Development 105, 11-16 (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
11
Biochemical research on oogenesis.
RNA accumulation in the oocytes of the newt Pleurodeles waltl
HILDE VAN DEN EYNDE1, ANDRE MAZABRAUD2 and HERMAN DENIS2
1
Depanement Biochemie, Universiteit Antwerpen (VIA), Universiteitsplein, B-2610 Antwerpen, Belgium
Centre de Gininque MoUculaire, Laboratoire propre du CNRS associe" d I'University P. et M. Curie (Pans VI), F-91198 Gif-sur-Yvette
Cedex, France
2
Summary
We have compared the accumulation of 5S RNA and
tRNA in oocytes of Pleurodeles waltl with the corresponding process previously studied in Xenopus laevis.
5S RNA synthesis is regulated similarly in both species
since different families of 5S RNA genes are transcribed
in oocytes and in somatic cells of P. waltl, as in those of
X. laevis. Previtellogenic oocytes of P. waltl contain only
one prominent kind of storage particles (thesaurisomes).
In contrast, X. laevis oocytes of the same size contain two
major classes of thesaurisomes, sedimenting at 42S and
7S. The more abundant particles found in P. waltl
oocytes are homologous to the larger thesaurisomes
(42S) of X. laevis, but they have a lower sedimentation
coefficient and a higher tRNA/5S RNA molar ratio than
their X. laevis counterparts. Small amounts of particles
which we think to be homologous to the 7S particles of X.
laevis are present in previtellogenic oocytes of P. waltl.
Therefore, the storage function of the 7S particle protein
(TFIIIA) is only marginal in this species. In X. laevis
oocytes TFIIIA has a second function. It acts as a
positive transcription factor involved in the developmentally regulated expression of the 5S RNA genes. In X.
laevis expression of the oocyte-type 5S RNA genes is
accompanied by a massive accumulation of TFIHA. This
is not the case in P. waltl.
Introduction
oocytes are stored in two kinds of nucleoprotein particles (thesaurisomes), sedimenting at 42S and 7S
(Ford, 1971; Denis & Mairy, 1972; Picard & Wegnez,
1979).
The main features of RNA accumulation in oocytes
first described in X. laevis (gene amplification and
derepression; presence of two kinds of thesaurisomes),
have also been found in several teleost species (Vincent
et al. 1969; Mazabraud et al. 1975; Denis & Wegnez,
1977; Denis et al. 1980; Mashkova et al. 1981; Denis & le
Maire, 1983). Less is known about RNA accumulation
in the oocytes of urodeles. This is surprising since many
studies have been devoted to RNA transcription in
these cells because of the large size of their chromosomes. No information is available concerning the
existence of a dual 5S RNA gene system in urodeles,
although the lampbrush chromosome loops which transcribe 5S RNA have been identified in Notophthalmus
viridescens (Pukkila, 1975). Urodele oocytes are known
to amplify their ribosomal genes (Brown & Dawid,
1968), and to contain thesaurisomes similar in composition to the 42S particles of X. laevis (Kloetzel et al.
1981). However, it has been reported that the oocytes
of the newts Triturus vulgaris and T. cristatus do not
contain 7S particles (Barrett et al. 1984).
Mature amphibian oocytes contain large amounts of
ribosomes and tRNA. Early studies have shown that
growing oocytes of the anuran Xenopus laevis accumulate the components of their protein-synthesizing machinery in an unusual fashion. In this respect, a clear
distinction must be made between 28S, 18S and 5-8S
RNA on the one hand and 5S RNA and tRNA on the
other hand. The genes coding for the former types of
RNA are amplified at the beginning of the oocyte
growth (Brown & Dawid, 1968; Gall, 1968). The 5S
RNA and tRNA genes are not amplified (Brown &
Dawid, 1968). However, the oocytes increase their
production capacity for 5S RNA by activating 5S RNA
genes which are repressed in somatic cells (Wegnez et
al. 1972; Ford & Southern, 1973). The amplified genes
are not actively transcribed during the early phase of
the oocyte growth, called previtellogenesis (Ford, 1971;
Mairy & Denis, 1971). In contrast, the 5S RNA and
tRNA genes are active from the beginning of oogenesis.
