MOLECULAR REPRODUCTION AND D E V E L O P M E N T 33:172-181(1992)
F-Actin in Guinea Pig Spermatozoa: Its Role in Calmodulin Translocation During Acrosome Reaction LETICIA MORENO-FIERROS, ENRIQUE OTHON HERNANDEZ, ZAIRA 0. SALGADO, ADELA MUJICA Departamento de Biologia Celular, Centro de Inuestigacion y de Estudios Avanzados del Znstituto Politkcnico Nacional, Mkxico D.F., M&xico AND
Key Words: F-actin in guinea pig spermatozoa, Calmodulin, Cytochalasin D, Phalloidin-rhodamine,Acrosomal reaction
Eddy, 1988; Cowan et al., 1987; Aguas and Pinto Da Silva, 1989). Some of those proteins have been characterized (Cowan et al., 1987). Our group (Trejo and MUjica, 1990) showed that during acrosome reaction in guinea pig spermatozoa, the localization of calmodulin changes. Saxena et al. (198613) communicated that migration of a protein was inhibited by cytochalasin D in boar spermatozoa, and proposed actin participation in this sperm protein translocation. Actin have been detected in mature mammalian spermatozoa by immunofluorescence technique (Clarke and Yanagimachi, 1978; Campanella et al., 1979; Clarke et al., 1982; Virtanen et al., 1984; Camatini et al., 1987; Flaherty et al., 1986, 19881, immunoelectronmicroscopy (Lora-Lamia et al., 1986; Camatini et al., 1986a,b), and biochemical methods (Ochs, 1984; Flaherty et al., 1986).There is no common actin pattern among species. Moreover, actin localization varies between laboratories studying spermatozoa of the same species (cf. Clarke and Yanagimachi, 1978; Virtanen et al., 1984; Flaherty et al., 1986). Sites where actin has been commonly found are the acrosome, the tail, the neck region, the fibrous sheath, and the postacrosomal region. In general, F-actin has not been observed in noncapacitated spermatozoa, neither from the epididymis or ejaculated, nor in those with acrosome reaction. F-actin has been detected only in a characteristic head structural component in a rodent (hidromynos) (Flaherty et al., 1983; Flaherty, 1987); in in vitro capacitated boar spermatozoa (Saxena et al., 198613); and recently in plasma membrane and along the outer acrosomal membrane of noncapacitated boar spermatozoa (Peterson et al., 1990). In guinea pig spermatozoa, using immunofluorescence, actin has been found in the postacrosomal region, in the flagellum (Clarke and Yanagimachi, 1978), and in the neck (Flaherty et al., 1986). But in this cell
INTRODUCTION In mammalian spermatozoa, migration and redistribution of external surface plasma membrane proteins occur during capacitation and acrosome reaction (Friend et al., 1977; Friend, 1982; Saxena et al., 1986a;
Received July 15,1991; accepted April 16,1992. Address reprint requests to Adela Mujica, Departamento de Biologia Celular, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, Apdo. Postal 14740, 07000 Mexico, D.F. Mexico.
The presence of actin has been deABSTRACT termined in mammalian spermatozoa. However, its function in these cells is still almost unknown. Only in boar spermatozoa has evidence for F-actin and a possible function for it been presented. In this work, actin distribution and F-actin were determined in uncapacitated, capacitated, and acrosomal-reacted guinea pig spermatozoa, by means of monoclonal and polyclonal antibodies, using an indirect immunoperoxidase technique, and by the use of rhodamine-phalloidin. With the last probe we found filamentous actin in these cells. By both techniques,actin was detected in the acrosome and in the entire tail. In some cells with acrosomal reaction, actin was also detected in the equatorial and in the postacrosomal regions. SDSPAGE and Western blots irnmunostained with monoclonal and polyclonal anti-actin antibodies confirmed the presence of actin in extracts of guinea pig spermatozoa.Actin was also detected in preparations of Percoll-purified spermatozoa. We have communicated that guinea pig spermatozoa show a change on calmodulin location during the acrosome reaction. They present it first in the equatorial region and later in the postacrosomal region. To determine if F-actin participates in this calmodulin translocation, we studied the effect of cytochalasin D. It was found that the number of cells with calmodulin in the equatorial region increased in the presence of cytochalasin D while the number of cells with calmodulin in the postacrosomal region decreased. We also found that after cytochalasin D treatment acrosome loss was increased and sperm motility was slightly inhibited. Our results suggest that actin participate in calmodulin translocation to the postacrosoma1 region during acrosome reaction, in maintaining the acrosome structure, and perhaps also in sperm motility. 0 1992 Wiley-Liss, Inc.
0 1992 WILEY-LISS, INC.
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A
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Fig. 1. Presence of actin in spermatozoa separated by one-dimensional SDS-PAGE (11% and 10% for A and B, respectively) and in immunoblot. A Lanes 1 , 2 and, 3 represent Coomassie Brilliant Bluestained proteins. Lanes 4-9 represent immunoblot. Lanes 1,5, and 8: SDS-solubilized whole guinea pig spermatozoa. Lanes 2, 4, and 7, guinea pig muscle actin. Lanes 3, 6, and 9, SDS-solubilized whole guinea pig spermatozoa and guinea pig muscle actin. Monoclonal and polyclonal antibodies were used for lanes 4-6 and lanes 7-9, respec-
tively. B Lanes 1 and 2 represent Ponceau S-stained proteins. Lanes 3 and 4 represent immunoblot, stained with the culture medium of a hybridoma producing monoclonal anti-actin antibody. Lanes 1 and 3, guinea pig muscle actin. Lanes 2 and 4, SDS-solubilized Percoll purified guinea pig spermatozoa. A single 43 KD band stains in standard actin samples, and a reactive protein of identical molecular weight is present in each spermatozoa extract, from non- or Percoll purified cells. No degradation of exogenous actin was observed.
there is still controversy with respect to actin localization, and furthermore in relation to the presence of the protein (Halenda et al., 1987). Here we show the presence of actin in guinea pig spermatozoa by Western blot, F-actin was detected by staining with rhodamine-phalloidin, and spermatozoa actin distribution by indirect immunoperoxidase. We also studied the effect of cytochalasin D on spermatozoa, to determine if F-actin participates in calmodulin translocation during acrosome reaction, and found that the drug inhibited CaM translocation to the postacrosoma1 region.
sin-D, horseradish peroxidase RZ-3, phenyl-Sepharose, protein-A-Sepharose, monoclonal anti-a-smooth muscle actin clone 1A4, Percoll, and Ponceau S from Sigma Chemical Co., St. Louis, MO; isopropanol, N,N-dimethylformamide, and cyanogen bromide (BrCN) from Merck Schuchardt, FRG; Sepharose 4B from Pharmacia, Uppsala, Sweden; EDTA from J.T. Baker de Mexico, Mexico; sodium dodecyl sulfate from BDH Chemical Ltd, England; and Amido Black 10 B from Bio-Rad, Richmond, CA.
MATERIALS AND METHODS Chemicals Chemicals were purchased from the following sources: leupeptin, phenylmethylsulfonylfluoride (PMSF), Na-p-tosyl-L-lysine chloromethyl ketone, p-hydroxymercuribenzoic acid, N-ethylmaleimide, diisopropylfluorophosphate, 2-mercaptoethanol, glutaraldehyde, aluminum chloride, 3',3'diaminobenzidine, nickel chloride, cobalt chloride, phalloidin-TRITC, cytochala-
Sperm Preparation and Incubation Spermatozoa from the vas deferens of adult guinea pigs were collected and prepared as previously described (Trejo and Mujica, 1990). Incubations were started by the addition of prewarmed (37°C) minimal culture medium (MCM-PL), as was used by Mujica and Valdes Ruiz (1983), to washed fluffy pelleted spermatozoa. The final sperm concentration was 35 x lo6 spermatozoa per ml (except when indicated) and incubated in a water bath at 37°C under air. Parallel incubations with and without the assayed compound(s) (cytochala-
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Fig. 2. Controls of the indirect immunoperoxidase technique used for actin detection. a, Positive control, NaCl washed guinea pig spermatozoa treated, as described in Methods, with a polyclonal anti-actin antibody, showing stain in the acrosome and the flagella. b, c, and d, Negative controls, MCM-incubated spermatozoa stained as in a but
with the following modifications: b, treated without the first antiactin antibody; c, treated with anti-actin antibody preadsorbed with a fivefold excess of actin; d, stained with preimmune rabbit IgG instead of the anti-actin antibody. In b,c , and d we can appreciate that there is no staining of the cells.
sin D, A23187) were run from the same washed sperm suspension, when indicated.
deferens with 0.154 M NaCl plus 10 pM cytochalasin D and the cell concentration adjusted to 5 x 106/ml and maintained during the treatment. Spermatozoa were washed in NaCl plus cytochalasin D and incubated in MCM-PL medium with cytochalasin D. Samples were stained at different times during incubation with rhodamine-phalloidin. For assaying the cytochalasin effect on calmodulin location during capacitation and acrosome reaction, citochalasin D (10 or 50 pM) was added from the start of incubation in MCM-PL sperm (5 x lo6 cells/ml). Sample aliquots of the sperm suspensions were observed under light microscopy until acrosome reaction was ob-
A23187 Sperm Treatment Washed guinea pig spermatozoa (35/106 cells/ml) were incubated for 20 min in MCM-PL, then supplemented with A23187 in DMSO. The final concentration was 1.3 pM and 0.1% for A23187 and DMSO, respectively.
