ELECTRON
MICROSCOPE
IN SEA URCHIN
PATRICIA
STUDY
OF MITOSIS
BLASTOMERES
HARRIS
From the Department of Zoology, University of California, Berkeley
ABSTRACT
The fine structure of cells at different stages of the mitotic cycle was studied in the blastomeres of 6-hour-old embryos of the sea urchin Strongylocentrotus purpuratus. The material
was fixed in 1 per cent osmium tetroxide in sea water, buffered with veronal-acetate to
p H 7.5, embedded in Araldite, and sectioned with glass knives. The aster, as it forms around
the centriole, has the appearance of the endoplasmic reticulum, with elements oriented
radially from the centrosphere to the periphery of the cell. Anaphase structures described
include the kinetochores, with bundles of fine filaments extending toward the centrioles,
as well as continuous filaments passing between the chromosomes. Two cylindrical centrioles
composed of parallel rods are present in each of the anaphase asters. At late anaphase,
elements of the endoplasmic reticulum condense on the surface of the chromosomes to
form a double membrane which already at this stage possesses pores or annuli. At telophase bundles of continuous filaments can be seen in the interzonal region. 3-hese filaments,
as well as those associated with the chromosomes, have a diameter of approximately 15 m~t,
and appear physically different from the astral structure.
The literature on mitosis, covering the span of
years since the development of adequate histological techniques for electron microscopy, includes
among the early work many studies which suffer
from the use of fixation methods borrowed from
light microscopy. More recently, with the use of
better techniques, the increasing number of
papers dealing with limited aspects of mitotic
structures in a large variety of materials has
contributed greatly to the general picture of cell
division. Thus, the morphology of the centriole
has been described (Bernhard and de Harven,
1956; de Harven and Bernhard, 1956) and confirmed by other workers (Bessis et al., 1958, and
others) in various materials, and deviant forms
have been described by Amano (1957) and De
Robertis and Franco Raffo (1957). M a n y studies
have covered the structure of the nuclear membrane both in non-dividing cells and during the
mitotic cycle, and have been reviewed by Watson
(1955), Bernhard (1958), and Whaley et al. (1960).
The relationship of the endoplasmic reticulum to
division structures in onion root tip was studied by
Porter and Machado (1960), and lamellar systems
were especially considered by Ito (1960) in his
work on Drosophila spermatogenesis. Fibrous
elements have been described by several authors
(Porter, 1954; Bernhard and de Harven, 1958;
and others), and R u t h m a n n (1958) demonstrated
the existence not only of what he termed tubular
filaments, but of oriented elements of the endoplasmic reticulum as well, in the meiotic spindle
of the crayfish.
Studies by light microscopy, polarization
microscopy, interference microscopy, microscopic
cytochemistry, isolation and chemical analysis of
the mitotic apparatus, and physiological experimentation furnish a background for the interpretation of electron microscope findings from
which such interpretation cannot stray very far.
419
For example, studies with polarized light such as
those of Inoufi (1953) and observations on isolated
mitotic apparatus support the "existence" of the
classical "fibers" in the spindle without, however,
making any statement concerning the actual fine
structure. The question as to whether these fibers
are solid linear units, an oriented network of
linear units, tubular, lamellar, or the like, remains
in the d o m a i n of the electron microscopist.
Similarly, the available accurate measurements on
cells in anaphase indicate that in most forms
studied there are two components of movement, a
shortening of the chromosome to pole distance
and a lengthening of the pole to pole distance,
without specifying that any class of real fibers
actually lengthens or shortens. Studies on the
staining characteristics of the centrioles and their
behavior during the division cycle indicate that
these bodies duplicate themselves and somehow
move apart, but give no details as to the mode of
duplication or mechanism of movement. O t h e r
studies have made use of mitotic poisons or
blocking agents to dissociate the various processes
involved in cell division, but the morphological
changes related to the physiological effects have
yet to be revealed. The present study of fine
structure changes during the mitotic cycle in sea
urchin blastomeres was initiated as part of a study
to correlate morphology with the physiological
results of mercaptoethanol blocking experiments
recently conducted in this laboratory (Mazia
et al., 1960).
MATERIALS
AND
METHODS
In the previous work on mercaptoethanol blockage,
a timetable was worked out for the mitotic stages of
the first and second divisions of fertilizcd sea urchin
eggs by fixing samFlcs in Carnoy's fixative at 5 to l0
minute intervals and making light microscope
observations. Since the timetable was already
available for these divisions, a preliminary study was
made here on the first division by taking corresponding time samples for electron microscopy. The
results of this study indicated that the first division
has some rather special features as a result of the
fertilization process, pronuclear fusion, etc., details of
which are now being investigated in this laboratory.
