The effect of luteinizing hormone releasing hormone and anti-luteinizing hormone releasing hormone antibodies on chorionic gonadotropin and progesterone secretion by human placental villiin vitro

Gonadotropin releasing hormone has been located and found to be secreted by the human placenta in culture. Addition of the releasing hormone upto 1μg concentration in the placental cultures brings about stimulation of chorionic gonadotropin and progesterone secretion. Higher amounts of the decapeptide has an inhibitory influence on both the gonadotropin and the steroid production. The action of the releasing hormone on the placenta could be blocked by the anti-luteinizing hormone releasing hormone monoclonal antibodies indicating a possible site of action of the antibodies for control of fertility     

GnRH in non-hypothalamic reproductive tissues

Gonadotropin releasing hormone (GnRH) is a hypothalamic neuronal secretory decapeptide that plays a pivotal role in mammalian reproduction. GnRH and its analogues are used extensively in the treatment of hormone dependent diseases and assisted reproductive technology. Fourteen structural variants and three different forms of GnRH, named as hypothalamic GnRH or GnRH-I, mid brain GnRH or GnRH-II and GnRH-III across various species of protochordates and vertebrates have been recognised. The hormone acts by binding to cell surface transmembrane G protein coupled receptors (GPCRs) and activates Gq/11 subfamily of G proteins. Although hypothalamus and pituitary are the principal source and target sites for GnRH, several reports have recently suggested extra-hypothalamic GnRH and GnRH receptors in various reproductive tissues such as ovaries, placenta, endometrium, oviducts, testes, prostrate, and mammary glands. GnRH-II appears to be predominantly expressed in extra pituitary reproductive tissues where it produces its effect by PLC, PKA2, PLD, and AC cell signalling pathways. In these tissues, GnRH is considered to act by autocrine or paracrine manner and regulate ovarian steroidogenesis by having stimulatory as well as inhibitory effect on the production of steroid hormones and apoptosis in ovarian follicle and corpus luteum. In male gonads, GnRH has been shown to cause a direct stimulatory effect on basal steroidogenesis and an inhibitory effect on gonadotropin-stimulated androgen biosynthesis. Recent studies have shown that GnRH is more abundantly present in ovarian, endometrial and prostrate carcinomas. The presence of type-II GnRH receptors in reproductive tissues (e.g. gonads, prostrate, endometrium, oviduct, placenta, and mammary glands) suggests existence of distinct role(s) for type-II GnRH molecule in these tissues. The existence of different GnRH forms indicates the presence of distinctive cognate receptors types in vertebrates and is a productive area of research and may contribute to the development of new generation of GnRH analogues with highly selective and controlled action on different reproductive tissues and the target-specific GnRH analogues could be developed.     

Gonadotropin-releasing hormone and ovarian cancer: a functional and mechanistic overview

The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) is well known for its role in the control of pituitary gonadotropin secretion, but the hormone and receptor are also expressed in extrapituitary tissues and tumor cells, including epithelial ovarian cancers. It is hypothesized that they may function as a local autocrine regulatory system in nonpituitary contexts. Numerous studies have demonstrated a direct antiproliferative effect on ovarian cancer cell lines of GnRH and its synthetic analogs. This effect appears to be attributable to multiple steps in the GnRH signaling cascade, such as cell cycle arrest at G(0)/G(1). In contrast to GnRH signaling in pituitary gonadotropes, the involvement of G(alpha q), protein kinase C and mitogen-activated protein kinases is less apparent in neoplastic cells. Instead, in ovarian cancer cells, GnRH receptors appear to couple to the pertussis toxin-sensitive protein G(alpha i), leading to the activation of protein phosphatase, which in turn interferes with growth factor-induced mitogenic signals. Apoptotic involvement is still controversial, although GnRH analogs have been shown to protect cancer cells from doxorubicin-induced apoptosis. Recently, data supporting a regulatory role of GnRH analogs in ovarian cancer cell migration/invasion have started to emerge. In this minireview, we summarize the current understanding of the antiproliferative actions of GnRH analogs, as well as the recent observations of GnRH effects on ovarian cancer cell apoptosis and motogenesis. The molecular mechanisms that mediate GnRH actions and the clinical applications of GnRH analogs in ovarian cancer patients are also discussed.     

