Generation of Kisspeptin Cell Lines to Investigate the Sex Steroid-Mediated Initiation of Puberty Public Deposited

http://ir.library.oregonstate.edu/concern/undergraduate_thesis_or_projects/gf06g469c

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  • Mammalian female reproduction requires preovulatory surges of gonadotropin-releasing hormone (GnRH) from neurons in the preoptic area (POA) of the hypothalamus, initiated by elevated estradiol (E₂). Rising E₂ activates a subset ofsexually dimorphic Kisspeptin neurons in the female, located in the anteroventralperiventricular nuclei (AVPV). Conversely, E₂ negative feedback is mediated by a neuroanatomically separate population of Kisspeptin neurons in the arcuate (Arc) nuclei. Kisspeptin is a regulator of GnRH neuronal activity in vivo, and the development of the Kisspeptin system is critical for the initiation of puberty.To elucidate how phenotypically similar neuronal populations react in a dramatically different fashion to the same E₂ exposure, we have begun generating two kisspeptin-expressing cell lines from kiss1-GFP postpubertal female mice. AVPV and Arc explants were removed from 9-wk old females using a vibratome, and dissociated enzymatically, with neurite process extension occurring within four to five days after harvest. Cells were re-suspended, sorted for GFP fluorescence, and re-plated in DMEM with 20% FBS. We found that AVPV neurons retain differential expression patterns when exposed to E₂, but interestingly we observed that E₂-stimulated increased in GFP occurred within 20 minutes of exposure, suggesting a rapid time course of kiss1 stimulation. In contrast, Arc-derived primary neurons exhibited a slightly higher baseline level of GFP fluorescence, which diminished within 4h of E₂ exposure, recapitulating decreases in kiss1 expression observed previously. We are using a third generation lentiviral packaging system to infect both AVPV and Arc kisspeptin neurons with SV40-large T antigen, to immortalize both populations via suppression of tumor-suppressing gene p53. Four weeks post-infection, AVPV neurons began proliferating,retaining their unique expression pattern when treated with E₂. These cell lines will be useful tools to probe the molecular mechanisms underlying this differential regulation of kiss1 expression in response to sex steroids.
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  • description.provenance : Made available in DSpace on 2016-01-05T15:40:09Z (GMT). No. of bitstreams: 2 JacobsBRR Final Thesis.pdf: 1895461 bytes, checksum: 3c5aabac3dcfe8736695ed741cc09ea2 (MD5) Jacobs Seminar.pdf: 2654814 bytes, checksum: cc684d258008508cb0445a7ab39fbb98 (MD5)
  • description.provenance : Approved for entry into archive by Patricia Black(patricia.black@oregonstate.edu) on 2016-01-05T15:40:09Z (GMT) No. of bitstreams: 2 JacobsBRR Final Thesis.pdf: 1895461 bytes, checksum: 3c5aabac3dcfe8736695ed741cc09ea2 (MD5) Jacobs Seminar.pdf: 2654814 bytes, checksum: cc684d258008508cb0445a7ab39fbb98 (MD5)
  • description.provenance : Submitted by Wanda Crannell (brr@oregonstate.edu) on 2016-01-01T21:09:17Z No. of bitstreams: 2 JacobsBRR Final Thesis.pdf: 1895461 bytes, checksum: 3c5aabac3dcfe8736695ed741cc09ea2 (MD5) Jacobs Seminar.pdf: 2654814 bytes, checksum: cc684d258008508cb0445a7ab39fbb98 (MD5)

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