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Testis; |
Section of Experimental Endocrinology,3 Department of Pharmacology, and Department of Biochemistry,4 Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo 04044-020, Brazil
Department of Morphology,5 Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais 31270-901, Brazil
ABSTRACT
The aim of the present study was to determine the mechanisms involved in estrogen actions in cultured rat Sertoli cells. RT-PCR detected transcripts for the estrogen receptors ESR1 and ESR2 in cultured immature Sertoli cells and in the testis of 15-, 28-, and 120-day-old rats. The expression of ESR1 and ESR2 was confirmed in Sertoli cells by immunofluorescence and Western blot. Immunohistochemistry with cryosections of testes from immature and adult rats revealed that ESR1 is present in Sertoli, Leydig, and some peritubular myoid cells, and ESR2 is present in multiple cell types, including germ cells. Treatment of Sertoli cells with 17beta-estradiol (E2) induced a translocation of ESR1 and ESR2 to the plasma membrane and a concomitant phosphorylation of MAPK3/1. Both effects reached a maximum after 10 min and were blocked by PP2, an inhibitor of the SRC family of protein tyrosine kinases, and by the antiestrogen ICI 182,780 (ICI). MAPK3/1 phosphorylation was also decreased in the presence of AG 1478, an inhibitor of the epidermal growth factor receptor (EGFR) kinase, and in the presence of MAP2K1/2 inhibitor UO126. Treatment with E2 for 24 h increased the incorporation of [methyl-3H]thymidine, which was blocked by ICI. These results indicate that E2 activates an SRC-mediated translocation of estrogen receptors to the plasma membrane, which results in the activation of EGFR and the mitogen-activated protein kinase signaling pathway. In addition, activation of ESR1 and/or ESR2 by E2 is involved in proliferation of immature Sertoli cells. The estrogen actions in Sertoli cells might be a key step mediating cellular events important for spermatogenesis and fertility.
estradiol, estradiol receptor, kinases, mechanisms of hormone action, Sertoli cells
Estrogen plays an important role in the development, differentiation, and growth of the male reproductive system [1–6]. Estrogens are synthesized from androgens by the aromatase complex, which contains the cytochrome P450 enzyme encoded by the CYP19 gene [6, 7]. Aromatase expression is detected in Leydig and Sertoli cells, spermatogonia, spermatocytes, elongate spermatids, and spermatozoa in adult mice and rats [3, 7] and in Sertoli cells isolated from immature rat testis [8].
In order to mediate its biological effects, estrogen interacts with specific receptors ESR1 and ESR2 (also known as ER
and ERβ, respectively), which are widely distributed in the male reproductive tract [4, 9–11]. In the testis of adult rats and mice, ESR1 immunostaining is detected in Leydig and peritubular myoid cells [3, 4, 9, 11–13]. Several studies reported the absence of ESR1 in germ cells, but Pelletier et al. [14] detected ESR1 in spermatocytes and spermatids. Although most of the studies did not find a positive ESR1 immunostaining in Sertoli cells, a faint and diffuse staining, interpreted as an artifact, was detected in seminiferous cords from immature rats [9] and baboon testis [15], and a positive immunostaining was detected in Sertoli cells of immature and mature boars [16, 17]. ESR2 expression is detected in multiple cell types, including the Sertoli cells, and in some but not all germ cells [12, 13]. Recently, ESR1 mRNA and protein expression has been shown in the SK11 cell line derived from Sertoli cells of 10-day-old H2Kb-tsA58 transgenic mice [18].
The mechanisms by which estrogens influence male fertility remain uncertain. Estrogen effects on reproductive function have been studied using mice with targeted disruption of estrogen receptors (Esr1–/–, Esr2–/–, Esr1–/–/Esr2–/–), aromatase enzyme (Cyp19–/–), and animals treated with the antiestrogen ICI 182,780 (ICI) [3, 5, 11, 19, 20]. The studies with knockout animals have shown that the spermatogenesis, steroidogenesis, and fertility of Esr1–/–, Esr1–/–/Esr2–/–, and Cyp19–/– animals are affected, but none of these parameters is affected in Esr2–/– animals. In fact, infertility in Esr1–/– seems to be mainly due to disruption of fluid reabsorption in efferent ductules, and the backpressure of the accumulating luminal fluids leads to a progressive degeneration of the testicular tissue and to dilation of seminiferous tubules [3, 21]. There is evidence that estrogen also affects Sertoli cell proliferation and may suppress differentiation [19], but no changes were observed in the number of Sertoli cells and spermatogonia in Esr1–/– mice [20]. On the other hand, ESR2 inactivation increases the number of spermatogonias by more than 50% in neonatal mice [22]. It is surprising that, in spite of the evidence that ESR2 regulates mitosis of spermatogonia, disturbances of sperm production were not evident in Esr2–/– mice [23]. Paradoxically, spermatogenic arrest occurs in Esr1–/– mice [21], suggesting that testicular cells regulate Sertoli cell support of germ cell development through unidentified ESR1-mediated mechanisms. This line of evidence is supported by experiments in which germ cells from donor males homozygous for the mutation Esr1–/– were transplanted to testes of wild-type Esr1+/+ recipient mice depleted of germ cells. When mated to wild-type females, the recipients sired offspring heterozygous for the mutation Esr1+/–, but retained the coat-color marker of Esr1–/– donor mice. This finding confirmed that somatic cells in the testis, but not germ cells, require ESR1 in order to support the process of spermatogenesis [24, 25].
