Expression of Aryl Hydrocarbon Receptor and Aryl Hydrocarbon ...

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Endocrinology 144(3):767–776 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2002-220642

Expression of Aryl Hydrocarbon Receptor and Aryl Hydrocarbon Receptor Nuclear Translocator Messenger Ribonucleic Acids and Proteins in Rat and Human Testis ¨ DIGER SCHULTZ, JANNE SUOMINEN, TANJA VA ¨ RRE, HARRI HAKOVIRTA, MARTTI PARVINEN, RU JORMA TOPPARI, AND MARKKU PELTO-HUIKKO Department of Developmental Biology, Tampere University (R.S., T.V., M.P.-H.), FIN-33014 Tampere, Finland; Departments of Dermatology (R.S.) and Pathology (M.P.-H.), Tampere University Hospital, FIN-33520 Tampere, Finland; Department of Pediatrics and Physiology, Turku University Hospital (J.S., H.H., J.T.), and Department of Anatomy, University of Turku (M.P.), FIN-20520 Turku, Finland Dioxins, e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), use the aryl hydrocarbon receptor (AHR)/aryl hydrocarbon receptor nuclear translocator (ARNT) receptor complex to mediate their toxic actions. In addition to interaction with environmental pollutants, several transcription factors, steroid receptors, and growth factors are capable interacting with the AHR/ARNT complex, which suggests a constitutive role for the receptor complex. The testis has been reported to be among the most sensitive organs to TCDD exposure. Our experiments revealed a complex distribution of AHR and ARNT mRNAs and proteins in rat and human testis. AHR and ARNT immunoreactivities could

be detected in the nuclei of interstitial and tubular cells. The incubation of seminiferous tubules in a serum-free culture medium resulted in up-regulation of AHR mRNA, which could be depressed by adding FSH to the culture medium. Furthermore, the incubation of tubular segments with a solution of 1 or 100 nM TCDD resulted in a 2- to 3-fold increase in apoptotic cells. Thus, up-regulation of AHR in cultured tubular segments and consecutive depression by FSH suggest a role for AHR in controlled cell death during spermatogenesis. We suggest that AHR and ARNT mediate effects by direct action on testicular cells in the rat and human testis. (Endocrinology 144: 767–776, 2003)

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OLYAROMATIC hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are toxic xenobiotics that are well known for their teratogenic and carcinogenic effects. They mediate their toxic effects by the dimeric aryl hydrocarbon receptor (AHR)/AHR nuclear translocator (ARNT) complex, which binds to the xenobiotic response element on target DNA (1). AHR and ARNT are members of the basic-helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family of heterodimeric transcription factors. Besides activation by xenobiotics, several enzymes and transcription factors have been demonstrated to interact with the AHR/ ARNT complex (2– 4). Additionally, AHR/ARNT is ubiquitously expressed in mammalian tissues and may play a critical role in early embryonic development (5– 8). Thus, besides mediating the toxic effects of TCDD, a constitutive role for AHR may be postulated. However, the naturally occurring ligand for AHR remains to be elucidated. Mammalian spermatogenesis is characterized by a complicated cascade of processes that allows the development of highly differentiated spermatozoa from diploid stem cells. This process includes the action of hormones, and paracrine and transcription factors (9). As observed during recent decades, any disturbance of this complicated system may lead to spermatogenic cancer, disruption of spermatogenesis, and reduction in semen quality (10). The possible role of dioxins

Materials and Methods Experimental animals

Abbreviations: AHR, Aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; IR, immunoreactivity; SDS, sodium dodecyl sulfate; SSC, standard saline citrate; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TdT, terminal deoxynucleotidyl transferase.

