtellogenic oocytes synthesize mostly low molecular weight RNA (4 S and 5 S RNA) and nonribosomal, high.molecular weight RNA. The metabolism of these two RNA classes is discussed. Synthesis of rRNA seems to be very low.

INTRODUCTION

Mature Xenopus laevis oocytes contain about 4 #g of RNA, about 95% of which is rRNA (Davidson et al., 1964; Brown and Littna, 1964b; Mairy and Denis, 1971). The main part of this RNA is synthesized during maximal lampbrush stage (David- son et al., 1964; Scheer, 1973) and is conserved throughout oogenesis. The high rate of rRNA synthesis is due to a specific rDNA amplification, which occurs early in oogenesis, at the pachytene stage (Brown and Dawid, 1968; Gall, 1968; Macgregor, 1968; Ficq, 1968).

In contrast, during previtellogenesis very few ribosomes are present in the cytoplasm of Xenopus oocytes. Indeed, at this stage, cytoplasmic RNA is visible as fine fibrils (Thomas, 1967, 1969) and more than 80% of the total oocyte RNA consists of 4 S and 5 S RNA (Thomas, 1970; Mairy and Denis, 1971; Ford, 1971). The growth of the dip- lotene oocyte, during previtellogenesis is thus characterized by a preferential accu- mulation of low molecular weight RNA. Despite these data, no conclusive informa- tion exists concerning RNA synthesis in the previtellogenic oocytes, since the previ- ous studies (Mairy and Denis, 1971; Thomas, 1970; Ford, 1971) were performed on RNA from whole ovaries. Autoradio-

~The author is Charge de Recherches du Fonds national de la Recherche scientifique.

Copyright �9 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

graphs show a very high labeling of the follicle cells, after uridine incorporation (Ficq, 1960; Mairy and Denis, 1971; Van Gansen and Schram, 1974). It is thus impossible to distinguish, in the labeled RNA from whole previtellogenic ovaries, which kind of RNA, other than 4 S and 5 S, is synthesized, in particular whether there is any rRNA synthesis during previtel- logenesis.

Recent autoradiographic observations (Van Gansen and Schram, 1974) show that in early previtellogenic oocytes (100 #m or less) 7 hr incubation with 3H-uridine pro- duces heavy labeling of germinal vesicles and the nucleoli. I have analvzed RNA synthesis in these 100 #m oocytes after removal of follicle cells and separation of germinal vesicle and cytoplasm by mi- cromanipulation. The results of these ex- periments are reported in this paper.

MATERIAL AND METHODS

Xenopus laevis ovaries containing only previtellogenic oocytes, were dissected from animals sacrificed 3-4 months after metamorphosis. Pieces of ovary were incu- bated, at 22~ for 24 hr, in 0.1 ml TC 199 medium (Gall, 1966) containing 300 #Ci [5,6-3H]uridine (50 Ci/mmole) and 200 tzCi [8-SH]guanosine (10 Ci/mmole) (Ra- dioisotopes were obtained from the Radio- chemical Centre, Amersham). RNA me- tabolism in vitro resembles normal in vivo

191

192 DEVELOPMENTAL BIOLOGY VOLUME 39, 1974

metabolism in the following ways (observa- tions by Thomas, Van Gansen, and Schram):

1. Autoradiographic observations show a continuous increase in the labeling of the oocytes during the whole incubation pe- riod.

2. The ultrastructure of the oocytes is normal at the end of the incubation.

The ovaries were fixed with ethanol-ace- tic acid (3:1), at 4~ and washed 3 times in 70% ethanol. They were then transferred to a solution of ethanol and glycerol (1:1) and stored at 20~ until microdissected (Daneholt and Hosick, 1973).

Isolation of RNA

Small fragments of ovaries were mi- crodissected in an oil chamber, using a de Fonbrune micromanipulator (de Fon- brune, 1949). A sufficient number (usually about 30) of germinal vesicles (Fig. 1) or cytoplasm was collected from oocytes hav- ing about the same diameter. The compo- nents were digested for about 30 min at 37~ in a hanging drop in the oil chamber, with a protease-SDS solution (0.05 M Tris buffer containing 0.5% SDS, 0.1 M NaC1 and 1 mg/ml predigested protease (type 6, Sigma). After addition to the digest of unlabeled carrier RNA (isolated from Xenopus ovaries, according to Brown and Littna, 1964a), RNA was precipitated with ethanol.

