Distribution of laminin,type IV collagen,and fibronectin in the cell columns and trophoblastic shell of early macaque placentas

Summary The cytotrophoblastic cell columns and trophoblastic shell of macaque placentas accumulate progressively greater amounts of intercellular material during early gestation. We studied the composition of this material in placentas collected from 22–34 days of gestation by using immunoperoxidase techniques directed to the extracellular matrix molecules fibronectin, type IV collagen, and laminin. These antigens co-localized within the intercellular deposits at all stages studied. At day 22 the proximal cell columns were composed of cells with narrow interstices and which lacked immunoreactivity for the 3 antigens. Distally the cells were vacuolated and the intercellular spaces increased in size and contained dense matrix deposits. The trophoblastic shell consisted of closely packed, non-vacuolated cytotrophoblast cells with only a delicate meshwork of matrix. By day 27 the matrix deposits of the distal cell columns increased markedly in size. The trophoblastic shell contained larger numbers of vacuolated cells and was occupied by accumulations of matrix. By 34 days the matrix deposits of the cell columns expanded substantially along the longitudinal axes of the columns. These deposits were often continuous with a matrix-dense, cell-deficient layer in the trophoblastic shell. This matrix-rich zone lay between a cellular layer adjacent to the intervilous space and a similar, but discontinuous, cell layer that formed the junctional zone with the endometrium.     

Immunofluorescent localization of collagen types I,III and IV,fibronectin, laminin,and basement membrane proteoglycan in developing mouse skin

Summary The distribution of various extracellular matrix components was studied in frozen sections of embryonic (14–18 days) and early postnatal (birth and 4 days post parturn) dorsal mouse skin using monospecific antibodies and indirect immunofluorescence. Basement membrane zone components — type IV collagen, laminin and heparan sulphate proteoglycan — were found to be uniformly and unchangingly distributed along the dermal-epidermal junction. In contrast, the distribution of interstitial matrix components — types I and III collagen, and fibronectin — was heterogeneous and varied with the stages of hair development. Collagens became sparse and were eventually completely removed from the prospective dermal papilla and from a one-cell-thick sheath of dermal cells around hair buds. They remained absent from the dermal papilla throughout hair organogenesis. Fibronectin was always present around dermal papilla cells and was particularly abundant along the dermal-epidermal junction of hair rudiments, as well as underneath hair buds. In contrast, in interfollicular skin, collagens accumulated in increasing density, while fibronectin became progressively sparser. It thus appears that interstitial collagens and fibronectin are distributed in a manner which is related to hair morphogenesis. In morphogenetically active regions, collagen density is low, while that of fibronectin is high. Conversely, in histologically stabilized zones, collagen is abundant and fibronectin is sparse. This microheterogeneous distribution of interstitial collagens and of fibronectin might thus constitute part of the morphogenetic message that the dermis is known to transmit to the epidermis during the development of skin and of cutaneous appendages.     

Electron-microscopic localization of type II,IX, and V collagen in the organ of Corti of the gerbil

Summary The presence of types II, IX and V collagen was probed in the organ of Corti of the adult gerbil cochlea by use of immunocytochemistry at the light- and electron-microscopic levels. Type II collagen is found in the connective tissues of the osseous spiral lamina and spiral limbus. In the region of the sensory hair cells it is present in the tectorial membrane and antibodies bind to the thick unbranched radial fibers. Type IX collagen co-localizes with type II collagen in the tectorial membrane, where antibodies bind to the thick unbranched radial fibers. Type V collagen is present in the connective tissue of the spiral limbus, the osseous spiral lamina, the eighth nerve, and the tectorial membrane. In the tectorial membrane, the staining with antibodies to type V collagen is more diffuse than that seen for types II and IX collagen and antibodies to type V bind to the thin, highly branched fibers in which the thick fibers are embedded. The results indicate that collagens characteristic of cartilage are localized in the organ of Corti. Within the tectorial membrane, types II and IX collagen form heterotypic thick fibers embedded in a reticular network of type V collagen fibers. These collagens form a highly structured matrix which contributes to the rigidity of the tectorial membrane and allow it to withstand the physical stresses associated with transmission of the stimuli necessary for sensory transduction.     

