LIBRARY

OF THE

UNIVERSITY OF CALIFORNIA.

QtltAJJa

GIFT OF

Class

A CYTOLOGICAL STUDY OF ARTIFICIAL

PARTHENOGENESIS IN STRONGYLO-

GENTROTUS PURPURATUS

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE

UNIVERSITY OF CALIFORNIA

BY

EDWARD HINDLE

BERKELEY, CALIFORNIA
May, 1910

A Cytological Study of Artificial Parthenogenesis in
Strongylocentrotus purpuratus

by

Edward Hindle

With one plate

Sonderabdruck aus dem

Archiv fur Entwicklungsmechanik der Organismen

Herausgegeben von Prof. Dr. Dr. Wilh. Roux in Halle a/S.
XXXI. Band, 1. Heft

Ausgegeben am 8. November 1910

Of THE

UNIVERSITY

OF

UFOR

Leipzig

"Wilhelm Engelmann
1910

Das

Archiv fiir Entwicklungsmechanik der Organismen

steht offen jeder Art von exakten Forschungen iiber die ,,Ursachen" der
Entstehung, Erhaltimg und Riickbildung der organischen Gestaltungen*).

Bis auf weiteres werden auch kntische Referate und zusammen-
fassende Ubersichten Uber andern Orts erschienene Arbeiten gleichen
Zieles, sowie Titelubersichten der beziiglichen Literatur aufgenommen.

Das Archiv erscheint zur Ermb'glichung rascher Veroffent-
lichung in zwanglosen Heften sowohl in bezug auf den Umfang, wie
auch auf die Zeit des Erscheinens ; mit etwa 40 Druckbogen wird ein
Band abgeschlossen.

Die Herren Mitarbeiter erhalten unentgeltlich 40 Sonderdrucke
ihrer Arbeiten; eine grb'Bere Anzahl Sonderdrucke wird bei Voraus-
bestellung gegen Erstattung der Herstellungskosten geliefert unter
der Voraussetzung, da6 die Exemplare nicht fiir den Handel be-
stimmt sind. Referate, Besprechungen und Autoreferate werden
mit Ji 40. — fur den Druckbogen nach Ablauf des Bandes honoriert.

Die Zeichnungen der Textfiguren sind im Interesse der
rascheren Herstellung womoglich in der zur Wiedergabe durch
Zinkatzung geeigneten Weise auszufiihren**). Die Textfiguren sind
vom Texte gesoudert beizulegen; an den Einfiigungsstellen im
Texte sind die Nummern der bezUglichen Figuren anzubringen. Sind
die eigentlich fiir den Text bestimmten in linearer bzw. punk-
tierter Manier hergestellten Figuren sehr zahlreich, so werden sie
besser auf Tafeln beigegeben. Tafeln sind in der Ho he dem
Format des Archivs anzupassen; fiir jede Tafel ist eine Skizze iiber
die Verteilung der einzelnen Figuren beizufiigen.

Die Erklarungen der Tafel- sowie auch der Textfiguren sind
auf besonderen Blattern beizufiigen.

Die Einsendutig von Manuskripten wird an den Herausgeber
erbeten.

Der Herausgeber: DerVerleger:

Prof. Dr. Wilh. Roux. Wilhelm Engelmann,

HALLE a/S. (Deutschland). LEIPZIG.

*) Den in nichtdeutscher : in englischer, italienischer oder franzb'-
sischer Sprache zu druckenden Originalabhandlungen ist eine kurze Ztt-
sammenfassung der Ergebnisse, sei es in der Sprache des Originals oder in
deutscher Sprache beizufiigen.

**) Dies geschieht in line ar erbzw. pun ktierte rZeichnung mit tiefschwarzer
Tinte oder Tusche, kann aber leicht auch durch nachtragliches Uberzeichnen der
Bleistiftzeichnung mit der Tuschfeder hergestellt werden. Wer jedoch im
Zeichnen mit Feder nicht geiibt ist, kann die einfache Bleistiftzeichnung ein-
senden. wonach sie von technischer Seite uberzeichnet wird. Die Bezeichnungen
(Buchstaben oder Ziffern) sind bloB schwach mit Bleistift einzutragen, sofern sie
der Autor nicht kalligraphisch herzustellen vermag: Anweisungen fiir die
Herstellung wissenschaftlicher Zeichnungen zu Textfiguren mit Aus-
fiihrungen iiber die einzelnen Herstellungsarten und Proben derselben stellt die
Verlagsbuchhandlung den Herren Mitarbeitern gern unentgeltlich zur Verfiigung.

A Cytological Study of Artificial Parthenogenesis
in Strongylocentrotus purpuratus1).

By
Edward Kindle.

With Plate V.

Eingegangen am 14. Mai 1910.

Introduction.

The following investigation was undertaken mainly with the
object of tracing the cytological changes that follow the chemical
fertilization2) of sea-urchin eggs by treatment with a monobasic
fatty acid, followed by treatment with a hypertonic salt solution
(LOEB, '05, '09).

With this method LOEB has shown that it is possible to obtain
from the unfertilized eggs of echinoids practically 100 per cent of
larvae, a large percentage of which are normal. In external features
the process of development is almost identical with that occurring
in normally fertilized eggs and, therefore, it is of interest to deter-
mine whether the cytological changes are also similar to those that
ordinarily follow the entrance of a sperm into the ovum.

The cytological effects of various solutions, mainly on the eggs
of echinoderms, have been investigated by a number of authors.

R. HERTWIG ('96) studied the effect of dilute solutions of strychnine
on the unfertilized eggs of Echinus and SphaerecMnus, and found that
the nucleus, after this treatment, may give rise to a typical mitotic
spindle in which division of the chromosomes takes place. The

!) A thesis submitted in partial fulfillment of the requirements for the
degree of Doctor of Philosophy at the University of California. Berkeley,
May, 1910.

2) For the sake of brevity the term > Chemical fertilization* is employed
to denote the starting of development in unfertilized eggs by chemical means.

