Simultaneous measurement of gonadotropin-releasing hormone, luteinizing hormone, and follicle-stimulating hormone in the orchidectomized rat

In the present study two recently developed techniques have been combined to enable the simultaneous in vivo determination of pulsatile release of GnRH, LH, and FSH in the orchidectomized rat. The first of these techniques involves the implantation of two vascular catheters and collecting serial blood samples through one while simultaneously infusing a replacement blood mixture through the other; consequently, blood samples can be collected for an extended period of time, and detailed plasma LH and FSH release profiles can be established for individual animals. The second technique involves push-pull perfusion of the pituitary gland to determine changes in GnRH concentration as might be perceived by the gonadotropes. For each animal (n = 6), blood (150 microliters) and push-pull perfusate (200 microliters) samples were collected at 5- and 10-min intervals, respectively, for approximately 6 h, and the hormone release profiles were determined by RIA. All of the rats showed a clear pulsatile release pattern for GnRH, LH, and FSH. Moreover, the interpulse interval was remarkably similar for each of these hormones (36.9, 41.5, and 43.5 min, respectively, as determined by PULSAR). The percentage of GnRH pulses associated with a gonadotropin pulse was 72% for LH and 76% for FSH; only 14% of the pulses were silent for both gonadotropins. These results demonstrate that in the orchidectomized rat the pulsatile pattern of GnRH release is reflected in the pulsatile pattern of not only LH but also FSH. They may, therefore, be construed to support the concept that the pulsatile secretion of both gonadotropins is primarily orchestrated by a single hypothalamic releasing hormone. Alternatively, if two separate hypothalamic releasing hormones do indeed exist (LHRH and FSH-releasing hormone), it would appear that in the orchidectomized rat their episodic release is tightly coupled to the same hypothalamic pulse generator.     

Inhibition of hypothalamic gonadotropin-releasing hormone release by endogenous opioid peptides in the female rabbit

Recent evidence suggests that the endogenous opioid peptides (EOPs) inhibit luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by suppression of hypothalamic gonadotropin-releasing hormone (GnRH) release, and that the feedback inhibition by EOPs is influenced by ovarian steroids. In the present studies, intact (INT) and ovariectomized (OVX) adult female rabbits were fitted with femoral vein catheters and mediobasal hypothalamic (MBH) push-pull perfusion (PPP) cannulae. One week after brain cannulation, does were subjected to 6 h of PPP and sequential blood sampling. In experiment I, INT (n = 6) and OVX (n = 5) does were infused intravenously with saline for 4 h followed by 2 h of infusion of the opiate antagonist naloxone (NAL; 10 micrograms/min/kg) while the MBH was simultaneously perfused with media. In experiment II, INT (n = 5) and OVX (n = 5) does were perfused with media for 4 h followed by 2 h of intrahypothalamic (IHP) NAL perfusion (0.2 microgram/min). The GnRH in push-pull perfusates and LH and FSH in plasma samples collected at 10-min intervals were measured by specific radioimmunoassays. In INT does, neither intravenous infusion nor IHP perfusion of NAL altered pulsatile parameters of GnRH or LH release. In contrast, both intravenous and IHP NAL administration stimulated GnRH and LH release within 30-50 min in OVX does by marked increases in both GnRH and LH pulse amplitudes. Neither route of NAL administration affected FSH secretion in any of the treatment groups. We conclude that EOPs are involved in the inhibition of hypothalamic GnRH secretion in OVX does; the feedback inhibition by ovarian steroids on the hypothalamic-pituitary axis in the rabbit is sufficient to compromise the effects of EOPs, and under these experimental conditions, the hypothalamic mechanisms which regulate the secretion of pituitary LH and FSH may be independent.     

Acute inhibitory effects of antiserum to gonadotropin-releasing hormone in ovariectomized rats. Evidence for pulsatile secretion of gonadotropin-releasing hormone.

