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Cytochemistry of Sea Urchin Gametes
© 1968 by Academic Press Inc,
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J, ULTRASTRUCTURE RESEARCH 2 4 , 398-411 (1968)
Cytochemistry of Sea Urchin Gametes I. Intramitochondrial Localization of Glycogen, Glucose-6-phosphatase, and Adenosine Triphosphatase Activity in Spermatozoa of Paracentrotus livJdus WINSTON A. ANDERSON1
Laboratoire de Biologie Cellulaire 4, Facultd des Sciences, 91, Orsay, France~ Received April 26, 1968 Dense aggregates of granular material, ranging from 400 to 1600 A in diameter, are present in the mitochondria of spermatozoa of Paraeentrotus lividus. These intramitochondrial aggregates are intensely stained with lead citrate, are enhanced by lead stains after oxidation in H202 or periodic acid solutions, extracted with e-amylase or saliva, and are composed of 40 A subunits. Based on these cytochemical tests the dense intramitochondrial aggregates are thought to consist of glycogen particles. Glucose-6-phosphatase and adenosine triphosphatase activity are present in the middle piece of sea urchin spermatozoa. The enzyme reaction product lies next to the cristae and on the mitochondrial envelope. The presence of glucose6-phosphatase activity and glycogen in the mitochondria of spermatozoa of the sea urchin implies that glycolytic sequences are probably active in their metabolism. Mammalian spermatozoa obtain metabolic energy through anaerobic and aerobic glycolysis; polysaccharides in the semen furnish the necessary glycolyzable substrates (22). Exogenous polysaccharides, including fructose, glucose, and mannose, are preferentially utilized by glycolytic pathways assumed to exist in the middle piece (22, 43). Marine invertebrate spermatozoa however, are considered to be incapable of utilizing exogenous glycolyzable substrates and depend chiefly on nonglycolytic aerobic metabolism (40, 42). Under aerobic conditions and in the absence of exogenous glycolyzable substrates most vertebrate and invertebrate marine spermatozoa metabolize endogenous phospholipids as sources of energy for motility and maintenance of cellular integrity (2, 19, 22, 42, 43). Vertebrate spermatozoa are therefore provided with the necessary enzyme systems for metabolizing endogenous phos1 Supported by Postdoctoral Fellowship (PF No. 342) from the American Cancer Society Inc., USA. 2This work has been partially supported by grants from the CNRS (LA 86 and RCP 21) and from the DRME (contract 500/67).
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pholipids or exogenous polysaccharides, depending upon the prevailing anaerobic or aerobic conditions and on the presence or absence of exogenous glycolyzable substrates. Few highly specialized invertebrate spermatozoa are however capable of metabolizing polysaccharides. Unlike the situation in mammals, the metabolizable polysaccharide substrates are not exogenously located, but are located within the mitochondria of these spermatozoa. Thus the complex paracrystalline mitochondrial derivative of spermatozoa of slugs (34, 36) and of snails (3, 7) contain glycogen reserves specifically relegated to the membrane-bound compartment named the principal helix (6). Presumably the glycogen reserves provide glycolyzable substrates for energy production (7). The absence of exogenous substrates in the seminal fluid and in seawater suggest that primitive spermatozoa of the sea urchin and of other marine invertebrates depend wholly on metabolizable endogenous reserves (40-42). Such endogenous sources of energy are assumed to arise from the oxidation of phospholipids located mainly in the middle piece (24, 25, 42). The disappearance of endogenous phospholipids coincides with the breakdown of mitochondrial cristae in aged sea urchin spermatozoa (2) suggesting that cristae-components are consumed in order to furnish energy for spermatozoan metabolism. It is probable therefore, that oxidation of fatty acids derived from phospholipids built into the structure of the mitochondrion provides the principal source of energy for these endogenously respiring spermatozoa (2, 22,
42). On the other hand, the intracellular degradation of glycogen or a glycogen-like material may provide energy for metabolism in some species of sea urchins, Strongylocentrotus purpuratus and Lytechinus pictus (16, 46). In Echinus eseulentus, the carbohydrate content of spermatozoa was estimated to be 110 mg glycogen-like material per 100 ml of spermatozoa (41). The existence of glycogen-like material in sea urchin spermatozoa implies the possible utilization of this substrate in energy production, and the probable presence of glycolytic enzymes. The purpose of this investigation is therefore to demonstrate the intramitochondrial localization of glycogen as well as glucose-6-phosphatase and adenosine triphosphatase activities in spermatozoa of Paracentrotus lividus. MATERIALS AND METHODS
Collection of gametes Gametes of the sea urchin Paracentrotus lividus were employed in these experiments. Spermatozoa were obtained either from animals stimulated to spawn by injection of 0.55 M KCI or by collecting "dry" spermatozoa (seminal fluid) from dissected gonads. After mild centrifugation in filtered seawater, the compact pellet containing the spermatozoa was sus-
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pended in glutaraldehyde fixative. To obtained "aged" spermatozoa, one drop of seminal fluid was diluted 1:25 in filtered seawater. After 4-5 hours of aging at approximately 20°C most of the cells were immofile and lost their ability to fertilize eggs. The spermatozoa were subsequently collected by centrifugation and fixed in glutaraldehyde.
