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A M . ZOOLOGIST, 3:109-126(1963). THE MANIPULATIONS OF MAGROMOLECULAR SUBSTANCES DURING FERTILIZATION AND EARLY DEVELOPMENT OF ANIMAL EGGS Ai-BERT TYLER Division of Biology, California Institute of Technology Pasadena In considering the role of various macromolecular substances in the developmental and other physiological processes of fertilized eggs and embryos, it is customary to think largely in terms of transactions within the cell. Ordinarily, there is a tendency to assume arbitrarily that macromolecular substances do not enter cells except under unusual circumstances, or in special form such as viruses, transforming DNA, or spermatozoa, or in certain specialized cells that are termed phagocytes. Where intercellular influences of large molecular substances, such as the protein hormones, are known, one is inclined a priori to think of these in terms of interactions at the surface of the cell rather than in terms of penetration into the intimate interior of the cell. In recent years the general outlook has altered considerably. This change is largely due to the work and writings of the late Professor A. M. Schechtman, to whom this symposium and this paper are dedicated. For example, in experiments with chickens, he demonstrated that large molecular substances were incorporated in essentially unaltered form into the growing oocyte. This was basic to the concept, developed since the early experiments of Hevesy and Hahn (1938) that much of the material of the mature egg is not manufactured by the growing oocyte itself but is supplied to it in essentially "finished" form from other cells (cf Tyler, 1955). Schechtman, also, speculated about the possible developmental role of such naturally-supplied substances, and others The preparation of this paper and the original experiments of the author reported herein have been supported by grants from the National Institutes of Health, U.S. Public Health Service (RG6965 and H-3103) and from the National Science Foundation (GB-28). that might be introduced artificially, speculations which, in a sense, bear fruit in experiments reported in several articles of this symposium. Through the activities of Holter (1961) and his associates and many others, the incorporation of large molecular substances into various kinds of cells (by mechanisms such as pinocytosis) and under various conditions, has been amply documented. One may, then, consider the role of macromolecular substances in various developmental processes in terms of both intercellular and intracellular transactions. The papers of the present symposium provide a number of examples of the discovery of new embryological phenomena and their analysis (first-order, or more detailed) in such terms. I shall present here a brief account of some pertinent current work of our laboratory. This work deals largely with interactions that are termed immunological in that they involve (a) immunologically produced antibodies against various antigenic constituents of eggs and embryos, or (b) naturally occurring substances whose interactions are of the "immunologic" type that depend on mutually complementary molecular configurations. The dis-. tinction between (a) and (b) essentially disappears if one accepts the clonal selection concepts of antibody formation proposed by Burnet (1959), Lederberg (1959), and Talmage (1959). They propose, in effect, that there is no such thing as an immunologically produced antibody, in the sense of a novel substance that the cell manufactures under instructions, direct or indirect, of the introduced foreign antigen. Rather, it is thought that antigen simply stimulates proliferation of those (109) 110 ALBERT TYLER cells of an organism that are normally producing a substance (antibody) capable of preferential reaction with the antigen. I shall not attempt to discuss here the relative merits of this theory, as contrasted with the instructional (template) types of theories originally proposed by Breinl and Haurowitz (1930), Mudd (1932) and Pauling (1940) and more recently developed by Karush (1961). I wish only to remark that the clonal selection theories do not eliminate the assumption of template mechanisms, but simply restrict these to the "DNA makes RNA makes Protein" part of the process. The current work that I shall report also deals with another kind of macromolecular substance, now known as Messenger RNA, that has molecular configurations complementary both to correlative DNA and correlative protein. Many years ago I (1940, 1946) had demonstrated the occurrence, within single cells (sea-urchin eggs), of macromolecular substances capable of interaction with one another in the manner of antigen with antibody, and I had adduced from the literature evidence of other reactions that could be interpreted in the same way (cf Tyler, 1956, 1957). On this basis I had proposed a "Natural Auto-Antibody Concept" to express this feature of the interrelations of the macromolecular constituents of cells and its relation to growth and differentiation. The concept stated simply that the macromolecular constituents of cells bore the same relationship to one another as did antigen with antibody, and that this came about by one serving as the basis for the construction of another, in the same way as antibody globulin was considered to have a structure regionally complementary to specific structures of the substances present at the site of synthesis of the globulin. As expressed at that time antibody production was considered a special case of the general manner in which macromolecular substances are manufactured by cells. The "Natural Auto-Antibody Concept" was a brief predecessor of what is now encompassed by "Template Theories," the development of which is largely due to Pauling (1948, 1954). In modern terms it is generally expressed as a unidirectional influence of DNA on the structure of (Messenger) RNA which, in turn, specifies the structure of protein. The Auto-Antibody Concept did not specify restrictions as to direction nor as to the chemical nature of the specific macromolecular substance that could serve as a template. SPECIFIC INTERACTING SUBSTANCES OF EGGS AND SPERM The union of egg and sperm in the initial steps of the process of fertilization has been interpreted (Tyler, 1957, 1959, 1961a, b, 1962a) in terms of interactions of certain substances carried on and in the respective gametes. F. R. Lillie (1913) applied the term fertilizin to a spermagglutinating material obtained from eggs. Fertilizin and the complementary antifertilizin from sperm are