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Departments of Pharmaco-Biology and Cell Biology (V.P., R.S., A.C., M.M., S.A.), University of Calabria, Arcavacata di Rende 87036 (CS), Italy; Prince Henry’s Institute of Medical Research (E.R.S., C.D.C.), Melbourne, Victoria 3168, Australia; and Department of Biochemistry (S.B., C.D., S.C.), University, Unité Pour Recherche Enseignement Supérieur Equipe Associeé 2608, USC Institut National de la Recherche Agronomique, 14032-Caen, France
Address all correspondence and requests for reprints to: Dr. Vincenzo Pezzi, Department of Pharmaco-Biology, University of Calabria, Arcavacata di Rende 87036 (CS), Italy. E-mail: v.pezzi{at}unical.it.
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In the rat testis, there is an age-related pattern of aromatase activity. Activity is present mainly in Sertoli cells of immature animals and in Leydig cells of adults (6). In pig, ram, and human, however, aromatase is mainly present in Leydig cells (7). In germ cells, the amount of P450arom mRNA changes with the stage of maturation: it is twice as high in pachytene spermatocytes (PS) than in round spermatids (RS), and 20-fold higher in RS than in spermatozoa (8). Conversely, aromatase activity appears to be 4- to 5-fold higher in spermatozoa than in either PS or RS (4). Similar patterns of aromatase expression have been reported in mouse Leydig, Sertoli, and germs cells (9). Moreover, the presence of aromatase in Leydig cells of primates and humans is well established (7), and we have recently identified aromatase expression in ejaculated human spermatozoa (10).
Aromatase expression is regulated by tissue-specific promoters (11, 12, 13, 14). A promoter proximal to the translation start site, termed promoter II (PII), regulates the expression of P450arom in ovaries of several species (15, 16), in fetal gonads (13) and in two rat Leydig tumor cells (R2C and H540) (17, 18). Recently we have demonstrated that PII is the principal promoter that is active in rat Sertoli, Leydig, and germ cells (19). This promoter contains several cAMP response element (CRE)-like motifs that mediate the effects of the cAMP transduction pathway that potentiates aromatase expression and activity. Basal and, in part, cAMP-induced transcription of CYP19 also requires the presence of a nuclear receptor half site [nuclear receptor element (NRE)] located at –90 relative to the start of transcription of the rat gene that has been proposed to bind the orphan nuclear receptor steroidogenic factor-1 [SF-1 (nuclear receptor 5A1)] (19, 20). However, the intratesticular sites of expression of SF-1 and aromatase do not correlate; in adult rats, SF-1 is mainly expressed in Sertoli cells (21), whereas aromatase is mainly in Leydig and germ cells. In seeking an alternative factor that might account for aromatase expression in these SF-1 negative sites, we have focused the present study on the liver receptor homolog-1 (LRH-1).
LRH-1 [nuclear receptor 5A2, also known as CYP7A promoter binding factor (CPF), 
-fetoprotein transcription factor (FTF), and hB1f (22, 23, 24, 25)] and SF-1 are the two mammalian homologues of the Drosophila nuclear receptor Fushi tarazu F1 (26) and share common DNA binding and transactivation properties. LRH-1 is expressed at high levels in liver, where it regulates expression of genes involved in cholesterol metabolism and bile acid synthesis including cholesterol 7-hydroxylase (CYP7A) (27, 28), sterol 12-hydroxylase (CYP8B1) (29), and the cholesteryl ester transfer protein (30). Initially, LRH-1 expression was thought to be limited only to the nonsteroidogenic tissues pancreas, intestine, and colon (31). Recently, however, LRH-1 was shown to be expressed in horse and rat ovary (32, 33, 34); whereas, in collaboration with Rainey and co-workers (35), we have quantified mRNA levels of SF-1 and LRH-1 in different human steroidogenic tissues, showing high expression in the human ovary and testis. We also described the effect of LRH-1 on transcription of the genes encoding the enzymes involved in steroidogenesis, suggesting that this transcription factor may play a role not only in bile acid production but also in steroidogenesis (35). Our recent results (36), suggesting that LRH-1 regulates aromatase gene transcription in preadipocytes acting on aromatase PII, led us to hypothesize a role for this factor in regulation of CYP19 gene in the testis. In the current study, we demonstrate expression of LRH-1 in several rat testicular cell types and investigate the role of LRH-1 in regulating aromatase expression in these cells.