In addition to the classic genomic mechanism of estrogen action, mediated by ESR1 and ESR2, there is now convincing evidence that the steroid also exerts rapid, nongenomic actions initiated at the cell surface. The rapid action of estrogen can be divided into two major categories: classical receptor-mediated responses, which involve translocation of estrogen receptors from nuclei to the plasma membrane [26, 27], and nonclassical responses, mediated through proteins other than estrogen receptors, such as the G-protein coupled receptor GPR30 [28–32]. Estrogen nongenomic signaling involves a series of cell type-dependent events that includes mobilization of second messengers, interaction with membrane receptors such as insulin-like growth factor 1 receptor (IGF1R) and epidermal growth factor receptor (EGFR), and stimulation of effector molecules, such as the SRC family of tyrosine kinases and the phosphatidylinositol 3-Kinase, the serine/threonine protein kinase (AKT), and the mitogen-activated protein kinases (MAPKs) [26, 27, 33–35].
Because the physiological role of estrogen in Sertoli cells is not completely understood and there is some controversy in the literature about ESR1 expression in these cells, the aim of the present study was to investigate the expression of ESR1 and ESR2 and the effects of 17β-estradiol (E2) on the subcellular distribution, signaling, and function of these receptors in rat Sertoli cells.
Primary cultures of Sertoli cells were obtained from 15-day-old male Wistar rats housed in the Animal Facility at Instituto de Farmacologia e Biologia Molecular (INFAR), Universidade Federal de São Paulo-Escola Paulista de Medicina (UNIFESP-EPM), and maintained on a 12L:12D lighting schedule at 23°C with food and water ad libitum. The experimental procedures were conducted according to guidelines for the care and use of laboratory animals as approved by the Research Ethical Committee from UNIFESP-EPM. The testes were removed and decapsulated, and Sertoli cells were prepared as previously described [36–38]. Cells were plated at a density of approximately 4 x 106 cells per milliliter in phenol-red free Ham F12/Dulbecco modified Eagle medium (F12/DMEM 1:1; Gibco; Invitrogen, Grand Island, NY) containing 0.02 g/L gentamicin (pH 7.2–7.4; Sigma Chemical Co., St. Louis, MO) and supplemented with 10 µg/ml insulin, 10 µg/ml transferrin, 10 ng/ml sodium selenite, and 10 ng/ml epidermal growth factor (EGF; Sigma). The cells were grown in a humidified atmosphere of 5% CO2:95% air at 35°C and, after 48 h, treated with 20 mM Tris-HCL (pH 7.4) to lyse residual germ cells [39], and allowed to grow for another 24 h. Culture medium was replaced by another one without supplements 20 h before the experiments with Sertoli cells. At this stage, the cells were 90%–95% confluent, and the amount of viable cells in each culture, as determined by trypan blue exclusion, was more than 90%. For proliferation assays, Sertoli cells were prepared as described above and plated at a low density, and at the day of experiments the confluence was 50%–60%.
The presence of other cell types in the primary culture of Sertoli cells was evaluated by several criteria. Cells were grown on coverslips coated with gelatin (0.1%) for both morphological and immunocytochemistry analyses. Under a phase-contrast microscope, Sertoli cells were the predominant cell type after hypotonic shock (data not shown). Hematoxylin-eosin staining showed that, under the described culture conditions, the cells presented the characteristic rounded nuclei and irregular polygonal shape with well-formed cytoplasmic extensions (Fig. 1A), as previously reported for cultured Sertoli cells [40]. Further analyses included the immunocytochemical detection of two major proteins secreted by Sertoli cells: transferrin and sulfated glycoprotein 2, also known as clusterin (CLU) [41, 42]. Transferrin and CLU were detected by using a rabbit polyclonal antibody against human transferrin (1:50 dilution; DAKO, Carpinteria, CA) and a mouse monoclonal antibody against rat CLU (1:50 dilution, clone 6E9) [43], and secondary antibodies Alexa Fluor 488-labeled anti-rabbit and Alexa Fluor 594-labeled anti-mouse, respectively (1:300 dilution; Molecular Probes, Eugene, OR; Fig. 1, B and C). All these analyses confirmed the absolute predominance of Sertoli cells in the primary culture.
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Total RNA was extracted with the TRIzol reagent (Invitrogen, Carlsbad, CA) from testes of 15-, 28-, and 120-day-old rats and from Sertoli cells cultured for 5 days, according to the standard protocol [44], and checked for ribosomal RNA integrity by agarose gel electrophoresis. A 2:1 ratio of sharp and clear ethidium-bromide stained 28S:18S ribosomal RNA bands was observed for all samples. Total RNA (5 µg) was used to synthesize the first strand cDNA at 42°C with the ThermoScritp RT-PCR kit (Invitrogen) and the random hexamers supplied with the kit. Reactions in the absence of reverse transcriptase were also included for each RNA tested in order to check for genomic contamination. The resulting cDNA was used in PCRs with primers (Invitrogen) designed to amplify specific regions of the different estrogen receptors (Table 1). Esr2_v2 (ERβ2) refers to the splice variant 2 of Esr2 found only in rodents, which differs from the full-length wild-type variant 1, Esr2_v1 (ERβ1), because it contains an insertion of 54 bp in the ligand-binding domain [45]. This variant has also been called βins [46] and differs from the ERβcx/β2 splice variant found in humans [47], which presents a truncation at the carboxyterminal region that leads to the loss of 61 amino acids. The amplification of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal control using the following primers: forward 5'-CGGGAAGCTTGTGATCAATGG-3' and reverse 5'-GGCAGTGATGCCATGGACTG-3' [48]. The PCR mixture consisted of 2 µl of cDNA, 1.5 mM MgCl2, 0.2 mM dNTP, 0.5 µM of each primer, and 2.5 U of Taq DNA polymerase (Invitrogen) in the appropriate PCR buffer. Samples were amplified in an Applied Biosystem 9600 Fast Thermal Cycler (Applied Biosystems, Foster City, CA). The thermal cycles for amplification of the Esr1 transcript were: a denaturation cycle at 94°C for 5 min, a touch-down protocol of three cycles at annealing temperature 75°C for 1 min, three cycles at annealing temperature 70°C for 1 min, 35 cycles at annealing temperature 65°C for 1 min, and a final extension of 72°C for 7 min. For amplification of Esr2_v1 and Esr2_v2 transcripts, the following thermal cycles were used: a denaturation cycle at 94°C for 5 min, a touch down protocol of three cycles at annealing temperature 75°C for 1 min, three cycles at annealing temperature 70°C for 1 min, three cycles at annealing temperature 65°C for 1 min, 35 cycles at annealing temperature 60°C for 1 min, and a final extension of 72°C for 7 min. For amplification of the Gapdh transcript, a denaturation cycle at 94°C for 5 min, 35 cycles at 94°C for 5 min, 62°C for 1 min, 72°C for 2 min, and a final extension cycle of 72°C for 7 min were used. The PCR products were resolved onto agarose gels (1.5%) containing ethidium bromide (0.5 µg/ml) and visualized under UV transillumination. After extraction from the gel (gel extration kit; QIAgen, Hilden, Germany), the identity of each product was confirmed by direct nucleotide sequencing in an ABI PRISM 377 automated sequencer (Applied Biosystems) using the DYEnamic ET Terminator Sequencing kit (Amersham Biosciences, Piscataway, NJ).
