Androgen Receptors in Gonadotrophs in Pituitary Cultures from Adult Male Monkeys and Rats
Division of Endocrinology and Metabolism, University of Louisville, Louisville, Kentucky 40202+, http://www.100md.com
Abstract+, http://www.100md.com
Top+, http://www.100md.com
Abstract+, http://www.100md.com
Introduction+, http://www.100md.com
Materials and Methods+, http://www.100md.com
Results+, http://www.100md.com
Discussion+, http://www.100md.com
References+, http://www.100md.com
There is substantial evidence demonstrating that the principal feedback action of androgens to decrease LH secretion in male primates, including man, is to slow the GnRH pulse generator, whereas in male rats androgens not only decrease GnRH but also suppress LH synthesis and secretion through a direct pituitary effect. Previous experiments in our laboratory revealed that testosterone (T) suppresses LH secretion and decreases
-subunit mRNA levels in male rat pituitary cell cultures perifused with pulses of GnRH but not in pituitary cells from adult male monkeys. In the present study, we sought to determine whether the lack of responsiveness of gonadotrophs to androgens in the primate is androgen receptor (AR) related. Primary cultures were prepared from the anterior pituitary glands of adult male monkeys and rats. Cells were identified as gonadotrophs if they were immunoreactive for LH-ß or FSH-ß. Of these cells in the monkey, 80% contained both gonadotropins, 17% contained only LH-ß, and 3% contained only FSH-ß. AR immunoreactivity (IR) was nuclear in 22% and 15%, respectively, of monkey and rat FSH-ß-positive cells in the absence of T. Following T treatment, nuclear AR IR was identified in 79% of monkey and 81% of rat gonadotrophs. T treatment similarly intensified AR IR in mouse gonadotroph T3-1 and LßT2 cells and in monkey and rat fibroblasts. Single-cell RT-PCR confirmed coexpression of LH-ß and AR mRNA as well as LH-ß and GH mRNA in monkey gonadotrophs. Our data reveal that most monkey, as well as rat, gonadotrophs are AR-positive with nuclear localization in the presence of T. GH expression is not required for AR expression in gonadotrophs. We conclude that the failure of T to inhibit LH secretion and decrease -subunit mRNA expression in the male primate is not due a disturbance in AR nuclear shuttling.
Introduction5i}}&r, 百拇医药
Top5i}}&r, 百拇医药
Abstract5i}}&r, 百拇医药
Introduction5i}}&r, 百拇医药
Materials and Methods5i}}&r, 百拇医药
Results5i}}&r, 百拇医药
Discussion5i}}&r, 百拇医药
References5i}}&r, 百拇医药
ANDROGENS RESTRAIN GONADOTROPIN secretion in adult male primates, including men, by slowing the GnRH pulse generator (1, 2, 3) but appear not to influence gonadotrophs directly. This idea is supported by experiments in rhesus monkeys rendered gonadotropin deficient with radio-frequency lesions and stimulated with pulses of GnRH; in these monkeys, LH secretion increased little after bilateral orchidectomy until GnRH pulse frequency was increased (4). In clamp experiments in men with congenital hypogonadotropic hypogonadism treated with pulsatile GnRH, suppression of LH secretion by testosterone (T) was blocked by the aromatase inhibitor testolactone, suggesting that inhibition by T was through bioconversion to estradiol (5), whereas the nonaromatizable androgen dihydrotestosterone (DHT) had no effect in this human model (6). Furthermore, in normal men, infusion of DHT for 4 d suppressed LH pulse frequency but increased, rather than decreased, the LH response to exogenous GnRH stimulation (7). In adult male rats, on the other hand, androgen-negative feedback appears to be partly GnRH mediated (8, 9) but is also through direct inhibition of GnRH-stimulated LH release (10, 11).
Cultured pituitary cells are a useful model in the effort to understand the species-specific cellular mechanisms regulating gonadotropin secretion and subunit gene expression. We have compared the effects of T on LH synthesis and secretion using dispersed pituitary cells from male monkeys and rats that were perifused with pulses of GnRH (12). In this model, T down-regulated GnRH-stimulated-subunit mRNA levels and suppressed LH secretion by rat pituitary cells but not by pituitary cultures from monkeys. DHT also failed to suppress GnRH-induced LH secretion, suggesting that the absence of a direct androgenic-negative feedback effect in the primate was not due to T metabolism. The explanation and significance for this species difference in the pituitary action of androgens are unknown.'y?@&, http://www.100md.com
The first step in androgen action is binding of androgen to its receptor, followed by shuttling of the activated androgen receptor (AR) to the cell nucleus, where it stimulates or represses gene transcription (13). To explore the hypothesis that a disturbance in AR shuttling explains the lack of responsiveness of primate gonadotrophs to androgens, we first determined whether primate gonadotrophs are monohormonal or bihormonal by localizing FSH-ß and LH-ß protein to gonadotrophs using double-labeled immunofluorescence staining. Second, we used double-labeled immunoperoxidase staining to localize AR in gonadotrophs in pituitary cell cultures from adult male monkeys and rats in the presence or absence of T. And, third, we studied LH-ß and AR mRNA coexpression in monkey pituitary cultures using single-cell RT-PCR techniques.
Materials and Methods|95w}qx, 百拇医药
Top|95w}qx, 百拇医药
Abstract|95w}qx, 百拇医药
Introduction|95w}qx, 百拇医药
Materials and Methods|95w}qx, 百拇医药
Results|95w}qx, 百拇医药
Discussion|95w}qx, 百拇医药
References|95w}qx, 百拇医药
Reagents|95w}qx, 百拇医药
DMEM, Dulbecco’s PBS (DPBS; without CaCl2 and MgCl2), Hanks’ balanced salt solution (HBSS), HEPES, penicillin G and streptomycin sulfate, fetal calf serum (FCS), calf serum (CS), BSA fraction V, pancreatin, oligo[deoxythymidine (dT)] primer, reverse transcriptase (RT) kit and SuperScript II ribonuclease (RNase) H-RT, terminal deoxynucleotidyl transferase (TdT) recombinant, 100 mM deoxynucleotide triphosphate (dNTP), and ultra-pure distilled water (H2O) were purchased from Invitrogen (Carlsbad, CA). FCS and CS were treated with dextran-charcoal (DCC) to remove steroids. Deoxyribonuclease I, poly-L-lysine, trypsin, EDTA, diaminobenzidine (DAB), diethyl pyrocarbonate, T, and DHT were purchased from Sigma (St. Louis, MO); fluconizole from Pfizer, Inc. (New York, NY); collagenase from Roche Molecular Biochemicals (Mannheim, Germany); a 100-bp DNA ladder from New England Biolabs, Inc. (Beverly, MA); cyanine 2-conjugated goat antirabbit IgG and cyanine 3-conjugated goat antiguinea pig IgG from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA); 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) from MolecularProbes (Eugene, OR); avidin-biotin-peroxidase complex, biotinylated antirabbit IgG, and SG substrate kit from Vector Laboratories (Burlingame, CA); prime RNase inhibitor from Fisher Scientific (Pittsburgh, PA); RNA guard from Amersham Biosciences (Piscataway, NJ); and Taq DNA polymerase and 10 mM dNTP mix from Promega Corp. (Madison, WI).
Preparation of anterior pituitary cells and cell cultures2rt%:), http://www.100md.com
Freshly removed anterior pituitary glands from adult male rhesus monkeys (Macaca mulatta) were obtained from Covance Laboratories, Inc. Research Primates (Alice, TX) and shipped on ice in HBSS containing 44 mM HEPES. Rat pituitary cells were prepared from 7-wk-old male Sprague Dawley rats (Harlan, Indianapolis, IN). All media contained 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, and 2 mg/ml fluconizole. The methods used for the preparation of pituitary cell cultures were similar to those described previously (14). Briefly, anterior pituitaries were minced and treated for 60 min with 0.33% collagenase and 0.003% deoxyribonuclease in HBSS (pH 7.3) containing 0.4% BSA fraction V, 0.2% sucrose, and 44 mM HEPES. Cells were then treated with 0.25% pancreatin in HBSS for 8 min and washed three times with DMEM containing 5% DCC-FCS and 5% DCC-CS. Dispersed cells were cultured in DMEM with 10% DCC-FCS on poly-L-lysine-coated glass chamber slides at a density of 1 x 105 cells per chamber for immunocytochemistry, or on 60-mm tissue culture dishes at a density of 1 x 104 cells per dish for single-cell RT-PCR analysis. After 24 h of preculture, media in the chamber slides were changed to include 20 nM T or vehicle for 24 h. Cells were then fixed with 4% paraformaldehyde and stored at -20 C. Mouse gonadotroph cell linesT3-1 and LßT2 (kindly provided by Dr. Pamela Mellon, University of California-San Diego, La Jolla, CA) were plated on chamber slides, treated with 20 nM T or vehicle for 24 h, and then fixed for immunocytochemistry. For RNA extraction, T3-1 and LßT2 cells were plated in DMEM with 10% DCC-FCS in six-well plates at a density of 1.2 x 106 cells per well. After 24 h of preculture, cells were treated with 10 nM T, 10 nM DHT, or vehicle for 72 h and were then harvested. Monkey pituitary fibroblast and rat genital skin fibroblast cultures were produced as previously reported (15). Cells were plated in DMEM with 10% DCC-FCS on chamber slides and treated with 20 nM T or vehicle for 24 h and then fixed for immunocytochemistry.
