| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
androgen receptor, mechanisms of hormone action, Sertoli cells, testis, testosterone
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-reduced product of T, dihydrotestosterone (DHT), produced both gonadally and locally, is the major androgen directing the development of Wolffian duct structures and is responsible for normal function of the epididymis, vas deferens, and prostate [2, 9]. The action of both T and DHT is mediated via the nuclear AR. This receptor belongs to a large superfamily of nuclear receptors and is a ligand-activated transcription factor. Androgens regulate gene expression via several known mechanisms. First, primary response genes are transcriptionally activated/suppressed rapidly following binding of ligand-activated AR to androgen-response elements (AREs) in their 5'-flanking regulatory regions. This is a direct response that does not require de novo protein synthesis. Second, the delayed primary response genes with AREs respond to androgen stimulation via AR binding, but the maximal response requires interaction with cofactors and ongoing protein synthesis. Third, secondary response genes lacking AREs also can respond indirectly to androgen stimulation via activation or suppression of transcription by other factors directly regulated by androgens. This response requires de novo protein synthesis and, therefore, has a temporal delay [3, 10].
The testis is not only the major site of T production but also one of the main targets of T action [2]. The AR has been localized exclusively in testicular somatic cells in a number of animal species and humans during development and in adulthood. These data have been obtained by various techniques, such as immunohistochemistry (IHC), in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) [11, 12]. The data concerning receptor localization is in good agreement with the results of a germ cell transplantation study that clearly demonstrated murine germ cells do not require functional ARs to complete spermatogenesis [13]. Therefore, the indispensable action of androgens to support spermatogenesis is mediated through the testicular somatic cells rather than being mediated by directly regulating germ cells.
In the testis, three types of somatic cells are under direct androgen regulation: Sertoli cells, peritubular myoid cells, and Leydig cells [11]. Expression of AR in Sertoli cells is stage-specific, but how this expression pattern and T action correlate with the function of Sertoli cells is not well understood. Only a limited number of androgen-regulated genes in Sertoli cells have been studied [14–16]. Further studies are required for a better understanding of T regulation in this important nursing cell. In Leydig cells, T is synthesized under LH control in adult males. However, Leydig cells also may be the target cells of T action by an autocrine regulation. It generally is accepted that circulating T (and/or locally converted estrogen) regulates the synthesis/secretion of LH and FSH. This feedback regulation of T production also may occur locally in the testis, and T may directly regulate the transcription of certain steroidogenic enzymes in Leydig cells, such as CYP17a1 (P450C17), which has been well-characterized in vitro [17]. However, more studies are needed to understand the autocrine regulation in Leydig cells. At present, little is known about T effects in peritubular myoid cells.
In summary, the importance of T for spermatogenesis has been substantiated, but the underlying molecular mechanism of T regulation (i.e., the genes that are regulated and their functions), particularly in each testicular somatic cell type, is largely unknown. Here, we describe using the neonatal male mouse without advanced germ cells to study T regulation examined by oligonucleotide microarray technology. The similarity of this in vivo model with adults concerning the aspects of androgen regulation of the testes also is investigated and discussed.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
All animal experiments were approved by Washington State University Animal Care and Use committees and were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals of the National Institutes of Health. A BL/6-129 mouse colony was maintained in a temperature- and humidity-controlled room with food and water provided ad libitum. Neonatal male mice at 8 days postpartum (dpp; day of birth = Day 0) were separated into groups of five or six animals. In all treatments, injections were made s.c. in 50 µl of sesame oil vehicle. The mice in T-treated groups received an injection of 0.5 mg of T propionate (TP) or DHT (Sigma Chemical Co., St. Louis, MO) [16, 18]. The mice in the control groups received an injection of vehicle alone. At 4, 8, and 16 h after injection, animals were killed, and testes were harvested and immediately placed in TRIzol reagent (Invitrogen, San Diego, CA). For each independent RNA sample applied to the microarray, RNA was pooled from testes of five to six mice. Two independent RNA replicates were used for microarray analysis. In a separate study, flutamide, an antagonist of the AR, was used in adult male mice to block androgen action. In the flutamide-treated experiment, adult mice (age, 70–90 dpp) were given either one injection of 100 µl of flutamide (2 mg/mouse) or control vehicle (sesame oil). The mice were killed at 4, 16, and 24 h after injection, and testes were harvested and placed in TRIzol reagent.
