Gonadotrofinomas: análisis multicéntrico y retrospectivo de 66 pacientes / Gonadotrophinomas: multicenter and retrospective analysis of 66 patients

Objetivo: Analizar la presentación clínica, radiológica, bioquímica y el comportamiento posquirúrgico de una cohorte de pacientes portadores de gonadotrofinomas. Pacientes y Métodos: Se evaluaron pacientes con gonadotrofinomas estudiados en nueve centros endocrinológicos de la ciudad de Bs.As. durante el período 1983 a 2003. El criterio de inclusión fue la inmunohistoquímica (IH) positiva para hormona luteinizante (LH), folículoestimulante (FSH) y/o alfa subunidad (ASU). Los adenomas plurihormonales fueron excluidos. Resultados: Fueron analizados 66 pacientes de 51,8 ± 12,1 (X +/- DS) años (39 varones). Los síntomas mas frecuentemente observados fueron las alteraciones visuales (72,8%), seguidas por el hipogonadismo y las cefaleas. El 10,6% se diagnosticaron en forma incidental. El 98,5% fueron macroadenomas, 56,9% de los cuales correspondieron a un estadio Hardy (EH) 3 y 29,6% a un EH 4. El tiempo de seguimiento fue de 47,8 meses (r: 5-168). El hipogonadismo definido bioquímicamente se presentó en el 82,4% de los pacientes. En su mayoría presentaban niveles bajos o inapropiadamente normales de gonadotrofinas, pero 4 mujeres y 3 varones presentaron niveles séricos elevados y disociados de FSH y LH. La hiperprolactinemia por desconexión fue observada en 45,2% de la población (X: 65.6 ng/ml r: 30-172). El hipopituitarismo se detectó en 25,7% de los casos. La cirugía fue transeptoesfenoidal (TSE) en 80%; una segunda operación fue realizada en el 28% de la población. La IH fue positiva por orden de frecuencia para LH, FSH y ASU o las 3 combinaciones. La evolución posquirúrgica evidenció mejoría en el campo visual (CV) en el 41%. La presencia de restos tumorales y/o recidiva fue del 84%. Se indicó radioterapia en 37% y la sustitución hormonal fue necesaria en el 65% de los pacientes.

The aim of our study was to describe the clinical-biochemical and radiologic presentation and the post surgery outcome in a cohort of patients with gonadotrophinomas. Patients were selected from nine Endocrinology Units of the city of Buenos Aires from 1983 at 2003. The inclusion criteria was defined by nonfunctinoning pituitary adenomas with positive innmunohistochemical (IH) for luteinizing hormone (LH), follicle-stimulating hormone (FSH) and/or alpha subunit (ASU). Innmunohistochemically plurihormonal adenomas were excluded. Sixty six patients were analyzed, aged 51,8 ± 12,1 (X +/- DS) years; (39 men). More prevalent symptoms were visual alterations (72,8%), hypogonadism and headaches. Eleven percent was diagnosed as incidentalomas. Ninety eight percent were macroadenomas, 56,9% was Hardy stage (HS) 3 and 29,6% was HS 4. The patients were followed up for 47,8 months (r: 5-168). Hypogonadism was biochemically found in 82,4%. The majority showed low or inappropriately normal levels of gonadotrophins except for 4 women and 3 men that had high and dissociated levels. Hyperprolactinemia was observed in 45,2% and was interpreted as an interference with normal dopamine inhibition of prolactin secretion (X+/-DS: 65.6+/- ng/ml, r: 30-172). Hypopituitarism was found in 25,7% of the patients. Transsphenoidal surgery was carried out in 80% and in 28% a second surgery was needed. The IH was positive for LH, FSH and ASU in this order of frequency or its combinations. Tumor persistency and/or recurrency were found in 84% of the patients. Forty one percent showed improvement of visual defects. Radiotherapy was indicated in 37% and hormonal replacement was needed in 65% of the patients.

Immunocytochemical localization and ultrastructural study of gonadotroph cells in the female desert lizard Uromastyx acanthinura.

