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Molecular Endocrinology, doi:10.1210/me.2005-0113
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Molecular Endocrinology 20 (2): 321-334
Copyright © 2006 by The Endocrine Society

The Effect of a Sertoli Cell-Selective Knockout of the Androgen Receptor on Testicular Gene Expression in Prepubertal Mice

Evi Denolet, Karel De Gendt, Joke Allemeersch, Kristof Engelen, Kathleen Marchal, Paul Van Hummelen, Karen A. L. Tan, Richard M. Sharpe, Philippa T. K. Saunders, Johannes V. Swinnen and Guido Verhoeven

Laboratory for Experimental Medicine and Endocrinology, Catholic University of Leuven (E.D., K.D.G., J.V.S., G.V.), and Microarray Facility, Flanders Interuniversity for Biotechnology (P.V.H.), Gasthuisberg, B-3000 Leuven, Belgium; Department of Electrical Engineering, Faculty of Engineering (J.A., K.E., K.M.), and Center for Microbial and Plant Genetics, Faculty of Applied Bioscience and Engineering (K.M.), Catholic University of Leuven, B-3001 Heverlee, Belgium; and Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology, Queen’s Medical Research Institute, University of Edinburgh (K.A.L.T., R.M.S., P.T.K.S.), Edinburgh EH16 4TJ, Scotland, United Kingdom

Address all correspondence and requests for reprints to: Dr. Guido Verhoeven, Laboratory for Experimental Medicine and Endocrinology, Onderwijs en Navorsing, Gasthuisberg, Herestraat 49 bus 902, B-3000 Leuven, Belgium. E-mail: guido.verhoeven{at}med.kuleuven.be.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To unravel the molecular mechanisms mediating the effects of androgens on spermatogenesis, testicular gene expression was compared in mice with Sertoli cell-selective androgen receptor knockout (SCARKO) and littermate controls on postnatal d 10. Microarray analysis identified 692 genes with significant differences in expression. Of these, 28 appeared to be down-regulated and 12 up-regulated at least 2-fold in SCARKOs compared with controls. For nine of the more than 2-fold down-regulated genes, androgen regulation was confirmed by treatment of wild-type mice with an antiandrogen (flutamide). Some of them were previously described to be androgen regulated or essential for spermatogenesis. Serine-type protease inhibitors were markedly overrepresented in this down-regulated subgroup. A time study (d 8–20), followed by cluster analysis, allowed identification of distinct expression patterns of differentially expressed genes. Three genes with a pattern closely resembling that of Pem, a prototypical androgen-regulated gene expressed in Sertoli cells, were selected for confirmation by quantitative RT-PCR and additional analysis. The data confirm that the SCARKO model allows identification of novel androgen-regulated genes in the testis. Moreover, they suggest that protease inhibitors and other proteins related to tubular restructuring and cell junction dynamics may be controlled in part by androgens.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DESPITE THE OVERWHELMING evidence that androgens play a key role in the control of spermatogenesis (1, 2, 3, 4, 5, 6), the cellular and molecular mechanisms underlying this control remain poorly understood. Germ cells, peritubular myoid cells, and Sertoli cells (SC) have been mentioned as potential androgen target cells, but their relative contributions to the control of spermatogenesis remain unclear. Germ cells probably do not express the classical androgen receptor (AR) (7), and they certainly do not need it for normal development (8, 9). However, alternative mechanisms of androgen action, for instance, via conversion into estrogens (10) or via nongenomic pathways (11, 12), cannot be excluded. Peritubular myoid cells are located in the vicinity of spermatogonia. They do express the AR (13); moreover, they depend on androgens for their normal development (14). Furthermore, androgen-modulated interactions between peritubular cells and SC have been described (15, 16). SC display intimate interactions with germ cells at all stages of spermatogenesis (17, 18) and express the AR in a stage-dependent manner (13). Therefore, they are usually considered to be the most likely mediators of androgen action on spermatogenesis.

Because attempts to use isolated and cultured SC to study the molecular mechanisms of androgen action in the control of spermatogenesis have been largely unsuccessful (19, 20), we and others have recently developed in vivo models in which the AR gene is selectively inactivated in SC during embryonic development (from d 15 post coitum), more than 1 wk before immunoexpression becomes evident (postnatal d 3–5) (21, 22, 23). In our laboratory, knockout of the AR gene was achieved by crossing mice with a floxed exon 2 of the AR with mice expressing Cre under control of the anti-Mullerian hormone promoter (AMH-Cre) (24). These mice (SCARKO mice) have normally descended testes, virtually absent levels of Pem (a prototypical gene expressed in SC under control of androgens), normal AR expression in Leydig and peritubular cells, and normal circulating levels of androgens and LH. Spermatogenesis, however, is arrested in meiosis.

In the present study we have used the GeneChip Mouse Expression Array 430a 2.0 to identify genes that are differentially expressed in the testis of SCARKO and control (AMH-Cre) littermates on d 10, a time point at which differences in AR expression have not yet resulted in noticeable differences in cellular composition of the testis. A subset of 692 genes, including the Pem gene, was differentially expressed in a statistically significant manner. Among these differentially expressed genes, serine-type protease inhibitors were clearly overrepresented. Analysis of testicular gene expression in SCARKO and control mice between d 8 and 20 allowed cluster analysis and identification of some typical gene expression patterns of androgen-regulated genes. A subset of genes with an expression pattern resembling that of Pem was selected for additional analysis. Furthermore, functional analysis of the data suggested that androgen action in SC may affect tubular remodeling and junction dynamics.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The microarray datasets generated in this study are available through National Center for Biotechnology Information via the Gene Expression Omnibus (GEO) data repository (http://www.ncbi.nih.gov/geo/), GEO accession nos. GSE2259 and GSE2260.

Identification of Differentially Expressed Transcripts in Control vs. SCARKO Testes on d 10
To avoid confounding effects of differences in testicular cell composition related to the meiotic arrest occurring in SCARKO mice, gene expression patterns in SCARKO and control littermates were compared on d 10. At this age, testicular weights were identical in SCARKO and control mice [mean ± SEM, 4.55 ± 0.18 mg (n = 25) and 4.58 ± 0.16 mg (n = 17), respectively]. Moreover, tubular and germ cell complements were still comparable, as confirmed by hematoxylin/eosin staining and immunostaining for Dazl, a germ cell-specific RNA-binding protein. Nonetheless, on d 10, the AR was clearly immunoexpressed in SC nuclei of control mice, whereas it was completely absent in SC of SCARKO mice (Fig. 1Go). As expected, AR staining in interstitial and peritubular myoid cells was comparable for SCARKO and control mice (21).


