Specific Gonadotropin-releasing Hormone Receptor Research Articles

Autocrine production of reproductive axis neuropeptides affects proliferation of canine osteosarcoma in vitro

BackgroundOsteosarcoma strikes hundreds of people each year, of both advanced and younger ages, and is often terminal. Like many tumor types, these bone tumors will frequently undergo a neuroendocrine transition, utilizing autocrine and/or paracrine hormones as growth factors and/or promoters of angiogenesis to facilitate progression and metastasis. While many of these factors and their actions on tumor growth are characterized, some tumor-derived neuropeptides remain unexplored.MethodsUsing validated canine osteosarcoma cell lines in vitro, as well as cells derived from spontaneous tumors in dogs, we explored the autocrine production of two neuropeptides typically found in the hypothalamus, and most closely associated with reproduction: gonadotropin-releasing hormone (GnRH) and kisspeptin (Kiss-1). We evaluated gene expression and protein secretion of these hormones using quantitative RT-PCR and a sensitive radioimmunoassay, and explored changes in cell proliferation determined by MTS cell viability assays.ResultsOur current studies reveal that several canine osteosarcoma cell lines (COS, POS, HMPOS, D17, C4) synthesize and secrete GnRH and express the GnRH receptor, while COS and POS also express kiss1 and its cognate receptor. We have further found that GnRH and kisspeptin, exogenously applied to these tumor cells, exert significant effects on both gene expression and proliferation. Of particular interest, kisspeptin exposure stimulated GnRH secretion from COS, similarly to the functional relationship observed within the neuroendocrine reproductive axis. Additionally, GnRH and kisspeptin treatment both increased COS proliferation, which additionally manifested in increased expression of the bone remodeling ligand rankl within these cells. These effects were blocked by treatment with a specific GnRH receptor inhibitor. Both neuropeptides were found to increase expression of the specific serotonin (5HT) receptor htr2a, the activation of which has previously been associated with cellular proliferation, suggesting that production of these factors by osteosarcoma cells may act to sensitize tumors to circulating 5HT of local and/or enteric origin.ConclusionsHere we report that kisspeptin and GnRH act as autocrine growth factors in canine osteosarcoma cells in vitro, modulating RANKL and serotonin receptor expression in a manner consistent with pro-proliferative effects. Pharmacological targeting of these hormones may represent new avenues of osteosarcoma treatment.

Synthesis and Evaluation of Novel Gonadotropin-Releasing Hormone Receptor-Targeting Peptides

The purpose of this study was to develop novel radiolabeled gonadotropin-releasing hormone (GnRH) receptor-targeting peptides for breast cancer imaging. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated GnRH peptides were designed and synthesized. The radiometal chelator DOTA was conjugated to the epsilon or alpha amino group of D-lysine, or the epsilon amino group of L-lysine via an Ahx {aminohexanoic acid} linker to generate DOTA-Ahx-(D-Lys(6)-GnRH1), DOTA-Ahx-(D-Lys(6)-GnRH2) and DOTA-Ahx-(L-Lys(6)-GnRH3), respectively. The conjugation of the DOTA to the epsilon amino group of D-lysine (rather than alpha amino group of D-lysine nor epsilon amino group of L-lysine) maintained the nanomolar GnRH receptor binding affinity. The IC(50) values of DOTA-Ahx-(D-Lys(6)-GnRH1), DOTA-Ahx-(D-Lys(6)-GnRH2) and DOTA-Ahx-(L-Lys(6)-GnRH3) were 36.1 nM, 10.6 mM and 4.3 mM, respectively. Since only DOTA-Ahx-(D-Lys(6)-GnRH1) displayed nanomolar receptor binding affinity, the specific GnRH receptor binding of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) was determined in human GnRH receptor membrane preparations. Furthermore, the biodistribution and tumor imaging properties of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) were examined in MDA-MB-231 human breast cancer-xenografted nude mice. (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) exhibited specific GnRH receptor binding and rapid tumor uptake (1.76 ± 0.58% ID/g at 0.5 h postinjection) coupled with fast whole-body clearance through the urinary system. The MDA-MB-231 human breast cancer-xenografted tumor lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT at 1 h postinjection of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1). The profound impact of DOTA position on the binding affinity of the GnRH peptide provided a new insight into the design of novel radiolabeled GnRH peptides. The successful imaging of MDA-MB-231 human breast cancer-xenografted tumor lesions using (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) suggested its potential as a novel imaging probe for human breast cancer imaging.

