The Organism as a Whole, from a Physicochemical Viewpoint
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Title: The Organism as a Whole, from a Physicochemical Viewpoint
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THE ORGANISM AS A WHOLE
From a Physicochemical Viewpoint
by
JACQUES LOEB, M.D., Ph.D., Sc.D.
Member of the Rockefeller Institute for Medical Research
With 51 Illustrations
G. P. Putnam's Sons
New York and London
The Knickerbocker Press
Copyright, 1916
by
Jacques Loeb
The Knickerbocker Press, New York
To
THE MEMORY OF
DENIS DIDEROT
Of the _Encyclopédie_ and the _Système de la nature_
“He was one of those simple, disinterested, and intellectually
sterling workers to whom their own personality is as nothing in the
presence of the vast subjects that engage the thoughts of their
lives.”
JOHN MORLEY.
(Article Diderot, _Encyclopædia Britannica_.)
PREFACE
It is generally admitted that the individual physiological processes,
such as digestion, metabolism, the production of heat or of
electricity, are of a purely physicochemical character; and it is also
conceded that the functions of individual organs, such as the eye
or the ear, are to be analysed from the viewpoint of the physicist.
When, however, the biologist is confronted with the fact that in the
organism the parts are so adapted to each other as to give rise to a
harmonious whole; and that the organisms are endowed with structures
and instincts calculated to prolong their life and perpetuate their
race, doubts as to the adequacy of a purely physicochemical viewpoint
in biology may arise. The difficulties besetting the biologist in this
problem have been rather increased than diminished by the discovery of
Mendelian heredity, according to which each character is transmitted
independently of any other character. Since the number of Mendelian
characters in each organism is large, the possibility must be faced
that the organism is merely a mosaic of independent hereditary
characters. If this be the case the question arises: What moulds these
independent characters into a harmonious whole?
The vitalist settles this question by assuming the existence of a
pre-established design for each organism and of a guiding “force”
or “principle” which directs the working out of this design. Such
assumptions remove the problem of accounting for the harmonious
character of the organism from the field of physics or chemistry. The
theory of natural selection invokes neither design nor purpose, but it
is incomplete since it disregards the physicochemical constitution of
living matter about which little was known until recently.
In this book an attempt is made to show that the unity of the organism
is due to the fact that the egg (or rather its cytoplasm) is the future
embryo upon which the Mendelian factors in the chromosomes can impress
only individual characteristics, probably by giving rise to special
hormones and enzymes. We can cause an egg to develop into an organism
without a spermatozoön, but apparently we cannot make a spermatozoön
develop into an organism without the cytoplasm of an egg, although
sperm and egg nucleus transmit equally the Mendelian characters. The
conception that the cytoplasm of the egg is already the embryo in the
rough may be of importance also for the problem of evolution since
it suggests the possibility that the genus- and species-heredity are
determined by the cytoplasm of the egg, while the Mendelian hereditary
characters cannot contribute at all or only to a limited extent to
the formation of new species. Such an idea is supported by the work
on immunity, which shows that genus- and probably species-specificity
are due to specific proteins, while the Mendelian characters may be
determined by hormones which need neither be proteins nor specific or
by enzymes which also need not be specific for the species or genus.
Such a conception would remove the difficulties which the work on
Mendelian heredity has seemingly created not only for the problem of
evolution but also for the problem of the harmonious character of the
organism as a whole.
Since the book is intended as a companion volume to the writer’s former
treatise on _The Comparative Physiology of the Brain_ a discussion of
the functions of the central nervous system is omitted.
Completeness in regard to quotation of literature was out of the
question, but the writer notices with regret, that he has failed to
refer in the text to so important a contribution to the subject as
Sir E. A. Schäfer’s masterly presidential address on “Life” or the
addresses of Correns and Goldschmidt on the determination of sex.
Credit should also have been given to Professor Raymond Pearl for the
discrimination between species and individual inheritance.
The writer wishes to acknowledge his indebtedness to his friends
Professor E. G. Conklin of Princeton, Professor Richard Goldschmidt
of the Kaiser Wilhelm Institut of Berlin, Dr. P. A. Levene of the
Rockefeller Institute, Professor T. H. Morgan of Columbia University,
and Professor Hardolph Wasteneys of the University of California who
kindly read one or more chapters of the book and offered valuable
suggestions; and he wishes especially to thank his wife for suggesting
many corrections in the manuscript and the proof.
The book is dedicated to that group of freethinkers, including
d’Alembert, Diderot, Holbach, and Voltaire, who first dared to follow
the consequences of a mechanistic science--incomplete as it then
was--to the rules of human conduct and who thereby laid the foundation
of that spirit of tolerance, justice, and gentleness which was the hope
of our civilization until it was buried under the wave of homicidal
emotion which has swept through the world. Diderot was singled out,
since to him the words of Lord Morley are devoted, which, however, are
more or less characteristic of the whole group.
J. L.
The Rockefeller Institute
for Medical Research,
_August, 1916_
CONTENTS
PAGE
CHAPTER I
Introductory Remarks 1
CHAPTER II
The Specific Difference between Living and
Dead Matter and the Question of the
Origin of Life 14
CHAPTER III
The Chemical Basis of Genus and Species: 40
I.--The Incompatibility of Species not Closely Related 44
II.--The Chemical Basis of Genus and Species and of
Species Specificity 53
CHAPTER IV
Specificity in Fertilization 71
CHAPTER V
Artificial Parthenogenesis 95
CHAPTER VI
Determinism in the Formation of an Organism from an Egg 128
CHAPTER VII
Regeneration 153
CHAPTER VIII
Determination of Sex, Secondary Sexual Characters,
and Sexual Instincts:
I.--The Cytological Basis of Sex Determination 198
II.--The Physiological Basis of Sex Determination 214
CHAPTER IX
Mendelian Heredity and its Mechanism 229
CHAPTER X
Animal Instincts and Tropisms 253
CHAPTER XI
The Influence of Environment 286
CHAPTER XII
Adaptation to Environment 318
CHAPTER XIII
Evolution 346
CHAPTER XIV
Death and Dissolution of the Organism 349
Index 371
The Organism as a Whole
CHAPTER I
INTRODUCTORY REMARKS
1. The physical researches of the last ten years have put the atomistic
theory of matter and electricity on a definite and in all probability
permanent basis. We know the exact number of molecules in a given
mass of any substance whose molecular weight is known to us, and we
know the exact charge of a single electron. This permits us to state
as the ultimate aim of the physical sciences the visualization of
all phenomena in terms of groupings and displacements of ultimate
particles, and since there is no discontinuity between the matter
constituting the living and non-living world the goal of biology can be
expressed in the same way.
This idea has more or less consciously prevailed for some time in the
explanation of the single processes occurring in the animal body or
in the explanation of the functions of the individual organs. Nobody,
not even a scientific vitalist, would think of treating the process
of digestion, metabolism, production of heat, and electricity or even
secretion or muscular contraction in any other than a purely chemical
or physicochemical way; nor would anybody think of explaining the
functions of the eye or the ear from any other standpoint than that of
physics.
When the actions of the organism as a whole are concerned, we find
a totally different situation. The same physiologists who in the
explanation of the individual processes would follow the strictly
physicochemical viewpoint and method would consider the reactions of
the organism as a whole as the expression of non-physical agencies.
Thus Claude Bernard,[1] who in the investigation of the individual
life processes was a strict mechanist, declares that the making of a
harmonious organism from the egg cannot be explained on a mechanistic
basis but only on the assumption of a “directive force.” Bernard
assumes, as Bichat and others had done before him, that there are two
opposite processes going on in the living organism: (1) the phenomena
of vital creation or organizing synthesis; (2) the phenomena of death
or organic destruction. It is only the destructive processes which give
rise to the physical manifestations by which we judge life, such as
respiration and circulation or the activity of glands, and so on. The
work of creation takes place unseen by us in the egg when the embryo
or organism is formed. This vital creation occurs always according to
a definite plan, and in the opinion of Bernard it is impossible to
account for this plan on a purely physicochemical basis.
[1] Bernard C., _Leçons sur les Phénomènes de la Vie_. Paris, 1885, i.,
22-64.
There is so to speak a pre-established design of each being and of
each organ of such a kind that each phenomenon by itself depends
upon the general forces of nature, but when taken in connection
with the others it seems directed by some invisible guide on the
road it follows and led to the place it occupies....
We admit that the life phenomena are attached to physicochemical
manifestations, but it is true that the essential is not explained
thereby; for no fortuitous coming together of physicochemical
phenomena constructs each organism after a plan and a fixed
design (which are foreseen in advance) and arouses the admirable
subordination and harmonious agreement of the acts of life....
We can only know the material conditions and not the intimate
nature of life phenomena. We have therefore only to deal with
matter and not with the first causes or the vital force derived
therefrom. These causes are inaccessible to us, and if we believe
anything else we commit an error and become the dupes of metaphors
and take figurative language as real.... Determinism can never be
but physicochemical determinism. The vital force and life belong to
the metaphysical world.
In other words, Bernard thinks it his task to account for individual
life phenomena on a purely physicochemical basis--but the harmonious
character of the organism as a whole is in his opinion not produced
by the same forces and he considers it impossible and hopeless
to investigate the “design.” This attitude of Bernard would be
incomprehensible were it not for the fact that, when he made these
statements, the phenomena of specificity, the physiology of development
and regeneration, the Mendelian laws of heredity, the animal tropisms
and their bearing on the theory of adaptation were unknown.
This explanation of Bernard’s attitude is apparently contradicted by
the fact that Driesch[2] and v. Uexküll,[3] both brilliant biologists,
occupy today a standpoint not very different from that of Claude
Bernard. Driesch assumes that there is an Aristotelian “entelechy”
acting as directing guide in each organism; and v. Uexküll suggests
a kind of Platonic “idea” as a peculiar characteristic of life which
accounts for the purposeful character of the organism.
v. Uexküll supposes as did Claude Bernard and as does Driesch
that in an organism or an egg the ultimate processes are purely
physicochemical. In an egg these processes are guided into definite
parts of the future embryo by the Mendelian factors of heredity--the
so-called genes. These genes he compares to the foremen for the
different types of work to be done in a building. But there
must be something that makes of the work of the single genes a
harmonious whole, and for this purpose he assumes the existence
of “supergenes.”[4] v. Uexküll’s ideas concerning the nature of a
Mendelian factor and of the “supergenes” are expressed in metaphorical
terms and the assumption of the “supergenes” begs the question. The
writer is under the impression that this author was led to his views
by the belief that the egg is entirely undifferentiated. But the
unfertilized egg is not homogeneous, on the contrary, it has a simple
but definite physicochemical structure which suffices to determine the
first steps in the differentiation of the organism. Of course, if we
suppose as do v. Uexküll and Driesch that the egg has no structure, the
development of structure becomes a difficult problem--but this is not
the real situation.
[2] Driesch, H., _The Science and Philosophy of the Organism_. 2 vols.
The Gifford Lectures, 1907 and 1908.
[3] v. Uexküll, J., _Bausteine zu einer biologischen Weltanschauung_.
München, 1913.
[4] v. Uexküll, J., _Bausteine zu einer biologischen Weltanschauung_.
München, 1913, p. 216.
2. Claude Bernard does not mention the possibility of explaining
the harmony or apparent design in the organism on the basis of the
theory of evolution, he simply considers the problem as outside of
biology. It was probably clear to him as it must be to everyone with an
adequate training in physics that natural selection does not explain
the origin of variation. Driesch and v. Uexküll consider the Darwinian
theory a failure. We may admit that the theory of a formation of new
species by the cumulative effect of aimless fluctuating variations
is not tenable because fluctuating variation is not hereditary;
but this would only demand a slight change in the theory; namely a
replacement of the influence of fluctuating variation by that of
equally aimless mutations. With this slight modification which is
proposed by de Vries,[5] Darwin’s theory still serves the purpose of
explaining how without any pre-established plan only purposeful and
harmonious organisms should have survived. It must be said, however,
that any theory of life phenomena must be based on our knowledge of
the physicochemical constitution of living matter, and neither Darwin
nor Lamarck was concerned with this. Moreover, we cannot consider any
theory of evolution as proved unless it permits us to transform at
desire one species into another, and this has not yet been accomplished.
[5] de Vries, H., _Die Mutationstheorie_. Leipzig, 1901.
It may be of some interest to point out that we do not need to make any
definite assumption concerning the mechanism of evolution and that we
may yet be able to account for the fact that the surviving organisms
are to all appearances harmonious. The writer pointed out that of all
the 100,000,000 conceivable crosses of teleost fish (many of which
are possible) not many more than 10,000, _i. e._, about one-hundredth
of one per cent., are able to live and propagate. Those that live and
develop are free from the grosser type of disharmonies, the rest are
doomed on account of a gross lack of harmony of the parts. These latter
we never see and this gives us the erroneous conception that harmony
or “design” is a general character of living matter. If anybody wishes
to call the non-viability of 99-99/100 per cent. of possible teleosts
a process of weeding out by “natural selection” we shall raise no
objection, but only wish to point out that our way of explaining the
lack of design in living nature would be valid even if there were no
theory of evolution or if there had never been any evolution.
3. v. Uexküll is perfectly right in connecting the problem of design
in an organism with Mendelian heredity. The work on Mendelian heredity
has shown that an extremely large number of independently transmissible
Mendelian factors help to shape the individual. It is not yet proven
that the organism is nothing but a mosaic of Mendelian factors, but no
writer can be blamed for considering such a possibility. If we assume
that the organism is nothing but a mosaic of Mendelian characters it
is difficult indeed to understand how they can force each other into a
harmonious whole[6]; even if we make ample allowance for the law of
chance and the corresponding wastefulness in the world of the living.
