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BOR - Papers in Press, published online ahead of print April 2, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.011817
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BIOLOGY OF REPRODUCTION 69, 322–330 (2003)
DOI: 10.1095/biolreprod.102.011817
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

{gamma}-Tubulin Overexpression in Sertoli Cells In Vivo. II: Retention of Spermatids, Residual Bodies, and Germ Cell Apoptosis1

Shawna L. Fleming3, Peter R. Shank4, and Kim Boekelheide2,3

Departments of Pathology and Laboratory Medicine3 Molecular Microbiology and Immunology,4 Brown University, Providence, Rhode Island 02912


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The degree of germ cell dependence on Sertoli cell-mediated activities has been a subject of considerable attention. Sertoli cell secretory pathways have been extensively studied both in an effort to understand their normal physiologic roles and as targets for pharmacologic and toxicant activity. To determine the degree to which normal spermatogenesis depends on key functions of the Sertoli cell microtubule network, adenoviral vectors that overexpress the microtubule nucleating protein, {gamma}-tubulin, were delivered to Sertoli cells in vivo. {gamma}-Tubulin overexpression disrupts the Sertoli cell microtubule network (as described in the companion article); leads to gross disorganization of the seminiferous epithelium, inducing retention of spermatids and residual bodies; and causes germ cell apoptosis. These data are consistent with earlier studies in which toxicants and pharmacologic agents were used to disrupt microtubule networks. These data confirm that Sertoli cell microtubule networks play an important role in maintaining the organization of the seminiferous epithelium and that in the absence of an intact Sertoli cell microtubule network, germ cell viability is impaired.

Sertoli cells, spermatogenesis, testis, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The spatial distribution of germ cells in the Sertoli cell cytoplasm is highly ordered. The layers of germ cells in the seminiferous epithelium form a gradient, in which less mature germ cells occupy basal positions and more mature germ cells, are positioned apically. This pattern is not invariant, however, because during the final cycle of the seminiferous epithelium, spermatids become positioned toward the basement membrane and are returned to the lumenal surface, where they are released [1]. Thus, the mobility of germ cells in the seminiferous epithelium is primarily basal to apical, parallel to the long axis of the Sertoli cell, although their trajectory is not a simple, linear one.

Sertoli cells secrete numerous substances into the lumen of the seminiferous epithelium and engage in transcytosis of materials from the basal to the apical compartment, both in vitro and in vivo [2–7]. Many Sertoli-derived products are necessary for germ cell functions and have been demonstrated to be taken up by germ cells [5, 6]. The pattern of cell and secretory movement coincides with the distribution of Sertoli cell microtubules [8–11].

Sertoli cell microtubules are prominent features of the seminiferous epithelium. The distribution of microtubules in Sertoli cells is most dense along the trunk, with fine extensions observed in the vicinity of apically-embedded elongate or elongating spermatids during some stages [12, 13]. Immunostaining for tubulin in cross-sections of seminiferous tubules reveals a dense, spoke-like pattern that radiates from the base of the seminiferous epithelium toward the lumen [13–15]. The conspicuousness of these structures, and their localization along the major route of cellular and secretory movement have led to speculation about their roles in spermatid and intracellular transport processes [8–12, 15–18].

Microtubules are involved in such diverse functions as maintenance of cellular shape, locomotion, polarized secretion, and positioning of organelles. The notion that microtubules are involved in intracellular transport is not unique to Sertoli cells. Indeed, many cells with distinct apicobasal axes exhibit polarized secretion, which has been demonstrated to be microtubule dependent by a variety of techniques [19–22]. Loss of microtubule polymers is associated with reduced rates of transport and mislocalization of organelles and secreted proteins [20, 22].

Data supporting the importance of microtubule-based functions in the testis have been largely derived from studies with toxicants and pharmacologic agents that target microtubules. 2,5-Hexanedione (2,5-HD) is one testicular toxicant for which a considerable database of information is available. 2,5-HD induces testicular injury, which is correlated with alterations in Sertoli cell morphology [23] and testicular microtubule assembly dynamics [24–27], reduction in seminiferous tubule fluid secretion [28], and increased germ cells apoptosis [29]. Although the data suggest that 2,5-HD-induced testicular injury is the result of actions on Sertoli cell microtubule networks, it is not possible to rule out that effects are due, at least in part, to direct effects on germ cells.

