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Parts of human seminiferous tubules containing late spermatids with condensed nuclei (Stage II) absorbed more transmitted light than did parts at other stages. Spermatogenic stages I, III, IV and VI were identifiable by phase-contrast microscopy.
The functional state of living Sertoli cells can be assessed by using transillumination and phase-contrast microscopy. In tissue from hypospermatogenic men the Sertoli cells contained more lipid droplets than in normal cases and sometimes whole degenerative cells.
The acrosomal matrix of hamster spermatozoa was enriched and characterized. Acrosomal matrices were released from spermatozoa with shaking in a pH 5.2 buffer containing Triton X-100 and protease inhibitors, and enriched on a glass-bead column. Phase-contrast microscopy indicated that 70-80% of the acrosomal matrices were released from the spermatozoa and only minor contamination from sperm heads was detected. Transmission electron microscopy confirmed the low level of contamination in the preparation and revealed a bilaminar structure similar but not identical to that of guinea-pig acrosomal matrix. One- and two-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the acrosomal matrix to be a complex structure enriched for several polypeptides. Proteinase activity was demonstrated by gelatin-SDS-PAGE. The major activity corresponded to bands of relative molecular masses (Mr) of 56,000, 51,000 and 48,000 with two minor bands of Mr 30,000 and 28,000. The lectin Pisum sativum agglutinin (PSA) bound to the anterior head of spermatozoa and isolated acrosomal matrix as judged by fluorescence microscopy using FITC-PSA. Western blots of spermatozoa and acrosomal matrices followed by overlay with biotinylated PSA indicated that there are at least two PSA-binding glycoproteins of Mr 60,000 and 72,000.
Rat outer dense fibres were isolated from cauda epididymal spermatozoa using mechanical and chemical dissection methods. Sperm tail isolation procedures were monitored by phase-contrast microscopy and the purity of the outer dense fibres was verified by electron microscopy. SDS-PAGE of isolated outer dense fibres revealed at least nine Coomassie brilliant blue stained bands, and 12 silver staining bands. The most abundant proteins were a large band between 26.5 and 32.5 kDa, and 84 kDa, 21.5 kDa and 15.5 kDa bands. The amino acid composition of the total rat outer dense fibres and seven isolated proteins showed similar compositions, being abundant in aspartic and glutamic acid, serine, glycine and leucine. However, the content of cysteine and proline was highly variable among the isolated proteins. Immunofluorescence microscopy demonstrated that a polyclonal antiserum to isolated rat outer dense fibres showed positive staining localized to the mid-piece of rat and rabbit spermatozoa. However, there was crossreactivity in the principal piece as well as the mid-piece of the human spermatozoa. The antiserum also showed crossreactivity in the perforatorium of rat sperm heads and the acrosome and equatorial segment of rabbit sperm heads. These data indicate that it is technically possible to isolate proteins from the outer dense fibres that will enable further studies of the amino acid sequences of sperm tail proteins.
The objective of this work was to characterize, and compare different morphological types of hemocytes of Rhodnius prolixus, Rhodnius robustus, Rhodnius neglectus, Triatoma infestans, Panstrongylus megistus, and Dipetalogaster maximus. This information provides the basis for studying the cellular immune systems of these insects. Seven morphological hemocyte types were identified by phase-contrast microscopy: prohemocytes, plasmatocytes, granular cells, cystocytes, oenocytoids, adipohemocytes and giant cells. All seven types of hemocytes are not present in every species. For example, adipohemocytes and oenocytoids were not observed in P. megistus and P. infestans, and giant cells were rarely found in any of the species studied. The hemocytes of Rhodnius and Dipetalogaster are more similar to each other than those from Triatoma and Panstronglus which in turn closely resemble each other. Emphasis is placed on methodological problems arising in this work which are discussed in detail.
