Exposure to Phytoestrogens in the Perinatal Period Affects Androgen ...

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Endocrinology 148(9):4475– 4488 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2007-0327

Exposure to Phytoestrogens in the Perinatal Period Affects Androgen Secretion by Testicular Leydig Cells in the Adult Rat Benson T. Akingbemi, Tim D. Braden, Barbara W. Kemppainen, Karen D. Hancock, Jessica D. Sherrill, Sarah J. Cook, Xiaoying He, and Jeffrey G. Supko Department of Anatomy, Physiology and Pharmacology (B.T.A., T.D.B., B.W.K., K.D.H., J.D.S.), Auburn University, Auburn, Alabama 36849; and Massachusetts General Hospital (S.J.C., X.H., J.G.S.), Harvard Medical School, Boston, Massachusetts 02114 The use of soy-based products in the diet of infants has raised concerns regarding the reproductive toxicity of genistein and daidzein, the predominant isoflavones in soybeans with estrogenic activity. Time-bred Long-Evans dams were fed diets containing 0, 5, 50, 500, or 1000 ppm of soy isoflavones from gestational d 12 until weaning at d 21 postpartum. Male rats in all groups were fed soy-free diets from postnatal d 21 until 90 d of age. The mean ⴞ SD concentration of unconjugated (i.e. biologically active) genistein and daidzein in serum from the group of dams maintained on the diet containing the highest amount of isoflavones (1000 ppm) were 17 ⴞ 27 and 56 ⴞ 30 nM, respectively, at d 21 postpartum. The concentrations were considerably greater in male offspring (genistein: 73 ⴞ 46 nM; daidzein: 106 ⴞ 53 nM). Although steroidogenesis was de-

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XOGENOUS COMPOUNDS THAT interfere with the endocrine axis are classified as endocrine disruptors (EDs). In the male, estrogen receptors (ER)-␣ and ER␤, along with androgen receptors, are expressed in fetal, neonatal, and adult tissues such as the hypothalamus, pituitary, testis, and the excurrent duct system, indicating that estrogen supports multiple activities in male reproduction (1, 2). Indeed, poor semen quality is a consistent finding in male subjects with mutations in ER␣ and those suffering from aromatase deficiency (3). Isoflavones occur predominantly as glycoside conjugates (genistin and daidzin) in soybeans and soy-based infant formulas (4). Genistin and daidzin are hydrolyzed in the gastrointestinal tract to the aglycone compounds genistein and daidzein, which are biologically active agents that bear a structural similarity to endogenous estrogen [17␤estradiol (E2); Fig. 1]. Feeding genistein to ovariectomized rats enhances lobular-alveolar mammary gland development, increases uterine weight, and induces pituitary prolactin secretion (5). There is also evidence that genistein and First Published Online June 14, 2007 Abbreviations: ANG, Anogenital distance; AO, androsterone; E2, 17␤-estradiol; ED, endocrine disruptor; ER, estrogen receptor; GD, gestational day; 3␤-HSD, 3␤-hydroxysteroid dehydrogenase; p, phosphorylated; PND, postnatal day; T, testosterone, TSPO, mitochondrial translocator protein; StAR, steroidogenic acute regulatory protein; T, testosterone; TBST, Tris-buffered saline containing Tween 20. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

creased in individual Leydig cells, male rats from the highest exposure group (1000 ppm diet) exhibited elevated serum levels of the sex steroid hormones androsterone at 21 d (control: 15 ⴞ 1.5 vs.28 ⴞ 3.5 ng/ml; P < 0.05) and testosterone at 90 d of age (control: 7.5 ⴞ 1 vs.17 ⴞ 2 ng/ml; P < 0.05). Testosterone secretion by immature Leydig cells, isolated from 35-d-old male rats, decreased on exposure to 0.1 nM genistein in vitro (control: 175 ⴞ 5 vs. 117 ⴞ 3 ng/106 cells per 24 h; P < 0.05), indicative of direct phytoestrogen action. Thus, phytoestrogens have the ability to regulate Leydig cells, and additional studies to assess potential adverse effects of dietary soy-based products on reproductive tract development in neonates are warranted. (Endocrinology 148: 4475– 4488, 2007)

daidzein induce transactivation of ERs in vitro (6). After ingestion of soybeans and soy-based products, the reproductive organs and liver are major sites of isoflavone accumulation in the body and are therefore potential targets of toxicity (7). Soy-based infant formulas contain isoflavones at concentrations exceeding 100 ␮m and are the greatest dietary source of phytoestrogens consumed by humans. Blood concentrations of isoflavones that are 10 to 20,000 times higher than endogenous E2 levels have been reported in infants fed soybased formulas (8). For example, it is estimated that a 4-month-old infant consuming soy formula as directed by manufacturer’s instructions will ingest 6 –9 mg of isoflavones per kilogram of body weight per day (9). Therefore, infants fed soy-based formulas ingest considerably greater relative amounts of isoflavones than adults who eat soy-based products (10). Although there is much evidence that hormonally active chemicals exhibit greater potency during sexual differentiation in rodents and humans (11), there is little epidemiological data describing the long-term effects of soybased formulas and soy-enriched diets in children. To address this issue, the Center for the Evaluation of Risks to Human Reproduction (National Toxicology Program) recently constituted an expert panel to review the scientific data on genistein. The panel affirmed that studies are required to discriminate between the developmental and reproductive toxicity of genistein and concluded that experimental animal data are relevant to the assessment of human risk (12).

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Akingbemi et al. • Phytoestrogens Regulate Leydig Cells

FIG. 1. Chemical structure of endogenous estrogen (17␤-estradiol) and the major isoflavones present in soybeans. Genistein and daidzein exhibit estrogenic properties and are classified as phytoestrogens.

A diverse profile of male reproductive tract anomalies has been attributed to phytoestrogens in studies of laboratory animals and nonhuman primates. For example, neonatal rats sustained on a diet containing 5 and 300 ppm genistein exhibited decreased testis size and serum testosterone (T) levels (13). It has also been shown that sc administration of genistein at a daily dose of 2.5 mg/kg䡠d for 9 d resulted in reduced serum and testicular T concentrations and decreased prostate gland weight in adult mice (14). Neonatal marmosets fed soy-based formula, which provided an estimated daily intake of isoflavones of 1.6 –3.5 mg/kg from d 5 to d 45 postpartum subsequently had serum T levels ranging from 1.2 to 2.6 ng/ml; these concentrations were significantly lower than the levels measured in paired animals fed cow milk formula, which range from 2.8 to 3.1 ng/ml (15). In contrast to inhibitory effects, testis weights were increased in ICR mice treated neonatally with genistein (1 mg/pup) (16), and sc administration of genistein at 4 mg/kg䡠d to neonatal rats stimulated germ cell development relative to control animals (17). Similarly, feeding infant marmosets soy-based formula resulted in increased testis size, serum T concentrations, and Leydig cell numbers in adult animals (18). However, other reports have suggested that phytoestrogens cause only modest effects on male reproductive activity (19). Despite disparities in results from different laboratories, the bulk of the data demonstrates that phytoestrogens possibly regulate male reproduction. Development and maintenance of the male phenotype is stimulated by T, which is produced almost exclusively by Leydig cells in the testis. The primary trophic factor regulating Leydig cells is LH secreted by the pituitary gland in response to stimulation by hypothalamic GnRH (reviewed in 20). However, Leydig cells are subject to regulation by estrogen because they express ERs, and administration of E2 suppresses Leydig cell regeneration in rats treated with the cytotoxin, ethane dimethylsulfonate (21). The levels of T in the blood and testis are dependent on two factors: the numbers of Leydig cells and T production rate per Leydig cell. It is not clear whether phytoestrogen-induced changes in serum T levels are related to differences in T production rates and/or Leydig cell numbers. Moreover, the perinatal period of reproductive tract development is a particularly sensitive window of exposure to exogenous estrogens or xenoestrogens (22). Therefore, the present study was designed to: 1) describe the relationship between T production and phytoestrogen concentrations in the blood and testis; and 2) characterize the effects of perinatal phytoestrogen exposure on Leydig cell differentiation as measured by T production. The results show that perinatal exposures of male rats to