Short‐term fasting leads to inhibition of responsiveness to LH‐stimulated testosterone secretion in the adult male bonnet monkey

A variety of stressors including fasting profoundly inhibit reproductive function in mammals. Although the effect of short-term fasting on gonadotropic axis is well established, the direct effects of fasting on gonads have not been reported. The objectives of the present experiments were to examine the effect of short-term fasting on circulating luteinizing hormone (LH) and testosterone (T) secretion, and to determine the responsiveness of testis to exogenous recombinant human (rh) LH treatment in male bonnet monkeys. In addition, an experiment was carried out to examine whether brief inhibition of endogenous LH secretion causes alteration in testicular responsiveness. Adult male monkeys were fasted for 1 day for examining the circulating endocrine hormone concentrations and challenged with rhLH injection 1 day after fasting. Food withdrawal for 1 day resulted in significant (P<0.05) decrease in LH, T and increase in cortisol concentrations. Surprisingly, T secretion in response to direct stimulation of Leydig cells by LH was not observed in fasted monkeys. In fed monkeys, treatment with Antide (a specific gonadotropin releasing hormone receptor antagonist to inhibit pituitary LH secretion) for 1 day did not compromise T secretion stimulated by rhLH, suggesting that loss of responsiveness of testis to exogenous LH treatment in fasted monkeys was not because of interruption in pituitary LH stimulation of the testis. The results indicate that short-term fasting in adult male monkeys cause inhibition of LH and T secretion, and inhibition of responsiveness of testis to LH stimulation.

Gonadotropin-Releasing Hormone (GnRH) Receptors in Tumors: a New Rationale for the Therapeutical Application of GnRH Analogs in Cancer Patients?

Gonadotropin-Releasing Hormone (GnRH) is the hypothalamic decapeptide which plays a key role in the control of reproductive functions. By binding to specific receptors present on the pituitary gonadotropes, GnRH regulates gonadotropin release and, consequently, steroid hormone secretion from the gonads. When given continuously and at high doses, GnRH agonists suppress the pituitary gonadal axis through the down-regulation and desensitization of its own receptors. Based on this rationale, pituitary GnRH receptors represent the target for the successful utilization of GnRH agonists (that are more stable than the native peptide) for the treatment of hormone-dependent tumors (e.g., prostate, breast, endometrial, ovarian cancers). The observation that GnRH receptors are expressed in steroid-dependent tumors, and that their activation reduces cell proliferation and metastatic behavior of cancer cell lines, both in vitro and in vivo (when inoculated into nude mice), indicates a possible additional and more direct antitumor activity for these compounds. Interestingly, GnRH receptors have been shown to be expressed also in androgen-independent prostate carcinoma, as well as in tumors that are not classically considered hormone-related (e.g., melanoma), suggesting a clinical utility of the administration of GnRH analogs also in these tumors. More recently, GnRH agonists have been proposed as useful carriers to target cytotoxic drugs or toxins to cancer cells displaying the specific GnRH receptors. A second form of GnRH (designated GnRH-II) has been discovered in most vertebrates, including humans. GnRH-II has been suggested to act through a 'putative' cognate type II GnRH receptor, which is distributed in different tissues, both normal and tumoral. In humans, a full-length functional type II GnRH receptor has not been found. Therefore, its functions as well as its possible utility as a molecular target for a GnRH-II based therapy in oncology still has to be clarified. This review will focus on the role of GnRH receptors in the control of tumor growth, progression and dissemination. It will also be discussed whether the presence of these receptors might represent an additional rationale for the clinical utility of GnRH analogs as anticancer drugs.