ArcMv f. Entwicklungsmechanik. XXXI. 10

218262

146 Edward Hindle

cleavage centrosomes were supposed to arise from the achromatic
part of the nucleus and the chromosomes from the nucleolus.

MORGAN ('96, '99, 1900) found that the cytoplasm of eggs of
Arbacia, when treated with hypertonic solutions of NaCl and MgCl2,
or strychnine, became filled with » artificial astrospheres« each con-
taining a centrosome. The centrosomes were therefore considered
to arise de novo and MORGAN expressed agreement with HERT-
WIG'S view that they may develop out of the achromatic part of the
nucleus.

In 1901 appeared WILSON'S important paper on the cytological
changes following the chemical fertilization of the- eggs of Toxo-
pmustes variegatus by means of LOEB'S older method, in which
magnesium chloride was the agent employed. He found that, in
accordance with LOEB'S observations, the eggs vary considerably in
their response to the hypertonic solution, even when derived from the
same female and lying side by side in the same solution. He goes
on to say (p. 531): » First, even those eggs that segment and give
rise to swimming larvae show wide differences in both the rate and
character of the internal changes and in the ensuing form of cleavage.
Second, a great variety of asymmetrical mitoses and other patholog-
ical phenomena, leading to the production of monstrous forms always
occur in a considerable number of the eggs. Third, numerous gra-
dations of the mitotic processes occur, the incomplete mitoses show-
ing innumerable modifications ranging from a hardly perceptible
change up to a nearly complete division. « As a result of his exam-
ination, WILSON showed that both cleavage-asters and cytasters to-
gether with their centrosomes may arise de novo. Further, the
cleavage aster arises from the nucleus and its centrosome is more
distinct than that of the cytasters. The chromosomes were found to
arise in two different ways; in one case, they arise in the ordinary
way direct from the reticulum, whereas in the other case they are
built up by the nucleolus. Moreover only eighteen chromosomes were
found instead of the normal number of thirty six.

In the same year DELAGE (1901) published the results of his
cytological study of artificial parthenogenesis in the eggs of Strongylo-
centrotus lividus. His results differed markedly from those of WILSON,
for the full number of chromosomes was stated to be restored in the
artificial embryos, DELAGE having found nine chromosomes in the
unfertilized ovum of this species and eighteen in the cells of the
parthenogenetic larvae.

A Cytological Study of Artificial Parthenogenesis etc. 147

BOVERI ('02) and PETRUNKEWITSCH ('04) have both shown that
the number of chromosomes normally occurring in the cells of this
species is 36, and that the number occurring in the unfertilized egg
is 18. The latter is the number of chromosomes found by DELAGE in
the parthenogenetic larvae, and therefore his own observations support
the idea that after chemical fertilization the full number of chromo-
somes is not restored during subsequent development. In spite of
this, however, in a recent paper DELAGE ('08) still adheres to his
former view that the number of chromosomes constant for the species
is restored in the cells of the parthenogenetic larvae. This view
seems to be based mainly on theoretical considerations for no ob-
servations are brought forward in support of it1).

WASSILIEFF ('02) and PETRUNKEWITSCH ('04) have studied the
cytological effects of artificial fertilization in echinoids, but in both
cases their methods and results were imperfect, as they used the
older method of treatment with a hypertonic salt solution in order
to cause development.

The only complete account of the cytological details of artificial
parthenogenesis in echinoids is WILSON'S ('01), for the later studies
of HERBST ('07, '09) and other authors, do not concern the finer
cytology of development. As GODLEWSKI ('09) has given a summary
of this subject it will be unnecessary to discuss in detail the various
studies of artificial parthenogenesis in other groups that have ap-
peared during the last few years. In all cases where the number
of chromosomes in the parthenogenetically developing eggs was deter-
mined the reduced number was found to occur, but these observa-
tions have been confined to the first few divisions. On these grounds,
therefore, DELAGE considers the question undecided, as the restoration
to the full number is supposed to take place gradually.

In the following account it will be seen that the reduced number
of chromosomes persists in the cells of parthenogenetic embryos of
Strongylocentrotus purpuratus as far as the free swimming blastula,
and beyond this stage it is impossible to count them owing to the
small size of the cells. These observations, therefore, agree with
those of KOSTANECKI ('04, '08) on Mactra, LEFEVRE ('07) on Tha-

*) >0n voit que, si les partisans de la non-regulation du nombre des chromo-
somes avaient raison, leur nombre devait se reduire de moitie" a chaque gene-
ration, jusqu'a ce que la reduction devienne impossible en presence d'un nombre
impair ou, finalement, de 1'unite* (DELAGE, p. 497). The logic of this remark is
not very forcible.

10*

148 Edward Hindle

lassema, and other investigators, but are opposed to the theory of
DEL AGE.

LOEB ('09) has shown that in sea-urchins, and many other classes
of animals, the developmental effects of fertilization consist essenti-
ally of two processes; first, in an alteration of the cortical layer of
the egg, presumably a cytolysis, resulting in the formation of a mem-
brane; and, secondly, in a modification of the processes of oxidation,
probably by the destruction of some substance within the egg that
is injurious to the synthetic processes started by the membrane
formation.

The first process may be effected by any cytolytic agent and if
cytolysis is not carried too far the egg begins to develop. Among
the agents that are able to cause membrane formation in the egg
the following may be mentioned: In the first place we have those
substances having a specific cytolic action on cells in general, such
as saponin, solanin, digitalin, bile -salts and soap; then we have
the simple fat solvents such as benzol, toluol, amylen, and chloro-
form, which cause membrane formation very rapidly and, in addition,
completely cytolize the eggs in a short time; alcohol, ether, and
even distilled water are also effective ; but the most convenient agents
to employ are solutions of alkalies, or of acids, especially monobasic
fatty acids.

