Human Gonadotropin-releasing Hormone Research Articles

Total solid-phase synthesis and prolactin-inhibiting activity of the gonadotropin-releasing hormone precursor protein and the gonadotropin-releasing hormone associated peptide.

The human gonadotropin-releasing hormone precursor protein, pHGnRH (Met-23-Ile69) (preproGnRH), and three of its fragment peptides, pHGnRH (Asp14-Ile69) (gonadotropin-releasing hormone associated peptide--GAP), pHGnRH (Phe38-Ile69), and pHGnRH (Ser47-Ile69), were assembled in a stepwise solid-phase cosynthesis employing Boc/Bzl tactics and an optimized acylation schedule which included recoupling steps with hexafluoro-2-propanol to help overcome the aggregation of the pendant peptide chains of the peptidoresin during difficult couplings. Reversed-phase high-performance liquid chromatography (HPLC) purification yielded products which were characterized by analytical reversed-phase HPLC, ion-exchange chromatography, capillary zone electrophoresis, SDS-polyacrylamide gel electrophoresis, and ion-spray mass spectrometry to reveal a high degree of homogeneity. Biological characterization demonstrated that only GAP stimulated luteinizing hormone and follicle-stimulating hormone release from primary cultures of rat anterior pituitary cells, while GAP, pHGnRH (Phe38-Ile69), and preproGnRH all inhibited prolactin release, with the latter being the most potent at concentrations comparable to bromocryptine. However, only GAP and pHGnRH (Phe38-Ile69) were able to displace a labeled gonadotropin-releasing hormone agonist from binding to rat pituitary membrane preparations. This first demonstration of significant biological activity with a precursor protein also suggests that the gonadotropin-releasing and prolactin release-inhibiting functions of GAP are not mediated through the same pituitary receptors.

Evidence for direct estrogen regulation of the human gonadotropin-releasing hormone gene.

This study is an attempt to determine whether estrogen could directly regulate human gonadotropin-releasing hormone (GnRH) gene expression. Human GnRH expression vectors were constructed by fusing various 5' flanking regions of the human GnRH gene upstream of the luciferase reporter gene (LUC) or the thymidine kinase promoter linked to the chloramphenicol acetyltransferase reporter gene (CAT). These constructs were transiently transfected into a human choriocarcinoma cell line (JEG-3) and LUC or CAT activity was measured after either no treatment or treatment with various concentrations of estradiol. A stimulatory estrogen response element (ERE) was localized to a 32-bp region between -547 and -516 bp. To determine whether estrogen receptor bound to this region of the gene, we performed DNase I footprinting using purified calf uterine estrogen receptor. DNase I footprinting demonstrates a strong footprint between -567 and -514 bp of the human GnRH gene. In addition, an avidin-biotin complex DNA-binding assay demonstrated that a biotinylated DNA fragment containing -541 to -517 bp of the human GnRH gene bound 35S-labeled estrogen receptor as well as a biotinylated DNA fragment containing the xenopus vitellogenin ERE. On the other hand, the negative control biotinylated DNA fragment derived from adenovirus 5 bound insignificant amounts of 35S-labeled estrogen receptor. Both the GnRH ERE and vitellogenin ERE bound 35S-labeled estrogen receptor with high affinity (approximately 1 nM). These data indicate that the human GnRH gene contains an ERE sufficient to mediate a stimulatory response to estrogen in heterologous cells. Based upon these data we hypothesize that the human GnRH gene might also be directly regulated by estrogen in the hypothalamus, and that this regulation may explain the GnRH hypersecretion observed at the time of the preovulatory luteinizing hormone (LH) surge.

Migratory arrest of gonadotropin-releasing hormone neurons in transgenic mice.

Gonadotropin-releasing hormone (GnRH) is important in reproduction, although the mechanism of central hypogonadism in humans remains unclear. Because the GnRH neuron originates from the olfactory placode and migrates to the hypothalamus during development, central hypogonadism in humans could be caused by failure in normal migration of GnRH neurons to the hypothalamus. We report that in transgenic mice expression of the simian virus 40 T antigen, driven by the promoter of human GnRH gene, resulted in central hypogonadism due to an arrest in neuronal migration during development and tumor formation along the migratory pathway. This system appears to be an important animal model of hypogonadotropic hypogonadism in humans. Additionally, olfactory bulb tumors from these animals were dispersed, and a GnRH-secreting neuronal cell line (GN cell line) was established.