Cytochemical procedures Demonstration of glycogen. Thin Epon-Araldite sections were oxidized for 10 min in 5 % of 110 volumes of H202 solution or in 5-10% periodic acid solution, rinsed thoroughly in distilled water and stained for 10-30 rain in undiluted lead citrate solution (23, 35). For light microscopy, 0.5/~ thick sections mounted on glass slides were subjected to the periodic acid Schiff (PAS) procedure for polysaccharides according to Lane and Europa (18). Digestion of glycogen. The procedure according to Monneron and Bernhard (27) was used to extract glycogen from thin sections. Thin sections were oxidized for 15-20 min in 10 % of 110 volumes of HsO2 solution. After oxidation the sections were washed in distilled water and then immersed for 3-6 hours at 37°C in 0.5 % c~-amylase solution in 0.02 M p h o s phate buffer at pH 6.9 or in several changes of saliva for 3-12 hours at 37°C. Control sections were oxidized in HzO~, or periodic acid solutions and subsequently incubated in distilled water or phosphate buffer for the same time and at the same temperature and pH as in experimentals. Demonstration of glucose-6-phosphatase activity. Procedures according to Chiquione (12) and Wachstein and Meisel (50) were employed to demonstrate glucose-6-phosphatase activity. Unfixed spermatozoa and spermatozoa fixed for 10-15 min in cold 2% cacodylatebuffered glutaraldehyde at pH 7.4 were rinsed in several changes of distilled water and subsequently incubated for 5-30 rain at 37°C in medium containing glucose 6-phosphate and lead citrate at pH 6.7. After incubation the specimens were washed in distilled seawater and postfixed either in cacodylate-buffered glutaraldehyde or in 2 % osmium tetroxide in seawater. I n control experiments, spermatozoa that were fixed in glutaraldehyde for 6-12 hours were incubated in medium containing substrate and capture agent. Unfixed and glutaraldehyde-fixed spermatozoa were also incubated in complete medium lacking only glucose 6phosphate. Demonstration of adenosine triphosphatase activity. Unfixed and glutaraldehyde-fixed (15-30 min) spermatozoa were used to demonstrate ATPase activity. Subsequent to fixation, specimens were rinsed in 3 changes (10 rain per change) or overnight in Tris-maleate buffer at pH 7.2 and then incubated for 10-60 min at 37°C in medium containing ATP, magnesium sulfate, lead nitrate, and Tris-maleate buffer at pH 7.2 (31, 51). In control exFIG. 1. A transverse section through the middle piece of a spermatozoon is illustrated in this micrograph. Aggregates of glycogen granules (G) occupy the matrix of the mitochondrion. Uranyl acetate/ lead citrate staining, x 105,000. Fro. 2. Aggregates of glycogen granules ranging from 400 to approximately 1600 A in diameter are shown in the mitochondrion of a spermatozoon. /3 and )' subunits constitute the glycogen stores (arrow). Uranyl acetate/lead citrate staining. × 120,000. FIG. 3. After oxidation in H202 solution, uranyl acetate and lead citrate staining, glycogen granules (G) are shown in the matrix of a mitochondrion, x 88,000. FIG. 4. After oxidation in H~O~ solution and incubation for 4 hours in e-amylase, the regions previously occupied by dense granular material are replaced by clear areas (s). Uranyl acetate/lead citrate staining. × 75,000.
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periments, unfixed and glutaraldehyde-fixed cells were incubated in medium from which only ATP was omitted; or heat-inactivated cells were incubated in complete medium. Heatinactivation consisted of placing a suspension of cells in a water bath for 5 min at 80°C prior to fixation or incubation. After incubation, washed specimens wele postfixed for 60 rain either in 2 % glutaraldehyde or in 2 % osmium tetroxide.
Electron Microscope Techniques Spermatozoa were fixed in medium containing 5.6% biological grade glutaraldehyde (36.4%), 1.5 g sucrose, 50 ml 0.1 M sodium cacodylate in filtered seawater at p H 7.4, and 44.4 ml filtered seawater. Tissues were rinsed rapidly in medium containing 5 g of sucrose in 100 ml of 0.1 M sodium cacodylate at p H 7.4, and were postfixed for 30-60 rain in 2% osmium tetroxide in seawater. After fixation samples were dehydrated rapidly in acetonewater solutions and embedded in an Epon-Araldite mixture containing 1.5 % DMP-30 as catalyst (5). Sections cut with glass knives were stained first for 10 rain in a saturated solution of uranyl acetate in 40% ethanol and then for 10-15 rain in undiluted lead citrate solution (38). OBSERVATIONS I t is well established t h a t a single m i t o c h o n d r i o n which s u r r o u n d s the p r o x i m a l p o r t i o n of the flagellum a n d is j u x t a p o s e d to the flattened p o s t e r i o r surface of the nucleus, f o r m s the m i d d l e piece in s p e r m a t o z o a of the sea urchin (1, 10). I n these primitive s p e r m a t o z o a (15) the m i t o c h o n d r i o n is relatively unmodified, c o n t a i n i n g typical cristae e m b e d d e d in m o d e r a t e l y dense matrix. Paracentrotus s p e r m a t o z o a also possess a single m i t o c h o n d r i o n , however, l i p o i d globules are a b s e n t a n d cristae are n o t organized as in m i t o c h o n d r i a of Arbacia s p e r m a t o z o a (10). I n m a n y cases, m i t o c h o n d r i a of s p e r m a t o z o a collected directly f r o m the dissected testis, c o n t a i n aggregates of dense material. These aggregates, r a n g i n g f r o m 400 to 1600/~, stain intensely with lead solutions (Figs. 1 a n d 2) a n d are usually s u r r o u n d e d b y a clear space. W h e n thin sections are oxidized in H~O2 o r p e r i o d i c acid solution, in o r d e r to solubilize the b o u n d osmium, lead stains enhance the c o n t r a s t of glycogen particles (23, 35). W h e n thin E p o n - A r a l d i t e sections are t r e a t e d in this m a n n e r , the i n t r a m i t o c h o n d r i a l aggregates are intensely stained a n d their subunit structure is e m p h a s i z e d FIG. 5. Aggregates of dense material are removed after oxidation for 10 rain in 5 % periodic acid solution and incubation for 4 hours in e-amylase. Clear areas (s) now occupy the matrix of the mitochondrion (M). Uranyl acetate/lead citrate staining. × 70,000. Fro. 6. Dense glycogen aggregates (arrows) persist in a mitochondrion (M), after oxidation in H~O2 solution and incubation for 4 hours at 37° in water, x 60,000. FI~. 7. Subsequent to oxidation in H202 solution and incubation for several hours in saliva, the dense granular material is partly removed. Arrows indicate that some dense material persists in the clear areas. Uranyl acetate/lead citrate staining. × 80,000. FIG. 8. After 5 hours aging in filtered sea water, the mitochondrion (M) of a spermatozoon contains clear areas (s). × 90,000.
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(Fig. 3). The aggregates are composed of small, rod-like subunits, each approximately 40 A in diameter. The subunits are similar in dimension to v-glycogen particles (nomenclature according to Drochmans, 1962), though fl-particles, approximately 300 A in diameter, are also detectable (Fig. 2). The aggregates are not comparable to c~-glycogen particles often observed in other tissues. Aggregates of dense intramitochondrial material totally disappear and are replaced by clear regions when oxidized thin sections are incubated for 3-6 hours in 0.5 % s-amylase solution (Figs. 4 and 5). Dense aggregates persist in mitochondria when oxidized thin sections are incubated in distilled water for the same time and at the same temperature and pH as sections incubated in amylase (Fig. 6). These aggregates are largely cleared up but do not wholly disappear even after several hours' incubation in saliva (Fig. 7); dense filamentous material still remains. Based on these cytochemical tests, it is suggested that aggregates of glycogen-like material are present in the mitochondria of spermatozoa of Paracentrotus. Biochemical evidence indicates that glycogen-like material disappears from sea urchin spermatozoa during aging (46). After 12 hours aging at 19°C, glycogen-like material decreased from 29 mg per gram dry weight of spermatozoa to 8.5 mg/g dry weight. Four to 5 hours after a 1:25 dilution in filtered seawater at 20°C, Paracentrotus spermatozoa are largely immotile, possessing disorganized flagella but intact mitochondria. Intramitochondrial dense aggregates are replaced by clear compartments in "aged" spermatozoa of Paracentrotus (Fig. 8). Since glucose 6-phosphate plays a key role in glycolytic pathways in tissues using glucose for energy, the existence of glycogen-like material in mitochondria of some sea urchin spermatozoa suggests the participation of this intermediate in spermatozoan metabolism. When unfixed and glutaraldehyde-fixed (less than 20 min) spermatozoa are incubated in the presence of glucose 6-phosphate and lead nitrate, enzyme reaction product appears in the mitochondria of approximately 30 % of the cells examined. Lead phosphate deposits are located next to the cristae that appear as negative images in glutaraldehyde-fixed tissues (Fig. 9). Lead phosphate deposits are similarly arranged adjacent to the cristae and on the mitochondrial envelope in spermatozoa that were fixed in glutaraldehyde after incubation. Fixation in glutaraldehyde for long periods (approximately 45 rain prior to incubation) eliminates the intramitochondrial appearance of enzyme-reaction product. Glutaraldehyde-fixed FIG. 9. Glucose-6-phosphatase end product in the mitochondrion of a sea urchin spermatozoon is shown. Dense, lead phosphate deposits are located next to the negatively stained cristae (arrow), on the mitgchondrial envelope, and on the spermatozoon plasma membrane. Prefixed in glutaraldehyde (10 rain), incubated (30 rain), postfixed in glutaraldehyde (45 min). N, nucleus. × 90,000. FIG. 10. After 60 min fixation in glutaraldehyde, incubation in substrate-free medium and postfixation in 2 % osmium tetroxide, enzyme reaction product is absent from the mitochondrion (M) and flagellum (F) of the spermatozoon. N, nucleus. × 112,000.