In this study, adenoviral vectors that overexpress {gamma}-tubulin were delivered to Sertoli cells in adult testes. Adenoviral vectors have proven useful for selectively targeting transgene expression to Sertoli cells when delivered to the lumen of the seminiferous epithelium [30–32]. Because toxicants and pharmacologic agents that target ubiquitous molecules such as tubulin cannot be selectively delivered to particular cell types, an adenoviral vector was used to specifically target expression of a microtubule nucleating protein to Sertoli cells. Overexpression of {gamma}-tubulin has been demonstrated to perturb microtubule nucleation patterns in cultured cells by promoting ectopic nucleation of microtubules [33, 34]. Similar to Taxol or 2,5-HD, {gamma}-tubulin promotes microtubule nucleation by lowering the concentration at which free tubulin subunits assemble into polymers [24, 35–39], although the mechanistic basis of this is presumably distinct from these compounds. This method was used to test the hypothesis that disruption of Sertoli cell microtubule networks in vivo leads to disruption of spermatogenesis.

The first of these companion articles describes the effects of {gamma}-tubulin overexpression on the microtubule structure of Sertoli cells in infected tissues. The second article details the outcomes of these effects on the germ cells associated with infected Sertoli cells and describes the histopathologic changes that resulted from the microtubule disruption.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
General

Young adult male Fischer rats (150–175 g) were obtained from Charles River Laboratories (Wilmington, MA) and housed in hanging wire cages at a constant temperature (70° ± 2C), with 35–70% humidity and a 12L:12D schedule. Rats were acclimatized for at least 3 days prior to experimental manipulation and treated according to the NIH Guide for the Care and Use of Laboratory Animals.

Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified. The control viral vector, AdGFP, was purchased from QbioGene (Montreal, Canada). Microscopy was performed using an Eclipse E800 microscope equipped for epifluorescence (Nikon, Melville, NY). Digital photomicroscopy was performed using a SpotRT camera (Diagnostic Instruments, Sterling Heights, MI). Photoshop (Adobe, San Jose, CA) was used for post-acquisition image processing.

Delivery of Adenoviral Vectors to the Seminiferous Epithelium in Vivo

The viral vectors used in this study expressed {gamma}-tubulin either co-ordinately with EGFP or expressed a c-terminal fusion of enhanced green fluorescent protein (EGFP) to {gamma}-tubulin (as described in the companion article). Viral constructs were driven by tetracycline-regulatable promoters, which require coadministration of AdtTA, the viral vector that expresses tTA, the "Tet-Off" transactivator protein (a generous gift of Drs. Thomas C. Harding and James Uney, University of Bristol, Bristol, UK) [40–42].

Between 50 and 100 µl of adenoviral stock, containing 1% blue food dye, was introduced into the testis (as described in the companion article).

Histological Evaluation

Histologic processing and staining and immunofluorescence methods were performed as described in the companion article.

To determine the effect of viral infection on spermatid and residual body position, periodic acid-Schiff/hematoxylin (PAS/H)-stained tissue was examined by bright-field microscopy. To determine the effect of {gamma}-tubulin overexpression on localization of step 19 spermatids, seminiferous tubules in stages VII–VIII were analyzed for the presence of basally localized spermatids. Those seminiferous tubules in which one or more spermatid heads were positioned in the lower 50% of the seminiferous epithelium were considered to have lagging spermatids. Data are presented as the proportion of stage VII–VIII seminiferous tubules containing basally positioned spermatids divided by the number of stage VII–VIII seminiferous tubules examined.

To analyze the effect of viral infection on spermatid release, seminiferous tubules in stages IX–XIV were examined for the presence of step 19 spermatids. Stage IX–XIV seminiferous tubules having attached or embedded step 19 spermatids were considered to have retained spermatids. Similarly, retention of residual bodies was defined by the presence of apically positioned residual bodies in stage IX–XIV seminiferous tubules. The proportion of seminiferous tubules containing retained step 19 spermatids or residual bodies was calculated by dividing the number of stage IX–XIV seminiferous tubules containing step 19 spermatids or residual bodies by the total number of IX–XIV seminiferous tubules examined. For all three histopathologic analyses, seven testes from each treatment and five uninfected control testes were examined.

