Stimulated Luteinizing Hormone Release Research Articles

Hypothalamic Neurons Preferentially Respond to Female Nest Coo Stimulation: Demonstration of Direct Acoustic Stimulation of Luteinizing Hormone Release

Avian vocalizations are generally understood to play a pivotal role in reproductive functions. The role of the hypothalamus in gonadotropin release in higher vertebrates including birds is well established. To date, however, a direct linkage between the neuronal processing of vocal input and the contingent luteinizing hormone (LH) response has not been demonstrated. In this study, using female ring doves, we recorded neuronal activity from hypothalamic nuclei that, as we have shown previously, receive acoustic inputs from the auditory thalamic relay. Concurrently with recording single-unit responses to stimulation with species-specific coo vocalizations, we sampled LH levels in blood from the pituitary veins. LH concentration in the plasma was significantly elevated in birds hearing species-typical coos but not in birds exposed to experimentally altered coos or white noise or in birds that received no vocal stimulation. We found two types of neurons in the preoptic and anterior hypothalamus that selectively responded to the female nest coo: excitatory units and inhibitory units. Among the excitatory neurons are units characterized by two bursts separated by a period of slow spiking or complete silence, in a pattern approximately corresponding temporally to the two-note coo. We designate them as female-nest-coo-specific units. Most neurons in the posterior hypothalamus were nonselective in their response. Female nest coo and male nest coo stimulation evoked an equal magnitude of discharge changes from responsive units in the preoptic-anterior hypothalamic area. We found, however, that the LH increment was three times greater for birds hearing female nest coos than for birds hearing male nest coos. These observations suggest that feature-detecting neurons such as the female-nest-coo-specific units are involved in gonadotropin-releasing hormone output. The present findings are consistent with the well established behavioral evidence that female nest coos mediate ovarian growth.

Fasting suppresses pulsatile luteinizing hormone (LH) secretion and enhances orderliness of LH release in young but not older men.

Pulsatile gonadotropin secretion and sex-steroid concentrations are suppressed reversibly in young fasted or malnourished human subjects. In this study, we investigated the impact of age on the dynamic neuroendocrine mechanisms underlying this stress response in healthy young (age, 28 +/- 3 yr, n = 8) vs. older (age 67 +/- 2 yr, n = 8) men with similar body mass indices (mean, 26 +/- 0.6 vs. 26 +/- 1.3 kg/m2, respectively). Serum LH concentrations were measured by immunoradiometric assay (IRMA) in blood collected at 10-min intervals over 27 h on a control (fed) day and on the third day of a 3.5-day fast (water only) assigned in randomized order. After 24 h of basal sampling, GnRH (10 micrograms i.v. bolus) was administered to test gonadotrope responsiveness. Cortisol, dehydroepiandrosterone sulfate, androstenedione, testosterone, FSH, GH, and PRL were measured in 24-h pooled serum as positive and negative control hormones. Approximate entropy was used to quantitate the orderliness of LH release over 24 h, and a multiple-parameter deconvolution method was applied to quantify pulsatile LH secretion and LH half-life. In the fed state, older men exhibited elevated mean (24-h pooled) serum FSH and cortisol concentrations compared with young controls but equivalent serum LH concentrations and reduced serum GH, free testosterone, androstenedione, and dehydroepiandrosterone sulfate concentrations. Fed older men also manifested a lower frequency and amplitude of 24-h pulsatile LH secretion, and, by approximate entropy calculations, a more disorderly pattern of basal LH release than younger individuals. Three- and one-half days of fasting evoked 40% and 47% increases in mean (24-h) serum cortisol concentrations in young and older men, respectively (P < 0.01 vs. fed, but P = not significant for percentage rise in older vs. young men). Concurrently, fasting induced a 2.1-fold fall in the 24-h endogenous LH production rate in young men (fed 36 +/- 9.7 vs. fasted 17 +/- 2.0 IU/L of distribution volume/day, P < 0.01), but did not significantly affect the daily LH secretion rate in older men (fed 27 +/- 4.5 vs. fasted 21 +/- 3.4 IU/day). The reduced LH production rate in fasting young men was accounted for by a 1.7-fold decline in the mass of LH secreted per burst (fed 2.5 +/- 0.45 vs. fasted 1.5 +/- 0.16 IU/L, P < 0.05), whereas LH burst mass in older men remained unchanged (and low) during fasting. In addition, in young men, during the 3.5-day fast the number of computer-resolved LH secretory bursts per 24 h decreased (fed 15 +/- 0.7 vs. fasted 11 +/- 0.7, P < 0.01), and the interburst interval increased (fed 94 +/- 4.2 vs. fasted 125 +/- 8.7 min, P < 0.05). In contrast, in older men in the fed state, basal LH peak frequency and serum free testosterone concentrations were reduced compared with corresponding values in young men, and did not decline further with fasting. Whereas the orderliness of LH release patterns increased significantly during fasting in the young men, the approximate entropy measure failed to change significantly in unfed older subjects. By cosinor analysis, young men showed lower 24-h mesor (mean of nyctohemeral rhythm of) serum LH concentrations than older volunteers during fasting. Moreover, young but not older men manifested preserved 24-h variations in LH interpulse intervals when fasting. Exogenously stimulated LH release (mean 3-h serum LH concentration or calculated mass of LH secreted) following a single i.v. injection of 10 micrograms GnRH was independent of age and fasting status. We conclude that the metabolic stressor of short-term fasting unmasks specific age-related neuroendocrine contrasts in the stress-responsive control of both the pulsatile and nyctohemeral regulation of the male hypothalamo-pituitary-gonadal-axis.

