Interpubic Ligament Research Articles

Multiparity and Aging Impact Chondrogenic and Osteogenic Potential at Symphyseal Enthesis: New Insights into Interpubic Joint Remodeling

Pregnancy and childbirth cause adaptations to the birth canal to allow for delivery and fast recovery. To accommodate delivery through the birth canal, the pubic symphysis undergoes changes that lead to the interpubic ligament (IpL) and enthesis formation in primiparous mice. However, successive deliveries influence joint recovery. We aimed to understand tissue morphology and chondrogenic and osteogenic potential at symphyseal enthesis during pregnancy and postpartum in primiparous and multiparous senescent female mice. Morphological and molecular differences were found at the symphyseal enthesis among the study groups. Despite the apparent incapacity to restore cartilage in multiparous senescent animals, the symphyseal enthesis cells are active. However, these cells have reduced expression of chondrogenic and osteogenic markers and are immersed in densely packed collagen fibers contiguous to the persistent IpL. These findings may indicate alterations of key molecules in the progenitor cell population maintenance of the chondrocytic and osteogenic lineages at the symphyseal enthesis in multiparous senescent animals, possibly compromising the mouse joint histoarchitecture recovery. This sheds light on the distention of the birth canal and the pelvic floor that may play a role in pubic symphysis diastasis (PSD) and pelvic organ prolapse (POP), both in orthopedic and urogynecological practice in women.

An exceptional case of bladder exstrophy with pubic symphysis diastasis in a 3-year-old male child

Bladder exstrophy is a rare birth defect in which the anterior wall of the abdomen over the bladder is not closed in the intrauterine period, hence the child born with exposed bladder. Incidence of bladder exstrophy is 1/10,000 childbirths. Complaints would vary from exposed bladder, incontinence or leakage of urine, defect in genitals and pelvic bones as well as defect in intestine and reproductive organs. Mostly patient get operated with bladder exstrophy repair in infant age. If it gets neglected, it needs stage wise surgery in this case. Parents of a 3-year-old male child came to Paediatric surgeon OPD with congenital bladder exstrophy. Orthopaedic surgeons´ opinion was taken in view of pubic diastasis. In infant age, there is no need of additional concern of pubic diastasis. At age of 3 acetabulo-pelvic surgeon advice for External fixator application if pelvic wings to decrease tension on reconstructed bladder wall and inter-pubic ligament. Patient managed as neglected bladder exstrophy. Reconstruction of abdominal wall and urethra reconstruction done and application of external fixator application done.

Evidence of macrophage modulation in the mouse pubic symphysis remodeling during the end of first pregnancy and postpartum

In mouse pregnancy, pubic symphysis (PS) remodels into an elastic interpubic ligament (IpL) in a temporally regulated process to provide safe delivery. It restores at postpartum to assure reproductive tract homeostasis. Recently, macrophage localization in the IpL and dynamic changes in the expression of inflammatory mediators observed from the end of pregnancy (D18, D19) to early days postpartum (1dpp, 3dpp) highlighted the necessity of the identification of the key molecules involved in innate immune processes in PS remodeling. Therefore, this study uses morphological and high-sensitivity molecular techniques to identify both macrophage association with extracellular matrix (ECM) remodeling and the immunological processes involved in PS changes from D18 to 3dpp. Results showed macrophage association with active gelatinases and ECM components and 25 differentially expressed genes (DEGs) related to macrophage activities in interpubic tissues from D18 to 3dpp. Additionally, microarray and proteomic analysis showed a significant association of interpubic tissue DEGs with complement system activation and differentially expressed proteins (DEPs) with phagocytosis, highlighting the involvement of macrophage-related activities in mouse PS remodeling. Therefore, the findings suggest that PS ECM remodeling is associated with evidence of macrophage modulation that ensures both IpL relaxation and fast PS recovery postpartum for first labor.

Recruitment of monocytes and mature macrophages in mouse pubic symphysis relaxation during pregnancy and postpartum recovery†.