Localization of integrin subunits α6 and β1 during somitogenesis in the long-tailed macaque (M. fascicularis)

The distribution of integrin subunits 6 and 1, and the 61 integrin ligand, laminin, was examined during somitogenesis in developmental stages 11, 13, and 16 in the long-tailed macaque, using peroxidase immunocytochemistry. Within differentiating somites in stage 11, 6 expression was observed in the sclerotome, basal surface of dermamyotomal cells adjacent to the basal lamina and on scattered cells throughout the dermamyotome. In further advanced somites in stages 13 and 16, 6 immunoreactivity become restricted to the myotome, 6 was expressed on mesenchymal core cells within the myocele of undifferentiated epitheliod somites and the ventromedial wall of somites commencing differentiation at each stage. 1 distribution resembled that of 6 in stage 11 somitic tissue, however, it remained present on myotome and sclerotome cells in the later stages, and was also expressed on dermatomal cells in stage 16. Laminin immunoreactivity, while more intense and prevalent than 6 and 1 in each stage examined, occurred on the same somite cell populations as the 2 integrin subunits. These results show a defined distribution of 6 on somitic tissue, and suggest this integrin is involved in somite differentiation. They also support a possible role for 6 in myoblast formation and migration. Overlapping of 1 and laminin immunoreactivity with that of 6 further suggests that 6 paris with 1 as a functional heterodimer for laminin in defined somitic regions.     

Cytochemical variations in the nucleolus during spermiogenesis in man and monkey

Summary The fine structure, nature and fate of the components of the nucleolus were studied in young (steps 1, 2), intermediate (steps 3, 4, 5) and mature spermatids (steps 6, 7, 8) of man and monkey, by use of several cytochemical techniques (alcoholic PTA; sodium tungstate; EDTA; HAPTA; nuclease-gold complexes; NOR silver staining). As controls, comparative ultrastructural and cytochemical observations of the nucleolus in spermatids and Sertoli cells were made in the same sections of seminiferous tubules. In the young spermatids of the two species studied, the nucleolar masses exhibited identical features. Segregation of the nucleolar components took place in the nuclei of step 1 spermatids. No typical fibrillar center was observed. In spermatids at steps 1 and 2, the nucleolar masses appeared to be made up of two fibrillar components of equal density, one spherule-shaped, the other forming cords, both surrounded by clusters of 15–20 nm-diameter granules. Alcoholic PTA and sodium tungstate yielded a selective positive contrast of the two fibrillar components whereas EDTA and RNase-gold reacted with the peripheral granular material. Treatment with RNase-gold and DNase-gold complexes resulted in preferential labeling at the periphery of the fibrillar components. After NOR silver staining, numerous small silver grains were localized over the fibrillar cords, suggesting the persistence of specific acidic non-histone proteins. On the contrary, the spherule was never stained. In intermediate spermatids, when the nucleolar components were dissociated, scattered clusters of granules stained by EDTA and HAPTA remained in the entire nucleoplasm. Nucleolar disintegration was accompanied by dispersion of argyrophilic material. In mature spermatids granular material revealed by PTA and silver staining methods was found in the nuclear pockets bounded by the redundant nuclear envelope.     

Immunocytochemical localization of collagen types I,III, IV,and fibronectin in the human dermis

Summary The distribution of collagen types I, III, IV, and of fibronectin has been studied in the human dermis by light and electron-microscopic immunocytochemistry, using affinity purified primary antibodies and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies. Type I collagen was present in all collagen fibers of both papillary and reticular dermis, but collagen fibrils, which could be resolved as discrete entities, were labeled with different intensity. Type III collagen codistributed with type I in the collagen fibers, besides being concentrated around blood vessels and skin appendages. Coexistence of type I and type III collagens in the collagen fibrils of the whole dermis was confirmed by ultrastructural double-labelling experiments using colloidal immunogold as a probe. Type IV collagen was detected in all basement membranes. Fibronectin was distributed in patches among collagen fibers and was associated with all basement membranes, while a weaker positive reaction was observed in collagen fibers. Ageing caused the thinning of collagen fibers, chiefly in the recticular dermis. The labeling pattern of both type I and III collagens did not change in skin samples from patients of up to 79 years of age, but immunoreactivity for type III collagen increased in comparison to younger skins. A loss of fibronectin, likely related to the decreased morphogenetic activity of tissues, was observed with age.     

