Gametes and Spores
Ideas About Sexual Reproduction 1750-1914
John Farley, 1982
This chapter is reproduced here through the generous permissions of the author and The Johns Hopkins University Press, Baltimore, MD.
Experimentation and statistical research may well be indispensable, Edmund Wilson noted in 1909, "but it is well to remember that the sex problem was first attacked by such methods, and that they long gave inconclusive or wholly misleading results." On the contrary, he reminded his readers, 'the most fruitful suggestions for its solution were first given by morphological studies. . . [and] the newer experimental work is bringing complete demostration to these suggestions.'" Wilson was referring, of course, to the experimental work of Boveri, Morgan, and himself; he was not alluding to Jacques Loeb's experiments, which called into question the whole morphological approach to the problems of sexual reproduction.
Those who looked for physical and chemical explanations of sexual reproduction did not accept the answers provided by the cytologists. Somewhat eclipsed by the cytological discoveries of the late nineteenth century, they reemerged at the turn of the century to seek once more a true physicochemical explanation of the events surrounding fertilization. Such an outlook, for example, permeated a series of articles entitled "Biological Problems of Today," which appeared in the 1898 volume of Science. Among the contributors to this series were Jacques Loeb and Thomas Hunt Morgan.
Jacques Loeb was motivated by the desire to account for all living phenomena in physicochemical terms. Physical chemistry-in particular, stereochemistry, van't Hoff's theory of osmotic pressure, and the theory of electrolytic dissociation-he argued in his 1898 paper, all provide tools with which to effect an understanding of the constituents of living matter. (2) Born in 1859, Loeb had studied at the Universities of Berlin and Strasbourg before moving to the University of Wurzburg in 1886 to become Adolph Fick's assistant. Fick had been a student of Carl Ludwig, who in 1847 had joined forces with Hermann von Helmholtz, Emil du Bois-Reymond, and Ernst Brucke to forrn the Physical Society. These four had "imagined that we should constitute physiology on a chemico-physical foundation, and give it equal rank with Physics." They took as their premise von Helmholtz's statement that "physiologists must expect to meet with an unconditional conformity to the law of the forces of nature in their inquiries respecting the vital processes; they will have to apply themselves to the investigation of the physical and chemical processes going on within the organism." (3)
In the late 1880s Loeb joined the ranks of the developmental mechanists and made the acquaintance of the American pioneers in the field. Feeling that his career would be seriously jeopardized in Germany because he was a Jew and a socialist, he emigrated to the United States and took up a position at Bryn Mawr in 1891. Thomas Hunt Morgan was appointed to Bryn Mawr that same year, bringing with him a classical training in descriptive embryology under William Brooks at Johns Hopkins University. In 1894 Morgan spent ten months at the Naples Station, met Hans Driesch, and returned to Bryn Mawr committed to the new experimental, analytical embryology.
During the 1890s these embryologists discovered that when placed in various salt solutions, unfertilized sea urchin eggs actually began to develop. Thomas Hunt Morgan described such events in 1899, but he refrained from taking a totally mechanistic view of the process. To reveal that physicochemical stimuli trigger an egg to divide, he argued, "is not in itself a mechanical explanation of the principal changes that take place."
To speak of a mechanical explanation of development, when we mean only a study of the responses to stimuli from without, gives an exaggerated and erroneous idea of the entire problem. It is the vital structure of the egg on which the result largely depends, for if one kind of stimulus is as capable as another of starting the development of the egg, then we have accomplished very little in way of explanation if we have only determined what these stimuli may be. (4)
Morgan was conscious that a complete explanation involved both an understanding of fertilization per se and an analysis of development and inheritance.
No such qualms were felt by Loeb, however. Totally committed to the belief that physical chemistry provided the answer to all biological riddles, he announced in 1899 that "former researchers [have] led me to suspect that changes in the state of matter (liquefactions and solidifications) might play an important role in the mechanics of life phenomena." Arguing that some constituents of normal sea water may prevent parthenogenesis, he set out to show that "by making two changes in the constitution of sea water the eggs of the sea urchin might be able to produce perfect embryos without being fertilized." He reported that unfertilized eggs left in a mixture of magnesium chloride and sea water for two hours before being returned to normal sea water formed normal larvae. This mixture, he concluded dramatically, "was able to bring about the same effect as the entrance of a spermatozoon." (5)
Loeb's conclusions were sweeping and totally countered Boveri's morphological theory. The unfertilized egg lacked nothing; all the"essential elements" for complete development were present in it. Parthenogenesis was prevented under normal circumstances only by the unfavorable constituents of sea water (or blood in the case of mammals). The sperm, whose role in fertilization had only recently been resurrected after two centuries of neglect, was once again relegated to a minor function. "All the spermatozoon needs to carry into the egg for the process of fertilization are ions to supplement the lack of one or counteract the effects of the other class of ions in the sea water or both. " As Loeb triumphantly exclaimed, ions, not nuclei, were essential to fertilization, "which may interest those who believe with me that physiologists ought to pay a little more attention to inorganic chemistry. " (6) "I consider the chief value of the experiments on artificial parthenogenesis," he concluded in a lecture given at Woods Hole in 1899, "to be the fact that they transfer the problem of fertilization from the realm of morphology into the realm of physical chemistry." (7)
