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Physico-chemical properties of the fertilizins of the sea urchin Arbacia punctulata and the sand dollar Echinarachnius parma
Experimental
Cell Research, 10, 3iS-38G (1956)
377
PHYSICO-CHEMICAL PROPERTIES OF THE FERTILIZINS OF THE SEA URCHIN ARBACIA PUNCTULATA AND THE SAND DOLLAR
ECHINARACHNI
US PARMA’
A. TYLER Kerckhoff
Laboratories
of Biology, California
Institute
of Technology, Pasadena, Calif., U.S.A.
THE: fertilizins of eggs of sea urchins and other animals are characterized ordinarily by their ability, when in solution, to cause an agglutination of the homologous sperm. Lillie [ 1 l] originally considered that the unfertilized eggs continuously secreted fertilizin as they remained in sea water. However, later experiments by Tyler and Fox [30, 311 and Tyler [20, 21, 221 demonstrated that the source of fertilizin is the gelatinous coat of the egg. The coat slowly dissolves as the unfertilized eggs remain in sea water, giving the false impression of the substance being secreted by the egg. If the gelatinous coat is rapidly dissolved, as by means of sea water acidified to between pH 3 and 5, the amount of fertilizin obtained is as great, or greater, than can be obtained by permitting eggs to “secrete” in sea water for a prolonged period of time. The evidence shows also that there is no macromolecular constituent other than fertilizin constituting the gelatinous coat of the egg. The view that the gelatinous coat of the egg is the source of fertilizin has been corroborated by experiments of Hartmann [7], Evans et 01. [4], Runnstriim and Lindrall [IS], \Tasseur and Hagstrom [37], and Monroy et al. [15]. It has been reported by hlotomura [ 16, 171 that a “cytofertilizin” can also be extracted in relatively lo\\- titer by special methods from eggs that hare been deprived of their gelatinous coat. It has been suggested [l] that the eggs had not been completely denuded. Assuming that proper precautions had been taken by Motomura completely to denude the eggs, this obervation would be of interest in connection with such questions as whether the gelatinous coat is originally formed by the follicle cells of the ovary or is a product of the oocyte itself. The presence of a small amount of fertilizin within the egg would tend to favor the former view, which has been adopted by Tyler [23, 25, 271 rather than the latter which Vasseur [35, 361 considers the more likely. It should be noted, in any case, that the growing oocyte can absorb large molecular substances from its surroundings, as illustrated in 1 Submitted in comlection .Ipril 26-30, 1955.
with the Symposium
on the Physiology
of Fertilization,
Experimental
Palermo,
Cell Research 10
the experiments of Hevesy and Hahn [8, 91 Chargaff [2], Lorenz et al. [12] and others. There is, as yet, no evidence that this may be the case for fertilizin, which is not found in the body fluids or tissues other than the ovary of sea urchins. The finding by Vasseur [35, 361 that ovaries of post-spawning animals yield some fertilizin may well be due to the previous presence of the eggs which have left some gelatinous coat material behind upon spawning or, in many instances, have undergone atresia. Solutions of sea urchin fertilizin that are electrophoretically homogeneous and from which practically all nitrogen containing substance is absorbable by homologous sperm are rather easily prepared. Such solutions can be obtained by collecting the eggs in cold sea water, washing with several changes of cold sea water and then extracting with acidified (pH 3 to 5, depending on the species) sea water, precautions being taken to avoid damage to the eggs. Further processing and concentration can be done by dialysis and precipitation with alcohol or ammonium sulphate, as earlier described [25, 271. Preparations of fertilizin of sea urchins were early found to give both protein and carbohydrate reactions [30, 31, 22, 191 and from later work [23, 24, 25, 10, 33, 34, 361 it has become clear that fertilizin belongs to the class of large molecular substances that may be termed glycoproteins. There are roughly equal amounts of sugar, amino acid and sulphate present in the molecule. In most species that have been examined there are two kinds of sugars and twelve or more different amino acids. et al. [19] and by Vasseur [33, 34, 361 the In the reports by Runnstriim term “jelly coat substance” rather than fertilizin is employed. However, in view of the above mentioned evidence of identity of the two, it seems reasonable to assume that their analyses apply to what is termed fertilizin. It should also be noted that fertilizins may be readily converted into a nonagglutinating form (termed univalent) by relatively mild treatments and that, in certain species, the fertilizin-sperm system may be ordinarily non-agglutinating but still specifically interacting [21, 22, 23, 13, 141. It seems reasonable to suppose, then, that in investigations such as those of Runnstrijm et al. [19] on the non-agglutinating jelly coat substance of Psammechinus miliaris, the analyses apply to what may be termed the fertilizin of the species. Runnstriim et al. [19] subjected a solution of jelly coat substance of Psammechinus miliaris to ultracentrifugation and obtained a main component sedimenting with a sharp boundary and a sedimentation constant of 2.9 x lo-l3 along with a small amount of extremely rapidly sedimenting substance. This solution contained 0.26 mg N per ml which, assuming an approximately 5 per cent nitrogen content, would mean that it contained about 0.5 per cent Experimental
Cell Research 10