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AtaTCA) to produce pII-688mNRE. Both these plasmids were generously provided by Michael McPhaul (University of Texas Southwestern Medical Center, Dallas, TX). For all transfections, empty pGL2-Basic was used as the control vector to measure basal activity. The coding region of mouse LRH-1 (provided by Dr. David Mangelsdorf, UT Southwestern Medical Center) and mouse SF-1 (provided by Dr. William Rainey, UT Southwestern Medical Center) were inserted into pcDNA3.1 zeo (Invitrogen, Carlsbad, CA) eukaryotic expression vector and used for transfections and in vitro transcription/translation reactions.
Cell culture and transfection
Primary cultures of purified adult rat Leydig cells, Sertoli cells, and testicular mixed germ cells (enriched PS or RS, respectively) were established as described previously (37, 38). All animal studies were conducted in accordance with the Guide for Care and Use of Laboratory Animals (National Institutes of Health). TM3 and TM4 cells (immature mouse Leydig and Sertoli cell lines) were cultured in DMEM/Ham’s F12 (Invitrogen, S.R.L., San Giuliano Milanese, Italy) supplemented with 5% NU Serum (Collaborative Biom, Bedford, MA) and antibiotics in a 24-well plate. For transfection experiments, Fugene6 (Roche, Indianapolis, IN) was used, as directed by the manufacturer, to transfect the reporter plasmid and the indicated amounts of expression vectors. pcDNA3.1 Zeo empty vector was used to ensure constant amounts of DNA per well for each transfection. To normalize luciferase activity, cells were cotransfected with 50 ng/well of TK Renilla luciferase plasmid (Promega). Where indicated, cells were treated with forskolin (FSK) (10 µM) for the indicated time, 18–24 h after the beginning of transfection, and then assayed for activity using the Dual Luciferase assay system (Promega).
RNA extraction, cDNA synthesis, and real-time RT-PCR
The RNAgents total RNA isolation system (Promega, Charbonnieres, France) was used to extract total RNA from primary cells. Total cellular RNA was extracted from TM3 and TM4 cells using Total RNA Isolation System kit (Promega). All RNA was treated with DNase (Ambion, Austin, TX), and purity and integrity of the RNA was confirmed spectroscopically and by gel electrophoresis before use. Four micrograms of total RNA were reverse transcribed in a final vol of 100 µl using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) and stored at –20 C. Primers for the amplification were based on published sequences for the rat and mouse LRH-1 and SF-1. The nucleotide sequences of the primers are shown in Table 1
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Ct method, determined as follows: n-fold = 2–(Ctsample – Ctcalibrator), where Ct values of the sample and calibrator are determined by subtracting the average Ct value of the nuclear receptor gene from the average Ct value of the 18S rRNA reference gene. Before using the Ct method for relative quantification, we perform validation experiments to demonstrate that efficiencies of target and reference are approximately equal, following instructions of Applied Biosystems [http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf (page 14)].
Western blot analysis
Nuclear extracts were prepared from cultured cells as previously described (39). Briefly, cells plated into 60-mm2 dishes were scraped into 1.5 ml cold PBS. Cells were pelleted for 10 sec and resuspended in 400 µl cold buffer A (10 mM HEPES-KOH, pH 7.9, at 4 C; 1.5 mM MgCl2; 10 mM KCl; 0.5 mM dithiothreitol; 0.2 mM phenylmethylsulfonylfluoride) by flicking the tube. The cells were allowed to swell on ice for 10 min and then vortexed for 10 sec. Samples were centrifuged for 10 sec, and the supernatant fraction was discarded. The pellet was resuspended in 50 µl cold buffer C (20 mM HEPES-KOH, pH 7.9; 25% glycerol; 1.5 mM MgCl2; 420 mM NaCl; 0.2 mM EDTA; 0.5 mM dithiothreitol; 0.2 mM phenylmethylsulfonylfluoride) and incubated in ice for 20 min for high-salt extraction. Cellular debris was removed by centrifugation for 2 min at 4 C, and the supernatant fraction (containing DNA binding proteins) was stored at –70 C. The yield was determined by the Bradford method. The proteins (50 µg) were separated on sodium dodecyl sulfate-polyacrylamide (12%) gel and then electroblotted onto a nitrocellulose membrane. The blots were incubated overnight at 4 C with FTF-2 antiserum (polyclonal antibody generated against the mouse FTF extra DNA binding domain, provided by Dr. Luc Belanger, Laval University, Quebec Canada) or with anti-CPF antibody against the amino-terminal region of mouse LRH-1, (1:1000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or rabbit antiserum to adrenal 4 binding protein (Ad4BP)/SF-1 [Ad4BP is the bovine homolog of SF-1 (1:1000) kindly provided from Dr. Morohashi (National Institute for Basic Biology, myodaiji-cho, Okazaki, Japan)]. The antigen-antibody complexes were detected by incubation of the membranes at room temperature with antigoat (for CPF) or antirabbit for Ad4BP IgG coupled to peroxidase, developed using the ECL Plus Western blotting detection system (Amersham Biosciences, Cologno Monzese, Italy). In vitro transcribed and translated LRH-1 and SF-1 proteins were synthesized from the expression vectors described above using T7 polymerase in the rabbit reticulocyte lysate system as directed by the manufacturer (Promega). These proteins were used as positive controls in the immunoblot and EMSA experiments. The specificity of each antibody was tested using antisera preabsorbed with excess amount of antigens.
