The role of RXFP2 in mediating androgen-induced inguinoscrotal testis descent in LH receptor knockout mice.

LH receptor knockout (LhrKO) male mice exhibit a bilateral cryptorchidism resulting from a developmental defect in the gubernaculum during the inguinoscrotal phase of testis descent, which is corrected by testosterone replacement therapy (TRT). In vivo and in vitro experiments were conducted to investigate the roles of the androgen receptor (AR) and RXFP2 signals in regulation of gubernacular development in LhrKO animals. This study demonstrated that AR and RXFP2 proteins were expressed in the gubernaculum during the entire postnatal period. TRT normalized gubernacular RXFP2 protein levels inLhrKO mice. Organ and primary cell cultures of gubernacula showed that 5alpha-dihydrotestosterone (DHT) upregulated the expression of Rxfp2 which was abolished by the addition of an AR antagonist, flutamide. A single s.c. testosterone injection also led to a significant increase in Rxfp2 mRNA levels in a time-dependent fashion in LhrKO animals. DHT, natural and synthetic insulin-like peptide 3 (INSL3), or relaxin alone did not affect proliferation of gubernacular mesenchymal cells, while co-treatments of DHT with either INSL3 or relaxin resulted in an increase in cell proliferation, and they also enhanced the mesenchymal cell differentiation toward the myogenic pathway, which included a decrease in a mesenchymal cell marker, CD44 and the expression of troponin. These effects were attenuated by the addition of flutamide, siRNA-mediated Rxfp2 knockdown, or by an INSL3 antagonist. Co-administration of an INSL3 antagonist curtailed TRT-induced inguinoscrotal testis descent in LhrKO mice. Our findings indicate that the RXFP2 signaling pathway plays an important role in mediating androgen action to stimulate gubernaculum development during inguinoscrotal testis descent.

Synthesis and conformational analysis of the insulin-like 4 gene product.

Insulin-like 4 (INSL-4) is a protein expressed in the early placenta. Its primary structure is insulin-like with reference to the distribution of cysteine residues and the single chain pro-form. Insulin-like 4 was generated by solid-phase peptide synthesis of the two chains followed by the sequential synthesis of the three disulfide bonds. Two disulfide isomers were produced, one with an insulin-like disulfide bonding pattern and the other with a reversed chain orientation. The CD spectra of the two disulfide isomers were indistinguishable without any features produced by periodic structures. In addition, the hydrodynamic properties of the two isomers were identical which implied a very open structure of the disulfide-bonded two-chain molecules. It appears that insulin-likeness cannot be defined solely on the basis of the primary structure of cDNA.

[Omphalocele and gastroschisis. Outcome--complications--follow-up--quality of life]. / Omphalocele und Gastroschisis. Ergebnisse--Komplikationen--Verlauf--Lebensqualität.

From 1970 to 1998, 35 children with omphalocele (OC) and 31 with gastroschisis (GS) were treated at the Department of Paediatric Surgery at Lübeck Medical University. Forty of 43 survivors were examined in 1990, the data of 30 patients were renewed in 1999 and 12 new cases added. Total follow-up was 1-28 years. Primary closure was possible in 25 OCs and 20 GSs. Eighteen children with OC and 8 with GS suffered from additional abnormalities, which were treated simultaneously. Twenty percent of the babies with OC died mostly because of severe congenital anomalies and 12.9% of GS because of infectious complications in combination with other diseases. There were no more deaths in the last decade. Accordingly, there was a reduction in consecutive operations. Improvements were due to better operative and perioperative treatment as well as abortions following improved ultrasound examinations. The results of the literature and our own experience show the benefit of primary closure. A two-stage approach with dura/amnion or a silo procedure prevents high intra-abdominal pressure, therefore, indirect measurements of intra-abdominal pressure can be used exceptionally. Umbilical preservation offers better cosmetic results. Long-term follow-up reveals normal growth and development of the children except for those with severe congenital anomalies. All the others are participating without problems in normal activities and education without reduction in their quality of life. Today an isolated OC or GS is not an indication for abortion. If prenatal OC or GS is diagnosed, paediatric surgeons should be involved in the consultations.

The relaxin receptor-binding site geometry suggests a novel gripping mode of interaction.