Gonadotrophin-releasing activity of histones H2A and H2B.

We report that histones H2A and H2B possess gonadotrophin-releasing activity in vitro and assess the signal transduction pathways involved in these effects. Perifused and incubated rat anterior pituitary (AP) cells were used, and luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured by RIA. Perifusion of cells with histone H2A (30 muM) or histone H2B (30 muM), markedly stimulated LH release but failed to elicit any FSH response. Cells incubated with 6 or 30 muM histone H2A showed a dose- and time-dependent stimulatory effect on bot LH and FSH release which was blocked by 1 muM peptide MB35, an 86-120 amino acid fragment of histone H2A. Incubation of pituitary cells with gonadotrophin-releasing hormone (GnRH) and histones H2A or H2B showed a stimulatory effect on LH and FSH release which was similar to the sum of the separate effects. Trifluoperazine as well as ethylene glycol bis(b-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), alone or in the presence of the calcium ionophore A23187, significantly reduced the response of AP cells to histones. Various cyclic adenosine monophosphate (cAMP) enhancers had no effect on histone-stimulated release of gonadotrophins in incubated AP cells. Our results confirm previous evidence that histones may act as hypophysiotrophic signals. Calcium- and diacylglycerol-associated pathways, but not cAMP, appear to participate in these effects.

Regulation of Gonadotropin-Releasing Hormone and Luteinizing Hormone Secretion by AMPA Receptors

Recent work has demonstrated that glutamate functions as a major transmitter involved in the regulation of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion in female animals, although the specific receptors and mechanisms mediating its effects have not been completely worked out. The purpose of the present study, therefore, was to examine the role of the AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)-type glutamate receptor in the control of GnRH and LH secretion in female animals. Toward this end, the dose- and steroid-dependent effects of AMPA on GnRH and LH secretion in female rats were investigated using both in vitro and in vivo approaches, and the role of AMPA receptors in the production of the steroid-induced LH surge was also assessed. The results of the study revealed that central administration of AMPA resulted in a stimulation of LH release in the estrogen-primed ovariectomized adult rat. AMPA was also found to potently stimulate GnRH release in vitro from mediobasal hypothalamic (MBH) fragments obtained from estrogen-primed ovariectomized adult rats, and this effect was blocked by the selective AMPA receptor antagonist, NBQX. The mechanism of action of AMPA appeared to differ from that of N-methyl-D-aspartate (NMDA) as AMPA, in contrast to NMDA, failed to elevate nitric oxide synthase activity in the hypothalamus. The effect of AMPA on LH secretion was demonstrated to be steroid dependent, as central administration of AMPA stimulated LH release in estrogen-primed ovariectomized rats, but was inhibitory to LH release in non-estrogen-primed ovariectomized rats. In contrast, AMPA stimulated GnRH release equally well from MBH fragments obtained from estrogen-primed or non-estrogen-primed ovariectomized rats. The different effects of AMPA on LH release may be due to different pituitary sensitivities between the two models, or alternatively, AMPA may stimulate the release of LH inhibitory factors in the ovariectomized rat in the absence of estrogen. Finally, a physiological role for AMPA receptors in the production of the steroid-induced LH surge was suggested, based on the finding that central administration of the selective AMPA receptor antagonist, NBQX, into the third cerebroventricle significantly attenuated the steroid-induced LH surge in the ovariectomized adult female rat.

