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PubMed1981

Long term primary monolayer culture of adult murine magnocellular neurons.

Primary monolayer culture of adult murine hypothalamic cells has been carried out for 2 months. Neuron-like cells as well as fibroblast, glial, and ependymal-like cells demonstrated characteristic morphological features which distinguished them from one another. In addition, immunocytochemical identification of cytoplasmic substances cross-reactive with neurophysin and (8-arginine)vasopressin further characterized specific magnocellular neuronal populations throughout the culture period. This culture system should provide a basis for studying neurosecretion at the cellular and molecular levels.

Animals↗
PubMed1983

Hypothalamic and pituitary sites of action of oxytocin to alter prolactin secretion in the rat.

To determine whether oxytocin (OT) could alter the release of PRL and other hormones from the anterior pituitary gland, the effects of OT were examined in two in vitro and two in vivo test systems. Cells dispersed from anterior pituitary glands of intact adult male rats were incubated in medium containing OT at doses of 10(-8), 10(-7), 10(-6), and 10(-5) M in two trials. OT stimulated PRL release 1.5-fold (P less than 0.01) and 2- to 3-fold (P less than 0.001) above control levels at 10(-8) and 10(-7) M doses, respectively, thus indicating a dose-dependent relationship. Higher doses did not produce a further elevation above that obtained with 10(-7) M OT. Arginine vasopressin (AVP) caused a slight decrease in PRL release from dispersed cells while TRH produced a small (25%), significant, but nondose-related increase in PRL release. Hemipituitary glands from adult male rats, incubated with 10(-6) and 10(-5) M OT, released twice as much PRL (P less than 0.01) into the medium as paired controls, but 10(-7) M OT was ineffective. The iv injection of 1 or 10 micrograms OT into conscious male rats elevated plasma PRL by 50% (P less than 0.05) or 500% (P less than 0.001), respectively, above basal values at 5 min only. Vehicle or 0.1 microgram OT were without effect. When 0.1 microgram OT was microinjected into the third ventricle (3V) of conscious male rats, it paradoxically reduced plasma PRL by 40% at 30 min (P less than 0.05), whereas 1 microgram OT significantly lowered PRL at 5-60 min, with the maximum suppression (60%, P less than 0.001) occurring at 30 min. These latter findings may indicate that an ultrashort loop feedback mechanism exists whereby exogenous OT decreases hypothalamic OT secretion, thereby reducing the OT stimulus for PRL release. The specificity of the OT effect on PRL was attested to by the failure of OT to alter significantly FSH, LH, and TSH in each system. GH was unchanged except that 3V-injected OT (1 microgram only) elevated (P less than 0.001) plasma GH at 15-30 min. These results support the view that OT acts directly on the cells of the anterior pituitary gland at low to high doses to release PRL specifically and in a dose-related fashion. In contrast, 3V injection of OT reduces PRL secretion, thereby suggesting that OT may decrease its own neurosecretion by ultrashort loop feedback and thus reduce an OT stimulus for PRL release.

Animals↗
PubMed1985

Effects of intraventricular growth hormone-releasing factor on growth hormone release: further evidence for ultrashort loop feedback.

We examined the effects of cerebroventricular injection of synthetic human GH-releasing factor [hGRF-(1-44)] on regulation of GH release in conscious male rats. These results were compared with the direct effects of hGRF on hormone released from dispersed anterior pituitary cells. Administration of two higher doses of hGRF (200 and 2000 ng) into the third ventricle (3V) produced a dose-related increase in plasma GH levels (P less than 0.001). Injection of hGRF into the 3V at two lower doses actually reduced GH release. Infusion of 20 ng (5 pmol) hGRF reduced plasma GH from 5-60 min (P less than 0.005), with a maximum suppression of 66%. The 2-ng (0.5-pmol) dose decreased GH secretion by 45% (P less than 0.05). hGRF stimulated a significant and dose-dependent release of GH from dispersed pituitary cells at concentrations of 10(-10) and 10(-9) M (P less than 0.025). The specificity of GRF for GH control, whether stimulatory or inhibitory, was seen by the failure of GRF to modify PRL, TSH, or LH release. Our results indicate that injection of larger doses of GRF into the 3V produce GH release, but at lower doses, 3V GRF may exert an action centrally to inhibit GH release. We propose that hypothalamic GRF may decrease its own neurosecretion by negative ultrashort loop feedback.

Animals↗
PubMed1985

In vivo gonadotropin-releasing hormone release and serum luteinizing hormone measurements in ovariectomized, estrogen-treated rhesus macaques.