Analysis of RNA

Electrophoresis on 2% agarose gels. The RNA pellet was dissolved in 20 #1 of E buffer (0.02 M Tris buffer, pH 8, 0.02 M NaC1 and 0.002 M EDTA) with 0.5% SDS and analyzed by electrophoresis on 2% agarose gels, at 4~ (Ringborg et al., 1970). Carrier RNA was localized by methylene blue staining (Peacock and Dingman, 1967), and the gel was sliced with an apparatus consisting of parallel razor blades (Ringborg et al., 1970). The slices were transferred to 1 ml of Soluene 350

FIG. 1. Isolated germinal vesicles from Xenoput laevis oocytes about 120 ttm in d iameter , x 270.

(Packard) and left overnight at 40~ Ten milliliters of a toluene solution containing omnifluor (4 g/l) was added to each slice, and these were counted in a liquid scintil- lation spectrometer (Packard, Mode] 3380).

Filtration on Sephadex G-tO0. The RNA pellet was dissolved in 0.5 ml 0.01 M acetate buffer (pH 5) and eluted from a Sephadex G-100 column (about 110 x 1.2 cm) with the same buffer, at 4~ Frac- tions of 1 ml were collected and the posi- tion of carrier RNA was localized by optical density measurement. After TCA precipi- tation, RNA was collected on Millipore filters. The radioactivity was measured in the toluene-omnifluor scintillation me- dium.

Analysis of total RNA. Total RNA from small fragments of ovaries was analyzed by sucrose density gradient centrifugation. RNA extraction was performed under the same conditions as described before. RNA

CHRISTIAN THOMAS RNA Synthesis in Xenopus Oocytes 193

was layered over a 5 ml 15 to 30% linear sucrose gradient in E buffer, and centrifu- gation was performed at 4~ for 4 hr at 50,000 rpm with an SW 50 L rotor. Frac- tions were collected through a hole in the bo t tom of the tube, the carrier RNA was localized by UV absorption, RNA was prec ip i ta ted by TCA and collected on Mil- lipore filters, and the radioact ivi ty was measured.

RESULTS

Analysis of Labeled RNA from Young Ovaries

After 24 hr incorporation, the labeling pa t te rn of these ovaries shows a large amoun t of 18 S and 28 S rRNA, even though the most prominent peak consists of low molecular weight RNA (Fig. 2).

Analysis of Labeled RNA from Previtello- genic Oocytes about 120 #m Diameter

RNA from the Germinal Vesicles. Gel electrophoretic analysis (Fig. 3) shows tha t in the germinal vesicle, 75% of the RNA has a lower electrophoretic mobil i ty than 18 S RNA. A reproducible amount , about 30%, of the total counts did not enter the gel. A peak is always located at a position corresponding to the 30 S rRNA precursor (Gall, 1966; Rogers, 1968; Landesman and Gross, 1969). According to its molecular weight, the 40 S rRNA precursor should be located at the fraction indicated by the arrow in Fig. 32 (Birnstiel et al., 1968; Loening et al., 1969; Scheer, 1973). Of the total RNA appl ied to the gels, 25% moved in a position corresponding to 4 S and 5 S RNA, which are not resolved by this tech- nique.

Analysis of the labeled RNA on Sepha- dex G-100 columns (Fig. 2) revealed tha t 75% of the appl ied radioact ivi ty appeared in the exclusion volume, and became acid- soluble af ter t r ea tmen t with 0.3 N K O H at

2 A linear relationship between electrophoretic mo- bility and log molecular weight of RNAs was assumed for agarose gels (Daneholt et at., 1969).

37~ for 18 hr. Figure 4, shows that 4 S RNA cannot be detec ted in extracts of germinal vesicles, while the peak of 5 S RNA accounts for 25% of the radioact ivi ty applied to the column. The two analyt ical

r CPM xl0 4

5 /I 2~s 4

3

2

I

0 i i L

0 1 0 - - 20 30 frQction N~

FIG. 2. Analysis by sucrose gradient centrifugation of 24-hr labeled RNA from Xenopus laevis ovaries containing previtellogenic oocytes. The positions of carrier RNAs are indicated.

DPM x 10 2 ,I

10= l 4-5S 50.7

8 [ t 28S 6 L~ !

I \ A 4os '

0 10 20 30 40 50

\ fraction N ~

Fro. 3. Electrophoresis of 24-hr labeled RNA ex- tracted from 27 germinal vesicles of previtellogenic Xenopus laevis oocytes (120 urn). The positions of 18 S, 28 S and 4-5 S carrier RNAs are indicated. 30 S rRNA is visible at fraction No. 18. The position of 40 S rRNA precursor has been estimated according to its molecular weight.

