help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2008-0310
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Laguë, E.
Right arrow Articles by Tremblay, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Laguë, E.
Right arrow Articles by Tremblay, J. J.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*BIS(2-ETHYLHEXYL)PHTHALATE
*TESTOSTERONE
Endocrinology Vol. 149, No. 9 4688-4694
Copyright © 2008 by The Endocrine Society

Antagonistic Effects of Testosterone and the Endocrine Disruptor Mono-(2-Ethylhexyl) Phthalate on INSL3 Transcription in Leydig Cells

Éric Laguë and Jacques J. Tremblay

Department of Reproduction, Perinatal, and Child Health (E.L., J.J.T.), Centre Hospitalier Universitaire of Québec Research Centre, Québec City, Québec, Canada G1V 4G2; Centre for Research in Biology of Reproduction (J.J.T.), Department of Obstetrics and Gynecology, Faculty of Medicine, Université Laval, Québec City, Québec, Canada G1V 0A6

Address all correspondence and requests for reprints to: Dr. Jacques J. Tremblay, Reproduction, Perinatal, and Child Health, Centre Hospitalier Universitaire of Québec Research Centre, CHUL Room T1-49, 2705 Laurier Boulevard, Québec City, Québec, Canada G1V 4G2. E-mail: jacques-j.tremblay{at}crchul.ulaval.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-like 3 (INSL3) is a small peptide produced by testicular Leydig cells throughout embryonic and postnatal life and by theca and luteal cells of the adult ovary. During fetal life, INSL3 regulates testicular descent in males, whereas in adults, it acts as an antiapoptotic factor for germ cells in males and as a follicle selection and survival factor in females. Despite its considerable roles in the reproductive system, the mechanisms that regulate Insl3 expression remain poorly understood. There is accumulating evidence suggesting that androgens might regulate Insl3 expression in Leydig cells, but transcriptional data are still lacking. We now report that testosterone does increase Insl3 mRNA levels in a Leydig cell line and primary Leydig cells. We also show that testosterone activates the activity of the Insl3 promoter from different species. In addition, the testosterone-stimulating effects on Insl3 mRNA levels and promoter activity require the androgen receptor. We have mapped the testosterone-responsive element to the proximal Insl3 promoter region. This region, however, lacks a consensus androgen response element, suggesting an indirect mechanism of action. Finally we show that mono-(2-ethylhexyl) phthalate, a widely distributed endocrine disruptor with antiandrogenic activity previously shown to inhibit Insl3 expression in vivo, represses Insl3 transcription, at least in part, by antagonizing testosterone/androgen receptor action. All together our data provide important new insights into the regulation of Insl3 transcription in Leydig cells and the mode of action of phthalates.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE 3 (INSL3), ALSO known as relaxin-like factor, is a member of the insulin-IGF-relaxin family of growth factors and hormones (1, 2). This hormone is almost exclusively expressed in the gonads. During fetal life, INSL3 is produced by maturing Leydig cells of the male gonad, whereas in adults it is secreted by both the testis (Leydig cells) and the ovary (theca and luteal cells) (1, 2, 3, 4). Consistent with its sexually dimorphic expression pattern during fetal life, INSL3 was found to be a master regulator of gonadal descent, an essential step of the male sex differentiation process. Insl3–/– null mice have bilateral cryptorchid testes located high in the abdominal cavity close to the kidneys (5, 6). Further support for INSL3 as a regulator of testicular descent came from elegant transgenic experiments in which INSL3 was overexpressed in pancreatic β-cells of Insl3 null mice (7). In these animals, normal testis descent was restored. Even more convincing was the generation of female transgenic mice overexpressing INSL3, which led to descended ovaries (7, 8). In adults, INSL3 acts as a germ cell survival factor (9) and would also be involved in follicle selection and survival (10, 11).

Despite its important roles in reproductive development and function, surprisingly very little is known regarding the mechanisms regulating Insl3 expression in gonadal cells. At the molecular level, so far only two transcription factors have been shown to regulate INSL3 promoter activity in Leydig cells: the nuclear receptors steroidogenic factor-1 (SF1; Ad4BP/NR5A1) and NUR77 (NGFI-B/NR4A1) (12, 13, 14, 15, 16). At the hormonal level, Insl3 expression was found to require the pituitary LH in postnatal Leydig cells (3, 9, 17). This, however, is believed to be associated with the differentiation status of Leydig cells, which is dependent on LH, rather than the result of an acute response to LH as for steroidogenesis (3, 18). In addition to LH, there is mounting evidence that Insl3 expression would be regulated by androgens in several species. In testicular feminized mice, which harbor a mutation in the androgen receptor (AR) gene causing complete androgen insensitivity (19, 20, 21), Insl3 expression is severely decreased (22). In an AR hypomorphic mice model in which AR levels are reduced (23), Insl3 expression was down-regulated nearly 10-fold (24). In these mice, LH and testosterone levels were dramatically increased, consistent with normally differentiated and functioning pituitary gonadotrope and testicular Leydig cells (24). In adult rats, GnRH antagonists (chemical castration) down-regulate Insl3 expression (9). Treatment with human chorionic gonadotropin restored Insl3 mRNA levels in GnRH antagonist-treated animals (9) and hpg mice (3), which have a deletion of the Gnrh1 gene (25, 26). In humans, INSL3 and testosterone concentrations are significantly correlated (17, 18). Finally, endocrine disruptors exhibiting antiandrogenic activity, such as the widely distributed plasticizer phthalate, repress Insl3 expression in Leydig cells (27, 28, 29). The exact mechanisms of action of androgens on Insl3 expression, however, remain completely obscure.

