Expression of Androgen and Estrogen Receptors in Sertoli Cells: Studies Using the Mouse SK11 Cell Line
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《内分泌学杂志》
Medical Research Council Human Reproductive Sciences Unit (S.F.S., P.T.K.S.), Centre for Reproductive Biology, Edinburgh EH16 4TJ, Scotland, United Kingdom
Institute for Hormone and Fertility Research (N.W.), University of Hamburg, 22529 Hamburg, Germany
Abstract
Sertoli cells (Sc) play a major role in the establishment and maintenance of spermatogenesis. In the adult testis, Sc contain androgen receptor (AR) and estrogen receptor (ER)- but exhibit a loss of steroid responsiveness when maintained in primary culture. In the present study, we demonstrated that a transformed murine cell line (SK11) has retained a Sc phenotype and remains steroid responsive. SK11 cells expressed mRNAs found in Sc (aromatase, sulfated glycoprotein-1, sulfated glycoprotein-2, GATA-1, Sry-type high-mobility-group box transcription factor-9, testatin, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1) including those for AR and ER but not ER. AR and ER were immunolocalized to cell nuclei, and their ability to activate gene expression was investigated using transient transfections with reporter constructs containing either 3xERE or pem-androgen-responsive element promoters. Expression of the 3xERE reporter was induced after incubation with 17-estradiol (E2), 5-androstane-3-, 17-diol (3Adiol), or testosterone (T); up-regulation of the pem-androgen-responsive element reporter was detected only in the presence of T or dihydrotestosterone. Activation of the ERE reporter did not occur after targeted knockdown of ER mRNA. Expression of AR and ER mRNAs was increased after incubation of cells with T or E2, respectively. In conclusion, we have demonstrated that the SK11 Sc cell line contains functional AR and ER and that treatment of the cells with their respective steroids results in an increase in the amount of their mRNAs. Our results suggest that E2 or 3Adiol acting via ER might modulate Sc function in vivo and that SK11 cells provide a useful model that can be used to complement studies using Sc selective gene ablation.
Introduction
IN MAMMALS, the adult testis performs two essential functions; the synthesis and secretion of steroid hormones and the production of mature, haploid spermatozoa from immature diploid spermatogonia (spermatogenesis). The adult testis consists of two distinct compartments, the seminiferous tubules containing the germ cells and the Sertoli cells (Sc) and the interstitium that contains Leydig cells, peritubular myoid cells, macrophages, and blood vessels (1). Spermatogenesis is a complex process that involves numerous interactions between the Sc and germ cells that they support, overlaid with hormonal regulation of the Sc (reviewed in Refs.2, 3, 4). In rodents and the human, Sc proliferate during fetal life, but after birth their proliferation rate declines and during puberty they undergo a multistep process usually referred to as maturation or differentiation during which their morphology changes and adjacent Sc form tight junctions (reviewed in Ref.2). Among the changes that occur at this time are an increase in immunoexpression of sulfated glycoprotein 2 (SGP-2)/clusterin, (5) and p27kip (6).
Steroids are essential regulators of male fertility. The major secretory product of the Leydig cell is testosterone (T), and levels of T within the testis far exceed those within the peripheral circulation (7). T is metabolized to estrogens by the aromatase cytochrome P450 complex (8) and to dihydrotestosterone (DHT) by 5-reductase type 1 or 2 (9). In the intact testis, DHT can be further metabolized to 5-androstane-3-, 17-diol (3Adiol) by 3-hydroxysteroid dehydrogenase (3HSD) (10), which is expressed exclusively in Leydig cells (11). Expression of aromatase has been detected in Leydig cells, elongate spermatids, and immature Sc (12, 13); expression of 5 reductase has been detected in Leydig cells in rodents (9, 11).
In the testis, as in other tissues, steroids can influence cell function by binding to specific receptors that are members of a superfamily of ligand-activated transcription factors (14). To date one androgen receptor (AR), AR/NR3C4, encoded on the X-chromosome and two estrogen receptors (ERs), ESR1/ER and ESR2/ER, encoded on different autosomes, have been identified. The cell-specific pattern of expression of AR and ERs in the adult testes of rodents (15, 16, 17) and humans (18, 19, 20) has been elucidated using immunohistochemistry. In the adult testis, expression of AR in Sc nuclei is stage dependent in mouse, rat, and human (15, 16, 20). In mice, immunoexpression of AR in Sc is detectable on and after postnatal d 10 with a clear increase in intensity between d 12 and 20 (5, 21). The pattern of expression of ERs is distinct from that of AR. In rodents ER has been immunolocalized to Leydig cells and peritubular myoid cells but is not detectable in Sc or germ cells (15, 22, 23). ER has been immunolocalized to Sc, Leydig cells, and peritubular cells as well as some but not all germ cells (15, 24). Expression of ER protein does not appear to be stage dependent in adult rodent Sc, nor is there a clear age-dependent increase in expression during puberty as occurs with AR.
Because Sc make up a small proportion of the total cell number in the adult testis, attempts to identify the impact of hormones on Sc-specific gene expression have often used isolated primary Sc (25, 26, 27). However, we, and others, have found that freshly isolated Sc change their morphology and lose expression of steroid receptors (Sneddon, S. F., and P. T. K. Saunders, unpublished observations and Ref.28). The effort of constantly preparing fresh cell isolates has prompted investigators to prepare Sc cell lines from both rats and mice using a variety of methods (reviewed in Refs.29, 30). In the current study, we used the SK11 Sc cell line (31) that was originally derived from Sc isolated from 10-d-old H2Kb-tsA58 transgenic mice that express the temperature sensitive form of the Simian virus 40 (SV40) large T antigen (32). When these cells are incubated at 34 C (the permissive temperature), expression of the SV40 large T antigen is induced and the cells are mitotically active. However, at the nonpermissive temperature of 39 C, the SV40 antigen is inactivated, and the cells lose mitotic activity and undergo a change in cell morphology.
The current investigations were aimed at establishing whether the SK11 cell line might be used to complement other investigations in which the impact of steroids on Sc function have been advanced by the development of transgenic mice with a Sc-specific knockout of AR (33). We have demonstrated that the SK11 cells have retained the phenotype of murine Sc and that they express both AR and ER.
Materials and Methods
Steroids
17-Estradiol (E2), 3Adiol, T, and DHT were all obtained from Sigma (St. Louis, MO). Stock solutions were prepared by dissolving steroids in ethanol to a final concentration of 10–3 to 10–8 M and then added to the media to give final dilution in the range 10–6 to 10–11 M.
