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/3.

ON THE ORIGIN OP THE SO-CALLED "TEST-
CELLS" IN THE ASCIDIAN OVUM. By J. PLAY-
FAIR MoMURRICH, B. A., Assistant in the Biological Lahora-
lory, University of Toronto. With Plate X.

The following observations have been made in the hope of
elucidating to some extent the nature of the so-called " test-cells,"
so characteristic of the ova of Tunicates. These bodies have been
described by various authors as occurring in the eggs of most of
the commoner forms, and under normal circumstances probably
do not make their appearance until after fertilization. Lacaze-
Duthiers^^^ states that in Molgula a true layer of "test-cells" is
•wanting, and only the follicle-epithelium surrounds the newly
deposited ovum. Under abnormal, circumstances, however, they
are formed at a much earlier period, and thus in most eggs that
have been observed, " test-cells " were present or soon made their
appearance.

My observations have been carried on for the most part on ova
of Ascidia amphora, but I have also confirmed most of them by
similar experiments on eggs of Cynthia ocellata. I made use only
of mature or almost mature eggs, so that I am unable to give as
complete an account of some points as could be desired.

The mature eggs oi A. amphora (PI. X, Fig. 1) have an average
diameter of about .255 mm. and present on optical section two dis-
tinctly marked zones, enclosing a semi-transparent granular mass,
the yolk. The outer of the two zones is formed by the follicle-
epithelium, consisting of a single layer of cells surrounding the
whole egg, and presenting on a surface view a polygonal appear-
ance. On examining a single cell which has become separated
from the egg, with a rather iiigh magnifying power, its interior is
seen to be occupied almost entirely by a number of vacuoles, sepa-
rated from one another and surrounded by very delicate bands of
protopla&m, which, in some of the angles formed by the meeting
of the polygonal vacuoles, appear as dark spots. (PI. X, Fig. 2.)
I have not been able to observe the development of these cells, but
Semper ^^^ las described it as it occurs in Molgula nana, where, in.

147

148

J. PLAYFAIR McMURRIGB.

the earliest observed stages, they appear as a layer of flat cells on
the surface of the egg, which, later on, become prismatic, and in
the interior of which 2-4 yellow granules make their appear-
ance. These afterward disappear and large vacuoles take their
place, pressing the protoplasm and nucleus to one side. In his
figures, the formation of the vacuoles has not advanced as far as in
the eggs I studied, but, on comparing his Figure 5, Plate I, with
my Figure 2, Plate X, it will at once be recognized that the ap-
pearance I have observed is to be accounted for in the same
manner, the vacuoles having become exceedingly abundant, and
pressed the original contents of the cell to the periphery, small
portions only being left in the intervals between the vacuoles.

Fol,^^^ having succeeded in tracing tlie origin of these cells still
farther back in Phallusia intesiinalisy states that they are not
formed from the ovary, but from the interior of the egg at the
boundary between the yolk and the nucleus, and wander thence to
the surface, where they form an epithelial layer round the egg.
One would fancy at first that the eminent observer had accidentally
confused " test-cells " with the follicle-epithelium, but that he has
not done so is evident from his also describing the " test-cells " as
formed later on. This discovery is of great interest, both from its
upsetting all former theories as to the formation of these cells,
which have hitherto always been considered as being formed from
the ovary, and also from the singular manner in which Kowa-
lewsky's theory in regard to the formation of the " test-cells " from
these cells has been turned upside down, these bodies {i. e. the
** test-cells") being formed inlependently from the yolk (as will be
seen hereafter) from which at an earlier period the follicle-cells
had also been derived. If M. Fol's observations are correct, it
is evident that the term " follicle-cell " is entirely a misnomer,
as is also indeed that of " test-cell," both being to a high degree
misleading to one who has not studied the history of the ap-
pellations-

Within this layer of so-called " follicle-cells" comes the second
izone of the egg, consisting of a transparent, apparently homoge-
neous structure, which, however, when acted on by acetic acid,
becomes markedly granular. This is the egg-shell or "chorion"
of some authors.

In the interior of the egg-shell and filling it almost completely
in the fresh ovum, is the yolk. On the average it measured

3r^6££t

A8GIDIAN OVA.

