RESUMEN
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.
Asunto(s)
Catepsinas/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Periodo Posparto/metabolismo , Preñez/metabolismo , Sínfisis Pubiana/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Animales , Catepsinas/genética , ADN/química , ADN/genética , Femenino , Metaloproteinasas de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Reacción en Cadena de la Polimerasa , Embarazo , Sínfisis Pubiana/enzimología , Sínfisis Pubiana/ultraestructura , Inhibidores Tisulares de Metaloproteinasas/genéticaRESUMEN
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.
Asunto(s)
Granulocitos/ultraestructura , Preñez/fisiología , Sínfisis Pubiana/fisiología , Sínfisis Pubiana/ultraestructura , Animales , Western Blotting , Femenino , Inmunohistoquímica , Metaloproteinasa 8 de la Matriz/metabolismo , Ratones , Microscopía Electrónica , Embarazo , Sínfisis Pubiana/citologíaRESUMEN
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.
Asunto(s)
Glicosaminoglicanos/análisis , Glicosaminoglicanos/ultraestructura , Preñez/fisiología , Proteoglicanos/análisis , Proteoglicanos/ultraestructura , Sínfisis Pubiana/metabolismo , Sínfisis Pubiana/ultraestructura , Animales , Condroitín Liasas/metabolismo , Electroforesis en Gel de Agar , Femenino , Glicosaminoglicanos/química , Ácido Hialurónico/análisis , Inmunohistoquímica , Indoles/farmacología , Ratones , Compuestos Organometálicos/farmacología , Embarazo , Proteoglicanos/química , Azufre/químicaRESUMEN
Reference is usually made to the parallel orientation towards the main line of exerted tension at the pubic joint in mice, for supporting forces applied to the joint. Despite the wealth of morphological information about the extracellular matrix in this joint, little is known regarding the involvement of the crimp of collagen fibers in the dramatic transformations occurring in this region during the last 3 days of pregnancy. Examination of the collagenous architecture suggests that the biomechanical properties are directly related to fibril diameters, composition of ground substance and changes in the bundle morphology, particularly in the crimp structure. The purpose of this study was to further describe the transformation of the collagen fibers of the pubic symphysis during late mouse pregnancy. We examined the architecture of collagen fibers in the symphysis and pubic ligament through the Picrosirius-polarization method and also through scanning electron microscopy to directly visualize and measure the crimping from pregnant and virgin mice. The crimp angle and the length of five consecutive crimps were measured according to Patterson-Kane et al. [Connect. Tissue Res. 36 (1997) 253]. It could be demonstrated that the angles progressively decreased and the crimp length increased, denoting that the fibers have untwisted during the relaxation process. Our findings suggest that a disruption of the helical arrangement of the collagen containing fibers may contribute to explaining the rapid remodeling that occurs at the end of pregnancy and that is responsible for an increase in pliancy and length of the pubic ligament in mice.
Asunto(s)
Colágeno/ultraestructura , Preñez/metabolismo , Sínfisis Pubiana/ultraestructura , Animales , Colágeno/metabolismo , Femenino , Ratones , Microscopía Electrónica de Rastreo , Embarazo , Sínfisis Pubiana/citologíaRESUMEN
The expansion of the pubic symphysis during pregnancy in some rodet depends on the growth of the interpubic ligament, primarily through the biosynthesis of extracellular matrix. Although there is a significant metabolism of elastin in the female reproductive tract during pregnancy, little is known of the architectural and ultrastructural aspects of the elastic system fibers in the mouse pubic symphysis. In this study, the main characteristics of the elastic system of the mouse pubic symphysis during pregnancy, partum and post-partum were determined by light and electron microscopy. A distinct arrangement of microfibrils, elastin deposition and development of the extracellular matrix formed a supporting framework that uniformly distributed stress in order to provide adequate interpubic resilience during delivery. These changes support a role for elastic system fibers in symphyseal maturation and reconstruction during pregnancy and after delivery.
Asunto(s)
Animales , Embarazo , Ratas , Microscopía Electrónica/métodos , Fibras Musculares Esqueléticas , Sínfisis Pubiana/anatomía & histología , Sínfisis Pubiana/anomalías , Sínfisis Pubiana/ultraestructuraRESUMEN
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.
Asunto(s)
Proteoglicanos/análisis , Proteoglicanos/ultraestructura , Sínfisis Pubiana/química , Animales , Cartílago/química , Colorantes/química , Femenino , Inmunohistoquímica , Ratones , Microscopía Electrónica , Microscopía de Polarización/métodos , Sínfisis Pubiana/ultraestructuraRESUMEN
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.