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Molecular Endocrinology, doi:10.1210/me.2006-0113
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Molecular Endocrinology 21 (4): 895-907
Copyright © 2007 by The Endocrine Society

Transcriptional Profiling of Androgen Receptor (AR) Mutants Suggests Instructive and Permissive Roles of AR Signaling in Germ Cell Development

Stephen M. Eacker, James E. Shima, Charles M. Connolly, Manju Sharma, Robert W. Holdcraft, Michael D. Griswold and Robert E. Braun

Department of Genome Sciences (S.M.E., C.M.C., M.S., R.W.H., R.E.B.), School of Medicine, University of Washington, Seattle, Washington 98195-5065; and School of Molecular Biosciences (J.E.S., M.D.G.), Washington State University, Pullman, Washington 99164

Address all correspondence and requests for reprints to: Robert E. Braun, University of Washington School of Medicine, Department of Genome Sciences, Box 355065, 1705 NE Pacific, Foege Building, Room 133C, Seattle, Washington 98195-5065. E-mail: braun{at}u.washington.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 Materials and Methods
 REFERENCES
 
The androgen receptor (AR) is a transcription factor that plays a critical role in male sexual development, spermatogenesis, and maintenance of hormonal homeostasis. Despite the extensive knowledge of the phenotypic consequences of mutations in Ar, very little is known about the transcriptional targets of AR within the testis. To identify potential targets of androgen signaling in the testis, we have analyzed the transcriptional profile of adult testes from Ar hypomorphs alone or in combination with Sertoli cell-specific Ar ablation. Using Affymetrix MOE430A mouse genome arrays we interrogated more than 22,000 transcripts. We found the expression level of 62 transcripts in the Ar mutants differed by greater than 2-fold compared with wild type. We also found that more transcripts were up-regulated than down-regulated, highlighting AR’s role as a transcriptional repressor in the testis. Twelve transcripts were uniquely affected, and 16 transcripts were more severely affected in Sertoli cell-specific Ar ablation compared with hypomorphic Ar mutants. Using a comparative genomic approach, we analyzed the 6 kb around the transcriptional start sites of affected transcripts for conserved AREs (androgen response elements). We identified at least one conserved ARE in 65% of the genes misregulated in our microarray analysis where clear mouse-human orthologs were available. We used a reporter assay in cell culture to functionally verify the AREs for the kallikrein 27 gene. This suggests that the majority of the misregulated transcripts have a high probability of being direct AR targets. The transcripts affected by these Ar mutations encode a diverse array of proteins whose molecular functions support the contention that AR supports spermatogenesis in both a permissive and instructive fashion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 Materials and Methods
 REFERENCES
 
THE ANDROGEN RECEPTOR (AR) is a member of the nuclear hormone receptor family of ligand-dependent transcription factors. AR plays a critical role in the development of the testis and in maintenance of spermatogenesis in adult mammals. During development, testosterone (T) and dihydrotestosterone (DHT) act through AR to promote testicular descent and Leydig cell maturation. Extensive work in the rat has demonstrated that T is the critical hormone that supports spermatogenesis (1). A variety of models have been used to study the effects of T withdrawal on spermatogenesis. Many studies have used hypophysectomy to ablate gonadotropin secretion, and consequently T production (2, 3). Other studies using testosterone-estrogen (TE) pellets and selective destruction of Leydig cells by ethanedimethane sulfonate (EDS) also have been extremely useful in dissecting the steps of spermatogenesis, which require T signaling. These studies have largely concluded that T is essential for maintenance of spermatogenesis, although FSH is required for maximal sperm output (4).

AR is expressed in the somatic cells of the testis: the Sertoli cells, which make intimate contacts with germ cells, Leydig cells located within the interstitium, and peritubular myoid cells, which encompass the seminiferous tubule (5). Extensive data support a role for T in meiotic progression, retention of round spermatids, the transition of round spermatids to elongating spermatids, and the release of mature sperm (6, 7, 8, 9). The role of T in germ cell differentiation is mediated in large part by expression of AR in the Sertoli cell (10, 11, 12). Although these studies have been crucial in the identification of the cell type where AR is required for germ cell maturation, they have not yet defined the mechanism by which AR supports germ cell development. One possibility might be that AR acts in an instructive fashion, affecting the expression of signals that directly support germ cell maturation. Another possibility is that AR is required to create a permissive microenvironment within the seminiferous epithelium that allows for the progression of germ cell development. Identification of AR targets may help distinguish between these two models.

The function of AR in the Leydig cell has been the topic of a number of investigations. Work on the spontaneous Ar mutant Artfm/Y has shown that AR is required for the normal development of adult-type Leydig cells (13). The Artfm/Y mice fail to express key steroidogenic genes such as cytochrome P450 17a1 (Cyp17a1) and hydroxysteroid (17ß) dehydrogenase 3 (Hsd17b3). The absence of these enzymes in Leydig cells prevents high levels of T synthesis. Although a number of genes encoding steroidogenic enzymes are down-regulated in wild-type animals surgically rendered cryptorchid, it is clear that their expression is much lower in Artfm/Y mice. However, it is not clear whether transcription of these steroidogenic genes requires Ar function directly. In fact, a large body of evidence has shown AR can act as a negative regulator of Cyp17a1 in Leydig cells (14, 15). It has been shown that AR binds to the promoter of Cyp17a1 and can repress its expression (16). These seemingly contradictory data can be resolved by the proposed dual role of AR as a positive regulator of Leydig cell development and as a negative regulator of steroidogenesis (13, 17).