Involvement of protein phosphatases in gonadotropin releasing hormone regulated gonadotropin secretion

The role of persistent protein phosphorylation upon gonadotropin releasing hormone (GnRH) stimulated luteinizing hormone (LH) release was investigated by the use of the selective inhibitors of protein phosphatase type 1 (PP1) and 2A (PP2A), okadaic acid (OA) and calyculin A. Pre-incubation of cultured rat pituitary cells with OA (24 h) or calyculin A (30 min) resulted in inhibition of GnRH-stimulated LH release with significant inhibition being detected at 10 nM and 30 nM for OA and calyculin A, respectively. The inactive OA analog norokadone and the protein tyrosine phosphatase inhibitor vanadyl hydroperoxide had no significant effect on GnRH-induced LH release. The stimulatory effects of the protein kinase C (PKC) activator 12- O-tetradecanoylphorbol 13-acetate (TPA, 50 ng/ml) or the Ca 2+ ionophore, ionomycin (1 μm), upon LH release were also abolished by pretreatment with OA (10–20 nM) or calyculin A (30nM). Stimulation of LH release by high K + (28 mM) or residual LH release stimulated by GnRH in Ca 2+-free medium were also blocked by OA. These observations indicate that protein dephosphorylation is involved positively in GnRH-stimulated LH release. The site of action of the protein phosphatases PPI and PP2A is most likely downstream to Ca 2+ elevation and PKC activation by GnRH.

Failure of ethanol metabolites to alter gonadotropin secretion or luteinizing hormone synthesis in vitro.

The impact of ethanol on the male reproductive axis are multiple and varied, with both gonadal and control hypothalamic-pituitary pertubations being reported. There appears to be a discrepancy, however, between the in vivo and in vitro effects of ethanol on hypothalamic luteinizing hormones releasing hormone (LHRH) and the pituitary gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH). While in vivo data suggests a decrease in LHRH release after EtOH, in vitro studies find no effect on secretion. Similarly, in vivo acute EtOH profoundly diminishes LH synthesis and secretion, while in vitro impaired release with no alteration in the transcription of beta LH has been found. A potential exploration for these discrept results could be the in vivo metabolism of EtOH into acetaldehyde and acetate, or the subsequent formation of salsolinol, a product of acetate combining with dopamine. To test this possibility, a series of in vitro experiments were conducted exposing dispensed anterior pituitary cells from male rats to different doses of acetaldehyde, acetate or salsolinol for varying amounts of time for which gonadotropin secretion and beta LH mRNA levels were assessed. The results demonstrated no effect of either acetaldehyde or acetate on basal or LHRH stimulated LH release, FSH release or steady-state beta LH mRNA levels. These data suggest that the metabolites of EtOH, which occur in vivo but not in vitro, are not responsible for the discrepant gonadotropin changes reported between the in vivo and in vitro setting. Other potential mechanisms to explain this phenomenon include differences in the molarity of EtOH, hyperprolactinemia and suprapituitary influences including hypothalamic LHRH, catecholamines, excitatory amino acids, substance P and beta endorphin.

Laboratory monitoring of children with precocious puberty.

It is necessary not only to perform laboratory tests for the correct diagnosis of children with precocious puberty, but also to monitor laboratory tests to ensure adequacy of therapy. Careful laboratory testing is a requisite in the differentiation of central from peripheral precocious puberty. It is also required to determine whether the patient who presents with early physical changes of pubertal development with peripheral precocious puberty has evidence of pubertal hormonal secretion. The most useful single test is gonadotropin-releasing hormone (gonadorelin) stimulation of luteinizing hormone release. The same stimulation test is also indicated to verify the adequacy of suppression of gonadotropin secretion among patients with central precocious puberty who are being treated with gonadotropin-releasing hormone analogue. It is necessary to know which gonadotropin assay is being used and the range of normal levels to correctly interpret the tests.

Effects of taurine on basal and stimulated luteinizing hormone (LH) and LH-releasing hormone secretion in ovariectomized rats: in vitro studies.

Taurine (Tau), a putative inhibitory amino acid neurotransmitter, has been shown to inhibit luteinizing hormone (LH) release in vivo. We investigated the effect of this amino acid on LH secretion by cultured anterior pituitary cells. A 5-h incubation with Tau (10(-3)-10(-8) M) did not affect basal or LH-releasing hormone (LHRH)-stimulated LH release. Basal LHRH release from superfused mediobasal hypothalamic fragments was not affected by Tau (10(-3) M). However, this substance clearly diminished LHRH release after stimulation with KCl (50 mM) or N-methyl-D-aspartate (10(-4) M). It is concluded that Tau may exert an inhibitory effect on LH secretion acting at the hypothalamic level.