Gel mobility shift assay
Nuclear extracts were prepared from TM3 and TM4 as previously described (39). In vitro transcribed and translated LRH-1 and SF-1 proteins were synthesized using T7 polymerase in the rabbit reticulocyte lysate system as directed by the manufacturer (Promega). The probe was generated by annealing single-stranded oligonucleotides (Sigma Genosys, Cambridge, UK) and labeling with [
32P] ATP and T4 polynucleotide kinase, followed by purification using Sephadex G50 spin columns (Amersham Pharmacia Biotech). The DNA sequences used as probe or as cold competitors are the following (the nucleotide motifs of interest are underlined): 5'-CAG GAC CTG AGT CTC CCA AGG TCA TCC TTG TTT GAC TTG TA-3'. The protein binding reactions were carried out in 20 µl buffer [20 mM HEPES, pH 8; 1 mM EDTA; 50 mM KCl; 10 mM DTT; 10% glycerol; 1 mg/ml BSA] with 50,000 cpm of labeled probe, 20 µg nuclear proteins or 2 µl of transcribed and translated in vitro SF-1 protein or LRH-1 protein, and 5 µg poly (dI-dC) (Roche). The mixtures were incubated at 4 C for 30 min in the presence or absence of unlabeled competitor oligonucleotides or in vitro-translated protein in the presence or absence of rabbit antiserum to Ad4BP/SF-1 or FTF-1 antiserum [rabbit immunized with the peptide CLTSAIQNIHSSASKGL; rat position 142–156 (22), provided by Dr. Luc Belanger, Laval University]. For FTF-1 the reaction mixture was incubated with this antibody at 4 C for 2 h before addition of labeled probe. The entire reaction mixture was electrophoresed through a 6% polyacrylamide gel in 0.25x Tris borate-EDTA for 3 h at 150 V. Gels were dried and subjected to autoradiography at –70 C.
Statistical analysis
Data were analyzed using STATPAC software (Minneapolis, MN).
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). LRH-1 was expressed at appreciable levels in mature Leydig cells as well as in PS and RS (
50% and 75% of the levels in Leydig cells, respectively, but was undetectable in Sertoli cells of any age (Fig. 1A). The relatively lower LRH-1 mRNA levels measured in coculture of Sertoli and germ cells and in ST reflects the diluted quantity of LRH-1 mRNA expressed in germ cells, and confirms that LRH-1 expression is present only in germ cells but not in Sertoli cells (Fig. 1A). In contrast, we observed high levels of SF-1 mRNA in immature and mature Sertoli cells as well as in mature Leydig cells; whereas in germ cells, the SF-1 mRNA was present at negligible levels (Fig. 1B). This is confirmed by the relatively lower SF-1 mRNA levels measured in coculture of Sertoli and germ cells and in ST (Fig. 1B). SF-1 was undetectable in rat liver, confirming the lack of cross-reactivity of the primers used in the assay. The data obtained for the somatic cells was confirmed using RNA isolated from immature mouse Leydig cell line (TM3) and Sertoli cell line (TM4): LRH-1 was undetectable in TM4 cells, where SF-1 expression was high, whereas TM3 showed an opposite pattern with high LRH-1 expression and almost undetectable expression of SF-1 (Fig. 1C). Thus, the two orphan receptors show overlapping, but distinct, patterns of expression within the rat testis: Leydig cells express both SF-1 and LRH-1 mRNA, whereas Sertoli cells and germ cells (PS and RS) exclusively express SF-1, and LRH-1 mRNA, respectively.
