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This study was done to examine whether PRL, which appears in both active milk secretory cells (MSC) (1) and milk (1,2) during normal lactation, could also be detected in PRL target cells which do not transfer this hormone into an exocrine secretory product. Ovaries, adrenals, and mammary and pituitary glands were collected from actively nursing rats decapitated on day 15 post-partum. All four tissues were processed for light microscopic immunohistochemical identification of PRL. Immunoreactive PRL was again found in pituitary PRL cells and in MSC. It was also detected within both lutein and adrenal cortical cells (zona fasciculata). In all three target tissues, PRL was present in target cell cytoplasm, but, in MSC and adrenal cells, it was also found occasionally in nuclei. These findings, which seem to indicate that transfer into an exocrine secretory product cannot be the only possible explanation for the appearance of this protein hormone inside its target cells, extend the evidence suggesting that PRL may have intracellular sites of action.

The question of whether endogenous prolactin might be detectable in normally lactating rat mammary glands (LMG) was examined by light microscopic immunohistochemistry. In an initial study, sections of LMG were incubated sequentially with I) rabbit anti-rat prolactin (APRL); II) anti-rabbit gamma globulin: peroxidase conjugate (ARgammaG-per), prepared from goat anti-rabbit gamma globulin and horseradish peroxidase, and III) 3,3'-diaminobenzidine mixed with H2O2(DAB). Use of this three-step procedure led to staining of stromal, parenchymal and intraluminal elements. However, control procedures which ommitted step I produced similar staining, caused by direct binding of the ARgammaG-per to endogenous tissue components (possibly rat immunoglobulins). This precluded detection of indirect, APRL-linked, binding of ARgammaG-per. In subsequent experiments, direct binding of ARgammaG-per was prevented by preincubating LMG sections with goat anti-monkey gamma globulin (AMgammaG), following which, the three-step reaction sequence (APRL, ARgammaG-per and DAB) led to APRL-dependent staining, both within the alveolar lumina and in the apices of epithelial cells. This staining, presumably of endogenous prolactin, appeared to be specific, since it did not occur when absorbed APRL (APRL admixed with highly purified rat prolactin) was substituted for APRL in step I of the reaction sequence. In contrast, when an incubation with purified rat prolactin was interposed between the AMgammaG pretreatment and the three-step reaction sequence, staining of epithelial elements was intensified, indicating that these cells had contained unoccupied prolactin-binding sites. The apparent presence of both prolactin-binding sites and endogenous immunoreactive prolactin in LMG alveolar cells strongly suggests that this protein hormone can enter these target cells under physiological conditions.