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00/0 Molecular Endocrinology 21(4):895–907

Printed in U.S.A. Copyright © 2007 by The Endocrine Society
doi: 10.1210/me.2006-0113

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,

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University of Washington, Seattle, Washington 98195-5065; and School of Molecular Biosciences
(J.E.S., M.D.G.), Washington State University, Pullman, Washington 99164

The androgen receptor (AR) is a transcription fac- severely affected in Sertoli cell-specific Ar ablation
tor that plays a critical role in male sexual devel- compared with hypomorphic Ar mutants. Using a
opment, spermatogenesis, and maintenance of comparative genomic approach, we analyzed the 6
hormonal homeostasis. Despite the extensive kb around the transcriptional start sites of affected
knowledge of the phenotypic consequences of transcripts for conserved AREs (androgen re-
mutations in Ar, very little is known about the tran- sponse elements). We identified at least one con-
scriptional targets of AR within the testis. To iden- served ARE in 65% of the genes misregulated in
tify potential targets of androgen signaling in the our microarray analysis where clear mouse-human
testis, we have analyzed the transcriptional profile orthologs were available. We used a reporter assay
of adult testes from Ar hypomorphs alone or in in cell culture to functionally verify the AREs for the
combination with Sertoli cell-specific Ar ablation. kallikrein 27 gene. This suggests that the majority
Using Affymetrix MOE430A mouse genome arrays of the misregulated transcripts have a high proba-
we interrogated more than 22,000 transcripts. We bility of being direct AR targets. The transcripts
found the expression level of 62 transcripts in the affected by these Ar mutations encode a diverse
Ar mutants differed by greater than 2-fold com- array of proteins whose molecular functions sup-
pared with wild type. We also found that more port the contention that AR supports spermato-
transcripts were up-regulated than down-regu- genesis in both a permissive and instructive
lated, highlighting AR’s role as a transcriptional fashion. (Molecular Endocrinology 21: 895–907,
repressor in the testis. Twelve transcripts were 2007)
uniquely affected, and 16 transcripts were more

T HE ANDROGEN RECEPTOR (AR) is a member of

the nuclear hormone receptor family of ligand-
dependent transcription factors. AR plays a critical
have been used to study the effects of T withdrawal on
spermatogenesis. Many studies have used hypophy-
sectomy to ablate gonadotropin secretion, and con-
role in the development of the testis and in mainte- sequently T production (2, 3). Other studies using
nance of spermatogenesis in adult mammals. During testosterone-estrogen (TE) pellets and selective de-
development, testosterone (T) and dihydrotestoster- struction of Leydig cells by ethanedimethane sulfonate
one (DHT) act through AR to promote testicular de- (EDS) also have been extremely useful in dissecting
scent and Leydig cell maturation. Extensive work in the steps of spermatogenesis, which require T signal-
the rat has demonstrated that T is the critical hormone ing. These studies have largely concluded that T is
essential for maintenance of spermatogenesis, al-
that supports spermatogenesis (1). A variety of models
though FSH is required for maximal sperm output (4).
First Published Online January 23, 2007 AR is expressed in the somatic cells of the testis: the
Abbreviations: AR or Ar, Androgen receptor; ARE, andro- Sertoli cells, which make intimate contacts with germ
gen response element; 8-Br-cAMP, 8-bromo-cAMP; BTB, cells, Leydig cells located within the interstitium, and
blood-testis-barrier; cAREs, conserved AREs; Cyp17a1, cy- peritubular myoid cells, which encompass the semi-
tochrome P450 17a1; DHT, dihydrotestosterone; EDS,
ethanedimethane sulfonate; Hsd17b3, hydroxysteroid 17␤
niferous tubule (5). Extensive data support a role for T
dehydrogenase 3; Lhcgr, LH/chorionic gonadotropin recep- in meiotic progression, retention of round spermatids,
tor; LLR, log-likelihood ratio; MMTV-luc, mouse mammary the transition of round spermatids to elongating sper-
tumor virus-luciferase; PSA, prostate-specific antigen; RA, matids, and the release of mature sperm (6–9). The role
retinoic acid; T, testosterone; TE, testosterone-estrogen; TP, of T in germ cell differentiation is mediated in large part
testosterone propionate; TSS, transcriptional start site.
by expression of AR in the Sertoli cell (10–12). Although
Molecular Endocrinology is published monthly by The
Endocrine Society (http://www.endo-society.org), the
these studies have been crucial in the identification of the
foremost professional society serving the endocrine cell type where AR is required for germ cell maturation,
community. they have not yet defined the mechanism by which AR

895
896 Mol Endocrinol, April 2007, 21(4):895–907 Eacker et al. • Testicular Gene Expression in Ar Mutants

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supports germ cell development. One possibility might ate a permissive microenvironment within the seminifer-
be that AR acts in an instructive fashion, affecting the ous epithelium that allows for the progression of germ
expression of signals that directly support germ cell mat- cell development. Identification of AR targets may help
uration. Another possibility is that AR is required to cre- distinguish between these two models.
Eacker et al. • Testicular Gene Expression in Ar Mutants Mol Endocrinol, April 2007, 21(4):895–907 897

