Abstract
Claudin-11 and occludin are protein components in tight junctions (TJs) between Sertoli cells which are important for the maintenance of the blood–testis barrier. Barrier formation occurs during puberty, with evidence suggesting hormonal regulation of both claudin-11 and occludin. This study aimed to investigate the regulation of claudin-11 and occludin mRNA expression by testosterone (T) and FSH and their immunolocalisation at rat Sertoli cell TJs in vitro, and to correlate any steroid regulation with the functional capacity of TJs. Sertoli cells formed functional TJs within 3 days as assessed by transepithelial electrical resistance (TER). Both T and dihydrotestosterone significantly (P < 0.01) increased TER twofold and claudin-11 mRNA two- to threefold within 3 days. FSH partially stimulated TER and claudin-11 mRNA, but estradiol had no effect. T also promoted claudin-11 localisation into extensive intercellular contacts. In contrast to claudin-11, Tand FSH did not change occludin mRNA expression, however, T promoted localisation of occludin at cell contacts in a similar manner to claudin-11. Addition of flutamide to T-stimulated cells caused a twofold decrease in both TER and claudin-11 mRNA expression, and resulted in the loss of both proteins from cell contacts. This effect was reversible following flutamide removal. It is concluded that androgens i) co-regulate claudin-11 mRNA expression and TER, implicating claudin-11 in TJ formation and ii) promote the localisation of claudin-11 and occludin at Sertoli cell contacts. Hence, the ability of androgens to maintain spermatogenesis in vivo is partly via their effects on TJ proteins and regulation of the blood–testis barrier.
Introduction
The inter-Sertoli cell junctional complex forming the blood–testis barrier is located around the basal aspect of the seminiferous epithelium and is comprised of various types of cell junctions, including tight junctions (TJs), adherens junctions, desmosome-like junctions, gap junctions and a testis-specific actin-containing junctional structure known as the ectoplasmic specialisation (Russell & Peterson 1985, Pelletier & Byers 1992, Mulholland et al. 2001, Lui et al. 2003b, 2003c, Toyama et al. 2003, Mruk & Cheng 2004). TJs maintain a selectively permeable barrier separating the seminiferous epithelium into basal (outside the blood–testis barrier) and adluminal compartments (inside). This barrier prevents the bidirectional passage of molecules (Russell & Peterson 1985) and creates a specialised microenvironment required for germ cell meiosis and maturation in the adluminal compartment that is biochemically and immunologically distinct from the remainder of the testis (Maddocks & Setchell 1990). It is well established that disruption of TJ function leads to germ cell atresia, cessation of spermatogenesis and the potential exposure of sequestered antigens to immunological attack (Russell & Peterson 1985).
Several transmembrane proteins involved in TJs in various epithelia have been described, including claudins, occludin and junction adhesion molecules (JAMs; for reviews, see Gonzalez-Mariscal et al. 2003, Ebnet et al. 2004, Feldman et al. 2005, Koval 2006). Testicular TJs in mice and rats contain occludin (Saitou et al. 1997, Moroi et al. 1998, Cyr et al. 1999), some members of the claudin family (Morita et al. 1999a) including claudin-11 (Morita et al. 1999b, Hellani et al. 2000) and claudin-3 (Meng et al. 2005), and JAM-A and JAM-C (Aurrand-Lions et al. 2001, Gliki et al. 2004). The claudin-11 knockout male mouse is infertile and lacks functional Sertoli cell TJs and mature spermatozoa (Gow et al. 1999, Mitic et al. 2000), whilst the occludin knockout mouse testis contains a normal germ cell complement in animals of reproductive age (Saitou et al. 2000). However, the suppression of rat testicular occludin function caused the loss of germ cells in vivo and partially disrupted formation of Sertoli cell TJs in vitro (Chung et al. 2001), suggesting that occludin may contribute to TJ function. The JAM-A knockout is fertile (Cera et al. 2004), and the blood–testis barrier remains functional in JAM-C knockout mice (Gliki et al. 2004). Thus, evidence from these models suggests that claudin-11 is a major component involved in both the formation and function of Sertoli cell TJs, with additional input from occludin.
Although not well understood, an association exists between blood–testis barrier function and endocrine status. For example, Sertoli cell TJs and the resultant impermeable barrier first appear in the pubertal (15–20 days old) rat testis as follicle stimulating hormone (FSH) and luteinizing hormone increase (Russell & Peterson 1985, Russell et al. 1989), and TJ formation can be delayed (Vitale et al. 1973) or prevented in the absence of these hormones (Bressler 1976). Similarly, treatment of hypogonadotrophic hypogonadal men with gonadotrophins converts Sertoli cell TJs from a pre-pubertal phenotype to the mature junctional structure (de Kretser & Burger 1972).
Direct links between Sertoli cell TJ proteins and androgens have recently been established. Androgens upregulate claudin-11 mRNA expression in primary cultured mouse and rat Sertoli cells in vitro (Gye 2003, Florin et al. 2005), and also upregulate claudin-3 mRNA and protein in the Sertoli-like TM4 cell line (Meng et al. 2005). The regulation of occludin in these models is unclear, although in vivo treatment of adult rats with the androgen antagonist, flutamide, caused an ~40% decrease in testicular occludin mRNA expression (Gye & Ohsako 2003). Despite these studies, the mechanism(s) by which hormones (androgens, FSH) regulate both Sertoli cell TJ proteins and function remains to be clarified. Recently, we demonstrated that FSH is pivotal for the formation of two of the other junctional types in the inter-Sertoli cell junctional complex (adherens junctions and ectoplasmic specialisations), and that androgens were without apparent effect (Sluka et al. 2006). In the current study, we have used a well-established in vitro model of Sertoli cells in bicameral culture (Hadley et al. 1987, Handelsman et al. 1989, Onoda et al. 1990, Janecki et al. 1991a, Djakiew & Onoda 1993) to study the effects of androgens and FSH on TJs. TJ function in this culture system is readily monitored by the determination of transepithelial electrical resistance (TER; Janecki et al. 1991a, 1991b, 1992, Chung et al. 1999, Fanning et al. 1999, Chung & Cheng 2001, Li et al. 2001, Lui et al. 2003a, Siu et al. 2003). Testosterone and FSH, either alone or in combination, are known to increase rat Sertoli cell TER two to threefold in this model (Janecki et al. 1991a, 1991b, Steinberger & Klinefelter 1993) indicating that Sertoli cell TJ function can be regulated by gonadotrophins. Hence, the aim of this study was to further elucidate the hormonal regulation of key TJ components (claudin-11, claudin-3, occludin) by correlating the effects of steroid (T, DHT and estradiol (E2)) treatment and FSH with both the functional capacity of TJs (by monitoring TER), and with TJ protein mRNA expression and immunolocalisation.