Double-labeled immunofluorescence stainingm(#q2dd, http://www.100md.com
Anti-FSH-ß (rabbit antibody; batch 5, National Hormone and Pituitary Program, Torrance, CA) at 1:10,000, and antimonkey LH-ß (guinea pig antibody; AFP555194, National Hormone and Pituitary Program) at 1:20,000 were incubated overnight at 4 C. In the first reaction, the primary rabbit (FSH-ß) antiserum was localized using 1:200 cyanine 2-conjugated goat antirabbit IgG, and in the second reaction, the primary guinea pig anti-LH-ß serum was localized with 1:400 cyanine 3-conjugated goat antiguinea pig IgG (each for 1 h at 23 C). Nuclear counterstaining was performed with DAPI diluted 1:3000 in PBS for 5 min at 23 C. Appropriate filters were used to observe the green fluorescence of cyanine 2-labeled IgG and the red fluorescence of cyanine 3-labeled IgG. A total of 763 FSH-ß- or LH-ß-positive cells were counted from three separate monkey preparations. The percentage of double-labeled cells was calculated and expressed as mean ± SEM.
Single- and double-labeled immunoperoxidase staining:]z\/7c, http://www.100md.com
A rabbit polyclonal antibody to the AR (AR N-20 sc-816, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used at 1:200. Slides were incubated with antibodies overnight at 4 C. Staining was accomplished using biotinylated antirabbit IgG and the avidin-biotin-peroxidase complex. For monkey pituitary fibroblasts, rat genital skin fibroblasts, and LßT2 andT3-1 cells, AR was stained with DAB, and a brown color was developed. For primary pituitary cultures, AR was stained with DAB in the first reaction. Then FSH-ß was stained as described above using antiserum batch 5 at 1:5000. A blue color was developed using the SG substrate kit.:]z\/7c, http://www.100md.com
A total of 1180 and 954 FSH-ß-positive cells from three different cell preparations from monkeys and rats, respectively, were analyzed. The percentage of nuclear AR-positive cells was calculated and expressed as the mean ± SEM. No staining was observed when the first antibodies were omitted.
mRNA analysis by RT-PCR:*p3, http://www.100md.com
RNA was extracted by the guanidinium thiocyanate-phenol-chloroform procedure (16). The concentration of total RNA was determined by reading the OD at 260 nm. Reverse transcription of total pituitary RNA was performed from control and T-treated pituitary cell cultures from monkeys and rats that were stimulated with hourly pulses of GnRH (12). First-strand cDNA was synthesized with a RT kit using 1 µg of total RNA. The cDNAs, equivalent to 30 ng of RNA, were amplified in a 20-µl PCR containing Taq DNA polymerase, 1.5 mM MgCl2, and each primer. Primers used in the PCR were as follows: for monkey AR (GenBank accession no. U94179): forward, 5'-GACTCCGTGCAGCCTATTG-3'; reverse, 5'-GGGCACTTGCACAGAGATG-3'; product size 123 bp; for monkey glyceraldehyde phosphate dehydrogenase (GAPDH) (NM002046): forward, 5'-TCAACAGCGACACCCACTC-3'; reverse, 5'-CTTCCTCTTGTGCTCTTGCTG-3'; product size 201 bp; Ref. 17 ; for rat AR (NM012502): forward, 5'-GGATTCTGTGCAGCCTATTG-3'; reverse, 5'-GGGCACTTGCACAGAGATG-3'; product size 124 bp; for mouse AR (NM013476): forward, 5'-CAGCATTATTCCAGTGGATGG-3'; reverse, 5'-GGGCACTTGCACAGAGATG-3'; product size 274 bp; and mouse and rat GAPDH (AF106860, M32559): forward, 5'-GGCATTGCTCTCAATGACAA-3'; reverse, 5'-TGTGAGGGAGATGCTCAGTG-3'; product size 223 bp. Amplification was conducted for 24 cycles for monkey GAPDH, 20 cycles for rat GAPDH, 22 cycles for mouse GAPDH, and 35 cycles for monkey, rat, and mouse AR (94 C for 30 sec, 58 C for 75 sec, and 72 C for 90 sec). Each PCR product was separated on a 1.8% agarose gel in Tris-borate EDTA buffer and visualized by ethidium-bromide staining, digitized with a GS-700 Imaging Densitometer (Bio-Rad Laboratories, Inc., Hercules, CA), and analyzed using Molecular Analysis software (Bio-Rad Laboratories, Inc.). In preliminary experiments, a linear range of amplification was established by varying the number of PCR cycles. The amount of AR mRNA product was normalized to that of GAPDH in each sample.
Single-cell RT-PCR8, 百拇医药
Single-cell RT-PCR was based on previously published protocols (18). Primary pituitary cultures were dispersed in 60-mm tissue culture plates at a density of 1 x 104 cells per dish. Twenty-four hours after dispersal, cells were washed five times with DPBS (without Mg and Ca), and 1.5 ml of DPBS was added. Cells were observed under an inverted microscope. Single cells were isolated with a micromanipulator fitted with a pulled microcapillary. A single cell was expelled into a PCR tube filled with a reaction mixture containing 4.5 µl of lysis buffer [for 100 µl mix of 20 µl of 5x RT buffer, 76 µl of H2O, 0.5 µl of IGEPAL, 1 µl of prime RNase inhibitor, 1 µl of RNA guard, and 2 µl of a freshly made 1/24 dilution of the stock primer mix (10 µl of 100 mM each dNTP, 20 µl of 820 µg/ml phosphorylated oligo(dT)22, and 10 µl of H2O)]. The mixture was incubated at 65 C for 2 min to lyse the cells, placed on ice for 1 min, and incubated at 37 C for 2 min to anneal the primers. The reaction was initiated by adding 1 µl of RT enzyme at 37 C for 50 min, followed by 65 C for 10 min. Homopolymer deoxy-ATP was added to the 3' end of the first-strand cDNA using TdT enzyme in 4.5 µl of stock tailing buffer (100 µl of 5x TdT buffer, 3.75 µl of 100 mM deoxy-ATP, 146.25 µl of H2O) at 37 C for 15 min, followed by 65 C for 10 min, and placed on ice until PCR. PCR mix containing 10 µl of 10x PCR buffer, 10 µl of 25 mM MgCl2, 2 µl of 10 mM dNTP mix, 4 µl of 0.73 µg/µl oligo(dT)-X primer [5'-ATGTCGTCCAGGCCGCTCTGGACAAAATATGAATTC(T)24-3'], 2 µl of Taq DNA polymerase, and 62 µl of H2O was mixed on ice. PCR mix (90 µl) was added to each PCR tube with 10 µl of the template and then placed in a PTC-200 DNA Engine Thermal Cycler (MJ Research, Inc., Line Village, NV) for 25 cycles (94 C for 1 min, 42 C for 2 min, 72 C for 6 min with a 10-sec time extension at each cycle). After the first 25 cycles, 1 µl of Taq polymerase was added to each tube, and 25 additional cycles were performed with the same program but without the 10-sec extension at each cycle. After general amplification of cDNA, specific PCR was carried out using 1.5 µl of the general amplified PCR product as a template for 50 cycles in a 20-µl PCR containing 1 U Taq DNA polymerase, 1.0 mM (for AR) or 1.5 mM (for LH and GH) MgCl2, 0.15 mM of dNTP, and 0.2 µM of each primer. PCR primers (for LH-ß: forward, 5'-GTGTGCATCACCGTCAACAC-3'; reverse, 5'-CCACAGCGACAGCTGAGAG-3'; product size 200 bp; and for GH: forward, 5'-AGGCATCCAAACACTGATGG-3'; reverse, 5'-CAATGCGCAGGAATGTCTC-3'; product size 301 bp) were designed based on the published sequences of the monkey LH-ß (XM009418) and GH (L16556). A nested PCR was conducted for AR (outer forward, 5'-CAGCATTATTCCAGTGGATGG-3'; inner forward, 5'-GACTCCGTGCAGCCTATTG-3'; reverse, 5'-GGGCACTTGCACAGAGATG-3'; product sizes 274 bp and 123 bp, respectively). All primer pairs used span at least one intron so that the PCR products could be distinguished by size from contaminating genomic DNA. Total monkey pituitary cDNA was used as a positive control. After a prerun of 95 C for 8 min, specific amplifications were conducted for 50 cycles at 95 C for 30 sec; 50 C (for AR), 58 C (for GH), or 62 C (for LH-ß) for 90 sec; 72 C for 90 sec; and 72 C for 7 min post run. The nested PCR for an additional 40 cycles was performed for AR amplification using 1.5 µl of the first PCR products as template under the same reaction conditions, except the final concentration of MgCl2 was 2.0 mM. To minimize the chance of cross-contamination between reagents or individual PCR amplifications, the following controls were performed in each experiment. First, cells were dispersed in the culture dishes at a relatively low density. Second, for each cell harvested, a new pipette was used. Third, the pipette was held for a second in the close vicinity of a cell, and media without a cell were ejected into a test tube and subjected to RT-PCR as a negative control. Fourth, approximately 50 cells, which were amplified without the RT reaction, were used as a negative control.
