A cytochemical study of the Golgi apparatus of the spermatid during spermiogenesis in the rat

The reactivity of the various components of the Golgi apparatus of rat spermatids for three phosphatase activities (nicotinamide adenine dinucleotide phosphatase, NADPase; thiamine pyrophosphatase, TPPase; cytidine monophosphatase, CMPase) and the incorporation of ³H-fucose by the spermatids was analyzed at the 19 steps of spermiogenesis, i.e., during and after this organelle elaborated the glycoprotein-rich acrosomic system. During steps 1–3, the Golgi apparatus produced, in addition to the proacrosomic granules, multivesicular bodies that became associated with the chromatoid body. NADPase was located within the four or five intermediate saccules of Golgi stacks, and TPPase was found in the last one or two saccules on the trans aspect of the stacks from steps 1 to 17 of spermiogenesis. CMPase was located within the thick saccular GERL elements found in the trans region of the Golgi apparatus from steps 1 to 7 of spermiogenesis, but the CMPase-positive GERL disappeared from the Golgi apparatus after its detachment from the acrosomic system at step 8. The acrosomic system itself was reactive for CMPase and TPPase but was negative for NADPase, while the multivesicular bodies were CMPase and NADPase positive but unreactive for TPPase. Tritiated-fucose was readily incorporated within the Golgi apparatus of steps 1–17 spermatids; in steps 1–7 it was subsequently incorporated within the acrosomic system and multivesicular bodies. These various data indicated (1) that the Golgi apparatus of spermatids, although it loses its CMPase-positive GERL element in step 8, retains evidence of functional capacity until it degenerates in step 17; (2) that in early spermatids the various saccular components of the Golgi are specialized with respect to enzymatic activities; and (3) that each Golgi region may contribute in a coordinated fashion to the formation of the acrosomic system and multivesicular bodies.     

Morphological and cytochemical alterations of the Golgi apparatus and GERL in rat parotid acinar cells during ethionine intoxication and recovery

The present electron microscopic cytochemical investigation was undertaken to characterize the alterations in the Golgi apparatus and GERL of rat parotid acinar cells during ethionine intoxication and recovery. Although the Golgi apparatus and GERL were reduced in size, and some broadening of the Golgi saccules occurred as the result of ethionine treatment, the relative localization of thiamine pyrophosphatase (TPPase) activity in the Golgi saccules, and acid phosphatase activity (AcPase) in GERL, remained unchanged. Shortly after ethionine treatment was stopped, a dramatic redistribution of enzyme activities was noted. Within the first 24 hours of recovery, the Golgi apparatus began to enlarge, and the content of secretory granules increased. By day 3 of recovery, cisternae morphologically identifiable as GERL and forming secretory granules possessed TPPase activity, while AcPase activity was virtually undetectable. After seven days of recovery, the Golgi apparatus and GERL appeared both morphologically and cytochemically normal. The enzyme modulation observed during recovery may be correlated with increased secretory granule production. Furthermore, the presence of TPPase activity in GERL and forming secretory granules lends support to the suggestion that GERL may be derived from the trans Golgi saccule.     

Simultaneous visualization of the binding of nerve growth factor and epidermal growth factor to single rat pheochromocytoma (PC12) cells through indirect immunohistofluorescence

The rat pheochromocytoma cell line, PC12, which has receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF), was used to develop a technique for the simultaneous visualization of separate growth factor receptors by indirect immunohistofluorescence. The cells were incubated with saturating concentrations of nerve growth factor and epidermal growth factor. After fixation, the cells were treated with anti-NGF sheep antiserum and then with antisheep rabbit IgG conjugated with fluorescein; they also were treated with anti-EGF rabbit antiserum and then with anti-rabbit sheep IgG conjugated with rhodamine. Fluorescence microscopy showed that a single PC12 cell bound both NGF and EGF. The fluorescence due to EGF binding was reduced when the cells were grown in the presence of NGF. A similar reduction of fluorescence was observed after addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Both manipulations are known to reduce the specific binding of 125I-EGF to these cells. Subclones of PC12 cells, NR11 and NR20, reported not to have NGF receptors, did not demonstrate NGF binding when tested with this indirect immunohistofluorescence method. Thus, the binding of growth factors which is demonstrable by indirect immunohistofluorescence method seems to reflect the presence of the specific cell surface receptors for both peptides on individual PC12 cells.     