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Testes from 15- and 120-day-old rats were removed, embedded in Jung Tissue Freezing Medium (Leica Instruments, Nussloch, Germany), frozen in dry, ice-cold acetone, and maintained at –75°C until use. Frozen sections (8-µm thickness) of testis were cut at –20°C in a cryostat (Microm HM550; Microm International GmbH, Walldorf, Germany) and mounted on silanized slides (DAKO). Sections were air dried, fixed in 4% (v/v) paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) diluted in PBS (137 mM NaCl, 2.68 mM KCl, 6.03 mM Na2HPO4, and 1.47 mM KH2PO4; pH 7.4; Sigma) [10] for 20 min at room temperature, and then incubated with 0.1 M glycine (Sigma). In order to block the endogenous peroxidase activity, slides were treated with 3% (v/v) H2O2 and washed with PBS. Nonspecific antibody binding was minimized by incubation with blocking solution (PBS containing 0.01% saponin and 3% BSA, Sigma) for 1 h at room temperature, and the endogenous biotin was blocked by incubation with Avidin/Biotin Blocking Kit (Vector Laboratories, Inc., Burlingame, CA). Slides were incubated with one of the following primary antibodies (Santa Cruz Biotechnology, San Diego, CA): rabbit polyclonal antibody raised against the aminoterminal region of ESR1 of human origin (H184), rabbit polyclonal antibody raised against the carboxyterminal region of ESR1 of mouse origin (MC20), and goat polyclonal antibody raised against the aminoterminal region of ESR2 of mouse origin (Y19), at 1:50 dilution in blocking solution overnight at 4°C. Afterwards, slides were washed with PBS and blocking solution, and incubated with the appropriate biotinylated secondary antibody (Santa Cruz Biotechnology): donkey anti-rabbit IgG (for ESR1) or rabbit anti-goat IgG (for ESR2) at 1:300 dilution in blocking solution for 1 h at room temperature. The immunoreaction was visualized using avidin-biotin complex (Vector Laboratories) for 90 min at room temperature, followed by several washes with PBS and incubation with 0.05% (w/v) 3,3'diaminobenzidine containing 0.01% (v/v) H2O2 in PBS. Slides were mounted with Permount (Fisher Scientific, Fair Lawn, NJ). Negative control slides were performed either with the primary antibody preadsorbed with its respective blocking peptide or in the absence of primary antibody. The sections were visualized with a Nikon E800 microscope (Nikon, Melville, NY). Images were processed using a CoolSNAP-Pro CCD digital camera and the Image-Pro Express Software Program (Media Cybernetics, Silver Spring, MD).
Sertoli cells were grown as described above on coverslips coated with gelatin (0.1%) and placed into six-well plates. Cells were incubated in the absence (control, C) and presence of E2 (0.1 nM; Sigma) for 5, 10, 15, and 30 min. In another series of experiments, the cells were untreated or pretreated with ICI (1 nM; Faslodex; AstraZenica, São Paulo, Brazil) or the selective inhibitor of the SRC family of protein tyrosine kinases PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine; 5 nM; Calbiochem, Darmstadt, Germany) for 30 min at 35°C. Afterwards, the cells were stimulated with E2 (0.1 nM, 10 min). Concentrations of E2 and ICI were chosen from preliminary experiments, in agreement with previous reports [49, 50]. Medium was removed, the cells were washed with PBS, fixed in 2% paraformaldehyde for 20 min at room temperature, and washed with PBS containing 0.1 M glycine. Cells were then permeabilized with 0.01% saponin and blocked with PBS containing 1% BSA for 10 min at room temperature. After blocking, cells were incubated with one of the following primary antibodies: rabbit anti-ESR1 antibodies (H184 and MC20), mouse monoclonal antibody raised against the full-length human ESR1 (NCL-ER-6F11; Novocastra Laboratories, Newcastle, UK) and goat anti-ESR2 antibody (Y19), at 1:50 dilution in PBS containing 0.01% saponin and 1% BSA, for 1 h at room temperature. Afterwards, cells were washed with PBS containing 0.01% saponin and 1% BSA and incubated with the appropriate Alexa Fluor 594-labelled secondary antibody (Molecular Probes): anti-rabbit, anti-mouse, or anti-goat antibodies (1:300) for 30 min at room temperature. Coverslips were washed and mounted on slides with Fluoromount G (Electron Microscopy Sciences). Negative control slides were performed either with the primary antibody preadsorbed with its respective blocking peptide (Santa Cruz Biotechnology) or in the absence of primary antibody. The slides were also incubated with DAPI (4',6'-diamidino-2-phenylindole; Sigma) for cell nuclei visualization. Immunolocalization of the receptors was visualized under a Nikon E800 fluorescence microscope equipped with a short arc mercury lamp, respective exciter filters of 330–380 nm and 528–553 nm, and respective barrier filters of 435–485 nm and 600–660 nm (Nikon). Images were processed with a CoolSNAP-Pro CCD digital camera and the Image-Pro Express Software Program.
