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Endocrinology, doi:10.1210/en.2007-0689
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Endocrinology Vol. 148, No. 11 5507-5519
Copyright © 2007 by The Endocrine Society

Estrogen Receptor {alpha} Is a Major Contributor to Estrogen-Mediated Fetal Testis Dysgenesis and Cryptorchidism

Christopher R. Cederroth, Olivier Schaad, Patrick Descombes, Pierre Chambon, Jean-Dominique Vassalli and Serge Nef

Department of Genetic Medicine and Development (C.R.C., J.-D.V., S.N.), University of Geneva Medical School and Genomics Platform (O.S., P.D.), National Center of Competence in Research Frontiers in Genetic, University of Geneva, 1211 Geneva 4, Switzerland; and Institut de Génétique et de Biologie Moléculaire et Cellulaire and Institut Clinique de la Souris (P.C.), Collège de France, 67404 Illkirch Cedex, France

Address all correspondence and requests for reprints to: Serge Nef, Department of Genetic Medicine and Development University of Geneva Medical School 1, rue Michel-Servet, CH 1211 Geneva 4, Switzerland. E-mail: Serge.Nef{at}medecine.unige.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Failure of the testes to descend into the scrotum (cryptorchidism) is one of the most common birth defects in humans. In utero exposure to estrogens, such as 17β-estradiol (E2) or the synthetic estrogen diethylstilbestrol (DES), down-regulates insulin-like 3 (Insl3) expression in embryonic Leydig cells, which in turn results in cryptorchidism in mice. To identify the molecular mechanism whereby xenoestrogens block Insl3 gene transcription, we performed a microarray analysis of wild-type or estrogen receptor (ER) {alpha}-mutant testes exposed in utero to pharmacological doses of E2 or DES. Six and 31 genes were respectively down-regulated and up-regulated by estrogen exposure (≥4-fold). All six genes down-regulated by estrogen exposure, including Insl3 and the steroidogenic genes steroidogenic acute regulatory protein and cytochrome P450 17{alpha}-hydroxylase/17,20-lyase, were done so by an ER{alpha}-dependent mechanism. In contrast, up-regulation was mediated either by ER{alpha} for 12 genes or by an independent mechanism for the 19 remaining genes. Finally, we show that Insl3 gene expression and testicular descent were not affected by in utero exposure to E2 or DES in ER{alpha} mutant mice, whereas absence of ERβ did not influence the effect of these estrogens. Collectively, these data demonstrate that xenoestrogens inhibit the endocrine functions of fetal Leydig cells through an ER{alpha}-dependent mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MALE SEXUAL DIFFERENTIATION requires the secretion of three testicular hormones: testosterone, Müllerian inhibiting substance (MIS), and insulin-like 3 (Insl3). Both Insl3 and androgens are synthesized by Leydig cells and are required for a critical event in male sexual differentiation: testicular descent. In humans, failure of the testes to descend into the scrotum (cryptorchidism) is one of the most common birth defects in humans, affecting approximately 2–9% of newborn males and 1–3% of boys at 3 months of age (1, 2, 3). The intraabdominal temperature is toxic for germ cells, and cryptorchidism leads to infertility and is associated with an increased risk of testicular tumors (4).

Gonadal descent into the scrotum is one of the major aspects of male sexual differentiation. In mammals, the testes migrate from their initial intraabdominal position into the scrotal sac in two distinct hormonally regulated phases (4, 5, 6). First, during the transabdominal phase, the testis relocates from the high abdominal position next to the lower pole of the kidney to the base of the abdominal cavity. In the second inguinoscrotal phase, the testes migrate through the inguinal canal into the scrotum (7, 8). In mice the transabdominal phase occurs during embryonic d (E) 15.5–17.5, whereas the inguinoscrotal descent is completed in the second or third postnatal week. In humans, testicular descent is completed at birth, with the transabdominal descent occurring at 10- to 15-wk gestation, whereas the second phase occurs at 18–35 wk.

The first phase of testicular descent, the transabdominal phase, is mediated by both androgens and Insl3 (for review, see Refs. 9 and 10). In males, testosterone causes the regression of the cranial suspensory ligaments, whereas Insl3 promotes contraction and outgrowth of the gubernaculum, a mesenchymal tissue that connects the urogenital ridge to the inguinal abdominal wall. In females, the absence of testosterone and Insl3 allows the cranial suspensory ligaments to develop and retain the ovaries next to the kidneys, whereas the gubernaculum remains thin and elongated. The second phase of testicular descent, the inguinoscrotal phase, is androgen dependent because it is absent in humans or rodents with androgen resistance (11).

The pivotal role of Insl3 in mediating testicular descent has been revealed by mouse transgenesis. Mice lacking either Insl3 or its receptor, the leucine-rich G protein-coupled receptor 8 (LGR8), exhibit bilateral intraabdominal cryptorchidism with testes freely moving within the abdominal cavity (12, 13, 14, 15). Conversely, Insl3 overexpression causes gubernacular differentiation and contraction in females, which in turn leads to the descent of the ovary into the scrotal position (16, 17). These data indicate that Insl3 is not only necessary but also sufficient to promote gonadal descent in mice. Both Insl3 and LGR8 appear to play similar roles in humans, although mutations in these genes are not a common cause of human cryptorchidism (18). However, patients with maldescended testes and mutations in the Insl3 or LGR8 loci have been reported, indicating that in some rare cases, cryptorchidism is associated with alteration of Insl3/LGR8 signaling (12, 13, 19). Approximately 90% of cases of cryptorchidism occur spontaneously or from unknown causes. This suggests that environmental or lifestyle, rather than genetic, factors are plausible causes.

Relatively little is known concerning the regulation of Insl3 gene expression in gonads. In mice, Insl3 expression is male specific and coincides with the apparition of fetal Leydig cells at E13.5 in the developing testis (14, 20, 21, 22, 23). In contrast, no Insl3 transcripts could be detected in the developing prenatal ovary. The expression profile of Insl3 corresponds to the timing of testicular descent in rodents. A robust expression is observed before parturition and then decreases between postnatal d 0 (P0) and P7, and increases again around P10 (22, 24).

Only two transcription factors are known to affect Insl3 gene expression in vitro: the steroidogenic factor 1 (Sf-1) and the orphan nuclear receptor dosage-sensitive sex reversal/adrenal hypoplasia congenita critical region on the X chromosome (Dax)-1 (25, 26). In mice, the proximal Insl3 promoter contains three consensus sequences for Sf-1 binding (23, 25), whereas sequence comparison among human, mouse, rat, pig, and dog Insl3 promoters indicates that one of these Sf-1 binding sites is conserved in all five species (9). It has been shown, in vitro, that Sf-1 increases Insl3 gene expression by binding directly to the Sf-1 binding sites located within the 188-bp proximal region of the Insl3 promoter. Conversely, Dax-1 represses the Sf-1 mediated transactivation of the Insl3 promoter (25). Clearly, Sf-1 is a major regulator of Insl3 expression, although other factors may regulate its transcription. Sf-1 expression starts around E9.5 in both male and female genital ridges before sex determination, and then becomes restricted to male somatic cells that include Sertoli and Leydig cells. Sf-1 is a key regulator of endocrine development and function, and its expression has been detected in steroidogenic cells such as Leydig, theca, and adrenocortical cells but also in supporting cells such as the Sertoli and granulosa cells (27, 28). Sf-1 has also been expressed in the hypothalamus and in the gonadotrope cells of pituitary gland (29, 30). The expression of Sf-1 in numerous steroidogenic tissues, the pituitary gland, and hypothalamus compared with the specific expression of Insl3 in Leydig cells (20, 21) indicates that other factors in addition to Sf-1 regulate Insl3 transcription, either positively or negatively.