In the present study, we provide evidence that testosterone regulates Insl3 transcription in Leydig cells in an AR-dependent manner. We have also mapped an androgen-responsive region in the human INSL3 promoter. Finally, we found that mono-(2-ethylhexyl) phthalate (MEHP) represses Insl3 transcription, at least in part, by antagonizing the testosterone stimulating effects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
The –1137-, –920-, and –656- to +11-bp human INSL3 promoter fragment have been previously described (15). Additional deletion constructs to –132 and –85 bp were generated by PCR using the –1137-bp construct as template along with a common reverse primer containing a KpnI cloning site (underlined) 5'-GGG GTA CCG GTG GGT GGC GCC GGG GCC AAG C-3' and the following forward primers that contain BamHI cloning sites (underlined): –132 bp, 5'-CGG GAT CCC AGA AAG GCT CTG GCA C-3' and –85 bp, 5'-CGG GAT CCT GAA GAA TGT TCT G-3'. The –31- to +11-bp reporter construct was obtained by subcloning a double-stranded oligonucleotide: sense oligo 5'-CGG TGG GTG GCG CCG GGG CCA AGC GGG GAC CCC CTT TAT AGG CG-3' and antisense 5'-GAT CCG CCT ATA AAG GGG GAC CCC GCT TGG CCC CGG CGC CAC CCA CCG GTA C-3'.

Various trinucleotide mutant constructs in the context of the –1137- and –132-bp reporters were generated using the QuikChange XL mutagenesis kit (Stratagene, La Jolla, CA) along with the following oligonucleotides (only the sense oligonucleotide is shown) in which the mutations are in lowercase: M1, 5'-AAG GCC CTG GCC CTG GCC CTG GAA tcc AGG CTC CTC TGG CAC TAA CCC C-3'; M2, 5'-CCC TGG CCC TGG GAG AAA tta TCT GGC ACT AAC CCC ACC C-3'; M3, GCC CTG GGA GAA AGG CTa gtG CAC TAA CCC CAC CCT TGA C-3'; M4, 5'-CCT GGG AGA AAG GCT CTG Gac aTA ACC CCA CCC TTG ACC-3'; M5, 5'-AAG GCT CTG GCA CTA ACC aac CCC TTG ACC TTT TTC CTG GGC G-3'; M6, 5'-CTC TGG CAC TAA CCC CAC aag TGA CCT TTT TCC TGG GCG-3'; M7, 5'-GGC ACT AAC CCC ACC TTt caC TTT TTC CTG GGC GGT CC-3'; M8, 5'-CTA ACC CCA CCC TGA CCg ggT TCC TGG GCG GGT CCT GAA G-3'; M9, 5'-CCC CAC CCT GAC CTT TTg aaT GGG CGG GTC CTG AAG AAT G-3'; M10, 5'-CCA CCC TGA CCT TTT TCC Ttt tCG GGT CCT GAA GAA TGT TCT GTC C-3'. The –978- to +5-bp mouse Insl3 promoter fragment was isolated by PCR using mouse genomic DNA and the following primers: forward, 5'-GCG GAT CCG AAT GGG GAT ATT AAA TAT GTG-3' (BamHI cloning site is underlined) and reverse, 5'-GGG GTA CCG TGG CAG GAG GCA GTG GGC AG-3' (KpnI cloning site is underlined).

A similar PCR-based approach using rat genomic DNA was used to isolate the –508- to +5-bp of the rat Insl3 promoter along with the following primers: forward, 5'-CTG GAT CCG GGC AGA GGC AGT GC-3' (BamHI cloning site is underlined) and reverse, 5'-GGG GTA CCC GGA GGC AGC GGG TAA GGA C-3' (KpnI cloning site is underlined). All reporter constructs were subcloned in a modified pXP1 Luciferase reporter construct (30) and subsequently verified by sequencing (Centre de Génomique de Québec, Centre Hospitalier Universitaire of Québec Research Centre, Québec City, Canada).

Cell culture and transfections
The mouse Leydig cell line MA-10 (31) was provided by Dr. Mario Ascoli (University of Iowa, Iowa City, IA). Cells were grown in Waymouth’s media supplemented with 12% horse serum and 50 mg/liter of gentamicin and streptomycin sulfate, at 37 C in 5% CO2. Cells were plated in a 24-well plate at a density of 90,000 cells/well in Waymouth’s supplemented with 12% charcoal-treated horse serum and allowed to recover for 16–24 h before transfection. An amount of 0.2 picomoles of Insl3 Firefly luciferase reporter construct along with 8 ng of phRL-TK Renilla luciferase reporter, used as internal control for transfection efficiency, were transfected using Lipofectamine 2000 reagent (Invitrogen Canada, Burlington, Ontario, Canada) according to the manufacturer’s recommendations. Four hours later, media were replaced and cells were treated with either testosterone (concentrations indicated in the figure legends) with or without 30 µM di-2-ethylhexyl phthalate (DEHP) or MEHP (TCI America, Portland, OR) for 24–36 h. Cells were then lysed and luciferase activities measured using the dual-luciferase assay system (Promega Corp., Madison, WI) and the EG&G Berthold LB 9507 luminometer (Berthold Technologies, Oak Ridge, TN). Transfections were performed at least three times in duplicate using different preparations of plasmid DNA.

