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Background: The Endocrine Self-Assessment Program In-Training Examination (ESAP-ITE) has the novel formative approach of allowing open access to all questions and answers after secure examination administration is complete, resulting in the creation of an entirely new in-training examination annually., Objective: To determine whether scores on the novel ESAP-ITE predict pass/fail outcomes on the American Board of Internal Medicine Endocrinology, Diabetes, and Metabolism Certification Examination (ABIM-ECE)., Methods: All endocrine fellows-in-training who took the ESAP-ITE between 2016 and 2019 and then subsequently attempted the ABIM-ECE within the same calendar year were included (n = 982). Primary analyses used the ESAP-ITE score from the final year of fellowship training. Covariates included sex, age on date of ABIM-ECE, medical school country, fellowship program region, pass/fail outcomes on the ABIM Internal Medicine Certification Examination, and ESAP-ITE score. All variables were analyzed using multivariable logistic regression., Results: ESAP-ITE score ( P  < 0.001), ABIM Internal Medicine Certification Examination outcome ( P  < 0.001), and age ( P  = 0.005) were each significant predictors of passing the ABIM-ECE on the first attempt. ESAP-ITE score was the strongest predictor of passing the ABIM-ECE, and this relationship was such that a score of 75% correct yielded a 97% probability of passing the ABIM-ECE, whereas a score of 50% correct generated only a 70% probability of doing so. Sex, fellowship program region, and medical school country were not significant predictors of ABIM-ECE outcomes., Conclusions: In addition to serving as an important learning instrument for endocrine fellowship programs, ESAP-ITE is a robust predictive tool for pass/fail outcomes on the ABIM-ECE., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Gestational primary hyperparathyroidism (GPHPT) is a rare condition with fewer than 200 cases reported. We present the case of a 21-year-old woman who presented at 10 weeks' gestation with severe hypercalcemia. Laboratory investigation was consistent with primary hyperparathyroidism. Neck ultrasound did not reveal any parathyroid enlargement. Due to the persistence of severe hypercalcemia, she was treated with 4 weeks of cinacalcet therapy, which was poorly tolerated due to nausea and vomiting. At 14 weeks' gestation, she underwent neck exploration with right lower, left upper, and partial right upper parathyroid gland excision. Intra- and postoperative parathyroid hormone (PTH) and calcium levels remained elevated. After a thorough discussion of risks/benefits, the patient requested further treatment. A parathyroid sestamibi scan (PSS) revealed an ectopic adenoma in the left mediastinum. The adenoma was removed via video-assisted thorascopic parathyroidectomy with intraoperative PTH declining to nearly undetectable levels. She ultimately delivered a physically and developmentally normal infant at 37 weeks' gestation. Appropriate treatment of severe GPHPT may prevent the maternal and fetal complications of hypercalcemia. This case, in which cinacalcet therapy and PSS were used, adds to the body of literature regarding treatment of severe GPHPT.

Over 50 million Americans have low bone mass. Poor bone quality is known to complicate spinal fusion surgery, which relies on strong bony purchase to be effective. Unfortunately, many spine surgeons do not perform routine workups for either osteoporosis or osteomalacia. Effective screening and risk factor assessment can allow for appropriate medical management of osteoporosis in the perioperative setting, improving outcomes. Medical management can be grouped into several different categories: vitamins and minerals, bisphosphonates, recombinant parathyroid hormone, estrogen replacement or modification, inhibitors of receptor activator of nuclear factor κ-B ligand (RANKL), and calcitonin. Calcium and vitamin D supplements are the least expensive to prescribe, with minimal side effects and promising animal studies, and thus should be provided to most osteoporotic patients. Recombinant parathyroid hormone can also be considered, as clinical studies have demonstrated impressive results in spine fusion patients. Bisphosphonates, estrogen therapy or selective estrogen receptor modulators, and calcitonin should all be avoided in this patient population given unproven benefit and potentially harmful side-effect profiles. Denosumab is potentially an option, but may not be first line given the general lack of supporting data for its use in perioperative management of spine surgery patients.

Patients with malignancies commonly experience abnormalities in serum electrolytes, including hyponatremia, hypokalemia, hyperkalemia, hypophosphatemia, and hypercalcemia. In many cases, the causes of these electolyte disturbances are due to common etiologies not unique to the underlying cancer. However, at other times, these electrolyte disorders signal the presence of paraneoplastic processes and portend a poor prognosis. Furthermore, the development of these electrolyte abnormalities may be associated with symptoms that can negatively affect quality of life and may prevent certain chemotherapeutic regimens. Thus, prompt recognition of these disorders and corrective therapy is critical in the care of the patient with cancer., (Copyright © 2014 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Osteoporosis, a "silent disease," is often unrecognized until fracture. Lifestyle modification with nutritional counseling is recommended during menopausal transition. Bone density testing is recommended for women aged 65 years and older, younger postmenopausal women with risk factors, or to follow therapy. Bisphosphonates treat osteoporosis (prevent bone resorption). Raloxifene and hormone therapy prevent bone loss and fracture, with extraskeletal benefits. Denosumab treats osteoporosis, although bone effects reverse rapidly. Teriparatide (anabolic therapy) is considered for women at high risk of fracture. Bazedoxifene with conjugated estrogens, novel delivery of teriparatide, new parathyroid hormone proteins, anti-sclerostin antibodies, cathepsin K inhibitors, and stem cell therapies are in testing.

