Forty years ago, Lord Rothschild reviewed the status of work on sea urchin spermatozoa in an article filling 27 pages of Biological Reviews(’). Even in those early days, before the basic architectures of the sperm flagellum, mitochondria, and acrosome were established by electron microscopy, it was well recognized that anyone seeking a convenient sample of cells with uniform size and properties, easily dispersed and aliquoted for experiments, could hardly do better than to choose to study spermatozoa. For anyone wanting an excuse to get away to a marine biology laboratory during the appropriate reproductive season, sea urchin spermatozoa were the obvious choice, because of their abundant quantities and easy release. Now, with the routine availability of efficient air parcel service, working with sea urchin spermatozoa is feasible at inland laboratories, and the laboratory culture of sea urchins(’) is removing seasonal restrictions. Recent work has continued to emphasize biochemistry and physiology. While molecular biologists are no strangers to sea urchin eggs and their development, the molecular genetic study of sea urchin spermatozoa is only just beginning. My first serious introduction to the sea urchin spermatozoon was in 1954, in the laboratory of Albert Tyler’s embryology class at Caltech. As an exercise for that laboratory, we repeated some of the experiments and probability calculations of Rothschild and on collisions of sea urchin spermatozoa with sea urchin eggs, using various concentrations of spermatozoa. In part because of the interest that I took in that exercise, I had the opportunity to meet Victor Rothschild later that year, while he was spending some time at Caltech to work on his book, FertiZi~ation(~). As a result, in 1955, I went to Cambridge to work as a PhD student under his tutelage. In the same year, Professor Sir James Gray published his important papers on the movement and propulsion of sea urchin s p e r m a t o z ~ a ( ~ Rothschild’s .~). interest in sea urchin spermatozoa dated back to his attempts to measure fertilization-induced membrane voltage changes of sea urchin eggs for his PhD under Gray’s supervision(7). Nevertheless, although I remember Cambridge in the mid-1950’s as always being cold enough to keep sea urchins healthy without refriger-
ation, I did nothing more with sea urchin spermatozoa until I began work at Caltech’s Kerckhoff Marine Laboratory, in 1963. My first paper on sea urchin spermatozoa(8)was basically the result of the improved photographic resolution obtained by using electronic flash illumination with durations of loops or less, instead of the 2ms shuttered exposure used by Gray. Inspection of Fig. 1, taken with flashes separated by 2ms intervals, clearly indicates the amount of movement that will smear the image of the flagellum during a 2 ms exposure. The improved resolution obtained with short-duration flashes led to the observation that the bends on the sperm flagellum often approximated circular arcs, rather than the shape of a sinusoidal bending wave. Unfortunately for the theme of this article, the initial observation of circular bends was made neither with sea urchin spermatozoa, nor with Ceratium flagella(’), but with one chance photograph taken in 1962 of a spermatozoon from the mussel, Mytifus edulis. The detailed publication@),based on the sea urchin sperm photographs, was delayed by my slowness in working out the mathematical analysis of propulsion by bending waves constructed of circular arcs. These earliest flash photographs of swimming sea urchin spermatozoa were obtained using a flash illuminator modeled on the one used by Gray for his study of bull sperm motility(lO). It used a General Electric FT 230 flash tube, operated at around 2 kV and triggered with a spark coil. In later work in my lab, these lamps were operated at 4kV, using thyratron switching instead of spark triggering(”). Fortunately for
Fig. 1. A portion of a multiple exposure photomicrograph of a live sea urchin spermatozoon (Strongylocentrotus purpuratus) swimming in sea water, photographed on moving film with 500 flashes per second. Scale bar, 1Opm.
