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Department of Physiology (T.S., T.L., K.H., M.P.), Institute of Biomedicine, and Department of Pathology (H.K.), University of Turku, FIN-20144 Turku, Finland; and Medical Research Council Human Reproductive Sciences Unit (M.W., P.S.), Queens Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom
Address all correspondence and requests for reprints to: Matti Poutanen, Department of Physiology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20144 Turku, Finland. E-mail: matti.poutanen{at}utu.fi.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-hydroxysteroid dehydrogenase activity, HSD17B4 is involved in ß-oxidation of fatty acids, and HSD17B10 catalyzes the oxidation of branched and straight fatty acids (2, 3). Most of the HSD17Bs belong to the ketosteroid reductase family also known as short-chain alcohol dehydrogenases (2). The family consists of a group of oxidoreductases found in bacteria, plants, and animals, whereas the amino acid sequence identity between the short-chain dehydrogenase/reductase/ ketosteroid reductase family members may be less than 25% (1). Based on studies in cultured cells, human HSD17B1 has been demonstrated to catalyze the reduction of the biologically low active estrone (E1) to highly potent estrogen, estradiol (E2) (4). HSD17B1 in rodents and humans seems to be involved in E2 biosynthesis in the granulosa cells of the ovary, and in syncytiotrophoplasts of the human placenta (5, 6, 7, 8). The enzyme is down-regulated in luteinizing granulosa cells and is not expressed in rodent corpora lutea (5, 9, 10). The central role for the enzyme in E2 biosynthesis is also suggested by the high expression in human placenta, whereas it is not expressed in rodent placenta that lack E2 biosynthesis (6, 7). Thus, it is likely that, together with P450aromatase (cytochrome P450, family 19, subfamily A, polypeptide 1), HSD17B1 catalyzes the last steps in glandular E2 biosynthesis both in rodents and humans. However, there are two major differences between the human and rodent HSD17B1s. Based on data in vitro, human HSD17B1 is estrogen specific, whereas the rodent HSD17B1s convert estrogens and androgens with similar catalytic efficiencies (4, 6). Furthermore, the human enzyme has been detected in several sex steroid target tissues whereas there is less evidence for extragonadal expression of the rodent enzyme (4, 6).
The putative role of human HSD17B1 in regulating the availability of highly active ligand for estrogen receptors in steroid target tissues, such as mammary gland and endometrium, has been acknowledged, and altered HSD17B1 activity has been associated with certain endocrine-dependent human diseases such as breast cancer, endometrial adenocarcinoma, and endometriosis (11, 12, 13, 14). Furthermore, two HSD17Bs catalyzing the activation of E1 to E2 (HSD17B1) and inactivation of E2 to E1 (HSD17B2) are simultaneously expressed, for example, in normal endometrium and breast tissue (15). Based on these observations, it has been hypothesized that the relative activity of these two enzymes with opposite activities could regulate the amount of highly active E2 in target cells. Because local formation of sex steroids is likely to play a major physiological role in both normal and malignant hormone-sensitive tissues, the HSD17B enzymes are promising targets for drug discovery and development.
Almost all studies pointing to the function of HSD17B1 have been, so far, carried out in vitro and with cultured cells. Thus, in the present study we developed a transgenic (TG) mouse model (HSD17B1TG) in order to study the properties of the human enzyme in vivo and to study the putative role of HSD17B1 in enhancing sex steroid action in the target tissues. In the female mice, we observed marked, well-known, androgen-dependent phenotypes, many of them being indicators of prenatal androgen exposure.
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RESULTS |
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). Strongest expression was detected in line 013, in which the universal expression of the transgene was confirmed (Fig. 1B), and the line was used as a major tool in the present study. HSD17B1 activity for the transgene was confirmed by analyzing the conversion of E1 to E2 in vitro, and activity in different mouse lines correlated well with the level of transgene expression in various HSD17B1TG mouse lines (Fig. 1C). Although reductive activity from E1 to E2 was increased in male heart, HSD17B1TG males were fully fertile, and the only obvious phenotype observed was slightly decreased testis weight in adulthood (data not shown).