Isolation of primary Leydig cells
Primary Leydig cells were isolated as described previously (32) from 35-d-old Sprague Dawley rats obtained on site. Serum-free medium 199 with Earle’s salts (Sigma-Aldrich Canada, Oakville, Ontario, Canada), L-glutamine, 1.5 mM HEPES, 2.5 g/liter NaHCO3, and antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin) was used during preparation and culturing. After 2 d in culture, the purity of the Leydig cell preparation was evaluated to be about 95% enriched as assessed by histochemical staining for 3β-hydroxysteroid dehydrogenase activity (33). All experiments were conducted according to the Canadian Council for Animal Care and have been approved by the Animal Care and Ethics Committee of Laval University (protocol 2005-184).

RNA isolation, reverse transcription, and quantitative real-time PCR
Total RNA was isolated using the RNeasy Plus minikit (QIAGEN Canada, Mississauga, Ontario, Canada). First-strand cDNAs were synthesized from a 5-µg aliquot of the various RNAs using the Superscript III reverse transcriptase system (Invitrogen Canada). Quantitative real-time PCR for Insl3 and Rpl19 were performed as previously described (15, 34). Each amplification was performed in duplicate using at least three different preparations of cDNAs for each of the three different RNA extractions.

Statistical analysis
Statistical analyses were done using one-way ANOVA or ANOVA on ranks (when parameters were not met) followed by a Student-Newman-Keuls posttest. For all statistical analyses, P < 0.05 was considered significant. All statistical analyses were done using the SigmaStat software package (Systat Software Inc., San Jose, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Testosterone increases Insl3 mRNA levels in Leydig cells
To assess whether androgens could regulate Insl3 expression, we first treated MA-10 cells, a Leydig cell line known to express AR (35), with increasing doses of testosterone, doses that are within the physiological range found within the rodent testis (200–350 nM) (36, 37). As shown in Fig. 1AGo, Insl3 mRNA levels were significantly increased in response to testosterone as determined by quantitative real-time PCR. An even stronger response was observed when primary Leydig cell cultures were used (Fig. 1BGo). These results indicate that Insl3 expression is regulated by androgens in Leydig cells and that the MA-10 Leydig cell line constitutes an appropriate model to study the underlying mechanisms of androgen action.


Figure 1
View larger version (16K):
[in this window]
[in a new window]

  		FIG. 1. Testosterone stimulates Insl3 expression in Leydig cells. A, MA-10 Leydig cells were treated with either 1500 nM dimethylsulfoxide (open bar) or increasing doses of testosterone (T) (black triangle: 0.15, 1.5, 15, 150, and 1500 nM) for 36 h. B, MA-10 and primary cultures of Leydig cells from rats were treated with 15 nM dimethylsulfoxide (open bars) or testosterone (T) (black bars) for 36 h. Total RNA was next isolated and used in quantitative real-time PCR using primers specific for Insl3 cDNA as described in Materials and Methods. Results were corrected with the Rpl19 cDNA. Results are the mean of three individual experiments performed in duplicate (±SEM). *, Statistically significant difference from control.

 
Testosterone stimulates Insl3 promoter activity from different species
Next, we ought to determine whether the effects of testosterone could be due to an increase in Insl3 gene transcription. As shown in Fig. 2AGo, treatment of MA-10 Leydig cells with increasing doses of testosterone led to a dose-dependent activation of a –1137-bp human INSL3 reporter construct. Similar results were obtained with the rat and mouse Insl3 promoters (Fig. 2BGo). Thus, testosterone can activate the Insl3 promoter from different species.


Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		FIG. 2. The INSL3 promoter from different species is activated by testosterone. A, MA-10 cells were transfected with a –1137 bp human INSL3 promoter construct and treated with either 1500 nM dimethylsulfoxide (open bar) or increasing doses of testosterone (T) (black triangle: 0.15, 1.5, 15, 150, and 1500 nM) for 36 h. A different letter indicates a statistically significant difference. B, MA-10 cells were transfected with a –1137- to +11-bp human INSL3 promoter, –978- to +5-bp mouse Insl3 promoter, and –508- to +5-bp rat Insl3 promoter and treated with 15 nM dimethylsulfoxide (open bars) or testosterone (T) (black bars) for 36 h. *, Statistically significant difference from the respective control. Results are shown as fold activation over control (±SEM).

 
Testosterone acts in an AR-dependent manner to increase in Insl3 transcription
Although testosterone is known to act mainly through AR, some AR-independent responses to androgens have been reported (reviewed in Ref. 38). The involvement of AR in testosterone-induced Insl3 transcription was assessed by treating MA-10 Leydig cells with testosterone in the presence or absence of hydroxyflutamide, the biologically active form of flutamide and a potent AR antagonist. Testosterone-mediated increase in INSL3 promoter activity (Fig. 3AGo) and endogenous mRNA levels (Fig. 3BGo) were abolished in the presence of the antagonist. The AR antagonist had no effect when used alone (Fig. 3Go). All together, these results establish the requirement of AR for testosterone-dependent stimulation of Insl3 transcription.