our safety, these illuminators became obsolete in 1969 when the Chadwick-Helmuth Company in California was persuaded of the need to add a microscope illuminator to their line of stroboscopic illuminators, originally developed for examination of helicopter rotors. The widespread availability of these illuminators came just in time for the next major advance in the study of the flagellar motility of sea urchin spermatozoa. In 1972, Barbara and Ian Gibbons (a contemporary of mine at Cambridge in the mid 1950’s, but as a PhD student in Physics rather than Zoology) published flash photomicrographs showing the ATP-reactivated movements of sea urchin spermatozoa demembranated with the detergent, Triton X-1Oo(l2). Actually, papers on the ATP-reactivated movements of sea urchin spermatozoa had been published as early as 1958 by Kinoshita(13), using glycerinated sperm preparations similar to the original glycerinated grasshopper sperm preparations of H~ffmann-Berling(’~). However, Kinoshita was not able to photograph the reactivated movements in his preparations, and it is difficult to assess the quality of reactivation that he was able to obtain. My own efforts to work with glycerinated sea urchin spermatozoa were frustrating, because the quality of the reactivated movement was poor, both with respect to the percentage of motile spermatozoa and the high asymmetry of the movements. It was possible with these preparations to learn that the beat frequency showed a Michaelis-Menten type of relationship to ATP concentration(15) and that the ATP dephosphorylation by the demembranated spermatozoa was coupled to their moti1ity(l6), but the results were never very satisfying because of the poor quality of the movement. The Gibbons’ results showed nearly 100 % reactivation, with bending wave parameters very similar to those of live spermatozoa. It was therefore with great excitement that I tried demembranation with Triton X-100 as soon as I learned of this technique from Ian Gibbons. And it was very disappointing to find that the results, using two Californian sea urchin species, were not much better than previous results with glycerinated sea urchin spermatozoa, and not at all like the results that were being obtained in the Gibbons’ lab with Hawaiian sea urchin species. So I sent off some Californian sea urchins to Hawaii, where they were quickly confiscated by the authorities of the State of Hawaii as a threat to the indigenous biota of Hawaii - in spite of the fact that the warm ocean waters of Hawaii would have killed them within minutes. Fortunately, they allowed Ian Gibbons to come to the airport and collect spermatozoa from the urchins, before they destroyed them. In his lab, they performed beautifully, just as well as the Hawaiian species. It took many months before this discrepancy was resolved by realizing that my technique of collecting ‘dry’ spermatozoa, diluting them only with 0 . 5 ~ NaCl, in contrast to the Gibbons’ technique of diluting the spermatozoa with a small amount of sea
water, maintained a very low Ca2+ ion concentration during the demembranation with Triton X-100. Exposure to a higher Ca2+ ion concentration in the presence of Triton X-100 is required to prepare spermatozoa that will swim symmetrically when ATPrea~tivated(~’,l’).This has not been found to be necessary for species other than sea urchin spermatozoa, and we still do not fully understand it. My best guess at the moment is that something happens at the time of demembranation that sensitizes the demembranated flagellum to calmodulin, allowing calmodulin from the flagellar matrix to become tightly bound. This tightly bound calmodulin increases the asymmetry of the bending waves obtained with ATP-reactivation, but the calmodulin can be released when the calmodulin affinity of the axoneme is reduced by exposure to high Ca2+ ion concentrations in the presence of Triton x-100(19). This is not the only unique feature of sea urchin sperm reactivation. In attempting to apply the same methods to other spermatozoa, I and others have learned that most spermatozoa have to undergo a CAMP-dependent activation process before the axoneme can be reactivated by ATP. In sea urchin spermatozoa in vivo, an increase in intracellular pH may be directly effective in activating the flagellar ATPase and allowing motility, without requiring CAMP-dependent phosphorylation(20). However, the presence of a CAMP-dependent activation process in sea urchin spermatozoa also is suggested by observations showing an effect of CAMPon the bending wave parameters of Lytechinus spermatozoa(21) and by observations with Strongylocentrotus purpuratus spermatozoa showing an improvement in reactivation by incubation with CAMP, if the Triton-demembranation is carried out at p H 7 instead of pH8. Apparently something unique to sea urchin spermatozoa happens when they are demembranated, that bypasses the usual CAMP-dependent activation step. Whatever it is, it is a fortunate coincidence, and the history of work on ATPreactivation of sea urchin sperm flagellar motility would have been very different if these spermatozoa behaved like spermatozoa from other species. The easy demembranation and reactivation of high quality motility of sea urchin spermatozoa, as demonstrated by Fig. 2, made them a premier material for a large variety of experiments on flagellar motility. They are responsible for a long list of ‘firsts’, including the direct visualization of sliding between flagellar microtubules, first by observations during ATP-induced disintegration(22)and, more recently, during normal flagellar bending, by using old microbeads as markers of microtubule position 8 3 ) . For a while, sea urchin spermatozoa also appeared to be unique in showing no chemotaxis during fertilization. Sperm chemotaxis was the subject of my PhD work at Cambridge, motivated by Rothschild’s interest that we might in ‘sperm-egg interacting now call egg pheromones, but were then most
Fig. 2. Triton-demembranated sea urchin spermatozoon (Lytechinus pictus) reactivated at 8 0 p ~MgATP and photographed with 120 flashes per second. Scale bar, 10pm.
