Gonadotrophin-releasing Hormone Expression Research Articles

Mast Cell Dynamics in the Ovary Are Governed by GnRH and Prolactin.

Gonadotrophin releasing hormone (GnRH) facilitates the migration of mast cells (MCs) into the involuting mammary gland. As GnRH is also expressed in the ovary, we examined changes in ovarian MCs. MCs in the ovary were mainly in interstitial tissue and their number increased during the estrous cycle to produce 2 peaks, one at diestrus 2 (20:00 hours) and another at proestrus (17:00 hours). Laser microdissection demonstrated that GnRH mRNA is expressed throughout ovarian tissues (corpora lutea, follicles, and interstitial tissues). GnRH immunoreactivity was also ubiquitous, but MCs were the most strongly immunostained. Analysis of GnRH mRNA in the ovary showed it to fluctuate similarly to the variation in MC number during the estrous cycle, and MCs also expressed GnRH. Local administration of a GnRH agonist (GnRHa) into the hemilateral ovarian bursa increased MCs in the administered ovary. MC number and GnRH mRNA were significantly lowered in the pregnant ovary. Prolactin administration suppressed the normal peaks in MC number in the ovary at both diestrus and proestrus. By contrast, a dopamine agonist, administered when prolactin was elevated during pseudopregnancy, increased ovarian MC number. Furthermore, prolactin inhibited GnRHa-induced peritoneal MC migration in a Transwell assay. These data clearly demonstrate that ovarian MC number is regulated positively by local GnRH expression and negatively by prolactin. The suppressive effect of prolactin on GnRH and MCs would be part of its luteotrophic action.

Arcuate nucleus kisspeptin response to increased nutrition in rams.

Rams respond to acute nutritional supplementation by increasing the frequency of gonadotrophin-releasing hormone (GnRH) pulses. Kisspeptin neurons may mediate the effect of environmental cues on GnRH secretion, so we tested whether the ram response to nutrition involves activation of kisspeptin neurons in the arcuate nucleus (ARC), namely kisspeptin, neurokin B, dynorphin (KNDy) neurons. Rams were given extra lupin grain with their normal ration. Blood was sampled before feeding, and continued until animals were killed for collection of brain tissue at 2 or 11h after supplementation. In supplemented rams, LH pulse frequency increased after feeding, whereas control animals showed no change. Within the caudal ARC, there were more kisspeptin neurons in supplemented rams than in controls and a higher proportion of kisspeptin cells coexpressed Fos, regardless of the time the rams were killed. There were more Fos cells in the mid-ARC and mid-dorsomedial hypothalamus of the supplemented compared with control rams. No effect of nutrition was found on kisspeptin expression in the rostral or mid-ARC, or on GnRH expression in the preoptic area. Kisspeptin neurons in the caudal ARC appear to mediate the increase in GnRH and LH production due to acute nutritional supplementation, supporting the hypothesised role of the KNDy neurons as the pulse generator for GnRH.

Lipopolysaccharide reduces gonadotrophin-releasing hormone (GnRH) gene expression: role of RFamide-related peptide-3 and kisspeptin.

RFamide-related peptide (RFRP)-3 reduces luteinising hormone (LH) secretion in rodents. Stress has been shown to upregulate the expression of the RFRP gene (Rfrp) with a concomitant reduction in LH secretion, but an effect on expression of the gonadotrophin-releasing hormone (GnRH) gene (Gnrh1) has not been shown. We hypothesised that lipopolysaccharide (LPS)-induced stress affects expression of Rfrp, the gene for kisspeptin (Kiss1) and/or Gnrh1, leading to suppression of LH levels in rats. Intracerebroventricular injections of RFRP-3 (0.1, 1, 5 nmol) or i.v. LPS (15μgkg-1) reduced LH levels. Doses of 1 and 5 nmol RFRP-3 were then administered to analyse gene expression by in situ hybridisation. RFRP-3 (5 nmol) had no effect on Gnrh1 or Kiss1 expression. LPS stress reduced GnRH and Kiss1 expression, without affecting Rfrp1 expression. These data indicate that LPS stress directly or indirectly reduces Gnrh1 expression, but this is unlikely to be due to a change in Rfrp1 expression.

Kisspeptin-10 treatment generated specific GnRH expression in cells differentiated from rhesus monkey derived Lyon NSCs

Embryonic stem cells (ESCs) have enormous potential as novel cell-based therapies, but their effectiveness depends on stem cell differentiation and specific signaling regulators, which remain poorly understood. In this study, a kisspeptin peptide (KP-10) was used at different dosages to determine whether rhesus macaque-derived tau GFP-Lyon ES cells underwent kisspeptin-specific neuronal differentiation. It was found that KP-10 exhibited an anti-proliferative effect on the cells and led to morphological changes and cellular differentiation consistent with neuronal stem cell (NSC) development. The cells differentiated into Gonadotrophin Releasing Hormone (GnRH) neuronal-like cell types in response to the KP-10 treatment. There has been a previously observed connection between kisspeptin signaling, GnRH neurons and their dysfunction found in congenital disorders like idiopathic hypogonadotropic hypogonadism (IHH). Although therapeutics are a still a far-off goal, the formation and development of GnRH-positive neuronal-like cells following the application of KP-10 to Lyon NSC cells opens the door for future NSC-based therapies to treat specific reproductive disorders.

Peripheral and Central Mechanisms Involved in the Hormonal Control of Male and Female Reproduction.

Reproduction involves the integration of hormonal signals acting across multiple systems to generate a synchronised physiological output. A critical component of reproduction is the luteinising hormone (LH) surge, which is mediated by oestradiol (E2 ) and neuroprogesterone interacting to stimulate kisspeptin release in the rostral periventricular nucleus of the third ventricle in rats. Recent evidence indicates the involvement of both classical and membrane E2 and progesterone signalling in this pathway. A metabolite of gonadotrophin-releasing hormone (GnRH), GnRH-(1-5), has been shown to stimulate GnRH expression and secretion, and has a role in the regulation of lordosis. Additionally, gonadotrophin release-inhibitory hormone (GnIH) projects to and influences the activity of GnRH neurones in birds. Stress-induced changes in GnIH have been shown to alter breeding behaviour in birds, demonstrating another mechanism for the molecular control of reproduction. Peripherally, paracrine and autocrine actions within the gonad have been suggested as therapeutic targets for infertility in both males and females. Dysfunction of testicular prostaglandin synthesis is a possible cause of idiopathic male infertility. Indeed, local production of melatonin and corticotrophin-releasing hormone could influence spermatogenesis via immune pathways in the gonad. In females, vascular endothelial growth factor A has been implicated in an angiogenic process that mediates development of the corpus luteum and thus fertility via the Notch signalling pathway. Age-induced decreases in fertility involve ovarian kisspeptin and its regulation of ovarian sympathetic innervation. Finally, morphological changes in the arcuate nucleus of the hypothalamus influence female sexual receptivity in rats. The processes mediating these morphological changes have been shown to involve the rapid effects of E2 controlling synaptogenesis in this hypothalamic nucleus. In summary, this review highlights new research in these areas, focusing on recent findings concerning the molecular mechanisms involved in the central and peripheral hormonal control of reproduction.

Hyperglycaemic Hyperosmolar Nonketotic State as a Cause of Low Gonadotrophin Levels in Postmenopausal Diabetic Women: A Role for Severe Hypernatraemia

Hypogonadotrophic hypogonadism is associated with uncontrolled diabetes mellitus. Hyperglycaemia is a unique metabolic abnormality of the hyperglycaemic hyperosmolar nonketotic state (HHNKS) and, as glucose availability regulates gonadotrophin release, we investigated whether gonadotrophin release is inhibited in diabetic women with HHNKS, and whether hyperglycaemia, hypernatraemia or both inhibit in vitro gonadotrophin-releasing hormone (GnRH) expression in GT1-7 neurones. Three groups of postmenopausal women were studied: nine diabetics with HHNKS, nine hospitalised ill nondiabetics and 15 healthy women. In addition, the effects of glucose (5.55, 33.3, 66.6 mmol/l) and sodium chloride (150 and 170 mmol/l) on GnRH expression were investigated using GT1-7 neurones. Postmenopausal diabetics with HHNKS showed a decrease in serum levels of luteinising hormone (diabetic HHNKS 2.2 +/- 0.9 IU/l versus ill nondiabetic 21.0 +/- 2.3 IU/l and healthy controls 20.9 +/- 2.8 IU/l, P < 0.01), follicle-stimulating hormone (diabetic HHNKS 8.2 +/- 2.1 IU/l versus ill nondiabetic 50.4 +/- 9.1 IU/l and controls 60.2 +/- 6.9 IU/l, P < 0.01) and free 3,5,3'-triiodothyronine (diabetic HHNKS 1.48 +/- 0.57 pmol/l versus ill nondiabetic 4.28 +/- 0.26 pmol/l and controls 3.88 +/- 0.11 pmol/l, P < 0.01). The plasma cortisol level was higher in both diabetic (985 +/- 130 nmol/l) and ill nondiabetic (726 +/- 52 nmol/l) women than in healthy women (512 +/- 47 nmol/l), but no differences were observed in plasma oestradiol, thyroid-stimulating hormone or free thyroxine. In vitro GT1-7 neurones expressed three-fold less GnRH at 170 mmol/l than at 150 mmol/l NaCl, whereas changing glucose concentrations in the culture medium did not affect GnRH expression. In conclusion, postmenopausal diabetic women with HHNKS show decreased serum gonadotrophin levels, and severe hypernatraemia may participate in the hypogonadotropism observed in HHNKS.

Corticotrophin-releasing factor type 2 receptor-mediated suppression of gonadotrophin-releasing hormone mRNA expression in GT1-7 cells

Corticotrophin-releasing factor (CRF) released during stress has been implicated in the suppression of the hypothalamo–pituitary–gonadal (HPG) axis, especially the gonadotrophin-releasing hormone (GnRH) pulse generator, the central neural regulator of pituitary LH and FSH secretion, resulting in reproductive dysfunction. The gonadal steroid 17β-oestradiol (E2) has been shown to enhance CRF- and stress-induced suppression of pulsatile LH secretion. In the present study, we investigated the potential direct action of CRF on GnRH neurones by using GT1-7 cells, an established GnRH cell line. Furthermore, we investigated the modulatory influence of E2 on the effects of CRF and expression of CRF type 2 receptors (CRF-R2). Expression of CRF-R2 in the GT1-7 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). CRF produced a dose-dependent suppression of GnRH mRNA expression, an effect reversed by the selective CRF-R2 antagonist, astressin2-B (Ast2-B). E2 combined with CRF resulted in a greater suppression of GnRH expression compared with either treatment alone. E2 also increased CRF-R2 expression. These results demonstrate for the first time expression of CRF-R2 in the GT1-7 cells and suggest that CRF may directly regulate GnRH gene expression, an effect mediated, at least in part, by CRF-R2. They also raise the possibility that up-regulation of CRF-R2 may contribute to the sensitising influence of E2 on CRF- and stress-induced suppression of the GnRH pulse generator.

Endogenous regulation of the GnRH receptor by GnRH in the human placenta.

Gonadotrophin releasing hormone (GnRH) plays an important regulatory role in the function and growth of human placenta, but its placental expression sites and co-localization with GnRH receptor (GnRH-R) are not well known. GnRH and GnRH-R expression has been found in both placenta and cultured trophoblasts; however, cultured trophoblastic cells very quickly lose GnRH-R message and subsequently receptor protein. Speculating that endogenously released GnRH induced this down-regulation, we determined GnRH-R in cultures of trophoblastic cells in the presence of a neutralizing anti-GnRH antibody. Cells incubated with this antibody showed a strong signal for the GnRH-R, while those cultured without did not (as analysed by immunofluorescence, reverse transcription-polymerase chain reaction, protein 'dot blot' and Western blotting). Furthermore, addition of the GnRH agonist buserelin led to a reduction of the receptor protein. We have therefore shown that GnRH released from trophoblastic cells down-regulates GnRH-R in these trophoblastic cells. Having previously shown that trophoblast layers were synchronously positive for GnRH and GnRH-R, these new findings support the hypothesis of an ultrashort feedback regulation of trophoblasts by GnRH involving autocrine regulation of GnRH-R.

Detection of gonadotrophin releasing hormone and its receptor mRNA in human placental trophoblasts using in-situ reverse transcription-polymerase chain reaction.

Neuropeptides such as gonadotrophin releasing hormone (GnRH) are presumed to play an important role in the regulation of the function and growth of human placenta. Knowledge about the placental site of GnRH expression and the eventual co-localization of its peptide with the GnRH receptor (GnRH-R) is crucial for a better understanding of possible autocrine/paracrine mechanisms. We therefore investigated these questions by use of in-situ reverse transcription-polymerase chain reaction (RT-PCR) alone or in combination with immunocytochemistry in human first and third trimester placentae. Paraffin-embedded placental sections (7 microm thick), or single trophoblasts in monolayer cultures for up to 3 days, were treated with proteinase K. Following RT with GnRH or GnRH-R specific oligoprimers, PCR was performed employing primers with exon-exon overlaps to exclude non-specific DNA amplification. Detection of the amplicons was accomplished by nested PCR which was performed with digoxigenin-labelled dUTP and nitroblue tetrazolium/5-bromo-4-chloro-3-indoyl-phosphate (NBT/BCIP) for substrate visualization. The GnRH peptide was detected using a sandwich-antibody assay. GnRH and GnRH-R gene expression was found in all first and third trimester placentae, with abundant signals for the GnRH and GnRH-R message both in the cyto- and syncytiotrophoblasts. Single trophoblasts of different gestational ages in culture also displayed GnRH expression in individual cytotrophoblasts and in syncytiotrophoblast-like fusionates. Additional immunostaining revealed GnRH peptide to be co-localized with GnRH-R message in trophoblast layers. Since messages for GnRH and GnRH-R were found in virtually all trophoblasts, we infer that GnRH and GnRH-R are co-expressed in identical cells. These data strongly suggest that the trophoblasts are the source of GnRH, and that there is autocrine/ paracrine regulation by GnRH in human placenta.