MOLECULAR REPRODUCTION AND DEVELOPMENT 3399-107 (1992)
F-Actin in Acrosome-Reacted Boar Spermatozoa L. CASTELLANI-CERESA, M.F. BRIVIO, AND G. RADAELLI Department of Biology, University of Milan, Milan, Italy
ABSTRACT Biochemical and immunoelectron microscopic methods have been used to analyze the distribution of actin in boar spermatozoa and its state of aggregation before and after acrosome reaction F actin was detected on sperm head and tail by electron microscopy using an improved phalloidin probe. incubation with a fluorescein-phalloidin complex and an anti-fluorescein antibody, followed by labeling with protein A-gold complex. Gold particles, indicating the presence of F-actin, were localized on the sperm surface of the acrosome-reacted spermatozoa. Specific labeling was localized (1) between the outer acrosomal membrane and the plasma mernbrane in the equatorial region, (2) between the outer surface of the fibrous sheath and the plasma membrane in the postacrosomal region, (3) around the connecting piece and the neck region, and (4) on the external surface of the fibrous sheath in the principal piece of the tail. Furthermore, after NP-40 extraction, the SDS-PAGE revealed a difference in solubility between reacted and unreacted boar spermatozoa, reflecting actin polymerization. We conclude that most actin in the acrosome reacted boar spero 1992 Wiley-Liss, Inc matozoa is polymeric Key Words: Spermatozoa, F-actin, Acrosome reaction, Boar
INTRODUCTION Actin has been detected in several mammalian spermatozoa, including hamster (Clarke and Yanagimachi, 1978; Talbot and Kleve, 1978; Flaherty e t al., 1988; Fouquet et al., 19901, dog, rabbit, mouse and guinea pig (Clarke and Yanagimachi, 19781, r a t (Campanella et al., 19791, human (Clarke and Yanagimachi, 1978; Campanella et al., 1979; Clarke e t al., 1982; Virtanen et al., 1984; Flaherty e t al., 19881,boar (Tamblyn, 1980; Camatini et al., 1986; Castellani-Ceresa e t al., 19871, rabbit (Welch and O’Rand, 19851, plains mouse (Flaherty et al., 19831, and mole (Castellani-Ceresa et al., 1986). The localization of actin has been investigated by biochemical and immunological studies (light and electron microscopy). Although these studies confirmed the presence of actin in mammalian spermatozoa, there is still some confusion as to its localization (for review see Lora-Lamia et al., 1986; Flaherty et al., 1986). The relation between monomeric and oligo-polymeric forms of actin in mammalian sperm cells has been investigated by numerous investigators and the results are not conclusive. Many workers have indicated that 0 1992 WILEY-LISS, INC.
the monomeric form (G-actin) is the predominant or only aggregation state of the molecule in uncapacitated sperm (Ochs and Wolf, 1985; Virtanen, et al., 1984; Flaherty, et al., 1988). However, there is some controversy about the aggregation state of the protein after the acrosome reaction. While many investigators feel that actin has a significant role in spermiogenesis (Baccetti et al., 1980; Tamblyn, 1980; Welch and O’Rand, 1985; Halenda et al., 1987; Masri et al., 1987; Fouquet et al., 19891, the function of this protein in mature spermatozoa is still uncertain, mostly because of the differences in actin localization reported by various groups. In accord with Talbot and Kleve (1978) we think that actin might play a significant role in the events of mammalian fertilization, as a result of the ability of the sperm cytoskeleton, of which actin is a n important component, to regulate the maintenance or the mobility of the sperm surface domains during the processes of capacitation, acrosome reaction and fertilization. The involvement of actin in the migration of plasma membrane proteins would be strongly supported if the polymerization of G-actin to F-actin during capacitation and acrosome reaction, suggested by Saxena et al. (19861, is confirmed. In this report we describe a n improved immunological approach to recognition and localization of F-actin by a three-step indirect procedure, i.e., fluoresceinphalloidin, anti-f luorescein antibody, and protein A-gold complex, was used successively to label filamentous actin. In addition, a biochemical approach based on differential Nonidet P-40 extraction was employed to confirm the data. The aim of this study was to demonstrate, biochemically and immunologically, the presence of F-actin in acrosome-reacted boar spermatozoa.
MATERIALS AND METHODS Chemicals All chemicals used are commercially available and of analytical grade. They were purchased from Sigma Chemical Company (St. Louis, MO); Molecular Probes, Inc. (Eugene, OR) and BioRad Laboratories (Richmond, CA). Fluorescein-phalloidin and anti-fluorescein IgG were from Molecular Probes (code F-432 and A-889); polyclonal anti-actin antibodies were from Sigma Received January 14,1992; accepted April 6,1992. Address reprint requests to Dr. L. Castellani-Ceresa, Department of Biology, Section of Zoology and Cytology, University of Milan, via Celoria 26,20133 Milan, Italy.
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Chemical Company (code 59F-4851); rabbit antiserum was developed to actin isolated from chicken back muscle a s the immunogen. Protein A-gold complex (10 nm gold) was from Sigma Chemical Company (code 98F8105). Monoclonal anti-actin was from Amersham (code N-350; immunogen cytoskeletal proteins from chicken gizzard); a n anti-mouse Ig (Amersham code NA.931) was employed as secondary antibody. Biochemical reagents were from BioRad.
