ToxSci Advance Access originally published online on March 14, 2006
Toxicological Sciences 2006 91(2):456-466; doi:10.1093/toxsci/kfj156
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Effects of Neonatal Exposure to 4-Tert-Octylphenol, Diethylstilbestrol, and Flutamide on Steroidogenesis in Infantile Rat Testis
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* Laboratory of Animal Physiology, Department of Biology, Departments of Physiology and Pediatrics, Department of Anatomy, University of Turku, 20520 Turku, Finland
1 To whom correspondence should be addressed. Fax: +358 2-3337352. E-mail: paranko{at}utu.fi.
Received November 28, 2005; accepted March 14, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Exposure of neonatal testis, populated by fetal-type Leydig cells, to endocrine-active compounds may have far-reaching consequences. Our aim was to resolve the sensitivity of testosterone synthesis of infant rat (Sprague-Dawley) testis to diethylstilbestrol (DES; 0.1–1.0 mg/kg), 4-tert-octylphenol (OP; 10–100 mg/kg), and Flutamide (FLU; 2.0–25 mg/kg) given by daily sc injections from birth to postnatal day 4. Testes and serum were collected on day 14 when body and testis weight, testicular histology, circulating testosterone, LH and FSH levels, and steroidogenic acute regulatory protein (StAR) and 3ß-hydroxy-steroid-dehydrogenase (3ß-HSD) protein levels were determined. DES at each dose and FLU at 25 mg/kg dose reduced testis weight and the diameter of seminiferous cords. FLU caused some Leydig cell hyperplasia. Plasma testosterone was reduced in all DES animals, LH elevated in DES 0.5 mg/kg and FLU 25 mg/kg animals, and FSH reduced in the DES 1.0 mg/kg group. Basal testicular ex vivo progesterone and human chorionic gonadotropin (hCG)–stimulated testosterone production were decreased in DES animals. Despite a decrease in hCG-induced cyclic adenosine-3',5'-monophosphate (cAMP) production, intratesticular testosterone was increased in the FLU 10 and 25 mg/kg groups. OP 100 mg/kg elevated hCG-induced progesterone production only. No changes were seen in 3ß-HSD protein levels in any treatment group. StAR levels were reduced in DES animals. The results indicate the sensitivity of postnatal fetal-type Leydig cells to endocrine-active compounds. Suppression of StAR expression level was an early sign of the DES-induced steroidogenic lesion. FLU-induced changes suggest the importance of androgen receptor–mediated regulation of testosterone synthesis in the postnatal rat testis. Octylphenol appeared less effective in bringing about acute steroidogenic changes.
Key Words: diethylstilbestrol; octylphenol; Flutamide; testosterone; rat; testis; endocrine disruption.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testicular androgen synthesis in developing male mammals is regulated in a time-dependent manner. Abnormal androgen production profile combined with altered androgen-estrogen ratio has a great potency to demasculinize androgen-dependent differentiation, growth, and function of male reproductive organs and central nervous system (Casto et al., 2003
; Rivas et al., 2002; Sharpe et al., 2003). Events causing demasculinization can be traced back to changes in gonadal sex steroid synthesis (Majdic et al., 1996, 1997; Saunders et al., 1997) and in hypothalamic-pituitary-gonad axis (Goyal et al., 2003; Sweeney et al., 2000), and may involve altered expression pattern of androgen and estrogen receptors in androgen-dependent target tissues (Goyal et al., 2003; Gray et al., 1999; McKinnell et al., 2001; Mylchreest et al., 1999; Nishikawa et al., 1999; Sohoni and Sumpter, 1998; Williams et al., 2001). Androgen receptors in fetal and postnatal rat testis are expressed in nonsteroidogenic interstitial cells and Leydig cells and in postnatal Sertoli cells (Shan et al., 1997; Williams et al., 2001; You and Sar, 1998). Estrogen receptor 
is found in fetal-type Leydig cells (Fisher et al., 1997; Saunders et al., 1997; Williams et al., 2001) and type ß in Leydig and Sertoli cells and gonocytes (Fisher et al., 1997; Saunders et al., 1998; van Pelt et al., 1999; Williams et al., 2001). Therefore, a number of cell types in developing rat testis are potential targets of steroid hormone receptor–mediated endocrine disruption.