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, A and B). Furthermore, using estrogens as substrates, both the reductive (E1 to E2) and oxidative (E2 to E1) activities were significantly increased. However, similar to that shown previously in cultured cells, the conversion of E1 to E2 was markedly higher than the opposite reaction in TG mice in vivo (Fig. 2A). Interestingly, also in WT mice the basal HSD17B activity for reductive reaction was significantly higher than for the oxidation of E2 to E1. There is evidence in literature that HSD17B1 and HSD17B2 compensate each other at target tissue level, and that the final E2 concentration would be the sum of these two opposite activities (15). Because masculinized phenotypes were observed and HSD17B2 could possibly exert an antiestrogenic effect, we analyzed the putative alteration in the endogenous mouse HSD17B2 expression in the HSD17B1 TG mice in the two tissues with the highest level of HSD17B2, the liver and intestine. However, no significant induction of HSD17B2 expression was observed (Table 1).
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, the androgen-dependent phenotypic changes observed in HSD17B1TG females at the age of 4 months were the following: lack of upper nipples, male-like anogenital distance (AGD), lack of vaginal opening, partially unseparated vagina and urethra, and a phenotype resembling human benign ovarian serous cystadenoma. Several of the phenotypes detected in the two HSD17B1TG mouse lines with highest level of transgene expression (013, 050) were not observed in the lines with only a low level of transgene expression, indicating dose dependence of the phenotypes.
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). This indicated that the feminine pseudohermaphroditism observed was due to an increased exposure to T of fetal origin during fetal period. We, therefore, treated pregnant females with antiandrogen flutamide, and, consequently, the morphological alterations in the lower reproductive tract were efficiently rescued. The increased AGD in HSD17B1TG females was decreased from 9.3 ± 0.3 mm to 5.9 ± 0.2 mm (P < 0.001), and was not significantly different from that of WT mice after the treatment (Fig. 3, A and B). Also the development of upper nipples (Fig. 3C) and opening of vagina were induced in the flutamide-treated mice, but not in the placebo-treated group (Fig. 3, A and B). We also demonstrated an improper separation of vagina and urethra in HSD17B1TG females (Fig. 3E), which could be rescued to a morphology similar to that of WT mice by prenatal flutamide treatment (Fig. 3, D and F). Although differences in sex steroid concentrations were detected before birth, they were normalized by adulthood (Table 3). However, by the age of 4 months, HSD17B1TG females developed androgen-dependent benign ovarian serous cystadenomas characterized by hyperplasia of ovarian surface epithelium (OSE), and cysts surrounding the ovaries diagnosed by histological characterization of HE-stained sections (Fig. 4, A, E, and F). At 2 months of age these changes were absent. The mice were followed until the age of 12 months, but no overt ovarian cancer development was observed. Because there were no significant alterations in adult serum hormone concentrations, we analyzed the dependence of the ovary phenotypes on prenatal masculinization. The formation of benign serous cystadenomas was efficiently prevented by prenatal flutamide treatment (Fig. 4C), and development of OSE hyperplasia was also suppressed in WT ovaries prepubertally transplanted to HSD17B1TG females (Fig. 4B). The effect of WT ovary transplantation on ovarian cysts was not evaluated because the method itself induced cystic appearance of the ovaries. Because the ovarian phenotype of HSD17B1TG females appeared to be androgen dependent, the presence of androgen receptor (AR) in fetal ovaries was confirmed at the end of pregnancy (Fig. 4, G and H). Table 4 summarizes the data on studies aimed to rescue the androgen-induced phenotypes in HSD17B1TG mice by prenatal antiandrogen treatment and by transplanting WT ovaries into TG mice prepubertally.
