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a 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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Ectoplasmic specializations in mammals consist of regions of the Sertoli cell plasma membrane that are adherent to spermatid heads, a submembrane layer of actin filaments, and an attached cistern of endoplasmic reticulum [2]. The actin filaments are hexagonally packed [3] and uniformly polar [4], and are not associated with myosin II [5]. Intact ectoplasmic specializations remain attached to spermatids that have been mechanically dissociated from the seminiferous epithelium [6]. This result not only indicates a strong attachment between Sertoli cells and spermatids in regions of the junction plaque, but it is also consistent with the conclusion that the various components of the plaque function as a structural unit.
The observations that microtubules are abundant in Sertoli cell cytoplasm immediately surrounding the junction plaques and that these microtubules are arranged parallel to the direction of spermatid translocation in the epithelium have led to the hypothesis that spermatid translocation may be microtubule-based and involve the presence of motor proteins on the endoplasmic reticulum component of the junction plaques [7]. As the motor proteins move the cistern of endoplasmic reticulum along the microtubules, force is transferred through the plaques to adjacent spermatids, resulting in the observed translocation of these cells through the epithelium (see Fig. 1).
If this hypothesis is true, ectoplasmic specializations should support microtubule transport in vitro. To verify this prediction, we mechanically isolated spermatids with attached junction plaques from the seminiferous epithelium and assayed them for their ability to translocate fluorescently labeled microtubules.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
All animals used in this study were reproductively active Sprague-Dawley rats with an average weight of 372 g. They were obtained either from a colony maintained in the Animal Care Facility at the University of British Columbia or from Charles River (St. Constant, PQ, Canada). The animals were maintained and used in accordance with guidelines established by the Canadian Council on Animal Care.
Chemicals and Reagents
Unless otherwise indicated, all chemicals and reagents used in this study were purchased from Sigma Chemical Co. (St. Louis, MO). Motility kits were obtained from Cytoskeleton (Denver, CO), and the fluorescent phallotoxins and the DiOC6 (3,3'-dihexyloxacarbocyanine iodide) were obtained from Molecular Probes (Eugene, OR). Paraformaldehyde was purchased from Fisher Scientific (Vancouver, BC, Canada). The glutaraldehyde, sodium cacodylate, OsO4, and embedding resins were all obtained from J.B. EM Services (Dorval, PQ, Canada).
The basic working buffer (PEM) consisted of 80 mM PIPES, 1.0 mM EGTA, and 1.0 mM MgCl2 (adjusted to pH 6.8 with KOH). Epithelia were isolated in PEM containing 250 mM sucrose (PEM/250), and motility assays were performed using PEM/250. The various concentrations of sucrose used to make the step gradients were made by mixing appropriate amounts of PEM with a solution containing 60% (w:v) sucrose in PEM. All working solutions contained 10 µg/ml soybean trypsin inhibitor, 0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin, and 0.25 mM PMSF.
Motility buffers were made by adding to PEM/250 the appropriate amount of the required reagents from stock solutions. All motility buffers contained taxol (20.0 µM), antifade (0.1 mg/ml catalase, 0.03 mg/ml glucose oxidase, 10 mM glucose, 0.3% 2-mercaptoethanol), and a nucleotide-replenishing system (50.0 mM phosphocreatine, 10 U/ml creatine phosphokinase). Mg2+-ATP was added to a final working concentration of 5.0 mM. When used for a control, apyrase was added to a final dilution of 10 U/ml in motility buffer not containing any exogenously added ATP. Testicular supernatant was added in the appropriate volumes to bring the final working solution to the required protein concentration, which for our routine assays was 2 mg/ml (S1a).