now considered to represent the specific receptor substances located in the plasma membranes of egg and sperm respectively. Fertilizin also extends, in attenuated form, into a gelatinous coat in those species of animals whose eggs possess such a coat. These substances interact in antigen-antibody-like manner and the species-specificity of this interaction, along with the fact that these substances are detectably present only in the gametes, largely accounts for the speciesand tissue-specificity of fertilization. In addition to the specific adherence of sperm to egg by virtue of combination of fertilizin and antifertilizin of the respective plasma membranes, this interaction can also lead to incorporation of the sperm into the egg. A general scheme for this has been previously presented (Tyler, 1959), and its current version is represented in Fig. 1. The scheme proposes that, following initial local attachment, the progressive union of the plasma membranes of egg and sperm draws the sperm into the egg, by a sort of pinocytotic process, while the fused membranes break down as they enter into the egg cytoplasm. FERTILIZATION AND EARLY DEVELOPMENT The diagrams represent species in which the entire sperm enters the egg, but requires only slight modification for those cases in which the tail is left out. As the diagrams illustrate, there is a vitelline membrane that overlies the plasma membrane of the egg. However, as was earlier inferred and is now substantiated by electron miscroscopic observations, the vitelline membrane is perforated by microvillous extensions of the plasma membrane. It is with the fertilizin on protruding tips of these microvilli that the sperm is considered to make the first effective contact that attaches it to the eggDissolution of the intervening portion of the vitelline membrane is accomplished by a membrane-lysin contained in the acrosome of the sperm. The opening up of the acrosomal pocket is considered also to be a result of the stresses set up by the progressive fusion of the plasma membranes through interaction of the receptors, fertilizin and antifertilizin. This scheme also offers an explanation for the establishment of the block to polyspermy by a retraction of other microvilli after the initial effective contact has been made with the fertilizing sperm. Also, it can account for the refertilizability of mechanically demembranated fertilized eggs (Tyler, Monroy, and Metz, 1956) through the persistence of fertilizin on the plasma membrane. While there may be some observations that do not seem to be consistent with the scheme in its present form, such as the microvilli that remain protruding through the vitelline membrane in eggs of Mylilus that have evidently been fertilized (Dan, 1962), information on which to base any further substantial modifications is not available at present. Besides the three kinds of substances that have been mentioned there is also an antifertilizin that occurs within the egg (Tyler, 1940, 1946). The finding of such a substance, complementary to the fertilizin of the surface of the egg suggested the concept of Natural Auto-Antibodies, mentioned above. With respect to 111 processes of fertilization, however, there are no experiments yet on the basis of which one might reasonably assign a role to this material. Possibly it is involved in the activation process by interaction with the fertilizin of the plasma membrane that disappears as it involutes with that of the sperm. This could possibly account for the ability of eggs to be activated mechanically, for example, by puncture with a glass needle. One may speculate also on the basis that it may be ribonucleoprotein, but again experimental evidence is lacking. Experimental intervention with fertilization by manipulations of these substances, antibody-production against them in rabbits, and their chemical and physical properties, have been described in earlier publications (cf Metz, 1961a, b; Tyler, 1961a, b). Some further consideration will now be given to problems of activation and later development. ACTIVATION OF THE EGG I reviewed the subject of artificial parthenogenesis some time ago (1941) and, while there have been a number of interesting developments pertaining mainly to chromosomal aberrations in higher animals (cf Beatty, 1960), there has been no real advance in our knowledge of the physico-chemical basis for the process of activation. Considerable interest attaches to the reports of Perlmann (1956, 1957, 1959; Perlmann and Perlmann, 1957) that unfertilized sea-urchin eggs can be activated parthenogenetically by means of rabbit antisera prepared against egg-materials. He has indicated that the effective antibodies are directed against a specific "activation antigen" located in the egg. One can formulate a number of attractive hypotheses on this basis. For example, such an "activation antigen" might be an inhibitor for the ribosomes (see below) and its neutralization could then permit proteinbiosynthesis and other developmental activities to proceed. Other attempts (Tyler, 1959; Tyler, Seaton, and Signoret, 1961) to obtain parthenogenetic activation of ALBERT TYLER a i-c Perivitelline Space ine Layer FIG. 1. Diagram of union of sperm and egg based on fertilizin-antifertilizin interaction and specific pinocytosis. a, b, c and d represent successive stages. Outer surface of egg is toward top in each figure. The reactive chemical groups of antifertilizin (specific receptor substance of sperm) are shown as knob-shaped structures on the sperm. Those of fertilizin (specific receptors of egg) are shown as cup-shaped structures on the plasma membrane (which protrudes as microvilli through pores in the vitelline membrane in the unfertilized egg) and on the micelles of the gelatinous coat (which is an attenuated extension of the plasma membrane). These react by virtue of complementary configuration, as in antigen-antibody reactions, and this proceeds in rapid, zipper-like fashion, so that the several microvilli, that make initial contact with the sperm, extend over the surface of the sperm, drawing the latter into the egg. As this occurs the acrosomal vesicle opens up (assumed to result from stresses set up by fertilizin-antifertilizin interaction) and liberates lysin which dissolves the portion of FERTILIZATION AND EARLY DEVELOPMENT 113 the vitelline membrane enclosed within the microvilli that are attached to the sperm. As the sperm is, thus drawn into the egg, the fused plasma membranes of sperm and egg break down and disappear starling at the innermost portion, thus exposing the sperm nucleus to early