Human zona pellucida (ZP) is maintained up to the blastocyst stage prior to hatching. In in vitro fertilized (IVF) embryos, it eventually acts as a morphodynamic interface between the cultured embryo and its microenvironment. Ultrastructural data on the ZP of IVF blastocysts are scarce in humans. We employed correlated phase contrast microscopy (PCM) and scanning electron microscopy (SEM) to study retrospectively the ultrastructural morphology of the ZP outer surface of 20 IVF human blastocysts from 16 Japanese patients (28-44 years of age, average 36.7+/-4.2) with a history of infertility. Blastocysts were derived from conventional in vitro fertilization (cIVF) (n = 10) and from intracytoplasmic sperm injection (ICSI) (n = 10). Both cIVF and ICSI groups included "clear blastocysts" (n = 5) and "dark blastocysts" (n = 5). By PCM, the clear blastocysts exhibited a regular, round-shaped contour and consisted of clear and voluminous cells. By SEM, they displayed a spongy ZP with numerous fenestrations formed by networked filaments. By PCM, dark blastocysts appeared irregularly shaped and often collapsed, and comprised dark cells and debris. By SEM, their ZP were smooth with remnants of compact fenestrations. In conclusion, viable blastocysts presented a normal ZP outer surface ultrastructure, whereas unhealthy blastocysts showed an altered ZP outer surface, comparable to that of immature/atretic oocytes. Such alterations could reflect sub-optimal culture conditions and/or could be related to blastocyst degenerative processes. The blastocyst ZP surface ultrastructure was unaffected by the fertilization technique (cIVF or ICSI). These data suggest that blastocyst survival in vitro is related to ZP ultrastructure maintenance.
The presence of specific bacteria in subgingival plaque has been used as an indicator of active periodontal disease. The technique of subgingival sampling may conjecturally influence the identification and enumeration of microorganisms reported. In this study, paper point sampling and scaler sampling are compared. Subgingival samples using both methods were taken from three surfaces in each of 12 patients at the following time points: at each of two appointments one week apart before treatment and at each of two appointments 12 and 13 weeks following treatment. Microbiological analyses were undertaken to determine the total number of colony forming units, the proportions of suspected periodontal pathogens, and the number of spirochetes using phase contrast microscopy. Significantly higher numbers of colony forming units and spirochetes were found for paper point sampling both before and after treatment.
Aims of this study were to comprehensively describe the intraoral distribution of the spirochete morphogroup and of 7 antigenically distinct oral treponema, and to relate their presence to periodontal status. Periodontal tissues were evaluated at 4 sites on every tooth except third molars and 76 subjects were classified according to the worst periodontal condition at any one site: Group 1, gingivitis (n = 13); Group 2, early periodontitis (n = 38); and Group 3, advanced periodontitis (n = 25). Subgingival plaque was collected from each half of every tooth evaluated clinically. Spirochetes were identified with phase contrast microscopy and specific treponema were detected immunochemically using monoclonal antibodies to Treponema denticola serovars A-D, T. socranskii subspecies bucalle, T. socranskii subspecies socranskii, and T. pallidum (pathogen-related oral spirochetes, PROS). The counting protocol was conservative and probably underestimated the actual presence of organisms. Spirochetes were found at one or more sites in approximately 60% of subjects in all groups. PROS were found in approximately 40% of subjects in all groups while T. denticola (predominantly serotype B) and T. socranskii (exclusively T. socranskii subsp. buccale) were more frequently observed in Group 2 (roughly 25% for both treponema) than in Groups 1 or 3. Overall, spirochetes were detected in less than 15% of the 4,040 sites examined. Spirochetes were found at more sites of periodontitis (group mean range 20 to 40%) than of gingivitis (6 to 20%), and were only infrequently found at sites of periodontal health (4 to 10%). Spirochetes were identified most often in plaque from around molars and they were usually found in only one of two samples from individual teeth. Results of this study suggest that although spirochetes are most often found associated with periodontitis, their distribution is restricted and most periodontitis sites do not harbor spirochetes.
