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Endocrinology Vol. 144, No. 12 5530-5536
Copyright © 2003 by The Endocrine Society

Reduced Intratesticular Testosterone Concentration Alters the Polymerization State of the Sertoli Cell Intermediate Filament Cytoskeleton by Degradation of Vimentin

Matthew D. Show, Matthew D. Anway, Janet S. Folmer and Barry R. Zirkin

Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205

Address all correspondence and requests for reprints to: Matthew D. Show, Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205. E-mail: mshow{at}jhsph.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Sertoli cell intermediate filament cytoskeleton is composed of the type III family member vimentin. The distribution of Sertoli cell vimentin varies with the stage of spermatogenesis, with shortening of the filaments at stages VII–VIII, the stages of spermiation. Experimental reduction in intratesticular testosterone (T) concentration also results in the sloughing of advanced spermatids from the Sertoli cells, as well as in the apoptotic death of spermatocytes. We hypothesized that alteration of the distribution of Sertoli cell vimentin might play a role in the loss of germ cells that occurs in response to reduced intratesticular T. To test this hypothesis, intratesticular T was reduced by implanting LH-suppressive SILASTIC brand capsules containing T and estradiol into adult rats for 8 wk. Immunohistochemical analyses revealed that, in response to the implants, the vimentin cytoskeleton collapsed around the Sertoli cell nuclei at all stages of the cycle, losing the extensive branching and structure normally seen at most stages of the cycle. Western blots of isolated Sertoli cells revealed that protein levels did not differ significantly between control and T- and estradiol-treated rats. However, Sertoli cell fractions containing the vimentin monomer revealed that vimentin was cleaved into four to five fragments in Sertoli cells in response to the implants, suggestive of proteolysis. These results indicate that, in response to reduced intratesticular T, the vimentin cytoskeleton of the Sertoli cell collapses to a perinuclear localization, and suggest that this collapse is associated with, and perhaps caused by, the degradation of the vimentin monomer rather than by loss of its expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTERMEDIATE FILAMENTS ARE a large family of proteins that form polymers whose sizes fall in between those of microfilaments and microtubules (1). All intermediate filament monomers contain a variable N and C terminus plus a central helical rod domain believed to be important in polymerization (2). Many issues remain unresolved about intermediate filaments, including their function in particular cells and the mechanism of their polymerization/depolymerization. Intermediate filament polymerization seems to be ATP independent, though some data suggests that they are subject to phosphorylation on serine and threonine residues (3). It has been found that such phosphorylation can disassemble polymerized intermediate filament polymers into soluble forms (3).

The Sertoli cell is the principal structural element of the seminiferous epithelium, providing physical support and an environment conducive to germ cell development and maturation (4). Sertoli cells possess a cytoskeleton composed of three elements: actin, vimentin, and tubulin (5). Actin makes up the microfilament network, and tubulin comprises the microtubule cytoskeleton. Sertoli cells also possess an extensive intermediate filament cytoskeleton composed primarily of the type III family member vimentin (6). In a typical Sertoli cell, vimentin filaments, which are formed by polymerization of 57-kDa vimentin monomers, surround the nucleus, giving it a characteristic "halo" appearance (6, 7), and radiate out from the nucleus to the cell periphery, terminating near points of contact between the Sertoli cell and adjacent cells. The points of contact include the tight junctions found between neighboring Sertoli cells, the desmosome-like junctions located between the Sertoli cells and early germ cells (i.e. spermatagonia and spermatocytes), and the ectoplasmic specialization junctions found between Sertoli cells and more advanced germ cells (i.e. round to elongated spermatids). As is also true of intermediate filaments in other cell types, the function of vimentin in Sertoli cells is not well understood. Some have hypothesized that intermediate filaments serve functions as diverse as structural support, plasma membrane-nucleus communication, or nuclear positioning (5).

The vimentin filaments in the Sertoli cell of the rat have been reported to vary dynamically in length with the stages of the cycle of the seminiferous epithelium. Zhu and colleagues (8) showed that during stages I–V and XI–XIV, vimentin is extensively branched, with filaments extending into the apical region of the cell. These filaments decrease in length at stages VI–VII, and by stages VIII–X are localized to a halo surrounding the nucleus. The dramatic shortening of vimentin filaments during stages VI–VII corresponds to the so-called androgen-dependent stages of spermatogenesis, when high levels of androgen receptor are seen to immunolocalize to the Sertoli cell nucleus (9) and when mature spermatozoa are released by the Sertoli cell into the lumen of the seminiferous tubule.