The following year, having realized that the effect of the magnesium chloride could equally well be attributed to the higher osmotic pressure of the medium rather than to the individual ions per se, Loeb found that similar osmotic concentrations of sodium and potassium chloride and even urea produced the same effect on the eggs as did magnesium chloride. Substituting a theory of "osmotic parthenogenesis" for his earlier ionic theory, Loeb wrote: "The development of the unfertilized egg is produced through an increase in the concentration of the surrounding solution," and "there can be no more doubt that the essential feature in this increase in the osmotic pressure of the surrounding solution is a loss of water on the part of the egg." (8) Then, moving quickly from artificial fertilization to normal fertilization, Loeb claimed that an entering sperm was hypertonic to the egg (contained a higher concentration of ions than the egg), and thus water osmosed from the egg into the sperm, thereby stimulating the egg to develop. Once again he argued: "There is certainly no reason left for defining the process of fertilization as a morphological process."(9) Expanding his work shortly thereafter to include work on annelid eggs, Loeb found that these eggs, too, could be induced to develop, both by "osmotic fertilization"—increasing the osmotic concentration of the surrounding fluids—and by "chemical fertilization"—changing the concentration of individual ions without changing the total ionic concentration. (10)
Loeb's findings were greeted with rapturous praise in the popular press. The December 13, 1902, issue of Harper's Weekly carried a front-page picture of Loeb with the caption, "Americans of Tomorrow: Jacques Loeb, " and described him as "one of the three or four greatest biologists living. " Articles in Cosmopolitan and the Fortnightly Review even linked his experiments to the creation of life in the laboratory! "It was near to a realization of the dreams of Berthelot and Claude Bernard, aye, of every chemist who ever bordered the mysteries of life, the manufacture of life in the laboratory. In some ways, it was the most vital discovery in the history of physiology." (11)
By this time, however, Loeb was theorizing that the role of sperm in fertilization was more fundamental than merely acting as carriers of ions and hypertonic solutions. In his annelid paper in 1901 he suggested that sperm carry into the egg a "catalytic substance" which accelerates a process that would begin of its own accord, "but much more slowly." (12) This interpretation was far more "biological" than any offered previously; it implied that sperm manufactured and carried specific enzymes into the egg. Similar hypotheses had been proposed earlier in France. J.-B. Piri had argued that a liquid extract from echinoderm sperm contained a soluble ferment, ovulase, which upon diffusion into the egg "has the property of bringing about segmentation of ovules." In 1900 Raphael Dubois argued that fermentation involved the reciprocal activity of "spermase" from the sperm and "ovulose" from the egg, but that because the former was incapable of direct diffusion into the egg, it needed the mechanical activity of the sperm in order to gain entrance. That, Dubois concluded, was the "raison d'etre" of the spermatozoa. (13) A year later, however, William Gies, a student of Loeb's at Woods Hole, tested these enzyme theories and found them wanting. Studying liquid extracts from the sperm of Arbacia in which care had been taken to minimize osmotic differences, he found that they did not cause the eggs to divide. (14) Despite his attacks on morphological theories, Loeb now admitted that the sperm were needed to transmit hereditary qualities. "We must in future consider," he wrote, "the possible or probable separation of the fertilizing qualities of the spermatozoa from the transmission of hereditary qualities through the same." (15) This, of course, was a restatement of Newport's and Meissner's earlier views. It was also the opinion of Victor Hensen, who in 1881 had argued that fertilization involves both a physicochemical impetus and a morphological "moment." (16) In his 1902 review of the whole process of fecundation, Yves Delage took the same approach.
Born in 1854, Delage was by this time professor of zoology at the Sorbonne. He, like Loeb, had investigated artificial parthenogenesis and merogony (the artificial development of nonnucleated egg fragments containing sperm nuclei). Fecundation, he argued, had a double purpose: to put a ripe egg into a state capable of developing (embryogenesis), and to introduce male hereditary material into the egg (amphimixis). Studies on fecundation and artificial parthenogenesis, he noted, had shown that "the morphological phenomena of fecundation-in particular nuclear copulation-are essentially related to amphimixis, and that embryogenesis depends on concomitant physicochemical phenomena." (17) Such phenomena, he pointed out, "include the removal of cytoplasmic water by the male pronucleus, which absorbs it, dehydrates the cytoplasm, and thus communicates to the egg the capacity to divide" (18)
Thus, the explanation of fertilization proposed by the physiologists was at odds with that offered by the cytologists. Both groups agreed that the curious mobile spermatozoon, discovered over two centuries before, had two basic functions: to transmit through its nucleus and chromosomes the inherited male qualities and to initiate egg development. However, whereas the cytologists believed that egg development was initiated by the introduction of a missing centromere, the physiologists believed that the initiation of development involved some simple chemical reaction-a reaction so simple, in fact, that changing the osmotic pressure or ionic concentration could initiate egg development in the absence of spermatozoa.