There are numerous reports indicating that estrogenic molecules affect testicular descent both in rodents and humans (31, 32). In fact, before the identification of Insl3 and LGR8 mutant mice, the main model for intraabdominal cryptorchidism involved the exposure of pregnant rodents to exogenous estradiol (E2) (33, 34, 35, 36). In humans, the offspring of pregnant women treated with diethylstilbestrol (DES), a nonsteroidal estrogenic substance used during the 1950s to 1970s to prevent miscarriages, exhibited an increased incidence of cryptorchidism and hypoplastic testes (37, 38). In rodents, we and others have shown that administration of DES or E2 during the second half of gestation resulted in a drastic reduction of Insl3 gene expression and testosterone levels, even though the expression of Sf-1 was unaltered (22, 39, 40). Recently, it has been shown that exposure to mono-N-butyl phthalate, an endocrine disruptor with weak estrogenic activities, also impairs Insl3 gene expression and testicular descent in rat embryos (41, 42).

Collectively, these reports indicate that xenoestrogen-dependent cryptorchidism is caused by a down-regulation of Insl3 gene expression. However, the molecular mechanisms mediating estrogen-dependent inhibition of fetal Leydig cell function and cryptorchidism remain an important open question. The purpose of this study was to investigate the toxicogenomic effects of DES or E2 exposure on testicular gene expression, and to assess the contribution of the estrogen receptors (ERs) {alpha} or β in mediating these effects with an emphasis on Insl3 gene expression and testicular descent. We found 63 and 175 genes that were respectively down- or up-regulated by estrogen exposure in fetal testes; for more than half of these, this was mediated by ER{alpha}. The expression of Leydig-specific genes such as Insl3, cytochrome P450 17{alpha}-hydroxylase/17,20-lyase (Cyp17a1), steroidogenic acute regulatory protein (Star), and renin 1 (Ren1) was profoundly decreased upon exposure to E2 or DES but not affected in mutant testes lacking ER{alpha}. These data suggest that estrogenic exposure affects fetal Leydig cell endocrine functions, more precisely the steroidogenic function, Insl3 expression and testicular descent, via an ER{alpha}-dependent mechanism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and hormonal treatment
Adult female CD1 or C57/B6 mice were time mated and checked for the presence of vaginal plugs the next morning [day post coitum (dpc) 0.5]. At 13 dpc, pregnant females received a sc injection of 0.2 ml dimethylsulfoxide (DMSO) alone or containing 6 mg 17β-E2, as previously published (22). For DES exposure, pregnant females received a constant exposure of 10 µg/d DES from E10.5 onwards using an Alzet pump (DURECT Corp., Cupertino, CA). The time of hormonal treatment was selected because E13.5 corresponds to the apparition of fetal Leydig cells and also from previous similar studies (22, 33, 34, 39). Pregnant females were killed at E18.5, and the testes of pups were dissected out. Animal protocols used in these studies were approved by the Commission d’Ethique de l’Expérimentation Animale of the University of Geneva Medical School and the Geneva Veterinarian Office.

Histology
Freshly dissected tissues were fixed with a 4% paraformaldehyde, 1x PBS solution, washed and embedded in paraffin. Five-microgram sections were stained with hematoxylin and eosin. Images were captured with AxioCam (Carl Zeiss MicroImaging GmbH, Göttingen, Germany), and analysis was performed with the ImageJ software (National Institutes of Health, Bethesda, MD).

RNA extraction, RT-PCR, and real-time RT-PCR
Total RNAs were extracted using the RNeasy micro kit from QIAGEN (Hilden, Germany) according to the manufacturer’s protocol. RNA integrity and quantity were assessed using RNA 6000 nanochips with an Agilent 2100 bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA). RT-PCR assays were performed with 1 µg total RNA from E18.5 testes. Total RNAs were reverse transcribed with the Omniscript RT-kit from QIAGEN according to manufacturer’s instructions, and one-twentieth cDNA template was used as template for each PCR. cDNA was PCR amplified in a 7900HT Sequence Detection Systems (Applied Biosystems, Foster City, CA) using Power SYBR Green PCR master mix (Applied Biosystems). Raw threshold-cycle (Ct) values were obtained from Sequence Detection Systems 2.0 software (Applied Biosystems). Relative quantities (RQs) were calculated with the formula RQ = E – Ct, using efficiencies calculated for each run with the Data Analysis for Real-Time PCR (DART-PCR) algorithm, as described (43). A mean quantity was calculated from triplicate PCR for each sample, and this quantity was normalized to two similarly measured quantities of normalization genes (glyceraldehyde-3-phosphate dehydrogenase, G3pdh and transferring receptor 1, Trf1R), as described (44). Normalized quantities were averaged for three replicates for each data point and represented as the mean ± SD. The highest normalized relative quantity was arbitrarily designated as a value of 1.0. Fold changes were calculated from the quotient of means of these normalized quantities and reported as ± SEM. The statistical significance of fold changes was determined by a paired Student’s t test. The primers used for quantitative RT-PCR (qRT-PCR) are listed in supplemental Table 3, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org.

Microarray probe labeling and hybridization
Total RNAs from wild-type (WT) or ER{alpha} mutant testes exposed to either DMSO (vehicle) or 17β-E2 were extracted individually using the RNeasy micro kit from QIAGEN according to the manufacturer’s protocol. To minimize biological variability, ER{alpha} mutant and WT embryos originated from the same litter. We used a small-scale protocol from Affymetrix (High Wycombe, UK) to reproducibly amplify and label total RNA. In short, approximately 100 ng total RNA were converted into double-stranded cDNA using a cDNA synthesis kit (Superscript; Invitrogen Corp., Carlsbad, CA) with a special oligo(dT)_24 primer containing a T7 RNA promoter site added 5' to the poly(T) tract. After the first cRNA amplification by in vitro transcription using the Ambion MEGAscript T7 kit (Ambion, Austin, TX), 400 ng cRNA was once more reverse transcribed, and biotinylated cRNAs were generated from double-strand cDNAs using an in vitro transcription labeling kit from Affymetrix. For each probe, 20 µg of the second amplification biotinylated cRNA was fragmented and hybridized to Mouse Genome 430 2.0 Array (Affymetrix) following standard protocols. For each condition, three independent sets of total RNA were isolated and used as a template for probe generation. These triplicates were performed to minimize the effects of biological variability. GeneChips were incubated at 45 C for 16 h with biotin-labeled cRNAs probes, and then washed and stained using a streptavidin- phycoerythrin conjugate with antibody amplification as described in Affymetrix protocol, using Affymetrix GeneChip Fluidics Station 450. GeneChips were scanned on a GCS3000 scanner (Affymetrix).

Selection of differentially expressed genes
To identify differentially expressed transcripts, pairwise comparison analyses were performed with Affymetrix GCOS 1.2. Each of the experimental samples (n = 3) was compared with each of the reference samples (n = 3), resulting in nine pairwise comparisons. This approach, which is based on the Mann-Whitney U pairwise comparison test, allows the ranking of results by concordance, as well as the calculation of significance (P value) of each identified change in gene expression (45). Genes for which the concordance in the pairwise comparisons exceeded a threshold (i.e. 60%) were considered to be statistically significant. A 77% cutoff in consistency of change (at least seven of nine comparisons were either increased or decreased) was then applied to identify potential dimorphic-regulated genes. Only genes that satisfied the pair-wise comparison test and displayed at least a 2- or 4-fold change in expression were selected for further study. This conservative analytical approach was used to limit the number of false positives. Regulated genes were organized and visualized into using the GeneSpring software (Agilent Technologies, Inc.).

