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.

Fig. 1. Localization of LH (red) in gonadotrope-enriched primary pituitary cultures using confocal microscopy

Gonadotrope-enriched (panel A) and unfractionated cells (panel B) from the same pituitary dispersion were cultured for 3 days and fixed and immunostained for confocal microscopy. Red identifies LH-positive cells (gonadotropes). Dark blue (TOTO-3) identifies cell nuclei. Scale bar (20 μm) in panel A applies to both panels.

Fig. 2. Comparison of three RNA amplification protocols for microarray analysis

Scatter plots of microarray data obtained from three different RNA amplification methods. Lβ T2 cells were treated with 100 nM GnRH or vehicle (Control) for 1 h. RNA was isolated and aliquots were used for amplification procedures. Labeled probes were hybridized to Affymetrix GeneChip U74A microarrays. A, Amplification using a standard Affymetrix one-cycle T7 protocol with 40 μg total RNA. B, Hybrid DNA-RNA primer isothermal amplification with 100 ng total RNA. C, Amplification using a modified Affymetrix two-cycle T7 protocol with 100 ng total RNA. Copy numbers were estimated by intrapolation of C_T values against a serial dilution of known concentrations of plasmid DNA containing either mouse GAPDH or mouse β-actin cDNA. Ideally, copy numbers reported for the 3’ and 5’ assays should yield a ratio of 1.

Fig. 3. Validation of the early gene microarray data by real-time PCR

The expression levels of eight candidate genes were evaluated by real-time PCR in vehicle- vs. GnRH-treated primary rat gonadotropes. Represented are fold changes relative to the control conditions (i.e. with vehicle). Data from three independent experiments were calculated and plotted. Bars show standard deviations. All genes shown were significant at p<.05.

Fig. 4. GnRH activates Rap1b gene expression in single rat gonadotropes

Individual rat gonadotrope cells were perfused with a 15-min pulse of extracellular medium only (Ctrl, control) or with medium containing either 10 nM GnRH or 1 mM 8-bromo-cAMP. Perfusion with control medium only was continued, and 25 min later cells were harvested and immediately processed for reverse transcription, as described in Materials and Methods. Rap1b data were normalized to Ppia transcripts determined in the same gonadotrope. Single cell data for all groups were obtained from two separate pituitary dispersions. The number of individual gonadotropes is in parentheses. Compared to control: ** P < 0.001, *P < 0.02.

Fig. 5. GnRH activates Rap1 protein expression in primary gonadotropes

Rat pituitary cells were either incubated in the presence of vehicle for 60 min (A), challenged with a 15-min pulse of 1nM GnRH and fixed immediately (B) or 45 min later (C). Fixed cells were subjected to dual immunofluorescence staining for LH (red) and Rap1 (green) and optical sectioning. Panels A–C: Single 2 μm optical sections at maximum cell diameter (A and B) or at intermediate diameters maximum for the field (C). Inset of panel C, an expanded view of immunofluorescent punctuate Rap1-associated objects. Scale bars = 5 μm; the dimension of the expanded view (C) is approximately 9 x 3 μm. D, Rap1-associated object volume determined from 2 μm overlapping optical sections through each cell: Fold-change in Rap1 immunofluorescence in gonadotropes (GnRH-treated/vehicle-treated) fixed at either 15 min (open bar) or 60 min (hatched bar). * P < 0.03 compared to cells fixed at 15 min.