EXTRUSION
OF NUCLEOLI
PRONUCLEI
OF THE
DANIEL
FROM
RAT
S Z O L L O S I , Ph.D.
From the Department of Biological Structure, University of Washington, Seattle
ABSTRACT
Electron microscope observations of osmium tetroxide-fixed rat eggs indicate that small
nucleoli are extruded from pronuclei in a sharply demarcated time period after sperm penetration. Approximately 4 ~ hours after sperm penetration, fine fibrous material aggrcgated
in distinct loci along the inner surface of the nuclear envelope and condensed into small,
dense bodics. The term tertiary nucleolus or extrusion body is used to designate the forming bodics. The small tertiary nucleoli form distinct protrusions from the pronuclei during
the following developmental period and finally bud off into the cytoplasm, carrying with
them a small portion of the double nuclear envelope. The extrusion bodies can be observed
only in the vicinity of the pronuclei and have not been seen near the cell membrane. The
fate of the tertiary nucleoli is not known; apparently they transform or disappear after they
have passed into the cytoplasm. Eleven hours after sperm penetration, tertiary nucleoli are
not present near the nuclear membrane and the extrusion activity has apparently ceased.
Large and small nucleoli react similarly to cytochcmical reagcnts: thcy are Feulgen negative;
they are positive to the Millon, Sakaguchi, brom-phenol blue, and PAS reactions. Azure B
stain combined with nuclease extraction indicates the presence of small amounts of RNA
in the nucleoli.
INTRODUCTION
Extrusion of nucleoli from the germinal vesicle as
observed by light microscopy has been described
in different phases of oogenesis for representatives
of every phyla (36). In general, the extruded
nucleolar material has been implicated in controlling yolk formation but the evidence provided
for this hypothesis has not been conclusive. Several
observers have reported nucleolar extrusion also
in mammalian eggs. Nucleoli were reported by
Kremer (28) to be extruded as intact small bodies
from the germinal vesicle of mice and rats, but
other investigators concluded that the nucleolar
material passed across the nuclear membrane in
solution or in an unknown manner (3, 24). In the
mouse and rat, extrusion of nucleoli also occurs
from pronuclei in the form of small spherical
bodies (28, 38, 39). During later embryonic de-
velopment of the rat (2- to 8-cell stages), extrusion
of nucleoli from cleavage nuclei has been reported
(18).
Although some investigators have not observed
the extrusion of nucleoli as a distinct phenomenon,
extrusion of nucleolar material was inferred from
the decrease of staining reaction for nucleolar
material in the nucleus and the concomitant appearance of cytoplasmic granules with similar
staining properties (3).
In several electron microscope studies, fragmentation of the nucleolus was reported and these
fragments subsequently were thought to migrate
across nuclear pores into the cytoplasm (5, 1l, 33).
In a very recent electron microscope study of
oocytes from Thyone briareus, Kessel and Beams
(27) described the morphological changes of the
545
nuclear m e m b r a n e d u r i n g extrusion of small
nucleoli.
Several electron microscopists have also reported
o n nuclear " b l e b b i n g " (17, 23, 26) and actual
emission of large bodies from the nuclei (1, 2).
These processes were interpreted as the microscopically visible elements of nucleocytoplasmic
interactions. O n e report (25) described the extrusion of nuclear material into the perinuclear
space where it b e c a m e m e m b r a n e - b o u n d e d in
a n u n k n o w n m a n n e r a n d was distributed to the
cisternae of the endoplasmic reticulum.
Shortly after formation of pronuclei in m a m m a l i a n eggs, several nucleoli a p p e a r associated
with the chromosomal masses. These nucleoli
aggregate a n d fuse to form gigantic, p r i m a r y
nucleoli. D u r i n g pronuclear growth phase the
so called secondary nucleoli a p p e a r in association
with the nuclear m e m b r a n e . R e c e n t review articles
discuss these events in detail (7, 10, 14).
T h e present study on pronuclei in the r a t egg
provides electron microscope evidence for formation of a third, small set of nucleoli along the
nuclear envelopes and for the mode of their trans-
port into the cytoplasm. These nucleoli can be
defined only by their small size and by the time
of their appearance in relationship to the time of
sperm penetration. Morphologically a n d chemically (as d e t e r m i n e d by cytochemical methods)
they seem to be identical with the p r i m a r y a n d
secondary nucleoli. T h e t e r m tertiary nucleoli or
extrusion bodies will be used to designate these
small nucleoli.
MATERIAL
AND
METHODS
Eggs were flushed from oviducts of rats with Medium
T C 199 (Difco Laboratories, Inc., Detroit) containing 5 or 10 per cent serum between 11 a.m. and 6 p.m.
the day after mating (12, 40, 43). In most cases adult
female rats in estrus were mated with vigorous males
of proven fertility. In some instances ovulation was
induced in 28-day old rats by an intrapcritoneal injection of 30 I U pregnant mare serum (PMS) (Equinox,
A. P. L. Ayerst Laboratories, Inc., New York) followed
48 hours later with a subsequent intraperitoneal injection of 30 I U of chorionic gonadotrophin (HCG)
(A.P.L. Ayerst Laboratories, Inc.). The injections
were administered at 2 p.m. each day. The animals
were paired and checked for sperm the subsequent
morning.
