Effects of 4-tert-octylphenol on the testes and seminal vesicles in adult male bank voles

Reproductive Toxicology 31 (2011) 95–105

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Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox

Effects of 4-tert-octylphenol on the testes and seminal vesicles in adult male bank voles ´ Anna Hejmej ∗ , Małgorzata Kotula-Balak, Jerzy Galas, Barbara Bilinska Department of Endocrinology, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Krakow, Poland

a r t i c l e

i n f o

Article history: Received 2 February 2010 Received in revised form 19 May 2010 Accepted 21 August 2010 Available online 17 September 2010 Keywords: Octylphenol Testis Seminal vesicles Bank vole Androgens Estrogens Endocrine disrupters

a b s t r a c t The present study was designed to evaluate the effects of 4-tert-octylphenol (OP) on male testes and seminal vesicles of bank vole. Adult males kept under long or short photoperiod were orally administered OP (200 mg/kg bw) for 30 or 60 days. Treatment for 30 days had no discernible effect on the parameters examined. Treatment for 60 days adversely influenced weights and histological structure of the testes and seminal vesicles. In these tissues, expression of 3␤-hydroxysteroid dehydrogenase and androgen receptor and testosterone levels were reduced, whereas expression of aromatase and estrogen receptor ␣ and estradiol levels were increased. The alterations were more evident in voles kept in long photoperiod. Taken together, it is suggested that adverse changes in bank vole reproductive tissues induced by longterm OP-exposure result from disturbed androgen and estrogen synthesis and action. Moreover, there might be a subtle difference in the sensitivity to OP between voles kept in different light conditions. © 2010 Elsevier Inc. All rights reserved.

1. Introduction In recent years concern has been raised about the possibility that reproductive disorders reported in humans and wildlife populations might stem from exposure to substances present in the environment which mimic estradiol, the so-called xenoestrogens. Exposure to chemicals with estrogenic activity may have potential to adversely affect the endocrine system and reproductive organs in males and females [1–3]. 4-tert-octylphenol (OP) has been reported to exhibit weak estrogenic activity as demonstrated by its ability to bind and activate the estrogen receptor (ER) [4]. OP is the primary final biodegradation product of alkylphenol ethoxylates, non-ionic surfactants that are widely used as components of plastics, detergents, paints and pesticides [5]. Large amounts of OP have been detected in rivers, sewage treatment plants and drinking water [6,7]. Half-life of OP in the environment is estimated as 5 days [8]. In flounder OP accumulation was observed in liver, muscle and plasma up to 12 h whereas in testis 18 h post administration [9]. Even though alkylphenols are between 100 and 10000 times less estrogenic than 17␤-estradiol, the widespread use of these compounds and their ability to bioaccumulate in fats suggest that they contribute substantially to the environmental estrogen pool [10]. Studies on the exposure of U.S.

∗ Corresponding author. Tel.: +48 12 663 24 66; fax: +48 12 634 07 85. E-mail address: [email protected] (A. Hejmej). 0890-6238/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2010.08.007

population to OP revealed that this compound was present in the urine of 57% of persons >6 years of age with total concentrations ranging from 0.2 ␮g/L to 20.6 ␮g/L [11]. Currently, OP has been also found in human breast milk [12]. Little is known about the degree of exposure of human and other mammals to OP. Estimated daily intake is up to 300 pg/kg bw, but concentrations of OP in the aquatic environment range up to 200 ␮g/L [13,14]. In experimental studies on animal models diverse effects of OP have been obtained depending on the choice of animal species, age, routes of administration and dose levels. It was shown that in adult male rats OP at a high dose (400 mg/kg bw) reduced the size and function of the entire male gametogenic and accessory reproductive organs, decreased serum LH, FSH and testosterone concentrations, and reduced sperm count [10,15]. However, reports on the effects of lower doses (20–200 mg/kg bw) of OP administration on male reproductive system are contradictory [16,17]. Much of the data concerning the adverse effects of OP on male reproductive health were obtained following fetal and neonatal exposure [18–21]. Bank voles are seasonally breeding animals which are a useful model for studies of male reproduction since the recrudescence of spermatogenesis is physiologically regulated [22,23]. Earlier observations revealed that season and length of the light cycle regulate changes in hormonal and spermatogenic activities [24–27]. Bank voles kept under different light conditions in the laboratory show similar reproductive characteristics as that observed in the wild. They show different phases of reproduction, active or regressive,

