Immunoreactive gonadotropin and luteinizing hormone releasing hormone in the pituitary gland of neonatal platyfish

An immunoperoxidase method has been used to localize luteinizing hormone releasing hormone (LHRH) and gonadotropin (GTH) in neonatal (1-, 3-, 6-, and 9-day-old) platyfish (Xiphophorus maculatus). Immunoreactive (ir-) GTHβ and ir-LHRH are found in cells of the two extreme lateral regions of the caudal pars distalis (CPD), in a small number of the chromophobes comprising the single cell layer of the ventral CPD, and in many PAS+ cells of the pars intermedia (PI). Antiserum to the alpha subunit of GTH stains the same cells in the CPD but none in the PI. Neither ir-GTH nor ir-LHRH is found in the brain. Evidence that suggests that the PI is the source of a GTH which is not identical to the one in the CPD and which may function early in postnatal development is presented and discussed. The speculation that the cells of the lateral CPD give rise to the ventral GTH zone of the CPD which is characteristic of sexually mature fish is also presented.     

Pheromonal stimulation of spawning release of gametes by gonadotropin releasing hormone in the chiton,Mopalia sp

The chiton Mopalia sp., a mollusc, was exposed to various dilutions of gonadotropin releasing hormone (GnRH) in sea water to determine whether this peptide is capable of acting as a pheromone that could stimulate release of ripe gametes (spawning). Two of the peptides, lamprey GnRH-1 and tunicate GnRH-2, had this action at a higher concentration (1.0 mg/L) but dilutions to 50 microg/L no longer were effective. Three other GnRHs: lamprey GnRH-3, tunicate GnRH-1, and a modified chicken GnRH-2, had no such action under the same test conditions. Since the spawning response could be produced by some GnRHs and not by others, it would appear that some kind of molecular recognition is involved, possibly by specific binding to a receptor. In earlier preliminary experiments tunicate GnRH-2 rapidly stimulated gamete release in a hemichordate, Saccoglossus. Thus it is suggested that GnRHs, in at least some invertebrates, may function as pheromones, serving to stimulate simultaneous spawning of individuals in a population of animals, and in this way assure more successful fertilization in species that must release their gametes into the water in which they live.     

Luteinizing hormone pulsatile secretion and pituitary response to gonadotropin releasing hormone and to thyrotropin releasing hormone in male epileptic subjects on chronic phenobarbital treatment

Endocrine changes have been reported in treated epileptic subjects, who often exhibit sexual dysfunctions, but the endocrine effects of single antiepileptic drugs have not been completely elucidated. In this study we have investigated the influence of phenobarbital (PB) on adenopituitary function and on peripheral sexual steroid pattern in 8 epileptic males. Chronic PB treatment does not modify luteinizing hormone (LH) pulsatile secretion. In the same subjects, LH and follicle stimulating hormone (FSH) response to Gonadotropin Releasing Hormone was blunted with respect to healthy controls both in terms of absolute values and of secretion areas. No difference was found in prolactin (PRL) response to Thyrotropin Releasing Hormone. In the epileptic group a significant increase in the levels of sex hormone binding globulin and a consequent decrease of the percent free testosterone have been observed. PB treatment also significantly lowers 17-beta-estradiol mean levels. These data suggest that PB independently affects both gonadotropin secretion and peripheral steroid pattern.     

Requirement of extracellular calcium in fish pituitary gonadotropin release by gonadotropin hormone-releasing hormone

