(a) HETEROGENEOUS HYBRIDISATION.
It was held until recently that hybridisation is not possible except
between closely related species and that even among these a successful
hybridisation cannot always be counted upon. This view was well
supported by experience. It is, for instance, well known that the
majority of marine animals lay their unfertilised eggs in the ocean and
that the males shed their sperm also into the sea-water. The numerical
excess of the spermatozoa over the ova in the sea-water is the only
guarantee that the eggs are fertilised, for the spermatozoa are
carried to the eggs by chance and are not attracted by the latter. This
statement is the result of numerous experiments by various authors,
and is contrary to common belief. As a rule all or the majority of
individuals of a species in a given region spawn on the same day, and
when this occurs the sea-water constitutes a veritable suspension of
sperm. It has been shown by experiment that in fresh sea-water the sperm
may live and retain its fertilising power for several days. It is thus
unavoidable that at certain periods more than one kind of spermatozoon
is suspended in the sea-water and it is a matter of surprise that the
most heterogeneous hybridisations do not constantly occur. The reason
for this becomes obvious if we bring together mature eggs and equally
mature and active sperm of a different family. When this is done no egg
is, as a rule, fertilised. The eggs of a sea-urchin can be fertilised by
sperm of their own species, or, though in smaller numbers, by the sperm
of other species of sea-urchins, but not by the sperm of other groups of
echinoderms, e.g. starfish, brittle-stars, holothurians or crinoids, and
still less by the sperm of more distant groups of animals. The consensus
of opinion seemed to be that the spermatozoon must enter the egg
through a narrow opening or canal, the so-called micropyle, and that
the micropyle allowed only the spermatozoa of the same or of a closely
related species to enter the egg.
It seemed to the writer that the cause of this limitation of
hybridisation might be of another kind and that by a change in the
constitution of the sea-water it might be possible to bring about
heterogenous hybridisations, which in normal sea-water are impossible.
This assumption proved correct. Sea-water has a faintly alkaline
reaction (in terms of the physical chemist its concentration of hydroxyl
ions is about (10 to the power minus six)N at Pacific Grove, California,
and about (10 to the power minus 5)N at Woods Hole, Massachusetts).
If we slightly raise the alkalinity of the sea-water by adding to it a
small but definite quantity of sodium hydroxide or some other alkali,
the eggs of the sea-urchin can be fertilised with the sperm of widely
different groups of animals, possibly with the sperm of any marine
animal which sheds it into the ocean. In 1903 it was shown that if we
add from about 0.5 to 0.8 cubic centimetre N/10 sodium hydroxide to
50 cubic centimetres of sea-water, the eggs of Strongylocentrotus
purpuratus (a sea-urchin which is found on the coast of California)
can be fertilised in large quantities by the sperm of various kinds of
starfish, brittle-stars and holothurians; while in normal sea-water or
with less sodium hydroxide not a single egg of the same female could
be fertilised with the starfish sperm which proved effective in the
hyper-alkaline sea-water. The sperm of the various forms of starfish was
not equally effective for these hybridisations; the sperm of Asterias
ochracea and A. capitata gave the best results, since it was possible to
fertilise 50 per cent or more of the sea-urchin eggs, while the sperm of
Pycnopodia and Asterina fertilised only 2 per cent of the same eggs.
Godlewski used the same method for the hybridisation of the sea-urchin
eggs with the sperm of a crinoid (Antedon rosacea). Kupelwieser
afterwards obtained results which seemed to indicate the possibility of
fertilising the eggs of Strongylocentrotus with the sperm of a mollusc
(Mytilus.) Recently, the writer succeeded in fertilising the
eggs of Strongylocentrotus franciscanus with the sperm of a
mollusc--Chlorostoma. This result could only be obtained in sea-water
the alkalinity of which had been increased (through the addition of
0.8 cubic centimetre N/10 sodium hydroxide to 50 cubic centimetres
of sea-water). We thus see that by increasing the alkalinity of the
sea-water it is possible to effect heterogeneous hybridisations which
are at present impossible in the natural environment of these animals.
It is, however, conceivable that in former periods of the earth's
history such heterogeneous hybridisations were possible. It is known
that in solutions like sea-water the degree of alkalinity must increase
when the amount of carbon-dioxide in the atmosphere is diminished. If it
be true, as Arrhenius assumes, that the Ice age was caused or preceded
by a diminution in the amount of carbon-dioxide in the air, such a
diminution must also have resulted in an increase of the alkalinity
of the sea-water, and one result of such an increase must have been to
render possible heterogeneous hybridisations in the ocean which in the
present state of alkalinity are practically excluded.
But granted that such hybridisations were possible, would they have
influenced the character of the fauna? In other words, are the hybrids
between sea-urchin and starfish, or better still, between sea-urchin and
mollusc, capable of development, and if so, what is their character?