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, bands of the expected size (64 kDa and 56 kDa) were observed using TM3 Leydig cell nuclear extract and primary rat Leydig cells (TM3 and L) but not in TM4 Sertoli cells or primary rat Sertoli cells (TM4 and S). Strong expression was observed in rat PS and RS, consistent with the mRNA data above (Fig. 1
). Strong expression was also observed in the positive control tissue, liver. Using in vitro-translated SF-1 and LRH-1 as the source of protein, no staining was observed for SF-1 [SF-1ttp (transcripted translated protein)], indicating an absence of cross-reactivity of the LRH-1 antibody with SF-1, whereas bands of the expected size were observed using in vitro-translated LRH-1 (LRH1ttp). Note that the 64-kDa protein corresponds to full-length LRH-1, whereas the shorter isoform presumably arises through use of an alternative in-frame initiator methionine at position 62. This isoform is 61 amino acids smaller than the full-length product and has a size of 56 kDa. Alternatively, two LRH-1 isoforms with different length of the A/B region (hB1F and hB1F-2) have been identified in human hepatocytes (25), both of which are recognized by the antibody used in this study.
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). SF-1 was detected at similar low levels in TM3 Leydig cells, primary rat Leydig cells, TM4 Sertoli cells (TM4), and primary rat Sertoli cells, but was completely absent from PS, RS, or liver. Again, in vitro-translated proteins confirmed the specificity of the SF-1 antibody (SF-1ttp and LRH1ttp). Densitometric analysis of Western blot data combined from three independent experiments indicated that SF-1 protein is expressed at similar low levels in TM3, TM4, and primary Leydig and Sertoli cells. Further, the relative levels of LRH-1 protein in purified germ cells is approximately 50% of that seen in the positive control tissue liver (Fig. 2C). Therefore, the intratesticular patterns of expression of LRH-1 and SF-1 proteins mirror that of their respective mRNAs, and are distinct: whereas Leydig cells express both SF-1 and LRH-1 protein, Sertoli cells uniquely express SF-1, and germ cells (PS and RS) uniquely express LRH-1. Moreover, LRH-1 protein is present in these testicular cell types at significant levels.
Regulation of CYP19 PII by LRH-1
Aromatase activity and CYP19 mRNA expression in testis are strongly induced by LH in Leydig cells or by FSH in Sertoli cells, both of which act through the cAMP pathway to induce transcription from PII. Basal and, in part, cAMP-induced transcription of CYP19 has been shown to require SF-1 (40), which, in rat, binds to a nuclear receptor half site (NRE) located at –90 relative to the start of transcription. Because LRH-1 recognizes the same binding site as SF-1, we next assessed the potential of LRH-1 to induce transcription from PII.
TM3 and TM4 cells were cotransfected with the rat CYP19 PII reporter construct and increasing concentrations of either LRH-1 or SF-1 expression vectors. Cells were then incubated in the presence or absence of the adenylyl cyclase activator FSK for 24 h. Transfected into TM3 cells, in the absence of stimulation, LRH-1 dose-dependently increased PII activity, reaching a maximum of 8-fold over basal at 0.05 µg plasmid (Fig. 3A). Treatment with FSK increased basal promoter activity 3-fold (over basal level at 0.05 µg); however, in the presence of this agent, LRH-1 strongly induced PII activity, reaching a maximum of 20-fold at only 0.01 µg LRH-1 (Fig. 3A). Transcription was inhibited at higher concentrations of LRH-1. Analogous transfection experiments using SF-1 instead of LRH-1 revealed a similar pattern, although the maximum levels of induction were lower (8-fold in the presence of FSK and 5 ng SF-1 plasmid) (Fig. 3C). Interestingly, in TM4 cells in basal conditions, neither LRH-1 nor SF-1 increased luciferase activity (Fig. 3, B and D). However, once activated by FSK, both LRH-1 and SF-1 further increased luciferase activity, reaching a maximum of 9-fold at 0.05 µg LRH-1 and 13-fold at 0.05 µg SF-1. Treatment with PKC activators alone or in combination with FSK was ineffective (data not shown).
