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Endocrinology Vol. 149, No. 6 2917-2922
Copyright © 2008 by The Endocrine Society

Estrogen Receptor β Selective Ligand 5{alpha}-Androstane-3β, 17β-Diol Stimulates Spermatogonial Deoxyribonucleic Acid Synthesis in Rat Seminiferous Epithelium in Vitro

Aida Wahlgren, Konstantin Svechnikov, Mona-Lisa Strand, Kirsi Jahnukainen, Martti Parvinen, Jan-Åke Gustafsson and Olle Söder

Department of Woman and Child Health (A.W., K.S., M.-L.S., K.J., O.S.), Pediatric Endocrinology Unit, Astrid Lindgren Children’s Hospital and Department of Biosciences and Nutrition (J.-Å.G.), Novum, Karolinska Institutet and University Hospital, S-171 76 Stockholm, Sweden; Department of Pediatrics (K.J.), Turku University Hospital, Institute of Biomedicine, 20521 Turku, Finland; and Department of Physiology (M.P.), University of Turku, 20520 Turku, Finland

Address all correspondence and requests for reprints to: Aida Wahlgren, M.D., Ph.D., Department of Woman and Child Health, Pediatric Endocrinology Unit, Q2:08, Astrid Lindgren Children’s Hospital, Karolinska Institutet & University Hospital, S-171 76 Stockholm, Sweden. E-mail: aida.wahlgren{at}ki.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gonadotropins and testosterone are important regulators of spermatogenesis, even though gonadotropin receptors and the androgen receptor are not expressed by germ cells. However, a functional role for estrogens in connection with male reproduction has been postulated on the basis of the phenotypes of mice lacking estrogen receptor (ER) and cytochrome P-450 aromatase. This has further support by findings of ER expression in the testis, including that of ERβ in spermatogonia. 5{alpha}-Androstane-3β, 17β-diol (3βAdiol), a metabolite of testosterone produced via the intermediate potent androgen 5{alpha}-dihydrotestosterone (DHT), has been reported to selectively bind ERβ rather than ER{alpha}, but not androgen receptor. Here, we have characterized the influence of 17β-estradiol (E), the major physiological estrogen, 3βAdiol, and DHT on DNA synthesis in vitro by segments of the seminiferous epithelium at different stages of the seminiferous epithelial cycle in the rat. E and 3βAdiol exerted similar stimulatory effects on premitotic DNA synthesis in stage I segments, whereas other stages tested (V, VIIa, and XIII–IX) remained unresponsive. In contrast, DHT had no effect on this process. 5-bromo-2'-deoxyuridine labeling of stage I segments revealed a 30-fold higher labeling index in the presence than in the absence of E, and the labeled cells were identified as spermatogonia. Moreover, high levels of 3βAdiol were found in the testis of intact rats as well as in primary cultures of rat Leydig cells in response to human chorionic gonadotropin. We suggest that 3βAdiol may serve as a growth factor for germ cells stimulating premitotic DNA synthesis in connection with spermatogenesis via an ERβ-dependent pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WELL established that spermatogenesis is regulated by the pituitary gonadotropins and paracrine testosterone (T), although the developing male germ cells lack receptors for these hormones. In males, estrogens are synthesized primarily in the testis from T by the enzyme cytochrome P-450 aromatase (CYP19). Expression of CYP19 in Sertoli and Leydig cells is well documented (1), and it has also been detected in spermatocytes and spermatids of rodents (2, 3), and in gonocytes and spermatogonia of the human testis (4). The effects of estrogens are mediated via two nuclear receptors, the estrogen receptors (ERs) {alpha} and β. In the rat testis, ER{alpha} is present in Leydig cells and the efferent ductules. ERβ, on the other hand, has been localized to rat Sertoli cells, spermatogonia (5, 6), pachytene spermatocytes, and early round spermatids (5, 6, 7, 8). In a recent human study, ERβ, but not ER{alpha} and the androgen receptor (AR), was expressed by gonocytes and spermatogonia (4).

The role of estrogens in male reproduction is complex. Previous studies have shown that male mice lacking ER{alpha} are completely infertile, whereas male mice lacking ERβ are fertile (9, 10). Disruption of spermatogenesis in the ER{alpha} knockouts was due to the increased tubular pressure relating to reduced fluid reabsorption in the efferent ducts (11).

