Gonadotropin releasing hormone (GnRH): Neuropharmacological studies

The hypothalamic hormone GnRH was found to potentiate behavioral effects of DOPA and serotonin. In addition, GnRH was observed to reduce slightly audiogenic seizures as well as spontaneous motor activity. No significant effects were observed against footshock induced fighting or oxotremorine effects. These studies support our concept of the actions of hypothalamic peptides on the central nervous system.     

Gonadotropin releasing hormone (GnRH) agonists in male contraception

A potent gonadotropin releasing hormone (GnRH) agonist, D(Nal2)6 GnRH (Nafarelin) has been administered to two groups of normal men for 16 weeks by two routes in order to assess its effectiveness in suppressing spermatogenesis. In this report 400 micrograms of the GnRH agonist was given daily by constant subcutaneous infusion and the results compared to an earlier study in which 200 micrograms of the same agonist was given as a single daily subcutaneous injection. All subjects in both groups received an intramuscular injection of testosterone enanthate (200 mg) every two weeks to prevent symptoms of androgen deficiency. The higher dose infusion regimen was much more effective in suppressing spermatogenesis than the single daily injection. With infusion treatment, 3 of 7 subjects were azoospermic, a fourth subject had less than 1 million sperm per ml of semen and 5 of 7 subjects had sperm counts less than 5 million per ml. Because of the differences in GnRH dose it is unclear if the enhanced effect seen in the infusion group is the result of the route or dose of drug. Data from experimental animals and short term comparative studies with two routes and two doses suggest that both mechanisms may be operative. In either case, the results are the most promising to date and raise the possibility that constant delivery of a higher dosage of agonist could produce azoospermia in most or all subjects.     

Gonadotropin releasing hormone (GnRH) and gonadotropin inhibitory hormone (GnIH) in the songbird hippocampus: Regional and sex differences in adult zebra finches

Hypothalamic gonadotropin releasing hormone (GnRH) and gonadotropin inhibitory hormone (GnIH) are vital to reproduction in all vertebrates. These neuropeptides are also present outside of the hypothalamus, but the roles of extra-hypothalamic GnRH and GnIH remain enigmatic and widely underappreciated. We used immunohistochemistry and PCR to examine whether multiple forms of GnRH (chicken GnRH-I (GnRH1), chicken GnRH-II (GnRH2) and lamprey GnRH-III (GnRH4)) and GnIH are present in the hippocampus (Hp) of adult zebra finches (Taeniopygia guttata). Using immunohistochemistry, we provide evidence that GnRH1, GnRH2 and GnRH4 are present in hippocampal cell bodies and/or fibers and that GnIH is present in hippocampal fibers only. There are regional differences in hippocampal GnRH immunoreactivity, and these vary across the different forms of GnRH. There are also sex differences in hippocampal GnRH immunoreactivity, with generally more GnRH1 and GnRH2 in the female Hp. In addition, we used PCR to examine the presence of GnRH1 mRNA and GnIH mRNA in micropunches of Hp. PCR and subsequent product sequencing demonstrated the presence of GnRH1 mRNA and the absence of GnIH mRNA in the Hp, consistent with the pattern of immunohistochemical results. To our knowledge, this is the first study in any species to systematically examine multiple forms of GnRH in the Hp or to quantify sex or regional differences in hippocampal GnRH. Moreover, this is the first demonstration of GnIH in the avian Hp. These data shed light on an important issue: the sites of action and possible functions of GnRH and GnIH outside of the HPG axis.     

Gonadotropin releasing hormone (GnRH) neurones of triploid catfish,Heteropneustes fossilis (Bloch): an immunocytochemical study

Gonadotropin-releasing hormone (GnRH), a regulator of gonadal maturation in vertebrates, is primarily secreted by neurosecretory cells of the pre-optic area (POA) in the forebrain of teleosts. GnRH-immunoreactive (GnRH-ir) cells of this area demonstrate positive correlation in number and size of soma with gonadal maturity and directly innervate the pituitary in most teleosts. Gonadal development in triploid fish remains impaired due to genetic sterility. The gonadal immaturity in triploid fish may be due to low levels of gonadotropin and sex steroids during the vitellogenic phase of reproductive cycle. However, the nature of GnRH-ir cells in triploid fish is not yet known. Triploid catfish (H. fossilis) showed significant decrease (P<0.001) in size and number of immunoreactive-GnRH cells of POA and low immunoreactivity in pituitary in comparison to their diploid full-sibs during the late pre-spawning phase of ovarian cycle. This study suggests that low activity of GnRH-cells in triploid may be due to lack of positive feedback stimulation by sex steroids and/or reduced responsiveness of sensory cells to environmental cues required for gonadal maturation in teleosts.     