Distribution of opioidergic,sympathetic and neuropeptide Y-positive nerves in the sphincter of Oddi and biliary tree of the monkey,Macaca fascicularis

Summary The opioidergic, sympathetic and neuropeptide Y-positive innervation of the sphincter of Oddi (common bile duct sphincter and pancreatic duct sphincter), as well as other segments of the extrahepatic biliary tree was studied in the monkey by use of immunohistochemistry. Methionine-enkephalin-positive nerves were seen to innervate the smooth muscle of all portions of the sphincter of Oddi and also local ganglion cells. No methionine-enkephalin-positive nerves could be detected in the common bile duct, pancreatic duct or gallbladder. Tyrosine hydroxylase-positive nerves occurred between smooth muscle bundles and also ran to local ganglion cells as well as along the common bile duct. Neuropeptide Y-positive nerves were observed within smooth muscle of the sphincter of Oddi (all portions), common bile duct, pancreatic duct and gallbladder. No evidence of any differential innervation of the pancreatic duct and common bile duct sphincters could be detected with these markers.     

Heat-stable alkaline phosphatase as a marker for human and monkey type-I pneumocytes

Summary The expression of the heat-stable isoenzyme of alkaline phosphatase in the human and monkey (Macaca mulatta, M. fascicularis) lung was investigated at the light- and electron-microscopic level, using cytochemical techniques and immunocytochemical procedures based on monoclonal and polyclonal antibodies against human term-placental alkaline phosphatase. Both in man and monkey, the enzyme was present in type-I pneumocytes. In the monkey, the enzyme was found in all type-I cells. In man, strong staining was observed only in some type-I cells and in certain cuboidal respiratory bronchiolar cells. Staining was localized on the apical and basal plasma membrane, in apical and basal caveolae, and in the underlying basement membrane. The level of heat-stable alkaline phosphatase expression in the human lung was 10-fold lower than in the monkeys studied. In human fetal lung, the onset of heat-stable alkaline phosphatase expression was associated with the development of the alveolar epithelium from 17–20 weeks gestation onward. It is concluded that: (1) heat-stable alkaline phosphatase is a specific constitutent of type-I pneumocytes in man and monkeys; and (2) its subcellular localization may explain its rapid appearance in the circulation under certain conditions.This work was supported by grants from the Fonds voor Kankeronderzoek van de Algemene Spaar- en Lijfrentekas, Nationale Loterij-FGWO (Grant No. 9.0005.84), the National Program for Reinforcement of the Scientific Research (PREST/UIA 04) and a research grant from the University of Antwerp     

Feeding ecology of the long-tailed macaque(Macaca fascicularis) in Kalimantan Tengah,Indonesia

I studied long- tailed macaque (Macaca fascicularis)feeding behavior and ecology as part of a larger behavioral ecological study at the Natai Lengkuas Station, Tanjung Puting National Park, Kalimantan Tengah, Indonesia. I collected data on feeding behavior via scan sampling of all visible individuals in the focal group (approximately 800 observation hours). I established vegetational plots and monitored them monthly to determine food availability and abundance. I found long- tailed macaques to be primarily frugivorous;leaves, flowers, insects and bark provided the remainder of the diet. They used at least 33 plant species as food sources, but >60% of the diet was provided by only 5 species. Based on previous vegetational analyses, these tree species were among the highest in relative density. However, selection ratios for 19 food species indicate that 13 of them were selected more often than expected. Long- tailed macaques appear to be selective feeders but can exploit a variety of food sources during periods of food scarcity.     

