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Nebbsheimer, E. 1908 Ueber die Hohe histologischer Differenxieruog bei
heterotrichen Ciliaten. Arch. f. Protistenkunde ,vol. 2, pp. 305-325.

Prowazek, S. von 1903 Studien zur Biologie der Zelle. Zeitschr. f. Allg.
Phys., Bd. 2.

RossBACH, M. J. 1872 Die rhythmischen Bewegungserscheinungen der ein-
fachsten OrganiBmen und ihr Verhalten gegen physikalische AgentieD
und Arsneimittel. Verb. d. pbys. med. Gesell. zu Wtlrzburg, N.F.,
vol. 11, pp. 179-242.



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(Reprinted from Biological Bulletin, Vol. XXII., No. 6, May, 191 i.i



CERTAIN MEANS BY WHICH STARFISH EGGS NATU-

RALLY RESISTANT TO FERTILIZATION MAY BE

RENDERED NORMAL AND THE PHYSIOLOG-

ICAL CONDITIONS OF THIS ACTION.

RALPH S. LILLIE.

(From the Marine Biological Laboratory, Woods Hole, and the Physiological
Laboratory, Depeulment of TMlogy, University of Pennsylvania.)

Last summer at Woods Hole, while investigating the influence
of ether and other anaesthetics in protecting the unfertilized eggs
of starfish against the cytolytic action of pure isotonic sodium
chloride solution,^ I observed that toward the end of the breed-
ing season the eggs often proved abnormally resistant to this
solution, withstanding in spme instances exposure of more than
three hours to 0.55m NaCl without losing the power of develop*
ment on fertilization; in normal eggs this solution typically
causes complete cytolysis and coagulation of the protoplasm in
two hours or less. Other characteristic abnormalities of be-
havior were found to be associated with this abnormal resistance
to salt solutions. Usually a large proportion of such eggs failed
to undergo maturation in sea-water, and of those which matured
only a small proportion developed to a free swimming stage on
fertilization, and th^ resulting larvae were largely abnormal. Also
the mature eggs, if left unfertilized in sea-water, frequently failed
to die and break down within the usual time, but remained clear
and apparently normal in appearance for an unusually prolonged
period. It is well known that unfertilized mature starfish eggs
undergo spontaneously a characteristic cytolytic alteration, ac-
companied by a darkening or coagulation of the protoplasm,
which is typically complete within twelve to fifteen hours after
deposition;' in contrast to this behavior a considerable propor-
tion of the eggs under consideration often remained clear and
uncoagulated in sea-water for twenty-four and in some cases for
forty-eight hours. These several peculiarities, (i) failure of

^American Journal of Physiology, 191 2, Vol. 30, p. i.
> Cf . J. Loeb, Afchiv fUr dii fiammte PhysMogH, 190a* Vol. 03* P* 59^

328



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329 RALPH S. LILLIE.

maturation in a large proportion of eggs, (2) failure of those eggs
which did maturate to develop after fertilization, (3) marked
delay in the typical post-maturational cytolysis of unfertilized
eggs, and (4) unusual resistance to the cytolytic action of salt
solutions were found in more or less constant association with one
another in numerous lots of eggs. All of these peculiarities are
evidence of a certain inertia or resistance to change in the egg-
system; such eggs fail to react, or react slowly and imperfectly
to conditions which call forth a definite and regular response in
normal eggs. The fact of their concurrence, as well as their
general nature, suggests that some single structural or metabolic
abnormality, whose general effect is to lower reaction-velocities
in the egg-system, is responsible for all of these peculiarities of
behavior.

The further incidental observation was made in several in-
stances that such eggs after treatment for two or three hours
with sodium chloride solution containing a little ether or chloral
hydrate recovered to a considerable degree the power of develop-
ing to a normal larval stage on fertilization; i. ^., eggs so treated
yielded more numerous and more active larvae than the un-
treated eggs of the same lot, fertilized at the same time. Pure
sodium chloride solution showed a similar though less marked
action. The surprising result thus appeared that treatment with
solutions which are markedly injurious to normal eggs may bring
a certain proportion of these abnormal or "over-ripe** eggs into
a condition — as regards power of development on fertilization —
closely approaching the normal.

The following record will illustrate the above-described condi-
tions in detail.