Three-dimensional architecture of the Golgi apparatus in Sertoli cells of the rat

Glutaraldehyde-fixed testes were stained “en bloc” with the Ur-Pb-Cu technique of Thiéry and Rambourg ('76) or post-fixed and stained with the osmium tetroxide-potassium ferrocyanide method of Karnovsky ('71). Thin or thick (up to 3 μm) sections were examined with the Philips (301 or 400) EM or the high voltage EM. Stereopairs were prepared with photographs of tilted specimens (± 7°). At low magnification, in thick sections (0.5–3 μm) stained with Ur-Pb-Cu, the whole Golgi apparatus formed a single network of interconnected wavy ribbon or platelike structures extending from the juxtanuclear region toward the apex of the cell. At higher magnifications, with the two staining techniques, this Golgi network showed two distinct types of regions: the “saccular region” corresponding to the conventional stack of saccules and the “intersaccular connecting region” made up of anastomotic tubules which bridge adjacent stacks. In the saccular regions, there was, on the cis-face of the stack, a tight polygonal meshwork of anastomotic tubules (osmiophilic element). Underlying it there were three to seven closely apposed saccules perforated with pores of various diameters, and finally, on the trans-face, a network of tubules was usually connected to the last saccule of the stack, which seemed to peel off from the pile, The intersaccular connecting regions showed proximal and distal zones with regard to the associated stacks. The proximal zone was made up of superimposed and parallel polygonal networks of membranous tubules which were continuous with corresponding saccules of the stack. In the distal zone they interdigitated, intertwined, anastomosed and bridged adjacent saccular regions; others turned at right angles and established connections with tubular extensions arising at various levels of the same stack. While cisternae of endoplasmic reticulum were contiguous with tubules or saccules located on the transface of the Golgi apparatus, a close association between the ER cisternae and the cis-face of the stacks was not usually observed.     

Regulation of acetylcholinesterase by nerve growth factor in the pheochromocytoma PC12 cell line

In the PC12 clonal nerve cell line derived from a rat pheochromocytoma, the activity of acetylcholinesterase (AChE) is regulated by nerve growth factor (NGF). The specific activity of AChE was significantly increased 2 days after exposure of PC12 cells to NGF (8 × 10^?8 M), and reached its maximal increase of 94% after 3 days. Thereafter the specific activity of AChE did not significantly vary during the following 7 days. AChE cytochemistry showed the enzyme activity to be localized predominantly in cisternae of the rough endoplasmic reticulum, with a strongly enhanced activity after NGF treatment.     

Sensitive time-resolved fluoroimmunoassay of nerve growth factor and the disappearance of nerve growth factor from rat pheochromocytoma PC12 cell culture medium

A sensitive time-resolved fluoroimmunoassay of nerve growth factor (NGF) has been developed. The method is based on the unique property of the lanthanides for delayed fluorescence, which reduces substantially the endogenous fluorescence of biological substances, because the excitation of the sample and detection of the fluorescence signal are separated in time and in wavelength. Using the europium-conjugated antibodies to the NGF from Vipera lebetina (snake) venom and to the beta NGF from mouse submandibular gland in a solid-phase quantitative two-site fluoroimmunoassay, we obtained a maximal sensitivity of 10 pg/ml (0.38 pM)for mouse NGF and 40 pg/ml (1.2 pM) for snake NGF. Using this method, we investigated the disappearance of NGF from rat pheochromocytoma PC12 cell culture medium. Mouse beta NGF (5-10 ng/ml) disappeared completely after 12 h of incubation, whereas snake NGF was not substantially internalized even after 48 h.     