Several man-made chemicals have a potency to alter testicular and pituitary functions via steroid receptor–dependent and –independent pathways. Many of the known demasculinizing compounds possess binding affinity to estrogen receptors and are referred to as xenoestrogens. This group includes, for instance, alkylphenols, bisphenol A, and phytoestrogens. So far, only a few chemicals, such as herbicides linuron, procymidone, p,p'-dichlorodiphenyldichloroethylene (Gray et al., 1999), and viclozolin metabolites, have been identified as androgen receptor antagonists, but the number of chemicals identified as antiandrogens acting via androgen receptor is increasing (Korner et al., 2004; O'Connor et al., 2002; Vinggaard et al., 2005).
For the present study, three differently endocrine-active compounds, diethylstilbestrol (DES), Flutamide (FLU), and octylphenol, were selected to analyze their potency to acutely interfere with the growth and steroid hormone synthesis of infant rat testis. DES, a synthetic nonsteroidal estrogen introduced neonatally, has a high affinity to estrogen receptors, causes a variety of reproductive tract anomalies, and alters androgen status by modifying biosynthesis of sex steroids (Majdic et al., 1996, 1997) and the expression pattern of androgen and estrogen receptors (Goyal et al., 2003; McKinnell et al., 2001; Williams et al., 2001). FLU, a nonsteroidal antiandrogen drug, effectively prevents androgen action at receptor level and is used for the treatment of prostate cancer and acne, and as a model compound for developmentally induced demasculinization (Anahara et al., 2004; Miyata et al., 2002; O'Connor et al., 2002). In Leydig cells, FLU may inhibit androgen receptor–dependent autoregulation of testosterone synthesis (Houk et al., 2004; Ruiz de Galarreta et al., 1983). Octylphenol (4-tert-octylphenol, OP), a constituent of alkylphenol polyetoxylates, is used as a surfactant in detergents, paints, and herbicides and may accumulate in sewage sludge (White et al., 1994). In the fish, avian and mammalian species, OP has been shown to interfere with the development of reproductive system (Blake and Boockfor, 1997; Sweeney et al., 2000; White et al., 1994). OP binds to estrogen receptors, but compared to DES, the binding affinity is 5000- to 10,000-fold weaker (Gutendorf and Westendorf, 2001). In developing rat testis, OP may cause a prolonged suppression of testosterone synthesis (Yoshida et al., 2001).
The aim of the present study was to evaluate the sensitivity of fetal-type Leydig cells to neonatally induced endocrine disruption by measuring circulating testosterone and gonadotropin levels, testicular testosterone and progesterone production, and steroidogenic acute regulatory protein (StAR) and 3ß-hydroxy-steroid-dehydrogenase (3ß-HSD) type I protein expression levels in 14-day-old infant male rats. In the rat, fetal-type Leydig cells are the major androgen-producing cell population in infancy and become gradually replaced by adult-type Leydig cells from day 14 postpartum (Ariyaratne and Mendis-Handagama, 2000; Mendis-Handagama et al., 1998; Tapanainen et al., 1984). Compared to adult-type Leydig cells, the physiology of fetal-type Leydig cells differs in several aspects, including high sensitivity to LH/human chorionic gonadotropin (hCG), the fast recovery of LH receptors, and the lack of LH-induced steroidogenic desensitization until the age of 15–20 days (Huhtaniemi et al., 1984). In neonatal and infant male mammals, circulating gonadotropin and androgen levels are relatively low (Döhler and Wuttke, 1974) but sufficient to support the prepubertal masculinization and growth of reproductive system. If elevated, like in the male mice overexpressing hCG (Rulli et al., 2003), infertility and severe alterations in the urogenital tract may occur. In rats, high postnatal androgen levels may cause premature onset of puberty which negatively impinges on androgen status and spermatogenesis in adulthood (Chandrasekharam et al., 2003; Duckett et al., 1997; Pomerantz, 1984). Therefore, it is important to clarify the sensitivity of steroidogenesis in infantile testis to endocrine-active chemicals.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Pregnant Sprague-Dawley female rats (The Animal Center of Turku University, Turku, Finland) were housed individually under a 12 h light/12 h dark cycle in plastic cages with woodchips as the floor covering. The temperature of the breeding room was set at 22 ± 2°C and humidity at 55 ± 10%. The rats were fed ad libitum with standard laboratory animal food (Commercial RM3(E) SQC, Special Diet Services, Witham, England) and tap water. The day of birth was designated as postnatal day (PND) 0. Pups grew with the dams until the day of analysis. All animal experiments were approved by the Turku University Committee on the Ethics of Animal Experimentation.
