/ . Embryo!, exp. Morph. Vol. 66, pp. 127-140, 1981
Printed in Great Britain © Company of Biologists Limited 1981
127
Accumulation, spatial distribution
and partial characterization of poly(A)+RNA in the
developing oocytes of Xenopus laevis
ByM. WAKAHARA1
From the Zoological Institute, Hokkaido University
SUMMARY
In situ hybridization using [3H]poly(U) was applied to developing oocytes of Xenopus
laevis, which had beenfixedin Bouin's solution. Tissue sections were pretreated with DNase I,
annealed with [3H]poly(U) and post-treated with RNase A and TCA. After the autoradiographical processing, silver grains over the oocyte were counted. As a result of the control
experiments which included RNase A, RNase T2, DNase I and Pronase E hydrolysis and
Cordycepin (3'-deoxyadenosine) incubation before in situ hybridization, it was concluded
that the poly(U)-binding activity detected upon the oocytes was due to the possible presence
of poly(A)+RNAs. Spatial distribution of the poly(U)-binding sites changed during the
development of the oocytes; in a small oocyte before the pachytene stage, silver grains
developed over the nucleus, while in a larger oocyte after the diplotene the grains were
concentrated over the cytoplasm. After yolk platelets were deposited in the cytoplasm, two
types of poly(U)-binding activities were noted; a bound-type activity which was firmly
associated with the cytoplasm, so that the positions of the silver grains were not influenced
by fixation, and an unbound type which did not bind so firmly to the cytoplasm and was
therefore easily influenced by inflow of fixative. The bound-type activity persisted in the
cytoplasm throughout the oogenesis, but the unbound type appeared only after the vitellogenesis, especially in the yolky cytoplasm. The total poly(U)-binding activity per oocyte
increased continuously with the growth of the oocyte.
INTRODUCTION
Since Spirin (1966, 1969) proposed that embryonic development depends on
the utilization of masked maternal information stored in 'informosomes',
a body of experiments has supported the existence of masked maternal messenger in the oocytes and eggs of many species of animals (reviewed in Davidson,
1976). Extensive analysis using biochemical techniques demonstrates that the
maternal messenger stored in oocytes includes messenger RNA (mRNA)
molecules (Slater, Gillespie & Slater, 1973; Lovett & Goldstein, 1977; Ruderman & Pardue, 1977; Jackie, 1979). Although a molecular basis for the utilization of the masked mRNA is now becoming known in some species (Kawmeyer,
1
Author's address: Zoological Institute, Faculty of Science, Hokkaido University,
Sapporo 060, Japan.
5-2
128
M. WAKAHARA
Jenkins & Raff, 1978; Jenkins, Kawmeyer, Young & Raff, 1978), there is little
information concerning the synthesis, storage and especially the masking
mechanism of maternal information.
Many eukaryotic mRNA molecules contain a polyadenylic acid [poly(A)]
sequence at their 3'-terminus (Brawerman, 1974). The kinetics of the poly(A)containing RNA [poly(A)+RNA] have been intensively investigated in Xenopus
oocytes and eggs (Rosbash & Ford, 1974; Darnbrough & Ford, 1976; Levenson
& Marcu, 1976; Cabada, Darnbrough, Ford & Turner, 1977; Ford, Mathieson
& Rosbash, 1977; Ruderman & Pardue, 1977; Miller, 1978; Dolecki & Smith,
1979; Sagata, Shiokawa & Yamana 1980). Biochemical analysis indicates that
even the previtellogenic (stage 1, after Dumont, 1972) oocytes contain almost
the same amount of poly(A)+RNA as is stored in the full-grown (stage 6)
oocytes (Rosbash & Ford, 1974). Moreover, a substantial number of poly(A)+RNA molecules which have been synthesized in the previtellogenic oocyte
survive for lengths of time commensurate with the length of oogenesis (1-2
years) (Ford et al. 1977). Since the poly(A)+RNA stored in oocytes is able to
direct protein synthesis in the cell free system (Darnbrough & Ford, 1976;
Levenson & Marcu, 1976; Cabada et al. 1977; Ruderman & Pardue, 1977),
these must be the presumptive mRNA molecules which are utilized during the
early development after fertilization. On the other hand, the lampbrushchromosome-stage oocyte, which has meiotic diplotene chromosomes, is
believed to show a high transcriptional activity (Gall & Callan, 1962), but no
substantial increase of the poly(A)+RNA is observed during this stage of
oogenesis (Rosbash & Ford, 1974; Dolecki & Smith, 1979). In this respect, it
is important to study the synthesis, accumulation and possible degradation of
the poly(A)+RNA molecules during the oogenesis.
