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Molecular Endocrinology, doi:10.1210/me.2007-0144
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Molecular Endocrinology 21 (11): 2627-2636
Copyright © 2007 by The Endocrine Society

Activation of Androgens by Hydroxysteroid (17ß) Dehydrogenase 1 in Vivo as a Cause of Prenatal Masculinization and Ovarian Benign Serous Cystadenomas

Taija Saloniemi, Tarja Lamminen, Kaisa Huhtinen, Michelle Welsh, Philippa Saunders, Harry Kujari and Matti Poutanen

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.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hydroxysteroid (17ß) dehydrogenases (HSD17Bs) belong to the short-chain dehydrogenase/reductase family consisting of a diverse pool of enzymes with oxidoreductase activity. HSD17B enzymes catalyze the conversion between 17-keto and 17-hydroxy steroids, either activating or inactivating sex steroids. Previous studies have demonstrated a role for human HSD17B1 enzyme in estradiol (E2) biosynthesis both in gonads and extragonadal steroid target tissues and various estrogen-dependent diseases. In the present study, five transgenic (TG) mouse lines universally overexpressing human HSD17B1 were generated and characterized at fetal and adult ages, especially to study the enzyme function in vivo. Activity measurements in vivo indicated that in addition to activating estrone to E2, the enzyme is able to significantly reduce androstenedione to testosterone, and TG females presented increased testosterone concentration preceding birth. As a consequence, TG females suffered from several phenotypic features typical to enhanced fetal androgen exposure. Furthermore, the ovaries developed androgen-dependent ovarian benign serous cystadenomas at adulthood. Androgen dependency of the phenotypes was confirmed by rescuing them by antiandrogen treatment, or by transplanting wild-type ovaries to the TG females. In conclusion, the data evidently show that, in addition to activating estrone to E2, human HSD17B1 enhances androgen action in vivo. Thus, the relative amounts of androgenic and estrogenic substrates available partially determine the physiological function of the enzyme in vivo. The novel function observed for human HSD17B1 is likely to open new possibilities also for the use of HSD17B1-inhibitors as drugs against androgen-related dysfunctions in females.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HYDROXYSTEROID (17ß) DEHYDROGENASES (HSD17Bs) catalyze the reaction between the low active 17ß-ketosteroids and the highly active 17ß-hydroxysteroids. Currently, more than 12 different HSD17B enzymes having individual cell-specific expression profiles, substrate specificities, and unique regulatory mechanisms have been identified. In mammals, still increasing evidence is suggesting variable substrate specificities and differential physiological roles of these enzymes (1), and it is likely that some of the HSD17B enzymes act in multiple metabolic pathways. For example, HSD17B2 possesses 20{alpha}-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Five HSD17B1TG mouse lines were generated to study the relationship between TG expression in vitro and in vivo activities and phenotypes. Quantitative RT-PCR analysis revealed differential expression of the transgene in the different HSD17B1TG mouse lines (Fig. 1AGo). Strongest expression was detected in line 013, in which the universal expression of the transgene was confirmed (Fig. 1BGo), 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. 1CGo). 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).


Figure 1
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  		Fig. 1. Transgene Expression and HSD17B1 Activity in Vitro

A, Quantitative RT-PCR analysis of transgene expression from male heart in various TG mouse lines (013, 050, 012, 020, 016) and WT. Highest expression was detected in line 013, which was chosen for further experimentation. B, Transgene expression (line 013) was detected in all tissues studied by RT-PCR. C, HSD17B1 activity (E1 to E2) in male heart was determined in vitro. HSD17B activity in various TG mouse lines correlated with the quantitative RT-PCR analysis for HSD17B1 in various mouse lines.

 
To typify substrate specificity of human HSD17B1 expressed in the TG mice, enzyme activities using E1, E2, and androstenedione (A-dione) as substrate were determined in line 013 and wild-type (WT) mice in vivo. With a substrate concentration of 35 µg/kg, both the estrogenic and androgenic activities were significantly increased in HSD17B1TG mice compared with WT mice (Fig. 2Go, 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. 2AGo). 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 1Go).


Figure 2
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  		Fig. 2. HSD17B1 Activity Measurement in Vivo

A, Reductive (E1 to E2) and oxidative (E2 to E1) activity was determined in TG and WT males. Reductive activity was significantly higher than oxidative activity in both TG and WT mice (*, P < 0.05, Holm-Sidak method) and in TG mice the reductive activity was significantly higher than the oxidative conversion (***, P < 0.001, t test). B, The reductive androgenic activity (A-dione to T) was also determined from males and females and was shown to be significantly higher in the HSD17B1TG mice (P < 0.001, t test).

 

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  		Table 1. Compensatory HSD17B2 Induction in HSD17B1TG Mice

 
As summarized in Table 2Go, 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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  		Table 2. Androgen-Mediated Phenotypic Changes in HSD17B1TG Female Mice

