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Regular Article |
a Institute of Veterinary Physiology,
b University of Parma, 43100 Parma, Italy
c Animal Reproduction and Biotechnology Laboratory, Department of Physiology, Colorado State University, Fort Collins, Colorado 80523
ABSTRACT
In several physiological paradigms, secretion of FSH and LH are not coordinately regulated. Because these hormones appear to be produced by a single cell type in the anterior pituitary gland, their discordant regulation must be related to differential intracellular responses to various stimuli. Estradiol-17ß (estradiol) has been shown to influence secretion of both FSH and LH and some of its effects are mediated directly on the gonadotrope. Changes in expression of intrapituitary factors such as activin and follistatin may mediate effects of estradiol and account for discordant patterns of FSH and LH. The aims of this study were 1) to determine if estradiol alters expression of genes encoding activin, follistatin, or both in ovine pituitary cells; and 2) to observe the effects of immunoneutralizing activin B in vitro on gonadotropin secretion. Pituitary cells from five ewes in the anestrous season were cultured for 24 h with estradiol (0.01 or 1.0 nM). Estradiol reduced basal secretion of FSH in a dose-dependent manner (P < 0.001) and simultaneously increased basal secretion of LH (P < 0.001). Decreased secretion of FSH in estradiol-treated cultures was accompanied by suppressed levels of FSHß subunit mRNA (P < 0.001). Amounts of mRNA for activin ßB were reduced in a dose-dependent manner by estradiol (27% ± 4.9% at 0.01 nM, P < 0.02; and 46% ± 3.9% at 1.0 nM, P < 0.002). In contrast, mRNA for follistatin was not affected by treatment with estradiol. Treatment of pituitary cells with an antibody to activin B reduced secretion of FSH by 50% (P < 0.01) without influencing secretion of LH. These data lead us to conclude that discordant secretion of gonadotropins can be induced by estradiol acting directly at the pituitary level. The inhibitory effect of estradiol on FSH secretion may be mediated indirectly through decreased pituitary expression of the activin gene.
activin, estradiol, follicle-stimulating hormone, follistatin, luteinizing hormone, neuroendocrinology
INTRODUCTION
Reproductive activity of sheep varies with season. Female sheep in temperate latitudes become anestrus during late winter or early spring and resume reproductive activity in late summer or early fall [1]. Seasonal fluctuation in reproductive activity is most likely due to changes in photoperiod because similar changes in reproductive function are manifest in sheep exposed to differing artificial photoperiods in an otherwise constant environment [2]. Photoperiodic cues modulate reproductive function in sheep through steroid-dependent and steroid-independent mechanisms [3]. The steroid-dependent effect of photoperiod is clearly evident in sheep chronically treated with estradiol. In gonadectomized sheep, regardless of sex, the negative feedback of exogenous estradiol is most profound during the anestrous season [4–6]. The effect of photoperiod on secretion of gonadotropins may reflect a seasonal shift in the sensitivity of the hypothalamus or other neural centers to estradiol [7]. Indeed, secretion of GnRH is attenuated during the anestrous season in ovariectomized ewes treated with estradiol [8]. But estradiol, in addition to its action on the hypothalamus, has a direct effect on the anterior pituitary gland. Estradiol increases tissue concentration of GnRH receptor in ovine pituitary cells in culture [9] and hypothalamic-pituitary disconnected ewes [10]. On the other hand, estradiol may exert an inhibitory action on gonadotropin secretion directly at the pituitary gland [11–14]. Thus, it appears likely that estradiol acts on gonadotropin secretion at two different levels, hypothalamic and pituitary, probably through different molecular mechanisms.
Maintenance of normal reproductive function is dependent on the precise regulation of gonadotropin biosynthesis and secretion. Control of gene expression for the ß subunit of both gonadotropins is regulated by circulating gonadal steroids as well as by hypothalamic factors, principally GnRH. In addition to GnRH and gonadal steroids, expression of mRNA for FSHß and secretion of FSH are under the control of the peptide hormones inhibin, activin, and follistatin [15]. The heterodimeric inhibins, designated inhibin A (
ßA) and inhibin B (ßB) are produced primarily by gonads and act to reduce secretion of FSH [16]. Activins, which are homomeric or heteromeric dimers of the ßA and ßB chains, are produced in a wide variety of tissues and stimulate synthesis of FSH by direct action on gonadotropes [17]. The monomer follistatin probably regulates synthesis and secretion of FSH through its ability to bind activin [18]. Inhibin, activin, and follistatin are produced by the ovary and are present in the circulation, but activin and follistatin are also produced in the pituitary gland by folliculo-stellate cells and by gonadotropes in rats [19] and sheep [20], and may exert autocrine/paracrine actions on synthesis and secretion of FSH. Like its gonadal homologue [21], pituitary expression of the follistatin gene varies during the rat estrous cycle [22] and mRNAs for both ßB and follistatin increase after gonadectomy in rats [23, 24]. Thus, the local production of activin and follistatin appears to be important in regulating secretion of FSH and represents a potential intrapituitary modifier of hypothalamic or systemic endocrine signals.