Hypercalcemia complicates the course of 10%-30% of all patients with malignancies and can be a sign of very poor prognosis and advanced malignancy. Prompt recognition of the nonspecific signs and symptoms of hypercalcemia and institution of therapy can be lifesaving, affording the opportunity to address the underlying etiology. The mechanisms of malignancy-associated hypercalcemia generally fall into three categories: humoral hypercalcemia due to secreted factors (such as parathyroid-related hormone), local osteolysis due to tumor invasion of bone, and absorptive hypercalcemia due to excess vitamin D produced by malignancies. The mainstays of therapy for hypercalcemia are aggressive intravenous volume expansion with saline, bisphosphonate therapy, and perhaps loop diuretics. Adjunctive therapy may include calcitonin and corticosteroids. In refractory cases, gallium nitrate and perhaps denosumab are alternatives. In patients presenting with severe AKI, hemodialysis with a low-calcium bath can be effective. In most cases, therapy normalizes calcium levels and allows for palliation or curative therapy of the malignancy.

Background: A 62-year-old postmenopausal woman with a family history of breast cancer, mild gastroesophageal reflux disease, iron-deficient anemia and declining BMD was seen in a specialist center for the evaluation and management of osteoporosis., Investigations: Analysis of tissue transglutaminase IgA, endoscopic biopsy, serial BMD scans, FRAX calculation of osteoporotic fracture risk, Gail model calculation of breast cancer risk, assessment of blood vitamin D concentration and secondary evaluation for osteoporosis., Diagnosis: Osteoporosis, persistent after 12 years of hormone replacement therapy, and celiac disease., Management: The patient was initially treated for bone loss with postmenopausal hormone replacement therapy. DXA analyses showed a continued decline in BMD despite adequate replacement of calcium and vitamin D levels and withdrawal of gluten from the patient's diet. An oral bisphosphonate was recommended with plans to reassess BMD after 1 year.

Oncogenic (tumor-induced) osteomalacia is a rare paraneoplastic syndrome of phosphate wasting that is frequently associated with phosphaturic mesenchymal tumor (PMT). As the cytologic features of this tumor apparently have not been reported, we describe the fine-needle aspiration (FNA) findings for PMT that arose from the gluteal soft tissue in a patient with hypophosphatemia and multiple fractures secondary to osteomalacia. Smears from the computerized tomography (CT)-guided FNA showed groups of spindle cells having elongated nuclei, fine to moderately coarsely granular chromatin, inconspicuous nucleoli, and delicate cytoplasm. Marked nuclear atypia, mitotic figures, and necrosis were absent. The differential diagnosis included a variety of benign and malignant spindle cell neoplasms such as monophasic synovial sarcoma, leiomyoma, peripheral nerve sheath tumor, fibrosarcoma, and, less likely, metastatic melanoma and sarcomatoid carcinoma. The bland-appearing cytologic features of a spindle cell tumor in a patient with osteomalacia should suggest the diagnosis of PMT., ((c) 2008 Wiley-Liss, Inc.)

Combination therapy for osteoporosis has been tested in small trials of short duration with various combinations. Pertinent human and animal randomized clinical trial data were identified through Medline and reviewed with a focus on the risks and benefits of different types of combination therapies. Improvements in bone density were found in some, but not all, combinations. There are no large trials of adequate length or numbers to determine fracture efficacy. Consider combination therapy if monotherapy is unsuccessful, if there is an added nonskeletal benefit to the proposed combination or as sequential treatment with an anabolic agent followed by an antiresorptive agent. Although combination therapy, in general, has limitations based on cost, concern about potential oversuppression of bone, and lack of long-term safety and fracture efficacy, selected patients may benefit.