interaction with the egg c) topi asm. Rapid retraction of the microvilli elsewhere, along with elevation of the vitelline membrane, causes the receptors on the plasma membrane to become inaccessible to additional sperm (block to polyspermy). Mechanical removal of the vitelline (fertilization) membrane permits ready refertilization. The granules, that are released from the cortical vesicles in the plasma membiane, serve to "tan" the inner surface of the vilelline membrane, to elevate the latter by osmotic action, and, in time, to help cover up the receptors on the plasma membrane. sea-urchin eggs by similar and other kinds of antisera have not been successful. According to Perlmann's reports, most batches of eggs are not readily activatable by treatment with antiserum, and it is necessary to test many sea-urchins to obtain eggs that respond well. The attempts to confirm the reports were done on a large scale so as to take such factors into account, but obviously one can attribute the lailures of activation by this method to unsuitability of either the batches of eggs, or of the particular species of seaurchins. Also there were undoubtedly differences in the specific conditions of the experiments. On the other hand the changes that are reported by Perlmann are mostly not in the category that one would clearly designate as a type of parthenogenetic activation; many may be ordinary cytolytic types of changes. Possibly some represent parthenogenetic activation occurring on occasion in some eggs in response to a constituent present in the antiserum—a constituent that may be unrelated to the antibodies against sea-urchin material. As is well-known, sea-urchin eggs respond parthenogenetically to a large variety of simple chemical substances and physical agents. On the whole, then, one cannot consider the existence of an "activation-antigen" to have been securely established. While we find no clear developmental changes upon treatment of unfertilized eggs with antisera prepared against eggmaterials, cytolytic effects are commonly obtained. Such effects can be obtained with antisera that are prepared against purified fertilizin (Fig. 2). There have been two previous reports of stimulating action on cell proliferation by specific antisera. One was by Bogomolets (1943) using an antiserum prepared against reticulo-endothelial cells; this reportedly stimulated cell growth and activity, and had many remarkable therapeutic properties when used in low doses. Attempts by Pomerat (1945, 1946, 1949) and others to confirm some of the claims have been unsuccessful. The other report was by Weiss (1947) to the effect that there is a relative stimulation of growth of the homologous organ when hens' eggs receive at an early stage of incubation, rabbit antisera prepared against adult liver or kidney. More recently, however, Weiss (1953, 1955) has indicated that the effect was more likely due to hemorrhage caused by vascular damage, rather than to a stimulation of growth by the antisera. A report of another kind of stimulating effect of antibodies is cited and analyzed by Nace (1955) in his concise review of investigations of development in the presence of antibodies. This refers to the work of Asai and Umeda (1929) indicating that ciliary activity of isolates of pharyngeal epithelium of Rana esculenta is accelerated by a rabbit antiserum produced against saline extracts of this tissue. On examination of this report one would agree with Nace's conclusion that the effect may be only an apparent case of stimulation by antibody. In summary it can be stated that, while it seems theoretically possible that specific antibodies might stimulate various cellular processes, directly or by neutralization of an inhibitor, clear-cut demonstrations of such effects are lacking. 114 ALBERT TYLER FIGS. 2-5. Photomicrographs of eggs of Lytechinus pictus. Magnif. 200 x. Unfertilized eggs placed in rabbit antiserum versus fertilizin (Fig. 2) and in control (pre-injection) serum (Fig. 3) for 8 hours at 20°C. Fertilized eggs placed in rabbit antiserum versus fertilizin (Fig. 4) and in control (pre-injection) serum (Fig. 5) at 15 minutes after fertilization for 2 hours at 20°C. FERTILIZATION AND EARLY DEVELOPMENT 115 later stages corresponding to the extent of dilution. Our current concept of the arrangement Effectiveness of fertilizin-antisera. In con- of the surface layers of the unfertilized trast to the lack of evidence of cell stimu- and fertilized egg (Fig. 1) provides a basis lation by antibodies there is an extensive for understanding the effectiveness of ferliterature dealing with cytotoxic effects. tilizin in engendering the formation of Reference to some of this has been made antibodies that are inhibitory to the ferearlier (Tyler, 1957, 1959; cf Latta, 1959). tilized egg. Fertilizin is no longer thought Experiments with sea-urchin eggs have to represent simply the material of the permitted some further analysis. As one gelatinous coat of the egg; nor is it a part might expect, antisera that are prepared of the vitelline membrane (which elevates in rabbits against whole egg homogenates as the fertilization membrane). As a comcan block the development of fertilized ponent of the plasma membrane fertilizin eggs. Somewhat more surprising was the remains available for reaction with specific finding (Tyler and Brookbank, 1956a. b; antibodies in the fertilized egg. Generally, Tyler, 1957, 1959) that antisera prepared in these experiments the fertilization memagainst purified fertilizin could also block brane is mechanically removed shortly development of the fertilized egg, and after it elevates, since this membrane rethat they were generally more effective tards to some extent the access of the than were antisera prepared against whole antibodies to the plasma membrane. The egg homogenates, against sperm-materials, presence of fertilizin as a component of or against various tissues of the adult sea- the plasma membrane of the fertilized eggs is consistent also with the discovery urchin. (Tyler, Monroy, and Metz, 1956) that A typical example of blocking of cell mechanically demembranated eggs can be division of fertilized sea-urchin eggs by ex- refertilized. posure to an antiserum obtained from a It is, then, no longer surprising that rabbit immunized with homologous fertiantisera against fertilizin can block divilizin is shown in Fig. 4. The eggs were sion of the fertilized eggs. In fact this placed in the antiserum shortly after fertilization. As illustrated, there were no represents the first instance, in experidistinctive cytolytic changes occurring