Relaxin: a product of the human corpus luteum of pregnancy.

Plasma samples from peripheral and ovarian veins were obtains from women at cesarean section. A peptide that immunologically cross-reacts with a specific antiserum to porcine relaxin is present in all samples. Its concentration is four times higher in the ovarian vein draining the ovary, which contains the corpus luteum of pregnancy, than in either the peripheral vein or the contralateral ovarian vein. Secretion of ovarian relaxin correlates with secretion of ovarian progesterone, thus providing another index of luteal function.

Serum concentrations of relaxin, chorionic gonadotropin, estradiol-17 beta, and progesterone during the reproductive cycle of the chimpanzee (Pan troglodytes).

Levels of serum relaxin were measured by a specific RIA and correlated with serum patterns of estradiol-17 beta, progesterone, LH, or cCG during a single menstrual cycle in each of 10 female chimpanzees, and throughout 24 pregnancies in 21 chimpanzees. Significant concentrations of relaxin, higher than those reported for the human being, were detected in serum of nonpregnant chimpanzees during the late luteal phase of the menstrual cycle. During pregnancy in the chimpanzee, serum relaxin concentrations, exceeding levels found during the luteal phase, were highest during the first third of gestation, and declined thereafter. Although the absolute concentrations were higher, the patterns of relaxin secretion throughout the reproductive cycle in chimpanzees was qualitatively very similar to that observed in other primates, including the human being. The chimpanzee should thus provide a useful model for examining the role of relaxin in human reproduction.

Diurnal variation of serum progesterone, but not relaxin, prolactin, or estradiol-17 beta in the pregnant bitch.

The concentrations of progesterone (P), relaxin (Rlx), estradiol (E2) and PRL were measured by specific RIAs in serum samples collected twice daily at 8:00 am and 3:00 pm at weekly intervals after mating and until whelping in five Labrador Retriever bitches. From weeks 3 to 6 of pregnancy, P exhibited a conspicuous and statistically significant diurnal pattern (P less than 0.05), its serum concentration being approximately twice as high at 8:00 am as at 3:00 pm. A similar but nonsignificant trend was observed weeks 2, 7, and 8, and the overall ratio of the am/pm P concentrations was 2.4 +/- 0.28 (P less than 0.001). Rlx, E2, and PRL did not show a diurnal pattern at any time during pregnancy. The glandular sources and regulation of secretion of Rlx were further investigated. Rlx bioactivity was detected in canine placentas and ovaries, providing supportive evidence that these organs may be a dual source of the hormone in canine pregnancy. Injection of ovine PRL in three pseudopregnant bitches significantly increased serum P concentration as compared with seven untreated pseudopregnant controls, but Rlx was not detectable in serum before, during or after PRL treatment. The data support the view that Rlx and P are independently regulated in the bitch; PRL may be a regulator of P, but not of Rlx secretion. However, as PRL showed no diurnal changes in concentration, it does not appear to be directly responsible for the diurnal pattern exhibited by P.

The source of relaxin in pregnant Syrian hamsters.

Serum immunoreactive relaxin (IR) was measured on days 8, 10, and 14 of gestation in intact and ovariectomized (day 8 of pregnancy) hamsters. In intact hamsters, IR increased from 3-4 ng/ml on day 8 to 20 ng/ml by day 14 of pregnancy. After ovariectomy on day 8, pregnancy failed, and IR decreased rapidly to 0.29 ng/ml on day 14. However, when pregnancy was maintained in ovariectomized hamsters by daily injections of 0.1 microgram 17 beta-estradiol and 4 mg progesterone, serum IR rose to levels similar to those in intact hamsters on days 10 and 14 of pregnancy (i.e. 15 and 20 ng/ml, respectively). Placentas were obtained from other groups of hamsters on days 11, 14, and 15 of pregnancy and homogenized for bioassay by the classical guinea pig pubic symphysis palpation bioassay. Homogenates of placentas obtained on days 14 and 15 contained, respectively, 4 and 10 micrograms eq porcine relaxin/serum relaxin/g fresh tissue. The placenta, rather than the ovary, appears to be the source of during pregnancy in the hamster.