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specimens, incubated in substrate-free medium show no lead phosphate deposits (Fig. 10). Mg2+-activated ATPase is present in mitochondria of glutaraldehyde-fixed spermatozoa. The enzyme-reaction product is discretely localized adjacent to the cristae and on the mitochondrial envelope (Figs. 11 and 12). The ATPase end product disappears from within mitochondria after 45 min fixation in glutaraldehyde; however, enzyme-reaction product persists on the surface plasma membrane (Fig, 13). The glutaraldehyde-resistant end product probably reflects the activity of a nonspecific phosphatase located on the plasma membrane. Lead phosphate deposits are absent form cells incubated in Mg2+-free medium, from cells fixed in 2 % osmium tetroxide and from heat-inactivated cells that are incubated in complete medium containing A T P and capture agent (Fig. 14). DISCUSSION Aggregates of ~- and fi-glycogen particles (terminology according to Drochmans, 1962) exist in highly differentiated mitochondria of pulmonate spermatozoa (7, 34, 36). Glycogen particles are also present in the intracristae space or in membranebound compartments of typical mitochondria in digestive cells of Hydra (21), cells of the silk-worm prothoracic gland (9), in retinal receptor cells of rat (17), in h u m a n cancer cells (47), and in the mitochondrial matrix of hypobranchial gland cells of some molluscs (14). In pulmonate spermatozoa, the dense granules that have an affinity for lead stains and disappear after treatment with amylase or saliva are cytochemically characteristic of glycogen (34). In addition, the presence of intense phosphorylase activity in the vicinity of the glycogen reserves (33), the existence of glycolytic enzymes (4) and some of the intermediates of the electron transport chain in mitochondria of Helix aspersa (Ritter and Andr6, 39) strongly suggests that glycogen is the energetic reserve that furnishes energy for motility and metabolism of these cells (7). Similarly, the presence of glycogen in typical mitochondria suggests that FIG. 11. Lead phosphate deposits (indicating Mg~+-stimulatedATPase activity) lie next to the negatively stained cristae and on the mitochondrial envelope of the spermatozoon middle piece (M). Prefixed in glutaraldehyde (10 min), incubated (30 min), postfixed in glutaraldehyde (45 min). x 130,000. FIG. 12. After 20 rain prefixation in glutaraldehyde, 45 rain incubation at 37°C, and postfixation in glutaraldehyde for 30 rain, intense Mg2+-stimulated ATPase end-product is shown next to cristae, at random intermitochondrial sites and on the plasmalemma. N, nucleus, x 92,000. F~G. 13. After 60 rain fixation in glutaraldehyde, ATPase end product disappears from within the mitochondrion but lead phosphate deposits persist on the surface of the plasma membrane (arrow). N, nucleus, x 100,000. Fro. 14. After 15 rain fixation in glutaraldehyde, heating for 5 rain in a water bath at 80°C, and incubation in complete medium, lead phosphate deposits are absent from the mitochondrion of this spermatozoon. Postfixation in OsO4 (15 rain); uranyl acetate/lead citrate staining, x 66,000.
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these organelles may play some role in the metabolism of polysaccharides. The existence of glycogen in these highly specialized and in typical mitochondria is an unusual finding, suggesting that these organelles have the enzymatic capacities not only to synthesize and store glycogen but also to utilize and convert these endogenous reserves into energy production. Dense granular patches in the matrix of mitochondria in spermatozoa of Paraeentrotus, range from 400 to 1600 • and are composed of rod-like subunits 30-40 A in diameter. These subunits which correspond in size to y-glycogen particles are randomly arranged in the aggregate and are separated by clear material. In addition, the subunits are most apparent after oxidation in H202 or periodic acid solutions and staining with lead salts (23, 35). The individual aggregates are also removed by treatment with ~-amylase, but a residue subsists after saliva treatment. Based on these results the intramitochondrial aggregates are considered to be glycogen. The visualization of glycogen particles in the mitochondria of some spermatozoa of the sea urchin not only confirms the biochemical demonstration of glycogen-like material in spermatozoa of sea urchins (41, 46), but also suggests the existence of glycogen degradation and glycolytic pathways. Indeed, glutaraldehyde-resistant glucose-6-phosphatase activity is present in mitochondria of some spermatozoa of Paracentrotus. Enzyme-reaction product appears next to the cristae, on the outer mitochondrial membranes, and on the surface plasmalemma of the spermatozoa. The latter location of lead phosphate is thought to represent the action of a nonspecific phosphatase. The intramitochondrial location of glucose-l-phosphatase activity in muscle tissue (26, 49) and glucose-6-phosphatase activity in gastropod spermatozoa (4) have been reported; both these tissues appear to depend upon the rapid delivery of glycolytic energy from glycogen stores. Glucose-6-phosphatase activity is also characteristic of the isolated outer envelope of liver mitochondria (32). The mitochondrial localization by biochemical and cytochemical procedures of an enzyme normally relegated to the microsomal fraction and endoplasmic reticulum is of uncertain significance. These findings, however, definitely indicate the necessity to admit that Embden-Meyerhof enzymes may be located inside mitochondria. Glucose-6phosphatase plays an important role in glycolysis and may thus function in glycolytic sequences in the metabolism of sea urchin spermatozoa. Glucose-6-phosphatase is also related to glycogen catabolism, hydrolyzing glucose 6-phosphate to free glucose and phosphate. Rothschild and Mann (41) measured the carbohydrate content of sea urchin spermatozoa; 110 mg of glycogen-like material obtained from 100 ml of spermatozoa yielded 40 mg of glucose. It seems significant therefore that dense glycogen patches are replaced by clear compartments in mitochondria of spermatozoa after dilution and storage for 4 hours and that glycogen-like material disappears from sea urchin spermatozoa with aging (46), suggesting that glycogen degradation mecha-
