The source and secretion of immunoactive relaxin in rat milk.

The milk of many mammalian species contains hormones and growth factors in addition to nutrients and immunocompetent substances. These factors can be absorbed into the circulation of suckling neonates to exert important effects on metabolism and promote tissue and organ growth. Frequently, there is uncertainty as to whether such substances are gene products of the mammary glands themselves or are produced elsewhere and concentrated from the systemic circulation. The 6 kD polypeptide, relaxin, appears in milk of several mammalian species, including that of the rat, but proof of its source of secretion (corpus luteum vs. mammary gland) is so far lacking. The specific monoclonal anti-rat relaxin antibody MCA1 has previously been utilized successfully to investigate many of relaxin's actions in the rat, including those affecting the development of the mammary apparatus. In this report, MCA1 was utilized to aid in the identification of the source of relaxin in rat milk. Treatment of lactating rats with MCA1 completely neutralized the luteal relaxin circulating in serum but did not decrease the concentration of immunoactive relaxin secreted in milk. Moreover, the antibody did not appear to reach the mammary epithelium. The evidence thus supports the view that in the rat, the relaxin secreted in milk is primarily a product of the mammary glands and not concentrated from the systemic circulation.

Refinement of a commercial bench-top relaxin assay for pregnancy diagnosis using urine from domestic and nondomestic felids.

Relaxin, a 6-kDa polypeptide hormone, is excreted in the urine during pregnancy in several mammalian species. A recent study showed that detection of urinary relaxin using a bench-top serum assay (Witness relaxin kit, Synbiotics Corp., San Diego, California 92127, USA) can be diagnostic for pregnancy in domestic cats (Felis silvestris catus), but it is unknown whether the bench-top kit is applicable with urine across felid species. Our objectives were to 1) examine modifications in urine processing to improve kit reliability in pregnant cats, 2) evaluate the impact of concentrating urine via filtration on relaxin detection, 3) assess the effect of sample freezing on relaxin concentrations, and 4) begin quantifying urinary relaxin levels in nondomestic felids. Urine and serum were collected from domestic cats and nondomestic cat species (Pallas' cat, Otocolobus manul; sand cat, Felis margarita; cheetah, Acinonyx jubatus; and lion, Panthera leo) at several times after breeding. Urine samples, subjected to various processing methods, were tested using the bench-top kit, and relaxin levels were later quantified via radioimmunoassay. For domestic cat urine samples, filtration and addition of protein/phosphate buffer improved the consistency of the relaxin kit for early pregnancy diagnosis. Urine freezing caused a slight (approximately 13%) but significant decrease in relaxin concentrations, but frozen-thawed samples still tested positive with the bench-top kit. In nondomestic felids, urinary relaxin immunoreactivity during pregnancy was similar to or higher than that of pregnant domestic cats, suggesting that relaxin is a reliable cross-species marker of pregnancy. Urinary relaxin was detectable using the bench-top kit in pregnant Pallas' cats, but urine samples from other species tested negative, regardless of processing methods. Findings suggest that measurement of urinary relaxin is a promising approach for noninvasive pregnancy diagnosis in exotic felids, but further assessment of urinary relaxin profiles among cat species and modification of the bench-top relaxin kit are warranted to improve cross-species utility.

Expression of LGR7 and LGR8 by neonatal porcine uterine tissues and transmission of milk-borne relaxin into the neonatal circulation by suckling.

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.

Immuno-neutralization of circulating relaxin does not alter the breast cancer-protective action of parity in MNU-treated rats.

Early pregnancy and childbirth protects women against future development of breast cancer by an unknown mechanism. Parity likewise reduces mammary cancer incidence in rats exposed to the carcinogen, N-methyl-N-nitrosourea (MNU), providing a model for the human phenomenon. We hypothesized that relaxin, a 6KD luteal mammotropic hormone of pregnancy, might be the anti-cancer pregnancy factor, and that induced relaxin deficiency during rat gestation would restore carcinogen sensitivity. Forty-one pregnant (age 50 days) and 25 age-matched virgin Sprague-Dawley rats were used. Relaxin deficiency was induced by injecting mouse monoclonal anti-rat relaxin antibody (MCA1) days 12-18 of gestation. Pregnant controls were injected with vehicle or mouse IgG on the same schedule. Because MCA1 disrupts parturition, all rats underwent cesarean section on day 22. At age 100 days, all rats were injected i.v. with MNU (50mg/Kg) and examined daily for tumors until euthanized at age 240 days. Mammary tumor incidence and frequency were significantly (p<0.01) reduced and tumor latency was increased (p<0.001) in primiparous as compared with virgin rats. However, tumor incidence, type, size and latency were similar in MCA1-treated and control primiparous rats. Thus, luteal relaxin does not appear to be the factor responsible for resistance to breast cancer.