Pulsatile gonadotropin-releasing hormone stimulation of gonadotropin subunit transcription in rat pituitaries: evidence for the involvement of Jun N-terminal kinase but not p38.

We investigated whether Jun N-terminal kinase (JNK) and p38 mediate gonadotropin subunit transcriptional responses to pulsatile GnRH in normal rat pituitaries. A single pulse of GnRH or vehicle was given to female rats in vivo, pituitaries collected, and phosphorylated JNK and p38 measured. GnRH stimulated an increase in JNK phosphorylation within 5 min, which peaked 15 min after GnRH (3-fold). GnRH also increased p38 phosphorylation 2.3-fold 15 min after stimulus. Rat pituitary cells were given 60-min pulses of GnRH or media plus the JNK inhibitor SP600125 (SP, 20 microM), p38 inhibitor SB203580 (20 microM), or vehicle. In vehicle-treated groups, GnRH pulses increased LHbeta and FSHbeta primary transcript (PT) levels 3-fold. SP suppressed both basal and GnRH-induced increases in FSHbeta PT by half, but the magnitude of responses to GnRH was unchanged. In contrast, SP had no effect on basal LHbeta PT but suppressed the stimulatory response to GnRH. SB203580 had no effect on the actions of GnRH on either LH or FSHbeta PTs. Lbeta-T2 cells were transfected with dominant/negative expression vectors for MAPK kinase (MKK)-4 and/or MKK-7 plus a rat LHbeta promoter-luciferase construct. GnRH stimulated a 50-fold increase in LHbeta promoter activity, and the combination of MKK-4 and -7 dominant/negatives suppressed the response by 80%. Thus, JNK (but not p38) regulates both LHbeta and FSHbeta transcription in a differential manner. For LHbeta, JNK is essential in mediating responses to pulsatile GnRH. JNK also regulates FSHbeta transcription (i.e. maintaining basal expression) but does not play a role in responses to GnRH.

Testosterone stimulates follicle-stimulating hormone beta transcription via activation of extracellular signal-regulated kinase: evidence in rat pituitary cells.

This study investigated whether estradiol (E2) or testosterone (T) activate extracellular signal-regulated kinase (ERK) and calcium/calmodulin-dependent kinase II (Ca/CaMK II), as indicated by enzyme phosphorylation in rat pituitaries. In vivo studies used adult female rats given E2, T, or empty silastic capsules (vehicle controls). Twenty-four hours later, the rats were given a single pulse of GnRH (300 ng) or BSA-saline (to controls) and killed 5 min later. GnRH stimulated a two- to three-fold rise in activated Ca/CaMK II, and E2 and T had no effect on Ca/CaMK II activation. In contrast, both GnRH and T stimulated threefold increases in ERK activity, with additive effects seen following the combination of GnRH+T. E2 had no effect on ERK activity. In alpha T3 clonal gonadotrope cells, dihydrotestosterone did not activate ERK alone but enhanced and prolonged the ERK responses to GnRH, demonstrating direct effects on the gonadotrope. Thus, the ERK response to GnRH plus androgen was enhanced in both rat pituitary and alpha T3 cells. In vitro studies with cultured rat pituitary cells examined the effect of GnRH+/-T in the presence of the mitogen-activated protein (MAP) kinase kinase inhibitor, PD-098059 (PD). Results showed that PD suppressed ERK activational and FSH beta transcriptional responses to T. These findings suggest that one site of T regulation of FSH beta transcription is through the selective stimulation of the ERK pathway.

Regulation of gonadotropin subunit gene transcription.

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.

Gonadotropin-releasing hormone stimulation of gonadotropin subunit transcription: evidence for the involvement of calcium/calmodulin-dependent kinase II (Ca/CAMK II) activation in rat pituitaries.

