Endocrinology, Vol 136, 1367-1373, Copyright © 1995 by Endocrine Society

ARTICLES

Immunohistochemical localization of specific relaxin-binding cells in the cervix, mammary glands, and nipples of pregnant rats

MJ Kuenzi and OD Sherwood
Department of Physiology and Biophysics, University of Illinois-Urbana- Champaign 61801.

Previously, we demonstrated that endogenous circulating relaxin promotes the growth and softening of the cervix, the development of the mammary glands, and the growth and development of nipples. Due to the remarkably similar modifications in the histological appearance of the extracellular matrix in the cervix, mammary glands, and nipples, we hypothesized that there may be a common mechanism(s) of action of relaxin in these tissues. A fundamental step toward understanding this mechanism is to identify specific cells that contain relaxin receptors, that is to identify those cells that initiate relaxin's effects within relaxin target tissues. To identify specific relaxin-binding cells in the cervix, mammary glands, and nipples of the pregnant rat, a biologically active biotinylated relaxin probe was prepared. This probe for putative relaxin receptors was administered to intact rats on day 18 of pregnancy. After 1 h, the animals were killed, and tissues were fixed by immersion in 4% paraformaldehyde for 10 h. Fixed tissues were rinsed in 0.1 M phosphate buffer (pH 7.4) and cryoprotected in an ascending series of 5%, 10%, and 20% sucrose solutions. The tissues were frozen in Tissue-Tek O.C.T. compound and stored at -70 C until sectioning. Frozen sections (12 microns) were cut on a Tissue Tek II cryostat at -24 C and 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 subsequently visualized for light microscopy with silver intensification. Specific binding of the biotinylated relaxin was localized in the epithelial 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 specific relaxin binding probably contain relaxin receptors and, therefore, mediate relaxin's effects in these tissues. As relaxin bound specifically to epithelial cells in the cervix, mammary glands, and nipples, we postulate that the epithelial cells may initiate a common mechanism of action that brings about modifications of the extracellular matrix in all three tissues.

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