/. Embryo/, exp. Morph. Vol. 32, 2, pp. 515-532, 1974
Printed in Great Britain
5\ 5
Synthesis of RNA in oocytes of Xenopus laevis
during culture in vitro
By A. C O L M A N 1
From MRC Laboratory of Molecular
Biology, Cambridge
SUMMARY
RNA synthesis can be maintained in large oocytes of Xenopus laevis during periods of
in vitro culture of at least 10 days. A simple salt medium, modified Barth's solution, is
found to be as effective a culture medium for these oocytes as several other complex media.
The newly synthesized RNA is characterized electrophoretically and shown to consist
predominantly of ribosomal RNA precursor, 28S and 18S ribosomal RNA, and 4S RNA.
The distribution of this RNA within the oocyte is detected autoradiographically, where it
is found to be greatly concentrated over the nucleoli. No qualitative alterations in either
of these parameters are found during culture, within the limits of sensitivity of the assay
procedures.
INTRODUCTION
Somatic cell nuclei, when injected into the oocytes of Xenopus laevis, undergo
pronounced morphological changes and begin to synthesize RNA at a greatly
increased rate (Gurdon, 1968). The rate at which these responses occur in the
oocyte was found to be inversely related to the state of differentiation of the
cells from which the nuclei were taken and was also influenced by the RNAsynthetic activities of the particular oocytes into which the nuclei were injected
(Gurdon, 1968).
Recently several workers have reported attempts to effect, in vitro, tissue
specific transcription from isolated nuclei (Soil & Sussman, 1973), and isolated
chromatin (Paul & Gilmour, 1966; Gilmour & Paul, 1973; Axel, Cedar &
Felsenfeld, 1973). Since most of these experiments used hybridization to detect
specific RNA species, the fidelity of the transcripts cannot be accurately
estimated. The most sensitive assay of the product RNA would be to include
a coupled translational assay in such systems. Oocytes have been shown to
provide a very sensitive in vivo assay for many messenger RNAs (see review by
Gurdon, 1974), and particularly for globin 9s mRNAs (Gurdon, Lane, Woodland & Marbaix, 1971). It might then be of considerable interest to test nuclei
and chromatin from various haemopoietic cell lines, for the production of
1
Author's address: Department of Zoology, South Parks Rd., Oxford, U.K.
516
A. COLMAN
specific transcription and translation products after their injection into oocytes.
Such differentiated-cell nuclei and chromatin might take several days to develop
the responses to oocyte cytoplasm described above; conditions are needed
therefore in which oocytes continue to make RNA for several days in culture.
In this paper, the pattern of RNA synthesis in injected oocytes is examined
for several days after removal of the oocytes from the frog. It is concluded
that the ability of large oocytes to synthesize RNA species of a type and
cellular localization similar to that seen immediately after removing such
oocytes from the ovary (of Xenopus laevis), can be maintained in culture for
several days.
MATERIALS AND METHODS
Selection of oocytes
Oocytes were removed from large females of Xenopus laevis (Daudin)
immediately after decapitation. In some cases, the females were induced to
ovulate three days earlier by administration of the hormone 'Chorulon'
(Intervet Ltd); the unovulated oocytes from such frogs are termed 'postovulation' oocytes. In all other cases, the females used had been maintained
in the laboratory, without ovulating, for at least 10 months before killing; the
oocytes from these frogs are termed 'pre-ovulation' oocytes. Oocytes are
classified according to the morphological criteria elaborated by Dumont (1972),
i.e. large, white-banded oocytes (1-2-1-3 mm diameter) are referred to as Stage
VI oocytes; 1-0-1-2 mm diameter oocytes, as Stage V; and oocytes ranging
in diameter from 0-6 to 1-0 mm, as Stage IV.
Follicle cells and their removal
Wallace & Dumont (1968) distinguished three cellular layers surrounding
oocytes. The term 'follicle layers' as used in this text refers to all three cellular
layers. After most experiments, the follicle layers and vitelline membranes
surrounding the oocytes were manually removed: oocytes were exposed for
90 sec to 500 /tg/ml predigested pronase (Calbiochem Grade B) in 88 mM-NaCl,
50 mM-EDTA, 15 mM-tris/HCl, pH 7-6; this was followed by a 5 min immersion
in the same solution without pronase (a procedure modified after Masui,
1967; Smith & Ecker, 1969). This procedure removed all follicle cells as judged
by examining serial sections (7 /.im diameter) of 10 separate oocytes. Additionally
the electrophoretic profiles of nucleic acid extracted from oocytes with intact
follicle layers contain a band of DNA which is absent in the profiles of nucleic
acid extracted from defollicled oocytes or ovulated eggs (which no longer
possess follicle layers). Apart from the presence of grains over the follicle layers,
no differences were detected between the autoradiographs of untreated oocytes
and those of pronase-treated oocytes.