June 29, 191 1. The eggs were removed from a considerable number of starfish
at 10.00 A.M. A large proportion of these mixed eggs remained permanently
immature in sea-water (with intact germinal vesicles), but about half underwent
apparently normal maturation. On fertilization (at 3:30 P.M.) very few of the
eggs — a fraction of one per cent. — formed blastulse. modt died in early cleavage
stages, but many failed to cleave or even to form fertilization-membranes. The
unfertilized mature eggs after twenty-four hours in sea-water remained for the
most part decu: and translucent with no sign of coagulation; a good many, however,
showed apparently normal coagulation, while others showed an intermediate
condition. After forty-eight hours many unfertilized mature eggs were still un-
coagulated.

About two hours after removal from the animals the unfertilized eggs were



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RENDERING RESISTANT STARFISH EGGS NORMAL. 33O

placed in the following solutions: (i) pure 0.55m NaCl, and (2-10) o.ssm NaCl
containing anaesthetics as follows: (2-4) ether, 0.6, 0.45, and 0.3 volumes per cent..
(S-7) chloral hydrate, 0.6, 0.4 and 0.2 per cent., (8-9) chloroform, one sixth and
one tenth saturated, and (10) ethyl alcohol. 5 volumes per cent. After three hours
and fifteen minutes in these solutions the eggs were transferred to normal sea-water
and washed free of the anaesthetics by two changes of sea-water; spermatozoa were
then added. At the same time spermatozoa were added to the untreated eggs
which had remained in sea-water. The results were as follows: of the control eggs,
fertilized in sea- water without treatment, almost all died before reaching the blastula
stage; only a few feeble blastulse (a fraction of i per cent.) were found on careful
search; many eggs failed to cleave or even to form membranes. The eggs exposed
to pure 0.55m NaCl also formed few blastulse, but these were relatively somewhat
more numerous as well as more active than in the control; the eggs treated with
0.55m NaCl containing 0.45 and 0.3 vol. per cent, ether, especially the latter, showed
a more decided improvement over the control, though the proportion of blastulae
was still small. The eggs from the other solutions showed no improvement.

A similar result was observed in a second series of experiments
with eggs which showed similar peculiarities. Eggs treated
for three hours with 0.55W NaCl containing 0.3 vol. per cent,
ether gave about 5 per cent, of blastulae, while of the control
untreated eggs less than one per cent, reached this stage. Eggs
similarly treated with solutions containing a higher proportion
of ether (0.75, 0.6, and 0.45 vol. per cent.) showed no improve-
ment over the control. In another series eggs exposed for 3 h.
45 m. to 0.55m NaCl containing o.i per cent, chloral hydrate
gave considerably more blastulae than the control eggs.

It is to be noted that the improvement in the developmental
power of these abnormal or resistant eggs was produced only
by the pure salt solution or by solutions with a low concentration
of anaesthetic. The concentration of ether most favorable for
retarding the cytolytic action of 0.55W NaCl is considerably
higher — from 0.5 to 0.6 vol. per cent.^ The present effect, how-
ever, is not due to a simple prevention of cytolysis; the improve-
ment over eggs left in sea-water, none of which undergo cytolysis
within the time of exposure, cannot thus be explained. The effect
is different from a simply protective action; and since it seemed
to be favored by weak solutions of ether, the experiment was
tried of exposing a batch of similarly abnormal eggs to sea-water
containing 0.3 vol. per cent, ether. After three hours the eggs
were returned to normal sea-water and fertilized. Next day
it was found that the great majority of mature eggs had formed

1 Cf. R. S. LiUie. loc. cit., p. 6.



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531 KALPH S. ULUE.

active and vigorous blastube and gastnike, while of the untreated
control eggs left in sea-water and fertilized at the same time as
the others less than one per cent, formed larvae and these were
feeble and abnormal. It thus appeared that the abnormal condi-
tion which renders the egg incapable of responding f uUy to the
fertilizing action of the spermatozodn might be removed by
treatment with sea-water containing ether in certain concentra*
tions, which are considerably lower than those required for
typical anaesthetic or protective action.

