the formation of a membrane--the fertilization membrane. It might be
considered possible that this membrane formation or the alteration
underlying or accompanying it is responsible for the fact that an egg
once fertilized becomes immune against a spermatozoön. We shall see
in the next chapter that it is possible to call forth the membrane
in an unfertilized sea-urchin egg by treating it with butyric acid.
This membrane is so tough in the egg of _Strongylocentrotus_ that
no spermatozoön can get through it; in the egg of _Arbacia_ the
membrane is occasionally replaced by a soft gelatinous film. If no
second treatment is given to such eggs they will disintegrate in a
comparatively short time, but when sperm is added some or most of the
eggs will develop in the way characteristic of fertilized eggs.[75]
When the membrane is too tough to allow the spermatozoön to enter the
egg it can be shown that if the membrane is torn mechanically the egg
can still be fertilized by sperm.
[75] Loeb, J., _Science_, 1913, xxxviii., 749; _Arch. f.
Entwcklngsmech._, 1914, xxxviii., 277; Wasteneys, H., _Jour. Biol.
Chem._, 1916, xxiv., 281.
Should it be possible that the spermatozoön can no longer agglutinate
with the fertilized egg or that those phagocytotic reactions which we
suppose to play a rôle in the entrance of the spermatozoön into the egg
are no longer possible after a spermatozoön has entered? The mere fact
of development is apparently not the cause which bars a spermatozoön
from entering an egg already fertilized by sperm.
Lillie assumes that the egg loses its “fertilizin” in the process of
membrane formation since the sea water containing such eggs no longer
gives the agglutinin reaction with sperm, and he believes that the lack
of “fertilizin” in the fertilized egg or in the egg after membrane
formation is the cause of the block in the fertilized egg. But we have
seen that the artificial membrane formation does not create such a
block although it puts an end to the “fertilizin” reaction. In the
egg of _purpuratus_ the “fertilizin” reaction ceases when the jelly
surrounding the egg is dissolved by an acid and the eggs are repeatedly
washed; yet such eggs can easily be fertilized by sperm.
Lillie does not assume that the “fertilizin” causes an agglutination
between egg and spermatozoön--we should assent to such an
assumption--but that the “fertilizin” acts like an “amboceptor” between
egg and spermatozoön, the latter being the complement, the former the
antigen. The pathologist would probably object to this interpretation
since no “amboceptor” is needed for agglutination. The writer has had
some doubts concerning the value of Ehrlich’s side-chain theory which,
besides, can only be applied in a metaphorical sense to the mechanism
of the entrance of the spermatozoön into the egg.[76]
[76] Loeb, J., _Am. Naturalist_, 1915, xlix., 257.
The writer may be permitted to illustrate by a special case his reason
for declining to accept Ehrlich’s side-chain theory. Ehrlich and Sachs
found that if to a given mass of toxin small quantities of antitoxin
are added successively the first fraction added neutralized more
than the later fractions; and on the basis of this reasoning Ehrlich
concluded that ten different toxins were contained in the diphtheria
toxin. Arrhenius showed that the same phenomenon can be obtained when
a weak base like NH₄OH is neutralized by a weak acid (_e. g._, boric
acid); hence we should assume that NH₄OH consists of ten different
forms of ammonia. Both cases, the saturation of toxin with antitoxin
and ammonia with boric acid are equilibrium phenomena. (Arrhenius, S.,
_Quantitative Laws in Biological Chemistry_, London, 1915.)
6. The reason that an egg once fertilized with sperm cannot be
fertilized again may be found in a group of facts which we will now
discuss, namely, the self-sterility of many hermaphrodites. The fact
that hermaphrodites are often self-sterile, while their eggs can be
fertilized with sperm from a different individual of the same species
has played a great rôle in the theories of evolution. We are here only
concerned with the mechanism which determines the block to the entrance
of a spermatozoön into an egg of the same hermaphroditic individual.
Castle[77] observed and studied the phenomenon of self-sterility in an
Ascidian, _Ciona intestinalis_, which is hermaphroditic. Animals which
were kept isolated discharged both eggs and sperm into the surrounding
sea water. Often no egg was fertilized, but in some cases five, ten, or
as many as fifty per cent. of the eggs could be successfully fertilized
with sperm from the same individual; while if several individuals
were put into the same dish as a rule one hundred per cent. of the
eggs which were discharged segmented. Morgan[78] found that the eggs
of various females differ in their power of being fertilized by sperm
of the same individual while one hundred per cent. could usually be
fertilized with sperm of a different individual. He found in addition
that if the eggs of _Ciona_ are put for about ten minutes into a
two per cent. ether solution in sea water in a number of cases the
percentage of eggs fertilized by sperm of the same individual shows a
slight increase. Fuchs[79] has reported results similar to those of
Castle and Morgan.
[77] Castle, W. E., _Bull. Mus. Comp. Zoöl._, Harvard, 1896, xxvii.,
203.
[78] Morgan, T. H., _Jour. Exper. Zoöl._, 1904, i., 135; _Arch. f.
Entwcklngsmech._, 1910, xxx., 206.
[79] Fuchs, H. M., _Jour. Genet._, 1915, iv., 215.
A new point of attack has been introduced into the work of
self-sterility in plants by the consideration of heredity. Darwin
found that in _Reseda_ which is monœcious (or hermaphroditic)
certain individuals are either completely self-sterile or completely
self-fertile; and Compton showed that apparently self-fertility is a
Mendelian dominant to self-sterility.[80]
[80] Quoted from Fuchs.
According to Jost this self-sterility in hermaphroditic plants is due
to the fact that if pollen of the same plant is used the normal growth
of the pollen tube is inhibited, while this inhibition does not exist
for pollen from a different individual. Correns calls these substances
which prevent the adequate growth of pollen, “inhibitory” substances,
and finds that they can apparently be transmitted to the offspring. He
made experiments on _Cardamine pratensis_ which is self-sterile.[81] He
fertilized two individuals of _Cardamine_ crosswise and raised sixty
plants of the first generation. He compared the fertility of these
F₁ plants toward (_a_) their parents, and (_b_) foreign plants. All
the fertilizations with the foreign plants were successful, but the
fertilizations with the parents were only partly successful. According
to their reaction they could be divided into four groups:
(_A_) fertile with both parents. Type bg
(_B_) fertile with one (B), sterile with the other parent (G).
(_a_) fertile with B, sterile with G. Type bG
(_b_) fertile with G, sterile with B. Type Bg
(_C_) sterile with both parents. Type BG
[81] Correns, C., _Biol. Centralbl._, 1913, xxxiii., 389.
It was found that approximately fifteen of the sixty children belonged
to each of the four groups. This should be expected if the inhibitory
substance to each parent is transmitted to the children independently.
Half of the children will thus inherit the inhibitory substance of one
parent and the other half will inherit the inhibitory substance of the
other parent. This agrees with the assumption that there are definite
determiners for the inhibitory substances in the children which will be
transmitted to half of the children. Rather complicated assumptions are
needed to explain all the facts observed by Correns on this basis and
since the subject is still under investigation we need not go further
into the details.
To us the assumption and experimental support of the idea that
self-sterility is caused by the presence of a substance inhibitory to
the entrance of a spermatozoön is important. Should it be possible
that the block created by the entrance of a spermatozoön into the egg
is also due to an inhibitory substance carried by a spermatozoön into
the egg; and furthermore that the effect of the inhibitory substance
should be the prevention of further agglutination of the spermatozoön
with the egg or of the growth of the pollen tube in plants? On such
an assumption self-sterility would be due to a lack of agglutination
between the egg of a hermaphrodite and a spermatozoön of the same
individual. The experiments on the agglutinins have shown that while
isoagglutinins (_i. e._, agglutinins for other individuals of the same
species) are common auto-agglutinins (_i. e._, agglutinins for cells of
the same individual) rarely if ever occur.
7. A positive chemotropism of the spermatozoa toward an egg of the same
species has been demonstrated in a few cases, but it seems that this
phenomenon is not determined by that type of substances which give
rise to species specificity. The famous experiment of Pfeffer on the
spermatozoa of ferns inaugurates this line of investigation. He found
that such spermatozoa when moving in a straight line through the water
will be deviated in their course if they come near an archegonium;
they will then turn toward it, enter it, and enter the egg. Pfeffer
showed that 0.01 per cent. malic acid if put into a capillary tube will
attract the spermatozoa of ferns.
When the liquid in the tube contains only 0.01 per cent. malic acid
the spermatozoa of ferns very soon move toward the opening of the
capillary tube and within from five to ten minutes many hundreds of
spermatozoa may accumulate in the tube. The malic acid acts as well
in the form of a free acid as in the form of salts.[82]
[82] Pfeffer, _Untersuchungen aus dem botanischen Institut zu
Tübingen_, 1881-1885, i., 363.
These experiments were continued and amplified by Shibata.
Bruchmann[83] found that the spermatozoa of _Lycopodium_ are positively
chemotactic to citric acid and salts of this acid, although no citric
acid could be shown in the contents of the archegonia. They are also
positively chemotactic to the watery extract from archegonia.
[83] Bruchmann, H., _Flora_, 1909, ic., 193.
Dewitz, Buller, and the writer have vainly tried to prove the existence
of a positive chemotropism of spermatozoa to eggs of the same species.
Lillie claims to have proved a positive chemotropism of the sperm of
sea urchins to “fertilizin,” but such a conclusion is only justified
if a method similar to that of Pfeffer’s with capillary tubes, gives
positive results; such a method was not used in Lillie’s experiments.
It seems that the fertilization of the egg by sperm is rendered
possible by two facts; first that where fertilization takes place
outside the body egg and sperm are shed simultaneously by the two
sexes. This can be easily observed in the case of fish. But it is also
the case in invertebrates. Thus the writer has observed that the sea
urchins _Strongylocentrotus purpuratus_ at the shore of Pacific Grove
all spawn simultaneously. The examination extended over several miles
of shore. At such spawning seasons the sea water becomes a suspension
of sperm.
The second fact guaranteeing the fertilization of the eggs is the
overwhelming excess of spermatozoa over eggs. The enormous waste in
animated nature is in agreement with the idea of a lack of purpose;
since in this case the laws of chance must play a great rôle; and the
origin of durable organisms by laws of chance is only comprehensible
on the basis of an enormous wastefulness, for which evidence is not
lacking.
CHAPTER V
ARTIFICIAL PARTHENOGENESIS
1. The majority of eggs cannot develop unless they are fertilized,
that is to say, unless a spermatozoön enters into the egg. The
question arises: How does the spermatozoön cause the egg to develop
into a new organism? The spermatozoön is a living organism with a
complicated structure and it is impossible to explain the causation of
the development of the egg from the structure of the spermatozoön. No
progress was possible in this field until ways were found to replace
the action of the living spermatozoön by well-known physicochemical
agencies.[84] Various observers such as Tichomiroff, R. Hertwig, and
T. H. Morgan had found that unfertilized eggs may begin to segment
under certain conditions, but such eggs always disintegrated in
their experiments without giving rise to larvæ. In 1899 the writer
succeeded in causing the unfertilized eggs of the sea urchin _Arbacia_
to develop into swimming larvæ, blastulæ, gastrulæ, and plutei, by
treating them with hypertonic sea water of a definite osmotic pressure
for about two hours. When such eggs were then put back into normal sea
water many segmented and a certain percentage developed into perfectly
normal larvæ, blastulæ, gastrulæ, and plutei.[85] Soon afterward
this was accomplished by other methods for the unfertilized eggs of
a large number of marine animals, such as starfish, molluscs, and
annelids. None of these eggs can develop under normal conditions unless
a spermatozoön enters. These experiments furnished proof that the
activating effect of the spermatozoön upon the egg can be replaced by a
purely physicochemical agency.[86]
[84] The substitution of well-known physicochemical agencies
for the mysterious action of the spermatozoön was the task the
writer set himself in this work and not the explanation of natural
parthenogenesis, as the author of a recent text-book seems to assume.
[85] Loeb, J., _Am. Jour. Physiol._, 1899, iii., 135; 1900, iii., 434.
[86] Loeb, J., _Artificial Parthenogenesis and Fertilization_, Chicago,
1913. The reader is referred to this book for the literature on the
subject.
The first method used in the production of larvæ from the unfertilized
eggs did not lend itself to an analysis of the activating effect of the
spermatozoön upon the egg, since nothing was known about the action of
a hypertonic solution, except that it withdraws water from the egg; and
there was no indication that the entrance of the spermatozoön causes
the egg to lose water. No further progress was possible until another
method of artificial parthenogenesis was found. When a spermatozoön
enters the egg of a sea urchin or starfish or certain annelids,
the surface of the egg undergoes a change which is called membrane
formation; and which consists in the appearance of a fine membrane
around the egg, separated from the latter by a liquid (Figs. 4 and
5). O. and R. Hertwig and Herbst had observed that such a membrane
could be produced in an unfertilized egg if the latter was put into
chloroform or xylol, but such eggs perished at once. It was generally
assumed, moreover, that the process of membrane formation was of no
significance in the phenomenon of fertilization, except perhaps that
the fertilization membrane guarded the fertilized egg against a further
invasion by sperm. However, since the fertilized egg is protected
against this possibility by other means the membrane is hardly needed
for such a purpose.
[Illustration:
FIG. 4. Unfertilized egg surrounded by spermatozoa (whose flagellum is
omitted in the drawing).
FIG. 5. The same egg after a spermatozoön has entered. The
fertilization membrane is separated from the egg by a clear space.]
