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BOR - Papers in Press, published online ahead of print August 22, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.059204
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BIOLOGY OF REPRODUCTION 77, 1017–1026 (2007)
DOI: 10.1095/biolreprod.106.059204
© 2007 by the Society for the Study of Reproduction, Inc.

Effects of CDB-4022 on Leydig Cell Function in Adult Male Rats1

Yu-Chyu Chen 3, Renate K Cochrum 3, Michael T Tseng 4, Dushan T Ghooray 5, Joseph P Moore 4 5, Stephen J Winters 3 5 6, and Barbara J Clark 2 3 6

Departments of Biochemistry and Molecular Biology3 and Anatomical Sciences and Neurobiology,4 Division of Endocrinology, Metabolism and Diabetes,5 Department of Medicine, and The Center for Genetics and Molecular Medicine,6 University of Louisville, Louisville, Kentucky 40202

ABSTRACT

CDB-4022, an indenopryridine, suppresses spermatogenesis and decreases inhibin secretion in adult male rats. In the present study, we investigated the effects of CDB-4022 on Leydig cell function. A single oral dose of CDB-4022 (2.5 mg/kg) resulted in a 2-fold decrease in serum testosterone levels after 7 days that was paralleled by a decrease in Cyp17a1 mRNA and protein levels and 17alpha hydroxylase enzymatic activity compared with vehicle-treated rats. Consistent with the lower serum testosterone levels, pituitary Lhb and Fshb mRNA levels were increased 3.2- and 2.3-fold, respectively, by CDB-4022 treatment. Ultrastructural analysis of pituitary gonadotrophs showed distended endoplasmic reticulum (ER) and fewer secretory granules in CDB-4022-treated rats, characteristic of enhanced secretory activity. Conversely, CDB-4022 increased serum progesterone levels, testicular Star mRNA and protein expression, and the number of Leydig cells per testis. Serum inhibin B levels were undetectable in CDB-4022-treated rats, while serum activin A levels were similar to controls, indicating that the CDB-4022-treated rats have an elevated activin A:inhibin B ratio. In the presence of hCG stimulation, activin A directly suppressed testosterone secretion but enhanced progesterone secretion from rat Leydig cell primary cultures. Likewise, treatment of MA-10 cells with activin A was found to enhance cAMP-stimulated progesterone secretion and STAR expression. Together, our data indicate that CDB-4022 treatment inhibits CYP17A1 and stimulates STAR expression, thereby decreasing testosterone but increasing progesterone production. We propose that unopposed actions of activin A most likely contribute to the steroid profile in rats after CDB-4022 treatment. Our findings establish CDB-4022 as a new model to examine intratesticular control mechanisms that modulate Leydig cell gene expression and function.

activin, CDB-4022, CYP17A1, gene regulation, Leydig cells, steroid hormones, steroidogenesis, STAR, testis

INTRODUCTION

Disruption of Sertoli cell functions, e.g., by testicular radiation or chemotherapeutic agents, impairs spermatogenesis and decreases serum inhibin levels [1–3]. SANDOZ20–438, an indenopyridine, was initially developed as a histamine receptor antagonist for the treatment of allergic disorders but was found to suppress spermatogenesis [4]. Recently, SANDOZ20–438 and its derivatives, including RTI4758–056 and the more potent agent CDB-4022, have been proposed as potential nonsteroidal male contraceptives because their disruption of spermatogenesis are reversible by pretreatment with the GnRH analog, lupron, or the GnRH antagonist, acyline [5–8]. Also, these compounds had no effect on the libido in dogs or rats and were not mutagenic [6, 9]. A recent study in a rat model reported that CDB-4022 decreased spermatogenesis and reduced serum inhibin and epididymal androgen binding protein levels, two markers of Sertoli cell function [4, 8]. The effects of CDB-4022 may be related to the chemical induction of apoptosis in Sertoli cells and/or germ cells directly, leading to decreased spermatocyte and spermatid cell populations [4, 8].

The effects of CDB-4022 on Leydig cell function have not been studied in detail and are the focus of the current study. Initial studies reported no significant effect of CDB-4022 on serum testosterone or FSH levels [8] and no increase in the number of apoptotic Leydig cells in rats [4]. However, a large variation in serum levels between animals may have masked a decrease in serum testosterone. Furthermore, the same study reported elevated serum LH levels [8]. Therefore, we compared key gene expression profiles in the testis and pituitary of vehicle- and CDB-4022-treated rats to analyze the effects on Leydig cell function.

Leydig cell testosterone synthesis begins with the uptake of cholesterol into mitochondria. This initial step is classified as the acute phase of steroidogenesis and is dependent upon the synthesis of the steroidogenic acute regulatory (STAR) protein [10, 11]. Cholesterol is converted to pregnenolone by cytochrome P450scc complex [12]. Testosterone synthesis in rodents preferentially follows the {Delta}4-pathway in which pregnenolone is converted to progesterone by 3β-hydroxysteroid dehydrogenase, and progesterone is metabolized to androstenedione by two reactions that are catalyzed by CYP17A1 (Cyp17a1 gene encoding cytochrome P450 17{alpha}-hydroxylase/C17,20 lyase) [13]. Lastly, 17β-hydroxysteroid dehydrogenase (HSD17B) converts androstenedione to testosterone. Testosterone synthesis is primarily controlled by LH release from the anterior pituitary. LH binds to a seven-transmembrane G-protein coupled receptor and activates the cAMP-dependent protein kinase A signal transduction pathway. LH activates two temporally distinct transcriptional responses: first, STAR synthesis for cholesterol delivery to the mitochondria and second, activation of the genes encoding the steroid hydroxylase enzymes of the steroid hormone biosynthetic pathway, such as CYP17A1.