We have developed a unique model to investigate AR function in the testis of Mus musculus (10). A hypomorphic conditional allele of Ar was created by placing a neomycin resistance cassette within intron 1 of Ar and flanking exon 1 with inverted loxP sites. A cryptic splice acceptor in the neomycin resistance cassette results in stochastic splicing with exon 1, thereby reducing the amount of full-length Ar mRNA. This hypomorphic allele, termed Arinvflox(ex1-neo), results in a partial failure of terminal spermatid differentiation accompanied by elongating spermatid phagocytosis and a 95% reduction in epididymal sperm numbers. Using Cre recombinase driven by the Sertoli cell-specific Amh promoter [Tg(Amh-Cre)], we ablated Ar from Sertoli cells in an otherwise hypomorphic Ar background. This Sertoli cell-specific ablation results in a more severe phenotype characterized by near-complete failure in elongating spermatid differentiation and severe round spermatid sloughing. In the hope of identifying the AR targets responsible for these phenotypes, we compared the gene expression profiles of adult whole testis mRNA from wild-type, hypomorphic Ar mutants, and hypomorphic Ar mutants with Sertoli cell-specific Ar ablation. Using a computational approach, we determined which of the genes misregulated in Ar mutants contain consensus androgen-responsive elements (AREs) that are conserved between mice and humans, and thus may be direct targets of AR action.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 Materials and Methods
 REFERENCES
 
Identification of Genes Misregulated in Testes of Hypomorphic and Sertoli Cell-Specific Ar Mutants
In an effort to identify AR-regulated genes involved in spermatogenesis, we conducted an expression array analysis of mRNA from testes of 8-wk-old wild-type, Arinvflox(ex1-neo)/Y, and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) mice. The hypomorphic nature of Arinvflox(ex1-neo)/Y leads to a reduction of AR in all cells in the animal including those in the testis (10). Therefore, we expected that analysis of gene expression in Arinvflox(ex1-neo)/Y testis would reveal AR targets in all of the somatic cell types in the testis. We also expected that some gene expression in germ cells may be affected because of changes in paracrine signaling between the soma and germ line. When AR expression is ablated from Sertoli cells by the addition of Tg(Amh-Cre), we expected that some genes that are misregulated in Arinvflox(ex1-neo)/Y would be more severely affected. In many cases, this should reflect changes in gene expression in Sertoli cells.

Transcripts that demonstrated greater than 2-fold change compared with wild-type are listed in Table 1Go. In the testes of Arinvflox(ex1-neo)/Y, 31 transcripts were up-regulated and 15 down-regulated compared with wild type. When Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) testis mRNA was profiled, the levels of 40 transcripts were increased and 17 decreased relative to wild type. As discussed above, the differences between Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) likely reflect changes in Sertoli cell-specific gene expression. In all, nine transcripts were up-regulated and three transcripts down-regulated in Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) but not in Arinvflox(ex1-neo)/Y. Interestingly, there was one transcript uniquely up-regulated and four uniquely down-regulated in Arinvflox(ex1-neo)/Y but not Arinvflox(ex1-neo)/Y; Tg(Amh-Cre).


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  		Table 1.
 
To validate results obtained from our microarray analysis, we performed northern analysis for selected transcripts. Both Arinvflox(ex-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) animals demonstrate elevated serum T (~40x over wild type), LH (~25x over wild type), and FSH (~3x over wild type) (10); these hormones can have significant impacts on transcription in the testis. Therefore, we analyzed transcripts known to be regulated by T (Rhox5, Star), LH (Ren1, Star, Inha), and FSH (Ren1, Klf4, Inha) in addition to transcripts regulated by unknown factors (Adh1, Klk27, Lcn2). Of the transcripts that were misregulated in our microarray analysis, all (7/7) demonstrated similar magnitude of change by Northern blot (Fig. 1Go). With this sample of Northern blots, we validated the results for 11% of the transcripts misregulated in our microarray analysis. As indicated by our analysis, the level of Rhox5 message was reduced in Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre), consistent with a reduction in AR signaling. T is known to be a negative regulator of Star expression, whereas LH is a positive regulator. Accordingly, Star mRNA was also up-regulated in both mutants as predicted by our microarray analysis. The levels of neither Ren1 nor Klf4 appeared to be altered in our microarray analysis. However, despite its absence from the list of misregulated transcripts, Ren1 was up-regulated by Northern blot (Fig. 1Go). The variability observed in the levels of Ren1 message, as seen by Northern, could have resulted in exclusion from the list of 2-fold altered transcripts. In summary, these data provide evidence for changes in LH and AR signaling, but no clear perturbation in FSH signaling. The consistency between the results of the microarray analysis and Northern blot validation give us a high degree of confidence in the results listed in Table 1Go.


Figure 1
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  		Fig. 1. Verification of Microarray Results

Northern analysis of wild-type, Arinvflox(ex1-neo)/Y, Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) whole testis RNA from 8-wk-old animals. Selected transcripts that were up-regulated (Adh1, Inha, Star) and down-regulated (Rhox5, Lcn2, Klk27) were chosen for their known regulation patterns or for scientific interest. Rhox5 and Star are known to be under positive regulation by androgen and LH signaling, respectively. Klf4 is a FSH-regulated transcript and showed no change in expression level. Although not identified on the microarray analysis, Ren1, a FSH- and LH-regulated transcript was up-regulated in both Ar mutants. Actb was used as a loading control. These results are representative of results from Northern analyses of three to six individuals of each genotype.