As a result, previtellogenic oocytes accumulate 5S
RNA and tRNA in large molar excess with respect to
28S, 18S and 5-8S RNA (Ford, 1971; Mairy & Denis,
1971). 5S RNA and tRNA made in excess by small
Key words: oogenesis, urodele, 5S RNA sequence, storage
particles (thesaurisomes).
12
H. Van den Eynde and others
The latter observation deserves interest because the
protein component of the X. laevis 7S particles plays a
crucial role in the developmentally regulated transcription of 5S RNA. This protein is, in fact, a positive
transcription factor, called TFIIIA (Honda & Roeder,
1980; Pelham & Brown, 1980). TFIIIA binds to an
internal control region of both kinds of 5S RNA genes,
thereby increasing their transcription rate by RNA
polymerase III (Engelke et al. 1980; Sakonju et al.
1981). The amount of TFIIIA present in the cells is
thought to regulate the transcription of the 5S RNA
genes (Honda & Roeder, 1980). The oocyte-type 5S
RNA genes are transcribed only when TFIIIA is very
abundant. This is what happens in oocytes. When
TFIIIA is present in limited amounts, as in somatic
cells, only the somatic-type 5S RNA genes are transcribed. The mechanism by which TFIIIA selectively
activates the oocyte 5S RNA genes has been the subject
of many studies. According to a simple model, TFIIIA
is supposed to bind more tightly to the somatic 5S RNA
genes than to the oocyte ones (Sakonju & Brown, 1982;
Wormington et al. 1983; Brown & Schlissel, 1985a).
Therefore, only trace amounts of TFIIIA would be
sufficient to ensure transcription of the somatic 5S RNA
genes. Transcription of the oocyte 5S RNA genes would
require a much higher concentration of TFIIIA. However, this explanation is no longer acceptable since
recent studies have shown that TFIIIA has no preferential affinity in vitro for the somatic 5S RNA genes
(McConkey & Bogenhagen, 1988). Additional factors
such as TFIIIC are probably involved in the selective
transcription of the 5S RNA genes (McConkey &
Bogenhagen, 1988; Wolffe, 1988). The crucial factor in
this process is apparently the differential stability of the
transcription complexes, which in turn depends on the
concentration of TFIIIA and TFIIIC in the various
types of cells (Wolffe, 1988; Wolfe & Brown, 1988).
The absence of 7S particles in the oocytes of Triturus
(Barrett et al. 1984) is intriguing. We wished to confirm
this observation in another urodele species, Pleurodeles
waltl. We also wanted to know if different families of 5S
RNA genes are transcribed in oocytes and in somatic
cells of this animal. We found that in the latter respect
P. waltl does not differ from other amphibians. We also
report that previtellogenic oocytes of P. waltl contain
small amounts of particles that are most probably
homologous to the 7S particles of X. laevis (Picard &
Wegnez, 1979) and T. tinea (Denis et al. 1980).
Materials and methods
Previtellogenic oocytes were obtained from immature ovaries
of P. waltl (ll-13cm from snout to tailtip). The largest
oocytes in these animals were in late previtellogenic period
(200-300/an in diameter). Total RNA was purified from
whole ovaries and analysed as described (Mairy & Denis,
1971). Post-mitochondrial extracts of ovaries were fractionated by'pblyacrylamide gel electrophoresis or sucrose density
centrifugation (Denis & Mairy, 1972; Mazabraud et al. 1975).
Aliquots of the sucrose gradients were analysed for u.v.absorbance, RNA content (Mazabraud et al. 1975), protein
content (Laemmli, 1970), and cross-reactivity with an antiserum against X. laevis TFIIIA (Lagaye et al. 1988). The
thesaurin a/thesaurin b molar ratio in the storage particles
was determined by scanning the stained electrophoregrams in
visible light. When applied to X. laevis 42S particles, this
method gives a thesaurin a/thesaurin b absorbance ratio of
2-5, which corresponds to the expected stoichiometry (two
moles of thesaurin a per mole of thesaurin b; Picard et al.
1980).
Liver and ovary 5S RNA purified as described (Denis &
Wegnez, 1977) was end-labelled with pCp (Peattie, 1979) and
submitted to electrophoresis in 8% polyacrylamide gels
containing 7 M-urea. Autoradiography of the gels revealed the
presence of one major band in the case of liver 5S RNA and
five bands in the case of ovary 5S RNA. All visible bands were
eluted from the gels and sequenced according to Peattie's
procedure (Peattie, 1979).