The push-pull perfusion technique was used to measure GnRH release in unanesthetized female rhesus macaques (Macaca mulatta) and to examine the dynamic relationship between GnRH release and LH levels during the estrogen-induced LH surge. Each ovariectomized macaque was anesthetized and stereotaxically fitted with a push-pull cannula directed into the median eminence (ME). After at least 1 week of recovery, each animal received an estradiol benzoate (E2B) injection (42 micrograms/kg BW) or an oil (OIL) injection and underwent push-pull perfusion of the ME and blood sampling for at least 5 h between 28 and 56 h postinjection. Continuous 10-min push-pull perfusates were collected and prepared for GnRH RIA. Peripheral venous blood samples were obtained either hourly or every 10 min, and serum LH levels were determined by Leydig cell bioassay. GnRH release was detectable and pulsatile in areas in or adjacent to the ME or arcuate nucleus. In eight OIL monkeys, GnRH pulses were regular (approximately one pulse every 60 min) and of low amplitude (14.7 +/- 12.0 pg), with a mean GnRH release rate of 4.0 +/- 1.7 pg/10 min. In five E2B-treated monkeys, GnRH release during the rising phase of the LH surge occurred as an apparent burst of high amplitude GnRH pulses. The mean GnRH release rate (37.5 +/- 17.9 pg/10 min) and mean GnRH pulse amplitude (170.0 +/- 90.0 pg) during the 5 h before the peak LH level in E2B-treated monkeys were greater than OIL values (P less than 0.025, mean release; P less than 0.05, mean amplitude). Within individual E2B-treated monkeys, hourly mean GnRH release rates were significantly correlated with LH levels during the ascending limb of the LH surge (r = 0.75 +/- 0.11; P less than 0.025). We have concluded that an increase in GnRH neurosecretion occurs in E2B-treated monkeys and that it is associated with generation of the LH surge. On the basis of our observations, we hypothesize that the primate hypothalamus, through changes in GnRH secretion, actively participates in the E2B-induced LH surge.

Animals↗
PubMed1987

Steroid effects on the secretory modalities of gonadotropin-releasing hormone release.

GnRH neurons isolated immunochemically from the brain of adult male rats were used to determine whether testosterone (T), dihydrotestosterone (DHT), estradiol-17 beta (E(2)17 beta), 20H-estrone (OHE1), or progesterone (P4) have a direct effect on the spontaneous neurosecretion and/or cellular content of GnRH. Neurons harvested from individual rats were treated with a single steroid pulse; samples were collected before treatment, during the steroid pulse, and at 24 h post treatment. Androgen treatments of 100 pg/ml or 1 ng/ml media elicited an increase in GnRH pulse frequency 1-6 min after steroid exposure without affecting the amplitude of release; these modalities persisted at 2 h. The frequency of GnRH pulses was increased 24 h after the neurons received the brief 100 pg/ml or 1 ng/ml T or 1 ng/ml DHT treatments. Neurons exposed to androgen treatments also appeared to express large amplitude GnRH pulses infrequently at this time whereas there was no androgen affect on the cellular GnRH concentration. In contrast, E(2)17 beta, OHE1, and P4 treatments had no affect on the mean media GnRH concentration, baseline GnRH concentration, or on the frequency and amplitude of GnRH pulses at any time point. However, the 1 ng/ml P4 and the 1 ng/ml OHE1 treatments both reduced cellular GnRH content at 48 h post treatment. These results suggest that T and DHT may specifically interact with GnRH neurons to elicit immediate and/or long-term changes in the modalities of neuropeptide release and that under physiological conditions GnRH neurons of adult male rats are not directly influenced by E(2)17 beta, the catecholestrogen OHE1, or P4.

Animals↗
PubMed1989

Testosterone raises neuropeptide-Y concentration in selected hypothalamic sites and in vitro release from the medial basal hypothalamus of castrated male rats.