We hypothesized that experimental reduction of intratesticular testosterone (T) concentration, which can result in the apoptotic death of some germ cells and the sloughing of others, might also result in the perturbation of the intermediate filament cytoskeleton. To test this hypothesis, we investigated the effects of lowering intratesticular T on the distribution and biochemical characteristics of the Sertoli cell vimentin cytoskeleton, as well as on the microfilament and microtubule cytoskeletons. The results presented herein show that reduced intratesticular T indeed results in changes in the distribution of vimentin filaments but not in microfilaments or microtubules in Sertoli cells and, moreover, that the changes in vimentin result not from the loss of expression of the vimentin protein but from potential cleavage of the protein monomer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Sprague Dawley rats of 8–12 wk of age were purchased from Charles River (Kingston, MA). All rats were housed in a vivarium under a 14-h light, 10-h dark cycle and were provided water and rat chow ad libitum. To experimentally suppress LH-stimulated T production from Leydig cells, rats were administered subdermal 2.5-cm T- and 0.1-cm 17 ß-estradiol (E)-filled polydimethylsiloxane (SILASTIC brand, Dow Corning, Midland, MI) capsules for 56 d, and control rats received empty capsules for 56 d, according to methods previously described (10). This and all protocols used herein were approved by the Johns Hopkins University Animal Care and Use Committee.

Sertoli cell isolation
Sertoli cells were isolated following methods previously described (11) but omitting the 10-min trypsin digestion. Briefly, two decapsulated testes were incubated in 0.5 mg/ml collagenase in 1x Hanks’ solution (pH 7.4) at 34 C, shaken for 15 min to eliminate the interstitial cells, and then washed a total of three times. To separate the Sertoli and germ cells, the tubules were incubated in a mixture of enzymes (0.1% collagenase, 0.2% hyaluronidase, 0.04% deoxyribonuclease I, and 0.03% trypsin inhibitor in 1x Hanks’, pH 7.4) at 34 C, with shaking for 40 min. The Sertoli cells were pelleted by centrifugation, washed in 1x Hanks’, and repelleted a total of three times. The pellets were then resuspended in 1x Hanks’ and subjected to hypotonic shock in a dilute Hanks’ solution (1:3.5 Hanks’:water final dilution). Cells were collected by centrifugation, resuspended in 1x Hanks’, and filtered through 53-µm nylon mesh. The cells were then washed and resuspended in F12/DMEM (1:1) tissue culture media. Sertoli cell number and purity were estimated by hemocytometer and light microscopic analyses, as previously described (11). In each Sertoli cell preparation, an average of 7–8 million Sertoli cells per testis was obtained, with approximately 80% purity. Germ and myoid cells made up the contaminants.

Immunofluoresence microscopy
Immediately after determinations of cell purity and number, suspended Sertoli cells that had been isolated from control rats or rats implanted for 56 d were dried to microscope slides and fixed with neutral buffered formalin. Slides were blocked in diluted normal serum and then incubated with antimouse vimentin (1:200, V6630; Sigma, St. Louis, MO) for 1 h at room temperature. Bound primary antibodies were detected with an FITC-conjugated antimouse IgM secondary antibody (1:100, FI-2000; Vector Laboratories Inc., Burlingame, CA). Nuclei were stained with Vectashield Anti-Fade Mounting Medium containing DAPI (H-1200; Vector Laboratories). Vimentin is specifically expressed in Sertoli cells (12).

For immunofluorescence studies of testis sections, rats were anesthetized and whole-body perfused with neutral buffered formalin for 1 h at a rate of 7 ml per min. The testes were removed and immersed in neutral buffered formalin overnight at 4 C. The tissue was then dehydrated in ice-cold (4 C) 70%, 90%, and 99% ethanol for 1 h each, and then in absolute ethanol for 1 h at room temperature. Tissue was infiltrated with 50% polyester wax/50% ethanol for 2 h at 42 C followed by a 90% polyester wax/10% ethanol mixture for 1 h at 42 C. The tissue was then transferred into 90% wax/10% ethanol in plastic embedding dishes and chilled on ice for 30 min or until the wax solidified. Sections (5 µm) were cut and mounted on Hipure subbed glass slides. The slides were dewaxed by immersion into 100%, 90%, and 70% ethanol baths for 10 min each. Slides were blocked in diluted normal serum and then incubated with antimouse vimentin, antimouse tyrosine tubulin (1:500, T9028; Sigma), or antimouse ß-actin (1:500, A5441; Sigma) antibody for 1 h at room temperature. Bound primary antibodies were detected with a FITC-conjugated antimouse IgM secondary antibody (1:100, FI-2000). Nuclei were stained with Vectashield Anti-Fade Mounting Medium containing propidium iodide (H-1300). Images were obtained by a Nikon (Melville, NY) Microflex H-III automatic camera system with a x40 Zeiss (Thornwood, NY) PlanApo lens.

