Nuclear pores in luteal cells during pregnancy and after parturition and pup removal in the rat: a freeze-fracture study

In a previous paper we described a pronounced increase of apoptotic nuclei in rat corpus luteum of pregnancy whose programmed chromatin degeneration was induced by the progesterone antagonist mifepristone. Those observations encouraged us to study the apoptotic nuclear membrane during pregnancy and after parturition and pup removal, by using a freeze-fracture technique which allows us to observe 'en face' the nuclear envelop and also permits nuclear pore counting. This study was complemented with the TUNEL assay (TdT-mediated dUTP nick-end labelling). Changes in nuclear pores during pregnancy begin with an intense reduction in number but still showing an even distribution on the nuclear membrane, never forming aggregations sharply separated from pore-free areas, which are characteristic of other apoptotic models. Electron microscopy of thin-sections shows, coincidently with findings in the freeze-fracture replicas, a moderately irregular aggregation of marginal heterochromatin condensations. After nuclear fragmentation and micronuclear formation, pores behave in the usual manner in other apoptotic models, i.e., mainly showing migrations of nuclear pores toward the chromatin-free areas. The present results support the hypothesis that nuclear pore complexes are dynamic structures, which permit their migration toward nuclear membrane areas devoid of chromatin aggregations that might block the nucleocytoplasmic transport in such areas.

Long-term observation for osseointegriation of calcium 'phosphate sol-gel coating on Ti-6Al-4V implants / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To observe the long-term impact of calcium phosphate (CaP) sol-gel coating on bone growth around porous-surfaced implant.</p><p><b>METHODS</b>The porous-surfaced Ti-6Al-4V implants were prepared with the addition of a thin film of CaP sol-gel coating, and implanted into the tibiae of 8 rabbits, each with two implants. Implanted sites were allowed to heal for 2, 8, 12, and 24 weeks, after which specimens were obtained for scanning electron microscope analysis using the freeze-fracture technique.</p><p><b>RESULTS</b>The sol-gel coated implants recovered by freeze-fracture technique showed extensive bone growth from the endosteum along the implant surface. The bone was in direct contact with the CaP layer. The cement line-like layer was clearly demonstrated to be an intervening electron dense afibrillar layer between the CaP coat and the overlying newly deposited bone. The stability and osseointegration of the porous-surfaced implants seemed not to be affected by the osteoclastic resorption of CaP layer occurred during 24 weeks of healing.</p><p><b>CONCLUSION</b>Based on the findings in the long-term observation, the addition of a thin layer of CaP promotes an extensive osseointegrated interface between the porous-surfaced Ti-6Al-4V implants and the newly deposited bone.</p>

Ultrastructural study of the in vitro interaction between Biomphalaria glabrata hemocytes and Schistosoma mansoni miracidia

Biomphalaria glabrata and Schistosoma mansoni relationship was studied by light microscopy (LM) and freeze-fracture replica technique (FFR). We observed very thin cytoplasmic extensions of hemocytes in the LM, which then surround immobilize the miracidia. FFR images showed that the contact site between hemocytes cytoplasmic extensions and the external tegumentary coat involved only superficial layers of miracidia. Numerous vacuoles and filopodia were observed in the hemocyte cytoplasm, the latter binding with those from neighboring cells. In spite of the close interfilopodia contact, no cellular junctions were seen at these sites nor between filopodia-miracidia contact areas. The observed migration of hemocytes and their disposition in layers surrounding the miracidia in vitro correspond to previous studies.

Ultrastructural and cytochemical changes in the liver of primary biliary cirrhosis patients

The aim of this paper is to establish whether there are cytochemical or ultrastructural alterations in the hepatocytes of patients with primary biliary cirrhosis (PBC) at stages I and II compared with the biopsies from individuals with normal liver. Cytochemical technique with ATP as substrate, transmission electron microscopy (TEM) and freeze fracture were used for the studies. In the normal liver biopsies the ultrastructural cytochemical localization of the enzymatic activity was clearly shown in the bile canaliculi. In the PBC biopsies, the enzymatic activity is increased in the bile canaliculi and is also present in the lateral membranes of the hepatocyte. TEM of the lateral surface of the hepatocyte in normal livers showed a smooth surface without microvilli but in PBC livers a large number of microvilli were seen in the lateral membranes. The Golgi apparatus in these patients was localized not only near the canaliculi (normal livers) but also in front of the microvilli. Freeze-fracture showed normal features in the bile canaliculus junctions of the PBC patients. We suggest that the localization of the enzymatic reaction, microvilli and Golgi apparatus at the PBC hepatocyte lateral membranes may represent a compensatory mechanism for derivation of bile flow and other components from the hepatocyte to the intercellular space.