Oscar Hertwig completely misunderstood these explanations of the role of sperm. Physicochemical accounts of fertilization, he protested in a passionate rebuke of Loeb's work, do not increase our biological understanding. "Fertilization is an amphimixis," he exclaimed, "a mixing or fusing of the properties of the two parental generators. How can the properties of the father be transmitted to the egg through osmosis or through ions or through catalytic substances? In [the] face of this fact, how can one speak of osmotic or chemical fertilization? Fertilization is a biological process which, at this time, one cannot expect to be resolved into a chemical-physical process by the concepts and experimental methods of chemists and physicists."(19)
It quickly became apparent, however, that Loeb's simple explanations were inadequate. Eggs induced to develop by these means did so abnormally and, unlike eggs fertilized by sperm, failed to produce a fertilization membrane. (In normal fertilization the outer membranes of the egg become separated from the protoplasmic contents by a clear space.) In addresses to theInternational Zoological Congress and the International Medical Congress in 1907 and 1909, respectively, Loeb reported that a fertilization membrane could be induced by treating sea urchin eggs with a monobasic fatty acid, and that normal development would occur if these eggs were subsequently placed in hypertonic sea water. "We see," he reported, "that the formative stimulus in the artificial activation of the egg of the sea urchin consists of two phases,. . . first the artificial causation of the membrane formation and, second, the subsequent short treatment of the egg with a hypertonic solution." (20) Membrane formation, he continued, could be caused by any cytolytic agent, and "from this we draw the inference that the membrane formation depends upon the cytolysis of the surface layer of the egg," which, being dissolved, gives the appearance of a membrane becoming separated from the protoplasmic layers beneath. (21)
According to Loeb, the spermatozoa contained two essential chemical components that together activated the egg to develop normally. One, a cytolytic chemical, dissolved away the superficial layers of egg protoplasm, while the other acted like hypertonic sea water. Further experiments convinced Loeb that the spermatozoon "causes the development of the egg through two agencies; one of these agencies is a cytolytic substance, a so called lysin," which is situated at the surface of the sperm; the other, situated in the interior of the sperm, acts in a corrective manner to prevent general lysis. (22) Thus, sperm of the same species as the egg will cause the egg to develop normally because both substances are introduced into the egg. Sperm of a foreign species, however, will cause the egg to disintegrate because only the surface lysin is introduced. This hypothesis enabled Loeb to account for egg-sperm specificity, a problem that his earlier "osmotic" and "chemical" theories had failed to explain. What is more, Loeb's hypothesis suggested that the egg-sperm interaction might be related to the wider phenomenon of immunity .
In the early years of the twentieth century, scientists believed that blood contained lysins that destroyed the blood cells of foreign species but not those of the same species. Living cells, including egg cells, were thus thought to be immune to the Iysins produced by cells of the same species. In fact, Frank Lillie later realized that fertilization was an example of an immune reaction and thus could be explained by principles derived from immunology. As he put it, "No theory of fertilization which fails to include the factor of specificity as one of the prime elements can be true." (23) Loeb, however, simply used the egg-sperm interaction as a model to explain why tissue cells were immune to the lysins produced by their own blood cells. His interests lay elsewhere. His goal, as expressed in an address to the First International Congress of Monists in Hamburg, was to answer the basic question "whether our present knowledge gives us any hope that ultimately life, i.e., the sum of all life phenomena, can be unequivocally explained in physico-chemical terms." Because he viewed fertilization in these terms, he argued that the process of activation of the egg by the spermatozoon, which twelve years ago was shrouded in complete darkness, is today practically completely reduced to a physico-chemical explanation.... Individual life begins ... with the acceleration of the rate of oxidation in the egg, and this acceleration begins after the destruction of its cortical layer. (24)
In 1910 Lillie began his investigations of fertilization by observing the interaction between egg and sperm in Nereis, a polychaete annelid. This eariy work was clearly influenced by Loeb's findings (in 1900 both joined the University of Chicago faculty: Loeb as a full professor, Lillie as an assistant professor), and thus it was not surprising that Lillie came to much the same conclusion as Loeb had over the role of the sperm. The sperm, Lillie noted, functioned both to transfer paternal qualities to the egg and to stimulate egg development. Lillie's observations on Nereis also suggested that two phases were involved in the stimulation process. The first phase involved complex changes in the exterior layers of the egg by means of which the head of the sperm was drawn into the egg. The second phase, a long, continuous process extending to the time of nuclear fusion, involved, Lillie suggested, the creation of "a free oxidation in the interior of the egg. " Lillie's work showed that, contrary to the usual opinion, the sperm did not enter into the egg by simple mechanical boring. Instead, "the presence of the sperm calls forth a reaction on the part of the egg that leads to the absorption of the former. " By centrifuging the egg at various stages of the fertilization process, Lillie also showed that the failure to segment was due either to the failure of the sperm to penetrate or to the destruction of the sperm nucleus within the egg. However, since in the latter situation the nuclear material of the sperm was still present within the egg (although "existing only as so much chemical matter"), the fertilizing power of the sperm must be "in some way bound up with its organization and growth." Lillie may have essentially agreed with Loeb that "individual life begins with the acceleration of the rate of oxidation in the egg, and this acceleration begins after the destruction of its cortical layer," but he also believed that these processes required the presence of a completely formed spermatozoon. (25)
Even at this stage of his work, Lillie's approach differed from Loeb's. In the preface to his famous monograph on fertilization, which was published in 1919, Lillie noted that past work on the problem had revealed "the inevitable conflict between the strictly biological and the physico-chemical methods of analysis." (26) Describing his own work as a reconciliation of the two approaches, he urged physiologists to use the tools and methods of physics and chemistry without losing sight of the inherent biological unity of organization which characterizes living things. Fertilization was a physiological process, and thus could never be understood without reference to the sperm. On the other hand, any explanation of fertilization had also to account for the fact that eggs could sometimes develop in the absence of sperm. Lillie had clearly expressed these views in 1911, at the end of his first paper on the subject:
"The experiments on artificial parthenogenesis are sometimes regarded as involving the entire problem of fertilization. But if it be true, as many believe, that biological fertilization is fundamentally a sexual reaction, then the physico-chemical analysis of fertilization must compass the entire problem of sex, which is much wider than the problem of parthenogenesis.... From the zoological point of view, at least, parthenogenesis and fertilization are not interchangeable functions. There is a factor present in fertilization which is absent in parthenogenesis, and the latter is never the exclusive mode of reproduction among animals. The biological analysis of fertilization therefore involves problems that do not occur in the physico-chemical analysis of parthenogenesis." (27)
Lillie argued that Loeb had failed to explain the role of sperm in fertilization. As he pointed out in a 1913 lecture to the Zoology Club of the University of Chicago, the current vogue of attacking the problem through artificial parthenogenesis had turned up so many factors that were capable of invoking development that one had to view with suspicion any theory that attempted to account for these phenomena. (28) Lillie was convinced that any meaningful theory of fertilization must take into account the role of the sperm-something which Loeb's work on artificial parthenogenesis clearly had not done. "There are obviously fundamental problems of fertilization," he wrote, "that cannot be touched by methods of artificial parthenogenesis." (29)
It would be tempting to suggest that Lillie's criticism of artificial parthenogenesis and of physicochemical methods sprang from his religious background (just as Loeb's extreme physicochemical thinking seemed in tune with his left-wing political and social views). Lillie had gained his B.A. degree from the University of Toronto in 1891, having enrolled there "determined to make religion my life work." With two Scottish grandfathers, both of whom were Toronto-based Congregational ministers, and a religious mother of Loyalist stock, a young man would be expected to have such an ambition. After long and passionate discussions at Toronto about science, evolution, and theology, however, Lillie opted for a career in science. Because there were few prospects for serious graduate work in Canada, he enrolled in a summer course at Woods Hole: "[In] June 1891 I took a train to Boston, my father presented me with a fine Leitz microscope, which I took with me and used for many years." Lillie returned to Canada thereafter only for brief visits. His father retired in 1896 and moved to Berlin, where he died two years later; his mother escaped from Germany and emigrated to the United States at the outbreak of World War I. (30)
Available evidence suggests that Lillie's religious upbringing did not influence his scientific attitude; religion and Canada were the rejects of his youth. Thus, Lillie's objection was more to Loeb's experimental design than to his mechanistic principles. It was an objection that sprang from Lillie's pragmatic acceptance of a holistic physiological outlook. This outlook can be seen in his earliest writings, which reflected not his religious upbringing but the influence of his Ph.D. supervisor, Charles Whitman. Lillie met Whitman that first summer at Woods Hole and under his guidance began working on cell lineage in the fresh-water clam Unio. He later obtained a graduate fellowship to Clark University, where Whitman was a professor. Then, when the University of Chicago, flushed with its $10 million grant from J. D. Rockefeller, offered Whitman a lucrative post there, Lillie and Whitman's other students accompanied him. Lillie gained his Ph.D. from Chicago in 1894 and, after holding junior faculty positions at the University of Michigan and Vassar College, returned to Chicago as an assistant professor in 1900. There he remained for the rest of his life, succeeding Whitman to the chair of zoology in 1910. He also returned to Woods Hole every summer, becoming its director when Whitman resigned in 1908.
Figure 1 Woods Hole laboratory, 1893. Among those pictured are S. Watase (front row, far left), F. S. Conant (front row, third from right), F. R. Lillie (second row, second from left), K. Foot (second row, third from right), C. O. Whitman (second row, second from right), G. N. Calkins (third row, secondfrom left), E. G. Conklin (third row, sixth from left), J. Loeb (fourth row, second from left), and T. H. Morgan (fourth row, third from left). Reproduced by permission of the Woods Hole Marine Laboratory.
Lillie's assumptions about the nature of life and living organisms mirrored Whitman's. In 1893 Whitman had criticized the "cell dogma," as he called it, and the premise that organisms are basically colonies of cells. An embryo, Whitman argued, "is not a simple adhesion of independent cells," but is an "integral structural adhesion." "Organization precedes cell formation and regulates it," he stressed; "the organization of the egg is carried forward to the adult as an unbroken physiological unity."(31) Similarly, Lillie, following his work on Unio, argued that "the egg acts as an undivided organism controlling some events by orientation of cleavage planes or shifting of cells, and others by protoplasmic movements clearly independent of cell boundaries." (32) Other organismic concepts pervaded Lillie's lengthy paper on the embryology of Chaetopterus. He denied, for example, the assumption that physiological unity arose secondarily as previously independent cells began to act as one unit. Instead, he argued, "there are certain properties of the whole, constituting a principle of unity of organization, that are part of the original inheritance, and thus continuous through the cycles of the generations."(33)
Lillie, like Whitman, was not averse to thinking in teleological terms. This facet of his physiological approach is best seen in his early work on cell lineages. The problem of germ layers and cell lineages had become a contentious issue by the 1890s. (34) The basis of the germ layer theory had been laid down in the early nineteenth century. In all animals, it appeared, the embryo became differentiated into two or three layers that could be distinguished both by their location in the embryo and by the fact, so it seemed, that each gave rise to particular tissues and organs. With the advent of evolutionary thinking, Ernst Haeckel and others maintained that "quot;ontogeny is the short and rapid recapitulation of phylogeny. " Moreover, they postulated that the two-layered gastrula stage, which was present in all Metazoa, represented a gastraea, the ancestral form of all multicellular animals. As we have seen, the search for ancestry through homology dominated biology during the second half of the nineteenth century, and the germ layer theory proved to be a vital tool in these endeavors. Organs were assumed to be homologous if they could be derived from the same ancestral germ layer.