But it is doubtful whether this idea of the rôle of Mendelian factors
is correct. The facts of experimental embryology strongly indicate the
possibility that the cytoplasm of the egg is the future embryo (in the
rough) and that the Mendelian factors only impress the individual (and
variety) characters upon this rough block. This idea is supported by
the fact that the first development--in the sea urchin to the gastrula
stage inclusive--is independent of the nucleus, which is the bearer of
the Mendelian factors. Not before the skeleton or mesenchyme is formed
in the sea urchin egg is the influence of the nucleus noticeable. This
has been shown in the experiments of Boveri in which an enucleated
fragment of an egg was fertilized with a spermatozoön of a foreign
species. If this is generally true, it is conceivable that the generic
and possibly also the species characters of organisms are determined by
the cytoplasm of the egg and not by the Mendelian factors.
[6] This difficulty is also felt by mechanistic writers like Child,
who on page 12 of his recent book on _Senescence and Rejuvenescence_
(Chicago, 1915) makes the following remarks: “These theories of
Weismann do not account satisfactorily for the peculiarly constant
course and character of development and morphogenesis. If we follow
them to their logical conclusion, which their authors have not done,
we find ourselves forced to assume the existence of some sort of
controlling and co-ordinating principle outside the units themselves
and superior to them. If the units constitute the physicochemical
basis of life, as their authors maintain, then this controlling
principle, since it is an essential feature of life, must of necessity
be something which is not physicochemical in nature. In short these
theories lead us in the final analysis to the same conclusion as that
reached by the neovitalists. If we are not content to accept this
conclusion we must reject the theories.” These last sentences do not
exhaust all the possibilities, since the writer is trying to show
in this book that the widest acceptance of the chromosome theory of
heredity is compatible with a consistent physicochemical conception of
the organism as a whole.
In any case, we can state today that the cytoplasm contains the rough
preformation of the future embryo. This would show then that the idea
of the organism being a mosaic of Mendelian characters which have to be
put into place by “supergenes” is unnecessary. If the egg is already
the embryo in the rough we can imagine the Mendelian factors as giving
rise to specific substances which go into the circulation and start
or accelerate different chemical reactions in different parts of the
embryo, and thereby call forth the finer details characteristic of the
variety and the individual. The idea that the egg is the future embryo
is supported by the fact that we can call forth a normal organism
from an unfertilized egg by artificial means; while it is apparently
impossible to cause the spermatozoön to develop into an organism
outside the egg.
4. The influence of the whole on the parts is nowhere shown more
strikingly than in the field of regeneration. It is known that pieces
cut from the plant or animal may give rise to new growth which in many
cases will restore somewhat the original organism. Instead of asking
what is the cause of this so-called regeneration we may ask, why the
same pieces do not regenerate as long as they are parts of the whole.
In this form the mysterious influence of the whole over its parts
is put into the foreground. We shall see that growth takes place in
certain cells when certain substances in the circulation can collect
there. The mysterious influence of the whole on these parts consists
often merely of the fact that the circulating specific or non-specific
substances--we cannot yet decide which--will in the whole be attracted
by certain spots and that this will prevent them from acting on other
parts of the organism. If such parts are isolated the substances can
no longer flow away from these parts and the parts will begin to grow.
It thus becomes utterly unnecessary to endow such organisms with a
“directing force” which has to elaborate the isolated parts into a
whole.
5. The same difficulty which we have discussed in regard to
morphogenesis exists also in connection with those instincts which
preserve the life of the organism and of the race. The reader need
only be reminded of all the complicated instincts of mating by which
sperm and eggs are brought together; or those by which the young
are prevented from starvation to realize the apparently desperate
problems in store for a mechanist, to whom the assumption of design
is meaningless. And yet we are better off in regard to our knowledge
of the instincts than we are in regard to morphogenesis, as in the
former we can show that the apparent instincts in some cases obey
simple physicochemical laws with almost mathematical accuracy. Since
the validity of the law of gravitation has been proved for the solar
system the idea of design in the motion of the planets has lost its
usefulness, and this fact must serve us as a guide wherever we attempt
to put science beyond the possibility of mysticism. As soon as we
can show that a life phenomenon obeys a simple physical law there is
no longer any need for assuming the action of non-physical agencies.
We shall see that this has been accomplished for one group of animal
instincts; namely those which determine the relation of animals to
light, since these are being gradually reduced to the law of Bunsen and
Roscoe. This law states that the chemical effect of light equals the
product of intensity into duration of illumination. Some authors object
to the tendency toward reducing everything in biology to mathematical
laws or figures; but where would the theory of heredity be without
figures? Figures have been responsible for showing that the laws of
chance and not of design rule in heredity. Biology will be scientific
only to the extent that it succeeds in reducing life phenomena to
quantitative laws.
Those familiar with the theories of evolution know the extensive
rôle ascribed to the adaptations of organisms. The writer in
1889 called attention to the fact that reactions to light--_e.
g._, positive heliotropism--are found in organisms that never
by any chance make use of them; and later that a great many
organisms show definite instinctive reactions towards a galvanic
current--galvanotropism--although no organism has ever had or
ever will have a chance to be exposed to such a current except in
laboratory experiments. This throws a different light upon the
seemingly purposeful character of animal reactions. Heliotropism
depends primarily upon the presence of photosensitive substances
in the eye or the epidermis of the organism, and these substances
are inherited regardless of whether they are useful or not. It is
only a metaphor to call reactions resulting from the presence of
photosensitive substances “adaptation.” In this book other examples
are given which show that authors have too often spoken of adaptation
to environment where the environment was not responsible for the
phenomena. The blindness of cave animals and the resistance of certain
marine animals to higher concentrations of sea water are such cases.
Cuénot speaks of “preadaptation” to express this relation. The fact
is that the “adaptations” often existed before the animal was exposed
to surroundings where they were of use. This relieves us also of the
necessity of postulating the existence of the inheritance of acquired
characters, although it is quite possible that the future may furnish
proof that such a mode of inheritance exists.
6. We have mentioned that according to Claude Bernard two groups of
phenomena occur in the living organism: (1) the phenomena of vital
creation or organizing synthesis (especially in the egg and during
development); (2) the phenomena of death or organic destruction. These
two processes are briefly discussed in the first and last chapters.
These introductory remarks may perhaps make it easier for the reader to
retain the thread of the main ideas in the details of experiments and
tables given in this book.
CHAPTER II
THE SPECIFIC DIFFERENCE BETWEEN LIVING AND DEAD MATTER
AND THE QUESTION OF THE ORIGIN OF LIFE
1. Each organism is characterized by a definite form and we shall see
in the next chapter that this form is determined by definite chemical
substances. The same is true for crystals, where substance and form
are definitely connected and there are further analogies between
organisms and crystals. Crystals can grow in a proper solution, and can
regenerate their form in such a solution when broken or injured; it
is even possible to prevent or retard the formation of crystals in a
supersaturated solution by preventing “germs” in the air from getting
into the solution, an observation which was later utilized by Schroeder
and Pasteur in their experiments on spontaneous generation. However,
the analogies between a living organism and a crystal are merely
superficial and it is by pointing out the fundamental differences
between the behaviour of crystals and that of living organisms that
we can best understand the specific difference between non-living
and living matter. It is true that a crystal can grow, but it will
do so only in a supersaturated solution of its own substance. Just
the reverse is true for living organisms. In order to make bacteria
or the cells of our body grow, solutions of the split products of
the substances composing them and not the substances themselves
must be available to the cells; second, these solutions must not be
supersaturated, on the contrary, they must be dilute; and third,
growth leads in living organisms to cell division as soon as the mass
of the cell reaches a certain limit. This process of cell division
cannot be claimed even metaphorically to exist in a crystal. A correct
appreciation of these facts will give us an insight into the specific
difference between non-living and living matter. The formation of
living matter consists in the synthesis of the proteins, nucleins,
fats, and carbohydrates of the cells, from the split products. To give
an historical example, Pasteur showed that yeast cells and other fungi
could be raised on the following sterilized solution: water, 100 gm.,
crystallized sugar, 10 gm., ammonium tartrate, 0.2 gm. to 0.5 gm.,
and fused ash from yeast, 0.1 gm.[7] He undertook this experiment
to disprove the idea that protein or organic matter in a state of
decomposition was needed for the origin of new organisms as the
defenders of the idea of spontaneous generation had maintained.
[7] Pasteur, L., _Annal. d. Chim. et d. Physique_, 1862, 3 sér., lxiv.,
1.
2. That such a solution can serve for the synthesis of all the
compounds of living yeast cells is due to the fact that it contains
the sugars. From the sugars organic acids can be formed and these
with ammonia (which was offered in the form of ammonium tartrate) may
give rise to the formation of amino acids, the “building stones” of
the proteins. It is thus obvious that the synthesis of living matter
centres around the sugar molecule. The phosphates are required for the
formation of the nucleins, and the work of Harden and Young suggests
that they play also a rôle in the alcoholic fermentation of sugar.
Chlorophyll, under the influence of the red rays of light, manufactures
the sugars from the CO₂ of the air. This makes it appear as though
life on our planet should have been preceded by the existence of
chlorophyll, a fact difficult to understand since it seems more natural
to conceive of chlorophyll as a part or a product of living organisms
rather than the reverse. Where then should the sugar come from, which
is a constituent of the majority of culture media and which seems a
prerequisite for the synthesis of proteins in living organisms?
The investigations of Winogradsky on nitrifying,[8] sulphur and
perhaps also on iron bacteria have to all appearances pointed a way
out of this difficulty. It seemed probable that there were specific
micro-organisms which oxidized the ammonia formed in sewage or in
the putrefaction of living matter, but the attempts to prove this
assumption by raising such a nitrifying micro-organism on one of the
usual culture media, all of which contained organic compounds, failed.
Led by the results of his observations on sulphur bacteria it occurred
to Winogradsky that the presence of organic compounds stood in the way
of raising these bacteria, and this idea proved correct. The bacteria
oxidizing ammonia to nitrites were grown on the following medium; 1 gm.
ammonium sulphate, 1 gm. potassium phosphate, 1 gm. magnesium
carbonate, to 1 litre of water. From this medium, which is free from
sugar and contains only constituents which could exist on the planet
before the appearance of life, the nitrifying bacteria were able to
form sugars, fatty acids, proteins, and the other specific constituents
of living matter. Winogradsky proved, by quantitative determination,
that with the nitrification an increase in the amount of carbon
compounds takes place. “Since this bound carbon in the cultures can
have no other source than the CO₂ and since the process itself can have
no other cause than the activity of the nitrifying organism, no other
alternative was left but to ascribe to it the power of assimilating
CO₂.”[9] “Since the oxidation of NH₃ is the only source of chemical
energy which the nitrifying organism can use it was clear _a priori_
that the yield in assimilation must correspond to the quantity of
oxidized nitrogen. It turned out that an approximately constant ratio
exists between the values of assimilated carbon and those of oxidized
nitrogen.” This is illustrated by the results of various experiments as
shown in Table I.
[8] Winogradsky, S., “Die Nitrification,” _Handb. d. tech. Mykol._,
1904-06, iii., 132.
[9] Winogradsky, _loc. cit._, p. 163 and ff.
TABLE I
--------------+---------+---------+---------+---------
| _No. 5_ | _No. 6_ | _No. 7_ | _No. 8_
+---------+---------+---------+---------
| _mg._ | _mg._ | _mg._ | _mg._
Oxidized N | 722.0 | 506.1 | 928.3 | 815.4
Assimilated C | 19.7 | 15.2 | 26.4 | 22.4
--------------+---------+---------+---------+---------
Ratio N:C | 36.6 | 33.3 | 35.2 | 36.4
--------------+---------+---------+---------+---------
It is obvious that 1 part of assimilated carbon corresponds to about
35.4 parts oxidized nitrogen or 96 parts of nitrous acid.
These results of Winogradsky were confirmed in very careful experiments
by E. Godlewski, Sr.[10]
[10] Godlewski, E., _Anz. d. Akad. d. Wissensch. in Krakau_, 1892, 408;
1895, 178.
The nitrites are further oxidized by another kind of micro-organisms
into nitrates and they also can be raised without organic material.
Winogradsky had already previously discovered that the hydrogen
sulphide which is formed as a reduction product from CaSO₄ or in
putrefaction by the activity of certain bacteria can be oxidized by
certain groups of bacteria, the sulphur bacteria. Such bacteria,
_e. g._, _Beggiatoa_, are also commonly found at the outlet of sulphur
springs. They utilize the hydrogen sulphide which they oxidize to
sulphur and afterwards to sulphates, according to the scheme:
(1) 2H₂S + O₂ = 2H₂O + S₂
(2) S₂ + 3O₂ + 2H₂O = 2H₂SO₄
The sulphuric acid is at once neutralized by carbonates.
Winogradsky assumes that the oxidation of H₂S by the sulphur bacteria
is the source of energy which plays the same rôle as the oxidation
of NH₃ plays in the nitrifying bacteria, or the oxidation of carbon
compounds--sugar and others--in the case of the other lower and higher
organisms. Winogradsky has made it very probable that sulphur bacteria
do not need any organic compounds and that their nutrition may be
accomplished with a purely mineral culture medium, like that of the
nitrite bacteria. On the basis of this assumption they should also be
able to form sugars from the CO₂ of the air.
Nathanson[11] discovered in the sea water the existence of bacteria
which oxidize thiosulphate to sulphuric acid. They will develop if some
Na₂S₂O₃, is added to sea water. These bacteria can only develop if CO₂
from the air is admitted or when carbonates are present. For these
organisms the CO₂ cannot be replaced by glucose, urea, or other organic
substances. Such bacteria must therefore possess the power of producing
sugar and starch from CO₂ without the aid of chlorophyll. Similar
observations were made by Beijerinck on a species of fresh-water
bacteria.[12]
[11] Nathanson, _Mitteil. d. zool. Station_, Neapel, 1902.