Principal component analysis (PCA)
PCA was performed on the triplicate for WT DMSO, [ER{alpha} knockout ER{alpha}KO)] DMSO, and ER{alpha}KO E2. To facilitate the analysis of large data sets, PCA is used as a statistical method that transforms multidimensionality data sets into a new set of variables that illustrates the differences between the data. The variables are reduced in their complexity into lower dimensions, and this allows the visualization of multidimensional data sets and facilitates the biological interpretation of the variation in a data set. The analysis identifies complex relationships between conditions based on the variance. The greatest variance is projected on the first axis [principal component (PC) 1], whereas the second greatest variance is on the second axis (PC2). Because we were interested in the differential gene expression, we plotted position of each condition in triplicate against the PC1, PC2, and PC3 axis in three-dimensional space. By definition, the PC1 is the direction along which there is the greatest variation (i.e. the axis PC1 from supplemental Fig. 1Go explains 71.89% of the variability of all data sets). Thus, the distance between some of the triplicates along PCs is due to the variability. We selected genes that were above 70% in consistency of change. To reduce the variability that could affect the PCA, we selected genes for which a signal of expression was above 50 in both DMSO and E2 conditions, and excluded high values (>2000) for which the SD for the biological triplicate was higher than 0.65 times the mean (very high variability). We used the JMP 6 software (SAS Institute Inc., Cary, NC) to represent the PCA.


Figure 1
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  		FIG. 1. qRT-PCR using selected genes affected by in utero exposure to 17β-E2 and DES. mRNA expression levels of representative genes modulated by in utero exposure to 17β-E2 or DES compared with vehicle (control). Note that COMP, DCAM, Greb1, and Grem1 were up-regulated, whereas Cyp17, Ren1, Insl3, and Star were down-regulated both in E2- or DES-exposed testes. Sertoli markers (Mis), germ cell-specific markers (Oct4), and Sf-1 were unaffected. White bars represent DMSO animals, whereas gray and black bars symbolize E2 and DES-exposed animals. Results are mean ± SEM (n = 3 per group). *, P < 0.05, **, P < 0.01, ***, P < 0.001 vs. DMSO in the Student’s unpaired t test; and #, P < 0.05, ##, P < 0.01, ###, P < 0.001 in one-way ANOVA. REL, Relative expression level.

 
Statistical analysis
The statistical significance of the difference between the mean values for two different conditions or genotypes was evaluated using the Student’s unpaired t test. One-way ANOVA (nonparametric) was used for the comparison of data from more than two groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of fetal exposure to E2 on testicular gene expression
To identify genes affected by in utero exposure to E2, we compared mRNA expression profiles in testes of E18.5 embryos exposed to either vehicle or a supraphysiological dose of E2 (a single ip injection of 6 mg E2 at 13.5 dpc). As described previously, testes of E2-exposed animals were cryptorchid at E18.5. At the morphological and histological level, E2-exposed testes appeared comparable with the control group (data not shown), consistent with earlier observations (22, 33). Three pools of testicular RNA from E18.5 testes exposed to E2 or DMSO were compared by hybridization to Affymetrix oligonucleotide microarrays 430 2.0, representing virtually all protein-encoding mouse genes. The microarray data are accessible through ArrayExpress (http://www.ebi.ac.uk/arrayexpress/, accession no. E-TABM-275), a public repository for microarray data from the European Bioinformatics Institute. For simplicity the terms "gene" and "probe set" are used synonymously, although this is not totally accurate because some genes are represented by more than one probe set on the microarray. Table 1Go displays the results of pairwise comparisons.


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  		TABLE 1. Down-regulated genes in WT testis exposed to E2 (>2-fold change)

 
Testicular genes down-regulated by in utero estrogen exposure
Only six genes, namely Insl3, Star, Cyp17a1, Ren1, flavin-containing monooxygenase 2 (FMO2), and the endometrial bleeding-associated factor (Ebaf/Lefty-A), were down-regulated by more than 4-fold in testes of animals exposed to estrogens. A total of 55 additional genes displayed a 2- to 4-fold down-regulation, including the ER{alpha} (Table 1Go). In terms of gene ontology, the sets of genes down-regulated by estrogen exposure were almost exclusively classified in the categories of steroid and hormone biosynthesis, steroid and hormone metabolism, lipid transport, biosynthesis and metabolism, and male gonad development (data not shown). Insl3, Star, and Cyp17a1 gene expression have already been down-regulated by E2 or DES (22, 39, 46, 47). Interestingly, all these genes are specifically expressed in Leydig cells and require Sf-1 as a transcription factor. In silico analysis of Ren1 proximal promoter identifies three Sf-1 binding sites that share close similarities to those found in Insl3 and Cyp17a1.

Testicular genes up-regulated by in utero estrogen exposure
We observed that more genes were stimulated rather than inhibited by E2 exposure: 31 were up-regulated by more than 4-fold, and 144 additional genes displayed a 2- to 4-fold up-regulation in the testes of E18.5 embryos exposed to E2 (the complete list of genes can be found in supplemental Table 2). Among them, we identify some known E2-target genes such as gene regulated by estrogen in breast cancer 1 (Greb1), progesterone receptor, nuclear receptor interacting protein 1 (Nrip1), and cyclin D1 (50, 51, 52). Both Greb1 and Nrip1 cis-regulatory regions possess estrogen response elements (EREs) conserved in humans and mouse (51). The probe sets exhibiting the highest fold changes are for genes whose expression is extremely low if not absent in control testes. For example, this includes myomesin 2, a cytoskeletal protein (51), or the cartilage oligomeric matrix protein (Comp), a protein of the extracellular matrix primarily expressed in cartilage (52, 53). Some of the genes up-regulated by E2 [myomesin2, Comp2, doublecortin and CaM kinase-like 1 (DcamKl1)] have important roles in cell architecture and cytoskeletal regulation, in accord with the known effects of estrogens on the cytoskeleton of mammary and bone cells (54, 55).

Quantitative real-time RT-PCR verification of gene expression regulation in E2- or DES-exposed testes
To confirm the up- or down-regulation of genes identified with our microarray analysis, we selected 11 genes for subsequent validation by quantitative real-time RT-PCR (Fig. 1Go). We performed this experiment with E18.5 testis total RNA from either E2- or DES-exposed animals. For DES exposure, pregnant females received 10 µg/d from E10.5 onwards using an Alzet pump. As expected, the expression of Insl3, Cyp17a1, and Star and Ren1 were strongly down-regulated, whereas Dcamkl1, Comp, Greb1, and Gremlin1 (Grem1) were up-regulated both in E2 and DES-exposed testes. Finally, the expression of genes reported by our microarray analysis as being unaffected was not altered in either E2 or DES-exposed testes. Overall, we found a very good reproducibility between our qRT-PCR data and the microarray data. These results also suggest that DES and E2 induce similar transcriptional alterations in testes exposed to these estrogenic compounds.

Does ER{alpha} abundance play a role in the susceptibility to xenoestrogens?
Two ERs, ER{alpha} and ERβ, are known to mediate estrogen signaling. In fetal testis, immunohistological studies showed that ER{alpha} was restricted to Leydig cells, whereas ERβ was present in Leydig, Sertoli, and germ cells (for review, see Ref. 56). Interestingly, all the steroidogenic or endocrine genes negatively affected by E2 or DES exposure in fetal testis (Insl3, Star, and Cyp17a1) are specifically expressed in Leydig cell and under the control of the Sf-1 transcription factor. In contrast, Mis, another gene under Sf-1 control but expressed in Sertoli cells, remained unaffected by estrogenic exposure. Finally, the Star gene, which is expressed in all steroidogenic tissues, is not affected by DES or E2 exposure in the adrenal gland, a tissue with low levels of ER{alpha} and ERβ (see next paragraph). Together, these results suggest that the abundance of ERs might determine the susceptibility to xenoestrogens.

To verify this hypothesis, we compared the abundance of ER{alpha} and ERβ transcripts both in fetal testes and adrenal glands by classical and quantitative real-time RT-PCR (Fig. 2Go). Expression comparison indicated that ER{alpha} and ERβ were expressed at much higher levels in testis compared with adrenal gland (6- and 64-fold higher, respectively). DES or E2 exposure reduced ER{alpha} gene expression levels by approximately 50% in fetal testis but did not affect it in adrenal gland. ERβ expression remained unchanged in both steroidogenic tissues when exposed to xenoestrogens. In contrast to testis, E2 and DES exposure did not affect Star expression in the adrenal gland. This lack of inhibitory effect of xenoestrogens correlates with the dimorphic expression of ERs in steroidogenic tissues. Considering the findings that ERβ inactivation does not affect, even partially, the endocrine function of fetal Leydig cells (57, 58), these results are most consistent with the idea that the specific down-regulation of endocrine genes (e.g. Insl3, Star, and Cyp17a1) by xenoestrogens is mediated by ER{alpha}.