Apoptosis

To evaluate the effect of {gamma}-tubulin overexpression on germ cell viability, testes were infected with AdBiEGFP{gamma}T/AdtTA (the construct that overexpresses EGFP and {gamma}-tubulin separately), AdTet{gamma}TEGFP/AdtTA (the construct that expresses a C-terminal fusion of EGFP to {gamma}-tubulin), or AdGFP/AdtTA (a GFP-expressing control), and harvested at 30 h post infection. Similar evaluations were performed at 48 h; however, it was determined that the majority of germ cell death had already occurred and that many germ cells were eliminated by that time point. For that reason, an earlier time point was selected. Tissues were fixed for 48 h by immersion, as described in the companion article, and 1 mm-thick sections were embedded in OCT (Miles, Inc., Elkhart, IN) and frozen. Sections were cut (8 µm) and tissue was processed according to the manufacturer's instructions (Apoptag, Intergen, Norcross, GA). Peroxidase-labeled antidioxigenin antibodies were used in co-ordination with the diaminobenzidine (DAB) colorimetric substrate for detection of labeled cells [29]. Tissues were then counterstained with methyl green and examined by bright-field microscopy for the presence of apoptotic cells. Three testes per infection regimen were examined, and all seminiferous tubule cross-sections per testis were evaluated. Apoptotic data are expressed as the number of seminiferous tubules containing 0, 1, 2–3, 4–6, or 7+ apoptotic cells per seminiferous tubule cross-section, divided by the total number of seminiferous tubules examined. The 4–6 and 7+ categories were pooled for statistical analysis.

Statistical Analysis

Statistical analyses were performed using StatView software (Abacus Concepts, Berkeley, CA). The rates of apoptosis measured in control-infected testes were compared with those observed in tissues infected with {gamma}-tubulin overexpressing viruses. Data were analyzed by one-way ANOVA, followed by multiple pair-wise comparisons using the Fisher protected least significant difference test. For all analyses, the criterion for significance was set at P < 0.05. SEMs are given.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Control Infections: Histopathologic Outcomes of Infection with AdGFP/AdtTA Vectors

Viral infection does not appreciably alter the histological appearance of the seminiferous epithelium. Tissues infected with control vectors that express an EGFP reporter (AdGFP/AdtTA) appear intact, compared with uninfected tissues (Fig. 1B vs. Fig. 1A). Despite strong transgene expression (Fig. 1C), tissues infected with AdGFP contain normal complements of germ cells. EGFP distributes throughout infected cells, outlining the positions of embedded germ cells (Fig. 1C, asterisks).



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  		FIG. 1. Histological architecture remains intact following infection with control viruses. Little difference is noted between uninfected (A) and infected (B) seminiferous tubules. Infected Sertoli cells (B, C, arrows) can be seen to surround germ cells (*). C) The distribution of EGFP demonstrates Sertoli specificity of infection. Dark spots (*) are areas of germ cells that are embedded in EGFP-containing Sertoli cell cytoplasm. A and B are similarly staged seminiferous tubules from the same testis. C is a serial section of the same seminiferous tubule as B

{gamma}-Tubulin Overexpression: Histopathologic Outcomes of Infection with AdBiEGFP{gamma}T/AdtTA Vectors

In contrast to control-infected tissues, overexpression of {gamma}-tubulin in vivo was observed to produce substantial histopathology by 48 h post infection. Infected seminiferous tubules were observed to lack germ cells and contain large numbers of retained spermatids. Figure 2B illustrates one seminiferous tubule infected with an adenoviral construct that drives expression of separate {gamma}-tubulin and EGFP reporter transcripts (AdBiEGFP{gamma}T/AdtTA). Compared with uninfected tubules (Fig. 2A), those expressing AdBiEGFP{gamma}T/AdtTA contain gaps in the epithelium in which immature germ cells have been lost (Fig. 2B) and contain retained spermatids (arrowheads) and residual bodies (long arrows), despite the presence of the next generation of elongating spermatids (short arrows). Strong transgene expression, as evidenced by high levels of EGFP expression, can be observed in infected cells (Fig. 2C).