Materials and Methods
Animals
Male outbred Sprague–Dawley rats at 19–21 days of age were obtained from Monash University Animal Services, Monash University (Melbourne, Australia). Rats were killed by CO2 asphyxiation and testes were removed immediately for the isolation of Sertoli cells. The use of animals for this study was approved by the Monash Medical Centre Animal Ethics Committee.
Preparation of Sertoli cells for culture experiments
Primary Sertoli cells were isolated from 19–21-day-old Sprague–Dawley rats as previously described (Perryman et al. 1996, Sluka et al. 2006). Freshly isolated Sertoli cells were suspended in serum free Dulbecco’s Modified Eagle’s Medium (DMEM)/Hams F12 medium (1:1) supplemented with l-glutamine (1 mM, Trace Scientific, Melbourne, Australia), non-essential amino acids (1:100 dilution of 100× stock, Trace), NaHCO3 (1.4 mM, Trace), BSA (1% (w/v), Sigma Chemical Co.), HEPES (10 mM, Trace), insulin (5 μg/ml Novo-Nordisk, Sydney, NSW, Australia), transferrin (5 μg/ml, Sigma), sodium selenite (50 ng/ml, Sigma) and penicillin (200 U/ml)-streptomycin (200 μg/ml)-fungizone (0.5 μg/ml; CSL, Melbourne, Australia). Cells were plated at a density of 1.25×106 cells/cm2 into either 24-well culture plates (Nunc, Nalge Nunc International, Denmark) for total RNA isolation, or into Millicell PCF bicameral chambers (12 mm diameter, 0.4 μm pore size, 0.6 cm2 surface area; Millipore, Bedford, MA, USA) for measurement of TER and immunocytochemistry. All cell culture surfaces (24-well plates, bicameral chambers) were pre-coated 4 h before use with Matrigel (BD BioSciences, Bedford, MA, USA) at a 1:8 dilution in DMEM/F12 medium. Cells were then incubated at 37 °C in a humidified atmosphere of 95% air–5% CO2 (v/v) for periods of 5–13 days, with the day of isolation designated as day 0. Media were routinely replaced every 2 days. On day 3, cells were hypotonically shocked with 10% culture medium in water for 45 s to lyse the contaminating germ cells (Galdieri et al. 1983), after which the cells were washed once with DMEM/F12 and returned to the incubator for the remaining culture period. Sertoli cell cultures prepared by this method were typically of 92% purity, with the remaining cells being peritubular cells and residual germ cells (Perryman et al. 1996, Lampa et al. 1999).
Hormonal treatments were added at the following concentrations on day 0 unless otherwise stated: testosterone (T), 28 μg/ml (Sigma); human recombinant FSH, 2.35 IU/ml (Puregon, Organon, Oss, The Netherlands); DHT, 28 ng/ml (Sigma); E2, 28 ng/ml (Sigma); flutamide, 27.6 μg/ml (Sigma). Steroids were prepared from stock solutions in ethanol, and diluted in DMEM/F12 immediately prior to use, while an equivalent volume of ethanol (0.26% v/v final) was added to all other culture wells not receiving steroid treatments.
Measurement of TER
To assess the assembly of inter-Sertoli cell TJs, TER across the Sertoli cell epithelia was quantified using a Millipore Millicell-electrical resistance system (Millipore), with measurements taken daily commencing from the day of plating (day 0). To enhance the reproducibility of measurements, cultured cells were allowed to equilibrate at room temperature for 30 min, prior to the measurement of TER. The final resistance readings were calculated by subtracting the mean TER of bicameral chambers coated with Matrigel but containing no cells, and correcting for the surface area of the PCF bicameral chamber (0.6 cm2) to produce a value expressed as Ω/cm2. All TER values were calculated from triplicate culture wells.
Isolation of total RNA and RT
Cells were removed at specified time points for extraction of total RNA using a total RNA extraction kit (Qiagen) according to the manufacturer’s instructions. Any contaminating DNA was removed using the DNAse-free kit (Ambion, Austin, TX, USA), and samples were stored at −80 °C. Total RNA concentrations were determined using the Ribogreen fluorescence RNA assay (Molecular Probes, Eugene, OR, USA) as described elsewhere (Sluka et al. 2002).
RT was performed on 500 ng total RNA/sample using AMV- reverse transcriptase (8 U; Roche), random hexamer primers (200 ng; Amersham Biosciences), dNTPs (20 nmol each; Roche), RNasin (20 U; Promega) and 5× reaction buffer (Roche) in a final volume of 20 μl for 90 min at 46 °C, after which samples were heated for 2 min at 95 °C before storage at −20 °C.
Real-time PCR
Quantitative real-time PCR analysis was performed using the Roche Lightcycler (Roche) and the FastStart DNA Master SYBR-Green 1 system (Roche). Oligonucleotide primers for claudin-11 (forward 5′-TTAGACATGGGCACTCTTGG-3′, reverse 5′-ATGGTAGCCACTTGCCTTC-3′), occludin (forward: 5′-CTGTCTATGCTCGTCATCG-3′, reverse 5′-CATTCCCGATCTAATGACGC-3′) and claudin-3 (forward 5′-CGTTAGCGTGCTCCGTCCAT-3′, reverse 5′-CCCGAAGGCTGCCAGTAGGATA-3′) were obtained from published sources (Chung et al. 2001, Lui et al. 2001) or were designed using the Oligo program (version 6; Molecular Biology Insights, Cascade, CO, USA). For PCR analysis, sample cDNA was diluted 1:20 to 1:150 fold and PCR conditions including Mg2+ and primer concentrations, anneal time and extension time are summarised in Table 1. Standard curves for PCR analyses were generated using dilutions of an adult rat testicular cDNA preparation of arbitrary unitage. Unless otherwise noted, PCR of all samples was performed using triplicate reactions for 38 cycles, after which a melting curve analysis was performed to monitor PCR product purity (see Table 1). In initial experiments, amplification of a single PCR product was confirmed by agarose gel electrophoresis and DNA sequencing.
Immunocytochemistry
For immunocytochemical analyses, Sertoli cell monolayers on PCF filters were pre-extracted on ice with ice-cold PBS (PBS; 10 m 0.01 M PBS with 154 mM NaCl) containing 0.2% (v/v) Triton X-100 for 2 min and then fixed in 3% paraformaldehyde in PBS for 30 min at room temperature. Cells were then permeabilised with 0.05% (v/v) Triton X-100 in PBS for 5 min on ice and washed in PBS. Non-specific binding sites were blocked with CAS block (Zymed, San Francisco, CA, USA) containing 10% normal serum (sheep or rabbit) for 20 min. Primary antisera used were affinity-purified rabbit anti-occludin (2.5 μg/ml, Zymed) or rabbit anti-claudin-11 (1:400, CovalAb) diluted in PBS/bSA and were incubated with cells overnight at room temperature. Cell monolayers were then washed and secondary antisera (goat anti-rabbit Alexafluor-488 1:100 dilution; Molecular Probes or biotinylated sheep anti-rabbit immunoglobulin G (IgG) 1:100 dilution) were applied for a period of 1 h at room temperature. Claudin-11 immunostaining was then visualised with streptavidin –Alexafluor 488 (1:100, 30 min, Molecular Probes), and monolayers were counterstained with the fluorescent stains DAPI (90 μM) and TO-PRO-3 iodide (10 μm in PBS/bSA; Molecular Probes) for 5 min to allow nuclear visualisation. Monolayers were then rinsed thrice with PBS/bSA and mounted on glass slides with FluorSave reagent (Calbiochem, San Diego, CA, USA).
