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 CCa 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 COH or CCa.
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.
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]
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]
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 COH or CCa or both are increased within certain limits.
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]
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.
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 COH or CCa) 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]
TABLE V
Specificity of Activation of Sperm by Eggs
| Asterias♂ | Asterina♂ | Franciscanus♂ | Purpuratus♂ | |
|---|---|---|---|---|
| Asterias♀ (immature) | Immediately very motile. | No activation. | Moderately active. | Slight effect in immediate contact with egg. |
| Asterina♀ (immature) | Not motile. | Violent activity. | Violent activity. | Slight effect only near the egg. |
| Franciscanus♀ (mature) | Slightly motile. | No motility. | Immediately active. | Immediately active. |
| Purpuratus♀ (mature) | Slightly motile after some time. | Slight effect in immediate contact with eggs. | Immediately active. | Immediately active. |
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.
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]
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. 21⁄2 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]
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.
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]
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.
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]
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
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]
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.
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.