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Division of Endocrinology, Department of Internal Medicine, and the Center for Research in Reproduction, University of Virginia, Charlottesville, Virginia 22908
Address all correspondence and requests for reprints to: Laura L. Burger University of Virginia, Department of Internal Medicine P.O. Box 801412, Charlottesville, Virginia 22908. E-mail: llb3k{at}virginia.edu.
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Abstract |
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Both GnRH frequencies increased 
primary transcript (PT) 5-fold within 6 h, but only fast frequency GnRH increased mRNA. Only fast frequency GnRH pulses affected LHß PT, resulting in 6- to 9-fold increases between 1–24 h. Fast frequency GnRH pulses transiently increased FSHß PT at 1 and 6 h (4- and 2-fold, respectively); but by 24 h FSHß PT had returned to control levels and was correlated to a 5- to 9-fold increase in FS mRNA. In contrast, slow GnRH pulses increased FSHß PT 3- and 6-fold at 8 and 24 h, respectively, which was correlated with a decline in FS mRNA. Activin mRNA did not change significantly after either GnRH frequency, but tended to fall after fast pulses.
To test whether activin was required for the effects of GnRH on FSHß transcription, rats were treated with GnRH pulses every 240 min for 8 h ± FS. FS treatment alone markedly decreased basal FSHß PT. GnRH in the presence of FS increased FSHß PT 8-fold but did not restore FSHß transcription to control or GnRH alone values.
In summary, whereas -subunit transcription is independent of frequency, an increase in mRNA requires fast frequency GnRH pulses. Fast frequency GnRH pulses increased both LHß and FSHß transcription, but the response of FSHß was transient. The sustained rise in FSHß transcription and mRNA expression required slow frequency GnRH pulses and was correlated to low FS mRNA. Neutralization of pituitary activin by exogenous FS markedly reduced basal FSHß PT and mRNA but did not prevent the stimulation of FSHß transcription by slow frequency GnRH pulses. These studies suggest that the frequency regulation of FSHß transcription involves both direct actions of GnRH and indirect effects, via changes in pituitary FS expression.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit and a unique ß-subunit, and in the rat are coded for by independent genes located on different chromosomes (1). The gonadotropins subunit genes are regulated in both a coordinate and differential manner, primarily by pulsatile release of the hypothalamic decapeptide GnRH. GnRH differentially regulates LH and FSH synthesis via changes in both pulse frequency and amplitude (2). In GnRH-deficient castrate + testosterone (cast + T) replaced male rats, fast frequency GnRH pulses (8-min interval) favor and to a lesser extent LHß mRNA expression (3); fast physiological GnRH pulses (30-min interval) stimulate expression of all three subunit mRNAs, whereas slow frequency GnRH pulses (>120-min intervals) increase only FSHß mRNA. GnRH pulse amplitude had little effect on either or FSHß mRNA expression, but LHß mRNA expression was maximally stimulated by mid-physiological pulse doses (4).
Although there is some evidence that GnRH may regulate subunit mRNA concentrations via mRNA stability (5, 6), GnRH appears to exert its main action on subunit gene mRNA transcription. A pulsatile signal is required for GnRH-dependent actions; continuous GnRH in vivo (7) or in vitro (8, 9) does not increase ß-subunit transcription. Using nuclear run-on assays, we previously showed in male rats that , LHß, and FSHß transcription rates were increased 1 and 4 h after GnRH pulses every 30 min, with only transcription maintained through 24 h of pulses (7). Furthermore, pulsatile GnRH increases subunit transcription in a frequency-dependent manner. Slow frequency pulses (every 120 min) selectively increased FSHß transcription. The results of recent time course studies, using quantitative primary transcript (PT) RT-PCR assays to assess transcription, reveal that gonadotropin subunit transcription occurs in bursts in response to pulsatile GnRH, with maximal subunit PT concentrations occurring 5–15 min after a GnRH pulse and declining toward basal within 30 min (10).
