Pubic Ligament Research Articles

Structure of a porcine relaxin inactive on the mouse pubic ligament

In addition to B 31 (CM-a) and B 28 (CM-B) relaxins, acid-acetone extracts of ovaries of pregnant sows contain a third major relaxin species (relaxin C). The major components of relaxin C possess about half the activity of CM-a or CM-B in the guinea pig palpation assay, but are completely inactive in the mouse pubic ligament assay. Its uterotrophic and protein anabolic effects in ovariectomized, estrogen-primed mice, however, are comparable to those of CM-B. Sequence analysis indicates that the two major components of relaxin C, like the other porcine relaxins, consist of two polypeptide chains linked by disulfide bonds. The shorter (A) chains are identical to the A chains of the other porcine relaxins, except for the absence of the N-terminal arginine residue. The B chains display microheterogeneity; the B sequences of the two predominant species are the same as those of the other porcine relaxins through B 25, but terminate at valine residue B 25 or serine residue B 26, respectively.

Permanent chondrification in the pelvis and occurence of hernias in mice treated neonatally with tamoxifen

Male and female C57BL/Tw mice were given 5 daily subcutaneous injections of 100 μg tamoxifen (Tx), starting on the day of birth (Tx mice). In untreated fetal mice on day 18 of gestation, the greater part of the pubic and ischial bones were cartilaginous. At more than 30 days of age, however, untreated mice showed completely calcified pelvic bone, whereas in age-matched Tx mice the greater part of the junctional regions in the pelvis remained cartilaginous. Treatment with Tx starting within 5 days of age caused bladder hernia with or without cecum hernia. The pubic ligament in Tx mice at ages of 30–540 days was markedly expanded as compared with that in age-matched controls. The permanent chondrification in the pelvis was found in all mice given Tx starting within 10 days of age. By contrast, neonatal treatments of mice with other antiestrogens, clomiphene and nafoxidine (100 μg/day), induced neither permanent chondrification in the pelvis nor expansion of the pubic ligament nor hernia. These findings suggest that Tx has a specific effect on the symphysis pubis and some junctional regions of the developing pelvis in mice when given neonatally.

Effects of the hormone relaxin on the metabolism of the glycosaminoglycans in the mouse symphysis pubis.

The influence of the peptide hormone relaxin on the glycosaminoglycan (GAG) metabolism was investigated in the pubic ligament of the symphysis pubis and in serum of the virgin mouse. Fresh weight DNA and GAG content per 1 ligament is significantly increased, the level of water soluble protein is not affected. A shift in the electrophoretic GAG pattern by an increasing amount of hyaluronic acid and a decreasing amount of chondroitin sulfate and dermatan sulfate can be observed. Concerning GAG-splitting enzymes (N-acetylglucosaminidase, arylsulfatase, beta-glucuronidase) the N-acetylglucosaminidase reveals a significant increase of its activity in the interpubic ligament and in the serum. The data demonstrate that relaxin treatment induces some changes in the GAG metabolism.

Effects of porcine relaxins upon uterine hypertrophy and protein metabolism in mice.

Two variant forms of porcine relaxin (B and C) are active in producing relaxation of the guinea pig pubic symphysis and in effecting uterine growth in rats. Only relaxin B, however, is active in the mouse pubic ligament assay. These two hormones were compared in mice for their effects upon uterine growth and incorporation of radioactively labeled proline into soluble protein and collagen in vitro and in vivo. Both relaxin B and relaxin C produced an early (3-hr) elevation in in vitro protein synthesis and a later (6-hr) increase in collagen incorporation of proline at the time when the uterotrophic effect was maximal. In vivo effects of relaxin C on the uterus were in some cases greater than relaxin B in contrast to the complete inactivity of the former upon the pubic ligament of the mouse. These findings suggest a high degree of tissue specificity for relaxin stimulation, a variability in responsiveness among tissues in the same animal, and perhaps a primary role of relaxin in uterine function with pelvic relaxation representing a secondary function which has developed in certain species.