Although neuropeptide-Y (NPY)-containing neurons are widely distributed in the hypothalamus, castration decreased NPY concentrations only in the median eminence (ME), arcuate nucleus (ARC), and ventromedial nucleus (VMN). We have now examined the effects of testosterone (T) replacement in 2-week castrated male rats on NPY levels in hypothalamic and preoptic area regions and in vitro NPY release in three experiments. In the first experiment we studied the effect of T on NPY concentration in castrated rats. Two-week castrated rats were implanted sc with T-filled or empty Silastic capsules 30 mm in length. Ten days later rats were killed, and NPY levels were measured by RIA in microdissected sites. T implants raised serum T levels to the range found in gonad-intact rats and decreased serum LH levels to the basal range. Further, of the six brain sites examined, significant increases in NPY concentrations occurred selectively in the ME, ARC, and VMN of T-implanted rats. In the second experiment, the ability of T to reverse the effect of castration on NPY levels compared to those in intact (sham) rats was assessed. Again, castration decreased NPY levels in the ME, ARC, and VMN only, and replacement of physiological levels of T restored NPY levels approximately 100%, 127%, and 74% in the ARC, VMN, and ME, respectively. In the third experiment, the effect of castration and T implants (30-mm T capsules for 10 days) to 2-week castrated rats on the in vitro release of NPY from medial basal hypothalamus (MBH) was assessed. Basal NPY release was not significantly changed after castration and T replacement. However, in response to a 30-min pulse of KCl (45 mM) NPY release from the MBH of castrated rats was significantly reduced compared to that in intact and T-replaced castrated rats. These studies show that castration decreases and T replacement restores NPY levels selectively in three hypothalamic sites, viz. ME, ARC, and VMN, and KCl-induced NPY release from the MBH in vitro is decreased after castration and restored by T replacement, thereby suggesting that a local subset of androgen-concentrating neurons may regulate NPY levels and release in a site-specific manner. Further, these results are in line with our emerging view that gonadal steroids modulate neurosecretion not only of LHRH, but also of other functionally linked regulatory peptides.

Animals↗
PubMed1989

Blockade of growth hormone-releasing factor (GRF) activity in the pituitary and hypothalamus of the conscious rat with a peptidic GRF antagonist.

Microinjection of synthetic GRF into the cerebroventricles or hypothalamus of the rat produces a number of neural effects, including the suppression of GH secretion, possibly representing a negative ultrashort loop autoregulation of GRF and/or stimulation of somatostatin neurosecretion. To demonstrate that such neuromodulation acts physiologically through endogenous GRF activity, the peptidic GRF antagonist (N-Ac-Tyr1,D-Arg2)GRF-(1-29)-NH2 was used to block the action of GRF on its presumed receptors in the hypothalamus. First, to establish the efficacy of the antagonist to block GRF receptors in the anterior pituitary, we injected the antagonist iv at doses of 2, 20, and 50 micrograms or saline (controls) into conscious male rats fitted with jugular cannulae. Sequential blood sampling every 15 min for 6 h between 1000-1600 h showed that 50 micrograms antagonist, iv, significantly suppressed the two periods of spontaneous release of radioimmunoassayable GH in controls in the morning and afternoon. A dose of 20 micrograms, iv, lowered mean plasma GH between 1400-1500 h (P less than 0.025), while the 2-microgram dose was without effect. The GRF antagonist was then microinjected into the third ventricle (3V) of conscious male rats at doses of 0.5 and 8.0 ng in 2 microliter sterile saline. The 8.0-ng dose of 3V antagonist elicited a 3-fold increase in the morning peak of GH (nanograms per ml): 3V antagonist, 159.0 +/- 62.0; 3V control, 51.0 +/- 21.9 (P less than 0.05). The 0.5-ng dose was without effect. Finally, we observed that pretreatment with the GRF antagonist 3V (10 ng), followed 15 min later by 10 ng rat GRF administered 3V, completely blocked the GRF-induced suppression of pulsatile GH release observed earlier. Both the systemic and central effects of the antagonist were specific to the control of GH, since PRL concentrations were unaltered. These results 1) have demonstrated the ability of a peptidic GRF antagonist to specifically suppress pulsatile GH release after its systemic administration, presumably by acting on pituitary GRF receptors, and 2) support the notion that GRF receptors are also present in the hypothalamus and are available for the physiological mediation of GRF-induced inhibition of GH release by a central mechanism.

Animals↗
PubMed1990

Neuropeptide-Y concentration in microdissected hypothalamic regions and in vitro release from the medial basal hypothalamus-preoptic area of streptozotocin-diabetic rats with and without insulin substitution therapy.

