Immunohistochemical localization of procollagen types I and III during placentation in pregnant rats by type-specific procollagen antibodies.

To examine the sequential localizations of procollagen Types I (Pro I) and III (Pro III) during chorioallantoic placental formation in pregnant rats, we prepared polyclonal anti-rat Pro I- and III-specific antibodies. Biochemical analysis of a fraction containing [14C]-glycine-incorporated collagen from pregnant rat uteri showed that collagen Types I and III were actively synthesized during placental development. We examined 8-, 9.5-, 13-, and 20-day gestation rat uteri immunohistochemically. At Days 8 and 9.5, in the basal decidua facing the fetal cytotrophoblastic giant cell layer and implantation site, the immunoreactivity for Pro I was higher than that for Pro III. On Day 13, the enlarged myometrium and cytotrophoblastic cell layer showed increased immunoreactivity for Pro III. Unexpectedly, polygonal trophoblastic cells invading and modifying the maternal central artery showed intense immunoreactivity for Pro III. On Day 20, the fetal mesenchyme, large fetal blood vessels, and subendothelial stroma, including fetal blood capillaries, were more immunoreactive to Pro III antibody than to Pro I antibody in the labyrinth. Pro I and III synthesis and processing appear to be developmentally regulated and may be related to control of the microenvironment for supporting the fetus, control of the maternal blood supply stabilizing the fetoplacental physiological functions, and parturition.

Association of immunolocalization of matrix metalloproteinase 1 with ovulation in hCG-treated rabbit ovary.

Matrix metalloproteinase 1, MMP-1, which was previously called interstitial collagenase, is necessary for extracellular matrix reconstruction. The immunolocalization of the latent form of MMP-1 (proMMP-1) was examined in the ovulatory process in hCG-treated (100 iu per animal) rabbit ovaries. Immunoreactive products of proMMP-1 were identified by the avidin-biotin peroxidase complex formation using an anti-rabbit proMMP-1 polyclonal antibody. ProMMP-1 was distributed in the cytoplasm of theca interna cells, theca externa cells, interstitial glands and germinal epithelium throughout ovulation. However, at 9 or 10 h after hCG treatment, this enzyme was identified in several capillary lumina around the apex of preovulatory follicles. In addition, the staining density of immunoreactive products apparently increased in granulosa cells and theca interna cells around the orifice of the ruptured follicle 10 h after the stimulation. These results indicate that the spatiotemporal appearance of proMMP-1 in ovulation may be closely associated with the initiation of rupture of the follicle.

Influence of monocyte-macrophage lineage cells on alkaline phosphatase activity of developing osteoblasts derived from rat bone marrow stromal cells.

We have studied the osteogenic changes of the marrow stromal cells cultured from bone marrow cells of Wistar rats. After the stromal cells became confluent, several clusters of cuboidal cells appeared. These cuboidal cells showed strong alkaline phosphatase (ALP) activity and revealed some osteoblastic features electron-microscopically. In the culture, monocyte-macrophage-lineage cells (MMLC) proliferated in contact with the stromal cells. The MMLC exist near the bone cells in vivo. Yet the exact role of the MMLC is not clear. In order to evaluate how the MMLC influence the stromal cells, the ALP activity of the stromal cells in the culture condition containing a large number of MMLC (group A) was compared with that in the condition in a small number of MMLC (group B). The ALP activity in group A (11.28 +/- 1.04 Units/mg protein) was significantly higher than that in group B (6.58 +/- 0.38 Units/mg protein). This suggests that the MMLC stimulate the osteogenic differentiation of the stromal cells.

Immunohistochemical localization of basal lamina components in the developing rat epiphyseal cartilage canals.