[12] Beijerinck, M., _Folia Microbiologica_, 1914, iii., 91.
Finally the case of iron bacteria may briefly be mentioned though
Winogradsky’s views are not accepted by Molisch.
We may, therefore, consider it an established fact that there are a
number of organisms which could have lived on this planet at a time
when only mineral constituents, such as phosphates, K, Mg, SO₄, CO₂,
and O₂ besides NH₃, or SH₂, existed. This would lead us to consider it
possible that the first organisms on this planet may have belonged to
that world of micro-organisms which was discovered by Winogradsky.
If we can conceive of this group of organisms as producing sugar, which
in fact they do, they could have served as a basis for the development
of other forms which require organic material for their development.
In 1883 the small island of Krakatau was destroyed by the most violent
volcanic eruption on record. A visit to the islands two months after
the eruption showed that “the three islands were covered with pumice
and layers of ash reaching on an average a thickness of thirty metres
and frequently sixty metres.”[13] Of course all life on the islands
was extinct. When Treub in 1886 first visited the island, he found
that blue-green algæ were the first colonists on the pumice and on
the exposed blocks of rock in the ravines on the mountain slopes.
Investigations made during subsequent expeditions demonstrated the
association of diatoms and bacteria. All of these were probably carried
by the wind. The algæ referred to were according to Euler of the nostoc
type. Nostoc does not require sugar, since it can produce that compound
from the CO₂ of the air by the activity of its chlorophyll. This
organism possesses also the power of assimilating the free nitrogen of
the air. From these observations and because the _Nostocaceæ_ generally
appear as the first settlers on sand the conclusion has been drawn that
they or the group of _Schizophyceæ_ to which they belong formed the
first settlers of our planet.[14] This conclusion is not quite safe
since in the settlement of Krakatau as well as in the first colonizing
of sand areas the nature of the first settler is determined chiefly by
the carrying power of wind (or waves and birds).
[13] Ernst, A., _The New Flora of the Volcanic Island of Krakatau_,
Cambridge, 1908.
[14] Euler, H., _Pflanzenchemie_, 1909, ii. and iii., 140.
We may now return from this digression to the real object of our
discussion, namely that the nutritive solutions of organisms must
be very dilute and consist of the split products of the complicated
compounds of which the organisms consist. The examples given
sufficiently illustrate this statement.
The nutritive medium of our body cells is the blood, and while we
take up as food the complicated compounds of plants or animals, these
substances undergo a digestion, _i. e._, a splitting up into small
constituents before they can diffuse from the intestine into the blood.
Thus the proteins are digested down to the amino acids and these
diffuse into the blood as demonstrated by Folin and by Van Slyke. From
here the cells take them up. The different proteins differ in regard
to the different types of amino acids which they contain. While the
bacteria and fungi and apparently the higher plants can build up all
their different amino acids from ammonia, this power is no longer found
in the mammals which can form only certain amino acids in their body
and must receive the others through their food. As a consequence it is
usually necessary to feed young animals on more than one protein in
order to make them grow, since one protein, as a rule, does not contain
all the amino acids needed for the manufacture of all the proteins
required for the formation of the material of a growing animal.[15]
[15] This fact was thoroughly established by Mendel and Osborne. A
summary of their work is given in Underhill, F. P., _Physiology of the
Amino Acids_, 1916.
3. The essential difference between living and non-living matter
consists then in this: the living cell synthetizes its own complicated
specific material from indifferent or non-specific simple compounds of
the surrounding medium, while the crystal simply adds the molecules
found in its supersaturated solution. This synthetic power of
transforming small “building stones” into the complicated compounds
specific for each organism is the “secret of life” or rather one of the
secrets of life.
What clew have we in regard to the nature of this synthetic power?
We know that the comparatively great velocity of chemical reactions
in a living organism is due to the presence of enzymes (ferments) or
to catalytic agencies in general. Some of these catalytic agencies
are specific in the sense that a given catalyzer can accelerate
the reaction of only one step in a complicated chemical reaction.
While these enzymes are formed by the action of the body they can be
separated from the body without losing their catalytic efficiency. It
was a long time before scientists succeeded in isolating the enzyme of
the yeast cell which causes the alcoholic fermentation of sugar; and
this gave rise to the premature statement that it was not possible to
isolate this enzyme since it was bound up with the life of the yeast
cell. Such a statement was even made by a man like Pasteur, who was
usually a model of restraint in his utterances, and yet the work of
Buchner proved him to be wrong.
The general mechanism of the action of the hydrolyzing enzymes is
known. The old idea of de la Rive, that a molecule of enzyme combines
transitorily with a molecule of substrate; the further idea, which may
possibly go back to Engler, that the molecule of substrate is disrupted
in the “strain” of the new combination and that the broken fragments
fall off or are easily knocked off by collision from the ferment
molecule which is now ready to repeat the process, seems to be correct.
On the assumption that the velocity of enzyme reaction is proportional
to the mass of the enzyme and that de la Rive’s idea was correct,
Van Slyke and Cullen were able to calculate the coefficients of the
velocity of enzyme reactions for the fermentation of urea and other
substances, and the agreement between calculated and observed values
was remarkable.[16]
[16] Van Slyke, D. D., and Cullen, G. E., _Jour. Biol. Chem._, 1914,
xix., 141.
While the hydrolytic action of enzymes is thus clear the synthesis
in the cell is still a riddle. An interesting suggestion was made by
van’t Hoff, who in 1898 expressed the idea that the hydrolytic enzymes
should also act in the opposite direction, namely synthetically.
Thus it should not only be possible to digest proteins with pepsin
but also to synthetize them from the products of digestion with the
aid of the same enzyme. This expectation was based on the idea that
the enzyme did not alter the equilibrium between the hydrolyzed and
non-hydrolyzed part of the substrate but only accelerated the rate
with which the equilibrium was reached. Van’t Hoff’s idea omitted,
however, the possibility that in the transitory combination between
enzyme molecule and substrate a change in the molecular configuration
of the substrate or in the distribution of intramolecular strain may
take place. The first apparently complete confirmation of van’t Hoff’s
suggestion appeared in the form of the synthesis of maltose from grape
sugar by the enzyme maltase, which decomposes maltose into grape sugar.
By adding the enzyme maltase from yeast to a forty per cent. solution
of glucose Croft Hill[17] obtained a good yield of maltose. It turned
out, however, that what he took for maltose was not this compound
but an isomer, namely isomaltose, which has a different molecular
configuration and cannot be hydrolyzed by the enzyme maltase.
[17] Hill, C., _Jour. Chem. Soc._, 1898, lxxiii., 634.
Lactose is hydrolyzed from kephyr by an enzyme lactase into galactose
and glucose; by adding this enzyme to galactose and glucose a synthesis
was obtained not of lactose but of isolactose; the latter, however, is
not decomposed by the enzyme lactase.
E. F. Armstrong has worked out a theory which tries to account for
this striking phenomenon by assuming “that the enzyme has a specific
influence in promoting the formation of the biose which it cannot
hydrolyze.”[18] The theory is very ingenious and seems supported by
fact. This then would lead to the result that certain hydrolytic
enzymes may have a synthetic action but not in the manner suggested by
van’t Hoff.
[18] Armstrong, E. F., _Proc. Royal Soc._, 1905, B. lxxvi., 592.
The principle enunciated by Armstrong, that in the synthetic action of
hydrolytic enzymes not the original compound but an isomer is formed
which can not be hydrolyzed by the enzyme, may possibly be of great
importance in the understanding of life phenomena. It shows us how
the cell can grow in the presence of hydrolytic enzymes and why in
hunger the disintegration of the cell material is so slow. It was at
first thought that the formation of isomers contradicted the idea of
the reversible action of enzymes, but this is not the case; on the
contrary, it supports it but makes an addition which may solve the
riddle of what Claude Bernard called the creative action of living
matter. We shall come back to this problem in the last chapter.
Kastle and Loevenhart demonstrated the synthesis of a trace of
ethylbutyrate by lipase if the latter enzyme was added to the products
of the hydrolysis of ethylbutyrate, ethyl alcohol, and butyric acid
by the same enzyme.[19] Taylor[20] obtained the synthesis of a slight
amount of triolein
by the addition of the dried fat-free residue of the castor bean to
a mixture of oleinic acid and glycerine.... No synthesis occurred
with acetic, butyric, palmitic, and stearic acids with glycerine,
mannite, and dulcite, and the experiments with the last two
alcohols and oleinic acid likewise yielded no synthesis.
[19] Kastle, J. H., and Loevenhart, A. S., _Am. Chem. Jour._, 1900,
xxiv., 491.
[20] Taylor, A. E., _Univ. Cal. Pub._, 1904, _Pathology_, i., 33;
_Jour. Biol. Chem._, 1906, ii., 87.
This suggests possibly a specific action of the enzyme. If this slight
reversible action had any biological significance (which might be
possible, since in the organism secondary favourable conditions might
be at work which are lacking _in vitro_) there should be a parallelism
between masses of lipase in different kinds of tissues and fat
synthesis. Loevenhart indicated that this might be a fact, but a more
extensive investigation by H. C. Bradley has made this very dubious.[21]
[21] Bradley, H. C., _Jour. Biol. Chem._, 1913, xiii., 407.
Very little is known concerning the reversible action of the hydrolytic
protein enzymes. A. E. Taylor digested protamine sulphate with trypsin
and found that after adding trypsin to the products of digestion a
precipitate was formed after long standing; and we may also refer to
experiments of Robertson with pepsin on the products of caseinogen
to which we shall return in the next chapter. It therefore looks at
present as if van’t Hoff’s idea of reversible enzyme action might hold
in the modification offered by Armstrong. It remains doubtful, however,
whether this reversibility can explain all the synthetic processes in
the cell. No objection can be offered at present if any one makes the
assumption that each cell has specific synthetic enzymes or some other
synthetic mechanisms which are still unknown.
The mechanisms for the synthesis of proteins must have one other
peculiarity: they must be specific in their action. We shall see in the
next chapter that each species seems to possess one or more proteins
not found in any other but closely related species. Each organism
develops from a tiny microscopic germ and grows by synthetizing the
non-specific building stones (amino acids) into the specific proteins
of the species. This must be the work of the yet unknown synthetic
enzymes or mechanisms. The elucidation of their character would seem
one of the main problems of biology. Needless to say crystallography is
not confronted with problems of such a nature.
The fact that the living cell grows after taking up food has given
rise to curious misunderstandings. Traube has shown that drops of a
liquid surrounded with a semipermeable membrane may increase in volume
when put into a solution of lower osmotic pressure. This has led and
is possibly still leading to the statement that the process of growth
by a living cell has been imitated artificially. Only one feature has
been imitated, the increase in volume; but the essential feature of
the process in the living cell, _i. e._, the formation of the specific
constituents of the living cell from non-specific products, has of
course not been imitated.
4. The constant synthesis then of specific material from simple
compounds of a non-specific character is the chief feature by which
living matter differs from non-living matter. With this character
is correlated another one; namely, when the mass of a cell reaches
a certain limit the cell divides. This is perhaps most obvious in
bacteria which on the proper nutritive medium take up food, grow, and
divide into two bacteria, each of which takes up food, divides, and
grows _ad infinitum_, as long as the food lasts, provided the harmful
products of metabolism are removed. If it be true that specific
synthetic ferments exist in each cell it follows that the cell must
synthetize these also,[22] as otherwise the synthesis of specific
proteins would have to come to a standstill.
[22] This would lead to the idea that the enzymes in the cell also
synthetize molecules of their own kind, or that, in other words, the
synthetic processes in the cell are of the nature of autocatalysis.
Loeb, _Der chemische Character des Befruchtungsvorgangs_, Leipzig,
1908. Robertson, T. B., _Arch. f. Entwicklngsmech._, 1908, xxv., 581;
xxvi., 108; 1913, xxxvii., 497; _Am. Jour. Physiol._, 1915, xxxvii.,
1; Robertson and Wasteneys, H., _Arch. f. Entwicklngsmech._, 1913,
xxxvii., 485. Ostwald, Wo., _Über die zeitlichen Eigenschaften der
Entwicklungsvorgänge_, Leipzig, 1908.
This problem of synthesis leads to the assumption of immortality of the
living cell, since there is no _a priori_ reason why this synthesis
should ever come to a standstill of its own accord as long as enough
food is available and the proper outside physical conditions are
guaranteed. It is well known that Weismann has claimed immortality for
all unicellular organisms and for the sex cells of metazoa, while he
claimed the necessity of death for the body cells of the latter. Leo
Loeb was led by his investigations on the transplantation of cancer to
assume immortality not only for the cancer cell but also for the body
cell of the organism. He had found in transplanting a malignant tumor
from one individual to another that the tumor grew; that it was not the
cells of the host but the transplanted tumor cells of the graft which
grew and multiplied, and that this process could be repeated apparently
indefinitely so that it was obvious that the transplanted tumor cells
outlived the original animal. Such experiments have since been carried
on so long that we may now say that an individual cancer cell taken
from an animal and transplanted from time to time on a new host lives
apparently indefinitely. Leo Loeb had found that these tumor cells are
simply modified somatic cells. He therefore suggested that the somatic
cells might be considered immortal with the same right as we speak of
the immortality of the germ cells of such animals.[23]
[23] Loeb, Leo, _Jour. Med. Res._, 1901, vi., 28; _Arch. f.
Entwicklngsmech._, 1907, xxiv., 655.