A total of 25 adult male Sprague Dawley rats (aged 3–5 months) were used in this study. The animals were maintained in a controlled environment at 26 C and 60% humidity, with a constant 12-h light, 12-h dark cycle. The animals had free access to standard laboratory food and water. Two or three animals were kept in the same cage. All animal experiments

in testicular dysfunction has been studied in the past in several animal models. Even short-term exposure to TCDD has been shown to disturb intercellular signaling between Sertoli and neighboring germ cells, with a consequent disruption of spermatogenic cells. Additionally, a single exposure causes a dose-dependent reduction in the volume of Leydig cells (11, 12). In addition to direct toxic responses, the AHR/ARNT receptor complex is able to modify hormonal signals by interacting with their response elements on target DNA (3, 4). To understand the consequences of possible toxic or constitutive activation of AHR/ARNT in testicular cells, their exact localization is of great importance. Therefore, we studied the cell- and stage-specific expression of AHR and ARNT mRNAs and proteins in the testicular cells of Sprague Dawley rats employing Northern analysis, in situ hybridization, and immunocytochemistry. Additionally, we examined the testicular expression of AHR and ARNT mRNAs and proteins in the human testis. To investigate the possible roles of these receptors in the regulation of apoptosis, we studied the effects of TCDD on segments of seminiferous tubules at two different concentrations and carried out an apoptosis assay.

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were approved by the local ethical committee for animal research at University of Tampere. Ten adult male Sprague Dawley rats were used for in situ hybridization and immunocytochemistry. Six were used for the Northern analysis, and nine were used for the in vitro analysis of apoptosis.

Human testis Paraffin sections containing multiple human tissues (T1065, lot 9994A) were obtained from DAKO Corp. (Copenhagen, Denmark). In addition, routine testicular paraffin sections were used (Department of Pathology, Tampere University Hospital). For in situ hybridization fresh human testes were obtained from surgery performed due to prostatic malignancies and were frozen on dry ice.

In situ hybridization and immunocytochemistry Immunocytochemistry. For immunocytochemistry the animals were perfused through the ascending aorta under fentanyl-fluanisone anesthesia, first with physiological saline and then with a fixative containing 4% paraformaldehyde in 0.1 m PBS for 3 min. Subsequently, the testes were excised and further fixed by immersion at 4 C in the same fixative for 60 min. The samples were cryoprotected with 20% sucrose in PBS and frozen with carbon dioxide, and 10-␮m sections were cut with a Microm HM 500 cryostat (Microm, Heidelberg, Germany). The human testicular biopsies were immersion-fixed in Bouin’s fixative and embedded in paraffin. Thereafter the sections were deparaffinized with xylene and rehydrated through a graded series of ethanol. Subsequently, the sections were subjected to microwave antigen retrieval treatment as described by Shi et al. (13). Endogenous peroxidase activity was blocked by treating the sections with 0.1% hydrogen peroxide in PBS for 20 min. The sections were incubated for 12–24 h at 4 C with rabbit antiserum to rat AHR (14) and ARNT (15) (1:250 –500), followed by biotinylated goat antirabbit IgG and the avidin-biotin peroxidase complex (Vector Laboratories, Inc., Burlingame, CA). Diaminobenzidine was used as chromogen to visualize AHR/ARNT immunoreactivity (IR). Controls included omission of the primary antibody and staining with nonimmunized rabbit serum (1:100). In addition, presaturation was performed with the peptides used for generation of the primary antibodies (14, 15). No specific staining could be observed in the controls, and only immunoreactivities not seen in the controls were considered specific. Sections were examined under a Nikon FXA microscope equipped with a PCO Sensicam digital camera (PCO, Kelheim, Germany). The stages of the cycle were recognized using the morphological criteria described by Leblond and Clermont (16). Images were processed using Corel Draw software (Corel Corp. Ltd., Ontario, Canada) and printed with an HPDeskJet 995c printer (Hewlett-Packard Co., Palo Alto, CA). In situ hybridization. After decapitation of the animals, the testes were excised and frozen on dry ice. Serial 14 ␮m thick sections were cut with a Microm HM-500 cryostat, and the sections were thawed on Polysine glasses (Menzel-Gla¨ ser, Braunschweig, Germany). Nonfixed human testicular samples were sectioned similarly. Sections were stored at ⫺20 C until use. Four oligonucleotide probes directed against the rat AHR (nucleotides 138 –178, 645– 686, 739 – 840, and 1219 –1260) (17), two rat/ human ARNT (rat nucleotides 361– 407 and 1146 –1190; human nucleotides 409 – 455 and 1194 –1238) (18), and two human AHR (nucleotides 1722–1751 and 3409 –3438) (19) genes were used in this study. The sequences exhibited less than 60% homology with any other known gene compared with the known sequences in the GenBank database. Several control probes with the same length, similar GC content, and specific activity were used to ascertain the specificity of the hybridizations. The in situ hybridization was carried out as described in detail previously (20). The probes were labeled with [␣-33P]deoxy-ATP (NEN Life Science Products, Boston, MA) using terminal deoxynucleotidyl transferase (Tdt; Amersham International, Little Chalfont, UK) to a specific activity of 6 ⫻ 109 cpm/␮g. The sections were briefly air-dried and hybridized at 42 C for 18 h with 5 ng/ml of the probes in the hybridization cocktail. After hybridization, the sections were rinsed four times at 55 C in 1⫻ standard saline citrate (1⫻ SSC) for 15 min each and subsequently left to cool for 1 h at room temperature. The sections were dipped in distilled water, dehydrated with 60% and 90% ethanol, and air-dried. Thereafter,