IHC of AR and Germ Cell Nuclear Antigen 1
A rat anti-mouse immunoglobulin M for germ cell nuclear antigen (GNCA) 1 was a gift from George Enders (University of Kansas, Kansas City, KS), and Dr. Gail Prins (University of Illinois, Chicago, IL) kindly provided the AR-specific rabbit anti-human antibody PG21. The IHC of AR and GCNA1 was performed as previously described [11]. Briefly, testes of male mice (age, 8 days) were immersion-fixed with 10% neutral buffered formalin overnight at 4°C, transferred to 70% ethanol, and embedded in paraffin. Sections were cut (thickness, 5 µm) and dried at 37°C overnight. To unmask the AR protein, sections were microwaved in a 0.01 M citrate buffer solution (pH 6.0) for 20 min. Slides were incubated with either the AR-specific antibody PG21 or the GCNA1 antibody diluted 1: 500 with PBS. Results were visualized using the avidin-biotin complex (ABC Kit; Vector Laboratories, Burlingame, CA) and the diaminobenzidine chromogen. Hematoxylin (Sigma Chemical Co.) was applied as a counterstain. The AR21 (AR peptide) was used to perform antibody competition. Briefly, a 10- to 15-fold molar excess of peptide was incubated together with antibody overnight at 48°C, and then sections incubated with the AR21-preabsorbed primary antibody served as negative controls for color development on the same slide. Images were captured with an Olympus OLY-200 digital camera (Olympus America) using Olympus MagnaFire Camera Imaging and Control (version 1.0; Olympus America) and compiled using Adobe PhotoShop 7.0 (Adobe Systems, San Jose, CA).
Microarray Processing
Total RNA was extracted according to the manufacturer's instructions. Quality of RNA was determined by electrophoretic methods using a denaturing agarose gel or analysis using an Agilent Bioanalyzer 2100 (Palo Alto, CA) and by spectroscopy at 260 and 280 nm. The RNA with a 260: 280 ratio of 1.8 or higher was used for DNA microarray analysis. Ten micrograms of total RNA were used to create the target for the microarray. "Target" refers to biotinylated cRNA created from the total RNA, whereas "probe" refers to the oligonucleotides synthesized on the microarray platform. The biotinylated cRNA was generated using an oligo(dT) primer in an RT reaction followed by in vitro transcription using the MEGAScript kit (Ambion, Austin, TX) with biotinylated cytosine and uridine triphosphate. The labeled cRNA was fragmented, hybridized to MOE 430A and 430B arrays (Affymetrix, Santa Clara, CA), and stained in accordance with the manufacturer's standard protocol. The same procedure was performed on duplicate samples. The arrays were stained and washed by the Affymetrix GeneChip Fluidics Station 400 and scanned using a GeneArray Scanner 2500A (Agilent, Palo Alto, CA). The resulting data were viewed and a preliminary assessment made using Microarray Suite 5.0 (MAS) software (Affymetrix). All reactions and microarray hybridization procedures were performed in the Laboratory for Biotechnology and Bioanalysis I at Washington State University.
Absolute and Statistical Analysis
Microarray output was examined visually for excessive background noise and physical anomalies. The default MAS statistical values were used for all analyses, and all probe sets on each array were scaled to a mean target signal intensity of 125. An absolute analysis using MAS was performed to assess the relative abundance of the approximately 39 000 represented transcripts based on signal and detection (present, absent, or marginal). The absolute analysis from MAS was imported into GeneSpring 6.1 (Silicon Genetics, Redwood City, CA). The time-course data were normalized within GeneSpring using the default/recommended normalization methods. These included setting of signal values less than 0.01 to 0.01, total chip normalization to the 50th percentile, and normalization of each gene to the median. These normalizations allowed for the visualization of data based on relative abundance at any given time point rather than compared to a specific control value. Transcripts expressed differentially at a statistically significant level were determined by a one-way ANOVA parametric test using all available error estimates and a P-value cutoff of 0.05. Subsequently, certain criteria were applied in the comparison analysis. The criteria for "TP-regulated" genes included a raw signal value of no less than 50 in either the control or the treated samples, a twofold or higher change in signal between the control and treated samples, and statistically significant difference based on the one-way ANOVA test.