This view receives its support first from the fact that certain trees
like the _Sequoia_ live several thousand years and may therefore be
considered immortal and second, from the method of tissue culture. The
method of cultivating tissue cells in a test tube, in the same way as
is done for bacteria, was first proposed and carried out by Leo Loeb,
in 1897,[24] but his test-tube method did not permit the observation
of the transplanted cell under the microscope. This was made possible
by a modification of the method by Harrison, who established the fact
that the axis cylinder grows out from the ganglionic cell. Harrison
and Burrows then perfected the method for the cultivation of the cells
of warm-blooded, animals, and with the aid of these methods Carrel
succeeded in keeping connective-tissue cells of the heart of an early
chick embryo alive more than four years, and these cells are still
growing and dividing.[25] Only very tiny masses of cells can be kept
alive in this way since all the cells in the centre of a piece die on
account of lack of oxygen; and every two days a few cells from the
margin of the piece have to be transferred to a new culture medium.
[24] Loeb, Leo, _Über die Entstehung von Bindegewebe, Leucocyten und
rothen Blutkörperchen aus Epithel und über eine Methode isolierte
Gewebsteile zu züchten_. Chicago, 1897.
[25] While this has been demonstrated thus far only for
connective-tissue cells it may be true also for other cells.
This effect of lack of oxygen explains also why the immortality of
the somatic cells is not obvious. Death in a human being consists in
the stopping of heart beat and respiration, which also terminates the
action of the brain or at least of consciousness. Immediately after the
cessation of heart beat and respiration the cells of muscle and of the
skin and probably many or most other organs are still alive and might
continue to live if transferred to another body with circulation and
respiration. As a consequence of the lack of oxygen supply in the dead
body they will, however, die comparatively rapidly. It may be stated
that hearts taken out of the body after a number of hours can still
beat again when put into the proper solutions and upon receiving an
adequate oxygen supply.
The idea that the body cells are naturally immortal and die only if
exposed to extreme injuries such as prolonged lack of oxygen or too
high a temperature helps to make one problem more intelligible. The
medical student, who for the first time realizes that life depends upon
that one organ, the heart, doing its duty incessantly for the seventy
years or so allotted to man, is amazed at the precariousness of our
existence. It seems indeed uncanny that so delicate a mechanism should
function so regularly for so many years. The mysticism connected with
this and other phenomena of adaptation would disappear if we could be
certain that all cells are really immortal and that the fact which
demands an explanation is not the continued activity but the cessation
of activity in death. Thus we see that the idea of the immortality of
the body cell if it can be generalized may be destined to become one
of the main supports for a complete physicochemical analysis of life
phenomena since it makes the durability of organisms intelligible.
5. This generalized idea of the immortality of some or possibly most
or all somatic cells has a bearing upon the problem of the origin of
life on our planet. The experiments of Spallanzani, Schwann, Schroeder,
Pasteur, Tyndall, and all those who have worked with pure cultures of
micro-organisms, have proved that no spontaneous generation of living
from non-living matter can be demonstrated; and the statements to the
contrary were due to experimental errors inasmuch as the new organisms
formed were the offspring of others which had entered into the culture
medium by mistake.
In the last chapter of that most fascinating book _Worlds in the
Making_,[26] Arrhenius discusses the possibility of life being eternal
and of living germs of very small dimensions--_e. g._, the spores
of micro-organisms--being carried through space from one planet to
another or even from one solar system to another. If it be true that
there is no spontaneous generation; if it be true that all cells are
potentially immortal, we may indeed seriously raise the question: May
not life after all be eternal? Such ideas were advocated by Richter in
a rather phantastic way and more definitely by Helmholtz as well as
Kelvin. The latter authors assumed that in the collision of planets or
worlds on which there is life, fragments containing living organisms
will be torn off and these fragments will move as seed-bearing stones
through space. “If at the present instant no life existed upon this
earth, one such stone falling upon it might ... lead to its becoming
covered with vegetation.” Arrhenius points out the difficulties which
oppose such a view, as, _e. g._, the fact “that the meteorite in its
fall towards the earth becomes incandescent all over its surface and
any seeds on it would therefore be deprived of their germinating power.”
[26] Arrhenius, S., _Worlds in the Making_, London and New York, 1908,
p. 212.
Arrhenius suggests another and much more ingenious idea based on the
fact that for particles below a certain size the mechanical pressure
produced by light waves--the radiation pressure--can overcome the
attractive force of gravitation.
Bodies which according to Schwarzschild would undergo the strongest
influence of solar radiation must have a diameter of 0.00016 mm.
supposing them to be spherical. The first question is therefore:
Are there any living seeds of such extraordinary minuteness? The
reply of the botanist is that spores of many bacteria have a size
of 0.0003 or 0.0002 mm., and there are no doubt much smaller germs
which our microscopes fail to disclose.
This assumption is undoubtedly correct.
We will, in the first instance, make a rough calculation of what
would happen if such an organism were detached from the earth
and pushed out into space by the radiation pressure of our sun.
The organism would first of all have to cross the orbit of Mars;
then the orbits of the smaller and of the outer planets.... The
organisms would cross the orbit of Mars after twenty days, the
Jupiter orbit after eighty days, and the orbit of Neptune after
fourteen months. Our nearest solar system would be reached in nine
thousand years.
For the assumption of eternity of life only the transference of germs
from one solar system to another would have to be considered and the
question arises whether or not germs can keep their vitality so many
thousands of years. Arrhenius thinks that this is possible on account
of the low temperature (which must be below -220° C.) at which no
chemical reaction and hence no decomposition and deterioration are
possible in the spores; and on account of the absence of water vapour.
The question then arises: Have we any facts to warrant the assumption
that spores may remain alive for thousands of years under such
conditions and retain their power of germination? We know that seeds
have a very limited vitality, and the statement that grain found in
the Egyptian tombs was still able to germinate has long been recognized
as a myth. Miss White[27] found that in wheat grains, there appeared
a well-marked drop in their germinating power after about the fourth
year, reaching zero in eleven to seventeen years. In a drier climate
they last longer than in a moist climate. It is of importance that
the hydrolyzing enzymes in the seeds, such as diastase, erepsin,
remained unimpaired even after the germinating power of the seeds had
disappeared. The seeds were able to resist for two days the temperature
of liquid air, though the subsequent germination was delayed by this
treatment. Macfadyen[28] exposed non-sporing bacteria, viz., _B.
typhosus_, _B. coli communis_, _Staphylococcus pyogenes aureus_, and a
_Saccharomyces_ to liquid air.
The experiments showed that a prolonged exposure of six months
to a temperature of about -190° has no appreciable effect on the
vitality of micro-organisms. To judge by the results there appeared
no reason to doubt that the experiment might have been successfully
prolonged for a still longer period.
[27] White, J., _Proc. Roy. Soc._, 1909, B, lxxxi., 417.
[28] Macfadyen, A., _Proc. Roy. Soc._, 1903, lxxi., 76.
Paul Becquerel[29] found that seeds which possess a very thick
integument may live longer than the grain in Miss White’s experiments.
The thickness of the integument prevents the exchange of gases between
air and seed. Thus seeds of leguminoses (_Cassia bicapsularis_,
_Cytisus biflorus_, _Leucæna leucocephala_, and _Trifolium arvense_)
had retained their power of germination for eighty-seven years.
Becquerel has shown that the dryness of the membrane is very essential
for such a duration of life, since when dry it is impermeable for gases
and the slow chemical reactions inside the grain become impossible.
[29] Becquerel, P., _Revue générale des Sciences_, 1914, xxv., 559.
In the cosmic space there is no water vapour, no atmosphere, and a
low temperature, and there is hence no reason why spores should lose
appreciably more of their germinating power in ten thousand years than
in six months. We must therefore admit the possibility that spores may
move for an almost infinite length of time through cosmic space and
yet be ready for germination when they fall upon a planet in which all
the conditions for germination and development exist, _e. g._, water,
proper temperature, and the right nutritive substances dissolved in the
water (inclusive of free oxygen).
While thus everything is favourable to Arrhenius’s hypothesis,
Becquerel raises the objection that the spores going through space
would yet be destroyed by ultraviolet light. This danger would
probably exist only as long as the germ is not too far from a sun. The
difficulty is a real one since the ultraviolet rays have a destructive
effect even in the absence of oxygen. It is possible, however, that
there are spores which can resist this effect of ultraviolet light.
Arrhenius’s theory can not of course be disproved and we must agree
with him that it is consistent not only with the theories of cosmogony
but also with the seeming potential immortality of certain or of all
cells.
The alternative to Arrhenius’s theory is that living matter did
originate and still originates from non-living matter. If this idea is
correct it should one day be possible to discover synthetic enzymes
which are capable of forming molecules of their own kind from a simple
nutritive solution. With such synthetic enzymes as a starting point
the task might be undertaken of creating cells capable of growth and
cell division, at least in the apparently simple form in which these
phenomena occur in bacteria; viz., that after the mass has reached a
certain (still microscopic) size it divides into two cells and so on.
If Arrhenius is right that living matter has had no more beginning
than matter in general, this hope of making living matter artificially
appears at present as futile as the hope of making molecules out of
electrons.
The problem of making living matter artificially has been compared to
that of constructing a _perpetuum mobile_; this comparison is, however,
not correct. The idea of a _perpetuum mobile_ contradicts the first law
of thermodynamics, while the making of living matter may be impossible
though contradicting no natural law.
Pasteur’s proof that spontaneous generation does not occur in the
solutions used by him does not prove that a synthesis of living
from dead matter is impossible under any conditions. It is at least
not inconceivable that in an earlier period of the earth’s history
radio-activity, electrical discharges, and possibly also the action of
volcanoes might have furnished the combination of circumstances under
which living matter might have been formed. The staggering difficulties
in imagining such a possibility are not merely on the chemical
side--_e. g._, the production of proteins from CO₂, and N--but also
on the physical side if the necessity of a definite cell structure is
considered. We shall see in the sixth chapter that without a structure
in the egg to begin with, no formation of a complicated organism is
imaginable; and while a bacterium may have a simple structure, such a
structure as it possesses is as necessary for its existence as are its
enzymes.
Attempts have repeatedly been made to imitate the structures in the
cell and of living organisms by colloidal precipitates. It is needless
to point out that such precipitates are of importance only for the
study of the origin of structures in the living, but that they are
not otherwise an imitation of the living since they are lacking the
characteristic synthetic chemical processes.
CHAPTER III
THE CHEMICAL BASIS OF GENUS AND SPECIES
1. It is a truism that from an egg of a species an organism of this
species only and of no other will arise. It is also a truism that
the so-called protoplasm of an egg does not differ much from that
of eggs of other species when looked at through a microscope. The
question arises: What determines the species of the future organism?
Is it a structure or a specific chemical or groups of chemicals? In a
later chapter we shall show that the egg has a simple though definite
structure, but in this chapter we shall see that the egg must contain
specific substances and that these substances which determine the
“species” and specificity in general are in all probability proteins.
Since solutions of different proteins look alike under a microscope we
need not wonder that it is impossible to discriminate microscopically
between the protoplasm of different eggs.
The idea of definiteness and constancy of species, a matter of daily
observation in the case of man and higher animals in general, was
not so readily accepted in the case of the micro-organisms, which on
account of their minuteness and simplicity of structure are not so
easy to differentiate. There existed for a long time serious doubt
whether or not the simplest organisms, the bacteria, possessed a
definite “specificity” like the higher organisms, or whether they
were not endowed, as Warming put it, with an “unlimited plasticity,”
which forbade classifying them according to their form into definite
species as Cohn had done. An interesting episode in this discussion,
which was settled about twenty-five years ago arose concerning the
sulphur bacteria, which often develop in large masses on parts of
decaying plants or animals along the shore. Sir E. Ray Lankester
found collections of red bacteria covering putrefying animal matter
in a vessel and forming a continuous membrane along its wall. These
red bacteria were of very different shape, size, and grouping, but
they seemed to be connected by transition forms. They had a common
character, however, namely, their peach-coloured appearance. This
common character, together with their association in the same habitat,
led Lankester to the then justifiable belief that they all belonged to
one species which was protean in character and that the different forms
were only to be considered as phases of growth of this one species. The
presence of the same red pigment “_Bacterio-purpurin_” seemed justly
to indicate the existence of common chemical processes. Cohn, on the
contrary, considered the different forms among these red bacteria (they
are today called sulphur bacteria since they oxidize the hydrogen
sulphide produced by bacteria of putrefaction to sulphur and sulphates)
as definite and distinct species, in spite of their common colour and
their association. Later observations showed that Cohn was right.
Winogradsky[30] succeeded in proving by pure culture experiments that
each of these different forms of sulphur bacteria was specific and did
not give rise to any of the other forms of the same colour found in the
same conditions.
[30] Winogradsky, S., _Beiträge zur Morphologie und Physiologie der
Bacterien_. Leipzig, 1888.
The method of pure line breeding inaugurated by Johannsen[31] has shown
that the degree of definiteness goes so far that apparently identical
forms with only slight differences in size may breed true to this size;
but for reasons which will become clear later on we may doubt whether
they are to be considered as definite species.
[31] Johannsen, W., _Elemente der exacten Erblichkeitslehre_. 2d ed.,
1913.
The fact of specificity is supported by the fact of constancy of forms.
de Vries has pointed out that regardless of the possible origin of new
species by mutation the old species may persevere. Walcott has found
fossils of annelids, snails, crustaceans, and algæ in a precambrian
formation in British Columbia whose age (estimated on the rate of
formation of radium from uranium) may be about two hundred million
years and estimated on the basis of sedimentation sixty million years.
