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From the Department of Biology, Bucknell University, Lewisburg, Pennsylvania
| Correspondence to: Dr Sally E. Nyquist, Department of Biology, Bucknell University, Lewisburg, PA 17837 (e-mail: nyquist{at}bucknell.edu). |
| Received for publication February 10, 2003; accepted for publication June 20, 2003. |
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Abstract |
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 Top Abstract DIG Membrane...Rafts in Sertoli CellsMethodsResultsDiscussionReferences |
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Key words: Peritubular myoid cells, detergent-insoluble glycosphingolipid-enriched fractions
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DIG Membrane Fractions—Associated Structural Proteins |
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TopAbstractDIG Membrane...Rafts in Sertoli CellsMethodsResultsDiscussionReferences |
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A second family of proteins, the flotillin family, may also have a structural role in raft formation. Two isoforms of flotillin have been characterized, the 45-kd flotillin-1 and the 42-kd flotillin-2 (Bickel et al, 1997; Galbiati et al, 1998; Volonte et al, 1999). Flotillins are found in both cell types that contain and that lack caveolin expression (Bickel et al, 1997). Flotillin-1 possesses 2 hydrophobic regions that may interact with the plasma membrane; however, the conformation assumed by flotillin-1 is still under debate (Bickel et al, 1997; Volonte et al, 1999). Flotillin-2 shares structural homology with flotillin-1, although it lacks the N-terminal domain contained by flotillin-1 (Volonte et al, 1999).
Although it is known that caveolins and flotillins may interact in vitro through hetero-oligomerization (Lisanti et al, 1993; Scherer et al, 1997; Volonte et al, 1999), the mechanism by which these proteins facilitate vesicle formation has not been characterized. It has been shown, however, that the transfection of cells lacking caveolae either with caveolin-1 or flotillin-1 is sufficient to form plasmalemmal vesicles (Lipardi et al, 1998; Volonte et al, 1999). It is also believed that flotillins plays a structural role in vesicle formation in cells lacking caveolin expression, such as neuronal cells (Bickel et al, 1997; Volonte et al, 1999). Other studies, however, have implicated flotillin-2 in filopodia formation in neuronal cells that lack caveolae (Lang et al, 1998; Hazarika et al, 1999). In these cases, flotillin-2 is thought to localize to noncaveolar rafts rich in glycosylphosphatidylinositol (GPI)-anchored cell adhesion proteins (Lang et al, 1998).
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Rafts in Sertoli Cells |
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TopAbstractDIG Membrane...Rafts in Sertoli CellsMethodsResultsDiscussionReferences |
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Peritubular myoid cells (PMCs) are the major contaminant in Sertoli cell primary cultures; thus, this study also examined PMC lysates. Early studies (Lacy and Rotblat, 1960; Leeson and Leeson, 1963; Ross, 1967) of PMCs using transmission electron microscopy clearly identified a population of contractile cells possessing flask-shaped, pinocytotic vesicles on both surfaces of the cell. These vesicles structurally resemble caveolae, and we recently reported that PMCs contain caveolin distributed in a punctate pattern characteristic of caveolae. DIG fractions isolated from these PMCs were significantly enriched in caveolin (Shubert et al, 2001).
Little is known about the composition of Sertoli cell DIG fractions, and to our knowledge, the only previous study of Sertoli DIG fractions (Fortna et al, 1999) reported the concentration of a novel GPI-anchored form of the copper-binding protein ceruloplasmin in these fractions. In this study, we investigated the distribution of the structural proteins caveolin and flotillin-1 in Sertoli cell DIG fractions. The impact of primary Sertoli cell culture contaminants and peritubular myoid and SPGC on DIG fraction content was also evaluated.
