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a Prince Henry's Institute of Medical Research, Monash Medical Centre, Clayton, Victoria 3168, Australia
b Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611
ABSTRACT
The Sertoli cell ectoplasmic specialization is a unique junctional structure involved in the interaction between elongating spermatids and Sertoli cells. We have previously shown that suppression of testicular testosterone in adult rats by low-dose testosterone and estradiol (TE) treatment causes the premature detachment of step 8 round spermatids from the Sertoli cell. Because these detaching round spermatids would normally associate with the Sertoli cell via the ectoplasmic specialization, we hypothesized that ectoplasmic specializations would be absent in the seminiferous epithelium of TE-treated rats, and the lack of this junction would cause round spermatids to detach. In this study, we investigated Sertoli cell ectoplasmic specializations in normal and TE-treated rat testis using electron microscopy and localization of known ectoplasmic specialization-associated proteins (espin, actin, and vinculin) by immunocytochemistry and confocal microscopy. In TE-treated rats where round spermatid detachment was occurring, ectoplasmic specializations of normal morphology were observed opposite the remaining step 8 spermatids in the epithelium and, importantly, in the adluminal Sertoli cell cytoplasm during and after round spermatid detachment. When higher doses of testosterone were administered to promote the reattachment of all step 8 round spermatids, newly elongating spermatids associated with ectoplasmic specialization proteins within 2 days. We concluded that the Sertoli cell ectoplasmic specialization structure is qualitatively normal in TE-treated rats, and thus the absence of this structure is unlikely to be the cause of round spermatid detachment. We suggest that defects in adhesion molecules between round spermatids and Sertoli cells are likely to be involved in the testosterone-dependent detachment of round spermatids from the seminiferous epithelium.
FSH, Sertoli cells, spermatid, spermatogenesis, testosterone
INTRODUCTION
The process of sperm production is well known to be regulated by testosterone and FSH. We have shown that the suppression of testicular testosterone causes the detachment of haploid step 8 round spermatids from the adult rat seminiferous epithelium [1]. Normally, step 8 round spermatids associate with a specialized Sertoli cell structure called the ectoplasmic specialization (ES). Therefore, we reasoned that the testosterone-dependent loss of step 8 round spermatids may be due to the absence of this structure within the Sertoli cell.
The Sertoli cell ES is an important and unique junctional structure within the seminiferous epithelium [2–4] and is defined as hexagonally packed noncontractile actin filaments sandwiched between the Sertoli cell plasma membrane and underlying endoplasmic reticulum [3]. The ES is considered to be an adhesion junction [5–8], and adhesion molecules on the surface of the Sertoli cell have been postulated to interact with membrane-bound components of the ES. However, the identity of molecules in the putative "adhesive domain" is unclear [4]. The ES structure has therefore been described as an actin-associated adhesion junction that is postulated to stabilize an adhesive membrane domain [6]. Although the ES has been well characterized by electron microscopy [2, 3], the molecular composition is less well understood. Immunocytochemical and immunogold techniques have shown that the Sertoli cell ES contains actin [9], vinculin [10], and fimbrin [6]. Recently, a novel actin-binding protein, espin, was shown to be a component of the Sertoli cell ES [11].
The ES is observed by electron microscopy opposite germ cells in the adluminal compartment of the epithelium. Although focal patches of ES are observed opposite pachytene spermatocytes and early round spermatids, the structure is extensively associated with elongating spermatids [2]. In the rat, extensive ES first forms opposite step 8 round spermatids and is removed prior to the release of mature step 19 spermatids [2]. The spermatid-ES interface functions as an extremely tight adhesion junction [5], and this junction may be important for the translocation of spermatids through the epithelium as they elongate [12]. Recently, the ES was shown to promote the movement of microtubules [13], and this movement may involve the molecular motor protein dynein [14]. Thus, one of the functions of the ES may be to facilitate translocation of elongating spermatids through the epithelium. At the completion of elongation, ES is removed during spermatid release (spermiation), and this removal is thought to be facilitated by specialized structures called tubulobulbar complexes [15].
