Gonadotropin-releasing Hormone Pulse Amplitude Research Articles

12218 Gnrh Pulse Frequency And Irregularity Play A Role In Depression

Abstract Disclosure: J. Choi: None. M. Kim: None. Gonadotropin-releasing hormone (GnRH) has a role in hypothalamic control of depression, but the underlying patterns and relationship with downstream reproductive hormones are still unclear. Here we report that hypothalamic GnRH pulse frequency and irregularity increase before GnRH pulse amplitude slowly decreases during social defeated stress models (SDS). While mice castrated and testosterone inhibited induced depression by conducting SDS, these mice led to anti-depression effects and affected by GnRH signaling, indicating that high-frequency GnRH pulses. Regramming the endogenous GnRH pulses of depressed male mice via an optogenetic approach revealed that increasing GnRH pulses frequency causes depression acceleration, while lowering the frequency of and stabilizing GnRH pulses can slow down depression. In conclusion, GnRH pulses are important for depression in male mice. Presentation: 6/1/2024

Hypothalamic kisspeptin neurons as potential mediators of estradiol negative and positive feedback

Gonadal steroid feedback regulates the brain’s patterned secretion of gonadotropin-releasing hormone (GnRH). Negative feedback, which occurs in males and during the majority of the female cycle, modulates the amplitude and frequency of GnRH pulses. Positive feedback occurs in females when high estradiol induces a surge pattern of GnRH release. These two forms of feedback and their corresponding patterns of GnRH secretion are thought to be mediated by kisspeptin-expressing neurons in two hypothalamic areas: the arcuate nucleus and the anteroventral periventricular area. In this review, we present evidence for this theory and remaining questions to be addressed.

GnRH pulse frequency and irregularity play a role in male aging.

Gonadotropin-releasing hormone (GnRH) has a role in hypothalamic control of aging, but the underlying patterns and relationship with downstream reproductive hormones are still unclear. Here we report that hypothalamic GnRH pulse frequency and irregularity increase before GnRH pulse amplitude slowly decreases during aging. GnRH is inhibited by nuclear factor (NF)-κB, and GnRH pulses were controlled by oscillations in the transcriptional activity of NF-κB. Exposure to testosterone under pro-inflammatory conditions stimulated both NF-κB oscillations and GnRH pulses. While castration of middle-aged mice induced short-term anti-aging effects, preventing elevation of luteinizing hormone (LH) levels after castration led to long-term anti-aging effects and lifespan extension, indicating that high-frequency GnRH pulses and high-magnitude LH levels coordinately mediate aging. Reprogramming the endogenous GnRH pulses of middle-aged male mice via an optogenetic approach revealed that increasing GnRH pulses frequency causes LH excess and aging acceleration, while lowering the frequency of and stabilizing GnRH pulses can slow down aging. In conclusion, GnRH pulses are important for aging in male mice.

Regulation of GnRH pulsatility in ewes.

Early work in ewes provided a wealth of information on the physiological regulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion by internal and external inputs. Identification of the neural systems involved, however, was limited by the lack of information on neural mechanisms underlying generation of GnRH pulses. Over the last decade, considerable evidence supported the hypothesis that a group of neurons in the arcuate nucleus that contain kisspeptin, neurokinin B and dynorphin (KNDy neurons) are responsible for synchronizing secretion of GnRH during each pulse in ewes. In this review, we describe our current understanding of the neural systems mediating the actions of ovarian steroids and three external inputs on GnRH pulsatility in light of the hypothesis that KNDy neurons play a key role in GnRH pulse generation. In breeding season adults, estradiol (E2) and progesterone decrease GnRH pulse amplitude and frequency, respectively, by actions on KNDy neurons, with E2 decreasing kisspeptin and progesterone increasing dynorphin release onto GnRH neurons. In pre-pubertal lambs, E2 inhibits GnRH pulse frequency by decreasing kisspeptin and increasing dynorphin release, actions that wane as the lamb matures to allow increased pulsatile GnRH secretion at puberty. Less is known about mediators of undernutrition and stress, although some evidence implicates kisspeptin and dynorphin, respectively, in the inhibition of GnRH pulse frequency by these factors. During the anoestrus, inhibitory photoperiod acting via melatonin activates A15 dopaminergic neurons that innervate KNDy neurons; E2 increases dopamine release from these neurons to inhibit KNDy neurons and suppress the frequency of kisspeptin and GnRH release.