The purpose of this study was to investigate the relationship between the appearance of basal lamina components (Type IV collagen and laminin) and the development of cartilage canals. In the distal femoral epiphyses from developing rats, the distribution of basal lamina components in the cartilage canal was examined immunohistochemically. The formation of cartilage canals from the perichondrium was first observed on the fifth day after birth. By Day 8, a few cartilage canals penetrated the epiphyseal cartilage and considerably increased in size and length. By light microscopic immunohistochemistry, reticular structures stained with anti-Type IV collagen and antilaminin antibodies were observed in the cartilage canals. In the early development of cartilage canals, however, immunostaining by anti-Type IV collagen antibodies was weaker than that by antilaminin antibodies. In eight-day-old rats, the laminin-positive reticular structures were more densely colored and more widely distributed in the canal than the Type IV collagen-positive ones. Type IV collagen was found around the endothelial cells of the developing capillaries by electron microscopic immunohistochemistry. Laminin was observed in the cytoplasm of the mesenchymal fibroblastic cells and their pericellular matrix as well as in the capillary basal lamina. These immunohistochemical electron microscopic observations can explain the differences that are observed in Type IV collagen and laminin immunostaining patterns as cartilage canals develop. Laminin synthesized by the mesenchymal fibroblastic cells may promote the migration and the outgrowth of endothelial cells in the formation of cartilage canals.

Effects of RU486 on the interstitial collagenase in the process of cervical ripening in the pregnant rat.

Changes in interstitial collagenase activity in the rat uterine cervix during ripening were clarified in a time-dependent manner. Premature delivery was induced by an antiprogesterone agent, RU486, for rats in late pregnancy. The presence of interstitial collagenase in the extract from the rat cervical tissue was demonstrated, by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis using the natural and unaffected collagen as a substrate. The collagenase activity was determined as the release of digested peptides from the radio-labeled collagen. Our experiments with RU486 were performed in rats on the 18th day of pregnancy. A single administration of RU486 (15 mg/kg) resulted in the premature delivery of all treated rats within 30 h after the injection (average time was 23.9 h). The marked increase in cervical wet weight was observed up to the time to premature delivery along with a significant acceleration from 18 h after the administration of RU486. In this state, the cervical collagenase activity was enhanced, the highest levels being recorded at 21 h after the administration. The interstitial collagenase in the uterine cervix appears to play a significant role in the regulation mechanisms of cervical ripening in late pregnant rats.

A histologic and immunohistochemical study of calcium pyrophosphate dihydrate crystal deposition disease.

The articular cartilage, synovial membrane, and meniscus from ten patients who had calcium pyrophosphate dihydrate (CPPD) crystal deposition disease showed strong immunoreactivity for dermatan sulfate proteoglycan, Type I collagen, and S-100 protein in hypertrophic chondrocytes around the crystals, their pericellular matrix, and deposits of the crystals. Electron microscopy revealed that small crystals were formed around the hypertrophic chondrocytes, especially in the degenerated matrix containing electron-dense granular materials and cellular debris. Chondrocytes of this kind were never observed in the articular tissue from ten patients who had osteoarthrosis. These hypertrophic chondrocytes with several unique immunohistochemical characteristics may initiate the formation of CPPD crystals.

Dystrophin: localization and presumed function.

To determine the localization and functional significance of dystrophin, we studied various tissues from almost the entire body of control and mdx mice, and control rats, using polyclonal antibodies against dystrophin. We observed a dystrophin reaction in synaptic regions such as neuromuscular junctions, the equatorial region of intrafusal muscle fibers, the outer plexiform layer of the retina, the myoepithelial cell layer of salivary and sweat glands, tactile nerve endings, and neurons in the brain. These dystrophin-positive regions reportedly contain actin filaments as a common characteristic, which is compatible with the dystrophin cDNA sequence. Dystrophin was absent in these regions in mdx mice. These results suggest that dystrophin plays an important physiological and/or structural role in cell motility as a trigger for propagating contractile force in, for example, the conduction system, with some relationship between actin filaments.

Type VI collagen in healing rabbit corneal wounds.

A type-specific monoclonal antibody was used to examine the localization of type VI collagen in healing full-thickness corneal wounds (3 mm in diameter) in rabbits. By immunofluorescence, in 1-week-old wounds, type VI collagen was found only at the wound periphery. Subsequently, type VI collagen showed a rapid increase throughout the wounded area. In 3-week-old wounds, type VI collagen immunofluorescence had a laminar pattern as seen in normal corneal stroma, although it was irregular in arrangement. By electron-microscopic immunohistochemistry, type VI collagen was found in the pericellular region of fibroblastic cells possibly derived from keratocytes. These results suggest that type VI collagen might originate from the fibroblastic cells and play an important role in the regeneration of the corneal lamellar structure.

Possible systemic smooth muscle layer dysfunction due to a deficiency of dystrophin in Duchenne muscular dystrophy.