Backbone metal cyclization: Novel 99mTc labeled GnRH analog as potential SPECT molecular imaging agent in cancer

Gonadotropin-releasing hormone (GnRH) is a decapeptide secreted to the pituitary where it binds to specific receptors on the gonadotropes to regulate gonadotropic hormones (luteinizing hormone (LH) and follicle-stimulating hormone (FSH)) synthesis and secretion. Specific GnRH receptors are overexpressed in breast, prostatic, ovarian, and other tumors. The aim of this study was to synthesize a cyclic GnRH analog with high affinity to GnRH receptors that can be radiolabeled with 99mTc. A precyclic GnRH analog, [Cys-Gly] 1[D-Ala] 6[N α(η-Cys-amino hexyl)] 10GnRH (Gn-2), containing two hemi-chelator groups was synthesized. It was cyclized applying the recently reported backbone metal cyclization (BMC) approach, to obtain cyclo(Re(O)1-10)[Cys-Gly] 1[D-Ala] 6[N α(η-Cys-amino hexyl)] 10GnRH ( cyclo[Re(O)-Gn-2]). For comparative evaluations, Gn-2 was oxidized on-resin to yield cyclo(S-S,1-10)[Cys-Gly] 1[D-Ala] 6[N α(η-Cys-amino hexyl)] 10GnRH, ( cyclo[S-S-Gn-2]). The binding affinity of cyclo[Re(O)-Gn-2] to rat pituitary membranes showed IC 50 of 50nM, compared to IC 50 = 10 nM in the native GnRH. Cyclo( 99mTc(O)1-10)[Cys-Gly] 1[D-Ala] 6[N α(η-Cys-amino hexyl)] 10GnRH ( cyclo[ 99mTc(O)-Gn-2]) was synthesized from Gn-2 and showed similar chromatographic behavior to its rhenium surrogate.

Expression and Function of Gonadotropin-releasing Hormone (GnRH) Receptor in Human Olfactory GnRH-secreting Neurons: AN AUTOCRINE GnRH LOOP UNDERLIES NEURONAL MIGRATION

Olfactory neurons and gonadotropin-releasing hormone (GnRH) neurons share a common origin during organogenesis. Kallmann's syndrome, clinically characterized by anosmia and hypogonadotropic hypogonadism, is due to an abnormality in the migration of olfactory and GnRH neurons. We recently characterized the human FNC-B4 cell line, which retains properties present in vivo in both olfactory and GnRH neurons. In this study, we found that FNC-B4 neurons expressed GnRH receptor and responded to GnRH with time- and dose-dependent increases in GnRH gene expression and protein release (up to 5-fold). In addition, GnRH and its analogs stimulated cAMP production and calcium mobilization, although at different biological thresholds (nanomolar for cAMP and micromolar concentrations for calcium). We also observed that GnRH triggered axon growth, actin cytoskeleton remodeling, and a dose-dependent increase in migration (up to 3-4-fold), whereas it down-regulated nestin expression. All these effects were blocked by a specific GnRH receptor antagonist, cetrorelix. We suggest that GnRH, secreted by olfactory neuroblasts, acts in an autocrine pattern to promote differentiation and migration of those cells that diverge from the olfactory sensory lineage and are committed to becoming GnRH neurons.

The effect of gonadotropin-releasing hormone on growth hormone and gonadotropin subunit gene expression in the pituitary of goldfish, Carassius auratus

The goldfish brain contains at least two forms of gonadotropin-releasing hormone (GnRH): sGnRH and cGnRH-II. In goldfish sGnRH and cGnRH-II are present both in the brain and pituitary, and exert direct effects via specific GnRH receptors stimulating growth hormone (GH) and gonadotropin hormone (GtH) synthesis and secretion. In this study, we investigated the effects of sGnRH and cGnRH-II on GtH subunit (α, FSH-β and LH-β) and GH mRNA levels in the goldfish pituitary in vivo and in vitro. Injection of goldfish with sGnRH or cGnRH-II (4 μg/fish) stimulated GtH-α, FSH-β and LH-β mRNA levels after 24 h. For in vitro studies, goldfish pituitary fragments were treated continuously for 12 h with 10−7 M sGnRH or cGnRH-II. Both sGnRH and cGnRH-II stimulated GtH-α, FSH-β, LH-β and GH mRNA levels, however, cGnRH-II appeared to have a more pronounced effect. Similar experiments were carried out using cultured dispersed goldfish pituitary cells. In this study, treatments for 12 h with 10−7 M sGnRH or cGnRH-II also stimulated GtH and GH gene expression. The present results provide a basis for the investigation of the signal transduction pathways that mediate GnRH-induced changes in GtH subunit and GH mRNA levels in the goldfish pituitary.

