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Are Necessary to Repress the Formation of Morphological and Functional Leydig-Like Cells in the Female Gonad
Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology (J.F.C., M.M.Y., K.F.R., K.S.K.), Laboratory of Experimental Pathology (J.A.J.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; and Medicinal Chemistry Division (D.P.), Oncology and Molecular Endocrinology Research Center, Centre Hospitalier Universitaire de Quebec, Sainte-Foy, Quebec, Canada G1V 4G2
Address all correspondence and requests for reprints to: Dr. Kenneth S. Korach, Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, MD B3-02, P.O. Box 12233, Research Triangle Park, North Carolina 27709. E-mail: korach{at}niehs.nih.gov.
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Abstract |
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. ER-null (ERKO) female mice exhibit testis-like levels of Hsd17b3 expression in the ovaries and male-like levels of plasma T. Herein, we demonstrate that: 1) Hsd17b3 expression in ERKO ovaries is a primary effect of the loss of intraovarian ER actions; 2) ERKO ovarian cells produce substantial levels of T in vitro, and this is blocked by a HSD17B3-specific inhibitor; 3) Hsd17b3 expression in ERKO ovaries is LH regulated and localized to the secondary interstitial (SI)/TI cells; and 4) ERKO SI/TI cells possess Leydig-like ultrastructural features. These data indicate that intraovarian ER actions are required to repress Hsd17b3 expression in the ovary and may be important to maintaining a female phenotype in SI/TI cells. |
Introduction |
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The importance of sexual dimorphic expression of HSD17B1 and HSD17B3 in female and male gonads, respectively, is well appreciated yet poorly understood. This is especially true of the mechanisms that restrict HSD17B3 expression to testicular Leydig cells. Leydig cells constitutively express the LH receptor and rely on LH stimulation to maintain HSD17B3 expression and T synthesis (6, 9, 10). Thecal interstitial (TI) cells of the ovary, however, also constitutively possess LH receptor and rely on LH stimulation to maintain steroidogenesis (11, 12) but are limited to the secretion of C19 steroids of low biological activity because they lack HSD17B3 expression (13). This divergence between Leydig and TI cells, which are thought to arise from a common primordial cell during gonadal differentiation, suggests that the latter cell type either lacks an LH-stimulated transcription factor(s) specific to the induction of HSD17B3 expression or possesses a mechanism(s) to actively repress HSD17B3 expression. E2 has long been considered a leading candidate to fulfill this postulated inhibitory mechanism because it exists at significantly higher levels in ovaries than testes and inhibits Leydig cell function and proliferation in experimental studies (14, 15) but has received little investigative attention in this regard. However, our recent studies provide the first definitive evidence of E2-mediated repression of Hsd17b3 expression in the ovary and suggest that receptor-mediated actions of E2 may play an important physiological role during ovarian development and function. We have shown previously that the ovaries of estrogen receptor (ER)--null (ERKO) mice exhibit testis-like levels of Hsd17b3 expression and male-like levels of plasma T (16). These findings are corroborated by a report that female mice null for CYP19A1 and therefore lacking endogenous E2 synthesis exhibit comparable ovarian Hsd17b3 expression that is abolished by exogenous E2 treatments (17). These data indicate that ligand-dependent ER actions are fundamental to the repression of Hsd17b3 expression in the ovary. In the current study, we expand our description of this unique ERKO ovarian phenotype by demonstrating that: 1) ectopic Hsd17b3 expression is fundamentally due to the loss of intraovarian ER actions and not a secondary effect of the hypergonadotropism that follows the neuroendocrine loss of ER functions, 2) ovarian HSD17B3 activity in ERKO females is responsible for their characteristic male-like T burden, 3) Hsd17b3 expression in ERKO ovaries exhibits a pattern of LH regulation comparable with that of Leydig cells in the testes, 4) ectopic Hsd17b3 expression in ERKO ovaries is localized to the secondary interstitial (SI)/TI cells, and 5) these same cells exhibit ultrastructural features that are generally considered unique to Leydig cells.
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Materials and Methods |
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ERKO (Esr1–/–) mice were of the C57BL/6 strain and obtained from our colony at Taconic Farms (Germantown, NY). LH-CTP transgenic mice were a mixed background strain (C57BL/6 x CF-1) and obtained from our in-house colony established with breeders from the original colony at Case Western Reserve University (Cleveland, OH; as a gift from Dr. John H. Nilson). Compound hypogonadal Gnrhhpg/Esr1+/+ (wild-typehpg) or Gnrhhpg/Esr1–/– (ERKOhpg) mice were obtained from our colony that was established by crossing heterozygous Gnrhhpg males (C3H/HeH x 101/H; The Jackson Laboratory, Bar Harbor, ME) with Esr1+/– females (C57BL/6) and maintained at Charles River Laboratories (Wilmington, MA). All animals were genotyped by PCR on DNA extracted from tail biopsies using the Wizard SV 96 Genomic DNA extraction kit (Promega, Madison, WI). The procedures for genotyping Esr1–/– (16) and LH-CTP (18) mice have been described previously. All potential Gnrhhpg mice were genotyped using a three-primer PCR scheme based on the original description of the mutant Gnrhhpg allele (19). This PCR method used the following common forward primer and two reverse primers to differentiate the wild-type and mutant Gnrh alleles: forward, 5'-ATGATGCTGCCCACAATCG (bp 2446–2464) to reverse, 5'-TCCCAGACAGGAGTGAAGTGC (bp 2847–2827). These primers flank the mutant breakpoint at bp 2700 and produce a 402-bp amplimer from a wild-type Gnrh allele and no amplimer from a mutant Gnrh allele because sequences homologous to the reverse primer are deleted, and the above forward primer paired with the following reverse primer 5'-TTTCTACTCTCTTGAAACAGGCAAATT, which is homologous to sequences 47 bp beyond the mutant breakpoint and therefore produces a 301-bp amplimer that is unique to the mutant Gnrhhpg allele. All PCR results were evaluated by agarose gel electrophoresis.