Cytochalasin D Sperm Treatment When cytochalasin D was assayed to analize its effect on sperm F-actin, the cells were flushed from the vas
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Fig. 3. Immunolocalization of actin in guinea pig spermatozoa during incubation in MCM-PL. a: Spermatozoa incubated for 1 h showed the same actin localization pattern as did washed spermatozoa. b: Spermatozoa with acrosome reaction, after incubation for more than two h, showed actin localization in a belt in the equatorial region and along the flagella
served, and 70 min after samples of spermatozoa were fixed in 1.5% formaldehyde-PBS and calmodulin distribution was studied by immunoperoxidase.
Preparation of Sperm Extracts Spermatozoa washed in NaCl were collected by centrifugation. The sperm pellet (0.5-1 x 10' cells) was resuspended in 0.5 ml of 0.05 M Tris-HC1 pH 9 and 0.5 ml of inhibitors cocktail in the same buffer. The inhibitors used were 2 mM leupeptin, 6 mM phenylmethylsulfonylfluoride (dissolved in isopropanol), 2 mM N-a-p-tosyl-L-lysine chloromethyl ketone, 8 mM p-hydroxymercuribenzoic acid sodium salt, 5 mM N-ethylmaleimide, 5 mM diisopropyl fluorphosphate, and 2 mM EDTA. In order to solubilize the cells, 200 (*.110% SDS were added per 1 ml sperm suspension and left at room temperature for 10 min. After that, the samples were boiled for 10 min. Immediately, the samples were cooled on ice and sonicated twice (Branson Model B-12, microtip) at 50 mA for 30 sec each time. They were then centrifuged for 5 min at 600g. The supernatant fluid was withdrawn and kept on ice before sample buffer, without 2-mercaptoethanol (Laemmli, 1970), addition (3 vol sample11 vol sample buffer). The samples were boiled for 3 min, then 2%2-mercaptoethanol was added and boiled for another min before being electrophoresed. Electrophoreses and Western Blotting The sperm extracts were separated by SDS-PAGE (10 or 11% polyacrylamide) a s described by Laemmli (1970). The spermatozoa proteins were transferred to nitrocellulose for immunostaining (Towbin e t al., 1979). Immunostaining of blots, with two monoclonal and two polyclonal anti-actin antibodies, was performed as follows: the blots were blocked with 5%non-
fat dry milk in PBS (pH 7.4) containing 1%Triton X-100 (PBS-Triton) plus 20% preimmune serum, preabsorbed in a n actin-Sepharose column, and incubated with this solution for 2 h at 37°C. The blots were washed three times with PBS-Triton and incubated overnight at 4°C with the anti-actin antibody a t the appropriate dilution in PBS (1:50 for both polyclonal antibodies; 1:lOOO for the monoclonal anti alfa-smooth muscle actin antibody) or for 1h with non-diluted culture medium of a hybridoma producing monoclonal anti-actin antibody (donated by Dr. Manuel Hernandez), to which were added l.% Triton X-100 and 1-3% BSA and 5% preimmune serum. The blots were washed three times with PBS-Triton and incubated for 1h a t 37°C with the HRP labeled secondary antibody in PBSTriton with 3% nonfat milk and 10% preimmune serum. Blots were washed four times with PBS-Triton and twice with 50 mM phosphate buffer. HRP was developed with diaminobenzidine as described by De Blas and Cherwinski (1983).
Spermatozoa Purification on Percoll Gradient Sperm were purified on a n isotonic discontinuous gradient of Percoll, basically a s described by Lessley and Garner (1983). Vas deferens spermatozoa were recovered in 2 ml of 0.154 M NaCl solution. The sperm suspension (up to 2.5 x 10' cells) was layered over a Percoll gradient made in a 19 ml glass test tube (127 x 15 mm) by placing the following Percoll solutions: 3 ml of loo%, 2.8 ml each 75%, 65%, 55%, and 45%, then 0.5 ml each 30% and 15%. Percoll solutions were prepared in 0.154 M NaC1. The gradient was fractionated by aspiration, manually. The purified spermatozoa were obtained from 75-100% Percoll interface. Sperm extracts, electrophoreses, and Western blotting were performed as indicated above. Immunostain-
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Fig. 4. F-actin staining with rhodamine-phalloidin. Guinea pig spermatozoa from the vas deferens were formaldehyde (1.5% final concentration) fixed, permeabilized in absolute methanol, and stained with rhodamine-phalloidin (10 pgiml, see Methods). Spermatozoa: a, unwashed; b, NaC1-washed; c, MCM-PL capacitated; and d, capaci-
tated and acrosome reacted. Bright fluorescence throughout entire length of spermatozoa was observed, except in the postacrosomal region (a-d).Acrosome-reacted cells show fluorescence in the equatorial and postacrosomal regions and along the flagellum (d).
ing of blots was carried out with non-diluted culture medium of the hybridoma producing monoclonal antiactin antibody (see above).
solution and the addition of 0.1% SDS and 0.2 M KC1 (final concentrations) before the coupling. The antibody titer (1:250) was determined by indirect ELISA and by DOT-ELISA.
Actin Purification Actin was purified from guinea pig striated muscle as described by Pardee and Spudish (1982) but with two more cycles of polymerization-despolymerization. Actin purity was verified on sodium dodecyl sulfate-polyacrylamide (11%)gel electrophoresis (SDS-PAGE). Preparation of Polyclonal Anti-Actin Antibodies Purified actin was dissolved in sample buffer and then cross-linked with 0.1% glutaraldehyde and subsequently precipitated with 1% AlC1, a s described by Lazarides (1982). Rabbits were immunized with this actin preparation following the scheme used by Lazarides (1982), and the antibodies were purified by affinity chromatography in columns of a protein-A-Shepharose and Sepharose-actin. Purified actin was coupled to Br-CN activated Sepharose 4B a s described by Lazarides (1982), with modifications to avoid actin polimerization during coupling. The modifications consisted of two cycles of freezing and thawing the actin
Calmodulin and Anti-Calmodulin Antibody Purification Calmodulin was obtained from bovine testicular tissue a s described by Dedman and Kaetzel (1983) and purified by affinity chromatography on phenylSepharose as described by Gopalakrishna and Anderson (1982). Polyclonal antibodies against calmodulin were prepared in sheep following a common immunization program. They were purified by affinity chromatography on calmodulin-Sepharose column as described by Dedman et al. (1978). Antibody titer (150) was determined by ELISA (Voller and de Savigny, 1981). Preparation of Peroxidase Conjugates Rabbit IgG anti-mouse IgG and goat IgG anti-rabbit IgG: mouse and rabbit IgG were obtained by affinitychromatography on protein A-Sepharose, antibodies were rinsed in rabbit or goat, respectively, and purified
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room temperature and given a PBS rinse, the smears were blocked for 1h a t 37°C with 25 pl (viv) 3%bovine serum albumin in PBS-1% preimmune goat serum preabsorbed in a n actin-Sepharose column (when mouse anti-actin monoclonal antibody was used, only BSA was utilized to block). PBS washed smears were treated with 25 p1 microliters of the primary antibody (rabbit or mouse IgG anti-actin), mounted under a cover-glass slide, and incubated for 18 h a t 4°C in a humid atmosphere. Exhaustively PBS washed cells were incubated for 2 h at 37"C, in a humid atmosphere, with 25 pl of goat anti-rabbit or rabbit anti-mouse globulin tagged with peroxidase. Slides were washed thrice with PBS and stained for peroxidase following the procedure described by Johnson and Dorling (1981). Glycerol-PBS, 1 : l pH 8, and nail polish were used to mount and seal the preparations. Cells were viewed through a DOCUVAL Carl Zeiss microscope. Images were recorded on Kodak Tri-X film, 400 ASA.