In addition to regulation by LH, paracrine factors secreted by Sertoli cells play a role in the maintenance of steroidogenesis [14, 15]. As stated above, the Sertoli cell is a major target of CDB-4022 [4, 7, 8]; therefore, we were interested in evaluating potential Sertoli cell factors that might influence Leydig cell function. In particular, the suppression of inhibin B in CDB-4022-treated rats [4, 8], coupled with early literature that implicated a direct role for activin A as a suppressor of LH-stimulated testosterone synthesis in isolated rat Leydig cells [16, 17], prompted us to focus on activin A. Indeed, more recent studies have demonstrated both stimulatory and inhibitory actions of activin A and other transforming growth factor β (TGFβ) family members on hormone-activated steroid production in other tissue types [18–24]. Specifically, activin A has been shown to stimulate cAMP-stimulated Star mRNA expression as well as steroid production in both H295R human adrenal cells [24] and HOTT theca cells [25], but, to our knowledge, its effect on Leydig cells has not been studied. Therefore, we have tested the direct effects of activin A on Leydig cell steroid production and STAR expression in MA-10 Leydig cells.

MATERIALS AND METHODS

Materials

The Assay-on-Demand gene expression kits for StAR or 18sRNA and SYBR Green PCR Master Mix were obtained from PE Applied Biosystems (Foster City, CA). The RNeasy kit was purchased from Qiagen Inc. (Valencia, CA). Lysing Matrix-D was obtained from Qbiogene (Carlsbad, CA). Progesterone and testosterone enzyme immunoassay (EIA) kits were purchased form Cayman Chemical Company (Ann Arbor, MI). Active inhibin B ELISA kit was obtained from Diagnostic System Laboratories Inc. (Webster, TX), and the activin A ELISA kit was purchased from Serotec (Oxford, UK). Percoll, BSA, soybean trypsin inhibitor (SBI), collagenase 17{alpha}-hydroxyprogesterone (17OHP4), progesterone, androstenedione, hCG, isocitrate, isocitrate dehydrogenase, and monoclonal anti-β-actin serum were obtained from Sigma Chemical Company (St. Louis, MO). NADPH was obtained from Calbiochem (La Jolla, CA). [1,2,6,7 3H]-progesterone and Western Lighting Chemiluminescence Reagents were purchased from Perkin-Elmer Lab Inc. (Boston, MA). The thin layer chromatography (TLC) aluminum sheet Silica gel 60 F254 was obtained from EMD Chemical Inc. (Gibbstown, NJ). Araldite and propylene oxide were purchased from Ted Pella Inc. (Ridding, CA). Glutaraldehyde, cacodylate, osmium tetroxide, and potassium ferrocyanide were obtained from Electron Microscopy Sciences (Hatfield, PA). Recombinant activin A and inhibin B were purchased from R&D Biosystems (Minneapolis, MN) and Diagnostic Systems, respectively. Horse radish peroxidase (HRP)-donkey anti-rabbit secondary antibody was obtained from Amersham (Piscataway, NJ), and HRP-goat anti-mouse secondary antibody was purchased from Pierce (Rockford, IL). Bio-Rad polyvinylidene fluoride (PVDF) membrane was obtained from Bio-Rad (Hercules, CA). CDB-4022, [4aRS,5SR,9bRS]-2ethyl-2,3,4,4a,5,9b-hexahydri-8iodo-7-methyl-5-[carbomethoxy-phenyl]-1H-indeno[1,2-c] pyridine-hydrochloride, was kindly provided by Dr. Richard Blye (Contraceptive and Reproductive Health Branch, National Institute of Child Health and Human Development, Rockville, MD).

Animal Management and Treatment

Adult male Sprague-Dawley rats (60–90 days old) were purchased from Harlan (Indianapolis, IN) and housed in groups in the University of Louisville Animal Facility under a 12L:12D cycle with constant access to laboratory chow and tap water. Rats were given a single dose of 2.5 mg/kg of CDB-4022 via oral gavage. Control animals received one oral dose of vehicle (7.5% ethanol, 7.5% water, and 85% sesame oil). One week later, rats were killed by CO2 asphyxiation and then decapitated; trunk blood was immediately collected, and serum was separated and stored. Testes and pituitary were isolated and processed as outlined below. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC 05020) in agreement with the National Institutes of Health Assurance of Compliance with Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Isolation of Rat Leydig Cells

Testes from vehicle-treated or CDB-4022-treated adult rats were placed in cold Hanks balanced salt buffer with 0.1% BSA and SBI at 25 mg/L. The Leydig cell isolation procedure is based on previous protocols with slight modifications [26]. Testes were decapsulated and dispersed by shaking for 20 min at 80 cycles/min at 34°C in a 15-ml sterile tissue culture tube containing Dulbecco modified Eagle medium (DMEM)/F12 medium with collagenase at 0.25 mg/ml, 0.1% BSA, and SBI at 25 mg/L. The cell suspension was filtered through four layers of sterile gauze and pelleted by centrifugation at 800 x g for 5 min. The cell pellet was washed with DMEM/F12 medium and allowed to settle by gravity for 15 min at room temperature. Leydig cells were isolated by centrifugation at 20 000 x g for 60 min at 4°C in a 60% Percoll gradient. Percoll was removed from the cells by centrifugation at 1000 x g for 5 min. Cell pellets were resuspended in the DMEM/F12 medium, and RNA was isolated with the RNeasy kit. Also, 5 x 105 Leydig cells were seeded onto each well of a 24-well plate; treated with activin A at 10, 25, 50, or 100 ng/ml for 24 h; and then stimulated with hCG at 50 ng/ml for 4 h. Media were collected and frozen for testosterone and progesterone EIA assays.