Physico-chemical properties of fertilizins jelly coat substance. Runnstrijm et al. mention that the sedimentation constant of the chief component is highly concentration-dependent, a feature that is characteristic of elongate, gel-forming molecules. Tyler [24, 251 reported a sedimentation constant of 6.3 X lo-15 for fertilizin preparations of Stronyylocentrotus purpuratus which showed only a single boundary in the ultracentrifuge. The solutions were run at concentrations of 0.1 to 0.2 per cent. This higher sedimentation constant might well be due to the determinations having been made on more dilute solutions, as well as to species differences. From the latter value the molecular weight, calculated for spherical shape of the molecules, would be approximately 80,000. However, since the indications are that the molecule is rather elongate the actual molecular weight would be considerably greater. In order to obtain values for the actual molecular weight and shape of the molecule, measurements were therefore made of the diffusion constants along with the sedimentation constants of the fertilizins of Arbacia and of Echinarachnius. For further characterization of the substance measurements were also made of electrophoretic mobility and viscosity. Brief reports of these values have been previously presented [28, 291. MATERIAL
AND
METHODS
Eggs of the sea urchin Arbacia punctulata and of the sand-dollar Echinarachnius parma were obtained by the method of injection of the animals with isotonic KC1 [26]. They were collected in ice-cold filtered sea water and washed a minimum of four times with the cold sea water, at least 20 volumes of the sea water being used per volume of settled eggs for each washing. After the final washing the suspensions were usually concentrated to approximately 10 per cent by volume of settled eggs. The eggs were allowed to settle, the supernatant solution drawn off, centrifuged at 2000 to 3000 xg for about 20 minutes and filtered through rapid filter paper. The solution was then dialyzed against glass-distilled water in the cold. Some samples were precipitated with alcohol but most of the preparations employed were concentrated by evaporation of water from the dialysis bags suspended in front of a fan in a dry room. The bags were suspended on a motor driven “bumper” to reduce deposition of the fertilizin on the dried portions of the cellophane tubing. Samples were then removed for dry weight and Kjeldahl nitrogen determinations and dialysis of the remainder continued against the particular buffer solutions to be employed, precautions being taken to avoid further changes in the concentration of the fertilizin within the dialysis bags. RESULTS
Sedimentation velocity.-Determinations of sedimentation rate were made in a Spinco analytical ultracentrifuge. Runs were made on three samples of Arbacia fertilizin and one of Echinarachnius. The Arbacia samples were: Experimental
Cell Research 10
380
A. Tyler
(A) 0.92 per cent solution in M/l0 phosphate buffer at pH 6.95, (B) 0.46 per cent in M/l0 phosphate buffer at pH 6.95 (two-fold dilution of (A)), and (C) 0.21 per cent in M/l0 borate buffer at pH 9.02. The Echirxrachnius sample was a 0.39 per cent solution in M/l0 phosphate buffer at pH 7.02. Photographic records mere taken at the time that the refractive boundary was first distinctly visible and at four successive 16 minute intervals thereafter. The first four frames of each of these runs are reproduced in Fig. 1. The displacement of the peak was measured with a metal rule under a magnification of 30-fold, and the sedimentation coefficients were calculated from the equation:
where .rl and .zZ are the distances of the peak from the center of rotation at successive times t, and t, respectively (t,-t, =960 seconds), and w is the angular velocity (w2 =3.92 x lo5 radians2/sec2). The measurements of displacement of the peak for four successive 16-minute time intervals for each of the four runs gave the following values for the sedimentation coefficients: Time interval (minutes) . Arbacia fertilizin (A) . . . Arbczcia fertilizin (B) . . . Arbacia fertilizin (C) . . . Echinarachnius fertilizin (D)
O-16 4.46 5.76 6.73 4.80
16-32 4.40 5.77 6.73 4.80
32-48 4.33 5.76 6.73 4.77
48-64 4.29 5.78 6.73 4.60
Average 4.37 Svedbergs 5.77 ,) 6.73 ,, 4.74 ,,
The measurements on the Arbacia fertilizin vere not made over a sufficient range of concentration to warrant extrapolation to infinite dilution. They sho\v the marked increase in sedimentation constant with decreasing concentration typical of elongate macromolecules. The higher value obtained at the lower concentration is, of course, colser to what might be obtained by extrapolation to infinite dilution and will be used in subsequent calculations. This may be assumed to be not too far from what would be gotten by extrapolation. The value (szO =4.74 x lo-13) for the Echinarachnius preparation (0.39 per cent solution) is lower than that (szO =5.77 x 10-13) obtained for a more concentrated (0.46 per cent) solution of Arbacia fertilizin. This would indicate that the Echinarachnius fertilizin is of smaller molecular size or lower axial ratio or both. Determinations of the relative viscosities, in an Ostwald viscometer, of the four preparations, A, B, C and II, gave the values (cps) 8.22, 2.80, 1.52, and 2.26, respectively. Experimentd
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Physico-chemical properties of fertilizins
Fig. 1. Photographs of refractive index boundary of fertilizin preparations run in the Spinco analytical ultracentrifuge at 60,000 rpm. The pictures were taken at 16.minute intercals and the sequence is right to left, which is also the direction of sedimentation. Lateral Nag. = 1.48. A, B, and C: Arbncia fertilizin at 0.92 per cent in M/l0 phosphate at pH 6.95, 0.46 per cent in M/l0 phosphate at pH 6.95, and 0.21 per cent in .I\r/lO borate at pH 9.02. D: Echinnrachnius fertilizin at 0.39 per cent in .\I/10 phosphate at pH 7.02. Experimental
Cell Research Jr
382
A. Tyler