The number of 5S RNA and tRNA genes in somatic cells of
P. waltl was determined by hybridizing erythrocyte DNA with
saturating amounts of end-labelled 5S RNA or tRNA (Denis
& Wegnez, 1977). The gene number was calculated from the
hybridization values thus obtained and the DNA content of a
P. waltl somatic cell (25 pg; Olmo, 1973).
Results
The sequence of P. waltl somatic 5S RNA is presented
in Fig. 1 according to the secondary structure model of
De Wachter et al. (1982). The sequence shown is that of
the only component that could be obtained from liver
cells in sufficient amount to be analysed. As indicated in
Fig. 1, oocyte 5S RNA is a heterogeneous population of
molecules, differing by their length and/or by their
sequence. A minor component, amounting to approximately 1 % of the material purified from ovaries, has
the same sequence as somatic 5S RNA. Given its §mall
abundance, this component might in fact originate from
the accessory cells of the gonads. All other 5S RNA
molecules of ovary origin differ by their sequence from
somatic 5S RNA (Fig. 1). Among the four major
subtypes of 5S RNA that could be purified from ovaries
and sequenced, two components (Nos 3 and 4) are
shorter versions of component No 2 (Fig. 1). We
ascribe the length heterogeneity of oocyte 5S RNA to
post-transcriptional modifications similar to those occurring in X. laevis oocytes (Denis & Wegnez, 1973).
We conclude from the data presented in Fig. 1 that at
least three different families of 5S RNA genes are
transcribed in P. waltl oocytes. Another family is
transcribed in liver cells. The first three families of
genes differ from the fourth one at four positions (Nos
30, 40, 92 and 113), four positions (Nos 30, 40, 47 and
92) and five positions (Nos 30, 40, 47, 60 and 92),
respectively.
Previtellogenic oocytes of P. waltl contain relatively
little 5S RNA. The tRNA/5S RNA mass ratio ranges
from 2:1 to 4:1 in these oocytes, instead of 1:1 to 1-5:1
in those of X. laevis (Mairy & Denis, 1971). Fractionation of P. waltl ovary homogenates by sucrose density
centrifugation reveals the presence of one major class of
5S RNA- and tRNA-containing particles (Fig. 2A),
sedimenting like dimers of X. laevis thesaurisomes
(Picard et al. 1980). The 25S particles of P. waltl have
Biochemical research on oogenesis
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the same overall composition as the 42S particles of X.
laevis (Picard et al. 1980), since they contain four major
RNA and protein molecules (5S RNA, tRNA,
thesaurin a and thesaurin b; Fig. 2). We find two
molecules of thesaurin a per molecule of thesaurin b in
the particles of P. waltl as in those of X. laevis, T. tinea
and T. cristatus (Picard et al. 1980; Denis et al. 1980;
Kloetzel et al. 1981). However, the P. waltl particles
differ from the X. laevis ones in several respects. The
former particles have a higher tRNA/5S RNA molar
ratio (5:1 to 6:1 instead of 3:1; Fig. 2A), and a higher
protein/RNA mass ratio (2:1 instead of 1-4:1).
Thesaurins a and b of P. waltl have a slightly higher
molecular mass than the corresponding proteins of X.
laevis ( 5 3 x 1 0 ^ , instead of 50xlO 3 ^,. for thesaurin a;
41xl(PM r instead of 40X103 Mr for thesaurin b).
Approximately 5 % of 5S RNA present in an ovary
extract of P. waltl sediments in the top region of the
sucrose density gradients (Fig. 2A). This RNA is not
free in the cell extract since no u.v.-absorbing material
migrates at the same position as pure 5S RNA when the
extract is submitted to polyacrylamide gel electrophoresis (Fig. 3). Instead, a small peak with nearly the same
mobility as X. laevis 7S particles can be seen in the gel
(Fig. 3). This peak accounts for less than 0-5 % of the
260 nm-absorbance detected in the ovary extract. The
corresponding peak in extracts of X. laevis immature
ovaries contains approximately 40 times as much u.v.absorbing material (Fig. 3). Western blot analysis of the
4-7S fractions of the P. waltl extracts (Fig. 2A) reveals a
faint band reacting with an anti-TFIIIA antiserum. This
band is not visible in the stained electrophoregrams
(Fig. 2B). From these data, we conclude that extracts of
P. waltl previtellogenic oocytes contain small but detectable amounts of particles which are most probably
homologous to the 7S particles of X. laevis, since their
protein moiety cross-reacts with antibodies raised
against X. laevis TFIIIA. We estimate that the latter
protein is 40 times less abundant in oocytes of P. waltl
than in those of X. laevis (Fig. 3). TFIIIA makes up less
Fig. 1. Nucleotide sequence of somatic
5S RNA from P. waltl. The sequence is
arranged according to the secondary
structure model of De Wachter et al.