Selective FSH-releasing activity of [D-Trp 9]GAP 1–13: Comparison with gonadotropin-releasing abilities of analogs of GAP and natural LHRHs

We had previously shown that fragments of human gonadotropin-releasing hormone associated peptide (GAP) stimulated FSH and LH release in vivo. In particular, GAP 1−13 had a preferential FSH-releasing activity. To decrease enzymatic degradation, analogs of GAP 1–13 with D-amino acid substitutions were synthesized. The activities were tested in ovariectomized, estrogen-progesterone primed (OEP) rats and compared with those of GAP 1−13, mammalian (m), chicken II (eII), and lamprey (1) LHRH. The peptides were injected (IV) into conscious, OEP rats and blood samples were obtained via the jugular catheter. [D-Trp 9]GAP 1–13 selectively stimulated FSH release at a dose of 1 μg. Multiple injections of this analog (10 μg every 30 min for 5 injections) induced a marked elevation of plasma FSH values which peaked ( p < 0.001) after the third injection. By contrast, [D-Trp 9]GAP 1–13 had no effect on LH and prolactin (PRL) release after either single or multiple injections. These doses of [DAla 4] GAP 1–13 had no effect on the release of FSH, LH or PRL. Both human GAP 1–13 and its [D-Trp 9] analog exerted a selective FSH-releasing effect at a dose of 10 μg, however, the [D-Trp 9] analog was more potent than GAP 1–13 on FSH release. The potency of [D-Trp 9]GAP 1–13 in releasing FSH was approximately 1 100th that of mLHRH. Chicken II LHRH had slightly selective FSH-releasing activity with a potency 1 10th that of mLHRH. Lamprey LHRH had a preferential LH-releasing activity and a potency 1000 times less than mLHRH. In conclusion, [D-Trp 9]GAP 1–13 is a selective FSH-releasing peptide of potential clinical value.

Utility of Freeα-Subunit as an Alternative Neuroendocrine Marker of Gonadotropin-Releasing Hormone (GnRH) Stimulation of the Gonadotroph in the Human: Evidence from Normal and GnRH-Deficient Men*

To examine the hypothesis that the secretion of free alpha-subunit (FAS) can serve as an alternative to LH as a neuroendocrine marker of gonadotroph stimulation by GnRH in euthyroid humans, we have investigated the relationship of pulsatile FAS secretion in euthyroid GnRH-deficient men (n = 10) before and after exogenous GnRH stimulation and in normal men under the influence of endogenous GnRH secretion (n = 18). Before GnRH exposure, the GnRH-deficient men showed a complete absence of both LH and FAS pulses. During the initial 7 days of GnRH exposure, all GnRH-deficient men exhibited pulsatile release of FAS by the third day, whereas the appearance of pulsatile release of LH and FSH was more variable. Long term administration of GnRH led to pulses of LH and FAS that were 100% concordant with a demonstrable dose-response relationship between GnRH and FAS, which was quantitatively similar to but more exuberant than that for LH. All doses of GnRH that produced LH pulses within the normal adult range yielded supraphysiological FAS pulses. Analysis of distribution histograms of interpulse intervals and pulse amplitudes of LH and FAS in both normal and GnRH-deficient subjects demonstrated no significant difference between these glycoproteins in interpulse intervals in either the normal or GnRH-deficient groups or in the pulse amplitudes in the GnRH-deficient subjects. There was, however, a significant difference (P less than 0.01) between the distribution histogram of LH and FAS pulse amplitudes in normal men. We conclude that the pulsatile secretion of FAS in euthyroid men 1) is determined by GnRH secretion, 2) is the initial glycoprotein to be secreted in a pulsatile fashion from the gonadotroph during early GnRH exposure in GnRH-deficient men, 3) demonstrates a dose-response relationship to exogenous GnRH which is more robust than that of LH in GnRH-deficient men receiving GnRH, and 4) can, therefore, serve as a complementary and powerful tool with LH for the study of GnRH neurosecretory dynamics.

