Download A storage form of ribosomes in mouse oocytes

Experimental Cell Research 69 (1971) 361-371
A STORAGE FORM OF RIBOSOMES IN MOUSE OOCYTES
G. D. BURKHOLDER, D. E. COMINGS and T. A. OKADA
Department of Medical Genetics, City of Hope Medical Center, Duarte, Calf{. 91 0/0, USA
SUMMARY
By utilizing the technique of whole mount electron microscopy, large numbers of very
ordered lattice-like structures have been observed in mouse oocyte preparations, and min-section
studies have shown that these structures are of cytoplasmic origin. In some preparations, there
appeared to be a breakdown of the lattice with the release of particles having the dimensions of
ribosomes (225 A). This evidence, together with the results of enzyme digestion studies which
show that the lattices are composed of RNA and protein, leads to the conclusion that these
structures are highly ordered aggregates of ribosomes. The ribosomes are strung together to
form chains which are capable of regular cross-linkLrig with adjacent chains to form the charac­
teristic lattice. It is suggested that the ribosomes are held together by basic proteins and are
stored in an inactive form within the lattice until after fertilization, when they will be released
and used in protein synthesis during early cleavage, until the embryo makes its own functional
ribosomes. This premise is discussed in relation to the appearance and disappearance of the
lattice, the synthesis of ribosomal RNA, the existence of ribosome inactivating proteins, and the
possible formation of inactive maternal ribosome-mRNA complexes during oocyte maturation.
The nucleus of maturing mammalian oocytes
has been examined in extensive detail, how­
ever few detailed studies have been conducted
on the specialized features of the cytoplasm.
Thin-section studies [8, 13, 14, 20, 23, 25]
have shown that the cytoplasm of mature
oocytes from myomorph rodents (mouse, rat,
hamster) is packed with highly ordered arrays
of parallel chains, or lattice-like structures.
These specialized structures are only found
in maturing and mature oocytes of this group
of animals, and they completely disappear
from the cytoplasm during the early cleavage
stages after fertilization. They have not been
observed in oocytes of other mammals.
Relatively little is known concerning the
biochemical nature of these structures, and
they have been variously referred to as yolk
material [20], protein strands [9, 13, 22], or
aggregates of ribosomes [14, 25]. The develop­
ment of a technique for bursting and spread-
ing single oocytes on an air-liquid interface
(whole mount preparation) has permitted a
more detailed study to be made of these
peculiar lattices. The results of electron
microscopy and enzyme digestion experiments
suggest that these arrays are
of
ribosomes which are held together by proteins
and are stored in an inactive form in the
lattice until after fertilizai:io1G, when
will
be released and used in protein synthesis
during early cleavage, until the
makes its own functional ribosomes.
MATERIALS AND METHODS
Oocyte preparation
The ovaries were removed from mature female mice,
cleaned of fat and connective tissue, and placed a
depression slide containing oocyte culture medium
[6]. All further operations were performed under a
dissecting microscope with adjustable magnification
(10-40 x ). To release the oocytes, the
was held
follicles
in position with a 25 gauge needle, and
Exptl Ce!l Res 69
362 G. D. Burkholder et al.
were punctured using a second needle. After all the
visible follicles were punctured, repeated jabbing of
the ovary released a few more oocytes. Follicular
cells adhering to the oocytes were removed by gentle
pipetting. Oocytes having a zona pellucida and intact
nucleus were collected using a finely drawn Pasteur
pipette and were washed twice by transferring them
through fresh media.
Whole mount preparations
In order to burst and spread the oocytes on an air­
liquid interface, it was both necessary to remove the
zona pe!lucida and expose the oocytes to a hypotonic
solution. This was accomplished in one step by
transferring the occytes to a solution of 0.5 % pronase
made in distilled water. The oocytes were left in this
solution at room temperature until microscopic
observation revealed that all of the zona pellucida
had been removed and the oocytes appeared some­
what swollen. This usually took 10-15 min.
