Gonadotropin-releasing Hormone Decapeptide Research Articles

Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells

SummaryGonadotropin-releasing hormone (GnRH) neurons regulate human puberty and reproduction. Modeling their development and function in vitro would be of interest for both basic research and clinical translation. Here, we report a three-step protocol to differentiate human pluripotent stem cells (hPSCs) into GnRH-secreting neurons. Firstly, hPSCs were differentiated to FOXG1, EMX2, and PAX6 expressing anterior neural progenitor cells (NPCs) by dual SMAD inhibition. Secondly, NPCs were treated for 10 days with FGF8, which is a key ligand implicated in GnRH neuron ontogeny, and finally, the cells were matured with Notch inhibitor to bipolar TUJ1-positive neurons that robustly expressed GNRH1 and secreted GnRH decapeptide into the culture medium. The protocol was reproducible both in human embryonic stem cells and induced pluripotent stem cells, and thus provides a translational tool for investigating the mechanisms of human puberty and its disorders.

Anti-GnRH Receptor Monoclonal Antibodies, First-In-Class GnRH Analog

A monoclonal antibody (Mab) designated as GHR106 was generated against the extracellular domain (N1-29 synthetic peptide) of human gonadotropin releasing hormone (GnRH) receptor. It is a first-in-class GnRH analog and can serve as a drug candidate for potential applications in the treatment of human cancers and/or fertility regulations. Both Mabs in murine (mGHR106) or humanized (hGHR106) forms were shown to have comparable specificity and affinity to intact GnRH receptor on cancer cells or to N1-29 synthetic peptides from humans and monkeys. Similar to decapeptide GnRH analogs, both Mabs were shown to induce apoptosis to cultured cancer cells of various tissue origins, including those from the ovary, breast, prostate, and lung. However, both Mabs were also found to induce complement-dependent cytotoxicity (CDC) reaction for lysis of cancer cells, an immune property which is not shared by peptide analogs of GnRH. By using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), both GHR106 Mabs and the GnRH decapeptide antagonist, Antide, were shown to be bioequivalent in terms of their respective effects on genes involved in the proliferation and apoptosis of cancer cells. In addition, GHR106 Mabs have a much longer circulating half-life than GnRH peptide analogs (days versus hours). Based on the results of these studies, it can be concluded that both mGHR106 and hGHR106 are bioequivalent to the GnRH decapeptide antagonist, Antide, in many biological and functional properties. Therefore, hGHR106 can serve as a long-acting alternative to the current decapeptide GnRH antagonists for therapeutic treatments in the immunotherapy of human cancers, including those of gynecologic origin.

Molecular cloning, tissue distribution, and ontogeny of gonadotropin-releasing hormone III gene (GnRH-III) in half-smooth tongue sole (Cynoglossus semilaevis)

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that plays a vital role in hypothalamus-pituitary-gonad (HPG) axis. In the present study, the GnRH-III gene was isolated from half-smooth tongue sole (Cynoglossus semilaevis). In the 1160 bp genomic sequence, four exons, three introns, and 5'-/3'-flanking sequences were identified. The putative peptide was 92 residues long, including a putative signal peptide containing 23 amino acids, the GnRH decapeptide, a proteolytic cleavage site of three amino acids and a GnRH associated peptide of 56 amino acids. The overall amino acid sequence of C. semilaevis GnRH-III (csGnRH-III) was highly conserved with other teleost GnRH-III genes. Phylogenetic analysis showed the evolutionary relationships of csGnRH-III with other known GnRH genes. A 320 bp promoter sequence of the csGnRH-III was also analyzed, and several potential regulatory motifs were identified which were conserved in the GnRH promoters of other teleosts. Quantitative real-time PCR analysis indicated csGnRH-III was expressed only in brain and gonads. In C. semilaevis, the csGnRH-III transcript was maternally deposited and appeared to be developmentally regulated during embryogenesis and early larval development. Comparing sequence and expression patterns of csGnRH-III with other teleosts GnRH-IIIs suggested that the main function of GnRH-III might be conserved in teleosts.

Intron retention in a salmon gonadotropin releasing-hormone in three species of Chirostoma genus.

Intron retention in a salmon gonadotropin releasing-hormone in three species of Chirostoma genus.

