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In long bone development, the evolution of the cartilaginous anlagen into a secondary ossification center is initiated by the formation of canals. The excavation to create the canals is achieved through lysis of the two major cartilage components, aggrecan, and the type II collagen (COL2) fibril. The present study examines the lysis of the fibril. Because it is known that matrix metalloproteinases (MMPs) cleave COL2 in vitro at the Gly(775)-Leu(776) bond, it has been reasoned that, if such cleavage is detected in relation to the canals, it can be concluded that a collagenase is involved. Furthermore, because MMPs undergo change in domain structure with activation resulting in propeptide domain loss then, if such a loss is revealed in relation to the cleavage of COL2, this MMP is likely involved. The collective findings reveal that COL2 is attacked at the afore-described susceptible peptide bond at the surface of cartilage canals and, that MMP-13 cleaves it. Developmental Dynamics 238:1547-1563, 2009. (c) 2009 Wiley-Liss, Inc.

A principle of regulation of matrix metalloproteinase (MMP) activity has been introduced as the cysteine-switch mechanism of activation (Springman et al. 1990). According to this mechanism, a critical Cys residue found in the auto-inhibitory propeptide domain of latent proenzyme is important to determine whether or not activation is turned on or off. The mechanism further allows for multiple modes of activation. To determine whether or not activation is accomplished proteolytically within a rat test cartilage model, protease analysis by the neoepitope approach, which relies upon a set of antibodies, was applied. One is used to identify the MMP-9 proenzyme bearing the critical cysteine residue, the other to identify any enzyme present bearing a new NH2-terminus 89FQTFD. This is indicative of MMP-9 lacking the cysteine switch. The antibody set has been applied to frozen tissue sections and analyzed by light and electron microscopic methods. Results reveal that activation of the MMP-9 protease involves limited proteolysis resulting in propeptide domain release. Here we report the observed changes of protease form to indigenous cells and extracellular matrix, thereby making it possible to uncover the features of MMP-9 activation within a specified set of tissue circumstances where a cartilage model is transformed into definitive bone. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

In the human stomach, the oxyntic epithelium includes numerous tubular invaginations consisting of short pits opening into long glands. The pit is lined by pit cells, whereas the gland is composed of three regions: the base, containing zymogenic cells; the neck, containing neck cells; and the isthmus, composed of little known immature cells and of parietal cells, which are also scattered through the neck and base. The aim of this study was to examine the ultrastructure of the immature cells and to determine their relation to mature cells. To do so, normal oxyntic mucosal biopsies from subjects ranging from 20-43 years old were fixed in aldehydes and postfixed in reduced osmium for electron microscopy and morphometric analysis. The immature cells were sorted out into four classes, whose roles were clarified by comparison with the thoroughly investigated mouse oxyntic epithelium. The first class was composed of the least differentiated immature cells, which were rare and characterized by minute, dense, or cored secretory granules and were accordingly named mini-granule cells. Their function was not clarified. The second class consisted of pre-pit cells, which were characterized by few dense mucous granules and give rise to pit cells that ascend the pit wall and, after reaching the luminal surface, die or are extruded. Both pre-pit and pit cells underwent continuous renewal and, therefore, together constituted a renewal system referred to as pit cell lineage. The third class, or pre-neck cells, characterized by cored secretory granules, give rise to neck cells that descend toward the base region and differentiate further into pre-zymogenic cells, which finally become zymogenic cells. The latter eventually degenerate and die. Thus pre-neck cells and their progeny constitute a renewing system, designated zymogenic cell lineage. The fourth class, or pre-parietal cells, characterized by long microvilli and few tubulovesicles, differentiate into parietal cells that descend along the neck and base regions and eventually degenerate and die. Pre-parietal and parietal cells represent a renewing system referred to as parietal cell lineage. While the origin of the last three classes of progenitor cells has not been elucidated, it is likely that they arise either from an unidentified multipotential stem cell, possibly the mini-granule cell itself, or from the mitotic activity of pre-pit and pre-neck cells. In conclusion, the human oxyntic epithelium is composed of continually renewing cells organized in distinct cell lineages.

The formation of a secondary ossification center in the cartilaginous epiphysis of long bones requires the excavation of canals and marrow space and, therefore, the resorption of cartilage. On the assumption that its resorption requires the lysis of the major cartilage component aggrecan, it was noted that the core protein may be cleaved in vitro by proteinases from two subfamilies: matrix metalloproteinases (MMPs) and aggrecanases. Such cleavage results in aggrecan being replaced by a fragment of itself referred to as a "G1-fragment." To find out if this cleavage occurs in the developing epiphysis of the rat tibia, the approach has been to localize the G1 fragments. For this purpose two neoepitope antisera were applied, one capable of recognizing the MMP-generated G1-fragment that bears the C-terminus ...FVDIPEN341 and the other capable of recognizing the aggrecanase-generated G1-fragment that carries the C-terminus ...NITEGE373. With the aid of these antisera, we report here that aggrecan cleavage is localized to newly developed sites of erosion. Thus, at 6 days of age, canals allowing the entry of capillaries are dug out from the surface of the epiphysis in a radial direction (stage I), whereas immunostaining indicative of aggrecan cleavage by MMPs appears at the blind end of each canal. The next day, the canal blind ends fuse to create a marrow space in the epiphysis (stage II), whereas immunostaining produced by MMPs occurs along the walls of this space. By 9 days, clusters of hypertrophic chondrocytes are scattered along the marrow space wall to initiate the formation of the secondary ossification center (stage III), where the resorption sites are unreactive to either antiserum. From the 9th to the 21st day, the center keeps on enlarging and, as the distal wall of the marrow space recedes, it is intensely immunostained with both antisera indicating that both MMPs and aggrecanases are involved in this resorption. We conclude, that both enzyme subfamilies contribute to the lysis of aggrecan. However, the results suggest that the respective subfamilies target different sites and even stages of development in the tissue, suggesting some diversity in the mode of aggrecan lysis during the excavation of a secondary ossification center., (Copyright 2001 Wiley-Liss, Inc.)

In the transformation of the cartilaginous epiphysis into bone, the first indication of change in the surfaces destined for resorption is the cleavage of aggrecan core protein by unidentified matrix metalloproteinases (MMPs) (Lee et al., this issue). In cartilage areas undergoing resorption, the cleavage leaves as superficial, 6-microm-thick band of matrix, referred to as "pre-resorptive layer." This layer harbors G1-fragments of the aggrecan core protein within a framework of collagen-rich fibrils exhibiting various stages of degeneration. Investigation of this layer in every resorption area by gelatin histozymography and TIMP-2 histochemistry demonstrates the presence of an MMP whose histozymographic activity is inhibited by such a low dose of the inhibitor CT1746 as to identify it as gelatinase A or B. Attempts at blocking the histozymographic reactions with neutralizing antibodies capable of inhibiting either gelatinase A or B reveals that only those against gelatinase B do so. Immunostaining of sections with anti-gelatinase B IgG confirms the presence of gelatinase B in every pre-resorptive layer, that is, at the blind end of excavated canals (stage I; 6-day-old rats), at sites along the walls of the forming marrow space (stage II; 7days), at sites within the walls of this space as it becomes the ossification center (stage III; 9 days) and along the wall of the maturing center (stage IV; 10-21 days). We also report the presence of collagenase-3 in precisely the same sites, possibly as active enzyme, but this remains to be proven. Because the results reveal that collagenase-3 is present beside gelatinase B in every pre-resorptive layer and, because these sites exhibit various signs of degradation including fibrillar debris, reduction in fibril number, or overt loss, we propose that gelatinase B and collagenase-3 mediate the lysis of this pre-resorptive layer-most likely through a cooperative attack leading to the disintegration of the collagen fibril framework., (Copyright 2001 Wiley-Liss, Inc.)

