Analysis of the direct regulation of reproductive-associated genes in hypothalamic kisspeptin-expressing neuronal cell models

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 515-547-Female Reproductive Endocrinology
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-539
Zoey B Friedman*1 and Denise D Belsham2
1University of Toronto, Toronto, ON, Canada, 2Univ of Toronto, Toronto, ON, Canada
Fertility is a complex and highly regulated process dependent on the orchestration of hypothalamic neuropeptides and peripheral hormones. Signals converge on gonadotropin-releasing hormone (GnRH) neurons, positioned at the pinnacle of the hypothalamic-pituitary-gonadal (HPG) axis. Kisspeptin (Kiss) and its receptor, GPR54, have emerged as fundamental gatekeepers of reproduction, acting centrally upstream of GnRH neurons. It is well established that Kiss neurons express estrogen receptors, and estradiol-mediated regulation of these neurons is nuclei-specific. Further, subpopulations of Kiss neurons have been found to express GPR54 and the gonadotropin-inhibitory hormone (GnIH) receptor, GPR147, suggesting an additional level of regulation in Kiss neurons. Currently, in vitro studies focusing on the regulation of key genes expressed within hypothalamic Kiss neurons are limited. To address this issue, we have generated immortalized, clonal cell lines derived from rodent hypothalamic primary culture.  We have identified two cell lines, mHypoA-50 and mHypoA-55, which exhibit endogenous Kiss expression, as well as GPR54, GPR147, and the estrogen receptors (ERα, ERβ, GPR30). Using qPCR, we report a biphasic expression pattern with the suppression of GPR54, ERα and ERβ mRNA expression at 4 h, followed by an upregulation at 24 h in both cell lines. Interestingly, while Kiss-10 treatment (10 nM) induces GPR54, ERα and ERβ mRNA expression in the mHypoA-55 cell line, we measured no changes in the mHypoA-50 cell line, thus highlighting differences between hypothalamic Kiss neuronal populations. Current studies using estrogen receptor agonists and antagonists, as well as Western blot analyses, are being used to delineate the direct estrogen and Kiss-mediated mechanisms controlling transcription of the genes studied. Preliminary Western blot results suggest that Kiss-10 treatment activates the ERK1/2 MAP kinase pathway, thus second messengers to this pathway will be further analyzed. Future studies will be directed at characterizing several new cell models of Kiss-GFP neurons, mHypoA-Kisspeptin/GFP, generated in our lab from the Kiss-GFP transgenic mouse hypothalamus, in order to determine the specific gene expression profile of each line. We anticipate that these novel models will further define the mechanisms by which different populations of Kiss neurons are regulated and modulate reproductive function.

Nothing to Disclose: ZBF, DDB

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Sources of Research Support: CIHR; Canadian Foundation for Innovation; Canadian Research Chair.