In 1905 the writer found that membrane formation, or rather the change
of the surface of the egg underlying the membrane formation, is the
essential feature in the activation of the egg by a spermatozoön. He
observed that when unfertilized eggs of the Californian sea urchin
_Strongylocentrotus purpuratus_ are put for from one and a half to
three minutes into a mixture of 50 c.c. of sea water+2.6 c.c. N/10
acetic or propionic or butyric or valerianic acid and are then put
into normal sea water all or the majority of the eggs form membranes;
and that such eggs when the temperature is very low will segment
once or repeatedly and may even--if the temperature is as low as
4°C. or less--develop into swimming blastulæ[87]; but they will then
disintegrate. On the other hand, if they are kept at room temperature
they will develop only as far as the aster formation and nuclear
division and then begin to disintegrate. It should be mentioned
that the time which elapses between artificial membrane formation
and nuclear division is greater than that between the entrance of a
spermatozoön and nuclear division.
[87] The reader will find a description of the development of this egg
in the next chapter.
It was obvious, therefore, that artificial membrane formation induced
by butyric acid initiates the processes underlying development of the
egg but that for some reason the egg is sickly and perishes rapidly.
When, however, such eggs were given a short treatment with hypertonic
sea water or with lack of oxygen or with KCN they developed into normal
larvæ. This new or improved method of artificial parthenogenesis is as
follows: The eggs are put for from two to four minutes into 50 c.c.
sea water containing a certain amount of N/10 butyric acid (2.6 c.c.
in the case of S. _purpuratus_ in California and 2.0 c.c. in the case
of _Arbacia_ in Woods Hole). Ten or fifteen minutes later the eggs are
put into hypertonic sea water (50 c.c. sea water+8 c.c. 2-1/2 m NaCl
or Ringer solution or cane sugar) in which they remain, at 15° C. from
thirty-five to sixty minutes in the case of _purpuratus_, and from
17-1/2 minutes to 22-1/2 minutes at 23° in the case of _Arbacia_ at
Woods Hole. If the eggs are then transferred to normal sea water they
will develop. In making these experiments, which have been repeated and
confirmed by numerous investigators, it should be remembered that this
effect of the hypertonic solution has a high temperature coefficient
(about two for 10° C.) and that a slight overexposure to the hypertonic
sea water injures the eggs so that development is abnormal. By this
method it is possible to imitate the activating effect of the living
spermatozoön upon the egg in every detail and eggs treated in this way
will develop in large numbers into perfectly normal larvæ. We shall see
later that they can also be raised to the adult state.
2. The next task was to find out the nature of the action of the
two agencies upon the development of the egg. It soon became obvious
that the membrane formation (or the alteration underlying membrane
formation) was the more important of the two, since in the eggs of
starfish and annelids this was sufficient for the production of
larvæ, and that the second treatment had only the corrective effect,
of overcoming the sickly condition in which mere membrane formation
had left the eggs. It was, therefore, of great interest to ascertain
what substances or agencies caused membrane formation in the egg,
since it now became clear that the spermatozoön could only cause
membrane formation by carrying one such substance into the egg. These
investigations led the writer to the result that all those substances
and agencies which are known to cause cytolysis or hemolysis (see
Chapter III) will also induce membrane formation, and that the
essential feature in the causation of development is a cytolysis of
the superficial or cortical layer of the egg. As soon as this layer is
destroyed the development of the egg can begin.
The substances and agencies which cause cytolysis and hence, if their
action is restricted to the surface of the egg, will induce development
are, besides the fatty acids: (1) saponin or solanin or bile salts;
(2) the solvents of lipoids, benzol, toluol, amylene, chloroform,
aldehyde, ether, alcohols, etc.; (3) bases; (4) hypertonic or hypotonic
solutions; (5) rise in temperature, and (6) certain salts, _e. g._,
BaCl₂ and SrCl₂ in the case of the egg of _purpuratus_, and according
to R. Lillie, NaI or NaCNS in the egg of _Arbacia_. Whenever we submit
an unfertilized sea-urchin egg to any of these agencies and restrict
the cytolysis to the superficial or cortical layer of the egg (_i. e._,
if we transfer the egg to normal sea water before the cytolytic agent
has had time to diffuse into the main egg) the egg will form a membrane
and behave as if the membrane formation had been called forth by a
fatty acid, with this difference only, that the various agencies are
not all equally harmless for the egg.[88]
[88] The reader is referred for details to the writer’s book on the
subject.
If the idea was correct that the change underlying membrane formation
was essentially a cytolysis of the cortical layer of the egg, it was
to be expected (from the data contained in Chapter III) that the
blood serum or the cell extracts of foreign species would also cause
membrane formation and thus induce the development of the unfertilized
egg, while serum of animals of the same species or genus would have no
such effects. This was found to be correct. In 1907 the writer showed
that the blood serum of a Gephyrean worm, _Dendrostoma_, was able to
cause membrane formation in the egg of the sea urchin. When added in a
dilution of 1 c.c. of serum to 500 or 1000 c.c. of sea water to eggs of
_purpuratus_ a certain number formed fertilization membranes. It was
found later that the serum and tissue extracts of a large number of
animals, especially of mammals (rabbit, pig, ox, etc.), had the same
effect, though it was necessary to use higher concentrations, one-half
sea water and one-half isotonic blood serum. The eggs of every female
sea urchin, however, did not give the reaction and not all the eggs
even of sensitive females formed membranes. The writer found, however,
that it was possible to increase the susceptibility of the eggs against
foreign blood serum by putting them into a 3/8 m solution of SrCl₂ for
from five to ten minutes (or possibly a little longer) before exposing
them to the foreign blood serum. BaCl₂ acts similarly. The fact that
SrCl₂ alone can cause membrane formation in unfertilized eggs if they
are left long enough in the solution suggests that the sensitizing
effect of the substance consists in a modification of the cortical
layer similar to that underlying membrane formation; and that the
subliminal effect of a short treatment with SrCl₂ and the subliminal
effect of the foreign serum when combined suffice to bring about the
membrane formation.
Not only the watery extract of foreign cells but also that of foreign
sperm, induces membrane formation in the sea-urchin egg. The watery
extract of sperm of starfish is especially active, but the degree of
activity varies considerably with the species of starfish from which
the sperm is taken. The eggs of different species of sea urchins
also show a different degree of susceptibility for the sperm of
foreign species. Thus the eggs of _Strongylocentrotus purpuratus_
require a higher concentration of sperm extract than the eggs of _S.
franciscanus_. For the latter the amount of foreign cell constituents
which suffices to call forth membrane formation is so small that
contact with almost any foreign living spermatozoön produces this
effect; and as a rule no previous sensitizing action of SrCl₂ is
required. When we bring the unfertilized eggs of _S. franciscanus_ into
contact with the living sperm of starfish or shark or even of fowl,
the eggs form a fertilization membrane without previous sensitization.
A specific substance from the foreign spermatozoön causes membrane
formation before the spermatozoön has time to enter the egg. The effect
is the same as if artificial membrane formation had been called forth
with butyric acid, _i. e._, they begin to develop and then disintegrate
unless they receive a second short treatment.
When, however, we treat the eggs with the watery extracts from the
cells of their own or closely related species we find that these
extracts are utterly inactive, even if used in comparatively strong
concentrations. This agrees with the results given in Chapter III.
These phenomena lead to a very paradoxical result; namely that while
in the case of foreign sperm we can cause membrane formation by both
the living and the dead spermatozoön, only the living spermatozoön
of the same species can induce membrane formation. This might find
its explanation on the assumption that the active substance contained
in the foreign sperm or serum is water-soluble and a protein, while
the activating or membrane-forming substance in the spermatozoön is
insoluble in water but soluble in the egg (or in lipoids). If this
assumption is correct the two substances are essentially different.
Robertson[89] has succeeded in extracting a substance from the sperm
of the sea urchin which causes membrane formation of the sea-urchin
egg after the latter has been sensitized by a treatment with SrCl₂. It
seems to the writer that if the substance extracted by Robertson were
the real fertilizing agent contained in the spermatozoön it should
fertilize the egg without a previous sensitization of the egg with
SrCl₂ being required.
[89] Robertson, T. B., _Arch. f. Entwcklngsmech._, 1912, xxxv., 64.
3. The action of acids in the mechanism of artificial parthenogenesis
provides some interesting physiological problems. When unfertilized
sea-urchin eggs are left _in_ sea water containing any of the lower
fatty acids up to capronic, the eggs will form no membranes, while
_in_ such sea water, and they will show no outer signs of cytolysis
(swelling). When, however, the eggs are left in sea water containing
any of the fatty acids from heptylic upward the eggs will form
membranes while _in_ the acid sea water and soon afterward will
cytolyze completely and swell enormously. In solutions of the mineral
acids no membranes are formed and none are formed as a rule when the
eggs are transferred back to sea water. When both a mineral and a lower
fatty acid, _e. g._, butyric, are added to sea water the mineral acid
acts as if it were not present, _i. e._, the eggs form membranes when
transferred back to sea water if the concentration of the butyric acid
is high enough. All these data are comprehensible if we assume that
only that part of the acid causes membrane formation which is lipoid
soluble, while the water soluble part is not involved in the process
of membrane formation; and that the cytolysis or swelling of the whole
egg can only take place in the higher fatty acids (heptylic or above)
which are little soluble in water and very soluble in lipoids, while
the lower fatty acids, whose water solubility is comparatively high,
can only bring about a cytolysis and swelling in the cortical layer but
not in the rest of the egg. This makes it appear as though the part
undergoing an alteration in membrane formation was a lipoid; and this
would harmonize with the assumption that the specific membrane-inducing
substance in the spermatozoön is not soluble in water, but soluble in
fat.
4. These and other observations led the writer to the view that the
essential process which causes development might be an alteration
of the surface of the egg, in all probability an alteration of the
superficial layer probably of the nature of a superficial cytolysis.
The question remains: What could be the physicochemical nature of
this cytolysis? The writer had suggested in former papers that in the
cytolysis underlying membrane formation lipoids were dissolved, and he
supposed that the substance to be dissolved might be a calcium-lipoid
compound which might form a continuous layer under the surface of the
egg.[90] v. Knaffl, working on the cytolysis of eggs in the writer’s
laboratory, gave the following idea of the process:
Protoplasm is rich in lipoids; probably it is mainly an emulsion
of these and proteins. Any physical or chemical stimulus which
can liquefy the lipoids causes cytolysis of the egg. The protein
of the egg can really only swell or be dissolved if the condition
of aggregation of the lipoid is altered by chemical or physical
agencies. The mechanism of cytolysis consists in the liquefaction
of the lipoids and thereupon the lipoid-free protein swells or
is dissolved by taking up water.... Hence this supports Loeb’s
view that membrane formation is induced by the liquefaction of
lipoids.[91]
[90] Loeb, J., _Über den chemischen Charakter des
Befruchtungsvorgangs_, etc., Leipzig, 1908.
[91] v. Knaffl, E., _Arch. f. d. ges. Physiol._, 1908, cxxiii., 279.
The writer suggested that the destruction of an emulsion in the
cortical layer might possibly be the essential feature of the
alteration leading to membrane formation and development. It had been
long observed that unfertilized starfish eggs may begin to develop
apparently without any outside “stimulus,” and A. P. Mathews found
that slight mechanical agitation of these eggs in sea water increased
the number which developed. It has been shown in numerous experiments
by Delage, R. S. Lillie, and the writer, that the substances causing
development in the starfish egg are identical or closely related to
those which bring about this effect in the egg of the sea urchin and in
both cases the development is preceded by a membrane formation.
But how can membrane formation be produced by mere agitation? It
seems to me that this can be understood if we suppose that it
depends upon the destruction of an emulsion in the cortical layer
of the egg. It is conceivable that in the egg of certain forms the
stability of this emulsion is so small that mere shaking would
be enough to destroy it and thus induce membrane formation and
development.[92]
[92] Loeb, J., _Artificial Parthenogenesis and Fertilization_, p. 255.
The durability of emulsions varies, and where an emulsion is very
durable shaking has no effect, while where it is at the critical point
of separating into two continuous phases a slight shaking will bring
about the separation, and where the emulsion is still less durable we
observe the phenomenon of a “spontaneous” parthenogenesis. Eggs like
those of most sea urchins belong to the former, eggs like those of some
starfish and annelids belong to the second or third type.
It is impossible to state at present whether the fertilization
membrane is preformed in the fertilized egg and merely lifted off from
the egg or whether its formation is due to the hardening of a colloidal
substance separated from the emulsion (or excreted) and hardened in
touch with sea water. But we can be sure of one thing, namely, that
the liquid between egg and membrane contains some colloidal substance
which determines the tension and spherical shape of the membrane. The
membrane is obviously permeable not only to water but also to dissolved
crystalloids, while it is impermeable to colloids. When we add some
colloidal solution (_e. g._, white of egg, blood serum, or tannic
acid) to the sea water containing fertilized eggs of _purpuratus_, the
membrane collapses and lies close around the egg; while if the eggs are
put back into sea water or a sugar solution the membrane soon assumes
its spherical shape. This is intelligible on the assumption that in the
process of membrane formation (or in the destruction of the emulsion
in the cortical layer) a colloidal substance goes into solution which
cannot diffuse into the sea water since the membrane is impermeable to
the colloidal particles. The membrane is, however, permeable to the
constituents of sea water or to sugar. Consequently sea water will
diffuse into the space between membrane and egg until the tension of
the membrane equals the osmotic pressure of the colloid dissolved in
the space between egg and the membrane. If we add enough colloid to the
outside solution so that its osmotic pressure is higher than that of
the colloidal solution inside the membrane the latter will collapse.