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). TM3 (Fig. 4B) and TM4 (Fig. 4C) cells were transfected with LRH-1 and either a wild-type PII reporter construct (PII-688) or a promoter construct harboring a mutation in the NRE (AGGTCAAtaTCA pII-688m, see Fig. 4A). Under control conditions, 0.05 µg/well of LRH-1 plasmid increased PII activity 8-fold in TM3 cells, and this stimulation was completely abolished when the NRE was mutated (Fig. 4B). FSK treatment increased the activity of pII-688 approximately 3-fold, and this induction was augmented by a further 5-fold by LRH-1 cotransfection (Fig 4B). Mutation of NRE did not significantly affect the ability of the promoter to respond to FSK; however, LRH-1 did not increase activity of pII-688m (Fig. 4B). Similar results were obtained performing the same experiments in TM4 cells (Fig. 4C); although, as before, LRH-1 did not increase transcription in the absence of FSK. These data suggest that the NRE is required for induction of PII by LRH-1 acting as a basal transcription factor, whereas cAMP-dependent transcription occurs through other mechanisms.
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, lane 1). Formation of this complex was abolished by the addition of a 100-fold molar excess of nonradiolabeled wild-type probe (lane 2), but not by 100-fold molar excess of nonradiolabeled mutated probe (lane 3), confirming sequence-specific DNA binding. Similar binding profiles were observed using TM3 nuclear extracts (lanes 4–6), in vitro synthesized LRH-1 (lanes 7–9), and SF-1 (lanes 10–12). Because the use of a 6% polyacrylamide gel in EMSA does not permit discrimination of the different sizes of LRH-1 and SF-1 protein, as does Western analysis using a 12% sodium dodecyl sulfate-polyacrylamide gel, we confirmed the presence and identity of the two nuclear receptors using specific antibodies (Fig. 6). As before, a distinct band was observed using either TM3 or TM4 nuclear extracts, in vitro translated LRH-1 or SF-1 (lanes 1–4). This band was recognized and supershifted by a specific LRH-1 antibody in TM3, but not TM4, nuclear extracts (lanes 5 and 6) or in vitro-translated LRH-1 (lane 7). In contrast, a specific SF-1 antibody recognized the DNA/protein complex in both TM3 and TM4 nuclear extracts (lanes 8 and 9), as well as using in vitro-translated SF-1 (lane 10). Nonspecific IgG had no effect (lanes 11 and 12). These data confirm the mRNA and protein data above and further support the conclusion that TM3 Leydig cells express both active LRH-1 and SF-1, whereas TM4 Sertoli cells express only SF-1. Finally, we confirmed the cell line EMSA experiments using, as the source of protein, nuclear extracts from primary cultures of rat Leydig and Sertoli cells (Fig. 7). Primary Leydig cell nuclear extracts produced a protein/DNA complex of mobility similar to that formed with TM3 cell extracts (lanes 1 and 2) or in vitro-translated LRH-1 (lane 5), whereas primary Sertoli cell nuclear extracts produced a complex of mobility similar to that formed with TM4 extracts (lanes 3 and 4). The LRH-1 antibody supershifted the binding activity from both TM3 and primary rat Leydig cell extracts (lanes 6 and 7) but not from TM4 or primary rat Sertoli cells (lanes 8 and 9). Thus, LRH-1 DNA binding activity is present in rat Leydig, but not Sertoli, cells.
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and ERß are found in the testis at all ages, suggesting a role for estrogen in testicular development and function. Targeted disruption of the genes encoding the ER or aromatase indicates that estrogen is essential for normal male fertility (41, 42). On the other hand, estrogen excess stimulates Leydig cell hyperplasia in rodents and has been associated with cryptorchidism, testicular cancer, and impaired spermatogenesis (43). The molecular mechanisms controlling aromatase expression and the estrogen production in the different testicular cellular compartment are poorly understood. For this reason, it is particularly important to analyze this process. Previous studies have implicated SF-1 in regulating steroidogenic gene expression in Leydig cells (17, 18). Although aromatase is expressed in Leydig cells, it is also expressed in germ cells. Because germ cells do not express SF-1 (21), we hypothesized that other factors might regulate aromatase expression in the testis. Because LRH-1 is the closest relative of SF-1 and recognizes the same DNA response element, we have focused the current study on this protein. The first aim of this study was to investigate the cellular localization of LRH-1 in the testis. We show that LRH-1 mRNA and protein are expressed in Leydig and germ cells, but not in Sertoli cells, which, in contrast, express high levels of SF-1. Moreover, SF-1 is almost undetectable in germ cells. Quantitative real-time PCR revealed that LRH-1 mRNA is expressed approximately 5-fold higher than SF-1 in Leydig cells. Because LRH-1 can regulate many of the known SF-1 target genes in Leydig cells, including CYP11A, CYP17, 3ßHSD, and StAR (35), we suggest that LRH-1 may regulate these and other genes in vivo. This is significant because SF-1 and LRH-1 are differentially regulated by other nuclear receptors such as dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1) and short heterodimer partner (SHP) (unpublished observations). Our findings are consistent with immunostaining data reported previously by Morohashi and co-workers (21) showing a higher expression of SF-1 in Sertoli cells compared with Leydig cells in the prepubertal rat, decreased expression of SF-1 in Sertoli cells of mature animals, and constant expression in Leydig cells, with no detectable SF-1 expression in germ cells. Our data therefore indicate that LRH-1 could play a role in the regulation of estrogen-dependent testicular development and function, especially in those cellular types where SF-1 is expressed at very low concentration or not expressed (e.g. germ cells).
