Ultrastructural changes in mouse hepatocytes exposed to vinblastine sulfate with special reference to the intercellular junctions.

The effects of a single dose (0.5 mg) of the antimicrotubular drug vinblastine sulfate on the ultrastructure of mouse hepatocytes was studied by thin sectioning and freeze-fracturing after intravenous injection of the drug. The following cytoplasmic modifications occurred in the hepatocytes: storage of lipid droplets, heavy accumulation of autophagosomes and vacuoles with very low density lipoprotein (VLDL)-like vesicles, pathological changes in the mitochondria, and dilatation of the Golgi complexes. From 30 minutes onwards the bile canaliculi appeared altered. The tight junctions surrounding the bile canaliculi became permeable to lanthanum. This increased permeability was correlated with a disorganized arrangement of tight junctional strands and localized interruptions within the zonulae occludentes. In contrast to controls the gap junctions appeared more numerous and larger in size, exhibiting a high degree of pleomorphism. In the cytoplasm gap junctional vesicles could be observed indicating the removal of gap junctions from the surface by a process of internalization. We cannot establish whether the described changes are a direct or indirect effect of vinblastine sulfate treatment. In view of the antimicrotubular effect of vinblastine sulfate we hypothesize that normal formation of gap- and tight junctions is dependent directly or indirectly on intact microtubules.

Carbon tetrachloride induced proliferation of tight junctions in the rat liver as revealed by freeze-fracturing.

Application of carbon tetrachloride produced a progressive proliferation of tight junctions in the rat liver. This system proved to be rapid and highly reproducable and affords the opportunity for tracing the fate of tight junctions in freeze-fracture replicas, facilitating investigations on their formation and function. Beginning on day one carbon tetrachloride treatments resulted in the progressive loosening and fragmentation of the junctional meshwork. After three to four days the membrane outside the zonulae occludentes was extensively filled with proliferated discrete junctional elements often forming complex configurations. From the fifth day on the zonulae occludentes were restricted again predominantly around the bile canaliculus margins. But the junctional meshwork of the zonulae occludentes remained loosened in comparison to those in the control rats. It could be shown that tight junction proliferation on the lateral surface of the plasmalemma occurred both through de novo formation from discrete centers of growth by addition of intramembranous particles and through reorganization of preexistent junctional strands of the fragmented zonulae occludentes bodies. Whereas the large gap junctions close associated with the zonulae occludentes remained more or less unaffected during the experiments, small gap junctions increased in number after five days and were located at the margin or in the tight junction domain. It is assumed that the degeneration of the tight junctions served as a pool for intramembranous particles which form the gap junctions. The results of these observations are discussed in relation to those obtained in other systems.

Binding and degradation of proteoglycans by cultured arterial smooth muscle cells. II. Binding sites of proteoglycans on the cell surface.

Exogenous proteoglycans stained for electron microscopy with colloidal gold and/or cuprolinic blue bind to the surface of cultured arterial smooth muscle cells at two different sites. (I) About 20% of the proteoglycans adsorbed to the cells from the culture medium interact as monomeric and multimeric proteoglycans with smooth or coated membrane areas. (II) The bulk of exogenous proteoglycans exhibits high affinity binding to cell membrane-associated 10 nm fibrils containing or being closely associated with fibronectin and to collagen. It is suggested that the self association of proteoglycans and their binding to the cell membrane and to cell surface-associated fibronectin and collagen are important for maintaining an appropriate micro-environment for the cultured cells.

The correlation between tissue differentiation and production of mammary tumor virus (MTV) in transplanted murine mammary tumors. Electron microscopic observations.

The spontaneous mammary tumors of the NMRI mouse are well developed microcystic adenocarcinomas. Serial isologous transplantation of the tumors results in nearly complete dedifferentiation to a solid tumor, in which only electron-microscopically rudimentary acinus-like microlumina can be observed. The adenocarcinomas produce A and B particles in abundance, with the A particles appearing intracellularly in the adluminal cytoplasmic regions of the epithelial cells in association with typical cellular structures and the B particles being restricted to closed extracellular compartments such as vacuoles or acini alone. The loss of alveolar organization in the solid tumors is followed by an almost complete reduction in mature B particles, while A particles are still regularly observed and appear to be less reduced in number. This suggests that the production of extracellular B particles is dependent upon the secretory activity of the tumor cells and that in nonsecreting cells it is predominantly a late step in virus release that is inhibited, not the synthesis of intracellular precursors.