Schultz et al. • AHR and ARNT in Testis

the sections were covered with MR autoradiography film (Kodak, Rochester, NY). The films were developed using Kodak LX24 developer and AL4 fixative.

Northern analysis Transillumination-assisted microdissection of seminiferous tubules. The rats were killed by CO2 asphyxiation, and the testes were excised and decapsulated. The seminiferous tubules were teased free by fine forceps under a transilluminating stereomicroscope in DMEM-Ham’s F-12 medium (1:1; DMEM/F12; Life Technologies, Inc., Paisley, Scotland, UK) supplemented with 15 mm HEPES, 1.25 g/liter sodium bicarbonate, 10 mg/liter gentamycin sulfate, 60 mg/liter penicillin G, 1 g/liter BSA, and 0.1 mm 3-isobutyl-1-methylxanthine (Aldrich Chemie, Steinheim, Germany). The stages were recognized according to light absorption criteria (21). For Northern analysis, pools of stages II–VI, VII–VIII, IX–XII, and XIII–I, each containing a total of 100 5-mm seminiferous tubule segments, were collected. For the apoptosis assay we additionally collected 1-mm segments from stage XII of the cycle. Tissue culture and FSH and TCDD stimulation. One hundred pieces of 5-mm seminiferous tubule segments were incubated in 5 ml of the above-mentioned culture medium in the presence or absence of FSH (10,000 IU/mg; rh FSH, Org 32489, Organon, Oss, The Netherlands) in a concentration of 10 ng/ml for 30 h. The tubules were then collected for isolation of total RNA. One-millimeter seminiferous tubule segments were incubated in 100 ml of the above-mentioned culture medium in the presence or absence of TCDD at concentrations of 1 and 100 nm for 8 h. Isolation of polyadenylated mRNA and Northern blot hybridization. The extraction of total RNA was carried out using the single-step method (22). Polyadenylated RNA was isolated from total RNA with a NucleoTrap Nucleic Acid Purification Kit (CLONTECH Laboratories, Inc., Palo Alto, CA). The RNA was size-fractionated in denaturing 1% agarose gels, transferred onto a Hybond-N⫹ nylon membrane (Amersham International), and fixed to the membrane by UV cross-linking. The filters were prehybridized in 50% formamide, 3⫻ SSC, 5⫻ Denhart’s solution (1 mg/ml Ficoll, 1 mg/ml polyvinylpyrrolide, and 1 mg/ml BSA), 1.2% sodium dodecyl sulfate (SDS), 10% dextran sulfate, and 1 mm EDTA (Sigma-Aldrich, St. Louis, MO) containing 10 ␮g yeast transfer RNA at 66 C for 4 –16 h. Hybridization was performed at the same temperature for 14 –24 h by adding the 32P-labeled probe. The 713-bp sequence of AHR, spanning nucleotides 127– 839 of the published rat AHR cDNA, was cloned into pCR 2.1, and an antisense probe was made with T7 RNA polymerase. The filters were washed for 20 min with 2⫻ SSC/0.1% SDS at room temperature, followed by two 20-min washes in 0.2⫻ SSC/0.1% SDS in 65 C and two 20-min washes in 0.1 SSC/0.1% SDS at 65 C. The filters were stripped after hybridization with the AHR probe by pouring the boiling 0.1% SDS onto the filters. Control hybridization was made using a 1.3-kb BamHI fragment of a pI-19 cDNA clone of the mouse 28S rRNA at 42 C. In vitro transcription reactions were performed as recommended by the manufacturer (Promega Corp., Madison, WI). Filters were exposed to Rx 100 film (Fuji Photo Film Co., Ltd., Tokyo, Japan) at ⫺70 C between intensifying screens.