Recent reports suggest that androgens increase FSHbeta transcription directly via the androgen receptor and by modulating activin signaling. Estrogens may also regulate FSHbeta transcription in part through the activin system. Activin signaling can be regulated extracellularly via activin, inhibin, or follistatin (FS) or intracellularly via the Smad proteins. We determined the effects of androgen and estrogen on FSHbeta primary transcript (PT) concentrations in male and female rats, and we correlated those changes with pituitary: activin betaB mRNA, FS mRNA, the mRNAs for Smads2, -3, -4, and -7, and the phosphorylation (p) status of Smad2 and -3 proteins. In males, testosterone (T) increased FSHbeta PT two- to threefold between 3 and 24 h and was correlated with reduced FS mRNA, transient increases in Smad2, -4, and -7 mRNAs, and a six- to 10-fold increase in pSmad2, and activin betaB mRNA was unchanged. In females, T also increased FSHbeta PT twofold and pSmad2 threefold but had no effect on activin betaB, FS, or the Smad mRNAs. Androgen also increased Smad2 phosphorylation in gonadotrope-derived alphaT3 cells. In contrast, estradiol had no effect on FSHbeta PT but transiently increased activin betaB mRNA and suppressed FS mRNA before increasing FS mRNA at 24 h and increased Smads2, -3, and -7 mRNAs and pSmad2 threefold. In conclusion, T acts on the pituitary to increase FSHbeta PT in both sexes and modulates FS mRNA, Smad mRNAs, and/or Smad2 phosphorylation. These findings suggest that T regulates FSHbeta transcription, in part, through modulation of various components of the activin-signaling system.

This study investigated FSHbeta transcriptional responses to the suppression of endogenous follistatin (FST) production using FST antisense RNA (FST-AS) expressing adenovirus constructs in female rat pituitary cells in vitro. Adenoviral delivery systems were characterized and optimized using an adenovirus-green fluorescent protein construct, and maximal infection (85-90% of cells) was seen 48 h post adenovirus treatment. A 424 bp fragment, which included the translational start site and exons 1-3 of the rat FST gene, was subcloned in the reverse orientation into an adenovirus vector. Construct efficacy was tested using cultured rat pituitary cells infected with the adenovirus-FST-AS construct. Infection with adenovirus-FST-AS increased FST-AS mRNA expression in a dose-dependent manner, reduced FST protein expression to undetectable levels, and stimulated increases in FSHbeta primary transcript and FSH secretion. Treatment with testosterone alone stimulated FSHbeta primary transcript and FSH release, and responses were doubled in the presence of adenovirus- FST-AS. These results demonstrate the effectiveness of adenovirus FST-AS in suppressing pituitary FST protein expression and enhancing FSH biological responses at the transcriptional level. Thus, the FST-deficient rat gonadotrope cell is a model that allows for the investigation of factors regulating FSHbeta expression, which might otherwise involve the autocrine/paracrine actions of FST.

The neuropeptide pituitary adenylate cyclase activating polypeptide (ADCYAP 1, or PACAP) has been demonstrated to enhance gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion and regulate gonadotropin subunit gene expression in cultures of anterior pituitary cells. In the present study, we used in situ hybridization and real-time polymerase chain reaction to examine the expression of Pacap mRNA within the paraventricular nucleus (PVN) and anterior pituitary throughout the estrous cycle of the rat. Levels of luteinizing hormone in serum and pituitary gonadotropin subunit mRNAs were evaluated and displayed cyclic fluctuations similar to those reported previously. Pacap mRNA expression in the PVN and pituitary varied significantly during the estrous cycle, with the greatest changes occurring on the day of proestrus. Pacap mRNA levels in the PVN declined significantly on the morning of diestrus. During proestrus, PVN Pacap mRNA levels significantly increased 3 h before the gonadotropin surge and then declined. Pituitary expression of Pacap mRNA also varied on the afternoon of proestrus with a moderate decline at the time of the gonadotropin surge and a significant increase later in the evening. Expression of the mRNA species encoding the 288 amino acid form of follistatin increased significantly following the rise in pituitary Pacap mRNA, at the termination of the secondary surge in follicle-stimulating hormone beta (Fshb) gene expression. These results suggest that PACAP is involved in events before and following the gonadotropin surge, perhaps through increased gonadotroph sensitivity to GnRH and suppression of Fshb subunit expression through increased follistatin, as previously observed in vitro.

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.

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.

Follistatin (FS), along with the members of the transforming growth factor beta family activin and inhibin, are important regulators of FSH secretion and messenger RNA production. While activin and inhibin appear to function as tonic modulators of FSH (stimulatory and inhibitory, respectively), dynamic changes in FS are noted through the estrous cycle and under varying physiological experimental paradigms. This suggests that FS is a major contributor to the precisely coordinated secretion of FSH that maintains reproductive function. The aim of this study was to investigate changes in FS, in particular the early (<12 h) rise observed after ovariectomy (OVX), and to determine whether these changes were as a consequence of variations in gene transcription rates. FS primary transcript (PT) and mRNA were found to increase 3-fold 12 h post-OVX, indicating increased gene transcription during this time period. Replacement with estradiol and/or blockade of GnRH had only modest effects on FS PT concentration. Inhibin immunoneutralization of intact rats resulted in a 3-fold increase in FS PT 12 h after administration of inhibin alpha antisera. Significant increases in FS mRNA at both 2 and 12 h also suggested that inhibin also may have effects on message stability. After administration of recombinant human inhibin A, there was a prompt decline in both FS PT and mRNA. These results indicate that inhibin is a major regulator of FS, both by transcriptional and nontranscriptional mechanisms.