Cell Culture and Treatment
Sertoli cells were isolated from mice (age, 14–18 days) as described by Karl and Griswold [19]. Mouse Sertoli cells were cultured in tissue-culture dishes and maintained in serum-free media with 50% (v/v) Ham F-12 medium (Gibco BRL Life Sciences, Grand Island, NY) and 50% (v/ v) Dulbecco modified Eagle medium (Sigma-Aldrich, St. Louis, MO) at 32°C in an atmosphere of 95% air and 5% CO2. Cells were allowed to attach to the culture dishes for 2 days before use. The purity of the Sertoli cells was 95% or greater. After 2 days of culture, the medium was replaced with fresh medium containing TP or vehicle for controls. The TP used in all treatments was dissolved in absolute ethanol and added to cultures at a final concentration of 2 x 10–7 M. Three independent time-course experiments using three separate cell samples at each time point were performed. Treated and untreated control Sertoli cells were harvested at 2, 4, 8, 16, and 24 h. The RNA was purified as the manufacturer's instructions.
Real-Time Polymerase Chain Reaction (PCR)
A two-step, real-time reverse transcription (RT)-PCR was used to measure the expression of candidate genes as previously described [16]. Total RNA (2 µg) was reverse-transcribed into cDNA in a 20-µl reaction containing random primers and Superscript III reverse transcriptase (Invitrogen, San Diego, CA) according to the manufacturer's instructions. Reverse and forward oligonucleotide primers, specific to the chosen candidate genes, were designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA) as described by the manufacturer. The primer pairs were designed as follows: Pem forward primer, 5'-CAAAATCTCGGTGTCGCAAA-3'; Pem reverse primer, 5'-GCAACACCAGTCCCTGAACA-3'; Cyp17a1 forward primer, 5'-TTCTGATCGACCCTTTCAAAGTG-3'; Cyp17a1 reverse primer, 5'-GGATCCGGACGTTAGATTCG-3'; steroidogenic acute regulatory protein (StAR) forward primer, 5'-TGCAGGACTCAGGACCTTGAA-3'; StAR reverse primer, 5'-GGAGCTTCCAGCACACAGC-3'; Slc2a1 forward primer, 5'-CTCCCATGTGCGTGCATAAT-3'; Slc2a1 reverse primer, 5'-TCTTGGGTTCATTGCCTGC-3'; CAC forward primer, 5'-GGGACGGTAAAGGCTTGGAT-3'; CAC reverse primer, 5'-GCCACTGCCAACCTCCAG-3'; Amot-1 forward primer, 5'-CCGTGGAGAAGCAGGAGAAC-3'; Amot-1 reverse primer, 5'-CTCGCCATCTGGGAACTCA-3'; S2 forward primer, 5'-CTGACTCCCGACCTCTGGAAA-3'; and S2 reverse primer, 5'-GAGCCTGGGTCCTCTGAACA-3'. Real-time PCR was carried out in a 96-well plate using a 7000 ABI prism sequence detection system (Applied Biosystems). The previously synthesized cDNA was used as template. Reactions for each time point were performed in triplicate and contained approximately 15–30 ng of cDNA, 2x SYBR GREEN master mix (Applied Biosystems, Foster City, CA), and 600 nM of each reverse and forward primer specific for the candidate genes. Reactions were run for 40 cycles (95°C for 15 sec, 58°C for 1 min) following an initial 2-min step at 50°C for enzyme activation and a 10-min incubation at 95°C. The threshold cycle (CT), which indicates the relative abundance of a particular transcript, was calculated for each reaction by the 7000 ABI prism sequence detection system. Ribosomal protein S2 CT values were used as normalizing endogenous controls. The quantification of the candidate gene expression was calculated for the different T-treatment time points using the formula 
as described in the SYBR Green user manual:
 |
Real-time PCR quantification of gene expression level in each sample was the mean of triplicate real-time PCR experiments. For each time point, values are presented as the mean ± SEM of triplicated independent ex periments. All gene expression levels were normalized to ribosomal pro tein S2 expression levels.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Detection of AR in Neonatal Mouse Testis by IHC
At 8 dpp, seminiferous tubules within the mouse testis contained Sertoli cells and a layer of germ cells, mainly spermatogonia. A single layer of peritubular myoid cells surrounded the seminiferous tubules preventing the direct contact of Sertoli cells with Leydig cells located in the in terstitium. The localization of AR in the testis of neonatal mice was examined by IHC before and after TP treatment, whereas GCNA1 staining was used to identify germ cells. All positive reactivity was localized to the nucleus, and no nonspecific staining was observed in tissues incubated with peptide-preabsorbed primary antibodies. The IHC revealed AR in the three somatic cell types of 8-dpp animals as reported in the testis of adult mice (Fig. 1) [11]. The Sertoli cells are the only AR-positive cells within the seminiferous tubules, and as expected, no staining of germ cells within the neonatal mouse testis was evident. The administration of TP to these neonates up to 16 h did not change the AR expression intensity or pattern in a significant way (data not shown). These results suggested that the testis at this age should have the capability to respond to androgen stimulation.