Preparation of Elongate Spermatids with Attached Junction Plaques
A testicular fraction enriched for elongate spermatids with attached junction plaques was prepared using a protocol similar to one used previously to obtain material for a microtubule binding assay [6]. Animals were anesthetized with halothane, and their testes were removed and decapsulated. The masses of seminiferous tubules and interstitial tissue were placed in a Petri dish containing ice-cold PEM/250 and cut, with scalpels, into pieces. Small segments of seminiferous tubules that were free of interstitial tissue were collected with a pipette and transferred into a second Petri dish containing fresh PEM/250. Using a dissecting microscope fitted with darkfield optics, the seminiferous epithelium was separated from the tubule walls by anchoring the end of a tubule with a fine probe and, with a second probe, squeezing the epithelium out of the free end of the tubule. The isolated epithelium was collected using a pipette and transferred to a centrifuge tube on ice. Epithelium was collected over a period of approximately 30 min. The collected epithelium was centrifuged in a clinical centrifuge at setting 2 for 3 min; then the supernatant was removed and the pellet resuspended in 100–200 µl PEM/250. To fragment the epithelium and mechanically dissociate spermatids from Sertoli cells, the suspension was gently aspirated 5–7 times through a fine-bore pipette.
To obtain a testicular fraction enriched for elongate spermatids with attached junction plaques, suspensions of fragmented epithelia were centrifuged through step sucrose gradients (60%, 55%, 50%, 45%, 40%, 35%, 30% sucrose in PEM) in Ultra-Clear centrifuge tubes (5 x 41 mm) (Beckman, Fullerton, CA). Gradients were centrifuged for 7.5 min at 5000 rpm using a Beckman SW65 Ti rotor fitted with adapters. The band just below the 40–45% interface of each gradient was withdrawn through the wall of the centrifuge tube, using a 23-gauge needle mounted on a syringe. Bands from three tubes were pooled and placed into 1 ml of ice-cold PEM/250. The material was pelleted by centrifugation for 4 min at setting 6 on an Eppendorf (Hamburg, Germany) 5415C centrifuge, then gently resuspended in 50 µl of PEM/250.
Electron Microscopy
Bands withdrawn by syringe from three sucrose gradients were gently ejected into a 1.5-ml Eppendorf tube containing 1 ml of warm (33°C) fixative consisting of 80 mM PIPES, 1.0 mM EGTA, 1.0 mM MgCl2, 0.5% glutaraldehyde, and 2.5% paraformaldehyde (adjusted to pH 6.8 with KOH). The material was mixed by inverting the tube and left at room temperature for 1 h. The cells were sedimented by centrifugation at 11 000 rpm in an Eppendorf centrifuge, and the supernatant was replaced with 0.1 M sodium cacodylate (pH 6.9). Samples were washed twice with fresh buffer and then post-fixed for 1 h on ice in 0.1 M sodium cacodylate containing 1% (w:v) OsO4. Samples were washed three times (10 min each wash) with dH2O and then stained for 1 h with 1% aqueous uranyl acetate. The cells were washed three times with dH2O, dehydrated through a graded concentration series of ethyl hydroxide and then embedded in JEMBED 812 resin (J.B. EM Services Inc., Pointe-Claire, PQ, Canada). Sections were cut, stained with uranyl acetate and lead citrate, and photographed on a Philips 300 electron microscope (Eindhoven, The Netherlands) operated at 80 KV.
Preparation of Microtubules
Rhodamine-labeled microtubules were prepared using reagents from, and exactly as indicated by, Cytoskeleton. After the final centrifugation through the cushion buffer, the microtubules were resuspended in 100 µl of buffer. Before adding an aliquot to the motility chamber, the overall lengths of the microtubules were shortened by aspirating the preparation through a 30-gauge needle and diluting 1:4 with buffer. Preliminary experiments indicated that if microtubules were too long, they could not be resolved in a single plane of focus and their ends would anchor to glass adjacent to the spermatid head.
Preparation of Supernatants
Preparation of testicular supernatants was modeled after protocols used to obtain similar supernatants for motility assays in other systems [8]. For the studies reported here, we chose to use testis as our starting material rather than to try to obtain a pure population of Sertoli cells, mainly because the former was much simpler. Also, we felt that Sertoli cells removed from association with spermatogenic cells (either by culturing or by using Sertoli cell-only testis) might not contain the necessary factors to support motility at the specific sites we were interested in. The junction plaques that we proposed to be involved with spermatid translocation are formed only by morphologically differentiated Sertoli cells at specific times during spermatogenesis and in association with specific stages of differentiating spermatogenic cells.