The sterile abutments of 2-stage implants offer a unique model to study intraoral transmission and bacterial colonization patterns in the oral cavity. This study aimed to examine, by means of differential phase contrast microscopy, parameters that influence the intra-oral colonization of these abutments. In part one, 159 partially edentulous patients were examined to determine the influence on the microbial composition around implants of the following parameters: 1) the relative location of the teeth in relation to the implants; 2) the microbial composition of the subgingival plaque around these teeth; and 3) the frequency of deep pockets around the natural dentition. The results indicate that the subgingival flora around the implants harbored more spirochetes and motile rods when teeth were present in the same jaw (P < 0.05) and/or when the pockets around them harbored a pathogenic flora (P < 0.05). In part two, the impact of periodontitis around the remaining teeth and of probing depth around the implants on the composition of the peri-implant subgingival flora was investigated. Thirty-one partially edentulous implant patients were classified according to their periodontal condition into 3 groups: healthy (n = 17), chronic periodontitis (n = 11), and refractory periodontitis (n = 3). The samples from deep pockets (> or = 4 mm) around implants showed significant increases in the total proportion of spirochetes and motile organisms when compared to samples from healthy subjects (1.2%) or in chronic periodontitis patients (21.0%), or to patients suffering from refractory periodontitis (31.5%). For shallow pockets (< 4 mm) significant differences were only detected between subjects with a healthy periodontium (1.0%) or chronic periodontitis (2.4%), and refractory periodontitis cases (19.7%). The present findings confirm the transmission of microorganisms from teeth to implants, and thereby highlight the importance of periodontal health around the natural dentition before as well as after implant installation.
The purpose of this investigation was to determine whether the presence of disease-associated bacteria in health-associated plaque correlated with susceptibility to periodontitis over time. Sites of periodontal health were identified in 65 adults. Six months later (recall 1), plaque was collected from sites that remained in periodontal health, and specific bacteria were detected using monoclonal antibodies in a microscopic assay. The spirochete morphogroup was identified by phase contrast microscopy. The relationship between detection at recall 1 and development of periodontitis over two successive 6-month intervals (recalls 2 and 3) was evaluated by means of logistic regression using generalized estimating equations (GEE), from which odds ratios (OR) were estimated and tested for significance. Significant relationships were defined as those having ORs with P < 0.05. Ninety-three of 1,032 sites developed signs of early periodontitis over the 12-month interval between recall 1 and recall 3. The spirochete morphogroup (OR = 3.13, P < 0.001) and pathogen-related oral spirochetes (PROS) (OR = 3.68, P < 0.001) were significantly associated with healthy sites that developed periodontitis. The association of Treponema socranskii was not significant (OR = 3.62, P = 0.0918). Odds ratios for Campylobacter rectus, Eikenella corrodens, and Porphyromonas gingivalis were less than 2.0 and not significant. Treponema denticola was not detected in health-associated plaque from stable health sites and was detected in only three sites that progressed to periodontitis. These findings indicate that the presence of PROS and some unidentified spirochetes in health-associated plaque is associated with increased susceptibility to periodontitis.
BACKGROUND: The use of membranes in guided tissue regeneration (GTR) can limit the apical migration of gingival cells and favor the establishment of new attachment by periodontal ligament fibroblasts. However, gingival recession during healing following GTR has been described as a frequent complication. The purpose of this study was to determine if gingival fibroblasts are affected by the composition of the bioabsorbable membranes used in mucogingival surgery. METHODS: Two type of bioabsorbable regenerative materials were used as cell carriers. Wistar rat gingival fibroblasts (RGF) were obtained from attached gingiva, cut into small fragments, and placed in culture dishes. When confluent, cells were detached using trypsin and identified as "first transferred cells" (P1). At the third passage (P3), cell count, trypan blue exclusion test, acid phosphatase activity, DNA synthesis, phase contrast microscopy, and scanning electron microscopy were performed. The cells were then placed in wells containing the membranes and incubated for 72 hours. RESULTS: When examined under microscopy, the control wells (without membranes) showed one cell type with the elongated appearance characteristic of fibroblasts. The wells with membranes showed an altered cell morphology with a high proportion of cell fragments regardless of the type of membrane used. CONCLUSIONS: These results suggest that cell carrier membranes could affect RGF morphology and thus alter gingival tissue healing following GTR.