Immunohistochemical expression of gonadotropin releasing hormone receptor in human breast carcinoma

Gonadotropin releasing hormone (GnRH) analogs can cause regression of hormone-dependent breast carcinomas via the specific GnRH receptor (GnRH-R). In an attempt to obtain a better understanding of GnRH actions in human breast carcinoma, the expression of GnRH-R was examined immunohistochemically in 58 invasive ductal carcinomas and correlated with various clinicopathological parameters. GnRH-R was immunolocalized in the cytoplasm of carcinoma cells in 37 of 58 invasive ductal carcinoma cases (64%). Immunoreactivity for GnRH-R was also detected focally in the cytoplasm of morphologically normal glandular epithelia adjacent to the carcinoma. A significant correlation was observed between the immunohistochemical expression of GnRH-R and estrogen receptor labeling index (LI; P = 0.030) or progesterone receptor LI (P = 0.0074). There was a significant inverse correlation between GnRH-R immunoreactivity and Ki-67 LI (P = 0.012). No significant correlations were detected between GnRH-R and other clinicopathological parameters, including patient age, menopausal status, stage, tumor size, lymph node status, histological grade and prognosis. This study indicates that GnRH-R is widely distributed in human breast carcinoma cells and regulates GnRH actions locally. Breast carcinomas positive for GnRH-R maintain some hormonal regulatory mechanisms, and GnRH actions may lead to a low proliferative rate in human breast carcinoma.

Regulation of gonadotropin releasing hormone receptor mRNA expression in cultured rat granulosa cells

The homologous regulation of pituitary Gonadotropin Releasing Hormone Receptor (GnRH-R) mRNA expression by GnRH has been well demonstrated. However, the regulation of the ovarian GnRH-R is poorly understood. The present study was performed to demonstrate the presence of GnRH transcripts in addition to GnRH-R mRNA and the regulation of GnRH-R mRNA expression in the granulosa cells isolated from small antral follicles. The GnRH and GnRH-R mRNA levels were determined by a competitive reverse transcription-polymerase chain reaction (RT-PCR). The granulosa cells were obtained from immature rats implanted with diethylstilbestrol for 3 days. When GnRH transcript expression was examined in isolated granulosa cells by RT-PCR, the PCR products showed two bands. The larger band contained intronic sequences and the smaller band was a fully processed GnRH gene transcript identical to hypothalamic GnRH. This suggests that authentic GnRH gene transcripts are expressed in ovarian granulosa cells and may act on the granulosa cells in a paracrine or autocrine manner. Since GnRH action in the granulosa cells is mediated by specific GnRH-R, it is of interest to examine whether GnRH-R is synthesized in the granulosa cells. When the granulosa cells were cultured in media only, GnRH-R mRNA levels increased abruptly within 3 h and gradually decreased thereafter during the 24 h culture period. However, GnRH itself did not alter the GnRH-R mRNA expression levels in cultured granulosa cells. Interestingly, treatment with FSH decreased the GnRH-R mRNA levels in a dose-dependent manner. A time-course analysis revealed that the GnRH-R mRNA levels were significantly lower up to 9 h after FSH treatment, and returned to the basal level between 12 h-24 h. Activation of adenylate cyclase with forskolin also decreased the GnRH-R mRNA levels. It is therefore concluded that in the granulosa cells of the small antral follicles GnRH-R mRNA expression was not homologously regulated by GnRH, while FSH may negatively regulate GnRH-R mRNA expression in the granulosa cells possibly through a cAMP-protein kinase A pathway.