Subcellular fractionation
Soluble cytoplasmic proteins from isolated Sertoli cells were separated from insoluble cytoskeletal components according to Patterson et al. (13). Briefly, isolated Sertoli cells were gently lysed at 4 C in lysis buffer [1% Triton X-100, 20 mM HEPES-NaOH (pH 7.2), 100 mM NaCl, 1 mM sodium orthovanadate, and 0.5% protease inhibitor cocktail (P8340; Sigma)] by brief sonication. The lysate was centrifuged at 10,000 x g for 20 min at 4 C. Supernatants contained the soluble cytoplasmic proteins, and the pellets contained the insoluble proteins.

Western blot analyses
Sertoli cells were isolated from control rats and from rats implanted with T and E (TE) capsules for 56 d. The cells were homogenized in RIPA buffer [1% Triton X-100, 15 mM HEPES-NaOH (pH 7.5), 0.15 mM NaCl, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM sodium orthovanadate, 10 mM EDTA, and 0.5% protease inhibitor cocktail (Sigma)] and stored at -80 C until analyzed. Protein concentrations were determined by the bicinchoninic acid method (Pierce, Rockford, IL), according to the manufacturer’s specifications. Protein samples were added to an equal volume of 2x loading buffer [100 mM Tris (pH 6.8), 4% SDS, 0.2% bromophenol blue, and 20% glycerol]. Samples were reduced with 0.1% ß-mercaptoethanol, boiled for 2 min, and loaded on 12% SDS-PAGE as described by Laemmli (14). Protein was transferred to Protran Nitrocellulose (Schleicher & Schuell, Keene, NH) with a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA), according to manufacturer’s specifications.

Membranes were blocked for 1 h with 5% nonfat dry milk in PBS (blocking solution) at room temperature, followed by antivimentin (1:1000) overnight in blocking solution at room temperature. Membranes were washed three times with PBS for 5 min, and then incubated for 30 min at room temperature with secondary antimouse horseradish peroxidase-linked IgG (1:3000, NA 931; Amersham Pharmacia, Piscataway, NJ) in PBS. Signal was detected using the SuperSignal WestPico Chemiluminescent kit (Pierce), according to manufacturer’s specifications. Protein membranes were stripped using Restore Western Blot Stripping Solution (Pierce), according to manufacturer’s specifications. Membranes were then blocked in blocking solution for 1 h and probed with anti-tyrosine tubulin (1:1000) or anti-ß-actin (1:1000) for 3 h in blocking solution, followed by antimouse horseradish peroxidase-linked IgG (1:3000) for 1 h at room temperature.

RIAs
Trunk blood (serum) and testicular interstitial fluid (IF) were collected according to previously described methods (15). All samples were stored at -80 C until assay for T. Serum and IF T concentrations were determined in duplicate for each sample, by RIA, according to a previously described method (15). T was assayed with T antibody from ICN (Costa Mesa, CA) and 3H-testosterone from NEN Life Science Products (Boston, MA). The sensitivity of the assay was 10 pg/ tube.

Statistical analysis
Data were expressed as the mean ± SEM of three separate experiments. Sample differences were analyzed by Student’s t test. Means were considered significantly different at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Intratesticular T concentration
Adult rats were administered TE-containing SILASTIC brand capsules to suppress endogenous LH and thus Leydig cell T production. At 56 d, the implanted rats exhibited significant reductions in testicular weight (71%, P < 0.0001; Fig. 1A) and intratesticular T concentration (96.5%, P < 0.0001; Fig. 1BGo), compared with controls.



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  		FIG. 1. Effects of TE implants on testicular weight (A) and IF T concentration (B), compared with controls. Shown are the means ± SEM (n = 4). In both cases, there was a significant decrease, compared with control (P < 0.0001).