Figure 2
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  		FIG. 2. Disruption of steroidogenesis correlates with the abundance of ER{alpha}. A, RT-PCR performed with E18.5 testis and adrenal gland, two steroidogenic tissues, indicate that both ER{alpha} and ERβ are expressed at much higher levels in testicular tissues. B, RT-PCR of testis and adrenal glands from E18.5 embryos exposed to E2 or DES in utero. In contrast to the fetal testis, the expression of the Star gene, which is present in both organs, remained unaffected by E2 or DES exposure in the adrenal gland. C, Real-time quantitative RT-PCR of testis and adrenal gland shows higher abundance of ERs in the fetal testis compared with the adrenal gland. White bars represent DMSO animals, whereas gray and black bars symbolize E2 and DES-exposed animals. Results are mean ± SEM (n = 3 per group). *, P < 0.05, **, P < 0.01, ***, P < 0.001 vs. DMSO. G3pdh, glyceraldehyde-3-phosphate dehydrogenase; REL, relative expression level.

 
Toxicogenomic profile of ER{alpha} mutant mice exposed to xenoestrogens
To assess the extent to which ER{alpha} is required to mediate the estrogen-dependent alteration of testicular gene expression, we completed our Affymetrix microarray analysis by comparing the mRNA expression profiles of WT and ER{alpha} mutant testes exposed to E2 or vehicle. Figure 3AGo is a scatter plot representation of the transcriptome of WT testes with a particular emphasis on the 63 and 175 genes, respectively, up- and down-regulated by E2 exposure (≥2-fold change; Table 1Go and supplemental Table 2, respectively). In contrast, a similar scatter plot performed with ER{alpha} mutant testes (Fig. 3BGo and supplemental Table 2) revealed that only a small fraction of genes is affected by estrogen exposure in ER{alpha} mutant testes, suggesting that ER{alpha} mediates most of the effects during in utero exposure to E2 or DES. To confirm this hypothesis, we performed a PCA of all four transcriptome profiles (>2-fold, >4-fold; the transcriptome profiles can be found in supplemental Fig. 1AGo). Changes in gene expression profiles can be based on the distance between each condition, which is proportional to the variance of genes. Thus, the more differentially expressed the genes are from one condition compared with another one, the more distance there is between them. Our PCA indicates that the profile of expression of genes (>4-fold) from the ER{alpha}KO DMSO is similar to that of ER{alpha}KO E2, confirming that genes affected by E2 are mostly dependant on ER{alpha}.


Figure 3
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  		FIG. 3. Global expression analysis of the transcriptomes of WT or ER{alpha} mutant testes exposed to 17β-E2. Scatter plot analysis representing the systematic comparison of the log2-scaled expression signals from WT (A) or ER{alpha} mutant (B) testes exposed in utero to E2 or DMSO. Blue dots represent genes with expression levels statistically similar in both conditions, whereas red dots represent genes whose expression profiles were found to be affected by E2 exposure (≥2- and ≥4-fold change, black and blue diagonals, respectively). C, Hierarchical clustering performed with the 39 probes sets exhibiting a ≥4-fold change in expression upon E2 exposure in WT animals. Note that down-regulation of gene expression is achieved exclusively via an ER{alpha}-dependent mechanism, whereas up-regulation is mediated either via an ER{alpha}-dependent or independent mechanism. Red and green colors indicate increased and decreased expression respectively.

 
A broad overview of the expression profile of the 39 genes exhibiting at least a 4-fold change in expression after E2 exposure in WT testes is presented in Fig. 3CGo. This hierarchical clustering arranges the genes in tight clusters according to similar patterns of expression. Examination of the clusters shows first that all six genes (eight probe sets) that are down-regulated in WT animals exposed to E2 were either unchanged or slightly increased in similarly treated ER{alpha} mutant testes. This includes, in particular, Insl3, but also Star, Cyp17a1, and Ren1. This suggests that inhibition of expression is exclusively mediated by an ER{alpha}-dependent mechanism. Similarly, of the 31 genes up-regulated in E2-exposed WT testis, 12 were unaffected in similarly treated ER{alpha}KO testes, suggesting an ER{alpha}-dependent mechanism of up-regulation. In contrast, 19 genes remained overexpressed in ER{alpha} mutant testis exposed to E2, indicating that these genes are either affected by estrogens in an ER{alpha}-independent mechanism or up-regulated due to the absence of ER{alpha} itself.

Quantitative real-time RT-PCR validation of gene expression in E2- or DES-exposed testes lacking ER{alpha}
To confirm our microarray analysis, we performed quantitative real-time RT-PCR with a selection of genes previously identified as E2/DES sensitive to assess their expression either in WT or in ER{alpha} mutant testes exposed to E2 or DES or control vehicle (Fig. 4Go). As expected, Insl3 gene expression was fully restored in testes of ER{alpha} mutant mice. This was also the case for the three other Leydig-specific genes tested: Star, Cyp17a1, and Ren1. Up-regulation of Grem1 and Greb in WT testes exposed to DES or E2 was absent in mutant mice, whereas Mis or Sf-1 expression were not altered. Overall, these results indicate that E2/DES action on down-regulated genes, in particular Insl3, is exclusively mediated by ER{alpha}.


Figure 4
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  		FIG. 4. qRT-PCR using WT and ER{alpha} mutant testes as template. A, mRNA expression levels of representative genes modulated by in utero exposure to E2 and DES in WT and ER{alpha} mutant testes. White bars represent DMSO animals, whereas gray and black bars symbolize E2 and DES-exposed animals. Results are mean ± SEM (n = 3 per group). *, P < 0.05, **, P < 0.01, ***, P < 0.001 vs. DMSO. B, Table describing a one-way ANOVA analysis of the effect of ER{alpha} in mediating xenoestrogens effects on gene expression represented in A. #, P < 0.05. ##, P < 0.01. ###, P < 0.001. n.s., Nonsignificant; REL, relative expression level.

 
Absence of ER{alpha}, but not of ERβ, allows for normal testicular descent in E2- and DES-exposed animals
To assess the contribution of ER{alpha} and ERβ in estrogen-mediated cryptorchidism, we analyzed whether E2- or DES-mediated cryptorchidism could be prevented in ER mutant mice. In contrast to WT and ERβ mutant mice, testicular descent in ER{alpha} mutant mice was not affected by in utero exposure to E2 or DES (Fig. 5Go); in these animals, the gubernaculum was contracted and developed. The transabdominal descent appeared to be completed, and both E2- or DES-exposed ER{alpha} mutant testes were localized beneath the bladder neck and proximal to the inguinal region similar to control animals. Indeed, Insl3 transcription is fully restored in the absence of ER{alpha}, but not in the absence of ERβ, even in the presence of saturating levels of exogenous estrogens, strongly suggesting that ER{alpha} mediates Insl3 down-regulation and subsequent cryptorchidism upon exposure to xenoestrogens in utero.


