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Testis |
3 Acts at Sites Distinct from Classic Cadherins in Rat Testis and Sperm1
Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 and 4, isoform-specific polyclonal antibodies were generated against protocadherin 3. Western blotting of rat testis showed that protocadherin 3 was solubilized completely by Triton X-100, in contrast to the adhesion junction components N-cadherin, ß-catenin, and p120 catenin. Corroborating this data, protocadherin 3 was immunolocalized to the spermatid acrosomal area, intercellular bridge, and flagellum, but not classic cadherin-based adhesion junctions. Acrosome-associated protocadherin 3 was first detected at step 8 of spermiogenesis, and this association remained on cauda epididymal sperm. Acrosome immunostaining was reduced, but present, in acrosome-reacted sperm. Spermatid intercellular bridges became positive for protocadherin 3 coincident with the appearance of plectin, occurring at spermiogenic steps 8 to 9, and elongate spermatid bridges remained positive throughout spermatogenesis. The developing flagellum was uniformly immunostained for protocadherin 3 up to approximately spermiogenic step 17. Subsequently, flagellar immunostaining was confined to the principal piece, and this pattern continued in cauda epididymal sperm. These data show that protocadherin 3 performs functions unique from classic cadherins in spermatogenesis and suggest a role for protocadherin 3 in organizing germ cell-specific structures including the intercellular bridge, flagellum, and acrosome.
gamete biology, sperm, spermatid, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One mode of cell-cell interaction is via adhesive contact mediated by cadherin proteins. The cadherin superfamily consists of both the classic cadherin subfamily (such as N-cadherin and E-cadherin) and protocadherin subfamilies [1]. Classic cadherins localize to cell-cell adhesion junctions and are linked to the underlying cytoskeleton, providing structural continuity to all tissues [2]. Although the biology of classic cadherins is advanced, relatively little is known about the function of protocadherins. Protocadherins form a large group (more than 60 proteins in mammals) of transmembrane cell surface proteins with much diversity in their cytoplasmic domains, indicating interactions with a variety of cytoplasmic proteins [3]. In general, protocadherins exhibit modest or no homotypic cell-cell adhesion characteristics compared with classic cadherins, although certain protocadherins have been localized to cell-cell contacts in vitro [4, 5]. Unlike classic cadherins, structural motifs in protocadherin cytoplasmic domains suggest that protocadherins are primarily interacting with signaling proteins, much like typical receptors [6].
-Protocadherins comprise one protocadherin subfamily with approximately 15 members, and localization to neuronal synapses has been demonstrated for protocadherin 4 (PCDH4) [7]. Although -protocadherins are positioned at the synapse, a homotypic cell adhesion function has not been demonstrated for this protein family [7]. A receptor-like signaling function for -protocadherins in brain is suggested by data showing binding of the extracellular domain to the soluble ligand reelin and the intracellular domain to the tyrosine kinase fyn [7, 8]. Like brain, testis expresses many cadherins (over 20 cadherin superfamily members at the mRNA level) representing all cadherin subfamilies, suggesting that cadherins function in numerous processes of germ cell maturation and fertilization [9]. Although classic cadherins mediate inter-Sertoli and Sertoli-germ cell adhesion in testis [10, 11], the subcellular localization and function of testicular protocadherins are unknown. Given that protocadherins in other tissues may localize to cell-cell junctions, it was previously hypothesized that protocadherins localize to adhesion junctions within the seminiferous epithelium [9]. Here this hypothesis was tested by generating isoform-specific antibodies against protocadherin 3 (PCDH3) and determining PCDH3 distribution in rat testis cryosections and epididymal sperm and extracts derived from rat testis and sperm.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Biological material was obtained from Fisher 344 rats (Charles River Laboratories Inc., Wilmington, MA). Rats were given water and chow (Prolab Rat/Mouse/Hamster 3000; LabDiet, Purina Mills, St. Louis, MO) ad libitum until they were killed via carbon dioxide asphyxiation. All animals were treated according to the NIH Guide for the Care and Use of Laboratory Animals. Immunostaining and Western blotting experiments were repeated at least twice with consistent results.