Relaxin has a unique, clearly identifiable, mixed function receptor-binding region comprising amino acid residues that evolve sequentially from the central portion of the B chain alpha-helix. Two arginine residues in positions B13 and B17 that project like forefinger and middle finger from the helix provide the electrostatic element opposed by the hydrophobic (thumb) element isoleucine (B20), offset from the arginines by about 40 degrees. The binding intensity of relaxin to its receptor decreases by 3 orders of magnitude if alanine is substituted for the newly discovered binding component isoleucine in position B20. The arginine residues cannot be replaced by other positive charges, nor can the guanidinium group be presented on a longer or shorter hydrocarbon chain. In contrast, the hydrophobic interaction is incremental in nature, and the contribution to the total binding energy is roughly proportional to the number of hydrocarbon units in the side chain. It appears that a hydrophobic surface exists on the receptor that offers optimal van der Waals' interaction with beta-branched hydrophobic amino acids. The binding energy increases roughly 10-fold with each methylene group whereby beta-branching is more effective per surface unit than chain elongation. Aromatic side chains appear to demarcate the extent of the binding region in so far as residues larger than phenylalanine decrease receptor binding. The exceptional clarity of binding site geometry in relaxin makes for an excellent opportunity to design peptido-mimetics.

Porcine relaxin, a 500 million-year-old hormone? the tunicate Ciona intestinalis has porcine relaxin.

The fossil record of tunicates reaches back to the upper Cambrian period. Ascidians have mobile, tadpole-like juvenile forms with a notochord, which inspired the classification of tunicates as Urochordata, i.e., predecessors of vertebrates. The genome of the tunicate Ciona intestinalis contains a relaxin coding region that is organized like a mammalian gene, i.e., signal peptide, B-chain domain, connecting peptide domain, followed by the A-chain domain with a stop codon after cysteine A-22. RNA-derived cDNA encodes a relaxin that is identical to the circulating form of the porcine hormone. In contrast to the porcine gene, the ascidian gene has no intron in the C-peptide domain, and in that respect is similar to the bombyxin gene of the silkworm. During the spawning period, only enough relaxin could be extracted and isolated from gonads of C. intestinalis for a partial sequence analysis. Remarkable as it may be, these findings suggest that relaxin is identical in pigs, whales, and the tunicate C. intestinalis.

Antiporcine relaxin (antipRLX540) treatment decreases relaxin plasma concentration and disrupts delivery in late pregnant pigs.

Antibody against porcine relaxin (antipRLX540; 1:950,000) was produced in sheep and used to determine the effect on relaxin and progesterone secretion, and on parturition in late pregnant pigs. In group 1, Yorkshire gilts with normal estrous cycles were bred on the second observed estrus and fitted with an indwelling jugular cannula and an intraperitoneal cannula on day 100 of pregnancy. Gilts were infused at 6-h intervals with antipRLX540 (n = 10) or PBS (n = 10) beginning on day 103 until parturition. From days 103 to 120, daily blood samples (10 ml) were collected for RIA of relaxin, progesterone, and prolactin. In group 2, bred gilts were randomly assigned to antipRLX540 (n = 11), relaxin (n = 5), and PBS (n = 8) treatment on days 111, 113, and 115. Blood was collected twice daily from day 108 to 120, and every 20 min on days 111, 113, and 115 beginning 60 min before treatment and continuing 180 min. Parturition in gilts given antipRLX540 occurred on day 112.7 compared with day 114.0 in relaxin-treated gilts and day 114.3 in PBS controls (P < 0.05). Duration of delivery from first to last piglet was greatly delayed in antipRLX540 gilts (240 min) compared with PBS controls ([117 min] P < 0.005). Average number of stillborns was greater in antipRLX540- than in PBS-treated controls (2.4 vs. 1.0; P < 0.05). Relaxin concentration in peripheral plasma was lower in antipRLX540-treated gilts from day 105 to 110, but on day 113 the antipRLX540-treated group had a greater relaxin peak release compared with PBS-treated animals (P < 0.05). Plasma progesterone concentrations were similar in antipRLX540- and PBS-treated gilts throughout the period of the study. In group 2, by day 113, progesterone decreased in antipRLX540-treated gilts compared with relaxin- and PBS-treated gilts. Prolactin levels were similar in both antipRLX540- and PBS-treated gilts; however, from 1 to 3 days postpartum the antipRLX540 group had higher prolactin concentration (P < 0.05). The results indicate that antipRLX540 decreased circulating plasma concentrations of unbound or free relaxin during the last 10 days of pregnancy in Yorkshire gilts. AntipRLX540 markedly increased both the duration of delivery of piglets and the average number of stillbirths in this litter-bearing species compared with PBS-treated controls. This study provides strong evidence that increasing circulating concentrations of relaxin during late pregnancy is crucial for unimpaired parturition in the pig.

