Healthy Donors Exhibit a CD4 T Cell Repertoire Specific to the Immunogenic Human Hormone H2-Relaxin before Injection.

H2-relaxin (RLN2) is a two-chain peptide hormone structurally related to insulin with a therapeutic potential in multiple indications. However, multiple injections of human RLN2 induced anti-RLN2 Abs in patients, hampering its clinical development. As T cell activation is required to produce Abs, we wondered whether T cells specific for RLN2 might be already present in the human blood before any injection. We therefore quantified the RLN2-specific T cell repertoire using PBMCs collected from healthy donors. CD4 T cells were stimulated in multiple replicates by weekly rounds of stimulation by dendritic cells loaded with RLN2, and their specificity was assessed by IFN-γ ELISPOT. The number of specific T cell lines was used to estimate the frequency of circulating T cells. In vitro T cell response was demonstrated in 18 of the 23 healthy donors, leading to the generation of 70 independent RLN2-specific T cell lines. The mean frequency of RLN2-specific CD4 T cells was similar to that of T cells specific for known immunogenic therapeutic proteins. Using overlapping peptides, we identified multiple T cell epitopes hosted in the N-terminal parts of the α- and ß-chains and common to multiple donors, in agreement with their capacity to bind to multiple HLA-DR molecules. Our results provide important clues to the immunogenicity of RLN2 and highlight the weak central immune tolerance induced against this self-hormone.

Relaxin is essential for renal vasodilation during pregnancy in conscious rats.

Marked vasodilation in the kidney and other nonreproductive organs is one of the earliest maternal adaptations to occur during pregnancy. Despite the recognition of this extraordinary physiology for over four decades, the gestational hormone responsible has remained elusive. Here we demonstrate a key role for relaxin, a member of the IGF family that is secreted by the corpus luteum in humans and rodents. Using a gravid rodent model, we employ two approaches to eliminate relaxin or its biological activity from the circulation: ovariectomy and administration of neutralizing antibodies. Both abrogate the gestational elevation in renal perfusion and glomerular filtration, as well as preventing the reduction in myogenic reactivity of isolated, small renal arteries. Osmoregulatory changes, another pregnancy adaptation, are also abolished. Our results indicate that relaxin mediates the renal vasodilatory responses to pregnancy and thus may be important for maternal and fetal health. They also raise the likelihood of a role for relaxin in other cardiovascular changes of pregnancy, and they suggest that, like estrogen, relaxin should be considered a regulator of cardiovascular function.

Cryptocrine signaling in the thymus network and T cell education to neuroendocrine self-antigens.

Both during phylogeny and ontogeny the thymus appears as a nodal point between the two major systems of cell-to-cell signaling, the neuroendocrine and immune systems. This review presents the experimental observations which support a dual role in T cell selection played by the thymic repertoire of neuroendocrine polypeptide precursors. Through the mode of cryptocrine intercellular signaling thymic neuroendocrine-related precursors synthesized in thymic epithelial cells have been shown to influence the early steps in T cell differentiation. In addition, thymic neuroendocrine-related polypeptides are a source of self-antigens which are presented by the major histocompatibility system of the thymic epithelium. Preliminary data also suggest that the intrathymic T cell education to neuroendocrine self-antigens is not strictly superimposible to the antigen presentation by dedicated presenting cells. Insulin-like growth factor-II (IGF-II) was identified as one dominant member of the insulin family expressed by thymic epithelial and nurse cells. The intrathymic presentation of IGF-II or IGF-II derived self-antigens is under current investigation. If further confirmed, the central tolerogenic properties of IGF-II could be considered in the elaboration of a strategy for an efficient and safe prevention of insulin-dependent diabetes.

Prohormone convertase-1 will process prorelaxin, a member of the insulin family of hormones.