FSHß gene expression is also regulated by activin and follistatin (FS). Activin and FS are produced in a broad range of tissues, including the gonads and the pituitary. The activins are a member of the TGFß superfamily and are homo-/heterodimers of the inhibin ß-subunits (ßA or ßB). The activin ßB-subunit is produced by the gonadotropes (11), and pituitary-derived activin acts in an autocrine/paracrine fashion to stimulate FSHß gene expression via transcription (12) and/or mRNA stability (13). FS is a glycoprotein produced in both the gonadotropes and the folliculostellate cells (14). FS decreases FSHß gene expression by binding to and bioneutralizing activin (15).
Both pituitary FS and activin ßB mRNAs are differentially regulated by GnRH pulse frequency. In male rats, FS mRNA is increased only by rapid-interval GnRH pulses, in contrast to the effects of rapid GnRH on FSHß mRNA (16). Activin ßB mRNA expression is also regulated by GnRH pulse frequency; in female rats, both ßB and FSHß mRNA are maximal after GnRH pulses every 30 min (17). In this model, ßB and FS mRNA expression are inversely correlated; GnRH pulses every 30 min increased ßB but not FS mRNA, whereas pulses every 8 min increased FS mRNA but not ßB. These relationships suggest that GnRH regulates FSH expression at least in part through the modulation of pituitary activin.
In the present study, we used sensitive PT assays to investigate the actions of GnRH pulse frequency on subunit transcription and to correlate changes in subunit transcription, in particular FSHß, with changes in FS and ßB mRNA expression within the same pituitary. Finding that FSHß transcription was inversely correlated with FS mRNA expression, we then assessed the role of pituitary activin in mediating GnRH stimulation of FSHß transcription.
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Materials and Methods |
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All experiments used a GnRH-deficient cast + T replaced male rat model. Rats were castrated, and two 20-mm testosterone containing silicone tubing implants were inserted sc as previously described (18). Serum testosterone levels in these animals were 5.2 ± 0.2 ng/ml, which suppressed endogenous GnRH. An indwelling right jugular cannula was also inserted at the time of castration for iv drug administration. All experiments began 24 h after castration.
Experiment 1: effects of GnRH pulse frequency on gonadotropin subunit transcription
To determine the time course of GnRH pulse frequency actions on subunit transcription rates, 24 h cast + T male rats (n=4–8/group) were pulsed with 25 ng GnRH pulses (in 0.25 ml 0.9% saline-0.1% BSA) either every 30 min and killed at 1 h, 6 h, or 24 h; or every 240 min and killed at 4 h, 8 h, and 24 h. Rats were killed 10 min after the last GnRH pulse as we have previously found that subunit PT concentrations are greatest 5–15 min after a GnRH pulse (10). For each GnRH pulse frequency, a group of untreated 24 h cast +T males rats (groups 0 h) were included as controls. Subunit PTs and mRNAs, FS and activin ßB mRNAs, and serum LH and FSH concentrations were determined.
Experiment 2: effects of slow frequency GnRH pulses on FSHß transcription after activin neutralization via exogenous FS
Previously, Besecke et al. (19) reported that the stimulation of FSHß mRNA by slow frequency GnRH pulses was dependent on activin and could be blocked by FS in perifused rat pituitary cells. To determine whether activin is required to increase FSHß transcription after slow frequency GnRH pulses in this model, we measured FSHß PT in rats treated with slow frequency GnRH pulses ± FS. Twenty-four hours after castration and T replacement, groups of male rats (n = 6–8) were randomly assigned to one of 4 treatment groups: 1) controls (24 h cast + T only); 2) GnRH only; 3) FS only; or 4) GnRH + FS (see Fig. 1
). The duration of the experiment was 8 h as FSHß PT concentrations in experiment 1 were greatest following slow frequency GnRH pulses at this time. Pretreatment serum FSH levels were measure in blood (300 µl) collected at the beginning of the experiment. GnRH (25 ng/pulse in 0.25 ml 0.9% saline-0.1% BSA) was given every 240 min for 8 h (3 pulses). The dose and frequency of administration of FS are based on a previous study by Inouye et al. (20); they reported that 20 µg recombinant human (rh) FS-288 decreased serum FSH by 50% between 4 and 6 h in 7 d ovariectomized rats. Therefore, to maximize the suppression of pituitary activin activity we administered 40 µg (in 0.25 ml 0.9% saline-0.1% BSA) rh FS-288 iv at 0 h and 4 h. Recombinant human FS-288 was kindly provided by Dr. A. F. Parlow through the NIDDK-National Hormone and Pituitary Program. Saline-BSA (0.9%–0.1%) was used as vehicle for both GnRH and FS injections. Rats were killed 10 min after the last GnRH (or vehicle) pulse. FSHß and LHß PTs and mRNAs, ßB mRNA, and serum FSH and LH were measured. mRNA and PT were not measured because transcription did not appear to be differentially regulated by GnRH pulse frequency.