The aim of this study was to investigate the possible relationship between the direct effect of estradiol on pituitary gonadotropin secretion and pituitary expression of genes encoding activin (ßB) and follistatin. We focused our investigations on the activin ßB subunit and follistatin based on previous reports showing that changes in expression of these genes following ovariectomy may be modulated by estradiol [23–25]. Anestrous ewes were chosen for this study because they are more responsive to estradiol than ewes during the breeding season [4].
MATERIALS AND METHODS
Animals and Experimental Design
Five sexually mature, western-range ewes were used during the anestrous season (June) in Colorado. Anestrous status was confirmed by the absence of ovarian activity at slaughter. Anterior pituitary glands were collected following anesthesia with sodium pentobarbital and exsanguination. Tissues were removed and immediately placed in ice cold dissociation medium. All procedures involving animals were approved by the Colorado State University Animal Care and Use Committee and complied with National Institutes of Health (NIH) guidelines.
Cell Culture
Anterior pituitary cells were dissociated as described previously [9]. Briefly, the anterior pituitary gland was separated from the neurohypophysis and 0.5-mm slices were prepared using a Stadie-Riggs hand microtome. Slices were washed five times in dissociation medium (0.8% NaCl, 0.59% Hepes, 0.18% glucose, 0.02% KCl pH 7.3) and incubated, with gentle shaking, for 90 min with 1 mg/ml collagenase, 1 mg/ml hyaluronidase, and 0.1% DNase at 37°C. After dissociation, cells were washed five times in dissociation medium and, finally, dispersed in Dulbeccos minimal essential medium (DMEM) containing 30% wether serum. Cell viability, as assessed by trypan-blue-exclusion, was >90% and yield was 1.2 ± 0.05 x 108 cells/gland. Cells were seeded at a concentration of 4 x 105 cell/well in 24-well plates for secretion studies and 3 x 106 cells in 7-cm plates for analysis of mRNA. Cells were incubated for 72 h at 37°C in the presence of 95% O2 and 5% CO2. Chemicals and DMEM were purchased from Sigma Chemical Company (St. Louis, MO).
Treatments
Estradiol (0.01 or 1.0 nM) was added to the cells for the final 24 h of culture. After treatment, medium was collected from all cultures and stored at -20°C until hormone assays were performed.
To immunoneutralize activin, monoclonal antibody to activin B (anti-activin, 25 µg/ml, kindly supplied by Ralph Schwall, Genentech, Inc., San Francisco, CA) was added to individual wells (n = 5) of 24-well plates for the final 24 h of culture. At the end of treatment medium was collected and stored at -20°C until hormone assays were performed.
Reverse Transcription-Polymerase Chain Reaction Analysis
Total RNA was prepared from pituitary cells using the single-step method of Chomczynski and Sacchi [26]. One hundred nanograms of total RNA were reverse transcribed to cDNA using reverse transcription-polymerase chain reaction (RT-PCR) Ready-to-Go beads (Amersham Pharmacia, Piscataway, NJ). Briefly, total RNA and 0.5 µg oligo(dT) were incubated for 75 min at 42°C with beads, then for 10 min at 95°C. For the amplification step, [32P]dCTP (0.5 µCi/µl), primers and target-specific primers were added to each tube. Samples were withdrawn every two cycles (ranging from 14 to 34 cycles) to determine amplification kinetics of the reaction. Products were separated on 6% TBE polyacrylamide gels. Gels were dried and reaction products were visualized on a PhosphorImager Cassette (Molecular Dynamics, Sunnyvale, CA) for 24–48 h at room temperature and optical densitometry was performed using ImageQuant Software (Molecular Dynamics). The oligonucleotide primers used for the amplification of cDNAs specific to FSHß, activin ßB, follistatin, and ß-actin are shown in Table 1. Conditions were validated for all three transcripts and for ß-actin (used as the internal standard) as follows: denaturation at 95°C for 5 min (1 cycle), 30 sec at 95°C, 30 sec at 56°C, and 1 min at 72°C (30 cycles) and 10 min at 72°C for extension. To reduce variability the same master mix of reagents (including ß-actin primers) was used for each transcript (FSHß, activin ßB, and follistatin). For each PCR reaction, samples were run in duplicate.