Partial specific r/olonle.-The apparent partial specific volume was determined by means of a micro-balance and a 1 ml pycnometer. For this purpose a 11.585 mg sample of alcohol precipitated and dried Arbacia fertilizin was dissolved in ;V/lOOO SaOH. Calculation of the results (see :3], p. 562), gave a value of 0.725. Diffusion rate.-Measurements were made of the rate of diffusion of the 0.21 per cent solution of Arbacia fertilizin in M/l0 borate buffer at pH 9.02. This was done in a Perkin-Elmer electrophoresis apparatus at 20” C, and over a period of 10 hours. From the measurements of the change in shape of the refractive index boundary, calculations by the maximum ordinate-area method (Greenberg [6] p. 358) g ave a value for the diffusion coefficient, D, of 2.16 x lo-’ cm2/sec. Electrophoretic mobilities.-Determinations of electrophoretic mobilities were made in the Perkin-Elmer design of the Tiselius apparatus. Three in 0.2 per cent solutions at different pH runs wxe made on Arbacia fcrtilizin and one run was made on 0.2 per cent Echinarachnius fertilizin. The following values were obtained:
cm2/sec/rolt Arbacia fertilizin in ,U =O.l phosphate at pH 7.02 Arbacia fertilizin in p =O.l acetate at pH 4.84 Arbacia fertilizin in ,u =0.05 HCl-KC1 at pH 2.04 Echinarachnius fertilizin in ,LL=O.l phosphate at pH 7.02
1.99 1.57 2.02 1.82
x 10-4 x10-4 x 10-d x 1O-4
These values are in the general range reported previously by Runnstrijm et al. [19] for the gelatinous coat substance of Psammechinus miliaris and by Tyler [25, 271 for the fertilizin of Strongylocentrotus purpuratus, and the rapid mobility is indicative of the highly acidic character of the substance. However, the value for the Arbacin fertilizin at pH 2 seems anomalous with respect to those at the two higher pH’s. This is presumably not due to its having been run at 0.05 molar ionic strength rather than 0.1 molar, since the correction would entail only about 20 per cent reduction of the mobility. It is more likely due to the fact that the fertilizins are unstable below pH 3, as shown by loss of agglutinating activity (though specific reactivity -\vith sperm may be retained). There may then be a splitting-off of some uncharged portions of the molecule at this pH, \vhich would account for the failure of the mobility to decrease regularly with decrease in pH. Amount of fertilizin bound by sperm.-Two experiments were run to determine the amount of Arbacia fertilizin absorbed by homologous sperm. Samples of the fertilizin solutions at 0.2 per cent in sea \vater were absorbed for Experimental
Cell Research 10
Physico-chemical properties of fertilizins
383
one hour at room temperature with equal volumes of serial dilutions of sperm suspension of known sperm concentration. The sperm were removed by centrifugation at about 5000 g for three minutes and the supernatants titrated for sperm-agglutination activity. From the lowest concentration of sperm that just removed detectable fertilizin from the solutions in the two experiments it was calculated that 10’” sperm absorb 0.45 to 0.58 mg of this material. DISCUSSION
Molecular size and shape.-From the values determined above of the sedimentation coefficient, szaO=6.73 x lo-13, for the 0.2 per cent solution of Arbacia fertilizin, of the diffusion coefficient, D,,- =2.16 x 10-T for a similar solution, of the partial specific volume, V =0.725 and solvent density, Q =1.007 a molecular weight of 280,000 is obtained, using the standard equation M = R Ts/D (1 -VQ) in which R T is the product of the gas constant and absolute temperature. A detailed evaluation of the range of error of the various determinations has not been made. However, it appears that the value for the diffusion coefficient is subject to the greatest error, because of the small size of the refractive index boundaries measured, and it is estimated its value may be in error by as much as 20 per cent. The previously reported [28] figure of 300,000 represents what is considered to be a reasonable approximation when allowance is made for extrapolations to infinite dilution and most likely direction of errors. From the same data (giving a molecular weight of 280,000 for Arbacia fertilizin) a value for the frictional ratio (f/fJ can be calculated (see [6], p. 392). The value obtained, 2.275, corresponds to an axial ratio of the molecule of 28 to 1 calculated as an unhydrated prolate ellipsoid. Assuming 0.4 gram of water per gram of fertilizin, the frictional ratio corrected for hydration1 would be 1.96 and the corresponding axial ratio would be 20 to 1. In the absence of diffusion data for the Echinarachnius fertilizin no calculations were made of its molecular dimensions. Assuming that its diffusion 1 In the excellent chapter by Lundgren and Ward in Amino Acids and Proteins edited by Greenberg [S] there are evidently arithmetical errors in the calculations on the bottom of p. 393 of the factor f/l, in the Kraemer relation used to correct for effect of hydration. For r=O.2 the value of f/f, from this equation should be 1.085 instead of 1.01, and for r = 0.8 it is 1.28 instead of 1.04. The erroneous figures would indicate little effect of correction for water of hydration on the calculated axial ratios, whereas the effect is evidently quite large. Experimental
Cell Research 10
constant was similar to that of Arbacia and hydration about the same, the relatively lower sedimentation constant indicates smaller molecular size or lower axial ratio or both. Number of charged groups per molecule.-From the calculated molecular weight of the Arbacia fertilizin and the data on electrophoretic mobility, it is possible to estimate the number of charged groups per molecule, by making use of Stokes’ law for spherical particles and adopting a resistance factor to apply to cylindrical particles. Valences estimated in this way show 14.25 effective negatively charged groups per molecule at pH 7 and 11.25 at pH 4.85. The isoelectric points of fertilizins are evidently very low, as has been previously noted [19, 251. It seems doubtful that this can be reached without alterations of the molecule induced by low pH. The highly acidic nature of fertilizin is evidently due to the high content of ester-linked sulphate discovered by Vasseur [33]. Number of molecules of fertilizin bound per spermatozoon.