The gonadotropin beta-subunit mRNAs are differentially regulated by androgens. Testosterone (T) suppresses LH-beta and increases FSH-beta. We aimed to determine whether androgens regulate LH-beta and FSH-beta transcription [as measured by changes in primary transcript (PT)] and to determine whether androgens act directly on FSH-beta or via the intrapituitary activin/follistatin (FS) system. In castrate + GnRH antagonist-treated rats, T increased FSH-beta PT between 3 and 48 h. In contrast, T suppressed LH-beta PT. The increases in FSH-beta mRNA and PT were associated with reduced FS mRNA. Activin betaB mRNA was modestly suppressed. The increase in FSH-beta PT after T was androgen specific. Both T and dihydrotestosterone (DHT) increased FSH-beta PT 2-fold and decreased both FS and betaB mRNA. Estradiol suppressed FSH-beta PT 3-fold and had no effect on FS or betaB mRNAs. LH-beta PT was suppressed by DHT. To determine whether T stimulation of FSH-beta PT reflected a decrease in pituitary FS, we gave androgen in the presence of exogenous FS in vitro. T and DHT increased FSH-beta PT 2- to 3-fold. FS alone decreased FSH-beta PT 40% but did not diminish the increase FSH-beta PT in response to T. T, DHT, and FS did not affect FS mRNA, betaB mRNA, or LH-beta PT. In conclusion, androgens acting directly on the pituitary increase FSH-beta and decrease LH-beta transcription. The increase in FSH-beta PT in response to T was androgen specific and occurs in the presence of excess FS, suggesting that T stimulates FSH-beta transcription independently of modulation of FS.

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.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to differentially regulate the expression of the gonadotropin subunit genes in cultures of rat pituitary cells. PACAP is expressed within the hypothalamus, and concentrations of PACAP are 2- to 4-fold higher in the portal circulation than in the general circulation. Therefore, PACAP is a candidate regulator of pituitary function. In the present study, we examined the expression of PACAP mRNA within the paraventricular nucleus (PVN) during maturation (Days 20-60) in the male rat and compared this expression to the levels of the gonadotropin subunits, follistatin, and GnRH-receptor mRNAs within the anterior pituitary. Serum concentrations of FSH and LH confirm the established maturational pattern of divergent secretion of LH and FSH. Northern analysis of the gonadotropin subunit mRNAs revealed that FSHbeta expression parallels FSH secretion whereas LHbeta mRNA concentrations do not change during development. Expression of the GnRH receptor in the pituitary parallels that of FSHbeta. In situ hybridization revealed a developmental pattern of PACAP mRNA within the PVN that is reciprocal to that of FSHbeta. Competitive reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of total pituitary follistatin mRNA revealed no significant changes; however, semiquantitative RT-PCR analyses revealed the presence of two follistatin mRNA species, one of which, corresponding to follistatin-288, was developmentally regulated. These studies identified a reciprocal relationship between PVN PACAP and FSHbeta gene expression in maturing rats. We propose that PACAP contributes to the selective regulation of FSHbeta expression during maturation in the male rat, perhaps via regulation of follistatin.