And yet these invertebrates are so closely related to the forms
existing today that the systematists have no difficulty in finding
the genus among the modern forms into which each of these organisms
belongs. W. M. Wheeler, in his investigations of the ants enclosed
in amber, was able to identify some of them with forms living today,
though the ants observed in the amber must have been two million years
old. The constancy of species, _i. e._, the permanence of specificity
may therefore be considered as established as far back as two or
possibly two hundred millions of years. The definiteness and constancy
of each species must be determined by something equally definite and
constant in the egg, since in the latter the species is already fixed
irrevocably.
We shall show first that species if sufficiently separated are
generally incompatible with each other and that any attempt at fusing
or mixing them by grafting or cross-fertilizing is futile. In the
second part of the chapter we shall take up the facts which seem
destined to give a direct answer to the question as to the cause of
specificity. It is needless to say that this latter question is of
paramount importance for the problem of evolution, as well as for that
of the constitution of living matter.
_I. The Incompatibility of Species not closely Related_
2. It is practically impossible to transplant organs or tissues from
one species of higher animals to another, unless the two species
are very closely related; and even then the transplantation is
uncertain and the graft may either fall off again or be destroyed.
This specificity of tissues goes so far that surgeons prefer, when a
transplantation of skin in the human is intended, to use skin of the
patient or of close blood relations. The reason why the tissues of a
foreign species in warm-blooded animals cannot grow well on a given
host has been explained by the remarkable experiments of James B.
Murphy of the Rockefeller Institute.[32] Murphy discovered that it is
possible to transplant successfully any kind of foreign tissue upon
the early embryo of the chick. Even human tissue transplanted upon the
chick embryo will grow rapidly. This shows that at this early stage
the chick embryo does not yet react against foreign tissue. This lack
of reaction lasts until about the twenty-first day in the life of the
embryo; then the growth of the graft not only ceases but the graft
itself falls off or is destroyed. Murphy noticed that this critical
period coincides with the development of the spleen and of lymphatic
tissue in the chick and that a certain type of migrating cells, the
so-called lymphocytes, which develop in the lymphatic tissue, gather
at the edge of the graft in great numbers, and he suggested that these
lymphocytes (by a secretion of some substance?) rid the host of the
graft. He applied two tests both of which confirmed this idea. First he
showed that when small fragments of the spleen of an adult chicken are
transplanted into the embryo the latter loses its tolerance for foreign
grafts. The second proof is still more interesting. It was known that
by treatment with Roentgen rays the lymphocytes in an animal could be
destroyed. It was to be expected that an animal so treated would have
lost its specific resistance to foreign tissues. Murphy found that this
was actually the case. On fully grown rats in which the lymphocytes
had been destroyed by X-rays (as ascertained by blood counts) tissues
of foreign species grew perfectly well. These experiments have assumed
a great practical importance since they can also be applied to the
immunization of an animal against transplanted cancer of its own
species. Murphy found that by increasing the number of lymphocytes in
an animal (which can be accomplished by a mild treatment with X-rays)
the immunity against foreign grafts as well as against cancer from the
same species can be increased. It is quite possible that the apparent
immunity to a transplantation of cancer produced by Jensen, Leo Loeb,
and Ehrlich and Apolant through the previous transplantation of tissue
in such an animal was due to the fact that this previous tissue
transplantation led to an increase in the number of lymphocytes in the
animal. The medical side, however, lies outside of our discussion, and
we must satisfy ourselves with only a passing notice. The facts show
that each warm-blooded animal seems to possess a specificity whereby
its lymphocytes destroy transplanted tissue taken from a foreign
species.
[32] Murphy, J. B., _Jour. Exper. Med._, 1913, xvii., 482; 1914, xix.,
181; xix., 513; Murphy and Morton, J. J., _Jour. Exper. Med._, 1915,
xxii., 204.
A lesser though still marked degree of incompatibility exists also in
lower animals for grafts from a different species.[33] The graft may
apparently take hold, but only for a few days, if the species are not
closely related. Joest apparently succeeded in making a permanent union
between the anterior and posterior ends of two species of earthworms,
_Lumbricus rubellus_ and _Allolobophora terrestris_. Born and later
Harrison healed pieces of tadpoles of different species together. An
individual made up of two species _Rana virescens_ and _Rana palustris_
lived a considerable time and went through metamorphosis. Each half
regained the characteristic features of the species to which it
belonged. It seems, however, that if species of tadpoles of two more
distant species are grafted upon each other no lasting graft can be
obtained, _e. g._, _Rana esculenta_ and _Bombinator igneus_. These
experiments were made at a time when the nature and bearing of the
problem of specificity was not yet fully recognized. The rôle of
lymphocytes in these cases has never been investigated. The grafted
piece always retained the characteristics of the species from which it
was taken.
[33] The reader is referred to Morgan’s book on _Regeneration_ (New
York, 1901), for the literature on this subject.
Plants possess no leucocytes and we therefore see that they tolerate
a graft of foreign tissues better than is the case in animals. As
a matter of fact heteroplastic grafting is a common practice in
horticulture, although even here it is known that indiscriminate
heteroplastic grafting is not feasible and that therefore the
specificity is not without influence. The host is supposed to furnish
only nutritive sap to the graft and in this respect does not behave
very differently from an artificial nutritive solution for the raising
of a plant. The law of specificity, however, remains true also for the
grafted tissues: neither in animals nor in plants does the graft lose
its specificity, and it never assumes the specific characters of the
host, or _vice versa_. The apparent exceptions which Winkler believed
he had found in the case of grafts of nightshade on tomatoes turned
out to be a further proof of the law of specificity. Winkler, after
the graft had taken, cut through the place of grafting, after which
operation a callus formation occurred on the wound. In most cases
either a pure nightshade or a pure tomato grew out from this callus. In
some cases he obtained shoots from the place where graft and host had
united, which on one side were tomato, on the other side nightshade.
What really happened was that the shoots had a growing point whose
cells on the one side consisted of cells of nightshade, on the other
side of tomato.[34] We know of no case in which the cell of a graft has
lost its specificity and undergone a transformation into the cell of
the host.
[34] Baur, E., _Einführung in die experimentelle Vererbungslehre_.
Berlin, 1911, p. 232.
3. Another manifestation of the incompatibility of distant species
is found in the domain of fertilization. The eggs of the majority of
animals cannot develop unless a spermatozoön enters. The entrance
of a spermatozoön into an egg seems also to fall under the law of
specificity, inasmuch as in general only the sperm of the same or
a closely related species is able to enter the egg. The writer[35]
has found, however, that it is possible to overcome the limitation
of specificity in certain cases by physicochemical means, and by the
knowledge of these means we may perhaps one day be able to more closely
define the mechanism of specificity in this case. He found that the
eggs of a certain Californian sea urchin, which cannot be fertilized by
the sperm of starfish in normal sea water, will lose their specificity
towards this type of foreign sperm if the sea water is rendered a
little more alkaline, or if a little more Ca is added to the sea water,
or if both these variations are effected. Godlewski has confirmed the
efficiency of this method for the fertilization of sea-urchin eggs with
the sperm of crinoids.
[35] Literature on this subject in Chapter IV.
[Illustration: FIG. 1. Five-days-old larvæ from a sea urchin
(Strongylocentrotus purpuratus) ♀ and a starfish (Asterias) ♂. (Front
view.)]
[Illustration: FIG. 2. Five-days-old larvæ of Strongylocentrotus
purpuratus produced by artificial parthenogenesis. (Side view.) The
larvæ in Figs. 1 and 2 are identical in appearance, proving that
heterogeneous hybridization leads to a larva with purely maternal
characters.]
If such heterogeneous hybridizations are carried out, two striking
results are obtained. The one is that the resulting larva has only
maternal characteristics (Figs. 1 and 2), as if the sperm had
contributed no hereditary material to the developing embryo. This
result could not have been predicted, for if we fertilize the egg of
the same Californian sea urchin, _Strongylocentrotus purpuratus_, with
the sperm of a very closely related sea urchin, _S. franciscanus_,
the hereditary effect of the spermatozoön is seen very distinctly in
the primitive skeleton formed by the larva.[36] (Fig. 3.) In the case
of the heterogeneous hybridization the spermatozoön acts practically
only as an activating agency upon the egg and not as a transmitter of
paternal qualities.
[36] Loeb, J., King, W. O. R., and Moore, A. R., _Arch. f.
Entwicklngsmech._, 1910, xxix., 354.
[Illustration: FIG. 3. Five-days-old larvæ of two closely related forms
of sea urchins (S. purpuratus ♀ and S. franciscanus ♂). In this case
the larva has also paternal characters as shown by the skeleton.]
The second striking fact is that while the sea-urchin eggs fertilized
with starfish sperm develop at first perfectly normally they begin to
die in large numbers on the second and third day of their development,
and only a very small number live long enough to form a skeleton; and
these are usually sickly and form the skeleton considerably later than
the pure breed. It is not quite certain whether the sickliness of these
heterogeneous hybrids begins or assumes a severe character with the
development of a certain type of wandering cells, the mesenchyme cells;
it would perhaps be worth while to investigate this possibility. The
writer was under the impression that this sickliness might have been
brought about by a poison gradually formed in the heterogeneous larvæ.
He investigated the effects of heterogeneous hybridization also in
fishes, which are a much more favourable object. The egg of the marine
fish _Fundulus heteroclitus_ can be fertilized with the sperm of almost
any other teleost fish, as Moenkhaus[37] first observed. This author
did not succeed in keeping the hybrids alive more than a day, but
the writer has kept many heterogeneous hybrids alive for a month or
longer,[38] and found the same two striking facts which he had already
observed in the heterogeneous cross between sea urchin and starfish:
first, practically no transmission of paternal characters, and second,
a sickly condition of the embryo which begins early and which increases
with further development. The heterogeneous fish hybrids between,
_e. g._, _Fundulus heteroclitus_ ♀ and _Menidia_ ♂ have usually no
circulation of blood, although the heart is formed and beats and
blood-vessels and blood cells are formed; the eyes are often incomplete
or abnormal though they may be normal at first; the growth of the
embryo is mostly retarded. In exceptional cases circulation may be
established and in these a normal embryo may result, but such an embryo
is chiefly maternal.
[37] Moenkhaus, W. J., _Am. Jour. Anat._, 1904, iii., 29.
[38] Loeb, J., _Jour. Morphol._, 1912, xxiii., 1.
This incompatibility of two gametes from different species does not
show itself in the case of heterogeneous hybridization only, but also
though less often in the case of crossing between two more closely
related forms. The cross between the two related forms _S. purpuratus_
♀ and _S. franciscanus_ ♂ is very sturdy and shows no abnormal
mortality as far as the writer’s observations go. If, however, the
reciprocal crossing is carried out, namely that of _S. franciscanus_
♀ and _S. purpuratus_ ♂, the development is at first normal, but
beginning with the time of mesenchyme formation the majority of larvæ
become sickly and die; and again the question may be raised whether
or not the beginning of sickliness coincides with the development of
mesenchyme cells. If we assume that the sickliness and death are due to
the formation of a poison, we must assume that the poison is formed by
the protoplasm of the egg, since otherwise we could not understand why
the reciprocal cross should be healthy.
All of these data agree in this one point, that the fusion by grafting
or fertilization of two distant species is impossible, although the
mechanism of the incompatibility is not yet understood. It is quite
possible that this mechanism is not the same in all the cases mentioned
here, and that it may be different when two different species are
mixed and when incompatibility exists between varieties, as is the case
in the graft on mammals.
II. _The Chemical Basis of Genus and Species and of Species Specificity_
4. Fifty or sixty years ago surgeons did not hesitate to transfuse the
blood of animals into human beings. The practice was a failure, and
Landois[39] showed by experiment that if blood of a foreign species
was introduced into an animal the blood corpuscles of the transfused
blood were rapidly dissolved and the animal into which the transfusion
was made was rendered ill and often died. The result was different
when the animals whose blood was used for the purpose of transfusion
belonged to the same species or a species closely related to the animal
into which the blood was transfused. Thus when blood was exchanged
between horse and donkey or between wolf and dog or between hare and
rabbit no hemoglobin appeared in the urine and the animal into which
the blood was transfused remained well.[40] This was the beginning
of the investigations in the field of serum specificity which were
destined to play such a prominent rôle in the development of medicine.
Friedenthal was able to show later that if to 10 c.c. of serum of a
mammal three drops of defibrinated blood of a foreign species are added
and the whole is exposed in a test tube to a temperature of 38°C. for
fifteen minutes the blood cells contained in the added blood are all
cytolyzed; that this, however, does not occur so rapidly when the blood
of a related species is used. He could thus show that human blood serum
dissolves the erythrocytes of the eel, the frog, pigeon, hen, horse,
cat, and even that of the lower monkeys but not that of the anthropoid
apes. The blood of the chimpanzee and of the human are no longer
incompatible, and this discovery was justly considered by Friedenthal
as a confirmation of the idea of the evolutionists that the anthropoid
apes and the human are blood relations.[41]
[39] Landois, L., _Zur Lehre von der Bluttransfusion_. Leipzig, 1875.
[40] This is probably true only within the limits of exactness used in
these experiments.
[41] Friedenthal, H., “Experimenteller Nachweis der
Blutverwandtschaft.” _Arch. f. Physiol._, 1900, 494.
This line of investigation had in the meanwhile entered upon a new
stage when Kraus, Tchistowitch, and Bordet discovered and developed
the precipitin reaction, which consists in the fact that if a foreign
serum (or a foreign protein) is introduced into an animal the blood
serum of the latter acquired after some time the power of causing a
precipitate when mixed with the antigen, _i. e._, with the foreign
substance originally introduced into the animal for the purpose of
causing the production of antibodies in the latter; while, of course,
no such precipitation occurs if the serum of a non-treated rabbit is
mixed with the serum of the blood of the foreign species.