thoroughly studied in the form of the fertilizin and antifertilizin of sea urchin gametes(24).In the 1950’s, sperm chemotaxis had only been established to exist in lower plants. Subsequently, largely through the efforts of Richard Miller, the existence of sperm chemotaxis in many marine invertebrate groups, including echinoderms other than sea urchins, was demonstrated using various types of extracts from homologous eggs or female reproductive organs(25). In other laboratories, the peptides named speract and resact were purified from sea urchin egg jelly and sequenced, based on their ability to overcome the inhibition of sperm motility and respiration by low pH. In 1984, Gary Ward, a graduate student with Vic Vacquier at the Scripps Institution of Oceanography, came to my laboratory to examine the effects of synthetic resact on the motility parameters of Arbuciu spermatozoa. What we saw immediately suggested that we should look for chemotaxis, and this was easily demonstrated in our first attempts to inject a small droplet of resact solution into a suspension of swimming Arbuciu spermatozoa(26) - the first case, among animal spermatozoa, of chemotaxis to a chemically defined component released by eggs. In many ways, of all the observations I have made with sea urchin spermatozoa, this was the most satisfying, but in retrospect it is still difficult to understand why the chemotaxis of Arbucia spermatozoa to egg jelly extracts was not clearly demonstrated by numerous earlier
attempts at Woods Hole. Quite possibly it was overshadowed by agglutination reactions caused by other components of egg jelly(25).In spite of considerable information about the biochemical events resulting from binding of resact to its receptor in the sperm membrane and about the regulation of the asymmetry of sea urchin sperm flagellar bending by Ca2+ ion concentration, we still do not have a complete picture of the mechanism of sea urchin sperm chemotaxis. In fact, as Rothschild pointed out to me when I visited him for the last time in Cambridge in 1986, we still do not have adequate evidence that the degree to which the movements of these spermatozoa are directed by the concentration gradient of resact justifies use of the term ‘chemotaxis’, as originally defined, as opposed to ‘chemokinesis’ - some form of alteration of the parameters of random turns. There is no way that this short article could adequately review the whole field of current research on sea urchin spermatozoa, so I will not apologize for my emphasis on motility. However, I do want to give a brief idea of the breadth of research on these cells during the past few decades. Perhaps most important is to remind everyone that the name ‘tubulin’ for the ubiquitous microtubule protein of eukaryotic cells was introduced by Hideo Mohri on the basis of his work with sea urchin spermatozoa(27).In more recent work, the range of techniques applicable to these cells has grown to include direct mechanical measurements of the stiffness of flagella(28); real-time measurements of intracellular Ca2+ concentration(29);characterization of sperm membrane ion channels by direct patch clam ing and by incorporation into artificial membraned3 31); and determination of the sequence of the mRNA coding for bindin, an acrosomal protein that mediates species-s ecific adhesion of spermatozoa to the egg surface(3 ) . There are obviously many other workers who would name the sea urchin spermatozoon as ‘My Favourite Cell’.
t
Y
Acknowledgements My study of sea urchin spermatozoa has been generously supported by grants from the U. S. National Institutes of Health, primarily GM 18711.
