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 language | English (US) |
|---|---|
| Pages (from-to) | 10-19 |
| Number of pages | 10 |
| Journal | Molecular and Cellular Endocrinology |
| Volume | 350 |
| Issue number | 1 |
| DOIs | |
| State | Published - Mar 5 2012 |
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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 journal › Article
}
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
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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 -