GnRH-Induced Desensitization of in Vitro Luteinizing Hormone Secretion in the Turtle, Trachemys scripta

Gonadotropin-releasing hormone (GnRH)-induced desensitization of pituitary luteinizing hormone (LH) secretion in female turtles (Trachemys scripta) was investigated by in vitro superfusion of whole or hemipituitaries with the endogenous forms of the peptide. In sub-adults, LH secretion was rapidly attenuated desensitized) by both prolonged and pulsatile treatments of chicken II GnRH (cII-GnRH), but pulsatile GnRH administration was more effective than prolonged administration in stimulating LH release. Prolonged administration of chicken I GnRH (cI-GnRH) also desensitized LH secretion; cI-GnRH was slightly less potent than cII-GnRH. GnRH-induced attenuation of LH secretion was not the result of tissue deterioration or depletion of relesable LH: GnRH responsiveness of desensitized pituitaries was fully restored after 4 hr of in vitro recovery, and further LH secretion was induced by tetraethylammonium chloride, a membrane depolarizing agent, in the desensitized glands; TRH-stimulated TSH release did not show desensitization in the same glands. Pituitaries from most adult (vitellogenic) females failed to respond to GnRH challenges; however, when responsive, adult pituitaries also desensitized under prolonged GnRH stimulation and were more resistant to GnRH-induced desensitization under pulsatile GnRH treatments. Current results show that adult T. scripta typically display lower GnRH sensitivity than the subadults; the latter are like mammals, birds, and goldfish in their susceptibility to desensitization by both forms of endogenous GnRH.

Selected endocrine parameters of the adult male chimpanzee

This study provides baseline data on reproductive endocrine parameters of the male chimpanzee. A colony group of 47 male chimpanzees were classified by age as juvenile, ages 4-6 years (n = 7); adolescent, ages 7-9 (n = 9); and adult, ages 10-33 years (n = 31). Blood samples (n = 112) obtained from these animals [juveniles (n = ll), adolescents (n = 25), and adults (n = 76)] were analyzed for testosterone (T), luteinizing hormone (LH), and follicle stimulating hormone (FSH). There was a significant increase (mean ± SE) in T (ng/ml) between the juvenile (0.2 ± 0.1) and adolescent group (2.4 ± 0.3) and between the adolescent (2.4 ± 0.3) and the 10-14‒year-old adult age group (4.2 ± 0.2). T peaked at 15-24 years of age and then declined. There was a significant difference in T between animals aged 20-24 years (5.3 ± 0.4) and 25-29 years (3.0 ± 0.4). There was no significant change in serum LH (mlU/ml) with age. Serum FSH (mlU/ml) increased significantly between 4-6 years of age (90.0 ± 11.6) and 10-14 years of age (120.5 ± 8.3), plateaued between 10 and 19 years of age and was significantly lower in males older than 20 years (92.6 ± 4.2). Blood samples (n = 5) obtained from an experimental group of six adult male chimpanzees, ages 10 to 15 years, were analyzed for T, LH, and FSH, Pituitary response to challenge with exogenous GnRH and to hypotha-lamic stimulation with NMA also was assessed. T, LH, and FSH in the experimental group did not differ significantly from animals of equivalent age in the colony group. Challenge with 50, 100, 200, and 500μg GnRH stimulated LH release. The response was not directly dose related. Challenge with 3 and 6 mg/kg NMA stimulated LH release. The response was dose related. © 1993 Wiley-Liss, Inc.

Streptozotocin-induced diabetes blocks the positive feedback release of luteinizing hormone in the female rat

The effects of streptozotocin-induced (STZ) diabetes on the release of gonadotropins was studied in female rats. In the first experiment, rats were ovariectomized and 2 days later were injected with STZ. Three weeks later rats were treated with estrogen and progesterone and blood samples were taken via intraatrial cannulae for luteinizing hormone (LH) assay. Afternoon surges of LH were seen in 4 5 control but 0 8 STZ rats. Pituitary responses to LH-releasing hormone in vitro did not differ. In the 2nd experiment, ovariectomized estrogen-primed rats were killed prior to and during a progesterone-induced LH surge. As in Experiment 1, STZ-treatment inhibited the LH surge but did not effect the afternoon rise in median eminence norepinephrine turnover which has previously been shown to be important in stimulating LH release. Turnover of norepinephrine in the anterior hypothalamus was depressed in the diabetic rats both prior to and during the expected time of the LH surge. Dopamine turnover was depressed in all three brain regions studied. It can be concluded that the positive feedback control of LH release is severely attenuated in diabetic rats but the mechanism explaining the loss is not clear. Diabetes-induced alterations in hypothalamic catecholamine metabolism may be involved but further work is needed to more carefully define these relationships.

