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Regular Article |
a Biologie et Pathologie du Spermatozoïde Humain, EA 1719, Institut de Recherches sur le Cancer, 59045 Lille, France
b URA 1291 INRA-CNRS Institut National de la Recherche Agronomique, Station de Physiologie des Mammifères Domestiques, 37380 Monnaie, France
c Laboratoire de Cytologie et Biologie de la Reproduction, Hôpital Claude Huriez, CHRU de Lille, 59037 Lille, France
d Service d'Urologie, Hôpital Claude Huriez, CHRU de Lille, 59037 Lille, France
ABSTRACT
The nucleus of mammalian spermatozoa is surrounded by a rigid layer, the perinuclear theca, which is divided into a subacrosomal layer and a postacrosomal calyx. Among the proteins characterized in the perinuclear theca, calicin is one of the main components of the calyx. Its sequence contains three kelch repeats and a BTB/POZ domain. We have studied the association of boar calicin with F-actin and the distribution of boar and human calicin during spermiogenesis compared with the distribution of actin. Calicin was purified from boar sperm heads under nondenaturating conditions. The molecule bound actin with high affinity (Kd =
5 nM), and a stoichiometry of approximately one calicin per 12 actin monomers was observed. Gel filtration studies showed that calicin forms homomultimers (tetramers and higher polymers). According to immunocytochemical results, calicin is present (together with actin) in the acrosomal region of round spermatids and is mainly localized in the postacrosomal region of late spermatids and spermatozoa. Taken together, the results suggest that the affinity of calicin to F-actin allows targeting of calicin at the subacrosomal space of round spermatids, and that its ability to form homomultimers contributes to the formation of a rigid calyx.
sperm, spermatid, spermatogenesis, testes
INTRODUCTION
During mammalian spermiogenesis, many morphologic changes are observed: a cell with an ultrastructural organization similar to undifferentiated somatic cells, the round spermatid, is progressively transformed into a highly specialized, testicular spermatozoon. Changes leading to the mature sperm head are important for gamete function: formation of the acrosome, condensation of the nucleus, and assembly of a special cytoskeleton, the perinuclear cytoskeleton or perinuclear theca [1, 2], surrounding the nuclear membrane, both in the subacrosomal region and in the postacrosomal part of the head (named the calyx) [3]. Several proteins have been identified in the perinuclear theca (either in the subacrosomal layer, the calyx, or both). The molecular structure of some of these proteins is known: calicin [4]; cylicin I [5] and cylicin II [6], both belonging to the previously named group of multiple band polypeptides [3, 7]; CP
3 [8–10] and CPß3 [9], which are testis-specific isoforms of the and ß subunits of capping protein; and PERF 15 or TLBP [11, 12].
In addition to these proteins specifically expressed during spermiogenesis, a transcription factor also expressed in other cell types, Stat4, has been found in the perinuclear theca of mouse spermatids and spermatozoa [13]. Other proteins have also been described, but their molecular structure is yet unknown: three proteins of the perinuclear theca of mouse spermatids of 75, 77, and 80 kDa, named thecins [14]; a 90-kDa protein of the subacrosomal layer of human sperm [15]; and approximately 20 polypeptides of 15 to 60 kDa in bull sperm perinuclear theca [16, 17]. The protein composition of perinuclear theca is different according to the mammalian species. Bull sperm perinuclear cytoskeleton contains many proteins [16, 17] whereas mouse and rat sperm perinuclear theca contain one prominent protein, PERF 15 [18, 19].
The subacrosomal layer of mammalian spermatids is also filled with actin, one of the ubiquitous cytoskeletal proteins, in its filamentous form (F-actin) during the greater part of spermiogenesis [20–28]. In late spermatids and spermatozoa of many species, most or all of the F-actin is depolymerized to G-actin and seems to be redistributed in a species-specific pattern [2, 27, 28]. The role of F-actin and of its interaction with other perinuclear theca proteins is not known. Possible roles are anchorage of the acrosome [17, 22, 27, 29] or shaping of the head by capping the nuclear membrane [26, 30].