Synthesis of RNA in oocytes
517
Culture of oocytes
Small pieces of ovarian tissue were dissected with watchmakers' forceps
into small clumps, containing six or seven large oocytes, and transferred to
one of the following culture media: (1) a modified Barth's solution (M.B.S.)
(Gurdon, 1968) containing 88 mM-NaCl, 1 mM-KCl, 0-33 mM-Ca(N03)2,0-4 mMCaCl2, 0-8 mM-MgS04, 2-4 mM-NaHC03 with either 15 mM-tris/HCl, pH 7-6,
or 15 mM-iV-2-hydroxyethyl piperazine W-2-ethanesulphonic acid (H.E.P.E.S.)/
NaOH, pH 7-6; (2) Leibovitz L-15 medium (Leibovitz, 1963) with or without
10% foetal calf serum, pH7-6; (3) Dulbecco's modified Eagle's medium
(Dulbecco & Freeman, 1959), with or without 10 % foetal calf serum (maintained at pH 7-6 in a 5 % C 0 2 atmosphere). Both the Leibovitz and Dulbecco's
media were adjusted to 67 % of their original concentration by addition of
glass-distilled water (Balls & Ruben, 1966). Media also contained 0-01 g/1. of
both sodium penicillin and streptomycin sulphate.
In the initial experiments where both Leibovitz and Dulbecco's media were
used, oocytes were removed aseptically from the frog. These precautions were
found to be unnecessary in the later experiments which used modified Barth's
solution only. Oocytes were cultured at 19 °C in 4 cm diameter Petri dishes
(100 oocytes in each) and the culture media were changed every two days.
Depending on the particular experiment, oocytes were either injected with
10-30 nl. of various solutions using a 25 /.im diameter micropipette, pricked
with an empty micropipette, or left untouched before culturing.
Labelling of oocytes
5- H-uridine (29 Ci/mmol in H 2 0) and 8-3H-guanosine (16 Ci/mmol in H 2 0)
obtained from the Radiochemical Centre, Amersham were evaporated to
dryness and dissolved in culture medium at 0-45-0-9 mCi/ml.
Oocytes were incubated in batches of 20 in 100/A. of labelled medium,
washed three times in unlabelled medium and then either transferred to Perenyi's
fixative for autoradiography or divested of their follicle layers and stored at
- 2 0 °C.
3
Autoradiography
After fixing in Perenyi's fluid for 12-24 h at 4 °C, oocytes were embedded
in Paraplast wax (Raven, Ltd) and sectioned at 7 /im. Slides were dipped in
llford K2 nuclear emulsion, exposed for 2-21 days, and developed. Sections
were stained with Mayer's haemalum and then light green. Control sections
were digested with 0-5 mg/ml boiled RNase A (Worthington Ltd.) in 2 x SSC
(0-3 M-NaCl, 0-03 M-trisodium citrate) for 12 h before being coated with
emulsion. No silver grains could be found in the subsequent autoradiographs
of these controls. Similar digestion with 2 x SSC alone had a negligible effect.
Grain counting was performed in the following manner :
518
A. COLMAN
(1) nucleolar values were estimated by averaging the number of grains
within an area of (9 /mi)2 over each of the 10 highest labelled nucleoli in each
of 10 oocytes;
(2) germinal vesicle and cytoplasmic values were estimated by averaging
the number of grains over 300 (9 /on)2 areas over germinal vesicle (excluding
nucleoli) and cytoplasmic regions respectively in the same oocytes in which
nucleolar estimates were made. All values are background corrected and
expressed as grains per unit area (i.e. (9 /tm)2).
Biochemical analysis
(1) Extraction of RNA
Batches of 20 oocytes were homogenized in 2 ml of the following medium:
0-1 M-tris-HCl, 0-05 M-NaCl, 0-01 M - E D T A , 4 /*g/ml polyvinyl sulphate (Sigma),
0*5%, w/v, sodium dodecyl sulphate • (SDS), pH7-6; containing 500/tg/ml
pronase, predigested for 2 h at 37 °C (Miller & Knowland, 1970).