-In the experiments described above 10 lo Arbacia spermatozoa were found to combine with an average maximum of 0.5 mg of fertilizin. For fertilizin of 300,000 molecular weight this would be 1015 molecules or lo5 per spermatozoon. There is evidence [25] that the antifertilizin of the sperm is confined to the region of the head between acrosome and midpiece. This is estimated (as a 1 x 3 micron cylinder) to have a surface area of 9 square microns. Fertilizin of 300,000 molecular weight and 0.725 partial specific volume would have a molecular volume of 3.6 x lo-l9 cm3. As a prolate ellipsoid of 20 to 1 axial ratio its short axis would be 3.2 x lo-’ cm and its greatest cross-sectional area would be 8 x 10-B square microns. Attached end on, then, the 105 molecules of fertilizin bound by a spermatozoon would effectively cover about 10 per cent of the reactive surface of the head. It is of some significance that the sperm head is evidently richly provided with receptor groups. The figure of 100,000 such groups represents our first approximation, but it is clear that revisions of molecular weight within the range of error indicated will still give a high figure for the reactive sites on the sperm. It can also be assumed that this is a minimum figure since it seems likely that all reactive sites may not be available for combination with fertilizin, in the absorption experiments. Being so richly provided with reactive sites it now appears more readily understandable that the spermatozoon can effect the rapid union that is customarily observed upon its contact with the egg. Since, upon fertilization the entering sperm reacts with the fertilizin of the Experimental
Cell Research 10
Physico-chemical properties of fertilizins
385
gelatinous coat and surface of the egg it seems likely that the fertilizin thus bound may be brought inside the egg. The question may then be raised as to whether or not fertilizin thus transferred into an internal location may play any role in the activation of the egg. Professor Albert0 Monroy and the writer have undertaken some experiments to examine this by means of microinjection, but certain uncertainties inherent in micro-injection methods (see [32]) have so far frustrated these attempts. It is, however, of interest to note that an analagous proposal has been made [5] for virus penetration of susceptible cells, in that it is considered that the incorporation of a considerable portion of the cell surface material results in the production of complete as opposed to incomplete virus particles. SUMMARY
Measurements have been made of the sedimentation rates, diffusion, viscosity and electrophoretic mohilities of the fertilizins of the sea urchin Arbacia punctulata and the sand dollar Echinarachnius parma. From the data for Arbacia a molecular weight of 280,000 is calculated, and a value of 300,000 considered more probable. The axial ratio is estimated to be 28 to 1, calculated for an unhydrated prolate ellipsoid and 20 to 1 if 0.4 gram of water is assumed to be bound per gram of fertilizin. The Echinarachnius fertilizin sedimented more slowly than that of Arbacia. The measurements of electrophoretic mobilities show these fertilizins to be highly acidic as had been found in other species. This data also permitted calculations of valence for Arbacia fertilizin as 14.25 effective negatively charged groups per molecule at pH 7 and 11.25 at pH 4.85. Measurements of the amount of fertilizin absorbed show that a single spermatozoon binds approximately 100,000 molecules, and therefore possesses at least that many receptor sites. The relation of this to certain features of fertilization is briefly discussed. This investigation was supported by a research grant (C-2302) from the National Cancer Institute of the National Institutes of Health, Public Health Service. The experimental work was carried out at the Marine Biological Laboratory, Woods Hole, Massachusetts. I am indebted to Mr. James S. Tyler and Mr. Alan Burbank for technical assistance. REFERENCES 1. BYERS, H. L., Biol. Bull. 101, 218 (1951). 2. CHARGAFF, E., J. Biol. Chem. 142, 285 (1942). 3. EDSALL, J. T., The Proteins. Ed. by H. Neurath and K. Bailey,
Vol.
I, Part
Experimental
B, p. 549,
1953.
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A. Tyler 4. EVANS, T. C., BEAMS, I-I. W., and SMITH, M. E., Hal. Butt. 80, 363 (1941). S. and GRAHAM, D. M., Brit. J. Erptf. Pafhol. 36, 205 (1955). 5. FAZEKAS DE ST. GROTH, Theory, Methods, Applica6. GREENBERG, D. RI. (camp. & ed. by), Amino Acids and Proteins; 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.
tions. Springfield, Ill., 1951. HARTMANN, M., Naturwissenschaffen 28, 144 (1940). HEVESY, G., Aduances in Enzymol. 7, 111 (1947). HEVF.SY, G. and HAHN, L., Kgl. Danske Videnskabernes Sefskab. Biol. Medd. 14, (2), 3 (1938). KRAUSS, M., Biol. Bull. 96. 74 (1949). LILLIE, F. R., Problems of Fertilization. University of Chicago Press, 1919. LORENZ. F. W.. PERLMAN. I.. and CHAIKOFF, I. L.. Am. J. Phwiol. 138. 318 (1943). , METZ, c. B., L?iol. Bull. 82, 446 (1942). ~ Viol. Buff. 89, 84 (1945). MONROY, A., Tosr, L., GIARDINA, G., and MAGGIO, R., Biol. Bull. 106, 169 (1954). MOTOMURA, I., Sci. Rep&. Tdhoku Uniu., 4fh Ser., Biol. 18, 554 (1950). __ Sci. Rep&-. Tdhoku Uniu., 4th Ser., Biol. 20, 93 (1953). RUNNSTR~M, J. and LINDVALL, S., Arkiu Zool. 38 A, No. 10 (1946). RUNNSTRBY, J., TISELIUS, A., and LINDVALL, S., Arkiu Zool. 35A, No. 3 (1942). TYLER, A., Biof. Bull. 78, 159 (1940). __ Biol. Bull. 81, 190 (1941). -Western J. Surg., ObsfeL’and Gynecol. 50, 126 (1942). -Physiof. Reu. 28, 180 (1948 a). __ A&f. Rec. 101, 8 (1948 b). ~ Am. Naturalist 83, 195 (1949 a). ~ The Collecting Net 19: No. 1 (1949 b). -in Analysis of Development, p. 170. Ed. by B. H. Willier, P. A. Weiss and V. Hamburger. W. B. Saunders Co., Philadelphia, 1955. TYLER, A., BURBANK, A., and TYLER, J., Biof. Bull. 107, 303 (1954). __ Biof. Buff. 107, 304 (1954). TYLER, A. and Fox, S. W., Science 90, 516 (1939). -Mol. Buff. 79, 153 (1940). TYLER, A. and MONROY, A., Biol. Bull., 109, 370 (1955). VASSEUR, E., Acta them. Stand. 2, 900 (1948 a). __ Acfa them. Stand. 2. 693 (1948 b). ~ Acfa Borelia. A. Scientia.,‘No. 2’(1951). -The Chemistry and Physiology of the Jelly Coat of the Sea Urchin Egg. Stockholm, 1952. VASSEUR, E. and HAGSTR~~M, B., Arkiu Zool. 37A, No. 17 (1946).
