Gonadotropin-releasing Hormone Binding Sites Research Articles

Targeting Gonadotropin-releasing Hormone Receptor Inhibits the Early Step of Ovarian Cancer Metastasis by Modulating Tumor-mesothelial Adhesion

Ovarian cancer has a clear predilection to metastasize to the peritoneum, which represents one of the most important prognostic factors of poor clinical outcome. Gonadotropin-releasing hormone (GnRH) receptor is significantly overexpressed during the malignant progression of human ovarian cancer. Here, using lentiviral-based small interfering RNA (siRNA) technology to downregulate GnRH receptor in metastatic ovarian cancer cells, we show that GnRH receptor is an important mediator of ovarian cancer peritoneal metastasis. GnRH receptor downregulation dramatically attenuated their adhesion to the peritoneal mesothelium. By inhibiting the expression of GnRH receptor, we showed decreased expression of α2β1 and α5β1 integrin and adhesion to specific extracellular matrix (ECM) proteins. This was also associated with a reduction of P-cadherin. Furthermore, adhesion of ovarian cancer cells to different ECMs and the mesothelium were abrogated in response to β1 integrin and P-cadherin reduction, confirming that the effects were β1 integrin- and P-cadherin-specific. Using a mouse model of human ovarian cancer metastasis, we found that the inhibition of GnRH receptor, β1 integrin, and P-cadherin significantly attenuated tumor growth, ascites formation, and the number of metastatic implants. These results define a new role for GnRH receptor in early metastasis and offer the possibility of novel therapeutic targets.

Expression of gonadotrophin-releasing hormone binding sites in somatic tissues of the gilthead seabream (Sparus aurata): a quantitative autoradiographic study

In this study, we have analysed the expression of gonadotrophin-releasing hormone (GnRH) binding sites in somatic tissues (intestine, liver, gill, skeletal muscle, ovary, heart, stomach, kidney and spleen) of the gilthead seabream, Sparus aurata using 3-[125I]iodototyrosyl5-mammalian GnRH and auto-radiographic techniques. The qualitative and quantitative analysis showed the existence of a basal expression of specific GnRH binding sites in intestine, skeletal muscle, ovary, stomach and spleen. Furthermore, our data suggest that the level of expression of GnRH binding sites can be significantly enhanced by GnRH treatment in intestine, gill, heart, stomach, kidney and spleen. This study shows that GnRH can exert direct effects in both reproductive and non-reproductive somatic tissues of the gilthead seabream.

Gonadotropin-releasing hormone receptor expression in the human prostate.

Inhibitory effects of gonadotropin-releasing hormone (GnRH) analogs on prostate cancer cell proliferation, both in vivo and in vitro, indicate the presence of specific binding sites for GnRH on prostate cancer cells. To investigate this issue further, we examined the expression of GnRH receptor (GnRH-R) mRNA and protein in human prostate biopsies as well as in other extrapituitary tissues. The relative quantity of GnRH-R mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) in human prostate biopsies. Extrapituitary GnRH-R levels were determined by a semiquantitative PCR reaction. Using PCR, a relatively high expression level of GnRH-R mRNA was found in prostate tumor tissue followed by normal prostate, thymus, and kidney expression levels. The levels showed by heart, brain, placenta, lung, liver, skeletal muscle, pancreas, colon, ovary, small intestine, spleen, and testis were low but detectable, whereas peripheral blood leukocyte showed no demonstrable product. GnRH-R immunoreactivity was localized in both luminal and basal epithelial cells in benign and malignant prostate tissue, and GnRH-R were also observed in intraprostatic lymphocytes. The relative GnRH-R mRNA levels in prostate biopsies from 16 patients showed a wide range of individual differences, but these differences were not related to histological grade. Castration therapy did not significantly influence GnRH-R mRNA expression in normal and malignant prostate tissue. These results suggest that epithelial cells and infiltrating lymphocytes are targets for GnRH action in the human prostate. Comparative data show relatively high GnRH-R expression in human prostate tissue compared to other human tissues.

Embryo implantation and GnRH antagonists: the search for the human placental GnRH receptor.