At least two steps of spermatogenesis are, in part, regulated by estrogen: germ cell number and spermatid maturation (4). However, the observations that mouse germ cells contain only ERß but not ER, and that ERß knockout mice are fertile (44), raise questions as to the role of ERs in germ cells and point to a potential action of estrogen in germ cells via nonclassic receptors activating nongenomic pathways. In this scenario, the observation that male mice deficient in aromatase by the age of 1 yr develop abnormal spermatogenesis with a blockage of germ cell maturation at the RS stage (42) suggests that the regulation of expression and activity of aromatase is probably the most important step determining the role of estrogen in spermatogenesis. Interestingly, the expression of LRH-1 (but not SF-1) in PS and RS leads us to hypothesize that this factor could regulate key genes involved in spermatogenesis including aromatase, and opens a new field that will require further investigation.
The current observation that SF-1, but not LRH-1, is expressed in Sertoli cells is consistent with our previous study (20), showing that SF-1 plays a pivotal role in the regulation of aromatase expression in Sertoli cells. In these cells, aromatase activity is highest in prepubertal rats, declines as Sertoli cells mature, and is hormonally regulated, principally by FSH (45). It has been proposed that estrogen may participate in the FSH-mediated mitogenic activity on Sertoli cells via induction of TGF-ß (46, 47). However, several observations support the indication that estrogen also has negative effects on Sertoli cell differentiation and development (3). In fact, toward the end of the period of Sertoli cell proliferation, FSH-induced aromatase activity begins to decline. Thyroid hormone, which stimulates Sertoli cell differentiation (47), decreases aromatase activity (48, 49). We have recently demonstrated that the molecular mechanisms by which this inhibition occurs is through a competition of thyroid receptor ß and SF-1 for the same regulatory site NRE (20), demonstrating the crucial role of this site in regulating aromatase expression.
Our transfection studies clearly demonstrate that, in TM3 Leydig cells, very low concentrations of LRH-1 induce basal aromatase promoter activity, as well as potentiate cAMP-induced transcription. By contrast, neither LRH-1 nor SF-1 modulates basal aromatase transcription in TM4 Sertoli cells, but both potentiate cAMP-induced transcription. These differences possibly reflect differences in coregulator expression between Leydig and Sertoli cells. The two members of the NR0B family of nuclear receptors, SHP and DAX-1, inhibit transcriptional activity of LRH-1 and SF-1, respectively. It is therefore possible that Sertoli cells express higher levels of either or both of these receptors than Leydig cells, necessitating higher concentrations of cotransfected LRH-1 or SF-1 to overcome a tonic inhibitory action. This hypothesis has yet to be tested. Few other coregulators for LRH-1 have been identified; recently however, multiprotein-bridging factor-1 (MBF-1) has been shown to coactivate LRH-1, as well as LXR and PPAR (50). Of the tissues examined, the site of highest expression of MBF-1 was the testis (50). MBF-1 also coactivates members of the CRE-binding protein/ATF-1 family of transcription factors (51). Because aromatase PII is regulated by cAMP through at least two CRE-like sequences that bind CRE-binding protein/ATF-1, it follows that MBF-1 may facilitate the synergistic activation of PII by LRH-1 and cAMP in the testis.