Female ERβ knockout mice have reduced fertility compared with their wild types (12), and Gould et al. (13) have shown that the spermatogonial number per testis was increased in male ERβ knockout mice, indicating a role of this receptor in the regulation of fertility in both sexes.

In rats the concentration of estrogen in rete testis fluid is considerably higher than in serum from female animals (14, 15). However, until recently, the function(s) of estrogen in male reproductive organs remained obscure. In 2001, Weihua et al. (16) demonstrated that both estradiol (E) and the androgen 5{alpha}-androstane-3β, 17β-diol (3βAdiol), a metabolite of the potent androgen dihydrotestosterone (DHT), bind with high affinity to ERβ, which appears to play an important role(s) in the growth and differentiation of the prostate epithelium.

The aim of the present study was to determine whether E and 3βAdiol influence DNA synthesis by germ cells in connection with spermatogenesis. For this purpose we examined the effects of various sex steroids on DNA synthesis during specific stages of the seminiferous epithelial cycle in the rat, using isolated microdissected segments of seminiferous tubules. Moreover, the testicular level of 3βAdiol as well as the capacity of Leydig cells to produce this steroid were also investigated. In addition, we used 5-bromo-2'-deoxyuridine (BrdU) staining combined with morphological analysis to identify the proliferating germ cell in the seminiferous epithelium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals, hormones, and growth factors
Sixty-day-old adult male Sprague Dawley rats (B&K Universal, Sollentuna, Sweden) were maintained under standard laboratory conditions with access to standard chow and water ad libitum. 17β-E and DHT were obtained from Sigma-Aldrich Sweden AB (Stockholm, Sweden), whereas 3βAdiol was a kind gift from by Dr. Margaret Warner (Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden). Human cytosolic aldo-keto reductase (AKRICI) was a gift from Dr. Penning (University of Pennsylvania School of Medicine, Philadelphia, PA). All animal experiments were conducted in accordance with institutional guidelines and approved in advance by the local ethics committee for animal experimentation (N151/01; N218/05).

Staging and microdissection of segments of the seminiferous tubules
After decapsulation of the testes, microdissection was performed in PBS (modified according to Dulbecco, i.e. without calcium and magnesium; Life Technologies, Inc., BRL, Baisley, UK) under a transilluminating stereomicroscope (17). Using fine forceps the seminiferous tubules were carefully teased apart in a petri dish containing PBS. The stages of the seminiferous epithelium were identified on the basis of the light absorption criteria described previously (17, 18).

Stages I, V, VIIa, and VIII–IX were selected for study. These stages are characterized by harboring developing germ cells in distinct phases of mitotic and meiotic division. Stage I contains primarily type A4 spermatogonia in the S phase of the cell cycle, stage V contains type B spermatogonia in the S phase, stage VIIa, preleptotene spermatocytes exhibiting no DNA synthesis, and stages VIII–IX, these same spermatocytes in the S phase (19). Two-millimeter segments of tubule from these various stages were collected and transferred individually in 10 µl PBS to microwells for subsequent culture. For evaluation of labeling with BrdU, squash preparations were prepared from 0.5-mm stage I segments of seminiferous tubules.

Culture conditions
To maintain an environment as close to physiological as possible for the germ cells, whole segments of seminiferous tubules were cultured on 96-well tissue culture plates (Falcon 3072 Microtest III; Becton Dickinson, Lincoln Park, NJ), as described previously (20). All of these incubations were performed in 100 µl Modified Eagle’s MEM ({alpha}-MEM) containing Earle’s salts (Flow Laboratories Svenska AB, Solna, Sweden), and supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and {alpha}-thioglycerol (7.5 x 10–5 M) at 34 C, i.e. the physiological temperature of the testis.

Analysis of DNA synthesis using 3H-thymidine incorporation
Tubule segments were incubated on 96-well plates at 34 C and labeled for the last 4-h culture with 0.5 µCi 3H-thymidine per well (specific radioactivity 5.0 mCi/mmol; Amersham Pharmacia Biotech, Buckinghamshire, UK), and subsequently harvested at 24, 48, or 72 h culture (21). The radioactivity thus incorporated into DNA of proliferating cells in one single 2-mm tubule segment per culture well was quantified as counts per minute in a Beckman scintillation spectrometer (Beckman Coulter, Inc., Fullerton, CA). In each experiment six individual replicate preparations of tubule segments were used for each concentration of each hormone and each time point, and three such independent experiments were performed.

