Journal of Molecular Endocrinology (2004) 33, 559-584 DOI: 10.1677/jme.1.01600
© 2004 Society for Endocrinology
Regulation of gonadotropin subunit gene transcription
L L Burger,
D J Haisenleder,
A C Dalkin and
J C Marshall
Division of Endocrinology, Department of Internal Medicine and the Center for Research in Reproduction, University of Virginia, Charlottesville, VA 22908, USA
(Requests for offprints should be addressed to L L Burger; Email: lburger{at}virginia.edu)
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Abstract
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Reproductive function in mammals is regulated by the pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH are secreted by the gonadotrope cell and act on the gonad in a sequential and synergistic manner to initiate sexual maturation and maintain cyclic reproductive function. The synthesis and secretion of LH and FSH are regulated mainly by the pulsatile release of the hypothalamic decapeptide hormone gonadotropin-releasing hormone (GnRH). The control of differential LH and FSH synthesis and secretion is complex and involves the interplay between the gonads, hypothalamus and pituitary. In this review, the transcriptional regulation of the gonadotropin subunit genes is discussed in a physiologic setting, and we aimed to examine the mechanisms that drive those changes.
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Introduction
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Dynamic regulation of the pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is essential for mammalian reproduction. LH and FSH are comprised of two glycoprotein subunits, 
(common to both), and LHß and FSHß, which are coded by three genes located on separate chromosomes (Chin 1987, Gharib et al. 1990b). LH and FSH are secreted by the pituitary gonadotropes and act on the gonad in a sequential and synergistic manner to initiate sexual maturation and maintain cyclic reproductive function (Bäckström et al. 1982, Marshall & Kelch 1986, Wu et al. 1990). The synthesis and secretion of the gonadotropins are regulated primarily by the hypothalamic decapeptide gonadotropin-releasing hormone (GnRH), which is secreted into the hypophyseal-portal circulation in a pulsatile manner (Clarke & Cummins 1982, Levine & Ramirez 1982). The control of LH and FSH synthesis and secretion is complex and involves interplay between the gonads, pituitary and hypothalamus. LH and FSH act on the ovaries and the testes to regulate folliculogenesis, ovulation, spermatogenesis and steroidogenesis. Gonadal steroids and peptides, in turn, act at the hypothalamus and/or pituitary to regulate either positively or negatively LH and FSH synthesis and secretion. The aims of this review are to present the transcriptional regulation of the gonadotropin subunit genes in a physiologic setting and examine the mechanisms that drive those changes.
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Physiologic changes in subunit gene transcription
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Estrous cycle
Changes in gonadotropin subunit mRNA expression have been determined over the course of the estrous cycle in rats and mice. In rats, we have shown that LHß and FSHß mRNAs increase during the time of preovulatory gonadotropin surge. The increase in LHß mRNA precedes that of FSH ß; LHß mRNA began increasing around 1400 h, before the beginning of the LH surge; was maximal at 1700 h; and had returned to basal by 2200 h (Zmeili et al. 1986, Kerrigan et al. 1995). FSHß mRNA did not increase until about 2200 h, 2 h after the increase in serum FSH, and was maximal at 0200 h during estrus and had returned to basal by 0800 h (Ortolano et al. 1988). In contrast, mRNA did not change during the preovulatory surge (Zmeili et al. 1986). The periovulatory changes in the ß-subunit mRNAs are coincident with increased GnRH secretion (Levine & Ramirez 1982) and result from increased gene transcription. ß-subunit mRNA synthesis, as measured by nuclear run-on assays, was found to be greatest during proestrus for LHß, and during late proestrus and estrus for FSHß (Shupnik et al. 1989a). Similarly, FSHß promoter activity was increased on estrus (versus metestrus) in transgenic mice harboring an ovine FSHß promoter-luciferase (LUC) reporter gene (Huang et al. 2001a,b).
In addition to the changes in subunit mRNAs that occur around the preovulatory gonadotropin surge, FSHß mRNA expression increased at metestrus, and both and LHß mRNAs increased in parallel during diestrus, periods when serum LH and FSH levels are low. The mechanism(s) for the these changes are not well understood, but probably reflect the sensitivity of the subunit genes to differences in GnRH pulse frequencies and steroid milieu; and for FSHß, changes in serum and/or intrapituitary inhibin, activin or follistatin (FS).
Gonadectomy
Several studies have shown that gonadotropin subunit gene expression is differentially regulated after gonadectomy. The loss of negative feedback by sex steroids at the hypothalamus results in increased GnRH pulse amplitude and frequency (Levine & Ramirez 1982). This increase in GnRH drives expression of all three subunit genes, but there are significant differences in the magnitudes of change and timing, both among the subunits and between the sexes.
In male rats, castration (CAST) results in a rapid increase in serum LH, reflecting an increase in GnRH secretion. Coincident with the increase in GnRH, all three subunit mRNAs are elevated 24 h after CAST (Papavasiliou et al. 1986, Dalkin et al. 2001, Burger et al. 2004). Whereas and LHß mRNA concentrations continue to increase steadily after CAST (Papavasiliou et al. 1986, Dalkin et al. 1990), increases in FSHß mRNA are more modest and begin to decline by day 7 (Gharib et al. 1987, Dalkin et al. 1990).
The increases in subunit mRNA after CAST are regulated in part at the level of transcription. We have recently investigated the changes in subunit gene transcription after CAST by measuring subunit primary transcript (PT) concentrations (Fig. 1
). These transcripts are newly formed RNA that include both exon and intron sequences, prior to RNA splicing, and thus are closely linked to gene transcription and mRNA formation (Dalkin et al. 2001). After CAST, LHß PT increases within 8 h and remains elevated (Dalkin et al. 2001, Burger et al. 2004). Similarly, LHß promoter activity has been reported to increase after CAST in transgenic mice harboring either a rat LHß promoter-LUC construct (Fallest et al. 1995) or an ovine LHß-chloramphenicol acetyltransferase (CAT) reporter (McNeilly et al. 1996). Although FSHß mRNA was elevated within 24 h of CAST, we did not observe an acute increase in FSHß PT (Burger et al. 2004). However, FSHß transcription was increased 5–7 days after CAST, as measured by either nuclear run-on assays (Paul et al. 1990), PT (Dalkin et al. 2001) or FSHß promoter activity in transgenic mice carrying an ovine FSHß-LUC reporter gene (Huang et al. 2001a,b). In contrast, changes in endogenous gene transcription after CAST are not consistent; we have reported that PT was unchanged at day 7 (Dalkin et al. 2001), whereas mRNA synthesis was increased on day 5 (Paul et al. 1990). Additionally, promoter activity increased after CAST in transgenic mice carrying either a human or bovine promoter-CAT reporter (Clay et al. 1993); in vitro, the activity of a human promoter-LUC reporter was greater when transfected into pituitary cells from male rats that had been CAST for 7–21 days (versus intact male rats) (Colin et al. 1996).
