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Intracellular calcium in hamster spermatozoa in testis, epididymis and during the acrosome reaction
Animal Reproduction Science, 16 (1988) 145-153 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
145
Intracellular Calcium in Hamster Spermatozoa in Testis, Epididymis and During the Acrosome Reaction A. RUKNUDIN, J.P. DADOUNE and I.A. SILVER 1'2
Laboratoire d'Histologie, Embryologie et Cytogdndtique, Universitd de Paris VI, 45, rue des Saints-P~res, Paris (France) 1Department of Pathology, Medical School, University of Bristol, Bristol (Great Britain) 2To whom all correspondence should be addressed. (Accepted 29 October 1987)
ABSTRACT Ruknudin, A., Dadoune, J.P. and Silver, I.A., 1988. Intracellular calcium in hamster spermatozoa in testis, epididymis and during the acrosome reaction. Anim. Reprod. Sci., 16: 145-153. Hamster spermatozoa from the testis and cauda epididymis were examined for intracellular localization of calcium using a pyroantimonate technique. Calcium precipitates were found in spermatozoa from the testis but not in those from cauda epididymis. Spermatozoa from the epididymis which had been induced to undergo capacitation and the acrosome reaction in vitro, were also investigated. In these, the anterior region of the acrosome showed precipitates indicating calcium binding at specific sites, possibly prior to transport across the membranes. The additional presence of calcium precipitates in the postacrosomal region suggests that the appearance of free calcium in that region is an important step in the acrosome reaction.
INTRODUCTION
The involvement of calcium in the acrosome reaction in mammalian spermatozoa is well recognized (Fraser, 1984) and it has been shown that extracellular calcium is necessary for this process to occur (Iwamatsu and Chang, 1971; Yanagimachi and Usui, 1974 ). Hamster spermatozoa need extracellular calcium during initiation and maintenance of hyperactivated motility, penetration into the zona pellucida and fusion with eggs (Yanagimachi, 1982). Among the various processes in which spermatozoa require extracellular calcium, the acrosome reaction has been comparatively well studied and an influx of extracellular calcium associated with this reaction was demonstrated by Reed and Lardy (1972). Furthermore, Babcock et al. (1978) showed that bovine spermatozoa exposed to the ionophore A23187 accumulate intracellular cal0378-4320/88/$03.50
© 1988 Elsevier Science Publishers B.V.
147
cium and this ionophore has been used successfully to induce the acrosome reaction in vitro in spermatozoa of many species (Fraser, 1984). Thus the steps in sperm capacitation leading to fertilization appear to involve uptake of extracellular calcium. In the hamster, it has been shown that there are differences in the physiological status of spermatozoa from testis, epididymis and ejaculate (Mohri and Yanagimachi, 1980). Since the importance of intracellular Ca 2+ in the control of function in various cells is well established (Borle, 1981 ) we postulated that there might be parallel differences in the distribution of intracellular Ca 2+, in testicular, epididymal and capacitating spermatozoa. Such possible differences were analyzed using a cytochemical technique and the results are presented here. MATERIALS AND METHODS
Male hamsters (Mesocricetus auratus) were anaesthetized and small portions of testis removed immediately and placed in a fixative containing 2.5% potassium pyroantimonate ( Prolabo, Paris) in 1% osmium tetroxide at pH 7.6 (Gravis, 1979). One portion of the distal segment of the cauda epididymis was placed directly in the fixative while another portion was placed in TALP-1 medium (Yanagimachi, 1982) and the spermatozoa were squeezed out. This cell suspension was divided into three equal parts; the first was centrifuged immediately ( 600g for 5 min) and fixed in pyroantimonate-osmium solution. From the other two parts, the most highly motile spermatozoa were separated by the ascending technique (Roldan et al., 1986) and incubated at 37 ° C under an atmosphere of 5% CO2 in air, to induce capacitation and the acrosome reaction. Of these aliquots, the first was fixed after 1 h and the second after 4 h of incubation. All material was fixed for 1-2 h at 4°C and then washed quickly in 0.01 N potassium acetate. The fixative solutions were filtered through 0.2-/lm millipore filters prior to use. Dehydration and embedding were carried out by stan-
Figs. 1-8. Electron micrographs of sagittal sections through the head region of hamster spermatozoa fixed in pyroantimonate-osmium. Fig. 1. Spermatozoon in testis surrounded by sertoli cells (S). A thick deposit is seen at the postacrosomal lamina (PAL) region (arrow heads) and granules of precipitate are present at the P face of the plasma membrane (PM) of sertoli cells (arrows). × 21 000. Fig. 2. Spermatozoon in cauda epididymis. The characteristic pyroantimonate precipitate is absent. )< 25 000. Fig. 3. Spermatozoon fixed immediately after being released into capacitating medium. No pyroantimonate precipitate is present. X 20 500.