Fig. 5. Fluorescence of guinea pig spermatozoa, treated with A23187 (37 pmolesll x lo6 spermatozoa) and stained with rhodamine-phalloidin. a and b: Acrosome reacted sperm cells (after 3 and 4 min of A23187 treatment, respectively) showed fluorescence from the equatorial region (as a bright belt) to the tip of the flagellum. c: Cells after 7 min of A23187 treatment; fluorescence increased in the postacrosomal region with a less defined equatorial belt. Fluorescence is also present along the flagellum.
by chromatography on Sepharose-(mouse or rabbit) IgG. Rabbit and goat IgG anti-IgG were conjugated to peroxidase (HRP) as described by Avrameas and Ternyck (1971). Conjugate titers were (1:3,000) for anti-mouse IgG and (1:2,000) for anti-rabbit IgG.
Location of Calmodulin For calmodulin localization, indirect immunoperoxidase technique was done as described by Trejo and MUjica (1990) for indirect immunofluorescence. Peroxidase staining was carried out following the procedure described by Johnson and Dorling (1981) with diaminobenzidine as substrate. Immunocytochemical Localization of Actin Sperm suspension aliquots were fixed in 3% formaldehyde-PBS (1.5% formaldehyde, final concentration) for 60 rnin a t room temperature. Pelleted spermatozoa (600g, 4 min) were washed thrice by dilution in 15 vol PBS and centrifugation, then the cells were incubated in 0.05 M NH,C1 for 10 min and rinsed twice with PBS. The spermatozoa were permeabilized in absolute acetone at -20°C for 5 min. PBS wash was used to eliminate the solvent. Endogenous peroxidase was eliminated by 0.074% HC1-absolute ethanol treatment (Weir et al., 1974, see Johnson and Dorling, 1981) for 10 min. After two PBS washes, a final distilled water wash was done. Spermatozoa resuspended in distilled water were used to prepare the smears. After being air-dried a t
Rhodamine-Phalloidin Staining Sample spermatozoa were fixed in 0.75-1.5% formaldehyde final concentration in PBS for one h at room temperature. Pelleted cells (600g, 4 min) were resuspended in 50 mM NH,C1 for 10 min, followed by three washings by resuspension and centrifugation in PBS as above and one wash in distilled water, and finally resuspended in water. This suspension was spread on microscope slides and air dried, permeabilized in absolute methanol for 5 min at -2O"C, stained with rhodaminephalloidin (30 plislide, at a concentration of 10 Fgiml), and mounted under a cover-glass slide for 20 min in a humid atmosphere. The slides were rinsed in PBS thrice and mounted in a mixture of glycero1:PBS (1:l). Cells were observed in a Zeiss photomicroscope equipped with epifluorescence optics. Photographs were taken with Kodak Tri-X pan film (ASA 400), on exposure for 45 sec. RESULTS Actin Detection in Guinea Pig Sperm Extracts by SDS-PAGE and Western Blot Analysis The protein pattern of whole spermatozoa solubilized with SDS plus six protease inhibitors revealed the presence of a polypeptide with a n apparent molecular weight of 43 KD (Fig. l A , lane 1)that comigrated with reference actin (Fig. lA, lane 2). Western blot of spermatozoa proteins followed by overlay with a monoclonal or a polyclonal monospecific anti-actin antibody indicated a positive signal on the corresponding 43 KD band (Fig. l A , lanes 5, 8). No degradation of actin was observed when a measured amount of rabbit skeletal muscle actin was added to a sperm preparation, processed together, subjected to electrophoresis (Fig. 1A, lane 3), and revealed on Western blot (Fig. lA, lanes 6, 9). Reference actin is shown (Fig. lA, lanes 4,7). The protein pattern of whole purified (on Percoll gradient) spermatozoa with only 0.2% contaminating cells revealed the presence of a polipeptide with a n apparent
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Fig. 6. F-Actin detection by rhodamine-phalloidin in guinea pig spermatozoa treated with 10 (*.Mcytochalasin D. Cytochalasin D was added from the start of the cell extraction and the cell concentration was adjusted and maintained a t 5 x lo6 spermatozoa per ml. d and e,
Controls incubated in the absence of cytochalasin D for 2 and 5 h, respectively. a, b, and c, Spermatozoa incubated with cytochalasin D for 2 h, 4 h, and 5 h, respectively. In b and c a loss of fluorescence is evident while in a, as in the controls, there is intense fluorescence.
molecular weight of 43 KD (Fig. l B , lane 4) that comigrated with reference actin (Fig. lB, line 3), revealed with a monoclonal anti-actin antibody.
reacted spermatozoa, no actin stain was observed on the nucleus region where the acrosome was, but a n actin belt was apparent in the equatorial region (Fig. 3b).
Localization of Actin in Guinea Pig Spermatozoa Detection of actin by immunoperoxidase technique, using one monoclonal (Sigma) and two polyclonal antiactin antibodies, showed reaction with the whole acrosome (clearly observable in color pictures, apparently surrounding only the acrosome in the black and white pictures) and the flagella of NaC1-washed, formaldehyde-fixed guinea pig spermatozoa (Fig. 2a). Specificity of the immunoperoxidase technique was tested by performing three controls: (1)incubation with the second antibody without the primary (anti-actin) antibody (Fig. 2b); (2) preadsorbing the purified anti-actin antibody with a fivefold excess of homogeneous skeletal muscle actin (Fig. 2c); and (3)use of preimmune rabbit IgG as the primary antibody (Fig. 2d). There was no staining of the cells in any of the controls assayed. Spermatozoa incubated a t least one h in MCM-PL showed actin stain similar (Fig. 3a) to NaC1-washed cells (Fig. 2a) in the whole acrosome and flagella. In acrosome
Evidence of F-Actin Presence Rhodamine-labelled phalloidin was used to localize F-actin in guinea pig spermatozoa: in spermatozoa from the vas deferens fixed immediately in epididymal plasma; in the cells washed with NaC1; in MCM-PL capacitated spermatozoa with and without acrosome reaction; and also in noncapacitated acrosome-reacted spermatozoa by induction with A23187. Independent of being washed or not, spermatozoa from the vas deferens showed bright fluorescence (rhodaminel across their entire length, except in the postacrosomal region (Fig. 4a,b) as did the MCM-PL incubated spermatozoa (Fig. 4c,d). The fluorescence was associated with the flagellum, acrosome, and the equatorial region (Fig. 4c,d). Acrosome-reacted spermatozoa showed fluorescence in the equatorial and postacrosomal regions and throughout the flagellum (Fig. 4d). In spermatozoa with acrosome reaction, elicited by the ionophore A23187, a bright fluorescence in the equatorial region was ob-
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Effect of Cytochalasin D on Sperm F-Actin Ten micromolar cytochalasin D diminished the fluorescence produced by rhodamine-phalloidin, although cytochalasin D effect began to be apparent after 3 h of incubation (Fig. 6b,c). From the start of incubation, slightly weaker motility and increased acrosome loss (70% of the cells were without acrosome) was observed in cytochalasin-treated spermatozoa, compared with their controls.
Effect of Cytochalasin D, During Capacitation and Acrosome Reaction, on Calmodulin Location in Spermatozoa Without Acrosome Seventy minutes after the acrosome reaction started, it was found that cytochalasin D increased, by 26-35%, the loss of acrosome over that shown by control spermatozoa incubated without cytochalasin but with 1-5% dimethylformamide, used as a solvent for cytochalasin D. After cytochalasin D treatment, a higher percentage, with respect to the control, of spermatozoa without acrosome showed calmodulin in the equatorial region (Fig. 7a versus b) in a dose dependent manner (Fig. 8). Also during cytochalasin D-treatment, the percentage of acrosome-reacted spermatozoa stained in the postacrosomal region decreased (Fig. 7a, 8) in a dose dependent manner. The flagellum showed calmodulin stain in cells with and without cytochalasin D treatment (Fig. 7a,b). DISCUSSION
Fig. 7. Effect of cytochalasin D on calmodulin location in spermatozoa with acrosome reaction. Spermatozoa (5 x lo6 cellsiml) were treated with 50 KM cytochalasin D during incubation in MCM-PL, fixed 70 min after acrosome reaction started, and calmoldulin revealed by indirect immunoperoxidase. a: Cytochalasin D-treated spermatozoa. Calmodulin is localized as a belt in the equatorial region and along the flagellum. b: Control spermatozoa without cytochalasin D. Calmodulin is present in the postacrosomal region and along the flagellum.
served after a period of 3 min (Fig. 5a), and a t 4 and 7 min after A23187 treatment, a bright fluorescence in the postacrosomal region was observed (Fig. 5b,c). Control spermatozoa samples labelled with 300-fold phalloidin followed by rhodamine-phalloidin showed greatly diminished fluorescence; no spermatozoa fluorescence was observed with 500-fold phalloidin pretreatment (data not shown).