Experimental diabetes adversely affects hypothalamic control of gonadotropin secretion and sex behavior and induces hyperphagia accompanied by severe body weight loss. Neuropeptide-Y (NPY) stimulates pituitary gonadotropin release, inhibits sexual behavior, and stimulates robust feeding in rats by acting at different sites in the hypothalamus. Therefore, we tested the hypothesis that altered hypothalamic NPY neurosecretion may mediate the constellation of effects observed in streptozotocin-induced diabetic (STZ-D) rats. Adult male rats were made diabetic by a single injection of STZ (50 mg/kg). Five months later, in vitro NPY release from the hypothalamic fragment encompassing the medial basal hypothalamus and preoptic area and NPY concentrations in seven hypothalamic sites were assessed. Basal NPY release was not significantly changed after STZ treatment. However, in response to a 30-min pulse of KCl (45 mM), NPY release from the medial basal hypothalamus-preoptic area of STZ-D rats was significantly increased compared to that in age-matched controls. In the STZ-D rats, NPY concentrations in six of the seven microdissected nuclei, including those mediating control of pituitary gonadotropin, sexual, and feeding behaviors, were increased compared to control values. In an additional study similar increments in NPY concentrations in the hypothalamic sites were observed 6 months after STZ treatment. The effects of insulin on NPY levels in microdissected hypothalamic sites in STZ-treated and BB diabetic rats was next assessed. One group of rats was treated with STZ, and the other group of rats was additionally treated with insulin (6 U/kg.day) for 3 months after development of diabetes with STZ. Again, STZ treatment alone, even for 3 months, increased NPY levels in all seven nuclei, including the suprachiasmatic nuclei. Insulin therapy completely prevented the STZ-induced increments in NPY levels in all hypothalamic sites, and the blood glucose level was 233 +/- 22 mg/dl in insulin-treated STZ-D rats and 496 +/- 6 mg/dl in untreated STZ-D rats. Similarly, NPY concentrations in five of the seven nuclei were unchanged in spontaneously diabetic BB rats (blood glucose, 435 +/- 67 mg/dl) maintained on insulin (5-8 U/kg.day). These results demonstrate that STZ-D rats have a widespread increase in NPY levels in hypothalamic sites, and there is an increase in the evoked release of NPY from the hypothalamus.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Localization of neuropeptide-Y immunoreactivity in estradiol-concentrating cells in the hypothalamus.

Considerable evidence shows that gonadal steroids exert a facilitatory influence on levels and release of neuropeptide-Y (NPY) from the hypothalamus. However, it is not known whether gonadal steroids act directly on NPY-producing cells in the arcuate nucleus (ARC) of the hypothalamus to produce these facilitatory effects on NPY or whether they act on other cells that have a modulatory influence via synapses on ARC NPY cells. We applied the combined method of steroid autoradiography and immunocytochemistry to assess the localization of [3H]estradiol in relation to NPY-producing cells in the hypothalamus. Rats (n = 6) were bilaterally ovariectomized and injected intracerebroventricularly with colchicine. Twenty-four hours later each rat received an iv injection of 17 beta-[2,4,6,7,16,17(-3)H]estradiol (SA, 166 Ci/mmol) at a dose of 5.0 micrograms/kg BW. One hour after the injection of [3H]estradiol, the rats were perfused with 4% paraformaldehyde; brains were removed, frozen in isopentane precooled in liquid nitrogen (-190 C), sectioned, and processed for autoradiography. The autoradiograms were then incubated with specific antibodies for NPY immunostaining by the avidin-biotin-peroxidase method. The results revealed NPY-immunopositive cells in the ARC, striatum, hippocampus, amygdala, and cerebral cortex and a few cells in the median eminence. NPY-immunoreactive fibers were also detected in the internal layer of the median eminence. The largest number of neurons showing NPY immunoreactivity in the cytoplasm was detected in the ARC, and only in this nucleus did we observed colocalization of [3H]estradiol and NPY immunoreactivity in neurons. A population of NPY-immunopositive cells in the ARC (10-20%) exhibited nuclear [3H]estradiol; the majority of these cells were located in the lateral and ventral portions of the ARC. Since gonadal steroids stimulate hypothalamic NPY levels and release, this anatomical evidence of colocalization is suggestive of a direct genomic modulation of NPY neurosecretion by steroids in a subpopulation of hypothalamic NPY-immunopositive neurons.

Animals↗
PubMed1993

RU486 administration blocks neuropeptide Y potentiation of luteinizing hormone (LH)-releasing hormone-induced LH surges in proestrous rats.