The Symphysis Pubis Anatomic and Pathologic Considerations

The symphysis pubis is a nonsynovial amphiarthrodial joint that is situated at the confluence of the two pubic bones. A thick intrapubic fibrocartilaginous disc is sandwiched between thin layers of hyaline cartilage. The inferior pubic ligament provides most of the joint's stability. Anatomic sections demonstrate a symphysis by the end of the second month of gestation. Thick cartilaginous end-plates are present at birth but become thin by the time of skeletal maturity. Congenital diseases resulting in failure of symphysis formation include exstrophy of the bladder and cleidocranial dysostosis. Both pyogenic and tuberculous infectious diseases involve the symphysis. Metabolic disease, such as renal osteodystrophy, produces widening, while ochronosis results in calcific deposits in the symphysis. Inflammatory disease, such as ankylosing spondylitis, results in bony fusion of the symphysis. Osteitis pubis, the most common inflammatory disease, is treated with anti-inflammatory medication and rest. Degenerative joint disease of the symphysis, which can cause groin pain, results from instability or from abnormal pelvic mechanics. As is the case with most joints, the symphysis serves as a barrier to tumor invasion. The patterns of trauma include diastasis, straddle fracture, intraarticular fracture and overlapping dislocation, and combinations of injuries.

A0-Phenylalanyl] relaxin (porcine): An active intermediate

A [phenylalanyl A0] relaxin has been isolated as a byproduct during large scale porcine relaxin preparations, using ion exchange chromatography on CM-cellulose at pH 7.8 followed by high performance liquid chromatography on reversed phase columns. The elongation at the N terminus of the A-chain has been demonstrated by amino acid and sequence analyses of the isolated and carboxymethylated relaxin-A-chain. The phenylalanyl relaxin and B29 relaxin are indistinguishable by circular dichroism spectroscopy, in mouse pubic ligament assay, and radioimmunoassay. The occurrence of phenylalanyl relaxin may be caused by an incomplete conversion of prorelaxin to relaxin.

Isolation and characterization of porcine and rat relaxin.

Hisaw (1926) first reported that experimentally induced “relaxation” of the pubic ligament in guinea pigs was controlled by hormones. In 1930 Fevold, Hisaw, and Meyer (1930) showed that an aqueous extract of sow corpora lutea caused relaxation of the pubic ligament in estrogen-primed, ovariectomized guinea pigs. They named the active substance relaxin. Essentially all subsequent efforts to isolate relaxin employed ovaries obtained from pigs daring late pregnancy since this source of relaxin is known to have a high content of relaxin activity and is relatively easy to acquire. Progress toward the isolation of porcine relaxin was slow. It was not until the 1950’s and early 1960’s when new techniques for the isolation and characterization of proteins, as well as improved methods for relaxin bioassay (Steinetz et al.3 1960, 1969), became available that well documented advancement was made in relaxin purification efforts.

Relaxin localization in porcine and bovine ovaries by assay and morphologic techniques.

Relaxin is a polypeptide hormone produced primarily by the ovaries during pregnancy in several mammalian species. Investigations beginning more than 50 years ago on isolation of hormones from the corpus luteum eventually showed that the lipid-soluble fraction yields progesterone and estrogens, whereas the water-soluble fraction contains relaxin. A brief report by Hisaw in 1926 describes induced relaxation of the pubic ligament of virgin guinea pigs within 6 to 8 hours after the subcutaneous injection of blood serum from pregnant rabbits. Hisaw (1925) previously had documented the relaxation of the pubic bones during late pregnancy in the guinea pig by expansion of the connective tissue at the symphysis, allowing separation of the pubic bones. During the early postpartum period, the ligaments shortened, and the pubic symphysis again appeared similar to that found in unmated guinea pigs. In the spring-breeding pocket gopher, he noted that the pubic symphysis was lost, usually before pregnancy occurs, and that the interpubic ligament response was controlled by the ovary. Fevold et al. (1930) developed extraction and purification methods for this hormone and proposed the name relaxin. Several investigators questioned the existence of relaxin since pelvic relaxation could be produced in ovariectomized guinea pigs by estrogen alone or by combinations of estrogen and progesterone.