Recently, a technique has been developed to demonstrate the cytological
localization of poly(A)+RNAs by means of in situ hybridization using a [3H]poly(U) probe (Lamb & Laird, 1976). Capco & Jeffery (1978) have succeeded in
detecting a spatial distribution of the poly(A)+RNA in insect oocytes on
paraffin-embedded histological sections. In situ hybridization with [3H]poly(U)
has the merit of demonstrating accurate cytological localization of poly(A)+RNAs which are synthesized and in part degraded during oogenetic processes.
In the present investigation, in situ hybridization using [3H]poly(U) was applied
to Xenopus developing oocytes; the consequent accumulation and cytological
localization of poly(A)+RNA is here described.
MATERIALS AND METHODS
Animals
African clawed toads, Xenopus laevis, were used: they were reared routinely
and reproduced in our laboratory. Ovarian pieces were obtained from immature toads 1-6 months after metamorphosis.
Poly(A)+RNA in Xenopus oocytes
129
In situ hybridization
In situ hybridization with [3H]poly(U) was carried out according to the
method developed by Capco & Jeffery (1978) with some modifications.
Ovarian pieces were fixed in Bouin's solution, dehydrated, embedded in
paraffin and sectioned serially at a thickness of 6 /mi. Hydrated slides were
rinsed twice in DNase buffer (100 HIM Tris-HCl, 3 mM MgCl2, pH 7-6) and then
treated with 50/*g/ml of DNase (DNase I, grade III, Sigma) dissolved in
DNase buffer for 1 h at 37 °C. After rinsing with DNase buffer, the slides were
immersed in a hybridization buffer (10 mM Tris-HCl, 200 mM NaCl, 5 mM
MgCl2, pH 7-6). Annealing was performed by applying 60 /*1 aliquots of 2-5 /*Ci/
ml of [3H]poly(U) (17-61 Ci/mmole; 41-147 nucleotides in length, The Radiochemical Centre, Amersham) dissolved in hybridization buffer to each slide,
sealing with a clean cover slip (24 x 50 mm), and incubating the slide for 3-5 h
at 55 °C in a moist chamber. Following the incubation, the cover slip was carefully removed and the slide was rinsed twice with the hybridization buffer
at 20 °C.
In order to remove the unhybridized [3H]poly(U) from the tissue, the slide
was treated with pancreatic RNase. All slides were rinsed with RNase buffer
(50 mM Tris-HCl, 100 mM KC1, 1 mM MgCl2, pH 7-6) and then treated with
50 /*g/ml of RNase A dissolved in the RNase buffer for 30 min at 37 °C.
Finally, the slides were rinsed once in the RNase buffer and twice in distilled
water; they were then immersed in ice-chilled 5 % trichloroacetic acid (TCA)
for 15 min.
Autoradiography and grain count
Autoradiography was performed by coating the slides with Sakura NR II
liquid emulsion (Konishiroku, Tokyo). After exposure in the dark at 4°C
(14 days), the slides were developed with D-19 developer for 2-5 min at 20 °C.
The sections were stained through the emulsion with Delafield's haematoxylin
and eosin after development.
The grain density was determined by counting the number of grains over
a unit cytoplasmic square (100 /mi2). Silver grains over five given unit squares
from a previtellogenic oocyte or ten given unit squares from a postvitellogenic
oocyte were counted for one determination of the grain density. An average
grain density was calculated from the grain densities of 20 similar oocytes.
