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Endocrinology, doi:10.1210/en.2003-1237
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Endocrinology Vol. 145, No. 5 2487-2497
Copyright © 2004 by The Endocrine Society

Aberrant Cholesterol Transport and Impaired Steroidogenesis in Leydig Cells Lacking Estrogen Sulfotransferase

M. H. Tong, L. K. Christenson and W.-C. Song

Center for Experimental Therapeutics (M.H.T., W.-C.S.), Department of Pharmacology (W.C.S.), and Center for Research on Reproduction and Women’s Health (L.K.C.), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: W. C. Song, Ph.D., University of Pennsylvania School of Medicine, Room 1351 BRBII/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104. E-mail: song{at}spirit.gcrc.upenn.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfoconjugation and inactivation of estrogens. It is expressed abundantly in the mammalian testes in which it may modulate the activity of locally produced estrogen. We demonstrate here that testicular Leydig cells from mice rendered deficient in EST expression by targeted gene deletion acquire a phenotype of increased cholesterol ester accumulation and impaired steroidogenesis with natural aging or in response to estrogen challenge. Abnormal accumulation of cholesterol ester in the mutant Leydig cells correlated with induced expression of the scavenger receptor type B class I, and cultured EST-deficient but not wild-type Leydig cells avidly uptook high-density lipoprotein cholesterol ester ex vivo. EST-deficient Leydig cells in culture produced 50–70% less testosterone than wild-type cells. This deficiency was reversed by androstenedione but not progesterone supplementation, indicating that reduced activities of 17-{alpha}-hydroxylase-17, 20-lyase were responsible. This conclusion was corroborated by decreased expression levels of 17-{alpha}-hydroxylase-17, 20-lyase but not of other key steroidogenic enzymes in the mutant cells. These results suggest that EST plays a physiologic role in protecting Leydig cells from estrogen-induced biochemical lesions and provide an example of critical regulation of tissue estrogen sensitivity by a ligand-transformation enzyme rather than through estrogen receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OUR UNDERSTANDING OF the role of estrogen in many physiologic or pathophysiologic processes has greatly expanded in recent years. Studies of estrogen insufficiency from estrogen receptor (ER) {alpha}, ERß, ER{alpha}ß double-knockout, and aromatase knockout (KO) mice have revealed a critical role for estrogen in the regulation of male as well as female reproduction (1, 2, 3, 4, 5, 6). In addition, estrogen plays a physiologic role in a wide range of other organ systems such as the bone and the cardiovascular system (2, 7, 8). On the other hand, epidemiological and experimental animal studies have suggested that excessive or inappropriate exposure to estrogenic stimulation can produce serious adverse outcomes (9, 10, 11, 12). For example, there is a well-established connection between cumulative estrogen exposure and the incidence of breast and uterine cancers (9), and inappropriate prenatal exposure to the synthetic estrogen diethylstilbestrol (DES) markedly increases the risk of developing female reproductive tract abnormalities later in life (13, 14). There are also concerns over the potential impact of exposure to environmental chemicals with estrogenic activity on endocrine and reproductive functions of animals and humans (15, 16, 17, 18), although the mechanistic basis of these concerns is debated (19, 20).

In general, the response of tissues to estrogenic stimulation depends on the presence of ERs as well as receptor-active estrogen ligands in the local cellular environment. The level of free estrogen ligand in target tissues, in turn, is determined not only by plasma concentrations of the hormone but also by the local expression of enzymes capable of synthesizing and metabolizing estrogens. One of the notable estrogen transformation enzymes expressed in estrogen target tissues is estrogen sulfotransferase (EST, SULT1E1) (21, 22), a cytosolic enzyme that catalyzes the sulfoconjugation of estrogens at the 3-hydroxyl position. Sulfated estrogens do not bind to the ER and are therefore hormonally inactive (21, 22). EST is differentially expressed in normal and malignant breast epithelial cells, and increased estrogen stimulation as a result of EST down-regulation may contribute to the pathogenesis of breast cancers in humans (23, 24, 25).

Through the use of animal models, we investigated the tissue distribution of EST and described sites in the male reproductive system in which EST is specifically expressed and regulated (26, 27, 28, 29). In the mouse, EST is highly expressed in interstitial Leydig cells of the testis (26, 27, 28) and in the middle to distal regions of the epididymis (29). In both locations, its expression is regulated by the LH via the action of androgen (28, 29). Because the mammalian testes (Leydig cells, Sertoli cells, and germ cells in various stages of differentiation) and epididymal sperm express P450 aromatase and are significant sources of estrogen production in males (30, 31, 32, 33), the prominent expression of EST in these tissues suggests that it may play a physiologic role in protecting these tissues from excessive estrogen stimulation. Indeed, we previously showed that targeted disruption of the EST gene in the mouse resulted in age-dependent development of epididymal dysfunction and Leydig cell hyperplasia/hypertrophy (29, 34).

In the present report, we illustrate that EST-deficient mouse Leydig cells also develop major biochemical defects in cholesterol homeostasis and steroidogenesis with natural aging or in response to estrogen challenge. Specifically, we found that EST-deficient Leydig cells abnormally accumulated cholesterol ester as a result of increased expression of the scavenger receptor type B, class I (SR-BI). They also had decreased expression of 17-{alpha}-hydroxylase-17, 20-lyase (P450 17{alpha}), which led to significantly impaired steroidogenic potential. These findings provide biochemical evidence to support the conclusion that EST normally plays a physiologic role in protecting Leydig cells from estrogen-induced aberrations in cholesterol uptake and steroidogenesis. They suggest that inhibition of EST activity by environmental chemicals, as recently demonstrated in vitro for human EST by hydroxylated polychlorinated biphenyls (35, 36), may cause endocrine disruption by potentiating endogenous estrogen activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments
Wild-type male C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME). EST-deficient mice were generated by gene targeting and backcrossed (for nine generations) to have a C57BL/6 background as previously described (29, 34). Mice were either used directly at 18 months old or were treated at the age of 3 months with exogenous estrogen for 3 d and then used (3 wk releasing 10 µg estradiol pellet, sc; Innovative Research of America, Sarasota, FL). In some experiments, estrogen-treated mice also received human chorionic gonadotropin (hCG) injection (10 IU hCG/animal in 200 µl PBS, sc, Sigma, St. Louis, MO) 3 h before being killed, and sera were collected to determine testosterone and progesterone levels. Immediately after the mice were killed, testes were removed and either processed for histological studies or used for preparing Leydig cells for in vitro experiments. Blood was collected from the aortic artery at the time of killing and serum samples were prepared. Use of mice in this study was approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