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Figure 1 The effects of GnRH antagonist (A) and testosterone (T) on pituitary ß-subunit primary transcripts (PT) in CAST male rats. Rats (n=5–10/group) were CAST only or CAST and treated with GnRH antagonist LRF-147 (200 µg, s.c.) every 12 h with and without silastic T implants (designed to achieve T levels of 3.5 ng/ml). Rats were killed 0, 8 and 24 h later. All data are presented as percent 0-h (±S.E.) controls. Bars with different letters are significantly different (P<0.05). Reproduced with permission from Burger et al. 2004.
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The utilization of GnRH antagonists or androgens to suppress endogenous GnRH demonstrates that the increased subunit transcription after CAST is GnRH dependent. The synthesis rates of all three subunit mRNAs were no different between intact rats and 4-day CAST rats treated with a GnRH antagonist (Paul et al. 1990), and we have also shown that a GnRH antagonist suppresses both LHß and FSHß PT below intact levels within hours after CAST (Fig. 1
) (Burger et al. 2004). Similarly, in transgenic mice, either GnRH antagonist or testosterone suppressed the post-CAST increases in rat LHß-LUC (Fallest et al. 1995), ovine LHß-CAT (McNeilly et al. 1996), and both human and bovine -CAT promoter constructs (Clay et al. 1993). The elevated transcription rates seen after CAST require sustained GnRH input. Administration of a GnRH antagonist to 7-day CAST rats rapidly reduced LHß and FSHß PT, with half-disappearance times of 2.7 and 0.75 h respectively (Fig. 2) (Dalkin et al. 2001).
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Figure 2 Disappearance rates for the gonadotropin subunit primary transcripts (PT) in 7-day CAST rats. PT concentration (fmol PT/100 µg RNA) is shown on the vertical axis, and time after GnRH antagonist treatment (h) is shown along the horizontal axis. The shaded areas represent the range observed in intact animals. The calculated t1/2 for the LHß and FSHß subunits is displayed on the respective curves. Reproduced with permission from Dalkin et al. 2001.
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Gonadotropin subunit gene expression is also differentially regulated after ovariectomy (OVX). In female rats, serum LH rises more slowly after OVX and is not significantly elevated until days 2 or 3 (Dalkin et al. 1993, Burger et al. 2001). Coincident with the increase in GnRH, both and LHß mRNA expression begins to rise about day 3, and continues to rise through day 21 (Dalkin et al. 1990, 1993, Burger et al. 2001). In contrast, both serum FSH and FSHß mRNA increase rapidly after OVX; serum FSH doubles by 8 h and FSHß mRNA by 30–60 min (Dalkin et al. 1993), and they continue to rise through day 7 (Dalkin et al. 1990, 1993, Burger et al. 2001). As in males, increased subunit mRNA levels result from elevated transcription rates; mRNA synthesis rates were increased for all three subunits in 30-day OVX rats (Shupnik et al. 1988). Recently, we have examined the changes in subunit transcription in the 7 days after OVX during the dynamic period after the loss of gonadal feedback (Fig. 3) (Burger et al. 2001). We found that LHß PT concentrations were increased after day 3, paralleling changes in serum LH and LHß mRNA. In contrast, there was a biphasic change in serum FSH, FSHß mRNA and FSHß PT after OVX, with an acute increase at 12–24 h, followed by an additional increase after 72 h. Although mRNA was elevated after OVX, as in our earlier studies in males, -PT did not change.
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Figure 3 The changes in gonadotropin subunit primary transcript (PT) concentrations following OVX. Pituitaries were collected from intact rats or rats OVX for the indicated times. n=5–9/group. Each bar represents the mean±S.E. Bars with different letters are significantly (P < 0.05) different. Reproduced with permission from Burger et al. 2001.
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As in males, increased GnRH secretion after OVX is an important regulator of subunit transcription. Suppression/blockade of GnRH by administration of either a GnRH antagonist or estradiol (E2), in vivo, reduced and LHß mRNA synthesis rates significantly, but had either no effect or a modest suppression on FSHß in long-term OVX rats (Shupnik et al. 1988, 1990, Fallest & Shupnik 1994). Similarly, we found that GnRH blockade prevented the post-OVX increase in LHß-PT, but had no effect on the acute (12–24-h) increase in FSHß-PT and only partially suppressed the increase in FSHß PT after day 3 (Fig. 4). Sustained GnRH secretion is also required to maintain post-OVX increases in ß-subunit transcription; giving a single dose of GnRH antagonist to 7-day OVX rats rapidly reduced both LHß and FSHß PT, with half-disappearance times of 13 and 17 min respectively (Fig. 5).
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Figure 4 The effects of GnRH antagonist on gonadotropin subunit primary transcript (PT) concentrations after OVX. Pituitaries were collected from intact rats, and 12- and 72-h OVX rats treated either with the GnRH antagonist LRF-147 (+) or vehicle (–, BSA-saline) n=5–7/group. Each bar represents the mean±S.E. * Indicates means are significantly different (P<0.05) from intact rats. ** Indicates antagonist-treated group is significantly different (P<0.05) from OVX (vehicle, –) at the same time point. Reproduced with permission from Burger et al. 2001.
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Figure 5 The effects of GnRH antagonist on ß-subunit primary transcript (PT) half-disappearance times (t1/2). Pituitaries were collected from 7-day OVX rats treated with the GnRH antagonist LRF-147 (30 µg i.v.) and then killed 0, 30, 60 or 120 min later (n=5–6/time). Each point represents the mean±S.E. The shaded areas represent the mean ±S.E. observed in intact rats. The calculated t1/2 for LHß and FSHß primary transcripts is displayed on the respective curves. Reproduced with permission from Burger et al. 2001.
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While and LHß transcription after OVX appear to be largely regulated by GnRH, control of FSHß transcription is more complex. The modest effect of either GnRH antagonist or E2 in suppressing FSHß transcription in vivo, hinted at a role for inhibin, activin and/or FS. We previously showed that administration of inhibin- antisera to intact female rats mimics the GnRH-independent increases in FSH and FSHß mRNA that occur within hours after OVX (Dalkin et al. 1993, 1998). Similarly, we found that FSHß PT levels increased rapidly after inhibin immunoneutralization and were suppressed in OVX rats treated with recombinant human inhibin, suggesting that inhibin suppresses FSHß transcription (Burger et al. 2001). However, we also found that inhibin suppressed FSHß mRNA levels much faster than after a GnRH antagonist, suggesting that inhibin may also affect FSHß mRNA stability.
Several transgenic mouse models have been used to investigate the effects of OVX on subunit promoter activity. Hamernik et al.(1992) reported CAT that E2 suppressed activity of either human or bovine CAT constructs in OVX mice and that OVX levels could be restored with pulsatile GnRH. Activity of rat, ovine and bovine LHß promoter–reporter constructs increased after OVX in transgenic mice, and were suppressed by either GnRH antagonist (Fallest et al. 1995, Quirk et al. 2001) or E2 (Fallest et al. 1995, McNeilly et al. 1996). Huang et al. (2001a, b) reported that FSHß promoter activity is increased after OVX in transgenic mice harboring an ovine FSHß –LUC construct.