Incubation at 35 °C for 3 h was followed by two extractions with watersaturated phenol at room temperature. The aqueous phase was dialysed
against three changes of 2-5 1. of lOmM-tris/HCl, 50mM-NaCl, 1 mM-EDTA,
2/«g/ml polyvinyl sulphate, 0-5% (w/v) SDS pH 7-6, for 24 h at 4 °C. The
samples were adjusted to 3 % NaCl, two volumes of absolute ethanol were
added and the solutions left overnight at 4 °C. The precipitated RNA was
pelleted, washed twice with ethanol and air dried samples were kept at - 20 °C.
At several stages during the procedure, aliquots were taken for determination
of total radioactivity and for precipitation with 10 % TCA, followed by collection of precipitates on glass fibre filters. RNA was defined as that material
which was alkali-labile in the case of 5-3H-uridine labelling (aliquots were
boiled for 20 min, at 100 °C, in 0-1 N-KOH before precipitation). Since 8-3Hguanosine is itself alkali-labile (Wilt, 1969), RNA labelled by this precursor
was defined as that material which was released from filters after treatment
with 250 /tg/ml RNase A (boiled) in 2 x SSC at 37° for 12 h. All samples were
counted in 10 ml of a water-miscible scintillant consisting of 8 g PPO, 0-2 g
POPOP, 1250 ml toluene and 750 ml 2-ethoxy-ethanol. Addition to the samples,
before extraction, of 32P-labelled yeast RNA showed recoveries of total RNA
to be consistently greater than 80 %.
In order to avoid the objection that the above procedure would selectively
extract ribosomal RNA, as opposed to poly A-containing RNA (see review
by Brawerman, 1973), two other methods of extraction were tried:
(a) the pH of the pronase-SDS homogenate, described above, was adjusted
to pH 9-0 after the 3 h incubation at 35 °C. The homogenate was then twice
extracted with phenol/chloroform (1:1) at room temperature, and processed
as above (modified after Brawerman, Mendecki & Lee, 1972).
(b) labelled oocytes were homogenized in 0T M-NaCl, 10 mM-tris/HCl,
1 mM-EDTA, 0-5 % SDS, pH 7-4, at room temperature, twice extracted with
Synthesis of RNA in oocytes
519
phenol/chloroform (1:1) and then processed as above (Perry, La Torre, Kelly
& Greenberg, 1972).
All the methods used were compared by Polyacrylamide gel electrophoresis,
and no qualitative differences between the types of newly synthesized (i.e.
radioactive) RNA, recovered by each method, were detected. However, the
original method described, yielded the highest recovery of total RNA and
was the method routinely used.
(2) Polyacrylamide gel electrophoresis
RNA samples (20-60 fig RNA) were taken up in 50/*1. of sample buffer
(40 mM-tris, 20 mM sodium acetate, 2 mM-EDTA, acetic acid to pH 7-6, 25 %
glycerol) and applied to 10 cm 2-4 % acrylamide gels containing 0-5 %, (w/v)
SDS, and run for 2\ h at 5 mA/gel (Loening, 1967). Gels were scanned at
265 vufi on a Joyce-Loebl Uviscan, and occasionally also stained with 0-2 %
methylene blue. Gels were then sliced (1 mm slices) on a Mickle gel sheer and
dissolved overnight in 0-25 ml hydrogen peroxide and ammonia (48 parts : 2
respectively) at 50 °C in closed glass vials. 10 ml of scintillation fluid containing
2-4 ml/1, glacial acetic acid were added and the samples counted.
RESULTS
I. Optimal conditions of culture and analysis
(A) Choice of medium
Several different types of media have been developed or specifically modified
for use in amphibian cell culture (Barth & Barth, 1959; Auclair, 1961; Rugh,
1962; Wolf & Quimby, 1964; Balls & Ruben, 1966). With respect to amphibian
oocyte culture these media can be grouped in three categories: simple salt
media (Dettlaff, 1966; Merriam, 1966; Smith & Ecker, 1969), bicarbonatebuffered complex media (Jared & Wallace, 1969), and non-bicarbonate-buffered
complex media (Gurdon & Laskey, 1970; Thomas, 1970). The effects of media
from each of these categories, on the incorporation of 8-3H-guanosine into
RNA by Stage VI oocytes, is shown in Table 1. Both the uptake of 8-3Hguanosine and its incorporation into RNA decreases, in oocytes, with time
in culture. The absolute amounts of RNA synthesis cannot be calculated, since
pool sizes of guanosine are not measured. However, no significant difference
exists between the various media with regards to the incorporation of 8-3Hguanosine, by the oocytes, into both nucleolar RNA and total oocyte RNA.