phytoestrogens at levels relevant to real-world exposures affect Leydig cell function in adult rats, causing a decrease in testicular T concentrations at a low dose and an increase in serum and testicular T levels at the higher dose. Materials and Methods Animal studies Time-bred Long-Evans dams (n ⫽ 9), weighing approximately 250 g, were allowed to acclimate in the housing facility at Auburn University’s College of Veterinary Medicine Laboratory Animal Facility for 2 d before commencement of experiments. Each dam was housed individually in a standard plastic cage lined with wood chip bedding. Animals were maintained under constant conditions of light (12 h daylight, 12 h darkness) and temperature between 68 F and 74 F, with free access to pelleted food and water in glass bottles. Standard rodent diets typically contain as much as 100 –200 ppm genistein and higher amounts of daidzein (23), which have the potential to modulate endocrine responses in toxicological studies (24). For these reasons, diets containing casein (control) and whole soybean as sources of protein were used in the present study and taking care to ensure that diets were identical in micronutrients, cholesterol, calcium, and phosphorus (Tarlan-Heklad, Indianapolis, IN). The concentration of isoflavones in the experimental diets were 0, 5, 50, 500, or 1000 ppm based on the assayed content of genistein and daidzin and calculation of the equivalent aglycone as specified by the manufacturer. Pregnant dams were fed diets from gestational day (GD) 12 to postnatal day (PND) 21; this period of exposure does not exert toxicity on fetal development (25). Feed intake was similar for all groups of dams (data not shown), and the date of birth was designated PND 1. Animals from all groups were fed the soy-free control diet from PND 21–90. Therefore, male rats were exposed to different levels of phytoestrogens in the maternal diet only for the perinatal period (GD 12 to PND 21). Pregnancy outcome was assessed on PND 1: litter size, pup weight, and pup sex ratio. Body weights and anogenital distance (ANG) were measured in male rats on d 5 postpartum. To minimize within-group variations in treatment effect due to individual metabolic profiles of dams, male offspring were pooled together on PND 5 and reassigned to dams in the same group until weaning. Subsequently male rats were randomly selected from each group for analysis at 21 d of age: measurement of genistein and daidzein levels in the serum, liver, and testis and assessment of testicular steroidogenesis. Serum was separated from trunk blood collected at the time that animals were killed. Other rats were allowed to attain sexual maturity at 90 d when they were killed and analyzed for serum hormone concentrations (T, LH, and E2), epididymal and accessory sex organ weights, testicular and Leydig cell T production, and ER␣ gene expression in the prostate gland. The experimental protocols (Fig. 2A) and euthanasia procedures were approved by the Institutional Animal Care and Use Committee of Auburn University.

Determination of isoflavones in serum and tissues A validated analytical method based on reversed-phase HPLC with mass spectrometric detection was used to determine the concentration of genistein and daidzein in serum and tissue samples as previously described, with minor modifications (26). Calibration standards were made by adding both compounds to PBS at concentrations ranging from 2.5 to 100 ng/ml (9.3–370 nm genistein; 9.8 –393 nm daidzein). Frozen serum and tissue samples were thawed at ambient temperature. Each

Akingbemi et al. • Phytoestrogens Regulate Leydig Cells

Endocrinology, September 2007, 148(9):4475– 4488

A

ANIMAL STUDIES

Pregnant dams were fed diets containing soy isoflavones at 0, 5, 50, 500 or 1000 ppm from GD 12-PND 21

FIG. 2. Experimental protocol to investigate phytoestrogen regulation of testicular steroidogenesis. A, Animal studies. Time-bred LongEvans dams were maintained on soy-based diets from GD 12 to PND 21 (n ⫽ 9/group), whereas male offspring were fed soy-free diets from PND 21 to PND 90. Male rats were analyzed at 5 and 21 d of age and in adulthood at 90 d. B, In vitro analysis of genistein action in immature Leydig cells. I, Control untreated and genistein-treated Leydig cells were incubated in media containing a maximally stimulating dose of ovine LH (100 ng/ml) in the 3-h posttreatment period. II, Control untreated and genistein-treated Leydig cells were incubated in media containing steroid intermediates as T precursors in the 6-h posttreatment period. III, Leydig cells were incubated with the antiestrogen ICI 182,780 for 3 h before genistein treatment. For experiments I–III, Leydig cells were treated with genistein for a period of 24 h, and T production was assayed in aliquots of spent media by RIA. To investigate changes in gene expression, Leydig cells were harvested after genistein treatment and processed to obtain total RNA for analysis by RT-PCR and whole cell lysate for immunoblotting. IV, Leydig cells were cultured with and without genistein for 18 h followed by incubation in media containing [3H]thymidine in the 3-h posttreatment period.

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Weanling male rats were fed soy-free diets from PND 21–90

Measurements (PND 5):

Measurements (PND 21):

Measurements (PND 90):

- Pregnancy outcome

- Body and testis weight

- Reproductive organ weights

- Body and testis weight

- Tissue isoflavone levels (genistein, daidzein)

- Serum LH and E2 concentrations - Testicular steroidogenesis

- Anogenital distance - Testicular steroidogenesis

- Estrogen receptor (ERα) gene expression in prostate gland

B IN VITRO ANALYSIS OF GENISTEIN ACTION IN LEYDIG CELLS

(I)

Leydig cells incubated with genistein

Post-treatment incubation with LH (100 ng/ml)

3h

24 h

(II)

Leydig cells incubated with genistein

6h

24 h

(III)

Pretreatment incubation with Leydig cells incubated with genistein ICI 182,780

3h

(IV)

24 h

Leydig cells incubated with genistein 18 h

tissue sample was rinsed twice in ice-cold PBS, blotted on filter paper, and weighed. After adding ice-cold PBS at a volume equivalent to the tissue weight, assuming a density of 1.0 g/cm3, the tissue was homogenized using an Ultra-Turrax T8 disperser with an S8N-5G dispersing element (IKA Works, Inc., Wilmington, NC). The homogenate was sonicated for 5 min and subjected to three freeze-thaw cycles to completely lyse cells before removing an aliquot for analysis. Samples were prepared for analysis of the aglycone isoflavones by directly extracting a 100-␮l aliquot with tert-butyl methyl ether (3 ml) after adding the internal standard (10 ␮l of 1 ␮g/ml 7-hydroxyflavone in dimethylsulfoxide). To determine the total concentration of isoflavone glucuronides, 50 ␮l of each sample (serum, tissue homogenate, calibration standard) was mixed with 50 ␮l of a 10,000 U/ml suspension of ␤-glucuronidase (type B-1 from bovine liver; Sigma-Aldrich Co., St. Louis, MO) in 0.1 m ammonium acetate buffer (pH 5.0) and incubated for 2 h at 37 C before extraction. The organic phase was removed, the solvent evaporated, and the extract reconstituted with 150 ␮l of methanol-acetonitrile-10 mm ammonium formate [5:28:67 (vol/vol/vol)]. The sample solution (100 ␮l) was loaded onto a 15 cm ⫻ 4.6 mm Luna 5 ␮m

Post-treatment incubation with testosterone precursors

Post-treatment incubation with [3H] thymidine

3h

C18(2) HPLC column preceded by a 4 ⫻ 3 mm precolumn (Phenomenex, Torrance, CA). The column was eluted at ambient temperature using a ternary mobile phase, delivered at 1.0 ml/min, composed of methanolacetonitrile-10 mm ammonium formate at proportions that were changed from 5:28:67% at the beginning of the run to 10:56:34% linearly over 10 min. Flow from the analytical column was directed into the atmospheric pressure ionization-electrospray interface of an 1100 Series ion trap mass spectrometer (Agilent Technologies, Palo Alto, CA). Nitrogen was used as the nebulizing gas at 30 psi and as the drying gas at a flow rate of 12 liters/min and a temperature of 250 C. Operating parameters for the ion source and ion transfer optics were optimized for maximum response of the [M-H]⫺ ion for genistein (m/z 269). Negative ion detection was performed in the multiple reaction monitoring mode with a scan range of m/z 220 –275. Ions corresponding to the [M-H]⫺ ion of genistein, daidzein, and internal standard were isolated at m/z 269, 253 and 237, respectively, with a resolution of 1 m/z. Extracted ion chromatograms were integrated to provide peak areas. Study samples were assayed together with a series of seven calibration standards and three quality control samples of the analytes in PBS.

