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Monash Institute of Reproduction and Development (J.J.B., J.R.M.) and Department of Anatomy and Cell Biology (J.J.B., N.G.W.), Monash University, Clayton, 3168, Melbourne, Australia
Address all correspondence and requests for reprints to: Dr. John R Morrison, c/o Monash Institute of Reproduction and Development, 27-31 Wright Street, Clayton 3168, Victoria, Australia. E-mail: jmorrison{at}copyrat.com.au.
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Abstract |
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Introduction |
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In the rat, Sertoli cells divide during the fetal and early neonatal periods before differentiating at around d 15 postpartum (pp) (4, 5), after which no further proliferation can occur. The number of Sertoli cells present in the adult is therefore dependent upon both the duration of the proliferative phase and the rate of division during that phase. The rate of Sertoli cell division is influenced by the major Sertoli cell mitogens FSH (6, 7) and activin A (8, 9, 10). The duration of Sertoli cell division has been demonstrated to involve thyroid hormone (primarily T3). Experimentally induced neonatal hypothyroidism has been shown to lead to a delay in the differentiation of Sertoli cells (11), resulting in a significant increase in adult testis size and a concomitant increase in sperm production (12, 13, 14). Conversely, hyperthyroidism leads to early cessation of Sertoli cell proliferation, a decrease in testis size, and decreased sperm production (15, 16, 17, 18). Treatment of cultured proliferative phase rat Sertoli cells with thyroid hormone leads to suppression of proliferation, increased protein expression, and increased expression of clusterin, inhibin ßB-subunit, and decreased aromatase activity (19, 20), suggestive of differentiation. The demonstration of thyroid hormone receptor expression in Sertoli cells (20, 21) further verifies that thyroid hormone acts directly on Sertoli cells.
Although thyroid hormone is the best described modulator of Sertoli cell differentiation, two other factors have a suggested role: retinoic acid (RA) and testosterone (T). In organ cultures of fetal testis, treatment with RA leads to deposition of basement membrane components by the Sertoli cells (22), a function similar to that of thyroid hormone in the early postnatal testis (23). Treatment of cultured fetal and neonatal testes with RA also leads to the inhibition of seminiferous cord formation and suppression of FSH or epidermal growth factor-induced DNA synthesis (24) and cAMP production (25, 26). Treatment of cultured d 3 pp testes with RA causes Sertoli cell hyperplasia (27); methodical examination of these effects using specific RA receptor agonists shows that activation of RA receptor ß (RARß) causes this hyperplasia, whereas activation of RAR
leads to suppression of FSH-induced cAMP production and gonocyte number (26).
The first suggestion that T may be directly involved in Sertoli cell division stemmed from the finding that treatment of rat pups with T for 2 or 4 d leads to suppression of Sertoli cell DNA synthesis (28). As T is capable of suppressing FSH secretion by the pituitary (29), this effect has been attributed to decreasing levels of circulating FSH. Treatment of cultured testis fragments with T has been demonstrated to cause no significant change in Sertoli cell thymidine incorporation (30), which leads to the assumption that if T does suppress Sertoli cell proliferation, then it must be further regulated by other factors. In contrast, there is some evidence for abnormal differentiation and/or persistent mitotic activity of Sertoli cells in humans with androgen receptor mutations (31, 32, 33).
In addition to the functional evidence of a role for T and RA in Sertoli cell proliferation, these factors share a number of physical and biological properties with thyroid hormone. Firstly, they are lipophilic hormones (like T3) that act on ligand-dependent transcription factors that are members of the steroid/thyroid hormone receptor superfamily. Members of this superfamily generally dimerize with a RAR (primarily RXR, which is responsive to 9-cis-RA) to effect transcription (34). Secondly, receptors for RA, T, and T3 are present in proliferative phase Sertoli cells. Dufour and Kim (35) used immunohistochemistry to demonstrate that in vivo RAR is present in Sertoli cells throughout postnatal development, but markedly decreases on d 20 pp [a pattern strikingly similar to that of the thyroid hormone receptors (21)]. In contrast, RARß only becomes apparent in the postnatal testis by immunohistochemistry after d 15 pp. Bremner et al. (36) and You et al. (37) demonstrated that immunohistochemical staining for androgen receptor in Sertoli cells is first apparent on d 5 pp, increasing in intensity thereafter, before becoming spermatogenic stage specific (though still restricted to Sertoli cells) in the sexually mature testis. In apparent conflict with this, Weber et al. (38) demonstrated that androgen receptor only becomes detectable by immunohistochemistry in Sertoli cells after d 15 pp.