Animal treatments and tissue collection
All animals were 2–5 months of age at the time of use. Animals were killed via CO2 asphyxiation, and whole blood was immediately drawn from the inferior vena cava, mixed with heparin, and the plasma later separated by centrifugation; gonads were promptly removed, trimmed of surrounding tissue, and processed according to the intended downstream analysis (as described below). Two experiments required the treatment of animals with gonadotropins before tissue collection. In the first, animals were implanted (sc) with an Alzet osmotic pump (Durect Corp., Cupertino, CA) rated for drug delivery of 0.5 µl/h for 7 d. Pumps were filled with: 1) vehicle (0.85% saline), 2) purified human LH (hLH) at 0.83 IU/µl, or 3) recombinant human FSH (hFSH) at 0.83 IU/µl to provide a calculated delivery of 10 IU gonadotropin/d. Gonadal tissues were collected as described above on the morning of the 7th day. hLH and hFSH preparations were purchased from Dr. A. F. Parlow via the National Hormone and Peptide Program (Torrance, CA). In the second experiments involving gonadotropin treatment, animals were injected (sc) twice daily for 3 consecutive days with vehicle (0.85% saline) or 5 IU human choriogonadotropin (hCG) (Sigma, St. Louis, MO). Gonadal tissues were then collected as described above on the morning of the 6th day, approximately 1 h after the final treatment.
Granulosa-thecal cell isolation
Ovaries were immediately removed from adult wild-type and ERKO females upon death by CO2 asphyxiation and pooled according to genotype in 100-mm cell culture dishes containing ice-cold M199 medium (Invitrogen, Carlsbad, CA). Granulosa cells were then expressed into the medium by manual puncture of the ovaries with a 25-gauge needle and by applying slight pressure with a sterile spatula. The suspended granulosa cells in the medium were then concentrated via centrifugation at 250 x g for 5 min at 4 C and washed two times in M199 medium; the pellets were stored at –70 C for later RNA isolation. The remaining ovarian fragments were considered to represent an enriched stromal/TI cell fraction and as such were washed several times in M199 medium and concentrated by centrifugation; the pellets were stored at –70 C for later RNA isolation.
RNA isolation and gene expression assays
Total RNA was isolated from snap-frozen tissues or cell pellets using TRIZOL reagent (Invitrogen) according to the manufacturer’s protocol. The concentration and integrity of all final preparations was calculated via an A260 reading using a Molecular Devices Spectramax (Sunnyvale, CA) spectrophotometer and agarose gel electrophoresis of a 1-µg aliquot.
Northern blots were generated from 20 µg total RNA per sample using NorthernMax formaldehyde-based reagents and BrightStar (Ambion, Austin, TX) positively charged nylon membrane according to the manufacturer’s protocol. Blots were sequentially probed, stripped, and reprobed for serial gene expression analyses using StripEZ (Ambion)-generated riboprobes and stripping reagents. Radiolabeled antisense riboprobes for Hsd17b3 (bp 363–729 of U66827) and Cyp17a1 (bp 522–932 of M4863) mRNAs were generated from previously described cDNA clones (16) using the StripEZ transcription reagents (Ambion) and [32P]-UTP (Amersham Biosciences, Piscataway, NJ). Hybridizations were carried out in ULTRAhyb hybridization solution (Ambion) with approximately 1 x 107 cpm riboprobe/ml in a 68 C rotisserie oven (Thermo-Hybaid, Franklin, MA) and then washed according to the manufacturer’s protocol. Blots were exposed to a PhosphorImager screen, and the data were analyzed with a Storm 860 and accompanying ImageQuant Software (Molecular Dynamics, Sunnyvale, CA).
RT-PCR was carried out on total RNA that was first rid of contaminating DNA by use of the DNA-free reagents (Ambion) according to the manufacturer’s protocol. For each sample, cDNA was generated from 1 µg RNA in a 25-µl reaction using random hexamers and the Superscript cDNA synthesis system (Invitrogen) according to the manufacturer’s protocol. Traditional PCRs were then prepared from the equivalent of 1 µl cDNA per 15-µl reaction for each respective primer set using PCR reagents and Platinum Taq Polymerase (Invitrogen) as previously described. PCR was carried out in a Thermo Hybaid Multiblock System (Thermo-Hybaid) with the following cycling conditions: 95 C/30 sec (one time); 95 C/30 sec, 58 C/45 sec, and 72 C/30 sec (28–35 times); and 72 C/7 min. The primer sets for assessing Hsd17b3, Hsd17b1, and Actb mRNAs have been described previously (16). All samples were electrophoresed on an agarose gel (2% NuSieve/0.7% SeaKem, BMA Bioproducts, Rockland, ME) in 1x Tris-borate-EDTA buffer, stained with ethidium bromide, and photographed using an EC3 Imaging System (UVP, Upland, CA).