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that mimic spring or autumn photoperiods. The testes of bank voles submitted to long photoperiod are larger in size, express higher aromatase activity, produce higher levels of testosterone and estradiol and show higher concentration of the total antioxidant status than those kept in short photoperiod [28]. A previous study in this laboratory demonstrated that immature male bank voles are sensitive to estrogens; administration of low dose of 17␤-estradiol (0.1 ␮g/g bw) induced the acceleration of the onset of spermatogenesis. On the other hand, when voles were treated with a high dose of estradiol (10 ␮g/g bw), disruption of testicular structure and tubular atrophy were observed [29]. However, the impact of xenoestrogens on seasonally breeding mammals and relative sensitivities of animals of different reproductive status to toxicant exposure have not been studied. Thus, this study was designed to evaluate the in vivo effects of OP on the reproductive system of male bank voles in relation to the functional status of the gonads. In order to investigate structural integrity of reproductive tissues, histological examination of testes and seminal vesicles was performed. As potential markers of endocrine alteration, expression of key enzymes involved in the synthesis of steroids, hydroxysteroid 3␤-dehydrogenase (3␤-HSD) and aromatase, was assessed together with testosterone and estradiol levels in tissue homogenates. Additionally, to characterize the possible changes in tissue responsiveness to androgens and estrogens, expression of androgen receptor (AR) and estrogen receptor ␣ (ER␣) was investigated in the testes and seminal vesicles. The seminal vesicles were chosen because our preliminary findings and previously reported data [30] have suggested that estrogeninduced changes were clearly noticeable in this tissue. Since the bank voles are sensitive to changes in daylight, the animals were exposed chronically to oral gavage OP under both long and short photoperiods. 2. Materials and methods 2.1. Chemicals 4-tert-octylphenol (Sigma–Aldrich, St. Louis, MO, USA) was dissolved in a minimum amount of 100% ethanol and then a measured amount of sesame oil (Sigma–Aldrich, St. Louis, MO, USA) was added in 1:14 (v/v). The concentration of the stock solution was 0.2 mg/␮l. Ethanol without OP was added to the sesame oil to prepare a solution for the control group. The solutions were placed in a hood and stirred with magnetic stirring bars overnight in order to evaporate the ethanol. Fresh solutions were made each week. In order to maintain the stability of the stock solution it was freshly made each week [10]. 2.2. Animals and experimental design Bank voles (Clethrionomys glareolus, Schreber) were obtained from our own colony (Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland) which have been reared under short light cycles (6 h light and 18 h darkness; 6L:18D) or long (18 h light and 6 h darkness;18L:6D) light cycles for 10 generations. The animal rooms were maintained at a temperature of 18 ◦ C and a relative humidity of 55 ± 5%. Voles were housed in polyethylene cages (42 cm × 27 cm × 18 cm) furnished with sawdust and wood shavings for bedding. A standard pelleted diet (LSM diet, Agropol, Motycz, Poland) supplemented with seeds of wheat or oat, red beet, apples, and water was provided ad libitum. Twenty-four mature male bank voles (60–70-day-old) were dosed orally with OP (200 mg/kg bw) every Monday, Wednesday and Friday for 30 (OP30; n = 12) or 60 days (OP60; n = 12). A dose of exposure to OP was based on the literature [16,17] and it was finally selected upon our preliminary study in which lower doses (50 and 100 mg/kg bw) had no effects on bank vole testis and seminal vesicles weight and morphology. After the animals were sacrificed, testes, seminal vesicles and vasa deferentia were immediately excised. For all animals, body, testis and seminal vesicles weights were recorded at necropsy. The organ weights were reported as relative weights (organ weight/body weight). 2.3. Ethics of experimentation Experiments were performed in accordance with Polish legal requirements, under the license given by the Local Ethics Committee at the Jagiellonian University, Krakow, Poland.

2.4. Tissue preparation For morphological, immunohistochemical and TUNEL analysis, one testis and half the tissue of the seminal vesicles from each animal (n = 6/each group) were immersed immediately either in Bouin fixative or in 4% formaldehyde, respectively, and embedded in paraplast (Sigma–Aldrich, St. Louis, MO, USA). Sections of 5 ␮m in thickness were mounted on slides coated with 3-aminopropyltriethoxysilane (Sigma–Aldrich, St. Louis, MO, USA), deparaffinized, and rehydrated through decreasing alcoholic solutions. For morphology routine haematoxylin–eosin staining (H–E) was performed. For Western blot and radioimmunological analysis, contralateral testis and the remaining part of the seminal vesicles from each animal were frozen and stored in −80 ◦ C. 2.5. Isolation of spermatozoa and sperm morphology Sperm cell isolation has been described in detail [31]. In short, small drops of suspensions of sperm cells were transferred to microscope slides and smears were carefully made. After that smears were stained with eosin solution (1% eosin Y in water), allowed to dry and mounted with glycerol gel. The sperm were evaluated microscopically for head and tail abnormalities. At least 300 sperm were evaluated on each slide, with two slides assessed per vas deferens. Three animals per group were included in this analysis. Sperm from each vas deferens/animal was evaluated. 2.6. TUNEL assay The presence of apoptosis-related DNA strand breaks in testicular cells was evaluated by TUNEL assay using the In situ Cell Death Detection kit, POD (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, after deparaffinization and rehydration, the 5 ␮m paraffin tissue sections were immersed for 10 min in 3% H2 O2 in methanol at room temperature to quench endogenous peroxidase activity, rinsed in PBS and incubated with 10 ␮g/ml Proteinase K solution (Promega Corporation, Madison, WI, USA) for 15 min at 37 ◦ C. Thereafter, sections completely rinsed in PBS were incubated with TUNEL reaction mixture (terminal deoxynucleotidyl transferase and labeled nucleotide mixture) for 60 min at 37 ◦ C in a humidified atmosphere in the dark. Next, sections were rinsed in PBS and incubated with Converter-POD (anti-fluorescein antibody conjugated with horseradish peroxidase) in a humidified chamber for 20 min at 37 ◦ C. Peroxidase activity was then visualized by precipitation of 3,3 -diaminobenzidine (DAB Substrate, Roche, Mannheim, Germany). Cells containing fragmented nuclear chromatin characteristic of apoptosis would exhibit a brown nuclear stain. Apoptotic cells were observed under bright field optics in Leica DMR microscope (Leica Microsystems, Wetzlar, Germany). Sections from each animal were viewed under bright field using a 40× objective, and scored by an observer blinded to the treatment groups. For each testicular crosssection, all cross-sectional tubular profiles (∼150) were counted and the number of TUNEL-positive cells noted for each cross-sectioned tubule. 2.7. Immunohistochemistry Immunohistochemical staining results were evaluated qualitatively and quantitatively. Various testicular cells were considered immunopositive if brown reaction product was present; the cells without any specific immunostaining were considered immunonegative [32]. To optimize immunohistochemical staining, slices were immersed for 2–5 min in 10 mM citrate buffer (pH 6.0) and heated in a microwave oven (600 W). The whole procedure has been described in detail elsewhere [33]. In short, nonspecific staining was blocked twice, first with 3% H2 O2 in methanol for 15 min, to inhibit endogenous peroxidase activity, and second with 5% normal goat or horse serum for 30 min at room temperature to block nonspecific binding sites. Thereafter, sections were incubated overnight at 4 ◦ C in a humidified chamber in the presence of primary antibodies: (1) a polyclonal antibody against 3␤-HSD (1:2000; a gift from Prof. Anita H. Payne, Stanford University Medical Center, San Francisco, CA, USA); (2) a polyclonal antibody against human placental P450 aromatase (1:300; a gift from Dr. Y. Osawa, Hauptman-Woodward Medical Research Institute, Buffalo, NY, USA); (3) a rabbit polyclonal antibody against human AR (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA); (4) a mouse monoclonal antibody against human ER␣ (1:100; Dako, Glostrup, Denmark). Next, biotinylated secondary antibody, goat anti-rabbit IgG or horse anti-mouse IgG, respectively (dilution, 1:400; Vector Labs, Burlingame, CA, USA) was applied for 1 h. Finally, avidin–biotinylated horseradish peroxidase complex (Vectastain ABC Kit; dilution, 1:100; Vector Labs, Burlingame, CA, USA) for a further 30 min was used. After each step in these procedures, sections were carefully rinsed with Tris-buffered saline (TBS; 0.05 M Tris–HCl plus 0.15 M NaCl, pH 7.6). Bound antibody was visualized with TBS containing 0.05% 3,3 -diaminobenzidine and 0.07% imidazole for 3–4 min. The slides were processed immunohistochemically at the same time with the same treatment so that the staining intensities among testicular cells could be compared. Experiments were repeated three times on serial sections per animal. Control sections included omission of the primary antibody and substitution by an irrelevant IgG. The sections were examined with a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany). To evaluate the intensity of immunohistochemical reaction quantitatively, digital color images were obtained using a CCD Video Camera (KYF55, JVC) mounted on an optical