The presence of actin in guinea pig spermatozoa (from non- or Percoll purified samples) was confirmed by Western blot technique. A band of 43 KD was recognized using two monoclonal and two policlonal antiactin antibodies. By addition of six protease inhibitors proteolysis was stringently controlled. This condition differed from that used by Halenda et al. (19871, who did not use any protease inhibitors and did not detect actin in guinea pig spermatozoa from the epididymal cauda. Our results are in agreement with those of Flaherty et al. (19861, who using three protease inhibitors detected actin in each of eight species of spermatozoa, including those of guinea pig. In some guinea pig sperm extracts revealed by immunoblot with monoclonal anti-actin antibodies, besides the actin band (43 KD), a second light band (50KD) was detected (data not shown), similar to that described for rat and mouse sperm (Flaherty e t al., 1986). By indirect immunoperoxidase technique, actin was localized in the guinea pig spermatozoa in the whole acrosome, along the flagellum and in the equatorial region of acrosome reacted spermatozoa. Actin distribution pattern was maintained from NaC1-washed cells through capacitation in MCM-PL and in the acrosomereacted cells, but in the last cells no actin was observed in the region previously occupied by the acrosome (Fig. 3b). In guinea pig spermatozoa, Clarke and Yanagimachi (1978) detected actin only in the postacrosomal region, and Flaherty et al. (1986) in the neck, midpiece,
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Immunoperoxidase Patterns (%)
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Fig. 8. Calmodulin localization during acrosomal reaction in the presence of Cytochalasin D. Spermatozoa (5 x lo6 cellsiml) were incubated in MCM-PL with cytochalasin D, a t the concentrations indicated. Samples were fixed 70 min after acrosome reaction started and sperm calmodulin distribution was studied with indirect immunoperoxidase. Five hundred cells were assessed. The percentage of each calmodulin distribution pattern is indicated.
and principal piece. The presence of actin in the flagellum had been difficult to evaluate by immunofluorescence because of sperm autofluorescence (Flaherty et al., 1986).This problem was abolished by the immunoperoxidase technique. To our knowledge, we describe for the first time the presence of filamentous actin in guinea pig spermatozoa (Figs. 4,5) by using phalloidin-rhodamine which is a specific probe for actin filaments. Only in boar capacitated (Saxena et al., 1986a) and uncapacitated (Peterson et al., 1990) spermatozoa and in the sperm head of the plains mouse (Flaherty, 1987) has F-actin been shown. We found filamentous actin in the whole cell, except in the postacrosomal region, in both capacitated and uncapacitated cells with acrosome (Fig. 4). In acrosome-reacted spermatozoa, but not in cells with acrosome, F-actin was apparent in the postacrosomal region (Fig. 4d), suggesting actin polymerization in this region during acrosome reaction. Flaherty et al. (1986), using NDB-phallacidin, detected F-actin in the neck of guinea pig spermatozoa and a weak label in the flagellum indicating that it was unspecific. Under the experimental conditions for rhodamine observation, sperm autofluorescence was not seen. F-actin in the postacrosomal region was not detected by immunoperoxidase technique, but was positive by immunofluorescence, a higher sensitivity technique. Unsuccessful results (Halenda et al., 1987) on F-actin detection in guinea pig spermatozoa and negative results in F-actin observation in other sperm species could be due to techni-
cal problems such as non-permeabilization (Virtanen et al., 1984; Welch and O’Rand, 19851, excessive fixation (Halenda et al., 1987) (when we used these conditions, we observed diminished fluorescence in guinea pig spermatozoa [data not shown]), or sperm autofluorescence that is observed under conditions needed for microscopic observation of NBD-phallacidin. Cytochalasin gradually decreased the intensity of fluorescence detected with rhodamine-phalloidin after 3 h of incubation in MCM-PL, which suggests that cytochalasin depolymerize F-actin in guinea pig spermatozoa, a n observation which is in agreement with Saxena’s e t al. (1986b) results. During the acrosome reaction of guinea pig spermatozoa, calmodulin is apparently translocated from the equatorial region to the postacrosomal region. Our results showed t h a t cytochalasin D produced a n arrest of the calmodulin in the equatorial region, since we found a n increase of the number of cells with calmodulin located in this zone, and a decrease of the number of cells showing calmodulin in the postacrosomal region. This suggests that, for calmodulin translocation to the postacrosomal zone to take place, a n intact actin cytoskeleton is needed. This study suggests that F-actin is involved in calmodulin (an intracellular protein with known physiological properties) migration to the postacrosomal region, a zone involved in egg recognition, in a Ca2+dependent process. Our result is in agreement with Saxena’s et al. (1986a) report, since in both instances
F-ACTIN IN GUINEA PIG SPERMATOZOA the translocation of a protein was blocked by the microfilament toxin cytochalasin D. The presence of F-actin in the acrosome and in the flagellum and the changes elicited on spermatozoa by cytochalasin D treatment suggest actin participation in stabilization of the acrosome structure, as well a s a possible actin function in the spermatozoa motility, although we do not know if actin is directly involved in flagella beating, a s was suggested for Chlamydomonas (Baccetti et al., 1989). Other actin functions could be the maintenance of tail shape as proposed previously (Baccetti e t al., 1989) and protein translocation. In summary, like spermatozoa of many species, guinea pig spermatozoa contain actin. The presence of F-actin in spermatozoa allows a reconsideration on its physiological role in acrosome reaction, in movement, in the maintenance of tail shape (Baccetti et al., 19891, and in protein translocation.
ACKNOWLEDGMENTS We thank Dr. A. Huberman, Dr. S. Villa-Trevino, M. en C. 0. Velasco-Castaneda, and M.A. Torres-Vega for helpful comments on the manuscript. This research was partly supported by Consejo Nacional de Ciencia y Tecnologia, grant P228CCOX891518.
REFERENCES Aguas AP, Pinto Da Silva P (1989): Bimodal redistribution of surface transmembrane glycoproteins during Ca2 -dependent secretion (acrosome reaction) in boar spermatozoa. J Cell Sci 93:467-479. Avrameas S, Ternyck T (1971): Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration. Immunochemistry 8:1175-1179. Baccetti B, Burrini AG, Collodel G, Magnano AR, Piomboni P, Sensini C, Renieri T (1989): Human sperm cytoskeleton: Function and pathology. In M Serio (ed): “Perspectives in Andrology,” Vol 53. New York: Raven Press, pp 271-280. Camatini M, Anelli G, Casale A (1986a):Identification of actin in boar spermatids and spermatozoa by immunoelectron microscopy. Eur J Cell Biol42:311-318. Camatini M, Casale A, Anelli G, Cifarelli M (1986b): Immunoelectronmicroscopic localization of actin in ionophore treated boar sperm. Cell Biol Int Rep 10:231-238. Camatini M, Casale A, Cifarelli M (1987):Immunocytochemical identification of actin in rabbit spermiogenesis and spermatozoa. Eur J Cell Biol 45:27&281. Campanella C, Gabbiani G, Baccetti B, Burrini AG, Pallini V (1979): Actin and myosin in the vertebrate acrosomal region. J Submicrosc Cytol 11:53-71. Clarke GN, Clarke FM, Wilson S (1982):Actin in human spermatozoa. Biol Reprod 26:319-327. Clarke GN, Yanagimachi R (1978):Actin in mammalian sperm heads. J Exp Zool 205:125-132. Cowan AE, Myles DG, Koppel DE (1987): Lateral diffusion of the PH20 protein on guinea pig sperm: Evidence that barriers to diffusion maintain plasma membrane domains in mammalian sperm. J Cell Biol. 104:917-923. De Blas AL, Cherwinski HM (1983):Detection of antigens on nitrocellulose paper immunoblots with monoclonal antibodies. Anal Biochem 133:214-219. Dedman JR, Kaetzel MA (1983):Calmodulin purification and fluorescent labelling. In AR Means and BW O’Malley (eds): “Methods in Enzymology,” Vol 102. New York: Academic Press, pp 1-12. Dedman JR, Welsh MJ, Means AR (1978):Ca2’-dependent regulator: Production and characterization of monospecific antibody. J Biol Chem 253:7515-7521.