SK11 cell culture
SK11 cells were originally prepared as described in detail by Walther et al. (31) and have been intermittently cultured or frozen in the Edinburgh laboratory since 1997. They were cultured in DMEM (Sigma) containing 10% (vol/vol) heat-inactivated fetal bovine serum (Life Technologies, Inc., Paisley, UK) with the addition of 1% (wt/vol) penicillin/streptomycin (Life Technologies), 1% (wt/vol) nonessential amino acids (Life Technologies), 1% (wt/vol) Fungizone (Life Technologies), 1% (wt/vol) D(+)glucose (Sigma), and 1% 2 mM L-glutamine (Life Technologies). Cells were incubated at both permissive (34 C) and nonpermissive (39 C) temperatures. At 34 C the cells remained mitotically active, exhibiting undifferentiated characteristics. Functional differentiation, marked by the cells ceasing to divide, was achieved by culture at 39 C for at least 48 h. Steroid treatment of SK11 cells at 34 and 39 C was carried out by incubation with E2 or T for 48 h.
RT-PCR and quantitative RT-PCR
RNA was isolated from SK11 cells or freshly collected mouse testis using TRI reagent (Sigma) according to the manufacturer’s instructions. Total RNA was treated with DNase (Promega, Madison, WI), and oligo dT-primed cDNA was prepared using BioScript reverse transcriptase (Bioline, Randolph, MA) according to the manufacturer’s instructions. PCR was performed using specific primers for ER, ER, and AR (Table 1) in a reaction mix containing BioTaq DNA polymerase and buffers supplied with the enzyme (Bioline), an annealing temperature of 58 C, an extension temperature of 72 C, and 30 cycles of amplification
Random primed cDNA was prepared using the TaqMan reverse transcription kit (Applied Biosystems, Foster City, CA). Real-time PCR was performed with the ABI PRISM 7700 sequence detection system (Applied Biosystems). Expression of ER and AR mRNAs was determined by using specific primers and TaqMan probes (Table 2). Expression levels for both mRNAs were related to an internal control, 18S ribosomal RNA. Results are a mean of three separate experiments performed in duplicate.
Detection of protein in SK11 cells
Immunohistochemistry was performed on SK11 cells grown on 2-well glass chamber well slides (Nalgene Nunc International, Hereford, UK); 1 x 105 cells were plated in each well and allowed to grow until 70% confluency was reached. Medium was removed and cells were washed with PBS then fixed in ice-cold 100% methanol for 10 min. The cells were permeabilized with the addition of 0.2% Nonidet P-40 (Sigma), 1% BSA (Sigma), and 10% blocking serum in Tris-buffered saline for 20 min at room temperature. Before incubating cells with primary antibodies directed against ER and ER, they were subjected to heat-induced antigen retrieval (34) in 0.01 M citrate (pH 6) for 5 min. Cells were incubated with sheep anti-ER (1:1500) (35), rabbit anti-AR (1:200, Santa Cruz Biotechnology Inc., Santa Cruz, CA), or mouse anti-ER (1:20, Novocastra, Newcastle, UK) overnight at 4 C. Thereafter cells were washed in Tris-buffered saline and incubated with the appropriate biotinylated secondary antibodies for 1 h (ER: rabbit antisheep, Vector Laboratories, Peterborough, UK; AR: swine antirabbit, Dako, Cambridge, UK; rabbit antimouse, Dako), all used at a 1:500 dilution. Immunolocalization of receptors was visualized using diaminobenzidine according to standard methods (35); cells were counterstained with hematoxylin.
Transient transfections and luciferase assays
Jet-PEI (Q-Biogene, Cambridge, UK) was used for transient transfections according to the manufacturer’s instructions. SK11 cells (34 C) were plated in 12-well tissue culture plates at a density of 1 x 105 cells/ml per 24 h before transfection. Cells were maintained in DMEM phenol red-free medium (Sigma) supplemented with 10% charcoal-stripped fetal bovine serum (Life Technologies) and other additives (as above). In each well, cells were transfected with 1 μg of a reporter construct [either 3x vitellogenin estrogen response element (ERE) linked to TK luciferase reporter (36) or the promoter of the Pem gene that contains two androgen-responsive element (ARE) binding sites (37) prepared as detailed elsewhere (38) together with 100 ng control plasmid (pRL-CMV, Promega). Fresh media containing vehicle alone (ethanol) or ligand (E2, 3Adiol, T, or DHT) dissolved in ethanol were added to the cells 4 and 24 h after transfection; final ligand concentrations ranged from 10–11 to 10–6 M. Cells were harvested and assayed for gene activity using the dual-luciferase reporter gene assay (Promega) 48 h after transfection. Results were normalized against the Renilla internal control and expressed as a fold increase relative to the vehicle-treated control.
Construction of ER-specific short hairpin (sh) RNA-containing vector
The pSilencer 3.0-H1 vector (Ambion, Abingdon, UK) was used to express an ER-specific shRNA using the RNA polymerase III H1 promoter. Briefly, oligonucleotides with 3' single stranded overhangs were designed to contain a 19-mer hairpin sequence specific to the ER mRNA target, a loop sequence separating the two complementary domains and a transcription termination sequence (sense oligonucleotide, 5'-GATCCAAGATAATGGTCAAGCTTCTCAAGAGAAAGCTTGCCATTATCTTCTTTTTTCGGAAA-3', antisense oligonucleotide, 5'-AGCTTTTCCAAAAAAGAAGATAATGGTCAAGCTTTCTCTTGAGAAGCTTGACCATTATCTTCG-'). The oligonucleotides were annealed to form the hairpin, digested with BamH1 and HindIII and ligated into the pSilencer using standard methods. Plasmids were propagated in XL1-blue cells (Stratagene, La Jolla, CA), purified, and sequenced to ensure the correct orientation of the insert.
Statistical analysis
All experiments were repeated at least three times, and values are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni posttest using GraphPad Prism software (GraphPad Inc., San Diego, CA), with P < 0.05 deemed significant.
Results
Expression of Sc-specific mRNAs in SK11 cells
In previous studies we, and others, have noted rapid dedifferentiation of primary Sc in vitro. When the SK11 cells were originally prepared, they were reported to express mRNAs for FSH receptor (FSHR), AR, GATA-1, and SGP-2 but not the Leydig cell-specific marker 3HSD (31). Recent publications have suggested that over time expression of FSHR has been lost (39), and because we have maintained our SK11 cells in culture for a number of years, we reevaluated their pattern of gene expression using cells grown at both 34 C (permissive) and 39 C (nonpermissive) temperatures. The expression of mRNAs for SGP-2 (clusterin), dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1, testatin, GATA-1, and -actin, all of which are expressed by Sc in vivo, was confirmed. Expression of SGP-2 mRNA was increased in the cells grown at 39 C, compared with those at 34 C (not shown). Expression of the mRNA for SGP-1, a mRNA previously identified in mouse Sc (40), was detected in SK11 cells (Fig. 1A, lanes 3 and 4) as well as extracts of testes from d 10 and adult mice (Fig. 1A, lanes 1 and 2). The SK11 cells also expressed low levels of aromatase mRNA (Fig. 1B, lanes 3 and 4). Immunoexpression of SGP-1 and GATA-1 in cell monolayers was confirmed (not shown).