Rat Protocadherin cDNA Cloning
All polymerase chain reaction (PCR) reactions were performed with a proofreading polymerase (Expand Hi-Fidelity; Roche Diagnostics, Indianapolis, IN), and unless otherwise indicated, rat testis cDNA was the template. DNA sequences were confirmed by sequencing of both strands. Rat PCDH4 sequence from within the signal peptide to extracellular cadherin (EC) repeat 2 was amplified using a degenerate forward primer to a peptide conserved in mouse and human PCDH4 (MEFSWG) and a reverse primer within the published sequence of EC2 [9]. The intervening sequence from PCDH4 EC2 to the known sequence of EC5 was amplified using primers within each domain (accession number AB045585).
The intervening sequence from rat PCDH3 EC2 to EC4 was amplified using a specific forward primer and a degenerate reverse primer against an EC4 peptide conserved in human PCDH family members (HVPFKL). PCDH3 sequence from EC4 to the intracellular juxtamembrane region was amplified using the published sequence of the rat PCDH constant region [12].
Because the genes for PCDH3 and PCDH4 are repeated in tandem within the genome [13], the 3' sequence of PCDH3 found in the type O splice form and the 5' PCDH4 coding sequence including the start codon was amplified by using a forward primer in the PCDH3 juxtamembrane region and by a reverse primer within PCDH4 EC1 [14]. Rat genomic DNA was used as the template. Sequences for rat PCDH3 and PCDH4 have been submitted to Genbank (accession numbers AF539749 and AF539750).
PCDH3 Antibody Production
PCDH3 antigen was a bacterially expressed glutathione S-transferase (GST) fusion protein including the first 101 amino acids of rat PCDH3 EC3 (Figure 1). PCDH3 sequence was cloned into pGEX-6P-2 (Amersham Pharmacia Biotech, Piscataway, NJ) restricted with BamHI and EcoRI to generate the fusion protein (GST-PCDH3). In the PCDH3 EC3 reverse primer, an in-frame stop codon was placed just prior to an EcoRI site. After protein induction, GST-PCDH3 was isolated from the bacterial soluble fraction and purified using a glutathione-agarose column (Sigma Chemical Co., St. Louis, MO). Polyclonal rabbit antibodies against the purified fusion protein were generated in cooperation with Pocono Rabbit Farm and Laboratory (Canadensis, PA). PCDH3 EC3 antibodies were affinity purified against a column of maltose binding protein PCDH3 EC3 fusion protein (MBP-PCDH3) coupled to Affi-Gel 15 (Bio-Rad, Hercules, CA). Antibodies were eluted from the affinity column with 100 mM glycine, pH 2.5. MBP-PCDH3 was generated after cloning the PCDH3 EC3 sequence into EcoRI and XbaI restricted pMal-c2x (New England Biolabs, Beverly, MA). A stop codon was placed just prior to an XbaI site in the PCDH3 EC3 reverse primer. MBP-PCDH3 was purified from the bacterial soluble fraction using an amylose column according to the manufacturer's instructions (New England Biolabs, Beverly, MA). An MBP fusion with the EC3 domain of PCDH4 (MBP-PCDH4) was made similar to MBP-PCDH3.
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Lysate Preparation and Western Blotting
All lysate preparation steps were performed at 4°C. Triton X-100 soluble and insoluble testis fractions were prepared according to Maekawa et al. [15]. Testes from postnatal days 7, 21, 28, 40, and adult (six animals at Day 7 and two animals at all other time points) were decapsulated, pooled, weighed, and homogenized with three volumes of Triton X-100-containing lysis buffer (50 mM Tris, pH 7.5, 1% Triton X-100, 5 mM EDTA, 1 mM MgCl2) containing Complete EDTA-free Protease Inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) by 10 strokes of a glass Dounce homogenizer. Lysates were centrifuged at 1000 x g for 5 min to pellet cellular debris, and supernatants were centrifuged at 10 000 x g for 10 min. The resulting pellets (TX-100 insoluble fractions) were washed once with lysis buffer and solubilized in a volume of sample buffer equal to the supernatants (TX-100 soluble fractions). For adult rat cauda epididymal sperm lysates, sperm were isolated using a protocol adapted from Chayko et al. [16]. The cauda epididymis was minced with a razor blade and rotated in 1 ml of PBS for 15 min at room temperature. After settling for 1 min, the supernatant was centrifuged at 400 x g for 10 min at room temperature. From the resulting pellet, sperm were fractionated into TX-100 soluble and insoluble fractions as for decapsulated testes. Protein concentrations of the TX-100 soluble fractions were determined via Bio-Rad DC protein assay (Bio-Rad, Hercules, CA).