Defective gonadotropin-releasing hormone neuron migration in mice lacking SEMA3A signalling through NRP1 and NRP2: implications for the aetiology of hypogonadotropic hypogonadism

Kallmann syndrome (KS) is a genetic disease characterized by hypogonadotropic hypogonadism and impaired sense of smell. The genetic causes underlying this syndrome are still largely unknown, but are thought to be due to a developmental defect in the migration of gonadotropin-releasing hormone (GnRH) neurons. Understanding the causes of the disease is hampered by lack of appropriate mouse models. GnRH neurons are hypothalamic cells that centrally control reproduction in mammals by secreting the GnRH decapeptide into the portal blood vessels of the pituitary to stimulate the production of gonadotropins. During development, these cells are born in the nasal placode outside the brain and migrate in association with olfactory/vomeronasal axons to reach the forebrain and position themselves in the hypothalamus. By combining the analysis of genetically altered mice with in vitro models, we demonstrate here that a secreted guidance cue of the class 3 semaphorin family, SEMA3A, is essential for the development of the GnRH neuron system: loss of SEMA3A signalling alters the targeting of vomeronasal nerves and the migration of GnRH neurons into the brain, resulting in reduced gonadal size. We found that SEMA3A signals redundantly through both its classical receptors neuropilin (NRP) 1 and, unconventionally, NRP2, while the usual NRP2 ligand SEMA3F is dispensable for this process. Strikingly, mice lacking SEMA3A or semaphorin signalling through both NRP1 and NRP2 recapitulate the anatomical features of a single case of KS analysed so far, and may therefore be used as genetic models to elucidate the pathogenesis of KS.

GNRH1 mutations in patients with idiopathic hypogonadotropic hypogonadism

Idiopathic hypogonadotropic hypogonadism (IHH) is a condition characterized by failure to undergo puberty in the setting of low sex steroids and low gonadotropins. IHH is due to abnormal secretion or action of the master reproductive hormone gonadotropin-releasing hormone (GnRH). Several genes have been found to be mutated in patients with IHH, yet to date no mutations have been identified in the most obvious candidate gene, GNRH1 itself, which encodes the preprohormone that is ultimately processed to produce GnRH. We screened DNA from 310 patients with normosmic IHH (nIHH) and 192 healthy control subjects for sequence changes in GNRH1. In 1 patient with severe congenital nIHH (with micropenis, bilateral cryptorchidism, and absent puberty), a homozygous frameshift mutation that is predicted to disrupt the 3 C-terminal amino acids of the GnRH decapeptide and to produce a premature stop codon was identified. Heterozygous variants not seen in controls were identified in 4 patients with nIHH: 1 nonsynonymous missense mutation in the eighth amino acid of the GnRH decapeptide, 1 nonsense mutation that causes premature termination within the GnRH-associated peptide (GAP), which lies C-terminal to the GnRH decapeptide within the GnRH precursor, and 2 sequence variants that cause nonsynonymous amino-acid substitutions in the signal peptide and in GnRH-associated peptide. Our results establish mutations in GNRH1 as a genetic cause of nIHH.

Dependence of GnRH‐induced phosphorylation of CREB and BAD on EGF receptor transactivation in GT1‐7 neuronal cells

The hypothalamic neuropeptide, gonadotropin releasing hormone (GnRH), is a primary regulatory factor in the neuroendocrine control of reproduction. The GnRH decapeptide is released in an episodic manner from hypothalamic GnRH neurons, which are known to express GnRH receptors. Here we examined the signaling pathways by which autocrine GnRH stimulation generates cell survival and proliferative signals in hypothalamic GT1-7 cells. Both GnRH and epidermal growth factor (EGF) caused rapid phosphorylation of cyclic AMP response element binding protein (CREB) and BAD. The selective epidermal growth factor receptor (EGF-R) antagonist, AG1478, attenuates the phosphorylation of these proteins by GnRH and EGF. Inhibition of PKC and Src abolished the stimulatory effects of GnRH, but not that of EGF, consistent with a critical role of these signaling molecules upstream of the EGF-R. All of these effects of GnRH were mimicked by phorbol 12 myristate 13-acetate (PMA). Consistent with the prosurvival and mitogenic effects of phosphoinositide 3-kinase/Akt (P13-K/Akt) downstream of the EGF-R, inhibition of P13-K diminished the activation of these proteins following stimulation with GnRH, EGF, and PMA. Overexpression of dominant negative Akt attenuated agonist-induced phosphorylation of BAD, but not that of ERK1/2 and CREB. Moreover, overexpression of wild-type RSK-1 resulted in enhanced basal as well as agonist-induced phosphorylation of CREB and BAD, indicating a critical role of RSK-1 in activating cytosolic as well as nuclear proteins. These data reveal novel signaling mechanisms of GnRH-induced phosphorylation of CREB and BAD in GT1-7 neurons through transactivation of the EGF-R.