Additionally, the large standard deviation associated with each autoradiographic estimate reflects the considerable variation observed to exist between
individual Stage VI oocytes with regard to nucleolar RNA synthesis (i.e.
ribosomal RNA synthesis - see reviews by Brown, 1967; Macgregor, 1972).
520
A. COLMAN
Table 1. Incorporation of 8-3H-guanosine into pricked Stage VI oocytes
during extended culture in different media
Medium
category
Culture medium
Simple salt
Modified Barth's
solution (M.B.S.)
Simple salt
Hepes solution
Complex
bicarbonate
buffered
Dulbscco's Modified
Eagle's Medium*
(D.M.E.)
Complex
bicarbonate
buffered
D.M.E. containing
10 % undialysed
foetal calf serum*
Complex
non-bicarbonate
buffered
Leibovitz-15* (L-15)
Complex
non-bicarbonate
buffered
L-15 containing 10 %
foetal calf serum*
Period
of
labelling!
(h)
Total
cpm per
oocyte
0-24
75000
±24
41000
0-24
80000
38000
84000
21 ±10
56 ±28
30±11
56 ±34
206 ± 54
445 ±80
150 ±96
500 ±110
96-120
0-24
40000
110000
26 ±8
41 ±15
190±85
516 ±201
96-120
0-24
56000
83000
12±8
61 ± 21
312±168
377 ±101
96-120
0-24
41000
118000
25 ±13
48 ±16
123 ±66
494 ±131
96-120
58000
18±7
150±71
96-120
0-24
96-120
No.
No. of cpm
grains/unit incorporated
area over into RNA
nucleolij per oocyte§
412±49
* These media were adjusted to 67 % of their osmolarity (see Methods).
t Oocytes were labelled by 0-45 mCi/ml S-^H-guanosine.
% The autoradiographs were exposed for 4 days.
§ For each estimate 60 oocytes (3 batches of 20) were defollicled, homogenized and
processed (see Methods).
(B) Choice of radioactive precursor
Since DNA synthesis is absent in large oocytes ( > 1 -0 mm diameter) (Gurdon,
1967), radioactive guanosine can be used as a spécifie label for RNA synthesis
(Mairy & Denis, 1971). The following experiments examine whether 8-3Hguanosine or the more traditional precursor 5-3H-uridine gives rise to RNA
of the higher specific activity.
(a) Uptake. 8-3H-guanosine enters Stage VI oocytes faster and saturates at
a higher level than 5-3H-uridine (Fig. 1). The onset of saturation is not due
to exhaustion of exogenous labelled precursor since transfer of oocytes, after
200 h in culture, to fresh labelled medium causes no increase in uptake. If the
specific activity of the 5-3H-uridine is adjusted to equal that of 8-3H-guanosine,
uptake of 5-3H-uridine is further reduced (by about 50 %). Similar data were
obtained using pricked Stage V oocytes. From Table 2, it is evident that the
Synthesis of RNA. in oocytes
521
•
•
^ ^
•
5 --
y
A
Guanosine
—
O
3 -
O
J •
Uridine
2 -
-17
1 -
i
i
40
i
i
i
i
120
Hours
160
200
240
Fig. 1. Uptake of 5-3H-uridine (0-9 mCi/ml, 29 mCi/mmol in M.B.S.) and 8-:,Hguanosine (0-9 mCi/ml, 16Ci/mmol in M.B.S.), by pricked Stage VI oocytes.
Each point represents an average value obtained from 30 oocytes.
Table 2. Uptake of 8-^H-guanosine and 5-zH-uridine by
pricked Stage VI oocytes
Duration
(h)of
labelling
0-24
72-96
144-168
0-24
72-96
144-168
Total cpm/oocyte (includ ing
follicle cells) xlO- 3
Label*
3
0-9 mCi/ml 8- H-guanosine
in M.B.S.
Frog 1
Frog 2
Frog 3
242
317
214
234
290
152
71
41
46
3
72
0-9 mCi/ml 5- H-uridine
105
94
in M.B.S.