Supernatants were prepared as follows. Testes from six animals, anesthetized with halothane, were removed and briefly perfused, via the testicular arteries, with PBS to remove blood. The organs were decapsulated, placed in a Petri dish containing ice-cold PEM/250, and cut into small pieces with scalpels. The material was transferred to centrifuge tubes, and the tissue fragments were sedimented at setting 3 for 3–5 min on a clinical centrifuge. The pellets were resuspended in 0.9 vol of homogenization buffer (PEM/250 containing 1.0 mM DTT) and then aspirated 20–30 times, first through a 10-ml pipette and then through an 18-gauge needle on a 10-ml syringe. The resulting material was centrifuged at 6000 x g for 15 min at 4°C, the pellet was discarded, and the supernatant was spun at 200 000 x g for 30 min. The resulting supernatant (S1) was divided into aliquots and stored at -70°C, or processed further as follows. To 1 ml of S1, taxol and GTP were added to final concentrations of 20 µM and 1 mM, respectively. The solution was incubated at 33°C for 30 min and then centrifuged at 40 000 x g for 30 min. The final supernatant (S1a) was stored at -70°C. Protein concentrations in each of the preparations was determined using a Bio-Rad Protein Assay (Bio-Rad, Richmond, CA).
Supernatants were prepared three times during the course of the experiments reported here, and data recorded using each were similar.
Motility Assay
The motility assay was performed using commercially available flow chambers (Cytoskeleton) and a Zeiss Axiophot microscope (Carl Zeiss, Inc., Thornwood, NJ) fitted with a 100x lens and appropriate fluorescence filter sets. A neutral density filter was inserted between the lamp and filter sets to reduce excitatory light levels. Also, an external shutter (Uniblitz Shutter, Vincent Associates, Rochester, NY) was positioned in the same light path and controlled through the camera controller to ensure that excitation of fluorochromes occurred only when data were being recorded. Data were recorded using a CCD camera (Princeton Instruments, Trenton, NJ) through a Power Wave 604/120 computer and IPLab Spectrum software (Signal Analytics Corporation, Fairfax, VA).
For basic assays, motility chambers were prepared as follows. An aliquot (10 µl) of the testicular fraction enriched for elongate spermatid/junction plaque complexes was added to a chamber and then left to sit for 10 min at room temperature for cells to attach to the coverslip. PEM/250 containing casein (15 mg/ml) was drawn into the chamber to remove unbound cells and to block both excess binding sites on the coverslip and nonspecific binding sites on the junction plaques. After 10 min, a 10-µl aliquot of rhodamine-labeled and taxol-stabilized microtubules was drawn into the chamber and left to sit for 10 min for microtubules to bind to the junction plaques. The microtubule-containing buffer was replaced with a 10-µl aliquot of wash buffer (motility buffer not containing ATP or replenishing system), and the chamber was placed on the microscope.
Using phase microscopy, an elongate spermatid was identified in the chamber, and the camera was set, using a script written in IPLab software, to record a data set. Each data set consisted of 80 exposures (fluorescence) of 3 sec each. Exposures were separated by a 3-sec pause, and images were saved directly onto the hard disk of the computer. Total time of each run was recorded; times varied, depending on image size and the time taken for the computer to save each image, between 8 and 11 min. For orientation, the first and last two images of each data set were recorded using phase microscopy. After image 15 was recorded, the motility buffer in the chamber was replaced with the same buffer, but containing ATP. Exchange of the buffer was usually completed between images 17 and 20. The first 15 images served as internal controls for any microtubule movement recorded later in the data set. The data set was animated, using IPLab software, to detect movement.
Data were recorded from five cells from any single epithelial preparation (single animal).