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Standard curves were constructed by plotting the analyte to internal standard chromatographic peak area ratio against the known analyte concentration in each calibration standard. Linear least squares regression was performed with weighting in proportion to the reciprocal of the analyte concentration normalized to the number of calibration standards. Values of the slope and y-intercept of the best-fit line were used to calculate the analyte concentration in study samples. The concentration of the glucuronide metabolite(s) of genistein and daidzein was calculated as the difference between the molar concentrations of the compound on analysis of the sample with and without enzymatic hydrolysis. The glucuronide is the predominant metabolite of isoflavones (⬎90%) and only small amounts of sulfate conjugates are produced in the rat (27). The analyte concentration in tissue homogenates was multiplied by a factor of two to provide the tissue concentration, expressed as nanograms per gram tissue weight, to account for the PBS added to prepare the homogenate. Genistein was determined with an interday accuracy of 95.3% and a precision of 14.9% at the lowest concentration included in the calibration curves (9.3 nm). At all other concentrations in the calibration curve, accuracy ranged from 95.9 to 108.4% and the precision was 8.3–16.6%. Accuracy and precision for measuring daidzein at a concentration of 9.8 nm were 91.0 and 15.2%, respectively. Daidzein was assayed with an accuracy of 91.9 –110.0% and a precision of 3.8 – 14.4% in the higher concentration calibration standards.

Isolation of Leydig cells Leydig cells were isolated from 21- and 90-d-old male offspring of time-bred dams that were maintained on soy-based diets in the perinatal period (GD 12 to PND 21). To demonstrate direct phytoestrogen action, Leydig cells were isolated from 35-d-old Long-Evans male rats for studies in vitro (Fig. 2B). All animals were obtained from Harlan-Teklad. The animals were euthanized by CO2 asphyxiation. Leydig cells were isolated from testis by a combination of collagenase digestion and Percoll density centrifugation according to a method described previously but excluding the elutriation step (28). After testis digestion and before Percoll density centrifugation, seminiferous tubules were removed by passage of testicular fractions through nylon mesh (Leydig cells isolated from 21 and 35 d old rats) or were sedimented using BSA (adult Leydig cells from 90 d old rats). Cell yields were estimated with a hemocytometer and purity was assessed by histochemical staining for 3␤-hydroxysteroid dehydrogenase (3␤-HSD) using 0.4 mm etiocholan-3␤-ol17-one as the enzyme substrate (29). Progenitor Leydig cells (at 21 d) were 90% enriched for cells that stain weakly for the marker enzyme 3␤-HSD, and immature (at 35 d) and adult Leydig cell fractions (at 90 d) were typically 95–97% enriched for cells that stain intensely.

Hormone measurements To measure T production rates, testicular explants (0.1– 0.15 g) and aliquots of Leydig cells (0.5 to 1 ⫻ 106) were incubated in microcentrifuge tubes. The culture medium consisted of DMEM/F-12 buffered with 14 mm NaHCO3 and 15 mm HEPES (Sigma Chemical Co., St. Louis, MO), and containing 0.1% BSA (MP Biomedicals LLC, Aurora, OH) and 0.5 mg/ml bovine lipoprotein (Perbio, Logan, UT). Incubations were conducted with a maximally stimulating dose of 100 ng/ml ovine LH [(National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)] at 34 C for 3 h. The concentration of T was assayed in serum samples and aliquots of spent media by a previously described tritium-based RIA with an interassay variation of 7.8%. The lower limit of detection for this assay is 0.01 ng/ml (30). Although T is the primary androgen stimulating reproductive tract development, androsterone (AO) is the predominant steroid (⬃75%) secreted by rat Leydig cells at 21 d of age (31). Therefore, serum levels of AO and the rate of AO secretion by Leydig cells were measured in male rats at d 21 postpartum. Serum T and AO levels were calculated as ng/ml, and the rates of Leydig cell T and AO secretion were normalized to nanograms per 106 cells. Testicular steroidogenesis is stimulated by the pituitary hormone LH to maintain androgen levels in blood, and Leydig cells are the only binding sites for LH in the testis (32). Therefore, LH concentrations in the serum of 21- and 90-d-old male rats were measured using 125I rat LH, primary antibody (R15), and LH reference standards (NIDDK rLF-RP-3, National Hormone and Pituitary Program, NIDDK) and a secondary

Akingbemi et al. • Phytoestrogens Regulate Leydig Cells

immunoglobin G (A840, Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO). Each sample was assayed in duplicate. The lower limit of detection for this assay is 0.22 ng/ml, and LH values are expressed in relation to the RP-3 standards. Moreover, adult Leydig cells are the primary source of E2 in the rat and E2 is known to act as an autocrine regulator of Leydig cells (33). Conversion of androgen to estrogen in all tissues is catalyzed by the aromatase enzyme (34), and it has been suggested that phytoestrogens act as aromatase inhibitors to decrease E2 secretion (35). Therefore, the serum concentrations of E2 were measured in adult rats at 90 d of age by RIA using tritium-labeled E2 (NET 317; PerkinElmer Life and Analytical Sciences, Boston, MA) and antiestradiol-6-BSA (no. 244; Animal Reproduction and Biotechnology Laboratory, Colorado State University). This assay has an interassay variation of 8.5%, and the lower limit of detection is 0.005 ng/ml.

Analysis of ER␣ gene expression in the prostate gland in adult male rats Male accessory sex organs are subject to regulation by estrogen independently of changes in serum androgen or T levels, and there is evidence showing that ER␣ is the dominant ER subtype mediating developmental estrogenization of the prostate gland (36). Thus, ER␣ gene expression was analyzed in tissue from the ventral lobes of the prostate gland in the 0, 5, and 1000 ppm diet groups at 90 d of age. The 0, 5, and 1000 ppm diet groups were chosen to assess the dose-dependency of phytoestrogen action.

Analysis of genistein action in Leydig cells Experiments were performed to dissociate effects exerted on the pituitary gland from action occurring in the testis and thereby demonstrate that phytoestrogens have the ability to act directly in Leydig cells (Fig. 2B). Genistein (Indofine Chemical Co., Inc., Hillsborough, NJ) was used in these studies because of the two major isoflavones present in soybeans; the estrogenic properties of genistein have been the most characterized (37). Leydig cells, isolated from 35-d-old male rats, were used in these assays because they represent the intermediate stage of development and retain significant proliferative and steroidogenic capacity, which define Leydig cell differentiation. The culture medium consisted of DMEM/F-12 buffered with 14 mm NaHCO3 and 15 mm HEPES (Sigma) and containing 0.1% BSA (MP Biomedicals) and 0.5 mg/ml bovine lipoprotein (Perbio). Leydig cells were cultured in 6-well plates (0.5 to 1.0 ⫻ 106 cells/well) for an initial period of 1 h to stabilize and then in media containing genistein at 37 C and 5% CO2 under humidified conditions. Also, genistein treatment of Leydig cells was for a period of 24 h in all experiments designed to investigate steroid secretion in vitro. Genistein was dissolved in dimethyl sulfoxide (DMSO), which was also added to control untreated cultures (0.1%). In pilot experiments, Leydig cells were incubated with varying doses of genistein (10⫺12 to 106 m) in media containing ovine LH (10 ng/ml). Two doses of genistein (0.1 nm and 10 ␮m) were used in subsequent experiments, representing low and high doses, respectively. The viability of Leydig cells after treatment with genistein at the two doses was assessed by the trypan blue exclusion test and staining for the 3␤-HSD enzyme and did not differ from controls. Sensitivity to LH stimulation was assessed by incubation of untreated control and genistein-treated Leydig cells in microcentrifuge tubes containing media plus a maximally stimulating dose of 100 ng/ml ovine LH (38) in the 3-h posttreatment period and shaking in a water bath at 34 C. To identify the site(s) of lesion in the androgen biosynthetic pathway, untreated control and Leydig cells that were previously treated with genistein were incubated for 6 h in media containing steroid intermediates as T precursors: 5 ␮m hydroxycholesterol (22R-CHO) or 20 ␮m of pregnenolone, progesterone, and androstenedione. These steroid substrates are known to diffuse readily into Leydig cells and are used at high doses to determine the capacity of their respective enzymes (39): cytochrome P450 cholesterol side-chain cleavage [(22R-CHO), 3␤-HSD (pregnenolone), cytochrome P450 17␣-hydroxylase/17–20 lyase (progesterone), and 17␤-hydroxysteroid dehydrogenase (androstenedione)]. Pilot studies confirmed that the concentrations of steroids used were substrate saturating for these enzymes in immature Leydig cells. The transfer of cholesterol from outer to inner mitochondrial membrane is facilitated by steroidogenic acute