The MA-10 mouse Leydig tumor cell line was a generous gift from Dr. Mario Ascoli, University of Iowa College of Medicine (Iowa City, IA), and was grown and maintained as described previously [11, 27].

Quantitative Real-Time PCR

Testicular and pituitary RNA was prepared by homogenizing the frozen tissue samples in a FastPrep (Savant, Holbrook, NY) with Lysing Matrix-D and extracted with the RNeasy kit according to the manufacturer's protocol. For testicular and Leydig cell RNA, reverse transcription (RT) was performed by incubating 1 µg of total RNA with Moloney murine leukemia virus reverse transcriptase at 37°C for 1 h, and 30 ng of the resulting cDNA was used for quantitative PCR (q-PCR). To detect Star mRNA, cDNA was mixed with 100 nM of TaqMan probe and 300 nM primers supplied by the StAR Assay-on-Demand Gene Expression set (PE Applied Biosystems). To detect Cyp17a1 or Hsd17b, q-PCR was performed with SYBR Green PCR Master Mix and 300 nM forward and reverse primers. Cyp17a1 forward, 5'-ACTGAGGGTATCGTGGATGC-3' and reverse, 5'-TCGAACTTCTCCC TGCACTT-3'; Hsd17b3 forward, 5'-GTTCTCGCAGCACCTTTTTC-3' and reverse, 5'-ACAATCTTCACACCGCTTCC-3'. TaqMan human 18srRNA probe and primers were included in the reaction as a loading control. Reactions were performed at 94°C for 30 sec, 60°C for 20 sec, and 72°C for 30 sec for a total of 40 cycles with an ABI Prism 7700 Sequence Detector (Perkin-Elmer Applied Biosystems, Foster City, CA). Reactions were performed in duplicate, and the average threshold cycle (CT) was used in subsequent calculations to determine relative mRNA levels. In brief, for each target gene, duplicate reactions for RNA samples from individual rats (four vehicle and six CDB-4022-treated rats) were performed, and a {Delta}CT value was determined with 18S. {Delta}{Delta}CT values were determined for each gene target independently by selecting a single {Delta}CT value from the control group to use as the calibrator for all samples for that specific target gene (ABI PRISM 7700 Sequence Detection System User Bulletin 2). Thus, the error bars in the control group represent the interanimal variation in target gene expression.

For pituitary mRNA analysis, cRNA standard curves were constructed as previously described [28, 29]. Briefly, target specific primers were synthesized with and without a T7-promoter sequence (5'-GGATCCTAATACGACTCACTATAGGGAGG-3') at the 5'-end of the forward primer and with an oligo-dT(T12) at the 5'-end of the reverse primer. PCR was performed to produce cDNA containing the T7 promoter sequence. The PCR product (1 µg) was then used as template in an in vitro transcription reaction (MAXIscript T7 kit; Ambion, Austin, TX). The subsequent cRNA was quantified with a spectrophotometer and serially diluted to make a standard of known starting material containing 1011 to 104 molecules of cRNA. These standards were processed in parallel with pituitary RNA samples (1 µg), and oligo-dT(12–24) was used for RT and followed by quantitative real-time PCR with the primers lacking T7 and dT sequences for the sample set. PCR was performed on a Stratagene MX4000 Multiplex Quantitative PCR System with the Brilliant SYBR Green QPCR Master Mix (Stratagene, La Jolla, CA). Postamplification dissociation curves were performed to verify the presence of a single amplification product in the absence of DNA contamination. Sample values were determined by interpolation to the standard curve of known RNA amounts.

Serum Hormone Assays

Progesterone and testosterone levels were measured in duplicate in serum samples from vehicle- or CDB-4022-treated rats or in medium from MA-10 cells (progesterone only) with Progesterone and Testosterone EIA Kits. Serum inhibin B and activin A levels were measured with the Active Inhibin B and the Activin A ELISA kits, respectively.

Microsome Preparation and 17{alpha}-Hydroxylase Activity

Decapsulated testes (0.5 g) were homogenized in 50 mM potassium phosphate (pH 7.4), 0.25 M sucrose, 0.5 mM EDTA, and 0.1 mM PMSF with Lysing Matrix-D and FastPrep. The testicular lysates were subjected to sequential centrifugation steps at 1000 x g for 5 min 4°C to pellet the nuclear fraction, and the resultant supernatants were centrifuged at 10 000 x g at 4°C for 10 min. Microsomes were pelleted from the supernatants by centrifugation at 100 000 x g for 30 min 4°C in a Beckman L8-70M ultracentrifuge. The microsomal pellets were rinsed twice with homogenization buffer, resuspended in 50 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA and 20% glycerol, and stored at –80°C. Protein concentrations were measured with the BCA Protein Assay kit. Activity of 17{alpha}-hydroxylase was measured according to the published method of Auchus et al. [30] with slight modification. In brief, 200 µg of microsome protein was incubated with progesterone substrate (1 nmol [3H]-progesterone: 5 nmol of nonlabeled progesterone), 100 mM potassium phosphate (pH 7.4), 1 mM MgCl2, 1 U of isocitrate dehydrogenase, and 4 mM isocitrate in a final volume of 200 µl. The reaction was initiated by adding NADPH (final concentration, 5 mM) and was incubated at 37°C with shaking for 5, 10, 15, 20, or 30 min. The reaction was stopped at each time by adding 5 volumes of dichloromethane, and steroids were extracted, dried under nitrogen stream, and resuspended in ethyl acetate. Progesterone (substrate) and 17OHP4 (product) were separated by TLC with precoated Silica Gel 60 F254 in 90% chloroform and 10% ethyl acetate. Progesterone and 17OHP4 standards were run simultaneously, and their migration was visualized by short-wave ultraviolet light. To quantify the metabolites of [3H]-progesterone, each sample lane was divided evenly into eight sections to recover progesterone and 17OHP4 in independent sections, and the radioactivity was determined by liquid scintillation counting. Percent conversion to 17OHP4 was determined by the count per minute for 17OHP4 divided by the total count per minute per lane. Percent conversion averaged 7.6% for vehicle- and 4.5% for CDB-4022-treated animals at 30 min. The data were transformed to picograms of steroid on the basis of the specific activity of the [3H]-progesterone and the count per minute of the recovered 17OHP4 and expressed as picomoles of product per milligram of protein.