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Gonadotropin subunit regulation by GnRH
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Physiologic changes in subunit transcription by pulsatile GnRH
A central question in studying the physiology of the gonadotropins is how a single hormone (GnRH) acting on a single cell type (gonadotrope) can differentially regulate two hormones (LH and FSH). The answer is that GnRH differentially regulates LH and FSH synthesis via changes in the pattern of GnRH pulse secretion. Our laboratory and others have investigated the effects of GnRH pulse amplitude and frequency on subunit mRNA expression both in vivo and in vitro. In GnRH-deficient male rats, fast-frequency GnRH pulses (every 8 min) favored expression of and, to a lesser extent, LHß mRNA, fast-physiologic GnRH pulses (every 30 min, the approximate frequency of GnRH pulses found in CAST male rats or intact female rats on the afternoon of proestrus (Levine & Duffy 1988, Levin & Ramirez 1982) increased all three subunit mRNAs, and slow-frequency pulses (>120-min intervals) selectively increased FSHß mRNA (Haisenleder et al. 1988, Dalkin et al. 1989, Kirk et al. 1994). GnRH pulse amplitude also differentially regulates subunit mRNA, though to a lesser extent than pulse frequency; only LHß mRNA expression was sensitive to GnRH pulse amplitude and was maximally stimulated by GnRH pulse doses of 25 ng or less (Haisenleder et al. 1988, Iliff-Sizemore et al. 1990).
GnRH regulation of subunit mRNA is somewhat different in female rats. In initial studies, we reported that GnRH pulses increased and FSHß mRNA levels, but not LHß in GnRH-deficient OVX rats (Kerrigan et al. 1993), a finding which was consistent with the lack of LHß mRNA response to GnRH reported in cultured pituitary cells from OVX rats (Weiss et al. 1990). Subsequently, we found that testosterone was required for GnRH pulses to increase LHß mRNA in OVX rats, and that the optimal dose of testosterone was similar to levels seen at proestrus (Yasin et al. 1996). Although testosterone is required in females, it is not acutely required in males, as GnRH increased LHß mRNA in short-term CAST males with or without T (Yasin et al. 1996). On a background of proestrus T levels, we have found that GnRH pulse amplitude and frequency differentially regulate subunit mRNA expression in females similar to males. Alpha and FSHß mRNAs were increased by a wider range of GnRH pulse amplitudes, while LHß mRNA was maximally stimulated by lower GnRH pulse doses (Dalkin et al. 1999). Fast-physiologic GnRH frequencies (8–120-min intervals) stimulated all three subunit mRNAs, but only slow-frequency GnRH pulses (every 240 min) increased FSHß (Dalkin et al. 1999). The effects of GnRH pulse amplitude and frequency have also been examined in cultured pituitary cells. In female rat pituitary cells, only mRNA increased with high-amplitude GnRH pulses, whereas LHß and FSHß mRNAs responded to lower doses of GnRH (Haisenleder et al. 1993a). In male pituitary cells, ß mRNA was increased by all GnRH pulse frequencies examined, LHß mRNA was maximally stimulated by pulses every 30 min, and FSHß mRNA was greatest after 120-min pulses (Kaiser et al. 1997b).
Although there is some evidence that GnRH may regulate subunit mRNA concentrations via mRNA stability (Salton et al. 1988, Weiss et al. 1992), GnRH appears to exert its main action at the transcriptional level. While continuous GnRH stimulated transcription in vitro (Shupnik 1990), a pulsatile GnRH input was required to increase ß-subunit transcription both in vivo (Haisenleder et al. 1991) and in vitro (Shupnik 1990, Shupnik & Fallest 1994). Furthermore, pulsatile GnRH increased ß-subunit transcription in a frequency-dependent manner. Faster GnRH pulse frequencies (
60-min interpulse interval) preferentially increased LHß mRNA synthesis or PT levels both in vitro (Shupnik 1996) and in vivo (Haisenleder et al. 1991, Burger et al. 2002). In contrast, slower GnRH pulse frequencies (60-min intervals) preferentially increased FSHß transcription. Alpha-subunit transcription does not appear to be tightly regulated by GnRH pulse frequency, some studies indicating that transcription is favored by fast-frequency GnRH pulses (Haisenleder et al. 1991, Shupnik et al. 1996), and others showing that fast and slow frequencies are equally effective (Haisenleder et al. 2001, 2003a, Burger et al. 2002).
The effects of a GnRH pulse on subunit transcription are both rapid and transient. In male rats, LHß and FSHß PTs increased six- and fourfold 5 min after a GnRH pulse, and declined to basal levels by 30 min, with FSHß PT decreasing faster than LHß PT (Dalkin et al. 2001). Although PT tended to increase 5–15 min after a GnRH pulse, the rise was not statistically significant. The duration of GnRH pulsatile treatment also appears to be important in regulating subunit transcription rates. In earlier studies, we found that GnRH pulses given every 30 min to male rats increased , LHß and FSHß mRNA synthesis rates, but that the increases in LHß and FSHß returned to basal at 4–24 h (Haisenleder et al. 1991). In a more recent study, we found that only fast-frequency GnRH pulses (30 min) increased LHß PT levels, and that the increase in transcription was sustained after 1–24 h of pulses (Fig. 6) (Burger et al. 2003). Unexpectedly, we found that both fast- and slow-(240-min) frequency GnRH pulses increased FSHß PT, but with significantly different time courses. Fast-frequency GnRH pulses transiently increased FSHß PT after 1–6 h, but returned to control levels by 24 h. In contrast, slow-frequency GnRH pulses resulted in a delayed but sustained increase in FSHß PT at 8–24 h, and only 240-min pulses increased both FSHß PT and mRNA expression.
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Figure 6 The effects of fast- and slow-frequency GnRH pulses over 24 h on gonadotropin subunit primary transcripts (PT). CAST plus testosterone-replaced male rats were i.v. pulsed with 25 ng GnRH every 30 (fast) or 240 (slow) min for 1–24 h (n=4–8 rats/observation). Data are expressed as fold change versus controls (0 h). * Indicates significant differences (P<0.05) versus untreated, CAST plus testosterone (T) controls (0 h). ** Indicates significant differences between GnRH pulse regimens at 24 h. Reproduced with permission from Burger et al. 2002.
