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Notes: Immunohistochemical methods were used to determine whether type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan were present in diverse basement membranes. Antisera or antibodies against each substance were prepared, tested by enzyme-linked immunosorbent assay, and exposed to frozen sections of duodenum, trachea, kidney, spinal cord, cerebrum, and incisor tooth from rats aged 20 days to 34 months. Bound antibodies were then localized by indirect or direct peroxidase methods for examination in the light microscope.Immunostaiing for type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan was observed in all of the basement membranes encountered. Fibronectin was also found in connective tissue. In general, the intensity of immunostaining was strong for type IV collagen and laminin, moderate for heparan sulfate proteoglycan, and weak for fibronectin. The pattern was similar in the age groups under study. Very recently the sulfated glycoprotein, entactin, was also detected in the basement membranes of the listed tissues in 20-day-old rats.It is accordingly proposed that, at least in the organs examined, type IV collagen, laminin, fibronectin, heparan sulfate proteoglycan, and entactin are present together in basement membranes.

Notes: To examine whether collagen is assembled into fibrils within or outside fibroblasts, the connective tissue of the rat foot pad was investigated by electron microscopy and by radioautography at times varying from 4 min to 3 days after an intravenous injection of 3H-proline.The fibroblasts of the rat food pad are long polarized cells with the nucleus at one end, the Golgi apparatus in the center, and a region with long processes at the other end. This region contains secretory granules and is considered to be the secretory pole of the cell. In the Golgi apparatus the stacks of saccules are separated from rough endoplasmic reticulum (rER) by groups of “intermediate vesicles” including similarly structured tubules which may be over 300 nm long and are referred to as “intermediate tubules”. The Golgi saccules exhibit distended portions which differ at the various levels of the stack. On the cis side, the distentions tend to be spherical and contain fine looping threads; in the middle of the stack, they are cylindrical and present distinct straight threads; whereas on the trans side, they are again cylindrical, but the straight threads are grouped in parallel aggregates. Between these cylindrical distentions and the secretory granules, there are transitional forms within which thread aggregates are packaged more and more tightly. Finally, the fibroblasts are associated with two types of collagen fibrils: extracellular ones arranged into large groups between the cells and intracellular ones located within long intracytoplasmic channels.Quantitative radioautography after 3H-proline injection reveals that the number of silver grains per unit area reaches a peak over the rER at 4-10 min, Golgi apparatus at 40 min, secretory granules at 60 min, and extracellular collagen fibrils at 3 h. At no time are intracellular collagen fibrils labeled. Qualitative observations further indicate that spherical Golgi distentions are mainly labeled at 40 min, and cylindrical distentions, at 60 min. In addition, from 20 min to 3 hr, some lysosomal elements are labeled.The biogenetic pathway leading to the formation of collagen fibrils is interpreted as follows. Collagen precursors arise in the rER and, by way of intermediate tubules or vesicles, reach the spherical Golgi distentions. These seem to migrate in a trans direction, while they become cylindrical and their looping threads straighten out into rods which have been identified as procollagen. The cylindrical distention is then freed from its saccule to become a secretory granule. The procollagen content of the granule is released outside the cell and presumably transforms into collagen which then polymerizes into fibrils. Thus, except for a minor fraction of the labeled precursors deviated toward the lysosomal pathway, the bulk of them give rise to collagen, which is added to the extracellular fibrils and, therefore, provides for their growth. No collagen is incorporated into the intracellular collagen fibrils, which, therefore, do not grow, but often undergo degeneration. Thus, the assembly of collagen into fibrils does not occur within cells but is entirely extracellular.

Notes: The stomachs of adult CD1 mice were investigated by anatomical examination and light microscopy. Serial sections were prepared of entire stomachs; the various types of gastric glands were characterized; and, using every 30th or 60th serial section, maps of gland distribution were obtained through point-plotting serial reconstruction.Gross examination shows that the cephalic third of the stomach consists of a thin-walled, domelike structure, the forestomach. The rest of the organ, or stomach proper, is subdivided into two parts, the thick-walled corpus, which approximately occupies the middle third, and the less vascular pyloric antrum which forms the remaining caudal third of the organ.Histologically, glands are absent from the forestomach mucosa but are numerous throughout the stomach proper. They are of two main types, namely, zymogenic glands, which contain, among others, zymogenic cells, and mucous glands, which lack these cells but contain mucous cells. Both gland types show a few enteroendocrine cells. Moreover, some of the mucous glands include parietal cells (mucoparietal glands), while others do not (pure mucous glands).Mucosal maps reveal that the glands of each type are located in distinct areas of the mucosa. Thus a compact zymogenic region may be defined, occupying 56% of the glandular mucosa and containing only zymogenic glands. The mucous region, on the other hand, composed only of mucous glands, is extensive and divided into a narrow cephalic band (5.2% of the glandular mucosa) and a large caudal part (38.8%). Along the lesser curvature these parts are continuous, and together they encapsulate the zymogenic region. In proximity to the border of this region, and only there, do the mucous glands include parietal cells.A comparison of mucosal maps and gross features indicates that the corpus includes both the zymogenic region and the cephalic band of mucous glands, whereas the antrum is composed entirely of the mucous glands of the caudal part.