To evaluate the dependence of pulsatile secretion of luteinizing hormone (LH) on gonadotropin-releasing hormone (GnRH), the acute effects of immunoneutralization of endogenous GnRH on plasma LH were compared in ovariectomized rats with phenobarbital treatment and hypophysectomy. Anti-GnRH produced a rapid dose-dependent decrease in plasma LH and LH pulse amplitude. Pulsatile secretion of LH was eliminated in 6 of 12 rats treated with the highest dose of anti-GnRH, but plasma LH was still 50% of control values 3 h after treatment. Frequency of LH pulses was unchanged in animals which had persistence of pulsatile secretion of LH. Phenobarbital eliminated pulsatile secretion of LH transiently. Hypophysectomized rats displayed a striking decrease in plasma LH which could be resolved into two exponential components with half-lives of 16 and 70 min. The initial half-life of plasma LH from untreated rats determined after LH pulses was also 16 min. These studies support the hypothesis that pulses of GnRH induce the pulsatile pattern of plasma LH and may be responsible for all LH secretion in ovariectomized rats.     

Transplanted gonadotropin-releasing hormone neurons promote pulsatile luteinizing hormone secretion in congenitally hypogonadal (hpg) male mice

Congenitally hypogonadal (hpg) male mice are unable to synthesize biologically active gonadotropin-releasing hormone (GnRH). Implantation of normal fetal preoptic area tissue containing GnRH neurons into the third ventricle of adult hpg males significantly elevates pituitary levels of luteinizing hormone (LH) and corrects their hypogonadism. In all responding animals, immunoreactive GnRH neurons within the transplant innervate the median eminence of the host. To assess whether gonadal recovery in hpg hosts results from pulsatile secretion of GnRH from grafted neurons, we compared the pattern of variation in plasma LH levels in 19 hpg graft recipients with testicular growth to that of 10 normal adult mice. All animals were castrated prior to receiving an indwelling catheter in the jugular vein. Sequential blood samples were collected (t = 10 min) and assayed for LH. Pulsatile LH secretion was seen in 11 of 19 hpg hosts and in all control mice. While there was great variability between individual animals, measures of baseline LH, LH pulse amplitude and duration, interpulse interval, and LH pulse frequency revealed no difference between hpg graft recipients and normal castrates in their LH pulse pattern. Immunocytochemical analysis of the brain in hpg hosts suggested no correlation between any parameter of pulse activity and individual differences in GnRH cell number or GnRH fiber outgrowth into the median eminence. Sources of variation in LH secretion among graft recipients, and between hpg hosts and normal mice, are discussed. We suggest that transplanted GnRH neurons are capable of integration into a GnRH 'pulse generator' which can support a near-normal pattern of pulsatile LH secretion, leading to testicular growth and steroid production.     

Simultaneous induction of neuropeptide Y and gonadotropin-releasing hormone release in the rabbit hypothalamus

Neuropeptide Y (NPY) can induce the release of endogenous mediobasal hypothalamic gonadotropin-releasing hormone (MBH-GnRH) and pituitary gonadotropins, especially LH. In these studies, we monitored changes in endogenous NPY concentrations at 20-min intervals for 6-8 h during push-pull perfusion (PPP) in both the mediobasal hypothalamus (MBH) and the third cerebroventricle (3VT) of ovarian intact, conscious rabbits. Because previous studies had shown that copper ion can induce hypothalamic GnRH release, cupric acetate (CuAc) was administered either intravenously or intraventricularly during the PPP to manipulate changes in NPY concentrations. Our results show that NPY concentrations in both MBH and 3VT PPP samples were detectable by radioimmunoassay. Administration of CuAc sharply increased hypothalamic NPY release within the same time interval as that for induction of hypothalamic GnRH release. The results are consistent with the hypotheses that NPY may act as a neuromodulator for hypothalamic GnRH secretion, or that common mechanisms drive secretion of these two neuropeptides.     

The frequency of gonadotropin-releasing-hormone stimulation differentially regulates gonadotropin subunit messenger ribonucleic acid expression