Confocal Laser Scanning Microscopy
Cells grown on coverslips coated with gelatin (0.1%) were incubated in the absence (control, C) and presence of E2 (0.1 nM; Sigma) for 10 min, and immunofluorescence assays were performed as described above, with an additional staining step using either a green nuclear marker (Yo-Pro1; Molecular Probes) or FITC-conjugated lectin from Triticum vulgaris (Sigma), which is also known as wheat germ agglutinin (WGA) and selectively binds to N-acetylglucosamine and sialic acid residues, common to many membrane glycoproteins. Images were obtained using an inverted LSM 510 confocal laser scanning microscope (Carl Zeiss Inc., Oberkochen, Germany). An Ar-ion 488-nm and a He-Ne 543-nm laser were used for excitation of green and red fluorescences, respectively. Serial z-axis sections were collected at a 0.4-µm thickness with a 63X PlanAploChromat objective (Carl Zeiss Inc.). Analysis and reconstruction of confocal microscopy images were performed using the free software Image J 1.37 [51]. Images are presented as projections of optical images stacks.
Western Blot for Detection of Estrogen Receptors
Primary Sertoli cell cultures were washed with ice-cold PBS, lysed in ice-cold lysis buffer (50 mM Tris, 150 mM NaCl, 0.05% sodium deoxycholate, 1% NP-40, 1 mM EDTA, 0.1% SDS, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 mM PMSF; pH 8.0), and centrifuged at 11 200 x g for 30 min at 4°C. Proteins from supernatant (50 and 100 µg per lane) were incubated with sample buffer containing dithiothreitol and β-mercaptoethanol and subjected to 7.5% SDS-PAGE. Proteins were electrotransferred onto PVDF membranes (0.45-µm pore size; ImmobilonP; Millipore, Bedford, MA) overnight, 20 V at 4°C. Membranes were blocked in Tris-buffered saline (TBS; 10 mM Tris, 150 mM NaCl; pH 8.0) containing 0.2% Tween 20 and 5% nonfat dry milk for 2 h at room temperature. ESR1 and ESR2 were detected by probing membranes with rabbit anti-ESR1 antibodies and goat anti-ESR2 antibody diluted 1:200 in blocking solution, for 2 h at room temperature. Membranes were washed in TBS containing 0.2% Tween 20 (TBS-T), and the antigens bound to primary antibodies were detected with the appropriate horseradish peroxidase (HRP)-conjugated anti-IgG secondary antibodies (donkey anti-rabbit [1:5000] and rabbit anti-goat [1:4000]; Jackson Immunoresearch Lab, West Grove, PA) for 1 h at room temperature. Thereafter, the membranes were washed with TBS-T, and immunoreactive bands were visualized onto preflashed Biomax XAR film (Eastman Kodak Co., Rochester, NY) by enhanced chemiluminescence reagent (Luminol; PerkinElmer, Boston, MA). In negative controls, primary antibodies were preadsorbed with the respective peptide immunogen and processed as described above. Apparent molecular masses of protein bands were determined from molecular mass standards (prestained protein marker, broad range 6–175 kDa; New England Biolabs, Boston, MA).
Western Blot for Detection of MAPK3/1 and Phospho-MAPK3/1
Primary Sertoli cell cultures were incubated in the absence (control, C) and presence of E2 (0.1 nM; Sigma) for 5–30 min at 35°C. In another series of experiments, the cells were untreated or pretreated with ICI (1 nM), PP2 (5 nM; Calbiochemical, San Diego, CA), the MAP2K1/2 inhibitor U0126 (100 µM; Cell Signaling Technology, Beverly, MA) for 30 min, or the EGFR kinase inhibitor AG 1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline; 50 µM; Calbiochem) for 15 min at 35°C. Afterward, the cells were stimulated with E2 (0.1 nM, 10 min). EGF (10 ng/ml, 10 min, 35°C) was used as control. Medium was removed, the cells were washed with ice-cold PBS and lysed in ice-cold lysis buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM PMSF, 2 mM Na3VO4, 50 mM NaF, and 10 mM Na4 P207), as previously described [52]. Total cellular proteins (60 µg per lane) were incubated with sample buffer containing β-mercaptoethanol and subjected to 10% SDS/PAGE. Proteins were electrotransferred onto PVDF membranes overnight, 20 V at 4°C. Membranes were blocked in TBS-t containing 5% nonfat dry milk (pH 7.6) for 2 h at room temperature. After washes in TBS-T, membranes were incubated with a rabbit polyclonal antibody raised against a synthetic peptide derived from the sequence of rat MAPK3/1 (p44/p42 MAP kinase, Erk1/Erk2, #9102; Cell Signaling Technology) or anti-phospho-MAPK3/1 antibody (Thr202/Tyr204, #9101; Cell Signaling Technology) diluted in blocking solution (1:1000 and 1:2000, respectively) overnight at 4°C. Proteins were visualized after incubation with donkey anti-rabbit HRP-conjugated secondary antibody (Amersham Biosciences) diluted in TBS-T (1:3000) for 1 h at room temperature by enhanced chemiluminescence reagent (ECL; Amersham Biosciences). Apparent molecular masses of these proteins were determined from molecular mass standards (New England Biolabs).