149

.236 mm, and was of a yellowish gray color, due to the coloration
of the constituent granules. In the majority of the eggs of this
Asoidian I examined, no nuclei were visible either in the fresh
egg, or in those that had been subjected to the reaction of acetic
acid and glycerine, or osmic acid and carmine. In some, however,
a clear spot was noticeable, usually situated eccentrically (in ore
instance at the periphery of the egg), and measuring .020-.08(5
mm. One egg presented a rather peculiar abnormality, which I
deem worthy of being recorded. The peculiarity consisted in the
presence of two distinct nuclei, both situated eccentrically on the
same side of the egg and varying somewhat in size, the larger
measuring .06 mm. and the smaller .04 mm. I am certain that I
did not observe the male and female pronuclei, as the egg under
observation had just been removed from the ovary, so that it could
not have been impregnated any length of time, if at all, before
my observation of it.

These were all the points to be observed in a perfectly fresh
ovum, but in one that had been removed from the ovary for a
short time, or which had been subjected to the action of various
reagents, there was to be seen surrounding the yolk a layer of
bodies, which have received the name of " test-cells " from the
idea that they were the cells of the developing ovum, from which,
eventually, the characteristic test of the adult Ascidian was formed.
Kowalewsky ^*^ in his paper on the development of Aacidia canina
states his belief that such is the fate of these cells, which, he also
maintains, have their origin from the epithelial cells of the egg-
follicle. Later on, however, in his paper on the development of
Pyrosoma,^^^ he withdraws the statement that the mantle is formed
from the "test-cells," but still adheres to the opinion that these
are merely follicle-epithelial cells, which have migrated inwards
towards the yolk. Before the appearance of his first paper, how-
ever, KupflPer,^*^ after investigating the subject, came to the conclu-
sion that the " test-cells " formed at the surface of the egg itself,
which theory had been independently adopted by MetschnikoflP.^'^^
Before the publication of Kowalewsky's second papf-r, Hertwig^*^
shewed that the " test-cells " take no part in the formation of the
mantle, this being formed as a secretion of a homogeneous substance
from the epidermis, into which, later on, cells migrate from the
epidermis. Hertwig's observations were made on PhaUuaia mamil-
IcUa and P. virginea (?), and have been confirmed by Semper ^^^ by

150

J. PLATFAIB McMUBBICK

observations on Clavelina vitrea and Cynthia depresaa. In the
same year that Semper published his observations, a paper by
TJssow^'^ appeared, in which the old theories first advanced by
Kowalewsky are revived and most emphatically insisted upon.
He says : " The outer mantle of the Tunicates is developed, not as
a secretion product of the epidermal cells of the inner mantle,
(Hertwig, Arsenjew,) but by the increase in number and growth of
the so-called 'test-cells' (Kupffer, Kowalewski)," and again : "The
result of my observations on the formation of the so-called 'test-
cells' is in complete accord with that of A. Kowalewski. The
yellow bodies are in fact nothing but cells of the Graafian
follicle . . ; . "

Semper shews that in the several species in which he examined
the ova, the " test-cells " were formed in the interior of the egg,
and that by the action of reagents, or even by exposure to sea-
water, these bodies might be produced in immature eggs. He
holds that they are devoid of a nucleus and of a cell-wall, and dis-
carding the term "test-cells," substitutes instead that of "test-
drops." '

My own observations having been confined to mature or almost
mature eggs, I cannot confirm Professor Semper's statement as to
the production of these peculiar bodies in immature eggs by means
of reagents, but these have the effect of producing them in most
cases almost immediately in mature eggs, even the exposure to sea-
water for a short time being sufficient for the purpose. Produced
in this manner these bodies are : naall and roundish in shape, and
in their interior numerous clear highly-refractive granules are to
be seen. I could detect no nucleus either in the fresh or in the
stained "drop," and a limiting membrane was also apparently
wanting.

As regards their mode of origin I am in accord with the obser-
vations of Kupffer,^*^ Metschinkoflr,^^^ etc. When an egg has been
removed from the ovary for a few minutes, there appear in the
interior of the yolk, numerous clear spots situated nearer the
periphery than the centre. In no case did they make their ap-
pearance at the centre of the yolk, and though in Figure 3, (PI. X,)
some appear to be very close to it, these in reality are peripheral
and appear indistinctly when an optical section of the egg is made
and accordingly have been represented. I accordingly conclude
that their origin is peripheral as stated by MetschinkofiT.^^^ They

\

' v *

V*

A8GIDIAN OVA.

tftl

71

f'radually migrate outwards, until they form a layer at the periph**
ery of the yolk (PI. X, Fig. 4), and then pass outside of it alto-
gether. The yolk at the same time contracts and leaves a space
between its circumference and the egg-membrane, in which the
"test-cells" lie, forming at first a layer round the yolk (PI. X,
Fig. 6), but as the contraction of the yolk proceeds, and the space
becomes larger, they move away from the surface of the egg and
scatter themselves irregularly. (PI. X, Fig. 6.)

