New GnRH analogs in canine reproduction

Gonadotropin-releasing hormone (GnRH) analogs, which include agonists and antagonists, have been produced by amino acid substitutions of the native GnRH molecule to create greater potency and longer duration of effectiveness. The aim of this article was to review the pharmacological effects and the existing clinical literature of new GnRH analogs, namely agonists released from long-term formulations and third generation antagonists, in domestic dogs. Long-term administration of agonists functions through desensitization and down-regulation of GnRH pituitary receptors inhibiting gonadotropin production and release after an initial stimulation. Conversely, GnRH antagonists bind to gonadotrope GnRH receptors and compete successfully with endogenous GnRH for occupancy, thereby inhibiting the pituitary-gonadal axis immediately. There is a promising place for both agonists and antagonists in future canine reproduction. They can be used in the control of estrous cycle, hormone-dependent diseases as well as in contraception. Some information on the effectiveness and safety of these new analogs in canine reproduction is already available, yet further work is needed before they could be widely recommended. The increase in gonadotropins and gonadal steroids following administration of agonists might have adverse effects when used on hormone-dependent diseases. This initial "flare" should also be carefully managed in anestrous and prepubertal bitches. At present, the main application of antagonists seems limited to situations where an acute endocrine, inhibitory effect is required, e.g. proestrus or pregnancy termination. Future commercial availability of long-acting, single-dose antagonists could go far towards controlling pet population.     

Reversible suppression of pituitary-testicular function by a sustained-release formulation of a GnRH agonist (leuprolide acetate) in dogs

Pituitary-testicular function was studied in 15 dogs following treatment with a sustained-release formulation of a GnRH agonist, leuprolide acetate (LA). Adult male dogs were treated with a single subcutaneous injection of microencapsulated LA (0.1 or 1 mg/kg). Treatment with LA at a dose of 1 mg/kg resulted in decreased (P<0.001) ejaculatory volume and disappearance of morphologically normal spermatozoa within 8 wk and the effect persisted for 6 wk, while the 0.1 mg/kg dose was not adequate to effect suppression of spermatogenesis. The larger dose treatment (1 mg/kg) caused a transient rise in plasma levels of LH and testosterone followed by a marked decline to below the normal level by 2 wk, the low levels being maintained for at least 5 wk, indicating a prolonged effect of LA treatment on pituitary-gonadal axis. Twenty weeks after treatment with LA, a complete return to normal spermatogenesis was observed. The full reversibility of spermatogenesis in the dog after LA treatment suggests that this peptide could be used as a reversible method of male contraception.     

Antagonistic and agonistic GnRH analogue treatment of precocious puberty: tracking gonadotropin concentrations in urine

BACKGROUND: The pharmacodynamics of gonadotropin-releasing hormone (GnRH) agonists includes an initial 'flare-up' of the pituitary-gonadal axis, followed by reduced luteinizing hormone (LH) secretion. The question is if combining a short-acting antagonist with a long-acting agonist can diminish gonadotropin flare-up. METHODS: To achieve quick downregulation in patients with recently diagnosed central precocious puberty (CPP, 7 patients) or short stature with short predicted final height (3 patients), we combined the GnRH antagonist cetrorelix (3 subcutaneous injections every 72 h) at the beginning of GnRH agonist treatment (leuprorelin or triptorelin) in 6 patients and compared the effect to 4 patients treated solely with GnRH agonist. To monitor effects, we measured LH and FSH concentrations in urine collected from initial morning urination during the first month of treatment. RESULTS: In both treatment groups, gonadotropin flare-up could be detected in urine levels increased due to the flare-up phenomenon which was of short duration (<5 days) in the majority (5 of 6) of combined-treated patients and in the minority (1 of 4) of patients treated by agonist alone. During the first 10 days of treatment, mean LH concentration measured in urine was significantly lower in 4 CPP patients treated by the combined therapy compared to 3 CPP patients treated by the agonist only (mean LH combined therapy: 10.4 +/- 2.8 vs. 20.1 +/- 11.0 mU/ml in the agonist-only group, mean +/- SEM, p < 0.05). Significant correlations between stimulated serum LH in GnRH test prior to treatment and maximum urine LH after initiating GnRH analogue treatment (r = 0.547, p = 0.043), as well as basal serum LH and basal urine LH (r = 0.685, p = 0.014) were found. CONCLUSION: Combined GnRH agonist and antagonist treatment led to rapid gonadotropin suppression. Also, urine measurements of LH and FSH seemed suitable for monitoring gonadotropin-inhibiting or -stimulating properties of GnRH analogues in individual patients. However, a controlled trial of a larger patient cohort is required to decide which treatment is the most effective.     