The ES structure is also found on either side of the junctional complex that is formed between two Sertoli cells towards their bases. This inter-Sertoli cell complex [16], which also contains gap and occluding junctions, restricts the passage of macromolecules and forms the basis of the blood-testis barrier that divides the seminiferous epithelium into the basal and adluminal compartments. Given that the ES is an important structure at two sites within the seminiferous epithelium, it is of interest to study its formation, regulation, and composition.
To study the effects of the suppression of testicular testosterone on spermatogenesis, low doses of testosterone in combination with low doses of estradiol (TE treatment) are given to adult rats via Silastic implants for periods of 6–12 wk [1, 17–19]. The slightly supraphysiological serum androgen and estrogen levels feedback on the pituitary to suppress LH but not FSH release. Consequently, the level of testosterone in the testis is markedly suppressed [18], resulting in the absence of mature sperm in the seminiferous epithelium [18, 19]. When larger doses of testosterone are then given, testicular testosterone levels are partially restored, and sperm production returns to near normal [17, 19]. An important role of testosterone is in the conversion of step 7 to step 8 round spermatids, just prior to the elongation phase of spermiogenesis [1, 18]. After TE treatment, approximately 80% of step 8 round spermatids lose contact with the Sertoli cell and are released into the seminiferous tubule lumen before proceeding to the epididymis, where they degenerate [1]. The remaining step 8 spermatids degenerate within the seminiferous epithelium, and virtually no spermatids proceed through the elongation phase [18]. Within 4 days of higher dose testosterone treatment, all step 8 round spermatids reattach to the seminiferous epithelium in a dose-responsive manner [18], and elongation of spermatids then proceeds. Because the production of mature spermatids in the testis is dependent on the ability of round spermatids to adhere to the seminiferous epithelium, it becomes important to understand the control of adhesive junctions between step 8 round spermatids and Sertoli cells.
We hypothesized that the detachment of step 8 round spermatids from Sertoli cells may be due to the absence of the specialized Sertoli cell ES structure in the seminiferous epithelium. For the purpose of this study, we used the term ES structure to reflect the submembrane component of the ES, consisting of hexagonally packed filamentous actin bundles sandwiched between the Sertoli cell plasma membrane and smooth endoplasmic reticulum. In this case, the term ES structure does not include any intercellular adhesive elements that may be a part of the junction. The aim of this study was to investigate the presence of ES structures in the seminiferous epithelium of normal and hormone-treated adult rats. The ES was assessed by visualization of the structure by electron microscopy, and then light microscopy and confocal analysis of ES-associated molecules actin, vinculin, and espin were used to give further information on ES in the testis.
MATERIALS AND METHODS
Animals
Adult male Sprague-Dawley rats (80–100 days old) were obtained from the Monash Central Animal House and housed under a 12L:12D cycle with free access to food and water. Three-centimeter implants filled with testosterone powder (Sigma, St. Louis, MO) and 0.4-cm implants filled with estradiol powder (Sigma) were prepared as described previously [18]. Animals were anesthetized by ether inhalation, and implants were placed subcutaneously along the dorsal surface. Animals received either one 3-cm testosterone plus one 0.4-cm estradiol implant (TE implants) [18] or no implants (control) for 8 wk. At the end of the 8-wk period, control animals (n = 6) and TE-treated animals (n = 6) were killed. In addition, a further eight TE-treated animals had their TE implants removed and replaced with 3- x 8-cm testosterone implants (T24) for 2 (n = 4) or 4 (n = 4) days to partially restore testicular testosterone levels [18]. The study was approved by the Monash Medical Centre Animal Ethics Committee.
Tissue Preparation
At the end of the experiment animals were anesthetized and subjected to whole-body perfusion as previously described [18].
For electron microscopic analysis, two control and two TE-treated rats were perfused with 5% glutaraldehyde in 0.1 M cacodylate buffer. A systematic uniform random sampling scheme [20] was used to select three wedges of perfusion-fixed testis, which were postfixed in osmium tetroxide/potassium ferrocyanide, block stained in uranyl acetate, and embedded in Epon araldite [19]. One-micrometer sections were prepared from each wedge and stained with toluidine blue for examination of tubule stages. An area containing approximately three or four round tubule cross sections with tubules in stages VII and/or VIII was selected for further examination by electron microscopy. Ultrathin sections were then prepared and stained using standard techniques.
