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Endocrinology, doi:10.1210/en.2003-1628
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Endocrinology Vol. 145, No. 8 3950-3960
Copyright © 2004 by The Endocrine Society

The Effects of Estrogen on the Expression of Genes Underlying the Differentiation of Somatic Cells in the Murine Gonad

Kara L. Britt, Peter G. Stanton, Marie Misso, Evan R. Simpson and Jock K. Findlay

Prince Henry’s Institute of Medical Research (K.L.B., P.G.S., M.M., E.R.S., J.K.F.) and the Department of Biochemistry and Molecular Biology (K.L.B., M.M.), Monash University, Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Kara Britt, Prince Henry’s Institute of Medical Research, Monash Medical Centre Clayton, Block E, Level 4, Clayton, Victoria 3168, Australia. E-mail: kara.britt{at}phimr.monash.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen (17ß-estradiol, E2)-deficient aromatase knockout (ArKO) mice develop Sertoli and Leydig cells at puberty. We hypothesized that estrogen, directly or indirectly, regulates genes responsible for somatic cell differentiation and steroidogenesis. ArKO ovaries expressed estrogen receptors {alpha} and ß, and LH receptor, indices of estrogen responsiveness in the ovary. Wild-type (Wt) and ArKO mice received either E2 or placebo for 3 wk, from 7–10 wk of age. E2 decreased serum FSH and LH and increased uterine weights of 10-wk-old ArKO mice. We measured mRNA expression of Sertoli cell, Sry-like HMG box protein 9 (Sox9); three upstream transcription factors, liver receptor homolog-1 (Lrh-1), steroidogenic factor 1, and dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on the X chromosome gene 1; and one downstream factor, Müllerian-inhibiting substance. Placebo-treated ArKO ovaries have increased Sox9 (15-fold; P < 0.001), Müllerian-inhibiting substance (2.9-fold), Lrh-1 (7.7-fold), and dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on the X chromosome gene 1 (12-fold) expression compared with Wt at 10 wk. Steroidogenic factor 1 was similar to Wt. Consistent with increased serum T levels and Leydig cells in their ovaries, placebo-treated ArKO ovaries had increased 17{alpha}-hydroxylase, 17ß-hydroxysteroid dehydrogenase type-3, and 17ß-hydroxysteroid dehydrogenase type-1 expression compared with Wt at 10 wk. E2 treatment for 3 wk improved the ovarian phenotype, decreased development of Sertoli cells, decreased the expression of Sox9, Lrh-1, and the steroidogenic enzymes in ArKO ovaries, and induced ovulation in some cases. In conclusion, the expression of the genes regulating somatic cell differentiation is directly or indirectly responsive to estrogen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGEN PLAYS A VITAL role in the control of the cyclic pattern of ovarian follicular development essential for fertility. The exact roles of estrogen in the ovary have not been fully defined. Estrogens stimulate the proliferation of granulosa cells and also facilitate the differentiating actions of FSH and LH in granulosa cells (1, 2). Estrogen receptor (ER) knockout (KO) (i.e. ERKO) and aromatase null mouse (ArKO) models developed recently have been useful in advancing knowledge about the effects of estrogen (3, 4, 5, 6, 7, 8). The presence of Sertoli and Leydig cells in the ovaries of these models suggests that not only is estrogen required for complete folliculogenesis, but it also maintains the phenotype of the female somatic cells. The mechanism by which estrogen does this may be a direct effect via the ovarian ERs (reviewed in Ref.9) or indirect through its effect on gonadotropin and androgen levels (10, 11). The role played by the oocyte must also be considered because previous cases of premature oocyte loss also show transdifferentiation of granulosa cells into Sertoli-like cells (12, 13, 14).

In mammals, male somatic cell development normally occurs in utero within the gonad containing the Y chromosome, that encodes the testis or sex-determining gene (Sry) (15). Sry expression within the gonadal ridge of the embryo is short-lived but vital because it induces downstream targets, namely Sry-like homeobox transcription factor 9 (Sox9) and Müllerian-inhibiting substance (Mis), also known as anti-Müllerian hormone, responsible for the male phenotype. Previous mouse models of estrogen deficiency (4, 7, 16) reported an increase in Sox9 gene expression, consistent with the emergence of male somatic cells in the ovaries of these models. It is possible that the role of estrogen in determining somatic cell phenotype is as a repressor of gene(s) regulating the male somatic cell phenotype. Examination of the genes encoding nuclear transcription factors upstream and downstream of Sox9 in the ovaries of ArKO mice provides an opportunity to examine the role of estrogen in the regulation of the genes known to mediate somatic cell differentiation and function in utero. The genes in the regulatory pathways thought to be involved in the normal differentiation of the testis and ovary in mice in utero have been reviewed previously (17, 18). We therefore investigated the expression of steroidogenic factor 1 (Sf-1) and dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on the X chromosome gene 1 (Dax-1), together with Sox9 and MIS in ArKO and wild-type (Wt) ovaries. We also measured liver receptor homologue 1 (Lrh-1), a recently discovered member of the nuclear receptor superfamily (19) that is related to Sf-1 and is highly expressed in granulosa cells (20, 21, 22). Lrh-1 is also expressed in the embryonic but not adult testis, suggesting a role in early gonad differentiation (23).

Elevated testosterone levels in female ERKO and ArKO mice (3, 8) provide further evidence of the manifestation of the male phenotype and the presence of Leydig cells in ArKO females (7). Because 10-fold increases in testosterone are observed in young ArKO female mice (3), the steroid hydroxylases involved in androgen biosynthesis are likely up-regulated in ArKO mice as they are in ER{alpha}ßKO mice (8). We have correlated the age-dependent phenotype observed within the ArKO mice (5, 7) with changes in steroidogenic potential.

Although ArKO mice are deficient in functional aromatase, they do remain responsive to exogenous estrogen in terms of an increase in uterine weight (7). This suggests that ArKO mice are a powerful model with which to study the ER-mediated response of the ovaries to estrogen replacement. However, the ER status of the ovaries of ArKO mice and the effects of estradiol 17ß (E2) treatment on estrogen-sensitive ovarian and pituitary parameters of these mice have not been described previously. We therefore measured the ER and LH receptor (LHR) status as indices of estrogen action in the ovary, and serum FSH and LH as indices of estrogen action in the pituitary of ArKO and Wt mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
ArKO and Wt mice used in this study have been described previously (3, 5, 7). Mice were housed under specific pathogen-free conditions and, unless otherwise stated, were maintained ad libitum on a soy-free diet (Glen Forest Stockfeeders, Glen Forest, Western Australia), with undetectable levels of isoflavones (Soyfree). Mice were maintained on these diets because dietary phytoestrogens have been shown to delay the ovarian phenotype of ArKO mice (7). All animal procedures were approved by the Monash University Animal Ethics Committee. Animals were killed at 6, 10, or 52 wk by cervical dislocation. One ovary from each animal was immersion-fixed in Bouin’s fluid for 24 h, embedded in paraffin, and serially sectioned (3 µm). Sections were stained using a modified Masson’s Trichrome (24). The other ovary was frozen in liquid nitrogen and used for RNA extraction. Trunk blood was collected at the time of cervical dislocation, and serum was frozen at –20 C until assayed for testosterone.

E2 treatment
Twenty-one-day sc release pellets containing either E2 (0.05 mg) or placebo (Innovative Research of America, Sarasota, FL) were administered for 3-wk to 7-wk-old Wt and ArKO mice (n = 4–5 for gene expression, n = 3–10 for serum gonadotropin levels, n = 4–6 for serum testosterone levels) as described previously (25). These pellets were estimated to restore serum estradiol to 50–100 pg/ml (peak estrous levels). This treatment led to vaginal smears indicative of persistent estrus, an increase in uterine weight, and a decrease in serum gonadotropin levels in ArKO mice (see Results). These mice were killed at 10 wk of age.

