Bernard G. Steinetz

THE EFFECTS OF ESTROGENS, PROGESTAGENS, AND RELAXIN IN PREGNANT AND NONPREGNANT LABORATORY RODENTS

The role of relaxin in reproductive physiological processes has been the subject of considerable investigation. However, the biological relationships of this hormone to the estrogens and progestagens are as yet poorly understood. Treatment with progesterone and estrogen has been reported to stimulate the occurrence of a pubic symphysis-relaxing substance in the blood of ovaricctomized (but not hysterectomized) guinea pigs and However, Hall4 was unable to induce pubic symphyseal separation in mice with a variety of combinations of estrogen and Progesterone. Indeed, progesterone has been found to inhibit relaxin-induced interpubic ligament formation in mice.5-* Relaxin has been reported to have a synergistic action with estrogen and progesterone in stimulating development of mammary glands of mice and

Myometrial inhibiting activity of relaxin-containing extracts of human corpora lutea of pregnancy

Relaxin is a peptide hormone secreted by the human corpus luteum of pregnancy . Aqueous extracts of relaxin-rich pregnancy corpora lutea decreased the amplitude of spontaneous human myometrial strip contractions in vitro. Relaxin-poor extracts of menstrual cycle corpora lutea did not affect contractions. Antibody precipitation of relaxin from pregnancy luteal extracts eliminated the effect on myometrial strips. Relaxin removal was confirmed by bioassay. This demonstrates an inhibiting action of human relaxin on human myometrial tissue in vitro. This action suggests a mechanism for maintaining uterine quiesence in early pregnancy.

RELAXIN IN HUMAN SEMINAL PLASMA

While relaxin is known as a hormone of pregnant females, it has been identified in rooster l and boar testes.g A recent report described immunoreactive relaxin in human seminal p l a ~ m a . ~ Relaxin is a structural homologue of insulin and other growth factors.* Epidermal growth factor and nerve growth factor are found in secretions of male salivary glands and the genitourinary Seminal plasma is rich in proteins and contains other peptide hormones, including growth hormone,8 p ro la~ t in ,~ and insulin.10 In this report, we characterize the immunoreactive relaxin-like substance in seminal plasma as bioactive relaxin and determine its source of production.

Antiarthritic Effects of Relaxin, in Combination with Estrogen, in Rat Adjuvant-Induced Arthritis

The incidence and severity of rheumatoid arthritis (RA) are reduced during pregnancy. Estradiol-17β and relaxin (RLX), hormones of pregnancy, are implicated in decreased immune responsiveness. The aim of this study was to determine the effects of estrogen and RLX, alone or in combination, on the development of adjuvant-induced arthritis (AIA) in ovariectomized (OVX) Lewis rats. Arthritis was induced on day 0 by adjuvant injection in the left hind paw. Rats were treated with estradiol valerate (E), porcine RLX, E + RLX, or vehicle. Healthy OVX control animals were used for comparison. Treatment with RLX or E alone decreased adjuvant-induced inflammation in both the injected (primary) and noninjected (secondary) hind paws. Combined treatment with E and RLX was more effective than either hormone alone in blocking secondary paw inflammation. Furthermore, E plus RLX reduced changes to spleen and thymus weights induced by adjuvant injection. Both E and RLX alone decreased circulating tumor necrosis factor (TNF) α. The combination of E and RLX resulted in a greater decline in TNFα than treatment with either hormone alone. There was no effect of hormones on the proinflammatory cytokine, interleukin (IL)-1β. The anti-inflammatory cytokine IL-10 increased in response to E and E plus RLX. In conclusion, combined therapy with E and RLX was more effective than either hormone alone in reducing chronic inflammation, joint changes, and high circulating TNFα associated with AIA in rats. Accordingly, these hormones could play a role in reducing RA-induced inflammation during pregnancy by an effect on the immune system.

Hormonal changes accompanying cigarette smoke-induced preterm births in a mouse model.