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Eddy EM (1988):The spermatozoon. In E Knobil (ed):“The Physiology of Reproduction,” Vol 1. New York: Raven Press, pp 27-67. Flaherty SP (1987): Further ultrastructural observations on the sperm head of the plains mouse, Pseudomys australis (Rodentia: muridae). Anat Rec 217240-249. Flaherty SP, Breed WG, Serafis V (1983): Localization of actin in the sperm head of the plains mouse Pseudomys australis. J Exp Zool 225:497-500. Flaherty SP, Winfrey VP, Olson GE (1986): Localization of actin in mammalian spermatozoa: A comparison of eight species. Anat Rec 216:504-515. Flaherty SP, Winfrey VP, Olson GE (1988): Localization of actin in human, bull, rabbit and hamster sperm by immunoelectron microscopy. Anat Rec 221:599-610. Friend DS (1982): Plasma-membrane diversity in a highly polarized cell. J Cell Biol 93:243-249. Friend DS, Orci L, Perrelet A, Yanagimachi R (1977): Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa. J Cell Biol 74561-577. Gopalakrishna R, Anderson WB (1982): Ca2 -induced hydrophobic site on calmodulin: Application for purification of calmodulin by phenyl-sepharose affinity chromatography. Biochem Bioph Res Commun 1042330-836. Halenda RM, Primakoff P, Myles DG (1987): Actin filaments, localized to the region of the developing acrosome during early stages, are lost during later stages of guinea pig spermiogenesis. Biol Reprod 36:491499. Johnson GD, Dorling J (1981): Immunofluorescence and immunoperoxidase techniques. In RA Thomson (ed): “Techniques in Clinical Immunology.” London: Blackwell Scientific Publications, pp 106137. Laemmli UK (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. Lazarides E (1982): Antibody production and immunofluorescent characterization of actin and contractile proteins. In L Wilson (ed): “Methods in Cell Biology,” Vol 24, Part A. New York: Academic Press, pp 313-371. Lessley BA, Garner DL (1983): Isolation of motile spermatozoa by density gradient centrifugation in Percoll. Gamete Res 7:4941. Lora-Lamia C, Castellani-Ceresa L, Andreetta F, Cotelli F, Brivio M (1986): Localization and distribution of actin in mammalian sperm heads. J Ultrastruc Mol Struct Res 96:12-21. Mujica A, Valdes Ruiz M (1983):On the role of glucose incapacitation and acrosomal reaction of guinea pig sperm. Gamete Res 8:335-344. Ochs D (1984):Actin in post-ejaculatory human sperm. Biol Reprod 30 (suppl 1):78. Pardee JD, Spudish J A (1982): Purification of muscle actin. In L Wilson (ed): “Methods in Cell Biology. The Cytoskeleton Part A,” Vol 24. New York: Academic Press Inc, pp 271-289. Peterson RN, Bozzola JJ, Hunt WP, Darabi A (1990):Characterization of membrane-associated actin in boar spermatozoa. J Exp Zool 253:202-214. Saxena N, Petersen RN, Sharif S, Saxena NK, Russell LD (1986a): Changes in the organization of surface antigens during in vitro capacitation of boar spermatozoa as detected by monoclonal antibodies. J Reprod Fertil78:601-614. Saxena N, Peterson RN, Saxena NK, Russell LD (1986b): Microfilaments appear in boar spermatozoa during capacitation in vitro. J Exp Zool 293:423-427. Towbin H, Staenhelin T, Gordon J (1979): Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76:43504354. Trejo R, Mujica A (1990): Changes in calmodulin compartmentalization throughout capacitation and acrosome reaction in guinea pig spermatozoa. Mol Reprod Devel26:366376. Virtanen I, Badley RA, Paasivuo R, Lehto VP (1984): Distinct cytoskeletal domains revealed in sperm cells. J Cell Biol99:1083-1091. Voller A, de Savigny D (1981): Enzyme linked immunosorbent assay (ELISA). In RA Thompson (ed): “Techniques in Clinical Immunology.’’ Blackwell Scientific Publications, pp 157-169. Welch JE, O’Rand MG (1985): Identification and distribution of actin in spermatogenic cells and spermatozoa of the rabbit. Dev Biol 109:411417.
F-Actin in Guinea Pig Spermatozoa: Its Role in Calmodulin Translocation During Acrosome Reaction LETICIA MORENO-FIERROS, ENRIQUE OTHON HERNANDEZ, ZAIRA 0. SALGADO, ADELA MUJICA Departamento de Biologia Celular, Centro de Inuestigacion y de Estudios Avanzados del Znstituto Politkcnico Nacional, Mkxico D.F., M&xico AND
Key Words: F-actin in guinea pig spermatozoa, Calmodulin, Cytochalasin D, Phalloidin-rhodamine,Acrosomal reaction
Eddy, 1988; Cowan et al., 1987; Aguas and Pinto Da Silva, 1989). Some of those proteins have been characterized (Cowan et al., 1987). Our group (Trejo and MUjica, 1990) showed that during acrosome reaction in guinea pig spermatozoa, the localization of calmodulin changes. Saxena et al. (198613) communicated that migration of a protein was inhibited by cytochalasin D in boar spermatozoa, and proposed actin participation in this sperm protein translocation. Actin have been detected in mature mammalian spermatozoa by immunofluorescence technique (Clarke and Yanagimachi, 1978; Campanella et al., 1979; Clarke et al., 1982; Virtanen et al., 1984; Camatini et al., 1987; Flaherty et al., 1986, 19881, immunoelectronmicroscopy (Lora-Lamia et al., 1986; Camatini et al., 1986a,b), and biochemical methods (Ochs, 1984; Flaherty et al., 1986).There is no common actin pattern among species. Moreover, actin localization varies between laboratories studying spermatozoa of the same species (cf. Clarke and Yanagimachi, 1978; Virtanen et al., 1984; Flaherty et al., 1986). Sites where actin has been commonly found are the acrosome, the tail, the neck region, the fibrous sheath, and the postacrosomal region. In general, F-actin has not been observed in noncapacitated spermatozoa, neither from the epididymis or ejaculated, nor in those with acrosome reaction. F-actin has been detected only in a characteristic head structural component in a rodent (hidromynos) (Flaherty et al., 1983; Flaherty, 1987); in in vitro capacitated boar spermatozoa (Saxena et al., 198613); and recently in plasma membrane and along the outer acrosomal membrane of noncapacitated boar spermatozoa (Peterson et al., 1990). In guinea pig spermatozoa, using immunofluorescence, actin has been found in the postacrosomal region, in the flagellum (Clarke and Yanagimachi, 1978), and in the neck (Flaherty et al., 1986). But in this cell
INTRODUCTION In mammalian spermatozoa, migration and redistribution of external surface plasma membrane proteins occur during capacitation and acrosome reaction (Friend et al., 1977; Friend, 1982; Saxena et al., 1986a;
Received July 15,1991; accepted April 16,1992. Address reprint requests to Adela Mujica, Departamento de Biologia Celular, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, Apdo. Postal 14740, 07000 Mexico, D.F. Mexico.
The presence of actin has been deABSTRACT termined in mammalian spermatozoa. However, its function in these cells is still almost unknown. Only in boar spermatozoa has evidence for F-actin and a possible function for it been presented. In this work, actin distribution and F-actin were determined in uncapacitated, capacitated, and acrosomal-reacted guinea pig spermatozoa, by means of monoclonal and polyclonal antibodies, using an indirect immunoperoxidase technique, and by the use of rhodamine-phalloidin. With the last probe we found filamentous actin in these cells. By both techniques,actin was detected in the acrosome and in the entire tail. In some cells with acrosomal reaction, actin was also detected in the equatorial and in the postacrosomal regions. SDSPAGE and Western blots irnmunostained with monoclonal and polyclonal anti-actin antibodies confirmed the presence of actin in extracts of guinea pig spermatozoa.Actin was also detected in preparations of Percoll-purified spermatozoa. We have communicated that guinea pig spermatozoa show a change on calmodulin location during the acrosome reaction. They present it first in the equatorial region and later in the postacrosomal region. To determine if F-actin participates in this calmodulin translocation, we studied the effect of cytochalasin D. It was found that the number of cells with calmodulin in the equatorial region increased in the presence of cytochalasin D while the number of cells with calmodulin in the postacrosomal region decreased. We also found that after cytochalasin D treatment acrosome loss was increased and sperm motility was slightly inhibited. Our results suggest that actin participate in calmodulin translocation to the postacrosoma1 region during acrosome reaction, in maintaining the acrosome structure, and perhaps also in sperm motility. 0 1992 Wiley-Liss, Inc.
0 1992 WILEY-LISS, INC.