Data analysis and presentationh, http://www.100md.com
The percentage of double-labeled cells was calculated and expressed as the mean ± SEM. Two group comparisons were performed with the Student’s t test. Statistical significance was inferred at P < 0.05.h, http://www.100md.com
Resultsh, http://www.100md.com
Toph, http://www.100md.com
Abstracth, http://www.100md.com
Introductionh, http://www.100md.com
Materials and Methodsh, http://www.100md.com
Resultsh, http://www.100md.com
Discussionh, http://www.100md.com
Referencesh, http://www.100md.com
Double-labeled immunofluorescence to colocalize LH-ß and FSH-ß in gonadotrophsh, http://www.100md.com
Gonadotrophs from three separate monkey pituitary preparations were identified by immunofluorescence for LH-ß and/or FSH-ß content. Figure 1 illustrates representative monkey pituitary cells stained by double-labeled immunofluorescence. Phase contrast and nuclear staining with DAPI, which is stained blue, was used to identify all pituitary cells in the microscopic field. FSH-ß protein is stained green, and LH-ß protein is stained red. At high magnification (x400), both FSH-ß and LH-ß protein were found in the cell cytoplasm but were absent from nuclei. In the overlay image (FSH-ß + LH-ß), most cells were positive for both FSH-ß and LH-ß (yellow). At lower magnification (x200), some cells contained only FSH-ß or LH-ß, implying that monohormonal as well as bihormonal gonadotrophs are present in the monkey pituitary. Of 763 cells studied from three separate cell preparations, 80% ± 3% were positive for both LH-ß and FSH-ß, 17% ± 3% contained only LH-ß, and 3% ± 1% contained only FSH-ß.
fig.ommitteed*^e, 百拇医药
Figure 1. Colocalization of FSH-ß and LH-ß protein in monkey primary pituitary cultures. Anterior pituitary cells from adult male monkeys in chamber slides were fixed and subjected to double-labeled immunofluorescence for FSH-ß and LH-ß. The first column shows a phase contrast image. The second column shows nuclear staining with DAPI (blue). The third column shows FSH-ß staining (green), and the fourth column shows LH-ß staining (red). Combined image (FSH-ß + LH-ß) in the fifth column shows the coexpression of FSH-ß and LH-ß (yellow). The arrowhead points to the cell labeled only for FSH-ß, and the arrow points to the cell labeled only for LH-ß.*^e, 百拇医药
Double-labeled immunoperoxidase staining to detect AR in FSHß(+) cells in primary pituitary cultures*^e, 百拇医药
To investigate the effects of androgens on AR distribution in gonadotrophs, we first identified AR in two mouse gonadotroph cell lines,T3-1 and LßT2. AR immunoreactivity (IR) was uniformly recognized both in the cytoplasm and nucleus in both cell lines grown in media containing 10% DCC-FCS in the absence of T. When 20 nM T was added to the culture media, AR IR in the nucleus was intensified (not shown). We then extended the immunostaining method to primary pituitary cultures from adult male rats and monkeys. Multiple fields were investigated with a range of 256–653 gonadotrophs in each monkey pituitary culture and 263–397 gonadotrophs in each rat pituitary culture. Representative pituitary cells that were double-labeled for FSH-ß and AR protein are shown in Fig. 2A. FSH-ß-positive cells stained blue, and AR was stained brown. In the absence of T, AR IR was present in both the cytoplasm and nucleus of monkey as well as rat FSH-ß-positive cells. Adding T to the culture media strongly intensified the nuclear AR IR, which was homogeneous in appearance in both rat and monkey gonadotrophs. In the absence of T, AR IR was nuclear in 22 ± 3% and 15 ± 4% of monkey and rat FSH-ß-positive cells, respectively. With T treatment, nuclear AR IR was identified in 79 ± 5% and 81 ± 6% of monkey and rat FSH-ß-positive cells, respectively. Likewise, in monkey and rat fibroblasts, AR IR was present in both the cytoplasm and nucleus, and nuclear AR IR was intensified with T treatment (Fig. 2B).
fig.ommitteed1v1f7sp, 百拇医药
Figure 2. Localization of AR-IR. A, Colocalization of FSH-ß and AR IR in monkey and rat primary pituitary cultures. Anterior pituitary cells from adult male monkeys or 7-wk male rats were maintained in DMEM with 10% DCC-FCS and treated with vehicle or 20 nM T for 24 h. Representative pituitary cells that were double-labeled for FSH-ß and AR IR are shown. FSH-ß-positive cells stained blue with SG, and AR was stained brown with DAB. B, Localization of AR IR in monkey pituitary fibroblasts and rat genital skin fibroblasts. Cells were maintained in DMEM with 10% DCC-FCS and treated with vehicle or 20 nM T for 24 h.1v1f7sp, 百拇医药
AR mRNA expression in primary pituitary cultures and gonadotroph cell lines1v1f7sp, 百拇医药
To investigate whether pituitary AR gene regulation by androgens differs in monkeys and rats, AR mRNA concentrations were examined in monkey and rat primary pituitary cultures perifused with pulses of GnRH, as well as in monolayer cultures of T3-1 cells and LßT2 cells. In GnRH-stimulated primary pituitary cultures, the level of AR mRNA was unaffected by 10 nM T treatment for 56 h both in the monkey (109 ± 14% of control; P > 0.05) and in the rat (107 ± 13% of control; P > 0.05). Similarly, in the mouse gonadotroph cell lines, the level of AR mRNA was unaffected by 10-nM androgen treatment for 72 h (forT3-1 cells, 90 ± 6% of control with T and 88 ± 8% of control with DHT, P > 0.05; and for LßT2 cells, 100 ± 14% of control with T and 99 ± 11% of control with DHT, P > 0.05).
Single-cell RT-PCR to identify AR mRNA in gonadotrophs!v(4m-, 百拇医药
AR mRNA was also identified in monkey gonadotrophs using single-cell RT-PCR techniques. Of 138 single pituitary cells analyzed by RT-PCR, 7 cells (5.1%) were positive for LH-ß mRNA (L1–L7; Fig. 3). Coexpression of LH-ß and AR was observed clearly in four of these seven cells (57%). Cells L1 and L4 produced a smear-like diffuse pattern, and L6 was faintly positive. Thus, all LH-ß mRNA-positive cells may have been AR positive. Because rat gonadotrophs may coexpress GH (19), we sought to determine whether gonadotrophs that are AR-positive also express GH. Of the seven cells positive for LH-ß mRNA, five cells were also positive for GH mRNA. AR expression was observed in LH-ß-positive cells that were either GH positive or GH negative. PCR products from nearly 50 cells (P1) and total RNA from a primary culture of monkey pituitary cells (P2) represent positive controls. PCR products without RT reaction from 50 cells (N1) or an equivalent volume of cellular pipette-spent culture medium (N2) were negative controls.
fig.ommitteed4#, 百拇医药
Figure 3. RT-PCR products from single monkey pituitary cells. A total of 138 single cells were subjected to RT and general PCR amplification, followed by specific LH-ß PCR. Amplified fragments were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. Of 138 pituitary cells, 7 were positive for LH-ß (L1–L7). Specific PCR for GH and AR is also shown. P1, Fifty monkey pituitary cells as a positive control; P2, total RNA from monkey pituitary as a positive control; N1, 50 cells without RT as a negative control; N2, an equivalent volume of spent culture media as a negative control.4#, 百拇医药
Discussion4#, 百拇医药
Top4#, 百拇医药
Abstract4#, 百拇医药
Introduction4#, 百拇医药
Materials and Methods4#, 百拇医药
Results4#, 百拇医药
Discussion4#, 百拇医药
References4#, 百拇医药
The experiments described in this report were designed to determine whether the inability of androgens to negatively regulate gonadotrophs in higher primates may be explained by a disturbance in AR expression or shuttling. Previous studies have identified AR binding (20), mRNA (21), and protein (22) in the mixed cellular population of the anterior pituitary of adult male monkeys. There is also one study (23) using human pituitaries in which most FSH cells and LH cells, as well as some GH-positive cells, were noted to be immunoreactive for AR.