Western blotting was performed as previously described [17]. For testis and sperm lysates, 20 µg of TX-100 soluble protein or an equal volume of TX-100 insoluble protein was submitted to SDS-PAGE using a 7% gel. For Western blotting of MBP fusion proteins, 10 ng of protein was electrophoresed through a 10% gel. Following transfer to Immobilon-P membranes (Millipore Corp., Bedford, MA), membranes were probed with the following primary antibodies: 2 µg/ml affinity-purified rabbit anti-PCDH3; 1 µg/ml mouse anti-N-cadherin (clone 32, BD Transduction Laboratories, San Diego, CA); mouse anti-ß-catenin diluted 1:1000 (clone 6F9, Sigma); or 1 µg/ml mouse anti-p120 catenin (clone 8D11) [18]. As a negative control, the primary antibody was omitted or normal IgG was substituted. Secondary antibodies were horseradish peroxidase-coupled sheep anti-mouse IgG (NA 931; Amersham Pharmacia Biotech, Piscataway, NJ) or horseradish peroxidase-coupled donkey anti-rabbit IgG (NA 934; Amersham), both diluted 1:2500.
Acrosome Reaction
For the acrosome reaction, adult rat cauda epididymal sperm were isolated by puncturing the cauda epididymis several times with a 23-gauge needle and incubated for 45 min at 37°C with 5% CO2 in capacitation media (mTALP) [19]. The capacitation media (mTALP) contained 100.3 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 1.8 mM CaCl2, 4.3 mM glucose, 0.2 mM penicillin-G, 0.1 mM sodium pyruvate, 9 mM sodium lactate, 300 mg/ml bovine serum albumin, 0.3 mM NaH2PO4, and 35.7 mM NaHCO3. Epinephrine and hypotaurine were added to 1 and 40 mM, and sperm were incubated for another 3 h as before. To one aliquot, calcium ionophore A32187 was added to 10 µM. Following a 60-min incubation, sperm were processed for immunostaining. All reagents were obtained from Sigma. A large increase in acrosome-reacted sperm was noted with calcium ionophore treatment (data not shown).
Immunostaining
Immunostaining was performed as previously described [17]. Testes were frozen in liquid nitrogen, and 7 µm cryosections were dried onto positively charged slides. For all experiments except actin colocalization, cryosections were fixed with -20°C acetone for 3 min prior to blocking. For actin colocalization, cryosections were fixed with 4% paraformaldehyde for 15' at room temperature followed by -20°C acetone for 3 min. To immunostain cauda epididymal sperm, isolated sperm were air-dried onto a positively charged slide prior to fixation with -20°C acetone for 3 min. After fixation and blocking, samples were incubated with primary antibodies for 1 h at room temperature. After washing, secondary antibodies were applied for 45 min at room temperature. Primary antibodies and final concentrations were as follows: affinity purified rabbit anti-PCDH3 at 20 µg/ml; mouse anti-plectin (clone 7A8; Sigma) diluted 1:1000; mouse anti-flotillin-2 (clone 29; BD Transduction Laboratories, San Diego, CA) at 1 µg/ml, and rabbit anti-pericentrin (Covance Inc., Princeton, NJ) diluted 1:250. Cryosections immunostained with PCDH3 antibodies at 1 µg/ml still showed bright immunofluorescence associated with the spermatid flagellum and acrosome, but circular intercellular bridge immunostaining was absent (data not shown). Secondary antibodies and dilutions were Alexa488-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR) and Alexa594-conjugated goat anti-rabbit (Molecular Probes), both diluted 1:500. F-actin was detected with Alexa488-conjugated phalloidin (Molecular Probes) present in the secondary antibody solution and diluted 1:40. As negative controls, the following were performed: 1) preabsorption of the antibody with antigen prior to use; 2) omission of the primary antibody; and 3) substitution of normal rabbit IgG (product number I-1000; Vector Laboratories Inc., Burlingame, CA) at an identical concentration. For preabsorption experiments, affinity purified PCDH3 antibodies were incubated overnight at 4°C with a 10-fold molar excess of MBP-PCDH3 or MBP-PCDH4 in PBS or PBS alone. The acrosome was identified with Alexa488-conjugated peanut lectin (Molecular Probes) added to the secondary antibody solution and used at 25 µg/ml [20]. DNA was localized by incubating samples for 5 min with 50 ng/ml Hoescht 33258 (Sigma). Samples were viewed with epifluorescence using Nikon Eclipse E800 (Nikon, Tokyo, Japan) or Zeiss Axiovert 200 (Carl Zeiss, Oberkochen, Germany) microscopes, and digital images were captured with a Spot RT camera (Diagnostic Instruments Inc., Sterling Heights, MI). Seminiferous tubule staging criteria were based upon the tubule location and morphology of elongate spermatid heads [17].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 Antibody Characterization and Western Blotting