Four functional GnRH receptors in zebrafish: analysis of structure, signaling, synteny and phylogeny

Reproduction in all vertebrates requires the brain hormone gonadotropin-releasing hormone (GnRH) to activate a cascade of events leading to gametogenesis. All vertebrates studied to date have one to three forms of GnRH in specific but different neurons in the brain. In addition, at least one type of GnRH receptor is present in each vertebrate for activation of specific physiological events within a target cell. Humans possess two types of GnRH (GnRH1 and GnRH2) but only one functional GnRH receptor. Zebrafish, Danio rerio, also have two types of GnRH (GnRH2 and GnRH3), although in contrast to humans, zebrafish appear to have four different GnRH receptors in their genome. To characterize the biological significance of multiple GnRH receptors within a single species, we cloned four GnRH receptor cDNAs from zebrafish and compared their structures, expression, and cell physiology. The zebrafish receptors are 7-transmembrane G-protein coupled receptors with amino-acid sequence identities ranging from 45 to 71% among the four receptors. High sequence similarity was observed among the seven helices of zebrafish GnRHRs compared with the human GnRHR, the green monkey type II GnRHR, and the two goldfish GnRHRs. Also, key amino acids for putative ligand binding, disulfide bond formation, N-glycosylation, and G-protein coupling were present in the extracellular and intracellular domains. The four zebrafish receptors were expressed in a variety of tissues including the brain, eye, and gonads. In an inositol phosphate assay, each receptor was functional as shown by its response to physiological doses of native GnRH peptides; two receptors showed selectivity between GnRH2 and GnRH3. Each of the four receptor genes was mapped to distinct chromosomes. Our phylogenetic and syntenic analysis segregated the four zebrafish GnRH receptors into two distinct phylogenetic groups that are separate gene lineages conserved throughout vertebrate evolution. We suggest the maintenance of four functional GnRH receptors in zebrafish compared with only one in humans may depend either on subfunctionalization or neofunctionalization in fish compared with mammalian GnRH receptors. The differences in structure, location, and response to GnRH forms strongly suggests that the four zebrafish GnRH receptors have novel functions in addition to the conventional activation of the pituitary gland in the reproductive axis.     

Neonatal treatment of male rats with a gonadotropin-releasing hormone antagonist results in altered function of the pituitary-testicular axis in adult age