A better understanding of actin function in the subacrosomal layer implies a search for the actin-binding properties of proteins of the perinuclear cytoskeleton. Calicin may be an actin-binding protein. The sequence of calicin [4] contains three consecutive repeating units of a domain of approximately 50 amino acids, first described in Drosophila kelch protein [31], which has six of these kelch elements, but also in other proteins of the kelch family [32]. Among proteins of this family, -scruin, which is a protein with two repeats of six kelch domains, clearly binds actin [33–35]; the binding seems to depend on the special organization of the six kelch domains, which have compared to the blades of a propeller [33, 36]. The actin-binding properties of proteins with six kelch repeats, such as Drosophila kelch protein [37, 38], the Physarum polycephalum actin-fragmin kinase [39], ENC-1 protein [40], IPP [41], and mayven [42] proteins, are suggested mostly by indirect experiments (except for IPP and mayven, for which a direct binding to actin has been demonstrated). Whether calicin, a protein with only three kelch domains, is an actin-binding protein and whether its localization during spermiogenesis is superimposed on the one of actin were investigated in the present study.
MATERIALS AND METHODS
Materials
Human semen from fertile donors with normal semen characteristics according to World Health Organization criteria were obtained from the CECOS-Nord (Lille, France) and stored until use in liquid nitrogen. Human testicular tissues presenting a normal germinal epithelium as controlled by light microscopy were obtained from the testes of patients (all of whom granted informed consent) undergoing orchidectomy for carcinoma of the prostate. Spermatozoa from cauda epididymis and testes from freshly slaughtered boars were collected at the Station de Physiologie des Mammifères Domestiques (INRA, Nouzilly, France). Boar spermatozoa were stored at -80°C. Boar and human testes were treated immediately after collection.
Antibodies
A peptide with the sequence MKLEFTEKNYNSFVLQNLNRQRKR, corresponding to the amino-terminal sequence of human calicin from amino acid residues 1 to 24 [4], was used for immunization of rabbits. This sequence is identical to the amino-terminal sequence of bull calicin, except for a change from arginin (human sequence) to lysine (bull sequence) at position 20 [4]. The peptide was synthesized and coupled to keyhole limpet hemocyanin (KLH; Sigma-Aldrich, Saint Quentin Fallavier, France) using m-maleimidobenzoyl-N-hydroxysuccinimide ester. Rabbits were immunized by s.c. injection of peptide-KLH conjugate at intervals of 3 wk, with the first injection in Freund complete adjuvant (0.5 mg of conjugate) and the others in Freund incomplete adjuvant (0.5 mg at Day 21 and 0.25 mg for later injections). The animals were bled 10 days after each immunization, beginning with the third immunization. The immune sera were affinity purified, first by isolation of an IgG fraction by chromatography of serum on a protein A-agarose column (Sigma-Aldrich) and second by specific anti-peptide antibodies in the IgG fraction by chromatography on a column of peptide coupled to Sulfolink gel (Pierce Chemicals, Montluçon, France) according to the manufacturer's instructions. When necessary, residual antibody activity against KLH was removed by final immunoabsorption onto a column of KLH coupled to CNBr-activated Sepharose 4B (Amersham-Pharmacia, Les Ulis, France). Antibodies to actin were a mouse monoclonal antibody to -smooth muscle actin (Amersham-Pharmacia) and a rabbit polyclonal antiserum (Sigma). Secondary antibodies for immunofluorescence were fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) and tetramethylrhodamine-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Sigma). For immunoblotting, goat anti-rabbit IgG conjugated with peroxidase (Sigma) was used.
Preparation and Fractionation of Spermatogenic Cells
Spermatogenic cells were prepared from the testes of patients undergoing orchidectomy for carcinoma of the prostate. The protocol for preparation of cell suspensions was similar to that described by Blanchard et al. [43]. Briefly, minced testicular tissue was incubated with stirring for 5 min at 32°C with 0.25% trypsin and 0.0025% DNase I in 150 ml of PBS (pH 7.4) containing 0.5 mM MgCl2, 1 mM CaCl2, 0.1% glucose, and 6 mM pyruvate (PBSGP). After removal of the cell debris on successive nylon filters of 150, 75, and 25 µm, the cellular suspension (107 cells/ml) in PBSGP containing 0.5% BSA, 0.002% DNase I, and 5 mM 2-naphtol-6.8-disulfonic acid (NDA) was fractionated by sedimentation at 1 g in a unit sedimentation chamber (150-mm diameter) using a linear, 2% to 4% BSA gradient in PBSGP containing 5 mM NDA [44]. Ninety minutes after layering the cell suspension, 10-ml fractions were collected. The cells were recovered from the fractions by centrifugation at 700 x g; air-dried smears were made from each fraction, fixed with methanol-acetic acid (3:1), and stained with May-Grünewald-Giemsa.