PCDH3 was chosen as the first protocadherin to study in testis for two reasons: its mRNA expression increased when elongating spermatids were produced in postnatal testis, and the coding sequence for a unique antigenic region had been almost completely identified [9]. These data suggested that PCDH3 was produced by germ cells and that expression of a PCDH3-specific antigen would be relatively simple. For the antigen, a nearly complete cadherin repeat (EC3) from PCDH3 was chosen (Fig. 1). By Western blot, affinity purified PCDH3 antibodies detected a PCDH3 fusion protein but not a PCDH4 fusion protein containing the homologous EC3 region (Fig. 2A). Since PCDH4 shows the highest amino acid identity to PCDH3 among the -protocadherin family, these data provided evidence that PCDH3 antibodies were isoform-specific for PCDH3.
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Classic cadherin-based adhesion junction components are poorly solubilized by buffers containing Triton X-100 because of their cytoskeletal attachment [21]. To determine if testicular PCDH3 showed a similar insolubility, testes from Postnatal Day 23 were extracted with TX-100 and separated into soluble and insoluble fractions, and these fractions were immunoblotted for PCDH3 as well as components of testicular adhesion junctions [10, 17]. Although the adhesion junction components N-cadherin, ß-catenin, and p120 catenin were distributed in both the soluble and insoluble fractions, PCDH3 was exclusively found in the soluble fraction (Fig. 2B). PCDH3 antibodies detected a single protein of approximately 115 kDa in testis extracts, a molecular weight consistent with the expected molecular weight of -protocadherins based upon cDNA sequence [14]. This PCDH3 solubility pattern was observed throughout postnatal testis development (Fig. 2C). These data suggested that PCDH3 was not present at any of the numerous adhesion junctions found between cells of the testis. As described below, PCDH3 antibodies immunostained sperm isolated from the cauda epididymis. Similar to testis extracts, cauda epididymal sperm extracts showed a single 115 kDa band in the TX-100 soluble fraction after immunoblotting with PCDH3 antibodies (Fig. 2D).
PCDH3 Immunostaining Specificity
To determine the subcellular distribution of PCDH3 in rat testis, fresh frozen cryosections were immunostained with affinity-purified PCDH3 antibodies. Immunostaining specificity was assayed by preabsorbing the antibodies with either MBP-PCDH3 or MBP-PCDH4 recombinant proteins, omission of the primary antibody, or substitution of normal IgG for the primary antibody (Fig. 3). Adult testis cryosections immunostained with PCDH3 antibodies preabsorbed with either buffer alone or MBP-PCDH4 displayed identical results: immunostaining was seen in numerous areas of the seminiferous epithelium, including the flagellum and head of elongating spermatids, circular structures in the apical epithelium, and extremely bright elements in the basal half of the epithelium (Fig. 3, A and B). In contrast, antibodies preabsorbed with MBP-PCDH3 produced only faint background immunostaining of adult testis cryosections (Fig. 3C). Compared with PCDH3 antibodies, normal rabbit IgG as the primary antibody generated weak immunostaining of flagella, elongate spermatid heads, and what may be germ cell Golgi complexes (Fig. 3D). These data demonstrated that the PCDH3 immunostaining was specific and provided evidence that the antibodies were detecting PCDH3, rather than a different PCDH family member in the immunostaining protocol.