Formation of secretion granules in the Golgi apparatus of plasma cells in the rat

The three-dimensional structure of the components of the Golgi apparatus was analyzed in plasma cells of rat duodenum. The spheroidal juxtanu-clear Golgi apparatus was formed by a continuous rib-bonlike structure composed of the following stacked elements. On the cis-face of the Golgi stack, there was a tubular membranous network referred to as the cis-element and/or a slightly dilated saccule perforated with small pores. The two or three subjacent saccules, which showed few pores, were slightly dilated and contained a fluffy granulofilamentous material. They were also perforated in register by cavities or wells containing 80-nm vesicles. The next one or two underlying elements were fenestrated saccules showing flattened portions as well as distended portions containing a homogeneous material denser than that seen in the overlying saccules. The last two or three elements of the stack showed a partially separated or “peeling off” configuration. These last elements consisted of prosecretory granules attached to flattened, empty-looking saccules showing buds at their surface; detached, more-or-less fenestrated, flattened saccules; and shrivelled residual trans-tubular networks. In the trans-region of the stack, in addition to numerous small vesicles, short membranous tubules, detached prosecretory granules, and denser fully formed secretion granules were also seen. These images were interpreted to indicate that secretory material present in the trans-saccules flows toward the dilated portions which become prosecretory granules. The trans-most elements seemingly peel off the stack to yield prosecretory granules and fragmenting trans-tubular networks.     

Tridimensional structure of the Golgi apparatus in type A ganglion cells of the rat

The three-dimensional structure of the whole Golgi apparatus and of its components in type A ganglion cells was examined in thin and thick sections by low- and high-voltage electron microscopy. At low magnification, in 10-μm-thick sections of osmicated cells, the Golgi apparatus formed a broad, continuous perinuclear network. At higher magnification and in thinner sections of cells impregnated with uranyl acetate-lead-copper citrate or postfixed in K-ferrocyanide-reduced osmium, the Golgi apparatus appeared as a heterogeneous structure in which saccular regions characterized by stacks of saccules alternated with intersaccular regions made up of branching membranous tubules which bridged the saccules of adjacent stacks. The saccular regions consisted of the following superimposed elements: (1) a cis-osmiophilic element made up of anastomosing tubules; (2) two or three saccules negative for the phosphatases tested (i.e., nicotinamide adenine dinucleotide phosphatase = NADPase, thiamine pyrophosphatase = TPPase, and cytidine monophosphatase = CMPase); (3) two saccules showing TPPase activity; and (4) one to three trans-sacculotubular elements showing a “peeling-off” configuration, one of which showed CMPase activity. The saccules (phosphatase-negative) on the cis-side of the Golgi stacks showed, in addition to small circular pores, larger perforations in register. The cavities thus formed in the stacks of saccules, called “wells,” always associated with small 80-nm vesicles, had a pan shape with the mouth directed toward the cis-face and the bottom closed by a TPPase-positive saccule. In face views of the saccules, the smallest of these perforations showed either a crescent shape, due to the presence of a bud on one side of the perforation, or a circular shape with a single small 80-nm vesicle in the center which was occasionally attached to the saccule by a filiform stalk. Such smaller cavities were considered as the precursors of the larger perforations and eventually of the wells. The small 80-nm vesicles seen in the small cavities or in the wells appeared to form in situ and possibly migrate toward the cisternae of endoplasmic reticulum seen proximal to the cis-face of the stack of saccules. Small 80-nm vesicles were also numerous in the intersaccular regions, along the lateral- and trans-aspects of the Golgi stacks, while larger, 150-to 300-nm vesicles, coated and uncoated, were seen only on the trans-face of the Golgi stacks in proximity to the trans-sacculotubular elements which appear to “peel off” from the Golgi stacks.     