Neuropeptide Y potentiates luteinizing hormone (LH)-releasing hormone-induced LH secretion only under conditions leading to preovulatory LH surges.

We recently demonstrated that neuropeptide Y (NPY) potentiates the ability of pulsatile LHRH infusions to restore LH surges in pentobarbital (PB)-blocked, proestrous rats. In the present study we determined if specific endocrine conditions are necessary for the expression of these direct pituitary effects of NPY. Facilitatory actions of NPY were examined in the absence of gonadal feedback [ovariectomy (OVX)], in the presence of negative gonadal feedback (metestrus), after estrogen priming of the pituitary gland [OVX plus 30 micrograms estradiol benzoate (EB) 2 days before experiments], and after treatments which evoke preovulatory-like LH surges (OVX plus EB and 5 mg progesterone or P the morning of experiments). Rats received jugular catheter implants the day before experiments. On the day of experiments, hourly blood samples were taken from 1100-2100 h. At 1330 h, rats received injections of PB to block endogenous LHRH release, or saline. Every 30 min from 1400-1800 h, PB-treated rats received iv pulses of LHRH (15 ng/pulse) or saline, along with concurrent pulses of NPY (1 or 5 micrograms/pulse) or saline. Plasma samples were analyzed by LH RIA. In all cases, pulsatile administration of 15 ng LHRH resulted in plasma LH levels that were significantly elevated above saline-treated, PB-blocked controls. Only in the case of EB+P-treated rats did coadministration of 5 micrograms NPY along with LHRH significantly enhance LHRH-stimulated LH secretion (P < 0.001). NPY had no effect on LHRH-stimulated LH secretion in OVX, OVX + EB-treated, or metestrous rats. Pulsatile administration of either dose of NPY alone did not stimulate LH release in any of the four groups examined. These results demonstrate that the facilitatory effects of NPY on LHRH-stimulated LH secretion can be manifest only under the endocrine conditions required to produce full, preovulatory-like LH surges, i.e. after estrogen and P treatment.

Neuropeptide Y potentiates luteinizing hormone (LH)-releasing hormone- induced LH secretion only under conditions leading to preovulatory LH surges

We recently demonstrated that neuropeptide Y (NPY) potentiates the ability of pulsatile LHRH infusions to restore LH surges in pentobarbital (PB)-blocked, proestrous rats. In the present study we determined if specific endocrine conditions are necessary for the expression of these direct pituitary effects of NPY. Facilitatory actions of NPY were examined in the absence of gonadal feedback [ovariectomy (OVX)], in the presence of negative gonadal feedback (metestrus), after estrogen priming of the pituitary gland [OVX plus 30 micrograms estradiol benzoate (EB) 2 days before experiments], and after treatments which evoke preovulatory-like LH surges (OVX plus EB and 5 mg progesterone or P the morning of experiments). Rats received jugular catheter implants the day before experiments. On the day of experiments, hourly blood samples were taken from 1100-2100 h. At 1330 h, rats received injections of PB to block endogenous LHRH release, or saline. Every 30 min from 1400-1800 h, PB-treated rats received iv pulses of LHRH (15 ng/pulse) or saline, along with concurrent pulses of NPY (1 or 5 micrograms/pulse) or saline. Plasma samples were analyzed by LH RIA. In all cases, pulsatile administration of 15 ng LHRH resulted in plasma LH levels that were significantly elevated above saline-treated, PB-blocked controls. Only in the case of EB+P-treated rats did coadministration of 5 micrograms NPY along with LHRH significantly enhance LHRH-stimulated LH secretion (P < 0.001). NPY had no effect on LHRH-stimulated LH secretion in OVX, OVX + EB-treated, or metestrous rats. Pulsatile administration of either dose of NPY alone did not stimulate LH release in any of the four groups examined. These results demonstrate that the facilitatory effects of NPY on LHRH-stimulated LH secretion can be manifest only under the endocrine conditions required to produce full, preovulatory-like LH surges, i.e. after estrogen and P treatment.