Appropriate remodeling of the female lower reproductive tract and pelvic floor is essential during normal mammalian pregnancy, labor, and postpartum recovery. During mouse pregnancy, in addition to reproductive tract modifications, the pubic symphysis (PS) is remodeled into a soft interpubic ligament (IpL) to provide safe delivery of the offspring and fast postpartum recovery. Although temporal changes in the phenotypes of myeloid cells, such as mononuclear phagocytes, are crucial to remodeling the lower reproductive tract organs in preparation for a safe delivery, little is known about the involvement of recruited monocytes or macrophages in mouse PS remodeling. We used combined light microscopy, electron microscopy, and qPCR analysis to investigate the profile of recruited monocytes and macrophage polarization markers in C57Bl6 mouse interpubic tissues during pregnancy (D12, D18, and D19) and early days postpartum (1 dpp and 3 dpp) to better identify their presence in proper remodeling of the mouse PS. Our morphological data show that the number of recruited monocytes is increased in interpubic tissues and that recruited monocytes differentiate into proinflammatory or anti-inflammatory macrophage phenotypes from D18 to 3 dpp, which may contribute to dynamic changes in the gene expression of specific inflammatory mediators involved in interpubic tissue remodeling at these time points. Therefore, our morphological and quantitative gene expression data suggest that both differentiated macrophages from recruited monocytes and polarized macrophages may collaborate for IpL relaxation at labor and the appropriate repair of the PS after the first pregnancy.

Time-dependent regulation of morphological changes and cartilage differentiation markers in the mouse pubic symphysis during pregnancy and postpartum recovery.

Animal models commonly serve as a bridge between in vitro experiments and clinical applications; however, few physiological processes in adult animals are sufficient to serve as proof-of-concept models for cartilage regeneration. Intriguingly, some rodents, such as young adult mice, undergo physiological connective tissue modifications to birth canal elements such as the pubic symphysis during pregnancy; therefore, we investigated whether the differential expression of cartilage differentiation markers is associated with cartilaginous tissue morphological modifications during these changes. Our results showed that osteochondral progenitor cells expressing Runx2, Sox9, Col2a1 and Dcx at the non-pregnant pubic symphysis proliferated and differentiated throughout pregnancy, giving rise to a complex osteoligamentous junction that attached the interpubic ligament to the pubic bones until labour occurred. After delivery, the recovery of pubic symphysis cartilaginous tissues was improved by the time-dependent expression of these chondrocytic lineage markers at the osteoligamentous junction. This process potentially recapitulates embryologic chondrocytic differentiation to successfully recover hyaline cartilaginous pads at 10 days postpartum. Therefore, we propose that this physiological phenomenon represents a proof-of-concept model for investigating the mechanisms involved in cartilage restoration in adult animals.

Recovery of the pubic symphysis on primiparous young and multiparous senescent mice at postpartum.

It has been observed that parturition has a significant effect on female skeletal architecture and that age alters musculoskeletal tissues and their functions. We therefore hypothesized that multiparity affects the recovery of the pubic symphysis in senescent mice at postpartum and the morphology of the interpubic tissues. The pubic symphysis of primiparous young, virgin senescent (VS) and multiparous senescent (MS) Swiss mice was examined by light microscopy, transmission electron microscopy, morphometric analysis and immunohistochemistry. The mouse pubic symphysis was remodeled during the first pregnancy: the cellular phenotype and morphology changed to ensure a structurally safe birth canal, followed by recovery of the interpubic articulation after birth. The morphology of the pubic symphysis in the VS group was maintained in a state similar to that observed in virgin young mice. In contrast, MS mice exhibited an interpubic ligament characterized by extended fibrocyte-like cells, an opened interpubic articulation gap, compacted and thin collagen fibrils and scarce galectin-3-positive cells. Thus, we found that the cellular and extracellular characteristics of the pubic symphysis were altered by multiparity in senescent mice. These particular tissue characteristics of the MS group might be associated with an impaired recovery process at postpartum. Thus, a better understanding of the alterations that occur in the birth canal, including the pubic symphysis, due to multiparity in reproductively aged mice may contribute to our comprehension of the biological mechanisms that modify the skeleton and pelvic ligaments and even play a role in the murine model of pelvic organ prolapse.