Figure 3
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 3. Testosterone-dependent activation of Insl3 transcription requires the androgen receptor. A, MA-10 Leydig cells were transfected with a –1137-bp human INSL3 promoter construct and pretreated with 50 µM dimethylsulfoxide (open bars) or increasing doses of hydroxyflutamide (HF) (black triangle: 50 nM, 250 nM, 500 nM, 5 µM, 25 µM, and 50 µM) for 1 h followed by addition of vehicle (–) or 15 nM testosterone (T, +) for 36 h. *, Statistically significant difference from the respective control. Results are shown as fold activation over control (SEM). B, MA-10 cells were treated with either dimethylsulfoxide (open bar), 15 nM testosterone (T) (black bar), 5 µM hydroxyflutamide (HF) (gray bar), or a combination of hydroxyflutamide and testosterone (hatched bar). Total RNA was isolated and Insl3 expression was determined by quantitative real-time PCR. Results were corrected with the Rpl19 cDNA. Results are the mean of three individual experiments performed in duplicate (±SEM). *, Statistically significant difference from control.

 
Mapping of the testosterone-responsive element in the INSL3 promoter
To locate the testosterone-responsive element in the human INSL3 promoter, a series of 5' progressive deletion constructs were generated and transfected in MA-10 Leydig cells that were then treated with 15 nM testosterone. As shown in Fig. 4Go, deletion from –1137 to –132 bp had no effect on testosterone responsiveness (nearly 3-fold). Further deletion to –85 bp severely blunted the testosterone-responsiveness of the INSL3 promoter to 1.4-fold. The testosterone-activating effects were specific because a –31-bp minimal INSL3 promoter construct was no longer responsive (Fig. 4Go). These data indicate that the main testosterone-responsive element(s) are located between –132 and –85 bp. A survey of the 47-bp promoter sequence between –132 and –85 bp did not reveal the presence of a consensus androgen response element (GGTACAnnnTGTTCT). The 47-bp region, however, contains a half-site for the binding of nuclear receptors (AGGTCA), which we previously described as a binding site for the nuclear receptors NUR77 and SF1 (15, 16). To better define the element within the 47-bp region, a series of mutant constructs were generated in the context of the –1137-bp (Fig. 5AGo) and the –132-bp (Fig. 5BGo) INSL3 promoter. In the context of the –1137-bp promoter construct, mutations M1-M3 had no impact on the testosterone-responsiveness of the INSL3 promoter, whereas mutations M4-M7 caused a small but significant reduction in the testosterone-dependent activation (Fig. 5AGo). Mutations M8, M9, and M10, sequentially changing each of the underlined triplet in the sequence GACCTTTTTCC, had the most dramatic effects, decreasing testosterone responsiveness to the level observed with the –85-bp construct (from nearly 3-fold to 1.4-fold) (Fig. 5AGo). Interestingly, this sequence contains the NUR77/SF1 nuclear receptor binding site. Similar results were obtained in the context of the –132-bp reporter construct with mutation M9 eliminating the testosterone-responsiveness of the INSL3 promoter (Fig. 5BGo).


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 4. The testosterone-responsive area is located in the proximal INSL3 promoter. MA-10 cells were transfected with various 5' deletion constructs of the human INSL3 promoter (the 5' end point of each construct is indicated on the left of the graph) and treated with dimethylsulfoxide (open bars) or 15 nM testosterone (T) (black bars). *, Statistically significant difference from the respective control.

 

Figure 5
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 5. Fine mapping of the testosterone-responsive element in the human INSL3 promoter. MA-10 Leydig cells were transfected with either a wild-type (WT) or a series of human INSL3 promoter constructs containing trinucleotide mutations in the testosterone-responsive region either in the context of the –1137-bp (A) or –132-bp (B) reporter. The wild-type INSL3 promoter sequence is shown on top and the corresponding mutated nucleotides are shown underneath in lowercase. MA-10 cells were treated with either dimethylsulfoxide (open bars) or 15 nM testosterone (T) (black bars). A different letter indicates a statistically significant difference. Results are shown as fold activation over control (±SEM).

 
MEHP antagonizes testosterone-induced Insl3 expression
MEHP, the active metabolite of a widely used plasticizer, is known to repress Insl3 expression in vivo in Leydig cells (29). Although the exact molecular mechanisms remain unidentified, it is known that MEHP cannot bind to AR but nonetheless acts as an antiandrogen (39, 40, 41). Because we have shown that androgens activate Insl3 transcription, we tested whether MEHP could modulate testosterone action on Insl3 transcription. As shown in Fig. 6Go, 30 µM of MEHP significantly inhibited the testosterone-mediated increase in Insl3 mRNA in primary Leydig cells but had no effect in unstimulated cells. Similarly, MEHP, but not its inactive precursor DEHP, significantly reduced the testosterone-dependent activation of the human INSL3 promoter (Fig. 7Go). Thus, MEHP represses Insl3 expression, at least in part, by antagonizing testosterone action.


Figure 6
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 6. MEHP inhibits the testosterone-induced Insl3 expression. Rat primary Leydig cells were treated with vehicle or 30 µM MEHP in the absence (open bars) or presence of 15 nM testosterone (T) (black bars) for 36 h. Total RNA was isolated and Insl3 expression was determined by quantitative real-time PCR. Results were corrected with the Rpl19 cDNA. Results are the mean of three individual experiments performed in duplicate (±SEM). A different letter indicates a statistically significant difference.