dur- ments with animal cells, in which such ing the approximately two hours that the cytotoxic antibodies have been engendered eggs were in antiserum, while the control with an antigen that is readily prepared eggs (Fig. 5) had reached the four to eight in highly pure form (electrophoretically cell stages. Distinct cytolytic changes do, and ultracentrifugally homogeneous, and however, generally appear after another specifically absorbable by homologous two hours or so, depending upon the anti- sperm) and has been chemically characserum. Also, it is not necessary for the terized to a large extent (a glycoprotein, eggs to remain in the antiserum in order the molecules of which are comprised of for cleavage to be blocked. An exposure two kinds of sugars, some fourteen amino to the usual antisera, used full strength, acids, and sulphate in approximately equal for the equivalent of one-fourth of the amounts; and have weights of the order time of a division cycle (about 15 minutes of 300,000 with axial ratios of 20:1). in most species of sea-urchins) suffices to Additional questions of special interest block subsequent cleavage. When eggs are concern antibodies against other egg-conplaced in dilutions of antisera that permit stituents and the relation of the cleavageinitial cleavage the divisions are delayed, block effect in sea-urchins to other cytoare usually abnormal in form with sepa- toxic systems that have been studied. ration and frequent disintegration of blasIneffectiveness of antibodies against intomeres, and complete block sets in at ternal constituents of the egg. As noted INHIBITION OF CLEAVAGE AND DEVELOPMENT WITH ANTISERA 116 ALBERT TYLER TABLE 1. Reduction of cleavage-blocking action of anti-egg-homogenate-sera following absorption with fertilizin-lreated sperm in the sea-urchin Lytechinus pictus. Experiment A B C Serum absorbed with unabsorbed control sperm fertilizin-treated sperm unabsorbed control sperm fertilizin-treated sperm unabsorbed control sperm fertilizin-treated sperm Percentage of first division at I14 to \\/2 hours after fertilization in eggs placed iin following dilutiions of antiserum at 10-15 minutes 2x 4x 8x 16x 32x 64x 128x 25Gx 0 0 5 0 0 0 0 0 2 0 3 50 5 0 15 0 0 15 1 3 80 15 0 50 0 25 80 15 2 80 50 70 80 80 80 80 80 80 80 80 80 80 20 15 90 50 50 95 80 80 95 90 80 95 0 1 65 3 20 75 60 70 85 75 80 90 80 90 90 85 90 90 above, it has been found that antisera against whole egg homogenates can block cleavage of sea-urchin eggs. This occurs also when the antigen has been prepared from eggs in which the fertilizin of the gelatinous coat has been removed. However, such preparations are still likely to contain fertilizin-antigen derived from the plasma membrane. Possibly, then, cleavageblock by such antisera may be due simply to the antibodies against fertilizin rather than to those directed against other constituents of the egg. This has been examined in some absorption experiments that have been previously reported in abstract (Tyler, Seaton, and Signoret, 1961). The results indicate that antibodies directed against internal constituents of the egg are not particularly effective, or probably not at all effective, in blocking cell division. The principal experiments consist in absorbing such antisera with fertilizin. For technical reasons (since fertilizin solutions gelate at relatively low concentrations and this limits their use for absorbing antisera) the absorption was done with sperm that had reacted with fertilizin and were thus coated with it. Absorption with such sperm greatly reduces the cleavage-blocking action of anti-egg antisera, whereas the control, untreated sperm have no such effect. Table 1 illustrates an experiment of this type. In most other experiments, some of which are controlled also by absorption of anti-fertilizin antisera, it ap- pears that complete absorption of the fertilizin-antibodies was not effected. While the use of fertilizin-coated sperm has some advantages, it too has its limitations since large quantities must be employed and, when densely packed and centrifuged, even in normal sera, may release some materials that interfere with development of the eggs. The most reasonable conclusion from the present data is that antibodies directed against internal constituents of the egg are ineffective in blocking development. In fact, it appears that eggs may develop in the presence of large quantities of antibodies directed against internal constituents. The antibodies evidently do not manage to get inside the egg, or the cells of the embryo, in sea-urchins. This appears to be the general experience with other kinds of organisms but it is not usually clear if one is dealing with antibodies directed against surface constituents or internal constituents. However, the clearest example probably would be that of antibodies directed against viruses. It is well-known (Loffler, Henle, and Henle, 1962) that virus-neutralizing antibodies are ineffective against virus once it has entered the cell. Exploration of the extent to which this general situation may apply to developing organisms, and of methods for inducing cells of developing embryos to take in antibodies, denotes important areas of investigation for the further analysis of de- 117 FERTILIZATION AND EARLY DEVELOPMENT TABLE 2. Effect of complement on the blocking action of rabbit antisera vs fertilizin of Lytechinus pictus. Eggs placed in solutions at 15 minutes after fertilization Per cent cleaved at 60 minutes after fertilization Rabbit serum dilution 1/10 1/20 1/40 1/80 Guinea pig serum* 1/5 fresh heated fresh heated 80 80 20 20 80 80 50 50 80 80 Normal rabbit serum (JC 7 A) fresh heated 75 75 Antiserum vs fertilizin (JC 7 B) fresh heated 0 0 Normal rabbit serum (JC 2 A) Antiserum vs fertilizin (JC 2 BC) 1/160 80 80 80 80 80 80 80 80 80 80 80 80 80 80 75 80 80 80 80 80 80 80 80 80 15 15 50 50 80 80 80 80 80 80 • Guinea pig serum was reconstituted lyophilized preparation (Hyland Labs) used at a dilution of 1/20; complement titer 1/160 for 50% hemolysis of sensitized 2.5% sheep RBC. velopmental processes and of the experimental control of development in specific ways by appropriate manipulations with these macromolecular reagents. As mentioned in the introduction, the growing oocyte possesses incorporating-ability, and as indicated below, the ripe unfertilized egg may still retain it to some degree. It is not unreasonable to expect that under appropriate conditions various cells of the developing embryo may exhibit this property. Since tissue-specific antigens appear to be primarily subsurface constituents