Changes of cytochemical properties in the Golgi apparatus during in vivo differentation of the ameloblast in developing rat molar tooth germs

The cytochemical changes of the Golgi stacks occurring concomitantly with cell differentiation were examined in ameloblasts of developing rat molar tooth germs using osmium impregnation and cytochemistry with nicotinamide adenine dinucleotide phosphatase (NADPase), thiamine pyrophosphatase (TPPase), and acid phosphatase (Acpase). NADPase, TPPase, and Acpase activites were already present in the Golgi stacks of the inner enamel epithelial cells, the undifferentiated form of the ameloblast: NADPase activity existed in the medial Golgi cisternae, TPPase activity in the trans Golgi cisternae, and Acpase activity in almost all cisternae and strongly in the trans-most cisterna of the Golgi stack. At this stage, however, osmium deposits after impregnation were not observed in the cisterna of Golgi stacks but were present in some small vesicles. These vesicles were located throughout the cytoplasm. Osmiophilic cisternae in the Golgi stacks were apparent for the first time at the stage when the Golgi apparatus developed and migrated to the region distal to the nucleus with the progression of cell differentiation. These findings indicate that the cis subcompartment of the Golgi apparatus was incomplete in the inner enamel epithelial cells with regard to appearance of its cytochemical property, as compared with the medial and trans subcom partments. It is suggested that the cis compartment of the Golgi stack may be completed only in the last stage of the compartmentalized Golgi organization during differentiation of the ameloblast.© Willey-Liss, Inc.     

Fine structure of initial outgrowth of processes induced in a pheochromocytoma cell line (PC12) by nerve growth factor

Summary Cells of the PC12 line (which is derived from a rat pheochromocytoma) develop neuron-like processes upon exposure to nerve growth factor (NGF), and thus provide an opportunity to study this phenomenonde novo. We have used the transmission electron microscope to analyse the early stages of process outgrowth (1, 2, 3 and 7 days) to determine what organelles are involved and in what sequence they appear during development. Despite the non-synchronous response to NGF, we can derive three main stages in early process formation. (1) NGF-treated cells develop conical extensions similar to, but larger and more numerous than those of controls. Extensions terminate in bulbous expansions that contain large number of chromaffin-like granules and bear microspikes filled with microfilaments. (2) The extensions of NGF-treated cells then acquire membranous organelles indicative of transmitter packaging and/or recycling of cytoplasmic membranes, for example, tubular reticulum, clear and dense-cored vesicles, multivesicular bodies, and lysosomes. (3) As processes elongate, they develop a shaft that contains an array of microtubules and fine tubular reticulum dispersed in a filamentous matrix, and varicosities that exhibit the same organelles seen in stage 2. The discussion stresses the similarities in the outgrowth of processes in PC12 cells and neurons, and speculates that NGF causes a change in organization and/or quantity of organelles that already exist in non-treated control cells.     

Nerve growth factor immunohistochemistry and biological activity in the rat iris

Summary Nerve growth factor (NGF) production in the cultured rat iris was examined using immunohistochemistry and bioassay of irides and conditioned media. NGF immunoreactivity increased steadily with days in culture so that the intensity of staining was maximal after 6 days of culture. The localization was shown to be sensitive to the presence of cross-linking fixatives such as formaldehyde and glutaraldehyde and this effect was only partially alleviated by the use of very high concentrations of antibodies. NGF immunoreactivity was localized in Schwann cells and possibly nerve axons, but with no antigen detectable in smooth muscle fibres. Media conditioned over irides initially supported a high percentage of dissociated sympathetic neurons, but the number supported decreased with time in culture until day 4. Moreover, the use of antibodies to NGF allowed the detection of at least two types of neuronotropic activity, NGF accounting for at least 94% of the total trophic activity present after 4 days of culture. These findings provide support for the proposal that Schwann cells produce NGF and question the accepted hypothesis that the molecule is produced by smooth muscle fibres as a peripheral maintenance factor for sympathetic and sensory nerves. The results also suggest that two survival factors may be involved in the regulation of sympathetic function.     