For immunocytochemical analysis, control rats, TE-treated rats, and rats treated with TE + T24 for 2 or 4 days (n = 4 rats/group) were subjected to whole body perfusion with Bouin fluid. Following perfusion fixation, testes were removed and immersed in Bouin fluid for <5 h and then kept in 70% ethanol at 4°C until use. Wedges of testis were embedded in low-melting-point ribboning polyester wax [21, 22]; tissues were dehydrated in 70%, 90%, 99%, and absolute ethanol for 1 h each, infiltrated with 50% polyester wax (BDH, Poole, England) in ethanol for 1 h at 37°C and then with 90% polyester wax for 1 h at 37°C, and then tissues were embedded in 90% polyester wax and rapidly chilled on ice. Seven-micrometer sections were cut on a cryostat set at 0°C, floated onto a waterbath set at 32°C, and collected onto slides coated with 2% 3-aminopropyltriethoxy-saline (AAS; Sigma) and allowed to dry for 48–72 h at 4°C.
Electron Microscopy
Each section was examined at 60 kV using a JEOL 1200EX transmission electron microscope. For purposes of electron microscopic analysis, an ES structure was defined as hexagonally packed filamentous actin bundles sandwiched between Sertoli cell plasma membrane and smooth endoplasmic reticulum. In each tubule, the basal ES and the adluminal compartment was examined using a lower magnification (x3000–7000). Round spermatids were categorized as step 7 round spermatids, containing a central nucleus with an extensive acrosome [23], step 7–8 round spermatids, containing a nucleus polarized to one side of the cell and <25% of the spermatid's acrosome in contact with the plasma membrane, and step 8 round spermatids, with a polarized nucleus and 25–100% of the acrosome in contact with the spermatid plasma membrane. Following identification of a round spermatid, the entire circumference of the spermatid plasma membrane was examined on higher power (approximately x15 000–20 000). The presence of Sertoli cell ES was recorded when it was in apposition to spermatid plasma membrane. The percentage of spermatid acrosome in apposition to ES was also recorded. In addition, the entire junction between each spermatid and the Sertoli cell was examined for other structures, such as premature tubulobulbar complex formation and desmosome junctions. The number of cells examined in each grouping is indicated in Table 1. Three sections were examined per rat, and therefore approximately 3–5 stage VII and 3–5 stage VIII tubules were sampled per rat (n = 2 rats/group).
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Immunocytochemistry and Double-Label Immunofluorescence
Three ES-associated proteins, whose localization in the rat testis have been previously described, were immunolocalized for analysis of ES; 1) vinculin [21] detected by a mouse monoclonal antibody against human vinculin (clone h-Vin1; Sigma) at a dilution of 1:250, 2) actin [24] detected by a mouse monoclonal antibody that cross-reacts with all muscle and nonmuscle forms of actin (clone C4; ICN Biomedicals, Aurora, OH) at a dilution of 1:500, and 3) espin, detected by an immunoaffinity purified rabbit polyclonal antibody [11] used at a dilution of 1:50 for immunofluorescence and 1:200 for conventional light immunocytochemistry. Colocalization experiments involved combinations of either espin and actin or espin and vinculin antisera. Although vinculin and actin are known to be components of Sertoli cells, these molecules are also present at other sites within the seminiferous epithelium. Although espin has been shown to be located at basal and adluminal sites corresponding to the location of ES in the rat seminiferous epithelium [11], we reasoned that the colocalization of this molecule with the ES-associated molecules actin or vinculin would be a more convincing demonstration of ES structures than the identification of espin alone.