Expression of AR and ER in SK11 cells
DNA products corresponding to ER (339 bp) and AR (266 bp) were amplified from cDNA prepared from SK11 cells and testes recovered on d 10 and in adulthood (Fig. 1C). No ER mRNA was detected in SK11 cells (lanes 3 and 4), although mRNA was present in extracts prepared from d 10 and adult testes (lanes 1 and 2). ER and AR mRNAs were detected in testes from d 10 and adult mice (ER, lanes 6 and 7; AR, lanes 11 and 12) as well as SK11 cells. Levels of expression of ER mRNA were similar in cells grown at the nonpermissive temperature, compared with those that were cultured at 34 C (compare lanes 8 and 9). AR mRNA appeared higher in cells grown at 39 C (lane 14), compared with those that were growing at the permissive temperature (lane 13), but this was not confirmed when a more detailed analysis was carried using quantitative (Taqman) RT-PCR (not shown).
Immunohistochemistry was carried out to confirm that the SK11 cells had actively translated the mRNAs encoding AR and ER (Fig. 2). AR and ER proteins were both detected within the nuclei of the cells. Immunostaining of AR appeared more intense in the cells cultured at 39 C (Fig. 2B), compared with those maintained at 34 C (Fig. 2A). No striking difference was observed in the intensity of ER immunostaining, or the proportion of immunopositive cells between the two culture conditions. No ER protein was detected (not shown).
Activation of an ARE in SK11 cells
Cells were transfected with a reporter construct containing an androgen-responsive promoter-reporter prepared from the mouse Pem gene (37) and then incubated with androgenic (T, DHT) or estrogenic (E2, 3Adiol) ligands. An increase in reporter gene activity was measured when the cells were stimulated with T or DHT, but there was no change in the presence of Adiol or E2 (Fig. 3). Stimulation of the reporter gene was highest when the cells were incubated with DHT (6-fold, 10–7 M); maximal stimulation with T was approximately 2.5-fold (10–8 M).
Activation of an ERE reporter in SK11 cells
The transcriptional activity of ER in the SK11 cells was analyzed by introduction of a reporter construct containing three copies of the vitellogenin ERE (36). Stimulation of expression of the reporter construct was detected when cells were incubated with E2, 3Adiol, or T, but there was no increase in expression with DHT (Fig. 4). Maximal responsiveness (5-fold) was detected in the presence of 10–7 M E2 (Fig. 4A); at identical concentrations of 3Adiol and T, reporter gene expression was 3.6- and 2.2-fold, respectively (Fig. 4, B and C).
Impact of ER selective knockdown on ERE activation
Functional activity of the shRNA directed against mouse ER was validated by cotransfecting SK11 cells with a murine ER cDNA cloned into the DsRed expression vector (Clontech, Palo Alto, CA). After 48 h, expression of DsRed-mER was detected when an shRNA directed against an unrelated protein (green fluorescent protein) was present, but no ER-DsRed was detected when the shRNA was directed against ER (not shown). Transfection of SK11 cells with the shRNA directed against ER reduced the endogenous level of ER mRNA in the cells (Fig. 5A). Introduction of the shRNA also prevented activation of the ERE reporter construct when cells were incubated with increasing concentrations of E2, confirming that this was mediated via ER in the control cells (Fig. 5).
Impact of steroid hormones on expression of AR and ER mRNAs
Incubation of cells with E2 for 48 h (34 C) resulted in a significant increase in the amount of ER mRNA, compared with controls (vehicle alone). The impact of E2 was concentration dependent (Fig. 6A). Addition of T did not change levels of expression of ER mRNA (Fig. 6A). Similar results were obtained when the experiment was repeated using cells grown at the nonpermissive temperature (not shown).
Incubation of cells grown at 34 C with T resulted in a significant increase in the amount of AR mRNA (Fig. 6B). The increase was concentration dependent (4-fold at 10–8 M and 14-fold at 10–7 M). In cells grown at 39 C, incubation with T also resulted in increased expression of AR mRNA (14-fold at 10–8 M and 33-fold increase at 10–7 M T, not shown). This result would appear to be consistent with the observed increase in of the amount of AR protein in cells at the nonpermissive temperature. Incubation of cells with E2 had no impact on expression of AR mRNA at either 34 C (Fig. 6B) or 39 C (not shown).
Discussion
In the present study, we have demonstrated that SK11 cells, originally isolated from the testes of d 10 mice, have retained a Sc phenotype. In agreement with the original publication in which their isolation was described (31), they continued to express mRNAs for GATA-1, SGP-2, and AR, and we also detected expression of a range of other mRNAs that are expressed by Sc in vivo. At 34 C the cells were large with an even distribution of -actin, whereas after 48 h at 39 C, they become rounded and flattened with an accumulation of -actin around their nuclei (Sneddon, S., unpublished observations). Incubation at 39 C was also associated with a quantitative increase in the expression of SGP-2 mRNA. We have shown for the first time these cells express mRNA and protein for ER but not ER. Immunoexpression of AR in SK11 cells grown at the nonpermissive temperature appeared to be increased, compared with those that were grown at 34 C. There was no obvious difference in ER immunoexpression between cells grown at the different temperatures. This pattern of expression is consistent with that described after immunostaining of fixed sections from testes of mice (15). Notably, whereas the intensity of immunoexpression of AR in murine Sc increases during the first wave of spermatogenesis and is stage dependent in adulthood, no apparent increase in immunoexpression of ER in mature Sc, compared with their immature counterparts, has been reported (5, 24).
Sc lines have been derived from the testes of mice, rats, and sheep using a number of different immortalization strategies (reviewed in Ref.30) including the same method as that used for the SK11 cells, namely the expression of the SV40 T antigen. Several studies have explored whether these cell lines are responsive to FSH and/or cAMP (41, 42, 43), and they have been used to study FSH responsiveness after stable transfection with FSHR constructs (39, 44). In comparison, relatively little attention has been paid to the steroid hormone status of Sc lines (30). Mather (41) reported that the TM4 cell line, which was prepared from the testes of 11- to 13-d-old mice by repeated passaging, was growth inhibited by low levels of E2 (45). They demonstrated that TM4 contained low-capacity, high-affinity binding sites for E2 and T; binding of radioactive E2 was reduced by addition of 100-fold molar excess of unlabeled E2 or diethylstilbestrol but was unaffected by T, progesterone, or dexamethasone (46). In their cultures the content of both ERs and ARs increased as a function of cell density (46). These studies predate the cloning of the ER cDNA, and therefore, we cannot be sure whether expression of ER protein was responsible for the binding of estrogen by these cells. Konrad et al. (47) reported that an immortalized rat Sc cell line prepared using SV40-induced transformation lost expression of AR, and they did not examine estrogen responsiveness. In a recent study, the MSC-1 cell line (48) was used to confirm that expression of rHox genes was androgen regulated. Endogenous expression of AR was too low to induce significant gene expression; however, introduction of an AR expression construct resulted in a T-mediated induction of gene expression (49).