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DISCUSSION |
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There is a marked difference in the steroid specificity between the human and rodent HSD17B1 enzymes, whereas the catalytic efficacy for the rodent enzymes in vitro is similar for both androgens and estrogens. HSD17B1 enzyme is composed of 327 and 344 amino acids in humans and rodents, respectively (6, 17). The identity between the rat and human amino acid sequences is 63%, whereas the highest identity (81%) is located at the region composed of the 200 N-terminal amino acids (19). Of those, the first 100 amino acids make up the cofactor-binding region (20), whereas the next 50 are involved in the dimerization of the enzyme (19). Studies with chimeric enzymes composed of rat and human sequences have shown that the region between residues 148–266 determines most of the steroid specificity in the HSD17B1 enzyme, with a consistently decreasing E1:A-dione ratio along with more amino acid residues identical to the rat enzyme. At this region, Asn152His, Asp153Glu, and Pro187Ala variations were found to be closely related to steroid specificity (19). However, the difference in the steroid binding regions between human and rodent enzymes is relatively small, and present study indicates that also the human enzyme possesses physiologically significant androgenic activity converting A-dione to T in vivo.
Along with the significant androgenic activity, HSD17B1TG females presented well-known androgen-dependent phenotypes. The females suffered from feminine pseudohermaphroditism, induced by prenatal androgen exposure. Increased T level was also detected in the prenatal TG females. The pseudohermaphroditism could be effectively treated by prenatal antiandrogen administration, confirming the dependence of the phenotypes on androgens. Feminine pseudohermaphroditism results when a differentiating female fetus is exposed to androgens (21). In humans, masculinization of external genitalia begins with lengthening of AGD at wk 10 of pregnancy, followed by the fusion of the labioscrotal folds and closure of the rims of the urethral groove (22). In FVB/N mice this occurs at E13.5–E14.5, after which the genitalia continue to develop until the time of birth (23). Based on this information, flutamide administration was timed from E13.5 until birth, and this efficiently rescued the increase in AGD. It is acknowledged that as a response to androgens, the vagina fails to completely descend to the perineum, causing a common urogenital canal or sinus with incomplete separation of the vagina and urethra, as observed in HSD17B1TG-females (21). Nipple development has also been shown to be androgen dependent. Male rodents normally lack nipples but, for example, in rats the nipple development can be induced by undermasculinizing males by prenatal flutamide treatment (24, 25). Consequently, prenatal flutamide exposure could retain the nipple development and rescue vaginal morphology in masculinized TG females. These data unequivocally show that overexpression of human HSD17B1 led to increased androgen exposure during embryonic development.
In addition to the prenatal masculinization, biochemical and physiological indications of hyperandrogenism at adult age were observed. Biochemically the hyperandrogenism appeared in the form of increased conversion of A-dione to T, although not observed as increased serum T concentration. It is likely that the target tissue level of human HSD17B1 in HSD17BTG females was not high enough to alter systemic T and E2 concentration, whereas the serum sex steroid levels are determined by the ovarian production, which seems not to be altered significantly. The induction of endogenous HSD17B2 expression in HSD17B1TG females would partially explain the lack of increased serum E2 concentration, but no such induction was observed. Physiologically, the hyperandrogenism appeared as induction of nipple development in prepubertal HSD17B1TG females with transplanted WT ovaries. One more adult phenotype also appeared to be androgen dependent. Namely, between the age of 2 and 4 months, untreated HSD17B1TG females developed a benign cystadenoma resembling ovarian phenotype. The development of benign serous cystadenomas was prevented in HSD17B1TG mice by treating the mice prenatally with flutamide, or by transplanting the WT ovaries to HSD17B1TG females. The data thus provide evidence for a connection between androgen production, dysregulation of HSD17B1 expression in the ovaries, and the development of benign serous cystadenomas. In a developing human embryo, OSE undergoes epithelial transition at wk 10 of gestation [corresponding to d 13.5 in mouse pregnancy (26)]. Androgen exposure during the second half of pregnancy in our mouse model seems to program adult TG ovaries to develop benign serous cystadenomas. Interestingly, epithelial ovarian carcinomas make up more than 85% of human ovarian cancers, and OSE-lined surface invaginations and epithelial inclusion cysts are considered as a site for neoplastic progression (26, 27). Our HSD17B1TG model is a phenocopy of these early epithelial events described in the progression of ovarian cancer in humans.
