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Endocrinology, doi:10.1210/en.2006-1412
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Endocrinology Vol. 148, No. 6 2914-2924
Copyright © 2007 by The Endocrine Society

Altered Expression of Genes Involved in Regulation of Vitamin A Metabolism, Solute Transportation, and Cytoskeletal Function in the Androgen-Insensitive Tfm Mouse Testis

P. J. O’Shaughnessy, M. Abel, H. M. Charlton, B. Hu, H. Johnston and P. J. Baker

Institute of Comparative Medicine (P.J.O., B.H., H.J., P.J.B.), Division of Cell Sciences, University of Glasgow Veterinary School, Glasgow G61 1QH, Scotland, United Kingdom; and Department of Human Anatomy and Genetics (M.A., H.M.C.), University of Oxford, Oxford OX1 3QX, United Kingdom

Address all correspondence and requests for reprints to: P. J. O’Shaughnessy, Division of Cell Sciences, University of Glasgow Veterinary School, Bearsden Road, Glasgow G61 1QH, Scotland, United Kingdom. E-mail: p.j.oshaughnessy{at}vet.gla.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgens are essential for the development and maintenance of spermatogenesis, but the underlying mechanisms of androgen action in the testis remain unclear. To help clarify these mechanisms, gene expression was measured in testes of pubertal (20 d old), androgen-insensitive, testicular feminized (Tfm) mice and in normal controls. Using microarrays (Affymetrix chips 430A and 430B), initial data identified a large number of genes down-regulated in the Tfm testis (>4700). These genes were largely of germ cell origin, reflecting the arrest of spermatogenesis that is apparent in the 20-d-old Tfm testis. Subsequent screening in vitro and in silico of this gene set identified 20 genes of a somatic tubular origin that were significantly down-regulated in the Tfm testis and six genes that were significantly up-regulated. Altered expression of these genes was confirmed by real-time PCR, and genes down-regulated in the Tfm testis were shown to be up-regulated in testes of hypogonadal (hpg) mice treated with androgen. In a developmental study using real-time PCR most of the regulated genes showed normal expression during fetal and neonatal development and deviated from control only between 10 and 20 d. In all cases, expression was also reduced in the adult, although interpretation is more complex because of the inherent cryptorchidism in the adult Tfm mouse. Of the total number of somatic genes showing differential expression in the Tfm testis, 50% were associated with three separate groups of genes involved in regulation of vitamin A metabolism, solute transportation, and cytoskeletal function. Thus, effects of androgens on tubular function and spermatogenesis may be mediated in part through regulation of the tubular environment and control of retinoic acid concentrations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE ADULT male, spermatogenesis and subsequent fertility are critically dependent on androgens. Suppression of androgen levels will disrupt spermatogenesis (1), whereas androgens will rescue spermatogenesis in mice lacking circulating gonadotropins (2). In addition, inactivating mutations in the androgen receptor have been shown to block spermatogenesis in early meiosis (3, 4). The germ cells do not appear to be a direct target of androgen action (5, 6), and it is clear that androgen regulation of spermatogenesis is regulated, at least partly, through the Sertoli cell because specific knockout of the androgen receptor in these cells will disrupt spermatogenesis (7, 8, 9). Androgen receptors are also expressed, however, in the peritubular myoid cells of the testis and throughout fetal life, and up until a few days after birth the peritubular cells are the only identifiable site of androgen receptor expression in the testis (10, 11). In testicular feminized (Tfm) mice, which lack functional androgen receptors through a mutation in the receptor gene (12), Sertoli cell number is reduced at birth and remains significantly less than normal up to adulthood (13). This suggests that androgen action on Sertoli cell proliferation may be mediated through the peritubular cells, a hypothesis that is supported by the observation that Sertoli cell proliferation is normal in mice with a specific Sertoli cell loss of androgen receptor expression (7). Thus, androgen action on the function and development of the seminiferous tubule is likely to be mediated through actions on both Sertoli cells and peritubular myoid cells.