Insulin receptor signaling in the GnRH neuron plays a role in the abnormal GnRH pulsatility of obese female mice.

Infertility associated with obesity is characterized by abnormal hormone release from reproductive tissues in the hypothalamus, pituitary, and ovary. These tissues maintain insulin sensitivity upon peripheral insulin resistance. Insulin receptor signaling may play a role in the dysregulation of gonadotropin-releasing hormone (GnRH) secretion in obesity, but the interdependence of hormone secretion in the reproductive axis and the multi-hormone and tissue dysfunction in obesity hinders investigations of putative contributing factors to the disrupted GnRH secretion. To determine the role of GnRH insulin receptor signaling in the dysregulation of GnRH secretion in obesity, we created murine models of diet-induced obesity (DIO) with and without intact insulin signaling in the GnRH neuron. Obese control female mice were infertile with higher luteinizing hormone levels and higher GnRH pulse amplitude and total pulsatile secretion compared to lean control mice. In contrast, DIO mice with a GnRH specific knockout of insulin receptor had improved fertility, luteinizing hormone levels approaching lean mice, and GnRH pulse amplitude and total secretion similar to lean mice. Pituitary responsiveness was similar between genotypes. These results suggest that in the obese state, insulin receptor signaling in GnRH neurons increases GnRH pulsatile secretion and consequent LH secretion, contributing to reproductive dysfunction.

The effects of insulin on gonadotropin‐releasing hormone secretion (911.1)

The purpose of this study was to determine the role of insulin signaling in gonadotropin‐releasing hormone (GnRH) pulse generation. The Cre‐lox system was used to generate mice without insulin receptors in GnRH neurons (GnRH Cre+ IR fl/fl; GnRHIRKO). Hypothalami from female GnRHIRKO or littermate controls with intact insulin receptors in GnRH neurons (GnRH Cre‐), in the lean or diet induced obesity (DIO) state were incubated in static culture. Media collected every seven minutes was subjected to radioimmunoassay for GnRH. The pulse pattern in the control DIO state was visually less regular than other states. GnRH pulse amplitude was not different between GnRHIRKO or control mice in the lean or DIO state. Pulse frequency was trended lower in the control DIO state than in lean controls or GnRHIRKO lean or DIO hypothalami. However, this result was not statistically significant. Pulse interval was also longer in the control DIO state compared to the control lean or GnRHIRKO hypothalami. Insulin signaling in the GnRH neuron may play a role in GnRH pulse generation in the DIO but not the lean state.Grant Funding Source: Supported by: NIH K08HD056139 and APS Undergraduate Summer Research Fellowship

Pulsatile Hormonal Signaling to Extracellular Signal-regulated Kinase: EXPLORING SYSTEM SENSITIVITY TO GONADOTROPIN-RELEASING HORMONE PULSE FREQUENCY AND WIDTH

Gonadotropin-releasing hormone (GnRH) is secreted in brief pulses that stimulate synthesis and secretion of pituitary gonadotropin hormones and thereby mediate control of reproduction. It acts via G-protein-coupled receptors to stimulate effectors, including ERK. Information could be encoded in GnRH pulse frequency, width, amplitude, or other features of pulse shape, but the relative importance of these features is unknown. Here we examine this using automated fluorescence microscopy and mathematical modeling, focusing on ERK signaling. The simplest scenario is one in which the system is linear, and response dynamics are relatively fast (compared with the signal dynamics). In this case integrated system output (ERK activation or ERK-driven transcription) will be roughly proportional to integrated input, but we find that this is not the case. Notably, we find that relatively slow response kinetics lead to ERK activity beyond the GnRH pulse, and this reduces sensitivity to pulse width. More generally, we show that the slowing of response kinetics through the signaling cascade creates a system that is robust to pulse width. We, therefore, show how various levels of response kinetics synergize to dictate system sensitivity to different features of pulsatile hormone input. We reveal the mathematical and biochemical basis of a dynamic GnRH signaling system that is robust to changes in pulse amplitude and width but is sensitive to changes in receptor occupancy and frequency, precisely the features that are tightly regulated and exploited to exert physiological control in vivo.