A. Hejmej et al. / Reproductive Toxicology 31 (2011) 95–105 microscope (Microphot, Nikon, Japan) and connected to a video capture card (PVBT878P1, Prolink, Nei-Hu, Taipei, Taiwan) installed in a personal computer. Image processing and analyses were performed on 18 serial sections (three sections of six animals) from each control and experimental group using a public domain ImageJ software (National Institutes of Health, Bethesda, MD) as described in detail [34]. The intensity of histochemical reaction was expressed as relative optical density (ROD) [35]. 2.8. Western blot analysis Tissues were homogenized on ice with a cold Radio-Immunoprecipitation Assay Buffer (RIPA; 0.05 mmol/L Tris–HCl, 0.15 mol/L NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS, PH 8.0), sonicated, and centrifuged at 15,000 × g for 20 min at 41 ◦ C. The protein concentration for each sample was estimated using Bradford dye-binding procedure with BSA as a standard [36]. Homogenates containing 100 ␮g of protein were solubilized in a sample buffer (Bio-Rad Labs, GmbH, München, Germany) and heated at 99.91 ◦ C for 3 min. After denaturation the samples were subjected to electrophoresis on 8% or 12% SDS-PAGE gels. Separated proteins were transferred onto a nitrocellulose membrane using a wet blotter in the Genie Transfer Buffer (pH 8.4) for 90 min at a constant voltage of 135 V. Then, blots were blocked with 5% nonfat dry milk in TBS, 0.1% Tween 20, overnight at 4 ◦ C with shaking, followed by an incubation with appropriate primary antibody (as for immunohistochemistry; anti-3␤HSD, dilution 1:8000; anti-aromatase, dilution 1:3000; anti-ER␣, dilution 1:1000; anti-AR, dilution 1:500; anti-␤-actin (Sigma–Aldrich, St. Louis, MO, USA), dilution 1:3000), at room temperature for 1.5 h. The membranes were washed and incubated with a secondary antibody conjugated with the horseradishperoxidase labeled goat anti-rabbit IgG or horse anti-mouse IgG (Vector Labs., Burlingame, CA, USA) at a dilution 1:1000, for 1 h at room temperature. Then, bound antibody was revealed using 3,3 -diaminobenzidine as the substrate. Finally, the membranes were dried and then scanned using Epson Perfection Photo Scanner (Epson Corporation, CA, USA). Molecular masses were estimated by reference to standard proteins (Prestained SDS-PAGE Standards, Bio-Rad Labs, GmbH, München, Germany). Quantitative analysis was performed for three separately repeated experiments of six animals from each control and experimental group using a public domain ImageJ software (National Institutes of Health, Bethesda, MD) as described in detail [34]. The relative protein expressions were expressed as arbitrary units. 2.9. Radioimmunological analysis Homogenized testicular tissues from control and experimental groups were used for radioimmunological determination of testosterone [37] and estradiol [38] levels. For testosterone and estradiol assays 0.2 ml of each homogenate was extracted with 2.5 ml ethyl ether. Testosterone levels were assessed using [1,2,6,7-3H]-testosterone (The Radiochemical Centre, Nycomed Amersham, Buckinghamshire, England), specific activity 88.0 Ci/mmol, as a tracer and antibody raised in rabbit against testosterone-3-o-carboxymethylo-oxime-bovine serum albumin (BSA), whereas estradiol concentrations were determined using [2,4,6,73H]-estradiol (The Radiochemical Centre, Nycomed Amersham, Buckinghamshire, England), specific activity 140 Ci/mmol, as a tracer and an antibody raised in rabbit against estradiol-17␤-6-oxime-BSA. Both assays were validated by demonstrating parallelism between serial dilutions of testicular homogenates and standard curve. Coefficients of variation within and between testosterone assays were 7.6% and 9.8% respectively, while within and between estradiol assays they were 3.3% and 7.4% respectively. The recovery of unlabeled testosterone and estradiol was also assessed (never less than 90% and 88%, respectively). 2.10. Statistical analysis All statistical analyses were performed using one-way analysis of variance (ANOVA). Dunnet multiple comparison test was used to determine which values differed significantly from controls (p < 0.05). Data were presented as mean ± SD.