Epidemiologic evidence indicates that maternal smoking increases the risk of preterm birth. While a number of plausible mechanisms for early delivery have been offered, the role of gestational hormones in this smoke-induced outcome is uncertain. Thus, a toxicologic study was performed to examine the effects and underlying hormonal mechanisms of mainstream cigarette smoke (MCS) exposure on gestational duration. Pregnant B6C3F1 mice were exposed by inhalation to MCS for 5 days/week (4 hrs/day) from Gestational Day (GD) 4 to parturition. Smoke-induced effects on gestational length, interpubic ligament length, maternal hormone secretion patterns (estradiol-17β, progesterone, prolactin, and relaxin), body weight gain, postimplantation loss, litter size, and offspring sex ratio were examined. Dams exposed to MCS at a concentration equivalent to smoking less than one pack of cigarettes/day (carbon monoxide = 25 parts per million, total suspended particulates = 16 mg/m3) demonstrated a significant (P < 0.05) shortening of gestational duration (compared with pregnant, air-exposed mice). In addition, MCS-exposed mice sacrificed on GD 18 had significantly (P < 0.05) increased interpubic ligament length, elevated serum estrogen levels, and a reduced progesterone to estradiol-17β ratio (compared with air-exposed controls); levels of progesterone and prolactin were only modestly decreased and increased, respectively, in the MCS-exposed mice. Smoke exposure had no significant effects on maternal relaxin levels, body weight gain, postimplantation loss, litter size, or sex ratio. Results of this study demonstrate that inhalation exposure of pregnant mice to a low dose of MCS shortens gestation and alters hormone secretory patterns, which are important for maintaining pregnancy and inducing parturition. These findings support the view that pregnant women who smoke (even modestly) may be at increased risk for preterm birth, and that early delivery may be related (at least partly) to MCS-induced alterations in gestational hormones.

Development, Validation, and Application of a Urinary Relaxin Radioimmunoassay for the Diagnosis and Monitoring of Pregnancy in Felids

Abstract Many nondomestic felids are highly endangered, and captive breeding programs have become essential components of holistic conservation efforts for these species. The ability to diagnose pregnancy early in gestation is fundamental to developing effective breeding programs. The purpose of this study was to develop a radioimmunoassay (RIA) for the detection of urinary relaxin in felids and assess its applicability for early, noninvasive pregnancy diagnosis in domestic cats (Felis silvestris catus) and leopards (Panthera pardus). Urine was collected from pregnant and nonpregnant domestic cats and leopards at mating, and then weekly thereafter for the duration of gestation. Paired serum samples were also collected from the domestic cats. A RIA for relaxin that uses an antiserum against synthetic canine relaxin was validated for felid urine and shown to detect relaxin immunoreactivity in pregnant cat urine subjected to acid-acetone extraction. In the cat, urinary relaxin was first detected between Days 21 and 28 of gestation; levels peaked at 42–49 days, and the concentrations then declined over 2 wk prior to parturition. The urinary relaxin profiles of the cat mirrored those in serum. In the leopard, urinary relaxin was first detected at Day 25–28 of gestation; levels peaked at Day 60–64 and declined in the last 3–4 wk of pregnancy. These results indicate that measurement of urinary relaxin in the cat and leopard provides a reliable method for pregnancy determination from as early as 3–4 wk of gestation. This method of pregnancy diagnosis and monitoring may prove useful in the breeding management of domestic cats and other felid and canid species, and provides a foundation for future studies on pregnancy in captive exotic carnivores.

Characterization and biological activity of relaxin in porcine milk.

A lactocrine mechanism for delivery of maternally derived relaxin (RLX) into the neonatal circulation as a consequence of nursing was proposed for the pig. Immunoreactive RLX was detected in colostrum and in the serum of newborn pigs only if they were allowed to nurse. Milk-borne RLX concentrations are highest during early lactation (9-19  ng/ml), declining to <2  ng/ml by postnatal day 14. Whether milk-borne RLX is bioactive is unknown. Evidence that RLX concentrations in milk are higher than in maternal circulation in several species suggests the mammary gland as a site of local RLX production. It is unknown whether the porcine mammary gland is a source of RLX. Therefore, objectives were to evaluate RLX bioactivity in porcine milk during the first 2 weeks of lactation, identify the form of RLX in porcine milk, and determine whether mammary tissue from early lactation is a source of milk-borne RLX. Milk RLX bioactivity was determined using an in vitro bioassay in which cAMP production by human embryonic kidney (HEK293T) cells transfected with the human RLX receptor (RXFP1) was measured. RLX bioactivity was highest at lactation day (LD) 0, decreasing to undetectable levels by LD 4. Immunoblot analysis of milk proteins revealed an 18  kDa band, indicating proRLX as the primary form of RLX in porcine milk. ProRLX protein and transcripts were detected in porcine mammary tissue on LD 0 and 7. Results support the lactocrine hypothesis by defining the nature and a potential source for bioactive proRLX in porcine colostrum/milk.

