Radioautographic Analysis Research Articles

Radioautographic study of the seasonal distribution of leukocytes in turtles Phrynops hilarii (Chelonia Chelidae)

The aim of this study was to present a morphological description of the leukocytes of Phrynops hilarii turtles according to the seasonal distribution of these cells and to show their replacement in the blood circulation using a radioautographic method. Five animals of both sexes weighing 600–1200 g were used. The animal's blood was aspirated, smeared on glass slides, and stained with the Romanowsky stain, and 500 cells of each animal were counted during each season. The results obtained were analyzed statistically by analysis of variance followed by the Bonferroni test (NCSS), with the level of significance set at p < 0.05. The radioautographic analysis of turtle blood exposed to 1000 μCi of 3H-thymidine and developed after 30 days showed a large number of silver grains incorporated into the cells, except for basophils, with cell renewal occurring every seven days. Quantitative data demonstrated a seasonal influence on the distribution of some leukocyte types, with the following “ p” values: heterophils ( p = 0.0007), basophils ( p = 0.0002), monocytes ( p = 0.0016), eosinophils ( p = 0.0073). However, using this statistical method, it was not possible to detect a significant difference related to seasonal influence on lymphocytes ( p = 0.16295) or thrombocytes ( p = 0.1046). Using this experimental animal model, a seasonal influence on the distribution of some leukocyte types was observed, and the radioautographic method revealed a cell renewal system occurring every seven days, except for basophils.

Difference analysis of genome-wide DNA methylation status of CpG islands between the monozygotic twins with disconcordant phenotypes of chronic hepatitis B virus infection

To analyse the difference of genome-wide DNA methylation status of CpG island between the monozygotic twins with disconcordant phenotype of HBV infection and to discover possible differentially methylated genes. Modified AIMS (amplification of inter-methylated sites) method was adopted. According to the frequent sites of CpG islands (-CGCG- and -CCGG-), three groups of isochizomers with distinct methylation sensitivity (Sma I-Xma I, Hpa II-Msp I and BssH II-Pau I/BseP I) were used to modify the AIMS method. The modified AIMS method combined with Personal Molecular Imager FX system analysis and radioautographic analysis was used to make the global detection of the methylome of CpG islands. Multiple anonymous bands were compared between the twins with the Quantity One bio-soft. The different bands were cloned into T vectors and positive clones were sequenced. BLAST analysis of positive clone sequences was conducted to give the clues for the differential methylated genes between monozygotic twins with discordant phenotype of HBV infection. Nearly the same bands were found between one pair of twins with concordant phenotype of HBV infection. Different methylated bands were found not only between monozygotic twins with concordant phenotype but also between those with disconcordant phenotype. More differential methylated bands were found in the latter groups. By BLAST analysis with sequences of differential methylated bands, four possible genes were got. These genes might relate to the monozygotic twins with disconcordant phenotype of chronic HBV infection. Differential methylation of genes occurs in monozygotic twins with discordant phenotype of HBV infection. Whether these changes are involved in the pathogenesis of different phenotypes needs further elucidation.

Antibody targeting studies in a transgenic murine model of spontaneous colorectal tumors.

Monoclonal antibodies (mAbs) have been used to treat malignancies in humans with varying degrees of success. Progress has been hindered by the lack of suitable animal models, which would ideally consist of immunocompetent animals that are tolerant to tumor-associated antigens. Suitable models would allow the study and optimization of anti-tumor immunotherapy. We describe a murine model for the study of immunotherapy in colorectal cancers. Carcinoembryonic antigen (CEA) is a cell-surface glycoprotein that is expressed on normal human intestinal epithelium and that is overexpressed in intestinal tumors. Mice that are transgenic for the human CEA gene (CEA.Tg) were crossed with multiple intestinal neoplasia (MIN) mice. MIN mice carry a germline APC mutation and are prone to the development of intestinal adenomas. The offspring from the MIN x CEA.Tg cross developed intestinal adenomas that were shown by immunohistochemistry to overexpress CEA. Pharmacokinetic studies by using (125)I-labeled anti-CEA mAb PR1A3 showed rapid localization of antibody to tissues expressing CEA, especially the gastrointestinal tract. Macroscopic and microscopic radioautographic analysis of the gastrointestinal tracts from MIN/CEA.Tg mice indicated that PR1A3 targeted and was retained in tumors at levels higher than in areas of normal gut. These results demonstrate the utility of the MIN/CEA.Tg mouse as a model for the study of anti-CEA immunotherapy and, furthermore, demonstrate the efficiency of tumor localization by PR1A3.