Confocal analysis was performed using an Olympus Fluoview FV300 confocal system equipped with Fluoview version 4.2 software (Olympus) and attached to an Olympus IX 81 inverted microscope. A 60× water immersion lens was used, with confocal aperture set to 2, laser intensities set to 5%, software zoom set to ×2, and software filter mode set to Kalman with 4 scans. Excitation of Alexa Fluor 488 (green) was performed using the 488 nm line of the Argon laser, while TO-PRO-3 iodide (red) was excited using the 633 nm line of the red HeNe laser. Sertoli cell monolayers were initially visualised by epifluorescence microscopy, and an optic plane that contained inter-Sertoli cell junctions was selected for image capture. Confocal scanning of double label experiments was performed sequentially to prevent bleed-through between red and green output channels.
Immunoblot analysis for occludin and claudin-11 used the same antibodies as for immunocytochemistry, with cell extracts prepared in hot non-reducing sample buffer (125 mM Tris–HCl, 2.5% (w/v) SDS, 10% (w/v) glycerol, pH 6.8), electrophoresed on 10% polyacrylamide gels (Laemmli 1970) and blotted onto a PVDF membrane (Immobilon-P, Millipore). Detection was with a horseradish peroxidise conjugate in conjunction with an ECL western blot detection system (Amersham Biosciences).
Statistical analysis
TER values (n=3 wells/treatment) and PCR data (n=3 PCR estimations from total RNA pooled from two wells) were calculated as mean ± s.d. from a single culture. Each study was repeated 2–3 times with representative experiments presented in the Results section. Statistical analyses were performed using GB Stat (Dynamic Systems Inc., Silver Spring, MD, USA) with an initial assessment of homogeneity of variance for all groups. Homogeneous groups were assessed using one-way ANOVA, followed by the Newman–Keuls post hoc multiple comparisons test. When groups were non-parametric, data were log10 treated, and the above tests repeated. Remaining non-parametric data were analysed using the Kruskal–Wallis test followed by the Newman–Keuls analogue test (equal ns).
Results
Androgens stimulate Sertoli cell TER
When Sertoli cells were cultured on Matrigel-coated bicameral chambers in serum-free DMEM/F12, TJs typically formed within the first 2–3 days post-plating as assessed by the measurement of TER. Under control conditions, TER reached a plateau of 60–80 Ω/cm2 shortly after junction formation (days 2–3, Fig. 1) and increased slightly thereafter (up to day 7; Fig. 1), (or up to day 13 (see Fig. 4A)) reaching a maximum of ~130 Ω/cm2. A significant (P < 0.01) increase in TER was observed for Sertoli cells cultured with added T (28 ng/ml) compared with control wells, when a 1.5-to 2-fold stimulation of TER was observed after day 3 (Fig. 1, see also Fig. 4A and B). Similar findings were observed for Sertoli cells cultured with both T (28 ng/ ml) + FSH (2.35 IU/ml), with twofold differences to control which is evident after day 5 (Fig. 1). FSH alone produced a minor but significant (P < 0.05) increase in TER, but this was not observed until days 5 and 7 of culture (Fig. 1). E2 (28 ng/ml) alone had no effect on TER (data not shown). Cells treated with DHT (28 ng/ml) responded in a similar manner to T-treated cultures (data not shown). These results show that T, and to a lesser extent FSH, stimulates Sertoli cell TER.
Testosterone stimulates claudin-11 mRNA but not occludin or claudin-3 mRNA expression
The expression of three TJ transmembrane proteins, claudin-11, occludin and claudin-3 were examined using real-time RT-PCR. Under control conditions, claudin-11 mRNA expression showed a slight but significant increase (P=0.015) over the 7-day culture period (Fig. 1B), whereas T stimulated claudin-11 mRNA expression significantly (P < 0.01) within 3 days, and was two- to threefold higher than control at days 5–7. Cells treated with T plus FSH responded in a similar manner to T alone (Fig. 1B), while FSH alone elevated claudin-11 mRNA expression approximately twofold greater than control wells between days 3–7 (Fig. 2B). E2 had a marginal effect on claudin-11 expression with a significant increase only evident after 7 days of treatment (Fig. 1B), whereas DHT increased claudin-11 mRNA expression in a similar manner to T (data not shown).
In contrast to claudin-11, occludin mRNA expression levels remained relatively stable throughout the culture in all treatment groups (Fig. 1C), with no evidence for hormonal stimulation of mRNA expression. Although recent data suggest the presence of claudin-3 mRNA expression in newly forming Sertoli cell TJs in the mouse (Meng et al. 2005), we were unable to detect this protein by PCR at any time in this rat Sertoli cell culture system (data not shown). Subsequent studies showed positive claudin-3 immunostaining in the interstitium of the adult rat testis, but not in the seminiferous epithelium (Stanton unpublished observations).
These results show that T and FSH stimulate claudin-11 mRNA but not occludin mRNA expression in rat Sertoli cells whilst claudin-3 is not expressed in rat Sertoli cells.
Claudin-11 mRNA expression correlates with TER
Correlations were performed between occludin and claudin-11 mRNA expressions and inter-Sertoli cell TJ function as measured by TER to assess whether these variables were related. No significant relationship (r=0.212, n=32, ns) was observed between occludin mRNA expression and TER (data not shown). In contrast, a significant correlation (r=0.534, n=32, P < 0.01) was observed between claudin-11 mRNA expression and TER for pooled data from all treatment groups (Fig. 2A), which was maintained within the T-treated group (r=0.790, n=8, P < 0.02; Fig. 2B).
Testosterone stimulates localisation of claudin-11 and occludin at inter-Sertoli cell contacts in vitro
Claudin-11 protein was detected as a major band of 27 kDa in rat Sertoli cells (Fig. 3H) as reported elsewhere (Hellani et al. 2000, Florin et al. 2005). Under control conditions, punctate immunostaining of claudin-11 at Sertoli cell contacts and in Sertoli cell cytoplasm was apparent at day 1 (data not shown) and day 5 (Fig. 3A), while more extensive contacts containing claudin-11 were apparent in extended cultures at days 9 and 13 (Fig. 3B and C). In contrast, claudin-11 immunostaining was markedly upregulated in cells treated with T for 5 days (Fig. 3E), and was present in both the cytoplasm of the Sertoli cells and in extensive contacts between Sertoli cells. This pattern of immunostaining was preserved in cells continuously treated with T for 9 and 13 days (Fig. 3F and G).
