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.

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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.

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