Gene expression
RNA was extracted from individual ovaries using a phenol chloroform-based method (Ultraspec; Fisher Biotech, Subiaco, Western Australia, Australia) incorporating an additional chloroform extraction step. An aliquot of RNA was taken for reverse transcription (RT) using random primers (Roche, Mannheim, Germany) and AMV reverse transcriptase (Promega, Madison, WI). Complementary DNA was diluted 1:20 and amplified by real-time PCR in the Lightcycler (Roche) using Fast Start Master SYBR Green I (Roche) and specific oligonucleotide pairs that were designed to avoid DNA contamination. All PCR products exhibited a single peak in melting curves and were identified as single bands of the appropriate size on ethidium bromide gels. Amplified products were verified with sequencing. Experimental samples were quantified by comparison with cDNA standards of known concentration (0.01–1000 fg/µl). Primers for PCR are shown in Table 1Go.


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  		TABLE 1. Primer sequences and product size

 
ß-2 Microglobulin, glyceraldehyde-3-phosphate dehydrogenase, and 18S were assessed in pilot studies for suitability as housekeeping genes. It was found for all genes tested that ArKO mice had significantly lower levels of the housekeeping genes compared with Wt. This appears to be a consequence of the reduced cellularity of the ArKO ovaries resulting from apoptosis and fibrotic infiltration (5). We have expressed all RNA transcript levels relative to 18S because it was the most reproducible housekeeping gene and had acceptable variation within genotype [coefficient of variation (CV) less than 10%]. Three separate RT reactions per sample were subjected to PCR analysis, and then the mean value was calculated for the three RTs, each corrected for 18S. For each separate RT on a group of age-matched ovaries, all mice across genotype and treatment were analyzed in a single PCR. This allows comparisons of expression to be performed within, but not across, age groups.

Serum gonadotropins
An established RIA was used to measure LH and FSH levels in serum (5). The lower limits of detection were 1.05 ng/ml (FSH) and 0.08 ng/ml (LH). The intraassay CVs in the FSH and LH assays (n = 3) were less than 7% and less than 12%, respectively. The interassay CVs for LH and FSH assays (n = 3) were 1.4 and 12.1%, respectively, calculated using a pool of normal mouse serum.

Serum testosterone
Testosterone levels were determined using a double-antibody RIA in unextracted serum (26). The RIA uses iodinated histamine-testosterone (20,000 cpm/tube) as tracer (prepared inhouse) in combination with a low-pH buffer (5.1) to disrupt testosterone from its binding proteins. The assay buffer consists of 0.05 M citric acid, 0.01% sodium azide, and 0.02% {gamma}-globulins. A rabbit testosterone primary antiserum, TAS 0811 (Department of Clinical Biochemistry, Monash Medical Centre, Clayton, Victoria, Australia) was used at a concentration of 1:40,000; and the primary antiserum was diluted in 1:60 normal rabbit serum. The antiserum cross-reacted 39% with dihydrotestostetone, less than 2% with 17{alpha}-methyl-testosterone, {Delta}4-androstene-3,17-dione, and less than 0.1% with E2, pregnenolone, and 20-hydroxy-pregnen-3-one. Complexes were precipitated with goat antirabbit IgG at a concentration of 1:10 in conjunction with 2 ml of 6% polyethylene glycol 6000. Testosterone (Sigma, Castle Hill, New South Wales, Australia) dissolved in 100% ethanol and then diluted in assay buffer was used as standard (ranging from 12.8–3330 pg/ml). The total volume of the assay was 500 µl, and serum samples were diluted 1:20 before the assay. Charcoal-stripped female mouse serum was used to match serum content in the standard curve and samples. The detection limit for this assay was 116 pg/ml; the within-assay CV was 0.3%. All samples were assayed in duplicate across two assays with an interassay CV less than 5%.

Statistical analyses
Data are presented as mean ± SEM. Statistical analysis was performed using Sigmastat statistical software (version 2.0, Jandel Corp., San Rafael, CA,). If data was normally distributed, it was subjected to two-way ANOVA. If data was not normally distributed, it was log-transformed and then subjected to two-way ANOVA. The two-way ANOVA was used to assess the differences between genotype and treatment. If genotype or treatment had a significant effect on gene expression or testosterone levels, multiple pairwise comparisons were made using a Tukey test. If no effect was observed between treatments within the same genotype, the data were pooled and genotypes were compared. For this reason, the effect of E2 may not be shown. A value of P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reproductive organ weights (Table 2Go)
ArKO mice had decreased ovarian weight compared with Wt animals (P < 0.001). E2 treatment decreased ovarian weight in both Wt and ArKO animals (P = 0.025). ArKO mice also had decreased uterine weight (P < 0.001) compared with Wt mice. E2 treatment increased uterine weight in both Wt and ArKO animals (P < 0.05) and, in ArKO mice, returned uterine weights to those of Wt mice.


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  		TABLE 2. Effect of E2 replacement on reproductive organ weights in Wt and ArKO mice

 
Serum gonadotropin levels (Table 3Go)
FSH.
Placebo-treated ArKO mice had FSH levels 3.5-fold above those of Wt mice (P < 0.001) (Table 3Go). E2 treatment decreased FSH (P = 0.002) in both ArKO and Wt mice. ArKO mice treated with E2 had FSH levels comparable with Wt mice treated with either E2 or placebo pellets.


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  		TABLE 3. Effect of E2 replacement on serum gonadotropin levels

 
LH.
Placebo-treated ArKO mice had LH levels 3-fold above those of Wt mice (P < 0.001). E2 treatment decreased ArKO (P < 0.001) levels to the same level as Wt mice (Table 3Go). E2 treatment also decreased LH levels in Wt mice.

Morphology
E2 treatment did not cause any obvious changes in the ovarian morphology of Wt ovaries, although there does appear to be an increase in antral follicles and corpora lutea (Fig. 1Go, A–C). In ArKO mice, E2 treatment improved follicle development and decreased the incidence of Sertoli cell cords compared with placebo-treated controls (Fig. 1DGo). No hemorrhagic cysts were observed in ArKO mice treated with E2 (7). The ovaries of ArKO mice treated with placebo pellets did not possess many follicles beyond the primary stage of development, whereas those treated with E2 contained numerous antral follicles (Fig. 1Go, D–F). Although the ovaries of placebo-treated ArKO mice did not contain corpora lutea, as previously reported (7), there were corpora lutea present in the ovaries of some E2-treated ArKO mice (Fig. 1EGo), but not in all cases (Fig. 1FGo). The abundance of collagen fibrils in ArKO mice was reduced by E2 treatment, and the stroma appeared more densely populated with interstitial cells than the ovaries of ArKO mice treated with placebo (Fig. 1Go, D and E).



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  		FIG. 1. Ovarian morphology of Wt and ArKO mice at 10 wk of age after 3 wk treatment with either placebo (Plc) or 0.05 mg E2 (100x magnification). Wt mice ovaries (A–C) have a normal complement of growing follicles and CL (*). Ovaries of ArKO mice (D) show impaired follicular development, hemorrhagic cysts (HC), and Sertoli cell development (arrow). Follicular growth was improved when treated with E2 (E–F). The incidence of Sertoli cells was also decreased, and no hemorrhagic cysts were present. In some cases, ovulation was restored in ArKO mice, as evidenced by the presence of CL (*) (E). Not all ovaries of ArKO mice treated with E2 had CL present (F). At 1 yr of age, Wt mice possessed some follicles (arrow) and defined stroma, whereas the ArKO mice did not show much follicular or cord-like tissue (H), although collagen fibrils (blue) and macrophages (arrow) were abundant (G).