Chemicals and treatments.
DES, OP, and FLU were purchased from Sigma (Sigma Chemical Co., St. Louis, MO). DES and OP were dissolved in 33% dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany) in water giving 50 µl/kg as the final daily volume of 33% DMSO in the animals. FLU was dissolved in corn oil. Daily sc exposure doses were for DES 0.1, 0.5, and 1.0 mg/kg body weight, OP 10, 50, and 100 mg/kg, and FLU 2, 10, and 25 mg/kg. For each dam, four male pups were randomly allocated from different litters. In each group, one male served as a control treated with the vehicle only and the three were treated with the serial doses of one of the compounds. The first neonatal injection was given 6 h after the birth and then daily until the PND 4, i.e., five doses altogether. In the preliminary experiments, no differences were found in steroid hormone levels of oil-treated and DMSO-treated control animals. Total number of control and treated male pups is given in Table 1.
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Hormone assays.
Hormone measurements were carried out by time-resolved fluoroimmunoassays (DELFIA, Dissociation-Enhanced Lanthanide Fluoroimmuno-assay; PerkinElmer Life and Analytical Sciences Ltd., Wallac, Turku, Finland) as described earlier (Haavisto et al., 2001
, 2003). Testosterone was measured in ether-extracted plasma samples, testis homogenates of freshly isolated testes and testes cultured for 3 h, and in culture media. Progesterone was assayed from culture media. LH and FSH levels were measured in plasma samples. For plasma, the animals were anesthesized with carbon dioxide and killed by neck dislocation on PND 14. Blood was collected by heart puncture into heparinized syringes, kept on ice (> 1 h), and centrifuged at 1000 g at +4°C for 5 min. Plasma was stored at –85°C until analysis. Testicular testosterone was measured from the lysis supernatant after incubation for 30 min at 4°C in the lysis buffer (25mM Tris-HCl, 120mM NaCl, 0.5% NP-40, 4mM NaF, 100µM Na3VO4, 10 µg/ml aprotinin, 1mM phenylmethylsulphonyl fluoride (PMSF), and 10 µg/ml leupeptin). For the measurement of hCG-stimulated testosterone production, the testes were excised and one of them was incubated in the absence (control) and one in the presence of 50 ng/ml of recombinant hCG (4700 IU/mg, Akzo Nobel, N.V. Organon, Netherlands) for 3 h. Incubation was carried out in Dulbecco's modified Eagle's medium/HAM's F-12 supplemented with 15mM HEPES, 2 mg/l pyridoxine, 1.2 g/l NaHCO3 (Sigma-Aldrich, Irvine, United Kingdom), 0.365 g/l L-glutamine (Life Technologies, Gibco BRL, Paisley, Scotland), 0.1% bovine serum albumin (BSA) (Sigma Chemical Co.), and 0.1 g/l gentamycin (Biological Industries, BetHaEmek, Israel).
Testis culture ex vivo.
Testes, aseptically excised from PND 14 rats, were decapsulated and transferred into 4-well plastic culture dishes (NUNC, Roskilde, Denmark), one testis in a well containing 1 ml of Dulbecco's modified Eagle's medium/HAM's F-12 with the supplements described. After the incubation for 3 h at 37°C under 5% CO2 in air, culture media were collected and stored at –20°C for hormone measurements.
Futamide ex vivo cAMP assay.