We previously demonstrated that NPY potentiates LHRH-induced LH secretion specifically under endocrine conditions in which preovulatory LH surges are generated. The present study was designed to test the hypothesis that NPY's facilitatory actions are dependent upon preovulatory progesterone secretion. In Exp 1, female rats were fitted with atrial catheters on diestrus. On proestrus, hourly blood samples were collected from 1100-2100 h. At 1230 h, rats received a sc injection of the progesterone receptor antagonist RU486 (6 mg/kg BW) or oil. At 1330 h, rats received pentobarbital (40 mg/kg BW), to block hypothalamic LHRH release, or saline. Every 30 min from 1400-1800 h, pentobarbital-treated rats received iv pulses of LHRH (15 ng/pulse) or saline along with concurrent pulses of NPY (5 micrograms/pulse), or saline. In Exp 2, rats received jugular catheters on diestrus, but were sampled every hour throughout the morning (0700-1600 h), rather than the afternoon, of proestrus. In these morning groups, pentobarbital was injected at 0830 h, and peptides (LHRH or combined LHRH and NPY solutions) were administered as pulses at 30-min intervals between 0900-1300 h. Results from Exp 1 were as follows: administration of RU486 to rats given an ip injection of vehicle at 1330 h and pulses of saline from 1400-1800 h completely blocked the endogenous LH surge. In oil-treated pentobarbital-blocked rats, concurrent administration of NPY with LHRH significantly (P < 0.01) potentiated the ability of LHRH to restore LH surges. However, NPY was without any potentiating effects in animals pretreated with RU486 at 1230 h. RU486 also attenuated the ability of LHRH alone to restore LH surges in pentobarbital-blocked rats. In Exp 2, NPY was without effect on LHRH-induced LH secretion during the morning hours of proestrus. Our results demonstrate that 1) NPY facilitates LHRH-induced LH surges on the afternoon of proestrus; 2) presumptive progesterone receptor blockade by RU486 completely prevents NPY's potentiating effects; and 3) NPY is without effect on the morning of proestrus, before the afternoon surge of progesterone. These findings are entirely consistent with the idea that one function of preovulatory progesterone secretion is to up-regulate pituitary sensitivity to the facilitatory actions of NPY. It is hypothesized that these actions of progesterone together with an increase in NPY neurosecretion mediate the acute increase in pituitary sensitivity to LHRH that occurs just before the LH surge.

Animals↗
PubMed1995

Does estradiol induce the preovulatory gonadotropin-releasing hormone (GnRH) surge in the ewe by inducing a progressive change in the mode of operation of the GnRH neurosecretory system.

Estradiol profoundly influences GnRH secretion during the follicular phase of the estrous cycle of the sheep. Estradiol not only regulates the frequency and amplitude of GnRH pulses, but also produces qualitative changes in its pattern of release and induces a sustained GnRH surge during which discrete pulses are not readily evident. In this study, we tested the hypothesis that qualitative changes in GnRH secretion are an integral part of an estradiol-induced change in the mode of operation of the GnRH neurosecretory system that leads to generation of the GnRH surge. This was achieved by the measurement of GnRH in samples of pituitary portal blood collected at 1-min intervals for an 11-h period encompassing the pre- and early surge periods in an artificial follicular phase model. In each of the seven ewes studied, a highly characteristic alteration in the moment to moment pattern of GnRH was observed. This consisted of a progressive change from a strictly episodic pattern of GnRH release to one containing both episodic and nonepisodic components and, after amplification of both components, a period of extremely high values during which individual episodic increases were no longer readily recognizable. Preliminary mathematical modeling of the data suggested that these patterns could be produced by a change in GnRH from a predominantly low to a mixture of low and high amplitude inputs. Similar changes in minute to minute patterns of GnRH secretion were observed during the natural follicular phase. These findings are consistent with the hypothesis that estradiol induces the GnRH surge by altering the mode of neurosecretion, rather than by merely causing quantitative changes in the episodic pattern of release.

Animals↗
PubMed1995

Endotoxin-induced suppression of the somatotropic axis is mediated by interleukin-1 beta and corticotropin-releasing factor in the juvenile rat.