Band intensities of total MAPK3/1 and phosphorylated MAPK3/1 from individual experiments were quantified by densitometric analysis of linear-range autoradiograms by using the Epson Expression 1680 scanner (Epson America Inc., Long Beach, CA) and Quick Scan 2000 WIN software (Helena Laboratories, Beaumont, TX). Results were normalized based on total MAPK3/1 expression in each sample and plotted (mean ± SEM) in relation to control (C = 1).
[Methyl-3H]thymidine Incorporation Assays
Incorporation of [methyl-3H]thymidine into cell DNA was estimated as described by Guizzetti et al. [53]. Previous studies in our laboratory indicated that [methyl-3H]thymidine (0.074 MBq/ml, specific activity 2923 GBq/ml; Amersham Biosciences) incorporation in cultured Sertoli cells was time-dependent and linear from 2 to 10 h of incubation. All studies were performed using 6 h of [methyl-3H]thymidine incubation [38].
Primary Sertoli cell cultures were initially incubated with 0.074 MBq/ml [methyl-3H]thymidine for 6 h at 35°C. Incubation was continued in the absence (basal incorporation) and presence of E2 (0.1 nM; Sigma) for 24 h at 35°C. In another series of experiments, the cells were treated with ICI (1 nM) for 30 min at 35°C before addition of E2 (0.1 nM) for 24 h. The reaction was stopped by cooling the cells at 0°C. The medium was aspirated and the cells rinsed twice with ice-cold PBS and 5% trichloroacetic acid (Sigma). The cells were then solubilized with 0.5 N NaOH, collected with cotton-swabs, and transferred to 5 ml OptiPhase HiSafe-3 scintillation liquid (PerkinElmer Life Science Products). Bound radioactivity was determined in a scintillation β counter (LS 6000 IC; Beckman Coulter Inc., Palo Alto, CA). Results were expressed as percentage of [methyl-3H]thymidine incorporation above basal level.
Protein concentration was determined by Bio-Rad protein assay, using BSA as standard (Bio-Rad Laboratories, Hercules, CA).
Data were expressed as mean ± SEM. Statistical analysis was carried out by ANOVA followed by the Newman-Keuls test for multiple comparisons or by Student t-test to compare the differences between two data. P < 0.05 was considered significant.
Expression of ESR1 and ESR2 in Rat Testis and Cultured Sertoli Cells
RT-PCR experiments detected the presence of Esr1 and Esr2_v2-related transcripts in cultured Sertoli cells from 15-day-old rats and in testes from 15-, 28-, and 120-day-old rats (Fig. 2). A transcript related to Esr2_v1 was also detected in cultured Sertoli cells and in testis from 15-day-old animals (Fig. 2). The identity of all transcripts was confirmed by automated sequencing. No transcripts related to the smaller Esr2 isoforms were detected in testes or cultured Sertoli cells. A slightly smaller-than-expected product was detected during amplification of the Esr2_v2-related transcript, but was not related to any Esr isoform (Fig. 2). No PCR products were detected when reverse transcriptase was omitted from the RT-PCR, assuring that the amplified products were not from genomic DNA contamination (data not shown).
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In cryosections of testis from 15-day-old rats, a specific and intense immunostaining was detected in many Sertoli and Leydig cell nuclei as well as in some peritubular myoid cells, but not in germ cells, when using the MC20 antibody raised against the carboxyterminal region of ESR1 (Fig. 3B). Using the same antibody, positive immunostaining was also detected in Sertoli cells from 120-day-old rat testis (Fig. 3D). Although germ cells from immature rats were ESR1-negative, some spermatids were positive for this receptor in the adult rat testis (Fig. 3D). No immunostaining was observed when the primary antibody was preadsorbed with the respective blocking peptide (Fig. 3, A and B). Similar results were observed in testes from 15- and 120-day-old rats using the H184 antibody raised against the aminoterminal region of ESR1 (Supplemental Fig. 1, A and B available online at www.biolreprod.org). Using the Y19 antibody raised against the aminoterminal region of the ESR2, a specific immunostaining was detected in multiple testicular cell types, including Sertoli and Leydig cells, and in some but not all germ cells (Supplemental Fig. 1, C and D), confirming previous studies [9, 12, 54].
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ESR1 was also localized by immunofluorescence in the nuclei of cultured Sertoli cells obtained from 15-day-old rats, using the H184, MC20, or 6F11 antibody, against the full-length ESR1. Figure 4A (left panel) shows the results obtained using the MC20 antibody, and Figure 4A (right panel) shows the positive immunostaining for ESR2 using the Y19 antibody. The specificity of the antibodies was further confirmed by Western blot analysis using total protein extracts of cultured Sertoli cells. ESR1 and ESR2 were detected as single protein bands of about 66 and 55 kDa, respectively (Fig. 4B). These protein bands were absent when the membranes were incubated with primary antibody preadsorbed with the appropriate peptide immunogen (Fig. 4B).
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E2 Induces ESR1 and ESR2 Translocation to the Sertoli Cell Membrane Region
Under basal conditions, ESR1 and ESR2 immunostaining was detected in the nuclei of Sertoli cells (Fig. 5A). Treatment with E2 (0.1 nM) induced a time-dependent translocation of both estrogen receptors. After 10 min of E2 treatment, the immunostaining for ESR1 and ESR2 was detected not only in the nuclei, but also in other cell regions. Both ESRs were immunolocalized in the cell nuclei with E2 treatment for 15–30 min.