The androgen-mediated phenotypes observed in prenatal and adult HSD17B1TG mice demonstrate that, like the mouse ortholog, human HSD17B1 possesses significant androgenic activity in vivo when proper substrate is available. Taken together, our data suggest that the physiological function of human HSD17B1 in vivo is to be reevaluated and androgenic activity described is expected to lead to new roles for HSD17B1 in humans, particularly in females. Furthermore, the phenotypes observed represent novel disease indications for HSD17B1 inhibitors in disorders in the female reproductive organs associated with enhanced androgen action, such as ovarian serous cystadenomas.
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MATERIALS AND METHODS |
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For microinjection the actin-HSD17B1 transgene was cleaved from the plasmid backbone by XmnI/HindIII digestion, purified by a Quick-Pick Electroelution Capsule Kit (QIAGEN, Valencia, CA) and Elutip diethylaminoethyl columns (Schleicher & Schüell, Dassel, Germany), and diluted to the final concentration of 2 ng/µl. TG founder mice were generated in the genetic background of the FVB/N strain by microinjecting the DNA into pronuclei of fertilized oocytes using standard techniques (28). Integration of the transgene was verified by Southern blot analysis and PCR screening of DNA isolated from tail or ear biopsies by the salting-out method (29). The PCR consisted of denaturation cycle (5 min at 97 C, 1 min at 56 C, 30 sec at 72 C) 31 cycles of amplification (1 min at 95 C, 1 min at 56 C, 30 sec at 72 C) and termination cycle 10 min at 72 C. The following primer pairs were used for genotyping and RT-PCR. HSD1 forward 2: 5'-cttcagatccatcccagagc-3'-HSD1Ex32 5'-GCCCAGGCCTGCGTTACAC-3' and HSD1 forward 562, 5'-ACACCTTCCACCGCTTCTAC-3'-HSD1 reverse 562, 5'-GAACGTCGCCGAACACTT-3'. The founder mice were mated with WT FVB/N mice to create several HSD17B1TG mouse lines.
Animal Experimentation
All animal experimentation was conducted in accordance with the institutional animal care policies of the University of Turku (Turku, Finland). Animal experiments were approved by the respective authorities, and the institutional policies on animal experimentation fully meet the requirements as defined in the NIH Guide on animal experimentation. The animals were housed under controlled environmental conditions (12 h light/12 h darkness; temperature, 21 ± 1 C) at the Animal Facility of University of Turku. Soy-free SDS RM3 (Special Diet Service, Whitman, Essex, UK) and tap water were available ad libitum. For sample collecting, mice were terminally anesthetized with 400–800 µl 2.5% tribromoethanol [Avertin; Aldrich Chemical Co., Milwaukee, WI;30)] injection, ip, and blood was withdrawn from the heart followed by euthanasia by cervical dislocation. When relevant, macroscopic changes were observed at autopsy, and tissues were weighed and frozen at –80 C or processed for histology as described below.
Analysis of Transgene Expression and HSD17B2 Expression
For quantitative transgene expression analysis, total RNA was isolated from mice hearts from all mouse lines generated by RNeasy Mini Kit (QIAGEN). RNA samples were then DNAse treated (DNase I Amplification Grade Kit; Invitrogen, Life Technologies, Paisley, UK), and used for quantitative RT-PCRs (conditions described in supplemental Table 1). The data were normalized by relating HSD17B1 expression to mouse ribosomal protein L19. Primer sequences used to detect L19 were as follows: L19 forward, 5'-CTGAAGGTCAAAGGGAATGTG-3'; and L19 reverse, 5'-GGACAGAGTCTTGATGATCTC-3'. QuantiTect SYBR Green RT-PCR Kit (QIAGEN) was used following manufacturer’s instructions. To confirm ubiquitous expression of the transgene, RT-PCR was carried out from various tissues of mouse line 013. Total RNA for RT-PCR analysis was isolated from frozen tissues utilizing single-step phenol chloroform extraction. RNA samples were then DNase treated, and semiquantitative RT-PCR analysis was performed (conditions described in supplemental Table 2). For quantitative HSD17B2 analysis, total RNA was isolated using NucleoSpin RNA II (Macherey-Nagel, Düren, Germany) RNA isolation kit. Quantitative RT-PCR was performed utilizing DyNAmo HS qRT-PCR kit for two-step SYBR Green qRT-PCR (Finnzymes Oy, Espoo, Finland; reaction conditions are described in supplemental Table 3). Primer sequences used to detect mouse endogenous HSD17B2 expression were as follows: mHSD17B2 forward, 5'-GAGCGTCTTTCAGTGCTCCAG-3'; and mHSD17B2 reverse, 5'-CCTTGGACTTTCTAAGTAGAGGCA-3'. The data were normalized by relating HSD17B2 expression to mouse ribosomal protein L19.
