Relaxin Immunoactivity Research Articles

The Source and Secretion of Immunoactive Relaxin in Rat Milk

The milk of many mammalian species contains hormones and growth factors in addition to nutrients and immunocompetent substances. These factors can be absorbed into the circulation of suckling neonates to exert important effects on metabolism and promote tissue and organ growth. Frequently, there is uncertainty as to whether such substances are gene products of the mammary glands themselves or are produced elsewhere and concentrated from the systemic circulation. The 6 kD polypeptide, relaxin, appears in milk of several mammalian species, including that of the rat, but proof of its source of secretion (corpus luteum vs. mammary gland) is so far lacking. The specific monoclonal anti-rat relaxin antibody MCA1 has previously been utilized successfully to investigate many of relaxin's actions in the rat, including those affecting the development of the mammary apparatus. In this report, MCA1 was utilized to aid in the identification of the source of relaxin in rat milk. Treatment of lactating rats with MCA1 completely neutralized the luteal relaxin circulating in serum but did not decrease the concentration of immunoactive relaxin secreted in milk. Moreover, the antibody did not appear to reach the mammary epithelium. The evidence thus supports the view that in the rat, the relaxin secreted in milk is primarily a product of the mammary glands and not concentrated from the systemic circulation.

Relaxin Concentrations in Serum and Urine of Endangered and Crazy Mixed‐Up Species

The human population explosion has pushed many mammalian wildlife species to the brink of extinction. Conservationists are increasingly turning to captive breeding as a means of preserving the gene pool. We previously reported that serum immunoactive relaxin provided a reliable means of distinguishing between true and pseudopregnancy in domestic dogs, and this method has since been found to be a reliable indicator of true pregnancy in endangered Asian and African elephants and Sumatran rhinoceroses. Our canine relaxin radioimmunoassay (RIA) has now been adapted and validated to measure relaxin in the serum and urine of felids, including domestic and wild species. Moreover, a commercially available canine serum relaxin kit (Witness) Relaxin Kit; Synbiotics, San Diego, CA), has been adapted for reliable detection of relaxin in urine of some felid species. Our porcine relaxin RIA has also been utilized to investigate the role of relaxin in reproductive processes of the spotted hyena, a species in which the female fetuses are severely masculinized in utero. Indeed, this species might well now be extinct were it not for the timely secretion of relaxin to enable copulation and birth of young through the clitoris. Additional studies have suggested relaxin may be a useful marker of pregnancy in the northern fur seal and the maned wolf (the former species has been designated as "depleted" and the latter as "near threatened"). Given appropriate immunoassay reagents, relaxin determination in body fluids thus provides a powerful tool for conservationists and biologists investigating reproduction in a wide variety of endangered and exotic species.

Transmission of relaxin and estrogens to suckling canine pups via milk and possible association with hip joint laxity

To determine whether abnormal laxity of hip joints of canine pups with genetic predisposition to hip dysplasia (HD+) is related to ingestion of milk-borne hormones. 7 female Labrador Retrievers with HD+ and 8 with low predisposition to hip dysplasia (HD-) and their offspring. Immunoactive relaxin, estrogen, and estrogen precursor concentrations in milk of HD+ lactating bitches and in serum of their pups were compared with those of HD- bitches and pups. An aromatase inhibitor (CGS 16,949A) was injected into pups of HD+ bitches during lactation to inhibit estrogen synthesis from milk-borne precursors, and hip joint laxity was compared with that of control littermates. Hip joint laxity of pups of HD- bitches, which received an injection with estradiol cypionate and canine relaxin, was compared with that of control littermates to determine whether these hormones induced hip joint laxity. High concentrations of estrogens and relaxin were found in milk of HD+ and HD- bitches throughout lactation. Serum concentrations of milk-derived relaxin and total estrogens were similar in all pups, but estradiol-17B was detected only in pups of HD+ bitches. Hip joint laxity was reduced in pups that received CGS 16,949A. Hip joint laxity was INCREASED IN PUPS OF HD- BITCHES THAT RECEIVED ESTRADIOL CYPIONATE AND RELAXIN. Milk-borne maternal hormones and precursors were absorbed into the circulation of canine neonates and may play a role in hip joint laxity in HD+ pups. Phenotypic expression of hip dysplasia may therefore be preventable by antihormone treatment.

