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a Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Act and Inh are dimeric proteins sharing a common ß subunit, with antagonistic functions in the anterior pituitary, stimulating (Act) and inhibiting (Inh) the expression of the FSHß subunit gene and FSH secretion [3, 7, 8]. FS is a heterogeneously glycosylated cysteine-rich, single-chain protein that is structurally unrelated to Act and Inh. FS bioneutralizes Act by binding to the ß subunit, which in turn prevents its binding to the Act receptor, and thereby indirectly suppresses FSH synthesis and secretion [9–11].
The gonadal steroids, progesterone (P4) in the female and testosterone (T) in the male, and the adrenal steroid corticosterone (B), increase FSHß subunit mRNA levels and FSH release in vivo and in vitro [12–17]. In addition, a recent study from our laboratory demonstrated that the anti-progestin and anti-glucocorticoid RU-486 suppresses Act-induced FSH synthesis in vitro in the presence of estradiol (E2) in anterior pituitary cell culture from female rats [18]. This finding suggests that there is cross-talk between Act-induced Smad signaling and progesterone receptor (PR) activation. In the present study, we tested the hypothesis that Act mediates the effects of the steroids P4, B, and T on FSH secretion in anterior pituitary cell culture by taking advantage of the ability of FS to bind and neutralize Act. Furthermore, by examining the effects of these steroids on mRNA levels of FSHß, ActßB, and FS, we tested the hypothesis that P4 or B mimic the effects of Act in modulating an Act-Inh-FS paracrine-autocrine loop in the anterior pituitary gonadotroph to favor increased FSH secretion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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P4, B, T, and E2 (Sigma Chemical Co., St. Louis, MO) were dissolved in absolute ethanol at 1000 times the desired final concentration; the corresponding concentration of ethanol (0.1%) was used as the vehicle. Recombinant human Act A, provided by the National Hormone and Pituitary Program (NHPP; Rockville, MD), was diluted with 0.05 M Tris-HCl (pH 7.4)-0.15 M NaCl from a 730 µg/ml stock. Recombinant human FS, provided by the NHPP, was dissolved in H2O at a concentration of 100 µg/ml, and 50-µl (5 µg) aliquots were lyophilized for storage. On subsequent occasions, single aliquots were reconstituted in H2O; the process of lyophilization and reconstitution led to an appreciable loss of activity. Media, sera, and all other reagents for cell culture were obtained from Life Technologies (Grand Island, NY).
Animals
Female and male Sprague-Dawley rats (Crl: CD [SD] BR-CD; 55–60 days old) were obtained from Charles River (Portage, MI). Animals were housed under a 14L:10D schedule, with lights-on at 0500 h, and were provided with standard rat chow and tap water ad libitum. In some experiments, in which metestrous donor rats were used, estrous cycles were monitored by daily vaginal cytology; only rats that exhibited at least two consecutive 4-day estrous cycles were included. Protocols were approved by the animal care and use committee of Northwestern University (Evanston, IL) and animals were maintained in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory animals.
Cell Dissociation and Culture
Animals were killed by decapitation at 0900–1000 h on metestrus or at random stages of the estrous cycle. Anterior pituitaries were collected and enzymatically dissociated as previously described [19]. The final cell pellet was suspended in Dulbecco's modified Eagle's medium containing 10% charcoal-adsorbed fetal bovine serum and 10 nM E2 as indicated. Cells were plated in 24-well culture plates at a density of 3–5 x 105 cells/2-cm2 well in 1 ml medium for RIA or 12-well culture plates at a density of 1.5–2 x 106 cells/3.8-cm2 well in 2 ml medium for mRNA extraction. Primary cultures were incubated in a humidified atmosphere of 94% air-6% CO2 at 37°C for 48 h. Media were collected, fresh media containing the test agents were then added, and incubation was continued for an additional 48 h. Incubation with each test agent was performed in quadruplicate.
RNA Extraction and Reverse Transcription
Total RNA was extracted by a single-step acid guanidinium thiocyanate-phenol-chloroform extraction (Tri Reagent; Molecular Research Center, Inc., Cincinnati, OH). Approximately 10 µg total RNA for each sample was reverse-transcribed into cDNA with MMLV (Moloney murine leukemia virus) reverse transcriptase (5 U; Promega, Madison, WI), and random hexamers to prime the reaction, at 42°C for 75 min.