of cells, the importance of the solution to the penetration problem is further evident. Complement and cleavage-block. In most cytotoxic systems of most vertebrates the cytolytic effects have generally been found to be promoted by, or in many cases to require, the cooperation of a set of components of the serum, termed "complement" (cf Osier, 1961; Winn, 1962, for current reviews). In the earlier experiments with sea-urchin eggs it was found that the heated rabbit antisera were effective in blocking cleavage and in inducing the subsequent cytolytic changes. This has been investigated further in this laboratory by Dr. H. Timourian in experiments that also involved testing effects of unheated and heated guinea-pig serum on the blocking action of a heated rabbitanti-fertilizin-serum. Table 2 presents the part of an experiment of this type that contains data for the effect on the first division. The results for the quantitative examination of effects on later development are similar. It is clear, also, that added complement had no effect on the titers of the antisera with respect to these effects. This does not, however, mean that chemical components related to "complement" may not be involved in the action of the rabbit antisera on sea-urchin eggs. There is, in fact, some indication that the egg itself may provide a component of this sort. Some years ago, in an investigation (Tyler, 1942) of the possibility that the fertilizin-antifertilizin reaction might fix "complement," it was found that fertilizin itself was anticomplementary, binding C'4 of guinea pig "complement." Reaction with antifertilizin released the bound C'4 in quantitative fashion. The experiments indicated a similarity in some properties between antifertilizin and C'4. It is possible, then, that the subsurface antifertilizin of the egg might, in effect, be supplying the equivalent of an essential component of complement. PROTEIN BIOSYNTHESIS IN SEA URCHIN EGGS Incorporation of amino acids into protein before and after fertilization. The rate of incorporation of labelled constituents into protein by intact eggs of sea urchins increases considerably upon fertilization. This was shown originally in the experiments of Hultin (1950) with N1B-labelled 118 ALBERT TYLER ammonia, and of Hoberman, Metz, and Graff (1952) with deuterium. Hultin (1952) also obtained evidence that incorporation of N15 glycine and alanine into protein increased after fertilization but the results were obscured by possible changes in permeability. Nakano and Monroy (1958) overcame this difficulty by injecting S35 methionine into the body cavity of the female, thus preloading the eggs and effectively demonstrating that the increased incorporation upon fertilization was due to an intrinsic change in activity rather than to an increase in permeability. Hultin and Bergstrand (I960) found that the difference is exhibited also by cellfree amino-acid-incorporating systems prepared from unfertilized and fertilized eggs. By exchanging cell sap and particulate fractions of homogenates Hultin (1961) obtained evidence that the increased activity is largely due to modifications of the ribosomes. More recently, Nemer (1962), Tyler (1962b), and Wilt and Hultin (1962), upon following-up the discovery of Nirenberg and Matthaei (1961) on the E. coli cell-free system, reported that polyuridylic acid greatly stimulated incorporation of phenylalanine in homogenates of sea-urchin eegs. The preparations from unfertilized eggs responded as well as, or better than, those from fertilized eggs or later embryos. From this it would appear that, in contrast to Hultin's (1961) view of essentially inert ribosomes that are activated upon fertilization or artificial parthenogenesis, the inactivity of the ribosomes of the unfertilized egg is primarily attributable to lack of messenger RNA. A further test of this proposition has been undertaken in this laboratory, principally by Mr. Paul C. Denny,1 with homogenates prepared from non-nucleated seaurchin eggs before and after artificial parthenogenetic treatment. We have also explored other features of the cell-free preparations from sea-urchin eggs. I have also initiated some experiments on the effects of polyribonucleotides on amino-acid incorporation and development of the intact eggs, In these and certain other experi- ments on ion-composition of the medium we have been joined by Dr. Hector Timourian.- A brief account of some of these experiments is given here. Stimulation of phenylalanine-incorporation in homogenates by polyuridylic acid. Methods. The tests were performed with the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus. The cellfree suspensions were prepared with a motor-driven, Potter all-glass, or glass and teflon, homogenizer. In some experiments the homogenization was done by sonication. The homogenization- and incubationmedia were modifications of those employed by Nirenberg and Matthaei (1961) and by Hultin (1961). The TCA-precipitation and purification of proteins were done as described by Siekevitz (1952), with additional solution in N/l NaOH, reprecipitations and extraction with hot TCA and lipid solvents so as to obtain zero-time (t:l) radioactivity values close to that of the background. The final solutions were dried on filter paper and radioactivity measured in a scintillation counter with counting efficiencies near 50%. Some experiments were performed by the method of Mans and Novelli (1961) in which the incubation mixtures are placed directly on filter papers which are then all run through the various extraction procedures in a single beaker. Protein determinations were done by the biuret method of Ellman (1962). Results. Table 3 shows the results of an experiment in which polyuridylic (poly U) acid is added to cell-free preparations of unfertilized eggs and of embryos at the hatching blastula stage (ca. 500 cells) of the sea urchin Lytechinus pictus. The set of preparations termed Homogennte was made by use of the Potter homogenizer. The other set {Sonicate) was made by exposure of the egg-suspension (in an ice-bath) to ultrasonic waves (40 kc, 500watt Cavitator Mark I of Mettler Electronics Corp., Pasadena) for just enough time to reduce all cells to a suspension of particles. The tests were done in duplicate as indicated in the table. 