The Organism as a Whole, from a Physicochemical Viewpoint

8. The history of the egg shows a reversible condition of rest and of activity. The primordial egg cell multiplies actively until a large number of eggs are formed in the ovary which may reach into the millions in the case of sea urchins or certain annelids. These cell divisions then stop and the egg goes into the resting stage in which it deposits the reserve material for the development of the embryo. From this condition it can only be called into activity again by the spermatozoon or the agencies of artificial parthenogenesis.

It seemed of interest to find out whether or not the development of the egg may be reversed once more after it has been activated. From all that has been said in the chapter on artificial parthenogenesis, such a reversal should take place in the cortical layer. The result of these experiments seems to be that if a complete destruction or change in the cortical layer has once taken place — such as that caused by the entrance of a spermatozoon into the egg — no reversal is possible; although the development of the fertilized egg may be suppressed for a long time by either low temperature or lack of oxygen, or, in the case of seeds and spores, by lack of water. But as soon as the conditions for the chemical reactions in the egg are normal again, the development may go on unless the egg has suffered by the methods used to prevent development or by the long duration of the suppression. With an incomplete destruction of the cortical layer both development as well as reversal of development are possible. Thus the writer has shown that in the egg of Arbacia the effect of the cortical alteration of the egg induced by the butyric acid treatment or by the treatment with bases can be reversed. When unfertilized eggs of Arbacia are put for from two to five minutes into 50 c.c. sea water + 2.0 c.c. N/io butyric acid they will all form a gelatinous, somewhat atypical fertilization membrane; when put back into normal sea water all will perish in a few hours unless they are submitted to the short treatment with a hypertonic solution mentioned in the previous chapter, while if submitted to this treatment they will develop. If, however, these eggs are transferred from the butyric acid sea water not into normal sea water but into sea water containing some NaCN (10 drops of n, per cent. NaCN or KCN in 50 c.c. sea water), and if they remain here for some time (e. g. overnight) they will not perish

when subsequently transferred back to normal sea water. Such eggs will develop when fertilized with sperm. The activating effect of the membrane formation has, therefore, been reversed and the eggs have gone back into the resting stage.1 Wasteneys has found that the rate of oxidation which was raised considerably by the characteristic for the resting eggs after the reversal of their developmental tendency.2 Similar results were obtained in eggs activated with NH4OH. It appears from this as though the change in the cortical layer which leads to the development of the egg and the increase in the rate of oxidations were reversible in the egg of Arbacia.3

The writer had previously noticed that eggs of Strongylocentrotus purpuratus, which had been treated for two hours with hypertonic sea water, not infrequently began to divide into two, four, or eight cells (and sometimes more) and then went back into the resting state (except that they possessed the second factor required for development as stated in Chapter V). It may be 3 F. Lillie thinks that the KCN in this experiment merely inhibits the change of the cortical layer necessary for development. This is contradicted by two facts: first, the writer has shown in 1906 that KCN does not inhibit the membrane formation, and, second, the eggs will not return to the resting stage when put back into sea water too soon; in that case they will disintegrate. This shows that in the KCN something more happens than the mere block to disintegration.

remarked incidentally that such eggs at the time of cell division contained the centrosomes and astrospheres, and yet went back into a resting state, thus showing that the centrosomes are only transitory organs or organs which are only active under certain conditions. It is quite possible that in these phenomena of reversal not the whole of the cortical layer has undergone alteration. The writer must leave it undecided whether the changes from the resting to the active state in body cells can also be explained in analogy with these experiments.

9. In the formation of the lens we have already noticed an instance where the adjacent organ influences growth inasmuch as the optic cup controlled the formation of the lens. Such influences are quite commonly observed. A piece of Tubularia when cut out from a stem and suspended in water will regenerate at the aboral pole not a stolon but a polyp, so that we have an animal terminating at both ends of its body in a head. The writer called such cases in which an organ is replaced by an organ of a different kind heteromorphosis.

Contact with a solid body favours the formation of stolons. Fig. 36 shows a piece of a stem of Pennaria another hydroid, which was lying on the bottom of an aquarium and which formed stolons at both ends a and b. In Margelis, another hydroid, the writer observed that without any operation the apical ends of branches which were in contact with solid bodies continued to grow as stolons, while those surrounded by sea water continued to grow as stems.