 
The transcripts misregulated in the testes of Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) encode proteins encompassing a broad range of molecular functions (Table 1Go). These molecular functions were assigned based on their gene ontology annotations provided by The Jackson Laboratory Mouse Genome Informatics (http://www.informatics.jax.org). A graphical representation of the molecular functions of misregulated transcripts with known function clearly demonstrates an overrepresentation of genes involved in metabolic processes (Fig. 2Go). The transcripts encoding metabolic proteins are dominated by genes involved in cholesterol biosynthesis and steroidogenesis. The next most abundant class of protein functions is involved in signal transduction. Most striking among the signal transduction genes is the 14- to 17-fold up-regulation of the LH/chorionic gonadotropin receptor (Lhcgr, Table 1Go). Also of note is the significant up-regulation of inhibin {alpha} (Inha) mRNA. After signal transduction-related transcripts, proteases were most severely affected. Among these transcripts are four mRNAs encoding members of the kallikrein family of proteases. Data from previous studies and the growing information from cell type-specific microarray data (18) have allowed us to assign the cell types in which the misregulated transcripts are normally expressed (Table 1Go).


Figure 2
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  		Fig. 2. Classification of Molecular Functions of Proteins Encoded by Misregulated Transcripts in Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre)

A single molecular function was assigned for each transcript based on its gene ontology annotation provided by the Mouse Genome Informatics web site (http://www.informatics.jax.org). Transcripts associated with metabolic functions in the testis, primarily steroidogenesis and cholesterol biosynthesis, dominate the collection of misregulated transcripts. The next most abundant classification of transcripts is those of unknown function. Addition of the Tg(Amh-Cre) to the hypomorphic Arinvflox(ex1-neo)/Y background results in an increase the proportion of misregulated transcripts involved in cell adhesion, nucleic acid metabolism, and transcription. The transcripts comprising each functional classification are listed in Table 1Go.

 
Identification of Potential AREs
Identifying transcripts altered in our microarray analysis as being androgen regulated must be carefully considered because of the complex endocrine phenotype of Arinvflox(ex1-neo)/Y. High levels of FSH and LH could obscure the relative contribution of the absence of AR to the observed transcriptional phenotype. AR interacts with DNA sequences termed AREs that consist of two 6-bp half-sites separated by a 3-bp spacer. The sequence of these AREs has been experimentally determined in a variety of androgen-responsive promoters, many of which are listed in Table 2Go. The high degree of degeneracy at many of the sites within the 6-bp half-sites has made de novo prediction of AREs in promoter regions a difficult task. Also, studies using in vitro selection for sequences that bind the AR DNA binding domain have identified high-affinity sequences that are found rarely, if at all, in nature (19). To help identify transcripts that were more likely to be androgen-regulated, we examined 6000 bp encompassing the transcriptional start site (TSS) of each gene listed in Table 1Go. Of the 62 genes listed in Table 1Go, 59 genes had good evidence identifying the TSS and were therefore retained for further analysis. Using sequences listed in Table 2Go we generated a weight-matrix model for AREs and used it to identify potential AREs with a log-likelihood ratio (LLR) > 3. A graphical representation of the weight matrix used in this analysis is shown in Fig. 3AGo. The results of this analysis and details of the method are supplied in Supplemental Fig. 1 and Supplemental Table 1 (published as supplemental data on The Endocrine Society’s Journals Online web site http://mend.endojournals.org/). Using these criteria, we identified 3689 potential AREs in the 6 kb encompassing the TSS for 59 genes.


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  		Table 2. Experimentally Validated Androgen Response Elements

 

Figure 3
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  		Fig. 3. Computational Identification of cAREs in the 6 kb Surrounding the Transcriptional Start Site of Misregulated Transcripts

A, A logo representation of the frequencies of bases at each position in experimentally validated AREs used to generate the weight matrix model of AR binding sites. The heights of the letters represent the relative frequency of a nucleotide at each position in the ARE. The logo was generated using the sequences in Table 2Go with WebLogo (http://weblogo.berkeley.edu/logo.cgi). The distribution of sequences conserved between mouse and human in the promoters of genes misregulated in Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) are illustrated in panel B. Details of method used to identify the cAREs are described in Materials and Methods. C, The number of cAREs identified in the promoters of genes analyzed in (B). D, A diagram of the location and orientation of cAREs in misregulated genes validated in Fig. 2Go. Large black boxes indicate exons and a black arrow indicates the transcriptional start site (TSS). The gray arrows indicate the position of cAREs as defined in panel A. Right-pointing arrows indicate the cARE is on the (+) strand, whereas left-pointing arrows indicate the cARE is on the (–) strand.

 
To identify AREs that have a higher probability of being functional, we identified potential AREs conserved over the course of evolution. We examined 6000 bp of sequence encompassing the TSS of the human orthologs of the genes identified in Table 1Go. We used information provided in the "Mammalian Homology" section of Mouse Genome Informatics web site (http://www.informatics.jax.org) to identify the human orthologs of genes affected in our microarray analysis. The total number of genes with clear human orthologs reduced the number of promoter regions to 49 from our original 59 sequences. Using aligned mouse and human sequences, we used MONKEY (20) to identify conserved AREs (cAREs). We identified 108 potential cAREs within the 6 kb surrrounding the TSS (Supplemental Table 2). The distribution of these cAREs relative to TSS are shown in Fig. 3BGo. Most genes had at least one cARE, but 35% had no identifiable cAREs (Fig. 3CGo). Positions of the cAREs in the promoter regions of validated transcripts with clear human orthologs are illustrated in Fig. 3DGo. As a negative control we performed the same computational analysis on two genes that are expressed in the testis but are not under androgen regulation, Protamine 1 (Prm1), and testis nuclear RNA binding protein (Tenr). Neither of these genes contained a conserved ARE.