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The translocation of ESR1 and ESR2 induced by E2 was blocked by pretreatment of the cells with the antiestrogen ICI (1 nM) and with the selective inhibitor of the SRC family of protein tyrosine kinases PP2 (5 nM; Fig. 5B). Treatment with ICI or PP2, in the absence of E2, did not have any effect on ESR translocation (data not shown).
The ESR1 and ESR2 translocation to other cell regions after E2 treatment required further investigation by confocal microscopy. Colocalization of ESR1 and ESR2 with the nuclear marker Yo-Pro1 confirmed the nuclear localization of these receptors in the absence of E2, whereas after 10 min treatment with E2, the nuclear colocalization was poorly detected (Fig. 6).
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Binding sites for FITC-lectin WGA were observed not only in the plasma membrane, but also in the perinuclear region and cytoplasmic organelles of Sertoli cells. Colocalization of ESR1 and ESR2 with the FITC-lectin WGA was observed in the perinuclear region in both control and E2-treated cells (10 min), but colocalization of ESRs with the plasma membrane and cytoplasmic organelles was observed only upon 10-min E2 treatment (Fig. 7).
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E2 Induces MAPK3/1 Phosphorylation Through the Activation of Membrane-Associated ESR1 and ESR2 in the Sertoli Cells
MAPK3 and MAPK1 activity and expression in Sertoli cell lysates were measured by immunoblotting using phosphorylation state-dependent (top panels) and -independent (middle panels) antibodies (Fig. 8). The treatment with physiological concentrations of E2 (0.1 nM) induced a rapid and transient increase in the phosphorylation state of MAPK3/1 in the Sertoli cells. Peak MAPK3/1 phosphorylation levels occurred at 10 min (7- to 12-fold increase). The MAPK3/1 activity returned to control levels by 15–30 min (Fig. 8).
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The activation of MAPK3/1 induced by a 10-min treatment with E2 (0.1 nM) was blocked by pretreatment with ICI (Fig. 9), suggesting that ESRs are upstream components regulating MAPK3/1 activity in this E2 rapid action. PP2 or the MAP2K1/2 inhibitor U0126 also blocked the MAPK3/1 activation induced by E2 (0.1 nM) for 10 min (Fig. 9). The treatment with ICI (1 nM), PP2, or U0126 for 30 min, in the absence of E2, did not have any effect on MAPK3/1 phosphorylation (data not shown). To further confirm the involvement of EGFR, the EGFR kinase inhibitor AG 1478 was tested and markedly decreased E2-induced MAPK3/1 phosphorylation. EGF alone was used as a positive control and strongly activated MAPK3/1, and this effect was blocked by AG 1478 (Fig. 10). No differences were observed in total MAPK3/1 protein expression under any of these conditions (middle panels of Figures 8–10).
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E2 Stimulates Sertoli Cell Proliferation
E2 caused an enhancement of 41.3% ± 1.3% of [methyl-3H]thymidine incorporation after 24 h incubation (Fig. 11). In addition, the pretreatment of Sertoli cells with ICI markedly reduced the [methyl-3H]thymidine incorporation into DNA induced by E2 (Fig. 11). In the absence of E2, the antiestrogen did not have any effect on [methyl-3H]thymidine incorporation (Fig. 11).
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The present study indicates that estrogen promotes Sertoli cell proliferation and the activation of a signaling cascade that initiates at the plasma membrane and involves SRC, EGFR, and MAPK3/1 activation. Furthermore, it was identified that both ESR1 and ESR2 are present in Sertoli cells and the treatment with E2 induces the translocation of both to the plasma membrane region.
ESR1 and ESR2 are encoded by two different genes located on different chromosomes. Multiple RNA splicing variants for ESR1 [55] and alternative splicing variants [56–58] and polymorphism [59] for ESR2 have been reported for human. Such a molecular diversity due to alternative splicing mechanisms of the correpondent genes is also observed in rodents [45, 60, 61]. Esr1 splicing variants were not detected in cultured Sertoli cells or in testis, and only a single band for ESR1 was detected in cultured Sertoli cells by the Western blot analysis. On the other hand, two splicing variants were detected for Esr2 both in cultured Sertoli cells and in testis: the Esr2_v1 isoform, with high similarity to the human ESR2 [62, 63], and the Esr2_v2, which contains an in-frame insert of 54 nucleotides that has only been found in tissues from rodents [45]. Esr2_v2 encodes a protein that differs from the isoform 1 of ESR2 only by an insertion of 18 aminoacids in the ligand-binding domain. In fact, in Western blot analysis only one protein band was distinguished when an antibody that recognizes the aminoterminal region of the receptor common to both ESR2 isoforms was used. Petersen et al. [45] reported an identical migration pattern for the recombinant ESR2 isoforms 1 and 2 expressed in HEK293T cells. Interestingly, in the present study an Esr2_v1-related transcript was detected only in cultured Sertoli cells and in testes from 15-day-old animals but not from older animals, whereas an Esr2_v2-related transcript was detected in the testes of all studied ages. The physiological implication of the existence of several ESR2 isoforms has been investigated. The human ESR2 isoform 1 apparently is the only fully functional one. However, the other isoforms can heterodimerize with ESR2 isoform 1 under the stimulation of estrogens and enhance the transactivation [64]. Furthermore, the human ESR2 isoform 2 has been proposed to act as a dominant negative receptor, preventing estrogen action in gonocytes [58]. The differential distribution of ESR2 variants in human testicular cells may indicate that these variants have specific functions in spermatogenesis [57]. On the other hand, the rodent ESR2 isoform 2 seems to be functional, although much higher concentrations of estrogen are required for its activity [45].