The pars distalis from the pituitary gland of adult female desert lizards (Uromastyx acanthinura), captured during vitellogenesis (late may) and hivernal period, was studied with immunocytochemical methods using specific antisera against human FSH (hFSH) and LH (hLH). The immunostaining with anti-hLH and anti-hFSH allowed the identification of only FSH-like containing cells. The FSH-like immunoreactive cells were affected differently by a physiological stage and showed some heterogenous cytological characteristics. During vitellogenesis, four aspects of rostral FSH-like immunoreactive cells could be recognized. The expression of FSH-like in mainly immunoreactive cells was parallel to an intense synthetic activity and to the presence of ultrastructural features indicating an intense release of the hormone. This release was considerably altered in winter, the immunoreactive cells stored an important amount of secretion granules which increased in size and undergo a crinophagic process.

Antipituitary antibodies against gonadotropin-secreting cells in adult male patients with apparently idiopathic hypogonadotropic hypogonadism.

CONTEXT: Hypogonadotropic hypogonadism (HH) can occur at any stage of life as an isolated congenital or acquired abnormality or within a more generalized pituitary or hypothalamic impairment. However, the defect in patients with idiopathic HH is still unknown. OBJECTIVE: The aim of this study was to investigate the prevalence of antipituitary antibodies (APA) in a group of HH patients with or without Kallmann's syndrome and to characterize their pituitary target. DESIGN: We conducted a cross-sectional cohort study. SETTING: The study was performed at the Endocrinology Unit of the Second University of Naples. PATIENTS: Twenty-one HH patients with normal sense of smell (group 1), 10 patients with Kallmann's syndrome (group 2), 13 patients with HH associated with other pituitary hormone deficiencies (group 3), and 50 normal controls were studied. MAIN OUTCOME MEASURES: APA were evaluated in patients and in controls by indirect immunofluorescence. Moreover, a magnetic resonance imaging (MRI) of the hypothalamic-pituitary region was performed in all three groups of patients. RESULTS: APA were detected at high titer in eight out of 21 patients in group 1 (38%) and in five of 13 in group 3 (38.4%), and at low titers in two out of 10 in group 2 (20%) and in three of 50 controls (6%). In patients of group 1, APA immunostained selectively gonadotropin-secreting cells, whereas in those of group 3, they immunostained other pituitary hormone-secreting cells also. None of patients in group 1 showed alterations on MRI, whereas all patients in group 2 showed aplasia/hypoplasia of the olfactory bulbs/tracts and/or of olfactory sulci. Among the five APA-positive patients in group 3, three had normal MRI, one had findings of empty sella, and one had findings of autoimmune hypophysitis. CONCLUSIONS: Our results suggest that some apparently idiopathic cases of HH, both isolated and associated with other pituitary impairment, can be caused by an early autoimmune process involving the gonadotrophs at pituitary level. Future longitudinal studies are needed to clarify the natural history of this process and the possible effect of early corticosteroid therapy.

Preparation and use of specific antibodies to the beta-I and beta-II subunits of gonadotropic hormone from Fundulus heteroclitus pituitary.

Fundulus heteroclitus naturally spawns with a semilunar periodicity throughout most of the year in its southernmost habitat, an activity that can be maintained in the laboratory. The alpha and two beta subunits comprising F. heteroclitus gonadotropic hormones (GtHs) I and II have been sequenced, and antibodies have been raised against unique peptides found in each of the two beta subunits. On immunoblots of pituitary proteins, each antibody recognizes a single band with a molecular mass of 16-17 kDa, somewhat larger than the deduced sizes (11-13 kDa) of the unglycosylated subunits. Each antibody also recognizes a different subset of pituitary cells in the central (GtH I) and peripheral (GtH II) proximal pars distalis, regions that display the typical tinctorial properties of gonadotrops. The distribution and distinct separation of cells containing GtH beta subunits I and II thus differ from those found for previously described teleost species, most of which are salmonids that engage in a single spawning episode during the year. The availability of these antibodies thus makes F. heteroclitus an inexpensive, easily manipulated model system for studies on the hormonal regulation of fractional spawning common to a large class of commercially important species other than salmonids.

Immunological cross-reactivity between rainbow trout GTH I and GTH II and their alpha and beta subunits: application to the development of specific radioimmunoassays.