Isolation of Spermatozoa and Sperm Heads
All procedures were performed at 4°C, with all buffers containing the following proteinase inhibitors: aprotinin, 10 µg/ml; bestatin, 5 µg/ml; leupeptin, 50 µg/ml; p-aminobenzamidine, 0.1 mM; pepstatin A, 50 µg/ml; and PMSF, 1 mM. Human spermatozoa were recovered by centrifugation of semen at 700 x g for 10 min over 45% Percoll (Amersham-Pharmacia; 1 volume of semen for 1.2 volumes of 45% Percoll in PBS). The pellet of sperm cells was subsequently washed twice with PBS containing 25 mM EDTA. Boar epididymal spermatozoa were washed twice in PBS containing 25 mM EDTA and then pelleted by centrifugation at 600 x g for 10 min. Sperm heads were dissociated from tails by sonication on ice with a sonifier (Vibra-Cell 74234; Bioblock Scientific, Molsheim, France) set at maximum power: seven bursts of 10 sec separated by 30-sec intervals followed by three bursts of 30 sec separated by 1-min intervals. The suspension of heads and tails was made 50% sucrose and layered atop a sucrose gradient of 90%, 80%, 70%, 60% and 50% in PBS containing 25 mM EDTA and centrifuged for 90 min at 100 000 x g [9]. The pelleted sperm heads were collected and then washed in PBS containing 25 mM EDTA.
Extraction of Proteins from Sperm Heads
Successive extractions of proteins from sperm heads were performed essentially as described by Oko and Maravei [16], but with the following modifications: After the steps of extraction, first with 0.2% Triton X-100 and second with 1 M NaCl, additional steps were tried before extraction with 0.1 M NaOH, including treatment with 1 M Na2CO3 (pH 11.0) or with 8 M urea. The supernatants containing the proteins were dialyzed against PBS and concentrated by filtration on Diaflo PM 10 membrane (Amicon Bioseparation; Millipore, Saint Quentin en Yvelines, France), followed by filtration on Nanosep (10-kDa cutoff) centrifugal membrane (Pall Filtron, Northborough, MA). Protein concentration was determined by Coomassie blue protein-dye binding assay [45], and the material was stored as aliquots at -80°C.
Extraction of Proteins from Spermatogenic Cells
Proteins from spermatogenic cells were extracted first with PBS (pH 7.4) containing 0.2% Triton X-100 for 1 h at 4°C, followed by centrifugation at 27 000 x g for 30 min at 4°C. After two washes in PBS (pH 7.4) the pellets were extracted by 0.01 M Na-phosphate (pH 7.4) containing 1 M NaCl for 1 h at 4°C and then centrifuged at 27 000 x g. Final extraction of proteins from these cells was performed for 1 h at room temperature with 50 mM Tris-HCl, 2% SDS, and 5% 2-mercaptoethanol (pH 8.0).
Purification of Calicin
Calicin was purified from proteins extracted from boar sperm heads with 0.2% Triton X-100, 1 M NaCl, or 0.1 M NaOH. Triton X-100 extracts were incubated with Biobeads SM (Bio-Rad, Ivry sur Seine, France) before chromatography to remove detergent. Proteins (10–15 mg) were first loaded onto a Sepharose 4B column coupled to 30 mg of IgG purified from a rabbit serum containing anti-calicin 1–24 antibodies by protein A-agarose chromatography. Buffers successively used were as follows: PBS (pH 7.4), 10 mM Na-phosphate containing 0.5 M NaCl (pH 7.4), PBS (pH 7.4) containing 2 M guanidine hydrochloride; and PBS (pH 7.4) containing 3 M sodium thiocyanate. Calicin-containing fractions eluted with the fourth buffer were pooled, equilibrated in 0.05 M sodium acetate and 0.05 M NaCl buffer (pH 6.0), and then loaded onto a 1-ml HiTrap-SP column (Amersham-Pharmacia) equilibrated with the same buffer before elution with a 0.05–0.5 M NaCl gradient. The purity of pooled and concentrated calicin-containing fractions was controlled by SDS-PAGE. Purified calicin was stored at -80°C until use.