Hence, the choice of material for this study was the
6-hour-old embryo (16 to 32 cells), a stage where
division is proceeding very rapidly and the cells are
smaller and no longer in synchrony, so that a single
batch of embryos would include all division stages.
These stages could then be easily identified by
420
comparing them with the known first division stages
already determined.
The eggs of the sea urchin Strongylocentrotus purpuratus were collected by injecting the urchins with
0.5 M KC1 and allowing them to shed into sea water
at 15°C, their normal environmental temperature.
After washing by settling and decanting, the eggs
were fertilized by the addition of dilute sperm. For
purposes of better fixation and infiltration of embedding material, the fertilization membranes were
removed, using the technique generally followed in
this laboratory for obtaining the isolated mitotic
apparatus. At the time the membrane first appeared,
mercaptoethylgluconamide solution was added to
bring the final concentration in the egg suspension to
about 10 ~ M. The effect of this substance is to
prevent the hardening of the fertilization membrane,
which as a result becomes extremely thin and greatly
distended. The membranes were subsequently
removed by passing the eggs once or twice through
25 mesh silk screen. They were then returned to
fresh sea water at 15°C to develop, and were constantly stirred for adequate aeration. After 6 hours
lhe embryos were collected and fixed in 1 per cent
osmium tetroxide dissolved in sea water and buffered
with acetate-veronal buffer at pH 7.5. This fixative
was made up by using 5 parts of 2 per cent OsO4 in
sea water, 3 parts of sea water, and 2 parts of acetateveronal buffer stock made up in distilled water. This
mixture was then brought to pH 7.5 by the dropwise
addition of concentrated HC1. Fixation was carried
out for 4 hours at room temperature.
The embryos were then dehydrated in graded
alcohols and flat embedded in Araldite epoxy resin
(Glauert and Glauert, 1958; supplied in the United
States by Cargille Sons, Little Falls, New Jersey).
The choice of an epoxy embedding material was
necessitated by the uncertainty of polymerization
damage in methacrylate embedding, but difficulties
arose with the use of such a highly viscous material
with a finely particulate suspension of marine eggs or
embryos. A technique, based on suggestions by
Dr. W. E. Berg of this department, was finally
developed for flat embedding this material. It is
repeatable and provides blocks which section very
easily with glass and with diamond knives. Following
the final change of absolute ethanol in the dehydration procedure, the embryos were put into a mixture
of approximately 3 parts absolute ethanol to 1 part
of Araldite accelerator for 10 to 15 minutes. The
concentrated embryos were then pipetted into
small shallow polyethylene containers and excess
liquid was drawn off with bibulous paper until they
were almost, but not quite, dry. They were then
covered with the final Araldite embedding mixture,
made up in batches using the following quantities:
3 ml resin, 3.2 ml hardener, 0.2 ml accelerator. The
THE JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY • VOLUME 11, 1961
embryos were then stirred well into the Araldite
mixture, either covered tightly or put into a desiccator
for infiltration overnight (or about 15 hours), and
then put into the oven at 58-60°C for hardening.
During this procedure the specimens sink to the
bottom of the container, a distance of less than 1~
inch. The hardened sheet can then be examined
under the dissecting microscope, and desirable
embryos cut out and mounted on Araldite blocks
made by casting the surplus embedding resin in 00
gelatine capsules.
Sectioning was done on a Porter-Blum microtome
with Pyrex glass knives, and examined without
staining, with an RCA EMU-3F electron microscope.
OBSERVATIONS
Interphase
I n the rapidly dividing cells of the 6 hour
embryo the nucleus rarely attains a completely
rounded form in interphase, but remains irregular
in shape. T h e nuclear envelope, a double m e m brane structure characterized by regularly spaced
pores or annuli, surrounds a generally homogeneous nucleus containing a n u m b e r of dense
ovoid bodies of unknown significance. T h e cytoplasm at this stage of development is still filled with
Key to Abbreviations
a, aster
ar, astral ray
c, centriole
oh, chromosome
of, chromosome fiber
k, kinetochore
m, mitochondrion
n, nucleus
ne, nuclear envelope
p, pore or annulus
sf, spindle fiber
y, yolk
FIGURE 1
Part of a cell in interphase, showing a centriole in close proximity to the nucleus. Note fibers (between
arrows) diverging from the centrlole. X 28,000.