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Methods |
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TopAbstractDIG Membrane...Rafts in Sertoli CellsMethodsResultsDiscussionReferences |
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PMC cultures were obtained by pelleting the supernatant of the first
collagenase/dispase digestion of the seminiferous tubules during Sertoli cell
isolation. The pellet from one isolation (15 pups) was suspended in 25 mL of
DME/F-12 (1:1) supplemented with 10% newborn calf serum and allowed to
sediment for approximately 10 minutes to remove any cell clumps; then, the
upper 20 mL of supernatant was plated in a 75-cm2 flask. After 24 hours, the
medium was changed, and after 4 days, the cells were passed, split 1:10, grown
to confluence, passed again, and harvested as second-pass PMCs. The identity
of these cells as PMCs, fraction purity greater than 95%, was based on
phase-contrast morphology and immunofluorescent staining using antismooth
muscle 
-actin (Sigma Chemical Co, St Louis, Mo)
(Tung and Fritz, 1990).
Madin-Darby canine kidney (MDCK) and NIH-3T3 cell cultures were maintained in DME/F-12 (1:1) with 10% bovine calf serum and were grown to confluence prior to lysate preparation. Preelutriation SPGC fractions were prepared as previously described (Nyquist and Holt, 1986).
Isolation of Detergent-Insoluble Microdomains
Sertoli cell DIG fractions were isolated by a modification of the method
described by Sargiacomo et al
(1993). Four to six 225-cm2
flasks of primary Sertoli cells were used per isolation. All procedures were
carried out at 4°C. The cultures were washed with cold PBS, extracted by
scraping off the cells in 1 mL/flask of ice-cold 1% Triton X-100 in
morpholinoethanesulfonic acid-buffered saline (MBS; 25 mM
2-[N-morpholino]ethanesulfonic acid, pH 6.5, 0.15 M NaCl, 0.2 mM
phenylmethylsulfonyl fluoride [PMSF]), and homogenized with 10 passes of a
Potter-Elvehjem tissue homogenizer (Daigger, Vernon Hill, Ill). The homogenate
was adjusted to 40% sucrose and divided into 6 clear 5-mL ultracentrifuge
tubes and overlaid with a 25%/15%/5% discontinuous sucrose gradient in MBS.
Gradients were centrifuged for 18 hours at 46 000 rpm in an SW55Ti rotor
(Beckman Instruments Inc, Palo Alto, Calif). The DIG fraction appeared as a
light-scattering band at the 25%/15% sucrose interface. This insoluble
material was collected, diluted in MBS, and pelleted by centrifugation for 45
minutes at 46 000 rpm in an SW55Ti rotor. Pellets were resuspended in 100
µL of phosphate-buffered saline (PBS; 0.1 mM sodium phosphate, pH 7.2, and
0.15 M NaCl) containing 0.2 mM PMSF. A portion of the DIG fraction was saved
for protein quantification using the method of Lowry et al
(1951), and the remainder was
prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) by the addition of an equal volume of sample preparatory buffer
(0.125 M Tris, pH 6.8, 4% SDS, 20% glycerol, 10% ß-mercaptoethanol, and
0.002% bromophenol blue), which was followed by heating at 95°C for 3-5
minutes.
Preparation of Cell/Tissue Lysates
For the preparation of cell lysates (Sertoli, PMC, NIH-3T3, and MDCK),
cultures were washed twice with cold PBS. To each flask, 1 mL of lysis buffer
(1% SDS, 1.0 mM sodium vanadate, 10 mM Tris, pH 7.4, and 0.2 mM PMSF) was
added. Lysed cells were scraped and heated at 95°C for 5 minutes. For
tissue lysates, samples were obtained from an adult male Sprague-Dawley rat.
Tissues were washed twice with cold PBS, and 1 mL of lysis buffer per gram of
tissue was added. Tissue was homogenized in a Polytron tissue homogenizer
(Brinkman Instruments, Westbury, NY) for 1 minute. Lysates were sonicated
briefly to decrease viscosity, insoluble material was removed by
centrifugation, and a protease inhibitor cocktail (General Use, 20 µL/mL,
Sigma) and PMSF (0.2 mM) were added. Samples of all lysates were removed for
protein quantification by the method of Lowry et al
(1951), and the remainder was
prepared for SDS-PAGE as described above for DIG fractions. All protein
samples were stored at -80°C until use.