References 1 ROTHSCHILD, N. M. V. (1951). Sea urchin spermatozoa. Biol. Rev. 26, 1-27. 2 LEAHY,P. (1986). Laboratory culture of Strongylocentrotus purpuratus adults, embryos, and larvae. In: Echinoderm Gametes and Embryos (ed. T. E. Shroeder) (Meth. Cell Bid. 27), 1-13. Academic Press. 3 ROTHSCHILD, N. M. V. AND SWANN, M. M. (1951). The fertilization reaction in the sea urchin. The probability of a successful sperm-egg collision. J. Exp. Biol. 28, 403-416. 4 ROTHSCHILD, N . M. V. (1956). Fertilization. London, Methuen & Co., Ltd. 5 GRAY, .I. (1955). The movement of sea urchin spermatozoa. J . Exp. B i d . 32, 775-801. 6 GRAY,J . AND HANCOCK, G . J. (1955). The propulsion of sea urchin spermatozoa. J . Exp. Biol. 32, 802-814. 7 ROTHSCHILD, N. M. V. (1938). The biophysics of the egg surface of Echinus esculentus during fertilization and cytolysis. J . Exp. Biol. 15, 209-216. 8 BROKAW, C. J. (1955). Non-sinusoidal bending waves of sperm flagella. J. Exp. Biol. 43, 155-169.
9 BROKAW, C. J. AND WRIGHT, L. (1963). Bending waves of the posterior flagellum of Ceratium. Science 142, 1169-1170. 10 GRAY, J. (1958). The movement of spermatozoa of the bull. J . Exp. B i d . 35, 96-108. 11 GOLDSTEIN, S. F. (1969). Irradiation of sperm tails by laser microbeam. J . Exp. Biol. 51, 431-441. 12 GIBBONS, B. H. AND GIBBONS, I. R. (1972). Flagellar movement and adenosine triphosphatase activity in sea urchin sperm extracted with Triton X1M). J. Cell Biol. 54, 75-97. 13 KINOSHITA, S. (1958). The mode of action of metal-chelating substances on sperm motility in some marine forms as shown by glyerol-extracted sperm models. J . Fac. Sci. Univ. Tokyo, Sec. N 8, 219-228. 14 HOFFMANN-BERLING, H. (1955). Geisselmodelle und Adenosintriphosphat (ATP). Biochim. Biophys. Acra 16, 146-154. 15 BROKAW, C. J. (1967). Adenosine triphosphate usage by flagella. Science 156, 76-78. 16 BROKAW,C. J . AND BENEDICT, B. (1968). Mechanochemical coupling in flagella. I. Movement-dependent dephosphorylation of ATP by glycerinated spermatozoa. Arch. Biochem. Biophys. 125, 770-778. 17 BROKAW, C. J., JOSSLIN, R. AND BOBROW, L. (1974). Calcium ion regulation of flagellar beat symmetry in reactivated sea urchin spermatozoa. Biochem. Biophys. Res. Comm. 58, 795-800. 18 GIBBONS, B. H . AND GIBBONS, I. R. (1980). Calcium-induced quiescence in reactivated sea urchin sperm. J . Cell B i d . 84, 13-27. 19 BROKAW, C. J . AND NACAYAMA, S. M. (1985). Modulation of the asymmetry of sea urchin sperm flagellar bending by calrnodulin. J. Cell Biol. 100, 1875- 1883. 20 CHRISTEN, R . , SCHACKMANN, R. W. AND SHAPIRO, B. M. (1982). Elevation of the intracellular pH activates respiration and motility of sperm of the sea urchin, Strongylocenrrorus purpuratus. J . B i d . Chem. 257, 14 881-14 890. 21 BROKAW, C. J. (1987). A lithium-sensitive regulator of sperm flagellar oscillation is activated by CAMP-dependent phosphorylation. J. Cell B i d . 105, 1789-1798. 22 SUMMERS, K. E. AND GIBBONS, I. R. (1971). Adenosine triphosphate-