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Slot Blot Analysis
To corroborate the RT-PCR results, steady state amounts of FSHß mRNA were also measured by slot blot analysis as previously described [27]. Amounts of mRNA for activin ßB and follistatin were insufficient for detection by this method. Polyadenylated (poly[A]+) RNA was prepared [28] from pituitary cells immediately following incubation and its integrity was confirmed by Northern blot analysis [27]. Quantification of mRNA encoding the FSHß subunit was performed as described previously [29]. Samples of poly(A)+ RNA (600 ng) were applied to nylon membranes in duplicate using a slot blot apparatus and cross-linked by exposure to ultraviolet radiation. Membranes were hybridized to radiolabeled cDNA for bovine FSHß [30]. Membranes were exposed to film for 3 days, after which they were stripped by washing in boiling 0.1% SDS and probed with radiolabeled ß-actin cDNA (188 base pair-amplified PCR product; see above). This procedure allowed for normalization of unequal loading among RNA samples on the nylon membrane. Autoradiographs were analyzed using the NIH 1.52 image analysis program. Concentrations of mRNA are expressed as percent of control values.
Hormone Analyses
Concentrations of LH and FSH were assayed using previously described procedures [31, 32]. Reference preparations for LH and FSH were NIH-oLH-S24 and NIH-oFSH-S12, respectively. Mean limit of detection, intraassay coefficient of variation (CV), and interassay CV were 127 pg/ml, 11.4%, and 12.2%, respectively, for the LH assay; and 6.0 ng/ml, 10.3%, and 10.8%, respectively, for the FSH assay.
Statistical Analysis
Differences in media concentrations of gonadotropins and amounts of mRNA were determined by one-way ANOVA using the general linear model procedure of SAS [33]. Data obtained from multiple-culture wells were pooled for statistical analysis. When significant differences were found, means were compared by Tukey's test. All quantitative data are presented as mean ± SEM.
RESULTS
FSH and LH Release from Pituitary Cells after Estradiol Treatment
Estradiol reduced secretion of FSH in a dose-dependent manner (P < 0.001; Fig. 1A). In the presence of 0.01 and 1.0 nM estradiol, FSH concentrations were suppressed by 37% and 54%, respectively, of mean basal release in control cultures. In contrast, secretion of LH was significantly enhanced by estradiol (P < 0.001; Fig. 1B). In the presence of 0.01 and 1.0 nM estradiol, LH concentrations were increased to 237% and 267%, respectively, of mean basal release in control cultures.
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FSH and LH Release from Pituitary Cells after Treatment with Anti-Activin
Anti-activin reduced mean secretion of FSH by 51% (P < 0.01) but did not influence secretion of LH (Fig. 2).
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Changes in Amounts of mRNA
As revealed by RT-PCR analysis, levels of mRNA for FSHß in pituitary cells were reduced up to 70% (P < 0.001) following treatment with estradiol compared with untreated controls (Fig. 3). Similar results were obtained by slot-blot analysis (data not shown).
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Treatment with estradiol reduced the amount of mRNA for activin ßB (Fig. 4). The reduction in activin ßB expression was dose-dependent, with a decrease of 27% ± 4.9% (P < 0.02) and 46% ± 3.9% (P < 0.002), observed in response to 0.01 nM and 1.0 nM estradiol, respectively. There was no significant change in the amounts of mRNA for follistatin in response to treatment with estradiol (Fig. 5).
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DISCUSSION
Current concepts of regulation of gonadotrope function include the autocrine-paracrine regulation of FSH synthesis and secretion by activin and follistatin. Some reports have documented small but significant increases in mRNAs for activin ßB and inhibin after gonadectomy in rats [22, 23, 34]. Alternatively, effects of activin action on expression of FSHß may be mediated via changes in production of follistatin. The amount of pituitary mRNA for follistatin increases during proestrus in the rat [22]. This seems incongruous with the idea that follistatin sequesters activin and thereby reduces synthesis and secretion of FSH. Furthermore, pituitary content of follistatin and free activin are inversely related during the estrous cycle, and free activin peaks during the cycle and appears to be responsible for a large portion of the secondary surge of FSH in the rat [35]. We have demonstrated that activin has a specific effect on FSH secretion in sheep. In fact, treatment of cultured anterior pituitary cells with a monoclonal antibody to activin ßB decreased secretion of FSH, but did not alter release of LH. The antibody should neutralize activin B, the primary form of activin present in the anterior pituitary gland [23], and activin AB. This indicates that activin acts through a paracrine or autocrine mechanism to stimulate secretion of FSH even in absence of hypothalamic input. Furthermore, our data show that the genes for ßB and follistatin are expressed in ovine pituitary cells during anestrus.