At Woods Hole, however, Whitman argued that this embryological search for homologies should begin with the individual blastomeres, not merely with the gastrula stage. The germ layers, and even the organs that arose from them, he argued, "can be traced directly to special blastomeres. " This search for the cellular origins of the germ layers provided the major impetus for Lillie's earliest work on Unio, for by that time the value of germ lineage studies to the general problem of homology had been undermined to some extent by the discovery that the same germ layer in different animals might not have the same cell lineage. This became a crucial issue. As Edmund Wilson expressed it in 1892, 'The 'gastrula' cannot be taken as a starting point for the investigation of comparative ontogeny unless we are certain that the two layers are everywhere homologous." (35) A determination of whether the layers were truly homologous necessitated "tracing out the cell lineage or cytogeny of the individual blastomeres from the beginning of development." This program was carried out by Lillie, who argued that "the most striking feature is not the contradictions existing, but the wonderful agreements." Cells of the same lineage, he found, usually differentiated into the same tissues and organs, but discrepancies did arise, because of the future needs of particular organisms. A firm advocate of the biogenetic law, Lillie believed that any seeming irregularities reflected adaptations "to the needs of the future larva." Special needs, such as the early requirements for a functional shell gland in mollusks, may alter the order of appearance and character of segmentation, Lillie argued, "but that in no way renders invalid the value of cell lineage studies to homology. (36)
In a series of lectures delivered at Woods Hole in 1898, Lillie reminded his audience that however useful lineage studies might be in uncovering homologies, one must not lose sight of "the special features of the cleavage in each species, which are, I believe, so definitely adapted to the needs of the future larva as is the latter to the actual conditions of its environment. " It was Lillie's belief that differences in the cleavage patterns of organisms had "prospective significance" that could not be explained simply by "mechanical laws of cell division."(37) The mechanistic view of life, he again argued in a lecture given twenty-one years later to the Philosophy Club at the Universityof Chicago, "[is] not inconsistent with a general teleological point of view." After quoting such holistic thinkers as Hans Driesch, J. S. Haldane, and Lawrence Henderson, Lillie concluded this lecture as follows: "Many doctrines classed as vitalistic do not reject experimental determinism. They, therefore, interfere in no wise with the program of science."(38) This organismic, or holistic, approach typified Lillie's work on fertilization, particularly after 1911, when Lillie became dissatisfied with the methods he had been using to investigate the penetration of the egg by sperm.
How was an egg stimulated to begin development? This was the basic question that had been posed by nineteenth-century physiologists and more recently by Loeb and others. Although Lillie had contributed to the answering of this question, by showing, for example, that Boveri's centromere theory was invalid because only the head and not the middle piece of the sperm entered the egg, he began to feel that too much emphasis had been placed on this single question. The question, he argued, had become an "inhibiting factor." Because eggs could develop without the influence of sperm, their role in fertilization had been underplayed. More than that, "the problem of specificity had been left almost entirely out of account." Proponents of contact, ionic, and osmotic theories had never posed the question, Why can an egg be stimulated only by a sperm of the same species? To an organismic physiologist like Lillie, these problems of specificity were absolutely fundamental; the mysteries of fecundation could never be explained without an understanding of egg-sperm specificity.
To attack this very basic question, Lillie began to investigate how sperm responded to egg secretions. "It occurred to me," he remarked in a lecture to the Zoology Club at Chicago, "that the spermatozoa might prove better indicators of some of the processes taking place in fertilization than the slowly reacting egg. " (39) The sperm, he noted, might be able to indicate "the presence of an agent to which spermatozoa of the same species are positively chemotactic," if the union of the ovum and spermatozoon after they have come into contact operates not mechanically, but through some biochemical reaction between spermatozoon and ovum, the sea-water in which eggs have been standing should contain a substance also capable of reaction with the sperm, which should be an efficient indicator for it. (40)
The results of Lillie's early experiments were extremely significant. They showed that when a suspension of active sperm of Arbacia and Nereis was added to sea water in which unfertilized eggs had been allowed to stand, not only was a specific chemotactic response noted, but, more significantly, the sperm agglutinated. The sperm, he reported, became "sticky," and some change in their membranes took place such that the sperm adhered to each other and could not be separated by shaking. Eggs, he suggested, liberate an isoagglutinin which acts only on the sperm of the same species. It was now clear to Lillie that the reaction between egg and sperm was an immune reaction; the specificity of sperm and egg had become the crucial issue. "We must consider," he wrote, "the general fact that ova and spermatozoa of the same species do behave in a specific way with reference to one another in the process of fertilization. This must have some chemical basis and on the chemical side, the only reactions that exhibit a corresponding degree of specificity are those between antigens and antibodies in the field of immunity." (41)
At this stage Lillie must have read reports of some of the recent work in immunology, for in 1913 he proposed a theory of fertilization based on the work of the great immunologist Paul Ehrlich. It is certain that Ehrlich's Croonian lecture of 1900 was known to Lillie, and in 1912 Lillie had acquired the second edition of Ehrlich and Boldvan's Studies in Immunity. (42) The many marginal notations in Lillie's copy of this edition draw attention to the parallel between fertilization and Ehrlich's work. By the early twentieth century, all theories of immunity, and there were many, had been "overshadowed by that associated with the name of Ehrlich." (43)
It was thought at this time that there were two ways in which bacteria elicited a response from the host organism. The first was by way of poisons or toxins produced by the bacteria; the second, which was associated with inflammatory reactions, was elicited only by the actual bacterial organism and not by chemicals secreted by them. Immunity, whether natural or acquired, was associated with the formation of antitoxins to the first group of bacteria and bactericidal sera to the second. The beauty of Ehrlich's theory lay in its applicability to both groups, whereas Metchnikoff's older theory of phagocytosis had only partially explained the reactions to the second group.
