Relaxin is a potent renal vasodilator in conscious rats.

The kidneys and other nonreproductive organs vasodilate during early gestation; however, the "pregnancy hormones" responsible for the profound vasodilation of the renal circulation during pregnancy are unknown. We hypothesized that the ovarian hormone relaxin (RLX) contributes. Therefore, we tested whether the administration of RLX elicits renal vasodilation and hyperfiltration in conscious adult, intact female rats. After several days of treatment with either purified porcine RLX or recombinant human RLX 2 (rhRLX), effective renal plasma flow (ERPF) and glomerular filtration rate (GFR) increased by 20%-40%. Comparable renal vasodilation and hyperfiltration was also observed in ovariectomized rats, suggesting that estrogen and progesterone are unnecessary for the renal response to rhRLX. The nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester completely abrogated the increase in ERPF and GFR elicited by chronic administration of purified porcine RLX. In contrast, the renal vasoconstrictory response to angiotensin II was attenuated by the RLX treatment. Short-term infusion of purified porcine RLX to conscious rats over several hours failed to increase ERPF and GFR. Plasma osmolality was consistently reduced by the chronic administration of both RLX preparations. In conclusion, the renal and osmoregulatory effects of chronic RLX administration to conscious rats resemble the physiological changes of pregnancy in several respects: (a) marked increases in ERPF and GFR with a mediatory role for nitric oxide; (b) attenuation of the renal circulatory response to angiotensin II; and (c) reduction in plasma osmolality.

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.

The extent to which relaxin promotes proliferation and inhibits apoptosis of cervical epithelial and stromal cells is greatest during late pregnancy in rats.

Relaxin promotes marked growth of the cervix during the second half of rat pregnancy, and this growth is accompanied by an increase in both epithelial and stromal cells. The objective of this study was to test the hypothesis that the extent to which relaxin promotes proliferation and inhibits apoptosis of cervical cells is greatest during late pregnancy in rats. The influence of neutralization of circulating relaxin by iv injection of 5 mg monoclonal antibody against rat relaxin (MCA1) was examined at 3-d intervals throughout the second half of pregnancy. Controls were injected with either 5 mg monoclonal antibody against fluorescein or 0.5 ml PBS vehicle. To evaluate cell proliferation, 5'-bromo-2-deoxyuridine was injected sc 8 h before cervixes were collected. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling and electron microscopy were used to detect apoptotic cells. Neutralization of relaxin with MCA1 decreased the rate of proliferation and increased the rate of apoptosis of cervical cells by d 13. However, the extent to which relaxin influenced these processes was greatest and dramatic by late pregnancy. In MCA1-treated rats on d 22 of pregnancy, the rates of proliferation of both epithelial and stromal cells were less than 20% those in controls, and the rates of apoptosis in epithelial cells and stromal cells were more than 10- and 3-fold, respectively, greater than those in controls. In conclusion, this study provides evidence that the extent to which relaxin promotes proliferation and inhibits apoptosis of cervical epithelial and stromal cells is greatest during late pregnancy.

Porcine and human relaxin bioactivity: bioactivities of porcine relaxin and human relaxin do not differ in mice and rats.

This study compares the bioactivity of porcine relaxin-1 to that of recombinant human relaxin-2 in mice and rats. The effects of the two hormone preparations on elongation of the mouse interpubic ligament and both the wet weight and the extensibility of the rat cervix were compared. No difference in bioactivity was detected between porcine relaxin-1 and recombinant human relaxin-2 in either rodent. Therefore, decisions concerning which of the two available forms of relaxin to employ for in vivo experimentation in mice and rats can be made without concerns about relative bioactivity.

Clinical use of relaxin to facilitate birth: reasons for investigating the premise.

In the United States, both medical and nonmedical factors have driven the cesarean section rate to over 26% of all deliveries. In addition to questions of increased cost associated with operative delivery, some have questioned the ethics of performing cesarean section for nonmedical reasons. Reduction of both the duration and the pain associated with vaginal delivery would likely bring about a decline in the rate of both medical and nonmedical cesarean sections. This chapter summarizes recent findings that support the premise that through its growth-promoting and softening effects on the cervix, short-term subcutaneous administration of pharmacologic amounts of relaxin to women at term holds promise as a means of reducing the duration and discomfort associated with delivery. Two recent studies conducted in pregnant rats demonstrated that the cervix is highly responsive to relaxin during the antepartum period and that short-term subcutaneous administration of the hormone to relaxin-deficient animals not only promotes growth and softening of the cervix, but also reduces the duration of labor and delivery. Moreover, recent human clinical trials examining the influence of 24 weeks of continuous subcutaneous administration of recombinant human relaxin for the treatment of scleroderma provided evidence not only that the human reproductive tract is responsive to relaxin, but also that the administration of the hormone does not cause serious adverse side effects. It is concluded that recent findings provide an impetus for an investigation into relaxin's potential for cervical remodeling and facilitating birth in women.