 
We also examined ovaries of 1-yr-old Wt and ArKO mice (Fig. 1Go, G and H) to assess the age-dependent ovarian degeneration. Compared with Wt ovaries, which contain some follicles, corpus luteum (CL), and corpus albicans (Fig. 1GGo), the ovaries of ArKO mice contained few follicles and sparsely populated intersitium and an increased deposition of collagen (Fig. 1HGo).

Estrogen responsiveness
To confirm the capacity of ArKO ovaries to respond to E2, we measured ER mRNA levels in the ovaries. ArKO ovaries possessed transcripts for both ERs. ER{alpha} was decreased in ArKO ovaries compared with Wt (P < 0.001) (Fig. 2AGo), whereas ERß (total, ERß1 +ERß2) was increased compared with Wt (P < 0.001) (Fig. 2BGo). Estrogen treatment to either Wt or ArKO mice did not significantly alter the levels of expression of the ER (Fig. 2Go, A and B).



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  		FIG. 2. Expression of ER{alpha} (A) and ERß (B) in Wt and ArKO ovaries. Expression is shown relative to 18S RNA after treatment with either placebo (P) or E2 for 3 wk. Values represent mean ± SEM of four to five animals. Significance is shown by P < 0.001 (**).

 
LHR mRNA expression was examined in Wt and ArKO ovaries as an index of differentiation status (27, 28, 29, 30). Levels were decreased (P < 0.001) in ArKO ovaries compared with Wt (Fig. 3Go). E2 treatment of ArKO mice increased LHR (P < 0.001) to levels statistically similar to those of Wt placebo. Wt animals treated with E2, however, showed a decrease (P < 0.001) in LHR mRNA compared with Wt placebo-treated ovaries (Fig. 3Go).



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  		FIG. 3. Expression of LHR in Wt and ArKO ovaries. Expression is shown relative to 18S RNA after treatment with either P or E2 for 3 wk. Values represent mean ± SEM of four to five animals. Significance is shown by P < 0.001 (**).

 
Gene expression
There was a significant effect of genotype on the levels of the genes expressed by the ovarian somatic cells. The relative expression levels of Sox9 in ArKO ovaries were increased 15-fold (P < 0.001) compared with Wt (Fig. 4AGo). When ArKO mice were treated with E2, the expression levels of Sox9 were decreased compared with placebo (P < 0.001) but still remained elevated compared with E2-treated Wt. The expression of the downstream target, Mis, was also increased in ArKO ovaries (2.9-fold; P < 0.001) compared with Wt (Fig. 4BGo). E2 treatment did not alter the levels of Mis transcripts (data not shown) in either genotype. Sf-1 levels were similar (Fig. 5AGo) in ArKO and Wt ovaries. However, when mice of either genotype were treated with E2, Sf-1 expression levels were decreased (P = 0.049). Lrh-1 expression was increased 7.7-fold (P < 0.001) in ArKO ovaries (Fig. 5BGo); E2 treatment did not alter Lrh-1 expression (data not shown). Dax-1 levels in ArKO ovaries were increased 12-fold compared with Wt (P < 0.001) (Fig. 5CGo), and again there was no effect of estrogen treatment (data not shown).



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  		FIG. 4. Expression of Sox9 (A) and Mis (B) genes reflecting Sertoli cell differentiation in 10-wk Wt and ArKO ovaries. Expression is shown relative to 18S RNA after treatment with either P or E2 for 3 wk. Values represent mean ± SEM of four to five animals. Significance is shown by P < 0.001 (**). In the case of MIS (B), no significant difference was observed between placebo and E2 treatment, so the placebo and E2 groups were pooled within genotypes for analysis.

 


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  		FIG. 5. Expression of the Sf-1 (A), Lrh-1 (B), and Dax-1 (C) genes involved in somatic cell differentiation in 10-wk-old Wt and ArKO ovaries. Expression is shown relative to 18S RNA after treatment with either P or E2 for 3 wk. Values represent mean ± SEM of four to five animals. Significance is shown by P < 0.05 (*) and P < 0.001 (**). In the case of Lrh-1 (B) and Dax-1 (C), no significant difference was observed between placebo and E2 treatment, so the placebo and E2 groups were pooled within genotypes for analysis.

 
There was a significant effect of genotype on the mRNA transcript levels encoding steroidogenic enzymes within the ovary. 17{alpha}-Hydroxylase (17{alpha}-OHase) expression was increased in ArKO, compared with Wt, by 7.3-fold (P < 0.001) (Fig. 6AGo). E2 treatment decreased the expression of 17{alpha}-OHase within the ovaries of both Wt and ArKO mice (P = 0.002). 17ß-Hydroxysteroid dehydrogenase (17ß-Hsd) type-1 transcript levels were increased 3.8-fold (P < 0.001) in ArKO ovaries compared with Wt. The levels were decreased in ArKO, but not Wt mice, treated with E2 (Fig. 6BGo). The expression of 17ß-Hsd-3 was markedly increased (257-fold) in the ovaries of ArKO mice compared with Wt (P < 0.001). ArKO mice treated with E2 had decreased levels (P < 0.001) compared with placebo-treated ArKO mice (Fig. 6CGo).



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  		FIG. 6. Expression of 17{alpha}-OHase (A), 17ß-Hsd-1 (B), and 17ß-Hsd-3 (C), which regulate androgen biosynthesis, in 10-wk Wt and ArKO ovaries. Expression is shown relative to 18S RNA after treatment with either P or E2 for 3 wk. Values represent mean ± SEM of four to five animals. Significance is shown by P < 0.05 (*) and P < 0.001 (**).

 
Serum Testosterone
The levels of serum testosterone were increased in ArKO mice, compared with Wt, at each of the ages examined (Fig. 7Go). At 6 wk, testosterone levels were increased 61-fold above Wt (P < 0.001). Serum testosterone in ArKO mice was increased 8.2-fold above Wt (P < 0.001) at 10 wk; whereas at 52 wk, only a 4.1-fold increase (P < 0.001) was observed.



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  		FIG. 7. Serum testosterone concentrations in Wt and ArKO mice. Values represent mean ± SEM of four to 10 animals. Significance is shown by P < 0.001 (**). The shaded box shows the range of serum testosterone (mean ± SD) measured in male Wt littermates (3 103 ).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies show that: 1) the presence of Sertoli and Leydig cells in the ArKO ovary is accompanied by increased expression of genes known to be associated with the male cellular phenotype and with testosterone production; and 2) ArKO mice respond to treatment with E2 by a reduction in serum FSH and LH and increased uterine weight. The ovaries of ArKO mice are capable of responding to estrogen, as shown by expression of ER{alpha} and ERß mRNA, and E2 treatment of ArKO mice reduced levels of Sox9, Sf-1, and three steroidogenic enzymes needed for testosterone production. Consistent with the ability of some E2-treated ArKO mice to ovulate, LHR mRNA levels were also increased. E2 treatment of 7-wk-old ArKO mice for 3 wk did not significantly change the mRNA levels of Mis, Lrh-1, Dax-1, ER{alpha}, or ERß.