Expression of Four Members of the Kallikrein Family of Proteases Is Disrupted by Loss of AR
The Kallikrein family of serine proteases reside in a cluster on mouse chromosome 7 (Fig. 4AGo). This family has a dynamic evolutionary history that includes numerous species-specific expansions and contractions (21). Interestingly, two human Kallikrein family members, prostate-specific antigen (PSA) and KLK2, are well studied for their tissue-specific androgen responsiveness. Because androgen responsiveness is a common feature of members of the Kallikrein family, and four Klks were misregulated in our microarray analysis, Klks seem to be a good candidate for direct androgenic regulation. Indeed, the 2-kb encompassing the TSS of the four Klks affected contain numerous potential AREs (Fig. 4BGo). To test whether Klks respond to androgen signaling, we treated MA-10 Leydig tumor cells with DHT. MA-10 cells are an easily manipulated cell culture model of murine Leydig cells. After treatment, total RNA from the cell lines was analyzed by Northern blot (Fig. 4CGo). MA-10 cells alone or with 50 µM 8-bromo-cAMP (8-Br-cAMP) did not exhibit significant levels of Klk expression. However, when treated with 10 µM DHT, MA-10 cells expressed detectable message. Surprisingly, addition of 8-Br-cAMP to DHT-treated cells reduced the level of Klk27 mRNA. Although the probe used in this hybridization is from a Klk27 cDNA, the high degree of sequence identity between Klk-family members makes it impossible to determine which Klk members are being induced by DHT.


Figure 4
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  		Fig. 4. The Genomic Region Containing the Kallikrein Gene Family Cluster

A, Gray gene models represent non-kallikrein transcripts that border the gene cluster. Black and red gene models represent the non-pseudogene members of the kallikrein family. The red gene models indicate transcripts that are down-regulated in the testes of Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) testes. B, An alignment of potential AREs in the 5' end of the closely related Klk genes. Arrowheads indicate the location and orientation of potential AREs relative to the transcripts’ first exon (red box). The AREs in these alignments all have a LLR >7, the median LLR of sequences in Table 2Go. The blue arrowheads indicate the position of ARE sequences as defined in panel A of Fig. 3Go. Right-pointing arrows indicate the predicted ARE is on the (+) strand, whereas left-pointing arrows indicate the ARE is on the (–) strand. C, DHT-induced expression of Klk expression in MA-10 Leydig tumor cells. A northern blot of MA-10 mRNA indicates treatment with DHT up-regulates the expression of Klk27. This blot is representative of three separate experiments. D, A luciferase assay demonstrating the androgen responsiveness of the Klk27 promoter. The androgen-responsive MMTV-luc reporter shows little DHT-dependent activation in MA-10 cells. When MMTV-luc is cotransfected with an Ar expression vector, 10 nM DHT elicits an 8-fold increase in luc activity. In contrast, the small amount of AR present in MA-10 cells is sufficient to elicit a 3.4-fold increase of DHT-dependent luc activity. When the Ar expression construct is cotransfected with Klk27-luc, there is a 44-fold increase in DHT-dependent luc activity. E, Removing sequence containing three of the predicted AREs is sufficient to ablate Klk27-luc’s androgen responsiveness. Constructs labeled A–E were transfected into MA-10 cells and DHT responsiveness measured. The graph indicates the luciferase activity of each construct relative to a ß-gal internal control. All luciferase activity levels in panels D and E represent the mean relative values from three separate experiments and error bars represent SEM.

 
To determine whether the consensus AREs we identified in the promoter region of Klk27 confer androgen responsiveness, we fused 1.3 kb of sequence upstream of the Klk27 TSS to a luciferase reporter (Klk27-luc). When MA-10 Leydig tumor cells transfected with Klk27-luc are stimulated with DHT, we observed a 3.4-fold increase in reporter activation, demonstrating androgen-dependent activation (Fig. 4DGo). MA-10 cells express low levels of AR, as demonstrated by insignificant androgen-dependent activation of a mouse mammary tumor virus-luciferase (MMTV-luc) reporter (Fig. 4DGo) (16). However, upon cotransfection of MMTV-luc with an AR expression construct, we observed an 8-fold increase in luciferase activity. Similarly, when MA-10 cells were cotransfected with Klk-luc and an AR expression vector and treated with DHT we observed a 44-fold activation of the reporter. These data strongly suggest that the Klk27 promoter demonstrates both androgen- and AR-dependent activation.