For real-time RT-PCR assessment of gene expression, Applied Biosystems Primer Express (Applied Biosystems, Foster City, CA) software was used to select primers specific for the amplification of murine Hsd17b1, Hsd17b3, Cyp17a1, and Cyp19a1 cDNAs (Table 1
). All primer sets were: 1) designed to lie in separate exons to avoid erroneous amplification of contaminating genomic DNA, 2) confirmed to amplify a single product via dissociation analysis and gel electrophoresis, and 3) confirmed to amplify the expected sequences by restriction enzyme mapping of the amplified product. Each sample was assayed in duplicate using the equivalent of 0.01 µl cDNA (prepared as described above), 10 pmol of each primer, and 1x SYBR Green Master Mix (Applied Biosystems) in a total reaction volume of 25 µl. For normalization purposes, an identical set of reactions was prepared using primers specific for ribosomal 18S RNA (Rn18s) (Table 1). Amplification was carried out in an ABI PRISM 7700 Sequence Detection System (Applied Biosystems) as follows: 50 C/2 min and 95 C/10 min (1 time) and 95 C/15 sec and 60 C/30 sec (40 times). Quantitative differences in the cDNA target between samples were determined using the mathematical model of Pfaffl (20) in which an expression ratio was determined for each sample by calculating (Etarget)
Ct(target)/(ERn18s)Ct(Rn18s), where E is the efficiency of the primer set and Ct = Ct(normalization cDNA) – Ct(experimental cDNA). The amplification efficiency of each primer set was calculated from the slope of a standard amplification curve of log microliters of cDNA per reaction vs. Ct value over at least 4 orders of magnitude (E = 10–(1/slope)); Hsd17b3 primers, E = 1.97 (vs. testis cDNA); Hsd17b1 primers, E = 2.02 (vs. ovary cDNA); Cyp17a1 primers, E = 2.16 (vs. ovary cDNA); and Cyp19a1 primers, E = 2.13 (vs. ovary cDNA).
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ERKO animals immediately upon death, fixed overnight in Phylogeny’s proprietary fixative, then dehydrated and infiltrated with paraffin. Serial sections of 5–8 µm were mounted on gelatin-coated slides, deparaffinized in xylene, and rehydrated through a series of graded ethanol baths in 1x PBS (pH 7.5). The sections were treated with proteinase K, then triethanolamine/acetic anhydride, washed, and dehydrated. Radiolabeled sense and antisense riboprobes for Hsd17b3 mRNA (bp 363–729 of U66827) were generated from a previously described cDNA clone (16) using Maxiscript reagents (Ambion) and 35S-UTP (>1000 Ci/mmol; Amersham) according to the manufacturer’s protocol. Sections were hybridized overnight at 55 C in a solution of 50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 10 nM NaPO4, 10% dextran sulfate, 1x Denhardt’s, 50 µg/ml total yeast RNA, and radiolabeled riboprobe at 5–8 x 105 cpm/µl. The sections were then subjected to stringent washing at 65 C in a solution of 50% formamide and 2x sodium chloride/sodium citrate (SSC) buffer with 10 mM dithiothreitol and washed in 1x PBS, then treated with 20 µg/ml ribonuclease A at 37 C for 30 min. The slides were washed in 2x SSC and then 0.1x SSC for 10 min per wash at 37 C, dehydrated in a series of graded ethanol baths, dipped in Kodak NTB-2 nuclear track emulsion (Eastman Kodak, Rochester, NY), and exposed for 21 d in light-tight boxes with desiccant at 4 C. All slides were then developed in Kodak D-19, lightly counterstained with hematoxylin and eosin (H&E) and viewed and photographed under both light- and dark-field microscopy.
In vitro acute steroidogenic assays
The acute steroidogenic capacity of dispersed ovarian cells from wild-type, ERKO, and LH-CTP animals was assessed in vitro using a method first described by Magoffin and Erickson (11, 21), with some modifications. In brief, ovaries were collected from five to six untreated or hCG-treated adult females of each genotype and pooled according to genotype and in vivo treatment in medium 199 with 100 mg/liter L-glutamine and 25 mm HEPES (M199; Invitrogen) on ice. The pooled ovaries were minced into eight to 10 pieces each, and the fragments were gently washed in M199. The ovarian fragments were incubated in M199 supplemented with 4 mg/ml collagenase (Sigma), 10 mg/ml deoxyribonuclease I (Sigma), and 10 mg/ml BSA (Sigma) at 37 C for 90 min in an ambient atmosphere. Several times during this incubation, the fragments were flushed through sterile pipettes with successively smaller orifices to obtain a preparation of dispersed cells. Each cell preparation was washed three times in M199, and the final cell pellet was suspended in 0.16 ml M199 per pair of ovaries in the original pool.
Acute steroidogenic assays were conducted in sterile 12 x 75 polystyrene culture tubes (BD Falcon, Bedford, MA). Each assay consisted of 25 µl dispersed ovarian cells in a 500-µl final volume of M199 supplemented with 10 IU/ml hCG or an equivalent volume of vehicle (0.85% saline) and/or a HSD17B3 inhibitor, 3ß-((N-cyclohexylmethyl-N-cyclopropylcarbonyl)aminomethyl)-3-hydroxy-5-androstan-17-one) (DP3-3), or equivalent volume of vehicle (100% ethanol). The generation and characterization of DP3-3 has been previously described (22) as compound 213 and was provided for these studies by D.P. All assays were prepared at a minimum of duplicate per genotype, per in vivo treatment, per in vitro treatment. Immediately after preparation, all assays were incubated for 4 h in a 37 C water bath in an ambient atmosphere. The cells were then pelleted by centrifugation at 8000 x g for 5 min, and the medium was transferred to a fresh tube and stored at –70 C for later analyses of A4 and T content by RIA. These experiments were repeated in three independent trials.