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nisms are existent. In this manner an intramitochondrial glucose-6-phosphatase could participate in controlling the level of glycogen reserves, and the level of inorganic phosphate and of glucose 6-phosphate available for glycolysis. Free glucose if not glycolyzed could be released from the cell or could reenter the glycolytic pathway by way of the hexokinase-catalyzed reaction with ATP. The accumulation of lactic acid when sea urchin spermatozoa are incubated in the presence of glucose supports the existence of a glycolytic pathway (46). Rothschild and Tyler (unpublished, see 40) noticed that sea urchin spermatozoa under anaerobic conditions and in the presence of glycine remain motile for periods longer than 4 hours. An intramitochondrial glycogen reserve could equally provide the source for this anerobic energy production. Adenosine triphosphate is considered to be the major carrier of metabolic energy in cells. The presence of significant quantities of ATP in spermatozoa of sea urchin has been reported (41). Glycolytic and oxidative phosphorylation pathways existing in the mitochondria are most likely the sources of ATP in these cells. ATPase through its action on ATP is considered responsible for providing energy for spermatozoan metabolism and motility (I1, 22, 29). A Mg 2+ activated ATPase, located next to the cristae and on the mitochondrial envelope of sea urchin spermatozoa may serve in the control of A T P : A D P ratio and of respiration in these cells. ATPase activity is indeed located on the membranes isolated from ruptured mitochondria and not in the matrix (45). Mg2+-activated ATPase that is specifically localized in the mitochondria of rat diaphragm muscle has been termed "mitochondrial ATPase" by Padykula and Gauthier (31). Cytochemical evidence has shown Mg ~÷activated ATPase on cristae (8) over cristae and matrix (30, 44), on the surface of mitochondria and in the axial filament of vertebrate and invertebrate spermatozoa (4, 28). A specific mitochondrial ATPase is considered to be functionally related to oxidative phosphorylation (20, 37), and to energy-requiring changes during mitochondrial contraction and active ion transfers.
Conclusions Metabolites derived from the breakdown of polysaccharide reserves in mitochondria of spermatozoa of Paracentrotus lividus probably generate energy-producing glycolytic pathways and contribute to the survival of the spermatozoa under unfavorable conditions. Under anaerobic conditions or conditions of low oxygen tension, as in the overcrowded state in the semen, the degradation of polysaccharide reserves may not only provide ATP but may preserve the endogenous lipid reserves and contribute to fatty acid biosynthesis (48). Aerobically respiring and phosphorylating mitochondria of sea urchin spermatozoa probably have the necessary enzymes for energy transformations by both glycolytic and oxidative pathways of metabolism. These
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findings demonstrate the presence of small amounts of carbohydrate and of glucose 6-phosphatase activity within spermatozoan mitochondria. Phospholipids and glycogen stores located in the middle piece may provide the necessary energy for metabolism and motility during their brief but active life span prior to fertilization and loss of vitality. The disappearance of glycogen-like material from spermatozoa during aging also suggests that the carbohydrate material is utilized, even if endogenous phospholipids remain the major energy sources in these cells. I wish to thank Professor Drach for the provision of laboratory facilities during the summer of 1967 at Laboratoire Arago, Banyuls-sur-Mer, Pyr6n6es Orientales, France. I also thank Dr. Jerome Mangan, Department of Biochemistry, Albert Einstein Medical School, New York, for technical advice on some aspects of this work. The comments and advice of Dr. P. Drochmans, Laboratoire de Cytologic et de Canc6rologie Exp6rimentale, Universit6 Libre de Bruxelles, Bruxelles, Belgium, and the criticism of this manuscript by Dr. J. Andr6, Biologic Cellulaire 4, Facult6 des Sciences, 91-Orsay, France, are greatly appreciated. REFERENCES 1. AFZELIUS,B. A., Z. Zellforsch. Mikroskop. Anat. 42, 134 (1955). 2. AFZELIUS,B. A. and MOHPd, H., Exptl. Cell Res. 42, 10 (1966). 3. ANDERSON,W. A. and PERSONNE, P., J. Microscopic 6, 1053 (1967). 4. - ibid. 6, 75A (1967). 5. AFZELIUS,B. A., WEISSMAN,A. and ELLIS, R. A., J. CellBiol. 32, 11 (1967). 6. ANDRE, J., J. Ultrastruct. Res., Suppl. 3, 1 (1962). 7. - - - - Arch. BioL (Li@ge) 76, 277 (1965). 8. ASHWORT~I,C. T., LtJmEL, F. J. and STEWART, S. C., J. Cell Biol. 17, 1 (1963). 9. BEAULATON,J., Compt. Rend. Acad. Sci. 258, 4139 (1964). 10. BERNSTEIN,M. H., Exptl. Cell Res. 27, 197 (1962). 11. BISHOr, D. W., in BISHOP, D. W. (Ed.). Spermatozoan Motility, p. 251. Am. Assoc. Advance. Sci., Washington, D.C., 1962. 12. CHIQUIONE,A. D., J. Histoehem. Cytochem. 1, 429 (1953). 13. DROCHMANS,P., J. Ultrastruct. Res. 6, 141 (1962). 14. FAIN-MAUREL,M. A., Compt. Rend. Aead. Sei. 263, 1107 (1966). 15. FRANZEN, A., Zool. Bidrag., Uppsala. 31, 355 (1956). 16. HAYASHI,T., Biol. Bull. 90, 177 (1946). 17. ISHAKAWA,T. and PEI, Y. F., J. CellBiol. 25, 402 (1965). 18. LANE, B. P. and EUROPA, D. L., J. Histochem. Cytochem. 13, 579 (1965). 19. LARDY, H. A., WrNCHESTER,B. and PmLLreS, P. H., Arch. Biochem. 6, 33 (1945). 20. LEHNINGER,A. L., The Mitochondrion. Benjamin, New York, 1965. 21. LENTZ, T. L., J. Cell Biol. 29, 162 (1966). 22. MANN, T., in METZ, C. B. and MONROY, A. (Eds.), Fertilization, Vol. 1, p. 99. Academic Press, New York, 1967. 23. MARtNOZZI,V., J. Roy. Mieroscop. Soe. 81, 141 (1963). 24. MOHRL H., Sei. Papers Coll. Gen. Edue., Univ. Tokyo 11, 109 (1961). 25. MoHea, H. and HORILrCm,K., J. Exptl. Biol. 38, 249 (1961).