Western Blotting
Samples were separated by SDS-PAGE
(Laemmli, 1970) using 12%
(flotillin and caveolin) or 10% (total protein) acrylamide gels and
transferred to nitrocellulose. Transfer was monitored by Ponceau S staining.
Blots were blocked for 1 hour in Tris-buffered saline (TBS) containing 0.05%
Tween-20 (TBST) and 5% nonfat dried milk (TBST-MILK). All antibodies were
diluted in TBST-MILK, and incubations were carried out at room temperature.
All secondary antibodies were horseradish peroxidase (HRP) conjugates, and all
blots were visualized by enhanced chemiluminescence (ECL). For flotillin-1
detection, blots were incubated in a mouse antiflotillin-1 monoclonal antibody
(F65020; Transduction Laboratories, Newington, NH) at a 1:250 dilution for 1
hour, and goat anti-mouse immunoglobulin G (IgG) secondary antibody (Bio-Rad
Laboratories, Richmond, Calif) was used at a 1:1000 dilution for 30 minutes.
For caveolin detection, blots were incubated in a rabbit anticaveolin
polyclonal antibody (C13630; Transduction) at a 1:1000 dilution for 1 hour,
and a goat anti-rabbit IgG secondary antibody (Bio-Rad) was used at a 1:1000
dilution for 30 minutes. The immunogen for production of the antiflotillin-1
antibody (F65020) was the C-terminus of mouse flotillin-1; it recognizes a
48-kd protein and does not cross-react with flotillin-2. The immunogen used
for production of the anticaveolin antibody (C13630) was the N-terminus of
human caveolin-1. This antibody recognizes both the and ß forms
of caveolin-1 and cross-reacts with caveolin-2.
Immunofluorescence Microscopy
PMCs were plated on 18-mm coverslips and maintained for several days in
DME/F-12 medium containing 10% newborn calf serum prior to fixation. Coverslip
cultures were fixed for 10 minutes in a 0.1-M phosphate buffer, pH 6.8,
containing 4% paraformaldehyde, and washed with phosphate buffer. Cells were
permeabilized with a 2-minute incubation in phosphate buffer containing 0.05%
Triton X-100 and then washed in phosphate buffer.
Coverslips were blocked for 20 minutes in phosphate buffer containing 5% bovine serum albumin (BSA). Caveolin labeling experiments were conducted using polyclonal anticaveolin-1 antibody (C13630; Transduction) at a 1:100 dilution in phosphate buffer containing 1% BSA. Incubations in primary antibody were carried out for 30 minutes at room temperature and were followed by a second block for 20 minutes in 10% goat serum in 0.1 M phosphate buffer, pH 7.2. Fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG antibody (Calbiochem, San Diego, Calif) and rhodamine-conjugated donkey anti-mouse IgG antibody (Chemicon, Temecula, Calif) were used at a 1:100 dilution in phosphate buffer containing 1% BSA. Coverslips were washed once in water and mounted with Fluormount-G containing 2.5% 1,4-diazobicyclo(2,2,2)-octane. Slides were visualized by epifluorescence and confocal microscopy on a Nikon (Melville, NY) Eclipse E800 microscope, and images were processed using Simple PCI imaging software (Compix Inc, Cranberry Township, Pa). The anti-Golgi marker (GM130) antibody was a gift from Dr Carolyn Machamer.
Preparation and Staining of Tissue Sections
Rat testes were perfusion fixed with 4% paraformaldehyde in a 0.05-M
phosphate buffer, pH 7.4, and were then immersion fixed for 24 hours.
Paraffin-embedded sections were irradiated in the microwave at 800 W for 4-7
minutes to retrieve antigenicity. Sections were cooled, placed into PBS for 10
minutes, incubated in Immunopure Peroxidase Suppressor (Pierce Biotechnology
Inc, Rockford, Ill) for 10 minutes, and blocked for 20 minutes in 20% goat
serum in PBS. For caveolin staining, sections were incubated for 1 hour in a
polyclonal anticaveolin antibody (C13630; Transduction) at a 1:500 dilution,
which was followed by a 45-minute incubation in a biotin-conjugated
anti-rabbit antibody (Chemicon). For flotillin-1 staining, sections were
incubated for 1 hour with an antiflotillin-1 (F65020; Transduction) at a 1:75
dilution, which was followed by a 45-minute incubation in a biotin-conjugated
anti-mouse antibody (Chemicon). Sections were then washed and incubated with
streptavidin HRP conjugate (Zymed Laboratories Inc, South San Francisco,
Calif) at a 1:150 dilution, and staining was visualized using the aminoethyl
carbazole (AEC) substrate. Sections were counterstained with Mayer
hematoxylin.