Given that receptors for T3, RA, and T exist in Sertoli cells during their proliferative phase, and that some evidence exists suggesting a role for these factors in Sertoli cell proliferation, we examined their effects on the proliferation of high purity cultured rat Sertoli cells. Our data demonstrate that these factors are all capable of suppressing Sertoli cell proliferation in vitro and suggest that they may play a role during Sertoli cell development in vivo.
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Materials and Methods |
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Sertoli cells were isolated according to previously described methods (39), and cells were maintained in medium containing recombinant human FSH (390 IU/liter) and selenium-transferrin-insulin (41400-045, Life Technologies, Inc., Gaithersburg, MD). After 4 d of preculture, high purity Sertoli cells were replated, and medium was further supplemented with T3 (1 x 10-6 M), RA (1 x 10-6 M), T (3 x 10-7 M), vehicle (ethanol), or a combination of these.
[3H]Thymidine incorporation assay
[Methyl-3H]thymidine incorporation was used to measure proliferative activity. Prewarmed [methyl-3H]thymidine (2.0 Ci/mmol, 3 x 105 cpm/well; Amersham Pharmacia Biotech, Uppsala, Sweden) in DMEM/Ham’s F-12 was added to the culture medium for 18 h. Cells were rinsed and harvested with trypsin/versene solution using a Micromate 196 Cell Harvester (Packard Instrument, Meriden, CT), immobilizing them on a glass-fiber filter (Packard Instrument), which was then rinsed with ethanol. Filters were air-dried and placed into 1 ml liquid scintillant, and incorporated radionucleotide was measured using a liquid scintillation counter.
RNA isolation
Cells for RNA isolation were cultured in laminin (1 µg/cm2)-coated six-well culture dishes (BD Falcon, Boston, MA). Cells were harvested by treatment with trypsin/versene for 5 min at 37 C and counted on a hemocytometer, and 2 x 106 cells were pelleted at 500 x g. Poly(A) mRNA was isolated using oligo(deoxythymidine)25 magnetic Dynabeads (Dynal, Lake Success, CA) according to the manufacturer’s instructions. mRNA was eluted into 10 mM Tris-HCl (pH 7.5) and stored at -70 C.
RT
RT was performed using Superscript II (Life Technologies, Inc.) according to the manufacturer’s instructions, primed with oligo(deoxythymidine)10–15 (Amersham Pharmacia Biotech). For every sample a no-RT control was performed in which all incubations and buffers were identical, but no Superscript enzyme was added. This control verified the absence of contaminating genomic DNA in PCR reactions (data not shown).
PCR primers
PCR primers (sequences shown in Table 1
) were designed using primer3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) to previously described sequences for Id1, Id2, Id3, Id4, Dmrt1, Serz, Gata-1, Gata-4, and transferrin (see Table 2 for descriptions of these genes). Primers were synthesized by Sigma-Genosys (Sydney, Australia). For each primer set, annealing temperature was optimized by performing PCR on identical replicates in a gradient block thermocycler (PCR express with 96-well gradient block, Hybaid, Ashford, UK). PCR was performed in a 25-µl solution containing 0.5 U Taq polymerase (Amersham Pharmacia Biotech), 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM deoxy-NTPs (Biotech Australia, Sydney, Australia), 1 mM forward and reverse primers, and 1 µl cDNA from the RT reaction. The initial denaturation step was at 94 C for 3 min, followed by 35 cycles of denaturation for 30 sec at 94 C, annealing for 30 sec at 54–66 C (described below), and extension for 1–1.5 min (depending upon product size) at 72 C. The reaction was subjected to a final extension for 3 min at 72 C before being electrophoresed at 90 V on a 1% agarose gel stained with ethidium bromide. The annealing temperature used was the highest temperature at which a large amount of specific PCR product could be seen when run on an agarose gel. For all primer sets tested, PCR at this temperature yielded no products of a size other than that expected.
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) for 5 sec, and extension at 72 C for 5–20 sec (see Table 1). DNA content was measured by fluorometry at the end of each extension step. At the end of the PCR, melting curve analysis was performed to verify product specificity by increasing the temperature from 66 to 95 C at 0.1 C/sec, measuring fluorescence constantly. In each reaction a standard curve was prepared from serial dilutions of an arbitrary standard. PCR products were quantified using LightCycler software. These values were controlled for RT efficiency and cDNA loading by normalizing with an endogenous control (18S ribosomal RNA; this control was also correlated with ß-actin to verify efficacy, i.e. data were similar independent of the internal control used). Normalized values were then calibrated against values for untreated control Sertoli cells to give a rational value. All real-time RT-PCR experiments yielded a single amplicon band of the expected size. Melting point analysis demonstrated a single melting breakpoint in all experiments performed. Triplicate samples for real-time RT-PCR were derived from three separate preparations of Sertoli cells.