In order to determine which proteinases mediate the resorption of endochondral cartilage in the course of long bone development, a novel assay called "histozymography" has been developed. In this assay, frozen sections of tibial head from 21-day-old rats are placed for 4 hr at room temperature on light-exposed photographic emulsion (composed of silver grains embedded in gelatin). We report a localized but complete digestion of emulsion gelatin facing two tissue sites which are, therefore, presumed to contain an active proteinase. One of the sites is localized at the growth plate surface forming the epiphysis/metaphysis interface. The other consists of small patches located within the epiphysis at the edge of the marrow space. Both sites are engaged in the resorption of endochondral cartilage. In both sites, inhibitor tests have established that the involved proteinase is a gelatinase. Furthermore, the use of neutralizing antibodies against gelatinase A or B have demonstrated that only those that are specific for the latter block the reaction. That gelatinase B is present in the two sites has been confirmed by light microscopic immunohistochemistry. Finally, when immunoelectron microscopy is used for fine localization of the cartilage structures that form the epiphysis/metaphysis interface, the enzyme is detected within the 0.5-microm thick edge of the cartilage, and outside the cartilage, it is present in debris composed of type II collagen-rich fibrils in various states of digestion. It is concluded that gelatinase B attacks the edge of an endochondral cartilage and helps to solubilize the type II-collagen-rich fibrillar framework, which is then released as debris for further digestion. This final step opens the way to invasion by capillaries, thereby making possible the replacement of cartilage by bone. Dev Dyn 1999;215:190-205., (Copyright 1999 Wiley-Liss, Inc.)

Since we had subdivided the cell cycle into 11 stages--four for mitosis and seven for the interphase--and since we had experience in detecting DNA in the electron microscope (EN) by the osmium-amine procedure of Cogliati and Gauthier (Compt. Rend. Acad. Sci., 1973;276:3041-3044), we combined the two approaches for the analysis of DNA-containing structures at all stages of the cell cycle. Thin Epon sections of formaldehyde-fixed mouse duodenum were stained by osmium-amine for electron microscopic examination of the stages in the 12.3-hr long cell cycle of mouse duodenal crypt columnar cells. In addition, semi-thin Lowicryl sections of mouse duodenal crypts and cultured rat kidney cells were stained with the DNA-specific Hoechst 33258 dye and examined in the fluorescence microscope. The DNA detected by osmium-amine is in the form of nucleofilaments, seen at high magnification as long rows of 11 nm-wide rings (consisting of stained DNA encircling unstained histones). At all stages of the cycle as well as in nondividing cells, nucleofilaments are of three types: 'free,' 'attached' to chromatin accumulations, and 'compacted' in all chromatin accumulations, the form of dense spirals within. At stage I of the cycle, besides free and attached nucleofilaments, compacted ones are observed in the three heterochromatin forms (peripheral, nucleolus-associated, clumped). Soon after the S phase begins, chromatin 'aggregates' appear, which are small at stage II, mid-sized at stage III, and large at stage IV. Chromatin 'bulges' also appear at stage III and enlarge at stage IV, while heterochromatins disappear. At stage V, aggregates and bulges accrete into 'chromomeres,' a process responsible for the apparent chromosome condensation observed at prophase. The chromomeres gradually line up in rows and, at stage VIa (prometaphase), approach one another within each row and coalesce to build up the metaphase chromosomes which are fully formed at stage VIb (metaphase). Daughter chromosomes arising at stage VII (anaphase) are eventually packed into a chromosomal mass at each pole of the cell. During stage VIII (telophase), the chromosomal mass is split into large chunks. In the course of the G1 phase, the chunks thin out to give rise to irregular 'bands' at stage IX, the bands are then cleaved into central and peripheral fragments at stage X, and finally the central fragments are replaced by free nucleofilaments and clumps at stage XI, while the peripheral fragments are replaced by peripheral heterochromatin. The "nucleoli" at stages I-III are associated with stained heterochromatin but otherwise appear as unstained lucent areas, except for weakly stained patches composed of histone-free DNA filaments. During stage IV, nucleoli lose patches and associated heterochromatin, while weakly lucent, pale vesicles appear within nucleoli and in the nucleoplasm. By the end of substage VIa, nucleoli generally disappear, while pale vesicles persist around the chromosomes appearing at substage VIb. At stages VIII and IX, the vesicles seem to become strongly lucent and, at stages IX and X, they associate and fuse to yield homogeneous lucent areas, the 'prenucleolar bodies,' which include histone-free DNA patches. During stage XI, groups of these bodies associate to give rise to nucleoli. In conclusion, the cell cycle DNA changes can be classified into 4 broad periods (Fig. 6): 1) Stage I is a 2-hr long interphase "pause," during which the stained DNA shows no signs of either chromosome condensation or decondensation, while the overall nuclear pattern is similar to that in nondividing cell nuclei. Nucleoli are fully developed. 2) From stage II to VIa, the "chromosome condensation" period extends over about 7 hr, during which the events are interpreted as follows. Throughout the S phase (stages II-IV), newly-synthesized segments of nucleofilaments approach one another, adhere and thus build aggregates and later bulges on nuclear matrix sites. (ABSTRACT TRUNCATED)

In view of the extensive lysis of hyaline cartilage known to take place during endochondral bone formation, the current study was designed to test the hypothesis that metalloproteinases are the agents that mediate this lysis. Since these enzymes have been shown in vitro to cleave the core protein of the major proteoglycan of cartilage, aggrecan, at the Asn341-Phe342 bond, an immunohistochemical method has been developed to find out whether or not there are sites in the growth plate of the rat tibia where cleavage of this bond takes place. The cleavage of aggrecan by metalloproteinases is followed by the retention of the fragment known as G1, for it includes the G1 domain. Since the G1 fragment terminates in the amino acid residues ...FVDIPEN, we prepared an antiserum against FVDIPEN, confirmed its specificity, then applied it to the growth plate of 21-day-old rat tibia in the hope of localizing the G1 fragments. The antiserum specificity was shown by its recognition of the ...FVDIPEN sequence at the C-terminus of peptides and of G1 fragments produced by aggrecan cleavage. When the antiserum was applied to Western blots of guanidinium chloride extracts prepared from epiphyseal growth plate, it recognized two species (56 and 52 kDa), which differed only in the degree of glycosylation. These fragments were comparable in size to the G1 fragments generated by the action of recombinant metalloproteinase in vitro, thus confirming antiserum specificity for these fragments. Applying the antiserum to cryosections of 21-day-old rat tibiae revealed immunostaining at two intensities within the growth plate matrix: a strong staining was observed in a 1-5 microm-wide layer designated "peripheral" matrix, which borders the epiphyseal and metaphyseal marrow spaces as well as the perichondrium, while a weak staining was found in the rest of the plate, designated "central" matrix. The abundance of G1 fragments terminating in ...FVDIPEN in the peripheral matrix indicates that this is where the growth plate is lysed to achieve longitudinal and latitudinal bone growth. The site where metalloproteinases exert their main lytic activity is a thin layer of matrix separating central from peripheral matrix.

Background: Although the rate of RNA synthesis is known to drop at mitosis, the recent identification of 11 stages in the cell cycle (El-Alfy et al., 1994) makes it possible to measure the rate of this synthesis at each one of the stages and thus find out how it varies throughout the cell cycle., Methods: Mice were injected intravenously with the RNA precursor, 3H-uridine; the duodenum was fixed 5-15 minutes later for embedment in Epon, and the duodenal crypts were cut in semithin serial sections for study of the rapidly dividing crypt columnar cells. Using Feulgen-stained sections, each cell nucleus was assigned to one of the 11 stages described in the cell cycle, and the same nucleus was identified in the next serial section that had been processed for radioautography, so that the overlying silver grains were enumerated. The count was taken as an index of the rate of RNA synthesis by this nucleus., Results: Starting from stage I of the cell cycle (the period defined by the presence of a minimal amount of chromatin during which the S phase begins) and up to stage IV (when the S phase ends and the G2 phase begins), all or nearly all nuclei are synthesizing RNA with the rate peaking at stage III. During stages V to VIII (the period comprising the mitotic steps), the percentage of RNA-synthesizing nuclei decreases to over half at stage V (prophase), -10% at stages VIa (prometaphase) and VIb (metaphase) and none at stages VII (anaphase) and VIII (telophase). During stages IX-XI (which correspond to the G1 phase), the percentage rises sharply at stage XI to reach up to 100% at stages X and XI. Finally, on the average, 35% of nuclear silver grains are over the nucleolus (presumably representing ribosomal RNA precursors), whereas 65% are over the nucleoplasm (presumably representing mainly heterogeneous RNA precursors)., Conclusions: Cells synthesize RNA during the interphase, but at a variable rate with a peak in S. The synthesis proceeds in a majority of the cells at prophase, but only in a few of them at prometaphase and metaphase, and in none at anaphase and telophase.