We examined the time course of action of GnRH pulse frequency on gonadotropin subunit gene transcription and assessed the roles of GnRH, follistatin (FS), and activin on differential transcription of the LHbeta and FSHbeta genes. GnRH-deficient male rats were pulsed with 25 ng GnRH either every 30 min (fast frequency) or every 240 min (slow frequency) for 1-24 h. Both GnRH frequencies increased alpha primary transcript (PT) 5-fold within 6 h, but only fast frequency GnRH increased alpha mRNA. Only fast frequency GnRH pulses affected LHbeta PT, resulting in 6- to 9-fold increases between 1-24 h. Fast frequency GnRH pulses transiently increased FSHbeta PT at 1 and 6 h (4- and 2-fold, respectively); but by 24 h FSHbeta PT had returned to control levels and was correlated to a 5- to 9-fold increase in FS mRNA. In contrast, slow GnRH pulses increased FSHbeta PT 3- and 6-fold at 8 and 24 h, respectively, which was correlated with a decline in FS mRNA. Activin mRNA did not change significantly after either GnRH frequency, but tended to fall after fast pulses. To test whether activin was required for the effects of GnRH on FSHbeta transcription, rats were treated with GnRH pulses every 240 min for 8 h +/- FS. FS treatment alone markedly decreased basal FSHbeta PT. GnRH in the presence of FS increased FSHbeta PT 8-fold but did not restore FSHbeta transcription to control or GnRH alone values. In summary, whereas alpha-subunit transcription is independent of frequency, an increase in alpha mRNA requires fast frequency GnRH pulses. Fast frequency GnRH pulses increased both LHbeta and FSHbeta transcription, but the response of FSHbeta was transient. The sustained rise in FSHbeta transcription and mRNA expression required slow frequency GnRH pulses and was correlated to low FS mRNA. Neutralization of pituitary activin by exogenous FS markedly reduced basal FSHbeta PT and mRNA but did not prevent the stimulation of FSHbeta transcription by slow frequency GnRH pulses. These studies suggest that the frequency regulation of FSHbeta transcription involves both direct actions of GnRH and indirect effects, via changes in pituitary FS expression.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates alpha-subunit transcription and lengthens LH-beta mRNA transcripts, but reduces FSH-beta mRNA levels in rat pituitary cell cultures. PACAP also stimulates follistatin transcription, an effect which may explain the decrease in FSH-beta mRNA. To begin to investigate the cells in which PACAP activates the follistatin gene, quantitative in situ hybridization for follistatin mRNA combined with immunostaining for LHbeta and S100 protein was performed. In control cultures, follistatin mRNA was expressed in 70% of gonadotrophs and in 47% of folliculostellate cells (S-100+). PACAP increased (P<0.001) both the number of follistatin-expressing cells as well as the number of grains per cell in both gonadotrophs and folliculostellate cells, while GnRH only affected (P=0.01) gonadotrophs. Follistatin and FSH-beta gene expression in rat pituitary cultures were also measured by competitive quantitative RT-PCR and northern analysis, respectively. Both PACAP and GnRH increased (P<0.05) follistatin gene expression and suppressed (P<0.05) FSH-beta mRNA, and the effect of PACAP together with GnRH on follistatin exceeded that of GnRH alone. PACAP regulation of follistatin and FSH-beta gene expression was studied further in LbetaT2 cells that were found to express receptors for the specific PACAP receptor, PAC(1). Follistatin mRNA was undetectable in cultures exposed to control media, or stimulated with PACAP, GnRH or rh-activin-A. In contrast to the results in primary pituitary cultures, PACAP increased FSH-beta mRNA in these follistatin-deficient cells. Moreover, using transient transfection, PACAP stimulated transcription of ovine-FSH-beta-luciferase. GnRH likewise increased FSH-beta mRNA and stimulated FSH-beta gene transcription in LbetaT2 cells. Activin-A increased FSH-beta gene expression dose-dependently, and activin induction of FSH-beta mRNA was blocked completely by 3-fold excess follistatin. These results indicate that PACAP stimulates follistatin gene expression in both gonadotrophs and folliculostellate cells, and provide further evidence that follistatin is required for PACAP or continuous GnRH to down-regulate FSH-beta mRNA. These experiments suggest a mechanism by which PACAP influences FSH production selectively by an autocrine effect on gonadotrophs and by a paracrine mechanism through folliculostellate cells that involves follistatin.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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)

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)

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)

Glucose-6-phosphatase catalyzes the final step of glucose production by liver and kidney. Though its strategic position has sparked interest in its regulation, difficulty with isolating a pure, stable enzyme has slowed progress. Virtually all previous work examining the physiologic regulation of this enzyme has relied on estimates of glucose-6-phosphatase activity in crude microsome preparations. The recent cloning of human and murine glucose-6-phosphatase cDNAs has now allowed study of its mRNA expression. We studied the effect of acute, streptozotocin-induced diabetes on hepatic microsomal glucose-6-phosphatase activity and mRNA expression in young (89 +/- 3 g), juvenile (304 +/- 4 g) and adult (512 +/- 10 g) rats. In control rats, mRNA expression and enzyme activity was similar among the three age groups. Streptozotocin-induced diabetes significantly increased the enzyme activities in both intact and triton-treated microsomes in all groups of rats (p < 0.001). Glucose-6-phosphatase mRNA expression was increased in the diabetic rats as well (p < 0.0001). Blood glucose concentrations correlated significantly with glucose-6-phosphatase mRNA level (p < 0.005) and both intact (p < 0.002) and triton-treated (p < 0.001) microsomal glucose-6-phosphatase activity. Both intact and triton-treated microsomal glucose-6-phosphatase activity correlated with mRNA level (p < 0.001, for each). We conclude that acute streptozotocin-diabetes increase expression of glucose-6-phosphatase mRNA and this contributes to the increased glucose-6-phosphatase activity seen with diabetes mellitus.

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.

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.