Squash preparations, in situ 3⬘-end labeling, and quantification of germ cells The segments (1 mm) of mouse seminiferous epithelium from stage XII were incubated for 8 h in 100 ml Ham’s F-12/DMEM (Life Technologies, Inc.), supplemented as described above, in a humidified atmosphere (5% CO2, 34 C) in the presence and absence of TCDD and then transferred in 15 ml culture medium onto a microscope slide. The tubules were carefully squashed between microscope slides and coverslips, frozen in liquid nitrogen. Thereafter the coverslips were removed, and the slides were dipped briefly in ice-cold 96% ethanol and fixed in 10% formalin for 10 min. After fixation the slides were washed twice in PBS for 5 min, postfixed in ethanol/acetic acid (2:1, vol/vol), washed in PBS again, and finally dehydrated in ethanol solutions of increasing concentrations, air-dried, and stored at ⫺70 C. In situ 3⬘-end labeling was carried out as previously described (23) with modifications. Each incubation was performed in a humidified box. The squash preparations were rehydrated in ethanol solutions of decreasing concentrations. The

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samples were incubated for 10 min in 1⫻ TdT buffer (Roche Molecular Biochemicals, Mannheim, Germany) before the 3⬘-end labeling. Ten milliliters of labeling mixture containing 2 ml fresh 5⫻ TdT, 0.16 ml TdT (Roche Molecular Biochemicals), 0.05 ml digoxigenin-11-dideoxyuridine triphosphate (1 mmol/ml; Roche Molecular Biochemicals), 2 ml 25 mm CoCl2 (Roche Molecular Biochemicals), 0.09 ml 5 mm dideoxyATP (Amersham Pharmacia Biotech, Piscataway, NJ), and 5.7 ml water were applied to each sample, and the incubation was performed at 37 C for 1 h. After washing the slides three times for 10 min each time in a Tris buffer (150 mm NaCl and 100 mm Tris-HCl, pH 7.4), about 20 ml of the same buffer containing 2% (wt/vol) blocking reagent (Roche Molecular Biochemicals) were applied to each slide, and the samples were incubated at room temperature for 30 min. Twenty milliliters of 2% (wt/vol) blocking buffer containing diluted (1:4000) antidigoxigenin alkaline phosphatase conjugate (Roche Molecular Biochemicals) were applied to the same samples, which were then incubated at room temperature for 2 h. The slides were washed (three times for 10 min each time) in the Tris buffer and then left for 5–10 min in an alkaline phosphatase buffer (100 mm Tris-HCl, 100 mm NaCl, and 50 mm MgCl2, pH 9.5). Twenty milliliters of a substrate solution containing nitro blue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate (Roche Molecular Biochemicals) were diluted to 1 ml alkaline phosphatase buffer, and then about 20 ml of this solution were added to each sample in the dark at room temperature. After the color reaction was assumed to have fully developed, the slides were treated for at least 5 min in EDTA buffer (100 mm Tris-HCl and 1 mm EDTA, pH 8.0) to stop the reaction. The experiment was repeated three times; each time four parallel tubule segments were cultured, and the number of apoptotic cells in each segment was counted under a light microscope.

Densitometric analysis The x-ray films of Northern hybridization were first scanned by a UMAX scanner (Super Vista S-20, Binuscan, Inc., Mamaroneck, NY) and a Binuscan Photoperfect software package (Binuscan). The images were analyzed using a TINA 2.0 densitometric analytical system (Raytest Isotopenmessgera¨ te GmbH, Straubenhardt, Germany) according to the manufacturer’s instructions.

Replication of experiments and statistical analysis In all of the Northern hybridization analyses, the densitometric values of the signals of AHR mRNA were first normalized to 28S signals, and then the 0 h control from stages XIII–I was arbitrarily given the value 1. All other values were expressed in relation to this value. The Northern hybridization analyses were repeated twice with similar results. The experiments with TCDD-induced apoptosis in the cultured seminiferous tubules were repeated three times. In the tubules incubated with 1 or 100 nm TCDD, apoptotic cells were counted and compared with the control group. The values from all three experiments were pooled for calculation of the means and their ses, and for one-way ANOVA and Duncan’s new multiple range test to determine the significant differences between experimental groups using the StatView 4.51 statistical program (Abacus Concepts, Inc., Berkeley, CA). P ⬍ 0.05 was considered as statistically significant.