From Menarche to Regular Menstruation

Marked changes in hormone secretion occur from childhood to adulthood. Prior to puberty gonadotropin-releasing hormone (GnRH) secretion is markedly suppressed. At the onset of puberty, the hypothalamic gonadostat is derepressed and the amplitude of GnRH pulses increases. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels increase gradually during puberty stimulating follicle maturation and estrogen production in the ovaries. Only the negative feedback mechanism is powerful before puberty, while the positive feedback mechanism becomes active for the first time in late puberty. As a result, normal cyclicity is usually established at that time. During normal menstrual cycle, steroidal and nonsteroidal hormones mediate the effect of the ovaries on the hypothalamic-pituitary system. Estradiol and progesterone are important regulators of FSH and LH secretion, while inhibins play a role in the control of FSH secretion. Gonadotropin surge attenuating factor (GnSAF) is a nonsteroidal ovarian substance that controls the amplitude of the midcycle LH surge by antagonizing the sensitizing effect of estradiol on the pituitary.

Gonadotropin-Releasing Hormone (GnRH) Receptor Expression and Membrane Signaling in Early Embryonic GnRH Neurons: Role in Pulsatile Neurosecretion

The characteristic pulsatile secretion of GnRH from hypothalamic neurons is dependent on an autocrine interaction between GnRH and its receptors expressed in GnRH-producing neurons. The ontogeny and function of this autoregulatory process were investigated in studies on the properties of GnRH neurons derived from the olfactory placode of the fetal rat. An analysis of immunocytochemically identified, laser-captured fetal rat hypothalamic GnRH neurons, and olfactory placode-derived GnRH neurons identified by differential interference contrast microscopy, demonstrated coexpression of mRNAs encoding GnRH and its type I receptor. Both placode-derived and immortalized GnRH neurons (GT1-7 cells) exhibited spontaneous electrical activity that was stimulated by GnRH agonist treatment. This evoked response, as well as basal neuronal firing, was abolished by treatment with a GnRH antagonist. GnRH stimulation elicited biphasic intracellular calcium ([Ca2+]i) responses, and both basal and GnRH-stimulated [Ca2+]i levels were reduced by antagonist treatment. Perifused cultures released GnRH in a pulsatile manner that was highly dependent on extracellular Ca2+. The amplitude of GnRH pulses was increased by GnRH agonist stimulation and was diminished during GnRH antagonist treatment. These findings demonstrate that expression of GnRH receptor, GnRH-dependent activation of Ca2+ signaling, and autocrine regulation of GnRH release are characteristics of early fetal GnRH neurons and could provide a mechanism for gene expression and regulated GnRH secretion during embryonic migration.

Circadian Gene Expression Regulates Pulsatile Gonadotropin-Releasing Hormone (GnRH) Secretory Patterns in the Hypothalamic GnRH-Secreting GT1-7 Cell Line

Although it has long been established that episodic secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus is required for normal gonadotropin release, the molecular and cellular mechanisms underlying the synchronous release of GnRH are primarily unknown. We used the GT1-7 mouse hypothalamic cell line as a model for GnRH secretion, because these cells release GnRH in a pulsatile pattern similar to that observed in vivo. To explore possible molecular mechanisms governing secretory timing, we investigated the role of the molecular circadian clock in regulation of GnRH secretion. GT1-7 cells express many known core circadian clock genes, and we demonstrate that oscillations of these components can be induced by stimuli such as serum and the adenylyl cyclase activator forskolin, similar to effects observed in fibroblasts. Strikingly, perturbation of circadian clock function in GT1-7 cells by transient expression of the dominant-negative Clock-Delta19 gene disrupts normal ultradian patterns of GnRH secretion, significantly decreasing mean pulse frequency. Additionally, overexpression of the negative limb clock gene mCry1 in GT1-7 cells substantially increases GnRH pulse amplitude without a commensurate change in pulse frequency, demonstrating that an endogenous biological clock is coupled to the mechanism of neurosecretion in these cells and can regulate multiple secretory parameters. Finally, mice harboring a somatic mutation in the Clock gene are subfertile and exhibit a substantial increase in estrous cycle duration as revealed by examination of vaginal cytology. This effect persists in normal light/dark (LD) cycles, suggesting that a suprachiasmatic nucleus-independent endogenous clock in GnRH neurons is required for eliciting normal pulsatile patterns of GnRH secretion.

