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a Department of Environmental Toxicology, University of California, Davis, California 95616
b Department of Anatomy, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cytoskeletal structures in the mammalian testis are involved in many processes including Sertoli cell transport functions as well as germ cell mitosis/meiosis, nuclear shaping, and flagella formation. The Sertoli cell cytoskeleton is remarkably complex and morphologically related to a number of events involving spermatogenic cells. One of the more dramatic of these events is the movement or translocation of spermatids to the base and then back to the apex of the epithelium. This change in position occurs after spermatids have acquired an elongate shape and while they occur within, and are attached to, apical invaginations (crypts) of Sertoli cells. The physiological significance of this translocation event is not known. The process may serve to increase the amount of surface contact for exchange between Sertoli cells and spermatids, or it may simply function to increase support for spermatids during the elongation phase of spermiogenesis. Whatever the function, the mechanism for this translocation event has been proposed elsewhere [14–16] to involve the movement of unique intercellular adhesion plaques in Sertoli cells along adjacent microtubule tracts.
In areas of Sertoli cell crypts attached to the heads of elongate spermatids occurs a unique class of actin-related adhesion junctions termed ectoplasmic specializations [14]. These structures each consist of the plasma membrane in regions adherent to the spermatid head, a cistern of endoplasmic reticulum, and an intervening layer of hexagonally packed actin filaments [15]. The three elements of the plaque occur as a structural unit that remains attached to the spermatid when the latter is mechanically separated from the epithelium [17–19]. In vivo, the endoplasmic reticulum component of the plaque is related, on its cytoplasmic face, to microtubules [14].
Microtubules are prominent elements of the Sertoli cell cytoskeleton. They surround apical crypts containing elongate spermatids [20, 21], are generally arranged parallel to the axis of spermatid translocation, and have their positive ends positioned at the base of the epithelium [16].
Our working hypothesis of spermatid translocation is that motor proteins are anchored to the cytoplasmic face of the endoplasmic reticulum component of ectoplasmic specializations and that they move the junction plaques, and therefore the attached spermatids, along Sertoli cell microtubule tracts. The nature of the putative motor proteins involved in transport of the spermatid/junction complexes along microtubules is as yet unknown.
In this study, commercially available dynein and kinesin antibodies (BabCo, Berkeley, CA) were used to screen for immunoreactive motor proteins in testis tissue sections and in epithelial fragments. The cytoplasmic dynein antibody used was raised against the intermediate chain (IC74). Kinesin antibodies tested included those raised against kinesin heavy chain (SUK-4), kinesin light chain, kinesin-II, and a kinesin-related protein (HIPYR). Since preliminary data indicated that only the IC74 dynein antibody, the kinesin-II antibody, and the antibody to the kinesin heavy chain (previously immunolocalized to the manchette [9] and trans-Golgi network [10]) produced significant immunofluorescence, the work reported here involves only the former two antibodies, for which testis immunostaining has not previously been reported.
To explore further the possibility that motor proteins could be associated with the spermatid/junctional complexes, gelsolin, an actin-severing enzyme that has been used previously to remove actin filaments from permeabilized cells [22, 23], was used to disrupt the junction plaques, and low-speed centrifugation was used to remove spermatid heads from solution. We reasoned that if motor proteins were indeed associated with junction plaques, then the amount of these motors in supernatants collected from spermatid/junction complexes in which the junction plaques were artificially disassembled should be increased relative to controls. Supernatants from gelsolin-treated and control preparations were compared, on immunoblots, for their reactivity with antibodies to the motor proteins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals used in this study were mature Sprague-Dawley rats that ranged in weight from 240 g to 390 g. While animals were under deep halothane anesthesia, their testes were removed for fixation or epithelial isolation, and then the animals were killed.
Reagents
Most reagents were obtained from Sigma-Aldrich (Oakville, ON, Canada) unless otherwise indicated. Fluorescent phallotoxins were obtained from Molecular Probes (Eugene, OR). Paraformaldehyde was purchased from Fisher Scientific (Vancouver, BC, Canada).
Immunological reagents were obtained from two sources. Monoclonal antibodies to the intermediate chain of cytoplasmic dynein (clone 74.1) and kinesin II (clone K2.4) were obtained from Berkeley Antibody Company (Berkeley, CA). The antibodies were stored at concentrations of 1.0 mg/ml and 5.0 mg/ml, respectively. Secondary antibodies for immunofluorescence were obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA) and consisted of goat anti-mouse IgG conjugated to either Texas Red or fluorescein.