In the presence of free oxygen this membrane formation starts
the oxidations underlying the synthesis of nucleins and other pro-
cesses of development, and in some forms (e. g. Thalassema and
Asterina) the eggs continue to segment normally and develop into
free swimming larvae. But the eggs of sea-urchins seem to contain
some substance that has an injurious effect upon the syntheses under-
lying development. By the time that the first spindle is formed the
eggs begin to disintegrate and at ordinary temperatures (15° C.) the
majority do not complete the first division. It is true that by keep-
ing them at a low temperature (2 — 5° C.) they may segment normally
for some time, but only a very small percentage develop into blas-
tulae. Therefore a second process is necessary in order to eliminate
this disturbing factor of development. After membrane formation, the
subsequent treatment with hypertonic sea water, containing oxy-
gen, for from thirty to fifty minutes is sufficient to destroy this in-
jurious substance, and if the eggs are then transferred back into
ordinary sea water development proceeds regularly and a large per-
centage develop into normal larvae. Instead of employing a hyper-

A Cytological Study of Artificial Parthenogenesis etc. 149

tonic solution, the same result may be obtained by preventing the
oxidations in the eggs for about three hours, either by putting them
in sea water containing a little potassium cyanide, or simply by re-
moving the oxygen from the sea water by means of a stream of puri-
fied hydrogen.

Technique.

The eggs of Strongylocentrotus purpumtus were the only kind
employed, and in all cases were obtained by taking out the gonads
from a female and allowing them to remain in a dish of sterilized
sea water for a few hours. The ripe eggs gradually drop out of the
ovaries and fall to the bottom of the dish where they may be col-
lected by means of a pipette.

Membrane formation was effected by allowing the eggs to re-
main in a mixture of 50 cc.s. sea water -f- 2.9 cc.s. of N/10 butyric
acid from 90 to 150 seconds, according to the temperature. The eggs
were then transferred back into normal sea water and if they had
been in the butyric acid solution for the correct length of time all
of them formed membranes. As it was desired to study the cyto-
logical effects of butyric acid alone, some of these eggs were allowed
to develop without further treatment but at 15° C. very few of them
completed even the first division.

When the eggs from the butyric acid solution had remained in
normal sea water for about 20 minutes, they were then placed in a
mixture of 50 cc.s. sea water + 8 cc.s. of 2y2 N-NaCl and exposed
to the action of this solution for times varying from 30 to 60 mi-
nutes. The length of exposure necessary depends mainly upon the
temperature, but also on the length of time the eggs have remained
in the sea water after removal from the butyric acid solution. Finally,
the eggs were again transferred into normal seawater and, if the
time of exposure to the hypertonic solution had been correctly chosen,
practically all them developed and gave rise to free swimming larvae.

The various stages in the formation of spindles and segmentation
were followed under the low power of the microscope and embryos
were obtained at all stages of development.

The material was fixed in FLEMMING'S strong solution, and im-
bedded in paraffin in the usual way. Sections were generally cut
3 u in thickness and stained on the slide by one of the three follow-
ing methods:

150 Edward Hindle

a) ARNOLD'S Method : Mordant the sections for ten minutes in a
dark brown solution of Iodine and Kl in 70% alcohol. Rinse in 70%
alcohol and stain for two hours in a saturated solution of Basic Fuchsin
in 75% alcohol. Wash in water and then stain for ten minutes
in a 1% aqueous solution of Polychromatic Methylene Blue. Rinse
with water and pass up rapidly through the alcohols into a satur-
ated solution of Orange G in clove oil. Allow the sections to differ-
entiate in this solution until they appear more pink than blue, then
wash in clove oil, followed by xylol, and mount in Canada Balsam.

b) HEIDENHAIN'S Haematoxylin Method (slightly modified). Mord-
ant the sections for ten minutes in an alcoholic solution of Iodine
in Kl. Then mordant in a 3.5% aqueous solution of iron alum for
about twelve hours. Stain for an equal length of time in a 0.5%
aqueous solution of haematoxylin, artificially ripened by the addition
of a few drops of lithium carbonate solution; then differentiate and
mount in the usual way.

c) MALLORY'S Phospho-Tungstic Haematoxylin Method. Mordant
the sections for ten minutes in a 0.25 % aqueous solution of potassium
permanganate. Rinse in water and clear in a 5 % aqueous solution
of oxalic acid for twenty minutes. Wash thoroughly and then stain
for twelve to twenty four hours in a solution of phospho-tungstic
haematoxylin. Rinse in water, pass up rapidly through the alcohols
into xylol, and mount in Canada balsam.

ARNOLD'S stain was found to give the most reliable results but
for the sake of comparison sections were stained by all three me-
thods.

The experimental part of the work was performed at the Herz-
stein Research Laboratory, Pacific Grove, under the direction of Pro-
fessor LOEB, without whose assistance and kindly criticism it would
have been impossible to complete this study. The cytological exam-
ination of the eggs was completed in the Zoological Department of
the University of California, with Professor KOFOID, to whom it gives
me great pleasure to express my indebtedness for much help received
in this part of the work.

External Changes after Artificial Fertilization.

Under a low power of the microscope the unfertilized egg of
S. purpuratus appears as an opaque spherical body about 60 p in
diameter. During treatment with butyric acid it presents no morpho-

A Cytological Study of Artificial Parthenogenesis etc. 151

logical changes, but as soon as the egg is transferred hack into
normal sea water the process of membrane formation commences.
Small vesicles appear over the whole surface increasing in size until
they run together and form a clear transparent layer completely
surrounding the egg and bounded on the outside by the surface film
that has been raised up by this cytolytic process. The thickness of
this transparent outer layer is usually about 7 (.t but it may vary
considerably according to the amount of cytolysis that takes place
after the action of the butyric acid.

The membrane formation is complete a few minutes after trans-
ference to normal sea water. It is accompanied by the more definite
appearance of a light space in the cytoplasm, marking the position
of the nucleus. The greater distinctness of this clear space at this
stage is due to the dissolution of the cytoplasmic granules immedi-
ately surrounding the nucleus to form a clear perinuclear zone.