Relaxin is a polypeptide hormone involved in remodeling of the birth canal during parturition. It is synthesized as a preprohormone precursor, which undergoes specific processing to form the mature two-chain disulfide-linked active species that is secreted by the cell. A major part of this processing requires endoproteolytic cleavage at specific pairs of basic amino acid residues, an event necessary for the maturation of a variety of important biologically active proteins, such as insulin and nerve growth factor. Human type 2 preprorelaxin was coexpressed in human kidney 293 cells with the candidate prohormone convertase-processing enzymes mPC1 or mPC2, both cloned from the mouse pituitary tumor AtT-20 cell line, or with the yeast kex2 alpha-mating factor-converting enzyme from Saccharomyces cerevisiae. Prorelaxin expressed alone in 293 cells was secreted into the culture medium unprocessed. Transient coexpression with mPC1 or kex2, but not with mPC2, resulted in the secretion of a low mol wt species with an electrophoretic mobility very similar, if not identical, to that of authentic mature relaxin purified from human placenta. This species was precipitable by monoclonal antibodies specific for relaxin and had a retention time on reverse phase HPLC comparable to that of relaxin. Its analysis by both electrospray and fast atom bombardment mass spectrometry generated mass data that were consistent only with mature relaxin. The basic residues required for mPC1-dependent cleavage of prorelaxin are defined by site-directed mutagenesis.

Effect of anti-relaxin antiserum on sperm motility in vitro.

Rabbit anti-relaxin antisera, but not normal rabbit sera, causes a rapid decline of motility of washed human sperm. Preincubation of the antisera with relaxin eliminates this effect. This sperm immobilization effect can serve as a basis of a rapid screening test for anti-relaxin antisera and as a novel adjuvant to barrier contraceptive methods.

Development of a radioimmunoassay for porcine relaxin using 125I-labeled polytyrosyl-relaxin.

Tyrosine was incorporated into highly purified porcine relaxin employing the reagent N-carboxy-L-tyrosine anhydride. The resulting polytyrosyl-relaxin contained 1.67 mol of tyrosine per mol of relaxin, retained its original biological activity, and was readily radioiodinated to specific activities ranging from 80 to 100 muCi per mug. High affinity antibodies applicable in final dilutions ranging from 1:50,000 to 1:200,000 were developed in rabbits against unconjugated highly purified porcine relaxin. A double antibody radioimmunoassay for porcine relaxin sufficiently sensitive to routinely measure from 32 to 1000 pg of pig relaxin was developed. Using this radioimmunoassay, peripheral serum concentrations of porcine relaxin were found to be less than 1 ng/ml during early pregnancy. Serum concentrations of porcine relaxin were high during late pregnancy. The mean concentration of porcine relaxin one day before parturition was 38 ng per ml. Within a day following parturition relaxin concentrations fell to a mean concentration of 2.1 ng per ml.

Monoclonal antibodies specific for rat relaxin. VII. Passive immunization with monoclonal antibodies throughout the second half of pregnancy prevents development of normal mammary nipple morphology and function in rats.

We recently demonstrated that relaxin-dependent development of the mammary nipples during the second half of pregnancy is required for pup survival during lactation in the rat. The two related objectives of this investigation were to 1) characterize the effects of endogenous relaxin on the histological modifications that normally occur in the mammary nipples, and 2) test the hypothesis that the cause of lactational failure in relaxin-deficient rats is attributable to failure of the nipples to grow and develop during the second half of pregnancy. Endogenous relaxin was neutralized by daily iv injection of a highly purified monoclonal antibody specific for rat relaxin (MCA1) to intact rats from days 12-22 of pregnancy. Mammary nipples were collected on day 22 of pregnancy and routinely prepared for light microscopy. Tissue cross-sections (6 microns) obtained from the midpoint of mammary nipples were stained with either Gomori's trichrome stain (to visualize collagen) or orcein (to visualize elastin). Nipple size as well as histological characteristics of nipple cross sections were determined by morphometric analysis. MCA1-treated rats were significantly different from controls with the following parameters: shorter length of the nipples; smaller cross-sectional areas of the entire nipple, lactiferous duct lumen, and blood vessels; greater percentage of the analysis field composed of collagen; lower percentage of the analysis field composed of amorphous ground substance; and longer elastin fibers. To test the hypothesis that the cause of lactational failure in relaxin-deficient rats is attributable to the failure of nipples to grow and develop, MCA1 and control rats were cesarean sectioned between 2100-2400 h on day 22 of pregnancy, and lactation was examined using normal foster pups from intact donor females. Unlike pups fostered to controls, pups fostered to MCA1-treated dams failed to grasp the nipples, stimulate PRL release, or have milk in their abdomens. This study demonstrates that endogenous relaxin promotes not only growth, but also modifications of the histological characteristics of the nipple that are consistent with relaxin's effects on the cervix and mammary glands. Additionally, this study provides evidence that lactational failure in relaxin-deficient rats is attributable to the small size and different histology of the mammary nipples, which results in the inability of the pups to attach to the nipple, stimulate PRL release, and obtain milk from the dams.

