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PubMed1996

In vitro, follicle-stimulating hormone prevents apoptosis and stimulates deoxyribonucleic acid synthesis in the rat seminiferous epithelium in a stage-specific fashion.

The effects of FSH on stage-specific apoptosis and DNA synthesis in the adult rat seminiferous epithelium were studied in vitro. Seminiferous tubular segments from stages I, V, VIIa, and VIII-IX were cultured for 24, 48, and 72 h in different concentrations of FSH. Apoptotic cells were detected by in situ end labeling of DNA strands and quantified from squash preparations. After 48 h of culture, a FSH concentration of 2 ng/ml prevented apoptosis of early (steps 1-3) spermatids. In stage VIII-IX tubules cultured for 72 h, FSH decreased the apoptosis of pachytene spermatocytes. An apoptotic type of cell death of germ cells was confirmed by DNA laddering, electron microscopy, supravital acridine orange staining, and phase contrast microscopy of unstained living cells. The effects of FSH on stage-specific DNA synthesis were studied using the same culture system. FSH increased [3H]thymidine incorporation specifically at stages I and VIII-IX, and autoradiography confirmed stimulation of mitotic and meiotic DNA synthesis in type B spermatogonia and preleptotene spermatocytes, respectively. Increased thymidine incorporation also suggested that FSH stimulated DNA synthesis of type A and intermediate spermatogonia. Most effects exerted by FSH were seen in stages containing high levels of FSH receptors and FSH-stimulated cAMP production. In conclusion, the results suggest that FSH, probably acting via Sertoli cells, has a regulatory function in spermatogenic apoptosis and DNA synthesis in stages previously demonstrated to be preferentially dependent on FSH stimulation.

Acridine Orange↗
PubMed1985

[Visual classification of erythrocytes in blood smears from normal subjects and patients].

Erythrocytes on specially prepared and coloured blood smears can be classified not only by idcture analysis but also by phase contrast microscopy. Parameters of change for the peripheral zone area and pallor area (phase contrast value PW and index of erythrocyte changes Ev-I) serve as classificators of visual technique. 449 blood smears of the same number of male and female grown-up persons, whose diagnosis was unknown at the time of evaluation, were used for evaluation. In 145 cases these were healthy test persons in clinical and laboratory respect (group I). 163 test persons were ill, but without any malignous neoplasias (group II), and in 141 patients an ensured malignous neoplasia could be found (group III). From those 308 test persons or patients respectively in group I + II 11 blood smears were falsely classified as positive (= 3.6%). In 5 from 141 cases in group III blood smears were falsely evaluated as negative (= 3.6%). The findings were statistically ensured by the t-test. Clinical parameters of evaluation gained with visual techniques of classification are in accordance with those gained with automatic picture analysis. The reasons for the good separating ability of PW and Ev-I are discussed. Visual techniques of classification are suitable for institutions of all grades of equipment.

Adult↗
PubMed2004

Sperm selection for ICSI: shape properties do not predict the absence or presence of numerical chromosomal aberrations.

BACKGROUND: We hypothesize that the potential relationship between abnormal sperm morphology and increased frequency of numerical chromosomal aberrations is based on two attributes of diminished sperm maturity: (i) cytoplasmic retention and consequential sperm shape abnormalities; and (ii) meiotic errors caused by low levels of the HspA2 chaperone, a component of the synaptonemal complex. Because sperm morphology and aneuploidies were assessed in semen, but not in the same spermatozoa, previous studies addressing this relationship were inconclusive. We recently demonstrated that sperm shape is preserved following fluorescence in situ hybridization (FISH). Thus, we examined the shape and chromosomal aberrations in the same sperm. METHODS: We performed phase contrast microscopy and FISH, using centromeric probes for chromosomes X, Y, 10, 11 and 17 in 15 men. The fluorescence and respective phase contrast images were digitized using the Metamorph program. We studied 1286 sperm (256 disomic, 130 diploid and 900 haploid sperm) by three criteria: head and tail dimensions, head shape and Kruger strict morphology. Furthermore, in each analysis, we considered whether disomic or diploid sperm may be distinguished from haploid sperm. RESULTS: There was an overall, but not discriminative, relationship between abnormal sperm dimensions or shape and increased frequencies of numerical chromosomal aberrations. However, approximately 68 of the 256 disomic, and four of 130 diploid sperm showed head and tail dimensions comparable with the most normal, lowest tertile of the 900 haploid spermatozoa. Considering all 1286 sperm, among those with the most regular, symmetrical shape (n = 367), there were 63 and five with disomic and diploid nuclei, respectively. In line with these findings, among the 256 disomic sperm, 10% were Kruger normal. CONCLUSIONS: Sperm dimensions or shape are not reliable attributes in selection of haploid sperm for ICSI.