Changes of cytochemical properties in the Golgi apparatus during in vivo differentiation of the ameloblast in developing rat molar tooth germs.

The cytochemical changes of the Golgi stacks occurring concomitantly with cell differentiation were examined in ameloblasts of developing rat molar tooth germs using osmium impregnation and cytochemistry with nicotinamide adenine dinucleotide phosphatase (NADPase), thiamine pyrophosphatase (TPPase), and acid phosphatase (Acpase). NADPase, TPPase, and Acpase activities were already present in the Golgi stacks of the inner enamel epithelial cells, the undifferentiated form of the ameloblast: NADPase activity existed in the medial Golgi cisternae, TPPase activity in the trans Golgi cisternae, and Acpase activity in almost all cisternae and strongly in the trans-most cisterna of the Golgi stack. At this stage, however, osmium deposits after impregnation were not observed in the cisterna of Golgi stacks but were present in some small vesicles. These vesicles were located throughout the cytoplasm. Osmiophilic cisternae in the Golgi stacks were apparent for the first time at the stage when the Golgi apparatus developed and migrated to the region distal to the nucleus with the progression of cell differentiation. These findings indicate that the cis subcompartment of the Golgi apparatus was incomplete in the inner enamel epithelial cells with regard to appearance of its cytochemical property, as compared with the medial and trans subcompartments. It is suggested that the cis compartment of the Golgi stack may be completed only in the last stage of the compartmentalized Golgi organization during differentiation of the ameloblast.     

Formation of secretion granules in the Golgi apparatus of pancreatic acinar cells of the rat

The three-dimensional structure of the Golgi apparatus and its components has been analyzed in sections of pancreatic acinar cells by using stereopairs of electron microscope photographs. Pancreatic tissue fixed in glutaraldehyde was postfixed in reduced osmium, and the sections were stained with lead citrate. Tissues were also treated to demonstrate phosphatase activity (i.e., nicotinamide adenine dinucleotide phosphatase, NADPase; thiarnine pyrophosphatase, TPPase; cytidine monophosphatase, CMPase). The following stacked components were observed along the branching, anastomotic, continuous, ribbon like Golgi apparatus. (1) On the cis-face of the Golgi stack there was a tubular membranous network known to be osmiophilic and referred to as the cis-osmiophilic tubular network or cis-element. (2) A first, poorly fenes-trated saccule, unreactive for the phosphatases tested, was slightly distended in places and contained a fluffy granulofilamentous material. (3) The subjacent three or four saccules, reactive for NADPase and/or TPPase, showed dilated portions containing a granulofilamentous secretory material similar to that filling the rest of the saccule. They also showed nondilated portions perforated with large fenestrations, some of which were in register and formed wells containing 80-nm vesicles. The dilated portions of these saccules were present at random along the length of the saccules and were not located exclusively at their edges. (4) The remaining one or two elements of the stack, CMPase positive, showed dilated spheroidal portions or prosecretory granules containing a homogeneous secretory material and flattened fenestrated regions free of secretory material and having the appearance of networks of narrow membranous tubules. (5) Lastly on the trans-aspect of the stack there were detached prosecretory granules reactive for CMPase and surrounded by a corona of small vesicles, and smooth-surfaced spherical CMPase-negative granules having a denser content that were identified as fully formed secretion granules; there were also occasional free trans-tubular networks strongly reactive for CMPase that appeared to undergo fragmentation and numerous small vesicles free from acid-phosphatase activity. These various images were interpreted as indicating that prosecretory granules formed in relation to two or three fenestrated saccules on the trans-side of the stack. Such granules, following their detachment from the trans-face of the stack, their separation from trans-tubular networks, and condensation of their content, yielded mature secretion granules.     