Influence of extracellular calcium on gonadotropin hormone-releasing hormone (GnRH)-stimulated gonadotropin hormone (GtH) release from a teleostean fish (Channa punctatus) pituitary was examined in vitro by preparing enzymatically dispersed pituitary cell incubation. Effect of Ca2+ on GnRH-augmented GtH release was evaluated with partially purified C. punctatus GnRH (cGnRH) and synthetic mammalian GnRH (mGnRH). Cells were dispersed by 0.3% collagenase plus 0.05% trypsin in culture medium and a high yield of viable cells were obtained. Addition of cGnRH (10 micrograms/ml) to pituitary cells in Ca2+-free medium resulted in a significant increase in GtH release, but the addition of Ca2+ (2 mM) enhanced it to about four- and threefold over cGnRH and mGnRH, respectively. Increasing concentrations of Ca2+ (0.1-2.0 mM/well) with fixed concentrations of GnRH (10 micrograms/ml) or increasing doses of GnRH (2.5 to 20 micrograms/ml) with fixed amount of Ca2+ (2 mM/well) resulted in a dose dependent increase in GtH release. EDTA or EGTA (2 mM/well) completely suppressed the Ca2+-augmenting effect of GnRH-stimulated GtH release. Addition of lanthanum (La3+, 4 mM/well), a competitive inhibitor of Ca2+, reduced 60% of the Ca2+ (2 mM/well) stimulation. Verapamil, a specific Ca2+ channel blocker, when added in increasing concentrations (1-100 microM/well) to pituitary cell incubations containing GnRH-stimulated GtH release in Ca2+-free medium could be waived by EGTA (2 mM/well), indicating availability of extracellular calcium from tissue sources. The uptake of radioactive Ca2+ by pituitary cells was greatly enhanced by GnRH while the addition of verapamil (10 microM/well) not only inhibited the GnRH-stimulated uptake, but also reduced it below the control level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum sex hormone and gonadotropin concentrations in premenopausal women with multiple sclerosis

Dysfunctions within the hypothalamic-pituitary-gonadal axis occur frequently among women with multiple sclerosis (MS) and may induce menstrual disturbances and subsequent infertility. We have measured serum concentrations of prolactin. gonadotropins and sex hormone binding globulin (SHBG) as well as free and bound oestrogen and androgen levels in 14 women of fertile age with MS. These women all displayed regular cycles without having experienced fertility problems. As controls 14 normal women with regular periods and ideal body weight of 91% (range 80-101) were included. Serum from both groups was sampled during the early follicular phase. The MS-patients had significantly (P less than 0.05) higher concentrations of prolactin, LH, FSH, total and free testosterone (P less than 0.01) and a significantly lower serum concentration of oestrone sulphate (P less than 0.01). The abnormal hormone concentrations were not related to clinical status of the disease. We propose that the increased androgen levels are of ovarian origin as adrenal androgens were normal. The reason for the slight increase of prolactin and the marked increase of gonadotropins in women with MS is speculative. As oestradiol levels, however, were within normal range, we assume that a peripheral resistance to gonadotropins combined with an abnormal central regulation causes the increased pituitary secretion.     

Increased thyroid-stimulating hormone response to thyrotropin-releasing hormone in hyperprolactinemic women

The TSH response to TRH and basal thyroid function were studied in 21 unselected women with hyperprolactinemia. The mean serum free T4 (FT4) concentration was significantly lower in hyperprolactinemic women than in normal women. The mean basal TSH concentrations were similar, but the incremental TSH response to TRH was significantly greater in hyperprolactinemic women than in normal women. Increased dopaminergic inhibition of TSH release has been reported in hyperprolactinemic women. The low serum FT4 concentration in hyperprolactinemic women may be the result of increased dopaminergic inhibition of TSH release, and the increased pituitary TSH reserve may reflect increased TSH storage due to dopaminergic inhibition of basal TSH release. Alternatively, though less likely, the low serum FT4 concentration may be the result of direct action of PRL on the thyroid, and the low FT4 concentration may, in turn, lead to the increased pituitary TSH reserve.     

Binding affinity and biological activity of gonadotropin releasing hormone agonists in isolated pituitary cells

The relationships between binding affinity and the biological potency of eight GnRH agonist analogs were evaluated in isolated rat pituitary cells. For this purpose, binding affinity and biological activity were assayed under similar physiological conditions in medium 199, pH 7.4, and binding affinity was also measured under the standard conditions in hypotonic buffer at low temperature. Under physiological conditions, receptor binding affinity was consistently lower than when measured in the hypotonic Tris buffer usually employed for GnRH receptor studies. In the low temperature binding assay at 0 C, which provided a measure of the affinity constant without degradation, a difference of 20- to 30-fold was observed between native GnRH and its most potent analog, [D-Ser(t-Bu)6]des-Gly10-GnRH N-ethylamide. Modifications of the amino acid residues at both positions 6 and 10 of the decapeptide increased the binding affinity of GnRH analogs. When the receptor binding assay was performed at 37 C, the range of the apparent affinity constants was extended up to 60-fold. The affinity constants derived at 37 C were closely correlated with the biological potencies of the individual analogs measured in the same cell system. The effect of temperature on binding affinity was not significantly influenced by peptide metabolism, which was minor in the absence of horse serum from the incubation medium. At the pituitary level, the biological potency of the GnRH agonist analogs is predominantly determined by their higher receptor affinity, and reduced degradation is a less important aspect of the high biological activity of the superagonist analogs.     