About 30 minutes after the completion of the subsequent treat-
ment with hypertonic solution, the cleavage aster appears. As it
develops the nucleus becomes still more distinct and the various stages
in the formation of the spindle and subsequent division of the nucleus
can be observed. In those eggs that segment normally the first three
cleavages give rise to eight cells of approximately equal size. The
fourth cleavage results in the formation of four smaller cells at one of
the poles. After this stage the divisions become less regular but eventu-
ally a blastula is produced which develops cilia on its outer surface
and becomes free swimming. These blastulae swim about at the sur-
face of the water and behave in exactly the same way as normal
larvae1). The blastula undergoes the process of invagination giving
rise to a gastrula and from this is developed the pluteus with its
typical skeleton.

The Unfertilized Ovum.

In sections the unfertilized egg of S. purpuratus is rather opaque
owing to the large number of yolk granules scattered through the
cytoplasm. These granules are more abundant at the periphery,
thus forming a kind of ectoplasmic layer. The nucleus appears as
an oval, lightly-staining area, bounded by a definite membrane, and

*) Those that develop from eggs treated simply with a hypertonic solution
(LOEB'S first method) are unable to rise to the surface of the water but can
only swim about the bottom.

152 Edward Hindle

contains a fine meshwork of chromatin together with a deeply stain-
ing nucleolus. The latter usually contains numerous vacuoles, and
is not a mere aggregation of chromatin.

Internal Changes after Treatment with Butyric Acid.

After the membrane formation is complete the cytoplasmic
granules hecome aggregated together in clumps and therefore the
protoplasm loses its regular appearance.

A few minutes later the nucleolus shows unmistakable signs of
dissolution, as it stains much more lightly, and may even break
down into two or more smaller ones (Fig. 2,^). Meanwhile the granules
in the vicinity of the nucleus disappear, and thus it becomes sur-
rounded by a clear zone, from which faint radiations extend into
the cytoplasm (Fig. 2).

Certain of the granules immediately surrounding this region stain
much more deeply than others (Fig. 2, ch.gr.). Probably this ap-
pearance is due to the formation of acids, as a result of oxidation
processes starting from the nucleus and extending out into the cyto-
plasm, and LOEB ('06) has shown that acids are formed in develop-
ing eggs.

This is succeeded by a rapid growth of the nucleus which in
some cases increases to as much as three times its original diameter.
The chromatin then aggregates together and assumes the form of a
spireme which subsequently breaks up into 18 chromosomes.

In eggs that are developing at ordinary temperatures, about two
hours after treatment with butyric acid very distinct astral rays appear,
extending radially from the now indistinct nuclear membrane into
the cytoplasm and apparently centring in the nucleus. This radiation
does not usually resolve itself into a bipolar figure but persists as
a monaster. Frequently the chromosomes divide and become drawn
out along the rays so as to appear scattered throughout the cyto-
plasm (Fig. 14). This may be succeeded by a reduction of the rays
and a reconstruction of the nucleus with an increased number of
chromosomes. A redevelopment of the monaster may now follow and
the whole series of processes be repeated two or three times. The
changes observed agree, therefore, with WILSON'S description of the
behaviour of monasters in eggs that have been treated with mag-
nesium chloride ( WILSON, '01). Within 2 or 3 hours these eggs be-
gin to degenerate by a process of cytolysis (Fig. 13), by means of

A Cytological Study of Artificial Parthenogenesis etc. 153

which the protoplasm becomes broken up into a mass of small par-
ticles lying within the fertilization membrane.

If, after treatment with butyric acid, the eggs are kept at a
low temperature (2 — 5° C.) the first few divisions may be accomplished.
In this case the period of nuclear growth is succeeded by the almost
complete disappearance of the perinuclear zone, and the subsequent
development of a typical amphiaster in the region of the nucleus.
The succeeding changes are identical with those described below,
that take place in eggs that have been subsequently treated with
hypertonic solution.

We have never observed any cytasters to be developed in eggs
treated with butyric acid alone; usually a monaster appears which
may divide to form a typical cleavage -aster or, more often, re-
mains undivided. In all cases, therefore, the rays that develop in
these eggs originate from the nucleus and do not have an indepen-
dent cytoplasmic origin as in the case of cytasters.

Internal Changes after Treatment with Butyric Acid
followed by a Hypertonic Solution.

(LOEB'S improved method of artificial fertilization.)

The interval (about 20 minutes) between the transference of the
eggs from butyric acid to normal sea water and their subsequent
treatment with hypertonic salt solution is characterized by the
alterations in the appearance of the cytoplasm and nucleolus, and
the subsequent development of a perinuclear zone, as described above.
The nucleus then commences to grow and faint radiation can some
times be seen extending from the perinuclear zone into the surround-
ing cytoplasm (Fig. 2).

During immersion in the hypertonic solution there are no apparent
changes beyond a slight reduction of the clear zone of hyaloplasm
surrounding the nucleus (Fig. 3).

After the eggs are put back into normal sea water the internal
changes resulting in the first cleavage follow each other in quick
succession. The first change noticed is an increase in the development
of the perinuclear zone, followed by further growth of the nucleus.
Meanwhile, the meshwork of chromatin becomes coarser and more
aggregated together and the nucleolus gradually disappears. This
stage is succeeded by a reduction of the perinuclear zone together
with its radiations.

154 Edward Hindle

About half an hour after transference to normal sea water, from
one pole of the nucleus a definite aster begins to develop, its rays
focussing in a more or less indistinct centrosome situated on the
nuclear membrane (Fig. 4). By division of the centrosome a typical
amphiaster is formed in the nuclear area and as it develops the
nuclear membrane disappears (Fig. 15). At the same time the
chromatin assumes the form of a spireme, which subsequently breaks
up into about 18 long and slender chromosomes. At this stage it is
impossible to clearly distinguish their number, but, as the chromo-
somes are gradually drawn into the equator of the cleavage amphi-
aster (Fig. 6), they shorten considerably and become quite distinct
by the time that the equatorial plate is formed (Fig. 5).

At this stage we have made numerous counts of the chromo-
somes and invariably found it in the neighbourhood of 18, which is
half the number that is normally present in this species.