Experimental Procedures Boar-ejaculated sperm were obtained from Ente Lombard0 Potenziamento Zootecnico (Zorlesco, Milan, Italy). For all techniques used, the sperm were washed twice by suspension in 0.1 M phosphate-buffered saline (PBS), centrifuged for 15 min a t 500g, and then processed by the various techniques we used in this study. Acrosome reaction. Washed sperm were resuspended in a capacitation medium of the following composition: 139mM NaCl, lOmM Hepes, lOmM glucose, 2mM calcium chloride, and 0.5% BSA. The incubation was carried out for 1h r a t 37°C (pH of medium: 7.4). The acrosome reaction was initiated in capacitated sperm by the addition of the divalent metal cation ionophore A23187. The stock solution of A23187 (dissolved in DMSO) was 3.8 mM, and we added 2 pliml. The suspension was gently stirred for 2 h r at 37°C. After 1hr, we checked the acrosome reaction, the quality and the motility of the sperm suspension by phase-contrast microscopy. Immunoelectron microscopic labeling. Two different groups of spermatozoa were used in all experiments, uncapacitated spermatozoa and acrosomereacted spermatozoa. Washed spermatozoa were fixed in a mixture of 0.4% glutaraldehyde and 2% paraformaldehyde buffered with 0.1 M phosphate buffer, pH 7.4, for 45 min a t room temperature. After three washes in the same buffer, the pelletted material was embedded in agar (Castellani-Ceresa and Brivio, 1991) and cut into 40-pm sections with a Sorvall TC-2 tissue sectioner. The protocol issued by Sorvall with the Smith and Farquhar TC-2 Tissue Sectioner was followed. Briefly, 7% agar is liquefied in boiling water and stored in a n oven a t 60180°C (agar solidifies at about 40°C). A strip of fixed tissue or a pellet is placed on a polyethylene filter paper cushion and covered with agar at 50°C. Then the sample is placed in a refrigerator for 5 min to harden the agar. The cushion containing the tissue or the pellet is secured to the specimen table of the sectioner (provided with a razor blade) and sections ranging from 5 to 200 pm can be obtained. With this technique, about 40% of specimens 8 x 11.5 pm in size, which is the size of boar sperm, in a section of 40 pm thick should be cut through. The cut sections were washed overnight in PBS containing 0.1 M glycine to block free aldehyde groups. Incubation was carried out as follows: Step 1.The sections, washed in 0.1M glycine-NaOH, pH 7.4, then in 0.1M phosphate-buffered solution, pH
7.4, were incubated with fluorescein-phalloidin (diluted 1:lO in phosphate buffer containing 0.1% glycine and 1% BSA); Step 2. The specimens were incubated with a n antifluorescein IgG (diluted 1:lO in phosphate buffer containing 0.1%glycine and 1% BSA); Step 3. The anti-fluorescein IgG was visualized with the protein A-gold complex (protein A-gold diluted 1:20 in phosphate buffer with 0.5% BSA and 0.05% Tween 20). After two washes for 5 min each in 0.1M phosphate buffer, the sections were fixed with 2% glutaraldehyde and postfixed with 1% omium tetroxide. The sections were then dehydrated with a graded series of ethanol and embedded in Epon-Araldite. Thin sections (60-80 nm) were cut with a glass knife, using a Reickert-Jung E Ultramicrotome, mounted on copper grids, stained with uranyl acetate for 10 min and with lead citrate for 1 min, and examined in a Jeol lOOSX electron microscope. Many types of control were performed: (1) sections incubated with a nonimmune serum instead of the antifluorescein IgG, (2) phalloidin complex omitted, (3) anti-fluorescein antibody omitted, and (4) saturation of actin with phalloidin prior to incubation with fluorescein-phalloidin.
Biochemical Analysis Differential extraction. Extractions with detergent were performed by a slightly modified Ochs and Wolf technique (Ochs and Wolf, 1985). Sperm were washed as described under Experimental Procedures and then incubated for 10 min at room temperature with 0.3%Nonidet P-40 (NP-40) in 100mM phosphate buffer (PBS), pH 7.4, with slow shaking. After centrifugation (for 15 rnin a t 1,500g) supernatants were collected and recentrifuged at 18,OOOgfor 1rnin to remove contaminating cells. Aliquots of NP-40 soluble fractions (supernatants of the 18,OOOg centrifugation) were collected, dialyzed against 0.01M phosphate buffer for 24 h r and finally lyophilized in a Savant Speed Vac Concentrator; other aliquots were precipitated by addition of 20% TCA; washed twice in Diethylether and dried under a stream of nitrogen. Finally the samples were analyzed by SDS-PAGE followed by Western Blot. A23187 surnatant analysis. After ionophore treatments the incubation media were collected to analyze the protein contents. Aliquots of the surnatants were separated by centrifugation at 1,500g for 15 min and 10,OOOg for 10 rnin and finally treated with TCA (as described under Differential Extraction). The TCA-precipitated fractions were submitted to analytical electrophoresis. SDS-PAGE. Monodimensional electrophoresis was done by a slightly modified Laemmli technique (Laemmli, 1970). A 10% acrylamide gel was employed in the resolving gel for analytical purposes. The samples were resuspended and denatured for 5 min a t 100°C in sample buffer (0.01 M PB, pH 7, 1%SDS, 0.1 M dithiothrei-
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Fig. 1. Longitudinal section of uncapacitated boar sperm showing the acrosome (A) and the equatorial segment (ES).Only occasional gold particles are visible (arrow). ~ 5 6 , 0 0 0 . Fig. 2. lmmunogold localization of F-actin in acrosome-reacted boar sperm. Gold particles are present a t the equatorial (ES) and postacrosomal (PS) segments, but only where the plasma membrane is detached. ~ 5 5 , 0 0 0 .
tol, 10% glycerol, 0.001% bromophenol blue), loaded onto the gels and run overnight a t 60 volts (constant voltage) in a BioRad Protean 11 Cell. After electrophoresis the gels were stained with Coomassie-blue R-250. Western blot. Polypeptides were transferred onto nitrocellulose sheets by the procedure of Towbin et al. (1979). After transfer, the sheets were soaked in blocking solution (137 mM NaC1; 2.7 mM KC1; 1.5 mM KH,PO,; 8 mM Na,HPO,, pH 7.3 plus 3% bovine serum albumin and 0.1% Triton x-100)for 30 min a t room temperature and then incubated with monoclonal
anti-actin (1:1,000 in blocking solution) for 1 hour and with peroxidase-conjugated anti-mouse Ig (1:1,000 in blocking solution), finally revealed by DAB-Cobalt Chloride (Scopsi and Larsson, 1986). All treatments were performed at room temperature. In the controls, the first antibody was omitted.