Inositol Hexasulphate, a Casein Kinase Inhibitor, Alters Enamel Formation in Cultured Embryonic Mouse Tooth Germs

Post-translational modification of enamel proteins is regulated by casein kinases (CK) and results in binding sites for calcium ions that subsequently play a key role during the initial stages of mineralization. Phosphorylation may also influence the secretion and extracellular organization of enamel proteins. Previous studies indicated that inositol hexasulphate inhibited the activity of CK-I and/or CK-II in mouse tooth germs (Torres-Quintana et al., 1998). We hypothesized that inositol hexasulphate would also inhibit the activity of the specific casein kinase(s) identified in secretory ameloblasts, and would prove useful for determination of the extent to which phosphorylation might influence the organization of enamel proteins at early stages of enamel formation. To test this hypothesis, we dissected mandibular first molars from 18-day-old mouse embryos and cultured them for 11 days in the presence of 0-0.1 mM inositol hexasulphate. Ultastructural analysis revealed that the formation of enamel was largely impaired at an inhibitor concentration > or = 0.08 mM. Quantitative radioautographic analysis of [33P]phosphate incorporation indicated that radiolabeled phosphate normally secreted into forming enamel was retained within ameloblasts. In contrast, no significant difference was observed between control and inositol-hexasulphate-treated tooth germs when cultures were labeled with [3H]serine and [3H]proline. SDS-PAGE and Western blot analysis confirmed that while inositol hexasulphate inhibited CK-mediated phosphorylation, it did not significantly alter protein synthesis. We conclude that impairment of phosphorylation leads to intracellular accumulation of [3H]phosphate-containing material by ameloblasts. We also conclude that when non-phosphorylated enamel matrix proteins are secreted, they are either unable to form an enamel matrix that supports mineralization, or they diffuse throughout a poorly mineralized dentin.

Relationship of cellular proliferation to expression of osteopontin and bone sialoprotein in regenerating rat periodontium.

Cellular repopulation was studied in a model in which adjacent mineralising and soft connective tissue matrices are regenerated. Window wounds were created through alveolar bone, with either preservation or removal of periodontal ligament, in 30 male Wistar rats. Three animals per time period were killed on days 1, 3, 7, 14, and 28 after surgery for each wound type. Cellular proliferation in alveolar bone and periodontal ligament was assessed by 3H-thymidine labelling 1 h before death, followed by radioautographic analysis. Cellular differentiation was determined by the temporal expression of the bone-related markers osteopontin and bone sialoprotein, using immunohistochemical methods. In regenerating periodontium, osteopontin was expressed earlier than bone sialoprotein, which was restricted to alveolar bone. After wounding, transient expression of osteopontin was detected in the periodontal ligament at days 1 and 3. In general, wounding induced fivefold higher proliferation and clonal growth of periodontal ligament cells compared to the unwounded (control) side. Combined immunostaining and radioautography demonstrated colocalisation of osteopontin in sites with high numbers of labelled cells in both nascent periodontal ligament and regenerating alveolar bone at days 3 and 7. In contrast, bone sialoprotein, which appeared in regenerating alveolar bone on days 14-28 after wounding, was expressed much later than the peak of cellular proliferation. We conclude that (1) the intact periodontal ligament influences cell proliferation and osteopontin expression; (2) osteopontin is an early marker of periodontal tissue regeneration that is temporally and spatially associated with intensive cell proliferation and migration in osteogenic and periodontal ligament cell populations; and (3) bone sialoprotein is expressed after proliferation at sites of mineralising bone formation.