The hypothalamic decapeptide GnRH is known to regulate the synthesis and secretion of LH and FSH by pituitary gonadotrope cells. The frequency of pulsatile GnRH secretion changes and LH and FSH are differentially secreted in various physiological situations. To investigate the potential role of altered frequency of GnRH stimulation in regulating differential secretion of LH and FSH, we examined the effects of GnRH frequency on expression of the alpha, LH beta, and FSH beta genes. GnRH pulses (25 ng/pulse) were administered to castrate testosterone-replaced rats at intervals of 8-480 min to cover the range of physiological pulsatile GnRH secretion. Fast frequency GnRH pulses (8-min pulse intervals) increased alpha-subunit mRNA concentrations 3-fold above those in saline-pulsed controls (controls, 1.01 fmol cDNA bound/100 micrograms pituitary DNA) and LH beta mRNA by 50% (controls, 0.18 fmol cDNA bound), but FSH beta mRNA was unchanged (controls, 0.38 fmol cDNA bound). GnRH pulses given every 30 min increased all three subunit mRNAs (alpha, 3-fold, LHbeta, 2-fold; FSH beta, 2-fold), and acute LH release and serum FSH concentrations were maximal after this frequency. Slower frequency GnRH stimuli (120- to 480-min pulse intervals) did not change alpha and LH beta mRNA levels, but increased FSH beta mRNA 2- to 2.5-fold, and FSH secretion was maintained. Equalization of the total dose of GnRH given at different intervals over 24 h confirmed the frequency dependence of subunit mRNA expression. Fast frequency GnRH stimuli (8 min) increased alpha mRNA 1.5- to 2.5-fold, while the same total GnRH doses were ineffective when given at slow frequency (480 min). Similarly, LH beta mRNA was only increased by GnRH pulses given at 8-min intervals. In contrast, FSH beta mRNA increased 2-fold after pulses given every 480 min, and the 8-min pulse interval was ineffective. The data show that the frequency of GnRH stimulation can differentially regulate gonadotropin subunit mRNA expression and may be a mechanism that enables a single GnRH peptide to selectively regulate gonadotropin subunit gene expression and hormone secretion.     

Central electrophysiologic correlates of pulsatile luteinizing hormone secretion in the rhesus monkey

Characteristic increases in neuronal activity coincident with the pulsatile release of luteinizing hormone from the pituitary gland have been recorded from electrodes chronically implanted in the medial basal hypothalamus of the rhesus monkey. This electrophysiologic manifestation of the hypothalamic 'pulse generator' which governs the secretion of hypothalamic luteinizing hormone releasing hormone provides, for the first time, direct access to the central component of the neuroendocrine control system which regulates reproductive processes in this higher primate.     

Effects on plasma luteinizing hormone and follicle-stimulating hormone of varying the frequency and amplitude of gonadotropin-releasing hormone pulses in ovariectomized ewes with hypothalamo-pituitary disconnection

The effects on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion of various regimens of pulsatile gonadotropin-releasing hormone (GnRH) replacement were examined in ovariectomized (OVX) ewes after hypothalamo-pituitary disconnection (HPD). Hourly pulses of 500 ng GnRH restored gonadotropin secretion in OVX-HPD sheep. Replacement beginning 2 days after HPD gave consistent responses of LH and FSH within a week. Replacement beginning 61-96 days after HPD caused more gradual re-establishment of LH and FSH secretion with LH responses appearing immediately and FSH responses appearing 2 weeks later. When hourly GnRH pulses were increased in amplitudes from 250 to 500 ng the plasma LH baseline, peak values and pulse amplitudes were increased. There was no significant change in plasma FSH levels over 10 pulses at the higher dose. Decreases in GnRH pulse frequency led to increases in LH pulse amplitude and decreases in plasma LH baseline. In contrast, immediately after a change from a 2-hourly to an hourly mode, an increase in LH baseline occurred without an immediate reduction in LH pulse amplitude. Mean plasma FSH concentrations increased when the frequency was reduced from hourly to 2-hourly or 4-hourly. However, a change from 4-hourly to hourly pulses did not reduce FSH values within 7 days. It is concluded that changes in the pattern of LH secretion observed during the ovine estrous cycle could be accounted for, in part, by changes in GnRH pulse frequency.     

Hypothalamic dysfunction

A pulsatile GnRH stimulus is required to maintain gonadotropin synthesis and secretion. The frequency and amplitude of GnRH pulses determine gonadotropin subunit gene expression and secretion of pituitary LH and FSH. Rapid frequency (more than 1 pulse per h) GnRH pulses favor LH while slower frequencies favor FSH secretion. During ovulatory cycles, an increase in GnRH frequency during the follicular phase favors LH synthesis prior to the LH surge, while following ovulation, luteal steroids slow GnRH pulses to favor FSH synthesis. Thus, a changing frequency of GnRH stimulation of the gonadotrope is one of the mechanisms involved in differential gonadotropin secretion during ovulatory cycles. In hypothalamic amenorrhea a majority of women exhibit a persistent slow frequency of LH (GnRH) pulses, which reflects excess hypothalamic opioid tone and can be temporarily reversed by opioid antagonists. At the other end of the spectrum, in polycystic ovarian syndrome, LH (GnRH) pulses are persistently rapid and favor LH synthesis, hyperandrogenism and impaired follicular maturation. Administration of progesterone can slow GnRH pulse secretion, favor FSH secretion and induce follicular maturation. Thus, the ability to change the pattern of GnRH secretion is an important factor in the maintenance of cyclic ovulation, and loss of this function leads to anovulation and amenorrhea.     