Some oocytes were spread directly from the pronase
solution, however in most experiments the oocytes
were transferred to NMT (0.12 M NaCl, 0.005 M
MgCl2, 0.01 M Tris buffer pH 7.0) containing 10 %
fetal calf scrum, to avoid prolonged exposure to
pronase. For spreading, a depression slide was filled
with aqueous 10 % sucrose. In some experiments 0.1 M
sucrose was used, but the increased buoyancy of the
10 % solution was preferred. Single oocytes were
drawn, with a small amount of NMT + serum, just
into the tip of a fine pipette. Focusing on the surface
of the sucrose solution with the dissecting microscope,
the tip of the pipette, containing the oocyte, was
carefully touched to the liquid surface, and the oocyte
was gently expelled with a small amount of fluid
onto the sucrose. Extreme care must be taken not
to create bubbles during this step. The oocyte floats
for several seconds and then bursts, thereby releasing
its contents onto the surface of the sucrose solution.
Immediately after bursting, a Formvar- and carbon­
coated 75 mesh specimen grid was touched to the
surface where the oocyte had been. In this manner
each grid picked up only the contents of a sing!�
oocyte. The grids were then stained in 2 % uranyl
acetate for 10 min, dehydrated through a graded
series of ethanol washes, passed through amyl acetate
for 10-20 min, and air-dried. The specimens were
examined in an Hitachi HS-8-1 electron microscope
at 50kV. Some preparations were platinum-carbon
shadowed.
This same procedure was used for rat and Chinese
hamster oocytes.
Thin sectioning
Whole oocytes were fixed in 2 % glutaraldehyde at
4°C for 1 h, rinsed for 30 min in phosphate buffer
pH 7.2, fixed with I % osmic acid for 1-2 h, rinsed for
30 min with Ringer solution, passed through a graded
series of · ethanol washes into propylene oxide and
embedded in a mixture of Epon-Araldite. After
sectioning, specimens were stained with saturated
uranyl acetate in 50 % ethanol, and lead citrate.
Exptl Cell Res 69
Enzyme and chemical treatments
After being picked up from the surface of the sucrose
solution, some of the spread oocyte preparations
were treated by floating the grids on the surface of
the following enzymes or chemicals:
(1) DNase (Worthington) 200 µg/ml in 0.12 M
NaCl, 5 x 10-3 M MgC12, 0.01 M Tris buffer pH 7.0
(NMT) at 37°C for 5-10 min; (2) RNase-A (Sigma)
200 µg/ml and RNase T, (Calbiochem) 200 µg/ml
in 0.12 M NaCl, 0.01 M EDTA, 0.01 M Tris buffer
pH 7.0 (NET) at room temperature for 5 min. Both
ribonucleases were first heated to 80°C for 10 min
to destroy residual DNase; (3) 0.01 % or 0.1 % Trypsin
in NMT at 35 ° C for 1-5 min; (4) 0.2 M HCl, room
temperature for 1-5 min; (5) 8 M urea, room tem­
perature for 1�5 min.
Following these treatments, the grids were processed
as described above.
Some of the oocytes were treated with RNase
(concentrations as in (2) above) prior to spreading.
In these experiments, the oocytes were treated with
pronase, washed in NMT + 10 % fetal calf serum for
15 min, and then transferred to enzyme in NET at
30 °C for 4 h. Control oocytes were exposed to NET
under the same conditions. Following this treatment,
some oocytes were spread on sucrose while others
were prepared for sectioning.
Blastocysts
Mouse blastocysts were kindly provided by Dr John
Melnyk, Department of Biology, City of Hope
Medical Center. Superovulated mice were mated, and
embryos at the 8-cell stage were flushed from the
Fallopian tubes on the third day after mating. These
embryos were cultured under paraffin oil in the
medium described by Mintz [16]. After approximately
3 days, the embryos had progressed to the blastocyst
stage. The blastocysts were transferred to 0.5 %
pronase to remove the zona pellucida, and were then
spread on 10 % sucrose as previously described for
oocytes.
RESULTS
The mouse follicular oocytes used in these
experiments had a clearly recognizable zona
pellucida and intact nucleus. These oocytes
were in the dictyate stage of meiotic prophase
and had not yet begun the first maturation
division.
When the whole mount preparations of
these oocytes were examined with the electron
microscope, very highly ordered lattice-like
structures were commonly observed (fig.