 

Figure 7
View larger version (16K):
[in this window]
[in a new window]

  		FIG. 7. Testosterone-mediated induction of INSL3 promoter activity is repressed by MEHP. MA-10 Leydig cells were transfected with a –1137-bp human INSL3 reporter construct and treated with vehicle or 30 µM DEHP or MEHP in the absence (open bars) or presence of 15 nM testosterone (T) (black bars) for 36 h. A different letter indicates a statistically significant difference. Results are shown as fold activation over control (±SEM).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
INSL3, a small peptide of gonadal origin, plays important roles in reproductive function (42). In addition, its expression in males is highly correlated with the functional status of testicular Leydig cells and to circulating testosterone levels (17, 18, 43, 44, 45). In the present study, we found that androgens activate Insl3 transcription in Leydig cells and that this is partly antagonized by MEHP, a widely distributed endocrine disruptor with antiandrogenic activity.

Expression of Insl3 in Leydig cells
Although the Insl3 gene is located within the last intron of the Jak3 gene, the two genes have very divergent expression profile. Jak3 is restricted to lymphoid and hematopoietic cells (46, 47), whereas Insl3 is found exclusively in the gonads, mainly in testicular Leydig cells (1, 2, 3, 4). Jak3 and Insl3 are therefore not likely to share regulatory elements despite their close genomic association. Because of this atypical organization, it is believed that the regulatory motifs driving Insl3 expression in Leydig cells are located within a relatively short 5' flanking sequence. In agreement with this, the first 200 bp upstream of the transcription start site were found to be sufficient to confer activity to the mouse (12, 13), rat (14), and human (our data) Insl3 promoter in MA-10 Leydig cells. Within this fragment are found binding sites for the transcription factors SF1 and NUR77 (12, 13, 14, 15, 16). Consistent with an in vivo role for SF1, Insl3 expression was reduced in a Leydig cell-specific Sf1 knockout mouse model (48).

Mechanism of androgen action on Insl3 transcription
In addition to these two transcription factors, we found that testosterone also activates Insl3 expression in a mouse Leydig cell line and primary Leydig cells from rats. These data are supported by evidence from the literature whereby modulation of androgen production or action in animal models was shown to cause a concomitant alteration in Insl3 mRNA levels (3, 9, 17, 18, 22, 24, 49).

We found that testosterone directly regulates the activity of the Insl3 promoter from various species. This indicates that the mechanism of androgen action on Insl3 expression has been evolutionarily conserved despite the poor sequence conservation between the human and rodent Insl3 promoter. Testosterone action on Insl3 transcription was found to be AR dependent. The AR-responsive region that we mapped in the human INSL3 promoter, however, does not contain an androgen response element, suggesting that androgen-activated AR acts in a DNA binding-independent manner. AR has been shown to modulate gene expression and cell function without directly binding to DNA through nonclassical pathways (reviewed in Refs. 50 and 51).

Androgen-MEHP antagonism in Insl3 transcription
Phthalates are chemicals used to impart flexibility and durability to polyvinyl chloride and comprise up to 40% of the plastic volume. Phthalates are ubiquitous contaminants of the environment and humans, and animals are inevitably exposed to these chemicals (52, 53, 54, 55, 56, 57, 58). The primary targets of phthalates are testicular Leydig cells (59).

In the present study, we found that MEHP, a biologically active phthalate with endocrine disrupting activity, partly inhibited testosterone action on Insl3 expression in a Leydig cell line (MA-10) and primary cultures of rat Leydig cells. Our data confirm that MA-10 cells are an appropriate model to study phthalate-mediated effects, which is in agreement with a previous study by Dees et al. (60). It is known that in utero exposure to phthalates, including MEHP, causes several detrimental reproductive defects, including undescended testis, hypospadias, and decreased testosterone production, in males in various animal models (reviewed in Ref. 61). They are believed to have adverse effects on human reproductive health as well (reviewed in Ref. 62). Phthalate exposure results in decreased expression of Insl3 and genes involved in testosterone biosynthesis in Leydig cells (reviewed in Refs. 63 and 64). Although phthalates have antiandrogenic effects, they cannot interact with AR (39, 40, 41). Phthalate repressive effects in Leydig cells are believed to be mediated, at least in part, through activation of the peroxisome proliferator-activated receptor (PPAR) (65, 66). In the testis of PPAR{alpha}-deficient mice exposed to phthalates, expression of the peripheral-type benzodiazepine receptor, which is involved in cholesterol transport, was no longer repressed (67), and the overall testis structure was found to be predominantly normal (68). Activated PPAR can repress gene expression in a DNA-binding-independent manner through the recruitment of corepressors or by interfering with other DNA-associated transcription factors (65, 66). Recently phthalates were also reported to act by decreasing recruitment of the transcription factor CCAAT/enhancer-binding protein-β to DNA but having no effect on SF1 (69). Although the exact mechanism remains to be identified, we found that the phthalate MEHP antagonizes AR action through a region of the Insl3 promoter known to bind transcriptional activators, which suggests that disruption of transcription factor interactions might be involved.


   Acknowledgments
 
We thank Dr. Mario Ascoli (University of Iowa, Iowa City, IA) for providing the MA-10 cell line. We are also indebted to Sylvain Gauthier, Shankar Singh, and Robert Sullivan (Centre Hospitalier Universitaire of Québec Research Centre, Quebec City, Canada) for providing hydroxyflutamide and testosterone. We also thank Stéphanie Fiola and Luc Martin for technical assistance.


   Footnotes
 
This work was supported by Canadian Institutes of Health Research (CIHR) Grant MOP-77575 (to J.J.T.). J.J.T. holds a New Investigator Scholarship from the CIHR.

Disclosure Statement: E.L. and J.J.T. have nothing to declare.

