Cleavage of Blastomeres in the Absence
of Nuclei.
By
F. Gross
(Kaiser Wilhelm Institut f iir Biologie, Berlin-Dahlem, Abt. M. Hartmann,
and Galton Laboratory, University College, London.)
With Plate 1 and 8 Text-figures.
IN the following pages a brief account is given of observations
which touch upon two much discussed problems: (1) are eggs
or blastomeres capable of division if deprived of the nucleus?
(2) are divisions of blastomeres in the absence of chromosomes
and spindles to be regarded as cleavage, and the product
resulting from the divisions as corresponding to stages of normal
development, in this case, to a blastula?
The observations were made on eggs of parthenogenetic races
of A r t e m i a s a l i n a which at certain stages were exposed to
low temperature (+4° C). The experiments were concerned
mainly with the possibility of inducing polyploidy by suppression of polar body formation, some results of which have been
described in previous papers (1932, 1935). The latter paper
contains the description of the culture methods and details
concerning fixation (Camoy) and staining (Heidenhain's iron
haematoxylin).
Irregularities of mitosis such as triaster and tetraster formations are rather frequent in the early embryonic development
of A r t e m i a even under 'normal' conditions. Brauer (1894)
gave an account of some abnormalities, and points out the
sensitiveness of A r t e m i a eggs to changes of environmental
conditions. In my cultures, which were kept at room temperature, some abnormalities were observed without previous treatment, but the dates of fixation suggested that they might have
been caused by temperature changes. Later experiments with
low temperature were undertaken; they produced in fact not
only all those abnormalities of meiosis and mitosis which were
S8
F. GROSS
observed before but also many more. Some of them are of no
particular interest, such as irregular cleavage resulting in blastomeres with two or more nuclei or broad spindles with an
unusually large number of chromosomes. In most cases not all
eggs which were lying in the oviducts or the uterus were affected,
and, since normally all egg cells move together from the ovaries
TEXT-FIG. 1.
Irregular cleavage without previous treatment; a, section showing
cytasters, b, a triaster. x 280.
into the oviducts and from there into the uterus and develop
simultaneously, the unaffected eggs or embryos may be regarded
as controls.
The first case of cleavage of blastomeres in the absence of
nuclei was found in an untreated diploid parthenogenetic female
from Marguerita di Savoia (Italy). The eggs were in the uterus;
most of them in the blastula stage similar to Text-fig. 21 and
perfectly normal, some showing abnormal cleavage with fewer
blastomeres and spindles with a great number of chromosomes.
One egg, however (Text-fig. 1), possessed many chromosomes
near the surface, whereas the rest of the egg showed irregular
1
The text-figures were drawn by Erl. G. v. Levinski.
CLEAVAGE WITHOUT NUCLEI
59
cleavage furrows with cytasters but without any trace of nuclei
or chromosomes in the resulting cells.
The most interesting abnormalities were observed in a series
of sections obtained from another diploid parthenogenetic
female from Marguerita. This animal was put for 3 days 17
hours at a temperature of +4° C. At the time of exposure to
cold the eggs were in the oviducts (metaphase of the first
TEXT-FIG. 2.
Normal blastula. x 280.
meiotic division) and remained there until the shrimp was
brought back to room temperature.1 Then the eggs passed into
the uterus after a short time, and 10 hours later they were fixed
in Carnoy.
The normal stage of development is indicated by some
blastulae (Text-fig. 2 and fig. 1, PI. 1). These eggs, in spite
of the previous treatment with cold, have undergone normal
meiotic and cleavage divisions. There are several nuclei to be
seen in every section.
1
Normally they remain for about 4 hours in the oviducts. At low temperature, however, they remain there for days, and sometimes do not
move at all into the uterus. By the exposure to low temperature I originally
intended to inhibit this movement and to keep the oocytes in metaphase 1
overnight so as to be able to fix them in later stages on the following day.
The occurrence of abnormalities such as suppression of polar body extrusion
showed that low temperature not only arrested development but also
produced lasting effects upon further development.