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Results |
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TopAbstractDIG Membrane...Rafts in Sertoli CellsMethodsResultsDiscussionReferences |
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Membrane rafts, detergent-insoluble membrane microdomains, are frequently found to be enriched in the 21-kd protein caveolin, a marker protein for caveolae. For this reason, we explored the distribution of caveolin in testicular lysates, Sertoli cell lysates, and Sertoli cell DIG fractions. SDS-PAGE and Western blotting with a polyclonal anticaveolin-1 antibody suggested the absence of caveolins in Sertoli cells and Sertoli cell DIG fractions (Figure 2). To probe more rigorously for low levels of caveolin in Sertoli cell DIG fractions, exposure of the Western blot was increased. These results suggested the presence of trace amounts of caveolin in the heavily loaded Sertoli DIG sample in lane 3 and in the Sertoli cell lysate in lane 4 (data not shown). These bands migrated at a position on the gel identical to that of the positive caveolin control, MDCK cell lysate.
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To determine whether this low level of caveolin was endogenous to the
Sertoli cell or whether it was the result of the low levels of PMCs
traditionally found as the major contaminant in primary Sertoli cell cultures,
experiments were conducted to evaluate the caveolin content of the PMCs. PMC
cultures were established and evaluated by immunofluorescent staining for
smooth muscle -actin (Figure
3A). On the basis of antismooth muscle -actin staining, the
PMC cultures were exceptionally homogeneous, although there was evidence of a
phenotypic change to a more contractile cell with time in culture. These cells
were also double-stained for caveolin and smooth muscle actin
(Figure 3B and C). Caveolin
clearly exhibited a punctate image along the cell surface as would be expected
for a caveolar distribution. Caveolin was also heavily concentrated in the
perinuclear region of the cell consistent with a Golgi location. The smooth
muscle -actin, in addition to forming the large stress fibers, was
concentrated along the plasma membrane at the leading edge of the PMC.
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Other testicular cells were also examined for caveolin content. Lysates were prepared and examined for the presence of caveolin-1 by SDS-PAGE, Western blotting, and immunostaining using the same polyclonal anticaveolin-1 antibody (Figure 4). Among the testicular cell lysates examined, only the PMC lysates and the whole-testis fractions showed prominent caveolin bands.
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A cryostat-sectioned rat testis was also examined by immunofluorescent microscopy using the same anticaveolin-1 antibody (Figure 3D). The peritubular location of the immunofluorescence is consistent with caveolin localization within the PMCs. These results confirm the presence of caveolin in testicular cells, a result that conflicts with earlier reports of an absence of caveolin messenger RNA in the testis (Scherer et al, 1994) and suggests that PMCs contribute significantly to the caveolin content of whole-testis homogenates.
In addition to the peripheral ring of fluorescence around each seminiferous tubule, contributed by the PMCs, other spots of fluorescence were observed within the epithelium of the tubule, and the distribution and intensity of spots varied among tubules. Since caveolin is also endogenous to Golgi apparatus membranes and since both Sertoli and SPGC cells contain large quantities of Golgi membranes, cryostat sections of rat testis were subjected to a double stain with anticaveolin-1 and anti-GM130 (Golgi-specific) antibodies. No co-localization was apparent (Figure 3E), thus indicating that these fluorescent spots did not likely come from Golgi membranes. Nor was co-localization observed with developing acrosomes, although the immunofluorescent staining of spermatozoal (SPZOA) smears under identical conditions yielded false-positive caveolin reactions. The identity of these spots is under continued investigation.