Elastic Fiber Assembly in the Adult Mouse Pubic Symphysis During Pregnancy and Postpartum1

Impairment of pelvic organ support has been described in mice with genetic modifications of the proteins involved in elastogenesis, such as lysyl oxidase-like 1 (LOXL1) and fibulin 5. During pregnancy, elastic fiber-enriched pelvic tissues are modified to allow safe delivery. In addition, the mouse pubic symphysis is remodeled in a hormone-controlled process that entails the modification of the fibrocartilage into an interpubic ligament (IpL) and the relaxation of this ligament. After first parturition, recovery occurs to ensure pelvic tissue homeostasis. Because ligaments are the main supports of the pelvic organs, this study aimed to evaluate elastogenesis in the IpL during mouse pregnancy and postpartum. Accordingly, virgin, pregnant, and postpartum C57BL/6 mice were studied using light, confocal, and transmission electron microscopy as well as Western blots and real-time PCR. Female mice exhibited the separation of the pubic bones and the formation, relaxation, and postpartum recovery of the IpL. By the time the IpL was formed, the elastic fibers had increased in profile length and diameter, and they consisted of small conglomerates of amorphous material distributed among the bundles of microfibrils. Our analyses also indicated that elastin/tropoelastin, fibrillin 1, LOXL1/Loxl1, and fibulin 5 were spatially and temporally regulated, suggesting that these molecules may contribute to the synthesis of new elastic fibers during IpL development. Overall, this work revealed that adult elastogenesis may be important to assure the elasticity of the pelvic girdle during preparation for parturition and postpartum recovery. This finding may contribute to our understanding of pathological processes involving elastogenesis in the reproductive tract.

Changes of large molecular weight hyaluronan and versican in the mouse pubic symphysis through pregnancy.

During pregnancy, the mouse pubic symphysis undergoes expansion and remodeling resulting in formation of a flexible and elastic interpubic ligament allowing passage of a term fetus. In the current study, we sought to identify and characterize components of the extracellular matrix that likely play an important role in elongation and flexibility of the interpubic ligament during parturition. Mouse pubic symphyses and interpubic ligaments collected at time points during pregnancy and postpartum were utilized to evaluate collagen type, collagen content, processing and solubility, matricellular protein, and proteoglycan expression and quantitative assessment of all glycosaminoglycans. These studies revealed increased gene expression for hyaluronan synthase 1, hyaluronan synthase 2, and versican on Gestation Day 18 as well as a decline in protein expression for the versican-degrading protease a disintegrin-like and metalloprotease with thrombospondin type 1 (ADAMTS1) motif. These findings suggest that the primary mediators of increased elongation and flexibility of the interpubic ligament at term result from increased synthesis and reduced metabolism of viscoelasticity-promoting molecules such as high molecular weight hyaluronan and versican.

High iNOS mRNA and protein localization during late pregnancy suggest a role for nitric oxide in mouse pubic symphysis relaxation

Remodeling and relaxation of the mouse pubic symphysis (PS) are central events in parturition. The mouse PS remodels in a hormone-controlled process that involves the modification of the fibrocartilage into an interpubic ligament (IpL), followed by its relaxation prior to parturition. It is recognized that nitric oxide synthase (NOS) and consequently nitric oxide (NO) generation play important roles in extracellular matrix modification, and may promote cytoskeleton changes that contribute to the remodeling of connective tissue, which precedes the onset of labor. To our knowledge, no studies thus far have investigated inducible nitric oxide synthase (iNOS) expression, protein localization, and NO generation in the mouse PS during pregnancy. In this work, we used a combination of the immunolocalization of iNOS, its relative mRNA expression, and NO production to examine the possible involvement of iNOS in remodeling and relaxation of the mouse IpL during late pregnancy. The presence of iNOS was observed in chondrocytes and fibroblast-like cells in the interpubic tissues. In addition, iNOS mRNA and NO production were higher during preterm labor on Day 19 of pregnancy (D19) than NO production on D18 or in virgin groups. The significant increase in iNOS mRNA expression and NO generation from the partially relaxed IpL at D18 to the completely relaxed IpL at D19 may indicate that NO plays an important role in late pregnancy during relaxation of the mouse IpL.