1 (a, b)). These lattices are composed of
individual chains each of which resembles
a series of beads on a string. The diameter
Storage form of ribosomes
363
Fig. 1. Lattice-like configurations in mouse oocytes. (a) Whole mount preparation. x 53 400; (b) whole mount
preparation. x 84 000; (c) cytoplasm of mouse oocyte observed in sectioned material. x 53 400. Note the
similar appearance of the lattice structure in spread (a, b) and sectioned oocytes (c).
364 G. D. Burkholder et al.
pheral region of the cytoplasm tended to be
devoid of lattices.
In order to determine the biochemical
Controia Experimental
nature of these arrays, a series of enzyme
digestions was performed, the results of which
+
+
DNase
+
are shown in table 1 . The DNase and trypsin
RNase
+
Trypsin
digestions were performed on spread material.
Although some RNase digestions were con­
( +) indicates structures are preserved; ( - ) indicates
ducted on spread material, these enzyme
they are destroyed.
a Controls were exposed to buffer solutions without digestions were more easily performed on
enzyme.
intact whole oocytes which were then used
for making whole mount preparations or for
of the beads averaged 212 A while the con­ embedding and sectioning. DNase had no
nections between beads were approx. 1 25 A effect on the lattices, but they were readily
in diameter.
destroyed by either trypsin or RNase. In all
Sometimes the chains existed singly but cases, control preparations were unaffected.
they were often interconnected to one another It may therefore be concluded that these
in a highly regular manner. Crosslinks existed structures are composed of protein and RNA.
between chains with a periodicity of about
Brief exposure to weak trypsin solutions
360 A and were always such that the connec­ (0.01 %, 1-2 min) resulted in a partial dis­
tions occurred between two beads on adjacent solution of the chains. Although the charac­
chains. In this manner, the beads on one teristic beaded appearance was not as obvious
chain were always aligned with corresponding after this treatment (fig. 2(a)), the presence
beads on adjacent chains thereby giving rise of beads could still be demonstrated by
to the lattice configuration. The centre-to­ platinum-carbon shadowing (fig. 2(b)). The
centre distance between neighboring chains interconnections between the beads were not
was found to be somewhat variable, ranging obvious however, suggesting that these con­
from 235-300 A. The number of chains cross­ nections had been removed by the trypsin
linked to form a lattice varied from 2 to 8 and were therefore of a protein nature.
or more. Chain length was also highly variable
HCl treatment (0.2 M) destroyed the lattice
and lattices frequently branched or anasto­ configuration, but ribosomes were morpho­
mosed with one another. Sometimes the logically normal and clusters were common.
lattices appeared to form layers of intercon­ Treatment with 8 M urea completely dis­
rupted both lattices and ribosomes.
nected sheets.
Further evidence concerning the nature of
When oocytes were embedded and section­
ed, identical structures were found in the the lattices was obtained from some of the
cytoplasm (fig. I (c)). Under low magnification, untreated whole mount preparations in which
the lattices appear to be arranged in a rather there appeared to be a breakdown of the
haphazard manner and impart a whorl-like lattice-work. This was probably caused by
appearance on the cytoplasm. Many of the the physical forces occurring at the time of
shorter lattices are curved in a semi-circle bursting and spreading of the oocyte. This
while the longer ones tend to be straight. apparent breakdown was more commonly
In cross-section, they appear as clusters of observed in oocytes spread from NMT + 10 %
granules. As found by others [21], the peri- fetal calf serum than in those spread directly
Table 1 . Effect of enzymes on the lattice-like
conjigurations
Exptl Cell Res 69
Storage form of ribosomes 365
Fig. 2. Ribosomal chains exposed to 0.01 % trypsin for 2 min. (a) Whole mount preparation. Stained with
uranyl acetate. x 44 500; (b) whole mount preparation. Stained with uranyl acetate and platinum shadowed.
x 44 500. The beaded nature of the chains is obvious, but the connections between beads appear to have been
partially removed by the treatment.
from the pronase solution. In such prepara­
tions, many of the lattices had fallen apart
into single chains. In addition, there was
sometimes a breakdown in structure at the
ends of individual chains (fig. 3(a-i)), with
the apparent release of particles having the
dimensions of ribosomes (225 A). This evi­
dence, taken together with the results of the
enzyme digestion studies, suggests that the
lattices are composed of chains of ribosomes,
held together by some proteinaceous material.