Monoclonal antibodies specific for rat relaxin. V. Passive immunization with monoclonal antibodies throughout the second half of pregnancy disrupts development of the mammary apparatus and, hence, lactational performance in rats.

There were two related objectives to this study. The first was to determine the influence of relaxin on development of the mammary apparatus (nipples and glands) during the second half of pregnancy. The second was to determine whether the relaxin-dependent development of the mammary apparatus was required for normal postpartum lactational performance. Both objectives were accomplished by neutralizing endogenous relaxin throughout the second half of pregnancy with a monoclonal antibody specific for rat relaxin (MCA1). MCA1 was administered iv to rats daily from days 12-22 of pregnancy. On day 22 the morphology of the mammary apparatus of MCA1-treated rats differed from that of controls; nipples were dramatically smaller, collagen fibers had significantly greater mean density and consistency, and elastin fibers had greater mean density, length, and interdigitation. In addition, the mean number of alveoli surrounding lactiferous ducts was significantly smaller in MCA1-treated rats than in controls. There were no differences between MCA1-treated rats and controls in the mean thickness of connective tissue surrounding ducts, the height or density of luminal cells lining lactiferous ducts, or the sizes of either adipocytes or arteries. To examine lactational performance, MCA1-treated and control rats were cesarean sectioned between 2100-2400 h on day 22 of pregnancy and given foster pups born of untreated intact donors. Although both MCA1-treated rats and control rats exhibited a high incidence of maternal behavior after cesarean delivery, mean pup weight and incidence of live pups declined markedly during days 1-5 of fosterage in MCA1-treated rats compared to controls. Furthermore, unlike controls, there was no observable postpartum nipple development in MCA1-treated rats by day 5 of fosterage. Mammary glands obtained from MCA1-treated rats on day 5 of fosterage had markedly lower mean weight than controls. This study demonstrates that passive immunization of endogenous relaxin throughout the second half of pregnancy disrupts development of the nipples and mammary glands in the rat. Moreover, it establishes that relaxin's effects on the development of the mammary apparatus during pregnancy are essential for growth and survival of the young during lactation.

Monoclonal antibodies specific for rat relaxin. IX. Evidence that endogenous relaxin promotes growth of the vagina during the second half of pregnancy in rats.

It is established that endogenous relaxin promotes the growth and development of the cervix, mammary glands, and nipples in pregnant rats. Additionally, the observation that porcine relaxin promotes growth of the vagina in both nonpregnant and pregnant rats provides indirect evidence that endogenous relaxin may effect growth of the vagina during rat pregnancy. The purpose of this study was to determine whether endogenous relaxin promotes growth of the vagina in pregnant rats. To that end, a monoclonal antibody, specific for rat relaxin, designated MCA1, was used to passively neutralize endogenous relaxin throughout the second half of pregnancy in intact rats. Five milligrams of highly purified MCA1 were injected iv to rats daily from days 12-22 of pregnancy. Controls received either a monoclonal antibody for fluorescein or PBS. The vaginal wet weight, dry weight, length, diameter, inner surface area, DNA content, and percent water content were determined. No differences were found between monoclonal antibody for fluorescein and PBS controls for any of the measured vaginal parameters. In contrast, values for all physical parameters, except percent water content, were significantly lower in MCA1-treated rats than in controls. Vaginal DNA content was also lower in MCA1-treated rats than in controls; and this observation supports the view that relaxin induces vaginal growth at least in part by promoting cell proliferation. To examine the mechanism of relaxin's apparent action on the vagina, specific relaxin-binding sites were localized immunohistochemically. Relaxin-binding sites were found in epithelial and smooth muscle cells, and the binding was specific for relaxin. We conclude that endogenous relaxin promotes growth of the vagina in pregnant rats.