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)

The present study examined the effect of GnRH pulse amplitude on alpha, LH beta and FSH beta mRNAs using an in vitro perfusion model. Pituitaries from 30-day-old female rats were dissociated and the cells plated for 48 h to allow attachment to collagen-coated microcarrier beads. The beads were loaded into perifusion chambers, preperifused for 1 h, and then given GnRH pulses (17.5-175 pg/ml) every 30 min for 24 h. Perifusate LH was measured after 2 h and 22 h of perifusion and alpha LH and FSH beta messenger RNAs (mRNAs) were determined by hybridization to complementary DNA (cDNA) probes. All doses of GnRH acutely stimulated LH release, and responses were similar after 2 h and 22 h. LH release increased as a function of GnRH pulse dose with maximal increases seen following 70 pg/ml pulses. alpha mRNA levels (control = 0.73 +/- 0.1 fmol cDNA bound/100 micrograms pituitary DNA) were increased 30% and 40% after 24 h of 35 and 70 pg/ml pulses, respectively (P < 0.05 vs. media controls). LH beta mRNA concentrations (control = 0.44 +/- 0.08 fmol cDNA bound) were only elevated after 35 pg/ml GnRH pulses (36% increase). FSH beta mRNA showed the largest responses to GnRH pulses, increasing by 45% and 84% after 35 and 70 pg pulses, respectively (control = 0.14 +/- 0.02 fmol bound). The highest GnRH pulse dose (175 pg/ml) was ineffective in stimulating an increase in FSH beta mRNA levels. These results show that all three gonadotropin subunit mRNA concentrations increase after 24 h of GnRH pulses, but the pattern of individual subunit mRNA responses was dependent upon the amplitude of the GnRH pulse stimulus. These data support earlier results in vivo, in that the subunit responses to GnRH pulse dose were similar. Thus, alterations in the amplitude of pulsatile GnRH secretion from the median eminence may be one mechanism by which the expression of gonadotropin subunit genes are regulated.

After ovariectomy (ovx), FSH beta mRNA levels and serum FSH increase 2- to 3-fold within 12 h, and this persists in the presence of a GnRH antagonist. As a fall in plasma estradiol and progesterone appears to regulate FSH beta via increased GnRH secretion, it is thought that the acute (by 2 h) changes in FSH beta mRNA after ovx reflect falling levels of plasma inhibin. The current study addressed the following questions. 1) Does a reduction of circulating inhibin (via passive immunoneutralization or gonadectomy) increase FSH beta mRNA levels? 2) If so, are the acute increases in FSH beta mRNA associated with changes in the transcription rate? Adult male and female rats received 0.5 ml antiinhibin antiserum, iv, and were killed 2 or 12 h later. A second group of rats was gonadectomized; some received a GnRH antagonist and were killed at various intervals between 2 h and 7 days later. In adult males, no change in gonadotropin mRNA levels was observed after either addition of inhibin antiserum or removal of the testes. In contrast, in adult female rats, both ovx and inhibin antiserum increased FSH beta mRNA levels (2-fold) within 2 h, and a similar increase occurred in the presence of a GnRH antagonist. To determine if the increase in FSH beta resulted from increased mRNA synthesis, adult female rats were ovx, and half received a GnRH antagonist. Animals were killed 2 or 12 h later, and transcription rates were measured by nuclear run-off assay in pituitaries pooled from three rats. The transcription rate of the alpha-subunit, although not altered by ovx, was decreased in animals receiving the GnRH antagonist. Transcription of the LH beta gene was increased within 2 h after ovx, a change that was abolished by the GnRH antagonist. mRNA concentrations of either alpha or LH beta do not increase acutely after ovx, suggesting that GnRH regulates alpha and LH beta gene transcription and 12 h or more of mRNA synthesis are required to increase cytoplasmic concentrations. The FSH beta gene transcription rate was unchanged in both ovx and GnRH antagonist-treated animals, but serum FSH increased at 12 h. These data indicate that the rapid GnRH-independent increase in FSH beta mRNA levels seen immediately after ovx is not associated with altered mRNA synthesis and suggest that inhibin may also regulate FSH beta gene expression through nontranscriptional mechanisms.