We identified a number of potential AREs in the 1.3-kb upstream of the Klk27 TSS. To determine the minimal region of the promoter that confers androgen responsiveness, we generated a series of deletion constructs that remove portions of the Klk27-upstream region that contain potential AREs (Fig. 4EGo). We demonstrated that only the proximal 440 bp of the upstream sequence are required for androgen-dependent activation (Fig. 4EGo, construct D) and when the sequence containing the three predicted AREs is removed, the androgen responsiveness of the Klk27 promoter is eliminated (Fig. 4EGo, construct E). This suggests that these computationally predicted AREs are functional in the context of the Klk27 promoter.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
Materials and Methods
 REFERENCES
 
Despite the absolute requirement of androgen signaling for spermatogenesis, very few direct targets of AR in the testis are known. In our current study, we have characterized the transcriptional profile of the testes of adult animals that bear a hypomorphic allele of Ar alone or in combination with Sertoli cell-specific ablation of Ar. We identified a total of 62 transcripts that were altered greater than 2-fold compared with wild-type in the testes of Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) mice. The majority of these transcripts contain evolutionarily conserved androgen response elements, suggesting that many of these transcripts are direct targets of AR. For one of these transcripts, Klk27, we demonstrated that computationally predicted AREs in the promoter region are functional in a Leydig-cell culture model. We have therefore greatly expanded the pool of potential AR targets in the testis, providing novel insight into possible mechanisms by which T promotes spermatogenesis.

These animals provide significant advantages for the identification of targets of AR signaling in the whole testis as well as specifically in Sertoli cells. Unlike Artfm/Y mice, Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) mice have normally descended testes. Cryptorchidism alone has a strong effect on testicular gene expression making the use of Artfm/Y to identify targets of androgen signaling problematic (13). Additionally, unlike Artfm/Y and other Sertoli cell-specific Ar mutant mice, Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) animals have a nearly intact complement of transcriptionally active germ cells. Therefore, changes in gene expression observed in our analysis are not the result of a lack of major classes of germ cells within the testis. The presence of particular classes of germ cells are also required for the production proteins synthesized in Sertoli cells, making the nearly normal complement of germ cells even more important to our analysis (22).

The best-characterized target of AR signaling within the testis is Rhox5 (formerly known as Pem), a homeobox gene located on the X chromosome that is expressed exclusively in Sertoli cells. Our microarray analysis demonstrated the expected down-regulation of Rhox5 (Table 1Go) that was confirmed by Northern blot (Fig. 1Go). It has been shown that Rhox5 is responsive to androgens and that androgen-induced transcription requires two AREs located –233 and –69 relative to the transcriptional start site (23, 24). Male mice bearing a null mutation of Rhox5 are subfertile but do not have severe defects in spermatogenesis (25, 26). Because androgen withdrawal has a far more severe phenotype than mutation of Rhox5, this clearly suggests that Rhox5 is only one of several targets of AR required to support spermatogenesis.

Two recent investigations have used pharmacological treatment coupled with microarray expression analysis to identify transcripts that are regulated by androgens in an in vivo system. To isolate the effects of testosterone alone on testis gene expression, Sadate-Ngatchou and colleagues (28) used the spontaneous Gnrh mutant Gnrhhpg/hpg treated with testosterone propionate (TP). This work on the Gnrhhpg/hpg animal has the significant advantage of no confounding effects of LH and FSH on gene regulation during the treatments with TP. Total testis RNA was collected after short-term treatment with TP and microarray analysis revealed a small number of transcripts (234) that change relative to controls. This is consistent with our observation that a relatively small number of transcripts were significantly altered in both Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) testis. Also consistent with our observations, more transcripts were down-regulated than up-regulated with testosterone treatment. Our data further support the suggestion that AR functions primarily as a mediator of transcriptional repression within the adult testis. A second study to identify AR-regulated transcripts used wild-type prepubertal animals treated with TP (27). The prepubertal animals lack a functional HPG axis, thus removing the confounding effects of FSH and LH. In contrast to our findings and those in TP-treated adult Gnrhhpg/hpg mice, prepubertal animals treated with TP show a roughly equal number of up- and down-regulated transcripts. One intriguing possibility is that the difference in response to androgens in the prepubertal animal reflects a developmental change in the profile of AR coregulators in the testis. Rhox5 is also up-regulated by TP in prepubertal mice, making it the only commonly regulated gene between the TP-treated Gnrhhpg/hpg mice (28), TP-treated prepubertal mice, and the current study.

Two other groups have published studies of adult Sertoli-selective Ar knockouts (11, 12). With the exception of Rhox5, there is no overlap in published gene expression changes associated with these mutations and Arinvflox(ex1-neo)/Y;Tg(Amh-Cre). Certainly, this is partly due to the relatively small changes observed for the transcripts these groups have selected for analysis; we have chosen a 2-fold or greater change cut-off for the transcripts we have analyzed. In a recent study of prepubertal Sertoli cell-specific Ar mutant (SCARKO) mice, the authors identified 12 up-regulated and 28 down-regulated transcripts at a 2-fold or greater cut-off (29). Of these transcripts, only Rhox5 was commonly regulated gene between our study and that of Denolet et al. (29). There are several possible reasons for the lack of common transcriptional changes between the two studies. Prepubertal Sertoli cells are mitotically active, whereas adult Sertoli cells are mitotically quiescent, prepubertal Sertoli cells are not yet surrounded by the full-complement of germ cells, and AR coactivator and corepressor expression may be different between prepubertal and adult Sertoli cells.