Frequently the terminal ribosome appeared
to be firmly attached to the end of the chain
(fig. 3(a-c)), but occasionally, one or two
ribosomes were only connected to the chain
by a thin filament (fig. 3(d g
- )). In other cases,
there appeared to be clusters of ribosomes
in the vicinity of the end of the chain, but
without any apparent connection to the chain
(fig. 3(h, i)). Free ribosomes (fig. 3(h, i)), and
sometimes terminal ones (fig. 3(a g
- )) were
more electron-dense than ribosomes which
were still an integral part of the chain. In
addition, there was a difference in size bet­
ween these ribosomes. Those within the chain
averaged 2 1 0 A in diameter, terminal ribo­
somes were 220 A, and free ribosomes were
about 225 A in diameter.
In small pieces of chain there sometimes
appeared to be a simultaneous separation of
the component ribosomes from one another
(fig. 3(j-l)). It is possible that these ribosomes
could be uniting to form a chain but this
seems unlikely.
In all preparations of mouse oocytes, free
ribosomes were never found scattered at
random but were aiways observed in clusters
(fig. 3(m-p)). Undoubtedly some of these
associations are polyribosornes, however
most of the clusters are probably derived
from broken-down chains. The tendency of
the ribosomes within a cluster to have a
Exptl Ceil Res 69
366 G. D. Burkholder et al.
linear arrangement (fig. 3(n- p)) supports this
idea and suggests that there might be some
remnant of a physical bond remaining bet­
ween them. Occasionally a fine filament
could be seen connecting two of the ribosomes
in such a cluster (fig. 3(m)), but more often,
no connections could be demonstrated, even
after platinum-carbon shadowing.
No lattice-like arrays were observed in
whole mount preparations of mouse blasto­
cysts, however there were a few small single
chains which appeared to be in the process
of breaking down. The predominant char­
acteristic of these preparations was a very
large number of ribosomes. Some of the
ribosomes were attached to membranous
material, probably endoplasmic reticulum,
while others existed in clusters. The majority
however, appeared to be scattered at random
over large areas of the grid. This is in direct
contrast to the findings in mouse oocytes,
where comparatively few ribosomes were
always observed in clusters and were never
found scattered at random.
The existence of lattice-like configurations
in rat and Chinese hamster oocytes was
verified by thin sectioning. Whole mount
preparations of oocytes from these animals
did not reveal any obvious chains or lattice,
but close linear associations of ribosomes
were found in some of these preparations.
DISCUSSION
The application of whole mount electron
microscopy to single mouse oocytes at the
dictyate stage of meiosis has provided a new
means of examining speeific structural fea­
tures of these oocytes which heretofore could
only be studied in thin sections. Utilizing
this technique, large numbers of very highly­
ordered lattice-like structures have been
observed in spread oocyte preparations, and
thin-section studies have shown that these
structures are of cytoplasmic origin (fig. 1).
In favorable preparations, these lattices
appear to give rise to particles the size of
ribosomes (fig. 3), and this evidence, together
with the results of enzyme digestion studies
which show that the lattices are composed
of RNA and protein (table 1 ), leads to the
conclusion that these structures are highly
ordered aggregates of ribosomes. The ribo­
somes are strung together to form chains
which are capable of regular crosslinking
with adjacent chains to form the characteris­
tic lattice. It is suggested that the ribosomes
are combined with, and held together by,
proteins and are stored in an inactive form
in the lattice until after fertilization, when
they will be released and used in protein syn­
thesis during the early cleavage stage until
the embryo makes its own functional ribo­
somes.
The interconnections between the ribo­
somes of a chain are largely removed by brief
exposure to weak trypsin solutions (0.01 %,
1-2 min), indicating that these connections
are proteinaceous (fig. 2). Basic proteins may
be responsible for holding the ribosomes
together since there was a rapid deterioration
of the lattice in 0.2 M HCL The disintegration
of both lattice and free ribosomes during
treatment with 8 M urea suggests that
Fig. 3. Examples of the apparent breakdown of ribosomal chains in untreated whole mount preparations of
mouse oocytes. x 53 400.