Gonadotropin releasing hormone in first trimester human placenta: Isolation,partial characterisation andin vitro biosynthesis

Using a specific radioimmunoassay for gonadotropin releasing hormone, the presence of gonadotropin releasing hormone like material in the first trimester human placenta has been demonstrated. The material has been partially characterized using carboxy methyl cellulose chromatography, high pressure gel permeation chromatography and reverse phase C18 high pressure liquid chromatographic analysis. Analysis for bioactivity revealed that placental gonadotropin releasing hormone is much more active than synthetic gonadotropin releasing hormone inin vitro rat pituitary lutinising hormone release assay.In vitro biosynthetic studies using labelled precursors and immunoaffinity chromatography indicated that first trimester human placenta synthesizes gonadotropin releasing hormone like material.     

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-Ala17]pHGnRH 14-26 and [D-Trp22]pHGnRH 14-26, with D-amino acid substitutions inside and peripheral to this decapeptide sequence were chemically synthesized. pHGnRH 14-26 and the D-Ala17 analogue were inactive and GnRH, pHGnRH 14-36 and the D-Trp22 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.     

Steroid-protein interaction in human placenta

Human placenta produces a large variety of bioactive substances with endocrine and neural competence: pituitary and gonadal hormones, hypothalamic-like releasing or inhibiting hormones, growth factors, cytokines and neuropeptides. The most recent findings indicate that locally produced hormones regulate the secretion of other placental hormones supporting a paracrine/autocrine regulation. In placental endocrinology, a particular relevance is played by steroid hormones. In fact, a specific gonadotropin-releasing hormone (GnRH)-human chorionic gonadotropin (hCG) regulation of placental steroidogenesis has been proposed as a placental internal regulatory system acting on steroids production from human placenta. In addition, activin and inhibin have been proposed as further regulatory substances of the synthesis and secretion of steroids; the addition of activin A to placental culture augments GnRH, hCG and progesterone, and this effect can be significantly reduced by the addition of inhibins. Finally, a steroid-steroid interaction is suggested by the evidence that placental estrogen has a positive role in the regulation of progesterone biosynthesis. Other steroid-protein interactions have been observed in human placenta. In fact, recent data indicate that progesterone inhibits placental corticotropin-releasing factor (CRF) and estrogens act on placental conversion of cortisol to cortisone, activating cortisol secretion by the fetal adrenal and enhancing fetal adrenal function with advancing gestation.     

Effect of phorbol ester on stimulus-secretion coupling mechanisms in gonadotropin releasing hormone-stimulated pituitary gonadotrophs

The feedback regulatory control mechanism exerted by activated Ca2+/phospholipid-dependent protein C kinase upon gonadotropin releasing hormone (GnRH) binding, stimulation of phosphoinositide turnover and gonadotropin secretion was investigated in cultured pituitary cells. Addition of the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), at concentrations which activate pituitary protein C kinase, to cultured pituitary cells resulted in up-regulation of GnRH receptors (155% at 4 h). The stimulatory effect of GnRH on [3H]inositol phosphates (Ins-P) production in myo-[2-3H]inositol prelabeled pituitary cells was not inhibited by prior treatment of the cells with TPA (10(-9)-10(-7) M). Higher concentrations of TPA (10(-6)-10(-5) M) inhibited the effect of GnRH on [3H]Ins-P production. Increasing concentrations of TPA or the permeable analog of diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) stimulated luteinizing hormone (LH) release from cultured pituitary cells with ED50 values of 5 x 10(-9) M and 10 micrograms/ml, respectively. No consistent inhibition or additivity of LH release was observed when increasing doses of TPA or OAG were added with a submaximal dose of GnRH. These results suggest that protein C kinase might mediate the known homologous up-regulation of GnRH receptors during the reproductive cycle. Protein C kinase is positively involved in mediating the process of gonadotropin secretion. Unlike many other systems, activation of protein C kinase in pituitary gonadotrophs is not involved in negative feed-back regulation of stimulus-secretion-coupling mechanisms in GnRH-stimulated gonadotrophs.     

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 $\text{in vitro}$. A labeled GnRH agonist, [des-Gly¹⁰, D-Ser (TBu)⁶, Pro⁹-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.     

