A Quantitative Study of the Changes in the Cyto-some of Eggs of the Echinodermata during Early Cleavage

THE early stages in the development of the sea urchin, Arbacia punctulata, occur when the embryos are enclosed in a fertilization membrane through which there is an exchange of sea-water; but no food is taken in, the animal deriving energy for its rapid growth from the reserve food materials stored in the egg cytoplasm. It is well known that these eggs contain fat, yolk, and mitochondria (Morgan, 1927), from which, it may be supposed, that the larva obtains its supply of energy. It was thought that if changes in the quantities of fat and mitochondria occurred, these could be estimated quantitatively by counting the numbers of granules of these substances which appear in material prepared by the usual cytological methods. In addition, eggs preserved in this way and carefully sectioned were measured in order to find out if significant changes in volume occurred during the period of fertilization and cleavage up to the young blastula stage.

The eggs of Arbacia were collected at the Marine Biological Laboratory, Wood’s Hole, Mass, U.S.A., during July. The ovaries were removed from the sea urchins and the eggs were allowed to shed into finger bowls of sea-water (Just, 1928). After being washed, they were inseminated and the samples were collected at various intervals.

The eggs were collected from several finger bowls, and transferred at once to fixatives which preserve both fat and mitochondria, viz. Regaud, Flemming without acetic acid, and Champy-Kull (Lee, 1928). The material was imbedded in paraffin after having been dehydrated and cleared in butyl alcohol.

In order to estimate the volume of the larvae at different stages, and to count the granules of fat, as well as the mitochondria, it was necessary to cut sections of these eggs which were 1μ in thickness. This presented considerable difficulties, but finally many slides were produced with uncrushed sections which could be used for measurements.¹ It was not possible to obtain serial sections of single eggs, but in measuring diameters it was comparatively easy to judge which sections had gone through the centre, although this is no doubt a source of error.

The forms assumed by cleaving eggs do not, on the whole, admit of being subjected to comparison with geometrical figures. A mathematician² was asked to devise a formula for the two-cell and blastula stages, since these presented the greatest difficulties. Eggs tend to be spherical before and after fertilization, and also in the four-cell stage as may be seen in fig. 1, Pl. 27 ; thus the ordinary formula for a sphere is used for these stages. Table I gives the formula which is used in each stage and the volume obtained from the measurements made with a micrometer eyepiece which had been previously calibrated.

The errors involved in measuring the diameters or radii were of the order of 2·3 per cent., but other sources of error may account for the fact that the volumes vary markedly, but not in any one direction. The fixing fluids may cause different degrees of shrinking and swelling in the different stages, and changes in volume might be brought about by raising the temperature of the water in which the animals were reared, since the temperature during July of the laboratory in Wood’s Hole is about 21° C. or more. In this connexion it is interesting to compare Koehler’s (1912) figures for the variation in the volume of Strongylocentrotus larvae produced by changes in temperature which he estimated from whole mounts.

The estimation of the number of fat granules and mitochondria was carried out by means of a squared disc placed in the eye-piece of the microscope. Text-fig. 1 shows the appearance of a section of a four-cell stage and the squares of the disc drawn with a camera lucida at the two magnifications which were used in the counting. The fat-granules were quite distinct in unstained sections, and the counting was carried out with an apochromatic objective (3 mm.) and a compensating ocular (10×). The mitochondria are exceedingly small and can only be counted in perfectly cut and stained sections, using rather brilliant illumination with an oil immersion lens.

The appearance of the fat globules in unstained sections is illustrated in figs. 1, 2, and 3 in Pl. 27.

The fat appears as blackened granules irregularly spaced in the cytoplasm, with a tendency to clump in various parts of the cell. These granules were soluble in turpentine after immersion of the slide for a period of 2 hours or more.

In order to eliminate the personal element in counting the fat granules, my counts were repeated by an assistant¹ to whom my results were unknown. In this way it was found that these independent counts agreed very well, any doubtful cases being discarded. The percentage errors of all the counts were worked out, and the results are expressed within the limits of these errors in Table I (Text-fig. 2).

The counting of the mitochondria was extremely difficult owing to their small dimensions and the necessity for producing perfection of staining and illumination. It was not possible to have independent counts made by a second person, as in the case of the fat, but all the counts were repeated after an interval of time had elapsed, which allowed the figures to be forgotten.

The results of the counts for fat and mitochondria and the errors for each stage are given in Table II. The numbers are expressed as occurring in a unit volume of the egg, namely, 100μ³, so that the relative numbers of granules can be compared.

These figures are expressed graphically in Text-fig. 2.

The most striking point is the sharp decline in volume, fat, and mitochondria at the four-cell stage, although the fat is decreasing steadily before this, while the mitochondria show a distinct decrease between the unfertilized and fertilized egg with no change until the four-cell stage is reached, when a sharp increase follows. As the intervening stages have not been investigated as yet it is not possible at this time to draw any conclusions.

A similar investigation will shortly be completed on the developing eggs of Asterias, which are markedly different from those of Arbacia, not only in their lack of easily visible fat-globules in unstained preparations but also in the larger size of the mitochondria. Figs. 4 and 5, Pl. 27, show the details of the cytoplasm, containing numerous yolk-droplets and the spherical, evenly distributed mitochondria.

f., fat-granules; m., mitochondria; N., nucleus; n., nucleolus; y., yolk-granules.

All drawings were made with the camera lucida.

Fig. 1.—Section of Ar bacía larva, four-cell stage, showing fat-granules. Flemming without acetic fixation. Unstained.

Fig. 2.—Young blastula of Ar ba cia. Champy-Kull. Unstained.

Fig. 3.—Scale of magnification for figs. 1 and 2.

Fig. 4.—Oblique section of four-cell stage (Arbacia) Champy-Kull fixation and stain. The left half shows distribution of fat-granules only, other details of the cytoplasm being omitted. On the right-hand side, two areas have been drawn under the squared disc, showing actual numbers of fat and mitochondria.

Fig. 5.—Unfertilized egg of Asterias forbesi. Regaud fixation, Bensley-Cowdry stain.

Fig. 6.—Same as fig. 5, but with Flemming without acetic fixation, unstained, in which mitochondria appear as greyish refractive granules.

Fig. 7.—Scale for figs. 4, 5, and 6.