Monoclonal anti-gonadotropin releasing hormone (GnRH) antibodies reacting to sequence of GnRH preferentially recognize to native hormone

Monoclonal anti-GnRH antibodies (MoAbs) P862, P778, P813 and P764 reacted optimally to native GnRH and poorly to GnRH(OH) of sequence 4-6, 7-10, 4-10 and 1-10. The heptapeptide 4-10 showed maximum reactivity amongst the four peptides tested for immunoreactivity. Sepharose 4B-GnRH(OH)4-10 (H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-OH) column was therefore used to purify the fraction of MoAb reacting to this sequence. The affinity purified MoAbs (A-MoAbs) were further characterized for their binding to the different sequences and affinity with native GnRH. The binding, cross-reactivity and affinity characteristics of A-MoAbs were comparable with those of MoAbs. Immunoreactivity of A-MoAbs was also observed to be partly regained when GnRH(OH)1-10 was coupled to Lys, Lys-MDP or H-Ala-Ala-Thr-Lys-Pro-Arg-OH. These observations clearly demonstrate that MoAbs were neither contaminated nor were sequence specific but were directed against the conformation of the molecule.     

Restoration of responsiveness to gonadotropin releasing hormone (GnRH) in calcium-depleted rat pituitary cells.

GnRH-stimulated, but not basal, luteinizing hormone (LH) release from cultured pituitary cells requires extra-cellular calcium. The present studies were designed to show whether cells which had lost responsiveness to GnRH in the absence of extracellular calcium (“Ca²⁺-depleted cells”) could regain responsiveness by readdition of calcium to the media. The addition of calcium-containing medium to cells which were preincubated (75 min) in calcium-free medium resulted in elevated basal LH release. Addition of GnRH to the media in the presence of calcium did not cause additional stimulation of LH release above the elevated basal level. Incubation of Ca²⁺-depleted cells in calcium-containing media for 2 h before measuring responsiveness depressed the basal level to near that seen in control cells and GnRH was able to stimulate LH release, but not to as high a level as in control cells (which were preincubated in 1 mM Ca²⁺-containing media). After incubation of calcium depleted cells in calcium-containing media for 3 h or 5 h, the basal and stimulated levels of LH response were statistically indistinguishable from those seen in control cells.     

Gonadotropin releasing hormone enhances polyphosphoinositide hydrolysis in rat pituitary cells

Addition of gonadotropin releasing hormone to myo-[2-3H]inositol-prelabeled rat pituitary cells in primary culture evoked a dose-dependent increase of the accumulation of [3H]inositol phosphates with a rise of inositol triphosphate within 30 sec of stimulation, followed by a rise in inositol diphosphate and inositol monophosphate. Inositol phosphate accumulation was enhanced up to 5-to-8-fold and was time-dependent between up to 15 min incubation without further increase beyond this time period. Without preincubation with LiCl2, there was no measurable increase of GnRH-induced inositol phosphate accumulation compared to controls. The presence of calcium in the incubation medium did not affect the increase of inositol phosphates. These data give evidence, that polyphosphoinositide breakdown may be an early step in the action of gonadotropin releasing hormone on gonadotropin secretion.     

Gonadotropin-releasing hormone (GnRH) neurons and pathways in the rat brain

Summary Gonadotropin-releasing hormone (GnRH) neurons and their pathways in the rat brain were localized by immunocytochemistry in 6-to 18-day-old female animals, by use of thick frozen or vibratome sections, and silver-gold intensification of the diaminobenzidine reaction product. GnRH-immunoreactive perikarya were observed in the following regions: olfactory bulb and tubercle, vertical and horizontal limbs of the diagonal band of Broca, medial septum, medial preoptic and suprachiasmatic areas, anterior and lateral hypothalamus, and different regions of the hippocampus (indusium griseum, Ammon's horn). In addition to the known GnRH-pathways (preoptico-terminal, preoptico-infundibular, periventricular), we also observed GnRH-immunopositive processes in several major tracts and areas of the brain, including the medial and cortical amygdaloid complex, stria terminalis, stria medullaris thalami, fasciculus retroflexus, medial forebrain bundle, indusium griseum, stria longitudinalis medialis and lateralis, hippocampus, periaqueductal gray of the mesencephalon, and extracerebral regions, such as the lamina cribrosa, nervus terminalis and its associated ganglia. By use of the silver-gold intensification method we present Golgi-like images of GnRH perikarya and their pathways. The possible distribution of efferents from each GnRH cell group is discussed.     

Gonadotropin releasing hormone (GnRH) stimulates both secretion and synthesis of human chorionic gonadotropin (hCG) by first trimester human placental minces in vitro

An in vitro system using the minces of placental villi from first trimester human pregnancy (6-10 weeks) has been validated to examine the effect of addition of GnRH and its analogues on hCG secreted into the medium. Addition of low concentration of GnRH or its analogues (1 X 10(-8) M to 1 X 10(-6) M) resulted in an increase in the quantity of hCG in the medium, while addition of high concentrations of GnRH resulted in an inhibitory effect. Of the analogues tested, Buserelin was highly effective in exerting an inhibitory effect. A significant increase in 35S-methionine incorporation into immunoprecipitable hCG was noticed in the presence of GnRH. These results suggests that GnRH stimulates both synthesis and secretion of hCG by first trimester human placenta.     