Background: In the hope of understanding how chromosomes condense at mitosis, we took advantage of a subdivision of the cell cycle into 11 stages to examine the changes in DNA taking place during the stages preceding the emergence of metaphase chromosomes., Methods: To identify DNA changes, pieces of mouse duodenum were fixed in formaldehyde, and sections of the rapidly dividing cells of the crypts were stained by the osmium-ammine method, which is specific for the detection of DNA in the electron microscope., Results: Throughout the cell cycle, DNA is present in nucleofilaments composed of rows of 11-nm-wide nucleosomes. At stage I, during which the DNA-synthesizing or S phase of the cell cycle begins, some of the nucleofilaments are compacted in the heterochromatin accumulations associated with nuclear envelope and nucleoli, while the others are scattered in the nucleoplasm where they appear either "free" or "attached" to the heterochromatin. This DNA distribution is similar to that observed in the noncycling cells examined. After the beginning of the S phase, "free" nucleofilaments are seen to assemble into structures composed of compacted nucleofilaments and referred to as "aggregates"; these make their appearance at stage II and increase in size through stage III up to the end of S during stage IV. Meanwhile, the heterochromatin associated with nuclear envelope and nucleoli expands toward the nucleoplasm in the form of protrusions referred to as "bulges," which gradually enlarge during stages III and IV, while the heterochromatin shrinks and eventually vanishes. On average, a total of 1,171 aggregates and bulges are formed in the nucleus during the S phase. At the apparition of stage V, which corresponds approximately to prophase, aggregates and bulges are rapidly gathered into an average of 288 spheroidal bodies referred to as "chromomeres." These are connected to one another by nucleofilamentous bridges in such a way as to be lined up in rows. The formation of rows of chromomeres represents in the electron microscope the prophasic condensation observed in the light microscope. Finally, during stage VIa, which corresponds to prometaphase, the chromomeres approach one another within each row, make contact, and coalesce to become the 40 chromosomes of the mouse, which during stage VIb are organized in the equatorial plate of metaphase., Conclusions: The condensation of metaphase chromosomes occurs in three main steps. The first and longest takes place during the S phase, as nucleofilaments are assembled into aggregates, while the heterochromatin gives rise to bulges. The brief second step occurs toward the beginning of prophase, when the numerous aggregates and bulges are congregated into a limited number of chromomeres, which are lined up in rows. The third step takes place during the brief prometaphase, when the chromomeres of a row coalesce into a mitotic chromosome.

Background: Since it has been found that new chromatin structures make their appearance in the nucleus during the DNA-synthesizing or S phase of the cell cycle, the question arises as to how these structures are related to the nascent DNA., Methods: DNA-containing structures were detected in sections of mouse duodenal crypt cells by the DNA-specific osmium-ammine procedure. In the same sections, the nascent or newly-replicated DNA was localized during stages I-IV of the cell cycle (corresponding to four successive parts of the S phase) by immunogold labeling of the DNA precursor bromodeoxyuridine (BrdU) in mice sacrificed 10 min after its injection. Moreover, the fate of the nascent DNA with time was traced up to 6 hr after the injection. (The nomenclature of the DNA-containing structures is that proposed by El-Alfy et al., 1995.), Results: Ten minutes after BrdU injection, the gold particles indicative of nascent DNA are associated with discrete nucleofilaments scattered in the nucleoplasm, but not with the compacted nucleofilaments making up the heterochromatin or the new S phase structures named "aggregates." The gold-particle-associated discrete nucleofilaments are classified into three types: a) The "free" nucleofilaments have been given this name, since they appear to be independent of heterochromatin and aggregates; nearly all gold particles are over these at stage I; but the numbers of particles over them decreases from stage I to IV. b) The "aggregate-attached" nucleofilaments project from the surface of the aggregates; the number of particles over these is high at stages II and III but decreases at stage IV. c) The "heterochromatin-attached" nucleofilaments project from the surface of the heterochromatin; the number of particles over these increases from stage II to IV. By 1 hr after BrdU injection, gold particles can be over loose clumps of nucleofilaments at stages I and II, but are mostly over small aggregates at stage II, midsized aggregates and small heterochromatin-associated "bulges" at stage III and large aggregates and large bulges at stage IV. By 2-6 hr, virtually all particles are over aggregates and bulges, frequently deep within them., Conclusions: The distribution of the gold particles at 10 min reveals that DNA is synthesized in discrete nucleofilaments that are "free" or "aggregate-attached" or "heterochromatin-attached." In contrast, by one and especially two hours, the gold particles are present over aggregates and bulges, indicating that, after discrete nucleofilaments acquire nascent DNA, they are displaced to become part of these structures. More precisely, the aggregates arise from the repeated addition of replicated portions of "free" nucleofilaments, while the bulges arise from the repeated addition of replicated portions, of "heterochromatin-attached" nucleofilaments. Aggregates and bulges are the two initial building stones from which mitotic chromosomes are eventually formed.

The secretions of the mammalian stomach are produced by cells present in invaginations of the epithelium, which in the mouse are straight tubules referred to as "zymogenic units." These units comprise four regions, namely pit, isthmus, neck, and base, in which there are several cell lineages with different phenotypes and migratory pathways. In the isthmus, stem cells designated "undifferentiated granule-free cells" undergo division so as to maintain their own number and produce several differently oriented progenitors: (1) "Pre-pit cell precursors" are characterized by prosecretory Golgi vesicles with a uniform, fine particulate content. They give rise to "pre-pit cells" defined by the presence of few dense mucous granules. These cells migrate outward from the isthmus to the pit, where they become the dense granule-rich "pit cells" which populate the pit region and migrate to the gastric surface where they are lost. (2) "Pre-neck cell precursors" are identified by prosecretory Golgi vesicles containing an irregular dense center and a light rim. They give rise to "pre-neck cells" defined by a few mucous secretory granules with a clear-cut core. These cells migrate inward from the isthmus to the neck where they become "neck cells," which contain many such granules. Even though neck cells are mature mucus-producers, they are not end cells. As they enter the base region, they become "prezymogenic cells" whose phenotype gradually changes from mucous to serous. These cells eventually lose the ability to produce mucus and thus become the typical zymogenic cells that populate the base region. (3) "Pre-parietal cells" are classified into three variants, which probably come from three different sources, that is, pre-pit cell precursors, pre-neck precursors, and the undifferentiated granule-free cells themselves. The preparietal cells mature into parietal cells which migrate either outward to the pit or inward to the neck and base. As a result, parietal cells are scattered in the four regions of the unit. (4) Precursors of "entero-endocrine" and "caveolated" cells give rise in the isthmus to these cells, which may also migrate outward or inward.

The Feulgen reaction has been utilized to localize DNA in nuclei throughout the cycle of mouse duodenal crypt cells using Epon-embedded 1 micron thick sections. The observed changes indicate that the 12.3 h long mitotic cycle of these cells can be subdivided into eleven stages, seven of which take place during the interphase. Computer measurements of Feulgen-stained nuclei and previous radioautographic studies indicate that DNA synthesis begins during stage I and ends during stage IV. The staining pattern shows no distinctive feature in the nuclei of the 1.5 h long stage I. Thereafter, marked changes occur during the rest of the interphase--that is during the 6.3 h that precede karyokinesis and the 3.5 h that follow it. Thus, at stage II the background of the nuclei darkens; at stage III, there appear stained threads interpreted as densifying chromosomes and dots interpreted as chromomeres, both of which thicken from 0.2 to 0.4 micron; at stage IV they further thicken to about 0.5 micron and at stage V, to about 0.7 micron. At this stage, which approximately corresponds to prophase, the intensely stained, discrete dots are localized within the less intensely stained sausage-shaped threads. As the breakup of the nuclear envelope introduces stage VI, whose early part corresponds to prometaphase, the intensely stained dots become close to one another within the threads and eventually fuse. The staining of the threads thus intensifies, and, by the late part of the stage that corresponds to metaphase, they have become the homogeneously dense metaphase chromosomes. At stage VII, the anaphase chromosomes reach each pole where they associate into a compact mass.(ABSTRACT TRUNCATED AT 250 WORDS)