 
Vimentin filament structure in Sertoli cells in situ: effects of stage and of experimentally reduced intratesticular T concentration
Figure 2Go shows fluorescently stained vimentin filaments in Sertoli cells from sections of control rat testis. A cross-section of an untreated stage-II seminiferous tubule is shown in Fig. 2AGo that has been double-labeled for vimentin (green) and DNA (red). Figure 2BGo shows the same tubule with only the vimentin filaments labeled. In these figures, the vimentin filaments can be seen to be localized around the Sertoli cell nuclei and to extend toward the tubule lumen. Figure 2CGo-I shows vimentin in stage I Sertoli cells. The filaments extend almost to the tubule lumen. By stage V (Fig. 2CGo-V), the filaments appear to have shortened; and by stage VIII (Fig. 2CGo-VIII), the only vimentin staining appears as a halo surrounding the Sertoli cell nucleus. However, at stage XII (Fig. 2CGo-XII), the vimentin filaments begin again to extend toward the tubule lumen.



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  		FIG. 2. Distribution of vimentin filaments in Sertoli cells of control rat testes. A, Stage II seminiferous tubule showing vimentin (green) and propidium iodide-stained DNA (red); B, the same tubule showing only the distribution of vimentin; C, distribution of vimentin (green) and DNA (red) in stages I, V, VIII, and XII seminiferous tubules of control rats. Magnification, x40.

 
In contrast to the striking cycle-related variations in vimentin localization in Sertoli cells of control rats, vimentin filaments were seen to localize only around the nuclei of Sertoli cells of every tubule examined in the testes of rats administered TE capsules (Fig. 3Go, A and B); branching of the vimentin filaments in these tubules was almost nonexistent. Vimentin localization resembled the distribution pattern seen at stage VIII tubules in controls, though a few cells exhibited small "whisps" of unbranched filament that extended toward the lumen of the tubule.



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  		FIG. 3. Distribution of vimentin filaments in the Sertoli cells of 56-d TE-treated rat testes. A, Vimentin distribution (green) and propidium iodide-stained DNA (red); B, the same tubule as in A, showing only the distribution of vimentin. Magnification, x40.

 
Microtubule and microfilament structure in Sertoli cells in situ
The effects of TE treatment on the Sertoli microtubule cytoskeleton were examined using a primary antibody to tyrosine-{alpha}-tubulin, which is the predominant form of {alpha}-tubulin expressed in Sertoli cells (12). Similar to the vimentin filament structure, the microtubule cytoskeleton in Sertoli cells in the testes of control rats was highly branched and extended from the basal region of the Sertoli cell toward the tubule lumen (Fig. 4Go, A and B). In contrast to the changes in vimentin filament structure and distribution seen in the Sertoli cells after TE, the microtubule cytoskeleton seemed to be unaffected in localization with reduced T (Fig. 4Go, C and D). As with tubulin, the microfilament cytoskeleton, visualized with an antibody to ß-actin, also seemed to be unaffected in localization by the lowering of intratesticular T; actin in both control and TE-treated rats remained predominately around the periphery of the Sertoli cells (data not shown).



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  		FIG. 4. Distribution of microtubules in the Sertoli cells of control and 56-d TE-treated rat testis. A, Microtubule distribution (green) and propidium iodide-stained DNA (red); B, the same tubule as in A, showing only microtubule distribution in Sertoli cells of control rats; C, microtubule distribution (green) and propidium iodide-stained DNA (red); D, the same tubule as in C, showing only microtubule distribution in Sertoli cells of TE-treated rats. Magnification, x40.

 
Biochemical analyses of Sertoli cell vimentin structure and expression
Sertoli cells were isolated from control and 56-d TE-treated rats to examine vimentin filament morphology and protein levels in individual cells. When labeled with a primary antibody to vimentin, many of the Sertoli cells isolated from the testes of untreated rats exhibited intricate and highly branched vimentin filaments that radiated outward from the nucleus of the cell into its cytoplasmic extensions (Fig. 5AGo). In contrast, in most Sertoli cells isolated from TE-treated rats, the intermediate filament cytoskeleton was visible only around the nucleus (Fig. 5BGo). In some instances, small whisps of vimentin filaments were seen extending outward from the nucleus, but these structures lacked the intricate branching and length seen in the control cells (Fig. 5BGo). Thus, consistent with the in situ images, the vimentin cytoskeleton in isolated Sertoli cells from TE-treated rats exhibited a loss of the filament structure that characterized the untreated controls.