Physiological role of putative testicular gonadotrophin releasing hormone (GnRH)

Evidence suggests that exogenous GnRH and agonist analogues have short-term stimulatory effects on rat Leydig cell function - when administered intratesticularly. Since rat Leydig cells possess GnRH receptors and their endogenous ligand has not yet been identified the physiological importance of the observations for testis function is unknown. To address this issue we have determined the consequences of blockade of testis GnRH receptors on Leydig cell function under both normogonadotrophic and hypogonadotrophic stimulation of the testis in vivo. A GnRH antagonist (ANT) was used to achieve receptor blockade but during continuous systemic infusion ANT occupied pituitary GnRH receptors and markedly reduced serum LH, FSH, testosterone, and intratesticular testosterone in adult and 30 d old immature male rats. These results were similar to those obtained by administration of a GnRH antiserum which did not bind to testis GnRH receptors. Thus, blockade of testis GnRH receptors during hypogonadotrophism did not produce additional inhibition of steroidogenesis by Leydig cells. However, direct continuous infusion of ANT into one testis produced greater than 90% occupancy of GnRH receptors while reducing GnRH receptors by only 50% in the contralateral testis. Unilateral intratesticular infusion did not reduce serum LH, FSH, Prolactin or testosterone levels despite 75% occupancy of pituitary GnRH receptors. Thus, both ANT infused and saline infused testes were exposed to the same gonadotrophic stimulants but in the former GnRH-R were essentially non-existent. Compared to the control testis, the ANT infused testis showed a 20-30% reduction in LH, FSH, lactogen receptors and 30-40% fall in testosterone content. Identical results were obtained in adult and 30 d-old male rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypophysiotrophic thyrotropin releasing hormone (TRH) synthesizing neurons

Summary The neuropeptide thyrotropin releasing hormone (TRH) is capable of influencing both neuronal mechanisms in the brain and the activity of the pituitary-thyroid endocrine axis. By the use of immunocytochemical techniques, first the ultrastructural features of TRH-immunoreactive (IR) perikarya and neuronal processes were studied, and then the relationship between TRH-IR neuronal elements and dopamine--hydroxylase (DBH) or phenylethanolamine-N-methyltransferase (PNMT)-IR catecholaminergic axons was analyzed in the parvocellular subnuclei of the hypothalamic paraventricular nucleus (PVN). In control animals, only TRH-IR axons were detected and some of them seemed to follow the contour of immunonegative neurons. Colchicine treatment resulted in the appearance of TRH-IR material in parvocellular neurons of the PVN. At the ultrastructural level, immunolabel was associated with rough endoplasmic reticulum, free ribosomes and neurosecretory granules. Non-labelled axons formed synaptic specializations with both dendrites and perikarya of the TRH-synthesizing neurons. TRH-IR axons located in the parvo-cellular units of the PVN exhibited numerous intensely labelled dense-core and fewer small electron lucent vesicles. These axons were frequently observed to terminate on parvocellular neurons, forming both bouton- and en passant-type connections. The simultaneous light microscopic localization of DBH or PNMT-IR axons and TRH-synthesizing neurons demonstrated that catecholaminergic fibers established contacts with the dendrites and cell bodies of TRH-IR neurons. Ultrastructural analysis revealed the formation of asymmetric axo-somatic and axo-dendritic synaptic specializations between PNMT-immunopositive, adrenergic axons and TRH-IR neurons in the periventricular and medial parvocellular subnuclei of the PVN.These morphological data indicate that the hypophysiotrophic, thyrotropin releasing hormone synthesizing neurons of the PVN are directly influenced by the central epinephrine system and that TRH may act as a neurotransmitter or neuromodulator upon other paraventricular neurons.Supported by NIH research grants NS19266 and DK34540     

Gonadotropin secretion following pernasal stimulation with synthetic gonadotropin releasing hormone (GnRH) and a highly effective GnRH-analog in healthy men and prepubertal boys]

Potent long acting analogs of GnRH are of great interest especially in view of pernasal (p.n.) treatmen of hypogonadism of hypothalamic origin and of cryptorchidism. To find the necessary p.n. dosage of such a substance, serum LH and FSH were measured in 6 normal adult human males after p.n. application of various doses of D Leu6-des-Gly10-GnRH ethylamide. 50 microgram of the GnRH analog were necessary to obtain increased serum gonadotropins over a period of at least 8 hours. By repeated p.n. application of 200 microgram of synthetic GnRH every 2 hours in 6 normal adult males a considerable increase of serum gonadotropins could be demonstrated as well. Pernasal application of 200 microgram GnRH repeated at an interval of 1 hour in 3 cryptorchid boys produced a distinct increase of the serum gonadotropins. The intraindividual comparison of 200 microgram GnRH and 20 microgram of the GnRH analog in one boy showed equivalent net increases of the gonadotropins. With the analog the gonadotropin increase lasted for about 6 hours.     