When tissues are processed for electron microscopy by conventional methods, such as glutaraldehyde fixation followed by rapid dehydration in acetone, basement membranes show two main layers: the electron-lucent "lamina lucida". (or rara) and the electron-dense "lamina densa". In an attempt to determine whether this subdivision is real or artefactual, two approaches have been used. Firstly, rat and mouse seminiferous tubules, mouse epididymis and associated tissues, and anterior parts of mouse eyes were subjected to cryofixation by instant freezing followed by freeze substitution in a -80 degrees C solution of osmium tetroxide in dry acetone, which was gradually warmed to room temperature over a 3-day period. The results indicate that, in areas devoid of ice crystals, basement membranes consist of a lamina densa in direct contact with the plasmalemma of the associated cells without an intervening lamina lucida. Secondly, a series of tissues from mice perfused with 3% glutaraldehyde were cryoprotected in 30% glycerol, frozen in Freon 22 and subjected to a 3-day freeze substitution in osmium tetroxide-acetone as above. Under these conditions, no lamina lucida accompanies the lamina densa in the basement membranes of the majority of tissues, including kidney, thyroid gland, smooth and skeletal muscle, ciliary body, seminiferous tubules, epididymis and capillary endothelium. Thus, even though these tissues have been fixed in glutaraldehyde, no lamina lucida appears when they are slowly dehydrated by freeze substitution. It is concluded that the occurrence of this lamina in conventionally processed tissues is not due to fixation but to the rapid dehydration. However, in this series of experiments, the basement membranes of trachea and plantar epidermis include a lamina lucida along their entire length, while those of esophagus and vas deferens may or may not include a lamina lucida. To find out if the lamina lucida appearing under these conditions is a real structure or an artefact, the trachea and epidermis were fixed in paraformaldehyde and slowly dehydrated by freeze substitution. Under these conditions, no lamina lucida was found. Since this result is the same as observed in other tissues by the previous approaches, it is proposed that the lamina lucida is an artefact in these as in the other investigated basement membranes. Thus, basement membranes are simply composed of a lamina densa that closely follows the plasmalemma of the associated cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Entero-endocrine cells and the rare cells named caveolated or brush cells have been examined in light microscopic radioautographs of the mouse corpus after various periods of continuous 3H-thymidine infusion. Moreover a search for immature forms and mitoses of these cells was undertaken in the electron microscope. Entero-endocrine cells are present in the four regions of the epithelial units, but their number is low in the pit, intermediate in the isthmus and neck, and high in the base. The labeling pattern after continuous 3H-thymidine infusion indicates that these cells are produced in the isthmus from undifferentiated granule-free cells presumed to be the stem cells of the epithelium, and may retain a limited ability to divide. A few of the newly formed entero-endocrine cells migrate to the pit, but the majority goes to the neck and, from there, to the base where they are present in relatively high numbers. Little information is available on the dynamics of caveolated cells. Since immature forms are present in the isthmus and mature ones in the other regions, it is concluded that they arise in the isthmus and migrate away in both directions. Finally, concluding remarks are presented on the kinetics of each one of the cell lineages described in this and the four previous articles.

The pit cells (or surface mucous cells) present along pit walls and gastric surface have been investigated by electron microscopy and radioautography after a pulse or continuous infusion of 3H-thymidine. For these studies, the pit region has been subdivided into four segments: three of equal length along the pit wall, respectively named low pit, mid pit and high pit, and a last one at the surface named pit top. The pit region includes an average of 37 pit cells, characterized by dense mucous granules accumulated along the apical membrane in an organelle-free zone referred to as ectoplasm. Continuous 3H-thymidine infusion reveals that pit cells come from pre-pit cells, which are believed to arise in the isthmus region from the undifferentiated granule-free cells through a pre-pit cell precursor stage. The pre-pit cells, characterized by the presence of a few mucous secretory granules scattered in the cytoplasm, migrate outward (i.e., in the direction of the gastric lumen). When the secretory granules line up along the apical membrane in the ectoplasm, the pre-pit cell becomes pit cell. It is estimated that 87% of pit cells differentiate from pre-pit cells, while the remaining 13% come from their own mitoses. Observations at successive times after a 3H-thymidine pulse demonstrate that pit cells, like pre-pit cells, migrate toward the gastric surface where they are eventually lost. The continuous 3H-thymidine infusion results indicate that this migration takes 3.1 days on the average. Cells spend almost a day in each pit wall segment. In the low pit segment, cells produce more and larger mucous secretory granules than do pre-pit cells. In the mid and high pit segments, the number and size of the granules generally keeps on increasing, thus indicating that mucous differentiation is progressing. The secretory granules arising in the Golgi apparatus of pit wall cells are mostly spherical; they retain this shape during the few minutes taken to cross the cytoplasm and enter the apical ectoplasm. They spend about an hour in the ectoplasm, where they change to an ovoid shape as they approach the apical membrane to finally release their content by exocytosis. The mucous differentiation along the pit wall is associated with a progressive decline in the organelles: nucleoli and mitochondria decrease in size while the amount of free ribosomes diminishes. When pit cells reach the free surface, they produce fewer, smaller secretory granules and at a lower rate than in mid and high pit.(ABSTRACT TRUNCATED AT 400 WORDS)

In a recent study of the corpus epithelium in the mouse stomach, eleven cell types have been identified and enumerated (Karam and Leblond: Anat. Rec. 232:231-246, 1992). The dynamics of these cells will be examined in a series of five articles, of which this is the first. This article focuses on the proliferative ability of the cells, as measured by the labeling index in radioautographs from mice sacrificed 30 min after an intravenous injection of 3H-thymidine. Furthermore, the ultrastructure of the cells found to be proliferative was examined in the hope of finding features characteristic of stem cells. On the basis of their labeling index, the epithelial cells have been classified into four groups. The first includes three cell types which do not take up any label and accordingly are non-dividing: parietal or oxyntic cells, cells named pre-parietal as they are immature cells suspected of being parietal cell precursors, and the rare caveolated or brush cells. The second group is composed of three cell types which are only rarely labeled and, therefore, divide only occasionally: zymogenic or chief cells, entero-endocrine cells, and cells named pre-zymogenic cells as they are suspected of being zymogenic cell precursors. The third group includes two cell types which are always labeled at a low degree and, therefore, divide regularly, but at a low rate: surface mucous cells, herein called pit cells, whose labeling index is 0.8%, and mucous neck cells, simply known as neck cells, 1.8%. The final group consists of three immature cell types with high labeling indices indicating a high rate of division: granule-free cells, which are devoid of secretory granules and have the highest labeling index, 32.4%, pre-pit cells, which possess a few dense secretory granules similar to, but smaller than, those in pit cells, 24.6%, and pre-neck cells, with a small number of secretory granules similar to, but smaller than, those in neck cells, 11.3%. These three cell types, as well as pre-parietal cells, are rapidly renewed, with the turnover times estimated at 3.0 days for pre-neck and pre-parietal cells and less than 2.6 days for granule-free and pre-pit cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The neck cells (or mucous neck cells) present in the neck region and the zymogenic cells (or chief cells) present in the base region of the units in the mouse corpus were examined in the electron microscope (EM) and in radioautographs prepared after administration of 3H-thymidine by single or multiple injections or by continuous infusion for 1-52 days. For these studies, the neck region of the units has been subdivided into three equal segments, respectively named high neck, mid neck, and low neck, while the base region has been similarly subdivided into high base, mid base, and low base. The neck region includes an average of 12.6 neck cells, characterized in the EM by dark, mucous secretory granules that frequently exhibit a light, pepsinogenic core. Continuous 3H-thymidine infusion reveals that neck cells come from pre-neck cells, which are believed to arise in the isthmus region from the undifferentiated granule-free cells through a pre-neck cell precursor stage. The pre-neck cells, characterized by the presence of a few cored secretory granules, migrate inward (i.e., in the direction of the blind end of the units) and enter the neck region to become neck cells. It is estimated that 59% of the neck cells arise from differentiation of pre-neck cells, whereas the other 41% are derived from their own mitoses. Neck cells migrate inward in 1-2 weeks from the high through the mid and low neck segments, while they keep on producing more and larger secretory granules and thus further differentiate as mucus-producing cells. When neck cells reach the high base segment, they become pre-zymogenic cells that produce secretory granules in which appear light, irregular, pepsinogenic patches which encroach on the dark mucous content. With time, the pre-zymogenic cells, of which there are 5.0 per unit on the average, keep on producing new granules with larger and larger light patches, so that in the end the cells produce granules which are entirely filled by light, pepsinogenic material. At this stage, the cells are zymogenic cells. Zymogenic cells, which average 67.5 per unit, further migrate inward, while gradually enlarging and producing pepsinogenic granules of increasing size. In the low base segment, some zymogenic cells show signs of degeneration leading to death by either necrosis or apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Since conventional chemical fixation may extract tissue components and thus alter structural organization, cryofixation was used to reexamine the ultrastructure of three thick basement membranes: lens capsule, Reichert's membrane, and Engelbreth-Holm-Swarm (EHS) tumor matrix, and two thin basement membranes, those of epididymis and semi-niferous tubules. Cryofixation was achieved by slam freezing followed by either freeze substitution in dry acetone containing 1% osmium tetroxide and 0.05% uranyl acetate or freeze drying in a molecular distillation dryer. The results by both procedures demonstrate that thick basement membranes and the lamina densa of thin basement membranes are composed of a network of anastomosing strands referred to as cords. The cords vary in density and distinctiveness, but their thickness averages 3 to 5 nm in every tissue examined. The spaces separating the cords vary within wide limits, but their mean diameter is approximately 15 nm in every case. Two other common features are 1) the presence within the network of a few 1.5-3.0-nm-thick filaments and 2) 4.5-nm-wide sets of parallel lines referred to as double tracks. When these results are compared with those previously described after conventional fixation, no significant difference is observed in either the cord network or the associated filaments and "double tracks." However, in the thin basement membranes processed by cryofixation, the lamina densa is in direct contact with epithelial cells, whereas, after conventional fixation, the lamina densa is separated from the epithelial cells by a pale layer referred to as lamina lucida or lamina rara. Immunogold labeling of three basement membranes after cryofixation and freeze substitution in acetone containing 0.3% glutaraldehyde yields strong reactions for laminin, type IV collagen, and heparan sulfate proteoglycan. Comparison with previous results indicates that conventional formaldehyde fixation adequately preserves laminin and type IV collagen but causes the loss of some proteoglycan. It is concluded that, except for this loss and the absence of lamina lucida in cryofixed thin basement membranes, the morphological and antigenic features obtained after cryofixation are similar to those observed in the past after conventional fixation.