Effects of Relaxin on Relapse After Experimental Tooth Movement in Rats

Long-term stability is the major goal of orthodontic treatment. However, long-term observation of treated cases after retention often reveals a disturbing degree and frequency of relapse. Relaxin is a member of the insulin/relaxin family of structurally related hormones. This hormone is produced in many mammals during pregnancy, and has been shown to promote cervical softening and elongation of interpubic ligaments in mice and cattle. Furthermore, relaxin is related to many other physiologic processes, such as collagen turnover, angiogenesis, and antifibrosis in both males and females. So far, no study concerning the effect of relaxin on orthodontic tooth movement has been done. The purposes of this study were to investigate whether local administration of relaxin in rats has an effect on relapse after orthodontic tooth movement and to explore the molecular mechanism of its role. In the present study, the upper first molars were moved mesially in 16male, 6-week-old, Wistar strain rats using a coil spring with a force of 10g. After 14days, the appliance was removed, animals in the experimental group were given attachment gingivae injections of relaxin at 20μl for 7days, and animals in the control group received phosphate-buffered saline. The results were evaluated by micro-computed tomography (μCT). At days 0, 14 and 21 after tooth movement, histopathological features were examined by immunohistochemistry based on proliferating cell nuclear antigen (PCNA). Relapse distances and percentages were significantly lower in the experimental group compared to those of the control group. Relapse in the control group was greater and faster than that in the experimental group. The ratio of PCNA-positive cells increased at day 14 after tooth movement and then decreased at day 21. Our results indicated that relaxin inhibited the relapse of experimentally moved rat molars, and this hormone might prevent relapse following orthodontic treatment.

Development of Prepubertal Mouse Interpubic Joint Sensitivity to Estrogen and Relaxin.

Relaxin (RLX) induces uterine growth and causes the cartilaginous caps of the interpubic (IP) joint to resorb and form a flexible ligament, facilitating parturition in humans, guinea pigs and mice. In non-pregnant mice, estrogen priming is required for increasing sensitivity to RLX resulting in uterine growth and IP ligament lengthening. This RLX-induced growth of the IP ligament is the basis for a quantitative, in vivo bioassay. In 1955, Crelin and Levin reported that estrogen priming of prepubertal mice increased IP joint sensitivity to a crude extract of RLX in an age-dependent manner with sensitivity developing only after postnatal week (PNW) 3. However, the molecular basis for estrogen-dependent sensitivity of the IP joint during prepubertal development is unknown. Thus, objectives were to: 1) confirm when mouse IP joint sensitivity to RLX develops and 2) determine the effects of estrogens and RLX on estrogen receptor-alpha (ESR1) expression in the IP joint and uterus during prepubertal development. Since estrogens bind ESR1 in the reproductive tract, ESR1 was a marker of interest with respect to uterine and IP ligament growth. Mice were studied: 1) before (PNW 2 at d 14; n = 10/group) and 2) after (PNW 5 at d 35; n = 12/group) reported development of IP joint sensitivity to hormones. Mice were primed with estradiol cyclopentylpropionate (E) or peanut oil vehicle (C) and six days later treated with purified porcine RLX or benzopurpurine-4B vehicle (to facilitate slow release of RLX) 24 h before sacrifice. IP ligament lengths were measured using standard bioassay methods for RLX: a transilluminator equipped with a micrometer. Uterine and IP joint proteins were examined by immunoblotting and quantified by densitometry. At PNW 2 and 5, uterine weight increased (p < 0.05) in response to E and E+RLX when compared to controls. Crelin and Levin defined significant ligament lengthening as an IP separation of 0.5 mm or greater. In this study, eight out of nine E+RLX treated mice at PNW 2 met that criterion. Relaxin increased IP ligament length in E-primed mice when compared to controls (p < 0.05) at both PNW 2 and 5. At PNW 5, RLX alone reduced ESR1 protein in the uterus (p < 0.05) and IP ligament (p < 0.001). These studies suggest that the E-primed mouse uterus and IP joint are sensitive to purified porcine RLX as early as PNW 2. Detection of tissue sensitivity to E+RLX in mice after two weeks of life differs from the original Crelin and Levin observations and may be due to differences in methods of IP ligament measurement, type of estrogen used for priming and/or the purity of the RLX used in these two studies. This sensitivity of immature mouse tissues to E and RLX is consistent with hormone responsiveness reported in neonatal tissues of pigs and rats and may be important for understanding how neonatal exposure to such growth-promoting hormones affects long-term development. The significance of a decline in uterine and IP ligament ESR1 at PNW 5 in response to RLX is unclear. That E priming was effective in preventing the RLX-induced decline in ESR1 in both tissues points to the importance of estrogen in maintaining its own receptor. Estrogens increase uterine RLX receptor expression in rats and pigs and may play a similar role in mouse tissues. Thus, further studies are needed to evaluate expression of the RLX receptor during development of the mouse uterus and IP ligament. (Support: Aresty and GH Cook Undergraduate Research Programs) (poster)