The data reviewed in this chapter provide evidence that the pattern of GnRH secretion appears to be an important factor in regulating gonadotropin subunit gene expression, gonadotropin synthesis and hormone secretion. The data on gonadotropin synthesis were obtained in rodents and hence, must be interpreted with caution when applied to primates. Despite this reservation, the data suggest a similarity of regulatory mechanisms in mammalian species. The data also provide an explanation for the mechanisms whereby a single gonadotropin-releasing hormone can differentially regulate the three gonadotropin genes and allow differential hormone secretion. In overall agreement with this view, the observations during pubertal maturation reveal increasing GnRH pulsatile secretion during puberty with an evolution from predominant FSH to a predominant LH secretion by the gonadotropes. In males, the patterns of GnRH secretion appear to be fairly consistent throughout adult life, but in women cyclic changes occur which perhaps are important in maintaining cyclic ovulation. It is proposed that once pubertal maturation has been established, GnRH is secreted at a relatively fast frequency (one pulse per hour), and an essential feature of repeated ovulatory cycles is the slowing of this GnRH stimulus during the luteal phase: to allow subsequent preferential FSH release. This slowing of GnRH secretion appears to be effected by estradiol and progesterone acting to enhance hypothalamic opioid activity. Similar mechanisms involving increased opioid tone appear to be causally related to the reduced frequency and irregular GnRH stimulus seen in hypothalamic amenorrhea and hyperprolactinemia. In contrast, some forms of polycystic ovarian disease may reflect abnormalities of the estradiol-progesterone/opioid/GnRH neuron feedback mechanisms, with failure to establish slowing in the peripubertal anovulatory cycles. The resulting persistent GnRH stimulus increases LH with consequent effects of abnormal follicular maturation and enhanced ovarian androgen production. Present data are supportive of these hypotheses, but future studies will determine whether these views prove to be correct. However, current data provide strong support for the view that the pattern of GnRH secretion is a critical factor in the regulation of differential gonadotropin synthesis and secretion in mammalian species.

The influence of TRH pulse pattern on PRL and TSH alpha- and beta-subunit gene expression was examined in vitro. Pituitaries from adult female rats were dissociated and plated for 48 h to allow attachment to collagen-coated microcarrier beads. The beads were perifused for 24 h with TRH (pulses or continuous). To examine the effects of TRH pulse amplitude, TRH pulses (0.032-100 nM) were given every 60 min (controls received either medium pulses or continuous 100 nM TRH). PRL mRNA rose progressively to a peak at 4 nM TRH/pulse (93% increase vs. medium-pulsed controls), but higher TRH pulse amplitudes were less effective. alpha-Subunit mRNA also rose with increasing TRH pulse dose, with maximal (137%) elevations after 100-nM pulses. TSH beta mRNA concentrations were increased by TRH pulse doses between 0.8-20 nM, but a clear dose-response pattern was not seen. Continuous TRH (100 nM) resulted in PRL, TSH beta, and alpha mRNAs that were less than the values in medium-pulsed controls. To assess the effects of pulse frequency, 4-nM TRH pulses were given at intervals between 15-240 min (controls received medium pulses or continuous 4 nM TRH). PRL mRNA was increased (55-107% increase) after all pulse intervals, except 240 min, and rose to a similar degree after 4 nM TRH given continuously. alpha-Subunit mRNA concentrations increased by a lesser degree and also did not rise after the slowest (240 min) pulse interval. In contrast, TSH beta mRNA levels increased progressively as pulse intervals were increased, and maximal (85%) elevations were seen after 240-min pulses. Continuous 4 nM TRH did not alter alpha or TSH beta mRNAs. These results show that pulsatile TRH is more effective than continuous TRH in stimulating PRL, TSH beta, and alpha mRNAs. Further, the pattern of TRH pulsatile signals can influence the expression of these pituitary hormone genes in a differential manner.

