230
IS CLEAVAGE RATE A FUNCTION OF THE
CYTOPLASM OR OF THE NUCLEUS?
BY A. R. MOORE.
(From the Hopkins Marine Station, Pacific Grove, California.)
(Received 10th November, 1932.)
THERE are two views of the relative roles which the nucleus and the cytoplasm play
in forming the embryo. One view is the working hypothesis of the geneticist
according to which the cytoplasm of the fertilised egg is a passive and plastic
substrate on which the chromosomes of the nucleus work to mould the form of the
new being. In its extreme form this hypothesis ignores the facts of segregation and
localisation of formative substances in the egg. The other view holds that the cytoplasm is characteristic of the species and therefore determines the general form and
pattern of the embryo, that in the early stages of development the cytoplasm is
supreme in this respect, and only later do the chromosomes induce superficial
modifications in the developing individual.
The earliest determinable character of a species which is constant and which is
susceptible of modification by experiment is the rate of segmentation. Godlewski
(1906) was the first to make a study of the inheritance of the tempo of division in
crosses between two sea-urchins and a crinoid. He fertilised the eggs of Echinus and
Sphaerechinus with the sperm of Antedon, and reported that the cross-fertilised eggs
divided at the same rate as those fertilised with sperm of their own species.
Godlewski relied on the figures of Seeliger (1893) for the segmentation rate of
Antedon. However, neither Seeliger nor Godlewski took any account of the effect
of temperature on rate, and this as we now know from the work of Peter (1906)
and of Loeb (1908) is a factor of great importance, since segmenting eggs show a
temperature coefficient of 2-5-3 f° r IO ° C. Experiments done at different seasons
without temperature control would therefore yield enormously varying results, so
that for this reason alone a comparison of segmentation rates is meaningless unless
the temperatures are known.
A second difficulty in the way of interpretation of the results of the early workers
is that it was not then recognised that the eggs of a given female do not divide at the
same instant but, as a matter of fact, show a probability curve when the number of
eggs dividing is plotted against cleavage time. This is demonstrated in the work of
Loeb and Chamberlain (1915) on Arbacia. Consequently the best end-point for a
given segmentation stage is the time at which 50 per cent, of the eggs divide. The
earlier workers took only infrequent readings and give no information as to percentages of eggs which have divided at the points selected. It is therefore impossible
to assign any precise values to such published figures as those of Seeliger and
Godlewski.
Cleavage Rate as a Function of the Cytoplasm and of the Nucleus 231
Nevertheless, statements are frequently made on the basis of these old experiments that the cytoplasm alone determines the rate of segmentation. This conclusion
has been questioned by Newman (1910), who studied the effects of sperm on the
rate of cleavage in fish hybrids (Fundulus majalts $ x F. heteroctitus <J). Newman
treated his data statistically and concluded that the sperm accelerates the developmental rate of the egg when the sperm is from a more rapidly developing
species. The acceleration in development is slight, and the rate of division of the
hybridised eggs lacks much of being intermediate between the rates of the two
species.
In all the experiments referred to, entire eggs were used, so that both egg and
sperm nuclei were present. It may very well be that the maturated egg nucleus can
affect the rate of cleavage, since the nucleus may influence relatively remote parts of
the cell (Peters, 1930). The ideal solution of the problem is therefore to free the egg
cytoplasm of its nucleus and then fertilise it. The Hertwigs in 1887 were the first to
attempt this. They shook sea-urchin eggs in a test-tube until they were fragmented.
The pieces of cytoplasm, apparently non-nucleated, were then fertilised and
spindles formed, but no further development took place. Two years later Boveri
(1889) succeeded in raising plutei from such pieces. Delage in 1898 by means of a
micro-technique sectioned individual eggs under the microscope. He reported that
he had performed the operation on a dozen eggs (Strongylocentrotus lividus),
fertilised the two halves, nucleated and non-nucleated, with the result that both
divided more slowly than the entire egg, the fragment containing the egg nucleus
was next to the egg in rapidity of cleavage, and the non-nucleated fraction slowest of
the three. He says: "For example, when the non-nucleated fragment is at stage 2,
the nucleated fragment is at 4 and the whole egg at 8-16." Unfortunately no data
showing time intervals were published by Delage.
Tennent (1912), employing the Hertwig method, fragmented eggs of Toxopneustes
and found that non-nucleated pieces fertilised with sperm of the same species
segmented at the same rate as the whole egg. Tennent, Taylor and Whitaker (1929)
studied egg fragments of Lytechinus prepared by cutting eggs in two with a microneedle. This is essentially an adaptation of Delage's experiment and has the advantage over the Hertwig method in that the egg is all the time under observation and
the fate of the nucleus can be seen. In fact, in his last paper Boveri (1918) called in
question all experiments in which the eggs had been fragmented by shaking, for the
reason that parts of the nuclei might still remain in pieces which to all appearances
were non-nucleated. Tennent, Taylor and Whitaker have given precise readings of
temperature and time, and have shown that the nucleated pieces were 1-20 min.
slower than the whole egg in making the first division, while the non-nucleated ones
were 7-40 min. slower, the whole egg requiring but 35 min. after fertilisation to
complete its first segmentation at 29-5° C. The differences in time between the two
types of fragments could not be assigned to their size, nor to delayed activation,
since in both cases the membranes appeared at the same time after insemination.