Preparation of Tissue for Immunofluorescence
The spermatic arteries of isolated testes were cannulated using 26-gauge needles attached to a gravity-fed perfusion apparatus, and the organs were perfused for 1–2 min with warm (33°C) PBS (150 mM NaCl, 5 mM KCl, 3.2 mM Na2HPO4, 0.8 mM KH2PO4) and then perfused for 30 min with fixative (3% paraformaldehyde, PBS, pH 7.3, 33°C). After this, the testes were washed with PBS by perfusion for an additional 30 min.
Perfusion-fixed testes to be used for sectioning were frozen in OCT compound (Fisher Scientific). Sections of 5–10-µm thickness were cut on a cryostat, collected on polylysine-coated slides, immediately treated with cold acetone (-20°C) for 5 min, and then air-dried.
Fixed testes to be used for collecting epithelial fragments were decapsulated, and then the seminiferous tubule masses were cut into small pieces with scalpel blades. The pieces were aspirated through 18-gauge and 21-gauge syringe needles and then sedimented at low speed in a clinical centrifuge. Fragments in the upper layer of sedimented material were collected with a pipette and attached to polylysine-coated slides. Excess fluid was removed, and the slides were immediately treated with cold acetone (-20°C) for 5 min and air-dried.
Immunofluorescence
Air-dried sections and epithelial fragments were rehydrated with TPBS (PBS, 0.05% Tween-20, 0.1% BSA) containing 5% normal rabbit serum (NRS) for 20 min at room temperature. After this, the material was incubated for 1 h at 37°C with either a 1:250 dilution of the dynein antibody or a 1:50 dilution of kinesin II antibody in TPBS containing 1% NRS. The sections were washed 3 times (10 min each wash) with TPBS and then incubated for 1 h with a 1:100 dilution of goat anti-mouse IgG conjugated to either fluorescein isothiocyanate or Texas Red. Sections were washed 3 times with TPBS and then mounted in Vectashield (Vector Labs., Burlingame, CA). In some cases, the second wash contained rhodamine or Oregon green phalloidin to stain for filamentous actin in junction plaques. Slides were observed and photographed on a Zeiss Axiophot microscope (Carl Zeiss, Inc., Thornwood, NJ) fitted with filter sets for detecting rhodamine and fluorescein. Negatives were scanned into digital format, and the images were manipulated using Photoshop 4 (Adobe Systems Incorporated, San Jose, CA) without altering the integrity of the data.
Controls for specificity of staining included replacing the primary antibody with normal mouse IgG used at the same concentration as the antibody, replacing primary antibody with buffer alone, and replacing both the primary and secondary antibodies with buffer alone.
We also checked the IC74 and kinesin II antibodies for reactivity on immunoblots of isolated seminiferous epithelium. Epithelium, collected over a period of approximately 30 min as described elsewhere [19], was sedimented at low speed in a clinical centrifuge. The supernatant was discarded, and the pellet was resuspended in 250–500 µl of PEM/250 (80 mM PIPES, 1.0 mM EGTA, and 1.0 mM MgCl2 [adjusted to pH 6.8 with KOH] and containing 250 mM sucrose) with and without protease inhibitors. To this was added 500 µl of treatment buffer (0.125 M Tris-Cl pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol). Samples were sonicated for 2 sec at 65 kHz and then aliquoted and stored at -20°. Aliquots at a protein concentration of approximately 1 mg/ml were processed for SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane using standard techniques, blocked overnight in buffer (0.05 Trizma-base adjusted to pH 7.5 with HCl, 0.25 NaCl, 0.1% Tween 20) containing 10% skim milk and 4% BSA, and then reacted with the dynein antibody at a 1:1000 dilution of the 5 mg/ml stock. Controls included replacing the primary antibody with normal mouse IgG at the same concentration or with buffer alone.
Gelsolin Digestion of Testicular Fractions Enriched for Elongate Spermatids with Attached Sertoli Cell Junction Plaques
Testicular fractions enriched for spermatid/junction complexes were collected generally as described elsewhere [19,24]. In brief, the protocol involved manually stripping epithelium from tubule walls and then mechanically fragmenting the epithelium by aspiration through a fine-bore pipette tip. The epithelial fragments were then loaded onto step sucrose gradients and centrifuged, and fractions enriched in spermatids with attached junction complexes were collected at one of the interfaces.