Abbreviations for Figures
oh, chromatin aggregates
my, multivesiculate bodies
pl, perinuclear lacunae
pn, pronucleus
m, mitochondria
l, lysosome-likc bodies
nch, nucleolus-associated chromatin
sn, secondary nucleoli
nm, nuclear membrane
tn, tertiary nucleoli and extrusion bodies
no, primary nucleolus
v, cytoplasmic vesicles
p, nuclear pores
z, zona pellucida
The thin section in Fig. 6 has been stained with saturated aqueous uranyl acetate for 16
hours at room temperature. Sections in the remaining electron micrographs were stained
with Millonig's alkaline lead tartrate.
FIGURE 1 A large pronucleus (pn) approximately 41/~ hours after sperm penetration is
seen. The nucleus is enclosed by a double membrane (nm) demonstrating in places nuclear
pores (p). In certain areas the two components of the nuclear membrane separate, forming
large perinuc]ear lacmlac (pl). Occasionally a vesicle (v) protrudes into these spaces.
Within the pronucleus three large nucleoli (no), chromatin aggregates (ch), and an unidentified structure can be seen (arrow). The cytoplasm contains mitochondria (m), many single
vesicles (v), multivesiculate bodies (my) and irregular structures which may represent
lysosomes (l). X 7800.
FIGURE ~ The two components of the nuclear membrane (nm) have separated along
long distances forming perinuclear lacunae (pl). In places (arrows) the inner nuclear
membrane forms a small vesicle which projects into a lacuna. Fibrous, dense material
accumulates in well defined loci along the inner nuclear membrane and protrudes slightly
towards the cytoplasm. A mitochondrion (m), vesicles (v) and dense granules are seen in
the cytoplasm. The granules are interpreted as a polysaccharide component. M 43,000.
546
THE JOURNAL OF CELL BIOLOGY - VOLUME ~5, 1965
DAI~IEL SzoI~osl Extrusion of Nucleoli from Pronuclei
547
A short region of the fallopian t u b e was straightened o u t o n both ends, u s i n g w a t c h m a k e r forceps. A
b l u n t e d 30 g a u g e h y p o d e r m i c needle was inserted at
the i s t h m u s a n d eggs were flushed out toward the
fimbriated e n d by a gentle s t r e a m of flushing fluid.
Before use, the flushing fluid a n d the receptacles were
w a r m e d to 37°C. O v a were quickly transferred from
the flushing m e d i u m with a n a r r o w bore pipette into
cold 3.3 per cent o s m i u m tetroxide solution, adj u s t e d to p H 7.4 with s-collidine buffer (13) a n d kept
in a n ice b a t h d u r i n g fixation.
After 45 to 60 m i n u t e s ' fixation, the eggs were
w a s h e d in several c h a n g e s of 70 per cent ethanol a n d
were t h e n d e h y d r a t e d by pipetting t h e m t h r o u g h a
series of ethyl alcohol with increasing concentrations.
T h e eggs were t h e n e m b e d d e d in E p o n 812 (31).
T h e total infiltration time was at m i n i m u m 3 to 5
hours. T h e eggs were transferred into the final E p o n
m i x t u r e in a n a l u m i n u m foil boat for flat e m b e d d i n g .
T h e c u r i n g of the epoxy resin took place in three
steps: 12 h o u r s at 37°C, 24 h o u r s at 50°C, a n d 24
h o u r s at 65°C. T h e h a r d e n e d resin was c u t with a
d i a m o n d d e n t a l cutting tool into small blocks w h i c h
were oriented a n d m o u n t e d on m e t a l chucks for
sectioning.
Silver to pale gold sections were cut with a d i a m o n d
knife on a H u x l e y u l t r a m i c r o t o m e a n d the sections
were placed on c a r b o n - c o a t e d copper grids. T o increase contrast of the specimen the grids were stained
either with a s a t u r a t e d a q u e o u s u r a n y l acetate solution for 16 hours at r o o m t e m p e r a t u r e or for 12
m i n u t e s with a n alkaline lead stain (34). Electron
m i c r o g r a p h s were t a k e n in either a modified R C A
E M U 2C or an Siemens Elmiskop I microscope on d u
P o n t O r t h o A lithographic sheet film with C r o n a r
(polyester) base.
