Ecophysiological responses of the seminal vesicle of Libyan jird (Meriones libycus) to the Saharan conditions: histological, morphometric and immunohistochemical analysis.

The Libyan jird (Meriones libycus) is a nocturnal Saharan Rodent submitted to a seasonal cycle of reproduction characterized by a short active period during spring and beginning of summer, and a long phase of sexual quiescence from the end of summer until the end of winter. During this cycle, the male reproductive organs, and more particularly seminal vesicles, experience some important weight and histological variations. During the breeding period, the wall of each seminal vesicle describes several folds radiating inside a broad lumen filled with a very abundant secretion. The wall is limited with high columnar epithelial cells surrounded with extracellular matrix restricted to some connnective fibres located in the narrow axis of the folds and in the chorion. The fibro-muscular wall is narrow. During sexual quiescence, the seminal vesicles regress. No secretion has been observed inside the lumen. The wall of lumen is now surrounded with a single cubic epithelium. The persistent epithelial folds possess a wide axis. The hypertrophied extracellular matrix is constituted with a very tight and abundant connective tissue. The fibro-muscular wall is thick. A quantitative morphometric study was performed with automatic image analysis that allowed to quantify the seasonal variations of the histological components. The numerical values obtained agree with the histological images observed, the epithelial surface area (microm2) is high in spring and significantly weak during sexual quiescence. The stroma and the fibro-muscular wall occupy an important surface area on sections during the resting period compared with the value collected during the active phase. The study of the apoptosis by TUNEL method revealed the presence of a considerable number of apoptotic nuclei in the epithelial fraction during the resting phase. The indirect immunohistochemical method allowed us to visualize the presence of types I and III collagen in the extracellular matrix, weak during the period of breeding, intense and diffuse during the resting season like in castrated Meriones libycus.

Ultrastructural nuclear defects and increased chromosome aneuploidies in spermatozoa with elongated heads.

BACKGROUND: Cellular and molecular mechanisms leading to elongated sperm heads are not known. We have analysed the nuclear status of spermatozoa with elongated heads. METHODS: Fourteen men with at least 30% of spermatozoa with an elongated nucleus were studied and compared with five fertile men as controls. Sperm morphology was analysed by a quantitative ultrastructural analysis. Sperm chromosomal content was assessed by three-colour fluorescence in-situ hybridization (chromosomes X, Y, 18). Y chromosome microdeletion and karyotype were analysed. RESULTS: Elongated sperm head rates of the patients were 46.9% (30-75 versus 0-2% in the control group) by light microscopy and 34.4% by electron microscopy. In all patients, the chromatin was poorly condensed in elongated sperm heads (50% of elongated nuclei). No anomalies of sperm biochemical markers were found. All the men showed normal karyotype (46,XY) and absence of Y chromosome microdeletion. Aneuploidy rates of gonosomes and chromosome 18 were significantly increased in patients (1.64- and 3.6-fold, P = 0.006 and 0.026, respectively). CONCLUSIONS: This study demonstrates that impaired chromatin compaction and slightly increased chromosome aneuploidies are found in spermatozoa with an elongated head, suggesting possible mechanisms such as meiotic non-disjunctions or spermiogenesis anomalies.

Cytogenetic, molecular and testicular tissue studies in an infertile 45,X male carrying an unbalanced (Y;22) translocation: case report.

(Y;autosome) translocations have been reported in association with male infertility. Different mechanisms have been suggested to explain the male infertility, such as deletion of the azoospermic factor (AZF) on the long arm of the Y chromosome, or meiosis impairment. We describe a new case with a de novo unbalanced translocation t(Y;22) and discuss the genotype-phenotype correlation. A 36 year old male with azoospermia was found to have a mosaic 45,X/46,X, + mar karyotype. Fluorescence in situ hybridization (FISH) showed the presence of a derivative Y chromosome containing the short arm, the centromere and a small proximal part of the long-arm euchromatin of the Y chromosome and the long arm of chromosome 22. The unstable small marker chromosome included the short arm and the centromere of chromosome 22. This unbalanced translocation t(Y;22)(q11.2;q11.1) generated the loss of the long arm of the Y chromosome involving a large part of AZFb, AZFc and Yq heterochromatin regions. Testicular tissue analyses showed sperm in the wet preparation. Our case shows the importance of documenting (Y;autosome) translocations with molecular and testicular tissue analyses.