Rabbit placental relaxin: ultrastructural localization in secretory granules of the syncytiotrophoblast using rabbit placental relaxin antiserum.

Although relaxin has been isolated from the placenta of the human, rabbit, horse, and cat, this study represents the first ultrastructural localization of the hormone in placental tissue. Placentas were removed from rabbits on days 15, 23, and 30 of pregnancy, and the tissues were prepared for light and electron microscopies. The cytoplasm of the syncytiotrophoblast from all stages of pregnancy studied showed positive staining for the hormone at the light level using guinea pig antirabbit relaxin serum and the avidin-biotin technique. Ultrastructurally, the syncytiotrophoblast was found to contain membrane-bounded granules (150-400 nm in diameter) which formed at the Golgi and were seen in close association with the cell membrane. Exocytosis involving the incorporation of the granule membrane into the cell membrane was observed. These granules labeled positively for relaxin after treatment with guinea pig antirabbit relaxin serum and goat antiguinea pig immunoglobulin G-colloidal gold. Control sections in which the relaxin antiserum was absorbed with purified rabbit relaxin or substituted with normal guinea pig serum contained no gold-labeled granules. Cross-reactivity of the rabbit relaxin antiserum with porcine relaxin was demonstrated by labeling of the relaxin-containing granules in the pregnant pig corpus luteum with the rabbit relaxin antiserum and by inhibiting the labeling of rabbit placental and pig corpora luteal granules by absorbing the rabbit relaxin antiserum with porcine relaxin. We have previously described the labeling of rabbit placental relaxin with porcine relaxin antiserum. This study suggests that relaxin is synthesized and secreted from the syncytiotrophoblast of the rabbit placenta, with the subcellular site of storage being membrane-bounded granules.

Neutralization of relaxin within the brain affects the timing of birth in rats.

Experiments were performed to determine whether neutralization of relaxin in the brain, by injecting monoclonal antibodies to rat relaxin into the ventricular system of the brain, affected either the timing or the processes of birth in rats. Pregnant rats were injected daily through a chronically implanted intracerebroventricular cannula either with a specific monoclonal antibody raised against rat relaxin from days 12-22 of gestation or with an antibody raised against fluorescein as a control. The rats were watched closely from the afternoon of day 20 of pregnancy, and the process of birth was observed. No sign of dystocia was observed in any of the rats in the experiment. Neutralization of endogenous relaxin caused a significant decrease in the length of gestation (505.4 +/- 3.1 h) compared with that in rats treated with PBS (524.6 +/- 0.5 h) or that in rats treated with a nonspecific antibody (525.9 +/- 0.7 h). The time to the onset of delivery was also shorter in the relaxin-neutralized group (507.8 +/- 1.1 h) compared with that in either PBS-treated (526.5 +/- 0.6 h) or fluorescein antibody-treated (525.3 +/- 0.7 h) animals. In contrast, there was no significant effect of the relaxin antibody on length of straining, duration of parturition, delivery interval, live birth rate, or body weight of the neonates. Premature delivery in the relaxin-neutralized group was accompanied by a 24-h advance in the fall in plasma progesterone. These data support the hypothesis that there may be a central relaxin system that is independent of the peripheral relaxin system. Central relaxin may have a significant physiological role on the timing of pregnancy in the rat, but does not affect the course of labor once it has started.

Rabbit placental relaxin: purification and immunohistochemical localization.