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  		FIG. 2. Overexpression of {gamma}-tubulin leads to retention of spermatids and residual bodies and loss of germ cells. Compared with uninfected seminiferous tubules (A), those infected with AdBiEGFP{gamma}T (B) contain fewer germ cells. Newly elongating spermatids (short arrows) coincided with retained late spermatids (arrowheads), which were distributed throughout the epithelium and retained residual bodies (long arrows). Whereas uninfected tubules occasionally exhibited spermatid retention (arrowheads, A), they typically occurred at the lumenal edge, rather than throughout the epithelium, and were less numerous. Sertoli cells are marked with *. Infection resulted in high-level expression that was restricted to Sertoli cells (as can be observed in C). Arrowheads denote position of retained spermatids in C. Seminiferous tubules in A, B, and C are from the same testis; C is the same seminiferous tubule as B (serial section) and is a phase/fluorescence overlay to illustrate EGFP reporter localization and spermatid head position (arrowheads)

{gamma}-Tubulin Overexpression: Histopathologic Outcomes of Infection with AdTet{gamma}TEGFP/AdtTA ({gamma}-Tubulin-EGFP Fusion) Vectors

Similar to infection with AdBiEGFP{gamma}T, which expresses {gamma}-tubulin independently of EGFP, expression of {gamma}-tubulin fused at its C-terminus to EGFP (AdTet{gamma}TEGFP/AdtTA) results in retention of spermatids and residual bodies and depletion of germ cells. Compared with uninfected stage VII seminiferous tubules in which mature spermatids line the apical surface of the epithelium (Fig. 3A, arrowheads), spermatids in stage VII–VIII tubules infected with AdTet{gamma}TEGFP/AdtTA were seen throughout the epithelium (Fig. 3B, arrowheads). These spermatids were observed to co-associate with the next generation of early elongating spermatids (Fig. 3B, short arrows). Moreover, retained spermatids were observed to associate with residual bodies (Fig. 3B, long arrows). Similar to infection with AdBiEGFP{gamma}T, infection with AdTet{gamma}TEGFP led to loss of germ cells, indicated by the moth-eaten appearance of the seminiferous epithelium (Fig. 3A versus Fig. 3B). Interestingly, the {gamma}TEGFP fusion protein was observed to aggregate in the vicinity of many retained spermatids in infected seminiferous tubules (Fig. 3C, arrowheads).



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  		FIG. 3. Overexpression of the {gamma}TEGFP fusion protein leads to retention of spermatids and residual bodies and loss of germ cells. The pattern of germ cell loss, spermatid, and residual body retention in tissues that overexpress the {gamma}TEGFP fusion protein was similar to that observed following infection with AdBiEGFP{gamma}T. A) An uninfected stage VII tubule illustrates the normal position of spermatids (arrowheads) with their associated residual cytoplasms (long arrows). The next generation of spermatids (short arrows) was arranged in rows, and few spaces were noted in the seminiferous epithelium. In contrast, tissues infected with AdTet{gamma}TEGFP contained retained spermatids (arrowheads) and residual bodies (long arrows), despite the presence of step 9 spermatids (short arrows). Seminiferous tubules in A, B, and C were from the same testis; C is the same seminiferous tubule as B (serial section) and is a phase/fluorescence overlay to illustrate localization of {gamma}TEGFP transgene to spermatid heads (arrowheads)

Effect of {gamma}-Tubulin Overexpression on Spermatid Position

To quantitate the effect of {gamma}-tubulin expression on spermatid position, tissues were examined for the presence of lagging spermatids in stages VII–VIII. No significant differences were noted between uninfected tissues and those infected with control viruses (AdGFP/AdtTA) in the frequency of stage VII–VIII seminiferous tubules containing spermatids in the lower 50% of the epithelium. The proportion of seminiferous tubules in control-infected tissues containing basally positioned spermatids in stages VII–VIII was 2.9% (Fig. 4). Similarly, 3.4% of stage VII–VIII seminiferous tubules in uninfected tissues exhibited spermatid retention (Fig. 4).