For the analysis of ex vivo pattern of testicular testosterone production in FLU-treated pups, basal and hCG-stimulated cAMP production was measured from the testis of control and FLU-treated (25 mg/kg) animals. The highest dose of FLU was selected for the assay due to its potency to induce significant increase in plasma LH levels and testosterone production in vitro. For a 3-h tissue incubation of single testis in the supplemented Dulbecco's modified Eagle's medium/HAM's F-12, 250µM 3-isobutyl-1-methylxanthine (Sigma) was added to the culture medium as a phosphodiesterase inhibitor. hCG was added at concentrations of 0, 10, 50, or 100 ng/ml. After incubation, media (1 ml) and testes were taken into Eppendorf tubes containing 110 µl of 4.4.M perchloric acid and then processed as described earlier (El-Gehani et al., 1998; Haavisto et al., 2001, 2003). cAMP levels were measured by protein-binding assay (Nordstedt and Fredholm, 1990). Briefly, the samples and standards (0–320nM) were incubated for 2.5 h at 4°C with protein extract from bovine adrenal cortex containing cAMP-binding protein and [3H]-cAMP (specific activity 54 Ci/mmol, Amersham, Buckinghamshire, UK) diluted with distilled water to get approximately 15,000 cpm per well. The bound and free cAMPs were separated in Millipore MultiScreen filtration plates. The wells were washed three times with 200 µl of 50mM Tris-HCl, pH 7.4. After drying (+37°C, 1 h), 50 µl of SuperMix scintillation fluid (PerkinElmer Wallac Scintillation Products) was added to the well. Radioactivity was counted using a MicroBeta scintillation counter (Wallac, Turku, Finland). Standards were prepared in the range of 0–320nM. The intra- and interassay coefficients of variation were 5 and 10%, respectively.
Western blot analysis.
Testes lysate was centrifuged at 14,000 rpm for 25 min at +4°C, and protein concentration in the supernatant was determined. Protein samples (20–30 µg) were dissolved in 7.5 µl of 2x Laemmli sample buffer and set to 15 µl with buffer C (25% glycerol, 0.42 M NaCl, 1.5mM MgCl2, 0.2 mM EDTA, 20 mM HEPES, 0.5 mM DTT, and 0.5 mM PMSF). After electrophoresis in 12.5% sodium dodecyl sulfate–polyacrylamide gel (Mini Protean II system, Bio-Rad laboratories, Hercules, CA), proteins were transferred onto a nitrocellulose membrane (Hybond, Amersham). After incubation in blocking buffer (10 mM Tris-HCl, pH 8.0, 0.3% Tween 20, and 3% nonfat milk powder) for 1.5 h at room temperature, rabbit anti-human StAR antibodies (courtesy of Dr. J. F. Strauss III, Center for Research on Reproduction and Women's Health, University of Pennsylvania, University of Pennsylvania, PA) or rabbit anti-mouse recombinant 3ß-HSD type 1 antibodies (courtesy of Dr. A. Payne, Stanford University Medical Center) were added on separate strips at dilutions 1:2000 and 1:5000 in 1% BSA-PBS, respectively. Washed (10mM Tris-HCl, pH 8.0, 0.3% Tween 20) membranes were incubated with horseradish peroxidase–linked anti-rabbit IgG (Amersham Science) diluted 1:5000 for StAR and 1:20,000 for 3ß-HSD type 1. Antibody binding was visualized on X-ray film (Kodak, X-OMAT, Rochester, NY) with an ECL Western Blot Detection Kit (Amersham). Reprobing with mouse anti-ß-actin antibody (Sigma; 1:20 000) was done on the washed strips. Antibody binding was visualized by peroxidase-linked anti-mouse IgG (Amersham Pharmacia Biotech; 1:50 000) labeling. The densitometric data obtained by Chemi Imager 4400 program (version 5.5, Alpha Innotech Corporation, San Leandro, CA) was normalized for actin signal. For each experiment, Western blot analysis was repeated at least for three times.
Microscopy.
For histological analysis and for the measurement of the seminiferous cord diameter, the testes were dissected from pups on PND 14, one testis from four animals in each group. Tunica albuginea was punched with thin needles, and the testes were prefixed in 5% glutaraldehyde in 0.16 mol/l s-collidine-HCl buffer (pH 7.4) for 1 h at room temperature. Thereafter, the testis was cut into four horizontal slices with a razor blade and returned to the fixative for 24–48 h. After washing in s-collidine buffer, the tissues were postfixed in potassium ferrocyanide-osmium fixative. For toluidine blue staining and light microcopic analysis under Leica DMRXA light microscope, five dehydrated 1-mm3 samples from every horizontal testis slice were embedded in Epon and cut into 1-µm-thick semithin sections. From each 1-mm3 sample, a series of three semithin sections were cut at 250-µm intervals yielding in 240 sections from one testis. From each section, the diameter of 12 seminiferous cords was measured.