It should also be stated that the unfertilized eggs of many marine
animals are surrounded by a jelly (chorion) which is dissolved when
the egg is fertilized.[93] The writer has shown that the same chemical
substances which will induce membrane formation and artificial
parthenogenesis will as a rule also cause a swelling and liquefaction
of the chorion.
[93] It has been stated by several writers that the eggs of the sea
urchin can no longer form the fertilization membrane when the jelly
surrounding the egg is dissolved. The writer has found that if the
jelly surrounding the eggs of _Strongylocentrotus purpuratus_ is
dissolved by acid the eggs still form a fertilization membrane upon the
entrance of a spermatozoön.
We have devoted so much space to the mechanism of membrane formation
since it is likely to give a clearer insight into the physicochemical
nature of physiological processes than the phenomena of muscular
stimulation and contraction or nerve stimulation, upon which the
majority of physiologists base their conclusions concerning the
mechanism of life phenomena.
Before we come to the discussion of the second factor in the activation
of the egg it should be stated more definitely that for the eggs of
some forms the first factor, the process underlying membrane formation,
suffices for the development of the egg into a larva and that no
second factor is required in these cases. This is true for the eggs
of starfish and certain annelids. Thus in 1901 Loeb[94] and Neilson
showed that a short treatment with HCl and HNO₃ sufficed to cause some
eggs of _Asterias_ in Woods Hole to develop into larvæ without a second
treatment being needed, and Delage[95] showed the same for CO₂; and
in 1905 the writer found that the eggs of the Californian starfish
_Asterina_ can be induced to form a membrane by butyric acid treatment
and that ten per cent. of these eggs developed into normal larvæ.
Quite recently R. S. Lillie observed that the eggs of _Asterias_ at
Woods Hole can be caused to form membranes and develop into larvæ by a
treatment with butyric acid and that the time of exposure required to
get a maximal number of larvæ varies approximately inversely with the
concentration of the acid, within a range of 0.0005 to 0.006 N butyric
acid. If the exposure is too short membrane formation will occur
without normal development.[96]
[94] Loeb, J., _Artificial Parthenogenesis and Fertilization_, 1913, p.
250 and ff.
[95] Delage, Y., _Arch. d. Zoöl. expér. et gén._, 1902, x., 213; 1904,
ii., 27; 1905, iii., 104.
[96] Lillie, R. S., _Jour. Biol. Chem._, 1916, xxiv., 233.
All this leads us to the conclusion that the main effect of the
spermatozoön in inducing the development of the egg consists in an
alteration of the surface of the latter which is apparently of the
nature of a cytolysis of the cortical layer. Anything that causes
this alteration without endangering the rest of the egg may induce
its development. The spermatozoön, therefore, causes the development
of the egg by carrying a substance into the latter which effects an
alteration of its surface layer.
5. We will now discuss the action of the second, corrective factor,
in the inducement of development. When we cause membrane formation in
a sea-urchin egg by the proper treatment with butyric acid it will
commence to develop and segment but will disintegrate rapidly if kept
at room temperature and the more rapidly the higher the temperature.
If, however, the eggs are treated afterward for a certain length of
time (from thirty-five to sixty minutes at 15° C. for _purpuratus_ and
17-1/2 to 22-1/2 minutes for _Arbacia_ at 23° C.) in a solution which
is isosmotic with 50 c.c. sea water +8 c.c. 2-1/2 m NaCl,[97] they
will develop into larvæ, many of which may be normal. Any hypertonic
solution of this osmotic pressure, sea water, sugar, or a single salt,
will suffice provided the solution does not contain substances that are
too destructive for living matter. The hypertonic solution produces
its corrective effect only if the egg contains free oxygen; and in a
slightly alkaline medium more rapidly than in a neutral medium. The
time of exposure in the hypertonic solution diminishes in certain
limits with the concentration of OH ions in the solution.
[97] It is necessary to call attention to the fact that sugar solutions
of a high concentration (_e. g._, m solutions) have a much higher
osmotic pressure than that which they should have theoretically (Lord
Berkeley and Hartley). Delage by ignoring this fact has misinterpreted
his experiments with sugar solutions. See Lloyd, D. J., _Arch. f.
Entwcklngsmech._, 1914, xxxviii., 402.
It is strange that in the eggs of _purpuratus_ the corrective effect
can also be brought about by exposing the eggs after the artificial
membrane formation for about three hours to normal sea water free from
oxygen; or to sea water in which the oxidations have been retarded by
the addition of KCN. This method is not so reliable as the treatment
with hypertonic solution.
What does the hypertonic solution do to prevent the disintegration of
the egg after the artificial membrane formation? The writer suggested
in 1905 that the artificial membrane formation alone starts the
development but leaves the eggs usually in a sickly condition and that
the hypertonic solution or the lack of oxygen allows them to recuperate
from such a condition. The second factor is, according to this view,
merely a corrective or curative factor. The following observations will
explain the reasons for such an assumption.
The writer found that if we keep the unfertilized eggs after artificial
membrane formation in sea water deprived of oxygen the disintegration
of the egg following artificial membrane formation is prevented for
a day at least. The same result can be obtained by adding ten drops
of 1/10 per cent. KCN to 50 c.c. of sea water, and certain narcotics,
_e. g._, chloral hydrate, act in the same way. Wasteneys and the writer
found that chloral hydrate (and other narcotics) in the concentration
required do not suppress or even lower the oxidations in the egg to
any considerable extent,[98] but they prevent the processes of cell
division. Hence it seems that the egg disintegrates so rapidly after
artificial membrane formation because it is killed by those processes
leading to nuclear division or cell division which are induced by
the artificial membrane formation. If we suppress these phenomena of
development (for not too long a time) we give the egg a chance to
recover and if now the impulse to develop is still active we notice
a perfectly normal development. If the egg is kept too long without
oxygen it suffers for other reasons and cannot develop; the writer has
shown that if eggs fertilized by sperm are kept for too long a time
without oxygen they also will no longer be able to develop normally.
The short treatment with a hypertonic solution supplies the corrective
factor required, so that the egg can then undergo cell division at room
temperature without disintegrating.
[98] Loeb, J., and Wasteneys, H., _Jour. Biol. Chem._, 1913, xiv., 517;
_Biochem. Ztschr._, 1913, lvi., 295.
The correctness of this interpretation, which is in reality mainly a
statement of observations, is proved by the two following groups of
facts. The older observers had already noticed that the unfertilized
eggs of the sea urchin when lying in sea water will die after a day
or more, and that occasionally such eggs show nuclear division or
even the beginning of cell division shortly before disintegration
sets in. The writer has studied this phenomenon in the unfertilized
eggs of _purpuratus_ and found that only the eggs of certain females
show this cell division before disintegration and that the cell
division is preceded by an atypical form of membrane formation; the
eggs surrounding themselves by a fine gelatinous film comparable to
that produced in the egg of _Arbacia_ by a treatment with butyric
acid. It is difficult to state what induces the alteration of the
surface in the eggs that lie so long in sea water. It may be due to
the CO₂ formed by the eggs--since we know that CO₂ may induce membrane
formation--or it may be due to the alkalinity of the sea water or to
a substance originating from the jelly surrounding the eggs. It was
found that if such eggs are kept without oxygen their disintegration
(and cell division) will be delayed considerably. The presumable
explanation for this is that the lack of oxygen prevents the internal
changes underlying cell division and thus prevents the disintegration
of the egg. The direct proof that an egg in the process of cell
division is more endangered by abnormal solutions than an egg at rest
has been furnished by numerous observations of the writer. He showed
in 1906 that the fertilized egg of _purpuratus_ dies rather rapidly
in a pure m/2 NaCl or any other abnormal isotonic solution, while
the unfertilized egg can live for days in such solutions.[99] In a
series of papers, beginning in 1905, he showed that the fertilized egg
will live longer in hypertonic, hypotonic, and otherwise abnormally
constituted solutions when the cell divisions are suppressed by lack of
oxygen or by the addition of KCN or of chloral hydrate.[100] It is thus
obvious that coincident with the changes underlying nuclear division
or cell division alterations occur in the sensitiveness of the egg to
salt solutions of abnormal concentration or constitution, _e. g._,
NaCl+CaCl₂ isotonic with sea water, hypertonic, or hypotonic solutions.
[99] Loeb, J., _Biochem. Ztschr._, 1906, ii., 81.
[100] Loeb, J., _Arch. f. d. ges. Physiol._, 1906, cxiii., 487;
_Biochem. Ztschr._, 1910, xxvi., 279, 289; xxvii., 304; xxix., 80;
_Arch. f. Entwcklngsmech._, 1914, xl., 322.
We must, therefore, conclude that artificial membrane formation
induces development but that it leaves the egg in a sickly condition
in which the very processes leading to cell division bring about
its destruction; that if it is given time it can recover from this
condition and that the treatment with the hypertonic solution also
brings about this recovery rapidly and reliably.
Herlant[101] suggested that the corrective effect of the hypertonic
solution consisted in the proper development of the astrospheres
required for cell division. According to this author mere membrane
formation does not lead to the formation of sufficiently large
astrospheres and hence cell division may remain impossible.[102] The
writer has no _a priori_ objection to this suggestion which agrees with
earlier observations by Morgan except that it is at present difficult
to harmonize it with all the facts. Why should it be possible to
replace the treatment with the hypertonic solution by a suspension of
the oxidations in the egg for three hours while we know that lack of
oxygen suppresses the formation of astrospheres in the fertilized eggs?
What becomes of the astrospheres if the treatment with the hypertonic
solution precedes the membrane formation by a number of hours or a day
(which is possible as we shall see), and why do they not induce cell
division, if Herlant’s idea is correct? Nevertheless the suggestion of
Herlant deserves to be taken into serious consideration.
[101] Herlant, M., _Arch. de Biol._, 1913, xxviii., 505.
[102] It is also important to remember that the formation of
astrospheres after mere membrane formation occurs considerably more
slowly than if the egg has also received a treatment with a hypertonic
solution.
6. How can an alteration of the surface of the egg--_e. g._, a
cytolytic or other destruction of the cortical layer--lead to a
beginning of development? The answer is possibly given in the relation
of oxidation to development. The writer found in 1895 that if oxygen
is withdrawn from the fertilized sea-urchin egg it can not segment and
this seems to be the case for eggs in general.[103] In 1906 he found
that the rapid disintegration of the eggs of the sea urchin which
follows artificial membrane formation could be prevented when the eggs
were deprived of oxygen or when the oxidations were suppressed in the
eggs by KCN. This suggested a connection between the disintegration of
the egg after artificial membrane formation and the increase in the
rate of oxidations; and he found further that the formation of acid is
greater in the fertilized than in the unfertilized egg. He, therefore,
expressed the view in 1906 that the essential feature (or possibly one
of the essential features) of the process of fertilization was the
increase of the rate of oxidations in the egg and that this increase
was caused by the membrane formation alone.[104] These conclusions
have been since amply confirmed by the measurements of O. Warburg as
well as those of Loeb and Wasteneys, both showing that the entrance
of the spermatozoön into the egg raises the rate of oxidations from
400 to 600 per cent., and that membrane formation alone brings about
an increase of similar magnitude. Loeb and Wasteneys found that the
hypertonic solution does not increase the rate of oxidations in a
fertilized egg. It does do so, however, in an unfertilized egg without
membrane formation, but merely for the reason that in such an egg the
hypertonic solution brings about the cytolytic change in the cortex of
the egg underlying membrane formation.[105] According to Warburg it
is probable that the oxidations occur mainly if not exclusively at the
surface of the egg since NaOH, which does not diffuse into the egg,
raises the rate of oxidations more than NH₄OH which does diffuse into
the egg. And finally, the same author showed that the oxidations in
the sea-urchin egg are due to a catalytic process in which iron acts
as a catalyzer.[106] In view of all these facts and their harmony with
the methods of artificial parthenogenesis the suggestion is justifiable
that the alteration or cytolysis of the cortical layer of the egg is in
some way connected with the increased rate of oxidations.
[103] The writer found that the eggs of _Fundulus_ will segment a
number of times even if all the oxygen has apparently been removed.
[104] Loeb, J., _Biochem. Ztschr._, 1906, ii., 183.
[105] Thus the treatment of an unfertilized egg without membrane with a
hypertonic solution combines two effects, first the general cytolytic
alteration of the cortical layer of the membrane and the corrective
effect of the hypertonic solution. The former effect raises the rate of
oxidations in the egg, the latter does not.
[106] Warburg, O., _Sitzungsber. d. Heidelberger Akad. d. Wissnsch._,
B. 1914.
The question remains then: How can membrane formation or the alteration
of the cortical layer underlying membrane formation cause an increase
in the rate of oxidations? One possibility is that the iron (or
whatever the nature of the catalyzer may be) exists in the cortex of
the egg in a masked condition--or in a condition in which it is not
able to act--while the alteration of the cortical layer makes the iron
active. It might be that either the iron or the oxidizable substrate is
contained in the lipoid layer in the unfertilized condition of the egg
and that the destruction or cytolysis of the cortical layer brings both
the iron and the oxidizable substrate into the watery phase in which
they can interact.
