Prolactin Receptor (PRLR) Isoform Expression in the Murine Oviduct

Abstract

The most common and most deadly form of “ovarian”cancer is High-Grade Serous Carcinoma (HGSC), now appreciated to arise from epithelial cells of the fallopian tube/oviduct. The serum analyte with the greatest specificity and sensitivity that correlates with the presence or absence of HGSC is prolactin (PRL). However, unknown is whether elevated PRL initiates disease or is the result of disease, although once cancer is present,it is clear that PRL promotes disease. In order to examine the potential for disease initiation in normal animals, it was first important to determine which cells in the mouse oviduct respond to PRL. In many cell types, the long form of the Prlr mediates increased survival, proliferation and migration, whereas the short forms have the opposite effect. After establishing equivalent efficiencies of primers for the long form (LF) and 3 short forms (SF1-3) of the murine Prlr and the endogenous control gene, Gapdh, the mRNA expression profiles of these forms of the receptor were followed by RT-qPCR as a function of stage of the estrous cycle (proestrus, estrus, metestrus, diestrus - determined by vaginal cytology) and region of the oviduct (isthmus, ampulla and fimbria). Expression of SF1 was not detected. SF2 was essentially consistently expressed throughout the oviduct and at all stages of the estrous cycle. By contrast, the ratio of LF to SF3 varied by region of the tube, with more SF3 towards the fimbria and more LF towards the isthmus. The epithelium of the oviduct is primarily composed of multi-ciliated cells and secretory cells, with more ciliated cells towards the fimbria and more secretory cells towards the isthmus. The RT-qPCR results therefore suggested the possibility that a greater proportion of LF Prlr was present on secretory cells and a greater proportion of SF3 Prlr on ciliated cells. Using antibodies raised against intracellular peptide regions specificto the LF (aa 309-325) and SF3 (aa 281-296), both receptor isoforms were localized by immunofluorescence to apical regions of both epithelial cell types,but the presence of receptors on cilia (clearly demonstrated by 3Dreconstruction and rotation) complicated analysis of relative fluorescence by microscopy. Only the LF Prlr signals via Stat5 and so it was anticipated thatStat5 activation could serve as a substitute marker of the relative presence ofLF Prlr. Following in vivo intraperitoneal injection of PRL (5μg/g, 30 min),activated Stat5 was localized to epithelial cells at the base of, and in between, mucosal folds, thereby suggesting a further regionality to receptor distribution. For the fimbrial region only, which is where HGSC is thought to arise, expression of both the LF and SF3 Prlr changed as a function of the stage of the estrous cycle, with highest mRNA expression at diestrus/proestrus. Ongoing work includes flow cytometry of epithelial subpopulations and spatialanalysis of gene expression.