|
Absolute Analysis
In a preliminary study, control samples from two time points (8 and 16 h) were tested on the MOE430 arrays, and comparison analyses between them were performed in MAS. No significant differences were apparent between the two control samples. Similarly, at 16 h, TP- and DHT-treated samples were compared, and once again, no significant differences in gene expression were observed. The similarity between the TP- and DHT-treated samples indicated little or no difference between the effects of TP treatment and DHT treatment, despite the possible conversion of T to estrogen. Therefore, the following results of the analysis are comparisons between control and TP-treated samples. Correlation coefficients were calculated using Microsoft Excel (Redmond, WA), and all correlation coefficients between the replicates of the two treated samples were greater than 0.92 (range, 0.92–0.98).
Approximately 39 000 transcripts, including more than 34 000 well-substantiated mouse genes, are present on the MOE430 A and B arrays and represent a large percentage of the total murine genome. Based on analysis in MAS, 45.17% of the transcripts in the control sample were designated as present or marginally present, whereas 46.01%, 43.21% and 44.22% were designated as present or marginally present in the TP-treated testes at 4, 8, and 16 h, respectively. These percentages represent averages of duplicate samples, and they indicate that T treatment does not drastically change the number of transcripts that MAS designates as being present.
Comparison Analysis
To examine the time-dependent regulation of gene expression by T in neonatal testes, the raw data generated from the microarrays was imported into GeneSpring for comparison analysis. Expression data from treated animals at each time point (4, 8, and 16 h) were compared to gene expression in the vehicle-treated control, and the change in expression of each gene was monitored over time. Three criteria were applied to define what we designated "TP-regulated" transcripts as described in Materials and Methods. These included a raw signal value of no less than 50 in either the control or treated samples, a twofold or higher difference in signal between the control and treated samples, and statistically significant difference based on the one-way ANOVA test. Based on these criteria, 224, 218, and 220 transcripts were T-regulated at 4, 8, and 16 h, respectively (both A and B chips) (Fig. 2). Percentagewise, 62.9%, 54.6%, and 49.5% of the T-regulated transcripts were up-regulated at 4, 8, and 16 h, respectively, whereas 37.1%, 45.4%, and 50.5%, respectively, were down-regulated at those same time points. The majority of these T-regulated transcripts were expressed sequence tags, with well-characterized genes only occupying a small proportion of the total regulated transcripts at each time point (48/224 at 4 h, 53/218 at 8 h, and 53/220 at 16 h). Tables 1 and 2 list only a selected number of known genes that were T-induced or T-repressed at each time point.
|
|
|
Venn diagrams were used to detect transcripts that were commonly or uniquely regulated by T in the time-course study (Fig. 3). This analysis shows that 41 transcripts were commonly regulated. Twenty of them were consistently up-regulated at all three time points, with five of them being known genes, including Nkcc2 (a solute carrier), angiomotin-like 1 (a tight junction molecule), Cacna1a (a calcium channel), and H2-D1. Twenty of them were consistently down-regulated at all three time points, and only two of them were known genes (leiomorphic adenoma gene-like 1 and ADP-ribosylhydrolase-like 1). Names, and GenBank accession numbers of a few representative transcripts are listed in Table 3.
|
|
Confirmation of Experiment Model and Verification of Microarray Data
To verify that these neonatal mice responded to our TP treatments in an expected way, we monitored the expression of two well-characterized androgen-regulated genes in the testis. One is placentae and embryos oncofetal gene (Pem), which is a homeobox gene induced in Sertoli cells by androgens [14]. In the gene arrays, Pem was up-regulated 3.8-fold at 16 h based on the array data. The second gene is P450 17-hydroxylase/17,20-lyase (17–20) (Cyp17a1), a Leydig cell gene that is suppressed by T [20]. The Cyp17a1 was down-regulated 2.6- and 4.3-fold at 8 and 16 h, respectively, in the time-course study. The array results of these two positive-control genes were in agreement with those of previous in vivo and in vitro studies and indicated both that our T treatment is effective and that the neonatal testes responded to T in a fashion similar to that of adult animals.