Another possibility is that the act of fertilization increases the
permeability of the egg. This idea, which seems attractive, was first
suggested and discussed by the writer in 1906.[107] He had found that
when fertilized and unfertilized eggs were put into abnormal salt
solutions, _e. g._, pure solutions of NaCl, the fertilized eggs died
more rapidly than the unfertilized eggs and he pointed out that these
experiments suggested the possibility that fertilization increases the
permeability of the egg for salts. The reason for his hesitation to
accept this interpretation was, that the fertilized egg is also more
easily injured by lack of oxygen than the unfertilized egg and in this
case the greater sensitiveness of the fertilized egg was obviously due
to its greater rate of metabolism. Later experiments by the writer
showed that the fertilized egg can be made more resistant to abnormal
salt solutions if its development is suppressed by lack of oxygen or
by KCN or by certain narcotics. With our present knowledge it does not
seem very probable that lack of oxygen diminishes the permeability of
the egg, but we know that it inhibits the developmental processes.
Warburg has made it appear very probable that the fertilized egg
is impermeable for NaOH and if this is the case it should also be
impermeable for NaCl.[108]
[107] Loeb, J., _Biochem. Ztschr._, 1906, ii., 87.
[108] Unless the egg is left so long in the pure NaCl solution that its
permeability is increased.
The idea that fertilization and membrane formation cause an increase
in the permeability of the egg was later accepted and elaborated by R.
Lillie. This author assumes that the unfertilized egg cannot develop
because it contains too much CO₂ but that the CO₂ can escape from the
egg as soon as its permeability is increased through the destruction
of the cortical layer of the egg.[109] After the CO₂ has escaped, the
excessive permeability must be restored to its normal value and this
is the rôle of the hypertonic treatment. It is, however, difficult to
harmonize the assumption of an impermeability of the unfertilized egg
for CO₂ with the fact that if the unfertilized sea-urchin egg is cut
into two, as is done in merogony, no development takes place, while
such pieces will develop when a spermatozoön enters. The cortical
layer is removed along the cut surface and there is no reason why the
CO₂ should not escape. Besides, the experiments of Godlewski and the
writer prove that the cortical layer of the unfertilized sea-urchin
egg is apparently very permeable for CO₂ since the latter causes
membrane formation if contained in the sea water in sufficiently high
concentration.
[109] Lillie, R. S., _Jour. Morphol._, 1911, xxii., 695; _Am. Jour.
Physiol._, 1911, xxvii., 289.
Lillie assumes that the hypertonic treatment restores the permeability
raised to excess by the butyric acid treatment, but this assumption is
not in harmony with the following facts. The writer has shown that it
is immaterial whether the eggs are treated first with the hypertonic
solution and then with butyric acid or the reverse, if only the eggs
remain longer in the hypertonic solution when the hypertonic treatment
precedes the butyric acid treatment. It was stated in the beginning
of this chapter that the development of the egg can be induced by
hypertonic sea water, and we know the reason since hypertonic sea
water is a cytolytic agency. The writer found that when we expose
unfertilized eggs of _purpuratus_ for from two to two and a half-hours
to hypertonic sea water they will often not develop and only a few eggs
will undergo the first cell divisions, then going into a condition of
rest. When these eggs, both the segmented and unsegmented, were treated
twenty-four or thirty-six hours later with butyric acid, so that they
formed a membrane, they all developed into larvæ without further
treatment. It is impossible to apply Lillie’s theory to these facts,
for the simple reason that the treatment with hypertonic sea water was
just long enough to induce development in some eggs and hence according
to Lillie’s ideas must have increased the permeability of these eggs.
Yet these same eggs were induced to develop normally when subsequently
treated with butyric acid, which according to Lillie also acts by
increasing the permeability. Nothing indicates that the treatment of
the eggs with a hypertonic solution diminishes their permeability; the
reverse would be much more probable.
Lillie’s theory also fails to explain that mere treatment of the eggs
with a hypertonic solution can bring about their development into
larvæ. This, however, is intelligible on the assumption that the
hypertonic solution in this case has two different effects, first a
cytolysis of the cortical layer of the egg and second an entirely
different effect, possibly upon the interior of the egg, which
represents the second or corrective effect.
McClendon[110] has shown that the electrical conductivity of the egg
is increased after fertilization, and J. Gray[111] has found that this
increase in conductivity is only transitory and disappears in fifteen
minutes. This might indicate that the egg becomes transitorily more
permeable for salts after the entrance of the spermatozoön or after
membrane formation; although an increase in conductivity might be
caused by other changes than a mere increase in permeability of the
egg. The writer is of the opinion that it is necessary to meet all
these and other difficulties before we can state that the alteration
of the cortical layer, which is the essential feature of development,
acts chiefly or exclusively by an increase in the permeability of the
egg.[112]
[110] McClendon, J. F., Publications of the Carnegie Institution, No.
183, 125; _Am. Jour. Physiol._, 1910, xxvii., 240.
[111] Gray, J., _Proc. Cambridge Philosophical Society_, 1913, xvii., 1.
[112] R. Lillie has recently shown that in a hypotonic solution water
diffuses more rapidly into a fertilized than into an unfertilized
egg. This is exactly what one should expect since the unfertilized
egg is not only surrounded by the cortical layer but also by a thick
layer of jelly both of which are lacking in the fertilized egg. It is
difficult to understand how this observation can throw any light on the
mechanism of development, since water diffuses rapidly enough into the
unfertilized egg.
7. When the experiments on artificial parthenogenesis were first
published they aroused a good deal of antagonism not only among
reactionaries in general but also among a certain group of biologists.
O. Hertwig had defined fertilization as consisting in the fusion of two
nuclei, the egg nucleus and the sperm nucleus. No such fusion of two
nuclei takes place in artificial parthenogenesis since no spermatozoön
enters the egg, and it became necessary, therefore, to abandon
Hertwig’s definition as wrong. The objection raised that the phenomena
are limited to a few species soon became untenable since it has been
possible to produce artificial parthenogenesis in the egg of plants
(_Fucus_, according to Overton) as well as of animals, from echinoderms
up to the frog; and it may possibly one day be accomplished also in
warm-blooded animals. A second objection was that the eggs caused to
develop by the methods of artificial parthenogenesis could never reach
the adult stage and that hence the phenomenon was merely pathological.
There was no basis for such a statement, except that it is extremely
difficult to raise marine invertebrates. Delage[113] was courageous
enough to make an attempt to raise parthenogenetic larvæ of the sea
urchin beyond the larval stage and he succeeded in one case in carrying
the animal to the mature stage. It proved to be a male.
[113] Delage, Y., _Compt. rend. Acad. Sc._, 1909, cxlviii., 453.
Better opportunities were offered when a method was discovered which
induced the development of the unfertilized eggs of the frog. In
1907, Guyer made the surprising observation that if he injected
lymph or blood into the unfertilized eggs of frogs he succeeded
in starting development and he even obtained two free-swimming
tadpoles. “Apparently the white rather than the red corpuscles are the
stimulating agents which bring about development, because injections
of lymph which contains only white corpuscles produce the same effects
as injections of blood.” Curiously enough, Guyer thought that probably
the cells which he introduced and not the egg were developing. In
1910, Bataillon showed that a mere puncture of the egg with a needle
could induce development but he believes that for the full development
the introduction of a fragment of a leucocyte is required. Bataillon
has called attention to the analogy with the writer’s results on
lower forms, the puncturing of the egg corresponding to the cytolysis
of the surface layer of the egg and the introduction of a leucocyte
as the analogue of the second or corrective factor. The method of
producing artificial parthenogenesis by puncturing the egg has thus
far been successful only in the egg of the frog. The writer has tried
it in vain on the eggs of many other forms. He has at present seven
parthenogenetic frogs over a year old, produced by merely puncturing
the eggs with a fine needle (Fig. 6). These frogs have reached over
half the size of the adult frog. They can in no way be distinguished
from the frogs produced by fertilization with a spermatozoön.
This makes the proof conclusive that the methods of artificial
parthenogenesis can result in the production of normal organisms which
can reach the adult stage.
Bancroft and the writer tried to determine the sex of a parthenogenetic
tadpole and of a frog just carried through metamorphosis. Since in
early life the sex glands of both sexes in the frog contain eggs it is
not quite easy to determine the sex, except that in the male the eggs
gradually disappear and from this and other criteria we came to the
conclusion that both parthenogenetic specimens, which were four months
old, were males.
The writer has recently examined the gonads of a ten months old
parthenogenetic frog. Here no doubt concerning the sex was possible
since the gonads were well-developed testicles containing a large
number of spermatozoa of normal appearance, and no eggs.[114] (Figs. 7
and 8.) This would indicate that the frog belongs to those animals in
which the male is heterozygous for sex.
[114] Since this was written, two more of the parthenogenetic frogs
over a year old died. Both were males.
8. The fact that the egg of so high a form as the frog can be
made to develop into a perfect and normal animal without a
spermatozoön--although normally the egg of this form does not develop
unless a spermatozoön enters--corroborates the idea expressed in
previous chapters that the egg is the future embryo and animal; and
that the spermatozoön, aside from its activating effect, only transmits
Mendelian characters to the egg. The question arises: Is it possible
to cause a spermatozoön to develop into an embryo? The idea has
been expressed that the egg was only the nutritive medium on which
the spermatozoön developed into an embryo, but this idea has been
rendered untenable by the experiments on artificial parthenogenesis.
Nevertheless the question whether or not the spermatozoön can develop
into an embryo on a suitable culture medium remains, and it can only be
decided by direct experiments. It was shown by Boveri, Morgan, Delage,
Godlewski, and others, that if a spermatozoön enters an enucleated egg
or piece of egg it can develop into an embryo, but since the cytoplasm
of the egg is the future embryo this experiment proves only that
the egg nucleus may be replaced by the sperm nucleus; and also that
the sperm nucleus carries into the egg, the substances which induce
development. Incidentally these experiments on merogony also prove that
the mere mechanical tearing of the cortical layer,--which must happen
in the separation of the unfertilized egg into parts with and without
a nucleus,--by dissection or by shaking, is not sufficient to start
development in the sea-urchin egg.
J. de Meyer put the spermatozoa of sea urchins into sea water
containing an extract of the eggs of the same species but found only
that the spermatozoa swell in such a solution. Loeb and Bancroft made
extensive experiments in cultivating spermatozoa of fowl _in vitro_
on suitable culture media. In yolk and white of egg the head of the
spermatozoön underwent transformation into a nucleus, but no mitosis
or aster formation was observed.[115] These experiments should be
continued.
[115] Loeb, J., _Artificial Parthenogenesis and Fertilization_,
Chicago, 1913.
CHAPTER VI
DETERMINISM IN THE FORMATION OF AN ORGANISM FROM AN EGG
1. The writer in a former book (_Dynamics of Living Matter_, 1906, p.
1), defined living organisms as chemical machines consisting chiefly
of colloidal material and possessing the peculiarity of preserving and
reproducing themselves. Some authors like Driesch, and v. Uexküll seem
to find it impossible to account for the development of such machines
from an undifferentiated egg on a purely physicochemical basis. A
study of Driesch’s very interesting and important book[116] shows
that he assumes the eggs of certain animals, _e. g._, the sea urchin,
to consist of homogeneous material; and he concludes that nature has
solved, in the formation of highly differentiated organisms from such
undifferentiated material, a problem which does not seem capable of
a solution by physicochemical agencies alone. But the supposition
of a structureless egg is wrong, since Boveri has demonstrated the
existence of a very simple but definite structure in the unfertilized
egg of the sea urchin; and a similar simple structure has been
demonstrated by other authors, especially Conklin, in the eggs of other
forms.
[116] Driesch, H., _Science and Philosophy of the Organism_. London,
1908 and 1909.
In this chapter we shall attempt the task among others of showing
how, on the basis of the simple physicochemical structure of the
unfertilized egg, the main organ of self-preservation of the organism,
the intestine, is formed through the mere process of cell division and
growth. Cell division is the most general of the specific functions
of living matter and it is the basis underlying the differentiation
of the comparatively simple structure of the egg into a more complex
organism. If cell division and growth were equal in all parts of the
egg no differentiation would be possible, but the different regions of
the unfertilized egg contain different constituents and these, probably
on account of their chemical difference, do not all begin to grow or
divide simultaneously and equally.
Boveri[117] found that in the unfertilized egg of the sea urchin
_Strongylocentrotus lividus_ at Naples a definite structure is
indicated by the fact that the yellowish-red pigment is not equally
distributed over the whole surface of the egg but is arranged in a wide
ring from the equator almost to one of the poles. Thus three zones can
be recognized in the egg (Fig. 9), a small clear cap _A_ at one pole,
a pigmented ring _B_, and the rest again unpigmented _C_. Observation
has shown that each one of these regions gives rise to a definite
constituent of the egg: _A_ furnishes the mesenchyme from which the
skeleton and the connective tissue originate; _B_ is the material for
the formation of the intestine, and _C_ gives rise to the ectoderm.
[117] Boveri, Th., _Verhandl. d. physik.-med. Gesellsch._, Würzburg,
1901, xxxiv., 145.
[Illustration: FIG. 9]
The pigment is only at the surface of the egg, and its collection at
_B_ indicates only that the material in _B_ differs physicochemically
from _A_ and _C_. The real determiners of the three different groups
of organs are three different groups of substances whose distribution
is approximately but probably not wholly identical with the regions
indicated by distribution of pigment. The intestine-forming material is
probably not entirely lacking in _C_ but is contained here in a lower
concentration and probably the more so the greater the distance from
_B_; and the same may probably be said for the substances determining
mesenchyme and ectoderm formation. Hence the unfertilized egg contains
already a rough preformation of the embryo inasmuch as the main axis of
the embryo and the arrangement of its first organs are determined.