Figure 5
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  		FIG. 5. Normal testicular descent in mice lacking ER{alpha} despite xenoestrogen exposure. A, Schematic drawing of testicular positioning at E18.5 under normal conditions or after in utero exposure to xenoestrogens. At this stage, the transabdominal descent is completed, the testes (t) are located at the level of the bladder (b) neck, and the gubernaculum (gb) (red) is contracted and developed, whereas xenoestrogen exposure induces cryptorchidism characterized by intraabdominal testes usually located below the kidney (k) and a thin elongated gubernaculum. B, Photomicrographs of the urogenital system of E18.5 embryo exposed to E2, DES, or vehicle (DMSO) and that are mutant for ER{alpha} or ERβ. Note that the transabdominal descent is completed only in ER{alpha} mutant animals, despite exposure to E2 or DES (arrows). The bladder has been surgically removed to ensure better accessibility, and the position of the testes and kidneys has been encircled with dashed lines for better visibility. a, Adrenal; c, cecum; e, epididymis; vd, vas deferens.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study suggests that E2 or DES inhibit testicular descent and steroidogenesis via ER{alpha} by acting directly on fetal Leydig cells. Indeed, the effects of these estrogenic compounds correlate with ER{alpha} expression in Leydig cells. Numerous studies have investigated the localization of both ER{alpha} and ERβ in the developing rodent testis (for review, see Ref. 56). ER{alpha} is expressed in fetal Leydig cells until birth, whereas ERβ is present in gonocytes, Sertoli cells, and Leydig cells, and this receptor subtype appears to remain in these cells until birth. It has recently been shown that endogenous estrogen physiologically inhibits steroidogenesis via ER{alpha} by acting directly on fetal Leydig cells (57). ER{alpha}-deficient mice display higher levels of testicular testosterone due to hypertrophy of fetal Leydig cells, and increased expression of Star, Cyp17a1, and P450scc genes. Our comparative analysis of two steroidogenic tissues corroborates these findings. The Star gene, which is expressed at high levels both in adrenal and testicular tissues, is specifically inhibited in fetal Leydig cells exposed to E2 or DES (Fig. 2Go). The lack of inhibition in the fetal adrenal gland is probably a direct consequence of low levels of ER{alpha}.

In addition to the drastic reduction of Insl3 (–10.9-fold), Star (–10.7), and Cyp17a1 (–4.6), we observed also a significant decrease in transcripts levels for genes such as Ren1 (–9.9), FMO2, –18.9-fold), and the Ebaf/Lefty-A (–12.3-fold). FMO2 is part of a family of flavin-containing monooxygenases that plays an important role in the detoxification of foreign chemicals (59). E2 has down-regulated the expression of FMO in the coculture of male rat hepatocytes (60). Ebaf/Lefty-A, initially identified as an endometrial bleeding-associated factor, is a member of the TGF-β family, and has regulated the expression and activation by ovarian steroids of the matrix metalloproteinase-9 (MMP9) in the human endometrium (61). Interestingly, the down-regulation of Ebaf correlates with a significant inhibition of MMP9 gene expression (–3.09-fold). Finally, Ren1 is part of the renin-angiotensin system, and is expressed both in kidneys and Leydig cells (62), although its function in testis remains unclear. For all these three proteins, the potential consequences linked with a reduction in expression on testicular physiology remain unclear.

Pharmacological vs. environmental exposure to estrogenic substances: relevance to humans
There is growing evidence from clinical and epidemiological studies for an increasing incidence of male reproductive disorders that include undescended testes (63, 64), hypospadias (65), low semen quality (66, 67), and testicular cancer (68, 69). Because these changes in male reproductive health have occurred over a very short period of time, i.e. 50 yr, it appears that environmental or lifestyle factors, rather than genetic modifications, are most likely involved. Indeed, mutations in the Insl3 or LGR8 loci do not seem to represent a frequent cause of human cryptorchidism, although cryptorchidism is the most important congenital disorder in baby boys (18). In fact, the rapid increase in male reproductive disorders since World War 2 suggests that environmental factors acting as endocrine disruptors, and in particular environmental xenoestrogens, may be a plausible cause for sporadic cryptorchidism. However, there is currently very little evidence that low doses of environmental estrogens (at concentration relevant for human exposure to xenoestrogens) have any inhibitory effect on Insl3 gene transcription and cryptorchidism. We performed dose-response experiments to assess the minimal dose of DES required for Insl3 down-regulation and cryptorchidism, and found that at 0.25 µg/d DES, inhibition of Insl3 transcription was minimal and testicular descent normal (data not shown). This suggests that estrogen-dependent repression of Insl3 transcription is achieved only at high doses of xenoestrogens. In support of this hypothesis, fetal exposure to weaker xenoestrogens such as soy-derived phytoestrogens (i.e. isoflavones) or phthalate esters, a class of endocrine disrupter commonly used as plasticizers for polyvinyl chloride, did not affect testicular descent and Insl3 gene expression in mice offspring (unpublished data). In contrast to what we observed with mice, rat fetuses exposed to similar levels of phthalate esters exhibit cryptorchidism caused by reduced Insl3 gene expression (41, 42). These apparent contradictory results may be reconciled if the effects of phthalates are species specific.

Molecular mechanism of action
The molecular mechanism by which E2 and DES is able to specifically down-regulate Insl3 and the steroidogenic genes, including Star and Cyp17a1, is largely unknown. In particular, it remains to be determined whether ER{alpha}-mediated down-regulation is a direct effect on fetal Leydig cells or indirect through alteration in the hypothalamo-pituitary system. Evidence from previous studies support a direct effect. Embryonic exposure to DES in rat resulted in a decrease in testosterone production at E19.5 without affecting pituitary LH concentration (40). Similarly, 2-d-old pups lacking ER{alpha} display an increase in testosterone secretion without changes in circulating LH (57). These reports suggest strongly that ER{alpha}-mediated inhibition of steroidogenesis and testicular descent by estrogens in mouse embryos result from a direct effect of ER{alpha} on fetal Leydig cells. However, at this stage, the remaining puzzling question concerns the potential mechanisms of ER{alpha} direct action in fetal Leydig cells. At least three mechanisms are possible. First, estrogen-activated ER{alpha} binds directly to EREs of target genes via a classical mode of action and induces changes in gene expression. Alternatively, activated ER{alpha} may act indirectly by interacting with other non-ERE sites through protein-protein tethering with other transcription factors (AP1, SP1). Finally, estrogen-activated ER{alpha} may act indirectly (either through EREs or non-ERE binding sites) by modulating the level of expression of transcription factors that will subsequently regulate the expression of steroidogenic enzymes and Insl3.

So far, no experimental data indicate that ER{alpha} may act directly, via a classical mode of action, on the promoters of Insl3, Star, Cyp7a1, and Ren1. Analysis of the 190-bp Insl3 proximal promoter required for Leydig cell-specific transcription did not reveal any putative EREs (25, 26). However, in silico analysis using a program for identification of EREs in genomic DNA has revealed four putative EREs within the 10 kb of the Insl3 promoter region, respectively at 311, 1852, 4414, and 4430 nucleotides upstream of the transcription start site [Dragon ERE Finder version 2 (70)]. Using the same algorithm, multiple potential EREs were also found within the 5' region of Star, Cyp17a1, and Ren1. Whether these ERE binding sites are relevant, functional, and could negatively affect the expression of Insl3 and steroidogenic genes remain to be determined.

Concerning a possible interaction of ER{alpha} with other non-ERE binding sites, a report has shown that estrogen-activated ER{alpha} is able to bind directly to Sf-1 binding sites (71), which could potentially interfere with Sf-1-mediated transcription of the Insl3 gene. In addition, it has been shown that activated-ER{alpha} could act through nonclassical pathways by interacting with AP-1 and Sp1 transcription factor complexes at their binding sites (72, 73, 74). Interestingly, the 5' proximal promoter of the Insl3 gene contains both a TATA box and a Sp1-binding element upstream of the transcription site (25). In an attempt to determine whether ER{alpha} was able to bind directly on the Insl3 proximal promoter, we performed EMSA with in vitro translated ER{alpha}, ERβ, and Sf-1. In contrast to Sf-1, we failed to show that ER{alpha} and ERβ interact with the Insl3 190-bp promoter (data not shown).