Statistical analysis.
The results were analyzed by one-way ANOVA followed by Dunnett's post hoc two-sided t-test or independent-samples t-test using the SPSS for Windows 9.0 program. Values for animal body weights and testis weights were expressed as mean ± SD and all the other values as mean ± SEM. A probability p less than 0.05 was chosen as the limit of statistical significance.
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RESULTS |
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Body and Testis Weight
Body and testis weight data of 14-day-old male rat infants are shown in Table 1. Body weight was not affected by any of the treatments, but the testis weight was reduced approximately by 40% at every dose of DES (p < 0.001). The highest dose of FLU (25 mg/kg/day) suppressed testis weight by approximately 15% (p < 0.05). OP had no effect on testis weight.
Testis Histology and the Diameter of Seminiferous Cords
Testicular histology was analyzed in 14-day-old males neonatally exposed to 0.5 mg DES/kg, 100 mg OP/kg, and 25 mg FLU/kg. The average seminiferous cord diameter in the control testes was 85.51 ± 1.9 µm (Fig. 1). In the DES group, it was reduced by 17.1% (p < 0.001) and by 7.6% (p < 0.01) in the FLU-treated offspring. A nonsignificant (p < 0.069) reduction in the cord diameter was recorded in the animals treated with OP. Leydig cell hyperplasia/hypertrophy was seen in one out of four testes of FLU-treated males. Large Leydig cell islets shown in Figure 2, however, occurred only sporadically and comprised only 2–5% of the total number of Leydig cell islets. Compared to controls, the testes of DES- and OP-treated males showed no overt histological changes in Leydig cells and seminiferous cords.
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Plasma Testosterone, LH, and FSH
In males exposed to DES (0.1, 0.5, or 1.0 mg/kg/day), plasma testosterone (Fig. 3A) was suppressed by 82–91% (p < 0.05–0.01), but the levels were not significantly altered in the groups treated with OP and FLU. A twofold increase in LH (Fig. 3B) was seen in the animals treated with 0.1 and 1.0 mg DES/kg, but the change was not statistically significant due to a relatively high interindividual variation. However, a 2.8-fold increase in LH was significant (p < 0.05) in males exposed to 0.5 mg DES/kg. While OP treatment did not affect plasma LH levels, a dose-dependent increase was seen in the FLU-treated animals. A threefold increase at 25 mg FLU/kg dose was statistically significant (p < 0.01). Plasma FSH levels (Fig. 3C) were reduced by 46% (p < 0.01) in the 1.0 mg/kg DES group, but no effect was seen in the animals treated with OP or FLU.
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Testosterone and Progesterone Production Ex vivo
Basal testosterone secretion during a 3-h ex vivo culture of PND 14 testes was not significantly affected by any of the treatments (Fig. 4A). In control testes, basal testosterone secretion was stimulated by 2.3-fold in the presence of hCG. In the DES group, stimulation rate was 1.8- to 1.6-fold corresponding to approximately 40% decrease (p < 0.05) from the control levels. Testes of FLU-treated rats (25 mg/kg/day) showed a 5.2-fold increase (p < 0.05) in the hCG-stimulated secretion of testosterone. None of the changes in the OP-treated animals were statistically significant.
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As for intratesticular testosterone (Fig. 4B), the profile of basal and hCG-stimulated testosterone levels confirmed the pattern observed for secreted testosterone. In the absence of hCG, none of the compounds caused statistically significant changes in intratesticular testosterone levels. In the DES groups, the doses 0.1 and 0.5 mg/kg/day decreased hCG-stimulated testosterone content by 58.5% (p < 0.05) and the dose 1.0 mg/kg/day by 68.5% (p < 0.01). Testosterone concentration in the OP group remained at the control level throughout. In the FLU-treated animals, the highest dose (25 mg/kg/day) caused a significant (p < 0.05) increase in hCG-induced testosterone production exceeding 1.7-fold the level measured from the stimulated control testes. The elevation from the control basal level was 5.4-fold.
Progesterone secretion (Fig. 4C) of the control testes was not altered after hCG stimulation. In the testes of DES-exposed males, however, basal secretion was reduced in a dose-independent manner by 34–37% (p < 0.05–0.01). After hCG stimulation, progesterone levels in the culture media of the DES group remained slightly under the control levels. Basal progesterone secretion was not affected by OP. However, OP 100 mg/kg/day significantly (p < 0.05) elevated hCG-stimulated progesterone levels. In the FLU-exposed animals, progesterone remained at control level throughout.