148
Fig. 4. Anterior portion of acrosome of spermatozoon undergoing the acrosome reaction. Double granules between plasma membrane (PM) and outer acrosomal membrane (OAM) are indicated by arrows. × 31 000. Fig. 5. Deposits in the extramitochondrial region of the tail. × 42 000.
Fig. 6. A dense precipitate is seen in the PAL region in a spermatozoon undergoing the acrosome reaction. × 36 000. Fig. 7. Section as for Fig. 6 but exposed to EGTA and stained. No precipitates can be identified. × 35 000. Fig. 8. Acrosome-reacted spermatozoon after 4 h incubation showing absence of calcium precipitate. X 38 800.
8)
150 dard techniques for electon microscopy. Ultrathin sections, either unstained or stained with uranyl acetate and lead citrate, were observed in a Jeol 100CX electron microscope. Two types of controls for the cytochemical technique were carried out. One set of sections was collected on copper grids and incubated in 5 mM EGTA (pH 8 ) at 60 ° C for 45 rain. Simultaneously, another set was floated on distilled water and incubated under the same conditions. RESULTS Calcium precipitates were found in the postacrosomal region of spermatozoa fixed in the final stages of spermiogenesis and fine deposits were also observed on the cytoplasmic side of the plasma membrane (PM) of surrounding sertoli cells (Fig. 1). However, sperm within the epididymis did not show calcium pyroantimonate precipitates at the postacrosomal lamina (PAL), although fine deposits could be observed in lumen of the epididymal duct ( Fig. 2 ). Sperm from the epididymis showed no calcium precipitate in either the periacrosomal or postacrosomal regions immediately after dilution into the capacitating medium (Fig. 3). However, after incubation in the medium for 1 h, many spermatozoa from the epididymis exhibited precipitates; but in any single preparation there were spermatozoa at various stages of the acrosome reaction. During the initial stages of the reaction, calcium precipitates were found in the anterior region of the acrosome (Fig. 4), and at higher magnification they could be seen between the P face of the PM and the E face of the outer acrosomal membrane (OAM), i.e., at the intermembranous region. They were attached to the opposite sides of PM and OAM. These precipitates were removed by EGTA treatment. In the later stages of the acrosome reaction, when the hybrid vesicles of PM and OAM were found, the calcium pyroantimonate precipitates were seen inside the vesicles. In the postacrosomal region, a heavier deposit of calcium was observed after incubation of the spermatozoa for 1 h. These heavy precipitates which were localized posterior to the equatorial region and anterior to the plasma membrane in the postacrosomal region (Fig. 6 ), were also removed by EGTA digestion (Fig. 7 ). In the midpiece of spermatozoa undergoing the acrosome reaction, the precipitates were found to be intracellular but extramitochondrial (Fig. 5). Such intracellular precipitates were not found at the other stages of maturation. For instance, spermatozoa fixed after incubation for 4 h, by which time the acrosome reaction was completed, had very little or no calcium precipitate in the postacrosomal region (Fig. 8). DISCUSSION The pyroantimonate technique has been widely used to localize free and loosely bound calcium in cells and tissues and its selectivity for identifying
151
calcium is well recognized (see Wick and Hepler, 1982 ). The pyroantimonate precipitates observed during this study are from free or loosely bound calcium since they were removed by incubation with EGTA whereas sections kept in distilled water retained the deposits (Wick and Hepler, 1982 ). Conditions during the fixation procedure were suitable for localization of calcium as already shown by Franchi and Camatini (1985). Sections of testis show precipitates at the P face of the plasma membrane of sertoli cells and it is known that many enzymes and proteins bound to plasma membranes are Ca 2+-dependent (Dedman et al., 1979) and in addition, a Ca 2+activated adenylate cyclase in haploid germ cell membranes has already been reported (Adamo et al., 1981 ). It is possible that the precipitates seen in the postacrosomal region of developing spermatozoa might have been deposited from calcium bound to actin which is known to be involved in various aspects of spermiogenesis and also in condensing the nucleus by forming a contractile system around it (Russel, 1980). We cannot, however, eliminate the possibility that during development, sperm may accumulate calcium in the postacrosomal region for subsequent mobilization during capacitation, although