Hormone RIAs
Plasma and media androgen levels were assessed using the Active Androstenedione RIA and Active Testosterone RIA kits (Diagnostic Systems Laboratories, Webster, TX) according to the manufacturer’s protocol. All plasma hormone assays were performed in duplicate (when sample volume allowed) on samples collected from individual animals. All media hormone assays were performed on individual experimental samples and always in duplicate. Final assay samples were quantified using an Apex Automation 
-Counter (Micromedic Systems, Seattle, WA) and accompanying software. For the A4 RIA, the least detectable concentration was 0.03 ng/µl, the average intraassay coefficient of variation was 2.5%, and the interassay coefficient of variation was 9.2%. For the T RIA, the least detectable concentration was 0.08 ng/µl, the average intraassay coefficient of variation was 3.0%, and the interassay coefficient of variation was 11.6%. RIAs on an equivalent volume of M199 supplemented with 10 IU/ml hCG or an equivalent volume of vehicle (0.85% saline) and/or 10 µM HSD17B3 inhibitor (DP3-3) or equivalent volume of vehicle (100% ethanol) indicated that the A4 and T content was below the level of detection for each steroid.
Transmission electron microscopy (TEM)
Adult animals were administered a sufficient dose of pentobarbital to induce deep anesthetization and then promptly perfused (whole body) with a modified Karnovsky’s fixative containing 2% paraformaldehyde and 2.5% glutaraldehyde, buffered to pH 7.4 in 0.1 M sodium cacodylate (Electron Microscopy Sciences, Hatfield, PA). Gonads were excised from the animals, placed whole in the above fixative for approximately 2 h, cut into 1–2-mm cubes, and then stored in the above fixative at 4 C until further processing. The tissues were then washed in 0.1 m sodium cacodylate, postfixed in 0.1 M sodium cacodylate-buffered 1% OsO4 (Electron Microscopy Sciences), rinsed in distilled water, dehydrated through a series of graded ethanol baths followed by propylene oxide, and embedded in Polybed epoxy resin (Polysciences, Warrington, PA). Ultrathin (90 nm) sections were prepared and stained on-grid with 5% uranyl acetate and Reynold’s lead citrate. All sections were examined in a FEI Tecnai 12 (Hillsboro, OR) transmission electron microscope operated at 80 kV and equipped with Microsoft Windows 2000 (Redmond, WA) and the MegaView III Soft Imaging System (Lakewood, CO). Several electron micrographs were taken for each sample to give an adequate sampling and representative overview.
Statistical analysis
All data were analyzed for statistical significance (P < 0.05) using JMP software (SAS Institute, Cary, NC). Data sets were first tested for homoscedasticity of variance using the Levene’s test and if failed were log-transformed before further statistical analysis. All data sets were then evaluated by a one-way ANOVA followed by the Tukey-Kramer honestly significant differences post hoc test when applicable.
The T to A4 (T/A4) ratios were calculated for each individual animal or in vitro assay sample using the formula first described by Weibe and Morris (23) to evaluate the endocrine profiles of women diagnosed with ovarian androgen excess. This value more effectively compares the degree of elevated T above normal (vs. a simple ratio) and employs the following formula: (plasma T/95% upper limit normal T)/(plasma A4/95% upper limit normal A4). For determining the in vivo T/A4 ratios (Fig. 1), the individual plasma levels from a group of adult wild-type female mice (n = 24) were used to determine the 95% upper limit for T and A4 as 0.10 and 0.25 ng/ml, respectively. For determining the in vitro T/A4 ratios (Figs. 5 and 6), the acute in vitro steroid synthesis by dispersed cells from untreated wild-type ovaries (n = 3 replicates) was used to determine the 95% upper limit for T and A4 as 0.20 and 0.60 ng/ml, respectively.
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Results |
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ERKO females exhibit abnormally elevated plasma androgensERKO females possess levels of circulating A4 and T that are more than 3- and 50-fold that of wild-type females, respectively (Fig. 1
). Wild-type females treated with hCG twice daily for 3 consecutive days exhibited significantly increased plasma androgen levels, but only A4 and not T approached the levels characteristic of ERKO females (Fig. 1). Similar treatment of ERKO females had no additive effect on plasma androgen levels. Female LH-CTP mice, a transgenic line that possesses chronically elevated LH levels that are more comparable with those of ERKO females (16, 24), also exhibited significantly increased plasma A4 levels relative to wild-type and even ERKO females, but much lower T levels compared with the latter genotype (Fig. 1). Therefore, ERKO females largely differ from other models of ovarian LH hyperstimulation in their unique capacity to produce much greater levels of T. This male-like capacity of ERKO ovaries to synthesize T is especially revealed when T/A4 ratios among the different genotypes are compared (Fig. 1). Wild-type females exhibited a T/A4 ratio of 0.82, which was significantly increased to 1.17 (P < 0.05) after 3 d of hCG treatment. Remarkably, LH-CTP females exhibited a similar T/A4 ratio (1.11) despite a life-long elevation in circulating LH. In contrast, ERKO females exhibited a striking T/A4 ratio of 17.2 and showed no further increase after hCG treatment (17.0). These data indicate that chronic, excessive LH stimulation alone does not lead to male-like efficiency of T synthesis in murine ovaries but instead indicate that this phenotype is a primary effect of the loss of intraovarian ER-mediated actions.