Ultrastructural, radioautographic, and morphometric analysis of gastric mucosa in chronic gastritis

Biopsy specimens of gastric mucosa from patients with chronic gastritis are examined. The dynamics of structural changes occurring during the development of chronic inflammation is demonstrated and the ultrastructural changes in the mucosa cell populations are described, which together with the radioautographic analysis of biosynthetic reactions and morphometric data characterizes the complex structural and functional rearrangements in the gastric mucosa.

Interaction of the calcium ionophore A.23187 (Calcimycin) with Bacillus cereus and Escherichia coli

Ionophores isolated from bacterial strains, and especially A.23187, are efficient antibiotics against Gram‐positive bacteria and devoid of activity on Gram‐negative species. This difference in activity was attributed to the outer membrane of Gramnegative bacteria which is presumably impermeable to these very hydrophobic compounds. In this context, the partition of the calcium ionophore A.23187 between bacteria and the medium was studied on Escherichia coli (Gram‐negative) and Bacillus cereus (Gram‐positive) using, on the one hand, fluorimetric measurements and, on the other hand, radioautographic analysis of bacteria incubated with the [3H]‐labelled ionophore. Although the first method did not give a definitive answer, the second one clearly showed that the tritiated metabolite was only incorporated into B. cereus.

Sites of plasmalemmal expansion in growth cones

Studies on plasmalemmal expansion in isolated nerve growth cones identified large, clear vesicles characteristically found in growth cones as the plasmalemmal precursor. The present article examines these plasmalemmal precursor vesicles (PPVs) in greater detail in the intact cell. (a) Pulse-chase experiments with the phospholipid precursor, [ 3H]glycerol, followed by radioautographic analysis show that PPVs in distal neurites and growth cones are labeled prior to equilibration of the label with the plasmalemma. (b) Pulse-chase experiments with lectin-ferritin conjugates demonstrate that PPVs are not endocytotic, that they contain lectin receptors, and that, during growth, patches of lectin receptors appear on the plasmalemma covering PPV clusters. (c) Freeze-fracture studies show that this plasmalemma shares with PPVs a paucity of intramembrane particles. (d) Lectin labeling experiments and freeze-fracture analysis demonstrate, furthermore, that the plasmalemma forms a network of invaginations at the base of PPV clusters. (e) Correlative studies indicate that the refractive ‘vacuoles’ seen in growth cones by phase-contrast light microscopy correspond to the PPV clusters seen at the ultrastructural level. These results confirm the identity of the plasmalemmal precursor in the intact cell and demonstrate that PPV clusters form distinct, dynamic organelles specialized for plasmalemmal expansion in the growth cone.

Albumen gland of the snail Achatina fulica is the site for synthesis of AchatininH, a sialic acid binding lectin.

A sialic acid binding lectin, AchatininH was purified from the hemolymph of Achatina fulica snail. To identify the site of synthesis of AchatininH, in vitro incubation studies in presence of labelled amino acid precursor were performed. Different organs from the snail were sliced and incubated in methionine-deficient Eagle's minimum essential medium containing [35S]-methionine at 25 degrees C for 5 h. After termination of incubation, tissues were homogenized, centrifuged and the de novo synthesized protein was immunoprecipitated with specific AchatininH antibody, followed by protein-A. The precipitated antigen-antibody complex was analysed by SDS-PAGE. Data obtained from native gel electrophoresis and SDS-PAGE radioautographic analysis indicates that AchatininH is synthesized in the albumen gland.

Distribution of [ 3H]clonidine binding sites in the brain of the convulsive mutant quaking mouse: a radioautographic analysis

The radioautographic analysis of [ 3H]clonidine binding was performed on brain slices from the convulsive mutant mice quaking and their controls of the same strain. In the quaking mice significant increases were observed mostly in the brainstem and the cerebellum, but also in a few regions of the forebrain, such as the lateral and medial thalamic nuclei, the medial geniculate nucleus, the amygdala and the hypothalamus. Other regions, such as the cerebral cortex and the hippocampus, which are classically involved in various models of epilepsy, but not in the quaking mice, did not show any modification of [ 3H]clonidine binding. A high degree of correlation was found between the structures with an increased density of α 2-adrenoceptor binding sites and the distribution of regions from which seizures can be elicited by intracerebral electrical stimulation in head-restrained quaking mice. This comparison emphasizes the role of noradrenaline acting at the level of α 2-adrenoceptors in the epileptic syndrome of the quaking mutants.