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J, ULTRASTRUCTURE RESEARCH 2 4 , 398-411 (1968)
Cytochemistry of Sea Urchin Gametes I. Intramitochondrial Localization of Glycogen, Glucose-6-phosphatase, and Adenosine Triphosphatase Activity in Spermatozoa of Paracentrotus livJdus WINSTON A. ANDERSON1
Laboratoire de Biologie Cellulaire 4, Facultd des Sciences, 91, Orsay, France~ Received April 26, 1968 Dense aggregates of granular material, ranging from 400 to 1600 A in diameter, are present in the mitochondria of spermatozoa of Paraeentrotus lividus. These intramitochondrial aggregates are intensely stained with lead citrate, are enhanced by lead stains after oxidation in H202 or periodic acid solutions, extracted with e-amylase or saliva, and are composed of 40 A subunits. Based on these cytochemical tests the dense intramitochondrial aggregates are thought to consist of glycogen particles. Glucose-6-phosphatase and adenosine triphosphatase activity are present in the middle piece of sea urchin spermatozoa. The enzyme reaction product lies next to the cristae and on the mitochondrial envelope. The presence of glucose6-phosphatase activity and glycogen in the mitochondria of spermatozoa of the sea urchin implies that glycolytic sequences are probably active in their metabolism. Mammalian spermatozoa obtain metabolic energy through anaerobic and aerobic glycolysis; polysaccharides in the semen furnish the necessary glycolyzable substrates (22). Exogenous polysaccharides, including fructose, glucose, and mannose, are preferentially utilized by glycolytic pathways assumed to exist in the middle piece (22, 43). Marine invertebrate spermatozoa however, are considered to be incapable of utilizing exogenous glycolyzable substrates and depend chiefly on nonglycolytic aerobic metabolism (40, 42). Under aerobic conditions and in the absence of exogenous glycolyzable substrates most vertebrate and invertebrate marine spermatozoa metabolize endogenous phospholipids as sources of energy for motility and maintenance of cellular integrity (2, 19, 22, 42, 43). Vertebrate spermatozoa are therefore provided with the necessary enzyme systems for metabolizing endogenous phos1 Supported by Postdoctoral Fellowship (PF No. 342) from the American Cancer Society Inc., USA. 2This work has been partially supported by grants from the CNRS (LA 86 and RCP 21) and from the DRME (contract 500/67).
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pholipids or exogenous polysaccharides, depending upon the prevailing anaerobic or aerobic conditions and on the presence or absence of exogenous glycolyzable substrates. Few highly specialized invertebrate spermatozoa are however capable of metabolizing polysaccharides. Unlike the situation in mammals, the metabolizable polysaccharide substrates are not exogenously located, but are located within the mitochondria of these spermatozoa. Thus the complex paracrystalline mitochondrial derivative of spermatozoa of slugs (34, 36) and of snails (3, 7) contain glycogen reserves specifically relegated to the membrane-bound compartment named the principal helix (6). Presumably the glycogen reserves provide glycolyzable substrates for energy production (7). The absence of exogenous substrates in the seminal fluid and in seawater suggest that primitive spermatozoa of the sea urchin and of other marine invertebrates depend wholly on metabolizable endogenous reserves (40-42). Such endogenous sources of energy are assumed to arise from the oxidation of phospholipids located mainly in the middle piece (24, 25, 42). The disappearance of endogenous phospholipids coincides with the breakdown of mitochondrial cristae in aged sea urchin spermatozoa (2) suggesting that cristae-components are consumed in order to furnish energy for spermatozoan metabolism. It is probable therefore, that oxidation of fatty acids derived from phospholipids built into the structure of the mitochondrion provides the principal source of energy for these endogenously respiring spermatozoa (2, 22,
42). On the other hand, the intracellular degradation of glycogen or a glycogen-like material may provide energy for metabolism in some species of sea urchins, Strongylocentrotus purpuratus and Lytechinus pictus (16, 46). In Echinus eseulentus, the carbohydrate content of spermatozoa was estimated to be 110 mg glycogen-like material per 100 ml of spermatozoa (41). The existence of glycogen-like material in sea urchin spermatozoa implies the possible utilization of this substrate in energy production, and the probable presence of glycolytic enzymes. The purpose of this investigation is therefore to demonstrate the intramitochondrial localization of glycogen as well as glucose-6-phosphatase and adenosine triphosphatase activities in spermatozoa of Paracentrotus lividus. MATERIALS AND METHODS
Collection of gametes Gametes of the sea urchin Paracentrotus lividus were employed in these experiments. Spermatozoa were obtained either from animals stimulated to spawn by injection of 0.55 M KCI or by collecting "dry" spermatozoa (seminal fluid) from dissected gonads. After mild centrifugation in filtered seawater, the compact pellet containing the spermatozoa was sus-
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pended in glutaraldehyde fixative. To obtained "aged" spermatozoa, one drop of seminal fluid was diluted 1:25 in filtered seawater. After 4-5 hours of aging at approximately 20°C most of the cells were immofile and lost their ability to fertilize eggs. The spermatozoa were subsequently collected by centrifugation and fixed in glutaraldehyde.
Cytochemical procedures Demonstration of glycogen. Thin Epon-Araldite sections were oxidized for 10 min in 5 % of 110 volumes of H202 solution or in 5-10% periodic acid solution, rinsed thoroughly in distilled water and stained for 10-30 rain in undiluted lead citrate solution (23, 35). For light microscopy, 0.5/~ thick sections mounted on glass slides were subjected to the periodic acid Schiff (PAS) procedure for polysaccharides according to Lane and Europa (18). Digestion of glycogen. The procedure according to Monneron and Bernhard (27) was used to extract glycogen from thin sections. Thin sections were oxidized for 15-20 min in 10 % of 110 volumes of HsO2 solution. After oxidation the sections were washed in distilled water and then immersed for 3-6 hours at 37°C in 0.5 % c~-amylase solution in 0.02 M p h o s phate buffer at pH 6.9 or in several changes of saliva for 3-12 hours at 37°C. Control sections were oxidized in HzO~, or periodic acid solutions and subsequently incubated in distilled water or phosphate buffer for the same time and at the same temperature and pH as in experimentals. Demonstration of glucose-6-phosphatase activity. Procedures according to Chiquione (12) and Wachstein and Meisel (50) were employed to demonstrate glucose-6-phosphatase activity. Unfixed spermatozoa and spermatozoa fixed for 10-15 min in cold 2% cacodylatebuffered glutaraldehyde at pH 7.4 were rinsed in several changes of distilled water and subsequently incubated for 5-30 rain at 37°C in medium containing glucose 6-phosphate and lead citrate at pH 6.7. After incubation the specimens were washed in distilled seawater and postfixed either in cacodylate-buffered glutaraldehyde or in 2 % osmium tetroxide in seawater. I n control experiments, spermatozoa that were fixed in glutaraldehyde for 6-12 hours were incubated in medium containing substrate and capture agent. Unfixed and glutaraldehyde-fixed spermatozoa were also incubated in complete medium lacking only glucose 6phosphate. Demonstration of adenosine triphosphatase activity. Unfixed and glutaraldehyde-fixed (15-30 min) spermatozoa were used to demonstrate ATPase activity. Subsequent to fixation, specimens were rinsed in 3 changes (10 rain per change) or overnight in Tris-maleate buffer at pH 7.2 and then incubated for 10-60 min at 37°C in medium containing ATP, magnesium sulfate, lead nitrate, and Tris-maleate buffer at pH 7.2 (31, 51). In control exFIG. 1. A transverse section through the middle piece of a spermatozoon is illustrated in this micrograph. Aggregates of glycogen granules (G) occupy the matrix of the mitochondrion. Uranyl acetate/ lead citrate staining, x 105,000. Fro. 2. Aggregates of glycogen granules ranging from 400 to approximately 1600 A in diameter are shown in the mitochondrion of a spermatozoon. /3 and )' subunits constitute the glycogen stores (arrow). Uranyl acetate/lead citrate staining. × 120,000. FIG. 3. After oxidation in H202 solution, uranyl acetate and lead citrate staining, glycogen granules (G) are shown in the matrix of a mitochondrion, x 88,000. FIG. 4. After oxidation in H~O~ solution and incubation for 4 hours in e-amylase, the regions previously occupied by dense granular material are replaced by clear areas (s). Uranyl acetate/lead citrate staining. × 75,000.