For each of three experiments, and to ensure sufficient material for Western blot analysis, spermatid/junction complexes were collected and pooled from three animals. Three sucrose gradients were run for each animal, and material from the appropriate interfaces was pooled and stored on ice as wet pellets. When pellets had been obtained from all three animals, the pellets were resuspended in MES (2-(4 morpholino)-ethane-sulfonic acid) buffer (50 mM MES-KOH pH 6.3, 2 mM MgCl2, 0.1 mM CaCl2 0.5 mM dithiothreitol [DTT]) [23] and pooled. The spermatid/junction complexes were washed once in MES buffer, centrifuged at 4000 rpm for 2 min in an Eppendorf bench-top centrifuge (Hamburg, Germany), and resuspended in MES buffer. Two equal aliquots of cell suspensions were taken and again washed in MES buffer. One pellet was resuspended in MES buffer, and the other pellet was suspended in an equal volume of gelsolin dialysate (0.4 mg/ml gelsolin in MES buffer). Samples were incubated for 1 h at room temperature with occasional gentle agitation. After incubation, small aliquots (2 µl) of the MES control and gelsolin-treated cells were placed on polylysine-coated slides and stained with Oregon green-conjugated phalloidin. The slides were evaluated by fluorescence microscopy for the status of actin filaments in spermatid-associated Sertoli cell junction plaques. In this material, it was noted that the tails had separated from many of the spermatids. Remaining samples were centrifuged (4000 rpm for 2 min in Eppendorf centrifuge) to sediment out intact spermatids and spermatid heads, and the supernatants were frozen at -70°C in 10-µl aliquots.
Gel Electrophoresis and Immunoblots
Before electrophoresis, protein samples were diluted 1:1 with treatment buffer, boiled for 2 min, and then loaded into wells. Proteins were separated on 10% polyacrylamide gels [25] and transferred overnight to PVDF membranes. Blots were washed twice (10 min each wash) in TTBS (100 mM Tris-Cl pH 7.5, 0.9% NaCl, 0.1% Tween 20) and then blocked for 2 h in 10% nonfat dry milk plus 4% BSA in TTBS. The two 10-min washes were repeated, after which the blot was incubated for 1 h with freshly prepared primary antibodies (1:1000 dilution in TTBS) to cytoplasmic dynein and kinesin II. Mouse IgG controls at equivalent protein concentrations were run in parallel. Blots were washed (3 x 5 min, 3 x 10 min) and then treated with avidin-biotin-peroxidase complex (ABC) reagent (Vectastain ABC kit, Vector Labs) made up in a high-salt (0.5 M NaCL) TTBS solution for 30 min. Final washes were in TBST (3 x 5 min) and TBS (100 mM Tris-Cl pH 7.5, 0.9% NaCl) (3 x 10 min). Proteins were detected by chemiluminescence using ECL reagent (Amersham, Piscataway, NJ). Molecular weights were approximated against biotinylated protein molecular weight markers (Vector Labs).
Immunoblots were scanned into digital format and manipulated, using Adobe Photoshop 4, without altering the integrity of the data.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On immunoblots of seminiferous epithelium, antibodies to IC74 and kinesin-II reacted predominantly with bands of the appropriate molecular weight for each of the motor proteins (Fig. 1). When gels were overloaded with protein or when blots were overexposed, the IC74 antibody also reacted weakly with two minor bands (at a lower molecular mass than 74 kDa) that did not convincingly react with normal mouse IgG in control blots. Kinesin-II appeared on the blot as a characteristic doublet. When gels were overloaded with protein, minor bands also appeared on the blot; however, these bands were reactive with normal mouse IgG.