Experimental
Cell Research 10
Cell Research, 10, 3iS-38G (1956)
377
PHYSICO-CHEMICAL PROPERTIES OF THE FERTILIZINS OF THE SEA URCHIN ARBACIA PUNCTULATA AND THE SAND DOLLAR
ECHINARACHNI
US PARMA’
A. TYLER Kerckhoff
Laboratories
of Biology, California
Institute
of Technology, Pasadena, Calif., U.S.A.
THE: fertilizins of eggs of sea urchins and other animals are characterized ordinarily by their ability, when in solution, to cause an agglutination of the homologous sperm. Lillie [ 1 l] originally considered that the unfertilized eggs continuously secreted fertilizin as they remained in sea water. However, later experiments by Tyler and Fox [30, 311 and Tyler [20, 21, 221 demonstrated that the source of fertilizin is the gelatinous coat of the egg. The coat slowly dissolves as the unfertilized eggs remain in sea water, giving the false impression of the substance being secreted by the egg. If the gelatinous coat is rapidly dissolved, as by means of sea water acidified to between pH 3 and 5, the amount of fertilizin obtained is as great, or greater, than can be obtained by permitting eggs to “secrete” in sea water for a prolonged period of time. The evidence shows also that there is no macromolecular constituent other than fertilizin constituting the gelatinous coat of the egg. The view that the gelatinous coat of the egg is the source of fertilizin has been corroborated by experiments of Hartmann [7], Evans et 01. [4], Runnstriim and Lindrall [IS], \Tasseur and Hagstrom [37], and Monroy et al. [15]. It has been reported by hlotomura [ 16, 171 that a “cytofertilizin” can also be extracted in relatively lo\\- titer by special methods from eggs that hare been deprived of their gelatinous coat. It has been suggested [l] that the eggs had not been completely denuded. Assuming that proper precautions had been taken by Motomura completely to denude the eggs, this obervation would be of interest in connection with such questions as whether the gelatinous coat is originally formed by the follicle cells of the ovary or is a product of the oocyte itself. The presence of a small amount of fertilizin within the egg would tend to favor the former view, which has been adopted by Tyler [23, 25, 271 rather than the latter which Vasseur [35, 361 considers the more likely. It should be noted, in any case, that the growing oocyte can absorb large molecular substances from its surroundings, as illustrated in 1 Submitted in comlection .Ipril 26-30, 1955.
with the Symposium
on the Physiology
of Fertilization,
Experimental
Palermo,
Cell Research 10
the experiments of Hevesy and Hahn [8, 91 Chargaff [2], Lorenz et al. [12] and others. There is, as yet, no evidence that this may be the case for fertilizin, which is not found in the body fluids or tissues other than the ovary of sea urchins. The finding by Vasseur [35, 361 that ovaries of post-spawning animals yield some fertilizin may well be due to the previous presence of the eggs which have left some gelatinous coat material behind upon spawning or, in many instances, have undergone atresia. Solutions of sea urchin fertilizin that are electrophoretically homogeneous and from which practically all nitrogen containing substance is absorbable by homologous sperm are rather easily prepared. Such solutions can be obtained by collecting the eggs in cold sea water, washing with several changes of cold sea water and then extracting with acidified (pH 3 to 5, depending on the species) sea water, precautions being taken to avoid damage to the eggs. Further processing and concentration can be done by dialysis and precipitation with alcohol or ammonium sulphate, as earlier described [25, 271. Preparations of fertilizin of sea urchins were early found to give both protein and carbohydrate reactions [30, 31, 22, 191 and from later work [23, 24, 25, 10, 33, 34, 361 it has become clear that fertilizin belongs to the class of large molecular substances that may be termed glycoproteins. There are roughly equal amounts of sugar, amino acid and sulphate present in the molecule. In most species that have been examined there are two kinds of sugars and twelve or more different amino acids. et al. [19] and by Vasseur [33, 34, 361 the In the reports by Runnstriim term “jelly coat substance” rather than fertilizin is employed. However, in view of the above mentioned evidence of identity of the two, it seems reasonable to assume that their analyses apply to what is termed fertilizin. It should also be noted that fertilizins may be readily converted into a nonagglutinating form (termed univalent) by relatively mild treatments and that, in certain species, the fertilizin-sperm system may be ordinarily non-agglutinating but still specifically interacting [21, 22, 23, 13, 141. It seems reasonable to suppose, then, that in investigations such as those of Runnstrijm et al. [19] on the non-agglutinating jelly coat substance of Psammechinus miliaris, the analyses apply to what may be termed the fertilizin of the species. Runnstriim et al. [19] subjected a solution of jelly coat substance of Psammechinus miliaris to ultracentrifugation and obtained a main component sedimenting with a sharp boundary and a sedimentation constant of 2.9 x lo-l3 along with a small amount of extremely rapidly sedimenting substance. This solution contained 0.26 mg N per ml which, assuming an approximately 5 per cent nitrogen content, would mean that it contained about 0.5 per cent Experimental
Cell Research 10