The occludin protein was observed as a major band of immunoreactivity at ~57 kDa in rat Sertoli cells (Fig. 3L) consistent with published data (Furuse et al. 1993, Sakakibara et al. 1997), although other minor bands (87, 42 and 36 kDa, Fig. 3L) were also observed. Under control conditions, punctate occludin immunostaining was observed at Sertoli cell contacts at both day 1 (data not shown) and day 7 (Fig. 3I). In contrast, Sertoli cells treated with T for 7 days exhibited a marked increase in occludin localisation at inter-Sertoli cell contacts (Fig. 3J) compared with control cultures. Limited non-specific staining was observed for claudin-11 within the cell nuclei (Fig. 1D), however, non-specific staining was not observed for occludin (Fig. 1K) when the primary antibodies were substitituted for a non-specific rabbit IgG.
Flutamide antagonises testosterone-stimulated TER and claudin-11 mRNA expression in Sertoli cells
Sertoli cells were allowed to form TJs for 5 days in medium alone, after which the androgen receptor antagonist, flutamide, was added for 4 days. Under control conditions (Fig. 4A), a twofold decrease in TER was observed following the addition of flutamide by day 9 of culture compared with cells maintained in medium alone. Similarly, a significant (P < 0.01) greater than threefold decrease in TER to the same level as non-stimulated cells was observed when flutamide was added to Sertoli cells pre-stimulated with T for 5 days (Fig. 4B). This effect was reversible, as replacement of flutamide on day 9 of culture with either medium alone (Fig. 4A), or with T (Fig. 4B) restored TER to pre-treatment levels within 2 days. The TER of Sertoli cells constantly treated with T for 13 days continued to increase to ~350 Ω/cm2 and did not appear to reach a maximum, although this increase may have been biphasic with a plateau around days 3–5 (Fig. 4B).
Claudin-11 mRNA expression was also measured in cells treated with the same protocol as described above for TER (Fig. 4C). In accord with earlier experiments (see Fig. 1), claudin-11 mRNA expression in Sertoli cell monolayers continuously stimulated with T was significantly (P < 0.05) elevated compared with its respective control (medium alone – open circles) at days 5, 9 and 13. When T was removed at day 5 and replaced with flutamide (closed squares), a significant (P < 0.01) > threefold decrease in claudin-11 mRNA expression was observed at day 9, with claudin-11 mRNA expression not different from medium-alone cells similarly treated with antagonist (closed circles). Additionally, no difference in claudin-11 mRNA expression was detected between the medium-alone and medium-alone plus antagonist treated group between days 5 and 9. Following removal of flutamide from the cells at day 9 and replacement with either medium alone or testosterone, no significant changes in claudin-11 mRNA expression were observed at day 13.
In conjunction with the decreases in claudin-11 mRNA, flutamide treatment of T-stimulated cells also caused a marked loss of claudin-11 immunostaining at inter-Sertoli cell contacts at day 9 of culture, but some cytoplasmic staining remained apparent (compare Figs 3F with 4D). A similar action of flutamide on claudin-11 immunostaining in control cultures was also observed (data not shown). This effect appeared reversible, as replacement of the flutamide with T for a further 4 days (to day 13) caused the reappearance of claudin-11 immunostaining at inter-Sertoli cell contacts (Fig. 4E). A similar regulation of occludin immunostaining at cell contacts was also observed (data not shown).
Discussion
This study has established that testosterone and DHT significantly increased claudin-11 mRNA expression two- to threefold during TJ formation by rat Sertoli cells in vitro. This increase was androgen dependent as it was not reproduced by E2, but was inhibited by the non-steroidal androgen-receptor antagonist, flutamide. Androgens, and to a lesser extent FSH, also increased the ‘tightness’ of Sertoli cell TJs as quantitated by TER from a basal value of ~80 to ≥ 150 Ω/cm2. A significant correlation between TER and claudin-11 mRNA expression suggests that claudin-11 plays a major role in Sertoli cell TJ formation. Androgens also increased both the cytoplasmic staining of claudin-11 in Sertoli cells and its localisation to extensive inter-Sertoli cell contacts in vitro. In contrast, Sertoli cell occludin mRNA expression was not regulated by hormones (T, E2, FSH), although T did promote localisation of occludin to inter-Sertoli cell contacts in a similar manner to claudin-11. Hence, these studies show that androgens promote the synthesis and/or localisation of two key TJ proteins to regions of inter-Sertoli cell contact in vitro.
Our study demonstrates that rat Sertoli cell TJ function and TJ proteins (claudin-11 and occludin) are regulated in a biphasic manner in vitro, with a basal component and a second androgen-regulated component. In medium alone, TJs formed with an average TER of ~80 Ω/cm2, during which time both claudin-11- and occludin- mRNA expression were also evident, with punctate claudin-11 and occludin protein localisation at inter-Sertoli cell contacts. Hence, we propose that this level of expression and localisation comprises a basal component of Sertoli cell TJ function. Androgens (T or DHT) significantly increased both TER and claudin-11 mRNA two- to threefold compared with controls, and increased both cytoplasmic claudin-11 staining and incorporation of both proteins into extensive intercellular contacts. We therefore propose this to be an androgen-regulated component of TJ function. Addition of the androgen receptor antagonist flutamide to T-stimulated cells inhibited this component of claudin-11 mRNA expression, with a marked decrease in cell surface localisation of claudin-11 and occludin, and a concomitant decrease in TER to basal levels. This effect was partially reversible, as replacement of the antagonist with T restored both TER and junctional immunostaining of both proteins; however, no significant increase in claudin-11 mRNA expression was observed. While this result could indicate an adverse or toxic effect of flutamide in this study, we note that the antagonist concentration used (~100 μM) was mid-range compared with other in vitro rat Sertoli cell studies which have employed concentrations of 0.5 μM (Swift & Dias 1988), 1 μM (Lyng et al. 2000) or 3 mM (Gorczynska & Handelsman 1995).
Data from mice with the conditional knockout of the Sertoli cell androgen receptor (SCARKO) confirm our in vitro findings, by showing a significant 40% reduction in claudin-11 mRNA expression in both adult (Tan et al. 2005) and pubertal (Wang et al. 2006) mice. Other in vitro (Gye 2003, Florin et al. 2005) studies have also demonstrated that testosterone upregulates claudin-11 mRNA expression in Sertoli cells, although our study is the first to show a direct link between claudin-11 mRNA and TER, thereby suggesting that claudin-11 is a major contributor to Sertoli cell TJ function (Koval 2006). An additional androgen-regulated claudin, claudin-3, has recently been localised at the mouse blood–testis barrier, where it is potentially involved in newly formed TJs (Meng et al. 2005). Our inability to detect claudin-3 mRNA expression in the rat Sertoli cell culture system at any time during junction formation, or by immunohistochemical localisation in the adult rat testis, does not support a role for this protein in the rat testis.