Neuroendocrine control of reproductive aging: roles of GnRH neurons

The process of reproductive senescence in many female mammals, including humans, is characterized by a gradual transition from regular reproductive cycles to irregular cycles to eventual acyclicity, and ultimately a loss of fertility. In the present review, the role of the hypothalamic gonadotropin-releasing hormone (GnRH) neurons is considered in this context. GnRH neurons provide the primary driving force upon the other levels of the reproductive axis. With respect to aging, GnRH cells undergo changes in biosynthesis, processing and release of the GnRH decapeptide. GnRH neurons also exhibit morphologic and ultrastructural alterations that appear to underlie these biosynthetic properties. Thus, functional and morphologic changes in the GnRH neurosecretory system may play causal roles in the transition to acyclicity. In addition, GnRH neurons are regulated by numerous inputs from neurotransmitters, neuromodulators and glia. The relationship among GnRH cells and their inputs at the cell body (thereby affecting GnRH biosynthesis) and the neuroterminal (thereby affecting GnRH neurosecretion) is crucial to the function of the GnRH system, with age-related changes in these relationships contributing to the reproductive senescent process. Therefore, the aging hypothalamus is characterized by changes intrinsic to the GnRH cell, as well as its regulatory inputs, which summate to contribute to a loss of reproductive competence in aging females.

GnRH pre-mRNA splicing: solving the mystery of a nature’s knockout, hpg mouse

The hypogonadal ( hpg) mouse represents a unique animal model for hypogonadism. In this mutant the truncation of the gene encoding gonadotropin-releasing hormone (GnRH) leads to drastically lowered gonadotropin levels and prepubertal gonads. The deletional mutation encompasses only the distal half of the gene leaving the region encoding GnRH decapeptide intact. The partially deleted gene is transcriptionally active, but translationally inactive. Even though several aspects have been considered to account for the phenomenon, there is no satisfactory explanation so far. Recent reports showed that excision of the GnRH first intron is delicately regulated in a cell type- and developmental stage-specific manner mediated by putative-specific splicing factors acting on cis-acting elements located in exon 3 and 4, and is significantly decreased in hpg mouse whose exonic splicing enhancers are absent. Furthermore, the suppressing effect of intron A retention on the translational activity of downstream open reading frame was reported, giving an insight into the understanding the mystery of hpg mice.

GnRH pre-mRNA splicing: role of exonic splicing enhancer

Publisher Summary This chapter discusses gonadotropin-releasing hormone (GnRH) pre-mRNA splicing. GnRH plays a key role in the regulation of reproduction. It is synthesized in the preoptic area (POA) of the vertebrate brain and released from nerve terminals in the median eminence into the hypothalamic-pituitary portal vessel in a pulsatile fashion. GnRH-1 then acts on gonadotrope in the anterior pituitary to stimulate the biosynthesis and secretion of pituitary gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). The biosynthesis of GnRH is regulated at multiple levels, such as transcription rate, mRNA stability, translation rate of the mRNA into pro GnRH peptide, and processing of the precursor to the mature GnRH decapeptide. The GnRH gene consists of four short exons and three long introns. In the hypothalamus and GnRH-producing cell lines, all three introns are excised from the primary transcript, producing a mature mRNA of about 560 bases. The chapter also explains the presence of GnRH neuron-specific splicing factors acting on ESE4.