Localisation of substance P-like immunoreactivity in the ciliary ganglia of monkey (Macaca fascicularis) and cat: a light- and electron-microscopic study

The present study describes substance P-like immunoreactivity in the ciliary ganglia of monkey (Macaca fascicularis) and cat. About 60% of neurons in the monkey ciliary ganglion and 40% in the cat ciliary ganglion were substance P-like immunoreactive, ranging from faint to moderate staining. Substance P-like immunoreactivity was located in cell bodies, dendritic profiles and axons. In the monkey, substance P-like immunoreactive pericellular arborisations were associated with about 0.5%–3% of the ganglion cells, which were either negatively, faintly or moderately stained. An electron-microscopic study demonstrated the presence of either substance P-like immunoreactive positive or negative axon terminals synapsing or closely associated with positive dendritic profiles in both the monkey and cat ciliary ganglia. The results suggest that substance P plays an important role in the ciliary ganglion, perhaps as a modulator or transmitter.     

Structural analysis of potential barriers to bulk-flow exchanges between uvea and sclera in eyes of Macaque monkeys

Summary The uveal tract of the eyes of monkeys was examined by electron microscopy using both thin sections and freeze-fracture replicas. The ultrastructural features of the lamina fusca in the monkey resembled those previously described for rabbit. The lamina fusca was composed of numerous interleaved processes of fibroblastic and pigmented cells and contained tight junctions between fibroblastic cell processes that were predominantly discontinuous, as well as numerous fenestrations through the attenuated cell processes. There was no regional compaction of cellular processes traversing the entire uvea at the level of the ora serrata as reported previously in hamster eyes.     

Localization and identification of nuclear radioactivity in the pituitary gland and genital tract after administering 3H-testosterone, 3H-dihydrotestosterone,or 3H-estradiol to male rhesus monkeys

Summary Target cells for testosterone, dihydrotestosterone, and estradiol in the pituitary gland and genital tract of the male primate were localized by thaw-mount autoradiography, and high performance liquid chromatography was used to identify the metabolites of these steroids in cell nuclei. Castrated rhesus monkeys were injected with ³H-testosterone, ³H-dihydrotestosterone, or ³H-estradiol and killed 60 min later. In the anterior pituitary gland, fewer cells were labeled and less radioactivity was taken up by cell nuclei following the administration of either ³H-testosterone (4% of pars distalis cells and 5 dpm/g DNA) or ³H-dihydrotestosterone (5% of cells and 13 dpm/g DNA) than following the administration of ³H-estradiol (43% of cells and 214 dpm/g DNA). Most of the radioactivity in nuclei was in the form of the unmetabolized parent compound (78–94%). In prostate, seminal vesicles, and penis, ³H-dihydrotestosterone was the predominant form of nuclear radioactivity following both ³H-testosterone (67–90%) and ³H-dihydrostestosterone (94–97%) administration, and both androgens labeled epithelial and smooth muscle cells. In contrast, ³H-estradiol was taken up in unchanged form, by cell nuclei of the genital tract and it labeled connective tissue fibroblasts, but not epithelial cells. Thus, the distributions of target cells for androgens and estrogens were clearly different in all these tissues, and the uptake of testosterone resembled that of its androgenic rather than that of its estrogenic metabolite.     

Uveoscleral permeability to intracamerally infused ferritin in eyes of rabbits and monkeys

Summary The permeability of the uveoscleral outflow pathway from the anterior ocular chamber was examined in rabbit and monkey eyes using anionic ferritin as a tracer. Ferritin, infused intracamerally, had ready access to the choroidal interstitium, and the degree of penetration was generally correlated with the time and pressure relationships during infusion. In both species, there were accumulations of tracer in intercellular spaces at the lamina fusca, but tracer was also present in the sclera. Thus, in contrast to the situation in the eyes of hamsters, the uveoscleral outflow pathway in the eyes of rabbits and monkeys includes the choroidal connective tissue and allows passage of relatively large molecular weight substances.     

