Estrogen receptor-dependent organizational events between birth [postnatal day (PND) 0] and PND 14 affect development and function of porcine uterine tissues. Observations that uterotrophic effects of relaxin (RLX) in neonatal gilts were inhibited by the antiestrogen ICI 182,780 suggested that a RLX signaling system, capable of cross-talk with the estrogen receptor, evolves during a critical period for uterine programming (PND 0-14). Objectives were to determine 1) effects of age and estrogen exposure from birth on porcine uterine RLX/insulin-like 3 receptor (LGR7/LGR8) expression and 2) whether milk serves as a natural source of RLX in neonatal pigs. Uterine LGR7/LGR8 expression, detected by RT-PCR and in situ hybridization on PND 0, 7, and 14, was predominantly stromal for LGR7, myometrial for LGR8, and increased with age and after treatment with estradiol valerate (50 microg/kg body weight x d) from birth. Stromal expression of LGR7 was also detected immunohistochemically. Milk RLX concentrations declined (P < 0.001) from 17.3 +/- 1.4 ng/ml (lactation d 0) to 1.7 +/- 0.3 ng/ml (lactation d 14). RLX, present in the serum of nursing pigs on PND 0 and 1, was undetectable before nursing and in neonates fed RLX-free milk replacer for 12 h. Thus, a developmentally regulated, estrogen-sensitive LGR7 and LGR8 receptor system is present in the porcine uterus at birth and may be activated by milk-borne RLX delivered into the circulation during the first 48 h of postnatal life. Maternal lactocrine contributions to the neonatal hormonal milieu could affect the developmental programming of uterine and other somatic tissues.

Recent work from our laboratory led to the development of a radioimmunoassay (RIA) for porcine relaxin utilizing [ 12.51]polytyrosyl relaxin (1 ) . Addition of tyrosine to the relaxin molecule proved to be necessary as the purified native hormone does not contain tyrosine or histidine (2) and fails to iodinate when subjected to the Hunter and Greenwood method (1). An impure relaxin extract was used by other workers who claimed successful iodination of the hormone (3).We have previously demonstrated crossreactions of antisera to porcine relaxin with relaxin-con taining tissues of several species including rats, mice, chickens, whales, and human beings (4). Because of this broad range of immunological cross-reactivity of relaxin from different sources, it was of interest to develop a RIA for determination of blood levels of relaxin in species other than the pig. We have now assayed relaxin levels throughout pregnancy in rats, mice, and guinea pigs, and near term in dogs and primates.Materials and methods. Radioimmunoassay. The relaxin RIA has been modified since the publication of the original report Reagents. Two fractions of porcine relaxin prepared according to the procedure of Sherwood and O'Byrne (2) were used in the development of the method. A moderately pure fraction (G-50:1000 U/mg) was used for immunization and a highly purified fraction (CM-A + B: 2500 to 3000 U/mg) was used for standards and preparation of polytyrosyl-relaxin . The assay was equally sensitive and linear when we used a purified relaxin prepared by a different method and kindly supplied by Dr . Christian Schwabe, Medical College of South Carolina. Thus, the antigenic determinants of porcine relaxin do not appear to be altered by differing routes of biochemical purification. How-(1). ever, reduced-alkylated relaxin as well as isolated a and p subunits of relaxin did not compete with [1251]polytyrosyl-relaxin for binding sites on the antibody. Although relaxin precursors and/or metabolic products may be immunoreactive, these observations suggest a requirement for the overall structure of two peptide chains connected by disulfide bonds for antibody recognition. Polytyrosyl-relaxin ,[ 1251]polytyrosyl-relaxin, and relaxin antisera were prepared as previously described (1). Goat anti-rabbit gamma globulin was purchased from Antibodies Incorporated, Davis, California.RIA procedure. 1. One-hundred microliters of relaxin standard solutions [ l o to 2500 pg relaxin in phosphate buffered saline (PBS = 0.01 M sodium phosphate, pH 7.0, 0.14 M sodium chloride)-1% egg albumin] plus a volume of control serum or plasma (male or castrated) equivalent to that of the unknown samples were added to standard curve tubes.2. Unknown serum or plasma samples were added to other tubes.3 . Sufficient PBS-1% egg albumin was added to each tube to bring the volume to 500 pl. 4. One-hundred microliters of [12"I]polytyrosyl-relaxin in PBS-1% egg albumin (30,000 to 60,000 cpm) were added to each tube.5 . One-hundred microliters of relaxin antiserum diluted in 0.05 M EDTA-...

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The 6-kDa polypeptide hormone relaxin (Rlx) has been identified in human and bovine milk, and we recently reported its presence in canine milk. We postulated that Rlx might be transferred via suckling to the newborn pups, where, by virtue of its known effects to increase the distensibility of the pelvic connective tissues, it could play a role in causing the excessive laxity of the capsule and ligaments of the coxofemoral joint that precedes the development of hip dysplasia in genetically predisposed animals. Rlx was found in the serum of dysplastic (HD+) bitches for up to 6 wk of lactation, whereas it was detected in the serum of nondysplastic (HD-) bitches for only 1-2 wk of lactation. Rlx concentrations in milk were up to 60-fold greater than in serum. Milk Rlx levels varied markedly, but were highest during the first week of lactation and decreased thereafter. There were no significant differences in milk Rlx concentrations between HD+ and HD- bitches. Although the source of Rlx in milk is unknown, it cannot be the ovary or uterus, since hystero-ovariectomy performed at the time of cesarean section did not eliminate Rlx from milk during subsequent lactation. In serum samples taken from newborn pups before suckling, there were significant quantities of Rlx, demonstrating that the hormone enters the fetus in utero. However, Rlx rapidly disappears from serum of pups prevented from suckling for five hours.(ABSTRACT TRUNCATED AT 250 WORDS)