Estimation of cell viability in segments of seminiferous tubules using supravital staining
The supravital precursor dye C,N-diphenyl-N'-4,5-dimethylthiazol-2-yl tetrazolium bromide (MTT) (Sigma-Aldrich Sweden AB) was added to the segments (0.5 mg/well) the last 4 h of the total culture time of 24, 48, or 72 h (22). Thereafter, the segments were lysed with 10% sodium dodecyl sulfate in 0.01 M HCl (100 µl/well) and then incubated at 34 C for an additional 20 h. The resulting absorbance at 570 nm was used as a measure of the number of living cells present in the cultures (23). In each experiment five to eight individual preparations of tubules in the presence or absence of 17β-E were examined at each time point, and three such independent experiments were performed.

Detection and identification of cells in the S phase by BrdU labeling
To assess further cell proliferation and to identify the proliferating cells, labeling with the thymidine analog BrdU was performed. Squash preparations were prepared from 0.5-mm stage I tubule segments, treated and nontreated with 17β-E. The cultures were pulsed with BrdU and 5-fluoro-2'-deoxyuridine (cell proliferation kit RPN20; Amersham Pharmacia Biotech), final dilution 1:50, for the last 4 h of the total 24 or 48-h incubation. To identify labeled cells, the labeled seminiferous tubules were carefully squashed between microscope slides and coverslips, giving a monolayer of slightly flattened cells.

After freezing these slides in liquid nitrogen and removing the coverslips flipping with a scalpel, the frozen squash preparations were fixed in ice-cold ethanol, incubated for 10 min in 10% formalin, washed twice in PBS for 5 min each time, incubated for 5 min in ethanol/acetic acid (2:1), and finally washed twice again in PBS for 5 min each time. Immunocytochemical staining was subsequently performed according to the manufacturer’s instructions. The number of BrdU-positive cells (per 0.5 mm) in three independently prepared samples of segments after 48-h incubation was counted in each of two independent experiments. Using a phase contrast microscope, virtually all of the labeled, stained cells could be easily distinguished from unlabeled cells.

Determination of 3βAdiol in the testis
Because no commercial source of radiolabeled 3βAdiol is currently available, needed for quantitative measurement of this steroid, we synthesized 3H-3βAdiol using recombinant AKRICI. This enzyme catalyzes preferentially the reduction of DHT into 3βAdiol. After incubation of 3H-DHT (10 µCi) in the presence of AKRICI (10 µg protein) for 2 h at 37 C, the reaction was terminated by the addition of 500 µl ice-cold ethyl acetate and steroids extracted by continuous vortexing for 5 min. The organic phases were transferred into a tube, and the extraction step was repeated once. The combined organic phases were evaporated to complete dryness. The dried extracts were redissolved in 20 µl ethyl acetate and applied to Silica gel 60 F254 thin-layer chromatography plates (Merck, Darmstadt, Germany). In parallel, nonradioactive 3βAdiol and DHT were applied as reference steroids. Chromatograms were developed in chloroform/ethyl acetate (4:1). 3H-3βAdiol was isolated after identification of its position on the thin layer chromatography plate by autoradiography and by comparison to an authentic reference standard. The tracer thus obtained and a specific anti-3βAdiol polyclonal antibody (BioSite, Stockholm, Sweden) were further used for RIA of 3βAdiol. Anti-3βAdiol polyclonal antibodies demonstrated high specificity for 3βAdiol, the only cross-reacting steroids being DHT and T (10 and 5%, respectively). The sensitivity of the method was 80 pg/tube or 2 ng/ml. The interassay and intraassay precisions were 12 and 8%, respectively, at the actual levels of determination.

To assay intratesticular concentrations of 3βAdiol, testicular tissue obtained from individual rats was homogenized by sonication (2 x 20 sec) in a sodium phosphate buffer, and then steroids were extracted by ice-cold ethyl acetate (1:2 vol/vol). The organic phases were transferred into a tube, and the extraction step was repeated once. The combined organic phases were evaporated to complete dryness. The dried extracts were redissolved in 100 µl sodium phosphate buffer, and 3βAdiol concentrations in the supernatant were determined by RIA. In a separate experiment, concentrations of 3βAdiol in culture media from control and human chorionic gonadotropin (hCG)-stimulated rat Leydig cells in primary culture, as described previously (24), were also assayed.