Estrogen replacement
A primary aim of this study was to compare the ovarian phenotypes, at the morphological and molecular levels, of ArKO and Wt mice before and after E2 replacement. The dose of E2 replacement used in our study was able to lower gonadotropin levels into the normal range, increase uterine weights, and improve ovarian follicular profiles, in some cases restoring ovulation in ArKO mice. In this paradigm, it is not possible to distinguish direct from indirect effects of E2. However, the likelihood that some of the effects of E2 replacement of ArKO mice are direct is supported by the expression of both ER{alpha} and ERß mRNA in the ArKO ovary. Comparisons of the results of this study, particularly in terms of E2 effects on ovarian weights, with published studies using hypophysectomized rats and hamsters (31, 32, 33) is not warranted. The experimental paradigms are very different, e.g. estrogen dose and type, hypophysectomized vs. intact, no estrogen vs. estrogen deplete. Further studies in which the gonadotropin and testosterone levels in ArKO mice are reduced into the normal range will be needed to confirm a direct effect of E2 on gene expression in the ovary of this mouse model.

Estrogen treatment decreased ovarian weight in both Wt and ArKO mice. This agrees with decreased ovarian weights after prenatal diethylstilbestrol exposure (34). Ovarian weight has been shown to increase in response to estrogen treatment; however, these studies were performed in hypophysectomized rats with relatively high doses of estrogen (31).

ER expression levels were assessed within the ovaries of ArKO mice to determine whether they were capable of responding to exogenous estrogen treatment. ER{alpha} mRNA was decreased in ArKO mice compared with Wt mice, consistent with the loss of interstitial cells and theca-rich antral follicles (5). ERß, however, was increased in ArKO mice compared with Wt controls, reflecting either an up-regulation of expression per cell or possibly an increase secondary to the development of Sertoli cells within ArKO ovaries. Cellular localization of the respective ERs is needed to confirm this. Estrogen treatment did not affect ER expression within either genotype. Sharma and colleagues (32) have shown decreased ER{alpha} expression in rat granulosa cells 48 h after estrogen exposure, whereas Yang et al. (33), using cycling hamsters, showed the stimulatory effect of E2 on ER{alpha} and the inhibitory effect on ERß.

LHR expression was assessed in Wt and ArKO mice as a measure of ovarian differentiation. Because its expression is known to increase in antral follicles (27, 28, 29, 30), its low expression in ArKO placebo-treated mice is not surprising. Additionally, the increase is consistent with the improved ovarian morphology. It is possible that the changes in LHR expression may also reflect changes in serum LH levels, which are known to decrease LHR expression (29, 35).

Somatic cell gene expression
The expression of Sox9 in ArKO mice was significantly elevated compared with Wt controls. It is possible that, in the absence of estrogen, the ERKO and ArKO models lose a direct or indirect (estrogen-responsive) inhibitor of Sox9 expression. Treatment of ArKO mice with E2 for 3 wk significantly reduced expression of Sox9, coincident with the reduction in Sertoli cells and the appearance of a more normal ovarian phenotype. Granulosa cells of ArKO ovaries that have no detectable oocyte contact (medullary location or follicles with apoptotic/no oocytes) (7) differentiate into Sertoli cells. Dupont et al. (16), using in situ localization studies rather than PCR or ribonuclease protection assays, reported that the expression of Sox9 by granulosa cells precedes the appearance of Sertoli cells within the ovaries of ER{alpha}ßKO mutants. In addition, XX transgenic mice with chromosomal duplication or overexpression of Sox9 develop masculinized gonads (36, 37). The ability of Sox9 to induce Sertoli cell differentiation in the absence of Sry is also supported by findings of a female patient with duplication in the Sox9 gene causing sex reversal (36) and XX oddsex mice (38). These studies support the hypothesis that Sertoli cells present in adult ArKO and ER{alpha}ßKO ovaries are derived from granulosa cells aberrantly expressing Sox9.

Although the ovarian Sertoli cells within the ArKO ovary were nearly ameliorated by estrogen treatment, we believe that this is not due to the effect of E2 on serum gonadotropin levels. This is supported by the absence of Sertoli cell cords in LH-overexpressing mice (39) and the presence of Sertoli cell tumors in FSH receptor-deficient mice (40). The restoration of ovulation, as evidenced by corpora lutea within the ovaries of some E2-treated ArKO mice, may however involve indirect effects mediated by the normalization of gonadotropin levels. Although serial blood samples were not performed to define surges, previous data (41, 42) and the fact that ovulation was restored in some cases suggest that the estrogen treatment used was able to induce ovulatory gonadotropin surges. Estrogen replacement studies in a separate colony of ArKO mice (43) using an alternate regime of estrogen treatment (sc injections of 15 ug/25 µl E2 every 4th day, from 4–8 wk of age) failed to restore ovulation, highlighting the need for further studies into the threshold of estrogen treatment required to induce ovulation.

In the male, Mis is secreted by developing Sertoli cells in the fetal gonad and induces the regression of the Müllerian duct (44, 45). It remains at high levels in the male until puberty, at which time secretion falls below detection (46). In contrast, Mis is undetectable in the ovaries of newborn females (45, 47, 48), although Mis is measurable throughout reproductive life after puberty in females (49, 50). Mis transcript expression in ArKO mice increased 3-fold compared with Wt at 10 wk, coincident with the increased expression of Sox9 mRNA and the differentiation of Sertoli and Leydig cells within the ovaries. Female mice overexpressing Mis did not possess Müllerian ducts; their ovaries were completely devoid of germ cells, and they developed seminiferous cords holding Sertoli cells (51). It is hypothesized that the increase in Mis in the ArKO ovary occurred after puberty, because the Müllerian ducts are present at birth, and oocytes develop within follicles, and follicles are not lost until after puberty. Estrogen treatment did not affect Mis expression, unlike Sox9 mRNA. The continued expression of Mis after E2 treatment could be due to the raised levels of Sf-1 and Lrh-1 in ArKO mice even though Sox9 mRNA is reduced (see below).

Nuclear transcription factor gene expression
Sf-1 is known to induce the expression of many steroidogenic genes, including aromatase (52), and it also acts in synergy with Sox9 to induce Mis (53). Because Mis was increased in ArKO compared with Wt mice, we presumed that Sf-1 would also be increased in ArKO mice. Surprisingly, Sf-1 expression was similar in the ovaries of Wt and ArKO mice, although E2 reduced Sf-1 expression in both genotypes. However, Lrh-1, a homolog of Sf-1 that binds to the same DNA-binding site (19, 54, 55, 56, 57), was markedly increased in ArKO ovaries, and its expression was not affected by E2. Like Sf-1, Lrh-1 is highly expressed in the adult ovary (22, 58) and during embryological development of the gonad of both sexes (23). Sf-1 is known to be crucial for gonad development and steroidogenesis (59), suggesting that the closely related Lrh-1may also be important for ovarian development and function. Lrh-1 can regulate aromatase expression (60); and within the ovary, it is localized to granulosa cells (20, 21) where aromatase is also present (61). The apposing or complementary roles of these two nuclear transcription factors are still being unraveled. It is interesting to postulate that one may be more important than the other in estrogen action and somatic cell phenotype within the ovary, particularly because Lrh-1, and not Sf-1, mRNA expression was reduced by E2 treatment.