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A recent study investigated the effects of GnRH pulse frequency on subunit promoter activities in gonadotrope-derived LßT2 cells and found similar results to those seen in vivo or in cultured pituitary cells (Bedecarrats & Kaiser 2003). Fast-frequency GnRH pulses (60 min) stimulated expression of a rat LHß promoter-reporter to a greater degree than slower frequencies. In contrast, a rat FSHß promoter-reporter was preferentially stimulated by slower GnRH pulse frequencies (60 min), but a clear preference for a slower GnRH frequency was not observed until after 20 h of pulses, before which fast GnRH frequencies were as effective if not better in stimulating FSHß promoter activity (Bedecarrats & Kaiser 2003). Alpha promoter activity was also stimulated by GnRH in LßT2 cells but was less frequency dependent than the ß-subunits; human -LUC reporter activity increased with all GnRH frequencies but was greatest with more frequent GnRH pulses (Bedecarrats & Kaiser 2003).
Intracellular mechanisms of subunit gene transcription by GnRH
The GnRH receptor (GnRH-R) is a member of the seven-transmembrane receptor family, with receptor binding activating two specific GTP-binding proteins (Gq, G11) (Hsieh & Martin 1992, Stojilkovic & Catt 1995b, Kaiser et al. 1997a, Liu et al. 2002b). GnRH-R activation stimulates increased phosphoinositide turnover and a rise in intracellular diaclyglycerol levels, resulting in the activation of protein kinase C (PKC) (Andrews & Conn 1986, Stojilkovic & Catt 1995a). Activation of the GnRH-R also stimulates a transient rise in intracellular calcium, derived from inositol 1,4,5-triphosphate-induced release from intracellular storage pools and from influx via voltage-sensitive(L-type) channels (Naor 1990, Ando et al. 2001). GnRH also stimulates an increase in cAMP (Bourne 1988, Garrel et al. 1997) and activates members of the mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinase (ERK) (Mitchell et al. 1994, Roberson et al. 1995, Sundaresan et al. 1996, Reiss et al. 1997), c-Jun NH2-terminal kinase (JNK) (Levi et al. 1998, Roberson et al. 1999, Yokoi et al. 2000) and p38 (Roberson et al. 1999, Liu et al. 2002a) in several species.
As discussed previously, gonadotropin subunit gene transcription is regulated in a differential manner by alterations in GnRH pulse frequency. Recent data suggest that various mammalian species express a second GnRH isoform (GnRH II) and GnRH-R (GnRH-R II) in the hypothalamus and pituitary (Neill 2002). Although effects on sexual behavior and preferential actions on FSH secretion have been described, the physiologic roles of these isoforms remain to be determined. The evidence to date suggests that differential synthesis and secretion of LH and FSH is the product of altered patterns of pulsatile GnRH I secretion by activation of pituitary GnRH-R I. Although the intracellular messengers responsible for transmitting frequency-dependent signals from the plasma membrane to the nucleus within the gonadotrope have yet to be fully characterized, recent findings have provided insights into critical sites in the signal transduction pathways involved.
cAMP
Activation of the cAMP pathway stimulates (mouse, rat and human) promoter activity (Maurer et al. 1999), and cAMP may also have a general stimulatory response to GnRH signaling. The mouse GnRH-R promoter contains two cAMP responsive element-binding (CREB) sites, and increased cAMP/protein kinase A (PKA) activity enhanced mouse GnRH-R promoter activity in lactotropic GH3 cells stably transfected with rat GnRH-R cDNA (GGH3 cell lines) (Han & Conn 1999, Lin & Conn 1999). However, studies conducted in either gonadotrope-derived T3 cells or transgenic mice models reveal that PKC, MAPK and activator protein-1 (AP-1) play a more prominent role in GnRH regulation of the GnRH promoter (Norwitz et al. 1999, White et al. 1999, Ellsworth et al. 2003). In the human model, CREB plays an essential role in GnRH activation of the promoter, and GnRH stimulates the phosphorylation of CREB (Delegeane et al. 1987, Duan et al. 1999). The porcine FSHß promoter also contains a putative CREB site (Kato et al. 1999). We have conducted in vitro studies in rat pituitary cells to determine whether cAMP stimulates gonadotropin subunit mRNA (Haisenleder et al. 1992). The results demonstrated that a diffusible cAMP analog stimulated a rise in , but not LHß or FSHß mRNA. Of interest, we showed that the pattern of cAMP input was critical, as pulsatile, but not continuous, cAMP enhanced mRNA expression. More recent studies suggest that the cAMP/PKA pathway plays a role in cross-talk between specific intracellular messenger systems (that is, PKC and ERK) in response to GnRH stimulation (Garrel et al. 1997, Han & Conn 1999, Lin & Conn 1999, Fowkes et al. 2002).
Calcium
The GnRH-induced increases in intracellular calcium play an essential role in gonadotropin secretion (Naor 1990, Stojilkovic & Catt 1995a,b). Studies in various species and experimental models have also shown that , LHß and FSHß gene expression are regulated by increases in intracellu-lar calcium (Ben Menahem & Naor 1994, Saunders et al. 1998, Weck et al. 1998). In the rat, calcium stimulates a rise in , LHß and FSHß mRNA levels (Ben Menahem & Naor 1994, Haisenleder et al. 1993b, 1997). Other reports reveal that calcium stimulates gonadotropin subunit transcriptional activation, including increases in and LHß transcription (as measured by nuclear run-on assay or promoter–luciferase construct assay) (Weck et al. 2000) and , LHß and FSHß PT (Haisenleder et al. 2001). In contrast, other investigations using GH3 cells that express GnRH-R (GGH3–1 cells) have shown that the response to calcium is selective, stimulating but not LHß and FSHß (Saunders et al. 1998). Another report found that calcium suppresses GnRH- or PKC-induced rat LHß promoter activity in LßT4 cells (Vasilyev et al. 2002a). The reasons for these different gonado-tropin subunit responses to calcium remain to be determined. However, it is likely that factors such as differences in cell model, promoter constructs used, experimental paradigms and the end products measured play a role in the outcomes seen.
Gonadotrope cells express spikes and oscillations in intracellular calcium, the patterns of which are altered by GnRH and linked to LH secretion (Holl et al. 1988, Tse & Hille 1992, Stojikovic & Catt 1995b). These observations are of interest, as in other cell types, alterations in intracellular calcium can play a role in the regulation of gene expression and activation of downstream mediators of signal transduction pathways (that is, ERK and calcium/ calmodulin-dependent kinase II (Ca/CaMK II)) (Villalobos et al. 1988, Gu & Spitzer 1995, Dolmetsch et al. 1997, De Koninck et al. 1998, Durham & Russo 2000). We have investigated whether alterations in intracellular calcium are involved in transmission of frequency-dependent signals from the plasma membrane to the nucleus, and whether these signals mediate the , LHß and FSHß transcriptional responses to pulsatile GnRH (Haisenleder et al. 2001). The model used cultured rat pituitary cells and pulses of the calcium channel activator, Bay K 8644, plus potassium chloride (BK+KCl) given at intervals of 15, 60 or 180 min for 6 h. Gonadotropin subunit PT was measured to determine transcriptional responses to treatment. BK+KCl pulses stimulated release of both LH and FSH with a similar pattern and magnitude to those seen after pulsatile GnRH. Alterations in the frequency of calcium pulse signals revealed that was selectively stimulated by faster (every 15 min) pulses of BK+KCl, with slower pulses being ineffective (Fig. 7). LHß PT was stimulated by 15-or 60-min pulses, but not 180-min pulses, of BK+KCl. In contrast, FSHß PT was maximally stimulated by the slower (180-min) pulse interval. This pattern of divergent gonadotropin subunit responses to pulse frequency is similar to previous findings for GnRH (Haisenleder et al. 1997). Thus, intermittent changes in intracellular calcium appear to be important in the transmission of GnRH signals, and may mediate the differential actions of pulse frequency on gonadotropin subunit gene expression.