Since Xenopus oocytes increase in volume extensively during oogenesis, the
grain density per unit cytoplasmic area does not represent the actual [3H]poly(U)-binding activity per oocyte. The total labelling per oocyte was therefore
calculated from the volume of the oocytes at different diameters by treating the
oocyte as a series of 6 /mi-thick discs. The volume of the germinal vesicle was
deducted from the volume of oocyte because the silver grains upon it were
practically zero. The total grains per oocyte was estimated from the following
130
M. WAKAHARA
formula, in which rx is the radius of the oocyte and r2 that of the germinal
vesicle:
4 / 3 7 7 ( r ?2 r | ) / * m 3 x r a i n d e n s i t
6 fim (thickness) x 100 /*m (unit square)
Control experiments
In order to check the specificity of [3H]poly(U) binding to tissue sections,
several slides were pretreated with RNase and/or proteinase before in situ
hybridization. The enzymes used were 50 fig/m\ of RNase A, 2 units/ml of
RNase T2 (Sigma) and 50 or 1 /*g/ml of Pronase E (Tokyokasei, Tokyo).
Ovarian pieces, which contained about 100 oocytes, were cultured in vitro.
The culture medium was diluted Leibovitz L-15 (which was a mixture of L-15,
distilled water and foetal calf serum, in the ratio of 5:4:1, respectively) containing Cordycepin (3'-deoxyadenosine). After a short period of cultivation, the
ovarian pieces were processed for in situ hybridization as described above.
RESULTS
In situ hybridization with a [*H]poly(U) probe
The first experimental step was to examine the effect of increasing concentrations of the [3H]poly(U) on the grain density over sections of the developing
oocytes. Sixty ji\ of [3H]poly(U) at various concentrations were applied to
similar sections. Figure 1 shows the poly(U)-binding activities on the cytoplasm
of stage-1 oocytes (200 /*m in diameter) at different concentrations of [3H]poly(U). After applying higher concentrations (above 0-8 /*Ci/ml) a significant
increase of the grain density was observed, but no substantial increase was
observed above 2-5 /*Ci/ml. Therefore, the saturation of the poly(U)-binding
sites on histological sections seemed to be attained at a concentration between
1-0-2-5 /tCi/ml of [3H]poly(U). Subsequent experiments were carried out using
[3H]poly(U) at the concentration of 2-5 /tCi/ml in order to assure an excess
poly(U) hybridization.
The next experimental step was to establish the specificity of [3H]poly(U)
binding to tissue sections. The grain density over the tissues which had been
exposed to specific enzymes prior to annealing was examined. Table 1 is a summary of the effects of enzymatic digestions on the grain density upon the cytoplasm of stage-1 oocytes. RNase A was dissolved in two different buffer solutions
according to Capco & Jeffery (1978), who stated that the poly(A) was protected
from RNase hydrolysis in the presence of 0-1 M KC1, but not in the presence of
0-01 M KC1. However, both sections which had been pretreated with RNase
dissolved in either low or high K+ concentration buffers were labelled equally,
and no substantial reduction of grains was detected even after a 24 h digestion
period (Fig. 2b). On the other hand, RNase T 2 had a strong effect on the
poly(U)-binding activity. No grains developed on the sections after the RNase
T 2 digestion (Fig. 2 c).
Poly(A)+RNA in Xenopus oocytes
50
I I I I I II
131
I
I I I I I III
I
I I I I I II
I
I I I I I III
I
I I I I III
40
30
O 20
10
01
1
Concentration of [3H]poly(U) (/iCi/ml)
10
Fig. 1. The grain density on the cytoplasm of Xenopus laevis oocytes at 200 /*m in
diameter after in situ hybridization with increasing concentrations of [3H]poly(U).
Grain density ± SD.