Relaxin--a review.

Relaxin is a polypeptide hormone, similar in structure to insulin and has been found in the female of all species studied. The corpus luteum of pregnancy is the main source of relaxin in many species but in others the decidua is apparently of greater importance. It has also been found in other tissues; e.g. prostatic fluid, testis and ovary. First discovered and extracted from the corpora lutea of pregnant sows in an impure form in 1926, it was found to relax the pubic ligament of the oestrogen primed guinea-pig. It was named after this action, but has since been found to have many other possible roles, including preparation of the endometrium for implantation, inhibition of uterine activity in early pregnancy, remodelling of the uterine stroma during pregnancy, cervical ripening and the initiation of parturition. Relaxin's main cellular action in pregnancy may be to drive collagen biosynthesis in its target organs, thus facilitating the remodelling of the connective tissue. Due to the impurity of relaxin preparations used in clinical trials until the mid-1970's, the role of relaxin in the human has been in doubt. Porcine and rat relaxins have now been highly purified and their detailed structure is known. Human relaxin awaits adequate isolation, purification and characterization, and is not yet available for laboratory and clinical trials. However, the recent preparation of purified porcine relaxin for clinical trials and the availability of specific radioimmunoassays for this relaxin together with the identification of relaxin receptor sites, are rapidly helping to establish the concept that relaxin is indeed an important hormone both in human reproduction and in other physiological processes.

Relaxin immunoactivity in plasma during the reproductive cycle of the female guinea pig.

Plasma relaxin immunoactivity was measured in guinea pigs during the estrous cycle from midpregnancy to parturition as well as during acute suckling using a homologous porcine relaxin radioimmunoassay. Samples were collected at frequent intervals from an indwelling jugular vein catheter. The peak levels found during pregnancy, ∿12 ng/ml, were higher than the peak levels during the estrous cycle and suckling (4–5 ng/ml). During the estrous cycle, relaxin levels were low at estrus and early diestrus and were high at late diestrus and proestrus preceeding the opening of the vaginal membrane. During late pregnancy, episodic secretion of immunoreactive relaxin was common and the timing of this secretion varied. In some guinea pigs, relaxin levels increased in mid to late pregnancy; others showed only a rise before parturition or only the mid to late pregnancy rise. Spectral analysis of the data revealed clear periodicities in the results from several animals but others showed no evidence of periodicity. The most common period was 18 h. In most animals, but not all, the onset of lengthening of the pubic ligament as determined by palpation was preceded by a period of relaxin production. There was no evidence for an acute effect of suckling to elevate relaxin secretion in this species; indeed suckling decreased relaxin immunoactivity with mean levels of control animals significantly higher than those of animals suckling their young.

Hormonal requirements for interpublic ligament formation in hypophysectomized mice.

We have studied the effects of hypophysectomy on hormonally induced “relaxation” of the pubic symphysis of mice. Hypophysectomy prevented interpubic ligament formation due to injection of estradiol cyclopentylpropionate (ECP). Combinations of ECP and relaxin were likewise less effective in hypophysectomized than in unoperated mice. Administration of thyroid did not improve the effectiveness of ECP, but enhanced the activity of a combination of ECP and relaxin, in hypophysectomized mice. STH preparations restored pubic ligament response both to ECP and to the combination of ECP and relaxin. Evidence is presented which suggests, but does not prove, that the action of STH extracts on the symphysis pubis may be due to STH activity proper, and not to contaminants.

The enhancement of relaxin-induced growth of the pubic ligament in mice.

Using the x-ray measurement of interpubic distance as an assay procedure, it was found that purified relaxin which is of low potency in mice can be enhanced in activity by delaying its absorption from the site of injection. Of the depot media and binding agents which were tested, the most effective was a suspension of lyophilized relaxin in 5% beeswax and oil, which produced a 65-fold increase in assay potency. In contrast, the mouse-active fraction of sow ovaries, which is notabty less soluble than relaxin, was not potentiated by the depot medium. It was therefore concluded that the growth response of the pubic ligament can be obtained with depot relaxin, and that the mouse-active fraction represents a more slowly mobilized form of relaxin. The relationship of these findings to the interpretation of bioassay data was discussed, and a modified procedure was suggested to provide a quantitative assay of relaxin in mice.