Rabbit placentas were extracted with 0.7 N HCl-acetone (3:5, vol/vol) containing protease inhibitors. Gel filtration (Sephadex G-50) followed by ion exchange chromatography (carboxymethyl cellulose) separated a bioactive relaxin-like fraction with a specific activity of 8.5 U/mg protein as determined by the in vitro mouse uterus bioassay. Column chromatography using Sepharose CL-4B in 6 M guanidine HCl was employed to purify the bioactive sample. The yield of the purified relaxin-like protein was 12 micrograms/g placenta and the specific activity was 23 U/mg protein. The bioactive sample was also immunoreactive after being electrophoretically transferred to nitrocellulose paper and stained using rabbit antiporcine relaxin serum and peroxidase-antiperoxidase immunochemistry. Isoelectrofocusing of the purified relaxin-like protein revealed one band with an isoelectric point of approximately 6.5. The apparent molecular weight of the rabbit relaxin was approximately 7200 as determined by sodium dodecyl sulfate-urea slab gel electrophoresis. Upon reduction with 5.0% mercaptoethanol and electrophoresis on sodium dodecyl sulfate-urea polyacrylamide gels, both the 7200 dalton rabbit immunoreactive relaxin-like polypeptide and porcine relaxin migrated as a lower molecular weight protein. These results suggest that rabbit relaxin, like pig, rat, shark, and human relaxin, consists of two chains linked by disulfide bonds. At the light microscopy level, immunohistochemical staining with guinea pig antiporcine relaxin serum indicated that relaxin was located in the syncytiotrophoblast cells of the placental labyrinth of day-23 and day-30 pregnant rabbits. The syncytiotrophoblast cells from day 16 of pregnancy did not stain for relaxin.

Production of antisera against electrophoretically separated relaxin and immunofluorescent localization of relaxin in the porcine corpus luteum.

Antisera to porcine relaxin were produced in rabbits injected with different fractions that had been separated by polyacrylamide gel electrophoresis (PAGE). Analyses by agar double immunodiffusion demonstrated that the different fractions of relaxin separated by PAGE have similar antigenic sites and the individual fractions are indistinguishable from one another by this procedure. Antiserum to porcine relaxin inhibited the interpubic ligament forming ability of the hormone in vivo. Indrict fluorescent antibody studies demonstrated that the hormone was localized only in the corpus luteum of the pregnant sow ovary. Large ovoid or polyhedral cells, assumed to be granulosa lutein cells, exhibited the heaviest fluorescence.

Monoclonal antibodies specific for rat relaxin. I. Production and characterization of monoclonal antibodies that neutralize rat relaxin's bioactivity in vivo.

The physiological role of relaxin during pregnancy and at parturition in the rat is not absolutely established. There are limitations to the experimental approach used in the few studies that examined the influence of relaxin in the pregnant rat. These studies were unphysiological, since they involved administration of porcine relaxin as well as progesterone and estrogen to ovariectomized pregnant rats. A more physiological approach is to use antibodies to neutralize the biological actions of endogenous relaxin in the intact pregnant rat. The purpose of the present study was to produce and characterize monoclonal antibodies suitable for this approach. Six stable and rapidly growing hybridoma clones which produced monoclonal antibodies specific for rat relaxin (MCA-rR) were obtained after the fusion of NSO mouse myeloma cells with lymphocytes from the spleen of a BALB/c mouse immunized with rat relaxin. Five MCA-rR (MCA1-5; all immunoglobulin G1 kappa) inhibited the ability of exogenously administered rat relaxin to increase the interpubic ligament length in estrogen-primed mice. Of the five MCA-rR that neutralized rat relaxin's bioactivity in vivo, MCA1 exhibited the highest relative affinity for rat relaxin. MCA1 was also highly specific for rat relaxin. MCA1 demonstrated no cross-reactivity with rat insulin, rat insulin-like growth factor I and II, or porcine relaxin-proteins that are structurally related to rat relaxin. In view of its high affinity and high specificity for rat relaxin as well as its ability to neutralize rat relaxin's bioactivity in vivo, MCA1 was selected for use in subsequent studies aimed at the neutralization of endogenous relaxin in intact pregnant rats.

Monoclonal antibodies specific for rat relaxin. II. Passive immunization with monoclonal antibodies throughout the second half of pregnancy disrupts birth in intact rats.