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periments, unfixed and glutaraldehyde-fixed cells were incubated in medium from which only ATP was omitted; or heat-inactivated cells were incubated in complete medium. Heatinactivation consisted of placing a suspension of cells in a water bath for 5 min at 80°C prior to fixation or incubation. After incubation, washed specimens wele postfixed for 60 rain either in 2 % glutaraldehyde or in 2 % osmium tetroxide.
Electron Microscope Techniques Spermatozoa were fixed in medium containing 5.6% biological grade glutaraldehyde (36.4%), 1.5 g sucrose, 50 ml 0.1 M sodium cacodylate in filtered seawater at p H 7.4, and 44.4 ml filtered seawater. Tissues were rinsed rapidly in medium containing 5 g of sucrose in 100 ml of 0.1 M sodium cacodylate at p H 7.4, and were postfixed for 30-60 rain in 2% osmium tetroxide in seawater. After fixation samples were dehydrated rapidly in acetonewater solutions and embedded in an Epon-Araldite mixture containing 1.5 % DMP-30 as catalyst (5). Sections cut with glass knives were stained first for 10 rain in a saturated solution of uranyl acetate in 40% ethanol and then for 10-15 rain in undiluted lead citrate solution (38). OBSERVATIONS I t is well established t h a t a single m i t o c h o n d r i o n which s u r r o u n d s the p r o x i m a l p o r t i o n of the flagellum a n d is j u x t a p o s e d to the flattened p o s t e r i o r surface of the nucleus, f o r m s the m i d d l e piece in s p e r m a t o z o a of the sea urchin (1, 10). I n these primitive s p e r m a t o z o a (15) the m i t o c h o n d r i o n is relatively unmodified, c o n t a i n i n g typical cristae e m b e d d e d in m o d e r a t e l y dense matrix. Paracentrotus s p e r m a t o z o a also possess a single m i t o c h o n d r i o n , however, l i p o i d globules are a b s e n t a n d cristae are n o t organized as in m i t o c h o n d r i a of Arbacia s p e r m a t o z o a (10). I n m a n y cases, m i t o c h o n d r i a of s p e r m a t o z o a collected directly f r o m the dissected testis, c o n t a i n aggregates of dense material. These aggregates, r a n g i n g f r o m 400 to 1600/~, stain intensely with lead solutions (Figs. 1 a n d 2) a n d are usually s u r r o u n d e d b y a clear space. W h e n thin sections are oxidized in H~O2 o r p e r i o d i c acid solution, in o r d e r to solubilize the b o u n d osmium, lead stains enhance the c o n t r a s t of glycogen particles (23, 35). W h e n thin E p o n - A r a l d i t e sections are t r e a t e d in this m a n n e r , the i n t r a m i t o c h o n d r i a l aggregates are intensely stained a n d their subunit structure is e m p h a s i z e d FIG. 5. Aggregates of dense material are removed after oxidation for 10 rain in 5 % periodic acid solution and incubation for 4 hours in e-amylase. Clear areas (s) now occupy the matrix of the mitochondrion (M). Uranyl acetate/lead citrate staining. × 70,000. Fro. 6. Dense glycogen aggregates (arrows) persist in a mitochondrion (M), after oxidation in H~O2 solution and incubation for 4 hours at 37° in water, x 60,000. FI~. 7. Subsequent to oxidation in H202 solution and incubation for several hours in saliva, the dense granular material is partly removed. Arrows indicate that some dense material persists in the clear areas. Uranyl acetate/lead citrate staining. × 80,000. FIG. 8. After 5 hours aging in filtered sea water, the mitochondrion (M) of a spermatozoon contains clear areas (s). × 90,000.
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(Fig. 3). The aggregates are composed of small, rod-like subunits, each approximately 40 A in diameter. The subunits are similar in dimension to v-glycogen particles (nomenclature according to Drochmans, 1962), though fl-particles, approximately 300 A in diameter, are also detectable (Fig. 2). The aggregates are not comparable to c~-glycogen particles often observed in other tissues. Aggregates of dense intramitochondrial material totally disappear and are replaced by clear regions when oxidized thin sections are incubated for 3-6 hours in 0.5 % s-amylase solution (Figs. 4 and 5). Dense aggregates persist in mitochondria when oxidized thin sections are incubated in distilled water for the same time and at the same temperature and pH as sections incubated in amylase (Fig. 6). These aggregates are largely cleared up but do not wholly disappear even after several hours' incubation in saliva (Fig. 7); dense filamentous material still remains. Based on these cytochemical tests, it is suggested that aggregates of glycogen-like material are present in the mitochondria of spermatozoa of Paracentrotus. Biochemical evidence indicates that glycogen-like material disappears from sea urchin spermatozoa during aging (46). After 12 hours aging at 19°C, glycogen-like material decreased from 29 mg per gram dry weight of spermatozoa to 8.5 mg/g dry weight. Four to 5 hours after a 1:25 dilution in filtered seawater at 20°C, Paracentrotus spermatozoa are largely immotile, possessing disorganized flagella but intact mitochondria. Intramitochondrial dense aggregates are replaced by clear compartments in "aged" spermatozoa of Paracentrotus (Fig. 8). Since glucose 6-phosphate plays a key role in glycolytic pathways in tissues using glucose for energy, the existence of glycogen-like material in mitochondria of some sea urchin spermatozoa suggests the participation of this intermediate in spermatozoan metabolism. When unfixed and glutaraldehyde-fixed (less than 20 min) spermatozoa are incubated in the presence of glucose 6-phosphate and lead nitrate, enzyme reaction product appears in the mitochondria of approximately 30 % of the cells examined. Lead phosphate deposits are located next to the cristae that appear as negative images in glutaraldehyde-fixed tissues (Fig. 9). Lead phosphate deposits are similarly arranged adjacent to the cristae and on the mitochondrial envelope in spermatozoa that were fixed in glutaraldehyde after incubation. Fixation in glutaraldehyde for long periods (approximately 45 rain prior to incubation) eliminates the intramitochondrial appearance of enzyme-reaction product. Glutaraldehyde-fixed FIG. 9. Glucose-6-phosphatase end product in the mitochondrion of a sea urchin spermatozoon is shown. Dense, lead phosphate deposits are located next to the negatively stained cristae (arrow), on the mitgchondrial envelope, and on the spermatozoon plasma membrane. Prefixed in glutaraldehyde (10 rain), incubated (30 rain), postfixed in glutaraldehyde (45 min). N, nucleus. × 90,000. FIG. 10. After 60 min fixation in glutaraldehyde, incubation in substrate-free medium and postfixation in 2 % osmium tetroxide, enzyme reaction product is absent from the mitochondrion (M) and flagellum (F) of the spermatozoon. N, nucleus. × 112,000.