Notes: Cell populations may be classified into three groups: static, which do not divide in the adult (e.g., neurons), expanding, which may divide but at a decreasing rate with age (e.g., kidney cells), and renewing, which undergo active division throughout life (e.g., blood cells, intestinal and seminiferous epithelium). The first two groups consist of nonrenewing populations whose cells usually survive as long as the body itself. In contrast, the cells of renewing populations have a short life, of the order of 2 to 3 days in the intestinal epithelium of rodents and 7 to 8 weeks in their seminiferous epithelium. In the present work, the life of a nonrenewing cell - the proximal convoluted tubule cell of kidney - is compared to that of the renewing cells of small intestine and testis.The proximal convoluted tubule cell arises during embryonic development, when it has few organelles, many free ribosomes, a pale nucleus with diffuse chromatin, and a large open-network nucleolus. As the cell differentiates, organelles accumulate while free ribosomes decrease in number, chromatin masses appear in the nucleus, and the nucleolus becomes rather dense. With old age, some of the cells show features interpreted as senescence, particularly a small dense nucleolus.The short-lived columnar cells of small intestine in adult rats arise from stem cells present in the base of the crypts. These cells have features similar to those of the embryonic proximal tubule cell, particularly many free ribosomes and a large open-network nucleolus. The stem cells give rise to cells which ascend crypts and villi. Shortly after the cells reach the villus, they acquire a full set of organelles, suggesting maturity, while free ribosomes decrease in number and the nucleolus undergoes condensation. Meanwhile radioautographic tests indicate that the production by the cells of glycoproteins identified as intestinal enzymes reaches a high level. However, when cells arrive in the villus tip region, their activity decreases, as shown by reduced enzyme production, while the nucleolus appears atrophic. The cells are then lost to the lumen.The cells of the seminiferous epithelium also arise in the adult from stem cells with embryonic features. In the course of a complex differentiation, the nucleolus becomes atrophic and disappears. Soon thereafter the fragile spermatozoon stage is reached.Thus, the renewing cells of the adult show in rapid succession some of the features occurring slowly during the life of nonrenewing cells: origin from embryonic-like elements, maturation, and senescence indicated by a fragile state before life's end.

Notes: Electron microscopic immunostaining was used to examine the localization of type IV collagen, laminin, entactin, heparan sulfate proteoglycan, and fibronectin within the basement membranes of the rat kidney. In preliminary experiments, various methods of processing formaldehyde-fixed kidney were compared using antilaminin antiserum and the indirect immunoperoxidase method. Little or no laminin immunostaining of the glomerular basement membrane was present in sections unless they had been frozen-thawed; and even in this case, the immunostaining was light in comparison to that of basement membranes in adjacent tubules. However, when frozen-thawed sections were treated with 0.5% sodium borohydride, immunostaining was then as strong in glomerular as in tubular basement membranes. Accordingly, this treatment was applied to frozen-thawed sections before immunostaining for any of the substances under study.Immunostaining of the glomerular basement membrane for each of the five substances was fairly uniform throughout the lamina densa (also called basal lamina), but uneven in the lamina lucida interna and externa (also called lamina rara interna and externa) in which stained bands extended from the lamina densa. Similarly in the basement membranes of tubules, immunostaining for the five substances was localized to the lamina densa and bands extending into the lamina lucida.When the ultrastructure of the glomerular basement membrane was examined, three structures were found: (1) a network of 4-nm-thick “cords,” which seems to be the main component; the cords are closely packed in the lamina densa and more loosely arranged in the lamina lucida interna and externa; (2) straight, hollow 7-10-nm-thick structures referred to as “basotubules”; and (3) 3.5-nm elements composed of minute paired rods, referred to as “double pegs.” The distribution of the cords, but not that of the other two structures, was related to the immunostaining pattern.It is concluded that (1) to fully reveal the antigenicity of the glomerular basement membrane, frozen-thawed sections must be treated with sodium borohydride prior to immunostaining, possibly because this basement membrane is more compact than the others; and (2) in both glomerular and tubular basement membranes, type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin are colocalized in the lamina densa and its extensions to the laminae lucidae. Since the distribution of the cords corresponds to that of immunostaining, it is likely that the five substances are present within the cords.

Notes: The “round body,” a spherical structure typically associated with a nucleolus in male germ cells of the rat, has been examined in the electron microscope using routine and cytochemical methods to determine its structure, composition, and mode of development. Cytochemical analysis indicates that the round body includes neither nucleic acid nor lipid, but is composed of nonhistone protein which appears in the form of 1.6-nm-wide fibrils. Development begins in late leptotene, when a single round body appears in each spermatocyte as an irregular spheroid located along the inner surface of the nuclear envelope. During subsequent stages of the meiotic prophase, the round body leaves the nuclear envelope, becomes a regular sphere, and gradually enlarges from a diameter of 0.4 μm in leptotene to 1.6 μm in diplotene. Concurrently, lacunae appear within its substance and enlarge. At each maturation division, the amount of round-body material is decreased by about half, presumably because the constituent proteins are dissociated at metaphase, distributed between the two daughter cells at telophase, and reconstituted into half-sized round bodies. As spermiogenesis proceeds, the round body shrinks gradually and disappears at step 8.Soon after its appearance at leptotene, the round body becomes associated with and is surrounded by the pars granulosa of one of the nucleoli. Moreover, 3H-uridine incorporation into nucleolar RNA is high as long as the size of the round body increases, but is low or absent when it decreases. It is possible, therefore, that the round body exerts some control on nucleolar activity in meiotic cells.