Finally, estrogen-activated ER{alpha} may act indirectly by regulating other transcription factors potentially regulating the expression of Insl3 and steroidogenic enzymes. Our toxicogenomic analysis did not reveal any major alteration in expression levels of Sf-1 in testes exposed to E2 and DES, as already described by others (22, 39). However, a growing body of evidence indicates that Sf-1 regulates its target genes by interacting with other transcription factors, including Dax-1, Mip-2a, Sp-1, Rip140, and pituitary homeobox 1 (Pitx1) (75, 76, 77, 78, 79). Among these genes, two of them were modulated by DES or E2 exposure: Dax-1 and Pitx1. Dax-1, an orphan nuclear receptor known to directly interact with and inhibit Sf-1 transcriptional activity, was reduced by 1.5-fold in DES and E2-exposed testes (data not shown). Similarly, we found that Pitx1 was up-regulated by 5-fold in E2-exposed testes. Pitx1 is a bicoid-related homeobox transcription factor known to interact with Sf-1 in the regulation of pituitary gene expression (79) but also in the regulation of transcription of enzymes involved in adrenal steroidogenesis (80). The involvement of Pitx1 in the estrogen-induced inhibition of fetal Leydig cell function remains to be assessed. In summary, the mechanism regulating ER{alpha} down-regulation of Insl3 and steroidogenic genes remains unclear. We believe that estrogenic exposure, through ER{alpha}, alters the combinatorial code of transcription factors required for the activation of Insl3 and steroidogenic genes in fetal Leydig cells.

Conclusions
This study demonstrates that the inhibitory effects of estrogens on testicular descent and testicular steroidogenesis are mediated exclusively via ER{alpha}. Because Insl3 expression and androgen production by fetal Leydig cells are required for normal male differentiation and testicular functions, these findings provide important insights into the teratogenic effects of estrogenic exposure at pharmacological doses, and may help explain how DES impairs the masculinization of the male urogenital system and male fertility.


   Acknowledgments
 
We thank Françoise Kühne, Laurence Tropia, Clélia Bardonneau, Rime Abla, Alexandre Fort, Gregg Sealy, Mylène Docquier, Christelle Barraclough, and Didier Cholet for technical assistance, and all members of the Nef and Vassalli laboratories for helpful discussions.


   Footnotes
 
This research was supported by grants from the Swiss National Research Project 50 on endocrine disruptors, the Sir Jules Thorn Charitable Overseas Trust Reg., Schaan, and The Fondation Gertrude von Meissner.

Disclosure Statement: C.R.C., P.D., P.C., J.-D.V., and S.N. have nothing to declare. O.S. consults for Sectoral Asset Management Inc.

First Published Online August 2, 2007

Abbreviations: Comp, Cartilage oligomeric matrix protein; Cyp17A1, cytochrome P450 17{alpha}-hydroxylase/17,20-lyase; Dax-1, dosage-sensitive sex reversal/adrenal hypoplasia congenita critical region on the X chromosome; DES, diethylstilbestrol; dpc, days post coitum; DMSO, dimethylsulfoxide; E, embryonic day; Ebaf/Lefty-A, endometrial bleeding-associated factor; E2, estradiol; ER, estrogen receptor; ER{alpha}KO, ER{alpha} knockout; ERE, estrogen response element; FMO2, flavin-containing monooxygenase 2; Greb1, gene regulated by estrogen in breast cancer 1; Grem1, Gremlin1; Insl3, insulin-like 3; LGR8, leucine-rich G protein-coupled receptor 8; MIS, Müllerian inhibiting substance; MMP9, matrix metalloproteinase-9; P0, postnatal d 0; principal component; PCA, principal component analysis; Pitx1, pituitary homeobox 1; qRT-PCR, quantitative RT-PCR; Ren1, renin 1; Sf-1, steroidogenic factor 1; Star, steroidogenic acute regulatory protein; WT, wild type.

Received May 23, 2007.