During the metaphase each chromosome splits longitudinally, and
in the succeeding anaphase the halves move away from each other
along the spindle fibres, 18 going to each pole (Fig. 7). As they
approach the centrosomes the chromosomes swell up and become
indistinct, and finally fuse together to form two daughter nuclei.
These changes are accompanied by a division of the cytoplasm into
two equal parts, followed by complete disappearance of the spindle
fibres, and the first cleavage is complete (Fig. 8).

Each daughter nucleus remains partially surrounded by a clear
region that seems to represent the centrosome together with a
slight accumulation of cytoplasm, and here the cleavage aster of
the next division first makes its appearance. After the first cleav-
age is complete the succeeding divisions follow each other in re-
gular succession and present all the features of ordinary mitosis
(Fig. 9).

In each case the nucleus becomes completely surrounded by a
clear zone of hyaloplasm. This is followed by a period of nuclear
growth during which this perinuclear zone almost disappears. From
the centrosome, that has persisted at one pole of the nucleus, rays
now appear extending out into the cytoplasm (Fig. 10) and by the
subsequent division of the centre of attraction a typical amphiaster
is produced (Figs. 9 — 10). As it develops the nuclear membrane
disappears and the chromatin breaks up into 18 chromosomes which
arrange themselves about the equator of the amphiaster (Fig. 9).
In this way a typical mitotic spindle is formed in the nuclear area

A Cytological Study of Artificial Parthenogenesis etc. 155

and the succeeding stages in the division of the cell are identical
with those described above.

The cytoplasmic granules are gradually used up as development
proceeds and disappear before the gastrula stage is reached.

As the cells become smaller at each division, it becomes in-
creasingly more difficult to count the number of chromosomes with
any degree of accuracy, owing to their smaller size and also to the
smallness of the cells, which prevents the chromosomes separating
distinctly. But it is possible to obtain a close approximation of their
number as late as the free swimming blastula stage, in which there
are at least 512 cells, and we have invariably found it to be in the
neighbourhood of 18 (Fig. 11). The reduced number of chromosomes,
therefore, may reasonably be supposed to persist throughout the
further stages of development, for a multiplication of their number
has never yet been observed in any dividing cells.

In addition to the normal processes of development described
above, which occur in the majority of the eggs, abnormal pheno-
mena are always found in some of them. The reason for this
is evident, for it is obviously impossible to secure for all the eggs
exactly the same supply of oxygen and hence the effect of the
hypertonic solution cannot be the same for all the eggs taken out
at one time.

The most commonly occurring abnormality is the development
of a varying number of asters in the cytoplasm quite independently
of the cleavage aster, which arises from the nuclear region. These
cytasters develop in eggs that have been over-exposed to the action
of the hypertonic solution and have not been observed after treatment
with butyric acid alone.

Each cytaster consists of a number of fibres radiating from a
more or less indistinct central granule into the surrounding cytoplasm
(Fig. 15). As a rule they do not attain much development but remain
scattered around the periphery of the cell and disappear before the
completion of the first division. In certain cases, however, they may
become so well developed as to interfere with the normal division
of the egg, the rays of cytasters in the region of the nucleus be-
coming attached to the chromosomes and thus producing multipolar
mitoses. In these cases the chromosomes are divided irregularly
between the two poles of the cleavage amphiaster and the centres
of as many cytasters as form a connection with the nucleus.

A cell wall may develop between any two centres of attraction

156 Edward Hindle

connected by spindle fibres, and therefore, in the case of multipolar,
mitoses, the egg may divide into as many cells as there are centres
of attraction Cytoplasmic cleavage does not necessarily follow a
multipolar mitosis, but the resulting nuclei may remain scattered
through the undivided cytoplasm of the egg and redivide, thus pro-
ducing a multinucleate cell.

In addition to the formation of multipolar spindles the centres
of the cytasters may divide and thus produce a typical amphiaster
which may operate as a centre of cytoplasmic division independently
of the cleavage amphiaster.

Occasionally a cell wall is developed between the two centres
of one of these cyt-amphiasters and thus an enucleated portion of
cytoplasm may become divided off from the main mass.

In every case the formation of cytasters results in the production
of more or less irregular segmentation, or may completely stop
development.

These cytasters resemble those described by WILSON in the eggs
of Arbacia after treatment with hypertonic salt solutions. The pheno-
mena observed by us in the eggs of S.purpuratus entirely support
his conclusions that » there is no possibility of drawing any other
than purely an arbitarary distinction between cytasters and nuclear
asters in their relation to the nucleus « and »that the central bodies
of the cytasters are true centrosomes that are formed de nove in
the cytoplasm «.

Comparison of the Cytological Effects of Natural and Chemical

Fertilization.

In comparing these two processes it is necessary to avoid con-
sideration of those pathological phenomena that are always present
in a certain percentage of the chemically fertilized eggs. When the
sperm enters the ovum it introduces the exact amount of substances
(lysins, etc.) necessary to start normal development and there is little
chance of error. But when one attempts to imitate this process
chemically the possibilities of error are much greater. In the first
place the cytolytic substance may enter in excess; and similar
difficulties arise in the subsequent exposure of the eggs to the action
of the hypertonic solution, through the impossibility of ensuring
exactly the same exygen supply for all of the eggs.

Therefore, the embryos that present pathological features should

A Cytological Study of Artificial Parthenogenesis etc. 157

be ignored in comparing the processes of natural and chemical
fertilization.

In both cases the first effect of fertilization is to start a cytolytic
process resulting in the formation of a membrane. In naturally
fertilized eggs the sperm nucleus in its passage through the cytoplasm
is accompanied by a radiation which comes in contact with the egg
nucleus and there spreads out on one pole of it. The fusion of the
two nuclei which now takes place is followed by a period of nuclear
growth during which the radiations almost disappear. Meanwhile
the nucleolus exhibits unmistakeable signs of dissolution, as it
changes from being a dense spherical body to a faintly staining and
often indistinct mass.