Murine Monoclonal Antibodies Reactive with a Variety of Androgen Independent Dunning Rat Prostate Adenocarcinoma Sublines also Reactive with Human Prostate Adenocarcinoma

Murine hybrldoma-derived monoclonal antibody (MCA) to Dunning rat prostate adenocarcinoma R-3327HIS (androgen independent type) has been produced by fusing P3x63 Ag8-653 myeloma cells with splenocytes of BALB/c mice which were immunized with R-3327HIS tumor cell membranes. One monoclonal antibody designated MCA-R1 (IgG2a subtype) produced an intense im-munostaining of various androgen independent Dunning rat prostate tumor sublines (HIS, HIM, HIF, AT-1, AT-2, AT-3, MAT-Lu and MAY-Ly-Lu), but did not stain other tumors of rat origin or normal rat tissues. Marginal immunostaining was detected in the androgen responsive R-3327H and R-3327G tumor subline. Although MCA-R1 antibody did not react with the regressed prostate tumor of R-3327H or R-3327G, it strongly reacted with the relapsed prostate tumor from either R-3327H or R-3327G tumor derived from rats were treated with diethyl stilbestrol (DES) or castration. MCA-R1 antibody also produced a strong cross-reaction with human prostate adenocarcinoma. Like the Dunning rat tumor, human adenocarcinoma exhibited distinct immunostaining patterns with respect to intracellular localization among well differentiated, moderately differentiated and poorly differentiated tumor. Benign prostatic hyperplasia or other normal tissues did not stain. Immunofluorescence, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioautographic analysis revealed that the Dunning rat prostate tumor antigen recognized (m.wt. 50 and 120Kd) by MCA-R1 antibody are localized on both cell surface and in the cytoplasm. This MCA may represent a potential reagent for the study of tumor biology and immunotherapy of prostate tumor.

Subcellular distribution of [3H]-dexamethasone mesylate binding sites in leydig cells using electron microscope radioautography

The present view is that glucocorticoid hormones bind to their cytoplasmic receptors before reaching their nuclear target sites, which include specific DNA sequences. Although it is believed that cytoplasmic sequestration of steroid receptors and other transcription factors (such as NFKB) may regulate the overall activity of these factors, there is little information on the exact subcellular sites of steroid receptors or even of any other transcription factors. Tritiated (3H)-dexamethasone 21-mesylate (DM) is an affinity label that binds covalently to the glucocorticoid receptor (GR), thereby allowing morphological localization of the receptor at the light and electron microscope levels as well as for quantitative radioautographic (RAG) analysis. After injection of 3H-DM into the testis, a specific radioautographic signal was observed in Leydig cells, which correlated with a high level of immunocytochemically demonstrable GR in these cells at the light-microscope level. To localize the 3H-DM binding sites at the electron microscope (EM) level, the testes of 5 experimental and 3 control adrenalectomized rats were injected directly with 20 microCi 3H-DM; control rats received simultaneously a 25-fold excess of unlabeled dexamethasone; 15 min later, rats were fixed with glutaraldehyde and the tissue was processed for EM RAG analysis combined with quantitative morphometry. The radioautographs showed that the cytosol, nucleus, smooth endoplasmic reticulum (sER), and mitochondria were labeled. Since the cytosol was always adjacent to tubules of the sER, the term sER-rich cytosol was used to represent label over sER networks, which may also represent cytosol labeling due to the limited resolution of the radioautographic technique. Labeling was highest in sER-rich cytosol and mitochondria, at 53% and 31% of the total, respectively. Cytosol (exclusively of all organelles) and nucleus showed comparatively weak labeling, at 9% and 7%, respectively. This study thus clearly establishes with the electron microscope, the localization of glucocorticoid binding sites using DM. It remains to be determined whether or not these DM binding sites represent bona fide glucocorticoid receptors or nonreceptor proteins that bind DM. Whereas the functional significance of the subcellular distribution of DM is not known, the labeling of the cytosol may represent localization of the steroid and GR in their traditional compartment. The steroid antagonistic properties of DM may have prevented the DM-GR complexes from translocating to the nucleus. However, the significant labeling of the sER-rich cytosol and mitochondria was unexpected and raises intriguing questions that are being addressed in current studies.