Despite the clear evidence that androgen action is required to ensure normal development and function of the seminiferous tubules, the mechanisms by which androgen regulates these processes has remained unclear until recently. Androgens have been shown to affect Sertoli cell function in culture (14), but these effects are not generally marked, and in many studies androgens do not appear to have any direct effect in vitro. To identify androgen target genes in the somatic cells of the seminiferous tubule we have used Affymetrix GeneChip arrays to compare testicular gene expression in normal mice and in Tfm mice at 20 d. At this age, there is a clear morphological difference in the progress of spermatogenesis between normal and Tfm animals (13), and initial data from the arrays was overwhelmingly populated with germ-cell-specific genes. Through extensive post-array analysis, however, we have identified 26 genes, of likely Sertoli cell or peritubular cell origin, that are differentially regulated in the Tfm mouse. Within this gene set, solute carriers, regulators of vitamin A (retinol) metabolism, and cytoskeletal proteins are highly represented, indicating possible mechanisms through which androgens may act to regulate tubule function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, tissues, and treatments
Tfm mice were bred in Glasgow on a C3H/HeH-101/H genetic background from stock animals obtained originally from the Medical Research Council Radiobiology Unit (now the Medical Research Council, Mammalian Genetics Unit, Harwell, UK). The testes of normal mice undergo final descent to the scrotum at about 25 d, whereas the testes remain intraabdominal in Tfm mice into adult life. To control for the failure of testicular descent in adult Tfm mice, normal animals on the same background were surgically rendered cryptorchid at 21 d (15). Hypogonadal (hpg) mice (on the same background as the Tfm mice) were bred at the University of Oxford and were treated with testosterone through a sc 2-cm SILASTIC brand silicon implant (Dow Corning, Midland, MI) for 1 wk (16). Whole testes were recovered from all animals immediately after death and were either stored in liquid N2 or seminiferous tubules and interstitial tissue were separated mechanically as previously described (17) and isolated tissues stored in liquid N2. Studies of gene expression in normal mice at different developmental stages used animals on a C3H/HeH-101/H background. All animals were maintained as required under United Kingdom Home Office regulations.

RNA extraction
The RNA from whole testes or isolated tissues was extracted in TRIzol (Invitrogen Ltd., Paisley, Scotland, UK). In studies designed to measure transcript expression through real-time PCR, luciferase mRNA (Promega UK, Southampton, UK) was added to the samples at the time of RNA extraction and subsequently used as an external standard in some real-time assays (13, 18). For array studies, the RNA was further processed using RNeasy kits (QIAGEN Ltd., Crawley, UK) according to the manufacturer’s instructions.

Microarrays
Total RNA from testes of normal and Tfm mice aged 20 d was used in the array studies. The quality of the RNA was assessed with an Agilent RNA bioanalyzer, and samples of 10 µg total RNA were reverse transcribed and then in vitro transcribed according to Affymetrix standard protocols. The mouse Affymetrix gene chips 430A and 430B were used in all hybridizations. These arrays contain probes representing transcripts for over 34,000 characterized mouse genes. Microarray data from three independent samples from normal mice and three samples from Tfm mice were analyzed initially using the robust multichip average method in the BioConductor microarray analysis software (19), whereas differentially expressed genes were identified using RankProducts (20).

Real-time PCR
Data generated from arrays were confirmed and further assessed using real-time PCR. RNA was extracted as described above, and residual genomic DNA was removed by DNase treatment (DNA-free, Ambion Inc., supplied by AMS Biotechnology, Abingdon, UK). RNA was reverse transcribed using random hexamers and Moloney murine leukemia virus reverse transcriptase (Superscript II; Invitrogen) as described previously (21, 22). The real-time PCR approach used the SYBR green method in a 96-well plate format using a Stratagene MX3000 cycler. Reactions contained 5 µl 2x SYBR MasterMix (Stratagene, Amsterdam, The Netherlands), primer (100 nM), and template in a total volume of 10 µl. The thermal profile used for amplification was 95 C for 8 min followed by 40 cycles of 95 C for 25 sec, 63 C for 25 sec, and 72 C for 30 sec. At the end of the amplification phase, a melting-curve analysis was carried out on the products formed. All primers were designed by Primer Express 2.0 (Applied Biosystems, Warrington, UK) using parameters previously described (23). The primers used are described in supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org.

Statistics
Results from real-time PCR studies were analyzed using t tests or two-factor ANOVA followed by t tests using the pooled variance. Where necessary, data were log-transformed before analysis to avoid heterogeneity of variance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Screening of initial array data
Differentially expressed genes.
In this study, differences in gene expression between testes from normal mice and Tfm mice have been examined at 20 d. Statistical analysis of the data derived from the gene arrays identified 5976 genes significantly up-regulated and 4706 genes significantly down-regulated in the Tfm testis. Of these, 1747 were significantly up-regulated by more than 2-fold and 1988 significantly down-regulated more than 2-fold (supplemental Table 2, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Because of the different morphology of the tissues used in this study, it is necessary to apply a biological filter to the data and, in particular, to the genes apparently up-regulated in the Tfm samples. Samples hybridized to the gene chips are normalized according to the mass of the initial RNA sample, but this does not allow for differences in cell composition between normal and Tfm testes. The volume of the Tfm testis is 17.5% of normal at 20 d, whereas Sertoli cell number is 45% of normal (13). Thus, Sertoli cell number will be enriched by about 2.5-fold (per unit volume) in the Tfm samples. This means that a Sertoli-cell-specific gene that shows normal expression per cell in the Tfm testis would be expected to show a 2.5-fold increase in expression on the array assuming RNA content of the Tfm testis (per unit volume) is normal. Conversely, this reasoning implies that on the arrays a 2-fold reduction in expression in the Tfm testis equates to an actual reduction of 5-fold per cell for any Sertoli-cell-specific product. For these reasons, characterization of differentially expressed genes was limited initially to those genes showing greater than a 5-fold increase (42 genes) or 2-fold decrease (1990 genes) in expression in the Tfm samples.