Presence of immunoreactive gonadotropin releasing hormone (GnRH) and its receptor (GnRHR) in rat ovary during pregnancy

The present study aims at quantification of gonadotropin releasing hormone (GnRH) by radioimmunoassay, relative expression of its mRNA by real-time PCR accompanied by its cellular localization in the rat ovary by immunonohistochemistry (IHC) during different time points of pregnancy. To determine the involvement of endogenous ovarian GnRH in receptor mediated local autocrine/paracrine functions within the ovary, the cell specific localization of the classical receptor for GnRH (GnRHR) in the ovary by IHC and expression pattern of its mRNA were studied during pregnancy. Receptor expression during each time point within the ovary was reconfirmed by Western blot analysis accompanied by densitometric analysis of the signal intensity. Results reveal that the content of ovarian GnRH reaches its maximum on Day 20. The densitometric analysis of GnRHR receptor expression from Western blot study exhibits a decreasing trend by Day 20. Presence of GnRH and GnRHR mRNA in the ovary indicates the local synthesis of both ligand and receptor in the rat ovary. Differential expression of GnRH/GnRHR in the corpus luteum throughout pregnancy strengthens the hypothesis of the involvement of ovarian GnRH in local ovarian functions by receptor-mediated mechanisms. The expression of GnRH and GnRHR in the atretic antral follicles is indicative of the possible involvement of this decapeptide in processes like follicular atresia. The expression of GnRH/GnRHR in the nonatretic antral follicles and their oocytes requires further in-depth investigation. Collectively, this study for the first time reveals the presence of endogenous ovarian GnRH/GnRHR supporting their possible involvement in local autocrine/paracrine functions during pregnancy.     

Agonist-induced regulation of pituitary receptors for gonadotropin-releasing hormone. Dissociation of receptor recruitment from hormone release in cultured gonadotrophs

The regulation of receptors for gonadotropin-releasing hormone (GnRH) by the homologous decapeptide ligand was analyzed in cultured rat anterior pituitary cells. Assay of GnRH receptors in both intact and disrupted cells showed that GnRH binding to gonadotrophs was rapidly followed by dose-dependent loss of sites that was maximal within 1 h. This early loss of GnRH receptors was not dependent on protein synthesis, and was attributable to ligand-induced processing of the peptide binding sites. No loss of GnRH sites was observed after receptor occupancy by a GnRH antagonist, or after target cell activation by exposure to a depolarizing concentration of KCl to stimulate luteinizing hormone release. After their initial down-regulation, GnRH receptors returned to normal and subsequently increased in concentration after 6 h of incubation. The delayed phase of receptor up-regulation was prevented by treatment with cycloheximide or actinomycin D and was calcium-dependent, being induced by 50 mM KCl and by low concentrations of the calcium ionophore, A23187. Conversely, calcium antagonists such as verapamil and MgCl2 impaired the agonist-induced increase of GnRH receptor sites. These findings have demonstrated that pituitary GnRH receptors undergo two distinct phases of regulation after interaction with the homologous ligand. The initial phase of agonist-dependent receptor loss is followed by a postsecretory phase of receptor recruitment that is dependent on protein synthesis. The expression of GnRH receptors can be completely dissociated from gonadotropin secretion, indicating that fusion of luteinizing hormone secretory granules with the plasma membrane is not a major pathway for transport of GnRH receptors to the cell surface in cultured gonadotrophs. Such changes in cell surface GnRH receptors during activation by the peptide agonist are relevant to the alterations in gonadotroph sensitivity that occur in vivo during physiological regulation of the pituitary gland by GnRH.     

Regulation of gonadotropin secretion in the anterior pituitary

Studies on the regulation of gonadotropin secretion in dissociated pituitary cell cultures are described. Initial studies employing a ferritin-labelled analogue of gonadotropin hormone releasing hormone (GnRH) to localize its receptor sites on the gonadotropin cell surface that while these receptor sites initially have a random monodisperse distribution, binding of the ligand causes coarse aggregation and internalization of the GnRH receptor. These events are not due to the multivalency of the ligand and probably reflect redistributive events in vivo. By using an octapeptide analogue GnRH that binds to the GnRH receptor but lacks gonadotropin releasing activity in conjunction with sequence-specific antisera it is shown that antibodies that bind the octapeptide can induce the octapeptide to release gonadotropin. These data suggest that receptor aggregation is important in GnRH stimulation. Finally immunocytochemical studies are described in which golg-protein-A-antibody complexes are used to identify gonadotropins on ultrathin frozen sections of porcine pituitary cells. These studies indicate that in porcine gonadotropin cells the majority of the secretory granules contain both luteinizing hormone and follicle-stimulating hormone.     