Suppression of mediobasal hypothalamic gonadotropin-releasing hormone and plasma luteinizing hormone pulsatile patterns by phentolamine in ovariectomized rhesus macaques

In gonadectomized animals, pulses of LH are secreted concurrently with pulsatile hypothalamic GnRH and it is hypothesized that pulses of GnRH are either driven or modulated by episodes of catecholamine release. The objective of this study was to determine if the alpha-adrenergic antagonist phentolamine (PHEN) can simultaneously block the release of GnRH and LH in ovariectomized (OVX) rhesus macaques. In Exp 1, simultaneous peripheral blood and mediobasal hypothalamic push-pull perfusion (PPP) samples were collected remotely at 10-min intervals for 24 h via a swivel/tether device in eight conscious, freely moving OVX rhesus monkeys. Phentolamine was continuously infused iv for 6 h at the rate of 4 mg/kg BW.h in five animals and 20 mg/kg BW.h in three animals. Infusion started at 6 h after the commencement of PPP. Sampling of PPP and blood continued for 12 h after the cessation of PHEN infusion. Exp 2 was carried out to determine if PHEN affects pituitary responsiveness to exogenous GnRH under conditions similar to those in Exp 1. Exogenous GnRH (5 micrograms, iv) was injected as a single bolus at 10-h intervals before, during, and after either a saline (4 ml/h for 6 h) infusion or, 3 weeks later, a PHEN infusion (4 mg/kgBW.h for 6 h) in three OVX females. The results of Exp 1 show that pulsatile patterns of hypothalamic GnRH and LH were either dampened or abolished by PHEN infusion. During the recovery period after PHEN infusion, pulse amplitudes of LH were enhanced, but pulse amplitudes of endogenous GnRH did not differ, as compared to those of corresponding LH and GnRH before infusion of PHEN. Data from Exp 2 suggested that the alpha-adrenergic blocking agent had no effect on the pituitary LH response to exogenous GnRH administration. These results directly support the hypothesis that adrenergic neuronal activities are critical for the pulsatile release of hypothalamic GnRH which governs the pulsatile release of LH in gonadectomized animals.     

Pulsatile gonadotropin-releasing hormone release from the human mediobasal hypothalamus in vitro: opiate receptor-mediated suppression

An in vitro perifusion system was used to investigate pulsatile gonadotropin-releasing hormone (GnRH) release from the fetal (20-23 weeks of gestation) and adult human mediobasal hypothalamus (MBH). Fetal human MBHs released GnRH in discrete pulses, with a periodicity of approximately 1 h. Adult human MBHs also released GnRH in a pulsatile manner, with a periodicity of 60-100 min. The calcium-dependent pulsatile GnRH release from fetal human MBHs was suppressed by addition of morphine (10 microM) to the perifusion medium, and this suppression was reversed by addition of the opiate receptor antagonist naloxone (10 microM). These results indicate that the human hypothalamic GnRH pulse-generating mechanism is located entirely within the MBH, and that this pulse generator can maintain intrinsically pulsatile GnRH release independent of all innervation from outside this site. Our data also demonstrate that human hypothalamic pulsatile GnRH release can be suppressed by an opiate receptor-mediated mechanism located within the MBH.     

