Gonadotrope function in ovariectomized ewes actively immunized against gonadotropin-releasing hormone (GnRH)

The gonadotrope cells of the ovine anterior pituitary were insulated from hypothalamic inputs by imposing an immunologic barrier generated by active immunization of ovariectomized ewes against gonadotropin-releasing hormone (GnRH) conjugated to keyhole limpet hemocyanin (KLH) through a p-aminophenylacetic acid bridge. All GnRH-KLH animals immunized developed titers of anti-GnRH that exceeded 1:5000. The antisera were specific for GnRH and cross-reacted with GnRH agonists modified in position 10 to an extent that was less than 0.01%. Ewes actively immunized against GnRH-KLH displayed levels of basal and GnRH agonist-induced gonadotropin secretion that were markedly lower (p less than 0.05) than comparable parameters in ewes actively immunized against KLH. In contrast, basal and thyrotropin-releasing hormone (TRH)-induced prolactin (PRL) secretion were not compromised by active immunization. Immunization against the GnRH-KLH conjugate, but not KLH alone, prevented expression of the positive feedback response to exogenous estradiol (E2). Pituitary stores of immunoactive luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were significantly (p less than 0.001) reduced in ewes immunized against GnRH-KLH but stores of PRL were not affected by such immunization. Further, the biopotency of the residual LH stores in tissue of animals from the anti-GnRH group was significantly (p less than 0.05) lower than LH biopotency in anti-KLH animals. Serum levels of LH in anti-GnRH ewes were restored by circhoral administration of a GnRH agonist that did not cross-react with the antisera generated. Pulsatile delivery of GnRH agonist in anti-GnRH ewes significantly (p less than 0.05) elevated serum LH within 48 h and reestablished LH levels comparable to anti-KLH ewes within 6 days of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pattern of gonadotropin-releasing hormone (GnRH)-like stimuli sufficient to induce follicular growth and ovulation in ewes passively immunized against GnRH.

The pattern of GnRH-like stimuli capable of inducing follicular growth, ovulation, and luteal function was evaluated in ewes passively immunized against GnRH. The estrous cycles of 30 regularly cyclic sheep were synchronized using vaginal pessaries impregnated with a synthetic progestogen. Animals were passively immunized against GnRH (groups 2-5, n = 6) or the carrier protein, keyhole limpet hemocyanin (KLH; group 1, n = 6), at the time of pessary removal (PR). Circhoral delivery of saline (groups 1, 2, and 5) or low amplitude GnRH agonist (des-Gly10 GnRH ethylamide [100 ng/hourly pulse]; groups 3 and 4) was initiated at PR and continued for 3 (groups 4 and 5) or 12 days (groups 1-3). In groups 4 and 5, the amplitude of the GnRH-like stimulus was increased to 800 ng/hourly pulse (stimulus-shift) during the 24-h period beginning 72 h after PR. The amplitude of the hourly stimulus was adjusted to 100 ng/pulse 96 h after PR and continued at that level to Day 12. The endocrine changes associated with follicle growth and maturation (serum concentrations of estradiol [E2] above 10 pg/ml), ovulation (surge-like secretion of LH and FSH), and normal luteal function (serum concentrations of progesterone [P] above 2 ng/ml) were evident in ewes passively immunized against KLH (group 1). In this group, the preovulatory surge of gonadotropins was noted 48.7 +/- 1.2 h after PR. These endocrine events were blocked by passive immunization against GnRH (group 2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of gonadotropin-releasing hormone (GnRH) in ovarian cancer

The expression of GnRH (GnRH-I, LHRH) and its receptor as a part of an autocrine regulatory system of cell proliferation has been demonstrated in a number of human malignant tumors, including cancers of the ovary. The proliferation of human ovarian cancer cell lines is time- and dose-dependently reduced by GnRH and its superagonistic analogs. The classical GnRH receptor signal-transduction mechanisms, known to operate in the pituitary, are not involved in the mediation of antiproliferative effects of GnRH analogs in these cancer cells. The GnRH receptor rather interacts with the mitogenic signal transduction of growth-factor receptors and related oncogene products associated with tyrosine kinase activity via activation of a phosphotyrosine phosphatase resulting in downregulation of cancer cell proliferation. In addition GnRH activates nucleus factor κB (NFκB) and protects the cancer cells from apoptosis. Furthermore GnRH induces activation of the c-Jun N-terminal kinase/activator protein-1 (JNK/AP-1) pathway independent of the known AP-1 activators, protein kinase (PKC) or mitogen activated protein kinase (MAPK/ERK).     