(a-c) Terminal ribosomes (arrows) which are larger and more electron-dense than stored ribosomes within
the chain. These terminal ribosomes appear firmly connected to the end of the chain; (d-g) terminal ribosomes
which are connected to the chain by thin filaments. Arrows indicate connecting strands; (h-i) clusters of
ribosomes in the vicinity of the. end of a chain; (j-l) small chains in which there appears to be a simultaneous
separation of the component ribosomes from one another; (m-p) clusters of ribosomes. Arrow in (m) indicates
connecting filament between two ribosomes.
Exptl Cell Res 69
Storage form of ribosomes 367
Exptl Cell Res 69
368 G. D. Burkholder et al.
hydrogen bonds and/or hydrophobic bonds
play a significant role in both lattice and
ribosome structure.
The breakdown of the lattice and concomi­
tant release of ribosomes in the whole mount
preparations is probably caused by the
physical forces occurring at the time of
bursting and spreading of the oocyte, how­
ever it could also be a result of the release
of intracellular proteases during the prepa­
ration. In vivo, the release of ribosomes from
the lattice after fertilization is probably due
to the controlled activity of a specific
protease.
Free ribosomes and often terminal ribo­
somes were found to be more electron-dense
and somewhat larger than stored ribosomes
within a chain (fig. 3(a-i)). This suggests that
the association of ribosomes with lattice
proteins decreases the electron density of the
ribosomes and perhaps alters their configu­
ration. The ribosomes must become either
more compact or else slightly compressed
perpendicular to the long axis of the chains
to account for their smaller size within a
chain or lattice. During release from the
lattice, the original configuration and size
is restored as indicated by the fact that
terminal ribosomes were often intermediate
in size between stored and free ribosomes.
Large terminal ribosomes had the same
electron density as free ribosomes.
Lattice-like arrays are not restricted to the
mouse. Similar structures have also been
found in thin-sectioned oocytes of the rat
and Golden hamster f8, 1 3, 14, 20, 23]. They
have not been observed in other mammals
[23]. Morphological differences in the arrays
have been observed between the mouse,
hamster, and rat [9, 23]. In the mouse there
are many cross-linked chains forming a
typical lattice configuration, while in the
rat only single chains exist, and in the hamster
double chains are found resembling a ladderExptl Cell Res 69
like structure. These single and double chains
often form part of large parallel arrays.
Whole mount preparations of rat or Chinese
hamster oocytes did not reveal any obvious
chains or lattices, however close linear asso­
ciations of ribosomes were found in favour­
able spreads. The failure to find intact
chains in spread preparations of rat or
hamster oocytes may reflect a difference in
their stability between species.
Assuming that the inactivation and storage
of ribosomes in oocytes is a characteristic
phenomenon of many diverse animal species,
a more widespread distribution of lattice­
like arrays might be expected. It is quite
possible however, that ribosomes could be
stored in oocyte cytoplasm without the for­
mation of the elaborate configurations ob­
served in this study. Rabbit oocytes, which
do not exhibit lattice formations, have many
free ribosomes arranged in rosette-like clusters
in the cytoplasm during maturation [26].
Whether these ribosomes are active or stored
is not known. In comparison, free ribosomes
or polysomes are not commonly observed in
mature oocytes [2 1] or fertilized ova [19] of
the rodents with lattice configurations. Pre­
sumably the bulk of the ribosomes in these
oocytes are an integral part of the lattice
structure. Obviously more work is required
to determine if there is any morphological
or biochemical evidence for ribosome storage
in other species.
It has been suggested that the arrays are
primarily proteinaceous in nature [9, 22].
Evidence for this has largely been obtained by
testing the effect of various fixatives on these
structures. Glutaraldehyde, which is consi­
dered to be a protein fixative, was found to
preserve the lattice, however osmium tetro­
xide, another protein fixative, was not a good
preservative. The solubility of the lattice
structure in permanganate fixative led Enders
& Schlafke [9] to conclude that it was com-
Storage
posed of protein strands. They considered
permanganate to be a poor protein preserva­
tive, but according to Glauert [1 1], perman­
ganate preferentially destroys cytoplasmic
components having a high RNA content. Inter­
pretations of chemical composition based on
such studies must be considered tentative
at best, and in any case, do not seriously
disagree with the findings of the present
work in which the lattices are shown to b e
composed o f both protein and RNA.