Y-Chromosome and Mitochondrial Markers in Macaca fascicularis Indicate Introgression with Indochinese M. mulatta and a Biogeographic Barrier in the Isthmus of Kra

This is the first report of Y-chromosome introgression between primate species. We sequenced 3.1 Kb of Y-chromosome DNA and 1.5 Kb of mtDNA for 27 macaques of Fooden's (Folia Primatol. [1976] 25: 225–236) fascicularis species group and 5 outgroup taxa (Macaca sylvanus, Papio hamadryas, Theropithecus gelada, Allenopithecus nigroviridis, and Cercopithecus mona). Phylogenies constructed separately for the paternal and maternal data sets show a Y-chromosome paraphyly among lineages of Macacafascicularis, but a mitochondrial monophyly for the same individuals. The Y-chromosome topology depicts Indochinese Macaca fascicularis haplotypes joining with those of M. mulatta, followed by M. cyclopis and M. fuscata, before clustering with a clade of lineages of M. fascicularis from peninsular Malaysia, Indonesia, and the Philippines. These contrasting patterns of mitochondrial and Y-chromosome DNA, evaluated in the context of the evolutionary consequences of macaque sex-biased dispersal, present strong evidence for contemporary hybridization between Macaca fascicularis and M. mulatta in Indochina and a biogeographic barrier in the Isthmus of Kra.     

Immunohistochemical localization of laminin,neural cell adhesion molecule,collagen type IV and T-61 antigen in the embryonic retina of the Japanese quail by in vivo injection of antibodies

Summary Antibodies against laminin (LN), fibronectin (FN), collagen type IV (Col IV), neural cell adhesion molecule (N-CAM), T-61 antigen, actin, tubulin and neurofilament protein were injected into the eyes of quail embryos (Coturnix coturnix japonica) of different ages. Twenty h after injection, the heads of the embryos were fixed and the antibodies visualized in sections with the use of fluorescein-isothiocyanate (FITC) or peroxidase-labeled second antibodies by light- and electron microscopy. Antibodies against cell surface molecules, such as N-CAM, LN, Col IV and T 61, labeled matrix and membrane components of the retinal cells in different antigen-specific patterns. Antibodies against intracellular antigens, such as actin, tubulin and neurofilament protein labeled nonspecifically the vitreous body and the inner basal lamina of the retina, but resulted in only a very weak and diffuse labeling of retinal cells. N-CAM was detected in high concentration in the optic fiber layer on the surface of axons and on the membranes of all retinal cells. Col IV, LN and T 61 antigen were found predominantly in the optic fiber layer. LN and Col IV were located on the surface of axons and the endfeet of ventricular (neuroepithelial) cells in a patchy distribution. The T-61 antigen was found in early stages in the cell-free space of the optic fiber layer, on the surface of ventricular cells and axons, and at later stages also in high-density patches between nerve fibers. The distribution of LN and T-61 antigen together with data from in vitro experiments suggests a crucial role of these proteins in axon extension in the avian retina during early development of the optic fiber layer.     

Changes in the distribution of type IV collagen,laminin, proteoglycan,and fibronectin during mouse tooth development

The distribution of certain basement membrane (BM) components including type IV collagen, laminin, BM proteoglycan, and fibronectin was studied in developing mouse molar teeth, using antibodies or antisera specific for these substances in indirect immunofluorescence. At the onset of cuspal morphogenesis, type IV collagen, laminin, and BM proteoglycan were found to be present throughout the basement membranes of the tooth. Fibronectin was abundant under the inner enamel epithelium at the region of differentiating odontoblasts and also in the mesenchymal tissues. After the first layer of predentin had been secreted by the odontoblasts at the epithelial-mesenchymal interface, laminin remained in close association with the epithelial cells whereas type IV collagen, BM proteoglycan, and fibronectin were distributed uniformly throughout this area. Later when dentin had been produced and the epithelial cells had differentiated into ameloblasts, basement membrane components disappeared from the cuspal area. These matrix components were not detected in dentin while BM proteoglycan and fibronectin were present in predentin. The observed changes in the collagenous and noncollagenous glycoproteins and the proteoglycan appear to be closely associated with cell differentiation and matrix secretion in the developing tooth.     