This study extends the neuroendocrine role of central interleukin-1 beta (IL-1 beta) during the stress of lipopolysaccharide (LPS) challenge to include inhibition of the somatotropic [GH-releasing hormone (GHRH)-somatostatin (SRIF)-GH] axis in juvenile male rats and clarifies the role of CRF in the mediation of LPS/IL-1-induced changes in GHRH and SRIF neurosecretion. The results of the in vivo component of this study demonstrated that LPS treatment (2.5 mg/kg twice daily for 5 days) caused a significant attenuation of body weight gain for 2 days (2.4 +/- 1.7% vs. 10.3 +/- 1.8% BW/day in saline controls; P < 0.05) and failure of catch-up growth thereafter even though a small transient suppression of food intake returned to normal by the second of 4 days of treatment. Associated with the first day of growth attenuation was an acute suppression of all plasma GH parameters, including GH mass (area under the curve, 1.972 +/- 0.1837 vs. 6.402 +/- 1.7 micrograms/ml.6 h for saline controls; P < 0.05), in animals receiving an acute bolus of LPS, which was blocked by prior microinjection of IL receptor antagonist protein (IRAP) into the third ventricle. In contrast, GH parameters associated with the second day of LPS-suppressed body weight gain were increased (GH mass, 9.4 +/- 2.2 vs. 3.5 +/- 0.5 micrograms/ml.4 h in saline controls; P < 0.05). These increases were reversed after another 2 days of LPS treatment. In a series of in vitro experiments using medial basal hypothalamic (MBH) explants incubated with LPS [100 ng/ml alone or with 10(-7) M IRAP or 10(-6) M CRF antagonist (CRF-ANT)], GHRH release from MBH incubated with LPS was significantly greater than that in controls (231 +/- 79% vs. 71 +/- 34% of baseline release; P < 0.05), and this stimulation was antagonized by both IRAP and CRF-ANT. SRIF release was significantly increased by incubation with LPS (163 +/- 28% vs. 97 +/- 20% of the baseline for controls; P < 0.05) and blocked (to 88 +/- 14% of the baseline) by IRAP, but not by CRF-ANT. Finally, when MBH explants were incubated with IL-1 beta (10(-9) M), there was a significant inhibition of in vitro GHRH release (37.9 +/- 6.7% vs. 74.9 +/- 16.6% for controls), which was reversed by IRAP and CRF-ANT, and a significant stimulation of SRIF release (168.7 +/- 37.5% vs. 98.0 +/- 11.6% for controls), which was reversed by IRAP, but not CRF-ANT.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1996

Gonadotropin-releasing hormone regulation of pituitary follistatin gene expression during the primary follicle-stimulating hormone surge.

Follistatin is produced in the gonadotrope and folliculostellate cells of the pituitary gland and is thought to indirectly regulate FSH biosynthesis and secretion through its ability to bind activin. Recent measurements of follistatin gene expression during the rat estrous cycle revealed a marked increase in pituitary follistatin messenger RNA (mRNA) levels at the time of the preovulatory FSH surge. This finding suggests a role for follistatin in the regulation of FSH at this dynamic time of the cycle. The aim of the present study was to identify the hormonal control mechanisms responsible for stimulating follistatin gene expression on proestrus. In particular, the roles of estrogen (E) and GnRH were assessed using an in vivo ovariectomized (OVX) animal model in which steroid priming results in daily gonadotropin surges. Follistatin mRNA and serum FSH levels were unchanged throughout the day in untreated OVX rats. E priming of OVX rats elicited a 2-fold elevation in follistatin mRNA levels between 1600-2000 h coincident with the peak of the E-induced FSH surge. To determine whether this effect of E on follistatin mRNA levels was the result of the direct or indirect effects of E on the pituitary, follistatin mRNA levels were examined in E-primed OVX rats that had been treated with pentobarbital at 1430 h (to block hypothalamic neurosecretion). Pentobarbital treatment prevented the E-induced increase in follistatin mRNA levels, suggesting that the effects of E are mediated via GnRH or other hypothalamic factors. The effects of GnRH on follistatin gene expression were examined further using an in vitro perifusion model. Proestrous or metestrous pituitaries were perifused for 8 h with pulsatile GnRH (one pulse per h), continuous GnRH, or medium only. Continuous GnRH treatment resulted in a significant elevation in follistatin mRNA levels in both proestrous and metestrous pituitaries, whereas pulsatile GnRH had no effect at either cycle stage. These results suggest that the proestrous GnRH surge is responsible at least in part for the elevation in pituitary follistatin mRNA levels that is associated with the primary FSH surge. GnRH-induced follistatin production on proestrus probably plays a role in the dynamic regulation of FSH at this time of the ovulatory cycle.

Animals↗
PubMed1998

Absence of increased neuropeptide Y neuronal activity before and during the luteinizing hormone (LH) surge may underlie the attenuated preovulatory LH surge in middle-aged rats.