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To increase the efficiency of reverse transcription reaction, the location on the ßB mRNA quantitative RT-PCR assay was moved to the 3' untranslated (UT) region of the mRNA and a new competitive template was created. Oligonucleotides specific to the 3' UT from approximately 2042 bp (24) to the first polyadenylation signal (25) (upstream 5' CAC ACC ACA ATA GCA CTT GCA GGT 3'; downstream 5' ACT CTA CCT TCT GGG TGT ATA AGG 3') were used to RT-PCR amplify a 506-bp piece of the 3' flank of the ßB gene from rat ovarian RNA. The PCR product was subcloned into the PGEM-T Easy expression vector (Promega Corp., Madison, WI) and was bidirectionally sequenced. The PCR product corresponds to the published sequences by Esch et al. (25) and Feng et al. (26). The PCR fragment was removed from PGEM-T Easy by digestion with AatII, blunt ended, and cut again with SacI. The fragment was subcloned into PSP64A (Promega Corp.). To create the competitive template, a 170-bp fragment (HincII/PstII digest) of the ßB mRNA 3' UT was replaced with an unrelated 294-bp sequence from pBR 322 (Life Technologies, Inc., Pst/HincII digest). Competitive template RNA was generated by in vitro transcription (RiboMAX-T7, Promega Corp.). The conditions for the RT-PCR assay are the same as reported previously for the FS mRNA assay (16). The size of the RT-PCR assay products using these primers is 506 bp for the native RNA and 630 bp for the CT (GC correction factor = 0.67).
Analysis
Data examining the differential effects of fast and slow GnRH pulses on subunit transcription were analyzed by two-way ANOVA with GnRH pulse frequency and hours of GnRH pulses as the main effects. Changes in subunit transcription after slow frequency GnRH pulses ± FS were analyzed by one-way ANOVA. Significant differences (P < 0.05) were determined post hoc by Duncan’s multiple range test. Before analyses, all measurements were transformed to the logarithmic scale to attain equal variation among treatments. All data are presented as fold change vs. controls
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Results |
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. Untreated, 24 h cast + T, control concentrations for: PT, LHß PT, FSHß PT, mRNA, LHß mRNA, FSHß mRNA, FS mRNA, and ßB mRNA (± SE) were 0.39 ± 0.04, 0.07 ± 0.01, 0.10 ± 0.01, 52.78 ± 3.81, 33.68 ± 4.25, 15.77 ± 0.59, 0.82 ± 0.19, 2.62 ± 0.21 fmol/100 µg RNA, respectively. Serum LH and FSH were 0.21 ± 0.03 and 11.31 ± 0.62 ng/ml, respectively. Both 30- and 240-min GnRH pulses increased PT concentrations at all time points examined, and peak responses of 4- to 5-fold observed between 6 and 24 h were similar. Despite the rapid and sustained increases in PT with both GnRH pulse frequencies, mRNA was elevated only after 24 h of 30-min pulses. LHß transcription responses were dependent on GnRH pulse frequency. GnRH pulses every 240 min had no effect on either LHß PT or mRNA concentrations. In contrast, after 30-min GnRH pulses LHß PT was 5-, 9-, and 7-fold greater than controls at 1 h, 6 h, and 24 h, respectively. Changes in LHß mRNA levels lagged those of LHß PT, rising to values 1.5-fold greater than controls at 6 and 24 h. The 2- and 5-fold increases in and LHß PTs after 1 h of 30-min GnRH pulses are similar in magnitude to those reported previously (10).