Reports of Dax-1 expression profiles in gonads are not consistent. On the one hand, Dax-1 transcript levels have been reported to be expressed in rodent ovaries and testis at 12.5–15.5 dpc and then down-regulated in the ovary (62, 63). On the other hand, Swain and colleagues (64) reported Dax-1 expression in the genital ridges of both mouse sexes at 11.5 dpc, which was later down-regulated in the testis but not the ovary. We have shown Dax-1 expression was increased significantly in the ovaries of adult ArKO mice, and expression was not influenced by E2 treatment. Dax-1 is able to act as a corepressor of both Sf-1 and Lrh-1 (65, 66, 67), and both possess functional Dax-1 response elements (63, 65, 66, 67, 68, 69). However, functional Sf-1 binding sites also exist within the Dax-1 promoter (70), and Sf-1 null mice show reduced levels of Dax-1 expression (71, 72). Within the ovaries of ArKO mice, Sf-1 was unchanged and Lrh-1 was increased, suggesting that normal communication between these factors may be disrupted. The relative importance of each factor in this regulatory interaction and the direct and indirect effects of estrogen upon this system require further investigation.

Steroidogenic enzyme gene expression
17{alpha}-OHase and 17ß-Hsd-3 are two important enzymes in the production of testosterone. ArKO mice show increased levels of 17{alpha}-OHase at 10 wk of age, when Sertoli and Leydig cells are beginning to populate the ovary (7) and serum LH and FSH are high, which correlated with high serum testosterone levels (Ref.3 and current study). 17{alpha}-OHase is produced by the thecal cells, which mature and increase in number as the follicle grows. The levels of this enzyme are increased 5-fold between medium-sized and large follicles (73). Moreover, LH is known to increase the activity of 17{alpha}-OHase in the ovaries or follicles of rats (74, 75).

17ß-Hsd enzymes regulate the intracellular levels of biologically active androgens and estrogens in gonadal as well as extragonadal tissues (76). They interconvert low-activity sex steroids (i.e. estrone, androstenedione, and 5{alpha} androstanedione) to more potent derivatives/metabolites (E2, testosterone, and 5{alpha}-dihydrotestosterone). There are 11 isoenzymes of 17ß-Hsd described to date (77, 78, 79, 80). 17ß-Hsd-3 is expressed exclusively within the Leydig cells, whereas 17ß-Hsd-1 and -4 are expressed within the ovary (81, 82, 83). 17ß-Hsd-1 is abundantly expressed in the granulosa cells of developing follicles (84, 85, 86, 87). 17ß-Hsd-3 is specific for converting androgens, whereas 17ß-Hsd-1 is specific for the reduction of estrone to estradiol. Ovarian 17ß-Hsd-1 expression is up-regulated in the immature rat by FSH, with estrogen and androgens both enhancing FSH-induced stimulation of 17ß-Hsd-1 expression (86). ArKO mice showed an increase in 17ß-Hsd-1 expression, which suggests that estrogen acts to inhibit 17ß-Hsd-1 expression. There was a very marked increase in 17ß-Hsd-3, the Leydig cell-specific, testosterone-producing enzyme, which correlated with the increased serum testosterone (Ref.3 and the current study) and the presence of Leydig-like cells within ArKO ovaries (7). 17ß-Hsd-3 has been associated with somatic cell sex reversal in {alpha}ßERKO mice (8) and in mice with a deficiency of the Wnt4 signaling protein (88). Conversely, in males with pseudohermaphroditism, levels of 17ß-Hsd-3 are compromised (89, 90, 91). Treatment of ArKO mice with E2 reduced the expression of 17{alpha}-OHase, 17ß-Hsd-1, and 17ß-Hsd-3, consistent with a change in the morphology to a more ovarian phenotype and a reduction in the numbers of Leydig cells.

The patterns of expression of the steroidogenic enzymes responsible for androgen biosynthesis in the ArKO ovaries correlated with serum testosterone levels reported in this and previous studies (3, 43). The initial increase in serum testosterone levels in ArKO mice well above Wt male levels (Fig. 7Go) may reflect the onset of testicular development, which in the postnatal testis is associated with elevated testosterone production (92). The decrease in testosterone production observed in aging ArKO mice may be due to repression of androgen biosynthesis by Dax-1 (93).

Pathways of gene expression controlling somatic cell differentiation
Compared with normal embryonic male and female mice and Wt adult females, evidence to date shows that the ArKO mouse ovary has a largely male pattern of gene expression. Because ArKO mice have increased levels of Dax-1 and Mis, it would be interesting to determine the levels of Wilms’ tumor-1 (Wt-1), which is known to act with Dax-1 to regulate the expression of both Sf-1 and Mis (63, 94, 95, 96). Wt-1 is essential for gonad development (97, 98) and has a postulated role in both Sry and Dax-1 expression (94, 99). Future research is needed to describe the role of Lrh-1 in gonadal differentiation.

Our conclusions are as follows. In the absence of estrogen, the murine ovary develops normally in utero, together with the remainder of the reproductive tract. However, after the expected time of puberty, the ovaries of ArKO mice develop Sertoli and Leydig cells that express testicular differentiation genes. The functionality of these male somatic cells is highlighted in the masculinized steroidogenic profile of the ArKO ovary, which correlates with increased serum testosterone levels. Therefore, estrogen plays an important role in maintaining the somatic cell phenotype within the ovary after puberty. Estrogen treatment improved the ovarian phenotype in ArKO mice and inhibited the development of Sertoli cells, which was reflected, to a certain degree, by the molecular profile in ArKO ovaries. The induction of ovulation after E2 treatment shows that the ArKO ovaries are responsive to estrogen treatment and capable of undergoing the final maturation stages of the follicle. Thus, as in lower vertebrates (100, 101, 102), the maintenance of the expression of the genes regulating somatic cell differentiation is directly or indirectly responsive to estrogen. This data show that gonadal cells retain a degree of plasticity in their phenotype, which is regulated hormonally by estrogen and may also involve communication with the oocyte.


   Acknowledgments
 
The authors thank Wah Chin Boon and Yoko Murata for technical advice and Ann Drummond and Wah Chin Boon for assistance with primer design.


   Footnotes
 
This work was supported by National Health and Medical Research Council (NH&MRC) Program Grants 983212 and 241000 (to J.K.F.) and an NH&MRC Fellowship 198705 (to J.K.F.) and Grant R37AG08174 (to E.R.S.) from the National Institute on Aging.

Abbreviations: ArKO, Aromatase null; CL, corpus luteum; CV, coefficient of variation; Dax-1, dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on the X chromosome gene 1; E2, 17ß-estradiol; 17ß-Hsd, 17ß-hydroxysteroid dehydrogenase; ER, estrogen receptor; ERKO, estrogen receptor knockout; KO, knockout; LHR, LH receptor; Lrh-1, liver receptor homolog-1; Mis, Müllerian-inhibiting substance; 17{alpha}-OHase, 17{alpha}-hydroxylase; RT, reverse transcription; Sf-1, steroidogenic factor 1; Sox9, Sry-like HMG box protein 9; Wt, wild-type.

Received December 1, 2003.