60
F. GROSS
Sixteen embryos from the same uterus which could be studied
section by section were abnormal. One (No. 14) showed irregular
cleavage with a nucleus or chromosomes and spindle in each
blastomere. No. 10 possessed a number of cleavage furrows
dividing the egg into a corresponding number of irregular
blastomeres. Only three of these contained big nuclei, two at
prophase, one at interphase. The rest had cytasters only. The
common feature of the remaining fourteen embryos was the
presence of only one big resting nucleus which might show
several lobes, or a spindle with a large number of chromosomes
mostly at metaphase or anaphase. No. 4 showed only two
cleavage furrows, about twenty cytasters scattered through the
egg, and one abnormal metaphase spindle with a very great
number of chromosomes (fig. 6, PI. 1, right-hand egg). In
No. 11 only one slight furrow extends from the surface into the
egg for one-third of its diameter. There are many cytasters
scattered through the egg and one large lobed resting nucleus
is situated in the centre.
The remaining twelve eggs show more or less regular cleavage,
cytasters in all blastomeres, and only one nucleus or spindle
in one blastomere. The most striking cases of apparently quite
regular cleavage are shown in Text-fig. 3 b and on figs. 2 and 4,
PI. 1. Except for the absence of nuclei there is no difference
in the arrangement of blastomeres or the formation of the
cleavage cavity between these sections and those through normal
blastulae in an earlier stage than that represented by Text-fig. 2
and fig. 1, PI. 1. The number of blastomeres is estimated at
about sixteen to twenty in both cases. Pig. 3, PI. 1, shows a
section preceding 3 b with a blastomere containing a big lobed
resting nucleus. Text-fig. 3 a is the corresponding section of
the other series showing an abnormally wide metaphase spindle
with a great number of chromosomes. There are no other
nuclei or chromosomes present.
In these cases there can be no doubt that the blastomeres
resulted from a division process and not, as might be supposed
from less regular figures like Text-fig. 1, from disintegration or
post-mortem cleavage. In all eggs with abnormal cleavage those
blastomeres are biggest which contain the 'giant nucleus' or
CLEAVAGE WITHOUT NUCLEI
61
the chromosomes (Text-fig. 1 b, 3 a; fig. 3, PI. 1). In Text-fig. 3 b
the blastomere in the lower right-hand corner shows two
cytasters, the rest only one.
The same results, that is cleavage with only one nucleus in
one blastomere, were obtained from a diploid parthenogenetic
female from Cette which has been exposed to cold at the same
TEXT-FIG. 3.
Abnormal blastula from the same female as Text-fig. 2 resulting from
treatment with low temperature, a, the only blastomere with
spindle and chromosomes; b, another section showing regular
cleavage. In the right-hand corner two cytasters in one blastomere.
x280.
stage (eggs in the oviducts) for almost the same length of time
(3 days 15 hours).
In A r t e m i a no spindles occur in the absence of nuclei or
chromosomes. As has been shown in detail in a previous paper
(1935), an exclusively intranuclear spindle is formed in meiotic
as well as in cleavage divisions. The aster fibres take no part
morphologically in the formation of the cleavage spindle. In
the first cleavage the centrosphere or aster consists of a hyaloplasmic centre and well developed polar fibres. In the course
of cleavage the asters become gradually smaller and less distinct
until in the late blastoderm only small protoplasmic spots
without any polar fibres are found at the poles of the metaphase
spindles. In all stages the spindle is barrel-shaped and different
in structure from the polar fibres.
62
F. GEOSS
I have also dealt with the origin of the centrospheres in the
above-mentioned paper. Several facts led me to assume that
a relationship or even continuity might exist between the ectoplasm of the oocytes and the centrospheres of the first cleavage.
Briefly summarized these facts are as follows. (1) In parthenogenetic eggs the centrospheres must arise within the oocytes,
since no sperms are present. (2) The meiotic divisions take
place in the absence of any formed centres or polar fibres.
The spindles lie within the cortical plasm layer which is well
developed until telophase. (3) After the meiotic division (usually
only one) the egg nucleus appears surrounded by ectoplasm.
It moves towards the centre, and in many cases remains connected with the egg surface by a protoplasmic bridge. At this
time the ectoplasm has disappeared from the rest of the egg
surface and two centrospheres are being formed, apparently out
of the same material which represented the protoplasmic bridge
and the ectoplasm around the newly formed egg nucleus. In
other cases only one centrosphere is formed which by division
produces two. Therefore, it may be assumed that the cortical
plasm and the substance which gives rise to the centrospheres
are—perhaps only in part—morphologically homologous.