Histological studies
Histological studies were carried out on Leydig cells to detect neutral lipid deposition by oil red O or SR-BI expression by immunofluorescence staining. For oil red O staining, testes were snap frozen in OCT medium (Sakura Finetek U.S.A., Inc., Torrance, CA) and sectioned at 5 µm. The tissue sections were first stained with oil red O as described by Pickren (37) and then counterstained with hematoxylin. For immunofluorescence staining of SR-BI, testes were fixed overnight at 4 C in Bouin’s solution before dehydration and paraffin embedding and sectioning at 5 µm. Deparaffinized sections were washed in distilled water and 0.05% Tween in PBS and then incubated for 30 min at room temperature with 10% normal goat serum to block nonspecific binding. A rabbit polyclonal antimouse SR-BI antibody (Novus Biologicals, Inc., Littleton, CO) or preimmune rabbit serum was then added to the slides. After incubating for 30 min at 37 C, the slides were rinsed with PBS and fluorescein-conjugated goat antirabbit IgG (ICN Biomedicals, Inc., Aurora, OH) was added. After 30 min at 37 C, the sections were washed three times (5 min each) in PBS, rinsed quickly in pure ethanol, and then mounted in Mounting Medium (Sigma Diagnostics, St. Louis, MO).

Isolation and culture of primary Leydig cells
Leydig cells were isolated from mouse testes by following a modified procedure of Schumacher et al. (38). Briefly, after removing the testicular capsule, the testes were placed in dissociation buffer at 5 ml/testis [M199 medium containing 0.1% BSA, 25 mM HEPES buffer (pH 7.4)]. Tissues were mechanically dispersed by flushing them up and down 15 times into a 60-ml syringe. Seminiferous tubules from dispersed testes were allowed to settle for 5 min, and the supernatants were collected by aspiration with a pipette. The same procedure was repeated on the settled seminiferous tubules. The combined supernatants were centrifuged at 80 x g for 10 min to collect the Leydig cells. After washing, the crude Leydig cells were purified by continuous Percoll (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) density gradient centrifugation (1.01–1.126 kg/liter). Cells were separated on the basis of buoyant density, and those gathered at the zone equivalent to 1.07 kg/liter of Percoll were collected and washed. The purity of Leydig cells as assessed by 3ß-hydroxysteroid dehydrogenase staining (39) was shown to be greater than 80%. For steroidogenesis assays, purified Leydig cells (105 cells) were resuspended in 1 ml DMEM-Ham’s F12 medium containing 0.1% BSA (pH 7.2) and incubated for 3 h in a 34 C water bath with shaking, either with or without 100 µM dibutyryl-cAMP (dbcAMP), 1 ng/ ml hCG or exogenous steroidogenesis substrates as specified. At the end of the 3-h incubation period, the incubation mixtures were centrifuged at 1500 x g for 10 min at 4 C. Cell pellets were stored at –80 C for total RNA and protein extraction and supernatants were collected for testosterone and progesterone measurement.

In some experiments, purified Leydig cells were also assessed for the uptake of high-density lipoprotein cholesterol ester (HDL-CE) in vitro. Cells were placed on 25 x 25 mm glass coverslips coated with fibronectin (Roche Molecular Biochemicals, Indianapolis, IN) and cultured at 34 C, 5% CO2, and 95% O2 in the presence of HDL-BODIPY-CE (Molecular Probes, Inc., Eugene, OR) (50 µg/ml) for 12 h in DMEM-Ham’s F12 medium, supplemented with 0.1% BSA, 12 mg/ml gentamicin, 1.2 mg/ml sodium bicarbonate, and 20 mM HEPES (pH 7.2). After washing, cells were examined by confocal microscopy to evaluate the degree of internalization of the fluorescent HDL-BODIPY-CE.

Assays of steroid levels in the cell culture medium and serum
Levels of testosterone and progesterone in serum and cell culture medium were determined with RIA kits (Diagnostic System Laboratories, Inc., Webster, TX) by manufacturer’s instruction.

Northern blot analysis
Total RNA samples (20 µg per lane) were separated on 1% denaturing formaldehyde-agarose gels and capillary transferred onto Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Arlington Heights, IL). The membrane was crossed-linked under UV and hybridized with {alpha}-32P (dCTP)-labeled cDNA probes in QuickHyb solution (Stratagene, La Jolla, CA) at 68 C for 1 h. All cDNA templates used in probe preparation were amplified by RT PCR using testis RNA and primers designed based on sequences available in the GenBank. Details of the primers are as follows: for SR-BI cDNA (173 bp) forward primer, 5'-ATCTGCCAACTGCGCAGCCA-3', reverse primer, 5'-GGGCTTATAGTGTCT-TCAGGA; for P450 17 {alpha} cDNA (424 base pairs) forward primer, 5'-GCCTGACA-GACATTCTG-3', reverse primer, 5'-TCGTGATGCAGTGCCCAG-3'; for P450 side-chain cleavage enzyme (P450scc) cDNA (343 bp) forward primer, 5'-GCACACAACTTGAAGGTACAG-3', reverse primer, 5'-CAGCCAAAGCCCAAGTACCGGAAG-3'; for 3ß- hydroxysteroid dehydrogenase (3ß-HSD) cDNA (1360 bp) forward primer, 5'-AATCTGAAAGGTACCCAGAA-3', reverse primer, 5'-TCATCATAGCTTTGGTGAGG-3'; for steroidogenic acute regulatory protein (StAR) cDNA (231 bp) forward primer, 5'-AAGAGCTCAACTGGAGAGCAC-3', reverse primer, 5'-TACTTAGCACTTCGTCCCCGT-3'. Probes were radiolabeled using the Ready-To-Go DNA labeling kit (Amersham Pharmacia Biotech Inc.) and purified using the MicroSpin S-200 HR column (Amersham Pharmacia Biotech Inc.). After hybridization, the membrane was washed first in 2x saline sodium citrate/0.1% sodium dodecyl sulfate (SDS) at 60 C for 15 min and then twice in 0.1x saline sodium citrate/0.1% SDS at 60 C for 10 min and exposed to x-ray film at –80 C. In experiments with older mice in which the RNA samples were limited, membranes were stripped after the first hybridization and reused for other cDNA probes. In all other experiments, individual membranes were used for separate probes.