Characterization of the circulating form of porcine relaxin: biological activity and terminal amino acids.

Relaxin is structurally related to insulin, and it induces pregnancy-related changes in the reproductive tract of several mammalian species. Relaxin isolated from the ovaries of pregnant sows has been used for primary structure determination, for much of the biological characterization of the hormone, and for the development of RIAs. Immunoreactive (IR) relaxin is found in peripheral blood during pregnancy in pigs and other species, but it has not been established that the substance identified by RIA is structurally or biologically equivalent to the native ovarian hormone. IR relaxin was, therefore, isolated from peripheral plasma of late pregnant gilts (days 112-114) for bioassay and determination of terminal amino acid residues. IR relaxin was monitored by a specific homologous RIA during fractionation of plasma by gel filtration, cation exchange, and hydrophobic binding to octadecysilica. IR relaxin circulates unbound and is equipotent with ovarian relaxin in the mouse pubic ligament bioassay. Amino acids released from IR relaxin by pyroglutamic aminopeptidase and carboxypeptidase-Y were converted to their 4-dimethylamino-azo-4'-sulfonyl derivatives for identification by HPLC. The B-chain of IR relaxin had an amino-terminal pyroglutamic acid. Amino acids sequentially released from the carboxy-terminal indicated a chain length of 28-30 amino acids, suggesting a heterogeneity reminiscent of that of ovarian relaxin isolated by other methods. Arginine was released from the free amino-terminal by dimethylaminozaobenzene-isothiocyanate degradation, indicating an intact A-chain of 22 amino acids. Blood immunoreactive relaxin in pigs is, therefore, a secreted biologically active form of relaxin with an amino acid composition similar to that of the form stored in the corpus luteum.

Serum immunoreactive relaxin and softening of the uterine cervix in pregnant hamsters.

Serum immunoreactive relaxin levels and ripening of the cervix were measured throughout pregnancy in hamsters. RIA relaxin rose from an undetectable level on day 7 to a maximum value of 29 ng/ml on day 15 of gestation and then fell prior to parturition. The cervix became progressively more dilatable from the 12th to the 16th day of pregnancy. It is suggested that the endogenous relaxin measured by RIA may induce the cervical softening. The absolute levels of immunoreactive relaxin appear to be 10 to 15-fold higher than those previously observed in rats, mice and guinea pigs.

Placental control of ovarian immunoreactive relaxin secretion in the pregnant rat.

To investigate the control mechanisms for the secretion of relaxin in pregnant rats, the effects of the fetus, placenta, and uterus were studied. Plasma immunoreactive relaxin and progesterone were measured in pregnant rats, from days 8--1 post partum. On day 16 of pregnancy, groups of animals were subjected to removal of the fetuses, conceptuses (fetuses and placentae), or uteri. To determine whether there are extraovarian sources of circulating relaxin, a group of pregnant rats was ovariectomized on day 16. Immunoreactive relaxin was undetectable in the plasma of pregnant rats before day 10, and increased to a mean concentration of 0.52 +/- 0.01 (SEM) ng/ml on day 13. In control animals, immunoreactive relaxin levels remained at about this concentration throughout the remainder of pregnancy and declined rapidly post partum. The pattern of secretion of relaxin in fetectomized animals was similar to that in controls. In contrast, a significant decline in immunoreactive relaxin was seen, within 24 h after surgery, in those animals in which removal of the conceptuses or hysterectomy was performed. In these animals, immunoreactive relaxin was undetectable within 48 h after surgery and remained undetectable throughout the experimental period. In animals that were ovariectomized, immunoreactive relaxin was undetectable 24 h after surgery. Progesterone secretion in animals that had fetectomy or removal of the conceptuses performed was similar to that in controls. These groups showed a significant decline in progesterone on day 17 of pregnancy, and progesterone continued to decline until day 1 post partum. Progesterone in hysterectomized animals declined more abruptly than in either controls or other experimental groups. Ovariectomy resulted in a prompt fall in plasma progesterone. These results indicate that in the rat, the fetus is not needed for the maintenance of relaxin secretion throughout pregnancy, the placenta controls the ovarian secretion of relaxin. The uterus does not exert a tropic effect upon relaxin secretion, no extraovarian sources of circulating relaxin exist in the rat, and there is a divergence between progesterone and relaxin secretion during rat pregnancy.