Figure 1
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  		Fig. 1. Hematoxylin/Eosin (H&E) Staining and Immunoexpression Profiles for AR and Dazl in Testes of 10-d-Old Control and SCARKO Mice

H&E and Dazl staining show that testicular morphology and germ cell complement are still similar in control and SCARKO testes on d 10. Immunostaining of the AR is observed in SC nuclei of control mice (arrows) but is absent in SC nuclei of SCARKO mice. Scale bar, 50 µm.

 
Statistical analysis of microarray data from five independent samples of 10-d-old SCARKO and control mice (see Materials and Methods) generated a list of 848 differentially expressed transcripts corresponding to 692 genes after correction for the redundancy of oligonucleotide probes (supplemental Table 1, published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Of these genes, 296 were expressed at a higher level in SCARKOs compared with controls (referred to as up-regulated in SCARKOs) and 396 were expressed at a lower level in SCARKOs compared with controls (referred to as down-regulated in SCARKOs). Forty of the identified genes showed a difference in expression level of at least 2-fold. The majority of the latter genes (n = 28) displayed lower expression levels in SCARKOs (strongly down-regulated) compared with controls (Table 2Go).


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  		Table 2. Genes with a More than 2-Fold Difference in Expression Level between SCARKO and Control (Ctrl)

 
Using Onto-Express software (25), the list of 396 genes that were down-regulated in SCARKOs reflected a significant (P < 0.01) overrepresentation of genes related to MAPK activity (n = 4) and serine-type endopeptidase inhibitor activity (n = 6). Interestingly, most of these serine-type endopeptidase inhibitors [Eppin, Serpina3N, Serpina12, Pcskn1n, and protein C inhibitor (PCI)] were down-regulated more than 2-fold (Table 2Go). For the 296 genes that appeared up-regulated in SCARKOs, overrepresentation was observed for genes related to IGF binding (n = 5), Ca2+ binding (n = 16), Ca2+-dependent phospholipid binding (n = 5), regulation of cell cycling (n = 12) and cell growth (n = 8), cell adhesion (n = 13), and signal transduction (n = 43).

Confirmation of Androgen Regulation and Tubular Localization for a Selection of Down-Regulated Genes
To clarify the contribution of androgens to the differences in testicular gene expression identified by the microarray experiments, 7-d-old wild-type mice were treated for 3 or 5 d with the antiandrogen flutamide (Flut) or with testosterone proprionate (TP), and the expressions of nine of the strongly down-regulated genes were measured by quantitative RT-PCR (Table 3Go). The selected genes comprised the four genes most strongly down-regulated in SCARKOs (Pem, Eppin, Galgt1, and DRD4), a testis-specific X-linked gene (Tsx), a gene encoding an enzyme involved in lipid and glucose metabolism (Gpd1), and genes encoding a microtubular component (Tubb3), a serine protease inhibitor (PCI), and a tumor protein (Tpd52l1). After 3 d of treatment with Flut (d 10), the expression of all studied genes tended to be decreased, but a significant decrease was observed only for Pem, Eppin, Drd4, and Gpd1. After 5 d of Flut (d 12), the expressions of all studied genes were significantly decreased. With the exception of Galgt1, genes that were most strongly down-regulated in SCARKOs tended to be more sensitive to inhibition by Flut. Interestingly, on d 10, TP still increased the expression of three of the studied genes, whereas on d 12, stimulatory effects were absent, and inhibitory effects were observed for five of the selected genes, suggesting that, already at that age, intratesticular androgen levels may be decreased by treatment with TP.


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  		Table 3. Effect of (Anti)-Androgen Treatment on Testicular Gene Expression

 
Enzymatic digestion, followed by quantitative RT-PCR, were used to study the interstitial or tubular localization of the same subset of selected genes in 10-d-old wild-type mice. Pem and SCP3 were used as markers of tubular localization (for SC and spermatocytes, respectively), and P450c17 (Leydig cells) was used as a marker for interstitial localization. As shown in Table 4Go, the data point to a predominantly tubular localization for all selected genes.


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  		Table 4. Gene Expression in Interstitial and Tubular Fractions in Testes at d 10

 
Expression Patterns of Transcripts Differentially Expressed on d 10 in SCARKO and Control Mice during Prepubertal Development
To analyze the relationship of the differentially expressed genes, identified above, to the initiation of spermatogenesis and the development of the testis, gene expression was assessed in testis samples derived from SCARKO and control littermates at five different ages (d 8, 10, 12, 16, and 20).

The physiological relevance of the resulting data was supported by the expression pattern displayed by a number of typical meiotic marker genes (Fig. 2Go). Dmc1, for instance, and SCP3, which are known to be expressed during early stages of meiosis, showed a marked increase in expression from d 12 onward and displayed similar expression profiles in control and SCARKO testes. In contrast, genes such as heat shock protein 70-2 (Hsp70-2) and proacrosin-binding protein (Acrbp) displayed their steepest increase in expression later in meiosis (from d 16 on) and rose more markedly in controls than in SCARKOs, a finding compatible with the previously described progressive disruption of meiosis in SCARKO testes.


Figure 2
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  		Fig. 2. Representative Expression Profiles (d 8–20) of Early (Dmc1 and SCP3) and Late (Acrbp and Hsp70-2) Meiotic Marker Genes

Microarray signal values (RMA normalized) were normalized to the highest signal value observed for the studied gene in SCARKO or control testes, arbitrarily set at 100. The resulting values are designated the relative signal intensity for the studied gene at the indicated time points.

 
Cluster analysis of the transcripts found to be differentially expressed on d 10 resulted in the identification of 14 clusters encompassing 796 transcripts. Clusters containing at least 30 genes are shown in Fig. 3AGo, and the detailed data can be consulted in supplemental Table 2Go. The vast majority of the transcripts that showed a difference in expression level of at least 2-fold when comparing SCARKO and controls were found in clusters 3, 4, and 5. Clusters 1, 2, and 6 grouped genes with more limited differences in expression level on d 10. Cluster 1 genes were characterized by a progressive decline in expression from d 8 to 20. The decline was more pronounced in controls than in SCARKOs. Cluster 2 genes displayed a marked decline in expression in control testes from d 12 onward, whereas expression in SCARKOs remained relatively stable. Cluster 6 genes were characterized by a late-onset (d 16) increase in expression that was more pronounced in controls than in SCARKOs.