The data reported herein confirm that during anestrus, estradiol can regulate secretion of gonadotropins by a direct action on the anterior pituitary gland in the absence of any hypothalamic factors. These effects of estradiol appear to be mediated, at least in part, by intrapituitary activin. Inhibition of ovine pituitary ßB gene expression by estradiol is consistent with previous findings in rats in which the ovariectomy-induced increase in ßB mRNA was prevented by treatment with estradiol [23, 25]. There is a potential estrogen response element (AGGTAAnnnTGACCT) in the 3'-untranslated region of the published sequence of the human activin ßB gene [36] that differs from the consensus estrogen response element (AGGTCAnnnTGACCT) by a single base. Thus, the potential for direct estrogenic regulation of activin production exists. Alternatively, it is possible that estradiol may alter the transcription of other pituitary factors that reduce expression of activin ßB.
In addition to decreased secretion of FSH, we observed a specific reduction in expression of the FSHß subunit in response to treatment with estradiol in vitro. These results are in good agreement with previous observations made in vivo. During both breeding and nonbreeding seasons, estradiol reduced secretion of FSH [37] and decreased expression of mRNA for the FSHß subunit [38].
Increased secretion of LH stimulated by estradiol in the present study might have arisen from a direct effect on the gene for LHß because it harbors an estrogen response element (ERE) in the 5'-flanking region [39]. However, there does not appear to be an ERE in the 5'-flanking region of the FSHß gene [40, 41]. Thus, it is likely that estradiol acts at the pituitary level by stimulating other intracellular factors that modulate synthesis and secretion of FSH.
Analysis by RT-PCR failed to demonstrate a correlation between expression of FSHß and follistatin in response to treatment with estradiol. No increase in expression of follistatin was noted in cells from any of the five animals following treatment with estradiol even though there was a consistent decrease in expression of FSHß. The lack of increase in expression of follistatin is not surprising in light of the fact that activin induces expression of follistatin in the rat pituitary gland [42–44]. Because exposure of our cultures to estradiol led to a dose-dependent decrease in expression of ßB, there may not have been a stimulus for production of follistatin. These data lead us to believe that estradiol reduces expression of activin at the level of the pituitary gland, independent of GnRH. The effect of estradiol on expression of activin may only partially explain the reduction observed in expression of FSHß. The question arises whether the effect of estradiol on FSHß gene expression, which was very dramatic (up to 70%), may be mediated by the relatively lesser decrease (
46%) in expression of activin. We hypothesize that a small reduction in free activin concentration may be sufficient to inactivate second messenger cascades and thereby greatly reduce synthesis and secretion of FSH as reported for GnRH receptor expression mediated by GnRH [45].
Ovine anterior pituitary cells locally synthesize follistatin, and activin stimulates the production of follistatin, independently of inhibin [20]. However, there appear to be important differences among species with respect to effects of activin on FSH secretion. The activin concentration necessary to stimulate FSH secretion in ovine gonadotropes is three times that required in rat gonadotropes (EC50 = 0.3 and 0.1 nM, respectively), and the corresponding increase in FSH secretion was 150% and 500% (respectively) of control values [20]. Likewise, a more robust FSH response was obtained to lower concentrations of activin (EC50 = 0.18 nM) with cultured ovine pituitary cells from immature lambs [46]. Thus, it appears that factors such as age, sex, and gonadal status of the donor animals may affect pituitary production of FSH via the activin/follistatin intrapituitary feedback loop. The studies reported herein were performed with pituitary cells obtained from anestrous ewes. These pituitary cells appear to be particularly responsive to estradiol, and the endogenous concentrations of activin and follistatin may differ from those in the ovine pituitary gland at other times of the year. An investigation into seasonal changes in pituitary concentrations of activin and follistatin in sheep is needed.
In summary, we report that estradiol inhibits synthesis and secretion of FSH and stimulates secretion of LH in ovine pituitary cells during anestrus. These effects were independent of GnRH. Estradiol appears to reduce the intrapituitary production of activin ßB but does not affect production of follistatin. Furthermore, immunoneutralization of activin reduced FSH secretion but did not influence secretion of LH. We conclude that estradiol may directly influence gonadotropin secretion in the ewe through local mediators such as the activin-follistatin network. Further examination of molecular mechanisms is needed to better understand this intrapituitary system in sheep.
FOOTNOTES
First decision: 8 August 2000.
1 Supported by U.S. Department of Agriculture grant 94-37203-0716. 
2 Correspondence: Terry M. Nett, Animal Reproduction and Biotechnology Laboratory, Department of Physiology, Foothills Campus, Colorado State University, Fort Collins, CO 80523. FAX: 970 491 3557; tnett{at}cvmbs.colostate.edu
3 Current address: Department of Physiology, University of Arizona, Tucson, AZ 85750.
Accepted: October 4, 2000.
Received: July 17, 2000.
REFERENCES
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