Improved quantitative radioautography of rat brain δ-opioid binding sites using [3H]DSTBULET, a new highly potent and selective linear enkephalin analogue

The binding properties of [(3)H]DSTBULET, a new potent and specific ligand for ?-opioid binding sites, were characterized using slices from rat brain striatum. Specific [(3)H]DSTBULET binding reached steady-state within 60 min at 20 degrees C, was saturable and reversible and the ligand appeared to interact with a single class of binding sites, characterized as ? by competition experiments. Scatchard analysis of the saturation isotherm plots yielded a K(D) value of 5.73 nM. At this concentration the specific binding was 94% of the total binding. Unlabeled DSTBULET had a low affinity (K(1) = 422 nM) for ? binding sites labeled by [(3)H]DAGO and at 5 nM [(3)H]DSTBULET interacted with a maximum of 1.2% of total ? sites. [(3)H]DSTBULET was therefore used for a detailed quantitative radioautographic analysis of the distribution of rat brain ?-opioid binding sites in order to clarify some inconsistencies in the relative densities of these sites previously reported using either various non specific ligands or [(3)H]DPDPE, a cyclic enkephalin endowed with high ? selectivity but relatively low affinity. In addition to the claustrum, basolateral and posteromedial cortical amygdaloid nuclei, nucleus accumbens ahd caudate putamen, regions well-known to contain very high levels of ? sites, a [(3)H]DSTBULET labeling was also found in the intermediodorsal, rhomboid and mediodorsal (lat.) thalamic nuclei, cortex frontoparietal (layers II + III and V + VI), ventroposterior (parvocellular) and reuniens thalamic nuclei, and anterior olfactory nucleus (posterior part). In the thalamus, the mediodorsal, central medial and centrolateral nuclei and the posterior thalamic nucleus group were moderately labeled. The superior colliculus (superficial gray layer), the interpeduncular nucleus, the stria terminalis and the nucleus solitary tractus were moderately labeled. Lower labeling was found in the medial habenula, ventromedial, laterodorsal and ventroposterior (median) thalamic nuclei, dentate gyrus and the pyramidal cell layer of the hippocampus. In the globus pallidus, ventromedial hypothalamic nucleus, ventroposterior (lateralis) thalamic nucleus, substantia nigra, ventral tegmental area, periaqueductal gray matter and dorsal raphe the labeling was scarce but significant. At all levels in the spinal cord, a low labeling was found, restricted to the substantia gelatinosa. Interestingly ?-opioid receptors are highly concentrated in the more recently developed areas of the brain (cortex, neostriatum), where they could be involved in modulation of integrational processes mainly through regulation of aminergic (especially dopamine) pathways. Therefore, owing to its high affinity and good selectivity, [(3)H]DSTBULET seems to be the most appropriate radiolabeled probe currently available to investigate the ?-opioid receptors and to quantify these binding sites by radioautography with a high sensitivity and accuracy. Accordingly, labeling was found in several areas (ventral tegmental area, substantia nigra, nucleus tractus solitarius) where a mismatch between the site of action of ?-opioids and the presence of ? sites has been previously hypothesized.

The basalis of the primate endometrium: a bifunctional germinal compartment.