The first experiment made it appear doubtful whether these heterogeneous
hybrids could live. The sea-urchin eggs which were fertilised in the
laboratory by the spermatozoa of the starfish, as a rule, died earlier
than those of the pure breeds. But more recent results indicate that
this was due merely to deficiencies in the technique of the earlier
experiments. The writer has recently obtained hybrid larvae between the
sea-urchin egg and the sperm of a mollusc (Chlorostoma) which, in the
laboratory, developed as well and lived as long as the pure breeds of
the sea-urchin, and there was nothing to indicate any difference in the
vitality of the two breeds.
So far as the question of heredity is concerned, all the experiments on
heterogeneous hybridisation of the egg of the sea-urchin with the sperm
of starfish, brittle-stars, crinoids and molluscs, have led to the same
result, namely, that the larvae have purely maternal characteristics and
differ in no way from the pure breed of the form from which the egg
is taken. By way of illustration it may be said that the larvae of the
sea-urchin reach on the third day or earlier (according to species
and temperature) the so-called pluteus stage, in which they possess a
typical skeleton; while neither the larvae of the starfish nor those
of the mollusc form a skeleton at the corresponding stage. It was,
therefore, a matter of some interest to find out whether or not the
larvae produced by the fertilisation of the sea-urchin egg with the
sperm of starfish or mollusc would form the normal and typical pluteus
skeleton. This was invariably the case in the experiments of Godlewski,
Kupelwieser, Hagedoorn, and the writer. These hybrid larvae were
exclusively maternal in character.
It might be argued that in the case of heterogeneous hybridisation the
sperm-nucleus does not fuse with the egg-nucleus, and that, therefore,
the spermatozoon cannot transmit its hereditary substances to the
larvae. But these objections are refuted by Godlewski's experiments,
in which he showed definitely that if the egg of the sea-urchin is
fertilised with the sperm of a crinoid the fusion of the egg-nucleus
and sperm-nucleus takes place in the normal way. It remains for further
experiments to decide what the character of the adult hybrids would be.
(b). ARTIFICIAL PARTHENOGENESIS.
Possibly in no other field of Biology has our ability to control
life-phenomena by outside conditions been proved to such an extent as
in the domain of fertilisation. The reader knows that the eggs of the
overwhelming majority of animals cannot develop unless a spermatozoon
enters them. In this case a living agency is the cause of development
and the problem arises whether it is possible to accomplish the same
result through the application of well-known physico-chemical agencies.
This is, indeed, true, and during the last ten years living larvae
have been produced by chemical agencies from the unfertilised eggs
of sea-urchins, starfish, holothurians and a number of annelids and
molluscs; in fact this holds true in regard to the eggs of practically
all forms of animals with which such experiments have been tried long
enough. In each form the method of procedure is somewhat different and
a long series of experiments is often required before the successful
method is found.
The facts of Artificial Parthenogenesis, as the chemical fertilisation
of the egg is called, have, perhaps, some bearing on the problem of
evolution. If we wish to form a mental image of the process of evolution
we have to reckon with the possibility that parthenogenetic propagation
may have preceded sexual reproduction. This suggests also the
possibility that at that period outside forces may have supplied
the conditions for the development of the egg which at present the
spermatozoon has to supply. For this, if for no other reason, a brief
consideration of the means of artificial parthenogenesis may be of
interest to the student of evolution.
It seemed necessary in these experiments to imitate as completely as
possible by chemical agencies the effects of the spermatozoon upon
the egg. When a spermatozoon enters the egg of a sea-urchin or certain
starfish or annelids, the immediate effect is a characteristic change of
the surface of the egg, namely the formation of the so-called membrane
of fertilisation. The writer found that we can produce this membrane in
the unfertilised egg by certain acids, especially the monobasic acids
of the fatty series, e.g. formic, acetic, propionic, butyric, etc.
Carbon-dioxide is also very efficient in this direction. It was also
found that the higher acids are more efficient than the lower ones,
and it is possible that the spermatozoon induces membrane-formation by
carrying into the egg a higher fatty acid, namely oleic acid or one of
its salts or esters.
