IMMUNOHISTOCHEMICAL LOCALIZATION OF SPECIFIC RELAXIN-BINDING CELLS INTHE CERVIX, MAMMARY-GLANDS, AND NIPPLES OF PREGNANT RATS

Previously, we demonstrated that endogenous circulating relaxin promot es the growth and softening of the cervix, the development of the mamm ary glands, and the growth and development of nipples. Due to the rema rkably similar modifications in the histological appearance of the ext racellular matrix in the cervix, mammary glands, and nipples, we hypot hesized that there may be a common mechanism(s) of action of relaxin i n these tissues. A fundamental step toward understanding this mechanis m is to identify specific cells that contain relaxin receptors, that i s to identify those cells that initiate relaxin's effects within relax in target tissues. To identify specific relaxin-binding cells in the c ervix, mammary glands, and nipples of the pregnant rat, a biologically active biotinylated relaxin probe was prepared. This probe for putati ve relaxin receptors was administered to intact rats on day 18 of preg nancy. After 1 h, the animals were killed, and tissues were fixed by i mmerr sion in 4% paraformaldehyde for 10 h. Fixed tissues were rinsed in 0.1 M phosphate buffer (pH 7.4) and cryoprotected in an ascending s eries of 5%, 10%, and 20% sucrose solutions. The tissues were frozen i n Tissue-Tek O.C.T. compound and stored at -70 C until sectioning. Fro zen sections (12 mu m) were cut on a Tissue Tek II cryostat at -24 C a nd thaw mounted on slides coated with 0.01% poly-l-lysine (mol wt, 300 -6000). The biotinylated relaxin was localized in cryosections with an antibiotin immunoglobulin G conjugated to colloidal gold, which was s ubsequently visualized for light microscopy with silver intensificatio n. Specific binding of the biotinylated relaxin was localized in the e pithelial and smooth muscle cells of the cervix, the epithelial cells of the mammary glands, and the epithelial cells, smooth muscle cells, and skin of the nipples. We conclude that those cells exhibiting speci fic relaxin binding probably contain relaxin receptors and, therefore, mediate relaxin's effects in these tissues. As relaxin bound specific ally to epithelial cells in the cervix, mammary glands, and nipples, w e postulate that the epithelial cells may initiate a common mechanism of action that brings about modifications of the extracellular matrix in all three tissues.