The intracellular pathways mediating GnRH regulation of gonadotropin subunit transcription remain to be fully characterized, and the present study examined whether calcium/calmodulin-dependent kinase II (Ca/CAMK II) plays a role in the rat pituitary. Preliminary studies demonstrated that a single pulse of GnRH given to adult rats stimulated a transient 2.5-fold rise in Ca/CAMK II activity (as determined by an increase in Ca/CAMK II phosphorylation), with peak values at 5 min, returning to basal 45 min after the pulse. Further studies examined the alpha, LHbeta, and FSHbeta transcriptional responses to GnRH or Bay K 8644+KCl (BK+KCl) pulses in vitro in the absence or presence of the Ca/CAMK II-specific inhibitor, KN-93. Gonadotropin subunit transcription was assessed by measuring primary transcripts (PTs) by quantitative RT-PCR. In time-course studies, both GnRH and BK+KCl pulses given alone increased all three subunit PTs after 6 h (2- to 4-fold). PT responses to GnRH increased over time (3- to 8-fold over basal at 24 h), although BK+KCl was ineffective after 24 h. KN-93 reduced the LHbeta and FSHbeta transcriptional responses to GnRH by 50-60% and completely suppressed the alphaPT response. In contrast, KN-93 showed no inhibitory effects on basal transcriptional activity or LH or FSH secretion. In fact, KN-93 tended to increase basal alpha, LHbeta, and FSHbeta PT levels and enhance LH secretory responses to GnRH. These results reveal that Ca/CAMK II plays a central role in the transmission of pulsatile GnRH signals from the plasma membrane to the rat alpha, LHbeta, and FSHbeta subunit genes.

Gonadotropin subunit transcriptional responses to calcium signals in the rat: evidence for regulation by pulse frequency.

Alterations in the frequency of calcium influx signals to rat pituitary cells can regulate the expression of gonadotropin subunit mRNAs in a differential manner, producing effects that are similar to those previously found for GnRH. The present study was conducted to investigate whether this reflects a transcriptional response to calcium pulse frequency, as determined by alterations in primary transcript (PT) expression. Perifused rat pituitary cells were given pulses of the calcium channel-activator Bay K 8644 (BK; with 10 mM KCl in the injectate) for 6 h. The response to alterations in pulse dose was examined by giving pulses of 1, 3, or 10 microM BK at 60-min intervals. Maximal increases in LHbeta and FSHbeta PTs were obtained with the 3-microM BK pulse dose and with the 10-microM dose for alpha. To investigate the effect of calcium pulse frequency, 3-microM BK pulses were given at intervals of 15, 60, or 180 min. Alpha PT was selectively stimulated by 15-min pulses and LHbeta by 15- and 60-min pulses of BK. In contrast, FSHbeta PT was maximally stimulated by the slower, 180-min pulse interval. These findings reveal that pulsatile increases in intracellular calcium stimulate alpha, LHbeta, and FSHbeta transcription in a differential manner. Thus, intermittent changes in intracellular calcium appear to be important in the transmission of GnRH pulse signals from the plasma membrane to the gene, and they may mediate the differential actions of pulse frequency on gonadotropin subunit gene expression.

Androgen suppression of GnRH-stimulated rat LHbeta gene transcription occurs through Sp1 sites in the distal GnRH-responsive promoter region.

Steroids may regulate LH subunit gene transcription by modulating hypothalamic GnRH pulse patterns or by acting at the pituitary gonadotrope to alter promoter activity. We tested direct pituitary effects of the androgen dihydrotestosterone (DHT) to modulate the rat LHbeta promoter in transfected LbetaT2 clonal gonadotrope cells and in pituitaries of transgenic mice expressing LHbeta-luciferase. The LHbeta promoter (-617 to +44 bp)-luciferase construct was stimulated in LbetaT2 cells 7- to 10-fold by GnRH. Androgen treatment had little effect on basal promoter activity but suppressed GnRH stimulation by approximately 75%. GnRH stimulation of LHbeta was also suppressed by DHT in isolated pituitary cells from male or female mice with functional nuclear ARs, but not in male littermates with mutant AR. GnRH stimulation of the LHbeta promoter requires interactions between a complex distal response element containing two specificity protein-1 (Sp1) binding sites and a CArG box, and a proximal element with two bipartite binding sites for steroidogenic factor-1 and early growth response protein-1 (Egr-1). DHT effectively suppressed promoter constructs with an intact distal response element. The distal response element does not bind AR, but AR reduces Sp1 binding to this region. Glutathione-S-transferase pull-down studies demonstrated direct interactions of AR with Sp1, which requires the DNA-binding domain of AR, and weaker interactions with Egr-1. We conclude that androgen suppression of the rat LHbeta promoter occurs primarily through direct interaction of AR with Sp1, with some possible role through binding to Egr-1. These interactions result in interference with GnRH-stimulated gene transcription by reducing cooperation between the distal and proximal GnRH response elements.

Regulation of gonadotropin subunit transcription after ovariectomy in the rat: measurement of subunit primary transcripts reveals differential roles of GnRH and inhibin.