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Ectoplasmic Specializations and Germ Cell Intercellular Bridges
PCDH3 immunostaining of the elongating spermatid head and the circular nature of the apical pattern suggested localization to ectoplasmic specialization junctions between Sertoli cells and elongating spermatids and to intercellular bridges between elongating spermatids, respectively. Since actin is a component of both ectoplasmic specializations and germ cell intercellular bridges [22, 23], the codistribution of actin and PCDH3 was determined in adult rat testis cryosections. At the elongate spermatid head, actin and PCDH3 had closely apposed immunostaining patterns (Fig. 4A). In some cryosections, however, actin and PCDH3 exhibited clearly distinct patterns surrounding the elongate spermatid head (Fig. 4B). In these cases, PCDH3 immunostaining appeared most intensely in the caudal region of the elongate spermatid head, whereas actin-positive ectoplasmic specializations were concentrated in the apical area. Similarly, actin-containing ectoplasmic specialization junctions between Sertoli cells were negative for PCDH2 (data not shown). From these data, it was concluded that PCDH3 was not a component of ectoplasmic specializations. The circular PCDH3-positive structures in the apical epithelium colocalized precisely with actin (Fig. 4A). These structures were located apical to nonelongate spermatid germ cells, as indicated by localizing germ cell nuclei with Hoescht 33258 (Fig. 4A). In addition, PCDH3-positive circular structures were immunostained with antibodies against plectin (Fig. 4C), a cytoskeletal protein [24]. Since previous data has shown that these plectin-containing structures are associated with elongate spermatid intercellular bridges and actin is also a component of elongate spermatid intercellular bridges [25], it was concluded that PCDH3 is localized to intercellular bridges between elongating spermatids. This conclusion was corroborated further by the diameter of PCDH3 circular structures (approximately 2 µm) matching that of rat elongate spermatid intercellular bridges [22]. The colocalization of plectin and PCDH3 at elongate spermatid intercellular bridges was observed consistently throughout the seminiferous epithelial cycle (Fig. 5). Although PCDH3- and plectin-positive spermatid intercellular bridges were observed in stage VIII tubules close to round spermatids (Fig. 5, A and A'), these may represent degenerating intercellular bridges between elongating spermatids. In approximately stage IX tubules, intercellular bridges located near spermatids were weakly positive for PCDH3 and plectin (data not shown), suggesting that spermatid intercellular bridges acquired both PCDH3 and plectin at the transition from round to elongating spermatids.
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Acrosomal Area
Because PCDH3 did not colocalize with ectoplasmic specializations, elongate spermatid head-associated PCDH3 immunostaining may have represented an acrosomal localization. In some seminiferous tubule cryosections, the acrosome clearly was visible by phase contrast microscopy, and PCDH3 was distributed precisely with the phase-dense acrosome (Fig. 5, A and A''). The presence of acrosome-associated PCDH3 was first observed in approximately step-8 spermatids and continued throughout subsequent steps of spermiogenesis (Fig. 5). The intensity of PCDH3 immunostaining within different acrosomal regions appeared to vary during spermiogenesis (Figs. 4 and 5). Localization to the acrosome was explored further by immunostaining mature sperm isolated from the cauda epididymis (Fig. 6A). In these samples, the sperm head was positive for PCDH3, and colocalization with sperm nuclei showed that PCDH3 was associated with the acrosome, instead of the entire nuclear surface. Cauda sperm immunostained with normal rabbit IgG displayed a relatively weak acrosomal signal (Fig. 6B); unlike the PCDH3 pattern, this signal appeared as a brighter streak on the convex surface of the sperm head, similar to peanut lectin histochemistry (Fig. 7A'). In acrosome-reacted sperm, PCDH3 remained associated with the sperm head, but the immunostaining signal was greatly diminished and concentrated toward the anterior portion of the head (Fig. 7).
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Classic Cadherin-Based Adhesion Junctions
To determine if PCDH3 localized to adhesion junctions containing classic cadherins, PCDH3 and plectin colocalization was performed. Previous data from our laboratory and others have shown that plectin is a component of desmosome-like, classic cadherin-based adhesive junctions between Sertoli and nonelongate germ cells [17, 25]. In all stages of the seminiferous cycle, plectin-positive, classic cadherin-based adhesion junctions were negative for PCDH3 immunostaining (Figs. 4C and 5A', D', and E').