Hypothalamic gonadotrophin-releasing hormone (GnRH) plays a major role in the endocrine control of reproduction. Acting through its high affinity receptors on pituitary gonadotrophs, it regulates the secretion of gonadotrophins. In addition, GnRH also functions as a local regulator in a number of other cell lines and tissues, including the placenta. In a manner analogous to hypothalamic GnRH stimulation of LH and FSH from the anterior pituitary, GnRH was found to cause a dose-dependent release of human chorionic gonadotrophin (HCG) from the placenta. So began the search for a putative GnRH receptor in the human placenta. Although early radio-receptor studies reported specific binding, the properties of these 'putative' GnRH binding sites were found to differ significantly from those of their pituitary counterparts in several important respects. This was followed by a series of contradictory reports that led to more questions and opened up avenues for further investigations. Even after nearly two decades of research, the human placental GnRH receptor has not been characterized beyond all reasonable doubt. This review recalls the discovery, the controversies and unanswered questions concerning the human placental GnRH receptor.

Gene expression of gonadotropin-releasing hormone and its receptor in rat pancreatic cancer cell lines.

The regression of experimental and clinical pancreatic cancers by treatment with gonadotropin-releasing hormone (GnRH) agonists or antagonists has been repeatedly reported and is usually presumed to result from the creation of a sex steroid deficiency. There are, however, indications that GnRH analogs can also suppress the growth of the tumor cells in vitro and that specific binding sites for GnRH are present on membranes of these cells. The regulatory role of GnRH in rat pancreatic adenocarcinoma was investigated by examining the gene for GnRH and GnRH receptor (GnRH-R) in two pancreatic tumor cell lines (AR42J and ARIP). Reverse transcriptase polymerase chain reaction and Southern blot analysis indicated both GnRH-mRNA and GnRH-R-mRNA transcripts in the two cell lines. This is the first report raising the possibility of an autocrine/paracrine role for GnRH in rodent malignant pancreas.

GnRH action on luteal steroidogenesis during pregnancy 1

The results of our study presented here establishes that gonadotropin-releasing hormone (GnRH) acts directly on the corpus luteum, leading to suppressed production and release of progesterone and thus disrupting pregnancy. A GnRH-agonist (GnRH-Ag) treatment suppressed the luteal and serum progesterone levels. This suppression is neither mediated by a fall in ovarian testosterone production nor its conversion to estradiol. Although the treatment suppressed the nuclear estradiol-receptor content and binding sites for LH in the corpus luteum, it had no effect on the luteal binding sites for GnRH and prolactin within 24 h. GnRH-Ag augmented the plasma levels of luteinizing hormone, decreased the magnitude of nocturnal surges of prolactin, and had no effect on luteal cyclic adenosine 5′-monotriphosphate levels. Yet, the treatment had no effect on the luteal content of free cholesterol. We have also demonstrated, for the first time, the presence of steroidogenic acute regulatory protein and peripheral benzodiazepine receptor in the rat corpus luteum, and the suppression of these proteins by GnRH-Ag leads to reduced steroidogenesis by the corpus luteum. Concomitantly, P450 side-chain cleavage enzyme, its activity, and its mRNA content and 3β-hydroxy-steroid dehydrogenase content in the corpus luteum decreased. The treatment suppressed the plasma levels of pregnenolone and 20α-dihydroprogesterone. These data suggest that the suppression of luteal steroidogenesis by GnRH-Ag may be due to its inhibitory effect on the cholesterol transport and/or on the enzymes involved in the steroidogenic pathway. Furthermore, based on other observations made in our laboratory, we propose a hypothesis that an endogenous GnRH is present in the corpus luteum/ovary during pregnancy in the rat and that this GnRH may play a physiological role in the regulation, maintenance, and/or termination of pregnancy.

Delta Opiate Receptors Account for the Castration-Induced Unmasking of Gonadotropin-Releasing Hormone Binding Sites in the Rat Pituitary