Herbst discovered a very interesting form of heteromorphosis in certain crustaceans; namely, that in the place of an eye which was cut off, an entirely different organ could be formed, namely, an antenna. Pie showed that the experimenter has it in his power to determine whether the crustacean shall regenerate an eye or an antenna in place of the eye. The latter will take place when the optic ganglion is removed with the eye, the former when it is not removed. These experiments were carried out successfully on Palamon, Palamonetes, Sicyonia, Palinurus, and other crustaceans.

in stems of Bryophyllum placed horizontally the roots usually come out from the lower end of the callus. Such phenomena are not often found in animals but they exist here too as the following observation shows. If we cut a piece a b (Fig. 37), from the stem ss oi Antennularia antennina (Fig. 38), a hydroid, and put it into the water in a horizontal position, new stems cd (Fig. 37) may arise on its upper side. The small branches on the under side of the old stem a b begin suddenly to grow vertically downward.1 In appearance and function these downward-growing elements are entirely different from the branches of the normal Antennularia; they are roots. In order to understand better the transformation which thus occurs in these branches, it may be stated that under normal conditions they have

1 Loeb, J.f Untersuchungen zur physiologischen Morphologic def Tiere. II. Organbildung und Wachsthum. Wurzburg, 1892. a limited growth (see Fig. 38), are directed upward, and have polyps on their upper side. The parts which grow down (Fig. 37) have no polyps, but attach themselves like true roots to solid bodies. Thus the changed position of the stem alone, without any operation, suffices to transform the lateral branches, whose growth is limited, into roots with unlimited growth. The lateral branches on the upper side of the stem do not undergo such a transformation into roots except in the immediate surroundings of the place where a new stem arises. It seems that the formation of a new stem also causes an excessive growth of roots, possibly because the formation of new branches causes the removal of substances which naturally inhibit the formation of roots. If a piece from the stem be put vertically into the water with top downward, the uppermost point may continue to grow as a stem, while the lowest point may give rise to roots. In this case, therefore, a change in the orientation of organs has the effect of changing the character of organs.

There are only two ways by which we can account for these influences of gravitation. Either certain substances flow to the lowest level and collecting there induce growth and possibly changes in the character of growth (as in Antennularia) or if the cells have elements of different specific gravity the relative position of these elements may possibly change and influence in this way the conditions for growth. The influence of gravitation as well as of contact upon life phenomena are at present little understood.

/ In all these cases of heteromorphosis the original : form is not restored. It is needless to say that they are ^ incompatible with the theory of natural selection. The reader will have noticed that in this chapter one term has not been mentioned which is commonly met with in the literature, namely the "wound stimulus." As the writer had indicated in a former publication,1 the word "stimulus" is generally used to disguise our ignorance of (and also our lack of interest in) the causes which underlie the phenomena which we investigate. Regeneration very often does not take place near the wound but at some distance from it. But even when the regeneration takes place at the edge of the wound the latter only serves to create conditions for regeneration, and these conditions cannot be expressed by the word "stimulus."

1 Loeb, J., Die chemische Entwicklungserregung des lierischen Eies. Berlin, 1909. regeneration is incomplete in a great many details it seems that the known facts warrant the statement that the phenomena of regeneration belong as much to the domain of determinism as those of any of the partial phenomena of physiology. I. It is a general fact that both sexes appear in approximately equal numbers, provided a sufficiently large number of cases are examined. This fact has furnished the clue for the discovery of the mechanism which determines the relative number of the two sexes. The honour of having pointed the way to the solution of the problem belongs to McClung.1 It has been known that certain insects, e. g., Kemiptera and Orthoptera, possess two kinds of spermatozoa but only one kind of eggs. The two kinds of spermatozoa differ in regard to a single chromosome, which is either lacking or different in one-half of the spermatozoa.

The first one to recognize the existence of two kinds of spermatozoa was Henking, who stated that in Pyrrhocoris (a Hemipteran) one-half of the spermatozoa of each male possessed a nucleolus, while in the other half it was lacking. Montgomery afterward showed that Henking's nucleolus was an accessory chromosome. McClung was the first to recognize the importance of this fact for the problem of sex determination. He observed an accessory chromosome in one-half of the spermatozoa of two forms of Orthoptera, Brachystola and Hippiscus, and reached the following conclusion:

A most significant fact, and one upon which almost all investigators are united in opinion, is that the element is apportioned to but one-half of the spermatozoa. Assuming it to be true that the chromatin is the important part of the cell in the matter of heredity, then it follows that we have two kinds of spermatozoa that differ from each other in a vital matter. We expect, therefore, to find in the offspring two sorts of individuals in approximately equal numbers, under normal conditions, that exhibit marked differences in structure. A careful consideration will suggest that nothing but sexual characters thus divides the members of a species into two well-defined groups, and we are logically forced to the conclusion that the peculiar chromosome has some bearing upon the arrangement.