Frequency and amplitude of gonadotropin-releasing hormone stimulation and gonadotropin secretion in the rhesus monkey

In adult ovariectomized rhesus monkeys bearing hypothalamic lesions which reduced circulating LH and FSH to undetectable levels, sustained elevated gonadotropin concentrations were reestablished by the intermittent administration of gonadotropin-releasing hormone (GnRH) at the rate of 1 microgram/min for 6 min once every hour. The effects of varying either the frequency or the amplitude of these GnRH pulses on gonadotropin secretion were examined in such animals. Increasing the frequency of GnRH administration from the physiological one pulse per h to two, three, or five pulses h while maintaining a constant infusion rate and pulse duration resulted in gradual declines in plasma gonadotropin concentrations. These declines were most profound at the highest frequencies and the consequence of reduced pituitary responses to individual GnRH pulses. Decreasing the frequency of GnRH pulses from one per h to one every 3 h led to variable declines in plasma LH levels, but circulating FSH invariably rose. Reducing the GnRH infusion rate from 1 to 0.1 mg/min while maintaining constant frequency and pulse duration resulted in abrupt declines in plasma LH and FSH to immeasurable levels, although pulsatile increments in circulating GnRH concentrations without a concomitant reduction in plasma LH concentrations, which remained unchanged. An infusion rate of 0.5 microgram/min resulted in unstable plasma LH and FSH levels. These results demonstrate that changes in the frequency or amplitude of hypophysiotropic stimulation have profound effects on plasma gonadotropin levels as well as on FSH to LH ratios in the circulation. The physiological implications of these observations are discussed.     

Evidence for alpha 1-adrenergic involvement in neuropeptide Y-stimulated GnRH release in female rabbits.

Both neuropeptide Y (NPY) and norepinephrine stimulate gonadotropin-releasing hormone (GnRH) secretion in intact or ovariectomized (OVx) estradiol-treated rabbits. The mechanism by which NPY stimulates GnRH is currently unknown. We have tested the hypothesis that NPY increases GnRH release via an alpha-adrenergic pathway. Adult female rabbits were OVx and had Silastic capsules containing 17 beta-estradiol inserted subcutaneously that maintained plasma estradiol levels similar to those in ovarian intact rabbits. One week later, push-pull (PP) perfusion cannulae, with tips positioned in the mediobasal hypothalamus (MBH), and jugular vein catheters were placed in all does. Blood and PP perfusate samples were obtained every 20 min during 7 h perfusion of the MBH with Krebs-Ringer phosphate buffer (KRP). Intrahypothalamic treatment with NPY (n = 5), prazosin (alpha 1-adrenergic antagonist; n = 7), yohimbine (alpha 2-adrenergic antagonist; n = 7), NPY plus prazosin (n = 7) or NPY plus yohimbine (n = 6) dissolved in KRP occurred during hours 4 through 6. GnRH in hypothalamic perfusate and luteinizing hormone (LH) and prolactin (PRL) in peripheral plasma were measured by specific radioimmunoassays. As anticipated, NPY alone significantly increased MBH-GnRH secretion (0.93 +/- 0.24 vs. 2.46 +/- 0.37 pg/ml; p less than 0.05). In contrast, NPY infused concomitantly with prazosin did not increase MBH-GnRH release (1.26 +/- 0.50 vs. 0.78 +/- 0.19 pg/ml; p greater than 0.05) whereas NPY plus yohimbine did stimulate GnRH secretion (1.15 +/- 0.13 vs. 2.65 +/- 0.89 pg/ml; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of gonadotropin-releasing hormone pulse frequency on gonadotropin secretion and subunit messenger ribonucleic acids in perifused pituitary cells.

Slow frequency GnRH pulses have been proposed to preferentially increase circulating FSH levels by increasing FSH synthesis and pulsatile release. Examination of this proposal using various in vivo models, however, has produced conflicting results. To examine directly the effects of GnRH pulse frequency on the pituitary, we compared the effects of 2.5-nM GnRH pulses administered every 1 h or every 4 h vs. no GnRH, using perifused rat pituitary cells. FSH secretion (total area under the response curve) was 2-fold greater (P less than 0.01) with every hour than with every 4 h GnRH pulses. This difference resulted from the increased number of GnRH pulses and increased (P less than 0.05) interpulse FSH secretion, whereas FSH pulse amplitude was unchanged. FSH beta mRNA levels at the completion of the 11-h perifusion were increased 4.5-fold by GnRH every h (P less than 0.01) and 3.3-fold by GnRH every 4 h (P less than 0.05) above levels in untreated cells. FSH beta mRNA levels were greater (P less than 0.05) at the faster GnRH pulse frequency. Because more frequent stimulation delivered more GnRH during the study, cells were next stimulated with 2.5 nM GnRH every 1 h for nine pulses, 7.5 nM GnRH every 4 h for three pulses to equalize the GnRH dose, or 2.5 nM GnRH every 4 h for three pulses. Interpulse FSH secretion and FSH beta mRNA levels were again greater (P less than 0.05) with every hour than every 4 h GnRH pulses. Interpulse LH secretion, FSH and LH pulse amplitude, and LH beta and alpha-subunit mRNA levels were not different between the groups. GnRH doses of 0.1-10 nM every hour increased FSH and LH pulsatile secretion dose-dependently, but FSH beta, LH beta, and alpha-subunit mRNA levels were similar. In conclusion, our data reveal that reducing the frequency of GnRH pulses from every hour to every 4 h reduces both FSH beta mRNA levels and FSH interpulse secretion, but does not change GnRH-stimulated FSH pulsatile release. We suggest that the finding by others that slow frequency GnRH pulses increase circulating FSH levels under certain experimental conditions in vivo may instead be explained by complex hormonal interactions or changes in FSH clearance.     