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.     

蒙药乌力吉-18对大鼠下丘脑-垂体-卵巢轴相关激素及受体表达的影响

目的:探讨蒙药乌力吉-18对大鼠下丘脑-垂体-卵巢轴相关激素及受体的影响。方法:选取40只健康雌性未孕SD大鼠,随机分为空白组、对照组、乌力吉-18高、低2个剂量组,每组10只。空白组灌胃等体积蒸馏水,对照组灌胃逍遥丸,高、低剂量组分别灌胃2.0 g·kg^-1·d^-1、1.0 g·kg^-1·d^-1乌力吉-18,连续给药31学艺术d。采用酶联免疫吸附法测定血清促性腺激素释放激素(GnRH)、促卵泡生成素(FSH)、黄体生成素(LH)、雌二醇(E₂)及孕酮(PROG)的含量;免疫组化法检测下丘脑组织促性腺激素释放激素(GnRH)、垂体组织促性腺激素释放激素受体(GnRHR)的表达;以蛋白免疫印迹技术检测卵巢组织促卵泡生成素受体(FSHR)、黄体生成素受体(LHR)蛋白表达量。以实时荧光定量PCR检测卵巢组织中FSHR、LHR基因表达量。结果:与空白组比较,乌力吉-18低剂量组可明显升高血清LH含量(P<0.05),上调下丘脑组织GnRH、垂体组织GnRHR表达及卵巢组织FSHR、LHR蛋白表达(P<0.05);乌力吉-18高剂量组可显著升高血清FSH、LH、E₂含量(P<0.05),上调下丘脑组织GnRH表达及卵巢组织FSHR表达量(P<0.05),并可显著升高卵巢组织中FSHR、LHR基因表达量(P<0.05);对照组可明显升高血清E₂含量(P<0.05)。结论:蒙药乌力吉-18可明显升高血清FSH、LH及E₂的含量,促进下丘脑组织GnRH、垂体组织GnRHR及卵巢组织中FSHR、LHR的表达,表明乌力吉-18能够对下丘脑-垂体-卵巢轴相关激素及受体表达产生影响。     

Evolutionary aspects of gonadotropin-releasing hormone and its receptor

Summary 1. Gonadotropin-releasing hormone (GnRH) was originally isolated as a hypothalamic peptide hormone that regulates the reproductive system by stimulating the release of gonadotropins from the anterior pituitary. However, during evolution the peptide was subject to gene duplication and structural changes, and multiple molecular forms have evolved.2. Eight variants of GnRH are known, and at least two different forms are expressed in species from all vertebrate classes: chicken GnRH II and a second, unique, GnRH isoform.3. The peptide has been recruited during evolution for diverse regulatory functions: as a neurotransmitter in the central and sympathetic nervous systems, as a paracrine regulator in the gonads and placenta, and as an autocrine regulator in tumor cells.4. Evidence suggests that in most species the early-evolved and highly conserved chicken GnRH II has a neurotransmitter function, while the second form, which varies across classes, has a physiologic role in regulating gonadotropin release.5. We review here evolutionary aspects of the family of GnRH peptides and their receptors.     

Gonadotropin releasing hormone activation is mediated by dimerization of occupied receptors

A dinitrophenyl (DNP)-derivative of a gonadotropin releasing hormone (GnRH) antagonist was prepared by chemical modification of the epsilon amino group in position 6 of [D-pGlu1,D-Phe2,D-Trp3,D-Lys6]GnRH with 1-fluoro-2, 4-dinitrobenzene. The DNP-antagonist D-pGlu-D-Phe-D-Trp-Ser-Tyr-D-Lys(N epsilon-DNP)-Leu-Arg-Pro-Gly-NH2, retained high affinity binding to the GnRH receptor of pituitary membrane preparations and exhibited antagonistic activity when assayed in cultured pituitary cells. Both antibodies against DNP and their Fab fragments were able to bind the DNP-antagonist. However, only the addition of bivalent antibodies (and not the Fab fragments) converted the DNP-antagonist to an agonist. These results suggest that divalency is a critical factor in GnRH action.     

