Presence Of Gonadotropin-releasing Hormone Research Articles

Multiplicity of gonadotropin-releasing hormone in fish

Gonadotropin-releasing hormone (GnRH) is the major neuroendocrine hormone regulating reproduction in vertebrates, including finfish. In recent years, presence of GnRH and their function in reproduction has been confirmed in shellfish, crustaceans and molluscs. Interestingly, shellfish GnRH do not cluster within finfish GnRH, suggesting diversity in gnrh genes and their evolution in animal kingdom. GnRH analogues have been synthesized chemically and successfully used in induced reproduction of finfish. Studies on GnRH potency in induced maturation and spawning of shellfish are limited. The present paper report the recent information of shellfish GnRH cDNAs that have been successfully isolated in number of molluscan species, and would help in the farming of shellfish under controlled conditions. Also, the paper highlights the scintometric mapping of GnRH research in fish, suggesting rapid accumulation of information on fish GnRH and their importance in fish reproductive biology and endocrinology.

Effects of Dietary Fat on Mammary Gland Pyroglutamyl Aminopeptidase on Experimental Breast Cancer

Aim: To analyze the relationship between dietary fat and breast cancer mediated by the local functions of gonadotropin-releasing hormone (GnRH), analyzed by its regulating peptidase. The presence of GnRH and its receptors has been demonstrated in tumor tissue of the breast, although the functions of this peptide remain unclear. Pyroglutamyl aminopeptidase (PAP) is the enzyme involved in the local regulation of GnRH, and changes in its soluble and membrane-bound activities have been described in both women with breast cancer and animals with mammary tumors induced by N-methyl nitrosourea (NMU). Methods: We analyze here the effects of different normolipidic (4%) dietary fats (soybean oil (commercial), extra virgin olive oil (EVOO), refined sunflower oil (SO) and refined sunflower oil enriched with 50% oleic acid (OAESO)) on soluble and membrane-bound PAP specific activities in breast tissue of rats with mammary tumors induced by N-methyl-nitrosourea (NMU) administration. Results: We found that animals with breast cancer showed higher levels of soluble PAP than control healthy animals, but in a different degree depending on the type of dietary fat. On the contrary, membrane-bound mammary PAP specific activity was modified only in animals fed on EVOO. Conclusion: Dietary fat is involved in the regulation of GnRH functions mediated by PAP at mammary tumor tissue in different degree depending on its localization and the type of fat administrated.

Luteinizing hormone receptors are expressed in rat myenteric neurons and mediate neuronal loss.

Clinical observations have suggested repeated gonadotropin-releasing hormone (GnRH) exposure to cause intestinal dysfunction and loss of enteric neurons. This has been further studied and confirmed in a rat in vivo model involving iterated GnRH treatments. Mechanisms behind are enigmatic since no GnRH receptors are found to be expressed in enteric neurons neither in man nor rat. Both species, however, harbor substantial subpopulations of luteinizing hormone (LH) receptor-immunoreactive myenteric neurons which suggests that intestinal GnRH-induced neuropathy may be mediated by LH release. To reveal if exposures of GnRH or LH to rat myenteric neurons in vitro cause neuronal loss. Primary cultured adult rat myenteric neurons were exposed to single or repeated treatments of the GnRH analog buserelin or the LH analog lutrotropin alpha, and neuronal survival was determined by cell counting. Possible presence of GnRH- or LH receptor -immunoreactive neurons was determined by immunocytochemistry. Exposure to the LH, but not the GnRH, analog caused significantly reduced neuronal survival. LH, but not GnRH, receptors were found to be expressed on cultured myenteric neurons. Myenteric neurons express LH receptors in vitro and LH exposure causes reduced neuronal survival. This suggests that GnRH-induced enteric neuropathy in vivo is mediated by way of LH release and activation of enteric neuronal LH receptors.