Expression of the GnRH and GnRH receptor (GnRH-R) genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland of anestrous and luteal phase ewes

Data exists showing that seasonal changes in the innervations of GnRH cells in the hypothalamus and functions of some neural systems affecting GnRH neurons are associated with GnRH release in ewes. Consequently, we put the question as to how the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland is reflected with LH secretion in anestrous and luteal phase ewes. Analysis of GnRH gene expression by RT-PCR in anestrous ewes indicated comparable levels of GnRH mRNA in the preoptic area, anterior and ventromedial hypothalamus. GnRH-R mRNA at different concentrations was found throughout the preoptic area, anterior and ventromedial hypothalamus, stalk/median eminence and in the anterior pituitary gland. The highest GnRH-R mRNA levels were detected in the stalk/median eminence and in the anterior pituitary gland.During the luteal phase of the estrous cycle in ewes, the levels of GnRH mRNA and GnRH-R mRNA in all structures were significantly higher than in anestrous ewes. Also LH concentrations in blood plasma of luteal phase ewes were significantly higher than those of anestrous ewes.In conclusion, results from this study suggest that low expression of the GnRH and GnRH-R genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland, amongst others, may be responsible for a decrease in LH secretion and the anovulatory state in ewes during the long photoperiod.     

Gonadotropin secretion in ovariectomized ewes: effect of passive immunization against gonadotropin-releasing hormone (GnRH) and infusion of a GnRH agonist and estradiol

Gonadotropin secretion was examined in ovariectomized sheep after passive immunization against gonadotropin-releasing hormone (GnRH). Infusion of ovine anti-GnRH serum, but not control antiserum, rapidly depressed serum concentrations of luteinizing hormone (LH). The anti-GnRH-induced reduction in serum LH was reversed by circhoral (hourly) administration of a GnRH agonist that did not cross-react with the anti-GnRH serum. In contrast, passive immunization against GnRH led to only a modest reduction in serum concentrations of follicle-stimulating hormone (FSH). Pulsatile delivery of the GnRH agonist did not influence serum concentrations of FSH. Continuous infusion of estradiol inhibited and then stimulated gonadotropin secretion in animals passively immunized against GnRH, with gonadotrope function driven by GnRH agonist. However, the magnitude of the positive feedback response was only 10% of the response noted in controls. These data indicate that the estradiol-induced surge of LH secretion in ovariectomized sheep is the product of estrogenic action at both hypothalamic and pituitary loci. Replacement of the endogenous GnRH pulse generator with an exogenous generator of GnRH-like pulses that were invariant in frequency and amplitude could not fully reestablish the preovulatory-like surge of LH induced by estradiol.     

Interactions of gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH) in birds and mammals

Gonadotropin-releasing hormone (GnRH) regulates secretion of both of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone. Thus, it is a key hormone for vertebrate reproduction. GnRH was considered to be unusual among hypothalamic neuropeptides in that it appeared to have no direct antagonist, although some neurochemicals and peripheral hormones (opiates, GABA, gonadal steroids, inhibin) can modulate gonadotropin release to a degree. Five years ago, a vertebrate hypothalamic neuropeptide that inhibited pituitary gonadotropin release in a dose-dependent manner was discovered in quail by Tsutsui et al. (2000. Biochem Biophys Res Commun 275:661-667). We now know that this inhibitory peptide, named gonadotropin-inhibitory hormone, or GnIH, is a regulator of gonadotropin release in vitro and in vivo. Its discovery has opened the door to an entirely new line of research within the realm of reproductive biology. In our collaborative studies, we have begun to elucidate the manner in which GnIH interacts with GnRH to time release of gonadotropins and thus time reproductive activity in birds and mammals. This paper reviews the distribution of GnIH in songbirds relative to GnRHs, and our findings on its modes of action in vitro and in vivo, based on laboratory and field studies. These data are simultaneously compared with our findings in mammals, highlighting how the use of different model species within different vertebrate classes can be a useful approach to identify the conserved actions of this novel neuropeptide, along with its potential importance to vertebrate reproduction.     