Physico-chemical properties of fertilizins jelly coat substance. Runnstrijm et al. mention that the sedimentation constant of the chief component is highly concentration-dependent, a feature that is characteristic of elongate, gel-forming molecules. Tyler [24, 251 reported a sedimentation constant of 6.3 X lo-15 for fertilizin preparations of Stronyylocentrotus purpuratus which showed only a single boundary in the ultracentrifuge. The solutions were run at concentrations of 0.1 to 0.2 per cent. This higher sedimentation constant might well be due to the determinations having been made on more dilute solutions, as well as to species differences. From the latter value the molecular weight, calculated for spherical shape of the molecules, would be approximately 80,000. However, since the indications are that the molecule is rather elongate the actual molecular weight would be considerably greater. In order to obtain values for the actual molecular weight and shape of the molecule, measurements were therefore made of the diffusion constants along with the sedimentation constants of the fertilizins of Arbacia and of Echinarachnius. For further characterization of the substance measurements were also made of electrophoretic mobility and viscosity. Brief reports of these values have been previously presented [28, 291. MATERIAL
AND
METHODS
Eggs of the sea urchin Arbacia punctulata and of the sand-dollar Echinarachnius parma were obtained by the method of injection of the animals with isotonic KC1 [26]. They were collected in ice-cold filtered sea water and washed a minimum of four times with the cold sea water, at least 20 volumes of the sea water being used per volume of settled eggs for each washing. After the final washing the suspensions were usually concentrated to approximately 10 per cent by volume of settled eggs. The eggs were allowed to settle, the supernatant solution drawn off, centrifuged at 2000 to 3000 xg for about 20 minutes and filtered through rapid filter paper. The solution was then dialyzed against glass-distilled water in the cold. Some samples were precipitated with alcohol but most of the preparations employed were concentrated by evaporation of water from the dialysis bags suspended in front of a fan in a dry room. The bags were suspended on a motor driven “bumper” to reduce deposition of the fertilizin on the dried portions of the cellophane tubing. Samples were then removed for dry weight and Kjeldahl nitrogen determinations and dialysis of the remainder continued against the particular buffer solutions to be employed, precautions being taken to avoid further changes in the concentration of the fertilizin within the dialysis bags. RESULTS
Sedimentation velocity.-Determinations of sedimentation rate were made in a Spinco analytical ultracentrifuge. Runs were made on three samples of Arbacia fertilizin and one of Echinarachnius. The Arbacia samples were: Experimental
Cell Research 10
380
A. Tyler
(A) 0.92 per cent solution in M/l0 phosphate buffer at pH 6.95, (B) 0.46 per cent in M/l0 phosphate buffer at pH 6.95 (two-fold dilution of (A)), and (C) 0.21 per cent in M/l0 borate buffer at pH 9.02. The Echirxrachnius sample was a 0.39 per cent solution in M/l0 phosphate buffer at pH 7.02. Photographic records mere taken at the time that the refractive boundary was first distinctly visible and at four successive 16 minute intervals thereafter. The first four frames of each of these runs are reproduced in Fig. 1. The displacement of the peak was measured with a metal rule under a magnification of 30-fold, and the sedimentation coefficients were calculated from the equation:
where .rl and .zZ are the distances of the peak from the center of rotation at successive times t, and t, respectively (t,-t, =960 seconds), and w is the angular velocity (w2 =3.92 x lo5 radians2/sec2). The measurements of displacement of the peak for four successive 16-minute time intervals for each of the four runs gave the following values for the sedimentation coefficients: Time interval (minutes) . Arbacia fertilizin (A) . . . Arbczcia fertilizin (B) . . . Arbacia fertilizin (C) . . . Echinarachnius fertilizin (D)
O-16 4.46 5.76 6.73 4.80
16-32 4.40 5.77 6.73 4.80
32-48 4.33 5.76 6.73 4.77
48-64 4.29 5.78 6.73 4.60
Average 4.37 Svedbergs 5.77 ,) 6.73 ,, 4.74 ,,
The measurements on the Arbacia fertilizin vere not made over a sufficient range of concentration to warrant extrapolation to infinite dilution. They sho\v the marked increase in sedimentation constant with decreasing concentration typical of elongate macromolecules. The higher value obtained at the lower concentration is, of course, colser to what might be obtained by extrapolation to infinite dilution and will be used in subsequent calculations. This may be assumed to be not too far from what would be gotten by extrapolation. The value (szO =4.74 x lo-13) for the Echinarachnius preparation (0.39 per cent solution) is lower than that (szO =5.77 x 10-13) obtained for a more concentrated (0.46 per cent) solution of Arbacia fertilizin. This would indicate that the Echinarachnius fertilizin is of smaller molecular size or lower axial ratio or both. Determinations of the relative viscosities, in an Ostwald viscometer, of the four preparations, A, B, C and II, gave the values (cps) 8.22, 2.80, 1.52, and 2.26, respectively. Experimentd
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Physico-chemical properties of fertilizins
Fig. 1. Photographs of refractive index boundary of fertilizin preparations run in the Spinco analytical ultracentrifuge at 60,000 rpm. The pictures were taken at 16.minute intercals and the sequence is right to left, which is also the direction of sedimentation. Lateral Nag. = 1.48. A, B, and C: Arbncia fertilizin at 0.92 per cent in M/l0 phosphate at pH 6.95, 0.46 per cent in M/l0 phosphate at pH 6.95, and 0.21 per cent in .I\r/lO borate at pH 9.02. D: Echinnrachnius fertilizin at 0.39 per cent in .\I/10 phosphate at pH 7.02. Experimental
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Partial specific r/olonle.-The apparent partial specific volume was determined by means of a micro-balance and a 1 ml pycnometer. For this purpose a 11.585 mg sample of alcohol precipitated and dried Arbacia fertilizin was dissolved in ;V/lOOO SaOH. Calculation of the results (see :3], p. 562), gave a value of 0.725. Diffusion rate.-Measurements were made of the rate of diffusion of the 0.21 per cent solution of Arbacia fertilizin in M/l0 borate buffer at pH 9.02. This was done in a Perkin-Elmer electrophoresis apparatus at 20” C, and over a period of 10 hours. From the measurements of the change in shape of the refractive index boundary, calculations by the maximum ordinate-area method (Greenberg [6] p. 358) g ave a value for the diffusion coefficient, D, of 2.16 x lo-’ cm2/sec. Electrophoretic mobilities.-Determinations of electrophoretic mobilities were made in the Perkin-Elmer design of the Tiselius apparatus. Three in 0.2 per cent solutions at different pH runs wxe made on Arbacia fcrtilizin and one run was made on 0.2 per cent Echinarachnius fertilizin. The following values were obtained:
cm2/sec/rolt Arbacia fertilizin in ,U =O.l phosphate at pH 7.02 Arbacia fertilizin in p =O.l acetate at pH 4.84 Arbacia fertilizin in ,u =0.05 HCl-KC1 at pH 2.04 Echinarachnius fertilizin in ,LL=O.l phosphate at pH 7.02
1.99 1.57 2.02 1.82
x 10-4 x10-4 x 10-d x 1O-4
These values are in the general range reported previously by Runnstrijm et al. [19] for the gelatinous coat substance of Psammechinus miliaris and by Tyler [25, 271 for the fertilizin of Strongylocentrotus purpuratus, and the rapid mobility is indicative of the highly acidic character of the substance. However, the value for the Arbacin fertilizin at pH 2 seems anomalous with respect to those at the two higher pH’s. This is presumably not due to its having been run at 0.05 molar ionic strength rather than 0.1 molar, since the correction would entail only about 20 per cent reduction of the mobility. It is more likely due to the fact that the fertilizins are unstable below pH 3, as shown by loss of agglutinating activity (though specific reactivity -\vith sperm may be retained). There may then be a splitting-off of some uncharged portions of the molecule at this pH, \vhich would account for the failure of the mobility to decrease regularly with decrease in pH. Amount of fertilizin bound by sperm.-Two experiments were run to determine the amount of Arbacia fertilizin absorbed by homologous sperm. Samples of the fertilizin solutions at 0.2 per cent in sea \vater were absorbed for Experimental
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Physico-chemical properties of fertilizins
383
one hour at room temperature with equal volumes of serial dilutions of sperm suspension of known sperm concentration. The sperm were removed by centrifugation at about 5000 g for three minutes and the supernatants titrated for sperm-agglutination activity. From the lowest concentration of sperm that just removed detectable fertilizin from the solutions in the two experiments it was calculated that 10’” sperm absorb 0.45 to 0.58 mg of this material. DISCUSSION
Molecular size and shape.-From the values determined above of the sedimentation coefficient, szaO=6.73 x lo-13, for the 0.2 per cent solution of Arbacia fertilizin, of the diffusion coefficient, D,,- =2.16 x 10-T for a similar solution, of the partial specific volume, V =0.725 and solvent density, Q =1.007 a molecular weight of 280,000 is obtained, using the standard equation M = R Ts/D (1 -VQ) in which R T is the product of the gas constant and absolute temperature. A detailed evaluation of the range of error of the various determinations has not been made. However, it appears that the value for the diffusion coefficient is subject to the greatest error, because of the small size of the refractive index boundaries measured, and it is estimated its value may be in error by as much as 20 per cent. The previously reported [28] figure of 300,000 represents what is considered to be a reasonable approximation when allowance is made for extrapolations to infinite dilution and most likely direction of errors. From the same data (giving a molecular weight of 280,000 for Arbacia fertilizin) a value for the frictional ratio (f/fJ can be calculated (see [6], p. 392). The value obtained, 2.275, corresponds to an axial ratio of the molecule of 28 to 1 calculated as an unhydrated prolate ellipsoid. Assuming 0.4 gram of water per gram of fertilizin, the frictional ratio corrected for hydration1 would be 1.96 and the corresponding axial ratio would be 20 to 1. In the absence of diffusion data for the Echinarachnius fertilizin no calculations were made of its molecular dimensions. Assuming that its diffusion 1 In the excellent chapter by Lundgren and Ward in Amino Acids and Proteins edited by Greenberg [S] there are evidently arithmetical errors in the calculations on the bottom of p. 393 of the factor f/l, in the Kraemer relation used to correct for effect of hydration. For r=O.2 the value of f/f, from this equation should be 1.085 instead of 1.01, and for r = 0.8 it is 1.28 instead of 1.04. The erroneous figures would indicate little effect of correction for water of hydration on the calculated axial ratios, whereas the effect is evidently quite large. Experimental
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constant was similar to that of Arbacia and hydration about the same, the relatively lower sedimentation constant indicates smaller molecular size or lower axial ratio or both. Number of charged groups per molecule.-From the calculated molecular weight of the Arbacia fertilizin and the data on electrophoretic mobility, it is possible to estimate the number of charged groups per molecule, by making use of Stokes’ law for spherical particles and adopting a resistance factor to apply to cylindrical particles. Valences estimated in this way show 14.25 effective negatively charged groups per molecule at pH 7 and 11.25 at pH 4.85. The isoelectric points of fertilizins are evidently very low, as has been previously noted [19, 251. It seems doubtful that this can be reached without alterations of the molecule induced by low pH. The highly acidic nature of fertilizin is evidently due to the high content of ester-linked sulphate discovered by Vasseur [33]. Number of molecules of fertilizin bound per spermatozoon.