Immuno-electron-microscopic localization of types III pN-collagen and IV collagen,laminin and tenascin in developing and adult human spleen

The distribution of the extracellular matrix proteins types III pN-collagen and IV collagen, laminin and tenascin was investigated in fetal, infant, and adult human spleens by using immuno-electron microscopy. The presence of type III pN-collagen was assessed by using an antibody against the aminoterminal propeptide of type III procollagen. All the proteins other than type III pN-collagen were found in reticular fibers throughout development. In the white pulp of the fetus aged 16 gestational weeks, only an occasional type III pN-collagen-containing fibril was present, although type III pN-collagen was abundant in the reticular fibers of the red pulp. Conversely, in adults, most of the reticular fibers of the white pulp, but not of the red pulp, were immunoreactive for type III pN-collagen. Ring fibers, the basement membranes of venous sinuses, were well developed in both infant and adult spleens. The first signs of their formation could be seen as a discontinuous basement membrane, which was immunoreactive for type IV collagen, laminin, and tenascin in the fetus aged 20 gestational weeks. Intracytoplasmic immunoreactivity for all the proteins studied was visible in the mesenchymal cells of the fetus aged 16 gestational weeks and in the reticular cells of the older fetuses, which also showed labeling for type IV collagen and laminin in the endothelial cells. The results suggest that proteins of the extracellular matrix are produced by these stationary cells.     

A comparison of the immunofluorescent localization of collagen types I,III, and V with the distribution of reticular fibers on the same liver sections of the snow monkey (Macaca fuscata)

Summary Localizations of collagen types I, III, and V in monkey liver, as determined by the indirect immunofluorescence method, were photographically superimposed on the fibers revealed by silver-staining in the same tissue sections. Immunofluorescence for type I collagen was found to correspond with the brown collagen fibers and with some of the coarse reticular fibers, while that for type III collagen was found to correspond with most, but not all, reticular fibers of the liver as well as with the brown collagen fibers. The distribution of type V collagen coincides not only with the collagen fibers in the stroma of portal triads and around the central veins, but also with the coarse and fine reticular fibers in the liver lobules. By immuno-electron microscopy, reaction products with anti-type III and V collagens antibodies were demonstrated on cross-striated collagen fibrils, about 45 nm in diameter, in the space of Disse. From these observations, it is concluded that: (1) the fine reticular fibers are mainly composed of type III and type V collagens, and (2) the collagen fibers and coarse reticular fibers in the periphery of liver lobules are composed of type I, type III and type V collagens.     

Distal invaginations and the renewal of cone outer segments in anuran and monkey retinas

Summary Although it is now clear that the outer segments of mature vertebrate cones are regularly renewed, it is not known how a cone outer segment can maintain a tapered shape if its narrower tip is periodically lost by shedding. This problem was addressed by morphological examination of photoreceptors in retinas of anurans (Xenopus laevis) and monkeys (Macaca fascicularis). Light microscopy revealed a marked daily change in the shape of cone outer segments in X. laevis: at light offset they were long and conical, at light onset they had shed their narrow tips, were sharply truncated, and 40% shorter. Electron microscopy revealed previously undescribed fine-structural features in these mature cone outer segments, most notably the presence of many partial membrane infoldings within their distal lamellae. The growth of each of these distal invaginations apparently split 1 pre-existing distal lamella into 2 daughter lamellae of reduced width. The formation of distal invaginations at various heights within a cone outer segment would thus make it longer and narrower. Similar ultrastructural features were also found in cone outer segments of monkey retinas. These findings suggest that during outer segment renewal the tapered shape of mature cone outer segments is maintained via a remodelling process that accompanies the formation of distal invaginations.Portions of this work have been published in abbreviated or preliminary form (Eckmiller 1988, 1989b, c)