Figure 3
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  		Fig. 3. Expression Patterns of Transcripts, Differentially Expressed on d 10 in SCARKO and Control Mice, between d 8 and d 20

A, Adaptive quality-based clustering (AQBC) of transcripts differentially expressed on d 10. Cluster analysis was used to identify subsets of transcripts with a similar expression pattern. Fourteen clusters were identified, containing 796 transcripts in total. Only clusters containing at least 30 transcripts are shown. For each cluster, the representative expression profile is represented in bold. B, Left, Hierarchical clustering of all transcripts present in cluster 4. Columns indicate different genotypes (control and SCARKO) and ages (d 8–20), and rows show the expression patterns for individual genes. Expression levels are visualized using a color code in which expression decreases from dark red (highest expression level) to dark green (lowest expression level). Genes are grouped based on the resemblance of their expression pattern to that of neighboring genes. The panel on the right gives a detailed view of the transcripts with an expression pattern most strongly resembling that of Pem. C, Expression pattern of Pem. Relative signal intensities were calculated as explained in Fig. 2Go.

 
Cluster 5 contained transcripts with a decreasing expression pattern from d 8–20 and with higher expression levels in SCARKOs than in controls. The time course of the most representative transcript (minimal distance between expression profile and cluster center) for this cluster (RIKEN cDNA 6530411B15) is illustrated in Fig. 4Go. Five of the 12 transcripts with a more than 2-fold higher expression level in SCARKO testes (Table 2Go and supplemental Table 2Go) were found in this cluster.


Figure 4
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  		Fig. 4. Expression Patterns of the Most Representative Transcripts of Clusters 5, 3, and 4 in the AQBC Clustering of the Differentially Expressed Genes

RIKEN cDNA 6530411B15 gene (6530411B15Rik), phosphatidylinositol 4-kinase type 2 ß (Pi4k2b), and N-acetylglutamate synthase (Nags) represent clusters 5, 3, and 4, respectively. Relative signal intensities were calculated as explained in Fig. 2Go.

 
Eight and 12 of the genes that had a more than 2-fold lower expression level in SCARKO testes than in control testes (29% and 43% of this entire subgroup) were found in clusters 3 and 4, respectively (Table 2Go and supplemental Table 2Go). Prototypical genes of clusters 3 (phosphatidylinositol 4-kinase type 2ß) and 4 (N-acetylglutamate synthase) are shown in Fig. 4Go. The expression of the transcripts found in cluster 3 increased over the study period, more so in controls than in SCARKOs (Fig. 4Go). Transcripts in cluster 4 were characterized by a limited increase in the SCARKO testes, but a marked early increase in controls (up to d 12), followed by a slower decline (Fig. 4Go). Several of the genes with the largest decrease in expression level in SCARKO compared with controls (Pem, Eppin, Drd4, and Gpd1; Table 2Go) were found in cluster 4.

Because Pem was previously shown to be directly regulated by androgens in SC, and the main aim of our study was to identify additional androgen-regulated genes in SC, we specifically searched for genes whose expression pattern most strongly resembled that of Pem (Fig. 3CGo). To this end, pairwise Euclidian distances between all profiles in cluster 4 were calculated and visualized using hierarchical clustering as implemented in Expression Profiler (EPCLUST) (26). Figure 3BGo shows that the expression profiles of Eppin, Drd4, and Gpd1 most strongly resemble that of Pem. Therefore, these genes were retained for additional analysis. Mtap7 was represented on the array by several transcripts, and because not all of them shared the same expression pattern, this gene was not included.

Genes with an Expression Pattern Resembling that of Pem
The expressions of Pem, Eppin, Drd4, and Gpd1 were studied from d 2–20 using quantitative RT-PCR. The data summarized in Fig. 5Go confirm a similar expression pattern as a function of age for the four genes studied and noticeably higher expression in controls compared with SCARKOs at all ages. This difference in expression remained evident on d 50 (data not shown). In contrast to the expression pattern of these genes as studied by microarray analysis (illustrated for Pem in Fig. 3CGo) the expression pattern studied by quantitative RT-PCR did not show a decline after d 12. This may be explained by the fact that the quantitative RT-PCR data are normalized to exogenously added luciferase mRNA to obtain an estimate of the absolute number of transcripts per testis. The absence of such a normalization for the microarray data means that transcripts in a particular cell type (for instance, SC) will be diluted in the total amount of transcripts when another cell population (in this case, the germ cells) is making up an increasing proportion of the total testicular volume.


Figure 5
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  		Fig. 5. mRNA Levels of Placentae and Embryos Oncofetal Gene (Pem), Dopamine Receptor 4 (Drd4), Serine Protease Inhibitor with Kunitz and WAP Domains 1 (Eppin), and Glycerol-3-Phosphate Dehydrogenase 1 (Gpd1) in Control and SCARKO Testes from d 2–20, as Determined by Quantitative RT-PCR

The results confirm differential expression between SCARKO and control testes and similarity of the expression patterns of the studied genes. Expression values were corrected using luciferase mRNA as an external standard and are represented as the mean ± SEM of three independent experiments with triplicate measurements. Note that the expression levels of Eppin are much higher than those of Pem, Drd4, and Gpd1.

 
Effects of SC-Selective AR Inactivation on Genes Related to Tubular Remodeling and Junction Dynamics
Given the finding from the original d 10 study that transcripts encoding serine protease inhibitors and cell adhesion molecules are overrepresented in the list of genes differentially expressed in SCARKO and control testes, we explored whether androgen action in SC might play a role in tubular remodeling and junction dynamics. Time patterns were analyzed for transcripts differentially expressed on d 10 and encoding potentially relevant proteins, such as proteases and protease inhibitors, cytoskeletal elements, extracellular matrix molecules, and cell adhesion molecules. The data summarized in Fig. 6Go illustrate that differentially expressed transcripts were found for each of these categories of molecules, and that for all transcripts, differential expression was observed at several time points. For many genes, including Eppin, PCI, tissue-type plasminogen activator (tPA), tubulin ß3 (Tubb3), procollagen type IV {alpha}6 (Col4a6), brevican (Bcan), claudin 11 (Cldn11), and desmocollin (Dsc2), differential expression was already evident on d 8. For other genes, such as actinin {alpha}3 (Actn3), scinderin (Scin), thrombospondin 1 (Thbs1), and embigin (Emb), differential expression was absent or marginal on d 8, but increased toward d 12. As explained above, the decline in relative signal intensity observed in control testes after d 12 may be due at least in part to the increase in the relative proportion of germ cells in the testis.


Figure 6
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  		Fig. 6. Expression Pattern of Genes Related to Tubular Remodeling and Junction Dynamics: Eppin, PCI, Tissue-Type Plasminogen Activator (tPA), Tubulin ß3 (Tubb3), Actinin {alpha}3 (Actn3), Scinderin (Scin), Procollagen Type IV {alpha}6 (Col4a6), Thrombospondin 1 (Thbs1), Brevican (Bcan), Claudin 11 (Cldn11), Embigin (Emb), and Desmocollin (Dsc2)

Relative signal intensities were calculated as explained in Fig. 2Go.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Recently developed transgenic models in which the AR is selectively inactivated in SC support the contention that SC play a pivotal role in the control of spermatogenesis by androgens (21, 22, 23). In this study we used the combination of such a model, the SCARKO mouse, and microarray technology to analyze the molecular consequences of selective absence of androgen action in SC at the onset of spermatogenesis.