Androgen assay
Media collected from the cultures described previously were stored at –20 C before assaying T and DHT using a Coat-a-Count RIA kit for T (Diagnostic Products Corp., Los Angeles, CA) and an ELISA procedure for DHT (catalog no. 1940; Diagnostic Intl., San Antonio, TX), according to the manufacturer’s instructions. The reported cross-reactivity of the T RIA for DHT was 3.3% and that for T with the DHT ELISA was 8%.

Data processing and statistical analysis
Sigma plot 2000 (version 8.0; Jandel Scientific, Sausalito, CA) was used to obtain mean values ± SEM from the data, as well as to produce graphs. Statistical analysis was performed using ANOVA followed by Duncan’s test (Figs. 1Go and 2Go), the Student’s t test (Fig. 3Go) and ANOVA with supplementation by the Student-Newman-Keuls t test (Fig. 4Go) (Sigma Stat v 3.0 package; SPSS, Inc., Chicago, IL). P < 0.05 was considered statistically significant.


Figure 1
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  		FIG. 1. Stimulatory effects of 17β-E on DNA synthesis in stage I rat testis seminiferous tubule segments in vitro. E dose dependently stimulated the incorporation of tritiated thymidine after 48 h culture in stage I of seminiferous tubules at 34 C. No effect was demonstrated in cultures of tubule stages V, VIIa, and VII–IX after incubation with E. Data points are mean counts per min (cpm) ± SEM from six replicates of independently microdissected segments. Three separate experiments were performed with the same results. The asterisk (*) indicates statistically significant changes (P < 0.05) vs. control (0 ng/ml) as calculated by ANOVA, followed by Duncan’s test.

 

Figure 2
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  		FIG. 2. Effect of various steroids on DNA synthesis by testicular cells in cultures of microdissected stage I segments of seminiferous tubules from rat testes in vitro. 3βAdiol stimulated DNA synthesis of stage I seminiferous tubules in vitro (A), whereas DHT had no significant effect (B). Incubations were performed for 48 h at 34 C. No effects on DNA synthesis by segments of tubule stages V, VIIa, and VIII–IX were observed for both steroids tested (data not shown). Data points are mean counts per minute (cpm) ± SEM of six individually microdissected tubule segments. Three independent experiments were performed, showing the same results. The asterisk (*) indicates statistically significant changes (P < 0.05) vs. control (0 ng/ml) as calculated by ANOVA, followed by Duncan’s test.

 

Figure 3
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  		FIG. 3. 17β-E stimulated incorporation of BrdU by spermatogonia in rat stage I tubule segments in vitro. A, A micrograph showing BrdU-labeled cells (arrows) after incubation with E for 48 h at 34 C (magnification, x40). B, A micrograph of the living cells corresponding to the BrdU-labeled cells in A, illustrating their morphology. These cells can be identified as type A spermatogonia on the basis of their nuclear structure (heterochromatin). C, The number of cells per segment incorporating BrdU after a 48-h incubation at 34 C in the presence or absence of E (***, P < 0.001, Student’s t test).

 

Figure 4
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  		FIG. 4. Activation of 3βAdiol, DHT, and T production by adult Leydig in vitro in response to hCG. Primary cultures of Leydig cells were incubated with hCG (10 ng/ml) or standard medium alone (control) for 24 h. 3βAdiol, DHT, and T in the culture medium were then measured by RIA, and the results are expressed as ng per 105 cells per 24 h. Each experiment was performed three times independently, obtaining similar results. Statistically significant changes vs. control as calculated by ANOVA with supplementation by the Student-Newman-Keuls t test are expressed as **, P < 0.01 and *, P < 0.05.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of E, 3βAdiol, and DHT on DNA synthesis
After 24 (data not shown) and 48 h exposure at 34 C and pulse labeling with tritiated thymidine the last 4 h of culture, E stimulated DNA synthesis at stage I of the seminiferous tubules (Fig. 1Go). At the lowest dose (10 ng/ml), the level of DNA synthesis was slightly elevated compared with the control value, and a doubling in DNA synthesis was observed at the higher doses (100 and 1000 ng/ml). In contrast, no significant stimulation was detected at any time point in the case of the other stages (V, VIIa, and VIII–IX; see Fig. 1Go for data from 48 h) or after 72-h exposure at stage I (data not shown).