N. M. Stevens and E. B. Wilson1 have not only proved the correctness of this idea for a number of animals but have laid the foundation of our present knowledge of the subject. Wilson shewed that in those cases where there are two types of spermatozoa, one with and one without an accessory or as it is now called an X chromosome, all the cells of the female have one chromosome more than the cells of the male. From this he concludes correctly that in such species a female is produced when the egg is fertilized by a spermatozoon containing an X chromosome, while a male is produced when a spermatozoon without an X chromosome enters the egg.

Such a form is Protenor, one of the Hemiptera. Wilson made sure that all the eggs are alike in the number of chromosomes, each egg containing an X chromosome in addition to the six chromosomes characteristic of the species Protenor. There are two types of spermatozoa in equal numbers in this species, each with six chromosomes, but one with, the other without, an X chromosome. The two possible chromosome combinations between egg and spermatozoa are therefore as follows (see the diagrammatic Fig. 39) :

The egg which receives a spermatozoon without an X chromosome has after fertilization 12 + X chromosomes and develops into a male; while the egg into which a spermatozoon with an X chromosome enters gives rise to a female. Since all the body cells arise from the fertilized egg by nuclear division and the chromosomes remain constant in number in all cells, the consequence is that all the cells of a female Protenor have two X chromosomes; while all the cells of a male Protenor have only one X chromosome.

The chromosome situation in Protenor is a somewhat extreme case, inasmuch as one X chromosome is entirely lacking in the male. In other forms of Hemiptera, e.g., Lygaus, there are also two types of spermatozoa appearing in equal numbers differing in regard to the X chromosome, but here it is only a difference in size ; one-half of the spermatozoa having a large X chromosome, the other half instead a smaller chromosome. Calling this latter the Y chromosome, the sex determination in this form is as follows: leaving aside the chromosomes which are equal in both egg and spermatozoon we may say that there is one type of egg containing one large X chromosome; there are two types of spermatozoa in equal numbers, one possessing a large X chromosome, the other possessing a small Y chromosome. Wilson showed by a study of the chromosomes in males and females that when one of the spermatozoa containing a large X chromosome enters the egg, the egg will develop into a female; while when one of the spermatozoa containing a small Y chromosome enters it will give rise to a male. Leaving aside the common chromosomes of both sexes, a fertilized egg containing XX gives rise to a female, while one containing XY gives rise to a male. There is in this case as in that of Protenor a preponderance of chromosome material in the female, but this quantitative difference is not essential for the determination of sex, since in some species the Y chromosome may be as large as the X chromosome.

The main fact is that the female cells have the chromatin composition XX, the male cells the composition XY, where Y is apparently qualitatively different and often, but not necessarily, smaller than X, or entirely lacking. It may be mentioned in passing that indirect evidence exists indicating that in man there are also two kinds of spermatozoa and one kind of egg, and that sex depends on whether a male determining or a female determining spermatozoon enters the egg.

2. This mode of sex determination holds only for those animals in which there is one type of egg and two types of spermatozoa. Experimental evidence furnished first by Doncaster in 1908 on a moth, Abraxas, indicated that a number of other forms exists in which matters are reversed, inasmuch as there are two types of eggs and one type of spermatozoa. This condition of affairs exists not only in the moth Abraxas, but also in the fowl as shown by Pearl. In these forms it is assumed that all the spermatozoa have one sex chromosome X, while there are two types of eggs, one possessing the sex chromosome X, the other possessing Y. When a spermatozoon enters an egg with an X chromosome, the egg will give rise to a male, while if it enters a Y egg, a female will arise. The evidence pointing toward this result is chiefly contained in experiments on sexlimited or more correctly sex-linked heredity; i.e., a form of heredity which follows the sex in a peculiar way. Thus colour-blindness is a case of sex-linked inheritance, since this abnormality appears overwhelmingly in the male offspring of a colour-blind person. Doncaster crossed two varieties of Abraxas differing in one character which was sex-linked, and the

results of his crossings indicated that in this form there are two types of eggs and one type of spermatozoa.1 These observations on sex-linked heredity confirm the idea that the sex chromosomes determine the sex. The most extensive and conclusive experiments along this line are those by Morgan on the fruit fly Drosophila. In this form there are two kinds of spermatozoa and one kind of eggs ; the egg has one X chromosome, while one-half of the spermatozoa has an X the other a Y chromosome ; the entrance of the latter into an egg gives rise to a male, of the former to a female.