Role of Gamma-Aminobutyric Acid Neurons in the Release of Gonadotropin-Releasing Hormone in Cultured Rat Embryonic Olfactory Placodes

We recently established a primary cell culture system of gonadotropin-releasing hormone (GnRH) neurons originating from olfactory placodes of rat embryos at E13.5 and showed that cultured olfactory placodes released GnRH into the medium in a pulsatile fashion with an interpulse interval of about 30 min. Since the reported presence of γ-aminobutyric acid (GABA) neurons in the culture of rat olfactory placode raises questions as to the role played by these GABA neurons in the GnRH pulse generation, we immunostained GnRH neurons and GABA neurons in this culture system to examine the interrelationship between both types of neurons, and determined the effects of GABA and the GABA_A receptor antagonist, bicuculline, on GnRH release. The immunohistochemical study showed that GnRH neurons received fiber terminals from GABA neurons. GnRH neurons in culture released GnRH into the medium at intervals of 30–40 min, confirming our previous study. Treatment with 20 µM GABA prolonged the interpulse interval and decreased the amplitude of GnRH pulses. Bicuculline administered at 20 µM did not affect either parameter, but 50 µM bicuculline elevated the mean GnRH level, although it did not affect either the interpulse interval or the amplitude of GnRH pulses. In addition, 50 µM bicuculline increased the mean trough levels of GnRH pulses, although 20 µM bicuculline did not. In light of the in vivo studies performed previously, we suggest that the GnRH pulse generator, which probably consists of a small population of GnRH neurons in the culture, does not involve GABA neurons to generate the pulsatile GnRH release, although it may be responsive to the inhibitory transmitter GABA. We also found that there may be another population of GnRH neurons in the culture whose activity is strongly suppressed by the tonic inhibition of GABA neurons. Although it is speculative, these latter GnRH neurons may be responsible for the surge of GnRH release.

Electrical Activity of the Pulse Generator of Gonadotropin-Releasing Hormone in 26-Month-Old Female Rats

To know whether age-related changes occur in the activity of the pulse generator of gonadotropin-releasing hormone (GnRH), old (26 months) and young (3 months) female rats were examined by recording multiunit activity (MUA) in the median eminence region of the hypothalamus, concurrently with blood samplings through an intra-atrial cannula at 6-min intervals to determine serum luteinizing hormone (LH) concentrations. We have regarded the MUA showing intermittent increases (volleys) at 20–30 min intervals, followed by LH pulses, as the electrical activity of the GnRH pulse generator. We were successful in recording MUA in 18 (26%) of 69 old ovariectomized rats and in 8 (32%) of 25 young ovariectomized rats. The overall mean (±SE) of the interval between MUA volleys in old ovariectomized rats was 35.1 ± 2.0 min (n = 18), which was significantly longer than that of 22.5 ± 1.5 min (n = 8) in young ovariectomized rats. The mean interval between LH pulses in old ovariectomized rats was 32.2 ± 3.6 (n = 10), also being significantly longer than that of 23.3 ± 1.0 (n = 8) in young ovariectomized rats. Further, the LH pulse amplitude in old rats (0.95 ± 0.07 ng/ml) was significantly smaller than in young rats (3.40 ± 0.36 ng/ml). The present study also confirmed that the increase in serum LH after intravenous injection of 50 ng GnRH was much smaller in old ovariectomized rats. These results show that the electrical activity of the GnRH pulse generator is certainly reduced with age. Taken together with findings suggesting an age-dependent decrease in stimulated transmitter release, attenuation in both frequency and amplitude of GnRH pulses as well as in pituitary responsiveness to GnRH pulses may account for the decreased pulsatile LH secretion observed in aging rats.