-In the experiments described above 10 lo Arbacia spermatozoa were found to combine with an average maximum of 0.5 mg of fertilizin. For fertilizin of 300,000 molecular weight this would be 1015 molecules or lo5 per spermatozoon. There is evidence [25] that the antifertilizin of the sperm is confined to the region of the head between acrosome and midpiece. This is estimated (as a 1 x 3 micron cylinder) to have a surface area of 9 square microns. Fertilizin of 300,000 molecular weight and 0.725 partial specific volume would have a molecular volume of 3.6 x lo-l9 cm3. As a prolate ellipsoid of 20 to 1 axial ratio its short axis would be 3.2 x lo-’ cm and its greatest cross-sectional area would be 8 x 10-B square microns. Attached end on, then, the 105 molecules of fertilizin bound by a spermatozoon would effectively cover about 10 per cent of the reactive surface of the head. It is of some significance that the sperm head is evidently richly provided with receptor groups. The figure of 100,000 such groups represents our first approximation, but it is clear that revisions of molecular weight within the range of error indicated will still give a high figure for the reactive sites on the sperm. It can also be assumed that this is a minimum figure since it seems likely that all reactive sites may not be available for combination with fertilizin, in the absorption experiments. Being so richly provided with reactive sites it now appears more readily understandable that the spermatozoon can effect the rapid union that is customarily observed upon its contact with the egg. Since, upon fertilization the entering sperm reacts with the fertilizin of the Experimental
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Physico-chemical properties of fertilizins
385
gelatinous coat and surface of the egg it seems likely that the fertilizin thus bound may be brought inside the egg. The question may then be raised as to whether or not fertilizin thus transferred into an internal location may play any role in the activation of the egg. Professor Albert0 Monroy and the writer have undertaken some experiments to examine this by means of microinjection, but certain uncertainties inherent in micro-injection methods (see [32]) have so far frustrated these attempts. It is, however, of interest to note that an analagous proposal has been made [5] for virus penetration of susceptible cells, in that it is considered that the incorporation of a considerable portion of the cell surface material results in the production of complete as opposed to incomplete virus particles. SUMMARY
Measurements have been made of the sedimentation rates, diffusion, viscosity and electrophoretic mohilities of the fertilizins of the sea urchin Arbacia punctulata and the sand dollar Echinarachnius parma. From the data for Arbacia a molecular weight of 280,000 is calculated, and a value of 300,000 considered more probable. The axial ratio is estimated to be 28 to 1, calculated for an unhydrated prolate ellipsoid and 20 to 1 if 0.4 gram of water is assumed to be bound per gram of fertilizin. The Echinarachnius fertilizin sedimented more slowly than that of Arbacia. The measurements of electrophoretic mobilities show these fertilizins to be highly acidic as had been found in other species. This data also permitted calculations of valence for Arbacia fertilizin as 14.25 effective negatively charged groups per molecule at pH 7 and 11.25 at pH 4.85. Measurements of the amount of fertilizin absorbed show that a single spermatozoon binds approximately 100,000 molecules, and therefore possesses at least that many receptor sites. The relation of this to certain features of fertilization is briefly discussed. This investigation was supported by a research grant (C-2302) from the National Cancer Institute of the National Institutes of Health, Public Health Service. The experimental work was carried out at the Marine Biological Laboratory, Woods Hole, Massachusetts. I am indebted to Mr. James S. Tyler and Mr. Alan Burbank for technical assistance. REFERENCES 1. BYERS, H. L., Biol. Bull. 101, 218 (1951). 2. CHARGAFF, E., J. Biol. Chem. 142, 285 (1942). 3. EDSALL, J. T., The Proteins. Ed. by H. Neurath and K. Bailey,
Vol.
I, Part
Experimental
B, p. 549,
1953.
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A. Tyler 4. EVANS, T. C., BEAMS, I-I. W., and SMITH, M. E., Hal. Butt. 80, 363 (1941). S. and GRAHAM, D. M., Brit. J. Erptf. Pafhol. 36, 205 (1955). 5. FAZEKAS DE ST. GROTH, Theory, Methods, Applica6. GREENBERG, D. RI. (camp. & ed. by), Amino Acids and Proteins; 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.
tions. Springfield, Ill., 1951. HARTMANN, M., Naturwissenschaffen 28, 144 (1940). HEVESY, G., Aduances in Enzymol. 7, 111 (1947). HEVF.SY, G. and HAHN, L., Kgl. Danske Videnskabernes Sefskab. Biol. Medd. 14, (2), 3 (1938). KRAUSS, M., Biol. Bull. 96. 74 (1949). LILLIE, F. R., Problems of Fertilization. University of Chicago Press, 1919. LORENZ. F. W.. PERLMAN. I.. and CHAIKOFF, I. L.. Am. J. Phwiol. 138. 318 (1943). , METZ, c. B., L?iol. Bull. 82, 446 (1942). ~ Viol. Buff. 89, 84 (1945). MONROY, A., Tosr, L., GIARDINA, G., and MAGGIO, R., Biol. Bull. 106, 169 (1954). MOTOMURA, I., Sci. Rep&. Tdhoku Uniu., 4fh Ser., Biol. 18, 554 (1950). __ Sci. Rep&-. Tdhoku Uniu., 4th Ser., Biol. 20, 93 (1953). RUNNSTR~M, J. and LINDVALL, S., Arkiu Zool. 38 A, No. 10 (1946). RUNNSTRBY, J., TISELIUS, A., and LINDVALL, S., Arkiu Zool. 35A, No. 3 (1942). TYLER, A., Biof. Bull. 78, 159 (1940). __ Biol. Bull. 81, 190 (1941). -Western J. Surg., ObsfeL’and Gynecol. 50, 126 (1942). -Physiof. Reu. 28, 180 (1948 a). __ A&f. Rec. 101, 8 (1948 b). ~ Am. Naturalist 83, 195 (1949 a). ~ The Collecting Net 19: No. 1 (1949 b). -in Analysis of Development, p. 170. Ed. by B. H. Willier, P. A. Weiss and V. Hamburger. W. B. Saunders Co., Philadelphia, 1955. TYLER, A., BURBANK, A., and TYLER, J., Biof. Bull. 107, 303 (1954). __ Biof. Buff. 107, 304 (1954). TYLER, A. and Fox, S. W., Science 90, 516 (1939). -Mol. Buff. 79, 153 (1940). TYLER, A. and MONROY, A., Biol. Bull., 109, 370 (1955). VASSEUR, E., Acta them. Stand. 2, 900 (1948 a). __ Acfa them. Stand. 2. 693 (1948 b). ~ Acfa Borelia. A. Scientia.,‘No. 2’(1951). -The Chemistry and Physiology of the Jelly Coat of the Sea Urchin Egg. Stockholm, 1952. VASSEUR, E. and HAGSTR~~M, B., Arkiu Zool. 37A, No. 17 (1946).
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