induced sliding of tubules in trypsin-treated flagella of sea urchin sperm. Proc. Narl Acad. Sci. USA 68, 3092-3096. 23 BROKAW, C. J. (1989). Direct measurements of sliding between outer doublet microtubules in swimming sperm flagella. Science 243, 1593-1596. 24 TYLER,A. (1965). The biology and chemistry of fertilization. Amer. Naturalist 99, 309-334. 25 MILLER, R. L. (1985). Sperm chemo-orientation in the metazoa. In Biology of Fertilization, Vol. 2. (ed. Metz, C. B. and Monroy, A,), pp. 275-337. Academic Press. 26 WARD,G. E., BROKAW, C . J., GARBERS, D. L. AND VACQUIER, V. D. (1985). Chemotaxis of Arbacia punctuluta spermatozoa to resact, a peptide from the egg jelly layer. J. Cell Biol. 101, 2324-2329. 27 MOHRI,H. (1968). Amino-acid composition of ‘tubulin’ constituting microtubules of sperm flagella. Nature 217, 1053-1054. 28 OKUNO, M. AND HIRAMOTO, Y.(1979). Direct measurement of the stiffness of echinoderm sperm flagella. J . Exp. Biol. 79, 235-243. 29 SCHACKMANN, R. W. (1986). Ion measurements in sea urchin spermatozoa. In: Echinoderm Gametes and Embryos (ed. T . E. Shroeder) (Merh. Cell Biol. 27), pp. 57-71. Academic Press. 30 GUERRERO, A., SANCHEZ, J. A. AND DARSZON, A. (1987). Single-channel activity in sea urchin sperm revealed by the patch-clamp technique. FEBS Lerr. 220, 295-298. 31 LI~VANO, A , , VEGA-SAENZDEMIERA, E. C. AND DARSZON, A . (1990). Ca2+ channels from the sea urchin sperm plasma membrane. J . Gen. Physiol. 95, 273-296.
32 GAO, B., KLEIN,L. E., BRITTEN, R. J. AND DAVIDSON, E. H. (1986). Sequence of mRNA coding for bindin, a species-specific sea urchin sperm protein required for fertilization. Proc. Natl Acad. Sci. USA 83, 8634-8638
I
Charles J. Brokaw is at the Division of Biology, California Institute of Technolonv. Pasadena. CA 91125, USA.
ation, I did nothing more with sea urchin spermatozoa until I began work at Caltech’s Kerckhoff Marine Laboratory, in 1963. My first paper on sea urchin spermatozoa(8)was basically the result of the improved photographic resolution obtained by using electronic flash illumination with durations of loops or less, instead of the 2ms shuttered exposure used by Gray. Inspection of Fig. 1, taken with flashes separated by 2ms intervals, clearly indicates the amount of movement that will smear the image of the flagellum during a 2 ms exposure. The improved resolution obtained with short-duration flashes led to the observation that the bends on the sperm flagellum often approximated circular arcs, rather than the shape of a sinusoidal bending wave. Unfortunately for the theme of this article, the initial observation of circular bends was made neither with sea urchin spermatozoa, nor with Ceratium flagella(’), but with one chance photograph taken in 1962 of a spermatozoon from the mussel, Mytifus edulis. The detailed publication@),based on the sea urchin sperm photographs, was delayed by my slowness in working out the mathematical analysis of propulsion by bending waves constructed of circular arcs. These earliest flash photographs of swimming sea urchin spermatozoa were obtained using a flash illuminator modeled on the one used by Gray for his study of bull sperm motility(lO). It used a General Electric FT 230 flash tube, operated at around 2 kV and triggered with a spark coil. In later work in my lab, these lamps were operated at 4kV, using thyratron switching instead of spark triggering(”). Fortunately for
Fig. 1. A portion of a multiple exposure photomicrograph of a live sea urchin spermatozoon (Strongylocentrotus purpuratus) swimming in sea water, photographed on moving film with 500 flashes per second. Scale bar, 1Opm.