The extent to which the ‘tightness’ of Sertoli cell TJs can be regulated by androgens as observed here, and elsewhere (Janecki et al. 1991a, Gye 2003), is of interest in understanding how these junctions function. Under basal conditions, rat Sertoli cells at similar cell plating densities typically exhibit TERs of 60–100 Ω/cm2 (this study, Janecki et al. 1991a, 1991b, Chung & Cheng 2001, Li et al. 2001, Lui et al. 2001, Siu et al. 2003), which is greater than ‘leaky’ TJs formed by cells from mammalian kidney proximal tubules (6–7 Ω/cm2; Fanning et al. 1999), but less than kidney collecting ducts (300 Ω/cm2) or the very tight epithelium of the bladder (≥6000 Ω/cm2; Fanning et al. 1999). After T stimulation for 5–7 days, TER values increased from 150 to 250 Ω/cm2; however, values ~350 Ω/cm2 were observed after continuous culture for 13 days, and it was evident that a plateau had not been reached. This suggests that optimal conditions for the establishment of TJs in Sertoli cell cultures have not been attained, which is supported by up to tenfold higher TERs (~800 Ω/cm2) observed elsewhere (Janecki et al. 1991a, 1991b).
The role of FSH in Sertoli cell TJ regulation in vitro remains an issue. Our study demonstrated that while FSH could upregulate claudin-11 mRNA expression, TER was not altered to the same extent. Other studies have demonstrated a greater upregulation of TER by FSH (Janecki et al. 1991a), or an inhibitory effect of FSH on claudin-11 mRNA expression (Hellani et al. 2000), leading us to presume that differences between in vitro culture methods can lead to altered FSH dose–response characteristics. It has also been speculated that FSH may transiently stimulate protease activity to alter TER (Janecki et al. 1991a, Chung & Cheng 2001).
Several lines of evidence suggest that gonadotrophins are important for the formation and function of Sertoli cell TJs in vitro (this study, Janecki et al. 1991a, 1991b, Gye 2003) and in vivo (Vitale et al. 1973, Bressler 1976, Russell & Peterson 1985, Bergmann 1987, Russell et al. 1989, Gye & Ohsako 2003). In several species of hamster (Bergmann 1987, Bergmann et al. 1989) and mink (Pelletier 1988), TJs comprising the blood–testis barrier undergo a cyclic breakdown and reappearance in association with photoperiod and changes to circulatory gonadotrophins. We recently demonstrated an extensive disruption of the localisation of two TJ-associated proteins, claudin-11 and ZO-1, in the adult short-day Djungarian hamster (Tarulli et al. 2006) where serum gonadotrophins are low and there is a lack of functional TJs (Bergmann 1987). Exogenous FSH restored the organisation of these proteins to resemble the localisation observed in the functional TJs of the long-day hamster (Tarulli et al. 2006). In the mouse, ablation of the SCARKO resulted in an increased permeability of the blood–testis barrier to a biotin tracer (Meng et al. 2005), confirming a role for androgen in this species. In contrast, available data from the rat indicate that TJs remain morphologically present following short-term (6–8 days) androgen withdrawal by the selective Leydig cell toxicant, ethane dimethansulphonate (Kerr et al. 1993), or long-term (41 days) gonadotrophin withdrawal following hypophysectomy (Franca et al. 1998), although blood–testis barrier functionality was not tested in these models. It would be of interest to examine whether rat Sertoli cell TJs in vivo become ‘leaky’ following gonadotrophin or selective androgen withdrawal. Such studies may well be of importance in understanding why there is a non-uniform induction of azoospermia in men undergoing hormonal contraception following suppression of serum gonadotrophins (World Health Organization 1990, 1996).
During spermatogenesis, germ cells must translocate through the blood–testis barrier into the adluminal compartment. While the mechanism of this translocation is unknown, it stands to reason that remodelling of Sertoli cell TJs must occur (Russell & Peterson 1985, Pelletier & Byers 1992). In addition to the stimulatory effects of androgen presented in this study, rat Sertoli cell TER can be negatively regulated by a number of testicular cytokines and growth factors including TGF-β3 (Lui et al. 2001, 2003a, Siu et al. 2003) and TNFα (Lui et al. 2003c, Siu et al. 2003; for reviews see Wong & Cheng 2005, Xia et al. 2005), with TNFα also capable of downregulating occludin and claudin-11 mRNA expression (Hellani et al. 2000, Siu et al. 2003). Androgen receptor levels are highest in stages VII–VIII of spermatogenesis in the rat (Bremner et al. 1994), which precedes primary spermatocyte translocation into the adluminal compartment (Russell & Peterson 1985). Hence, Sertoli cell TJ function and TJ protein expression can be both up and down regulated by hormones and local factors in vitro.
In addition to these changes, our data also suggest that a TJ protein endocytosis or recycling process may be contributing to TJ function in rat Sertoli cells, as demonstrated by the depletion of claudin-11 and occludin from cell contacts after flutamide treatment. The removal of TJ structures, including claudin-3 (Matsuda et al. 2004) by endocytosis-related processes in various epithelial cells is well recognised (for review see Ben-Shaul & Ophir 2001); however, the extent to which Sertoli TJs proteins and function are regulated in this manner in vivo remains to be determined.
In summary, this study demonstrates that claudin-11 plays an important role in the establishment and function of Sertoli cell TJs in vitro, and that androgens promote the expression and localisation of claudin-11 to inter-Sertoli cell contacts. In addition, while not directly regulating occludin mRNA expression, this study demonstrates that androgens promote the localisation of occludin protein at inter-Sertoli cell contacts. In contrast to recent in vivo studies in the mouse, no evidence for a role for claudin-3 in the formation of rat Sertoli cell TJs in vitro could be found. Collectively, these data show that the ability of androgens to maintain spermatogenesis in vivo is partly via their effects on Sertoli cell TJ proteins and regulation of the blood–testis barrier.