In most mammals, pituitary-testicular hormone secretion is very active during the perinatal period, but the physiological significance of this function for later pituitary-gonadal interactions and sexual maturation is largely unknown. Short-term neonatal treatment with gonadotropin-releasing hormone (GnRH) antagonist results in delayed sexual maturation and infertility in male rats. We have now extended our earlier findings and studied in more detail the pituitary-gonadal function in adult rats after such neonatal treatment. In this study, the pituitary-testicular activity of newborn male rats was temporarily blocked by treatment with a GnRH antagonist analogue (N-Ac-4-Cl-D-Phe1, 4-Cl-D-Phe2, D-Trp3, D-Phe6, des-Gly10-GnRH-D-alanylamide; Organon 30039; 2 mg/kg s.c. twice daily) on Days 1-5 of life. Timing of puberty was slightly delayed in the treated rats (average: 2 days, p less than 0.05), as determined by the age of the balano-preputial separation. In adult rats (90-110 days), only 3 of the 17 rats treated neonatally with GnRH antagonist were fertile (14 of 17 controls, p less than 0.01), despite normal circulating androgen levels. Pituitary and serum follicle-stimulating hormone (FSH) levels were slightly but consistently elevated (20-30%; p less than 0.05) in antagonist-treated animals, whereas luteinizing hormone (LH) levels (both immunoreactive and bioactive) were unaffected. The pituitary contents of GnRH receptors were increased in antagonist-treated animals 85 +/- 6.6 (mean +/- SEM, n = 19) vs. 58 +/- 4.1 fmol/gland in controls (n = 20; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Seasonal variation in plasma testosterone, luteinizing hormone concentrations and LH-RH responsiveness in mature, male rusa deer (Cervus rusa timorensis)

Plasma testosterone and luteinizing hormone (LH) concentrations in immobilized or yarded rusa stags (Cervus rusa timorensis) were investigated over a two-year period. Testosterone concentrations showed a minor elevation in autumn (May) and reached maximal levels in late winter-early spring (August) coinciding with the rut. Luteinizing hormone in plasma was only detectable from January to May. Maximal responsiveness of the pituitary-gonadal axis to LH-RH stimulation was recorded in August. The combination of Fentaz (fentanylcitrate and azaperone) and Rompun (xylazine hydrochloride) for immobilizing deer influences hypothalamic function.     

Conserved amino acid residues that are important for ligand binding in the type I gonadotropin-releasing hormone (GnRH) receptor are required for high potency of GnRH II at the type II GnRH receptor

GnRH I regulates reproduction. A second form, designated GnRH II, selectively binds type II GnRH receptors. Amino acids of the type I GnRH receptor required for binding of GnRH I (Asp2.61(98), Asn2.65(102), and Lys3.32(121)) are conserved in the type II GnRH receptor, but their roles in receptor function are unknown. We have delineated their functions using mutagenesis, signaling and binding assays, immunoblotting, and computational modeling. Mutating Asp2.61(97) to Glu or Ala, Asn2.65(101) to Ala, or Lys3.32(120) to Gln decreased potency of GnRH II-stimulated inositol phosphate production. Consistent with proposed roles in ligand recognition, mutations eliminated measurable binding of GnRH II, whereas expression of mutant receptors was not decreased. In detailed analysis of how these residues affect ligand-dependent signaling, [Trp2]-GnRH I showed lesser decreases in potency than GnRH I at the Asp2.61(97)Glu mutant. In contrast, [Trp2]-GnRH II showed the same loss of potency as GnRH II at this mutant. This suggests that Asp2.61(97) contributes to recognition of His2 of GnRH I, but not of GnRH II. GnRH II showed a large decrease in potency at the Asn2.65(101)Ala mutant compared with analogs lacking the CO group of Gly10NH2. This suggests that Asn2.65(101) recognizes Gly10NH2 of GnRH II. GnRH agonists showed large decreases in potency at the Lys3.32(120)Gln mutant, but antagonist activity was unaffected. This suggests that Lys3.32(120) recognizes agonists, but not antagonists, as in the type I receptor. These data indicate that roles of conserved residues are similar, but not identical, in the type I and II GnRH receptors.     