The localization of dystrophin was examined immunohistochemically in various tissues from mice and rats as well as from biopsied human muscle specimens, using polyclonal antibodies against dystrophin. Although dystrophin was completely absent in biopsied muscle specimens from 3 male DMD patients, it was faintly observed in the surface membrane of almost all muscle fibers examined in a female DMD patient. In all controls, human and animal, a strong dystrophin reaction was observed in the surface membrane of intrafusal muscle fibers and at the neuromuscular junctions, rather than in the surface membrane of skeletal and cardiac muscle fibers. In addition, dystrophin was clearly localized in the surface membrane of smooth muscle fibers in the viscera, bronchial system, ureter, and tunica media of blood vessels, including arteries and veins, in all examined animal tissues. In mdx mice, dystrophin was absent in almost all muscle and smooth muscle fibers in various tissues and blood vessels. These results suggested a possible systemic dysfunction of smooth muscle layers, especially those of blood vessels, as well as skeletal muscle fibers, due to a deficiency of dystrophin in Duchenne muscular dystrophy.

Distribution of type VI collagen in the bovine cornea.

Using a specific polyclonal antibody against type VI collagen, the distribution of type VI collagen in the bovine cornea was investigated by indirect immunofluorescence microscopy and immunoelectron microscopy. By immunofluorescence, type VI collagen was detectable throughout the stroma. In addition to the uniform but relatively weak signals, a laminar pattern similar to the arrangement of stromal lamellae was clearly seen. Consistent with the immunofluorescent pattern, immunoelectron micrographs showed strong linear reactions located in the interlamellar spaces. The reactions were also observed in the pericellular region of the keratocytes. Epithelium, Descemet's membrane and endothelium were negative.

An assay of collagenase activity using enzyme-linked immunosorbent assay for mammalian collagenase.

A new method was developed for the measurement of collagenase activity using the enzyme-linked immunosorbent assay (ELISA). Rabbit colon wall collagenase, pepsin-soluble rat skin type I collagen, and its antisera were used in the present experiment. After the collagenase-degraded portion of the collagen coated on the microwell was released, the immunoreaction of the residual collagen on the microwell to anticollagen sera was determined by ELISA. This method was approximately 10 times more sensitive than the conventional assay procedure using [14C]-glycine-labeled reconstituted collagen fibrils as substrate. It was suitable for screening a large number of samples without radioisotopes.

Endosteal new bone formation in the long bones of adjuvant treated rats.

Histopathological changes were examined mainly in the diaphyseal parts of long bones, especially femur in adjuvant-treated male Lewis-SPF rats, with reference to clinical symptoms of chronic osteoarthritis. The diaphyseal bone marrow of long bones in these rats sequentially showed three different processes of chronic pathological changes, which, however, partly overlapped each other. Initially, multiple epitheloid cell granulomas or granulomatous lesions containing fibrin deposits began to appear in the marrow space about 22 days after adjuvant injection, when the joint score of arthritis reached a peak in severity. Secondly, about a week after appearance of the granulomas, there occurred the intramembranous endosteal new bone formation proceeding from the endosteum towards the granulomatous lesions. The bone formation reached a maximum about 64 days after the treatment, when the redness of joints of feet and hands was already sedated. Finally, about 40 days after occurrence of the second event, the newly growing bone matrix began to be actively resorbed simultaneously. On the other hand, in the bone marrow of metaphyseal parts of long bones in these rats, severe acute osteomyelitis was observed from an early stage, with marked destruction of bone trabeculae and simultaneous new bone formation. In the diaphyseal bone marrow of affected long bones, the epitheloid cell granulomas appear to induce the endosteal new bone formation.

Collagenase from the culture medium of rabbit colon wall with special reference to the latent type and substrate specificity.

Latent and active forms of collagenase were detected in culture media of the normal rabbit colon. During culture, the collagenase appeared to be produced by surviving and growing mucosas on the degenerated or necrotic colon wall. Type III collagen was most readily degraded by the collagenase, followed by type I and II collagens. The collagenase did not attack type IV or V collagens. The latent collagenase was activated by trypsin and chaotropic agents such as 3 M NaSCN or NaI, and autoactivated gradually during storage. Activated latent collagenase showed the properties of metalloproteinase as in the active collagenase. The apparent molecular weights, determined by calibrated Sephadex G-75, were 39,000 and 31,000 for the latent and active enzymes, respectively. After 12 h of tissue culture, the latent collagenase appeared in the culture media 10-20 h earlier than the active collagenase. The collagenase in the culture media of the early period was mainly the latent form, while the media of the late period contained a large amount of the active form.