63
85
92
56
78
55
* Oocytes were cultured in M.B.S. before labell ng.
Frog 4
340
283
89
160
171
154
differential uptake of 8-3H-guanosine relative to 5-3H-uridine is not maintained
after long periods in culture.
(b) RNA synthesis. Oocytes divested of their outermost cellular layers exhibit
abnormal morphological changes within 24 h of culture in many amphibian
cell-culture media (Jared & Wallace, 1969). During long-term culture, therefore,
it is essential to leave follicle cell layers intact. However, Ficq (1960) has shown,
by autoradiography, that the follicle layers are very active in RNA synthesis.
Fig. 2 shows one of four experiments with different frogs, where the amount
of labelled precursor, incorporated into the RNA of the follicle layer cells, is
compared with the amount incorporated into the RNA of the oocytes, contained within these layers. The rate of accumulation of 8-3H-guanosine or
522
A. C O L M A N
(rt) Guanosine
(b) Uridine
2
Ml '
z *
I i 0-8
yx>
Follicle cells
O
ii
o o
/
/
®f
0-4
0 //
Defollicled oocytes"
/ ^-— —
°§
00
^ — •
/
S
20
•
^-—-1
40
60
0
Hours
20
_ -•
1
i
40
60
Fig. 2. Incorporation of (a) 8-3H-guanosine; (b) 5-3H-uridine (both 0-9 mCi/ml in
M.B.S.) into RNA in the follicle cell layers (O) and the pricked Stage VI oocytes
they surround ( # ) . The removal of follicle cells is described in the Methods
section.
5-3H-uridine into RNA of the follicle cells varied in the four experiments
from one-third to double their rate of accumulation into oocyte RNA.
However, all four experiments gave results consistent with the following
conclusions :
(1) The amount of radioactive precursor found incorporated into the RNA
of the cellular layers around the oocyte constitutes a significant but variable
proportion of the total amount (of precursor) found in the RNA of the intact
oocyte. It is therefore essential to remove these layers prior to the assay
procedures.
(2) The percentage of acid-soluble label which is incorporated into RNA by
oocytes is the same whether 5-3H-uridine or 8-3H-guanosine are used.
(c) Conclusion. (1) The amount of incorporation of 8-3H-guanosine into the
RNA of cultured oocytes is not significantly influenced by the type of medium
used. However, since the risks of bacterial and fungal contamination are
greater using complex media, the simple salt medium, modified Barth's solution,
was adopted as the medium of choice.
(2) During the earlier stages of culture 8-3H-guanosine enters oocytes faster
and saturates at a higher level than 5-3H-uridine. It is also observed that RNA
of higher specific activity is obtained with 8-3H-guanosine. In the later stages
of culture (Table 2), 8-3H-guanosine penetration falls to, or below, the value
for 5-3H-uridine. However, it is evident that when oocytes are continually
exposed to labelled medium from the beginning of their culture, RNA of
higher specific activity will be obtained, using 8-3H-guanosine, rather than
5-3H-uridine, as the labelled precursor.
523
Synthesis of RNA in oocytes
5 /mi
.-> 5 [im
(A).
•?'•."/
e»)
: ' 4 * ^ 5 - , % '%
•i l
À'
^
^
'
0.
Fig. 3. Autoradiographs of nucleoli (and surrounding nucleoplasm) in oocytes of
Xenopus laevis: (a) Stage VI oocytes; (b) Stage V; (c) Stage IV. All oocytes were
incubated in 0-9 mCi/ml 8-3H-guanosine in M.B.S. for 24 h. Autoradiographs were
exposed for 3 days.
II. Characteristics of RNA synthesis
(A) Distribution of newly synthesized RNA in different regions of the oocyte
(a) Normal pattern. Autoradiographs of oocytes from 20 frogs all show,
that in Stage V and YI pre-ovulation and Stage V post-ovulation oocytes,
there are 50-100 times more grains per unit area over nucleoli than over the
germinal vesicle nucleoplasm (Figs. 3 a, b) or over the cytoplasm. The grain
densities over the cytoplasm and the germinal vesicle nucleoplasm are of the
524
A. COLMAN
Nucleol
Frog A
Frog B
Frog C
Germinal vesicle
H
^
Cytoplasm
1 3 5 7
1 3 5 7
Days in culture
1 1
1 3
f
f
5 7
Fig. 4. Distribution of newly synthesized RNA between different regions of pricked
Stage VI oocytes, pulse labelled for 24 h in 8-3H-guanosine (0-9 mCi/ml in M.B.S.)
during the days indicated in the figure. The autoradiographs were exposed for 68 h
(frogs A and B) and 26 h (frog C).
same order of magnitude but accurate comparison is precluded by the presence
of large yolk platelets in the cytoplasm. Similar analyses on small Stage IV
pre-ovulation oocytes (0-6-0-7 mm diameter) show that the number of grains
per unit area over the nucleoplasm is 3-8 times higher than in Stages V and
VI but that the nucleolar grain densities are similar (Fig. 3 c).