Table 1. Effects of specific enzyme treatments on [3H]poly(U)-binding activity on
Xenopus oocyte cytoplasm
Treatment
Enzyme
Condition
Grain density ± SD
DNaseI(50/*g/ml)
DNase-RNase A (50/tg/ml)
IOOITIMKCI, 24 h
DNase-RNase A (50/^g/ml)
10 HIM KC1, 24 h
50 mM KC1, 24 h
DNase-RNase T2 (2 u/ml)
DNase-Pronase E (50 /*g/ml)
50mMKCl, lOmin
DNase-Pronase E (1 /*g/ml)
50 mM KC1, 10 min
DNase-Buffer
100 mM KC1, 24 h
* The stainability to haematoxylin and eosin of tissues was greatly
37-3 ± 4 1
28-2±3-8
21-6±31
0-5 ±0-2
6-2 ±2-2*
33-2 ± 4 0
29-5 ±4-3
reduced.
Mild Pronase E digestion (1 /*g/ml for 10 min at 37 °C) did not alter the
labelling patterns. Even after very strong Pronase E digestion (50 /^g/ml) which
removed a substantial amount of material from the sections, so that the stainability of sections to haematoxylin and eosin was significantly reduced, a few
grains were still detected over the tissues.
132
M. WAKAHARA
Fig. 2. Autoradiographs of developing oocytes in Xenopus laevis after in situ
hybridization with [3H]poly(U). (a) Control section pretreated with DNase I (50 /ig/
ml, 60 min at 37 °C) and post-treated with RNase A (50/^g/ml, 30 min at 37 °QTCA (5 % 15 min at 0 °C). (b) Section pretreated with RNase A (50/tg/ml, 24 h at
37 °C). The presence of silver grains is comparable to that in the control, (c) Section
pretreated with RNase T2 (2 units/ml, 24 h at 37 °C). No grains are present. See
also Table 2. C, oocyte cytoplasm; GV, germinal vesicle. Haematoxylin and eosin
stained. Scale bars: 100/im.
Post-transcriptional polyadenylation in the oocyte
In order to examine the effects of Cordycepin on the poly(U)-binding activity
and on the uptake of [3H]uridine into acid-insoluble fractions, ovarian pieces
were cultured in vitro with or without Cordycepin. Cordycepin is reported to be
a specific inhibitor of the post-transcriptional adenylation of nuclear RNAs
Poly(A)+RNA in Xenopus oocytes
133
Table 2. Effect of Cordycepin on [3H]poly(U)-binding activity and
[3H]uridine uptake
Dose of
Cordycepin
(/ig/ml)
Duration
in vitro
(h)
Grain density ± SD
[3H]poly(U) binding*
[3H]uridine uptakef
0
0
331 ±4-4
0
12
34-6 ± 5 0
0
48
36-3 ±4-5
258 ±32
50
12
350±41
50
48
24-3 ±5-6
241 ±40
1000
12
30-3 ±5-8
100
48
226 ±35
24-6 + 4-2
* Grain density on 100 [im2 cytoplasmic area of 200/*m oocyte.
t Grain density on the germinal vesicle.
(Darnell, Philipson, Wall & Adesnik, 1971; Jelinek et al. 1973). Table 2 shows
the results of the poly(U)-binding activity of the sections and the uptake of
[3H]uridine after the Cordycepin incubation. Without Cordycepin, grain density
resulting from the in situ hybridization was not significantly reduced even after
48 h in vitro. In contrast to this, the poly(U)-binding activities decreased in
accordance with the duration of incubation and with the concentrations of
Cordycepin used. Since the [3H]uridine uptake into acid-insoluble fractions in
the germinal vesicle did not alter following the Cordycepin incubation, the
reduction of the poly(U)-binding activity cannot be due to the reduced rate of
the nuclear RNA synthesis.
