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.

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.

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.

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.

Evidence that pituitary adenylate cyclase activating polypeptide suppresses follicle-stimulating hormone-beta messenger ribonucleic acid levels by stimulating follistatin gene transcription.

There is accumulating evidence to suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) may be an important modulator ofgonadotrope function. One of the actions of PACAP identified previously is to decrease FSHbeta messenger RNA (mRNA) levels. In the present series of experiments we demonstrate that PACAP-induced suppression of FSHbeta mRNA correlates with a rise in follistatin mRNA levels in primary pituitary cell cultures. Transient transfection of gonadotrope-derived alphaT3-1 cells with a rat follistatin promoter-luciferase reporter plasmid reveals that PACAP stimulates follistatin gene transcription. PACAP stimulation of LUC activity was maximal at concentrations as low at 1 nM. Furthermore, in alphaT3-1 cells PACAP activation of the follistatin promoter appears to be via the cAMP-dependent protein kinase A pathway. Accordingly, we propose that PACAP stimulates follistatin transcription, which neutralizes activin activity and thereby reduces FSHbeta mRNA. Since PACAP and follistatin are colocalized in multiple tissues including the brain, adrenals, and gonads, our findings may reflect a broadly distributed autocrine/paracrine mechanism for modification of activin effects that is under PACAP control.

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.

Activin inhibition of prostate cancer cell growth: selective actions on androgen-responsive LNCaP cells.

Prostate epithelial cell growth is under the control of both steroid and peptide factors. Human prostate cancer cell lines have been used to investigate similar agents in malignancy. Activins are dimeric peptides structurally related to transforming growth factor-beta and produced in the gonads and a wide array of extragonadal tissues. The activins act at the pituitary to regulate the synthesis and secretion of FSH. At other sites, such as bone marrow, liver, and gonads, activin may play an important role in the regulation of cell growth and differentiation. It was the purpose of the current study to determine whether activin had similar actions on prostate cancer cells, specifically the androgen-responsive LNCaP and the androgen-resistant PC-3 cell lines. Using reverse transcription-PCR, messenger RNAs for type I and type II activin receptor subunits as well as the activin-binding protein follistatin were detected in both cell lines. Activin treatment rapidly (<24 h) inhibited LNCaP, but not PC-3, cell growth. The effects of activin were evident at low levels, with a concentration of 5 ng/ml being effective at 24 h, and a concentration of 0.5 ng/ml being effective at 48 h. These results contrasted with the actions of transforming growth factor-beta, which inhibited only PC-3 cells and required a greater treatment duration (96 h) to be effective. To determine whether these prostate cancer cell lines were also producing activin, LNCaP and PC-3 cells were treated with follistatin. Again, only the LNCaP cells responded, with growth acceleration noted by 24 h. As PC-3 cell responses to activin could be independent of cell proliferation, we transfected LNCaP and PC-3 cells with a known activin-responsive promoter/reporter gene construct (p3TP-Lux) and treated cells with activin. Only LNCaP cells produced a measurable response in luciferase activity. Finally, we attempted to determine whether the PC-3 cell resistance to activin was mediated via a transferable factor. PC-3 conditioned medium was added to LNCaP cells in the absence or presence of exogenous activin and had a small, but statistically nonsignificant (P < 0.09), action to blunt the actions of activin. We conclude that activin is a potent growth inhibitor of LNCaP cell growth. Moreover, these cells also produce activin, suggesting that locally derived activin may play a role in regulating cell proliferation. Despite expressing messenger RNAs for activin receptors, PC-3 cells are resistant to activin, perhaps the result of the production of an activin-blocking factor or a defective activin response system. These cell lines will thus serve as useful models in which to further study the cellular basis of activin action.

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.

Changes in myocardial A1 adenosine receptor and message levels during fetal development and postnatal maturation.