First Published Online May 22, 2008

Abbreviations: AR, Androgen receptor; DEHP, di-2-ethylhexyl phthalate; INSL3, insulin-like 3; MEHP, mono-(2-ethylhexyl) phthalate; PPAR, peroxisome proliferator-activated receptor; SF1, steroidogenic factor-1.

Received March 5, 2008.

Accepted for publication May 12, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Adham IM, Burkhardt E, Benahmed M, Engel W 1993 Cloning of a cDNA for a novel insulin-like peptide of the testicular Leydig cells. J Biol Chem 268:26668–26672[Abstract/Free Full Text]
  2. Pusch W, Balvers M, Ivell R 1996 Molecular cloning and expression of the relaxin-like factor from the mouse testis. Endocrinology 137:3009–3013[Abstract]
  3. 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]
  4. 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]
  5. Nef S, Parada LF 1999 Cryptorchidism in mice mutant for Insl3. Nat Genet 22:295–299[CrossRef][Medline]
  6. 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]
  7. 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]
  8. 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]
  9. Kawamura K, Kumagai J, Sudo S, Chun SY, Pisarska M, Morita H, Toppari J, Fu P, Wade JD, Bathgate RA, Hsueh AJ 2004 Paracrine regulation of mammalian oocyte maturation and male germ cell survival. Proc Natl Acad Sci USA 101:7323–7328[Abstract/Free Full Text]
  10. Irving-Rodgers HF, Bathgate RA, Ivell R, Domagalski R, Rodgers RJ 2002 Dynamic changes in the expression of relaxin-like factor (INSL3), cholesterol side-chain cleavage cytochrome p450, and 3β-hydroxysteroid dehydrogenase in bovine ovarian follicles during growth and atresia. Biol Reprod 66:934–943[Abstract/Free Full Text]
  11. Spanel-Borowski K, Schafer I, Zimmermann S, Engel W, Adham IM 2001 Increase in final stages of follicular atresia and premature decay of corpora lutea in Insl3-deficient mice. Mol Reprod Dev 58:281–286[CrossRef][Medline]
  12. 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]
  13. 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]
  14. Sadeghian H, Anand-Ivell R, Balvers M, Relan V, Ivell R 2005 Constitutive regulation of the Insl3 gene in rat Leydig cells. Mol Cell Endocrinol 241:10–20[CrossRef][Medline]
  15. Robert NM, Martin LJ, Tremblay JJ 2006 The orphan nuclear receptor NR4A1 regulates insulin-like 3 gene transcription in Leydig cells. Biol Reprod 74:322–330[Abstract/Free Full Text]
  16. Tremblay JJ, Robert NM 2005 Role of nuclear receptors in INSL3 gene transcription in Leydig cells. Ann NY Acad Sci 1061:183–189[CrossRef][Medline]
  17. Foresta C, Bettella A, Vinanzi C, Dabrilli P, Meriggiola MC, Garolla A, Ferlin A 2004 A novel circulating hormone of testis origin in humans. J Clin Endocrinol Metab 89:5952–5958[Abstract/Free Full Text]
  18. Ferlin A, Garolla A, Rigon F, Rasi CL, Lenzi A, Foresta C 2006 Changes in serum insulin-like factor 3 during normal male puberty. J Clin Endocrinol Metab 91:3426–3431[Abstract/Free Full Text]
  19. Charest NJ, Zhou ZX, Lubahn DB, Olsen KL, Wilson EM, French FS 1991 A frameshift mutation destabilizes androgen receptor messenger RNA in the Tfm mouse. Mol Endocrinol 5:573–581[Abstract/Free Full Text]
  20. He WW, Kumar MV, Tindall DJ 1991 A frame-shift mutation in the androgen receptor gene causes complete androgen insensitivity in the testicular-feminized mouse. Nucleic Acids Res 19:2373–2378[Abstract/Free Full Text]
  21. Yarbrough WG, Quarmby VE, Simental JA, Joseph DR, Sar M, Lubahn DB, Olsen KL, French FS, Wilson EM 1990 A single base mutation in the androgen receptor gene causes androgen insensitivity in the testicular feminized rat. J Biol Chem 265:8893–8900[Abstract/Free Full Text]
  22. O'Shaughnessy PJ, Johnston H, Willerton L, Baker PJ 2002 Failure of normal adult Leydig cell development in androgen-receptor-deficient mice. J Cell Sci 115:3491–3496[Abstract/Free Full Text]
  23. Holdcraft RW, Braun RE 2004 Androgen receptor function is required in Sertoli cells for the terminal differentiation of haploid spermatids. Development 131:459–467[Abstract/Free Full Text]
  24. Eacker SM, Shima JE, Connolly CM, Sharma M, Holdcraft RW, Griswold MD, Braun RE 2007 Transcriptional profiling of androgen receptor (AR) mutants suggests instructive and permissive roles of AR signaling in germ cell development. Mol Endocrinol 21:895–907[Abstract/Free Full Text]
  25. Cattanach BM, Iddon CA, Charlton HM, Chiappa SA, Fink G 1977 Gonadotrophin-releasing hormone deficiency in a mutant mouse with hypogonadism. Nature 269:338–340[CrossRef][Medline]
  26. Mason AJ, Hayflick JS, Zoeller RT, Young III WS, Phillips HS, Nikolics K, Seeburg PH 1986 A deletion truncating the gonadotropin-releasing hormone gene is responsible for hypogonadism in the hpg mouse. Science 234:1366–1371[Abstract/Free Full Text]