T h e localization of nucleic acids was carried out on
eggs fixed in 10 per cent neutral formalin, buffered
with 0.1 M p h o s p h a t e buffer. After w a s h i n g a n d deh y d r a t i n g , t h e eggs were e m b e d d e d either in m e t h a crylate or in paraffin. One-/z sections were c u t f r o m
the m e t h a c r y l a t e blocks with glass knives, while the
p a r a f f i n - e m b e d d e d material was sectioned 4 to 6 /z
thick. T h e preparations were stained, after r e m o v a l
of t h e m e t h a c r y l a t e or paraffin with xylene, with a n
0.5 per cent azure B solution, adjusted to p i t 4.2
with acetate buffer. A l t e r n a t i n g sections were incub a t e d overnight at 37°C with D N a s e (0.2 m g / m l )
solution in 0.033 M Veronal-acetate buffer at p H 7.2
c o n t a i n i n g 0.0025 ~t M g S O 4 ; R N a s e solution (1.0
m g / m l ) in distilled water at 37°C; 4 per cent perchloric acid solution at 4°C. S o m e slides were incub a t e d in both e n z y m e solutions in succession. Control
slides were i n c u b a t e d for the s a m e time periods in
0.0025 ra M g S O 4 solution or distilled water, respectively, at the s a m e t e m p e r a t u r e as the e n z y m e
solutions.
54g
T h e trichloroacetic a c i d - M i l l o n reaction for the
estimation of protein content (35) a n d the S a k a g u c h i
reaction for the presence of arginine (20) were performed. Alkaline fast g r e e n (4) a n d b r o m - p h e n o l
blue reactions (15) were e m p l o y e d for detection of
basic proteins. Localization of D N A was d e t e r m i n e d
by the Feulgen reaction on whole eggs fixed in alcohol vapor a n d posttreated with 95 per cent ethanol
(30). After hydration, the eggs were hydrolyzed in
saturated, a q u e o u s picric acid at 60°C for 1 h o u r (15)
a n d stained with the Feulgen r e a g e n t (29). T h e PAS
reaction was used as a test for polysaccharides with
acetylation or amylase extraction as controls.
T h e time of s p e r m p e n e t r a t i o n in m a m m a l i a n eggs
c a n n o t be d e t e r m i n e d accurately b u t m u s t be est i m a t e d in a separate e x p e r i m e n t for each strain in
the particular e n v i r o n m e n t a l conditions. I n the
Sprague-Dawley rats the majority of the ovulated
eggs were penetrated by s p e r m a t o z o a by 7:30 a.m.;
at earlier times the n u m b e r of eggs penetrated was
lower. T h i s time was taken t h e n as the average time
of s p e r m penetration. T h e experimental animals were
kept on a n a t u r a l d a y - n i g h t cycle. Most experiments
were carried out d u r i n g the m o n t h s of J u l y a n d
August. Sunset was between 6 : 5 0 a n d 8 : 0 0 p.m.
(Pacific s t a n d a r d time) a n d sunrise was between 4 : 0 0
a n d 5:15 a.m. (Pacific s t a n d a r d time). M a t i n g
usually occurred between 11 p.m. a n d m i d n i g h t .
Austin a n d B r a d e n (9), using a different strain of rats,
found the same average time for s p e r m penetration
u n d e r similar e n v i r o n m e n t a l conditions.
Detailed d a t a on the t i m i n g of ovulation i n d u c e d
by P M S a n d H C G t r e a t m e n t in 28-day old rats are
not available. It was f o u n d in this study that ovulation followed 10 to 12 h o u r s after H C G injection,
w h i c h is similar to t h a t in the m o u s e as reported by
E d w a r d s a n d Gates (21). G o n a d o t r o p h i n s were administered so t h a t m a t i n g a n d fertilization coincided
a p p r o x i m a t e l y with the estrus period of the a d u l t
a n i m a l s used in these experiments.
RESULTS
F o u r a n d o n e - h a l f h o u r s after t h e e s t i m a t e d t i m e
of s p e r m p e n e t r a t i o n , t h e p r o n u c l e i w e r e s p h e r i c a l
a n d 8 to 14/~ in d i a m e t e r . T h e y possessed a d o u b l e
nuclear membrane with widely dispersed nuclear
pores (37, 41, 42). I n Fig. 1 t h e f e m a l e p r o n u c l e u s
o f this d e v e l o p m e n t a l period c a n be seen w i t h t h r e e
l a r g e nucleoli. T h e t w o c o m p o n e n t s of t h e n u c l e a r
m e m b r a n e a r e s e p a r a t e d in regions, f o r m i n g l a r g e
perinuclear lacunae. From the inner membrane
s m a l l vesicles p r o t r u d e into this p e r i n u c l e a r space.
After formation of the large secondary nucleoli
along the nuclear membrane, during the pron u c l e a r g r o w t h p h a s e , fine f i b r o u s e l e c t r o n o p a q u e m a t e r i a l a g g r e g a t e d in d i s t i n c t l o c a t i o n s
THE JOUI~NAL OF CELL BIOLOGY • VOLUME 25, 1965
FIGURE 3 Tertiary nucleoli protrude more into the cytoplasm. Both nuclear membranes can be followed along them. An unidentified very dense structure (d) is seen in the pronucleus (pn). Several mitochondrla and a complex multivesiculate body are seen in the cytoplasm. X 36,500.
FIGURE 4 Three extrusion bodies, covered by both nuclear membranes, project clearly into the cytoplasm. A membrane-bounded membranous structure and vesicles are seen in the cytoplasm. X 44,500.