Studies on GnRH agonist suppression of estrogen production in patients with endometriosis

The chronic administration of GnRH agonists to women results in the reversible suppression of estrogen production by the ovary. In the present study, the mechanism of the GnRH agonist suppression of estrogen production was investigated in patients with endometriosis. During the treatment with intranasal buserelin spray, the concentration of serum estradiol-17 beta (E2) was suppressed to near-castrate levels. Despite this marked suppression of serum E2, immunoreactive LH and FSH levels in serum were not changed. On the other hand, serum bioactive LH was markedly reduced. It was also observed during the treatment that the pituitary LH pulse disappeared and pituitary response to exogenous GnRH was significantly suppressed. In contrast, ovarian response to human menopausal gonadotropin (hMG) was not altered during the treatment. These findings suggest that the GnRH agonist suppression of estrogen production in the patients with endometriosis is through both suppression of the secretion of biologically active LH and the reduction of the LH pulse, but not through a direct inhibitory effect on ovarian estrogen biosynthesis.     

Preparation and characterization of photoreactive derivatives of GnRH. Persistent activation of function by photoaffinity labeling

A photoreactive derivative of the highly potent gonadotropin releasing hormone (GnRH) agonist, D-Lys6-GnRH(1-9)-ethylamide, was prepared by selective modification of the epsilon-amino group with 2-nitro-4-azidophenyl sulfenyl chloride (2,4-NAPS C1). The modified peptide [D-Lys(NAPS)]6-GnRH-(1-9)-ethylamide was found to be a full agonist of LH release from rat pituitary cells with a relative potency 23 compared to GnRH. Covalent attachment of the photoreactive analog to rat pituitary cells resulted in prolonged activation of LH secretion which could not be inhibited by a potent GnRH antagonist. Persistent stimulation of pituitary gonadotrophs caused by covalently bound hormone led to desensitization of the LH releasing mechanism.     

Novel antitumor peptide hormones and their effect on signal transduction.

A series of novel gonadotropin releasing hormone (GnRH) and Somatostatin analogs have been developed in our laboratory and were screened for antiproliferative and signal transduction inhibitory effect. Our GnRH analog Folligen, had significant antitumor activity on DMBA induced mammary carcinomas in rats without blocking ovarian functions. The direct effect of Folligen and Buserelin has been compared on the human breast cancer cell line MDA-MB-231. Folligen was found to be more effective in inhibiting cell proliferation and significant differences were found in the signal transduction pathways activated by these analogs. Our novel Somatostatin analogs were screened for tyrosine kinase inhibition and for antiproliferative effect on human colon tumor cells and for growth hormone (GH) release inhibition in vitro and in vivo. The analog TT-2-50 was significantly more active inhibiting GH release in superfused rat pituitary cells and in vivo than native Somatostatin and it strongly inhibited tyrosine kinase and proliferation while it stimulated protein kinase C activity.     

Effects of chronic treatment with a GnRH agonist on oestrous behaviour and on the secretion of LH and progesterone in the ewe

A study was carried out to investigate a novel approach to oestrus synchronization in the ewe by treatment with a gonadotrophin releasing hormone (GnRH) agonist. Groups of ewes were initially treated on Day 2, 10 or 14 of the oestrous cycle with 10 mug GnRH analogue (D-Ser(Bu(t)) 6 des Gly GnRH ethylamide) per ewe per day for 14 days. Behavioural oestrus was inhibited during GnRH agonist treatment and recurred from 8 to 38 days after the treatment in an unsynchronized manner. Luteal activity during treatment was not impaired but reduced progesterone concentrations occurred in cycles after the treatment. The rhythm of ovarian function, generally characterized by prolonged follicular development, was impaired. During the treatment and subsequent recovery period, integrity of pituitary function was examined by measuring luteinizing hormone (LH) after GnRH agonist was injected, and after stimulation test doses of 150 ng natural GnRH were administered. During treatment there was, with time, a decline in pituitary response to the agonist which suggested that pituitary release of LH was exhausted. After the 14-day treatment the stimulation test with GnRH revealed a gradual return to normal responsiveness although this was not complete three weeks after the treatment when compared to control ewes. This lowered pituitary activity could cause the impaired ovarian function.