ERKO ovaries exhibit testis-like expression and regulation of the Hsd17b3 gene
Comparative Northern blot analyses of gonadal RNAs indicated that ERKO ovaries repeatedly possess levels of Hsd17b3 expression that are equal to or even greater than those observed in the testes of normal adult males, whereas levels in wild-type ovaries were below the level of detection (Fig. 2A). These data were confirmed by quantitative real-time RT-PCR, which indicated the level of Hsd17b3 expression in ERKO ovaries was 2-fold that of adult testes (Fig. 2C). Similar analyses of LH-CTP ovaries also indicated a lack of Hsd17b3 expression but increased Cyp17a1 expression (Fig. 2, B and C), the latter being a known ovarian marker of LH stimulation. ERKO ovaries also exhibited increased levels of Cyp17a1 expression that were 6- and 3-fold higher than those of wild-type and LH-CTP ovaries, respectively (Fig. 2C).
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ERKO ovaries exhibits a similar, testis-like pattern of gonadotropin regulation. These experiments were conducted by treating hypogonadal (hpg) wild-type (wild-typehpg) and ERKO (ERKOhpg) females, which lack circulating endogenous gonadotropins, with a continuous infusion of exogenous hFSH or hLH for a period of 1 wk. Wild-typehpg males were similarly treated to provide data on the expected response in testes. As shown in Fig. 3, hLH increased Hsd17b3 expression almost 40-fold in the testes of wild-typehpg males, whereas the response to hFSH was measurable but conspicuously lower (<5-fold). Surprisingly, ERKOhpg ovaries exhibited a similar response to hLH by increasing Hsd17b3 expression over 25-fold, whereas wild-typehpg ovaries exhibited a measurable but notably less robust increase in Hsd17b3 expression (Fig. 3). This difference in the induction of Hsd17b3 expression in the gonads of wild-type and ERKO females is not due to a disparity in the effectiveness of hLH treatment because Cyp17a1, a known LH-induced gene, was increased 23- and 80-fold in each genotype, respectively (data not shown). hFSH treatment had no effect on Hsd17b3 expression in the ovaries of either genotype and only a minimal effect in the testes. However, the effectiveness of hFSH treatment in the ovaries of wild-type and ERKO females was illustrated by induction of Hsd17b1 (Fig. 3), a known FSH regulated gene in the ovary (29, 30). FSH induced Hsd17b1 expression 4-fold in both wild-typehpg and ERKOhpg ovaries, whereas LH had minimal effect (Fig. 3). Another known FSH-induced gene in the ovary, Cyp19a1, was also increased over 200-fold in both wild-type and ERKO ovaries (data not shown). Neither gonadotropin induced Hsd17b1 expression in the testes of wild-typehpg males, demonstrating that this gene is distinctly expressed in ovaries.
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ERKO ovariesERKO ovaries, total RNA was prepared from ovaries that were first partitioned into fractions of enriched granulosa and stromal-interstitial cells. Northern blot analysis clearly indicated that Hsd17b3 mRNAs were concentrated in the stromal-interstitial compartment of ERKO ovaries with only nominal detection in the granulosa cell fraction, most likely due to cross-contamination by small amounts of stromal material (Fig. 4). The relative purity of the fractions was illustrated by reprobing the blot for Cyp19a1 mRNAs, which are generally localized to granulosa cells of large follicles (Fig. 4).
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ERKO ovaries. As shown in Fig. 4, Hsd17b3 mRNAs were distinctly localized to the stromal-interstitial regions of ERKO ovaries, whereas hybridization levels were below background in the granulosa cells of follicles of all sizes. More precisely, Hsd17b3 expression in ERKO ovaries was concentrated in the SI cells, which populate the areas between the follicles and medullary portions of the ovary. SI cells are thought to have once been TI cells, but after follicle atresia, they become no longer associated with growing follicles and populate the ovarian stroma (11). Because growing follicles in ERKO ovaries characteristically exhibit poorly organized layers of TI cells, whether Hsd17b3 expression was distinct to the SI or shared between the two interstitial cell types was difficult to discern. No substantial hybridization of the Hsd17b3 probe was observed among any cell types in wild-type ovaries (Fig. 4). As expected, parallel experiments in testes from adult wild-type males indicated significant Hsd17b3 expression that was distinctly localized to Leydig cells (Fig. 4). Furthermore, hCG-mediated induction of Hsd17b3 expression in the testis of an adult wild-typehpg male was again specifically localized to the interstitial (extratubular) compartments of the testis where Leydig cells are located.