To validate gene expression patterns identified by the microarray results, the expression of selected transcripts was examined using two-step, real-time RT-PCR in triplicate with the same total RNAs used in the array hybridization. Selected genes include Pem, Cyp17a1, StAR, an Na+/ K+-2Cl– cotransporter (Slc12a1), and a voltage-controlled Ca2+ channel (Cacna1a) (Fig. 4). The overall trends of expression by real-time PCR were in agreement with the array data, although the fold-changes did not always precisely match those of the array data.
|
Examination of Marker Genes for Cell Proliferation and Differentiation
To examine whether TP treatment can facilitate or inhibit cell proliferation and differentiation in the testis, some selected marker genes were assessed based on the array data. In adult rodent testes, some previous studies suggested that androgens positively regulate AR in Sertoli cells [21, 22]. Based on the array data, no change was found in the level of AR mRNA (Table 4), which agrees with another recent study using adult rats [23]. We chose c-myc and proliferating cell nuclear antigen (Pcna) as markers for cell proliferation [24, 25]. Neither gene showed change in expression level following T treatment. Three genes were selected as the differentiation markers for Sertoli cells, including clusterin, transferrin, and transferrrin receptor. None of these genes was modulated by TP treatment (Table 4). In the present study, no evidence was found to support the possibility that short-term TP treatment could modulate cell proliferation and differentiation in the neonatal testis.
|
Regulation of Androgen-Responsive Genes in Flutamide-Treated Adult Mouse Testis
To evaluate further whether the response to T stimulation detected in the neonatal mouse could be verified in the adult mouse testis, three genes, including Pem, StAR, and Amotl1, were selected and examined using real-time PCR in adult mice treated with the AR-antagonist flutamide. The level of Pem transcript showed a rapid, 20–30% reduction at 4 h after the flutamide injection and an additional 20% decrease at 16 h (Fig. 5). The Pem exhibited a more than twofold repression at 24 h, which was statistically significant (Fig. 5). Expression of Amotl1 increased in T-treated neonatal mice and showed a moderate but statistically significant reduction in the adult testis treated with flutamide for 24 h (Fig. 5). The StAR was repressed by TP treatment in the neonatal testis. However, expression was elevated at 4 and 16 h and fell back to the control level at 24 h after the flutamide treatment. These results collectively demonstrated that the expression changes of these genes observed in neonatal TP-treated testes also could be verified in adult testes treated with flutamide.
|
Regulation of Androgen-Responsive Genes in Testicular Somatic Cells
Three different types of somatic cells in the testis respond directly to T treatment. The StAR was selected from the microarray analysis results to evaluate whether its response to T also can be observed in specific cell types. Qualitative RT-PCRs were performed to evaluate which cell type predominantly expressed this gene (data not shown). Two-step, real-time RT-PCRs were used to examine the time-course response to TP. The StAR was detected in both Sertoli cells and myoid cells. In a time-course study of TP-treated primary Sertoli cells, a significant inhibition of StAR expression was observed at 8 and 16 h (Fig. 6). The same trend of repression was observed at 2 and 4 h, but it was not statistically significant in the three replicates.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The effects of androgen on the initiation and maintenance of spermatogenesis are better understood in the pubertal and adult males [2, 8, 29], but its physiological function in the neonatal testis is obscure [30–32]. To decipher the data correctly and for possible extrapolation, it is important to prove that the neonatal testes may respond to T in a way similar to that of adult testes. Under normal physiological condition, the intratesticular and serum T concentrations in the neonatal males remain at a relatively low level during this developmental stage and do not become elevated until puberty [33]. However, these neonates not only exhibited the same pattern of AR expression in their testes but also demonstrated the ability to respond to androgen stimulation. For instance, in humans, boys experiencing precocious puberty induced by exogenous and endogenous androgen exposure may have the ability to produce functional spermatozoa [34–38]. This strongly suggests that similar transcriptional and posttranscriptional events under androgen regulation occurred in these boys, similar to what normally occurs in pubertal and adult men. Perhaps the same set of genes are activated or suppressed to achieve the successful initiation of spermatogenesis. The present study demonstrated this similarity and suggested that the effects of androgen regulation obtained from this neonatal model can be extrapolated, to some extent, to the adult system.