Determining which of the misregulated transcripts are regulated directly by AR is a challenging problem. To address this question we chose to take a bioinformatic approach to identify evolutionarily conserved sequences in the promoter regions of the genes, we identified as misregulated in Ar mutants. We identified 108 cAREs in the 6 kb encompassing the transcriptional start site (TSS) of 49 of the misregulated genes. These cAREs show a modest enrichment in the –500- to +500-bp region relative to the TSS (Fig. 3AGo). This is significant because many experimentally validated AREs were found within this region (Table 2Go). Interestingly, there is a spike in the number of cAREs from +2501 to +3000 bp. The significance of these potential regulatory sequences is unclear, although the presence of AREs in this region are not without precedent. As is true for many androgen-responsive genes, most ARE-containing promoters have more than one cARE (Fig. 3BGo). In general, AREs in androgen-responsive promoters function in a cooperative manner where each binding site contributes to a portion of the promoter’s responsiveness. Although the presence of cAREs in the promoters of genes misregulated in Ar mutants is not proof that they are direct targets of AR, the localization of cAREs does provide fertile ground for hypothesis testing.

A potential complication to our analysis is the high level of gonadotropins in Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre). In both Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre), there is an approximately 25x increase in serum LH and an approximately 3x increase in FSH. As discussed previously, we believe that these increases reflect a lack of feedback regulation within the hypothalamic-pituitary-gonadal axis as a result of a reduction in AR signaling. Indeed, many known targets of LH signaling are up-regulated in our analysis of Ar mutants, including transcripts encoding steroidogenesis-related proteins. It is not clear to what extent, however, the high levels of steroidogenic gene expression are due to elevated LH levels or to the reduction of AR signaling. For instance, Star is a well characterized target of LH signaling and is up-regulated in Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre). However, it has been shown that T can suppress LH-induced Star transcription (30). Therefore, both the presence of high level of gonadotropin and the low levels of AR signaling could contribute to the levels of gene expression observed in Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre). In addition to the strikingly high levels of serum LH, we observed a 14- to 17-fold increase in Lhcgr transcript levels. Although we do not know whether this up-regulation of Lhcgr mRNA results in an increase in Lhcgr protein, it does suggest that there is an as-yet-uncharacterized role for AR or LH signaling in the transcription of Lhcgr.

Despite the 3-fold increase in FSH levels, we observed no clear transcriptional effect that could be ascribed to increases in FSH signaling in the testis of Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) animals. To confirm this observation made from the microarray expression profile, we measured levels of Klf4 and Ren1 message, both of which are targets of FSH signaling in vivo (31). We found no significant change in the levels of Klf4. Although not borne out in the microarray analysis, there was modest and variable up-regulation of Ren1 as determined by Northern blot (Fig. 1Go). However, Ren1 can be up-regulated by both LH and FSH signaling (31, 32). Transcription of Inha is also thought to be stimulated by FSH and is indeed up-regulated in both Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) testes. However, our analysis of serum Inhibin B levels thus far has demonstrated no significant difference between Arinvflox(ex1-neo)/Y, Arinvflox(ex1-neo)/Y; Tg(Amh-Cre), and wild-type animals (Eacker, S. M., unpublished observations). Therefore, it seems more likely that the extraordinarily high level of LH is stimulating the transcription of Inha in Leydig cells, which is a well-documented phenomenon (33).

Regardless of being direct or indirect targets of AR, there are a number of interesting misregulated transcripts that may shed light on gonadotropin and steroid action in the testis. The Leydig cell-specific transcript insulin-like 3 (Insl3) was down-regulated nearly 10-fold in Arinvflox(ex1-neo)/Y. Similar observations have been made in an investigation of Leydig cell development in Artfm/Y mice (13). The authors in this study concluded that the misregulation of Insl3 was due a failure in Leydig cell maturation in Artfm/Y. Treatment with GnRH antagonists can also down-regulate Insl3 in the adult male rat, suggesting that its transcription is hormonally regulated (34). Late-stage germ cell apoptosis observed in GnRH antagonist-treated rats is partially relieved when exogenous INSL3 is provided, suggesting that INSL3 can function to support germ cell development. We have also observed late-stage germ cell apoptosis in Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) mice, which could in part be due to the lack of INSL3 production by the Leydig cell (Meng, J., and R. E. Braun, unpublished observations). When hCG is provided to Gnrhhpg/hpg mice or to rats treated with a GnRH antagonist, Insl3 levels return to normal, suggesting either direct effects through LHCGR or secondary effects through androgen production (34, 35). Because LH levels in Arinvflox(ex1-neo)/Y mice are very high, AR signaling is reduced, and Insl3 expression is low, we suggest that Insl3 may be a target of AR. Alternatively, additional factors acquired during Leydig cell maturation may be required for proper hormonally induced Insl3 expression.

As previously noted, a number of steroidogenic genes are severely down-regulated in the Artfm/Y mouse. In particular, Hsd3b6, Hsd17b3, and Cyp17a1 were significantly down-regulated in adult Artfm/Y Leydig cells (17). This trend holds true in Arinvflox(ex1-neo)/Y for Hsd3b6 and Hsd17b3 but not for Cyp17a1. Surprisingly, despite the high levels of LH in Arinvflox(ex1-neo)/Y, Cyp17a1 expression was not significantly affected. Previous work demonstrated that reduction of 17-{alpha} hydroxylase activity associated with CYP17A1 is a significant contributor to the block in steroidogenesis observed in Artfm/Y testis (36, 37). The normal levels of Cyp17a1 in Arinvflox(ex1-neo)/Y may therefore be the reason that Arinvflox(ex1-neo)/Y mice are capable of synthesizing vast amounts of testosterone despite the apparent down-regulation of other steroidogenic enzymes.