Cell Shape↗
PubMed1978

The mechansim of antibody-dependent, eosinophil-mediated damage to schistosomula of Schistosoma mansoni in vitro: a study by phase-contrast and electron microscopy.

A characteristic sequence of events has been identified by phase-contrast and electron microscopy during antibody-dependent, eosinophil-mediated damage to schistosomula of Schistosoma mansoni in vitro. Human eosinophils initially adhere to the intact schistosomulum and then, in the presence of antibody, flatten and spread very intimately over the parasite's surface. Subsequently, dense material similar to the contents of the lysosomal granules of the eosinophils appears in the extracellular space between the eosinophil and the schistosomulum, probably following fusion of the granules with the plasma membrane of the cell. Eventually all the eosinophils adhering to the parasite are completely degranulated and large amounts of the dense material are observed on the surface of the schistosomulum. This release of granular material from the eosinophils is followed by structural changes in the schistosomulum, starting with vacuolation of the inner layer of the tegument, followed by removal of the tegument, often in the form of large sheets. Subsequently the tegument disintegrates and the fragments are phagocytosed by other eosinophils which have not degranulated. Eosinophils then attach to the exposed muscle layers of the schistosomula and participate in the further degradation of the parasites by phagocytosing fragments of muscle fibres and other cellular components. This sequence of events is compared with published observations of the damage induced by various combinations of antibody, complement and effector cells in vitro, and of cell-mediated damage to schistosomula in vivo, and it is concluded that the observations described in the present paper may reflect a process of destruction of schistosomula in the immune host.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1988

Fine structure of early human embryos frozen with 1,2 propanediol.

Previous studies by a French group (Fertil Steril 44:645-651, 1985) have shown that two- to eight-cell human embryos can survive slow freeze-thawing with propanediol in a biological freezer. These embryos were assessed for morphological appearance by phase-contrast microscopy. We assessed the structure of 25 frozen-thawed one- to 12-cell embryos, obtained from our in vitro fertilization (IVF) and GIFT programmes, by phase-contrast and electron microscopy, using the same method of cryopreservation. One-fourth of the embryos examined had all cells intact, and more than one-half the embryos had over 50% of their cells well preserved. Some of these embryos had unequal blastomeres and cytoplasmic fragments. Ultrastructural assessment revealed good preservation of fine structure in the intact blastomeres of all embryos and maintenance of cell-to-cell contacts. Most cytoplasmic organelles, cell membranes, and nuclei were well preserved compared to nonfrozen controls. The cells that were cryoinjured showed varying degrees of disorganization of the cell membrane, cytosol, and cellular membranes, including swelling and disruption of the nuclear envelope. Disruption of the zona was somewhat rare. Small cytoplasmic fragments were less prone to cryoinjury than blastomeres. The use of propanediol for embryo cryopreservation seems to be feasible; frozen embryos with more than 50% cells intact have produced 10 pregnancies after embryo transfer (Fertil Steril 46:268-272, 1986). Replacement of 17 frozen embryos in seven patients has resulted in a twin pregnancy in Singapore. However, the effects of freezing on the mitotic spindles of embryonic cells need to be investigated further.

Blastomeres↗
PubMed2004

[Differential diagnosis of urinary findings].

Classical urinalysis is extremely useful in the differential diagnosis of diseases of the kidneys and lower urinary tract. Although dipsticks are suitable for orientational purposes, when pathology is indicated, and when renal disease presents, they must be supplemented by urine microscopy or quantitative and qualitative protein electrophoresis to establish the diagnosis. Phase-contrast microscopy can distinguish glomerular from nonglomerular hematuria and thus guide the further diagnosis, and the detection of leukocytes and bacteria confirms the diagnosis of urinary tract infection. In asymptomatic patients microhematuria or proteinuria is detected more frequently with increasing age. A differentiated diagnostic strategy must be adapted to the individual risk of the patient, and must avoid overdiagnosis while not missing potentially serious pathology.

Acanthocytes↗
PubMed1958

Observations on the cytoplasmic membranes of testicular cells, examined by phase contrast and electron microscopy.