Results Northern blot analysis

Using Northern blot analysis we found two transcripts of 6.6 and 6.2 kb specific for AHR mRNA in all stages of the rat seminiferous epithelium. The initial signal seen in the freshly dissected tissues was very low during all stages of the cycle (Fig. 1, A and B). Stages IX–XII and XIII–I showed a slightly higher signal for AHR mRNA than stages II–VI and VII–VIII (Fig. 1B). After 30 h of incubation, all stages of the cycle showed a clear up-regulation of AHR mRNA, with the highest signal during stages IX–XII and XIII–I (Fig. 1, A and B). A slightly lower signal could be observed during stages II–VI, whereas the lowest signal was seen at stages VII–VIII.

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FIG. 1. Demonstration of two transcripts (6.6 and 6.2 kb) specific for AHR mRNA in the seminiferous tubules of the rat testis at different stages of the cycle. At 0 h, a slightly higher expression can be seen during stages I–IX of the cycle. A clear up-regulation of AHR mRNA is present after 30 h of incubation during stages VI–IX, whereas during stages VII-VIII only a slight increase can be observed. With the addition of FSH (10 ng/ml) up-regulation of AHR mRNA can be depressed. 28S cDNA was used for control hybridization. A, Quantitative analysis of the stage-specific expression of AHR mRNA in the rat seminiferous epithelium. The different levels for AHR mRNA are defined as arbitrary densitometric units (ADU); they were first normalized to 28S signals, and then the densitometric level of AHR mRNA after 0 h of incubation (stages I–XIII) was arbitrarily given the value 1. Other values were expressed in relation to the highest one. Each bar represents the mean ⫾ range of two independently performed experiments (B).

The presence of FSH inhibited the up-regulation of AHR mRNA during all stages of the cycle (Fig. 1, A and B). In situ hybridization of rat testis

In the rat seminiferous epithelium a clear signal for both AHR and ARNT mRNAs could be seen (Fig. 2, B and C). Both mRNAs exhibited variable levels in different tubules. Additionally, AHR and ARNT appeared in the interstitial cells (Fig. 2, B and C). No signal could be observed in the control hybridization using nonrelated probes of the same length, similar GC contents, and similar specific activities (Fig. 2A). In situ hybridization of human testis

Low expression of AHR and a moderate signal for ARNT mRNA were present in tubular cells of human testis, whereas no signal could be seen after hybridization with nonrelated probes (Fig. 3, A–C). Immunocytochemistry of AHR in the rat testis

In the adult rat testis, AHR-IR was present in the nuclei of cells of the seminiferous epithelium (Fig. 4 and Table 1) and in the interstitial cells (Fig. 4, A and B). In the sem-

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FIG. 3. In situ hybridization of AHR and ARNT mRNA in the human testis. A low signal for AHR (B) and a moderate signal for ARNT can be seen in the seminiferous epithelium (arrows; C). Low signal for both AHR (B) and ARNT (C) was detectable in the interstitium (arrowheads) of human testis. Control hybridization with a nonrelated probe exhibited no signal (A).

FIG. 2. In situ hybridization of AHR and ARNT mRNA in the rat testis. A slightly weaker expression for AHR can be seen in the seminiferous tubules (white arrows) and the interstitium (white arrowheads; B), whereas a stronger signal was seen for ARNT mRNA (C) in the seminiferous tubules (white arrows) and additionally in the interstitium (white arrowheads) of rat testis. No signal could be observed in the control hybridizations (A).

iniferous tubules AHR-IR appeared in a stage-specific pattern. At stages III–VI, the tubules were devoid of AHR-IR (Fig. 4, A and D). In the tubules at stages VII–XI, only primary pachytene spermatocytes showed immunoreactivity (Fig. 4, E and F), whereas at stages XIV–II of the cycle the tubules exhibited AHR-IR in round spermatids, primary pachytene spermatocytes, and some Sertoli cells (Fig. 4C). In the interstitium AHR-IR was seen in the nuclei of the Leydig cells and some smooth muscle and in the endothelial cells of blood vessels (Fig. 4B). In comparison with AHR-IR in the seminiferous tubules, only a few Leydig cells exhibited AHR-IR.