The aim of this study was to determine if the changes in gonadotropin subunit gene expression following ovariectomy reflect transcriptional and/or posttranscriptional regulation by GnRH or inhibin. Subunit transcription rates were determined by recently developed quantitative RT-PCR for subunit primary transcripts (as an indicator of gene transcription), which allow us to measure both mRNA and PT from RNA extracted from a single pituitary. Following ovariectomy, LHbeta PT concentrations increased 2- to 3-fold between 72 h and 7 d, paralleling changes in serum LH and LHbeta mRNA. In contrast, serum FSH, FSHbeta mRNA, and FSHbeta PT concentrations were 6- to 9-fold greater 12-24 h after ovariectomy followed by an additional 2.5-fold increase at 72 h. Although alpha RNA was elevated at 72 h after ovariectomy, alpha-primary transcript did not change. GnRH antagonist prevented the increase in LHbeta-PT at 72 h, but had no effect on the increase in FSHbetaPT at 12 h and was only partially effective at 72 h. The acute GnRH-independent increase in FSHbeta-primary transcript after ovariectomy could be duplicated by the administration of inhibin antiserum to intact rats; inhibin-alpha antiserum did not affect LHbeta-primary transcript, but increased FSHbeta-primary transcript concentrations 8- to 11-fold. The half-disappearance rates of LHbeta and FSHbeta primary transcripts were measured after GnRH blockade or administration of recombinant human inhibin A. The half-disappearance times for LHbeta and FSHbeta primary transcripts following GnRH blockade were 13 and 17 min, respectively; the mRNAs did not change. The effects of inhibin were specific for FSHbeta; 60 min after inhibin FSHbeta-primary transcript was undetectable with a half-disappearance time of 19 min, additionally FSHbeta mRNA levels also fell with a half-life of 94 min. In conclusion, these data support previous evidence that GnRH regulates gonadotropin gene expression primarily at the level of transcription. However, the acute increase in FSHbeta-primary transcript after ovariectomy or immunoneutralization of inhibin-alpha, and the rapid fall in FSHbeta-primary transcript following rh inhibin, provide novel evidence that inhibin suppresses FSHbeta gene transcription in addition to its action in regulating FSHbeta mRNA stability.

Regulation of gonadotropin subunit gene transcription by gonadotropin-releasing hormone: measurement of primary transcript ribonucleic acids by quantitative reverse transcription-polymerase chain reaction assays.

GnRH regulates the synthesis and secretion of the pituitary gonadotropins LH and FSH. One of the actions of GnRH on the gonadotropin subunit genes (alpha, LHbeta, and FSHbeta) is the regulation of transcription [messenger RNA (mRNA) synthesis]. Gonadotropin subunit transcription rates increase after gonadectomy and following exogenous GnRH pulses. However, prior studies of subunit mRNA synthesis were limited by the available methodology that did not allow simultaneous measurement of gene transcription and mature mRNA concentrations. The purpose of the current studies was to: 1) develop a reliable and sensitive method for assessing transcription rates by measuring gonadotropin subunit primary transcript RNAs (PT, RNA before intron splicing); 2) investigate the PT responses to GnRH following castration or exogenous GnRH pulses; 3) characterize the half-disappearance time for the three PT species after GnRH withdrawal; and 4) correlate changes in PT concentration with steady-state gonadotropin subunit mRNA levels measured in the same pituitary RNA samples. Using oligonucleotide primers that flanked intron-exon boundaries, quantitative RT-PCR assays for each subunit PT species were developed. These assays require only ng amounts of RNA to measure each gonadotropin subunit PT and allow us to measure both PTs and steady-state mRNAs in a single pituitary RNA sample. Primary transcript concentrations in intact male rats showed a relative abundance of alpha > LHbeta congruent with FSHbeta, similar to the relationship found previously for mRNA levels. Additionally, each PT species was only 1-2% as abundant as the corresponding mRNA. One week after castration, gonadotropin subunit PT levels were increased (alpha: 3-fold, LHbeta: 6-fold, and FSHbeta: 3-fold) in a pattern similar to subunit mRNAs. Administration of GnRH antagonist to 7-day castrate male rats resulted in a rapid decline in PT concentrations with a half-disappearance time of 2.7 h for LHbeta and 0.8 h for FSHbeta, significantly faster than earlier measurements of the half-disappearance time for mature mRNA. Finally, in a GnRH-deficient male rat model, LHbeta and FSHbeta PT concentrations increased 4- to 6-fold 5 min after a GnRH pulse and then declined toward levels seen in control animals. These data indicate that the effects of GnRH on subunit gene transcription are an important determinant of gonadotropin regulation. The appearance and disappearance of PT RNA occurs more rapidly than changes in mature mRNA. Additionally, concentrations are elevated in long term castrates, and following an exogenous GnRH pulse the transcriptional burst is rapid and brief.