In freshly isolated cells of the guinea pig germinal epithelium examined with phase contrast, dark contours are seen in the cytoplasm that appear to be optical sections of the cisternae of the endoplasmic reticulum. These increase in contrast, in number, and in linear extent with increasing time up to 4 hours after isolation of the cells from the testis. During this period, cisternae originally present in the cells are extended and new ones appear to be formed by coalescence of tubular and vesicular elements of the reticulum. The cisternae become associated in parallel array and ultimately form elaborate concentric systems resembling structures that have often been interpreted as intracellular "myelin figures." Until now our knowledge of the endoplasmic reticulum has been based largely upon electron micrographs. The observation that the cisternae are visible in certain cell types under phase contrast optics opens the way for experimental investigations on the behavior of this class of cytoplasmic membranes in living cells.

Animals↗
PubMed2003

Comparison of phase contrast transmission electron microscopy with optimized scanning transmission annular dark field imaging for protein imaging.

Henderson has already shown that electron microscopy should be superior to X-ray and neutron diffraction for determining protein structure with minimum radiation damage. Since the contrast for a molecule embedded in vitreous ice is very low, it is conceivable that dark field imaging would be superior to bright field phase contrast microscopy. A detailed analysis of contrast and signal/noise for both imaging modes is presented. Annular dark field scanning transmission microscopy gives improved contrast and equivalent signal/noise to phase contrast TEM when the molecule is the same thickness as a vitreous ice embedding medium. For a constant embedding medium thickness of 200 A the contrast is equivalent to phase contrast TEM but the signal/noise is 5 times worse. Even with an efficient detector that only excludes scattering less than 5 mrad there is insufficient signal at a dose of 5 electrons/A(2) to produce an image with more than 1 electron/per pixel. For larger molecules (>100 A thick which corresponds to 420 kDa for spherical molecules) the weak phase object approximation used to analyse a phase contrast image no longer applies at 100 kV. This limit could be extended to about 200 A (about 3 MDa) if a 400 kV microscope were used.

Algorithms↗
PubMed2001

Dimensional regulation of cell-cycle events in Escherichia coli during steady-state growth.

Two opposing models have been put forward in the literature to describe the changes in the shape of individual Escherichia coli cells in steady-state growth that take place during the cell cycle: the Length model, which maintains that the regulating dimension is cell length, and the Volume model, which asserts it to be cell volume. In addition, the former model envisages cell diameter as decreasing with length up to constriction whereas the latter sees it as being constrained by the rigid cell wall. These two models differ in the correlations they predict between the various cellular dimensions (diameter, length, volume) not only across the entire population of bacteria but also, and especially, within subpopulations that define specific cell-cycle events (division, for example, or onset of constriction); the coefficients of variation at these specific events are also expected to be very different. Observations from cells prepared for electron microscopy (air-dried) and for phase-contrast microscopy (hydrated) appeared qualitatively largely in accordance with the predictions of the Length model. To obtain a more quantitative comparison, simulations were carried out of populations defined by each of the models; again, the results favoured the Length model. Finally, in age-selected cells using membrane elution, the diameter-length and diameter-volume correlations were in complete agreement with the Length model, as were the coefficients of variation. It is concluded that, at least with respect to cell-cycle events such as onset of constriction and cell division, length rather than volume is the controlling dimension.

Cell Cycle↗
PubMed1980

Maturation of equine epididymal spermatozoa.

Spermatozoa from four regions of the epididymis and from ejaculated semen were evaluated for their resistance to cold shock, progressive motility, and structural stability. Spermatozoa were incubated at 38 C and their percentage of eosinophilia was compared with that of spermatozoa cooled to 0 C in 2 minutes, 10 C in 12 minutes, or 4 C in 22 minutes. Spermatozoa motility was estimated visually under phase-contrast microscopy and was recorded by cinematography. Structural stability of spermatozoa incubated in 0.05 M sodium borate buffer, 0.035 M sodium dodecyl sulfate (SDS), 0.002 M dithiothreitol (DTT), or SDS+DDT was evaluated by phase-contrast and electron microscopy. The percentage of eosinophilic spermatozoa did not differ among regions of the epididymis and was not increased by cold shock. Cooling ejaculated spermatozoa to 0 C in 2 minutes increased (P < 0.01) the occurrence of eosinophilia (32% vs 73%). Spermatozoa released from the caput or proximal corpus epididymidis into 0.154 M NaCl were not progressively motile; only 4% of the spermatozoa from the distal corpus were motile Cauda epididymal and ejaculated spermatozoa exhibited similar motility (41% vs 47%). Stability of chromatin was greater in spermatozoa from the distal corpus than those from the caput epididymis. Chromatin of spermatozoa from the distal corpus was resistant to 7.5 minutes of SDS+DTT treatment, whereas virtually all spermatozoal nuclei from the caput were decondensed by SDS alone. Tail organelles of spermatozoa acquired stability between the proximal corpus and the cauda epididymidis. All tail organelles of spermatozoa from the proximal corpus were dissolved by 7.5 minutes of SDS treatment, whereas tail organelles of distal corpus epididymal spermatozoa had dissolved after 7.5 minutes of SDS+DTT treatment. Stability of tail organelles in cauda epididymal and ejaculated spermatozoa was similar. Seemingly, equine spermatozoa are infertile until they enter the cauda epididymidis.