Transmission Electron Microscopy

Both vehicle- and CDB-4022-treated rats (n = 3 per study group) were anesthetized by injection with sodium pentobarbital, and tissues were fixed by perfusion with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) perfused through the left ventricle of the heart. The pituitary was isolated, postfixed in a 2% aqueous solution of osmium tetroxide, dehydrated in graded ethanols, and embedded in Araldite 502 resin. Ultrathin sections from pituitary blocks were cut with a diamond knife and mounted on nickel grids. The sections were doubly stained with saturated aqueous solution of uranyl acetate and lead citrate and examined with a Philips CM 10 electron microscope operated at 60 kV. Criteria for ultrastructural identification of adenohyphophyeal cell types are based on the size, shape, and distribution of secretory granules [31].

Quantitative Morphology

For stereological analysis of Leydig cells, the testes were fixed by whole body perfusion as described above. Prior to perfusion, one testis was removed and weighed. The fixed testis was embedded in Araldite resin and cut into blocks. Tissue sections (thickness, 3 µm) were cut from each embedded testis block with an LKB IV ultramicrotome (Pharmacia, LKB, Piscataway, NJ) and glass knives and mounted on glass slides. The testis sections were stained with 1% toluidine blue in 1% borax and examined with a Zeiss Hal 100 inverted microscope equipped with a Zeiss digital camera and AxioVision Rel 3.1 software. The criteria used to identify Leydig cells were based on the morphological characteristics as described [32–34]. To obtain a more accurate measure of testicular volume, the testicular capsule was excluded from the testis weight. As the density of the testis is approximately 1, testicular weight was transformed into volume. Leydig cells were counted in 10 separate sections cut from independent blocks for each animal, and the number was corrected for shrinkage during the fixation process. The total shrinkage, ST, was derived from the expression ST = S1 + S2 (1 – S1). S1 represents the difference between the unfixed and the fixed testis weight divided by the weight of the unfixed testis. S2 represents the shrinkage during the embedment by the expression, 1 – (L1/L2)3. The parameter L1 is the summed linear dimension (length plus width) of 10 blocks of fixed tissue before embedment, and L2 is the same measurement performed after embedding the tissue in the plastic. The total number of Leydig cells per testis was calculated by multiplication of the volume of the testis in the plastic-embedded state (Ve) by the numerical density (Nv). Ve was estimated by multiplication of the total number of sections obtained from the testis by the section thickness (T) and the mean area from 10 randomly selected sections obtained from the testis. Nv was calculated by the following method: Nv = (Q– AT). Here, Q– is the number of unique nuclear profiles in the test area (A). Section thickness (T) was estimated by differential focusing with a 63x oil-immersion objective and was found to be 2 µm on average. The Leydig cell number per testis per animal for three vehicle- and three CDB-4022-treated rats was determined, and the average value ± SEM was determined for each group.

Western Blot Analysis

Whole cell lysates were isolated from three vehicle- or three CDB-4022-treated rat testes, and 30 µg of protein for each sample was resolved by electrophoresis on a 12% SDS-PAGE gel and transferred to Bio-Rad PVDF membrane for Western blot analysis [35]. The membrane was incubated with a rabbit anti-human CYP17A1 antiserum (1:3000, a generous gift from Dr. Michael R. Waterman, Vanderbilt University, Nashville, TN) [36], and then incubated with a donkey anti-rabbit HRP antibody (1:5000). Proteins were visualized by Western Lighting Chemiluminescence Reagents and exposed to Kodak BioMax Light film. The membranes were stripped with Restore Western Blot Stripping Buffer and incubated with monoclonal anti-mouse β-actin serum (1:5000) and HRP-goat anti-mouse secondary antibody (1:10 000) or a rabbit {alpha}-GST-STAR serum (1:5000), followed by incubation with donkey anti-rabbit HRP secondary antibody at a 1:5000 dilution. Multiple exposure times were used, the films were scanned, and the integrated optical densities for STAR, CYP17, and actin were digitized and quantified by UN-SCAN-IT software (Silk Science Inc., Orem, UT). The STAR:actin and CYP17:actin ratios were determined within the linear range of the film.

MA-10 cells were seeded onto 24-well plates, incubated for 24 h with activin A at 10, 25, 50, and 100 ng/ml in serum-free medium containing 0.1% BSA (pH 7.4), and then treated with (Bu)2cAMP at 0.5 mg/ml for 4 h. Media were collected from triplicate treatments for progesterone measurements, and cell lysates were prepared for Western blotting by adding RIPA (RadioImmuno Precipitation Assay) lysis buffer to the cell monolayer. Thirty micrograms of cellular protein was resolved on a 12% SDS-PAGE gel, and the membranes were processed and analyzed as described above for STAR and β-actin. For MA-10 cells, the 37- and 30-kDa STAR bands were analyzed separately.