This assumption is supported by some observations on the
origin of the cytasters. Structures which closely correspond to
cytasters in appearance may arise without the meiotic division
having taken place. Text-fig. 4 is an example (this figure is a
combination of two adjacent sections). It represents an egg
from a diploid-parthenogenetic female which was exposed to
cold for 18 hours before the eggs moved into the oviducts. It
was then returned to room temperature when, after 3£ hours,
the eggs moved into the oviducts. After a further 3J hours
the eggs passed into the uterus and the animal was again
exposed to low temperature for 15£ hours and then fixed. No
egg had undergone the meiotic division.1 The spindle of the
metaphase 1 lies at the periphery of the oocytes (Text-fig. 4)
1
There are, however, twice as many 'tetrads' (42) as in the normal
Metaphase 1; a curious effect of cold, which I obtained in many experiments
with cold and chloroform and which will be dealt with in a separate paper
concerned with the origin of polyploidy in A r t e m i a .
CLEAVAGE WITHOUT NUCLEI
63
and no ectoplasm is to be seen either around the spindle or
elsewhere at the egg surface. Instead there are several cytasters
scattered through the egg. No cleavage has occurred in these
eggs. Here, and in similar experiments where the animals were
killed immediately after the treatment with cold, apparently
the meiotic division has been inhibited completely—except the
TEXT-FIG. 4.
Formation of cytasters independent of the meiotic division which has
been suppressed by low temperature. The metaphase spindle of
the first meiotic division is at the top of the drawing, x 280.
autonomous chromosome division; see footnote, p. 62—but not
the activity of the ectoplasm with regard to the formation
of centrospheres. There is no evidence as to whether all
cytasters have been formed simultaneously or by successive
divisions. It seems likely that such stages as Text-fig. 4 precede
the cleavage of blastomeres without nuclei as described above.
If the eggs represented by Text-fig. 4 had been allowed to
develop further at room temperature, it seems probable that
after the meiotic divisions—normal or with suppressed polar
body extrusion—the egg nucleus might have formed a cleavage
spindle near the surface and the cytasters have induced cleavage.
That would mean that the cytasters are formed in advance of
the activation of the meiotic and cleavage divisions or, in other
64
F. GROSS
words, low temperature seems to have a differential effect upon
different cytological processes; it has a greater inhibiting effect
upon the chromosome separation in meiosis than upon the
formation of centrospheres or cytasters.
But why does the egg nucleus not move to the centre of the
egg as normally, but remain at some distance from it, sometimes
rather close to the egg surface? That again seems to be connected with the cytasters previously formed and with their posi-
TEXT-FIG. 5.
Abnormal cleavage after exposure to low temperature resulting in
two blastomeres, the lower witli a number of nuclei, the upper with
a number of cytasters alone, x 280.
tion within the egg (Text-fig. 5 a and b). Thesefigureswere drawn
from sections of an egg from an animal exposed to cold for about
19 hours when the eggs were in the oviducts, and afterwards
transferred to room temperature when the eggs moved into the
uterus after about 2 hours. They were fixed 4£ hours later.
Most of the eggs showed normal development, and have undergone three cleavage divisions. Four eggs showed abnormalities
of which Text-fig. 5 serves as an example.
Here the egg is divided only into two blastomeres, the upper
containing a number of cytasters and no nuclei (in Text-fig. 5 b
one small nucleus lies in the centre where the furrow goes
CLEAVAGE WITHOUT NUCLEI
65
through the cell), whereas the lower blastomere contains a
number of nuclei lying close together and one bigger aster. It
may well be that the presence of the cytasters acted as an
obstacle to the movement of the egg nucleus which has undergone division in an abnormal position. The cleavage furrow has
apparently been produced as an effect of the interaction between
TEXT-FIG. 6.
Only two incomplete cleavage furrows formed after exposure to low
temperature. A group of nuclei lies in the central area, x 280.
the aster attached to the nuclei and the group of cytasters in
the upper part of the cell.