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  		FIG. 4. Overexpression of {gamma}-tubulin inhibits spermatid localization to the lumen of the seminiferous epithelium. Compared with uninfected and control infected tissues, overexpression of {gamma}-tubulin for 48 h led to spermatid retention at the basal aspect of stage VII–VIII seminiferous tubules in AdBiEGFP{gamma}T (*, ANOVA, P < 0.05). Data for AdTet{gamma}TEGFP ({gamma}TEGFP fusion construct) infections were marginally significant (P = 0.055)

In contrast, infection with AdBiEGFP{gamma}T/AdtTA was observed to impair spermatid mobility in the seminiferous epithelium. Compared with uninfected and control-infected tissue, tissues infected with AdBiEGFP{gamma}T/AdtTA exhibited increased numbers of misplaced spermatids in stage VII–VIII seminiferous tubules (Fig. 4). In tissues infected with AdBiEGFP{gamma}T/AdtTA, 19.1% of stage VII–VIII seminiferous tubules contained spermatid heads below 50% of the epithelial thickness (Fig. 4).

Similarly, the proportion of stage VII–VIII seminiferous tubules with spermatid heads in the lower 50% of the seminiferous epithelium rose to 18.4% in tissues overexpressing the {gamma}-tubulin-EGFP fusion protein (Fig. 4); however, these data were marginally significant (P = 0.055).

Effect of {gamma}-Tubulin Overexpression on Spermatid Retention

Infection with AdGFP/AdtTA did not perturb the release of spermatids from the seminiferous epithelium. The percentage of stage IX–XIV seminiferous tubules containing retained spermatids in AdGFP/AdtTA-infected tissues was 5.3%, which was not significantly different from that of uninfected tissues (6.9%, Fig. 5).



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  		FIG. 5. Overexpression of {gamma}-tubulin leads to retention of spermatids in stage IX–XIV seminiferous tubules. Expression of {gamma}-tubulin, either fused to EGFP (AdTet{gamma}TEGFP) or as a separate transcript (AdBiEGFP{gamma}T), led to retention of spermatids in the seminiferous epithelium of tissues infected for 48 h with AdBiEGFP{gamma}T or AdTet{gamma}TEGFP, compared with control-infected or uninfected tissues (*, P < 0.05)

In contrast, spermatid release was impaired by overexpression of {gamma}-tubulin. Tissues infected with AdBiEGFP{gamma}T/AdtTA were observed to contain retained spermatids in 29.1% of stage IX–XIV seminiferous tubules (Fig. 5). Spermatid retention was also observed in tissues infected with AdTet{gamma}TEGFP/AdtTA. The percentage of stage IX–XIV seminiferous tubules containing retained spermatids rose from 5.3% in control-infected tissues to 23.1% in tissues infected with AdTet{gamma}TEGFP/AdtTA (Fig. 5).

Effect of {gamma}-Tubulin Overexpression on Residual Body Elimination

Infection with AdGFP/AdtTA (control infections) did not increase the proportion of seminiferous tubules containing retained residual bodies. Residual body retention was noted in 1.0% of stage IX–XIV seminiferous tubules in uninfected testes, compared with 3.1% of stage IX–XIV seminiferous tubules in AdGFP/AdtTA-infected testes. This difference was not statistically significant (P > 0.52; Fig. 6).



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  		FIG. 6. Overexpression of {gamma}-tubulin leads to retention of residual bodies in stage IX–XIV seminiferous tubules. Expression of {gamma}-tubulin or {gamma}TEGFP fusion protein led to retention of residual bodies in testes infected for 48 h with either AdBiEGFP{gamma}T or AdTet{gamma}TEGFP, compared with control-infected (AdGFP) or uninfected tissues (*P < 0.01)

Overexpression of {gamma}-tubulin increased the frequency of residual body retention. In AdBiEGFP{gamma}T/AdtTA-infected tissues, 14.1% of stage IX–XIV seminiferous tubules were observed to contain retained residual bodies (Fig. 6). Similarly, overexpression of the {gamma}-tubulin-EGFP fusion protein (AdTet{gamma}TEGFP/AdtTA infection) also led to retention of residual bodies. The frequency of residual body retention in stage IX–XIV seminiferous tubules was 15.7% in testes infected with AdTet{gamma}TEGFP/AdtTA (Fig. 6).