Modifications of the gonadal function in the adult rat after fetal exposure to spironolactone

The effects of fetal exposure to spironolactone (SPL), an aldosterone antagonist with weak antiandrogen and gestagen properties, upon the pituitary-gonadal axis were studied in the offspring of rats that had been treated daily from gestation day 14 to day 20 with 10 or 20 mg SPL or the solvent vehicle (for controls). At 70-80 days of age, SPL-exposed rats showed no alterations in external genitalia or in body weight. However, males displayed a dose-dependent decrease in the weights of the ventral prostate and seminal vesicles. Whereas basal and gonadotropin-releasing hormone (GnRH)-induced plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, and 5 alpha-dihydrotestosterone levels were similar to controls, basal plasma and pituitary prolactin (Prl) levels were reduced (SPL-exposed 6.8 +/- 1.0 vs. controls 15.8 +/- 2.8 ng/ml and 6.1 +/- 1.2 vs. 11.6 +/- 1.8 microgram/anterior pituitary gland; mean +/- SEM). Cytosolic androgen receptors in ventral prostate were nonsignificantly decreased, but they increased after GnRH in contrast to controls. Nuclear androgen receptors were normal. Females displayed normal estrous cycles. Basal and GnRH-induced plasma FSH, Prl, estradiol, and progesterone concentrations were similar to controls, whereas plasma LH was elevated. Estrogen receptors in uterine cytosol were low and increased after GnRH. Ovaries and uteri were enlarged. The present study demonstrates that in utero exposure to SPL leads to endocrine dysfunctions that persist into adulthood. They are characterized in males by hypoprolactinemia, reduced weights of accessory sex organs, and a suggestion of functional modifications of androgen receptors. In females they are characterized by increased LH secretion, increased ovarian and uterine weights, and decreased uterine cytosol estrogen receptors, suggesting enhanced estrogenic action.     

Influence of a new slow-release GnRH analogue implant on reproduction in the Budgerigar (Melopsittacus undulatus, Shaw 1805)

The neuroendocrine conditioning of reproduction in birds could perform a very important role in captive breeding, especially in endangered species. Whereas in domestic and wild mammals pharmacological reproductive conditioning is well developed, in birds an effective method is not available. The aim of this study was to test the influence of a new slow-release GnRH analogue (buserelin acetate) implant on the reproductive activity of the Budgerigar (Melopsittacus undulatus), used as model species for captive-bred endangered birds. The effects were assessed by looking at reproductive parameters (egg-laying rate, egg fertility rate) and measuring excreted sex steroid metabolite concentrations in male and female birds. Modification of reproductive parameters and steroid metabolites excretion patterns were observed among birds administered with a GnRH analogue implant and maintained under artificial photoperiod (group I; 16L:8D). Implanted birds showed higher rates of egg-laying, potentially a higher proportion of fertile eggs and higher excreted steroid metabolite concentrations than birds maintained under natural photoperiod (group II; 10L:14D) and birds maintained under artificial photoperiod (group III; 16L:8D). Thus, it is concluded that the new slow-release GnRH analogue implant may represent an innovative and practicable treatment to rapidly induce reproductive activity in the Budgerigar, and that excreted sex hormone metabolites detection permits to monitor male and female gonadal activity.     

Role, mechanism of action and application of gonadoliberins in reproductive processes

Gonadoliberin (gonadotropin releasing hormone, GnRH) plays a central role in the regulation of reproductive functions as it regulates the release of both luteinizing hormone (LH) and follicle stimulating hormone (FSH). The isolation and structure determination of GnRH opened the possibility of its use for influencing reproductive processes. This possibility initiated a rapid development in the design of potent and long-acting GnRH agonists and antagonists. The most important structural modifications of GnRH leading to superagonists are the D-amino acid substitutions in position 6 combined with Pro9-ethylamide or azaGly10 at the C-terminus. We have synthesized several superagonists of GnRH according to these substitution principles. Furthermore, our L-isoaspartyl modification in position 6, as a new approach to GnRH agonist design, also resulted in superactive analogs. The recently discovered sequences of non-mammalian GnRH-s opened new routes for us to synthesize species specific GnRH agonists. All three groups of the above mentioned GnRH analogs have been successfully used for the treatment of sexual disorders of different animals (cattle, pigs, rabbits, etc.). Ovulation synchronization and a 30% increase in the fertility rate could be achieved by using GnRH agonists in cattle breeding. Analogs derived from species specific sequences could be applied for the induced artificial propagation of fish even out of the spawning season. It is known that superactive GnRH analogs can suppress the growth of certain hormone-dependent tumours. In vitro and in vivo tests of our analogs showed promising antitumour activity in breast cancer which might be explained by the mechanism of desensitization. Almost a hundred antagonist analogs of GnRH have been developed in our laboratory. The most effective ones contain 4 or 5 D-amino acids, and one of them is even orally active. The inhibition of ovulation can also be achieved by the administration of GnRH superagonists. This phenomenon might also be explained by the desensitization of LH-release. Radioactive analogs specifically labeled with tritium in different amino acid residues have been synthesized and used for studying tissue distribution and biodegradation of gonadoliberins. Analogs containing a photoreactive group have been prepared and applied for the trials of GnRH receptor isolation.     