Primer-specific LightCycler conditions used for PCR amplification of genes examined.
| Name | Product size (bp) | Primer concentration (pmol) | Mg2+ Concentration (mM) | Anneal temperature (°C) | Extension time (s) | Data acquisition temperature (°C)a | PCR product melting temperature (°C)b |
|---|---|---|---|---|---|---|---|
| aRefers to the temperature at which the fluorescence of the PCR product was quantified during LightCycler analysis. | |||||||
| bAs determined by melting temperature analysis on the LightCycler. | |||||||
| Claudin-11 | 624 | 40 | 3.0 | 68 | 25 | 85 | 91.7 |
| Occludin | 294 | 40 | 4.0 | 64 | 20 | 85 | 89.2 |
| Claudin-3 | 436 | 40 | 2.5 | 64 | 23 | 72 | 93.0 |
Effect of hormonal treatments on inter-Sertoli tight junction permeability and tight junction protein mRNA expression in vitro. (A) Transepithelial electrical resistance (TER) across the Sertoli cell epithelia was measured at daily intervals, beginning at day 0. Sertoli cells (1.25×106 cells/cm2) were cultured in DMEM/F12/1% BSA on Matrigel-coated bicameral chambers for a period of 7 days under basal conditions (medium alone, ○) or in the continuous presence of FSH (2.35 IU/ml; •), testosterone (T; 28 ng/ml; △) or T (28 ng/ml) + FSH (2.35 IU/ml; ▴), with medium and treatments replaced at 2-day intervals as indicated by the arrows (↓). On day 3, all cells were treated with a hypotonic solution to remove contaminating germ cells. Results are expressed as mean ± s.d. of triplicate wells from a single culture, and a representative from three cultures is shown. Asterisks indicate significant differences from basal cultures for each particular day by one-way ANOVA and Newman-Keuls post hoc analysis test (*P < 0.05, **P < 0.01). (B) Claudin-11 mRNA expression, and (C) occludin mRNA expression as measured by real-time PCR in Sertoli cells cultured (1.25×106 cells/cm2) in Matrigel-coated plastic 24-well culture plates with treatments as described in (A). Data are also included for the addition of estradiol (E2, 28 ng/ml, ▾) in (B). Results are expressed as mean ± s.d. of triplicate wells from a single culture, and a representative culture is shown.
Citation: Reproduction 133, 6; 10.1530/REP-06-0385
Relationship between TER and claudin-11 mRNA expression. Correlations between (A): TER established by Sertoli cells and mRNA expression data for all treatment groups (medium alone (○), FSH ( •),T (△),FSH + T (▴))and (B): T alone (△) for days 0–7 were constructed. A significant correlation was observed for the entire data set (A) (r=0.534, P < 0.01, n=32), and also for the T-treated group (B) (r=0.790, P < 0.02, n=8).
Citation: Reproduction 133, 6; 10.1530/REP-06-0385
Effect of androgen treatment on the localisation of claudin-11 and occludin at inter-Sertoli cell contacts in vitro. Sertoli cells were cultured on Matrigel-coated bicameral chambers under either basal conditions (medium alone), (panels A–C and I) or with added testosterone (28 ng/ml, Panels E–G and J) for time periods (days) as shown. Confocal immunofluoresence staining was then carried out for claudin-11 (panels A–G) or occludin (Panels I–J; green staining) with the nuclear counterstain TO-PRO-3 iodide (red-orange). Non-specific binding (panel D for claudin-11, and K for occludin) was assessed by substituting an equivalent concentration of non-specific rabbit IgG for each primary antibody. Bar=10 μm. Western blots for claudin-11 (panel H) or occludin (panel L) were probed with the same primary antibodies used for immunohistochemistry with extracts from cells cultured in the presence of testosterone for 5–7 days.
Citation: Reproduction 133, 6; 10.1530/REP-06-0385
Effect of the androgen receptor antagonist, flutamide, on TER and claudin mRNA expression and protein localisation. Sertoli cells were cultured in the presence of medium alone (A) or testosterone (28 ng/ml, B) as described in the legend to Fig. 1 and TER measurements were taken daily. On day 5, cells from each group were washed and placed in medium containing flutamide (28 μg/ml; • or ▪) for 4 days as shown by the vertical dashed lines. Open symbols (○, □) represent cells which remained in medium alone or medium plus testosterone and did not receive flutamide. On day 9, flutamide was removed and cells were returned to medium alone, or medium plus testosterone (28 ng/ml) until end of culture on day 13. Routine medium replacements were undertaken as shown by the arrows. At days 0, 5, 9 and 13 cells were removed and total RNA extracted for RT and real-time PCR. Panel C presents claudin-11 mRNA expression data for cells continuously treated either with medium alone (○) or testosterone (□), and data for control ( •) or testosterone-treated cells (▪), which received flutamide between days 5 and 9. Data are presented as mean ± s.d. (n=3 PCR estimations) from a single representative culture, and asterisks indicate significant differences compared with respective controls as determined by one-way ANOVA and Newman–Keuls post hoc analysis tests (*P < 0.05; **P < 0.01, ns, not significant). a versus b; P < 0.01, c versus d; ns. Immunohistochemistry for claudin-11 was carried out as described in Fig. 2 legend on testosterone-treated cells cultured with flutamide and sampled at day 9 (Panel D, equivalent to ▪ at day 9 in panel B), or on cells following testosterone replacement and sampled at day 13 (panel E, equivalent to ▪ at day 13 in panel B).
Citation: Reproduction 133, 6; 10.1530/REP-06-0385
The authors would like to thank Dr DM Robertson for helpful discussions during the course of this work, Drs L O’Donnell and RI McLachlan for critical reviews of the manuscript, and Ms S Panckridge for assistance with the figures. This study was funded by Program Grants from the National Health and Medical Research Council (Australia), Regkey no. 241000. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
References
Aurrand-Lions M, Duncan L, Ballestrem C & Imhof BA2001 JAM-2, a novel immunoglobulin superfamily molecule, expressed by endothelial and lymphatic cells. Journal of Biological Chemistry 276 2733–2741.
Ben-Shaul Y & Ophir I2001 Tight junctions and proteases. In Tight Junctions, pp 459–482. Eds M Cereijido & J Anderson. Boca Raton: CRC Press.
Bergmann M1987 Photoperiod and testicular function in phodopus sungorus. Advances in Anatomy, Embryology and Cell Biology 105 1–76.
Bergmann M, Nashan D & Nieschlag E1989 Pattern of compartmentation in human seminiferous tubules showing dislocation of spermatogonia. Cell and Tissue Research 256 183–190.
Bremner WJ, Millar MR, Sharpe RM & Saunders PT1994 Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens. Endocrinology 135 1227–1234.
Bressler RS1976 Dependence of Sertoli cell maturation on the pituitary gland in the mouse. American Journal of Anatomy 147 447–455.
Cera MR, Del Prete A, Vecchi A, Corada M, Martin-Padura I, Motoike T, Tonetti P, Bazzoni G, Vermi W, Gentili Fet al.2004 Increased DC trafficking to lymph nodes and contact hypersensitivity in junctional adhesion molecule-A-deficient mice. Journal of Clinical Investigation 114 729–738.
Chung NP & Cheng CY2001 Is cadmium chloride-induced inter-Sertoli tight junction permeability barrier disruption a suitable in vitro model to study the events of junction disassembly during spermatogenesis in the rat testis? Endocrinology 142 1878–1888.
Chung SS, Lee WM & Cheng CY1999 Study on the formation of specialized inter-Sertoli cell junctions in vitro.Journal of Cellular Physiology 181 258–272.
Chung NP, Mruk D, Mo MY, Lee WM & Cheng CY2001 A 22-amino acid synthetic peptide corresponding to the second extracellular loop of rat occludin perturbs the blood–testis barrier and disrupts spermatogenesis reversibly in vivo.Biology of Reproduction 65 1340–1351.