Another egg possessed a very big lobed nucleus lying in the
centre and four asters lying close to the nucleus. No cleavage
has occurred in this egg. A similar condition is shown in Textfig. 6, an abnormal stage resulting from a similar treatment of
a diploid-parthenogenetic female as above. Here several nuclei
of different sizes are present in the central area of the egg-cell
with four asters (only three are drawn in the figure; the fourth
lies in another section) attached to them. Only a small blastomere has been formed; another furrow is found in the right
upper part. Here apparently only one or two cytasters were
formed in advance of the formation and movement of the egg
NO.
313
F
66
F. GROSS
nucleus and the number of nuclei resulted probably from a
tri- or tetraster as is frequently found after treatment with
cold.
If one compares the division figures like Text-fig. 3 a,figs.5, 6,
PL 1, with normal metaphases of the first cleavage division
(see figures in my paper 1935), the most conspicuous difference
lies in the respective sizes of the spindles and the centrospheres.
Whereas the spindles and the number of chromosomes are much
larger in the abnormal eggs, the asters are much smaller. In
normal cleavages the amphiaster of the first cleavage division
almost fills the whole egg with polar rays; in the abnormal eggs
they are as large or a little larger than the cytasters. It seems,
therefore, likely that a number of cytasters were formed before
the formation of the metaphase spindle of the first cleavage
division, and thus only a small part of the ectoplasm which
normally forms two centrospheres was attached to the spindle.
The multiplication of the chromosomes and the occurrence of
single giant nuclei could then be explained by the assumption
that after every mitotic division the resulting daughter chromosomes or young interphase nuclei, owing to the weakness of the
asters, are not separated by a cleavage furrow, but fuse and form
a single nucleus, afterwards again a single metaphase spindle
and so on, behaving in this respect like the monasters.
It might be argued that in eggs which show cleavage and only
one nucleus in one blastomere there might be chromatin material
scattered through the egg and that this chromatin would then
be concerned in the cleavage. There were, in fact, granules in
a few of the eggs mentioned above, mostly situated at the
periphery of the cells, which were stained by iron haematoxylin
and gave the impression of small chromosomes or chromosome
fragments (Text-fig. 7). The sections were therefore restained
by Feulgen's method.1 Whereas the chromosomes lying in the
spindle gave a positive reaction, these granules did not show up
at all. Thus there is reason to believe that they do not represent
chromosomes.
One more observation may be recorded: Text-fig. 8 shows a
part of a section through an egg which showed, as in the cases
1
Dr. H. Bauer has kindly done the restaining.
CLEAVAGE WITHOUT NUCLEI
67
TEXT-FIG. 7.
Granules present at the periphery of eggs as in Text-fig. 3.
They give a negative Feulgen reaction, x 1,100.
TEXT-FIG. 8.
Part of an egg with abnormal cleavage showing two asters, a spindle
with chromosomes, and a nucleus-like body containing three yolk
spherules. X 1,100.
described above, cleavage of blastomeres without nuclei. Beside
the spindle lies a nucleus or at least a body which looks exactly
like a normal nucleus of a blastomere, containing, apart from
the granulated nucleoplasm, three yolk spherules. I do not
know of any other case where a nucleus contained foreign bodies.
DISCUSSION.
Some of the phenomena described in this paper have also
been observed in other species after various treatments. As early
as 1896 Boveri found that the formation and multiplication of
centres may occur in enucleated egg fragments which, however,
rarely showed any signs of cleavage. Later work of Ziegler
(1898), Wilson (1901ft), Yatsu (1905), McClendon (1908), and
others confirmed these results and showed that divisions of the
enucleated egg or blastomere also may occur after treatment
with agents like carbonated sea-water, MgCl2, &c. For a more
detailed discussion of this older work I refer to Wilson's book
(1925), from which the following conclusions may be quoted:
' Nevertheless there can be no doubt from all these observations
that the chromosomes play some part, indirect though it be, in
cleavage of the cell-body; and that we cannot, therefore, regard
the spindle or the astral rays as the sole agents involved.' As to
the cy tasters Wilson arrived at the conclusion that, since they are
often seen developing simultaneously and independently in large
numbers in all parts of the egg, they are formed d e n o v o .
