RESUMO
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.
Assuntos
Trabalho de Parto/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Prenhez/metabolismo , Sínfise Pubiana/metabolismo , RNA Mensageiro/metabolismo , Análise de Variância , Animais , Condrócitos/metabolismo , Feminino , Fibroblastos/metabolismo , Imuno-Histoquímica , Ligamentos/química , Ligamentos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/genética , Gravidez , Prenhez/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
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.
Assuntos
Tecido Elástico/metabolismo , Período Pós-Parto/metabolismo , Sínfise Pubiana/metabolismo , Aminoácido Oxirredutases/metabolismo , Animais , Tecido Elástico/citologia , Elasticidade , Elastina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibrilina-1 , Fibrilinas , Ligamentos/citologia , Ligamentos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Pelve , Gravidez , Sínfise Pubiana/citologia , Proteínas Recombinantes/metabolismoRESUMO
Remodeling and relaxation of the mouse pubic symphysis (PS) are central events in parturition. The involvement of endogenous proteins such as matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs), and cathepsins in these phenomena remains unclear. In this work, we used a combination of immunolocalization, protein expression/activity, and relative messenger RNA (mRNA) expression to examine the changes in selected MMPs (-2, -9, and -8), TIMPs (-1 and -2), and cathepsins (B and K) during pregnancy and postpartum in mice. Immunohistochemistry revealed the presence of all of these proteins in the cytoplasm of chondrocytes, fibrochondrocytes, and fibroblast-like cells in the interpubic tissues. Zymography showed increases in the active forms of MMP-2 and -9 primarily on days 15 to 19 of pregnancy. Western blotting showed enhanced expression of MMP-8 on days 12 to 15 of pregnancy, with no changes in cathepsins B and K. Matrix metalloproteinases 2, TIMP-1 and -2, and cathepsin B had significant relative gene expression throughout pregnancy. These findings indicate that during pregnancy and postpartum there are variations in the expression and activity of proteins that may have an important role in remodeling the pubic symphysis during these events.
Assuntos
Catepsinas/metabolismo , Metaloproteinases da Matriz/metabolismo , Período Pós-Parto/metabolismo , Prenhez/metabolismo , Sínfise Pubiana/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , Animais , Catepsinas/genética , DNA/química , DNA/genética , Feminino , Metaloproteinases da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , Gravidez , Sínfise Pubiana/enzimologia , Sínfise Pubiana/ultraestrutura , Inibidores Teciduais de Metaloproteinases/genéticaRESUMO
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.
Assuntos
Matriz Extracelular/metabolismo , Ácido Hialurônico/metabolismo , Período Pós-Parto/metabolismo , Prenhez/metabolismo , Sínfise Pubiana/metabolismo , Animais , Feminino , Camundongos , GravidezRESUMO
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.
Assuntos
Glicosaminoglicanos/análise , Glicosaminoglicanos/ultraestrutura , Prenhez/fisiologia , Proteoglicanas/análise , Proteoglicanas/ultraestrutura , Sínfise Pubiana/metabolismo , Sínfise Pubiana/ultraestrutura , Animais , Condroitina Liases/metabolismo , Eletroforese em Gel de Ágar , Feminino , Glicosaminoglicanos/química , Ácido Hialurônico/análise , Imuno-Histoquímica , Indóis/farmacologia , Camundongos , Compostos Organometálicos/farmacologia , Gravidez , Proteoglicanas/química , Enxofre/químicaRESUMO
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.
Assuntos
Actinas/metabolismo , Desmina/metabolismo , Fibroblastos/ultraestrutura , Sínfise Pubiana/citologia , Vimentina/metabolismo , Animais , Feminino , Colágenos Fibrilares/metabolismo , Colágenos Fibrilares/ultraestrutura , Fibroblastos/metabolismo , Imuno-Histoquímica , Trabalho de Parto/fisiologia , Camundongos , Microscopia Eletrônica , Gravidez , Osso Púbico/citologia , Osso Púbico/metabolismo , Sínfise Pubiana/metabolismoRESUMO
The pubic joint of male and female rats was studied at the light- and electron microscopical levels using methods that selectively disclose the extracellular matrix fibres and glycosaminoglycans. The interpubic tissue showed no difference between sexes (including pregnant and intrapartum females). The medial ends of the pubic bones were covered by articular caps of hyaline cartilage that blended in the midline. The whole articular cartilage was covered dorsally and ventrally (as well as craneally and caudally) by a typical perichondrium. The differential distribution of the fibres of the collagenous and elastic systems in the pubic joint agreed with the results reported in the literature for other rat cartilages. Collagen fibres, composed mainly of type-I collagen, were localised to the fibrous perichondrium and bone. Type-II collagen was localised to the central nucleus of hyaline cartilage, whereas reticulin fibres (rich in type-III collagen) were found in the adventitial loose connective tissue adherent to the most superficial layer of the perichondrium. The central nucleus of hyaline cartilage possessed the two types of elastic-related fibres: elaunin fibres were localised mainly to the chondrogenic layer of the perichondrium, whereas oxytalan fibres were found in the matrix that surrounded the chondrocytes. The bulk of the glycosaminoglycans present in the pubic joint cartilage corresponded to hyaluronic acid and chondroitin sulphate. The propriety of classification of the rat pubic joint as a true synchondrosis (instead of symphysis), and the fact that the unaltered pelvis of the rat seems to be adequate for normal parturition, are discussed.