Activation of gonadotropin-releasing hormone receptors induces a long-term enhancement of excitatory postsynaptic currents mediated by ionotropic glutamate receptors in the rat hippocampus

Whole-cell patch-clamp recordings were made from CA1 pyramidal neurons of the rat hippocampus to study the modulation of gonadotropin-releasing hormone (GnRH) on synaptic transmission mediated by ionotropic glutamate receptors. Leuprolide (10 −9–10 −7 M), a specific GnRH analog, concentration-dependently elicited a long-lasting potentiation of excitatory postsynaptic currents (EPSCs) mediated by ionotropic glutamate receptors. GnRH receptor-induced synaptic potentiation was blocked by 1 μM [Acetyl-3,4-dehydro-Pro 1,D-p-F-Phe 2,D-Trp 3,6]-LHRH, a specific GnRH receptor antagonist. Furthermore, GnRH receptor-induced synaptic potentiation was associated with the stimulation of protein kinase C (PKC), being considerably attenuated by a potent PKC inhibitor (30 μM H-7). The results suggest a long-term enhanced modulation of GnRH on synaptic transmission mediated by ionotropic glutamate receptors, possibly via the actions of PKC in the hippocampus that is an important integrative system in the regulation of reproductive processes.

Effects of gonadotropin-releasing hormone agonist on steroidogenesis in the rat ovary.

To assess the regulatory roles of gonadotropin-releasing hormone (GnRH) in ovarian function, the kinetics of the ovarian GnRH receptor and the effects of the GnRH superagonist buserelin on steroidogenesis in ovarian cell culture were examined. Scatchard analysis of buserelin-binding to crude ovarian cell membrane revealed a specific high-affinity GnRH receptor. Buserelin together with follicle-stimulating hormone stimulated estradiol (E2) production in immature follicles in hypophysectomized and DES-treated rats. On the other hand, applied to developing follicles of rats treated with pregnant-mare-serum gonadotropin buserelin suppressed E2 production to terminate follicle maturation and simultaneously stimulated progesterone (P4) production to induce luteinization. With ovarian cells luteinized by human chorionic gonadotropin in vitro, buserelin suppressed production of both P4 and E2, leading to luteolysis. Buserelin affected steroid production by modulating activities of key enzymes in steroid synthesis. These findings indicate that buserelin action depended on the gonadotropin priming of ovarian cells, and suggest the possible involvement of GnRH in the regulation of steroidogenesis throughout the ovulatory cycle.

Receptors for gonadotropin releasing hormone are present in rat oocytes.

Specific receptors for gonadotropin releasing hormone (GnRH) in the rat oocyte have been identified by using two independent methods. Light microscopic autoradiography, utilizing an iodinated biologically active photoaffinity derivative of GnRH, revealed specific binding of the neurohormone to rat oocytes. Furthermore, the presence of GnRH-receptor is also evident from indirect fluorescent immunocytochemistry that shows binding of GnRH-receptor antibodies to rat oocytes which is neither detected with non immune serum nor with antiserum depleted of GnRH-receptor antibodies. These antibodies to the GnRH-receptor, also bind to both cumulus and granulosa cells but not to rat basophilic leukemia cells. The presence of specific GnRH receptors on rat oocytes provides an experimental basis for understanding the molecular events involved in GnRH-induced oocyte maturation.