Akingbemi et al. • Phytoestrogens Regulate Leydig Cells

regulatory protein (StAR) in concert with the mitochondrial translocator protein (TSPO; previously known as peripheral benzodiazepine receptor) (40, 41). Incubation with 22R-CHO, which diffuses readily within Leydig cells not requiring facilitated transport across mitochondrial membranes, restored T secretion by genistein-treated Leydig cells. Therefore, we investigated the possibility that genistein interferes with cholesterol transfer into mitochondria by analysis of TSPO and StAR gene expression, i.e. measurement of mRNA and protein levels by RTPCR and immunoblot analysis, respectively. Furthermore, we asked whether genistein action was ER-mediated by incubation of Leydig cells with the pure antiestrogen ICI 182,780 ([7␣[9-(4,4,5,5,-pentafluoropentylsulfinyl)estra-1,3,5(10)-triene-3,17␤-diol; Tocris Cookson, Ellisville, MO) at 100 nm for 3 h before incubation with genistein. The ICI 182,780 compound is known to block transcriptional activity via ER␣ and ER␤ (42). Developing Leydig cells are mitotically active, and excessive exposure to agents with estrogenic activity induces proliferation of estrogensensitive tissues (43). Thus, experiments were performed to assess the potential for genistein to induce proliferative activity in immature Leydig cells. Leydig cells, isolated from 35-d-old male rats, were incubated in DMEM/F-12 (Sigma) containing LH (10 ng/ml) and genistein (0, 0.01, 0.1, 1.0 ␮m) for 18 h in 6-well plates (0.5 ⫻ 106 cells per well) at 37 C and 5% CO2 under humidified conditions. Dose selection was based on isoflavone levels that have been reported in human subjects (4, 8) and are comparable with the concentrations of genistein measured in the present study. After treatment, media were replaced with that containing 1 ␮Ci/ml [3H]thymidine (1 Ci ⫽ 37 GBq, specific activity, 104.7 Ci/mmol; DuPont-NEN Life Science Products, Boston, MA) for 3 h. Cells were then rinsed in Dulbecco’s PBS and harvested, counted, and divided into aliquots of 0.3– 0.5 ⫻ 106 cells in microcentrifuge tubes. Cells were lysed in 0.5 ml of hyamine hydroxide (ICN Radiochemicals, Irvine, CA), and incorporation of [3H]thymidine was quantified by liquid scintillation counting.

Total RNA extraction and semiquantitative RT-PCR Tissue was obtained from the ventral lobes of the prostate gland of adult rats in the 0, 5, and 1000 ppm diet groups at 90 d of age. Also, Leydig cells were harvested after genistein treatment and after a 5-min incubation in a solution of 0.05% collagenase and 0.05% dispase in media 199 buffered with 8.45 mm NaHCO3 and 8.8 mm HEPES, containing 0.1% BSA and 0.0025% trypsin inhibitor (pH 7.1–7.2) (Sigma). Total cellular RNA was extracted from tissues by the isothiocyanate-acid phenolchloroform method using Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH), and was quantified by measuring absorbance at 260 nm and stored at ⫺80 C until assayed. Oligonucleotides used for RT-PCR were based on published sequences and were selected from different exons of the corresponding genes to discriminate PCR products that possibly arise from chromosomal DNA contamination. Primers with the following sequences were sourced from Invitrogen Life Technologies (Carlsbad, CA): ER␣ (44), 5⬘-gctccaattctgacaatcgac-3⬘ (forward) and 5⬘tttcgtatcccgcctttcatc-3⬘ (reverse); ribosomal S16 (45), 5⬘aagtcttcggacgcaagaaa-3⬘ (forward) and 5⬘-gacaagacgaagacccgtt-3⬘ (reverse), 150 bp; TSPO (46): 5⬘aagagctgggaggtttcaca-3⬘ (forward) and 5⬘-ccaggccaggtaaggataca-3⬘ (reverse) (224 bp); StAR (47), 5⬘-ttgggcatactcaacaaccca-3⬘ (forward) and 5⬘-atgacaccgctttgctcag-3⬘ (reverse), 389 bp; ␤-actin (46), 5⬘caccatgtacccaggcatcgc-3⬘ (forward) and 5⬘-aggggccggactcatcgtac-3⬘ (reverse), 194 bp. First-strand cDNA was generated using 2 ␮g RNA and random hexamers (2 ng/␮l), RNAsin (1 U/␮l) avian myeloblastosis virus reverse transcriptase (200 U), and deoxynucleotide triphosphate (0.5 mm) at 37 C for 60 min. The reaction was stopped by heating at 95 C for 5 min. Target cDNAs were amplified in PCR Master Mix (Promega, Madison, WI) in 50-␮l volumes and using a PCR protocol as follows: initial denaturation at 94 C for 4 min, followed by a variable number of cycles of amplification as determined in pilot studies and defined by denaturation at 95 C for 1 min, annealing at 58 C or 60 C for 1 min, and extension at 72 C for 1 min. A final extension at 72 C for 10 min was included. Analogous assessments of ribosomal S16 or ␤-actin gene expression served as internal controls. PCR products were size fractionated through a 1% agarose gel followed by staining with ethidium bromide. The intensity of UV light-generated band fluorescence in each sample was analyzed using Doc-lt LS software (Ultra-Violet Products Ltd., Upland, CA).

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Protein extraction and Western blotting Tissue from the ventral lobes of the prostate gland in adult male rats (PND 90) from the 0, 5, and 1000 ppm diet groups and Leydig cells previously treated with genistein in vitro were homogenized in M-PER lysis buffer freshly supplemented with a protease inhibitor cocktail, HALT (Pierce Chemical Co., Rockford, IL). Sample protein concentrations were determined using the Bradford assay and BSA as standards (Bio-Rad Laboratories, Hercules, CA). Aliquots of whole-cell lysate (10 ␮g) were dissolved in Laemmli sample buffer containing 5% ␤-mercaptoethanol and boiled for 5 min at 95 C. The samples were applied to 10% glycine-SDS-PAGE gels in a Mini-Protean System (Bio-Rad Laboratories) and transferred by electrophoresis to nitrocellulose membranes (0.45 ␮m; Bio-Rad). To reduce nonspecific protein binding to nitrocellulose, membranes were preincubated for 60 min at room temperature in blocking buffer [5% whole milk in Tris-buffered saline containing 0.1% Tween 20 (TBST)]. The nitrocellulose blots were incubated overnight at 4 C with antibody against ER␣ (sc-7207; Santa Cruz Biotechnology, Santa Cruz, CA), TSPO, StAR, active or phosphorylated (p)StAR, and ␤-actin (A-2228; Sigma) at a dilution of 1:1000, 1:2000, 1:2000, 1:5000, and 1:2000, respectively. The antibodies for TSPO, StAR, and p-StAR were generously provided by Dr. Vassilios Papadopoulos (Georgetown University Medical Center, Washington, DC), Dr. Douglas Stocco (Texas Tech University Health Sciences Center, Lubbock TX), and Dr. Steve King (Baylor College of Medicine, Houston, TX), respectively. Blots were washed in TBST three times for 5 min each time and incubated with horseradish peroxidase-conjugated goat antirabbit IgG (sc-2004; Santa Cruz Biotechnology) at a dilution of 1:2000 in TBST for 90 min. Subsequently blots were rinsed and exposed (1 min) to a chemiluminescent horseradish peroxidase antibody detection reagent (HyGLO; Denville Scientific Inc., Metuchen, NJ) followed by exposure to autoradiography films. The films (Danville Scientific Inc., Metuchen, NJ) were scanned using the Epson 4180 Perfection scanning software (Epson-America, Long Beach, CA). The relative protein amounts in identified immunoblots were estimated by measuring the ODs of the bands on exposed Autorad films using Doc-lt LS software (Ultra-Violet Products Ltd., Upland, CA).