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A
1 2 3 4 5
6
7
8 9
1 2 3 4
Fig. 1. Presence of actin in spermatozoa separated by one-dimensional SDS-PAGE (11% and 10% for A and B, respectively) and in immunoblot. A Lanes 1 , 2 and, 3 represent Coomassie Brilliant Bluestained proteins. Lanes 4-9 represent immunoblot. Lanes 1,5, and 8: SDS-solubilized whole guinea pig spermatozoa. Lanes 2, 4, and 7, guinea pig muscle actin. Lanes 3, 6, and 9, SDS-solubilized whole guinea pig spermatozoa and guinea pig muscle actin. Monoclonal and polyclonal antibodies were used for lanes 4-6 and lanes 7-9, respec-
tively. B Lanes 1 and 2 represent Ponceau S-stained proteins. Lanes 3 and 4 represent immunoblot, stained with the culture medium of a hybridoma producing monoclonal anti-actin antibody. Lanes 1 and 3, guinea pig muscle actin. Lanes 2 and 4, SDS-solubilized Percoll purified guinea pig spermatozoa. A single 43 KD band stains in standard actin samples, and a reactive protein of identical molecular weight is present in each spermatozoa extract, from non- or Percoll purified cells. No degradation of exogenous actin was observed.
there is still controversy with respect to actin localization, and furthermore in relation to the presence of the protein (Halenda et al., 1987). Here we show the presence of actin in guinea pig spermatozoa by Western blot, F-actin was detected by staining with rhodamine-phalloidin, and spermatozoa actin distribution by indirect immunoperoxidase. We also studied the effect of cytochalasin D on spermatozoa, to determine if F-actin participates in calmodulin translocation during acrosome reaction, and found that the drug inhibited CaM translocation to the postacrosoma1 region.
sin-D, horseradish peroxidase RZ-3, phenyl-Sepharose, protein-A-Sepharose, monoclonal anti-a-smooth muscle actin clone 1A4, Percoll, and Ponceau S from Sigma Chemical Co., St. Louis, MO; isopropanol, N,N-dimethylformamide, and cyanogen bromide (BrCN) from Merck Schuchardt, FRG; Sepharose 4B from Pharmacia, Uppsala, Sweden; EDTA from J.T. Baker de Mexico, Mexico; sodium dodecyl sulfate from BDH Chemical Ltd, England; and Amido Black 10 B from Bio-Rad, Richmond, CA.
MATERIALS AND METHODS Chemicals Chemicals were purchased from the following sources: leupeptin, phenylmethylsulfonylfluoride (PMSF), Na-p-tosyl-L-lysine chloromethyl ketone, p-hydroxymercuribenzoic acid, N-ethylmaleimide, diisopropylfluorophosphate, 2-mercaptoethanol, glutaraldehyde, aluminum chloride, 3',3'diaminobenzidine, nickel chloride, cobalt chloride, phalloidin-TRITC, cytochala-
Sperm Preparation and Incubation Spermatozoa from the vas deferens of adult guinea pigs were collected and prepared as previously described (Trejo and Mujica, 1990). Incubations were started by the addition of prewarmed (37°C) minimal culture medium (MCM-PL), as was used by Mujica and Valdes Ruiz (1983), to washed fluffy pelleted spermatozoa. The final sperm concentration was 35 x lo6 spermatozoa per ml (except when indicated) and incubated in a water bath at 37°C under air. Parallel incubations with and without the assayed compound(s) (cytochala-
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Fig. 2. Controls of the indirect immunoperoxidase technique used for actin detection. a, Positive control, NaCl washed guinea pig spermatozoa treated, as described in Methods, with a polyclonal anti-actin antibody, showing stain in the acrosome and the flagella. b, c, and d, Negative controls, MCM-incubated spermatozoa stained as in a but
with the following modifications: b, treated without the first antiactin antibody; c, treated with anti-actin antibody preadsorbed with a fivefold excess of actin; d, stained with preimmune rabbit IgG instead of the anti-actin antibody. In b,c , and d we can appreciate that there is no staining of the cells.
sin D, A23187) were run from the same washed sperm suspension, when indicated.
deferens with 0.154 M NaCl plus 10 pM cytochalasin D and the cell concentration adjusted to 5 x 106/ml and maintained during the treatment. Spermatozoa were washed in NaCl plus cytochalasin D and incubated in MCM-PL medium with cytochalasin D. Samples were stained at different times during incubation with rhodamine-phalloidin. For assaying the cytochalasin effect on calmodulin location during capacitation and acrosome reaction, citochalasin D (10 or 50 pM) was added from the start of incubation in MCM-PL sperm (5 x lo6 cells/ml). Sample aliquots of the sperm suspensions were observed under light microscopy until acrosome reaction was ob-
A23187 Sperm Treatment Washed guinea pig spermatozoa (35/106 cells/ml) were incubated for 20 min in MCM-PL, then supplemented with A23187 in DMSO. The final concentration was 1.3 pM and 0.1% for A23187 and DMSO, respectively.
Cytochalasin D Sperm Treatment When cytochalasin D was assayed to analize its effect on sperm F-actin, the cells were flushed from the vas
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Fig. 3. Immunolocalization of actin in guinea pig spermatozoa during incubation in MCM-PL. a: Spermatozoa incubated for 1 h showed the same actin localization pattern as did washed spermatozoa. b: Spermatozoa with acrosome reaction, after incubation for more than two h, showed actin localization in a belt in the equatorial region and along the flagella
served, and 70 min after samples of spermatozoa were fixed in 1.5% formaldehyde-PBS and calmodulin distribution was studied by immunoperoxidase.
Preparation of Sperm Extracts Spermatozoa washed in NaCl were collected by centrifugation. The sperm pellet (0.5-1 x 10' cells) was resuspended in 0.5 ml of 0.05 M Tris-HC1 pH 9 and 0.5 ml of inhibitors cocktail in the same buffer. The inhibitors used were 2 mM leupeptin, 6 mM phenylmethylsulfonylfluoride (dissolved in isopropanol), 2 mM N-a-p-tosyl-L-lysine chloromethyl ketone, 8 mM p-hydroxymercuribenzoic acid sodium salt, 5 mM N-ethylmaleimide, 5 mM diisopropyl fluorphosphate, and 2 mM EDTA. In order to solubilize the cells, 200 (*.110% SDS were added per 1 ml sperm suspension and left at room temperature for 10 min. After that, the samples were boiled for 10 min. Immediately, the samples were cooled on ice and sonicated twice (Branson Model B-12, microtip) at 50 mA for 30 sec each time. They were then centrifuged for 5 min at 600g. The supernatant fluid was withdrawn and kept on ice before sample buffer, without 2-mercaptoethanol (Laemmli, 1970), addition (3 vol sample11 vol sample buffer). The samples were boiled for 3 min, then 2%2-mercaptoethanol was added and boiled for another min before being electrophoresed. Electrophoreses and Western Blotting The sperm extracts were separated by SDS-PAGE (10 or 11% polyacrylamide) a s described by Laemmli (1970). The spermatozoa proteins were transferred to nitrocellulose for immunostaining (Towbin e t al., 1979). Immunostaining of blots, with two monoclonal and two polyclonal anti-actin antibodies, was performed as follows: the blots were blocked with 5%non-
fat dry milk in PBS (pH 7.4) containing 1%Triton X-100 (PBS-Triton) plus 20% preimmune serum, preabsorbed in a n actin-Sepharose column, and incubated with this solution for 2 h at 37°C. The blots were washed three times with PBS-Triton and incubated overnight at 4°C with the anti-actin antibody a t the appropriate dilution in PBS (1:50 for both polyclonal antibodies; 1:lOOO for the monoclonal anti alfa-smooth muscle actin antibody) or for 1h with non-diluted culture medium of a hybridoma producing monoclonal anti-actin antibody (donated by Dr. Manuel Hernandez), to which were added l.% Triton X-100 and 1-3% BSA and 5% preimmune serum. The blots were washed three times with PBS-Triton and incubated for 1h a t 37°C with the HRP labeled secondary antibody in PBSTriton with 3% nonfat milk and 10% preimmune serum. Blots were washed four times with PBS-Triton and twice with 50 mM phosphate buffer. HRP was developed with diaminobenzidine as described by De Blas and Cherwinski (1983).
Spermatozoa Purification on Percoll Gradient Sperm were purified on a n isotonic discontinuous gradient of Percoll, basically a s described by Lessley and Garner (1983). Vas deferens spermatozoa were recovered in 2 ml of 0.154 M NaCl solution. The sperm suspension (up to 2.5 x 10' cells) was layered over a Percoll gradient made in a 19 ml glass test tube (127 x 15 mm) by placing the following Percoll solutions: 3 ml of loo%, 2.8 ml each 75%, 65%, 55%, and 45%, then 0.5 ml each 30% and 15%. Percoll solutions were prepared in 0.154 M NaC1. The gradient was fractionated by aspiration, manually. The purified spermatozoa were obtained from 75-100% Percoll interface. Sperm extracts, electrophoreses, and Western blotting were performed as indicated above. Immunostain-
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Fig. 4. F-actin staining with rhodamine-phalloidin. Guinea pig spermatozoa from the vas deferens were formaldehyde (1.5% final concentration) fixed, permeabilized in absolute methanol, and stained with rhodamine-phalloidin (10 pgiml, see Methods). Spermatozoa: a, unwashed; b, NaC1-washed; c, MCM-PL capacitated; and d, capaci-
tated and acrosome reacted. Bright fluorescence throughout entire length of spermatozoa was observed, except in the postacrosomal region (a-d).Acrosome-reacted cells show fluorescence in the equatorial and postacrosomal regions and along the flagellum (d).
ing of blots was carried out with non-diluted culture medium of the hybridoma producing monoclonal antiactin antibody (see above).
solution and the addition of 0.1% SDS and 0.2 M KC1 (final concentrations) before the coupling. The antibody titer (1:250) was determined by indirect ELISA and by DOT-ELISA.