Statistical Analyses

All experiments were repeated independently at least three times, unless otherwise indicated. Data are presented as mean ± SEM. The Student t-test (unpaired) was used for direct analysis between vehicle and CDB-4022 treatment groups, while an analysis of variance followed by the Dunnett post hoc test was used for multiple comparisons (GraphPad Prism, GraphPad Software Inc., San Diego, CA). P < 0.05 was considered significantly different.

RESULTS

Serum Steroid Hormone Levels in CDB-4022-Treated Male Rats

Adult male Sprague-Dawley rats were treated with a single dose of CDB-4022 (2.5 mg/kg) or vehicle, and 1 wk later, serum steroid levels were measured. As shown in Figure 1, CDB-4022 treatment significantly increased serum progesterone but decreased serum testosterone levels. The average serum progesterone level in CDB-4022-treated rats was 370 ± 60 pg/ml compared with 130 ± 40 pg/ml for vehicle, representing a 2.9-fold increase (Fig. 1A). Conversely, mean testosterone levels were 1.95 ± 0.30 ng/ml in CDB-4022-treated rats compared with 3.87 ± 0.42 ng/ml in vehicle-treated rats, a 2-fold decrease (Fig. 1B).


Figure 01
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  		FIG. 1 Serum steroid levels in CDB-4022-treated rats. Adult male rats received a single dose of 2.5 mg/kg of CDB-4022 by oral gavage (n = 12), while control animals received the vehicle (n = 6 or 7). One week later, blood was collected, and serum progesterone (A) and testosterone (B) levels were measured. Shown is a scatter plot of all individual values, and the mean is depicted by the horizontal line. *P < 0.05.

Testicular Gene Expression Profiles and 17{alpha}-Hydroxylase Activity in CDB-4022-Treated Rats

Star and Cyp17a1 mRNAs were measured by quantitative real-time PCR. As shown in Figure 2A, Star mRNA levels were increased 2.5-fold in whole testis tissue from CDB-4022-treated rats, while Cyp17a1 mRNA levels were decreased ~20%. These data suggest that changes in Leydig cell gene expression contribute to altered steroid production in CDB-4022-treated rats. However, a 50% reduction in testis weight was observed 1 wk after CDB-4022 treatment (2.02 ± 0.17 g per testis for vehicle (n = 8) vs. 0.92 ± 0.12 g for CDB-4022 (n = 16), similar to previous reports in which the testis weight loss was attributed to a decreased volume of seminiferous tubules and a decreased number of germ cells [7]. Because mRNA analysis for whole testis may be complicated by the difference in cell populations between the vehicle and CDB-4022 groups, we next calculated the number of Leydig cells in fixed testis tissue sections. CDB-4022 treatment resulted in a 17% increase in the number of Leydig cells per testis, with 15.9 ± 0.6 x 106 cells/testis vs. 18.7 ± 0.3 x 106 cells/testis for vehicle vs. CDB-4022-treated rats (P < 0.05). Next, Star and Cyp17a1 mRNA expression levels were determined in isolated Leydig cells; Star mRNA levels were increased ~2-fold, whereas Cyp17a1 was significantly decreased ~55% in the CDB-4022 treatment group compared with vehicle-treated controls (Fig. 2B). On the other hand, there was no significant difference in testicular Hsd17b3 mRNA expression between vehicle- and CDB-4022-treated rats (data not shown).


Figure 02
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  		FIG. 2 Testicular Star and Cyp17a1 mRNA expression in CDB-4022-treated rats. Total RNA was isolated from the testes of vehicle- (n = 4) or CDB-4022- (n = 6) treated rats (A) or from isolated Leydig cells (B) and subjected to real-time RT-PCR amplification for Star and Cyp17a1. Messenger RNA levels are expressed relative to values in vehicle-treated rats, and shown are the mean values ± SEM for the relative expression (CDB-4022 relative to vehicle) for Star and Cyp17a1 as indicated. The data for Leydig cells are from quadruplicate measurements from two independent isolations (n = 2). Thus, the samples represent a pool of isolated Leydig cells from two vehicle-treated and four CDB-4022-treated animals per experiment. *P < 0.05 vehicle vs. CDB-4022.

Western blot analyses of STAR and CYP17A1 in testicular lysates demonstrated that STAR protein was increased 36%, and CYP17A1 protein was reduced 70% in the CDB-4022-treated rats (Fig. 3, A and B). The decrease in CYP17A1 protein was paralleled by a decrease in 17{alpha}-hydroxylase reaction rates in vitro. As shown in Figure 3C, the cytochrome P450 17{alpha}-hydroxylase enzymatic activity was decreased by 44% in microsomal membranes isolated from CDB-4022-treated rats compared with the control counterparts. The reaction rates were 11.4 ± 0.47 and 6.47 ± 0.84 pmol of 17OHP4 per milligram of protein per minute for samples from vehicle- and CDB-4022-treated rats, respectively (Fig. 3C).