What are the conditions of this effect? In a recent paper^ I
have presented evidence indicating that the protective and
anaesthetic actions exerted by ether and other lipoid-solvents in
certain concentrations are due primarily to a modificuiian of the
plasma membranes of the cells or irritable elements, of such a
kind as to render these membranes vaore^ resistant toward
agencies that under the usual conditions rapidly increase their
permeability; cytolysis and stimulation, both of which depend
on such increase of permeabilit>% are hence checked or prevented.
Decrease in the readiness with which the permeability is in-
creased thus involves for an irritable tissue decreased irritability;
this effect is produced by \'arious salts, r. ^ ., of magnesium, and
by ether and other lipoid-solvent anaesthetics in certain (not too
high) concentrations. In lower concentrations it has been ob-
served that ether and other lipoid-soK-ents frequentiy heighten
irritability;* i. f., expressed in terms of the membrane theory
of stimulation, they increase the readiness with which the permea-
bility — ^and hence the electrical polarization — of the plasma mem-
brane undergoes change. It seems clear that for irritable tissues
the state of the lipoids in the plasma membrane largely determines
the readiness with which changes of permeability — ^and of the
dependent electrical polarization — are induced by external agen-
cies. Slight permeation of the lipoids with a lipoid-solvent like
ether apparendy often facilitates such changes and hence in-

I R. S. Lillle. Anurican Jovmol of Physiology, 1912. Vol. 29. p. 372.

> For various instances of this efifect cf . the references dted in my recent paper
in American Journal of Physiology, 19 1 2. Vol. 29, p. 374. The neuromuscular
system of marine animals also shows it; e. g,, Bethe found that alcohol (0.5 per cent,
in sea-water) decidedly increased the mechanical irritability of the isolated central
portion of the medusa Coialorrhita; cf. "Allgemeine Anatomic und Physiologie des
Ncrvensy stems." Leipzig, 1903. p. 359.



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RENDERING RESISTANT STARFISH EGGS NORMAL. 332

creases irritability; the presence of more lipoid-solvent renders
a change of permeability difficult,^ hejice the protective or an*
aesthetic action; while concentrated solutions of lipoid-solvents
disrupt the membrane and produce cytolytic or irreversible
alterations in the cells; hence such substances in higher concen*
trations are markedly toxic.

On the assumption that lipoid-solvents influence the plasma
membranes of egg cells in essentially the same manner as those of
irritable tissues, the above action of ether on abnormal egg cells
becomes more readily intelligible; it falls, in fact, into the same
essential category with the facts just cited. There is a close
analogy between the stimulation of irritable tissues and the initia-
tion of cleavage in egg cells; the primary or critical change in
both cases appears to be a temporary and reversible increase in
the permeability of the plasma membrane, with accompanying
changes in the electrical polarization of the latter.* This analogy
suggests that the irresponsive -condition of the above "over-
ripe" starfish eggs is essentially the symptom or expression of an
abnormal condition of the plasma membrane. Apparently the
latter has in these eggs become abnormally resistant to changes of
permeability; hence the eggs are irresponsive to the spermato-
zo5n (whose primary action is to increase permeability); hence
also they show heightened resistance to cytolytic action — which
also depends on increase in surface permeability; this is shown
by the slowness with which they undergo the typical post-
maturational cytolysis, and also by their increased resistance to
pure isotonic sodium chloride solution. If this interpretation is
correct, the favorable action of weak ether solution consists es-
sentially in altering the plasma membrane and rendering it more
susceptible to the action of permeability-increasing (and hence
depolarizing) agencies — t. «., more irritable, on the above-men-
tioned analogy with irritable tissues. Through this means the
plasma membrane is restored to an approximately normal condi-

1 Thia is very clearly shown in the larvae of Arenicola; cf. the paper just cited,
p. 380 flf.

< I have discussed the probable basis of this resemblance at some length in an
earlier paper in the Biological Bulletin, 1909, Vol. 17. pp. 20 ff. The title of
Loeb's recent book, " Entwicklungserregung des tierischen Eies," also emphasizes
this analogy.



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333 RALPH S. LILLIE.

tion of responsiveness; the sperm then exhibits its normal action.
It is evident that this hypothesis also implies that the other
changes in the egg expressive of increased permeability should,
after the ether treatment, also follow an approximately normal
course. This in in fact the case as regards the post-maturational
cytolysis; this change is delayed in the above abnormal eggs, as
already described; but it is found to take place in a normal
manner in the ether-treated unfertilized eggs. The following
descriptions will illustrate both of these effects.

It should first be noted that the degree to which eggs abnor-
mally resistant to fertilization may be rendered normal by the
ether treatment is variable. In some of my last summer's experi-
ments the difference between the ether-treated and the untreated
eggs of the same lot was slight; in others the contrast was most
striking. The degree of resistance to the post-maturational
cytolysis is similarly variable. In general it was observed that
eggs which showed the most pronounced delay in the onset of
this latter change were most readily brought into a normally
responsive condition — or '* rejuvenated" — by ether. The fol-
lowing series of experiments with three separate lots of starfish
eggs — all of which failed with a few exceptions to develop to a
blastula stage on simple fertilization without ether treatment —
illustrates this variability, as well as the correlation between
delay in the post-maturational cytolysis and the possibility of
rejuvenation^ by the ether treatment.