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FIG. 4. Demonstration of AHR protein in the rat testis. In the seminiferous epithelium a stage-specific expression could be observed (A–F). Some tubules are devoid of AHR-IR (A and D). During stages II–XIV of the cycle, strong labeling for AHR appeared in some Sertoli cells (arrowheads), the primary pachytene spermatocytes (arrows), and the round spermatids (triangles; C). During some stages of the cycle (VII–XI) only the primary pachytene spermatocytes exhibited AHR-IR (E and F). Strong staining for AHR could also be observed in the interstitial cells of the rat testis (arrows, A). Some Leydig cells exhibited AHR-IR (arrows, B). Additionally, labeling could be observed in epithelial cells (arrowheads) and smooth muscle cells (triangle) of the blood vessels (B).

TABLE 1. AHR-IR during the rat spermatogenic cycle

were intensely stained, whereas others exhibited no ARNT-IR (Fig. 5, A and D). At stages XIV–I only Sertoli cells were labeled (Fig. 5C), during stages VII–VIII most of the primary pachytene spermatocytes and round/elongating spermatids were strongly stained (Fig. 5E), whereas during stages IX–XI of the cycle, labeling for ARNT-IR could only be observed in the primary pachytene spermatocytes (Fig. 5F). In the interstitium smooth muscle cells, the endothelial cells of the blood vessels (not shown) and most Leydig cells showed strong staining for ARNT-IR (Fig. 5B). Immunocytochemistry of AHR in the human testis

Shaded area represents spermatogenic cells with AHR-IR.

Immunocytochemistry of ARNT in the rat testis

The adult rat testis showed a stage-specific distribution of ARNT-IR in the seminiferous epithelium (Fig. 5 and Table 2). Labeling was restricted to the nuclei of cells. Some tubules

Strong labeling for AHR-IR was present in almost all nuclei of cells of the seminiferous epithelium (Fig. 6). The peritubular cells also showed labeling for AHR (Fig. 6C). No clear stage-specific distribution of AHR protein could be seen (Fig. 6, A and B). The interstitial Leydig cells, smooth muscle cells, and endothelial cells of the blood vessels exhibited strong AHR-IR (Fig. 6C). No immunoreactivity for AHR could be observed after presaturation (Fig. 6D).

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FIG. 5. ARNT-IR in seminiferous tubules and interstitial cells (arrows) of rat testis (A). Expression of ARNT protein in the seminiferous tubules appeared in a stage-specific pattern (A). Some tubules were devoid of ARNT-IR (D), whereas during stages I–XIV only Sertoli cells exhibited labeling for ARNT (C). During stages VII–VIII the primary pachytene spermatocytes (arrows) and the round/elongating spermatids (small arrowheads) showed ARNT-IR (E). During stages IX–XI, only the primary pachytene spermatocytes were labeled (arrowheads; F). In the interstitium, strong labeling was observed in Leydig cells (arrows) and smooth muscle cells (arrowheads; B).

TABLE 2. ARNT-IR during the rat spermatogenic cycle

(Fig. 7, A–C). No stage-specific distribution could be detected, but all tubular and peritubular cells were heavily stained (Fig. 7, B and C). In the interstitium, ARNT-IR was present in the interstitial Leydig cells, smooth muscle cells, and endothelial cells of the blood vessels (Fig. 7C). After presaturation, no immunoreactivity for ARNT protein could be detected (Fig. 7D). In vitro analysis of TCDD-induced apoptosis in cultured seminiferous tubules

Shaded area represents spermatogenic cells with ARNT-IR.