Sertoli cells are the indispensable nursing epithelial cells within the seminiferous tubules that support germ cell differentiation [39]. The expression of AR in Sertoli cells is stage-specific, and a number of studies illustrate that stages VII and VIII of the spermatogenic cycle are androgen-dependent [8]. Recently, three independent studies using Sertoli cell-specific AR-knockout mice demonstrated that the action of androgen in Sertoli cells is an absolute requirement for the completion of spermatogenesis, particularly in the process of meiosis [40–42]. Moreover, Johnston et al. [32] showed that T also is important for Sertoli cell proliferation during testis development. However, a very limited number of genes in Sertoli cells were identified as being regulated by androgen at the transcriptional level [14, 15]. Using a variety of adult mouse models, including Tfm, WWv mutants, and T-treated hpg and hypophysectomized mice, Sutton et al. [14] demonstrated that the homeobox gene Pem is specifically expressed in Sertoli cells in an androgen-dependent and stage-specific manner [14]. Therefore, Pem was used as a positive control gene for regulation by androgen and to monitor our TP treatment and microarray analysis. By 16 h after injection with TP, the raw signal of Pem was elevated almost fourfold compared to that of control mice, indicating that the Sertoli cells in these neonatal mice were responsive to T stimulation. Additional analysis of microarray data are ongoing and will dissect out other Sertoli cell genes demonstrating T regulation.
Leydig cells are the steroidogenic cells under LH control in the adult testis [26], but they also are subject to autocrine regulation by T [31, 43]. Several genes coding for steroidogenic proteins/enzymes were inhibited by T, including CYP17a1 [17, 20]. The CYP17a1 (P450C17) converts C(21) steroids to C(19) steroids, and T represses cAMP-induced Cyp17a1 mRNA synthesis via binding of the AR to sequences in the cAMP-responsive region of the Cyp17 promoter. This has been shown in both primary cultures of mouse Leydig cells and the Leydig cell line, MA-10 [17]. The Cyp17a1 was used to monitor the microarray data as a potential Leydig cell gene repressed by T. In the neonatal testis treated with TP, the array revealed that the signal of Cyp17a1 was repressed 2.6- and 4.3-fold after 8 and 16 h, respectively. To our knowledge, this is the first evidence demonstrating TP repression of Cyp17a1 in vivo. This result clearly suggests that Leydig cells in neonatal testes responded to T in a manner similar to that in adults, despite the fact that adult Leydig cells start responding to a pubertal LH pulse to produce T approximately 2–3 wk after birth in mice, when the feedback inhibition by T becomes more physiologically meaningful.
To verify directly the T regulation identified by microarray data in an adult system, AR antagonist-treated adult mouse testis was used, and the gene expression was examined by real-time PCR. The Pem was strongly repressed at 24 h after the flutamide injection, indicating that the antagonism of T action by flutamide in this model was effective. Additionally, Amotl1 was repressed significantly by flutamide in the adult model, whereas it was consistently up-regulated by T at all time points in the neonatal time-course study. The Amotl1 is a recently identified tight junction protein with conserved coiled-coil and PDZ-binding domains. Exogenously expressed Amotl1 colocalized with ZO-1 and occludin at tight junctions in polarized Madin-Darby canine kidney cells. Endogenously, it is specifically expressed in exocrine cells [44]. To our knowledge, the present study is the first report that Amotl1 is expressed in the testis and regulated by T. It indicates that the T regulation identified in neonatal testes also can be confirmed directly in the adult testes. The StAR, which has been extensively studied in many steroidogenic tissues, encodes a carrier protein that transports cholesterol to the inner mitochondrial membrane [45]. Recently, Houk et al. [46] reported a feedback inhibition of StAR expression in vitro and in vivo by androgens and T-repressed StAR mRNA in Leydig cells at the transcriptional level. The results from both the array data and flutamide-treated adult testis further verified the inhibitory effect of T on StAR expression. Recent studies demonstrate that StAR is expressed not only in Leydig cells but also in Sertoli cells [47, 48]. In primary cultured rat Sertoli cells, FSH induces StAR expression significantly [27]. Stocco et al. [45] speculated that Sertoli cells might represent a homologue of granulosa cells with a severely decreased capacity to synthesize steroids from cholesterol, and the presence of StAR in Sertoli cells is a remnant of the formerly functional steroidogenic pathway. From a physiological standpoint, it is reasonable to speculate that the sufficient T supply from Leydig cells may play an inhibitory role on StAR expression to turn off unnecessary steroid production in Sertoli cells. However, it is noteworthy that StAR was detected in some other nonsteroidogenic cells, such as the epithelium lining the distal convoluted tubules in the kidney [49]. Therefore, others speculated that StAR might facilitate the cholesterol hydroxylation and transport of free fatty acids into mitochondria for oxidation, which is a major energy source for Sertoli cells [47, 49]. We confirmed the presence of StAR in primary cultured, immature Sertoli cells by RT-PCR (data not shown). It was found that T treatment strongly repressed the StAR expression in Sertoli cells. This trend of StAR repression by T in Sertoli cells is in agreement with the data obtained from studies in Leydig cells and the time-course, T-treated neonatal testis, implying that the T regulation of StAR is not a cell type-specific event. These data strongly suggest that some T regulation observed in our array results can be confirmed not only in adult mouse testis in vivo but also in isolated cell populations in vitro.