June 30, 191 1. Three separate lots of eggs (A, B, Q were used; each lot con-
sisted of the mixed eggs from several starfish. After remaining about one and a
half hours in normal sea-water, eggs from each lot were transferred to sea-water
containing 0.3 vol. per cent, ether. These eggs were kept in small tightly corked
flasks. After i hour and 10 minutes in this solution part of the eggs were transferred
from each flask to normal sea-water in finger bowls; after washing the eggs free
from ether spermatozoa were added. The remainder of the eggs in each flask
were similarly transferred to sea-water and fertilized after three and a quarter hours
in the ether solution. For each lot there was a fertilized control consisting of eggs
which had lain untreated in sea-water for about 2 hours and 45 minutes before
fertilization.

i I use this term because of the analogies it suggests. The eggs are in fact
brought by the ether treatment into a condition which is characteristic of eggs
produced in the earlier portion of the reproductive cycle. The production of hyper-
resistant eggs like the above occurs late in the breeding season, and the phenomenon
bears certain analogies to senescence. See below, page 345-



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RENDERING RESISTANT STARFISH EGGS NORMAL. 334

The characteristics and behavior of the eggs from these three lots were respec-
tively as follows:

Lot A. — The great majority of these eggs fail to maturate. A small proportion
undergo apparently normal maturation.

Unfertilized Eggs. — 22 hours after removal almost all of the mature eggs show
the typical opaque and coagulated protoplasm; t. e.. i>06t-maturational cytolysis
appears normal.

Fertilized Eggs. — Condition ca. 20 hours after fertilization.

1. Untreated (Control) Eggs. — Many of the immature eggs have typical fer-
tilization membranes; but are otherwise unchanged. The few mature eggs are
mostly dead; only one abnormal blastula was found.

2. Ether-treated Eggs. — (a) Exposed i hour 10 minutes. Four or five blastulse
are found in some hundred eggs; little difference from control. (&) Exposed 3}^
hours. Little or no improvement over control; a few blastuls as in (a).

Lot B. — Most of these eggs remain immature, but about 20 per cent, undergo
apparently normal maturation.

Unfertilized Eggs. — 22 hours after removal from the animals most of the mature
eggs are opaque and coagulated, but in many the coagulation is less advanced than
in normal eggs, and in some the protoplasm remains semi-translucent.

Fertilized Eggs. — Condition ca. 20 hours after fertilization.

1. Untreated (Control) Eggs. — ^Almost all of the mature eggs are dead. Many
immature eggs have fertilization membranes. Only two abnormal blastuls are
found in several hundred eggs.

2. Ether-treated Eggs. — (a) Exposed i hour 10 minutes. Improvement over
the control; a large proportion (about one third) of the mature eggs have formed
blastulse, many of which have begun to gastrulate. (b) Exposed 3>^ hours. Also
shows a marked improvement over the control, but the larvs are fewer and less
active than in 2a.

Lot C. — In this lot of eggs the majority show normal maturation, though a few
remain immature.

Unfertilized Eggs. — ^After 22 hours in sea-water most of the eggs are more or less
coagulated, but the degree of opacity is distinctly less than in normal eggs, and a
considerable proportion remain translucent — almost like freshly shed eggs.

Fertilized Eggs. — Condition ca. 20 hours after fertilization.

1. Untreated (Control) Eggs. — Nearly all are dead. Most have membranes and
show evidence of having cleaved or fragmented, but many have failed to cleave or
even to form membranes. A small proportion of eggs have formed larvae some of
which appear normal: the larvs though few are more numerous than in the controls
of A and B.

2. Ether-treated Eggs. — (a) Exposed i hour 10 minutes. Striking contrast to
control. Almost all of the mature eggs have formed active larvse, many in the
early gastrula stage and swimming at the surface of the water, (b) Exposed 3}i
hours. Here also the majority of eggs form larvae, but these are largely abnormal,
and relatively few gastrulse or surface swimmers are present.

The power of development after fertilization is thus greatly

increased after ether- treatment in Lots B and C, but not in Lot ^4.