RESULTS Immunogold Staining of F-Actin When freshly ejaculated non-capacitated spermatozoa were incubated with the fluorescein-phalloidin
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Figs. 3,4. Immunogold localization of F-actin after acrosome reaction; the plasma membrane has disappeared and a great number of gold particles are visible (arrows). ~ 4 1 , 0 0 0Inset: . high magnification of the boxed area showing gold particles (10 nm). ES, equatorial segment; PS, postacrosomal segment. x 135,000
complex and anti-fluorescein antibody, followed by labeling with protein A-gold complex, the gold particles that indicate the presence of F-actin were not detected on the sperm surface, although the plasma membrane was often partially detached (Fig. 1). (Only occasional gold particles were visible.) After the acrosome reaction had been induced by ionophore A23187, F-actin was clearly demonstrated. At the beginning of the acrosome reaction, the morphologic integrity of the spermatozoa was still sufficiently preserved, but the plasma membrane was largely detached; the gold particles were visible on the equatorial and postacrosomal segments
(Fig. 2). Figure 2 shows that in the postacrosomal segment the gold particles were present only on the right side of the spermatozoon, precisely on the external surface of the postacrosomal sheath, while on the left side of the postacrosomal segment, still covered by the plasma membrane, no gold particles were visible. It is interesting that the equatorial and postacrosomal regions are those in which the monomeric form of actin has been localized in uncapacitated sperm by various groups, including ours. With the completion of the acrosome reaction, the membranes are no longer detectable (Figs. 3, 4)and a great number of gold particles
F-ACTIN IN BOAR SPERMATOZOA
Fig. 5. Immunogold localization of F-actin in the neck region. N, neck; PS, postacrosomal segment. x 40,000. Fig. 6. Longitudinal section of the sperm tail showing the middle and the principal pieces. Gold can be observed only on the fibrous sheath of the principal piece (arrow). MD, middle piece; PP, principal
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piece. x30,OOO. Figs. 7,s. Longitudinal sections of the sperm tail showing the principal piece. Gold particles are visible on the ribs of the fibrous sheath, where the plasma membrane is absent (arrows). ~ 3 0 , 0 0 0 .
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Figs. 9,lO. Control experiments. Fig. 9. Results obtained when F-actin is saturated with phalloidin prior to the incubation with fluorescein-phalloidin. ES: equatorial segment. PS, postacrosomal segment. x27,OOO.
Fig. 10. Results obtained on spermatozoa incubated with a nonimmune serum instead of the anti-fluorescein IgG. ES, equatorial segment; PS, postacrosomal segment. X41,OOO. In both cases, practically no gold particles (arrows) are seen over the reacted sperm.
were present on the sperm head surface, confirming previous localization of actin (Lora Lamia et al., 1986) under the plasma membrane. The presence of actin in the sperm tail was also investigated. Gold particles were detected in the neck (Fig. 5) and in the principal piece of the flagellum (Figs. 6-8) on the fibrous sheath, mainly on the circumferential ribs that connect the two longitudinal columns. The fibrous sheath is present only in the principal piece of the flagellum; it is located immediately under the plasma membrane and surrounds the outer dense fibers. We did not find F-actin on the outer dense fibers, but only on the fibrous sheath. In the tail, as in the head, the gold particles were localized only in zones where the plasma membrane had been detached (Figs. 6-81.
tween 35 kDa and 90 kDa, with the unreacted sperm showing more striking components (Fig. 11, lane c). The 45 kDa actin band (Fig. 11, arrowhead) was easy to detect only in unreacted sperm samples, although a faint band could also be seen in the ionophore-reacted sperm (Fig. 11, lane b). Furthermore, in the immunoblotting analysis, the unreacted sperm’s 45 kDa band cross-reacted with monoclonal anti-actin antibodies (Fig. 12, lane b). The analysis of A23187-treatment supernatant showed a simple pattern with three main bands; however, the putative actin band was not observable (Fig. 13, lane a?, and we did not observe any reaction in immunoblotting of A23187 supernatants.
SDS-PAGE and Immunoblotting NP-40-treated samples from reacted and unreacted sperm were studied to see whether or not actin polymerization gives rise to a detergent-resistant fraction, reflecting a change in solubility of the cytoskeletal components. When analyzed by SDS-PAGE, both the reacted and unreacted sperm samples showed similar banding patterns, while molecular weights ranging from 15 kDa to 120 kDa (Fig. 11,lanes b,c). There were differences between reacted and unreacted sperm be-
DISCUSSION The localization of F-actin by electron microscopy can be achieved either with heavy meromyosin or phalloidin-gold complexes. Phalloidin, a cyclic peptide from the mushroom Amanita phalloides, is one of the family of phallotoxins, which specifically bind to filamentous actin (Wieland et al., 1975).To our knowledge, previous studies of the ultrastructural localization of F-actin with phalloidin had been performed by Lachapelle and Aldrich (1988) with Physarum polycephalum, by Faulstich e t al. (1989) with rat cultured fibroblasts and by
F-ACTIN IN BOAR SPERMATOZOA
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Fig. 11. PAGE pattern of Nonidet P-40 extracted boar spermatozoa (soluble fraction). Lane a, molecular-weight markers; lane b, reacted sperm; lane c, unreacted sperm. SDS-PAGE, under reducing condition, shows a pattern of bands ranging from 15 t o 120 kDa; arrowhead indicates the 45-kDa actin band.
us (Castellani-Ceresa et al., 1991) with boar sperm. Lachapelle and Aldrich (1988) directly complexed phalloidin to colloidal gold and suggested that these phalloidin-gold complexes are useful tools for studying the distribution of F-actin. Faulstich et al. (1989) described the preparation of four biotin derivatives of phalloidin and the use of these in combination with gold-labeled avidin. Both methods permit labeling of F-actin without using antibodies. In the present work, we used a fluorescein-phalloidin complex and a n anti-f luorescein antibody, followed by labeling with protein A-gold complex. We localized filamentous actin in both head and tail of boar spermatozoa after the acrosome reaction, and these results were supported by detergent extraction of reacted and unreacted sperm. The presence of F-actin in mammalian reacted sperm heads has been affirmed by some investigators and denied by others. Peterson et al. (1978) observed microfilaments in the equatorial and postacrosomal regions of boar spermatozoa after the acrosome reaction by electron microscopy. Schatten et al. (1983) localized microfilaments in the equatorial region of mouse and hamster sperm during capacitation and acrosome reaction by immunofluorescence. Recently, Peterson et al. (1990) demonstrated the presence of F-actin in boar sperm plasma membrane by
Fig. 12. Western blot of the soluble fraction from reacted (a) and unreacted (b) Nonidet P-40 extracted boar sperm. The immune anti actin complex was revealed by DAB-COCI,. Lane a, reacted sperm; lane b, unreacted sperm.