Brain Angiotensin II Receptor Subtypes and the Control of Luteinizing Hormone and Prolactin Secretion in Female Rats

The present experiments examined the role of the two recently identified angiotensin II (Ang II) receptor subtypes, AT, and AT(2) , in the central nervous system regulation of luteinizing hormone (LH) and prolactin secretion in estrogen- and progesterone-treated ovariectomized rats. In this animal model, intracerebroventricular (icv) injection of Ang II stimulates LH and inhibits prolactin release. The specific Ang II receptor subtype antagonists losartan (AT(1) ) or PD123177 (AT(2) ) were administered (icv) in various doses (10 ng to 1,000 ng) 10 min prior to icv injection of Ang II (100 ng). Control animals were pretreated with artificial cerebrospinal fluid prior to Ang II administration. Blood samples for LH and prolactin determinations were taken from conscious, freely-moving rats prior to and following injection of the antagonists and Ang II. Water intake was measured. Ang ll-induced water intake was attenuated 62% by 1,000 ng losartan; water intake was not affected by lower doses of losartan or by any dose of PD123177. Ang ll-induced stimulation of LH release was abolished by the 1,000 ng doses of losartan and PD123177 and attenuated by the 500 ng doses of both drugs. Lower doses did not affect Ang ll-induced LH secretion. Ang ll-induced inhibition of prolactin release was significantly reduced by the 1,000 ng doses of both losartan and PD123177. Lower doses of either drug did not affect the Ang II inhibition of prolactin release. Previous studies had shown that Ang II administration into the anterior hypothalamus-medial preoptic (AHPO) area stimulated LH release. This brain area contains AT(1) receptors. To investigate the potential brain site where the AT(2) receptor may influence LH release, Ang II was injected into the locus ceruleus, a brain nucleus which contains predominately the AT(2) receptor subtype. Ang II administration into the locus ceruleus was paired with an injection of artificial cerebrospinal fluid or Ang II into the AHPO area. Injection of Ang II into the AHPO area stimulated LH release. Injection into the locus ceruleus did not affect LH secretion, nor did it modify the rise in LH elicited by administration of Ang II into the AHPO area. Plasma levels of prolactin were not altered by any of these injections. Taken together, these data demonstrate that, in estrogen- and progesterone-treated female rats, icv Ang ll-induced water intake is mediated by the AT, receptor subtype, while Ang ll-induced changes in LH and prolactin secretion appear to be mediated by both the AT(2) and AT(2) receptor subtypes. The latter observations are one of the first suggesting a potential function for the AT(2) subtype in vivo, although the physiological relevance of this observation, as well as the site of action for the effects on LH and prolactin, remain to be established.

Luteinizing hormone response to an intravenous infusion of substance P in normal men

The effec of synthetic substance P (SP), infused intravenously in doses of 0.5, 1.0, or 1.5 pmol/kg −1/min −1 for 60 minutes, on gonadotropin secretion was evaluated in seven healthy men. SP tests and a control test with normal saline were randomly performed at weekly intervals. During the tests, SP infusion did not produce untoward side effects or changes in blood pressure. Plasma testosterone concentrations were normal in all subjects and remained unmodified during all tests, regardless of the infused dose of SP. Plasma luteinizing hormone (LH) levels were not modified when either normal saline or the lowest dose of SP were infused, whereas they were significantly increased in a dose-dependent fashion when larger amounts of SP were administered. In contrast, plasma follicle-stimulating hormone (FSH) concentrations did not change significantly during any test. These data demonstrate for the first time in normal men that the systemic infusion of SP stimulates LH release, without modifications of FSH secretion.

In Vivo Effect of Pituitary Adenylate Cyclase Activating Polypeptide 38 (PACAP 38) on the Secretion of Luteinizing Hormone (LH) in Male Rats.

In view of the recent demonstrations that pituitary adenylate cyclase activating polypeptide (PACAP) 38 stimulates the release of LH from superfused pituitary cells and that the hypothalamus and anterior pituitary have highly selective binding sites for the peptide, we have surveyed the effect of intraatrial injections of PACAP 38 and vasoactive intestinal peptide (VIP), which has 68% homology with PACAP 38, in intact adult male rats. Furthermore the effect of intracerebroventricular (icv) injection of PACAP 38 was investigated. Intraatrial (10, 30, 100 micrograms) and icv (8, 32 micrograms) administration of PACAP 38 stimulated LH release significantly (P less than 0.01) in a dose-related fashion. Icv injection at a dose of 0.8 microgram was ineffective. The time course pattern of LH release by intraatrial injection and that by icv injection was similar, but the LH levels increased by intraatrial injection were much higher than that by icv injection. Intraatrial administration of VIP had almost no effect on LH release. These findings suggested that PACAP 38 stimulates LH release in vivo.