Previously we have shown myocardial adenosine A1 receptors are up-regulated during the newborn period. The timing of the increase or the mechanism of the changes are not known. The purpose of the present study was to (1) determine the time course of increased A1 adenosine receptors during fetal development and (2) determine if A1 adenosine receptor regulation is secondary to changes in A1 receptor mRNA levels. A1 adenosine receptor density was determined in whole hearts from fetal rats at 14 and 19 days' gestation and from newborn and adult rats using standard receptor-binding techniques. A quantitative PCR assay was developed to measure A1 adenosine receptor mRNA using total RNA samples from the above ages. A1 receptor density (fmol receptor/mg protein) increased during late gestation (79 +/- 14 and 122 +/- 7 in 14 and 19 days' gestation respectively) peaked during the newborn period (136 +/- 12) and decreased in the adult rat (36 +/- 5). A1 receptor message levels (fg message/microgram total RNA) changed in parallel to receptor density (7.2 +/ 1.7, 15.6 +/- 1.8, 19.9 +/- 4.3 and 9.9 +/- 1.3 in 14 and 19 days' gestation, newborn and adult respectively). These results provide evidence for transcriptional control of A1 receptor density and the increased receptor density in the newborn heart supports a possible role for the A1 receptor in the transition to the extrauterine circulation.

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)

Gonadotropin-releasing hormone (GnRH) pulse pattern regulates GnRH receptor gene expression: augmentation by estradiol.

GnRH acts via a single cell surface receptor (GnRH-R), and the number of pituitary GnRH-R increases on proestrus, after gonadectomy, or in response to pulsatile GnRH in the rat. Estradiol (E2) is known to exert a transient positive action to increase GnRH-R number, and the rise in plasma E2 contributes to initiation of the midcycle LH surge. The present study was designed to determine the effect of GnRH pulse amplitude and frequency on GnRH-R messenger RNA (mRNA) levels and to assess the relative contributions of GnRH and gonadal steroids to increasing GnRH-R gene expression. These studies were conducted in vivo using previously characterized GnRH-deficient male (castrate testosterone-replaced) and ovariectomized phenoxybenzamine-treated female models. To investigate the effect of GnRH pulse amplitude, adult male and female rats received GnRH iv (5-250 ng/pulse at 30-min intervals; saline pulses to controls) for 12 or 24 h. In males, GnRH-R mRNA was increased by all pulse doses, with maximal effects (3-fold) at 5-25 ng/pulse. In contrast, only lower doses (5-10 ng/pulse) were effective in females (2-fold increase). In a subsequent study, GnRH pulses (25 ng for males; 10 ng for females) were given at 8-, 30-, or 240-min intervals for 12 or 24 h. Some animals received a continuous GnRH infusion (200 ng/h). In males, GnRH-R mRNA levels were stimulated by all GnRH pulse intervals (maximal after 30-min pulses), whereas continuous GnRH was ineffective. In females, only 30- and 240-min pulse intervals increased GnRH-R mRNA levels, with faster (8-min) pulses or continuous GnRH being ineffective. To determine the relative roles of ovarian steroids and GnRH, ovariectomized phenoxybenzamine-treated female animals received GnRH (10 ng/pulse, 30-min interval), E2 (via sc implants; plasma E2 levels, approximately 50 pg/ml), or their combination for 12-24 h (saline pulses to controls). In the absence of E2, GnRH-R concentrations fell by 70% between 12-24 h. E2 alone tended to increase GnRH-R mRNA at 12 h, with a 2-fold rise observed after 24 h. Pulsatile GnRH alone increased GnRH-R mRNA by 50% at 12 h (compared to saline-pulsed controls; P < 0.05) and by 6-fold after 24 h. When GnRH and E2 were combined, the magnitude of the increase (vs. saline controls) was greater than that seen for either GnRH or E2 alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Ovarian inhibin subunit gene expression: regulation by gonadotropins and estradiol.