Whitaker (1928) performed similar experiments on the egg of the starfish Patiria
miniata by means of the same method. As a result he has concluded that the diploid
232
A. R. MOORE
pieces divide in shorter time than the haploid ones, but suggests that "the higher
concentration of yolk in the haploid pieces is probably the effective agent" in
delaying cleavage in the latter. In a subsequent paper (1929) he has described
experiments with the eggs of Arbacia in which he similarly found a constant lag of a
few minutes in the cleavage of the non-nucleated pieces. This, however, he refers
entirely to nuclear content, and concludes that "the ratio of the amounts of nuclear
material and cytoplasm is a determining factor in the cleavage rate." And further:
"Half an egg containing both the egg nucleus and a sperm nucleus cleaves sooner
than the normal diploid egg in spite of the injury from cutting. The normal egg
cleaves sooner than half an egg containing only the sperm nucleus." It will be noted
that Whitaker's result differs from Delage's in that the latter found a slower rate of
division in the pieces than in the whole egg, while Whitaker found that a piece of egg
containing both nuclei cleaved sooner than the whole egg. Delage unfortunately does
not give figures, and the differences tabulated by Whitaker are small, between 6 and
7 min. The results of Tennent, Taylor and Whitaker are, however, unexceptionable
and agree with those of Delage.
It seemed possible to obtain new and unequivocal evidence on the question by
crossing two forms which have widely different segmentation rates, and in which the
hybrid larvae show the inheritance of characters from both parents. This condition
is furnished by two echinoderms which occur abundantly in Monterey Bay, namely,
the sea-urchin Strongylocentrotus franciscanus and the sanddollar Dendraster eccentricus. For example, in one experiment it was found that at 20° C. the sea-urchin
eggs had an average segmentation time of 95 min. between fertilisation, as shown by
membrane formation and first cleavage, and for subsequent stages an average time
of 47 min. The sanddollar eggs showed corresponding average times of 57 and
28 min. The sea-urchin egg therefore requires more than one and a half times as
long for segmentation as does the egg of the sanddollar (Moore, 1932).
The experiments were carried out in the following way. A few eggs of Dendraster
were put into a small drop on a cover-glass and arranged for cutting with a microdissection needle. The nucleus of one of the eggs was cut off with more or less
cytoplasm. A little sperm of the sea-urchin Strongylocentrotus franciscanus was then
added. Next the cover-slip was inverted and laid over a small Syracuse watch-glass
containing a few drops of water. With such an arrangement the whole eggs and
pieces in the hanging drop developed apparently normally for several hours, and
could be kept continuously under observation. At the outset the experimental
pieces were sketched and their segmentation followed. The whole eggs served as
controls. Another set of controls was made by fertilising pieces of egg with sperm of
their own species. The end-point for segmentation was taken as the moment the
furrow was completed. The segmentation of the eggs and pieces was followed up to
the 4th cleavage. This is important for the reason that the time interval between
fertilisation and the first segmentation seemed to be less regular than that for
subsequent segmentations. The results give confirmation to Tennent's observation
(1912) that if the pieces segment then the rate of segmentation is independent of the
size of the piece.
Cleavage Rate as a Function of the Cytoplasm and of the Nucleus 233
The following tables from typical experiments indicate the degree of constancy
of the results and the extent of variations. Readings were made to the nearest
minute. The letter t designates the time in minutes between two cleavages. The time
from fertilisation to the first cleavage equals zt and is always so considered in
calculating the average t.
Table I.
0
June 29. Temp. 19 C. Whole eggs. Time based on 50 per cent, cleavage.
Strongylocentrotus
franciscanus $ x £
Strongylocentrotus
franciscanus $
Dendraster
eccentricus 0
min.
A
min.
B
min.
Fertilisation to 2 cell
2-4 cell
4-8 cell
IOO
95
40
47
11
93
47
44
Average t
47
46
46
July 9. Temp. 200 C. Whole eggs. Time based on 50 per cent, cleavage.
Dendraster
eccentricus $ x 0*
Dendraster
eccentricus $
Strongylocentrotus
franciscanus 6*
A
min.
B
min.
A
min.
B
min.
Fertilisation to 2 cell
2-4 cell
55
57
28
29
57
28
59
31
Average t
28
29
28
3°
July 12. Temp. 18-5° C. Dendraster eccentricus
Non-nucleated
fragment
Fertilisation to 2 cell
2—16 cell
Average t
Normal egg
min.
69
78
61
90
295
3°
Nucleated piece did not divide.
It is clear from the facts given in the tables that there is no consistent difference
between the tempo of segmentation in whole eggs of Dendraster fertilised with the
sperm of Strongylocentrotus and that of pieces of eggs either with or without nucleus,
similarly fertilised. Controls in which the eggs of Dendraster and pieces were
fertilised with sperm of their own species did not differ significantly from each
other nor from hybridised eggs and pieces in tempo of cleavage.
