RNA metabolism in previtellogenic oocytes of Xenopus laevis

DEVELOPMENTAL BIOLOGY39, 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 electrophoresis 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.

graphs show a very high labeling of the follicle cells, after uridine incorporation Mature Xenopus laevis oocytes contain (Ficq, 1960; Mairy and Denis, 1971; Van about 4 #g of RNA, about 95% of which is Gansen and Schram, 1974). It is thus rRNA (Davidson et al., 1964; Brown and impossible to distinguish, in the labeled Littna, 1964b; Mairy and Denis, 1971). The RNA from whole previtellogenic ovaries, main part of this RNA is synthesized which kind of RNA, other than 4 S and 5 S, during maximal lampbrush stage (Davidis synthesized, in particular whether there son et al., 1964; Scheer, 1973) and is is any rRNA synthesis during previtelconserved throughout oogenesis. The high logenesis. rate of rRNA synthesis is due to a specific Recent autoradiographic observations rDNA amplification, which occurs early in (Van Gansen and Schram, 1974) show that oogenesis, at the pachytene stage (Brown in early previtellogenic oocytes (100 #m or and Dawid, 1968; Gall, 1968; Macgregor, less) 7 hr incubation with 3H-uridine pro1968; Ficq, 1968). duces heavy labeling of germinal vesicles In contrast, during previtellogenesis very few ribosomes are present in the cytoplasm and the nucleoli. I have analvzed RNA synthesis in these 100 #m oocytes after of Xenopus oocytes. Indeed, at this stage, removal of follicle cells and separation of cytoplasmic RNA is visible as fine fibrils germinal vesicle and cytoplasm by mi(Thomas, 1967, 1969) and more than 80% cromanipulation. The results of these exof the total oocyte RNA consists of 4 S and periments are reported in this paper. 5 S RNA (Thomas, 1970; Mairy and Denis, 1971; Ford, 1971). The growth of the dipMATERIAL AND METHODS lotene oocyte, during previtellogenesis is Xenopus laevis ovaries containing only thus characterized by a preferential accu- previtellogenic oocytes, were dissected mulation of low molecular weight RNA. from animals sacrificed 3-4 months after Despite these data, no conclusive informa- metamorphosis. Pieces of ovary were incution exists concerning RNA synthesis in bated, at 22~ for 24 hr, in 0.1 ml TC 199 the previtellogenic oocytes, since the previ- medium (Gall, 1966) containing 300 #Ci ous studies (Mairy and Denis, 1971; [5,6-3H]uridine (50 Ci/mmole) and 200 Thomas, 1970; Ford, 1971) were performed tzCi [8-SH]guanosine (10 Ci/mmole) (Raon RNA from whole ovaries. Autoradio- dioisotopes were obtained from the RadioINTRODUCTION

~The author is Charge de Recherches du Fonds national de la Recherche scientifique. 191 Copyright9 1974by AcademicPress,Inc. All rightsof reproductionin any formreserved.

chemical Centre, Amersham). RNA metabolism in vitro resembles normal in vivo

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metabolism in the following ways (observations by Thomas, Van Gansen, and Schram): 1. Autoradiographic observations show a continuous increase in the labeling of the oocytes during the whole incubation period. 2. The ultrastructure of the oocytes is normal at the end of the incubation. The ovaries were fixed with ethanol-acetic 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 microdissected in an oil chamber, using a de Fonbrune micromanipulator (de Fonbrune, 1949). A sufficient number (usually about 30) of germinal vesicles (Fig. 1) or cytoplasm was collected from oocytes having about the same diameter. The components 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 i a m e t e r , 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 scintillation 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~ Fractions of 1 ml were collected and the position of carrier RNA was localized by optical density measurement. After TCA precipitation, RNA was collected on Millipore filters. The radioactivity was measured in the toluene-omnifluor scintillation medium. 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

CHRISTIANTHOMAS RNA Synthesis in Xenopus Oocytes was layered over a 5 ml 15 to 30% linear sucrose gradient in E buffer, and centrifugation was performed at 4~ for 4 hr at 50,000 rpm with an SW 50 L rotor. Fractions were collected through a hole in the b o t t o m of the tube, the carrier RNA was localized by UV absorption, RNA was p r e c i p i t a t e d by TCA and collected on Millipore filters, and the radioactivity was measured.

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37~ for 18 hr. Figure 4, shows that 4 S RNA cannot be detected in extracts of germinal vesicles, while the peak of 5 S RNA accounts for 25% of the radioactivity applied to the column. T h e two analytical r

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Analysis of Labeled RNA from Ovaries

Young 3

After 24 hr incorporation, the labeling p a t t e r n of these ovaries shows a large a m o u n t of 18 S and 28 S rRNA, even though the most p r o m i n e n t peak consists of low molecular weight RNA (Fig. 2).

2

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Analysis of Labeled RNA from Previtellogenic Oocytes about 120 # m Diameter R N A from the Germinal Vesicles. Gel electrophoretic analysis (Fig. 3) shows t h a t in the germinal vesicle, 75% of the RNA has a lower electrophoretic mobility t h a n 18 S RNA. A reproducible a m o u n t , 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; L a n d e s m a n 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 applied to the gels, 25% moved in a position corresponding to 4 S and 5 S RNA, which are not resolved by this technique. Analysis of the labeled RNA on Sephadex G-100 columns (Fig. 2) revealed t h a t 75% of the applied radioactivity appeared in the exclusion volume, and b e c a m e acidsoluble after t r e a t m e n t with 0.3 N K O H at

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

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Fro. 3. Electrophoresis of 24-hr labeled RNA extracted 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 2A linear relationship between electrophoretic mo- rRNA is visible at fraction No. 18. The position of 40 S bility and log molecular weight of RNAs was assumed rRNA precursor has been estimated according to its for agarose gels (Daneholt et at., 1969). molecular weight.