(b) Change of pattern with time. From Fig. 4 it can be seen that the same
differential distribution of label in different regions of the oocyte is maintained
during 24 h pulse labelling periods on the first, third, fifth and seventh day of
culture.
(c) Effects of various treatments. Many experiments using Xenopus laevis
have involved the microinjection of various solutions into oocytes. In Table 3,
the effects of two of these solutions on RNA synthesis in Stage VI oocytes
are shown. Solution A has been used for the microinjection of nuclei into
oocytes (Gurdon, 1968), and solution B for messenger RNA injections (Lane,
Marbaix & Gurdon, 1971; Stevens & Williamson, 1972). No significant difference can be found between any of the injected oocytes and the pricked or
unpricked controls. Therefore the results with pricked oocytes, which constitute most of the results described here, may be assumed to have general
application. Similar results have been obtained regarding the lack of effect of
injecting these substances, when Stage VI oocytes are tested for protein synthesis
(Colman, unpublished results).
525
Synthesis of RNA in oocytes
Table 3. Effect of various treatments on the incorporation of
8-zH-guanosine into RNA in Stage VI oocytes
Treatment
Solution A injected (30 nl
per oocyte of 0-25 M sucrose,
2 mM-Mg2+Cl2)
Solution B injected (30 nl
per oocyte of 88 mM-NaCl,
15mM-tris/HClpH7-6)
Pricking only
(with empty micropipette)
Un injected and unpricked
Labelling
period*
(h)
No. of grains/unit
area over nucleoli!
cpm
incorporated into
RNAJ
0-24
33 ± 9
691
0-24
25 ± 7
741
0-24
34 ±12
610
0-24
0-24
26±4
662
1554
(oocytes not defollicled)
* Oocytes were labelled in 0-9 mCi/ml 8-3H-guanosine in M.B.S.
f Procedure for counting grains described in Methods section. Autoradiographs were
exposed for 60 h.
% Batches of 30 oocytes were defollicled after labelling, homogenized and processed (see
Methods).
{B) Electrophoretic characterization of oocyte RNA
(a) Absorbance. The absorbance profiles at 265 m/£, of total RNA extracted
from the Stage VI oocytes of 18 frogs (12 non-ovulated, 6 ovulated) have
shown no significant deviation from the profiles shown in Fig. 5. Polyacrylamide
gels stained with methylene blue exhibit four bands migrating between the
major 28S and 18S ribosomal species (Fig. 6); these bands are less clearly
resolved by ultraviolet scanning. The small amount of 4S and 5S RNA present
agrees with the calculations of Mairy-Von Frenckell (1970), showing that 4S
and 5S RNA account for only 4 % of total RNA in Stage VI oocytes. Similar
absorbance profiles are found for RNA from unpricked, pricked and sucroseinjected oocytes, and for all oocyte stages examined, i.e. Stages IV and V
post-ovulation oocytes, Stages IV, V and VI pre-ovulation oocytes. The profile
is not tissue-specific since RNA extracted from Xenopus adult liver and spleen
by the same method gives a similar profile.
RNA extracted from ribosomes (Stage VI oocytes), isolated on sucrose
gradients, also exhibits this profile, while 60S subunits contain mainly 28S
RNA, 5S RNA and the same four minor species seen in Fig. 6, migrating
between 28 and 18S RNA (Colman, unpublished results). RNA from 40S
subunits contains only an 18S band. Thus, the additional species are probably
specific degradation products of 28S RNA. Whether these minor species are
a product of the particular extraction procedure used is undetermined. Dawid
(1970) using a slightly different extraction procedure obtained a similar profile
526
A. COLMAN
(a)
28S
i Ribosomal
precursor
4S 5S
I
• 20 S
I
2 500
60
50
Slice number
28S
(b)
18S
Il Ribosomal
j l precursor
4S 5S
1 1
•guanosine)
1
oo
- 20
'
500
- 10
400
£
300
e
200
-
o.
o 100
"
80
i
70
60
"
-A.