Immunocytochemistry of ARNT in the human testis

Likewise for AHR, strong staining for ARNT could be observed in almost all nuclei of the cells of the seminiferous epithelium and in nuclei of the interstitial cells in the human testis

Compared with the control slides, a 2.5-fold increase in apoptotic cells could be seen in the seminiferous epithelium incubated for 8 h with 1 nm TCDD (Fig. 8, A and B). After incubation for 8 h in a medium supplemented with 100 nm TCDD, the increase in apoptotic cells was 3-fold (Fig. 8, B and C). Discussion Expression of AHR and ARNT mRNA in rat testis

With in situ hybridization a strong signal for AHR and ARNT mRNAs could be detected in the seminiferous epi-

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FIG. 6. Presentation of AHR-IR in human testis. Virtually all cells in the seminiferous tubules showed strong nuclear labeling for AHR, but stage specificity could not be detected (A and B). Additionally, the nuclei of the peritubular cells exhibited AHR-IR (double arrow; C). In the interstitium, clusters of Leydig cells (arrows), some endothelial cells (arrowheads), and the smooth muscle cells (white triangle) of the blood vessels showed AHR-IR (C). No immunoreactivity could be observed after preabsorption with the AHR protein (D).

FIG. 7. Expression of ARNT protein in tubules and interstitial cells (arrowheads) of the human testis (A). In the tubules all nuclei of cells were heavily stained (B). Also, peritubular cells exhibited ARNT-IR (triangle; C). In the interstitium the Leydig cells (arrows) and smooth muscle cells of the blood vessels (arrowhead) revealed labeling. The nuclei of the smooth muscle cells of the blood vessels (arrowhead) and the epithelial cells of the blood vessels (asterisk) clearly showed ARNT-IR (C). Preabsorption extinguished all immunoreactivity (D).

thelium of the rat testis. However, the signal for ARNT mRNA was stronger in both tubular and interstitial cells of the rat testis. Northern analysis revealed two different tran-

scripts for AHR of 6.6 and 6.2 kb, respectively. Similar observations were made in earlier investigations, where it could be shown that some transcripts, which normally are

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FIG. 8. Demonstration of apoptosis in seminiferous tubules. Cultured 1-mm segments of seminiferous tubules were squashed, and the cell monolayer was stained with the in situ end labeling technique using a substrate solution containing nitro blue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate. All apoptotic cells (examples marked by arrows) were quantified in these preparations. In the controls, a few apoptotic cells can be seen (A). A notable increase in apoptotic germ cells (arrows) was present in the seminiferous tubules incubated in 100 nM TCDD (B). Quantification of apoptotic germ cells in seminiferous tubules of rat testis. The mean amount of apoptotic germ cells in the controls was 330. After incubation in 1 nM TCDD, a 2.5-fold increase in apoptotic germ cells was observed. After incubation in 100 nM, the number of apoptotic germ cells increased 3-fold compared with that in controls. This increase was statistically significant (P ⬍ 0.05; C).

expressed as single ones, can be detected as two separate transcripts in the rat testis (24, 25). The strongest signal for AHR was seen after 30 h of incubation during stages IX–I of the cycle. During these stages spermatogenic cells finish their second meiotic division and are completely surrounded by Sertoli cells, which are then most sensitive to FSH (26). We observed that incubation of the seminiferous tubules in a serum-free medium resulted in an induction of AHR mRNA, which could be prevented by adding 10 ng/ml FSH to the culture medium. It is well known that germ cells undergo apoptosis under serum-free culture conditions (27). Recently, it was shown that AHR drives bax gene expression and apoptosis in germinal cells (28). Additionally, stem cell factor, FSH, and a natural competitive inhibitor of AHR, resveratrol, are able to protect germ cells from apoptosis (29 – 31). As spermatogenesis, under the control of FSH and testosterone, is accompanied by a wave of apoptosis (32), it may be suggested that AHR is able to induce or enhance apoptosis in the germ cells of the rat testis. This assumption gets additional support from our experiments with seminiferous tubules incubated in 1 or 100 nm TCDD. A significant increase in the amount of apoptotic cells could be observed after incubation in a culture medium containing 100 nm TCDD.

AHR was not coexpressed. However, differences in the protein expression of these receptors in the same tissue were also observed in earlier studies (33). In addition, the widest expression of AHR-IR was in concert with the strongest signal for AHR mRNA in Northern analysis. Despite differing expressions during the stages of the cycle, neither ARNT nor AHR could be observed in premeiotic cells of seminiferous epithelium. In addition to germ cells, AHR and ARNT-IR were detected in Sertoli cells, interstitial Leydig cells, smooth muscle cells, and endothelial cells of blood vessels. According to their localization, many constitutive and toxic functions for the AHR/ARNT complex can be taken into consideration. It has been reported that AHR/ARNT is able to suppress the function of other steroid receptors, thereby acting as an endocrine disruptor (3). Thus, some of the reported adverse effects of dioxins on the testis could be explained by a reduction of plasma androgens, resulting in an impairment of spermatogenesis (34, 35). However, the localization of receptor proteins in the nuclei of germ cells offers the possibility for some direct action of environmental toxins such as dioxins on spermatogenesis in rats, resulting in the well known toxic effects of these components, such as reduction in sperm number, weight of testes, and daily amount of sperm production (12, 36, 37).