Hormonal regulation of gonadotropin-releasing hormone receptors and messenger RNA activity in ovine pituitary culture

Previous studies demonstrate that gonadotroph responsiveness to GnRH, GnRH binding, and the apparent number of GnRH receptors are all increased by 17 beta-estradiol (E) or inhibin (IN) in ovine pituitary cultures. Progesterone (P) attenuates these effects. To explore differences between the effects of IN and E on GnRH binding, a detailed time-course study was performed. The results indicate that after 48 h, IN had a greater effect on binding of a GnRH agonist (5-fold increase) than E (3-fold increase), but was slower to act initially. A combined treatment of IN and E gave a partially additive effect at 48 h (6.5-fold increase). The mechanism of receptor regulation in this system is not known, but could involve synthesis, recycling, or modification of GnRH receptors. To investigate the contribution of altered receptor biosynthesis to the regulation of receptor levels, a functional Xenopus oocyte-based assay for GnRH receptor mRNA activity was employed. After 48 h of treatment, IN or E each led to a 7- to 8-fold increase in GnRH receptor mRNA activity. Treatment with both hormones led to a 19-fold increase. The increase in mRNA activity induced by either hormone was greatly attenuated by P. Modulation of GnRH receptor mRNA levels suggests that IN, E, and P regulate responsiveness to GnRH in the ovine pituitary at least in part by altering de novo synthesis of GnRH receptors. The differing time courses of action, as assayed by GnRH binding, and the additivity of effects at the mRNA level suggest that IN and E alter mRNA levels via different mechanisms.     

Relaxin-3 stimulates the hypothalamic-pituitary-gonadal axis

The hypothalamus plays a key role in the regulation of both energy homeostasis and reproduction. Evidence suggests that relaxin-3, a recently discovered member of the insulin superfamily, is an orexigenic hypothalamic neuropeptide. Relaxin-3 is thought to act in the brain via the RXFP3 receptor, although the RXFP1 receptor may also play a role. Relaxin-3, RXFP3, and RXFP1 are present in the hypothalamic paraventricular nucleus, an area with a well-characterized role in the regulation of energy balance that also modulates reproductive function by providing inputs to hypothalamic gonadotropin-releasing hormone (GnRH) neurons. Other members of the relaxin family are known to play a role in the regulation of reproduction. However, the effects of relaxin-3 on reproductive function are unknown. We studied the role of relaxin-3 in the regulation of the hypothalamo-pituitary-gonadal (HPG) axis. Intracerebroventricular (5 nmol) and intraparaventricular (540-1,620 pmol) administration of human relaxin-3 (H3) in adult male Wistar rats significantly increased plasma luteinizing hormone (LH) 30 min postinjection. This effect was blocked by pretreatment with a peripheral GnRH antagonist. Central administration of human relaxin-2 showed no significant effect on plasma LH. H3 dose-dependently stimulated the release of GnRH from hypothalamic explants and GT(1)-7 cells, which express RXFP1 and RXFP3, but did not influence LH or follicle-stimulating hormone release from pituitary fragments in vitro. We have demonstrated a novel role for relaxin-3 in the stimulation of the HPG axis, putatively via hypothalamic GnRH neurons. Relaxin-3 may act as a central signal linking nutritional status and reproductive function.     