Purification and characterization of rat relaxin.

Two highly purified forms of relaxin, designated CM1 and CM2, were obtained from rat ovaries collected on day 20 of gestation. The isolation procedure consisted of aqueous extraction, followed by fractionation with Sephadex G-50 and ion exchange chromatography. The yields of CM1 and CM2 were approximately 140 microgram/geq ovarian fresh tissue. No difference in biological potency between CM1 and CM2 was found when they were bioassayed with mouse pubic symphysis bioassays. Physicochemical analyses indicated that CM1 and CM2 were similar but not identical. The molecular weights of CM1 and CM2 were approximately 6000, as determined by ultracentrifugation. Analytical acrylamide disc gel electrophoresis at pH 4.3 demonstrated that CM1 and CM2 had different electrophoretic mobilities. Electrofocusing indicated the isoelectric points of CM1 and CM2 were pH 7.6 and pH 9.4, respectively. The amino acid compositions of CM1 and CM2 were similar but not identical. Slab gel electrophoresis in polyacrylamide gel with sodium dodecyl sulfate showed that both reduced rat relaxin and reduced porcine relaxin migrated farther than their unreduced forms. This observation supports the view that rat relaxin, like porcine relaxin, consists of two chains linked by disulfide bonds.

Effects of relaxin on in vivo uterine contractions in conscious and unrestrained estrogen-treated and steroid-untreated ovariectomized rats.

The effect of extended infusions of highly purified porcine relaxin on uterine activity was tested in vivo in conscious unrestrained rats. Relaxin was found to inhibit oxytocin- and prostaglandin F2 alpha-induced uterine contractions in estrogen-treated ovariectomized rats as well as spontaneous contractions in both estrogen-treated and steroid-untreated ovariectomized rats. The inhibition of both induced and spontaneous uterine contractions was more effective in the early phases of relaxin infusion than after prolonged exposure to the hormone. However, this desensitization was not complete, and the inhibition was effective even with extended (up to 72 h in the oxytocin experiments) infusion of relaxin. The results indicate that relaxin may be important in controlling uterine activity in the rat near the end of gestation, and that estrogen priming in ovariectomized rats is not necessary for relaxin to exert its biological effects.

Immunohistochemical localization of specific relaxin-binding cells in the cervix, mammary glands, and nipples of pregnant rats.

Previously, we demonstrated that endogenous circulating relaxin promotes the growth and softening of the cervix, the development of the mammary glands, and the growth and development of nipples. Due to the remarkably similar modifications in the histological appearance of the extracellular matrix in the cervix, mammary glands, and nipples, we hypothesized that there may be a common mechanism(s) of action of relaxin in these tissues. A fundamental step toward understanding this mechanism is to identify specific cells that contain relaxin receptors, that is to identify those cells that initiate relaxin's effects within relaxin target tissues. To identify specific relaxin-binding cells in the cervix, mammary glands, and nipples of the pregnant rat, a biologically active biotinylated relaxin probe was prepared. This probe for putative relaxin receptors was administered to intact rats on day 18 of pregnancy. After 1 h, the animals were killed, and tissues were fixed by immersion in 4% paraformaldehyde for 10 h. Fixed tissues were rinsed in 0.1 M phosphate buffer (pH 7.4) and cryoprotected in an ascending series of 5%, 10%, and 20% sucrose solutions. The tissues were frozen in Tissue-Tek O.C.T. compound and stored at -70 C until sectioning. Frozen sections (12 microns) were cut on a Tissue Tek II cryostat at -24 C and thaw mounted on slides coated with 0.01% poly-l-lysine (mol wt, 300-6000). The biotinylated relaxin was localized in cryosections with an antibiotin immunoglobulin G conjugated to colloidal gold, which was subsequently visualized for light microscopy with silver intensification. Specific binding of the biotinylated relaxin was localized in the epithelial and smooth muscle cells of the cervix, the epithelial cells of the mammary glands, and the epithelial cells, smooth muscle cells, and skin of the nipples. We conclude that those cells exhibiting specific relaxin binding probably contain relaxin receptors and, therefore, mediate relaxin's effects in these tissues. As relaxin bound specifically to epithelial cells in the cervix, mammary glands, and nipples, we postulate that the epithelial cells may initiate a common mechanism of action that brings about modifications of the extracellular matrix in all three tissues.