Accepted for publication July 18, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Boisen KA, Kaleva M, Main KM, Virtanen HE, Haavisto AM, Schmidt IM, Chellakooty M, Damgaard IN, Mau C, Reunanen M, Skakkebaek NE, Toppari J 2004 Difference in prevalence of congenital cryptorchidism in infants between two Nordic countries. Lancet 363:1264–1269[CrossRef][Medline]
  2. Kaleva M, Toppari J 2005 Cryptorchidism: an indicator of testicular dysgenesis? Cell Tissue Res 322:167–172[CrossRef][Medline]
  3. Toppari J, Kaleva M 1999 Maldescendus testis. Horm Res 51:261–269[CrossRef][Medline]
  4. Hutson JM, Baker M, Terada M, Zhou B, Paxton G 1994 Hormonal control of testicular descent and the cause of cryptorchidism. Reprod Fertil Dev 6:151–156[Medline]
  5. Hutson JM 1985 A biphasic model for the hormonal control of testicular descent. Lancet 2:419–421[Medline]
  6. Satokata I, Benson G, Maas R 1995 Sexually dimorphic sterility phenotypes in Hoxa10-deficient mice. Nature 374:460–463[CrossRef][Medline]
  7. Hudson PA, Promes SB 1997 Abdominal ultrasonography. Emerg Med Clin North Am 15:825–848[CrossRef][Medline]
  8. Kersten W, Molenaar GJ, Emmen JM, van der Schoot P 1996 Bilateral cryptorchidism in a dog with persistent cranial testis suspensory ligaments and inverted gubernacula: report of a case with implications for understanding normal and aberrant testis descent. J Anat 189(Pt 1):171–176
  9. Adham IM, Agoulnik AI 2004 Insulin-like 3 signalling in testicular descent. Int J Androl 27:257–265[CrossRef][Medline]
  10. Nef S, Parada LF 2000 Hormones in male sexual development. Genes Dev 14:3075–3086[Free Full Text]
  11. Hutson JM 1986 Testicular feminization: a model for testicular descent in mice and men. J Pediatr Surg 21:195–198[Medline]
  12. Bogatcheva NV, Ferlin A, Feng S, Truong A, Gianesello L, Foresta C, Agoulnik AI 2007 T222P mutation of the insulin-like 3 hormone receptor LGR8 is associated with testicular maldescent and hinders receptor expression on the cell surface membrane. Am J Physiol Endocrinol Metab 292:E138–E144
  13. Gorlov IP, Kamat A, Bogatcheva NV, Jones E, Lamb DJ, Truong A, Bishop CE, McElreavey K, Agoulnik AI 2002 Mutations of the GREAT gene cause cryptorchidism. Hum Mol Genet 11:2309–2318[Abstract/Free Full Text]
  14. Nef S, Parada LF 1999 Cryptorchidism in mice mutant for Insl3. Nat Genet 22:295–299[CrossRef][Medline]
  15. Zimmermann S, Steding G, Emmen JM, Brinkmann AO, Nayernia K, Holstein AF, Engel W, Adham IM 1999 Targeted disruption of the Insl3 gene causes bilateral cryptorchidism. Mol Endocrinol 13:681–691[Abstract/Free Full Text]
  16. Adham IM, Steding G, Thamm T, Bullesbach EE, Schwabe C, Paprotta I, Engel W 2002 The overexpression of the insl3 in female mice causes descent of the ovaries. Mol Endocrinol 16:244–252[Abstract/Free Full Text]
  17. Koskimies P, Suvanto M, Nokkala E, Huhtaniemi IT, McLuskey A, Themmen AP, Poutanen M 2003 Female mice carrying a ubiquitin promoter-Insl3 transgene have descended ovaries and inguinal hernias but normal fertility. Mol Cell Endocrinol 206:159–166[CrossRef][Medline]
  18. Baker LA, Nef S, Nguyen MT, Stapleton R, Nordenskjold A, Pohl H, Parada LF 2002 The insulin-3 gene: lack of a genetic basis for human cryptorchidism. J Urol 167:2534–2537[CrossRef][Medline]
  19. Ferlin A, Simonato M, Bartoloni L, Rizzo G, Bettella A, Dottorini T, Dallapiccola B, Foresta C 2003 The INSL3-LGR8/GREAT ligand-receptor pair in human cryptorchidism. J Clin Endocrinol Metab 88:4273–4279[Abstract/Free Full Text]
  20. Balvers M, Spiess AN, Domagalski R, Hunt N, Kilic E, Mukhopadhyay AK, Hanks E, Charlton HM, Ivell R 1998 Relaxin-like factor expression as a marker of differentiation in the mouse testis and ovary. Endocrinology 139:2960–2970[Abstract/Free Full Text]
  21. McKinnell C, Sharpe RM, Mahood K, Hallmark N, Scott H, Ivell R, Staub C, Jegou B, Haag F, Koch-Nolte F, Hartung S 2005 Expression of insulin-like factor 3 protein in the rat testis during fetal and postnatal development and in relation to cryptorchidism induced by in utero exposure to di (n-Butyl) phthalate. Endocrinology 146:4536–4544[Abstract/Free Full Text]
  22. Nef S, Shipman T, Parada LF 2000 A molecular basis for estrogen-induced cryptorchidism. Dev Biol 224:354–361[CrossRef][Medline]
  23. Zimmermann S, Schottler P, Engel W, Adham IM 1997 Mouse Leydig insulin-like (Ley I-L) gene: structure and expression during testis and ovary development. Mol Reprod Dev 47:30–38[CrossRef][Medline]
  24. Boockfor FR, Fullbright G, Bullesbach EE, Schwabe C 2001 Relaxin-like factor (RLF) serum concentrations and gubernaculum RLF receptor display in relation to pre- and neonatal development of rats. Reproduction 122:899–906[Abstract]
  25. Koskimies P, Levallet J, Sipila P, Huhtaniemi I, Poutanen M 2002 Murine relaxin-like factor promoter: functional characterization and regulation by transcription factors steroidogenic factor 1 and DAX-1. Endocrinology 143:909–919[Abstract/Free Full Text]
  26. Zimmermann S, Schwarzler A, Buth S, Engel W, Adham IM 1998 Transcription of the Leydig insulin-like gene is mediated by steroidogenic factor-1. Mol Endocrinol 12:706–713[Abstract/Free Full Text]
  27. Ikeda Y, Lala DS, Luo X, Kim E, Moisan MP, Parker KL 1993 Characterization of the mouse FTZ-F1 gene, which encodes a key regulator of steroid hydroxylase gene expression. Mol Endocrinol 7:852–860[Abstract/Free Full Text]
  28. Ingraham HA, Lala DS, Ikeda Y, Luo X, Shen WH, Nachtigal MW, Abbud R, Nilson JH, Parker KL 1994 The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev 8:2302–2312[Abstract/Free Full Text]
  29. Asa SL, Bamberger AM, Cao B, Wong M, Parker KL, Ezzat S 1996 The transcription activator steroidogenic factor-1 is preferentially expressed in the human pituitary gonadotroph. J Clin Endocrinol Metab 81:2165–2170[Abstract]
  30. Roselli CE, Jorgensen EZ, Doyle MW, Ronnekleiv OK 1997 Expression of the orphan receptor steroidogenic factor-1 mRNA in the rat medial basal hypothalamus. Brain Res Mol Brain Res 44:66–72[Medline]
  31. Fry DM, Toone CK 1981 DDT-induced feminization of gull embryos. Science 213:922–924[Abstract/Free Full Text]
  32. Newbold RR, Hanson RB, Jefferson WN, Bullock BC, Haseman J, McLachlan JA 2000 Proliferative lesions and reproductive tract tumors in male descendants of mice exposed developmentally to diethylstilbestrol. Carcinogenesis 21:1355–1363[Abstract/Free Full Text]
  33. Grocock CA, Charlton HM, Pike MC 1988 Role of the fetal pituitary in cryptorchidism induced by exogenous maternal oestrogen during pregnancy in mice. J Reprod Fertil 83:295–300[Abstract/Free Full Text]
  34. Hadziselimovic F, Girard J 1977 Pathogenesis of cryptorchidism. Horm Res 8:76–83[CrossRef][Medline]
  35. Khan SA, Ball RB, Hendry 3rd WJ 1998 Effects of neonatal administration of diethylstilbestrol in male hamsters: disruption of reproductive function in adults after apparently normal pubertal development. Biol Reprod 58:137–142[Abstract/Free Full Text]
  36. Perez-Martinez C, Garcia-Iglesias MJ, Ferreras-Estrada MC, Bravo-Moral AM, Espinosa-Alvarez J, Escudero-Diez A 1996 Effects of in-utero exposure to zeranol or diethylstilboestrol on morphological development of the fetal testis in mice. J Comp Pathol 114:407–418[CrossRef][Medline]
  37. Gill WB, Schumacher GF, Bibbo M, Straus 2nd FH, Schoenberg HW 1979 Association of diethylstilbestrol exposure in utero with cryptorchidism, testicular hypoplasia and semen abnormalities. J Urol 122:36–39[Medline]
  38. Toppari J, Larsen JC, Christiansen P, Giwercman A, Grandjean P, Guillette Jr LJ, Jegou B, Jensen TK, Jouannet P, Keiding N, Leffers H, McLachlan JA, Meyer O, Muller J, Rajpert-De Meyts E, Scheike T, Sharpe R, Sumpter J, Skakkebaek NE 1996 Male reproductive health and environmental xenoestrogens. Environ Health Perspect 104(Suppl 4):741–803
  39. Emmen JM, McLuskey A, Adham IM, Engel W, Verhoef-Post M, Themmen AP, Grootegoed JA, Brinkmann AO 2000 Involvement of insulin-like factor 3 (Insl3) in diethylstilbestrol-induced cryptorchidism. Endocrinology 141:846–849[Abstract/Free Full Text]
  40. Haavisto T, Nurmela K, Pohjanvirta R, Huuskonen H, El-Gehani F, Paranko J 2001 Prenatal testosterone and luteinizing hormone levels in male rats exposed during pregnancy to 2,3,7,8-tetrachlorodibenzo-p-dioxin and diethylstilbestrol. Mol Cell Endocrinol 178:169–179[CrossRef][Medline]