Of the more recent investigations, those of Jollos and Peterfi
(1923), Seiler (1924), and Fankauser (1925, 1929) will be
briefly discussed. Jollos and Peterfi removed the egg nucleus
of fertilized Axolotl eggs by means of a micropipette. There
followed an irregular cleavage, mostly near the animal pole,
which lead to morula- and blastula-like configurations. There
were no centres present at all and the sperm nucleus disintegrated. The authors drew two conclusions from their observations: (1) that cell and nuclear divisions are independent
processes, and (2) that the protoplasm, at time of fertilization, is
already differentiated so as to be capable of repeated division
and even of differentiation to the blastula stage in the absence
of nuclei and centrosomes.
Seiler confirmed these results by observations on unfertilized
eggs of P h r a g m a t o b i a fuliginosa (a butterfly). After
the meiotic divisions the nucleus moves towards the centre of
the egg and disintegrates. In the ectoplasmic layer, however,
small, spherical, protoplasmic areas are formed, surrounded by
yolk granules. This process is extremely similar to that of normal
CLEAVAGE WITHOUT NUCLEI
69
cleavage, except of course that, in the normal blastoderm,
nuclei pass into those protoplasmic areas. There are no cell
limits formed at this stage, either in normal or in the abnormal
blastoderm.
Fankhauser found in enucleated parts of constricted fertilized
T r i t o n eggs, containing one or more sperm nuclei, cytaster
formation and subsequent cleavage of cells without nuclei.
This, however, appeared to be abnormal from the beginning.
In one case a blastula resulted of which the dorsal and lateral
parts were formed entirely by non-nucleated cells.
Schleip (1927), reviewing some of the papers mentioned above,
suggested that in the case of Axolotl eggs the cleavage was
only apparent and the furrows represented a post-mortem
phenomenon.
In this respect my observations seem to confirm the results
of Jollos and Peterfi. The resemblance of cleavage resulting
from treatment with cold and the normal one is so close, and
the arrangement of the blastomeres around the cleavage cavity
in some cases so regular, that the 'living' character of this
cleavage cannot be doubted. There is, however, some difference
between my material and the Axolotl- and A s t e r i a s eggs
(McClendon) inasmuch as these were deprived of their nuclei,
whereas in A r t e m i a the nucleus was present in one blastomere. Here, as in the T r i t o n eggs (Fankhauser), the possibility of an indirect effect of the nucleus and the chromosomes
respectively has not been eliminated. However, a conclusion
which may be safely drawn from the experiments on A r t e m i a
seems to be that normal cleavage of parts of the egg may result
in the absence of chromosomes and spindles. For the actual
division of blastomeres and formation of a blastula with cleavage
cavity the combined activity of cytasters and protoplasm
appears to be sufficient. An indirect effect of chromosomes
could not be excluded with certainty, even if perfectly normal
cleavage occurred in eggs deprived of all nuclear material. In
this case substances might have been released from the nucleus
in an early stage long before the operation, and these substances
may have determined the cleavage and the subsequent form and
arrangement of the blastomeres.
70
F. GBOSS
The possibility of separating nuclear and cell division byexperimental means has been demonstrated by many investigators. The observations on A r t e m i a differ from their results
in that stages were obtained with anuclear blastomeres showing
cleavage and a single nucleated blastomere unable to divide
although the chromosomes did divide. Without considering the
mechanism of cleavage division, it appears that the mere presence
of chromosomes, spindle, and asters in a blastomere is not
sufficient to cause cell division; but that probably the respective
size of spindle, aster, and cell is of some importance. Asters
of a certain size present in anuclear blastomeres are sufficient
to induce cleavage, whereas asters of the same or somewhat
larger size do not show the same effect in the presence of spindle
and chromosomes. It is difficult to say whether this is due to
the small size of the asters, as compared with those of the normal
first cleavage, or to the small size of the cell as compared with
the normal or doubled number of chromosomes caused by the
treatment with low temperature.
As to the widely discussed question of the origin of cytasters,
my observations suggested a connexion between the cortical
plasm and the cytasters. But, since I worked with parthenogenetic eggs, and it appears that also in normal development the
asters or centrospheres are formed by the ectoplasm, the alternatives are not continuity or de n o v o formation of asters but
rather the activation of aster-formation dependent on or
independent of the cycle of nuclear division. My observations
seem to show that aster formation may be induced in spite of
the meiotic division being inhibited.
SUMMARY.