we were not able to detect free calcium in epididymal spermatozoa by the pyroantimonate technique and further studies would be needed to confirm or refute this suggestion. However, Berruti et al. (1986) have identified intracellular Ca 2+ in the ejaculated spermatozoa of boar. Spermatozoa from within the cauda epididymis were devoid of pyroantimonate precipitates and little deposit was seen in the lumen of the epididymis. Epididymal fluid contains very little calcium and it has been suggested that this lack of extracellular Ca 2+ in the lumen of epididymis could be one reason for the lack of motility of spermatozoa in the mammalian epididymis ( Morton et al., 1974). When epididymal spermatozoa are incubated in calcium-containing medium, the calcium binds to them initially in certain specific regions. Ram spermatozoa, fixed during the acrosome reaction, show intracellular calcium precipitates between PM and OAM ( Watson and Plummer, 1986). In hamster spermatozoa, these precipitates were observed in the anterior region which seems to transport calcium into the acrosome ( Ruknudin and Dadoune, 1986). In a number of other mammals, the anterior region of the spermatozoa appears to be the site of the calcium transport mechanism (Gordon, 1977; Gordon et al., 1978). Before calcium is transported across the membranes, binding occurs as has been demonstrated in muscles (Sulakhe et al., 1973). During the progress of the acrosome reaction, precipitates were also noticed in the postacrosomal region where they were found beneath the plasma membrane. This region has been shown to contain actin ( Clarke and Yanagimachi, 1978) which could provide binding sites for calcium. Actin and other cytoskeletal proteins may play important roles during capacitation. In boar spermatozoa, microtubules have been demonstrated in the postacrosomal region dur-
152 ing the acrosomal reaction and calcium binding occurs in this region during microtubule formation ( Saxena et al., 1986). However, these microtubules have not been observed in hamster spermatozoa b u t the proper fixation procedures may yet visualize t h e m ( Clark and Yanagimachi, 1978). Since calmodulin has been identified in the postacrosomal region of guinea pig spermatozoa (Yamamoto, 1985 ), there is the possibility that a calcium-binding protein may also be present in hamster spermatozoa. The increase in intracellular calcium in the middle region of hamster spermatozoa as observed in this study during capacitation is similar to the increase in calcium in bovine spermatozoa reported after ionophore t r e a t m e n t (Babcock et al., 1978). This study shows that during maturation and also during capacitation and the acrosome reaction, free or loosely b o u n d calcium localizes at specific sites in the head of spermatozoa. T h e binding of calcium in the anterior region may well be involved in its transport across the plasma membrane during the acrosome reaction to induce fusion between the outer acrosomal membrane and the plasma membrane ( W a t s o n and Plummer, 1986). The appearence of free calcium in the postacrosomal region seems to be a preliminary step in capacitation leading to the acrosome reaction. However, the precise role of the calcium that binds in the postacrosomal region of hamster spermatozoa remains to be investigated. ACKNOWLEDGEMENTS We t h a n k Mr. X. Marchi and Mrs. Y. Meyer for their expert technical assistance. One of the authors (A.R.) is grateful to J a m a l M o h a m e d College, Tiruchirapalli, India, for having granted him leave during the course of study.
REFERENCES Adamo, S., Conti, M., Geremia, R. and Monesi, V., 1981. Adenylate cyclase of male germ cells. Adv. Cyclic Nucleotide Res., 14: 657-659. Babcock, D.F., Stamerjohn, D.M. and Hutchinson, T., 1978. Calcium redistribution in individual cells correlated with ionophore action on motility. J. Exp. Zool., 204: 391-400. Berruti, G., Franchi, E. and Camatini, M., 1986. Ca2+ localization in boar spermatozoa by the pyroantimonate technique and X-ray microanalysis. J. Exp. Zool., 237: 257-262. Borle, A.B., 1981. Control, modulation and regulation of cell calcium. Rev. Physiol. Biochem. Pharmacol., 90: 13-153. Clarke, G.N. and Yanagimachi, R., 1978. Actin in mammalian sperm heads. J. Exp. Zool., 205: 125-132. Dedman, J.R., Brinkly, B.R. and Means, A.R., 1979. Regulation of microfilaments and microtubules by calcium and cyclic AMP. Adv. Cyclic Nucleotide Res., 11: 131-174. Franchi, E. and Camatini, C., 1985. Morphological evidence for calcium stores at sertoli-sertoli and sertoli-spermatid interrelations. Cell Biol. Int. Rep., 9: 441-446.