AR and ER expressed in the SK11 cells were capable to inducing expression of reporter gene constructs when the cells were incubated with a range of steroid ligands. Previous investigators have been able to demonstrate functional AR in primary Sc prepared from immature rats using a mouse mammary tumor virus promoter construct (50), but to date no other studies have used ERE reporters to study the impact of estrogens on Sc. In our studies the reporter construct used to investigate functional activity of AR was prepared from the promoter of the Pem oncogene (37). Previous studies have identified sequences for two AREs in this promoter, and there is evidence that the proximal promoter directs expression in the Sc and epididymis in mice (51). Recently we detected expression of Pem mRNA in the SK11 cells using RT-PCR (Hooley, R. P., and P. T. K. Saunders, unpublished observations). Treatment of cells with either T or DHT resulted in an increase in expression of the pem reporter, but there was no expression above control levels in cells treated with E2 or 3Adiol. Activation with DHT was approximately 3-fold higher than that with T, consistent with reports that the AR displays higher affinity for DHT and that this ligand has a slower dissociation rate than T (52). To date there is no evidence that estrogens can have an impact on expression of the Pem gene, and the data obtained in the current study using the Pem reporter construct would suggest this does not occur. Previous investigators have demonstrated that ER can bind both E2 and 3Adiol (53), and in the present study, both ligands were able to induce expression of the 3xERE reporter in Sc. Significant expression of the reporter was also induced when cells were treated with T, but no activation was noted with DHT. This result suggests that some of the T was metabolized to E2 and is consistent with detection of aromatase mRNA in the SK11 cells. The SK11 cells do not express 3HSD (31), and this means they are unable to synthesize 3Adiol, consistent with the lack of reporter gene activation after incubation with DHT. The key role played by ER in mediating the induction of the ERE reporter expression was confirmed by introduction of an ER-specific shRNA that resulted in ablation of the E2-mediated response.
A key finding from the present study was that expression of ER and AR mRNAs were increased after incubation of cells for 48 h with E2 or T, respectively. Examination of sequences up to 10 kb upstream of the transcriptional start site in the Ar and Esr2 genes cloned from mice reveals the existence of a weak ARE consensus site in the Ar and several AP-1 binding sites but no consensus ERE in the Esr2. Additional studies will be required before we can establish whether the impact of steroid treatment on the amount of mRNA is due to a direct effect on gene transcription. However, it is notable that in cultures of primary Sc from immature rats, a transient effect on AR mRNA expression was observed after the addition of FSH, and results from a transcriptional run-on assay demonstrated that the short-term effect of FSH on AR mRNA expression reflected a change in mRNA stability (54). Androgens have also been shown to autoregulate expression of their receptors by altering mRNA stability in some cell types (reviewed in Ref.55). To date the impact of steroids on the stability of AR and ER mRNAs in mouse Sc has not been investigated, and the SK11 cells provide a model system in which to examine this. It would also be interesting to establish whether SK11 cells express other members of the recently identified Rhox gene family (49) in addition to Pem (rhox5) and to examine whether all, or some, are androgen regulated in SK11 cells. Further studies are underway using SK11 cells to investigate the regulation of genes recently identified using array analysis of testes from 10-d-old mice with Sc selective ablation of AR (known as SCARKO) (5, 33).
Whereas a role for expression of AR in Sc in supporting the process of spermatogenesis has been established using cell-specific knockouts of AR (33, 56), a role for ER in Sc function is less certain. To date targeted deletions of the Esr2 gene have not resulted in development of a testicular phenotype (57), and no Sc selective ablation of ER has been reported. In females ER is the predominant ER in the ovary, and ER protein is expressed in granulosa cells in both growing and mature follicles (35). Furthermore, in the absence of E2, granulosa cells adopt a phenotype that resembles that of Sc (58). In mice lacking a fully functional ER gene, the granulosa cells have an attenuated response to FSH-induced differentiation, resulting in a reduced ovulation rate (59). In the present study, activation of ERE reporter expression occurred when cells were incubated with the DHT metabolite 3Adiol. Studies in the mouse have demonstrated that 3Adiol is synthesized within the prostate, and it has been proposed that ER, probably as a complex with 3Adiol, is involved in regulating the AR content of the rodent prostate and restraining epithelial growth (60, 61). 3Adiol has also been shown to be a potent modulator of ER-mediated gene transcription in neuronal cells (62). The enzymes required for synthesis of 3Adiol from T (5-reductase and 3HSD) are both expressed in the Leydig cells within the rodent testis (9, 63), and 3Adiol has been measured in the venous drainage of the rat testis (64). Studies in the human have confirmed that 3Adiol is synthesized from DHT in testicular tissue and have reported that a significant decrease the amount of steroid is found in men with defects in spermatogenesis (65, 66, 67). Taken together these data suggest that this ligand may play a role in activation of the ER protein that is expressed in testicular Sc as well as other cells types including germ cells (15, 18). Ligand-dependent modulation of ER-mediated signaling by 3Adiol, which may be elevated in the testes of mice with targeted deletion of cyp19, may explain why the phenotype of these animals (68) differs from that of the ER/ER knockout mice (7).
In conclusion, SK11 cells have retained the phenotypic characteristics of mouse Sc including the expression of the steroid receptors AR and ER. These cells therefore provide a model system in which to explore the potential cross-talk between AR and ER signaling pathways in the Sc and may reveal new roles for ER in modulation of testis function.
Acknowledgments
We are grateful to the laboratories of Professors D. P. McDonnell (Durham, NC) and F. Claessens (Leuven, Belgium) for the generous gifts of plasmids containing 3xERE-luc and Pem-luc constructs, respectively. We thank Dr. Kevin Morgan for assistance with analysis of gene promoter sequences.
Footnotes
Present address for N.W.: Institute for Hormone and Fertility Research, University of Hamburg, Falkenried 88, 20251 Hamburg, Germany.
First Published Online September 15, 2005
Abbreviations: 3Adiol, 5-androstane-3-, 17-diol; AR, androgen receptor; ARE, androgen-responsive element; DHT, dihydrotestosterone; E2, 17-estradiol; ER, estrogen receptor; ERE, estrogen response element; FSHR, FSH receptor; 3HSD, 3-hydroxysteroid dehydrogenase; Sc, Sertoli cells; SGP-2, sulfated glycoprotein 2; SV40, Simian virus 40; T, testosterone.