The four members of the kallikrein family of serine proteases, Klk9, Klk21, Klk24, and Klk27, are present among the down-regulated transcripts. Within the testis, expression of Klk21, 24, and 27 is limited to the Leydig cells (38, 39, 40). The genes encoding the Kallikreins reside in a large cluster on mouse chromosome 7 (Fig. 4Go) (21). The best-studied member of the kallikrein gene family is the human PSA (41). We have demonstrated that one or more members of the Klk gene family are under T regulation in the Leydig tumor line MA-10. Previous investigations have shown that Klk21 is up-regulated by testosterone in immature Leydig cells in culture (39). Taken together with our data showing a severe down-regulation in the Klk transcripts under conditions of reduced androgen signaling, we propose that the four Klks down-regulated in our array analysis represent good markers for androgen signaling within the Leydig cell. It is also worth noting that the expression of all the Klk transcripts further decreased in the presence of Tg(Amh-Cre). This further reduction may represent Sertoli-Leydig cell interaction, a phenomenon described in a study of another Sertoli cell-specific Ar mutant (42).

Among the most severely affected transcripts identified in our microarray analysis is alcohol dehydrogenase 1 (Adh1), which was up-regulated >13-fold in both Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre). Aside from catalyzing the conversion of ethanol to acetaldehyde for detoxification, ADH1 is the rate-limiting enzyme in the conversion of vitamin A to the potent signaling molecule retinoic acid (RA) (43). Depletion of vitamin A from the diets of mammals has severe consequences for spermatogenesis (44). Also of interest, two candidate retinoid carrier molecules, prostaglandin D2 synthase and lipocalin 2, are among the most severely down-regulated transcripts (45, 46). These changes in gene expression offer the intriguing possibility of cross talk between the androgenic signaling and the RA pathway. Recently, RA signaling has been implicated in promoting meiotic progression in the murine gonad (47, 48). Although no meiotic phenotype has been observed in our Ar mutants, our results suggest that regulation of RA synthesis and transport could be the mechanism by which T promotes meiotic progression in other models of AR signaling.

In both Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) we observed a severe decrease in epididymal sperm. However, we observed sloughing of round spermatids from the seminiferous epithelium and observed round spermatids in the epididymis only in Arinvflox(ex1-neo)Y;Tg(Amh-Cre) males. A number of genes with putative roles in cell adhesion are either exclusively or more severely affected in Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) compared with Arinvflox(ex1-neo)/Y. The transcript encoding the tight junction component Cldn3 is exclusively down-regulated when the Tg(Amh-Cre) is present. CLDN3 is a component of newly forming tight junctions that make up the blood-testis-barrier (BTB) (49). Loss of CLDN3 correlates with a failure of the BTB, which could lead to the disorganization and disruption of germ-Sertoli cell contacts. It is possible that the dysfunction of the cell adhesion in the absence of CLDN3 leads directly to the sloughing of round spermatids. However, the improper establishment of the BTB could also lead to a disruption of a permissive microenvironment necessary for germ cell development.

Together, the alterations in the expression of transcripts identified in this study contribute to the overall phenotype observed in Arinvflox(ex1-neo)/Y andArinvflox(ex1-neo)/Y; Tg(Amh-Cre). Identification of cAREs in the promoters of affected genes will also be useful in the identification of genes under direct AR regulation. The potential targets of AR signaling identified in this analysis suggest that AR could promote germ cell development by providing both instructive signals (i.e. RA signaling) as well as by creating a permissive environment (i.e. maintenance of the BTB). The relative contribution of individual targets of AR identified in this study will provide insight into the mechanisms by which T supports the ongoing developmental process of spermatogenesis.


   Materials and Methods
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 Materials and Methods
REFERENCES
 
Animals
All animals were housed in a specific pathogen-free environment and cared for under Institutional Animal Care and Use Committee guidelines. Generation of the hypomorphic Ar allele Arinvflox(ex1-neo) has been previously described (10). The generation and characterization of Tg(Amh-Cre) mice will be described in a forthcoming publication. Animals bearing Arinvflox(ex1-neo) and/or Tg(Amh-Cre) were identified by PCR from tail DNA using methods described previously (10). Animals were euthanized by CO2 asphyxiation in accordance with Institutional Animal Care and Use Committee guidelines. All animals used in this study were maintained on a 129Sv/JaeSor background.

Preparation of Total Testis mRNA and Microarray Analysis
The testes from wild-type, Arinvflox(ex1-neo)/Y, and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) 8-wk-old adults were dissected for RNA isolation. Total RNA was extracted from whole testis that was homogenized in 1 ml Trizol (Invitrogen, Carlsbad, CA) using a Dounce (Wheaton, Milville, NJ) homogenizer and prepared in accordance with the manufacturer’s protocol. RNA from two animals of each genotype were used in separate array experiments. The quality of the RNA was assessed by gel electrophoresis and measuring the A260:A280 ratio. Biotinylated cRNA was generated from an oligo-deoxythymidine-primed reverse transcription reaction with 10 µg of total RNA using MEGAscript (Ambion, Austin, TX). Labeled cRNA was hybridized to Affymetrix (Santa Clara, CA) MOE430A microarrays following the manufacturer’s protocol. Microarrays were stained and washed using the GeneChip Fluidics Station 400. The arrays were then scanned with a GeneArray Scanner 2500A (Agilent, Palo Alto, CA) and analyzed using Microarray Suite 5.0 (Affymetrix) and Genespring 6.1 (Silicon Genetics, Redwood City, CA). Transcripts demonstrating at least a 2-fold change in expression and a minimal signal strength greater than 50 in any of the comparisons were considered for further analysis. All resulting transcripts demonstrated a statistically significant difference in at least one comparison.