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IC74 Immunoreactivity Was Concentrated in Sertoli Cell Regions Surrounding Apical Crypts
The probe for the intermediate chain of cytoplasmic dynein (IC74) reacted intensely with Sertoli cell regions surrounding apical crypts. This was particularly evident in fixed frozen sections and at stages of spermatogenesis when elongate spermatids were positioned deep within the epithelium (Fig. 2). Intense tracts of fluorescence followed the contour of adjacent spermatid heads and the related junction plaques (arrowheads in Fig. 2, A–E). Although the dynein probe was reactive with regions related to spermatid heads, the fluorescence signal was not restricted to these sites. Distinct tracts of fluorescence often extended beyond areas containing junction plaques into more apical regions of the Sertoli cell cytoplasm (arrows in Fig. 2, A–C and E), and a diffuse fluorescence often was present in the basal half of Sertoli cells.
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Immunoreactivity in Sertoli cell cytoplasm adjacent to spermatid heads was dramatically evident in epithelial fragments (Figs. 3 and 4). The cytoplasm associated with junction plaque regions was strongly immunoreactive when clusters of spermatids were observed (Fig. 3, A–C) and was also evident in Sertoli cell regions surrounding single spermatids. Although this fluorescence was diffuse in many cases (Fig. 3, D and E), the pattern was more linear in others (Fig. 3, F–H). This linear pattern of dynein immunoreactivity is particularly striking in Sertoli cell regions associated with the spermatid head shown in Figure 4, A and B, where the linear tracts of fluorescence are oriented parallel to the long axis of the adjacent head. Interestingly, the probe was also very reactive with Sertoli cell regions surrounding tubulobulbar processes (asterisk in Fig. 3H).
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Distribution of IC74 Immunoreactivity in Sertoli Cells Changed during Spermatogenesis
The overall pattern of cytoplasmic dynein distribution in Sertoli cells was different in sections of seminiferous tubules at different stages of spermatogenesis. At stages when spermatids were in the early phases of elongation (stage IX, Fig. 5A), the fluorescence signal emitted by Sertoli cells was weak and appeared diffuse. This pattern was in contrast to the signal emitted from germ cells themselves, in which an intense fluorescence was emitted by the manchette. At later stages (stage XII, Fig. 5B), linear tracts of fluorescence became apparent in the cytoplasm of Sertoli cells and were generally oriented parallel to the long axis of the cells. The signal associated with manchettes became less evident and eventually disappeared. The linear tracts of fluorescence in Sertoli cells were well established at stages when spermatids were at their deepest point in the epithelium (stage V, Fig. 5C). At stage VII (Fig. 5D), when spermatids had completed their translocation to the apex of the epithelium, intense fluorescence persisted in Sertoli cell cytoplasm surrounding spermatid heads and was also present more basally in columnar portions of the cells. After sperm release, this fluorescence pattern disappeared.
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Distribution of IC74 Immunoreactivity in Spermatogenic Cells
The immunological probe for the intermediate chain of cytoplasmic dynein reacted with two regions in spermatogenic cells. The first was the manchette (Fig. 6, A and B, Fig. 7, A and B), and the second was an area along the dorsal curvature of the spermatid head. The latter was not consistently evident in sections, but was better resolved in epithelial fragments (Fig. 3C, arrows) and in isolated spermatids (Fig. 7, A and B). This staining was clearly distinct from that in attached or associated Sertoli cell cytoplasm and was present in spermatids where junction plaques had detached. It was present in elongate spermatids but was absent in mature cells. The exact position of this signal within spermatids was not established. Although the IC74 antibody has been shown previously to react with a 67-kDa isoform of the intermediate chain in blots of mammalian flagella [26], staining in sperm tails was inconsistently observed by us and was either weak or not detectable.
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Controls for IC74 Staining
The pattern of fluorescence described above (Fig. 8A) was not present when the primary antibody was replaced by normal mouse IgG (Fig. 8B), nor was it present when the primary antibody was replaced by buffer alone (Fig. 8C). It was also absent in the control for autofluorescence (both the primary and the secondary antibodies replaced by buffer; Fig. 8D).
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Kinesin-II Was Localized in Spermatid Tails
The probe for kinesin-II was reactive only with spermatogenic cells and was concentrated in the tails (Fig. 9). In early elongate spermatids, staining was patchy along the tails (Fig. 9, A and B), while at later stages the signal was more uniformly distributed (Fig. 9, C–H). There also occurred intense foci of fluorescence apparently associated with most proximal tail regions (small arrows in Fig. 9, C and E).
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Controls for Kinesin-II Staining
Specific fluorescence was not observed when the primary antibody was replaced by normal mouse IgG, nor was it observed in other controls (Fig. 10).