3. Results 3.1. Relative organ weight The relative testis weight (mg/g bw) was reduced only in OP60 voles kept in long light cycles when compared to the respective control, whereas the relative weights of seminal vesicles were reduced in both OP30 and OP60 groups kept in both photoperiods, however this was more pronounced in OP60 voles (for details see Table 1). 3.2. Testis and sperm morphology Control animals showed a compact and regular arrangement of cells in the seminiferous epithelium (Fig. 1A). Administration

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of OP for 30 days had no detectable effect on testicular morphology (not shown), whereas OP60 males showed a disruption of normal epithelial organization. In testes of the latter animals either degenerating tubules or the tubules with disorganized seminiferous epithelium were visible. Occasionally, conglomerates of spermatocytes and spermatids were found in the lumen of seminiferous tubules (Fig. 1B). In sperm smears no differences in sperm head abnormalities were detected between treated and control groups, whereas the frequency of sperm tail abnormalities increased in OP60 males. The major tail abnormalities assessed were coiled tails (Fig. 1C). 3.3. TUNEL assay TUNEL analysis was used to quantify apoptosis of distinct cell types. Although TUNEL-labeled germ cells were detected in control testes (Fig. 1A, insert), the number of apoptotic cells markedly increased (∼2-fold) in the OP60 groups versus the respective controls (Fig. 1B, insert, Fig. 2). Apoptotic cells were mostly accounted for as degenerating spermatocytes. In contrast, such cells were infrequent in the OP30 groups (Fig. 2). Immunoreactive cells were absent in negative control sections (Fig. 1A, small image in top of insert A). 3.4. Immunolocalization of 3ˇ-HSD, aromatase and steroid hormone receptors’ in the testes In the testes of control and OP-treated animals immunostaining for 3␤-HSD was restricted to the cytoplasm of Leydig cells (Fig. 1D). The intensity of immunostaining was dependent on the length of light being stronger in animals kept under long-day conditions than in those kept in short ones (not shown). Independently of light cycles, in testes of OP30 voles no obvious changes in 3␤-HSD immunoexpression have been found, whereas OP60 treatment resulted in a significant decrease in the staining intensity (Figs. 1E and 3A). In control and OP30 males moderate to strong immunostaining for aromatase was detected in Leydig, Sertoli and germ cells (Fig. 1F). The staining was always stronger in animals reared under long light cycles (not shown). Irrespectively of the photoperiod, OP60 treatment resulted in significantly increased aromatase immunoexpression in seminiferous tubules, but not in the Leydig cells (Figs. 1G and 3B, C). In control animals immunostaining for AR was localized to the nuclei of Leydig, Sertoli and peritubular cells. The intensity of staining in Sertoli cells (from weak to moderate or strong), was different in individual tubules, indicating stage-dependent expression of ARs (Fig. 1H). Immunoexpression was stronger in tissue sections obtained from animals kept in long photoperiod than that of a short-day conditions (not shown). Administration of OP for 30 days did not affect AR expression, whereas OP60 exposure decreased the intensity of staining only in Sertoli cells of voles reared under long light cycles (Figs. 1I and 3E). Whatever the group of voles, testicular ER␣ immunoreactivity was confined to Leydig cell nuclei (Fig. 1J). In controls, intensity of the staining was dependent on the length of light being stronger in animals kept under long-day conditions (not shown). OP30 exposure did not alter the expression of ER␣ (Fig. 3F), whereas OP60 treatment resulted in the increased staining intensity (Figs. 1K and 3F). The effect was more pronounced in males reared under long light cycles (Fig. 3F). In all the negative control tissue sections, prepared individually for each slide, no immunopositive signal was found when the incubation was performed without the respective primary antibody (Fig. 1E, G, I and K, inserts).