The experiments prove that for the egg of Dendraster the cytoplasm of the
maturated egg without any chromatin material determines the rate of cleavage.
JKB-liii
16
A. R. MOORE
234
The sperm of the sea-urchin enters the cytoplasm of the sanddollar egg and initiates
a series of reactions in this cytoplasm which in turn acts upon the nucleus. This
results in the normally slow sea-urchin sperm nucleus performing the complicated
operations of mitosis in a little more than half the time normal to it. The sperm
nucleus in this cross is therefore without any effect on segmentation time which is
determined by the cytoplasm alone.
Table II.
July 4. Temp. 200 C.
Dendraiter cccentricw ? x Strongylocentrotus francitcama o •
Non-nucleated pieces
Control whole egg
min.
A
min.
B
min.
Fertilisation to 2 cell
2-4 cell
4-8 cell
8-16 cell
16-32 cell
55
24
25
27
52
24
25
26
26
50
25
24
29
25
Average /
26
25-5
25-5
July 12. Temp. 200 C.
Fertilisation to 2 cell
2-4 cell
4-8 cell
8-16 cell
Whole egg
Non-nucleated
piece*
Nucleated
piece*
min.
min.
A
min.
B
min.
56
56
56
32
25
26
—
27
25
16—32 cell
28
27
2S
26
—
Average t
It
—
—
27
28
27
55
29
• The nucleated and non-nucleated pieces were from the same egg.
In view of the results obtained it becomes of interest to consider the experiments
of Delage, of Tennent, Taylor and Whitaker, and of Whitaker, all of whom obtained
evidence which showed that the nucleated fragment segmented at a faster rate than
the non-nucleated piece from the same egg. It is not surprising that Whitaker was
led to suggest that the chromatin content of the piece is of significance in determining the tempo of cleavage. It does not, however, in view of our results seem
reasonable to suppose that such is the case.
The view that the chromatin content of an egg piece has nothing to do with
segmentation tempo is rendered secure by the recent experiments of Ethel Browne
Harvey (1932) on centrifuged pieces of Arbacia eggs. She has found it possible to
centrifuge these eggs into two pieces, one containing pigment and yolk but not the
nucleus, the other half fairly clear and with the egg nucleus. Both pieces when
fertilised form membranes, segment in normal time and develop into early larvae.
Cleavage Rate as a Function of the Cytoplasm and of the Nucleus 235
The non-nucleated half is a little slower in its development than the clearer nucleated
piece. If now the nucleated fragment before fertilisation be again centrifuged at
high speed, about 10,000 r.p.m., it again separates into two parts, one of which is
perfectly clear and contains the nucleus, the other contains a few granules and is of
course without nucleus. Upon fertilisation both of these quarters form membranes,
but in the nucleated quarter segmentation is enormously delayed—even up to 7
hours; the non-nucleated fragment, which contains some granules, segments in
slightly slower than normal time and forms a good early larva. The experiment
proves that in Arbacia the egg nucleus has nothing at all to do with cleavage time,
but suggests that the light granules are on the other hand concerned. In this
connection Loeb and Chamberlain accounted for differences in segmentation
tempo in the same lot of eggs by supposing a substance in the egg (enzyme?) to be
the material which determined cleavage. They suggested that a slightly unequal
distribution of this material as between the different eggs of a given female would
account for the fact that such eggs show differences in segmentation time. May it
not be then that the light granules of the non-nucleated quarter in Dr Harvey's
experiment are the "segmentation stuff" of which Loeb and Chamberlain spoke?
A slight degree of localisation of this material about the nucleus in the unfertilised
eggs of Paracentrotus, Arbacia and Lytechinus would account for the results of
Delage, Whitaker, and Tennent, Taylor and Whitaker. My experiments show that
there could be no similar localisation of "segmentation stuff" in the unfertilised
eggs of Dendraster. It is therefore possible to account for the facts of the cleavage
time of egg fragments without supposing that the chromatin of either egg or sperm
nucleus has anything to do with it. As a matter of fact the experimental evidence
excludes such a possibility. The reactions of the cytoplasm alone determine the
rate of cleavage, even when the fertilising sperm is from an animal of different order.
SUMMARY.
Advantage was taken of the fact that the eggs of Dendraster can readily be fertilised with the sperm of Strongylocentrotus. Such cross-fertilised eggs have the
cleavage tempo of their own species, and are unaffected in this respect by the sperm.
Eggs were fragmented by cutting them in two with the micro-needle and fertilised,
some lots with Dendraster sperm, some with that of Strongylocentrotus. Subsequently
nucleated and non-nucleated fragments showed the same time intervals for cleavage
in both lots, intervals identical to those of whole eggs. Whence it is concluded that
neither sperm nor egg nucleus has any effect on segmentation tempo, but that the
reactions of the cytoplasm alone determine it. The suggestion is made that the
cleavage reaction depends upon a substance of granular character in the cytoplasm.
16-2
236
A. R. MOORE
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