Isolation and characterization of relaxin from the sand tiger shark (Odontaspis taurus).

A peptide with relaxin activity in guinea pigs but not in mice has been extracted from the ovaries of pregnant sand tiger sharks (Odontaspis taurus). The structural similarity of this peptide to porcine relaxin includes molecular size approximately 6000 daltons), number of chains, and possibly, disulfide cross-links. The relaxin-type peptide isolated from shark ovaries contains the amino acid residues tyrosine, proline, and histidine, which are absent in the porcine hormone. The amino acid composition of shark relaxin, therefore, resembles that of porcine insulin to a greater extent than does the amino acid composition of porcine relaxin. This finding supports the idea that shark relaxin may be a primitive relaxin that has undergone fewer mutations than porcine relaxin since the putative duplication of the insulin gene. The data presented here suggest that the putative duplication of the insulin gene, which might have given rise to relaxin, has occurred much earlier than the separation of sharks from the general branch of animals that eventually gave rise to mammals.

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.

Relaxin bioactivity and immunoactivity in human corpora lutea.

Relaxin-like activity in extracts of corpora lutea (CL) from pregnant and non-pregnant women was determined by radioimmunoassay and by guinea pig pubic symphysis palpation assay. The biologically determined activity paralled the immunoactivity of extracts of CL of pregnancy. The relaxin content of CL of non-pregnant women was too low for detection by the bioassay.

Plasma immunoreactive relaxin levels in pregnant and nonpregnant women.

Immunoreactive relaxin was measured in plasma samples obtained from human volunteers utilizing the RIA procedure of Sherwood et al., as modified by O'Byrne and Steinetz for heterologous plasma samples. Immunoreactive hormone was not detected in samples obtained from men, and only rarely in plasma of nonpregnant women. Immunoreactive relaxin was present as early as the fourth week of pregnancy and was detectable throughout the course of gestation. Immunoreactive relaxin tended to be higher early in pregnancy, and there was no peak just before parturition as occurs in many other species. Our results are at variance with those of Bryant and coworkers, who reported high levels of immunoreactive relaxin in men and nonpregnant as well as pregnant women. The possible reasons for this discrepancy are presented.

Identification of potential antiatherosclerotic/hypolipidemic agents by their effect on hepatic conversion of androst-4-ene-3,17-dione to etiocholanolone and androsterone.

Clinical observations have shown that hypercholesterolemia is associated with abnormal androgen metabolism, viz. an increased excretion of etiocholanolone (E) relative to androsterone (A). Substances which restore the A/E ratio to normal likewise lower serum cholesterol. Postulating that the abnormal steroid and sterol metabolism may be either causally related or dependent on the same metabolic defect, we have developed in vitro and in vivo models to select drugs which favorably effect the ratio of A to E produced from [4-14C]androst-4-ene-3,17-dione [4-14C]A-dione). The in vitro model employs a mixture of rat liver microsomal delta 4-3-ketosteroid-5 alpha-reductase and cytosolic 3 alpha-hydroxysteroid dehydrogenase and delta 4-3-ketosteroid-5 beta-reductase. Kinetic and mechanistic studies have been performed on active compounds using this in vitro assay. The in vivo model employs i.v. injection of [4-14C]A-dione followed by collection of bile in anesthetized, hypophysectomized female rats. Many compounds preselected in the in vitro assay likewise reduced the A/E ratio in vivo. One of these compounds (CGS 10614A) also lowered serum cholesterol and reduced the incidence and severity of atherosclerotic lesions in aortas of cholesterol-fed rabbits.

Effects of altered endocrine function on biliary metabolites of [4-14C]androst-4-ene-3,17-dione in rats. Possible utility as a model for identifying anti-atherosclerotic agents.