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IC74 Was Enriched in Supernatants From Gelsolin-Treated Spermatid/Junction Complexes
In each of the three experiments, actin filaments, present in controls, were virtually eliminated from plaques attached to spermatid/junction complexes incubated in gelsolin (Fig. 11). Significantly, the intermediate chain of cytoplasmic dynein (74 kDa) was obviously and consistently enriched, relative to controls, in supernatants collected from this material after removal of spermatids by slow-speed centrifugation (compare lanes 1 and 2 in each of the dynein immunoblots in Fig. 11).
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The content of kinesin-II (presumably from contaminating tails or protein solubilized from the sperm tails during the protocol) in supernatants was qualitatively much less affected by gelsolin treatment than was dynein (compare lanes 1 and 2 in each of the kinesin-II immunoblots in Fig. 11). Levels of this motor appeared to be noticeably elevated in only one of the three experiments.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results provide a morphological correlate for the high levels of cytoplasmic dynein detected previously in testis [2] and in Sertoli cells [1]. The data indicate that an isotype of cytoplasmic dynein may be present in Sertoli cell regions associated with apical crypts containing attached elongate spermatids. They also support the conclusion that a form of cytoplasmic dynein is most likely related to, but not entirely restricted to, unique intercellular attachment plaques that occur in these regions and that have been proposed to translocate spermatids in the seminiferous epithelium [14–16].
A number of observations indicate that the IC74 antibody used in this study is reasonably specific for the intermediate chain of cytoplasmic dynein when used to probe rat testis. On immunoblots of isolated seminiferous epithelium, the antibody reacted predominantly with a single band at the appropriate molecular mass for the intermediate chain of cytoplasmic dynein (74 kDa). Moreover, a specific pattern of fluorescence was not observed when the antibody was replaced with normal mouse IgG, nor was the pattern observed in the second antibody control or in the control for autofluorescence. In addition, specific staining was observed in association with manchettes, microtubule-rich structures previously shown, using a different antibody, to contain an isoform of cytoplasmic dynein [7–9]. Finally, staining in Sertoli cells occurred in regions known to be rich in microtubules and thought to be sites of active microtubule-based organelle (endoplasmic reticulum, mitochondria, vesicles) motility during spermatogenesis [27].
Although the antibody reacted strongly with a band at the molecular size for the intermediate chain of cytoplasmic dynein on immunoblots, it did react weakly with two minor bands of lower molecular size that could not be convincingly accounted for in normal mouse IgG controls. These bands were of a lower molecular mass than the 67-kDa isoform of the intermediate chain of cytoplasmic dynein known to be present in mammalian flagella [26], which has been demonstrated to cross-react on blots with the 74.1 antibody. These could be proteolytic fragments of IC74. Alternatively, these minor antigens may be responsible for the enigmatic staining associated within dorsal regions of the spermatid head—a location in which microtubules are not known to be present.
In Sertoli cells, intense staining with the IC74 antibody was concentrated in columnar regions of the cell surrounding apical crypts containing attached spermatids. Although results from immunofluorescence and from experiments with gelsolin suggest an association of the immunoreactive protein with unique intercellular adhesion plaques related to spermatid heads, the staining pattern in fixed frozen sections of rat testis indicates that the protein is certainly not restricted to these areas. It occurs diffusely elsewhere in the cytoplasm and in linear tracts along structures, possibly cisternae of endoplasmic reticulum, projecting apically from regions containing the plaques.
There is now a growing body of evidence consistent with the microtubule-based hypothesis of spermatid translocation. In Sertoli cells, microtubules are arranged parallel to the axis of translocation and are morphologically related to the junction plaques attached to spermatid heads [28]. In vitro, exogenous microtubules bind to the endoplasmic reticulum component of the plaques in a nucleotide-dependent fashion [19], and in motility assays, the junction plaques support microtubule movement [24]. Results presented here demonstrate that Sertoli cell regions surrounding apical crypts and containing the unique intercellular adhesion junctions proposed to be related to spermatid translocation are strongly reactive with an immunological probe for the 74-kDa cytoplasmic dynein subunit. Significantly, changes in the distribution of this immunoreactivity during spermatogenesis indicate further that an isoform of dynein may be recruited to these regions just before spermatid translocation. In fixed frozen sections of rat testis, the probe for IC74 accumulated in cytoplasmic areas adjacent to the crypts as spermatids moved deep into the crypts and was dramatically present in these regions just before movement of spermatids apically.