The function of AR in the Leydig cell has been the analysis of mRNA from testes of 8-wk-old wild-type,
topic of a number of investigations. Work on the spon- Arinvflox(ex1-neo)/Y, and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre)
taneous Ar mutant Artfm/Y has shown that AR is re- mice. The hypomorphic nature of Arinvflox(ex1-neo)/Y leads
quired for the normal development of adult-type Ley- to a reduction of AR in all cells in the animal including
dig cells (13). The Artfm/Y mice fail to express key those in the testis (10). Therefore, we expected that anal-
steroidogenic genes such as cytochrome P450 17a1 ysis of gene expression in Arinvflox(ex1-neo)/Y testis would
(Cyp17a1) and hydroxysteroid (17␤) dehydrogenase 3 reveal AR targets in all of the somatic cell types in the
(Hsd17b3). The absence of these enzymes in Leydig testis. We also expected that some gene expression in
cells prevents high levels of T synthesis. Although a germ cells may be affected because of changes in
number of genes encoding steroidogenic enzymes are paracrine signaling between the soma and germ line.
down-regulated in wild-type animals surgically ren- When AR expression is ablated from Sertoli cells by

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dered cryptorchid, it is clear that their expression is the addition of Tg(Amh-Cre), we expected that some
much lower in Artfm/Y mice. However, it is not clear genes that are misregulated in Arinvflox(ex1-neo)/Y would
whether transcription of these steroidogenic genes be more severely affected. In many cases, this should
requires Ar function directly. In fact, a large body of reflect changes in gene expression in Sertoli cells.
evidence has shown AR can act as a negative regu- Transcripts that demonstrated greater than 2-fold
lator of Cyp17a1 in Leydig cells (14, 15). It has been change compared with wild-type are listed in Table 1.
shown that AR binds to the promoter of Cyp17a1 and In the testes of Arinvflox(ex1-neo)/Y, 31 transcripts were
can repress its expression (16). These seemingly contra- up-regulated and 15 down-regulated compared with
dictory data can be resolved by the proposed dual role of wild type. When Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) testis
AR as a positive regulator of Leydig cell development mRNA was profiled, the levels of 40 transcripts were in-
and as a negative regulator of steroidogenesis (13, 17). creased and 17 decreased relative to wild type. As dis-
We have developed a unique model to investigate cussed above, the differences between Arinvflox(ex1-neo)/Y
AR function in the testis of Mus musculus (10). A and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) likely reflect changes
hypomorphic conditional allele of Ar was created by in Sertoli cell-specific gene expression. In all, nine tran-
placing a neomycin resistance cassette within intron 1 scripts were up-regulated and three transcripts down-
of Ar and flanking exon 1 with inverted loxP sites. A regulated in Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) but not in
cryptic splice acceptor in the neomycin resistance Arinvflox(ex1-neo)/Y. Interestingly, there was one transcript
cassette results in stochastic splicing with exon 1, uniquely up-regulated and four uniquely down-regulated in
thereby reducing the amount of full-length Ar mRNA. Arinvflox(ex1-neo)/Y but not Arinvflox(ex1-neo)/Y; Tg(Amh-Cre).
This hypomorphic allele, termed Arinvflox(ex1-neo), re- To validate results obtained from our microarray
sults in a partial failure of terminal spermatid differen- analysis, we performed northern analysis for selected
tiation accompanied by elongating spermatid phago- transcripts. Both Arinvflox(ex-neo)/Y and Arinvflox(ex1-neo)/Y;
cytosis and a 95% reduction in epididymal sperm Tg(Amh-Cre) animals demonstrate elevated serum T
numbers. Using Cre recombinase driven by the Sertoli (⬃40⫻ over wild type), LH (⬃25⫻ over wild type), and
cell-specific Amh promoter [Tg(Amh-Cre)], we ablated FSH (⬃3⫻ over wild type) (10); these hormones can
Ar from Sertoli cells in an otherwise hypomorphic Ar have significant impacts on transcription in the testis.
background. This Sertoli cell-specific ablation results Therefore, we analyzed transcripts known to be regu-
in a more severe phenotype characterized by near- lated by T (Rhox5, Star), LH (Ren1, Star, Inha), and FSH
complete failure in elongating spermatid differentiation (Ren1, Klf4, Inha) in addition to transcripts regulated
and severe round spermatid sloughing. In the hope of by unknown factors (Adh1, Klk27, Lcn2). Of the tran-
identifying the AR targets responsible for these phe- scripts that were misregulated in our microarray anal-
notypes, we compared the gene expression profiles of ysis, all (7/7) demonstrated similar magnitude of
adult whole testis mRNA from wild-type, hypomorphic change by Northern blot (Fig. 1). With this sample of
Ar mutants, and hypomorphic Ar mutants with Sertoli Northern blots, we validated the results for 11% of the
cell-specific Ar ablation. Using a computational ap- transcripts misregulated in our microarray analysis. As
proach, we determined which of the genes misregulated indicated by our analysis, the level of Rhox5 message
in Ar mutants contain consensus androgen-responsive was reduced in Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y;
elements (AREs) that are conserved between mice and Tg(Amh-Cre), consistent with a reduction in AR sig-
humans, and thus may be direct targets of AR action. naling. T is known to be a negative regulator of Star
expression, whereas LH is a positive regulator. Ac-
cordingly, Star mRNA was also up-regulated in both
RESULTS mutants as predicted by our microarray analysis. The
levels of neither Ren1 nor Klf4 appeared to be altered
Identification of Genes Misregulated in Testes in our microarray analysis. However, despite its ab-
of Hypomorphic and Sertoli Cell-Specific sence from the list of misregulated transcripts, Ren1
Ar Mutants was up-regulated by Northern blot (Fig. 1). The vari-
ability observed in the levels of Ren1 message, as
In an effort to identify AR-regulated genes involved in seen by Northern, could have resulted in exclusion
spermatogenesis, we conducted an expression array from the list of 2-fold altered transcripts. In summary,
898 Mol Endocrinol, April 2007, 21(4):895–907 Eacker et al. • Testicular Gene Expression in Ar Mutants

lecular functions (Table 1). These molecular functions

were assigned based on their gene ontology annota-
tions provided by The Jackson Laboratory Mouse Ge-
nome 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. 2). The transcripts encod-
ing metabolic proteins are dominated by genes in-
volved in cholesterol biosynthesis and steroidogene-