DANIEL SZOLLOSI Extrusion of Nucleoli from Pronucld
549
along the inner surface of the nuclear membrane,
forming clumps. At these locl there are slight
evaginations of the nuclear m e m b r a n e toward the
cytoplasm (Figs. 2, 13). In other eggs of the same
rats or in eggs of different animals at a slightly
later stage, the fibrous material becomes more
compact, forming small dense bodies or tertiary
nucleoli which form distinct protrusions from the
pronucleus. Both nuclear membranes can be
traced along the surface of tertiary nucleoli (Figs.
2 to 4). At a later developmental stage, the fibrous,
tertiary nucleoli have protruded farther into the
cytoplasm (Figs. 5 and 6), and only a narrow
connection with the nucleus is visible. Both nuclear
membranes can be followed around the dense
tertiary nucleoli also at this stage, and in these
regions nuclear pores are lacking. In the cytoplasm, in the proximity of the pronuclei, several
extrusion bodies can be seen which are surrounded
by two membranes (Figs. 6 to 8) and are very
similar to the protruding tertiary nucleoli. Although the final separation was not seen, the
observed sequence indicates strongly that the
tertiary nucleoli are indeed pinched off and released into the cytoplasm.
Frequently, several tertiary nucleoli are observed projecting into a large perinuclear lacuna
(Fig. 7). In these cases the outer nuclear membrane
separates widely from the inner one but the inner
component remains closely applied to the surface
of the tertiary nucleoli.
T h e nuclear surface is quite smooth in eggs
collected at 6 p.m. (Fig. 9). Small tertiary nucleoli
are lacking completely. At this stage components
of the nuclear membrane follow each other closely
but the frequency of nuclear pores is low. Large
secondary nucleoli lose their association with the
nuclear membrane but remain at the periphery of
the pronuclei. The nuclear extrusion activity apparently has ceased by this time.
Two approaches were followed in an attempt
to confirm that a classification of the extrusion
bodies as small nucleoli is justified: (a) morpho-logical comparison of the small dense protruding
bodies with the larger, primary and secondary
nucleoli of the same pronuclci; and (b) a comparison of the cytochemical staining reactions of
extrusion bodies and nucleoli.
In Figs. 10 and 11 it is clearly seen that morphologically the protruding smaller bodies are similar
to the primary and secondary nucleoli. Every one
of these nuclear organelles contains densely packed,
thin, electron-opaque fibrils, although the fibrils
found in the tertiary nucleoli seem to be more
firmly packed. The granular component described
in nucleoli of many animal and plant cells cannot
be found. It is quite difficult to measure the width
of individual fibers with great accuracy, but they
seem to be about 100 A in diameter. These fibrils,
thus, cannot be clearly distinguished from chromosomal fibrils on the basis of morphological criteria
alone. A differentiation of these two components
becomes even more difficult, because the nucleoli
have no membrane surrounding them, and along
their entire periphery small fibers penetrate the
fibrillar nucleolar mass from the adjacent nucleoplasm. The chromosomes are in such a dispersed
state at this time that they cannot be identified
either in the electron micrographs or by light
microscopy. No specific chromosomal region can
thus be related to thc site of nucleolus formation
or of nuclcolar attachment.
The primary and secondary nucleoli and the
small extruding bodies show the same cytochemical
FmURES 5 and 6 At the short arrows in Fig. 5, narrow connections can be observed between the pronucleus and the extrusion bodies or tertiary nucleoli, while in Fig. 6 the
inner nuclear membrane apparently is closed over, entrapping the tertiary nucleoli in the
perinuclear saccules. At the long arrow in Fig. 5, an extrusion body in the cytoplasm is
seen to be bounded by two membranes. The inner membrane is applied closely to the
fibrous, central mass while the outer membrane is separated. In Fig. 6 three tertiary
nucleoli are membrane-bounded and are entrapped in perinuclear space. Note the absence of the small granules in the cytoplasm of Fig. 6. Fig. 5, )< 44,500; Fig. 6, X 40,000.
FIGURE 7 Several extrusion bodies protrude into a perinuclear lacuna. Centrally, a
membrane closely adheres to an extrusion body which is still located in the perinuclear
lacmaa. In the cytoplasm close to the nuclear membrane an extrusion body is seen encircled by two membranes. X 43~000.
550
THE ~OURNAL OF CELL BIOLOGY • VOLUME
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1965
DANIEL SZOLLOSI E~ru~on of Nucleoli from Pronuclei
551
FIGURE 8 Portions of both pronuclei are seen with several tertiary nucleoli located in perinuclear lacunae.
Three extrusion bodies are found in the cytoplasm between the pronuclei. × 18,500.
FmvR~ 9 A pronucleus approximately le hours after the approximated time of sperm penetration.
No tertiary nucleoli nor swollen perinuclear lacunae can be seen in this pronucleus. The large secondary
nucleolus lost its contact with the nuclear membrane. In the cytoplasm several mitochondria (m), vesicles
of different sizes, lysosome-like bodies and a multivesiculate body are found. X 14,000.