Glucagon-producing cell lines were established by fusing pancreatic islet cells of adult hamster and 6-thioguanine-resistant hamster insulinoma cells. Under phase-contrast microscopy, the morphology of cultured hybrid cells was intermediate between those of the parental cells. The hybrid cells contained A-like granules, though few in number, and were stained with anti-glucagon antibody. The mode of chromosome number decreased to 78 or 79 by 3 mo after hybridization in comparison with the expected chromosome number of the heterokaryon of 104, and showed a minute decrease in 4 of 6 cell lines after 6 mo. The population doubling time ranged from 24 to 38 h, while that of parental insulinoma cells was 22.8 h. There was no correlation between the expression of cellular function and the stability of chromosome number or the length of population doubling time. The capacity of glucagon secretion was between that of the parental cells. The glucagon secreted into the medium, as assayed by the glucagon-specific antibody, was 0.6-2.5 ng/10(6) cells for 2 h, which was about 40% of total glucagon-like immunoreactivity secreted. Secretion of glucagon was not affected by high concentration of glucose, was markedly increased by theophylline, and was suppressed by exogenous insulin. All of the hybrid cells produced tumors on transplantation 6 mo after hybridization. The tumor-bearing hamsters exhibited high levels of plasma glucagon and blood glucose as well as a high level of serum insulin.
Detailed histological findings on all the regularly occurring cochlear vessels in the normal guinea pig are presented. Vessels were examined using a surface prepration technique and phase-contrast microscopy. Particular attention was paid to the density of red blood corpuscles, the thickness of the vascular wall, the occurrence of endothelial cells and pericytes, together with their possible influence on the vessel lumen and the existence of perivascular spaces and vessels lacking blood corpuscles. The data are compared with some previous findings of "vascular pathology" following noise stimulation. The possible functional significance of some of the morphological findings is also discussed.
This study was performed to design a method for the culture of rat middle-ear epithelium and to apply the method to investigate the characteristics of this epithelium. Culture of explants of middle-ear epithelium in the presence of the epidermal growth factor was successful, whereas serial cultivation required 3T3 feeder cells in addition to the epidermal growth factor. Cultured middle-ear epithelium was studied by phase-contrast microscopy, transmission and scanning electron microscopy, and combined light and scanning electron microscopy (LM/SEM). These techniques showed similarity between the cultured and the natural middle-ear epithelium. Explants and outgrowths showed both flat polygonal and ciliated epithelial cells. In serial cultivation, however, only the first of these cell types was observed. Frequently, a single primary cilium was found on the cell surface. Transmission electron microscopy showed cross-linked envelopes whose formation was promoted by ionophore X537A. Cytokeratin was demonstrated by immunoblotting, immunofluorescence, and immunoperoxidase methods, using an anti-cytokeratin monoclonal antibody. The model described here permits study of the differentiation of middle-ear epithelium in vitro and may be of future value for the study of chronic middle-ear diseases.
This study examined the kinetics of sperm nuclear decondensation induced by the action of physiological concentrations of heparin and glutathione in hamster sperm nuclei as a chromatin model that contains protamine P1 and P2. Sperm suspension was incubated at different temperatures (37, 40, 43, and 46 degrees C) in media, keeping constant the concentration of either heparin or GSH and increasing concentrations of the other reagent. Spermatozoa nuclei without any treatment, incubated for 72 h, appear densely condensed. Swelling of hamster spermatozoa nuclei was observed after 30 min of incubation in the presence of efficient concentrations of heparin-GSH. The extent of this time lag was significantly reduced at higher temperatures. DNA presence was verified by the use of ethidium bromide, acridine orange, and Feulgen stain. Phase-contrast microscopy shows that nuclear decondensation begins at the equatorial levels, with DNA highly condensed at the acrosome pole, and the basal pole as the DNA attachment point. Electron microscopy observations showed that hamster sperm nuclei initiates its decompaction at the peripheral regions and this behavior remains until late stages of decondensation, nevertheless, the chromatin is organized into "hub-like" nuclear bodies that measured 10-100 nm in diameter, joined by a network of chromatin fibers with apparent reduction in number. At the decondensation full stage, the network seems to be wide open with a reduced number of hub-like nuclear bodies present in the interlace. DNA is not organized into topologically constrained loop domains and is attached to the basal plate instead of to the nuclear matrix or any other structure.