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sis. 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 recep-
tor (Lhcgr, Table 1). Also of note is the significant
up-regulation of inhibin ␣ (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 grow-
ing information from cell type-specific microarray data
(18) have allowed us to assign the cell types in which

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. Se-
lected 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-reg-
ulated transcript and showed no change in expression level.
Although not identified on the microarray analysis, Ren1, a Fig. 2. Classification of Molecular Functions of Proteins En-
FSH- and LH-regulated transcript was up-regulated in both coded by Misregulated Transcripts in Arinvflox(ex1-neo)/Y and
Ar mutants. Actb was used as a loading control. These results Arinvflox(ex1-neo)/Y; Tg(Amh-Cre)
are representative of results from Northern analyses of three A single molecular function was assigned for each tran-
to six individuals of each genotype. script 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
these data provide evidence for changes in LH and AR the testis, primarily steroidogenesis and cholesterol biosyn-
thesis, dominate the collection of misregulated transcripts.
signaling, but no clear perturbation in FSH signaling.
The next most abundant classification of transcripts is those
The consistency between the results of the microarray
of unknown function. Addition of the Tg(Amh-Cre) to the
analysis and Northern blot validation give us a high hypomorphic Arinvflox(ex1-neo)/Y background results in an in-
degree of confidence in the results listed in Table 1. crease the proportion of misregulated transcripts involved in
The transcripts misregulated in the testes of cell adhesion, nucleic acid metabolism, and transcription.
Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) The transcripts comprising each functional classification are
encode proteins encompassing a broad range of mo- listed in Table 1.
Eacker et al. • Testicular Gene Expression in Ar Mutants Mol Endocrinol, April 2007, 21(4):895–907 899

the misregulated transcripts are normally expressed these criteria, we identified 3689 potential AREs in the
(Table 1). 6 kb encompassing the TSS for 59 genes.
To identify AREs that have a higher probability of
Identification of Potential AREs being functional, we identified potential AREs con-
served over the course of evolution. We examined
Identifying transcripts altered in our microarray analy- 6000 bp of sequence encompassing the TSS of the
sis as being androgen regulated must be carefully human orthologs of the genes identified in Table 1. We
considered because of the complex endocrine pheno- used information provided in the “Mammalian Homol-
type of Arinvflox(ex1-neo)/Y. High levels of FSH and LH ogy” section of Mouse Genome Informatics web site
could obscure the relative contribution of the absence (http://www.informatics.jax.org) to identify the human
of AR to the observed transcriptional phenotype. AR orthologs of genes affected in our microarray analysis.

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interacts with DNA sequences termed AREs that con- The total number of genes with clear human orthologs
sist of two 6-bp half-sites separated by a 3-bp spacer. reduced the number of promoter regions to 49 from
The sequence of these AREs has been experimentally our original 59 sequences. Using aligned mouse and
determined in a variety of androgen-responsive pro- human sequences, we used MONKEY (20) to identify
moters, many of which are listed in Table 2. The high conserved AREs (cAREs). We identified 108 potential
degree of degeneracy at many of the sites within the cAREs within the 6 kb surrrounding the TSS (Supple-
6-bp half-sites has made de novo prediction of AREs mental Table 2). The distribution of these cAREs rela-
in promoter regions a difficult task. Also, studies using tive to TSS are shown in Fig. 3B. Most genes had at
in vitro selection for sequences that bind the AR DNA least one cARE, but 35% had no identifiable cAREs
binding domain have identified high-affinity se- (Fig. 3C). Positions of the cAREs in the promoter re-
quences that are found rarely, if at all, in nature (19). To gions of validated transcripts with clear human or-
help identify transcripts that were more likely to be thologs are illustrated in Fig. 3D. As a negative control
androgen-regulated, we examined 6000 bp encom- we performed the same computational analysis on two
passing the transcriptional start site (TSS) of each genes that are expressed in the testis but are not
gene listed in Table 1. Of the 62 genes listed in Table under androgen regulation, Protamine 1 (Prm1), and
1, 59 genes had good evidence identifying the TSS testis nuclear RNA binding protein (Tenr). Neither of
and were therefore retained for further analysis. Using these genes contained a conserved ARE.
sequences listed in Table 2 we generated a weight-
matrix model for AREs and used it to identify potential Expression of Four Members of the Kallikrein
AREs with a log-likelihood ratio (LLR) ⬎ 3. A graphical Family of Proteases Is Disrupted by Loss of AR
representation of the weight matrix used in this anal-
ysis is shown in Fig. 3A. The results of this analysis and The Kallikrein family of serine proteases reside in a
details of the method are supplied in Supplemental cluster on mouse chromosome 7 (Fig. 4A). This family
Fig. 1 and Supplemental Table 1 (published as sup- has a dynamic evolutionary history that includes nu-
plemental data on The Endocrine Society’s Journals merous species-specific expansions and contractions
Online web site http://mend.endojournals.org/). Using (21). Interestingly, two human Kallikrein family mem-