Many in vivo models have been used to study T action, but each has certain intrinsic advantages and drawbacks. For instance, knockout and mutant mouse models, such as Tfm and hpg, demonstrate more developmental effects rather than short-term, isolated T action. Moreover, the cryptorchidism in Tfm mice and the alteration of FSH in hpg mice make it difficult to separate the effects of T from those of other factors. The underlying and unknown contribution by FSH exists in several other animal models widely used to study T effects, including hypophysectomized animals, immunization against GnRH, or treatment with a GnRH antagonist [8]. A previous microarray study used the hpg mouse to investigate T regulation [16]. Theoretically, both hpg and neonatal males have undescended testes, immature somatic cells, and a lack of advanced germ cells. More similarities are expected regarding their response to T stimulation. However, preliminary comparison of T-regulated transcripts in the hpg and neonatal models revealed only a few similarities between two systems when the same criteria were applied to analyze the data. Among the few transcripts commonly regulated by T in both models were Pem and Cyp17a1. We speculate that lack of FSH in all developmental stages in these adult hpg males is one of the important factors making them different from normal neonates. Moreover, the immature testes of neonatal males may be incapable of fully responding to the presence of an unphysiological level of exogenous T in our experimental regime, even though this dose of T may generate reasonable responses in the adult males [14]. Further data analyses are ongoing, and more studies should be done to decipher the mechanism shaping the hormonal responsiveness of the testis during development.
Investigations concerning the hormonal regulation of male reproductive tissues using microarrays have become extensive [16, 18, 27], but the global profile of T regulation in the testis is still not as detailed as the descriptions in other organs [16, 50–52]. The present study attempted to fill the void by providing information concerning T regulation in the testis using a novel model. However, the analysis of the data is by no means conclusive and final but, rather, offers a limited expression profile of androgen-regulated genes in the testis. Because of the stringent nature of the analysis and the limited amount of T regulation overall, some genes under androgen regulation that are required for spermatogenesis may be omitted in the present report, either because they have a lesser fold-change (less than twofold) or because they are unable to pass the statistical tests performed in the present analysis. Another possibility is that certain genes are cell-type specific and the effect of T regulation may be diluted in the assays by using the whole testis. Thus, further analyses of the array data by employing different approaches, cutoff criteria, and incorporation of cell-specific datasets are necessary to use fully the wealth of information contained in the time-course study.
These data provide insights regarding what might occur in normal androgen-supported spermatogenesis in the adult testis and allow for further hypotheses to be generated concerning the action of T in testicular gene regulation. However, it is noteworthy that the data obtained from the present in vivo experimental model represent an integrated response by at least three major testicular somatic cells after T treatment. Each of these somatic cells exhibits different physiological functions in an in vivo environment with respect to androgen regulation [53]. Each testicular cell type could have unique answers to the T exposure and correspond to their specialized functions. Further work to classify additional transcripts and the androgen-regulated transcripts described here into specific cell types is ongoing and could provide information about how unique genes are responding to T in each specific somatic cell. In addition, more arrays could be screened with individual somatic cell lines, in combination with this time-course, to deepen our understanding of T regulation in a more cell type-specific manner.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Michael D. Griswold, 531 Fulmer Hall, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660. FAX: 509 335 9688; mgriswold{at}wsu.edu
Received: 8 September 2004.
First decision: 28 September 2004.