Effects of bovine follicular fluid on gonadotrophin secretion in intact and chronically ovariectomized ewes before and after desensitization of pituitary gonadotrophs to gonadotrophin-releasing hormone

Intact and chronically ovariectomized ewes were treated for 4 days with charcoal-treated bovine follicular fluid (FF) or charcoal-treated bovine serum during the late-anoestrous period, and the effects on basal and gonadotrophin-releasing hormone (GnRH)-induced secretion of LH and FSH observed. Subsequently, ewes received s.c. implants containing a sustained-release formulation of a potent GnRH agonist D-Ser(But)6-Azgly10-LHRH (ICI 118630) to desensitize pituitary gonadotrophs to hypothalamic stimulation, and the effects of bovine FF and bovine serum were re-assessed 2 weeks later. Chronic exposure (for 2-3 weeks) to ICI 118630 significantly reduced basal levels of LH and FSH in both intact and ovariectomized ewes and completely abolished both spontaneous LH pulses as well as exogenous GnRH-induced acute increases in plasma LH and FSH levels. Treatment with bovine FF significantly reduced plasma FSH levels, but not LH levels, in both intact and ovariectomized ewes before and after chronic exposure to ICI 118630. In intact ewes before exposure to ICI 118630, treatment with bovine FF actually enhanced pulsatile LH secretion and raised mean plasma LH levels by 240% (P less than 0.05). No such stimulatory effect of bovine FF on LH secretion was observed in intact ewes exposed to ICI 118630 or in ovariectomized ewes before or after exposure to ICI 118630, suggesting that the effect probably involved an alteration in ovarian steroid feedback affecting hypothalamic GnRH output. Treatment with bovine FF did not significantly affect the magnitude of GnRH-induced surges of LH or of FSH observed in either intact or ovariectomized ewes before exposure to ICI 118630.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that pulsatile follicle-stimulating hormone secretion is independent of endogenous luteinizing hormone-releasing hormone

Although LHRH can stimulate the release of both LH and FSH from the pituitary, there are a number of instances in which the secretion of LH and FSH are divergent. Previous studies from our laboratory have indicated that pulsatile LH and FSH secretion are independently regulated by gonadal factors. We have, therefore, reexamined the role of LHRH in regulating pulsatile gonadotropin secretion by evaluating the effect of passive LHRH immunoneutralization on LH and FSH secretion in castrate adult male rats. Injection of 500 microliters ovine anti-LHRH serum no. 772 (LHRH-AS) into 2-week-castrate rats caused an 85% suppression of mean plasma LH levels by 2 h, which lasted through 48 h. Mean plasma FSH, however, was reduced by only 19% after 2 h and by only 59% after 48 h. When cannulated 2-week-castrate rats were bled every 10 min, both LH and FSH were secreted in a pulsatile manner. Injection of 500 microliters LHRH-AS caused an immediate abolishment of LH pulses and a rapid reduction in mean plasma LH through 24 h. Pulsatile FSH secretion, as characterized by the parameters of pulse frequency and amplitude, was unaffected by LHRH-AS, although mean plasma FSH levels were significantly reduced. Collectively, the results suggest that pulsatile FSH secretion is regulated by a separate factor(s) distinct from LHRH, but that LHRH is required for the maintenance of elevated FSH levels.     

Does cortisol inhibit pulsatile luteinizing hormone secretion at the hypothalamic or pituitary level?