Under control incubation conditions, gonadotropin-releasing hormone (GnRH) binds only a fraction of its receptors in rat-cultivated pituitary cells. Unmasking of the remaining receptors, which have been termed ‘cryptic’, requires drug- or peptide-induced protein kinase activation. Spontaneous masking however is not observed on pituitary cells sampled from castrated male rats, suggesting the presence of an intrinsic unmasking factor. Many endogenous factors could theoretically account for the effect. Here we attempted to identify the factor involved by taking advantage of their differential dependency upon second messengers and transduction cascades. Spontaneous unmasking of GnRH binding was found reversed by pertussis toxin (PTX), an inhibitor of α_i and α_o subunits of heterotrimeric G proteins, and by U73122, a phospholipase C (PLC) inhibitor. In contrast, desensitization of protein kinase C (PKC) or inhibition of tyrosine kinase by herbimycin were ineffective. Among endogenous pituitary factors able to unmask GnRH receptors in pituitary cells from normal male rats, as EGF, NPY or opiate peptides, only the latter were found to correspond to this transduction profile. In an attempt to characterize the pharmacology of opiate effects, naloxone (10 µM), a poorly selective opiate antagonist, restored masking of GnRH binding in cells from castrates. Only the δ antagonist naltrindole (1 µM) was able to mimick the action of naloxone. Conversely, when tested on cells from intact animals, morphine (10 µM), as well as dslet (1 µM) and met-ENK (10 nM), preferential δ agonists, but not dago and β-endorphin or U50488 H and dynorphin, respectively µ and ĸ agonists, were able to suppress masking. Among opioid peptides endogenous to the pituitary, only met-ENK was able to unmask cryptic receptors, an effect antagonized by naltrindole. We conclude that an opiate δ receptor subtype is endogenously activated in the pituitary of castrated male rats to prevent masking of GnRH binding.

Adenocarcinoma Cells Are Targeted by the New GnRH-PE66 Chimeric Toxin through Specific Gonadotropin-releasing Hormone Binding Sites

Luteinizing hormone-releasing hormone, also termed gonadotropin-releasing hormone (GnRH), accounts for the hypothalamic-pituitary gonadal control of human reproduction. The involvement of GnRH has been demonstrated in several carcinomas of hormone-responsive tissues. Exploiting this common feature, we constructed a Pseudomonas exotoxin (PE)-based chimeric toxin (GnRH-PE66) aimed at targeting those cancer cells bearing GnRH binding sites. We report here the strong growth inhibition and killing of a surprisingly wide variety of cancers, confined to the adenocarcinoma type. These cancer cells arising from hormone-responsive tissues, as well as non-responsive ones, express specific GnRH binding sites as indicated by the marked killing of ovarian, breast, endometrial, cervical, colon, lung, hepatic, and renal adenocarcinoma. This cytotoxicity is specific as it could be blocked upon addition of excess GnRH. The specificity of GnRH-PE66 chimeric toxin was also confirmed by GnRH binding assays, and its ability to prevent the formation of colon cancer xenografts in nude mice is presented. Although the functional role of specific GnRH binding sites in human carcinomas remains obscure, GnRH-PE66 displays considerable targeting potential and its use as a therapeutic agent for cancer should be considered.

Gonadotrophin-releasing hormone and triptorelin inhibit the follicle stimulating hormone-induced response in human primary cultured granulosa-lutein cells.

Cyclic AMP (c-AMP)-dependent cell shape changes can be used to study the gonadotrophin response in cultured human granulosa-lutein cells. The same approach has been developed here to determine the direct potential antigonadotrophic effect of gonadotrophin-releasing hormone (GnRH) and a GnRH agonist (triptorelin) on human granulosa-lutein cells. Treatment with triptorelin or GnRH alone for 1 h did not affect granulosa-lutein cell morphology. However, in the presence of stimulatory doses of follicle stimulating hormone (FSH), triptorelin (5 x 10(-7)-5 x 10(-6) M) and GnRH (10(-11)-10(-9) M) inhibited the FSH-induced c-AMP-dependent response. The antigonadotrophic effect of triptorelin was prevented by two GnRH antagonists, indicating that triptorelin acts via specific GnRH binding sites. On the other hand, triptorelin failed to inhibit human chorionic gonadotrophin- and forskolin-mediated morphological changes. Our results suggest that the GnRH agonist interacts specifically with the FSH-induced c-AMP-dependent cascade of events, at a site located ahead of that of c-AMP generation. In conclusion, GnRH and triptorelin strongly inhibit FSH-mediated function in human granulosa-lutein cells in culture. This inhibition might play a role in the low follicular development rates observed in some patients treated with GnRH agonist + gonadotrophins for ovarian stimulation.

Measurement of luteal and placental gonadotrophin-releasing hormone (GnRH) binding sites: role of inactivation of GnRH tracer.