Western blot analysis
Western blot analysis was performed with the ECL Western blotting detection system (Amersham Pharmacia Biotech). Leydig cells were homogenized at 4 C in radioimmunoprecipitation assay buffer [50 mM Tris buffer containing 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholic acid, 1 mM EDTA (pH 8.0)] in the presence of a protease inhibitor cocktail (Sigma). The homogenate was centrifuged at 15,000 x g for 2 min, and the resulting supernatant was used for Western blot analysis (50 µg per lane). Concentrations of the protein samples were determined by the Bradford method using a protein assay kit (Pierce Chemical, Rockford, IL). Samples were electrophoresed on 10% SDS polyacrylamide gels and blotted onto nitrocellulose membranes (0.45 µm, Bio-Rad Laboratories, Inc., Hercules, CA). The membranes were probed with antibodies against SR-BI, P450 17{alpha}, StAR, P450scc, and 3ß-HSD. In most cases, the membranes were stripped after use and reprobed with a ß-actin antibody to confirm equal protein loading. Sources of the antibodies used are as follows: a rabbit antimouse 3ß-HSD polyclonal antibody was kindly provided by Dr. A. Payne (Stanford University, Palo Alto, CA); a rabbit antimouse P450 17{alpha} polyclonal antibody was kindly provided by Dr. D. B. Hales (University of Illinois, Chicago, IL); a rabbit antihuman StAR polyclonal antibody that cross-reacts with mouse StAR was kindly provided by Dr. J. Strauss (University of Pennsylvania, Philadelphia, PA); a rabbit antimouse P450scc polyclonal antibody was obtained from Chemicon International (Temecula, CA). A rabbit polyclonal antimouse SR-BI antibody was from Novus Biologicals Inc. Monoclonal anti-ß-actin clone AC-15 antibody was obtained from Sigma.

Total lipid extraction and thin-layer chromatography (TLC)
Total lipid was extracted from purified Leydig cells as described by Asmis and Jelk (40). Briefly, the volume of cell suspension was adjusted to 800 µl with water, and 3ml methanol/dichloromethane (2:1, vol/vol) containing 0.001% BHT (2,6-di-tert-butyl-4-methylphenol) was added. The sample was vortexed vigorously before 1 ml dichloromethane and 500 µl water were added to achieve phase separation. The aqueous phase was reextracted twice with 1 ml dichloromethane, and the organic phases were combined, evaporated under a stream of nitrogen to dryness, and stored at –20 C until TLC analysis. TLC analysis was performed according to a modified method described by Christie (41). Briefly, samples were resuspended in methanol/dichloromethane (2:1, vol/vol), spotted onto TLC plates (Whatman International Ltd., Kent, UK) and developed sequentially in two solvent systems. Free cholesterol, cholesterol ester, and triglyceride standards (Sigma) were also applied onto the TLC plates in separate lanes. The plates were first developed in heptane/diethyl ether/acetic acid (70:20:4, vol/vol/vol) until the solvent front migrated approximately 10 cm from the origin. After drying at room temperature, the plates were redeveloped in heptane until the solvent front migrated 15 cm from the origin. This second step allowed a better separation of cholesteryl esters from triglycerides. TLC spots were visualized by spraying cupric sulfate followed by heating the TLC plates at 120 C for 5 min. To reduce background staining, all TLC plates were predeveloped in methanol before use.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Abnormal accumulation of cholesterol ester in EST-deficient mouse Leydig cells
We previously found that Leydig cells from old (>12 months old) EST KO mice or from young adult KO mice treated with exogenous estrogen exhibited morphological abnormalities (34). They appeared to be hypertrophic (increased cell volume), and their cytoplasm did not stain well with hematoxylin and eosin staining (34). The general appearance of these Leydig cells is reminiscent of differentiated adipocytes. Their appearance prompted us to investigate if the mutant mouse Leydig cells might have abnormal lipid accumulation as a result of either a blockage in the steroidogenic pathway or deregulated cholesterol transport. To test this hypothesis, we stained testicular sections of 18-month-old wild-type and EST KO mice with oil red O. Figure 1Go, A–D, shows that Leydig cells of EST KO mice stained prominently with oil red O, suggesting that there was indeed accumulation of neutral lipid in the mutant mouse Leydig cells. Similar to the morphological changes described in a previous study (34), the accumulation of lipid in the KO mouse Leydig cells was age dependent. Thus, testes of 2- to 3-month-old KO mice appeared normal with only scattered lipid deposits (data not shown), whereas in 18-month-old KO mouse testes, more than 90% of the interstitial area stained positive (Fig. 1CGo). In contrast, testes of 2- to 3-month-old wild-type mice were devoid of lipid deposition (data not shown) and less than 20% of the interstitial area in 18-month-old wild-type mice stained positive with oil red O (Fig. 1AGo). To determine whether the abnormal lipid deposition in the KO mouse Leydig cells resulted from increased estrogen stimulation, we treated 3-month-old wild-type and KO mice with exogenous estradiol for 3 d at an equivalent dose of 0.5 µg/d and then examined lipid accumulation in their Leydig cells. Figure 1Go, E–H, shows that strong oil red O staining was detected in almost 100% of the interstitial areas in the treated KO mouse testes, whereas no staining was apparent in similarly treated wild-type mouse testes.