Identification of somatic tubular genes.
Because of the blocked development of spermatogenesis in the Tfm mice, it was expected that the overwhelming majority of genes shown to be reduced in expression in the Tfm testis would be of germ cell origin. The data from the arrays were, therefore, taken through a series of screens, initially to identify somatic genes and finally to identify genes of a likely Sertoli cell or peritubular cell origin that are differentially expressed in the Tfm testis. The first screens were applied in silico and identified those genes shown previously in the published literature to be germ-cell derived (e.g. Soggy-1) (24). For genes that are less well characterized, Unigene (http://www.ncbi.nlm.nih. gov/entrez/query.fcgi?db=unigene) was used to determine whether the Unigene clusters for each of the genes in question contained expressed sequence tag (EST) sequences arising from germ cell libraries. Expression profiles in GEO (http://www.ncbi.nlm.nih.gov/geo) and, in particular, GDS401, -409, and -410 were also screened for genes with a likely germ cell origin. Finally, genes were screened against SAGE libraries generated from isolated germ cell populations (25). None of these in silico screens is infallible because all depend upon isolation of different cellular components that will have varying degrees of contamination. Nevertheless, they served to reduce the number of genes of interest to 232 (supplemental Table 1).

Additional analysis of gene expression was by real-time PCR. Initially, each gene was screened for expression in the germ cell component by comparing expression in testes from adult normal mice and adult mice treated 60 d earlier with busulfan to eliminate the germ cell component (26). Most genes tested showed little or no expression in the busulfan-treated testis, indicating that they are likely to be predominantly of a germ cell origin, although Sertoli cell genes markedly down-regulated in the absence of germ cells would also be lost at this part of the screen. Genes showing expression in the busulfan-treated testes that was 20% (or higher) of control values (78 genes) were retained for the second screen. This compared expression in testes from normal 20-d-old mice with those from 20-d-old Wv/Wv mice that lack germ cells because of a mutation in the c-kit receptor (26, 27). From this study, 42 genes were identified that showed expression at 20 d in the Wv/Wv mice that was equal to or greater than that seen in the control mice. To localize expression of these genes in the testis, expression was measured in isolated interstitial cells and isolated tubules. From the 42 genes tested, 26 genes were identified (20 down-regulated and six up-regulated in the Tfm) that showed primarily tubular expression (Table 1Go), and additional analysis was confined to these genes. Two of the genes included in Table 1Go (Drd4 and Adh1) are likely to be predominantly of tubule origin, but the ratio of expression in tubules and interstitial tissue suggests that they also have some interstitial expression. This has already been confirmed directly for Adh1 (28). The remaining 16 genes, which showed predominantly interstitial expression, are listed in supplemental Table 3, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org.


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  		TABLE 1. Somatic tubular genes differentially expressed in 20-d-old Tfm mouse

 
Analysis of differentially expressed genes
Confirmation of array data.
Androgen dependence of genes identified above was tested by real-time PCR in control and Tfm mice at 20 d and in adult hypogonadal (hpg) mice treated for 1 wk with testosterone (Table 2Go). The hpg mouse lacks GnRH and, thus, circulating gonadotropins and testosterone (29, 30). The real-time PCR data generated in this study were expressed relative to the housekeeping gene Wbscr1 (31) and would be expected to generate data similar to that obtained using the arrays. Most of the genes identified by the arrays as down-regulated in the Tfm were confirmed to be down-regulated by real-time PCR, and most of these genes were also up-regulated by testosterone in the hpg (Table 2Go). There were only two genes identified from the arrays as down-regulated in the Tfm (Ciz1 and Bnip1) that subsequently showed no significant differences by real-time PCR in either the Tfm or hpg model. All but one of the genes showing increased expression in the Tfm testis using arrays also showed an increase by real-time PCR in the Tfm, although this group of genes showed no significant changes in expression in the hpg model (Table 2Go). It is noticeable that gene expression levels in the control hpg testes are generally higher than in the 20-d controls. This is likely to be because the hpg testis contains a significantly greater number of Sertoli cells per unit volume than the normal 20-d testis (up to 12-fold) (32).