The role of the individual amino acids of a GnRH-tandem-dimer peptide used as an antigen for immunocastration of male piglets determined with systematic alanine replacements

Immunocastration targeting gonadotropin releasing hormone (GnRH) can be obtained in male piglets using native GnRH conjugates. However, due to insufficient efficacy of these conjugates, improved GnRH antigens, like peptides existing of repeats of the GnRH amino acid sequence, have been designed. We previously reported about a dimerised GnRH-tandem peptide with a d-Lys at position 6 of the native GnRH sequence (G6k-TD) being highly effective. To evaluate the contribution of each individual amino acid of the GnRH decapeptide to the efficacy of the G6k-TD peptide, each amino acid was replaced consecutively by alanine (Ala-scan). The G6k-TD peptides were conjugated to ovalbumin, used for immunisation and tested for their ability to elicit GnRH antibodies and to immunocastrate male piglets. The results show that four out of nine amino acids (pGlu-1, Ser-4, Arg-8 and Gly-10) can be replaced by alanine without negatively affecting immunocastration efficacy. Replacement of amino acids in other positions (Tyr-5, Leu-7 and Pro-9) gave partial decrease of efficacy, respectively, five, six and six out of seven piglets were immunocastrated. Replacements at two other positions (His-2 and Trp-3) completely negated immunocastration activity. Thus, seven out of nine amino acid positions in the basic unit of G6k-TD can be substituted by alanine without affecting immunocastration efficacy.

Expression of Two Gonadotropin-Releasing Hormone (GnRH) Precursor Genes in Various Tissues of the Japanese Eel and Evolution of GnRH

Abstract We isolated and characterized two distinct cDNAs for mammalian gonadotropin-releasing hormone (mGnRH) and chicken GnRH-II (cGnRH-II) precursors from the Japanese eel by rapid amplification of cDNA ends. Each GnRH precursors were composed of a signal peptide, a GnRH decapeptide, a processing site and a GnRH-associated peptide. Northern blot and reverse transcription-polymerase chain reaction analysis revealed that the mGnRH precursor gene is expressed in all tissues tested including the brain, pituitary, eye, olfactory epithelium, ovary, testis, liver, kidney, spleen, heart, gill, intestine, pancreas, muscle, skin, fin and peripheral blood leukocyte. In contrast, the cGnRH-II precursor gene expression was detected only in the brain, pituitary, olfactory epithelium, ovary and testis. These findings suggest unknown physiological function(s) for mGnRH besides the well-documented role in the pituitary gonadotropin synthesis and release. The eel mGnRH and cGnRH-II precursors have high amino acid homolo...

An Alternative Gonadotropin-releasing Hormone (GnRH) RNA Splicing Product Found in Cultured GnRH Neurons and Mouse Hypothalamus

Gonadotropin-releasing hormone (GnRH) is encoded by the proGnRH gene which contains four exons and three introns. In this study, two immortalized GnRH-expressing cell lines (Gn11 and NLT) were characterized. The NLT and Gn11 cells, derived from a same brain tumor in a transgenic mouse, display neuronal morphology and neuron-specific markers. However, NLT cells secrete much higher levels of GnRH than Gn11 cells. To delineate the mechanism underlying this difference, reverse transcriptase-polymerase chain reaction and RNase protection assays were performed to examine proGnRH gene expression. While the mature proGnRH mRNA was predominately expressed in NLT cells, Gn11 cells express an abundant short transcript. Sequence analysis revealed that this short transcript contains exons 1, 3, and 4, but not exon 2, which encodes the GnRH decapeptide. RNase protection assays demonstrated that NLT cells express much higher levels of mature proGnRH mRNA than Gn11 cells. The lower level of GnRH secreting capacity in Gn11 cells is due, in part, to decreased expression of mature proGnRH mRNA. When proGnRH gene expression in the mouse brain was examined, the same short splicing variant was observed in the olfactory area and preoptic area-anterior hypothalamus. But the prevalent transcript in these regions was the mature proGnRH mRNA. In contrast, only the mature proGnRH mRNA was found in the caudal hypothalamus. These results suggest that alternative splicing may be one of the mechanisms regulating proGnRH gene expression in the animal brain.