[Illustration: FIG. 10]
[Illustration: FIG. 11]
After the egg is fertilized the cell divisions begin. The first
division is as a rule at right angles to the stratification of the
egg, each of the two cells contains one-half of the pigment ring (and
of each of _A_ and _C_) (Fig. 10), and after the next division each
contains one-fourth of the pigmented part. Each of the four cells
is a diminutive whole egg since each contains the three layers in
the normal arrangement (Fig. 11). The next divisions bring about an
unequal division of the material. Four cells will be formed of ectoderm
material _C_ and only little intestine material _B_, the other four
cells containing _B_ and _A_. These latter form at the next division
four very small colourless cells, the so-called micromeres, _A_ (Fig.
12), from which the mesenchyme, skeleton, and connective tissue are
formed, four larger cells, _B_, from which the intestine is formed, and
eight cells, _C_, from which the ectoderm will arise. The separation
of the three groups of substances is probably not as complete as our
purely diagrammatic drawing (Fig. 12) indicates.
[Illustration: FIG. 12]
The cell division proceeds and the cells become smaller and smaller and
all gather at the surface of the egg, thus forming a hollow sphere.
It is not known what brings about this gathering of the cells at the
surface, whether it is protoplasmic creeping or streaming or whether
the cells are held by a jelly-like layer which covers the surface of
the egg (hyaline membrane) (Fig. 13). Then the cilia are formed at the
external surface of these cells and the egg begins to swim; we say
it has reached the first larval, the so-called blastula stage. This
happens according to Driesch after the tenth series of cell divisions,
when the number of cells is theoretically 1024, in reality not quite
so many (between 800 and 900). The next step consists in the cells
derived from the material _A_ (mesenchyme and micromeres) gliding into
the hollow sphere, where they form a ring, the physicochemical process
responsible for this gliding being yet unknown. At the opening of this
ring an active growing of the cells of the entoderm into the hollow
sphere takes place and the hollow cylinder formed by this growth is the
intestine (Fig. 14). Why the cells grow into the hollow sphere and not
into the opposite direction is unknown. The next step is the formation
of a skeleton by the formation of crystals consisting of the CaCO₃ by
the mesenchyme cells surrounding the intestine. For the establishment
of the principle in which we are interested the description of
morphogenesis need not be carried farther.
[Illustration: FIG. 13]
[Illustration: FIG. 14]
This principle which is under discussion here is the development of
a purposeful arrangement of organs out of the egg. If we assume that
the egg consists of homogeneous material we are indeed confronted with
a riddle. Since the facts contradict such an assumption but show, as
Boveri has pointed out, a prearrangement which allows us to indicate
in the unfertilized egg already the exact spot where the intestine
will grow into the blastula cavity, we are on solid physicochemical
ground, although many questions of detail cannot yet be answered.
Such a preformation as Boveri has demonstrated is only conceivable if
the material of the egg has not too high a degree of fluidity; we may
consider it as consisting essentially of a semi-solid gel which is not
homogeneous throughout the egg but divided into three strata.
2. Lyon[118] tried to ascertain whether by centrifuging the sea-urchin
egg it was possible to modify its structure and thereby affect the
later embryo. He and subsequent experimenters found that it only is
possible to change the position of the nucleus and the distribution
of the pigment in the egg. It follows from this that the nucleus and
the pigment are suspended in rather fluid material, the former in
the centre, the pigment at or near the surface. The position of the
nucleus determines the first plane of segmentation, since the nuclear
division precedes the division of the cytoplasm of the egg and the
plane of nuclear division becomes also the plane of the division of
the whole egg--a point which need not be discussed here. It was found,
however, by Lyon and the subsequent investigators that the place where
the micromeres are formed and where the intestine of the embryo later
originates is little influenced by the centrifuging of the egg. The
localization of this spot must therefore be determined by a structure
sufficiently solid not to be shifted by the centrifugal force. The
intestinal stratum in the egg contains the forerunners of the tissues
which secrete hydrolyzing enzymes, _e. g._, trypsin into the digestive
tract.
[118] Lyon, E. P., _Arch. f. Entwcklngsmech._, 1907, xxiii., 151;
Morgan, T. H., and Spooner, G. B., _ibid._, 1909, xxviii., 104; Morgan,
_Jour. Exper. Zoöl._, 1910, ix., 594; Conklin, E. G., _ibid._, 1910,
ix., 417; Lillie, F. R., _Biol. Bull._, 1909, xvi., 54.
When the surrounding solution is altered in constitution or when the
temperature is too high, the intestine instead of growing into the
hollow sphere grows outside, we get an evagination instead of an
invagination of the intestine. Such larvæ may live for a few days but
they cannot grow into a living organism. The forces which make the
intestine grow into the hollow sphere are unknown; it may possibly be
only the difference between the tension on the external and internal
surfaces of the hollow sphere; under normal conditions, the resistance
on the inner surface being smaller, the intestine grows into the hollow
sphere.
The intestine is one of the organs required for the self-preservation
of a more complicated organism, in fact a higher organism without a
digestive tract is not capable of living for any length of time. In the
gastrula--_i. e._, the blastula with an intestine--we have an organism
which is durable, but the processes leading up to the formation of the
intestine are so simple that it is difficult to understand why the
assumption of a “supergene” should be required in this case.
3. Driesch[119] was the first to show that if we isolate one of the
first two cells of a dividing egg each develops into a whole embryo of
half size. This is perfectly intelligible, since each of the two cells
contains all the three layers in the normal arrangement (Fig. 10). The
cells divide and the cells having the tendency to creep to the surface
of the mass arrange themselves in a hollow sphere, the blastula.
Since micromeres and intestine material are present and in their
normal position an intestine will grow into the blastula and a whole
organism will result. All of this is as necessary as is the formation
of one embryo from the whole egg material. Yet the two half-embryos
betray their origin from two cleavage cells of the same egg, in that
the two gastrulæ formed are often if not always symmetrical to each
other (Fig. 15), as the writer had a chance to observe in the egg of
_Strongylocentrotus purpuratus_[120] in the following experiment.
The eggs of the sea urchin _Strongylocentrotus purpuratus_ are put soon
after fertilization into solutions which differ from sea water in two
points; namely that they are neutral or very faintly acid (through
the CO₂ absorbed from the air) instead of being faintly alkaline, and
second, that one of the following three constituents of the sea water
is lacking; namely: K, Na, or Ca. When the eggs are allowed to segment
in such a solution the first two cleavage cells are as a rule in a
large percentage of cases--often as many as ninety per cent.--separated
from each other, and when the eggs are put into normal sea water (about
twenty minutes after the cell division) each cell develops into a
normal embryo. In a number of cases the embryos remained inside the
egg membrane and did not move until after the invagination of the
intestine was far advanced; in such cases it was found quite often that
the invagination began at the plane of cleavage at symmetrical points
of the two embryos, and the growth of the intestine was symmetrical in
both embryos.
[119] Driesch, H., _Ztschr. f. wissnsch. Zoöl._, 1891, liii., 160.
[120] Loeb, J., _Arch. f. Entwcklngsmech._, 1909, xxvii., 119.
[Illustration: FIG. 15]
This symmetry is probably due to the following fact: the first cleavage
plane goes through that spot where the intestine grows into the
blastula cavity. If the micromere material does not change its position
after the two cleavage cells are separated and the new blastulæ do not
become completely spherical the symmetry which we observed is bound
to occur. The occurrence is a confirmation of Boveri’s observation.
It is natural that Driesch also found that each cell in the four-cell
stage should give rise to a full embryo, since each of these cells is
in reality a diminutive egg containing the three strata in the right
arrangement. When, however, the cells of the eight- or sixteen-cell
stage were isolated Driesch’s results were different. In this case
the isolated cells from the ectoderm material did no longer all form
a gastrula; when such a cell still formed a gastrula it was probably
due to the fact that it contained some entoderm material; while the
cells taken from the entoderm region all formed embryos and therefore
contained ectoderm material.[121] The isolated ectoderm cells of a
blastula could no longer form an intestine; they were lacking the
entoderm material. It looks as if a gradual migration of all the
entoderm material from the ectoderm into the entoderm took place during
the blastula formation.
[121] Driesch, H., _Arch. f. Entwcklngsmech._, 1900, x., 361.
When the contents of the egg are displaced by pressure the result
will be determined by the location of the main mass of the
intestine-forming material; where the main mass of this body is
located the invagination of the intestine will take place. In his
earlier work Driesch assumed from pressure experiments that the egg
had a great power of “regulation.” In a later paper[122] he expressed
to a large extent his agreement with Boveri who denied this power of
“regulation” and showed that the existence of the structure of the
egg--_i. e._, a division into three strata, one forming the ectoderm,
the second the entoderm, and the third the mesoderm--was sufficient
to explain the various phenomena of apparent “regulation.” Driesch’s
idea of a regulation in this case has often been used to insist upon
the non-explicability of the phenomena of development from a purely
physicochemical viewpoint. It is, therefore, only fair to point out
that Boveri[123] has furnished the facts for a simpler explanation,
which seems to have escaped the notice of antimechanists.[124]
[122] Driesch, H., _Arch. f. Entswcklngsmech._, 1902, xiv., 500.
[123] Boveri, Th., _Verhandl. d. physik. med. Gesellsch._, Würzburg,
N.F., 1901, xxxiv., 145.
[124] v. Uexküll makes in his last book (_Bausteine zu einer
biologischen Weltanschauung_, München, 1913, p. 24) the following
statement: “Driesch succeeded in showing that the germ cell has no
trace of a machine-like structure but consists entirely of equivalent
parts.” This is not correct.
The objection may be raised that in accepting Boveri’s facts and
interpretation we pushed the miracle only one step farther and that we
now have to explain the origin of the structure in the unfertilized
egg. This Boveri has done by showing that the egg grows from the wall
of the ovary and that that part of the egg which is connected with the
wall of the ovary gives rise to the ectoderm layer, while the opposite
part gives rise to the mesenchyme and the intestine. This shows a
connection between the orientation of the egg in the wall of the ovary
and its stratification. While this does not solve the problem of
stratification in the egg it gives the clue to its solution.
The ultimate origin of stratification probably goes back to the fact
of the presence of watery and water-immiscible substances, such as
fats. The experiments by Beutner and the writer have shown that the
electromotive forces which are observed in living tissues originate
at the boundaries between a watery and a water-immiscible phase, like
oleic acid or lecithin.[125] In his earlier writings[126] the writer
had thought that the colloids had special significance and this idea
seems to prevail today; but the actual observations have shown that the
phase boundary fat-water is of greater importance. Needless to say the
fats if not present in the cell from the beginning can be formed in the
metabolism.
[125] Loeb, J., and Beutner, R., _Biochem. Ztschr._, 1912, xli., 1;
xliv., 303; 1913, li., 288; li., 300; 1914, lix., 195.
[126] Loeb, J., _The Dynamics of Living Matter_. New York, 1906.
Introductory Remarks.
4. All the “regulation” in the egg is of a purely physicochemical
character; it consists essentially of a flow of material. If this idea
is correct, the apparent power of “regulation” of the blastomeres
should differ according to the degree of fluidity and the possibility
of different layers separating, and this assumption is apparently
supported by facts. The first plane of segmentation of the egg is
usually the plane of symmetry of the later organism and where the
degree of fluidity is less than in the sea-urchin egg, a separation of
the two first blastomeres should easily result in the formation of two
half-embryos instead of two whole embryos.
This is the case for the frog’s egg as Roux showed in a classical
experiment. Roux destroyed one of the two first cleavage cells of a
frog’s egg with a hot needle and found that as a rule the surviving
cell developed into only a half-embryo.[127] The frog’s egg consists
of two substances, a lighter one which is on top and a heavier one
below. Although viscous, the two substances are not too viscous to
prevent a flow if the egg is turned upside down. O. Schultze found that
if a normal egg is turned upside down in the two-cell stage and held
in that position, two full embryos arise, one from each of the two
blastomeres. Through the flow of the lighter liquid in the egg upwards
the two halves of the protoplasm on top become separated and develop
independently into two whole embryos instead of into two half-embryos.
In Roux’s experiment this flow of protoplasm was avoided. Morgan showed
that if Roux’s experiment is repeated with the modification that the
egg is put upside down after the destruction of the one cell, the
intact cell will give rise not to a half but to a whole embryo.[128]
These experiments prove that each of the first two cleavage cells of
the frog’s egg represents one-half of the embryo and that a whole
embryo can develop from each half only when a redistribution of
material takes place, which in the egg of the frog can be brought about
by gravitation since the egg consists of a lighter and a heavier mass.
[127] Roux, W., _Virchow’s Archiv_, 1888, cxiv., 113.
[128] Morgan, T. H., _Embryology of the Frog_. New York.
When, therefore, in the egg of the sea urchin each of the first two
blastomeres naturally gives rise to a whole embryo it is due to a
greater degree of fluidity of the protoplasm and not to a lack of
preformation of the embryo in the cytoplasm. This idea is confirmed
by the observations on the egg of _Ctenophores_ whose cytoplasm seems
to be more solid than that of most other eggs. Chun found that the
isolated blastomere of the first cell division produced a half-larva,
possessing only four instead of the eight locomotor plates of the
normal animal.
It seems that in the egg of molluscs, also, the simple symmetry
relations of the body are already preformed. It is well known that
there are shells of snails which turn to the right while others turn
in the opposite direction. The shells of _Lymnæus_ turn to the right,
those of _Planorbis_ to the left. It was observed by Crampton[129],
Kofoid, and Conklin that the eggs of right-wound snails do not segment
in a symmetrical, but in a spiral, order, and that in left-handed
snails the direction of the spiral segmentation is the reverse of that
of the segmentation in the right-handed snails. Conklin was able to
show that the asymmetrical spiral structure is already preformed in the
egg before cleavage. The asymmetry of the body in snails is therefore
already preformed in the egg.[130]
[129] Crampton, H. E., _New York Academy of Sciences_, 1894; Kofoid, C.