P. HAa~t~s Mitosis in Sea Urchin Blastomeres
421
yolk granules and lipid droplets. Cytoplasmic
membranes are for the most part confined to
specific regions which have previously been
described as Golgi elements (Afzelius, 1956).
Centrioles can be seen at this stage lying in the
vicinity of the nucleus, usually in a region devoid
of other cytoplasmic structures. In structure the
centrioles, similar to those described by Bernhard
and de Harven (1956), are short cylinders with a
diameter of about 0.15 micron and a length of
about 0.33 microns, made up of a circular array of
approximately nine rods or tubules. Often associated with them are fine fibers, about 150 A in
width, a p p e a r i n g - - i n favorable sections--as a
pair of parallel osmiophilic lines. Fig. 1 shows an
interphase centriole cut in cross-section, situated
near the nucleus, with some of the fine fibers
radiating from it.
Aster Formation
At a slightly later stage when the nuclear membrane is still intact and before there is any visible
condensation of chromosomal material in the
nucleus, asters begin to form, comprising a great
number of profiles of tubules and vesicles, possibly
connected, to form a dense endoplasmic reticulum
around the centrioles. These areas appear at
separated points along the periphery of the
nucleus and are devoid of large cytoplasmic
particles, but extending from them are finger-like
projections of the endoplasmic reticulum which
find their way around and between the yolk,
mitochondria, and lipid to reach the outer membrane of the cell. Fig. 2 shows this early stage of
aster formation.
As these astral areas grow, the chromosomes
condense and the nuclear membrane breaks up
into small fragments, very much as described by
Moses (1958).
spindle region, elongated vesicles or lamellae can
be seen extending between the asters and may be,
in part, remnants of the nuclear membrane.
Elements of the reticulum penetrate the spindle
region from the asters, and already at early
anaphase they appear between the chromosomes
and begin to condense on their surfaces, especially
those surfaces closest to the aster toward which the
chromosome is moving. Fig. 3 a shows an early
anaphase stage, the chromosomes appearing less
dense than their surroundings. Osmiophilic
bodies, presumably the kinetochores, can be seen
on the leading edge of the chromosomes, and a
number of straight fibers, about 150 A in diameter,
can be seen radiating from them in the direction
of the asters (cf. Fig. 3 b). These fibers look very
similar to those radiating from the interphase
centriole, and continuous fibers of the same
appearance can be seen between the chromosomes,
apparently extending from pole to pole of the
spindles.
As the chromosomes approach the aster, elements of the reticulum oriented toward the center
of the aster become very much compacted at the
polar ends, while in their wake the center of the
spindle remains devoid of large cytoplasmic
particles and elements of the reticulum, but has a
network of fine fibers oriented parallel to the
spindle axis. In the center of the aster are two
cylindrical centrioles similar to those described by
Bernhard and de Harven, oriented at right angles
to each other, and already at this time beginning
to separate (see Fig. 4). Within the mass of
oriented vesicles of the endoplasmic reticulum can
be found bundles of straight fibers extending from
the chromosomes toward the center of the asters,
but no visible connection to the centrioles is
apparent in these sections (Fig. 5). The individual
centrioles in the aster are sometimes seen surrounded by small dots or clumps of material of
the same density as the centrioles themselves.
Metaphase and Anaphase
At metaphase the chromosomes have assumed
positions at the equator of the spindle, midway
between the two asters. At the periphery of the
Telophase
In late anaphase and telophase, pores are
already present in the condensing membranes
FIGURE
Beginning of aster formation. A condensation of membranous elements forms around
the centriolc, excluding yolk, mitochondria, and other large particles from the astral
region. >( 20,000.
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1961
P. H•Rms
Mitosis in Sea Urchin Blastomeres
423
a r o u n d the vesicular chromosomes (Fig. 6 a).
W h e n the vesicles are cut tangentially, the pores
can be distinguished by the dense annuli, which
h a v e a n outer d i a m e t e r of a b o u t 90 to 100 m #
a n d a n inner d i a m e t e r of a b o u t 50 m#, a n d are
spaced more or less evenly with a n average distance
from center to center of a b o u t 150 m # (Fig. 6 b).
These measurements are in close a g r e e m e n t with
those found by Afzelius (1955) in the nuclear
m e m b r a n e of the sea u r c h i n oocyte.