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Figure 7 Effect of the calcium channel activator Bay K 8644 (BK) pulse interval (15, 60 or 180 min) on LHß, and FSHß primary transcript (PT) levels. Pulse treatments were given for 6 h, and a GnRH pulse group (60-min interval) was included for comparison. Results are expressed as the percent change versus vehicle-pulsed controls (Con). Means±S.E.M. are shown; note the different scale for each panel. The number of chambers per group was 10–12, derived from four or five separate experiments. *P<0.05 versus control, #P<0.05 versus 15' pulse BK group; +P<0.05 versus peak BK treatment group. Reproduced with permission from Haisenleder et al. 2001.
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The downstream mediators of calcium action within the gonadotrope have yet to be characterized. Among the putative candidates is Ca/CaMK II, which is an important mediator of calcium signaling in several cell types, including the pituitary (Miller & Kennedy 1986, Hanson & Schulman 1992, Cui et al. 1994, 1996, Schulman et al. 1995, Nowakowski et al. 1997). We have examined this issue in both primary pituitary and LßT2 cells (Haisenleder et al. 2003a,b). GnRH stimulated Ca/CaMK II activity, and activation was mediated via increases in intracellular calcium derived from cellular storage pools and influx from plasma membrane calcium channels. Moreover, inhibition of Ca/CaMK II activation by administering a specific blocker, KN-93, partially suppressed GnRH-induced increases in , LHß and FSHß PT (Fig. 8) or rat and LHß promoter activity. These finding support the hypothesis that Ca/CaMK II is a downstream mediator of GnRH/calcium signaling within the gonadotrope.
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Figure 8 , LHß and FSHß primary transcript (PT) responses to pulsatile GnRH with and without the Ca/CAMK II-specific inhibitor, KN-93 (10 µM). Rat pituitary cells received pulses of GnRH (200 pM, 60-min intervals) for 24 h. Results are expressed as the percent change versus medium-pulsed controls (Con). Note the different scale for each panel. Groups marked with different letters (a, b or c) are statistically different (P<0.05). Reproduced with permission from Haisenleder et al. 2003a.
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Protein kinase C (PKC)
Several investigations have shown that GnRH activates PKC, and that cross-talk between PKC, calcium and other intracellular pathways regulates gonadotropin secretion and mRNA expression (Naor 1990, Stojilkovic & Catt 1995b, Kaiser et al. 1997a, Ando et al. 2001). Studies in rat pituitary cells reveal that PKC stimulates a rise , LHß and FSHß mRNAs and ß transcription (Ben Menahem & Naor 1994, Weck et al. 1998, Halvorson et al. 1999, Vasilyev et al. 2002a). We have shown that PKC stimulates and LHß gene expression in rat pituitary cells in vitro; however, unlike results seen for cAMP and calcium, a pulsatile signal pattern does not play a significant role (Haisenleder et al. 1995). Data from other investigations in gonadotrope-derived LßT2, LßT4 or T3 cells, or lactotrope-derived GGH3–1 cells, demonstrate that PKC mediates the , LHß and FSHß transcriptional response to GnRH (Sundaresan et al. 1996, Weck et al. 1998, Vasilyev et al. 2002a,b). Kaiser et al.(2000) reported that PKC stimulation of the rat LHß gene is mediated via actions on Egr-1 binding within the proximal GnRH responsive region of the LHß promoter. An investigation of the bovine LHß promoter showed that interactions between Egr-1, SF-1 and Ptx-1 mediate the response to PKC (Tremblay & Drouin 1999). PKC has also been shown to stimulate the expression of the Egr-1 gene (Wolfe & Call 1999). In contrast, -subunit transcriptional responses to PKC reflect activation of the MAPK pathway (as discussed below). Characterization of the ovine FSHß promoter showed that PKC activation is mediated through multiple responsive regions, including 2 AP-1 sites (Strahl et al. 1998). However, these sites do not appear to play an important role in regulating the ovine FSHß promoter in transgenic mice (Huang et al. 2001b). Of interest, responses of individual gonadotropin subunits to PKC stimulation have been shown to differ between published reports, posssibly reflecting differences in cell model or experimental paradigm (Saunders et al. 1998, Weck et al. 1998, Vasilyev et al. 2002a,b).
Mitogen-activated protein kinase (MAPK)
A number of investigators have shown that GnRH stimulates activation of various members of the MAPK family, including ERK 1 and 2 (Mitchell et al. 1994, Roberson et al. 1995, Sundaresan et al. 1996, Reiss et al. 1997), JNK (Levi et al. 1998, Roberson et al. 1999, Yokoi et al. 2000) and p38 (Roberson et al. 1999, Liu et al. 2002a). GnRH-induced activation of ERK is mediated through both PKC-dependent and PKC-independent pathways, including calcium (Roberson et al. 1995, Sundaresan et al. 1996, Reiss et al. 1997, Mulvaney et al. 1999). Moreover, ERK phosphorylates various transcription factors that may play roles in the regulation of gonadotropin subunit gene expression (such as c-Fos, c-Jun and the Ets protein – ELK-1 (Seger & Krebs 1995, Maurer et al. 1999, Liu et al. 2002a).
We conducted studies to determine whether ERK plays a role in the differential gonadotrope responses to physiologic GnRH stimulation (Haisenleder et al. 1998). Initial work examined whether a pulsatile signal pattern is required to maintain ERK responsiveness to GnRH over longer durations. Groups of adult GnRH-deficient rats were given pulses of GnRH every 60 min for 1–8 h in vivo, and were compared with animals that received a continuous GnRH infusion for the same duration. Pulsatile GnRH stimulated a two- to fourfold increase in ERK activity, which was maintained over the 8-h experimental duration. However, continuous GnRH stimulated a transient rise in ERK only for the initial 2 h (Fig. 9). Follow-up studies showed that ERK activity is sensitive to GnRH pulse frequency, with slower (120-min) interval pulses being more effective than faster (30- or 60-min) interval pulses. To determine the role of ERK in mediating GnRH-induced stimulation of gonadotropin subunit gene expression, GnRH pulses were administered to rat pituitary cells in vitro, with or without the ERK inhibitor, PD-098059 (PD). As shown in Fig. 10, PD blocked the GnRH-induced rise in , FSHß and GnRH-R mRNAs, but not LHß. This suggests that GnRH-induced stimulation of LHß utilizes a distinct and different intracellular pathway or pathways. It is interesting to note that ERK is maximally stimulated by slower GnRH pulse intervals, and plays an important role in regulating the expression of two gonadotrope genes that are also maximally stimulated by slower GnRH pulse intervals (FSHß and GnRH-R).