Polymerase Chain Reaction (PCR) Amplification
Three separate PCR protocols were used to detect mRNA for FSHß, ActßB, and FS. For FSHß, a 384-base pair (bp) sequence was amplified using the oligonucleotide primers 5'-ATGAAGTCGATCCAGCTTTG-3' (sense) and 5'-TCATTTCACCGAAGGAGCAG-3' (antisense) [20]. A 306-bp fragment of Act ßB was amplified with sense 5'-TATGTCCTGGAGAAGGGCAGC-3' and antisense 5'-GCGATGTCTGCTATCGCCCAG-3' primers. For FS, the sense, 5'-TCTGCCAATTCATGGAAGAC-3' and antisense 5'-CTGGAGTAATCACACCATC-3' primers were used to amplify a 596-bp sequence [20]. For the FSHß and ActßB PCRs, primers (5'-CCATCCACCCGGCAGCCG-3' and 5'-GTAGTCGACAGAGAGCCT-3') that amplified a 514-bp cDNA for 
-tubulin [20] were included to control for reaction efficiency and variations in concentrations of mRNA in the reverse transcription (RT) reaction. Primers (5'-CTGAAGGTCAAAGGGAATGTG-3' and 5'-GGACAGAGTCTTGATGATCTC-3') that amplified a 191-bp cDNA for ribosomal protein L19 (RPL19) were used in the PCR amplification of FS mRNA [21]. PCR amplifications were performed in a single step for FSHß and in two steps for ActßB and FS to obtain similar intensities for the product of interest and the more abundant internal control. For the one-step PCR for FSHß, the reaction mixture contained 1.25 mM MgCl2, 0.2 mM of each of the four dNTPs, 12.5 pmol of each primer, 0.5 µCi [32P]dCTP (Amersham, Arlington Heights, IL), single-strength Taq polymerase buffer, and 1.25 U Taq DNA polymerase (Perkin Elmer Cetus, Norwalk, CT) in a final volume of 25 µl. For the two-step protocol, the first reaction mixture contained 2 mM MgCl2, 0.2 mM of each of the four dNTPs, 12.5 pmol of primer, 0.5 µCi [32P]dCTP, single-strength Taq polymerase buffer, and 1.25 U Taq DNA polymerase in a final volume of 25 µl, overlaid with 45 µl mineral oil. After the appropriate number of cycles with the first set of primers, 20 µl of a cocktail containing 12.5 pmol each of the second set of primers and 1.25 U additional Taq DNA polymerase was added in single-strength PCR buffer with 2 mM MgCl2 for the remaining cycles. Amplifications were carried out for successive cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 45 sec, and extension at 72°C for 45 sec, with an initial denaturation at 94°C for 5 min and a final extension at 72°C for 10 min. The optimal number of cycles, determined experimentally to yield a linear relationship between signal intensity and input cDNA, and an exponential one with respect to cycle number was 23 for FSHß, 26 for ActßB, and 29 for FS. Amplifications with the primers for -tubulin and RPL19 were performed for 23 and 25 cycles, respectively. Radiolabeled PCR products were separated by electrophoresis on 6% nondenaturing polyacrylamide gels in Tris borate-EDTA buffer. Signal intensity was quantified using PhosphorImage (Molecular Dynamics, Sunnyvale, CA).
FSH Release and Cell Content
Culture media were sampled for FSH concentration at 48 and 96 h. At the end of the incubation, as indicated, cell monolayers were lysed in 1 ml Dulbecco's PBS containing 0.3% BSA and 1% each of Triton X-100 and sodium deoxycholate for determination of intracellular hormone content as indicated. All media and lysates were stored at -20°C until RIA.
Hormone Assays
FSH concentration in the culture media and cell lysates was determined by a double-antibody RIA described previously [22], with reagents supplied by the NHPP.