In 1897 Ehrlich had discovered that, although the toxicity of bacterial toxins decreased with time, exactly the same amount of antitoxin was needed to neutralize the "young" and the "old" toxins. He suggested, therefore, that the toxins included both a "haptophorous" group, which combined with the antitoxin molecules, and a "toxophorous" group, which exerted the poisonous action on host cells. The former fixed the toxin to the cells and thereby allowed the toxophorous group to exert its effect. If, then, a series of toxins was injected into the host, the host cells would attach to the haptophorous group by their "receptors" or "side chains," which, being necessary for the normal metabolism of the cells, would be generated anew as more and more were used up in the attachment process. Eventually, Ehrlich postulated, so many receptors would be produced in this way that many would be secreted into the serum. These cast-off receptors would then act as antitoxic agents in the immune serum. By saturating the haptophorous group of toxins, they would prevent them from attaching to the host cell, and thus the toxophorous group would be unable to function (Fig. 9.2 [1-6]).
Figure 2 Paul Ehrlich's side-chain theory of immunity: (1-4) side chain of the cell attaches to black, toxin molecules with haptophore group; (s) excess side chains are liberated to become the antitoxin molecules; (6) free antitoxin molecules unite with toxins and thus protect the cell; (7) side chain of cell (bottom) unites with immune body (middle), which in turn links with the complement (black); (8) not relevant to the present discussion. Diagram reproduced from The Collected Papers of Paul Ehrlich (London: Pergamon Press, 1957), by permission of the publisher.
Ehrlich explained the immune reaction in the second group of bacteria along much the same lines. It was known that the immune serum of guinea pigs would dissolve the red blood cells of rabbits, but if heated to 55°C for 30 minutes, the serum lost this capability. On the other hand, when the serum of a nonimmunized guinea pig was added to the heated serum, the hemolytic activity returned. These results suggested to Ehrlich that two distinct bodies were also involved in this activity: a "complement," which was susceptible to heat and present in all serum, and a stable "immune body," which was present in the immune serum. He visualized the immune body as being analogous to the haptophorous group of the bacterial toxins in that it had two receptor sites, one of which linked to the red blood cells and the other to the complement. The complement, on the other hand, was analogous to the toxophorous group in that it lysed the cells only through the intermediation of the immune body. Thus, when red blood cells or other foreign bodies such as bacteria were passed into nonimmune sera, they would link to body-cell receptors. However, since these receptors performed a normal function in the body, they would be produced in excess and become liberated into the serum as immune bodies. Any red blood cells or bacteria entering this immune serum would then be destroyed as they linked with the immune bodies and complements. The key to immunity in both groups of bacteria lay in the activity of a double-sited haptophorous group, or "amboceptor" molecule. As James Ritchie, professor of pathology at Oxford, summarized the theory in 1902:
The methods by which bacteria are dealt with in the body are similar to those which obtain when many kinds of foreign cells gain an entrance into the latter. The development of artificial immunity against such bacteria depends on the latter being introduced either in a form not strong enough to cause death or, if virulent, not in sufficient numbers to cause death. In either case, the affected animal probably resists infection because it can develop in its body or already possesses a substance-immune body-which attached itself to the bacterial protoplasm and, in virtue of this attachment, permits another body-the complement-which exists normally in the animal's body to act on the bacteria with a fatal result to the latter. (44)
It would have taken little imagination for Lillie to realize that a parallel existed between the action of immune sera on bacteria and the action of a sperm on an egg. He would have noted, in particular, the discovery made by Karl Landsteiner and others that when spermatic fluid is injected into a guinea pig, the serum acquires the capacity to immobilize fresh sperm. The parallel with bactericidal activity became even clearer when it was discovered that this property, which was destroyed by heating the serum, returned when nonimmune serum was subsequently added. "The essential conclusion," Lillie wrote in 1914, "is that fertilization is a reaction between three bodies of which one is borne by the sperm and one by the egg; the third body, which is secreted by the egg, reacts with both the others. The spermatozoon functions especially as an activator of the third body, which I propose to name 'Fertilizin'; the latter, when activated, enters into certain reactions in the cortex of the egg which leads to membrane formation." (45) Fertilizin, in other words, was a typical "amboceptor" molecule with two reactive sites: a spermophile group that reacted with the sperm, and an ovophile group that reacted with the egg (Fig. 9.3).