Immunocytochemistry and double label immunofluoresence was performed on polyester wax-embedded sections. Sections were dewaxed and rehydrated in a graded series of ethanol concentrations and then washed in PBS (0.01 M PBS, 0.154 M NaCl, pH 7.4, no sodium azide). Sections were then subjected to microwave antigen retrieval [25] in 0.01 M citric acid pH 6.0 for 10 min at 650 W and left undisturbed for 20 min. Sections for conventional immunocytochemistry were incubated in 0.3% hydrogen peroxide. All sections were then washed in PBS, blocked with 300 mM glycine in PBS for 10 min and 0.1% Triton X-100 in PBS for 10 min, and incubated in CAS Block (Zymed, South San Francisco, CA) containing 10% normal sheep serum for 20 min. Primary antibodies diluted in PBS were incubated for 2 h or overnight at room temperature. For conventional immunocytochemistry, sections were incubated for 1 h with an appropriate biotinylated second antibody followed by streptavidin-horseradish peroxidase ABC complex (Vectastain Elite; Vector Laboratories, Burlingame, CA). The pink substrate used was Vector VIP (Vector Laboratories), and the sections were counterstained with Mayer hematoxylin (Sigma) prior to dehydrating through ethanol and histolene and mounting in DPX (BDH). Sections for double-label immunofluorescence were incubated for 1 h with the appropriate fluorescent goat anti-mouse or goat anti-rabbit IgG (fluorophores 488 and 546, Alexa; Molecular Probes, Eugene, OR) diluted 1:100 in PBS. Sections were then washed and blocked again with CAS block, and the second primary antibody was added, followed by the appropriate fluorescent antibody. Sections were counterstained with Mayer hematoxylin (Sigma), washed in PBS, and mounted in Fluorosave (Calbiochem, San Deigo, CA). Specificity of all primary antibodies was verified by substitution of vinculin or actin primary antibodies with an equivalent dilution of normal mouse serum and substitution of espin primary antibody with an equivalent dilution of preimmune rabbit immunoaffinity purified IgG. In all cases, no significant staining or fluorescence was observed for control antisera in any seminiferous tubules.
Confocal Analysis
Immunofluorescent sections were examined using a Nikon Diaphot 300 (Japan) microscope and a Biorad MRC 1000 confocal system (Biorad, Hertfordshire, UK) consisting of a krypton/argon laser and pinhole aperture linked to a computer and COMOS software. Specific tubule stages were selected by viewing the hematoxylin counterstain by transillumination. Tubules were classified into 1 of 14 stages as previously described [23]. Portions of staged seminiferous tubule were scanned for fluorescence using a x60 oil immersion objective. For colocalization analysis, a depth of approximately 4 µm was optically scanned at intervals of approximately 0.3–0.4 µm to produce a z series containing approximately 10–12 images in each series. At each optical plane, red and green immunofluoresence was scanned sequentially. Each z series for red or green fluorescence was first examined separately, and then the z series for red fluorescence was merged with the z series for green fluorescence using the Confocal Assistant 4.02 software package. Z series were played in order to appreciate the spatiotemporal relationships of the colocalized proteins. For each z series, one image was selected to give a two-dimensional view for the purposes of publication.
RESULTS
Electron Microscopic Analysis of Sertoli Cell ES
ES was examined by electron microscopy in the seminiferous epithelium of stage VII and VIII tubules from control and TE-treated rats. To study the ES as it forms opposite the round spermatid acrosome during the transition to step 8, round spermatids were subdivided into the various developmental steps, and the extent to which ES was associated with the spermatids was scored.
Ectoplasmic specializations in control testis Ectoplasmic specializations opposite round spermatids were first investigated in control animals to understand the normal formation of ES during stages VII to VIII. The morphology of ES opposite round spermatids in a control animal is shown in Figure 1A.
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In stage VII, the junctions between step 7 round spermatids and Sertoli cells were examined and, as shown in Table 1, 9% of step 7 spermatids examined were associated with ES. Ectoplasmic specializations associated with step 7 spermatids were not extensive, usually covering an area less than one-sixth of the circumference of the cell. Desmosomelike junctions were occasionally observed, where two opposing subsurface densities could be visualized at the plasma membrane of both the Sertoli cell and the spermatid (not shown).
As tubules progress to stage VIII, the round spermatid nucleus polarizes to one side of the cell and the acrosome becomes closely associated with the plasma membrane. Ectoplasmic specializations were observed more frequently opposite spermatids when the nucleus began to polarize, such that 32% of step 7–8 spermatids had ES opposite their acrosome (Table 1). As the nucleus polarized further, i.e., when >25% of the acrosome was polarized to the plasma membrane, 86% of spermatids had ES covering most of the acrosome (see Table 1). Generally, Sertoli cell ES was associated with step 8 spermatids at the site of the acrosome (see Fig. 1A), but occasionally ES extended beyond the acrosomal region.