(1982). Four distinct components
(numbered 1 to 4) have been detected
in oocytes, differing from somatic 5S
RNA at the positions shown in the
inset. S stands for G or C. The
nonstandard base pair U:U is indicated
by a lozenge instead of a dot for the
G: C, A: U and G: U base pairs.
than 1 % of total soluble protein in extracts of P. waltl
oocytes (Fig. 2B), instead of several per cent in extracts
oiX. laevis oocytes (Denis & le Maire, 1983).
Given the high C value of P. waltl cells (Olmo, 1973),
we found it interesting to compare the number of 5S
RNA and tRNA genes in this species with the corresponding gene number in other amphibians. According
to our measurements, a diploid cell of P. waltl contains
approximately 100000 5S RNA genes (instead of 50000
in X: laevis and 300000 in N. viridescens; Brown &
Weber, 1968; Pukkila, 1975), and 300000 tRNA genes
(instead of 16000 in X. laevis; Clarkson et al. 1973). The
high tRNA gene redundancy in P. waltl probably
explains why the oocytes of this animal accumulate
more tRNA than 5S RNA (Fig. 2).
Discussion
P. waltl is the first urodele in which different 5S RNA
genes are shown to be transcribed in oocytes and in
somatic cells. Each anuran and teleost species studied
so far differs from all others by the position of the
nucleotide substitutions occurring in the two types of 5S
RNA (Erdmann & Wolters, 1986; Nietfeld et al. 1988).
This is also the case for P. waltl since three nucleotide
substitutions that we have detected in oocyte and
somatic 5S RNAs (at residue Nos 60, 92 and 113; Fig. 1)
have not yet been observed in other 5S RNAs. The
remaining substitutions (C—>U at residue Nos 30 and
40 and G—>A at residue No 47) occur at identical
positions in somatic and oocyte 5S RNAs of other
species (X. laevis for transition at residue No 30; T.
tinea and M. fossilis for transition at residue No 40; X.
laevis for transition at residue No 47). All nucleotide
replacements except one (at residue No 40) are located
in regions of the 5S RNA molecule which are likely to
be base-paired (Fig. 1). Significantly, these substitutions tend to destabilize the secondary structure of
oocyte 5S RNA (Fig. 1).
14
H. Van den Eynde and others
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Fig. 2. RNA and protein content of a post-mitochondrial
extract of P. waltl immature ovaries. The ovaries from five
juvenile females were homogenized in 750/il of 50 mMTris-HCl, pH7-5, 25ITIM KC1, 5mM MgCl2. The
supernatant of a low-speed centrifugation (lOOOOrpm for
lOmin) was layered on top of a 15-30% sucrose density
gradient made up in the homogenization buffer and spun at
38000rpm for 5hr in a SW41 rotor. Aliquots of the gradient
fractions were either extracted with phenol and analysed for
RNA content in 7-2 % polyacrylamide gels (A) or analysed
for protein content in 10-4 % polyacrylamide gels containing
0 1 % sodium dodecyl sulphate (B). The position of
thesaurins a and b in fractions 13-16 of the gradient is
indicated. The position of TFIIIA in fraction 18 is marked
by an arrow. This protein can be revealed by an antiserum
against X. laevis TFIIIA (not shown). Volume of the
fractions: 0-6ml.