3βAdiol exerted a similar stimulatory effect on DNA synthesis in stage I segments of seminiferous tubules after a 48-h incubation (Fig. 2AGo), and no such stimulation was observed at any of the other stages tested (data not shown). DHT had no influence on DNA synthesis upon exposure at stage I for 48 h (Fig. 2BGo), or at any other stage or time point tested (data not shown).

Enumeration and identification of BrdU-labeled cells
When the morphology of living cells was compared with the pattern of staining (Fig. 3Go, A and B), the nuclear structure (heterochromatin) of the stained cells indicated that they were type A spermatogonia. Labeling with BrdU was significantly increased in segments treated for 48 h with E (P < 0.001) in comparison to the control (Fig. 3CGo).

Estimation of cell viability by MTT staining
The cell viability in cultured segments of seminiferous tubules was estimated on the basis of MTT staining. No statistical difference between control and E (100 ng/ml) treated cultures was observed (data not shown).

Production of 3βAdiol, DHT, and T
The concentration of 3βAdiol in testis tissue extracts from intact adult 60-d-old rats was 103 ± 9.1 ng/g tissue (n = 6). Further experiments showed that adult Leydig cells secreted detectable levels of 3βAdiol at basal conditions in primary cultures in vitro, and that stimulation with hCG significantly increased production of 3βAdiol and DHT (8- and 3-fold over control, respectively). Moreover, 3βAdiol and DHT production comprised 13 and 20%, respectively, of total steroid production by hCG stimulated Leydig cells when including T as a reference (Fig. 4Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here, we demonstrate that the major physiological estrogen 17β-E and the ERβ selective agonist 3βAdiol, but not the potent androgen DHT, are able to stimulate DNA synthesis by spermatogonia in cultures of microdissected stage I segments of rat seminiferous tubules in vitro. Furthermore, we also demonstrate that 3βAdiol is produced in substantial amounts by the intact rat testis and that its secretion by Leydig cells in vitro is under gonadotropin control. Apart from 3βAdiol, production of DHT, a precursor in the biosynthesis of 3βAdiol, was also increased in response to hCG by adult rat Leydig cells in vitro.

ERβ rather than ER{alpha} mRNA has been detected in germ cells in human (7, 25), rat (6, 26), and mouse (3, 27), and ERβ protein has been found in the same cells in the rat (5, 7) and mouse (28). E and 3βAdiol both bind ERβ and may exert agonist action on this receptor. 3βAdiol binds more selectively to ERβ than ER{alpha} (8), which makes the expression of ERβ on male germ cells highly relevant in the present context. Because ER but not AR is expressed by germ cells, our data suggest that the important role of T for the maintenance of spermatogenesis may be to serve as a paracrine substrate for production of estrogen. E may thus be locally synthesized from T (but not DHT) via action of CYP19, and 3βAdiol from T over DHT by sequential actions of 5{alpha}-reductase and 3β-hydroxysteroid dehydrogenase, respectively. ERs are also expressed by somatic cells in the testis in multiple species, including man (4, 5, 6, 7, 8), and it cannot be excluded that E and 3βAdiol also act indirectly via effects exerted on testicular somatic cells. Such action has been implicated for androgen, which is supported by observations of high levels of AR expression by somatic cells (29, 30, 31) rather than germ cells, although previous studies were contradictory (32, 33).

The increasing interest in the role(s) played by estrogens in the male also occurs from findings that ERs are expressed in the testis and male reproductive tract from prenatal life throughout adulthood. Of significance in this context is evidence that male fertility is impaired in mice lacking ER{alpha} (9, 10, 34, 35) or aromatase (36, 37, 38). Furthermore, certain reports indicate that exposure to environmental estrogens can result in a range of abnormalities in male reproductive development and function (39, 40). The present findings that estrogens directly affect male germ cells add to the possible mechanisms of action of such endocrine disrupting compounds displaying estrogen agonist or antagonist activity.

The significance of local conversion of T into E in the testis is further demonstrated by aromatase-deficient mice, which lack a functional gene for aromatase (37). In this model there is evidence of disrupted spermatogenesis and a reduction of testis weight. Further on elongated spermatids are not seen due to a disruption in early spermiogenesis in which degenerating round spermatids and multinucleated cells are frequently found.