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Figure 9 ERK responses to pulsatile or continuous GnRH after 1, 2, 4 or 8 h. GnRH was administered to adult male rats either in a pulsatile (50 ng/pulse, 60-min interval) or continuous manner (25 ng/min; this dose was selected to provide sustained levels of circulating GnRH similar to peak levels obtained after a single 50-ng pulse (that is, 200 pg/ml)). Controls (C) received BSA-saline pulses or a continuous infusion for 8 h. The means±S.E.M. are shown (n=3/group, except in the pulsatile control group, in which n=4. * P<0.05 versus controls. Reproduced with permission from Haisenleder et al. 1998.
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Figure 10 Gonadotrope mRNA responses to pulsatile GnRH (peak chamber concentration, 100 pg/ml) given every 60 min for 24 h in the presence of PD-098059 (PD) or vehicle in perifused male pituitary cells (n=4/group). The data are presented as the percent change versus medium-pulsed controls (Con). Reproduced with permission from Haisenleder et al. 1998.
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GnRH activation of the JNK pathway is mediated through PKC activation of the protein tyrosine kinase – cSrc (Levi et al. 1998). Recent findings suggest that JNK plays a role in the regulation of the rat LHß promoter, via actions on the proximal GnRH-responsive region of the gene (Yokoi et al. 2000). The p38 pathway has also been implicated in the activation of various transcription factors, including c-Jun, c-Fos and Elk-1 (Ham et al. 1997, Whitmarsh et al. 1997, Roberson et al. 1999). Experiments using T3 cells revealed that PKC mediates the GnRH-induced activation of p38 (Roberson et al. 1999). However, while p38 activates downstream pathways known to stimulate gene expression (Maurer et al. 1999), published reports suggest that the signal transduction protein does not stimulate promoter activity (Roberson et al. 1999). Whether p38 regulates the expression of the LHß and FSHß genes remains to be determined.
In summary, present information suggests that differential regulation of gonadotropin subunit gene expression by GnRH involves several mechanisms. GnRH may exert selective actions on specific signal transduction pathways (such as ERK regulation of and FSHß, but not LHß). Alternatively, the responses may reflect differential sensitivity to intracellular messenger systems (for example, specific gonadotropin subunit genes may be more sensitive to calcium or PKC pathways than other subunit genes). Furthermore, it is likely that the role played by GnRH pulse frequency is selectively to activate one or more intracellular signal transduc-tion pathways that are optimal for a specific gonadotropin subunit gene.
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Regulation of subunit gene transcription by steroids
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Steroids regulate gonadotropin subunit gene expression by acting either at the hypothalamus to alter GnRH pulsatility or directly on the pituitary gonadotropes. Differential regulation of the subunit genes by steroids was first reported in gonadectomized (GDX) animals. Both E2 and testosterone suppressed the post-GDX increases in pituitary and LHß mRNAs. In contrast, the post-OVX increase in FSHß mRNA was only partially reduced by E2or E2 plus progesterone (P4), reflecting the importance of ovarian inhibin in suppressing FSHß expression (Gharib et al. 1987, Dalkin et al. 1990, 1993); in CAST rats, testosterone either had no effect or enhanced FSHß responses in a dose-dependent manner (Gharib et al. 1987, 1990a, Wierman et al. 1988, 1990, Iliff-Sizemore et al. 1990). Experiments in GDX plus GnRH antagonist-treated rats, to eliminate the effects of steroids on GnRH secretion, largely show that in vivo steroids inhibit both and LHß gene expression by suppressing GnRH. In contrast, the regulation of FSHß by steroids appears to be more complex.
-subunit
In vivo, E2 markedly suppresses -subunit gene transcription. It inhibited the post-OVX increase in -subunit mRNA synthesis (Shupnik et al. 1988), and suppressed the post-OVX or -CAST increases in promoter activity in transgenic mice harboring either a human or bovine promoter-reporter construct (Keri et al. 1991, Hamernik et al. 1992, Clay et al. 1993). However, there is evidence that the suppression of subunit transcription by E2 is largely indirect. Shupnik et al. determined that E2 reduces transcription in vivo largely by suppressing hypothalamic GnRH; E2 had no effect on -subunit mRNA synthesis in rat pituitary cells in vitro (Shupnik et al. 1989a, Shupnik 1996) or in OVX rats treated with GnRH antagonist (Shupnik & Fallest 1994). Additionally, a functional estrogen response element (ERE) in the human promoter has not been identified (Keri et al. 1991), and E2 does not affect human promoter-reporter activity in T3 cells, even in the presence of an exogenous human estrogen-receptor (ER) (Clay et al. 1993). Although it appears that E2 regulates transcription largely via hypothalamic GnRH, it may also have a direct effect on the pituitary. Colin et al.(1996) reported that E2 suppressed basal activity of a human -promoter-LUC construct in the pituitaries of OVX transgenic mice, but enhanced GnRH responsiveness. We and others have also observed that E2 potentiates GnRH stimulation of gene expression, but it is unknown whether the effect is direct or indirect (that is, increasing GnRH receptor numbers) (Mercer et al. 1989, Dalkin et al. 1990, Kerrigan et al. 1993, Turgeon et al. 1996, Kawakami & Winters 1999).
In vivo, progesterone (P4) potentiates the suppressive effects of E2 on -subunit mRNA, largely via decreased GnRH secretion (Simard et al. 1988, Dalkin et al. 1990). The effects of P4 directly on the mRNA in pituitary are mixed; P4 (+E2) reduces OVX, hypothalamically disconnected ewes (Di Gregorio & Nett 1995), and either reduces (Dalkin et al. 1990) or has no effect in the rat (Kerrigan et al. 1993). Little is known about P4 actions on -subunit transcription; however, neither P4 nor P4 the receptor antagonist RU486 modulated E2 ± GnRH-induced changes in human -LUC activity in pituitary cells from transgenic mice (Colin et al. 1996).