A., _Proc. Am. Acad. Arts and Sciences_, 1894, xxix.
[130] Conklin, E. G., _Anat. Anzeig._, 1903, xxiii., 577; _Heredity and
Environment in the Development of Man_. Princeton, 1915, p. 171.
E. B. Wilson[131] has found a marked differentiation in the eggs of
some annelids and molluscs. He isolated the first two blastomeres of
the egg of _Lanice_, an Annelid. These two blastomeres are somewhat
different in size; from the larger one of the first two blastomeres,
the segmented trunk of the worm originates. Wilson found that
when either cell of the two-cell stage is destroyed, the
remaining cell segments as if it still formed a part of an entire
embryo.[132] The later development of the two cells differs in
an essential respect, and in accordance with what we should
expect from a study of the normal development. The posterior cell
develops into a segmented larva with a prototroch, an asymmetrical
pre-trochal or head region, and a nearly typical metameric
seta-bearing trunk region, the active movements of which show that
the muscles are normally developed. The pre-trochal or head region
bears an apical organ, but is more or less asymmetrical, and, in
every case observed, but a single eye was present, whereas the
normal larva has two symmetrically placed eyes. The development of
the anterior cell contrasts sharply with that of the posterior.
This embryo likewise produces a prototroch and a pre-trochal
region, with an apical organ, but produces no post-trochal region,
develops no trunk or setæ, and does not become metameric. Except
for the presence of an apical organ, these anterior embryos are
similar in their general features to the corresponding ones
obtained in _Dentalium_. None of the individuals observed developed
a definite eye, though one of them bore a somewhat vague pigment
spot.
This result shows that from the beginning of development the
material for the trunk region is mainly localized in the posterior
cell; and, furthermore, that this material is essential for the
development of the metameric structure. The development of this
animal is, therefore, to this extent, at least, a mosaic work
from the first cleavage onward--a result that is exactly parallel
to that which I earlier reached in _Dentalium_, where I was able
to show that the posterior cell contains the material for the
mesoblast, the foot, and the shell; while the anterior cell lacks
this material. I did not succeed in determining whether, as in
_Dentalium_, this early localization in _Lanice_ pre-exists in the
unsegmented egg. The fact that the larva from the posterior cell
develops but a single eye, suggests the possibility that each of
the first two cells may be already specified for the formation of
one eye; but this interpretation remains doubtful from the fact
that the larva from the anterior cell did not, in the five or six
cases observed, produce any eye.
[131] Wilson, E. B., _Science_, 1904, xx., 748; _Jour. Exper.
Zoöl._, 1904, i., 1, 197.
[132] The reader will notice the absence of “regulation.”
Conklin has established the existence of a definite structure in the
unfertilized eggs of Ascidians, Amphioxus, and many molluscs. In all
cases the results of the isolation of the first blastomeres seem to
agree with the demonstrable structure of the unfertilized egg.
5. These examples may suffice to show that the egg has from the
beginning a simple structure, and we will now point out by which
means further differentiation may come about. Sachs suggested that
all differentiation and the formation of every organ presupposes
the previous existence of specific substances responsible for the
formation. These substances which are now called internal secretions or
hormones develop gradually during embryonic development. What exists
first is a jelly-like block of protoplasmic material with a varying
degree of viscosity and with just enough differentiation to indicate
head and tail end, a right and left, and a dorsal and ventral side of
the future embryo.
Aside from such simple differences phenomena of protoplasmic streaming
contribute to the further differentiation. Such streaming begins,
according to Conklin,[133] in the egg just before fertilization when
the surface layer of the egg protoplasm
streams to the point of entrance of the sperm, and these movements
may lead to the segregation of different kinds of plasma in
different parts of the egg and to the unequal distribution of these
substances in different regions of the egg.
[133] Conklin, E. G., _Heredity and Environment in the Development
of Man_. Princeton University Press, 1915. The reader is referred to
this book for the literature and main facts on the structure of the
egg; it should also be stated that Conklin’s book is one of the best
introductions to modern biology in the English literature.
One of the most striking cases of this is found in the Ascidian
Styela in which there are four or five different kinds of substances
in the egg which differ in colour, so that their distribution to
different regions of the egg and to different cleavage cells may be
easily followed and even photographed while in the living condition.
The peripheral layer of protoplasm is yellow and when it gathers at
the lower pole of the egg where the sperm enters it forms a yellow
cap. This yellow substance then moves following the sperm nucleus,
up to the equator of the egg on the posterior side and there forms a
yellow crescent extending around the posterior side of the egg just
below the equator. On the anterior side of the egg a grey crescent
is formed in a somewhat similar manner and at the lower pole between
these two crescents is a slate-blue substance, while at the upper
pole is an area of colourless protoplasm. The yellow crescent goes
into cleavage cells which become muscle and mesoderm, the grey
crescent into cells which become nervous system and notochord, the
slate-blue substance into endoderm cells, and the colourless
substance into ectoderm cells.
Thus within a few minutes after the fertilization of the egg and
before or immediately after the first cleavage, the anterior and
posterior, dorsal and ventral, right and left poles are clearly
distinguishable, and the substances which will give rise to
ectoderm, endoderm, mesoderm, muscles, notochord, and nervous system
are plainly visible in their characteristic positions.[134]
[134] Conklin, E. G., _loc. cit._, p. 117.
We may finally allude briefly to the fact that when once a number of
tissues are differentiated each one may influence the other by calling
forth tropistic reactions. Thus the writer showed that in the yolk
sac of the fish _Fundulus_ the pigment cells lie at first without any
definite order but that they gradually are compelled to creep entirely
on the blood-vessels and form a sheath around them with the result
that the yolk sac assumes a tiger-like marking.[135] Driesch[136] has
pointed out that the mesenchyme cells are directed in their migration;
and it seems that the direction of the growth of the axis cylinder is
determined by the tissues into which it grows. The idea of tropistic
reactions in the formation of organs has been discussed by Herbst.[137]
[135] Loeb, J., _Jour. Morphol._, 1893, xiii., 161; _The Mechanistic
Conception of Life_. Chicago, 1912, p. 106.
[136] Driesch, H., _Science and Philosophy of the Organism_, i., p. 104.
[137] Herbst, C., _Formative Reize in der tierischen Ontogenese_.
Leipzig, 1901.
6. As a consequence of further changes definite anlagen or buds
originate later in the embryo which are destined to give rise to
definite organs. Thus in the tadpole early mesenchyme cells are
formed which are the anlagen for the four legs, which will grow out
under the proper conditions. These anlagen are specific inasmuch as
from the anlage of a foreleg only a foreleg, and from the anlage for
a hindleg only a hindleg, will develop. Braus[138] has proved this
by transplanting the anlage of a foreleg to different parts of the
body. No matter into which part of the body they are transplanted the
mesenchyme cells for the foreleg will give rise to a foreleg only; even
if they are transplanted into the spot from which the hindlegs grow
out under natural conditions. There is therefore nothing to indicate
“regulation.”
[138] Braus, H., _Münchener Med. Wochnschr._, 1903, 1 (II.), No. 47, p.
2076.
The same is true for the formation of the eye and probably in general.
We have to consider the formation of the various organs of the body as
being due to the development of specific cells in definite locations
in the organisms which will grow out into definite organs no matter
into which part of the organism they are transplanted. It is at present
unknown what determines the formation of these specific anlagen. They
may lie dormant for a long time and then begin to grow at definite
periods of development. We shall see later that we know more about the
conditions which cause them to grow.
7. The fact that the egg, and probably every cell, has a definite
structure should determine the limits of the divisibility of living
matter. In most cases the complete destruction of a cell means
the cessation of life phenomena. A brain or kidney which has been
ground to a pulp is no longer able to perform its functions; yet we
know that such pulps can still perform some of the characteristic
chemical processes of the organ; _e. g._, the alcoholic fermentation
characteristic of yeast can be caused by the press juice from yeast;
or characteristic oxidations can be induced by the ground pulp of
organs. The question arises as to how far the divisibility of living
matter can be carried without interfering with the total of its
functions. Are the smallest particles of living matter which still
exhibit all its functions of the order of magnitude of molecules
and atoms, or are they of a different order? The first step toward
obtaining an answer to this question was taken by Moritz Nussbaum,[139]
who found that if an infusorian be divided into two pieces, one
with and one without a nucleus, only the piece with a nucleus will
continue to live and perform all the functions of self-preservation
and development which are characteristic of living organisms. This
shows that at least two different structural elements, nucleus and
cytoplasm, are needed for life. We can understand to a certain extent
from this why an organ after being reduced to a pulp, in which
the differentiation into nucleus and protoplasm is definitely and
permanently lost, is unable to accomplish all its functions.[140]
[139] Nussbaum, M., _Arch. f. mikroscop. Anat._, 1886, xxvi., 485.
[140] It must not be overlooked that in bacteria and the blue algæ no
distinct differentiation into nucleus and protoplasm can be shown.
To these organisms, therefore, the experiments of Nussbaum cannot be
applied.
The observations of Nussbaum and those who repeated his experiments
showed that although two different structures are required, not the
whole mass of an infusorian is needed to maintain its life. The
question then arose: How small a fraction of the original cell is
required to permit the full maintenance of life? The writer tried
to decide this question in the egg of the sea urchin. He had found
a simple method by which the eggs of the sea urchin (_Arbacia_)
can easily be divided into smaller fragments immediately after
fertilization. When the egg is brought from five to ten minutes
after fertilization (long before the first segmentation occurs) into
sea water which has been diluted by the addition of equal parts of
distilled water, the egg takes up water, swells, and causes the
membrane to burst. Part of the protoplasm then flows out, in one egg
more, in another less. If these eggs are afterward brought back into
normal sea water those fragments which contain a nucleus begin to
divide and develop.[141] It was found that the degree of development
which such a fragment reaches is a function of its mass; the smaller
the piece, the sooner as a rule its development ceases. The smallest
fragment which is capable of reaching the pluteus stage possesses the
mass of about one-eighth of the whole egg. Boveri has since stated that
it was about one twenty-seventh of the whole mass. Inasmuch as only
the linear dimensions are directly measurable, a slight difference in
measurement will cause a great discrepancy in the calculation of the
mass. Driesch’s results disagree with the statement of Boveri and
support the observation of the writer.
[141] Loeb, J., _Arch. d. f. ges. Physiol._, 1893, lv., 525.
If we raise the question why such a limit exists in regard to the
divisibility of living matter, it seems probable that only those
fragments of an egg are capable of development into a pluteus which
contain a sufficient amount of material of each of the three layers. If
this be correct, it would certainly not suffice to mix the _chemical_
constituents of the egg in order to produce a normal embryo; this would
require besides the proper chemical substances a definite arrangement
or structure of this material. The limits of divisibility of a cell
seem therefore to depend upon its physical structure and must for this
reason vary for different organisms and cells. The smallest piece of a
sea-urchin egg that can reach the pluteus stage is still visible with
the naked eye, and is therefore considerably larger than bacteria or
many algæ, which also may be capable of further division.
8. The most important fact which we gather from these data is that the
cytoplasm of the unfertilized egg may be considered as the embryo in
the rough and that the nucleus has apparently nothing to do with this
predetermination. This must raise the question suggested already in the
third chapter whether it might not be possible that the cytoplasm of
the eggs is the carrier of the genus or even species heredity, while
the Mendelian heredity which is determined by the nucleus adds only
the finer details to the rough block. Such a possibility exists, and if
it should turn out to be true we should come to the conclusion that the
unity of the organism is not due to a putting together of a number of
independent Mendelian characters according to a “pre-established plan,”
but to the fact that the organism in the rough existed already in the
cytoplasm of the egg before the egg was fertilized. The influence of
the hereditary Mendelian factors or genes consisted only in impressing
the numerous details upon the rough block and in thus determining its
variety and individuality; and this could be accomplished by substances
circulating in the liquids of the body as we shall see in later
chapters.
CHAPTER VII
REGENERATION
1. The action of the organism as a whole seems nowhere more pronounced
than in the phenomena of regeneration, for it is the organism as a
whole which represses the phenomena of regeneration in its parts, and
it is the isolation of the part from the influence of the whole which
sets in action the process of regeneration. The leaf of the Bermuda
“life plant”--_Bryophyllum calycinum_--behaves like any other leaf as
long as it is part of a healthy whole plant, while when isolated it
gives rise to new plants. The power of so doing was possessed by the
leaf while a part of the whole, and it was the “whole” which suppressed
the formative forces in the leaf. When a piece is cut from the branch
of a willow it forms roots near the lower end and shoots at the upper
end, so that a tolerably presentable “whole” is restored. How does the
“whole” prevent the basal end of the shoot from forming roots as long
as it is part of the plant? A certain fresh-water flatworm has the
mouth and pharynx in the middle of the body. When a piece is excised
between the head and the pharynx a new head is formed at the oral end,
a new tail at the opposite end, and in the middle of the remaining old
tissue a new mouth and pharynx is formed. How does the “whole” suppress
all this formative power in the part before the latter is isolated? It
almost seems as if the isolation itself were the emancipation of the
part from the tyranny of the whole. The explanation of this tyranny or
of the correlation of the parts in the whole is to be found, however,
in a different influence. The earlier botanists, Bonnet, Dutrochet, and
especially Sachs,[142] pointed out that the phenomena of correlation
are determined by the flow of sap in the body of a plant. These authors
formulated the idea that the formation of new organs in the plant is
determined by the existence of specific substances which are carried by
the ascending or descending sap. Specific shoot-producing substances
are carried to the apex, while specific root-producing substances are
carried to the base of a plant. When a piece is cut from a branch of
willow the root-forming substances must continue to flow to the basal
end of the piece, and since their further progress is blocked there
they induce the formation of roots at the basal end. Goebel[143] and
de Vries have accepted this view and the writer made use of it in his
first experiments on regeneration and heteromorphosis in animals.[144]
At that time the idea of the existence of such specific organ-forming
substances was received with some scepticism, but since then so many
proofs for their existence have been obtained that the idea is no
longer questioned. Such substances are known now under the name of
“internal secretions” or “hormones”; their connection with the theory
of Sachs was forgotten with the introduction of the new nomenclature.