Effects of FLU on Acute Intratesticular Testosterone and cAMP Production
In order to assess whether the stimulatory effect of FLU observed in testicular testosterone production ex vivo could be acutely seen in the freshly isolated testes, testosterone was measured on PND 14 from the groups exposed to 2, 10, or 25 mg FLU/kg/day (Fig. 5). In the 10-mg/kg/day exposure group, a significant (p < 0.001) 1.8-fold increase and in the 25-mg/kg/day group a 2.2-fold increase (p < 0.001) in intratesticular testosterone content were recorded. In the cAMP assay carried out after a 3-h ex vivo culture of the testes neonatally exposed to 25 mg FLU/kg/day, basal cAMP production did not differ from the controls (Fig. 6). However, in the presence of hCG (10 or 50 ng/ml), a significant (p < 0.01–0.001) decrease in the cAMP levels was observed in the FLU-exposed group.
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Expression of StAR and 3ß-HSD 1 Proteins
In the Western blot analysis of the tissue homogenates from freshly isolated PND 14 rat testis, a major band at 30 kDa was recognized with anti-StAR antibody (Fig. 7A). In DES-treated groups, testicular StAR protein expression was suppressed by 41–44% (p < 0.05) from controls (Fig. 7B). OP did not alter the pattern of StAR protein expression. In the FLU group, StAR values remained at or slightly above the control level. For 3ß-HSD type 1 protein expression, no significant changes were found in any of the exposure groups (Figs. 7A and 7C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results indicate the sensitivity of infantile rat testis to neonatally introduced estrogens and antiandrogens. Given that postnatal steroidogenic profile based on the activity of fetal Leydig cells (Ariyaratne and Mendis-Handagama, 2000
) is of a pivotal importance for the prepubertal and pubertal sexual maturation, the observed changes may be early events in the chain that leads to the manifestation of reproductive lesions described in adult progeny exposed neonatally to endocrine-active compounds. The most striking, albeit opposing, steroidogenic effects were obtained in the male infant rats exposed to DES and FLU. That the highest concentration of octylphenol, a weak estrogenic compound, increased hCG-stimulated progesterone production is in line with the steroidogenic changes seen in the prenatal rat testis exposed to octylphenol in vitro (Haavisto et al., 2003). Otherwise, the effects of octylphenol on postnatal testis seem to remain less severe. The observed decrease in testis weight in male infants treated with DES and FLU is a characteristic feature of demasculinization and interference of androgen-dependent growth (Goyal et al., 2003; Miyata et al., 2002). The decrease in tissue weight, however, does not solely explain steroidogenic effects observed.
In the DES-exposed infants, the observed steep suppression of plasma testosterone levels is in line with the reported results in 18-day-old male rats submitted for a longer postnatal exposure period (Williams et al., 2001). From the present study, it appears that steroidogenic changes are induced relatively fast and after a short neonatal exposure period. Strongly altered steroidogenic profile in infancy apparently is involved in the origin of reproductive anomalies described in neonatally estrogenized adult male rats, including the reduction in pubertal testis size, Sertoli cell numbers, and lumen formation of the seminiferous epithelium, retarded spermatogenesis, and abnormal development of rat testis (Atanassova et al., 1999, 2000; Goyal et al., 2003; Rivas et al., 2002) and expression pattern of androgen and estrogen receptors (Tena-Sempere et al., 2000). Described morphologic changes due to estrogenization and altered estrogen-androgen balance may include reduced testicular cell proliferation, changes in cell differentiation program, increased rate of apoptosis (Williams et al., 2001), and downregulation of androgen receptors (Fritz et al., 2003; McKinnell et al., 2001; Williams et al., 2001).