The ability of radiolabelled gonadotrophin-releasing hormone (GnRH) and GnRH analogues to bind to homogenates and membranes of rat, rabbit and sheep pituitary and to luteal homogenates and membranes from a number of species was measured. In addition, inactivation of tracer during binding incubation was estimated by measurement of the ability of the unbound tracer fraction to bind to fresh GnRH-receptor or anti-GnRH antibody. Following incubation of rat, sheep and rabbit pituitary gland with a radiolabelled GnRH agonist tracer, appreciable amounts of specific binding of GnRH agonist to fresh rat pituitary or human placental GnRH receptors could still be demonstrated. In contrast, no specific binding of [125I]-labelled GnRH analogues were measured following incubation of the tracer with homogenates or membranes of bovine, sheep and porcine luteal tissue, nor with rabbit and rat placental homogenates. However, during incubation of GnRH tracers with these tissues, almost complete inactivation of GnRH tracers occurred. There was an inverse relationship between binding and inactivation of [125I]-labelled GnRH for a number of human corpora lutea. We conclude that degradation of GnRH tracers may prevent the measurement of specific GnRH binding sites in some tissues.

Expression of gonadotropin-releasing hormone receptor in human epithelial ovarian carcinoma.

We have previously demonstrated the presence of gonadotropin-releasing hormone (Gn-RH) messenger ribonucleic acid (mRNA) in epithelial ovarian carcinoma. In this study, the expression of Gn-RH receptor (Gn-RHR) was investigated in human ovarian carcinoma and human ovarian carcinoma cell line. Gn-RHR was determined by [3H]Gn-RH binding assay. Gn-RHR mRNA was determined by reverse transcription-polymerase chain reaction using oligonucleotide primers synthesized based on published human Gn-RHR sequence. Specific Gn-RH binding sites were shown to be present in plasma membrane isolated from five ovarian mucinous cystadenocarcinoma samples and one serous cystadenocarcinoma (Kd = 15.3 +/- 8.08 nmol/L). Gn-RHR mRNA was detected in four mucinous cystadenocarcinoma specimens, one serous cystadenocarcinoma, and SK-OV-3 cells, but not in white blood cells. These results suggest that Gn-RH may play an autocrine regulatory role in the growth of ovarian carcinoma.

Presence of Gonadotropin-Releasing Hormone Receptor and Its Messenger Ribonucleic Acid in Endometrial Carcinoma and Endometrium

In support of a possible clinical use of gonadotropin-releasing hormone (Gn-RH) analogs in the treatment of the endometrial carcinoma, this study was undertaken to establish the presence and characteristics of Gn-RH receptor on endometrial cancer. Materials were human endometrial carcinomas surgically removed and endometrial carcinoma cell lines. Gn-RH receptor was characterized by [ 3H]Gn-RH binding to plasma membrane preparations. Gn-RH receptor messenger ribonucleic acid (mRNA) was determined by reverse transcription-polymerase chain reaction (PCR) using oligonucleotide primers synthesized according to the published human Gn-RH receptor sequence. Specific Gn-RH binding sites were shown to be present in 16 of 18 well-differentiated and 4 of 7 poorly differentiated adenocarcinoma specimens ( K d = 5.89 ± 3.59 n M, B max , = 1.80 ± 0.95 pmol/mg protein) and cell lines RL95-2 and HHUA with K d of 2.38 ± 0.86 n M. The high-affinity binding sites were also detected in six proliferative-phase endometrium ( K d = 4.24 ± 2.32 n M, B max = 2.73 ± 1.12 pmol/mg protein). Gn-RH receptor mRNA was detected in all endometrial carcinoma and endometrial specimens and cell lines where the specific binding sites were detected, but not in adenomyosis or myometrial samples. The expression of Gn-RH receptor provides a possible point of attack for therapeutic approaches using Gn-RH analogs in this malignancy.

Presence of gonadotropin-releasing hormone and its messenger ribonucleic acid in human ovarian epithelial carcinoma