Flagella
PCDH3 localization to the developing flagellum showed a stage-dependent pattern. Immunostaining was weak to non-existent in step-13 spermatid flagella (Fig. 5B); however, step-16 spermatids showed positive PCDH3 immunostaining in both the neck and flagellum (Fig. 5C). At this developmental time point of spermiogenesis, PCDH3 was distributed throughout the flagellum, and neck immunostaining was strong and punctate. The intensity of step-17 spermatid flagellar immunostaining increased compared with earlier time points but was similar in morphology, and the spermatid neck remained PCDH3-positive (Fig. 5D). By step 19 of spermiogenesis, the spermatid neck was negative, and a clear redistribution of PCDH3 immunostaining to the distal end of the flagellum was observed (Fig. 5E). This pattern reflected that found in cauda epididymal sperm (Fig. 6), suggesting a localization of PCDH3 to the flagellar principal piece. Unlike PCDH3 antibodies, normal rabbit IgG produced a dim fluorescence pattern throughout the flagellum length (Fig. 6B).
Lipid Rafts and Centrosomes
Previously, it was hypothesized that protocadherins, including -protocadherins, localized to plasma membrane lipid rafts [26]. To determine if PCDH3 was present in testicular lipid rafts, rafts were isolated from adult rat testis using the common technique of flotation in a sucrose gradient following Triton X-100 extraction [27]. Using Western analysis, testicular lipid rafts were found to contain abundant flotillin-2, a known lipid raft component [28]; however, the vast majority of PCDH3 was detected in nonraft fractions (data not shown). To extend these results, PCDH3 and flotillin-2 were colocalized in rat testis cryosections. In the seminiferous epithelium, the basal, punctate PCDH3-positive structures colocalized invariably with flotillin-2 immunostaining (Fig. 8, A and A'). Recently, flotillin-2 was found at centrosomes in peripheral blood cells [29]. In the seminiferous epithelium, the intense, punctate flotillin-2 immunostaining colocalized with antibodies against the centrosomal protein pericentrin (Fig. 8, B and B'). From these data, it was concluded that PCDH3 is associated with centrosomes but not lipid rafts in testis, and the basal location of these elements in the seminiferous epithelium suggested an association with centrosomes of spermatogonia and spermatocytes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3) at nonjunctional sites within the rat seminiferous epithelium, indicating that testicular classic cadherins and protocadherins are functionally divergent.
Testicular adhesion junctions are diverse in both morphology and protein content [10, 11]. Since a similarly diverse set of cadherins (both classic cadherins and protocadherins) were expressed in testis at the mRNA level, it was hypothesized that protocadherins would localize to testicular adhesion junctions [9]. This hypothesis is supported by localization of certain non--protocadherins to intercellular junctions in vitro and localization of -protocadherins to the neuronal synapse in vivo [4, 5, 7]. Despite localization to cell contact sites in some nontesticular experimental systems, protocadherins generally show weak, if any, cell adhesion activity using standard in vitro adhesion assays [5]. In contrast to the hypothesized localization of PCDH3 to testis adhesion junctions, two sets of data showed that PCDH3 is not functioning as a cell adhesion protein in testis. First, PCDH3 was not found in testis detergent insoluble extracts containing known junctional components (classic cadherins and catenins). Similarly, PCDH3 immunostaining did not colocalize with markers of desmosome-like adhesion junctions between Sertoli and germ cells (identified by plectin antibodies) or actin-containing ectoplasmic specialization adhesion junctions. Although the localization of other -protocadherins has not been determined in testis, these data suggest that -protocadherins do not mediate cell adhesion in the testis.
Despite these results, PCDH3 was localized to areas crucial for intercellular communication (Fig. 9). One such site was intercellular bridges between elongating spermatids. Germ cell intercellular bridges form from incomplete cytokinesis and function as conduits of mRNA and protein between syncytial germ cells [30–32]. It is believed that bridges synchronize maturation within a germ cell syncytium and make possible the movement of factors between haploid spermatids, making them functionally diploid. The following data indicated that PCDH3 was a component of these bridges: 1) PCDH3 immunostaining colocalized with both plectin and actin, markers of elongate spermatid bridges [22, 25], and 2) the circular nature and size (diameter of approximately 2 µm) of these structures coincided with the known morphology of elongate spermatid bridges [33]. Although all nonstem germ cells are connected by intercellular bridges, PCDH3 immunostaining was not associated with bridges between spermatogonia, spermatocytes, or round spermatids during the early steps of spermiogenesis. A similar result was obtained with plectin immunostaining [25]; furthermore, intercellular bridges between elongate spermatids differ in size and morphology compared with intercellular bridges between less-mature germ cells [33]. Although the appearance of PCDH3 immunostaining at intercellular bridges coincided with changes in protein components and morphology, any functional relationship is unknown.