{gamma}-Tubulin Overexpression: Germ Cell Apoptosis

Histological images of infected seminiferous tubules reveal the extent of germ cell loss that occurred by 48 h post infection in {gamma}-tubulin-overexpressing seminiferous tubules, compared with tubules expressing control (EGFP) transgenes (Fig. 1 vs. Figs. 2 and 3). At this time point, however, cells were already lost from the seminiferous epithelium. To measure the rate of germ cell apoptosis, tissues were harvested at an earlier time point, prior to germ cell depletion. Germ cell apoptosis was measured in tissues infected for 30 h with AdBiEGFP{gamma}T/AdtTA, AdTet{gamma}TEGFP/AdtTA, or AdGFP/AdtTA (control). As suggested by the histological appearance of tissues, germ cell apoptosis was elevated in {gamma}-tubulin-overexpressing tissues relative to control-infected tissues (Fig. 7). At 30 h post infection, 1% of seminiferous tubules in testes infected with control viruses, versus 3.4% of seminiferous tubules in testes infected with AdBiEGFP{gamma}T/AdtTA were observed to have more than three apoptotic cells per tubule cross-section (Fig. 7). Similarly, in tissues infected with AdTet{gamma}TEGFP/AdtTA, 5.0% of seminiferous tubules contained more than three apoptotic cells (Fig. 7).



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  		FIG. 7. Overexpression of {gamma}-tubulin leads to increased germ cell apoptosis. The proportion of seminiferous tubules containing more than 3 apoptotic cells increased in both {gamma}-tubulin overexpression regimens, compared with control-infected tissues (*P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The role of Sertoli cell microtubules in supporting spermatogenesis has been a source of interest for a number of years. Studies with toxicants and pharmacologic agents that target microtubules have suggested that microtubule-based functions in Sertoli cells are important for their ability to support germ cell development. Depolymerization of microtubules with colchicine or vinblastine leads to a stage-dependent circumferential sloughing of the germinal epithelium with attached Sertoli cell cytoplasm into the lumen of the tubule [14, 16]. This reveals a structural vulnerability in the seminiferous epithelium and suggests that the columnar microtubule network is important in providing lateral stability, perhaps against the hydrostatic force generated by flow of seminiferous tubule fluid. More subtle alterations included altered positioning of germ cells in the seminiferous epithelium, perhaps including retraction of apically located spermatids [18]. Long-term treatment with colchicine caused failure of elongate spermatids to localize apically in the seminiferous epithelium, accumulation of smooth endoplasmic reticulum (SER) and abnormal acrosome formation in spermatid heads [18]. These results support the notion that Sertoli cell microtubules function in vesicle transport and that they are involved in the process of histological organization and shaping of spermatid heads.

Interestingly, however, many of these effects are also observed following treatment of testes with Taxol, a microtubule-stabilizing compound [17]. Effects included failure of advanced spermatids to become positioned near the lumen of the tubule, failure of residual bodies to be phagocytized, and/or failure of phagocytized residual bodies to be transported to lysosomes [17].

Similarly, models of toxicant-mediated injury in the testis have suggested that microtubules play an important role in the function of the testis. Chronic dosing with 2,5-HD leads to a slowly progressive injury culminating in a state of irreversible testicular trophy in rats [23, 25]. In vitro, polymers formed from 2,5-HD-treated tubulin exhibit reduced rates of kinesin-based transport, and treated tubulin disrupts spindle formation when injected into sea urchin embryos [43, 44]. In vivo, 2,5-HD exposure is associated with alterations in Sertoli cell cytoskeletal staining patterns, mislocalization of microtubule-associated motor proteins, abnormalities in spermatid head formation, and reduction in seminiferous tubule fluid formation [45–47]. These data suggest that 2,5-HD mediates its effect by subtly altering the dynamics of Sertoli cell microtubule networks.

