Immunochemical and in situ hybridization analyses of retinoic acid receptor alpha, beta, and gamma in murine Harderian and submandibular glands

Abstract

Retinoic acid (RA), through its cognate receptors (retinoic acid receptors, RARs), plays an important role in the ontogenesis and maintenance of the normal function of murine Harderian and submandibular glands. In the present study, autoradiography was used to study RA binding to these glands. Both glands showed high radioactive labelling after [14C]-RA administration in normal and partially vitamin A-deficient (VAD) mice. The peak uptake was at 6 h after [14C]-RA administration in normal mice and at 0.5 h in VAD mice. At 24 h, RA binding remained high in normal mice, while it decreased significantly in VAD mice. In western blots with an antibody recognizing all forms of RARs, a band of molecular weight 51 kDa was seen in homogenates of both glands. Immunohistochemically, RAR staining was found in the nuclei of the glandular cells. The Harderian gland exhibited more intense staining than the submandibular gland. In the latter, the most intense staining was seen in the acinar cells, followed by the intercalated duct cells. The granular convoluted tubule showed weak immunostaining and the striated duct was negative. In the Harderian gland, RAR immunostaining was observed in both type I and II cells, but only part of them stained with RAR antibody. The expression of RAR alpha, beta, and gamma transcripts was studied by in situ hybridization using specific oligonucleotide probes. The cell-specific expression of RAR alpha mRNA in the submandibular gland corresponded to the RAR proteins detected by immunohistochemistry, while the RAR beta transcript was mainly seen in the striated duct. The transcripts of RAR alpha and beta were evenly distributed in type I and II glandular cells of the Harderian gland. RAR gamma labelling was below detectable levels in both glands. This result suggests that RA and RARs regulate the functions of Harderian and submandibular glands in a cell-specific manner.