Search

Objective: We examined how the sex steroids influence the synthesis of gonadotropins. Materials and Methods: The effects of sex steroids estradiol (E2), progesterone (P4), and dihydrotestosterone (DHT) in pituitary gonadotroph cell model (LβT2 cells) in vitro and ovary-intact rats in vivo were examined. The effects of sex steroids on Kiss1 gene expression in the hypothalamus were also examined in ovary-intact rats. Results: In LβT2 cells, E2 increased common glycoprotein alpha (Cga) and luteinizing hormone beta (Lhb) subunit promoter activity as well as their mRNA expression. Although gonadotropin subunit promoter activity was not modulated by P4, Cga and Lhb mRNA expression was increased by P4. DHT inhibited Cga and Lhb mRNA expression with a concomitant decrease in their promoter activity. During the 2-week administration of exogenous E2 to ovary-intact rats, the estrous cycle determined by vaginal smears was disrupted. P4 or DHT administration completely eliminated the estrous cycle. Protein expression of all three gonadotropin subunits within the pituitary gland was inhibited by E2 or P4 treatment in vivo; however, DHT reduced Cga expression but did not modulate Lhb or follicle-stimulating hormone beta subunit expression. E2 administration significantly repressed Kiss1 mRNA expression in a posterior hypothalamic region that included the arcuate nucleus. P4 and DHT did not modulate Kiss1 mRNA expression in this region. In contrast, P4 administration significantly inhibited Kiss1 mRNA expression in the anterior region of the hypothalamus that included the anteroventral periventricular nucleus. The expression of gonadotropin-releasing hormone (Gnrh) mRNA in the anterior hypothalamic region, where the preoptic area is located, appeared to be decreased by treatment with E2 and P4. Conclusion: Our findings suggest that sex steroids have different effects in the hypothalamus and pituitary gland.

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.

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 Kiss1rmRNA 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.

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.NT, CRH and RFRP-3 might be involved in E2-induced negative feedback mechanisms.

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.