The expression of ESR1 and ESR2 in the fetal testis occurs very early in development, and their distribution in the different testicular cells has been extensively studied in mammals [5, 11, 65]. Immunohistochemical data have shown that ESR1 is present in the undifferentiated gonad and in the fetal Leydig cells until birth in rodents [19]. The most consistent data across species has been the presence of ESR1 in the Leydig cells. There are conflicting reports of ESR1 in Sertoli cells, germ cells, and spermatozoa [4, 9, 13, 16, 17, 66–68]. Our results demonstrated the presence of ESR1 in Sertoli cells from immature and adult rats by using antibodies raised against the carboxyterminal (presented data) or aminoterminal region of ESR1 (Supplemental Fig. 1). It was also confirmed that germ cells from immature rats are ESR1-negative, though some spermatids were positive for this receptor in the adult animal. Indeed, the presence of ESR1 in rat and human spermatozoa has been reported recently [3, 67, 68]. ESR1 was also detected by immunofluorescence in the nuclei of cultured Sertoli cells obtained from 15-day-old rats. Colocalization of ESR1 and CLU further confirmed that ESR1 was in fact expressed by Sertoli cells (data not shown). One possibility to explain the apparent discrepancies observed in the literature regarding to the presence of ESR1 in Sertoli cells could be related to the methodologies used. We examined various fixing protocols and antibody sources to obtain a suitable immunohistochemical procedure for ESR1 detection in rat testes. In fact, using testes from adult rats perfused with 10% neutral buffer formalin embedded in paraffin and the antibodies described above, we were not able to detect any immunoreaction for ESR1 in Sertoli cells, but Leydig cells and peritubular myoid cells were immunostained (data not shown).
There is more agreement in the literature concerning the immunolocalization of the ESR2 protein. We detected ESR2-specific immunostaining in multiple cell types, including Sertoli and Leydig cells, and in some but not all germ cells, which confirms previous studies [9, 12, 54]. It is worth mentioning that ESR2 is coexpressed with ESR1 in tumoral seminoma cells, where it may counteract the tumor cell proliferation mediated by ESR1 [69].
A series of E2-induced, membrane-initiated steroid signaling has been described in benign and malignant cells of various origins. These effects occur from seconds to minutes after administration of E2 and involve rapid activation of many signaling molecules [20, 26, 27, 34, 35]. In the present study, treatment with E2 induced a translocation of ESR1 and ESR2 from the nuclei to the plasma membrane of Sertoli cells. In addition to posttranslation modifications of ESR1 by palmitoylation [70], other mechanisms have been proposed to explain the translocation of ESR1 to the cell membrane in estrogen-stimulated cells [26, 27]. For example, in human mammary cancer MCF-7 cells, the translocation of the ESR1 to the cell membrane involves SHC1 as a transporter. In response to E2, SHC1 is phosphorylated and binds to ESR1. At the same time, the SHC1 binding sites on the IGF1R are phosphorylated, allowing SHC1 to bind to the IGF1R. ESR1 can then be tethered to the membrane through SHC1 interaction with IGF1R [50, 71, 26]. SHC1 is a substrate of activated SRC and, once phosphorylated, interacts with other proteins [26], suggesting a possible role of SRC as a signal integrator in estrogen-stimulated MCF-7 cells, since estrogen stimulates MAPK activation through the interaction of SRC with ESR1 [72–74]. There is evidence that ESR2 could also localize in caveolae and participate in nongenomic signaling through the interaction of SRC with ESR2 [75–76]. As presently found, the E2-induced translocation of ESR1 and ESR2 to the plasma membrane region of Sertoli cells was blocked by pretreatment of the cells with a selective inhibitor of the SRC family of protein tyrosine kinases, suggesting that SRC plays a crucial role in the translocation of ESR1 and ESR2 from the nucleus to the cell membrane. Fan et al. [77] have recently shown a similar mechanism of SRC- and EGFR-mediated translocation of ESR1 out of the nucleus induced by tamoxifen in MCF-7 cells resistant to tamoxifen, and they suggest that this mechanism could contribute to acquired tamoxifen resistance.
Membrane-associated ESR1 transduces estrogen rapid signals, leading to the activation of many signaling molecules, such as MAPK, AKT, KRAS, RAF1, and protein kinase C, alteration of maxi-K+ channels, increase in intracellular Ca++ levels, release of nitric oxide, and stimulation of prolactin secretion [26, 27, 78]. In addition to the activation of these signaling molecules, membrane receptors for growth factors, such as IGF1R and EGFR, can also be activated by estrogen rapid action, leading to activation of downstream MAPK and AKT signaling pathways [26, 27, 79, 80]. The translocation of ESR1 and ESR2 to the Sertoli cell membrane occurred after E2 treatment in the same time frame as MAPK3/1 activation, and these effects were also blocked by a selective inhibitor of the SRC. Furthermore, the antiestrogen ICI blocked E2-induced ESR1 and ESR2 translocation to the membrane and MAPK3/1 phosphorylation, indicating the direct involvement of ESRs in these events. These estrogen actions and induced pathways have also been demonstrated in many other cell types, including MCF-7 breast cancer cells [72], osteoblasts [81], rat brain [82], human colon carcinoma-derived Caco-2 cells [83], and LNCaP prostate cancer cells [84]. The evidence of a relationship between the EGFR and estrogen has been previously provided by data showing that neutralizing antibodies against EGF inhibited estrogen-mediated proliferation in the mice uterus [85]. In Sertoli cells, an EGFR kinase inhibitor decreased the E2-induced MAPK3/1 phosphorylation, confirming the involvement of EGFR in this activity. Whether a linear pathway involving a sequential activation of IGF1R, metalloproteinases, and EGFR by E2 is also present in Sertoli cells, such as recently demonstrated in MCF-7 breast cancer cells [80], still remains to be determined. Furthermore, in a wide variety of cells, besides the localization in nucleus and plasma membranes, ESR1 and ESR2 have also been localized in the mitochondria [86]. The relative subcellular localization of the receptors is altered in cancer cells, and ESR2 has been recently shown to shift from the mitochondria to the nucleus during neoplastic transformation of epithelial breast cells [87]. It would be interesting to determine whether, in Sertoli cells, ESRs may also localize in mitochondria and the influence of estrogen on their subcellular distribution.
