Electron microscopic autoradiographic localization of prolactin mRNA in rat pituitary

Recent immunoelectron microscopic studies have shown that immunoreactive prolactin (PRL) in rat pituitary can be detected not only in typical PRL cells, characterized by large secretory granules, but also in another type of cell, which contains small secretory granules. To determine whether or not these two cell types are involved in PRL biosynthesis, we developed a procedure to investigate PRL gene expression by using in situ hybridization at the ultrastructural level. Rat pituitary was fixed and vibratome sections were incubated with a PRL [35S]-cDNA probe and subsequently flat-embedded in Araldite. Semi-thin and ultra-thin sections were processed for autoradiography. The results indicate that only the two PRL cell types were labeled. When immunolabeling for PRL was applied to ultra-thin sections, only immunopositive cells were seen to contain silver grains. In these cells the silver grains were associated with the rough endoplasmic reticulum and nucleus. When a growth hormone (GH) [35S]-cDNA probe was used as a control, only GH-secreting cells were labeled. This study confirms that the two PRL cell types are involved in biosynthesis of PRL. Moreover, this simple in situ hybridization technique provides a new approach to accurately localize mRNA in complex tissue and to investigate the subcellular distribution of mRNA under differing experimental conditions.