Objective: The purpose of this study was to investigate the expression of gonadotropin-releasing hormone messenger ribonucleic acid and the presence of gonadotropin-releasing hormone in human ovarian carcinoma known to have gonadotropin-releasing hormone binding sites and to be affected by gonadotropin-releasing hormone analog. Study Design: Human ovarian carcinomas surgically removed and human ovarian carcinoma cell lines were examined. Gonadotropin-releasing hormone was determined by a radioimmunoassay and a bioassy. Gonadotropin-releasing hormone messenger ribonucleic acid was determined by reverse transcription polymerase chain reaction using oligonucleotide primers synthesized according to the published human gonadotropin-releasing hormone sequence. Results: Gonadotropin-releasing hormone was shown to be present in extracts of ovarian mucinous cystadenocarcinoma sample (0.8 ± 0.12 pg/mg of protein) and ovarian adenocarcinoma cell line SK-OV3 (0.92 ± 0.17 pg/mg of protein) but not in the normal ovary and placenta. Two of two extract samples from individual cases evoked dose-dependent phosphoinositide breakdown in rat granulosa cells similar to that caused by authentic gonadotropin-releasing hormone. Gonadotropin-releasing hormone messenger ribonucleic acid was detected in two of two mucinous cystadenocarcinoma specimens, one of one serous cystadenocarcinoma, and SK-OV3 cells but not in the dysgerminoma, mucinous cystadenoma, and normal ovary and placenta. Conclusion: The demonstration of gonadotropin-releasing hormone and its messenger ribonucleic acid raises the possibility that gonadotropin-releasing hormone may play an autocrine regulatory role in the growth of ovarian carcinoma.

Presence of gonadotropin-releasing hormone (GnRH) binding sites and compounds with GnRH-like activity in the ovary of African catfish, Clarias gariepinus.

GnRH binding was characterized in the African catfish ovary by use of an analog of salmon GnRH (sGnRH- ; [D-Arg6, Trp7, Leu8, Pro9-NEt]-GnRH) as a labeled ligand. Binding of sGnRH-A to catfish ovarian membrane preparation was found to be saturable, displaceable, reversible, and dependent on time, temperature, and tissue concentration. Optimal binding was achieved after 70 min of incubation at room temperature (approximately 22 degrees C) at pH 7.6. Addition of unlabeled sGnRH-A displaced the bound 125I-sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis indicated the presence of one class of high-affinity binding sites with a equilibrium dissociation constant (Kd) of 0.27 +/- 0.036 nM. Bound 125I-sGnRH-A was also found to be displaceable by catfish GnRH (cfGnRH; [His5, Leu7, Asn8]-GnRH), chicken GnRH-II (cGnRH-II; [His5, Trp7, Tyr8]-GnRH), and salmon GnRH (sGnRH; [Trp7, Leu8]-GnRH); all the peptides were found to bind with lower affinities than sGnRH-A to the catfish ovarian GnRH binding sites. Further experiments using ovarian extracts indicated the presence of compounds with GnRH-like activity in the ovary of African catfish. The crude ovarian extract was found to stimulate pituitary gonadotropin release from goldfish pituitary, as well as displacing 125I-sGnRH-A binding in the catfish ovary. HPLC analysis of the catfish ovarian extract revealed the presence of two fractions that bind specifically to the catfish ovary and release gonadotropin from cultured goldfish pituitary. These fractions include an early eluting peak that does not correspond with the retention time of known GnRH forms in addition to a fraction that co-elutes with the mammalian GnRH. Overall, the study provided characterization of GnRH binding sites in the catfish ovary, and evidence for the presence of compounds with GnRH-like activity in the catfish ovary.

Gonadotropin‐releasing hormone (GnRH) binding characteristics in the testis of goldfish (Carassius auratus)

AbstractGonadotropin‐releasing hormone (GnRH) binding sites were characterized in the goldfish testis using an analog of salmon GnRH ([D‐Arg6, Trp7, Leu8, Pro9‐NEt]‐GnRH; sGnRH‐A) as a labeled ligand. Binding of sGnRH‐A to goldfish testicular membrane homogenate was found to be saturable, reversible, and dependent on time, temperature, and tissue concentration. Addition of unlabeled sGnRH‐A displaced the bound 125 I‐sGnRH‐A in a dose‐related manner. Hill plot as well as Scatchard analysis indicated the presence of two classes of binding sites, a high affinity/low capacity site and a low affinity/high capacity site in the goldfish testis. Bound 125I‐sGnRH‐A was also found to be displaceable by sGnRH ([Trp7, Leu8]‐GnRH) and cGnRH‐II ([His5, Trp7, Tyr8,]‐GnRH) which occur naturally in the goldfish brain. sGnRH‐A and cGnRH‐II were found to bind with greater affinity than sGnRH to the goldfish testicular GnRH binding sites. The findings provide the first characterization of testicular GnRH binding sites in a teleost species. © 1993 Wiley‐Liss, Inc.