Staining for Filamentous Actin
To label filamentous actin in ectoplasmic specializations attached to spermatids, cells were treated with fluorescent phallotoxins. After a data set was recorded, buffer in the motility chamber was replaced with PEM/250 containing either fluorescein phalloidin, Oregon green phalloidin, or coumarin phalloidin. After 5 min, the chamber was washed once with PEM/250, and a single image was collected using the appropriate filter sets on the microscope. The pattern of actin fluorescence from labeled ectoplasmic specializations in rat is known [10] and can be distinguished easily from actin staining in the spermatogenic cells themselves.
Staining for Endoplasmic Reticulum
Buffer in the motility chamber was replaced with PEM/250 containing 0.5 µg/ml DiOC6 and left to sit for 1–10 min. The chamber was washed once with PEM/250, and as many as five single images were recorded of the staining pattern associated with the spermatids from which motility assay data sets had been recorded. DiOC6 has been used by others as a probe to label endoplasmic reticulum [11].
Controls
For the assay system As a control for the assay system itself, we applied purified bovine brain kinesin (2 µg/ml; Cytoskeleton) to the chamber and assayed for microtubule motility using our PEM/250 buffer both with and without supernatant (2 mg/ml). Also, as a control for the system and to assay for the presence of motor proteins in the testicular supernatants, we applied supernatant (S1a; 2–20 mg/ml) in place of the purified kinesin to the chambers and assayed for microtubule motility.
A motility chamber loaded either with purified kinesin or with supernatant usually was included as a control for the system with the set of motility assays run on any given day.
For the presence of nucleotide Controls for the presence of nucleotide were done in two ways. First, the initial 15 images of each data set were recorded from the chamber containing motility buffer without any exogenous ATP. These images served as controls for the presence of nucleotide in motility buffer that was drawn into the chamber after the first 15 frames. Second, a series of data sets were obtained from chambers in which apyrase was added to motility buffer not containing any exogenously added ATP that was drawn into the chamber after the first 15 frames were recorded. Apyrase is an ATPase that we used to deplete any endogenous ATP. This also served to control for any microtubule movement that was due to fluid or to mechanical effects of adding the buffer rather than to added nucleotide.
For microtubule binding to plasma membranes As a control for nonspecific binding of microtubules to membranes in general, we isolated epididymal spermatozoa from rats used to obtain spermatid preparations. The cauda epididymides were removed from the animals and minced in PEM/250 on ice. During the mincing process, spermatozoa were released into the buffer and collected with a pipette. The cells were concentrated by centrifugation, resuspended in a small volume of PEM/250, and added to sucrose gradients as described above for fragmented epithelia. The largest band of cells was collected from the gradient and processed in the same fashion as were the spermatid-enriched fractions of the epithelial preparation. The spermatozoa were added to the motility chamber and treated in the same fashion as were spermatids.
For the presence of supernatant Spermatid/junction plaque complexes were assayed for their ability to support translocation of microtubules both in the presence and absence of testicular supernatant (S1a). We also ran a single pilot study to test the effects of a range of concentrations of the supernatants on motility (2–20 mg/ml).
For temperature We performed most assays at room temperature (approximately 22–24°C); however, we did do some assays at 33°C. To regulate chamber temperature, we constructed a thin (2 mm) aluminum plate (7.5 x 4.5 cm) with a central aperture (2-cm diameter). On the underside of the plate, and surrounding the aperture, we attached a circular heating element. A thermistor probe was cemented into the aluminum plate approximately midway between the aperture and the corner of the plate. Small circular polytetrafluoroethylene discs (1-cm diameter, 2-mm thick) were attached to each corner of the underside of the plate to support it on, and insulate it from, the microscope stage. The heating element and the thermistor probe were attached to a temperature controller that was mounted on the body of the microscope. The temperature controller, the heating element, and the thermistor probe were purchased from Cell MicroControls (Wellesley Hills, MA). The glass slide containing the motility chamber was attached to the aluminum plate with heat-seal grease. The system was calibrated using an independent thermistor placed on the glass slide near the motility chamber.