For comparison, ES structures at the basal Sertoli cell–Sertoli cell junction are also shown (Fig. 1A, inset). The basal ES was observed as two opposing ES structures, with the plasma membranes of adjacent Sertoli cells in close apposition.
Ectoplasmic specializations in TE-treated testis Next, ES structures were investigated in the adluminal compartment of TE-treated animals, where few elongated spermatids beyond step 8 are observed. Ectoplasmic specializations were frequently observed in the seminiferous epithelium and in particular were often observed in the apical Sertoli cell cytoplasm that projected into the lumen of stage VII and stage VIII tubules (see Fig. 1B). This ES was not associated with spermatids and had a normal appearance in terms of actin bundles in close association with Sertoli cell plasma membrane and smooth endoplasmic reticulum.
The formation of ES opposite spermatids in transition from stage VII to stage VIII was examined as for control animals. As shown in Table 1, 21% of step 7 spermatids were associated with normal appearing ES (see Fig. 1C). As the nucleus polarized towards one side of the cell, ES was observed more frequently, and of the 41 step 8 round spermatids examined (Table 1), 90% of them were associated with an extensive ES covering their acrosome (see Fig. 1D). As in control animals, Sertoli cell ES was generally observed opposite the round spermatid acrosome but could also be observed opposite spermatid cytoplasm. The ES appeared to be of normal morphology (Fig. 1, C and D). No unusual structures were noted at sites of spermatid-Sertoli cell junctions. Specifically, no tubulobulbar complex-like structures could be observed at sites of spermatid-Sertoli cell contact, suggesting that ES were not prematurely removed from step 8 round spermatids by tubulobulbar complex formation.
For comparison, the morphology of basal ES was examined in the TE-treated testis. An intact basal ES was observed in the stages examined (not shown). Occasionally, vacuoles were seen in the basal compartment of stage VII and stage VIII tubules, and a single intact ES structure could be seen on either side of the vacuole.
Confocal Analysis of ES-Associated Proteins
In addition to electron microscopy, immunolocalization of the ES-associated proteins vinculin, actin, and espin was performed, and confocal analysis allowed assessment of the colocalization of these proteins within ES structures and their relationship to various cell types.
Localization of ES-associated molecules in control testis In stage VII tubules, no ES structures, as evidenced by espin-vinculin (Fig. 2A), or espin-actin (Fig. 2B) could be seen in association with step 7 round spermatids. However, tubulobulbar complexes, which facilitate removal of ES and cytoplasm from step 19 spermatids [15], were strongly labeled with antibodies to espin, vinculin, and actin (Fig. 2, A and B). Vinculin staining was also observed around the Sertoli cell nucleus, extending up towards the lumen in the central Sertoli cell cytoplasm (Fig. 2A). This vinculin staining of Sertoli cell cytoplasm was observed in all stages; however, actin was not observed at this site (Fig. 2B).
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During stage VIII, the step 8 round spermatid nucleus polarizes to one side of the cell, and the acrosomes gradually become oriented so that they face towards the base of the tubule. The formation of ES opposite the step 8 spermatid was illustrated by espin and vinculin localization (Fig. 2C). Espin staining at the site of the step 8 round spermatid acrosome was localized on either side of the vinculin-stained central Sertoli cell cytoplasm as the spermatids became oriented within the epithelium. Espin-actin colocalization (Fig. 2D) was also seen at the site of the round spermatid acrosome, indicating ES formation. At the ES opposite spermatids, espin and actin often colocalized, producing a yellow color (Fig. 2D), whereas espin and vinculin were closely associated but not strictly colocalized (Fig. 2C). Residual bodies were surrounded by both vinculin (Fig. 2C) and actin (Fig. 2D) but not by espin.
In later spermatogenic stages, the ES extends as the spermatid nucleus condenses and elongates during the subsequent steps of spermiogenesis so that elongated spermatids are associated with extensive areas of Sertoli cell ES. The extensive ES opposite elongated spermatids is demonstrated by espin-vinculin (Fig. 2E) and espin-actin (Fig. 2F) staining.