In the chemically fertilized eggs the changes are very similar.
The egg nucleus becomes surrounded by a perinuclear zone with
faint radiations extending into the cytoplasm. This stage probably
corresponds with the radiation that appears near the sperm nu-
cleus and forms a clear zone at one end of the cleavage-nucleus
after the two germ nuclei have fused. Moreover, just as in the
normal eggs, it is succeeded by a period of nuclear growth during
which the perinuclear zone with its radiations practically disappear
and also the nucleolus shows signs of dissolution. The appearance
of this radiation is probably the result of oxidation processes starting
from the nucleus, which result in the solution of the yolk granules
and is followed by the flowing of these solutions towards the nucleus.
They are used up in the synthesis of chromatin,, and hence this
process is followed by an increase in the size of the nucleus. With
the cessation of nuclear activities preparatory to division cytoplasmic
solutions no longer flow towards the nucleus and hence the radia-
tions caused by these centripetal currents disappear. From the
alteration in its appearance it is probable that the nucleolus also
takes some part in the nuclear syntheses.

In both kinds of eggs the period of nuclear growth is succeeded
by an apparent cessation of nuclear activities preparatory to division.

In naturally fertilized eggs a distinct aster (cleavage aster)
appears at one pole of the nucleus, its rays centering in a clear
area which represents a diffuse centrosome. This area divides and
the two halves move apart until they come to lie at opposite sides
of the nucleus and form the poles of a typical amphiaster which is
developed in the nuclear region. Meanwhile the chromatin assumes
the form of a spireme, which breaks up into 36 chromosomes that

158 Edward Hindle

arrange themselves about the equator of this amphiaster to form a
nuclear spindle. In the chemically fertilized eggs a nuclear spindle
arises in a similar way and the chromatin assumes the form of a
spireme preparatory to breaking up into chromosomes, but, instead
of 36, only 18 of these latter bodies appear. The subsequent changes
are identical in both kinds of eggs. The chromosomes split longitu-
dinally and each half moves along the spindle fibres towards its
respective pole. As they approach the poles the chromosomes swell
up and eventually fuse together to form a single nucleus in the
region occupied by each of the diffuse centrosomes. Meanwhile a
cell wall develops between the two nuclei dividing the cytoplasm
into two, and finally the spindle fibres disappear. The succeeding
processes of development, both internal and external, are similar
in both naturally and chemically fertilized eggs, with the exception
that at each succeeding division only 18 chromosomes appear in the
latter instead of the normal number, 36.

Summary of Results.

A cytological study of the changes occurring in the eggs of
Strongylocentrotus purpuratus after chemical fertilization by means
of LOEB'S improved method.

(A.) Effect of treatment with butyric acid.

1) The first change is the starting of a process of cytolysis
resulting in the formation of a fertilization membrane.

2) This is accompanied by an alteration in the appearance of
the nucleolus, which, from being a dense mass of chromatic substance,
changes to a lightly-staining body of somewhat indefinite shape.

3) A dissolution of the cytoplasmic granules in the immediate
neighbourhood of the nucleus results in the appearance of a clear
perinuclear zone. Probably as a result of currents flowing centri-
petally from the cytoplasm towards the nucleus, radiations appear
in and around this zone.

4) The appearance of these radiations is succeeded by a period
of growth during which there is an increase in the size of the
nucleus.

5) In eggs that are developing at ordinary temperatures a large
monaster is now developed, it's rays centering in the nucleus. The
nuclear membrane disappears and the chromatin breaks up into 18
chromosomes. These may undergo division and be drawn out of the

A Cytological Study of Artificial Parthenogenesis etc. 159

nuclear area along the rays, in which case, they appear scattered
through the cytoplasm. Such eggs never divide but simply disinte-
grate by a process of cytolysis.

6) In eggs that are developing at a low temperature (2 — 5° C.)
the first few divisions may be accomplished, in which case the period
of nuclear growth is succeeded by exactly the same changes as
those occurring in eggs that have been subsequently treated with
a hypertonic solution, described below.

7) Cytasters are not developed.

(B.) Effect of treatment with butyric acid followed by treatment
with hypertonic solution.

8) The interval (15 — 20 mins.) between treatment with butyric
acid and with hypertonic salt solution is characterized by the
membrane formation and an alteration in the staining properties of
the nucleolus. These changes are accompanied by the appearance
of a clear perinuclear zone as described above.

9) During the treatment with hypertonic salt solution there is
a slight increase in the size of the nucleus and the clear zone almost
disappears.

10) After transference of the eggs back into normal sea water
the perinuclear zone reappears, and is followed by further growth
of the nucleus.

11) A typical cleavage aster develops in the nuclear region, its
rays being focussed in two areas, one at each pole, that probably
represent diffuse centrosomes.

12) A varying number of asters, quite independent of the
cleavage aster sometimes appear in the cytoplasm. If excessively
developed they interfere with normal division of the cell and multi-
polar spindles are often formed. In eggs that have not been exposed
too long to the action of the hypertonic solution, the cytasters dis-
appear before the completion of the first division.

13) The chromatin assumes the form of a spireme and then
breaks up into 18 chromosomes, which is exactly half the number
occurring in normally fertilized eggs. They arrange themselves in
the cleavage aster, and divide in the usual way forming two daughter
nuclei each with 18 chromosomes. This process is accompanied by
division of the cytoplasm.

14) The reduced number of chromosomes, 18, persists in the
cells of the parthenogenetic larvae at least as far as the blastula,

160 Edward Hindle

and beyond this stage it is impossible to count them owing to the
small size of the cells.

Zusammenfassung der Ergebnisse,

Die cytologischen Anderungen, welche nach der Behandlung des Eies von
Strongylocentrotus purpuratus mit LOEBS verbesserter Methode der kunstlichen
Parthenogenese eintreten, wurden untersucht mit folgendem Resultat:

A. Behandlung der Eier mit Buttersaure allein.

1) Die erste Anderung im Ei nach der Behandlung mit Buttersaure ist ein
cytolytischer ProzeC an der Oberflache des Eies, der zur Bildung einer Befruch-
tungsmembran fiihrt.