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  		TABLE 2. Gene expression in 20-d-old Tfm mouse and in adult hpg mice treated with androgen

 
Developmental characteristics of differentially expressed genes.
Developmental changes in the expression of androgen-regulated genes are shown in Figs. 1Go and 2Go. Results show expression in the normal mouse and at selected times in the Tfm mouse. The real-time PCR data for these studies were expressed relative to an external standard (luciferase), and the results are therefore comparable as expression per testis (18, 31). The adult Tfm mouse is cryptorchid, and an adult cryptorchid control group is included for comparison. For 14 of the 18 genes in the down-regulated group, expression was normal in Tfm mice up to 10 d (Fig 1Go). Thereafter, there was an increase in expression in the normal in most cases that was not replicated in the Tfm so that expression was significantly lower at 20 d in the Tfm mice. For all genes in this group, there was significantly less expression in the adult Tfm testis compared with normal, although these data are complex because in many cases, there was an effect of cryptorchidism. For the remaining four genes (Rhox5, Drd4, Slc38a5, and P45026b1) expression in the Tfm testis was reduced at 10 d or earlier and remained lower than normal up to adulthood.


Figure 1
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  		FIG. 1. Developmental expression of genes showing reduced expression in the Tfm mouse. Data show changes in testicular gene expression during development in normal mice (solid line) and in Tfm mice (gray bars). Expression in adult cryptorchid control mice is shown by the solid black bar. Expression was measured by real-time PCR, and data are expressed relative to the external control luciferase. Results show mean ± SEM for three to five animals per group. *, Significantly different (P < 0.05) from normal at the same age (adult cryptorchid mice are considered as the adult control group).

 

Figure 2
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  		FIG. 2. Developmental expression of genes showing increased expression in the Tfm mouse. Data show changes in testicular gene expression during development in normal mice (solid line) and in Tfm mice (gray bars). Expression in adult cryptorchid control mice is shown by the solid black bar. Expression was measured by real-time PCR, and data are expressed relative to the external control luciferase. Results show mean ± SEM for three to five animals per group. *, Significantly different (P < 0.05) from normal at the same age (adult cryptorchid mice are considered as the adult control group).

 
Data in Fig. 2Go show developmental changes in expression of genes up-regulated in the Tfm testis. Expression of Adh1 was clearly increased in the Tfm testis from about d 10, and this increase was sustained up to adulthood. In contrast, there were no significant differences in expression between normal and Tfm animals for other genes in this group. It should be emphasized, however, that results in Figs. 1Go and 2Go are expressed per testis (in contrast to Tables 1Go and 2Go, which are expressed relative to a housekeeping gene) and must be seen, therefore, in the context of cell numbers in the two groups (see Discussion).

Expression of genes involved in vitamin A metabolism
Three of the genes identified by the arrays with altered expression in the Tfm mouse testis are involved in vitamin A metabolism (Adh1, Cyp26b1, and Crbp1). Genes encoding other factors potentially involved in vitamin A metabolism in the testis are mostly not present on the gene chips used in this study, and their expression in the Tfm testis was, therefore, measured directly by real-time PCR (Fig. 3Go). The enzymes alcohol dehydrogenase and retinal dehydrogenase are required for synthesis of retinoic acid (RA) from vitamin A. Expression of Adh1 was increased, markedly, in the Tfm testes (Fig. 2Go), whereas expression of the two isoforms of retinal dehydrogenase expressed in the testis (Aldh1a1 and Aldh1a2) was decreased although not markedly in the case of the Aldh1a1 (Fig. 3Go). RA is metabolized by three cytochrome P450 hydroxylases (CYP26A1, CYP26B1, and CYP26C1). Expression of Cyp26b1 was significantly reduced in the Tfm testis (Fig. 1Go), whereas Cyp26a1 also showed reduced expression in the Tfm, although the difference was less marked (Fig. 3Go). Expression of Cyp26c1 was not detected by real-time PCR in normal or Tfm testes. In control animals, the expression of Cyp26b1 was about 10-fold higher than Cyp26a1, suggesting that this may be the predominant isoform in the testis.