The proportion of primate gonadotrophs that express LH-ß and/or FSH-ß protein had not been reported previously and was determined at the start of these experiments. In primary pituitary cultures within 48 h of death, 80% of gonadotrophs were found to express both LH-ß and FSH-ß, and 20% contained LH-ß or FSH-ß solely. These proportions are similar to results found in cultured rat pituitary cells (24). The cellular localization of LH and FSH has also been determined in other species. In the human (25) and the frog (26), approximately two thirds of gonadotrophs are bihormonal, whereas in ewes (27), pigs (28) and lizards (29) all gonadotrophs appear to contain both LH and FSH. On the other hand, in some species, such as the chicken (30) and bovine (31), LH and FSH reside almost exclusively in separate populations of gonadotrophs. Although the significance of monohormonal gonadotrophs is unknown, the percentage of mono- and bihormonal gonadotrophs in the female rat varies with the reproductive cycle (32, 33, 34). Factors known to regulate FSH-ß and LH-ß gene expression differentially include GnRH pulse frequency (35), the activin-follistatin-inhibin system (36), pituitary adenylate cyclase-activating polypeptide (37), androgens (38), and glucocorticoids (39). Each of these factors could influence selectively the subpopulations of monkey gonadotrophs identified in this study. In addition, structural variants of GnRH appear to act through unique receptors (40) that could preferentially regulate monohormonal gonadotrophs.
We next studied AR gene expression and protein distribution in gonadotrophs and began by using the mouse gonadotroph-derived cell linesT3-1 and LßT2. Both cell lines stained for AR protein, and the intensity of nuclear AR IR increased with T treatment, whereas AR mRNA expression was not regulated by androgens under the experimental conditions of this study. Nuclear AR IR was also increased by T treatment in normal monkey and rat fibroblasts. Using green fluorescent protein ligated to the AR, Tyagi et al. (41) localized green fluorescent protein-AR primarily to the cytoplasm in cells grown in serum-free media, but after androgen treatment, AR overexpressed in PC3, HeLa, or COS1 cells moved rapidly to the nuclear compartment. Moreover, upon androgen withdrawal, the labeled AR migrated back to the cytoplasmic compartment and maintained its ability to reenter the nucleus on subsequent exposure to androgen. Our results are consistent with those findings. AR mRNA was recently identified by RT-PCR in LßT2 cells in which androgens activate the mouse mammary tumor virus promoter (42). We further document AR protein expression and shuttling from the cytoplasm to nucleus in LßT2 cells as well as in T3-1 cells treated with androgens.
We extended these methods to primary pituitary cultures using FSH-ß immunostaining to identify gonadotrophs. AR IR was observed in monkey as in rat gonadotrophs, and nuclear AR IR was similarly intensified during T treatment of cells from both species. These observations imply that AR shuttling from the cytoplasm to the nuclear compartment is comparable in gonadotrophs in primates and rats. We also found that T treatment of pituitary cultures stimulated hourly with pulses of GnRH did not affect AR mRNA expression in either species. Similarly, neither castration nor castration together with T replacement affected AR mRNA levels in anterior pituitary glands from adult male rhesus monkeys (43) or rats (44), implying that AR mRNA in the monkey and rat pituitary is not regulated transcriptionally by androgens.yo?a, http://www.100md.com
Single-cell RT-PCR was performed to examine cell-specific gene expression. Coexpression of LH-ß and AR mRNA in single monkey pituitary cells was documented, proving that monkey gonadotrophs express AR mRNA as well as AR protein. We also showed that five of seven of LH-ß-positive cells coexpressed GH mRNA. This finding is noteworthy because there is a growing awareness of the physiological importance of GH in reproductive function and its influence on the hypothalamic-pituitary-gonadal axis (45). GH mRNA was found in 37% of LHß mRNA-positive pituitary cells from female rats using single-cell RT-PCR techniques (46), and 50–55% of cells positive for LH-ß or FSH-ß antigen also expressed GH mRNA in the male rat pituitary using cytochemical in situ hybridization (19). Moreover, GnRH-R and GH have been colocalized in normal rat (47, 48, 49) and human (50) pituitary cells. Our finding of GH mRNA in monkey gonadotrophs indicates that these are multihormonal cells. Although the functional significance of these cells in the primate pituitary is unknown, our data suggest that there is no requirement for GH mRNA expression in order for LH-ß mRNA-positive gonadotrophs to express AR.
The mechanism by which androgens inhibit LH secretion from rat gonadotrophs directly is only partly understood. Basal secretion of stored LH was suppressed by androgens in female rat pituitary cultures (51), although our earlier study in male rat gonadotrophs did not demonstrate this effect (52). Androgens consistently suppress GnRH-stimulated LH secretion in vitro with a lag of 6–12 h (14, 53), suggesting a requirement for stimulation or suppression of protein synthesis. Whether androgens affect GnRH receptors directly has been controversial (12, 54). Inasmuch as LH release induced by phorbol ester-activated protein kinase C is also suppressed by T (55), the androgenic effect is partly distal to the GnRH receptor. Moreover, T treatment attenuated potassium-induced LH secretion without concomitant suppression of intracellular calcium levels, suggesting an androgenic effect beyond calcium elevation (51). T for 48 h decreased [35S]-methionine incorporation into newly synthesized-subunit and LH-ß proteins in primary pituitary cultures from castrated male rats (53), and -subunit as well as LH-ß mRNA levels are suppressed in T-treated rat pituitary cultures (38). Experiments in which the proximal promoter of the human -subunit gene was transfected into T3-1 cells (56, 57, 58), or the LH-ß gene was inserted into LßT2 cells (59), suggest that DHT can directly suppress -subunit and LH-ß transcription. However, these results must be interpreted cautiously in that AR overexpression was needed to demonstrate transcriptional inhibition by DHT. AR suppression of LH-ß basal transcription appears to involve the interaction with steroidogenic factor-1 (59), whereas interaction of AR with specificity protein-1 was shown to mediate suppression of GnRH-stimulated LH-ß transcription (60). AR suppression of -subunit basal transcription has been reported to involve binding to c-Jun and activation transcription factor 2 (58). Other transcriptional factors such as activated protein-1 (61), nuclear factor-
B (RelA; Ref. 62), and SMAD3 (63) can block transcriptional activation by the AR. More understanding of the interaction between AR and each of these cofactors is needed to unravel the differences between primate and rat gonadotrophs.
In summary, the concept of monohormonal and bihormonal expression of the gonadotropin subunit genes, already well established for rats and other species, is applicable to the adult male primate. Monkey gonadotrophs express both AR mRNA and AR protein, and most monkey gonadotrophs were AR positive with nuclear localization in the presence of T. Therefore, we conclude that the failure of T to negatively regulate LH secretion and -subunit mRNA expression in the adult male primate pituitary is not due a disturbance in AR expression or nuclear shuttling. Instead, coactivator proteins that are required for AR-mediated repression of gene transcription may be expressed in rodent but not in monkey gonadotrophs, or may fail to bind to AR in this species. Experiments to pursue these hypotheses are underway.kz44!, 百拇医药
Acknowledgmentskz44!, 百拇医药
The authors acknowledge the expert technical assistance provided by Mr. Dushan Ghooray and Mr. Alan Icard. We also thank Dr. Tony M. Plant for providing monkey pituitary glands for the initial phase of this project.