Regulation of Gonadotropin-Releasing Hormone Gene Expressionin Vivoandin Vitro

The pulsatile release of gonadotropin-releasing hormone (GnRH) into the portal vasculature is responsible for the maintenance of reproductive function. Levels of GnRH decapeptide available for this process can be regulated at transcriptional, posttranscriptional, and posttranslational levels. In the immortalized neuronal GT1 cell lines which synthesize and secrete GnRH, regulation of GnRH biosynthesis has been studied using activators of the protein kinase A (PKA), protein kinase C (PKC), and calcium second messenger systems. These substances, while stimulating GnRH release, cause a universal inhibition of all biosynthetic indices measured to date, including decreases in transcription of the proGnRH gene, GnRH mRNA levels, mRNA stability, and translational efficiency. In contrast, in the animal, the mechanism for the regulation of GnRH gene expression appears to be primarily posttranscriptional, since changes in GnRH mRNA levels often occur in the absence of changes in GnRH primary transcript levels, an index of GnRH gene transcription. For example, GnRH mRNA levels increase in response to stimulation with glutamate analogs, while GnRH primary transcript levels are unchanged. However, parallel changes in GnRH mRNA and primary transcript have been observed on proestrus prior to the LH/GnRH surge, suggesting that the regulation of GnRH mRNA levelsin vivoinvolves a complex interplay of transcriptional and posttranscriptional processes.

GnRH mRNA expression in the forebrain of a teleost fish with alternative male morphs

Multiple forms of gonadotropin releasing hormone (GnRH) are found within several species of teleost fishes. Within the infradivision Euteleostei, the superorder Paracanthopterygii represents one of the last major groups to be examined with respect to the GnRH mRNA sequence. The plainfin midshipman, Porichthys notatus , is a common member of this superorder which is intermediate between the ancestral euteleost taxa and the more derived Acanthopterygians (percomorphs). The goals of this study were to: (1) determine the cDNA sequence of prepro-GnRH in the plainfin midshipman, (2) address the anatomical localization of midshipman prepro-GnRH gene expression, and (3) perform a cladistic analysis using all currently known cDNA sequences of prepro-GnRH. We report 460 base pair of cDNA sequence containing the entire protein coding region, and 5′- and 3′-untranslated regions. The deduced amino acid sequence indicates that this cDNA encodes a GnRH decapeptide identical in sequence to that originally isolated in salmon (Trp7, leu8- GnRH). Northern analysis demonstrated transcripts in brain, ovary, and testis (600-700 nucleotides). PCR showed that the ovarian prepro-GnRH was identical to that found in brain. In situ hybridization labeled neurons in the ganglion of the terminal nerve and the preoptic area, forebrain areas previously observed to contain GnRH-like immunoreactivity. Last, a phylogenetic analysis of 18 prepro-GnRH sequences grouped the Paracanthopterygii with the Acanthopterygii. However, this recent clade was distinct from two separate and more ancestral lineages, the Paracanthopterygii (salmonids) and Ostariophysi (represented by catfish).

Structure, Localization, and Molecular Phylogeny of a GnRH cDNA from a Paracanthopterygian Fish, the Plainfin Midshipman ( Porichthys notatus)

Multiple forms of gonadotropin releasing hormone (GnRH) are found within several species of teleost fishes. Within the infradivision Euteleostei, the superorder Paracanthopterygii represents one of the last major groups to be examined with respect to the GnRH mRNA sequence. The plainfin midshipman, Porichthys notatus , is a common member of this superorder which is intermediate between the ancestral euteleost taxa and the more derived Acanthopterygians (percomorphs). The goals of this study were to: (1) determine the cDNA sequence of prepro-GnRH in the plainfin midshipman, (2) address the anatomical localization of midshipman prepro-GnRH gene expression, and (3) perform a cladistic analysis using all currently known cDNA sequences of prepro-GnRH. We report 460 base pair of cDNA sequence containing the entire protein coding region, and 5′- and 3′-untranslated regions. The deduced amino acid sequence indicates that this cDNA encodes a GnRH decapeptide identical in sequence to that originally isolated in salmon (Trp7, leu8- GnRH). Northern analysis demonstrated transcripts in brain, ovary, and testis (600-700 nucleotides). PCR showed that the ovarian prepro-GnRH was identical to that found in brain. In situ hybridization labeled neurons in the ganglion of the terminal nerve and the preoptic area, forebrain areas previously observed to contain GnRH-like immunoreactivity. Last, a phylogenetic analysis of 18 prepro-GnRH sequences grouped the Paracanthopterygii with the Acanthopterygii. However, this recent clade was distinct from two separate and more ancestral lineages, the Paracanthopterygii (salmonids) and Ostariophysi (represented by catfish).

New prostate cancer drugs: few CV effects?