Previous investigators have examined mitochondrial DNA (mtDNA) in the electron microscope (EM) after extraction from mitochondria and rotary shadowing. We have observed mtDNA in situ by the osmiumammine procedure for specific staining of DNA in the EM. The procedure was modified to improve the regularity of the staining and then applied to the rapidly dividing cells present in mouse duodenal crypts. In the stained sections of these cells, 25% of the mitochondria exhibited discrete reactive filaments. The filaments, whether observed directly or in stereopairs, appeared either irregular or arranged into distinct patterns, some of which were similar to those previously described after rotary shadowing of duplicating mtDNA: namely, simple and double circular figures, displacement loops and supercoiled forms. The filaments could be traced in serial sections of the same mitochondria and, therefore, were not artifacts. Moreover, their disappearance after DNase digestion demonstrated that they were composed of DNA. It is concluded that mtDNA can be visualized by the modified osmium-ammine technique and may show patterns that can be interpreted as phases in its replication.

The epithelial cells lining the oxyntic mucosa in the stomach "corpus" were identified, localized, and counted in 2-month-old male C57BL-6 mice, using glutaraldehyde-formaldehyde fixation and osmium tetroxide postfixation for studies in the light microscope (LM) while adding tannic acid to the fixative and postfixing in ferrocyanide-osmium for studies in the electron microscope (EM). The cells form a single epithelium, which invaginates into blind tubular units. Each unit is divided into four successive regions: pit, isthmus, neck, and base. On the average, a unit contains 194.2 cells. The cells have been classified into three groups totaling 11 types, listed with their mean number per unit. The first group is composed of three well-characterized cell types, each restricted to a region: (1) 37.0 surface mucous cells, hereafter called pit cells, in the "pit" region, (2) 12.6 mucous neck cells, simply called neck cells, in the "neck" region, and (3) 67.4 zymogenic cells in the "base" region. The second group is also composed of three well-characterized cell types, distributed over the four regions: (1) 26.0 parietal cells, (2) 13.2 entero-endocrine cells, and (3) 0.6 caveolated cell. The third group consists of five cell types, which have been little or not characterized in the past. Four are located in the "isthmus" region and show EM features indicative of immaturity, that is, a nucleus with mainly diffuse chromatin and large reticulated nucleoli, and a scanty cytoplasm rich in free ribosomes: (1) 17.2 cells are the least differentiated in the epithelium; they are devoid of secretory granules and accordingly named granule-free cells, (2) 10.0 cells contain a few dense secretory granules smaller than, but otherwise similar to, those in pit cells; they are referred to as pre-pit cells, (3) 1.8 cells possess a few marbled secretory granules that often exhibit a pale core and are smaller than, but otherwise similar to, those in neck cells; they are called pre-neck cells, (4) 0.6 cells display long microvilli and/or small canaliculi similar to those in parietal cells; they are named pre-parietal cells, and (5) 5.6 cells restricted to the base region are characterized by secretory granules with features intermediate between those of neck and zymogenic cells; they are named pre-zymogenic cells. The observations suggest the following hypothesis on cell filiation.(ABSTRACT TRUNCATED AT 400 WORDS)

The mandibular condyle from 20-day-old rats was examined in the electron microscope with particular attention to intracellular secretory granules and extracellular matrix. Moreover, type II collagen was localized by an immunoperoxidase method. The condyle has been divided into five layers: (1) the most superficial, articular layer, (2) polymorphic cell layer, (3) flattened cell layer, (4) upper hypertrophic, and (5) lower hypertrophic cell layers. In the articular layer, the cells seldom divide, but in the polymorphic layer and upper part of the flattened cell layer, mitosis gives rise to new cells. In these layers, cells produce two types of secretory granules, usually in distinct stacks of the Golgi apparatus; type a, cylindrical granules, in which 300-nm-long threads are packed in bundles which appear "lucent" after formaldehyde fixation; and type b, spherical granules loaded with short, dotted filaments. The matrix is composed of thick banded "lucent" fibrils in a loose feltwork of short, dotted filaments. The cells arising from mitosis undergo endochondral differentiation, which begins in the lower part of the flattened cell layer and is completed in the upper hypertrophic cell layer; it is followed by gradual cell degeneration in the lower hypertrophic cell layer. The cells produce two main types of secretory granules: type b as above; and type c, ovoid granules containing 300-nm-long threads associated with short, dotted filaments. A possibly different secretory granule, type d, dense and cigar-shaped, is also produced. The matrix is composed of thin banded fibrils in a dense feltwork. In the matrix of the superficial layers, the "lucency" of the fibrils indicated that they were composed of collagen I, whereas the "lucency" of the cylindrical secretory granules suggested that they transported collagen I precursors to the matrix. Moreover, the use of ruthenium red indicated that the feltwork was composed of proteoglycan; the dotted filaments packed in spherical granules were similar to, and presumably the source of, the matrix feltwork. The superficial layers did not contain collagen II and were collectively referred to as perichondrium. In the deep layers, the ovoid secretory granules displayed collagen II antigenicity and were likely to transport precursors of this collagen to the matrix, where it appeared in the thin banded fibrils. That these granules also carried proteoglycan to the matrix was suggested by their content of short dotted filaments. Thus the deep layers contained collagen II and proteoglycan as in cartilage; they were collectively referred to as the hyaline cartilage region.

The ultrastructure of nucleoli was examined in developing rat spermatocytes and spermatids, with the help of serial sections. In addition, the radioautographic reaction of nucleoli as examined in rats sacrificed 1 hr after intratesticular injection of 3H(5')-uridine and taken as an index of the rate of synthesis of ribosomal RNA (rRNA). Primary spermatocytes from preleptotene to zygotene have small nucleoli typically composed of fibrillar centers, a fibrillar component, and a granular component, within which are narrow interstitial spaces. During early and mid-pachytene, nucleoli enlarge to about nine times their initial size, with the fibrillar and granular components forming an extensive network of cords--a nucleolonema--within which are wide interstitial spaces. Meanwhile, there appear structures identical to the granular component but distinct from nucleoli; they are referred to as extranucleolar granular elements. Finally, from late pachytene to the first maturation division, nucleoli undergo condensation, as shown by contraction of fibrillar centers into small clumps, while fibrillar and granular components condense and segregate from each other, with a gradual decrease in interstitial spaces. In secondary spermatocytes, nucleoli are compact and rather small, while in young spermatids they are also compact and even smaller. Nucleoli disappear in elongating spermatids. In 3H-uridine radioautographs, nucleolar label is weak in young primary spermatocytes, increases progressively during early pachytene, is strong by the end of mid pachytene, but gradually decreases during late pachytene up to the first maturation division. In secondary spermatocytes and spermatids, there is no significant nucleolar label. In conclusion, rRNA synthesis by nucleoli is low in young spermatocytes. During pachytene, while nucleoli enlarge and form a lacy nucleolonema, rRNA synthesis increases gradually to a high level by the end of mid pachytene. However, during the condensation and segregation of nucleolar components occurring from late pachytene onward, the synthesis gradually decreases and disappears. The small, compact spermatids arising from the second maturation division do not synthesize rRNA.

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.