Figure 3
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  		FIG. 3. Expression of genes involved in vitamin A metabolism but not included on Affymetrix gene chips. Data show testicular expression in adult and 20-d-old normal and Tfm mice of retinal dehydrogenase isoforms 1a1 (Aldh1a1) and 1a2 (Aldh1a2) and cytochrome P45026a1 (Cyp26a1). Expression of Cyp26c1 was not detectable in any group. Gene expression was measured by real-time PCR, and data are expressed relative to the external control luciferase. Results show mean ± SEM for three to five animals per group. *, Significantly different (P < 0.05) from normal at the same age (adult cryptorchid mice are considered as the adult control group).

 
Expression of solute carriers
In addition to the solute carriers shown to be down-regulated in Table 1Go and Fig. 1Go (Slc38a5, Slc39a8, Slc7a4, Slc25a10, and Slc35a7), there are a number of other carrier genes significantly altered in the Tfm testis but with a fold difference on the arrays of less than 2 (supplemental Table 2). We screened the expression profile of each of these genes, and two (Slc4a8 and Slc7a7) were shown to be likely to be expressed in the somatic cells and to be of a tubule origin. As with the other solute carriers shown in Fig. 1Go, expression of these two genes was significantly reduced in the Tfm at both 20 d and adulthood (Fig. 4Go).


Figure 4
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  		FIG. 4. Testicular expression of solute carriers 4a8 (Slc4a8) and 7a7 (Slc7a7) in adult and 20-d-old normal and Tfm mice. Gene expression was measured by real-time PCR, and data are expressed relative to the external control luciferase. Results show mean ± SEM for three to five animals per group. *, Significantly different (P < 0.05) from normal at the same age (adult cryptorchid mice are considered as the adult control group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the Tfm mouse, a single base deletion in the N-terminal domain results in a frameshift mutation (33) and generation of a truncated protein lacking DNA- and ligand-binding domains. The mutants are, therefore, completely androgen insensitive and develop with an external female phenotype and cryptorchid testes but without internal Wolffian or Müllerian duct derivatives. In this study, we have used these mice to examine the effect of complete androgen insensitivity on gene expression in the somatic tubular cells of the testis using oligoarrays.

At birth and up to 5 d, germ cell development in the testis of the Tfm mouse appears normal, but by 20 d, when the array study described here was carried out, there are clear differences in the progression of spermatogenesis between normal and Tfm testes (13). This age was chosen for the array studies because the mouse testis at 20 d is starting to show functional characteristics of the adult testis [e.g. meiosis has started and Sertoli cells have stopped dividing (32, 34)], but full spermatogenesis has not developed and the testes have not descended. We anticipated that most adult, testicular, androgen-dependent genes would be showing differential expression in the Tfm testis by 20 d. We also anticipated that a large number of germ-cell-specific genes would be present in the dataset, but fewer than would be found using adult tissue. Subsequent data confirmed that a large number of germ cell genes showed differential hybridization on the arrays, necessitating a number of subsequent data screens. These screens were conservative in nature, and it is likely that a number of somatic genes of interest will have been screened out. For example, any Sertoli cell genes that show a marked reduction in expression after germ cell ablation will have been screened out as likely germ cell genes.

Other array studies have been carried out to identify androgen-dependent genes in the testis using androgen-treated neonatal (8 d old) mice or hpg mice (35–45 d old) (35, 36) or 10-d-old Sertoli-cell-specific androgen receptor knockout (SCARKO) mice (37). With the exception of Rhox5, which is identified in all studies, there is little overlap of androgen-regulated genes between this study and earlier ones using androgen treatment of neonatal or hpg mice. The most likely explanation for this apparent discrepancy is that in the earlier studies, animals were treated for up to 24 h with androgen and the changes in gene expression measured are likely to represent genes that show an early response to androgen. In the Tfm model used here, there has been longer-term deprivation of androgens and a different set of genes appears to be affected.