Pattern and source of secretion of relaxin in the reproductive cycle of the spotted hyena (Crocuta crocuta).

Female spotted hyenas are highly masculinized at birth and have no external vagina. Copulation with males and birth of young are accomplished through the central urogenital canal of the clitoris. This unusual adaptation requires remarkable changes in the elasticity of the connective tissues of the clitoris, without which neither copulation nor birth would be possible. We hypothesized that relaxin, a hormone that increases the extensibility of the connective tissues of the uterus and cervix of many other mammalian species, plays a role in the clitoral changes observed in hyenas. Serum relaxin was determined by specific RIA. Relaxin was not detected in serum of males, pubertal or nonpregnant adult females, or ovariectomized females. Immunoactive relaxin was detected in serum of juveniles at the time of initial growth of the urogenital meatus. High concentrations of immunoactive relaxin appeared in the serum of pregnant hyenas in the 2 wk preceding parturition. Immunoassays of extracts of hyena tissues and serum obtained from uterine and ovarian veins indicated that the placenta was the predominant source of relaxin, with possible ovarian contributions. Circulating relaxin decreased promptly following cesarean section near term. We conclude that relaxin secretion coincides with changes in extensibility of clitoral connective tissues 1) during growth of the clitoris in juveniles and 2) near the time of parturition in adults.

Purification and sequence determination of canine relaxin.

Relaxin immunological activity has been observed in the plasma of pregnant bitches, and preliminary studies in our laboratory indicated that the highest relaxin concentrations were found in placentas. Therefore, canine placentas were collected at term and also from spay and relaxin was purified by methods developed for equine relaxin. Tissue was prepared by homogenization and purification on a C18 column. The preparation was further purified by stepwise elution ion-exchange chromatography, gel filtration, and gradient elution ion-exchange chromatography. One predominant peak in relaxin immunoactivity was collected. Canine relaxin was found to be larger than either porcine or equine relaxin as determined by SDS-PAGE. It migrated faster under reducing conditions, indicating a subunit structure. Purified canine relaxin was used for tracer and standard in a canine radioimmunoassay (RIA) using an antiporcine relaxin antibody. Concentrations of relaxin immunoactivity using the canine assay were up to 300-fold higher in placental preparations than those measured in the porcine relaxin assay. Sequence analysis of canine relaxin revealed a structure similar to other relaxins in the presence and placement of cystine residues.

Ultrastructural morphology and relaxin immunolocalization in giant trophoblast cells of the golden hamster placenta

Relaxin immunoreactivity was previously demonstrated in three cell types within the hamster placenta; fetal primary and secondary giant trophoblast cells (GTCs) and maternal endometrial granulocytes. The objectives of the present research were to examine the ultrastructure of the GTCs and identify the intracellular relaxin storage site. Primary GTCs, first present on day 8 of gestation, were characterized by numerous polyribosomes and large heterogeneous cytoplasmic inclusions suggesting phagocytic activity. Primary and secondary GTCs from days 10, 14, and 15 of gestation contained numerous polyribosomes, mitochondria with tubular cristae, and extensive Golgi complex, and abundant rough endoplasmic reticulum, all characteristics of a cell actively involved in protein synthesis. Membrane-bound secretory granules were not present. Relaxin was immunolocalized within the Golgi complex of primary and secondary GTCs using the avidin-biotin-peroxidase method. Following differential centrifugation of hamster placental homogenates and radioimmunoassay (RIA) of subcellular fractions, the majority of relaxin immunoactivity was detected in the postmicrosomal fraction; however, the majority of relaxin immunoactivity from similarly treated pig corpora lutea was present in the mitochondrial/granule fraction. These data indicate that hamster placental relaxin is not stored in membrane-bound secretory granules but is contained within the extensive Golgi complex of the GTC.