119 FERTILIZATION AND EARLY DEVELOPMENT TABLE 3. Influence of polyuridylic acid on incorporatioti of C"-L-phenylalanine into protein with homogenates and sonicates of eggs and embryos of Lytechinus pictus. Counts per minute (minus t0; blanks = 37, 40, 36, 34) Without poly U With poly U Increase Homogenates of: Unfertilized eggs Blastulae (just hatching) Sonicates of: Unfertilized eggs Blastulae (just hatching) 53,56 avg55 (to = 50) 274,261 avg268 (to = 73) 34,40 avg37 (to = 79) 123, 124 avg 124 (t« = 67) 987, 916 avg 952 897 903, 891 avg 897 629 849, 888 avg 869 832 1049, 1014 avg 1032 908 Incubation mixtures = 0.225 ml homogenate or sonicate (derived from 1.5 X 105 eggs in 0.01 M tris, 0.01 M MgAc, 0.275 M KC1), 0.025 ml of Reaction Mixture (0.8 ml M/8 PEP; 0.1 ml of 0.0038 M C"-L-phenylalanine at 9.8 curies/mole; 0.1 ml M / 1 0 ATP, with or without poly U at 0.08 molar calculated as uridylic acid). Both types of preparations gave similar large increases in the incorporation of phenylalanine into protein upon the addition of polyuridylic acid. With the homogenate the increase in activity averaged somewhat less for the blastulae than for the unfertilized eggs. In the case of the sonicates the figures for the poly U-induced increases are more closely alike when the unfertilized eggs are compared with the embryos. An interesting feature of the sonicates, as indicated in Table 3, is that the endogenous activity of the preparations can be reduced considerably while the ability of the ribosomes to respond to poly U remains as high as in the homogenates, or higher. Unfortunately, the exposures are not readily reproducible and many preparations have shown considerably reduced ribosomal activity, which evidently means that inactivation of ribosomes proceeds soon after cell destruction by this method. Four other sets of experiments of this type have given similar results. The polyuridylic acid-stimulated average increase in uptake of phenylalanine for the preparations from the fertilized eggs and from the embryos was within about 20% of the average for the unfertilized eggs. This differs somewhat from the results reported by Nemer (1962) who found the poly Ustimulated incorporation of phenylalanine by preparations from blastulae to be less than half that obtained with preparations from unfertilized eggs of Arbncia punctulata. It seems unlikely that the difference in results represents species differences. Nemer mentions that absolute levels of response to poly U seem to depend upon the batch of unfertilized eggs used, but that the relative decreases in the stimulation, upon fertilization and development, occur regularly regardless of batch. With regard to the latter statement it does not seem reasonable to me to express, as Nemer does, the poly U-stimulated incorporation of phenylalanine into protein as a multiple of the value obtained without poly U and then to conclude that since this ratio decreases markedly upon fertilization and development, this means that the ability of the ribosomes to respond decreases correspondingly. It seems to me that the comparisons for this purpose should be simply with respect to the absolute amount of the poly U-stimulated increase in incorporation of phenylalanine into protein. On this basis, as noted above, Nemer's results still show a lower activity on the part of blastulae, preparations from which exhibit about half the activity of those from unfertilized eggs. In our experiments the differences are considerably less and, for the present, our findings do not demonstrate any substantial falling-off, with development, in ribosomal activity with respect to 120 ALBERT TYLER their ability to be stimulated by poly U. Apart from this, the results of the poly U experiments on preparations from seaurchin eggs, in the three different laboratories, agree in showing that the unfertilized egg contains a considerable number of ribosomes capable of responding to poly U. The data on incorporation of labelled phenylalanine into protein may give a misleading impression of the extent of the response. Since it is a homopolymeric protein—namely, polyphenylalanine (Nirenberg and Matthaei, 1961—that is presumably formed upon addition of poly U in the presence of the C14 phenylalanine, this would be expected to show much greater radioactivity than the ordinary sea-urchin egg proteins produced in response to endogenous messenger RNA, in which the labelled phenylalanine would presumably represent a small fraction of the different amino acids present in the molecules. In any case it appears that in preparations from unfertilized eggs, and also from developing embryos, most, or many of the ribosomes are "unprogrammed" or can be "reprogrammed." One could then attribute, as does Nemer (1962), the increase in activity upon fertilization primarily to an initiation of synthesis of new messenger RNA and its attachment to the ribosomes, rather than to a neutralization of some inhibitor of ihe ribosomes or other alteration in properties of the ribosomes, as Hultin (1961) proposed. There is evidence (Gross and Cousineau, 1963) that new RNA is produced shortly after fertilization and this would accord with the above-stated view. Artificial activation of non-nucleated fragments. This proposition is being explored further in this laboratory mainly by Mr. Paul Denny. In these experiments sea-urchin eggs are separated into nucleated (light) and non-nucleated (heavy) fragments by centrifugation in tubes containing sucrose-sea-water layers of different densities. As the eggs elongate and separate into two fragments under the influence of the centrifugal force, the heavy fragments form a band on the surface ot the lowermost sucrose-sea-water laver and the TABLE 4. Incorporation of C'-L-valine into protein of liomogenates of artificially activated eggs of Strongylocentrotus purpuratus. Counts per minute* Fragment Untreated Treated t Activation non-nucleated 19 17 98 92 50% 50% nucleated 19 91 100% 14 (to = 50) 94 100% * 0.075 ml packed egg-fragments; background = 32 cpm. t One minute in 5 X 10"3 M butyric acid in sea water. light fragments collect on an upper layer. Generally, when properly performed, the procedure yields suspensions of the two kinds of fragments that are negligibly crosscontaminated. The suspensions are then exposed to butyric acid or other parthenogenetic agents, and homogenates prepared for tests of their ability to incorporate amino-acid into protein. At present there are four sets of experiments in which the conditions appeared satisfactory with respect to absence of extraneous influences and in which the eggs responded reasonably well to the artificial activation. Tn all of these the homogenates prepared from the treated non-nucleated fragments were more active than those from the untreated non-nucleated fragments with respect to the incorporation of labelled amino acid into protein