Figure 3 Frank Lillie's theory of fertilization. Successive sectors of the egg show the mechanism of fertilization and the blocks to that mechanism as follows: (1) The arrangement of substances in the unfertilized egg and in the spermatozoon that are active in fertilization (see symbols). (2) The mechanism of normal fertilization. The sperm receptor unites with the spermophilic group of the fertilizin and the egg receptors unite with the ovophile group of the fertilizin owing to activation of the latter by the sperm (a). Molecules of the antifertilizin combine with the spermophilic group of the adjacent fertilizin (b and c ) and thus block the way for supernumerary spermatozoa. This is the postulated mechanism for prevention of polyspermy. At the same time, molecules b and c of the fertilizin have also united with the egg receptors. (3), (4), (5), and (6) not relevant to the present discussion. Reproduced from Frank Lillie, "Studies on Fertilization," J. Exp. Zool. 16 (1914), by permission of Alan R. Liss, Inc., New York.
By means of this theory, Lillie was able to explain not only the crucial issue of egg-sperm specificity but also why polyspermy was prevented. Sperm agglutination stopped, he discovered, when the eggs disintegrated. This suggested that the eggs contained within themselves a substance that could react with the spermophile side chain of fertilizin and thereby block the entry of additional sperm. As Lillie put it, "When we consider the extraordinary activity of the unfertilized egg in its secretion, and the equally extraordinary activity of the spermatozoa for it, one cannot escape the conviction that it must be a link in the normal fertilization process. When, moreover, one finds that the egg contains a more centrally located substance that can occupy the same combining group as the sperm, one seems to have before one, a view of a mechanism for preventing polyspermy."(46) The egg was thus assumed to carry egg-receptor molecules and "antifertilizin" molecules. The former attached to the ovophile group of fertilizin as soon as the fertilizin was activated by union with the sperm. The antifertilizin molecules immediately combined with the spermophilic group of adjacent fertilizin molecules, thereby preventing the entry of additional sperm.
The assumed presence of spermophilic and ovophilic side chains on the fertilizin molecules also enabled Lillie to propose a simple explanation for parthenogenesis-something that Loeb was unable to do except by undermining the role played by the sperm. The binding of the sperm with the spermophilic side chain of fertilizin, Lillie wrote:
activates the ovophile-combining group of the fertilizin which then seizes upon egg receptors, and it is the latter union which results in membrane formation. If this were so, it is obvious that the spermatozoon is only secondarily a fertilizing agent, in the sense of initiating development, and that the egg is in reality self-fertilizing, an idea that agrees well with the facts of parthenogenesis and with the amazing multiplicity of means by which parthenogenesis may be effected. For the agents need only facilitate the union of fertilizin and egg receptor. (47)
Loeb continued to criticize Lillie's theory and to postulate a more chemical hypothesis, but Lillie's theory quickly became the standard explanation, presumably because its application was so wide. (48)
The problem of fertilization, like that of sex determination, had been redefined by basically ignoring the developmental issue. Explanations of sex determination now rested on the inheritance of sex chromosomes and ignored the puzzle of how such chromosomes could determine sex in an embryological sense. Similarly, the issue of how sperm initiated development had faded. Lillie expressed this very forcibly in his text Problems of Fertilization:
It is commonly said that there are two main problems in the physiology of fertilization, viz.: the initiation of development, or activation, and biparental inheritance; but these are more properly results of fertilization. Indeed, so long as we regard fertilization primanly as a function of prospective significance in the life of the organism, we shall miss the more specific aspects of the process. Once fertilization is accomplished, development and inheritance may be left to look after themselves. (49)
Literature Cited, Including Footnotes
1. Edmund Wilson, "Recent researches on the determination and heredity of sex," Science 29 (1909): 70.
2. Jacques Loeb, "The biological problems of today: Physiology, " ibid . 7 (1898): 154-56.
3. Hermann von Helmholtz, "Aim and progress of physical science, " in Popular lectures on scientific subjects, English trans. E. Atkinson (New York, 1873), p. 384. For details of Loeb's life and work, see Donald Fleming's introduction to The mechanistic conception of life, ed. D. Fleming (Cambridge, Mass.: Harvard University Press, 1964).
4. Thomas Hunt Morgan, "The action of salt-solutions on the unfertilized and fertilized eggs of Arbacia and other animals," Arch. Entwick. 8 (1899): 527.
5. Jacques Loeb, "On the nature of the process of fertilization and the artificial production of normal larvae (plutei) from the unfertilized eggs of the sea urchin," Amer. J. Physiol. 3 (1899): 135-38.
6. Ibid., p. 138.
7. Loeb, "On the nature of the process of fertilization" (1899), in Mechanistic conception of life, ed. Fleming, p. 115.
8. Jacques Loeb, "Further experiments on artificial parthenogenesis and tbe nature of the process of fertilization," Amer. J. Physiol. 4 (1900): 181.
9. Jacques Loeb, "On the artificial production of normal larvae from the unfertilized eggs of the sea urchin (Arbacia)," Amer. J. Physiol. 3 (1900): 468.
10. Jacques Loeb, "Artificial parthenogenesis in annelids," Science 12 (1900): 170; idem, "Experiments on artificial parthenogenesis in annelids (Chaetopterus) and the nature of the process of fertilization," Amer. J. Physiol. 4 (1901): 423-59.