Gonadotropin-releasing hormone (GnRH) receptors of cattle aggregate on the surface of gonadotrophs and are increased by elevated GnRH concentrations

The presence of gonadotropin-releasing hormone (GnRH) receptors (GnRHRs) on gonadotrophs in the anterior pituitary (AP) is an important factor for reproduction control. However, little is known regarding GnRHR gene expression in gonadotrophs of cattle owing to the lack of an appropriate anti-GnRHR antibody. Therefore, an anti-GnRHR antibody for immunohistochemistry, flow cytometry, and immunocytochemistry assays was developed to characterize GnRHR gene expression in gonadotrophs. The anti-GnRHR antibody could suppress GnRH-induced LH secretion from cultured AP cells of cattle. The GnRHR, luteinizing hormone (LH), and follicle stimulating hormone (FSH) in the AP tissue was analyzed by fluorescence immunohistochemistry. The GnRHRs were aggregated on a limited area of the cell surface of gonadotrophs, possibly localized to lipid rafts. The LH secretion was stimulated with increasing amounts of GnRH; however, excessive concentrations (>1nM) resulted in a decrease in LH secretion. A novel method to purify gonadotrophs was developed using the anti-GnRHR antibody and fluorescence-activated cell sorting. Flow cytometric analysis using the anti-GnRHR antibody for cultured bovine AP cells, however, failed to support the hypothesis that GnRH induces GnRHR internalization and decreases GnRHR on the surface of GnRHR-positive AP cells. In contrast, immunocytochemistry using primary antibodies for cultured bovine AP cells showed that 10nM (P<0.05) and 100nM (P<0.01) GnRH, but not 0.01–1nM GnRH, increased GnRHR in the cytoplasm of LH-positive cells. In conclusion, these data suggested that GnRHRs were aggregated on the surface of gonadotrophs and GnRHR inside gonadotrophs increased with elevated concentrations of GnRH.

Histological organization of the central nervous system and distribution of a gonadotropin-releasing hormone-like peptide in the blue crab, Portunus pelagicus

We present a detailed histological description of the central nervous system (CNS: brain, subesophageal ganglion, thoracic ganglia, abdominal ganglia) of the blue crab, Portunus pelagicus. Because the presence of gonadotropin-releasing hormone (GnRH) in crustaceans has been disputed, we examine the presence and localization of a GnRH-like peptide in the CNS of the blue crab by using antibodies against lamprey GnRH (lGnRH)-III, octopus GnRH (octGnRH) and tunicate GnRH (tGnRH)-I. These antibodies showed no cross-reactivity with red-pigment-concentrating hormone, adipokinetic hormone, or corazonin. In the brain, strong lGnRH-III immunoreactivity (-ir) was detected in small (7-17 μm diameter) neurons of clusters 8, 9 and 10, in medium-sized (21-36 μm diameter) neurons of clusters 6, 7 and 11 and in the anterior and posterior median protocerebral neuropils, olfactory neuropil, median and lateral antenna I neuropils, tegumentary neuropil and antenna II neuropil. In the subesophageal ganglion, lGnRH-III-ir was detected in medium-sized neurons and in the subesophageal neuropil. In the thoracic and abdominal ganglia, lGnRH-III-ir was detected in medium-sized and small neurons and in the neuropils. OctGnRH-ir was observed in neurons of the same clusters with moderate staining, particularly in the deutocerebrum, whereas tGnRH-I-ir was only detected in medium-sized neurons of cluster 11 in the brain. Thus, anti-lGnRH-III shows greater immunoreactivity in the crab CNS than anti-octGnRH and anti-tGnRH-I. Moreover, our functional bioassay demonstrates that only lGnRH-III has significant stimulatory effects on ovarian growth and maturation. We therefore conclude that, although the true identity of the crab GnRH eludes us, crabs possess a putative GnRH hormone similar to lGnRH-III. The identification and characterization of this molecule is part of our ongoing research.

Nervous control of reproduction in Octopus vulgaris: a new model

The classic study of Wells and Wells on the control of reproduction in Octopus demonstrated that the activity of the subpedunculate lobe of the brain and environmental illumination both inhibit the release of an unknown gonadotropin from the optic gland. This inhibitory control may be exerted by the neuropeptide Phe-Met-Arg-Phe-NH₂ (FMRFamide). It was later demonstrated that the olfactory lobe is also likely to be involved in the control of optic gland activity. The presence of gonadotropin-releasing hormone in the olfactory lobe suggested that it might exert an excitatory action on optic gland activity. Other neuropeptides have now been localised in the olfactory lobe: neuropeptide Y, galanin, corticotropin-releasing factor, Ala-Pro-Gly-Trp-NH₂ (APGWamide), as well as steroidogenic enzymes and an oestrogen receptor orthologue. This supports the hypothesis that this lobe may also play a part in the control of reproduction in Octopus. The olfactory lobe receives distant chemical stimuli and also appears to be an integrative centre containing a variety of neuropeptides involved in controlling the onset of sexual maturation of Octopus, via the optic gland hormone. This review attempts to summarise current knowledge about the role of the olfactory lobe and optic gland in the control of sexual maturation in Octopus, in the light of new findings and in the context of molluscan comparative physiology.

Identification and distribution of gonadotropin-releasing hormone-like peptides in the brain of horseshoe crab Tachypleus tridentatus

Gonadotropin-releasing hormone (GnRH) is a crucial peptide for the regulation of reproduction. Using immunological techniques, we investigated the presence of GnRH in horseshoe crab Tachypleus tridentatus. Octopus GnRH-like immunoreactivity, tunicate GnRH-like immunoreactivity, and lamprey GnRH-I-like immunoreactivity were detected in the neurons and fibers of the protocerebrum. However, no mammal GnRH-like immunoreactivity or lamprey GnRH-III-like immunoreactivity was observed. Our results suggest that a GnRH-like factor, an ancient peptide, existed in the brain of T. tridentatus and may be involved in the reproductive endocrine system.

2,3,7,8-Tetrachlorodibenzo-p-dioxin potentially attenuates the gene expression of pituitary gonadotropin .BETA.-subunits in a fetal age-specific fashion: a comparative study using cultured pituitaries

Our previous studies have demonstrated that maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a reduction in gonadotropin biosynthesis in the fetal pituitary, resulting in the attenuated expression of steroidogenic proteins in the fetal gonads and the impairment of sexual behaviors in adulthood. However, the mechanism of the attenuation remains unknown. To address this issue, we investigated whether TCDD affects the pituitary production of gonadotropins, using cultured pituitary. In the absence of gonadotropin-releasing hormone (GnRH), a regulator of gonadotropin biosynthesis, TCDD did not affect the expression of gonadotropin mRNAs both in fetal and postnatal pituitaries. On the other hand, in the presence of GnRH, TCDD interfered with the synthesis of gonadotropin β-subunit mRNAs only in the fetal pituitary. A protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) and a PKA activator (8-bromoadenosine-3' 5'-cyclic monophosphate) induced the expression of gonadotropin mRNAs in the fetal pituitary. Among the subunits, only the induction of β-subunit was reduced by TCDD treatment. These results suggest that TCDD reduces gonadotropin biosynthesis via damage to GnRH-stimulated PKC and PKA signaling in a β-subunit- and fetal age-specific manner.

GnRH-induced PACAP and PAC1 receptor expression in pituitary gonadotrophs: A possible role in the regulation of gonadotropin subunit gene expression

We examined the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and the PACAP type 1 receptor (PAC1-R) mRNA following gonadotropin-releasing hormone (GnRH) stimulation using the gonadotroph cell line LβT2. GnRH stimulation increased PACAP and PAC1-R mRNA expression in a static culture. Increase in the cell surface density of the PAC1-R following transfection with PAC1-R expression vectors significantly increased gonadotropin LHβ and FSHβ subunit promoter activities following 100 nM PACAP stimulation. In addition, increasing concentrations of PACAP stimulation augmented the promoter activities for both LHβ and FSHβ in PAC-1R overexpressing cells. In the cells with PAC1-R, the effect of GnRH was further potentiated in the presence of PACAP from 5.31 ± 0.93 to 9.89 ± 0.38-fold for LHβ and for FSHβ subunit, respectively; from 2.58 ± 0.31-fold by GnRH alone to 10.90 ± 2.79-fold with PACAP. The combination treatment with GnRH and PACAP did not augment the ERK phosphorylation induced by GnRH alone. PACAP expectedly increased cAMP accumulation and this effect was significantly attenuated in the presence of GnRH. PACAP gene expression was more prominent following lower frequency GnRH pulses (every 120 min) in a perifused culture. Our results suggest that PACAP and PAC1-R are produced locally within the gonadotrophs following GnRH stimulation. They subsequently affect the gonadotrophs in an autocrine manner and modulate the GnRH pulse-dependent specific regulation of gonadotropin subunits.

Hypothalamic gonadotropin-releasing hormone receptor activation stimulates oxytocin release from the rat hypothalamo-neurohypophysial system while melatonin inhibits this process

The present study was undertaken to investigate the influence of gonadotropin-releasing hormone (GnRH) and its agonist and antagonist on oxytocin (OT) release from the rat hypothalamo-neurohypophysial (H-N) system. An additional aim was to determine whether the possible response of oxytocinergic neurons to these peptides could be modified by melatonin through a cAMP-dependent mechanism. The results show that the highly selective GnRH agonist (i.e., [Des-Gly 10, d-His(Bzl) 6,Pro-NHEt 9]-LHRH; Histrelin) stimulates the secretion of OT from an isolated rat H-N system. Melatonin significantly inhibited basal and histrelin-induced release of OT in vitro, and displayed no significant influence on OT release in the presence of GnRH or its antagonist. Addition of melatonin to a medium containing forskolin resulted in significant reduction of OT secretion from the H-N system. On the other hand, addition of forskolin to a medium containing both histrelin and melatonin did not further alter the inhibitory influence of melatonin on the histrelin-dependent secretion of OT in vitro. Intracerebroventricular ( icv) infusion (experiment in vivo) of a GnRH antagonist resulted in substantial inhibition of OT release, thus revealing the stimulatory action of endogenous GnRH. In melatonin-treated animals, blood plasma OT levels were not changed in comparison to the vehicle. Our present data strongly suggests that activation of the GnRH receptor in the hypothalamus is involved in stimulation of OT secretion from the rat H-N system. It has also been shown, under experimental in vitro conditions, that melatonin fully suppresses the response of oxytocinergic neurons to the GnRH agonist – histrelin. The effect of melatonin on OT release is mediated by the cAMP-dependent mechanism, although other mechanisms of action are also possible.

Bovine C-terminal octapeptide of RFamide-related peptide-3 suppresses luteinizing hormone (LH) secretion from the pituitary as well as pulsatile LH secretion in bovines

Gonadotropin-inhibiting hormone (GnIH), observed in quail as a member of the RFamide neuropeptide family, suppresses luteinizing hormone (LH) secretion from the avian pituitary. Rats and cattle have an active gene of another member of the RFamide neuropeptide family, termed RFamide-related peptide-3 (RFRP-3), although bovine RFRP-3 is different from that of rats in both length and amino-acid sequence. A single injection of GnIH or RFRP-3 inhibited LH secretion in rodents, which continued for various periods. This study was conducted to evaluate the effects of bovine C-terminal octapeptide of RFRP-3 (RFRP-3-8) on LH secretion from cultured anterior pituitary (AP) cells of cattle, and the effects of RFRP-3-8 injections on pulsatile LH secretion in castrated male calves. The suppressive effect of RFRP-3-8 on LH secretion from AP cells was observed in the presence of gonadotropin-releasing hormone (GnRH), but not in the absence of GnRH in culture media. In another experiment collecting blood samples serially from castrated male calves with repeated intravenous injections of RFRP-3-8 ( n = 6) or saline ( n = 6), the RFRP-3-8 group showed suppressed LH pulse frequency during the injection period ( P < 0.05); however, the RFRP-3-8 group showed no difference from the saline group in all measures of LH secretion in the postinjection period. In conclusion, our results suggested that RFRP-3-8 suppresses LH secretion from cultured AP cells, as well as LH pulse frequency in cattle.

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

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

The presence and distribution of gonadotropin-releasing hormone-liked factor in the central nervous system of the black tiger shrimp, Penaeus monodon

The distribution and presence of gonadotropin-releasing hormone (GnRH) in the central nervous system (CNS) of Penaeus monodon were examined by immunocytochemistry, high performance liquid chromatography (HPLC), and radioimmunoassay (RIA). We demonstrated the existence of octopus (oct)GnRH-liked immunoreactivity (ir-octGnRH) and lamprey (l)GnRH-III-liked immunoreactivity (ir-lGnRH-III) in cell bodies of medium-sized neurons of the anterior part (protocerebrum) of the supraesophageal ganglion (brain). In addition, only the ir-octGnRH was detected in the nerve fibers located in the brain and segmental ganglia (subesophageal, thoracic, and abdominal ganglia). Moreover, some branches of these fibers also innervated the neurons in the middle (deutrocerebrum), posterior (tritocerebrum) brain and segmental ganglia. There was no ir-lGnRH-I and ir-salmon (s)GnRH detected in the shrimp CNS. The results from HPLC and RIA showed ir-GnRH in the CNS using anti-lGnRH-III, but not with anti-mammalian (m)GnRH. The data from immunocytochemistry, HPLC and RIA suggest that ir-GnRH in shrimp may be more similar to octGnRH and lGnRH-III than the other forms. These findings support the hypothesis that GnRH-liked factor(s) may be an ancient peptide that also exists in this decapod crustacean.

The naris muscles in tiger salamander. II. Innervation as revealed by enzyme histochemistry and immunocytochemistry.

The naris muscles control the aperature of the external naris in tiger salamanders, Ambystoma tigrinum, and may contribute to glandular secretion. Autonomic neurons of the palatine ganglion and possibly neurons associated with the nervus terminalis innervate these muscles. To elucidate the neural control of the naris muscles, neurotransmitters in nerve fibers supplying the naris muscles and in neurons of the palatine ganglion were examined using acetylcholinesterase enzyme histochemistry and immunocytochemistry to visualize possible peptide candidates for muscle innervation. The naris muscles, autonomic neurons, and associated nerve fascicles demonstrated strong acetylcholinesterase labeling, and the muscles were innervated by substance P fibers passing through the palatine ganglion from the trigeminal ganglion. Gonadotropin-releasing hormone and molluscan cardioexcitatory peptide-like immunoreactivities were found in secretory cell bodies and/or fibers in the palatine ganglion, and gonadotropin-releasing hormone was found in fiber projection pathways into the muscles. Vasoactive intestinal peptide was found in cell bodies and fibers of the palatine ganglion but appeared to provide a sparse innervation to the naris dilator muscle only. These findings suggest a typical autonomic cholinergic and sensory innervation of the naris muscles with some variations in peptide innervation. The presence of gonadotropin-releasing hormone in palatine ganglion and naris constrictor muscle suggests a potential modulation of autonomic neurons and perhaps even muscle fibers by this neuropeptide. We hypothesize that this reproductive hormone may modulate the activity of the naris constrictor muscle during reproductively appropriate events in order to provide access of pheromones to the vomeronasal organ.

The effect of stimulators and blockers of adrenergic receptors on LH secretion and cyclic nucleotide (cAMP and cGMP) production by porcine pituitary cells in vitro

The direct effects of α- and β-adrenergic agents on luteinizing hormone (LH) secretion in vitro by porcine pituitary cells and the participation of secondary messengers, adenosine 3′5′-monophosphate (cAMP) and guanosine 3′5′-monophospate (cGMP), in transduction of signals induced by adrenergic agents and gonadotropin-releasing hormone (GnRH) in these cells have been investigated. Pituitary glands were obtained from mature gilts, which were ovariectomized (OVX) 1 month before slaughter. OVX gilts, assigned to four groups, were primed with: (1) vehicle (OVX); (2 and 3) estradiol benzoate (EB; 2.5 mg/100 kg b.w.) at 30–36 h (OVX+EB I) or 60–66 h (OVX+EB II) before slaughter, respectively; (4) progesterone (P 4; 120 mg/100 kg b.w.) for 5 consecutive days before slaughter (OVX+P 4). Anterior pituitaries were dispersed with trypsin and then pituitary cells were cultured (10 6 per well) in McCoy’s 5a medium containing horse serum (10%) and fetal calf serum (2.5%) for 3 days, at 37 °C and under the atmosphere of 95% air and 5% CO 2. On day 4 of the culture, the cells were submitted to 3.5 h incubation in the presence of GnRH (a positive control), α- and β-adrenergic agonists (phenylephrine (PHEN) and isoproterenol (ISOP), respectively), and α- and β-adrenergic blockers (phentolamine (PHENT) and propranolol (PROP), respectively). The culture media were assayed for LH (experiment I) and cyclic nucleotides (experiment II). In experiment I, addition of GnRH (100 ng/ml) increased LH secretion by pituitary cells taken from gilts of all experimental groups. The effects of α- and β-adrenergic agents on LH secretion by the cells depended on hormonal status of gilts. The LH secretion by pituitary cells of OVX gilts was potentiated in the presence of PHEN (10, 100 nM, and 1 μM) and PHENT (1 μM), alone or in combination with PHEN (100 nM) and by the cells derived from OVX+EB I and OVX+P 4 animals in response to PHEN (100 nM) and ISOP (1 μM). ISOP (1 μM) also stimulated LH secretion by the cells taken from OVX+EB II gilts. In experiment II, GnRH (100 ng/ml) increased cGMP production by pituitary cells obtained from all groups of gilts and cAMP secretion by the cells taken from OVX and OVX+P 4 animals. PHEN (100 nM) decreased and PROP (1 μM) enhanced cAMP production by pituitary cells derived from OVX+EB I and OVX gilts, respectively. Moreover, PHEN (100 nM) reduced, while PHENT (1 μM) stimulated the release of cGMP by pituitary cells taken from OVX+EB II animals. In turn, ISOP (100 nM) decreased and increased cGMP production by the cells derived from OVX+EB II and OVX+P 4 gilts, respectively. PROP (1 μM) potentiated cGMP accumulation by pituitary cells taken from OVX+EB I and OVX+P 4 animals. In conclusion, our results suggest that adrenergic agents can modulate LH release by porcine pituitary cells acting through guanyl and adenylyl cyclase and in a manner dependent on hormonal status of gilts.

Stage-dependent and alternative splicing of sGnRH messengers in rainbow trout testis during spermatogenesis.

The gonadotropin releasing hormone (GnRH) has long been considered as a neuropeptide involved in the control of the reproductive cycle. However, the presence of GnRH and its receptors in various tissues, including ovary and testis, suggests a role as autocrine/paracrine factor. In the present study, we report the expression of the sGnRH-1 and sGnRH-2 genes encoding salmon GnRH in rainbow trout testis throughout testicular development and spermatogenesis. We demonstrate that both sGnRH mRNA are expressed prior of sexual differentiation. In adult, northern blot analysis indicates that sGnRH-2 transcripts are expressed in the testis at higher levels than sGnRH-1 messengers. Moreover, we observed that the expression of sGnRH-2, and not sGnRH-1, messengers was stage-dependent. sGnRH-2 mRNA expression decreases at the onset and progressively rebounds at the end of spermatogenesis. In addition, we demonstrate that a complex stage-dependent and differential splicing of the sGnRH-2 messengers occurs throughout spermatogenesis. We isolated five transcripts corresponding to sGnRH-2 messengers. Two of them may encode a novel and shortened GnRH-associated peptide containing 18 residues instead of 46. Our data provide new insight in the putative role of GnRH and GAP peptides as autocrine/paracrine factors of spermatogenesis.

Presence of gonadotropin-releasing hormone (GnRH) and its mRNA in rat pancreas

Gonadotropin-releasing hormone (GnRH) has been reported to exist in many non-hypothalamic tissues, such as the placenta, gonads, and mammary glands, while there still have been no reports concerning the existence and expression of GnRH in the mammalian digestive system. Immunocytochemistry and in situ hybridization results show that GnRH molecule and GnRH mRNA are both exclusively distributed in exocrine pancreas, and RT-PCR result further proves that GnRH transcription units do exist in the pancreas, which possess the same sequence as the hypothalamus GnRH mRNA. Quantitative analysis indicates that mRNA levels in rat pancreas remain at a low level (less than 10% of that in hypothalamus) without sexual or developmental difference. This is the first report suggesting the existence and gene expression of GnRH in rat pancreas.

Immunoreactivity for Gonadotropin-Releasing Hormone in Microdialyzed Perfusates of Bovine Mature Follicles In Vitro.

