Ultrastructural and immunohistochemical analysis of proteoglycans in mouse pubic symphysis

Proteoglycans were accurately localized in mouse pubic symphyseal tissues using the cuprolinic blue method. Specific glycosaminoglycans degradative enzymes, together with chondroitin sulfate and decorin antibodies, allowed the identification of glycosaminoglycans. Chondroitin sulfate proteoglycans were the main proteoglycans observed in hyaline cartilage, fibrocartilage, and dense connective tissue. Ultrastructurally, they were seen as electron-dense granules and filaments. The granules, rich in chondroitin sulfate chains, were exclusively found in hyaline cartilage, whereas filaments were present in cartilage, fibrocartilage, and dense connective tissue. The latter were classified by size and susceptibility to enzyme digestion into F1, F2 and F3 filaments: F1 filaments were small, thin, and collagen fibril-associated; F2 filaments were thick, heavily stained, and localized around individual collagen fibrils and between bundles of collagen fibrils; and F3 filaments were scattered throughout elastic fiber surfaces. Considering their localization, susceptibility to chondroitinase AC and immunohistochemical detection, the symphysial F1 filaments were found to be preferentially decorin substituted with chondroitin sulfate side chains. The F2 filaments were also susceptible to chondroitinase AC treatment, whereas F3 filaments could be digested by heparitinase.The data thus obtained on the localization and identification of pubic symphyseal proteoglycans in virgin mice may be useful in the study of structural modifications that occur throughout pregnancy.     

The mouse pubic symphysis as a remodeling system: morphometrical analysis of proliferation and cell death during pregnancy, partus and postpartum

Marked changes in mice pubic symphysis occur by the end of pregnancy. Tissue remodeling involves a dynamic balance between cell proliferation and programmed cell death as well as changes in the extracellular matrix components. Therefore, it is important to consider both of these cellular behaviors when investigating the mechanism that regulates interpubic tissue remodeling, growth during late pregnancy and partus ensuring involution during the postpartum period. Proliferating and programmed death cells were identified by immunohistochemistry (proliferating cell nuclear antigen and TUNEL detection, respectively) and the rates at which these processes occurred were determined by morphometric analysis. The results demonstrated that cellular proliferation was intense during the period of ligament formation, from D15 to D18, thereafter abruptly declining on D19. From parturition (D19) onwards, an ever-increasing decline in the cellular proliferation levels could be observed. The quantitative analyses of cellular death showed opposite results when compared to cellular proliferation. During early pregnancy the cycle of cellular renovation was clearly proliferative and during late mouse pregnancy the cycle was directed by programmed cellular death. Although the high levels of cellular death during postpartum involution could be shown by the TUNEL-positive cells, we were unable to observed picnotic nucleus at the light microscopy.     

Estimation of age from the pubic symphysis by means of multiple regression analysis.

This study has been carried out to assess the age from the pubic symphysial surface employing a multiple regression analysis and a quantification theory model I analysis. Using partial regression coefficients and/or normalized scores obtained from the analyses, ages of skeletal remains can be quantitatively estimated with a fairly high reliability. The use of this method is, however, limited to the samples between 18 and 38 years of age, because age changes in the symphysial surface show large variations after about 40 years. The reliability of this method was also examined.     

Age changes in the pubic symphysis of Macaca mulatta.

Age changes in the pubic symphyses of 142 Cayo Santiago rhesus monkeys (known age, sex and maternal genealogy) are described. Symphyseal development is sexually dimorphic. Males generate a ridge and furrow system which is gradually replaced by a solid ankylosis at the mid-sagittal plane. Female development parallels that of the male until puberty. With pregnancy and delivery, relaxation and reaggregation of pelvic ligaments binding the symphysis destroy the symphyseal face. Continued bearing of offspring inhibits mid-sagittal ankylosis. Limited estimates of skeletal age can be made by using symphyseal development as an indicator of senescence.     

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 -smooth muscle actin (-SMA). During pregnancy, the pubic symphysis cells always expressed -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.This work was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). This paper is part of a thesis submitted to the Department of Histology and Embryology of the Biology Institute State University of Campinas, in partial fulfillment of the requirements for the Masters degree.     

Hyaluronan involvement in the changes of mouse interpubic tissue during late pregnancy and post-partum

The present work quantifies hyaluronan (HA) during the late pregnancy and post-partum in order to provide a better understanding of the role of HA in the adaptations that occur in the pubic symphysis during this period. HA was quantified in situ (histochemically) and in interpubic tissue extracts by fluorimetric assay. Samples were taken from virgin mice and from pregnant animals at various stages of pregnancy: 12th-18th days into pregnancy, the day of delivery (D19) and the 3rd and 5th day post-partum. The quantitative fluorimetric analysis indicated a gradual increase of HA in the interpubic tissue throughout late pregnancy (2.4-14.6 microg/mg dry weight). This was followed by a decrease beginning on D19 (12.4 microg/mg), reaching close to virgin levels (2.2 microg/mg) on the 5th day post-partum. The same optical density changes could be seen in the HA staining. Furthermore, the histochemical analysis demonstrated the presence of HA both in the extracellular matrix of the tissue and within its cells. Such results indicate that the extracellular presence of HA may contribute to the transformation of the symphysis into a flexible structure. In addition, HA's intracellular presence (until the 18th day of pregnancy) may contribute to cellular proliferation. Finally, during parturition and on the 5th day post-partum, HA may contribute to the maintenance of the myofibroblastic phenotype of ligament cells, aiding the ligament involution after parturition.     