under the influence of the endogenous messenger RNA. Data of one of these experiments, performed jointly, is given in Table 4. In this experiment an approximately five-fold increase in incorporating activity was obtained. Since the radioactivities for the preparations from the untreated eggs were low (as is usual) with respect to the t0 values (near background), the data can only be taken to give an order of magnitude of the response. The data in the table also show a response to artificial activation of the same order on the part of the nucleated fragments. However, the percentage of eggs that responded visibly (by membrane elevation) to the artificial activation in the case of the norv-nucleated fragments FERTILIZATION AND EARLY DEVELOPMENT 121 was only about half of that obtained with sibly by release of hydrolytic enzymes of the nucleated fragments. It would appear the lysosomes. then that the latter are not as well enPossibly, also, the DNA that has been dowed as are the activated non-nucleated reported (cf Brachet, 1960) to be present fragments with one or another of the com- in the cytoplasm of the unfertilized egg ponents of the protein-synthesizing system. may play a role in the initiation of protein In other experiments with the sea urchin synthesis. However, there are considerable Lytechinus pictus, apart from the stimula- differences in the amounts of cytoplasmic tion resulting from butyric acid treatment DNA that different workers have reported of the non-nucleated fragments, prepara- to be present, even in one and the same tions from the latter (untreated) consis- species. Also, there is considerable uncertently showed higher incorporating activity tainty about the nature of the cytoplasmic than did those from the nucleated frag- DNA and whether it really is a proper DNA at all. This is, then, an area that ments. requires considerable further exploration, In general, the results of the experiments the results of which should provide inforwith non-nucleated fragments would tend mation of great value not only with respect to favor Hultin's (1961) view of blocked to the initiation of development but for ribosomes being responsible for the in- the analysis of subsequent ontogenetic activity of the unfertilized egg. In another changes. similar experiment, Hultin (1961) exEffect of polyuridylic acid on amino acid, plored the possibility of activating the microsomes from unfertilized eggs in vitro incorporation by intact eggs and embryos. by various agents. He obtained some ac- Since, as noted at the beginning of this tivation which was relatively modest com- article, macromolecules of various kinds pared with that shown by microsomes from have been known to get into cells of varthe fertilized, or parthenogenetically acti- ious types, it seemed worthwhile to examine the possibility that the synthetic vated, eggs. There appears, then, to be evidence in polyribonucleotides might enter sea-urchin favor of both hypotheses that have been eggs at various stages of development. The advanced to account for the initiation of fact that free nucleic acids of various active protein synthesis upon fertilization. viruses (both RNA- and DNA-types) can The poly U experiments support the view infect cells is further encouragement for that messenger RNA becomes available. this kind of investigation. The effect of polyuridylic acid was exThe experiments with non-nucleated fragments, and possibly also Hultin's with rib- amined in five sets of experiments, all of osomal preparations, support the view that which showed a marked stimulation of inan inhibitor of the ribosomes is removed. corporation of phenylalanine into protein Naturally, one might also formulate com- on the part of the intact unfertilized eggs. binations of these hypotheses along with In the fertilized eggs and embryos, howother modifications involving changes, ever, poly U was generally somewhat inupon fertilization, in distribution of var- hibitory. ious ions or other constituents, or in the The results of one of these sets of exaggregation of the ribosomes that are periments is given in Table 5. The unknown, from experiments on cell-free sys- fertilized eggs had been freed of their gelatems to affect protein biosynthesis. Also, tinous coat by repeated washing. One one should take into account changes in fourth of the egg suspension was fertilized protein degradation, since after the initial on the previous evening and allowed to small supply of free amino acids and pep- develop to the hatching blastula stage, tides is utilized, the newly synthesized pro- while the remainder was kept in the cold. teins must be obtained from the partial An aliquot of one-third of the suspension or complete splitting of yolk proteins, pos- was removed and washed to provide the 122 ALBERT TYLER u TABLE 5. Influence of polyuridylic iacid on the incorporation of C -phenylalanine into protein in whole eggs and embryos of Strongylocentrotus purpuratus. Unfertilized eggs — poly U + poly U 605 768 8,485 6,235 (to = 43) Counts per minute (blanks = 47,44) De-membranated 2-4-cell stages 2-4-cell stages 20,726 20,584 13,298 17,634 (t. = 38) 20,166 23,110 11,404 15,073 (to = 44) "Hatching" blastulae 26,614 25,264 27,197 22,095 (t0 = 3 8 ) Incubated 2 hours at 20°C; suspensions contained in 2 ml ca. 4 X 10* eggs or embryos, 0.18 of C"-L-phenylalanine and 1.67 nig polyuridylic acid. unfertilized egg samples. The remainder was fertilized and divided into two equal suspensions one of which was mechanically de-membranated (passage through a pipette with a narrow bore at the tip within two minutes after insemination). Care was exercised to insure uniform distribution of eggs among the four different lots and in the incubation tubes. Incubation was terminated by addition of an equal volume of 10% trichloracetic acid (TCA), and the egg-material was washed and extracted thoroughly with hot and cold TCA and lipid solvents, dissolved in N/l NaOH and re-precipitated as described above. The effectiveness of this washing procedure is indicated by the fact that contents of the tubes (t0) that received TCA at the start of incubation gave radioactivity values that were essentially the same as the background. The differences shown by some of the duplicate tubes can be attributed mainly to losses of material occurring during the washing procedure. In the unfertilized eggs of this experiment, poly U caused a marked stimulation of incorporation of phenylalanine into protein. The difference between duplicates is relatively small. The other four experiments likewise show a stimulating effect on the