Transmission of relaxin and estrogens to suckling canine pups via milk and possible association with hip joint laxity

OBJECTIVE To determine whether abnormal laxity of hip joints of canine pups with genetic predisposition to hip dysplasia (HD+) is related to ingestion of milk-borne hormones. ANIMALS 7 female Labrador Retrievers with HD+ and 8 with low predisposition to hip dysplasia (HD-) and their offspring. PROCEDURES Immunoactive relaxin, estrogen, and estrogen precursor concentrations in milk of HD+ lactating bitches and in serum of their pups were compared with those of HD- bitches and pups. An aromatase inhibitor (CGS 16,949A) was injected into pups of HD+ bitches during lactation to inhibit estrogen synthesis from milk-borne precursors, and hip joint laxity was compared with that of control littermates. Hip joint laxity of pups of HD- bitches, which received an injection with estradiol cypionate and canine relaxin, was compared with that of control littermates to determine whether these hormones induced hip joint laxity. RESULTS High concentrations of estrogens and relaxin were found in milk of HD+ and HD- bitches throughout lactation. Serum concentrations of milk-derived relaxin and total estrogens were similar in all pups, but estradiol-17B was detected only in pups of HD+ bitches. Hip joint laxity was reduced in pups that received CGS 16,949A. Hip joint laxity was INCREASED IN PUPS OF HD- BITCHES THAT RECEIVED ESTRADIOL CYPIONATE AND RELAXIN. CONCLUSIONS AND CLINICAL RELEVANCE Milk-borne maternal hormones and precursors were absorbed into the circulation of canine neonates and may play a role in hip joint laxity in HD+ pups. Phenotypic expression of hip dysplasia may therefore be preventable by antihormone treatment.

Milk-Borne Relaxin and the Lactocrine Hypothesis for Maternal Programming of Neonatal Tissues

The fact that all newborn mammals drink milk extends the time frame of maternal influence on development into neonatal life. While the nutritional and immunological benefits of milk are clear, the role of milk as a conduit for bioactive factors with the potential to affect neonatal development is less well defined. Porcine and canine milk contain immunoreactive relaxin (RLX) that is transmitted into the circulation of nursing offspring. In the pig, a window of opportunity for transmission of milk‐borne RLX is open at birth and remains so for about the first 3 days of neonatal life. Recent studies have shown that pro RLX is the major form of RLX in milk and that milk‐borne porcine pro RLX is biologically active. Moreover, RLX receptor (RXFP1) expression is detectable in the porcine female reproductive tract and other somatic tissues at birth. The lactocrine hypothesis for maternal programming of neonatal development was proposed as a mechanism whereby RLX, a prototypical milk‐borne growth factor in the pig, is delivered to nursing offspring, where it can affect development of RXFP1‐positive target tissues. Data indicating that treatment of newborn gilts with RLX increased estrogen receptor‐α expression in the uterus and cervix by postnatal day 2 support a role for RLX in lactocrine programming of the female reproductive tract. Effects of RLX on Wnt/β‐catenin expression in neonatal porcine cardiac tissue support a role for RLX in developmental programming of nonreproductive target tissues as well. Ongoing studies will test the lactocrine hypothesis for maternal programming of development by RLX and related milk‐borne factors.

The effect of relaxin and progesterone on rat uterine contractions

The effect of porcine relaxin on electrically stimulated in vitro contractions of isolated uterine horn segments from estrogen-pretreated immature rats was studied. Relaxin decreased the amplitude of contractions. A mean of 8.3 ng/ml of relaxin produced a 90% decrease in contraction amplitude. There was a minimal effect of 1.0 microgram/ml of progesterone on contraction amplitude. In vitro pretreatment of the isolated uterine segment with this dose of progesterone for 15 minutes did not significantly affect the dose of relaxin needed to decrease the amplitude of contractions. In contrast, pretreatment with progesterone for 45 minutes significantly decreased the concentration of relaxin needed to decrease contraction amplitude. Only 4.7 ng/ml of relaxin was needed to produce a 90% decrease in amplitude after progesterone pretreatment for 45 minutes (p less than 0.005). Relaxin and progesterone synergize in decreasing the amplitude of uterine contractions in vitro. A similar effect may occur in vivo.