I should imagine that there is in a manner a separation of the
egg into two portions ; an outer, consisting of protoplasm with
comparatively few yolk granules, and an inner, containing most of
the yolk granules and a small amount of protoplasm. The outer
zone is of no further use in the process of development, and gradu*
ally splits up into these "test-drops," their formation commencing
at the inner part of the zone and proceeding outwards, until we
have numerous "test-drops" and nothing left of the egg but a
dense mass of food-granules, closely packed together in the re*
maining protoplasm, from which the embryo is formed. Metschino
koff ^'^ describes this separation of the egg into two portions. He
says : " In the greenish protoplasm of a young egg of Asddia
intestinalia, fine yolk-granules collect round the nucleus; the
number of these becomes continually greater, whereby only the
peripheral portion of the protoplasm retains its greenish colora*
tion. This layer now separates itself distinctly from the central
granular portion and splits up into a great number of round
bodies which are the first * Tunic-cells.' " From this description
one would imagine that the author implied that the "Tunic-cells''
were formed at the extreme periphery of the egg, which, however,
is not the case, for they make their appearance in its interior.

On treating a fresh ovum with a dilute solution of acetic acid
(1 or 2 drops of commercial acid to a watch-glassful of water) for
about half an hour, its appearance becomes considerably changed.
(PI. X, Fig. 6.) The interglobular protoplasm of the " follicle-
cells" becomes much more distinct, and, in consequence, the
globules themselves become more plainly marked o^. The trans*
parent, apparently homogeneous egg-membrane becomes, as men-'
tioned above, distinctly granular. The yolk contracts very much,
measuring on the average about .116 mm., half its original size.
This contraction leaves a clear space between the yolk and the
egg-membrane, which, however, is larger in one-half of its oir»
3

152

J. PLAYFAIR McMURRIGE.

cumference than in the other, owing to the eccentric position
assumed by the contracted yolk. In this clear space are numerous
" test-cells," not forming a layer round the yolk, as they usually
do in an egg that has been subjected for a short time only to the
action of acetic acid or sea-water, but scattered irregularly around
the yolk. The "test-cells" measure .008 mm. and present the
appearance described above. In eggs that have been left in acetic
acid for a much longer period (6-20 hours) no further changes
occur, showing that the acid has exerted its full influence on
them.

After exposure to sea-water for six hours, very much the same
appearance is presented as with dilute acetic acid. The " follicle-
cells," however, shew a tendency to separate from the egg-mem-
brane, which, on its part, does not present a granular appearance.
(Pi. X, Fig. 7.) .

Upon running some strong picro-carmine under a cover-glass,
below which were some ova in sea-water, very important changes
occurred. At first no "test-cells" were to be seen, but, as the
picro-carmine gradually reached the egg, and the picric acid
exerted its action upon it, it gradually assumed a yellow hue,
whil'j, at the same time, there appeared at its periphery many
small spherical bodies of a round or oval shape, the same size as
the " test-cells," and containing in their interior several highly
refractive granules, which, in fact, render them apparent. No
"test-cells" appear outside the yolk, which retains its original size.
The egg-membrane assumes a pink hue, and, after some time,
becomes distinctly granular. The "follicle-cells" do not stain for
some time and show a tendency to separate from the egg-mem-
brane. (PI. X, Fig. 8.) The reaction produced by very dilute
picro-carmine is also rather important. After being subjected to
this reagent for about half an hour, the eggs presented ai. appear-
ance intermediate between that produced by the continued action
of dilute acetic acid and that following the employment of strong
picro-carmine. (PI. X, Fig. 9.) The yolk contracts to a slight
degree, and "test-cells" make their appearance, filling up the
small space between the paitly contracted yolk and the egg-
membrane.

I also employed osmic acid in the following manner. The eggs
were placed in a watch-glass containing sea-water, to which 1 or
2 drops of I per cent, osmio acid had previously been added, and

< >

y

I I i

, .v-^

< p»

ASOIDIAN OVA.