Statistical Analysis
All data presented are the mean ± SE of quadruplicate incubations from a single representative experiment. The effects of Act and steroids in combination with FS (E2 in experiment 5) were evaluated by two-way ANOVA. When these treatments were found to produce significant overall effects and interactions, data obtained in the absence or presence of FS, or at each level of Act or P4 in experiments 3 and 4, were reanalyzed by one-way ANOVA to allow post-hoc comparisons of individual treatment groups. Post-hoc comparisons of the effects of test agents were performed using the Neuman-Keuls test. P values of < 0.05 were considered significant.
Experimental Protocols
Experiment 1 Anterior pituitaries from female rats (n = 8) at random stages of the estrous cycle were enzymatically dissociated, and cells were plated. The culture medium contained 10 nM E2 throughout the 96-h incubation. After preincubation for 48 h, cells were incubated with Act (10 ng/ml), B (1 µM), or P4 (1 µM), or combinations of Act, B, and P4 without or with FS (20 ng/ml) for an additional 48 h. Media were collected, and cell monolayers were lysed. FSH in the medium and intracellular FSH content were determined by RIA.
Experiment 2 Anterior pituitaries of male rats (n = 4) were collected and dissociated, and cells were plated. After incubation for 48 h, cells were treated with Act (10 ng/ml), B (1 µM), or T (10 nM) without or with FS (50 ng/ml; the concentration of FS was increased compared to that used in experiment 1 to compensate for the loss of activity due to lyophilization and reconstitution) for an additional 48 h. Media were collected, and FSH concentration was determined by RIA.
Experiment 3 Anterior pituitaries from cycling female rats (n = 10) were collected at 0900 h on the morning of metestrus and dissociated, and cells were plated in the presence of E2 (10 nM). At 48 h, media were replaced with fresh media containing E2, Act (3, 10, 30, or 100 ng/ml), and FS (20, 50, or 100 ng/ml). At 96 h, media were collected, and FSH was measured by RIA.
Experiment 4 Anterior pituitaries (n = 12) were collected from female rats at random stages of the estrous cycle and dissociated, and cells were plated in the presence of 10 nM E2. After 48 h, media were changed, and cells were treated with E2, P4 (1, 10, 100, or 1000 nM), and FS (20, 50, or 100 ng/ml) for an additional 48 h. FSH released into the media was measured by RIA.
Experiment 5 Anterior pituitaries from cycling female rats (n=21) were collected at 0900 h on the morning of metestrus and dissociated, and cells were plated. At 48 h, cells were treated with P4 (100 nM), B (1.7 µM), or Act (100 ng/ml); concentrations were chosen to ensure maximal responses. Duplicate sets of primary cultures were incubated with these agents in the absence or the presence of 10 nM E2. At 96 h, media were collected, and FSH was assayed by RIA. Messenger RNA levels were analyzed by semiquantitative RT-PCR for FSHß, FS, and ActßB.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment with Act, B, or T elicited a similar FSH response in anterior pituitary cell culture from male rats (Fig. 2; experiment 2). Act, B, or T significantly increased FSH release (P < 0.01, 0.05, and 0.001, respectively), although to a lesser degree than in anterior pituitary cell cultures from female rats. Coincubation with 50 ng/ml FS significantly suppressed FSH release to approximately 50% below basal (P < 0.0001), and there was a significant interaction between FS and the stimulators (P = 0.001).
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The effects of incremental concentrations of FS on Act- or P4-enhanced FSH release were compared in anterior pituitary cell cultures from female rats to determine whether the effects of the two stimulators were qualitatively similar (experiments 3 and 4). Act increased FSH release in a dose-dependent manner during 48 h of incubation (Fig. 3). The highest concentration of Act that we used did not appear to be maximally stimulatory. With increasing concentrations of Act, the FS-induced suppression of FSH was attenuated and eventually abolished. At the lowest concentration of Act tested, all concentrations of FS were inhibitory, whereas at the highest concentration of Act, all concentrations of FS were ineffective in suppressing FSH release. The effects of both Act and FS on FSH release were highly significant (P < 0.0001) and there was a highly significant interaction between the treatments (P < 0.0001).
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P4 at concentrations ranging from 1 nM to 1000 nM increased FSH release (Fig. 4); the effect of 100 nM P4 was maximal, i.e., not significantly different from that of 1000 nM. Addition of FS suppressed FSH release in a concentration-dependent manner at all concentrations of P4. As in experiment 3, the effects of both the stimulator and FS were highly significant, as was the interaction between the treatments (P < 0.0001). The interaction of FS with P4, however, differed markedly from that with Act, as even the highest concentration of P4 did not overcome the FS-induced suppression of FSH release.
