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 journalArticle

7 Citations (Scopus)

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

Fingerprint

Single-Cell Analysis
Gonadotropin-Releasing Hormone
Amplification
Rats
Chemical activation
Genes
Monomeric GTP-Binding Proteins
Microarray Analysis
Microarrays
Gonadotropins
Sedimentation
Gene expression
Real-Time Polymerase Chain Reaction
Assays
Immunohistochemistry
RNA
Gene Expression

Keywords

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

ASJC Scopus subject areas

  • Endocrinology
  • Molecular Biology
  • Biochemistry

Cite this

Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes. / Yuen, Tony; Choi, Soon Gang; Pincas, Hanna; Waring, Dennis W.; Sealfon, Stuart C.; Turgeon, Judith L.

In: Molecular and Cellular Endocrinology, Vol. 350, No. 1, 05.03.2012, p. 10-19.

Research output: Contribution to journalArticle

Yuen, Tony ; Choi, Soon Gang ; Pincas, Hanna ; Waring, Dennis W. ; Sealfon, Stuart C. ; Turgeon, Judith L. / Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes. In: Molecular and Cellular Endocrinology. 2012 ; Vol. 350, No. 1. pp. 10-19.
@article{c3ea2789c0e9490ebc02dfda492177de,
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.",
keywords = "Early gene, GnRH, Gonadotrope enrichment, Rap1b, Rat primary pituitary cultures, Single-cell gene expression",
author = "Tony Yuen and Choi, {Soon Gang} and Hanna Pincas and Waring, {Dennis W.} and Sealfon, {Stuart C.} and Turgeon, {Judith L}",
year = "2012",
month = "3",
day = "5",
doi = "10.1016/j.mce.2011.11.017",
language = "English (US)",
volume = "350",
pages = "10--19",
journal = "Molecular and Cellular Endocrinology",
issn = "0303-7207",
publisher = "Elsevier Ireland Ltd",
number = "1",

}

TY - JOUR

T1 - Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes

AU - Yuen, Tony

AU - Choi, Soon Gang

AU - Pincas, Hanna

AU - Waring, Dennis W.

AU - Sealfon, Stuart C.

AU - Turgeon, Judith L

PY - 2012/3/5

Y1 - 2012/3/5

N2 - 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.

AB - 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.

KW - Early gene

KW - GnRH

KW - Gonadotrope enrichment

KW - Rap1b

KW - Rat primary pituitary cultures

KW - Single-cell gene expression

UR - http://www.scopus.com/inward/record.url?scp=84856061390&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84856061390&partnerID=8YFLogxK

U2 - 10.1016/j.mce.2011.11.017

DO - 10.1016/j.mce.2011.11.017

M3 - Article

C2 - 22127306

AN - SCOPUS:84856061390

VL - 350

SP - 10

EP - 19

JO - Molecular and Cellular Endocrinology

JF - Molecular and Cellular Endocrinology

SN - 0303-7207

IS - 1

ER -