A large body of evidence suggests that the neuroendocrine axis plays a major role in the reproductive aging of female rats. Since increased hypothalamic neuropeptide Y (NPY) neurosecretion is crucial in the preovulatory LH discharge in young rats, we tested the hypothesis that diminution in the preovulatory LH surge in middle-aged (MA) rats may be due to altered neurosecretory activity in NPYergic neurons. In Exp 1, we examined NPY levels in six microdissected hypothalamic nuclei, including median eminence (ME), arcuate nucleus (ARC), and medial preoptic area (MPOA), at 1000, 1200, 1400, 1600, 1800, 2000, or 2200 h on the day of proestrus in young (2.5- to 3-month old) and MA (7- to 9-month old) regularly cycling rats. At 1000 h, ME NPY levels in young rats were significantly lower than those in MA rats. In young rats, the ME NPY levels were significantly increased at 1400 h before the LH surge in the afternoon and thereafter decreased progressively during the interval of the LH surge. In MA rats, however, ME NPY levels decreased in the afternoon in association with an attenuated LH surge. In addition, in the ARC and MPOA, the other hypothalamic sites associated with induction of LH surge, NPY levels increased before and during the LH surge in young rats, no change in NPY levels in these nuclei was observed in association with the attenuated LH surge in MA rats. Also, NPY levels in the ARC and MPOA during the afternoon were significantly lower in MA compared with those in young animals. These results demonstrated the absence of an antecedent increase in NPY levels, specifically in the ME and ARC, during the afternoon of proestrus in MA animals. In a second experiment, we evaluated whether the absence of dynamic changes in NPY levels in the ME and ARC in MA rats was due to altered hypothalamic NPY gene expression. Regularly cycling young (2.5- to 3-month-old) and MA (8- to 10-month-old) rats were killed at 1000, 1200, 1400, 1600, 1800, 2000, or 2200 h on the day of proestrus. The medial basal hypothalamus was processed for prepro-NPY messenger RNA (mRNA) measurement by ribonuclease protection assay. In young rats, prepro-NPY mRNA levels were significantly increased at 1200 h and remained elevated throughout the afternoon. In contrast, in MA rats prepro-NPY mRNA levels remained unchanged before and during the attenuated LH surge. These results clearly indicate that the augmentation in NPY neuronal activity before and during the LH surge seen in young rats fails to manifest itself in middle-aged rats. As hypothalamic NPY participates in the induction of LHRH surge, our results suggest that reduced LHRH and LH surges in MA rats may be due to diminution in NPY secretion in these animals.

Aging↗
PubMed1999

Pulsatile release of luteinizing hormone-releasing hormone (LHRH) in cultured LHRH neurons derived from the embryonic olfactory placode of the rhesus monkey.

To study the mechanism of LH-releasing hormone (LHRH) pulse generation, the olfactory pit/placode and the migratory pathway of LHRH neurons from monkey embryos at embryonic age 35-37 were dissected out, under the microscope, and cultured on plastic coverslips coated with collagen in a defined medium for 2-5 weeks. First, we examined whether cultured neurons release the decapeptide into media. It was found that LHRH cells release LHRH in a pulsatile manner at approximately 50-min intervals. Further, LHRH release was stimulated by depolarization with high K+ and the Na+ channel opener, veratridine. However, whereas the Na+ channel blocker, tetrodotoxin suppressed the effects of veratridine, tetrodotoxin did not alter the effects of high K+. Subsequently, the role of extracellular and intracellular Ca2+ in LHRH release was examined. The results are summarized as follows: 1) exposing the cells to a low Ca2+ (20 nM) buffer solution suppressed LHRH release, whereas exposure to a normal Ca2+ solution (1.25 mM) maintained pulsatile LHRH release; 2) LHRH release from cultured LHRH cells was stimulated by the voltage-sensitive L-type Ca2+ channel agonist, Bay K 8644 (10 microM), whereas it was suppressed by the L-type Ca2+ channel blocker, nifedipine (1 microM), but not by the N-type channel blocker, omega-conotoxin GVIA (1 microM); 3) the intracellular Ca2+ stimulant, ryanodine (1 microM), stimulated LHRH release, whereas the intracellular Ca2+ transporting adenosine triphosphatase antagonist, thapsigargin (1 and 10 microM), did not yield consistent results; and 4) carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (1 microM), a mitochondrial Ca2+ mobilizer, stimulated LHRH release, whereas ruthenium red, a mitochondrial Ca2+ uptake inhibitor, did not induce consistent results. These results indicate that: 1) the presence of extracellular Ca2+ is essential for LHRH neurosecretion; 2) Ca2+ enters the cell via L-type channels but not N-type channels; and 3) mobilization of intracellular Ca2+ from inositol 1,4,5-triphosphate-sensitive stores, as well as mitochondrial stores, seem to contribute to LHRH release in these cells.