Table 2. Experimentally Validated Androgen Response Elements

Promoter Position (bp) Sequence Ref.
Probasin ARE1 ⫺243 to ⫺228 ATAGCATCTTGTTCT 53
Probasin ARE2 ⫺137 to ⫺122 AGTACTCCAAGAACC 53
PSA ARR ⫺390 to ⫺375 GGATCAGGGAGTCTC 54
PSA ARE ⫺167 to ⫺154 AGAACAGCAAGTGCT 54
KLK2 ⫺157 to ⫺144 GGAACAGCAAGTGCT 55
C3-intron ⫹1359 to ⫹1374 AGTACGTGATGTTCT 56
C1 ⫹207 to 222 AGGACACAAAAATCC 56
Slp ARE 1 ⫹112 to ⫹127 GTAATTATCTGTTCT 57
Slp ARE 2 ⫹128 to ⫹143 TGGTCAGCCAGTTCT 57
Slp ARE 3 ⫹144 to ⫹159 AGAACAGGCTGTTTC 57
Factor IX ⫺37 to ⫺22 AGCTCAGCTTGTACT 58
JRE ⫺111 to ⫺96 ATTACACCAAGTACC 59
Aldose reductase ⫺111 to ⫺96 TGAAGTTCCTGTTCT 60
GUS ⫹7833 to ⫹7848 AGTACTTGTTGTTCT 61
AR-ARE1 ⫹2378 to ⫹2393 CTTTCTGAATGTCCT 62
AR-ARE2 ⫹2576 to ⫹2591 AGTACTCCTGGATGG 62
Pem ARE2 ⫺247 to ⫺233 AGCACATCGTGCTCA 63
PSCA ARE1 ⫺2796 to ⫺2782 AGGACGGAAAGTTCC 64
Many of the AREs collected in this table were collected in Ref. 19.
900 Mol Endocrinol, April 2007, 21(4):895–907 Eacker et al. • Testicular Gene Expression in Ar Mutants

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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 2 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. 2. 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.

bers, prostate-specific antigen (PSA) and KLK2, are Klk27 cDNA, the high degree of sequence identity
well studied for their tissue-specific androgen respon- between Klk-family members makes it impossible to
siveness. Because androgen responsiveness is a determine which Klk members are being induced by
common feature of members of the Kallikrein family, DHT.
and four Klks were misregulated in our microarray To determine whether the consensus AREs we iden-
analysis, Klks seem to be a good candidate for direct tified in the promoter region of Klk27 confer androgen
androgenic regulation. Indeed, the 2-kb encompass- responsiveness, we fused 1.3 kb of sequence up-
ing the TSS of the four Klks affected contain numerous stream of the Klk27 TSS to a luciferase reporter (Klk27-
potential AREs (Fig. 4B). To test whether Klks respond luc). When MA-10 Leydig tumor cells transfected with
to androgen signaling, we treated MA-10 Leydig tumor Klk27-luc are stimulated with DHT, we observed a
cells with DHT. MA-10 cells are an easily manipulated 3.4-fold increase in reporter activation, demonstrating
cell culture model of murine Leydig cells. After treat- androgen-dependent activation (Fig. 4D). MA-10 cells
ment, total RNA from the cell lines was analyzed by express low levels of AR, as demonstrated by insig-
Northern blot (Fig. 4C). MA-10 cells alone or with 50 nificant androgen-dependent activation of a mouse
␮M 8-bromo-cAMP (8-Br-cAMP) did not exhibit signif- mammary tumor virus-luciferase (MMTV-luc) reporter
icant levels of Klk expression. However, when treated (Fig. 4D) (16). However, upon cotransfection of MMTV-
with 10 ␮M DHT, MA-10 cells expressed detectable luc with an AR expression construct, we observed an
message. Surprisingly, addition of 8-Br-cAMP to DHT- 8-fold increase in luciferase activity. Similarly, when
treated cells reduced the level of Klk27 mRNA. Al- MA-10 cells were cotransfected with Klk-luc and an
though the probe used in this hybridization is from a AR expression vector and treated with DHT we ob-
Eacker et al. • Testicular Gene Expression in Ar Mutants Mol Endocrinol, April 2007, 21(4):895–907 901

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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 2. The blue arrowheads indicate
the position of ARE sequences as defined in panel A of Fig. 3. 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 ␤-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.