HSD17B Activity Measurements
Analyzing HSD17B1 Activity in Vitro.
HSD17B1 activity was determined in the heart tissue of the various mouse lines and WT mice using the method described by Tseng and Gurpide (31), with minor modifications. In brief, tissues were homogenized in 10 mM KH2PO4 (pH 7.5), 1 mM EDTA, 0.02% NaN3, and protein concentrations of the homogenates were determined (Bio-Rad protein assay; Bio-Rad Laboratories, Inc., Hercules, CA). Different amounts of protein (10–800 µg) were mixed with [3H]E1 (PerkinElmer, Life Sciences; 
500 000 cpm) and unlabeled E1 to a final concentration of 37 µmol/liter. The reaction was started by adding 50 µl of NADPH (Sigma-Aldrich, Inc., St Louis, MO), to a final concentration of 1.4 mmol/liter, and the tubes were incubated at +37 C for 3, 6, 9, and 12 min. The reactions were stopped by freezing the reaction tubes in dry ice-ethanol bath. Thereafter, the steroids were extracted twice with 2 ml of diethyl ether (Merck, Darmstadt, Germany), evaporated under nitrogen flow, and dissolved in 150 µl of acetonitrile-water (48%:52%, vol:vol). The amount of E1 converted to E2 was analyzed by separating the [3H]E1and [3H]E2 using HPLC (Waters 2695, Waters Corp.), connected to an online ß-counter.
Analyzing HSD17B1 Activity in Vivo.
HSD17B1 activity in the TG mice was measured in vivo in anesthetized (400–1000 µl 2.5% tribromoethanol ip) 2–3 months old mice (n = 5–6). [3H]E1 and [3H]A-dione and corresponding unlabeled substrates (PerkinElmer, Life Sciences) in a final concentration of 35 µg/kg were used as substrate. The substrate was dissolved in 25% ethanol in water, and an iv injection of 2.5 µl/g was given via the tail vein. Blood was collected by heart puncture from anesthetized mice 2 min after the substrate injection, and the mice were euthanized by cervical dislocation. The steroids were extracted from the blood by ether extraction, dissolved in acetonitrile-water (48:52), and E1/E2- and A-dione/T separation was performed as described above.
Antiandrogen Treatment
Pregnant female mice were treated with antiandrogen flutamide (Sigma-Aldrich) from pregnancy d 13.5 until the day of delivery. Dosing was performed once a day by injecting 50 mg/kg flutamide in 100 µl volume sc. Polyethylene glycol 400 (PEG-400; VWR International, West Chester, PA) was used as a vehicle. Six HSD17B1TG females were treated with flutamide, whereas five TG females received the vehicle only. Similarly, a group of four and three pregnant WT females were daily treated with flutamide or PEG-400 only, respectively. The macroscopic anatomy of the offspring was analyzed at the age of 4 months.