Radioautographic analysis of epithelial and stromal cell proliferation in the primate endometrial functionalis and basalis (rhesus monkey) has identified horizontal zonal patterns of mitotic activation and inhibition during natural menstrual cycles. At 1 h after a single i.v. injection of [3H]thymidine, mitotic activity in endometrial biopsies (hysterotomy) was determined on 9 days from the late proliferative to the late luteal phase (-2 days to + 14 days relative to the estrogen [E2]peak). Labeling indices (LIs) were determined within glandular segments of the 4 horizontal endometrial zones: Transient functionalis Zone I (luminal epithelium) and Zone II (uppermost gland); Germinal basalis: Zone III (middle gland) and Zone IV (basal gland). The size of the dividing epithelial populations (LI) differed zonally. During E2 dominance (-2 days to +3 days), the epithelial LIs of functionalis I (10 +/- 0.3%) and II (9.8 +/- 1.0%) were greater than those of basalis III (5.8 +/- 0.2%) and basalis IV (3.7 +/- 0.8%). During progesterone (P) dominance (+5 days to +14 days), epithelial mitosis was strongly inhibited in functionalis I (4.3 +/- 1.9%), functionalis II (0.8 +/- 0.2%), and basalis III (1.4 +/- 0.5%). Thus germinal basalis III was linked functionally with transient functionalis I and II by periovulatory uniformity in epithelial proliferation and postovulatory mitotic inhibition. A unique mitotic pattern set basalis IV apart from other zones by a steady rise in LI from 1% (-2 days) to 11% (+10 days). The LIs for stromal fibroblasts remained quite uniform in basalis IV but varied in other zones. Thus the postovulatory primate basalis was a distinct bipartite compartment in which the mitotic rate in basalis IV glandular epithelium increased steadily whereas that of basalis III was strongly inhibited. The remarkable enhancement of epithelial mitotic activity in basalis IV may reflect expansion of the stem-progenitor cell population for gestational growth or for post-menstrual regeneration.

Cell proliferation in chick oral membrane lags behind that of adjacent epithelia at the time of rupture.

Radioautographic analysis showed that ectoderm and endoderm cells in chick oral membrane continued to label with tritiated thymidine through the period of rupture, but their frequency of labeling was significantly lower than those of adjacent epithelia. Frequency of labeling increased in adjacent ectoderm and endoderm, while oral membrane rates remained relatively low, suggesting that growth in the membrane lags relative to adjacent epithelia. Relatively greater proliferation in adjacent epithelia could generate tension and pull apart the thinned oral membrane. Differentials in rates of cell proliferation, when considered along with knowledge of cellular rearrangements following changes in basal lamina and matrical components, suggest that differential growth is an important force in rupture of the avian oral membrane.

Evidence for a single class of somatostatin receptors in ground squirrel cerebral cortex

In the present study we characterized high-affinity somatostatin (SRIF) binding sites (Kd = 2.06 +/- 0.32 nM and Bmax = 295 +/- 28 fmol/mg protein) in cerebral cortex membrane preparations of European ground squirrel using 125I-[Tyr0-D-Trp8]-SRIF14 as a radioligand. The inhibition of radioligand specific binding by SRIF14, as well as by its agonists (SRIF28, Tyr0-D-Trp8-SRIF14, SMS 201 995) was complete and monophasic, thus revealing a single population of somatostatinergic binding sites. Radioautographic analysis of 125I-[Tyr0-D-Trp8]-SRIF14 labeled brain sections confirmed the results of our biochemical study. The homogeneity of SRIF binding sites in the ground squirrel neocortex was not dependent on the animal's life-cycle phase.

Ab Interno Sclerostomy With a High-Powered Argon Endolaser

We used a high-energy argon blue-green laser (15-W maximum power output) to create full-thickness sclerostomies from the region of the anterior chamber angle to the subconjunctival space in pigmented rabbits using an ab interno approach. One to four laser pulses delivered through a 300-micron noncontact fiberoptic probe produced patent sclerostomies in all 20 eyes treated using 0.1-second pulse duration and 5 to 14 W of power. No intraoperative complications were encountered. Intraocular pressure, measured in 12 animals, decreased an average of 12 mm Hg in the treated eye relative to the fellow eye on the first postoperative day. The drop in intraocular pressure was associated with formation of a functioning filtration bleb. Intraocular pressure returned to preoperative levels in ten of 12 (83%) of the animals by the fourth postoperative day, and there was an associated flattening of the filtration bleb. Histologic and radioautographic analysis indicated that the effect of the laser was focal. Tissue damage and cellular proliferative response were limited to within approximately 200 micron of the wound margin.