Espin, vinculin, and actin staining was also evident in basal ES structures (see Fig. 2 for examples) in a scalloped pattern over basal germ cells.
Localization of ES-associated molecules in TE-treated testis In stage VII tubules of the TE-treated testis, vinculin staining was more obvious (see Fig. 3, A and C), presumably because the seminiferous tubule volume has shrunk, and thus there are more cross sections through Sertoli cell cytoplasm. Patches of ES opposite spermatids are indicated by espin closely associated with vinculin adjacent to many step 7 spermatids (Fig. 3A) and patches of colocalized espin-actin (Fig. 3B).
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Stage VIII tubules contained round spermatids with polarized nuclei, and there were noticeably fewer round spermatids in these tubules than in stage VII tubules. The presence of ES in these tubules was indicated by espin-stained regions in close association with vinculin opposite round spermatids (Fig. 3C). Furthermore, patches of colocalized espin and actin were associated with round spermatids (Fig. 3D).
The observation of "free" ES structures in stage VII and stage VIII tubules by electron microscopy (see Fig. 1B) was further demonstrated by espin and vinculin (Fig. 3E) and espin and actin (Fig. 3F) staining in Sertoli cell cytoplasm projecting into the tubule lumen.
For comparison, basal ES structures showed vinculin, actin, and espin staining (see Fig. 3). The vacuoles that appeared in stage VII and stage VIII were associated with espin-actin (see Fig. 3D) and espin-vinculin staining (not shown).
Light Microscopic Immunocytochemical Localization of Espin
Espin staining was highly correlated with sites of Sertoli cell ES. Therefore, the localization of ES was further explored by espin immunocytochemistry using the ABC detection system, thus allowing the light microscopic evaluation of ES structures in the testis using a more sensitive method than the fluorescence microscopy technique.
In control late stage VII/early stage VIII tubules, espin staining was seen in thin fibers in the adluminal compartment (Fig. 4A). These fibers were not observed in the adluminal compartment in earlier stage VII tubules (refer to Fig. 2, A and B); rather, they first appeared in tubules in late stage VII (see Fig. 4A) just prior to the formation of ES opposite step 8 spermatids. These fibers appearing during late stage VII were also observed by confocal fluorescence microscopy (not shown). In stage VIII tubules, step 8 round spermatids showed extensive espin staining associated with the acrosome (Fig. 4B).
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Strong espin staining was seen in TE-treated stage VII and stage VIII tubules. Figure 4C shows a tubule in transition between stages VII and VIII, i.e., some spermatids have polarized nuclei. There was abundant espin staining in the adluminal compartment, with espin staining associated with most spermatids and with adluminal Sertoli cell cytoplasm. In later tubules that have lost all round spermatids (Fig. 4D), espin immunoreactivity was present in the adluminal compartment and in Sertoli cell cytoplasm adjacent to the tubule lumen.
When T24 implants were given to animals to promote the attachment of step 8 round spermatids to Sertoli cells [18], many step 9–10 spermatids could be seen in the epithelium by 2 days, and these spermatids displayed extensive espin staining associated with their acrosomes (Fig. 4E). After 4 days of T24 treatment, later spermatids appeared in the epithelium, and these spermatids also had ES, as evidenced by espin staining (Fig. 4F).
DISCUSSION
In this study, we used electron microscopy and confocal and light immunocytochemical localization of ES-associated proteins to investigate Sertoli cell ES structures within the seminiferous epithelium of normal and testosterone-treated rats. Although the ES has been studied by electron microscopy in normal rat testis [2–4], analysis of this structure at the light microscope level has not been possible because of a lack of specific marker proteins. When compared with electron microscopic analysis, immunolocalization of the newly discovered actin-binding protein espin [11] was a highly specific marker of ES, either alone or in combination with actin or vinculin. Ectoplasmic specialization structures, both basal and adluminal, are present in the TE-treated rat at a time when step 8 round spermatids are detaching from the Sertoli cell, suggesting that this process of detachment is not due to an absence of ES in the seminiferous epithelium.