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specimens, incubated in substrate-free medium show no lead phosphate deposits (Fig. 10). Mg2+-activated ATPase is present in mitochondria of glutaraldehyde-fixed spermatozoa. The enzyme-reaction product is discretely localized adjacent to the cristae and on the mitochondrial envelope (Figs. 11 and 12). The ATPase end product disappears from within mitochondria after 45 min fixation in glutaraldehyde; however, enzyme-reaction product persists on the surface plasma membrane (Fig, 13). The glutaraldehyde-resistant end product probably reflects the activity of a nonspecific phosphatase located on the plasma membrane. Lead phosphate deposits are absent form cells incubated in Mg2+-free medium, from cells fixed in 2 % osmium tetroxide and from heat-inactivated cells that are incubated in complete medium containing A T P and capture agent (Fig. 14). DISCUSSION Aggregates of ~- and fi-glycogen particles (terminology according to Drochmans, 1962) exist in highly differentiated mitochondria of pulmonate spermatozoa (7, 34, 36). Glycogen particles are also present in the intracristae space or in membranebound compartments of typical mitochondria in digestive cells of Hydra (21), cells of the silk-worm prothoracic gland (9), in retinal receptor cells of rat (17), in h u m a n cancer cells (47), and in the mitochondrial matrix of hypobranchial gland cells of some molluscs (14). In pulmonate spermatozoa, the dense granules that have an affinity for lead stains and disappear after treatment with amylase or saliva are cytochemically characteristic of glycogen (34). In addition, the presence of intense phosphorylase activity in the vicinity of the glycogen reserves (33), the existence of glycolytic enzymes (4) and some of the intermediates of the electron transport chain in mitochondria of Helix aspersa (Ritter and Andr6, 39) strongly suggests that glycogen is the energetic reserve that furnishes energy for motility and metabolism of these cells (7). Similarly, the presence of glycogen in typical mitochondria suggests that FIG. 11. Lead phosphate deposits (indicating Mg~+-stimulatedATPase activity) lie next to the negatively stained cristae and on the mitochondrial envelope of the spermatozoon middle piece (M). Prefixed in glutaraldehyde (10 min), incubated (30 min), postfixed in glutaraldehyde (45 min). x 130,000. FIG. 12. After 20 rain prefixation in glutaraldehyde, 45 rain incubation at 37°C, and postfixation in glutaraldehyde for 30 rain, intense Mg2+-stimulated ATPase end-product is shown next to cristae, at random intermitochondrial sites and on the plasmalemma. N, nucleus, x 92,000. F~G. 13. After 60 rain fixation in glutaraldehyde, ATPase end product disappears from within the mitochondrion but lead phosphate deposits persist on the surface of the plasma membrane (arrow). N, nucleus, x 100,000. Fro. 14. After 15 rain fixation in glutaraldehyde, heating for 5 rain in a water bath at 80°C, and incubation in complete medium, lead phosphate deposits are absent from the mitochondrion of this spermatozoon. Postfixation in OsO4 (15 rain); uranyl acetate/lead citrate staining, x 66,000.
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these organelles may play some role in the metabolism of polysaccharides. The existence of glycogen in these highly specialized and in typical mitochondria is an unusual finding, suggesting that these organelles have the enzymatic capacities not only to synthesize and store glycogen but also to utilize and convert these endogenous reserves into energy production. Dense granular patches in the matrix of mitochondria in spermatozoa of Paraeentrotus, range from 400 to 1600 • and are composed of rod-like subunits 30-40 A in diameter. These subunits which correspond in size to y-glycogen particles are randomly arranged in the aggregate and are separated by clear material. In addition, the subunits are most apparent after oxidation in H202 or periodic acid solutions and staining with lead salts (23, 35). The individual aggregates are also removed by treatment with ~-amylase, but a residue subsists after saliva treatment. Based on these results the intramitochondrial aggregates are considered to be glycogen. The visualization of glycogen particles in the mitochondria of some spermatozoa of the sea urchin not only confirms the biochemical demonstration of glycogen-like material in spermatozoa of sea urchins (41, 46), but also suggests the existence of glycogen degradation and glycolytic pathways. Indeed, glutaraldehyde-resistant glucose-6-phosphatase activity is present in mitochondria of some spermatozoa of Paracentrotus. Enzyme-reaction product appears next to the cristae, on the outer mitochondrial membranes, and on the surface plasmalemma of the spermatozoa. The latter location of lead phosphate is thought to represent the action of a nonspecific phosphatase. The intramitochondrial location of glucose-l-phosphatase activity in muscle tissue (26, 49) and glucose-6-phosphatase activity in gastropod spermatozoa (4) have been reported; both these tissues appear to depend upon the rapid delivery of glycolytic energy from glycogen stores. Glucose-6-phosphatase activity is also characteristic of the isolated outer envelope of liver mitochondria (32). The mitochondrial localization by biochemical and cytochemical procedures of an enzyme normally relegated to the microsomal fraction and endoplasmic reticulum is of uncertain significance. These findings, however, definitely indicate the necessity to admit that Embden-Meyerhof enzymes may be located inside mitochondria. Glucose-6phosphatase plays an important role in glycolysis and may thus function in glycolytic sequences in the metabolism of sea urchin spermatozoa. Glucose-6-phosphatase is also related to glycogen catabolism, hydrolyzing glucose 6-phosphate to free glucose and phosphate. Rothschild and Mann (41) measured the carbohydrate content of sea urchin spermatozoa; 110 mg of glycogen-like material obtained from 100 ml of spermatozoa yielded 40 mg of glucose. It seems significant therefore that dense glycogen patches are replaced by clear compartments in mitochondria of spermatozoa after dilution and storage for 4 hours and that glycogen-like material disappears from sea urchin spermatozoa with aging (46), suggesting that glycogen degradation mecha-