It has been suggested [19] that the weak
fluorescence in oocyte cytoplasm following
acridine orange staining [2], and the weak
ultraviolet absorption in this region [ l ] makes
it unlikely that large quantities of RNA could
be stored in the cytoplasm. Actually, signifi­
cant fluorescence and UV absorption were
associated with granular elements but it
was thought that this was due largely to the
presence of mononucleotides [2]. These results
are not easily reconciled with the present
results. One possibility however, is that the
association of the lattice protein with the
ribosome alters the configuration of the
ribosome such that the RNA is masked so
that it can n o longer be detected using these
techniques.
Mazanek [ 1 4} initially suggested that
the lattice-like configurations found in rat
oocytes, and cells of the early cleavage divi­
sions, might be aggregates of linearly­
arranged ribosomes. Zamboni [25] has indi­
cated that the lattices are formed from poly­
somes in mouse oocytes. He found that the
ribosomes became aligned in a curvilinear
pattern and then fused to form chains and
he concluded that the resulting structures
were "lattices of fibrillar RNA". In rat and
Golden hamster oocytes, Weakley [23] found
a close association between "lamellae" (lat­
tices) and large ribosomes.
The lattices first appear during oocyte
maturation. In a study of oocyte development
of ribosomes 369
in the Golden hamster, Weakley
found
no such structures in
follicles sur­
rounded by a single layer of flattened
granulosa ceHs. They first appeared as single
chains i n the inner portion of the cytoi,1asm
cuboida1
when the oocyte was surrounded
granulosa cells. Mature oocytes contained
many stacks of double chains . .r r,ee 1rmos,Jm: es.
or dusters of ribosomes, were quite abundant
in immature oocytes, but were uncomrnon
after maturation, although some appe,art::a
be e mbedded i n an amorphous matrix.
Similar findings have been obtained
in the mouse, where the lattices
Zamboni
increase in number during oocyte maturation
and are abundant i n preovulatory
and tubal ova.
If the lattices are composed
ribosomes to be used for protein <svr1th,�<:1 ,
during the early cleavage stages after ferti­
lization, the lattices should disappear
early development
with a concomitant
appearance of free ribosomes and po1ysorr1es.
No lattice-like arrays were found in blasto­
cyst preparations, but there were
numbers of ribosomes, existing either
in clusters, or attached to membranous mate­
contrast
rial. These
with
those from mouse oocytes, where arrays were
in abundance and there were
few
ribosomes. Corroborating evidence has been
m the
obtained by Schlafke & Enders
rat. They found densely packed
arrays of chains in the fertilized ovum,
no granular endoplasmic reticulum nor any
free ribosomes. A few small clusters of ribo­
somes appeared at the 2-4 cell stage.
8-cell stage, some ribosomes were attached to
endoplasmic reticulum and a few were pn:seiu
on the outer nuclear membrane.
no noticeable disappearance of the arrays
had yet been observed, this would not be
clearly apparent at first because of the
numbers initially present. In the cells
Exptl Cell Res 69
370
G. D. Burkholder et al.
blastocyst however, the arrays had started to maternal ribosomes would become available
disappear and had become more disorganized. for protein synthesis.
The association of protein with ribosomes
At this time there was considerable granular
endoplasmic reticulum and "clusters of poly­ in the lattice configuration very likely renders
ribosomes filled much of the background the ribosomes inactive in protein synthesis.
cytoplasm". The arrays had largely disap­ The existence of ribosome inactivating pro­
peared by the time of implantation [10]. The teins has previously been demonstrated i n
disappearance of arrays and correlated in­ sea urchin eggs. Monroy e t al. [181 found
crease in number of ribosomes or polysomes that ribosomes from unfertilized eggs are
during early development would be expected inactive as sites for protein synthesis in in
if, as the present study suggests, the arrays vitro systems, however they could be activated
by prior exposure to trypsin. This suggests
are a storage form of ribosomes.