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, and the relationship of FSHß PT to FS mRNA and activin ßB mRNA is shown in Fig. 3. Both GnRH frequencies increased FSHß PT, but the pattern of transcriptional activity was different over 24 h. Slow frequency GnRH pulses increased FSHß PT maximally (3- to 6-fold) between 8 and 24 h, which was associated with a reduction in FS mRNA to 30% of basal concentrations. Similar to our earlier report (10) we found a 4-fold increase in FSHß PT after 1 h of 30-min GnRH pulses, but the increase was transient declining to 2.5-fold vs. controls at 6 h and returning to basal by 24 h. The decrease in FSHß PT at 6 h was temporally correlated with a 5- to 9-fold increase in FS mRNA at 6 and 24 h. The differential changes in FSHß PT to fast and slow frequency GnRH pulses was reflected by similar changes in FSHß mRNA; after 24 h, FSHß mRNA concentrations were 2-fold greater in rats treated with GnRH every 240 min vs. every 30 min. Activin ßB mRNA did not change significantly over time with either GnRH pulse frequency but were significantly lower at 24 h in the 30-min GnRH pulse group vs. the 240 min.
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Experiment 2: effects of slow frequency GnRH pulses on FSHß transcription after activin neutralization via exogenous FS
Two lines of evidence from experiment 1 would suggest that the frequency regulation of FSHß transcription by GnRH lies in the regulation of pituitary FS, and hence available pituitary activin. First, the decline in the transient increase in FSHß PT after fast GnRH pulses was temporally correlated to a 5- to 9-fold increase in FS mRNA. Second, slow GnRH pulses increased FSHß PT while decreasing FS mRNA. Therefore, to determine whether activin was required for the effects of GnRH on FSHß transcription, we treated rats with slow frequency GnRH pulses, which increases FSHß PT but not FS mRNA, alone or in combination with FS to block activin action. The results of GnRH ± FS on ß-subunit PTs, mRNAs, and serum gonadotropins are shown Fig. 4. Untreated, 24 h cast + T control concentrations for LHß PT, FSHß PT, LHß mRNA, and FSHß mRNA (± SE) were 0.33 ± 0.03, 0.14 ± 0.02, 0.10 ± 0.01, 8.59 ± 0.73, 13.01 ± 0.59 fmol/100 µg RNA respectively. Serum LH and FSH were 0.14 ± 0.03 and 9.93 ± 0.54 ng/ml, respectively.
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Neither GnRH nor FS, alone or in combination, increased LHß PT or mRNA concentrations vs. controls. However, there was a small but significant reduction in LHß PT concentrations in rats given GnRH + FS compared with GnRH only. Additionally, FS suppressed GnRH-driven LH secretion; FS did not affect basal LH, but the increase in serum LH in rats treated with GnRH + FS was only one third of rats treated with GnRH alone.