Dogfish shark (Squalus acanthias) testes contain a relaxin.

Relaxin is a 6-kd polypeptide that exerts important hormonal effects in many female mammals. Relaxin is produced by the ovary, placenta, or uterus in many mammalian species. The functions of relaxin in the male mammal are not yet firmly established, but there is some evidence suggesting an exocrine effect on sperm motility and fertilizability. In the male mammals that have been studied, relaxin is produced by the prostate gland (human) or seminal vesicles (boar). However, in the bird, the testis is the likely source of relaxin. Among the elasmobranchs, ovaries obtained from dogfish sharks have been shown to contain a polypeptide hormone that is structurally, biologically, and immunologically similar to mammalian relaxins, but the male reproductive tract of this species has not previously been investigated as a potential source of relaxin. Extracts of testes obtained from mature dogfish sharks have now been tested by a specific relaxin bioassay and by a homologous porcine radioimmunoassay for the presence of relaxin. Both crude and partially purified testicular extracts contained unmistakable guinea pig pubic symphysis-"relaxing" activity and relaxin-like immunoactivity. Following immunoaffinity purification, the shark testis polypeptide had an apparent specific activity of 88 microg porcine relaxin equivalents per milligram in the radioimmunoassay, which is similar to the immunoactivity of pure shark ovarian hormones. These data, therefore, strongly support the view that in dogfish sharks, the male as well as the female gonad produces relaxin. Furthermore, as the dogfish shark has existed as a species for about 200 million years, the data suggest that testicular relaxin appeared early in vertebrate evolution.

Identification of a glycosylated relaxin-like molecule from the male Atlantic stingray, Dasyatis sabina.

The alkaline gland fluid of the Atlantic stingray (Dasyatis sabina) contains a molecule that cross-reacts weakly to anti-porcine relaxin antibodies. This material was isolated and purified to homogeneity by reversed-phase high-performance liquid chromatography. In SDS gel electrophoresis, the molecule showed an apparent molecular mass of 13 kDa which upon reduction formed two polypeptide chains of 4 and 9 kDa, respectively. Sequence analyses revealed a 27-amino acid residue A chain and a 54-amino acid residue B chain which contained an N-glycosylation site in position B37. The distribution of the six cysteines and possibly the disulfide bonding is identical to that found in insulins and most relaxins. Although the stingray relaxin-like molecule contains the structurally relevant glycine residues within the A chain, in the midregion of the B chain it has only one of the two requisite binding site arginines, which explains the lack of relaxin bioactivity in standard mammalian assays. Stingray relaxin is the first member of the relaxin family identified in a nonhomeotherm male. Carbohydrate analysis of relaxin revealed an N-linked asialo, agalacto, bisected biantennary, and a core-fucosylated oligosaccharide in the position of Asn B37 which makes it the first reported glycosylated relaxin-like molecule.

Continuous infusion of relaxin on periparturient progesterone, oxytocin and relaxin plasma concentrations and time of parturition in beef heifers.

These studies were designed to determine whether continuous i.v. infusion of increasing dosages of porcine relaxin during late pregnancy in beef heifers would influence circulating blood concentrations of relaxin, progesterone and oxytocin, and time of onset of parturition. Beef heifers were bred by artificial insemination and, on Day 277, fitted with indwelling jugular cannulas for hormone infusion and blood sampling from Day 277 to Day 286. Intravenous infusion of purified porcine relaxin (pRLX, 3000 U mg-1) was started in heifers (n = 8) at increasing dosages (200 U h-1 on Days 277 and 278, 300 U h-1 on Days 279 and 280, 500 U h-1 on Day 281, 600 U h-1 on Day 282, and 700 U h-1 on Days 283-286). Phosphate-buffered saline (PBS, 10 ml h-1) was infused during these same times to control animals (n = 6). Relaxin treatment steadily increased the circulating plasma concentration of immunoreactive relaxin to more than 120 ng ml-1 compared with less than 0.5 ng ml-1 in PBS-treated controls. Relaxin infusion in increasing dosages over the treatment time was associated with a significant decrease (P < 0.01) in plasma progesterone concentration compared with the PBS controls. The rate of change in progesterone levels between pRLX and PBS groups differed (P < 0.05) at 300 U h-1, 600 U h-1 and 700 Uh-1 dosage intervals, respectively. Plasma levels of oxytocin at 4 h intervals remained similar (P > 0.05) during the pretreatment period and throughout continuous infusion of pRLX and PBS. Mean concentrations of oxytocin in PBS control heifers peaked at 0.95 pgml-1 during the corresponding infusion of 700 Uh-1 pRLX, which peaked at 0.77 pgml-1. Although continuous i.v. infusion of relaxin resulted in a decrease in circulating blood levels of progesterone, it did not significantly reduce the interval between the beginning of pRLX treatment and parturition compared with the PBS-infused control heifers. These results indicate that continuous i.v. infusion of high levels of porcine relaxin resulted in a decrease in progesterone secretion in late pregnant beef heifers.