In fetal-type Leydig cells, inhibition of androgen synthesis by DES and other estrogens seems to be mediated via estrogen receptor alpha (Delbès et al., 2005). By binding to estrogen receptor, DES may inhibit steroidogenic pathway at multiple steps (Fielden et al., 2002; Majdic et al., 1996, 1997). According to the observed response to hCG stimulation and our previous results in the in utero DES-exposed testes (Haavisto et al., 2003), steroidogenic lesion apparently does not involve LH receptor–mediated signaling. Also LH secretion from the infant pituitary seems to be resistant to DES-induced defects. Instead, as indicated in the present and earlier studies in developing testis (Guyot et al., 2004), StAR protein synthesis seems to be among the most sensitive targets of the adverse effects of DES. Significant decrease in StAR protein levels in Western blot analysis suggests that the steroidogenic changes in the testis of DES-treated offspring can not only be explained by the reduced testis weight. Also in Leydig MA-10 cells, estrogens strongly inhibit StAR expression (Houk et al., 2004). Conversely, in estrogen receptor alpha knockout neonate male mice, StAR expression is upregulated (Delbès et al., 2005). Therefore, that the testes of DES-exposed male offspring do not react positively to elevated plasma LH levels and only marginally to hCG stimulation could be due to the upregulation estrogen receptors and estrogen receptor–mediated enzymatic inhibition of androgen synthesis, the cascade shown to occur in Leydig cells (Tena-Sempere et al., 2000; Tsai-Morris et al., 1986). 3ß-HSD enzyme converting pregnenolone to progesterone remained unchanged at protein level, suggesting that the production of the enzyme is not a potential target of DES action, as not in the prenatal rat testes either (Saunders et al., 1997). Studies on fetal mouse (Delbès et al., 2005; Guyot et al., 2004) and rat (Majdic et al., 1996; Saunders et al., 1997) testis propose 17- hydroxylase C17,20 lyase (P450c17) as a target of DES action. Inhibited transcription of P450c17 apparently is a key factor causing steroidogenic lesion in gonadotropin-induced adult rat Leydig cells (Lu et al., 1991). As for steroidogenic factor 1, a transcription factor known to regulate the expression of P450 enzyme genes, the effects of DES in developing testes have remained controversial (Guyot et al., 2004; Saunders et al., 1997).
Similar to DES used at the dose of 1.0 mg/kg (present study), treatment of newborn male rats with estradiol benzoate significantly reduces serum testosterone and FSH levels in 15-day-old and older rats (Tena-Sempere et al., 2000). As a regulator of Sertoli cells, reduced FSH levels could adversely affect the function and proliferation of Sertoli cells, testis size, and sperm production capacity as reported in estrogenized males (Atanassova et al., 2000; Sharpe, 1998). Under prevailing low aromatase activity and low enstradiol synthesis in the infant rat testis (George and Ojeda, 1987; Pomerantz, 1980), the altered FSH level apparently has no significant effect on intratesticular estradiol levels. Due to a low affinity to steroid hormone carrier proteins (Milligan et al., 1998), DES alone has a great potency to cause the physiological effects indicated also in the present study.
Confirming the androgen dependence of testis growth, FLU (25 mg/kg) reduced testis growth, even though not as effectively as DES. At higher doses (100 mg/kg), in utero-introduced FLU has been shown to interfere with body growth and masculinization of male sex organs (Mylchreest et al., 1999). The results from the perinatal exposure studies at 10–100 mg FLU/kg (Miyata et al., 2002), in turn, have confirmed the irreversibility of FLU-induced reproductive anomalies. Circulating testosterone levels in the present pups were not significantly elevated but this apparently would occur at higher perinatal exposure doses (Miyata et al., 2002). In the present study, intratesticular testosterone levels and circulating LH, however, were increased. In line with the above findings, in 15-day screening assay in adult rats, exposure to FLU (5-100 mg/kg/day) significantly elevates testosterone, estradiol, and dihydrotestosterone levels and causes hypergonadotropism (O'Connor et al., 2002). The changes reported exclude LH-mediated regulation as a negative target of FLU action. As we showed ex vivo, hCG-induced intratesticular and secreted testosterone levels were significantly elevated in 14-day-old rat testis. The ex vivo-induced increase was not seen in progesterone production, which apparently is due to the rapid turn over of the hormone.