Corticotropin-releasing hormone and arginine vasopressin: mRNA and secretion are differentially regulated according to the pattern of exposure to noradrenaline in rat hypothalamic neurones.

Hypothalamic corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) are secreted from the median eminence in a pulsatile manner and regulated by noradrenaline during stress. This study investigated the effect of pulsatile noraderanaline on CRH/AVP mRNAs and secretion. Foetal hypothalamic neurones were cultured on plastic coverslips, inserted into perifusion chambers and noraderanaline pulses given at various doses or pulse intervals for 24 h. CRH and AVP release rose in a dose dependant manner; however, maximal increases in mRNAs were seen with an intermediate noraderanaline pulse dose. The effect of noraderanaline pulse frequency was determined by giving noraderanaline pulses at intervals of 15-120 min vs continuous noraderanaline. Both pulsatile and continuous noraderanaline increased CRH and AVP release, but secretion was reduced after 22 h of treatment in the continuous noraderanaline and rapid pulse groups. CRH mRNA levels were maximally increased by medium interval pulses and AVP mRNA by rapid interval pulses. Neither CRH nor AVP mRNAs were stimulated by continuous noraderanaline. To determine noraderanaline specificity, pulses of veratridine (VER; 15-120 min intervals) vs continuous VER were examined. Only pulsatile VER increased CRH and AVP mRNAs, with maximal effects seen with the 60 min pulse interval for both. Thus, noraderanaline pulse pattern regulates CRH and AVP gene expression in both a coordinate and differential manner. Since noraderanaline plays an important role during stress, the pattern of noraderanaline signals may be critical to the observed changes in CRH and AVP expression.

Gonadotropin-releasing hormone regulation of gonadotropin subunit gene expression in female rats: actions on follicle-stimulating hormone beta messenger ribonucleic acid (mRNA) involve differential expression of pituitary activin (beta-B) and follistatin mRNAs.

GnRH is the primary stimulus in the regulation of gonadotropin subunit mRNA expression. Additionally, local (pituitary) production of activin and follistatin appear to modulate the expression of FSH beta mRNA. The current studies aimed to determine whether GnRH regulation of pituitary activin (beta-B) and follistatin mRNAs could play a role in the differential actions of GnRH pulse pattern on gonadotropin mRNA expression in female rats. In response to altered GnRH pulse amplitude, the expression of FSH beta and follistatin mRNAs followed an inverse pattern. Only high dose GnRH increased expression of follistatin whereas, in contrast, beta-B and FSH beta expression were increased following lower doses of GnRH. To determine whether increased follistatin mRNA expression was correlated with FSH beta mRNA responses, we examined their temporal relationship following high dose GnRH. Both FSH beta and follistatin mRNAs were increased within 2 h and remained increased through 6 h. However, by 12 h FSH beta mRNA levels returned to values seen in controls, suggesting that increased follistatin requires 6-12 h to reduce FSH beta mRNA. In response to altered GnRH pulse frequency, FSH beta expression was increased at all pulse intervals (8-240 min) examined. Rapid GnRH pulse frequencies (8-min intervals) increased follistatin expression, whereas beta-B mRNA was only increased after 30-min pulse intervals, which also resulted in maximal FSH beta mRNA concentrations. These results suggest that changes in pituitary activin (beta-B) and follistatin mRNA expression may be important components of gonadotrope responses to pulsatile GnRH, and potentially imply that GnRH stimulation of activin and follistatin peptide production provides regulatory control over the production of FSH.

Steroid feedback on gonadotropin release and pituitary gonadotropin subunit mRNA in mice lacking a functional estrogen receptor alpha.