fluorescence microscopy using the specific probe NBDphallacidin. They also used S1 myosin subfragments attached to colloidal gold to localize F-actin ultrastructurally. Moreover, F-actin is probably a component of the cross filaments described by Escalier (1984) in human spermatozoa. Confirming the presence and localization of F-actin in the postnuclear and tail regions of human spermatozoa, Clarke et al. (1982) suggested that actin might undergo changes of state during capacitation and acrosome reaction. NBD-phallacidin was used by Saxena et al. (1986) to localize F-actin in boar spermatozoa after capacitation, and they suggested that polymerization of actin could play a significant role in the capacitation. Several other investigators have reported that actin is present in mature spermatozoa only in monomeric form (Tamblyn, 1980; Virtanen et al., 1984; Welch and O’Rand, 1985; Flaherty et al., 1986). However, most of these workers did not look for F-actin in spermatozoa after acrosome reaction, although lack of NBD-phallacidin staining in acrosome-reacted boar spermatozoa was reported by Camatini et al. (1986) and in acrosome-reacted hamster spermatozoa by Fouquet e t al. (1990). In this paper, we present immunoelectron microscopic and biochemical data useful to ascertain the possibility of a change in the state of aggregation of actin after capacitation and acrosome reaction. The results presented here show that when the acrosome-reacted boar spermatozoa are labeled by the
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I
0 Y
-97
-31
-21 a
b
Fig. 13. Protein composition of the supernatant from A23187 treated sperm analyzed by SDSPAGE. The actin putative band is absent. Lane a, A23187 supernatant polypeptides; lane b, molecularweight markers.
three step procedure, F-actin is localized in the equatorial or postacrosomal regions. Recent data (Yanagimachi, 1988; Talansky et al., 1991) have suggested that the plasma membrane over either the equatorial or the postacrosomal segments might be the site of fusion with the egg plasma membrane. The sperm plasma membrane over these regions became able to fuse with the egg plasma membrane as a result of change in distribution and composition of cell surface components. Earlier studies (Ochs e t al., 1986; Olson et al., 1987) demonstrated cytoskeletal proteins, including actin, in mammalian spermatozoa. The hypothesis that actin polymerizes before sperm penetration is supported by the data on reduced penetration of eggs by sperm preincubated with cytochalasin D (an inhibitor of the polymerization of actin) (Rogers, 1987, 1989; our unpublished data). Possibly this reduction of penetration could be related to the hypothesis that actin, together with other cytoskeletal components, may regulate the changes in sperm membrane domains that occur during the various events leading to fertilization (Virtanen et al., 1984). In fact, Saxena et al., (1986) observed that cytochalasin D prevents movements of the membrane proteins. If, as Rogers et al. (1989) suggest, cytochalasin D inhibits fertilization, it could be due to its ability to prevent actin polymerization.
The presence of actin in the sperm tail has been widely demonstrated, although there are still some discrepancies about its exact localization and state of aggregation. According to Clarke et al. (1982) it is probable that actin is in filamentous form in the connecting piece, middle piece and principal piece of the human sperm tail, while Talbot and Kleve (1978) say that there is actin in the principal piece and not in the middle piece in hamster. Baccetti et al. (1984) found actin in the fibrous sheath and the pericentriolar region of bull sperm. Flaherty et al. (1988) localized actin on the external surface of the fibrous sheath in the principal piece of several mammalian spermatozoa, but without specifying its aggregation form. In acrosome-reacted boar spermatozoa we localized F-actin in both the neck region and the fibrous sheath of the principal piece of the tail. The disagreement about the changes in the state of aggregation of actin after the acrosome reaction is probably mainly attributable to the lack of biochemical data to compare with the immunocytochemical assays. In this work, our biochemical approach was based on the differences in detergent solubility of the monomeric and polymeric forms of actin. The hypothesis of a change in the state of aggregation of actin before fertilization seems to be confirmed by the electrophoretic behaviors of the reactedhonreacted sperm samples. In unreacted sperm, the higher solubility of the monomeric form of the actin produced a more conspicuous band of about 45 kDa, which in addition was positive to anti-actin monoclonal antibodies. Since we observed some other bands indicating greater solubility by nonionic detergents, we suppose that, in reacted sperm, the exocytotic process gives rise to the differences in the SDS-PAGE patterns. The hypothesis that actin is released along with the acrosome vescicle components was verified by analysis of the acrosome reaction supernatants; the actin band could no longer be detected by SDS-PAGE or immunolocalization after Western blot. However we cannot exclude a priori that a small amount of monomeric actin is released, perhaps attributable to the physiological turnover of the molecule, during the metabolic changes before fertilization. In conclusion, this study suggests that there is polymerization of actin during capacitation and/or acrosome reaction. Additional work is necessary to further define the exact period during which this change of the state of aggregation of actin occurs.
ACKNOWLEDGMENTS This study was supported by CNR and MURST grants. REFERENCES Baccetti B, Bigliardi E, Burrini AG, Gabbiani G, Jockusch BM, Leoncini P (1984): Microfilaments and intermediate sized filaments in sperm tail. J Submicrosc Cytol16:79-84. Baccetti B, Bigliardi E, Burrini AG, Pallini V (1980):Actin filaments and mitochondria1 movement in vertebrate spermiogenesis. Gamete Res 3203-209.