The presence of six substances--laminin, type IV collagen, heparan sulfate proteoglycan, entactin, fibronectin, and the amyloid P component--was investigated immunohistochemically in the matrix of the Engelbreth-Holm-Swarm (EHS) mouse tumor after it had been fixed in formaldehyde (with or without a brief preliminary glutaraldehyde fixation), embedded in Lowicryl K4M, and sectioned for processing through the protein A-gold sequence. Enumeration of the number of gold particles per square micrometer of matrix sections demonstrated that the six substances were present in distinct amounts. The results for each substance were fairly consistent throughout the matrix in three experiments. Furthermore, the available evidence indicated that, with the exception of the amyloid P component, the substances were associated with the cord network of the tumor matrix. Finally, the use of a reconstituted basement membrane containing known amounts of laminin, type IV collagen, and heparan sulfate proteoglycan as a standard, led to the conclusion that, in the tumor matrix, the relative content of laminin to type IV collagen to the proteoglycan was in a ratio of 1:0.6:0.03, suggesting molar ratios of approximately 1:1:0.2, respectively.

Basement membranes were divided into two types: 1) thin basement membranes, such as those of the epidermis, trachea, jejunum, seminiferous tubule, and vas deferens of the rat, the ciliary process of the mouse, and the seminiferous tubule of the monkey, and 2) thick basement membranes, such as the lens capsule of the mouse and Reichert's membrane of the rat. High-magnification electron microscopy was used to examine both types after fixation either in glutaraldehyde followed by postosmication or in potassium permanganate. The basic structure of thin and thick basement membranes was found to be a three-dimensional network of irregular, fuzzy strands referred to as "cords"; the diameter of these cords was variable, but averaged 4 nm in all cases examined. The spaces separating the cords differed, however. In the lamina densa of thin basement membranes, the diameter of these spaces averaged about 14 nm in every case, whereas in the lamina lucida it ranged up to more than 40 nm. Intermediate values were recorded in thick basement membranes. Finally, the third, inconstant layer of thin basement membranes, pars fibroreticularis, was composed of discontinuous elements bound to the lamina densa: i.e., anchoring fibrils, microfibrils, or collagen fibrils. In particular, collagen fibrils were often surrounded by processes continuous with the lamina densa and likewise composed of a typical cord network. Finally, two features were encountered in every basement membrane: 1) a few cords were in continuity with a 1.4- to 3.2-nm thick filament or showed such a filament within them; the filaments became numerous after treatment of the seminiferous tubule basement membrane with the proteolytic enzyme, plasmin, since cords decreased in thickness and could be reduced to a filament, and 2) at the cord surface, it was occasionally possible to see 4.5-nm-wide sets of two parallel lines, referred to as "double tracks." On the basis of evidence that the filaments are type IV collagen molecules and the double tracks are polymerized heparan sulfate proteoglycan, it is proposed that cords are composed of an axial filament of type IV collagen to which are associated glycoprotein components (laminin, entactin, fibronectin) and the double tracks of the proteoglycan.

Young rats given an intravenous injection of [3H]proline were killed at successive times from 4 to 80 min later. Fibroblasts from the front foot pad were radioautographed ; silver grains were counted over several of the organelles and the results were expressed as percent radiolabel per unit volume. These percentages reached a peak over rough endoplasmic reticulum cisternae at 4 min, intermediate vesicles and tubules at 10 min, spherical distensions of cis-side Golgi saccules at 20 min, cylindrical distensions of trans-side saccules between 40 and 60 min, and secretory granules at 60 min. It is proposed that the succession of peaks corresponds to the migration pathway of collagen precursor proteins within fibroblasts; that is, the proteins synthesized in rough endoplasmic reticulum are delivered by intermediate vesicles and/or tubules to the spherical distensions of cis-side saccules, somehow pass from there to the cylindrical distensions of trans-side saccules and, finally, are carried by secretory granules to the extracellular space.

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 micron in leptotene to 1.6 micron 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.

The cell populations of the occipital cortex were examined in young rats subjected to different sensory experiences. In one series recently weaned animals were reared in enriched, impoverished or control environments. The enriched environment was obtained by keeping the animals among "toys" and other rats; the impoverished environment, by rearing the animals one per cage in a darkened, quiet room; and the control environment, by housing the animals three per cage under usual animal room conditions. Six recently weaned rats were kept in each environment for 30 days and ten, for 80 days. In a second series suckling rats were handled daily. Handling consisted of touching, holding and rubbing rat pups for 15 minutes per day during the first ten days after birth; twelve rats were studied, six handled and six unhandled controls. In the two series, the animals were sacrificed under anesthesia by perfusion with mixed aldehydes. Semithin epon sections of occipital cortex were stained with toluidine blue; neurons and the three main types of glia were enumerated. In addition, the thickness of the cortex was measured and the glial cells of corpus callosum counted in the animals exposed to the three environments for 80 days. Under the influence of the enriched environment, the occipital cortex enlarged, the number of oligodendrocytes increased over the controls by 27-33% in the 30- and 80-day groups and the number of astrocytes, by 13% in the 80-day group. Within the cortex, only certain layers showed the increase in glial numbers. In the corpus callosum, however, the numbers of glial cells did not differ from those in controls. In the animals exposed to the impoverished environment, neither the size of the cortex nor the number of oligodendrocytes and astrocytes differed from controls. The animals subjected to handling also showed evidence of cortical enlargement, but the only significant change in glial cells was a 12% increase in astrocytes. It is concluded that handling and enrichment produce changes in anatomical indices of neural function including depth of cortex and numbers of glial cells. The glial response was specific to the type.of manipulation since astrocytes were predominantly affected by handling and oligodendrocytes, by enrichment. The effect of handling on astrocytes may be attributed to the stimulation being applied at a time of astrocyte proliferation, whereas the effect of enriched environment on oligodendrocytes occurred at a time of active production of these cells. The differences in cell numbers were explained by changes in the rate of cell population growth; since the impoverished did not differ from the control animals, the changes probably consisted of growth acceleration in the enriched animals rather than diminution in the impoverished ones.

Enamel formation was reviewed by morphology and radioautography in rat incisors. Labeled amino acids and sugars were used as matrix precursors whereas labeled calcium monitored mineral deposition. All ameloblasts synthesize organic material, but only cells in the zone of secretion release labeled matrix. The pattern of matrix deposition indicates that enamel rods are elaborated by Tomes' processes within cavities formed by interrod partitions. The latter are elaborated by cytoplasmic projections from adjacent ameloblasts. Initially-labeled matrix is added as a band near the cells. With time the label randomizes throughout the entire immature enamel and most of it is lost in the zone of maturation. However, a glycoprotein component attributed to remnants of Tomes' process membrane persists in mature enamel. Labeled calcium is incorporated into crystals which grow at a uniform rate throughout the entire layer of enamel in the zone of secretion and up to the middle of the zone of maturation. The ribbon-like crystals are built close to the cell membrane and elongate as the cell recedes. Crystal elongation occurs in the same location as new matrix is deposited; that is, rod crystals are related to Tomes' processes and interrod crystals, to cytoplasmic projections. The crystals grow to full size mainly by thickening and this growth presumably displaces the organic matrix.

The ultrastructure of connective tissue microfibrils was examined in two sites: the ciliary zonule of the eye and the foot pad, in 20-day-old mice perfused with glutaraldehyde. The microfibrils were classified into two categories, referred to as typical and atypical. Typical microfibrils predominate in both sites; they are unbranched, straight or gently curving, tubular structures of indefinite length with an overall diameter of 12.8 +/- 1.7 nm in the zonule and 13.8 +/- 2.8 nm in the foot pad. They are composed of two parts: tubule proper and surface band. The tubule is 7- to 10-nm wide and characterized in cross section by an approximately pentagonal wall and an electron-lucent lumen containing a 1- to 2-nm bead referred to as a spherule. When longitudinal sections of microfibrils are examined at high magnification, the wall of the tubule does not appear as a continuous line but as a series of successive dots. The interpretation of these findings is that the tubule is composed of successive annular segments with an approximately pentagonal outline. The surface band is a 3-nm-wide, ribbon-like structure wrapped around the tubule. The band has dense borders called tracks. Along the tracks, densely stained, 4.6-nm-long "spikes" are attached at 4.0-nm intervals. The wrapping of the bands is somewhat irregular. They may be in a transverse position across single or several microfibrils, in which case each band might constitute a distinct belt; more frequently, the bands are oblique and appear to form a continuous helix. It is proposed that surface bands play a role in holding together the juxtaposed segments making up a tubule. A model has been constructed to represent the association of tubule and band into a typical microfibril. Atypical microfibrils, which are more common in foot pad than in ciliary zonule, appear wavy, lack a definite tubule, and are characterized by distorted, irregular surface bands. They are attributed to proteolysis of typical microfibrils.