Animals↗
PubMed2004

Importance of platelets and fibrinogen in neutrophil-endothelial cell interactions in septic shock.

OBJECTIVE: To examine the role of platelets, fibrin, and adhesion molecules in mediating neutrophil-endothelial cell interactions in septic shock. DESIGN: Controlled experiments using phase contrast microscopy to examine neutrophil, platelet, and endothelial cell interactions in flowing cell suspensions under simulated physiologic conditions. SETTING: University research laboratory. PATIENTS: Adult patients with septic shock and normal volunteers. INTERVENTIONS: Microslides were coated with human umbilical vein endothelial cells. Neutrophils were removed from control subjects and patients in septic shock and were perfused over endothelial cells at rates representing a range of physiologic shear stresses. In an attempt to examine the effects of fibrin deposition on neutrophil-endothelial cell interactions, neutrophils, with and without platelets, were suspended in plasma and serum was removed from patients in septic shock. In addition, blocking monoclonal antibodies against the platelet receptor P-selectin and neutrophil receptor CD11b/CD18, and a platelet glycoprotein IIb/IIIa inhibitor, were incubated with cells suspended in plasma. Phase contrast video microscopy was used to count the number of neutrophils/mm adherent to endothelial cells during cessation of flow. Neutrophil rolling velocity was calculated as the time required for neutrophils to move across a 1-mm field (mm/sec). Leukoaggregation was defined as the number of neutrophils in aggregates (three or more nuclei) across a 1-mm field. MEASUREMENTS AND MAIN RESULTS: Normal neutrophils exposed to plasma from patients with septic shock demonstrated significant increases in aggregation and endothelial cell adherence with associated decreases in neutrophil rolling velocity. These changes were significantly enhanced in the presence of platelets and significantly attenuated in the presence of serum, which is fibrinogen depleted. Preincubation with antibodies to the surface receptors P-selectin, CD11b/CD18, and glycoprotein IIb/IIIa abrogated the changes in neutrophil aggregation, adhesion, and rolling velocity. CONCLUSIONS: These data suggest that platelets and fibrinogen play an important role in mediating neutrophil-endothelial cell adherence in septic shock.

Blood Platelets↗
PubMed1965

Phase-contrast and electron microscopy of murine strains of Mycoplasma.

Nelson, John B. (The Rockefeller University, New York, N.Y.), and Michael J. Lyons. Phase-contrast and electron microscopy of murine strains of Mycoplasma. J. Bacteriol. 90:1750-1763. 1965.-Two strains of Mycoplasma pulmonis (associated with infectious catarrh) on examination in fluid culture (20% horse serum-bouillon) by phase microscopy were highly pleomorphic, with many bacilliform elements and fewer coccoid ones. Motility, characterized by gliding of rods and spinning of spherical forms, was observed through the 9th subculture of one strain and the 15th of the second. Motile elements were not seen in later transfers and pleomorphism was reduced. One strain of M. neurolyticum (associated with conjunctivitis and encephalitis) was much less pleomorphic and showed neither bacilliform elements nor motility at any time. When examined by negative-contrast electron microscopy, organisms of this strain were found to have an average diameter of 0.7 mu and to possess a concentrated peripheral layer of cytoplasm and a central mass which may represent the cells' nuclear equivalent. The latter feature was not prominent in spherical forms of M. pulmonis. These cells, when observed after 48 hr of culture, showed evidence of the generation of new progeny cells in their central area. The filamentous or bacilliform cells of M. pulmonis were frequently serpentine in appearance, 2.0 to 3.0 mu in length and 80 to 250 mmu in width. They appeared to generate new cells from terminal buds from which outpouchings initially developed. Older cells, in the stationary phase, showed evidence of undergoing multipolar germination. Microtubules, about 60 mmu wide, were found in association with most filamentous cells from 48-hr cultures; fragments of membrane, studded with closely packed ribosomelike particles, were also found. There was no evidence of flagella or any specialized structure that could account for the observed motility of the organisms.

Animals↗