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Moore HDM, Bedford JM (1983): The interaction of mammalian gametes in the female. In YF Hartmann (ed):“Mechanism and Control of Animal Fertilization.” New York: Academic Press, pp 453-497. Ochs D, Wolf DP (1985): Actin in ejaculated human sperm cells. Biol Reprod 33:1223-1226. Ochs D, Wolf DP, Ochs RL (1986): Intermediate filament proteins in human sperm heads. Exp Cell Res 167:495-504. Olson GE, Hamilton DW, Fawcett DW (1976): Isolation and characterization of the perforatorium of rat spermatozoa. Reprod Fertil 47:293-297. Olson GE, Winfrey VP, Flaherty SP (1987):Cytoskeletal assemblies of mammalian spermatozoa. In MC Orgebin-Crist, Danzo BJ (eds): Ann NY Acad Sci 413:222-261. Peterson R, Russel L, Bundman D, Freund M (1978): Presence of microfilaments and tubular structures in hoar spermatozoa after chemically inducing the acrosome reaction. Biol Reprod 19:459466. Peterson RN, Bozzola JJ, Hunt WP, Darabi A (1990): Characterization of membrane-associated actin in boar spermatozoa. J Exp Zool 253:202-214. Rogers BJ, Bastias C, Russell LD, Peterson RN (1987):Cytochalasin D inhibition of guinea pig sperm actin reduces fertilization. Ann NY Acad Sci 513:566-568. Rogers BJ, Bastias C, Coulson RL, Russell LD (1989):Cytochalasin D inhibits penetration of hamster eggs by guinea pig and human spermatozoa. J Androl 10:275-282. Saxena N, Peterson RN, Sharif S, Saxena NK, Russell LD (1986): Changes in the organization of surface antigens during in-vitro capacitation of boar spermatozoa as detected by monoclonal antibodies. J Reprod Fertil78:601-614. Schatten G, Simerly C, Cline C, Schatten H (1983): Microtubules and microfilaments in mouse oocytes and sperm during fertilization: Immunofluorescence and fluorescence localization. J Cell Biol 97:534h. Scopsi L, Larsson LI (1986):Increased sensitivity in peroxidase immunocytochemistry. A comparative study of a number of peroxidase visualization methods employing a model system. Histochemistry 81:221-230. Talansky BE, Malter HE, Cohen J 11991):A preferential site for sperm egg-fusion in Mammals. Mol Reprod Dev 28:183-188. Talbot P, Kleve MG (1978): Hamster sperm cross-react with antiactin. J Exp Zool 204:131-136. Tamblyn TM (1980): Identification of actin in boar epididymal spermatozoa. Biol Reprod 22:727-734. Towhin H, Staehlin T, Gordon J (1979): Electrophoretic transfer of proteins from polyacrilamide gels to nitrocellulose sheets, procedure and some applications. Proc Natl Acad Sci USA 76:43504354. Virtanen I, Badley RA, Paasivuo R, Lehto VP (1984): Distinct cytoskeletal domains revealed in sperm cells. J Cell Biol 99:10831091. Welch J E , ORand MG (1985): Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit. Dev Biol 109:411-417. Wieland TH, de Vries JX, Schaffer A, Faulstich H (1975): Spectroscopic evidence for the interaction of phalloidin with actin. FEBS Lett 54(1):73-77. Yanagimachi R (1988): Mammalian fertilization. In E Knobil, J Neil1 (eds): “Physiology of Reproduction.” New York: Raven Press, pp 135-185.
F-Actin in Acrosome-Reacted Boar Spermatozoa L. CASTELLANI-CERESA, M.F. BRIVIO, AND G. RADAELLI Department of Biology, University of Milan, Milan, Italy
ABSTRACT Biochemical and immunoelectron microscopic methods have been used to analyze the distribution of actin in boar spermatozoa and its state of aggregation before and after acrosome reaction F actin was detected on sperm head and tail by electron microscopy using an improved phalloidin probe. incubation with a fluorescein-phalloidin complex and an anti-fluorescein antibody, followed by labeling with protein A-gold complex. Gold particles, indicating the presence of F-actin, were localized on the sperm surface of the acrosome-reacted spermatozoa. Specific labeling was localized (1) between the outer acrosomal membrane and the plasma mernbrane in the equatorial region, (2) between the outer surface of the fibrous sheath and the plasma membrane in the postacrosomal region, (3) around the connecting piece and the neck region, and (4) on the external surface of the fibrous sheath in the principal piece of the tail. Furthermore, after NP-40 extraction, the SDS-PAGE revealed a difference in solubility between reacted and unreacted boar spermatozoa, reflecting actin polymerization. We conclude that most actin in the acrosome reacted boar spero 1992 Wiley-Liss, Inc matozoa is polymeric Key Words: Spermatozoa, F-actin, Acrosome reaction, Boar
INTRODUCTION Actin has been detected in several mammalian spermatozoa, including hamster (Clarke and Yanagimachi, 1978; Talbot and Kleve, 1978; Flaherty e t al., 1988; Fouquet et al., 19901, dog, rabbit, mouse and guinea pig (Clarke and Yanagimachi, 19781, r a t (Campanella et al., 19791, human (Clarke and Yanagimachi, 1978; Campanella et al., 1979; Clarke e t al., 1982; Virtanen et al., 1984; Flaherty e t al., 19881,boar (Tamblyn, 1980; Camatini et al., 1986; Castellani-Ceresa e t al., 19871, rabbit (Welch and O’Rand, 19851, plains mouse (Flaherty et al., 19831, and mole (Castellani-Ceresa et al., 1986). The localization of actin has been investigated by biochemical and immunological studies (light and electron microscopy). Although these studies confirmed the presence of actin in mammalian spermatozoa, there is still some confusion as to its localization (for review see Lora-Lamia et al., 1986; Flaherty et al., 1986). The relation between monomeric and oligo-polymeric forms of actin in mammalian sperm cells has been investigated by numerous investigators and the results are not conclusive. Many workers have indicated that 0 1992 WILEY-LISS, INC.
the monomeric form (G-actin) is the predominant or only aggregation state of the molecule in uncapacitated sperm (Ochs and Wolf, 1985; Virtanen, et al., 1984; Flaherty, et al., 1988). However, there is some controversy about the aggregation state of the protein after the acrosome reaction. While many investigators feel that actin has a significant role in spermiogenesis (Baccetti et al., 1980; Tamblyn, 1980; Welch and O’Rand, 1985; Halenda et al., 1987; Masri et al., 1987; Fouquet et al., 19891, the function of this protein in mature spermatozoa is still uncertain, mostly because of the differences in actin localization reported by various groups. In accord with Talbot and Kleve (1978) we think that actin might play a significant role in the events of mammalian fertilization, as a result of the ability of the sperm cytoskeleton, of which actin is a n important component, to regulate the maintenance or the mobility of the sperm surface domains during the processes of capacitation, acrosome reaction and fertilization. The involvement of actin in the migration of plasma membrane proteins would be strongly supported if the polymerization of G-actin to F-actin during capacitation and acrosome reaction, suggested by Saxena et al. (19861, is confirmed. In this report we describe a n improved immunological approach to recognition and localization of F-actin by a three-step indirect procedure, i.e., fluoresceinphalloidin, anti-f luorescein antibody, and protein A-gold complex, was used successively to label filamentous actin. In addition, a biochemical approach based on differential Nonidet P-40 extraction was employed to confirm the data. The aim of this study was to demonstrate, biochemically and immunologically, the presence of F-actin in acrosome-reacted boar spermatozoa.
MATERIALS AND METHODS Chemicals All chemicals used are commercially available and of analytical grade. They were purchased from Sigma Chemical Company (St. Louis, MO); Molecular Probes, Inc. (Eugene, OR) and BioRad Laboratories (Richmond, CA). Fluorescein-phalloidin and anti-fluorescein IgG were from Molecular Probes (code F-432 and A-889); polyclonal anti-actin antibodies were from Sigma Received January 14,1992; accepted April 6,1992. Address reprint requests to Dr. L. Castellani-Ceresa, Department of Biology, Section of Zoology and Cytology, University of Milan, via Celoria 26,20133 Milan, Italy.