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Fig. 1. The effect of 60-day treatment with 4-tert-octylphenol on testicular (A, B) and sperm morphology (C), TUNEL staining (inserts in A, B) and immunolocalization of 3␤-HSD (D, E), aromatase (F, G), AR (H, I) and ER␣ (J, K) in the testes of bank voles kept under long light cycles (18L:6D). Bright field illumination (A–C), Nomarski interference contrast (D–K). Scale bars represent 25 ␮m. Testis morphology. H–E staining. (A) In seminiferous tubules of control males all germ cell types are seen. (B) Degenerating tubules or the tubules with disorganized seminiferous epithelium are visible in testis sections of OP-exposed voles. Note conglomerates of immature germ cells in the lumen (arrows). (C) Sperm with coiled tail are present in the sample obtained from an OP-treated animal; eosin Y staining (arrow). TUNEL staining. (Insert in A) In control testis apoptotic cell (arrow) is visible. (Insert in B) Increased number of TUNEL-positive germ cells, mainly spermatocytes (arrows) are seen in testis sections of OP-exposed males. No positive staining is visible in the negative control sections (small image in the top of insert A). Immunolocalization of 3␤-HSD. (D, E) The staining is localized to the cytoplasm of Leydig cells (arrows). (D) In control testis strong staining is visible (arrows). (E) Note decreased intensity of the staining in Leydig cells of OP-treated voles (arrows). Immunolocalization of aromatase. (F) In control testis moderate staining is visible in Sertoli cells (open arrows), germ cells (arrowheads) and Leydig cells (arrows). (G) Note a clearly stronger staining in Sertoli cells (open arrows) and germ cells (arrowheads) of OP-treated voles. Immunolocalization of AR. (H) In control testes, moderate to strong staining is noted in the nuclei of Sertoli cells (open arrows), Leydig cells (arrows), and peritubular-myoid cells (arrowheads). (I) Note, decreased intensity of the staining only in Sertoli cells (open arrows) of OP-treated voles. Immunolocalization of ER␣. (J, K) ER␣ staining is confined to the nuclei of Leydig cells (arrows). (J) In control testis moderate staining is visible (arrows). (K) Note, increased intensity of the staining following OP treatment (arrows), (at higher magnification, see insert in K). No positive staining is visible in the negative control sections (insert in E, G, I and K).

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columnar epithelium. Stromal compartment consisted of the layers of smooth-muscle cells (Fig. 4A and C). In OP60 voles, an increase in the stromal volume and reduction or lack of secretions in the lumen was visible (Fig. 4B and D). The mucosal folds surrounding the luminal areas were lined with cuboidal rather than columnar epithelium. In voles kept under short light cycles more marked changes were visible (Fig. 4D). 3.6. Immunolocalization of aromatase and steroid hormone receptors’ in the seminal vesicles

Fig. 2. The effect of 4-tert-octylphenol treatment on the number of apoptotic germ cells in the testis. Values represent the mean ± SD number of TUNEL-positive germ cells per 100 tubule sections. Data are from one testis from each of the six animals in each treatment group. Asterisks indicate significant differences between control animals (control) and males exposed to OP for 30 and 60 days (OP30; OP60). Statistical significance: *p < 0.05; **p < 0.01.

Quantitative evaluation of the intensity of immunohistochemical staining, expressed as relative optical density (ROD), confirmed qualitative data (Fig. 3). 3.5. Seminal vesicles morphology In the seminal vesicles of control and OP30 animals, acini contained secretory material and were lined by an extensively folded

In the seminal vesicles of control voles kept under short light cycles, moderate immunostaining for aromatase in the cytoplasm of epithelial cells was present (Fig. 4G). In the sections derived from voles kept under long photoperiod the immunostaining was always stronger (Fig. 4E). Whatever the light regime, OP30 treatment had no discernible effect on aromatase immunoexpression, whereas in OP60 males significantly stronger immunostaining was detected when compared to that of the control groups (Figs. 4F, H and 5A). In control males, ARs were localized in the nuclei of most epithelial cells and numerous stromal cells. Keeping voles under long light cycles resulted in significantly stronger intensity of immunostaining in both cell types (Fig. 4I and K). In OP60 males kept under long-day conditions, the intensity of staining was reduced compared to control animals (Figs. 4J and 5B). In control males, weak nuclear staining for ER␣ was observed in the nuclei of most epithelial cells and many stromal cells. No significant difference in staining intensity was found in the seminal vesicles derived from animals kept in both light regimes (Fig. 4M and O). In OP30 males no changes in ER␣ immunostaining were evident, whereas after OP60 exposure increased intensity of staining was observed in short-day animals (Figs. 4P and 5C).

Fig. 3. Quantitative analysis of the intensity of immunohistochemical staining for (A) 3␤-HSD, (B, C) aromatase, (D, E) AR and (F) ER␣ in testicular cross-sections of bank voles kept in either long light cycles (18L:6D) or short light cycles (6L:18D) expressed as relative optical density (ROD) of diaminobenzidine brown reaction products. Values are means ± SD. Asterisks indicate significant differences between control animals (control; n = 10) and males exposed to OP for 30 (OP30, n = 12) and 60 days (OP60; n = 12). Statistical significance: *p < 0.05; **p < 0.01.

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Fig. 4. The effect of 60-day treatment with 4-tert-octylphenol on seminal vesicle morphology (A, B, C, D) and immunolocalization of aromatase (E, F, G, H), AR (I, J, K, L), and ER␣ (M, N, O, P). Left panels (A, B, E, F, I, J, M, N) show seminal vesicles of bank voles kept under long light cycles (18L:6D), whereas right panels (C, D, G, H, K, L, O, P) show seminal vesicles of those kept under short light cycles (6L:18D). Bright field illumination (A–D), Nomarski interference contrast (E–P). Scale bars represent 25 ␮m. Seminal vesicle morphology. H–E staining. (A, C) Extensively folded glandular epithelium and stromal compartment consisted of smooth-muscle cells are visible. (B, D) Note increase in the stromal volume (arrows) and reduction or lack of secretions accompanied by alterations in epithelial organization (open arrows) in voles following OP-exposure. More marked changes are visible in 6L:18D voles (D). Immunolocalization of aromatase. (E, G) In controls weak to moderate staining in cytoplasm of epithelial cells is seen (open arrows). (F, H) Strong staining for aromatase is visible in the seminal vesicles of both 18L:6D and 6L:18D OP-treated males (open arrows). Immunolocalization of AR. (I, J, K, L) AR staining in the nuclei of epithelial cells (open arrows) and stromal cells (arrows) is visible. (J) Clearly reduced staining for AR in 18L:6D males following OP exposure. (L) The weak intensity of the staining in OP-treated 6L:18D males is similar to that of the respective control (K). Immunolocalization of ER␣. (M, N, O, P) Irrespectively of the photoperiod, ER␣ staining restricted to the nuclei of epithelial cells (open arrows) and stromal cells (arrows) is visible. Note either increased intensity of the staining in OP60 6L:18D voles versus control (P, O), or no changes are evident in 18L:6D males following exposure to OP and the control ones (N, M). No positive staining is visible in the negative control sections (insert in H, L and P).