168

allowed to remain there for from five to ten minutes, when they
were removed and stained with Beale's carmine. In most cases
no change occurred, the yolk remaining of its origii.il size, and no
"test-cells" or clear spots made their appearance in the yolk, with
the exception of one instance, in which I did perceive a number
of clear spots in the periphery of the yolk.

By these results two questions are suggested: Ist. What are
these " test-cells ? " 2d. How are the various phenomena caused
by the various reagents to be explained ? I shall give the second
question priority. The explanation that seems to me to be the
simplest, and that which bears the stamp of probability most dis-
tinctly, is, that these phenomena are caused by the varying effects
of the different reagents in producing a contraction of the proto-
plasm of the yolk. Thus, osmic acid, which "fixes" the proto-
plasm immediately, allows of little or no contraction, and hence no
"test-cells" appear; with picric acid (which evidently is the con-
stituent of the picro-carmine that is active in producing the phe-
nomenon) a slight contraction takes place before the protoplasm
becomes "fixed," whereby the "test-cells" are formed, but the
contraction is not sufficient to cause them to pass outside the yolk;
and, in the last place, with acetic acid and sea-water there is no
fixing of the protoplasm, and the contraction goes on to such an
extent that the "test-cells" are driven completely outside the
yolk. Strong evidence in support of this theory is afforded by
the variation in the action of picric acid, according to the strength
in which it is used. For, as we have seen, in a dilute solution so
much contraction of the yolk is produced, that the "test-cells" do
partly pass out. ^ •

Accordingly, then, the "test-cells" are formed by a contraction
of the protoplasm of the egg, and thus we can readily understand
their formation in a developing egg, where the contraction pro-
duced by the process of cleavage would be quite sufficient to cause
their extrusion from the yolk.

We are now in a position to discuss the question as to the nature
of these " test-cells." Semper ^^^ regards them as merely polar glob-
ules, comparing them, in respect to their number, with those of
the Mollusca. This theory is, however, untenable, for by the re-
searches of Hertwig on the formation of the polar globules in the
eggs of Hsemopis, Nephelis,^'"^ Asteracanthion, Mytilus,^"^ and
other forms, we know that the polar globules are formed by a true

154

J. PLAYFAIR McMURBICH.

cell-division, and arc themselves true cells, containing a nucleus,
whereas no such process has been observed during the formation
of the "test-cells," and I for my part am sure that it does not ob-
tain, and, as Semper himself insists, the "test-cells" are not true
cells, but merely "drops." Fol,^**^ too, states that in Phallusia
intestinalia polar globules (two in number) are formed after the
disappearance of the original nucleus and after the formation of
"test-cells." Accordingly then, there is no morphological ho-
mology between the polar globules and the "test-cells." In the
eggs of certain forms, however, such as, in the Amphibia, Rana,^'"^
and in the Pisces, the Trout,^'^> after the disappearance of the ger-
minal vesicle, peculiar bodies are extruded from the yolk without
any spindle-formation or cell-division, for which Hertwig proposes
the name of excreted bodies (Excretkorper) in contradistinction to
the polar globules formed by cell-division. These structures have
been supposed by the various authors to be the remains of the ger-
minal vesicle, and thus, as far as their mode of formation is con-
cerned, probably do not allo\7 of comparison with the "test-cells,"
but since they resemble these latter in being bodies whose presence
in the egg is not necessary to its further development, and since
the cause of their appearance is evidently the same, viz : the con-
traction of the yolk induced by a stimulus, I think there can be
DO objection to classifying the "test-cells" with them as Excret-
korper.

Wyville Thomson,^'^^ however, has described bodies as occurring
in Antedon roaaoeua which bear a closer homology to "test-cells"
than even these structures. He says : " Consequently on the con-
traction of the yolk, a number of minute spherical pale yellow
oil-globule£i are apparently pressed out into the space within the
Vitelline membrane." These bodies differ from "test-cells"
only in the fact that they are oil-globules, whereas "test-cells" are
distinctly protoplasmic in their nature, and contain in their interior
several oil-globules usually. This distinction, however, is of com-
paratively little moment, and both in their mode of formation and
general appearance these Excretkorper — for so they also may be
denominated — are evidently closely related to "test-cells" and
.perhaps identical with them.

I consider these "test-cells" to be simply masses of albuminous
material containing two or three granules of the food-yolk, and
presume that they are in reality only poriions of the protoplasm

A8CIDIAN OVA.