11. Harper's Weekly, December 13, 1902, p. 1936; and Garrett Serviss, "Artificial creation of life," Cosmopolitan 39 (1905): 459-68. Quotation from Carl Snyder, "The mysteries of life and mind: Dr. Loeb's researches and discoveries," Fortnightly Rev. 77 (1902): 1016.
12. Loeb, "Experiments on annelids (Chaetopterus)," p. 456.
13. J.-B. Piri, "Un nouveau ferment soluble: L'ovulase," Arch. Zool. Exper. Gen. 7 (1899): 29-30; Raphael Dubois, "Sur la spermase et l'ovulose," Compres-rendus Soc Biol. 52 (1900): 197-99.
14. William Gies, "Do spermatozoa contain enzymes having the power of causing development of mature ova?" Amer. J. Physiol. 6 (1901): 53-76.
15. Loeb, "Artificial production of normal larvae," p. 469.
16. Victor Hensen, Physiologie der Zeugung (Leipzig, 1881), p. 126.
17. Yves Delage, Les Theories de la fecondation (Jena: Gustav Fischer, 1902), p. 14.
18. Ibid., p. 17.
19. Oscar Hertwig, "Kritische Betrachtungen uber neuere Erklarungsversuche auf dem Gebiete der Befruchtungslehre," Sitz. Preuss. Akad. Wiss. 17 (1905): 375.
20. Jacques Loeb, "On the nature of formative stimulation (artificial parthenogenesis), " in Mechanistic conception of life, ed. Fleming, p. 121.
21. Ibid., p. 125.
22. Ibid., p. 128.
23. Frank Lillie, Problems of fertilization (Chicago: University of Chicago Press, 1919), p. 26.
24. Jacques Loeb, "The Mechanistic conception of life" (1911), in Mechanistic conception of life, ed. Fleming, pp. 5-6, 16.
25. Frank Lillie, "Function of the spermatozoon in fertilization from observations on Nereis," Science 31 (1910): 836; idem, "Studies on Fertilization in Nereis I and II," J. Morphol. 22 (1911): 389.
26. Lillie, Problems offertilization, p. vii.
27. Frank Lillie, "Studies on fertilization in Nereis, " J. Morphol. 22 (1911): 389.
28. Frank Lillie, "Fertilization" (lecture to Zoology Club, University of Chicago, October 1913), F. R. Lillie Papers, Marine Biological Laboratories, Woods Hole, Mass. (hereafter cited as Lillie Papers).
29. Lillie, Problems of fertilization, p. 22.
30. Frank Lillie, "My early life" (a biography written for Lillie's children in 1944), Lillie Papers.
31. Charles O . Whitman, "The inadequacy of the cell-theory of development, " J. Morphol . 8 (1893): 649, 658.
32. Frank Lillie, "The organization of the egg in Unio, based on a study of its maturation, fertilization, and cleavage," ibid. 17 (1901): 265.
33. Frank Lillie, "Observations and experiments concerning the elementary phenomena of embryonic development in Chaetopterus," J. Exp. Zool. 3 (1906): 251.
34. For excellent accounts of cell-lineage work, see Alice Baxter, "Edmund Beecher Wilson and the problem of development" (Ph.D. diss., Yale University, 1974), pp. 12-19; and Jane Maienschein, "Cell lineage, ancestral reminiscence, and the biogenetic law," J. Hist. Biol. 11 (1978): 129-58.
35. Edmund Wilson, quoted in Baxter, "Edmund Beecher Wilson," p. 90.
36. Frank Lillie, "The embryology of the Unionidae: A study in cell-lineage," J. Morphol. 10 (1895): 38-39.
37. Frank Lillie, "Adaptation in cleavage," Biological lectures at Woods Hole, 1897-1898 (Boston, 1899), pp. 43-56.
38. Frank Lillie, "The mechanistic view of vital phenomena" (lecture to Philosophy Club, University of Chicago, December 1919), Lillie Papers.
39. Lillie, "Fertilization," Lillie Papers.
40. Frank Lillie, "Studies on fertilization: V. The behaviour of the spermatozoa of Nereis and Arbacia, with special reference to egg-extractives," J. Exp. Zool. 14 (1913): 549.
41. Ibid., p. 565.
42. Paul Ehrlich, "On immunity, with special reference to cell-life" (Croonian Lecture, Royal Society of London, 1900), reprinted in Collected papers of Paul Ehrlich, vol. 2, ed. F. Himmelweit (New York: Permagon Press, 1957), pp. 178-95.
43. James Ritchie, "A review of current theories regarding immunity," J. Hyg. 2 (1902): 221.
44. Ibid., p. 261.
45. Frank Lillie, "Studies on fertilization: Vl. The mechanism of fertilization in Arbacia, " J. Exp. Zool. 16 (1914): 524.
46. Ibid., p. 549.
47. Ibid., p. 563.
48. Jacques Loeb, "On some non-specific factors for the entrance of the spermatozoon into the egg," Science 40 (1914): 316-18; idem, "Cluster formation of spermatozoa caused by specific substances from eggs," J. Exp. Zool. 17 (1914): 123-40; Frank Lillie, "Recent theories of fertilization and parthenogenesis," Trans. Illinois Acad. Sci. 7 (1914): 1-10; idem, "Sperm agglutination and fertilization," Biol. Bull. 28 (1915): 18-33.
49. Lillie, Problems of fertilization, p. 129.