Actin Purification Actin was purified from guinea pig striated muscle as described by Pardee and Spudish (1982) but with two more cycles of polymerization-despolymerization. Actin purity was verified on sodium dodecyl sulfate-polyacrylamide (11%)gel electrophoresis (SDS-PAGE). Preparation of Polyclonal Anti-Actin Antibodies Purified actin was dissolved in sample buffer and then cross-linked with 0.1% glutaraldehyde and subsequently precipitated with 1% AlC1, a s described by Lazarides (1982). Rabbits were immunized with this actin preparation following the scheme used by Lazarides (1982), and the antibodies were purified by affinity chromatography in columns of a protein-A-Shepharose and Sepharose-actin. Purified actin was coupled to Br-CN activated Sepharose 4B a s described by Lazarides (1982), with modifications to avoid actin polimerization during coupling. The modifications consisted of two cycles of freezing and thawing the actin
Calmodulin and Anti-Calmodulin Antibody Purification Calmodulin was obtained from bovine testicular tissue a s described by Dedman and Kaetzel (1983) and purified by affinity chromatography on phenylSepharose as described by Gopalakrishna and Anderson (1982). Polyclonal antibodies against calmodulin were prepared in sheep following a common immunization program. They were purified by affinity chromatography on calmodulin-Sepharose column as described by Dedman et al. (1978). Antibody titer (150) was determined by ELISA (Voller and de Savigny, 1981). Preparation of Peroxidase Conjugates Rabbit IgG anti-mouse IgG and goat IgG anti-rabbit IgG: mouse and rabbit IgG were obtained by affinitychromatography on protein A-Sepharose, antibodies were rinsed in rabbit or goat, respectively, and purified
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room temperature and given a PBS rinse, the smears were blocked for 1h a t 37°C with 25 pl (viv) 3%bovine serum albumin in PBS-1% preimmune goat serum preabsorbed in a n actin-Sepharose column (when mouse anti-actin monoclonal antibody was used, only BSA was utilized to block). PBS washed smears were treated with 25 p1 microliters of the primary antibody (rabbit or mouse IgG anti-actin), mounted under a cover-glass slide, and incubated for 18 h a t 4°C in a humid atmosphere. Exhaustively PBS washed cells were incubated for 2 h at 37"C, in a humid atmosphere, with 25 pl of goat anti-rabbit or rabbit anti-mouse globulin tagged with peroxidase. Slides were washed thrice with PBS and stained for peroxidase following the procedure described by Johnson and Dorling (1981). Glycerol-PBS, 1 : l pH 8, and nail polish were used to mount and seal the preparations. Cells were viewed through a DOCUVAL Carl Zeiss microscope. Images were recorded on Kodak Tri-X film, 400 ASA.
Fig. 5. Fluorescence of guinea pig spermatozoa, treated with A23187 (37 pmolesll x lo6 spermatozoa) and stained with rhodamine-phalloidin. a and b: Acrosome reacted sperm cells (after 3 and 4 min of A23187 treatment, respectively) showed fluorescence from the equatorial region (as a bright belt) to the tip of the flagellum. c: Cells after 7 min of A23187 treatment; fluorescence increased in the postacrosomal region with a less defined equatorial belt. Fluorescence is also present along the flagellum.
by chromatography on Sepharose-(mouse or rabbit) IgG. Rabbit and goat IgG anti-IgG were conjugated to peroxidase (HRP) as described by Avrameas and Ternyck (1971). Conjugate titers were (1:3,000) for anti-mouse IgG and (1:2,000) for anti-rabbit IgG.
Location of Calmodulin For calmodulin localization, indirect immunoperoxidase technique was done as described by Trejo and MUjica (1990) for indirect immunofluorescence. Peroxidase staining was carried out following the procedure described by Johnson and Dorling (1981) with diaminobenzidine as substrate. Immunocytochemical Localization of Actin Sperm suspension aliquots were fixed in 3% formaldehyde-PBS (1.5% formaldehyde, final concentration) for 60 rnin a t room temperature. Pelleted spermatozoa (600g, 4 min) were washed thrice by dilution in 15 vol PBS and centrifugation, then the cells were incubated in 0.05 M NH,C1 for 10 min and rinsed twice with PBS. The spermatozoa were permeabilized in absolute acetone at -20°C for 5 min. PBS wash was used to eliminate the solvent. Endogenous peroxidase was eliminated by 0.074% HC1-absolute ethanol treatment (Weir et al., 1974, see Johnson and Dorling, 1981) for 10 min. After two PBS washes, a final distilled water wash was done. Spermatozoa resuspended in distilled water were used to prepare the smears. After being air-dried a t
Rhodamine-Phalloidin Staining Sample spermatozoa were fixed in 0.75-1.5% formaldehyde final concentration in PBS for one h at room temperature. Pelleted cells (600g, 4 min) were resuspended in 50 mM NH,C1 for 10 min, followed by three washings by resuspension and centrifugation in PBS as above and one wash in distilled water, and finally resuspended in water. This suspension was spread on microscope slides and air dried, permeabilized in absolute methanol for 5 min at -2O"C, stained with rhodaminephalloidin (30 plislide, at a concentration of 10 Fgiml), and mounted under a cover-glass slide for 20 min in a humid atmosphere. The slides were rinsed in PBS thrice and mounted in a mixture of glycero1:PBS (1:l). Cells were observed in a Zeiss photomicroscope equipped with epifluorescence optics. Photographs were taken with Kodak Tri-X pan film (ASA 400), on exposure for 45 sec. RESULTS Actin Detection in Guinea Pig Sperm Extracts by SDS-PAGE and Western Blot Analysis The protein pattern of whole spermatozoa solubilized with SDS plus six protease inhibitors revealed the presence of a polypeptide with a n apparent molecular weight of 43 KD (Fig. l A , lane 1)that comigrated with reference actin (Fig. lA, lane 2). Western blot of spermatozoa proteins followed by overlay with a monoclonal or a polyclonal monospecific anti-actin antibody indicated a positive signal on the corresponding 43 KD band (Fig. l A , lanes 5, 8). No degradation of actin was observed when a measured amount of rabbit skeletal muscle actin was added to a sperm preparation, processed together, subjected to electrophoresis (Fig. 1A, lane 3), and revealed on Western blot (Fig. lA, lanes 6, 9). Reference actin is shown (Fig. lA, lanes 4,7). The protein pattern of whole purified (on Percoll gradient) spermatozoa with only 0.2% contaminating cells revealed the presence of a polipeptide with a n apparent
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Fig. 6. F-Actin detection by rhodamine-phalloidin in guinea pig spermatozoa treated with 10 (*.Mcytochalasin D. Cytochalasin D was added from the start of the cell extraction and the cell concentration was adjusted and maintained a t 5 x lo6 spermatozoa per ml. d and e,
Controls incubated in the absence of cytochalasin D for 2 and 5 h, respectively. a, b, and c, Spermatozoa incubated with cytochalasin D for 2 h, 4 h, and 5 h, respectively. In b and c a loss of fluorescence is evident while in a, as in the controls, there is intense fluorescence.
molecular weight of 43 KD (Fig. l B , lane 4) that comigrated with reference actin (Fig. lB, line 3), revealed with a monoclonal anti-actin antibody.
reacted spermatozoa, no actin stain was observed on the nucleus region where the acrosome was, but a n actin belt was apparent in the equatorial region (Fig. 3b).
Localization of Actin in Guinea Pig Spermatozoa Detection of actin by immunoperoxidase technique, using one monoclonal (Sigma) and two polyclonal antiactin antibodies, showed reaction with the whole acrosome (clearly observable in color pictures, apparently surrounding only the acrosome in the black and white pictures) and the flagella of NaC1-washed, formaldehyde-fixed guinea pig spermatozoa (Fig. 2a). Specificity of the immunoperoxidase technique was tested by performing three controls: (1)incubation with the second antibody without the primary (anti-actin) antibody (Fig. 2b); (2) preadsorbing the purified anti-actin antibody with a fivefold excess of homogeneous skeletal muscle actin (Fig. 2c); and (3)use of preimmune rabbit IgG as the primary antibody (Fig. 2d). There was no staining of the cells in any of the controls assayed. Spermatozoa incubated a t least one h in MCM-PL showed actin stain similar (Fig. 3a) to NaC1-washed cells (Fig. 2a) in the whole acrosome and flagella. In acrosome
Evidence of F-Actin Presence Rhodamine-labelled phalloidin was used to localize F-actin in guinea pig spermatozoa: in spermatozoa from the vas deferens fixed immediately in epididymal plasma; in the cells washed with NaC1; in MCM-PL capacitated spermatozoa with and without acrosome reaction; and also in noncapacitated acrosome-reacted spermatozoa by induction with A23187. Independent of being washed or not, spermatozoa from the vas deferens showed bright fluorescence (rhodaminel across their entire length, except in the postacrosomal region (Fig. 4a,b) as did the MCM-PL incubated spermatozoa (Fig. 4c,d). The fluorescence was associated with the flagellum, acrosome, and the equatorial region (Fig. 4c,d). Acrosome-reacted spermatozoa showed fluorescence in the equatorial and postacrosomal regions and throughout the flagellum (Fig. 4d). In spermatozoa with acrosome reaction, elicited by the ionophore A23187, a bright fluorescence in the equatorial region was ob-
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Effect of Cytochalasin D on Sperm F-Actin Ten micromolar cytochalasin D diminished the fluorescence produced by rhodamine-phalloidin, although cytochalasin D effect began to be apparent after 3 h of incubation (Fig. 6b,c). From the start of incubation, slightly weaker motility and increased acrosome loss (70% of the cells were without acrosome) was observed in cytochalasin-treated spermatozoa, compared with their controls.