For microtubule movement on glass To verify that any microtubule movement recorded was associated with the spermatid/junction complex and not due to any motors (from the supernatant or spermatid suspension) anchored to the glass, a single image of microtubules in the focal plane of the coverslip was obtained after the acquisition of a motility data set. This image was compared with the data set to verify that any moving microtubules were attached to the spermatid/junction complex and not to the coverslip.
Data Analysis
To produce still images for publication, data files were copied into and manipulated, without compromising data integrity, in Photoshop 4 (Adobe Systems Incorporated, San Jose, CA).
Rates of movement were calculated as follows. A sequence of frames over which a microtubule exhibited continuous progressive movement was chosen, and the speed or rate was calculated as distance moved divided by the time over which the sequence was collected. Taken into account in each time calculation were exposure time, pause time, and computer-processing time for images within the sequence. Values are expressed as mean (µm/second) ± standard error.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We routinely observed microtubule translocation when purified bovine brain kinesin (Cytoskeleton) was used a substrate in the motility chamber in both the absence (Fig. 3) and presence of 2 mg/ml testicular supernatant (S1a). The average rates calculated for these when assays were done at room temperature were 0.119 ± 0.004 µm/sec (n = 4) and 0.078 ± 0.001 µm/sec (n= 3), respectively. When the concentration of S1a was increased to 16.5 mg/ml in the motility buffer, the calculated rate was 0.031 ± 0028 µm/sec (n = 12). When assays were run at 33°C and 2 mg/ml S1a was included in the motility buffer, the rate was 0.111 ± 0.004 µm/sec (n = 4).
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Supernatant
Testicular supernatant (S1a), when used as a substrate in the motility chambers (2–20 mg/ml), supported microtubule transport (Fig. 4). Assays were done in both the presence and absence of supernatant (2 mg/ml) in the motility buffer. Interestingly, when assays were done at 33°C rather than at room temperature, progressive motility was observed, but not nearly as consistently as at room temperature; hence, we have not included rate values for assays run at this temperature. When 11 mg/ml S1a was applied to the motility chamber and assays were run at room temperature in the presence of 2 mg/ml S1a in the motility buffer, the rate of transport was 0.079 ± 0.006 µm/sec (n = 19). When 2 mg/ml was applied to the chamber and included at the same concentration in the motility buffer, the rate was 0.119 ± 0.006 µm/sec (n = 6).
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Spermatid/Junction Complexes Supported Microtubule Transport In Vitro
We routinely observed microtubule movement in association with spermatids mechanically isolated from the seminiferous epithelium; however, not all microtubules associated with any given cell moved, nor was microtubule movement observed in association with all cells. When motility events were observed, the nature of the movement varied. In some cases, the movement was saltatory, while in others, microtubules moved smoothly over the surface of the spermatid. In the example shown in Figure 5, a microtubule moved in an "inch-worm"-like fashion along the dorsal surface of the spermatid/junction complex, and then became anchored into position at the caudal end of the head. In another example (shown in Fig. 6), a microtubule moved in a saltatory fashion along the dorsal surface of the head, deviated slightly away from the cell along a phase dense structure that had torn away from the surface of the spermatid, and then ceased movement. The fragment was found to label heavily with fluorescent phallotoxin, indicating that it was part of the Sertoli cell junction plaque. A somewhat similar case is shown in Figure 7, in which a microtubule generally moved in a saltatory fashion along the dorsal surface of the head and assumed a position slightly removed from the surface of the spermatid in a region where DiOC6- and phallotoxin-positive material appeared to separate from the head.
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Microtubule motility events were detected in experiments in which testicular cytosol was omitted from the motility buffer (Fig. 7), and when assays were run both at room temperature and at 33°C. On the basis of a single pilot study in which we observed no motility at S1a concentrations above 4 mg/ml, a working concentration of 2 mg/ml of S1a was routinely used in most of the experiments.
No motility events were recorded before ATP was added, or when buffer containing apyrase was substituted for the motility buffer.