153 Fraser, L.R., 1984. Mechanisms controlling mammalian fertilization. Oxford Rev. Reprod. Biol., 6: 174-225. Gordon, M., 1977. Cytochemical analysis of the membranes of the mammalian sperm head. In: R.D. Yates and M. Gordon (Editors), Male Reproductive System. Masson Publishing U.S.A. Inc., New York, NY, pp. 15-33. Gordon, M., Dandekar, P.V. and Eager, P.R., 1978. Identification of phosphatases on the membranes of guinea pig sperm. Anat. Res., 191: 123-134. Gravis, C.J., 1979. Cytochemical localization of calcium ions in the testis of the Syrian hamster, utilizing potassium pyroantimonate. Am. J. Anat., 154: 245-266. Iwamatsu, T. and Chang, M.C., 1971. Factors involved in the fertilization of mouse eggs in vitro. J. Reprod. Fertil., 26: 197-208. Mohri, H. and Yanagimachi, R., 1980. Characteristics of motor apparatus in testicular, epididymal and ejaculated spermatozoa. Exp. Cell Res., 127: 191-196. Morton, B., Harrigan-Lum, J., Albagli, L. and Jooss, T., 1974. The activation of motility in quiescent hamster sperm from the epididymis by calcium and cyclic nucleotides. Biochem. Biophys. Res. Commun., 56: 372-379. Reed, P.W. and Lardy, H.A., 1972. A23187: a divalent cation ionophore. J. Biol. Chem., 247: 6970-6977. Roldan, E.R.S., Shibata, S. and Yanagimachi, R., 1986. Effect of Ca 2+ antagonists on the acrosome reaction of guinea pig and golden hamster spermatozoa. Gamete Res., 13: 281-292. Ruknundin, A. and Dadoune, J.P., 1986. Cytochemical localization of calcium in hamster spermatozoa during acrosomal reaction. Arch. Biol., 97 Suppl. 1: 180. Russel, L.D., 1980. Sertoli-germ cell interactions: a review. Gamete Res., 3: 179-202. Saxena, N., Peterson, R.N., Saxena, N.K. and Russel, L.D., 1986. Microfilaments appear in boar spermatozoa during capacitation in vitro. J. Exp. Zool., 239: 423-427. Sulakhe, P.V., Drummond, G.I. and Ng, D.C., 1973. Calcium binding by skeletal muscle sarcolemma. J. Biol. Chem., 248: 4150-4157. Watson, P.F. and Plummer, J.M., 1986. Relationship between calcium binding sites and membrane fusion during the acrosome reaction induced by ionophore in ram spermatozoa. J. Exp. Zool., 238: 113-118. Wick, S.M. and Hepler, P.K., 1982. Selective localization of intracellular Ca 2+ with potassium antimonate. J. Histochem. Cytochem., 30: 1190-1204. Yamamoto, N., 1985. Immunoelectron microscopic localization of calmodulin in guinea pig testis and spermatozoa. Acta Histochem. Cytochem., 18: 199-211. Yanagimachi, R., 1982. Requirement of extracellular calcium ions for various stages of fertilization and fertilization-related phenomena in the hamster. Gamete Res., 5: 323-344. Yanagimachi, R. and Usui, N., 1974. Calcium dependence of the acrosome reaction and activation of guinea pig spermatozoa. Exp. Cell Res., 89:161-174
145
Intracellular Calcium in Hamster Spermatozoa in Testis, Epididymis and During the Acrosome Reaction A. RUKNUDIN, J.P. DADOUNE and I.A. SILVER 1'2
Laboratoire d'Histologie, Embryologie et Cytogdndtique, Universitd de Paris VI, 45, rue des Saints-P~res, Paris (France) 1Department of Pathology, Medical School, University of Bristol, Bristol (Great Britain) 2To whom all correspondence should be addressed. (Accepted 29 October 1987)
ABSTRACT Ruknudin, A., Dadoune, J.P. and Silver, I.A., 1988. Intracellular calcium in hamster spermatozoa in testis, epididymis and during the acrosome reaction. Anim. Reprod. Sci., 16: 145-153. Hamster spermatozoa from the testis and cauda epididymis were examined for intracellular localization of calcium using a pyroantimonate technique. Calcium precipitates were found in spermatozoa from the testis but not in those from cauda epididymis. Spermatozoa from the epididymis which had been induced to undergo capacitation and the acrosome reaction in vitro, were also investigated. In these, the anterior region of the acrosome showed precipitates indicating calcium binding at specific sites, possibly prior to transport across the membranes. The additional presence of calcium precipitates in the postacrosomal region suggests that the appearance of free calcium in that region is an important step in the acrosome reaction.
