title: Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes.
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.
authors: Center for Translational Systems Biology and Department of Neurology, Mount Sinai School of Medicine, New York, NY 10029, United States.
authors: Tony
authors: T
authors: Yuen
authors: Soon Gang
authors: SG
authors: Choi
authors: Hanna
authors: H
authors: Pincas
authors: Dennis W
authors: DW
authors: Waring
authors: Stuart C
authors: SC
authors: Sealfon
authors: Judith L
authors: JL
authors: Turgeon
mesh_terms: D000818:Animals; D004789:Enzyme Activation; D015870:Gene Expression; D020869:Gene Expression Profiling; D052681:Gonadotrophs / Q000378:metabolism*; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology / Q000502:physiology*; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis; D061251:Primary Cell Culture; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D022142:Secretory Vesicles / Q000378:metabolism; D059010:Single-Cell Analysis; D015854:Up-Regulation; D020668:rap GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D007987:Gonadotropin-Releasing Hormone; C492135:Rap1b protein, rat; D020668:rap GTP-Binding Proteins