Ectopic HSD17B3 activity mediates T synthesis in ERKO ovaries
Adult wild-type and ERKO females were first treated with vehicle or hCG for 3 d to stimulate ovarian androgen production in vivo. Dispersed ovarian cell preparations were then generated, and their acute steroidogenic capacity was assessed during a 4-h in vitro incubation in the presence or absence of hCG and/or a HSD17B3 inhibitor (DP3-3). The results of these experiments are summarized in Fig. 5. As expected, ERKO ovarian cells produced significantly more A4 and T compared with wild-type cells, regardless of in vivo or in vitro hCG exposure (Fig. 5A). In vivo hCG treatments enhanced in vitro A4 and T synthesis in both genotypes; however, only ERKO ovarian cells exhibited an additive increase in overall androgen synthesis during in vitro hCG exposure. More importantly, these experiments provided two critical findings. First, consistent with the in vivo phenotype, T was clearly the predominant androgen produced by ERKO ovarian cells in vitro, yielding an average T/A4 ratio of 3.32 ± 0.5 vs. 0.47 ± 0.1 in wild-type cells (untreated both in vivo and in vitro). Secondly, inclusion of the HSD17B3-specific inhibitor (DP3-3) consistently reduced T synthesis by 50–60% in ERKO ovarian cells and decreased the average T/A4 ratio to 0.73 ± 0.2, providing strong evidence that T synthesis in ERKO ovaries is largely due to ectopic HSD17B3 activity. In contrast, the minimal but detectable T synthesis by wild-type cells was not affected by the HSD17B3 inhibitor (T/A4 ratio = 0.45 ± 0.02), indicating that it is likely mediated by the androgenic activity of rodent HSD17B1, which is innate to ovarian granulosa cells. A representative dose-response curve for HSD17B3 inhibition of T synthesis by ERKO ovarian cells in vitro is shown in Fig. 5B.
Similar experiments were conducted to compare the steroidogenic capacity of dispersed ovarian cells from LH-CTP and ERKO females because these two models are more comparable in terms of chronic LH hyperstimulation of the ovary. Acute in vitro steroidogenic assays on dispersed cells from LH-CTP ovaries indicated substantial A4 production that was 2-fold that observed in ERKO ovarian cells (Fig. 6), consistent with their in vivo androgen profile (Fig. 1). Only when ERKO females were treated with exogenous hCG before tissue collection did the in vitro level of A4 synthesis become comparable with that of LH-CTP cells. This overabundance of A4 in LH-CTP cells is indicative of the increased Cyp17a1 expression shown earlier (Fig. 2). Still, even the highest level of in vitro A4 synthesis achieved in LH-CTP cells did not provide for ERKO-like levels of T production, which were 3-fold higher in the latter genotype. Furthermore, the HSD17B3 inhibitor had no measurable effect on T synthesis in LH-CTP cells, indicating it is likely mediated by the androgenic activity of HSD17B1.
ERKO ovaries harbor stromal Leydig-like cells
To determine whether the interstitial portions of ERKO ovaries possessed Leydig-like ultrastructural features, we employed TEM to compare normal Leydig cells of mouse testes with stromal-interstitial cells of adult ERKO ovaries. The typical whorl-arranged smooth endoplasmic reticulum (SER) that is innate to Leydig cells of mouse testes (31) is illustrated in Fig. 7, A and B. The Leydig cell shown possesses multiple whorls of SER, one of which surrounds a lipid droplet. Also prominent are random tubular SER and numerous mitochondria, the latter possessing the tubular cristae that are characteristic of steroidogenic cells (Fig. 7B) (31). Remarkably, a parallel survey of stromal-interstitial regions of multiple ERKO ovaries identified cells that possessed similarly arranged SER, clearly organized as whorls and interspersed with large lipid droplets and tubular SER (Fig. 7, C–E). The whorl-like SERs found in ERKO ovarian stroma-interstitial cells usually occupied a smaller intracellular area and were less structured relative to that in testicular Leydig cells (Fig. 7, C and D) but were clearly Leydig-like in appearance. In addition, the whorled SERs observed among ERKO ovarian stromal-interstitial cells were often vesiculated (Fig. 7F), remarkably similar to that which occurs during the natural involution of Leydig cells in fetal or aged testes. These vesiculated structures in ERKO ovarian stromal-interstitial cells are likely sites of lipid degradation and may account for the substantial intracellular prevalence of lipofuscin observed in H&E-stained tissue sections (Fig. 7H), which is also a common cytoplasmic component of Leydig cells in multiple species (31). Furthermore, the cells possessing Leydig-like SER and lipofuscin were limited to the interstitial regions in ERKO ovaries, the normal site of SI cells and correlating with the areas of Hsd17b3 expression indicated by in situ hybridization. No similar structures were found in wild-type ovaries (data not shown).