Rat testes were also paraffin embedded, sectioned, subjected to antigen retrieval techniques, and stained for caveolin content (Figure 5A and B). Reaction product was seen clearly only within the PMCs of the seminiferous tubules. Within the interstitium, the walls of the blood vessels also showed a strong positive reaction for caveolin. Control sections not subjected to microwave antigen retrieval showed little deposition of reaction product. Unfortunately, the antigen retrieval process negatively affected section quality.
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Recently, another purported structural protein, the 48-kd flotillin-1, was also shown to be enriched in both caveolar and noncaveolar rafts, where it has been implicated in raft organization. Therefore, Sertoli cell lysates and Sertoli cell DIG fractions were examined for the presence of flotillin-1. This protein, in contrast to caveolin, appears to be present in the Sertoli cell and highly localized in the DIG fraction (Figure 2). The content of flotillin-1 in the Sertoli whole-cell lysate (lane 4) was similar to that noted in brain whole-cell lysate (lane 6). Flotillin-1 distribution was also examined among a number of testicular cell lysates (Figure 4). Flotillin-1 was abundant in both Sertoli cells and PMCs, but lesser quantities were also seen in the SPGC and SPZOA fractions (Figures 2 and 4). Although flotillin-1 is abundantly present in PMC and Sertoli cell cultures, its concentration varies depending on culture conditions.
Rat testes were also paraffin embedded, sectioned, subjected to microwave antigen retrieval techniques, and stained for flotillin-1 content. Flotillin-1 concentration within the seminiferous tubules was clearly stage-dependent, with intense labeling observed around the spermatid heads prior to spermiation (Figure 5D and E). Reaction product began to accumulate within the adlumenal compartment of the seminiferous tubules during stages 5/6. Less intense deposition of reaction product was also observed in the basal compartment of the seminiferous tubules in the late spermatogonia and early spermatocytes. For reasons not understood, little-to-no reaction product was visible in the PMCs. Without microwave antigen retrieval, no reaction product was visible for flotillin-1 (Figure 5C).
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Discussion |
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TopAbstractDIG Membrane...Rafts in Sertoli CellsMethodsResultsDiscussionReferences |
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A major issue raised by this study concerns the functional roles these Sertoli cell rafts play. Cell signaling is the function most widely attributed to both caveolae and noncaveolar rafts. We have, however, demonstrated that Sertoli DIG fractions lack caveolin, the membrane protein often responsible for interacting directly with membrane microdomain signaling components and thus regulating activity (Li et al, 1995, 1996; Mastick et al, 1995; Mastick and Saltiel, 1997). Since our studies have shown that flotillin-1 is a major structural protein of the Sertoli DIG fraction and since only limited data exist relative to flotillin's involvement in cell signaling, a more careful examination of flotillin's function in the testis may be instructive to the cell biology community at large. Of possible relevance is the very recent report of Baumann et al (2000) showing the involvement of flotillin-1 containing lipid rafts in insulin action.
The flotillin family of proteins currently consists of 2 isoforms. Flotillin-1, also known as reggie-2, is a protein originally characterized in fish retinal neurons that has been shown to have analogous forms in the rat. In addition, flotillin-2, also called reggie-1, has been described (Lang et al, 1998). Lisanti's research group suggests that flotillin-2 is homologous to epidermal surface antigen (ESA) (Bickel et al, 1997; Volonte et al, 1999), although subsequent studies have demonstrated significant differences between ESA and flotillin-2 (Hazarika et al, 1999). Flotillin-1 has been implicated in caveolar organization along with caveolin (Bickel et al, 1997; Volonte et al, 1999), yet recent studies suggest alternative functions (Lang et al, 1998; Hazarika et al, 1999). Flotillin-2 is thought to play a role in filopodia formation in fish retinal cells and in neuronal cells (Lang et al, 1998; Hazarika et al, 1999). In addition, in studies of fish retinal cells, flotillins segregate into patches of GPI-anchored cell adhesion proteins at the leading edge of filopodia formation in a cell type that lacks observable caveolae (Lang et al, 1998).