Relaxation of the mouse pubic symphysis during late pregnancy is not accompanied by the influx of granulocytes

In some animals, such as mice and guinea pigs, a hormonally controlled mechanism increases the flexibility of the pubic symphysis and enhances the cervical remodeling necessary for safe delivery. Cervical ripening during pregnancy is associated with a paradoxical influx of leukocytes. However, the changes in cell metabolism during relaxation of the mouse pubic symphysis for delivery have not been extensively studied. In this work, we used light microscopy and transmission and scanning electron microcopy, as well as immunohistochemistry and Western blotting for MMP-8, to investigate the involvement of granulocytes or resident stromal cells in the relaxation of the virgin pubic symphysis during late pregnancy (days 18 and 19, before delivery) in vivo and in explanted joints. MMP-8 was studied because this collagenase is a hallmark for cervical ripening associated with the influx of granulocytes during late pregnancy. Extensive dissolution and disorganization of the extracellular matrix was seen around fibroblastic-like cells in late pregnancy. In contrast to the cervix (positive control), morphological and immunohistochemical analyses revealed that there was no characteristic cellular inflammatory response in the interpubic tissue. Staining for MMP-8 was observed in chondroid and fibroblastic-like cells of virgin and relaxed interpubic ligament, respectively. However, no granulocytes were seen during the extensive remodeling of the pubic joint in late pregnancy. These results indicate that constitutive stromal cells may have an important role in tissue relaxation during remodeling of the pubic symphysis in pregnancy.

Hormonal Changes Accompanying Cigarette Smoke-Induced Preterm Births in a Mouse Model

Epidemiologic evidence indicates that maternal smoking increases the risk of preterm birth. While a number of plausible mechanisms for early delivery have been offered, the role of gestational hormones in this smoke-induced outcome is uncertain. Thus, a toxicologic study was performed to examine the effects and underlying hormonal mechanisms of mainstream cigarette smoke (MCS) exposure on gestational duration. Pregnant B6C3F1 mice were exposed by inhalation to MCS for 5 days/week (4 hrs/day) from Gestational Day (GD) 4 to parturition. Smoke-induced effects on gestational length, interpubic ligament length, maternal hormone secretion patterns (estradiol-17beta, progesterone, prolactin, and relaxin), body weight gain, postimplantation loss, litter size, and offspring sex ratio were examined. Dams exposed to MCS at a concentration equivalent to smoking less than one pack of cigarettes/day (carbon monoxide = 25 parts per million, total suspended particulates = 16 mg/m3) demonstrated a significant (P < 0.05) shortening of gestational duration (compared with pregnant, air-exposed mice). In addition, MCS-exposed mice sacrificed on GD 18 had significantly (P < 0.05) increased interpubic ligament length, elevated serum estrogen levels, and a reduced progesterone to estradiol-17beta ratio (compared with air-exposed controls); levels of progesterone and prolactin were only modestly decreased and increased, respectively, in the MCS-exposed mice. Smoke exposure had no significant effects on maternal relaxin levels, body weight gain, postimplantation loss, litter size, or sex ratio. Results of this study demonstrate that inhalation exposure of pregnant mice to a low dose of MCS shortens gestation and alters hormone secretory patterns, which are important for maintaining pregnancy and inducing parturition. These findings support the view that pregnant women who smoke (even modestly) may be at increased risk for preterm birth, and that early delivery may be related (at least partly) to MCS-induced.