Figure 03
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  		FIG. 3 Testicular STAR and CYP17A1 protein expression in CDB-4022-treated rats. A) Testicular lysates were prepared from vehicle- or CDB-4022-treated rats and subjected to Western blot analysis for the expression STAR and CYP17A1 proteins and β-actin (labeled actin). Lanes 1–3, samples from three independent control rats; lanes 4–6, samples from three independent CDB-4022-treated rats. B) StAR and CYP17A1 protein levels were normalized to β-actin, and the value for vehicle was set to 1.0. Shown are the mean ± SEM from three rat testes. *P < 0.05. C) Cytochrome P450 17{alpha}-hydroxylase activity was determined by incubating testicular microsomal protein (200 µg) with 30 mM steroid (1:5 [3H]:nonlabeled-progesterone) at 37°C for the indicated time periods. Steroids were extracted from the reaction mixture, progesterone metabolites were resolved by thin layer chromatography (TLC), and progesterone- and 17{alpha}-hydroxyprogesterone (17OHP4)-specific spots were recovered; their radioactivities were measured as detailed in Materials and Methods. The data are expressed as picomoles of 17OHP4 produced per milligram of protein, and shown are the mean values ± SEM for n = 3 separate microsomal preparations from three vehicle- and three CDB-4022-treated rats. The data were analyzed by linear regression and analyzed statistically by a two-way analysis of variance followed by the Dunnett post hoc test. *P < 0.05 CDB-4022 vs. vehicle.

Pituitary RNA Profiles and Ultrastructure in CDB-4022-Treated Rats

The transcript copy number for pituitary Lhb and Fshb was determined by q-PCR. Lhb expression was 16 ± 1.6 and 51 ± 5.0 x 108 transcripts per microgram of RNA in control and CDB-4022-treated rats, respectively (Fig. 4). Similarly, Fshb expression was increased from 3.9 ± 0.2 to 8.1 ± 0.63 x 108 transcripts per microgram of RNA by CDB-4022 treatment. These are the first quantitative measurements for rat pituitary gonadotropin subunit gene expression and show a ~4:1 Lhb:Fshb ratio in the adult male rat pituitary. Furthermore, CDB-4022 treatment resulted in a 3.2- and 2.1-fold increase in Lhb and Fshb mRNA levels, respectively (Fig. 4). Examination of the ultrastructure of the adenhypophyseal parenchyma from vehicle-treated rats revealed normal rough endoplasmic reticulum (RER) profiles with abundant and well-dispersed secretory granules (Fig. 5, A and C). Following CDB-4022 treatment, however, the RER is distended with fewer numbers of secretory granules in gonadotrophs when compared with controls (compare Fig. 5, A and C with B and D). These changes are characteristic of cells with increased protein synthesis and secretion [37]. The selective influence of CDB-4022 on gonadotrophs is illustrated by the lack of this effect on a neighboring somatotroph that contains normal-appearing stacks of RER (Fig. 5D).


Figure 04
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  		FIG. 4 The effect of CDB-4022-treatment on Lhb and Fshb mRNA levels. Pituitary RNA was prepared from vehicle- (n = 4) or CDB-4022- (n = 8)-treated rats, and the transcript copy number for Lhb or Fshb was determined by qRT-PCR. Shown is the mean mRNA transcript number per microgram of total RNA ± SEM (x108). *P < 0.05.


Figure 05
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  		FIG. 5 Effects of CDB-4022 treatment on the ultrastructure of gonadotrophs. Sections shown are representative electron micrographs from vehicle- (A and C) or CDB-4022 (B and D)-treated rats. Magnification is x9800 (A), x4900 (B), x18 000 (C), and x14 000 (D). The arrows indicate normal profiles of endoplasmic reticulum (ER) in the cytoplasm from vehicle-treated rats (A and C) and a somatotrope from a CDB-4022-treated animal (D). The asterisks denote the vacuole-like distended ER in the gonadotrophs from CDB-4022-treated rats (B and D). Arrowheads indicate secretory granules. G, Golgi apparatus; N, nucleus; M, mitochondria.

Serum Activin A and Inhibin B Levels in CDB-4022-Treated Male Rats

Serum inhibin B levels were 106 ± 23.7 pg/ml in vehicle-treated rats but were undetectable in CDB-4022-treated rats, consistent with previously published findings by Hild et al. [7, 8] and supporting disruption of Sertoli cell function. Serum activin A levels, however, were not different between the two groups: 2.45 ± 0.47 and 2.29 ± 0.2 ng/ml for vehicle- and CDB-4022-treated rats, respectively.

Activin A Effects on Testosterone and Progesterone Production by Rat Leydig Cell Primary Culture

Our data show that CDB-4022 suppresses testosterone secretion via decreased CYP17A1 expression and enzymatic activity. Conversely, progesterone levels are elevated, and Star mRNA and protein expression are increased. A similar expression pattern for these two proteins was previously reported following activin A treatment of a human ovarian theca cell line [25]. Because CDB-4022 treatment did not affect activin A levels but dramatically decreased serum inhibin B levels, we tested the possibility that activin A directly modulates testosterone and progesterone secretion by Leydig cells. Primary rat Leydig cell cultures were treated with increasing concentrations of activin A for 24 h and then stimulated with hCG for 4 h. Testosterone production was increased ~2-fold by hCG treatment, and pretreatment with activin A resulted in a dose-dependent suppression of hCG-stimulated testosterone production (Fig. 6A). Progesterone production in primary Leydig cell cultures was increased ~3-fold by hCG treatment, and activin A at doses of 25 or 50 ng/ml potentiated the stimulatory effect of hCG (Fig. 6B). Activin A alone had no effect on either testosterone or progesterone production.


Figure 06
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  		FIG. 6 Effects of activin A on steroid production by primary rat Leydig cell cultures. The cells were treated in triplicate wells for 24 h with the indicated amount of activin A, followed by treatment with hCG at 50 ng/ml for 4 h. The media were collected, and testosterone (A) or progesterone (B) levels were measured by enzyme immunoassay (EIA). Shown are the mean values ± SEM from three independent experiments. *Significantly increased when compared with basal activity (P < 0.05). **Significantly different from hCG alone (P < 0.05) as determined by a two-way analysis of variance followed by the Dunnett post hoc test.

