Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes
Copyright policy )Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes
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.
- Icahn School of Medicine at Mount Sinai United States
- University of California, Davis United States
Gene Expression Profiling, Secretory Vesicles, Primary Cell Culture, Gene Expression, Gonadotrophs, Real-Time Polymerase Chain Reaction, Rats, Up-Regulation, Enzyme Activation, Gonadotropin-Releasing Hormone, rap GTP-Binding Proteins, Animals, Single-Cell Analysis, Nucleic Acid Amplification Techniques, Oligonucleotide Array Sequence Analysis
Gene Expression Profiling, Secretory Vesicles, Primary Cell Culture, Gene Expression, Gonadotrophs, Real-Time Polymerase Chain Reaction, Rats, Up-Regulation, Enzyme Activation, Gonadotropin-Releasing Hormone, rap GTP-Binding Proteins, Animals, Single-Cell Analysis, Nucleic Acid Amplification Techniques, Oligonucleotide Array Sequence Analysis
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