Ultrastructural, immunohistochemical and biochemical analysis of glycosaminoglycans and proteoglycans in the mouse pubic symphysis during pregnancy

During pregnancy, an interpubic ligament is formed in the mouse pubic symphysis. In late stages, this ligament undergoes "relaxation" to allow proper delivery, which is expected on the 19th day. Proteoglycans and hyaluronic acid play an important role in the remodeling of the extracellular matrix in these tissues. Glycosaminoglycans and proteoglycans were studied by electron microscopic, immunohistochemical and biochemical methods in samples of mouse pubic symphysis from the 12th to 18th day of pregnancy. At the ultrastructural level, using cuprolinic blue and enzymatic digestion by chondroitin lyases, two types of proteoglycan filaments were observed in the fibrocartilage on the 12th day, as well as in D 15, D 17 and D 18 pubic ligaments. The only sulfated glycosaminoglycan in these filaments was chondroitin sulfate, as shown by chondroitin lyase treatment. Their electrophoretic mobility, before and after enzymatic degradation, corroborated this inference. The ratio of chondroitin sulfate/dry weight of symphysis showed two phases of increase: between D12 and D 15, and between D 17 and D 18. We suggest that the first corresponds mainly to an increase in decorin when the ligament is formed, and the second to versican, during "relaxation". Versican and hyaluronic acid, working as water holding molecules would be responsible for the hydration of the ligament at the end of pregnancy, allowing an increase in resiliency. The presence of hyaluronic acid was confirmed by labeling with HA-probe in the perichondrium, fibrocartilage and ligament. The role of collagen fibers as physical restrictors of the complete expansion of glycosaminoglycans and hyaluronic acid in tissue is discussed.

Porcine and Human Relaxin Bioactivity: Bioactivities of Porcine Relaxin and Human Relaxin Do Not Differ in Mice and Rats

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

Nutrition pathways to the symphysis pubis.

The blood supply of the symphysis pubis is still the subject of some debate. Classic anatomy books state that this joint is avascular, whereas some published works have shown blood vessels in young specimens. As several articular discs such as the knee menisci are known to have blood vessels in their peripheries, we decided to investigate the possible nutrition pathways to the interpubic disc and ligaments. We used 60 Wistar rats, male and female, aged between 28 and 32 days, or between 90 and 100 days. Samples were processed using a variety of techniques: regular histology, immunohistochemestry, India ink injection and corrosion casting. The interpubic disc consisted of an inner bearing portion and an outer fibrous rim. The interpubic ligaments and the fibrous rim were well vascularized in all groups. Marrow contacts between the interpubic disc and the subchondral bone were also observed. Blood vessels formed an authentic arterial circle embracing the joint, from which blood vessels branched into capillary loops facing the avascular inner bearing portion of the disc. These results confirm the need for future studies on the human symphysis pubis, to provide more details on its structure, which would enable clinicians such as physiotherapists to improve prognosis and treatment design. Future studies may also explain the pathways down which the hormone relaxin reaches its targets within this joint.

Phenotypic modulation of fibroblastic cells in mice pubic symphysis during pregnancy, partum and postpartum.

In many species, the cartilaginous pubic symphysis of the pregnant female is gradually replaced by a fibrous connective tissue, forming a flexible and elastic interpubic ligament. This newly formed ligament is responsible for the separation of the pubic bones, enabling safe delivery of the young. Following labor, the ligament undergoes rapid involution. To our knowledge, no previous work has focused on the phenotypic modulation that is responsible for the changes present at the interpubic ligament throughout the relaxation and closing of the symphysis. The purpose of this study was to investigate the ultrastructural features and immunophenotype of the peculiar cell type found in the pubic symphysis of cycling, pregnant and postpartum mice. In particular, immunohistochemistry studies were conducted on the expressions of the cytoskeletal proteins desmin, vimentin and alpha-smooth muscle actin (alpha-SMA). During pregnancy, the pubic symphysis cells always expressed alpha-SMA, whereas the expression of vimentin and desmin was transient from early pregnancy to postpartum. Furthermore, the expression patterns of these three cytoskeletal proteins were distinct. Cells present in the medial region of the mouse symphysis in cycling and at D12 displayed ultrastructural features characteristic of a typical fibroblast. In contrast, during the last week of pregnancy and in postpartum these cells acquired ultrastructural features representative of a myofibroblast; for example, a fibronexus and a contractile apparatus were found to be present lying in close contact with the extracellular collagenous and elastic system fibrils. Taken together, these results strongly suggest a contractile function for these cells which might contribute to support of the varying mechanical stresses present during pubic bone movement.