Effect of Cytochalasin D, During Capacitation and Acrosome Reaction, on Calmodulin Location in Spermatozoa Without Acrosome Seventy minutes after the acrosome reaction started, it was found that cytochalasin D increased, by 26-35%, the loss of acrosome over that shown by control spermatozoa incubated without cytochalasin but with 1-5% dimethylformamide, used as a solvent for cytochalasin D. After cytochalasin D treatment, a higher percentage, with respect to the control, of spermatozoa without acrosome showed calmodulin in the equatorial region (Fig. 7a versus b) in a dose dependent manner (Fig. 8). Also during cytochalasin D-treatment, the percentage of acrosome-reacted spermatozoa stained in the postacrosomal region decreased (Fig. 7a, 8) in a dose dependent manner. The flagellum showed calmodulin stain in cells with and without cytochalasin D treatment (Fig. 7a,b). DISCUSSION
Fig. 7. Effect of cytochalasin D on calmodulin location in spermatozoa with acrosome reaction. Spermatozoa (5 x lo6 cellsiml) were treated with 50 KM cytochalasin D during incubation in MCM-PL, fixed 70 min after acrosome reaction started, and calmoldulin revealed by indirect immunoperoxidase. a: Cytochalasin D-treated spermatozoa. Calmodulin is localized as a belt in the equatorial region and along the flagellum. b: Control spermatozoa without cytochalasin D. Calmodulin is present in the postacrosomal region and along the flagellum.
served after a period of 3 min (Fig. 5a), and a t 4 and 7 min after A23187 treatment, a bright fluorescence in the postacrosomal region was observed (Fig. 5b,c). Control spermatozoa samples labelled with 300-fold phalloidin followed by rhodamine-phalloidin showed greatly diminished fluorescence; no spermatozoa fluorescence was observed with 500-fold phalloidin pretreatment (data not shown).
The presence of actin in guinea pig spermatozoa (from non- or Percoll purified samples) was confirmed by Western blot technique. A band of 43 KD was recognized using two monoclonal and two policlonal antiactin antibodies. By addition of six protease inhibitors proteolysis was stringently controlled. This condition differed from that used by Halenda et al. (19871, who did not use any protease inhibitors and did not detect actin in guinea pig spermatozoa from the epididymal cauda. Our results are in agreement with those of Flaherty et al. (19861, who using three protease inhibitors detected actin in each of eight species of spermatozoa, including those of guinea pig. In some guinea pig sperm extracts revealed by immunoblot with monoclonal anti-actin antibodies, besides the actin band (43 KD), a second light band (50KD) was detected (data not shown), similar to that described for rat and mouse sperm (Flaherty e t al., 1986). By indirect immunoperoxidase technique, actin was localized in the guinea pig spermatozoa in the whole acrosome, along the flagellum and in the equatorial region of acrosome reacted spermatozoa. Actin distribution pattern was maintained from NaC1-washed cells through capacitation in MCM-PL and in the acrosomereacted cells, but in the last cells no actin was observed in the region previously occupied by the acrosome (Fig. 3b). In guinea pig spermatozoa, Clarke and Yanagimachi (1978) detected actin only in the postacrosomal region, and Flaherty et al. (1986) in the neck, midpiece,
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Immunoperoxidase Patterns (%)
e
d
20.0
I
43.2
59.6
25.8
66.8
12.9
Fig. 8. Calmodulin localization during acrosomal reaction in the presence of Cytochalasin D. Spermatozoa (5 x lo6 cellsiml) were incubated in MCM-PL with cytochalasin D, a t the concentrations indicated. Samples were fixed 70 min after acrosome reaction started and sperm calmodulin distribution was studied with indirect immunoperoxidase. Five hundred cells were assessed. The percentage of each calmodulin distribution pattern is indicated.
and principal piece. The presence of actin in the flagellum had been difficult to evaluate by immunofluorescence because of sperm autofluorescence (Flaherty et al., 1986).This problem was abolished by the immunoperoxidase technique. To our knowledge, we describe for the first time the presence of filamentous actin in guinea pig spermatozoa (Figs. 4,5) by using phalloidin-rhodamine which is a specific probe for actin filaments. Only in boar capacitated (Saxena et al., 1986a) and uncapacitated (Peterson et al., 1990) spermatozoa and in the sperm head of the plains mouse (Flaherty, 1987) has F-actin been shown. We found filamentous actin in the whole cell, except in the postacrosomal region, in both capacitated and uncapacitated cells with acrosome (Fig. 4). In acrosome-reacted spermatozoa, but not in cells with acrosome, F-actin was apparent in the postacrosomal region (Fig. 4d), suggesting actin polymerization in this region during acrosome reaction. Flaherty et al. (1986), using NDB-phallacidin, detected F-actin in the neck of guinea pig spermatozoa and a weak label in the flagellum indicating that it was unspecific. Under the experimental conditions for rhodamine observation, sperm autofluorescence was not seen. F-actin in the postacrosomal region was not detected by immunoperoxidase technique, but was positive by immunofluorescence, a higher sensitivity technique. Unsuccessful results (Halenda et al., 1987) on F-actin detection in guinea pig spermatozoa and negative results in F-actin observation in other sperm species could be due to techni-
cal problems such as non-permeabilization (Virtanen et al., 1984; Welch and O’Rand, 19851, excessive fixation (Halenda et al., 1987) (when we used these conditions, we observed diminished fluorescence in guinea pig spermatozoa [data not shown]), or sperm autofluorescence that is observed under conditions needed for microscopic observation of NBD-phallacidin. Cytochalasin gradually decreased the intensity of fluorescence detected with rhodamine-phalloidin after 3 h of incubation in MCM-PL, which suggests that cytochalasin depolymerize F-actin in guinea pig spermatozoa, a n observation which is in agreement with Saxena’s e t al. (1986b) results. During the acrosome reaction of guinea pig spermatozoa, calmodulin is apparently translocated from the equatorial region to the postacrosomal region. Our results showed t h a t cytochalasin D produced a n arrest of the calmodulin in the equatorial region, since we found a n increase of the number of cells with calmodulin located in this zone, and a decrease of the number of cells showing calmodulin in the postacrosomal region. This suggests that, for calmodulin translocation to the postacrosomal zone to take place, a n intact actin cytoskeleton is needed. This study suggests that F-actin is involved in calmodulin (an intracellular protein with known physiological properties) migration to the postacrosomal region, a zone involved in egg recognition, in a Ca2+dependent process. Our result is in agreement with Saxena’s et al. (1986a) report, since in both instances
F-ACTIN IN GUINEA PIG SPERMATOZOA the translocation of a protein was blocked by the microfilament toxin cytochalasin D. The presence of F-actin in the acrosome and in the flagellum and the changes elicited on spermatozoa by cytochalasin D treatment suggest actin participation in stabilization of the acrosome structure, as well a s a possible actin function in the spermatozoa motility, although we do not know if actin is directly involved in flagella beating, a s was suggested for Chlamydomonas (Baccetti et al., 1989). Other actin functions could be the maintenance of tail shape as proposed previously (Baccetti e t al., 1989) and protein translocation. In summary, like spermatozoa of many species, guinea pig spermatozoa contain actin. The presence of F-actin in spermatozoa allows a reconsideration on its physiological role in acrosome reaction, in movement, in the maintenance of tail shape (Baccetti et al., 19891, and in protein translocation.
ACKNOWLEDGMENTS We thank Dr. A. Huberman, Dr. S. Villa-Trevino, M. en C. 0. Velasco-Castaneda, and M.A. Torres-Vega for helpful comments on the manuscript. This research was partly supported by Consejo Nacional de Ciencia y Tecnologia, grant P228CCOX891518.
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