unfertilized eggs, the increase in rate of incorporation ranging from 3- to 15-fold. The data for the fertilized eggs and embryos of this experiment show a decrease which is more marked in the earlier stages than in the blastulae. The results of the other experiments accord with this except for one that showed a 40% increase in eggs that were exposed at 30 minutes after fertilization. One of the experiments was run also with labelled valine (Table 6). As the data show, in the unfertilized eggs poly U stimulates incorporation of valine into protein almost to the same extent as it does that of phenylalanine. In the fertilized egg the inhibiting effect of poly U is manifested for both ami no acids. Temporary unavailability of supplies of poly U has prevented the performance of many other obvious tests concerning the mechanism of its action here. If it is acting by getting into the egg then it is not simply specifying the incorporation into protein of the single amino acid, phenylalanine, for which it contains the code. One would have to assume that, in that situation, it could interact with endogenous nucleotides and other constituents of the cell so as to form additional messenger RNA's specifying other amino acids. On the other hand the poly U might exert its effect by action on the surface of the cell, so as, for example, to alter its permeability to some constituents. The effect could not, however, be explained as an alteration of permeability to the phenylalanine and the valine since experiments with the homogenates have shown that the activity of preparations from unfertilized eggs is low even when there is complete access to the amino acids. If poly U is operating by an effect on permeability in these experiments it would seem more reasonable to consider changes in various ions known to affect activity of homogenate systems. As an example of 123 FERTILIZATION AND EARLY DEVELOPMENT TABLE 6. Influence of polyuridylic acid on the incorporation of amino acids into protein in whole unfertilized and fertilized eggs of Stiongylocentrotus purpuratus. Counts per minute C'-L-phenylalanine Unfertilized eggs Fertilized eggs -polyU +polyU 1,260 1,008 (to = 48) (t. = 47) 15,635 29,759 (bk — 39) (bk = 39) 7,808 7,795 (to = 45) (to = 44) 14,372 12,131 (bk=35) (bk = 34) C"-L-valine - poly U -fpoly U 2,240 1,499 8,765 7,921 26,470 25,672 (t0 = 38) (t0 = 35) 14,896 15,286 (to = 41) (to = 40) Incubated li/| hrs at 20°C; fertilized eggs started 15 minutes after insemination. Mixtures contained in 2 ml 1.1 X 105 eggs; 0.5 fiC of the labeled amino acids and 1.67 mg polyuridylic acid. this, experiments were performed (mainly by Dr. H. Timourian) in which the magnesium ion concentration of the medium is varied (Table 7). As the data show, incorporation of amino acid into protein falls off sharply as the Mg+ + concentration is raised. The optimum in this experiment is less than no added Mg+ + . In the unfertilized eggs that have been stimulated by poly U to incorporate amino acid into protein at a greatly accelerated rate there are no visible signs of activation even after several hours in the medium. These eggs can still be fertilized and can develop normally to the blastula stage. Fertilization can take place in the presence of the added poly U and phenylalanine. Detailed investigations of effects on later development have not been made yet, but preliminary examinations of treated cultures show greater frequencies of abnormal development starting at the late blastula stage. CONCLUDING REMARKS The present article has given an account of some experiments dealing with interactions of certain macromolecular substances, both naturally occurring and synthetic, in developmental processes. The interactions are primarily of the type characterized by complementarity in configuration of regions of the reactant molecules, as originally exemplified by antigen-antibody reactions. A general term "alleloplasts" was proposed some time ago by my col- league Dr. Sterling Emerson, to designate such substances as fertilizins and antifertilizins, blood group antigens and antibodies, or other such naturally occurring systems, as well as the immunologically produced antibodies and homologous antigens. The DNA-RNA-protein relationship would fit also into this category. Primarily interactions of alleloplasts are characterized by a high degree of specificity and thus provide a basis for the specificity, both with respect to tissues and species, of the biological processes in which they may be involved. This has been illustrated by the scheme for sperm to egg attachment, a scheme that involves interaction of the respective receptors, fertilizin and antifertilizin. The scheme also includes a possible mechanism whereby the sperm is drawn into the egg by continuation of the same process. However, as yet, there is no indication that these alleloplasts TABLE 7. Influence of Mg** on incorporation of C'1L-valine into protein of an homogenate of fertilized (1 hr) eggs of Strongylocentrotus purpuratus. ^moles of added Mg++ Counts per minute .0* 0.5 1.0 5.0 10.0 20.0 300, 315 236, 243 214, 230 57, 64 11, 17 8, 15 * Eggs themselves contain 12 micromoles per ml. Homogenate is 1 vol eggs to 2.3 vols solution. Incubation mixture in millimoles per ml: 0.04 Tris, 0.075 sucrose, 0.23 KC1, 0.01 PEP, 0.001 ATP, 0.OOOO75 C"-L-valine (4.8 C/mole); nineteen amino acids (0.00005) and 20 micrograms PEP kinase. 124 ALBERT TYLER are involved in the further processes in the initiation of development of the egg. In the present article the action of immunologically produced antibodies on unfertilized and fertilized eggs is discussed particularly with respect to experiments on sea-urchins performed in this laboratory and elsewhere. Reports of activation of unfertilized eggs by certain antisera, corresponding to a so-called "activation-antigen" have not been substantiated. Instead cytolytic and inhibitory effects have been described in experiments with unfertilized and fertilized eggs. With respect to the latter, an account has been given of experiments showing that cleavage and development can be blocked by antisera prepared against purified fertilizin but not by antisera containing only antibodies against internal constituents of the cell. The limitations that this imposes on attempts to influence development in specific ways are discussed, and it is suggested that at present specific antibodies may find their most effective use as analytical tools for help in unravelling the components of specific processes in differentiation. 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