Elastic fiber assembly in the adult mouse pubic symphysis during pregnancy and postpartum

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.     

人血清脂蛋白与糖胺聚糖及人主动脉蛋白聚糖的相互作用

本文报道了在[Ca~(2+)]=30mmol/L时,人血清或人血清脂蛋白与各种糖胺聚糖(GAG)及人主动脉两种蛋白聚糖(PG)的相互作用。GAG与血清的作用能力为6—硫酸软骨素(C6—S)>肝素(Hep)>4—硫酸软骨素(C4—S)>透明质酸(HA)>硫酸皮肤素(DS)。极低密度脂蛋白(VLDL)及低密度脂蛋白(LDL)可与肝素作用形成不溶性复合物,而高密度脂蛋白(HDL)则不能。人主动脉硫酸软骨素—PG(CS—PG)、硫酸皮肤素—硫酸软骨素—PG(DS—CS—PG)与血清形成不溶性复合物的曲线类型不同,后者的类型似有利于DS—CS—PG与血清脂蛋白结合从而使之在动脉壁沉积。     

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.     

Evolution of the mandibular symphysis in Notharctinae (Adapidae,Primates)

The Eocene Notharctinae provide a record of increasing fusion of the mandibular symphysis. The two sympatric genera,Notharctus andSmilodectes, differed through time in two respects.Notharctus increased in body size and evolved a partially fused mandibular symphysis.Smilodectes changed little in body size and retained an unfused symphysis. Similarities in molar morphology between these two genera and extant leaf-eating mammals suggest thatNotharctus andSmilodectes were specialized for folivory, a dietary regime correlated with partial symphyseal fusion in many extant mammals. It is concluded that the presence and the extant of symphyseal fusion is a function of body size, diet, and jaw mechanics, complicated by lineagespecific factors that vary among higher mammalian taxa.     

Synthesis of proteoglycans and hyaluronic acid by long-term cultures of testicular cells from immature and pubertal rats

Long-term cultures of somatic testicular cells derived from immature and pubertal rats were used to study the synthesis of proteoglycans (PG) and hyaluronic acid (HA). Labelled PG and HA in the culture medium, membrane-associated and intracellular pools were characterized by gel filtration, ion exchange chromatography and selected enzymatic and chemical treatments. Somatic cells synthesize a PG containing both heparan and chondroitin/dermatan sulfate (CS/DS) chains and a PG containing only CS/DS chains. No major qualitative changes in the type of PG were observed in cells derived from immature and pubertal animals. However, significant age-dependent differences in the cell distribution pattern of PG and HA were determined. This may have implications in the regulation of spermatogenesis.     

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.     

The pubic scars of gestation and parturition in a group of pre-Columbian and colonial Peruvian mummies

The pubic scars of pregnancy and parturition as reported in the anthropological literature are a variant of the recognized clinical entity, osteitis pubis. The os pubis from 86 pre-Columbian and colonial Peruvian mummies were examined for this entity. Osteitis pubis was found in 13 pre-Columbian females (72.3%) and 19 colonial Indians (57.6%). Four of the colonial women were buried with newborn children and one had a four-month-old fetus in utero. This type of study has application in paleopathology and forensic medicine to show possible frequency of pregnancy in a population, but must be refined and quantitated to achieve any more than this. This study is of further interest as it demonstrates that racial groups have apparently a wide difference in frequency of this particular lesion of the pubic symphysis, a matter for further investigation.     

Changes in the chondroitin sulfate-rich region of articular cartilage proteoglycans in experimental osteoarthritis

The chondroitin sulfate-rich region was cleaved from cartilage proteoglycans of experimental osteoarthritic canine joints to establish whether changes in this region of the molecule contribute to the well-documented increase in the chondroitin sulfate to keratan sulfate ratio in osteoarthritis. Experimental osteoarthritis was induced in eight dogs by severance of the right anterior cruciate ligament, the left joint serving as a control. Proteoglycans were extracted from the femoral cartilage of both joints, isolated as A1 fractions by associative density gradient centrifugation and cleaved with hydroxylamine. The chondroitin sulfate-rich region was isolated by either gel chromatography or dissociative density gradient centrifugation. The chondroitin sulfate-rich region from the proteoglycans of the experimental osteoarthritic joints was slightly larger in hydrodynamic size and had both a higher uronate/protein weight ratio and galactosamine/glucosamine molar ratio than the corresponding control. We conclude that the chondroitin sulfate-rich region of proteoglycans in articular cartilage of experimental osteoarthritic joints is larger and has more chondroitin sulfate than that of proteoglycans of normal cartilage.