Atherosclerosis, coronary artery disease and elevated serum cholesterol are frequently associated with an abnormal pattern of androgen metabolites, especially an elevation of etiocholanolone (E) and/or epiandrosterone (EA) relative to androsterone (A). Therapeutic correction of these metabolic defects may lower serum cholesterol. We have attempted to reproduce this metabolic syndrome in rats by altering their endocrine status. Intact male rats excreted very little A or E in their bile; more than 80% of the [4-14C]A-dione was excreted as unknown polar compounds. Adrenalectomy, thyroidectomy or streptozotocin diabetes induced little or no change in the excretion of both E and A and did not alter the A/E ratio. Hypophysectomy (hypox), however, resulted in a huge increase in E excretion and a 10-fold decrease in the A/E ratio. Treatment of hypophysectomized males with bovine growth hormone (bGH) but not testosterone or thyroxine restored the pattern of androgen metabolites to that of intact male rats. Intact female rats excreted mainly A, and this was decreased by ovariectomy. Hypophysectomy, however, resulted in a marked increase in E and a corresponding large decrease in A excretion. Treatment of hypox females with estradiol or triiodothyronine did not correct the metabolic defects in A and E production, whereas GH resulted in a pattern of A-dione metabolism resembling that of intact males; i.e., primarily polar metabolites with low A and E. Hypophysectomy thus results in a dramatic increase in 5 beta-reductase activity in male and female rats. GH therapy restores the metabolic pathway to that seen in intact males. Our objective had been to find a model capable of detecting substances which would increase A and decrease E production. The male rat (regardless of endocrine status) has little 5 alpha-reductase activity. The intact female rat, however, has high 5 alpha-reductase activity, and retains significant 5 alpha-reductase in the absence of the ovaries. In hypox females, 5 alpha-reductase was much reduced while 5 beta-reductase was increased. Furthermore, serum cholesterol was elevated in hypox females but could be lowered by exogenous androsterone. Thus the hypox female rat appears to offer the best model for identifying non-hormonal agents which could enhance the production of A and/or decrease the production of E. Such agents might favorably influence cholesterol metabolism.

Contraceptive agents from cycloaddition reactions of diarylcyclopropenones and diarylthiirene 1, 1-dioxides.

The potential for compounds with antifertility activity from the reactions of diphenylcyclopropernone (1) and 2, 3-diphenylthiirene 1, 1-dioxide (2) with enamines is described. In certain instances, a marked dissociation of antifertility from estrogenic activity was possible. Two series were studied extensively, one was stilbene amides (7) and the other stilbene amino ketones (8). The latter series (8) afforded several materials from which, on further biological work-up, was singled out compound 21 as a potent antifertility agent in rats and hamsters.

PIP: The antifertility activity from the reactions of diphenylcyclopropene (1) and 2,3-diphenylthiirene 1,1 dioxide (2) with enamines was investigated in laboratory rodents. A considerable dissociation of antifertility from estrogenic activity was observed in certain instances. Stilbene amides (7) and stilbene aminoketones (8) we re extensively studied. The stilbene aminoketone group provided several materials of which compound 21 was found to be a highly potent antifertility agent. The preparation of the materials is described.

Relaxin and its role in pregnancy.

Relaxin is a 6000-d polypeptide, structurally related to insulin and the insulin-like growth factors. Unlike insulin, the structure of which is remarkably well conserved among the vertebrates, relaxin sequences can vary by more than 50% between different species. Despite these large sequence variations, relaxins (with few exceptions) have very similar biologic activities in animal test systems. The reason for this has recently come to light: the receptor binding region of the B chain, in contrast to the rest of the molecule, is highly conserved between species. Relaxin is measured by bioassays employing interpubic ligament formation in mice and guinea pigs, and by inhibition of uterine motility. A more sensitive and efficient bioassay is urgently needed. In women, the target organs for relaxin are the uterine cervix, myometrium, endometrium, and decidua. Other presumptive but unproven targets are the pubic symphysis and sacroiliac joints, mammary glands, and pituitary gland. Circulating relaxin is secreted by the corpus luteum. The placenta, decidua, or both also produce relaxin, which does not enter the circulation but may act in an autocrine or paracrine fashion. hCG is a stimulus to luteal relaxin secretion. Other regulatory factors are poorly defined. Aluteal women are hyporelaxinemic, and yet are capable of normal vaginal delivery of their infants. Local effects of placental or decidual relaxin cannot be discounted in such subjects. Hyperrelaxinemia may occur in women with multiple gestations and ovarian stimulation, and may be associated with increased premature births. Serum relaxin also is elevated in pregnant diabetics, but its role in this condition has not been defined. Clearly, further investigations are needed to delineate the precise role of relaxin in human pregnancy.