Measurement of plasma and tissue relaxin concentrations in the pregnant hamster and fetus using a homologous radioimmunoassay.

A homologous hamster relaxin RIA was developed to evaluate plasma and tissue concentrations of relaxin in the latter half of pregnancy in this species. Relaxin protein and mRNA were localized using antibodies developed to synthetic hamster relaxin and gene-specific molecular probes, respectively. Molecular weight and isoelectric point of the synthetic and native hormones were identical by electrophoretic methods, and synthetic hamster relaxin was active in the mouse interpubic ligament bioassay. Synthetic hormone was used as tracer and standard with rabbit antiserum to the synthetic hormone in the RIA. Relaxin was assayed in blood samples recovered from the retro-orbital plexus on Days 6, 8, 10, 12, 14, 15, and 16 of gestation and on Days 1 and 5 postpartum. Relaxin was first detected on Day 8 of gestation (3.7 +/- 0.6 ng/ml), increased to reach a maximum in the evening of Day 15 (826.0 +/- 124.0 ng/ml), and decreased by Day 16 (day of parturition). Relaxin concentrations were assayed in aqueous extracts of implantation sites (Days 6, 8, and 10) and chorioallantoic placentae (Days 12, 14, and 15). Concentrations were low on Day 6 (0.02 +/- 0.001 microg/g tissue), increased to Day 15 (6.96 +/- 0.86 microg/g tissue), and subsequently declined by the evening of Day 15. Relaxin protein and mRNA were localized to primary and secondary giant trophoblast cells in the chorioallantoic placental trophospongium. However, relaxin protein was not localized in ovaries of pregnant animals or oviductal tissues of cycling animals. Significant quantities of relaxin were detected in the serum of fetal hamsters recovered on Day 15.

Mice without a Functional Relaxin Gene Are Unable to Deliver Milk to Their Pups

We have used gene targeting to generate relaxin (rlx)-deficient mice. The majority (15 of 17) of homozygous (rlx−/−) mice are fertile and produce normal litters. However their mammary development is deficient; pups are unable to suckle and die within 24 h of birth unless cross-fostered to a wild-type (rlx+/+) foster mother. The nipples of rlx−/− animals do not enlarge significantly during pregnancy, and their histology retains the appearance of the virgin state. Breast parenchyma is somewhat underdeveloped at term even though milk is produced. Mammary ducts become grossly dilated in these animals. Heterozygous (rlx+/−) mice lactate normally. The interpubic ligament does not relax during pregnancy in rlx−/− mice. Plasma osmolality during late gestation was significantly higher (P < 0.001) in rlx−/− mice than in wild-type controls.

Mice without a functional relaxin gene are unable to deliver milk to their pups.

We have used gene targeting to generate relaxin (rlx)-deficient mice. The majority (15 of 17) of homozygous (rlx-/-) mice are fertile and produce normal litters. However their mammary development is deficient; pups are unable to suckle and die within 24 h of birth unless cross-fostered to a wild-type (rlx+/+) foster mother. The nipples of rlx-/- animals do not enlarge significantly during pregnancy, and their histology retains the appearance of the virgin state. Breast parenchyma is somewhat underdeveloped at term even though milk is produced. Mammary ducts become grossly dilated in these animals. Heterozygous (rlx+/-) mice lactate normally. The interpubic ligament does not relax during pregnancy in rlx-/- mice. Plasma osmolality during late gestation was significantly higher (P < 0.001) in rlx-/- mice than in wild-type controls.