552
Ta~ JOURNAL OF CELL BxoLoOY • VOLUME ~5, 1965
FmtraE 10 A secondary (sn) and tertiary nucleolus (tn) are found in the same field. Both are composed
of small fibrils and the only difference seems to be the denser packing of the fibrous contents in the tertiary
nucleolus (tn). X 54,000.
F~GrJRE 11 A portion of the large, centrally located primary nucleolus. The fibrous contents are demonstrated. Two fibrous masses can be seen which probably represent the nucleolus-associated chromatin
(nch). X 56,000.
reactions. They are negative to the Feulgen reaction (Fig. 12) even though they are frequently
surrounded by a more positively reacting chromatin ring. The rest of the pronucleus stains faintly
but uniformly with this reaction. The proteinaceous nature of all nucleoli is demonstrated by the
trichloroacetic acid-Millon reaction (Fig. 13).
The intensity of the color reaction was similar for
the nucleoli and extrusion bodies, whereas the
chromatin stained with a lower intensity. The
Sakaguchi reaction also stained intensely the large
centrally placed nucleoli and secondary nucleoli,
as well as the small extrusion bodies near the
nuclear membrane (Fig. 14). The results with the
brom-phenol blue stain were variable. Most
frequently, only the chromosomal material, corresponding to the Feulgen-positive area, stained
faintly blue, but on occasion the nucleoli stained
intensely instead (Fig. 15). T h e results were con-
sistently negative with the alkaline fast green stain
during the developmental period under investigation.
Results of azure B staining for the demonstration of R N A gave the most consistent and clearest
results of the several basic dyes tried. In 4 /~ paraffin sections of pronuclei the chromosomal
material stained orthochromatically. In order to
achieve better resolution, 1 # sections of methacrylate-embedded eggs were used for the enzyme
extraction procedures. The nucleoli stained distinctly blue in the control preparations (Fig. 16).
Following DNase extraction, the nucleoli stained
similar to the control preparation (Fig. 17), while
after RNase or perchloric acid treatment the
nucleoli lost their stainability (Fig. 18). After a
double extraction with DNase and RNase, only a
metachromatic stain of the extracellular matrix
remained between the follicle cells outside the egg
DANIEL SZOLLOS~ Extrusion of Nucleoli from Pronuclei
553
(Fig. 19). Following formalin fixation of rat eggs,
the time for the enzymatic extraction of D N A a n d
R N A must be extended. After 1 to 2 hours' treatm e n t , the staining intensity decreased, to be sure,
b u t some of the stain was always still observable.
All pronuclear inclusion bodies, b o t h large a n d
small, thus contain a small a m o u n t of RNA.
W i t h the PAS reaction the nucleoli stain uniformly a n d this reaction c a n n o t be abolished b y
acetylation or amylase extraction (Fig. 20). T h e
staining, therefore, is not due to glycogen or to any
other simple polysaccharide. (Table I summarizes
the staining reactions.)
Both lines of evidence, the ultrastructural comparison as well as the similarities of the stain reactions, indicate that the small bodies p r o t r u d i n g
from the pronuclei are similar to the large p r i m a r y
a n d secondary nucleoli a n d t h a t one is justified
in regarding the extrusion bodies as small nucleoli.
Large n u m b e r s of tertiary nucleoli were found
p r o t r u d i n g from pronuclei which h a d reached their
full size. T h e d i a m e t e r of these nucleoli ranged
between 200 a n d 500 m~, b u t in two cases they
were 1 ~ or more. I n one plane of section approximately forty protrusions could be counted in
a single large male pronucleus. T h e approximate
d i a m e t e r of the male a n d female pronuclei at their
m a x i m a l size was 25 a n d 18 ~, respectively. T h e
d u r a t i o n of the nuclear surface activity lasts approximately 6 hours and, although there is no
indication of the d u r a t i o n of the extrusion process
for each individual nucleolus, the n u m b e r of
tertiary nucleoli extruded from b o t h pronuclei
d u r i n g this interval m a y be very high. It is assumed
t h a t the extrusion occurs uniformly over the entire
p r o n u c l e a r surface in the rat, a n d there is evidence
from e x a m i n a t i o n of nearly fully serially sectioned
pronuclei to support this assumption.
DISCUSSION
General Considerations
F r o m light microscope observations of nucleolar
extrusions from the germinal vesicle of different
animals, four different mechanisms have been
postulated for the extrusion process (36):
1. "Diffusion of nucleolar substances t h r o u g h
the nuclear m e m b r a n e in solution . . . . "
2. "Passage of nucleolar products t h r o u g h the
m e m b r a n e in fluid or semifluid state . . . . "
3. " T h e nucleolar extrusions are said to b r e a k
bodily
through
the
nuclear
membrane .... "
4. " F i n a l l y , . . . the nucleolar buds first come
to lie in protrusions of the nuclear m e m brane, after which either the m e m b r a n e
breaks t h r o u g h at these spots a n d the
nucleoli pass into the cytoplasm or the
whole nuclear evagination constricts off
from the nucleus, so t h a t the nucleolar
extrusions in the cytoplasm are at first
still enveloped by the m e m b r a n e , w h i c h
later disappears . . . . "
T h e electron microscopic observations in this
study indicate that, in the case of rat pronuclei,
nucleoli pass into the cytoplasm, being s u r r o u n d e d
b y a double m e m b r a n e which originally was an
integral p a r t of the nuclear envelope. This extrusion thus fails into the fourth category of mechanisms proposed above.