Effects of Activin A on Progesterone Production and STAR Expression in MA-10 Mouse Leydig Tumor Cells

To further investigate the potential direct effects of activin A on testicular steroidogenesis, we studied STAR expression in MA-10 mouse Leydig tumor cells. MA-10 cells were pretreated with activin A for 24 h, and the effect on (Bu)2cAMP-stimulated progesterone production was determined. As shown in Figure 7A, activin A increased (Bu)2cAMP-stimulated progesterone secretion by MA-10 cells with a maximal 2.2-fold increase in activin A at 50 ng/ml. Inhibin B alone had no effect on progesterone production; however, inhibin B blocked completely the activin A-induced increase in (Bu)2cAMP-stimulated progesterone secretion.


Figure 07
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  		FIG. 7 Effects of activin A on MA-10 cell progesterone synthesis and STAR expression. For A–C, MA-10 cells were incubated in triplicate for 24 h with the indicated amounts of activin A or inhibin B alone or in combination as indicated and then stimulated with (Bu)2cAMP at 0.5 mg/ml for 4 h. A) Progesterone levels in the media were measured by enzyme immunoassay (EIA). Shown are the mean values ± SEM from three independent experiments. *Significantly increased (P < 0.05) when compared with (Bu)2cAMP treatment alone. **Significantly decreased (P < 0.05) when compared with (Bu)2cAMP plus activin A treatment. B) Cell lysates were prepared for immunoblotting, and shown is a representative Western blot for STAR and β-actin. The 37- and 30-kDa STAR forms were analyzed separately, and each was expressed relative to the value for cAMP treatment alone that was set to 1.0. Shown are the mean values ± SEM of three independent experiments. * and ** represent a significant increase in the 30- and 37-kDa STAR, respectively, compared with (Bu)2cAMP alone, P < 0.05. C) Total mRNA was isolated and subjected to real-time RT-PCR amplification as described in Materials and Methods. The Star mRNA levels were expressed relative to (Bu)2cAMP treatment alone. Shown are the mean values ± SEM from a minimum of four experiments. *Significantly increased compared with (Bu)2cAMP alone, P < 0.05. D) MA-10 cells were incubated in triplicate with the indicated amounts of CDB-4022 or vehicle for 24 h, followed by treatment with (Bu)2cAMP at 0.5 mg/ml for 4 h. The medium was removed, and progesterone was measured by EIA. Total protein concentrations were determined for each well, and the data were expressed as picograms of progesterone per microgram of protein. Shown are the mean values ± SEM from a representative experiment repeated twice. Basal indicates progesterone levels after a 24-h CDB-4022 treatment alone. *P < 0.01 and **P < 0.05 based on an analysis of variance followed by the Dunnett post hoc test.

Western blot analysis of STAR demonstrated that activin A increased both the precursor forms (37 kDa) and mature forms (30 kDa) of STAR protein ~2.0- and 1.5-fold, respectively, compared with cells stimulated with (Bu)2cAMP alone (Fig. 7B). Star steady-state mRNA levels following (Bu)2cAMP treatment were also increased ~1.5- to 2.0-fold by activin A as determined by qRT-PCR (Fig. 7C). Together, these data indicate that activin A enhances (Bu)2cAMP-stimulated progesterone production by MA-10 cells by increasing STAR expression, most likely via a transcriptional mechanism.

Lastly, MA-10 mouse Leydig tumor cells were treated with increasing doses of CDB-4022 to test whether CDB-4022 has a direct toxic effect on Leydig cell function. As shown in Figure 7D, (Bu)2cAMP increased MA-10 cell progesterone production 10-fold, and this response was not affected by CDB-4022 treatment. Basal progesterone production, however, appears to be stimulated with high concentrations of CDB-4022. Together, these data indicate that that MA-10 cell steroidogenesis is not directly inhibited by CDB-4022.

DISCUSSION

We demonstrate that CDB-4022 treatment of adult male rats significantly decreases serum testosterone levels. The elevated pituitary Lhb and Fshb mRNA levels indicate a decrease in the negative feedback inhibition of gonadotropin synthesis, while the distended ER structure and fewer secretory vesicles in gonadotrophs from CDB-2044-treated rats are characteristic of increased GnRH stimulation and support the finding of elevated Lhb and Fshb levels [37].

Previous studies demonstrated that CDB-4022-treated rats had no statistically significant change in serum testosterone or FSH levels, although serum LH levels were increased [4, 7, 8]. Differences in experimental design and high between-animal variations in hormone levels may have contributed to the apparent differences between our findings. It is unlikely that the decline in serum testosterone levels we observed is part of this antispermatogenic action of CDB-4022; for example, the mean serum testosterone values for the CDB-4022-treated animals fall within the lower range of the vehicle-treated animals. More specifically, suppression of inhibin B supports Sertoli cell dysfunction ([8] and data herein). Indeed, the major damage to the testis appears to be with the spermatocytes and differentiating spermatogonia, perhaps as a result of Sertoli cell dysfunction [4, 7].

Although CDB-4022 decreased serum testosterone levels, serum progesterone levels were increased. These unique findings were explained by a decrease in Leydig cell CYP17A1 protein expression, accompanied by a decrease in CYP17A1 enzymatic activity, while at the same time, STAR expression was increased. The lower level of Cyp17a1 was more apparent in isolated Leydig cells than in RNA from testicular homogenates because the loss of germ cells enriched the CDB-4022-treated testis with interstitial cells. A decrease in CYP17A1 expression per Leydig cell, as suggested by the enzymatic activity, would be partially compensated for by the increase in Leydig cell number. There was a small increase (~17%) in Leydig cell number in CDB-4022-treated animals. Leydig cell hyperplasia most likely resulted from increased LH stimulation and may explain why Star mRNA was increased more in whole testis than in isolated Leydig cells. Thus, the increased STAR and progesterone production can be explained, in part, by the elevated LH and increased Leydig cell number. However, the increase in STAR and Leydig cell number did not restore serum testosterone levels to normal because of reduced CYP17A1 expression.