Comparison of genes showing altered expression in the 10-d SCARKO testes with those altered in this study of 20-d Tfm testes showed that there are 409 genes that show a significant difference in expression in both models (82 down-regulated and 327 up-regulated in the androgen-insensitive animal) although only seven of these genes showed a 2-fold or greater difference in both Tfm and SCARKO testes (37). These seven genes include Rhox5 and Drd4, but the other five genes [Serine protease inhibitor-like, with Kunitz and WAP domains 1 (eppin), Serine (or cysteine) peptidase inhibitor, clade A, member 5, low density lipoprotein receptor-related protein 4, Testis-specific X-linked gene, and Serine (or cysteine) peptidase inhibitor, clade A, member 3N] were not further examined in this study because they were considered likely, from published studies or from EST data, to be expressed either in germ cells or in the interstitial tissue. Failure of both studies to identify a larger common set of genes showing a high level of androgen dependence is likely to be related to the age of the animals and to the animal models themselves. Most of the genes in this study that showed significantly altered expression in the Tfm testis at 20 d showed essentially normal expression at 10 d (Fig. 2Go) and would, therefore, not be detected in the study by Denolet et al. (37). In addition, the Tfm mouse lacks androgen receptors in both peritubular myoid cells and Sertoli cells, whereas the SCARKO mouse is more specific and lacks only Sertoli cell androgen receptor expression. Thus, androgen-dependent genes expressed predominantly in the peritubular cells (e.g. Cyp26b1) (38) would only be detected using the Tfm mouse. Finally, Tfm mice have high circulating FSH levels because of a lack of negative feedback (20-d Tfm serum FSH 94.4 ± 9.6 ng/ml vs. control 23.4 ± 2.4, unpublished), which may affect Sertoli cell gene expression and lead to further differences with the SCARKO mouse. It should be noted that in mice lacking FSH or the FSH receptor, expression of a number of Sertoli cell markers is reduced, and none are increased (13). Although this earlier study did not include a comprehensive review of all Sertoli cell genes, it suggests that the effect of elevated FSH would be to increase Sertoli cell activity. It is likely, therefore, that any confounding effect of elevated FSH in the current study will be on genes up-regulated in the Tfm testis. Additional studies to examine global gene expression in the FSH receptor knockout mouse will identify genes regulated by both androgen and FSH.

Overall, the different sets of genes identified in all these array studies should be considered complementary because it is likely that they identify genes directly or indirectly sensitive to androgens in the testis under different physiological and developmental conditions.

It is noticeable in this study that cryptorchidism in the adult acts to inhibit expression of many of the genes shown to be androgen dependent at 20 d. This means that in most cases, a significant effect of the Tfm mutation cannot be shown in the adult and this is clearly a drawback of this particular model. Interestingly, there are similarities in the effects of cryptorchidism and Sertoli cell androgen receptor ablation with respect to germ cell progression through spermatogenesis, although the effects of cryptorchidism are more severe (7, 13). Given the high coincidence of cryptorchid effects on androgen-sensitive genes shown in Fig. 1Go, it is possible that the effects of cryptorchidism and androgen receptor ablation may be mediated through a similar gene set. Alternatively, cryptorchidism may lead to a general depression of Sertoli cell function that affects expression of many genes including those identified in Fig. 1Go.

The genes showing altered expression in the testis of the Tfm mouse have a variety of postulated functions, but there are three clear subfamilies of genes grouped with respect to their overall cellular function: solute carriers and genes involved in vitamin A metabolism and cytoskeletal function.

Vitamin A metabolism
Vitamin A (retinol) is essential for male fertility, and deficiency leads to degeneration of spermatogenesis, probably through arrest of normal spermatogonial differentiation (39). Excess vitamin A also causes testicular lesions and spermatogenic disorders (40), and it is clear that exposure of the germ cells to RA (the active metabolite of vitamin A) must be carefully regulated. RA is inhibited from crossing the Sertoli cell barrier, and testicular RA comes largely from vitamin A metabolism in the Sertoli cells (41, 42). Production of RA from vitamin A is a two-step process; the first, rate-limiting, step is oxidation by alcohol dehydrogenase, while the second step is catalyzed by retinaldehyde dehydrogenase. Metabolism of RA is via hydroxylation catalyzed by three cytochrome P450 hydroxylases (CYP21A1, CYP21B1, and CYP21C1). In testes from Tfm mice, there was altered expression in a number of genes involved in vitamin A metabolism. The most notable difference was a marked increase in expression of Adh1, which encodes the type 1 isoform of alcohol dehydrogenase. This gene is expressed in Sertoli cells and some Leydig cells and is the predominant isoform expressed in the somatic cells of the testis (28). In addition to increased Adh1 expression, there were marked declines in expression of Cyp26b1 and Aldh1a2 and lesser changes in Cyp26a1 and Aldh1a1 in the Tfm testis. Both Cyp26b1 and Cyp26a1 are expressed in the peritubular cells, whereas Aldh1a1 is predominantly in the Leydig cells with some Sertoli cell expression, and Aldh1a2 is expressed in germ cells (38). Overall, the increased expression of Adh1 and reduced expression of RA-metabolizing enzymes would be expected to increase RA levels in the tubules of the Tfm testis. This may be ameliorated to some extent by reduced expression of retinaldehyde dehydrogenase isoforms, although expression of the predominant isoform (1a1) was not markedly affected, at least in the adult.