Isolation and Characterization of the Human Gonadotropin-Releasing Hormone Gene in the Hypothalamus and Placenta

The GnRH gene has been cloned in several species, but the location of the promoter and the exact start of transcription have not previously been determined. To characterize the low abundance human GnRH mRNA in the hypothalamus and placenta, we have employed the polymerase chain reaction. The hypothalamus was found to have a 61-base pair first exon, and its transcriptional start site was determined. The human hypothalamic GnRH cDNAs isolated thus far have all contained a short 5' untranslated region which would correspond to this start site. However, all human placental GnRH cDNAs reported to date have a long 5' untranslated region, which extends more than 140-base pairs 5' to this start site in the hypothalamus, suggesting the utilization of an alternative promoter in the placenta. In addition, the human GnRH gene undergoes differential splicing in these tissues. The first intron is removed from the hypothalamic, but retained in the placental, GnRH mRNA. Thus, the placenta has a very long first exon, while the hypothalamus has a comparatively short first exon, followed by a long first intron. This characterization of the human GnRH gene will now allow hormonal regulatory studies to be performed using gene transfer techniques.

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.

The complete nucleotide sequence of the human gonadotropin-releasing hormone gene

The complete nucleotide sequence of the human gonadotropin-releasing hormone gene

Immunohistochemical localization of gonadotropin‐releasing‐hormone‐associated peptide in the brain of the frog

Gonadotropin-releasing-hormone (GnRH)-associated peptide (GnRH-AP) is a 56 amino acid neuropeptide derived from the GnRH prohormone. GnRH-AP corresponds to the C-terminal fragment flanking the GnRH peptide. Using an antiserum raised against human GnRH-AP [1-56], or against human GnRH, we have investigated the neuronal systems containing either peptide in the central nervous system of the frog Rana ridibunda by immunohistological techniques. A main group of GnRH-AP-containing perikarya was found in a dorsoventral orientation of the supra anterior preoptic area (SAPA) just in front of the preoptic recess. Fibers originating from these perikarya projected rostrally toward the medial septal nucleus and the diagonal band of Broca. A network of GnRH-AP-immunoreactive (ir) fibers runs caudally from the SAPA toward the ventral hypothalamus. A high density of GnRH-AP-ir terminals was found in the median eminence. A few positive fibers were detected in the neural lobe of the pituitary, particularly in the region bordering the pars intermedia. Labelling of consecutive sections by either GnRH-AP or GnRH antibodies showed that GnRH and GnRH-AP-like irs were contained in the same cells of the SAPA. The double-staining technique with electrophoretic elution confirmed the colocalization of GnRH and GnRH-AP within the same neurons. Such a coexistence indicates that frog GnRH originates from a high molecular weight precursor which is closely related to rat pro-GnRH. The relative preservation of the C-terminal sequence of the pro-GnRH during evolution suggests that GnRH-AP may possess intrinsic biological activity. The high density of GnRH-AP-containing neurons projecting through the external zone of the median eminence would support the concept that GnRH-AP is involved in the modulation of pituitary hormone secretion.

Assessment of the effects of a synthetic gonadotropin-releasing hormone associated peptide on hormone release from the in situ and ectopic pituitaries in adult male rats

The in vivo effects of synthetic human gonadotropin-releasing hormone associated peptide (GAP) were evaluated in adult male rats. In normal rats, intracerebroventricular (III ventricle) injection of 5 ng GAP significantly increased plasma LH levels after 60 minutes. Intracerebroventricular administration of 5, 25 or 125 ng of GAP elevated circulating LH levels also at 120 minutes of injection but did not alter plasma FSH, prolactin or testosterone concentrations. In hypophysectomized-pituitary-grafted rats injection of 125 ng GAP directly into the ectopic pituitary induced no changes in peripheral hormone levels. However, intrapituitary graft injection of 25 ng of GnRH significantly elevated circulating levels of LH and testosterone. These results indicate that the ectopic pituitary graft can respond to acute exogenous GnRH stimulation and that the commercially available synthetic GAP fails to inhibit prolactin release in adult male rats.

Polygenic expression of gonadotropin-releasing hormone (GnRH) in human?

A non-mammalian lamprey-like gonadotropin-releasing hormone (lGnRH) has been detected in human hypothalami using a combination of immunocytochemistry, high performance liquid chromatography and radioimmunoassay. The hypothalamic distribution of immunopositive lGnRH neurons is similar to that observed for those containing the mammalian gonadotropin-releasing hormone (mGnRH), indicating a possible role for this newly identified peptide in the regulation of pituitary function. Our data suggest the existence of a separate gene for lamprey-like GnRH in humans. Confirmation of the exact nature and role of this newly detected form of GnRH will require future isolation and sequence analysis. The possibility that polygenic expression of a given peptide may be a common phenomenon even in higher mammals is discussed.

