[From the Proceedings of the American Association for the Advancement of Science, 
Yol. xxvii, St. Louis Meeting, August, 1878.] 
Changes Preliminary to Cleavage in the Egg of Clepsine. 
By Charles O. of Newton Highlands, Mass. 
[ABSTRACT.] 
The embryology of one of the higher animals may be said to 
embrace the history of two successive periods, viz., the preem- 
hryonic and the embryonic. 
The pre-embryonic period includes the growth and maturation 
of the egg, impregnation, nuclear changes preparatory to cleavage, 
the cleavage, the formation of a hollow sphere (Blastula, Hiickel) 
from the cleavage products, and the transformation of this sphere 
into a double-walled sac (Gastrula, Hiickel; Planula, Lankester). 
The second, or embryonic period, is introduced by the formation 
of a primitive stripe, formed by the concrescence and concomitant 
thickening of the two halves of the Gastrula-orifice. During this 
period the parts of the future animal are sketched out and more 
or less completely developed. 264 
CHANGES IN THE EGG OF CLEPSINE ; 
Older embryologists devoted their attention almost exclusively 
to the embryonic phases of development; it is only within the 
last seven or eight years that pre-embryonic phenomena have 
begun to receive the attention they deserve. It is some of these 
phenomena to which I shall now ask your attention, making the 
egg of Clepsine the point of departure. 
Clepsine marginata, as many of you know, belongs to the leech 
family. It is a fish parasite, measuring about one inch when 
fully grown. It is found in ponds or small brooks where the cur- 
rent is gentle or imperceptible, attached to leaves, stones, fallen 
branches, etc. The eggs of this species vary in number from 
twenty to two hundred, according to the size of the specimen 
and the amount of food it has taken, and are generally attached 
to the leaves of living plants. 
This leech, like others of the genus, has the interesting habit of 
remaining over its eggs till they hatch. At the time of hatching 
the young are little developed, and entirely helpless. Their pro- 
tection is very curiously provided for. Soon after hatching they 
become attached to the ventral side of the parent, and are thus 
borne about until fully developed. During this time they receive 
no food from the parent, as shown by the fact that the}'- develope 
quite as well when separated from the mother ; their only food is 
the nutritive yolk furnished by the egg. 
When, at the end of from two to three weeks from the time of 
hatching, this store of food is exhausted and the young are 
developed, the parent seeks to attach itself to a fish. This done, 
the young leave the mother and creep over the fish and take their 
first meal. 
Origin and Growth of the Egg.—The egg of Clepsine, like that 
of any other animal, is derived from a simple cell. This primary 
egg-cell is a spherical body inclosed within a thin transparent 
membrane, and is composed of three concentric parts, viz.— the 
protoplasmic body, the nucleus, and the nucleolus. The first step 
in the development of this cell into the proper ovum, is signalized 
by the accumulation of fine, opaque granules in the body of the 
cell, around the nucleus. These granular precipitations are white 
when seen by reflected light, and exhibit a lively tremulous motion 
when brought into contact with water the so-called Brownian 
movement. They multiply rapidly with the growth of the egg, 
and soon render the entire ovum opaque. When the egg is about BY CHARLES O. WHITMAN. 
265 
half-grown some of these granules begin to expand by a simple 
process of growth, thus giving rise to the yolk-spheres. The color 
of the egg, which may be either white, yellow or green, depends 
upon the color of the yolk-spheres. As these granules and spheres 
have a secondary origin, as compared with the semi-fluid proto- 
plasm in which they are imbedded, they have been termed by Van 
Beneden, deutoplasm. The origin of the deutoplasmic elements 
in the bird’s egg or in the reptile’s egg is, according to the investi- 
gations of Gegenbaur, precisely the same as in the egg of Clepsine, 
the only difference being, that in the former the yolk undergoes a 
farther differentiation into white and yellow yolk. 
The full-grown egg of Clepsine measures about *80mm about 
the size of a small pin-head the nucleus about *OB to •09mm, and 
the nucleolus only about "01mm. 