Cyr DG, Hermo L, Egenberger N, Mertineit C, Trasler JM & Laird DW1999 Cellular immunolocalization of occludin during embryonic and postnatal development of the mouse testis and epididymis. Endocrinology 140 3815–3825.
Djakiew D & Onoda M1993 Multichamber cell culture and directional secretion. In The Sertoli Cell, pp 181–194. Eds L Russell & MD Griswold. Clearwater, FL: Cache River Press.
Ebnet K, Suzuki A, Ohno S & Vestweber D2004 Junctional adhesion molecules (JAMs): more molecules with dual functions? Journal of Cell Science 117 19–29.
Fanning AS, Mitic LL & Anderson JM1999 Transmembrane proteins in the tight junction barrier. Journal of the American Society of Nephrology 10 1337–1345.
Feldman GJ, Mullin JM & Ryan MP2005 Occludin: structure, function and regulation. Advanced Drug Delivery Reviews 57 883–917.
Florin A, Maire M, Bozec A, Hellani A, Chater S, Bars R, Chuzel F & Benahmed M2005 Androgens and postmeiotic germ cells regulate claudin-11 expression in rat Sertoli cells. Endocrinology 146 1532–1540.
Franca LR, Parreira GG, Gates RJ & Russell LD1998 Hormonal regulation of spermatogenesis in the hypophysectomized rat: quantitation of germ-cell population and effect of elimination of residual testosterone after long-term hypophysectomy. Journal of Andrology 19 335–340.
Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S & Tsukita S1993 Occludin: a novel integral membrane protein localizing at tight junctions. Journal of Cell Biology 123 1777–1788.
Galdieri M, Zani BM, Monaco L, Ziparo E & Stefanini M1983 Changes of Sertoli cell glycoproteins induced by removal of the associated germ cells. Experimental Cell Research 145 191–198.
Gliki G, Ebnet K, Aurrand-Lions M, Imhof BA & Adams RH2004 Spermatid differentiation requires the assembly of a cell polarity complex downstream of junctional adhesion molecule-C. Nature 431 320–324.
Gonzalez-Mariscal L, Betanzos A, Nava P & Jaramillo BE2003 Tight junction proteins. Progress in Biophysics and Molecular Biology 81 1–44.
Gorczynska E & Handelsman DJ1995 Androgens rapidly increase the cytosolic calcium concentration in Sertoli cells. Endocrinology 136 2052–2059.
Gow A, Southwood CM, Li JS, Pariali M, Riordan GP, Brodie SE, Danias J, Bronstein JM, Kachar B & Lazzarini RA 1999 CNS myelin and Sertoli cell tight junction strands are absent in Osp/claudin-11 null mice. Cell 99 649–659.
Gye MC2003 Changes in the expression of claudins and transepithelial electrical resistance of mouse Sertoli cells by Leydig cell coculture. International Journal of Andrology 26 271–278.
Gye MC & Ohsako S2003 Effects of flutamide in the rat testis on the expression of occludin, an integral member of the tight junctions. Toxicology Letters 143 217–222.
Hadley MA, Djakiew D, Byers SW & Dym M1987 Polarized secretion of androgen-binding protein and transferrin by Sertoli cells grown in a bicameral culture system. Endocrinology 120 1097–1103.
Handelsman DJ, Spaliviero JA, Kidston E & Robertson DM1989 Highly polarized secretion of inhibin by Sertoli cells in vitro.Endocrinology 125 721–729.
Hellani A, Ji J, Mauduit C, Deschildre C, Tabone E & Benahmed M2000 Developmental and hormonal regulation of the expression of oligodendrocyte-specific protein/claudin 11 in mouse testis. Endocrinology 141 3012–3019.
Janecki A, Jakubowiak A & Steinberger A1991a Regulation of transepithelial electrical resistance in two-compartment Sertoli cell cultures: in vitro model of the blood–testis barrier. Endocrinology 129 1489–1496.
Janecki A, Jakubowiak A & Steinberger A1991b Effects of cyclic AMP and phorbol ester on transepithelial electrical resistance of Sertoli cell monolayers in two-compartment culture. Molecular and Cellular Endocrinology 82 61–69.
Janecki A, Jakubowiak A & Steinberger A1992 Effect of cadmium chloride on transepithelial electrical resistance of Sertoli cell monolayers in two-compartment cultures – a new model for toxicological investigations of the ‘blood–testis’ barrier in vitro.Toxicology and Applied Pharmacology 112 51–57.
Kerr JB, Savage GN, Millar M & Sharpe RM1993 Response of the seminiferous epithelium of the rat testis to withdrawal of androgen: evidence for direct effect upon intercellular spaces associated with Sertoli cell junctional complexes. Cell and Tissue Research 274 153–161.
Koval M2006 Claudins–key pieces in the tight junction puzzle. Cell Communication and Adhesion 13 127–138.
de Kretser DM & Burger HG1972 Ultrastructural studies of the human Sertoli cell in normal men and males with hypogonadotropic hypogonadism before and after gonadotropic treatment. In Gonadotropins, pp 640–656. Eds BB Saxena, CG Beling & HM Gandy. New York: Wiley-Interscience.
Laemmli UK1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 680–685.
Lampa J, Hoogerbrugge JW, Baarends WM, Stanton PG, Perryman KJ, Grootegoed JA & Robertson DM1999 Follicle-stimulating hormone and testosterone stimulation of immature and mature Sertoli cells in vitro: inhibin and N-cadherin levels and round spermatid binding. Journal of Andrology 20 399–406.
Li JC, Mruk D & Cheng CY2001 The inter-Sertoli tight junction permeability barrier is regulated by the interplay of protein phosphatases and kinases: an in vitro study. Journal of Andrology 22 847–856.
Lui WY, Lee WM & Cheng CY2001 Transforming growth factor-beta3 perturbs the inter-Sertoli tight junction permeability barrier in vitro possibly mediated via its effects on occludin, zonula occludens-1, and claudin-11. Endocrinology 142 1865–1877.
Lui WY, Lee WM & Cheng CY2003a Transforming growth factor- 3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase. Biology of Reproduction 68 1597–1612.
Lui WY, Mruk DD, Lee WM & Cheng CY2003b Adherens junction dynamics in the testis and spermatogenesis. Journal of Andrology 24 1–14.
Lui WY, Mruk D, Lee WM & Cheng CY2003c Sertoli cell tight junction dynamics: their regulation during spermatogenesis. Biology of Reproduction 68 1087–1097.
Lyng FM, Jones GR & Rommerts FF2000 Rapid androgen actions on calcium signaling in rat sertoli cells and two human prostatic cell lines: similar biphasic responses between 1 picomolar and 100 nanomolar concentrations. Biology of Reproduction 63 736–747.
Maddocks S & Setchell BP1990 Recent evidence for immune privilege in the testis. Journal of Reproductive Immunology 18 9–18.