  41. Shono T, Shima Y, Kondo T, Suita S 2005 In utero exposure to mono-n-butyl phthalate impairs insulin-like factor 3 gene expression and the transabdominal phase of testicular descent in fetal rats. J Pediatr Surg 40:1861–1864[CrossRef][Medline]
  42. Wilson VS, Lambright C, Furr J, Ostby J, Wood C, Held G, Gray Jr LE 2004 Phthalate ester-induced gubernacular lesions are associated with reduced insl3 gene expression in the fetal rat testis. Toxicol Lett 146:207–215[CrossRef][Medline]
  43. Peirson SN, Butler JN, Foster RG 2003 Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Res 31:e73
  44. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F 2002 Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034
  45. Hubbell E, Liu WM, Mei R 2002 Robust estimators for expression analysis. Bioinformatics 18:1585–1592[Abstract/Free Full Text]
  46. Guyot R, Odet F, Leduque P, Forest MG, Le Magueresse-Battistoni B 2004 Diethylstilbestrol inhibits the expression of the steroidogenic acute regulatory protein in mouse fetal testis. Mol Cell Endocrinol 220:67–75[CrossRef][Medline]
  47. Majdic G, Sharpe RM, O’Shaughnessy PJ, Saunders PT 1996 Expression of cytochrome P450 17{alpha}-hydroxylase/C17–20 lyase in the fetal rat testis is reduced by maternal exposure to exogenous estrogens. Endocrinology 137:1063–1070[Abstract]
  48. Altucci L, Addeo R, Cicatiello L, Dauvois S, Parker MG, Truss M, Beato M, Sica V, Bresciani F, Weisz A 1996 17beta-Estradiol induces cyclin D1 gene transcription, p36D1-p34cdk4 complex activation and p105Rb phosphorylation during mitogenic stimulation of G(1)-arrested human breast cancer cells. Oncogene 12:2315–2324[Medline]
  49. Lin CY, Strom A, Vega VB, Kong SL, Yeo AL, Thomsen JS, Chan WC, Doray B, Bangarusamy DK, Ramasamy A, Vergara LA, Tang S, Chong A, Bajic VB, Miller LD, Gustafsson JA, Liu ET 2004 Discovery of estrogen receptor {alpha} target genes and response elements in breast tumor cells. Genome Biol 5:R66
  50. Yu WC, Leung BS, Gao YL 1981 Effects of 17 beta-estradiol on progesterone receptors and the uptake of thymidine in human breast cancer cell line CAMA-1. Cancer Res 41:5004–5009[Abstract/Free Full Text]
  51. Van Der Ven PF, Obermann WM, Weber K, Furst DO 1996 Myomesin, M-protein and the structure of the sarcomeric M-band. Adv Biophys 33:91–99[CrossRef][Medline]
  52. Hedbom E, Antonsson P, Hjerpe A, Aeschlimann D, Paulsson M, Rosa-Pimentel E, Sommarin Y, Wendel M, Oldberg A, Heinegard D 1992 Cartilage matrix proteins. An acidic oligomeric protein (COMP) detected only in cartilage. J Biol Chem 267:6132–6136[Abstract/Free Full Text]
  53. Newton G, Weremowicz S, Morton CC, Copeland NG, Gilbert DJ, Jenkins NA, Lawler J 1994 Characterization of human and mouse cartilage oligomeric matrix protein. Genomics 24:435–439[CrossRef][Medline]
  54. Stossi F, Barnett DH, Frasor J, Komm B, Lyttle CR, Katzenellenbogen BS 2004 Transcriptional profiling of estrogen-regulated gene expression via estrogen receptor (ER) {alpha} or ERβ in human osteosarcoma cells: distinct and common target genes for these receptors. Endocrinology 145:3473–3486[CrossRef][Medline]
  55. Vic P, Vignon F, Derocq D, Rochefort H 1982 Effect of estradiol on the ultrastructure of the MCF7 human breast cancer cells in culture. Cancer Res 42:667–673[Abstract/Free Full Text]
  56. O’Donnell L, Robertson KM, Jones ME, Simpson ER 2001 Estrogen and spermatogenesis. Endocr Rev 22:289–318[Abstract/Free Full Text]
  57. Delbes G, Levacher C, Duquenne C, Racine C, Pakarinen P, Habert R 2005 Endogenous estrogens inhibit mouse fetal Leydig cell development via estrogen receptor {alpha}. Endocrinology 146:2454–2461[Abstract/Free Full Text]
  58. Delbes G, Levacher C, Pairault C, Racine C, Duquenne C, Krust A, Habert R 2004 Estrogen receptor β-mediated inhibition of male germ cell line development in mice by endogenous estrogens during perinatal life. Endocrinology 145:3395–3403[Abstract/Free Full Text]
  59. Cashman JR 2000 Human flavin-containing monooxygenase: substrate specificity and role in drug metabolism. Curr Drug Metab 1:181–191[CrossRef][Medline]
  60. Coecke S, Debast G, Phillips IR, Vercruysse A, Shephard EA, Rogiers V 1998 Hormonal regulation of microsomal flavin-containing monooxygenase activity by sex steroids and growth hormone in co-cultured adult male rat hepatocytes. Biochem Pharmacol 56:1047–1051[CrossRef][Medline]
  61. Cornet PB, Galant C, Eeckhout Y, Courtoy PJ, Marbaix E, Henriet P 2005 Regulation of matrix metalloproteinase-9/gelatinase B expression and activation by ovarian steroids and LEFTY-A/endometrial bleeding-associated factor in the human endometrium. J Clin Endocrinol Metab 90:1001–1011[Abstract/Free Full Text]
  62. Deschepper CF, Mellon SH, Cumin F, Baxter JD, Ganong WF 1986 Analysis by immunocytochemistry and in situ hybridization of renin and its mRNA in kidney, testis, adrenal, and pituitary of the rat. Proc Natl Acad Sci USA 83:7552–7556[Abstract/Free Full Text]
  63. Campbell DM, Webb JA, Hargreave TB 1987 Cryptorchidism in Scotland. Br Med J (Clin Res Ed) 295:1235–1236[Medline]
  64. Chilvers C, Pike MC, Forman D, Fogelman K, Wadsworth ME 1984 Apparent doubling of frequency of undescended testis in England and Wales in 1962–81. Lancet 2:330–332[CrossRef][Medline]
  65. Matlai P, Beral V 1985 Trends in congenital malformations of external genitalia. Lancet 1:108[CrossRef][Medline]
  66. Andersen AG, Jensen TK, Carlsen E, Jorgensen N, Andersson AM, Krarup T, Keiding N, Skakkebaek NE 2000 High frequency of sub-optimal semen quality in an unselected population of young men. Hum Reprod 15:366–372[Abstract/Free Full Text]
  67. Irvine S, Cawood E, Richardson D, MacDonald E, Aitken J 1996 Evidence of deteriorating semen quality in the United Kingdom: birth cohort study in 577 men in Scotland over 11 years. BMJ 312:467–471[Abstract/Free Full Text]
  68. Adami HO, Bergstrom R, Mohner M, Zatonski W, Storm H, Ekbom A, Tretli S, Teppo L, Ziegler H, Rahu M, Gurevicus R, Stengrevics A1994 Testicular cancer in nine northern European countries. Int J Cancer 59:33–38
  69. Moller H, Cortes D, Engholm G, Thorup J 1998 Risk of testicular cancer with cryptorchidism and with testicular biopsy: cohort study. BMJ 317:729[Free Full Text]
  70. Bajic VB, Tan SL, Chong A, Tang S, Strom A, Gustafsson JA, Lin CY, Liu ET 2003 Dragon ERE Finder version 2: a tool for accurate detection and analysis of estrogen response elements in vertebrate genomes. Nucleic Acids Res 31:3605–3607[Abstract/Free Full Text]
  71. Vanacker JM, Pettersson K, Gustafsson JA, Laudet V 1999 Transcriptional targets shared by estrogen receptor-related receptors (ERRs) and estrogen receptor (ER) alpha, but not by ERbeta. EMBO J 18:4270–4279[CrossRef][Medline]
  72. Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM, Webb P 2000 Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol 74:311–317[CrossRef][Medline]
  73. Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J, Kushner PJ, Scanlan TS 1997 Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites. Science 277:1508–1510[Abstract/Free Full Text]
  74. Porter W, Saville B, Hoivik D, Safe S 1997 Functional synergy between the transcription factor Sp1 and the estrogen receptor. Mol Endocrinol 11:1569–580[Abstract/Free Full Text]
  75. Ghosh AK, Steele R, Ray RB 2003 Modulation of human luteinizing hormone beta gene transcription by MIP-2A. J Biol Chem 278:24033–24038[Abstract/Free Full Text]
  76. 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]
  77. Sugawara T, Abe S, Sakuragi N, Fujimoto Y, Nomura E, Fujieda K, Saito M, Fujimoto S 2001 RIP 140 modulates transcription of the steroidogenic acute regulatory protein gene through interactions with both SF-1 and DAX-1. Endocrinology 142:3570–3577[Abstract/Free Full Text]
  78. Sugawara T, Saito M, Fujimoto S 2000 Sp1 and SF-1 interact and cooperate in the regulation of human steroidogenic acute regulatory protein gene expression. Endocrinology 141:2895–2903[Abstract/Free Full Text]
  79. Tremblay JJ, Lanctot C, Drouin J 1998 The pan-pituitary activator of transcription, Ptx1 (pituitary homeobox 1), acts in synergy with SF-1 and Pit1 and is an upstream regulator of the Lim-homeodomain gene Lim3/Lhx3. Mol Endocrinol 12:428–441[Abstract/Free Full Text]
  80. Hiroi N, Kino T, Bassett M, Rainey WE, Phung M, Abu-Asab M, Fojo T, Briata P, Chrousos GP, Bornstein SR 2003 Pituitary homeobox factor 1, a novel transcription factor in the adrenal regulating steroid 11beta-hydroxylase. Horm Metab Res 35:273–278[CrossRef][Medline]



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