Leptin effects on pulsatile gonadotropin releasing hormone secretion from the adult rat hypothalamus and interaction with cocaine and amphetamine regulated transcript peptide and neuropeptide Y

Leptin may act as a negative feedback signal to the hypothalamic control of appetite through suppression of neuropeptide Y (NPY) secretion and stimulation of cocaine and amphetamine regulated transcript (CART). We aimed at studying the effects of leptin, CART and NPY on the hypothalamic control of the pituitary–gonadal system. Pulsatile gonadotropin-releasing hormone (GnRH) secretion was studied in vitro using retrochiasmatic hypothalamic explants from adult rats. In the female, GnRH pulse amplitude was significantly increased by leptin (10 −7 M) and CART (10 −6 M) irrespective of the estrus cycle phase while no such effects were seen in the male. The GnRH interpulse interval was not affected in both sexes. Passive immunoneutralization against CART caused a reduction in GnRH pulse amplitude in the female. A slight but significant increase in GnRH pulse amplitude was caused by NPY (10 −7 M) in the female. However, GnRH pulse amplitude was not affected by a Y5-receptor antagonist (10 −6 M) while the interpulse interval was significantly increased as shown previously in the male. The increase in GnRH pulse amplitude caused by leptin was totally prevented by coincubation with an anti-CART antiserum whereas it was not affected by coincubation with the NPY Y5-receptor antagonist (10 −7 M). In conclusion, leptin and NPY show separate permissive effects on GnRH secretion in the adult rat hypothalamus. In both sexes, NPY is prominently involved in the control of the frequency of pulsatile GnRH secretion through the Y5 receptor subtype. Leptin causes a female-specific facilitatory effect on GnRH pulse amplitude which is mediated by CART and which occurs irrespective of the estrus cycle phase.

Concentration-dependent effects of muscimol to enhance pulsatile GnRH release from GT1–7 neurons in vitro

Immortalized GT1–7 neurons were used to characterize the effect of muscimol, a GABA A receptor agonist, to enhance pulsatile gonadotropin-releasing hormone (GnRH) release. GT1–7 neurons were grown on Cytodex-3 beads and placed in special superfusion microchambers. The cells were superfused at a rate of 6.2 ml·h −1 with Media 199 (pH 7.35) using a commercially available perfusion system. After a pre-muscimol period of 120 min, the cells were exposed for 5 min to 0.35, 1, 5 or 10 μM muscimol or 5 μM muscimol+20 μM of the GABA A receptor antagonist, bicuculline. Following removal of the muscimol (and bicuculline, in the case of the latter experiment), the superfusion was continued for another 115 min. Sample fractions were collected at 5 min intervals throughout the perfusion. Basal GnRH release from the GT1–7 neurons was pulsatile with an average interpulse interval of 45.4±0.5 min and an average pulse amplitude of 191.5±22.6 pg·min·ml −1. Our results also demonstrated that the GABA A receptor agonist, muscimol, enhances pulsatile GnRH release from GT1–7 neurons in culture. The response to muscimol was saturable and concentration-dependent with an EC 50 of 0.47 μM. The effects of 5 μM muscimol to increase GnRH pulsatility were blocked by co-exposure to the GABA A receptor antagonist, bicuculline. The average GnRH interpulse intervals were 41.7±1.8 min, 32.5±2.9 min, 30.6±0.7 min and 25.5±0.4 min in the period following exposure to 0.35, 1, 5 and 10 μM of muscimol, respectively (post-muscimol period). GnRH pulse amplitude (mean-area for each pulse) was increased during exposure to muscimol but not during the pre- or post-muscimol periods. The GABA A receptor antagonist, bicuculline, itself had no effect on pulsatile GnRH release. These results are consistent with previously published reports suggesting that activation of the GABA A receptor stimulates hypothalamic GnRH release in embryonic and neonatal animals.

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

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

Orchidectomy and NMDA increase GnRH secretion as measured by push-pull perfusion of rat anterior pituitary.