Animals↗
PubMed2005

Autosomal dominant neurohypophyseal diabetes insipidus with linkage to chromosome 20p13 but without mutations in the AVP-NPII gene.

CONTEXT: Autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) has been known as a rare disorder transmitted as an autosomal dominant trait, characterized by polyuria and polydipsia, and caused by deficient neurosecretion of arginine vasopressin precursor (AVP-NPII). We reported an ADNDI family with linkage to chromosome 20p13 but without mutations in the AVP-NPII gene. OBJECTIVE: The objective of this study was to identify the corresponding locus responsible for ADNDI in a family without AVP-NP II gene mutations. SUBJECTS AND METHODS: Two families with ADNDI were diagnosed by water deprivation test. The AVP-NPII gene was amplified by PCR and sequenced. A genomewide scan was performed in one family using 400 microsatellite markers covering 22 autosomes. RESULTS: A 3-bp deletion (1827-1829delAGG) of AVP-NPII gene was identified in the affected individuals in one family. Although no mutations could be detected in the coding, the promoter, and intronic regions of AVP-NPII gene in the other family, a maximum LOD score of 1.202999 (theta = 0.00) was obtained at marker D20S889 by genomewide scan, and a 7-cM interval on chromosome 20p13 was defined by fine mapping with markers D20S199-D20S849. Furthermore, the intragenic region that regulates AVP-NPII and oxytocin expression as an enhancer element and the UBCE7IP5 gene that participates in prohormone degradation were sequenced. No alterations could be detected either. CONCLUSION: The corresponding locus responsible for ADNDI is possibly heterogeneous regarding the slightly different clinical features in these two families.

Adolescent↗
PubMed1999

Clinical and molecular evidence of abnormal processing and trafficking of the vasopressin preprohormone in a large kindred with familial neurohypophyseal diabetes insipidus due to a signal peptide mutation.

The autosomal dominant form of familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disease characterized by postnatal onset of polyuria and a deficient neurosecretion of the antidiuretic hormone, arginine vasopressin (AVP). Since 1991, adFNDI has been linked to 31 different mutations of the gene that codes for the vasopressin-neurophysin II (AVP-NPII) precursor. The aims of the present study were to relate the clinical phenotype to the specific genotype and to the molecular genetic effects of the most frequently reported adFNDI mutation located at the cleavage site of the signal peptide of AVP-NPII [Ala(-1)Thr]. Genetic analysis and clinical studies of AVP secretion, urinary AVP, and urine output were performed in 16 affected and 16 unaffected family members and 11 spouses of a Danish adFNDI kindred carrying the Ala(-1)Thr mutation. Mutant complementary DNA carrying the same mutation was expressed in a neurogenic cell line (Neuro2A), and the cellular effects were studied by Western blotting, immunocytochemistry, and AVP measurements. The clinical studies showed a severe progressive deficiency of plasma and urinary AVP that manifested during childhood. The expression studies demonstrated that the Ala(- 1)Thr mutant cells produced 8-fold less AVP than wild-type cells and accumulated excessive amounts of 23-kDa NPII protein corresponding to uncleaved prepro-AVP-NPII. Furthermore, a substantial portion of the intracellular AVP-NPII precursor appeared to be colocalized with an endoplasmic reticulum antigen (Grp78). These results provide independent confirmation that this Ala(-1)Thr mutation produces adFNDI by directing the production of a mutant preprohormone that accumulates in the endoplasmic reticulum, because it cannot be cleaved from the signal peptide and transported to neurosecretory vesicles for further processing and secretion.

Adolescent↗
PubMed1997

Neuronal loss and plasticity in the supraoptic nucleus in Parkinson's disease.

Disturbed circadian control of renal water excretion and blood pressure adaptation in Parkinson's disease (PD) suggest impaired hypothalamic magnocellular neurosecretion. To test the hypothesis that this may relate to specific hypothalamic pathology in PD, we studied morphometrically the neuronal population of the supraoptic nucleus (SON) in PD patients and controls. Neuronal loss in the SON of PD patients was associated with increased somatic, nuclear, and nucleolar size of remaining neurons, suggesting compensatory response of these cells. We conclude that SON pathology is a feature of PD and may account for specific signs of neurohumoral dysfunction.

Aged↗