Our data support previous observations highlighting the importance of nuclear receptors in regulating aromatase gene expression in testis. The finding that, in DAX-1 knockout mice, aromatase is overexpressed selectively in Leydig cells (52) underscores the importance of this type of transcription factor in local testicular estrogen production in vivo. Although this Leydig cell phenotype of DAX-1 –/– mice might seem to argue against a role for LRH-1 in Leydig cell aromatase expression in vivo (because DAX-1 antagonizes SF-1 action), DAX-1 has recently been shown to inhibit LRH-1 transcriptional activity as well as SF-1 (53). Indeed, we have found that DAX-1 is a very potent inhibitor of LRH-1-induced aromatase PII activity in Leydig cells (data not shown). This, together with the fact that LRH-1 is expressed at higher levels than SF-1 in Leydig cells, raises the possibility that the overexpression of aromatase in Leydig cells of DAX-1 –/– mice arises through lack of repression of LRH-1, rather than SF-1.
Given that the DNA binding domains of SF-1 and LRH-1 are highly conserved, and both proteins recognize the same DNA sequence, the question arises as to the relative importance of each protein in testicular function. Both proteins are expressed in Leydig cells and therefore potentially share target genes; however, Sertoli cells and germ cells exclusively express SF-1 and LRH-1 respectively, suggesting cell-specific functions for each transcription factor. The critical role of SF-1 in testis development is clear from the dramatic phenotype of testicular agenesis seen in SF-1 –/– mice (54). Knockout of LRH-1, however, is embryonic-lethal at an early stage (D. Russell, personal communication). Identification of the physiological roles of LRH-1 in the testis will therefore await the development of tissue-specific or conditional transgenic animals.
In conclusion, we have provided evidence that LRH-1 regulates aromatase expression in Leydig cells. Because the regulation of aromatase expression in testis has been investigated mainly in relation to SF-1 and DAX-1, future studies focusing on LRH-1 in addition to SF-1 would enhance our understanding of the mechanisms regulating the age-specific expression of aromatase in the testis. The roles of LRH-1 in testis maturation and testicular carcinogenesis also warrant further study.
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Acknowledgments |
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Footnotes |
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Abbreviations: AD4BP, Adrenal 4 binding protein; CPF, CYP7A promoter binding factor; CRE, cAMP response element; Ct, threshold cycle; DAX-1, dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1; FSK, forskolin; FTF, fetoprotein transcription factor; LRH-1, liver receptor homolog-1; MBF-1, multiprotein-bridging factor-1; NRE, nuclear receptor element; PII, promoter II; PS, pachytene spermatocytes; RS, round spermatids; SF-1, steroidogenic factor 1; SHP, short heterodimer partner; ST, seminiferous tubules; ttp, transcripted translated protein.
Received October 10, 2003.
Accepted for publication January 6, 2004.
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1-fetoprotein locus is activated by a nuclear receptor of the Drosophila FTZ-F1 family. Mol Cell Biol 16:3853–3865[Abstract]
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C. Stocco, J. Kwintkiewicz, and Z. Cai Identification of regulatory elements in the Cyp19 proximal promoter in rat luteal cells J. Mol. Endocrinol., October 1, 2007; 39(4): 211 - 221. [Abstract] [Full Text] [PDF]
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 R. Sirianni, A. Chimento, R. Malivindi, I. Mazzitelli, S. Ando, and V. Pezzi Insulin-Like Growth Factor-I, Regulating Aromatase Expression through Steroidogenic Factor 1, Supports Estrogen-Dependent Tumor Leydig Cell Proliferation Cancer Res., September 1, 2007; 67(17): 8368 - 8377. [Abstract] [Full Text] [PDF]
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D. Silandre, C. Delalande, P. Durand, and S. Carreau Three promoters PII, PI.f, and PI.tr direct the expression of aromatase (cyp19) gene in male rat germ cells J. Mol. Endocrinol., August 1, 2007; 39(2): 169 - 181. [Abstract] [Full Text] [PDF]
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 C. Labelle-Dumais, J.-F. Pare, L. Belanger, R. Farookhi, and D. Dufort Impaired Progesterone Production in Nr5a2+/ Mice Leads to a Reduction in Female Reproductive Function Biol Reprod, August 1, 2007; 77(2): 217 - 225. [Abstract] [Full Text] [PDF]