Relaxin-like peptide in ascidians: I. Identification of the peptide and its mRNA in ovary of Herdmania momus

The ovaries of the ascidian Herdmania momus were extracted for relaxin. Relaxin immunoactivity was eluted from Sephadex G-50 in the position of authentic porcine ovarian relaxin and these fractions were parallel to porcine relaxin in an homologous porcine relaxin radioimmunoassay. Poly(A) + RNA from ascidian ovaries hybridized to a 32P-labeled porcine relaxin B-chain 48 mer oligonucleotide probe. There was no hybridization with 32P-labeled probes of equal length but was rather directed to the B-chain of human relaxin or to two different regions of the C-peptide of porcine relaxin. We conclude that the ascidian ovary produces a relaxin-like molecule, possibly with greater homology in the B-chain to mammalian relaxins than in the C-peptide region.

Immunoactive relaxin in boar seminal plasma and its correlation with sperm motility

Relaxin in boar seminal plasma ( n = 43) was assayed by an homologous radioimmunoassay. The seminal plasma levels of relaxin averaged 1.1 ± 0.38 ng/ml (±s.e.). The relaxin concentrations in seminal plasma were correlated with sperm motility ( n = 32, r = 0.33, P < 0.05) and the transmembrane migration ratio ( n = 11, r = 0.81, P < 0.01) of boar spermatozoa. Relaxin levels in seminal plasma of Yorkshire ( n = 19), Landrace ( n = 5), Duroc ( n = 10), and Poland China ( n = 4) boars were 1.3 ± 0.07, 0.9 ± 0.13, 0.8 ± 0.13, and 0.7 ± 0.03 ng/ml (mean ± s.e.), respectively. Seminal plasma relaxin levels differed among these breeds ( P < 0.05). The results in this study indicate the presence of immunoactive relaxin in boar seminal plasma which shows high positive correlations with sperm motility of fresh semen.

Localization of relaxin in the pig follicle during preovulatory development.

The avidin-biotin immunoperoxidase method and antisera to purified porcine relaxin were used to localize relaxin in sections of follicles from pregnant mare's serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-primed pigs during preovulatory development. Prepubertal pigs were treated i.m. with PMSG (750 IU) and 72 h later with hCG (500 IU) to induce follicular development and ovulation. Follicles were collected from untreated gilts or from gilts 24, 48, 60, 72, 84, 96, or 108 h after PMSG treatment. Light immunostaining in the theca interna was observed early in follicular development, at 48 and 60 h post-PMSG. At 72 h post-PMSG, relaxin immunostaining in the theca interna of the preovulatory follicle was more intense. After hCG treatment, the intense thecal immunostaining persisted and was apparent 84 and 96 h after PMSG. At about 6 h prior to expected ovulation (108 h post-PMSG), there was thinning of the follicle wall and a reduction in relaxin immunostaining in the theca interna. Immunoactive relaxin was not detected in follicles from untreated gilts, follicles 24 h post-PMSG, small healthy or atretic follicles, or in granulosa cells, theca externa or ovarian stroma, at any of the time points studied. These studies support the hypothesis that the theca interna is the primary source of follicular relaxin and provide further evidence for a paracrine role for relaxin in the ovulatory process.

Human semen relaxin and its correlation with the parameters of semen analysis

The relaxin content of 92 normal semen samples and 85 semen samples selected at random was correlated to the parameters measured on routine semen analysis in those samples. The concentration of immunoactive relaxin in a sample of semen plasma did not correlate with any of the parameters commonly used for semen analysis: count, percent motility, grade of forward progression, volume, and morphology.

Influence of the conceptuses and the maternal pituitary on the distribution of multiple components of serum relaxin immunoactivity during pregnancy in the rat.