Inhibin and FSH maintain a dynamic inverse relationship throughout the rat estrous cycle. In particular, inhibin alpha- and beta A-subunit messenger RNAs (mRNAs) have been shown to be maximally expressed immediately after the midcycle gonadotropin surge, when both circulating estradiol (E2) and inhibin are also elevated. The current study was designed to investigate the regulation of inhibin subunit gene expression and secretion in vivo by recombinant human FSH (rhFSH) and estradiol. Initially, we determined if physiological levels of rhFSH regulated ovarian inhibin subunit gene expression and secretion. Hypophysectomized (HYPOX) adult female rats received hCG (10 IU, sc) and were then treated for 24 h with either rhFSH (0.5-20 IU every 6 h, i.v.) or saline. Hypophysectomy reduced inhibin subunit mRNAs as well as serum inhibin and estradiol. Although 0.5 IU rhFSH was ineffective in increasing inhibin subunit mRNAs, all doses between 2.5-20 IU increased inhibin subunit gene expression and inhibin secretion. Inhibin alpha-, beta A-, and beta B-subunit mRNAs were increased to a similar degree (3- to 5-fold) by all rhFSH doses of 5 IU or more. Similarly, serum E2 and inhibin were increased 2- and 3-fold, respectively, above HYPOX values after all doses of rhFSH of 5 IU or more. To investigate the role of a pure FSH signal in a physiological dose on inhibin subunit gene expression, HYPOX rats were given either rhFSH (5 IU, i.v., every 6 h for 24 or 48 h), hCG (10 IU, sc), or their combination. Neither gonadotropin when given alone altered inhibin subunit gene expression or serum E2 concentrations. Inhibin secretion rose in response to rhFSH alone, but not to hCG. The combination of hCG and rhFSH resulted in increased inhibin subunit mRNAs (3- to 5-fold) as well as circulating E2 and inhibin concentrations. We next studied the effects of E2 replacement in HYPOX rats at both physiological (serum approximately equal to 40 pg/ml) and higher doses (serum approximately equal to 800 pg/ml, to mimic intraovarian concentrations) in the presence or absence of exogenous gonadotropins (for 24 and 48 h). Although not as effective as gonadotropins, both E2 regimens increased inhibin alpha to a similar degree (2-fold), whereas beta-subunit mRNAs were unchanged at 24 h. Serum inhibin concentrations were increased only 48 h after high dose E2 treatments. As the actions of E2 and gonadotropins on alpha-subunit mRNA were not additive, E2 appears to mediate gonadotropin regulation of alpha-subunit gene expression.(ABSTRACT TRUNCATED AT 400 WORDS)

Gonadotropin-releasing hormone pulse frequency regulates expression of pituitary follistatin messenger ribonucleic acid: a mechanism for differential gonadotrope function.

Follistatin (FS) is a monomeric glycoprotein that selectively inhibits both secretion of FSH and expression of FSH beta messenger RNA (mRNA), presumably via its ability to bind activin. FS mRNA and protein are present in the gonadotrope, suggesting a local action in regulating FSH beta. Pituitary FS mRNA increases after gonadectomy and at the midcycle gonadotropin surge of the estrous cycle, times of increased GnRH secretion. Thus, the purpose of the present studies was to assess the role of GnRH secretion on the regulation of pituitary FS. To confirm GnRH regulation of FS and to study the role of gonadal steroids, adult male rats were gonadectomized (2-36 h), with some animals receiving either testosterone (T) replacement, LRF-147 (a GnRH antagonist, AC-DTrp1-pCl-DPhe2-DTrp3-Ser4-Tyr5-DArg6-L eu7-Arg8-Pro9-DAla10), or both for 36 h (from the time of castration). Pituitary FS mRNA increased rapidly after castration, with levels rising 3-fold by 12 h and 4-fold by 36 h when compared to intact animals (P < 0.05). This rise was completely abolished by administration of LRF-147 and prevented by T replacement. Because GnRH pulse frequency can selectively regulate FSH beta mRNA expression, we next examined the effect of GnRH pulse interval (8-480 min) on FS mRNA expression. Fast frequency GnRH pulses (8 min), which did not increase FSH beta mRNA, were associated with an increase in FS mRNA (2.5-fold). The 30-min interval increased FS and gonadotropin subunit mRNAs. Slower pulse frequencies (> or = 120 min), which selectively stimulated a rise in FSH beta mRNA, did not increase FS mRNA. These results indicate that pituitary FS mRNA is regulated by GnRH. In addition, GnRH frequency modulation of pituitary FS provides a mechanism whereby a single hypothalamic GnRH can differentially regulate the gonadotropins, LH and FSH.

Ovarian regulation of pituitary inhibin subunit and activin receptor type II gene expression: evidence for a nonsteroidal inhibitory substance.