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D.-S. Wang, T. Kobayashi, L.-Y. Zhou, B. Paul-Prasanth, S. Ijiri, F. Sakai, K. Okubo, K.-i. Morohashi, and Y. Nagahama Foxl2 Up-Regulates Aromatase Gene Transcription in a Female-Specific Manner by Binding to the Promoter as Well as Interacting with Ad4 Binding Protein/Steroidogenic Factor 1 Mol. Endocrinol., March 1, 2007; 21(3): 712 - 725. [Abstract] [Full Text] [PDF]
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J. Guo, S.-X. Tao, M. Chen, Y.-Q. Shi, Z.-Q. Zhang, Y.-C. Li, X.-S. Zhang, Z.-Y. Hu, and Y.-X. Liu Heat Treatment Induces Liver Receptor Homolog-1 Expression in Monkey and Rat Sertoli Cells Endocrinology, March 1, 2007; 148(3): 1255 - 1265. [Abstract] [Full Text] [PDF]
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W. Zheng, J. Yang, Q. Jiang, Z. He, and L. M Halvorson Liver receptor homologue-1 regulates gonadotrope function J. Mol. Endocrinol., February 1, 2007; 38(2): 207 - 219. [Abstract] [Full Text] [PDF]
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 D. H. Volle, R. Duggavathi, B. C. Magnier, S. M. Houten, C. L. Cummins, J.-M. A. Lobaccaro, G. Verhoeven, K. Schoonjans, and J. Auwerx The small heterodimer partner is a gonadal gatekeeper of sexual maturation in male mice Genes & Dev., February 1, 2007; 21(3): 303 - 315. [Abstract] [Full Text] [PDF]
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G. Galmiche, N. Richard, S. Corvaisier, and M.-L. Kottler The Expression of Aromatase in Gonadotropes Is Regulated by Estradiol and Gonadotropin-Releasing Hormone in a Manner that Differs from the Regulation of Luteinizing Hormone Endocrinology, September 1, 2006; 147(9): 4234 - 4244. [Abstract] [Full Text] [PDF]
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R. Safi, A. Kovacic, S. Gaillard, Y. Murata, E. R. Simpson, D. P. McDonnell, and C. D. Clyne Coactivation of Liver Receptor Homologue-1 by Peroxisome Proliferator-Activated Receptor {gamma} Coactivator-1{alpha} on Aromatase Promoter II and Its Inhibition by Activated Retinoid X Receptor Suggest a Novel Target for Breast-Specific Antiestrogen Therapy Cancer Res., December 15, 2005; 65(24): 11762 - 11770. [Abstract] [Full Text] [PDF]
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M. F. Bouchard, H. Taniguchi, and R. S. Viger Protein Kinase A-Dependent Synergism between GATA Factors and the Nuclear Receptor, Liver Receptor Homolog-1, Regulates Human Aromatase (CYP19) PII Promoter Activity in Breast Cancer Cells Endocrinology, November 1, 2005; 146(11): 4905 - 4916. [Abstract] [Full Text] [PDF]
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L. J. Martin, H. Taniguchi, N. M. Robert, J. Simard, J. J. Tremblay, and R. S. Viger GATA Factors and the Nuclear Receptors, Steroidogenic Factor 1/Liver Receptor Homolog 1, Are Key Mutual Partners in the Regulation of the Human 3{beta}-Hydroxysteroid Dehydrogenase Type 2 Promoter Mol. Endocrinol., September 1, 2005; 19(9): 2358 - 2370. [Abstract] [Full Text] [PDF]
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 F.-Q. Yu, C.-S. Han, W. Yang, X. Jin, Z.-Y. Hu, and Y.-X. Liu Activation of the p38 MAPK pathway by follicle-stimulating hormone regulates steroidogenesis in granulosa cells differentially J. Endocrinol., July 1, 2005; 186(1): 85 - 96. [Abstract] [Full Text] [PDF]
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 W. Wang, C. Zhang, A. Marimuthu, H. I. Krupka, M. Tabrizizad, R. Shelloe, U. Mehra, K. Eng, H. Nguyen, C. Settachatgul, et al. The crystal structures of human steroidogenic factor-1 and liver receptor homologue-1 PNAS, May 24, 2005; 102(21): 7505 - 7510. [Abstract] [Full Text] [PDF]
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L. J. Martin and J. J. Tremblay The Human 3{beta}-Hydroxysteroid Dehydrogenase/{Delta}5-{Delta}4 Isomerase Type 2 Promoter Is a Novel Target for the Immediate Early Orphan Nuclear Receptor Nur77 in Steroidogenic Cells Endocrinology, February 1, 2005; 146(2): 861 - 869. [Abstract] [Full Text] [PDF]
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C. Stocco In Vivo and in Vitro Inhibition of cyp19 Gene Expression by Prostaglandin F2{alpha} in Murine Luteal Cells: Implication of GATA-4 Endocrinology, November 1, 2004; 145(11): 4957 - 4966. [Abstract] [Full Text] [PDF]
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