Chemical synthesis of a Zwitterhormon, insulaxin, and of a relaxin-like bombyxin derivative.

The structural motif of insulin and relaxin is frequently seen in molecules of divergent functions and origins. The insect developmental factor bombyxin, the relaxin-like factor from Leydig cells, and the insulin-like factor 4 (INSL4) all are made of two disulfide-linked chains and have one disulfide bond within the A-chain. The polyclonal antibody R6, which was raised against porcine relaxin, reacts with a wide variety of naturally occurring relaxins from primates, marine and terrestrial mammals, and elasmobranchs but does not recognize insulin. The antibody binds mainly to the arginines that occur in the N, N+4 positions in the B-chains of all relaxins which also constitute the receptor-binding site. The receptor-binding haptens were incorporated by total synthesis into human despentapeptide insulin and bombyxin II, a developmental factor from the silk moth Bombyx mori. In the process the insect factor became a perfect antigen for the anti-relaxin antibody, whereas the human insulin was transformed into a bona fide relaxin. The conversion was affected by changing four critical residues so that the insulin activity was retained to the extent of 10% of the original level. This, to the best of our knowledge, is the first designer protein to incorporate two unrelated biological functions in one primary sequence, and we are therefore proposing that, analogous to zwitterion, the generic name "Zwitterhormon" (German spelling) be used for this type of construct.

Use of synthetic canine relaxin to develop a rapid homologous radioimmunoassay.

Canine relaxin (cRlx) was synthesized by a combination of solid-phase methods and sequential site-directed disulfide bond formation. Proof that the intended molecule had been synthesized was obtained by analytical HPLC of the intact and reduced molecule, by amino acid and sequence analysis, and by receptor binding and in vivo mouse interpubic ligament bioassays. Antisera to synthetic cRlx were raised in six male rabbits; these cross-reacted with relaxins of other species, but not with insulin, LH, FSH, hCG, or prolactin (PRL). Three of the antisera neutralized relaxin-induced interpubic ligament formation in estrogen-primed mice in vivo. A new homologous cRlx RIA was developed through the use of rabbit antiserum 79888, synthetic cRlx for standards and 125l-labeled trace, and a goat anti-rabbit lgG-polyethylene glycol precipitant. The new RIA can be completed in 26 h and has a sensitivity of 0.195-0.39 ng cRlx/tube. Intra- and interassay coefficients of variation were 3% and 12.5%. During pregnancy in bitches, serum cRlx rose to about 10 micrograms/ml. Immunoactive cRlx was also detected in serum, colostrum, and milk of lactating bitches, but not in large volumes (100-300 microliters) of serum of pseudopregnant or estrous bitches. Immunoreactive cRlx was also found in seminal plasma, but not in serum, of male dogs. The new homologous cRlx RIA is simple, rapid, sensitive, and specific, and will be used in future studies of canine relaxin physiology.

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae).

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q0, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K m-values for both pyridine nucleotides were in the µmolar range (K m[NADH]=9.8 µM, K m[NADPH]=3.2 µM calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5-8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP(+) reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP(+) reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.

Structural contribution of the A-chain loop in relaxin.

Site-directed sequential disulfide bond formation has been used to synthesize relaxin analogs with modifications in the A chain loop (A10-A15). In the four different derivatives either the amino acid residues between the cysteines (A12-A14) were replaced or the intrachain disulfide bond (A10-A15) was eliminated. The substitution of the human relaxin II sequence (His-Val-Gly; A12-14) by the corresponding insulin sequence (Thr-Ser-Ile) or the hydrocarbon chain of omega-aminooctanoic acid (Aoc) caused significant loss of biological activity. Similar observations were made when the pair of cysteines (A10-A15) was replaced by either alanine or serine, whereby serine disturbs more than alanine. It is suggested that the structural features of the A chain loop not only make important contributions to the active conformation of relaxin but also that the structural requirements of insulin and relaxin are different.

Attenuation of antepartum relaxin surge and induction of parturition by antiprogesterone RU 486 in sheep.