Cosedimentation Assay with F-Actin
The procedure described by Matsudaira [46] was used. Rabbit skeletal muscle F-actin was purified from muscle acetone powder (Sigma) according to the procedure described by Pardee and Spudich [47] using two final cycles of depolymerization-repolymerization of F-actin and was stored (
3 wk) on ice in F-buffer (10 mM Tris-HCl, 30 mM NaCl, 1 mM MgCl2, and 0.3 mM NaN3 [pH 8.0]) [46]. Both whole proteins extracted from boar sperm heads with 1 M NaCl and purified calicin were studied. Proteins were centrifuged at 150 000 x g for 30 min at 4°C to remove any insoluble material immediately before each actin-binding assay. F-Actin in F-buffer (5 µl) was mixed with different amounts of protein in PBS (pH 7.4; 10 µl) and the volume adjusted to 50 µl with pelleting assay buffer (50 mM NaCl, 1 mM MgCl2, 10 mM PIPES, 0.1 mM EGTA, 0.1 mM ATP, and 0.1 mM 1,4-dithiothreitol [pH 7.0]). After an incubation of 12 h at 4°C under gentle stirring, the mixture was pelleted by centrifugation at 150 000 x g for 20 min. Controls without actin were performed for each protein concentration. Equal volumes of solubilized pellets, supernatants, and uncentrifuged mixture were loaded on gels for SDS-PAGE and quantitative immunoblotting. Binding assays with whole proteins from 1 M NaCl fraction were performed with 4 µM (final concentration) F-actin and with protein concentrations in the range of 0.1–1 mg/ml (final concentration of calicin determined by quantitative immunoblotting, 0.15–1.5 µM). Binding assays with purified calicin were performed with 1 µM F-actin and 0.05–0.1 µM calicin.
Gel Filtration HPLC
Calicin purified from either Triton X-100 or 1 M NaCl extracts was studied. Protein was loaded onto two (in series) 7.8- x 300-mm Bio-Sil SEC 250-5 gel filtration columns (Bio-Rad) equilibrated in 0.1 M Na-phosphate containing 0.01% NaN3 (pH 7.0) at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected, and protein was detected at 280 nm.
Gel Electrophoresis and Immunoblotting
The SDS-PAGE was performed on 10% or 12% polyacrylamide gels using the Laemmli buffer system [48], and immunoblotting was performed as described elsewhere [44]. For two-dimensional gel electrophoresis, the protein extracts were treated according to the method described by Longo et al. [3]. Two-dimensional gel electrophoresis was performed using, for the first dimension, either isoelectric focusing [49] or nonequilibrium pH gradient gel electrophoresis [50]. Staining of the proteins after SDS-PAGE was performed either with Coomassie blue or with the silver staining procedure [51]. To develop the horseradish peroxidase reaction in immunoblotting experiments, the enhanced chemiluminescence system (Amersham-Pharmacia) was used, with exposure times of the films (Hyperfilm ECL; Amersham-Pharmacia) ranging from 30 sec to 5 min. Alternatively, the chemiluminescence Supersignal Ultra (Pierce Chemicals) was used for quantitative analysis of blots, with exposure times ranging from 1 min to 16 h. Quantitative analysis of calicin was performed using standards of purified calicin from 0.05 to 1 µg. Films were analyzed with the Kodak Digital Science 1D Analysis system (Eastman Kodak, Rochester, NY).
Immunofluorescence Microscopy
Immunofluorescence microscopy was performed on spermatozoa and on testicular tissue. Suspensions of washed spermatozoa were spread as droplets on glass slides, air-dried, and fixed with methanol at -20°C for 5 min and then acetone at -20°C for 2 min. Testicular cells were either inprints of testis or tissue sections. Inprints of testis were prepared by pressing a freshly retrieved testis onto poly-L-lysine-coated slides; cells adhering to slides were fixed with methanol and acetone, as described earlier, before immunostaining. Tissue sections were obtained as follows: Testes were cleared from blood cells by several washes in lactated Ringer solution, cut into small blocks, immersed in Bouin fixative for 18 h to 5 days, thoroughly washed in 70% ethanol, dehydrated in graded ethanol, and embedded in paraffin. Sections (4–8-µm thickness) were mounted onto silanized slides, which was followed by deparaffinization, hydration, and then incubation for 1 h at room temperature in PBS with 0.2% Triton X-100.