2) Gleichzeitig findet eine Anderung im Aussehen des Nucleolus statt. der
vorher eine dichte Masse chromatischer Substanz war und nun sich in einen
Kb'rper umwandelt, der sich nur leicht farbt und etwas unbestimmte Form hat,

3) Spater erfolgt eine Auflosung der cytoplasmischen Kb'rner in der un-
mittelbaren Umgebung des Kerns, so daC eine helle perinucleare Zone sichtbar
wird. Strahlungen erscheinen in und um diese Zone.

4) Auf die Bildung dieser Strahlung folgt eine Periode der Volumzunahme
des Kerns.

5) In Eiern, die sich bei Zimmertemperatur entwickeln, bildet sich jetzt
ein Monaster, dessen Strahlen ihren Mittelpunkt im Kern haben. Die Kern-
membran verschwindet, und das Chromatin sammelt sich in 18 Chromosomen.
Solche Eier gehen an einem cytolytischen ProzeC zugrunde.

6) Bei niederer Temperatur (2 — 5° C.) kann es zu einer Reihe von Zelltei-
lungen kommen. In diesem Falle folgen auf die Periode des Kernwachstums
dieselben Anderungen, welche sich bei Eiern finden, welche nach der Butter-
saurebehandlung fur kurze Zeit in eine hypertonische Losung gebracht werden
und die sub B. erwahnt werden.

7) Durch die Buttersaurebehandlung werden keine Cytaster hervorgerufen.

B. Veranderungen, welche bei Eiern eintreten, die erst mit Buttersaure
und dann mit hypertonischem Seewasser behandelt werden.

8) In dem Zeitraum zwischen Membranbildung (durch Buttersaurebehand-
lung) und der Anwendung der hypertonischen Lb*sung (15—20 Minuten) findet
eine Anderung in der Farbbarkeit des Nucleolus statt, und die Bildung einer
hellen perinuclearen Zone.

9) Wahrend dem Verweilen der Eier in der hypertonischen Salzlosung
findet eine kleine Volumzunahme des Kerns statt, und die helle Zone verschwin-
det fast ganzlich.

10) Nachdem die Eier aus der hypertonischen Losung in Seewasser zu-
ruckgebracht sind, erscheint die perinucleare Zone wieder, und der Kern ent-
wickelt sich.

11) Eine typische Spindelfigur mit zwei Strahlensystemen bildet sich in
der Kernregion; in jedem der beiden Centren ist vermutlich ein diffuses Cen-
trosom.

12) In manchen Eiern bildet sich unabhangig von der Kernspindel eine
variierende Zahl von Astrospharen im Protoplasma. Wenn sie ungewohnlich
stark entwickelt sind, stb*ren sie die normale Zellteilung, indem es zur Bildung

A Cytological Study of Artificial Parthenogenesis etc. 161

multipolarer Spindeln kommt. In Eiern, die nicht zu lange dem hypertonischen
Seewasser ausgesetzt gewesen sind, verschwinden aber diese Cytaster, ehe die
erste Furchung vollendet ist.

13) Das Chromatin nimmt die Form eines Spirems an und trennt sich dann
in 18 Chromosomen, also in halb soviel, wie in normal befruchteten Eiern vor-
handen sind. Diese ordnen und teilen sich in der Spindel in der gewohnlichen
Weise, indem sie zwei Tochterkerne mit je 18 Chromosomen bilden. Dieser
ProzeC wird von einer Teilung des Cytoplasmas begleitet.

14) Die reduzierte Chromosomenzahl, namlich 18, bleibt dauernd in den
Zellen bestehen, zum mindesten bis zum Blastulastadium ; in spateren Stadien
gelang es nicht, dieselben zu zahlen, wegen der geringen Zellgrb'Ce.

Literature,

BOVERI, TH.

1895. Uber die Befruchtungs- und Entwicklungsfahigkeit kernloser Seeigel-

eier und Uber die Moglichkeit ihrer Bastardierung. Arch. f. Entw.-
Mech. Vol.2, pp. 394-443.

1900. Zellenstudien. Heft IV. Jena.
1905. Zellenstudien. Heft V. Jena.

1907. Zellenstudien. Heft VI. Jena.
DELAGE, YVES.

1901. Etudes Expe"rimentales sur la Maturation Cytoplasmique et sur la

Parthenogenese artificielle chez les Echinodermes. Arch. Zool. Exp.
3rd series. Vol. 9. pp. 285-326.

1902. Nouvelles Recherches sur la Parthenogenese Experimentale chez Aste-

rias glacialis. Arch. Zool. Exp. 3rd series. Vol. 10. pp. 213—236

1904. La Parthenogenese par TAcide Carbonique obtenue chez les oeufs

apres remission des Globules Polaires. Arch. Zool. Exp. 4th series.
Vol. 2. pp. 43—46.

1905. Nouvelles Experiences de Parthenogenese Experimentale. Arch. Zool.

Exp. 4th series. Vol. 3. Notes et Revue CLXIV— CLXVIII.

1908. Les vrais facteurs de la parthenogenese experimentale. Elevage des

larves parthenogenetiques jusqu'a la forme parfaite. Arch. Zool. Exp.
4th series. Vol. 7. pp. 445-506.
GODLEWSKI, E., jun.

1909. Das Vererbungsproblem. Leipzig.
HERBST, C.

1909. Vererbungsstudien. VI. Die cytologischen Grundlagen der Verschie-
bung der Vererbungsrichtung nach der mutterlichen Seite. 1. Mitteil.
Arch. f. Entw.-Mech. Vol. 27. pp. 266—308.
HERTWIG, R.

1896. Uber die Entwicklung des unbefruchteten Seeigeleies. Festschrift f.

GEGENBAUR. Vol.2, pp. 21-86.
KOSTANECKI, K.

1904. Cytologische Studien an kiinstlich parthenogenetisch sich entwickeln-

den Eiern von Mactra. Arch. f. mikr. Anat. Vol. 64. pp. 1—98.
1908. Zur Morphologie der kunstlichen parthenogenetischen Entwicklung bei

Mactra. Zugleich ein Beitrag zur Kenntnis der vielpoligen Mitosen.

Arch. f. Anat. Vol. 72. pp. 327—355.