Accepted for publication September 7, 2005.
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Institute for Hormone and Fertility Research (N.W.), University of Hamburg, 22529 Hamburg, Germany
Abstract
Sertoli cells (Sc) play a major role in the establishment and maintenance of spermatogenesis. In the adult testis, Sc contain androgen receptor (AR) and estrogen receptor (ER)- but exhibit a loss of steroid responsiveness when maintained in primary culture. In the present study, we demonstrated that a transformed murine cell line (SK11) has retained a Sc phenotype and remains steroid responsive. SK11 cells expressed mRNAs found in Sc (aromatase, sulfated glycoprotein-1, sulfated glycoprotein-2, GATA-1, Sry-type high-mobility-group box transcription factor-9, testatin, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1) including those for AR and ER but not ER. AR and ER were immunolocalized to cell nuclei, and their ability to activate gene expression was investigated using transient transfections with reporter constructs containing either 3xERE or pem-androgen-responsive element promoters. Expression of the 3xERE reporter was induced after incubation with 17-estradiol (E2), 5-androstane-3-, 17-diol (3Adiol), or testosterone (T); up-regulation of the pem-androgen-responsive element reporter was detected only in the presence of T or dihydrotestosterone. Activation of the ERE reporter did not occur after targeted knockdown of ER mRNA. Expression of AR and ER mRNAs was increased after incubation of cells with T or E2, respectively. In conclusion, we have demonstrated that the SK11 Sc cell line contains functional AR and ER and that treatment of the cells with their respective steroids results in an increase in the amount of their mRNAs. Our results suggest that E2 or 3Adiol acting via ER might modulate Sc function in vivo and that SK11 cells provide a useful model that can be used to complement studies using Sc selective gene ablation.
Introduction
IN MAMMALS, the adult testis performs two essential functions; the synthesis and secretion of steroid hormones and the production of mature, haploid spermatozoa from immature diploid spermatogonia (spermatogenesis). The adult testis consists of two distinct compartments, the seminiferous tubules containing the germ cells and the Sertoli cells (Sc) and the interstitium that contains Leydig cells, peritubular myoid cells, macrophages, and blood vessels (1). Spermatogenesis is a complex process that involves numerous interactions between the Sc and germ cells that they support, overlaid with hormonal regulation of the Sc (reviewed in Refs.2, 3, 4). In rodents and the human, Sc proliferate during fetal life, but after birth their proliferation rate declines and during puberty they undergo a multistep process usually referred to as maturation or differentiation during which their morphology changes and adjacent Sc form tight junctions (reviewed in Ref.2). Among the changes that occur at this time are an increase in immunoexpression of sulfated glycoprotein 2 (SGP-2)/clusterin, (5) and p27kip (6).
Steroids are essential regulators of male fertility. The major secretory product of the Leydig cell is testosterone (T), and levels of T within the testis far exceed those within the peripheral circulation (7). T is metabolized to estrogens by the aromatase cytochrome P450 complex (8) and to dihydrotestosterone (DHT) by 5-reductase type 1 or 2 (9). In the intact testis, DHT can be further metabolized to 5-androstane-3-, 17-diol (3Adiol) by 3-hydroxysteroid dehydrogenase (3HSD) (10), which is expressed exclusively in Leydig cells (11). Expression of aromatase has been detected in Leydig cells, elongate spermatids, and immature Sc (12, 13); expression of 5 reductase has been detected in Leydig cells in rodents (9, 11).
In the testis, as in other tissues, steroids can influence cell function by binding to specific receptors that are members of a superfamily of ligand-activated transcription factors (14). To date one androgen receptor (AR), AR/NR3C4, encoded on the X-chromosome and two estrogen receptors (ERs), ESR1/ER and ESR2/ER, encoded on different autosomes, have been identified. The cell-specific pattern of expression of AR and ERs in the adult testes of rodents (15, 16, 17) and humans (18, 19, 20) has been elucidated using immunohistochemistry. In the adult testis, expression of AR in Sc nuclei is stage dependent in mouse, rat, and human (15, 16, 20). In mice, immunoexpression of AR in Sc is detectable on and after postnatal d 10 with a clear increase in intensity between d 12 and 20 (5, 21). The pattern of expression of ERs is distinct from that of AR. In rodents ER has been immunolocalized to Leydig cells and peritubular myoid cells but is not detectable in Sc or germ cells (15, 22, 23). ER has been immunolocalized to Sc, Leydig cells, and peritubular cells as well as some but not all germ cells (15, 24). Expression of ER protein does not appear to be stage dependent in adult rodent Sc, nor is there a clear age-dependent increase in expression during puberty as occurs with AR.
Because Sc make up a small proportion of the total cell number in the adult testis, attempts to identify the impact of hormones on Sc-specific gene expression have often used isolated primary Sc (25, 26, 27). However, we, and others, have found that freshly isolated Sc change their morphology and lose expression of steroid receptors (Sneddon, S. F., and P. T. K. Saunders, unpublished observations and Ref.28). The effort of constantly preparing fresh cell isolates has prompted investigators to prepare Sc cell lines from both rats and mice using a variety of methods (reviewed in Refs.29, 30). In the current study, we used the SK11 Sc cell line (31) that was originally derived from Sc isolated from 10-d-old H2Kb-tsA58 transgenic mice that express the temperature sensitive form of the Simian virus 40 (SV40) large T antigen (32). When these cells are incubated at 34 C (the permissive temperature), expression of the SV40 large T antigen is induced and the cells are mitotically active. However, at the nonpermissive temperature of 39 C, the SV40 antigen is inactivated, and the cells lose mitotic activity and undergo a change in cell morphology.
The current investigations were aimed at establishing whether the SK11 cell line might be used to complement other investigations in which the impact of steroids on Sc function have been advanced by the development of transgenic mice with a Sc-specific knockout of AR (33). We have demonstrated that the SK11 cells have retained the phenotype of murine Sc and that they express both AR and ER.
Materials and Methods
Steroids
17-Estradiol (E2), 3Adiol, T, and DHT were all obtained from Sigma (St. Louis, MO). Stock solutions were prepared by dissolving steroids in ethanol to a final concentration of 10–3 to 10–8 M and then added to the media to give final dilution in the range 10–6 to 10–11 M.