Steroid hormones regulate levels of gonadotropin mRNA in the pituitary, and gonadotropic hormones in plasma. To determine whether estrogen receptor alpha (ERalpha) mediates steroid negative feedback, wild type (WT) and estrogen receptor alpha knockout (ERalphaKO) mice of both sexes were gonadectomized and implanted with a Silastic capsule containing either estradiol (E2), dihydrotestosterone (DHT), testosterone, or a blank capsule. Ten days later, plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were measured. Pituitary mRNA levels of gonadotropin subunit (alpha, LHbeta, FSHbeta) and prolactin (PRL) were quantified. LH levels in gonad-intact ERalphaKO females were elevated, similar to values seen following gonadectomy. By contrast, serum LH concentrations in gonad-intact ERalphaKO males were low and rose following gonadectomy, suggesting androgen feedback. Estradiol treatment significantly decreased plasma LH in WT animals, but not in ERalphaKOs. In fact, in female ERalphaKOs, our dose of E2 increased plasma levels of LH as compared with untreated, ovariectomized ERalphaKOs. All the steroid treatments suppressed LH in WT animals whereas only DHT consistently suppressed LH concentrations in ERalphaKO mice. The postgonadectomy rise in plasma FSH was prevented by steroid treatments in WT females, but not in any of the other groups. Gonadotropin subunit and PRL mRNA responses to E2 treatment (both inhibitory and stimulatory) were absent in ERalphaKO mice, suggesting a critical role for ERalpha. Although E2 can exert negative feedback effects on LH release in both males and females by actions at the ERalpha, the androgen receptor plays the primary physiological role in the male mouse.

Regulation of pituitary follistatin and inhibin/activin subunit messenger ribonucleic acids (mRNAs) in male and female rats: evidence for inhibin regulation of follistatin mRNA in females.

The regulation of FSHbeta messenger RNA (mRNA) expression is complex and involves signals from the hypothalamus and gonads. Additionally, the local (pituitary) production of activin and follistatin appears to serve as an important modulator of endocrine signals for FSHbeta regulation. The purpose of these studies was to identify factors controlling pituitary activin/inhibin subunit and follistatin mRNA production in male and female rats. Both males and females expressed the follistatin, inhibin alpha, and betaB mRNAs, whereas the betaA mRNA was not detected. In males, levels of FSHbeta and follistatin were higher than those in females. After gonadectomy, levels of FSHbeta and follistatin increased in both sexes, whereas betaB rose only in females. In males, blockade of GnRH action from the time of castration prevented the increase in FSHbeta and follistatin, suggesting that GnRH is the primary stimulus for these gene products. In females, treatment with a GnRH antagonist only partially prevented the rise in FSHbeta, follistatin, and betaB expression, suggesting that other factors were also important. Passive immunoneutralization of circulating inhibin increased FSHbeta and follistatin (but not betaB), providing evidence that inhibin is a physiological regulator of follistatin. Replacement of estradiol at the time of ovariectomy prevented the increase in betaB mRNA, suggesting that gonadal steroids may also act via local factors to regulate FSHbeta. In summary, these studies provide evidence that GnRH, gonadal steroids, and gonadal peptides probably regulate FSHbeta expression at least in part via the intrapituitary activin/follistatin system.

Gonadotropin-releasing hormone pulses are required to maintain activation of mitogen-activated protein kinase: role in stimulation of gonadotrope gene expression.

The present study examined the effect of alterations in GnRH signal pattern (pulsatile vs. continuous; pulse frequency) on mitogen-activated protein kinase (MAPK) activity and whether MAPK plays a role in regulating gonadotrope gene expression. Pituitary MAPK activity was measured by immunoblot, using a phospho-specific MAPK antibody, corrected to the amount of total MAPK per sample. In vivo studies were conducted in adult castrate testosterone-replaced male rats (to suppress endogenous GnRH). Animals received pulsatile or continuous GnRH (or BSA-saline for controls) via jugular cannulas. Initial studies revealed that pulsatile GnRH stimulated a dose-dependent rise in MAPK activity (30 ng, 2-fold increase; 100 ng, 4-fold; 300 ng, 8-fold) 4 min after the pulse. The effect of pulsatile vs. continuous GnRH was examined by administering 50-ng pulses (60-min interval) or a continuous infusion (25 ng/min) for 1, 2, 4, or 8 h. Pulsatile GnRH stimulated a 2- to 4-fold rise in MAPK activity (P < 0.05 vs. controls) that was maintained over the 8-h duration. In contrast, continuous GnRH only increased MAPK activity (2- to 3-fold; P < 0.05 vs. controls) for 2 h, with MAPK activity returning to baseline at later time points. The effect of GnRH pulse frequency on MAPK activation was determined by giving GnRH pulses (50 ng) at 30-, 60-, or 120-min intervals for 8 h. Maximal increases (3-fold vs. controls; P < 0.05) were seen after 120-min pulses, with faster (30- to 60-min interval) pulses stimulating 2-fold increases in MAPK activity (P < 0.05 vs. controls and 120-min GnRH pulse group). The role of MAPK activation on gonadotrope (alpha, LHbeta, FSHbeta, and GnRH receptor) gene expression was determined in vitro. Preliminary studies demonstrated that the MAPK inhibitor, PD-098059 (50 microM), completely blocked GnRH-induced increases in MAPK activity in adult male pituitary cells. Further studies revealed that PD-098059 blocked gonadotrope messenger RNA (mRNA) responses to pulsatile GnRH (100 pg/ml, 60-min interval, 24-h duration) in a selective manner, with alpha, FSHbeta, and GnRH receptor (but not LHbeta) mRNA responses being suppressed. These results show that a pulsatile GnRH signal is required to maintain MAPK activation for durations of longer than 2 h, and that slower frequency pulses are more effective. Further, MAPK plays a crucial role in alpha, FSHbeta, and GnRH receptor mRNA responses to pulsatile GnRH. Thus, divergent MAPK responses to alterations in GnRH signal pattern may be one mechanism involved in differential regulation of gonadotrope gene expression.