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nisms are existent. In this manner an intramitochondrial glucose-6-phosphatase could participate in controlling the level of glycogen reserves, and the level of inorganic phosphate and of glucose 6-phosphate available for glycolysis. Free glucose if not glycolyzed could be released from the cell or could reenter the glycolytic pathway by way of the hexokinase-catalyzed reaction with ATP. The accumulation of lactic acid when sea urchin spermatozoa are incubated in the presence of glucose supports the existence of a glycolytic pathway (46). Rothschild and Tyler (unpublished, see 40) noticed that sea urchin spermatozoa under anaerobic conditions and in the presence of glycine remain motile for periods longer than 4 hours. An intramitochondrial glycogen reserve could equally provide the source for this anerobic energy production. Adenosine triphosphate is considered to be the major carrier of metabolic energy in cells. The presence of significant quantities of ATP in spermatozoa of sea urchin has been reported (41). Glycolytic and oxidative phosphorylation pathways existing in the mitochondria are most likely the sources of ATP in these cells. ATPase through its action on ATP is considered responsible for providing energy for spermatozoan metabolism and motility (I1, 22, 29). A Mg 2+ activated ATPase, located next to the cristae and on the mitochondrial envelope of sea urchin spermatozoa may serve in the control of A T P : A D P ratio and of respiration in these cells. ATPase activity is indeed located on the membranes isolated from ruptured mitochondria and not in the matrix (45). Mg2+-activated ATPase that is specifically localized in the mitochondria of rat diaphragm muscle has been termed "mitochondrial ATPase" by Padykula and Gauthier (31). Cytochemical evidence has shown Mg ~÷activated ATPase on cristae (8) over cristae and matrix (30, 44), on the surface of mitochondria and in the axial filament of vertebrate and invertebrate spermatozoa (4, 28). A specific mitochondrial ATPase is considered to be functionally related to oxidative phosphorylation (20, 37), and to energy-requiring changes during mitochondrial contraction and active ion transfers.
Conclusions Metabolites derived from the breakdown of polysaccharide reserves in mitochondria of spermatozoa of Paracentrotus lividus probably generate energy-producing glycolytic pathways and contribute to the survival of the spermatozoa under unfavorable conditions. Under anaerobic conditions or conditions of low oxygen tension, as in the overcrowded state in the semen, the degradation of polysaccharide reserves may not only provide ATP but may preserve the endogenous lipid reserves and contribute to fatty acid biosynthesis (48). Aerobically respiring and phosphorylating mitochondria of sea urchin spermatozoa probably have the necessary enzymes for energy transformations by both glycolytic and oxidative pathways of metabolism. These
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findings demonstrate the presence of small amounts of carbohydrate and of glucose 6-phosphatase activity within spermatozoan mitochondria. Phospholipids and glycogen stores located in the middle piece may provide the necessary energy for metabolism and motility during their brief but active life span prior to fertilization and loss of vitality. The disappearance of glycogen-like material from spermatozoa during aging also suggests that the carbohydrate material is utilized, even if endogenous phospholipids remain the major energy sources in these cells. I wish to thank Professor Drach for the provision of laboratory facilities during the summer of 1967 at Laboratoire Arago, Banyuls-sur-Mer, Pyr6n6es Orientales, France. I also thank Dr. Jerome Mangan, Department of Biochemistry, Albert Einstein Medical School, New York, for technical advice on some aspects of this work. The comments and advice of Dr. P. Drochmans, Laboratoire de Cytologic et de Canc6rologie Exp6rimentale, Universit6 Libre de Bruxelles, Bruxelles, Belgium, and the criticism of this manuscript by Dr. J. Andr6, Biologic Cellulaire 4, Facult6 des Sciences, 91-Orsay, France, are greatly appreciated. REFERENCES 1. AFZELIUS,B. A., Z. Zellforsch. Mikroskop. Anat. 42, 134 (1955). 2. AFZELIUS,B. A. and MOHPd, H., Exptl. Cell Res. 42, 10 (1966). 3. ANDERSON,W. A. and PERSONNE, P., J. Microscopic 6, 1053 (1967). 4. - ibid. 6, 75A (1967). 5. AFZELIUS,B. A., WEISSMAN,A. and ELLIS, R. A., J. CellBiol. 32, 11 (1967). 6. ANDRE, J., J. Ultrastruct. Res., Suppl. 3, 1 (1962). 7. - - - - Arch. BioL (Li@ge) 76, 277 (1965). 8. ASHWORT~I,C. T., LtJmEL, F. J. and STEWART, S. C., J. Cell Biol. 17, 1 (1963). 9. BEAULATON,J., Compt. Rend. Acad. Sci. 258, 4139 (1964). 10. BERNSTEIN,M. H., Exptl. Cell Res. 27, 197 (1962). 11. BISHOr, D. W., in BISHOP, D. W. (Ed.). Spermatozoan Motility, p. 251. Am. Assoc. Advance. Sci., Washington, D.C., 1962. 12. CHIQUIONE,A. D., J. Histoehem. Cytochem. 1, 429 (1953). 13. DROCHMANS,P., J. Ultrastruct. Res. 6, 141 (1962). 14. FAIN-MAUREL,M. A., Compt. Rend. Aead. Sei. 263, 1107 (1966). 15. FRANZEN, A., Zool. Bidrag., Uppsala. 31, 355 (1956). 16. HAYASHI,T., Biol. Bull. 90, 177 (1946). 17. ISHAKAWA,T. and PEI, Y. F., J. CellBiol. 25, 402 (1965). 18. LANE, B. P. and EUROPA, D. L., J. Histochem. Cytochem. 13, 579 (1965). 19. LARDY, H. A., WrNCHESTER,B. and PmLLreS, P. H., Arch. Biochem. 6, 33 (1945). 20. LEHNINGER,A. L., The Mitochondrion. Benjamin, New York, 1965. 21. LENTZ, T. L., J. Cell Biol. 29, 162 (1966). 22. MANN, T., in METZ, C. B. and MONROY, A. (Eds.), Fertilization, Vol. 1, p. 99. Academic Press, New York, 1967. 23. MARtNOZZI,V., J. Roy. Mieroscop. Soe. 81, 141 (1963). 24. MOHRL H., Sei. Papers Coll. Gen. Edue., Univ. Tokyo 11, 109 (1961). 25. MoHea, H. and HORILrCm,K., J. Exptl. Biol. 38, 249 (1961).
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