Relaxin as a hormonal aid to evaluate pregnancy and pregnancy loss in bottlenose dolphins (Tursiops truncatus).

This study was conducted to critically evaluate weekly and monthly circulating concentrations of immunoreactive relaxin throughout pregnancies that resulted in live births, stillbirths, and abortions in aquarium-based bottlenose dolphins. A relaxin RIA was used to analyze serum collected during 74 pregnancies involving 41 dolphins and 8 estrous cycles as well as 8 non-pregnant dolphins. Pregnancies resulted in live births (n=60), stillbirths (n=7), or abortions (n=7). Relative to parturition (Month 0), monthly changes (P<0.0001) in relaxin was indicated by relatively low concentrations during early pregnancy (Months -12 to -9) which subsequently increased (P<0.05) during mid- (Months -8 to -5) to late (Months -4 to -1) pregnancy; relaxin was highest (P<0.05) at the time of parturition. Post-parturition (Month 1), concentrations decreased (P<0.05). During the first 4weeks post-ovulation, relaxin concentrations were not different between pregnant and non-pregnant dolphins (status-by-week interaction, P=0.59). Status-by-month interaction (P<0.0002) involving different pregnancy outcomes was due, impart, to an increase in relaxin during early pregnancy (P<0.05) that was comparable among dolphins with live births, stillbirths, and abortions except concentrations were lower (P<0.05; 52%) at mid-pregnancy in association with pregnancy loss. Thereafter, concentrations increased (P<0.05) during late pregnancy in dolphins with stillbirths but not in dolphins with abortions. In conclusion, this study provided new information on the pregnancy-specific nature of relaxin, critical evaluation of the fundamental characteristics of relaxin during pregnancy and pregnancy loss, and clarification on the strengths and limitations of relaxin as a diagnostic aid to determine pregnancy status and assess maternal-fetal health in bottlenose dolphins.

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.

Relaxin and progesterone during pregnancy and the post-partum period in association with live and stillborn calves in bottlenose dolphins (Tursiops truncatus).

The objectives of this study were to validate a relaxin and progesterone RIA for use in bottlenose dolphins, and quantify and characterize both hormones in extracts of placental tissue and serum collected during pregnancy and the post-partum period, and compare the results between dolphins with live and stillborn calves. In Experiment 1, validation of a heterologous relaxin and progesterone RIA involved specific displacement of antibody-bound radiolabeled human relaxin or progesterone in response to increasing volumes of pooled pregnant dolphin serum and amounts of respective hormone standards added to a fixed volume of serum. The displacement curves were considered parallel and additive relative to respective standard curves. In Experiment 2, immunoreactive relaxin and progesterone were detected in placental extracts and, in corresponding serum samples, concentrations of both hormones were higher during the pre-partum than post-partum periods. Circulatory concentrations of progesterone decreased (P < 0.05) from relatively high concentrations during early and mid-pregnancy to intermediate concentrations by late pregnancy (month effect, P < 0.0001) in dolphins with live births, whereas, in dolphins with stillbirths, the decrease in progesterone began earlier (month-by-birth status interaction, P < 0.007); mean concentrations were lower at mid- (37%, P < 0.06) and late (25%) pregnancy. Temporally, relaxin increased (P < 0.05) progressively from relatively low concentrations during early pregnancy to high concentrations during late pregnancy (month effect, P < 0.0001) and was not different between birth statuses (birth status effect, P = 0.76; month-by-birth status interaction, P = 0.17). Even though the interaction did not reach significance, mean relaxin concentrations were 42%, 29%, and 34% lower at early, mid-, and late pregnancy, respectively, in dolphins with stillbirths than in those with live births. In conclusion, the pregnancy-specific increase in serum concentrations of relaxin and lower concentrations of both relaxin and progesterone in association with stillbirths suggest the potential for relaxin to be used diagnostically to determine pregnancy status, and one or both hormones to be used to assess placental function, and, perhaps, fetal well-being in bottlenose dolphins and other cetaceans.