The physico-chemical process which underlies the formation of the
membrane seems to be the cause of the development of the egg. In all
cases in which the unfertilised egg has been treated in such a way as
to cause it to form a membrane it begins to develop. For the eggs of
certain animals membrane-formation is all that is required to induce a
complete development of the unfertilised egg, e.g. in the starfish and
certain annelids. For the eggs of other animals a second treatment is
necessary, presumably to overcome some of the injurious effects of
acid treatment. Thus the unfertilised eggs of the sea-urchin
Strongylocentrotus purpuratus of the Californian coast begin to develop
when membrane-formation has been induced by treatment with a fatty acid,
e.g. butyric acid; but the development soon ceases and the eggs
perish in the early stages of segmentation, or after the first nuclear
division. But if we treat the same eggs, after membrane-formation, for
from 35 to 55 minutes (at 15 deg C.) with sea-water the concentration
(osmotic pressure) of which has been raised through the addition of a
definite amount of some salt or sugar, the eggs will segment and develop
normally, when transferred back to normal sea-water. If care is taken,
practically all the eggs can be caused to develop into plutei, the
majority of which may be perfectly normal and may live as long as larvae
produced from eggs fertilised with sperm.
It is obvious that the sea-urchin egg is injured in the process of
membrane-formation and that the subsequent treatment with a hypertonic
solution only acts as a remedy. The nature of this injury became clear
when it was discovered that all the agencies which cause haemolysis,
i.e. the destruction of the red blood corpuscles, also cause
membrane-formation in unfertilised eggs, e.g. fatty acids or ether,
alcohols or chloroform, etc., or saponin, solanin, digitalin, bile
salts and alkali. It thus happens that the phenomena of artificial
parthenogenesis are linked together with the phenomena of haemolysis
which at present play so important a role in the study of immunity. The
difference between cytolysis (or haemolysis) and fertilisation seems to
be this, that the latter is caused by a superficial or slight cytolysis
of the egg, while if the cytolytic agencies have time to act on the
whole egg the latter is completely destroyed. If we put unfertilised
eggs of a sea-urchin into sea-water which contains a trace of saponin we
notice that, after a few minutes, all the eggs form the typical
membrane of fertilisation. If the eggs are then taken out of the saponin
solution, freed from all traces of saponin by repeated washing in normal
sea-water, and transferred to the hypertonic sea-water for from 35 to
55 minutes, they develop into larvae. If, however, they are left in
the sea-water containing the saponin they undergo, a few minutes after
membrane-formation, the disintegration known in pathology as CYTOLYSIS.
Membrane-formation is, therefore, caused by a superficial or incomplete
cytolysis. The writer believes that the subsequent treatment of the egg
with hypertonic sea-water is needed only to overcome the destructive
effects of this partial cytolysis. The full reasons for this belief
cannot be given in a short essay.
Many pathologists assume that haemolysis or cytolysis is due to a
liquefaction of certain fatty or fat-like compounds, the so-called
lipoids, in the cell. If this view is correct, it would be necessary to
ascribe the fertilisation of the egg to the same process.
The analogy between haemolysis and fertilisation throws, possibly,
some light on a curious observation. It is well known that the blood
corpuscles, as a rule, undergo cytolysis if injected into the blood of
an animal which belongs to a different family. The writer found last
year that the blood of mammals, e.g. the rabbit, pig, and cattle, causes
the egg of Strongylocentrotus to form a typical fertilisation-membrane.
If such eggs are afterwards treated for a short period with hypertonic
sea-water they develop into normal larvae (plutei). Some substance
contained in the blood causes, presumably, a superficial cytolysis of
the egg and thus starts its development.
We can also cause the development of the sea-urchin egg without
membrane-formation. The early experiments of the writer were done in
this way and many experimenters still use such methods. It is probable
that in this case the mechanism of fertilisation is essentially the same
as in the case where the membrane-formation is brought about, with
this difference only, that the cytolytic effect is less when no
fertilisation-membrane is formed. This inference is corroborated by
observations on the fertilisation of the sea-urchin egg with ox blood.
It very frequently happens that not all of the eggs form membranes in
this process. Those eggs which form membranes begin to develop, but
perish if they are not treated with hypertonic sea-water. Some of the
other eggs, however, which do not form membranes, develop directly into
normal larvae without any treatment with hypertonic sea-water, provided
they are exposed to the blood for only a few minutes. Presumably some
blood enters the eggs and causes the cytolytic effects in a less degree
than is necessary for membrane-formation, but in a sufficient degree to
cause their development. The slightness of the cytolytic effect allows
the egg to develop without treatment with hypertonic sea-water.
Since the entrance of the spermatozoon causes that degree of cytolysis
which leads to membrane-formation, it is probable that, in addition to
the cytolytic or membrane-forming substance (presumably a higher fatty
acid), it carries another substance into the egg which counteracts the
deleterious cytolytic effects underlying membrane-formation.
The question may be raised whether the larvae produced by artificial
parthenogenesis can reach the mature stage. This question may be
answered in the affirmative, since Delage has succeeded in raising
several parthenogenetic sea-urchin larvae beyond the metamorphosis into
the adult stage and since in all the experiments made by the writer
the parthenogenetic plutei lived as long as the plutei produced from
fertilised eggs.