 
Several of the phenotypes in HSD17B1TG females were present at birth, indicating enhanced androgen production during fetal life. These included increased AGD, morphological alterations at the lower reproductive tract, such as lack of vaginal opening and improper separation of vagina and urethra, and suppressed nipple development. In line with these morphological changes, intratissular testosterone (T) and E2 were significantly increased in TG female fetuses at embryonic d 17.5 (E17.5) (WT: T, 3.36 ± 0.57 pg/mg protein; TG: T, 9.48 ± 1.66 pg/mg protein, P < 0.01; WT: E2, 1.56 ± 0.59 pg/mg protein; TG: E2, 6.2 ± 1.21 pg/mg protein, P < 0.05), whereas placental T was not increased (Table 3Go). 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. 3Go, A and B). Also the development of upper nipples (Fig. 3CGo) and opening of vagina were induced in the flutamide-treated mice, but not in the placebo-treated group (Fig. 3Go, A and B). We also demonstrated an improper separation of vagina and urethra in HSD17B1TG females (Fig. 3EGo), which could be rescued to a morphology similar to that of WT mice by prenatal flutamide treatment (Fig. 3Go, D and F). Although differences in sex steroid concentrations were detected before birth, they were normalized by adulthood (Table 3Go). 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. 4Go, 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. 4CGo), and development of OSE hyperplasia was also suppressed in WT ovaries prepubertally transplanted to HSD17B1TG females (Fig. 4BGo). 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. 4Go, G and H). Table 4Go 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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  		Table 3. Hormone Concentrations in HSD17B1TG and WT Females

 

Figure 3
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  		Fig. 3. Feminine Pseudohermaphroditism

A, Prenatally flutamide-treated TG female, with normal AGD and vaginal opening (arrow). B, Placebo-treated TG female with increased AGD and closed vagina (arrow). C, Nipple (arrows) development could be induced by prenatal flutamide treatment, whereas nipples were absent in placebo-treated TG females. D, Combination of vagina and urethra in HSD17B1TG females could be recovered by prenatal flutamide treatment, and separate cavities are shown for vagina and urethra. E, The vaginal and urethral cavities were combined in placebo-treated HSD17B1TG female. F, Separate vaginal and urethral cavities are shown for WT female. Flut, Flutamide; Pla, placebo.

 

Figure 4
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  		Fig. 4. Ovarian Benign Serous Cystadenomas in HSD17B1TG Females at the Age of 4 Months

A, The OSE of untreated HSD17B1TG ovary was hyperplastic (arrow). B, Development of epithelial hyperplasia in TG females was prevented by prepubertal transplantation of WT ovaries and (C) by prenatal flutamide treatment. D, In placebo-treated TG female mice the hyperplasia was observed (arrow). E, The appearance of ovarian cysts in HSD17B1TG females (arrowhead) with the ovarian surface shown by dotted line (arrow). F, A closer view of cystic ovaries of HSD17B1TG females (cyst wall indicated by arrow). Androgen receptor was present in fetal WT (G) and HSD17B1TG (H) ovaries at E17.5. Flut, Flutamide; Pla, placebo.

 

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  		Table 4. Phenotype Rescuing in HSD17B1TG Female Mice

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Based on studies in vitro, human HSD17B1 has been considered to be highly estrogen specific with a markedly higher Michaelis-Menten constant (Km) values for androgenic substrates (4, 13, 15). Furthermore, the enzyme catalyzes both oxidative and reductive HSD17B activity with a proper cofactor added in vitro. However, in cultured cells, human HSD17B1 has been shown to catalyze predominantly reductive reaction. In cultured COS m6 cells the expression of human HSD17B1 decreased the oxidative activity (converting E2 to E1), whereas the reductive activity converting E1 to E2 was markedly increased. Furthermore, with A-dione as a substrate, the activity obtained was 20% of that observed with E1 (4). This suggests that the human enzyme possesses considerable androgenic activity with the preference for estrogenic substrates. Our recent studies have further provided evidence for a reductive estrogenic activity of human HSD17B1 in physiological conditions in vivo. Xenografting HSD17B1-transfected MCF7-cells into immunodeficient nude mice results in enhanced estrogen-dependent tumor growth in the presence of E1, whereas no such effect was detected in the absence of HSD17B1 (16).

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Establishment of HSD17B1 TG Mouse Lines
A cDNA fragment of the human HSD17B1, encoding for the full-length enzyme, was cloned under the chicken ß-actin promoter (kindly provided by Jun-ichi Miyazaki, Osaka University, Osaka, Japan (published as supplemental Fig. 1 on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Reductive HSD17B activity for the transgene construct was verified in cultured cells as previously described (15), with minor modifications. Briefly, human embryonic kidney-293 cells were transfected with the transgene construct using lipofectamine method (Invitrogen, Carslbad, CA). Transfected cells (4 x 105 cells per well) were applied in 12-well plates in DMEM/F12–10% fetal calf serum, including penicillin and streptomycin. After overnight culture, the medium was aspirated from the wells, and 2 ml of serum-free medium containing 200 nM [3H]E1 (2 x 105 cpm/ml; PerkinElmer Life Sciences, Boston, MA) was applied to the cells. The cells were then incubated at 37 C for 1, 2, 4, 8, and 24 h in the cell culture conditions. After incubation, E1 and E2 were separated on HPLC system (Waters 2695; Waters Corp., Milford, MA). The radioactivities were measured in an online ß-counter and the HSD17B activities were presented as percentages of the E2 produced.

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.


   ACKNOWLEDGMENTS
 
We thank the research assistants Nina Messner, Erja Mäntysalo, Heli Niittymäki, Tarja Laiho, Taina Kirjonen, Katja Suomi, Jonna Palmu, Johanna Lahtinen, Hannele Rekola, and Chris McKinnell for technical assistance. Heikki Hiekkanen is acknowledged for statistical advice. Dr. Sari Mäkelä and Dr. Saija Savolainen are acknowledged for histological advice.


   FOOTNOTES
 
This work was supported by The Academy of Finland (Project 211480), Hormos Medical Ltd., Solvay Pharmaceuticals B.V., Turku Graduate School for Biomedical Sciences (TUBS), Finnish Cultural Foundation, and British Medical Research Council.

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.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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