Immunostaining was performed essentially as described elsewhere [44]. For the double labeling with two primary antibodies raised in the same species (i.e., affinity-purified rabbit anti-calicin antibodies and rabbit anti-actin immune serum), sequential incubations were performed essentially according to the method described by Kroeber et al. [52].
RESULTS
Characterization of Calicin in Boar and Human Spermatozoa
An antiserum against a synthetic peptide corresponding to the sequence 1–24 of human calicin was raised in rabbits and affinity purified. The purified antibodies reacted with a polypeptide in the proteins extracted from boar sperm heads (Fig. 1A). The protein has the molecular characteristics of calicin, that is, a molecular weight of approximately 60 kDa and a pI of approximately pH 8 (pI = 7.9 in equilibrium two-dimensional gel electrophoresis). Similar results were obtained with proteins extracted from human sperm heads (data not shown). Immunofluorescence microscopy with the anti-peptide antibodies showed the labeling of the calyx structure both in boar and in human sperm cells (Fig. 1B).
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Successive protein extractions from boar sperm heads were performed according to the method described by Oko and Maravei [16]. Several proteins were obtained at each step (Fig. 2A). Controls by immunoblotting showed that calicin is mainly present in the proteins of the final NaOH extraction step (corresponding to the perinuclear theca proteins; Fig. 2B, lane 4), but immunoreactive material was also found in proteins extracted with 1 M NaCl (Fig. 2B, lane 2). Loading of more protein material onto the electrophoresis gels allowed detection of calicin in proteins extracted with Triton X-100 (data not shown). No calicin was found in an additional step using 1 M Na2CO3 (pH 11.0; Fig. 2B, lane 3), but 8 M urea extracted some calicin before the final NaOH extraction step (data not shown). Essentially, the same results were observed with human sperm heads, except that, in most experiments, no protein was released by 1 M NaCl treatment (data not shown).
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Calicin Forms Homomultimers In Vitro
Gel filtration HPLC was performed with calicin purified from proteins extracted from boar sperm heads either with 0.2% Triton X-100 or with 1 M NaCl. Figure 3 shows the elution profile obtained with calicin purified from the 1 M NaCl fraction. Most of the calicin was eluted in the void volume of the column, thus behaving as polymers with an apparent molecular weight of greater than 300 000 daltons; a small amount eluted as a tetramer of calicin (Mr, 240 000). With calicin purified from the Triton X-100 fraction, essentially the same results were obtained, except that the proportion of calicin with an apparent molecular weight of 240 000 (tetramer) and 120 000 (dimer) was greater (data not shown).
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Binding of Calicin to Actin
Attempts to demonstrate binding of calicin to F-actin using the actin blot-overlay technique [53] after two-dimensional electrophoresis of human or boar sperm head proteins were unsuccessful. Moreover, by actin pelleting assays, the binding to F-actin of calicin present in the protein fractions extracted from boar sperm heads with 0.1 M NaOH or 8 M urea could not be demonstrated, which probably relates to denaturation of the protein. However, with calicin purified from the 1 M NaCl fraction, results of actin cosedimentation assays (Fig. 4A) clearly showed that calicin binds F-actin. As purified calicin was not present in sufficient amounts to determine a binding curve, binding analysis was performed using whole protein from the 1 M NaCl fraction. A roughly linear Scatchard plot was obtained (Fig. 4B). A dissociation constant of approximately 5 nM and a capacity of one calicin per 12 actin monomers at saturation were calculated.