[142] v. Sachs, J., “Stoff und Form der Pflanzenorgane,” _Gesammelte
Abhandlungen_, 1892, ii., 1160. Arbeiten a. d. bot. Inst. Würzburg,
1880-82.
[143] Goebel, K., _Einleitung in die experimentelle Morphologie der
Pflanzen_, 1908.
[144] Loeb, J., Untersuchungen zur physiologischen Morphologie der
Tiere. I. Heteromorphose. Würzburg, 1891. II. Organbildung und
Wachsthum. 1892. Reprinted in _Studies in General Physiology_. Chicago,
1906.
It may be well to enumerate some of the cases in which the influence
of specific substances circulating in the blood upon phenomena of
growth has been proven. One of the most striking observations in this
direction is the one made by Gudernatsch on the growth of the legs of
tadpoles of frogs and toads.[145] The young tadpoles have no legs,
but the mesenchyme cells from which the legs are to grow out later
are present at an early stage. From four months to a year or more may
elapse before the legs begin to grow. Gudernatsch found that legs
can be induced to grow in tadpoles at any time, even in very young
specimens, by feeding them with the thyroid gland (no matter from
what animal). No other material seems to have such an effect. The
thyroid contains iodine, and Morse[146] states that if instead of the
gland, iodized amino acids are fed to the tadpole the same result can
be produced. We must, therefore, draw the conclusion that the normal
outgrowth of legs in a tadpole is due to the presence in the body of
substances similar to the thyroid in their action (it may possibly be
thyroid substance) which are either formed in the body or taken up in
the food.
[145] Gudernatsch, J. F., _Zentralbl. f. Physiol._, 1912, xxvi., 323;
_Arch. f. Entwcklngsmech._, 1912, xxxv., 457; _Am. Jour. Anat._, 1914,
xv., 431.
[146] Morse, M., _Jour. Biol. Chem._, 1914, xix., 421.
Thus we see that the mesenchyme cells giving rise to legs may lie
dormant for months or a year but will grow out when a certain type of
substances, _e. g._, thyroid, circulates in the blood. There may exist
an analogy between the activating effect of the thyroid substance and
the activating effect of the spermatozoön or butyric acid (or other
parthenogenetic agencies) upon the egg, but we cannot state that the
thyroid substance activates the mesenchyme cells by altering their
cortical layer.
The fact that the substance of the thyroid may induce general growth
in the human is too well known to require more than an allusion in
this connection. When growth stops in children as a consequence of a
degeneration of the thyroid, feeding of the patient with thyroid again
induces growth. It may also suffice merely to call attention to the
connection between acromegaly and the hypophysis.
It was formerly believed that the nervous system acted as a regulator
of the phenomena of metamorphosis in animals, but it was possible to
show by simple experiments that the central nervous system does not
play this rôle and that the regulator must be the blood or substances
contained therein. In the metamorphosis of the _Amblystoma_ larva the
gills at the head and tail undergo changes simultaneously, the gills
being absorbed completely. The writer showed that in larvæ in which the
spinal cord was cut in two, no matter at which level,--the sympathetic
nerves were in all probability also cut--the two organs continued to
undergo metamorphosis simultaneously.[147] Uhlenhuth found that if
the eye of a salamander larva is transplanted into another larva the
transplanted eye undergoes its metamorphosis into the typical eye of
the adult form, simultaneously with the normal eyes of the individual
into which it was transplanted.[148] These and other observations of
a similar character leave no doubt that substances circulating in
the blood and not the central nervous system are responsible for the
phenomena of growth and metamorphosis.
[147] Loeb, J., _Arch. f. Entwcklngsmech._, 1897, iv., 502.
[148] Uhlenhuth, E., _ibid._, 1913, xxxvi., 211.
An interesting observation on the rôle of internal secretion in growth
was made by Leo Loeb.[149] When the fertilized ovum comes in contact
with the wall of the uterus it calls forth a growth there, namely the
formation of the maternal placenta (decidua). This author showed that
the corpus luteum of the ovary gives off a substance to the blood which
alters the tissues in the uterus in such a way that contact with any
foreign body induces this deciduoma formation. The case is of interest
since it indicates that the substance given off by the corpus luteum
does not induce growth directly, but that it allows mechanical contact
with a foreign body to do so while without the intervention of the
corpus luteum substance no such effect of the mechanical stimulus would
be observable. The action of the substance of the corpus luteum is
independent of the nervous system, since in a uterus which has been cut
out and retransplanted the same phenomenon can be observed.
[149] Loeb, Leo, _Zentralbl. f. allg. Path. u. path. Anat._, 1907,
xviii., 563; _Zentralbl. f. Physiol._, 1908, xxii., 498; 1909, xxiii.,
73; 1910, xxiv., 203; _Arch. f. Entwcklngsmech._, 1909, xxvii., 89,
463; _Jour. Am. Med. Assoc._, 1908, l., 1897; 1909, liii., 1471.
Bouin and Ancel[150] have shown that the corpus luteum, which
in the case of pregnancy continues to exist for a long time, is
responsible for the changes in the mammary gland in the first half
of pregnancy, when an active cell proliferation takes place in the
gland. This process can be interrupted by destroying the corpus luteum
artificially. During the second half of gravidity no further cell
proliferation takes place, but the cells begin to secrete milk while
during the period of cell proliferation such secretions do not occur.
[150] Quoted from M. Caullery, _Les Problèmes de la Sexualité_, Paris,
1913, p. 126.
Claude Bernard and Vitzou had shown that the period of growth and
moulting of the higher crustacea is accompanied by a heaping up of
glycogen in the liver and subdermal connective tissue. Smith[151]
found that during the period between two moultings, when there is no
growth, the storage cells are seen to be filled with large and numerous
fat globules instead of with glycogen. He also found that in the
_Cladocera_ “the period of active growth is accompanied by glycogen--as
opposed to fat--metabolism.” He observed, moreover, that if _Cladocera_
are crowded at a low temperature the fat metabolism (with inhibition to
growth) is favoured, while at high temperatures and with no crowding
of individuals the glycogen metabolism is favoured. In the latter
case a purely parthenogenetic mode of propagation is observed, while
in the former sexual reproduction takes place. The effect of crowding
of individuals is possibly due to products of excretion, which then
act on growth and reproduction indirectly by changing the “glycogen
metabolism” to “fat metabolism.”
[151] Smith, Geoffrey, _Proc. Roy. Soc._, B. 1915, lxxxviii., 418.
All these cases agree in this, that apparently specific substances
induce or favour growth, not in the whole body, but in special parts of
the body. Sachs suggested that there must be in each organism as many
specific organ-forming substances as there are organs in the body.
We will now show that the assumption of the existence of such
“organ-forming” substances (which may or may not be specific) and of
their flow in definite channels explains the inhibitory influence of
the whole on the parts as well as the unbridled regeneration of the
isolated parts.
2. We have seen that the resting egg can be aroused to development and
growth by substances contained in a spermatozoön or by certain other
substances mentioned in the preceding chapter. We will assume that
plants contain a large number of cells or buds which are comparable to
the resting egg cell, but which can be aroused to action by certain
substances circulating in the sap; and that the same is effected for
animal cells by substances in the blood. In plants the cells which can
be aroused to new growth have very often a rather definite location
while in lower animals they are more ubiquitous. For experimental
purposes organisms where these buds have a definite location are more
favourable, since we are better able to study the mechanism underlying
the process of activation and inhibition (correlation). When a leaf
of the plant _Bryophyllum calycinum_ is cut off and put on moist
sand or into water or even into air saturated with water vapour, new
plants will arise from notches of the leaf. This is the usual way of
propagating the plant and in no other part of the leaf except the
notches will new plants arise. These notches therefore contain cells
comparable to seeds or to unfertilized eggs or to the mesenchyme
cells which give rise to legs in the tadpole of the frog. The question
arises: Why do notches in the leaf never begin to grow while the
leaf is attached to an intact plant, and why do they grow when the
leaf is isolated? To this we are inclined to give an answer in the
sense of Bonnet, Sachs, de Vries, and Goebel, namely that the flow of
(specific?) substances in the plant determines when and where dormant
buds or anlagen shall begin to grow. Such substances may originate
or may be present in the leaf; but as long as it is connected with a
normal plant they will be carried by the circulation to the growing
points of the stem and of the roots and they cannot reach the notches;
while when we detach the leaf, either a new distribution or a new flow
of liquids will be established whereby the substances reach some of the
notches; and in these notches new roots and a new shoot will be formed.
When we cut off a leaf and put it into moist air, not all but only a
few of the notches will, as a rule, grow out (Fig. 16); but when we
isolate each notch leaving as much of the rest of the leaf as possible
attached to it, each notch will give rise to a new plant.[152] (Fig.
17.) We see, therefore, that it does not even require a whole plant
to cause inhibition but that we may observe the tyranny of the whole
over the parts in a single leaf. The explanation is as follows: When
we isolate a leaf, some of the notches will commence to grow into new
plants and this growth will arrest the development of the other notches
of the leaf in the same way as their development was suppressed by the
whole plant.
[152] Loeb, J., _Bot. Gazette_, 1915, lx., 249.
[Illustration: FIG. 16. Growth of roots and shoots in a few notches of
an isolated leaf of _Bryophyllum calycinum_]
[Illustration: FIG. 17. If all the notches of a leaf are isolated from
each other each notch will give rise to roots and a shoot, but the
growth will be less rapid than in Fig. 16. Figs. 16 and 17 were two
leaves taken from the same node of a plant.]
The explanation is the same; those notches which begin to grow first
will attract the flow of substances to themselves, thus preventing the
other notches from getting those substances. This idea is supported by
the fact that if all the notches are isolated from the leaf each notch
will give rise to a slowly growing plant, while if the leaf is not cut
into pieces, and a few notches only grow out, their growth is much more
rapid.
In all these experiments the idea that the “isolation” in itself is
responsible for the growth still presents itself. It can be disposed
of by the following experiment which never fails. Three leaves of
_Bryophyllum calycinum_ are suspended in an atmosphere saturated with
water vapour but their tips are submersed in water (Figs. 18, 19, 20).
The first leaf, Fig. 20, is entirely separated from its stem, the
second leaf, Fig. 19, remains connected with the adjacent piece of
stem, and the third leaf, Fig. 18, remains also connected with this
piece of stem but the latter still possesses both leaves. The first
leaf, Fig. 20, produces new roots and shoots in the submerged part in
a few days; the second leaf, Fig. 19, produces no roots or shoots for
a long time. This might find its explanation by the assumption that
the first leaf, being more isolated than the second, regenerates more
quickly. But this explanation becomes untenable owing to the fact that
the third leaf, Fig. 18, being less isolated than both (possessing a
second leaf in addition to the stem), forms new roots and shoots also
more quickly than the second leaf. The phenomena become intelligible in
the following way. The fact that in the second leaf shoots and roots
are formed very late, if at all, finds its explanation not in the
lessened isolation of this leaf, but in the fact that the formation
of a new shoot or of a callus in the piece of stem takes place more
quickly than the formation of roots and shoots in the notches of a
completely isolated leaf. The stem acts therefore as a centre of
suction for the flow of substances from the leaf and this prevents
or retards the formation of roots and shoots in the notches. In the
isolated leaf of _Bryophyllum calycinum_ no callus formation takes
place and hence no flow of the sap away from the leaf will occur. This
will allow one or more of the notch buds of this leaf to grow out and
then a flow will be established towards these growing buds.
[Illustration: FIG. 18 FIG. 19 FIG. 20]
In the third specimen, Fig. 18, the presence of two leaves suppresses
or, as a rule, retards the growth of a shoot on the stem and possibly
also the flow from one leaf may block to some extent the flow from the
opposite leaf if the piece of stem is very short. This puts the leaves
in a condition not as good as that in leaf Fig. 20, but better than in
leaf Fig. 19.[153]
[153] With larger leaves the experiment may also succeed in moist air.
In the normal plant the buds in the notches of the leaf remain dormant
since the flow of the “stimulating” substances takes place towards the
tips of the stem and root, and because these substances are retained
there in excess. This is probably the real basis of the mysterious
dominance of the “whole” over its “parts” or of the anlagen of the
tip of the stem over those farther below. When a piece of the stem of
_Bryophyllum_ is cut off and its leaves are removed, the two apical
buds will grow out first. This “dominance” finds its explanation
probably in the anatomical structure and the mechanism of sap flow
which tend to bring the “stimulating” substances first to the anlagen
in the tip. In _Laminaria_ Setchell has been able to show directly that
regeneration always starts from that tissue which conducts nutritive
material.