Abbreviations: AR, Androgen receptor; CS, calf serum; DAB, diaminobenzidine; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; DCC, dextran-charcoal; DHT, dihydrotestosterone; dNTP, deoxynucleotide triphosphate; DPBS, Dulbecco’s PBS; dT, deoxythymidine; FCS, fetal CS; GAPDH, glyceraldehyde phosphate dehydrogenase; HBSS, Hanks’ balanced salt solution; IR, immunoreactivity; RNase, ribonuclease; RT, reverse transcriptase; T, testosterone; TdT, terminal deoxynucleotidyl transferase.c+g!5!, 百拇医药
Received July 31, 2002.c+g!5!, 百拇医药
Accepted for publication October 8, 2002.c+g!5!, 百拇医药
Referencesc+g!5!, 百拇医药
Topc+g!5!, 百拇医药
Abstractc+g!5!, 百拇医药
Introductionc+g!5!, 百拇医药
Materials and Methodsc+g!5!, 百拇医药
Resultsc+g!5!, 百拇医药
Discussionc+g!5!, 百拇医药
Referencesc+g!5!, 百拇医药
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Abstract+, http://www.100md.com
Top+, http://www.100md.com
Abstract+, http://www.100md.com
Introduction+, http://www.100md.com
Materials and Methods+, http://www.100md.com
Results+, http://www.100md.com
Discussion+, http://www.100md.com
References+, http://www.100md.com
There is substantial evidence demonstrating that the principal feedback action of androgens to decrease LH secretion in male primates, including man, is to slow the GnRH pulse generator, whereas in male rats androgens not only decrease GnRH but also suppress LH synthesis and secretion through a direct pituitary effect. Previous experiments in our laboratory revealed that testosterone (T) suppresses LH secretion and decreases
-subunit mRNA levels in male rat pituitary cell cultures perifused with pulses of GnRH but not in pituitary cells from adult male monkeys. In the present study, we sought to determine whether the lack of responsiveness of gonadotrophs to androgens in the primate is androgen receptor (AR) related. Primary cultures were prepared from the anterior pituitary glands of adult male monkeys and rats. Cells were identified as gonadotrophs if they were immunoreactive for LH-ß or FSH-ß. Of these cells in the monkey, 80% contained both gonadotropins, 17% contained only LH-ß, and 3% contained only FSH-ß. AR immunoreactivity (IR) was nuclear in 22% and 15%, respectively, of monkey and rat FSH-ß-positive cells in the absence of T. Following T treatment, nuclear AR IR was identified in 79% of monkey and 81% of rat gonadotrophs. T treatment similarly intensified AR IR in mouse gonadotroph T3-1 and LßT2 cells and in monkey and rat fibroblasts. Single-cell RT-PCR confirmed coexpression of LH-ß and AR mRNA as well as LH-ß and GH mRNA in monkey gonadotrophs. Our data reveal that most monkey, as well as rat, gonadotrophs are AR-positive with nuclear localization in the presence of T. GH expression is not required for AR expression in gonadotrophs. We conclude that the failure of T to inhibit LH secretion and decrease -subunit mRNA expression in the male primate is not due a disturbance in AR nuclear shuttling.Introduction5i}}&r, 百拇医药
Top5i}}&r, 百拇医药
Abstract5i}}&r, 百拇医药
Introduction5i}}&r, 百拇医药
Materials and Methods5i}}&r, 百拇医药
Results5i}}&r, 百拇医药
Discussion5i}}&r, 百拇医药
References5i}}&r, 百拇医药
ANDROGENS RESTRAIN GONADOTROPIN secretion in adult male primates, including men, by slowing the GnRH pulse generator (1, 2, 3) but appear not to influence gonadotrophs directly. This idea is supported by experiments in rhesus monkeys rendered gonadotropin deficient with radio-frequency lesions and stimulated with pulses of GnRH; in these monkeys, LH secretion increased little after bilateral orchidectomy until GnRH pulse frequency was increased (4). In clamp experiments in men with congenital hypogonadotropic hypogonadism treated with pulsatile GnRH, suppression of LH secretion by testosterone (T) was blocked by the aromatase inhibitor testolactone, suggesting that inhibition by T was through bioconversion to estradiol (5), whereas the nonaromatizable androgen dihydrotestosterone (DHT) had no effect in this human model (6). Furthermore, in normal men, infusion of DHT for 4 d suppressed LH pulse frequency but increased, rather than decreased, the LH response to exogenous GnRH stimulation (7). In adult male rats, on the other hand, androgen-negative feedback appears to be partly GnRH mediated (8, 9) but is also through direct inhibition of GnRH-stimulated LH release (10, 11).
Cultured pituitary cells are a useful model in the effort to understand the species-specific cellular mechanisms regulating gonadotropin secretion and subunit gene expression. We have compared the effects of T on LH synthesis and secretion using dispersed pituitary cells from male monkeys and rats that were perifused with pulses of GnRH (12). In this model, T down-regulated GnRH-stimulated
-subunit mRNA levels and suppressed LH secretion by rat pituitary cells but not by pituitary cultures from monkeys. DHT also failed to suppress GnRH-induced LH secretion, suggesting that the absence of a direct androgenic-negative feedback effect in the primate was not due to T metabolism. The explanation and significance for this species difference in the pituitary action of androgens are unknown.'y?@&, http://www.100md.comThe first step in androgen action is binding of androgen to its receptor, followed by shuttling of the activated androgen receptor (AR) to the cell nucleus, where it stimulates or represses gene transcription (13). To explore the hypothesis that a disturbance in AR shuttling explains the lack of responsiveness of primate gonadotrophs to androgens, we first determined whether primate gonadotrophs are monohormonal or bihormonal by localizing FSH-ß and LH-ß protein to gonadotrophs using double-labeled immunofluorescence staining. Second, we used double-labeled immunoperoxidase staining to localize AR in gonadotrophs in pituitary cell cultures from adult male monkeys and rats in the presence or absence of T. And, third, we studied LH-ß and AR mRNA coexpression in monkey pituitary cultures using single-cell RT-PCR techniques.
Materials and Methods|95w}qx, 百拇医药
Top|95w}qx, 百拇医药
Abstract|95w}qx, 百拇医药
Introduction|95w}qx, 百拇医药
Materials and Methods|95w}qx, 百拇医药
Results|95w}qx, 百拇医药
Discussion|95w}qx, 百拇医药
References|95w}qx, 百拇医药
Reagents|95w}qx, 百拇医药
DMEM, Dulbecco’s PBS (DPBS; without CaCl2 and MgCl2), Hanks’ balanced salt solution (HBSS), HEPES, penicillin G and streptomycin sulfate, fetal calf serum (FCS), calf serum (CS), BSA fraction V, pancreatin, oligo[deoxythymidine (dT)] primer, reverse transcriptase (RT) kit and SuperScript II ribonuclease (RNase) H-RT, terminal deoxynucleotidyl transferase (TdT) recombinant, 100 mM deoxynucleotide triphosphate (dNTP), and ultra-pure distilled water (H2O) were purchased from Invitrogen (Carlsbad, CA). FCS and CS were treated with dextran-charcoal (DCC) to remove steroids. Deoxyribonuclease I, poly-L-lysine, trypsin, EDTA, diaminobenzidine (DAB), diethyl pyrocarbonate, T, and DHT were purchased from Sigma (St. Louis, MO); fluconizole from Pfizer, Inc. (New York, NY); collagenase from Roche Molecular Biochemicals (Mannheim, Germany); a 100-bp DNA ladder from New England Biolabs, Inc. (Beverly, MA); cyanine 2-conjugated goat antirabbit IgG and cyanine 3-conjugated goat antiguinea pig IgG from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA); 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) from MolecularProbes (Eugene, OR); avidin-biotin-peroxidase complex, biotinylated antirabbit IgG, and SG substrate kit from Vector Laboratories (Burlingame, CA); prime RNase inhibitor from Fisher Scientific (Pittsburgh, PA); RNA guard from Amersham Biosciences (Piscataway, NJ); and Taq DNA polymerase and 10 mM dNTP mix from Promega Corp. (Madison, WI).