Gonadotropin-Releasing Hormone Associated Peptide (GAP) and Putative Processed GAP Peptides Do Not Release Luteinizing Hormone or Follicle-Stimulating Hormone or Inhibit Prolactin Secretion in the Sheep

A series of experiments was performed to monitor plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin responses to human gonadotropin-releasing hormone (GnRH) associated peptide (GAP) and related peptides. Ovariectomized hypothalamo-pituitary disconnected (HPD) ewes were challenged with injections (1-10 micrograms i.v.) of GAP, or given, with and without estradiol, hourly 500- or 1,000-ng pulses of GAP for 5-7 days. In all cases GAP failed to cause the release of LH or FSH from the pituitary gland or to alter mean plasma prolactin concentrations. When the same HPD ewes were given hourly or 2-hourly pulses of 250 ng GnRH, LH responded in a a pulsatile manner, and FSH secretion was maintained, thus confirming the functional integrity of the pituitary gland after HPD. Fragments of the GAP molecule (pro-GnRH 14-36, 28-36, 38-49, and 51-66) and GAP dimer did not stimulate LH or FSH or inhibit prolactin release in HPD ewes. GAP and GAP dimer did not affect pituitary responsiveness to GnRH administration. GAP also failed to inhibit the thyrotropin-releasing hormone-induced rise in prolactin. Finally, GAP injections (100 micrograms i.v.) given to lactating ewes did not cause any change in plasma prolactin concentrations. These data show that human GAP, GAP dimer, or putative processed GAP peptides do not act on the sheep pituitary gland in a variety of physiological states to regulate gonadotropin or prolactin secretion.

LH-releasing activity and receptor binding of pHGnRH 14–26 analogues

The human gonadotropin-releasing hormone (GnRH) precursor consists of the GnRH sequence followed by a cleavage and amidation site and a 56-amino acid carboxyl-terminal extension (pHGnRH — precursor human GnRH) which has been shown to stimulate gonadotropin release. This activity has been localized to a decapeptide sequence (corresponding to pHGnRH 17–26) in its amino-terminal region using human pituitary cell cultures. To further characterize the structural features required for gonadotropin release, two analogues, [D-Ala 17]pHGnRH 14–26 and [D-Trp 22]pHGnRH 14–26, with D-amino acid substitutions inside and peripheral to this decapeptide sequence were chemically synthesized. pHGnRH 14–26 and the D-Ala 17 analogue were inactive and GnRH, pHGnRH 14–36 and the D-Trp 22 analogue stimulated luteinizing hormone release from cultured rat pituitary cells in a calcium-dependent, dose-responsive manner. Experiments and receptor binding studies with the active pHGnRH peptides in conjunction with GnRH or a GnRH antagonist suggest that the active pHGnRH peptides act through the GnRH receptor.

The delineation of a decapeptide gonadotropin-releasing sequence in the carboxyl-terminal extension of the human gonadotropin-releasing hormone precursor.

The human gonadotropin-releasing hormone (GnRH) precursor consists of the GnRH sequence followed by a 59-amino acid carboxyl-terminal extension. A 56-amino acid peptide within this extension has been shown to stimulate gonadotropin release, and this activity has been localized to its amino-terminal region. A series of seven overlapping peptide fragments corresponding to the first 24 amino acids of the carboxyl-extension of the GnRH precursor were synthesized and tested for their ability to stimulate luteinizing hormone and follicle-stimulating hormone release from cultured human anterior pituitary cells. All active peptide fragments were found to incorporate the decapeptide sequence Asn-Leu-Ile-Asp-Ser-Phe-Gln-Glu-Ile-Val which is regarded as a minimal structural requirement delineated for gonadotropin-releasing activity. A further flanking sequence extending this active region from its carboxyl terminus was found to enhance gonadotropin-releasing activity although the flanking sequence itself was inactive. The gonadotropin release stimulated by the active peptides wa shown to be a dose- dependent, specific, and calcium-dependent phenomenon which occurred independently of the GnRH receptor on the pituitary gonadotrophs as a GnRH antagonist did not inhibit activity.