ArcMv f. Entwicklungsmechanik. XXXI. 11

162 Edward Kindle

LEFEVRE, GEORGE.

1907. Artificial Parthenogenesis in Thalassema Mellita. Journ. Exp. Zool.

Vol. 4. pp. 91—149.
LOEB, J.

1905. On an Improved Method of Artificial Parthenogenesis. Univ. Cal.

Publ., Phys. Vol. 2. pp. 113—123.

1906. Versuche tiber den cheinischen Charakter des Befruchtungsvorganges.

Biochemische Zeitschrift. Vol.1, pp. 183— 207.

1906. Weitere Beobachtungen uber den EinfluB der Befruchtung und der
Zahl der Zellkerne auf die Saurebildung im Ei. Biochemische Zeit-
schrift. Vol.2, pp. 34-43.

1909. Die chemische Entwicklungserregung des tierischen Eies. Berlin.

(This book contains a complete summary of LOEB'S previous
work on artificial parthenogenesis.)

1909. Uber das Wesen der formativen Reizung. (Vortrag gehalten auf dem

XVI. International Medizinischen KongreC in Budapest.) Berlin.
MORGAN, T. H.

1896. The Production of Artificial Astrospheres. Arch. f. Entw.-Mech. Vol. 3.
pp. 339—361.

1899. The Action of Salt-Solutions on the Unfertilized and Fertilized Eggs

of Arbacia. Arch. f. Entw.-Mech. Vol. 8. pp. 448—539.

1900. Further Studies in the Action of Salt-Solutions and other Agents on

the Eggs of Arbacia. Arch. f. Entw.-Mech. Vol. 10. pp. 489—524.
PETRUNKEWITSCH, A.

1904. Kiinstliche Parthenogenese. Zool. Jahrb. Suppl. 7. Festschrift fur

WEISMANN. pp. 77—138.
SCOTT, J. W.

1906. Morphology on the Parthenogenetic Development of Amphitrite. Journ.

Exp. Zool. Vol. 3. pp. 49—97.
TENNENT, D. H., and HOGUE, M. J.

1906. Studies on the Development of the Starfish Egg. Journ. Exp. Zool.

Vol.3, pp. 517-540.
WASSILIEFF, 0.

1902. Uber kiinstliche Parthenogenesis des Seeigeleies. Biol. Centralblatt.

Vol.22, pp. 758-772.
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1901. Experimental Studies in Cytology. I. Arch. f. Entw.-Mech. Vol. 12.

pp. 529—596.

Explanation of the Figures,

The figures are drawn to a magnification of 750 diameters with the ex-
ception of Fig. 11, which is magnified 1500 diameters. In all cases the drawings
were made from sections stained by one of the three methods described above.

Plate V.

Figs. 1—11. Various stages in the development of eggs of S. purpuratus
chemically fertilized by means of butyric acid, followed by treatment with a
hypertonic salt solution.

A Cytological Study of Artificial Parthenogenesis etc. 163

Fig. 1. Unfertilized ovum. Std. ARNOLD.

Fig. 2. Ovum, 15 minutes after treatment, for 120 sees., with a solution of

butyric acid, showing the appearance of a clear perinuclear zone (p.*.),

surrounded by chromatic granules (ch.gr.}. Std. HEID.
Fig. 3. Ovum 5. minutes after transference from hypertonic salt solution to normal

sea water (treatment undergone: butyric acid 2 mins., water 20 mins., hyper-
tonic salt solution 45 mins., water 5 mins.), showing the appearance of the

perinuclear zone. Std. MALL.

Fig. 4. Appearance of cleavage aster 30 mins. after transference from hyper-
tonic solution. (But. 2 mins., water 20 mins., hyper. 45 mins., water 30 mins.)

Std. HEID.
Fig. 5. Polar view of equatorial plate showing 18 chromosomes. (But. 2 mins.,

water 20 mins., hyper. 45 mins., water 50 mins.) Std. ARNOLD.
Fig. 6. Lateral view of nuclear spindle, drawn from same slide as Fig. 5.
Fig. 7. Anaphase of first division of egg. (But. 2 mins., water 20 mins., hyper.

45 mins, water 80 mins.) Std. MALL.
Fig. 8. Completion of first division. (But. 2 mins., water 20 mins., hyper.

45 mins., water 100 mins.) Std. HEID.
Fig. 9. Anaphase of second division. Std. ARNOLD.
Fig. 10. 8-cell stage of chemically fertilized egg showing stages in aggregation

of the chromatin and development of the cleavage aster. Std. ARNOLD.
Fig. 11. Parthenogenetic blastula, 24 hrs. old, one cell showing equatorial plate

containing 18 chromosomes. Std. HEID.
Fig. 12. Normally fertilized embryo, 4- to 8-cell stage; showing equatorial

plate containing 36 chromosomes. Std. HEID.
Fig. 13. Ovum 3 hours after treatment for 2 mins. with butyric acid (Temp.

16° C.), showing the cytolytic nature of degeneration. Std. MALL.
Fig. 14. Large monaster developed 2y2 hours after treatment with butyric acid

alone. Std. HEID.
Fig. 15. Abnormal embryo showing the development of cytasters (cyt.) 60 mins.

after chemical fertilization. (But. 2 mins., water 20 mins., hyper. 50 mins.,

water 60 mins.) Std. HEID.

11*

Archiv fiirEntwicklungsmechanik. Bd. XXXI.

Edward, ffindte d&l.

Taf. V.

5.

7(9.

elma/tn in, Leipzig.

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HEINRICH LEYPOLDT, Transplantationsversuche an Lumbriciden. Zur Be-
einflussung der Regeneration eines kleinen Pfropfstiickes durch einen
groBeren Komponenten. (Mit 17 Figuren im Text) 1

HEINRICH LEYPOLDT, Transplantationsversuche an Lumbriciden. Trans-
plantation kleiner Hautstiickchen. (Mit 48 Figuren im Text und
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J. F. McCLENDON, On the Dynamics of Cell Division. I. The Electric Charge
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