Spatial distribution of the [zH]poly(U)-binding sites
Cytological localization of the [3H]poly(U)-binding sites seemed to change
according to the developmental stages of oocytes. Even in very small oocytes
during the earlier stages of the meiotic prophase (leptotene and zygotene),
silver grains were already recognized over the nucleus, but not the cytoplasm
(Fig. 3 a, b). In the pachytene oocyte, silver grains were more concentrated over
the nucleus (Fig. 3 c) than during the former stages, but somewhat fewer over
the extrachromosomal 'cap', which corresponded to an accumulation of the
ribosomal DNA molecules (Ficq, 1970; Van Gansen & Schram, 1974). When
the oocytes grew to the diplotene stage, the labelling pattern changed drastically;
the silver grains over the nucleus diminished and appeared over the cytoplasm
(Fig. 3d). This pattern of labelling - heavy silver grains over the cytoplasm and
no or few grains over the germinal vesicle - was not altered thereafter. The
cytoplasm was evenly labelled except for the mitochondrial mass or Balbiani
body, which was a peculiar cytoplasm specific to the previtellogenic oocyte and
composed of mitochondria (Dumont, 1972). The silver grains over the mitochondrial mass were somewhat fewer than over the other cytoplasmic region
(Fig. 3e,f).
134
M. WAKAHARA
Poly(A)+RNA in Xenopus oocytes
135
After vitellogenesis began in the oocyte cytoplasm, the labelling pattern was
modified: there were relatively dense silver grains over the yolk-free cytoplasm
and more scattered grains over the yolky peripheral cytoplasm (Fig. 3g, h). It
is worth noting that the poly(U)-binding sites on the yolky cytoplasm seemed
influenced by the inflow of the fixative. On the other hand, the silver grains over
the yolk-free cytoplasm were firmly fixed regardless of the direction of fixative
inflow. The direction of the fixative invasion into oocytes was assumed from the
location of the detached site of the germinal vesicle from the cytoplasm (Fig. 4 a),
resulting from the different rate of shrinkage of the cytoplasm and the germinal
vesicle caused by the fixative. The yolky cytoplasm at the 'distal' end (Fig. 4 b)
represented a much higher silver-grain density than that at the 'proximal' end
(Fig. 4e) in terms of the direction of the fixative inflow.
After vitellogenesis, the presumptive animal-vegetal axis of oocyte was
assumed on the histological sections according to the distribution of melanin
granules and the size of yolk platelets; animal pole cytoplasm contained dense
melanin granules and small yolk platelets while vegetal pole cytoplasm had few
melanin and large yolk platelets. However, accurate spatial distribution of the
poly(U)-binding sites along the animal-vegetal axis could not be determined,
since the grain density over the yolky cytoplasm was influenced by the fixative
invasion regardless of the animal-vegetal axis of the oocyte.
Changes of poly(U)-binding activities during oogenesis
Figure 5 shows changes of the grain density upon the cytoplasm of oocytes
after in situ hybridization during oogenesis. The data from three different
experiments using different ovaries were combined. During the early phase of
oocyte growth (up to 200 fim in diameter) the average grain density over the
cytoplasm rapidly increased in accordance with the growing diameter of the
oocyte. The highest grain density was attained over the cytoplasm of oocytes of
200 jim diameter. Once the oocyte grew larger than 200 fim, the average grain
density dropped and thereafter gradually decreased with increasing size of
oocyte. Although the silver grains were uniformly distributed over the cytoplasm during the previtellogenic stage (35-300 fim in diameter), the distribution
of grains showed a bimodal pattern after the vitellogenesis: relatively higher
Fig. 3. Autoradiographs of developing oocytes in Xenopus after in situ hybridization
with [3H]poly(U). (a and b) Very small oocytes at leptotene or zygotene stages (*).
The focus is adjusted to the oocytes (a) and to the silver grains (b), on the same
section, (c) Pachytene oocyte showing extrachromosomal cap (arrow). The silver
grains are concentrated over the nucleus, (d) Early diplotene oocytes showing silver
grains over the cytoplasm, but not the germinal vesicle, {e and / ) Section through
the cytoplasm of a diplotene oocyte, focused on the silver grains over the cytoplasm
{e) and the mitochondrial mass (M) or Balbiani body (/), on the same section,
(g and h) Section through the cytoplasm of a vitellogenic oocyte, focused on the
silver grains over the yolk-free (g) and yolky (//) cytoplasm, on the same section.
Haematoxylin and eosin stained. Scale bars: 20/«n.
136
M. W A K A H A R A
Fig. 4. Autoradiographs of developing oocytes of Xenopus after in situ hybridization
with [3H]poly(U). (a) Low magnification photograph showing a vitellogenic oocyte.