SK11 cell culture
SK11 cells were originally prepared as described in detail by Walther et al. (31) and have been intermittently cultured or frozen in the Edinburgh laboratory since 1997. They were cultured in DMEM (Sigma) containing 10% (vol/vol) heat-inactivated fetal bovine serum (Life Technologies, Inc., Paisley, UK) with the addition of 1% (wt/vol) penicillin/streptomycin (Life Technologies), 1% (wt/vol) nonessential amino acids (Life Technologies), 1% (wt/vol) Fungizone (Life Technologies), 1% (wt/vol) D(+)glucose (Sigma), and 1% 2 mM L-glutamine (Life Technologies). Cells were incubated at both permissive (34 C) and nonpermissive (39 C) temperatures. At 34 C the cells remained mitotically active, exhibiting undifferentiated characteristics. Functional differentiation, marked by the cells ceasing to divide, was achieved by culture at 39 C for at least 48 h. Steroid treatment of SK11 cells at 34 and 39 C was carried out by incubation with E2 or T for 48 h.
RT-PCR and quantitative RT-PCR
RNA was isolated from SK11 cells or freshly collected mouse testis using TRI reagent (Sigma) according to the manufacturer’s instructions. Total RNA was treated with DNase (Promega, Madison, WI), and oligo dT-primed cDNA was prepared using BioScript reverse transcriptase (Bioline, Randolph, MA) according to the manufacturer’s instructions. PCR was performed using specific primers for ER, ER, and AR (Table 1) in a reaction mix containing BioTaq DNA polymerase and buffers supplied with the enzyme (Bioline), an annealing temperature of 58 C, an extension temperature of 72 C, and 30 cycles of amplification
Random primed cDNA was prepared using the TaqMan reverse transcription kit (Applied Biosystems, Foster City, CA). Real-time PCR was performed with the ABI PRISM 7700 sequence detection system (Applied Biosystems). Expression of ER and AR mRNAs was determined by using specific primers and TaqMan probes (Table 2). Expression levels for both mRNAs were related to an internal control, 18S ribosomal RNA. Results are a mean of three separate experiments performed in duplicate.
Detection of protein in SK11 cells
Immunohistochemistry was performed on SK11 cells grown on 2-well glass chamber well slides (Nalgene Nunc International, Hereford, UK); 1 x 105 cells were plated in each well and allowed to grow until 70% confluency was reached. Medium was removed and cells were washed with PBS then fixed in ice-cold 100% methanol for 10 min. The cells were permeabilized with the addition of 0.2% Nonidet P-40 (Sigma), 1% BSA (Sigma), and 10% blocking serum in Tris-buffered saline for 20 min at room temperature. Before incubating cells with primary antibodies directed against ER and ER, they were subjected to heat-induced antigen retrieval (34) in 0.01 M citrate (pH 6) for 5 min. Cells were incubated with sheep anti-ER (1:1500) (35), rabbit anti-AR (1:200, Santa Cruz Biotechnology Inc., Santa Cruz, CA), or mouse anti-ER (1:20, Novocastra, Newcastle, UK) overnight at 4 C. Thereafter cells were washed in Tris-buffered saline and incubated with the appropriate biotinylated secondary antibodies for 1 h (ER: rabbit antisheep, Vector Laboratories, Peterborough, UK; AR: swine antirabbit, Dako, Cambridge, UK; rabbit antimouse, Dako), all used at a 1:500 dilution. Immunolocalization of receptors was visualized using diaminobenzidine according to standard methods (35); cells were counterstained with hematoxylin.
Transient transfections and luciferase assays
Jet-PEI (Q-Biogene, Cambridge, UK) was used for transient transfections according to the manufacturer’s instructions. SK11 cells (34 C) were plated in 12-well tissue culture plates at a density of 1 x 105 cells/ml per 24 h before transfection. Cells were maintained in DMEM phenol red-free medium (Sigma) supplemented with 10% charcoal-stripped fetal bovine serum (Life Technologies) and other additives (as above). In each well, cells were transfected with 1 μg of a reporter construct [either 3x vitellogenin estrogen response element (ERE) linked to TK luciferase reporter (36) or the promoter of the Pem gene that contains two androgen-responsive element (ARE) binding sites (37) prepared as detailed elsewhere (38) together with 100 ng control plasmid (pRL-CMV, Promega). Fresh media containing vehicle alone (ethanol) or ligand (E2, 3Adiol, T, or DHT) dissolved in ethanol were added to the cells 4 and 24 h after transfection; final ligand concentrations ranged from 10–11 to 10–6 M. Cells were harvested and assayed for gene activity using the dual-luciferase reporter gene assay (Promega) 48 h after transfection. Results were normalized against the Renilla internal control and expressed as a fold increase relative to the vehicle-treated control.
Construction of ER-specific short hairpin (sh) RNA-containing vector
The pSilencer 3.0-H1 vector (Ambion, Abingdon, UK) was used to express an ER-specific shRNA using the RNA polymerase III H1 promoter. Briefly, oligonucleotides with 3' single stranded overhangs were designed to contain a 19-mer hairpin sequence specific to the ER mRNA target, a loop sequence separating the two complementary domains and a transcription termination sequence (sense oligonucleotide, 5'-GATCCAAGATAATGGTCAAGCTTCTCAAGAGAAAGCTTGCCATTATCTTCTTTTTTCGGAAA-3', antisense oligonucleotide, 5'-AGCTTTTCCAAAAAAGAAGATAATGGTCAAGCTTTCTCTTGAGAAGCTTGACCATTATCTTCG-'). The oligonucleotides were annealed to form the hairpin, digested with BamH1 and HindIII and ligated into the pSilencer using standard methods. Plasmids were propagated in XL1-blue cells (Stratagene, La Jolla, CA), purified, and sequenced to ensure the correct orientation of the insert.
Statistical analysis
All experiments were repeated at least three times, and values are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni posttest using GraphPad Prism software (GraphPad Inc., San Diego, CA), with P < 0.05 deemed significant.
Results
Expression of Sc-specific mRNAs in SK11 cells
In previous studies we, and others, have noted rapid dedifferentiation of primary Sc in vitro. When the SK11 cells were originally prepared, they were reported to express mRNAs for FSH receptor (FSHR), AR, GATA-1, and SGP-2 but not the Leydig cell-specific marker 3HSD (31). Recent publications have suggested that over time expression of FSHR has been lost (39), and because we have maintained our SK11 cells in culture for a number of years, we reevaluated their pattern of gene expression using cells grown at both 34 C (permissive) and 39 C (nonpermissive) temperatures. The expression of mRNAs for SGP-2 (clusterin), dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1, testatin, GATA-1, and -actin, all of which are expressed by Sc in vivo, was confirmed. Expression of SGP-2 mRNA was increased in the cells grown at 39 C, compared with those at 34 C (not shown). Expression of the mRNA for SGP-1, a mRNA previously identified in mouse Sc (40), was detected in SK11 cells (Fig. 1A, lanes 3 and 4) as well as extracts of testes from d 10 and adult mice (Fig. 1A, lanes 1 and 2). The SK11 cells also expressed low levels of aromatase mRNA (Fig. 1B, lanes 3 and 4). Immunoexpression of SGP-1 and GATA-1 in cell monolayers was confirmed (not shown).