Accepted: 29 November 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
expressed in porcine Sertoli cells is under the control of follicle-stimulating hormone and testosterone. Biol Reprod 2002 66:1734-1742-hydroxylase/C17–20 lyase gene (Cyp17) by androgens. Mol Endocrinol 1997 11:87-96-hydroxylase activity. J Endocrinol 1991 131:443-449This article has been cited by other articles:
|
|
 |
|
  Y. Li, Q. Zhou, R. Hively, L. Yang, C. Small, and M. D. Griswold Differential Gene Expression in the Testes of Different Murine Strains Under Normal and Hyperthermic Conditions J Androl, May 1, 2009; 30(3): 325 - 337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. S. Maywood, S. Chahad-Ehlers, M. L. Garabette, C. Pritchard, P. Underhill, A. Greenfield, F. J. P. Ebling, R. A. Akhtar, C. P. Kyriacou, M. H. Hastings, et al. Differential Testicular Gene Expression in Seasonal Fertility J Biol Rhythms, April 1, 2009; 24(2): 114 - 125. [Abstract] [PDF]
|
|
|
|
 |
|
 R.-S. Wang, S. Yeh, C.-R. Tzeng, and C. Chang Androgen Receptor Roles in Spermatogenesis and Fertility: Lessons from Testicular Cell-Specific Androgen Receptor Knockout Mice Endocr. Rev., April 1, 2009; 30(2): 119 - 132. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Abel, P. J. Baker, H. M. Charlton, A. Monteiro, G. Verhoeven, K. De Gendt, F. Guillou, and P. J. O'Shaughnessy Spermatogenesis and Sertoli Cell Activity in Mice Lacking Sertoli Cell Receptors for Follicle-Stimulating Hormone and Androgen Endocrinology, July 1, 2008; 149(7): 3279 - 3285. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Zhou, R. Nie, Y. Li, P. Friel, D. Mitchell, R. A Hess, C. Small, and M. D Griswold Expression of Stimulated by Retinoic Acid Gene 8 (Stra8) in Spermatogenic Cells Induced by Retinoic Acid: An In Vivo Study in Vitamin A-Sufficient Postnatal Murine Testes Biol Reprod, July 1, 2008; 79(1): 35 - 42. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Zhou, Y. Li, R. Nie, P. Friel, D. Mitchell, R. M. Evanoff, D. Pouchnik, B. Banasik, J. R. McCarrey, C. Small, et al. Expression of Stimulated by Retinoic Acid Gene 8 (Stra8) and Maturation of Murine Gonocytes and Spermatogonia Induced by Retinoic Acid In Vitro Biol Reprod, March 1, 2008; 78(3): 537 - 545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Rocha, M. S. Bonkowski, L. R. Franca, and A. Bartke Mild Calorie Restriction Does Not Affect Testosterone Levels and Testicular Gene Expression in Mutant Mice Experimental Biology and Medicine, September 1, 2007; 232(8): 1050 - 1063. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. O'Shaughnessy, M. Abel, H. M. Charlton, B. Hu, H. Johnston, and P. J. Baker Altered Expression of Genes Involved in Regulation of Vitamin A Metabolism, Solute Transportation, and Cytoskeletal Function in the Androgen-Insensitive Tfm Mouse Testis Endocrinology, June 1, 2007; 148(6): 2914 - 2924. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Schmidt, J. M. d. Avila, and D. J. McLean Analysis of Gene Expression in Bovine Testis Tissue Prior to Ectopic Testis Tissue Xenografting and During the Grafting Period Biol Reprod, June 1, 2007; 76(6): 1071 - 1080. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Eacker, J. E. Shima, C. M. Connolly, M. Sharma, R. W. Holdcraft, M. D. Griswold, and R. E. Braun Transcriptional Profiling of Androgen Receptor (AR) Mutants Suggests Instructive and Permissive Roles of AR Signaling in Germ Cell Development Mol. Endocrinol., April 1, 2007; 21(4): 895 - 907. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Xia, D. D Mruk, W. M Lee, and C Y. Cheng Unraveling the molecular targets pertinent to junction restructuring events during spermatogenesis using the Adjudin-induced germ cell depletion model J. Endocrinol., March 1, 2007; 192(3): 563 - 583. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Domanskyi, F.-P. Zhang, M. Nurmio, J. J. Palvimo, J. Toppari, and O. A. Janne Expression and localization of androgen receptor-interacting protein-4 in the testis Am J Physiol Endocrinol Metab, February 1, 2007; 292(2): E513 - E522. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. He, W.-Y. Chan, and M. Dym Microarray technology offers a novel tool for the diagnosis and identification of therapeutic targets for male infertility Reproduction, July 1, 2006; 132(1): 11 - 19. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. L. Tan, K. De Gendt, N. Atanassova, M. Walker, R. M. Sharpe, P. T. K. Saunders, E. Denolet, and G. Verhoeven 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, June 1, 2005; 146(6): 2674 - 2683. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