Using light microscopic and confocal analysis of ES markers and the visualization of ES structures by electron microscopy, we investigated the TE-treated epithelium, in which approximately 80% of step 8 spermatids detach from the Sertoli cell and are unable to complete the elongation process [1]. Our observations suggest that ES structures are qualitatively normal in the Sertoli cell when testicular testosterone levels are reduced. Several observations support the presence of ES in TE-treated rats. First, free ES was often observed by electron microscopy and confocal analysis in the Sertoli cell adluminal cytoplasm during and after round spermatid detachment. Also, light microscopic analysis of espin revealed strong staining in tubules at stage VII and beyond, also suggesting that there is abundant ES available in the seminiferous epithelium. Of the step 8 round spermatids remaining in the seminiferous epithelium, 90% of them had extensive ES structures in apposition to the acrosome, as revealed by electron microscopy, and espin, actin, and vinculin staining was associated with these remaining step 8 round spermatids. The observation of free ES structures in the adluminal portion of the Sertoli cell argues against the proposition that the spermatids that remained within the epithelium did so because they formed an ES; rather, it suggests that ES structures were present within the Sertoli cell at a time when round spermatids would normally associate with them, but the round spermatids still lost contact with the epithelium. In addition, we do not believe that ES structures are formed opposite step 8 spermatids and then prematurely removed, because tubulobulbar complex formation was not observed in association with ES structures and/or step 8 round spermatids. Basal ES structures also appeared normal in all stages, except for occasional vacuoles that have been previously described as fluid-filled focal dilations between two ES structures [26]. Given that the basal and adluminal ES structures within a Sertoli cell are structurally similar, it is difficult to envision how ES structures could be present in one part of the cell but disrupted in another. Finally, within 2–4 days of T24 replacement, all post-step 8 spermatids showed ES structures, as evidenced by espin staining, suggesting that ES readily extend around elongating spermatids as soon as they appear in the epithelium. Taken together, these observations suggest that ES structures are present and qualitatively normal in the TE-treated seminiferous epithelium.
The finding that suppression of testicular testosterone does not impair Sertoli cell ES structures is supported by the findings of Cameron and colleagues [27]. These authors used ethane dimethanesulphonate to acutely suppress testicular testosterone during the first wave of spermatogenesis in 26-day-old rats and also noted detachment of step 8 round spermatids from Sertoli cells, yet ES structures were observed within the epithelium. In adult rats, hypophysectomy promoted disorganization of ES, as revealed by electron microscopy, and of actin distribution, as revealed by immunocytochemistry on epithelial sheets prepared from seminiferous tubules [28]. However, delayed testosterone replacement could not restore actin distribution, suggesting that testosterone does not regulate this structure within the seminiferous epithelium [28]. Further studies by these authors suggested that FSH may maintain and restore junctional structures in the seminiferous epithelium [29], and these findings are in line with our observations that ES structures are present in the TE-treated rat, where circulating FSH is near normal [18]. Our future studies will explore the regulation of ES structures by FSH using in vivo models of FSH manipulation.
Our findings that ES is present in the Sertoli cell at a time when round spermatids are detaching from the epithelium, together with the previous studies of Muffly and colleagues suggesting that testosterone does not regulate Sertoli cell junctional structures [28, 29], lead us to the conclusion that testosterone-dependent detachment of round spermatids [1] is not caused by an absence of the Sertoli cell ES. Based on the analysis of this structure by electron microscopy, there does not appear to be a defect in the bundling of actin nor of the association of actin bundles with either the endoplasmic reticulum or the Sertoli cell plasma membrane. The finding that ES structures are present in the TE-treated epithelium raises the question of why step 8 round spermatids detach from the Sertoli cell. Although we believe that the ES structure associated with the plasma membrane is normal, we propose that there may be a deficiency in adhesive structures that normally form between a step 8 spermatid and the Sertoli cell ES. Therefore, there could be defects in the "adhesive domain" of the ES junction [4]. Linkages between the spermatid acrosome and the Sertoli cell ES have been observed by electron microscopy [7], and mechanical disruption of the seminiferous epithelium results in spermatids with attached fragments of Sertoli cell cytoplasm containing ES [5]. The adhesion between the spermatid and the attached fragment of ES can be disrupted by incubation with trypsin [5], suggesting that cell adhesion molecules are involved in the spermatid-ES interaction. Although focal ES can be associated with earlier germ cell types [2], the strong adhesive property of the ES as described above is first acquired as spermatids begin their elongation, because hypertonic fluids can separate ES from earlier germ cells but not from spermatids from step 8 onwards [8]. Therefore it is likely that step 8 round spermatids associate with the ES via specific cell adhesion and associated molecules that are not involved in the interaction between pre-step 8 spermatids and Sertoli cells. One candidate cell adhesion molecule could be 
6ß1-integrin, which has been localized to sites of elongated spermatid–Sertoli cell interactions [30]. Another candidate is N-cadherin, because it has been shown to be involved in interactions between Sertoli cells and round spermatids in vitro [31]. Further studies are required to identify key cell adhesion and integral membrane molecules that are involved in the step 8 spermatid-ES interaction and to investigate whether such molecules are regulated by testosterone.
