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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.

Kisspeptin and gonadotropin-releasing hormone (GnRH) are central regulators of the hypothalamic-pituitary-gonadal axis and control female reproductive functions. Recently established mHypoA-50 and mHypoA-55 cells are immortalized hypothalamic neuronal cell models that originated from the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) regions of the mouse hypothalamus, respectively. mHypoA-50 or mHypoA-55 cells were stimulated with kisspeptin-10 (KP10) and GnRH, after which the expression of kisspeptin and GnRH was determined. Primary cultures of fetal rat brain cells were also examined. mHypoA-50 and mHypoA-55 cells expressed mRNA for Kiss-1 (which encodes kisspeptin) and GnRH as well as receptors for kisspeptin and GnRH. We found that Kiss-1 mRNA expression was significantly increased in mHypoA-50 AVPV cells by KP10 and GnRH stimulation. Kisspeptin protein expression was also increased by KP10 and GnRH stimulation in these cells. In contrast, GnRH expression was unchanged in mHypoA-50 AVPV cells by KP10 and GnRH stimulation. In mHypoA-55 ARC cells, kisspeptin expression was also significantly increased at the mRNA and protein levels by KP10 and GnRH stimulation; however, GnRH expression was also upregulated by KP10 and GnRH stimulation in these cells. KP10 and estradiol (E2) both increased Kiss-1 gene expression in mHypoA-50 AVPV cells, but combined stimulation with KP10 and E2 did not potentiate their individual effects on Kiss-1 gene expression. On the other hand, E2 did not increase Kiss-1 gene expression in mHypoA-55 ARC cells, and the KP10-induced increase of Kiss-1 gene expression was inhibited in the presence of E2 in these cells. KP10 and GnRH significantly increased c-Fos protein expression in the mHypoA-50 AVPV and mHypoA-55 ARC cell lines. In primary cultures of fetal rat neuronal cells, KP10 significantly increased Kiss-1 gene expression, whereas GnRH significantly increased GnRH gene expression. We found that kisspeptin and GnRH affected Kiss-1- and GnRH-expressing hypothalamic cells and modulated Kiss-1 and/or GnRH gene expression with a concomitant increase in c-Fos protein expression. A mutual- or self-regulatory system might be present in Kiss-1 and/or GnRH neurons in the hypothalamus.

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 LβT2 cells. In these cells, the expression of FSHβ- but not α- and LHβ-subunits increased significantly as the concentration of AMH increased. LβT2 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 LβT2 cells did not alter the basal expression levels of α-, LHβ-, and FSHβ-subunits. In LβT2 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 FSHβ-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.

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.

Clomiphene citrate (CC) and letrozole stimulate the hypothalamic-pituitary-ovarian axis and are used widely as oral fertility drugs to induce folliculogenesis. We examined whether these drugs increase Kiss-1 expression in hypothalamic cell models. We utilized two hypothalamic cell models, mHypoA-50 and mHypoA-55, which originated from Kiss-1 neurons in the anteroventral periventricular (AVPV) nucleus and arcuate (ARC) nucleus of the mouse hypothalamus, respectively. The cells were stimulated with CC or letrozole, after which Kiss-1 mRNA expression was determined. CC stimulated Kiss-1 gene expression in mHypoA-50 and mHypoA-55 cells. The basal expression of Kiss-1 was significantly increased in the presence of estradiol (E2) in mHypoA-50 cells, and the CC-induced increase in Kiss-1 expression was not observed in the presence of E2 in these cells. In contrast, E2 did not modify the basal expression of Kiss-1 in mHypoA-55 cells, and CC-induced Kiss-1 expression was still observed in the presence of E2. The significant increase in Kiss-1 gene expression in mHypoA-50 and mHypoA-55 cells was blunted in the presence of estrogen receptor antagonists. Aromatase was expressed in mHypoA-50 and mHypoA-55 cells. Letrozole, an aromatase inhibitor, increased Kiss-1 expression in mHypoA-55 ARC cells but not in mHypoA-50 AVPV cells. Although the basal expression of Kiss-1 was increased by E2, letrozole did not modulate Kiss-1 expression in mHypoA-50 cells. Letrozole-induced Kiss-1 gene expression in mHypoA-55 cells was not modulated in the presence of E2. The fertility drugs CC and letrozole modulated Kiss-1 expression in hypothalamic cell models.