Progesterone secretion by induced corpora lutea (CL) of anestrous bitches after administration of gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (HOG)

Sixteen bitches were treated with pregnant mare serum gonadotropin (PMSG; 44 IU/kg bwt) intramuscularly (i.m.) for nine consecutive days and each was given 500 IU HOG i.m. on the tenth day or on the first day of induced estrus (Day 0). On Day 12, each bitch was randomly assigned to three groups and treated as follows: Group 1-six bitches, each given 10 mcg GnRH i.m.once every 24 hr; Group 2-six bitches, each given 500 IU HOG i.m. once every 24 hr; and Group 3-four bitches, each given 5 ml 0.9% saline i.m. once every 24 hr. Treatments were continued until Day 55. Blood samples were obtained prior to treatment and every other day until Day 55. Plasma progesterone concentrations were determined by radioimmunoassay (RIA) and data arranged using a split-plot design, with treatment as the main plot and days of sampling as subplots. Analysis of data was by Duncan's Multiple Range Test and treatment-by-day interaction determined by the least-squares method. After treatment, progesterone concentrations from Group 2 were higher (P<0.05) than those of the other groups. A significant effect was seen on Day 20 (P<0.01), while on Day 50 the difference approached significance. These findings suggest that HOG is capable of stimulating the production of progesterone by induced CL in the bitch. However, neither HOG nor GnRH prevented premature regression of these induced CL. Results of this study suggest that premature luteolysis of induced CL is probably not due to lack of gonadotrophic support from either the hypothalamus or the anterior pituitary gland.     

Ovarian responses of seasonally anestrous ewes administered progesterone,PMS, HCG and(or) GnRH

One hundred and sixty ewes were assigned to sixteen groups in a 2 × 2 × 4 factoral design and were treated during the anestrous season. The main effects were progesterone pretreatment (non-implanted and implanted for 14 days), PMS pretreatment (no pretreatment and pretreatment with 500 IU at the time of progesterone implant removal) and treatments (none, GnRH in saline, GnRH in gelatin capsules and HCG). GnRH in saline (250 μg) and HCG (500 IU) were administered intramuscularly and GnRH in gelatin capsules (250 μg) was administered subcutaneously 24 hours after the time of progesterone implant removal.Ewes were classified into one of four progesterone response categories: cyclic, transient, prolonged and no response. An injection of GnRH in saline induced a prolonged progesterone response in only one ewe (13%) which was similar to the response in the untreated ewes (0%). More ewes administered GnRH in gelatin capsules (56%) and more ewes administered HCG (89%) had a prolonged progesterone response than GnRH (in saline) treated or untreated ewes. A higher percentage of ewes that were pretreated with PMS and treated with GnRH in saline (78%) had a prolonged progesterone response than ewes treated with either PMS (22%) alone or with GnRH (in saline; 13%) alone.     

The role of GABA(A) and GABA(B) receptors in the control of GnRH release in anestrous ewes

The paper reviews data concerning the involvement of GABA(A) and GABA(B) receptors in the control of GnRH secretion in anestrous ewes. Generally, GABA influences the GnRH release through GABA(A) and GABA(B) receptors located on perikaria of the GnRH neurons in the preoptic area (MPOA) or through the influence on beta-endorphinergic and catecholaminergic systems activity in MPOA and in ventromedial-infundibular region of the hypothalamus (VEN/NI). Stimulation of GABA(A) receptors in VEN/NI and MPOA attenuates GnRH release, while activation of GABA(B) receptors in MPOA decreases GnRH secretion, and in VEN/NI increases concentration of GnRH. The different neural mechanisms could be involved in this process: direct ligand action on the GABA(A) and GABA(B) receptors located on GnRH cells and axon terminals or indirect effect through the changes in the beta-endorphinergic and catecholaminergic systems activity in these structures of the brain.     

Testicular modulation of luteinizing hormone response to gonadotropin-releasing hormone (GnRH)-agonist treatment