SCARKO mice develop spermatogenic arrest in meiosis. Accordingly, to avoid confounding effects due to the marked differences in tissue composition once spermatogenesis proceeds beyond the spermatocyte stage, we selected d 10 as the main time point to search for genes differentially expressed in SCARKO and control testes. Earlier experiments on the temporal mRNA expression of Pem suggested a marked increase between d 7 and 9, reflecting the onset of androgen action in SC (27). Moreover, the present data show that 1) there are no differences in testis weight between SCARKO testes and controls on d 10; 2) apart from the absence of AR expression in SC from SCARKO testes, the morphology of the testes is comparable in SCARKO and control mice; and 3) the expression of typical meiotic genes (Dmc1, SCP3, Hsp70-2, Acrbp, etc.) is identical in d 10 SCARKO and control testes.

Microarray analysis identified a subset of 692 genes differentially expressed in SCARKO and control testes. Of these genes, 396 were apparently down-regulated in SCARKOs, and 296 were up-regulated. Although the list covered a variety of functions, analysis using Onto-express software revealed overrepresentation of some potentially important genes/functions, including genes encoding serine-type protease inhibitors (down-regulated in SCARKOs) and genes related to cell cycling, cell growth, cell-cell adherence, and signal transduction (up-regulated in SCARKOs). When the list was limited to genes with an at least 2-fold difference in expression level between SCARKOs and controls, 28 down-regulated and 12 up-regulated genes were retained, with genes encoding serine-type endopeptidase inhibitors prominently present in the subset of genes down-regulated in SCARKOs.

The detailed mechanisms responsible for the differential expression of individual genes in SCARKOs and controls require further investigation. Because the primary difference between SCARKO and control testes, however, resides in the absence or presence of a functional AR in SC, genes that are down-regulated in SCARKOs may reasonably be expected to depend on androgen action in SC for their physiological expression, whereas genes that are up-regulated in SCARKOs may be repressed by androgen action. Physiologically relevant levels of androgens are present in the testis and the circulation on d 10 (28, 29, 30, 31, 32). Moreover, the two genes showing the most pronounced difference in expression in the present experiments, Pem and Eppin, have both previously been identified as androgen target genes. Pem is a member of the Rhox cluster of homeobox genes (33). Its regulation by androgens in SC is well documented, and the androgen response elements (AREs) mediating regulation by the AR have been extensively characterized (34). Eppin is a member of a highly conserved family of protease inhibitors expressed in the testis and epididymis. The presence of an ARE in the promoter region and immunoexpression in SC from d 12 onward have been interpreted as indirect evidence of androgen regulation (35). Finally, for a subset of nine of the genes strongly down-regulated in SCARKO testes (Pem, Eppin, Galgt1, Drd4, Tsx, Gpd1, Tubb3, PCI, and Tpd52l1), we could demonstrate by quantitative RT-PCR that treatment with the antiandrogen flutamide for 5 d significantly decreased expression on d 12. Whether all of these genes are directly or indirectly regulated by androgens remains to be investigated. Similarly, the exact cell(s) expressing the identified genes remains to be identified. Quantitative RT-PCR measurements after enzymatic separation of tubular and interstitial tissue, however, suggest that the studied subset of nine genes is expressed predominantly in the tubular compartment.

Other experimental paradigms have been used to identify androgen-regulated genes in the testis. It is striking that apart from Pem, the differentially expressed genes detected in the present study show little overlap with androgen-regulated genes identified in two other studies recently performed in hypogonadal (hpg) and neonatal mice (36, 37). There may be several explanations for this apparent discrepancy. The SCARKO model may be expected to identify specifically genes whose expression depends on AR action in SC, whereas in the other models, effects induced by androgen administration may be mediated not only by SC, but also by other AR-positive testicular cells, such as peritubular myoid and Leydig cells. Moreover, in both the hpg model and the neonatal mouse studies, gene expression was studied early (4–24 h) after androgen administration. In our experimental setup, gene expression was studied on d 10. Because AR expression in SC is observed from d 3–5, it is conceivable that some of the observed effects are indirect and do not necessarily imply direct interaction of the activated AR with an ARE in the relevant gene. Finally, in the SCARKO model we are looking at the effects of physiological amounts of endogenous androgens, whereas in the other models high amounts of exogenous androgens are administered. The absence of FSH could be an additional factor affecting the results in the hpg model.

The physiological relevance of the genes identified by use of the SCARKO model is underscored by the finding that three of the genes found to be markedly down-regulated in SCARKOs (Galgt1, Eppin, and PCI) have already been shown to be essential for normal spermatogenesis and fertility. Eppin is a serine protease inhibitor discussed above. Immunization of male monkeys against Eppin results in reversible contraception, but the mechanism underlying this effect remains to be defined (38). Gene inactivation of ß1,4-N-acetylgalactosaminyltransferase (Galgt1) or PCI results in severe disruption of spermatogenesis. Galgt1 encodes an enzyme involved in the formation of complex gangliosides, and the infertility of Galgt1–/– mice may be related to a disturbance in the production of a specific class of fucosylated glycosphingolipids that are apparently essential for spermatogenesis (39, 40). PCI encodes a serine-type protease inhibitor, and spermatogenic failure in PCI–/– mice has been related to disruption of the blood-testis barrier (41).

To search for genes with a particular relevance to the onset of spermatogenesis and the presence or absence of AR activation in SC we also studied gene expression in SCARKO and control testes between d 8 and 20 using microarray techniques and cluster analysis. Cluster analysis of the subset of genes known to be differentially expressed on d 10 revealed three clusters that may be particularly relevant. Two of these (clusters 3 and 4) identified subgroups of genes displaying higher expression levels in mice with an active AR in SC, but showing a different pattern of change with time. Interestingly, these two clusters encompassed 71% of the genes down-regulated more than 2-fold in SCARKOs. Cluster 5 encompassed 42% of the genes identified as strongly up-regulated in SCARKOs. Pem, which can be considered a lead gene in the search for androgen-regulated genes in SC, was found in cluster 4. A more detailed analysis of cluster 4, using hierarchical clustering, revealed that several of the genes showing the highest up-regulation on d 10 (Eppin, Drd4, and Gpd1) clustered in the immediate vicinity of Pem. Quantitative RT-PCR confirmed that the expression of these genes strongly depended on activation of the AR in SC and that their expression pattern mimicked that of Pem. Eppin has been discussed above, and the present data are the first to show directly that androgen action in SC enhances its expression in the testis. We can only speculate about the roles of Drd4 and Gpd1 in the testis. Drd4 encodes a G protein-coupled receptor that is strongly expressed in the testis, but no testicular phenotype was reported in mice lacking Drd4 (42, 43). Gpd1 encodes a reduced nicotinamide adenine dinucleotide-dependent enzyme involved in glycerol synthesis. Gpd1 is ubiquitously expressed, but its promoter appears to be highly methylated, specifically in sperm (44). Additional research will be required to clarify the roles and the relationship of these genes to androgen action in SC.