INTRODUCTION
The involvement of calcium in the acrosome reaction in mammalian spermatozoa is well recognized (Fraser, 1984) and it has been shown that extracellular calcium is necessary for this process to occur (Iwamatsu and Chang, 1971; Yanagimachi and Usui, 1974 ). Hamster spermatozoa need extracellular calcium during initiation and maintenance of hyperactivated motility, penetration into the zona pellucida and fusion with eggs (Yanagimachi, 1982). Among the various processes in which spermatozoa require extracellular calcium, the acrosome reaction has been comparatively well studied and an influx of extracellular calcium associated with this reaction was demonstrated by Reed and Lardy (1972). Furthermore, Babcock et al. (1978) showed that bovine spermatozoa exposed to the ionophore A23187 accumulate intracellular cal0378-4320/88/$03.50
© 1988 Elsevier Science Publishers B.V.
147
cium and this ionophore has been used successfully to induce the acrosome reaction in vitro in spermatozoa of many species (Fraser, 1984). Thus the steps in sperm capacitation leading to fertilization appear to involve uptake of extracellular calcium. In the hamster, it has been shown that there are differences in the physiological status of spermatozoa from testis, epididymis and ejaculate (Mohri and Yanagimachi, 1980). Since the importance of intracellular Ca 2+ in the control of function in various cells is well established (Borle, 1981 ) we postulated that there might be parallel differences in the distribution of intracellular Ca 2+, in testicular, epididymal and capacitating spermatozoa. Such possible differences were analyzed using a cytochemical technique and the results are presented here. MATERIALS AND METHODS
Male hamsters (Mesocricetus auratus) were anaesthetized and small portions of testis removed immediately and placed in a fixative containing 2.5% potassium pyroantimonate ( Prolabo, Paris) in 1% osmium tetroxide at pH 7.6 (Gravis, 1979). One portion of the distal segment of the cauda epididymis was placed directly in the fixative while another portion was placed in TALP-1 medium (Yanagimachi, 1982) and the spermatozoa were squeezed out. This cell suspension was divided into three equal parts; the first was centrifuged immediately ( 600g for 5 min) and fixed in pyroantimonate-osmium solution. From the other two parts, the most highly motile spermatozoa were separated by the ascending technique (Roldan et al., 1986) and incubated at 37 ° C under an atmosphere of 5% CO2 in air, to induce capacitation and the acrosome reaction. Of these aliquots, the first was fixed after 1 h and the second after 4 h of incubation. All material was fixed for 1-2 h at 4°C and then washed quickly in 0.01 N potassium acetate. The fixative solutions were filtered through 0.2-/lm millipore filters prior to use. Dehydration and embedding were carried out by stan-
Figs. 1-8. Electron micrographs of sagittal sections through the head region of hamster spermatozoa fixed in pyroantimonate-osmium. Fig. 1. Spermatozoon in testis surrounded by sertoli cells (S). A thick deposit is seen at the postacrosomal lamina (PAL) region (arrow heads) and granules of precipitate are present at the P face of the plasma membrane (PM) of sertoli cells (arrows). × 21 000. Fig. 2. Spermatozoon in cauda epididymis. The characteristic pyroantimonate precipitate is absent. )< 25 000. Fig. 3. Spermatozoon fixed immediately after being released into capacitating medium. No pyroantimonate precipitate is present. X 20 500.