Statistical analysis Data are described as mean ⫾ sd. Data were analyzed by one-way ANOVA followed by Dunnett’s test for multiple group comparisons (GraphPad, Inc., San Diego, CA). Differences of P ⬍ 0.05 were considered significant.

Results General observations

Feeding soy-based diets to pregnant dams did not affect litter size or the pup weights and sex ratios of offspring (Table 1). However, male rats obtained from dams fed the 5 and 50 ppm diets were heavier on d 5 postpartum than control (P ⬍ 0.05). Also, the ANG was greater in 5-d-old male rats from the 5, 50, and 500 ppm diet groups (P ⬍ 0.05); however, differences were not apparent when adjusted for body weight (P ⬎ 0.05). All male rats except the 500 ppm diet group exposed to phytoestrogens in the maternal diet were heavier than control at 21 d of age (P ⬍ 0.05), but these differences were no longer apparent at 90 d (P ⬎ 0.05). Also, testis weight was increased in 21-d-old male rats from all treatment groups, compared with control (P ⬍ 0.05); this effect was significant only in animals from the 50, 500, and 1000 ppm diet groups after adjusting for body weight (P ⬍ 0.05). At 90 d, there were no differences in body weights between male rats from all groups, but absolute testis weight was increased in the 50, 500, and 1000 ppm diet groups (P ⬍ 0.05) and was significantly lower in the 50 ppm diet group when corrected for body weight (P ⬍ 0.05). However, ex-

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TABLE 1. Pregnancy outcome and reproductive organ parameters in male ratsa Isoflavone levels in the maternal diet (ppm)

Litter size (number of pups) Pup weight (g) Pup sex ratio (male:female) Body weights (g, PND 5) Anogenital distance (mm, PND 5)c Relative anogenital distance, PND 5 (%)d Body weights (g, PND 21) Paired testis weight (g, PND 21)c Relative testis weight, PND 21 (%)d Body weight, PND 90 (g) Paired testis weight, PND 90 (g)c Relative testis weight, PND 90 (%)d

0

5

50

500

1000

12.1 ⫾ 2.9 5.76 ⫾ 1.2 1.1 ⫾ 0.7 22.5 ⫾ 3.4 7.28 ⫾ 0.6 0.33 ⫾ 0.05 50.8 ⫾ 6.9 0.23 ⫾ 0.04 4.62 ⫾ 0.6 431 ⫾ 4 3.75 ⫾ 0.7 8.7 ⫾ 1.5

10.8 ⫾ 2.5 6.72 ⫾ 1.7 1.2 ⫾ 0.6 25.7 ⫾ 2.5b 7.9 ⫾ 0.7b 0.31 ⫾ 0.02 59.2 ⫾ 5b 0.29 ⫾ 0.05b 4.97 ⫾ 0.7 418.6 ⫾ 4 3.39 ⫾ 0.3 8.2 ⫾ 1.0

9.1 ⫾ 2.6 7.63 ⫾ 3.3 1.2 ⫾ 0.9 28.6 ⫾ 2.5b 8.6 ⫾ 0.8b 0.30 ⫾ 0.02 63.9 ⫾ 4b 0.33 ⫾ 0.04b 5.11 ⫾ 0.5b 431 ⫾ 5 3.24 ⫾ 0.3b 7.6 ⫾ 0.7b

9.9 ⫾ 4.3 6.30 ⫾ 0.9 0.8 ⫾ 0.5 24.6 ⫾ 4.7 7.9 ⫾ 1.0b 0.32 ⫾ 0.03 54.9 ⫾ 10 0.30 ⫾ 0.06b 5.49 ⫾ 0.3b 417.9 ⫾ 4 3.28 ⫾ 0.2b 7.9 ⫾ 0.6

10.6 ⫾ 2 5.69 ⫾ 0.4 1.4 ⫾ 1.1 22.6 ⫾ 1.5 7.4 ⫾ 0.5 0.33 ⫾ 0.02 56.3 ⫾ 4.6b 0.32 ⫾ 0.03b 5.63 ⫾ 0.4b 411.5 ⫾ 4 3.22 ⫾ 0.3b 7.9 ⫾ 0.9

a Pregnant dams were fed soy-based diets from GD 12 to PND 21. Male offspring were fed soy-free diets from 21 to 90 d of age (n ⱖ 10). Data are expressed as mean ⫾ SD. b P ⬍ 0.05 vs. control. c Before adjustment for body weight. d Adjusted for body weight.

posure of male rats to isoflavones as present in the maternal diet during the perinatal period did not affect weights of the epididymis, dorsolateral and ventral prostate, bulbourethral glands, and seminal vesicles (including coagulating glands) in adult rats at 90 d of age (data not shown). Isoflavones in the maternal diet are present in male offspring

The serum levels of unconjugated genistein and daidzein and glucuronide metabolites in dams fed diets containing different levels of isoflavones and male offspring at 21 d of age are shown in Table 2. There were large individual variations in serum levels of genistein and daidzein (unconjugated or glucuronide metabolite) within groups, whereas liver and testis concentrations were less variable (Fig. 3). As expected, the concentrations of genistein and daidzein glucuronides were severalfold

higher than the unconjugated aglycone compounds. For example, in male rats from dams maintained on the diet containing the highest amount of phytoestrogens (1000 ppm), the levels of conjugated genistein were about 25 times greater than the free aglycone (⬃18-fold difference for daidzein). The levels of unconjugated genistein and daidzein in the serum of 21-d-old male rats were below assay detection limits in the 5 ppm diet group, i.e. 9.3 and 9.8 nm, respectively. Moreover, the concentrations of unconjugated genistein and daidzein in the liver were about 10-fold higher than their levels in the testis, and higher daidzein levels than genistein were detected in both tissues (Fig. 3). Altogether, these observations imply that phytoestrogens in the maternal diet have the capacity to cross maternal tissue barriers to reach the fetus and neonate. The use of casein, a dairy protein that may contain trace amounts of isoflavones, is probably responsible for

TABLE 2. Serum concentrations (nanomoles) of free and conjugated isoflavones in male rats and dams at d 21 postpartuma Isoflavone levels in the maternal diet (ppm)

Male rats Parameter

Dams

Genistein Free

0

No. of samples Mean conc.

0/10 n.d.

5

No. of samples Mean conc.

0/8 n.d.

50

No. of samples Mean conc.

500

No. of samples Mean conc.

1000

No. of samples Mean conc.

5/10 3.8 (5.7) 10/10 45.2 (19) 9/9 72.7 (46)

Daidzein

Conb

3/10 2.1c (2.7)d 8/8 17.1 (8.9) 10/10 156 (117) 10/10 1291 (611) 9/9 1900 (982)

Free

1/10 n.d. 0/8 n.d. 3/10 2.5 (3.5) 10/10 65.6 (23) 9/9 106 (53)

Genistein

Daidzein

Con-

Free

Con-

1/10 1.3 (1.1) 6/8 9.6 (15.4) 10/11 146 (112) 10/10 1087 (352) 9/9 1912 (965)

0/9 n.d.

0/9 n.d.

0/9 n.d.

0/9 n.d.

0/6 n.d.

1/6 1.5 (1.5) 7/9 19.4 (41) 7/7 1014 (448) 8/8 1695 (818)

0/6 n.d.

1/6 1.6 (1.6) 6/9 13.8 (30.3) 7/7 1113 (423) 8/8 1422 (707)

0/9 n.d. 7/7 22.8 (18.8) 8/8 17.3 (26.7)

Free

0/9 n.d. 7/7 60.6 (33.8) 8/8 55.8 (29.8)

Con-

n.d., Undetectable; Free, aglycone; Con-, glucuronide metabolite. a Dams were fed soy-based diets from GD 12 to PND 21, and serum was obtained from male offspring and dams at d 21 postpartum. b Number of samples with a measurable analyte concentration per total number of samples. c Samples with an unmeasurable analyte concentration (conc.) were assigned a value of 0.1 times the lower limit of quantitation for calculating the geometric mean. d Numbers in parentheses, SD.