There is increasing evidence that the interplay between proteases and protease inhibitors plays an important role in the restructuring of testicular tubules at the onset of spermatogenesis and in the assembly and functional dynamics of tight and adhesion junctions between SC and germ cells (45, 46). Our data seem to support this contention and provide preliminary evidence that the AR in SC may play a more prominent role in the control of these processes than previously suspected. Both protease inhibitors and cell adhesion molecules were overrepresented in the genes differentially expressed in SCARKO and control testes. Moreover, the time study showed that not only protease inhibitors and cell adhesion molecules, but also cytoskeletal and extracellular matrix elements, showed divergent expression patterns at the onset of spermatogenesis.

In conclusion, the present study has identified a subset of genes that are differentially expressed in d 10 testes with or without an active AR in SC. Some of the identified genes have previously been shown to be relevant for spermatogenesis or to be controlled by androgens. Of particular interest is the finding that some genes that may be related to tubular remodeling and junction dynamics are affected by androgen action in SC. Moreover, three genes with an expression pattern strongly resembling that of the lead gene, Pem, have been identified. These findings provide a basis for additional dissection of the molecular mechanisms by which androgen action in SC affects spermatogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Tissue Sampling and RNA Extraction
To obtain SCARKO animals, female mice (129/Swiss) heterozygous for the floxed AR allele (ARflox/+) (21) were mated to male mice (C57BL/6) homozygous for the AMH-Cre transgene (24). These AMH-Cre transgene animals were also used as a control for SCARKO animals. All animals were treated according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all experiments were approved by the local ethical committee. Whole testes were obtained from animals at different ages depending on the experiment. Immediately after removal, testis samples were snap-frozen and stored in liquid nitrogen. Before RNA extraction, testis samples were weighed and homogenized in a Dounce homogenizer (Kontes Co., Vineland, NJ). Thereafter, RNA was isolated with the RNeasy Kit (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions, encompassing an on-column deoxyribonuclease I (DNase I) treatment of the RNA. For quantitative RT-PCR analyses, 5 ng luciferase mRNA (Promega Corp., Madison, WI) were added to each testis sample before RNA preparation as an external standard (47).

Immunohistochemistry
Standard procedures were used for hematoxylin and eosin staining. A rabbit polyclonal antibody (N20) raised against a peptide within the N-terminal domain of the human AR (sc-816, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used at a 1:500 dilution. Rabbit anti-Dazl antibody was a gift from Dr. H. J. Cooke (Medical Research Council Human Genetics Unit, Edinburgh, UK) and was used at a 1:1000 dilution. Testes from d 10 control and SCARKO males were removed, weighed, fixed in Bouin’s fluid for 4–6 h, and transferred into 70% (vol/vol) ethanol before processing into paraffin wax using standard methods. Immunohistochemistry was performed on dewaxed 5-µm sections in conjunction with heat-induced antigen retrieval for 5 min in 0.01 M citrate buffer (pH 6.0; Sigma-Aldrich Corp., Dorset, UK) using a pressure cooker. A swine antirabbit biotinylated second antibody (E0353, DakoCytomation, Cambridge, UK) was used, and details of the immunohistochemistry protocol were reported previously (21).

To enable comparative evaluation of the immunostaining, sections of tissues from control and knockout animals were processed in parallel on at least three occasions to ensure reproducibility of results; on each occasion, tissue sections from four to six animals in each group were run. To ensure direct comparability of staining intensities, one section each from control and SCARKO mouse was mounted on the same slide for the immunostaining procedure.

Microarray Target Synthesis and Processing
The quality of all testis RNA samples was monitored by measuring the 260/280 and 260/230 nm ratios with a NanoDrop spectrophotometer (NanoDrop Technologies, Centreville, DE) and by means of the Agilent 2100 BioAnalyzer (Agilent, Palo Alto, CA). Only RNA showing no signs of degradation or impurities (260/280 and 260/230 nm ratios, >1.8) was considered suitable for microarray analysis.

Before target synthesis, RNA extracts from one testis of three control or three SCARKO littermates were pooled, creating paired control and SCARKO samples optimally adjusted for potential variation between litters. Five micrograms of polyadenylated RNA from each pool was reverse transcribed into double-stranded DNA with a polydeoxythymidine-T7 primer using SuperScript II RT, ribonuclease H, and DNA polymerase I (Invitrogen Life Technologies, Inc., Carlsbad, CA). From this double-stranded DNA, biotin-labeled target aRNA was generated in a T7 in vitro transcription reaction using the Affymetrix IVT Labeling Kit (Affymetrix, High Wycombe, UK). After purification (GeneChip Sample Cleanup Module, Affymetrix), yield (30–120 µg) and purity (260/280 and 260/230 nm ratios, >1.8) of the labeled amplified RNA were analyzed, 20 µg quality-controlled amplified RNA was fragmented by alkaline hydrolysis and resuspended with control spikes in 300 µl hybridization buffer (Eukaryotic Hybridization Control Kit, Affymetrix). Of this probe solution, 200 µl was used for hybridization in a rotisserie oven at 45 C.

The mouse expression array 430a 2.0 from Affymetrix was used in all hybridizations. This array contains 22,690 transcripts from Mus musculus, each represented by 11 probe pairs. Each pair consists of a perfect match and a mismatch (with a single base point mutation) 25-mer oligonucleotide probe.

After hybridization, gene chips were washed and stained in the GeneChip Fluidics Workstation 400 (Affymetrix) using the EukGE-WS2v4 protocol and subsequently scanned with the GeneChip Scanner 3000 (Affymetrix). Image analysis was performed by use of Affymetrix GeneChip operating software.