served a 44-fold activation of the reporter. These data minimal region of the promoter that confers androgen
strongly suggest that the Klk27 promoter demon- responsiveness, we generated a series of deletion
strates both androgen- and AR-dependent activation. constructs that remove portions of the Klk27-up-
We identified a number of potential AREs in the stream region that contain potential AREs (Fig. 4E). We
1.3-kb upstream of the Klk27 TSS. To determine the demonstrated that only the proximal 440 bp of the
902 Mol Endocrinol, April 2007, 21(4):895–907 Eacker et al. • Testicular Gene Expression in Ar Mutants

upstream sequence are required for androgen-depen- a null mutation of Rhox5 are subfertile but do not have
dent activation (Fig. 4E, construct D) and when the severe defects in spermatogenesis (25, 26). Because
sequence containing the three predicted AREs is re- androgen withdrawal has a far more severe phenotype
moved, the androgen responsiveness of the Klk27 than mutation of Rhox5, this clearly suggests that
promoter is eliminated (Fig. 4E, construct E). This sug- Rhox5 is only one of several targets of AR required to
gests that these computationally predicted AREs are support spermatogenesis.
functional in the context of the Klk27 promoter. Two recent investigations have used pharmacolog-
ical treatment coupled with microarray expression
analysis to identify transcripts that are regulated by
androgens in an in vivo system. To isolate the effects
DISCUSSION of testosterone alone on testis gene expression, Sa-

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date-Ngatchou and colleagues (28) used the sponta-
Despite the absolute requirement of androgen signal- neous Gnrh mutant Gnrhhpg/hpg treated with testoster-
ing for spermatogenesis, very few direct targets of AR one propionate (TP). This work on the Gnrhhpg/hpg
in the testis are known. In our current study, we have animal has the significant advantage of no confound-
characterized the transcriptional profile of the testes of ing effects of LH and FSH on gene regulation during
adult animals that bear a hypomorphic allele of Ar alone the treatments with TP. Total testis RNA was collected
or in combination with Sertoli cell-specific ablation of Ar. after short-term treatment with TP and microarray anal-
We identified a total of 62 transcripts that were altered ysis revealed a small number of transcripts (234) that
greater than 2-fold compared with wild-type in the testes change relative to controls. This is consistent with our
of Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) observation that a relatively small number of transcripts
mice. The majority of these transcripts contain evolution- were significantly altered in both Arinvflox(ex1-neo)/Y and
arily conserved androgen response elements, suggest- Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) testis. Also consistent
ing that many of these transcripts are direct targets of with our observations, more transcripts were down-
AR. For one of these transcripts, Klk27, we demon- regulated than up-regulated with testosterone treat-
strated that computationally predicted AREs in the ment. Our data further support the suggestion that AR
promoter region are functional in a Leydig-cell culture functions primarily as a mediator of transcriptional re-
model. We have therefore greatly expanded the pool pression within the adult testis. A second study to
of potential AR targets in the testis, providing novel identify AR-regulated transcripts used wild-type pre-
insight into possible mechanisms by which T pro- pubertal animals treated with TP (27). The prepubertal
motes spermatogenesis. animals lack a functional HPG axis, thus removing the
These animals provide significant advantages for confounding effects of FSH and LH. In contrast to our
the identification of targets of AR signaling in the whole findings and those in TP-treated adult Gnrhhpg/hpg
testis as well as specifically in Sertoli cells. Unlike mice, prepubertal animals treated with TP show a
Artfm/Y mice, Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; roughly equal number of up- and down-regulated tran-
Tg(Amh-Cre) mice have normally descended testes. scripts. One intriguing possibility is that the difference
Cryptorchidism alone has a strong effect on testicular in response to androgens in the prepubertal animal
gene expression making the use of Artfm/Y to identify reflects a developmental change in the profile of AR
targets of androgen signaling problematic (13). Addi- coregulators in the testis. Rhox5 is also up-regu-
tionally, unlike Artfm/Y and other Sertoli cell-specific Ar lated by TP in prepubertal mice, making it the only
mutant mice, Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; commonly regulated gene between the TP-treated
Tg(Amh-Cre) animals have a nearly intact complement Gnrhhpg/hpg mice (28), TP-treated prepubertal mice,
of transcriptionally active germ cells. Therefore, and the current study.
changes in gene expression observed in our analysis Two other groups have published studies of adult
are not the result of a lack of major classes of germ Sertoli-selective Ar knockouts (11, 12). With the ex-
cells within the testis. The presence of particular ception of Rhox5, there is no overlap in published gene
classes of germ cells are also required for the produc- expression changes associated with these mutations
tion proteins synthesized in Sertoli cells, making the and Arinvflox(ex1-neo)/Y;Tg(Amh-Cre). Certainly, this is
nearly normal complement of germ cells even more partly due to the relatively small changes observed for
important to our analysis (22). the transcripts these groups have selected for analy-
The best-characterized target of AR signaling within sis; we have chosen a 2-fold or greater change cut-off
the testis is Rhox5 (formerly known as Pem), a ho- for the transcripts we have analyzed. In a recent study
meobox gene located on the X chromosome that is of prepubertal Sertoli cell-specific Ar mutant
expressed exclusively in Sertoli cells. Our microarray (SCARKO) mice, the authors identified 12 up-regu-
analysis demonstrated the expected down-regulation lated and 28 down-regulated transcripts at a 2-fold or
of Rhox5 (Table 1) that was confirmed by Northern blot greater cut-off (29). Of these transcripts, only Rhox5
(Fig. 1). It has been shown that Rhox5 is responsive to was commonly regulated gene between our study and
androgens and that androgen-induced transcription that of Denolet et al. (29). There are several possible
requires two AREs located ⫺233 and ⫺69 relative to reasons for the lack of common transcriptional
the transcriptional start site (23, 24). Male mice bearing changes between the two studies. Prepubertal Sertoli
Eacker et al. • Testicular Gene Expression in Ar Mutants Mol Endocrinol, April 2007, 21(4):895–907 903