Using push-pull perfusion to measure concentrations of gonadotropin-releasing hormone (GnRH) in the extracellular fluid of the anterior pituitary gland of the male rat, we have measured GnRH release at specific times before and after castration and in response to acute administration of N-methyl-D-aspartate (NMDA). After castration (7 days), mean GnRH levels were substantially increased (4.3-fold) compared with intact controls (0.94 +/- 0.16 vs. 0.22 +/- 0.08 pg/10 min, respectively, P < 0.05) due to an increase in both the frequency and amplitude of GnRH pulses. Testosterone partially reduced GnRH release (0.62 +/- 0.10 pg/10 min). NMDA induced a rapid increase in plasma luteinizing hormone (LH) in both intact and castrated rats and increased GnRH concentrations in the perfusion samples (P < 0.05). There was no change in LH release induced by two doses of injected GnRH (5 and 25 ng/100 g body wt) 2 days after castration, but by 6 days after castration the response to both doses was significantly increased. These results demonstrate that GnRH release in the male rat is acutely increased by NMDA and is chronically increased after orchidectomy. Increased pituitary sensitivity to GnRH also contributes to the hypersecretion of LH after castration, particularly at longer times after removal of testosterone negative feedback.

N-methyl-D-aspartate (NMDA) receptors mediate the release of gonadotropin-releasing hormone (GnRH) by NMDA in a hypothalamic GnRH neuronal cell line (GT1-1).

Previous studies demonstrated that an excitatory amino acid analog, N-methyl-D-aspartate (NMDA), stimulates GnRH secretion in the rat, prepubertal primate, and ovine fetus at the hypothalamic level. It is not known if this stimulatory effect of NMDA is mediated directly on the GnRH neurosecretory neuron. A hypothalamic GnRH neuronal cell line (GT1-1) provided a useful model system to study the effect of NMDA on GnRH release by both superfusion and static incubation techniques. Studies with GT1-1 cells indicate that GnRH neurons exhibit spontaneous autorhythmicity and function intrinsically as a neuronal oscillator for the synchronous discharge of GnRH pulses. In static incubation studies, 10(-4) and 10(-3) M NMDA increased GnRH release, whereas 10(-6), 10(-5), and 10(-2) M NMDA had no effect. A competitive NMDA receptor antagonist, AP-5 (10(-4)-10(-2) M), and a noncompetitive NMDA receptor antagonist, MK-801 (10(-12)-10(-5) M), inhibited the action of NMDA. Superfusion of GT1-1 cells after a 90-min control period followed by either continuous NMDA or intermittent NMDA (10(-4) and 10(-3) M) for 90 min increased GnRH pulse amplitude by 100-400%, but had no effect on the interpulse interval (17 min by Cluster); 10(-6), 10(-5), and 10(-2) M NMDA had no effect on either pulse amplitude or interpulse interval. MK-801 (10(-5) M) attenuated the stimulatory effect of NMDA on GnRH pulse amplitude. Incubation in glycine-free and high magnesium medium abolished the action of NMDA on GnRH release. Hybridization analysis of GT1-1 mRNA with an NMDA R1 receptor cDNA showed that this pure neuronal cell line expressed NMDA receptor transcripts observed as a 4.2-kilobase band. The results demonstrate that NMDA stimulates GnRH neurons directly to secrete GnRH through their NMDA receptors by increasing pulse amplitude without affecting pulse frequency.

Failure of gonadotropin-releasing hormone (GnRH) pulses to increase luteinizing hormone beta messenger ribonucleic acid in GnRH-deficient female rats.