The production of type I collagen by fibroblasts, odontoblasts, and osteoblasts is reviewed on the basis of results obtained by electron microscopy, 3H-proline radioautography, and immunostaining for type I procollagen. In the three cell types, the precursors of type I collagen are processed along the rough endoplasmic reticulum (rER)-Golgi-secretory granule pathway in the same manner as secretory proteins, but the available evidence suggests a few special features: 1) From the rER site of synthesis, the initial collagen precursors, known as pro-alpha chains, are transported to the Golgi apparatus within tubular structures, referred to as intermediate tubules, rather than within vesicles. 2) The pro-alpha chains coil into a triple helix within spherical distensions present along the saccules on the cis side of Golgi stacks. 3) The resulting procollagens are fairly rigid and form bundles that cause spherical distensions to lengthen into cylindrical ones, whereas by an unknown mechanism these distensions become part of the saccules on the trans-side of Golgi stacks. 4) The procollagen-containing cylindrical distensions are released from trans-saccules to become secretory granules, and some procollagen material finds its way into lysosomes. 5) The secretory granules release their procollagen content by exocytosis at the cell surface. 6) The released procollagen is transformed into collagen before or, more probably, after associating with the surface of a collagen fibril.

The ultrastructure of Reichert's membrane, a thick basement membrane in the parietal wall of the yolk sac, has been examined in 13-14-d pregnant rats. This membrane is composed of more or less distinct parallel layers, each one of which resembles a common basement membrane. After routine fixation in glutaraldehyde followed by osmium tetroxide, the layers appear to be mainly composed of 3-8-nm thick cords arranged in a three-dimensional network. Loosely scattered among the cords are unbranched, straight tubular structures with a diameter of 7-10 nm, which mainly run parallel to the surface and to one another; they are referred to as basotubules. Permanganate fixation emphasizes the presence of a thick feltwork of irregular material around basotubules. Finally, minute dot-like structures measuring 3.5 nm and referred to as double pegs are present within the meshes of the cord network. Reichert's membranes have been treated for 2-48 h at 25 degrees C with plasmin, a proteolytic enzyme known to rapidly digest laminin and fibronectin. After a 2-h treatment, most of the substance of the cords is digested away leaving a three-dimensional network of 1.5-2.0-nm thick filaments. The interpretation is that the cords are formed of a plasmin-resistant core filament and a plasmin-extractable sheath. When plasmin treatment is prolonged for 15 h or longer, the filaments are dissociated and disappear, while basotubules are maintained. Plasmin digestion also reveals that basotubules are composed of two parts: a ribbon-like helical wrapping and tubule proper. Further changes in the tubule under plasmin influence are interpreted as a dissociation into pentagonal units suggestive of the presence of the amyloid P component. After 48 h of plasmin treatment, basotubules are further disaggregated and dispersed, leaving only linearly arranged double pegs. Reichert's membranes with or without a 2-hr plasmin treatment have been immunostained by exposure to antibodies against either laminin or type IV collagen with the help of peroxidase markers. The results indicate that the sheath of the cords contains laminin antigenicity, while the core filament contains type IV collagen antigenicity. It is proposed that Reichert's membrane consists mainly of a three-dimensional network of cords composed of a type IV collagen filament enclosed within a laminin-containing sheath. Also present are basotubules--which may contain the amyloid P component--and double pegs whose nature is unknown.

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. Immunostaining 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.

Microglial cells are absent from the corpus callosum of newborn rats. In the hope of finding out when and how microglial cells appear with age, 3H-thymidine was given intraperitoneally as single or three shortly spaced injections to 5-day-old rats weighing about 15 g; and these animals were sacrificed at various time intervals from 2 hours to 35 days later. Pieces of corpus callosum were taken near the superior lateral angle of the lateral ventricles; and semithin sections were radioautographed and stained with toluidine blue. The corpus callosum of 5-day-old rats is composed of loosely arranged unmyelinated fibers and scattered cells. Among these cells, microglia are rare; there are a few astrocytes, many immature glial cells, rare pericytes, and 6--7% of phagocytic "ameboid cells" consisting of a few monocytes and many macrophages. In the animals sacrificed two hours after 3H-thymidine administration, label is present only in immature cells and "ameboid cells." As time elapses and the fibers of corpus callosum become myelinated, oligodendrocytes and, later, microglial cells appear. At the age of 12 days, microglial cells are present in substantial number; and by 19 days, the number doubles to reach a plateau. Many of the new microglial cells are labeled, e.g., 78.1% in 12-day-old animals (7 days after 3H-thymidine administration). The labeled microglial cells must have come from the transformation of cells that acquired label early, that is, from the immature cells or the "ameboid cells." The height of the peaks of labeling--59.8% at nine days for immature cells and 77.8% at 12 days for "ameboid cells"--points to the latter as precursors of the highly labeled microglial cells. Furthermore, the "ameboid cells" disappear as microglial cells appear and there are transitional elements between these two cell types. Cell counts suggest that about a third of the "ameboid cells" transform into microglial cells, while the others degenerate and die. Thus, the microglial cells which appear in the corpus callosum during the first three weeks of life result from transformation of the "ameboid cells"--a group of macrophages showing various stages of transition from monocytes. As for the occasional microglial cell appearing after the third week or in the adult, they presumably come directly from monocytes. In either case, monocytes would be the initial precursors.

The formation and turnover of the glycoproteins of the plasma membrane have been investigated by quantitative radioautography in the kidney tubules of young rats and adult mice killed at various time intervals after an intravenous injection of 3H-fucose. In young (40 g) rats killed five to ten minutes after the injection, radioautographs of distal tubule cells show that the Golgi apparatus contained about 85% of the cell label. By 30 hours, only 8% of the label remained in this organnele, whereas 67% was in the plasma membrane, indicating that most of the label had migrated from Golgi apparatus to this membrane. Similarly, in proximal tubule cells, about 82% of the label was initially in the Golgi apparatus, but less than 2% remained at 30 hours, at which time 78% was in the plasma membrane. In the latter cells, the apical tubules and vacuoles became heavily labeled before the apical microvilli did and, therefore, may be involved in the transit of label from the Golgi apparatus to the microvillous membrane. The results are interpreted to mean that, in kidney tubule cells, the Golgi apparatus is the site of a continuous incorporation of fucose into glycoproteins and that these migrate to the plasma membrane. In fully formed cells, such a conclusion would imply a continuous turnover of plasma membrane glycoproteins. However, in the rapidly growing kidney of young rats many new cells are added daily, the growth of which might involve net addition as well as turnover of glycoproteins. Accordingly, the experiment has been repeated in adult mice, in which the cells are assumed to be fully formed. Furthermore, since turnover implies eventual decrease of incorporated label, some of the animals have been killed at longer intervals, up to 27 days after injection. In these adult mice, as in young rats, prompt Golgi uptake and subsequent migration of label to the plasma membrane were observed in distal and proximal tubules cells. With time the label content of the plasma membrane decreased gradually, and by 27 days had virtually disappeared. From grain counts, it is concluded that the mean half-life of glycoproteins in the apical membrane of distal tubule cells is about two days, whereas in both the apical and basal membranes of proximal tubule cells, it is slightly over three days.

In this series of two articles, the duration of mitosis and that of the cell cycle were examined in a group of proliferating cells located in the mouse pyloric antrum and known as isthmal cells. However, before measuring the duration of mitosis, as described in the second article, it is necessary to identify the early and late steps of the mitotic process. This is attempted in the present article, in which the four phases of mitosis and the interphase are described in semithin (0.5 micron thick) Epon serial sections stained with hemalun. The frequency of these phases is then estimated. The beginning of prophase is indicated by the appearance in the nucleus of numerous 0.2-0.3 micron thick basophilic threads. The threads gradually increase in thickness to become the typical chromosomes (about 0.7-micron thick) observed at the end of prophase. Metaphase and anaphase show no remarkable features. At telophase, chromosomes separate from one another, gradually acquire pale segments along their length eventually to look like rows of alternating dark and light patches, and finally vanish. When prophases and telophases are defined in this manner, the enumeration of isthmal cells yields a high proportion of prophases (28%) and telophases (31%), but a low proportion of metaphases (1%) and anaphases (0.3%). Forty per cent of the cells are in interphase.