Preparation of anterior pituitary cells and cell cultures2rt%:), http://www.100md.com
Freshly removed anterior pituitary glands from adult male rhesus monkeys (Macaca mulatta) were obtained from Covance Laboratories, Inc. Research Primates (Alice, TX) and shipped on ice in HBSS containing 44 mM HEPES. Rat pituitary cells were prepared from 7-wk-old male Sprague Dawley rats (Harlan, Indianapolis, IN). All media contained 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, and 2 mg/ml fluconizole. The methods used for the preparation of pituitary cell cultures were similar to those described previously (14). Briefly, anterior pituitaries were minced and treated for 60 min with 0.33% collagenase and 0.003% deoxyribonuclease in HBSS (pH 7.3) containing 0.4% BSA fraction V, 0.2% sucrose, and 44 mM HEPES. Cells were then treated with 0.25% pancreatin in HBSS for 8 min and washed three times with DMEM containing 5% DCC-FCS and 5% DCC-CS. Dispersed cells were cultured in DMEM with 10% DCC-FCS on poly-L-lysine-coated glass chamber slides at a density of 1 x 105 cells per chamber for immunocytochemistry, or on 60-mm tissue culture dishes at a density of 1 x 104 cells per dish for single-cell RT-PCR analysis. After 24 h of preculture, media in the chamber slides were changed to include 20 nM T or vehicle for 24 h. Cells were then fixed with 4% paraformaldehyde and stored at -20 C. Mouse gonadotroph cell lines
T3-1 and LßT2 (kindly provided by Dr. Pamela Mellon, University of California-San Diego, La Jolla, CA) were plated on chamber slides, treated with 20 nM T or vehicle for 24 h, and then fixed for immunocytochemistry. For RNA extraction, T3-1 and LßT2 cells were plated in DMEM with 10% DCC-FCS in six-well plates at a density of 1.2 x 106 cells per well. After 24 h of preculture, cells were treated with 10 nM T, 10 nM DHT, or vehicle for 72 h and were then harvested. Monkey pituitary fibroblast and rat genital skin fibroblast cultures were produced as previously reported (15). Cells were plated in DMEM with 10% DCC-FCS on chamber slides and treated with 20 nM T or vehicle for 24 h and then fixed for immunocytochemistry.Double-labeled immunofluorescence stainingm(#q2dd, http://www.100md.com
Anti-FSH-ß (rabbit antibody; batch 5, National Hormone and Pituitary Program, Torrance, CA) at 1:10,000, and antimonkey LH-ß (guinea pig antibody; AFP555194, National Hormone and Pituitary Program) at 1:20,000 were incubated overnight at 4 C. In the first reaction, the primary rabbit (FSH-ß) antiserum was localized using 1:200 cyanine 2-conjugated goat antirabbit IgG, and in the second reaction, the primary guinea pig anti-LH-ß serum was localized with 1:400 cyanine 3-conjugated goat antiguinea pig IgG (each for 1 h at 23 C). Nuclear counterstaining was performed with DAPI diluted 1:3000 in PBS for 5 min at 23 C. Appropriate filters were used to observe the green fluorescence of cyanine 2-labeled IgG and the red fluorescence of cyanine 3-labeled IgG. A total of 763 FSH-ß- or LH-ß-positive cells were counted from three separate monkey preparations. The percentage of double-labeled cells was calculated and expressed as mean ± SEM.
Single- and double-labeled immunoperoxidase staining:]z\/7c, http://www.100md.com
A rabbit polyclonal antibody to the AR (AR N-20 sc-816, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used at 1:200. Slides were incubated with antibodies overnight at 4 C. Staining was accomplished using biotinylated antirabbit IgG and the avidin-biotin-peroxidase complex. For monkey pituitary fibroblasts, rat genital skin fibroblasts, and LßT2 and
T3-1 cells, AR was stained with DAB, and a brown color was developed. For primary pituitary cultures, AR was stained with DAB in the first reaction. Then FSH-ß was stained as described above using antiserum batch 5 at 1:5000. A blue color was developed using the SG substrate kit.:]z\/7c, http://www.100md.comA total of 1180 and 954 FSH-ß-positive cells from three different cell preparations from monkeys and rats, respectively, were analyzed. The percentage of nuclear AR-positive cells was calculated and expressed as the mean ± SEM. No staining was observed when the first antibodies were omitted.
mRNA analysis by RT-PCR:*p3, http://www.100md.com
RNA was extracted by the guanidinium thiocyanate-phenol-chloroform procedure (16). The concentration of total RNA was determined by reading the OD at 260 nm. Reverse transcription of total pituitary RNA was performed from control and T-treated pituitary cell cultures from monkeys and rats that were stimulated with hourly pulses of GnRH (12). First-strand cDNA was synthesized with a RT kit using 1 µg of total RNA. The cDNAs, equivalent to 30 ng of RNA, were amplified in a 20-µl PCR containing Taq DNA polymerase, 1.5 mM MgCl2, and each primer. Primers used in the PCR were as follows: for monkey AR (GenBank accession no. U94179): forward, 5'-GACTCCGTGCAGCCTATTG-3'; reverse, 5'-GGGCACTTGCACAGAGATG-3'; product size 123 bp; for monkey glyceraldehyde phosphate dehydrogenase (GAPDH) (NM002046): forward, 5'-TCAACAGCGACACCCACTC-3'; reverse, 5'-CTTCCTCTTGTGCTCTTGCTG-3'; product size 201 bp; Ref. 17 ; for rat AR (NM012502): forward, 5'-GGATTCTGTGCAGCCTATTG-3'; reverse, 5'-GGGCACTTGCACAGAGATG-3'; product size 124 bp; for mouse AR (NM013476): forward, 5'-CAGCATTATTCCAGTGGATGG-3'; reverse, 5'-GGGCACTTGCACAGAGATG-3'; product size 274 bp; and mouse and rat GAPDH (AF106860, M32559): forward, 5'-GGCATTGCTCTCAATGACAA-3'; reverse, 5'-TGTGAGGGAGATGCTCAGTG-3'; product size 223 bp. Amplification was conducted for 24 cycles for monkey GAPDH, 20 cycles for rat GAPDH, 22 cycles for mouse GAPDH, and 35 cycles for monkey, rat, and mouse AR (94 C for 30 sec, 58 C for 75 sec, and 72 C for 90 sec). Each PCR product was separated on a 1.8% agarose gel in Tris-borate EDTA buffer and visualized by ethidium-bromide staining, digitized with a GS-700 Imaging Densitometer (Bio-Rad Laboratories, Inc., Hercules, CA), and analyzed using Molecular Analysis software (Bio-Rad Laboratories, Inc.). In preliminary experiments, a linear range of amplification was established by varying the number of PCR cycles. The amount of AR mRNA product was normalized to that of GAPDH in each sample.
Single-cell RT-PCR8, 百拇医药
Single-cell RT-PCR was based on previously published protocols (18). Primary pituitary cultures were dispersed in 60-mm tissue culture plates at a density of 1 x 104 cells per dish. Twenty-four hours after dispersal, cells were washed five times with DPBS (without Mg and Ca), and 1.5 ml of DPBS was added. Cells were observed under an inverted microscope. Single cells were isolated with a micromanipulator fitted with a pulled microcapillary. A single cell was expelled into a PCR tube filled with a reaction mixture containing 4.5 µl of lysis buffer [for 100 µl mix of 20 µl of 5x RT buffer, 76 µl of H2O, 0.5 µl of IGEPAL, 1 µl of prime RNase inhibitor, 1 µl of RNA guard, and 2 µl of a freshly made 1/24 dilution of the stock primer mix (10 µl of 100 mM each dNTP, 20 µl of 820 µg/ml phosphorylated oligo(dT)22, and 10 µl of H2O)]. The mixture was incubated at 65 C for 2 min to lyse the cells, placed on ice for 1 min, and incubated at 37 C for 2 min to anneal the primers. The reaction was initiated by adding 1 µl of RT enzyme at 37 C for 50 min, followed by 65 C for 10 min. Homopolymer deoxy-ATP was added to the 3' end of the first-strand cDNA using TdT enzyme in 4.5 µl of stock tailing buffer (100 µl of 5x TdT buffer, 3.75 µl of 100 mM deoxy-ATP, 146.25 µl of H2O) at 37 C for 15 min, followed by 65 C for 10 min, and placed on ice until PCR. PCR mix containing 10 µl of 10x PCR buffer, 10 µl of 25 mM MgCl2, 2 µl of 10 mM dNTP mix, 4 µl of 0.73 µg/µl oligo(dT)-X primer [5'-ATGTCGTCCAGGCCGCTCTGGACAAAATATGAATTC(T)24-3'], 2 µl of Taq DNA polymerase, and 62 µl of H2O was mixed on ice. PCR mix (90 µl) was added to each PCR tube with 10 µl of the template and then placed in a PTC-200 DNA Engine Thermal Cycler (MJ Research, Inc., Line Village, NV) for 25 cycles (94 C for 1 min, 42 C for 2 min, 72 C for 6 min with a 10-sec time extension at each cycle). After the first 25 cycles, 1 µl of Taq polymerase was added to each tube, and 25 additional cycles were performed with the same program but without the 10-sec extension at each cycle. After general amplification of cDNA, specific PCR was carried out using 1.5 µl of the general amplified PCR product as a template for 50 cycles in a 20-µl PCR containing 1 U Taq DNA polymerase, 1.0 mM (for AR) or 1.5 mM (for LH and GH) MgCl2, 0.15 mM of dNTP, and 0.2 µM of each primer. PCR primers (for LH-ß: forward, 5'-GTGTGCATCACCGTCAACAC-3'; reverse, 5'-CCACAGCGACAGCTGAGAG-3'; product size 200 bp; and for GH: forward, 5'-AGGCATCCAAACACTGATGG-3'; reverse, 5'-CAATGCGCAGGAATGTCTC-3'; product size 301 bp) were designed based on the published sequences of the monkey LH-ß (XM009418) and GH (L16556). A nested PCR was conducted for AR (outer forward, 5'-CAGCATTATTCCAGTGGATGG-3'; inner forward, 5'-GACTCCGTGCAGCCTATTG-3'; reverse, 5'-GGGCACTTGCACAGAGATG-3'; product sizes 274 bp and 123 bp, respectively). All primer pairs used span at least one intron so that the PCR products could be distinguished by size from contaminating genomic DNA. Total monkey pituitary cDNA was used as a positive control. After a prerun of 95 C for 8 min, specific amplifications were conducted for 50 cycles at 95 C for 30 sec; 50 C (for AR), 58 C (for GH), or 62 C (for LH-ß) for 90 sec; 72 C for 90 sec; and 72 C for 7 min post run. The nested PCR for an additional 40 cycles was performed for AR amplification using 1.5 µl of the first PCR products as template under the same reaction conditions, except the final concentration of MgCl2 was 2.0 mM. To minimize the chance of cross-contamination between reagents or individual PCR amplifications, the following controls were performed in each experiment. First, cells were dispersed in the culture dishes at a relatively low density. Second, for each cell harvested, a new pipette was used. Third, the pipette was held for a second in the close vicinity of a cell, and media without a cell were ejected into a test tube and subjected to RT-PCR as a negative control. Fourth, approximately 50 cells, which were amplified without the RT reaction, were used as a negative control.