Stimulation of Gonadotropin Release by a Non-GnRH Peptide Sequence of the GnRH Precursor

The human gonadotropin-releasing hormone (GnRH) precursor comprises the GnRH sequence followed by an extension of 59 amino acids. Basic amino acid residues in the carboxyl terminal extension may represent sites of processing to biologically active peptides. A synthetic peptide comprising the first 13 amino acids (H X Asp-Ala-Glu-Asn-Leu-Ile-Asp-Ser-Phe-Gln-Glu-Ile-Val X OH) of the 59-amino acid peptide was found to stimulate the release of gonadotropic hormones from human and baboon anterior pituitary cells in culture. The peptide did not affect thyrotropin or prolactin secretion. A GnRH antagonist did not inhibit gonadotropin stimulation by the peptide, and the peptide did not compete with GnRH for GnRH pituitary receptors, indicating that the action of the peptide is independent of the GnRH receptor. The GnRH precursor contains two distinct peptide sequences capable of stimulating gonadotropin release from human and baboon pituitary cells.

Stimulation of gonadotropin release by a non-GnRH peptide sequence of the GnRH precursor.

The human gonadotropin-releasing hormone (GnRH) precursor comprises the GnRH sequence followed by an extension of 59 amino acids. Basic amino acid residues in the carboxyl terminal extension may represent sites of processing to biologically active peptides. A synthetic peptide comprising the first 13 amino acids (H X Asp-Ala-Glu-Asn-Leu-Ile-Asp-Ser-Phe-Gln-Glu-Ile-Val X OH) of the 59-amino acid peptide was found to stimulate the release of gonadotropic hormones from human and baboon anterior pituitary cells in culture. The peptide did not affect thyrotropin or prolactin secretion. A GnRH antagonist did not inhibit gonadotropin stimulation by the peptide, and the peptide did not compete with GnRH for GnRH pituitary receptors, indicating that the action of the peptide is independent of the GnRH receptor. The GnRH precursor contains two distinct peptide sequences capable of stimulating gonadotropin release from human and baboon pituitary cells.

A prolactin-inhibiting factor within the precursor for human gonadotropin-releasing hormone.

The cloned complementary DNA sequence encoding the human gonadotropin-releasing hormone (GnRH) precursor protein was used to construct an expression vector for the bacterial synthesis of the 56-amino acid GnRH-associated peptide (GAP). GAP was found to be a potent inhibitor of prolactin secretion and to stimulate the release of gonadotropins in rat pituitary cell cultures. Active immunization with peptides corresponding to GAP sequences led to greatly increased prolactin secretion in rabbits.

Thyrotropin and gonadotropin releasing hormones (TRH and GnRH) do not alter levels of oxytocin and oxytocin does not change the response of luteinizing or follicle stimulating hormones to GnRH in humans.

Four hundred micrograms of synthetic thyrotropin releasing hormone (TRH) were given intravenously to 4 normal men and 4 normal women, and four weeks later, 1000 micrograms of TRH were administered intravenously to 4 of the 8 individuals and oxytocin (OT) was measured in plasma on both occasions. Following injection of either dose of TRH, OT did not change significantly from baseline. Likewise, synthetic gonadotropin releasing hormone (GnRH), 100 micrograms, administered intravenously to 6 normal men did not alter the levels of OT from baseline. Synthetic OT, 300 mU/minute, administered 30 minutes before and for 90 minutes after injection of GnRH, was without effect on the GnRH-induced rise of luteinizing hormone (LH) or follicle stimulating hormone (FSH) in normal men. We conclude that continuous infusion of OT in pharmacologic concentrations does not alter the pituitary release of LH or FSH in response to GnRH in humans. TRH and GnRH given intravenously do not alter basal levels of OT in the plasma of humans, thus a physiologic role for GnRH or TRH in the neuroendocrine control of OT secretion in humans is unlikely.

MEASUREMENT OF PLASMA LH, FSH, ESTRADIOL AND PROGESTERONE IN DISORDERS OF THE HUMAN MENSTRUAL CYCLE: THE SHORT LUTEAL PHASE

ABSTRACT Blood levels of LH, FSH, estradiol (E2) and progesterone (P) were determined daily during 11 cycles in 4 women with a luteal phase of less than 10 days. In all cycles peak preovulatory estradiol levels were subnormal. Luteal phase secretion of both estradiol and progesterone was more than 2 sem below normal and mean, midfollicular phase FSH was lower than found in a group of normal cycles. In 2 individuals the defect was found to occur repetitively in cycles analyzed for up to 1 yr. All cycles were of normal total duration. The menstrual cycle with a short luteal phase may be a distinct and common form of infertility. When identified, it may be amenable to specific treatment with human chorionic gonadotropin or gonadotropin-releasing hormone.