Changes In GnRH Secretion Research Articles

Progesterone Directly and Rapidly Inhibits GnRH Neuronal Activity via Progesterone Receptor Membrane Component 1

GnRH neurons are essential for reproduction, being an integral component of the hypothalamic-pituitary-gonadal axis. Progesterone (P4), a steroid hormone, modulates reproductive behavior and is associated with rapid changes in GnRH secretion. However, a direct action of P4 on GnRH neurons has not been previously described. Receptors in the progestin/adipoQ receptor family (PAQR), as well as progesterone receptor membrane component 1 (PgRMC1) and its partner serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1) mRNA binding protein 1 (SERBP1), have been shown to mediate rapid progestin actions in various tissues, including the brain. This study shows that PgRMC1 and SERBP1, but not PAQR, are expressed in prenatal GnRH neurons. Expression of PgRMC1 and SERBP1 was verified in adult mouse GnRH neurons. To investigate the effect of P4 on GnRH neuronal activity, calcium imaging was used on primary GnRH neurons maintained in explants. Application of P4 significantly decreased the activity of GnRH neurons, independent of secretion of gamma-aminobutyric acidergic and glutamatergic input, suggesting a direct action of P4 on GnRH neurons. Inhibition was not blocked by RU486, an antagonist of the classic nuclear P4 receptor. Inhibition was also maintained after uncoupling of the inhibitory regulative G protein (G(i/o)), the signal transduction pathway used by PAQR. However, AG-205, a PgRMC1 ligand and inhibitor, blocked the rapid P4-mediated inhibition, and inhibition of protein kinase G, thought to be activated downstream of PgRMC1, also blocked the inhibitory activity of P4. These data show for the first time that P4 can act directly on GnRH neurons through PgRMC1 to inhibit neuronal activity.

Aging Attenuates the Pituitary Response to Gonadotropin-Releasing Hormone

Complex changes in GnRH secretion occur with aging in women, but little is known about the effect of aging on the pituitary per se. The aim of the study was to determine whether pituitary responsiveness to GnRH is attenuated with aging. A GnRH antagonist and graded doses of GnRH were used to isolate pituitary responsiveness in Clinical Research Center studies at an academic medical center. Subjects were healthy postmenopausal women (PMW) aged 48-57 yr (n = 10) or 70-77 yr (n= 9). A suppressive dose of the NAL-GLU GnRH antagonist (150 microg/kg sc) was administered and was followed by GnRH doses of 25, 75, 250, or 750 ng/kg iv every 4 h. The LH response to GnRH was attenuated with aging (P = 0.05) with an interaction between age and dose (P = 0.01) such that the LH amplitude was less in older PMW at the higher doses (250 ng/kg, 50 +/- 9 vs. 29 +/- 4.9 IU/liter, for young and old PMW, respectively, P = 0.02; and 750 ng/kg, 97.7 +/- 11 vs. 70.2 +/- 9.3 IU/liter, P = 0.002), but not the lower doses of GnRH. The FSH response to GnRH was also attenuated with aging in PMW (P = 0.005). In studies that isolated the pituitary from endogenous GnRH stimulation, aging attenuated the LH and FSH responses to exogenous GnRH in PMW. These studies indicate that the pituitary plays a role in the decline in gonadotropin levels with aging, further supporting the potential contribution of age-associated changes in both hypothalamic and pituitary function to reproductive senescence.

Steroids Modulate Transcript and Protein Levels of Large-Conductance Ca2+-Activated Potassium (BK) Channels in GT1-7 Cells, Correlated with Changes in GnRH Secretion In Vitro.

Steroids Modulate Transcript and Protein Levels of Large-Conductance Ca2+-Activated Potassium (BK) Channels in GT1-7 Cells, Correlated with Changes in GnRH Secretion In Vitro.

Neuroendocrine control of follicle-stimulating hormone (FSH) secretion: II. Is follistatin-induced suppression of FSH secretion mediated via changes in activin availability and does it involve changes in gonadotropin-releasing hormone secretion?