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FIG. 3. Unconjugated genistein and daidzein concentrations in the liver and testis in prepubertal male rats exposed to phytoestrogens in the perinatal period. Time-bred Long-Evans dams were maintained on soy-based diets from GD 12 to d 21 postpartum. Isoflavone levels in tissue from the liver and testis, obtained from male rats in each diet group at weaning on d 21 postpartum, were analyzed using a validated analytical method based on reverse-phase HPLC with mass spectrometric detection (n ⫽ 8 –11/group). Study samples were assayed together with a series of seven calibration standards and three quality control samples of the analytes in PBS. Genistein and daidzein concentrations in the liver are described in the upper panels (A and B), and levels in the testis are shown in the lower panels (C and D). *, P ⬍ 0.05 vs. control.

the low concentrations of conjugated isoflavones measured in a number of male rats from the control diet group (Table 1) (48). Perinatal exposure to phytoestrogens affects steroid secretion by Leydig cells in prepubertal male rats

Compared with control, serum T levels were elevated in 21-d-old male rats exposed to the 5 ppm diet (P ⬍ 0.05; Fig. 4A), presumably due to increased Leydig cell T production (P ⬍ 0.05; Fig. 4B). Serum T concentrations were decreased, whereas serum AO levels were elevated in animals from the 500 and 1000 ppm diet groups (P ⬍ 0.05; Fig. 4, A and C). However, the rates of AO secretion by Leydig cells were decreased in the 50, 500, and 1000 ppm diet groups (P ⬍ 0.05; Fig. 4D). Interestingly, serum T and AO levels were unchanged in the 50 ppm diet group.

Exposures to low and high phytoestrogen doses in the perinatal period cause opposite effects on testicular T production in adult male rats

Serum T concentrations and testicular and Leydig cell T production measured at 90 d of age are shown in Fig. 5. Adult male rats from dams fed the 5 ppm diet showed reduced testicular T production (P ⬍ 0.05; Fig. 5B), although these were not reflected in serum T levels (Fig. 5A). It is possible that suppression of androgen biosynthesis was not profound enough to lower serum T levels or that changes in serum T levels were masked by the pulsatile and circadian rhythm of androgen secretion in vivo. In contrast, animals from the 1000 ppm diet group exhibited elevated serum T levels and increased testicular T secretion (P ⬍ 0.05; Fig. 5, A and B), although T production per Leydig cell was decreased (P ⬍ 0.05; Fig. 5C). The 50 ppm diet did not affect serum T concentrations and Leydig cell T production.

Serum LH and E2 concentrations in adult male rats

Serum LH concentrations were similar in male rats from all groups at 21 d of age (data not shown) and were increased only in the 50 ppm diet group at 90 d (P ⬍ 0.05; Table 3). On the other hand, perinatal exposures to phytoestrogens at levels achieved in the present study did not affect Leydig cell aromatase activity in adult rats because serum E2 levels were similar in all groups at 90 d (Table 3).

Phytoestrogens regulate ER␣ gene expression in the prostate gland of adult male rats

Feeding pregnant dams soy-based diets containing 5 and 1000 ppm soy isoflavones caused a decrease in ER␣ mRNA levels in the adult ventral prostate at 90 d of age (P ⬍ 0.05; Fig. 6A). Modulation of gene expression was confirmed by

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FIG. 4. Effect of exposure to phytoestrogens in the perinatal period on steroid hormone production in prepubertal male rats. Time-bred Long-Evans dams were maintained on soy-based diets from GD 12 to PND 21. Serum was separated from blood collected from nine rats per group at weaning (PND 21). Testes for Leydig cell isolation were pooled from at least 30 rats per group. Isolated Leydig cells were incubated in triplicate in microcentrifuge tubes containing serum-free DMEM-Ham’s F-12 medium in the presence of ovine LH (100 ng/ml) for 3 h. The concentrations of T and AO were measured in aliquots of serum and spent media by RIA. Serum T levels and T secretion by Leydig cells are shown in the upper panels (A and B), and serum AO concentrations and AO secretion by Leydig cells are described in the lower panels (C and D). *, P ⬍ 0.05 vs. control.

immunoblot analysis, demonstrating a decrease in ER␣ protein (P ⬍ 0.05; Fig. 6B). Genistein acts directly in Leydig cells

When tested over a wide dose range, genistein caused a biphasic decrease in T secretion by Leydig cells (Fig. 7A), presumably associated with a decrease in Leydig cell sensitivity to LH stimulation (P ⬍ 0.05; Fig. 7B). Prior treatment with the antiestrogen ICI 182,780 ameliorated genistein inhibition at the low dose (0.1 nm) and only partially at the high dose (10 ␮m; Fig. 7C), implying that genistein action is mediated, at least in part, via ERs. When incubated with cholesterol and steroid intermediates used in T biosynthesis, untreated control and genistein-treated Leydig cells produced similar amounts of T, meaning that genistein did not affect inherent steroidogenic

enzyme capacity at doses tested in the present study (Table 4). The results of proliferation assays showed that genistein caused an increase in [3H]thymidine incorporation by immature Leydig cells (P ⬍ 0.05; Fig. 7D), indicating that genistein has the ability to regulate Leydig cell division. Expression of TSPO and StAR is LH dependent, and TSPO and StAR associate directly with cholesterol to move it across mitochondrial membranes within Leydig cells. Thus, diminished Leydig cell sensitivity to LH stimulation (Fig. 7B), expectedly, had the effect of decreasing TSPO and StAR mRNA levels (P ⬍ 0.05; Fig. 8, A and B). However, greater StAR protein was measured in genistein-treated Leydig cells (P ⬍ 0.05), whereas the amounts of TSPO protein were unchanged (Fig. 8, C–E). Paradoxically, greater StAR protein was associated with a decrease in phosphorylation, i.e. syn-

TABLE 3. Serum luteinizing hormone and 17␤-estradiol levels (nanograms per milliliter) in adult male ratsa Isoflavone levels in the maternal diet (ppm)

LH E2

0

5

50

0.51 ⫾ 0.33 0.13 ⫾ 0.03

0.75 ⫾ 0.32 0.11 ⫾ 0.01

1.07 ⫾ 0.47 0.15 ⫾ 0.02

b

500

1000

0.55 ⫾ 0.11 0.15 ⫾ 0.02

0.88 ⫾ 0.48 0.14 ⫾ 0.01

a Pregnant dams were fed soy-based diets from GD 12 to d 21 postpartum, and male offspring were fed soy-free diets from 21 to 90 d of age. Blood was collected to obtain serum at 90 d (n ⫽ 9). Hormone concentrations were measured by RIA and data are expressed as mean ⫾ SD. b P ⬍ 0.05 vs. control.

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FIG. 6. Effect of exposure to phytoestrogens in the perinatal period on ER␣ gene expression in prostate gland of adult male rats. Timebred Long-Evans dams were maintained on soy-based diets from GD 12 to d 21 postpartum, and male offspring were fed the control soy-free diet from 21 to 90 d of age. Tissue was obtained from the ventral lobes of the prostate gland from four rats in the 0, 5, and 1000 ppm diet groups at 90 d. ER␣ mRNA levels were analyzed by RT-PCR and densitometry, and proteins were separated by SDS-PAGE and identified by immunoblot analysis. The left panel is representative of the ratio of ER␣ mRNA to ribosomal S16 as internal control (A). The right panel shows ER␣ protein normalized to Ponceau-stained protein bands (B). *, P ⬍ 0.05 vs. control.