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ERKO ovarian SI cells. As illustrated in Fig. 7, C and G, numerous crystalline-like cytoplasmic inclusions were found to repeatedly coexist in cells that harbored whorled, Leydig-like SER. These inclusions were long, cylindrical bodies of 0.1–2 µm in length that were often present in great numbers and almost always associated with whorled SER. Crystalline cytoplasmic inclusions are a common feature of normal Leydig cells in the testes of several species and are considered a definitive attribute of human Leydig cells and Leydig cell tumors, within which they are referred to as crystals of Reinke (31). However, there are no reports of similar cytoplasmic crystalline inclusions in Leydig cells or ovarian SI cells of mice. |
Discussion |
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Our assertion that ERKO ovaries inappropriately possess functional Leydig-like cells is based on their exhibiting traits that are regarded as exclusive to testicular Leydig cells, including: 1) substantial Hsd17b3 expression and enzymatic activity, 2) whorl-like SER, and 3) crystalline-like structures comparable with crystals of Reinke. Furthermore, the ectopic Hsd17b3 expression in ERKO ovaries is LH dependent and thus exhibits a pattern of gonadotropin regulation that is comparable with that of mature testicular Leydig cells. Similar Hsd17b3 expression and cells possessing Leydig-like features are reportedly present in the ovaries of mice lacking endogenous E2 due to targeted disruption of the Cyp19a1 gene, and the former phenotype is abolished by exogenous E2 treatments (17). However, because E2 treatment of CYP19A1-null females also results in normalization of the heightened LH levels that are present in these animals (17), these experiments do not discern whether the loss of ovarian Hsd17b3 expression is due to activation of intraovarian ER or decreased gonadotropin stimulation. In the current study, we provide definitive evidence that ectopic Hsd17b3 expression in ERKO females is due solely to the loss of ER-mediated actions within the ovary and simply requires LH stimulation to manifest. For example, the ovaries of transgenic LH-CTP female mice categorically lack Hsd17b3 expression and activity (18 and Figs. 2 and 6 herein) despite being chronically exposed to substantially high levels of circulating LH (34, 35). The ovaries of ERß-null female mice also lack ectopic Hsd17b3 expression (16) even when harboring the LH-CTP transgene to increase circulating LH levels (18). Likewise, reports of transgenic mice that constitutively express hCG, an LH analog, describe ovaries exhibiting TI cell hypertrophy but make no reference to Hsd17b3 expression or Leydig-like cells (36, 37). In contrast, Heikkilä et al. (38) recently reported that the ovaries of newborn Wnt4-null female mice exhibit ectopic induction of Hsd17b3 expression that is concurrent with an 8-fold reduction in ER expression but no change in ERß levels. All of these findings support our conclusion that ER is critical to repressing the expression of this Leydig cell-specific steroidogenic gene in the ovary.
Evidence supporting the presence of substantial HSD17B3 enzymatic activities in ERKO ovaries was revealed by several findings. Foremost was the remarkably high T/A4 ratio in the plasma of adult ERKO females that is more comparable with that of normal male mice (39, 40) and adult men (41) as well as women clinically diagnosed with ovarian androgen excess (23). We have shown previously that increased androgen levels in ERKO females are at least partially due to hyperstimulation of the ovary by LH (16). Still, when wild-type female mice were forced to possess a comparable hypergonadotropic condition via either daily injections with hCG, an LH analog, or transgenic methods, as achieved in LH-CTP mice, the resulting androgenic phenotype was markedly less severe and qualitatively different from that of ERKO females because A4, and not T, was the predominant steroid produced in the former models. Secondly, we conducted specific experiments to discern the individual contributions of type 1 and type 3 activities to the overall T load in ERKO females. This was necessary because the rodent ortholog of HSD17B1, which is normally expressed in the ovary and mediates the reduction of estrone to E2 (2, 42, 43), is equally efficient at reducing A4 to T (44, 45). Furthermore, Hsd17b1 expression is increased almost 2-fold above normal in ERKO ovaries (16). By employing a method first described by Magoffin and Erickson (21, 46) that allows for the evaluation of the acute steroidogenic capacity of dispersed ovarian cells in vitro with the use of a specific inhibitor of HSD17B3 enzymatic activities (22), we were able to successfully discriminate type 1- and type 3-mediated HSD17B T synthesis in dispersed wild-type and ERKO ovarian cells. The resulting data strongly indicated that the male-like capacity for T synthesis in ERKO ovaries is due predominantly to the presence of substantial HSD17B3 activities, whereas the minimal level of T synthesis observed in hCG-treated wild-type or LH-CTP ovaries is due to the androgenic activities of HSD17B1 that occur in the presence of increased A4. T synthesis in ERKO females is undoubtedly further promoted by a parallel increase in CYP17A1 activities that provide for ample synthesis of A4, the substrate for HSD17B3.
The mechanisms by which ER actions suppress the testicular pathway of T synthesis in the ovary are unclear. Ligand-activated ER may act in a dynamic manner within TI/SI cells to persistently inhibit Hsd17b3 expression. ER is indeed amply expressed in both testicular Leydig cells (47, 48) and ovarian TI/SI cells (49, 50, 51). Furthermore, estrogens are known to inhibit T synthesis in both thecal and Leydig cells in vitro, although most evidence indicates that the upstream steroidogenic enzyme, CYP17A1, is the primary target (11, 14, 52). Indeed, CYP17A1 expression and activity are also increased in the ovaries (Fig. 2) (16), in vitro cultured follicles (Taniguchi, F., J. F. Couse, and K. S. Korach, unpublished data) and testes (53) of ERKO but not ßERKO mice. However, ours is the first study to demonstrate that ER represses ovarian T synthesis via robust negative modulation of Hsd17b3 expression as well. Interestingly, a recent report that ERKO males exhibit a 2-fold increase in Hsd17b3 expression and type 3 activity (53) indicates that ER may play an analogous role in testes.