Results of the immunofluorescence experiments, particularly of those involving spermatids mechanically dissociated from the epithelium, and of the gelsolin experiments are consistent with the proposal that an isoform of cytoplasmic dynein is related to the specialized junction plaques associated with spermatid translocation. The finding that staining with the dynein probe occurs in a linear pattern that is in some cases correlated with the distribution of actin filaments within junction plaques is very suggestive of a morphological relationship with the plaques. In testicular sections, staining in regions that are directly associated with elongate and mature spermatid heads, and that label heavily with fluorescent phallotoxins for actin in Sertoli cell adhesion junctions, also is consistent with dynein's localization to junction plaques.
The finding that disassembly of the junction plaques with gelsolin increased the amount of dynein (IC74) detected in supernatants collected from epithelial fractions enriched for spermatid/junction complexes also supports the argument that the motor is located on the plaques; however, the data from these experiments are equivocal. Although less likely than the release of dynein from the actin-containing junction plaques, it is possible that IC74 in the supernatants from gelsolin-treated material was in some way released from contaminating manchettes in gelsolin-treated material. This alternative explanation has some merit because kinesin-II, found in sperm tails, also increases in supernatants collected from material treated with gelsolin, although to a lesser extent and with less consistency than dynein. The presence of kinesin-II in the low-speed supernatants could have been due to contaminating spermatid tails that had separated from the heads and remained suspended in solution, or to protein that had been solubilized from spermatid tails during the protocol. Together with the fluorescence data, results from the gelsolin experiments are generally consistent with the proposal that a dynein is associated with the specialized junction plaques involved with spermatid translocation.
The observation that IC74 co-sediments mainly with dynein heavy chain 1a (DHC1a) but not with DHC1b isoforms [6] suggests that the dynein isotype that may be associated with spermatid translocation is the DHC1a form.
The most likely position of any dynein related to the junction plaques is on the cytoplasmic face of the endoplasmic reticulum component of the structures. The endoplasmic reticulum is the component of each plaque that is related to microtubules in vivo [14] and is the component of the plague that binds microtubules in vitro [19]. Moreover, there is much precedent for motor proteins being related to endoplasmic reticulum in general in cells [29–31]. Localization of the motor at the ultrastructural level is needed to settle this issue and to determine other organelles with which the motor may be associated in Sertoli cells.
As reported for invertebrate sperm [11], kinesin-II in rat spermatids is localized to the flagellum. Striking immunofluorescence was seen in the developing flagella of spermatids when tissue sections were exposed to the kinesin-II antibody. Intensely staining structures observed near the base of the spermatid tails may be the flagellar homologues of basal bodies in cilia; however, this remains to be confirmed electron-microscopically. No immunostaining was apparent until the onset of spermatid elongation, at which time the early flagellar structures showed patchy but distinct staining. In invertebrates, kinesin-II has been implicated in intraflagellar transport and, therefore, in the process of flagellar assembly and maintenance [32]. In addition, KIF3 family members, of which kinesin-II is one, are thought to be involved with transport of a subset of membrane-bounded vesicles in neurons [33]. Although KIF3 occurs at high levels in testis [3], the specific antibody (clone K2.4) used by us to probe rat testis did not detectably react with any structures within Sertoli cells.
Our data provide the first morphological evidence for the presence of kinesin-II in mammalian spermatid tails and for the intermediate chain of cytoplasmic dynein in Sertoli cell regions involved with spermatid transport. Results with the IC74 antibody are generally consistent with the microtubule-based model of spermatid translocation and indicate that at least one of the motor proteins involved in the process may be a cytoplasmic dynein. If involved, because microtubules are arranged with their plus ends at the base of the Sertoli cells [16], this motor would most likely be responsible for the apical movement of elongate spermatids that occurs just before sperm release.
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FOOTNOTES |
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2 Correspondence: Wayne Vogl, Department of Anatomy, 2177 Wesbrook Mall, The University of British Columbia, Vancouver, BC, Canada V6T 1Z3. FAX: 604 822 2316; vogl{at}unixg.ubc.ca
3 These authors contributed equally to this work.
Accepted: December 1, 1998.
Received: October 15, 1998.
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