The purpose of this investigation was to use an approach targeted specifically on endogenous relaxin to determine the influence of relaxin on birth in the rat. To that end, a monoclonal antibody specific for rat relaxin, designated MCA1, was used to passively neutralize endogenous relaxin in intact pregnant rats. MCA1 was administered iv to intact rats daily from days 12-22 of pregnancy. Animals were observed for birth continuously from 2100 h on day 22 until 1200 h on day 24. MCA1-treated rats exhibited significantly prolonged durations of straining and litter delivery as well as reduced incidence of live pups compared with controls. Whereas approximately 20-25% of fetuses and placentae were retained in utero at 1200 h on day 24 in MCA1-treated rats, neither fetuses nor placentae were retained in control rats. Passive immunization with MCA1 throughout the second half of pregnancy had no apparent influence on normal ovarian function. The time of occurrence of the antepartum decline in serum progesterone levels (functional luteolysis) as well as the time interval between the attainment of basal progesterone levels and the onset of litter delivery in MCA1-treated and control rats that delivered on day 23 were in close agreement with previous reports. In conclusion, the prolonged durations of straining and litter delivery, reduced incidence of live pups, and increased incidence of retained fetuses and placentae after passive immunization with MCA1 establish the physiological need for endogenous relaxin to attain normal delivery of the young in the rat.

Monoclonal antibodies specific for rat relaxin. III. Passive immunization with monoclonal antibodies throughout the second half of pregnancy reduces cervical growth and extensibility in intact rats.

Relaxin is required for normal delivery in the rat. The mechanism(s) whereby relaxin contributes to rapid and safe delivery of the fetuses, however, has not been established. The purpose of this investigation was to determine if relaxin enables normal delivery by promoting the growth and modifying the tensile properties of the uterine cervix. To that end, a monoclonal antibody specific for rat relaxin, designated MCA1, was used to passively neutralize endogenous relaxin in intact pregnant rats. MCA1 or PBS vehicle was administered iv to rats daily from days 12-22 of pregnancy. Cervices were removed at 1200 h on day 22. Cervices obtained from MCA1-treated rats were much smaller and far less extensible than cervices obtained from control rats. Moreover, cervices from MCA1-treated rats were less able to accommodate stress created by extension than cervices from control rats. Passive neutralization of relaxin had no influence on 1) the weights of other reproductive tissues (uterus, placenta, and ovary), 2) the number of fetuses, and 3) the viability of fetuses. The present study indicates that in the rat endogenous relaxin is required for promoting cervical growth and softening during the second half of pregnancy. This work supports the hypothesis that the influence of endogenous relaxin on birth is attributable, at least in part, to its effects on the cervix.

Monoclonal antibodies specific for rat relaxin. X. Endogenous relaxin induces changes in the histological characteristics of the rat vagina during the second half of pregnancy.

This study employed morphometric analysis to evaluate changes in the histological characteristics that accompany relaxin-induced growth and softening of the vagina during the second half of rat pregnancy. There were three treatment groups (N = 4/group). Five milligrams of a monoclonal antibody for rat relaxin, designated MCA1, were injected i.v. daily on days 12-21 of gestation to treatment group MCA1. Control groups received either 5 mg of monoclonal antibody for fluorescein (MCAF; monoclonal antibody control) or 0.5 ml PBS (vehicle control). Vaginas were removed on day 22 of pregnancy, fixed in 10% neutral-buffered formalin, and embedded in paraffin. Tissue sections (5 microm) were stained with Gomori's trichrome to visualize collagen, or orcein to visualize elastin. Measurements were performed with a light microscope equipped with a video camera connected to a computer. Within the vaginal stroma, the density of collagen fiber bundles was lower, the length of elastin fibers was shorter, and the cross-sectional area and wall thickness of arteries were greater in relaxin-replete control rats than in relaxin-deficient MCA1-treated rats. These relaxin-induced changes in the stroma appear to account, at least in part, for the hormone's softening effect on the vagina. Within the epithelium, there were approximately 2-fold more basal and mucus-secreting cells in relaxin-replete control rats than in MCA1-treated rats. The relaxin-induced accumulation of epithelial cells appears to contribute to vaginal growth. We conclude that relaxin plays a role in preparing the vagina as well as the cervix for rapid and safe delivery in pregnant rats.

Monoclonal antibodies specific for rat relaxin. IV. Passive immunization with monoclonal antibodies during the antepartum period reduces cervical growth and extensibility, disrupts birth, and reduces pup survival in intact rats.