Relaxin isolated from the ovaries of pregnant rats has a mol wt of 6,500; however, relaxin immunoactivity (RI) in the peripheral serum of pregnant rats is associated with three major components. Essentially all RI is associated with a large component (C1, mol wt approximately 60,000) on day 15 of pregnancy (day 15), but the distribution shifts progressively so that about 50% of the RI is associated with two smaller components (C2, mol wt approximately 13,000; C3, mol wt approximately 6,500) by day 19. The present study examined factors that might influence the distribution of RI among these three components. The distribution of RI in sera from ovarian vein and abdominal aorta on days 16 and 19 was compared after gel filtration through Sephacryl S-200. The distribution of RI among the three major components was similar for sera collected from the two sources. It is concluded that the progressive shift in the distribution of RI among the three major components during late pregnancy is attributable to dynamic changes in their secretion, rather than peripheral metabolism of relaxin after its release from the ovary. This study also determined the influence of the conceptuses and the maternal pituitary on the distribution of RI. The distribution of RI in peripheral sera obtained on days 16 and 19 from intact and hypophysectomized full complement-bearing rats as well as pituitary-intact and hypophysectomized one conceptus-bearing rats was compared after filtration through Sephacryl S-200. The shift in distribution of RI from C1 to smaller components C2 and C3 was markedly attenuated in rats bearing one conceptus, whereas hypophysectomy was without effect. It is concluded that the progressive shift in the distribution of serum RI among the three major components during late pregnancy is influenced by the number of conceptuses but not the maternal pituitary.

Development of a homologous equine relaxin radioimmunoassay.

Equine relaxin (eRlx) immunoactivity has previously been measured in the mare during pregnancy using the porcine relaxin (pRlx) RIA (pRlx-RIA). This was not the optimal system for measurement of eRlx because the dose-response curve obtained with equine plasma was not parallel to the pRlx standard curve. A homologous eRlx-RIA has been developed and used to measure relaxin immunoactivity during pregnancy and parturition in the mare. Highly purified eRlx was used for the generation of antiserum in rabbits, preparation of tracer, and as assay standards. A double antibody eRlx RIA (eRlx-RIA) was developed which was highly specific and sensitive (0.023 ng relaxin/tube). The dose-response curve obtained with eRlx plasma was parallel to the equine standard curve while there was no parallelism noted between the pRlx and eRlx standard curves. This assay was utilized to measure eRlx in samples collected during pregnancy which were previously measured for relaxin content in the pRlx-RIA. It was found that the two assay systems gave almost identical patterns of secretion throughout pregnancy. The two assays differed in the amount of relaxin measured, with the eRlx-RIA measuring considerably higher amounts during gestation than the pRlx assay. In oxytocin-induced foalings, the differences became greater with the equine assay measuring as much as 10-fold greater concentrations.

Purification and characterization of equine relaxin.

It has been previously determined that the equine placenta is the sole significant source of relaxin during pregnancy and that relaxin immunoactivity is also present in term placentas. Therefore, placentas obtained at the time of foaling were selected for starting material for purification of equine relaxin. Frozen whole placentas were ground and then extracted with 0.5 N HCl-85% acetone. Relaxin was precipitated by raising the acetone concentration to 97%. Equine relaxin was further purified by stepwise elution ion exchange, gel filtration, and gradient elution ion exchange chromatographies and trichloroacetic acid precipitation. Equine relaxin purified in this manner was shown to be heterogeneous with one major form (R-1) and several minor forms (two of which are identified as R-2 and R-3). About 1.5 mg R-1 were obtained per kg placenta. Purity was assessed by slab and disc gel electrophoresis and dansyl end group analysis. R-1 had a potency of 28 U/mg, as measured in the mouse inter-pubic ligament bioassay and displayed a dose-response curve parallel with porcine relaxin. Amino acid analysis indicated the presence of tyrosine, histidine, and proline, amino acids absent in porcine relaxin. Dansyl end group analysis indicated the blockage of N-terminal groups on R-1 and the presence of Lys on R-3. A lower molecular weight was indicated by the electrophoretic migration of relaxin reduced with 2-mercaptoethanol suggesting that equine relaxin consists of two chains.

