Unurjargal Sukhbaatar

Kisspeptin induces expression of gonadotropin-releasing hormone receptor in GnRH-producing GT1-7 cells overexpressing G protein-coupled receptor 54.

Kisspeptin signaling through its receptor is crucial for many reproductive functions. However, the molecular mechanisms and biomedical significance of the regulation of GnRH neurons by kisspeptin have not been adequately elucidated. In the present study, we found that kisspeptin increases GnRH receptor (GnRHR) expression in a GnRH-producing cell line (GT1-7). Because cellular activity of G protein-coupled receptor 54 (GPR54) and GnRHR was limited in GT1-7 cells, we overexpressed these receptors to clarify receptor function. Using luciferase reporter constructs, the activity of both the serum response element (Sre) promoter, a target for extracellular signal-regulated kinase (ERK), and the cyclic AMP (cAMP) response element (Cre) promoter were increased by kisspeptin. Although GnRH increased Sre promoter activity, the Cre promoter was not significantly activated by GnRH. Kisspeptin, but not GnRH, increased cAMP accumulation in these cells. Kisspeptin also increased the transcriptional activity of GnRHR; however, the effect of GnRH on the GnRHR promoter was limited and not significant. Transfection of GT1-7 cells with constitutively active MEK kinase (MEKK) and protein kinase A (PKA) increased GnRHR expression. In addition, GnRHR expression was further increased by co-overexpression of MEKK and PKA. The Cre promoter, but not the Sre promoter, was also further activated by co-overexpression of MEKK and PKA. GnRH significantly increased the activity of the GnRHR promoter in the presence of cAMP. The present findings suggest that kisspeptin is a potent stimulator of GnRHR expression in GnRH-producing neurons in association with ERK and the cAMP/PKA pathways.

Circulating kisspeptin and pituitary adenylate cyclase-activating polypeptide (PACAP) do not correlate with gonadotropin serum levels.

Abstract Kisspeptins are known to be the principle regulators of the hypothalamic-pituitary gonadal (HPG) axis. In addition, the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of pituitary gonadotropins has been elucidated. We measured plasma concentrations of kisspeptin and PACAP and determined whether the levels of these peptides varied in proportion to circulating gonadotropin levels. Plasma luteinizing hormone (LH) levels were higher in postmenopausal women and in patients with premature ovarian failure (POF) and lower in patients with idiopathic hypogonadotropic hypogonadism (IHH) compared with the LH level in normally menstruating women. Similarly, serum follicle-stimulating hormone levels were higher in postmenopausal women and in patients with POF but lower in pregnant women and patients with IHH compared with normally menstruating women. Plasma levels of kisspeptins were significantly higher in pregnant women compared with normally menstruating women. However, no significant differences were observed in postmenopausal women, patients with POF, and patients with IHH. On the other hand, plasma levels of PACAP were significantly lower in pregnant women, patients with POF, and in IHH patients when compared with normally menstruating women. No significant differences were observed in PACAP concentration between postmenopausal women and in normally menstruating women. Our observations suggest that the serum levels of kisspeptins and PACAP did not correlate with variations in serum gonadotropin levels.

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases expression of the gonadotropin-releasing hormone (GnRH) receptor in GnRH-producing GT1-7 cells overexpressing PACAP type I receptor

The present study demonstrates the action of pituitary adenylate cyclase-activating polypeptide (PACAP) on gonadotropin-releasing hormone (GnRH)-producing neuronal cells, GT1-7. Because we found the expression levels of PACAP type 1 receptor (PAC1R) to be low in these cells, we transfected them with PAC1R expression vector and observed the outcome. PACAP increased the activity of the serum response element (Sre) promoter, a target of extracellular signal-regulated kinase (ERK), as well as the cAMP response element (Cre) promoter in GT1-7 cells overexpressing PAC1R. We also observed ERK phosphorylation and cAMP accumulation upon PACAP stimulation. PACAP stimulated the promoter activity of GnRH receptor (GnRHR) with increasing levels of GnRHR proteins. Notably, the increase in GnRHR promoter activity from kisspeptin was potentiated in the presence of PACAP. A similar increasing effect of PACAP on the action of kisspeptin was observed for Cre promoter activity. On the other hand, the Sre promoter activated by kisspeptin was inhibited by co-treatment with kisspeptin and PACAP. Likewise, kisspeptin-increased GnRHR promoter activity and Cre promoter activity were both potentiated in the presence of cAMP, whereas the Sre promoter activated by kisspeptin was inhibited in the presence of cAMP. Our observations show that PACAP increases GnRHR expression and stimulates kisspeptins effect on GnRHR expression in association with the cAMP/PKA signaling pathway in GT1-7 cells overexpressing PAC1R. In addition, PACAP was shown to have an inhibitory effect on ERK-mediated kisspeptin action.

Interactions between Two Different G Protein-Coupled Receptors in Reproductive Hormone-Producing Cells: The Role of PACAP and Its Receptor PAC1R

Gonadotropin-releasing hormone (GnRH) and gonadotropins are indispensable hormones for maintaining female reproductive functions. In a similar manner to other endocrine hormones, GnRH and gonadotropins are controlled by their principle regulators. Although it has been previously established that GnRH regulates the synthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH)—both gonadotropins—from pituitary gonadotrophs, it has recently become clear that hypothalamic GnRH is under the control of hypothalamic kisspeptin. Prolactin, which is also known as luteotropic hormone and is released from pituitary lactotrophs, stimulates milk production in mammals. Prolactin is also regulated by hypothalamic factors, and it is thought that prolactin synthesis and release are principally under inhibitory control by dopamine through the dopamine D2 receptor. In addition, although it remains unknown whether it is a physiological regulator, thyrotropin-releasing hormone (TRH) is a strong secretagogue for prolactin. Thus, GnRH, LH and FSH, and prolactin are mainly regulated by hypothalamic kisspeptin, GnRH, and TRH, respectively. However, the synthesis and release of these hormones is also modulated by other neuropeptides in the hypothalamus. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a hypothalamic peptide that was first isolated from sheep hypothalamic extracts based on its ability to stimulate cAMP production in anterior pituitary cells. PACAP acts on GnRH neurons and pituitary gonadotrophs and lactotrophs, resulting in the modulation of their hormone producing/secreting functions. Furthermore, the presence of the PACAP type 1 receptor (PAC1R) has been demonstrated in these cells. We have examined how PACAP and PAC1R affect GnRH- and pituitary hormone-secreting cells and interact with their principle regulators. In this review, we describe our understanding of the role of PACAP and PAC1R in the regulation of GnRH neurons, gonadotrophs, and lactotrophs, which are regulated mainly by kisspeptin, GnRH, and TRH, respectively.

How is GnRH regulated in GnRH-producing neurons? Studies using GT1-7 cells as a GnRH-producing cell model

Hypothalamic secretion of gonadotropin-releasing hormone (GnRH) has been established as a principle pathway for initiating and integrating female reproductive function. GnRH stimulates the release of two gonadotropins-luteinizing hormone and follicle-stimulating hormone-from the anterior pituitary, which eventually stimulate the synthesis of sex steroids in association with follicular growth and ovulation. This reproductive control of the hypothalamic-pituitary-gonadal (HPG) axis also mediates gonadal feedback mechanisms. Although GnRH neurons certainly play a pivotal role in the HPG axis, the detailed mechanisms of their functional network, including regulatory systems, remain unknown. After the discovery of the indispensable role of kisspeptin in the development of human reproductive functions, our understanding of the neuroendocrine regulation of the HPG axis was revolutionized, and it is now recognized that kisspeptin acts upstream of GnRH and is responsible for sex steroid feedback mechanisms. Kisspeptin can stimulate gonadotropin release from the pituitary gland by stimulating GnRH release and GnRH antagonists prevent kisspeptin-induced gonadotropin release. Furthermore, it has been shown that GnRH neurons express kisspeptin receptors. Nevertheless, the detailed mechanisms underlying the regulation of homogeneous populations of GnRH neurons are still largely unknown because of the limitations of experimental models used for investigation. The hypothalamus consists of a complex network of distinct neuronal cells, and it is difficult to isolate single-cell populations of GnRH neurons. The establishment of GnRH-expressing cell lines has allowed us to examine the events happening at the single-cell level. In this review, we describe in vitro studies using a GnRH-producing cell model, GT1-7 cells, which have been used to examine how GnRH-producing cells respond to hypothalamic factors and how they are involved in GnRH synthesis.

Mutual regulation by GnRH and kisspeptin of their receptor expression and its impact on the gene expression of gonadotropin subunits

Hypothalamic kisspeptin plays a pivotal role in the regulation of the hypothalamic-pituitary-gonadal axis by stimulating gonadotropin-releasing hormone (GnRH) release into the portal circulation, with the subsequent release of gonadotropins. Kisspeptin and its receptor, the kisspeptin 1 receptor (Kiss1R), are also expressed in the pituitary gland. This study demonstrates the interaction between GnRH and kisspeptin within the pituitary gonadotrophs by altering their individual receptor expression. Our results show that kisspeptin and Kiss1R are expressed in the mouse pituitary gonadotroph cell line LβT2. Endogenous Kiss1R did not respond to kisspeptin and failed to stimulate gonadotropin LHβ and FSHβ expression in LβT2 cells; however, kisspeptin increased both LHβ and FSHβ promoter activity in Kiss1R-overexpressing LβT2 cells. Stimulating the cells with GnRH significantly increased Kiss1R expression, whereas kisspeptin increased the expression of the GnRH receptor (GnRHR) in these cells. Elevating the Kiss1R concentration led to an increase in the basal activities of gonadotropin LHβ- and FSHβ-subunit promoters. In addition, the level of kisspeptin-induced LHβ promoter activity, but not that of FSHβ, was significantly increased when a large number of Kiss1R expression vectors was introduced into the cells. The level of induction of GnRH-induced gonadotropin promoter activities was not significantly changed by increasing Kiss1R expression. Increasing the amount of GnRHR by overexpressing cellular GnRHR did not potentiate basal gonadotropin promoter activities; however, kisspeptin- and GnRH-stimulated increases in gonadotropin promoter activities were significantly potentiated (except GnRH-induced LHβ promoters). The activities of serum response element-containing promoters were also modified in cells overexpressing Kiss1R or GnRHR. Our current observations demonstrate that GnRH and kisspeptin affect each others function to stimulate gonadotropin subunit gene expression by reciprocally increasing the expression of their receptors.

Stimulation of δ subunit-containing GABAA receptor by DS1 increases GnRH receptor expression but reduces GnRH mRNA expression in GnRH-producing GT1-7 cells

Acting via ionotropic GABAA receptors, the neurotransmitter γ-aminobutyric acid (GABA) is an important modulator of gonadotropin-releasing hormone (GnRH) neurons. In the present study, we examined the effect of DS1, a GABAA α4β3δ receptor agonist, on a strain of mouse hypothalamic immortalized GnRH neuronal cells, the GT1-7 cell line. DS1 increased the activities of serum-response element (SRE) and cAMP-response element (CRE) promoters, which reflect the activities of extracellular signal-regulated kinase and cAMP/protein kinase A (PKA) pathways, respectively. In G protein-coupled receptor 54 (GPR54)-overexpressing GT1-7 cells, both DS1 and kisspeptin-10 stimulated SRE promoter activity, and combined treatment with DS1 and kisspeptin further increased SRE promoter activity compared with DS1 or kisspeptin alone. Pituitary adenylate cyclase-activating polypeptide (PACAP) increased CRE promoter activity in PACAP type I receptor-overexpressing GT1-7 cells, with an effect similar to that of DS1 alone, and combined stimulation with PACAP and DS1 potentiated their individual effects. DS1 stimulated the transcriptional activity of GnRH receptor, and DS1 induced GnRH receptor mRNA and protein expression. PACAP-increased GnRH receptor expression was enhanced in the presence of DS1. However, DS1 significantly inhibited the basal expression of GnRH mRNA in GT1-7 cells. Our current observations suggest that DS1 exerts its stimulatory effect on the intracellular signal transduction system via GABAA α4β3δ receptors in GnRH-producing neurons. Stimulation with DS1 increased the expression of GnRH receptor but decreased the basal expression of GnRH mRNA.

DS1, a Delta Subunit-Containing GABAA Receptor Agonist, Increases Gonadotropin Subunit Gene Expression in Mouse Pituitary Gonadotrophs

ABSTRACT 4-Chloro-N-[6,8-dibromo-2-(2-thienyl)imidazo[1,2-alpyridine-3-yl] (DS1) is a GABAA receptor agonist that selectively binds to delta subunit-containing GABAA alpha4beta3delta receptors. In the present study, we examined the effect of DS1 on pituitary gonadotropin subunit gene expression using the mouse pituitary gonadotroph cell line LbetaT2. DS1 increased the promoter activity of the gonadotropin subunits luteinizing hormone beta (LHbeta), follicle-stimulating hormone beta (FSHbeta), and alpha. Gonadotropin-releasing hormone (GnRH) receptor promoters were also activated by DS1. The effects of DS1 on gonadotropin subunit promoters were obvious, but they were less than those induced by stimulation with GnRH. GnRH-stimulated gonadotropin subunit promoters were enhanced in the presence of DS1. A prototypic specific agonist for GABAA receptors, muscimol, failed to increase LHbeta and FSHbeta subunit promoter activity and had no effect on GnRH-increased LHbeta and FSHbeta promoter activity. In addition, SKF97541, a specific agonist for GABAB receptors, did not modulate basal or GnRH-induced LHbeta and FSHbeta promoter activity. A natural GABA compound failed to increase gonadotropin promoter activity and potentiated the effect of GnRH on the FSHbeta promoter. DS1 increased the activity of serum response element (SRE) and cAMP response element (CRE) promoters, which reflect the activity of the extracellular signal-regulated kinase and cAMP/protein kinase A (PKA) pathways, and GnRH-increased SRE and CRE promoter activity was enhanced in the presence of DS1. A specific inhibitor of the ERK signaling pathway, U0126, prevented DS1-induced LHbeta and FSHbeta promoter activity almost completely; however, H89, a PKA inhibitor, did not modulate the effect of DS1. Our current observations demonstrate that the GABAA alpha4beta3delta receptor agonist DS1 can stimulate gonadotropin subunit gene expression in association with the ERK signaling pathway.

Regulation of kisspeptin and gonadotropin-releasing hormone expression in rat placenta: study using primary cultures of rat placental cells.

BackgroundGonadotropin-releasing hormone (GnRH) and kisspeptin in the hypothalamus are thought to be crucial components of the hypothalamic-pituitary-gonadal (HPG) axis and maintain reproductive function. These neuropeptides are also expressed in the placenta, where they may contribute to placental physiology. In this study, we examined how these peptides are regulated within the placenta.MethodsWe used primary cultures of placental tissue from rats of 16–18 days gestation. After stimulation with estradiol, GnRH, kisspeptin, and neurokinin B (NKB), changes in placental GnRH, kisspeptin, and human chorionic gonadotropin (hCG) mRNA expression were evaluated by real-time quantitative RT-PCR analysis.ResultsImmunocytochemical analysis showed that rat placental cells contained cells expressing kisspeptin or GnRH. GnRH and kisspeptin mRNA expression was significantly increased in placental cells in the presence of estradiol; NKB mRNA expression was also stimulated by estradiol. Stimulation of the cells with kisspeptin failed to stimulate GnRH mRNA expression. Conversely, both GnRH itself and NKB increased GnRH mRNA expression. Kisspeptin mRNA expression was not increased by kisspeptin itself; however, GnRH and NKB significantly increased kisspeptin mRNA expression. hCG expression was increased in the presence of estradiol. In addition, kisspeptin, GnRH, and NKB could stimulate the expression of hCG mRNA in placental cells.ConclusionsOur experiments using primary cultures of rat placental cells showed that GnRH, kisspeptin, and NKB expression was enhanced by estradiol, and unlike in the hypothalamus, kisspeptin did not control the expression of GnRH in placental cells. NKB might be located upstream of kisspeptin and GnRH, and these neuropeptides might be involved in the induction of hCG expression in placental cells.

Expression of GnRH and Kisspeptin in Primary Cultures of Fetal Rat Brain

Genetic studies in humans or in vivo studies using animals have shown that kisspeptin released from the hypothalamus controls secretion of gonadotropin-releasing hormone (GnRH) from GnRH neurons, and subsequently GnRH induces gonadotropin secretion from the anterior pituitary. Kisspeptindid not stimulate GnRH expression in the GnRH-producing cell line GT1-7. Thus, we cultured GnRH and kisspeptin neurons from whole fetal rat brain and examined the regulation of GnRH and kisspeptin. Expression of GnRH messenger RNA (mRNA) was unchanged by estradiol (E2) treatment in these primary cultures. In contrast, kisspeptin mRNA expression was increased 2. 00 + 0. 23-fold by E2 treatment. When these cultures were stimulated by kisspeptin-10, GnRH mRNA was significantly increased up to 1. 51 + 0. 35-fold. Expression of GnRH mRNA was also stimulated 1. 84 + 0. 33-fold by GnRH itself. Interestingly, kisspeptin mRNA was significantly increased up to 2. 43 + 0. 40-fold by kisspeptin alone. In addition, kisspeptin mRNA expression was significantly increased by stimulation with GnRH (1. 46 + 0. 21-fold). Our observations demonstrated that kisspeptin, but not GnRH, was upregulated by E2 and that kisspeptin stimulates GnRH mRNA expression in primary cultures of whole fetal rat brain. Furthermore, GnRH and kisspeptin stimulate their own neurons to produce GnRH or kisspeptin, respectively.Genetic studies in humans or in vivo studies using animals have shown that kisspeptin released from the hypothalamus controls secretion of gonadotropin-releasing hormone (GnRH) from GnRH neurons, and subsequently GnRH induces gonadotropin secretion from the anterior pituitary. Kisspeptin did not stimulate GnRH expression in the GnRH-producing cell line GT1-7. Thus, we cultured GnRH and kisspeptin neurons from whole fetal rat brain and examined the regulation of GnRH and kisspeptin. Expression of GnRH messenger RNA (mRNA) was unchanged by estradiol (E2) treatment in these primary cultures. In contrast, kisspeptin mRNA expression was increased 2.00 ± 0.23-fold by E2 treatment. When these cultures were stimulated by kisspeptin-10, GnRH mRNA was significantly increased up to 1.51 ± 0.35-fold. Expression of GnRH mRNA was also stimulated 1.84 ± 0.33-fold by GnRH itself. Interestingly, kisspeptin mRNA was significantly increased up to 2.43 ± 0.40-fold by kisspeptin alone. In addition, kisspeptin mRNA expression was significantly increased by stimulation with GnRH (1.46 ± 0.21-fold). Our observations demonstrated that kisspeptin, but not GnRH, was upregulated by E2 and that kisspeptin stimulates GnRH mRNA expression in primary cultures of whole fetal rat brain. Furthermore, GnRH and kisspeptin stimulate their own neurons to produce GnRH or kisspeptin, respectively.