When we cut out a piece of a stem of _Bryophyllum_, and remove all the
leaves, new shoots will be formed from the two apical buds of the stem,
and roots will arise from the most basal nodes; provided that the stem
is suspended in air saturated with water vapour. The growth in such a
stem deprived of all leaves is slow. If we remove all the leaves on
such a piece of stem except the two at the apical end, the stem will
form only roots, but these will develop much more rapidly than on a
stem without leaves. If we remove all the leaves except the two at
the basal end, the stem will only form shoots (at the apical end) but
these will develop much more rapidly than in a leafless stem. Hence the
leaves accelerate the growth of roots towards the basal end and inhibit
it towards the apical end; and they favour the growth of shoots towards
the apical end and inhibit it in the nodes located nearer the base.
We thus see that while the stem inhibits the growth of the leaves
connected with it, the latter accelerate the growth in the stem. Both
facts can probably be explained on the same basis; namely, on the
assumption that it is the flow of substances from the leaf to the stem
which inhibits the growth of the notches and accelerates the growth
of the buds in the stem. On this assumption it would also follow
that the leaves send root-forming substances towards the basal and
shoot-forming substances towards the apex of the stem. It also seems
to follow from recent as yet unpublished experiments by the writer
that the root-forming substances are associated or identical with the
substances which cause geotropic curvature in the stem.
These observations show that the phenomena of correlation or of the
influence of the whole over the parts is due to peculiarities of
circulation or the flow of sap; and that the isolation prevents the sap
from flowing away to other parts of the plant. There is no need for
assuming the existence of a mysterious force which directs the piece to
grow into a whole.
[Illustration: FIG. 21]
3. Phenomena of inhibition or correlation such as we have described
in _Bryophyllum_ are not lacking in the regeneration of animals, as
experiments on _Tubularia_ show.[154] _Tubularia mesembryanthemum_
(Fig. 21) is a hydroid consisting of a long stem terminating at one
end in a stolon which attaches itself to solid bodies such as rocks,
at the other end in a polyp. The writer found that if we cut a piece
from a stolon and suspend it in an aquarium it forms as a rule a polyp
at either end (Fig. 22), but the velocity with which the two polyps
are formed is not the same, the polyp at the oral end of the piece
being formed much more rapidly--a day or one or two weeks sooner--than
the aboral polyp. The process of polyp regeneration at the aboral pole
could, however, be accelerated and its velocity made equal to that of
the regeneration of the oral polyp by suppressing the formation of the
latter. This was accomplished by depriving the oral pole of the oxygen
necessary for regeneration, _e. g._, by merely putting the oral end
of the piece of stem into the sand. It was, therefore, obvious that
the formation of the oral polyp retarded the formation of the aboral
polyp. This inhibition might have been due to the fact that a specific
organ-forming material needed for the formation of a polyp existed in
sufficient quantity in the stem for the formation of one polyp only at
a time. This idea, however, was found to be incorrect since when the
stem was cut into two or more pieces each piece formed a polyp at once
at its oral pole and regenerated the aboral polyps also, but again
with the usual delay. It seemed more probable then that the cause of
the difference in the rapidity of polyp formation at both ends lay
in the fact that certain material flowed first to the oral pole and
induced polyp formation here but that this flow was reversed as soon as
the polyp at the oral pole was formed or as soon as the formation of
the oral polyp was inhibited by lack of oxygen. The partial or full
completion of the formation of the oral polyp acted as an inhibition
to the further flow of material to this pole. This idea was supported
by an observation made independently by Godlewski and the writer that
if a piece of stem be cut out of a _Tubularia_, and if the piece be
ligatured somewhere between the two ends, the oral and the aboral
polyps are formed simultaneously. This would be comprehensible on the
assumption that the retarding effect which the formation of the oral
has on the aboral polyp was indeed of the nature of a flow of material
towards the oral pole.
[154] Loeb, J., Untersuchungen zur physiologischen Morphologie. I.
Heteromorphose. 1891. II. Organbildung und Wachsthum. Würzburg, 1892.
[Illustration: FIG. 22]
[Illustration: FIG. 23]
Miss Bickford[155] found that the difference in time between the
formation of the two polyps disappears also when the piece cut from the
stem becomes so small that it is of the order of magnitude of a single
polyp. In that case two incomplete polyps are formed simultaneously at
each end (Fig. 23). The new head in the regeneration of _Tubularia_
arises, as Miss Bickford observed, from the tissue near the wound. At
some distance from the wound in the old tissue two rows of tentacles
arise, which are noticeable as rows of longitudinal lines inside the
stem before the head is formed. Driesch noticed that the newly
formed head is the smaller the smaller the whole piece. (This is
true, however, only in rather small pieces.) There is, therefore, in
small pieces a rough proportionality between size of head and size of
regenerating piece. Driesch[156] uses this interesting fact to prove the
existence of an entelechy, while we are inclined to see in it an analogue
to the observation of Leo Loeb, that the velocity of the process of
healing in the case of a deficiency of the epithelium decreases when
the size of the uncovered area diminishes. While we do not wish to
offer any suggestion concerning the mechanism of these quantitative
phenomena--they may be related in some way with the velocity of certain
chemical reactions--we see no reason for assuming that they cannot be
explained on a purely physicochemical basis.
[155] Bickford, E. E., _Jour. Morphol._, 1894, ix., 417.
[156] Driesch, H., _Science and Philosophy of the Organism_, i., 127.
The writer noticed that certain pigmented cells from the entoderm of
the organism always gather at that end where a new polyp is about to
be formed. These red or yellowish cells always collect first at the
oral end of a piece of stem. It may be that certain substances given
off by the pigmented cells at the cut end are responsible for the polyp
formation, but this is only a surmise.
Another suggestion made by Child,[157] is that there exists an axial
gradient in the stem whereby the cells regenerate the more quickly
the nearer they are to the oral pole. If this were correct, and we cut
a long piece from the stem of a _Tubularia_ and bisect the piece, the
oral pole of the anterior half should regenerate more quickly than the
oral pole of the posterior half. According to the writer’s observations
on a Tubularian (_T. crocea_) growing in the estuaries near Oakland,
California, both oral ends regenerate equally fast in such cases.
[157] Child, C. M., “Die physiologische Isolation von Teilen des
Organismus,” Roux’s _Vorträge und Aufsätze_, Leipzig, 1911.
4. The phenomena of regeneration in _Cerianthus membranaceus_, a sea
anemone, can be easily understood from the experiments on Tubularians,
if we imagine the body wall of _Cerianthus_ to consist of a series of
longitudinal elements running parallel to the axis of symmetry of the
animal from the tentacles to the foot. The number of these elements
may be supposed to correspond to the number of tentacles in the outer
row of the normal animal. Each such element behaves like a Tubularian,
with this difference, however, that the elements in _Cerianthus_
are more strongly polarized than in _Tubularia_, and that each one
is able to form a tentacle at its oral pole only. This fact can be
nicely illustrated in the following way: if a square or oblong piece
(_a b c d_, Fig. 24) be cut from the body wall of a _Cerianthus_ in
such a way that one side, _a c_, of the oblong is parallel to the
longitudinal axis of the animal, tentacles will grow on one of the four
sides only; namely, on the side _a b_.[158] (Fig. 25.) The other three
free edges are not able to produce tentacles. If an incision be made in
the body wall of a _Cerianthus_, tentacles will grow on the lower edge
of the incision (Fig. 26).
[158] Loeb, J., “Untersuchungen zur physiologischen Morphologie der
Tiere.”
[Illustration: FIG. 24]
[Illustration: FIG. 25]
[Illustration: FIG. 26]
The writer tried whether or not by tying a ligature around the middle
of a piece of an Actinian this polarity could be suppressed; but the
experiments did not succeed, inasmuch as the cells compressed by the
ligature died, and were liquefied through bacterial action so that the
pieces in front and behind the ligature fell apart. It is therefore
impossible to decide whether or not a current or a flow of substances
in a certain direction through these elements is responsible for this
polarity, though this may be possible. The writer found, however, that
one condition is necessary for the growth and regeneration of tentacles
which also plays a rôle in the corresponding phenomena in plants,
namely turgidity. The tentacles of _Cerianthus_ are hollow cylinders
closed at the tip, and by liquid being pressed into them they can be
stretched and appear turgid. If, however, an incision is made in the
body, the tentacles above the incision can no longer be stretched out.
In one experiment the oral disk of a _Cerianthus_ was cut off; very
soon new tentacles began to grow at the top, and after having reached
a certain size, an incision was made in the animal. The tentacles
above the incision collapsed in consequence and ceased to grow, while
growth of the others continued. On the lower edge of the incision new
tentacles began to grow.
[Illustration: FIG. 27]
It seems also possible that Morgan’s well-known experiment on
regeneration in _Planaria_ can be explained by a flow of substances.
He[159] found that if a piece _a c d b_ be cut out of a fresh-water
Planarian at right angles to the longitudinal axis (Fig. 27), at the
front end a new normal head, at the back end a new tail, will be
regenerated (Fig. 28); but that if a piece _a c d b_ be cut from
a Planarian obliquely (Fig. 29) instead of at right angles to the
longitudinal axis a tiny head is formed at the foremost corner of the
piece _a_ and a tiny tail at the hindmost corner _b_ (Fig. 30). Why is
it that in the oblique piece the head is formed in the corner and not
all along the cut surface as is the case when the cut is made at right
angles to the longitudinal axis? The writer is inclined to believe
that the right answer to this question has been given by Bardeen.[160]
This author has pointed out the apparent rôle that the circulatory (or
so-called digestive) canals in Planarians play in the localization of
the phenomena of regeneration, inasmuch as the new head always forms
symmetrically at the opening of the circulatory vessel or branch which
is situated as much as possible at the foremost end of the regenerating
piece of worm. He assumes that through muscular action the liquids of
the body are forced to stream toward this end, and that this fact has
some connection with the formation of a new head. There can be no doubt
that the facts here mentioned agree with Bardeen’s suggestion. The
oblique pieces in Morgan’s experiments which at first have the heads and
tails outside the line of symmetry of the middle piece, gradually assume
a normal position (Figs. 31, 32). The writer is inclined to believe
that this is due to mechanical conditions. The head _a e c_ of such an
oblique piece is asymmetrical, the one side _a e_ being less stretched
than the other _e c_. The higher tension of the piece _e c_ will have
the effect of bringing _e_ nearer _c_, since we know that acid formation
and hence energy production increases in proportion to surface, _i. e._,
it must be the greater the more it is stretched. The reverse is true for
the tail _d f b_, and the effect here will be that _f_ will be pulled
nearer _d_. In this way purely mechanical conditions are responsible for
the fact that the soft tissues of the animal are gradually restored to
their true orientation.
[159] Morgan, T. H., _Regeneration_, New York, 1901.
[160] Bardeen, C. R., _Am. Jour. Physiol._, 1901, v., 1; _Arch. f.
Entwcklngsmech._, 1903, xvi., 1.
[Illustration: FIG. 28]
[Illustration: FIG. 29]
[Illustration: FIG. 30]
[Illustration: FIG. 31]
[Illustration: FIG. 32]
As a final possible example of the influence of internal secretion
or substances contained in the blood may be mentioned the following
curious observation of Przibram.[161] In a crustacean, _Alpheus_, the
two chelæ (pincers) are not equal in size and form, one being very
much larger than the other. Przibram found that when he cut off the
larger pincer in such crustaceans the remaining pincer assumes in the
next moulting the size and shape of the removed large pincer; while in
place of the removed pincer one of the small type is produced. Hence
a reversal of the two pincers is thus brought about. If later on the
large pincer is again cut off the process is repeated and the original
dissymmetry is restored. Przibram was able to show that the nervous
system has no connection with this phenomenon.
[161] Przibram, H., _Arch. f. Entwcklngsmech._, 1901, xi., 329.
The elements which have entered into the discussion thus far are,
first, the flow of substances in preformed channels; second, the
existence of general or specific substances required for the growing
or regenerating organ. A third element is to be added; namely the
“suction” effect upon these substances of a developing organ. Thus we
see that if one or a few of the notches in a leaf of _Bryophyllum_
grow out the other notches of the leaf are inhibited from growing.
There is enough material present in the leaf for all the notches to
grow into shoots as is proved by the fact that all will grow out if
they are isolated from each other. This was explained on the assumption
that the notches of a whole which happen to develop first, create a
flow of these substances from the rest of the leaf to themselves and
thus prevent any getting to the other notches. We stated that this is
supported by the fact that the few notches growing out in an undivided
leaf grow more rapidly than the many shoots growing from each notch of
a divided leaf. But why should a growing shoot or a growing point in
general produce such a suction? I think this may be possible on the
assumption that the consumption of these substances by the growing
organs causes a low osmotic pressure of these substances in the growing
region and this fall of osmotic potential will act as a cause for the
further flow. This brings about the apparent “suction” effect of the
growing elements upon the flow of substances.
5. We mentioned that when a piece is cut from a _Planaria_ between
pharynx and head a new mouth is formed in the middle. It should also
be mentioned that according to Child the piece after regeneration is
smaller than it was before.[162] This indicates that material in the
old cells has been digested or has undergone hydrolysis in order
to furnish the nutritive material for the new head and tail, since
the piece cannot take up any food from the outside before a mouth is
formed. These phenomena of autodigestion--the process itself will be
discussed in the last chapter--seem to occur in many (if not all)
phenomena of regeneration. It may be that the collecting of red cells
at the end in a Tubularian where regeneration is about to begin has
to do with the furnishing of material by self-digestion, since these
cells are partly at least destroyed in the process. It is of interest
to look for more examples of autodigestion accompanying phenomena of
regeneration.