神经内分泌因子调控鱼类生殖和生长的相互作用

脊椎动物的生长与生殖活动有着密切的联系并相互作用。许多调节生长和代谢活动的内分泌因子对青春期或者性腺的发育产生影响。同样,调节生殖活动的许多激素亦同时对生长和代谢产生影响。近年来,我们和其他学者对鱼类生长和生殖的神经内分泌调节的相互作用进行了研究,主要的进展是：①在促进性腺的激素影响生长方面,发现促性腺激素释放激素（ＧｎＲＨ）和多巴胺都能和脑垂体生长激素细胞的特异性受体结合而刺激生长激素释放,并能     

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.     

Adrenocorticotropin administration increases testosterone secretion in adult male rats

It appears that the effect of acute administration of pituitary-adrenal hormones on the pituitary-gonadal axis is species-dependent. However, no information is available with regard to the effect of acute adrenocorticotropin (ACTH) administration on testosterone secretion in rats. The present data indicate that acute ACTH administration can increase serum testosterone levels without modifying luteinizing hormone (LH) levels. Since this rise was not observed in castrated rats, it must be assumed that increased serum testosterone was of gonadal origin. The action of ACTH on testosterone secretion was likely an indirect one since there is no evidence at present for a direct, short-term action of the pituitary-adrenal axis on Leydig cell function.     

Analysis of ultrashort feedback regulation in human placenta: synthesis and secretion of GnRH by human trophoblastic cells.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) presumably controls placental growth and functions by autocrine/paracrine mechanisms, and is therefore an important part of the neuroendocrine network in human placenta. AIM: Our earlier work had indicated that GnRH was expressed in human placenta; in extension to these findings, we wanted to analyse synthesis and release of GnRH by trophoblastic cells. GnRH-associated peptide, co-linearly synthesised with GnRH, was used as indicator of actual peptide synthesis. METHOD: First, we immunised rabbits with lipopeptides containing partial sequences of GnRH-associated peptide (GAP) and developed antibodies for immunohistochemical staining. Second, we set up a competitive enzyme immunoassay to measure GnRH: Non-biotinylated GnRH, GnRH analogues or trophoblastic cell culture supernatants were used to inhibit binding of biotinylated des-pGlu1-GnRH to a monoclonal anti-GnRH antibody. RESULTS: a) Placental sections stained positive for GAP in the layers of trophoblastic cells. b) GnRH could be detect by a competitive EIA in supernatants of placental cultures in concentrations between 200 and 5 nM. CONCLUSIONS: GnRH is synthesised and released by trophoblastic cells.     

Hormonal regulation of female reproduction

Reproduction is an event that requires the coordination of peripheral organs with the nervous system to ensure that the internal and external environments are optimal for successful procreation of the species. This is accomplished by the hypothalamic-pituitary-gonadal axis that coordinates reproductive behavior with ovulation. The primary signal from the central nervous system is gonadotropin-releasing hormone (GnRH), which modulates the activity of anterior pituitary gonadotropes regulating follicle stimulating hormone (FSH) and luteinizing hormone (LH) release. As ovarian follicles develop they release estradiol, which negatively regulates further release of GnRH and FSH. As estradiol concentrations peak they trigger the surge release of GnRH, which leads to LH release inducing ovulation. Release of GnRH within the central nervous system helps modulate reproductive behaviors providing a node at which control of reproduction is regulated. To address these issues, this review focuses on several critical questions. How is the HPG axis regulated in species with different reproductive strategies? What internal and external conditions modulate the synthesis and release of GnRH? How does GnRH modulate reproductive behavior within the hypothalamus? How does disease shift the activity of the HPG axis?     