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Table 1 Relative testes and seminal vesicles weights of bank voles exposed to OP. Relative organ weight (mg/g) Testis

Control OP30 OP60

Seminal vesicles

18L:6D

6L:18D

18L:6D

6L:18D

15.83 ± 1.28 13.36 ± 3.14 11.80 ± 2.47*

12.73 ± 0.90 13.41 ± 0.44 12.36 ± 1.47

17.56 ± 1.32 10.53 ± 1.27* 6.28 ± 2.15**

13.98 ± 1.27 9.23 ± 1.95* 2.99 ± 0.41**

Animals kept in long (18L:6D) or short photoperiod (6L:18D) were treated either with vehicle (control, n = 10) or 4-tert-octylphenol (OP; 200 mg/kg bw) for 30 (OP30, n = 12) or 60 days (OP60, n = 12). Each value represents the mean ± SD. Significant differences from the control values are denoted as: * p < 0.05. ** p < 0.01.

In all the negative control tissue sections no immunopositive signal was found when the incubation was performed without the respective primary antibody (Fig. 4H, L and P). Quantitative evaluation of the intensity of immunohistochemical staining, expressed as relative optical density (ROD), confirmed qualitative data (Fig. 5). 3.7. Western blot analyses The results from Western blot analysis of testicular and seminal vesicles homogenates confirmed the major observations from immunohistochemistry. A distinct decrease in the levels of 3␤-HSD and AR and an increase in the levels of aromatase and ER␣ were found in OP60 animals when compared to the respective controls (Figs. 6 and 7). Moreover, due to differences in photoperiod the protein variations between control groups were observed in the testes as reported previously [26,27]. 3.8. Steroid hormone levels Radioimmunological analysis of testosterone and estradiol levels revealed differences in both steroid hormone synthesis in the testes and seminal vesicles. Hormone level alterations were observed only in OP60 males (Fig. 8). Irrespectively of the light regime, testosterone levels were lower in OP60 males when compared to the respective controls (Fig. 8A and C). In detail, in the testes of OP60 males (18L:6D versus 6L:18D) testosterone concentrations were 1.27 ± 0.13 ng versus 0.65 ± 0.02 ng/100 mg tissue, respectively, whereas in control groups it was 3.06 ± 0.36 ng versus 1.52 ± 0.19 ng/100 mg tissue. In the seminal vesicles of OP60 voles testosterone levels were 0.10 ± 0.01 ng versus 0.04 ± 0.02 ng/100 mg tissue, respectively, whereas in control groups it was 0.22 ± 0.03 ng versus 0.15 ± 0.03 ng/100 mg tissue.

A distinct increase in estradiol levels was observed in testicular homogenates of OP60 voles that were kept in either long or short light cycles (60.12 ± 8.29 pg versus 14.49 ± 3.18 pg/100 mg tissue) when compared to the respective controls (37.77 ± 7.02 pg versus 4.22 ± 0.74 pg/100 mg tissue) (Fig. 8B). Estradiol levels in the seminal vesicles showed a similar trend to that found in the testes (Fig. 8D). In OP60 males it was 31.08 ± 1.53 pg versus 7.03 ± 1.50 pg/100 mg tissue, whereas in control group 12.53 ± 1.56 pg versus 3.70 ± 0.74 pg/100 mg tissue.

4. Discussion Our results clearly demonstrate that exposure to OP alters the structure of bank vole testes and seminal vesicles and affects endocrine functions of these tissues. Significant changes in the parameters studied were, however, observed mainly in the OP60 group. Thus, it is suggested that in adult bank voles long-term exposure is necessary to affect male reproductive functions. The relative weights of the testes and seminal vesicles were significantly decreased following OP administration. However, reduction in testis weight was observed only in voles kept in long photoperiod. It may suggest that sexually active animals are more sensitive to OP than reproductively regressed ones. A decrease in testis and seminal vesicle weight following chronic exposure to OP (for 1–2 months at doses 400–450 mg/kg bw) was also observed in adult rats [15,16,39]. On the other hand, in adult mice treated with OP at a dose of 200 mg/kg bw for 5 days, no significant changes in testis weights were found when compared to the control [17]. Treatment of adult rats with OP at doses 125–250 mg/kg bw for longer period (21–60 days) resulted in reduced testis and body weight, abnormal distribution of heterochromatin and vacuolization of mitochondria in spermatocytes, decreased sperm motility and decreased mRNA expression of 3␤-HSD in testes [16,17,40].