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To determine whether the steroids modulated Act and FS mRNA levels to favor increased FSH synthesis, FSHß, Act ßB, and FS mRNA levels were analyzed by semiquantitative RT-PCR (experiment 5). Primary anterior pituitary cell cultures from female rats were incubated with Act, B, or P4 in the presence or absence of E2 for 48 h. Act, B, or P4 significantly increased FSH release (P < 0.0001; Fig. 5A); the effect of E2 was also significant (P = 0.001); there was not, however, a significant interaction between the treatments. Act and B, but not P4, significantly increased FSHß mRNA levels (Fig. 5B) in the presence or absence of E2 (P < 0.001), consistent with previous results from our laboratory and others [19, 23]. Treatment with E2 significantly lowered FSHß subunit mRNA (P = 0.0001). Act increased FS mRNA significantly only in the absence of E2 (P < 0.01; Fig. 5C); however, neither B nor P4 had a significant effect on FS mRNA. None of the treatments significantly affected ActßB mRNA levels (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Neither B nor P4 affected FS mRNA levels during a 48-h incubation in vitro, while under the same conditions, Act up-regulated FS mRNA levels. Thus, B and P4 at concentrations that increased release and intracellular content of FSH did not mimic the effects of Act on FS mRNA, suggesting either that the steroids do not act solely through accumulation of endogenous Act in stimulating FSH secretion or that they were not present in sufficient concentrations to activate FS gene transcription. Our failure to demonstrate increased ActßB mRNA levels in the pituitary cell culture upon addition of the steroids does not rule out an intermediary role of endogenous Act in their action on FSH secretion; inappropriate timing and concentrations may account for this finding.
Few previous studies have addressed the effects of the gonadal and adrenal steroids on the Act pathway in rat anterior pituitary cell culture. Miyake et al. [13] demonstrated that the effects of P4 or T were additive with those of exogenous Act A in stimulating FSH secretion from female rat anterior pituitary cell culture, as confirmed in the present study. In a different study, T increased ActßB mRNA and suppressed FS mRNA in anterior pituitary cell culture from male rats [25]. To date, no data are available on the interaction between glucocorticoids and Act in the gonadotroph.
The present results in conjunction with our previous finding that RU-486 suppresses Act-induced FSH release suggest that there may be cross-talk between Act-mediated and steroid-mediated pathways in the anterior pituitary. In a recent study, Yanagisawa et al. [26] demonstrated similar cross-talk between transforming growth factor-ß (TGFß)-mediated and vitamin D-mediated signaling. The TGFß signal transduction pathway, like that of Act, utilizes Smad proteins, whereas vitamin D, like P4, B, and T, binds to a nuclear receptor. Specifically, Smad 3, activated by TGFß, was found to form a complex with a member of the steroid receptor coactivator-1 protein family and thus enhance the actions of vitamin D. Likewise, it is possible that coactivators recruited by E2 bind to Act-activated, phosphorylated Smad proteins and associate with transcriptional complexes activated by PR in the nucleus. Moreover, the FSHß promoter area contains P4 response elements (PRE) [27, 28] as well as an AP-1 site [29]. The promoter area of the early intermediate gene junB, which activates AP-1 sites, is a target for Act and contains an Act response element [30, 31]. In the presence of E2, simultaneous activation of the PRE and AP-1 sites on the FSHß promoter may be required to promote transcriptional activation of the FSHß gene.
Our results suggest that in the gonadotroph, the steroids B, P4, and T increase FSH release in part by modulating the Act-FS-Inh autocrine paracrine loop. We conclude that endogenous Act contributes to the stimulatory action of steroids on FSH secretion.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH grants P01-HD-21921 and P30-HD-278048. 
2 Correspondence: Neena B. Schwartz, Department of Neurobiology and Physiology, Northwestern University, 2153 N. Campus Dr., Evanston, IL 60208–3520. FAX: 847 491 5211; n-schwartz{at}nwu.edu
Accepted: October 25, 1999.
Received: July 26, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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