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Chemical Company (code 59F-4851); rabbit antiserum was developed to actin isolated from chicken back muscle a s the immunogen. Protein A-gold complex (10 nm gold) was from Sigma Chemical Company (code 98F8105). Monoclonal anti-actin was from Amersham (code N-350; immunogen cytoskeletal proteins from chicken gizzard); a n anti-mouse Ig (Amersham code NA.931) was employed as secondary antibody. Biochemical reagents were from BioRad.
Experimental Procedures Boar-ejaculated sperm were obtained from Ente Lombard0 Potenziamento Zootecnico (Zorlesco, Milan, Italy). For all techniques used, the sperm were washed twice by suspension in 0.1 M phosphate-buffered saline (PBS), centrifuged for 15 min a t 500g, and then processed by the various techniques we used in this study. Acrosome reaction. Washed sperm were resuspended in a capacitation medium of the following composition: 139mM NaCl, lOmM Hepes, lOmM glucose, 2mM calcium chloride, and 0.5% BSA. The incubation was carried out for 1h r a t 37°C (pH of medium: 7.4). The acrosome reaction was initiated in capacitated sperm by the addition of the divalent metal cation ionophore A23187. The stock solution of A23187 (dissolved in DMSO) was 3.8 mM, and we added 2 pliml. The suspension was gently stirred for 2 h r at 37°C. After 1hr, we checked the acrosome reaction, the quality and the motility of the sperm suspension by phase-contrast microscopy. Immunoelectron microscopic labeling. Two different groups of spermatozoa were used in all experiments, uncapacitated spermatozoa and acrosomereacted spermatozoa. Washed spermatozoa were fixed in a mixture of 0.4% glutaraldehyde and 2% paraformaldehyde buffered with 0.1 M phosphate buffer, pH 7.4, for 45 min a t room temperature. After three washes in the same buffer, the pelletted material was embedded in agar (Castellani-Ceresa and Brivio, 1991) and cut into 40-pm sections with a Sorvall TC-2 tissue sectioner. The protocol issued by Sorvall with the Smith and Farquhar TC-2 Tissue Sectioner was followed. Briefly, 7% agar is liquefied in boiling water and stored in a n oven a t 60180°C (agar solidifies at about 40°C). A strip of fixed tissue or a pellet is placed on a polyethylene filter paper cushion and covered with agar at 50°C. Then the sample is placed in a refrigerator for 5 min to harden the agar. The cushion containing the tissue or the pellet is secured to the specimen table of the sectioner (provided with a razor blade) and sections ranging from 5 to 200 pm can be obtained. With this technique, about 40% of specimens 8 x 11.5 pm in size, which is the size of boar sperm, in a section of 40 pm thick should be cut through. The cut sections were washed overnight in PBS containing 0.1 M glycine to block free aldehyde groups. Incubation was carried out as follows: Step 1.The sections, washed in 0.1M glycine-NaOH, pH 7.4, then in 0.1M phosphate-buffered solution, pH
7.4, were incubated with fluorescein-phalloidin (diluted 1:lO in phosphate buffer containing 0.1% glycine and 1% BSA); Step 2. The specimens were incubated with a n antifluorescein IgG (diluted 1:lO in phosphate buffer containing 0.1%glycine and 1% BSA); Step 3. The anti-fluorescein IgG was visualized with the protein A-gold complex (protein A-gold diluted 1:20 in phosphate buffer with 0.5% BSA and 0.05% Tween 20). After two washes for 5 min each in 0.1M phosphate buffer, the sections were fixed with 2% glutaraldehyde and postfixed with 1% omium tetroxide. The sections were then dehydrated with a graded series of ethanol and embedded in Epon-Araldite. Thin sections (60-80 nm) were cut with a glass knife, using a Reickert-Jung E Ultramicrotome, mounted on copper grids, stained with uranyl acetate for 10 min and with lead citrate for 1 min, and examined in a Jeol lOOSX electron microscope. Many types of control were performed: (1) sections incubated with a nonimmune serum instead of the antifluorescein IgG, (2) phalloidin complex omitted, (3) anti-fluorescein antibody omitted, and (4) saturation of actin with phalloidin prior to incubation with fluorescein-phalloidin.
Biochemical Analysis Differential extraction. Extractions with detergent were performed by a slightly modified Ochs and Wolf technique (Ochs and Wolf, 1985). Sperm were washed as described under Experimental Procedures and then incubated for 10 min at room temperature with 0.3%Nonidet P-40 (NP-40) in 100mM phosphate buffer (PBS), pH 7.4, with slow shaking. After centrifugation (for 15 rnin a t 1,500g) supernatants were collected and recentrifuged at 18,OOOgfor 1rnin to remove contaminating cells. Aliquots of NP-40 soluble fractions (supernatants of the 18,OOOg centrifugation) were collected, dialyzed against 0.01M phosphate buffer for 24 h r and finally lyophilized in a Savant Speed Vac Concentrator; other aliquots were precipitated by addition of 20% TCA; washed twice in Diethylether and dried under a stream of nitrogen. Finally the samples were analyzed by SDS-PAGE followed by Western Blot. A23187 surnatant analysis. After ionophore treatments the incubation media were collected to analyze the protein contents. Aliquots of the surnatants were separated by centrifugation at 1,500g for 15 min and 10,OOOg for 10 rnin and finally treated with TCA (as described under Differential Extraction). The TCA-precipitated fractions were submitted to analytical electrophoresis. SDS-PAGE. Monodimensional electrophoresis was done by a slightly modified Laemmli technique (Laemmli, 1970). A 10% acrylamide gel was employed in the resolving gel for analytical purposes. The samples were resuspended and denatured for 5 min a t 100°C in sample buffer (0.01 M PB, pH 7, 1%SDS, 0.1 M dithiothrei-
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Fig. 1. Longitudinal section of uncapacitated boar sperm showing the acrosome (A) and the equatorial segment (ES).Only occasional gold particles are visible (arrow). ~ 5 6 , 0 0 0 . Fig. 2. lmmunogold localization of F-actin in acrosome-reacted boar sperm. Gold particles are present a t the equatorial (ES) and postacrosomal (PS) segments, but only where the plasma membrane is detached. ~ 5 5 , 0 0 0 .
tol, 10% glycerol, 0.001% bromophenol blue), loaded onto the gels and run overnight a t 60 volts (constant voltage) in a BioRad Protean 11 Cell. After electrophoresis the gels were stained with Coomassie-blue R-250. Western blot. Polypeptides were transferred onto nitrocellulose sheets by the procedure of Towbin et al. (1979). After transfer, the sheets were soaked in blocking solution (137 mM NaC1; 2.7 mM KC1; 1.5 mM KH,PO,; 8 mM Na,HPO,, pH 7.3 plus 3% bovine serum albumin and 0.1% Triton x-100)for 30 min a t room temperature and then incubated with monoclonal
anti-actin (1:1,000 in blocking solution) for 1 hour and with peroxidase-conjugated anti-mouse Ig (1:1,000 in blocking solution), finally revealed by DAB-Cobalt Chloride (Scopsi and Larsson, 1986). All treatments were performed at room temperature. In the controls, the first antibody was omitted.