Estradiol and progesterone effects on relative luteinizing hormone and follicle stimulating hormone release induced from superfused anterior pituitary cell cultures by defined LHRH pulse regimens

These studies examined the capacity of estradiol and progesterone to modulate relative luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion from superfused anterior pituitary cells when stimulated with luteinizing hormone releasing hormone (LHRH) pulse regimens of specific amplitude, duration and frequency. There was particular interest in whether such steroid and LHRH treatments induced evidence of divergent LH or FSH secretion. Pituitaries were recovered from adult, 2 week ovariectomized rats and cultured for 48 h with collagen coated Cytodex microcarrier beads. Cultures were preincubated either with or without estradiol (1 or 10 nM) for 48 h and were subsequently incubated for 3, 6 or 12 h with 100 nM progesterone. All groups were then pulsed with 1 of 3 LHRH regimens; regimen 1 delivered 8 ng in a single 100 μl bolus once/h; regimen 2 divided the 8 ng dose of regimen 1 into 3 equal doses administered at 4 min intervals thereby maintaining the 8 ng mass of regimen 1 while extending the duration of exposure; regimen 3 was the same as regimen 2 except that the 3 equal doses were administered at a pulse frequency of 1 per 2 h rather than 1 per h thereby not only maintaining the duration of exposure as in regimen 2 but also reducing the pulse frequency. 1 nM estrogen alone for 48 h had no effect on LHRH stimulated LH release regardless of regimen; however, FSH was increased when hourly pulses of increased duration were applied (regimen 2). When estrogen was increased to 10 nM, regardless of regimen, LH was predominantly inhibited while FSH was unaffected. When 1 nM estrogen was followed by progesterone, both LH and FSH were elevated at 6 h progesterone in response to regimen 2; with 10 nM estrogen however, a divergent response was observed in that LH release was elevated at 6 h while FSH was elevated at 3 h in response to regimens 2 and 3. These results first of all confirm that progesterone in combination with estrogen is capable of exerting both inhibitory and stimulatory effects on gonadotropin secretion; secondly, these studies show that, as a direct pituitary effect, the LHRH regimen and the gonadal steroid milieu are capable of interacting to significantly influence the relative secretion of LH and FSH. The data therefore suggest that the divergent gonadotropin secretion seen in various physiological states in vivo is due likely in part to a combination of estrogen and progesterone priming in combination with the hypothalamic LHRH secretory pattern.

Galanin: a hypothalamic-hypophysiotropic hormone modulating reproductive functions.

Galanin (GAL) is widely distributed in the peripheral and the central nervous systems. In the brain, the highest GAL concentrations are observed within the hypothalamus and, particularly, in nerve terminals of the median eminence. This location, as well as GAL actions on prolactin, growth hormone, luteinizing hormone (LH), and LH-releasing hormone (LHRH) secretion, suggest the possibility that GAL may act as a putative hypothalamic-hypophysiotropic hormone. To establish this, GAL and LHRH levels were measured in hypophyseal portal plasma samples using specific radioimmunoassays. Rat galanin (rGAL) concentrations in portal blood were approximately 7-fold higher than those observed in peripheral plasma in male and female (estrus, diestrus) rats, indicating an active secretory process of rGAL into the portal vasculature. Frequent (10 min) sampling revealed that rGAL and LHRH were secreted into the portal circulation in a pulsatile manner with a pulse frequency of one pulse per hour. Interestingly, both hormone series depicted a high degree of coincident episodes. In fact, the probability of random coincidence, calculated by the algorithm HYPERGEO, was less than 0.01. Moreover, the retrograde tracer Fluoro-Gold, when given systemically, was taken up by GAL neurons in the hypothalamus, including a subset of neurons expressing rGAL and LHRH, strengthening the notion of the existence of a GAL neuronal system connected to the hypophyseal portal circulation. These observations reinforce the concept that GAL regulates pituitary hormone secretion. To analyze this in further detail, the effects of rGAL on LH secretion were evaluated under basal and stimulated conditions. rGAL induced a small but dose-dependent increase in LH secretion from cultured, dispersed pituitary cells. Interestingly, rGAL enhanced the ability of LHRH to stimulate LH release. The tight link between GAL and LHRH neuronal systems is strengthened by the observation that during the estrous cycle of the rat, rGAL and LHRH contents in the median eminence show an identical profile (r = 1.00). These data indicate that GAL should be considered as a hypothalamic-hypophysiotropic hormone and as an important neuromodulator of LHRH secretion and action. The colocalization and cosecretion of GAL and LHRH and the cooperative action at the level of the anterior pituitary afford important evidence for the functional significance of coexistence of neurotransmitters in neurons of the central nervous system.