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  		FIG. 1. Marked lipid accumulation in EST-deficient mouse Leydig cells. Cryostat sections of testes from 18-month-old and estrogen-treated 3-month-old wild-type or EST KO mice were stained with oil red O (A and B, 18-month-old wild-type; C and D, 18-month-old KO; E and F, estrogen-treated 3-month-old wild-type; G and H, estrogen-treated 3-month-old KO. Magnification, x100 for left panels; x400 for right panels). Most Leydig cells in the KO mouse testes stained positive for neutral lipid (C, D, G, and H), whereas only isolated Leydig cells in 18-month-old wild-type mice (A and B) were positive. Leydig cells from estrogen-treated wild-type mice were essentially devoid of lipid accumulation (E and F). Results are representative of at least six independent experiments.

 
Because oil red O staining does not differentiate between triglycerides and cholesterol esters, we next sought to determine the identity of the neutral lipid(s) accumulated in the KO mouse Leydig cells. Total lipids extracted from Leydig cells of 18-month-old or estradiol-treated 3-month-old wild-type and KO mice were analyzed by TLC. Figure 2Go shows that on this analysis, free cholesterol and triglyceride and cholesterol ester standards were clearly separated. It is evident that there was no detectible triglyceride accumulation in any of the Leydig cells, whereas substantial amounts of cholesterol ester were detected in the Leydig cells of both 18-month-old and estrogen-treated 3-month-old EST KO mice (Fig. 2Go). No cholesterol esters were detected in Leydig cells of estrogen-treated 3-month-old wild-type mice, although a small amount was detected in the cells of 18-month-old wild-type mice (Fig. 2Go). These results are consistent with the oil red O staining data, and they identify the abnormally accumulated neutral lipids in the knockout mouse Leydig cells as cholesterol esters.



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  		FIG. 2. Identification by TLC of the neutral lipid(s) accumulated in EST-deficient mouse Leydig cells. Commercial standards of free cholesterol, triacyglycerols (TAG), and cholesterol esters were used to indicate the positions on TLC plate of different classes of lipid. No significant amount of TAG was detected in any of the Leydig cells. In contrast, high levels of cholesterol esters were detected in Leydig cells of 18-month-old and estrogen-treated 3-month-old KO mice. There was also an appreciable amount of cholesterol esters in the Leydig cells of 18-month-old but not estrogen-treated 3-month-old wild-type mice. Leydig cells isolated from three to four mice were pooled in each group for total lipid extraction. Results are representative of three independent experiments.

 
Decreased steroidogenic potential in EST KO mouse Leydig cells
Estrogen may directly inhibit Leydig cell steroidogenesis via paracrine and intracrine mechanisms (42). We therefore wondered whether the abnormal accumulation of cholesterol ester in the EST KO mouse Leydig cells was due to a blockage in the steroidogenic pathway, leading to decreased cholesterol metabolism. To test this possibility, we isolated Leydig cells from estrogen-treated 3-month-old wild-type and EST KO mice and cultured them in vitro, either with or without cAMP or hCG stimulation. After 3 h of culture, testosterone secretion into the cell culture medium was determined by RIA. Figure 3Go, A and B, shows that EST-deficient Leydig cells synthesized 50–70% less testosterone than wild-type Leydig cells. The reduction in testosterone production in the mutant cells was especially evident when cells were stimulated with cAMP (Fig. 3BGo), a second messenger for LH whose acute effect is to increase cholesterol mobilization. Stimulation with hCG produced result similar to that of cAMP stimulation (data not shown). These findings suggested that the defect in steroidogenesis in the mutant Leydig cells was downstream of LH signaling. In similar experiments using Leydig cells from nontreated mice, we did not detect any significant differences in testosterone production between 3-month-old wild-type and KO mice (data not shown).



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  		FIG. 3. EST-deficient mouse Leydig cells have impaired steroidogenesis but retain normal expression of StAR. Isolated Leydig cells from estrogen-treated 3-month-old wild-type or KO mice were cultured ex vivo (1 x 105 cells in 1 ml medium), and testosterone secretion into the culture medium was determined 3 h later. Cells were either untreated (basal, A), treated with 100 µM dbcAMP with or without 100 µM 22-hydroxycholesterol (22-OH) as an exogenous substrate (B and D) or were treated with 22-OH only (C). Leydig cells from six mice in each group were pooled, and values shown in A–D are mean ± SD of four replicate incubations [P < 0.05 (A) and P < 0.001 (B–D; Student’s t test)]. E and F, StAR mRNA (E) and protein (F) levels were not changed in Leydig cells of 18-month-old or 3-month-old estrogen-treated KO mice. Pooled cells from three 18-month-old mice were used directly and those from estrogen-treated 3-month-old mice (pooled from six mice) were cultured for 3 h with or without cAMP treatment (collected from the steroidogenesis experiments). As expected, cAMP treatment significantly increased StAR expression in wild-type mouse Leydig cells (43 ). A similar response to cAMP treatment was also observed with EST-deficient mouse Leydig cells. Three different StAR mRNA species and two StAR protein isoforms (43 ) were detected. Equal RNA and protein loading was confirmed by RNA and ß-actin levels. Results in A–F are representative of at least three independent experiments except the 18-month-old group, which was performed twice.

 
To investigate whether the steroidogenic lesion in the KO mouse Leydig cells arose from a defect in cholesterol metabolism, we studied the expression of StAR in wild-type and EST-deficient Leydig cells by Northern and Western blot analysis. StAR is a mitochondrial membrane protein whose expression is acutely stimulated by LH/cAMP (43). It plays an essential role in cholesterol metabolism by facilitating cholesterol transport across the mitochondrial membrane to initiate the first and rate-limiting step in steroidogenesis, catalyzed by P450scc (43, 44). As shown in Fig. 3Go, E and F, we found no significant difference in Leydig cell StAR mRNA and protein levels between 18-month-old or estrogen-treated 3-month-old wild-type and EST KO mice [Fig. 3EGo: average densitometry ratios of StAR and 18S rRNA (mean ± SEM); for 18-month-old mice, WT: 0.31 ± 0.02, KO: 0.35 ± 0.02, n = 2; for estrogen-treated 3-month-old mice and cells stimulated with cAMP, WT: 0.63 ± 0.08, KO: 0.72 ± 0.14, n = 3; for nonstimulated cells, WT: 0.35 ± 0.07, KO: 0.31 ± 0.02, n = 3. Fig. 3FGo: ratios of StAR and ß-actin: for 18-month-old mice, WT: 0.80 ± 0.10, KO: 0.74 ± 0.18, n = 3; for estrogen-treated 3-month-old mice and cells stimulated with cAMP, WT: 0.78 ± 0.01, KO: 0.73 ± 0.09, n = 3; for nonstimulated cells, WT: 0.17 ± 0.08, KO: 0.20 ± 0.08, n = 3]. These results argued against the possibility of a StAR-related defect in cholesterol metabolism and suggested that the steroidogenic lesion may lie somewhere downstream of the substrate mobilization step.