Accepted for publication May 5, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Robker RL, Richards JS 1998 Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1. Mol Endocrinol 12:924–940[Abstract/Free Full Text]
  2. Robker RL, Richards JS 1998 Hormonal control of the cell cycle in ovarian cells: proliferation versus differentiation. Biol Reprod 59:476–482[Free Full Text]
  3. Fisher CR, Graves KH, Parlow AF, Simpson ER 1998 Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene. Proc Natl Acad Sci USA 95:6965–6970[Abstract/Free Full Text]
  4. Couse JF, Hewitt SC, Bunch DO, Sar M, Walker VR, Davis BJ, Korach KS 1999 Postnatal sex reversal of the ovaries in mice lacking estrogen receptors {alpha} and ß. Science 286:2328–2331[Abstract/Free Full Text]
  5. Britt KL, Drummond AE, Cox VA, Dyson M, Wreford NG, Jones ME, Simpson ER, Findlay JK 2000 An age-related ovarian phenotype in mice with targeted disruption of the Cyp 19 (aromatase) gene. Endocrinology 141:2614–2623[Abstract/Free Full Text]
  6. Dupont S, Krust A, Gansmuller A, Dierich A, Chambon P, Mark M 2000 Effect of single and compound knockouts of estrogen receptors {alpha} (ER{alpha}) and ß (ERß) on mouse reproductive phenotypes. Development 127:4277–4291[Abstract]
  7. Britt KL, Kerr J, O’Donnell L, Jones ME, Drummond AE, Davis SR, Simpson ER, Findlay JK 2002 Estrogen regulates development of the somatic cell phenotype in the eutherian ovary. FASEB J 16:1389–1397[Abstract/Free Full Text]
  8. Couse JF, Yates MM, Walker VR, Korach KS 2003 Characterization of the hypothalamic-pituitary-gonadal axis in estrogen receptor (ER) null mice reveals hypergonadism and endocrine sex reversal in females lacking ER{alpha} but not ERß. Mol Endocrinol 17:1039–1053[Abstract/Free Full Text]
  9. Britt KL, Findlay JK 2002 Estrogen actions in the ovary revisited. J Endocrinol 175:269–276[Abstract]
  10. Magoffin DA, Erickson GF 1982 Direct inhibitory effect of estrogen on LH-stimulated androgen synthesis by ovarian cells cultured in defined medium. Mol Cell Endocrinol 28:81–89[CrossRef][Medline]
  11. Hotchkiss J, Knobil E 1996 The hypothalamic pulse generator: the reproductive core. New York: Lippincott-Raven
  12. Engle E 1946 Tubular adenomas and testis-like tubules of the ovaries of aged rats. Cancer Res 6:578–582[Free Full Text]
  13. Crumeyrolle-Arias M, Scheib D, Aschheim P 1976 Light and electron microscopy of the ovarian interstitial tissue in the senile rat: normal aspect and response to HCG of ‘deficiency cells’ and ’epithelial cords’. Gerontology 22:185–204[Medline]
  14. McLaren A 1991 Development of the mammalian gonad: the fate of the supporting cell lineage. Bioessays 13:151–156[CrossRef][Medline]
  15. Greenfield A, Koopman P 1996 SRY and mammalian sex determination. Curr Top Dev Biol 34:1–23[Medline]
  16. Dupont S, Dennefeld C, Krust A, Chambon P, Mark M 2003 Expression of Sox9 in granulosa cells lacking the estrogen receptors, ER{alpha} and ERß. Dev Dyn 226:103–106[CrossRef][Medline]
  17. Morrish BC, Sinclair AH 2002 Vertebrate sex determination: many means to an end. Reproduction 124:447–457[Abstract]
  18. Harley VR, Clarkson MJ, Argentaro A 2003 The molecular action and regulation of the testis-determining factors, SRY (sex-determining region on the Y chromosome) and SOX9 [SRY-related high-mobility group (HMG) box 9]. Endocr Rev 24:466–487[Abstract/Free Full Text]
  19. Nitta M, Ku S, Brown C, Okamoto AY, Shan B 1999 CPF: an orphan nuclear receptor that regulates liver-specific expression of the human cholesterol 7{alpha}-hydroxylase gene. Proc Natl Acad Sci USA 96:6660–6665[Abstract/Free Full Text]
  20. Falender AE, Lanz R, Malenfant D, Belanger L, Richards JS 2003 Differential expression of steroidogenic factor-1 and FTF/LRH-1 in the rodent ovary. Endocrinology 144:3598–3610[Abstract/Free Full Text]
  21. Hinshelwood MM, Repa JJ, Shelton JM, Richardson JA, Mangelsdorf DJ, Mendelson CR 2003 Expression of LRH-1 and SF-1 in the mouse ovary: localization in different cell types correlates with differing function. Mol Cell Endocrinol 207:39–45[CrossRef][Medline]
  22. Liu DL, Liu WZ, Li QL, Wang HM, Qian D, Treuter E, Zhu C 2003 Expression and functional analysis of liver receptor homologue 1 as a potential steroidogenic factor in rat ovary. Biol Reprod 69:508–517[Abstract/Free Full Text]
  23. Hinshelwood MM, Mendelson CR, Pattern of liver receptor homologue 1 (LRH-1) expression during gonadal development. Proc 36th Annual Meeting of the Society for the Study of Reproduction, Cincinnati, 2003, p 124
  24. Gurr E 1973 Biological staining methods. Buckinghamshire, UK: Searle Diagnostics
  25. Jones ME, Thorburn AW, Britt KL, Hewitt KN, Wreford NG, Proietto J, Oz OK, Leury BJ, Robertson KM, Yao S, Simpson ER 2000 Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity. Proc Natl Acad Sci USA 97:12735–12740[Abstract/Free Full Text]
  26. O’Donnell L, McLachlan RI, Wreford NG, Robertson DM 1994 Testosterone promotes the conversion of round spermatids between stages VII and VIII of the rat spermatogenic cycle. Endocrinology 135:2608–2614[Abstract]
  27. Kammerman S, Canfield RE, Kolena J, Channing CP 1972 The binding of iodinated hCG to porcine granulosa cells. Endocrinology 91:65–74[Medline]
  28. Channing CP, Kammerman S 1974 Binding of gonadotropins to ovarian cells. Biol Reprod 10:179–198[Abstract]
  29. Nimrod A, Bedrak E, Lamprecht SA 1977 Appearance of LH-receptors and LH-stimulable cyclic AMP accumulation in granulosa cells during follicular maturation in the rat ovary. Biochem Biophys Res Commun 78:977–984[CrossRef][Medline]
  30. Jaaskelainen K, Markkanen S, Rajaniemi H 1983 Internalization of receptor-bound human chorionic gonadotrophin in preovulatory rat granulosa cells in vivo. Acta Endocrinol (Copenh) 103:406–412[Abstract/Free Full Text]
  31. Richards JS 1975 Estradiol receptor content in rat granulosa cells during follicular development: modification by estradiol and gonadotropins. Endocrinology 97:1174–1184[Abstract]
  32. Sharma SC, Clemens JW, Pisarska MD, Richards JS 1999 Expression and function of estrogen receptor subtypes in granulosa cells: regulation by estradiol and forskolin. Endocrinology 140:4320–4334[Abstract/Free Full Text]
  33. Yang P, Kriatchko A, Roy SK 2002 Expression of ER-{alpha} and ER-ß in the hamster ovary: differential regulation by gonadotropins and ovarian steroid hormones. Endocrinology 143:2385–2398[Abstract/Free Full Text]