Gonadotropin-releasing hormone receptors in human breast cancer tissue

Analogues of gonadotropin-releasing hormone (GnRH) are presently being investigated for the treatment of metastatic breast cancer. Although their effects are thought to be mediated via the suppression of gonadotropins and gonadal steroids, they could possibly also act directly on the tumour. The binding of a GnRH-agonist to membrane fractions of 97 primary human breast carcinomas was investigated. In 43 cases (44%) the presence of specific GnRH receptor binding sites (above 3 fmol/mg membrane protein) was shown. GnRH receptor content was 6.2 fmol/mg membrane protein in estradiol receptor (ER) positive tissues and in ER negative tissues 2.0 fmol/mg membrane protein (p less than 0.05). Progesterone receptor (PR) positive contained 5.2 fmol/mg protein and PR negative carcinoma contained 2.0 fmol/mg membrane protein. In postmenopausal women GnRH receptor concentration was 5.4 and 2.9 fmol/mg membrane protein, respectively, in ER positive and negative tissues (p less than 0.05) whereas 4.4 and 5.2 fmol/mg membrane protein, respectively, in PR positive and negative samples. Binding of GnRH was positive (above 3 fmol/mg protein) in 33% premenopausal and in 54% of postmenopausal cases. Further clinical studies with GnRH analogues will clarify the therapeutic value of GnRH receptor determination in breast cancer.

Gonadotropin-releasing hormone specific binding sites in uterine leiomyomata

Gonadotropin-releasing hormone (GnRH) analogs can cause regression of uterine leiomyomata. This effect is thought to be mediated by the inhibition of gonadotropin release and steroid synthesis. In the present study we examined the possibility that these analogs may also act directly on uterine leiomyomata. Specific binding sites for GnRH are present in myoma membranes, as 125I-Buserelin binding was displaced with equal efficiency by the superagonists, Buserelin and D-Trp6-GnRH, and by the antagonist Organon 30276, but not by unrelated peptides such as thyrotropin releasing hormone and oxytocin. A nonlinear Scatchard curve obtained for Buserelin specific binding suggests the presence of at least two binding sites, one of which exhibits a relatively high affinity for GnRH analogs (Kd of approximately 10(-8) M). Western blotting with a specific GnRH receptor antibody revealed the presence of a 60 kDa protein in myoma membranes. This protein has a similar molecular weight to the purified pituitary GnRH receptor. These results indicate, for the first time, the presence of specific binding sites for GnRH in uterine leiomyomata, suggesting a direct effect of GnRH analogs on this tissue.

Characterization and distribution of receptors for gonadotropin-releasing hormone in the rat hippocampus.

Distribution and properties of receptors for gonadotropin-releasing hormone (GnRH) were analyzed in the brain of adult male rats. Binding of the iodinated GnRH agonist Des-Gly10-(D-Ala6)-GnRH ethylamide was studied in hippocampus and anterior pituitary using three convergent approaches: quantitative autoradiography on frozen tissue, binding to fresh slices, and binding to crude membrane preparations. In all cases, binding was specific, saturable, and time, pH, and temperature dependent. Quantitative autoradiography revealed that the density of binding sites was high in the stratum oriens and stratum radiatum of the CA1-CA4 regions of Ammon's horn. The pyramidal cell layer was faintly labelled. Binding was almost undetectable in the dentate gyrus. The highest density of sites (Bmax = 11.6 +/- 1.0 fmol/mg protein) was observed in the stratum radiatum of the CA3 region. Under the same conditions the value obtained for pituitary tissues was 20.7 +/- 2.8 fmol/mg protein. Analysis of saturation curves indicated only one class of high-affinity sites for the hippocampus (CA3; Kd = 0.28 +/- 0.03 nM) and for the pituitary (Kd = 0.29 +/- 0.08 nM). Both native GnRH and GnRH antagonist were potent competitors of binding. Fresh slices and membrane preparations from whole hippocampus confirmed these autoradiographic data and yielded affinity constants of 0.28 +/- 0.01 and 0.52 +/- 0.08 nM, respectively. In addition, a very high binding density was present in the amygdaloid complex, while binding was barely detectable in the hypothalamus. These results demonstrate that high densities of specific GnRH receptors are present in areas concerned with the regulation of behavioral functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotropin-releasing hormone as a paracrine hormone and neurotransmitter in extra-pituitary sites