Microarray Data Analysis
A first experiment was set up to identify differentially expressed genes in the testis of 10-d-old control vs. SCARKO mice. To this end, five control and five SCARKO samples were compared. The data were considered paired because the samples consisted two-by-two of testicular RNA from littermate mice (see microarray target synthesis and processing section). In an initial phase, results were analyzed using Affymetrix Microarray Suite (MAS) 5.0 software. The amount of transcripts in a sample that scored present, absent, or marginally detectable was assessed. MAS 5.0 software was also used to generate expression ratios after comparison of a control dataset with its littermate SCARKO dataset. For additional statistical analysis, all data were first normalized using the robust multiarray analysis algorithm (RMA) (48, 49) as implemented in the BioConductor packages (www.bioconductor.org) (50). Three statistical approaches, all part of the BioConductor software, were used to identify differentially expressed genes in testis of control vs. SCARKO mice: 1) significance analysis for microarrays (51), 2) a paired t test controlling the false discovery rate using the Benjamini and Hochberg correction (52), and 3) the moderated t test as described in linear models for microarray data (53) also encompassing the Benjamini and Hochberg correction. A threshold was set for each test, generating three lists of differentially expressed transcripts (significance analysis for microarrays: false discovery rate, P ≤ 0.05; paired t test and Benjamini-Hochberg correction, P ≤ 0.05; linear models for microarray data and Benjamini-Hochberg correction, P ≤ 0.05). A cross-section of these lists was obtained in Access (Microsoft Corp., Redmond, WA), yielding a summary of transcripts that were depicted as differentially expressed in a statistically significant way by all three tests. This list, which contained 848 transcripts, was used for further analysis. Onto-Express software (http://vortex.cs.wayne.edu/Projects.html#Onto-Express) (25) was used to check for statistical overrepresentation of certain molecular or biological functions among the differentially expressed transcripts.

In a second experiment, we tried to identify typical expression patterns during initiation of spermatogenesis for the list of differentially expressed transcripts generated. Hence, gene expression was assessed in one control and one littermate SCARKO sample at five different ages (d 8, 10, 12, 16, and 20). After a first data analysis using MAS 5.0 software, as described above, data were again RMA normalized for additional statistical analysis. An adaptive quality-based clustering using a heuristic iterative two-step algorithm (54), as implemented in the INCLUSive package of web-based tools (www.esat.kuleuven.ac.be/~dna/BioI/Software.html), was used in the search for expression patterns shared by genes that were differentially expressed in d 10 animals. This method is similar to k-means clustering, but has two advantages. First, no prior choice of the number of clusters is required, and second, not every gene is forced into a cluster. Only significantly coexpressed genes are retained in a cluster after derivation of its optimal radius. One of the resulting clusters was further studied by calculating and visualizing pairwise Euclidian distances between centered profiles using hierarchical clustering as implemented in Expression Profiler (http://ep.ebi.ac.uk) (26). Functional analysis of the clusters was performed based on Onto-Express statistics.

In Vivo Treatment with Androgens and Antiandrogens
Seven-day-old C57BL/6@Rj mice (Janvier, Le Genest-St-Isle, France) were treated for 3 or 5 d with TP (50 µg/animal·d), Flut (50 µg/animal·d), or vehicle only. TP and Flut were purchased from Fluka Chemie AG (Buchs, Switzerland) and Sigma-Aldrich Corp. Stock solutions were made in ethanol and then further diluted in arachis oil for injection. In all conditions a volume of 50 µl containing 10% ethanol was injected sc. Treatment groups consisted of five animals. Mice were killed on d 10 or 12, and testes were removed and additionally processed for quantitative RT-PCR as described above.

Enzymatic Separation of Interstitial and Tubular Fractions
Interstitial and tubular fractions were prepared through enzymatic digestions essentially as described previously (55). Twenty testes, derived from 10-d-old C57BL/6@Rj mice (Janvier) were decapsulated and digested for 10 min with trypsin (0.67 mg/ml) and DNase (6.7 µg/ml) in a shaking water bath (120 oscillations/min) at 32 C. The action of trypsin was stopped by the addition of 2 mg/ml soybean trypsin inhibitor. After sedimentation of the tubular fragments, the supernatant was collected as the interstitial fraction, and the sedimented tubular fragments were treated for an additional 5 min at 32 C with collagenase (0.67 mg/ml), hyaluronidase (0.67 mg/ml), and DNase (10 µg/ml). The remaining tubular fragments were treated once more with hyaluronidase (0.67 mg/ml) and DNase (10 µg/ml) for about 15 min in a shaking water bath (120 oscillations/min) to remove the remaining peritubular cells. The resulting tubular fraction as well as the above-generated interstitial fraction were centrifuged, snap-frozen in liquid nitrogen, and stored at –80 C until RNA preparation. RNA was isolated using the Qiagen RNeasy kit with an on-column DNase I treatment as described above.

Quantitative RT-PCR
cDNA was synthesized using SuperScript II reverse transcriptase and random hexamer primers (Invitrogen Life Technologies, Inc.). Primer sequences for Eppin, Galgt1, Drd4, Tsx, Gpd1, Tubb3, PCI, Tpd52l1, and SCP3 are described in Table 1Go. Pem primer and probe sequences and 17{alpha}-hydroxylase (P450c17) primer sequences were described previously (21, 31). For quantification of gene expression, the ABI PRISM 7700 sequence detector PCR detection system (Applied Biosystems, Foster City, CA) was used with a two-step quantitative RT-PCR protocol. Components for real-time PCR, apart from primers and probes (Eurogentec, Sar-Tilman, Belgium) and SYBR Green (Sigma-Aldrich Corp.), were obtained from Applied Biosystems. For Eppin, Galgt1, Drd4, Tsx, Gpd1, Tubb3, PCI, Tpd52l1, SCP3, and P450c17, each 25-µl real-time PCR contained 1x buffer A, 5 mM MgCl2, 400 µM deoxy-NTPs, 200 nM of each primer, 0.4x SYBR Green, and 0.025 U/µl AmpliTaq Gold enzyme. The uniqueness of the amplified band was checked by PAGE. For Pem, no SYBR Green was used; instead, 100 nM of each primer and 400 nM probe were added to the reaction. The quantity of measured mRNA was expressed relative to the external luciferase mRNA standard added before RNA preparation or to the 18S rRNA present in the same sample. All samples and standard curves were run in triplicate.


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  		Table 1. Oligonucleotide Primers Used for Quantitative RT-PCR

 


   ACKNOWLEDGMENTS
 
We thank Ludo Deboel, Ruth Maes, Kirsten De Schouwer, and Tom Bogaerts for their excellent technical assistance.


   FOOTNOTES
 
This work was supported by a Concerted Research Action (Research Fund, Katholieke Universiteit Leuven) and the Fund for Scientific Research Flanders (Belgium).

First Published Online September 15, 2005

Abbreviations: AMH-Cre, Cre expression under control of the anti-Mullerian hormone promoter; AR, androgen receptor; ARE, androgen response element; Dazl, deleted in azoospermia-like; DNase, deoxyribonuclease; Flut, flutamide; hpg, hypogonadal; Hsp, heat shock protein; MAS, Microarray Suite; PCI, protein C inhibitor; RMA, robust multi-array analysis algorithm; SC, Sertoli cell; SCARKO, Sertoli cell-selective androgen receptor knockout; TP, testosterone proprionate.