Androgens, like estrogens, suppress subunit expression either by reducing hypothalamic GnRH or by direct action on the pituitary. In vivo, testosterone suppressed mRNA and mRNA synthesis rates in male rats even in the presence of GnRH antagonist (Paul et al. 1990). Similarly, testosterone or dihydrotestosterone (DHT) suppressed the post-CAST increases in promoter activity in transgenic mice harboring either a human or bovine promoter-CAT reporter gene (Clay et al. 1993). The human -CAT reporter gene was also repressed by DHT when transiently transfected into T3 cells along with androgen receptor (AR), with suppression requiring both AR and ligand (Clay et al. 1993). A putative androgen response element (ARE) has been identified in the proximal promoter of the human gene between tandem cAMP response elements (CRE) and a CCAAT element (Clay et al. 1993). However, this binding site does not mediate the suppressive effect of androgen on transcription; instead, AR interferes with the proteins that bind an upstream -basal element and the CRE (Heckert et al. 1997). Specifically, AR suppresses promoter activity by protein–protein interactions with the two CRE-binding transcription factors cJun and ATF2 (Jorgensen & Nilson 2001a).
The effects of glucocorticoids on gene expression are inconclusive. In vivo, corticosterone (B) either increases (Ringstrom et al. 1991), decreases (Kilen et al. 1996), or had no effect (McAndrews et al. 1994) on subunit mRNA in rats. In pituitary-derived cell lines, dexamethasone suppressed -subunit mRNA in T3 cells (Akerblom et al. 1990), had no effect on mRNA in LT2 cells (Turgeon et al. 1996) or increased promoter activity in GH3 cells transiently transfected with a human -CAT construct (Gurr & Kourides 1989).
LHß
Estrogens rapidly suppress the post-OVX increases in LHß mRNA synthesis (Shupnik et al. 1988). This reflects a hypothalamic action, as the post-OVX increases in LHß transcription were abolished by a GnRH antagonist (Dalkin et al. 1993, Shupnik & Fallest 1994, Fallest et al. 1995, Burger et al. 2001), and additional treatment with E2 was no more effective than antagonist only (Shupnik & Fallest 1994). However, estrogens also exert direct action on the pituitary, and E2 rapidly increased LHß mRNA synthesis rates in pituitary cells from OVX female rats (Shupnik et al. 1989a, Shupnik 1996), and in pituitary cells from cycling rats, with the greatest effect in cells from rats in proestrus (Shupnik et al. 1989a). Shupnik and coworkers found that ER bound the rat LHß promoter at –1388 to –1105 bp, an area containing a 15 bp imperfect ERE; moreover, promoter-reporter constructs of the rat LHß gene containing the 284 bp estrogen-responsive region, transiently transfected into GH3 cells, were estrogen responsive in an ER-dependent manner (Shupnik et al. 1989b, Shupnik & Rosenzweig 1991). However, the estrogen responsiveness of the LHß promoter may not be conserved across the species. While E2 suppressed LHß promoter activity in OVX transgenic mice harboring a –776 to +10 bp bovine LHß promoter CAT reporter, a high-affinity binding site for the ER was not found in this portion of the promoter (Keri et al. 1994). In addition, E2 had no effect on bovine LHß CAT activity when transiently transfected into LßT2 cells, even when cotransfected with the human ER (Jorgensen & Nilson 2001b). It remains to be determined whether an estrogen-responsive region of the bovine LHß promoter may be more 5' than the area previously investigated. Alternatively, the ER may modulate promoter activity by interacting with other cis-acting elements, as described for androgens on the and LHß promoters.
There is little information on the regulation of LHß gene expression by either P4 or glucocorticoids. Progesterone alone had no effect on LHß mRNA levels in OVX rats and when combined with E2 was no more effective than E2 alone (Dalkin et al. 1990, Kerrigan et al. 1993). Similarly, while E2 blocked bovine LHß CAT promoter activity in OVX transgenic mice, the combination of E2 and P4 was no more effective than E2 alone (McNeilly et al. 1996). In vitro, neither mRNA levels in female P4 nor E2+ P4 altered LHß rat pituitary cells, but P4 alone or in combination with E2 augmented the GnRH-induced increase in LHß mRNA, perhaps by blocking LHß mRNA degradation (Park et al. 1996). Glucocorticoids do not appear to regulate LHß mRNA; B had no effect on LHß mRNA levels in cultured rat pituitary cells (Kilen et al. 1996) and dexamethasone did not alter the activity of the rat LHß promoter (Shupnik & Rosenzweig 1991).
Androgens rapidly suppress the post-CAST rise in LHß transcription. Testosterone inhibited LHß mRNA synthesis in CAST rats (Paul et al. 1990) and suppressed the post-CAST increases in LHß promoter activity in transgenic mice (Keri et al. 1994, Fallest et al. 1995). We examined the effects of testosterone on LHß transcription in vivo; testosterone rapidly suppressed LHß PT below basal values in GnRH-deficient CAST rats (Fig. 11) (Burger et al. 2004). The suppression of LHß PT in GnRH-deficient rats in vivo suggests that testosterone acts directly at the pituitary, but we found no effect of testosterone on LHß PT in cultured rat pituitary cells (Burger et al. 2004). Similarly, DHT had little effect on rat LHß promoter LUC reporter activity in LßT2 cells, but did suppress GnRH-induced LHß promoter activity (Curtin et al. 2001). This action required the AR even though a high-affinity binding site for the AR was not found in this construct. Curtin et al.(2001) determined that the AR suppressed rat LHß promoter activity by protein–protein interactions with the specificity protein-1 (Sp1) transcription factor, and, to a lesser extent, with the early growth response protein (Erg-1), which are required for GnRH stimulation of LHß transcription (Kaiser et al. 2000, Weck et al. 2000). In contrast to the rat, the bovine LHß promoter was directly responsive to testosterone (Jorgensen & Nilson 2001b). The AR, in a ligand-dependent manner, suppressed the activity of bovine LHß CAT when transfected into LßT2 cells. The bovine LHß promoter also lacks an AR binding site, and the AR suppressed bovine LHß transcription by protein–protein interactions with steriodogenic factor 1 (SF-1), blocking its interaction with Pitx1 and the transcriptional initiation complex (Jorgenson & Nilson 2001b).
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Figure 11 The time course of testosterone (T) action on pituitary ß-subunit primary transcripts (PT) and mRNAs in GnRH antagonist (GnRH-A)-treated CAST male rats. Castrated rats (n=4–7/group) were treated with GnRH-A LRF-147 (100 µg, s.c.) every 12 h. Four days after CAST, rats received T implants and were killed 0, 3, 8, 24 and 48 later. All data are presented as percent 0-h T (±S.E.) controls. Intact rat (±S.E.) control values are represented by shading. Points with different letters are significantly different (P<0.05).
