Is Cleavage Rate a Function of the Cytoplasm or of the Nucleus?

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 cyto-plasm 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.

Cleavage Rate as a Function of the Cytoplasm and of the Nucleus 231

Nevertheless, statements are frequently made on the basis of these old experi-ments 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 develop-mental 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 micro-needle. This is essentially an adaptation of Delage's experiment and has the ad-vantage 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

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

eccen-tricus. 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).

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.

June 29. Temp. 190 _{C. Whole eggs. Time based on 50 per cent, cleavage.}

Fertilisation to 2 cell 2-4 cell 4-8 cell

Average t

Strongylocentrotus franciscanus $ x £

min. IOO 40 47 47 Strongylocentrotus franciscanus $ A min. 95

11

July 9. Temp. 200 _{C. Whole eggs. Time based on 50 per cent, cleavage.}

Fertilisation to 2 cell 2-4 cell

Average t

Dendraster eccentricus $ x 0*

A min. 55 28 28 B min. 57 29 29 Dendraster eccentricus $ A min. 57 28 28 Strongylocentrotus franciscanus 6* B min. 59 31 3°

July 12. Temp. 18-5° C. Dendraster eccentricus

Fertilisation to 2 cell

2—16 cell Average t Non-nucleated fragment 69 78 295 Normal egg min. 61 90 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.

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 •

Fertilisation to 2 cell 2-4 cell 4-8 cell 8-16 cell 16-32 cell Average / Non-nucleated pieces A min. 55 24 25 27 26 B min. 52 24 25 26 26 25-5

Control whole egg min. 50 25 24 29 25 25-5

July 12. Temp. 200 C.

Fertilisation to 2 cell 2-4 cell 4-8 cell 8-16 cell 16—32 cell Average t Non-nucleated piece* min. 55 2 8 27 2S 26 27 Nucleated piece* min. 56 32 25 26 — 28 Whole egg A min. 56 27 25 — 27 B min. 56

It

—

—

• 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 deter-mining the tempo of cleavage. It does not, however, in view of our results seem reasonable to suppose that such is the case.

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 fer-tilised with the sperm of Strongylocentrotus. Such cross-ferfer-tilised 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.

REFERENCES.

BOVERI, T . (1889). Sitzungsber. d. Ges.f. Morph. u. Phys. MUnchen, 5, 73. • (1918). Arch. f. Entw.lt, 417.

DELAGE, Y. (1898). Comptes Rend. Acad. Set. 127, 528. GODLEWSKI, E. (1906). Arch.f. Entie. 20, 610. HARVEY, E. B. (1932). Biol. Bull. 62, 155.

HERTWIG, R. and O. (1887). Zeitschr.f. Mtd. u. Naturwiu. Jena, 20, 107. LOEB, J. (1908). Arch.f. d. get. Phytiol. 124, 411.

LOEB, J. and CHAMBERLAIN, M. M. (1915). Journ. Exp. Zool. 19, 559. MOORE, A. R. (1932). Collecting Net, 7, 253.

NEWMAN, H . H . (1910). Journ. Exp. Zool. 8, 143. PETER, K. (1906). Arch.f. Enttv. 20, 130.

PETERS, R. A. (1930). Trans. Faraday Soc. 26, 797. SEELIGER, O. (1893). Zool. Jahrbuch. 6, 161. TENNENT, D . H. (1912). Journ. Exp. Zool. 12, 391.

TENNKNT, D. H., TAYLOR, C. V. and WHITAKER, D. M. (1929). Carnegie Inst. Publ. 26, 1. WHITAKER, D. M. (1928). Physiol. Zool. 1, 63.