Confocal laser scanning, conventional scanning and transmission electron microscopy of vertebrate cerebellar granule cells

Confocal laser scanning microscopy of hamster cerebellar granular layer showed in montages of z-series the presence of small, medium and large granule cells. A granule cell Golgi cell ratio of 50/4 was observed surrounding glomerular regions. Field emission high resolution scanning electron microscopy of mouse cerebellar granular and molecular layers showed SE-I images of the outer and inner surfaces of nuclear and cytoplasmic compartments of chromium coated granule cells and the axo-spinodendritic synapses of parallel fibers with Purkinje cell dendrites. Conventional scanning electron microscopy of teleost fish cerebellar cortex showed three dimensional morphology of granule cell soma and processes and the synaptic relationship with mossy and climbing fibers, Golgi cell axonal ramifications and dendrites of stellate neurons, by means of SE-II and SE-III signal image mode, in sagittally and transversally cryofractured cerebellar cortex. SE-II images of the non-synaptic segments and synaptic varicosities of parallel fiber outer surface were characterized in the molecular layer. Ultrathin sections of transmission electron microscopy (TEM) revealed somato-somatic, dendro-somatic and dendro-dendritic like-desmosomal and like-hemidesmosomal junctions in human cerebellar granule cells. Freeze-etching replicas of mouse cerebellar cortex displayed granule cell intramembrane morphology, cytoplasmic fractured face and the Bergman glial cell cytoplasm completely surrounding the parallel fibers in the molecular layer. The mossy fiber-granule cell dendrite synaptic relationship was observed in sagittally and transversally cryofractured cerebellar cortex and correlated with TEM images. SE-II images of the climbing fiber synaptic connections with granule cell dendrites were obtained in teleost fish cerebellar cortex. One to one axo-dendritic synaptic contacts between Golgi cell axonal ramifications and granule cell dendrites were also seen. The above findings provide new vistas for future studies dealing with intracortical circuits and information processing in the cerebellar cortex.

Early myelin breakdown following sural nerve crush: a freeze-fracture study

In this study we describe the early changes of the myelin sheath following surgical nerve crush. We used the freeze-fracture technique to better evaluate myelin alterations during an early stage of Wallerian degeneration. Rat sural nerves were experimentally crushed and animals were sacrificed by transcardiac perfusion 30 h after surgery. Segments of the nerves were processed for routine transmission electron microscopy and freeze-fracture techniques. Our results show that 30 h after the lesion there was asynchrony in the pattern of Wallerian degeneration, with different nerve fibers exhibiting variable degrees of axon disruption. This was observed by both techniques. Careful examination of several replicas revealed early changes in myelin membranes represented by vacuolization and splitting of consecutive lamellae, rearrangement of intramembranous particles and disappearance of paranodal transverse bands associated or not with retraction of paranodal myelin terminal loops from the axolemma. These alterations are compatible with a direct injury to the myelin sheath following nerve crush. The results are discussed in terms of a similar mechanism underlying both axon and myelin breakdown

Double replicas allow the obtention of longitudinally freeze-fractured Trypanosoma cruzi

The double replica device was used to obtain freeze-fracture replicas of gently pressed cells, allowing the visualization of a large number of longitudinally fractured epimastigote and trypomastigote forms of Trypanosoma cruzi. This technique revealed large areas of the plasma membrane, the region of attachment of the flagellum to the cell body and the branched mitochondria.

A rotary shadow freeze-drying replica method for the three dimensional view of virus ultrastructure

A new rotary-shadowing process to obtain freeze-drying replicas is described for the analysis of virus ultrastructure, using the inner capsid of human rotavirus as a model. The findings corroborate the icosahedral symmetry with an arrangement pattern of capsomers of T=13L

Freeze-fracture study of Trichomonas vaginalis

The freeze-fracture technique was used to analyse the organization of the plasma membrane, as well as membranes of cytoplasmic organelles, of the pathogenic protozoan Trichomonas vaginalis. Rosettes formed by 4 to 14 intramembranous particles were seen on the fracture faces of the membrane lining the anterior flagella as well as in fracture faces of the plasma membrane enclosing the anterior region of the protozoan and in cytoplasmic organelles. Special organization of the membrane particles were also seen in the region of association of the recurrent flagellum to the cell body

Effect of freezing on ultrastructure of chimpanzee sperms as revealed by ethanol cryofracture and scanning electron microscope.