our safety, these illuminators became obsolete in 1969 when the Chadwick-Helmuth Company in California was persuaded of the need to add a microscope illuminator to their line of stroboscopic illuminators, originally developed for examination of helicopter rotors. The widespread availability of these illuminators came just in time for the next major advance in the study of the flagellar motility of sea urchin spermatozoa. In 1972, Barbara and Ian Gibbons (a contemporary of mine at Cambridge in the mid 1950’s, but as a PhD student in Physics rather than Zoology) published flash photomicrographs showing the ATP-reactivated movements of sea urchin spermatozoa demembranated with the detergent, Triton X-1Oo(l2). Actually, papers on the ATP-reactivated movements of sea urchin spermatozoa had been published as early as 1958 by Kinoshita(13), using glycerinated sperm preparations similar to the original glycerinated grasshopper sperm preparations of H~ffmann-Berling(’~). However, Kinoshita was not able to photograph the reactivated movements in his preparations, and it is difficult to assess the quality of reactivation that he was able to obtain. My own efforts to work with glycerinated sea urchin spermatozoa were frustrating, because the quality of the reactivated movement was poor, both with respect to the percentage of motile spermatozoa and the high asymmetry of the movements. It was possible with these preparations to learn that the beat frequency showed a Michaelis-Menten type of relationship to ATP concentration(15) and that the ATP dephosphorylation by the demembranated spermatozoa was coupled to their moti1ity(l6), but the results were never very satisfying because of the poor quality of the movement. The Gibbons’ results showed nearly 100 % reactivation, with bending wave parameters very similar to those of live spermatozoa. It was therefore with great excitement that I tried demembranation with Triton X-100 as soon as I learned of this technique from Ian Gibbons. And it was very disappointing to find that the results, using two Californian sea urchin species, were not much better than previous results with glycerinated sea urchin spermatozoa, and not at all like the results that were being obtained in the Gibbons’ lab with Hawaiian sea urchin species. So I sent off some Californian sea urchins to Hawaii, where they were quickly confiscated by the authorities of the State of Hawaii as a threat to the indigenous biota of Hawaii - in spite of the fact that the warm ocean waters of Hawaii would have killed them within minutes. Fortunately, they allowed Ian Gibbons to come to the airport and collect spermatozoa from the urchins, before they destroyed them. In his lab, they performed beautifully, just as well as the Hawaiian species. It took many months before this discrepancy was resolved by realizing that my technique of collecting ‘dry’ spermatozoa, diluting them only with 0 . 5 ~ NaCl, in contrast to the Gibbons’ technique of diluting the spermatozoa with a small amount of sea
water, maintained a very low Ca2+ ion concentration during the demembranation with Triton X-100. Exposure to a higher Ca2+ ion concentration in the presence of Triton X-100 is required to prepare spermatozoa that will swim symmetrically when ATPrea~tivated(~’,l’).This has not been found to be necessary for species other than sea urchin spermatozoa, and we still do not fully understand it. My best guess at the moment is that something happens at the time of demembranation that sensitizes the demembranated flagellum to calmodulin, allowing calmodulin from the flagellar matrix to become tightly bound. This tightly bound calmodulin increases the asymmetry of the bending waves obtained with ATP-reactivation, but the calmodulin can be released when the calmodulin affinity of the axoneme is reduced by exposure to high Ca2+ ion concentrations in the presence of Triton x-100(19). This is not the only unique feature of sea urchin sperm reactivation. In attempting to apply the same methods to other spermatozoa, I and others have learned that most spermatozoa have to undergo a CAMP-dependent activation process before the axoneme can be reactivated by ATP. In sea urchin spermatozoa in vivo, an increase in intracellular pH may be directly effective in activating the flagellar ATPase and allowing motility, without requiring CAMP-dependent phosphorylation(20). However, the presence of a CAMP-dependent activation process in sea urchin spermatozoa also is suggested by observations showing an effect of CAMPon the bending wave parameters of Lytechinus spermatozoa(21) and by observations with Strongylocentrotus purpuratus spermatozoa showing an improvement in reactivation by incubation with CAMP, if the Triton-demembranation is carried out at p H 7 instead of pH8. Apparently something unique to sea urchin spermatozoa happens when they are demembranated, that bypasses the usual CAMP-dependent activation step. Whatever it is, it is a fortunate coincidence, and the history of work on ATPreactivation of sea urchin sperm flagellar motility would have been very different if these spermatozoa behaved like spermatozoa from other species. The easy demembranation and reactivation of high quality motility of sea urchin spermatozoa, as demonstrated by Fig. 2, made them a premier material for a large variety of experiments on flagellar motility. They are responsible for a long list of ‘firsts’, including the direct visualization of sliding between flagellar microtubules, first by observations during ATP-induced disintegration(22)and, more recently, during normal flagellar bending, by using old microbeads as markers of microtubule position 8 3 ) . For a while, sea urchin spermatozoa also appeared to be unique in showing no chemotaxis during fertilization. Sperm chemotaxis was the subject of my PhD work at Cambridge, motivated by Rothschild’s interest that we might in ‘sperm-egg interacting now call egg pheromones, but were then most
Fig. 2. Triton-demembranated sea urchin spermatozoon (Lytechinus pictus) reactivated at 8 0 p ~MgATP and photographed with 120 flashes per second. Scale bar, 10pm.