Transformation of the germinal vesicle.— Shortly after the ma- 
turity of the egg, the nucleus, or germinal vesicle as it is usually 
called, undergoes a remarkable transformation. The cause of this 
change is as yet unknown. It is simply known that the membrane 
of the germinal vesicle dissolves, the nucleolus becomes invisible, 
and that the whole or a part of the vesicle assumes the form of a 
spindle, around the two poles of which the protoplasm of the yolk 
becomes arranged in radial lines. This form of the nucleus, the 
precise origin of which was first made known bj7 Auerbach, in 
1874, was called by him “ karyolytic figure,” but has since been 
more appropriately termed amphiaster, or double star, by a dis- 
tinguished naturalist of Geneva, Dr. Hermann Fol. 
The spindle of the amphiaster, or bistellate figure, is composed 
of fibres curving outward, and converging to a point in each pole 
of the amphiaster. These spindle-fibres appear to differ in no 
essential respect from the radial polar lines ; but in the eggs of 
some other animals and in the eggs of plants, according to 
Butschli and Strasburger, they form the most conspicuous part of 
the figure. 
The entire figure bears a most striking resemblance to the picture 
produced in iron-dust under the influence of a magnet. This 
resemblance was at once remarked by Leuckart and others who 
have seen my preparations ; but it is not quite perfect in one par- 
ticular— that no curves appear in the radial polar lines. But it is 
to be remembered that these phenomena have thus far been studied 
in microscopic sections of eggs that have been treated with various 266 
CHANGES IN THE EGG OF CLEPSINE ; 
acids. It is quite possible that such curves do exist in the living 
egg, and that they become obscured or obliterated by treatment 
with acids and staining fluids. That we have here an electrical 
phenomenon is suggested not only by the general appearance of 
the amphiaster, but also by the deportment of the two poles 
during the process of cleavage. This amphiastral form of the 
nucleus is, as we shall see farther on, one stage in the process of 
self-division. It is a transitional phase by which one nucleus 
becomes two nuclei. This division of the nucleus generally 
results in the division of the egg, or the cell. As often as the 
cell divides, the amphiastral figure recurs. It is plain, therefore, 
that we have here a life-phenomenon of very great importance. It 
is a phenomenon of the deepest interest to the biological student, 
inasmuch as it stands at the very threshold of all organic life. It 
is common not only to all eggs, whether the eggs of plants or 
of animals, but also to all cells, whether embryonic cells or the 
cells of fully developed tissues. Its universality testifies to the 
unity of life. 
Quiescent state.— After the formation of the amphiaster, the egg 
appears to maintain a quiescent condition, until, at the time of 
deposit, it is brought in contact with water. The proof of this 
fact was obtained in the following manner. I disturbed a leech 
that was laying its eggs, and, in consequence of the interruption, 
three eggs were retained in the ovary. The leech manifested no 
disposition to part with the remaining three eggs. At the end of 
forty-eight hours I cut the leech through the middle and thus lib- 
erated three eggs. To my surprise, I found them in precisely the 
same condition in which I had found the others two days before. 
The eggs were kept in both cases and developed in the normal 
manner, I have observed cases in which the eggs have been 
retained in the ovary four to five days after they were ripe for 
deposit. I am the more certain on this point, as 1 have been 
able to recognize this time with sufficient accuracy to exclude 
failure in every case where I made the experiment of cutting the 
leech to obtain eggs. 
This quiescent condition, which can be maintained for at least two 
days in the egg of Clepsine, and which in the egg of the hen, accord- 
ing to Colasanti, may continue three weeks, and, in rare cases, even 
four weeks, without fatal injury, reminds one of the analogous fact 
that seeds can be preserved for years in a similar condition. BY CHARLES O. WHITMAN. 
267 
Polar Figure.— Soon after deposit, the amphiaster moves 
towards the periphery of the egg in such a manner that one of its 
poles comes to lie exactly in the surface. This pole appears on 
the living egg as a small white spot; examined under a low 
magnifying power it shows a distinct radial structure. This polar 
figure is the outward expression of the radial lines around the 
external pole of the amphiaster. About twenty minutes after 
deposit there appears exactly in the centre of this figure a pellucid 
spot. This spot is free from deutoplasmic elements, but appears 
dark in consequence of the opaque background. This spot is the 
centre of the outer pole of the amphiaster. The polar figure not 
only reveals the position of the amphiaster, but also determines 
the main axis of the egg and at the same time the axis of the 
future embryo. It marks very nearly, at least, the place where 
the future mouth forms, and this pole of the egg may be therefore 
called the oral pole, and the opposite the aboral pole. 