T h e passage of the nucleoli out of the nucleus
as well defined bodies was not observed in living
eggs by light microscopists. T h e a p p e a r a n c e of
structures in the cytoplasm with staining characteristics similar to those of nuclcoli was t a k e n as
sufficient evidence to conclude t h a t nucleoli or
nucleolar fragments were actually extruded. T h e
FmURE 1~ Feulgen stain of a rat egg whole mount preparation. Nucleoli of different
sizes are negative to this dye but they are surrounded by diffusely staining chromatin
within both pronuclei. The head of a supplementary spermatozoon stains intensely. In
this photograph only the large nucleoli can be recognized (arrows).)< 1130.
FIGURE 13 Nucleoli of all sizes stain strongly with the trichloroacetic acid-Millon reaction. The chromatin stains less strongly. X ~150.
FIGURE 14 The large and small nucleoli stain intensely with tile Sakaguchi reaction.
The chromatin of the pronucleus stains less strongly. )< ~150.
FIGURE 15 Brom-phenol blue stain of a rat egg whole mount. Large and small nucleoli
stain intensely with this procedure. × 1130.
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THE JOURNAL OF CELL BZOLOGY • VOLUME
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1965
DANIEL SZOLLOSI Extrusion of Nucleoli from Pronuclel
555
stainability of these structures was usually lost
soon after migration into the cytoplasm.
Kremer (28) was the only investigator who
described previously the bodily extrusion of
nucleoli from the germinal vesicle of m a m m a l i a n
eggs. He is the only investigator, however, who
described by light microscopy the passage of
nucleoli from pronuclei of any species in the form
of such well defined, small chromatic bodies. He
thought that the nuclear membrane breaks at a
point over the protruding nucleoli so that they can
pass into the cytoplasm as a distinct small chromatin granule.
T h e formation of tertiary nucleoli and the extrusion process can be timed exactly in pronuclei
of the rat and can be studied adequately only by
electron microscopy due to their small size. The
first indication pointing to the appearance of
tertiary nucleoli is the slight aggregation of fine
fibriUar components in close apposition to the
internal nuclear membrane. Further compacting
of these fibrils into distinct bodies, their protrusion
into the cytoplasm, and their final pinching off
can be observed in detail. The probable sequence
of events is summarized diagrammatically in
Fig. 21 below. Extruded nucleoli were recognized
only in the vicinity of the pronuclei and were
never observed near the plasma membrane. They
must either disperse rapidly or transform immediately into an unrecognizable form.
The electron microscopic observations do not
allow at this time the drawing of any conclusions
as to the site of origin of the fibrillar components
of the tertiary nucleoli. The dimensions of the
fibrillae are very similar to those of the dispersed
chromosomal fibrils as well as to the components
of the larger nucleoli. Whether this new set of
nucleoli originates at any chromosomal locus or
possibly from one of the secondary nucleoli (or
other alternatives) must remain open at the moment. It should be stated, however, that, in contrast to some invertebrate eggs, fragmentation of
the primary or secondary nucleoli was not observed (5, 11, 33).
The morphological characterization of the
small extrusion bodies as nucleoli by electron
microscopy seems clear cut. The localization of
R N A and basic proteins at the site of tertiary
nucleoli lends further support to this idea. In
case of mammalian eggs, then, the extrusion
bodies can be referred to justifiably as nucleoli.
Cytochemical Characterization of Substances
Extruded from Nuclei
In most studies dealing with nucleolar extrusion,
it is assumed that the nuclear inclusion bodies are
nucleoli. By definition, a nucleolus should contain, among other components, ribonucleic acid.
Cytochemical methods, however, often have failed
to demonstrate clearly an R N A content in nucleoli
of oocytes. This was the case for nucleoli of mammalian pronuclei (8, 16) and the same held true
also for the snail, Helix aspersa (19). Cytochemical
staining characteristics of the so called nucleoli
and of nucleolar extrusion material were not
FIOUUES16 to 19 Sections of rat eggs stained with azure B. Fig. 16, control preparation:
nucleoli of rat egg pronuclei stain strongly blue. The stain over the chromatin material
is nearly imperceptible in a 1 ~ section of formalin-fixed, methac~ylate-embedded eggs.
X 1940.
Fig. 17, DNase treatment: the chromatin of pronuclei does not stain but the nucleoli
still stain with the same intensity. 1/~ section, formalin fixation, methacrylate embedding.
X 1940.
Fig. 18, RNase extraction: the nucleoli have lost their stain but the chromatin stains
similarly to the control preparation. 4 /~ section, formalin fixation, paraffin embedding.
X ~°150.