Expression of AHR and ARNT protein in rat testis

Expression of AHR and ARNT mRNA and protein in the human testis

Both proteins were abundantly present in seminiferous epithelium and interstitial tissue of adult rat testis. In seminiferous epithelium, AHR and ARNT-IR occurred in a stagespecific manner. Slight differences in the expression of these proteins could be observed. Although the widest distribution of AHR-IR could be seen at stages XIV–II, ARNT-IR was most abundantly present during stages VII–VIII of the cycle (Tables 1 and 2). Thus, ARNT as the nuclear translocator for

Only a low signal for AHR and a moderate one for ARNT mRNA could be observed in human testis. In contrast to these observations, strong AHR- and ARNT-IR was detected in the human testis, at a level that even exceeded the number of immunoreactive cells in the rat testis. Virtually all tubular and interstitial cells of the human testis revealed strong immunoreactivity for AHR and ARNT, including peritubular

Schultz et al. • AHR and ARNT in Testis

cells and fibroblasts. Although the human testis bears six different stages of the spermatogenic cycle, no clear stagespecific expression of AHR or ARNT-IR could be detected. The observed variation between expression in rat and human testes may be explained by differences between species. However, as discussed with the findings in rat testis, the wide and most abundant distribution of AHR and ARNT proteins in human testis explains how dioxins can interfere directly with human spermatogenesis and fertility, resulting in cell death and a reduction of testicular weight and sperm count (11, 37, 38). As, in contrast to the distribution in the rat testis, AHR and ARNT were found in pre- and postmeiotic cells of the seminiferous epithelium, the activation of AHR/ ARNT in the human testis may lead to an even wider extent of biological consequences than in the rat testis. In conclusion, the present study depicts a wide distribution of the aryl-hydrocarbon receptor and its nuclear translocator in both rat and human testes. Whereas the distribution of ARNT-IR in rat testis has been reported in one earlier study (33), we report the localization of AHR and ARNT mRNAs and proteins in rat and human testis here for the first time. The induction of AHR mRNA in cultured seminiferous tubules of Sprague Dawley rats could be down-regulated by adding FSH to the culture medium, whereas a culture medium supplemented with TCDD resulted in a significant increase in apoptotic cells in seminiferous epithelium. The most probable explanation for this fact is that the seminiferous epithelium undergoes controlled cell death, accompanied by an up-regulation of AHR. These considerations are supported by the findings of other investigators (28). Thus, one constitutive role for dioxin receptors during spermatogenesis could be the induction or enhancement of apoptosis. Recent research on the mechanisms of environmental pollutants on fertility have revealed putative molecules induced by AHR/ARNT that may interfere with spermatogenesis (39). Together with the results of the present study this may explain how environmental dioxins can damage spermatogenesis directly through the activation of AHR and the consecutive induction of apoptosis in target cells.

Acknowledgments The skillful technical assistance of Ulla Jukarainen is gratefully acknowledged. We are very thankful to Prof. Raimo Pohjanvirta, M.D., Ph.D. (National Public Health Institute, Division of Environmental Health, Department of Toxicology, Kuopio, Finland) for providing us with the AHR-cDNA. Prof. Lorenz Poellinger (Karolinska Institute, Solna, Sweden) is gratefully acknowledged for supplying us with the AHR and ARNT antibodies. Received June 21, 2002. Accepted November 13, 2002. Address all correspondence and requests for reprints to: Markku Pelto-Huikko, M.D., Ph.D., Department of Developmental Biology, Tampere University Medical School, FIN-33014 Tampere, Finland. Email: [email protected] This work was supported by grants from the Research Fund of Tampere University Hospital, Tampere University, Academy of Finland, Finnish Research Program on Environmental Health, European Union Quality of Life Program Contract QLK4-1999-01422, and Turku University Central Hospital.

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