We and others have observed that the response of serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) to chronic gonadotropin-releasing hormone-agonist (GnRH-A) treatment is substantially different in normal compared to hypogonadal males. These data suggested that products of the testes determine the gonadotropin response to GnRH-A. The present studies were designed to determine whether this effect is mediated by products of the interstitial (steroids) or the tubular compartment. To create experimental states with selective impairment of interstitial, tubular, or both compartments, 100 male sexually mature Wistar rats were divided into five groups: I, intact; II, castrated; III, castrated with 20-mm testosterone (T) implants; IV, bilaterally cryptorchid; and V, ketoconazole-treated animals. Cryptorchid animals have been shown to have impairment of tubular function while ketoconazole inhibits T biosynthesis. Each of the 5 groups was divided into 2 subgroups to receive daily injections of either saline or 1 microgram of a potent GnRH agonist, D-leu6 des-Gly10 GnRH N-ethylamide, for 4 wk. Unlike the intact animals, which showed an elevation of basal serum LH concentration after 4 wk of GnRH-A treatment, the castrated animals showed significant suppression below baseline. Animals with preferential impairment of tubular function (cryptorchid and castrated + T) also showed significant suppression of LH after GnRH-A treatment. However, the ketoconazole-treated animals (with inhibition of T biosynthesis and intact tubular function), behaved similarly to intact animals and demonstrated an elevation of LH after GnRH-A treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of novel GnRH analogues

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His⁵,Trp⁷,Leu⁸]-GnRH, dogfish (df) GnRH; [His⁵,Asn⁸]-GnRH, catfish (cf) GnRH; and [His⁵,Trp⁷,Tyr⁸]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro ( )-isomers -arginine ( -Arg) or -naphthylalanine ( -Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of -Nal⁶ into [His⁵,Asn⁸]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of -Nal⁶ or -Arg⁶ into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [ -Nal⁶,Pro⁹NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [ -Nal⁶,Pro⁹NEt]-mGnRH (k_d = 0.40 ± 0.04 and 0.55 ± 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.     

Development of gonadotropin-releasing hormone (GnRH) neuron regulation in the female rat

Summary 1. After reaching its final destination the GnRH neuronal network develops under the influence of both excitatory and inhibitory inputs.2. In the first 2 weeks of life, the immaturity of the GnRH neuronal system is reflected in sporadic unsynchronized bursts of the decapeptide, which determine the pattern of serum gonadotropin levels observed in female rats: high FSH levels and transient bursts of LH. The main inhibitory neuronal systems that operate in this period are the opioid and dopaminergic systems. A decrease in their inhibitory effectiveness may not be sufficient correctly to activate and synchronize the GnRH neuronal system.3. There is a concomitant increase in excitatory inputs, mainly noradrenaline, excitatory amino acids, and NPY, which increase the synthesis and release of GnRH at the beginning of the juvenile period and participate in the coupling of GnRH neural activity to the ongoing rhythmic activity of a hypothalamic circadian oscillator.4. The morphological changes of GnRH neurons which take place during the third and fourth weeks of life, and which are probably related to increasing estradiol levels, reflects the increasing complexity of the GnRH neuronal network, which establishes synaptic contacts to enable the expression of pulsatility and of the positive feedback of estradiol, both necessary components for the occurrence of puberty.     

Regulation of gonadotropin-releasing hormone (GnRH) gene expression in hypothalamic neuronal cells

Summary 1. Gonadotropin-releasing hormone (GnRH) is the hypothalamic releasing factor that controls pituitary gonadotropin subunit gene expression and indirectly gametogenesis and steroidogenesis from the gonad, which results in reproductive competence.2. GnRH is synthesized in only about 1000 neurons in the hypothalamus and released in an episodic fashion down the median eminence to regulate gonadotropin biosynthesis.3. Although much is known about the secretory dynamics of GnRH release, little is known about the pretranslational control of GnRH biosynthesis due to lack of appropriate model systems. The recent availability of immortalized neuronal cell lines that produce GnRH allows investigators for the first time to begin to dissect the factors that directly regulate GnRH gene expression.4. This article reviews the current state of knowledge concerning the mechanisms that direct tissue-specific and peptide hormone control of GnRH biosynthesis.     

Proteolytic degradation of gonadotropin-releasing hormone (GnRH) by rat ovarian fractions in vitro

GnRH in physiological concentrations is highly degradable by both soluble and particulate fractions of rat ovarian homogenate in vitro. The two proteolytic enzyme activities differ strongly by the soluble activity showing a dithiothreitol optimum, high inhibition by diisopropyl fluorophospate (k_i=0.7 μM), and a relatively high affinity (K_m=1.1 μM) as opposed to the particulate fraction (K_i=3.5 mM and K_m=150 μM, respectively). The results of this study show that the rat ovary is differently endowed with GnRH-degrading activity at different sites. The involvement of these in terminating the biological activity of the hormone on the ovary may possibly depend on its exact pathway in this GnRH-target organ.