Gonadotropin subunit and gonadotropin-releasing hormone receptor gene expression are regulated by alterations in the frequency of calcium pulsatile signals.

Previously, we have shown that intermittent calcium (Ca2+) stimuli increase alpha, LHbeta, and FSHbeta messenger RNAs (mRNAs), and only LHbeta mRNA was increased by continuous Ca2+. As gonadotropin subunit and GnRH receptor (GnRH-R) mRNAs are differentially regulated by alterations in GnRH pulse interval, we aimed to determine whether changes in the frequency of Ca2+ signals play a role in this effect. Cultured adult female rat pituitary cells in perifusion were given pulses of the Ca2+ channel activator BayK 8644 (10 microM; with 10 mM KCl in the injectate), at intervals of 16, 60, or 180 min for 24 h (vehicle pulses or 100 pM GnRH to controls). Pulsatile Ca2+ influx stimulated a rise in all mRNAs examined (P < 0.05 vs. vehicle controls); however, optimal pulse intervals differed. Alpha and LHbeta mRNAs were maximally stimulated by 16- or 60-min pulses (57% and 74% increases, respectively), with 180-min pulses being less effective. In contrast, FSHbeta and GnRH-R mRNAs were selectively stimulated by 180-min pulses (51% and 41% increases, respectively). Pulsatile GnRH produced similar increases in GnRH-R and subunit mRNAs (53-78% vs. controls). These results reveal that alterations in the frequency of Ca2+ signals can regulate gonadotrope gene expression in a differential manner, producing effects similar to previous findings for GnRH. Thus, intermittent increases in intracellular Ca2+ may be an important step in the transmission of GnRH pulse signals from the plasma membrane to the gene.

Ovarian activin receptor subtype and follistatin gene expression in rats: reciprocal regulation by gonadotropins.

The production of activin, follistatin (FS), and inhibin, proteins present in the ovary and involved in mammalian reproduction, is regulated by gonadotropins and estradiol. We report here gonadotropin regulation of ovarian activin receptor (ActR) subtype and FS mRNAs. Expression of ActRI, ActRIIA, ActRIIB, and FS mRNA was measured on the afternoon of proestrus (1800 h) and the morning of estrus (0800 h). ActRI and ActIIA subtype mRNA concentrations fell by approximately 50% (p < 0.05) following the proestrous gonadotropin surge (ActRIIB mRNA was undetectable), while FS mRNA was unchanged. To define the contribution of gonadotropins, hypophysectomized (HYPOX) female rats were given recombinant human (rh) FSH and hCG, which decreased both ActR mRNAs (by approximately 70% and aproximately 50% for ActRI and IIA, respectively) and increased FS mRNA by 2-fold. As gonadotropins could act via estradiol (E2), HYPOX rats were given E2; ActRI was decreased, but ActRIIA mRNA was increased. The actions of gonadotropins were preferential, as the combination of rhFSH and hCG with E2 reduced ActRIIA mRNA. FS mRNA was increased to a similar degree by E2 and/or gonadotropins. These data suggest that gonadotropins regulate ActR and FS gene expression via multiple mechanisms. Both a direct action on ActRIIA (inhibition) and an indirect action through E2 on ActRI (inhibition) and FS (stimulation) suggest potential physiologic mechanisms for the reciprocal regulation of ActR subtype and FS mRNAs.

GnRH regulates steroidogenic factor-1 (SF-1) gene expression in the rat pituitary.