In 1897 Kraus discovered that if the filtrates from cultures of
bacteria (_e. g._, typhoid bacillus) are mixed with the serum of an
animal immunized with the same serum (_e. g._, typhoid serum) it causes
a precipitate; and that this precipitin reaction is specific. This fact
was confirmed and has been extended by the work of many authors.
Tchistowitch in 1899 observed that the serum of rabbits which had
received injections of horse or eel serum caused a precipitate when
mixed with the serum of these latter animals.
Bordet found in 1899 that if milk is injected into a rabbit the serum
of such a rabbit acquires the power of precipitating casein, and Fish
found that this reaction is specific inasmuch as the lactoserum from
cow’s milk can precipitate only the casein of cow’s milk but not that
of human or goat milk. Wassermann and Schütze reached the same result
independently of each other.
Myers and later Uhlenhuth showed that if white of egg from a hen’s egg
is injected into a rabbit, precipitins for white of egg are found in
the serum of the latter, and Uhlenhuth[42] found, by trying the white
of egg of different species of birds, that the precipitin reaction
called forth by the blood of the immunized animal is specific, inasmuch
as the proteins from a hen’s egg will call forth the formation of
precipitins in the blood of the rabbit which will precipitate only the
white of egg of the hen or of closely related birds.
[42] Uhlenhuth, P., and Steffenhagen, K., Kolle-Wassermann, _Handb. d.
pathol. Mikroorg._, 2nd Ed., 1913, iii., 257.
To Nuttall[43] belongs the credit of having worked out a quantitative
method for measuring the amount of precipitate formed, and in this
way he made it possible to draw more valid conclusions concerning
the degree of specificity of the precipitin reaction. He found by
this method that when the immune serum is mixed with the serum or
the protein solution used for the immunization a maximum precipitate
is formed, but if it is mixed with the serum of related forms a
quantitatively smaller precipitate is produced. In this way the degree
of blood relationship could be ascertained. He thus was able to show
that when the blood of one species, _e. g._, the human, was injected
into the blood of a rabbit, after some time the serum of the rabbit
was able to cause a precipitate not only with the serum of man, or
chimpanzee, but also of some lower monkeys; with this difference,
however, that the precipitate was much heavier when the immune serum
was added to the serum of man. The method thus shows the existence
of not an absolute but of a strong quantitative specificity of blood
serum. This statement may be illustrated by the following table from
Nuttall. The antiserum used for the precipitin reaction was obtained by
treating a rabbit with human blood serum. The forty-five bloods tested
had been preserved for various lengths of time in the refrigerator with
the addition of a small amount of chloroform.
[43] Nuttall, George H. F., _Blood Immunity and Blood Relationship_,
Cambridge Univ. Press, 1904.
TABLE II
QUANTITATIVE TESTS WITH ANTI-PRIMATE SERA
_Tests with Antihuman Serum_
---------------------------+--------------+-------------------------
Blood of | _Precipitum_ | _Percentage_
| _Amount_ |
---------------------------+--------------+-------------------------
_Primates_ | |
Man | .031 | 100
Chimpanzee | .04 | 130 (loose precipitum)
Gorilla | .021 | 64
Ourang | .013 | 42
Cynocephalus mormon | .013 | 42
Cynocephalus sphinx | .009 | 29
Ateles geoffroyi | .009 | 29
| |
_Insectivora_ | |
Centetes ecaudatus | .0 | 0
| |
_Carnivora_ | |
Canis aureus | .003 | 10 (loose precipitum)
Canis familiaris | .001 | 3
Lutra vulgaris | .003 | 10 (concentrated serum)
Ursus tibetanus | .0025 | 8
Genetta tigrina | .001 | 3
Felis domesticus | .001 | 3
Felis caracal | .0008 | 3
Felis tigris | .0005 | 2
| |
_Ungulata_ | |
Ox | .003 | 10
Sheep | .003 | 10
Cobus unctuosus | .002 | 7
Cervus porcinus | .002 | 7
Rangifer tarandus | .002 | 7
Capra megaceros | .0005 | 2
Equus caballus | .0005 | 2
Sus scrofa | .0 | 0
| |
_Rodentia_ | |
Dasyprocta cristata | .002 | 7 (concentrated
| | serum clots)
Guinea-pig | .0 | 0
Rabbit | .0 | 0
| |
_Marsupialia_ | |
Petrogale xanthopus } | |
Petrogale penicillata } | |
Onychogale frenata } | |
Onychogale unguifera } | .0 | 0
Onychogale unguifera } | |
Macropus bennetti } | |
Thylacinus cynocephalus} | |
---------------------------+--------------+-------------------------
Among the Primate bloods that of the Chimpanzee gave too high a
figure, owing to the precipitum being flocculent and not settling
well, for some reason which could not be determined. The figure
given by the Ourang is somewhat too low, and the difference between
Cynocephalus sphinx and Ateles is not as marked as might have been
expected in view of the qualitative tests and the series following.
The possibilities of error must be taken into account in judging of
these figures; repeated tests should be made to obtain something
like a constant. Other bloods than those of Primates give small
reactions or no reactions at all. The high figures (10%) obtained
with two Carnivore bloods can be explained by the fact that one
gave a loose precipitum, and the other was a somewhat concentrated
serum.[44]
[44] Nuttall, _Blood Immunity and Blood Relationship_, pp. 319 and 320.
We have mentioned that even the proteins of the egg are specific
according to Uhlenhuth. Graham Smith, one of Nuttall’s collaborators,
applied the latter’s quantitative method to this problem and confirmed
the results of Nuttall. A few examples may serve as an illustration.
TABLE III
TESTS WITH ANTI-DUCK’S-EGG SERUM
--------------------------------+--------------+--------------
_Material tested_ | _Amount of | _Percentage_
| precipitum_ |
--------------------------------+--------------+--------------
Duck’s egg-albumin | .0384 | 100
Pheasant’s " | .0328 | 85
Fowl’s " | .0234 | 61
Silver Pheasant’s " | .0140 | 36
Blackbird’s " | .0065 | 15
Crane’s " | .0051 | 14
Moorhen’s " | .0046 | 12
Thrush’s " | .0046 | 12
Emu’s " | .0018 | 5
Hedge-Sparrow’s " | trace | ?
Chaffinch’s " | . | 0
Tortoise serum | trace | ?
Turtle serum | " | ?
Alligator serum | . | 0
--------------------------------+--------------+--------------
Frog, Amphiuma, and Dogfish sera, as well as Tortoise and Dogfish
egg-albumins, were also tested, with negative results.
TABLE IV
TESTS WITH ANTI-FOWL’S-EGG SERUM
--------------------------------+--------------+--------------
_Material tested_ | _Amount of | _Percentage_
| precipitum_ |
--------------------------------+--------------+--------------
Fowl’s egg-albumin (old) | .0159 | 100
Fowl’s " (fresh) | .0140 | 88
Silver Pheasant’s " | .0075 | 47
Pheasant’s " | .0075 | 47
Crane’s " | .0046 | 29
Blackbird’s " | .0046 | 29
Duck’s " | .0037 | 23
Moorhen’s " | .0028 | 18
--------------------------------+--------------+--------------
Thrush, Emu, Greenfinch, and Hedge-sparrow egg-albumins were tested
and gave traces of precipita, as also did Tortoise and Turtle sera.
The egg-albumins of the Tortoise, Frog, Skate, and two species of
Dogfish did not react. Alligator, Frog, Amphiuma, and Dogfish sera
also yielded no results.[45]
[45] Nuttall, pp. 345 and 346.
By improving the quantitative method in various ways, Welsh and
Chapman[46] were able to explain why the precipitin reaction
with egg-white was not strictly specific but gave also, though
quantitatively weaker, results with the egg-white of related birds.
They found that by a new method devised by them “it is possible
to indicate in an avian egg-white antiserum the presence of a
general avian antisubstance (precipitin) together with the specific
antisubstance.”
[46] Welsh, D. A., and Chapman, H. G., _Jour. Hygiene_, 1910, x., 177.
The Bordet reaction was not only useful in indicating the specificity
and blood relationship for animals but also among plants. Thus Magnus
and Friedenthal[47] were able to demonstrate with Bordet’s method the
relationship between yeast (_Saccharomyces cerevisiæ_) and truffle
(_Tuber brumale_).
[47] Magnus, W., and Friedenthal, H., _Ber. d. deutsch. bot.
Gesellsch._, 1906, xxiv., 601.
5. We must not forget, while under the spell of the problem of
immunity, that we are interested at the moment in the question of the
nature of the specificity of living organisms. It is only logical to
conclude that the fossil forms of invertebrate animals and of algæ
and bacteria, which Walcott found in the Cambrian and which may be two
hundred million years old, must have had the same specificity at that
time as they or their close relatives have today; and this raises the
question: What is the nature of the substances which are responsible
for and transmit this specificity? It is obvious that a definite answer
to this question brings us also to the very problem of evolution as
well as that of the constitution of living matter.
There can be no doubt that on the basis of our present knowledge
proteins are in most or practically all cases the bearers of this
specificity. This has been found out not only with the aid of the
precipitin reaction but also with the anaphylaxis reaction, by
which, as the reader may know, is meant that when a small dose of a
foreign substance is introduced into an animal a hypersensitiveness
develops after a number of days or weeks, so that a new injection of
the same substance produces serious and in some cases fatal effects.
This hypersensitiveness, which was first analysed by Richet,[48] is
specific for the substance which has been injected. Now all these
specific reactions, the precipitin reaction as well as the anaphylactic
reaction, can be called forth by proteins. Thus Richet, in his earliest
experiments, showed that only the protein-containing part of the
extract of actinians, by which he called forth anaphylaxis, was able
to produce this phenomenon, and later he showed that it was generally
impossible to produce anything resembling anaphylaxis by non-protein
substances, _e. g._, cocain or apomorphin.[49] Wells isolated from
egg-white four different proteins (three coagulable proteins and one
non-coagulable) which can be distinguished from each other by the
anaphylaxis reaction, although all come from the same biological
object.[50] Michaelis as well as Wells found that the split products of
the protein molecule are no longer able to call forth the anaphylaxis
reaction. Since peptic digestion has the effect of annihilating the
power of proteins to call forth anaphylaxis, we are forced to the
conclusion that the first cleavage products of proteins have already
lost the power of calling forth immunity reactions.
[48] Richet, C., _L’anaphylaxie_. Paris, 1912.
[49] Quoted from Wells, H. G., _Jour. Infect. Diseases_, 1908, v., 449.
[50] _Ibid._, 1911, ix., 147.
A pretty experiment by Gay and Robertson[51] should be mentioned in
this connection. Robertson had shown
that a substance closely resembling paranucleins both in its
properties and its C, H, and N content can be formed from the
filtered products of the complete peptic hydrolysis of an
approximately four per cent. neutral solution of potassium
caseinate by the action of pure pepsin at 36°C.
[51] Gay, F. P., and Robertson, T. B., _Jour. Biol. Chem._, 1912, xii.,
233.
He considered this a case of a real synthesis of proteins from
the products of its hydrolytic cleavage. This interpretation was
not generally accepted and received a different interpretation by
Bayliss and other workers. Gay and Robertson were able to show that
paranuclein when injected into an animal will sensitize guinea-pigs
for anaphylactic intoxication for either paranuclein or casein
and apparently indiscriminately. The products of complete peptic
digestion of casein had no such effect, but the synthetic product of
this digestion obtained by Robertson’s method has the same specific
antigenic properties as paranuclein, thus making it appear that
Robertson had indeed succeeded in causing a synthesis of paranuclein
with the aid of pepsin from the products of digestion of casein by
pepsin.
There are a few statements in the literature to the effect that
the specificity of organisms might be due to other substances than
proteins. Thus Bang and Forssmann claimed that the substances
(antigens) responsible for the production of hemolysis were of a lipoid
nature, but their statements have not been confirmed, and Fitzgerald
and Leathes[52] reached the conclusion that lipoids are non-antigenic.
Ford claims to have obtained proof that a glucoside contained in the
poisonous mushroom _Amanita phalloides_ can act as an antigen. But
aside from this one fact we know that proteins and only proteins can
act as antigens and are therefore the bearers of the specificity of
living organisms.
[52] Fitzgerald, J. G., and Leathes, J. B., _Univ. Cal. Pub._, 1912,
“Pathology,” ii., 39.
Bradley and Sansum[53] found that guinea-pigs sensitized to beef or dog
hemoglobin fail to react or react but slightly to hemoglobin of other
origin. The hemoglobins tried were dog, beef, cat, rabbit, rat, turtle,
pig, horse, calf, goat, sheep, pigeon, chicken, and man.
[53] Bradley, H. C., and Sansum, W. D., _Jour. Biol. Chem._, 1914,
xviii., 497.
6. It would be of the greatest importance to show directly that the
homologous proteins of different species are different. This has been
done for hemoglobins of the blood by Reichert and Brown,[54] who have
shown by crystallographic measurements that the hemoglobins of any
species are definite substances for that species.
The crystals obtained from different species of a genus are
characteristic of that species, but differ from those of other
species of the genus in angles or axial ratio, in optical
characters, and especially in those characters comprised under the
general term of crystal habit, so that one species can usually be
distinguished from another by its hemoglobin crystals. But these
differences are not such as to preclude the crystals from all
species of a genus being placed in an isomorphous series (p. 327).
[54] Reichert, E. T., and Brown, A. P., “The Differentiation and
Specificity of Corresponding Proteins and other Vital Substances in
Relation to Biological Classification and Organic Evolution.” Carnegie
Institution Publication No. 116, Washington, 1909.
As far as the genus is concerned it was found that the hemoglobin
crystals of any genus are isomorphous.
In some cases this isomorphism may be extended to include several
genera, but this is not usually the case, unless as in the case of
dogs and foxes, for example, the genera are very closely related.