Pregnant ewes were injected with either the antiprogesterone, RU 486 (4 mg kg-1 body weight, i.m.; n = 5), 3000 iu relaxin (i.m.; n = 9), or diluent (n = 8) at 12:00 h on days 144 and 145, to determine its effect on progesterone and relaxin secretion, and on induction of lambing. RU 486 induced earlier lambing (P < 0.01) compared with diluent treatment, but relaxin treatment did not significantly reduce the interval to parturition. Mean injection-lambing intervals were 31 +/- 2, 109 +/- 23 and 121 +/- 27 h for the RU 486, relaxin and diluent groups, respectively. There was no incidence of difficult birth (dystocia); all lambs were vigorous at birth; and placenta delivery was rapid (within 207 min) with RU 486 and relaxin treatments compared with diluent treated controls. Plasma progesterone concentrations averaged 11 ng ml-1 during the pretreatment period for all animals. RU 486 had a biphasic effect on progesterone concentrations, causing an initial increase (P < 0.05) within 2 h, and then an abrupt drop (P < 0.01) to 6 ng ml-1 by 18:00 h on day 145. Progesterone concentrations remained consistently lower (P < 0.05) in relaxin-treated ewes than in diluent-treated controls from days 144 to 147 and then began a steady decrease to 4 ng ml-1 on the day of parturition (days 149 and 150) in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The receptor-binding site of human relaxin II. A dual prong-binding mechanism.

Recent structure/function studies on human relaxin II have led to the conclusion that the arginines B13 and/or B17 are important for biological activity. These studies have been confirmed and extended with the help of chemically synthesized derivatives, i.e. dicitrulline (B13, B17), two monocitrulline (B13 and B17), a dilysine (B13, 17), and alanine (B17) relaxins. The CD spectra of synthetic human relaxin and of the derivatives are indistinguishable. Yet, only the native human relaxin II is biologically active and binds strongly to relaxin receptor preparations in vitro. The inactivation is strictly due to side chain functions, in particular the replacement of either or both arginines in the positions B13 or B17. Binding is mediated by a two-prong electrostatic and hydrogen-binding interaction via arginines B13 and B17. Neither B13 nor B17 alone are sufficient and a positive charge equidistant from the B chain helix is equally insufficient. This binding mechanism appears to be unique, as concerns hormone receptor interaction.

Relaxin receptors in mice: demonstration of ligand binding in symphyseal tissues and uterine membrane fragments.

A monocomponent, high specific activity, carrier-free porcine relaxin tracer (125I) has made it possible for us to demonstrate relaxin receptors in the symphysis pubis, uterus, and ovary via autoradiography. The receptors are concentrated in the symphyseal ligament and the peripheral layers of uterus and ovary. Specific relaxin binding was observed in crude membrane preparations of uteri, ovaries, and brain, whereas crude membranes of leg muscle and kidney showed only nonspecific binding. Uterine membranes prepared from estrogen-primed mice showed tracer binding, which could be significantly inhibited by porcine relaxin in a dose-dependent manner, but not by insulin. A linear Scatchard plot suggested the presence of only one kind of receptor and a dissociation constant of 5 x 10(-10) M, which is commensurate with an electrostatic double ion pair binding mechanism.

Purification from human pregnancy serum of a low molecular weight mitogen similar to placental lactogen and growth hormone.

Recently, we isolated from the serum of pregnant women a factor that induced rapid proliferation of a lactogen-dependent rat lymphoma cell line (Nb2). This mitogenic factor is reasonably specific to pregnancy, since it was present in serum samples from second trimester as well as term-pregnant women, but not in those of adult men or cycling females. It is unlikely that this mitogenic activity (referred to as pregnancy mitogen [PM]) is due to contamination by classical lactogens, since acetone fractionation of serum yielded a preparation devoid of placental lactogen and prolactin, as determined by radioimmunoassays. Further purification of acetone precipitates from term-pregnant serum by ion exchange chromatography and gel filtration yielded a mitogenic activity with a relative mol wt of approximately 10,000. PM activity in the NB2 cell bioassay was not affected by the presence of prolactin antiserum. However, its activity was immunoneutralized by coincubation with anti-placental lactogen serum and, to a lesser extent, anti-growth hormone serum. It appears that PM was not generated by our extraction procedure, since gel filtration of whole serum also yielded a bioactive fraction of approximately 10 kDa. PM was further purified to homogeneity by high-performance liquid chromatography. Examination of the preliminary amino acid composition of PM revealed differences from that of a bioactive fragment of growth hormone and a corresponding portion of placental lactogen, suggesting that PM could be either a molecular variant of these hormones or a novel protein.