Recent studies have demonstrated that the nuclear receptor, steroidogenic factor 1 (SF-1) plays a role in the regulation of pituitary gonadotropin gene expression. As GnRH is critical to stimulating LH and FSH gene expression, the present study was conducted to determine whether GnRH also regulates pituitary SF-1 mRNA. Pituitary SF-1 mRNA levels were measured in individual animals by RNase protection assay. In the first study, adult male and female rats were gonadectomized (GDX) for 7 days and some received testosterone (T) to prevent the post-GDX rise in GnRH, and compared to intact animals. Pituitary SF-1 mRNA levels increased significantly (3 fold in males, 2 fold in females; p < 0.05 vs intacts) after gonadectomy, which was blocked by exogenous T. Similar changes were observed in serum LH. To directly test whether GnRH stimulates SF-1 mRNA, we used a GnRH-deficient rat model (phenoxybenzamine-treated, ovariectomized females) and administered GnRH pulses for 6h (5ng at 30 min intervals; saline pulses to controls). Pulsatile GnRH stimulated a 51-64% increase in SF-1 mRNA levels (p < 0.05 vs controls). These results show that GnRH stimulates SF-1 gene expression, which may be a critical component in GnRH stimulation of gonadotropin subunit transcription.

Testosterone is required for gonadotropin-releasing hormone stimulation of luteinizing hormone-beta messenger ribonucleic acid expression in female rats.

Pulsatile GnRH stimulates the synthesis and secretion of LH and FSH in both male and female rats. In the male rat, exogenous GnRH pulses increase alpha, LH and FSH beta messenger RNAs (mRNAs) 3-fold within 24 h. In contrast, the results of recent in vivo and in vitro studies have shown that GnRH stimulates an increase in alpha and FSH beta mRNAs, but not LHbeta. However, during the estrous cycle, LHbeta mRNA increases during the GnRH-induced LH surge on proestrus afternoon. This increase in LHbeta mRNA appears to be coincident with a transient rise in serum testosterone (T). Therefore, the present study was conducted to determine whether T has a role in facilitating GnRH stimulation of LHbeta mRNA expression. In the first group of studies, adult female rats were ovariectomized, and T implants were inserted sc 7 days before the study (serum T, 1.86 ng/ml). Animals received iv pulses of GnRH (25 ng; 30-min interval) for 6-24 h (saline pulses to controls). The data showed that in the presence of T, GnRH stimulated a significant increase in LHbeta (as well as alpha and FSH beta) mRNAs within 6 h (P < 0.05 vs. saline-pulsed controls). Other results revealed that T treatment was critical to the stimulatory effect of GnRH on LH beta mRNA. A second group of studies examined the time course and dose effects of T on LH beta mRNA expression. Maximal LH beta mRNA responses to GnRH (3-fold increase vs. saline controls; P < 0.05) were seen after pretreatment with the lowest dose of T examined (serum T, 0.42 ng/ml), which is similar to T concentrations on proestrus. Higher doses of T suppressed LH release, as well as LH mRNA responses to GnRH. The T-induced LHbeta mRNA response to pulsatile GnRH was seen within 24 h of exposure to T and was the result of an androgenic action, as similar results were observed in rats that received dihydrotestosterone. These findings suggest that T is required to facilitate GnRH stimulation of LHbeta mRNA in the female rat. Moreover, in the presence of the concentrations of T seen on proestrus, LHbeta mRNA increases within 6 h, which is similar to the time course seen during the LH surge. Thus, the present results also suggest that the combined effects of the rise in serum T and increased GnRH secretion induce the rapid rise in LHbeta mRNA expression on the afternoon of proestrus.

Pituitary activin receptor subtypes and follistatin gene expression in female rats: differential regulation by activin and follistatin.

The activins, hormones produced in the gonads and extragonadal tissues (including the pituitary), rapidly increase FSH beta messenger RNA (mRNA) and FSH secretion. In the rat, activin acts via a family of activin receptor (ActR) subunits that includes at least one type I (ActRI or ALK-2) and two homologous type II (IIA and IIB) subunits. We have previously reported that ActRIIA mRNA rises after ovariectomy (OVX). Potentially, the OVX-induced increases in ActR mRNAs could result from altered activin or the activin-binding protein follistatin. It was the purpose of the current studies to determine whether activin and/or follistatin regulated activin receptor subunit mRNAs. Adult female rat pituitaries were dissociated and plated for 48 h, transferred to wells containing follistatin or activin for 2 or 24 h, then RNA extracted for measurement of ActRI, IIA, and IIB and follistatin mRNAs. All three ActR mRNAs were easily detectable in pituitary RNA, with the relative abundance of ActRI > IIA >> IIB (18:9:1). Between 2-24 h, levels of all three ActR mRNAs increased 2- to 3-fold in wells containing medium alone, whereas levels of follistatin mRNA were unchanged. Follistatin significantly reduced FSH secretion and follistatin mRNA, but not the ActR mRNAs. Activin increased ActRI (4-fold, at 2 h), ActRIIB (2-fold, at 24 h), and follistatin (2-fold, at 24 h) mRNAs and FSH release (2-fold, at 24 h), but did not alter ActRIIA mRNA levels. We conclude that 1) pituitary ActR mRNA expression is under inhibitory tone in vivo, as suggested by the effect of pituitary removal and cell dispersion and an earlier report after OVX. 2) Pituitary-derived activin stimulates follistatin (but not ActR) mRNA production, and additional increases in follistatin mRNA can be induced by exogenous activin. 3) Higher concentrations of activin differentially regulate pituitary ActR mRNA expression, suggesting that activin exerts a positive feedback effect on its own receptor.