166

of the egg, which have been forced out by the contraction. If
an egg, in which the "test-cells" have passed outside the yolk,
be subjected to pressure sufficient to rupture the yolk-raembrane,
allowing the yolk to come into contact with the "test-cells," and
at the same time leaving the egg-shell intact, the "test-celis"
commingle completely with the yolk and cannot be distingi^shed
again. The granules to be observed in a "test-coll" have a perfect
resemblance, both in shape and appearance, to those remaining in
the yolk as food, so that it may be presumed that they are in
reality the same, and were originally situated in the yolk, in that
portion of the protoplasm which formed the " test-cell," and were
extruded with it. •

The reason why portions of the yolk, originally of use to the
embryo, have become useless and are extruded, must remain unde-
cided until the life-histories of more of the lower types of Ascidians
have been fully worked out, but in all probability the explanation
is to be sought for in a change in the life of an ancestral form,
whereby the development became more rapid aufl less food-yolk
was required, while, at the same time, little or no diminution in
the amount of yolk in the egg was produced.

TABLE OF REFERENCES.

1. Lacaze-Duthiers. Les ascidies simpler des c6le» de la France.
Arch, de zool. exper. Vol. III. 1874. .

2. Semper. Ueher die Entstehung der geschichteten cellulose-
epidermis der Ascidien. Arb. aus dem zool.-zoot. Inst, zu Wiirz-
burg. Bd. II. 1875.

8. Fol. Sur la formation des ceufs chez les Aacidies. Journ,
de Micographie. T. I. 1877.

4. Kowalev/sky. Weitere Studien uber die Entwickelnng der
einfachen Ascidien. Arctiiv fur mikr. Anat. Bd. VII. 1870.

5. Kowalewsk). Ueber die Entwickelungsgeschichte von Pyro^
soma. Archiv fur mikr. Anat. Bd. XL 1875.

6. Kupflfer. Die Stammverwandtschaft zwischen Ascidien und
Wirbelthieren nach Untersuchungen Uber die Entwichelung der
Ascidia canina. Archiv fur mikr. Anat. Bd. VI. 1870.

166

J. PLA7FAIR McMUBRICH.

T. Metschinkoff. Zur Entioickelungsgeschichte der einfachen
Ascidien. Zeit. fur wissen. Zool. Bd. XXII. 1872.

8. Hertwig. Untersuchungen iiber den Bau und die Entwicke-
lung des CelMlosemantels der Tunicaten. Jen. Zeit. VII. 1872.

9. Ussow. Zoologischembryologische Untersuchungen: Die
Mantelthiere. Archiv/ur Naturg. Jahrg. XLI. 1875.

10. Hertwig. Beitrdge zur Kennfnias der Bildung, Befruchtung
und Theilung des thierischen Eies. 2ter Theil. Morph. Jahrb.
Bd. III. 1871

1 1. Hertwig. Beitrage zur Kenntniss der Bildung, Befruchtung
und Theilung des thierischen Eies. Ster Theil. Morph. Jahrb.
Bd. IV. 1878.

12. Oellachcr. Beitrage zur Entwickelungsgeschichte der Kno-
chenfische. Zeit. fur wissen. Zool. Bd. XXII. 1872.

13. Wyville Thomson. On the Embryogeny of Antedon rosaoeus,
Jjinck {Oomatula rosacea of Lamarck). Phil. Trans. 1865.

i

i ».

EXPLANATION OF FIGURES.

Figure 2
' drawn with

Figure L—

Figure 2.—

Figure 3.—

Figure 4.—

Figure 5.—

Figure 6.—

« Figure 7.—
Figure 8.—
Figure 9.-

is drawn with Hartnack obj. 9, oc. 2; all the rest are
Hartnack obj. 7, oc. 2.

-Fresh egg of Ascidia amphora.

-Pomcle-cell. ^ '

-Egg after short exposure to sea<water. "^

-Egg after longer exposure to sea-water.

-Egg after still longer exposure to sea-water.

-Egg after exposure for half an hour to the action of dilute
acetic acid.

-Egg after exposure to sea-water for six hours.

-Egg after the action of strong picro- carmine.

-Egg after the action of very dilute picro-carmine.

,»,<

' li

STUDIES FROM BIOI, LAB.

VOT, IT, - PLATE X

9

rhr HeUoty^^ Pnn*mo Co ^jyernortSi ff/tstdn.