The objective of the present study was to determine to what extent activin participates in setting the level of FSH secretion and if this regulation includes mediation via changes in GnRH secretion. We administered follistatin, the high-affinity binding protein for activin, to five ovariectomized sheep; we reasoned that the resultant binding of follistatin to activin should lower activin bioavailability and FSH secretion. Hypophyseal portal and peripheral blood samples were collected simultaneously at 10-min intervals for 18 h to measure GnRH, LH, FSH, and both activin-free and total follistatin. Six hours into collection, each ewe received 150 microg/kg i.v. of recombinant human follistatin-288. A week later, the same ewes were subjected to a second series of blood collections of similar length (time control). The FSH levels in pituitary portal blood were approximately 8-fold higher than those in the peripheral circulation. The FSH secretory patterns changed minimally during the time-control period. In contrast, follistatin had profound suppressive effects on FSH secretion. Maximal FSH suppression after FS-288 administration occurred at 5-6 h in the pituitary portal (65% suppression) and 9-10 h in the peripheral (48% suppression) circulation. Follistatin had no effect on GnRH or LH secretory patterns. Disappearance of total follistatin (i.e., free follistatin plus activin-bound follistatin) from the circulation was slower (P < 0.05) than that of free follistatin alone, suggesting that some of the follistatin was complexed with circulating activin, thus reducing the bioavailability of activin. The slower clearance of total follistatin and the lack of follistatin effects on GnRH secretion suggest that changes in activin bioavailability dictate the level of pituitary FSH secretion and that this is a pituitary-specific effect.

Photoperiodic modulation of GnRH mRNA in the male Syrian hamster

Male Syrian hamsters ( Mesocricetus auratus) are seasonal breeders. They show marked testicular regression when exposed to short autumnal photoperiods, and then remain sexually quiescent for several months. By mid-winter, however, they show a loss in responsiveness to the inhibitory influence of short photoperiods and their testes begin to recrudesce. To shed light on the neuroendocrine mechanism responsible for mediating these reproductive changes, we examined the influence of photoperiod on the expression of GnRH mRNA in the hamster forebrain. Adult males were either exposed to short photoperiods (6L:18D) for 16 weeks or were maintained under long photoperiods (14L:10D); additional animals were exposed to short or long photoperiods for 22 weeks. As expected, exposure to short photoperiods for 12 weeks resulted in a marked decrease ( P<0.01) in testicular mass and serum testosterone levels, but after 22 weeks these reproductive parameters were once again significantly elevated ( P<0.01). In contrast, quantitative in situ hybridization histochemistry revealed no difference ( P>0.05) between the GnRH mRNA levels of the short-photoperiod hamsters and their aged-matched long-photoperiod controls, although an age-related decrease ( P<0.05) was evident in both photoperiod-treatment groups. These data emphasize that GnRH mRNA is highly expressed in hamsters even when their reproductive axis has been rendered sexually quiescent by exposure to short photoperiods, and that photoperiod-induced changes in GnRH secretion, rather than synthesis, are more likely to regulate the timing of the breeding season. On the other hand, the data indicate that GnRH mRNA levels show an aging-related decrease, regardless of photoperiod, suggesting that in the long term a decrease in GnRH gene expression may contribute to the reduced fertility of old hamsters.

GnRH secretion into CSF in rams treated with a GnRH antagonist.

The equilibrium of the brain-pituitary-testicular axis is controlled by negative feedback exerted primarily through changes in the circulating concentrations of gonadal steroids. This is usually studied in gonadectomised animals treated with single large doses or constant low levels of exogenous steroid. However, the feedback system probably also contains dynamic components, perhaps expressed as delays to changes in GnRH secretion following a change in steroid concentration. These delays must be measured without interference from surgical procedures, including anaesthesia, bias associated with changes in pituitary responsiveness (which affect the efficiency of pulse detection), and chronic side-effects of gonadectomy. We used a GnRH antagonist ['Antarelix': Ac-D-Nal, D-Cpa, D-Pal, Ser, Tyr, D-Hci, Leu, Lys-(iPr), Pro, D-Ala-NH2] to transiently block LH and steroid secretion (in effect, inducing and reversing castration) in mature male sheep, and measured GnRH secretion into cerebrospinal fluid (CSF) in the third cerebral ventricle. The CSF was withdrawn with a peristaltic pump at a rate of 2 ml/h and pooled every 20 min. Jugular plasma was sampled every 20 min and analysed for testosterone and LH pulses. The antagonist (500 microg i.v.) was injected after 6 h of baseline sampling and the study continued for a further 24 h. The pulses of LH and testosterone disappeared shortly after antagonist injection, with delays of 20 +/- 12 min for LH and 80 +/- 29 min for testosterone. This led to an increase in GnRH pulse frequency, starting 300 +/- 54 min after antagonist injection. Secretion of LH and testosterone pulses resumed at 553 +/- 38 and 530 +/- 30 min (after antagonist injection), and GnRH pulse frequency returned to baseline values after 170 +/- 42 min (relative to LH) and 117 +/- 35 min (relative to testosterone). The consistent nature of these responses across the group of animals suggests that this can be used to test the effects of exteroceptive factors on the dynamics of negative feedback.