Immunological cross-reactivities between rainbow trout GTH I and GTH II and their alpha and beta have been studied using highly purified rainbow trout gonadotropins and subunits and antibodies raised against beta subunits. From these observations radioimmunoassays have been developed for rainbow trout GTH I and GTH II. The GTH II RIA was highly specific and cross-reacted only with GTH II and its beta 1 subunits, with beta 2 being less potent than beta 1 in competing GTH II binding. There was no cross-reactivity with GTH I. Its sensitivity varied between 0.1 and 0.2 ng/ml, allowing GTH II measurement early in the reproductive cycle. Variations between and within assays were less than 10%. There was a lack of specificity of GTH I RIA (44% cross-reactivity with GTH II, when using labelled native GTH I). Reasons for this lack of sensitivity were studied. It cannot be attributed to beta subunits (less than 1.2% cross-reactivity). However, the cross-reactivity of alpha subunits was very important. This suggests that the presence of free alpha subunits in the medium can be responsible for the lack of specificity. Labelling native GTH I resulted in conformational change in molecular weight and dissociation of the hormone into subunits, whereas iodination did not induce GTH II dissociation. This dissociation can be avoided by labelling the stable form of GTH I. Using this radio-tracer, the specificity and the sensitivity of the assay were greatly improved (GTH II cross-reactivity was decreased to 3.7, mean sensitivity 0.87 +/- 0.072 ng/ml). The sensitivity of the assay diminished with ageing of labelled GTH I. The assay variation was 4.6% within an assay and 9.8% between assays. The use of labelled beta GTH I still increases the specificity (2.3% GTH II cross-reactivity), but with a 2.4-fold loss of sensitivity. In both GTH I and GTH II RIA plasma and spiked plasma with purified GTHs gave displacement curves parallel to standard. These assays were used to study pituitary responsiveness to a GnRH analogue in female rainbow trout prior to oocyte maturation. The effects of GnRH on GTH II secretion were confirmed. The peptide did not significantly stimulate GTH I secretion.

Gonadotropic and thyrotropic cells from the Mediterranean yellowtail (Seriola dumerilii; Risso, 1810): immunocytochemical and ultrastructural characterization.

BACKGROUND: Gonadotropins GTH I and GTH II from the pituitary of Mediterranean (M.) yellowtail (Seriola dumerilii) were isolated and characterized, and antisera to the whole GTH II molecule (anti-My alpha,betaGTH II) and to its beta-subunit (anti-My betaGTH II) were obtained. At the light microscopic level, anti-My alpha,betaGTH II reacted with My betaGTH II-immunoreactive cells (GTH II cells), thyroid-stimulating hormone (TSH) cells, and a third cell population, which could have been GTH I cells. The aim of this study was the ultrastructural characterization of GTH and TSH cells in M. yellowtail using the immunogold method in order to provide a basis for future research into reproduction of this species. METHODS: Pituitaries from mature male and female specimens reared in captivity were dissected out and processed for electron microscopy. The immunogold method was carried out by using anti-My alpha,betaGTH II, anti-My alpha,betaGTH II preabsorbed with the alpha subunit of the M. yellowtail GTH (My alphaGTH-subunit), anti-My betaGTH II, anti-human (h) alpha,betaTSH, and anti-h betaTSH sera to reveal gonadotropic and thyrotropic cells. RESULTS: M. yellowtail gonadotropic cells were very heterogeneous with regard to their size, shape, and ultrastructural features. Cells were found with numerous, round, variably electron-dense, secretory granules and globules; others were found with their cytoplasm occupied mostly by dilated cisternae of rough endoplasmic reticulum (RER) and scarce secretory granules; and other intermediate cell forms were found that showed varying proportions of secretory granules and dilated RER. The secretory granules and globules were immunogold labeled with anti-My alpha,betaGTH II, and the reaction was weaker in the latter. A similar immunogold-labeling pattern was found with anti-My betaGTH II and with anti-My alpha,betaGTH II preabsorbed with the My alphaGTH-subunit, although some cells that showed the same ultrastructural features described above were not immunogold labeled and could have been GTH I cells. Thyrotropic cells had small, round, secretory granules of medium or high electron density that were immunogold labeled with anti-My alpha,betaGTH II, anti-h alpha,betaTSH, and anti-h betaTSH sera, but not with anti-My betaGTH II or anti-My alpha,betaGTH II serum preabsorbed with the My alphaGTH-subunit. All of the cell forms described for gonadotropes and thyrotropes were also found in a state of involution. CONCLUSIONS: Gonadotropes that are of a single morphological type but that vary in ultrastructure are present in the pituitary of captive M. yellowtail. GTH II- and putative GTH I-producing cells were distinguishable from one another and from TSH cells by their different reactions to anti-My alpha,betaGTH II, anti-My betaGTH II, and anti-My alpha,betaGTH II preabsorbed with the My alphaGTH-subunit.