Under normal physiological conditions, androgens are known to reduce testosterone biosynthesis in prepubertal and adult Leydig cells and Leydig cell lines in an autoregulative way, apparently by the receptor-mediated inhibition of StAR expression (Houk et al., 2004; Ruiz de Galarreta et al., 1983). Androgen-dependent regulation of Leydig cell function seems to become functional postnatally (O'Shaughnessy et al., 2002) and, therefore, fetal testicular testosterone production is not markedly affected by FLU (Mylchreest et al., 2002). The present observation of the increased ex vivo testosterone production in the presence of relatively high StAR protein levels in the testes of FLU-treated infant rats is in line with the observation of Houk et al. (2004): in mouse Leydig cell cultures, the inhibition of testosterone binding to androgen receptors prevents the normal downregulation of StAR. Inhibition of transcriptional activity or the expression of androgen receptors (McKinnell et al., 2001; Mylchreest et al., 2002; Ohsako et al., 2003) seems to have a great impact on Leydig cell steroidogenesis.
In order to further resolve the mechanisms of FLU action in postnatal rat Leydig cell population, hCG-stimulated cAMP production was measured from the testis of control and FLU-treated (25 mg/kg) pups. Despite hCG-induced stimulation of testosterone production, testicular ex vivo cAMP levels were lower in FLU-treated than in control animals. The mechanism behind the phenomenon remains to be elucidated. The combination of high levels of StAR with the exceptional high sensitivity of fetal-type leydig cells to LH/hCG (El-Gehani et al., 1998) could explain the elevated testosterone production. Compared to adult Leydig cells, surprisingly high testosterone production under extremely low cAMP levels has been demonstrated in neonatal rat Leydig cells (Huhtaniemi et al., 1984).
Leydig cell hyperplasia/hypertrophy could, in part, explain the elevation of hCG-induced intratesticular testosterone levels, but due to a relative low incidence of enlarged Leydig cell islets this seems less evident. Hyperplasia/hypertrophy of Leydig cells has been described in adult rat testes exposed to FLU in utero (Mylchreest et al., 1999) or in adulthood (O'Connor et al., 2002). Some Leydig cell hyperplasia also occurs in the prenatal Leydig cells exposed in utero to100 mg/kg FLU (Mylchreest et al., 2002). Hypertrophy apparently is due to the disturbance of endocrine hormonal homeostasis, a consequence of the absence of negative testosterone-mediated feedback of LH secretion. Transgenic mice with the overproduction of human chorionic gonadotropin also develop hypertrophy of fetal-type Leydig cells (Ahtiainen et al., 2005). Whether hypertrophy of gonadotropin-sensitive fetal-type Leydig cells leads to the extended survival of the cell population is an intriguing question. Leydig cell adenomas have been described in rat testes exposed in utero to antiandrogen di(n-butyl)phathalte (Barlow et al., 2004).
Octylphenol, a putative estrogen receptor agonist, had no or only marginal effects in neonatally exposed infant male rat testis. The only significant change was the observed elevation of hCG-induced progesterone production ex vivo at the highest dose of OP (100 mg/kg). The change was similar to that described in the prenatal rat testis exposed to OP (100 mg/l) in vitro (Haavisto et al., 2003) and may be an indication of the reduced expression of P450c17 shown to occur in fetal rat testis maternally exposed to octylphenol and DES (Majdic et al., 1996). Marginal changes observed in the present study may be a matter of dosing. Prolonged suppression of testosterone levels has been reported in male rats exposed neonatally to OP 100 mg/kg every other day until PND 15 (Yoshida et al., 2001).
The purpose of the present study was to resolve the sensitivity of infant testis and its fetal-type Leydig cell population to DES, octylphenol, and antiandrogen FLU. Both DES and FLU induced acute changes in testis growth but via different steroidogenic mechanisms. While DES caused a strong downregulation of StAR protein expression and hCG-induced testosterone production, opposite effects were seen in the testes of FLU-treated rats. Further studies are needed to resolve the developmental maturation of androgen receptor–mediated autoregulation of testosterone synthesis in Leydig cells and its role in the neonatal regulation of testis growth and development.
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ACKNOWLEDGMENTS |
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This work was supported by the Academy of Finland, Turku University Foundation, Sigrid Juselius Foundation, Turku University Central Hospital, Finnish Cultural Foundation, Maj and Tor Nessling Foundation, and the European Commission under the framework of the "Quality of Life" program (Contracts: QLK4-CT1999-01422 ENVIR.REPROD.HEALTH, QLK4-2001-00269 EXPORED, and QLK4-CT2002-00603 EDEN).
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