SERUM RELAXIN IN PREGNANCY

Serum relaxin immunoactivity was measured by means of a porcine radioimmunoassay in a cross-sectional study of 302 normal singleton pregnancies. Concentrations in the third trimester were lower than in early and mid pregnancy. At term, relaxin levels in patients who went into spontaneous labour within a week of sampling were significantly lower than in those who did not. However, relaxin levels were highest during labour and fell almost to non-pregnant levels by the third postnatal day. Levels in twin pregnancies in the third trimester were higher than those in singleton pregnancies. Serial samples from 4 patients with a history of premature labour showed declining, very low levels in the only patient who subsequently had a preterm delivery. These results are compatible with the proposed roles of relaxin during pregnancy: namely, to maintain myometrial quiescence, facilitate uterine stromal remodelling during uterine growth, and promote cervical ripening at the onset of parturition.

Influence of litter size on antepartum serum relaxin and progesterone immunoactivity levels and on birth in the rat.

In the rat, the antepartum elevation of serum relaxin levels consists of two phases separated by a 24-h interval. The second phase, which occurs between 36 and 24 h before birth, is temporally closely associated with functional luteolysis. Relaxin levels then decline throughout the last approximately 24 h of pregnancy. We have postulated that the two phases in the antepartum elevation of serum relaxin levels may be indicative of an increasingly effective endogenous circadian luteolytic process. There is limited evidence that both luteolysis and birth are delayed in rats with small litters. The present study investigated in detail the relationship between litter size and the timing of both functional luteolysis and birth in rats. The number of conceptuses (C) in Sprague-Dawley-derived rats was surgically adjusted on day 8 of pregnancy (day 8) so that rats bore one, two, three, five, or a full complement (FC) of eight or more C. Rats were maintained under a photoperiod regimen of 14 h of light and 10 h of darkness (lights on from 2100-1100 h) beginning on day 8 and observed for birth at 10-min intervals from 2100 h on day 22. Serum levels of both relaxin and progesterone were determined in blood samples obtained at 4-h intervals from 2400 h on day 19 until birth. Ninety-five percent of the rats that had five or more C gave birth during the light phase on day 23, which was designated the normal birth interval. However, only 20% of the rats with three C or less, gave birth during the normal birth interval, and 47% gave birth about 24 h later during the light phase on day 24, which was designated the late birth interval. The 24-h delay in birth of rats with small litters which delivered during the late birth interval appears to be attributable to a delay in functional luteolysis; the antepartum decline in serum relaxin and progesterone levels occurred about 24 h later in these rats than in rats that delivered during the normal birth interval. It is concluded that the C may be associated with the luteolytic process and thereby influence the time of birth in rats. Additionally, the results of this study are consistent with our hypothesis that there is an endogenous circadian luteolytic process in rats during the antepartum period.

Relaxin activity in the pregnant cat.

Plasma relaxin activity was measured by radioimmunoassay (RIA) in the domestic cat utilizing two different antisera developed against highly purified porcine relaxin. One was the 5858 antiserum from our laboratory and the other was the R6 antiserum of Dr. Bernard Steinetz. Relaxin activity could not be detected during the estrous cycle or during pseudopregnancy. Relaxin immunoactivity during early gestation was not detected by either antiserum. Plasma relaxin immunoactivity was first detected by both antisera on about Day 25 of gestation. Relaxin concentrations then increased rapidly, with a plateau reached between Days 30 and 35 that was maintained until 10-15 days prepartum. Relaxin concentrations then declined gradually until parturition. No prepartum increase was observed. Relaxin concentrations were undetectable within 24 h of delivery. Although amounts of immunoactivity measured with the R6 antiserum were consistently higher than measurements with the 5858 antiserum, the patterns of secretions observed were similar for both antisera.

Dynamic changes of multiple forms of serum immunoactive relaxin during pregnancy in the rat.