Taken together, the data from these diverse models of altered testicular tubulin physiology suggest that microtubule disruption, whether because of stabilization or depolymerization, has a profound effect on transport processes in the testis. Because of the observed interaction between ectoplasmic specializations (ESs) and microtubules [10, 11], it is expected that disruption of microtubule networks in Sertoli cells would lead to failure of elongate spermatids to migrate toward the apical surface as they mature and prepare for release.

Overexpression of {gamma}-tubulin has been demonstrated to affect the distribution of microtubules in cells in vitro, leading to ectopic nucleation of microtubules [33, 34]. This effect has been observed in a range of eukaryotic cells, including human and yeast [33, 34]. Hyperpolymerization of microtubules following overexpression of {gamma}-tubulin is similar, although mechanistically unrelated, to the effect of Taxol or 2,5-HD treatment on testicular cells in vivo [17, 48].

The question of whether germ cell survival and/or differentiation depends on key aspects of Sertoli cell cytoskeletal networks was addressed by construction of adenoviral vectors that overexpress the microtubule nucleating protein {gamma}-tubulin, with the goal of evaluating the effect of microtubule disruption in Sertoli cells of adult animals on the seminiferous epithelium, using a defined, molecular mechanism. {gamma}-Tubulin overexpression elicited a number of alterations in the seminiferous epithelium, including mislocalization of spermatids and retention of residual bodies. Unlike previous experiments aimed at addressing questions of Sertoli cell microtubule function, overexpression of {gamma}-tubulin in Sertoli cells has the advantage of avoiding indirect effects on germ cell microtubule networks. The observation that residual bodies persist beyond stage IX and that late spermatids are retained throughout the seminiferous epithelium in testes overexpressing {gamma}-tubulin, suggests that Sertoli cell microtubule networks are directly responsible for these aspects of seminiferous epithelial function.

The complexity of infection likely underlies the complex pattern of injury manifest by these treatments. Lesions in seminiferous tubules infected with {gamma}-tubulin-overexpressing viruses ranged from moderate to severe, which correlated with the level of fluorescence observed in the tissue. Seminiferous tubules upon which detailed tubulin immunostaining and histological analysis was performed were selected conservatively and included those with representative alterations but with relatively intact architecture. These criteria were believed to facilitate mechanistic understanding of the injury process while providing insight into the specific injuries observed. Moreover, because of the potential for direct toxicity to Sertoli cells via {gamma}-tubulin overexpression and the knowledge that levels of expression correlate directly with manifestations of toxicity, those seminiferous tubules exhibiting moderate expression levels are likely to yield informative data about the role of microtubule disruption on Sertoli cells, independent of a directly toxic effect of {gamma}-tubulin.

Notwithstanding the variability in expression levels, however, the lesions induced were specific to {gamma}-tubulin overexpression and were not simply the result of viral infection. Histological architecture, where affected in controls, was observed to be more consistent with damage resulting from the injection procedure than damage caused by the presence of viral vectors. The most frequent injury noted in seminiferous tubules infected for 48 h with the control vector, AdGFP/AdtTA, was the occurrence of sloughed materials, consisting chiefly of prematurely detached elongating and round spermatids.

The histological perturbations observed in tissues expressing the {gamma}-tubulin transgenes were markedly different from those expressing GFP/tTA alone (control infections). {gamma}-Tubulin overexpression resulted in retention of spermatids in the seminiferous epithelium. This pattern of spermatid retention has been observed previously with application of microtubule disrupters to the testis [16–18]. The presumption in those studies was that inhibition of the Sertoli cell microtubule network led to inhibition of spermatid transport. The fact that microtubule networks in tubules overexpressing {gamma}-tubulin were spatially disrupted, compared with uninfected control seminiferous tubules at similar stages of spermatogenesis, lends support to the notion that selective disruption of Sertoli cell microtubule networks impairs the ability of the Sertoli cell to organize the seminiferous epithelium.

The proportion of seminiferous tubules that contained high numbers of apoptotic germ cells, and in which spermatids and residual bodies were retained, was observed to increase in tubules infected with {gamma}-tubulin-overexpressing viruses, compared to either control-infected or uninfected tissues. Numerically, however, effects were small, compared with treatments that act globally. This undoubtedly is due to the fact that not all seminiferous tubules become infected. The quantitation of effect is therefore diluted by the presence of a large number of uninfected tubules.