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  		FIG. 5. Freshly isolated Sertoli cells showing the distribution of vimentin (green) and DAPI-stained DNA (blue) from control (A) and 56-d TE-treated rats (B). Magnification, x40. C, Western blots showing protein expression of vimentin, ß-actin, and tyrosine (tyr)-tubulin in freshly isolated Sertoli cells from untreated control (no treatment) and 56-d TE-treated (TE) rats.

 
Analysis of vimentin protein in Sertoli cells isolated from control and TE-treated rats by Western blot revealed that the expression of the 57-kDa vimentin protein monomer did not change, relative to the total (Fig 5CGo). This was surprising, given the extensive changes in vimentin filament structure observed in situ. However, we observed that protein isolated from the Sertoli cells of TE-treated rats consistently exhibited a prominent doublet when immunoblotted for vimentin, with one band representing the 57-kDa vimentin monomer and the second corresponding to a slightly smaller (~54 kDa) fragment (Fig. 5CGo). Western blot analysis also revealed relatively constant levels of the 55-kDa tyrosine-tubulin and the 42-kDa ß-actin protein monomers in Sertoli cells from control and TE-treated rats (Fig. 5CGo).

To pursue further the apparent difference between vimentin protein from control and TE-treated rats, isolated Sertoli cells were then lysed and the protein separated into soluble and insoluble protein fractions by differential centrifugation. The soluble fraction contained cytoplasmic protein, including the soluble vimentin and tubulin protein monomers. Soluble proteins were isolated from Sertoli cells from control and TE-treated rats and were probed, by Western blot, with antibodies to tyrosine-tubulin and then vimentin (Fig. 6Go). Consistent with Fig. 5Go, the levels of tubulin monomer remained constant between control and TE-treated Sertoli cells. When the same immunoblot was probed with an antibody to vimentin, the soluble fraction of Sertoli cell protein isolated from TE-treated rats exhibited multiple banding patterns (Fig. 6Go), whereas the soluble protein isolated from control Sertoli cells showed a doublet. The multiple bands seen in Sertoli cells from TE rats consisted of the 57-kDa vimentin monomer and three lower-molecular-mass bands, the smallest of which was approximately 45 kDa in size. As discussed below, the multiple banding was suggestive of proteolytic degradation of the vimentin protein monomer. The doublet in the control protein sample corresponded to the 57-kDa vimentin monomer and a smaller, approximately 54-kDa fragment. These results were found consistently in five separate studies, using five separate sets of rats.



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  		FIG. 6. Representative Western blot of vimentin and tyr-tubulin protein from soluble cytoplasmic fractions of freshly isolated Sertoli cells from control (no treatment) and 56-d TE-treated (TE) rats.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown herein that the Sertoli cell intermediate filament cytoskeleton responds to the lowering of intratesticular T by exhibiting a marked change in filament structure. This was in contrast to the microfilament and microtubule cytoskeletons, neither of which seemed to change in localization by the withdrawal of T. In the case of actin (microfilaments), this was expected, because previous research has shown that lowering intratesticular T had no effect on the actin (based junctions that adhere germ cells to Sertoli cells) (16).

The change in the distribution of vimentin filaments with reduced intratesticular T was not the result of reduced vimentin expression; vimentin protein expression remained constant in Sertoli cells from control and TE-treated animals relative to levels of tyrosine-tubulin and ß-actin. However, Western blot analyses of Sertoli cell fractions enriched for soluble protein gave evidence of cleavage of the vimentin protein monomer to multiple bands in the Sertoli cells from TE-treated rats. This result is not without precedent. Byun et al. (17) showed in vitro that vimentin can be cleaved by multiple members of the cysteine-aspartate protease (caspase) family of apoptotic proteins, notably effector caspases 3, 6, and 7. Additionally, vimentin has also been shown to be a substrate for proteolytic cleavage by the initiator caspase 9 (18). Although the cleavage of vimentin by activated caspases has been shown to yield a similar banding pattern on Western blots as was observed in the present study for the Sertoli cell vimentin from TE-treated rats in vivo, active caspases are found in cells undergoing programmed cell death, whereas Sertoli cells do not undergo apoptosis when placed under conditions of lowered intratesticular T (19). Thus, it seems unlikely that caspases are responsible for the cleavage of Sertoli cell vimentin after TE administration to rats. However, this does not preclude the possibility that another testis-specific aspartyl protease may be responsible for this phenomenon.