194 DEVELOPMENTAL BIOLOGY VOLUME 39, 1974

F CPM x10 2

5s

I

. J ~ lo 2'0

fraction N ~

FIG. 4. Fi l t ra t ion on Sephadex G-100 of 24-hr labeled R N A ext rac ted from about 120 germinal vesicles. The posit ions of carrier R NAs are indicated.

TABLE 1

PERCENTAGE OF THE DIFFERENT LABELED R NA SPECIES PRESENT IN THE GERMINAL VESICLE AND CYTOPLASM OF PREVITELLOGENIC OOCYTES (120 tiM DIAMETER) AFTER

24 HR OF INCUBATION a

Germina l vesicle Total RNA High molecular

weight RNA (larger t h a n 5 S RNA) b

rRNA 30 S 5 S RNA 4 S R N A

% of % of germinal oocyte vesicle or

label ing cytoplasmic label ing

36 100 c 27 75 (30% does not

enter the gel)

1-2 9 Undetec~

able Cy top la sm

Tota l RNA 64 High molecular 24

weight RNA (larger t h a n 5 S RNA) ~

rRNA, 18 S + 28 S 1.7 5 S RNA 25 4 S R N A 15

2-6 26

100 37

2.7 39 24

Da t a of th is table are ob ta ined from electropho- retic and Sephadex chromatograph ic pa t te rns , by in tegrat ion under the curves.

b Inc lud ing rRNA. c Inc lud ing the 30% t h a t does not en ter the gel.

I DPM x 10 2

10

8

6

4

28S

l

91 18 S

/ ' , 1 I I .2

!

4-5s /

0 lO 20 30 hO 50 6 fraction N ~

FIG. 5. Electrophoresis of 24-hr labeled RNA ex t rac ted from the cy top lasm of 27 previtellogeni, oocytes. The cy top lasm (used in Fig. 5) and th~ germinal vesicles (used in Fig. 3) were isolated fron the same oocytes. The posit ions of carrier RNAs an indicated.

CPM xlO 2

5s

I 0 0 10 20 30

froction N ~

FIG. 6. Fi l t ra t ion on Sephadex G-100 co lumn o 24-hr labeled R N A ext rac ted from the cy top lasm of 3t previtellogenic oocytes. The posit ions of carrier RNA: are indicated.

methods thus agree in the relative propor tions of the different kinds of RNA presen in germinal vesicles. The proportions of th, different newly synthesized nuclear RNA: are shown in Table 1.

RNA from the Cytoplasm. Low molecu lar weight RNA species (4 S and 5 S) ar, predominant, as shown by gel electropho resis (Fig. 5) or Sephadex (Fig. 6) analysis All the 4 S RNA in oocytes of this stage i localized in the cytoplasm (Fig. 6), an,

CHRISTIAN THOMAS RNA Synthesis in Xenopus Oocytes 195

some radioactive 18 S and 28 S rRNA is detectable superimposed on the heterodis- perse pattern (Fig. 5). Table 1 gives the proportions of the newly synthesized RNAs in the cytoplasm.

DISCUSSION

Ribosomal RNA Synthesis

The comparison between the labeled RNA present in young ovaries and in oocytes shows that almost all the 18 S and 28 S rRNA which is synthesized by ovaries is present in the follicle cells.

Although some rRNA is present in the cytoplasm of the oocytes, a part of it may still be a contaminant from follicle cells since it is very difficult to be absolutely sure that all the follicle cells have been removed by micromanipulation. The 30 S RNA present in germinal vesicles may be a precursor of 28 S rRNA. It is concluded that rRNA synthesis in previtellogenic oo- cytes is very low.

Although the nucleoli are the most densely labeled structures in the early previtellogenic oocytes (Van Gansen and Schram, 1974), autoradiographic observa- tions of centrifuged oocytes (unpublished observations by Van Gansen, Schram, and Thomas) show that most of the label cen- trifuges with the chromosomes, not with the nucleoli. The number of nucleoli in these oocytes is much lower than during vitellogenesis, as there is a continuous increase in the number of nucleoli during the previtellogenesis period (Perkowska et al., 1968; Van Gansen and Schram, 1972; Thomas, 1972).