RELAXATION OF THE PELVIS IN GUINEA-PIGS FOLLOWING IMPLANTATION OF PITUITARY GLANDS

Relaxation of the pubic symphysis and sacro-iliac ligaments in guinea-pigs was obtained with implants from pituitary glands of rats and guinea-pigs. The extent of relaxation was more pronounced than any previously described for other methods. These changes were not produced unless the ovaries were present.

Influence of Temperature on the Distensibility of the Pubic Ligament.

SummaryThe effect of temperature on the distensibility of the pubic ligament of the guinea pig was investigated in intact and isolated preparations using a constant load. Both types of preparations exhibited a marked: dependence of stretching on temperature between 30 to 40°C with the mode lying in the, range 34-35°C under conditions of moderate loading.Distension at 30°C is negligible, while at temperatures above 34 it is rapid. Preparations from pregnant animals show a lower threshold both for temperature and weight.

THE RÔLE OF THE FEMALE SEX HORMONE

To describe the rôle of the female sex hormone ten years ago would have been a simple matter, as this hormone alone would have had to be considered, the main attention then being focused on the corpus luteum.¹Four years ago the situation became more complicated when Philip Smith,²and almost simultaneously Zondek and Aschheim,³showed that the prepituitary was necessary to activate the ovaries. A further complication was added when Hisaw⁴discovered an aqueous corpus luteum factor which affected the pubic ligaments of the gopher, and Corner⁵concentrated "progestin," the special lipoid extract of the corpus luteum. Today, therefore, when analyzing the processes of the sex physiology in the female, it is necessary for one to consider all these chemical messengers. The structures to be analyzed now include the follicle,⁶which excretes female sex hormone alone except in pregnancy, at which time prepituitary hormone also

Inhibition of Ovulation and Associated Histological Changes.

The idea that the corpus luteum is responsible for the inhibition of ovulation seems to have been first elaborated by Beard and by Prenant. The experimental proof of the inhibitory action of the corpus luteum has been attacked in many different ways. The two most general methods have been based on anatomical and physiological modifications of the reproductive tract, correlated with the presence or absence of a functional corpus luteum, and, changes produced by the injection of extracts of lutein tissue. Corner and Hurni reported negative results in an attempt to inhibit ovulation by injecting corpus luteum extracts in normal white rats. Loeb failed to obtain consistent positive results in the guinea pig, while Papanicalaou recently reported inhibition of ovulation in the same animal. Pearl and Surface were able to stop hens from laying by injections of water extracts of a dried commercial preparation. Kennedy described positive results on the rabbit for extracts made from a desiccated commercial preparation which were injected for a period of time before mating, resulting in the inhibition of ovulation. Parkes and Bellerby reported inhibition of ovulation in mice by injections of an emulsion of an ether extract of the corpus luteum of the cow. They noted that not all of their extracts were potent, and that those that were soon deteriorated. Many different kinds of extracts have been employed by various investigators but the results obtained through their use are so contradictory that they cannot be discussed in this brief report. An acid alcohol extract of the fresh corpus luteum of the sow has been prepared in this laboratory and refined to the extent that very little protein remains. This preparation is capable of sensitizing the uterus of spayed rats and guinea pigs so that placentomata may be produced (Weichert). It will relax the pubic ligaments of virgin guinea pigs in oestrum (Hisaw)10; and will inhibit ovulation and oestrum in the normal female albino rat. Inhibition of oestrum was best effected by subcutaneous injections of an amount equivalent to 3 gm. of fresh tissue given every 4 or 5 hours from the beginning of oestrum until the experiment was ended. Rats injected in this manner did not come into oestrum for 6 to 12 days, the length of time for which ovulation was inhibited depending upon the amount of the extract injected. Attempts are now being made to inhibit ovulation for a greater period and to determine at what stage of the dioestrous interval injections may be most effective.