Each cytoplasmic region enclosed by squares is enlarged in Figs. Ab-e. Large
arrow indicates a presumed direction of fixative inflow, judged from the location of
detached site (*) of the germinal vesicle from the cytoplasm. The silver grains are
much more concentrated over the yolky cytoplasm at the 'distal' end (b) than at the
'proximal' end (e\ in terms of the direction offixativeinflow. The silver grain density
is the same upon the yolk-free cytoplasm of 'distal' (c) and 'proximal' (d) regions.
Haematoxylin and eosin stained. Scale bars: 100 fim (a), 20/im (b-e).
Poly(A)+RNA in Xenopus oocytes
137
§ 3 0 -
100
200
300
400
Diameter of oocyte (/im)
500
Fig. 5. The grain density on the cytoplasm (solid line) and the total silver grains per
oocyte (broken line), determined in Xenopus developing oocytes after in situ
hybridization with [3H]poly(U). Closed circles, SD represent grain density upon the
yolk-free cytoplasm and open circles represent grain density upon the yolky
cytoplasm after the vitellogenesis.
density over the yolk-free cytoplasm and lower density over the yolky peripheral cytoplasm. The grain densities over both yolk-free and yolky cytoplasms
decreased gradually with the growth of the oocyte.
The broken line shown in Fig. 5 represents changes of total silver grains per
oocyte during the oogenesis. The silver grains per oocyte increased almost
linearly in accordance with growth of the oocytes, so far as these were examined.
DISCUSSION
3
The [ H]poly(U)-binding activities on the developing oocytes of Xenopus
laevis after in situ hybridization with DNase pretreatment and RNase-TCA
post-treatment are equivalent to the activity which has been seen in insect
oocytes (Capco & Jeffery, 1978, 1979). We conclude that the poly(U)-binding
activity described in this paper is a result of the presence of poly(A)+RNAs in
the oocyte. We offer the following reasons.
First, the poly(U)-binding activity is sensitive to RNase T2 hydrolysis (which
preferentially attacks the adenylic acid bonds), but it is insensitive to DNase 1,
138
M. WAKAHARA
RNase A and Pronase E treatments (Table 1). Second, after Cordycepin
incubation labelling after in situ hybridization with [3H]poly(U) decreases considerably (Table 2), but the uptake of [3H]uridine into acid-insoluble fractions
is not altered. This agent acts at the level of post-transcriptional addition of
poly(A) to mRNA (Darnell et al. 1971; Mendecki, Lee & Brawerman, 1972;
Jelinik et al. 1973). Cytological localization of the poly(U)-binding activities
changes significantly during development of the oocyte. Before entering the
diplotene stage, the poly(U)-binding sites were concentrated in the nucleus, but
after the diplotene were concentrated in the cytoplasm. The polyadenylation of
presumptive mRNAs in the nucleus has been demonstrated in mammalian cells
(Mendecki et al. 1972; Jelnik et al. 1973). In Xenopus, also, it has been reported
that presumptive vitellogenin mRNA is polyadenylated in the nucleus just after
transcription (Ryffel et al. 1980). When applying in situ hybridization using
[3H]poly(U) onto Xenopus liver and kidney, almost all silver grains are concentrated over the nucleus (M. Wakahara, unpublished). Therefore, it seems
that nuclear RNAs are polyadenylated in the nucleus of differentiated or differentiating cells. In Xenopus oocytes after diplotene, however, the silver grains
become concentrated over the cytoplasm. This implies two possibilities. The
first is that nuclear RNAs are polyadenylated in the germinal vesicle and
released into the cytoplasm. However, the rapid release of poly(A)+ RNA into
the cytoplasm and rapid increase in the volume of the germinal vesicle, causes
the poly(U)-binding activity upon the germinal vesicle to be hardly detectable.
The second is that nuclear RNAs are instantly released into the cytoplasm
without polyadenylation and then polyadenylated in the cytoplasm. Although
the results of Cordycepin incubation (Table 2) demonstrate conclusively that
the post-transcriptional adenylation of RNAs occurs in the oocyte, we could
not determine the cytological location of the polyadenylation, in either nucleus
or cytoplasm, after the diplotene stage.