Western analysis
For Western analysis, cells were removed from the plate by treatment with a volume of trypsin/versene solution containing 0.1% (wt/vol) trypsin (T2021, Sigma-Genosys) and 0.5 mM tetrasodium EDTA (E6511, Sigma-Genosys) in PBS for 5 min at 37 C, followed by 2 vol 0.05% (wt/vol) soybean trypsin inhibitor (T6522, Sigma-Genosys) in DMEM/Ham’s F-12. Cells were counted on a hemocytometer using trypan blue exclusion to verify viability, and an aliquot containing 1 x 106 cells was removed for protein extraction. Cells were pelleted by centrifugation at 900 x g and rinsed once in PBS. The cell pellet was then homogenized in 1 ml PBS containing 1% (wt/vol) nonylphenol polyoxyethylene ether (Nonidet P-40, Sigma-Genosys), 0.5% (wt/vol) polyoxyethylene sorbitan monolaurate (Tween-20, P1379, Sigma-Genosys), 0.1% (wt/vol) sodium dodecyl sulfate, and Complete protease inhibitors (1697498, Roche; diluted according to the manufacturer’s instructions). The homogenate was incubated at 0 C for 30 min before being centrifuged at 18,000 x g for 20 min. The supernatant was transferred to a new tube and again centrifuged at 18,000 x g for 20 min. The total protein concentration in the supernatant was assayed using the Bio-Rad Protein Assay Kit I (500-0001, Bio-Rad Laboratories, Richmond, CA), and supernatants were frozen at -75 C until required.
Fifty micrograms of protein were run in each well of a 15% polyacrylamide gel alongside 5 µl BenchMark protein molecular weight standard (10748-010, Life Technologies, Inc.). Protein was transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA), which was then blocked for 15 min in blocking solution (2% skim milk powder and 0.1% Tween 20 in Tris-buffered saline) before incubation overnight with 1:5000 primary antibody (rabbit anti-p27, sc-528, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; mouse monoclonal anti-p21, MS-891-PO, Labvision (Fremont, CA); or 1:10000 mouse monoclonal anti-ß-tubulin, MAB3408, Chemicon, Temecula, CA). The membranes were rinsed three times before being incubated for 2 h at 37 C with second antibodies (horseradish peroxidase-conjugated sheep antirabbit, RAH, Silenus, Melbourne, Australia; or horseradish peroxidase-conjugated rat antimouse, 04-6020, Zymed Laboratories, South San Francisco, CA) diluted 1:10,000. Bound antibody was visualized using the enhanced chemiluminescence ECL Plus Kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Densitometry was performed using NIH Image (NIH, Bethesda, MD).
Immunohistochemistry
In some experiments, 0.65 nM 5-bromo-2'-deoxyuridine (BrdU; B5002, Sigma-Genosys) was added to the culture medium for 2 h to label S phase cells. Sertoli cells were rinsed in PBS and fixed in Bouin’s fixative for 2 h before immunofluorescent detection of incorporated BrdU. Cells were treated with 2 N HCl in PBS with 0.1% (wt/vol) polyoxyethylene isooctylphenyl ether (Triton X-100, Sigma-Genosys) for 15 min to denature DNA. This was followed by treatment with 0.1 M sodium borate in PBS with 0.1% Triton X-100 for 10 min. Cells were then washed, nonspecific protein binding was blocked with 2% milk powder in PBS, and monoclonal anti-BrdU (0.25%, vol/vol; Sigma-Genosys) was incubated overnight at 4 C. The cells were again washed and then incubated with 1.7% (vol/vol) fluorescein isothiocyanate-conjugated antimouse immunoglobulin G (DAKO, Glostrup, Denmark) for 1 h. A drop of Vectashield mounting medium with 4',6-diamido-2-phenylindole hydrochloride (Vector Laboratories, Inc., Burlingame, CA) was placed on the cells and then mounted with a coverslip.
Statistical analyses
All statistical analyses involved analysis of triplicate samples by one-way ANOVA, followed by Tukey’s post hoc test. Differences between groups were deemed significant if P < 0.05. All experiments were repeated at least three times (with the exception of BrdU immunohistochemistry, which was performed once) and yielded similar data.