The most important question for us is the following: Are the
differences between the corresponding hemoglobin crystals of different
species of the same genus such as to warrant the statement that they
indicate chemical differences? If this were the case we might say
that blood reactions as well as hemoglobin crystals indicate that
differences in the constitution of proteins determine the species
specificity and, perhaps, also species heredity. The following
sentences by Reichert and Brown seem to indicate that this may be true
for the crystals of hemoglobin.
The hemoglobins of any species are definite substances for
that species. But upon comparing the corresponding substances
(hemoglobins) in different species of a genus it is generally
found that they differ the one from the other to a greater or
less degree; the differences being such that when complete
crystallographic data are available the different species can be
distinguished by these differences in their hemoglobins. As the
hemoglobins crystallize in isomorphous series the differences
between the angles of the crystals of the species of a genus are
not, as a rule, great; but they are as great as is usually found
to be the case with minerals or chemical salts that belong to an
isomorphous group (p. 326).
As Professor Brown writes me, the difficulty in answering the question
definitely, whether or not the hemoglobins of different species
are chemically different, lies in the fact that there is as yet no
criterion which allows us to discriminate between a species and a
Mendelian mutation except the morphological differences. It is not
impossible that while species differ by the constitution of some or
most of their proteins, Mendelian heredity has a different chemical
basis.
It is regrettable that work like that of Reichert and Brown cannot be
extended to other proteins, but it seems from anaphylaxis reactions
that we might expect results similar to those in the case of the
hemoglobins. The proteins of the lens are an exception inasmuch as,
according to Uhlenhuth, the proteins of the lens of mammals, birds, and
amphibians cannot be discriminated from each other by the precipitin
reaction.[55]
[55] Uhlenhuth, _Das biologische Verfahren zur Erkennung und
Unterscheidung von Menschen und Tierblut_, Jena, 1905, p. 102.
7. The serum of certain humans may cause the destruction or
agglutination of blood corpuscles of certain other humans. This fact
of the existence of “isoagglutinins” seems to have been established
for man, but Hektoen states that he has not been able to find any
isoagglutinins in the serum of rabbits, guinea-pigs, dogs, horses,
and cattle. Landsteiner found the remarkable fact that the sera of
certain individuals of humans could hemolyze the corpuscles of certain
other individuals, but not those of all individuals. A systematic
investigation of this variability led him to the discovery of three
distinct groups of individuals, the sera of each group acting in a
definite way towards the corpuscles of the representatives of each
other group. Later observers, for example Jansky and Moss, established
four groups. These groups are, according to Moss,[56] as follows:
Group 1. Sera agglutinate no corpuscles.
Corpuscles agglutinated by sera of Groups 2, 3, 4.
Group 2. Sera agglutinate corpuscles of Groups 1, 3.
Corpuscles agglutinated by sera of Groups 3, 4.
Group 3. Sera agglutinate corpuscles of Groups 1, 2.
Corpuscles agglutinated by sera of Groups 2, 4.
Group 4. Sera agglutinate corpuscles of Groups 1, 2, 3.
Corpuscles agglutinated by no serum.
[56] Moss, W. L., _Johns Hopkins Hospital Bulletin_, 1910, xxi., 62.
The relative frequency of the four groups follows from the following
figures. Of one hundred bloods tested by Moss in series of twenty there
were found:
10 belonging to Group 1.
40 belonging to Group 2.
7 belonging to Group 3.
43 belonging to Group 4.
Groups 2 and 4 are in the majority and in overwhelming numbers, which
indicates that, as a rule, the sera agglutinate the blood corpuscles
of individuals of the other groups, but not those of individuals
belonging to the same group. The phenomenon that a serum agglutinates
no corpuscles (Group 1), or that the corpuscles are agglutinated by
no serum (Group 4), are the exceptions. It is obvious that, as far as
our problem is concerned, only Groups 2 and 3 are to be considered.
There is no Mendelian character which refers only to one half of the
individuals except sex. Since nothing is said about a relation of
Groups 2 and 3 to sex such a relation probably does not exist.
8. The facts thus far reported imply the suggestion that the heredity
of the genus is determined by proteins of a definite constitution
differing from the proteins of other genera. This constitution of the
proteins would therefore be responsible for the genus heredity. The
different species of a genus have all the same genus proteins, but the
proteins of each species of the same genus are apparently different
again in chemical constitution and hence may give rise to the specific
biological or immunity reactions.
We may consider it as established by the work of McClung, Sutton, E.
B. Wilson, Miss Stevens, Morgan, and many others, that the chromosomes
are the carriers of the Mendelian characters. These chromosomes occur
in the nucleus of the egg and in the head of the sperm. Now the latter
consists, in certain fish, of lipoids and a combination of nucleinic
acid and protamine or histone, the latter a non-coagulable protein,
more resembling a split product of one of the larger coagulable
proteins.
A. E. Taylor[57] found that if the spermatozoa of the salmon are
injected into a rabbit, the blood of the animal acquires the power of
causing cytolysis of salmon spermatozoa. When, however, the isolated
protamines or nucleinic acid or the lipoids prepared from the same
sperm were injected into a rabbit no results of this kind were
observed. H. G. Wells more recently tested the relative efficiency of
the constituents of the testes of the cod (which in addition to the
constituents of the sperm contained the proteins of the testicle).
From the testicle he prepared a histone (the protein body of the
sperm nucleus), a sodium nucleinate, and from the sperm-free aqueous
extract of the testicles a protein resembling albumin was separated by
precipitation.[58]
The albumin behaved like ordinary serum albumin or egg albumin,
producing typical and fatal anaphylactic reactions and being
specific when tried against mammalian sera. The nucleinate did not
produce any reactions when guinea-pigs were given small sensitizing
and larger intoxicating doses (0.1 gm.) in a three weeks’
interval; a result to be expected, since no protein is present in
the preparation. The histone was so toxic that its anaphylactic
properties could not be studied.
[57] Taylor, A. E., _Jour. Biol. Chem._, 1908, v., 311.
[58] Wells, H. G., _Jour. Infect. Diseases_, 1911, ix., 166.
It is not impossible that protamines and histones might be found to act
as specific antigens if they were not so toxic. The positive results
which Taylor observed after injection of the sperm might have been due
to the proteins contained in the tail of the spermatozoa, which in
certain animals at least does not enter the egg and hence can have no
influence on heredity.
It is thus doubtful whether or not any of the constituents of the
nucleus contribute to the determination of the species. This in its
ultimate consequences might lead to the idea that the Mendelian
characters which are equally transmitted by egg and spermatozoön,
determine the individual or variety heredity, but not the genus or
species heredity. It is, in our present state of knowledge, impossible
to cause a spermatozoön to develop into an embryo,[59] while we can
induce the egg to develop into an embryo without a spermatozoön. This
may mean that the protoplasm of the egg is the future embryo, while the
chromosomes of both egg and sperm nuclei furnish only the individual
characters.
[59] Loeb, J., and Bancroft, F. W., _Jour. Exper. Zoöl._, 1912, xii.,
381.
CHAPTER IV
SPECIFICITY IN FERTILIZATION
1. We have become acquainted with two characteristics of living
matter: the specificity due to the specific proteins characteristic
for each genus and possibly species and the synthesis of living matter
from the split products of their main constituents instead of from
a supersaturated solution of their own substance, as is the case in
crystals. We are about to discuss in this and the next chapter a third
characteristic, namely, the phenomenon of fertilization. While this
is not found in all organisms it is found in an overwhelming majority
and especially the higher organisms, and of all the mysteries of
animated nature that of fertilization and sex seems to be the most
captivating, to judge from the space it occupies in folklore, theology,
and “literature.” Bacteria, when furnished the proper nutritive medium,
will synthetize the specific material of their own body, will grow
and divide, and this process will be repeated indefinitely as long
as the food lasts and the temperature and other outside conditions
are normal. It is purely due to limitation of food that bacteria
or certain species of them do not cover the whole planet. But, as
every layman knows, the majority of organisms grow only to a certain
size, then die, and the propagation takes place through sex cells or
gametes: a female cell--the egg--containing a large bulk of protoplasm
(the future embryo) and reserve material; and the male cell which in
the case of the spermatozoön contains only nuclear material and no
cytoplasmic material except that contained in the tail which in some
and possibly many species does not enter the egg. The male element--the
spermatozoön--enters the female gamete--the egg--and this starts the
development. In the case of most animals the egg cannot develop unless
the spermatozoön enters. The question arises: How does the spermatozoön
activate the egg? And also how does it happen that the spermatozoön
enters the egg? We will first consider the latter question. These
problems can be answered best from experiments on forms in which the
egg and the sperm are fertilized in sea water. Many marine animals,
from fishes down to lower forms, shed their eggs and sperm into the sea
water where the fertilization of the egg takes place, outside the body
of the female.
The first phenomenon which strikes us in this connection is again a
phenomenon of specificity. The spermatozoön can, as a rule, only enter
an egg of the same or a closely related species, but not that of one
more distantly related. What is the character of this specificity?
The writer was under the impression that a clue might be obtained if
artificial means could be found by which the egg of one species might
be fertilized with a distant species for which this egg is naturally
immune. Such an experiment would mean that the lack of specificity
had been compensated by the artificial means. It is well known that
the egg of the sea urchin cannot as a rule be fertilized with the
sperm of a starfish in normal sea water. The writer tried whether this
hybridization could not be accomplished provided the constitution of
the sea water were changed. He succeeded in causing the fertilization
of a large percentage of the eggs of the Californian sea urchin,
_Strongylocentrotus purpuratus_, with the sperm of various starfish
(_e. g._, _Asterias ochracea_) and _Holothurians_ by slightly raising
the alkalinity of the sea water, through the addition of some base
(NaOH or tetraethylammoniumhydroxide or various amines), the optimum
being reached when 0.6 c.c. N/10 NaOH is added to 50 c.c. of sea water.
It is a peculiar fact that this solution is efficient only if both egg
and sperm are together in the hyperalkaline sea water. If the eggs
and sperm are treated separately with the hyperalkaline sea water and
are then brought together in normal sea water no fertilization takes
place as a rule, while with the same sperm and eggs the fertilization
is successful again if both are mixed in the hyperalkaline solution.
From this the writer concluded that the fertilizing power depends on
a rapidly reversible action of the alkali on the surface of the two
gametes. It was found that an increase of the concentration of calcium
in the sea water also favoured the entrance of the _Asterias_ sperm
into the egg of _purpuratus_; and that if C_{Ca} was increased it was
not necessary to add as much NaOH.
The spermatozoön enters the egg through the so-called fertilization
cone, _i. e._, a protoplasmic process comparable to the pseudopodium of
an amœboid cell. The analogy of the process of phagocytosis--_i. e._,
the taking up of particles by an amœboid cell--and that of the
engulfing of the spermatozoön by the egg presents itself. We do not
know definitely the nature of the forces which act in the case of
phagocytosis--although surface tension forces and agglutination have
been suggested; both are surface phenomena and are rapidly reversible.
We should then say that the specificity in the process of fertilization
consists in a peculiarity of the surface of the egg and spermatozoön
which in the case of _S. purpuratus_ ♀ and _Asterias_ ♂ can be supplied
by a slight increase in the C_{OH} or C_{Ca}.
By this method fifty per cent. or more of the eggs of _purpuratus_
could be fertilized with the sperm of the starfish _Asterias ochracea,
capitata_, Ophiurians, and Holothurians, while with the sperm of
another starfish, _Pycnopodia spuria_, only five per cent., and with
the sperm of _Asterina_ only one per cent. could be fertilized.[60]
Godlewski succeeded by the same method in fertilizing the eggs of a
Naples starfish with the sperm of a crinoid.[61] The writer did not
succeed in bringing about the fertilization of the egg of another sea
urchin in California, _Strongylocentrotus franciscanus_, with the sperm
of a starfish. Although these eggs formed a membrane in contact with
the sperm, the latter did not enter the egg; nor has the writer as
yet succeeded in causing the sperm of _Asterias_ to enter the egg of
_Arbacia_.
[60] Loeb, J., _Arch. f. d. ges. Physiol._, 1903, xcix., 323; 1904,
civ., 325; _Arch. f. Entwcklngsmech._, 1910, xxx., II., 44; 1914, xl.,
310; _Science_, 1914, xl., 316.
[61] Godlewski, E., _Arch. f. Entwcklngsmech._, 1906, xx., 579.
Kupelwieser[62] observed that the spermatozoön of molluscs may
occasionally enter into the egg of _S. purpuratus_ in normal sea
water and later, at Naples, he observed the same for the sperm of
annelids. In these cases no development took place. In teleost fishes
the spermatozoön can enter the eggs of widely different species but
with rare exceptions all the embryos will die in an early stage of
development.[63]
[62] Kupelwieser, H., _Arch. f. Entwcklngsmech._, 1909, xxvii., 434;
_Arch. f. Zellforsch._, 1912, viii., 352.
[63] See Chapter II.
2. The fact that an increase in the alkalinity or in the concentration
of calcium allowed foreign sperm to enter the egg of the sea urchin,
suggested the idea that a diminution of alkalinity or calcium in the
sea water might block the entrance of the sperm of sea urchin into
eggs of their own species. This was found to be correct; when we put
eggs and sperm of the same species of sea urchin into solutions whose
concentration of Ca or of OH is too small, the sperm, although it may
be intensely active, cannot enter the egg.
For the purpose of these experiments the ovaries and testes of the
sea urchins were not put into sea water, but instead into pure m/2
NaCl and after several washings in this solution were kept in it
(they remain alive for several days in pure m/2 NaCl). Several drops
of such sperm and one drop of eggs were in one series of experiments
put into 2.5 c.c. of a neutral mixture of m/2 NaCl and 3/8 m MgCl₂
in the proportion in which these two salts exist in the sea water.