Gonadotropin subunit gene expression is regulated by gonadal, hypothalamic, and locally derived hormones. In particular, activin rapidly (within hours) acts at the gonadotrope to selectively increase the expression of FSH beta messenger RNA (mRNA). A family of activin receptors (ActRI, ActRII, and ActRIIB) has been identified, which is expressed in the pituitary as well as numerous other tissues in which activin is thought to act. As alterations in activin sensitivity could modulate activin action and, thereby, FSH beta mRNA, the purpose of this study was to determine whether ovariectomy (OVX), which results in rapid (< 2 h) increases in FSH beta, is associated with changes in ActRII gene expression. Adult female rats were ovariectomized, and some animals also received a GnRH antagonist from the time of OVX. Animals were killed between 2 h and 7 days later, and ActRII mRNA levels were measured by a quantitative reverse transcriptase-polymerase chain reaction assay. Although levels were unchanged at 2 h, ActRII mRNA levels increased 5- to 6-fold by 8 h and remained increased through 7 days after OVX. These changes were not altered by GnRH blockade. To determine whether ActRII was regulated by gonadal steroids, female rats were ovariectomized, and some animals were replaced with estradiol and progesterone (Silastic implants) for 2 days. Again, ActRII mRNA levels increased significantly after OVX, and gonadal steroid replacement had no effect. Finally, to investigate whether pituitary ActRII mRNAs are regulated by circulating inhibin, intact female rats were treated with an inhibin antiserum or nonimmune sheep serum as a control and killed 12 h later. Despite its action to increase FSH beta mRNA and FSH secretion, selective removal of inhibin did not alter ActRII mRNA levels. Based on these results we conclude the following. 1) Pituitary ActRII mRNAs increase rapidly after OVX, although increases in FSH beta precede changes in ActRII. These data suggest that changes in activin sensitivity may be a factor involved in the regulation of FSH beta. 2) An ovarian factor, other than inhibin, estradiol, and progesterone, acting independently of GnRH maintains an inhibitory tone on pituitary ActRII gene expression in adult rats.

Failure of gonadotropin-releasing hormone (GnRH) pulses to increase luteinizing hormone beta messenger ribonucleic acid in GnRH-deficient female rats.

Gonadotropin subunit gene transcription and messenger RNA (mRNA) levels are differentially regulated by GnRH pulse frequency and amplitude in the male rat. The rapid changes of subunit mRNA levels and LH and FSH secretion during the estrous cycle, particularly the rapid rise in LH-beta subunit mRNA on proestrus afternoon, suggest that physiological changes in the pattern of GnRH action may also be important in female rats. However, in the absence of a GnRH-deficient female model the role of varying GnRH stimulation remains to be determined. We have characterized a GnRH-deficient model by administering the alpha-adrenergic antagonist phenoxybenzamine (PBZ) to ovariectomized (OVX) rats. Initial experiments showed that PBZ given 24 h earlier abolished the afternoon LH surge in OVX estradiol (E2) replaced rats whereas LH responses to exogenous GnRH were preserved. A PBZ regimen of 15 mg/kg ip at OVX followed by 10 mg/kg at 24 h and 5 mg/kg at 48 h prevented the increase in alpha, LH-beta, and FSH-beta mRNAs and LH and FSH secretion for 72 h post-OVX. LH and FSH responses to GnRH pulses were preserved suggesting that PBZ blocked the post-OVX increase in hypothalamic GnRH secretion. The suppressive effect of PBZ appeared to be specific to the hypothalamic-pituitary-ovarian axis as plasma PRL, TSH, and corticosterone were not decreased compared to controls. We have used this GnRH-deficient OVX female model to investigate the effects of exogenous GnRH pulses on subunit mRNA expression. GnRH pulses (5-250 ng/30 min for 12-24 h) were administered via an intraatrial catheter beginning 24 h after OVX and the first PBZ injection (OVX+PBZ+saline pulses to controls). Expression of alpha and FSH-beta mRNAs and LH and FSH secretion were increased by GnRH pulse doses of 5-25 ng to values similar to or greater than those in OVX controls though the higher doses of GnRH/pulse did not increase FSH-beta mRNA or plasma FSH. However, LH-beta mRNA levels were not increased by GnRH pulses. GnRH pulses were also given to rats replaced with proestrus concentrations of estradiol alone or in combination with progesterone (P). Again, no demonstrable increases in LH-beta mRNA expression were observed. alpha-mRNA concentrations were further increased in the presence of E2 alone, and P in combination with E2, produced an augmented response of FSH-beta subunit mRNA. These data suggest that ovarian steroid hormones act directly on the gonadotrope to augment alpha and FSH-beta mRNA responses to GnRH.(ABSTRACT TRUNCATED AT 400 WORDS)