Single frames of microtubules in the plane of the glass (see Figs. 5–7 and 9) were recorded after collection of the motility data set to verify that microtubules that had moved had done so in association with the spermatid/junction complexes and not on motors attached nonspecifically to the glass. Aspirating microtubules through a 30-gauge needle reduced their length before they were added to the motility chamber and improved the probability that both ends of the microtubule could be visualized in the same plane of focus and that one or both of the ends were not associated with the glass.
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Using only data sequences in which microtubule movement was uniform, S1a (2 mg/ml) was used in the motility buffer, and assays were run at room temperature, a rate of 0.046 ± 0.01 µm/sec (n = 9) was calculated. When S1a was omitted from the motility buffer, the rate was 0.054 ± 0.017 µm/sec (n = 3). A rate value calculated from an assay run at 33°C in which 2 mg/ml S1a was included in the motility buffer was 0.05 µm/sec.
Junction Plaques Were Attached to Spermatids Used in the Motility Assay
Intact junction plaques remained attached to the heads of elongate spermatids that were mechanically dissociated from the seminiferous epithelium and separated from most other epithelial material by centrifugation through step sucrose gradients. The Sertoli cell plasma membrane, the attached layer of actin filaments, and the associated cistern of endoplasmic reticulum were clearly evident in electron micrographs of the junction plaques before spermatids were loaded into the motility chambers (Fig. 8).
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To verify that the junction plaques were present on spermatids with which microtubule transport was recorded, cells were stained with fluorescent phallotoxins immediately after a data set was recorded. Actin bundles characteristic of those known to be in Sertoli cell junction plaques were clearly evident surrounding spermatid heads (Figs. 5–7 and 9). Cells were also stained with DIOC6 in some cases, to verify the presence of the junction-related cistern of endoplasmic reticulum. Although interpretation of data from these preparations was difficult because of a high level of nonspecific spermatid staining, lamellar and vesicular elements were often observed in association with many cells in areas related to the actin bundles stained with the fluorescent phallotoxin, in which microtubule movement was detected (Fig. 9).
When spermatozoa collected from the epididymis were used in the assay, no microtubules bound to the cells, no actin bundles characteristic of ectoplasmic specializations were detected, and no vesicular or lamellar staining with DIOC6 was recorded (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results of the motility assay are consistent with the known structure of the epithelium and with data obtained from an in vitro binding assay. Microtubules, in Sertoli cells, are aligned parallel to the long axis of the cells and to the direction of spermatid translocation [12], and are oriented with their minus ends at the apex of the epithelium [7]. Also, the microtubules are closely associated with apical crypts containing attached spermatids and are specifically related to the specialized adhesion junction plaques surrounding the crypts [1]. Moreover, spermatid/junction complexes similar to those used in the motility assay bind microtubules in a nucleotide-dependent fashion [6, 13], an observation that is consistent with the presence of motor proteins on the plaques.
The conclusion that it is the attached junction plaques that support microtubule binding and movement in the in vitro motility assay and not motors bound nonspecifically to the plasma membrane of the spermatids, or to the glass coverslip around the spermatids, is supported by a number of observations. First, the actin pattern associated with the spermatid heads was characteristic of the known actin pattern of the junction plaques and not of spermatids themselves [10]. Second, in samples in which junction plaques partially dissociated from spermatid heads, movement of microtubules followed the contour of junction plaques and not the contour of the heads. Third, spermatozoa from the epididymis did not bind microtubules in the assay system, indicating that the plasma membranes of these cells did not bind, and hence transport, microtubules in the assay system. Finally, although microtubules did bind to the glass around spermatid heads and although testicular supernatant, when adsorbed onto the glass, did support microtubule motility, the entire lengths of microtubules that translocated in association with junction plaques could be verified to be in a different plane of focus than those on the glass, therefore eliminating the possibility that motors bound nonspecifically to the chamber were actually moving the microtubules over the plaques.
The observation that microtubule movement did occur in association with plaques in the absence of any added cytosol supports the conclusion that active motors do occur intrinsically on isolated plaques and that at least some of the motors and/or factors supporting motor activities are not removed during the isolation procedure.