As the chromosomes come together and fuse to
re-form the nucleus, sections of m e m b r a n e are
often t r a p p e d within the nucleus. Also at this
stage fibers, presumably continuous, c a n be seen
in the spindle region between the two sets of
chromosomes, a n d these fibers are straight, often
occurring in bundles. These can be seen in Fig.
7 a; Fig. 7 b shows the fibers in a n o t h e r spindle at
a higher magnification. Continuous fibers remaining in the spindle region after the chromosomes
h a v e separated are finally gathered in by the
encroaching furrow a n d concentrated at the last
physical connection between the two d a u g h t e r
cells. These r e m n a n t s often r e m a i n long into the
next division cycle.
DISCUSSION
Interphase
I n the sections so far examined, the centriole at
interphase appears as a single structure. T h e fact
t h a t only single structures have been seen, however, does not preclude the possibility t h a t a
double structure m a y exist. F r o m late a n a p h a s e or
telophase of the previous division until the nuclei
h a v e reconstituted following division, the centrioles
are generally associated with clumps of g r a n u l a r
dense material. Since this time period is essentially
the same as t h a t described by M a z i a et al. (1960)
as the period wherein the functional duplication
of the centriole takes place, it is t e m p t i n g to
surmise, as B e r n h a r d a n d de H a r v e n (1958) h a v e
done in regard to "satellite" or pericentriolar
structures, t h a t this picture is related to the
duplication of the centrioles.
Aster Formation
T h e earliest indication of the initiation of
mitosis is the formation of a sphere of dense
reticulum a r o u n d the centrioles, which are located
at more or less widely separated points on the
periphery of the nucleus. Two previous workers
have described the relationship of the endoplasmic
reticulum to the division structures. Ito (1960), in
his study of lamellar systems in Drosophila meiosis,
found t h a t the central region of the aster is m a d e
u p of t u b u l a r or canalicular elements, while the
outer astral region is formed of radiating m e m branous lamellae. F r o m his studies of different
stages in the d e v e l o p m e n t of this system, he
concluded that the endoplasmic reticulum contributed to the formation of the parafusorial
lamellae which s u r r o u n d the spindle, a n d also to
the astral structure, while the dictyosomes or
Golgi complex contributed mainly to the formation of the asters. H e also found no basophilia
associated with the division structures. R e b h u n
(1960), in a study of aster-associated particles in
m a r i n e invertebrate eggs, d e m o n s t r a t e d the
presence of oriented t u b u l a r elements of the
endoplasmic reticulum in the aster of dividing
Spisula eggs. I n the centrifuged eggs at first
cleavage, the asters r e m a i n e d in the clear zone
away from particulate material, a n d the rays
could be plainly distinguished as greatly elongated
vesicles. In the present work on sea urchin
blastomeres, finger-like projections from the
FIGURE 3
a. Low magnification picture showing early anaphase, with the two sets of chromosomes
at an early stage of motion toward their respective poles. The chromosomes in these
preparations appear less dense than the surrounding spindle material. Note oriented
endoplasmic reticulum at the periphery of the spindle (arrow), which appears to be
continuous from pole to pole, and the vesicular elements of the reticulum in the asters
themselves. M 9400.
b. Enlarged portion of a, showing kinetochore with chromosomal fibers (cf, arrows) extending in the direction of the aster. Continuous spindle fibers (sf, arrow) which appear
to run from pole to pole can be seen between the chromosomes. X 31,000.
424
T H E J O U R N A L OF BIOPHYSICAL A N D B I O e H E ~ C A L C Y T O L O G Y • V O L U M E 11, 1961
compacted reticulum of the aster centers, very
similar in appearance to those shown by R e b h u n
in SpisuIa, are seen to wind outward from the
center of the aster toward the cell surface.
T h e b r e a k d o w n of the nuclear m e m b r a n e at
late prophase in the sea u r c h i n blastomere is not
essentially unlike that described in other forms,
a l t h o u g h there is no a p p a r e n t close relationship
between the m e m b r a n e a n d m i t o c h o n d r i a as
found by Barer et al. (1958), nor on the other h a n d
any relation between the m e m b r a n e and attached
chromosomes as described by Moses (1958).