To confirm that StAR was not involved, we incubated Leydig cells isolated from estrogen-treated 3-month-old wild-type and EST KO mice with 22R-hydroxycholesterol, a hydrophilic cholesterol derivative that is a known substrate for P450scc (44, 45). Unlike endogenous cholesterol, 22R-hydroxycholesterol can readily diffuse across the mitochondrial membrane and thus can bypass the requirement of a StAR-facilitated cholesterol transport mechanism (44, 45). Figure 3Go, C and D, shows that when 22R-hydroxycholesterol was added as a substrate, testosterone production by EST-deficient Leydig cells was significantly lower than that of wild-type cells. Additionally, measurement of pregnenolone levels in medium from wild-type (13.5 ± 0.3 ng/ml, mean ± SEM) and EST KO (14.1 ± 0.4 ng/ml) Leydig cells indicated no perturbation in StAR or P450scc activity. This provided further evidence that the steroidogenic defect in estrogen-treated EST KO mouse Leydig cells occurred after the cholesterol mobilization.

Reduced expression of P450 17{alpha} accounts for the steroidogenic lesion in EST KO mouse Leydig cells
After cholesterol is delivered to the inner space of mitochondria, steroidogenesis proceeds via the actions of P450scc, 3ß-HSD, and P450 17{alpha}. To determine whether reduced expression of any of these enzymes was responsible for the steroidogenic lesion in the EST KO mouse Leydig cells, we performed Northern and Western blot analysis to compare the mRNA and protein levels of these enzymes in wild-type and EST KO mice. Figure 4Go, A–D, shows that Leydig cells from estrogen-treated 3-month-old EST KO mice contained similar levels of P450scc and 3ß-HSD to cells of similarly treated wild-type mice, irrespective of whether the cells were stimulated with cAMP in culture [Fig. 4AGo, average densitometry ratios of P450scc and 18S rRNA (mean ± SEM): for cAMP-treated cells, WT: 0.63 ± 0.08, KO: 0.72 ± 0.05, n = 3; for nonstimulated cells, WT: 0.36 ± 0.08, KO: 0.38 ± 0.01, n = 3. Fig. 4BGo, ratios of P450scc and ß-actin: for cAMP-treated cells, WT: 0.53 ± 0.06, KO: 0.60 ± 0.09, n = 3; for nonstimulated cells, WT: 0.56 ± 0.09, KO: 0.56 ± 0.08, n = 3. Fig. 4CGo, ratios of 3ß-HSD and 18S rRNA: for cAMP-treated cells, WT: 0.58 ± 0.09, KO: 0.55 ± 0.04, n = 3; for nonstimulated cells, WT: 0.50 ± 0.07, KO: 0.53 ± 0.10, n = 3. Fig. 4DGo, ratios of 3ß-HSD and ß-actin: for cAMP-treated cells, WT: 0.57 ± 0.06, KO: 0.55 ± 0.06, n = 3; for nonstimulated cells, WT: 0.55 ± 0.10, KO: 0.53 ± 0.10, n = 3]. In contrast, as shown in Fig. 4Go, E and F, the mRNA and protein levels of P450 17{alpha} in Leydig cells of 18-month-old or estrogen-treated 3-month-old EST KO mice were significantly reduced, compared with that of age- and treatment-matched wild-type mice [Fig. 4EGo, ratios of P450 17{alpha} and 18S ribosomal RNA (mean ± SEM): estrogen-treated 3-month-old mice and cells stimulated with cAMP, WT: 0.78 ± 0.07, KO: 0.50 ± 0.10, n = 3; nonstimulated cells, WT: 0.71 ± 0.05, KO: 0.50 ± 0.02, n = 3; 18-month-old mice, WT: 1.16 ± 0.11, KO: 0.72 ± 0.09, n = 3; Fig. 4FGo, ratios of P450 17{alpha} and ß-actin: for cAMP-treated cells, WT: 0.91 ± 0.01, KO: 0.44 ± 0.06, n = 3; for nonstimulated cells, WT: 0.94 ± 0.11, KO: 0.55 ± 0.09, n = 3; 18-month-old mice, WT: 0.83 ± 0.05, KO: 0.26 ± 0.05, n = 3]. Thus, estrogen-induced inhibition of P450 17{alpha} was potentially responsible for the steroidogenic lesion observed in the EST-deficient mouse Leydig cells.



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  		FIG. 4. Northern (A, C, and E) and Western blot analysis (B, D, and F) of P450scc, 3ß-HSD, and P450 17{alpha} in EST-deficient mouse Leydig cells. Compared with wild-type mouse Leydig cells (WT), EST-deficient Leydig cells (KO) had similar levels of P450scc (A and B) and 3ß-HSD (C and D) but had reduced expression of P450 17{alpha} (E and F). Leydig cells from estrogen-treated 3-month-old mice were used for P450scc and 3ß-HSD analysis. Pooled Leydig cells from both 18-month-old (three mice) and estrogen-treated 3-month-old mice (six mice) were used for P450 17{alpha} analysis. Cells from 18-month-old mice were used directly and those from estrogen-treated 3-month-old mice were cultured for 3 h either with (cAMP) or without (basal) dbcAMP before RNA and protein extraction. Leydig cells isolated from three to four mice were pooled in each group, and result shown is representative of three independent experiments. Equal RNA and protein loading was confirmed by RNA and ß-actin levels.