A steroidal pheromone and spawning stimuli act via different neuroendocrine mechanisms to increase gonadotropin and milt volume in male goldfish Carassius auratus.

In goldfish (Carassius auratus), pheromonal 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) and spawning stimuli (interaction with a sexually active female releasing prostaglandin pheromone) both increase gonadotropin-II (GtH-II) and milt volume. In the goldfish pituitary, GtH-II release is stimulated by gonadotropin-releasing hormone (GnRH) and inhibited by dopamine (DA). In this study, we investigated the possibility that 17,20beta-P and spawning stimuli act via separate neuroendocrine mechanisms by determining whether their effects on GtH-II could be selectively disrupted by injection of DA type-2 receptor (D-2) agonists (bromocryptine and LY171555) or a goldfish GnRH antagonist, [Ac-Delta3-Pro1, 4FD-Phe2, d-Trp3,6]-mGnRH (analog E). D-2 agonists blocked 17,20beta-P-induced increases in GtH-II and milt volume but did not affect spawning-induced responses. GnRH antagonist blocked 17,20beta-P-induced increases in GtH-II and milt volume, and spawning-induced GtH-II increase, but did not affect spawning-induced increase in milt volume. These results suggest that (1) pheromonal 17,20beta-P and spawning stimuli increase GtH-II increase via distinct neuroendocrine mechanisms, (2) the effect of pheromonal 17,20beta-P on increasing milt volume is GtH-II-dependent, and (3) the effect of spawning stimuli on increasing milt volume is GtH-II-independent.

The adenohypophysis of the swamp eel, Synbranchus marmoratus, an immunocytochemical analysis.

The adenohypophyseal cell types of the protogynous fish Synbranchus marmoratus were studied by histochemical and immunocytochemical staining with antisera raised against piscine and human pituitary hormones to ascertain their distribution. The prolactin (PRL) cells were distributed in the rostral pars distalis and showed specific binding to antisera to carp and chum salmon prolactin. No reaction was observed with antiserum to human prolactin. The corticotrops showed strong immunoreactivity with anti-human ACTH, these cells bordered the neurohypophysis and islets between PRL cells in the rostral pars distalis. Growth hormone (GH) cells were densely distributed and associated with the neurohypophysis only in pars distalis proximal. They reacted with antisera to piscine GH but not with antisera to human growth hormone. The thyrotrops were scattered in the proximal pars distalis and showed strong immunoreactivity to the human thyrotropin Beta subunit antiserum. Gonadotrops were located in the central area of the proximal pars distalis and in the external border of the pars intermedia. These cells were alcian blue and PAS positive, and reacted with anti-croaker GTH and anti-coho GTH I and GTH II. The PAS positive cells from the pars intermedia bound specifically to anti-chum somatolactin.

The adenohypophysis of the swamp eel, Synbranchus marmoratus, an immunocytochemical analysis

The adenohypophyseal cell types of the protogynous fish Synbranchus marmoratus were studied by histochemical and immunocytochemical staining with antisera raised against piscine and human pituitary hormones to ascertain their distribution. The prolactin (PRL) cells were distributed in the rostral pars distalis and showed specific binding to antisera to carp and chum salmon prolactin. No reaction was observed with antiserum to human prolactin. The corticotrops showed strong immunoreactivity with anti-human ACTH, these cells bordered the neurohypophysis and islets between PRL cells in the rostral pars distalis. Growth hormone (GH) cells were densely distributed and associated with the neurohypophysis only in pars distalis proximal. They reacted with antisera to piscine GH but not with antisera to human growth hormone. The thyrotrops were scattered in the proximal pars distalis and showed strong immunoreactivity to the human thyrotropin Beta subunit antiserum. Gonadotrops were located in the central area of the proximal pars distalis and in the external border of the pars intermedia. These cells were alcian blue and PAS positive, and reacted with anti-croaker GTH and anti-coho GTH I and GTH II. The PAS positive cells from the pars intermedia bound specifically to anti-chum somatolactin.(AU)

Brain-pituitary-gonadal axis during early development and sexual differentiation in the rainbow trout, Oncorhynchus mykiss.