Fig. 5. Quantitative analysis of the intensity of immunohistochemical staining for (A) aromatase, (B) AR, and (C) ER␣ in seminal vesicles cross-sections of bank voles kept in either long light cycles (18L:6D) or short light cycles (6L:18D) expressed as relative optical density (ROD) of diaminobenzidine brown reaction products. Values are means ± SD. Asterisks indicate significant differences between control animals (control; n = 10) and males exposed to OP for 30 (OP30, n = 12) and 60 days (OP60; n = 12). Statistical significance: *p < 0.05.

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Fig. 6. (A) Representative Western blot analysis of 3␤-HSD, aromatase, AR and ER␣ protein expression levels in testicular homogenates of bank voles kept in either long light cycles (18L:6D) or short light cycles (6L:18D) treated with vehicle (n = 6) or with 4-tert-octylphenol for 30 and 60 days (OP30, n = 6; OP60, n = 6). Anti-␤-actin labeling served as an internal protein loading control. (B) The relative amount of 3␤-HSD, aromatase, AR and ER␣ protein normalized to ␤-actin. Data obtained from three separate analyses is expressed as mean ± SD. Significant differences from control values are denoted as *p < 0.05 and **p < 0.01.

It is conceivable that this discrepancy exists because of the short time of exposure in the former study. Reduction in testes and seminal vesicles weights was accompanied by an alteration of their normal histological structure. In the testes of OP60 voles, impaired spermatid maturation and presence of immature germ cells in the lumen were found in some tubules. Although decreased testis weight was restricted to long-day animals, a disruption of the seminiferous epithelium was observed in males derived from both light regimes. Such alterations indicate that OP affects germ cell differentiation. Other studies with 400 mg/kg bw of OP showed even more marked changes in the histological structure of rat testis: reduction of seminiferous tubules diameters, irregular and disordered arrangement of cells, complete lack of elongated spermatids and spermatozoa or spermatid retention, and the presence of multinucleated germ cells [19,41]. In the present study, besides degenerating or impaired tubules, numerous tubules showed normal epithelial organization and complete spermatogenesis possibly because of the relatively low doses of OP used. Germ cell apoptosis is a part of the mechanism involved in tubular degeneration and testis atrophy [42]. Our findings that the number of apoptotic germ cell was increased in animals treated with OP for 60 days resemble those reported previously for rats exposed to OP and nonylphenol [41,43,44]. Thus, it is suggested that OP reduces the size and/or function of the testis due to an increased apoptosis of germ cells. However, these data, and especially the

Fig. 7. (A) Representative Western blot analysis of aromatase, AR and ER␣ protein expression levels in seminal vesicle homogenates of bank voles kept in either long light cycles (18L:6D) or short light cycles (6L:18D) treated with vehicle (n = 6) or with 4-tert-octylphenol for 30 and 60 days (OP30, n = 6; OP60, n = 6). Anti-␤-actin labeling served as an internal protein loading control. (B) The relative amount of aromatase, AR and ER␣ protein normalized to ␤-actin. Data obtained from three separate analyses is expressed as mean ± SD. Significant differences from control values are denoted as *p < 0.05 and **p < 0.01.

conclusions, are somewhat at odds with other published results on the effects of high doses of bisphenol-A (BPA) and OP or low doses of potent estrogens, in which a decrease in germ cell apoptosis was reported [39,45]. Our previous studies on bank voles also revealed only few apoptotic germ cells after treatment with the low dose of estradiol (0.1 ␮g/g bw) [29]. These discrepancies could be due to the experimental design differences, including different age of animals and different timing of xenoestrogen exposure. Moreover, it can be hypothesized that some effects of OP may differ from that of the native estrogen or from other weak estrogenic chemicals and may result from non-estrogenic action of OP. Indeed, besides estrogenic nature of OP, its proapoptotic effect in vitro has been documented previously [46–48]. The effects of OP on sperm morphology have been demonstrated previously in rats following subcutaneous injection of 20 or 80 mg of OP for 1 month and following the addition of OP in the drinking water for 4 months [15,49]. The authors suggested that OP may directly influence flagellar structures of sperm. In the present study, many of the spermatozoa that were formed were also adversely influenced by OP treatment, as revealed by an increase in tail abnormalities. Induction of morphologic changes to the seminal vesicles depended on the period of OP exposure, as it was demonstrated for the testes. Altered stromal and epithelial organization and the reduction or lack of secretions in the lumen observed in OP60 voles indicate that OP administration may lead to the atrophy of the seminal vesicles and to the disruption of its exocrine function. Similar but more pronounced changes in seminal vesicles morphology were also evident in rodents treated neonatally with ethinyl estradiol and diethylstilbestrol (DES), but not with BPA or OP [30,50].

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Fig. 8. The effect of 4-tert-octylphenol treatment on testosterone (A, C) and estradiol (B, D) levels in the testes (A, B) and seminal vesicles (C, D) of 18L:6D and 6L:18D bank voles. Values are expressed as mean ± SD. Asterisks indicate significant differences between control animals (control; n = 10) and males exposed to OP for 30 (OP30, n = 12) and 60 days (OP60; n = 12). Statistical significance: *p < 0.05; **p < 0.01.