1962; Isawa et al., 1963). Recently, Som- merville (1973) isolated ribonucleoprotein particles derived from the lampbrush chro- mosomes of newt oocytes, which contain RNA with a distribution of sedimentation coefficients from 10 S to greater than 50 S. The synthesis of this chromosomal RNA commences probably very early in oogene- sis. Nonribosomal, heterogeneous high mo- lecular weight RNA is also present in the cytoplasm of the oocyte. Even though most of this RNA is composed of smaller RNAs than in the germinal vesicle, some of the cytoplasmic RNA has an apparent molecu- lar weight higher than that of the 40 S rRNA precursor.

The total nonribosomal, high molecular weight RNA synthesized in these oocytes represents about 50% of the RNA labeled in 24 hr. The analysis of the RNA synthesized in previtellogenic oocytes of different diameters (50 and 200 #m) shows that the percentage of labeled nonribosomal, high molecular weight RNA after 24 hr incorpo- ration is very high during all the previtel- logenesis period (personal observation). In contrast, about 75-80% total RNA content of oocytes of this stage is low molecular weight RNA (Thomas, 1970; Mairy and Denis, 1971; Ford, 1971). The kinetics of the accumulation of nonribosomal, high molecular weight RNA and of low molecu- lar weight RNA are thus different and suggest turnover of at least parts of the nonribosomal, high molecular weight RNA. However, a portion of this RNA seems to persist during previtellogenesis (Mairy and Denis, 1971).

Nonribosomal, High Molecular Weight RNA

The majority of labeled RNA in the germinal vesicles has an apparent molecu- lar weight greater than the 40 S rRNA precursor. Since most of the nuclear label- ing sediments with the chromosomes, this RNA could be analogous to the RNA asso- ciated with the lampbrush chromosomes from vitellogenic oocytes (Gall and Callan,

Low Molecular Weight RNA

All labeled 4 S RNA is localized in the cytoplasm. The 4 S RNA in Xenopus previtellogenic oocytes is present in a ribo- nucleoprotein particle (Thomas, 1970; Ford, 1971; Denis and Mairy, 1972). A fraction of the labeled 5 S RNA is present in the germinal vesicles. This might per- haps correspond to the nuclear 5 S RNA pool detected by Knight and Darnell (1967)

196 DEVELOPMENTAL BIOLOGY VOLUME 39, 1974

in H e L a ce l l s .

I thank Drs. J. E. Edstrom and R. Tencer for introduction into micromanipulation and electropho- resis techniques. I am also grateful to Drs. P. Van Gansen, D. D. Brown, I. B. Dawid, R. H. Reeder, R. D. Brown, and M. Lunt for critically reading the manuscript.

REFERENCES

BIRNSTIEL, M., SPEIRS, J., PURDOM, I., JONES, K., and LOENING, U. E. (1968), Properties and composition of the isolated ribosomal DNA satellite of Xenopus laevis. Nature (London) 219, 454-463.

BROWN, D. D., and DAWlD, I. B. (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. 8,669-687.

BROWN, D. D., and LITTNA, E. (1964b). Variations in the synthesis of stable RNAs during oogenesis and development of Xenopus laevis. J. Mol. Biol. 8, 688-695.

DANEHOLT, B., and HOSlCK, H. (1973). Evidence for transport of 75 S RNA from a discrete chromosome region via nuclear sap to cytoplasm in Chironomus tentans. Proc. Nat. Acad. Sci. U.S. 70, 442-446.

DANEHOLT, B., EDSTR~M, J. E., EGYHAZI, E., LAMBERT, B., and RINGEORG, U. (1969). Physico-chemical properties of chromosomal RNA in Chironomus tentans polytene chromosomes. Chromosoma 28, 379-398.

DAVIDSON, E. H., ALLFREY, V. G., and MIRSKY, A. E. (1964). On the RNA synthesized during the lamp- brush phase of amphibian oogenesis. Proc. Nat. Acad. Sci. U.S. 52, 501-508.

DE FONBRUNE, P. (1949). Techniques de micromanipu- lation. Monographies de l'Institut Pasteur, Masson, Paris.

DENIS, H., and MAIRY, M. (1972). Recherches bio- chimiques sur l'oogenbse. Distribution intracel- lulaire du RNA dans les petits oocytes de Xenopus laevis. Eur. J. Biochem. 25, 524-534.

FICQ, A. (1960). M~tabolisme de l'oog~nbse chez les amphibiens. In "Symposium on Germ Cells and Development", pp. 121-140. Institut d'embryologie and Fondazione A. Baseli, Milan.

FmQ, A. (1968). Synthesis and detection of DNA in early oogenesis. Exp. Cell Res. 53, 691-693.