Presence of gonadotropin-releasing hormone and its messenger ribonucleic acid in human ovarian epithelial carcinoma

The purpose of this study was to investigate the expression of gonadotropin-releasing hormone messenger ribonucleic acid and the presence of gonadotropin-releasing hormone in human ovarian carcinoma known to have gonadotropin-releasing hormone binding sites and to be affected by gonadotropin-releasing hormone analog. Human ovarian carcinomas surgically removed and human ovarian carcinoma cell lines were examined. Gonadotropin-releasing hormone was determined by a radioimmunoassay and a bioassay. Gonadotropin-releasing hormone messenger ribonucleic acid was determined by reverse transcription polymerase chain reaction using oligonucleotide primers synthesized according to the published human gonadotropin-releasing hormone sequence. Gonadotropin-releasing hormone was shown to be present in extracts of ovarian mucinous cystadenocarcinoma sample (0.8 +/- 0.12 pg/mg of protein) and ovarian adenocarcinoma cell line SK-OV3 (0.92 +/- 0.17 pg/mg of protein) but not in the normal ovary and placenta. Two of two extract samples from individual cases evoked dose-dependent phosphoinositide breakdown in rat granulosa cells similar to that caused by authentic gonadotropin-releasing hormone. Gonadotropin-releasing hormone messenger ribonucleic acid was detected in two of two mucinous cystadenocarcinoma specimens, one of one serous cystadenocarcinoma, and SK-OV3 cells but not in the dysgerminoma, mucinous cystadenoma, and normal ovary and placenta. The demonstration of gonadotropin-releasing hormone and its messenger ribonucleic acid raises the possibility that gonadotropin-releasing hormone may play an autocrine regulatory role in the growth of ovarian carcinoma.

Extrapituitary gonadotropin-releasing hormone (GnRH) binding sites in goldfish

In teleosts, as in other vertebrates, the secretion of pituitary gonadotropin (GTH) is mediated by the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH). Recent findings in teleosts indicate that GnRH receptors are not restricted to the pituitary gonadotropes and are also associated with somatotropes as well as being present in a number of other tissues. In the present study, we provide novel information on GnRH binding in a number of extrapituitary tissues in goldfish. However, we do not intend to provide full characterization of GnRH binding sites in various extrapituitary tissues in goldfish as this would clearly be outside the scope of this paper. In this study we examined GnRH binding in a number of extrapituitary tissues in goldfish and observed specific binding in ovary, testis, brain, liver and kidney. No specific GnRH binding was observed in muscle, skin, gut, gill and heart. In general, the present findings together with the results of other studies carried out in our laboratory demonstrate that mature goldfish ovary and testis contain two classes of GnRH binding sites, high affinity/low capacity and low affinity/high capacity sites with binding characteristics similar to those of the pituitary GnRH receptors. The brain of goldfish was also found to contain two classes of GnRH binding sites, a super-high affinity/low capacity and a low affinity/high capacity sites. Furthermore, study of goldfish liver and kidney demonstrated the presence of a single class of GnRH binding sites with characteristics different from those of pituitary, ovary, testis and brain. Overall, it is evident that goldfish contains a family of GnRH binding sites which can be classified into four groups based on binding affinities: 1) A class of high affinity binding sites present in the pituitary, ovary and testis, 2) a class of super high affinity sites so far only detected in the brain, 3) a class of intermediate-affinity GnRH binding sites in the liver and kidney, and 4) a class of low affinity binding sites present in all the tissues containing specific GnRH binding sites except for liver and kidney.

Characterization of gonadotropin-releasing hormone receptors in goldfish ovary: variation during follicular development

Gonadotropin-releasing hormone (GnRH) receptors were characterized in the goldfish ovary using an analogue of salmon GnRH ([D-Arg6,Trp7,Leu8,Pro9-NEt]GnRH; sGn-RH-A) as a labeled ligand. Binding of sGnRH-A to goldfish follicular membrane preparation was found to be saturable, reversible, and dependent on time, temperature, tissue concentration, and pH. Addition of unlabeled sGnRH-A displaced the bound 125I-labeled sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis indicated the presence of two classes of binding sites, a high-affinity/low-capacity site and a low-affinity/high-capacity site, in the fully mature and less mature goldfish ovary. However, immature goldfish ovary contained only a single class of low-affinity binding sites. The equilibrium association constants (Ka) of the low-affinity sites were found to be similar in all follicular groups. However, there was a tendency for a higher Ka value of the high-affinity sites in the less mature follicles (0.5-0.95 mm) compared with the fully mature follicles (1.11-1.48 mm), although the differences were not statistically significant. Bound 125I-sGnRH-A was also found to be displaceable by sGnRH ([Trp7,Leu8]GnRH) and chicken GnRH-II (cGnRH-II; [His5,Trp7,Tyr8]GnRH), which occur naturally in the goldfish brain. sGnRH-A and cGnRH-II were found to bind with greater affinity than sGnRH to the goldfish ovarian GnRH binding sites.