  34. Haney AF, Newbold RR, McLachlan JA 1984 Prenatal diethylstilbestrol exposure in the mouse: effects on ovarian histology and steroidogenesis in vitro. Biol Reprod 30:471–478[Abstract]
  35. Richards JS, Ireland JJ, Rao MC, Bernath GA, Midgley Jr AR, Reichert Jr LE 1976 Ovarian follicular development in the rat: hormone receptor regulation by estradiol, follicle stimulating hormone and luteinizing hormone. Endocrinology 99:1562–1570[Abstract]
  36. Huang B, Wang S, Ning Y, Lamb AN, Bartley J 1999 Autosomal XX sex reversal caused by duplication of SOX9. Am J Med Genet 87:349–353[CrossRef][Medline]
  37. Vidal VP, Chaboissier MC, de Rooij DG, Schedl A 2001 Sox9 induces testis development in XX transgenic mice. Nat Genet 28:216–217[CrossRef][Medline]
  38. Bishop CE, Whitworth DJ, Qin Y, Agoulnik AI, Agoulnik IU, Harrison WR, Behringer RR, Overbeek PA 2000 A transgenic insertion upstream of sox9 is associated with dominant XX sex reversal in the mouse. Nat Genet 26:490–494[CrossRef][Medline]
  39. Risma KA, Clay CM, Nett TM, Wagner T, Yun J, Nilson JH 1995 Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors. Proc Natl Acad Sci USA 92:1322–1326[Abstract/Free Full Text]
  40. Danilovich N, Roy I, Sairam MR 2001 Ovarian pathology and high incidence of sex cord tumors in follitropin receptor knockout (FORKO) mice. Endocrinology 142:3673–3684[Abstract/Free Full Text]
  41. Legan SJ, Coon GA, Karsch FJ 1975 Role of estrogen as initiator of daily LH surges in the ovariectomized rat. Endocrinology 96:50–56[Medline]
  42. Legan SJ, Karsch FJ 1975 A daily signal for the LH surge in the rat. Endocrinology 96:57–62[Abstract]
  43. Toda K, Takeda K, Okada T, Akira S, Saibara T, Kaname T, Yamamura K, Onishi S, Shizuta Y 2001 Targeted disruption of the aromatase P450 gene (Cyp19) in mice and their ovarian and uterine responses to 17ß-oestradiol. J Endocrinol 170:99–111[Abstract]
  44. Jost A, Vigier B, Prepin J, Perchellet JP 1973 Studies on sex differentiation in mammals. Recent Prog Horm Res 29:1–41
  45. Munsterberg A, Lovell-Badge R 1991 Expression of the mouse anti-müllerian hormone gene suggests a role in both male and female sexual differentiation. Development 113:613–624[Abstract]
  46. Hudson PL, Dougas I, Donahoe PK, Cate RL, Epstein J, Pepinsky RB, MacLaughlin DT 1990 An immunoassay to detect human müllerian inhibiting substance in males and females during normal development. J Clin Endocrinol Metab 70:16–22[Abstract]
  47. Hirobe S, He WW, Lee MM, Donahoe PK 1992 Müllerian inhibiting substance messenger ribonucleic acid expression in granulosa and Sertoli cells coincides with their mitotic activity. Endocrinology 131:854–862[Abstract]
  48. Taketo T, Saeed J, Manganaro T, Takahashi M, Donahoe PK 1993 Müllerian inhibiting substance production associated with loss of oocytes and testicular differentiation in the transplanted mouse XX gonadal primordium. Biol Reprod 49:13–23[Abstract]
  49. Lee MM, Donahoe PK, Hasegawa T, Silverman B, Crist GB, Best S, Hasegawa Y, Noto RA, Schoenfeld D, MacLaughlin DT 1996 Müllerian inhibiting substance in humans: normal levels from infancy to adulthood. J Clin Endocrinol Metab 81:571–576[Abstract]
  50. Rajpert-De Meyts E, Jorgensen N, Graem N, Muller J, Cate RL, Skakkebaek NE 1999 Expression of anti-müllerian hormone during normal and pathological gonadal development: association with differentiation of Sertoli and granulosa cells. J Clin Endocrinol Metab 84:3836–3844[Abstract/Free Full Text]
  51. Behringer RR, Cate RL, Froelick GJ, Palmiter RD, Brinster RL 1990 Abnormal sexual development in transgenic mice chronically expressing müllerian inhibiting substance. Nature 345:167–170[CrossRef][Medline]
  52. Parker KL, Schimmer BP 1997 Steroidogenic factor 1: a key determinant of endocrine development and function. Endocr Rev 18:361–377[Abstract/Free Full Text]
  53. De Santa Barbara P, Bonneaud N, Boizet B, Desclozeaux M, Moniot B, Sudbeck P, Scherer G, Poulat F, Berta P 1998 Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-müllerian hormone gene. Mol Cell Biol 18:6653–6665[Abstract/Free Full Text]
  54. Wilson TE, Fahrner TJ, Milbrandt J 1993 The orphan receptors NGFI-B and steroidogenic factor 1 establish monomer binding as a third paradigm of nuclear receptor-DNA interaction. Mol Cell Biol 13:5794–5804[Abstract/Free Full Text]
  55. Ellinger-Ziegelbauer H, Hihi AK, Laudet V, Keller H, Wahli W, Dreyer C 1994 FTZ-F1-related orphan receptors in Xenopus laevis: transcriptional regulators differentially expressed during early embryogenesis. Mol Cell Biol 14:2786–2797[Abstract/Free Full Text]
  56. Galarneau L, Pare JF, Allard D, Hamel D, Levesque L, Tugwood JD, Green S, Belanger L 1996 The {alpha}1-fetoprotein locus is activated by a nuclear receptor of the Drosophila FTZ-F1 family. Mol Cell Biol 16:3853–3865[Abstract]
  57. Lee MH, Rabe A 1999 Infantile handling eliminates reversal learning deficit in rats prenatally exposed to alcohol. Alcohol 18:49–53[CrossRef][Medline]
  58. Boerboom D, Pilon N, Behdjani R, Silversides DW, Sirois J 2000 Expression and regulation of transcripts encoding two members of the NR5A nuclear receptor subfamily of orphan nuclear receptors, steroidogenic factor-1 and NR5A2, in equine ovarian cells during the ovulatory process. Endocrinology 141:4647–4656[Abstract/Free Full Text]
  59. Ozisik G, Achermann JC, Meeks JJ, Jameson JL 2003 SF1 in the development of the adrenal gland and gonads. Horm Res 59(Suppl 1):94–98
  60. Clyne CD, Speed CJ, Zhou J, Simpson ER 2002 Liver receptor homologue-1 (LRH-1) regulates expression of aromatase in preadipocytes. J Biol Chem 277:20591–20597[Abstract/Free Full Text]
  61. Ishimura K, Yoshinaga-Hirabayashi T, Tsuri H, Fujita H, Osawa Y 1989 Further immunocytochemical study on the localization of aromatase in the ovary of rats and mice. Histochemistry 90:413–416[CrossRef][Medline]
  62. Ikeda Y, Swain A, Weber TJ, Hentges KE, Zanaria E, Lalli E, Tamai KT, Sassone-Corsi P, Lovell-Badge R, Camerino G, Parker KL 1996 Steroidogenic factor 1 and Dax-1 colocalize in multiple cell lineages: potential links in endocrine development. Mol Endocrinol 10:1261–1272[Abstract]
  63. Nachtigal MW, Hirokawa Y, Enyeart-VanHouten DL, Flanagan JN, Hammer GD, Ingraham HA 1998 Wilms’ tumor 1 and Dax-1 modulate the orphan nuclear receptor SF-1 in sex-specific gene expression. Cell 93:445–454[CrossRef][Medline]