The stage-specific accumulation of flotillin-1 in the adlumenal compartment of the seminiferous tubules prior to spermiation raises some major questions. Unfortunately, the resolution available from microwave retrieval on paraffin-embedded sections (at least in our hands) does not provide adequate resolution to determine the precise cellular location of flotillin-1. Immunohistochemical studies offering better resolution need to be conducted. Additionally, work to locate the Sertoli cell rafts in situ would provide important information, as would the identification of other proteins present within these rafts. It is clear, however, that flotillin-1 possesses a more ubiquitous distribution among the testicular cell types than does caveolin.
Flotillin is also known to associate with endocytotic vesicles. Endocytotic vesicles have been described by several authors on the apical surface of the Sertoli cell (Nagano and Suzuki, 1978; Morales et al, 1985); however, to the best of our knowledge, no biochemical analyses of these vesicles have been completed. In neuronal cells, flotillin has been reported to reside in endocytotic vesicles slightly larger than the characteristic caveolin containing caveolae (Volonte et al, 1999), whereas flotillin has also been reported to reside in flat, nonvesicular microdomains (Lang et al, 1998). Immunohistochemical localization studies of flotillin in seminiferous tubule cross sections, as well as ultrastructural studies using immunogold labeling and electron microscopy, need to be conducted.
Although considerable evidence exists for the involvement of caveolae and other membrane microdomains in cell signaling, other possible roles need to be considered. Flotillin-dependent microdomains may have a role in the vesicular transport of materials between Sertoli and SPGC. Also, since members of the flotillin family of proteins have been reported to be involved in filopodia formation (Hazarika et al, 1999), it is possible that flotillin-associated microdomains play a role in the morphogenetic movements of the Sertoli and SPGC cells within the seminiferous tubule. Not only does extensive transport of materials between cells occur at cell-cell contact points, but extensive cytoskeletal networks immediately underlie the plasma membrane of the Sertoli cell. The extensive forces necessary to facilitate the massive remodeling of cell shape that is an integral part of spermatogenesis may be mediated through some of these specialized membrane microdomains. If the latter suggestion is correct, it is likely that cytoskeletal proteins would be physically associated with flotillin. If this is the case, flotillin-associated microdomains may also represent centers of contact between SPGC and Sertoli cells. Studies of possible physical interactions between flotillin and other proteins of the DIG fractions could be instructive.
The observation of abundant caveolin present in the PMC has been shown by the immunoblotting of PMC lysates, the immunofluorescent microscopy of whole-testis cryosections, and the immunohistochemical staining of paraffin-embedded sections of whole testis following microwave antigen retrieval techniques. This, along with previous data of Shubert et al (2001) showing a punctate distribution of caveolin in PMC cultures and an enrichment of caveolin in PMC DIG fractions, strongly supports the idea that the micropinocytotic vesicles observed by early microscopists (Lacy and Rotblat, 1960; Leeson and Leeson, 1963; Ross, 1967) are caveolae. Attention now needs to be focused on functional studies of these vesicles.
The distribution of caveolin observed in whole-testis sections yielded some unidentified foci of fluorescence within the seminiferous tubules. The origin of this fluorescence remains unknown, but this study suggests that the Golgi membranes are not the source. Since Sertoli cell cultures, isolated nonflagellated SPGC fractions, and washed epididymal spermatozoa all lack caveolin, the source of this fluorescence must reside either in a Sertoli cell component lost with time in culture or in a later developmental stage of the SPGC. Although we have been unable to co-localize these fluorescent foci with the acrosomes of developing spermatids, we have noted an artifactual staining of acrosomes on washed epididymal spermatozoa after immunofluorescent staining for caveolin.
In conclusion, with the recognition and isolation of specialized microdomains, rafts, on the Sertoli cell surface, the reproductive cell biologist will possess an additional tool for the study of cell signaling, and perhaps other interactions, between various cells of the seminiferous tubule. The data reported in this study also argue for the necessity of using Sertoli cell cultures as free as possible from peritubular myoid and SPGC contamination when preparing Sertoli cell DIG fractions.
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Footnotes |
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