Fig 2: Combined single cell RNA fluorescentin situhybridization (scRNA FISH) and IF analyses in tissue sections detect frequent co-expression ofGhandPrland sexually dimorphic expression of hormonal genes in the mouse pituitary. (A) Correlation of Gh mRNA and GH protein detection. Combined single cell RNA FISH and IF analyses (scRNA FISH/IF) were performed in adult pituitaries (See METHODS). Left panel: detection of Gh mRNA by scRNA FISH. Scale bar = 5 µm. Right panel: detection of GH proteins by IF analysis in the same field. Scale bar: 5 µm. The white arrows indicate the cells with robust signals for both Gh mRNA and GH protein. The yellow arrows indicated cells with trace levels of Gh mRNA and lack of GH protein signal. The orange arrow indicates a cell lacking Gh mRNA and GH protein signals. (B) Correlation of Prl mRNA and PRL protein detection. Combined single cell RNA FISH and IF analyses (scRNA FISH/IF) were performed in female adult pituitaries (See METHODS). Left panel: detection of Prl mRNA by scRNA FISH analysis. Scale bar = 5 µm. Right panel: detection of PRL protein by IF analysis in the same field. Scale bar: 5 µm. The white arrows indicate four cells with robust signals for both Prl mRNA and PRL protein. The orange arrows indicate two cells lacking Prl mRNA and PRL protein signals. (C) ScRNA FISH analysis of female adult mouse pituitary tissue sections: concordance of Gh with Prl expression in vivo. Left panel: Prl mRNA detection. Prl mRNAs were detected by FISH using an array of anti-sense oligos (See METHODS). Bars = 5 µm. Right panel: Gh mRNA detection. GH mRNA was detected by FISH using an array of anti-sense oligos (See METHODS). Bars = 5 µm The white arrows in the two frames indicate three examples of Gh and Prl dual positive cells. The orange arrows indicate two cells with Gh mRNA expression but without Prl mRNA expression. The yellow arrows indicate three cells with Prl mRNA expression but without Gh mRNA expression. Histogram: analysis of female pituitary cells for Gh and/or Prl mRNA. Pituitary tissue sections from 8-week old, sexually naïve adult female mice (n = 2) were analyzed by RNA FISH. Cells from 5 randomly selected images from each pituitary were analyzed (>600 cells per pituitary). The histogram displays the representation of cells positive for Gh but not Prl mRNA (Gh+Prl-; 27%), cells positive for Prl but not Gh Mrna (Gh-Prl+; 45.2%), and those dual positive cells (Gh+Prl-; 27.8%) in Gh and/or Prl expressing cells. (D) scRNA FISH analysis of male adult mouse pituitary tissue sections reveals a concordance of Gh with Prl expression in vivo. Left panel: Prl mRNA detection. Prl mRNAs were detected by FISH using an array of anti-sense oligos as described in Methods. Bars = 5 µm. Right panel: Gh mRNA detection. GH mRNA was detected by FISH using an array of anti-sense oligos as described in Methods. Bars = 5 µm. The white arrows indicate three representatives of the Gh and Prl dual positive cells. The orange arrows indicate two cells with Gh mRNA expression but without Prl mRNA expression. Histogram: analysis of male pituitary cells. Pituitary tissue sections from 8-week old, sexually naïve male mice (n = 2) were analyzed by scRNA FISH for Gh and Prl mRNA expression as described in (A) The histogram represents the percent of the total cell population positive for Gh but not Prl mRNA (Gh+Prl-; 52.3%), positive for Prl but not Gh mRNA (Gh-Prl+; 16.7%), and positive for both Gh and Prl mRNA (Gh+Prl+ 31%) in Gh and/or Prl expressing cells. (E) IF analysis performed on 8-week old female mice confirms co-expression of GH and PRL proteins in pituitary cells. Left panel: IF detection of PRL protein (green). The IF was. Scale bar = 5 µm. Middle panel: IF detection of GH protein (red) in the same field as in left panel. Scale bar = 5 µm. Right panel: The merged images of the PRL IF and GH IF. The white arrows indicate the cells with robust levels of GH protein and PRL protein. The yellow arrows indicate cells with robust level of PRL but without GH protein signal. The orange arrows indicate cells with robust level of GH protein but without PRL protein signal. Histogram: co-expression of GH and PRL in the pituitaries of male or female mice. The IF analysis was performed in pituitary tissue sections of one male mouse (499 GH and/or PRL expressing cells were analyzed) and one female mouse (664 GH and/or PRL expressing cells were analyzed)