cells are mitotically active, whereas adult Sertoli cells Despite the 3-fold increase in FSH levels, we ob-
are mitotically quiescent, prepubertal Sertoli cells are served no clear transcriptional effect that could be
not yet surrounded by the full-complement of germ cells, ascribed to increases in FSH signaling in the testis of
and AR coactivator and corepressor expression may be Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre)
different between prepubertal and adult Sertoli cells. animals. To confirm this observation made from the
Determining which of the misregulated transcripts microarray expression profile, we measured levels of
are regulated directly by AR is a challenging problem. Klf4 and Ren1 message, both of which are targets of
To address this question we chose to take a bioinfor- FSH signaling in vivo (31). We found no significant
matic approach to identify evolutionarily conserved change in the levels of Klf4. Although not borne out in
sequences in the promoter regions of the genes, we the microarray analysis, there was modest and vari-
identified as misregulated in Ar mutants. We identified able up-regulation of Ren1 as determined by Northern

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108 cAREs in the 6 kb encompassing the transcrip- blot (Fig. 1). However, Ren1 can be up-regulated by
tional start site (TSS) of 49 of the misregulated genes. both LH and FSH signaling (31, 32). Transcription of
These cAREs show a modest enrichment in the ⫺500- Inha is also thought to be stimulated by FSH and is
to ⫹500-bp region relative to the TSS (Fig. 3A). This is indeed up-regulated in both Arinvflox(ex1-neo)/Y and
significant because many experimentally validated Arinvflox(ex1-neo)/Y; Tg(Amh-Cre) testes. However, our
AREs were found within this region (Table 2). Interest- analysis of serum Inhibin B levels thus far has dem-
ingly, there is a spike in the number of cAREs from onstrated no significant difference between
⫹2501 to ⫹3000 bp. The significance of these poten- Arinvflox(ex1-neo)/Y, Arinvflox(ex1-neo)/Y; Tg(Amh-Cre),
tial regulatory sequences is unclear, although the and wild-type animals (Eacker, S. M., unpublished
presence of AREs in this region are not without pre- observations). Therefore, it seems more likely that
cedent. As is true for many androgen-responsive the extraordinarily high level of LH is stimulating the
genes, most ARE-containing promoters have more transcription of Inha in Leydig cells, which is a well-
than one cARE (Fig. 3B). In general, AREs in androgen- documented phenomenon (33).
responsive promoters function in a cooperative man- Regardless of being direct or indirect targets of AR,
ner where each binding site contributes to a portion of there are a number of interesting misregulated tran-
the promoter’s responsiveness. Although the pres- scripts that may shed light on gonadotropin and ste-
ence of cAREs in the promoters of genes misregulated roid action in the testis. The Leydig cell-specific tran-
in Ar mutants is not proof that they are direct targets of script insulin-like 3 (Insl3) was down-regulated nearly
AR, the localization of cAREs does provide fertile 10-fold in Arinvflox(ex1-neo)/Y. Similar observations have
ground for hypothesis testing. been made in an investigation of Leydig cell develop-
A potential complication to our analysis is the high ment in Artfm/Y mice (13). The authors in this study
level of gonadotropins in Arinvflox(ex1-neo)/Y and concluded that the misregulation of Insl3 was due a
Arinvflox(ex1-neo)/Y; Tg(Amh-Cre). In both Arinvflox(ex1-neo)/Y and failure in Leydig cell maturation in Artfm/Y. Treatment
Arinvflox(ex1-neo)/Y; Tg(Amh-Cre), there is an approximately with GnRH antagonists can also down-regulate Insl3
25⫻ increase in serum LH and an approximately 3⫻ in the adult male rat, suggesting that its transcription is
increase in FSH. As discussed previously, we believe hormonally regulated (34). Late-stage germ cell apo-
that these increases reflect a lack of feedback regulation ptosis observed in GnRH antagonist-treated rats is par-
within the hypothalamic-pituitary-gonadal axis as a result tially relieved when exogenous INSL3 is provided, sug-
of a reduction in AR signaling. Indeed, many known gesting that INSL3 can function to support germ cell
targets of LH signaling are up-regulated in our analysis of development. We have also observed late-stage germ
Ar mutants, including transcripts encoding steroidogen- cell apoptosis in Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y;
esis-related proteins. It is not clear to what extent, how- Tg(Amh-Cre) mice, which could in part be due to the
ever, the high levels of steroidogenic gene expression lack of INSL3 production by the Leydig cell (Meng, J.,
are due to elevated LH levels or to the reduction of AR and R. E. Braun, unpublished observations). When
signaling. For instance, Star is a well characterized target hCG is provided to Gnrhhpg/hpg mice or to rats treated
of LH signaling and is up-regulated in Arinvflox(ex1-neo)/Y with a GnRH antagonist, Insl3 levels return to normal,
and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre). However, it has been suggesting either direct effects through LHCGR or
shown that T can suppress LH-induced Star transcrip- secondary effects through androgen production (34,
tion (30). Therefore, both the presence of high level of 35). Because LH levels in Arinvflox(ex1-neo)/Y mice are
gonadotropin and the low levels of AR signaling could very high, AR signaling is reduced, and Insl3 expres-
contribute to the levels of gene expression observed in sion is low, we suggest that Insl3 may be a target of
Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-Cre). In AR. Alternatively, additional factors acquired during
addition to the strikingly high levels of serum LH, we Leydig cell maturation may be required for proper
observed a 14- to 17-fold increase in Lhcgr transcript hormonally induced Insl3 expression.
levels. Although we do not know whether this up-regu- As previously noted, a number of steroidogenic genes
lation of Lhcgr mRNA results in an increase in Lhcgr are severely down-regulated in the Artfm/Y mouse. In
protein, it does suggest that there is an as-yet-unchar- particular, Hsd3b6, Hsd17b3, and Cyp17a1 were signif-
acterized role for AR or LH signaling in the transcription icantly down-regulated in adult Artfm/Y Leydig cells (17).
of Lhcgr. This trend holds true in Arinvflox(ex1-neo)/Y for Hsd3b6 and
904 Mol Endocrinol, April 2007, 21(4):895–907 Eacker et al. • Testicular Gene Expression in Ar Mutants