(c). ON THE PRODUCTION OF TWINS FROM ONE EGG THROUGH A CHANGE IN THE
CHEMICAL CONSTITUTION OF THE SEA-WATER.
The reader is probably familiar with the fact that there exist two
different types of human twins. In the one type the twins differ as much
as two children of the same parents born at different periods; they
may or may not have the same sex. In the second type the twins have
invariably the same sex and resemble each other most closely. Twins
of the latter type are produced from the same egg, while twins of the
former type are produced from two different eggs.
The experiments of Driesch and others have taught us that twins
originate from one egg in this manner, namely, that the first two cells
into which the egg divides after fertilisation become separated from
each other. This separation can be brought about by a change in the
chemical constitution of the sea-water. Herbst observed that if the
fertilised eggs of the sea-urchin are put into sea-water which is freed
from calcium, the cells into which the egg divides have a tendency
to fall apart. Driesch afterwards noticed that eggs of the sea-urchin
treated with sea-water which is free from lime have a tendency to give
rise to twins. The writer has recently found that twins can be produced
not only by the absence of lime, but also through the absence of sodium
or of potassium; in other words, through the absence of one or two of
the three important metals in the sea-water. There is, however, a second
condition, namely, that the solution used for the production of twins
must have a neutral or at least not an alkaline reaction.
The procedure for the production of twins in the sea-urchin egg consists
simply in this:--the eggs are fertilised as usual in normal sea-water
and then, after repeated washing in a neutral solution of sodium
chloride (of the concentration of the sea-water), are placed in a
neutral mixture of potassium chloride and calcium chloride, or of sodium
chloride and potassium chloride, or of sodium chloride and calcium
chloride, or of sodium chloride and magnesium chloride. The eggs must
remain in this solution until half an hour or an hour after they have
reached the two-cell stage. They are then transferred into normal
sea-water and allowed to develop. From 50 to 90 per cent of the eggs of
Strongylocentrotus purpuratus treated in this manner may develop into
twins. These twins may remain separate or grow partially together and
form double monsters, or heal together so completely that only slight or
even no imperfections indicate that the individual started its career
as a pair of twins. It is also possible to control the tendency of such
twins to grow together by a change in the constitution of the sea-water.
If we use as a twin-producing solution a mixture of sodium, magnesium
and potassium chlorides (in the proportion in which these salts exist in
the sea-water) the tendency of the twins to grow together is much
more pronounced than if we use simply a mixture of sodium chloride and
magnesium chloride.
The mechanism of the origin of twins, as the result of altering the
composition of the sea-water, is revealed by observation of the first
segmentation of the egg in these solutions. This cell-division is
modified in a way which leads to a separation of the first two cells.
If the egg is afterwards transferred back into normal sea-water, each
of these two cells develops into an independent embryo. Since normal
sea-water contains all three metals, sodium, calcium, and potassium, and
since it has besides an alkaline reaction, we perceive the reason why
twins are not normally produced from one egg. These experiments suggest
the possibility of a chemical cause for the origin of twins from one egg
or of double monstrosities in mammals. If, for some reason, the liquids
which surround the human egg a short time before and after the first
cell-division are slightly acid, and at the same time lacking in one
of the three important metals, the conditions for the separation of the
first two cells and the formation of identical twins are provided.
In conclusion it may be pointed out that the reverse result, namely,
the fusion of normally double organs, can also be brought about
experimentally through a change in the chemical constitution of the
sea-water. Stockard succeeded in causing the eyes of fish embryos
(Fundulus heteroclitus) to fuse into a single cyclopean eye through the
addition of magnesium chloride to the sea-water. When he added about 6
grams of magnesium chloride to 100 cubic centimetres of sea-water and
placed the fertilised eggs in the mixture, about 50 per cent of the
eggs gave rise to one-eyed embryos. "When the embryos were studied the
one-eyed condition was found to result from the union or fusion of the
'anlagen' of the two eyes. Cases were observed which showed various
degrees in this fusion; it appeared as though the optic vessels were
formed too far forward and ventral, so that their antero-ventro-median
surfaces fused. This produces one large optic cup, which in all cases
gives more or less evidence of its double nature." (Stockard, "Archiv f.
Entwickelungsmechanik", Vol. 23, page 249, 1907.)
We have confined ourselves to a discussion of rather simple effects of
the change in the constitution of the sea-water upon development. It
is a priori obvious, however, that an unlimited number of pathological
variations might be produced by a variation in the concentration and
constitution of the sea-water, and experience confirms this statement.
As an example we may mention the abnormalities observed by Herbst in the
development of sea-urchins through the addition of lithium to sea-water.
It is, however, as yet impossible to connect in a rational way the
effects produced in this and similar cases with the cause which produced
them; and it is also impossible to define in a simple way the character
of the change produced.