We investigated phagocytosis of biodegradable microspheres containing a drug by retinal pigment epithelial (RPE) cells and drug release within the cells to evaluate the potential usefulness of microspheres for intracellular drug delivery. The biodegradable polymers used were L-lactic acid, and DL-lactic acid with different molecular weights or the copolymers of different monomer compositions. The microspheres containing a non-bioactive fluorescent dye (rhodamine 6GX) as a model drug, were prepared by a solvent evaporation method. The in vitro release of the dye from the microspheres was examined. Phagocytosis of the microspheres by RPE cells was conducted to evaluate the extent of phagocytosis by phase-contrast microscopy and transmission electron microscopy. The RPE cells ingesting the microspheres at different stages were examined by fluorescent microscopy to estimate the intracellular release of the dye. The dye was released with time from every microsphere and the release was controlled by changing the type of polymers constituting microspheres. The microspheres containing the dye were phagocytosed by RPE cells and the dye was released intracellularly with time. The present study indicates that the drug incorporated in the microspheres was delivered into RPE cells by way of phagocytosis and released within the cells. It is concluded that this microsphere system is a promising delivery form capable of drug targeting to RPE cells.
Vascular endothelial cells play an important role in progression or healing of various retinal or choroidal diseases and they have a broad organ specificity. We have grown and studied human choroidal endothelial cells (CECs) cultured in a collagen gel, where they form tube-like structures. CECs were differentially isolated from choroidal tissues and cultured to near homogeneity. The cells were embedded in a type I collagen gel in 24-well culture dishes and incubated with M199 medium containing 10% fetal calf serum. Gels were observed using phase contrast microscopy and were evaluated histologically by light and electron microscopy. The CECs were strongly positive for factor VIII-related antigen and actively ingested diI-acetylated LDL, indicating their endothelial nature. Ultrastructural analysis of the tube-like structures revealed a central lumen surrounded by cells joined apically by junctions and showing prominent pinocytotic activity, fenestrations and basement membrane formation; these features are typical of CECs in vivo. This preparation should be a useful tool to study the development of physiology and pathology of choroidal endothelium.
PURPOSE: To evaluate some RPE cell functions, such as wound healing, in a preparation more similar to in situ conditions, we developed a method to obtain and culture retinal pigment epithelial (RPE) cells as a sheet. And we assessed the effects of fetal bovine serum (FBS) on the rate of RPE wound healing. METHODS: We prepared RPE sheet cultures by incubating rat eyes in 0.1% proteinase K for 13 min, peeling away the neural retina-RPE complex, and then incubating the tissue for 1 h to promote spontaneous separation of the RPE sheet from the retina. After several days of incubation, the cultured sheets of RPE cells were examined by phase-contrast microscopy, scanning and transmission electron microscopy and immunocytochemistry. We made round defects 1 mm in diameter in cultured RPE sheets and estimated the rate of wound closure in media with different concentrations of FBS (0 to 10%). RESULTS: The RPE cells cultured in sheets retained their in situ features, including microvilli, tight junctions and gap junctions, and the distribution of actin and cytokeratin filaments. A wound was noted to close with restoration of a polygonal configuration. The rate of wound closure depended on serum concentration in the culture medium; when supplemented with 10% fetal bovine serum, wound closure was complete in approximately 40 h. CONCLUSIONS: The RPE sheet-culture technique we developed thus provides a suitable model for studying such RPE cell functions as wound healing or phagocytosis.