Hsd17b3 but not for Cyp17a1. Surprisingly, despite the round spermatids from the seminiferous epithelium
high levels of LH in Arinvflox(ex1-neo)/Y, Cyp17a1 expres- and observed round spermatids in the epididymis only
sion was not significantly affected. Previous work dem- in Arinvflox(ex1-neo)Y;Tg(Amh-Cre) males. A number of
onstrated that reduction of 17-␣ hydroxylase activity as- genes with putative roles in cell adhesion are either ex-
sociated with CYP17A1 is a significant contributor to the clusively or more severely affected in Arinvflox(ex1-neo)/Y;
block in steroidogenesis observed in Artfm/Y testis (36, Tg(Amh-Cre) compared with Arinvflox(ex1-neo)/Y. The tran-
37). The normal levels of Cyp17a1 in Arinvflox(ex1-neo)/Y script encoding the tight junction component Cldn3 is
may therefore be the reason that Arinvflox(ex1-neo)/Y mice exclusively down-regulated when the Tg(Amh-Cre) is
are capable of synthesizing vast amounts of testosterone present. CLDN3 is a component of newly forming tight
despite the apparent down-regulation of other steroido- junctions that make up the blood-testis-barrier (BTB)
genic enzymes. (49). Loss of CLDN3 correlates with a failure of the BTB,

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The four members of the kallikrein family of serine which could lead to the disorganization and disruption of
proteases, Klk9, Klk21, Klk24, and Klk27, are present germ-Sertoli cell contacts. It is possible that the dysfunc-
among the down-regulated transcripts. Within the tes- tion of the cell adhesion in the absence of CLDN3 leads
tis, expression of Klk21, 24, and 27 is limited to the directly to the sloughing of round spermatids. However,
Leydig cells (38–40). The genes encoding the Kal- the improper establishment of the BTB could also lead to
likreins reside in a large cluster on mouse chromo- a disruption of a permissive microenvironment neces-
some 7 (Fig. 4) (21). The best-studied member of the sary for germ cell development.
kallikrein gene family is the human PSA (41). We have Together, the alterations in the expression of tran-
demonstrated that one or more members of the Klk scripts identified in this study contribute to the over-
gene family are under T regulation in the Leydig tumor all phenotype observed in Arinvflox(ex1-neo)/Y and
line MA-10. Previous investigations have shown that Arinvflox(ex1-neo)/Y; Tg(Amh-Cre). Identification of
Klk21 is up-regulated by testosterone in immature cAREs in the promoters of affected genes will also
Leydig cells in culture (39). Taken together with our be useful in the identification of genes under direct
data showing a severe down-regulation in the Klk tran- AR regulation. The potential targets of AR signaling
scripts under conditions of reduced androgen signal- identified in this analysis suggest that AR could
ing, we propose that the four Klks down-regulated in promote germ cell development by providing both
our array analysis represent good markers for andro- instructive signals (i.e. RA signaling) as well as by
gen signaling within the Leydig cell. It is also worth creating a permissive environment (i.e. maintenance
noting that the expression of all the Klk transcripts of the BTB). The relative contribution of individual
further decreased in the presence of Tg(Amh-Cre). targets of AR identified in this study will provide
This further reduction may represent Sertoli-Leydig insight into the mechanisms by which T supports the
cell interaction, a phenomenon described in a study of ongoing developmental process of spermatogenesis.
another Sertoli cell-specific Ar mutant (42).
Among the most severely affected transcripts iden-
tified in our microarray analysis is alcohol dehydroge-
nase 1 (Adh1), which was up-regulated ⬎13-fold in Materials and Methods
both Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; Tg(Amh-
Cre). Aside from catalyzing the conversion of ethanol Animals
to acetaldehyde for detoxification, ADH1 is the rate-
All animals were housed in a specific pathogen-free environ-
limiting enzyme in the conversion of vitamin A to the ment and cared for under Institutional Animal Care and Use
potent signaling molecule retinoic acid (RA) (43). De- Committee guidelines. Generation of the hypomorphic Ar
pletion of vitamin A from the diets of mammals has allele Arinvflox(ex1-neo) has been previously described (10). The
severe consequences for spermatogenesis (44). Also generation and characterization of Tg(Amh-Cre) mice will be
of interest, two candidate retinoid carrier molecules, described in a forthcoming publication. Animals bearing Arin-
vflox(ex1-neo) and/or Tg(Amh-Cre) were identified by PCR from
prostaglandin D2 synthase and lipocalin 2, are among tail DNA using methods described previously (10). Animals
the most severely down-regulated transcripts (45, 46). were euthanized by CO2 asphyxiation in accordance with
These changes in gene expression offer the intriguing Institutional Animal Care and Use Committee guidelines. All
possibility of cross talk between the androgenic sig- animals used in this study were maintained on a 129Sv/
JaeSor background.
naling and the RA pathway. Recently, RA signaling has
been implicated in promoting meiotic progression in
the murine gonad (47, 48). Although no meiotic phe- Preparation of Total Testis mRNA and Microarray
Analysis
notype has been observed in our Ar mutants, our
results suggest that regulation of RA synthesis and The testes from wild-type, Arinvflox(ex1-neo)/Y, and Arinvflox(ex1-neo)/Y;
transport could be the mechanism by which T pro- Tg(Amh-Cre) 8-wk-old adults were dissected for RNA isolation.
motes meiotic progression in other models of AR Total RNA was extracted from whole testis that was homoge-
signaling. nized in 1 ml Trizol (Invitrogen, Carlsbad, CA) using a Dounce
(Wheaton, Milville, NJ) homogenizer and prepared in accor-
In both Arinvflox(ex1-neo)/Y and Arinvflox(ex1-neo)/Y; dance with the manufacturer’s protocol. RNA from two animals
Tg(Amh-Cre) we observed a severe decrease in epi- of each genotype were used in separate array experiments. The
didymal sperm. However, we observed sloughing of quality of the RNA was assessed by gel electrophoresis and
Eacker et al. • Testicular Gene Expression in Ar Mutants Mol Endocrinol, April 2007, 21(4):895–907 905

