Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes

Tony Yuen, Soon Gang Choi, Hanna Pincas, Dennis W. Waring, Stuart C. Sealfon, Judith L Turgeon

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

Identifying the early gene program induced by GnRH would help understand how GnRH-activated signaling pathways modulate gonadotrope secretory response. We previously analyzed GnRH-induced early genes in LβT2 cells, however these lack GnRH self-potentiation, a physiological attribute of gonadotropes. To minimize cellular heterogeneity, rat primary pituitary cultures were enriched for gonadotropes by 40-60% using a sedimentation gradient. Given the limited number of gonadotropes, RNA was amplified prior to microarray analysis. Thirty-three genes were up-regulated 40. min after GnRH stimulation. Real-time PCR confirmed regulation of several transcripts including fosB, c-fos, egr-2 and rap1b, a small GTPase and member of the Ras family. GnRH stimulated rap1b gene expression in gonadotropes, measured by a sensitive single cell assay. Immunocytochemistry revealed increased Rap1 protein in GnRH-stimulated gonadotropes. These data establish rap1b as a novel gene rapidly induced by GnRH and a candidate to modulate gonadotropin secretion in rat gonadotropes.

Original languageEnglish (US)
Pages (from-to)10-19
Number of pages10
JournalMolecular and Cellular Endocrinology
Volume350
Issue number1
DOIs
StatePublished - Mar 5 2012

Keywords

  • Early gene
  • GnRH
  • Gonadotrope enrichment
  • Rap1b
  • Rat primary pituitary cultures
  • Single-cell gene expression

ASJC Scopus subject areas

  • Endocrinology
  • Molecular Biology
  • Biochemistry

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