148
Fig. 4. Anterior portion of acrosome of spermatozoon undergoing the acrosome reaction. Double granules between plasma membrane (PM) and outer acrosomal membrane (OAM) are indicated by arrows. × 31 000. Fig. 5. Deposits in the extramitochondrial region of the tail. × 42 000.
Fig. 6. A dense precipitate is seen in the PAL region in a spermatozoon undergoing the acrosome reaction. × 36 000. Fig. 7. Section as for Fig. 6 but exposed to EGTA and stained. No precipitates can be identified. × 35 000. Fig. 8. Acrosome-reacted spermatozoon after 4 h incubation showing absence of calcium precipitate. X 38 800.
8)
150 dard techniques for electon microscopy. Ultrathin sections, either unstained or stained with uranyl acetate and lead citrate, were observed in a Jeol 100CX electron microscope. Two types of controls for the cytochemical technique were carried out. One set of sections was collected on copper grids and incubated in 5 mM EGTA (pH 8 ) at 60 ° C for 45 rain. Simultaneously, another set was floated on distilled water and incubated under the same conditions. RESULTS Calcium precipitates were found in the postacrosomal region of spermatozoa fixed in the final stages of spermiogenesis and fine deposits were also observed on the cytoplasmic side of the plasma membrane (PM) of surrounding sertoli cells (Fig. 1). However, sperm within the epididymis did not show calcium pyroantimonate precipitates at the postacrosomal lamina (PAL), although fine deposits could be observed in lumen of the epididymal duct ( Fig. 2 ). Sperm from the epididymis showed no calcium precipitate in either the periacrosomal or postacrosomal regions immediately after dilution into the capacitating medium (Fig. 3). However, after incubation in the medium for 1 h, many spermatozoa from the epididymis exhibited precipitates; but in any single preparation there were spermatozoa at various stages of the acrosome reaction. During the initial stages of the reaction, calcium precipitates were found in the anterior region of the acrosome (Fig. 4), and at higher magnification they could be seen between the P face of the PM and the E face of the outer acrosomal membrane (OAM), i.e., at the intermembranous region. They were attached to the opposite sides of PM and OAM. These precipitates were removed by EGTA treatment. In the later stages of the acrosome reaction, when the hybrid vesicles of PM and OAM were found, the calcium pyroantimonate precipitates were seen inside the vesicles. In the postacrosomal region, a heavier deposit of calcium was observed after incubation of the spermatozoa for 1 h. These heavy precipitates which were localized posterior to the equatorial region and anterior to the plasma membrane in the postacrosomal region (Fig. 6 ), were also removed by EGTA digestion (Fig. 7 ). In the midpiece of spermatozoa undergoing the acrosome reaction, the precipitates were found to be intracellular but extramitochondrial (Fig. 5). Such intracellular precipitates were not found at the other stages of maturation. For instance, spermatozoa fixed after incubation for 4 h, by which time the acrosome reaction was completed, had very little or no calcium precipitate in the postacrosomal region (Fig. 8). DISCUSSION The pyroantimonate technique has been widely used to localize free and loosely bound calcium in cells and tissues and its selectivity for identifying
151
calcium is well recognized (see Wick and Hepler, 1982 ). The pyroantimonate precipitates observed during this study are from free or loosely bound calcium since they were removed by incubation with EGTA whereas sections kept in distilled water retained the deposits (Wick and Hepler, 1982 ). Conditions during the fixation procedure were suitable for localization of calcium as already shown by Franchi and Camatini (1985). Sections of testis show precipitates at the P face of the plasma membrane of sertoli cells and it is known that many enzymes and proteins bound to plasma membranes are Ca 2+-dependent (Dedman et al., 1979) and in addition, a Ca 2+activated adenylate cyclase in haploid germ cell membranes has already been reported (Adamo et al., 1981 ). It is possible that the precipitates seen in the postacrosomal region of developing spermatozoa might have been deposited from calcium bound to actin which is known to be involved in various aspects of spermiogenesis and also in condensing the nucleus by forming a contractile system around it (Russel, 1980). We cannot, however, eliminate the possibility that during development, sperm may accumulate calcium in the postacrosomal region for subsequent mobilization during capacitation, although we were not able to