Expression of AR and ER in SK11 cells
DNA products corresponding to ER (339 bp) and AR (266 bp) were amplified from cDNA prepared from SK11 cells and testes recovered on d 10 and in adulthood (Fig. 1C). No ER mRNA was detected in SK11 cells (lanes 3 and 4), although mRNA was present in extracts prepared from d 10 and adult testes (lanes 1 and 2). ER and AR mRNAs were detected in testes from d 10 and adult mice (ER, lanes 6 and 7; AR, lanes 11 and 12) as well as SK11 cells. Levels of expression of ER mRNA were similar in cells grown at the nonpermissive temperature, compared with those that were cultured at 34 C (compare lanes 8 and 9). AR mRNA appeared higher in cells grown at 39 C (lane 14), compared with those that were growing at the permissive temperature (lane 13), but this was not confirmed when a more detailed analysis was carried using quantitative (Taqman) RT-PCR (not shown).
Immunohistochemistry was carried out to confirm that the SK11 cells had actively translated the mRNAs encoding AR and ER (Fig. 2). AR and ER proteins were both detected within the nuclei of the cells. Immunostaining of AR appeared more intense in the cells cultured at 39 C (Fig. 2B), compared with those maintained at 34 C (Fig. 2A). No striking difference was observed in the intensity of ER immunostaining, or the proportion of immunopositive cells between the two culture conditions. No ER protein was detected (not shown).
Activation of an ARE in SK11 cells
Cells were transfected with a reporter construct containing an androgen-responsive promoter-reporter prepared from the mouse Pem gene (37) and then incubated with androgenic (T, DHT) or estrogenic (E2, 3Adiol) ligands. An increase in reporter gene activity was measured when the cells were stimulated with T or DHT, but there was no change in the presence of Adiol or E2 (Fig. 3). Stimulation of the reporter gene was highest when the cells were incubated with DHT (6-fold, 10–7 M); maximal stimulation with T was approximately 2.5-fold (10–8 M).
Activation of an ERE reporter in SK11 cells
The transcriptional activity of ER in the SK11 cells was analyzed by introduction of a reporter construct containing three copies of the vitellogenin ERE (36). Stimulation of expression of the reporter construct was detected when cells were incubated with E2, 3Adiol, or T, but there was no increase in expression with DHT (Fig. 4). Maximal responsiveness (5-fold) was detected in the presence of 10–7 M E2 (Fig. 4A); at identical concentrations of 3Adiol and T, reporter gene expression was 3.6- and 2.2-fold, respectively (Fig. 4, B and C).
Impact of ER selective knockdown on ERE activation
Functional activity of the shRNA directed against mouse ER was validated by cotransfecting SK11 cells with a murine ER cDNA cloned into the DsRed expression vector (Clontech, Palo Alto, CA). After 48 h, expression of DsRed-mER was detected when an shRNA directed against an unrelated protein (green fluorescent protein) was present, but no ER-DsRed was detected when the shRNA was directed against ER (not shown). Transfection of SK11 cells with the shRNA directed against ER reduced the endogenous level of ER mRNA in the cells (Fig. 5A). Introduction of the shRNA also prevented activation of the ERE reporter construct when cells were incubated with increasing concentrations of E2, confirming that this was mediated via ER in the control cells (Fig. 5).
Impact of steroid hormones on expression of AR and ER mRNAs
Incubation of cells with E2 for 48 h (34 C) resulted in a significant increase in the amount of ER mRNA, compared with controls (vehicle alone). The impact of E2 was concentration dependent (Fig. 6A). Addition of T did not change levels of expression of ER mRNA (Fig. 6A). Similar results were obtained when the experiment was repeated using cells grown at the nonpermissive temperature (not shown).
Incubation of cells grown at 34 C with T resulted in a significant increase in the amount of AR mRNA (Fig. 6B). The increase was concentration dependent (4-fold at 10–8 M and 14-fold at 10–7 M). In cells grown at 39 C, incubation with T also resulted in increased expression of AR mRNA (14-fold at 10–8 M and 33-fold increase at 10–7 M T, not shown). This result would appear to be consistent with the observed increase in of the amount of AR protein in cells at the nonpermissive temperature. Incubation of cells with E2 had no impact on expression of AR mRNA at either 34 C (Fig. 6B) or 39 C (not shown).
Discussion
In the present study, we have demonstrated that SK11 cells, originally isolated from the testes of d 10 mice, have retained a Sc phenotype. In agreement with the original publication in which their isolation was described (31), they continued to express mRNAs for GATA-1, SGP-2, and AR, and we also detected expression of a range of other mRNAs that are expressed by Sc in vivo. At 34 C the cells were large with an even distribution of -actin, whereas after 48 h at 39 C, they become rounded and flattened with an accumulation of -actin around their nuclei (Sneddon, S., unpublished observations). Incubation at 39 C was also associated with a quantitative increase in the expression of SGP-2 mRNA. We have shown for the first time these cells express mRNA and protein for ER but not ER. Immunoexpression of AR in SK11 cells grown at the nonpermissive temperature appeared to be increased, compared with those that were grown at 34 C. There was no obvious difference in ER immunoexpression between cells grown at the different temperatures. This pattern of expression is consistent with that described after immunostaining of fixed sections from testes of mice (15). Notably, whereas the intensity of immunoexpression of AR in murine Sc increases during the first wave of spermatogenesis and is stage dependent in adulthood, no apparent increase in immunoexpression of ER in mature Sc, compared with their immature counterparts, has been reported (5, 24).
Sc lines have been derived from the testes of mice, rats, and sheep using a number of different immortalization strategies (reviewed in Ref.30) including the same method as that used for the SK11 cells, namely the expression of the SV40 T antigen. Several studies have explored whether these cell lines are responsive to FSH and/or cAMP (41, 42, 43), and they have been used to study FSH responsiveness after stable transfection with FSHR constructs (39, 44). In comparison, relatively little attention has been paid to the steroid hormone status of Sc lines (30). Mather (41) reported that the TM4 cell line, which was prepared from the testes of 11- to 13-d-old mice by repeated passaging, was growth inhibited by low levels of E2 (45). They demonstrated that TM4 contained low-capacity, high-affinity binding sites for E2 and T; binding of radioactive E2 was reduced by addition of 100-fold molar excess of unlabeled E2 or diethylstilbestrol but was unaffected by T, progesterone, or dexamethasone (46). In their cultures the content of both ERs and ARs increased as a function of cell density (46). These studies predate the cloning of the ER cDNA, and therefore, we cannot be sure whether expression of ER protein was responsible for the binding of estrogen by these cells. Konrad et al. (47) reported that an immortalized rat Sc cell line prepared using SV40-induced transformation lost expression of AR, and they did not examine estrogen responsiveness. In a recent study, the MSC-1 cell line (48) was used to confirm that expression of rHox genes was androgen regulated. Endogenous expression of AR was too low to induce significant gene expression; however, introduction of an AR expression construct resulted in a T-mediated induction of gene expression (49).