Encouraging alternative approaches to orchiectomy and estrogen therapy for advanced prostatic cancer were highlighted in a series of presentations at the recent XIIIth International Congress of Chemotherapy in Vienna. Among the most prevalent malignant neoplasms worldwide, prostate cancer may be difficult to treat because it has often metastasized to bone and other organs by the time of diagnosis. One new investigational approach involves the use of synthetic analogs of gonadotropin-releasing hormone (GnRH), while the other employs drugs designed to destroy prostatic tumor tissue selectively. Most of the synthetic GnRH agents now under study result from substitution of a different amino acid in the 6 position of the naturally occurring GnRH decapeptide. Unlike natural GnRH, which is released in pulsatile fashion from the hypothalamus and acts through the pituitary to ultimately stimulate testosterone production by the gonads, the synthetic agonists apparently occupy the pituitary GnRH receptors continuously, desensitizing them and thereby

Binding of gonadotropin releasing hormone agonist to rat ovarian granulosa cells

We have previously shown that gonadotropin releasing hormone (GnRH) and its agonists inhibit ovarian functions by a direct action on ovarian granulosa cells in vitro . A labeled GnRH agonist, [des-Gly 10, D-Ser (TBu) 6, Pro 9-NHEt]GnRH, was used here to examine the possibility that these inhibitory actions of GnRH were mediated through specific receptors which recognize GnRH. Ovarian membrane fractions obtained from immature, hypophysectomized diethylstilbesterol-treated rats were incubated with the 125I-GnRH agonist and specific binding was determined by a filtration assay. Stereospecific, high affinity binding was detected in the ovarian membranes; the dissociation constant for the labeled GnRH agonist was determined to be 0.84 ± 0.33 × 10 −10 M and the binding capacity was calculated to be 12.9 fmol/mg protein, or 0.142 fmol/μg DNA. The binding affinity for the GnRH decapeptide was 3.3 times lower than that of the GnRH agonist whereas two GnRH partial peptides did not compete for the 125I-agonist binding. After sequential treatment with FSH, LH and prolactin to the hypophysectomized female rats, the ovarian GnRH binding capacity increased per ovary, but decreased per mg ovarian protein. Furthermore, ovarian granulosa cells were isolated and their binding capacity was determined to be 25.2 fmol/mg protein, or 0.133 fmol/μg DNA, suggesting that the granulosa cells contain GnRH binding sites. Thus, this report demonstrates the presence of stereospecific, high affinity GnRH binding sites in the rat ovarian granulosa cells.

Receptor-binding affinity of gonadotropin-releasing hormone analogs: analysis by radioligand-receptor assay.

A recently developed radioligand-receptor assay for pituitary gonadotropin-releasing hormone (GnRH) receptors, employing a radioiodinated superagonist analog as the ligand, was used to analyze the receptor-binding properties of several GnRH analogs. This receptor assay is specific for GnRH and related peptides with agonist or antagonist activity. All superagonist analogs had equilibrium association constants (Ka) between 4- and 8-fold greater than that of the natural GnRH decapeptide (Ka = 6.6 × 108 m−1). Conversely, weak agonists and inactive analogs were bound with affinities 0.003–0.05 times that of GnRH. Weak antagonists, such as [D-Phe2]GnRH and [DPhe2, D-Phe6]GnRH, had lower binding constants, while the most potent antagonist, [D-pGlu1, D-Phe2, D-Trp3,6]GnRH, exhibited 8-fold higher affinity than GnRH for pituitary receptors. There was a general positive correlation between receptor-binding affinity and relative biological activity, whether agonist or antagonist, for the several GnRH analogs tested. However, the observed increases in receptor affinity (4- to 8-fold) did not account for the 30- to 100-fold greater LH-releasing activity of the superagonists. The relatively high bioactivity of such analogs in comparison to their binding constants is attributable to their increased resistance to degradation in addition to their increased binding affinity. Comparison of receptor binding data with structural features of the GnRH analogs revealed that the three Nterminal amino acids are critical for receptor recognition, while the C-terminal des-Gly10 ethylamide modification does not enhance binding affinity. The radioligand assay provides a rapid and reliable method for evaluating the contribution of receptorbinding affinity to the agonist or antagonist actions of new GnRH analogs. This procedure also permits systematic determination of the structural requirements for GnRH receptor recognition. (Endocrinology106: 1154, 1980)