Mj. Kuenzi et Od. Sherwood, IMMUNOHISTOCHEMICAL LOCALIZATION OF SPECIFIC RELAXIN-BINDING CELLS INTHE CERVIX, MAMMARY-GLANDS, AND NIPPLES OF PREGNANT RATS, Endocrinology, 136(4), 1995, pp. 1367-1373
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.