Since the in situ hybridization procedure employed in this study did not
allow us to assess the length of the poly(A) sequence, the presence of two size
classes of poly(A) with different lengths, short and long poly(A), designated as
poly(A)s and poly(A)L (Cabada et al. 1977), could not be confirmed. But, as
a result of behaviour during fixation, two types of poly(U)-binding activities
were demonstrated; one type is firmly bound to the cytoplasm, so that the silver
grains are not influenced by fixative inflow, while the other may not be so
firmly bound to the cytoplasm, and so is easily influenced by the fixative invasion. The former, bound-type poly(U)-binding activity, persists throughout
oogenesis, and the latter, unbound type, appears after vitellogenesis. Since the
appearance, accumulation and persistence of the bound and unbound types
correspond to those of the poly(A)L and poly(A) s respectively, the bound-type
activity may correspond to the poly(A)L and unbound type to the poly(A)s.
Furthermore, it is possible to assume that the bound-type activity reflects the
poly(A)+RNA associated with the polysomes and the unbound type represents
Poly(A)+RNA in Xenopus oocytes
139
+
a poly(A) RNA in the form of mRNP particles which are presumed to represent
a store of mRNAs (Rosbash & Ford, 1974). At present, however, no evidence
is available to allow further characterization of these two types of poly(U)binding activities.
This work was supported in part by a grant (No. 457564) from the Japan Ministry of
Education, Science and Culture.
REFERENCES
BRAWERMAN, G. (1974). Eukaryotic messenger RNA. Ann. Rev. Biochem. 43, 621-642.
CABADA, M. O., DARNBROUGH, C , FORD, P. J. & TURNER, P. C. (1977). Differential accumulation of two size classes of poly(A) associated with messenger RNA during oogenesis in
Xenopus laevis. Devi Biol. 57, 427-439.
CAPCO, D. C. & JEFFERY, W. R. (1978). Differential distribution poly(A)-containing RNA
in the embryonic cells of Oncopeltusfasciatus. Devi Biol. 67, 137-151.
CAPCO, D. C. & JEFFERY, W. R. (1979). Origin and spatial distribution of maternal messenger
RNA during oogenesis of an insect, Oncopeltus fasciatus. J. Cell Sci. 39, 63-76.
DARNBROUGH, C. & FORD, P. J. (1976). Cell-free translation of messenger RNA from
oocytes of Xenopus laevis. Devi Biol. 50, 285-301.
DARNELL, J. E., PHILIPSON, L., WALL, R. & ADESNIK, M. (1971). Polyadenylic acid sequences:
role in conversion of nuclear RNA into messenger RNA. Science 11A, 507-510.
DAVIDSON, E. H. (1976). Gene Activity in Early Development. New York: Academic Press.
DOLECKI, G. J. & SMITH, L. D. (1979). Poly(A)+RNA metabolism during oogenesis in
Xenopus laevis. Devi Biol. 69, 217-236.
DUMONT, J. N. (1972). Oogenesis in Xenopus laevis (Daudin) I. Stages of oocyte development in laboratory maintained animals. J. Morph. 136, 153-180.
FICQ, A. (1970). RNA synthesis in early oogenesis of Xenopus laevis. Expl Cell Res. 63,
453^57.
FORD, P. J., MATHIESON, T. & ROSBASH, M. (1977). Very long-lived messenger RNA in
ovaries of Xenopus laevis. Devi Biol. 57, 417-426.
GALL, J. G. & CALLAN, H. G. (1962). 3H-uridine incorporation in lampbrush chromosomes.
Proc. natn. Acad. Sci., U.S.A. 48, 562-570.
JACKLE, H. (1979). Degradation of maternal poly(A)-containing RNA during early embryogenesis of an insect (Smittia spec, Chironomidae, Diptera). Wilhelm Roux1 Arch, devl Biol.
187, 179-193.