In vivo distribution of parathyroid hormone receptors in bone: evidence that a predominant osseous target cell is not the mature osteoblast.

Previous studies in vitro and in vivo have demonstrated the presence of receptor sites for PTH on cells of the osteoblast phenotype. Nevertheless, it is unclear whether the diverse functions of this hormone in bone can all be attributed to its interaction with a single cell type. In this study, we have used a radioautographic method to examine the competitive binding of 125I-labeled rat PTH-(1-34) to the long bones of rats in vivo. Our studies confirm the presence of competitive binding to mature osteoblasts and the absence of significant competitive binding to multinucleated osteoclasts. However, by light and electron microscopic radioautographic analysis, the majority of specific competitive PTH binding was present over a cell in the intertrabecular space of the metaphyseal region, which was distinct from the mature osteoblast. This large mononuclear cell with multiple cytoplasmic extensions appeared to interface with both the bone matrix and the microvascular osseous circulation and may provide an additional target to mediate hormonal effects on the skeleton.

L'endocytose de l'« androgen-binding protein » (ABP) par les cellules principales de l'épididyme chez le rat

The present study is based on the comparison between the radioautographic analysis of the fate of the androgen-binding protein purified from rat testes (HPLC) subsequently iodinated and injected into the epididymal lumen using a micromanipulator, and the biochemical analysis of the binding capacities of this molecule to soluble epididymal membrane extracts using HPLC and ultracentrifugation. The various experimental conditions used here allowed to demonstrate that ABP was internalized by the epididymal epithelium and to state that this internalization was not a non specific fluid phase endocytosis but a receptor-mediated-mechanism. Indeed, from a morphological stand point, the labeled ABP was associated rather with the membranes of the endocytic apparatus than with its content. In addition, from the two lumenal cell types able to resorb seminal fluid products, only the principal cells took up the labeled ABP. Our results clearly showed that this internalization was correlated with the presence of a 125I.ABP binding protein. Since the binding of this protein molecule to ABP was saturable and Calcium and pH dependent, it is strongly suggested that this molecule behaves as a receptor, the ligand (or one of the ligands) of which could be ABP.

Effect of monensin on cell ultrastructure and glycoprotein migration in adult mouse jejunal epithelium in organ culture.

Explants from adult mouse jejunum were cultured for 3 h in a medium which contained both 3H-fucose (10 or 25 microCi/ml) and monensin (100 microM) or 3H-fucose only (control). Radiochemical analysis of cell fractions showed that 3H-fucose labelling of the brush border fraction decreased 42% in monensin-treated explants, suggesting that in absorptive cells the intracellular transport of newly synthesized glycoproteins to the apical plasma membrane had been inhibited. Electron-microscopic examination of treated explants revealed a variation in response to the drug from region to region. In some areas, both absorptive and goblet cells exhibited little alteration. In others, the Golgi cisternae of both absorptive and goblet cells were entirely replaced by large vacuoles, and in the latter cell type, the cisternae of the rough endoplasmic reticulum were greatly distended. Electron-microscopic radioautographic analysis showed that in absorptive and goblet cells exhibiting little morphological change, intracellular transport of newly synthesized glycoproteins was similar to that in controls. In regions where absorptive cells exhibited extensive Golgi modifications, intracellular transport remained normal in some cases; more often-however, there was a marked inhibition (over 70%) of transport of labelled glycoproteins to the apical surface. Transport to the basolateral membrane was never affected. In goblet cells exhibiting modifications of the Golgi apparatus and rough endoplasmic reticulum, no incorporation of 3H-fucose label in the Golgi apparatus occurred, suggesting a block of intracellular transport proximal to the site at which 3H-fucose is added. In absorptive cells, this does not appear to be the case, since the level of 3H-fucose incorporation in all treated cells remained similar to that in controls.