Given information from other studies with microtubule disrupting agents in the testis, it is presumed that the mechanistic basis for the histological alterations resides in the inherent capacity of overexpressed {gamma}-tubulin to redirect patterns of microtubule organization in cells [33, 34]. However, a direct effect of {gamma}-tubulin on spermatid transit in the seminiferous epithelium cannot be ruled out. That {gamma}-tubulin is seen to aggregate in the vicinity of spermatid heads (as shown in the companion article) suggests that the overexpressed {gamma}-tubulin may itself impair movement of the spermatid in the seminiferous epithelium by effectively cementing it in position. Whether {gamma}-tubulin impairs spermatid transit in the seminiferous epithelium directly or indirectly, via its action on Sertoli cell microtubule polymers, is unclear.

A potential mechanistic explanation for how Sertoli cell microtubule disruption leads to spermatid retention in the seminiferous epithelium derives from a well-studied model of spermatid transport [8, 10, 11, 49–51]. Data in support of this model suggest that elongate spermatids form attachments to the Sertoli cell microtubule network via ESs. These structures occur on the Sertoli cell cytoplasmic face and are formed at sites of elongate spermatid attachment to the Sertoli cell plasma membrane. The structure of ESs, and the observation that elongate spermatids travel in the seminiferous epithelium along a path that coincides with the orientation of the microtubules attached to ESs, led to the proposition that ESs support transit of elongate spermatids along the apical to basal axis by means of associated motor proteins [8, 10, 11]. According to this model, microtubules are nucleated from the lumenal surface of seminiferous tubules [9], and spermatid-attached ESs hook onto vertically oriented microtubule networks in the Sertoli cell cytoplasm. Throughout the maturation cycle, spermatids are pushed and pulled through the thickness of the seminiferous epithelium with the assistance of microtubule-dependent motors that associate with ESs in the Sertoli cell [10, 11, 49, 51, 52]. That this structural reorganization occurs invariably throughout the spermatogenic cycle suggests that these circuitous vertical movements in the seminiferous epithelium are of biological significance for germ cells.

Microtubule-based mobility is known to be important to cells; however, the interdependence of one cell type on the microtubule-based processes of another is a level of complexity that has not been extensively examined outside the testis. The dependence of germ cells on Sertoli cell microtubule-mediated functions, such as secretion of nutrients and binding proteins (lactate, transferrin, and androgen binding protein) and transportation of spermatids in the seminiferous epithelium, suggests that Sertoli cell microtubules are extremely important to the function of the tissue. That {gamma}-tubulin overexpression disrupts the distribution of microtubules in Sertoli cells (as shown in the companion article) suggests that secretory function is important for germ cell viability, although the mechanism of germ cell loss is still unknown.

These data support notions that have been long suggested by pharmacologic disruptions of testicular microtubules: that Sertoli cells are themselves responsible for vertical movement of spermatids in the seminiferous epithelium, and that inhibition of spermatid transport can occur when Sertoli cell microtubule networks are altered. These data provide the first direct in vivo evidence for the role of the Sertoli cell microtubule networks in the maintenance of the epithelial architecture, a role that has been proposed for many decades.


   ACKNOWLEDGMENTS
 
The authors wish to thank the laboratory of Dr. Bert Vogelstein for the gift of the pShuttle and pAdEasy plasmids and the BJ5183 strain of Escherichia coli; Dr. Berl Oakley for the gift of the pH3-16 plasmid containing the {gamma}-tubulin transgene; Dr. David Brown, University of Ottawa, for the gift of the monoclonal antitubulin antibody, 5A6; and Drs. Thomas Harding and James Uney, University of Bristol, Bristol, UK, for the gift of the AdtTA viral stock. Thanks also are due to Susan Hall for her assistance with histology and in devising the injection apparatus.


   FOOTNOTES
 
1 This work was supported in part by RO1 ES08956 from the National Institutes of Environmental Health Sciences. Back

2 Correspondence. Kim Boekelheide, Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912. FAX: 401 863 9008; Kim_Boekelheide{at}Brown.edu Back

Received: 26 September 2002.

First decision: 13 November 2002.

Accepted: 3 March 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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