Kisspeptin, encoded by the Kiss-1 gene, plays a crucial role in reproductive function by governing the hypothalamic-pituitary-gonadal axis. The recently established Kiss-1-expressing cell model mHypoA-50 displays characteristics of neuronal cells of the anteroventral periventricular (AVPV) region of the mouse hypothalamus. Because Kiss-1 gene expression in these cells is upregulated by estradiol (E2), mHypoA-50 cells are regarded as a valuable model for the study of Kiss-1-expressing neurons in the AVPV region. These cells also express RFamide-related peptide-3 (RFRP-3), a mammalian homolog of gonadotropin inhibitory hormone. The RFRP-3 expression in mHypoA-50 cells was increased by melatonin stimulation. In addition, E2 stimulation increased RFRP-3 expression in these cells. Treatment of the mHypoA-50 cells with exogenous RFRP-3 resulted in the increase of Kiss-1 messenger RNA expression within the cells; however, RFRP-3 did not modify gonadotropin-releasing hormone or kisspeptin-induced Kiss-1 gene expression in these cells. In addition, we found that RFRP-3 stimulation increased the expression of corticotropin-releasing hormone, which may be involved in E2-induced positive feedback in mHypoA-50 cells. Our observations suggest that RFRP-3 might be involved in positive feedback regulation by directly or indirectly increasing Kiss-1 gene expression.

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 βA and βB 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.

Purpose: Anti-Mullerian 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 ex...

Hypothalamic kisspeptin, encoded by the Kiss-1 gene, governs the hypothalamic-pituitary-gonadal axis by directly regulating the release of gonadotropin-releasing hormone. In this study, we examined the roles of activin, inhibin, and follistatin in the regulation of Kiss-1 gene expression using primary cultures of fetal rat neuronal cells, which express the Kiss-1 gene and kisspeptin. Stimulation with activin significantly increased Kiss-1 gene expression in these cultures by 2.02 ± 0.39-fold. In contrast, a significant decrease in Kiss-1 gene expression was observed with inhibin A and follistatin treatment. Inhibin B did not modulate Kiss-1 gene expression. Activin, inhibin, and follistatin were also expressed in fetal rat brain cultures and their expression was controlled by estradiol (E2). The inhibin α, βA, and βB subunits were upregulated by E2. Similarly, follistatin gene expression was significantly increased by E2 in these cells. Our results suggest the possibility that activin, inhibin, and follistatin expressed in the brain participate in the E2-induced feedback control of the hypothalamic-pituitary-gonadal axis., Highlights • ・We examined the roles of activin, inhibin, and follistatin in the regulation of Kiss-1 gene expression in primary cultures of fetal rat neuronal cells. • ・Activin increased Kiss-1, whereas it was decreased by inhibin A and follistatin. • ・Intracerebral inhibin α, βA, and βB subunits were upregulated by estradiol. • ・Intracerebral activin, inhibin, and follistatin may participate in the estradiol-induced feedback control of Hypothalamic-pituitary gonadal axis.

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 βA and βB subunits and follistatin was much higher in mHypoA-55 ARC cells. Furthermore, we found that expression of the inhibin αsubunit 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.

Aim: We examined the effect of anti-Müllerian hormone (AMH) on the expression of gonadotropin subunits in pituitary gonadotrophs.Methods: The mouse pituitary gonadotroph cell line LβT2 was stimulated with AMH and the expression levels of gonadotropin subunits were determined by real-time PCR. We also examined the involvement of the Kiss-1 gene (encoding kisspeptin) and the kisspeptin receptor (Kiss-1R) in LβT2 cells. Results: A significant increase was observed in the expression level of the FSHβ subunit with AMH but not in the expression levels of gonadotropin α and LHβ subunits. A significant decrease was observed in the expression of Kiss-1 and Kiss-1R genes in LβT2 cells with AMH stimulation. Kiss-1 gene knockdown by siRNA did not alter the basal expression of gonadotropin subunits. When LβT2 cells overexpressing Kiss-1R were stimulated with kisspeptin, there was a significant increase in the gene expression levels of the gonadotropin subunits α, LHβ, and FSHβ. This inductive effect of kisspeptin was almost completely inhibited by AMH pretreatment. The GnRH-induced increase in gonadotropin subunit genes was unchanged in the presence of AMH. Conclusions: AMH can increase FSHβ subunit gene expression in pituitary gonadotroph cells. However, AMH decreases Kiss-1 and Kiss-1R gene expression within the gonadotrophs. Because AMH pretreatment abolishes kisspeptin-induced expression of gonadotropin subunit genes, AMH may control kisspeptin-regulated gonadotropin expression by inhibiting the expression and function of Kiss-1R within gonadotrophs.