AR and ER expressed in the SK11 cells were capable to inducing expression of reporter gene constructs when the cells were incubated with a range of steroid ligands. Previous investigators have been able to demonstrate functional AR in primary Sc prepared from immature rats using a mouse mammary tumor virus promoter construct (50), but to date no other studies have used ERE reporters to study the impact of estrogens on Sc. In our studies the reporter construct used to investigate functional activity of AR was prepared from the promoter of the Pem oncogene (37). Previous studies have identified sequences for two AREs in this promoter, and there is evidence that the proximal promoter directs expression in the Sc and epididymis in mice (51). Recently we detected expression of Pem mRNA in the SK11 cells using RT-PCR (Hooley, R. P., and P. T. K. Saunders, unpublished observations). Treatment of cells with either T or DHT resulted in an increase in expression of the pem reporter, but there was no expression above control levels in cells treated with E2 or 3Adiol. Activation with DHT was approximately 3-fold higher than that with T, consistent with reports that the AR displays higher affinity for DHT and that this ligand has a slower dissociation rate than T (52). To date there is no evidence that estrogens can have an impact on expression of the Pem gene, and the data obtained in the current study using the Pem reporter construct would suggest this does not occur. Previous investigators have demonstrated that ER can bind both E2 and 3Adiol (53), and in the present study, both ligands were able to induce expression of the 3xERE reporter in Sc. Significant expression of the reporter was also induced when cells were treated with T, but no activation was noted with DHT. This result suggests that some of the T was metabolized to E2 and is consistent with detection of aromatase mRNA in the SK11 cells. The SK11 cells do not express 3HSD (31), and this means they are unable to synthesize 3Adiol, consistent with the lack of reporter gene activation after incubation with DHT. The key role played by ER in mediating the induction of the ERE reporter expression was confirmed by introduction of an ER-specific shRNA that resulted in ablation of the E2-mediated response.
A key finding from the present study was that expression of ER and AR mRNAs were increased after incubation of cells for 48 h with E2 or T, respectively. Examination of sequences up to 10 kb upstream of the transcriptional start site in the Ar and Esr2 genes cloned from mice reveals the existence of a weak ARE consensus site in the Ar and several AP-1 binding sites but no consensus ERE in the Esr2. Additional studies will be required before we can establish whether the impact of steroid treatment on the amount of mRNA is due to a direct effect on gene transcription. However, it is notable that in cultures of primary Sc from immature rats, a transient effect on AR mRNA expression was observed after the addition of FSH, and results from a transcriptional run-on assay demonstrated that the short-term effect of FSH on AR mRNA expression reflected a change in mRNA stability (54). Androgens have also been shown to autoregulate expression of their receptors by altering mRNA stability in some cell types (reviewed in Ref.55). To date the impact of steroids on the stability of AR and ER mRNAs in mouse Sc has not been investigated, and the SK11 cells provide a model system in which to examine this. It would also be interesting to establish whether SK11 cells express other members of the recently identified Rhox gene family (49) in addition to Pem (rhox5) and to examine whether all, or some, are androgen regulated in SK11 cells. Further studies are underway using SK11 cells to investigate the regulation of genes recently identified using array analysis of testes from 10-d-old mice with Sc selective ablation of AR (known as SCARKO) (5, 33).
Whereas a role for expression of AR in Sc in supporting the process of spermatogenesis has been established using cell-specific knockouts of AR (33, 56), a role for ER in Sc function is less certain. To date targeted deletions of the Esr2 gene have not resulted in development of a testicular phenotype (57), and no Sc selective ablation of ER has been reported. In females ER is the predominant ER in the ovary, and ER protein is expressed in granulosa cells in both growing and mature follicles (35). Furthermore, in the absence of E2, granulosa cells adopt a phenotype that resembles that of Sc (58). In mice lacking a fully functional ER gene, the granulosa cells have an attenuated response to FSH-induced differentiation, resulting in a reduced ovulation rate (59). In the present study, activation of ERE reporter expression occurred when cells were incubated with the DHT metabolite 3Adiol. Studies in the mouse have demonstrated that 3Adiol is synthesized within the prostate, and it has been proposed that ER, probably as a complex with 3Adiol, is involved in regulating the AR content of the rodent prostate and restraining epithelial growth (60, 61). 3Adiol has also been shown to be a potent modulator of ER-mediated gene transcription in neuronal cells (62). The enzymes required for synthesis of 3Adiol from T (5-reductase and 3HSD) are both expressed in the Leydig cells within the rodent testis (9, 63), and 3Adiol has been measured in the venous drainage of the rat testis (64). Studies in the human have confirmed that 3Adiol is synthesized from DHT in testicular tissue and have reported that a significant decrease the amount of steroid is found in men with defects in spermatogenesis (65, 66, 67). Taken together these data suggest that this ligand may play a role in activation of the ER protein that is expressed in testicular Sc as well as other cells types including germ cells (15, 18). Ligand-dependent modulation of ER-mediated signaling by 3Adiol, which may be elevated in the testes of mice with targeted deletion of cyp19, may explain why the phenotype of these animals (68) differs from that of the ER/ER knockout mice (7).
In conclusion, SK11 cells have retained the phenotypic characteristics of mouse Sc including the expression of the steroid receptors AR and ER. These cells therefore provide a model system in which to explore the potential cross-talk between AR and ER signaling pathways in the Sc and may reveal new roles for ER in modulation of testis function.
Acknowledgments
We are grateful to the laboratories of Professors D. P. McDonnell (Durham, NC) and F. Claessens (Leuven, Belgium) for the generous gifts of plasmids containing 3xERE-luc and Pem-luc constructs, respectively. We thank Dr. Kevin Morgan for assistance with analysis of gene promoter sequences.
Footnotes
Present address for N.W.: Institute for Hormone and Fertility Research, University of Hamburg, Falkenried 88, 20251 Hamburg, Germany.
First Published Online September 15, 2005
Abbreviations: 3Adiol, 5-androstane-3-, 17-diol; AR, androgen receptor; ARE, androgen-responsive element; DHT, dihydrotestosterone; E2, 17-estradiol; ER, estrogen receptor; ERE, estrogen response element; FSHR, FSH receptor; 3HSD, 3-hydroxysteroid dehydrogenase; Sc, Sertoli cells; SGP-2, sulfated glycoprotein 2; SV40, Simian virus 40; T, testosterone.
Accepted for publication September 7, 2005.
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