The localization of espin, combined with actin and vinculin, in normal testis is correlated with the known sites of ES in the seminiferous epithelium, as has been previously characterized [2–4]. The observation that espin often colocalized with actin, producing a yellow color in the confocal images, is in agreement with the role of espin as an actin-bundling protein in rat Sertoli cell ES [11]. The confocal analysis of espin-vinculin colocalization, which showed vinculin staining within the central Sertoli cell cytoplasm, provided a unique illustration of how ES formation facilitates the orientation of newly formed step 8 spermatids and the subsequent positioning of elongating spermatids in "crypts" deep within a particular Sertoli cell [3]. Although vinculin is known to be a component of the Sertoli cell ES, possibly functioning to anchor the structure to the Sertoli cell plasma membrane [32], the role of vinculin in the central Sertoli cell cytoplasm is unclear but has been shown by others [33]. Confocal analysis showed that espin-stained regions opposite spermatids first appeared in the epithelium in late stage VII/early stage VIII when the process of spermiation was nearing completion. The observation that ES first appears when spermiation is nearing completion is in agreement with the hypothesis proposed by Russell [15], suggesting that ES forming opposite newly elongating spermatids are, at least in part, recycled from spermiating step 19 spermatids within the same Sertoli cell.
Quantitation of ES structures was not attempted because of the large potential for bias associated with counting structures at the electron microscopic level. Such sources of bias include difficulties in the effective sampling of epithelium in the small section required for electron microscopy and particularly the "lost caps" source of bias; because such thin sections are required for transmission electron microscopy, it is highly likely that a proportion of ES would not be sampled for counting because of grazing sections of the structure that could therefore not be identified. Instead, we have used immunocytochemical analysis of ES markers and electron microscopic analysis to show that the ES is qualitatively normal in TE-treated rats.
In summary, we used electron microscopy and immunolocalization of ES-associated proteins to study the Sertoli cell ES in normal and hormone-treated testis. The confocal analysis of immunofluorescent ES markers and light microscopic analysis of espin immunolocalization for the first time permits the evaluation of Sertoli cell ES at the light microscope level and will be a valuable tool for studying ES formation under a variety of hormonal conditions to investigate its regulation. The ES was present in TE-treated rats at a time when step 8 round spermatids detach from the epithelium, suggesting that the detachment of round spermatids is not caused by an absence of ES structures in the seminiferous epithelium and that this structure is not regulated by testosterone. Further studies will include an analysis of the adhesion molecules involved in the step 8 round spermatid-ES interaction and will incorporate other models of hormonal treatments to study the regulation of ES structures.
ACKNOWLEDGMENTS
The authors thank Robyn Mayberry for preparation of the tissue sections for electron microscopy.
FOOTNOTES
First decision: 4 January 2000.
1 L.O. is supported by Wellcome Trust Research Training Fellowship in Reproductive Biology grant 050387, J.R.B. is supported by grants R01 HD35280 and K02 HD01210 from the National Institutes of Health, P.G.S. and D.M.R. are supported by Program Grant 983212 from the National Health and Medical Research Council of Australia. 
2 Correspondence: Liza O'Donnell, Prince Henry's Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. FAX: 61 3 9594 6125; liza.odonnell{at}med.monash.edu.au
Accepted: February 11, 2000.
Received: December 2, 1999.
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