The effects of age on the postreceptor regulation of luteinizing hormone secretion by gonadotropin-releasing hormone

We studied the effects of age on the roles of phosphoinositide (PI) and protein kinase C (PKC) in luteinizing hormone (LH) release by gonadotropin-releasing hormone from mouse pituitaries. Pituitary cells from intact and 14-day ovariectomized (OVX) mice aged 4–8 months, 10–12 months and 14–18 months were cultured at a dilution of 3×10 5 cells/ml of M199-bovine serum albumin medium for 3 days prior to stimulation with either buserelin or phorbol ester (phorbol myristate acetate, PMA), while LH was assayed by radioimmunoassay using anti-rat LH antibody (NIDDK-5-10). In intact young mice, buserelin and PMA specifically induced time- and dose-dependent increases in LH release with specific mean ED 50 of 0.82×10 −11 M (buserelin) and of 1.6×10 −8 M (PMA) and a maximal LH release of 138±15 ng/10 6 cells after a 3 h stimulation period. Age did not affect the ED 50 of either agonist but significantly reduced their ability to release LH. This reduction was more pronounced for buserelin than for PMA and was evident as early as middle-age. OVX resulted in a significant increase in both basal and stimulated LH release, but did not affect the age-related reduced secretion rate of LH by either agonist. Buserelin stimulated the incorporation of [ 3H]inositol into [ 3H]inositol phosphates (IP) in a dose-dependent manner, which was unaffected by either age or OVX. We conclude that, with aging, there occurs a reduced LH release rate to both buserelin and PKC stimulations, uncoupled to changes in PI-IP cycle. Furthermore our results suggest that these cellular defects are first manifested at middle-age and are unaffected by the status of the gonad.

Involvement of Catecholaminergic Systems in the Zona incerta in the Steroidal Control of Gonadotrophin Release and Female Sexual Behaviour

Previous reports have shown that dopamine (DA) in the zona incerta (ZI) has a stimulatory effect on gonadotrophin release. We have now investigated the possibility that steroids exert their feedback effects on the release of luteinizing hormone (LH) via catecholamine systems in the ZI. Since the same steroid regimes also stimulate female sexual behaviour, the possibility that the ZI is also involved in the control of sexual activity was investigated. Lesions in the ZI increased proceptive and receptive behaviour in oestrogen-primed ovariectomised rats that exhibited a low level of lordosis in a pre-lesion test and had no effect in receptive animals. Turnover rates (TR) of DA, noradrenaline (NA) and adrenaline were measured in the ZI, preoptic area (POA), arcuate nucleus (ARC), median eminence (ME) and ventromedial nucleus in ovariectomised rats treated 54 h before with either oil, oestradiol benzoate (OB) at 5, 10 or 50 micrograms/rat, or 5 micrograms/rat OB followed by 0.5 mg/rat progesterone (P) 48 h later. The TR as measured from the decline in concentration after alpha-methyltyrosine and changes in DOPAC concentration were correlated with the effect of the steroids on plasma LH and lordotic activity. Confirming previous reports, NA turnover in the POA and ME was increased, and DA turnover in the ME was decreased by steroids when they enhanced LH release. DA turnover in the ARC and ME was reduced when lordosis behaviour increased. Treatment with OB plus P stimulated LH release and sexual receptivity and at the same time significantly increased DA and NA turnover in the ZI. There was no correlation between these parameters after OB alone. This report shows that the DA system in the ZI may mediate the stimulatory effect of P on LH release in OB-primed rats but is not involved in the feedback effects of oestradiol alone. NA activity in the ZI is increased after OB plus P and may therefore also be concerned with stimulating LH release. The ZI is involved in the control of sexual behaviour, as electrolytic lesions in this area enhance receptivity and proceptivity, but the catecholamine systems in the ZI do not appear to mediate this control.