The parietal layer of the rat yolk sac includes a 5 microliter thick sheet known as Reichert's membrane that exhibits properties of basement membranes. Its inner side is lined by a single layer of loosely distributed cells referred to as endodermal cells. Both Reichert's membrane and endodermal cells were examined at 13-14 days' gestation with emphasis on the ultrastructure of the Golgi apparatus, the identification of its component parts by specific phosphatase activities, and its possible role in the cells' secretory process. Reichert's membrane is composed of a series of stacked layers similar to basal laminae and composed of a network of fibrils with a diameter of 2-8 nm along which dots are located at irregular intervals. The endodermal cells contain the usual organelles, including interconnected rough endoplasmic reticulum (rER) cisternae and a prominent Golgi apparatus. With the help of phosphatase reactions, the stacks of Golgi saccules were divided into a) "phosphatase-free" saccules, the first ones on the cis or forming side, b) one or two "intermediate" saccules in the middle of the stacks, containing nicotinamide adenine dinucleotide phosphatase activity, c) one or two "last" saccules rich in thiamine pyrophosphatase activity on the trans or mature side, and d) continuing beyond the trans side, the GERL element displaying acid phosphatase activity. The latter is associated with profiles equally rich in acid phosphatase and tentatively considered to be prosecretory granules. Finally, the ectoplasm adjacent to Reichert's membrane displays large, acid phosphatase-containing structures tentatively considered to be secretory granules. Thus, the extensive rER network, the well-compartmentalized Golgi apparatus, and the presence of structures which may be prosecretory and secretory granules indicate that the endodermal cells are well-equipped for the secretion of the components of Reichert's membrane.

When periodontal capillaries of rat incisor tooth were immunostained for four basement membrane components (laminin, collagen IV, fibronectin, heparan sulfate proteoglycan), all four were detected in the secretory organelles of endothelial cells located within 3 mm of the tooth's proximal end, but only the proteoglycan was observed in cells located 4 mm away and beyond (Experiment I). [3H]-Thymidine autoradiography revealed that the endothelial cells located at the tooth's proximal end were young and actively dividing, whereas those located 4 mm or more away were older and generally quiescent (Experiment II). Since immunostaining of a cell's secretory organelles for a given substance indicates production of this substance, the first experiment shows that endothelial cells at the proximal end produce the four basement membrane components. The second experiment discloses that these cells are young. As for the endothelial cells located 4 mm or more beyond the proximal end, the first experiment reveals that they produce only heparan sulfate proteoglycan, while the second shows that they are relatively old. Production of laminin, collagen IV, and fibronectin only by young cells implies that these substances are long-lived and stable components of basement membrane, whereas production of the proteoglycan by both young and old cells implies that it is labile and continually replaced.

The phases of mitosis were examined in the columnar cells at the base of duodenal crypts in adult male mice given an intravenous injection of 3H-thymidine and sacrificed 20 min later. The duodenum was fixed by immersion into glutaraldehyde-formaldehyde, and the cells were examined in the electron microscope, with or without processing for radioautography. Interphase nuclei are characterized by the distribution of chromatin; aside from the cortical chromatin spread along nuclear envelope and nucleolus, there are chromatin accumulations that belong mainly in two different classes: 1) numerous chromatin "specks" ranging in size from about 5 to 70 nm and averaging 47 nm; 2) a few roughly circular or elongated chromatin "packets" measuring from 70 to 230 nm. Early prophase nuclei differ mainly by a large increase in the number of chromatin packets to 20-30 or more per nuclear profile; their average diameter is 128 nm. During mid-prophase, the chromatin packets enlarge gradually to an average 221 nm diameter. Between mid- and late prophase, there is a further increase in diameter to 679 nm. At metaphase, the packets take on the appearance of mature chromosomes, and their diameter increases to 767 nm. At anaphase, daughter chromosomes migrate to each pole, where they fuse into a compact chromatin mass. At telophase, nucleoplasmic areas progressively enlarge within the chromatin mass and separate strands of chromatin, which gradually become segmented into chromatin clumps. Counts of mitotic cells show a high proportion of prophase and telophase nuclei. Calculation from the counts yields the duration of the phases, that is, 5.6, 0.2, 0.1, and 1.6 hr, respectively, for pro-, meta-, ana-, and telophase. Finally, radioautography 20 min after 3H-thymidine injection shows labeling in 54% of the interphase nuclei, 85% of early prophase nuclei, and 73% of mid-prophase nuclei, while there is no label in late prophase, metaphase, anaphase and telophase nuclei. In confirmation of previous light microscopic work, the S stage of the cycle begins when a cell is in interphase and continues through the early prophase and part of mid-prophase. Moreover, the main sites of DNA synthesis are the chromatin specks during interphase and the cortical chromatin during early and mid-prophase. The chromosome condensation taking place in the meantime may be separated into two main steps: 1) a slow, moderate condensation of the chromatin packets during early and mid-prophase and 2) a rapid, pronounced one during late prophase and prometaphase when the packets become chromosomes.

Adult male mice were given a continuous infusion of about 0.5 muCi of 3H-thymidine per gram body weight per day for periods varying from 1 to 60 days. Semithin sections of descending colon were cut from/plastic-embedded blocks and stained by a method combining silver impregnation and iron hematoxylin, by which argentaffin entero-endocrine cells and caveolated cells could be identified. From radioautographs, the labeling index of these cells was determined. One to three days after the beginning of 3H-thymidine infusion, label is observed in some of the stained entero-endocrine cells in the bottom of the crypts; the apices of these cells reach the crypt lumen and are joined to neighboring cells by terminal bars (junctional complexes). After five to seven days, labeled entero-endocrine cells are seen on the sides of the crypts, where their base stretches along the basement membrane and their apex has lost its terminal bar connections to neighboring cells. Finally, by 13 and 24 days, labeled cells are observed within the epithelium at the mucosal surface. The turnover time, which is taken to be equal to the mean time required for migration from site of origin to site of loss on the mucosal surface, has been estimated at 23.3 days. This is much longer than the 4.6 days required by the two main cell types of the epithelium -- vacuolated-columnar and mucous cells -- to travel the same route. It is likely that, after entero-endocrine cells lose their terminal bar attachment to other epithelial cells, they migrate independently and very slowly. Labeled caveolated cells are first seen in the crypt bottom one day after the beginning of 3H-thymidine infusion. By three to five days, they are on the sides of the crypts; their base is stretched along the basement membrane, but their apex retains its attachment to neighboring cells by terminal bars. By seven days, labeled caveolated cells are on the mucosal surface. Their turnover time has been assessed at 8.2 days. This is, again, longer than for the two main types to which they are bound by terminal bars throughout migration. The discrepancy is explained by the caveolated cells arising deeper in the crypts than most vacuolated-columnar and mucous cells.

The isthmus of typical mucous units of the pyloric antrum was investigated in 3- to 4-month-old CD1 mice using light and electron microscopy as well as 3H-thymidine radioautography. On the average, the isthmus measured 25 microns in length and was composed of 36 isthmal cells and two enteroendocrine cells. Isthmal cells generally displayed features found in embryonic cells, such as many free ribosomes, scant organelles, and a large reticulated nucleolus, and were, therefore, at an immature stage of development. Isthmal cells could be devoid of secretory granules ("granule-free cells," 2%) or contain a few small, spherical, PA-Schiff-positive, mucous granules in their apex. The granules in some of the cells had a variegated appearance and a diameter averaging 235 nm ("mottled granule cells," 39%); in other cells, the granules had a large diameter, 278 nm, with a pale background and a dense core ("core granule cells," 28%); while in still others they were homogeneously dark and measured 264 nm ("dense granule cells," 12%). Finally, some cells included a mixture of core and dense granules ("mixed granule cells," 14%). One hour after a single injection of 3H-thymidine, 37% of the isthmal cells were labeled. Each of the five isthmal cell types could acquire label and, therefore, divide. After one or more days of continuous 3H-thymidine infusion, all isthmal cells were labeled. Their turnover time was estimated to be 16.1 hr (t1/2 = 11.2 hr). The isthmus is thus composed of several cell types which are turning over rapidly. While all are relatively immature, the various types are thought to represent different developmental stages in the life history of an isthmal cell. A model devised on this basis proposes that the granule-free cells are stem cells, from which mottled granule cells are derived. These in turn evolve into either the dense granule cells of the upper isthmus or the core granule cells of the lower isthmus, or into the mixed granule cells (which are believed to develop eventually into dense granule cells or core granule cells). Maintenance of a steady state requires that the rapid production of isthmal cells be associated with rapid emigration; the dense granule cells presumably going to the pit and the core granule cells to the gland. The turnover of isthmal cells is accordingly described as following a "bidirectional pattern" of renewal.