Electron-microscopic morphology of virus replication in cells of the murine Borstel-leukemia X 429.

Intraperitoneal injections of cell-free filtrates of Borstel-leukemia X 429 (immature-cell myeloid leukemia induced by 200 rad whole-body X-irradiation) were given to 330 1-week-old NMRI mice of both sexes. Leukemia developed in 21% after injection of cell-free filtrates from leukemic spleens and in 20% after injection of cell-free filtrates from leukemic lymph nodes. The mean time lapse from injection to the appearance of the leukemia was 12.8 months. The virus replication in the newly formed leukemic cells, was studied by electron microscopy. Special attention was given to the relation between A-particles and mature type-C viruses. The leukemia cells contain intracytoplasmic and intracisternal A-particles consisting of two concentric spherical shells with an external diameter of 70 nm. A-particles present mainly as closed "virus fields" in the vicinity of the cell nucleus, frequently in the Golgi area. The intracisternal A-particles appear inside the lumen of expanded vesicles of the cytoplasma. Typ-C viruses develop on the cytoplasmic membrane and/or on the outer cell membrane by condensation of crescent-shaped electron-dense zones. All layers of the later mature virus are recognisable in these early morphological stages. After the virus has been detached from the cell it collapses, and the outer membrane appears wrinkled. No spatial correlation between the storage sites of the A-particles and the sites of formation of mature type-C viruses are demonstrable.

Tissue connections in a transplantable virus-producing sebaceous adenoma of the mouse.

In this study a transplantable sebaceous adenoma is presented, which maintained its histological structure with similarities to normal sebaceous glands over a long series of transplantations. From the periphery toward the center a basal lamina, an outer layer of undifferenciated stem cells, loosely connected secreting tissue, a tightly packed zone of close cell attachment, and finally a central sebaceous zone can be distinguished. Special reference is made to the belt-like region around the zone of cell lysis and sebum accumulation, which forms a barrier to the surrounding tissue by means of numerous cell adhesions in the form of desmosomes. Relatively few virus particles of the retrovirus group, particularly type A particles, were found in any of the examined tumors.

Tissue connections in a transplantable virus-producing sebaceous adenoma of the mouse. II. A freeze-fracture study in conjunction with filipin.

The distribution, size, and configuration of intercellular junctions in the sebaceous tumor of mice were examined using the freeze-fracture technique. Three types of junctions were observed: desmosomes, gap, and tight junctions. Tight junctions in general consisted of short linear unbranched fibrils, and macular or complex tight junctional patterns were present only occasionally. Gap junctions ranged from small sports of 0.9 x 10(-3) micrometer2 to areas of approximately 0.46 micrometers2. Desmosomes were the most frequent junctional specializations, and it is concluded that they are at least partially responsible for this tumor not being metastatic. Filipin in conjunction with freeze-fracture showed filipin-sterol complexes on the plasma membrane, the nuclear envelope, and the membranes of the endoplasmic reticulum and mitochondria. The intercellular junctions were devoid of these complexes indicating that these regions are low in cholesterol.

Reversibility of long-term effects of GnRH agonist administration on testicular histology and sperm production in the nonhuman primate.