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Discussion |
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Although GnRH is considered the primary regulator of gonadotropin synthesis, FSH synthesis is also regulated by intrapituitary activin and FS as well as gonadal inhibin in females (17, 28). The differential regulation of FSHß and FS mRNA by GnRH pulse frequency are recognized, but this is the first study to correlate changes in the pattern of FSHß transcriptional activity to changes in FS and activin ßB mRNAs. Fast frequency GnRH pulses increased FS mRNA 5- to 9-fold beginning at 6 h, coincident with the decline in FSHß PT. Slow frequency GnRH pulses caused a robust increase in FSHß PT and mRNA that was correlated with a 3-fold reduction in pituitary FS mRNA over 24 h. Both the different patterns of FSHß transcription over time and the relationship to FS mRNA in response to fast and slow frequency GnRH correlate well with studies in perifused rat pituitary cells by Besecke et al. (19). They found that continuous GnRH increased both FSHß mRNA and FS mRNA in parallel during the first 4 h of treatment, but at 10 h only FS mRNA continued to rise, whereas FSHß mRNA had returned to basal. Whereas 60-min GnRH pulses slowly increased FSHß mRNA, FS mRNA remained unchanged. The inverse relationship between FSHß transcription and mRNA expression with FS mRNA strongly suggests that available intrapituitary activin plays a significant role in FSHß transcription.
Activin has been previously shown to increase FSHß PT in primary pituitary cell cultures (12) and to stimulate an ovine FSHß promoter-reporter construct transiently transfected into the gonadotrope derived LßT2 cell line (29, 30). To determine whether activin was required for FSHß transcription, we measured FSHß PT after slow frequency GnRH in the presence or absence of exogenous FS. FS treatment reduced basal FSHß PT concentrations to nearly undetectable levels. Despite this marked reduction, GnRH still increased FSHß PT 8-fold in FS-treated rats, a larger magnitude of response than GnRH alone, but we did not see a coordinate increase in FSHß mRNA. In a parallel in vitro study by Besecke et al. (19), FS profoundly blunted basal FSHß mRNA but did not prevent a 4-fold increase in response to 60-min GnRH pulses. The difference in results between these studies may reflect the method used to measure FSHß mRNA, we used a RNA dot blot assay and Bescke et al. used an RT-PCR assay, which may be more sensitive. These studies suggest that, although activin is important in regulating basal FSHß transcription and mRNA expression, it is not required for the effects of GnRH to be manifest.
The concept that activin/FS may act more as volume control, rather than an on-off switch, contrasts recent work in vitro suggesting that the actions of GnRH on FSHß transcription require activin. In those studies, FS eliminated GnRH-induced increases in expression of an ovine FSHß promoter-reporter construct transiently transfected into LßT2 cells (30). Several explanations may account for these differences between our studies. Foremost are the differences between GnRH pulse stimulation in vivo models and in vitro systems using transfected cell lines. Second, Pernasetti et al. (30) pretreated cells with FS for 24 h before exposure to GnRH; and as activin increases GnRH receptors (31, 32) the LßT2 cells may be relatively GnRH-R deficient. In the present study, rats were cotreated with FS and GnRH for only 8 h, which may not have reduced gonadotrope GnRH-R to the same degree. Finally, both activin and FS have been reported to alter FSHß mRNA half-life (degradation rate) with activin stabilizing (13) and FS destabilizing (33) the mRNA. It is possible that the FSHß PT half-life is regulated in a similar manner. A reduction in FS after slow frequency GnRH stimuli together with increased FSHß PT formation could have additive actions on PT concentration and explain different results between promoter activity strategies and PT assays.
Consistent with earlier reports by Bilezikjian et al. (34) we found that administration of FS only moderately suppressed ßB mRNA. Moreover, GnRH at either pulse frequency did not change ßB mRNA, although concentrations tended to fall after rapid pulses. We have previously reported that ßB mRNA does not change after castration in male rats (24), but FS mRNA increases after both castration and fast frequency GnRH pulses. This contrasts observations in female rats, where ßB mRNA increases following ovariectomy (24) and 30-min GnRH pulses (17). The absence of significant changes in pituitary ßB mRNA in males suggests that intrapituitary activin availability is regulated primarily via FS rather than through activin production.