 
To test the hypothesis that reduced expression of P450 17{alpha} was responsible for the steroidogenic defect in EST-deficient Leydig cells, we measured progesterone levels in the Leydig cell culture media. Progesterone is a major substrate of P450 17{alpha} in the steroidogenic pathway and a reduction in P450 17{alpha} enzyme activity would be expected to result in abnormal accumulation of unmetabolized progesterone in the cell culture medium. Figure 5AGo demonstrates that the level of progesterone in the cell culture media of EST-deficient Leydig cells was greatly elevated, compared with that of wild-type Leydig cells, both when the cells were stimulated with cAMP and incubated with 22R-hydroxycholesterol as a substrate. To confirm this finding, we directly tested Leydig cell P450 17{alpha} activity by adding 100 µm progesterone or 17- hydroxyprogesterone as a substrate to the cell culture. Figure 5BGo shows that testosterone production by EST-deficient Leydig cells from either progesterone or 17-hydroxyprogesterone was similarly reduced, compared with that of wild-type Leydig cells. In contrast, no difference was found between wild-type and EST-deficient Leydig cells in their ability to convert androstenedione to testosterone, a step that is distal to P450 17{alpha} activity (Fig. 5BGo). Collectively, these results indicated that steps before and after P450 17{alpha} in the steroidogenic cascade were normal in EST-deficient Leydig cells and that the estrogen-induced steroidogenic lesion in these cells resulted solely from reduced P450 17{alpha} expression.



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  		FIG. 5. Reduced P450 17{alpha} activity was responsible for the steroidogenic lesion in EST-deficient mouse Leydig cells. Pooled cells from six estrogen-treated 3-month-old wild-type (WT) or EST KO mice (KO) were cultured ex vivo for 3 h either without any treatment (basal) or with treatment of 100 µM dbcAMP, 22-hydroxy-cholesterol (22-OH), progesterone (P4), 17-hydroxyprogesterone (17-OH-P4), or androstenedione (Andro). A, Levels of progesterone, a substrate for P450 17{alpha}, were significantly elevated in the cell culture media of EST-deficient Leydig cells. Values shown are mean ± SD of four replicate incubations. (P > 0.05 for basal and P < 0.001 for all others, Student’s t test). B, Testosterone levels in the cell culture media with or without supplementation of steroidogensis intermediates. Supplementation in the culture media with Andro but not with two P450 17{alpha} substrates, progesterone (P4), and 17-hydroxy-progesterone (17-OH-P4) corrected the steroidogenic lesion. Values shown are mean ± SD of three replicate incubations (P < 0.05 for basal and 17-OH-P4 incubations, P < 0.001 for progesterone incubation, and P = 0.5 for androstenedione incubation, Student’s ttest). Results in A and B are representative of two to three independent experiments.

 
To determine whether the steroidogenic lesion detected in in vitro cultures is also present in vivo, we measured serum testosterone and progesterone levels in estrogen-treated 3-month-old wild-type and EST-deficient mice. We found that EST-deficient mice had lower serum testosterone and higher serum progesterone levels, both under basal condition and when stimulated for 3 h with a bolus hCG injection [basal testosterone (mean ± SEM), WT: 1.10 ± 1.07 ng/ml, KO: 0.22 ± 0.22 ng/ml, n = 8 for both groups; basal progesterone, WT: 1.47 ± 0.98 ng/ml, KO: 3.04 1 ± 0.82 ng/ml, n = 12 for both groups; hCG stimulated testosterone, WT: 27.5 ± 6.5 ng/ml, KO: 14.2 ± 8.5 ng/ml, n = 6 for WT and n = 9 for KO; hCG stimulated progesterone, WT: 6.7 ± 3.23 ng/ml, KO: 24.3 ± 5.49 ng/ml, n = 6 for WT and n = 9 for KO; P < 0.05 for all comparisons between WT and KO groups, Student’s t test].

EST-deficient Leydig cells have increased expression of SR-BI in response to estrogen challenge or with natural aging
Because we detected no impairment in cholesterol metabolism, we investigated the possibility that EST-deficient Leydig cells exhibited increased cholesterol uptake to explain the abnormal accumulation of cholesterol ester in these cells. In steroidogenic tissues and the liver, the scavenger receptor SR-BI has been implicated as a major cell surface HDL receptor involved in cholesterol influx, and previous studies have demonstrated that SR-BI expression in the rat adrenal cortex and testis was induced by estrogen treatment (46, 47, 48). As shown in Fig. 6Go, A and B, Northern and Western blot analysis revealed that SR-BI mRNA and protein levels were indeed significantly elevated in Leydig cells of 18-month-old or estrogen-treated 3-month-old EST KO mice, compared with that of age- and treatment-matched wild-type mice [Fig. 6AGo: average densitometry ratios of SR-BI and 18S ribosomal RNA (mean ± SEM), for 18-month-old mice, WT: 0.34 ± 0.02, KO: 1.47 ± 0.06, n = 3; for estrogen-treated 3-month-old mice, WT: 0.16 ± 0.02, KO: 0.83 ± 0.06, n = 3. Fig. 6BGo, ratios of SR-BI and ß-actin, for 18-month-old mice, WT: 0.09 ± 0.001, KO: 0.98 ± 0.03, n = 3; for estrogen-treated 3-month-old mice, WT: 0.08 ± 0.01, KO: 1.02 ± 0.06, n = 3]. The increased expression of SR-BI on EST-deficient Leydig cells was further confirmed by immunofluorescence analysis of testicular sections (Fig. 6CGo). Finally, when isolated Leydig cells were cultured for 12 h ex vivo with HDL-BODIPY-CE, a fluorescent HDL-CE, cells from estrogen-treated 3-month-old EST-deficient mice internalized significant amount of cholesterol ester, whereas cells from similarly treated wild-type mice failed to do so (Fig. 6DGo). Thus, increased SR-BI expression on EST-deficient mouse Leydig cells correlated with enhanced uptake of HDL-CE by these cells.