Hypothalamic kisspeptin is a known principal activator of gonadotropin-releasing hormone neurons and governs the hypothalamic-pituitary-gonadal axis. Previous reports have shown that kisspeptin is also released into the hypophyseal portal circulation and directly affects the anterior pituitary. In this study, we examined the direct effect of kisspeptin on pituitary prolactin-producing cells. The rat pituitary somatolactotroph cell line GH3 expresses the kisspeptin receptor (Kiss1R); however, in these cells, kisspeptin failed to stimulate prolactin-promoter activity. When GH3 cells overexpressed Kiss1R, kisspeptin clearly increased prolactin-promoter activity, with a concomitant increase in extracellular signal-regulated kinase (ERK) and cAMP/protein kinase A (PKA) signaling pathways. In the experiments using GH3 cells overexpressing Kiss1R, kisspeptin did not potentiate thyrotropin-releasing hormone (TRH)-induced prolactin-promoter activity, but it potentiated the pituitary adenylate cyclase-activating polypeptide-induced prolactin-promoter activity, with a concomitant enhancement of ERK and PKA signaling pathways. Although the basal and TRH-induced prolactin-promoter activities were not modulated by increasing amounts of Kiss1R expression in GH3 cells, kisspeptin-stimulated prolactin-promoter activity was increased by the amount of Kiss1R overexpression. Endogenous Kiss1r mRNA expression in GH3 cells was significantly increased by treatment with estradiol (E2) but not by TRH. In addition, kisspeptin's ability to stimulate prolactin-promoter activity was restored after E2 treatment in non-transfected GH3 cells. Our current observations suggest that kisspeptin might have a direct effect on prolactin expression in the anterior pituitary prolactin-producing cells under the influence of E2, which may regulate Kiss1R expression and function.

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 hormoneβ‐, and follicle‐stimulating hormone β‐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.

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 17β-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.

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.

Aim 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.

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.

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.

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 other's function to stimulate gonadotropin subunit gene expression by reciprocally increasing the expression of their receptors.

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 LβT2 were used. Expression of Kiss‐1, GnRH, and luteinizing hormone (LH)/follicle‐stimulating hormone (FSH) β‐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 LβT2, both LHβ‐ and FSHβ‐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.

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 17β-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.

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 ± 0.93-fold and 4.89 ± 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.

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.

Postpartum uterine bleeding is not uncommon and is caused by a variety of obstetrical and gynecological disorders, such as retained placenta, dysfunctional bleeding, and endometrial polyps. Placental polyps and uterine arteriovenous malformation are disorders often encountered in cases of abnormal uterine bleeding in the late puerperal period. These patients may experience life-threatening bleeding and require prompt intervention based on the correct differential diagnosis. The optimal treatments for both diseases differ as follows: intrauterine curettage or transcervical resection are chosen for placental polyps, while total abdominal hysterectomy or uterine artery embolization is preferred for uterine arteriovenous malformation since intrauterine curettage or transcervical resection has the risk of massive bleeding. However, since placental polyp and uterine arteriovenous malformation have similar clinical characteristics, it is important to accurately identify and differentiate between them to ensure optimal therapy. We report here cases that were suggestive of placental polyp or uterine arteriovenous malformation. We discuss the differential diagnoses and treatments for both diseases based on a literature review and propose a novel algorithm for managing such patients.