Studies in lower vertebrate animals have shown that E enhances spermatogonial proliferation (41, 42). This is in line with the present observations and also indicates an evolutionary conserved regulation. E has also been shown to induce c-fos activity in the testis, and to regulate storage and phosphorylation of Fos proteins in the cytoplasm and nucleus of germ cells, respectively, thus potentially contributing to the regulation of testicular function. This indicates that E may be involved in the mechanisms inducing spermatogonial proliferation (43, 44), which concords with our results. The testicular phenotype of ERβ knockout mice showing increased spermatogonial number (13) is difficult to interpret but indicates that ERβ may be of importance for spermatogonial control.

From the present data, we suggest that 17β-E and 3βAdiol may act as paracrine growth factors stimulating replicative DNA synthesis of developing male germ cells. It was previously demonstrated that E may act as a germ cell survival factor exerting antiapoptotic effects in the human testis in vitro (45). Such action may add to the present findings, although we were not able to detect any difference in cell survival between E treated and control cultures. Increased survival detected by the presently used method has accompanied antiapoptotic actions on testicular cells (46). However, we cannot exclude small differences of apoptotic activity in the cultures because we did not look specifically for such effects. Our present data are also consistent with the observation that E, DHT, and 3βAdiol concentrations are readily measurable in human testicular tissue fluid (40).

Our study is the first to show that 3βAdiol is produced and secreted by Leydig cells at high levels in response to stimulation with hCG and that the testicular concentration of this steroid is in the range of T after stimulation. Moreover, we observed that hCG-stimulated Leydig cells are able to produce significant levels of DHT that can further be converted to 3βAdiol by 3β-hydroxysteroid dehydrogenase (47, 48). Recent studies have shown that 3βAdiol is a potent modulator of ERβ-mediated gene transcription in neuronal (49) and Sertoli cell lines (50).

From our data we hypothesize that because the germ cells lack AR, the effects of androgens on spermatogenesis may be mediated to germ cells through 3βAdiol, a natural endogenous ligand for ERβ. We speculate that the androgen 3βAdiol having estrogenic action works in concert with E, and controls ERβ-mediated cellular processes in the reproductive organs. T shows very high paracrine concentrations within the testis (>100 x plasma levels) and may be converted to DHT in substantial amounts as reported previously (51, 52). In the human, 3βAdiol has been the major metabolite of DHT in the testis (53), and a significant decrease in the amount of this steroid in seminal plasma is found in men with defects in spermatogenesis (54). Interestingly, recent data indicate that ERβ may also play a role in the fetal testis (55). This finding has implications for male prenatal sex differentiation, including set points for future spermatogenesis and its susceptibility to perturbation by estrogen-acting endocrine disrupting chemicals.

Together, our data suggest that 17β-E and 3βAdiol may work as spermatogonial growth factors and, via modulation of ER{alpha} and ERβ activities, are involved in controlling the function of spermatogonia and spermatogenesis.


   Footnotes
 
This work was supported by the European Union Commission (CASCADE NoE FOOD-CT-2004-506319; PIONEER STREP FOOD-CT-2005-513991), by grants from the Swedish Research Council (Project No. 2005-8282), the Swedish Children’s Cancer Fund, the Foundation Frimurare Barnhuset in Stockholm, Crown Princess Lovisa’s Society of Pediatric Health Care, the Society for Child Care, the Wera Ekström’s Fund for Pediatric Research, Arbetsmarknadens Försäkringsaktiebolag (AFA) Sickness Insurance Research Fund, the Sigfrid Jusélius Foundation, and Karolinska Institutet.

Disclosure Statement: A.W., K.S., M.-L.S., K.J., and M.P. have nothing to declare. J.-Å.G. has consulted for and has equity interests in Karo Bio. O.S. has consulted for Eli-Lilly & Co.

First Published Online February 21, 2008

Abbreviations: AKRICI, Human cytosolic aldo-keto reductase; AR, androgen receptor; 3βAdiol, 5{alpha}-androstane-3β, 17β-diol; BrdU, 5-bromo-2'-deoxyuridine; CYP19, cytochrome P-450 aromatase; DHT, dihydrotestosterone; E, estradiol; ER, estrogen receptor; hCG, human chorionic gonadotropin; T, testosterone.

Received August 20, 2007.

Accepted for publication February 13, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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