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Results |
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). This suppression was first statistically significant (P < 0.05) after 2 d of treatment with T3 or RA; after 4 d of treatment, suppression was highly significant (P < 0.001) in all treatment groups. After 8 d of treatment, rates of Sertoli cell proliferation were suppressed to approximately 20% of control values. As RA and T stocks were dissolved in ethanol, we included a control to verify that suppression was not due to the small amounts of ethanol added as vehicle. Ethanol caused no significant change in Sertoli cell proliferation (Fig. 1A). When added in various combinations, T3, RA, and T suppression was not additive (Fig. 1B). Combinatorial treatment caused no significant suppression compared with treatment with one factor alone.
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, A, B, C, and D, respectively). Sertoli cells treated with T, thyroid hormone, or RA had an obvious decrease in BrdU-positive nuclei compared with control cultures.
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; see Table 2 for a description of these factors). Treatment with T3 caused a transient suppression of p21Cip1 levels (to 50% of control levels) after 1 d of treatment, with levels returning to 175% of control after 4 d of treatment. T3 caused a 195% up-regulation of p27Kip1 levels after 4 d of treatment (Fig. 3B). Treatment with RA induced a strong progressive up-regulation of p21Cip1 levels after 1, 2, and 4 d of treatment (peaking at 360% of control) and a similarly strong up-regulation of p27Kip1 levels after 2 and 4 d of treatment (peaking at 475% of control; Fig. 3C).
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for descriptions) as determined by quantitative fluorometric RT-PCR.
Id1 expression was only significantly (P < 0.05) induced by treatment with RA and T in combination (Fig. 4A), whereas Id4 expression was not significantly affected by any of the treatments (Fig. 4D). Id2 expression was induced approximately 4-fold (P < 0.05) by treatment with T3 or RA, both alone and in combination. Treatment with T did not significantly affect Id2 expression, and when applied in combination with T3, it suppressed the induction of Id2 seen with T3 treatment alone. Combinations of T3, RA and T did not appear to have additive or synergistic effects on Id2 expression (Fig. 4B).
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).
Thyroid hormone, RA, and T induce markers of Sertoli cell differentiation
Dmrt1 expression was unaffected by treatment with T3 or T. Treatment with RA, both alone and in combination with T3, and/or T, induced a statistically insignificant suppression of Dmrt1 expression (to 10–45% of control levels). Although this suppression was insignificant on a group to group basis due to the high variability of Dmrt1 expression, when treatment groups were pooled into RA-treated and untreated groups, regardless of other hormones, RA significantly suppressed Dmrt1 expression (P < 0.001) compared with cultures that were not exposed to RA (Fig. 5A).
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). Gata-1 expression was significantly induced (P < 0.05) by treatment with T or with T3 and RA in combination. Gata-1 expression was not significantly affected in any other treatment group. Gata-4 expression was significantly suppressed (P < 0.05) by treatment with RA, both alone and in combination with T3 or T. Transferrin expression was significantly induced (P < 0.05) by treatment with RA, both alone and in combination with T3 or T. |
Discussion |
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We have demonstrated that T3, RA, and T are each capable of suppressing Sertoli cell proliferative activity to a similar degree. To validate that the suppression of Sertoli cell proliferation that we observed with these treatments was not simply due to toxic effects of these hormones, we examined the expression of a number of factors that are known to be widely involved in cell cycle arrest and differentiation (p27Kip1 and p21Cip1) in addition to a selection of genes that have been specifically implicated in Sertoli cell differentiation. The degree of induction of p27Kip1 and p21Cip1 demonstrated in this paper is comparable to that shown to induce a significant suppression of cell proliferation in prostate carcinoma cell lines (40). It should be noted, however, that in addition to the induction of p27Kip1 and p21Cip1, it is possible that T, T3, or RA treatment modulates the Sertoli cell cycle via one or more of the other cell cycle regulators (such as the INK4 proteins or other Kip family members) (41) or via direct regulation of cyclins at the transcriptional level.
The hormonal environment in which Sertoli cells develop is only moderately defined. Circulating (and hence also testicular) T3 levels have been clearly demonstrated to dramatically increase between d 3 and 10 pp (42). Both serum and testicular T levels are suppressed between d 6 and 14 pp and only begin to significantly increase after d 14 pp (43, 44). Local RA levels are not entirely clear; however, they are likely to correlate to circulating vitamin A levels (which are, in turn, strongly dependent upon dietary intake) (45). Given these hormonal profiles, it is likely that T3 represents the first major hormonal signal to suppress Sertoli cell proliferation, whereas T (and perhaps RA) may be involved in later stages of suppression. This hypothesis is consistent with extant data demonstrating a prolongation of Sertoli cell division in the absence of thyroid hormone (12, 14, 17).