In addition to changes in expression of the vitamin A-metabolizing enzymes, there was increased expression of cellular retinol-binding protein 1 (Crbp1) in the Tfm testis. CRBP1 is the only retinol-binding protein expressed in the testis (43), and its expression is largely restricted to the Sertoli cells (44, 45). It is likely that CRBP1 plays a role in vitamin A storage and metabolism, and in CRBP1-null mice, storage is defective and animals are more sensitive to a vitamin A-deficient diet (46). This would suggest that in androgen-resistant Tfm mice, the storage capacity for vitamin A (and, thus, the available substrate for RA synthesis) may be increased.

Overall, these results show that androgens act to regulate synthesis and metabolism of RA in the tubules. Although the different genes identified in this study are expressed in a number of different cell types, the overall effect of androgen action may be to decrease RA levels.

It is possible that some of the differences in gene expression observed in the Tfm testis may not be due to loss of direct effects of androgen but may be indirect through, for example, loss of germ cells. It is unlikely, however, that all effects can be explained in this way. In cryptorchid mice, there is a similar loss of germ cells (13) and increased circulating FSH (47), but Adh1 levels are normal and Cyp26b1 is not markedly affected. Increased expression of Crbp1 in the adult Tfm testis is matched by a similar increase in the cryptorchid testis, although Sertoli cell number in the Tfm testis is only about 30% of normal, and expression per Sertoli cell is, therefore, significantly higher in the Tfm compared with the cryptorchid control (13).

Solute carriers
Solute carriers regulate the uptake and efflux of crucial compounds such as sugars, amino acids, nucleotides, and inorganic ions, and 43 different solute carrier families have now been identified (48). Tight junctions between adjacent Sertoli cells prevent free movement of water-soluble factors into the lumen of the seminiferous tubules and create the Sertoli cell barrier (49). This generates a unique intratubular environment that supports germ cell differentiation and development (50). The solute carriers, along with other transport proteins, will regulate the movement of solutes across the Sertoli cells and, thereby, maintain the composition of the tubule fluid. Our results show that in the absence of androgen receptor activity, the expression of genes regulating seven different solute carriers is disrupted. Five of these carriers (SLC38A5, SLC39A8, SLC7A4, SLC4A8, and SLC7A7) are associated with the plasma membrane and function to regulate transport of amino acids (SLC38A5, SLC7A4, and SLC7A7) (48), metal ions (SLC39A8) (51, 52, 53), and bicarbonate/chloride ions (SLC4A8) (54, 55).

The individual roles of each of these solute carriers is uncertain in terms of Sertoli cell function and spermatogenesis, although SLC38A5 transports three of the four predominant amino acids in seminiferous tubule fluid (48, 56, 57), and other substrates such as ornithine (SLC7A4) and zinc (SLC39A8) may also play a role in the development and maintenance of spermatogenesis (58, 59, 60). In addition, it is likely that the overall effect of disrupting expression of the seven solute carriers identified here will be an alteration in the composition of the fluid within the seminiferous tubule. The tight junctions between adjacent Sertoli cells normally ensure the integrity of the Sertoli cell barrier, and it is of interest that a recent publication has reported increased permeability of the Sertoli cell barrier in a mouse model for androgen insensitivity [Arinvflox(ex1-neo)/Y;Tg (Amh-Cre)] that may be associated with a change in expression of claudin 3 (61). This is consistent with earlier studies showing that the Sertoli cell barrier is disrupted in adult Tfm mice (62). Claudin 3 encodes a transient component of newly formed tight junctions (61), and expression was significantly reduced by 1.8-fold in our array studies (supplemental Table 2). This is less than the 10-fold difference reported for Arinvflox(ex1-neo)/Y;Tg (Amh-Cre) mice (61), although these array studies used adult animals, indicating that the androgen dependency of this gene may increase with age. It is of interest to note that Rai14, which is down-regulated 2.5-fold in the Tfm testis, is a cytoskeletal protein that is postulated to play a role in barrier formation (63). Altered expression of this gene may, therefore, contribute to the altered permeability of the Sertoli barrier in androgen-insensitive mice.

It should be noted that the altered expression of solute carriers in Tfm mice could be a secondary result of increased permeability of the Sertoli cell barrier in these mice, although it is not certain that the Sertoli cell barrier is in place in normal mice at 20 d (61, 64). Certainly, the reduced levels of Slc38a5 from d 5 in the Tfm testis shows that this particular carrier is affected by something other than changes in the Sertoli cell barrier. It might, in addition, be expected that disruption of the barrier would increase, rather than decrease, expression of the solute carriers that function to maintain the specialized composition of the tubular fluid.