Matsuda M, Kubo A, Furuse M & Tsukita S2004 A peculiar internalization of claudins, tight junction-specific adhesion molecules, during the intercellular movement of epithelial cells. Journal of Cell Science 117 1247–1257.
Meng J, Holdcraft RW, Shima JE, Griswold MD & Braun RE2005 Androgens regulate the permeability of the blood–testis barrier. PNAS 102 16696–16700.
Mitic LL, Van Itallie CM & Anderson JM2000 Molecular physiology and pathophysiology of tight junctions I. Tight junction structure and function: lessons from mutant animals and proteins. American Journal of Physiology. Gastrointestinal and Liver Physiology 279 G250–G254.
Morita K, Furuse M, Fujimoto K & Tsukita S1999a Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proceedings of the National Academy of Sciences of the United States of America 96 511–516.
Morita K, Sasaki H, Fujimoto K, Furuse M & Tsukita S1999b Claudin-11/OSP-based tight junctions of myelin sheaths in brain and Sertoli cells in testis. Journal of Cell Biology 145 579–588.
Moroi S, Saitou M, Fujimoto K, Sakakibara A, Furuse M, Yoshida O & Tsukita S1998 Occludin is concentrated at tight junctions of mouse/rat but not human/guinea pig Sertoli cells in testes. American Journal of Physiology 274 C1708–C1717.
Mruk DD & Cheng CY2004 Sertoli–Sertoli and Sertoli-germ cell interactions and their significance in germ cell movement in the seminiferous epithelium during spermatogenesis. Endocrine Reviews 25 747–806.
Mulholland DJ, Dedhar S & Vogl AW2001 Rat seminiferous epithelium contains a unique junction (Ectoplasmic specialization) with signaling properties both of cell/cell and cell/matrix junctions. Biology of Reproduction 64 396–407.
Onoda M, Suarez-Quian CA, Djakiew D & Dym M1990 Characterization of Sertoli cells cultured in the bicameral chamber system: relationship between formation of permeability barriers and polarized secretion of transferrin. Biology of Reproduction 43 672–683.
Pelletier RM1988 Cyclic modulation of Sertoli cell junctional complexes in a seasonal breeder: the mink (Mustela vison). American Journal of Anatomy 183 68–102.
Pelletier RM & Byers SW1992 The blood–testis barrier and Sertoli cell junctions: structural considerations. Microscopy Research and Technique 20 3–33.
Perryman KJ, Stanton PG, Loveland KL, McLachlan RI & Robertson DM1996 Hormonal dependency of neural cadherin in the binding of round spermatids to Sertoli cells in vitro.Endocrinology 137 3877–3883.
Russell LD & Peterson RN1985 Sertoli cell junctions: morphological and functional correlates. International Review of Cytology 94 177–211.
Russell LD, Bartke A & Goh JC1989 Postnatal development of the Sertoli cell barrier, tubular lumen, and cytoskeleton of Sertoli and myoid cells in the rat, and their relationship to tubular fluid secretion and flow. American Journal of Anatomy 184 179–189.
Saitou M, Ando-Akatsuka Y, Itoh M, Furuse M, Inazawa J, Fujimoto K & Tsukita S1997 Mammalian occludin in epithelial cells: its expression and subcellular distribution. European Journal of Cell Biology 73 222–231.
Saitou M, Furuse M, Sasaki H, Schulzke JD, Fromm M, Takano H, Noda T & Tsukita S2000 Complex phenotype of mice lacking occludin, a component of tight junction strands. Molecular Biology of the Cell 11 4131–4142.
Sakakibara A, Furuse M, Saitou M, Ando-Akatsuka Y & Tsukita S1997 Possible involvement of phosphorylation of occludin in tight junction formation. Journal of Cell Biology 137 1393–1401.
Siu MK, Lee WM & Cheng CY2003 The interplay of collagen IV, tumor necrosis factor-alpha, gelatinase B (matrix metalloprotease-9), and tissue inhibitor of metalloproteases-1 in the basal lamina regulates Sertoli cell-tight junction dynamics in the rat testis. Endocrinology 144 371–387.
Sluka P, O’Donnell L & Stanton PG2002 Stage-specific expression of genes associated with rat spermatogenesis: characterization by laser-capture microdissection and real-time polymerase chain reaction. Biology of Reproduction 67 820–828.
Sluka P, O’Donnell L, Bartles JR & Stanton PG2006 FSH regulates the formation of adherens junctions and ectoplasmic specialisations between rat Sertoli cells in vitro and in vivo.Journal of Endocrinology 189 381–395.
Steinberger A & Klinefelter G1993 Sensitivity of Sertoli and Leydig cells to xenobiotics in in vitro models. Reproductive Toxicology 7 23–37.
Swift TA & Dias JA1988 Testosterone suppression of ornithine decarboxylase activity in rat Sertoli cells. Endocrinology 123 687–693.
Tan KA, De Gendt K, Atanassova N, Walker M, Sharpe RM, Saunders PT, Denolet E & Verhoeven G2005 The role of androgens in Sertoli cell proliferation and functional maturation: studies in mice with total or Sertoli cell-selective ablation of the androgen receptor. Endocrinology 146 2674–2683.
Tarulli GA, Stanton PG, Lerchl A & Meachem SJ2006 Adult Sertoli cells are not terminally differentiated in the Djungarian hamster: effect of FSH on proliferation and junction protein organization. Biology of Reproduction 74 798–806.
Toyama Y, Maekawa M & Yuasa S2003 Ectoplasmic specializations in the Sertoli cell: new vistas based on genetic defects and testicular toxicology. Anatomical Science International 78 1–16.
Vitale R, Fawcett DW & Dym M1973 The normal development of the blood–testis barrier and the effects of clomiphene and estrogen treatment. Anatomical Record 176 331–344.
Wang RS, Yeh S, Chen LM, Lin HY, Zhang C, Ni J, Wu CC, di Sant’Agnese PA, deMesy-Bentley KL, Tzeng CRet al.2006 Androgen receptor in sertoli cell is essential for germ cell nursery and junctional complex formation in mouse testes. Endocrinology 147 5624–5633.
Wong CH & Cheng CY2005 The blood–testis barrier: its biology, regulation, and physiological role in spermatogenesis. Current Topics in Developmental Biology 71 263–296.
World Health Organization Task Force on Methods for the Regulation of Male Fertility1990 Contraceptive efficacy of testosterone-induced azoospermia in normal men. Lancet 336 955–959.
World Health Organization Task Force on Methods for the Regulation of Male Fertility1996 Contraceptive efficacy of testosterone-induced azoospermia and oligozoospermia in normal men. Fertility and Sterility 65 821–829.
Xia W, Mruk DD, Lee WM & Cheng CY2005 Cytokines and junction restructuring during spermatogenesis – a lesson to learn from the testis. Cytokine & Growth Factor Reviews 16 469–493.