Phagocytosis of cytoplasmic blebs derived from smooth muscle cells by fibroblast-like cells and macrophages in the post-partum rat uterus.

Numerous blebs were observed in contact with smooth muscle cells (SMC) by light microscopy in the myometrium of the rat uterus after parturition. Electron-microscopically the cell surface of SMC showed bulbous protrusions, which often lacked a basement membrane and were less electron-dense than the surrounding cytoplasm or sometimes nearly electron-lucent. Many bulbous protrusions were separated from SMC and became the isolated structures which we called cytoplasmic blebs. These bulbous protrusions and cytoplasmic blebs were often found to be phagocytosed by fibroblast-like cells and macrophages. A series of these tissue changes in the uterine myometrium after delivery, possibly due to hypoxic conditions, contribute to a rapid involution of SMC which have enlarged during pregnancy.

A freeze-fracture study of two types of collagen-phagocytosing cell in the post-partum rat endometrium.

In the post-partum rat endometrium, ultrastructural distinction could be made between stromal cells (fibroblast-like cells) and macrophages, especially by the freeze-fracture technic. The stromal cells were characterized by a well-developed rough-surfaced endoplasmic reticulum (RER) and intercellular junctions, while the macrophages had many vacuoles and vesicles, but no intercellular contact with each other. The freeze-fracture image showed that the stromal cells had many low linear elevations and gap junctions on the cleaved plane of the cell membranes, while the macrophages had no linear elevations or intercellular junctions. The cell membranes of the stromal cells had more intramembranous particles (IMP) (P-face 697 +/- 63/micrometers 2, E-face 303 +/- 52/micrometers 2) than those of the macrophages (P-face 467 +/- 50/micrometers 2, E-face 217 +/- 35/micrometers 2). It was confirmed that these two types of cell phagocytosed collagen fibrils.

An ultrastructural study on the ear cartilage of rabbits after the administration of papain. Appearance of cross-striated collagen segments of an atypical FLS-type.

Crude papain was administered intravenously to young rabbits and the cartilage of the collapsed ear was examined electron-microscopically. Degeneration and recovery of chondrocytes, and decrease in and recovery of the electron-density of elastic fibers, were observed during the collapse and restoration of the ear. Some samples were stained with ruthenium red. In the collapsed ear, with a marked decrease of proteoglycan in the cartilage, loss of ruthenium red-positive granules was observed in the extracellular matrix. Collagen fibrils in the cartilage appeared to be somewhat increased in number, some of their diameters became slightly greater, and a part were assembled into bundles, occasionally accompanied by periodic cross-striation. Decrease of proteoglycan in the cartilage matrix probably brought about the unmasking and the assembly of collagen fibrils. In one of the experimental animals, collagen fibrous segments of an atypical fibrous long spacing (FLS-)type with symmetrical cross-striation were found around the chondrocytes in the ear cartilage, during the period of recovery. Some kind of the endogenous sulfated carbohydrate may have acted to affect the arrangement of type II collagen or procollagen molecules newly produced by the recovering chondrocytes.

Electron microscopical studies on the genesis of white adipocytes: differentiation of immature pericytes into adipocytes in transplanted preadipose tissue.

In order to clarify the relationships between perivascular cells, capillaries and fat cells, with a special reference to the origin of fat cells, we have made a light and electron microscopical study on the developing epididymal adipose tissue of newborn to 5-week-old rats, and also on the differentiating, transplanted epididymal preadipose tissue from 6-day-old rats. Development of epididymal preadipose tissue progressed rapidly 6 or 7 days after birth. The preadipose tissue on the 5th day after transplantation consisted of differentiated areas with many mature fat cells, and of undifferentiated areas in which these cells were scanty. In the differentiated areas of developing epididymal preadipose tissue, both in situ and transplanted, many fat cells seemed to develop in the area immediately adjacent to growing capillaries, but cells intermediate between perivascular cells and preadipocytes were seldom observed. However, in undifferentiated areas of transplanted tissue, we found ultrastructural evidence that immature pericytes of capillaries can differentiate into preadipocytes.