The species-specificity of the nucleotide substitutions
occurring in oocyte and somatic 5S RNA genes raises
questions concerning the evolution of these genes
(Denis & Wegnez, 1978), and the mechanism of their
expression. The somatic 5S RNA genes do not differ
considerably from one species to another (Denis &
Wegnez, 1978; Erdmann & Wolters, 1986). In contrast,
Fig. 3. Electrophoretic analysis of X. laevis and P. waltl
ovary extracts. Two 70-/il aliquots of ovary extracts,
containing the same amount of u.v.-absorbing material
(0-54 A260nm unit) were layered on two 7-5 %
polyacrylamide gels made up in 40 mM Tris, 20mM sodium
acetate, pH8-4. The gels were submitted to a voltage
gradient of 8 volts cm"1 during 2h and scanned at 265 nm.
The arrows show the position of tRNA and 5S RNA in a
gel run in parallel. The 7S peaks contain 12% and 0-3%,
respectively, of the u.v.-absorbing material present in the
X. laevis and P. waltl extracts.
the oocyte 5S RNA genes have evolved much more
rapidly. Oocyte and somatic 5S RNAs of the six species
studied so far differ in a total of 22 positions, scattered
on the whole length of the molecule (Erdmann &
Wolters, 1986; Nietfeld etal. 1988; this paper). Among
these substitutions 14 are species-specific. Clearly, the
somatic 5S RNA genes have undergone a higher
selective pressure than the oocyte ones. We ascribe this
difference to be the constraints imposed by the interactions between the genes and the transcription factor(s)
involved in their activation. The key factor is probably
the need to integrate the somatic 5S RNA genes into
stable transcription complexes (Wolffe & Brown,
1988). This requires a tight structural fit between the
somatic 5S RNA genes and the transcription factors.
The constraint is less stringent for the oocyte 5S RNA
genes. These are engaged in less stable transcription
complexes (Wolffe & Brown, 1988). This implies that
the oocyte 5S RNA genes have a lower affinity for some
element(s) of the transcription machinery. Such a
reduction in affinity can be obtained by a variety of
mutations in the 5S RNA genes, leading to the observed
interspecific and intraspecific heterogeneity in the sequence of oocyte 5S RNA.
P. waltl previtellogenic oocytes contain only one
prominent kind of storage particles (Fig. 2). The stoi-
Biochemical research on oogenesis
chiometry of these particles is not the same as in X.
laevis. The main difference lies in the tRNA/5S RNA
molar ratio which significantly exceeds 3:1 in P. waltl
(Fig. 2). In several teleost species, the larger thesaurisomes have also been reported to contain more than
three molecules of tRNA per molecule of 5S RNA
(Mazabraud et al. 1975). It follows that the RNAprotein interaction in the vertebrate thesaurisomes do
not conform to a single model.
P. waltl previtellogenic oocytes contain relatively few
7S particles (Fig. 2). Therefore, the storage function of
the 7S particles is only marginal in P. waltl oocytes. In
fact, the 7S particle concentration in these oocytes is
intermediate between that found in X. laevis and T.
tinea oocytes (Picard et al. 1980; Denis et al. 1980) and
that found in somatic cells (Lagaye et al. 1988). These
cells contain only trace amounts of 5S RNA in association with a TFIIIA-related protein (Lagaye et al.
1988). A much larger amount of 5S RNA is associated
with a ribosomal protein (L5; Steitz et al. 1988). This
complex appears to be a precursor to ribosome assembly (Steitz etal. 1988).
As stated in the introduction, the 7S particle protein
is also involved in regulation of 5S RNA transcription.
What is thought to be crucial in this respect is the
number of TFIIIA molecules per 5S RNA gene copy
(Brown & Schlissel, 1985b; McConkey & Bogenhagen,
1988). In X. laevis oocytes this number is very large
(SxlC^-lO7; Shastry et al. 1984). Since P. waltl oocytes
contain twice as many 5S RNA genes, but 40 times as
few 7S particles as X. laevis oocytes, we estimate that
the TFHIA/5S RNA gene ratio is 80 times lower in P.
waltl oocytes than in X. laevis ones. Therefore, P. waltl
oocytes of any size probably contain several thousand
TFIIIA molecules per 5S RNA gene copy. This should
be enough to activate the oocyte 5S RNA genes, since
in living cells of X. laevis a 10-fold molar excess of
TFIIIA is apparently sufficient for this activation to
occur (Brown & Schlissel, 19856).
We thank Prof. De Wachter for his constant interest in this
work, Dr M. le Maire and A. Viel for many helpful discussions and A. Gomez de Garcia for skillful technical
assistance.
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{Accepted 20 January 1989)