In order to provide further evidence of the importance of estrogen on Sertoli cell biology, we examined the effects of E2 on Sertoli cell proliferation. After 24 h of E2 incubation, an increase of 41% of [methyl-3H]thymidine incorporation was observed. Moreover, ICI blocked this effect, suggesting that the E2 plays a role in the Sertoli cell proliferation.
In the developing testis, estrogen plays a significant role in establishing Sertoli cell function [19] and potentially even in establishing Sertoli cell-germ cell adhesion [88]. Immature, undifferentiated Sertoli cells may generate estrogen themselves to act in an autocrine manner [3]. Current evidence suggests that the effect of E2 on Sertoli cells depends on the concentration of the hormone. Administration of high doses (10 µg per animal during 10 days) of diethylstilbestrol to neonatal rats suppressed FSH plasma levels and induced changes that were consistent with a permanent impairment of functional maturation of Sertoli cells [89], whereas lower doses (0.1 µg per animal) decreased Sertoli cell numbers without interfering with FSH levels [90]. On the other hand, in immature bank voles, administration of low doses of E2 (0.1 µg/g body weight) accelerated the onset of spermatogenesis, and high doses (10 µg/g body weight) caused a disruption of testicular structure and tubular atrophy [91]. The present in vitro study shows that, in culture conditions, physiological concentrations of E2 (0.1 nM) can induce the proliferation of Sertoli cells from 15-day-old rats, and corroborates previous findings that low concentrations of E2 may have positive effects on testicular function. It is very likely that estrogens not only suppress Sertoli cell differentiation, but also act as a mitogen in this cell proliferation during the neonatal period.
It is interesting that in the Esr1–/– testis there is significantly less seminiferous tubular secretion than in the wild-type testis [1]. The same effect was suggested for the Cyp19–/– testis [92]. Mice subjected to a long-term treatment with ICI showed hypospermatogenesis and abnormal germ cell development, but no seminiferous tubular dilation or increase in testis weight was observed [93]. Taken together, these data suggest that estrogen-ESRs play a role in Sertoli cell function. Thus, the signaling mechanism elicited by estrogen involving translocation of ESRs to the plasma membrane and activation of MAPK signaling may have important implications for Sertoli cell function and spermatogenesis. A recent study described a similar signaling pathway elicited in Sertoli cells by testosterone acting on plasma membrane-associated androgen receptors [94].
A sequential event induced by estrogen and involving activation of G proteins, matrix metalloproteinases 2 and 9, release of heparin-binding EGF, and finally EGFR activation on the cell membrane has also been reported to lead to activation of MAPK and AKT [28–31]. The present data show that the E2 effect on MAPK3/1 activation involves the classical ESRs because it can be blocked by the antagonist ICI. Similarly, the E2-induced proliferation of Sertoli cells was blocked by ICI, demonstrating the involvement of ESRs in this process. It is not clear yet whether the proliferative effect of E2 on cultured Sertoli cells involves a genomic and/or a non-genomic action of ESR1 and ESR2.
Our studies do not exclude the possibility that the G protein-coupled receptor GPR30 may contribute to E2-induced MAPK3/1 activation and Sertoli cell proliferation, and the relative contribution of each receptor to control E2 action in Sertoli cells still remains to be determined. In ovarian cancer cells, it has been recently demonstrated that both GPR30 and ESR1 must be present to observe E2-mediated activation of EGFR signaling, MAPK3/1 activation, and cell proliferation [32]. On the other hand, in MCF-7 breast cancer cells, E2 stimulated cell proliferation via sequential activation of IGF1R and EGFR, and knockdown or blockade of ESR1, but not of GPR30, blocked E2-induced MAPK activation [80]. The apparent discrepancies found in the literature seem to result from differences in cellular context. Further studies are underway to determine the role of GPR30 in rat Sertoli cells.
In conclusion, these results indicate that the effect of E2 on Sertoli cell function involves the following steps: i) translocation of ESR1 and ESR2 to the plasma membrane mediated by the non-receptor tyrosine kinase SRC and ii) activation of EGFR and MAPK signaling pathway. In addition, E2 induces Sertoli cell proliferation. These actions of E2 might be one of the key steps that mediate the estrogen-dependent activation of cellular events in Sertoli cells. Additional studies are now required to further elucidate the role of each ESR in Sertoli cell biology and, consequently, in spermatogenesis and male (in)fertility.
ACKNOWLEDGMENTS
We thank Espedita M.J. Silva Santos, Maria Damiana Silva, and Sabrina A. Souza e Silva for technical assistance.
FOOTNOTES
1Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant 2004/01152-0, to C.S.P.). Research fellowship (C.S.P.) was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnológico, CNPq. Doctoral fellowships supported by CNPq (T.F.G.L.) and FAPESP (E.R.S.). Master fellowship supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES (C.A.E.). 
Correspondence: 2Maria Fatima M. Lazari, Section of Experimental Endocrinology, Department of Pharmacology, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Três de maio 100, INFAR, Vila Clementino, São Paulo, SP 04044-020, Brazil. FAX: 5511 5576 4448; e-mail: lazari{at}farm.epm.br
Received: 2 July 2007.
First decision: 28 July 2007.
Accepted: 8 October 2007.
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