Data analysis and presentationh, http://www.100md.com
The percentage of double-labeled cells was calculated and expressed as the mean ± SEM. Two group comparisons were performed with the Student’s t test. Statistical significance was inferred at P < 0.05.h, http://www.100md.com
Resultsh, http://www.100md.com
Toph, http://www.100md.com
Abstracth, http://www.100md.com
Introductionh, http://www.100md.com
Materials and Methodsh, http://www.100md.com
Resultsh, http://www.100md.com
Discussionh, http://www.100md.com
Referencesh, http://www.100md.com
Double-labeled immunofluorescence to colocalize LH-ß and FSH-ß in gonadotrophsh, http://www.100md.com
Gonadotrophs from three separate monkey pituitary preparations were identified by immunofluorescence for LH-ß and/or FSH-ß content. Figure 1 illustrates representative monkey pituitary cells stained by double-labeled immunofluorescence. Phase contrast and nuclear staining with DAPI, which is stained blue, was used to identify all pituitary cells in the microscopic field. FSH-ß protein is stained green, and LH-ß protein is stained red. At high magnification (x400), both FSH-ß and LH-ß protein were found in the cell cytoplasm but were absent from nuclei. In the overlay image (FSH-ß + LH-ß), most cells were positive for both FSH-ß and LH-ß (yellow). At lower magnification (x200), some cells contained only FSH-ß or LH-ß, implying that monohormonal as well as bihormonal gonadotrophs are present in the monkey pituitary. Of 763 cells studied from three separate cell preparations, 80% ± 3% were positive for both LH-ß and FSH-ß, 17% ± 3% contained only LH-ß, and 3% ± 1% contained only FSH-ß.
fig.ommitteed*^e, 百拇医药
Figure 1. Colocalization of FSH-ß and LH-ß protein in monkey primary pituitary cultures. Anterior pituitary cells from adult male monkeys in chamber slides were fixed and subjected to double-labeled immunofluorescence for FSH-ß and LH-ß. The first column shows a phase contrast image. The second column shows nuclear staining with DAPI (blue). The third column shows FSH-ß staining (green), and the fourth column shows LH-ß staining (red). Combined image (FSH-ß + LH-ß) in the fifth column shows the coexpression of FSH-ß and LH-ß (yellow). The arrowhead points to the cell labeled only for FSH-ß, and the arrow points to the cell labeled only for LH-ß.*^e, 百拇医药
Double-labeled immunoperoxidase staining to detect AR in FSHß(+) cells in primary pituitary cultures*^e, 百拇医药
To investigate the effects of androgens on AR distribution in gonadotrophs, we first identified AR in two mouse gonadotroph cell lines,
T3-1 and LßT2. AR immunoreactivity (IR) was uniformly recognized both in the cytoplasm and nucleus in both cell lines grown in media containing 10% DCC-FCS in the absence of T. When 20 nM T was added to the culture media, AR IR in the nucleus was intensified (not shown). We then extended the immunostaining method to primary pituitary cultures from adult male rats and monkeys. Multiple fields were investigated with a range of 256–653 gonadotrophs in each monkey pituitary culture and 263–397 gonadotrophs in each rat pituitary culture. Representative pituitary cells that were double-labeled for FSH-ß and AR protein are shown in Fig. 2A. FSH-ß-positive cells stained blue, and AR was stained brown. In the absence of T, AR IR was present in both the cytoplasm and nucleus of monkey as well as rat FSH-ß-positive cells. Adding T to the culture media strongly intensified the nuclear AR IR, which was homogeneous in appearance in both rat and monkey gonadotrophs. In the absence of T, AR IR was nuclear in 22 ± 3% and 15 ± 4% of monkey and rat FSH-ß-positive cells, respectively. With T treatment, nuclear AR IR was identified in 79 ± 5% and 81 ± 6% of monkey and rat FSH-ß-positive cells, respectively. Likewise, in monkey and rat fibroblasts, AR IR was present in both the cytoplasm and nucleus, and nuclear AR IR was intensified with T treatment (Fig. 2B).fig.ommitteed1v1f7sp, 百拇医药
Figure 2. Localization of AR-IR. A, Colocalization of FSH-ß and AR IR in monkey and rat primary pituitary cultures. Anterior pituitary cells from adult male monkeys or 7-wk male rats were maintained in DMEM with 10% DCC-FCS and treated with vehicle or 20 nM T for 24 h. Representative pituitary cells that were double-labeled for FSH-ß and AR IR are shown. FSH-ß-positive cells stained blue with SG, and AR was stained brown with DAB. B, Localization of AR IR in monkey pituitary fibroblasts and rat genital skin fibroblasts. Cells were maintained in DMEM with 10% DCC-FCS and treated with vehicle or 20 nM T for 24 h.1v1f7sp, 百拇医药
AR mRNA expression in primary pituitary cultures and gonadotroph cell lines1v1f7sp, 百拇医药
To investigate whether pituitary AR gene regulation by androgens differs in monkeys and rats, AR mRNA concentrations were examined in monkey and rat primary pituitary cultures perifused with pulses of GnRH, as well as in monolayer cultures of T3-1 cells and LßT2 cells. In GnRH-stimulated primary pituitary cultures, the level of AR mRNA was unaffected by 10 nM T treatment for 56 h both in the monkey (109 ± 14% of control; P > 0.05) and in the rat (107 ± 13% of control; P > 0.05). Similarly, in the mouse gonadotroph cell lines, the level of AR mRNA was unaffected by 10-nM androgen treatment for 72 h (for
T3-1 cells, 90 ± 6% of control with T and 88 ± 8% of control with DHT, P > 0.05; and for LßT2 cells, 100 ± 14% of control with T and 99 ± 11% of control with DHT, P > 0.05).Single-cell RT-PCR to identify AR mRNA in gonadotrophs!v(4m-, 百拇医药
AR mRNA was also identified in monkey gonadotrophs using single-cell RT-PCR techniques. Of 138 single pituitary cells analyzed by RT-PCR, 7 cells (5.1%) were positive for LH-ß mRNA (L1–L7; Fig. 3). Coexpression of LH-ß and AR was observed clearly in four of these seven cells (57%). Cells L1 and L4 produced a smear-like diffuse pattern, and L6 was faintly positive. Thus, all LH-ß mRNA-positive cells may have been AR positive. Because rat gonadotrophs may coexpress GH (19), we sought to determine whether gonadotrophs that are AR-positive also express GH. Of the seven cells positive for LH-ß mRNA, five cells were also positive for GH mRNA. AR expression was observed in LH-ß-positive cells that were either GH positive or GH negative. PCR products from nearly 50 cells (P1) and total RNA from a primary culture of monkey pituitary cells (P2) represent positive controls. PCR products without RT reaction from 50 cells (N1) or an equivalent volume of cellular pipette-spent culture medium (N2) were negative controls.
fig.ommitteed4#, 百拇医药
Figure 3. RT-PCR products from single monkey pituitary cells. A total of 138 single cells were subjected to RT and general PCR amplification, followed by specific LH-ß PCR. Amplified fragments were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. Of 138 pituitary cells, 7 were positive for LH-ß (L1–L7). Specific PCR for GH and AR is also shown. P1, Fifty monkey pituitary cells as a positive control; P2, total RNA from monkey pituitary as a positive control; N1, 50 cells without RT as a negative control; N2, an equivalent volume of spent culture media as a negative control.4#, 百拇医药
Discussion4#, 百拇医药
Top4#, 百拇医药
Abstract4#, 百拇医药
Introduction4#, 百拇医药
Materials and Methods4#, 百拇医药
Results4#, 百拇医药
Discussion4#, 百拇医药
References4#, 百拇医药
The experiments described in this report were designed to determine whether the inability of androgens to negatively regulate gonadotrophs in higher primates may be explained by a disturbance in AR expression or shuttling. Previous studies have identified AR binding (20), mRNA (21), and protein (22) in the mixed cellular population of the anterior pituitary of adult male monkeys. There is also one study (23) using human pituitaries in which most FSH cells and LH cells, as well as some GH-positive cells, were noted to be immunoreactive for AR.