Validation of a homologous canine relaxin radioimmunoassay and application with pregnant and non-pregnant Northern fur seals (Callorhinus ursinus).

The primary objectives of this study were to validate a canine relaxin RIA for use in otariids and phocids and consider practical applications. For 6 captive Northern fur seal females, serum samples were grouped and examined according to pregnancy (n=13), post-partum (n=8) and non-pregnancy (n=6), and, for 2 captive Northern fur seal males, serum samples were grouped and examined together regardless of age (2 mo-15 yrs, n=6). Placental tissue was available for examination from one Northern fur seal, Steller sea lion and harbor seal. The validation process involved several steps using an acid-acetone extraction process to isolate a relaxin-containing fraction in pools of serum from each group of fur seals and placental tissue from each seal species. A relaxin-like substance was detected in extracts of pregnant, non-pregnant and male serum and placental tissue in a dose-responsive manner as increasing volumes of respective extracts or amounts of canine relaxin were introduced into the assay. In raw serum samples, mean immuno-reactive relaxin concentrations were higher (P<0.05) during pregnancy than post-partum and non-pregnancy, and lower (P<0.05) in male than female fur seals. During pregnancy, mean serum concentrations of relaxin progressively increased (P<0.05) over Months 4-10 and, in serial samples collected from the same fur seals before and after parturition, mean concentrations were higher (P<0.06) pre-partum than post-partum. In conclusion, validation of a homologous canine relaxin RIA for use in otariids and phocids resulted in the discovery of a relaxin-like substance in extracted and raw serum and placental tissue from Northern fur seals, a Steller sea lion and harbor seal. Distinctly higher immuno-reactive concentrations during pregnancy indicated the potential for relaxin to serve as a hormonal marker to differentiate between pregnant and non-pregnant or pseudopregnant pinnipeds.

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.

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.

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.

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-17beta, 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-17beta 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.

Relaxin concentrations in serum and urine of endangered species: correlations with physiologic events and use as a marker of pregnancy.

Many mammalian species are facing extinction due to problems created by human encroachment, agriculture, pollution, and willful slaughter. Among those at risk are the Asian and African elephant, Sumatran rhinoceros, and giant panda. Conservation groups try to save species in the wild by preserving habitat and limiting animal-human conflicts, often with limited success. Another alternative is to preserve the extant gene pool through captive breeding as a hedge against extinction. Measurement of circulating reproductive hormones is impractical for most wildlife species; determination of urinary or fecal hormone metabolites provides a more viable approach. To aid breeding management, one important tool is the ability to diagnose and monitor pregnancy, especially in species with long gestations (e.g., rhinos over 15 mo and elephants over 20 mo). Unfortunately, measuring progestins often is not useful diagnostically, because concentrations are similar during at least part of the pregnancy and the nonpregnant luteal phase in some species (e.g., elephants, rhinoceroses, and giant pandas). As serum relaxin reliably distinguishes between pregnancy and pseudopregnancy in bitches, relaxin measurement might also provide a method for detecting a successful pregnancy in endangered species. Appropriate immunoassay reagents have enabled the estimation of relaxin concentrations in the serum of elephants and rhinos and the determination of pregnancy establishment and the outcome. Relaxin was also detected in panda serum and urine. However, the extreme variability of the time between observed mating and parturition and the confounding factors of delayed implantation, pseudopregnancy, and frequent fetal resorptions made it impossible to use the panda relaxin data as a specific marker of pregnancy.

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.

Abnormal relaxin secretion during pregnancy in women with type 1 diabetes.