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The properties of cadherin extracellular domains suggest a role for PCDH3 in the organization of the intercellular bridge. Like classic cadherin-based adhesion junctions, intercellular bridges contain an electron-dense submembrane plaque, likely composed of numerous proteins forming a large complex [30]. Prior to interaction on an apposing cell, classic cadherins form lateral dimers on the same cell surface mediated by their extracellular domains [34, 35]. By analogy, PCDH3 extracellular domains may form lateral dimers or multimers and act cooperatively with intracellular binding partners to generate a stable intercellular bridge structure. No PCDH3 immunostaining was detected at cytokinetic furrows in rat Sertoli and kidney cell lines (data not shown), suggesting that PCDH3 is not a general component of the cytokinetic furrow. Because PCDH3 was not detected at nonspermatid germ cell intercellular bridges, perhaps one of the numerous other protocadherins expressed in testis performs a similar function at these intercellular bridges.
Another site of intercellular interaction where PCDH3 immunostaining was detected is the acrosomal area. Although a change in immunostaining intensity across different regions of the acrosome during spermiogenesis was noted, the significance of this change is unclear. Acrosome-associated PCDH3 immunostaining persisted in sperm isolated from the cauda epididymis, suggesting a function for PCDH3 in sperm/egg interactions or the acrosome reaction during fertilization. In acrosome-reacted sperm, PCDH3 immunostaining was present (although diminished), suggesting that PCDH3 is localized to both the inner and outer acrosomal membranes. A series of binding events between the sperm and egg surfaces occurs, leading to fusion of the two cells, and the molecular mechanisms of these interactions are still being elucidated [36]. It is hypothesized that PCDH3 functions in one or more of these events, and studies examining the ultrastructural localization of PCDH3 within the acrosomal area in addition to genetic manipulations will be needed to ascertain the role of PCDH3 in fertilization.
PCDH3 immunostaining of the sperm tail implies a role in sperm motility. During spermiogenesis, the pattern of PCDH3 immunostaining changed from a positive signal over the entire flagellum through step 17 to being restricted to the distal region of the flagellum at step 19 and in epididymal sperm. This changing pattern has been observed for other flagellar plasma membrane proteins and is likely due to movement of the annulus from the spermatid neck region to the junction between the flagellar midpiece and principal piece [37]. During fertilization, vigorous flagellar movements termed hyperactivation are induced, which facilitate penetration of egg investments such as the cumulus and zona pellucida [38]. Hyperactivation involves transduction of an extracellular signal via a plasma membrane component [39–41]. Since -protocadherin cytoplasmic domains bind the signaling protein kinase fyn in brain [7], flagellar-associated PCDH3 may be functioning as a receptor involved in hyperactivation.
Although PCDH3 was localized to the germ cell centrosomal area and spermatid neck, the significance of these results is difficult to determine. Because the PCDH3 protein structure predicts a transmembrane protein, it is likely that PCDH3 is a component of membranous structures juxtaposed to the centrosome, and not the centrosome itself. In model cells, endocytotic vesicles congregate at the centrosome, suggesting that centrosomal PCDH3 may represent a concentration of plasma membrane-derived endocytotic vesicles near the centrosome [42].
In conclusion, PCDH3 may function in generating stable germ cell intercellular bridges or in one of many events related to fertilization, including sperm/egg interactions and sperm hyperactivation. In addition, our results, in combination with those previously published, show that PCDH3 and classic cadherins are components of distinct structures within the testis, indicating unique functions for classic cadherins and -protocadherins in spermatogenesis. Because numerous additional protocadherins are expressed in testis, including ß- and 
-protocadherins, it will be important to examine if other protocadherins share properties similar to PCDH3. Based on the results presented here, protocadherins may perform a diverse, protocadherin-specific set of functions in spermatogenesis and fertilization.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Kamin J. Johnson, CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park, NC 27709. FAX: 919 558 1300; kjohnson{at}ciit.org
3 Current address: CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park, NC 27709
Received: 31 July 2003.
First decision: 23 August 2003.
Accepted: 23 September 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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