Profiles of testosterone, 11-ketotestosterone, androstenedione, and estradiol were determined by RIA, and immunocytochemical techniques were employed to identify gonadotropin (GTH) I and II and gonadotropin releasing hormone (GnRH) in monosex and mixed sex populations of rainbow trout from 1 to 126 days postfertilization (dpf). Steroid levels were relatively high at 1 dpf and declined until 25 dpf. At 30 and 48 dpf (hatching) steroid levels increased slightly before they fell by 78 dpf and remained relatively constant thereafter. Trends toward differences in steroid content between males and females became evident around the time gonadal differentiation was histologically discernible (78 and 90 dpf). GTH I was present in the proximal pars distalis at all dates (48-126 dpf), whereas GTH II was not detectable. GnRH was found at all dates (48-126 dpf) and was distributed in several areas of the brain including the nucleus preopticus periventricularis, nucleus lateralis tuberis, and the pituitary in the region where GTH I was found. No differences were seen between males and females in the timing of appearance, localization, or intensity of staining of these peptide hormones. Given that the brain-pituitary-gonadal axis seems to be intact during the process of sexual differentiation and the fluctuations of steroid levels during this process, sex steroids may play the driving role for sexual differentiation of rainbow trout.

Brain-periphery connections: do they play a role in mediating the effect of centrally injected interleukin-1 beta on gonadal function?

The immune system and several endocrine axes communicate with each other through a network of molecules which collectively produce a coordinated response to immune challenges. This phenomenon, necessary for the survival of the organism, is thought to involve the release, by activated cells in the periphery, of proteins, called cytokines, which inform the brain about immune activation. The brain then organizes a series of neuroendocrine responses which participate in the regulation of the host response. With regard to the influence of cytokines on the hypothalamic-pituitary-gonadal axis, we know that the injection of these proteins lowers gonadotropin-releasing hormone release, which in turn inhibits luteinizing hormone (LH) secretion. These changes would be expected to decrease sex steroid production and, indeed, estrogens and testosterone are low in female and male rats, respectively, following acute intracerebroventricular (i.c.v.) injection of interleukin (IL)-1 beta. There is, however, another possibility that central cytokines could alter ovarian and testicular function independently of changes in gonadotropin levels. Prolonged i.c.v. infusion of the cytokine into the female rat brain produced a dramatic rise in progesterone levels. The absence of a comparable change in the progesterone release rate of males infused with IL-1 beta, and the presence of marked surges of prolactin (PRL) in the females, suggests that IL-1 beta altered ovarian function, and that the persistence of large corpora lutea induced PRL release. The possibility that the cytokine might stimulate the brain circuits that regulate PRL release, while possible, appears remote, because male rats injected with IL-1 beta showed significantly blunted PRL levels. In intact adult male rats, i.c.v. IL-1 beta administration caused the expected decrease in LH and testosterone levels, but was also accompanied by a loss of testicular responsiveness to gonadotropins. Though elevated levels of corticosteroids are known to interfere with normal gonadal steroidogenesis, blockade of IL-1-induced corticosterone release did not reverse the inhibitory influence of the cytokine. One mechanism that deserves attention is the possibility that i.c.v. injection of IL-1 beta might increase circulating cytokine levels, and indeed plasma IL-6 concentrations were significantly elevated in rats treated with IL-1 beta. This humoral mechanism may disrupt testicular function through the documented inhibitory effects of blood-borne cytokines on Leydig cell function. In addition, brain cytokines might influence a variety of peripheral events through direct (neural?) connections. This brief review discusses the hypothesis that there are brain-to-gonad connections that bypass the pituitary, and presents results that might support the possibility that central injection of IL-1 beta decreases testosterone secretion independently of blunted LH levels.