Are immediate early genes involved in gonadotropin-releasing hormone receptor gene regulation? Characterization of changes in GnRH receptor (GnRH-R), c-fos, and c-jun messenger ribonucleic acids during the ovine estrous cycle.

GnRH regulates the secretion and synthesis of gonadotropins by binding to specific receptors located in the plasma membrane of the pituitary gonadotroph. Like the concentration of the signaling ligand GnRH, the number of GnRH receptors (GnRH-R) varies dynamically with the changing endocrine milieu during the ovine estrous cycle. With the recent success in cloning of the mammalian GnRH-R gene, it is becoming increasingly evident that some of the changes in GnRH-R numbers may be mediated at least in part via changes in GnRH-R gene transcription. However, the regulatory steps involved in the GnRH-R transcription are unknown. The present studies were conducted to 1) characterize in detail the changes in GnRH-R gene expression during the 16-day ovine estrous cycle, 2) determine whether or not changes in GnRH-R gene expression during the estrous cycle are paralleled by alterations in the expression of c-fos and c-jun mRNAs, and 3) determine whether GnRH can induce expression of c-fos and c-jun mRNAs. Results revealed that concentrations of GnRH-R mRNA were highest on the day before estrus, when circulating LH concentrations were still low. GnRH-R mRNA concentrations declined steadily starting at 5 h postestrus, the time of the preovulatory LH surge, reaching their lowest levels by 24 h after estrus. Changes in c-jun mRNA levels, in general, paralleled changes in GnRH-R mRNA concentrations, being highest on the day before estrus and declining thereafter. c-Fos mRNA followed a different time course than c-jun mRNA, remaining elevated from Day 8 prior to estrus until the onset of estrus.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of gonadotropin subunit messenger ribonucleic acid expression in gonadotropin-releasing hormone (GnRH)-deficient female rats: effects of GnRH, galanin, GnRH-associated peptide, neuropeptide-Y, and thyrotropin-releasing hormone.

Gonadotropin subunit mRNA expression is differentially regulated during the 4-day estrous cycle in rats, with LH-beta and FSH-beta mRNA expression rapidly increasing on proestrus. Studies in an ovariectomized (OVX) GnRH-deficient female rat model have shown that GnRH pulses can increase alpha and FSH-beta mRNA concentrations, but LH-beta mRNA is unchanged. Thus, the factors required for physiologic regulation of the LH-beta gene are not fully understood. To determine whether or not the proestrous ovarian hormone environment is required to allow increased expression of the LH-beta gene, GnRH pulses were administered to GnRH-deficient (phenoxybenzamine-treated) intact female rats on proestrus. Both LH and FSH secretion and alpha and FSH-beta mRNA concentrations were increased, but LH-beta mRNA expression was unaltered. The effect of co-administration of GnRH and specific neurohormones (GnRH-associated peptide [GAP], galanin, neuropeptide-Y [NPY], and thyrotropin-releasing hormone [TRH] was also examined in OVX rats receiving estradiol (E2) and progesterone (P) replacement. Alpha and FSH-beta mRNA concentrations increased 2-fold in response to pulsatile GnRH, and no further increase was seen after the addition of GAP, galanin, or TRH. It was of interest that NPY blocked the GnRH-induced rise in alpha and FSH-beta mRNA. LH-beta mRNA expression was not increased by GnRH pulses alone or by addition of any of the neuropeptides. Further studies determined that continuous GnRH was no more effective than pulsatile GnRH in stimulating a rise in LH-beta mRNA. The results indicate that GnRH pulses are not sufficient to enhance LH-beta mRNA expression in the GnRH-deficient female rat.(ABSTRACT TRUNCATED AT 250 WORDS)