The mature eggs of Lots B and C show marked delay in the post-

maturational cytolysis; in Lot C this delay is greater, and the



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335 RALPH S. LILLIE.

action of the ether is correspondingly more favorable, than in Lot
B, I have already presented evidence that eggs showing this
abnormal behavior are characterized by the possession of hyper-
resistant plasma membranes. It should be noted that variations
in the degree of resistance of this membrane occur regularly in
normal eggs. Various facts indicate that the process of matura-
tion is constantly associated with a change in the properties of
the plasma membrane. This is shown by the fact that mature
eggs undergo cytolysis in 0.55m NaCl solution more rapidly
than immature eggs*/ also by the fact that contact with sperma-
tozoa and various forms of artificial treatment cause the separa-
tion of the surface-film of mature eggs in the form of a fertilization
membrane, a change indicating a superficial cytolytic or perme-
ability-increasing action; while immature eggs are not normally
subject to this change.* This difference between immature and
mature eggs is a constant or physiological feature in the life
history of these eggs. The difference between normal eggs and
the resistant eggs under consideration is in many respects similar.
For some reason the maturation process fails to bring these eggs
into the normally sensitive condition in which the permeability
of the membrane is readily increased. Hence fertilization is
imperfect, even fertilization-membranes failing to form in some
cases; in others membranes are formed and cleavage begins, but
the latter is characteristically irregular and fails to proceed far.
According to this view the failure of development is due not to
defective organization of the protoplasm, but simply to the
existence of an abnormally resistant plasma membrane. ^The
action of ether consists in restoring the membrane to its normal
condition. The response to the spermatozoon then becomes
normal.

The experimental evidence in favor of this hypothesis consists
at present simply in the fact that such abnormal eggs are rendered
normal by ether treatment not only in regard to their response
to fertilization, but also in regard to the rate and character of

» R. S. Lillie. American Journal of Physiology, 191 2, Vol. 30.

* Cf. J. Loeb's experiments on the eggs of Asterina, University of California
Publications, Physiology, 1905. Vol. 2, p. 1 50. Spermatozoa and artificial membrane-
forming agencies may however produce typical membranes in abnormal immature
starfish eggs. Cf. Journal of Experimenlal Zodlogy, 190a. Vol. 5, p. 407.



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RENDERING RESISTANT STARFISH EGGS NORMAL. 336

the spontaneous post-maturational cytolysis; i. e,, they undergo
complete coagulation within eighteen hours or less, precisely as
do normal eggs. Whether the resistance to cytolysis by salt
solutions is also decreased I have not yet determined; but the
decrease in the resistance to the post-maturational cytolysis —
a change supposedly due to the action of certain protoplasmic
oxidation products upon the plasma membrane' — ^is clear evidence
that the membrane has been brought into a condition more
nearly approaching the normal. The experiments to be described
show that a return of the normal responsiveness to the spermato-
zoon is closely correlated with a return of the normal behavior
with respect to this spontaneous oxidative cytolysis. In other
words, the plasma membranes of the ether-treated "reju-
venated" eggs undergo breakdown in the manner and at the
time characteristic of normal eggs.

The following record gives the description of two typical
experiments.

July 6, 191 1. Eggs were removed at 11:00 A.M. from two lots of starfish, A
and B, In both lots a good proportion of eggs underwent apparently normal mat-
uration. Eggs from each lot were divided into two portions. One portion re-
mained in sea-water; and about four hours after removal from the animals part of
these eggs were fertilized; the rest remained unfertilized. The other portion was
transferred, two hours after removal, to sea-water containing 0.3 vol. per cent,
ether; in this solution they remained for one hoiir and thirty-five minutes; they were
then returned to sea-water; to part of these eggs spermatozoa were added, the rest
remained unfertilized. The ether-treated and the untreated eggs were fertilized
at the same time. The two lots A and B were treated alike so far as possible. The
results of these experiments were as follows:

Lot a. The following was the condition of the eggs ca. 22 hours after removal:

1. Untreated Eggs, (a) Unfertilized, — Most mature eggs are coagulated but
to a varying degree; some are only slightly darkened, and in a fair proportion the
protoplasm remains pemi-translucent.

(&) Fertiliud,'-^Mi of the mature eggs have formed membranes and most have
undergone cleavage or irregular fragmentation; but many remain uncleaved; no
blastulx are present.

2. Ether-treated Eggs, — (In 0.3 vol. per cent, ether from i.oo to 2.35 P.M.)



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