Metaphase, Anaphase, and Telophase
T w o of the structural aspects of these division
stages, other t h a n the presence of oriented endoplasmic reticulum in the aster regions, are of
interest here. T h e r e is a condensation of vesicles
of the endoplasmic reticulum onto the surfaces of
the a n a p h a s e chromosomes, forming the earliest
nuclear envelope (or m e m b r a n e ) , and already at
this very early stage of nuclear re-formation this
m e m b r a n e has a porous structure similar to t h a t
of the nuclear m e m b r a n e of the resting stage. It is
also at this stage t h a t the fibrous elements of the
mitotic a p p a r a t u s become most easy to d e m o n strate. Fine straight fibers, usually in bundles,
extend from pole to pole, while other bundles of
fibers connect the chromosomes with the poles.
Attempts to demonstrate the fibrous structure of
the mitotic a p p a r a t u s h a v e previously met with
varying success, although within the past several
years a n u m b e r of investigators have reported fine
straight fibers similar to those described here
( R u t h m a n n , 1958; B e r n h a r d a n d de Harven,
1958; R o t h et al., 1960; Yasuzumi et al., 1961; a n d
others).
O t h e r workers have not been so successful in
d e m o n s t r a t i n g the fibrous elements in the mitotic
structures without the use of r a t h e r severe fixation
methods, such as the p r e t r e a t m e n t with - 1 0 ° C
ethanol used by Gross et al. (1958) on sea u r c h i n
eggs. T h e fact that fibrous structures can be d e m o n strated with neutral buffered osmium fixation
u n d e r certain circumstances a n d in certain
materials leads one to wonder just w h a t these
favorable conditions may be. Some of the factors
which m i g h t be considered are the osmolarity of
the fixative, salt concentrations, and the role of
specific ions such as Ca ++ or Mg++. A n o t h e r
factor m a y involve the e m b e d d i n g material, the
possible extraction of certain cytoplasmic structures by the solvents used, or the polymerization
d a m a g e evident in m e t h a c r y l a t e embedding.
T h o u g h almost every laboratory has experimented
to some extent with fixation methods to meet the
needs of the material at h a n d , it would seem t h a t
more systematic studies such as those of Palade
(1952) or Luft (1956) or Caulfield (1957) should
be made, to d e t e r m i n e just w h a t it is that fixation
does to a cell.
In considering the a p p e a r a n c e of the mitotic
structure as a whole, one is struck by the observation t h a t there seem to be two different structural
elements involved: the coarser material of the
astral center a n d the astral rays, which are composed of t u b u l a r elements and vesicles of the
endoplasmic reticulum; a n d the chromosomal a n d
continuous fibers, which are straighter, finer, a n d
more regular. T h e relationship between these
structures remains a problem for further investigation.
This work was supported by a grant (RG-6025) to
Dr. Daniel Mazia from the National Institutes of
Health, United States Public Health Service.
I wish to thank Dr. Mazia for his encouragement
and helpful suggestions during the course of this
work.
Receivedfor publication, March 23, 1961.
FIGURE 4
Anaphase, showing a pair of centrioles in the center of the aster, and radially oriented
vesicles of the endoplasmic reticulum extending from the aster toward the cell surface
to form the astral rays. Note that the interzonal region in the wake of the chromosomes
is largely devoid of endoplasmic reticulum. X 16,000.
426
' r i l e JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY • VOLUME 11, 1961
P. HARaIs Mitosis in Sea Urchin Blastomeres
427
FIGURE 5
A n a p h a s e chromosomes, showing bundles of fibers extending from the c h r o m o s o m e s t o w a r d the pole.
X 18,000.
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FIGURE 6
a. T e l o p h a s e c h r o m o s o m e s in cross-section, showing the e n d o p l a s m i c r e t i c u l u m condensing on the surfaces of the c h r o m o s o m e s to form the nuclear envelope. Pores (or
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P. HARRIS Mitosis in Sea Urchin Blastomeres
429
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27. YASUZUMI, G., KAYE, G. I., PAPPAS, G. D.,
YAMAMOTO, H., and Tsuso, I., Nuclear and
cytoplasmic differentiation in developing
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Zellforsch., 1961, 53, 141.
FIGURE 7
a. Part of a telophase spindle showing bundles of continuous fibers (arrows) in the interzonal region, with vesiculating chromosomes at the edge of the astral region at the
right. >( 15,000.
b. Another telophase figure at higher magnification, showing continuous fibers in the
interzonal region. >( 40,000.
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THE JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY • VOLUME 11, 1961
P. HARam Mitosis in Sea Urchin Blaatomeres
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