In contrast to the above-postulated dynamic mechanism for ER-mediated inhibition of Hsd17b3 expression and T synthesis in the ovary is a potential organizational role by which ER might repress the development of a Leydig cell phenotype among the SI/TI cells. Support for such an organizational role for ER action during gonadal differentiation comes from multiple descriptions of permanently disrupted Leydig cell function in adult rodents after developmental exposure to estrogens as well as estrogen-mediated inhibition of Leydig cell regeneration and proliferation in ethane dimethylsulfonate-exposed testis (14). Herein, we employed TEM to produce evidence of Leydig-like cells in ERKO ovaries. The "single most identifying characteristic of the Leydig cell" is their unique and often species-specific arrangements of SER (31). The Leydig cells of adult mouse testes, as well as other species, characteristically exhibit a portion of SER that is arranged as whorls often surrounding a lysosome or lipid droplet (Ref. 31 and references therein). This swirled form of SER is continuous with the tubular form and may comprise up to 7% of the total SER within a single Leydig cell (31). In the current study, we found that ERKO ovaries possess SI cells that exhibit similar whorl-arranged SER as well as other Leydig cell features, including crystalline-like cytoplasmic inclusions and an accumulation of lipofuscin. Furthermore, the ERKO ovarian cells exhibiting these Leydig-like features were located in the same ovarian regions that possessed significant levels of Hsd17b3 expression as indicated by in situ hybridization. These findings raise the prospect that a loss of ER actions during SI/TI cell differentiation in the ovary may allow for the inappropriate development of cells possessing a Leydig-like phenotype. The capacity of adult hypogonadal ERKO (ERKOhpg) ovaries to rapidly acquire Hsd17b3 expression after acute exogenous hLH treatment suggests that the Leydig-like cells may develop in an environment void of gonadotropins and are present as early as birth or before puberty.
Our finding that the Leydig-like cells in adult ERKO ovaries were located in the stroma and often bordered by SI/TI cells distinguishes them from hilar interstitial (HI) cells, which are natural to the ovary and considered structurally and functionally similar to testicular Leydig cells (11). HI cells are normally limited to the connective tissues in the hilus of the ovary (11), whereas the Leydig-like cells observed in ERKO ovaries are located in the stroma, surrounded by TI/SI cells and usually in close proximity to a follicle. Admittedly, poor organization of the medullary regions is characteristic of adult ERKO ovaries (54, 55) and may have altered the natural position of HI cells. Still, there is no evidence of Hsd17b3 expression in the HI cells of normal ovaries (8, 11), providing further evidence that the Leydig-like cells in ERKO ovaries are not of hilar origin.
The stromal location of the Leydig-like cells in ERKO ovaries in conjunction with their close proximity to hypertrophied SI/TI cells suggests that they may have once been part of the latter cell population but have since acquired a male-like phenotype via trans-differentiation. This phenomenon is thought to underlie the formation of stromal-Leydig cell tumors and nonhilar, pure Leydig cell tumors in women (56, 57). Although extremely rare, these ovarian tumors are invariably androgenic and often bordered by hypertrophied ovarian stroma (56, 57, 58, 59). Furthermore, there are clinical reports of HSD17B3-like activity in the ovaries of women suffering from ovarian androgen excess (60, 61), androgenic HSD17B immunoreactivity in a pure Leydig cell tumor (62), and HSD17B3 transcripts in a Sertoli-Leydig cell tumor and surrounding luteinized thecal tissue (63). In contrast, ovarian tissue and cell lines from women diagnosed with polycystic ovarian syndrome have been found to lack detectable HSD17B3 expression (64, 65, 66), indicating that this malady is not likely related to the ER-null phenotype described here.
Thecal cell androgen synthesis is critical to ovarian function because androgens serve as both important hormonal signals during granulosa cell differentiation in the early stages of folliculogenesis and later as substrates for E2 synthesis in mature preovulatory follicles (67). However, when androgen levels and activity are allowed to surpass that required to support E2 synthesis during the latter stages of folliculogenesis, the follicle invariably becomes atretic (68, 69). This condition is likely exacerbated if T, rather than A4, is the predominant androgen present because the former steroid is a more potent androgen receptor agonist. Therefore, stringent regulation of the quantity and nature of TI/SI cell androgen synthesis is critical to the generation of healthy follicles capable of ovulation and fertilization. Our data provide strong evidence that ligand-dependent ER actions are fundamental to this physiology of TI/SI cells by repressing their acquisition of HSD17B3 activities and perhaps their differentiation to a Leydig-like phenotype. Our description of ectopic Hsd17b3 expression and Leydig-like cells in the ovaries of ER-null female mice represents the first data to support an integral intraovarian role for ER. Further studies are required to discern whether ER operates via activational or organizational mechanisms to repress the acquisition of a testis-like phenotype in the SI/TI cells of the ovary. A similar phenotype of granulosa to Sertoli cell trans-differentiation occurs in the ovaries of compound ER-null (ER-null; ERß-null) female mice (70, 71). From these collective data, we may infer that ER-mediated E2 signaling is critical to the development and/or maintenance of the female phenotype in the endocrine somatic cell types of the ovary.
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Acknowledgments |
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-counter, Dr. Abraham Nyska for consultation on pathology issues, and Drs. E. Mitch Eddy and William Schrader for their careful review of the manuscript. |
Footnotes |
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J.F.C., M.M.Y., K.F.R., J.A.J., D.P., and K.S.K. have nothing to declare.
First Published Online April 20, 2006
Abbreviations: A4, Androstenedione; DP3-3, 3ß-((N-cyclohexylmethyl-N-cyclopropylcarbonyl)aminomethyl)-3-hydroxy-5-androstan-17-one); E2, estradiol; ER, estrogen receptor; ERKO, ER-null; hCG, human choriogonadotropin; hFSH, human FSH; H&E, hematoxylin and eosin; HI, hilar interstitial; hLH, human LH; HSD17B, hydroxy-steroid (17ß) dehydrogenase; SER, smooth endoplasmic reticulum; SI, secondary interstitial; SSC, sodium chloride/sodium citrate; T, testosterone; TEM, transmission electron microscopy; TI, thecal interstitial.
Received March 1, 2006.
Accepted for publication April 13, 2006.
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