In such a neutral solution eggs of _Arbacia_ or _purpuratus_ are
not fertilized no matter how long they remain in it, although the
spermatozoa swim around the eggs very actively. That no spermatozoön
enters the eggs can be shown by the fact that the eggs do not divide
(although they can segment in such a solution if previously fertilized
in sea water or some other efficient solution). When, however, eggs
and sperm are put into 2.5 c.c. of the same solution of NaCl+MgCl₂,
containing in addition one drop of a N/100 solution of NaOH (or NH₃ or
benzylamine or butylamine) or eight drops of m/100 NaHCO₃, most, and
often practically all of the eggs at once form fertilization membranes
and segment at the proper time, indicating that fertilization has
been accomplished. The same result can be obtained if the eggs are
transferred into a neutral mixture of NaCl+MgCl₂+CaCl₂ (in the
proportion in which these salts exist in the sea water) or into a
neutral mixture of NaCl+MgCl₂+KCl+CaCl₂. In such neutral mixtures the
eggs form fertilization membranes and begin to segment. The eggs are
not fertilized in a neutral solution of NaCl or of NaCl+KCl.[64]
[64] Loeb, J., _Science_, 1914, xl., 316; _Am. Naturalist_, 1915,
xlix., 257.
It is, therefore, obvious that if we diminish the alkalinity of the
solution surrounding the egg and deprive this solution of CaCl₂ we
establish the same block to the entrance of the spermatozoön of
_Arbacia_ into the egg of the same species as exists in normal sea
water for the entrance of the sperm of the starfish into the egg of
_purpuratus_.
The “block” created in this way, to the entrance of the sperm of
_Arbacia_ into the egg of the same species is also rapidly reversible.
We reach the conclusion, therefore, that the specificity which allows
the sperm to enter an egg is a surface effect which can be increased
or diminished by an increase or diminution in the concentration of
OH as well as of Ca. The writer has shown that an increase in the
concentration of both substances may cause an agglutination of the
spermatozoa of starfish to the jelly which surrounds the egg of
_purpuratus_.[65] It is thus not impossible that the specificity which
favours the entrance of a spermatozoön into an egg of its own species
may consist in an agglutination between spermatozoön and egg protoplasm
(or its fertilization cone); and that this agglutination is favoured if
the C_{OH} or C_{Ca} or both are increased within certain limits.
[65] Loeb, _Arch. f. Entwcklngsmech._, 1914, xl., 310.
Godlewski discovered a very interesting form of block to the entrance
of the spermatozoön into the egg which takes place if two different
types of sperm are mixed. He had found that the sperm of the annelid
_Chætopterus_ is able to enter the egg of the sea urchin and that in so
doing it causes membrane formation. The egg, however, does not develop
but dies rapidly, as is the case when we induce artificial membrane
formation, as we shall see in the next chapter.
Godlewski found that if the sperm of _Chætopterus_ and the sperm of
sea urchins are mixed the mixture is not able to induce development or
membrane formation, since now neither spermatozoön can enter; blood
has the same inhibiting effect as the foreign sperm. The mixture does
not interfere with the development of the eggs if they are previously
fertilized.[66]
[66] Godlewski, E., _Arch. f. Entwcklngsmech._, 1911, xxxiii., 196.
The phenomenon was further investigated by Herlant[67] who found that
if the sperm of a sea urchin is mixed with the sperm of certain
annelids (_Chætopterus_) or molluscs, and if after some time the eggs
of the sea urchin are added to the mixture of the two kinds of sperm no
egg is fertilized. If, however, the solution is subsequently diluted
with sea water or if the egg that was in this mixture is washed in sea
water, the same sperm mixture in which the egg previously remained
unfertilized will now fertilize the egg. From these and similar
observations Herlant draws the conclusion that the block existed at the
surface of the egg, inasmuch as a reaction product of the two types of
sperm is formed after some time which alters the surface of the egg and
thereby prevents the sperm from entering. This view is supported not
only by all the experiments but also by the observation of the writer
that foreign sperm or blood is able to cause a real agglutination after
some time if mixed with the sperm of a sea urchin or a starfish.[68]
We can imagine that the precipitate forms a film around the egg and
acts as a block for the agglutination between egg and spermatozoön. The
block can be removed mechanically by washing.
[67] Herlant, M., _Anat. Anzeiger_, 1912, xlii., 563.
[68] Loeb, J., _Jour. Exper. Zoöl._, 1914, xvii., 123.
3. The fact has been mentioned that the most motile sperm will not be
able to enter into the egg if certain other conditions (specificity
or C_{OH} or C_{Ca}) are not fulfilled. On the other hand, living but
immobile sperm cannot enter the egg under any conditions. If we add a
trace of KCN to the sperm of _Arbacia_ so that the spermatozoön becomes
immobile no egg is fertilized even if the eggs and the sperm are
thoroughly shaken together; while the same spermatozoa will fertilize
these eggs as soon as the HCN has evaporated and they again become
motile. It was formerly thought that the spermatozoön had to bore
itself into the egg, being propelled by the movements of the flagellum.
It is, however, more probable that only a certain energy of vibration
is needed on the part of the spermatozoön to make the latter stick
to the surface of the egg and agglutinate and that later forces of a
different character bring the spermatozoön into the egg. The fact that
under normal conditions a very slight degree of motility on the part of
the spermatozoön allows it to enter the egg of its own species seems to
favour such a view.
It is a common experience that spermatozoa become very active when they
reach the neighbourhood of an egg. v. Dungern assumed that only foreign
sperm became thus active, but F. R. Lillie[69] has pointed out that
this may be a specific effect. The writer tested this idea on the sperm
and eggs of two species of starfish and of sea urchins. It should be
mentioned that the eggs of the starfish used in this experiment were
completely immature and could not be fertilized, while the eggs of the
sea urchins were mature. The testicles and ovaries had been kept in
NaCl and all the sperm was immotile. Eggs and sperm were mixed together
in a pure m/2 NaCl solution where the sperm was only rendered motile by
the proximity of eggs. The following table gives the result.[70]
[69] Lillie, F. R., _Jour. Exper. Zoöl._, 1914, xvi., 523.
[70] Loeb, J., _Am. Naturalist_, 1915, xlix., 257.
TABLE V
SPECIFICITY OF ACTIVATION OF SPERM BY EGGS
--------------+------------+-------------+---------------+-------------
|_Asterias_♂ | _Asterina_♂ |_Franciscanus_♂|_Purpuratus_♂
| | | |
--------------+------------+-------------+---------------+-------------
_Asterias_ |_Immediately|No |Moderately |Slight effect
♀ (immature) | very | activation. | active. | in immediate
| motile._ | | | contact with
| | | | egg.
| | | |
_Asterina_ | Not motile.|_Violent |_Violent |Slight effect
♀ (immature) | | activity._ | activity._ | only near
| | | | the egg.
| | | |
_Franciscanus_|Slightly |No motility. |_Immediately |_Immediately
♀ (mature) | motile. | | active._ | active._
| | | |
| | | |
_Purpuratus_ |Slightly |Slight effect|_Immediately |_Immediately
♀ (mature) | motile | in immediate| active._ | active._
| after some | contact | |
| time. | with eggs. | |
--------------+------------+-------------+---------------+-------------
The spermatozoa of starfish show a marked specificity inasmuch as they
are strongly activated only by the eggs of their own species, although
in this experiment these were immature, and to a slight degree only
by the eggs of the sea urchin _purpuratus_. But it is also obvious
that the specificity is far from exclusive since the immature eggs
of _Asterina_ activate the sperm of the sea urchin _franciscanus_
as powerfully as is done by the mature eggs of the sea urchin
_purpuratus_ and _franciscanus_. In studying these results the reader
must keep in mind first that all these experiments were made in a NaCl
solution and second that it requires a stronger influence to activate
the spermatozoa of the starfish, which are not motile at first even in
sea water, than the sea urchin spermatozoa which are from the first
very active in such sea water, and which may therefore be considered as
being at the threshold of activity in pure NaCl solution.
Wasteneys and the writer (in experiments not yet published) did not
succeed in demonstrating an activating effect of the eggs of various
marine teleosts upon sperm of the same species.
4. F. R. Lillie[71] has studied the very striking phenomenon of
transitory sperm agglutination which takes place when the sperm of a
sea urchin or of certain annelids is put into the supernatant sea water
of eggs of the same species. If we put one or more drops of a very
thick sperm suspension of the Californian sea urchin _S. purpuratus_
carefully into the centre of a dish containing 3 c.c. of ordinary
sea water and let the drop stand for one-half to one minute and then
by gentle agitation mix the sperm with the sea water the mass of
thick sperm which is at first rather viscous is distributed equally
in sea water in a few seconds and the result is a homogeneous sperm
suspension. When, however, the same experiment is made with the sea
water which has been standing for a short time over a large mass of
eggs of the same species, the thick drop of sperm seems to be less
miscible and instead of a homogeneous suspension we get, as a result,
the formation of a large number of distinct clusters which are visible
to the naked eye and which may possess a diameter of 1 or 2 mm. The
rest of the sea water is almost free from sperm. These clusters of
spermatozoa may last for from two to ten minutes and then dissolve by
the gradual detachment of the spermatozoa from the periphery of the
cluster.
[71] Lillie, F. R., _Science_, 1913, xxxviii., 524; _Jour. Exper.
Zoöl._, 1914, xvi., 523; _Biol. Bull._, 1915, xxviii., 18.
This phenomenon seems to occur in sea urchins and annelids. The writer
has vainly looked for it in different forms of the Californian starfish
or molluscs and in fish at Woods Hole. Lillie failed to find it in the
starfish at Woods Hole.
The writer found that the sperm of the Californian sea urchin
_Strongylocentrotus purpuratus_ will form clusters with the egg sea
water of _purpuratus_ but not with that of _franciscanus_; while the
sperm of _franciscanus_ will agglutinate with the egg sea water of both
species, but the clusters last a little longer with the eggs of its own
species.
He also found that the clusters are more durable in a neutral than in
a slightly alkaline solution and that the agglutination disappears the
more rapidly the more alkaline the solution. The presence of bivalent
cations, especially Ca, also favours the agglutination.
It was also found that this agglutination occurs only when the
spermatozoa are very motile; thus if a trace of KCN is added to a mass
of thick sea-urchin sperm so that the spermatozoa become immotile a
drop of this sperm will not agglutinate when put in egg sea water of
the same species; while later, after the HCN has evaporated, the same
sperm will agglutinate when put into such sea water.
The writer suggests the following explanation of the phenomenon. The
egg sea water contains a substance which forms a precipitate with a
substance on the surface of the spermatozoön whereby the latter becomes
slightly sticky. This precipitate is slowly soluble in sea water and
the more rapidly the more alkaline (within certain limits). Only when
the spermatozoa run against each other with a certain impact will
they stick together, as Lillie suggested. Lillie assumes that this
agglutinating substance contained in egg sea water is required to
bring about fertilization and he therefore calls it “fertilizin.”[72]
But this assumption seems to go beyond the facts inasmuch as the
existence of such an agglutinating substance can only be proved in a
few species of animals (sea urchins and annelids); and as, moreover,
sea-urchin sperm can fertilize eggs which will not cause the sperm to
agglutinate, _e. g._, the egg of _franciscanus_ can be fertilized by
sperm of _purpuratus_, although the egg sea water of _franciscanus_
causes no agglutination of the sperm of _purpuratus_. When the jelly
surrounding the egg of the Californian sea urchin _S. purpuratus_ is
dissolved with acid and the eggs are washed, the eggs will not cause
any more sperm agglutination; and yet one hundred per cent. of such
eggs can be fertilized by sperm.[73]
[72] Lillie, F. R., _loc. cit._
[73] Loeb, J., _Jour. Exper. Zoöl_., 1914, xvii., 123; _Am.
Naturalist_, 1915, xlix., 257.
5. It is well known that if an egg is once fertilized it becomes
impermeable for other spermatozoa. This cannot well be due to the fact
that the egg develops; for the writer found some time ago that eggs of
_Strongylocentrotus purpuratus_ which are induced to develop by means
of artificial parthenogenesis can be fertilized by sperm. The following
observation leaves no doubts in this respect. When the unfertilized
eggs of _purpuratus_ are put for two hours into hypertonic sea water
(50 c.c. of sea water+8 c.c. 2-1/2 m NaCl) and then transferred into
sea water it occasionally happens that a certain percentage of the eggs
will begin to divide into 2, 4, 8 or more cells, without developing
any further. When to such eggs after they have remained in the resting
stage for a number of hours or a day, sperm is added, some or all
of the blastomeres form a fertilization membrane and now begin to
develop into larvæ; and if the spermatozoön gets into a blastomere of
the 2- or 4-cell stage normal plutei will result. When the sperm is
added while the eggs are in active parthenogenetic cell division the
individual blastomeres into which a spermatozoön enters will also form
a fertilization membrane, but such blastomeres perish very rapidly. It
is not yet possible to state why it should make such a difference for
the possibility of development whether the spermatozoön enters into
a blastomere when at rest or when it is in active nuclear division,
although the idea presents itself that in the latter case an abnormal
mix-up and separation of chromosomes and other constituents may be
responsible for the fatal result. Whatever may be the explanation
of this phenomenon it proves to us that it is not the process of
development in itself which acts as a block to the entrance of a
spermatozoön into an egg which is already fertilized.[74]
[74] Loeb, J., _Arch. f. Entwcklngsmech._, 1907, xxii., 479;
_Artificial Parthenogenesis and Fertilization_, Chicago, 1913, p. 240.
When the spermatozoön enters the egg of the sea urchin it calls forth
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 cell
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