The present findings also correlate well that the ribosomes of unfertilized eggs are
with what is known about the synthesis of i nactivated by a protein coat. Some doubts
ribosomal RNA (rRNA) during oogenesis have been expressed (see [4, 1 2] for reviews)
and during the early cleavage stages. Unfor­ concerning these results because untreated
tunately , practically nothing is known about ribosomes from unfertilized eggs are actually
rRNA synthesis in mammalian oocytes, how­ capable of synthesizing proteins using arti­
ever in Xenopus [5], there is a tremendous ficial mRNA, and are therefore not inactive,
build up of rRNA during the diplotene lamp­ at least in systems using the synthetic tem­
brush stage of meiosis. This RNA is retained plates. It was suggested that the maternal
by the oocyte for several months. After mRNAs, rather than the ribosomes, were
fertilization, rRNA synthesis first begins inactivated by proteins. Recently however,
during the 4-cell stage in the mouse [24), and Metafora et al. [15] have clearly shown that
undergoes a tremendous increase after the the ribosomes from unfertilized sea urchin
8-cell stage [7], however these changes may eggs are less efficient than those from ferti­
also reflect an increase in the uptake of RNA lized eggs in supporting polypeptide synthesis
precursors into the cells at this time [3]. Mintz using synthetic mRNA. Furthermore, they
[17] has shown that protein synthesis occurs have obtained a protein factor from the
in the mouse embryo shortly after fertili­ ribosomes of unfertilized eggs which is
zation, i.e. before rRNA synthesis begins, capable of inhibiting polypeptide synthesis i n
and this suggests that maternal ribosomes are a poly(U)-directed cell-free system containing
functional during these early stages of devel­ active ribosomes from fertilized eggs. This
opment. Although rRNA is synthesized protein inhibits the binding of mRNA and
quite early during embryogenesis, this does aminoacyl tRNA to the ribosomes but whe­
not, in itself, indicate that functioning ribo­ ther it affects the binding sites for these
somes are being produced. Little is known molecules or alters the configuration of the
concerning the synthesis of ribosomal pro­ ribosome as a whole is not known. It is
teins during preimplantation nor how quickly obviously necessary to exercise caution i n
new ribosomes are formed or are capable of extrapolating from sea urchins t o mice, but
functioning in protein synthesis. Fully func­ this example does set a precedent for the
tional ribosomes may not be produced for existence of ribosome inactivating proteins
some time after rRNA synthesis has been in oocytes.
initiated and in the meantime the stored
One intriguing question raised by the
Exptl Cell Res 69
Storage
of rib,,so111es
present work concerns the possible association lattice structures from the oocytes, biochemical
mRNA prior to their assembly into lattices. tion on the RNA
studies using diverse animal species
(see [4,
for review) have shown that stable
maternal mRNA is stored in the egg for
use after fertilization. These maternal messen�
gers are prevented from participating in
protein synthesis until after fertilization, i.e.
the message is "masked". It is generally
believed that this masking is effected by the
interaction of protein with mRNA, however
there is some debate as to the site of template
storage. In this regard, evidence obtained
from sea urchin eggs [18] suggests that during
oogenesis, a ribosome-mRNA complex is
formed which is subsequently inactivated by
a protein coat. It was postulated that, after
fertilization, proteases were released which
removed the protein coat and thereby ren­
dered the ribosome-mRNA complex active
in protein synthesis. Other evidence (reviewed
in [4, 12]) suggests that cytoplasmic particles
other than ribosomes may be the site of
maternal template inactivation. Neither of
these two alternatives has been established
definitively but it appears reasonable that
messenger inactivation might occur at both
ribosomal and non-ribosomal sites. Should
this prove to be the case, there is a distinct
possibility that maternal mRNA might also
be associated with the ribosomes stored in
the lattice configuration of mouse oocytes.
When the individual ribosomes are released
from the lattice after fertilization, the asso­
ciated messenger would be immediately
available for translation.
The present results, which rely heavily on the
interpretation of morphological data, suggest
that a biochemical approach might be very
informative. If it proves possible to isolate the
This work was supported a
ship from the Medical Research Council of Canada
to G. D. B., and by NIH grant GM-15886.
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Received June 21 , 1971