Gonadotropin subunit gene transcription and messenger RNA (mRNA) levels are differentially regulated by GnRH pulse frequency and amplitude in the male rat. The rapid changes of subunit mRNA levels and LH and FSH secretion during the estrous cycle, particularly the rapid rise in LH-beta subunit mRNA on proestrus afternoon, suggest that physiological changes in the pattern of GnRH action may also be important in female rats. However, in the absence of a GnRH-deficient female model the role of varying GnRH stimulation remains to be determined. We have characterized a GnRH-deficient model by administering the alpha-adrenergic antagonist phenoxybenzamine (PBZ) to ovariectomized (OVX) rats. Initial experiments showed that PBZ given 24 h earlier abolished the afternoon LH surge in OVX estradiol (E2) replaced rats whereas LH responses to exogenous GnRH were preserved. A PBZ regimen of 15 mg/kg ip at OVX followed by 10 mg/kg at 24 h and 5 mg/kg at 48 h prevented the increase in alpha, LH-beta, and FSH-beta mRNAs and LH and FSH secretion for 72 h post-OVX. LH and FSH responses to GnRH pulses were preserved suggesting that PBZ blocked the post-OVX increase in hypothalamic GnRH secretion. The suppressive effect of PBZ appeared to be specific to the hypothalamic-pituitary-ovarian axis as plasma PRL, TSH, and corticosterone were not decreased compared to controls. We have used this GnRH-deficient OVX female model to investigate the effects of exogenous GnRH pulses on subunit mRNA expression. GnRH pulses (5-250 ng/30 min for 12-24 h) were administered via an intraatrial catheter beginning 24 h after OVX and the first PBZ injection (OVX+PBZ+saline pulses to controls). Expression of alpha and FSH-beta mRNAs and LH and FSH secretion were increased by GnRH pulse doses of 5-25 ng to values similar to or greater than those in OVX controls though the higher doses of GnRH/pulse did not increase FSH-beta mRNA or plasma FSH. However, LH-beta mRNA levels were not increased by GnRH pulses. GnRH pulses were also given to rats replaced with proestrus concentrations of estradiol alone or in combination with progesterone (P). Again, no demonstrable increases in LH-beta mRNA expression were observed. alpha-mRNA concentrations were further increased in the presence of E2 alone, and P in combination with E2, produced an augmented response of FSH-beta subunit mRNA. These data suggest that ovarian steroid hormones act directly on the gonadotrope to augment alpha and FSH-beta mRNA responses to GnRH.(ABSTRACT TRUNCATED AT 400 WORDS)

Failure of gonadotropin-releasing hormone (GnRH) pulses to increase luteinizing hormone beta messenger ribonucleic acid in GnRH-deficient female rats

Gonadotropin subunit gene transcription and messenger RNA (mRNA) levels are differentially regulated by GnRH pulse frequency and amplitude in the male rat. The rapid changes of subunit mRNA levels and LH and FSH secretion during the estrous cycle, particularly the rapid rise in LH-beta subunit mRNA on proestrus afternoon, suggest that physiological changes in the pattern of GnRH action may also be important in female rats. However, in the absence of a GnRH-deficient female model the role of varying GnRH stimulation remains to be determined. We have characterized a GnRH-deficient model by administering the alpha-adrenergic antagonist phenoxybenzamine (PBZ) to ovariectomized (OVX) rats. Initial experiments showed that PBZ given 24 h earlier abolished the afternoon LH surge in OVX estradiol (E2) replaced rats whereas LH responses to exogenous GnRH were preserved. A PBZ regimen of 15 mg/kg ip at OVX followed by 10 mg/kg at 24 h and 5 mg/kg at 48 h prevented the increase in alpha, LH-beta, and FSH-beta mRNAs and LH and FSH secretion for 72 h post-OVX. LH and FSH responses to GnRH pulses were preserved suggesting that PBZ blocked the post-OVX increase in hypothalamic GnRH secretion. The suppressive effect of PBZ appeared to be specific to the hypothalamic-pituitary-ovarian axis as plasma PRL, TSH, and corticosterone were not decreased compared to controls. We have used this GnRH-deficient OVX female model to investigate the effects of exogenous GnRH pulses on subunit mRNA expression. GnRH pulses (5-250 ng/30 min for 12-24 h) were administered via an intraatrial catheter beginning 24 h after OVX and the first PBZ injection (OVX+PBZ+saline pulses to controls). Expression of alpha and FSH-beta mRNAs and LH and FSH secretion were increased by GnRH pulse doses of 5-25 ng to values similar to or greater than those in OVX controls though the higher doses of GnRH/pulse did not increase FSH-beta mRNA or plasma FSH. However, LH-beta mRNA levels were not increased by GnRH pulses. GnRH pulses were also given to rats replaced with proestrus concentrations of estradiol alone or in combination with progesterone (P). Again, no demonstrable increases in LH-beta mRNA expression were observed. alpha-mRNA concentrations were further increased in the presence of E2 alone, and P in combination with E2, produced an augmented response of FSH-beta subunit mRNA. These data suggest that ovarian steroid hormones act directly on the gonadotrope to augment alpha and FSH-beta mRNA responses to GnRH.

Regulation of gonadotropin subunit messenger ribonucleic acid expression by gonadotropin-releasing hormone pulse amplitude in vitro

Regulation of gonadotropin subunit messenger ribonucleic acid expression by gonadotropin-releasing hormone pulse amplitude in vitro