Ovary Transplantations
Prepubertal HSD17B1TG females were anesthetized with 100–300 µl 2.5% tribromoethanol (Avertin; n = 7) given ip, and TG ovaries on each side were replaced with half of the ovary of a WT mouse. As a control, WT ovaries were similarly transplanted to another WT female (n = 10). Anesthetic analgesia was achieved by giving 0.2–0.5 mg/kg buprenorphine (Temgesic; Schering Plough, Brussels, Belgium) preoperatively. Postoperative analgesia was performed similarly when needed. Ovary donors were euthanized by cervical dislocation. Females with transplanted ovaries were analyzed at the age of 4 months.
Histological Analysis
For histological analysis, tissues were fixed in 4% paraformaldehyde at room temperature for 15–20 h. After fixation, tissues were dehydrated and paraffin embedded. Sections cut at 5 µm were stained with hematoxylin and eosin for microscopic analysis. For histological analyses, at least six mice per group were analyzed.
Immunohistochemistry
Whole embryos were fixed in 4% paraformaldehyde at room temperature for 1 wk. Fetal ovaries were dissected, dehydrated in 70% ethanol, and processed for 17.5 h in an automated Leica TP1050 processor (Leica, Inc., Deerfield, IL). Fixed ovaries were embedded in paraffin wax, sectioned (5 µm), floated onto slides coated with 2% 3-aminopropyltriethoxy-silane (Sigma-Aldrich), and dried overnight at 50 C. Three randomly selected sections from each ovary were immunostained for androgen receptor (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) using a Bond-X automated immunostaining machine (Vision Biosystems, Newcastle, UK), with reagents purchased from Vision Biosystems. Sections were deparaffinized in xylene and rehydrated through a series of alcohols. High-temperature antigen retrieval was performed using 0.01 M citrate buffer, pH 6.0. For immunostaining the slides were peroxidase blocked for 5 min, incubated for 2 h with the primary antibody (diluted 1:200 in the diluent supplied), and then incubated with the postprimary reagent for 15 min. Control sections were incubated with the diluent alone to confirm antibody specificity. Sections were then incubated with the polymer reagent for 15 min to increase sensitivity of detection before 3,3'-diaminobenzidine detection for 10 min. Sections were counterstained in hematoxylin for 5 min, dehydrated, and mounted. To ensure reproducibility of results, the sections of ovaries from WT and TG animals were processed in parallel on at least two occasions.
Hormone Measurements
Blood samples were incubated at +4 C for 15–24 h. Serum was separated by centrifugation and stored at –20 C until hormone measurements. Organic extracts were prepared by diethyl ether extraction from serum or tissue samples for T and E2 determinations. E2 concentrations were measured by immunofluorometric assay, using the human estradiol Delfia kit (PerkinElmer Wallac, Inc., Turku, Finland) adapted for mouse samples (32). Serum and tissue T levels were measured by RIA (33). Concentrations of LH and FSH were measured by using time-resolved immunofluorometric assays as previously described (18, 34).
Protein Determinations
Protein concentrations from embryonic tissue homogenates were determined using the BCA Protein Assay Kit (Pierce Chemical Co., Rockford, IL) according to manufacturer’s instructions.
Statistical Analysis
Statistical analyses were carried out using SigmaStat 3.1. program (SPSS, Inc., Chicago, IL) using the following tests: Student’s t test or Mann-Whitney test when applicable to compare two groups and one-way ANOVA or Kruskal-Wallis one-way ANOVA of Ranks when applicable to analyze many groups. Significance was set as P < 0.05, and mean values ± SEM are presented.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Disclosure Statement: T.S., T.L., K.H., M.W., P.S., H.K., and M.P. have nothing to declare.
First Published Online July 31, 2007
Abbreviations: A-dione, Androstenedione; AGD, anogenital distance; E1, estrone; E2, estradiol; E17.5, embryonic d 17.5; HSD17B, hydroxysteroid (17ß) dehydrogenase; OSE, ovarian surface epithelium; T, testosterone; TG, transgenic; WT, wild type.
Received for publication March 16, 2007. Accepted for publication July 18, 2007.
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REFERENCES |
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-dihydroprogesterone dehydrogenase activities in human endometrium during the menstrual cycle. Endocrinology 94:419–423NURSA Molecule Pages Link:
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