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Characterization of Calicin in Spermatogenic Cells
Spermatogenic cells purified by sedimentation at 1 g were successively extracted with 0.2% Triton X-100, 1 M NaCl, and 2% SDS with 5% 2-mercaptoethanol. Immunoblots with anti-calicin antibodies (Fig. 5) showed the absence of calicin in the proteins extracted from a cell fraction containing approximately 70% spermatocytes, together with Leydig cells and multinucleate cells (Fig. 5A); in a cell fraction with a large majority of round spermatids (80%), calicin was detected in the proteins extracted with Triton X-100 (Fig. 5B, lane 1), but not in further extracts performed with 1 M NaCl or with 2% SDS and 5% 2-mercaptoethanol (Fig. 5B, lanes 2 and 3). In a cell population containing approximately 60% late spermatids together with cells of earlier stages, calicin was detected both in 1 M NaCl and SDS extracts (Fig. 5C, lanes 2 and 3).
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Immunolocalization of Calicin in Testis Cells
Immunofluorescent staining of boar testis sections showed the presence of calicin in a region corresponding to the acrosome of round or elongating spermatids (Fig. 6, A–F). In elongated spermatids (Fig. 6, G–I), immunostaining was either around the entire nucleus, both in the acrosomal and in the postacrosomal region, or only in the postacrosomal region. No staining was found in spermatocytes.
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Double-labeling experiments (Fig. 7) clearly showed the localization of both calicin and actin in the acrosomal region of spermatids. The immunofluorescent staining of actin was superimposed on the calicin labeling (Fig. 7, A, B, D, and E).
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Similar results were observed with human testis cells (Fig. 8). Actin and calicin have the same acrosomal localization in round spermatids (Fig. 8, A and B). In late spermatids, both the acrosomal region and the postacrosomal region were labeled with anti-calicin and anti-actin antibodies; alternatively, immunostaining was restricted either to the equatorial segment or to the postacrosomal region (Fig. 8, D and E).
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DISCUSSION
One of the main goals of this study was to define whether calicin, a protein of the mammalian sperm perinuclear theca, is an actin-binding protein. Calicin is the only protein of the kelch family that contains three "kelch repeats" and a BTB/POZ domain [4]. Most of the proteins in this family described so far have six kelch repeats and a BTB/POZ domain, except for proteins found in poxviruses, which have two to six repeats [32], and -scruin, which contains two distinct sets of six kelch repeats and no BTB/POZ domain [32, 35, 36]. We first attempted to isolate calicin from the boar sperm perinuclear theca to study its actin-binding properties. However, use of the protocol described by Oko and Maravei [16], which solubilizes proteins of the insoluble and rigid perinuclear theca in highly alkaline conditions (0.1 M NaOH), or the partial solubilization of thecal proteins in 8 M urea, failed to produce actin-binding calicin molecules. This is reminiscent of the results reported for -scruin, which loses most of its ability to bind actin either in 8 M urea or in highly acidic conditions [35]. Immunodetection of calicin in the fractions released from boar sperm heads according to the procedure described by Oko and Maravei [16] showed that in addition to most of the calicin present in the 0.1 M NaOH fraction, a small amount was also present in the fraction extracted with 0.2% Triton X-100, and a larger quantity was present in the proteins released with 1 M NaCl. This last fraction was used for calicin purification, actin-binding assays, and for a study of the association of calicin molecules in solution. A high affinity of calicin for F-actin (Kd 5 nM) was demonstrated with a stoichiometry calicin:actin (monomer) of approximately 1:12. Controls of cosedimentation assays without actin showed a tendency of calicin to form aggregates as found in the pellets after ultracentrifugation at 150 000 x g. Moreover, studies by gel filtration of soluble calicin obtained in the supernatants of ultracentrifugation demonstrated that this protein is present in solution in a multimeric form, in part corresponding to tetramers but mostly to polymeric material of greater size than tetramers. The association with actin has been well characterized for -scruin. This protein cross-links actin filaments through its two regions of six kelch repeats; each region interacts with one of two subdomains (subdomains 1 and 3) of an actin monomer [33, 34]. Kelch proteins may associate through BTB/POZ domains to form a dimer resembling -scruin that can bind and cross-link actin filaments through paired kelch repeat domains [32, 38, 41]. Our observations of the multimeric organization of calicin suggest that this sperm thecal protein, with only three kelch repeats and a BTB/POZ domain, may bind and cross-link actin filaments through an association of at least four calicin molecules providing the elements of one scruin motif.