The biological role that vimentin plays in Sertoli cells and in other cells in which it is expressed is poorly understood. It has long been assumed that, as with the microfilament and microtubule cytoskeletons, intermediate filaments provide mechanical resiliency and strength to cells (20, 21, 22). However, these traditional notions are being challenged with the proposal that vimentin may play a role in cell signaling (23, 24). The vimentin knockout mouse has been shown to be fertile (25), suggesting that vimentin changes may follow germ cell loss rather than being the cause. However, subsequent analysis of these animals revealed that they possess problems associated with wound healing (26, 27) and defects in actin and focal adhesion distribution (28), as well as other deficiencies (reviewed in Ref.24). As yet, there have been no reports of analyses of the testes of these animals; and therefore, nothing is known about daily sperm count, gross testicular morphology, or how the testes respond to stressors such as heat, hormonal withdrawal, and/or toxic compounds.

Spermatogenesis is an androgen-dependent process. Lowering intratesticular T levels below 20 ng/ml in the rat results in the failure of spermatogenesis and the apoptotic death of germ cells (19, 29, 30). Because germ cells do not express the androgen receptor, the signal to die, after T withdrawal, must be communicated to them by the Sertoli cell through a biological mechanism that has yet to be characterized. Loss of the Sertoli cell intermediate filament cytoskeleton could be one mechanism by which the Sertoli cell communicates an apoptotic signal to germ cells after T withdrawal. The intricate branching of vimentin filaments in Sertoli cells makes direct contact with the desmosome-like structures that are present at sites of Sertoli cell-spermatogonia/spermatocyte adhesion (5) and at the specialized junctional complexes that bind Sertoli cells to round and elongating spermatids (5) in control animals.

Normally, vimentin filament structure varies dynamically in Sertoli cells throughout the stages of spermatogenesis, with filaments dramatically shortened before release of step 19 spermatids during the androgen-dependent stages VII and VIII (8). The present study demonstrates that a similar loss of vimentin filament structure is seen in all tubules in rats with intratesticular T levels below what is physiologically required to maintain spermatogenesis (29). Whether changes in vimentin structure cause or result from the release of germ cells is not known.

The collapse of the Sertoli intermediate filament cytoskeleton has also been observed in cryptorchid testes of immature rats (31); in such testes, immunostaining of vimentin revealed loss of intermediate filament extensions and filament collapse to a perinuclear localization, coinciding with massive germ cell apoptosis. Additionally, administration of the toxins mono-(2-ethylhexyl) phthalate and colchicine has been shown to result in collapse of the vimentin cytoskeleton of Sertoli cells, followed by germ cell apoptosis and sloughing (32, 33, 34). Similarly, the toxin 2,5-hexanedione and the fungicide benomyl also have been shown to alter the distribution of the Sertoli cell vimentin cytoskeleton (35, 36). Thus, loss of normal Sertoli intermediate filament dynamics has been shown to occur in concert with the failure of spermatogenesis after hormonal withdrawal (present study), increased temperature (cryptorchidism) (31), and chemical insult (32, 33, 34, 35, 36). However, it remains unclear, in all these instances, whether the loss of vimentin filament structure is a cause of germ cell apoptosis after testicular insult, or is a secondary effect resulting from loss of germ cell adhesion that occurs by another, undefined mechanism. It has been reported, for the human, that vimentin localization remained unaffected despite significant deficiencies in germ cells (37). If this is generally the case, it would mean that loss of vimentin structure may be the result of reduced T and not loss of germ cells.

In conclusion, this study has demonstrated that the reduction of intratesticular T, to levels below that required to maintain spermatogenesis, results in a loss of Sertoli cell intermediate filament structural integrity and of normal vimentin distribution in the cell. This was brought about not by the loss of expression of the vimentin protein, but rather by fragmentation of the vimentin protein monomer, perhaps by proteolytic degradation. Changes in the biochemical properties of vimentin may represent a general mechanism used by the Sertoli cell to release adherent germ cells or to communicate a death signal to germ cells after reduction of intratesticular T concentration and perhaps other testicular insults as well.


   Footnotes
 
This work was supported by National Institutes of Health (NIH)/National Institute of Child Health and Human Development, through Cooperative Agreement U54-HD36209, as part of the Specialized Cooperative Centers Program in Reproduction Research, and by NIH Grant HD44258.

Abbreviations: E, Estradiol; IF, interstitial fluid; SDS, sodium dodecyl sulfate; T, testosterone; TE, T and E.

Received June 11, 2003.

Accepted for publication August 26, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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