Discussion

FIG. 5. Effect of exposure to phytoestrogens in the perinatal period on testicular steroidogenesis in adult male rats. Time-bred LongEvans dams were maintained on soy-based diets from GD 12 to d 21 postpartum, and male offspring were fed the control soy-free diet from 21 to 90 d of age. Blood and testicular explants were obtained from nine rats per group, and testes for Leydig cell isolation were pooled from at least eight rats per group at 90 d of age. Testicular explants and isolated Leydig cells were incubated in triplicate in microcentrifuge tubes containing serum-free DMEM-Ham’s F-12 medium in the presence of ovine LH (100 ng/ml) for 3 h. The concentration of T was measured in aliquots of serum and spent media by RIA. Serum T concentrations are shown in the upper panel (A) and testicular and Leydig cell T secretion are described in the middle and lower panels (B and C). *, P ⬍ 0.05 vs. control.

thesis of functional StAR (p-StAR), at the 0.1 nm dose (P ⬍ 0.05; Fig. 8, C and F), which presumably is responsible for decreased T secretion at this dose. On the other hand, the amount of p-StAR at the 10 ␮m dose was similar to untreated controls and higher than at the 0.1 nm dose (Fig. 8, E and F), implying that other factors, besides cholesterol availability, are involved in genistein inhibition of steroidogenesis in Leydig cells at higher doses.

Maintenance of pregnant dams on soy-based diets as used in the present study did not affect pregnancy outcome. However, the data indicate that exposure to phytoestrogens in the perinatal period modulates androgen biosynthesis in the adult rat testis. This finding has public health relevance because the levels of genistein and daidzein in the blood of male offspring exposed to phytoestrogens (Table 2) compare favorably with levels that were measured in the fetuses of mothers eating Western diets (49) and infants fed soy-based formulas (8). Perinatal exposure to phytoestrogens also altered ER␣ gene expression in the ventral lobes of the prostate gland in adult rats, validating our observations on testicular steroidogenesis and demonstrating that the blood levels of genistein and daidzein aglycone achieved in the present study were biologically active. The finding of higher concentrations of genistein and daidzein and glucuronide metabolites in the serum of male rats than their dams agree with previous reports in rodents and humans (50, 51). These levels are presumably due to continuous exposure of fetuses to compounds in the amniotic fluid, less binding of xenoestrogens to serum proteins, and a decrease in the activity of xenobiotic enzymes (e.g. uridine diphosphate glucuronyltransferase) plus reduced metabolic clearance of isoflavones and metabolites in the fetus and neonate, compared with the adult (52). Equol, a metabolic product of daidzein and an isoflavan, is produced in large quantities in rodents and monkeys and in about 30% of humans who consume soybased products (53). The tissue levels of equol were not

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FIG. 7. Effect of genistein treatment on androgen biosynthesis. Immature Leydig cells were isolated from 35-d-old Long-Evans male rats. A, Initial experiments involved incubation of Leydig cells with a range of doses of genistein (10⫺12 to 105 M) in DMEM-Ham’s F-12 medium containing ovine LH (10 ng/ml). At the end of treatment, Leydig cell T secretion was measured in aliquots of spent media by RIA. Results shown are means ⫾ SD from four independent experiments, each conducted in triplicate. B, Control and genistein-treated Leydig cells were harvested, counted, and incubated further in media containing a maximally stimulating dose of ovine LH (100 ng/ml) for 3 h. The concentrations of T in aliquots of spent media were measured by RIA. Results shown are means ⫾ SD from four independent experiments, each conducted in triplicate. C, Leydig cells were incubated first in media containing ovine LH (10 ng/ml) and the antiestrogen ICI 182,780 (100 nM) for 3 h followed by incubation in media containing LH (10 ng/ml) and genistein. At the end of treatment, the rates of T secretion were measured in aliquots of spent media by RIA, and the results represent means ⫾ SD from four independent experiments conducted in triplicate. Leydig cells were cultured in media plus and minus genistein for a period of 24 h (A–C). D, After Leydig cells were incubated in media containing ovine LH (10 ng/ml) plus genistein for 18 h, incubation was continued in media containing LH (10 ng/ml) plus 1 ␮Ci/ml [3H]thymidine for 3 h. At the end of the 3-h period, Leydig cells were harvested and analyzed for [3H]thymidine incorporation by liquid scintillation counting. Data represent the results of three independent experiments each conducted in triplicate. CON, Control; ICI, ICI 182,780; Gen, genistein. ⫽, P ⬍ 0.05 vs. genistein-treated groups; *, P ⬍ 0.05 vs. control.

measured in the present study, and it is not clear how much of equol was produced from daidzein. Reports in the literature indicate that phytoestrogens suppress androgen biosynthesis (13) or cause an increase in serum T levels (18, 54). Therefore, a major objective of the present study was to define the relationship between serum and testicular T production and steroidogenic capacity per

individual Leydig cell. Feeding pregnant dams with a low phytoestrogen diet (5 ppm) stimulated Leydig cell T production in prepubertal male rats, whereas the 500 and 1000 ppm diets caused a decrease in serum T and an increase in AO levels. The increase in Leydig cell T secretion may be the result of enhanced sensitivity to LH stimulation, although this hypothesis will need to be tested in follow-up experi-

TABLE 4. Leydig cell T secretion by Leydig cells after genistein treatmenta

LH (10 ng/ml) 22R-hydroxycholesterol (5 ␮M) Pregnenolone (20 ␮M) Progesterone (20 ␮M) Androstenedione (20 ␮M)

Control

0.1 nM

10 ␮M

190 ⫾ 14 480 ⫾ 60 880 ⫾ 115 950 ⫾ 112 2800 ⫾ 210

121 ⫾ 11b 420 ⫾ 55 790 ⫾ 105 930 ⫾ 120 2500 ⫾ 250

94 ⫾ 19b 490 ⫾ 52 940 ⫾ 90 1150 ⫾ 140 3200 ⫾ 180

a Immature Leydig cells were incubated in media in the absence and presence of genistein for 24 h and then in media plus or minus steroid intermediates as T precursors in the 6-h posttreatment period; T secretion was measured by RIA. Data are expressed as mean ⫾ SD from four independent experiments, each conducted in triplicate. b P ⬍ 0.05 vs. control.

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FIG. 8. Effect of genistein treatment on TSPO and StAR gene expression. Control untreated and genistein-treated Leydig cells (0.1 nM, 10 ␮M, 24 h) were harvested and processed to obtain total RNA and whole-cell lysate. TSPO and StAR mRNA levels were analyzed by RT-PCR followed by densitometry. TSPO, StAR, p-StAR, and ␤-actin protein were identified by immunoblot analysis after separation by SDS-PAGE. A and B, Ratios of TSPO and StAR mRNA to ␤-actin as internal control. C, Representative immunoblots for TSPO, StAR, p-StAR, and ␤-actin. D and E, Ratios of TSPO and StAR to ␤-actin. F, Ratio of total StAR to p-StAR. Data were collected from four independent experiments each conducted in triplicate. *, P ⬍ 0.05 vs. control.

ments. On the other hand, increased serum AO levels were not related to enhanced AO secretion by Leydig cells. Furthermore, there were large within-group variations in serum LH levels measured at d 21 postpartum (our unpublished observations), and it is not clear whether changes in LH stimulation contribute to differences in Leydig cell steroidogenesis in the prepubertal period. Interestingly, increased Leydig cell T production by the 5 ppm diet may explain, at least in part, previous observations that exposures to low genistein doses stimulate spermatogenesis in prepubertal male rats as well as alleviate disturbances of spermatogenesis in mice deficient in E2 production (17, 55). Pregnant and nursing dams fed the low phytoestrogen diet (5 ppm) resulted in decreased testicular T production in adult male rats at 90 d of age, whereas the 1000 ppm diet caused an increase in serum T levels and testicular T secretion, which, paradoxically, was associated with a decrease in the rates of Leydig cell T production. Serum LH levels were elevated only in the 50 ppm diet group at 90 d, and it is possible that the absence of phytoestrogen effect on T secretion in this group is related to increased serum LH levels, which potentially ameliorates deficits in T se-

cretion. Moreover, it has been suggested that changes in steroid metabolism and homeostasis play a role in the toxicity of EDs and confound measurements of blood steroid hormone levels (56). Hepatic steroid metabolizing enzyme activity was not evaluated in the present study, and it remains unclear whether impaired ability of the liver to metabolize steroids contributed to differences in serum T levels. However, male rats were not exposed to isoflavones in the period from d 21 postpartum, and the levels of genistein and daidzein measured in the livers of prepubertal rats at 21 d of age were probably the highest levels achieved in the present study. If that is true, it is perhaps reasonable to speculate that isoflavone levels in the liver (and perhaps other tissues) will decline over time (i.e. between 21 and 90 d of age) and not influence hepatic metabolism of sex steroids. Thus, the present results support the inference that there were exposure-dependent phytoestrogen effects in adult male offspring of dams that were maintained on soy-based diets during pregnancy and nursing. In addition, isoflavone concentrations in the liver and testis were related to levels in the maternal diet, and the changes seen in androgen biosynthesis were associated