Relaxin isolated from the ovaries of pregnant rats has a mol wt of 6,500. This study determined the size(s) of the relaxin-immunoactive component(s) in the serum of rats during pregnancy. Peripheral sera from rats on days 15, 17, 19, and 21 of pregnancy were filtered through Sephacryl S-200 columns at 37 C and 4 C. Relaxin immunoactivity eluted in three components designated C1, C2, and C3, which had mol wts of approximately 60,000, 13,000 and 6,500, respectively. The distribution of relaxin immunoactivity among C1, C2, and C3 changed with the day of pregnancy. On day 15 of pregnancy essentially all relaxin immunoactivity was associated with C1. By day 17 approximately 20% of the relaxin immunoactivity was in C2 plus C3, and by day 21 the percentage in each of these two components increased to about 25%. C1 appears to possess relaxin bioactivity since the frequency of intrauterine pressure cycles in nonpregnant rats declined during cross-circulation with day 15 pregnant rats. C1 may not be attributable to the binding of 6,500 mol wt relaxin to a binding protein(s) since relaxin immunoactivity remained associated with a component(s) having a mol wt greater than 28,000 after incubation and gel filtration of day 15 serum in the presence of 2.0 M potassium thiocyanate at 37 C. Moreover, the levels of C1 as well as those of C2 and C3 were markedly increased in the peripheral serum of day-21 rats within 15 min of LH administration. C3 may be the 6,500-mol wt form of relaxin and C2 may be an intermediate in the processing of the 20,000-mol wt rat relaxin precursor to the 6,500-mol wt form of relaxin. The physiological significance, if any, of the multiplicity of relaxin-immunoactive components is unknown.

Immunohistochemical demonstration of relaxin in the genital tract of men.

The biotin-avidin immunoperoxidase staining method and antisera against highly purified porcine relaxin were utilized to localize relaxin in the male genital tract. Formalin-fixed tissue specimens from the prostate gland, the testis, epididymis, vas deferens and the seminal vesicle were studied. Specific relaxin immunoactivity was seen in the glandular epithelium of the prostate and also in the glandular epithelium of the seminal vesicle and in the ampullary part of the vas deferens. The testis and epididymis were negative.

Influence of light-dark cycle on antepartum serum relaxin and progesterone immunoactivity levels and on birth in the rat.

Relaxin and progesterone are produced by the corpora lutea in the pregnant rat. Relaxin immunoactivity levels are elevated in peripheral sera during the last 12 days of pregnancy. In rats maintained under a conventional photoperiod of 14 h of light (0600-2000 h) and 10 h of darkness (2000-0600 h), there is an antepartum elevation in serum relaxin to maximal levels coincident with the rapid decline in serum progesterone to basal levels during the light phase of the photoperiod 1 day before birth. Therefore, we postulated that this maximal elevation in serum relaxin levels may be temporally associated with functional luteolysis and linked to the photoperiod. In the present study the photoperiod was advanced near midpregnancy in order to examine further the relationship of the antepartum elevation in serum relaxin levels with both functional luteolysis and the photoperiod. Three groups of rats were maintained under a conventional photoperiod of 14 h of light (0500-1900 h) and 10 h of darkness until days 7 and 8 of pregnancy when the photoperiod was advanced 8 h in group 2 (G2) and advanced 18 h in G3 relative to the conventional photoperiod that was maintained in G1. Serum relaxin and progesterone levels were determined in blood samples obtained at 4-h intervals from 2000 h on day 19 of pregnancy until birth. The times of occurrence of birth, maximal relaxin levels, and decline of progesterone to basal levels in G2 and G3 were generaly advanced 50-60% of the advancement of the photoperiod. There was a close temporal association between the attainment of maximal relaxin levels and basal progesterone levels; they both occurred during the light phase of the photoperiod, approximately 24 h before birth in all three groups. We conclude that the antepartum elevation of serum relaxin to maximal levels may be associated with functional luteolysis and that its time of occurrence is influenced by the photoperiod. This study also provides evidence that the antepartum elevation of relaxin levels consists of two phases which occur at a 24-h interval. It is proposed that these two phases in the elevation of relaxin levels may be indicative of an increasingly effective endogenous circadian luteolytic process whose time of occurrence is influenced by the light-dark schedule.

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.