Antiprogesterone, RU 486, facilitates parturition in cattle.

RU 486, a potent progesterone antagonist with high affinity for progesterone receptor, was used alone or in combination with relaxin in late pregnant cattle to determine its effect on induction of parturition. Cross-bred beef cattle were bred by artificial insemination. An indwelling cannula was inserted into a jugular vein on day 269 (expected term = day 283) for repeated blood sample collection. On day 277, the cattle were assigned randomly to three groups (n = 6 each): group 1 received RU 486 (2 mg/kg BW, im) at 0800 h on days 277 and 278; group 2 received the same RU 486 treatment plus 3000 U relaxin, injected sc at 0800 h on day 278; and group 3 served as controls and received vehicle injection. Parturition occurred 55 h after treatment in group 1 and 53 h after treatment in group 2 compared with 210 h in the controls (P less than 0.01). The calves from treated groups were vigorous at birth, and their birth weights (32 and 33 kg in groups 1 and 2) were less than those of control calves (38 kg; P less than 0.01). There was no incidence of difficult birth (dystocia) with RU 486 treatment compared with that in the controls. Placenta delivery averaged 6.5 h after birth in both RU 486-treated groups and did not differ from the control value (5 h). Plasma progesterone concentrations averaged 8.2 ng/ml during the pretreatment period for all animals. Progesterone started to decrease markedly by 1200 h on day 278, dropped to about 4 ng/ml by 2400 h that same day, and was at basal levels on day 279, the day of calving, in two hormone-treated groups. In sharp contrast, progesterone was maintained at about 6 ng/ml in placebo-treated controls during this period and did not decrease to basal levels until 2 days before parturition on day 286 (P less than 0.01). Peak RU 486 in plasma was 7.2 ng/ml after the first injection and 14.3 ng/ml after the second injection, and averaged 7.9 ng/ml on the day of induced calving (day 279). Peak relaxin was 4.1 ng/ml after hormone injection. The results indicate that RU 486 alone or combined with relaxin precisely controlled the time of parturition in cattle in late pregnancy. Such treatment can be used to facilitate parturition and increase survival rates of neonatal calves without detrimental effects of dystocia, retention of placenta, and delayed postpartum fertility.

Effect of relaxin on facilitation of parturition in dairy heifers.

Purified pig relaxin (3000 U/mg) was injected i.m. into pregnant Holstein dairy heifers on Day 276 or 277 to determine its effect on parturition and sequential measurements of the pelvic area, cervical dilatation, and peripheral blood-plasma concentrations of progesterone and relaxin. Treatments included phosphate-buffer saline (2 ml, Group C, N = 7), relaxin once (1 mg, Group 1R, N = 7), and twice (2 mg, 12 h apart; Group 2R, N = 7). Intervals (mean +/- s.e.) between the first injection of relaxin or PBS and calving were 64 +/- 17, 80 +/- 19 and 125 +/- 34 h for Groups 2R, 1R and C, respectively. The calving intervals were reduced in Groups 2R (P less than 0.01) and 1R (P less than 0.05) compared with Group C. The incidence of dystocia was 29% (2 of 7) in Group 2R and 43% (3 of 7) in Group 1R compared with 57% (4 of 7) in Group C (P less than 0.01). Body weights and ratios of males to females of the calves were similar (P greater than 0.05) between groups. Progesterone plasma concentrations decreased (P less than 0.01) earlier in Groups 1R and 2R compared with Group C, and this acute decrease began within 6 h of treatment. At 24 h after relaxin or PBS injection, progesterone concentrations were 2.7 +/- 1.1 ng/ml for Group 2R, 3.5 +/- 0.9 ng/ml for Group 1R, and 6.0 +/- 0.1 ng/ml for Group C. Relaxin reached peak blood-plasma levels of 19 +/- 2.2 ng/ml 1 h after injection of relaxin, but remained unchanged, 0.3 +/- 0.01 ng/ml, in Group C. Pelvic area was increased 26%, 22% and 14% and cervical dilatation was increased 109%, 76% and 53% 48 h after injection in Groups 2R, 1R and C, respectively, but these responses were similar among groups at the time of parturition. We conclude that two i.m. injections of relaxin facilitated earlier calving, acutely decreased progesterone secretion, increased cervical dilatation and pelvic area expansion, and decreased the incidence of dystocia in dairy heifers.