Polar Globules.— About thirty minutes after deposit an inter- 
esting change takes place. A well defined constriction appears in 
the equatorial plane of the egg, and passes by a sort of peristaltic 
movement toward that pole of the egg which bears the polar figure. 
In the course of ten minutes it passes off' at this pole, leaving only 
a nipple-like protuberance. In a few minutes this protuberance is 
seen to be a transparent spherical body in process of liberation. 
Soon it is fully eliminated and lies between the yolk and the egg- 
membrane. Thirty minutes later a second body, like the first, is 
expelled, though accompanied by a much Jess clearly defined con- 
striction. These two bodies are the so-called “polar globules,” or 
“ directive corpuscles.” They are of very general occurrence in the 
eggs of animals as well as the eggs of plants, but the constriction 
attending their exit above described, has not been observed in any 
egg save that of Clepsine. 
Whence come these polar globules? If we make sections of 
the egg, at the time the constriction is passing, parallel to 
the axis of the egg, we shall, if successful, obtain one section 
which includes this axis. In this section we shall find the am- 
phiaster in process of division, and we shall see that the polar 
globule represents the external half of the amphiaster, together 
with a little of the protoplasm of the egg. It contains then 
nuclear substance and cell-protoplasm, in other words, is a genuine 
cell. The remaining half of the amphiaster is soon transformed 268 
into a complete amphiaster, which divides, and produces the second 
polar globule. The half of the last amphiaster which still remains 
in the egg is not changed into a third amphiaster, but assumes the 
form of one of the amphiastral poles. This stellate figure is the 
female pronucleus. Near the opposite pole of the egg a second 
stellate figure is seen. This is the male pronucleus. It owes its 
origin to a spermatozoon which has previously entered the yolk. 
CHANGES IN THE EGG OF CLEPSINE ; 
Cleavage-nucleus.— In the female pronucleus two small bodies 
are seen in close apposition, and in the male pronucleus, one such 
body. These bodies are nucleoli. The two pronuclei begin to 
approach each other, as if by reciprocal attraction, meet near the 
centre of the egg and here blend into a single nucleus the 
cleavage-nucleus, so called because it is the nucleus which intro- 
duces the cleavage. The three nucleoli also meet in the centre of 
the cleavage-nucleus, but do not blend until about the time the 
cleavage begins. From this it appears that impregnation consists 
in the complete fusion of corresponding parts of two sexually differ- 
entiated cells the egg-cell and the sperm-cell. 
Polarity.'—While the phenomena thus far described polar 
figure, pellucid spot, and polar globules—have been confined to 
one pole of the egg, those which are to follow are repeated on both 
poles of the egg. A short period of uni-polar activity is followed 
by a longer period of bi-polar activity. In the latter period the 
pole thus far active, still asserts its preeminence by taking the 
lead in actions that repeat themselves later and more sluggishly 
on the opposite pole. 
Polar rings.— I have now to describe a phenomenon thus far 
known only in the egg of Clepsine. About one hour and thirty min- 
utes after deposit, a shallow groove forms around the oral pole, 
and in this groove a transparent fluid collects, thus forming a 
conspicuous polar ring. Soon the equatorial side of this ring 
becomes denticulate or raved. Ten minutes later a similar ring 
forms around the aboral pole, with rays from its equatorial side. 
The life of the egg at this time seems to be concentrated in this 
outward display of polarity. These rings begin to advance 
slowly, each toward the pole it incloses; meanwhile the rays have 
reached their maximum intensity and begin to dwindle. The oral 
ring advances in such a manner that it forms a constriction about 
the inclosed yolk, forming of this a kind of flat cap, or calotte. 
The aboral ring does not strike deep enough to form a calotte, BY CHARLES O. WHITMAN. 
269 
but seems to pass over the pole, forcing the inclosed yolk in 
towards the centre of the egg. This concentration of the rings 
continues until the aboral ring has the form of a disc, and the 
oral ring is pierced only by a slender column of yolk, on 
which rests the calotte. If we make a median section of the 
egg at this time, the aboral disc will present an oblong elliptical 
form, while the oral ring will appear as two separate parts, di- 
vided by the slender column of yolk which supports the calotte. 