50
40
Slice number
30
20
10
Fig. 5. Electrophoretic profiles of total RNA from pricked Stage VI oocytes
incubated in 0-9 mCi/ml 8-3H-guanosine in M.B.S. between (a) 0-24 h ; (b) 144168 h (
, Absorbance at 265 nm; • • • • • , cpm).
from Xenopus oocytes. However, Loening, Jones & Birnstiel (1969) using
RNA from cultured cells of Xenopus kidney observed no distinct species
migrating between 28S and 18S RNA. Nevertheless, RNA extracted from
oocytes maintained in culture for up to 19 days has given the same profile
as RNA extracted from oocytes immediately upon their removal from a frog ;
this demonstrates that the conditions of culture do not cause any degradation
of ribosomal RNA within this period of time.
(b) Radioactivity. During the first 24 h of culture, 8-3H-guanosine is incorporated into the ribosomal RNA precursor (Loening et al. 1969) 28S, 18S, 4S
and possibly 5S RNA (Fig. 5a). Good resolution between 4S and 5S species
cannot be obtained on 2-4 % Polyacrylamide gels. The small peak of radioactivity migrating between 28 and 18S species has also been reported by
Mairy & Denis (1971). A similar pattern of incorporation is obtained from
Synthesis of RNA in oocytes
527
Fig. 6. RNA from pricked Stage VI oocytes. 50/*g oocyte RNA was applied to
a 2-4 % Polyacrylamide gel. (Running conditions described in Methods section.)
Gel is stained with methylene blue.
oocytes labelled from 144 to 168 h (Fig. 5b), the only difference being the
lower level of incorporation of label. In two experiments, oocytes (Stage VI)
have, after 10 days in culture, incorporated 8-3H-guanosine into the RNA
species described above.
Pre-ovulation Stage IV (0-9-1-0 mm diameter) and Stage V oocytes were
34
E M B 32
A. C O L M A N
528
found to incorporate as much 8-3H-guanosine into RNA as Stage VI oocytes
from the same frog. The largest post-ovulation oocytes (i.e. Stage V) also
incorporated 8-3H-guanosine into RNA at a similar rate and into comparable
types of RNA. However, such oocytes invariably began to lose pigment in
their animal poles, and showed morphological changes in their nucleoplasm
within 3-4 days of culture.
Autoradiographic experiments performed in conjunction with the electrophoretic characterization discussed, nearly always showed that the relative
changes in incorporation of label into RNA, as assayed on gels, were paralleled
by similar changes in the density of silver grains over different regions of the
oocyte. In the one exceptional case where this did not occur, it could be shown
by adding E. coli RNA markers that some of the newly synthesized RNA seen
on the gels was of bacterial origin.
DISCUSSION
Culture conditions
For many cell types, serum is an essential supplement for successful longterm in vitro culture. However, this paper shows that serum neither enhances
nor significantly retards the ability of large oocytes to incorporate label into
RNA in culture. Serum did however increase the uptake of 8-3H-guanosine
into large oocytes (Table 1), an effect noted in the case of certain mammalian
cells with regard to uridine uptake (Cunningham & Pardee, 1969). The results
shown indicate that a non-nutrient medium consisting of only inorganic salts
(modified Barth's solution) can maintain large oocytes in a synthetically active
state with respect to RNA for up to 10 days in culture. This conclusion is
based on both the unchanging profiles of the types of newly synthesized RNA
as assayed on gels, and the unaltered distribution of newly synthesized RNA
within the oocyte as seen autoradiographically. Gurdon, Lingrel & Marbaix
(1973) have recently demonstrated that the protein synthesis directed by both
endogenous and injected messenger-RNA templates persists for up to 14 days
in oocytes incubated in this same medium. However, it should be emphasized
that substantial variation exists between the RNA synthetic abilities during
culture of different batches of oocytes and also between individual oocytes
within the same batch. Generally, pre-ovulation oocytes survived for at least
7 days in a synthetically active state (as detected by incorporation of 8-3Hguanosine into RNA). Large (1-0-1-2 mm diameter) post-ovulation oocytes
have been reported to be more active in incorporating 8-3H-guanosine in RNA
than similarly sized pre-ovulation oocytes (Mairy & Denis, 1971). These
observations are not confirmed in this report; however, this lack of agreement
might be due to the longer incubation periods used here, and the poorer
survival, in culture, of post-ovulation oocytes, when compared to pre-ovulation
oocytes.