  27. 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]
  28. 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]
  29. 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]
  30. Tremblay JJ, Viger RS 1999 Transcription factor GATA-4 enhances Müllerian inhibiting substance gene transcription through a direct interaction with the nuclear receptor SF-1. Mol Endocrinol 13:1388–1401[Abstract/Free Full Text]
  31. Ascoli M 1981 Characterization of several clonal lines of cultured Leydig tumor cells: gonadotropin receptors and steroidogenic responses. Endocrinology 108:88–95[Abstract/Free Full Text]
  32. Khan S, Teerds K, Dorrington J 1992 Growth factor requirements for DNA synthesis by Leydig cells from the immature rat. Biol Reprod 46:335–341[Abstract]
  33. Klinefelter GR, Hall PF, Ewing LL 1987 Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure. Biol Reprod 36:769–783[Abstract]
  34. Guigon CJ, Coudouel N, Mazaud-Guittot S, Forest MG, Magre S 2005 Follicular cells acquire Sertoli cell characteristics after oocyte loss. Endocrinology 146:2992–3004[CrossRef][Medline]
  35. Houk CP, Pearson EJ, Martinelle N, Donahoe PK, Teixeira J 2004 Feedback inhibition of steroidogenic acute regulatory protein expression in vitro and in vivo by androgens. Endocrinology 145:1269–1275[Abstract/Free Full Text]
  36. Comhaire FH, Vermeulen A 1976 Testosterone concentration in the fluids of seminiferous tubules, the interstitium and the rete testis of the rat. J Endocrinol 70:229–235[Abstract/Free Full Text]
  37. Turner TT, Jones CE, Howards SS, Ewing LL, Zegeye B, Gunsalus GL 1984 On the androgen microenvironment of maturing spermatozoa. Endocrinology 115:1925–1932[Abstract/Free Full Text]
  38. Walker WH 2003 Nongenomic actions of androgen in Sertoli cells. Curr Top Dev Biol 56:25–53[Medline]
  39. Borch J, Metzdorff SB, Vinggaard AM, Brokken L, Dalgaard M 2006 Mechanisms underlying the anti-androgenic effects of diethylhexyl phthalate in fetal rat testis. Toxicology 223:144–155[CrossRef][Medline]
  40. Gray Jr LE, Wolf C, Lambright C, Mann P, Price M, Cooper RL, Ostby J 1999 Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol Ind Health 15:94–118[Abstract/Free Full Text]
  41. Parks LG, Ostby JS, Lambright CR, Abbott BD, Klinefelter GR, Barlow NJ, Gray Jr LE 2000 The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the male rat. Toxicol Sci 58:339–349[Abstract/Free Full Text]
  42. Ferlin A, Arredi B, Zuccarello D, Garolla A, Selice R, Foresta C 2006 Paracrine and endocrine roles of insulin-like factor 3. J Endocrinol Invest 29:657–664[Medline]
  43. Bay K, Hartung S, Ivell R, Schumacher M, Jurgensen D, Jorgensen N, Holm M, Skakkebaek NE, Andersson AM 2005 Insulin-like factor 3 serum levels in 135 normal men and 85 men with testicular disorders: relationship to the luteinizing hormone-testosterone axis. J Clin Endocrinol Metab 90:3410–3418[Abstract/Free Full Text]
  44. Ivell R, Hartung S, Anand-Ivell R 2005 Insulin-like factor 3: where are we now? Ann NY Acad Sci 1041:486–496[CrossRef][Medline]
  45. Wikström AM, Bay K, Hero M, Andersson AM, Dunkel L 2006 Serum insulin-like factor 3 levels during puberty in healthy boys and boys with Klinefelter syndrome. J Clin Endocrinol Metab 91:4705–4708[Abstract/Free Full Text]
  46. Gurniak CB, Berg LJ 1996 Murine JAK3 is preferentially expressed in hematopoietic tissues and lymphocyte precursor cells. Blood 87:3151–3160[Abstract/Free Full Text]
  47. Kawamura M, McVicar DW, Johnston JA, Blake TB, Chen YQ, Lal BK, Lloyd AR, Kelvin DJ, Staples JE, Ortaldo JR 1994 Molecular cloning of L-JAK, a Janus family protein-tyrosine kinase expressed in natural killer cells and activated leukocytes. Proc Natl Acad Sci USA 91:6374–6378[Abstract/Free Full Text]
  48. Jeyasuria P, Ikeda Y, Jamin SP, Zhao L, de Rooij DG, Themmen AP, Behringer RR, Parker KL 2004 Cell-specific knockout of steroidogenic factor 1 reveals its essential roles in gonadal function. Mol Endocrinol 18:1610–1619[Abstract/Free Full Text]
  49. De RG, Aniello F, Branno M, Izzo G, Minucci S 2006 The expression level of frog relaxin mRNA (fRLX), in the testis of Rana esculenta, is influenced by testosterone. J Exp Biol 209:3806–3811[Abstract/Free Full Text]
  50. Hammes SR, Levin ER 2007 Extranuclear steroid receptors: nature and actions. Endocr Rev 28:726–741[Abstract/Free Full Text]
  51. Rahman F, Christian HC 2007 Non-classical actions of testosterone: an update. Trends Endocrinol Metab 18:371–378[CrossRef][Medline]
  52. Sathyanarayana S 2008 Phthalates and children’s health. Curr Probl Pediatr Adolesc Health Care 38:34–49[CrossRef][Medline]
  53. Gaudin R, Marsan P, Robert A, Ducos P, Pruvost A, Levi M, Bouscaillou P 2008 Biological monitoring of occupational exposure to di(2-ethylhexyl) phthalate: survey of workers exposed to plastisols. Int Arch Occup Environ Health 81:959–966[CrossRef][Medline]