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VOLUME39, 1974 I

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FIG. 4. Filtration on S e p h a d e x G-100 of 24-hr labeled R N A e x t r a c t e d from a b o u t 120 germinal vesicles. T h e positions of carrier R N A s are indicated.

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FIG. 5. Electrophoresis of 24-hr labeled R N A ex t r a c t e d from t h e c y t o p l a s m of 27 previtellogeni, oocytes. T h e c y t o p l a s m (used in Fig. 5) a n d th~ germinal vesicles (used in Fig. 3) were isolated fron t h e s a m e oocytes. T h e positions of carrier R N A s an indicated.

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TABLE 1 5s

PERCENTAGE OF THE DIFFERENT LABELED R N A SPECIES PRESENT IN THE GERMINAL VESICLE AND CYTOPLASMOF PREVITELLOGENIC OOCYTES (120 tiM DIAMETER) AFTER 24 HR OF INCUBATIONa % of oocyte labeling

G e r m i n a l vesicle Total R N A High molecular weight R N A (larger t h a n 5 S RNA) b r R N A 30 S 5 S RNA 4 S RNA Cytoplasm Total RNA High molecular weight R N A (larger t h a n 5 S RNA) ~ rRNA, 18 S + 28 S 5 S RNA 4 S RNA

36 27

% of germinal vesicle or cytoplasmic labeling

100 c 75 (30% does not enter t h e gel)

1-2 9 Undetec~ able

2-6 26

64 24

100 37

1.7 25 15

2.7 39 24

D a t a of this table are o b t a i n e d from electrophoretic a n d S e p h a d e x c h r o m a t o g r a p h i c p a t t e r n s , by integration u n d e r t h e curves. b I n c l u d i n g rRNA. c I n c l u d i n g t h e 30% t h a t does not e n t e r t h e gel.

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FIG. 6. Filtration on S e p h a d e x G-100 c o l u m n o 24-hr labeled R N A e x t r a c t e d from t h e c y t o p l a s m of 3t previtellogenic oocytes. T h e positions 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

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1962; Isawa et al., 1963). Recently, Sommerville (1973) isolated ribonucleoprotein particles derived from the lampbrush chromosomes of newt oocytes, which contain RNA with a distribution of sedimentation coefficients from 10 S to greater than 50 S. DISCUSSION The synthesis of this chromosomal RNA commences probably very early in oogeneRibosomal RNA Synthesis The comparison between the labeled sis. Nonribosomal, heterogeneous high moRNA present in young ovaries and in lecular weight RNA is also present in the oocytes shows that almost all the 18 S and cytoplasm of the oocyte. Even though most 28 S rRNA which is synthesized by ovaries of this RNA is composed of smaller RNAs than in the germinal vesicle, some of the is present in the follicle cells. Although some rRNA is present in the cytoplasmic RNA has an apparent molecucytoplasm of the oocytes, a part of it may lar weight higher than that of the 40 S still be a contaminant from follicle cells rRNA precursor. The total nonribosomal, high molecular since it is very difficult to be absolutely weight RNA synthesized in these oocytes sure that all the follicle cells have been represents about 50% of the RNA labeled in removed by micromanipulation. The 30 S 24 hr. The analysis of the RNA synthesized RNA present in germinal vesicles may be a precursor of 28 S rRNA. It is concluded in previtellogenic oocytes of different that rRNA synthesis in previtellogenic oo- diameters (50 and 200 #m) shows that the percentage of labeled nonribosomal, high cytes is very low. Although the nucleoli are the most molecular weight RNA after 24 hr incorpodensely labeled structures in the early ration is very high during all the previtelprevitellogenic oocytes (Van Gansen and logenesis period (personal observation). In Schram, 1974), autoradiographic observa- contrast, about 75-80% total RNA content tions of centrifuged oocytes (unpublished of oocytes of this stage is low molecular observations by Van Gansen, Schram, and weight RNA (Thomas, 1970; Mairy and Thomas) show that most of the label cen- Denis, 1971; Ford, 1971). The kinetics of trifuges with the chromosomes, not with the accumulation of nonribosomal, high the nucleoli. The number of nucleoli in molecular weight RNA and of low molecuthese oocytes is much lower than during lar weight RNA are thus different and vitellogenesis, as there is a continuous suggest turnover of at least parts of the increase in the number of nucleoli during nonribosomal, high molecular weight the previtellogenesis period (Perkowska et RNA. However, a portion of this RNA al., 1968; Van Gansen and Schram, 1972; seems to persist during previtellogenesis (Mairy and Denis, 1971). Thomas, 1972).

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

Nonribosomal, RNA

High Molecular Weight

The majority of labeled RNA in the germinal vesicles has an apparent molecular weight greater than the 40 S rRNA precursor. Since most of the nuclear labeling sediments with the chromosomes, this RNA could be analogous to the RNA associated 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 ribonucleoprotein 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 perhaps correspond to the nuclear 5 S RNA pool detected by Knight and Darnell (1967)

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in H e L a c e l l s . I thank Drs. J. E. Edstrom and R. Tencer for introduction into micromanipulation and electrophoresis 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 R N A synthesized during the lampbrush phase of amphibian oogenesis. Proc. Nat. Acad. Sci. U.S. 52, 501-508. DE FONBRUNE,P. (1949). Techniques de micromanipulation. Monographies de l'Institut Pasteur, Masson, Paris. DENIS, H., and MAIRY, M. (1972). Recherches biochimiques sur l'oogenbse. Distribution intracellulaire 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.

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RNA Synthesis in Xenopus Oocytes

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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 oogenesis: a high-resolution autoradiographic study. J. Cell Sci. 14, 85-103.