measuring the A260:A280 ratio. Biotinylated cRNA was generated Madison, WI). For luciferase assay, MA-10 cells were trans-
from an oligo-deoxythymidine-primed reverse transcription re- fected at 70% confluence in 24-well plates using Lipo-
action with 10 ␮g of total RNA using MEGAscript (Ambion, fectamine 2000 (Invitrogen). Reporter constructs (200 ng
Austin, TX). Labeled cRNA was hybridized to Affymetrix (Santa MMTV-luc or Klk27-luc) were cotransfected with pSV-␤gal
Clara, CA) MOE430A microarrays following the manufacturer’s (20 ng; Promega) alone or in combination with 10 ng of Ar
protocol. Microarrays were stained and washed using the expression vector. Cells were allowed to recover overnight in
GeneChip Fluidics Station 400. The arrays were then scanned RPMI1640 supplemented with charcoal-dextran-stripped fe-
with a GeneArray Scanner 2500A (Agilent, Palo Alto, CA) and tal bovine serum. The next day, cells were treated with 10 nM
analyzed using Microarray Suite 5.0 (Affymetrix) and Genespring DHT for 24 h before luciferase assay. All assay were per-
6.1 (Silicon Genetics, Redwood City, CA). Transcripts demon- formed in triplicate using Bright-Glo and Beta-Glo systems
strating at least a 2-fold change in expression and a minimal (Promega).
signal strength greater than 50 in any of the comparisons were
considered for further analysis. All resulting transcripts demon-

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strated a statistically significant difference in at least one Acknowledgments
comparison.
We thank Drs. Martin Tompa and William Noble for helpful
Northern Analysis discussion and comments during analysis of cAREs and De-
rek Pouchnik for microarray hybridization and staining. We
also thank Debra Sprague for assistance in preparation of this
For Northern analysis, 15 ␮g of total RNA was run on a 1.5%
manuscript and Kelly Tysseling for technical assistance.
agarose formaldehyde gel. The RNA was then transferred
overnight in 20⫻ SSC to Genescreen Hybridization Mem-
brane (PerkinElmer, Foster City, CA) and then UV cross-
linked. The membranes were hybridized overnight with Received March 7, 2006. Accepted January 16, 2007.
probes generated using randomly incorporated ␣32P-deoxy- Address all correspondence and requests for reprints to:
ATP using standard methods. Membranes were washed for Robert E. Braun, University of Washington School of Medi-
30 min in 2⫻ SSC, 0.5% sodium dodecyl sulfate, 0.1% cine, Department of Genome Sciences, Box 355065, 1705
sodium pyrophosphate at 65 C followed by a 30-min wash in NE Pacific, Foege Building, Room 133C, Seattle, Washington
0.5⫻ SET (10 mM Tris, pH 7.5; 5 mM EDTA; 1% sodium 98195-5065. E-mail: [email protected].
dodecyl sulfate), 0.1% sodium pyrophosphate at 42 C.
This work was funded by the Specialized Cooperative
Centers Program in Reproductive Research (U54 HD12629)
Computational Prediction of AREs (to R.E.B.) and the Contraceptive Centers Program (U54
HD42454) (to M.D.G.).
Conserved AREs were found using MONKEY, a program that Current address for J.E.S.: Department of Biopharmaceu-
uses a sequence alignment, the phylogeny of the sequences, tical Sciences, University of California, San Francisco, San
and a weight matrix describing a transcription factor binding Francisco, California 94143
site to identify conserved transcription factor binding sites Current address for R.H.W.: Department of Surgery, Col-
(20). The frequencies of the bases at each position in the ARE lege of Medicine (R.H.W.), University of Cincinnati, Cincinnati,
were determined from the AREs listed in Table 2. Mouse- Ohio 45267.
human alignments were determined using sequences from The authors have nothing to disclose.
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 con- REFERENCES
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