Gonadotropin-releasing hormone (GnRH), in addition to its classical releasing action at the pituitary level, acts on multiple extrapituitary sites to regulate various reproductive functions. In the rat ovary, specific high affinity GnRH receptors have been identified in granulosa and theca cells. These binding sites mediate the inhibitory effects of GnRH and its agonists on gonadotropin-stimulated estrogen, progestin and androgen biosynthesis. At the granulose cell level, GnRH treatment decreases aromatase activity as well as the biosynthesis of pregnenolone and progesterone via inhibition of cholesterol side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase enzymes. High concentrations of GnRH also stimulate low but significant levels of various steroids. In addition, treatment with high concentrations of GnRH induces ovulation and oocyte maturation in hypophysectomized rats. This is associated with the ability of GnRH to stimulate plasminogen activator activity in cultured granulosa cells. In the rat testis, GnRH receptors have been identified in Leydig but not Sertoli cells. Treatment with GnRH inhibits gonadotropin-stimulated androgen biosynthesis by the cultured Leydig cells. The inhibitory effect of GnRH on testicular androgen production occurs at sites distal to the formation of cyclic AMP and pregnenolone and may be due to decreases in the activity of the enzyme 17 alpha-hydroxylase and 17-20 desmolase. Since hypothalamic GnRH is unlikely to act at the gonadal level, several laboratories have attempted to isolate gonadal GnRH-like peptide which may serve as the ligand for specific gonadal GnRH receptors. Although the presence of ovarian GnRH-like substance still remains elusive, testicular GnRH-like substance has been identified. This gonadal peptide(s) may be an important local paracrine hormone. In addition to its action at the gonadal level, GnRH or GnRH-like peptides may play an important role as a neurotransmitter in the central nervous system. Exogenous administration of GnRH in selected brain areas has been shown to modulate sexual behavior in experimental animals, while neural pathways containing GnRH-like immunoreactive substances have been identified in several brain areas. We have recently synthesized a bioluminescent GnRH analog capable of serving as a specific GnRH ligand for a bioluminescent ligand receptor assay which is more sensitive than classical 125I-ligand assays. We have identified GnRH receptors in small, discrete brain regions. Thus, GnRH and GnRH-like peptides may play important paracrine and neurotransmitter roles in the regulation of various reproductive functions in extra-pituitary sites.

Absence of gonadotropin-releasing hormone receptors in human gonadal tissue.

The antifertility nature of the hypothalamic peptide gonadotropin releasing hormone (GnRH) and its synthetic agonist analogue (GnRH-A) has been repeatedly demonstrated in rodents, monkeys and humans (see ref. 1 for review). In all species studied, this action of GnRH was initially attributed to ‘desensitization’ of gonadal steroidogenesis due to loss of gonadotropin receptors in the testis and ovary, induced by high circulating levels of endogenous gonadotropins achieved following repeated administration of GnRH or GnRH-A2–5. Recently, an additional, direct inhibitory action of these peptides on ovarian granulosa6,7 and luteal8–10 and testicular Leydig11–13 cells has been defined. This direct inhibition of gonadal steroidogenesis is mediated through specific high-affinity GnRH receptors in the respective target tissues8,9,12,14. These receptors exhibit identical characteristics to GnRH receptors (GnRH-R) in rodent anterior pituitary tissue9,12,14,15. In the search for a naturally occurring ligand for gonadal GnRH-R, a biologically active ‘GnRH-like’ peptide has been isolated from rat ovary16 and testis17,18, and from monkey testis interstitial fluid18. These findings suggest the production in rodent gonads of a GnRH-like peptide which, following interaction with gonadal GnRH-R, serves as a local modulator of steroidogenesis, and hence of reproductive capacity. If a similar situation exists in humans, GnRH-R should be detectable also in the human testis and ovary. We report here that we have been unable to detect any binding of radiolabelled GnRH-A to human corpus luteum or testis, or to the monkey testis. In contrast, GnRH-R were detected in adult and fetal human anterior pituitary tissue. Thus, we suggest that a direct inhibitory action of GnRH on primate reproductive function is unlikely. Furthermore, this important species difference highlights the need for a re-evaluation of certain rodent models designed to investigate primate, and especially human, reproductive physiology.