Evidence that cellular proliferation contributes to relaxin-induced growth of both the vagina and the cervix in the pregnant rat.

It is well established that cervical growth during rat pregnancy is relaxin dependent. The first objective of this study was to determine if relaxin also promotes vaginal growth in the pregnant rat. Finding that this is the case, the second objective of this study was to determine if cell proliferation accompanies relaxin-dependent vaginal and cervical growth during rat pregnancy. Primiparous pregnant rats were ovariectomized (O) or sham ovariectomized (group C) on day 9 (D9) of pregnancy, before relaxin (R) is detectable in the peripheral circulation. After ovariectomy, rats were treated continuously with progesterone (P) and estrogen (E, group OPE), or P, E, and porcine R (group OPER) in doses that restored normal pregnancy and parturition parameters. P and E were administered via silicon tubing implants. R was administered from miniature osmotic pumps. Vaginas and cervices were collected on D9 and D22 from group C, and on D22 from groups OPE and OPER (n = 6/group). Vaginas and cervices were weighed, frozen, and lyophilized until dry. Dried tissues were weighed, homogenized, and their DNA contents were determined. In sham-operated controls (group C), the wet weight, dry weight, and DNA contents of both the vagina and cervix increased 50-300% from D9-D22. On D22, vaginal and cervical wet and dry weights were significantly lower than controls in R-deficient group OPE; whereas, they were greater than controls in group OPER. Similarly, on D22, vaginal and cervical DNA content did not differ from D9 controls in group OPE; whereas they exceeded D22 controls in group OPER. In conclusion, this study demonstrates that vaginal growth during the second half of rat pregnancy is R dependent. Additionally, this study provides evidence that R may contribute to both vaginal and cervical growth by promoting cellular proliferation.

The physiological role of relaxin in the pregnant rat. I. The influence of relaxin on parturition.

Length of gestation, duration of labor and delivery, and fetal survival were determined in control intact pregnant rats (group C). Pregnant rats were bilaterally ovariectomized on day 9 and given progesterone (P) implants and, in addition, one of the following injection regimens; estrogen (E; group OPE), E and porcine relaxin (R; group OPER), or porcine R (group OPR). Hormone treatments were given in doses designed to produce serum levels of these hormones similar to those observed in intact pregnant rats. The P implants were removed during the evening of day 21 to mimic the decline in serum P levels that normally occurs as a result of luteolysis. Animals in groups OPE and OPR exhibited significantly prolonged gestation, prolonged duration of labor and delivery, and reduced fetal survival compared with controls. Group OPER animals exhibited slightly but not significantly shorter length of gestation, similar duration of labor and delivery, and similar rate of fetal survival compared to control values. Group OPER animals showed normal maternal behavior and were able to suckle their young.

The physiological role of relaxin in the pregnant rat. II. The influence of relaxin on uterine contractile activity.

In control intact rats (group C), the frequency of intrauterine pressure cycles (IUPC) declines steadily during pregnancy from 80-130 cycles/3 h on day 10 (D10) to 20-30 cycles/3 h on D20. The decline in frequency is due to increasingly prolonged periods of myometrial quiescence, which increase from 30-90 min/3 h on D10 to 120-150 min/3 h by D20. During the 24 h preceding labor, the frequency of IUPC remains at less than 15 cycles/h until 3 h prepartum, when there is an abrupt increase to 30 cycles/h. Ovariectomized pregnant animals treated with progesterone (P) and estrogen (E; group OPE) exhibit significantly greater frequency of IUPC and significantly lower incidence of myometrial quiescence than intact pregnant rats from D12 and for the remainder of pregnancy. Ovariectomized pregnant rats that received P, E, and porcine relaxin (R: group OPER) or P and porcine R only (group OPR) exhibited declining frequency of IUPC similar to that observed in group C animals. Group OPER rats exhibited prolonged periods of myometrial quiescence of similar duration to those observed in control intact rats. In group OPR animals, however, the periods of myometrial quiescence were considerably diminished during late pregnancy. Group OPER animals exhibited a pattern of myometrial activity during labor and postpartum similar to that of control animals. The results suggest an important role for R in the control of myometrial activity during the second half of pregnancy and parturition in the rat.

The physiological role of relaxin in the pregnant rat. III. The influence of relaxin on cervical extensibility.