After exposure to low temperature, at the metaphase stage
of the first meiotic division, parthenogenetic eggs of A r t e m i a
sal in a may show abnormal cleavage resulting in blastulae
with a very big nucleus in only one blastomere. The rest of the
blastomeres contain cytasters alone which are apparently
sufficient to determine cell division. In shape and arrangement
around the cleavage cavity they look very like those of normal
blastulae.
CLEAVAGE WITHOUT NUCLEI
71
If the oocytes are fixed immediately after the exposure to
low temperature they show, in some cases, a number of cytasters
scattered near the surface, although the meiotic division has
not yet taken place. It is suggested that the cytasters originated
from the ectoplasmic layer, and that their formation and
division is independent of nuclear divisions.
I am greatly indebted to Prof. J. P. Hill for having read the
manuscript and made some corrections.
EEFERENCES.
Boveri, Th. (1896).—"Physiologie der Kern- und Zellteilung", 'Sitz.-Ber.
phys.-med. Ges. Wurzburg.'
Brauer, A. (1894).—"Reifung des parthenogenetisoh sich entwickelnden
Eies von Artemia salina", 'Arch, mikrosk. Anat.', 43.
Fankhauser, G. (1925).—"Analyse der physiologischen Polyspermie des
Triton-Eies auf Grand von Schnurungsexperimenten", 'Arch. Entwickl.
mech.', 105.
(1929).—"Beteiligung kernloser Strahlungen (Cytaster) an der
Furchung geschntirter Triton-Eier", 'Revue suisse zool.', 36.
Gross, F. (1932).—"Polyploidie und Variabilitat bei Artemia salina".,
'Die Naturwissenschaften', 20.
(1935).—"Reifungs- und Furehungsteilungen von Artemia salina:
Problem des Kernteilungsmechanismus ", 'Z. Zellforsoh.', 23.
Jollos, V., und Peterfl, T. (1923).—"Furchung von Axolotleiem ohne
Beteiligung des Kerns", 'Biol. Centralbl.', 43.
McClendon, J. E. (1908).—"Segmentation of eggs of Asterias forbesii
deprived of chromatin", 'Arch. Entwickl. mech.', 26.
Schleip, W. (1929).—"Entwicklungsmechanik und Vererbung bei Keren",
'Handbuch der Vererbungswissenschaft', III.
Seiler, J. (1924).—"Furchung des Schmetterlingseies ohne Beteiligung des
Kernes", 'Biol. Centralbl.', 44.
Wilson, E. B. (1901 a).—"Experimental Studies in Cytology I. Artificial
Parthenogenesis in Sea-urchin eggs", 'Arch. Entwickl. mech.', 12.
(1901 6).—"II. Some Phenomena of Fertilisation and Cell-division in
etherized eggs. III. Effect on cleavage of artificial obliteration of the
first cleavage-furrow", ibid., 13.
(1925).—'The cell in development and heredity.' New York.
Yatsu, N. (1905).—"Formation of centrosomes in enucleated egg-fragments", 'Journ. Exper. Zool.', 2.
Ziegler, H. E. (1898).—"Exper. Studien ti. d. Zellteilung. I. Zerschniirung
der Seeigeleier. II. Furchung ohne Chromosomen", 'Arch. Entwickl.
mech.', 6.
72
EXPLANATION OF PLATE 1.
Microphotographs taken with 4 mm. Zeiss apochromatic objective and
Zeiss Photo-ocular 2. All eggs from the same female. Approx. 240 x .
Pig. 1.—Normal blastula corresponding to Text-fig. 2.
Fig. 2.—Abnormal blastula with anuclear blastomeres containing
cytasters. From the same egg as Text-fig. 3 b.
Figs. 3 and 4.—Two sections of an abnormal blastula: fig. 3 (the righthand egg) showing a big lobed nucleus in one blastomere; fig. 4, a later
section, showing blastomeres of regular shape with cytasters but no nuclei
or chromosomes.
Fig. 5.—A metaphase spindle with a very large number of chromosomes.
The rest of this egg shows less regular cleavage than fig. 4.
Fig. 6.—Eggs with very broad spindles containing a large number of
chromosomes. There were only two cleavage furrows in the right-hand egg.
Quart. Journ. Micr. Sci.
Vol. 79, N. S., PL 1