The effect of changing somatostatin tone on the pituitary growth hormone and thyroid-stimulating hormone responses to their respective releasing factor stimuli.

Somatostatin (SS) inhibits GH and TSH secretion, but its role in modulating their pulsatility is unclear. We studied GH and TSH responses to GH-releasing hormone (GHRH) and TRH stimulation upon a variable background infusion of saline, SS-(1-14) at 20 and 100 micrograms/m2.h, and oral pyridostigmine (30 and 60 mg) in six adult males. Basal GH levels were unaffected by SS-(1-14). Deconvolution analysis of serum GH values demonstrated that the pituitary responded to two GHRH stimuli 90 min apart without attenuation of the second response. The higher dose of SS-(1-14) significantly blunted the first GH response; second GH responses were further attenuated by both SS-(1-14) doses. Maximum GH release and "switch-off" rates for both stimuli were reduced without changes in the 50% secretion time. Pyridostigmine enhanced the first GH response to GHRH with an increase in the GH release rate; second GH responses were not augmented. GH secretion was prolonged by pyridostigmine, although the 50% secretion time remained unchanged. Peak stimulated serum TSH was attenuated by both SS-(1-14) doses, but pyridostigmine had no effect. All other TSH parameters examined were unaffected. We conclude that the GH response to GHRH is dependent on SS tone, but that the thyrotroph is not tonically inhibited by SS. SS attenuates the rate of GH release without changing the duration of secretion and appears important in terminating GH secretion.     

Pregnancy in women with systemic sclerosis

We assessed fetal morbidity and mortality in women with systemic sclerosis (SSc). Women with a history of SSc and a concomitant pregnancy completed a detailed questionnaire about the pregnancy. These 48 subjects were age-matched and race-matched to 2 other groups of women (a rheumatoid arthritis group and a control group from the same neighborhood), all of whom had been pregnant at least once. There were no differences in the frequencies of miscarriage or perinatal death in the SSc group compared with the 2 control groups. Preterm births occurred slightly more frequently in both SSc patients and rheumatoid arthritis patients compared with the neighborhood control subjects. There were significantly more small full-term infants born to women with SSc. Interestingly, the increase in preterm births and small full-term babies occurred with equal frequency prior to and after the onset of disease. Although close monitoring for premature birth and intrauterine growth retardation is necessary, we conclude that an uneventful, healthy pregnancy is possible for women with SSc. Those with early, rapidly progressive, diffuse skin thickening should avoid becoming pregnant since, intrinsically, they are at higher risk of developing renal crisis.     

Participation of arachidonic acid metabolism in gonadotropin-releasing hormone stimulation of goldfish gonadotropin release

Two intraperitoneal injections of a mammalian gonadotropin-releasing hormone (GnRH) analog, [D-Ala6, Pro9-N-ethylamide]-GnRH (mGnRHa; 0.1 micrograms/g), at 12-hr intervals increased serum gonadotropin (GTH) levels in sexually mature and sexually regressed female goldfish 2 and 6 hr after the second injection. This serum GTH response was decreased by the coinjection of a lipoxygenase enzyme inhibitor, nordihydroguaiaretic acid (NDGA: 0.1 to 10 micrograms/g) at the time of the second mGnRHa application. In static cultures of dispersed goldfish pituitary cells, 1-100 microM arachidonic acid (AA) and 0.1-1000 nM [Trp7, Leu8]-GnRH (salmon GnRH, sGnRH) and [D-Arg6, Pro9-N-ethylamide]-sGnRH (sGnRHa) caused dose-dependent increases in GTH release. Additions of 1-40 microM NDGA reduced the sGnRH-stimulated GTH release in a dose-dependent manner, and completely inhibited the GTH response to increasing concentrations of AA. NDGA 40 microM also decreased the elevated GTH levels induced by sGnRHa treatment. Exposure to 10 microM 5,8,11,14-eicosatetraynoic acid, an inhibitor with mixed action on lipoxygenase and cyclooxygenase enzymes, reduced the dose-dependent GTH response to sGnRH and AA. In contrast, coincubation with another cyclooxygenase blocker, indomethacin, at 10 microM, did not alter AA and sGnRH-induced GTH release. These results provide in vivo and in vitro evidence for the participation of AA metabolism in mediating GnRH-stimulated GTH release in the goldfish. The importance of AA metabolism through the lipoxygenase pathway is also indicated.     