The most likely component of the junction plaques that supports microtubule transport is the endoplasmic reticulum. This is the component of the plaques that is related to microtubules in vivo [1] and is the element that binds microtubules in a nucleotide-dependent fashion in binding assays [6]. Moreover, all elements of the plaques, including the endoplasmic reticulum, are linked together, forming structural units that remain attached to spermatids mechanically dissociated from the epithelium [6]. Finally, the presence, in regions of the junction plaques where microtubule transport occurred in the motility assay, of DiOC6-labeled vesicular and lamellar elements is consistent with the endoplasmic reticulum being the junction component to which motor proteins are anchored.
The argument that the endoplasmic reticulum is the component of junction plaques to which the motor proteins are bound is not without precedent. The endoplasmic reticulum in general is codistributed with [14], and is distributed along [15], microtubules. Moreover, motor proteins [16–18] and at least one of their "receptors" [19] have been localized to this membranous organelle in other systems; hence, the endoplasmic reticulum appears to be preadapted for use as a bridging element between microtubule-based transport processes and structural elements in the cell such as junction plaques.
The nature of the observed microtubule movement and the lack of movement by the majority of microtubules bound to junction plaques probably reflects an underlying complexity in the association between microtubules and the junction plaques in vivo. In Sertoli cells, the crypts containing attached spermatids are structurally supported by the cell throughout spermatogenesis, whereas translocation of the crypts occurs only at specific times. In addition to control of the motors themselves, it also is possible that there exists a regulated balance between the action of nonmotor binding elements and motors on the plaques that changes during the process of spermatogenesis. A role for nonmotor linking elements in motility has been suggested in other systems [9, 20].
The identities of the motors involved with the spermatid translocation process are not known. Because spermatid translocation occurs only at specific times during spermatogenesis, it is possible that the motors themselves and/or the way in which they are controlled may be specific to the endoplasmic reticulum of the junction plaques. The presence of numerous novel kinesin molecules in the testis [21, 22], the abundance of cytoplasmic dynein both in the testis as a whole [23] and specifically in Sertoli cells [24], and the presence of at least four isoforms of cytoplasmic dynein in rat testis [25] make members of these two major motor families likely candidates for involvement in spermatid translocation. Moreover, the finding that Sertoli cell regions associated with spermatid translocation are highly reactive with an immunological probe for the intermediate chain of cytoplasmic dynein (74 kDa) [26] certainly indicates that an isotype of cytoplasmic dynein may be at least one of the motors involved with the process.
In our hands, the rates calculated for microtubule transport on junction plaques, and indeed for microtubule transport on purified bovine brain kinesin, are slow relative to those reported in the literature (see [27]). The reasons for the observed slow rates are difficult to assess at this point. More recent control experiments, with a different batch of kinesin, have resulted in much faster rates; therefore, we suspect that the slow rates reported here for kinesin may have been due to problems with the motor itself. In the case of the junction plaques, we are dealing with a crude system; that is, one in which none of the elements (motors, nonmotor binding elements, or controlling factors) have been identified or characterized. Moreover, our assay was done with microtubules of variable length, which could also influence rates. Although the rate issue remains to be clarified, the important observation reported here is that junction plaques attached to isolated spermatid heads support microtubule movement.
Results presented here are consistent with the spermatid translocation hypothesis and are prerequisite to determining the polarity of movement, and to identifying and characterizing the motor(s) and binding elements present and the control pathways involved. The novel feature of this system is that Sertoli cells have used cisternae of endoplasmic reticulum to couple intercellular actin-associated adhesion junctions to intracellular membrane trafficking machinery in order to move adjacent cells.
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FOOTNOTES |
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2 Correspondence: A. 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
Accepted: December 8, 1998.
Received: October 15, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 B. Chen, A. Li, D. Wang, M. Wang, L. Zheng, and J. R. Bartles Espin Contains an Additional Actin-binding Site in Its N Terminus and Is a Major Actin-bundling Protein of the Sertoli Cell-Spermatid Ectoplasmic Specialization Junctional Plaque Mol. Biol. Cell, December 1, 1999; 10(12): 4327 - 4339. [Abstract] [Full Text]
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