Cervices from day 18 (D18) intact pregnant rats show significantly greater extensibility and ability to accommodate to extension than cervices from D9 pregnant rats. On D22, cervices from intact pregnant rats show even greater extensibility and accommodation to stretch. Cervices from ovariectomized pregnant rats treated with estrogen (E) and progesterone (P; group OPE) show markedly reduced extensibility and ability to accommodate to stretch compared with intact pregnant cervical tissue (group C) on both D18 and D22. Extensibility of group OPE cervices resembles that of cervices from D9 intact pregnant rats. Cervices from ovariectomized pregnant rats treated with E, porcine relaxin (R), and P (group OPER) or porcine R and P (group OPR) exhibit similar extensibility and ability to accommodate to stretch as cervices from intact pregnant rats on both D18 and D22. The importance of R for cervical softening during pregnancy and its interaction with E near term and during parturition are discussed.

Development of a homologous radioimmunoassay for rat relaxin.

Highly purified rat relaxin has been radioiodinated to specific activities of approximately 100 micro Ci/microgram with the Bolton and Hunter reagent [N-succinimidyl 3-(4-hydroxy-5-[125I]iodophenyl) propionate]. A rabbit antirat relaxin serum, applicable in a final dilution of 1:100,000, was developed in a rabbit using unconjugated highly purified rat relaxin. A specific and precise double antibody RIA for rat relaxin sufficiently sensitive to routinely measure from 32--2000 pg rat relaxin was developed. Using this RIA, relaxin immunoactivity levels in extracts of pregnant rat ovaries were found to rise from 0.8 microgram/geq ovarian fresh tissue on day 8 of pregnancy to 723 microgram/geq ovarian fresh tissue on day 20 of pregnancy and then to drop precipitously to 6 microgram/geq ovarian fresh tissue on day 1 of lactation. Consistent with the occurrence and relative levels of relaxin in the ovarian extracts, levels of relaxin in pregnant rat serum were less than 2 ng/ml on day 10 of pregnancy, approximately 150 ng/ml on days 20 and 22 of pregnancy, and 12 ng/ml on day 2 of lactation.

Control of corpus luteum function during the second half of pregnancy in the rat: a direct relationship between conceptus number and both serum and ovarian relaxin levels.

The effects of conceptus or fetus removal on several parameters of luteal activity (serum and ovarian relaxin immunoactivity levels, serum progesterone immunoactivity levels, and corpus luteum weights) were investigated during the second half of pregnancy in the rat. Conceptuses were aspirated from the uteri of pregnant rats on day 8 (d8) of pregnancy so that they carried 0, 1, 2, 5, or 10 or more conceptuses until they were autopsied on d10, d12, d14, d16, d18, or d20 of pregnancy. A direct relationship existed between the number of conceptuses and the rate and/or degree of increase in all of the parameters of corpus luteum activity. The conceptuses had no local effect on ovarian relaxin levels or on luteal weights when the conceptus side ovary was compared with the nonconceptus side ovary. In pregnant rats in which all but two fetuses were removed (all placentas were left undisturbed), there were no significant differences on d18 or d20 in any parameter of corpus luteum activity between fetectomized and sham-operated rats. It appears, therefore, that the placenta plays the dominant role in the regulation of all of the above parameters of corpus luteum activity during the second half of pregnancy in the rat. The specific factor(s) or mechanism(s) by which the placenta promotes relaxin synthesis, progesterone secretion, and corpus luteum growth have yet to be clearly and unequivocally characterized in the rat. The fact that conceptus number and the rate and/or degree of increase of all of the parameters of luteal activity measured from d12 through d18 are directly related is consistent with the possibility that the placental support of these activities is mediated through a common mechanism during this period in the rat.

The physiological role of relaxin in the pregnant rat. IV. The influence of relaxin on cervical collagen and glycosaminoglycans.

Changes in the collagen and glycosaminoglycan components of cervical connective tissue were studied in nonpregnant, intact pregnant, and ovariectomized hormone-treated pregnant rats. Collagen concentration and solubility and the concentration of the glycosaminoglycans dermatan sulfate (DS), heparan sulfate, and hyaluronic acid (HA) in cervices taken from day 9 (D9) pregnant rats were similar to those in cervices from nonpregnant rats. In cervical tissue from late pregnant intact control rats on D18 and D22, the collagen concentration decreased, collagen solubility increased, and there was no significant change in total glycosaminoglycan concentration. In cervices from ovariectomized pregnant rats treated with progesterone and estrogen, collagen and glycosaminoglycan parameters resembled those of D9 and nonpregnant cervices on both D18 and D22. However, treatment of ovariectomized pregnant rats with progesterone, estrogen, and porcine relaxin (R) restored cervical collagen concentrations to those of intact controls on both D18 and D22. On D18 of pregnancy, cervical collagen solubility was partially increased by R treatment, and by D22, it was similar to that of intact D22 pregnant controls. R treatment also resulted in a significantly increased cervical concentration of HA. It is concluded that the decrease in collagen concentration, increase in collagen solubility, and increase in HA concentration resulting from R action may contribute in part to the increased extensibility of the cervix that occurs during late pregnancy 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.