Northern Analysis
For Northern analysis, 15 µg of total RNA was run on a 1.5% agarose formaldehyde gel. The RNA was then transferred overnight in 20x SSC to Genescreen Hybridization Membrane (PerkinElmer, Foster City, CA) and then UV cross-linked. The membranes were hybridized overnight with probes generated using randomly incorporated {alpha}32P-deoxy-ATP using standard methods. Membranes were washed for 30 min in 2x SSC, 0.5% sodium dodecyl sulfate, 0.1% sodium pyrophosphate at 65 C followed by a 30-min wash in 0.5x SET (10 mM Tris, pH 7.5; 5 mM EDTA; 1% sodium dodecyl sulfate), 0.1% sodium pyrophosphate at 42 C.

Computational Prediction of AREs
Conserved AREs were found using MONKEY, a program that uses a sequence alignment, the phylogeny of the sequences, and a weight matrix describing a transcription factor binding site to identify conserved transcription factor binding sites (20). The frequencies of the bases at each position in the ARE were determined from the AREs listed in Table 2Go. Mouse-human alignments were determined using sequences from the November 2003 mouse genome assembly (build 32) and the November 2003 human genome assembly (build 34). Sequences encompassing the 6 kb around transcriptional start sites were obtained using EZ-Retrieve (50). Alignments were performed using ClustalW (51). We considered a conserved site to be significant if the LLR of the ARE was greater than 3 in both mouse and human sequences, and the P value produced by MONKEY was less than 0.01. We chose an LLR cutoff of 3 because the lowest LLR for experimentally validated AREs listed in Table 2Go was 3.81.

MA-10 Leydig Tumor Cell Culture and cAMP/Androgen Responsiveness
An isolate of mouse Leydig tumor line MA-10 was a generous gift of Dr. Mario Ascoli (University of Iowa, Iowa City, IA) (52). The MA-10 cells were cultured in RPMI 1640 supplemented with 25 mM HEPES and 15% normal horse serum (Invitrogen) at 37 C and 5% CO2. To test responsiveness of the cells to DHT and cAMP, 107 cells were transfected with 25 µg of mouse Ar expression vector (a gift of Dr. Donald Tindall, Mayo Clinic, Rochester, MN) using a Gene Pulser electroporator (Bio-Rad, Hercules, CA). The transfected cells were divided four ways and plated on 60-mm tissue culture plates. Cells were allowed to recover for 16 h and then were exposed to normal media or media supplemented with DHT, 8-Br-cAMP, or both. Cells to be exposed to DHT were pretreated with 10 µM DHT (Sigma, St. Louis, MO) for 2 h. After pretreatment, cells were then exposed to 50 µM 8-Br-cAMP for 24 h. Afterward cells were rinsed twice with PBS and total RNA was harvested using Trizol as described above.

Klk27-luc was generated by introducing 1.3 kb of sequence proximal to the Klk27 TSS into pGl3-Basic (Promega, Madison, WI). For luciferase assay, MA-10 cells were transfected at 70% confluence in 24-well plates using Lipofectamine 2000 (Invitrogen). Reporter constructs (200 ng MMTV-luc or Klk27-luc) were cotransfected with pSV-ßgal (20 ng; Promega) alone or in combination with 10 ng of Ar expression vector. Cells were allowed to recover overnight in RPMI1640 supplemented with charcoal-dextran-stripped fetal bovine serum. The next day, cells were treated with 10 nM DHT for 24 h before luciferase assay. All assay were performed in triplicate using Bright-Glo and Beta-Glo systems (Promega).


   ACKNOWLEDGMENTS
 
We thank Drs. Martin Tompa and William Noble for helpful discussion and comments during analysis of cAREs and Derek Pouchnik for microarray hybridization and staining. We also thank Debra Sprague for assistance in preparation of this manuscript and Kelly Tysseling for technical assistance.


   FOOTNOTES
 
This work was funded by the Specialized Cooperative Centers Program in Reproductive Research (U54 HD12629) (to R.E.B.) and the Contraceptive Centers Program (U54 HD42454) (to M.D.G.).

Current address for J.E.S.: Department of Biopharmaceutical Sciences, University of California, San Francisco, San Francisco, California 94143

Current address for R.H.W.: Department of Surgery, College of Medicine (R.H.W.), University of Cincinnati, Cincinnati, Ohio 45267.

The authors have nothing to disclose.

First Published Online January 23, 2007

Abbreviations: AR or Ar, Androgen receptor; ARE, androgen response element; 8-Br-cAMP, 8-bromo-cAMP; BTB, blood-testis-barrier; cAREs, conserved AREs; Cyp17a1, cytochrome P450 17a1; DHT, dihydrotestosterone; EDS, ethanedimethane sulfonate; Hsd17b3, hydroxysteroid 17ß dehydrogenase 3; Lhcgr, LH/chorionic gonadotropin receptor; LLR, log-likelihood ratio; MMTV-luc, mouse mammary tumor virus-luciferase; PSA, prostate-specific antigen; RA, retinoic acid; T, testosterone; TE, testosterone-estrogen; TP, testosterone propionate; TSS, transcriptional start site.

Received for publication March 7, 2006. Accepted for publication January 16, 2007.


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NURSA Molecule Pages Link:

Nuclear Receptors:   AR
Ligands:   Dihydrotestosterone



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