Elevations in glucocorticoids suppress pulsatile LH secretion in sheep, but the neuroendocrine sites and mechanisms of this disruption remain unclear. Here, we conducted two experiments in ovariectomized ewes to determine whether an acute increase in plasma cortisol inhibits pulsatile LH secretion by suppressing GnRH release into pituitary portal blood or by inhibiting pituitary responsiveness to GnRH. First, we sampled pituitary portal and peripheral blood after administration of cortisol to mimic the elevation stimulated by an immune/inflammatory stress. Within 1 h, cortisol inhibited LH pulse amplitude. LH pulse frequency, however, was unaffected. In contrast, cortisol did not suppress either parameter of GnRH secretion. Next, we assessed the effect of cortisol on pituitary responsiveness to exogenous GnRH pulses of fixed amplitude, duration, and frequency. Hourly pulses of GnRH were delivered to ewes in which endogenous GnRH secretion was blocked by estradiol. Cortisol, again, rapidly and robustly suppressed the amplitude of GnRH-induced LH pulses. We conclude that, in the ovariectomized ewe, cortisol suppresses pulsatile LH secretion by inhibiting pituitary responsiveness to GnRH rather than by suppressing hypothalamic GnRH release.     

Intensive direct cavernous sinus sampling identifies high-frequency, nearly random patterns of FSH secretion in ovariectomized ewes: combined appraisal by RIA and bioassay.

Analyses of FSH secretion suggest pulsatile, nonpulsatile, or compositely pulsatile and nonpulsatile release modes. This may reflect the reduced signal-to-noise ratio inherent in FSH pulse estimation procedures and/or immunological-biological assay inconsistencies. To address these issues, we sampled cavernous sinus and jugular venous blood concomitantly from ovariectomized sheep at either 5-min or 1-min intervals. Samples from the former were assayed by RIA, and those from the latter by RIA and bioassay. Waveform-independent peak detection revealed FSH pulses occurring at high frequency. Pulsatile FSH secretion accounted for 28% of total secretion. Approximate entropy analysis showed that FSH secretion was nearly random. There was synchronous release of LH and FSH, but most FSH secretion was not associated with LH release; 13% of discrete FSH and LH pulses were concordant. We infer that FSH secretion exhibits pulsatile and basal/nonpulsatile features, with high-entropy features. Linear and nonlinear statistical measures revealed joint sample-by-sample synchrony of FSH and LH release, indicating pattern coordination despite sparse synchrony of pulses. We postulate that pattern synchrony of FSH and LH release is effected at the level of the gonadotrope. Concordant FSH and LH pulses probably result from pulsatile GnRH input, but other mechanisms could account for independent FSH pulses.     

Pulsatile administration of gonadotrophin-releasing hormone stimulates, in oestrogen-treated anaesthetized ovariectomized ewes, a surge release of LH qualitatively and quantitatively different from that induced by oestradiol in conscious ovariectomized ewes

The nature of the gonadotrophin-releasing hormone (GnRH) stimulus of the pituitary necessary for the oestrogen-induced plasma LH surge was studied in ovariectomized ewes. The sheep were treated with oestradiol benzoate (50 micrograms i.m.) at 0 h, and the hypothalamic contribution to the LH surge was blocked by pentobarbitone anaesthesia over the time during which the surge was expected (11-31 h). Pituitary responsiveness to exogenous GnRH (100 ng) administered i.v. in a pulsatile mode (once per hour or once per 20 min) over the period 15-30 h was assessed from plasma concentrations of LH. Neither of the GnRH treatments induced patterns of LH secretion similar to those seen in conscious ovariectomized ewes given oestrogen only. Plasma LH secretion in response to hourly GnRH pulses was less (P less than 0.01) than that associated with oestrogen-induced plasma LH surges in conscious control ewes. With pulses of GnRH administered every 20 min the amount of LH released was greater (P less than 0.05) than that in oestrogen-treated conscious control ewes. In contrast to the single surge induced by oestradiol in conscious ewes, GnRH pulses given every 20 min elicited phasic patterns of LH secretion consisting of two or three distinct surges. The failure of GnRH treatment to elicit an LH surge similar to an oestrogen-induced surge could reflect inappropriate GnRH treatment regimens, and/or inadequate priming of the pituitary with GnRH after induction of anaesthesia but before GnRH treatment.