To test the hypothesis that relaxin may play a role in the fetal abnormalities associated with pregnancy in type 1 diabetic women, we previously compared gestational relaxin concentrations in diabetic and clinically normal women using a porcine relaxin radioimmunoassay (RIA): Serum immunoactive relaxin was significantly (P < 0.001) elevated in the diabetic women. To confirm and extend this work in a larger group of subjects, we have now used an enzyme-linked immunosorbent assay (ELISA) specific for human H2 relaxin (the normal human gene product) to determine immunoactive serum relaxin concentrations in serial samples from 61 Type 1 diabetic and 21 normal pregnant women. Samples from 22 of the diabetic and nine of the normal women were also directly compared in the porcine relaxin RIA. ELISA-determined serum relaxin was higher (P < 0.001) at 24 and 36 weeks of pregnancy in type 1 diabetic women than in controls, confirming previous findings. However, the geometric mean increase in immunoactive relaxin concentration in identical samples from pregnant diabetic women over that of controls was significantly greater with the RIA than with the ELISA (271% vs 44%; P < 0.001). To investigate this discrepancy, the specificity and epitope selectivity of the RIA and the ELISA were compared using several synthetic polypeptides, including human relaxins H1 and H2, and relaxin and insulin derivatives. Both assays showed great specificity, but the porcine RIA selectively identified the epitopes of the receptor-binding domain of the relaxin B chain and cross-reacted strongly with H1 and H2 relaxins. In contrast, only the H2 peptide was detected by the ELISA antiserum. Therefore, the marked discrepancy between the RIA and the ELISA could be due to the presence in the diabetic samples of another relaxin-like molecule in addition to the normal H2 relaxin. The biological consequences of elevated serum relaxin in diabetic pregnancy remain to be elucidated.

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-alpha 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/beta-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.

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 has been proposed for the pig. Consistently, immunoreactive porcine RLX was detected in colostrum as well as in the serum of nursing pigs. Concentrations of porcine RLX in milk are highest during early lactation (9-19 ng/mL) and decline to less than 2 ng/mL by postnatal day 14. However, RLX bioactivity has not been described in porcine milk. Therefore, this study was designed to establish an assay for RLX bioactivity in porcine milk and to determine if milk RLX bioactivity was related to RLX concentrations in milk collected at parturition (lactation day 0) and on lactation day 14. To assess milk RLX bioactivity, an in vitro bioassay using human embryonic kidney (HEK293T) cells transfected with the human RLX receptor (LGR7) was developed. Milk RLX bioactivity was confirmed by documentation of a systematic increase in cAMP production by HEK293T-LGR7 cells in response to increasing volumes of day 0 milk. Addition of lactation day 14 milk, porcine insulin, or human insulin-like growth factor 1 to HEK293T-LGR7 cells, or porcine RLX treatment of nontransfected HEK293T cells, failed to elicit a cAMP response. Western blot analysis of milk proteins revealed an 18-kDa protein band, indicating that pro RLX is the primary form of bioactive RLX in porcine milk. Data support the lactocrine hypothesis and suggest a role for milk-borne pro RLX in porcine neonatal development.

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-17beta 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) alpha. The combination of E and RLX resulted in a greater decline in TNFalpha than treatment with either hormone alone. There was no effect of hormones on the proinflammatory cytokine, interleukin (IL)-1beta. 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 TNFalpha 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.

Development, validation, and application of a urinary relaxin radioimmunoassay for the diagnosis and monitoring of pregnancy in felids.

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.

The parity-related protection against breast cancer is compromised by cigarette smoke during rat pregnancy: observations on tumorigenesis and immunological defenses of the neonate.