Fig. 19: After a double enzymatic digestion with DNase and RNase, no stain is observed in developing rat eggs nor in the surrounding follicle cells. The intercellular matrix
between follicle cells stains metachromatically. By phase-contrast microscopy, both
pronuclei could be located (circles with interrupted lines). X 1870.
556
THE JOURNAL OF CELL BIOLOaY - VOLUME 25, 1965
DA~
SZOLLOS: Extrusion of Nucleoli from Pronuclei
557
pronuclear existence from a b o u t 4 a/~ to 11 hours
after the estimated time of sperm penetration. T h e
n u m b e r of extruded nucleoli m a y be very high.
T h e size of the extruded nucleoli varies, but, in
general, they are close to the limit of resolution
ot the light microscope. In K r e m e r ' s (28) drawings, m a n y nucleoli measured between 1 a n d 2 #.
T h e r e is no d o u b t in this writer's opinion t h a t the
nucleolar extrusion described in the c u r r e n t study
is identical with t h a t observed by K r e m e r ; the
size difference m a y be due to different strains of
animals used or to different preparation procedures.
Electron Microscopy of Nucleolar Extrusion
FIGURE ~0 Nucleoli of all sizes and the zona pellueida
stain with the PAS reaction. After acetylation or amylase extraction, the staining characteristics were not
altered. The cytoplasm also stains faintly. X 1940.
T h e r e are only a few studies by electron microscopy which would indicate extrusion of nuclear
materials by b u d d i n g off of m e m b r a n e - b o u n d e d
organelles. D u r i n g oogenesis, nucleolar fragmentation within the nucleus is frequent and, in some
cases, small dense masses a p p e a r in the cytoplasm.
Dense materials on either side of the nuclear
m e m b r a n e were interpreted as evidence for a n
active nucleolar extrusion process (5, l l, 33).
T h e r e is m u c h greater similarity of the nucleolar
extrusion, as observed in this study, to the " h l e b b i n g " of the nuclear m e m b r a n e s of salivary gland
TABLE
I
Summary of Staining Reactions
Azure
Chromatin
Feulgen
Millon
+
+
Sakaguehl
Brom-phenol
blue
+
+
F.G.
Azure
B
RNase
B
Double
enz.
DNase
ext.
PAS
AW
-~-
--
--
--
+
+
+
-
+
+
+
-
+
+
+
occasl.
Primary nucleoli
Secondary nucleoli
Protruding bodies
----
--}-+
++
+ +
+ +
++
+ +
studied in most cases by specific staining techniques. T h e azure B staining experiments with
R N a s e a n d cold percbloric acid extraction in the
present study demonstrate the presence of R N A
in the primary, secondary, a n d tertiary nucleoli.
T h e nuclear surface activity associated with
extrusion of material is sharply d e m a r c a t e d in
time. Its d u r a t i o n occupies the middle period of
558
THE JOURNAL OF CELL BIOLOGy • VOLUME
~5,
+
"
"
cells reported by G a y (23, 26), to the formation of
" h e a v y bodies" in sea u r c h i n eggs (1), a n d to the
interesting i n t r a n u c l e a r a n n u l a t e d vesicles in
Noctiluca described by Afzelius (2) which are app a r e n t l y transported t h r o u g h the nuclear m e m branes. G a y reported t h a t nuclear m e m b r a n e
activity does not seem to be r a n d o m in occurrence
b u t seems to occur near chromosomal bands. T h e
1965
sequence of events observed suggested to her that
the blebs are detached from the nucleus and are
liberated into the cytoplasm. Although in Drosoptn'la, Feulgen-positive material was found in the
blebs while still attached to the nucleus, after their
detachment this was not the case. No definite
statement was made about the chemical nature
of the extruded material but an RNA content can
be inferred from its basophilia and lack of Feulgen
stain.
The formation of the heavy bodies described by
Afzelius (1) in sea urchin eggs is morphologically
similar to the nucleolar extrusion process found in
the pronuclei of the rat. Afzelius demonstrated
that they are basophilic, RNA-containing bodies.
Figs. 7 and 8 in Afzelius' paper represent different
I~Gt~RE ~1 The aggregation and fate of tertiary nueleoli; a diagrammatic representation.
phases of heavy body formation which are very
similar to phases of formation and extrusion of
nucleoli from rat pronuclei but the behavior of
membrane differs in later extrusion stages. Heavy
body formation may be similar in general to
nucleolar extrusion reported in this study but some
of the details seem different. One of the most
significant points of difference is that the annulated membranes surrounding the heavy bodies are
not continuous around the granular matrix.
Recently, nucleolar extrusion has been described
by electron microscopy to occur from oocytes of
Thyone briareus (27). The protrusion phase of the
nucleoli in this egg is very similar to that observed
in rat pronuclei. Kessel and Beams suggested that
the final extrusion in their material occurs by a
focal dissolution of the nuclear membrane over the
extruding portion of the nucleolus and that after
extrusion of the nucleolus the membrane is rapidly
reconstituted. The particles liberated would accordingly not be membrane-bounded. The mere-
brane reconstitution is not documented sufficiently
by electron micrographs. In case of Thyone, apparently the RNA content of the extruded nucleoli
was assumed from the granular appearance of the
nucleolar material, and the scattering of ribosome-like particles in the cytoplasm. The passage
of 150 A diameter nucleolar particles through the
nuclear pores was suggested for Thyone and other
species (6, 7, 27, 33). The granular nature is, however, not convincingly documented in the presented electron micrographs. More recent studies
question whether particles can pass through the
nuclear pores (22). In pronuclei of mammalian
eggs the granular component of nucleoli is lacking; only fibrous elements are consistently found.