RESULTS Immunogold Staining of F-Actin When freshly ejaculated non-capacitated spermatozoa were incubated with the fluorescein-phalloidin
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Figs. 3,4. Immunogold localization of F-actin after acrosome reaction; the plasma membrane has disappeared and a great number of gold particles are visible (arrows). ~ 4 1 , 0 0 0Inset: . high magnification of the boxed area showing gold particles (10 nm). ES, equatorial segment; PS, postacrosomal segment. x 135,000
complex and anti-fluorescein antibody, followed by labeling with protein A-gold complex, the gold particles that indicate the presence of F-actin were not detected on the sperm surface, although the plasma membrane was often partially detached (Fig. 1). (Only occasional gold particles were visible.) After the acrosome reaction had been induced by ionophore A23187, F-actin was clearly demonstrated. At the beginning of the acrosome reaction, the morphologic integrity of the spermatozoa was still sufficiently preserved, but the plasma membrane was largely detached; the gold particles were visible on the equatorial and postacrosomal segments
(Fig. 2). Figure 2 shows that in the postacrosomal segment the gold particles were present only on the right side of the spermatozoon, precisely on the external surface of the postacrosomal sheath, while on the left side of the postacrosomal segment, still covered by the plasma membrane, no gold particles were visible. It is interesting that the equatorial and postacrosomal regions are those in which the monomeric form of actin has been localized in uncapacitated sperm by various groups, including ours. With the completion of the acrosome reaction, the membranes are no longer detectable (Figs. 3, 4)and a great number of gold particles
F-ACTIN IN BOAR SPERMATOZOA
Fig. 5. Immunogold localization of F-actin in the neck region. N, neck; PS, postacrosomal segment. x 40,000. Fig. 6. Longitudinal section of the sperm tail showing the middle and the principal pieces. Gold can be observed only on the fibrous sheath of the principal piece (arrow). MD, middle piece; PP, principal
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piece. x30,OOO. Figs. 7,s. Longitudinal sections of the sperm tail showing the principal piece. Gold particles are visible on the ribs of the fibrous sheath, where the plasma membrane is absent (arrows). ~ 3 0 , 0 0 0 .
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Figs. 9,lO. Control experiments. Fig. 9. Results obtained when F-actin is saturated with phalloidin prior to the incubation with fluorescein-phalloidin. ES: equatorial segment. PS, postacrosomal segment. x27,OOO.
Fig. 10. Results obtained on spermatozoa incubated with a nonimmune serum instead of the anti-fluorescein IgG. ES, equatorial segment; PS, postacrosomal segment. X41,OOO. In both cases, practically no gold particles (arrows) are seen over the reacted sperm.
were present on the sperm head surface, confirming previous localization of actin (Lora Lamia et al., 1986) under the plasma membrane. The presence of actin in the sperm tail was also investigated. Gold particles were detected in the neck (Fig. 5) and in the principal piece of the flagellum (Figs. 6-8) on the fibrous sheath, mainly on the circumferential ribs that connect the two longitudinal columns. The fibrous sheath is present only in the principal piece of the flagellum; it is located immediately under the plasma membrane and surrounds the outer dense fibers. We did not find F-actin on the outer dense fibers, but only on the fibrous sheath. In the tail, as in the head, the gold particles were localized only in zones where the plasma membrane had been detached (Figs. 6-81.
tween 35 kDa and 90 kDa, with the unreacted sperm showing more striking components (Fig. 11, lane c). The 45 kDa actin band (Fig. 11, arrowhead) was easy to detect only in unreacted sperm samples, although a faint band could also be seen in the ionophore-reacted sperm (Fig. 11, lane b). Furthermore, in the immunoblotting analysis, the unreacted sperm’s 45 kDa band cross-reacted with monoclonal anti-actin antibodies (Fig. 12, lane b). The analysis of A23187-treatment supernatant showed a simple pattern with three main bands; however, the putative actin band was not observable (Fig. 13, lane a?, and we did not observe any reaction in immunoblotting of A23187 supernatants.
SDS-PAGE and Immunoblotting NP-40-treated samples from reacted and unreacted sperm were studied to see whether or not actin polymerization gives rise to a detergent-resistant fraction, reflecting a change in solubility of the cytoskeletal components. When analyzed by SDS-PAGE, both the reacted and unreacted sperm samples showed similar banding patterns, while molecular weights ranging from 15 kDa to 120 kDa (Fig. 11,lanes b,c). There were differences between reacted and unreacted sperm be-
DISCUSSION The localization of F-actin by electron microscopy can be achieved either with heavy meromyosin or phalloidin-gold complexes. Phalloidin, a cyclic peptide from the mushroom Amanita phalloides, is one of the family of phallotoxins, which specifically bind to filamentous actin (Wieland et al., 1975).To our knowledge, previous studies of the ultrastructural localization of F-actin with phalloidin had been performed by Lachapelle and Aldrich (1988) with Physarum polycephalum, by Faulstich e t al. (1989) with rat cultured fibroblasts and by
F-ACTIN IN BOAR SPERMATOZOA
NP-40 I
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Fig. 11. PAGE pattern of Nonidet P-40 extracted boar spermatozoa (soluble fraction). Lane a, molecular-weight markers; lane b, reacted sperm; lane c, unreacted sperm. SDS-PAGE, under reducing condition, shows a pattern of bands ranging from 15 t o 120 kDa; arrowhead indicates the 45-kDa actin band.