Development of radioimmunoassay for bullfrog thyroid-stimulating hormone (TSH): effects of hypothalamic releasing hormones on the release of TSH from the pituitary in vitro

A bullfrog (Rana catesbeiana) thyroid-stimulating hormone (TSH) beta-subunit (TSHbeta) antiserum was produced by employing a C-terminal peptide synthesized on the basis of the amino acid sequence deduced from bullfrog TSHbeta cDNA. Immunohistochemical studies revealed that the bullfrog adenohypophyseal cells that immunologically reacted with the anti-bullfrog TSHbeta corresponded to those positively stained with an antiserum against human (h) TSHbeta. The antiserum was used for the development of a specific and sensitive radioimmunoassay (RIA) for the measurement of bullfrog TSH. The sensitivity of the RIA was 0.75+/-0.07ng TSH/100microl assay buffer. The interassay and intraassay coefficients of variation were 7.6 and 5.3%, respectively. Several dilutions of pituitary homogenates of larval and adult bullfrogs, or medium in which bullfrog pituitary cells were cultured, yielded dose-response curves that were parallel to the standard curve. Bullfrog prolactin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and alpha-subunit derived from glycoprotein hormones did not react in this assay. Immunoassayable TSH in the pituitary culture medium was confirmed to exist in the form of TSHbeta coupled with the alpha-subunit by an immunoprecipitation experiment using the TSHbeta antiserum and an alpha-subunit antiserum. TSH released from pituitary cells into the medium was also confirmed to possess a considerable activity in stimulating the release of thyroxine from the thyroid glands of larval bullfrogs in vitro.The effects of hypothalamic hormones such as mammalian gonadotropin-releasing hormone (mGnRH), ovine corticotropin-releasing hormone (oCRH), and thyrotropin-releasing hormone (TRH) on the release of TSH by dispersed anterior pituitary cells of the bullfrog larvae and adults were also studied. CRH markedly stimulated the release of TSH from both adult and larval pituitary cells. Both TRH and GnRH moderately stimulated the release of TSH from adult pituitary cells but not from the larval cells. This is the first report on the development of an RIA for amphibian TSH, which has provided the direct evidence that the release of TSH from the amphibian pituitary is enhanced by the hypothalamic releasing hormones such as CRH, TRH, and GnRH.     

Sexual dimorphism in the synaptic input to gonadotropin releasing hormone neurons

GnRH neurons form the final common pathway regulating the secretion of gonadotropins from the anterior pituitary. Since the patterns of gonadotropin release display profound sexual dimorphism among mammals including the rodent, we undertook an ultrastructural analysis to determine whether these neurosecretory cells were differentially innervated between the sexes. As a further exploration of the organization of the neurocircuitry integrating GnRH neurons with the central nervous system, we also determined the degree to which GnRH cells and their processes were innervated by terminals containing either the endogenous opiate, beta-endorphin (BE) or GnRH itself. Sections from the diagonal band of Broca and the preoptic area of adult male and diestrus II female rats were immunocytochemically processed for dual localization of GnRH and BE. GnRH neurons cut through the plane of the nucleus were identified in 1 micron sections. Serial ultrathin sections were made and analyzed for 1) total synaptic input to both cell bodies and dendrites; 2) BE input; and 3) input arising from GnRH itself. We report that GnRH neuronal cell bodies in females received approximately twice the number of synapses as did those of males. The input to the GnRH dendrites, when measured as percent of plasma membrane in synaptic contact, also showed a profound sexual dimorphism with the female having a larger proportion of the dendrite in synaptic apposition. BE innervation contributed to this dimorphism at the level of both the cell body and dendrite. In contrast, the distribution and number of GnRH terminals did not differ between the sexes. In both they were confined to the dendritic arbor. We hypothesize that the capacity of the female rodent GnRH system to show neurogenic derived alterations in GnRH output not seen in the male may be due in part to these anatomical differences.     