There are very few nuclear pores during the extrusion period and, therefore, it is not likely that
this mechanism plays an important role in transport of substances between the nucleus and cytoplasm in the recently penetrated rat egg.
In trophoblast cells of the 6-day old rabbit
embryo (as well as several other cell types), Hadek
and Swift (25) described membrane-bounded
intracisternal bodies which are quite similar to the
extruded nucleoli reported in the present study.
The interpretation offered by these investigators is
somewhat different and involved the extrusion of
nuclear material through "annuli" which penetrate only the inner, but not the outer, nuclear
membrane. The annuli observed in their study
may represent the final stages of the pinching off
process of nucleoli shortly before the membrane
closes over the extruding nucleolus (similar to
Figs. 5, 6, and 7 in this study). In rabbit trophoblast cells, the gradual formation and protrusion
of the nucleoli and the evagination of the nuclear
membrane could not be followed nor timed easily
and, thus, the details may have escaped these
investigators.
Functional Considerations of the Extruded
Nuclear Material
In the literature there are only vague suggestions as to the function of nucleolar extrusion.
Because the extrusion process usually precedes or
coincides with vitellogenesis, the extrusion material
was thought to control yolk formation. The proposed functional schemes, however, could not be
substantiated by subsequent studies. A generalized
statement by Kremer (28) concerning the significance of the extruded nucleoli foreshadows
the importance of the chromatic substances (which
D A m ~ SZOLLOSI Extrusion of Nucleoli from Pronudei
559
we would call R N A today) in the economy of the
egg. He postulated that the periodic accumulation
and dispersion of chromatic substances and their
exchange between nucleus and cytoplasm reflects
the morphological expressions of the metabolic
transformations in the egg.
A similar functional scheme was developed by
Dalcq (18) who suggested that nucleolar materials
pass into the cytoplasm in the form of small
nucleoli and influence the synthesis of mucopolysaccharides and plasmalogenes in the cytoplasm.
After the latter substances reach a certain concentration in the cytoplasm, they become precursors
for D N A synthesis for the daughter nuclei. "Thus,
there would be two linked cycles of major activity;
one directed outward from the nucleus and leading to the manufacture of the precursors, and the
other directed toward the nucleus and leading to
the synthesis of D N A . . . . "
The function of nucleolar extrusion remains
obscure. It is of great interest, however, that R N A
and basic proteins are apparently localized in
them. It should be pointed out in this respect that
only a few ribosomes are present in the cytoplasm
of tubal eggs of several mammals (Szollosi, unpublished studies) although enzyme-extractable
R N A can be demonstrated by cytochemical
methods. Ribosomes are present, however, in
large numbers in trophoblast cells of rabbits (25)
and rats (Szollosi, 1964, unpublished studies). It
has been proposed that ribosomes may either
increase slowly in numbers during t h e successive
cleavage divisions (32) or that they may appear
suddenly in the cells of the morula or blastocyst.
It is tempting to suggest that the extruded
nucleoli mediate the influence or control of the
nucleus over certain cytoplasmic events. The low
R N A content and the high content of basic pro-
teins of the extruded nucleoli indicate that we are
dealing with the extrusion of a large and complex
molecular system. Extruded material from the
pronuclei could be of importance in establishing
the machinery for the future protein synthetic
activity of blastomeres. Genetically, it may be of
importance that both male and female pronuclei
extrude materials simultaneously. Nucleolar extrusion observed in the cleavage stages by light
microscopy (18) indicates further that nucleolar
extrusion is of general and continued importance
to the developing rat embryo.
It is possible that the nucleolar extrusion or the
transfer of nucleolar materials observed from
germinal vesicles and pronuclei is an extreme case
of a phenomenon found generally in cells. T h e
extrusion of particles from pronuclei may be
limited to certain functional periods of cell life in
which large particles are being extruded. In
somatic cells nuclear membrane bubbling occasionally observed may suggest the transport of
nuclear or nucleolar material into the cytoplasm.
T h e fate of the extruded nucleoli has not been
determined as yet either by light or by electron
microscope studies and all suggestions offered are
speculative.
Some aspects of these studies represent part of the
author's doctoral dissertation presented to the Zoology Department of the University of Wisconsin.
Many thanks are due to Dr. Hans Ris for his interest,
supervision and stimulus during my student days.
The technical assistance of Miss Sandra Hastings is
gratefully acknowledged.
Financial support by the Institutional Cancer Re
search Grant and National Institutes of Health Grant
HE-02083 is gratefully acknowledged.
Received for publication, July 28, 1964.
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