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with low levels of genistein and daidzein in the testis, i.e. in the parts per billion range. Data describing serum hormone levels and androgen secretion by Leydig cells indicate that exposures to phytoestrogens early in life affect testicular steroidogenesis in adulthood. However, the surge in serum levels of steroid hormones in male rats, AO at 21 d, and T at 90 d, which was not related to increased rates of secretion by Leydig cells, raises the question of whether elevated serum steroid hormone levels result from greater Leydig cell population in the testis. In this regard, a recent report demonstrated that Leydig cell numbers were increased in adult marmosets fed soy formula as infants vs. animals fed standard milk formula (18). Although phytoestrogen induction of Leydig cell proliferative activity in vivo and regulation of Leydig cell numbers in the testis were not investigated in the present study, genistein stimulated Leydig cell division in vitro. Besides the effect of direct phytoestrogen action on Leydig cells, the concentrations of the two factors known to stimulate Leydig cell mitosis, i.e. LH and E2 (57, 58), were equivalent in the blood of adult animals in the control and highest exposure groups (0, 500, and 1000 ppm diets). Therefore, further studies are required to investigate phytoestrogen regulation of Leydig cell proliferative activity in the prepubertal period and effects on Leydig cell numbers in the adult testis. ER agonists are thought to exert direct effects in accessory sex glands because developmental estrogenization regulates androgen receptor-mediated stimulation of the prostate (36). Thus, the present data are consistent with previous observations that early-life exposures to genistein in the diet alter steroid hormone receptor gene expression in the adult prostate gland (50). These findings have clinical relevance because the prostate gland is thought to have the capacity to accumulate isoflavones (59, 60), and estrogen signaling was variably associated with induction of neoplasia and suppression of hyperplasia and carcinogenesis in this organ (61). Given the occurrence of feedback mechanisms between the levels of the hypothalamus-pituitary-gonadal axis and the confounding effects of LH secretion, experiments were performed to confirm that phytoestrogens act directly in Leydig cells. The results showed that genistein decreases Leydig cell responsiveness to LH stimulation, which impacts androgen biosynthesis. However, two pieces of evidence suggest that phytoestrogen regulation of Leydig cells is related to multiple mechanisms of action. First, the antiestrogen ICI 182,780 only partially alleviated genistein action at the 10-␮m dose. It is possible that the dose of ICI 182,780 (100 nm) was inadequate to prevent genistein action and use of higher doses of the antiestrogen was precluded by altered T secretion when acting alone. Otherwise, failure of the antiestrogen to block genistein action could be interpreted to mean that genistein action is mediated in part by ER-independent mechanisms. Second, the 10-␮m dose of genistein increased StAR protein in Leydig cells associated with diminished LH stimulation, although total StAR to p-StAR ratios were similar to untreated controls. This observation suggests that genistein action at higher doses is possibly due to mechanisms that are not related to cholesterol availability, e.g. induction of transcriptional activity through growth fac-

Akingbemi et al. • Phytoestrogens Regulate Leydig Cells

tor receptors (62), which may affect signaling pathways that regulate androgen biosynthesis. Nonlinear or nonmonotonic dose-response curves are typical of estrogens and have been described for several EDs (63). For example, Wisniewski et al. (13) reported that decreases in serum T levels were most pronounced in adult male rats exposed to 5 vs. 300 ppm genistein in the perinatal period. The mechanisms responsible for nonmonotonic doseresponse curves remain the subject of investigation and are probably related to ER biology. Low receptor occupancy appears to be adequate for induction of ER signaling, and it is thought that the activities of estrogenic compounds at high doses use both receptor and nonreceptor-mediated pathways (62, 64). In this regard, exposure to phytoestrogens in the maternal diet increased ER␤ gene expression in the pituitary gland of prepubertal male rats (our unpublished observations), and studies in vitro using isolated Leydig cells demonstrate that genistein acts in part via ER␣, the ER subtype expressed in rat Leydig cells (30). Thus, the effects of phytoestrogen action in the present study are likely due to estrogenic activity. However, the nonlinear actions of hormones in target tissues makes the extrapolation of data collected in rodents to humans problematic because EDs do not exert biphasic responses in all tissues (65). Nevertheless, it is established that children are at risk of sex steroid hormone disturbances because endogenous E2 levels are lower at this stage of development than previously assumed and exogenous sources of endocrine-active agents will have the effect of amplifying hormonal activity (66). In conclusion, the present study provides evidence that exposure to phytoestrogens in the perinatal period affects Leydig cell function in adult rats, causing a decrease in testicular T secretion at a low dose and an increase in serum and testicular T concentrations at a higher dose. The intermediate dose did not affect serum androgen levels. The phytoestrogen effects have implications for male reproductive function. For example, androgen insufficiency during the period of reproductive tract development is thought to be associated with congenital malformations of the male urogenital tract, e.g. hypospadias and cryptorchidism, which are now collectively described as testicular dysgenesis syndrome (67). Although there was no assessment of sperm quality and fertilizing capacity in the present study, the ER␤ is expressed in several cells of the seminiferous epithelium. Therefore, the possibility that genistein and daidzein exert direct effects on germ cells cannot be discounted. Indeed, genistein induced expression of the heat shock protein 90 in gonocytes, and heat shock protein 90 forms part of the ER cytosolic complex involved in the translocation of the ER-ligand complex from the cytosol to the nucleus to regulate transcriptional activity (68). On the other hand, phytoestrogen-induced increases in serum T levels have potential implications for pubertal development. Although definitive data are lacking on a secular trend toward accelerated puberty in male subjects, there is evidence that prepubertal males are taller at younger ages more than ever before, suggesting earlier maturity (69). Elevated serum T levels have also been linked to increased risk of testicular germ cell tumors in human subjects (70). The incidence of germ cell tumors has increased 3– 6% annually for the last 40 yr in Western countries (71) in tandem with

Akingbemi et al. • Phytoestrogens Regulate Leydig Cells

testicular dysgenesis syndrome (72). In summary, data from the present study indicate that the perinatal period is a sensitive window for phytoestrogen regulation of Leydig cell differentiation and testicular steroidogenesis. Further studies are required to identify the mechanisms of phytoestrogen action in testicular cells in order to identify phytoestrogen doses that may exert reproductive toxicity, especially after early-life exposures. Acknowledgments The authors gratefully acknowledge Dr. Chuck Benton (Harlan Teklad, Madison, WI) for help with diet formulation; Mr. Byron Harris (Department of Anatomy, Physiology, and Pharmacology, Auburn University) for technical assistance; and staff of the Division of Laboratory Animal Health (College of Veterinary Medicine, Auburn University) for the care and maintenance of animals under study. The authors also thank Ms. Chantal Manon Sottas (Center for Biomedical Research, Population Council, Rockefeller University, New York, NY) for help with the androsterone RIA. Received March 9, 2007. Accepted June 5, 2007. Address all correspondence and requests for reprints to: Benson T. Akingbemi, D.V.M., Ph.D., Department of Anatomy, Physiology and Pharmacology, 109 Greene Hall, Auburn University, Auburn, Alabama 36849. E-mail: [email protected]. This work was supported by an Auburn University fund for faculty development (to B.T.A.). Data were presented in preliminary form at the 88th Annual Meeting of The Endocrine Society, Boston, MA, June 21–26, 2006, and at the 39th Annual Meeting of the Society for the Study of Reproduction, Omaha, NE, July 21–25, 2006. Disclosure Statement: The authors have nothing to disclose.

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