FORD, P. J. (1971). Non coordinated accumulation and synthesis of 5S ribonucleic acid by ovaries of Xenopus laevis. Nature (London) 233, 561-564.

GALL, J. G. (1966). Nuclear RNA of the salamander oocyte. Nat. Cancer Inst. MonOgr. 23, 475-488.

GALL, J. G. (1968). Differential synthesis of the genes for ribosomal RNA during amphibian oogenesis. Proc. Nat. Acad. Sci. U.S. 60, 553-560.

GALL, J. G., and CALLAN, H. G. (1962). H3-Uridine incorporation in lampbrush chromosomes. Proc. Nat. Acad. Sci. U.S. 48,562-570.

ISAWA, M., ALLFREY, V. G., and MIRSKY, A. E. {1963). The relationship between RNA synthesis and loop structure in lampbrush chromosomes. Proc. Nat. Acad. Sci. U.S. 49, 544-551.

KNIGHT, E. JR., and DARNELL, J. E. (1967). Distribu- tion of 5 S RNA in HeLa cells, d. Mol. Biol. 28, 491-502.

LANDESMAN, R., and GROSS, P. R. (1969). Patterns of macromolecules synthesis during development of Xenopus laevis. Identification of the 40 S precursor to ribosomal RNA. Develop. Biol. 19, 244-260.

LOENING, U. E., JONES, K. W., and BIRNSTIEL, M. L. {1969}. Properties of the ribosomal RNA precursor in Xenopus laevis: comparison to the precursor in mammals and in plants. J. Mol. Biol. 45,353-366.

MAIBY, M., and DENIS, H. (1971). Recherches bio- chimiques sur l'oogen~se. I. Synthbse et accumula- tion du RNA pendant l'oogenbse du crapaud sud- africain Xenopus laevis. Develop. Biol. 24, 143-165.

MACGREGOR, n . C. (1968). Nucleolar DNA in oocytes of Xenopus laevis. J. Cell Sci. 3, 437-444.

PEACOCK, A. C., and DINGMAN, C. W. (1967). Resolu- tion of multiple ribonucleic acid species by poly- acrylamide gel electrophoresis. Biochemistry 6, 1818-1827.

PERKOWSKA, E., MACGREGOR, H. C., and BIRNSTIEL, M. L. (1968). Gene amplification in the oocyte nucleus of mutant and wild-type Xenopus laevis. Nature (London) 217, 649-650.

PvINGBORG, U., DANEHOLD, B., EDSTR'()M, J. E., EGY- HAZI, E., and LAMBERT, B. (1970). Electrophoretic characterization of nucleolar RNA from Chironornus tentans salivary gland cells. J. Mol. Biol. 51, 327-340.

ROGERS, M. E. (1968). Ribonucleoprotein particles in the amphibian oocyte nucleus. J. Cell Biol. 36, 421-432.

SCHEER, U. (1973). Nuclear pore flow rate of ribo- somal RNA and chain growth rate of its precursor during oogenesis of Xenopus laevis. Develop. Biol. 30, 13-28.

SOMMERVILLE, J. (1973). Ribonucleoprotein particles derived from the lampbrush chromosomes of newt oocytes. J. Mol. Biol. 78,487-503.

THOMAS, C. (1967). Evolution des structures riboso- miales au cours de l'oogenbse chez Zenopus laevis. Arch. Biol. 78, 347-369.

THOMAS, C. (1969). Etude cytochimique, au micro- scope ~lectronique des structures h ARN et du glycog~ne dans le cytoplasme des oocytes de Xenopus laevis. J. Embryol. Exp. Morphol. 21, 165-176.

THOMAS, C. (1970). Ribonucleic acids and ribonucleo- proteins from small oocytes of Xenopus laevis.

CHRISTIAN THOMAS RNA Synthesis in Xenopus Oocytes 197

Biochim. Biophys. Acta 224, 99-113. THOMAS, C. (1972). Correlation between ultrastruc-

tural aspect of nucleoli a~d inhibition of ribosomal RNA synthesis in Xenopus laevis oocytes. Exp. Cell Res. 74, 547-551.

VAN GANSEN, P., and SCHRAM, A. (1972). Evolution of the nucleoli during oogenesis in Xenopus laevis

studied by electron microscopy. J. Cell Sci. 10, 339 367.

VAN GANSEN, P., and SCHRAM, A. (1974). Incorporation of 3H-uridine and 3H-thymidine during the phase of nucleolar multiplication in Xenopus laevis oogene- sis: a high-resolution autoradiographic study. J. Cell Sci. 14, 85-103.