IDENTIFICATION OF SPECIFIC RELAXIN-BINDING CELLS IN THE CERVIX, MAMMARY-GLANDS, NIPPLES, SMALL-INTESTINE, AND SKIN OF PREGNANT PIGS

We previously demonstrated that relaxin promotes growth and softening of the cervix and development of the mammary glands in the pregnant pi g. An important aspect of understanding relaxin's mechanism of action in these tissues is to identify the specific cell type(s) that contain s relaxin receptors, that is, to identify those cells that initiate re laxin's effects. The objective of the present study was to identify re laxin-binding cells in tissues known to respond to relaxin (cervix and mammary gland) as well as in tissues suspected of being responsive to relaxin (nipple, small intestine, and skin) in the pregnant pig. To a ccomplish that objective we developed an in vitro modification of an i mmunohistochemical technique recently developed for identification of relaxin-binding cells. Two groups of pregnant gilts were used: intact control (group C) and ovariectomized progesterone-treated (group OF). Group OP was ovariectomized on Day 40 of gestation (Day 40) and treate d with progesterone (50 mg/2 ml corn oil i.m., twice daily) until Day 110 to maintain pregnancy. On Day 110, tissues from both groups were r emoved, cut into cubes (2-3 cm(3)), frozen in liquid nitrogen, and cry osectioned (8 pm). Specific cell types that bind relaxin were identifi ed by sequential application of a biotinylated relaxin probe, antibiot in immunoglobulin G conjugated to 1 nm colloidal gold, and silver for signal amplification. The study demonstrates for the first time that r elaxin binds with specificity to 1) blood vessels (cervix, mammary gla nds, nipples, small intestine); 2) smooth muscles in small intestine ( circular, longitudinal, muscularis mucosa); and 3) skin from sites oth er than the mammary nipples (back, ear, thigh, leg). In addition, cons istent with previous findings in the rat, prominent labeling was obser ved in epithelial cells in the cervix, mammary glands, and nipples; in smooth muscle cells in the cervix and mammary nipples; and in the ski n of the nipples. There were no apparent differences in relaxin bindin g between group C and group OF. We conclude that the specific relaxin- binding cells in the cervix, mammary glands, nipples, small intestine, and skin of the pregnant pig probably contain relaxin receptors and, therefore, mediate relaxin's effects in these tissues.