us (Castellani-Ceresa et al., 1991) with boar sperm. Lachapelle and Aldrich (1988) directly complexed phalloidin to colloidal gold and suggested that these phalloidin-gold complexes are useful tools for studying the distribution of F-actin. Faulstich et al. (1989) described the preparation of four biotin derivatives of phalloidin and the use of these in combination with gold-labeled avidin. Both methods permit labeling of F-actin without using antibodies. In the present work, we used a fluorescein-phalloidin complex and a n anti-f luorescein antibody, followed by labeling with protein A-gold complex. We localized filamentous actin in both head and tail of boar spermatozoa after the acrosome reaction, and these results were supported by detergent extraction of reacted and unreacted sperm. The presence of F-actin in mammalian reacted sperm heads has been affirmed by some investigators and denied by others. Peterson et al. (1978) observed microfilaments in the equatorial and postacrosomal regions of boar spermatozoa after the acrosome reaction by electron microscopy. Schatten et al. (1983) localized microfilaments in the equatorial region of mouse and hamster sperm during capacitation and acrosome reaction by immunofluorescence. Recently, Peterson et al. (1990) demonstrated the presence of F-actin in boar sperm plasma membrane by
Fig. 12. Western blot of the soluble fraction from reacted (a) and unreacted (b) Nonidet P-40 extracted boar sperm. The immune anti actin complex was revealed by DAB-COCI,. Lane a, reacted sperm; lane b, unreacted sperm.
fluorescence microscopy using the specific probe NBDphallacidin. They also used S1 myosin subfragments attached to colloidal gold to localize F-actin ultrastructurally. Moreover, F-actin is probably a component of the cross filaments described by Escalier (1984) in human spermatozoa. Confirming the presence and localization of F-actin in the postnuclear and tail regions of human spermatozoa, Clarke et al. (1982) suggested that actin might undergo changes of state during capacitation and acrosome reaction. NBD-phallacidin was used by Saxena et al. (1986) to localize F-actin in boar spermatozoa after capacitation, and they suggested that polymerization of actin could play a significant role in the capacitation. Several other investigators have reported that actin is present in mature spermatozoa only in monomeric form (Tamblyn, 1980; Virtanen et al., 1984; Welch and O’Rand, 1985; Flaherty et al., 1986). However, most of these workers did not look for F-actin in spermatozoa after acrosome reaction, although lack of NBD-phallacidin staining in acrosome-reacted boar spermatozoa was reported by Camatini et al. (1986) and in acrosome-reacted hamster spermatozoa by Fouquet e t al. (1990). In this paper, we present immunoelectron microscopic and biochemical data useful to ascertain the possibility of a change in the state of aggregation of actin after capacitation and acrosome reaction. The results presented here show that when the acrosome-reacted boar spermatozoa are labeled by the
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I
0 Y
-97
-31
-21 a
b
Fig. 13. Protein composition of the supernatant from A23187 treated sperm analyzed by SDSPAGE. The actin putative band is absent. Lane a, A23187 supernatant polypeptides; lane b, molecularweight markers.
three step procedure, F-actin is localized in the equatorial or postacrosomal regions. Recent data (Yanagimachi, 1988; Talansky et al., 1991) have suggested that the plasma membrane over either the equatorial or the postacrosomal segments might be the site of fusion with the egg plasma membrane. The sperm plasma membrane over these regions became able to fuse with the egg plasma membrane as a result of change in distribution and composition of cell surface components. Earlier studies (Ochs e t al., 1986; Olson et al., 1987) demonstrated cytoskeletal proteins, including actin, in mammalian spermatozoa. The hypothesis that actin polymerizes before sperm penetration is supported by the data on reduced penetration of eggs by sperm preincubated with cytochalasin D (an inhibitor of the polymerization of actin) (Rogers, 1987, 1989; our unpublished data). Possibly this reduction of penetration could be related to the hypothesis that actin, together with other cytoskeletal components, may regulate the changes in sperm membrane domains that occur during the various events leading to fertilization (Virtanen et al., 1984). In fact, Saxena et al., (1986) observed that cytochalasin D prevents movements of the membrane proteins. If, as Rogers et al. (1989) suggest, cytochalasin D inhibits fertilization, it could be due to its ability to prevent actin polymerization.
The presence of actin in the sperm tail has been widely demonstrated, although there are still some discrepancies about its exact localization and state of aggregation. According to Clarke et al. (1982) it is probable that actin is in filamentous form in the connecting piece, middle piece and principal piece of the human sperm tail, while Talbot and Kleve (1978) say that there is actin in the principal piece and not in the middle piece in hamster. Baccetti et al. (1984) found actin in the fibrous sheath and the pericentriolar region of bull sperm. Flaherty et al. (1988) localized actin on the external surface of the fibrous sheath in the principal piece of several mammalian spermatozoa, but without specifying its aggregation form. In acrosome-reacted boar spermatozoa we localized F-actin in both the neck region and the fibrous sheath of the principal piece of the tail. The disagreement about the changes in the state of aggregation of actin after the acrosome reaction is probably mainly attributable to the lack of biochemical data to compare with the immunocytochemical assays. In this work, our biochemical approach was based on the differences in detergent solubility of the monomeric and polymeric forms of actin. The hypothesis of a change in the state of aggregation of actin before fertilization seems to be confirmed by the electrophoretic behaviors of the reactedhonreacted sperm samples. In unreacted sperm, the higher solubility of the monomeric form of the actin produced a more conspicuous band of about 45 kDa, which in addition was positive to anti-actin monoclonal antibodies. Since we observed some other bands indicating greater solubility by nonionic detergents, we suppose that, in reacted sperm, the exocytotic process gives rise to the differences in the SDS-PAGE patterns. The hypothesis that actin is released along with the acrosome vescicle components was verified by analysis of the acrosome reaction supernatants; the actin band could no longer be detected by SDS-PAGE or immunolocalization after Western blot. However we cannot exclude a priori that a small amount of monomeric actin is released, perhaps attributable to the physiological turnover of the molecule, during the metabolic changes before fertilization. In conclusion, this study suggests that there is polymerization of actin during capacitation and/or acrosome reaction. Additional work is necessary to further define the exact period during which this change of the state of aggregation of actin occurs.
ACKNOWLEDGMENTS This study was supported by CNR and MURST grants. REFERENCES Baccetti B, Bigliardi E, Burrini AG, Gabbiani G, Jockusch BM, Leoncini P (1984): Microfilaments and intermediate sized filaments in sperm tail. J Submicrosc Cytol16:79-84. Baccetti B, Bigliardi E, Burrini AG, Pallini V (1980):Actin filaments and mitochondria1 movement in vertebrate spermiogenesis. Gamete Res 3203-209.
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