Synthesis of RNA in oocytes
529
Newly synthesized RNA
Large Stage IV (0-9-1-0 mm diameter), Stage V, and Stage VI pre-ovulation
oocytes, and Stage V post-ovulation oocytes, all incorporated similar amounts
of 8-3H-guanosine into RNA. The types and pattern of distribution of this
newly synthesized RNA are also similar in all the above-mentioned categories.
These results differ, in part, from the findings of Brown & Littna (19646)
and Mairy & Denis (1971); they are, however, consistent with the findings
of La Marca, Smith & Strobel (1973) who discuss possible explanations for
the failure of the above-mentioned workers to find RNA synthesis in Stage VI
oocytes. La Marca et al. (1973) determined the actual rate of RNA synthesis
in Stage VI oocytes to be ~ 2ng/h. However, after ovulation in Xenopus
laevis females, a new population of Stage VI oocytes arises within 40 days
(Scheer, 1972; Dumont, 1972). Since all the non-ovulated frogs used in the
experiments reported in this paper had not laid eggs for at least 10 months,
then a minimal estimate of the * age ' of Stage VI oocytes, in these frogs, would
be nearly 9 months. During this time, when the figures of La Marca et al.
(1973) are used, Stage VI oocytes would have accumulated 13 fig RNA per
oocyte, in addition to the 4 fig of RNA (approximately) reported by Scheer
(1972) to be already present in 1-1 mm diameter (i.e. Stage V) oocytes. However,
Stage VI oocytes contain only 4-5 fig RNA per oocyte (Brown & Littna,
1966; Scheer, 1972; Colman, unpublished observations); therefore some
explanation for this apparent anomaly must be sought. Several possibilities
exist :
(a) Stage VI oocytes gradually cease synthesizing RNA, at some time after
reaching this full grown stage: such an explanation would lend credence to
the report by Crippa (1970) of the presence of a ribosomal RNA inhibitor in
Stage VI oocytes. However, in all Stage VI oocytes the author examined,
RNA synthesis was detected when these oocytes were cultured in vitro, even
though they were obtained from frogs which had not ovulated within the last
10 months;
(b) Considerable turnover of RNA occurs: this RNA would have to be
mostly ribosomal since this is the major species of RNA present (Mairy-von
Frenckell, 1970) and being synthesized (La Marca et al. 1973) in Stage VI
oocytes. However, rRNA is conserved from early oogenesis to at least the
hatching stage of development (Brown & Littna, 1964Ö; Davidson, Allfrey &
Mirsky, 1964; Brown & Gurdon, 1966);
(c) Atresia, i.e. the resorption of large oocytes by the ovary: although no
data is available on the rate of resorption of oocytes in Xenopus laevis, the
ovaries used in the experiments reported in this paper contained a very low
proportion (~ 2 %) of large atretic oocytes and a very high rate of resorption
would be necessary to account for the unchanging amount of RNA present
in Stage VI oocytes. A continually changing population of Stage VI oocytes
34-2
530
A. C O L M A N
could however explain the variability seen in the in vitro synthetic abilities
between Stage VI oocytes obtained from the same frog;
(d) Removal of oocytes from the ovarian environment alters the nature of
synthetic events: Smith (1972) found evidence for this in regard to protein
synthesis in oocytes of Rana pipiens and also (Smith & Ecker, 1970) found
that the uterine environment could suppress biochemical and morphogenetic
events occurring after in vivo maturation of oocytes, a condition relieved by
environmental change. To the author's knowledge there is no evidence of
analogous environmental effects affecting Xenopus oocytes.
In the absence of further information which might distinguish the above
possibilities, the developmental significance of the RNA synthesis seen in Stage
VI oocytes remains unclear. However, the fact that this RNA synthesis continues for several days under the conditions described in this paper, makes
the testing in oocytes of nuclei and chromatin derived from haemopoietic
cells, a viable proposition.
The author is grateful to Dr J. B. Gurdon for helpful suggestions during the course of
this investigation and also to Drs R. M. Benbow, C. C. Ford and J. S. Knowland for
critically reading the manuscript. This work was supported by the Medical Research
Council.
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(Received 26 February 1974, revised 27 May 1974)