Pituitary Receptor Site Blockade by a Gonadotropin-Releasing Hormone Antagonist in Vivo: Mechanism of Action

Administration of a potent gonadotropin-releasing hormone (GnRH) antagonist [Nac-L-Ala1,pCl-D-Phe2,D-Trp3,6]GnRH as a single subcutaneous injection to castrated adult male rats reduced, by more than 90 percent, both serum luteinizing hormone concentrations and specific pituitary GnRH receptor binding. This effect persisted for 24 hours. The dissociation rate of the antagonist from pituitary membrane homogenates was fourfold slower than the dissociation rate of a potent agonist. The prolonged in vivo inhibition of pituitary GnRH receptor binding and luteinizing hormone secretion by the GnRH antagonist may be mediated by the slower dissociation rate of the antagonist from its specific pituitary membrane receptor site.

Ovarian gonadotropin-releasing hormone receptors. I. Properties and inhibition of luteal cell function.

Gonadotropin-releasing hormone (GnRH) and its agonist analogs exert paradoxical antifertility effects in several species by causing regression of ovarian function. In collagenase-dispersed rat luteal cells, the agonist analog [D-Ser(tBu)6]des-Gly10-GnRH-N-ethylamide (GnRHa) caused inhibition of hCG-stimulated progesterone production. Epinephrine- stimulated progesterone production in isolated luteal cells was also partially inhibited by the GnRH analog. On the other hand, there was no effect of the analog on the steroid doseresponse curve to dibutyryl cAMP. The effect of the GnRH analog on the steroidogenic response to both hormones was to shift the dose-response curve to the right without changing the maximum production of progesterone. Natural sequence GnRH also inhibited steroid production, with a half-maximal effect occurring at 5.10-9 M in agreement with the binding affinity of GnRH for its gonadal receptors (Ka = 5 × 108 M-1). GnRHa also caused a shift to the right in the dose-response curve for cAMP production stimulated by hCG. However, no direct inhibitory effects of GnRH and its agonist analogs could be detected on hCG- or epinephrine-stimulated adenylate cyclase activity measured in ovarian homogenates. GnRH did not affect the binding affinity or capacity of rat ovarian receptors for hCG. Specific GnRH receptors in luteinized rat ovaries were identified with [l25I]GnRHa and found to have high affinities for the analog (Ka = 5 × 109 M-1). A potent antagonist analog ([D-pGlu1,D-Phe2,DTrp36]GnRH) was also bound with high affinity (Ka = 3 times; 109 M-1) by the ovarian receptors. GTP, guanosine 5′-(β,γ-imido) triphosphate, and ATP as well as prostaglandin F2a and epinephrine did not alter binding of the analog to GnRH receptors. These studies demonstrate that the ovary contains specific, high affinity receptors for GnRH, through which GnRH and its gonist analogs can inhibit steroidogenic responses to hormonal stimulation. Since there was no inhibition of dibutyryl cAMPstimulated progesterone production and no direct action on hCG binding, it is suggested that GnRH interferes with the mechanism of hormonal stimulation of cAMP production.

Direct inhibition of testicular function by gonadotropin-releasing hormone: mediation by specific gonadotropin-releasing hormone receptors in interstitial cells.

Agonist analogs of gonadotropin-releasing hormone (GnRH) have been shown to exert antigonadal effects in male and female animals. In hypophysectomized male rats treated with follicle-stimulating hormone, administration of a potent GnRH agonist caused depletion of luteinizing hormone and prolactin receptors and marked suppression of serum testosterone levels. The possibility that such direct effects of GnRH agonists on testicular function could be expressed through specific receptors located in the interstitial cells of the testis was supported by the selective concentration of a 125I-labeled GnRH agonist by the testis in vivo. Specific receptors for the releasing hormone were demonstrated in testis particles and dispersed interstitial cells by direct binding analysis with the 125I-labeled GnRH agonist. The binding affinity (Ka = G X 10(9) M-1) and peptide specificity of the testicular GnRH binding sites were similar to those of anterior pituitary and ovarian GnRH receptors. The presence of GnRH receptors in the testis indicates that these sites mediate the direct inhibitory actions of GnRH agonists upon testicular endocrine function.