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  		FIG. 6. Induced expression of SR-BI and enhanced uptake of HDL-CE in EST-deficient mouse Leydig cells. Northern blot (A), Western blot (B), and immunofluorescence (C) analysis show that SR-BI expression was significantly induced in both 18-month-old and estrogen-treated 3-month-old EST-deficient (KO) mice, compared with age- and treatment-matched wild-type (WT) mice. Western blot results with estrogen-treated 3-month-old mice (B) were from individual mice. All other results shown in A and B were from Leydig cells pooled from three to four mice. Equal RNA and protein loading was confirmed by RNA and ß-actin levels. Designations in C are: a, 18-month-old wild-type; b, 18-month-old KO; c, estrogen-treated 3-month-old wild-type; d, estrogen-treated 3-month-old KO. When cultured ex vivo, Leydig cells from estrogen-treated 3-month-old EST-deficient (a in D) but not estrogen-treated 3-month-old wild-type mice (c in D) avidly uptook fluorescent HDL-BODIPY-CE. Dark-field microscopy of the same cells is shown (b and d in D). Magnification, x400 for pictures in C and x200 for pictures in D. Results are representative of at least three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
EST catalyzes the sulfoconjugation and inactivation of estrogens. Previous studies have conclusively demonstrated that EST is capable of metabolizing estrogen at a physiologic concentration (24, 49, 50) and suggested that it may play a highly relevant role in determining estrogen sensitivity in tissues in which it is expressed. In mammalian species, one of the major sites in which EST is present at high levels is the male reproductive system (26, 27, 29). Its expression there is regulated by androgen and the pituitary hormone LH (27, 28, 29). Our prior studies showed that targeted deletion of the EST gene in the mouse led to age-dependent development of testicular abnormalities including Leydig cell hyperplasia, hypertrophy, and seminiferous tubule atrophy (34). There was also an age-dependent decline in epididymal function in the EST KO mice with the consequence that sperm isolated from 1-yr-old KO mice had greatly reduced motility (29). In the present study, we characterized two major biochemical defects in the KO mouse Leydig cells associated with the age-dependent morphological changes previously observed. We found that, with natural aging or in response to estrogen challenge, EST-deficient mouse Leydig cells abnormally accumulated cholesterol esters as a result of up-regulation of the HDL cholesterol receptor SR-BI. They also had a selective decrease in P450 17{alpha} expression, leading to a steroidogenic lesion as reflected by reduced testosterone production and progesterone accumulation. These results support the conclusion that EST plays a physiologic role by protecting Leydig cells from potential adverse effects caused by locally produced estrogen in the testis.

It is now well recognized that estrogen plays an indispensable role in the reproductive systems of both male and female animals (2, 51). Targeted deletion of ER{alpha} in the mouse resulted in progressive damage of seminiferous tubules due to impairment of the absorptive function of the efferent ductules in the proximal epididymis, ultimately producing a male infertility phenotype (1, 52). Likewise, disruption of the gene encoding the key enzyme in estrogen biosynthesis, P450 aromatase, caused an age-dependent decline in spermatogenesis that eventually rendered male P450 aromatase KO mice sterile (5, 6). These interesting phenotypes of the two KO mice are consistent with earlier findings that ER{alpha} and P450 aromatase are expressed in the testis, germ cells, and the epididymides of multiple animal species (2, 30, 31, 32), and they suggest that estrogen exerts a key regulatory role in male reproduction through an ER-mediated mechanism.

In addition to serving the local estrogen needs in the male reproductive system, the estrogen biosynthetic pathway in the testis may also contribute significantly to the systemic estrogen pool from which estrogens required by extragonadal tissues like the bone is derived (51). Indeed, it was determined that the concentration of estrogen in the human spermatic vein is 50 times higher than that in the systemic circulation (53), and it is well accepted that, in male animals and man, the testis is a significant source for peripheral estrogens (33, 53). The prominent expression of EST in Leydig cells and other parts of the male reproductive tract may, therefore, constitute a protective mechanism to prevent these cells and tissues from excessive stimulation by the locally produced estrogen. Presumably, testis-derived estrogen sulfates are hydrolyzed by the steroid sulfatase (54) to generate free and receptor-active hormones once they are transported to periphery tissues.

The present study demonstrates potential consequences in Leydig cell function when such a protective mechanism is abrogated. Mice engineered to be EST-deficient abnormally accumulated cholesterol ester in their Leydig cells as they aged or in response to exogenous estrogen challenge. This aberrant biochemical event is likely responsible for the hypertrophic appearance and weak cytoplasmic eosin staining previously observed with these cells (34). Several lines of evidence suggest that the increased cholesterol ester accumulation in the mutant Leydig cells resulted from enhanced uptake of cholesterol ester rather than decreased use of cholesterol as a precursor in the steroidogenic pathway. First, by Northern blot, Western blot, and immunofluorescence studies, we showed that SR-BI, a major HDL receptor that mediates the selective uptake of cholesterol ester in liver and steroidogenic cells, was significantly up-regulated in EST-deficient Leydig cells, compared with wild-type Leydig cells. This finding was consistent with a previously published study in which estrogen administration was shown to induce SR-BI expression in the rat adrenal gland and ovary (48), although similar inductions were not observed in mice, suggesting a species difference in the sensitivity of SR-BI induction in steroidogenic tissues by estrogen (48). It appears that EST plays a more effective role in the wild-type mouse than in the rat.

Second, through the use of a fluorescent HDL-CE, we demonstrated directly that EST-deficient Leydig cells had rapid cholesterol ester uptake ex vivo, whereas the same phenomenon was not observed with wild-type cells. Finally, we detected no significant change in Leydig cell StAR expression in aged or estrogen-treated EST KO mice. StAR is a protein that facilitates cholesterol transport across the mictochondrial membrane and is essential for initiating the steroidogenic cascade (43). Mutations of the StAR gene in humans and mice caused lipoid adrenal hyperplasia, a condition characterized by abnormal cholesterol build-up in adrenal cortical cells due to the lack of cholesterol metabolism (44, 55). Because we observed no change in StAR expression, we concluded that the phenotype of abnormal cholesterol ester accumulation in EST-deficient mouse Leydig cells arose by a mechanism that is different from that operating in lipoid adrenal hyperplasia. Whereas supporting a major contributory role of increased cholesterol ester uptake, our data did not exclude the possibility that other mechanisms, such as altered intracellular cholesterol biosynthesis and/or cholesterol ester hydrolysis, and abnormal expression or function of sterol carrier protein 2 and peripheral benzodiazepine receptor might also have played a role in the pathogenesis of the mutant mouse Leydig cell phenotype.