Relaxin increases the accumulation of new epithelial and stromal cells in the rat cervix during the second half of pregnancy.

Both cervical and vaginal growth are relaxin dependent during rat pregnancy. We recently reported a relaxin-dependent 1.5-fold increase in cervical and vaginal DNA content from midpregnancy until term. This finding indicated that relaxin probably promotes cervical and vaginal growth at least in part by promoting cellular proliferation. The objective of this study was to identify and quantify cells in the cervix and vagina that proliferate during the second half of rat pregnancy in response to relaxin. Primiparous pregnant rats were ovariectomized or sham ovariectomized (group C; n = 8) on day 9 of pregnancy (D9). Ovariectomized rats were then treated with physiological doses of progesterone plus estrogen (n = 7) or progesterone, estrogen, and porcine relaxin (n = 7). Cellular proliferation was determined by continuously administering a low dose of 5-bromo-2'-deoxyuridine (BrdU) via miniature osmotic pump from D9-D22. On D22, cervices and vaginas were collected, fixed in formalin, paraffin embedded, and serially sectioned (4 microm). Adjacent serial sections were either immunostained for BrdU to assess cell proliferation or stained with hematoxylin to determine total cell number. Cell proliferation was evaluated by counting BrdU-positive nuclei and total nuclei in the same area on adjacent sections. Cell counts were determined using computerized digital morphometric analysis at x575. In control rats, nearly 75% of the epithelial cells and 55% of the stromal cells within the cervix at term had proliferated during the second half of pregnancy. The accumulation of approximately half of the new cells was relaxin dependent. Within the cervical stroma, relaxin increased the accumulation of cells associated with blood vessels and also the number of isolated cells (probably fibroblasts). Relaxin did not appear to affect smooth muscle cell proliferation in the cervix. In contrast to the cervix, a minority of vaginal epithelial cells (45%) and stromal cells (20%) proliferated during the second half of pregnancy. Although relaxin appeared to have a tendency to increase the accumulation of new vaginal epithelial and stromal cells, morphometric analysis did not provide support for such an effect. In conclusion, this study demonstrates that relaxin promotes a marked increase in the accumulation of new epithelial cells and stromal cells within the cervix. The relaxin-induced increase in new epithelial and stromal cells probably contributes to relaxin's effects on growth and remodeling of the cervix that are required for rapid and safe delivery.

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.

Effect of preparturient 17 beta-estradiol and relaxin on parturition and pup survival in the rat.

This study examined the requirement for 17 beta-estradiol (E) and relaxin (R) during the preparturient period (approximately the last 30 h of gestation) for normal parturition and pup survival in the rat. On the evening of day 21 of pregnancy, primiparous Sprague-Dawley rats were bilaterally ovariectomized or sham operated and fitted with Silastic capsules containing one of four doses of E (6.0, 11.4, 45.6, or 68.4 micrograms in sesame oil) or sesame oil only. These rats were also injected sc with either 200 micrograms highly purified porcine R dissolved in 200 microliter 5% beeswax in corn oil or R vehicle three times (approximately 8 to 10-h intervals) beginning at ovariectomy. The durations of straining and delivery were increasingly prolonged with increasing doses of E in the ovariectomized groups that received E but no R. In rats that received the two highest doses of E there was a reduction in the percentage of pups born alive and a reduction in pups surviving on day 2 postpartum, and one or two undelivered fetuses were retained in utero in some animals 3 days postpartum. The administration of R with the two highest doses of E restored all parturition parameters to values not different from those of intact controls. In the absence of both E and R, all measured parturition parameters were abnormal, and administration of R in the absence of E only partially restored them to control values. Two major conclusions are drawn. First, E treatment alone to pregnant rats ovariectomized on day 21 fails to elicit normal parturition; indeed, with increasing doses of E, parturition parameters and pup survival become increasingly adversely affected. Second, R treatment of E-treated ovariectomized rats during the immediate preparturient period restores parturition parameters and pup survival to the levels of intact sham-operated controls.

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.