As stated above, Sertoli cells appear to be a major target for CDB-4022 [4, 7, 8]; however, it remains to be proven whether the decline in serum testosterone by CDB-4022 treatment is due to a direct or indirect effect on Leydig cells. With MA-10 Leydig cells, we found no direct inhibition by CDB-4022 of (Bu)2cAMP-stimulated progesterone synthesis. These data suggest that the stimulatory actions on the early step of the steroidogenic pathway, e.g., STAR, are not due to a direct action of CDB-4022 on Leydig cells. Therefore, so far as paracrine factors secreted by Sertoli cells are known to play a role in testicular steroidogenesis, our data are consistent with the idea that the CDB-4022 effect on Leydig cell steroidogenesis is through effects on Sertoli cells [4].

Since CDB-4022 treatment completely suppressed the circulating levels of inhibin B but did not change serum activin A, the intratesticular activin A:inhibin ratio would be predicted to be elevated in CDB-4022-treated rats. Similar results for activin A levels remaining constant over time in CDB-4022-treated rats were recently presented [38]. Our data confirm that hCG-stimulated testosterone secretion is reduced by activin A [16] while demonstrating for the first time an increase in progesterone secretion. Thus, the steroid hormone profile for activin A effects in vitro resembles our in vivo data for CDB-4022-treated rats. In MA-10 cells, activin A increases (Bu)2cAMP-stimulated Star mRNA and protein levels as well as progesterone synthesis. Thus, the activin A effect on progesterone production in primary Leydig cell cultures is most likely due to a direct effect on STAR expression. The effect appears to be biphasic, with maximal activation at lower activin A concentrations. These results are consistent with previous reports showing that activin A or BMP-6 increases basal and ACTH-stimulated Star mRNA levels and reporter gene activity as well as aldosterone production in H295R human adrenal cells [24]. Likewise, in human ovarian thecal-like tumor cell culture model system (HOTT theca cells), cAMP-stimulated progesterone production and Star mRNA and protein levels were increased by activin A and BMP-4 [25]. Cyclic AMP-stimulated Star expression and progesterone synthesis in MA-10 cells were also recently shown to be enhanced by the TGFβ family member Müllerian inhibitory substance (MIS) [19, 21]. Furthermore, MIS was reported to directly suppress Cyp17a1 expression, and the resulting decrease in androgen production was shown to elevate Star expression [39]. According to that logic, decreased testicular testosterone in CDB-4022-treated rats may contribute to the increases in STAR and progesterone synthesis in Leydig cells. Although the mechanism for activin A-mediated suppression of testosterone remains to be clarified, BMP-4 or activin A decreased forskolin-stimulated Cyp17a1 mRNA expression treatment in HOTT theca cells [25]. Thus, the increased activin A:inhibin ratio in CDB-4022-treated rats could potentially enhance the STAR expression while suppressing the CYP17A1 expression. Future studies will focus on the potential regulation of CYP17A1 in Leydig cells by activin A signaling.

There are many examples of hypospermatogenesis, including human male infertility, in which Leydig cell function as well as sperm production are compromised, and it is unclear how the dysfunctions of the two testicular compartments are related [40]. We present the first evidence for the modulation of Leydig cell gene expression and function by the hypospermatogenic agent CDB-4022. Although direct effects of CDB-4022 on Leydig cells cannot be completely excluded at this time, our results with MA-10 cells support previous studies showing that CDB-4022 is not overtly toxic to Leydig cells. In fact, Leydig cell number was increased in CDB-4022-treated rats. Rather, our data support the idea that CDB-4022 affects Leydig cell gene expression and function via an indirect mechanism(s) possibly involving a decrease in inhibin B that results in unopposed activin A signaling. One consequence of activin A is enhanced hCG-stimulated STAR expression and progesterone production; however, the steroid biosynthetic pathway is also disrupted between progesterone and testosterone in CDB-4022-treated rats because CYP17A1 expression and activity are decreased. Whether this mechanism for Leydig cell dysfunction is applicable to other models of seminiferous tubular damage remains to be determined.

ACKNOWLEDGMENTS

We thank Drs. Sheri Hild and Barbara Attardi (BIOQUAL, Inc., Rockville, MD) for their valuable discussions regarding this study and sharing data on CDB-4022 effects on serum activin A levels. We thank Dr. Richard Blye (Contraceptive and Reproductive Health Branch, National Institute of Child Health and Human Development, Rockville, MD) for kindly providing the racemic CDB-4022 mixture. We also thank Dr. Michael R. Waterman (Vanderbilt University, Nashville, TN) for kindly providing the CYP17A1 antiserum.

FOOTNOTES

1Supported by a KY Collaborative Research Development Grant to S.J.W. and a National Institutes of Health grant (DK 51656) to B.J.C. Back

Correspondence: 2FAX: 502 852 6222; e-mail: bjclar01{at}gwise.louisville.edu

Received: 7 December 2006.

First decision: 4 January 2007.

Accepted: 16 August 2007.

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S. Koduri, S. A. Hild, L. Pessaint, J. R. Reel, and B. J. Attardi
Mechanism of Action of l-CDB-4022, a Potential Nonhormonal Male Contraceptive, in the Seminiferous Epithelium of the Rat Testis
Endocrinology, April 1, 2008; 149(4): 1850 - 1860.
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