While the eyes of the wild fruit fly Drosophila ampelophila are red, Morgan2 noticed in one of his cultures a male that had white eyes. This white-eyed male was mated to a red-eyed female. The offspring, the Fz generation, were all red eyed, males as well as females. These were inbred and now gave in the F3 generation the following three types of offspring : The character white eye was therefore transmitted only to half the grandsons; it was a sex-linked character. It is known from a study of the pedigrees of colour-blind individuals that if the corresponding ex-

periment had been carried out with them, instead of with white-eyed flies, the same proportions of normal and colour-blind would have been found: namely, normal colour vision in the Ft generation, in both males and females, and half of the males of the F3 generation colour-blind, the other half and all the females with normal vision. Of course, in man, intermarriage between two different Ft strains would have been required in place of the inbreeding of the Fj generation, which took place in Morgan's experiments. Morgan interprets his experiments as follows. The normal redeyed Drosophila has one kind of eggs, each possessing one X chromosome. This X chromosome has also the factor for the development of red-eye pigment. The white-eyed male has two kinds of spermatozoa, one with an X chromosome, the other with a Y chromosome, both lacking the factor for red-eye pigment. If we designate the X chromosome with the factor for redeye pigment by X and the X and Y chromosomes lacking the factor for redness with X and Y the following combinations must result if we cross a normal redeyed female with a white-eyed male:

It is obvious that all the offspring of the first generation (the Fx generation) must be red eyed, since all the eggs have one X chromosome with the factor for red. According to the results obtained from cytological studies which will be explained in the next chapter, the females with the chromatin constitution XX will form two types of eggs in equal numbers: namely, eggs with an X and eggs with an X, i. e., all eggs have one X chromosome, but in fifty per cent, of the eggs the X has the factor for red, in fifty per cent, this factor is lacking (X). The males having the chromosome constitution XY form two types of spermatozoa, one with an X possessing the factor for red pigment and one, the Y chromosomes, lacking this factor. If inbred the next F2 generation will give rise to the following four types of offspring: (i) XX, (2) XX, (3) XY, (4) XY, all four types in equal numbers.

(i) and (2) give females, both red eyed, since both contain a red-factored X chromosome. (3) and (4) give males, (3) giving rise to red-eyed males, since it contains a red-factored X chromosome, (4) producing males with white eyes since this X chromosome is lacking the factor for red eyes. Since all four combinations must appear in equal numbers (provided the experimental material is ample enough, which was the case in these experiments), in the Fz generation both males and females should have red eyes and in the F2 generation all the females should have red eyes and half of the males should have red, half white eyes. These results were obtained.

The experiments were carried further. No white-eyed females had appeared thus far. On the same assumptions of the relation of the X, X, and Y chromosomes to the heredity of sex as well as to eye colour it was possible to predict under what conditions and in which proportions white-eyed females should arise. Thus if a red-eyed female of the F x generation (a cross between white-eyed male and normal female) be mated with a white-eyed male the result should be an equal number of white-eyed males and white-eyed females if the chromosome theory of sex determination were correct. The reasoning would be as follows :

The red-eyed female, having the chromosome constitution XX should form two kinds of eggs in equal numbers with the constitution X and X; the whiteeyed male having the chromosome constitution XY should form two kinds of spermatozoa X and Y. The following four types of individuals must then be produced in equal numbers: In this case (2) must give rise to white-eyed females and (4) to white-eyed males, while (i) must give rise to red-eyed females and (3) to red-eyed males. Hence white-eyed males and females and red-eyed males and females are to be expected in this case in equal numbers, and this was actually observed.

experiments between the expected and observed result cannot well be an accident. The fact that the inheritance of sex-linked characters in man follows the same laws as in Drosophila is a strong argument in favour of the assumption that in man, also, sex is determined by two kinds of spermatozoa. Morgan and his students discovered no less than thirty-six sex-linked characters in Drosophila, and each behaved in a similar way to the red and white eye colour in regard to sex-linked inheritance, so that the chromosome theory of sex determination rests on a safe basis. That sex is merely determined by the number of X chromosomes, not by the Y chromosome, is proved by the facts that the Y chromosome may be completely absent as in Protenor and that Bridges1 has found a type of female Drosophila with a chromosome formula XXY whose sex was not affected by the supernumerary Y.

3. On the basis of all these experiments and theories it is comparatively easy to explain a number of phenomena concerning sex ratios which before had been very puzzling. In bees it had been shown many years ago by Dzierzon that the males develop from unfertilized eggs while the females, queens and workers, develop from fertilized eggs. This is intelligible on the assumption that the unfertilized egg contains only one X chromosome while the spermatozoon carries into the