In order to explain the exact mechanisms underlying the induction of histological abnormalities in the testes and seminal vesicles, steroid hormone production and expression of its receptors were investigated herein. Since androgens play a key role in maintaining the normal structure and function of the testes and accessory glands, we assume that the disruption of spermatogenesis could have resulted, at least in part, from the reduction of testosterone production following OP exposure. In fact, we demonstrated that testosterone concentration in the testes and seminal vesicles was markedly reduced in OP60 males kept in both light regimes. This is in agreement with earlier studies by Blake and Boockfor [10] and Han et al. [43] who demonstrated that adult exposure to OP or nonylphenol decreased testosterone production in male rats. On the contrary, other studies revealed no alterations in serum testosterone concentration in rats and mice exposed to high doses of OP [17,39]. It should be stressed, however, that in the present study, testosterone levels were measured in tissue homogenates, not in serum. Thus, our results reflect local testosterone concentrations that differ from those in the blood. In a further step of our study we demonstrated that OP60 exposure significantly decreased 3␤-HSD protein expression in the testes of voles kept in both photoperiods. This is in accord with other in vivo and in vitro studies showing that OP inhibits 3␤-HSD mRNA expression and the activity of 3␤-HSD protein in adult rat Leydig cells [51,52]. In contrast, 3␤-HSD enzyme remained unchanged at protein level in rats neonatally exposed to OP [17]. These findings, together with our present observations, may suggest that the enzyme is a potential target of OP action in adult males but perhaps not at earlier stages of testicular development. Treatment with OP for 60 days markedly reduced AR immunoexpression in Sertoli cells of the testes and in both epithelial and stromal cells of the seminal vesicles in males kept under long

photoperiod. The persistence of AR expression in peritubular and Leydig cells and its down-regulation in Sertoli cells appear to be due to cell-specific differences in the hormonal regulation of the AR. An alternative explanation for our findings is that Sertoli and germ cells are more susceptible to the toxic effects of OP than Leydig cells [10,46]. Inhibition of androgen action via the suppression of AR expression may be interpreted as in vivo evidence that OP is acting functionally as an anti-androgen. This is consistent with previous in vitro findings indicating that OP exerts antagonistic effect on AR [53–55]. Alterations in AR expression following OP exposure were detected only in animals reared under long-day conditions that correspond to sexually active rodents from the spring generation. Thus, it seems likely that reproductively regressed animals are less sensitive to the antiandrogenic effects of OP than reproductively active males. It is now widely accepted that not only androgens, but also estrogens play a vital role in the control of reproductive function in the adult male (reviewed in Carreau et al. [56,57] and Akingbemi [58]). Estrogens are the end products obtained from the irreversible transformation of androgens by aromatase. During the last two decades, the presence of cytochrome P450 aromatase expression has been shown in the testis of various mammalian species, including bank voles. As reported previously, in bank vole testes aromatase was expressed not only in Leydig and Sertoli cells but also in germ cells [26,27]. In the present study, aromatase was detected for the first time in the epithelium of seminal vesicles suggesting a putative role of locally produced estrogens in the control of seminal vesicle function. No report in the literature describes OP effects on estrogen biosynthesis in male reproductive tissues, however other estrogenic chemicals have been reported as adversely acting; genistein reduced aromatase activity and mRNA expression for aromatase

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in rat testicular homogenates [59], BPA administration decreased serum 17␤-estradiol levels and inhibited Leydig cell aromatase activity in rats [60], whereas in chickens it increased testicular aromatase mRNA expression [61]. Additionally, our earlier studies showed that treatment with low doses of estradiol enhanced aromatase expression in bank vole testis [29]. Herein, we demonstrated that OP60 exposure also increased aromatase expression and estradiol levels in the testes and seminal vesicles. It may be a critical finding, because hyperaromatization can strongly influence bank vole reproductive system. As reported by Li et al. [62], an over-expression of aromatase leads to an increase of estrogen production resulting in both histological and functional abnormalities within male reproductive tissues. The results of lastly published study on AROM(+) mice crossed with ER␣KO and ER␤KO mice indicate that the structural and functional disorders caused by overexposure to estrogens were mediated via ER␣, whereas ER␤ was not involved [63]. Thus, it was interesting to examine the expression of ER␣ in the testes and seminal vesicles following OP administration. Earlier papers that have addressed the effects of other estrogenic chemicals on ER␣ expression in these tissues have yielded conflicting results; some authors reported that ER␣ was down-regulated [64–66], whereas others demonstrated increased ER␣ expression [61,67–69]. Herein, we observed that OP60 treatment enhanced the expression of ER␣ in both the testes and seminal vesicles. Interestingly, Williams et al. [30] reported no effect of short-term OP administration on ER␣ expression in the seminal vesicles, whereas treatment with potent estrogens, DES or estradiol, resulted in increased ER␣ expression in this tissue. Therefore, it is suggested that upregulation of ER␣ following chronic OP exposure resulted from increased endogenous estradiol levels in the testes and seminal vesicles rather than from direct OP action. 5. Conclusion Taken together, our findings suggest that long-term OP exposure of adult bank voles results in male reproductive tissue abnormalities. Chronic OP administration decreased testosterone synthesis in adult males concomitantly with the increase in endogenous estrogen production, and expression of ER␣ was increased and the expression of AR was down-regulated. Thus, it seems likely that male reproductive tissue abnormalities result from altered androgen–estrogen balance but the exact mechanism needs to be investigated. It is worth noting that many chemicals lacking or showing low hormonal activity can modify the synthesis, metabolism, or elimination of hormones and thereby disrupt the endocrine system [70]. We cannot rule out, however, the possibility that OP may have also exerted direct effects, e.g. on germ cell apoptosis or sperm morphology in bank voles. Finally, it is also concluded that there might be a subtle difference in the sensitivity to OP between the voles kept in different light conditions with long-day males being more sensitive as demonstrated by the more progressive effects on testis weight and steroid hormone receptors’ expression. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgement This work was financially supported by the Academic Grants for Professors 2008 from The Foundation for Polish Science (to B.B.) and, in part, by a grant BW/IZ/54a/2008.

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