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FSHß
The effects of E2 on FSHß transcription have not been extensively studied, and there appear to be differences among species. In vivo , E2 suppressed the post-OVX increase in FSHß mRNA synthesis in the rat (Shupnik et al. 1988). The site of E2 action in the rat is the hypothalamus, as E2 did not suppress FSHß transcription in OVX rats treated with a GnRH antagonist (Shupnik et al. 1989a) and had no effect on FSHß mRNA synthesis in female rat pituitary fragments (Shupnik & Fallest 1994). In contrast to the rat, E2 suppressed both steady-state FSHß mRNA and mRNA synthesis rates in cultured ovine pituitary cells (Phillips et al. 1988, Baratta et al. 2001), and suppressed the activity of an ovine FSHß promoter-LUC construct trans-fected into ovine pituitary cells (Miller & Miller 1996). The estrogen-responsive region of the ovine FSHß promoter is located at –105 to –84 bp, but does not contain an estrogen-response element or bind ER (Miller & Miller 1996). Instead, this estrogen-responsive region contains two AP-1 sites and is a GnRH enhancer region of the ovine FSHß promoter (Strahl et al. 1998). Although E2 inhibits FSHß transcription in sheep, it had no effect on an ovine FSHß promoter-LUC construct in female transgenic mice, and the authors suggest that mouse gonadotropes lack some factor making them less responsive to negative feedback by estrogen (Huang et al. 2001a). We recently investigated the effects of steroids on FSHß transcription in male rats. Contrary to previous results in females, we PT, but found that E2 markedly suppressed FSHß not FSHß mRNA, and this may suggest that E2 differentially regulates FSHß transcription between the sexes in rodents (Burger et al. 2004).
FSHß transcription is also regulated by P4, and, like estrogens, there appear to be differences between species. Progesterone, in combination with mRNA expression in E2, increased FSHß immature rats (Attardi et al. 1990) and increased the stimulatory effects of GnRH on FSHß mRNA (versus E2 alone) (Kerrigan et al. 1993). Moreover, the preovulatory FSH surge and the accompanying increase in FSHß mRNA can be suppressed by antiprogestins (Ringstrom et al. 1997). Three progesterone-response element (PRE)-like sequences have been identified in the proximal promoter of the rat FSHß gene; these sites bind P4 receptor (PR) and render a heterologous promoter–reporter construct P4 responsive (O’Conner et al. 1997, 1999). In contrast, P4 suppressed endogenous FSHß mRNA synthesis in ovine pituitary cell cultures (Phillips et al. 1988). The ovine FSHß promoter contains six PRE-like sequences that bind PR and render a heterologous promoter reporter construct P4 responsive (Webster et al. 1995). However, contrary to the suppressive effects of P4 on endogenous FSHß transcription, P4 stimulated an ovine FSHß promoter-LUC reporter construct transfected into ovine pituitary cells (Webster et al. 1995).
Glucocorticoids affect FSHß gene expression, and B selectively upregulated FSHß mRNA levels (Ringstrom et al. 1991, McAndrews et al. 1994). These effects are at the level of the gonadotrope, as B or dexamethasone increased FSHß mRNA in rat pituitary cells (Kilen et al. 1996, Bohnsack et al. 2000, Leal et al. 2003) and in rats treated with a GnRH antagonist (McAndrews et al. 1994). Moreover, the effects of B appear to be at the level of transcription, as it increased FSHß mRNA in female rat pituitary cells, without changing post-transcriptional mRNA decay rates (Kilen et al. 1996).
The effects of testosterone on FSHß gene expression in the rat are intriguing. As mentioned previously, testosterone does not suppress the post-GDX increase in FSHß mRNA; instead, it either has no effect or further increases FSHß mRNA expression (Gharib et al. 1987, 1990a, Wierman et al. 1988, 1990, Iliff-Sizemore et al. 1990). Studies in GnRH-deficient rats or in pituitary cell cultures demonstrated that the stimulatory effects of testosterone on FSHß gene expression are at the level of the pituitary (Paul et al. 1990, Wierman & Wang 1990, Dalkin et al. 1992, Winters et al. 1992). Earlier we proposed that testosterone regulated FSHß mRNA stability, since testosterone increased the half-disappearance time of FSHß mRNA from 20 to 50 h, but did not significantly increase FSHß mRNA synthesis (Paul et al. 1990). However, we recently reinvestigated the effects of testosterone on FSHß transcription, and found that it rapidly and specifically increases FSHß PT levels at 3–48 h in male rats (Fig. 11) and that the stimulatory effects on FSHß transcription are androgen specific (Burger et al. 2004). Testosterone also suppresses FS mRNA, which suggests that testosterone’s actions on FSHß transcription may be indirect via activin/FS. Others have reported that the effects of testosterone on FSHß mRNA either require activin (Leal et al. 2003) or are blunted by FS (Bohnsack et al. 2000). To determine whether testosterone stimulation of FSHß PT reflected the fall in pituitary FS, we treated male rat pituitary cells with testosterone with or without exogenous FS. FS alone suppressed FSHß PT, but did not reduce the testosterone-stimulated increase in FSHß PT, suggesting that testosterone increases transcription by direct action on the FSHß gene. Recently, testosterone (or DHT) was reported to increase the activity of an ovine FSHß promoter-reporter transfected into LßT2 cells (Spady et al. 2004). Spady et al.(2004) identified three candidate androgen-response elements (AREs) at –245/–231, –212/–198 and –153/–139 bp of the ovine FSHß promoter, which are the same steroid-binding elements identified by Webster et al.(1995) as PREs. Only the –245/–231 bp putative ARE bound the AR, although mutations in either the –245/–231 or –153/–139 ARE disrupted the stimulation of the promoter by DHT (Spady et al. 2004). In contrast to the rat, the effects of testosterone on the ovine FSHß promoter are activin dependent. The ability of DHT to stimulate the ovine FSHß promoter is abolished by FS treatment, is increased synergistically by activin, and requires an activin-responsive Smad-binding element (SBE) at –138/–124 bp (Spady et al. 2004). Although the AREs identified in the ovine FSHß promoter are well conserved across mammalian species (Spady et al. 2004), the stimulatory effects of testosterone on FSHß expression are not. Testosterone suppressed FSHß mRNA in male rhesus pituitary cells (Kawakami et al. 2002) and a human FSHß promoter construct in transgenic mice (Kumar et al. 1992, Kumar & Low 1993, 1995).
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Regulation of FSHß gene transcription by activin, FS and inhibin
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Differential synthesis and secretion of LH and FSH, while in part dependent on the hypothalamic GnRH signal pattern, also appears to result from the actions of multiple local and gonadal peptide hormones. The hormones inhibin, activin and FS are produced both in the gonads and, at least for activin and FS, in the pituitary gland. Alterations in the balance of activin and inhibin concentrations, either directly or via changes in FS (for activin), selectively alter FSH secretion by regulating FSHß gene expression (Mather et al. 1997).
The inhibins and activins are members of the transforming growth factor-ß (TGFß) superfamily. The inhibins, heterodimers of an inhibin -subunit and one of two ß-subunits, ßA (inhibin A) or ßB (inhibin B), are produced primarily by the gonads and act in an endocrine manner to suppress pituitary FSH secretion without affecting LH (Burger et al. 1997). The activins are dimers of two inhibin ß-subunits. There are at least four forms of the ß subunit, ßA–ßD, although the ßA and ßB subunits are important in the regulation of FSH. The activins were discovered because of their ability to stimulate FSH synthesis and release
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Abstract
Introduction