  64. Swain A, Zanaria E, Hacker A, Lovell-Badge R, Camerino G 1996 Mouse Dax1 expression is consistent with a role in sex determination as well as in adrenal and hypothalamus function. Nat Genet 12:404–409[CrossRef][Medline]
  65. Ito M, Yu R, Jameson JL 1997 DAX-1 inhibits SF-1-mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita. Mol Cell Biol 17:1476–1483[Abstract]
  66. Suzuki T, Kasahara M, Yoshioka H, Umesono K, Morohashi K 2002 LXXLL motifs in Dax-1 have target specificity for the orphan nuclear receptors Ad4BP/SF-1 and LRH-1. Endocr Res 28:537[CrossRef][Medline]
  67. Suzuki T, Kasahara M, Yoshioka H, Morohashi K, Umesono K 2003 LXXLL-related motifs in Dax-1 have target specificity for the orphan nuclear receptors Ad4BP/SF-1 and LRH-1. Mol Cell Biol 23:238–249[Abstract/Free Full Text]
  68. Zazopoulos E, Lalli E, Stocco DM, Sassone-Corsi P 1997 DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis. Nature 390: 311–315
  69. Crawford PA, Dorn C, Sadovsky Y, Milbrandt J 1998 Nuclear receptor DAX-1 recruits nuclear receptor corepressor N-CoR to steroidogenic factor 1. Mol Cell Biol 18:2949–2956[Abstract/Free Full Text]
  70. Yu RN, Ito M, Jameson JL 1998 The murine Dax-1 promoter is stimulated by SF-1 (steroidogenic factor-1) and inhibited by COUP-TF (chicken ovalbumin upstream promoter-transcription factor) via a composite nuclear receptor-regulatory element. Mol Endocrinol 12:1010–1022[Abstract/Free Full Text]
  71. Kawabe K, Shikayama T, Tsuboi H, Oka S, Oba K, Yanase T, Nawata H, Morohashi K 1999 Dax-1 as one of the target genes of Ad4BP/SF-1. Mol Endocrinol 13:1267–1284[Abstract/Free Full Text]
  72. Hoyle C, Narvaez V, Alldus G, Lovell-Badge R, Swain A 2002 Dax1 expression is dependent on steroidogenic factor 1 in the developing gonad. Mol Endocrinol 16:747–756[Abstract/Free Full Text]
  73. Rodgers RJ, Rodgers HF, Hall PF, Waterman MR, Simpson ER 1986 Immunolocalization of cholesterol side-chain-cleavage cytochrome P-450 and 17 {alpha}-hydroxylase cytochrome P-450 in bovine ovarian follicles. J Reprod Fertil 78:627–638[Abstract/Free Full Text]
  74. Fukuda S, Terakawa N, Sato B, Imori T, Matsumoto K 1979 Hormonal regulation of activities of 17 ß-ol-dehydrogenases, aromatase and 4-ene-5 {alpha}-reductase in immature rat ovaries. J Steroid Biochem 11:1421–1427[CrossRef][Medline]
  75. Bogovich K, Richards JS 1982 Androgen biosynthesis in developing ovarian follicles: evidence that luteinizing hormone regulates thecal 17 {alpha}-hydroxylase and C17–20-lyase activities. Endocrinology 111:1201–1208[Medline]
  76. Labrie F, Simard J, Luu-The V, Pelletier G, Belghmi K, Belanger A 1994 Structure, regulation and role of 3 ß-hydroxysteroid dehydrogenase, 17 ß-hydroxysteroid dehydrogenase and aromatase enzymes in the formation of sex steroids in classical and peripheral intracrine tissues. Baillieres Clin Endocrinol Metab 8:451–474[CrossRef][Medline]
  77. Andersson S, Geissler WM, Patel S, Wu L 1995 The molecular biology of androgenic 17ß-hydroxysteroid dehydrogenases. J Steroid Biochem Mol Biol 53:37–39[CrossRef][Medline]
  78. Luu-The V 2001 Analysis and characteristics of multiple types of human 17ß-hydroxysteroid dehydrogenase. J Steroid Biochem Mol Biol 76:143–151[CrossRef][Medline]
  79. Baker ME 2001 Evolution of 17ß-hydroxysteroid dehydrogenases and their role in androgen, estrogen and retinoid action. Mol Cell Endocrinol 171: 211–215
  80. Baker ME 2001 Hydroxysteroid dehydrogenases: ancient and modern regulators of adrenal and sex steroid action. Mol Cell Endocrinol 175:1–4[CrossRef][Medline]
  81. Mustonen MV, Poutanen MH, Isomaa VV, Vihko PT, Vihko RK 1997 Cloning of mouse 17ß-hydroxysteroid dehydrogenase type 2, and analyzing expression of the mRNAs for types 1, 2, 3, 4 and 5 in mouse embryos and adult tissues. Biochem J 325:199–205
  82. Sha JA, Dudley K, Rajapaksha WR, O’Shaughnessy PJ 1997 Sequence of mouse 17ß-hydroxysteroid dehydrogenase type 3 cDNA and tissue distribution of the type 1 and type 3 isoform mRNAs. J Steroid Biochem Mol Biol 60:19–24[CrossRef][Medline]
  83. Baker PJ, Sha JH, O’Shaughnessy PJ 1997 Localisation and regulation of 17ß-hydroxysteroid dehydrogenase type 3 mRNA during development in the mouse testis. Mol Cell Endocrinol 133:127–133[CrossRef][Medline]
  84. Tremblay Y, Ringler GE, Morel Y, Mohandas TK, Labrie F, Strauss 3rd JF, Miller WL 1989 Regulation of the gene for estrogenic 17-ketosteroid reductase lying on chromosome 17cen—-q25. J Biol Chem 264:20458–20462[Abstract/Free Full Text]
  85. Ghersevich SA, Poutanen MH, Martikainen HK, Vihko RK 1994 Expression of 17ß-hydroxysteroid dehydrogenase in human granulosa cells: correlation with follicular size, cytochrome P450 aromatase activity and oestradiol production. J Endocrinol 143:139–150[Abstract/Free Full Text]
  86. Ghersevich S, Poutanen M, Tapanainen J, Vihko R 1994 Hormonal regulation of rat 17ß-hydroxysteroid dehydrogenase type 1 in cultured rat granulosa cells: effects of recombinant follicle-stimulating hormone, estrogens, androgens, and epidermal growth factor. Endocrinology 135:1963–1971[Abstract]
  87. Sawetawan C, Milewich L, Word RA, Carr BR, Rainey WE 1994 Compartmentalization of type I 17ß-hydroxysteroid oxidoreductase in the human ovary. Mol Cell Endocrinol 99:161–168[CrossRef][Medline]
  88. Vainio S, Heikkila M, Kispert A, Chin N, McMahon AP 1999 Female development in mammals is regulated by Wnt-4 signalling. Nature 397:405–409[CrossRef][Medline]
  89. Geissler WM, Davis DL, Wu L, Bradshaw KD, Patel S, Mendonca BB, Elliston KO, Wilson JD, Russell DW, Andersson S 1994 Male pseudohermaphroditism caused by mutations of testicular 17ß-hydroxysteroid dehydrogenase 3. Nat Genet 7:34–39[CrossRef][Medline]
  90. Andersson S, Geissler WM, Wu L, Davis DL, Grumbach MM, New MI, Schwarz HP, Blethen SL, Mendonca BB, Bloise W, Witchel SF, Cutler Jr GB, Griffin JE, Wilson JD, Russel DW 1996 Molecular genetics and pathophysiology of 17ß-hydroxysteroid dehydrogenase 3 deficiency. J Clin Endocrinol Metab 81:130–136[Abstract]
  91. Moghrabi N, Hughes IA, Dunaif A, Andersson S 1998 Deleterious missense mutations and silent polymorphism in the human 17ß-hydroxysteroid dehydrogenase 3 gene (HSD17B3). J Clin Endocrinol Metab 83:2855–2860[Abstract/Free Full Text]
  92. Corpechot C, Baulieu EE, Robel P 1981 Testosterone, dihydrotestosterone and androstanediols in plasma, testes and prostates of rats during development. Acta Endocrinol (Copenh) 96:127–135[Abstract/Free Full T