Effects of nutrition on testicular growth in mature Merino rams actively immunized against GnRH

Nutrition-induced changes in testicular size in Merino rams appear to involve both GnRH-dependent and -independent pathways. This hypothesis was tested by feeding mature Merino rams that had been actively immunized against BSA or GnRH conjugated to BSA a diet that maintained initial body weight or the same diet supplemented daily with 1.5 kg of lupin grain. Blood was sampled every 20 min for 24 h on days-1, 19 and 70 relative to the change in diet. The plasma was used to assess the effects of treatments on changes in LH, FSH and testosterone concentrations. In the group immunized against BSA, FSH increased in lupin-supplemented rams compared with maintenance-fed rams, while LH and testosterone were not affected by diet. In comparison, the concentrations of LH, FSH and testosterone were significantly lower in the group immunized against GnRH than in rams immunized against BSA, but none of these endocrine variables was affected by nutrition. With both immunization treatments, the testes were significantly larger in lupin-supplemented than in maintenance-fed rams. In the group immunized against BSA, this difference was caused by testicular growth in lupin-supplemented rams, whereas in the group immunized against GnRH, lupin supplementation effectively maintained testicular mass, rather than allowed the regression observed in maintenance-fed rams. In conclusion, differences in testicular growth that were induced by dietary treatments in rams immunized against GnRH were not associated with changes in gonadotrophin or testosterone secretion. This supports the hypothesis that part of the effect of nutrition on testicular growth is independent of changes in GnRH secretion. The differences in testicular size observed in control rams were of similar magnitude to those observed in treated rams, but associated with large differences in plasma FSH concentrations, suggesting that this hormone plays an important role in this effect.

Maturation of the Hypothalamic Control of Pulsatile Gonadotropin-Releasing Hormone Secretion at Onset of Puberty: II. Reduced Potency of an Inhibitory Autofeedback*

At the time of onset of puberty in the male rat, between 15 and 25 days of age, we have reported that pulsatile GnRH secretion by hypothalamic explants showed an increased frequency as indicated by the reduction of the mean interpulse interval from 66 to 40 min (P less than 0.05). This study aimed to evaluate whether these changes in GnRH secretion involved a self-regulatory mechanism. A 7.5-min exposure of explants obtained at 50 days to 0.1 microM superagonist D-TRP6-PRO9-N-Et-GnRH (GnRH-A) resulted in a delay of the next GnRH secretory pulse so that the mean interpulse interval increased from 35 to 67 min (P less than 0.001). In addition, after a 7.5-min exposure to GnRH-A, there was a 15-min period with absent or reduced release of GnRH in response to 50 microM veratridine, a depolarizing agent. A similar refractory period of 15 min was observed using explants obtained at 25 and 50 days whereas, at 15 days, the period of refractoriness lasted for 52.5 min. The inhibitory effect of GnRH-A on the subsequent response to veratridine occurred at similar concentrations of GnRH-A at the three studied ages and the inhibition was prevented using an antagonist of GnRH together with GnRH-A. The involvement of GnRH itself in an autofeedback mechanism was evaluated by studying the period of refractoriness separating two GnRH pulses elicited by 7.5-min exposures to veratridine. The initial responsiveness to veratridine was recovered after a refractory period of 52.5, 22.5, and 15 min when studied at 15, 25, and 50 days, respectively. While refractoriness occurred during repeated depolarization with K+ or veratridine, such an effect was not observed using N-methyl-D-aspartate (NMDA). During exposure to GnRH-A, the NMDA-induced release of GnRH was only reduced by 38% whereas veratridine-induced secretion showed a 94% reduction. Thus, exogenous activation of NMDA-sensitive receptors could bypass the inhibitory autofeedback. We conclude that: 1) pulsatile GnRH secretion is controlled by an inhibitory autofeedback involving NMDA sensitive receptors, 2) the increased frequency of pulsatile GnRH secretion at onset of puberty may be related to a reduced sensitivity of the hypothalamic pulse generator to an inhibitory autofeedback.

Bioactive Follicle-Stimulating Hormone Release in Nutritionally Growth-Retarded Ovariectomized Lambs: Regulation by Nutritional Repletion*