Received for publication March 7, 2005. Accepted for publication September 8, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Sharpe RM 1994 Regulation of spermatogenesis. In: Knobil E, Neill JD, eds. The physiology of reproduction. 2nd ed. New York: Raven Press; 1363–2434
  2. McLachlan RI 2000 The endocrine control of spermatogenesis. Baillieres Best Pract Res Clin Endocrinol Metab 14:345–362[CrossRef][Medline]
  3. Singh J, O’Neill C, Handelsman DJ 1995 Induction of spermatogenesis by androgens in gonadotropin-deficient (hpg) mice. Endocrinology 136:5311–5321[Abstract]
  4. Kumar TR, Wang Y, Lu N, Matzuk MM 1997 Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nat Genet 15:201–204[CrossRef][Medline]
  5. Dierich A, Sairam MR, Monaco L, Fimia GM, Gansmuller A, LeMeur M, Sassone-Corsi P 1998 Impairing follicle-stimulating hormone (FSH) signaling in vivo: targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance. Proc Natl Acad Sci USA 95:13612–13617[Abstract/Free Full Text]
  6. Abel MH, Wootton AN, Wilkins V, Huhtaniemi I, Knight PG, Charlton HM 2000 The effect of a null mutation in the follicle-stimulating hormone receptor gene on mouse reproduction. Endocrinology 141:1795–1803[Abstract/Free Full Text]
  7. Grootegoed JA, Peters MJ, Mulder E, Rommerts FF, Van der Molen HJ 1977 Absence of a nuclear androgen receptor in isolated germinal cells of rat testis. Mol Cell Endocrinol 9:159–167[CrossRef][Medline]
  8. Lyon MF, Glenister PH, Lamoreux ML 1975 Normal spermatozoa from androgen-resistant germ cells of chimaeric mice and the role of androgen in spermatogenesis. Nature 258:620–622[CrossRef][Medline]
  9. Johnston DS, Russell LD, Friel PJ, Griswold MD 2001 Murine germ cells do not require functional androgen receptors to complete spermatogenesis following spermatogonial stem cell transplantation. Endocrinology 142:2405–2408[Abstract/Free Full Text]
  10. O’Donnell L, Robertson KM, Jones ME, Simpson ER 2001 Estrogen and spermatogenesis. Endocr Rev 22:289–318[Abstract/Free Full Text]
  11. Silva FR, Leite LD, Wassermann GF 2002 Rapid signal transduction in Sertoli cells. Eur J Endocrinol 147:425–433[Abstract]
  12. Fix C, Jordan C, Cano P, Walker WH 2004 Testosterone activates mitogen-activated protein kinase and the cAMP response element binding protein transcription factor in Sertoli cells. Proc Natl Acad Sci USA 101:10919–10924[Abstract/Free Full Text]
  13. Bremner WJ, Millar MR, Sharpe RM, Saunders PT 1994 Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens. Endocrinology 135:1227–1234[Abstract]
  14. Schlatt S, Weinbauer GF, Arslan M, Nieschlag E 1993 Appearance of {alpha}-smooth muscle actin in peritubular cells of monkey testes is induced by androgens, modulated by follicle-stimulating hormone, and maintained after hormonal withdrawal. J Androl 14:340–350[Abstract/Free Full Text]
  15. Skinner MK, Fetterolf PM, Anthony CT 1988 Purification of a paracrine factor, P-Mod-S, produced by testicular peritubular cells that modulates Sertoli cell function. J Biol Chem 263:2884–2890[Abstract/Free Full Text]
  16. Verhoeven G, Cailleau J 1988 Testicular peritubular cells secrete a protein under androgen control that inhibits induction of aromatase activity in Sertoli cells. Endocrinology 123:2100–2110[Abstract]
  17. Jégou B, Pineau C 1995 Current aspects of autocrine and paracrine regulation of spermatogenesis. Adv Exp Med Biol 377:67–86[Medline]
  18. Griswold MD 1995 Interactions between germ cells and Sertoli cells in the testis. Biol Reprod 52:211–216[Abstract]
  19. Roberts K, Griswold MD 1989 Testosterone induction of cellular proteins in cultured Sertoli cells from hypophysectomized rats and rats of different ages. Endocrinology 125:1174–1179[Abstract]
  20. Sharpe RM, Maddocks S, Millar M, Kerr JB, Saunders PT, McKinnell C 1992 Testosterone and spermatogenesis. Identification of stage-specific, androgen-regulated proteins secreted by adult rat seminiferous tubules. J Androl 13:172–184[Abstract/Free Full Text]
  21. De Gendt K, Swinnen JV, Saunders PT, Schoonjans L, Dewerchin M, Devos A, Tan K, Atanassova N, Claessens F, Lécureuil C, Heyns W, Carmeliet P, Guillou F, Sharpe RM, Verhoeven G 2004 A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis. Proc Natl Acad Sci USA 101:1327–1332[Abstract/Free Full Text]
  22. Holdcraft RW, Braun RE 2004 Androgen receptor function is required in Sertoli cells for the terminal differentiation of haploid spermatids. Development 131:459–467[Abstract/Free Full Text]
  23. Chang C, Chen YT, Yeh SD, Xu Q, Wang RS, Guillou F, Lardy H, Yeh S 2004 Infertility with defective spermatogenesis and hypotestosteronemia in male mice lacking the androgen receptor in Sertoli cells. Proc Natl Acad Sci USA 101:6876–6881[Abstract/Free Full Text]
  24. Lécureuil C, Fontaine I, Crepieux P, Guillou F 2002 Sertoli and granulosa cell-specific Cre recombinase activity in transgenic mice. Genesis 33:114–118[CrossRef][Medline]
  25. Draghici S, Khatri P, Martins RP, Ostermeier GC, Krawetz SA 2003 Global functional profiling of gene expression. Genomics 81:98–104[CrossRef][Medline]
  26. Vilo J, Kivinen K 2001 Regulatory sequence analysis: application to the interpretation of gene expression. Eur Neuropsychopharmacol 11:399–411[CrossRef][Medline]
  27. Lindsey JS, Wilkinson MF 1996 Pem: a testosterone- and LH-regulated homeobox gene expressed in mouse Sertoli cells and epididymis. Dev Biol 179:471–484[CrossRef][Medline]
  28. Jean-Faucher C, Berger M, de Turckheim M, Veyssiere G, Jean C 1978 Developmental patterns of plasma and testicular testosterone in mice from birth to adulthood. Acta Endocrinol (Copenh) 89:780–788[Medline]
  29. Al Attar L, Noel K, Dutertre M, Belville C, Forest MG, Burgoyne PS, Josso N, Rey R 1997 Hormonal and cellular regulation of Sertoli cell anti-Mullerian hormone production in the postnatal mouse. J Clin Invest 100:1335–1343[Medline]