The present investigation evaluates the long-term effects of GnRH agonist treatment on testicular histology, sperm production and the subsequent recovery of these parameters. Four adult rhesus monkeys (M. mulatta) were treated with the GnRH agonist nafarelin (D-Nal(2)6-GnRH), released from i.m.--injected poly-D,L-lactic-co-glycolide microspheres for 20 months. Monthly injection of the GnRH agonist preparation uniformly suppressed serum levels of bioactive LH and testosterone. The size of the testis was reduced to about 30% of pretreatment. Sperm counts were suppressed to azoospermia for a total period of 53 and 77 weeks, respectively, in two monkeys and the other two animals were extremely oligozoospermic. Evaluation of testicular biopsy material after 6, 12 and 20 months of treatment revealed decreased seminiferous tubule diameter, spermatogenic disruption at the level of spermatogonia or spermatocytes, accumulation of lipid droplets and secondary lysosomes in the Sertoli cell cytoplasm, and increased thickness of the tubular wall compared with pretreatment histology. Electron microscopic examination revealed that the increased wall thickness was due to an enlargement of the inner collagen layer. No evidence of fibrosis or calcification could be obtained. Leydig cells were atrophic. Serum hormones, testis size and sperm counts returned to pretreatment values within 5 to 8, 13 to 16, and 18 weeks, respectively, after termination of treatment. Testicular histology, assessed 8 months after cessation of treatment, was indistinguishable from pretreatment. It is concluded that GnRH agonist-containing microspheres are a feasible modality for sustained administration of GnRH agonists and GnRH agonist-induced suppression of pituitary and testicular function is reversible following withdrawal of treatment. Thus, GnRH agonists may have a potential for regulation of male fertility and, presumably, also for treatment of precocious puberty.

Changes in mouse hepatocytes caused by vincamin. A thin-sectioning and freeze-fracture study.

Mouse liver was examined following a single intraperitoneal application of 25 mg/kg body weight vincamine. The studies were made using the technique of thin-sectioning for electron microscopy as well as the freeze-fracture method. The thin-sectioning technique was useful for observing discrete changes such as an increase in the mitochondria and alterations in the bile canaliculi, e.g. a loss of microvilli and dilatation of the lumen. No other cytoplasmic changes could be observed. It was only with the aid of the freeze-fracture method that alterations in the cell contacts became visible. In contrast to the control animals the tight junctions in the liver tissue of mice treated with vincamin were disorganized and irregularly arranged. The gap junctions showed a very irregular contour as well. The question arises as to the extent to which the freeze-fracture method should be applied in the testing of pharmaceuticals, so as to exclude the possibility of damage to membranes.

Quantitative analysis of intramembranous particles in the membranous system of rat liver cells at different stages of development and aging.

The density of intramembranous protein particles was studied by freeze-fracture. Particle density on the fracture faces of the plasmalemma and the rough endoplasmic reticulum (RER), as well as the outer and inner membranes of the nucleus and the mitochondria in rat hepatocytes were quantified. Comparison among different age groups sampled days postcoitum (dpc), days postpartum (dpp), and months postpartum (mpp) showed age-related changes in particle density in each membrane system. With the exception of the RER, particle densities increased after the 16th dpc, reached a maximum at birth, and then decreased with increasing age. Simultaneously, the number of nuclear pores shows a positive correlation with the particle density of the nuclear membranes. The particle density on the membranes of the RER shows a maximum on the 16th dpc and on the 6th dpp. Thereafter, the density of the RER decreases slightly. In all membrane systems, the density of the particles on the external fracture faces is more variable than density of the particles on the protoplasmic fracture faces.

Ultrastructural morphometric investigations on human platelets after long-term ingestion of oral contraceptives.

Platelets from healthy females, who were taking oral contraceptives over a long period of time, were investigated by ultrastructural morphometry. The size, shape, relative volumes, numerical and surface densities of the platelets and their organelles were measured. In 'long-term pill users' the platelets showed a significant increase in the area of section profile. Further changes as, for example, increased volume of platelet granules, reduced number of mitochondria, decreased volume and surface of the open canalicular system, and increased surface of the dense tubular system are to be noted.

PIP: Ultrastructural morphometry was used to measure the size, shape, relative volumes, and numerical and surface densities of platelets and their organelles from 10 longterm (up to 5 years) oral contraceptive (OC) users. Platelet materials from 14 non-OC users were also examined as a basis for comparison. The value for the area of section profile was 27% larger in OC users than in controls and the volume density of granules was 24% larger. The length of the platelet major axis, the axial ratio, and the boundary length of platelet profile did not differ significantly between the 2 groups. The surface density of granules was 2.743 mcgm2/mcgm3 in cases compared with 2.313 mcgm2/mcgm3 in controls. The numerical density of mitochondria was 46% smaller in OC users and the surface density of the dense tubular system was 97% larger. Overall, these finding suggest deviations in form and structure of platelets in OC users as well as an increase in platelet volume. It is recommended that morphology be correlated to an altered storage and transport function in platelet in OC users.