The nutritionally growth-retarded ovariectomized lamb provides a model system to investigate the regulation of immunoreactive and bioactive FSH release in the absence of feedback effects of ovarian products. We examined the acute effects of nutritional repletion on FSH release in this model. Five March-born lambs were weaned at 7 weeks of age and placed on a limited diet. All were ovariectomized at 31 weeks. At 37 weeks, they were fed ad libitum for 14 days. Blood samples were collected at 12 min intervals for 4 h on day -1 (restricted) and days 2, 7, and 14 of ad libitum feeding, and serum FSH concentrations were measured by both RIA and a Sertoli cell aromatase in vitro bioassay. Mean concentrations of serum immunoreactive FSH (I-FSH) and the bioactive FSH (B-FSH) increased 2- and 3-fold, respectively, after ad libitum feeding for 14 days. Pulse analyses by two objective computerized programs, DETECT and CLUSTER, revealed the presence of FSH pulses (predominantly B-FSH) during the restricted phase, as well as ad libitum fed phases, at times where I-LH pulses were not detected. In contrast to the increases in I-LH pulse frequency during ad libitum fed states, FSH pulse frequency remained stable whereas pulse amplitude increased. In addition, the overall B-FSH pulse amplitudes were 57 +/- 7% (mean +/- SE of three different pulse analyses) greater than I-FSH pulse amplitudes, implying preferential enrichment of FSH bioactivity. The results suggest that the mechanisms governing both the quantitative and qualitative changes in circulating FSH concentrations are extremely sensitive to changes in level of nutrition. The asynchronous occurrence of FSH pulses in the absence of I-LH pulses suggests that FSH may be more sensitive to small changes in GnRH secretion than I-LH, or that a hypothalamic releasing factor specific for FSH may be released.

Time-related neuroendocrine manifestations of puberty: a combined clinical and experimental approach extracted from the 4th Belgian Endocrine Society lecture.

The neuroendocrine manifestations of puberty converge on changes in GnRH secretion. Their appraisal through the assay of GnRH-like material in 24-hour urine extracts shows an increased excretion of this material in the late prepubertal period. The most striking pubertal changes in GnRH secretion occur on a circadian and ultradian basis. In man, they can be evaluated only indirectly. The circadian variations in LH and FSH secretion characteristic of puberty may be observed in timed fractions of 24-hour urine with some delay when compared to the variations of plasma levels. Studies on the frequency of pulsatile LH secretion and during chronic intermittent administration of GnRH support the existence of an increased frequency of GnRH secretory episodes at puberty. LH response to synthetic GnRH is directly related to the frequency of stimulation by endogenous GnRH pulses and provides a very useful index of neuroendocrine maturation in patients with delayed or precocious puberty. A direct evaluation of pulsatile GnRH secretion is possible using the rat hypothalamus in vitro. In these experimental conditions, the frequency of pulsatile GnRH release increases during very early stages of sexual maturation in the male rat. GnRH itself and beta-endorphin are inhibitory regulators of GnRH secretion in vitro and may participate in the mechanisms restraining the pulse-generating machinery in the hypothalamus before puberty.

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.

Effect of ovariectomy on the hypothalamic content of immunoreactive gonadotropin-releasing hormone in the female mouse as revealed by quantitative immunocytochemistry and radioimmunoassay.

AbstractOur radioimmunoassay data reveal that ovariectomy for either one or two months resulted in a significant decrease in the GnRH content of the mouse basal hypothalamus, while in mice ovariectomized for two weeks the GnRH levels did not differ significantly from controls. Parallel immunocytochemical analyses gave similar results. Tissue sections from specific regions of the median eminence in intact and ovariectomized mice were immunolabeled for GnRH with the peroxidase‐antiperoxidase procedure of Sternberger. Differences in area and average unit density of the reaction product were measured with a Quantiment 720 image analyzer. At two weeks after ovariectomy, a majority of matched sections from intact and ovariectomized animals, taken in each sampled region of the median eminence, showed no visually apparent difference in GnRH immunoreactivity. Quantitative image analysis of these preparations revealed no statistically significant difference in either the area of neural tissue covered by deposit or the average unit density of this material in any given region. In contrast, in mice ovariectomized for either one or two months, immunolabeled GnRH was decreased throughout the cephalo‐caudal extent of the median eminence. In each region, a reduction in reaction deposit was observed over most of the area in which GnRH is localized, especially in the medial aspects of this distribution. Image analysis of these preparations revealed a significant reduction of both specimen area immunolabeled for GnRH and the average unit density of reaction product in each region of the median eminence. While the decrease in density was uniform throughout the median eminence, the greatest reduction in area covered by deposit occurred in the infundibular region.The demonstration of a decrease in basal hypothalamic GnRH in the chronically ovariectomized mouse suggests that GnRH neurosecretion is increased in response to the absence of the ovaries and supports the premise that tonic secretion of this neurohormone, in the mouse, may be subject to regulation by the gonads. At two weeks after ovariectomy, it is possible that changes in GnRH secretion occur without a concomitant alteration in hypothalamic hormone content. In addition, the quantitative immunocytochemical data suggest that GnRH neurons responsive to ovariectomy terminate throughout the rostro‐caudal extent of the mouse median eminence, especially in the medial sites of localization. A proportionally greater number of these endings appear to be present within the infundibular region of the median eminence, relative to the other regions sampled.