  54. Frederiksen H, Skakkebaek NE, Andersson AM 2007 Metabolism of phthalates in humans. Mol Nutr Food Res 51:899–911[CrossRef][Medline]
  55. Wittassek M, Wiesmuller GA, Koch HM, Eckard R, Dobler L, Muller J, Angerer J, Schluter C 2007 Internal phthalate exposure over the last two decades—a retrospective human biomonitoring study. Int J Hyg Environ Health 210:319–333[CrossRef][Medline]
  56. Pak VM, McCauley LA 2007 Risks of phthalate exposure among the general population: implications for occupational health nurses. AAOHN J 55:12–17[Medline]
  57. Pan G, Hanaoka T, Yoshimura M, Zhang S, Wang P, Tsukino H, Inoue K, Nakazawa H, Tsugane S, Takahashi K 2006 Decreased serum free testosterone in workers exposed to high levels of di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP): a cross-sectional study in China. Environ Health Perspect 114:1643–1648[Medline]
  58. Hauser R, Calafat AM 2005 Phthalates and human health. Occup Environ Med 62:806–818[Free Full Text]
  59. Ge RS, Chen GR, Tanrikut C, Hardy MP 2007 Phthalate ester toxicity in Leydig cells: developmental timing and dosage considerations. Reprod Toxicol 23:366–373[CrossRef][Medline]
  60. Dees JH, Gazouli M, Papadopoulos V 2001 Effect of mono-ethylhexyl phthalate on MA-10 Leydig tumor cells. Reprod Toxicol 15:171–187[CrossRef][Medline]
  61. Fisher JS 2004 Environmental anti-androgens and male reproductive health: focus on phthalates and testicular dysgenesis syndrome. Reproduction 127:305–315[Abstract/Free Full Text]
  62. Matsumoto M, Hirata-Koizumi M, Ema M 2008 Potential adverse effects of phthalic acid esters on human health: a review of recent studies on reproduction. Regul Toxicol Pharmacol 50:37–49[CrossRef][Medline]
  63. Foster PM 2006 Disruption of reproductive development in male rat offspring following in utero exposure to phthalate esters. Int J Androl 29:140–147[CrossRef][Medline]
  64. David RM 2006 Proposed mode of action for in utero effects of some phthalate esters on the developing male reproductive tract. Toxicol Pathol 34:209–219[Abstract/Free Full Text]
  65. Corton JC, Lapinskas PJ 2005 Peroxisome proliferator-activated receptors: mediators of phthalate ester-induced effects in the male reproductive tract? Toxicol Sci 83:4–17[Abstract/Free Full Text]
  66. Latini G, Scoditti E, Verrotti A, De FC, Massaro M 2008 Peroxisome proliferator-activated receptors as mediators of phthalate-induced effects in the male and female reproductive tract: epidemiological and experimental evidence. PPAR Res 2008:359267[Medline]
  67. Gazouli M, Yao ZX, Boujrad N, Corton JC, Culty M, Papadopoulos V 2002 Effect of peroxisome proliferators on Leydig cell peripheral-type benzodiazepine receptor gene expression, hormone-stimulated cholesterol transport, and steroidogenesis: role of the peroxisome proliferator-activator receptor {alpha}. Endocrinology 143:2571–2583[Abstract/Free Full Text]
  68. Ward JM, Peters JM, Perella CM, Gonzalez FJ 1998 Receptor and nonreceptor-mediated organ-specific toxicity of di(2-ethylhexyl)phthalate (DEHP) in peroxisome proliferator-activated receptor {alpha}-null mice. Toxicol Pathol 26:240–246[Abstract/Free Full Text]
  69. Kuhl AJ, Ross SM, Gaido KW 2007 CCAAT/enhancer binding protein β, but not steroidogenic factor-1, modulates the phthalate-induced dysregulation of rat fetal testicular steroidogenesis. Endocrinology 148:5851–5864[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Toxicol SciHome page
A. Lehraiki, C. Racine, A. Krust, R. Habert, and C. Levacher
Phthalates Impair Germ Cell Number in the Mouse Fetal Testis by an Androgen- and Estrogen-Independent Mechanism
Toxicol. Sci., October 1, 2009; 111(2): 372 - 382.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
R. Anand-Ivell, K. Heng, B. Hafen, B. Setchell, and R. Ivell
Dynamics of INSL3 Peptide Expression in the Rodent Testis
Biol Reprod, September 1, 2009; 81(3): 480 - 487.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
R. Ivell and R. Anand-Ivell
Biology of insulin-like factor 3 in human reproduction
Hum. Reprod. Update, July 1, 2009; 15(4): 463 - 476.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
E. Rijntjes, H. J. M. Swarts, R. Anand-Ivell, and K. J. Teerds
Prenatal induced chronic dietary hypothyroidism delays but does not block adult-type Leydig cell development
Am J Physiol Endocrinol Metab, February 1, 2009; 296(2): E305 - E314.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Laguë, E.
Right arrow Articles by Tremblay, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Laguë, E.
Right arrow Articles by Tremblay, J. J.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*BIS(2-ETHYLHEXYL)PHTHALATE
*TESTOSTERONE

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals