The role of KNDy neurons in human reproductive health.

In the early 2000s, metastin, an endogenous ligand for G protein-coupled receptor 54 (GPR54), was discovered in human placental extracts. In 2003, GPR54 receptor mutations were found in a family with congenital hypogonadotropic hypogonadism. Metastin was subsequently renamed kisspeptin after its coding gene, Kiss1. Since then, studies in mice and other animals have revealed that kisspeptin is located at the apex of the hypothalamic-pituitary-gonadal axis and regulates reproductive functions by modulating gonadotropin-releasing hormone (GnRH). In rodents, kisspeptin (Kiss1) neurons localize to two regions, the hypothalamic arcuate nucleus (ARC) and the anteroventral periventricular nucleus (AVPV). ARC Kiss1 neurons co-express neurokinin B (NKB) and dynorphin and are thus termed KNDy neurons. Kiss1 neurons in humans are concentrated in the infundibular nucleus (equivalent to the ARC), with few Kiss1 neurons localized to the preoptic area (equivalent to the AVPV), and the mechanisms underlying GnRH surge secretion in humans are poorly understood. However, peripheral administration of kisspeptin to humans promotes gonadotropin secretion, and administration of kisspeptin to patients with hypothalamic amenorrhea or congenital hypogonadotropic hypogonadism restores the pulsatile secretion of GnRH/luteinizing hormone. Thus, kisspeptin undoubtedly plays an important role in reproductive function in humans. Studies are currently underway to develop kisspeptin receptor agonists or antagonists for clinical application. Modification of KNDy neurons by NKB agonists/antagonists is also being attempted to develop therapeutic agents for various menstrual abnormalities, including polycystic ovary syndrome and menopausal hot flashes. Here, we review the role of kisspeptin in humans and its clinical applications.

Impact of current and previous sperm findings on outcomes of intrauterine insemination.

Purpose: To examine the association between semen characteristics and outcomes of intrauterine insemination (IUI). Methods: This retrospective analysis examined 1380 IUI procedures involving 421 couples. The association of clinical pregnancy with pre- and post-wash sperm characteristics was assessed. Results: Pre- and post-wash sperm characteristics did not differ between IUI cycles that resulted in pregnancy and those that did not. When the motility of pre-wash sperm was below the normal range (<42%) established by the World Health Organization (WHO), the pregnancy rate was significantly lower. In the IUI cycles when post-wash sperm motility was below the WHO standard, pregnancy was not achieved. The frequency of improvement in post-wash sperm motility in repeated IUI cycles appeared to correlate with the success of future IUI cycles. At the fourth IUI cycle, pregnancy was not achieved unless the post-wash sperm motility was normal in at least two of three attempts. When post-wash sperm concentration was below the normal range, the woman's age did not affect the IUI outcomes. Conclusions: Sperm motility above the lower limit of the WHO criteria in post-wash semen samples is an important factor in IUI outcomes.

Kisspeptin induces Kiss-1 and GnRH gene expression in mHypoA-55 hypothalamic cell models: Involvement of the ERK and PKA signaling pathways.

mHypoA-55 cells are kisspeptin-expressing neuronal cells originating from the arcuate nucleus of the mouse hypothalamus. These cells are called KNDy neurons because they co-express kisspeptin, neurokinin B, and dynorphin A. In addition, they express gonadotropin-releasing hormone (GnRH). Here, we found that kisspeptin 10 (KP10) increased Kiss-1 (encoding kisspeptin) and GnRH gene expression in kisspeptin receptor (Kiss-1R)-overexpressing mHypoA-55 cells. KP10 greatly increased serum response element (SRE) promoter activity, which is a target of extracellular signal-regulated kinase (ERK) (20.0 ± 2.54-fold). KP10 also increased cAMP-response element (CRE) promoter activity in these cells (2.32 ± 0.36-fold). KP10-increased SRE promoter activity was significantly prevented in the presence of PD098095, a MEK kinase (MEKK) inhibitor, and KP10-induced CRE promoter activity was also inhibited by PD098059. Similarly, H89, a protein kinase A (PKA) inhibitor, significantly inhibited the KP10 induction of SRE and CRE promoters. KP10-induced Kiss-1 and GnRH gene expressions were inhibited in the presence of PD098059. Likewise, H89 significantly inhibited the KP10-induced increase in Kiss-1 and GnRH. Transfection of mHypoA-55 cells with constitutively active MEKK (pFC-MEKK) increased SRE and CRE promoter activities by 9.75 ± 1.77- and 1.36 ± 0.12-fold, respectively. Induction of constitutively active PKA (pFC-PKA) also increased SRE and CRE promoter activities by 2.41 ± 0.42- and 40.71 ± 7.77-fold, respectively. Furthermore, pFC-MEKK and -PKA transfection of mHypoA-55 cells increased both Kiss-1 and GnRH gene expression. Our current observations suggest that KP10 increases both the ERK and PKA pathways and that both pathways mutually interact in mHypoA-55 hypothalamic cells. Activation of both ERK and PKA signaling might be necessary to induce Kiss-1 and GnRH gene expressions.

Hyperandrogenism induces proportional changes in the expression of Kiss-1, Tac2, and DynA in hypothalamic KNDy neurons.

BACKGROUND: Kisspeptin released from Kiss-1 neurons in the hypothalamus plays an essential role in the control of the hypothalamic-pituitary-gonadal axis by regulating the release of gonadotropin-releasing hormone (GnRH). In this study, we examined how androgen supplementation affects the characteristics of Kiss-1 neurons. METHODS: We used a Kiss-1-expressing mHypoA-55 cell model that originated from the arcuate nucleus (ARC) of the mouse hypothalamus. These cells are KNDy neurons that co-express neurokinin B (NKB) and dynorphin A (DynA). We stimulated these cells with androgens and examined them. We also examined the ARC region of the hypothalamus in ovary-intact female rats after supplementation with androgens. RESULTS: Stimulation of mHypoA-55 cells with 100 nM testosterone significantly increased Kiss-1 gene expression by 3.20 ± 0.44-fold; testosterone also increased kisspeptin protein expression. The expression of Tac3, the gene encoding NKB, was also increased by 2.69 ± 0.64-fold following stimulation of mHypoA-55 cells with 100 nM testosterone. DynA gene expression in these cells was unchanged by testosterone stimulation, but it was significantly reduced at the protein level. Dihydrotestosterone (DHT) had a similar effect to testosterone in mHypoA-55 cells; kisspeptin and NKB protein expression was significantly increased by DHT, whereas it significantly reduced DynA expression. In ovary-intact female rats, DTH administration significantly increased the gene expression of Kiss-1 and Tac3, but not DynA, in the arcuate nucleus. Exogenous NKB and DynA stimulation failed to modulate Kiss-1 gene expression in mHypoA-55 cells. Unlike androgen stimulation, prolactin stimulation did not modulate kisspeptin, NKB, or DynA protein expression in these cells. CONCLUSIONS: Our observations imply that hyperandrogenemia affects KNDy neurons and changes their neuronal characteristics by increasing kisspeptin and NKB levels and decreasing DynA levels. These changes might cause dysfunction of the hypothalamic-pituitary-gonadal axis.

Effect of anti-Müllerian hormone on the regulation of pituitary gonadotropin subunit expression: roles of kisspeptin and its receptors in gonadotroph LßT2 cells.

Anti-Müllerian hormone (AMH) is primarily produced by ovarian granulosa cells and contributes to follicle development. AMH is also produced in other tissues, including the brain and pituitary; however, its roles in these tissues are not well understood. In this study, we examined the effect of AMH on pituitary gonadotrophs. We detected AMH and AMH receptor type 2 expression in LßT2 cells. In these cells, the expression of FSHß- but not α- and LHß-subunits increased significantly as the concentration of AMH increased. LßT2 cells expressed Kiss-1 and Kiss-1R. AMH stimulation resulted in decreases in both Kiss-1 and Kiss-1R. The siRNA-mediated knockdown of Kiss-1 in LßT2 cells did not alter the basal expression levels of α-, LHß-, and FSHß-subunits. In LßT2 cells overexpressing Kiss-1R, exogenous kisspeptin stimulation significantly increased the expression of all three gonadotropin subunits. However, kisspeptin-induced increases in these subunits were almost completely eliminated in the presence of AMH. In contrast, GnRH-induced increases in the three gonadotropin subunits were not modulated by AMH. Our observations suggested that AMH acts on pituitary gonadotrophs and induces FSHß-subunit expression with concomitant decreases in Kiss-1 and Kiss-1R gene expression. Kisspeptin, but not GnRH-induced gonadotropin subunit expression, was inhibited by AMH, suggesting that it functions in association with the kisspeptin/Kiss-1R system in gonadotrophs.

Effect of anti-Müllerian hormone in hypothalamic Kiss-1- and GnRH-producing cell models.

Purpose: Anti-Müllerian hormone (AMH) is one of the local factors involved in follicle development. In addition, AMH and its receptor are broadly expressed throughout the body. In this study, we examined how AMH modifies gene expression of Kiss-1 and GnRH.Materials and methods: mHypoA-50 and mHypoA-55 cells were originated from the hypothalamic anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC), respectively, and these cells are known as Kiss-1 (which encodes kisspeptin) expressing cell models. These cells also express gonadotropin-releasing hormone (GnRH) genes. Our experiments were performed useing these cell models.Results: Both mHypoA-50 and mHypoA-55 hypothalamic cells expressed AMH and AMH receptor type 2 (AMHR2). Exogenous AMH failed to alter the expression levels of the Kiss-1 gene in both cell models but significantly increased GnRH gene expression by 1.73 ± 0.2-fold at 100 pM in mHypoA-50 AVPV cells and by 1.74 ± 0.17-fold at 1 nM in mHypoA-55 ARC cells. AMH also augmented GnRH protein expression in both cell models. Similar to the phenomenon observed in the hypothalamic cell lines, 100 pM AMH significantly increased GnRH, but not Kiss-1, mRNA expression in primary cultures of fetal rat brain cells. Kisspeptin-10 (KP10) increased Kiss-1 gene expression in mHypoA-55 ARC cells but this was blocked by AMH. AMH did not alter the expression of the kisspeptin receptor (Kiss1R) or that of neurokinin B or dynorphin A in mHypoA-55 ARC cells.Conclusions: It was demonstrated that AMH participates in hypothalamic-pituitary-gonadal axis control by stimulating GnRH expression. In addition, AMH might be a potent repressor of Kiss-1 gene expression induced by KP10.

Reproductive prognosis of patients with hypogonadotropic hypogonadism: Retrospective review of 16 cases with amenorrhea.

AIM: The aim of this study was to evaluate the general characteristics, menstruation status, and fertility outcomes of patients with hypogonadotropic hypogonadism (HH). METHODS: We evaluated 16 patients with HH who visited our institution between April 2012 and March 2016 with a complaint of amenorrhea. RESULTS: Four (25%) patients had primary amenorrhea and the remaining 12 (75%) cases had secondary amenorrhea. Among the patients with primary amenorrhea, weight loss was considered to be the underlying cause in one (25%) patient, whereas the remaining three (75%) cases were idiopathic HH. Among HH cases with secondary amenorrhea, six (50%) developed amenorrhea following weight loss, whereas the remaining six cases were of unknown etiology. Among the 16 patients with HH, we observed the sporadic restart of the menstrual cycle in four (25%) women during follow-up. Infertility treatment was administered to nine patients with HH who wished to become pregnant. Clomiphene citrate was effective in four patients with secondary amenorrhea and induced follicular development. Seven of nine patients with HH (77.8%) became pregnant following infertility treatment. In some cases of HH, the serum levels of gonadotropin increased sporadically during follow-up, regardless of the recovery of menstruation. We followed one patient with HH for more than 20 years. Although her gonadotropin levels were generally low and sometimes fluctuated without spontaneous menstruation, they increased dramatically to menopausal levels at 50 years of age. However, they again decreased to hypogonadotropic levels. CONCLUSION: As the pathophysiology varied widely among patients, the etiologic factors underlying HH might also vary.

Role of activin, follistatin, and inhibin in the regulation of Kiss-1 gene expression in hypothalamic cell models†.

Kisspeptin (encoded by the Kiss-1 gene) in the arcuate nucleus (ARC) of the hypothalamus governs the hypothalamic-pituitary-gonadal (HPG) axis by regulating pulsatile release of gonadotropin-releasing hormone (GnRH). Meanwhile, kisspeptin in the anteroventral periventricular nucleus (AVPV) region has been implicated in estradiol (E2)-induced GnRH surges. Kiss-1-expressing cell model mHypoA-55 exhibits characteristics of Kiss-1 neurons in the ARC region. On the other hand, Kiss-1 expressing mHypoA-50 cells originate from the AVPV region. In the mHypoA-55 ARC cells, activin significantly increased Kiss-1 gene expression. Follistatin alone reduced Kiss-1 expression within these cells. Interestingly, activin-induced Kiss-1 gene expression was completely abolished by follistatin. Inhibin A, but not inhibin B reduced Kiss-1 expression. Activin-increased Kiss-1 expression was also abolished by inhibin A. Pretreatment of the cells with follistatin or inhibin A significantly inhibited kisspeptin- or GnRH-induced Kiss-1 gene expression in mHypoA-55 cells. In contrast, in the mHypoA-50 AVPV cell model, activin, follistatin, and inhibin A did not modulate Kiss-1 gene expression. The subunits that compose activin and inhibin, as well as follistatin were expressed in both mHypoA-55 and mHypoA-50 cells. Expression of inhibin ßA and ßB subunits and follistatin was much higher in mHypoA-55 ARC cells. Furthermore, we found that expression of the inhibin α subunit and follistatin genes was modulated in the presence of E2 in mHypoA-55 ARC cells. The results of this study suggest that activin, follistatin, and inhibin A within the ARC region participate in the regulation of the HPG axis under the influence of E2.

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of hypothalamic kisspeptin expression.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor are broadly distributed in the brain, and PACAP is known to work as a multifunctional peptide. However, it is still largely unknown how PACAP affects the hypothalamic-pituitary-gonadal (HPG) axis. In this study, we examined the effect of PACAP on hypothalamic kisspeptin expression, a known regulator of gonadotropin-releasing hormone. We used two hypothalamic cell models, mHypoA-50 and mHypoA-55, which were originated from kisspeptin-expressing neuron in anterioventral periventricular nucleus and arcuate nucleus regions in the hypothalamus, respectively. Expression of Kiss-1 gene, which encodes kisspeptin, was significantly increased by PACAP stimulation in both mHypoA-50 and mHypoA-55 cells, by up to 2.69 ±â€¯0.93-fold and 4.89 ±â€¯1.13-fold, respectively. PACAP6-38, a PACAP receptor antagonist did not antagonize the action of PACAP on Kiss-1 gene expression but increased Kiss-1 gene by itself in these cells. PACAP-induced Kiss-1 gene expression in both mHypoA-50 and mHypoA-55 cells was almost completely prevented in the presence of H89, a protein kinase A inhibitor. PACAP was expressed in both these hypothalamic cell models and its expression was up-regulated by estradiol in mHypoA-50 cells but not in mHypoA-55 cells. Stimulation of mHypoA-50 and mHypoA-55 cells with PACAP increased the expression levels of corticotropin-releasing hormone and neurotensin, both of which could modulate HPG axis. Our present observations suggest that hypothalamic PACAP might modulate the HPG axis by directly or indirectly modulating Kiss-1 gene expression.

Effect of relaxin-3 on Kiss-1, gonadotropin-releasing hormone, and gonadotropin subunit gene expression.

PURPOSE: Relaxin-3 is a hypothalamic neuropeptide that belongs to the insulin superfamily. We examined whether relaxin-3 could affect hypothalamic Kiss-1, gonadotropin-releasing hormone (GnRH), and pituitary gonadotropin subunit gene expression. METHODS: Mouse hypothalamic cell models, mHypoA-50 (originated from the hypothalamic anteroventral periventricular region), mHypoA-55 (originated from arcuate nucleus), and GT1-7, and the mouse pituitary gonadotroph LßT2 were used. Expression of Kiss-1, GnRH, and luteinizing hormone (LH)/follicle-stimulating hormone (FSH) ß-subunits was determined after stimulation with relaxin-3. RESULTS: RXFP3, a principle relaxin-3 receptor, was expressed in these cell models. In mHypoA-50 cells, relaxin-3 did not exert a significant effect on Kiss-1 expression. In contrast, the Kiss-1 gene in mHypoA-55 was significantly increased by 1 nmol/L relaxin-3. These cells also express GnRH mRNA, and its expression was significantly stimulated by relaxin-3. In GT1-7 cells, relaxin-3 significantly upregulated Kiss-1 expression; however, GnRH mRNA expression in GT1-7 cells was not altered. In primary cultures of fetal rat neuronal cells, 100 nmol/L relaxin-3 significantly increased GnRH expression. In pituitary gonadotroph LßT2, both LHß- and FSHß-subunit were significantly increased by 1 nmol/L relaxin-3. CONCLUSIONS: Our findings suggest that relaxin-3 exerts its effect by modulating the expression of Kiss-1, GnRH, and gonadotropin subunits, all of which are part of the hypothalamic-pituitary-gonadal axis.

Action of neurotensin, corticotropin-releasing hormone, and RFamide-related peptide-3 in E2-induced negative feedback control: studies using a mouse arcuate nucleus hypothalamic cell model.

The recently established immortalized hypothalamic cell model mHypoA-55 possesses characteristics similar to those of Kiss-1 neurons in the arcuate nucleus (ARC) region of the hypothalamus. Here, we show that Kiss-1 gene expression in these cells was downregulated by 17ß-estradiol (E2) under certain conditions. Both neurotensin (NT) and corticotropin-releasing hormone (CRH) were expressed in these cells and upregulated by E2. Stimulation of mHypoA-55 cells with NT and CRH significantly decreased Kiss-1 mRNA expression. A mammalian gonadotropin-inhibitory hormone homolog, RFamide-related peptide-3 (RFRP-3), was also found to be expressed in mHypoA-55 cells, and RFRP-3 expression in these cells was increased by exogenous melatonin stimulation. E2 stimulation also upregulated RFRP-3 expression in these cells. Stimulation of mHypoA-55 cells with RFRP-3 significantly increased the expression of NT and CRH. Furthermore, melatonin stimulation resulted in the increase of both NT and CRH mRNA expression in mHypoA-55 cells. On the other hand, in experiments using mHypoA-50 cells, which were originally derived from hypothalamic neurons in the anteroventral periventricular nucleus, Kiss-1 gene expression was upregulated by both NT and CRH, although E2 increased both NT and CRH expression, similarly to the mHypoA-55 cells. Our observations using the hypothalamic ARC cell model mHypoA-55 suggest that NT and CRH have inhibitory effects on Kiss-1 gene expression under the influence of E2 in association with RFRP-3 expression. Thus, these neuropeptides might be involved in E2-induced negative feedback mechanisms.

Role of pituitary adenylate cyclase-activating polypeptide in modulating hypothalamic-pituitary system.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional peptide that is isolated and identified from the ovine hypothalamus, whose effects and mechanisms have been elucidated in numerous studies. The PACAP and its receptor are widely expressed, not only in the hypothalamus but also in peripheral organs. METHODS: The studies on the role of PACAP in the hypothalamic-pituitary system, including those by the authors, were summarized. RESULTS: In the pituitary gonadotrophs, PACAP increases the gonadotrophin α-, luteinizing hormoneß-, and follicle-stimulating hormone ß-subunit expression and the expression of gonadotropin-releasing hormone (GnRH) receptor and its own receptor, PAC1R. Moreover, a low-frequency GnRH pulse increases the expression of PACAP and PAC1R more than a high-frequency GnRH pulse in the gonadotrophs. The PACAP stimulates prolactin synthesis and secretion and increases PAC1R in the lactotrophs. In the hypothalamus, PACAP increases the expression of the GnRH receptors, although it is unable to increase the expression of GnRH in the GnRH-producing neurons. CONCLUSION: The PACAP not only acts directly in each hormone-producing cell, it possibly might regulate hormone synthesis via the expression of its own receptors or those of other hormones.

Characterization of oocyte retrieval cycles with empty zona pellucida.

Purpose: To identify the factors that characterize cycles with empty zona pellucida (EZP). Methods: Thirty-six oocyte retrieval cycles from which EZP were collected and another 36 cycles from which no EZP was collected were compared. The patients were divided into three groups: those with no EZP collected during any cycle, those with EZP collected during all cycles, and those experiencing cycles both with and without EZP. Results: The mean number of oocytes collected per cycle was higher in the cycles with EZP than without EZP. The fertilization rate of the collected oocytes and the rate of good embryo formation were significantly lower in the cycles with EZP. No significant difference was observed between the three groups in terms of age, number of oocytes collected, or hormone levels before and after the oocyte retrieval. The fertilization and pregnancy rates were highest in the patients with no EZP being collected during any cycle, followed by those experiencing cycles both with and without EZP, and then by those with EZP collected during all cycles. Conclusion: The observation of lower fertilization, poor embryo formation, and a low pregnancy rate in the patients with EZP suggests the poor quality of oocytes that were collected with EZP in the same cycle.

Interaction between kisspeptin and adenylate cyclase-activating polypeptide 1 on the expression of pituitary gonadotropin subunits: a study using mouse pituitary lbetaT2 cells.

We examined direct effect of kisspeptin on pituitary gonadotrophs. Kisspeptin-10 (KP10) significantly increased the promoter activities of the gonadotropin subunits, common alpha-glycoprotein (Cga), luteinizing hormone beta (Lhb), and follicle-stimulatinghormone beta (Fshb) in LbetaT2 cells overexpressing kisspeptin receptor (Kiss1r). KP10 and gonadotropin-releasing hormone (GnRH) increased gonadotropin subunit levels to similar degrees and combined treatment with GnRH and KP10 did not potentiate their individual effects. Adenylate cyclase-activating polypeptide 1 (ADCYAP1) also stimulates all three gonadotropin subunits. When cells were stimulated with both KP10 and ADCYAP1, expression of gonadotropin subunits was further increased compared to KP10 or ADCYAP1 alone. KP10 and GnRH dramatically increased serum response element (Sre) promoter levels but only slightly increased cAMP response element (Cre) promoter levels. Combined stimulation with KP10 and GnRH further increased Sre promoter levels. In contrast, ADCYAP1 slightly increased Sre promoter expression but did not modify the effect of KP10. However, ADCYAP1 increased Cre promoter to greater levels than KP10 alone, and combined treatment with KP10 and ADCYAP1 further increased Cre promoter expression. KP10 increased the expression of ADCYAP1 type I receptor (Adcyap1r) and the basal activity of the Cga promoter was increased at a higher Adcyap1r transfection level. The KP10-induced fold increase in all three gonadotropin subunit promoters was not altered by transfection with a higher amount of Adcyap1r vector. Our findings using model cells show that distinct signaling activation by ADCYAP1 potentiates the action of KP10. We also found that KP10 increases Adcyap1r expression.

GLP-1 increases Kiss-1 mRNA expression in kisspeptin-expressing neuronal cells.

Feeding-related metabolic factors exert regulatory influences on the hypothalamic-pituitary-gonadal axis. Glucagon-like peptide-1 (GLP-1) is an anorexigenic hormone synthesized from the ileum in response to food intake. The purpose of this study was to examine the direct effect of GLP-1 on hypothalamic kisspeptin and gonadotropin-releasing hormone (GnRH) expression using the rat clonal hypothalamic cell line rHypoE-8. GLP-1 significantly increased Kiss-1 mRNA expression in rHypoE-8 cells up to 1.94 ± 0.22-fold. This effect of GLP-1 on Kiss-1 gene expression was also observed in GT1-7 GnRH-producing neurons and in primary cultures of fetal rat brain. GLP-1 increased cAMP-mediated signaling, as determined by cAMP response element activity assays, but failed to activate extracellular signal-regulated kinase pathways. Another anorexigenic factor, leptin, similarly increased Kiss-1 mRNA levels up to 1.34 ± 0.08-fold in rHypoE-8 cells. However, combined treatment with GLP-1 and leptin failed to potentiate their individual effects on Kiss-1 mRNA expression. Gnrh mRNA expression was not significantly increased by GLP-1 stimulation in rHypoE-8, but kisspeptin significantly stimulated the expression of Gnrh mRNA in these cells. Our current observations suggest that the anorexigenic peptide GLP-1 directly regulates Kiss-1 mRNA expression in these hypothalamic cell lines and in neuronal cells of fetal rat brain and affects the expression of Gnrh mRNA.

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 LßT2. Endogenous Kiss1R did not respond to kisspeptin and failed to stimulate gonadotropin LHß and FSHß expression in LßT2 cells; however, kisspeptin increased both LHß and FSHß promoter activity in Kiss1R-overexpressing LßT2 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 LHß- and FSHß-subunit promoters. In addition, the level of kisspeptin-induced LHß promoter activity, but not that of FSHß, 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 LHß 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 other's function to stimulate gonadotropin subunit gene expression by reciprocally increasing the expression of their receptors.

Retinoic acid and retinaldehyde dehydrogenase are not involved in the specific induction of the follicle-stimulating hormone ß subunit by trichostatin A, a selective inhibitor of histone deacetylase.

The selective histone deacetylase inhibitor, trichostatin A (TSA), increases follicle-stimulating hormone ß subunit (FSHß) mRNA expression but not α- and luteinizing hormone ß (LHß)-subunits in both the pituitary gonadotrophic cell line LßT2 and primary cultures of rat anterior pituitary cells. TSA increased histone acetylation in whole cell lysates in both cells. In addition, retinaldehyde dehydrogenases (RALDHs), which are retinoic acid (RA)-synthesizing enzymes, were induced by TSA in these cells. Anacardic acid, a histone acetyltransferase inhibitor that prevents histone acetylation, significantly inhibited TSA-induced FSHß mRNA expression as well as TSA-induced RALDH2 and RALDH3 mRNA expression. Similar to the effect of TSA, gonadotropin-releasing hormone stimulated RALDH expression in LßT2 cells. RA directly applied to the pituitary cells stimulated the transcriptional activity of the FSHß promoter. In addition, α- and LHß-subunit promoters were also activated by RA. Our results suggest that TSA specifically increases FSHß expression with a concomitant increase in RALDHs; however, RALDH and RA are not directly involved in the specific regulation of FSHß by TSA.

Pulsatile kisspeptin effectively stimulates gonadotropin-releasing hormone (GnRH)-producing neurons.

Hypothalamic kisspeptin is integral to the hypothalamic-pituitary-gonadal axis by stimulating gonadotropin-releasing hormone (GnRH) release. GnRH is released from the hypothalamus in a pulsatile manner and determines the output of the gonadotropins. However, the effect of kisspeptin on GnRH-secreting cells remains unknown. In an experiment using static cultures of GT1-7 cells, kisspeptin did not significantly increase GnRH mRNA expression. However, when kisspeptin was administered to the cells in a pulsatile manner, GnRH mRNA expression was significantly increased. Primary cultures of fetal rat brain containing GnRH-expressing neurons responded to kisspeptin and increased GnRH mRNA expression by 1.65 ± 0.27-fold in the static condition. When cells were stimulated with kisspeptin in a pulsatile manner, GnRH mRNA expression was increased by up to 2.40 ± 0.21-fold. In perifused GT1-7 cells, pulsatile, but not continuous kisspeptin stimulation, effectively stimulated GnRH mRNA expression. To assess the level of stimulation of GnRH neurons by kisspeptin, the expression of c-fos was examined. In GT1-7 cells, kisspeptin stimulation in the static condition failed to increase c-fos mRNA expression. However, pulsatile kisspeptin stimulation increased c-fos mRNA by 2.31 ± 0.47-fold. Similar to the phenomenon observed in GT1-7 cells, pulsatile, but not static, kisspeptin stimulation significantly increased c-fos mRNA expression in the primary cultures of fetal rat brain. These observations suggest that pulsatile kisspeptin more effectively stimulates GnRH-producing cells to increase the production of GnRH.

Gamma-aminobutyric acidA receptor agonist, muscimol, increases KiSS-1 gene expression in hypothalamic cell models.

Purpose: Accumulating evidence indicates that hypothalamic kisspeptin plays a pivotal role in the regulation of the hypothalamic-pituitary-gonadal (HPG) axis. In this study, the direct action of the gamma-aminobutyric acid (GABA)A receptor agonist on kisspeptin-expressing neuronal cells was examined. Methods: A hypothalamic cell model of rat hypothalamic cell line R8 (rHypoE8) cells and primary cultures of neuronal cells from fetal rat brains were stimulated with a potent and selective GABAA receptor agonist, muscimol, to determine the expression of the KiSS-1 gene. Results: Stimulation of the rHypoE8 cells with muscimol significantly increased the level of KiSS-1 messenger (m)RNA expression. The ability of muscimol to increase the level of KiSS-1 mRNA also was observed in the primary cultures of the neuronal cells from the fetal rat brains. The muscimol-induced increase in KiSS-1 mRNA expression was completely inhibited in the presence of the GABAA receptor antagonist. Although muscimol increased the expression of KiSS-1, the natural compound, GABA, failed to induce the expression of KiSS-1 in the rHypoE8 cells. Muscimol did not modulate gonadotropin-releasing hormone expression in either the rHypoE8 cells or the primary cultures of the fetal rat brains. Conclusions: This study's observations suggest that the activation of the GABAA receptor modulates the HPG axis by increasing kisspeptin expression in the hypothalamic neurons.