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 pig. An important aspect of understanding relaxin's mechanism of action in these tissues is to identify the specific cell type(s) that contains relaxin receptors, that is, to identify those cells that initiate relaxin's effects. The objective of the present study was to identify relaxin-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 accomplish that objective we developed an in vitro modification of an immunohistochemical 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 treated 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 removed, cut into cubes (2-3 cm(3)), frozen in liquid nitrogen, and cryosectioned (8 pm). Specific cell types that bind relaxin were identified by sequential application of a biotinylated relaxin probe, antibiotin immunoglobulin G conjugated to 1 nm colloidal gold, and silver for signal amplification. The study demonstrates for the first time that relaxin binds with specificity to 1) blood vessels (cervix, mammary glands, nipples, small intestine); 2) smooth muscles in small intestine (circular, longitudinal, muscularis mucosa); and 3) skin from sites other than the mammary nipples (back, ear, thigh, leg). In addition, consistent with previous findings in the rat, prominent labeling was observed in epithelial cells in the cervix, mammary glands, and nipples; in smooth muscle cells in the cervix and mammary nipples; and in the skin of the nipples. There were no apparent differences in relaxin binding 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.