Cellular characteristics of immunolabeled luteinizing hormone releasing hormone (LHRH) neurons

This study utilized the preembedding immunocytochemical technique in order to identify LHRH-containing neurons in rat brain and define their ultrastructural characteristics. LHRH-containing neurons in the vertical limb of the diagonal band of Broca, medial septum, triangular nucleus of the septum and other regions were studied by taking ultrathin serial sections. These neurons had scant cytoplasm surrounding a centrally-located, spheroid, euchromatic nucleus. Neurosecretory granules were evenly distributed throughout the cell, but many tended to lie directly under the plasmalemma. The cytoplasm was organized in such a way that the most extensive portion of the rough endoplasmic reticulum was polar opposite to areas having high concentrations of Golgi complex, lysosome-like bodies, smooth endoplasmic reticulum and ribosomes. The perikarya had no axosomatic synapses but functional interaction via unspecialized appositions to the plasmalemma cannot be discounted. Many of the perikarya bore at least one cilium. Processes from immunonegative cells were occasionally observed to penetrate the cytoplasm of the LHRH perikaryon or its processes. At their points of origin, dendrites were found to be broadened processes containing many elements common to the cytoplasm: ribosomes, smooth endoplasmic reticulum, cristal and lamellar mitochondria, neurotubules, and an occasional alveolate caveola. Infrequently, some of the LHRH axons were partially myelinated. This method of studying serial-sectioned immunocytochemically-identified cells is suggested as a means of describing the cellular and subcellular characteristics of other specific peptide-containing cells.     

Effect of thyrotropin releasing hormone injection on blood growth hormone (GH), TSH and growth hormone releasing hormone (GHRH) concentrations in cancer patients

In order to investigate whether endogenous GHRH and somatostatin were involved in the mechanism of the paradoxical GH rise after TRH injection, changes in serum GH and plasma GHRH were examined before and after TRH injection in 12 cancer patients and changes in serum TSH and GH were similarly studied in 76 cancer patients including 31 GH-responders and 45 GH-nonresponders to TRH. TRH stimulated GH secretions without altering the circulating GHRH concentration in 4 of the 12 cancer patients. There was neither a significant correlation between the increase from the basal to maximum GH and GHRH after TRH injection in the 12 cancer patients nor a reciprocal relationship between the increase in GH and TSH after TRH injection in the 76 cancer patients. These findings suggested that the paradoxical GH rise after TRH injection in cancer patients was exerted by its direct action at the pituitary level, and not mediated through the hypothalamus.     

Increased growth hormone responses to growth hormone releasing hormone and thyrotropin releasing hormone in patients with metastatic testicular cancer

In 16 patients with metastatic testicular cancer and 10 age matched male control subjects growth hormone (GH) responses to growth hormone releasing hormone (GHRH; 1 microgram/kg body weight iv.) and thyrotropin releasing hormone (TRH; 200 micrograms iv.) were measured. Basal GH levels and GH levels following stimulation with GHRH or TRH were significantly increased in cancer patients compared to control subjects. 9 patients with testicular cancer were studied both in the stage of metastatic disease and after they had reached a complete remission. In complete remission GH responses to GHRH tended to decrease but the differences did not reach statistical significance. Our data suggest an alteration of hypothalamic and/or pituitary regulation of GH secretion in patients with metastatic testicular cancer.     

Pulsatile infusion of gonadotrophin releasing hormone (GnRH): investigative and therapeutic applications

Normal gonadotrophin secretion, and therefore normal ovarian function, depend on delivery to the pituitary of the hypothalamic neuropeptide gonadotrophin releasing hormone (GnRH) in a pulsatile pattern. In the mid-follicular phase of the menstrual cycle, for example, discrete pulses of luteinizing hormone (LH) can be observed at approximately 90 min intervals. Many disorders of ovulation are caused by abnormalities of this natural pulsed signal. We have developed and used a small portable infusion pump to deliver GnRH to women with hypothalamic amenorrhoea; our studies, and those of other groups, have shown that successful ovulation and pregnancy result from such treatment. The results of treatment at St Mary's Hospital show that 16 women with hypogonadotrophic amenorrhoea received a total of 31 cycles of treatment with pulsatile GnRH; 25 (81%) of these cycles were ovulatory and 11 of the 14 women who were trying to conceive became pregnant. There was only one multiple pregnancy (twins).