Failure to detect gonadotrophin-releasing hormone receptors in human benign and malignant breast tissue and in MCF-7 and MDA-MB-231 cancer cells

We have measured the binding of radiolabelled analogues of gonadotrophin-releasing hormone (GnRH) to homogenates of human breast cancer and benign breast tissue, and to MCF-7 and MDA-MB-231 cell lines. Although incubation of breast cancer homogenates with the 125I-labelled GnRH agonist analogues, buserelin [(D-Ser tBU 6)GnRH 1–9 ethylamide] and tryptorelin [(D-Trp 6) GnRH 1–9 ethylamide] appeared to show significant though low, specific GnRH agonist binding in a high proportion of breast cancers ( 32 42 for buserelin; 15 32 for tryptorelin) and benign breast tissues ( 13 16 for buserelin; 10 12 for tryptorelin), after correction for displaceable binding in control assay tubes, GnRH agonist binding to breast tissue was no longer apparent. The lack of specific binding was not due to inactivation of GnRH agonist tracers, as >86% of the unbound tracer was still capable of rebinding to fresh placental membranes after incubation with breast cancer homogenates. GnRH agonist did not bind to MCF-7 and MDA-MB-231 cells, however GnRH agonist tracer inactivation following exposure to these cells was very high. We have shown recently that human placental receptors bound salmon GnRH and chicken GnRH II as well as GnRH agonists, but not other isoforms of GnRH. However, no isoform of GnRH bound significantly to human breast tumour tissue. In summary, we could not confirm the presence of specific GnRH binding sites in homogenates and membranes from human breast tissues in this study. Low levels of apparently specific binding of GnRH agonist tracers could be accounted for entirely by displacement of tracer from assay tubes. Inability to demonstrate specific binding was not due to extensive inactivation of GnRH tracers (although this may be a factor in the failure to demonstrate GnRH binding to MCF-7 and MDA-MB-231 cell lines).

Human placental gonadotrophin-releasing hormone (GnRH) binding sites: II. Comparison of binding and inactivation of 125I-labelled GnRH agonist to subcellular fractions following density gradient centrifugation

Human placental homogenates and membrane fractions were centrifuged on continuous sucrose density gradients, with or without buoyant density perturbation ofplasma-membranes by digitonin, and aliquots of each gradient fraction were assayed for a range of plasma-membrane and intracellular organelle markers, and for specific binding and inactivation of radiolabelled GnRH agonist (GnRH A), Buserelin ([D-Ser(tBu) 6] GnRH 1–9 ethylamide). GnRH agonist (GnRH A), binding equilibrated in the same regions of control gradients as the plasma-membrane markers, EGF-receptor and alkaline phosphatase. Moreover, binding of 125I-labelled GnRH A and 125I-labelled chicken GnRH II (ch GnRH II) was enriched in the same regions of the gradients, indicating that both bound to the same membrane fractions. Digitonin pretreatment increased the buoyant density of all three placental plasma-membrane markers to a similar degree. Intracellular organelle markers (and hCG content) equilibrated in different regions of the gradient to placental surface-membranes, and were not perturbed appreciably by digitonin. In contrast, inactivation of 125I-labelled (GnRH A), was associated largely with the soluble (cytosol) fraction which failed to enter the gradient, and little tracer inactivation was observed infractions enriched in GnRH A binding activity. Similar results were obtained with fractionated rat pituitary membranes. We conclude that: (a) placental GnRH binding sites do not represent binding of radiolabelled ligand to GnRH-degrading enzymes, (b) degradation of radiolabelled ligand is associated largely with placental cytosol fractions, and (c) GnRH binding activity appears to be associated largely with placental plasma-membranes.