The cyclin-dependent kinase inhibitor (CDKI) family include a number of proteins that directly interact with and suppress fundamental cell cycle processes (41). In the rat testis, the CDKI p27Kip1 becomes detectable in Sertoli cells after the proliferative phase is complete (46). Furthermore, mouse p27Kip1 knockouts display multiorgan hyperplasia, including the testis (47, 48, 49). Our data are further validated by the findings presented in a companion paper demonstrating that experimentally induced hyper- and hypothyroidism lead to precocious and delayed expression, respectively, of p27Kip1 in Sertoli cells of male mice (50).
Although p21Cip1 knockout mice have no overt testicular or reproductive phenotype (51), p21 has been implicated in the control of differentiation in other systems (52). In the present study we have demonstrated that T3, RA, and T all induce the expression of p27Kip1, and T3 and RA induce the expression of p21Cip1, whereas T has no obvious effect on this protein. This suggests that the suppressive effects of T3, RA, and T on Sertoli cell proliferation are mediated (at least in part) by direct suppression of the cell cycle.
Id proteins (Id1–4) are classically considered to be inhibitors of differentiation that act by sequestering basic HLH differentiation factors in an inactive complex (for reviews, see Refs.53, 54, 55, 56). We expected Id factors to be suppressed by T3 (which is considered to be a Sertoli cell differentiation promotor); however, none of these factors was suppressed, and some appeared to be up-regulated by treatment with T3, RA, and T. Recent evidence has emerged to suggest that in some systems, Id proteins may, in fact, be involved in promoting differentiation toward particular lineages (57, 58, 59, 60). Postmitotic (d 20 pp) Sertoli cells have been demonstrated to express all four Id proteins (61), and their levels can be modulated by FSH, cAMP, or serum treatment. Antisense oligonucleotides directed against Id1 stimulate, whereas those directed against Id2 suppress, FSH- and serum-induced transferrin promoter activation (61). These data suggest that Sertoli cells, like some immune cells, do not use Id proteins in the classical inhibition of differentiation manner. Immunohistochemical studies have demonstrated that Id2 is present in postmitotic Sertoli cell nuclei, and Id3 is present in Sertoli cell cytoplasm; however, Id1 and Id4 were undetectable in Sertoli cells (62). In this context, the relatively small change in the expressions of Id1 and Id4 observed in this study may be due to the very low levels present (and may suggest that Id2 and Id3 are the only Id factors that play a functional role in Sertoli cell maturation).
We have examined the expression of a number of genes that have been implicated in the Sertoli cell differentiation process; these genes are outlined in Table 2. Interestingly, T3, which has been previously demonstrated to act directly on Sertoli cells to induce differentiation, did not significantly affect the expression of any of these factors. T caused a small induction of Dmrt1 and Gata-1 expression, but had no significant effect on any of the other genes examined. RA significantly suppressed Dmrt1, Serz, and Gata-4 expression and induced transferrin expression. These findings contrast with our demonstration that T3, RA, and T have similar effects on Sertoli cell proliferation, suggesting that these hormones have some common effects and some differential effects on Sertoli cell growth.
In conclusion, this paper provides evidence that T3, RA, and T have similar effects on the process of Sertoli cell differentiation. In vivo, when Sertoli cells differentiate they undergo cell cycle arrest; we demonstrate that T3, RA, and T are each capable of suppressing Sertoli cell proliferation in vitro to a similar degree. Furthermore, we show that these factors are capable of modulating the expression of cell cycle inhibitors, p27Kip1 and p21Cip1, a likely mechanism for the cell cycle suppression observed. In vivo, postmitotic Sertoli cells have been demonstrated to express Id proteins, suggesting that Id proteins function beyond their classical antidifferentiation role in Sertoli cells. This study demonstrates that T3, RA, and T are capable of inducing the expression of Id2 and Id3, consistent with an emerging function for Id proteins playing a stimulatory role in Sertoli cell differentiation. Finally, we show that T3, RA, and T have quite different effects on the expression of Dmrt1, Serz, Gata-1, Gata-4, and transferrin, suggesting that despite the common antiproliferative effects of these hormones, they may have subtly different functions during the differentiation from proliferative to postmitotic Sertoli cell.
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Acknowledgments |
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Footnotes |
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Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; CDKI, cyclin-dependent kinase inhibitor; HLH, basic helix-loop-helix; pp, postpartum; RA, retinoic acid; RAR, retinoic acid receptor; T, testosterone.
Received March 25, 2003.
Accepted for publication June 4, 2003.
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