Early pregnancy is a powerful negative risk factor for breast cancer (BCa) in women. Pregnancy also protects rats against induction of BCa by carcinogens such as N-methyl-N-nitrosourea (MNU), making the parous rat a useful model for studying this phenomenon. Smoking during early pregnancy may lead to an increased risk of BCa in later life, possibly attributable to carcinogens in cigarette smoke (CS), or to reversal of the parity-related protection against BCa. To investigate these possibilities, 50-day-old timed first-pregnancy rats were exposed to standardized mainstream CS (particle concentration = 50 mg/m3) or to filtered air (FA) 4 h/day, Day 2-20 of gestation. Age-matched virgin rats were similarly exposed to CS or FA. At age 100 days, the CS or FA-exposed, parous and virgin rats were injected s.c. with MNU (50 mg/kg body wt), or with MNU vehicle. Mammary tumors (MTs) first appeared in virgin rats 9 weeks post-MNU injection. While no MTs were detected in FA-exposed parous rats until 18 weeks post-MNU, MTs appeared in the CS-exposed parous rats as early as 10 wks (P < 0.02). As no MTs developed in CS-exposed rats not injected with MNU, CS did not act as a direct mammary carcinogen. Serum prolactin concentration on Day 19 of pregnancy in CS-exposed dams was reduced by 50% compared with FA-exposed dams (P < 0.005). CS exposure during a pregnancy may thus 'deprotect' rats, enhancing their vulnerability to MNU-induced BCa. Prenatal CS exposure had no detectable effect on the immune responses of the pups examined at 3, 8 or 19 weeks of age. However, prolactin concentration in stomach contents (milk) of 3-day-old pups suckled by CS-exposed dams was decreased when compared with that of FA-exposed dams (P < 0.032). As milk-borne prolactin modulates development of the central nervous and immune systems of neonatal rats, CS exposure of the dams could adversely affect later maturation of these systems by reducing milk prolactin.

Serum relaxin concentrations and reproduction in male bonnethead sharks, Sphyrna tiburo.

Relaxin is a 6-kd polypeptide hormone that is responsible for regulating several reproductive processes in female vertebrates, but its role in male reproduction remains unclear. To aid in clarifying this role, the objective of the present study was to investigate changes in endogenous relaxin levels associated with reproductive events in male elasmobranchs, which represent one of only three vertebrate groups known to possess this hormone. Serum relaxin concentrations were measured in 27 immature and 66 mature male bonnethead sharks (Sphyrna tiburo), a species with a well-characterized, seasonal reproductive cycle. Temporal changes in serum relaxin concentrations of immature male S. tiburo were not observed. In contrast, a temporal cycle in serum relaxin concentrations of mature male S. tiburo was observed in individuals from two sampling locations. Significant increases (P<0.05) in serum relaxin concentrations of mature male S. tiburo from both collection sites occurred during late spermatogenesis and the mating period, two critical stages of the reproductive cycle. The results from this study suggest that relaxin may play an important role in regulating semen quality, or other aspects of reproduction in male sharks. This is the first study to demonstrate a temporal pattern in endogenous serum Rlx concentrations associated with reproductive events in feral vertebrates. As such, it strengthens earlier hypotheses that suggested a role for this hormone in regulating male vertebrate fertility and copulatory success.

Species and fetal gender effects on the endocrinology of pregnancy in elephants.

Quantitative and temporal progestin profiles vary during gestation in the elephant, sometimes making it difficult to determine if a pregnancy is progressing normally. The aim of the present study was to determine if circulating progestin variability was related to species or fetal gender effects. A similar comparison also was conducted for secretory profiles of prolactin, relaxin, and cortisol. Overall mean progestin concentrations during gestation in Asian (n = 19) and African (n = 8) elephants were similar; however, the temporal profiles differed (P < 0.001). Concentrations were higher in African elephants during the first half of pregnancy, but then declined to levels below those observed in Asian elephants (P < 0.05). There also was a fetal gender effect in Asian, but not African elephants. Progestin concentrations were higher in Asian cows carrying male calves (n = 9) as compared to those carrying females (n = 10) (P < 0.001). Overall prolactin concentrations were higher in Asian than in African elephants between 8 and 15 months of gestation ( P< 0.001). There were no species differences in the secretory patterns of relaxin. Cortisol was relatively stable until the end of gestation when significant surges were observed, mainly between 8 and 11 days before parturition, and again on the day of birth. In sum, a comparison of progestin patterns between Asian and African elephants identified notable differences related to species and fetal gender. A role for cortisol in the initiation of parturition also was inferred from these data. From a practical standpoint, understanding the factors affecting gestational hormone characteristics and recognizing what the species differences are will help ensure that data used in diagnosing and monitoring elephant pregnancies are properly interpreted.