In addition to the disturbance in cholesterol homeostasis, we also found that EST-deficient mouse Leydig cells are susceptible to developing estrogen-induced steroidogenic lesion. It is unlikely, however, that this steroidogenic defect is caused by abnormal cholesterol mobilization because addition of 22R-hydroxycholesterol, an exogenous substrate that can bypass the need of a StAR-mediated cholesterol transport mechanism, failed to correct the defect. The conclusion that abnormal cholesterol transport is not responsible for the steroidogenic lesion is also supported by the finding that StAR is expressed normally in these cells. Investigation by Northern blot and Western blot analysis of the downstream steroidogenic enzyme system revealed no changes in the expression levels of P450scc and 3ß-HSD. On the other hand, P450 17{alpha} expression was found to be significantly reduced. Decreased P450 17{alpha} expression appeared to be the sole underlying cause of the steroidogenic lesion because supplementation in the cell culture medium with androstenedione (an intermediate in testosterone biosynthesis distal to P450 17{alpha} action) but not with two P450 17{alpha} substrates, progesterone, and 17-hydroxyprogesterone corrected the deficiency in testosterone biosynthesis. Furthermore, concomitant with reduced testosterone production, progesterone level in cell culture medium was abnormally elevated.

Our finding of estrogen-induced P450 17{alpha} inhibition in the EST-deficient mouse Leydig cells is consistent with previous published studies that demonstrated similar effects of exogenous estrogen on Leydig cells. It was shown almost 40 yr ago that the synthetic estrogen DES inhibited Leydig cell P450 17{alpha} activity in BALB/c mice (56). This observation was subsequently extended to a hypophysectomized rat model in which both DES and 17ß-estradiol were found to inhibit Leydig cell P450 17{alpha} expression and activity (57, 58). Leydig cells from hypophysectomized rats may have partially or completely inhibited EST expression. Our previous studies in the mouse have established that EST expression in Leydig cells is totally dependent on LH (28). Additionally, the estrogen sensitivity of wild-type Leydig cells in these settings was likely related to the fact that DES is a relatively poor substrate of EST (50). Thus, lack of EST-dependent modulation of estrogen activity was likely responsible as well for the observed estrogen sensitivity in these earlier studies.

It is of interest that, despite the reduced P450 17{alpha} expression and impaired steroidogenesis by isolated Leydig cells ex vivo, we previously found that serum testosterone levels in 18-month-old EST KO mice were not significantly depressed (34). One potential explanation is that reduced steroidogenesis by individual Leydig cells was compensated by an increase in total Leydig cell number in vivo as a result of Leydig cell hyperplasia, another phenotype that develops in older EST KO mice (34). Indeed, under the short-term estrogen treatment regimen used in the current study, which did not cause Leydig cell hyperplasia in either wild-type or KO mice, EST-deficient mice were found to have lower serum testosterone and higher serum progesterone levels, both under basal condition and when stimulated for 3 h with a bolus hCG injection.

Our results exemplify in a physiological setting the concept that tissue estrogen sensitivity can be regulated by a ligand-transformation enzyme rather than through the ERs. Recent enzymatic and crystallographic studies have identified human EST as a target of potent inhibition by hydroxylated polychlorinated biphenyls (35, 36), a class of environmental chemicals whose potential as endocrine disrupters in humans and animal species has caused concern but whose mechanism of action is not well understood (17, 19, 20). The biochemical defect in EST-deficient mouse Leydig cells described here suggests that inhibition of EST activity by environmental agents such as hydroxylated polychlorinated biphenyls may produce dysfunction of Leydig cells as well as cause abnormality in other estrogen target tissues in which EST is expressed. Thus, by inhibiting estrogen transformation enzymes such as EST, an otherwise nonestrogenic chemical may become highly estrogenic in vivo by potentiating endogenous estrogen activity. This may constitute a previously unrecognized mechanism of action for certain endocrine disrupting chemicals.


   Acknowledgments
 
We thank Drs. Dan Rader and Jane Glick for advice on lipid analysis and Drs. Anita Payne, Buck Hales, and Jerome Strauss for providing antibodies.


   Footnotes
 
This work was supported by National Institutes of Health Grant HD-42767 (to W.C.S.) and a Mellon Foundation grant (to L.K.C.).

Abbreviations: dbcAMP, Dibutyryl-cAMP; DES, diethylstilbestrol; ER, estrogen receptor; EST, estrogen sulfotransferase; hCG, human chorionic gonadotropin; HDL-CE, high-density lipoprotein cholesterol ester; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; KO, knockout; P450 17{alpha}, 17-{alpha}-hydroxylase-17, 20-lyase; P450scc, P450 side-chain cleavage enzyme; SDS, sodium dodecyl sulfate; SR-BI, scavenger receptor type B, class I; StAR, steroidogenic acute regulatory protein; TLC, thin-layer chromatography.

Received September 17, 2003.

Accepted for publication January 21, 2004.


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Mol. Endocrinol., August 1, 2007; 21(8): 1781 - 1790.
[Abstract] [Full Text] [PDF]

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E. Gershon, A. Hourvitz, S. Reikhav, E. Maman, and N. Dekel
Low expression of COX-2, reduced cumulus expansion, and impaired ovulation in SULT1E1-deficient mice
FASEB J, June 1, 2007; 21(8): 1893 - 1901.
[Abstract] [Full Text] [PDF]

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X. Li, L. Strauss, A. Kaatrasalo, A. Mayerhofer, I. Huhtaniemi, R. Santti, S. Makela, and M. Poutanen
Transgenic Mice Expressing P450 Aromatase as a Model for Male Infertility Associated with Chronic Inflammation in the Testis
Endocrinology, March 1, 2006; 147(3): 1271 - 1277.
[Abstract]