Pituitary adenylate cyclase-activating polypeptide (PACAP) was first identified as a hypophysiotropic factor that regulates pituitary cell functions and has been subsequently shown to be widely distributed and have multiple functions. The PACAP is known to be expressed in placental tissues and is suggested to have a critical role in physiological function of the placenta. In addition to PACAP, the hypothalamic peptides kisspeptin and gonadotropin-releasing hormone (GnRH) are also expressed in placental cells. In this study, we used primary cultures of placental tissues from rats of 16 to 18 days gestation and examined the regulation and function of PACAP. The PACAP messenger RNA (mRNA) expression and PACAP-immunoreactive cells were detected in primary cultures of rat placental cells. The PACAP mRNA expression in placental cells was upregulated in the presence of the sex steroids estradiol and progesterone; however, their combined treatment failed to enhance their individual effects. When the cells were stimulated with kisspeptin, PACAP mRNA expression was increased. Similarly, GnRH had a stimulatory effect on PACAP expression. Conversely, kisspeptin expression in placental cells was increased by PACAP stimulation, whereas PACAP failed to stimulate GnRH mRNA expression in these cells. Finally, we found that PACAP had a stimulatory effect on human chorionic gonadotropin expression in placental cells. Our current observations suggest that the hypothalamic peptides PACAP, kisspeptin, and GnRH are interrelated and maintain placental functions.

We report a case of a woman who was incidentally diagnosed with acromegaly after referral for prolonged post-partum amenorrhea. A 25-year-old woman, gravida 2 para 1, had a normal transvaginal delivery and breastfeeding had been discontinued more than a year after delivery. Thereafter, spontaneous menstruation did not restart and she underwent hormonal therapy. Subsequently, she was referred to our university hospital for prolonged amenorrhea. Hormonal examination revealed severe hypogonadotropic hypogonadism (luteinizing hormone 0.5 mIU/mL, follicle-stimulating hormone < 1.0 mIU/mL, estradiol 13 pg/mL), along with a modest increase in serum prolactin levels (43.2 ng/mL). She also complained of milk ejection on nipple stimulation. Magnetic resonance imaging of the head was performed, because an organic abnormality in the central nervous system was suspected to be causing the amenorrhea, which revealed mass lesions extending from the pituitary fossa to the suprasellar area with similar signal intensity as the gray matter. In addition, bitemporal hemianopsia was observed on campimetry. After further examination, the patient was diagnosed with acromegaly.

Congenital anomalies of the female reproductive organs vary widely and the patients often have no symptoms related to them. We report an exceedingly rare case of complicated anomalies in the Mullerian duct and urogenital sinus. The patient was a 21-year-old woman evaluated for infertility, and the examination revealed the presence of a complete septate uterus. We found dual vaginal canals with right incomplete hymenal fenestration when we performed an examination during the patient's menstrual period. Laparoscopic findings showed bilateral undescended ovaries, absent utero-ovarian ligaments, and partial atresia of the left fallopian tube. We performed hymenotomy of the right vagina, resection of the vaginal septum and salpingostomy of the left fallopian tube. To the best of our knowledge, this is the first report to describe this complex combination of anomalies in the Mullerian duct and urogenital sinus. This case might provide us with information about the development of the female reproductive organs. © 2016 Japan Society of Obstetrics and Gynecology.

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 ± 0.93-fold and 4.89 ± 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.

Trichostatin A (TSA) is a selective inhibitor of mammalian histone deacetylase and is widely used to modify the ability of DNA transcription factors to bind DNA within chromatin by interfering with histone deacetylation. In the GnRH-producing neuronal cell line GT1-7, TSA significantly reduced expression of GnRH mRNA. Kisspeptin, a known regulator of GnRH release, failed to increase GnRH mRNA expression and did not modify TSA-induced reduction of GnRH expression. TSA, but not kisspeptin, increased histone acetylation in whole-cell lysates and significantly stimulated the expression of retinaldehyde dehydrogenase (RALDH), a retinoic acid (RA)-synthesizing enzyme that is known to be involved in cell differentiation. In addition, treatment of the GT1-7 cells with RA dose-dependently inhibited the expression of GnRH mRNA. Whereas, TSA-induced reduction of GnRH mRNA was not modulated by treatment with the pan-RA receptor inverse agonist BMS493 or the RA metabolism inhibitor liarozole. Our current results suggest that the RALDH and RA might not be directly involved in the reduction of GnRH expression induced by TSA, however these substances could be a novel regulator of GnRH.

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.

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.