The proportion of primate gonadotrophs that express LH-ß and/or FSH-ß protein had not been reported previously and was determined at the start of these experiments. In primary pituitary cultures within 48 h of death, 80% of gonadotrophs were found to express both LH-ß and FSH-ß, and 20% contained LH-ß or FSH-ß solely. These proportions are similar to results found in cultured rat pituitary cells (24). The cellular localization of LH and FSH has also been determined in other species. In the human (25) and the frog (26), approximately two thirds of gonadotrophs are bihormonal, whereas in ewes (27), pigs (28) and lizards (29) all gonadotrophs appear to contain both LH and FSH. On the other hand, in some species, such as the chicken (30) and bovine (31), LH and FSH reside almost exclusively in separate populations of gonadotrophs. Although the significance of monohormonal gonadotrophs is unknown, the percentage of mono- and bihormonal gonadotrophs in the female rat varies with the reproductive cycle (32, 33, 34). Factors known to regulate FSH-ß and LH-ß gene expression differentially include GnRH pulse frequency (35), the activin-follistatin-inhibin system (36), pituitary adenylate cyclase-activating polypeptide (37), androgens (38), and glucocorticoids (39). Each of these factors could influence selectively the subpopulations of monkey gonadotrophs identified in this study. In addition, structural variants of GnRH appear to act through unique receptors (40) that could preferentially regulate monohormonal gonadotrophs.
We next studied AR gene expression and protein distribution in gonadotrophs and began by using the mouse gonadotroph-derived cell lines
T3-1 and LßT2. Both cell lines stained for AR protein, and the intensity of nuclear AR IR increased with T treatment, whereas AR mRNA expression was not regulated by androgens under the experimental conditions of this study. Nuclear AR IR was also increased by T treatment in normal monkey and rat fibroblasts. Using green fluorescent protein ligated to the AR, Tyagi et al. (41) localized green fluorescent protein-AR primarily to the cytoplasm in cells grown in serum-free media, but after androgen treatment, AR overexpressed in PC3, HeLa, or COS1 cells moved rapidly to the nuclear compartment. Moreover, upon androgen withdrawal, the labeled AR migrated back to the cytoplasmic compartment and maintained its ability to reenter the nucleus on subsequent exposure to androgen. Our results are consistent with those findings. AR mRNA was recently identified by RT-PCR in LßT2 cells in which androgens activate the mouse mammary tumor virus promoter (42). We further document AR protein expression and shuttling from the cytoplasm to nucleus in LßT2 cells as well as in T3-1 cells treated with androgens.We extended these methods to primary pituitary cultures using FSH-ß immunostaining to identify gonadotrophs. AR IR was observed in monkey as in rat gonadotrophs, and nuclear AR IR was similarly intensified during T treatment of cells from both species. These observations imply that AR shuttling from the cytoplasm to the nuclear compartment is comparable in gonadotrophs in primates and rats. We also found that T treatment of pituitary cultures stimulated hourly with pulses of GnRH did not affect AR mRNA expression in either species. Similarly, neither castration nor castration together with T replacement affected AR mRNA levels in anterior pituitary glands from adult male rhesus monkeys (43) or rats (44), implying that AR mRNA in the monkey and rat pituitary is not regulated transcriptionally by androgens.yo?a, http://www.100md.com
Single-cell RT-PCR was performed to examine cell-specific gene expression. Coexpression of LH-ß and AR mRNA in single monkey pituitary cells was documented, proving that monkey gonadotrophs express AR mRNA as well as AR protein. We also showed that five of seven of LH-ß-positive cells coexpressed GH mRNA. This finding is noteworthy because there is a growing awareness of the physiological importance of GH in reproductive function and its influence on the hypothalamic-pituitary-gonadal axis (45). GH mRNA was found in 37% of LHß mRNA-positive pituitary cells from female rats using single-cell RT-PCR techniques (46), and 50–55% of cells positive for LH-ß or FSH-ß antigen also expressed GH mRNA in the male rat pituitary using cytochemical in situ hybridization (19). Moreover, GnRH-R and GH have been colocalized in normal rat (47, 48, 49) and human (50) pituitary cells. Our finding of GH mRNA in monkey gonadotrophs indicates that these are multihormonal cells. Although the functional significance of these cells in the primate pituitary is unknown, our data suggest that there is no requirement for GH mRNA expression in order for LH-ß mRNA-positive gonadotrophs to express AR.
The mechanism by which androgens inhibit LH secretion from rat gonadotrophs directly is only partly understood. Basal secretion of stored LH was suppressed by androgens in female rat pituitary cultures (51), although our earlier study in male rat gonadotrophs did not demonstrate this effect (52). Androgens consistently suppress GnRH-stimulated LH secretion in vitro with a lag of 6–12 h (14, 53), suggesting a requirement for stimulation or suppression of protein synthesis. Whether androgens affect GnRH receptors directly has been controversial (12, 54). Inasmuch as LH release induced by phorbol ester-activated protein kinase C is also suppressed by T (55), the androgenic effect is partly distal to the GnRH receptor. Moreover, T treatment attenuated potassium-induced LH secretion without concomitant suppression of intracellular calcium levels, suggesting an androgenic effect beyond calcium elevation (51). T for 48 h decreased [35S]-methionine incorporation into newly synthesized
-subunit and LH-ß proteins in primary pituitary cultures from castrated male rats (53), and -subunit as well as LH-ß mRNA levels are suppressed in T-treated rat pituitary cultures (38). Experiments in which the proximal promoter of the human -subunit gene was transfected into T3-1 cells (56, 57, 58), or the LH-ß gene was inserted into LßT2 cells (59), suggest that DHT can directly suppress -subunit and LH-ß transcription. However, these results must be interpreted cautiously in that AR overexpression was needed to demonstrate transcriptional inhibition by DHT. AR suppression of LH-ß basal transcription appears to involve the interaction with steroidogenic factor-1 (59), whereas interaction of AR with specificity protein-1 was shown to mediate suppression of GnRH-stimulated LH-ß transcription (60). AR suppression of -subunit basal transcription has been reported to involve binding to c-Jun and activation transcription factor 2 (58). Other transcriptional factors such as activated protein-1 (61), nuclear factor-B (RelA; Ref. 62), and SMAD3 (63) can block transcriptional activation by the AR. More understanding of the interaction between AR and each of these cofactors is needed to unravel the differences between primate and rat gonadotrophs.In summary, the concept of monohormonal and bihormonal expression of the gonadotropin subunit genes, already well established for rats and other species, is applicable to the adult male primate. Monkey gonadotrophs express both AR mRNA and AR protein, and most monkey gonadotrophs were AR positive with nuclear localization in the presence of T. Therefore, we conclude that the failure of T to negatively regulate LH secretion and -subunit mRNA expression in the adult male primate pituitary is not due a disturbance in AR expression or nuclear shuttling. Instead, coactivator proteins that are required for AR-mediated repression of gene transcription may be expressed in rodent but not in monkey gonadotrophs, or may fail to bind to AR in this species. Experiments to pursue these hypotheses are underway.kz44!, 百拇医药
Acknowledgmentskz44!, 百拇医药
The authors acknowledge the expert technical assistance provided by Mr. Dushan Ghooray and Mr. Alan Icard. We also thank Dr. Tony M. Plant for providing monkey pituitary glands for the initial phase of this project.
Abbreviations: AR, Androgen receptor; CS, calf serum; DAB, diaminobenzidine; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; DCC, dextran-charcoal; DHT, dihydrotestosterone; dNTP, deoxynucleotide triphosphate; DPBS, Dulbecco’s PBS; dT, deoxythymidine; FCS, fetal CS; GAPDH, glyceraldehyde phosphate dehydrogenase; HBSS, Hanks’ balanced salt solution; IR, immunoreactivity; RNase, ribonuclease; RT, reverse transcriptase; T, testosterone; TdT, terminal deoxynucleotidyl transferase.c+g!5!, 百拇医药
Received July 31, 2002.c+g!5!, 百拇医药
Accepted for publication October 8, 2002.c+g!5!, 百拇医药
Referencesc+g!5!, 百拇医药
Topc+g!5!, 百拇医药
Abstractc+g!5!, 百拇医药
Introductionc+g!5!, 百拇医药
Materials and Methodsc+g!5!, 百拇医药
Resultsc+g!5!, 百拇医药
Discussionc+g!5!, 百拇医药
Referencesc+g!5!, 百拇医药
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