Our study also investigated the localization of calicin in spermatogenic cells. Most of the calicin was extracted from human round spermatids in proteins soluble in 0.2% Triton-X100. Immunocytochemical studies detected calicin in the acrosomal region of early spermatids, with a localization superimposed on the one of actin and a main localization in the postacrosomal region of late spermatids and spermatozoa. These results are reminiscent of those reported by Oko and Maravei [17] on the distribution of the proteins of sperm perinuclear theca during bovine spermiogenesis, of Bellvé et al. [14] on the distribution of thecins during mouse spermiogenesis, and of Hurst et al. [10] on the distribution of capping protein subunit CP3 during rat spermiogenesis. They are somewhat different from those reported by Longo et al. [3], who described a localization of calicin to a subacrosomal ring (near the equatorial segment) in round spermatids and a postacrosomal localization in late spermatids and testicular spermatozoa. In our study, the exact association of calicin within the developing acrosome of early spermatids could not be determined because of the limited resolution provided by immunofluorescence. Nevertheless, differential extraction of these isolated cells showed that most of the calicin was present in proteins released by extraction of the cells with the detergent Triton X-100. This suggests either that calicin is mostly localized in the acrosomal membrane fraction or that the subacrosomal region of early spermatids is not as condensed and insoluble as the perinuclear theca of late spermatids and spermatozoa. Oko and Maravei [17], using an immune serum to whole perinuclear theca proteins for immunogold labeling at the electron microscopic level, showed that perinuclear theca proteins surround the entire acrosomic system of round spermatids. This may explain why calicin, which is one of these thecal proteins, is mainly extracted in proteins released from early spermatids with Triton X-100. What could be the role of actin-calicin interaction during the phases of acrosomal attachment onto the nucleus and of acrosomic development? Studies with mutants of Drosophila kelch, which is a structural protein of the ring canals linking nurse cells to the oocyte, revealed that the kelch repeat domain is necessary and sufficient for ring canal localization [38]; interaction with actin likely is involved. Moreover, kelch does not reach the ring canals until a maximum of actin filaments has been recruited to the ring canal [37], suggesting that kelch is not involved in actin localization to the ring canal but, rather, that actin-binding properties of kelch are used for ring canal localization. Such a situation may also be suggested for calicin: The actin-binding property of calicin may be one of the factors that first allows targeting of this thecal protein to the subacrosomal space containing a large amount of F-actin. Later, in elongated spermatids and spermatozoa, interactions between calicin molecules via their BTB/POZ domains may be sufficient to explain incorporation of additional calicin, leading to the formation of a rigid calyx in spermatozoa. However, a main difference between the localization of kelch in ring canals and the one of calicin in sperm calyx is that ring canals contain both F-actin and kelch, whereas actin, in its filamentous form, is absent from the calyx of spermatozoa that are rich in calicin. This is a puzzling situation previously stressed by Von Bülow et al. [4]. Other molecular mechanisms, and probably other proteins, are necessary to explain removal of F-actin at the end of spermiogenesis when, at the same time, calicin accumulates in the calyx structure.
ACKNOWLEDGMENTS
We thank Mr. L. Calvet and Mr. D. Deffrasnes for their technical help. We thank Dr. C. Bailly for helpful corrections of the revised manuscript. We are also grateful to the Spermothèque de la Fédération Nationale des CECOS and, most especially, to the CECOS-Nord (Director, Dr. P. Saint-Pol) for providing the human semen samples from donors.
FOOTNOTES
1 Supported in part by a grant from the CHRU de Lille and by the Université de Lille II. 
2 Correspondence: Roselyne Rousseaux-Prévost, Institut de Recherches sur le Cancer, Place de Verdun, F-59045 Lille, France. FAX: 33 320169229.
Accepted: August 1, 2000.
Received: February 25, 2000.
REFERENCES
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 L. M. Zipper and R. T. Mulcahy Erk Activation Is Required for Nrf2 Nuclear Localization during Pyrrolidine Dithiocarbamate Induction of Glutamate Cysteine Ligase Modulatory Gene Expression in HepG2 Cells Toxicol. Sci., May 1, 2003; 73(1): 124 - 134. [Abstract] [Full Text] [PDF]
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L. M. Zipper and R. T. Mulcahy The Keap1 BTB/POZ Dimerization Function Is Required to Sequester Nrf2 in Cytoplasm J. Biol. Chem., September 20, 2002; 277(39): 36544 - 36552. [Abstract] [Full Text] [PDF]
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