Cytoskeleton
Actin cytoskeleton structures are essential for a wide variety of cell functions, including cell shape change, cell motility, cell adhesion, cell polarity, and cytokinesis. Among the genes showing differential expression in the Tfm testis are five (Rims2, Bin3, Actn3, Cnn3, and Rai14) associated with cytoskeletal function and regulation of endocytosis and exocytosis. In particular, four of these genes (Bin3, Actn3, Cnn3, and Rai14) have been linked to regulation of the actin cytoskeleton. BIN3 is a member of the Bin/amphiphysin/Rvs (BAR) domain protein group, which has been suggested to regulate endocytosis, actin organization, apoptosis, and transcription (65), ACTN3 is a cross-linking protein that binds actin to intracellular structures (66), CNN3 regulates the organization of the actin cytoskeleton by stabilizing actin networks (67), whereas RAI14 may be involved in maintenance and/or reorganization of actin cytoskeletal structures (68). In addition, the RIMS2 protein is involved in exocytosis in the pancreas and neuroendocrine cells by regulating fusion of vesicles to the plasma membrane (69). Although there is no specific information on testicular function of these genes, we can, nevertheless, postulate that androgen may be involved in the regulation of cytoskeletal structure and endo-/exocytosis in the Sertoli cell. It has been shown previously that the actin cytoskeleton of the Sertoli cell and the actin-based junctional complexes found at points of Sertoli-germ cell adhesion become disorganized after hypophysectomy and that androgen treatment prevents this effect (70). This would be consistent, therefore, with a role for androgens in regulating the Sertoli cell cytoskeleton.

Other genes altered in the Tfm mouse testis
In addition to the three groups of genes discussed above, there are nine other genes that show altered expression in the Tfm testis. This includes Wnt5a, which is a member of the WNT family of secreted proteins. This group of proteins control essential developmental processes such as embryonic patterning, cell growth, migration, and differentiation. WNT-5A has been shown to be able to activate or inhibit canonical Wnt signaling (71), and during development, it is important for cellular adhesion, migration, and polarity. No role for WNT5a has been described in the testis, although an EST has been identified in a Sertoli cell library (GenBank CB318227), suggesting that this may be the primary cell of origin. Results from the arrays indicate that androgens act to inhibit Wnt5a expression, suggesting that overexpression may be deleterious to normal testicular function in the adult.

The remaining genes with altered expression in the Tfm testis have either no known function at this time (Cdkal1, 1810021J13Rik, Bcl7b, and 3300001G02Rik) or have a known function (Drd4, Ltzfl1, Sbds, and Nudt19) but few data with which to postulate a role in the testis. Drd4 encodes a G protein-coupled receptor, but there is no reported testicular phenotype in mice lacking Drd4 (72), and it may, therefore, play a redundant role in testicular function. Lztfl1 is a putative transcription factor but without a known function, and there are no reported knockout studies currently available. Mutations in the Sbds gene are responsible for Shwachman-Bodian-Diamond syndrome in humans, which is characterized by exocrine pancreatic insufficiency, hematological abnormalities, and skeletal abnormalities (73). The function of SBDS is not clear, but it may be involved in RNA metabolism. There are no reported testicular defects in Shwachman-Bodian-Diamond syndrome, and SBDS function in the testis is not clear. Nudt19 is a member of the Nudix superfamily, which are mainly pyrophosphohydrolases that act upon substrates with the general structure of nucleoside diphosphate linked to another moiety. It is predicted that NUDT19 is a CoA hydrolase (74), although any role in testicular function is uncertain. Interestingly, NUDT19 has been shown to be androgen regulated in the kidney (74).

In summary, this study has generated a list of genes that are expressed in the somatic cells of the testicular tubules and are regulated by androgen either directly or indirectly. Other studies, using different animal models, have produced different lists of genes, and it is clear, therefore, that this is a subset of all androgen-responsive genes reflecting insensitivity to androgen at early puberty. This subset of genes is likely to be of physiological significance because the period of early puberty is when a morphological difference between normal and Tfm mice is first seen. The three families of genes that have been identified by this study offer potential mechanisms by which androgens may act to regulate spermatogenesis in the testis, at least during the critical developmental stages ongoing through puberty.


   Footnotes
 
This work was supported by the Wellcome Trust.

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 1, 2007

Abbreviations: CRBP1, Cellular retinol-binding protein 1; EST, expressed sequence tag; RA, retinoic acid; SCARKO, Sertoli cell-specific androgen receptor knockout.

Received October 19, 2006.

Accepted for publication February 16, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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