Inhibin-like and gonadotropin-like immunoreactivity in pituitary cells of male monkeys (Macaca fascicularis, Macaca mulatta).

Inhibin-like immunoreactivity was detected by immunocytochemistry in the pituitaries of untreated male crab-eating macaques (cynomolgus monkey) and rhesus monkeys, in rhesus monkeys actively immunized against FSH, and in one orchidectomized crab-eating macaque. Localizations were performed by the immunogold-silver staining with 5-nm colloidal gold-conjugated second or third antibodies and by the alkaline phosphatase-anti-alkaline-phosphatase technique. Two different inhibin-specific antisera, raised against the alpha-subunit or the entire inhibin molecule, provided identical staining patterns. Positive label was confined to the pars distalis of the pituitary and occurred exclusively in the cytoplasm of morphologically different cell types throughout the pars distalis in all pituitaries. Staining was most prominent in clusters of chromophobic cells. The presence of inhibin-like activity in the pituitary of an orchidectomized monkey with undetectable serum inhibin levels suggests that inhibin is produced within the pituitary gland. Co-localization studies for the beta-subunits of the gonadotropic hormones revealed that on average 82% of the gonadotropes were bihormonal. Using the same protocol, co-localization of inhibin-like activity with gonadotropin-like immunoreactivity revealed only a small degree of common distribution (less than 15%). Inhibin-positive cells were frequently in close proximity to gonadotropic cells and, thus, paracrine effects of inhibin on gonadotropin-synthesizing cells are conceivable.

Isolated adrenocorticotropin deficiency associated with an autoantibody to a corticotroph antigen that is not adrenocorticotropin or other proopiomelanocortin-derived peptides.

A 44-yr-old man with hypocortisolism was shown to have an undetectable basal plasma ACTH level and absent or subnormal ACTH and beta-lipotropin responses to provocative testing with insulin, vasopressin, and CRH. Endocrine function after glucocorticoid replacement was otherwise normal, thus establishing the diagnosis of isolated ACTH deficiency. This patient's serum was tested immunohistochemically for the presence of an antipituitary antibody by indirect immunofluorescence of rat pituitary tissue. Positive immunostaining was observed in stellate-shaped cells in the anterior and intermediate lobes. Immunopositive cells were shown by immunoelectron microscopy to have ultrastructural characteristics of corticotrophs. Immunoreactivity was concentrated in secretory granules 120-170 nm in diameter. In a double immunolabeling procedure, staining by the patient's serum was shown to colocalize with rabbit antiserum to ACTH, but not with antisera to PRL, GH, beta TSH, or beta LH. Immunoabsorption of the patient's serum with ACTH-(1-24), ACTH-(1-39), gamma MSH, corticotropin-like intermediate lobe peptide, beta-endorphin, or beta-lipotropin failed to diminish immunolabeling in the pituitary. We conclude that the antipituitary antibody in this patient's serum shows immunohistochemical specificity for a rat corticotroph antigen located in secretory granules that is neither ACTH nor any of the proopiomelanocortin (POMC)-derived peptides tested. The autoantigen could be a cell-specific granular factor involved in the posttranslational processing of POMC or secretion of ACTH. We postulate that an autoimmune process may account for this patient's disease, and that his antipituitary antibody could play a pathogenic role by either inhibiting a POMC-processing enzyme or initiating an antibody-dependent cell-mediated cytotoxicity reaction, resulting in the selective destruction of corticotrophs.

Pituitary steroids in two teleost species: immunohistological and biochemical studies.

Antisera raised against steroid hormones [estradiol-17 beta (E2), testosterone (T), 11 beta-hydroxyandrostenedione (OHA)] were used to localize immunoreactive material in fixed and paraffin-embedded pituitaries of the African catfish and the rainbow trout. Organic extracts of pituitary homogenates were analyzed for steroid hormones by radioimmunoassay and gas chromatography-mass spectrometry (E2 in female catfish only). With the exception of an E2-positive cell type in the catfish neurointermediate lobe, steroid immunoreactivity was found to be restricted to the cytoplasm of adenohypophyseal cells, which were also labeled after incubation with catfish alpha,beta-gonadotropin and salmon gonadotropin antisera, respectively. Steroid levels determined by radioimmunoassay in the catfish ranged between 85 and 628 pg/pituitary, while lower levels (2-8 pg/pituitary) were found in the rainbow trout. E2 was identified by gas chromatography-mass spectrometry at a level of 84 pg/pituitary. The observation that immunolabeling after steroid antiserum incubation is confined mainly to gonadotrops provides morphological evidence for direct steroid effects on this particular cell type.