  30. O’Shaughnessy PJ, Baker P, Sohnius U, Haavisto AM, Charlton HM, Huhtaniemi I 1998 Fetal development of Leydig cell activity in the mouse is independent of pituitary gonadotroph function. Endocrinology 139:1141–1146[Abstract/Free Full Text]
  31. O’Shaughnessy PJ, Baker PJ, Heikkila M, Vainio S, McMahon AP 2000 Localization of 17ß-hydroxysteroid dehydrogenase/17-ketosteroid reductase isoform expression in the developing mouse testis: androstenedione is the major androgen secreted by fetal/neonatal Leydig cells. Endocrinology 141:2631–2637[Abstract/Free Full Text]
  32. Okamoto S, Takatsuka D, Tateishi K, Ogasawara Y, Yamane T, Kitamura Y, Matsumoto K 1982 Proliferative pattern of seminal vesicle cells and the production of testosterone and 5{alpha}-androgens from birth to adulthood in mice. Endocrinology 111:1230–1234[Abstract]
  33. Maclean JA, Chen MA, Wayne CM, Bruce SR, Rao M, Meistrich ML, Macleod C, Wilkinson MF 2005 Rhox: a new homeobox gene cluster. Cell 120:369–382[CrossRef][Medline]
  34. Barbulescu K, Geserick C, Schuttke I, Schleuning WD, Haendler B 2001 New androgen response elements in the murine pem promoter mediate selective transactivation. Mol Endocrinol 15:1803–1816[Abstract/Free Full Text]
  35. Richardson RT, Sivashanmugam P, Hall SH, Hamil KG, Moore PA, Ruben SM, French FS, O’Rand M 2001 Cloning and sequencing of human Eppin: a novel family of protease inhibitors expressed in the epididymis and testis. Gene 270:93–102[CrossRef][Medline]
  36. Sadate-Ngatchou PI, Pouchnik DJ, Griswold MD 2003 Identification of testosterone regulated genes in testes of hypogonadal mice using oligonucleotide microarray. Mol Endocrinol 18:422–433
  37. Zhou Q, Shima JE, Nie R, Friel PJ, Griswold MD 2004 Androgen-regulated transcripts in the neonatal mouse testis as determined through microarray analysis. Biol Reprod 72:1010–1019
  38. O’Rand MG, Widgren EE, Sivashanmugam P, Richardson RT, Hall SH, French FS, VandeVoort CA, Ramachandra SG, Ramesh V, Jagannadha RA 2004 Reversible immunocontraception in male monkeys immunized with eppin. Science 306:1189–1190[Abstract/Free Full Text]
  39. Takamiya K, Yamamoto A, Furukawa K, Zhao J, Fukumoto S, Yamashiro S, Okada M, Haraguchi M, Shin M, Kishikawa M, Shiku H, Aizawa S, Furukawa K 1998 Complex gangliosides are essential in spermatogenesis of mice: possible roles in the transport of testosterone. Proc Natl Acad Sci USA 95:12147–12152[Abstract/Free Full Text]
  40. Sandhoff R, Geyer R, Jennemann R, Paret C, Kiss E, Yamashita T, Gorgas K, Sijmonsma TP, Iwamori M, Finaz C, Proia RL, Wiegandt H, Grone HJ 2005 Novel class of glycosphingolipids involved in male fertility. J Biol Chem 280:27310–27318[Abstract/Free Full Text]
  41. Uhrin P, Dewerchin M, Hilpert M, Chrenek P, Schofer C, Zechmeister-Machhart M, Kronke G, Vales A, Carmeliet P, Binder BR, Geiger M 2000 Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility. J Clin Invest 106:1531–1539[Medline]
  42. Rubinstein M, Phillips TJ, Bunzow JR, Falzone TL, Dziewczapolski G, Zhang G, Fang Y, Larson JL, McDougall JA, Chester JA, Saez C, Pugsley TA, Gershanik O, Low MJ, Grandy DK 1997 Mice lacking dopamine D4 receptors are supersensitive to ethanol, cocaine, and methamphetamine. Cell 90:991–1001[CrossRef][Medline]
  43. Falzone TL, Gelman DM, Young JI, Grandy DK, Low MJ, Rubinstein M 2002 Absence of dopamine D4 receptors results in enhanced reactivity to unconditioned, but not conditioned, fear. Eur J Neurosci 15:158–164[CrossRef][Medline]
  44. Gwynn B, Lyford KA, Birkenmeier EH 1990 Sequence conservation and structural organization of the glycerol-3-phosphate dehydrogenase promoter in mice and humans. Mol Cell Biol 10:5244–5256[Abstract/Free Full Text]
  45. Mruk DD, Cheng CY 2004 Sertoli-Sertoli and Sertoli-germ cell interactions and their significance in germ cell movement in the seminiferous epithelium during spermatogenesis. Endocr Rev 25:747–806[Abstract/Free Full Text]
  46. Odet F, Guyot R, Leduque P, Magueresse-Battistoni B 2004 Evidence for similar expression of protein C inhibitor and the urokinase-type plasminogen activator system during mouse testis development. Endocrinology 145:1481–1489[Abstract/Free Full Text]
  47. Tan KA, De Gendt K, Atanassova N, Walker M, Sharpe RM, Saunders PT, Denolet E, Verhoeven G 2005 The role of androgens in Sertoli cell proliferation and functional maturation: studies in mice with total or Sertoli cell-selective ablation of the androgen receptor. Endocrinology 146:2674–2683[Abstract/Free Full Text]
  48. Bolstad BM, Irizarry RA, Astrand M, Speed TP 2003 A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19:185–193[Abstract/Free Full Text]
  49. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP 2003 Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4:249–264[Abstract]
  50. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J 2004 Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5:R80
  51. Tusher VG, Tibshirani R, Chu G 2001 Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98:5116–5121[Abstract/Free Full Text]
  52. Benjamini Y, Hochberg Y 2004 Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B 57:289–300
  53. Smyth G 2004 Linear models and empirical based methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3:3
  54. De Smet F, Mathys J, Marchal K, Thijs G, De Moor B, Moreau Y 2002 Adaptive quality-based clustering of gene expression profiles. Bioinformatics 18:735–746[Abstract/Free Full Text]
  55. Hoeben E, Swinnen JV, Heyns W, Verhoeven G 1999 Heregulins or neu differentiation factors and the interactions between peritubular myoid cells and Sertoli cells. Endocrinology 140:2216–2223[Abstract/Free Full Text]

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