thoroughly studied in the form of the fertilizin and antifertilizin of sea urchin gametes(24).In the 1950’s, sperm chemotaxis had only been established to exist in lower plants. Subsequently, largely through the efforts of Richard Miller, the existence of sperm chemotaxis in many marine invertebrate groups, including echinoderms other than sea urchins, was demonstrated using various types of extracts from homologous eggs or female reproductive organs(25). In other laboratories, the peptides named speract and resact were purified from sea urchin egg jelly and sequenced, based on their ability to overcome the inhibition of sperm motility and respiration by low pH. In 1984, Gary Ward, a graduate student with Vic Vacquier at the Scripps Institution of Oceanography, came to my laboratory to examine the effects of synthetic resact on the motility parameters of Arbuciu spermatozoa. What we saw immediately suggested that we should look for chemotaxis, and this was easily demonstrated in our first attempts to inject a small droplet of resact solution into a suspension of swimming Arbuciu spermatozoa(26) - the first case, among animal spermatozoa, of chemotaxis to a chemically defined component released by eggs. In many ways, of all the observations I have made with sea urchin spermatozoa, this was the most satisfying, but in retrospect it is still difficult to understand why the chemotaxis of Arbucia spermatozoa to egg jelly extracts was not clearly demonstrated by numerous earlier
attempts at Woods Hole. Quite possibly it was overshadowed by agglutination reactions caused by other components of egg jelly(25).In spite of considerable information about the biochemical events resulting from binding of resact to its receptor in the sperm membrane and about the regulation of the asymmetry of sea urchin sperm flagellar bending by Ca2+ ion concentration, we still do not have a complete picture of the mechanism of sea urchin sperm chemotaxis. In fact, as Rothschild pointed out to me when I visited him for the last time in Cambridge in 1986, we still do not have adequate evidence that the degree to which the movements of these spermatozoa are directed by the concentration gradient of resact justifies use of the term ‘chemotaxis’, as originally defined, as opposed to ‘chemokinesis’ - some form of alteration of the parameters of random turns. There is no way that this short article could adequately review the whole field of current research on sea urchin spermatozoa, so I will not apologize for my emphasis on motility. However, I do want to give a brief idea of the breadth of research on these cells during the past few decades. Perhaps most important is to remind everyone that the name ‘tubulin’ for the ubiquitous microtubule protein of eukaryotic cells was introduced by Hideo Mohri on the basis of his work with sea urchin spermatozoa(27).In more recent work, the range of techniques applicable to these cells has grown to include direct mechanical measurements of the stiffness of flagella(28); real-time measurements of intracellular Ca2+ concentration(29);characterization of sperm membrane ion channels by direct patch clam ing and by incorporation into artificial membraned3 31); and determination of the sequence of the mRNA coding for bindin, an acrosomal protein that mediates species-s ecific adhesion of spermatozoa to the egg surface(3 ) . There are obviously many other workers who would name the sea urchin spermatozoon as ‘My Favourite Cell’.
t
Y
Acknowledgements My study of sea urchin spermatozoa has been generously supported by grants from the U. S. National Institutes of Health, primarily GM 18711.