The presence of gonadotropin-releasing hormone (GnRH)-like protein has been previously demonstrated in the bovine follicle. This GnRH-like protein was purified and concluded to be histone H2A. However, neither GnRH peptide nor specific GnRH-immunoreactivity (GnRH-IR) has been demonstrated in the bovine follicle so far. Thus, this study focused on the detection of specific GnRH-IR using the second-antibody enzyme immunoassay in isolated bovine mature follicles, and on an examination of the direct effect of luteinizing hormone (LH), endothelin-1 (ET-1), and cytokines on the GnRH-IR using an in vitro microdialysis system (MDS). We further examined a cross-reactivity of the GnRH antibody with bovine histone H2A. GnRH-IR was detected in microdialyzed perfusate from isolated bovine mature follicles at 4.40 ± 0.35 pg/ml (mean ± SEM). Bovine histone H2A showed no GnRH-IR at all. Heat treatment of the extract (100 C for 10 min) did not affect the GnRH-IR. Single infusion of LH, ET-1, or cytokines into the MDS did not affect the GnRH-IR. However, infusion of ET-1 after LH exposure increased the GnRH-IR. These results demonstrate the presence of specific GnRH-IR, that is different from histone H2A, in microdialyzed perfusate of isolated bovine mature follicles in vitro, suggesting that the GnRH-IR may reflect a role of GnRH-like peptide or some peptide structurally similar to GnRH in the local regulation of mature follicles.

Expression of the messenger RNA for gonadotropin-releasing hormone and its receptor in human cancer cell lines

The presence of gonadotropin-releasing hormone (GnRH) binding sites in biopsy samples of human epithelial ovarian cancer and ovarian tumor cell lines as well as the demonstration of the inhibitory effects of GnRH analogues on the growth of these cells raised the possibility that GnRH is produced locally by ovarian cancer cells. In order to investigate an autocrine/paracrine regulatory mechanism in human carcinomas, we have studied the expression of GnRH and GnRHR mRNA in human ovarian epithelial cell lines (OVCAR-3 and SKOV-3), human choriocarcinoma cell line (JEG-3) and human hepatocarcinoma cell line (HepG 2). Using primers corresponding to published human GnRH and GnRHR cDNA sequences, predicted PCR products were obtained from these cell lines by reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by Southern hybridization. Sequencing analysis of GnRH PCR products showed that their sequences have 100% identity to the published human GnRH cDNA sequence. These results indicated that GnRH and GnRHR genes are expressed in all the cell lines tested in the present study, and strengthen the concept that GnRH may act as an autocrine regulator on the growth of cancer cells.

Rat gonadotropin-releasing hormone receptor expressed in insect cells induces activation of adenylyl cyclase

Increasing evidence exist that multiple G proteins mediate the effects of gonadotropin-releasing hormone (GnRH) on the synthesis and release of pituitary gonadotropins. In the present study, we have expressed the rat GnRH receptor (GnRH-R) in insect cells, by infection with a recombinant baculovirus. Under the conditions used, insect cells expressed, 48 h post-infection, a maximum of 7800±650 receptors/cell which bound GnRH agonist [ d-Trp 6]GnRH with a K d=0.52±0.06 nM indicating characteristics similar to those of the natural receptor. No binding was observed in non-infected cells or cells infected with wild-type baculovirus. In presence of GnRH, GnRH-R expressing cells elicited a time- and dose-dependent production of inositol trisphosphate, with a maximum level reached within 30 min and an EC 50=5 nM. These recombinant insect cells also produced cAMP in response to GnRH. However, in contrast to other heterologous systems, or rat pituitary gonadotropes wherein GnRH induced a weak and delayed elevation of cAMP, in insect cells the rise of cAMP was comparatively rapid, attaining a maximum level after 2 h, and the EC 50 was 5 nM. Finally, a clear activation of adenylyl cyclase (AC) in response to GnRH was shown for the first time by measuring the conversion of [ α- 32P]ATP into labeled cAMP, using membrane preparations from GnRH-R expressing insect cells. These data demonstrate that rat GnRH-R has the potential for dual coupling to both phosphoinositidase C and AC and suggest a major influence of the host cell for this coupling and/or its expression, probably in relation with the G protein repertoire and preference. This notion could be extended to several target cells other than pituitary gonadotropes that normally express the GnRH-R in mammals, including hippocampal, Leydig, granulosa, placental and GnRH-secreting hypothalamic cells.