神经生长因子干预实验性自身免疫性脑脊髓炎幼年大鼠脑神经干细胞的变化

BACKGROUND:Nerve growth factor is a neurotrophic factor that is involved in the cell regulation and promotes the proliferation of neural stem cells. OBJECTIVE:To observe the effect of nerve growth factor on neural stem cells of experimental autoimmune encephalomyelitis rats. METHODS:Wistar rats, 3 weeks of age, were randomly divided into control group, encephalomyelitis model group, nerve growth factor treatment group. Rat models of experimental autoimmune encephalomyelitis were made in the latter two groups. On the day of onset, rats in the nerve growth factor treatment group were given intraperitoneal injection of 1 000 U/kg nerve growth factor for 7 continuous days, and rats in the other two groups given no treatment. Effects of nerve growth factor on clinical symptoms of model rats were observed. Expression of Brdu in the subependymal zone and Brdu⁺/GFAP⁺ expression in the cortex were detected. RESULTS AND CONCLUSION:Rat models of experimental autoimmune encephalomyelitis appeared to have encephalomyelitis symptoms successively at the beginning of 10-day immunization, while rats in the control group had no symptoms of encephalomyelitis. In the model group, glial cell hyperplasia, inflammatory cell infiltration, damage to vascular endothelial cell proliferation, and perivascular aggregation of inflammatory cells in the rat brain were found, while no abnormal changes in the rat brain were found in the control group. Compared with the model group, the expression of Brdu in the subependymal zone and Brdu⁺/GFAP⁺ expression in the cortex were both higher in the model group (P < 0.05), and these expressions were also higher in the nerve growth factor treatment group than the model group at 7 and 21 days after onset (P < 0.05). To conclude, these findings suggest that the rat model of autoimmune encephalomyelitis can be successfully established, and nerve growth factor treatment can improve clinical symptoms of experimental autoimmune encephalomyelitis rats by promoting the proliferation and differentiation of neural stem cells into astrocytes.     

Prenatal development of growth hormone-producing cells in the rat anterior pituitary as studied by immunogold electron microscopy

The existence of three morphologically different types of GH cells in the rat anterior Pituitary glands has been reported previously. Among these three types, Type III which contains the smallest granules has been considered to be immature, because this type occurs more frequently in neonatal rats than in adults. In this study, the prenatal development of GH cells in the rat fetus was observed by immunoelectron microscopy. In the rat fetus at 18.5 days of gestation, Type III cells were most numerous (48.5%), followed by Type II cells (45.5%). Type I cells were almost absent from these fetal pituitaries. At 20.5 days Type II cells exceeded Type III in frequency, and Type I cells also increased to about 35%. These results indicate that Type III cells are the most immature type of GH cells, and might transform into Type II and, in turn, to Type I, as the rats mature. Images indicating active secretory functions such as granule formation in the Golgi apparatus and/or GERL, exocytosis and crinophagy were observed in the GH cells even in the fetal stage.     

Sequential changes in epidermal growth factor receptor/ligand function in cultured rat liver epithelial cells transformed chemically in vitro

Diploid WB rat liver epithelial cells contain abundant, rapidly internalized epidermal growth factor receptors, and respond pleiotropically to ligand binding. Signal transduction pathways downstream from the EGF receptor involve activation of elements that are both dependent on and independent of protein kinase C activation. Neoplastic transformation of wild-type WB rat liver epithelial cells by exposure to N-methyl-N'-nitro-N-nitrosoguanidine is associated with progressive alterations in the responses of affected cells to binding of EGF to EGF receptors, including heightened cell proliferation and the expression of several other phenotypic properties. Tumorigenic rat liver epithelial cells acquire the ability to express transforming growth factor-alpha (TGF-alpha), and to secrete this growth factor in a regulated and then unregulated manner. TGF-alpha expression, together with the presence of abundant EGF receptors, provides affected cells with an autocrine growth cycle. The ability of transformed WB rat liver epithelial cells to produce tumors cosegregates clonally with TGF-alpha expression and with heightened expression of c-myc, c-Ha-ras and c-Ki-ras proto-oncogenes.