LHß transcription was stimulated only by fast frequency GnRH pulses and was maintained through 24 h. The rapid induction of LHß PT after 30-min GnRH pulses agrees with our earlier findings, using nuclear run on assays, where LHß mRNA synthesis rates increased 3- to 4-fold at 1 and 4 h (7). However, the increases in LHß transcription were transient and returned to basal by 24 h. The differences at 24 h between these studies is unknown and may reflect the method of measuring transcription; nuclear run-on assays measure nascent RNA formation, whereas the PT assays measure fully transcribed, though unprocessed, RNA transcripts that may be stabilized or destabilized via posttranscriptional mechanisms. Important to the cast + T rat model are the findings that both GnRH and testosterone have been shown increase LHß mRNA stability; GnRH increases the length of the LHß mRNA poly A tail (6), and testosterone modestly increases LHß mRNA half-life (26). The half-life of the LHß PT in castrate rats has been estimated to be to be 2.7 h after GnRH antagonist (10), but data in the presence of GnRH and/or testosterone are not available. Thus, it is possible that although transcription may have diminished by 24 h, significant amounts of PT remain. Interestingly, FS treatment also decreased both LHß PT and LH secretion in response to slow frequency GnRH pulses vs. GnRH alone, suggesting a role for activin in both LHß transcription and secretion. Pernasetti et al. (30) also observed that FS attenuated the actions of GnRH and activin on LHß promoter activity in transiently transfected LßT2 cells. Additionally, activin alone or in combination with GnRH, increased LH secretion in rat pituitary cells (35) and in vivo in monkeys (36, 37). The mechanism for the actions of activin on LHß expression and secretion are unknown; the promoter-based studies indicate that part of activin’s action is at the level of transcription (30), but as noted earlier activin also increases GnRH-R number. Therefore, in the present study, the reduction in LHß PT and serum LH in rats treated with GnRH + FS vs. GnRH only may also reflect reduced sensitivity to GnRH.
The increase in transcription after 30-min GnRH pulses is consistent with our earlier measurements using nuclear run-on assays (7), but in that study 120-min pulses were ineffective. The differences in transcription after slow frequency GnRH pulses between these two studies may reflect different experimental paradigms and the enhanced sensitivity of the PT assays in assessing transcription. Previously, we examined transcription only after 4 h of slow frequency GnRH pulses, and a small but statistically insignificant increase in transcription was observed. In the present study, PT concentrations were moderately elevated at 4 h and continued to increase through 24 h. The earlier study may simply have assessed transcription at too early a time point. Of interest, however, is the lack of change in mRNA even though PT increased 4-fold after slow frequency GnRH pulses. This may reflect the fact that we are measuring peak PT concentrations after a GnRH pulse, and transcriptional bursts may occur too infrequently to increase mRNA. Alternatively, the lack of increased mRNA may indicate posttranscriptional regulation of the mRNA and/or PT. GnRH has been shown to increase mRNA levels, independent of transcription, by increasing mRNA stability in part via increased polyadenylation (6, 38), which may not be altered by slow frequency GnRH pulses.
In conclusion, the current data define the time course of action of GnRH pulse frequency in regulating transcription of the gonadotropin subunit genes. Although mRNA is elevated only by fast frequency GnRH, PT is increased by both pulse frequencies examined, suggesting a role for frequency specific posttranscriptional regulation of mRNA. Fast frequency GnRH pulses increased both LHß and FSHß transcription, but only LHß transcription was sustained for 24 h, FSHß transcription was transient; and elevated FSHß PT declined to control levels coincident with increased FS mRNA. Persistent FSHß transcription and mRNA expression required slow frequency GnRH pulses, which was temporally correlated to low FS mRNA. Neutralization of pituitary activin by exogenous FS markedly reduces basal FSHß PT and mRNA but did not prevent the stimulation of FSHß transcription by slow frequency GnRH pulses. This suggests that activin is not required for GnRH driven FSHß transcription but does play a major role in maintaining basal FSHß transcription.
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Acknowledgments |
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Footnotes |
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Abbreviations: ßA and ßB, Inhibin ß-subunits; cast + T, 24 h castrate + testosterone; CT, competitive template; FS, follistatin; PT, primary transcript; rh, recombinant human; UT, untranslated.
Received February 22, 2002.
Accepted for publication May 2, 2002.
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