Each of these parts has a sharply outlined cordate form. At this 
time the nucleus will be found a little eccentric, lying nearer the 
oral than the aboral pole. The three nucleoli have maintained 
their identity and relative positions thus far. A few minutes later 
the ring-substance loses its sharp outlines and begins to flow slowly 
towards the centre of the egg. At this moment the three nucleoli 
become invisible, and the nucleus assumes the amphiastral form 
already described. This amphiaster is much larger than those 
before mentioned, and its division will result, not in the production 
of a diminutive cell, or polar globule, but in the sundering of the 
egg into two nearly equal parts. It is interesting to note the 
behavior of the two poles of the amphiaster while the egg is di- 
viding. The cleavage-groove begins first on the oral side of the 
egg—on that side nearest to which the nucleus lies. At the 
moment it begins we find that the two amphiastral poles are much 
farther apart than at first, and that the radial polar lines are much 
more intense. As the cleavage advances the two poles move still 
farther apart and the radial lines reach their maximum intensity. 
It is plain to see that the power which cleaves the egg resides in 
the nucleus. During the cleavage the two poles of the amphiaster 
act as if repelling each other, moving farther and farther apart 
and pulling the passive yolk elements with them. Thus they 
appear as centres of attraction for the yolk. 
Approach of the two cleavage spheres.—As soon as the cleavage 
is completed, the two parts, instead of parting company, begin to 
approach, flattening against each other, until at length they to- 
gether form a perfect sphere —as perfect as the undivided egg. 
The line of division is not, however, obliterated. Why do the 
two parts of the egg approach in this manner? This fact has 
been often noted and has been generally explained as the result of 
pressure exerted by the elastic membrane of the egg. This expla- 
nation, however, cannot apply to those cases where the egg lies at 
a great distance from its membrane, as in the case of many Mol- 270 
luscan eggs. I have good evidence that it does not apply in any 
case. I once liberated an egg from its membrane, and thus had 
an opportunity to follow the cleavage unhindered by the membrane. 
At the close of the cleavage the two parts touched each other only 
at a common point. Subsequently they began to approach, flat- 
tening on their contiguous faces, and finally formecj together a 
perfect sphere. If we follow the two nuclei that have resulted 
from the division of the amphiaster, during the approach, we find 
that they take the lead in the movement. The cleavage as well as 
the subsequent approach of the cleavage-spheres are therefore due 
to nuclear influence. 
CHANGES IN THE EGG OF CLEPSINE ; BT C. O. WHITMAN. 
“ Faltenkranz.”—ln conclusion I will call attention to some 
interesting radial phenomena which have never been satisfactorily 
explained. 
Kleinenberg, in his well known work on the development of 
Hydra, states that the cleavage begins on one side of the egg, and 
that pseudopodial processes arise from the walls of the cleavage- 
groove, on this side. Metschnikoff observed folds on the walls 
of the cleavage-groove in the eggs of certain Siphonophores. As 
long ago as 1824 similar folds (“Faltenkranz,” Reichert) were 
observed by Provost and Dumas on the Amphibian egg. These 
folds were seen by von Baer, and more recently by Gbtte. They 
have been made a subject of special study by Max Schultze and 
Reichert. According to every explanation thus far offered of these 
folds, they should appear at right angles to the plane of cleavage 
in its entire circuit. This, however, is not the case. They appear 
only on one side of the egg, and instead of being at right angles 
to the plane of cleavage, they are at right angles only at the 
centre, diverging more and more towards either end of the groove, 
thus forming a kind of long star. From what has before been 
said, it is evident that the radial influence of the two poles of the 
amphiaster is greatest at the moment the cleavage appears, and 
that the cleavage begihs on one side, rather than on all sides 
simultaneously, because the nucleus lies eccentrically—always 
nearer the side on which the cleavage begins. It seems, therefore, 
highly probable that these “processes” and “folds” are merely 
the outward expression of the radial influence of the nucleus.1 
1 For a more detailed account of the above phenomena, see Quart. Journ, Mic. Sci., 
J aly, 1878. 
[Printed at the Salem Press, February, 1879.]