JEFFERY, W. R. & CAPCO, D. C. (1978). Differential accumulation and localization of
maternal poly(A)-containing RNA during early development of the ascidian, Styela. Devi
Biol. 67, 152-166.
JELINEK, W., ADESNIK, M., SALNITT, M., SHEINESS, D., WALL, R., MOLLOY, G., PHILIPSON, L.
& DARNELL, J. E. (1973). Further evidence on the nuclear origin and transfer to the cyto-
plasm of polyadenylic acid sequences in mammalian cell RNA. /. mol. Biol. 75, 515-532.
N. A., KAWMEYER, J. F., YOUNG, E. M. & RAFF, R. A. (1978). A test for masked
message: the template activity of messenger ribonucleoprotein particles isolated from sea
urchin eggs. Devi Biol. 63, 279-298.
KAWMEYER, J. F., JENKINS, N. A. & RAFF, R. A. (1978). Messenger ribonucleoprotein
particles in unfertilized sea urchin eggs. Devi Biol. 63, 266-278.
LAMB, M. M. & LAIRD, C. D. (1976). Increase in nuclear poly(A)-containing RNA at
syncitial blastoderm in Drosophila melanogaster embryos. Devi Biol. 52, 31-42.
LEVENSON, R. G. & MARCU, K. B. (1976). On the existence of polyadenyted histone mRNA
in Xenopus laevis oocytes. Cell 9, 311-322.
LOVETT, J. A. & GOLDSTEIN, E. S. (1977). The cytoplasmic distribution and characterization
of poly(A)+RNA in oocytes and embryos of Drosophila. Devi Biol. 61, 70-78.
JENKINS,
140
M. WAKAHARA
J., LEE, S. Y. & BRAWERMAN, G. (1972). Characteristics of the polyadenylic acid
segment associated with messenger ribonucleic acid in mouse sarcoma 180 ascites cells.
Biochemistry 11, 792-798.
+
MILLER, L. (1978). Relative amounts of newly synthesized poly(A) and poly(A)~ messenger
RNA during development of Xenopus laevis. Devi Biol. 64, 118-129.
PEARLMAN, S. & ROSBASH, M. (1978). Analysis of Xenopus laevis ovary and somatic cell
polyadenylated RNA by molecular hybridization. Devi Biol. 63, 197-212.
ROSBASH, M. & FORD, P. J. (1974). Polyadenylic acid-containing RNA in Xenopus laevis
oocytes. J. mol. Biol. 85, 87-101.
RUDERMAN, J. V. & PARDUE, M. L. (1977). Cell-free translation analysis of messenger RNA
in echinoderm and amphibian early development. Devi Biol. 60, 48-68.
MENDECKI,
RYFFEL, G. V., WYLER, T., MUELLENER, D. B. & WEBER, R. (1980). Identification, organiz-
ation and processing intermediates of the putative precursors of Xenopus vitellogenin
messenger RNA. Cell 19, 53-61.
SAGATA, N., SHIOKAWA, K. & YAMANA, K. (1980). Appearance of RNA with a specific size
class of poly(A) in oocytes and eggs in HCG-stimulated Xenopus laevis female. J. exp. Zool.
213, 117-122.
SLATER, I., GILLESPIE, D. & SLATER, D. W. (1973). Cytoplasmic adenylation and processing
of maternal RNA. Proc. natn. Acad. ScL, U.S.A. 70, 406-411.
SPIRIN, A. S. (1966). On 'masked' forms of messenger RNA in early embryogenesis and in
other differentiating system. In Current Topics in Developmental Biology (ed. A. Monroy
& A. A. Moscona), vol. 1, pp. 1-38. New York: Academic Press.
SPIRIN, A. S. (1969). Informosomes. Europ. J. Biochem. 10, 20-35.
8
3
VAN GANSEN, P. & SCHRAM, A. (1974). Incorporation of [ H]uridine and [ H]thymidine
during the phase of nucleolar multiplication in Xenopus laevis oogenesis: a high resolution
autoradiographic study. J. Cell Sci. 14, 85-103.
(Received 6 February 1981, revised 28 May 1981)