detect free calcium in epididymal spermatozoa by the pyroantimonate technique and further studies would be needed to confirm or refute this suggestion. However, Berruti et al. (1986) have identified intracellular Ca 2+ in the ejaculated spermatozoa of boar. Spermatozoa from within the cauda epididymis were devoid of pyroantimonate precipitates and little deposit was seen in the lumen of the epididymis. Epididymal fluid contains very little calcium and it has been suggested that this lack of extracellular Ca 2+ in the lumen of epididymis could be one reason for the lack of motility of spermatozoa in the mammalian epididymis ( Morton et al., 1974). When epididymal spermatozoa are incubated in calcium-containing medium, the calcium binds to them initially in certain specific regions. Ram spermatozoa, fixed during the acrosome reaction, show intracellular calcium precipitates between PM and OAM ( Watson and Plummer, 1986). In hamster spermatozoa, these precipitates were observed in the anterior region which seems to transport calcium into the acrosome ( Ruknudin and Dadoune, 1986). In a number of other mammals, the anterior region of the spermatozoa appears to be the site of the calcium transport mechanism (Gordon, 1977; Gordon et al., 1978). Before calcium is transported across the membranes, binding occurs as has been demonstrated in muscles (Sulakhe et al., 1973). During the progress of the acrosome reaction, precipitates were also noticed in the postacrosomal region where they were found beneath the plasma membrane. This region has been shown to contain actin ( Clarke and Yanagimachi, 1978) which could provide binding sites for calcium. Actin and other cytoskeletal proteins may play important roles during capacitation. In boar spermatozoa, microtubules have been demonstrated in the postacrosomal region dur-
152 ing the acrosomal reaction and calcium binding occurs in this region during microtubule formation ( Saxena et al., 1986). However, these microtubules have not been observed in hamster spermatozoa b u t the proper fixation procedures may yet visualize t h e m ( Clark and Yanagimachi, 1978). Since calmodulin has been identified in the postacrosomal region of guinea pig spermatozoa (Yamamoto, 1985 ), there is the possibility that a calcium-binding protein may also be present in hamster spermatozoa. The increase in intracellular calcium in the middle region of hamster spermatozoa as observed in this study during capacitation is similar to the increase in calcium in bovine spermatozoa reported after ionophore t r e a t m e n t (Babcock et al., 1978). This study shows that during maturation and also during capacitation and the acrosome reaction, free or loosely b o u n d calcium localizes at specific sites in the head of spermatozoa. T h e binding of calcium in the anterior region may well be involved in its transport across the plasma membrane during the acrosome reaction to induce fusion between the outer acrosomal membrane and the plasma membrane ( W a t s o n and Plummer, 1986). The appearence of free calcium in the postacrosomal region seems to be a preliminary step in capacitation leading to the acrosome reaction. However, the precise role of the calcium that binds in the postacrosomal region of hamster spermatozoa remains to be investigated. ACKNOWLEDGEMENTS We t h a n k Mr. X. Marchi and Mrs. Y. Meyer for their expert technical assistance. One of the authors (A.R.) is grateful to J a m a l M o h a m e d College, Tiruchirapalli, India, for having granted him leave during the course of study.
REFERENCES Adamo, S., Conti, M., Geremia, R. and Monesi, V., 1981. Adenylate cyclase of male germ cells. Adv. Cyclic Nucleotide Res., 14: 657-659. Babcock, D.F., Stamerjohn, D.M. and Hutchinson, T., 1978. Calcium redistribution in individual cells correlated with ionophore action on motility. J. Exp. Zool., 204: 391-400. Berruti, G., Franchi, E. and Camatini, M., 1986. Ca2+ localization in boar spermatozoa by the pyroantimonate technique and X-ray microanalysis. J. Exp. Zool., 237: 257-262. Borle, A.B., 1981. Control, modulation and regulation of cell calcium. Rev. Physiol. Biochem. Pharmacol., 90: 13-153. Clarke, G.N. and Yanagimachi, R., 1978. Actin in mammalian sperm heads. J. Exp. Zool., 205: 125-132. Dedman, J.R., Brinkly, B.R. and Means, A.R., 1979. Regulation of microfilaments and microtubules by calcium and cyclic AMP. Adv. Cyclic Nucleotide Res., 11: 131-174. Franchi, E. and Camatini, C., 1985. Morphological evidence for calcium stores at sertoli-sertoli and sertoli-spermatid interrelations. Cell Biol. Int. Rep., 9: 441-446.
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