References 1 ROTHSCHILD, N. M. V. (1951). Sea urchin spermatozoa. Biol. Rev. 26, 1-27. 2 LEAHY,P. (1986). Laboratory culture of Strongylocentrotus purpuratus adults, embryos, and larvae. In: Echinoderm Gametes and Embryos (ed. T. E. Shroeder) (Meth. Cell Bid. 27), 1-13. Academic Press. 3 ROTHSCHILD, N. M. V. AND SWANN, M. M. (1951). The fertilization reaction in the sea urchin. The probability of a successful sperm-egg collision. J. Exp. Biol. 28, 403-416. 4 ROTHSCHILD, N . M. V. (1956). Fertilization. London, Methuen & Co., Ltd. 5 GRAY, .I. (1955). The movement of sea urchin spermatozoa. J . Exp. B i d . 32, 775-801. 6 GRAY,J . AND HANCOCK, G . J. (1955). The propulsion of sea urchin spermatozoa. J . Exp. Biol. 32, 802-814. 7 ROTHSCHILD, N. M. V. (1938). The biophysics of the egg surface of Echinus esculentus during fertilization and cytolysis. J . Exp. Biol. 15, 209-216. 8 BROKAW, C. J. (1955). Non-sinusoidal bending waves of sperm flagella. J. Exp. Biol. 43, 155-169.
9 BROKAW, C. J. AND WRIGHT, L. (1963). Bending waves of the posterior flagellum of Ceratium. Science 142, 1169-1170. 10 GRAY, J. (1958). The movement of spermatozoa of the bull. J . Exp. B i d . 35, 96-108. 11 GOLDSTEIN, S. F. (1969). Irradiation of sperm tails by laser microbeam. J . Exp. Biol. 51, 431-441. 12 GIBBONS, B. H. AND GIBBONS, I. R. (1972). Flagellar movement and adenosine triphosphatase activity in sea urchin sperm extracted with Triton X1M). J. Cell Biol. 54, 75-97. 13 KINOSHITA, S. (1958). The mode of action of metal-chelating substances on sperm motility in some marine forms as shown by glyerol-extracted sperm models. J . Fac. Sci. Univ. Tokyo, Sec. N 8, 219-228. 14 HOFFMANN-BERLING, H. (1955). Geisselmodelle und Adenosintriphosphat (ATP). Biochim. Biophys. Acra 16, 146-154. 15 BROKAW, C. J. (1967). Adenosine triphosphate usage by flagella. Science 156, 76-78. 16 BROKAW,C. J . AND BENEDICT, B. (1968). Mechanochemical coupling in flagella. I. Movement-dependent dephosphorylation of ATP by glycerinated spermatozoa. Arch. Biochem. Biophys. 125, 770-778. 17 BROKAW, C. J., JOSSLIN, R. AND BOBROW, L. (1974). Calcium ion regulation of flagellar beat symmetry in reactivated sea urchin spermatozoa. Biochem. Biophys. Res. Comm. 58, 795-800. 18 GIBBONS, B. H . AND GIBBONS, I. R. (1980). Calcium-induced quiescence in reactivated sea urchin sperm. J . Cell B i d . 84, 13-27. 19 BROKAW, C. J . AND NACAYAMA, S. M. (1985). Modulation of the asymmetry of sea urchin sperm flagellar bending by calrnodulin. J. Cell Biol. 100, 1875- 1883. 20 CHRISTEN, R . , SCHACKMANN, R. W. AND SHAPIRO, B. M. (1982). Elevation of the intracellular pH activates respiration and motility of sperm of the sea urchin, Strongylocenrrorus purpuratus. J . B i d . Chem. 257, 14 881-14 890. 21 BROKAW, C. J. (1987). A lithium-sensitive regulator of sperm flagellar oscillation is activated by CAMP-dependent phosphorylation. J. Cell B i d . 105, 1789-1798. 22 SUMMERS, K. E. AND GIBBONS, I. R. (1971). Adenosine triphosphate-
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Charles J. Brokaw is at the Division of Biology, California Institute of Technolonv. Pasadena. CA 91125, USA.