Relaxin secretion in early pregnancy.

Relaxin is a peptide hormone produced solely by the corpus luteum of pregnant women. Extracts of human pregnancy corpora lutea have relaxin activity in bioassay systems. This activity can be measured in a heterologous porcine radioimmunoassay (RIA). Immunoreactive relaxin was undetectable in the RIA in 51 nonpregnant women. In conception cycles, relaxin immunoactivity is detectable in peripheral blood by the time of the missed menses. Relaxin detection may be used as a pregnancy test. Relaxin may be an important luteal factor, together with progesterone, in early pregnancy maintenance.

Distribution of relaxin in women during pregnancy.

The purpose of this study was to determine by RIA the concentrations of relaxin in various compartments and tissues in pregnant women. Ten pairs of maternal venous and cord blood were studied. The mean relaxin concentrations, in immunoreactive equivalents of porcine relaxin, were 0.683 ng/ml in maternal serum and 0.009 ng/ml in cord serum. Relaxin was undetectable in 8 of 9 samples of amniotic fluid. Mean concentrations of relaxin in pg immunoreactive equivalents of porcine hormone per mg protein, from maternal tissues at term pregnancy were as follows: fat 96 (N = 5), myometrium 47 (N = 4), skin 62 (N = 5), placenta 51 (N = 9), and corpus luteum 13,000 (N = 8). These data suggest that little relaxin crosses the placenta and little is produced in the fetus. Contrary to prior suggestions that relaxin may also be a placental product, relaxin appears to be solely produced in the corpus luteum, making it the only peripherally measured hormone that can be used as an index of luteal activity in pregnancy.

Secretion of progesterone and relaxin by the humancorpus luteum at midpregnancy and at term.

PIP: The secretion of progesterone (P) and relaxin (R):by the human corpus luteum at midpregnancy and at term was studied in 19 women, 6 of whom had their pregnancies terminated by hysterotomy at 14-18 weeks and 13 whose pregnancies went to term and were delivered by Caesarean section. Luteectomies were performed on 6 of the women at the time of Caesarean section. P and R levels were measured. R section correlated well with luteal P secretion. After Caesarean section at term the decline in serum P was parallel to that observed after hysterotomya at midtrimester. Absolute levels of P are higher at term. P decline after luteectomy was precipitous compared with Caesarean section. Results indicate that the corpus luteum in the human remains active throughout pregnancy.^ieng

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.

Relative importance of 5alpha reduction for the androgenic and LH-inhibiting activities of delta-4-3-ketosteroids.

The significance of 5alpha reduction of c19, delta1,4-3-ketosteroids in regulating growth of the rat ventral prostate (VP) was examined. The androgenic and LH-inhibiting activities of a C19 delta1,4-3-detosteroid which does not undergo appreciable 5alpha reduction were compared with those of its 5alpha reduced analogue and those of testosterone (T). In intact rats M (17beta-hydroxy-17alpha-methyl-androsta-1:4-dien-3-one) caused a suppression of VP weights and plasma testosterone concentrations, and in castrated rats suppressed plasma LH concentrations. M was considerably less androgenic and moderately less potent as an inhibitor of LH secretion than either T or the 5alpha reduced analogue of M [17beta-hydroxy-17alpha-methyl-5alpha-androst-1-ene-3-one; (5alphaM)]. 5alphaM was found to be at least as androgenic and as active as an inhibitor of LH as T, suggesting that the weak activity of M may be attributable to a lack of reduction to 5alphaM. Following incubation of 3H-M with VP minces, over 96% of the radioactivity recovered corresponded with M by TLC. Under identical conditions 32-48% of the radioactivity recovered from incubations with 14C-T corresponded with 5alpha reduced metabolites of T. This study demonstrates the importance of 5alpha reduction for both the androgenic and LH-inhibiting activities of delta4-3-ketosteroids.