Binding properties of solubilized gonadotropin-releasing hormone receptor: role of carboxylic groups

The interaction of 125I-buserelin, a superactive agonist of gonadotropin-releasing hormone (GnRH), with solubilized GnRH receptor was studied. The highest specific binding of 125I-buserelin to solubilized GnRH receptor is evident at 4 degrees C, and equilibrium is reached after 2 h of incubation. The soluble receptor retained 100% of the original binding activity when kept at 4 or 22 degrees C for 60 min. Mono- and divalent cations inhibited, in a concentration-dependent manner, the binding of 125I-buserelin to solubilized GnRH receptor. Monovalent cations require higher concentrations than divalent cations to inhibit the binding. Since the order of potency within the divalent cations was identical with that of their association constants to dicarboxylic compounds, it is suggested that there are at least two carboxylic groups of the receptor that participate in the binding of the hormone. The carboxyl groups of sialic acid residues are not absolutely required for GnRH binding since the binding of 125I-buserelin to solubilized GnRH receptor was only slightly affected by pretreatment with neuraminidase and wheat germ agglutinin. The finding that polylysines stimulate luteinizing hormone (LH) release from pituitary cell cultures with the same efficacy as GnRH suggests that simple charge interactions can induce LH release. According to these results, we propose that the driving force for the formation of the hormone-receptor complex is an ionic interaction between the positively charged amino acid arginine in position 8 and the carboxyl groups in the binding site.     

Structure-function studies of linear and cyclized peptide antagonists of the GnRH receptor.

Structurally new analogs of the peptidic GnRH receptor antagonist Cetrorelix as well as conformationally constrained cyclized deca- or pentapeptides were synthesized and selected peptides evaluated comprehensively. To understand how structural variations of the antagonistic peptide effect pharmacodynamic properties, binding affinities and antagonistic potencies toward the human and rat GnRH receptor were determined. Whereas large substituents in position 6 of linear peptides are compatible with high binding affinity (K(D) < 0.5 nM), all cyclized peptides except the cyclo[3-10] analog D-52391 depicted low binding affinity (K(D) > 10 nM). Binding affinity and antagonistic potency in vitro correlated for all peptides and surprisingly no discrimination between human and rat receptor proteins was observed. Since receptor residues W(101) and N(102) are involved in agonist and antagonist binding, equally potent but structurally different antagonists were tested for binding to the respective W(101)A and N(102)A mutants. In contrast to linear decapeptides, residues N(102) and W(101) are not involved in binding of D-23938 and W(101) is the critical residue for D-52391 binding. We conclude that although equally potent, peptidic GnRH receptor antagonists do have distinct interactions within the ligand binding pocket. Finally, selected antagonists were tested for testosterone suppression in male rats. The duration of testosterone suppression below castration levels differed largely from 1 day for Ganirelix to 27 days for D-23487. Systemic availability became evident as the most important parameter for in vivo efficacy.     

Impact of growth hormone administration on other hormonal axes

Growth hormone regulates several other hormonal systems and vice versa. The present review focusses on the effect of GH administration in adults on selected hormonal systems. Growth hormone treatment has been linked to development of central hypothyroidism in hypopituitary children. We now know that GH enhances the extra-thyroidal conversion of T(4) to T(3). Lowering of T(4) during GH treatment therefore reflects biochemical unmasking of subclinical central hypothyroidism. In normal adults GH administration does not affect the pituitary-gonadal axis. There is, however, evidence to suggest that GH substitution in hypopituitary adults enhances peripheral actions of sex steroids (males) and stimulates gonadal function (females). Both increased, unchanged and reduced basal and ACTH stimulated glucocorticoid levels have been reported during GH treatment. Several groups have recorded reduced levels of cortisol binding globulin with unchanged free cortisol concentrations. Regular assessment of thyroid and glucocorticoid status during GH substitution in GH-deficient patients is recommended.