Estradiol has inverse effects on pituitary glycoprotein hormone alpha-subunit messenger ribonucleic acid in the immature European eel and the gonadectomized rat.

In teleosts, the pituitary contains a single glycoprotein gonadotropic hormone (GTH) composed of two dissimilar alpha- and beta-subunits. The European eel, Anguilla anguilla L, is sexually immature at the silver stage due to a deficiency in GTH synthesis and secretion. In previous studies we (S.D., YA.F.) have demonstrated a strong stimulatory action of estradiol (E2) on eel pituitary GTH content. In contrast, we (R.C., M.J.) have shown that in the rat E2 negatively regulates gonadotropin subunit synthesis via changes in specific mRNA levels. The purpose of our present work was to check for such effects of E2 on the synthesis of GTH alpha- and beta-subunits in the European eel. Eel pituitary mRNA was translated in a cell-free system in the presence of [35S]Met + Cys. We demonstrate that one of the translated polypeptides, characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, cross-reacts with an antiserum to denatured bovine alpha-subunit. Its apparent mol wt (18.5K), which is slightly higher than that of the corresponding rat alpha-precursor, suggests that it represents the precursor of the alpha-subunit of eel glycoprotein hormones. The specificity of immunoprecipitation was confirmed by competition with ovine alpha (but not with ovine LH beta or bovine TSH beta). Quantitative evaluation of the putative eel alpha-subunit precursor showed that it represents 0.2% of the total protein translated by RNA from the normal silver eel. Chronic treatment of eels for 3 weeks with 17 beta-E2 increased by 8.0- to 8.5-fold the proportion of the putative alpha-subunit precursor among translation products. Due to the lack of cross-reactivity with the presumed GTH beta precursor, no radioactive material could be specifically detected in translation medium of eel pituitary mRNA using antisera to either denatured bovine LH beta or ovine FSH beta. Our data suggest that E2, depending on vertebrate group and probably on sexual status, may exert either positive or negative control on gonadotropin synthesis by opposite effects on the levels of specific mRNA.

Homologous desensitization with gonadotropin-releasing hormone (GnRH) also diminishes gonadotrope responsiveness to maitotoxin: a role for the GnRH receptor-regulated calcium ion channel in mediation of cellular desensitization.

Maitotoxin (MTX) stimulates gonadotropin release from pituitary cell cultures. The time course and efficacy of LH release in response to GnRH and to MTX are similar; both secretagogues require extracellular Ca2+ and are inhibited by the selective Ca2+ ion channel antagonist methoxyverapamil (D600). LH release in response to either GnRH or MTX is not measurably inhibited by two other chemical classes of Ca2+ ion channel inhibitors represented by nifedipine and by diltiazem. The two secretagogues are nonadditive in their action on LH release when presented at high doses and prior studies indicate that MTX has no endogenous ionophoretic activity. These observations indicate that MTX likely stimulates LH release due to activation of the GnRH receptor associated Ca2+-ion channel in the gonadotrope. We have therefore assessed the functional state of this channel during the development of homologous desensitization of the gonadotrope to GnRH by measuring the ability of MTX to stimulate LH release. Cells were desensitized with GnRH in the presence of 3 mM EGTA. Under these conditions, the cells become refractory to GnRH in the absence of gonadotropin release since the latter process, but not the former, requires extracellular Ca2+. Accordingly, this approach allows assessment of the degree of desensitization in the absence of the influence of gonadotropin depletion. Such desensitized cells are less responsive to GnRH. Desensitized pituitary cells also respond with diminished efficacy and potency to MTX three or more hours after GnRH treatment but not at an earlier time (1 h) when GnRH receptors are diminished. These data are consistent with a model in which homologous desensitization is viewed as developing in two phases.(ABSTRACT TRUNCATED AT 250 WORDS)