Ultrastructural features of fresh chimpanzee spermatozoa were studied following ethanol cryofracture of sperm pellets. Fresh and freeze preserved semen samples used for artificial insemination were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer. The centrifuged cells in the form of a pellet were post fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer. The ethanol dehydrated pellets were cryofractured, critical point dried from liquid CO2, mounted on stubs and decorated with 5-6 nm gold palladium. When compared with fresh samples the ultrastructural features of spermatozoa showed the following changes. The most conspicuous changes were noted in the acrosomal membrane region. The acrosomal membrane becomes swollen wavy and broken at several points. The plasma membrane around the neck region was thinned out with mitochondrial sheath being pulled back. The general 9 + 2 arrangement of the axoneme was unchanged. Thus critical changes in the membrane structure and mid piece morphology may account for the present failure of artificial insemination procedure using frozen sperm in this primate species.

Glucose modulation of gap junctions in the islets of Langerhans of protein malnourished rats.

It is believed that protein deficiency causes decreased insulin release in response to a glucose stimulus. We have recently shown that in prolonged protein deficiency, decreased insulin response to glucose is directly proportional to a decrease in the islet volume, suggesting no apparent defect in the insulin secretory mechanism in protein deficiency. It is documented that glucose-stimulated insulin release is closely related to an increase in the gap junctions of stimulated islet beta cells. In the present study, we show that the gap junctions of islets obtained from rats fed on a 4% protein diet were increased both in number and size following glucose treatment. This provided further proof that the mechanism to respond to glucose is not compromised in the endocrine tissue of the severely protein malnourished rats.

Thin-section and freeze-fracture study of post-mortem chenges in dog myocardium

Fragments of dog hearts submitted to 1,6,10,24 and 48 h of autolysis at 20-C were studied with freeze-fracture and thin-section techniques under the transmission electron microscope. The freeze-fracture replicas revealed maximal reduction in the mean number and clustering of intramembrane particles at 6 h post mortem, indicating irreversible cellular damage. However, sings of lethal damage (intramitochondrial amorphous dense bodies) were not observed in thin sections of the same material. The present study indicates that signs of irreversible damage similar to that occuring in in vivo ischemic alterations can be detected earlier by the freeze-fracture technique than by the thin-section technique

Scanning Electron Microscopy of Human Drusen

Drusen are small, yellowish deposits that form under the retinal pigment epithe lium with senescence or under certain pathological conditions. The present study examined these structures under the scanning electron microscope. Tissue came from the eyes of four donors, who were 22, 56, 60 and 61 years of age and who demonstrated widespread drusen of the posterior fundus. which was noted on postmortem examination. Specimens were prepared by detaching the retinal pig ment epithelium from Bruch's membrane and freeze fracturing the tissue. Drusen appeared as follows: 1. Distinct spherical masse, 10 X 9 um and 9 X 7 um in size, were situated between basement membrane of the retinal pigment epithelium and Bruch's membrane. The surface of the spherical masses were smooth. 2. Indistinct globular dome-like masses, with a harsh surface, were situated bet ween basement membrane of the retinal pigment epithelium and Bruch's membrane. These masses varied greatly in size. 3. Localized of dispersed small granular deposits of the inner collagen layer of Bruch's membrane were noted.

How many channels should there be in a gap junction?

La resistencia de las electrotónicas (uniones comunicantes) formadas entre los axones laterales gigantes del cangrejo de río ha sido medicda en preparaciones acopladas y desacopladas. Por otra parte el número de partículas sinápticas se ha contado en ambas condiciones. De estos dos grupos de datos se puede concluir que las partículas, agrupadas en placas, que se observan mediante criofractura representan canales comunicantes y que estos están abiertos. Basándonos en estos datos y en resultados publicados obtenidos en pares aislados de células acopladas se ha encontrado que existe una relación inversa entre la resistencia de entrada de las células y el número estimado de canales comunicantes abiertos entre ellas. Esta relación se aplica tanto a células excitables como a las no excitables

A freeze-fracture study on the developing satellite cells of spinal ganglia in the chick embryo

Foi feita análise pelo método da crio-fratura de células satélites em 8 diferentes fases do desenvolvimento do embriäo de galinha, do quinto dia à eclosäo. A disposiçäo laminar característica das células foi observada inicalmente no sétimo dia. Foram encontradas junçöes do tipo <> no 20- dia de incubaçäo. Pequenos grupos ou agregados de partículas foram descritos no sétimo e oitavo dias, mas näo típicas junçöes <>. Vesículas de pinocitose foram apontadas nos diferentes estágios considerados