Neuropeptide Y stimulates growth hormone and gonadotropin release from the goldfish pituitary in vitro

The effects of neuropeptide Y (NPY) on release of growth hormone (GH) and gonadotropin (GTH) from the goldfish pituitary in vitro were investigated. Exposure of perifused pituitary fragments, taken from female goldfish at late stages of gonadal recrudescence, to 5-min pulses of human NPY resulted in a rapid dose-dependent stimulation of GH and GTH release, with half-maximal effective dosages of 0.51 +/- 0.24 and 2.37 +/- 1.05 nM for GH and GTH, respectively. Repeated treatments with pulses of NPY (10 nM for GH, 5 nM for GTH) at 55-min intervals did not significantly alter the responsiveness of pituitary fragments to NPY; however, prior exposure of pituitary fragments to pulses of higher doses of NPY (50 nM GH, 10 nM for GTH) significantly reduced the subsequent hormone responses. When given at 85-min intervals repeated treatment with NPY did not blunt hormone responses to the second and third stimulations at these higher dosages. These results indicate that NPY acts at the pituitary level to stimulate GH and GTH secretion in female goldfish. The GTH response and, to a lesser extent, the GH response become desensitized to further stimulation by NPY in dose- and time-dependent manners. NPY should be considered as one element in the multifactorial systems regulating the GH and GTH secretion in goldfish.     

Effect of morphine on the release of thyroid-stimulating hormone stimulated by exposure to cold, thyroidectomy and the administration of thyrotrophin releasing hormone in male rats

Morphine reduced the release of thyroid-stimulating hormone (TSH) which stimulated by exposure to cold and by thyroidectomy as well as reducing the basal level of TSH in the serum of male rats. Thi inhibitory effect of morphine was antagonized by naloxone which did not enhance the basal or cold-induced TSH release. Pretreatment with morphine did not reduce the release of TSH induced by exogenous thyrotrophin-releasing hormone (TRH) but enhanced it. This effect of morphine was also antagonized by naloxone. The above results suggested that the effect of morphine in reducing levels of serum TSH was not mediated by blocking the effect of TRH on the anterior pituitary gland, but that it was probably mediated by the inhibition of the release of TRH.     

Local changes in immunoreactive gonadotropin releasing hormone in the rat median eminence during the estrous cycle. Correlation with the pituitary luteinizing hormone.

The immunocytochemical approach was used to explore the possibility of local changes in immunoreactive gonadotropin releasing hormone (GnRH) of the median eminence (ME) during the estrous cycle in rats, and an attempt was made to correlate these changes with immunoreactive luteinizing hormone (LH) content in gonadotropic cells. On the day of proestrus, between 9:00 and 18:00 h, a severe depletion of GnRH from the medial palisade zone (MPZ) of the rostral part of the ME was observed whenever any marked changes in the density of GnRH present in the lateral palisade zone and in organum vasculosum of the lamina terminalis (OVLT) were visible. The depletion of GnRH from the MPZ was accompanied by a degranulation of LH-cells (stained with anti-pLH-beta) in the pituitary which occurred over the same period. It is postulated that the MPZ of the rostral part of the ME contains the majority of nerve endings originating from the perikaya responsible for the cyclic surge of LH.     

Modulation of pituitary gonadotropin response to gonadotropin-releasing hormone by estradiol

The effects of 17beta-estradiol on the responsiveness of the pituitary to gonadotropin-releasing hormone (GnRH) and on the rate of disappearance of GnRH were studied in 15 healthy nulliparous women aged 18-21 years. The women were divided into 3 groups: Group 1 received no estradiol, Group 2 received the amount of estradiol needed to achieve a circulating level comparable with that in the late follicular phase, and Group 3 received enough estradiol to achieve a concentration similar to that at midcycle. Following administration of GnRH, a marked increase in both LH and FSH was seen in Group 1 subjects. A smaller increase in LH level was observed in Group 2, and virtually no LH response occurred in Group 3. There was no significant increase in FSH level in either group treated with estradiol. The infusion of estradiol did not affect the maximal plasma concentration of exogenously administered GnRH or its disappearance rate in 4 women studied.