Gonadal steroids can act both indirectly via gonadotrophin-releasing hormone (GnRH) and directly on the pituitary to regulate gonadotrophin subunit gene expression. Recent studies to assess a possible direct action at the pituitary have shown that testosterone, when given to males in the absence of endogenous GnRH action, selectively increases FSH-beta mRNA concentrations. Conversely, in females, oestradiol appears to regulate gonadotrophin subunit mRNAs primarily via GnRH. The present study was designed to determine whether these differing results reflect specific actions of the gonadal steroids themselves or different responses of the pituitary gonadotroph cells in males and females. Rats which had been castrated 7 days earlier were given silicone elastomer implants (s.c.) containing oestradiol (plasma oestradiol 68 +/- 4 ng/l) in males or testosterone (plasma testosterone 3.5 +/- 0.3 micrograms/l) in females in the absence or presence of a GnRH antagonist. Seven days later pituitaries were removed and steady-state mRNA concentrations measured by dot-blot hybridization. In males, oestradiol reduced LH-beta and FSH-beta but not alpha mRNA. The antagonist reduced levels of all three subunit mRNAs in males and the addition of oestradiol had no further effect, suggesting that oestradiol regulates gonadotrophin subunit gene expression in males by suppressing GnRH secretion. In females, testosterone reduced all three subunit mRNAs though FSH-beta remained threefold higher than in intact animals. The GnRH antagonist was as effective as testosterone alone and reduced alpha and LH-beta to levels found in intact animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The data reviewed present evidence that the pattern of GnRH secretion is an important factor in the regulation of gonadotropin subunit gene expression, gonadotropin synthesis, and secretion. The information on regulation of mRNA expression by GnRH pulses should be considered with some caution, as the experiments were performed in male rats and may not accurately reflect events in female primates or humans. However, an overall pattern emerges which suggests that common factors may be involved in all mammalian species. If current evidence is correct, and only a single gonadotropin-releasing hormone exists, then mechanisms to differentially regulate the three gonadotropin genes may involve changes in GnRH secretion. Alterations in GnRH pulse frequency and amplitude are recognized by the pituitary gonadotrope cell and could be the mechanism used to effect differential expression of the gonadotropin subunit genes. Differential regulation of subunit gene expression would be expected to be critically important in the establishment of pubertal maturation, and subsequently in the maintenance of ovulatory cycles in women. Our hypotheses, proposing a major role of pulsatile GnRH secretion in the regulation of human reproduction, are summarized in schematic form in Fig. 14 for men and Fig. 15 for women. In utero and during the first few months of life, GnRH is secreted at a relatively fast frequency (approximately 1 pulse/hour). During the first year, GnRH secretion is inhibited and both the amplitude and apparent frequency of pulsatile release is markedly reduced. The mechanisms involved in inhibiting GnRH release remain unclear in humans. Similarly, the mechanisms involved in the disinhibition of GnRH secretion, which first occurs during sleep at the initiation of puberty, are unclear, but in humans do not appear to involve opiates. In males, the increased frequency and amplitude of GnRH secretion favor LH synthesis and release, which in turn stimulates testosterone secretion (Fig. 14). Testosterone acts at the hypothalamus, perhaps through opioid mechanisms, to inhibit GnRH pulse frequency and to maintain a regular pattern of pulses occurring approximately every 90-110 min in adult males. In females, the mechanisms involving alterations in the patterns of GnRH secretion to regulate reproduction appear more complex. This may reflect the need to differentially synthesize and secrete FSH and LH at different times during reproductive cycles to allow orderly follicular maturation and ovulation. As shown in Fig. 15, we hypothesize that the events during the first decade of life and through the initiation of nocturnal GnRH secretion at puberty are similar in both sexes.(ABSTRACT TRUNCATED AT 400 WORDS)

Previous results have shown that the pattern of GnRH pulses (amplitude and frequency) can differentially regulate expression of gonadotropin subunit cytoplasmic messenger RNA (mRNA) concentrations. The present study examined the effect of GnRH pulses on alpha, LH-beta and FSH-beta transcription rates as determined by nuclear runoff transcription assay. GnRH pulses (saline to controls) were given to castrate, testosterone-replaced male rats, and the rate of subunit gene transcription was measured in isolated pituitary nuclei. The effect of GnRH treatment duration was examined by giving GnRH pulses (25 ng/pulse at 30-min intervals) for 1, 4, or 24 h. The basal transcription rates [expressed as parts per million (ppm)] were 82 +/- 25 for alpha; 39 +/- 19 for LH-beta and 27 +/- 6 ppm for FSH-beta, and transcription rates of all 3 subunits were elevated at 1 h (3-5-fold vs. saline controls). After 4 h of GnRH pulses, alpha and FSH-beta transcription rates were reduced vs. 1 h, but LH-beta mRNA synthesis rate was maintained. At 24 h, the alpha transcription rate was still increased (66%), but LH-beta and FSH-beta transcription rates had fallen to basal levels despite the continuing pulsatile GnRH stimulus. The second experiment investigated the effect of the duration of GnRH pulses (25 ng/pulse, every 30 min for 4 h or 24 h), on cytoplasmic subunit mRNA concentrations to assess if the initial 4-h increase in transcription rate would induce a rise in cytoplasmic mRNAs. After 4 h of GnRH pulses, alpha and LH-beta mRNAs were unchanged, but FSH-beta mRNA had increased by 36% (P less than 0.05) compared to controls. All 3 subunit mRNAs were increased (approximately 2-fold) by 24 h of GnRH pulses. Administering GnRH pulses for 4 h followed by 20 h of saline pulses did not increase alpha mRNA; LH-beta was slightly increased (P less than 0.05), but FSH-beta mRNA concentrations were similar to levels seen after 24 h of continued GnRH pulses. The third experiment examined the effects of a continuous GnRH infusion and different GnRH pulse frequencies on gonadotropin subunit transcription rates. GnRH (25 ng/pulse) was given at intervals of 8, 30, or 120 min for 4 h (saline to controls). The continuous GnRH infusion (200 ng/h) did not increase the transcription rate of any of the three subunit mRNAs. alpha-subunit transcription rate was increased 2.7- or 4-fold by GnRH pulses given every 8 or 30 min, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)