The purpose of this investigation was to use an approach targeted specifically on endogenous relaxin to determine the influence of antepartum (days 20-22) relaxin on cervical modifications and birth in the rat. To that end, a monoclonal antibody specific for rat relaxin, designated MCA1, was used to neutralize endogenous relaxin in intact pregnant rats. MCA1 or PBS vehicle was administered iv to intact rats daily from days 20-22 of pregnancy. Cervices were removed at 1200 h on day 22. Cervices obtained from MCA1-treated rats were less extensible than cervices obtained from PBS-treated control rats. Furthermore, wet weight, dry weight, water content, and uronate content were lower in cervices obtained from MCA1-treated rats than in cervices from PBS-treated controls. Birth and maternal behavior of MCA1-treated and PBS-treated control rats were observed continuously from 2100 h on day 22 until day 2 postpartum (d2PP). MCA1-treated rats exhibited significantly prolonged durations of litter delivery as well as reduced incidences of live pups on d2PP compared with controls. There were lower incidences of normal maternal behavior observed at birth and on d1PP with MCA1-treated rats than with control rats. In addition, little or no milk was observed in the abdomen of most live pups of MCA1-treated rats on d2PP, whereas abundant milk was observed in the abdomen of all live pups of control rats. The mean live pup weight on d2PP was lower in the litters of MCA1-treated rats than in control litters. The present study indicates that in the rat endogenous relaxin is needed during the antepartum period for normal cervical growth and extensibility, normal litter delivery, and high postpartum pup survival. This work supports the hypothesis that the influence of endogenous relaxin on birth is attributable, at least in part, to its effects on the cervix.

Monoclonal antibodies specific for rat relaxin. VIII. Passive immunization with monoclonal antibodies throughout the second half of pregnancy reduces water consumption in rats.

Recent studies demonstrated that exogenous relaxin promoted drinking in nonpregnant rats. The purpose of this investigation was to determine the influence of endogenous relaxin on water consumption in pregnant rats. To that end, a monoclonal antibody specific for rat relaxin, designated MCA1, was used to passively neutralize endogenous relaxin throughout the second half of pregnancy in intact rats. Five milligrams of highly purified MCA1 were administrated iv to rats daily from days 12-22 of pregnancy. Controls received either a monoclonal antibody for fluorescein (monoclonal antibody control) or PBS (vehicle control). The amount of water consumed and both the total duration of water consumption and the total number of episodes when water was consumed were determined daily during both dark and light periods for all treatment groups. From days 13-22 of pregnancy, all three of these parameters of water consumption increased during the 10-h dark period (P < 0.01), but not during the 14-h light period. The mean daily water consumption in MCA1-treated rats was significantly less than that in controls (P < 0.05). Relaxin's effects on water consumption were limited to the 14-h light period (P < 0.01). No difference was found in daily water consumption between the MCA1-treated and control groups during the 10-h dark period. There was a tendency during the light period for both the total duration of water consumption (P = 0.06) and the total number of episodes when water was consumed (P = 0.13) to be less in MCA1-treated rats than in controls. Food consumption and body weight increased as pregnancy progressed, but no differences were found among the three treatment groups. We conclude that endogenous relaxin has effects on water consumption. It promotes water consumption during the daily light period in the second half of pregnancy in rats. Thus, relaxin may be a dipsogenic agent.

Relaxin-dependent adenosine 6',5'-monophosphate concentration changes in the mouse pubic symphysis.

The amount of cAMP in the pubic symphyses of estrogen-primed mice increases after injection of the hormone relaxin. This relaxin-induced increase in symphyseal cAMP was observed in immature, mature, and ovariectomized mature mice and could be prevented by prior ip injection of rabbit antibodies to porcine relaxin or by treatment of relaxin with dithiothreitol. The highest level of cAMP was measured 30 min after relaxin injection; the level of measurable cMAP then diminished rapidly. Estrogen-priming of the mice was not a prerequisite for a relaxin-induced response to occur. Relaxin administration did not increase the level of cAMP in a non-target tissue such as liver, nor could an increase in cAMP in the pubic symphysis be elicited by injection of insulin, a protein of similar size and structure, or glucagon, a known stimulator of liver cAMP levels.