Effect of L-fucose and fucose-rich oligo- and polysaccharides (FROP-s) on skin aging: penetration, skin tissue production and fibrillogenesis.

Skin thickness is decreasing with age. This loss concerns both dermis and epidermis, cells and extracellular matrix. We could show here that percutaneous application of an L-fucose-containing preparation produced an increase of skin thickness and a densification of collagen bundles. We also could show that 3H-L-fucose penetrates in the dermis, a prerequisite for the above mentioned favorable pharmacological activities. These results, together with the previous favorable activities on the downregulation of matrix-degrading enzymes, free radical scavenging and increased cell proliferation confirm the favorable action of fucose and fucose-rich polysaccharides (FROP-s) on the skin by slowing down its aging.

Weightlessness acts on human breast cancer cell line MCF-7.

Because cells are sensitive to mechanical forces, weightlessness might act on stress-dependent cell changes. Human breast cancer cells MCF-7, flown in space in a Photon capsule, were fixed after 1.5, 22 and 48 h in orbit. Cells subjected to weightlessness were compared to 1 g in-flight and ground controls. Post-flight, fluorescent labeling was performed to visualize cell proliferation (Ki-67), three cytoskeleton components and chromatin structure. Confocal microscopy and image analysis were used to quantify cycling cells and mitosis, modifications of the cytokeratin network and chromatin structure. Several main phenomena were observed in weightlessness: The perinuclear cytokeratin network and chromatin structure were looser; More cells were cycling and mitosis was prolonged. Finally, cell proliferation was reduced as a consequence of a cell-cycle blockade; Microtubules were altered in many cells. The results reported in the first point are in agreement with basic predictions of cellular tensegrity. The prolongation of mitosis can be explained by an alteration of microtubules. We discuss here the different mechanisms involved in weightlessness alteration of microtubules: i) alteration of their self-organization by reaction-diffusion processes, and a mathematical model is proposed, ii) activation or deactivation of microtubules stabilizing proteins, acting on both microtubule and microfilament networks in cell cortex.

Quantitative analysis of cytokeratin network topology in the MCF7 cell line.

BACKGROUND: In the MCF7 human breast cancer cell line, several patterns of cytokeratin networks are observed, depending on the intracellular localization. Our hypothesis is that architectural variations of cytokeratin networks depend on local tensions or forces appearing spontaneously in the cytoplasm. The aim of this work was to discriminate between the different patterns and to quantitate these variations. MATERIALS AND METHODS: Image analysis procedures were developed to extract cytokeratin filament networks visualized by immunofluorescence and confocal microscopy. Two methods were used to segment sets of curvilinear objects. The first, the "mesh-approach," based on classical methods of mathematical morphology, takes into account global network topology. The second, the "filament-approach" (novel), is meant to account for individual element morphology. These methods and their combination allow the computation of several features at two levels of geometry: global (network topology) and local (filament morphology). RESULTS: Variations in cytokeratin networks are characterized by their connectivity, density, mesh structure, and filament shape. The connectivity and the density of a network describe its location in a local "stress-force" zone or in a "relaxed" zone. The mesh structure characterizes the intracellular localization of the network. Moreover, the filament shape reflects the intracellular localization and the occurrence of a "stress-force" zone. CONCLUSIONS: These features permitted the quantitation of differences within the network patterns and within the specific filament shapes according to the intracellular localization. Further experiments on cells submitted to external forces will test the hypothesis that the architectural variations of intermediate filaments reflect intracytoplasmic tensions.

Changes in hormonal balance and meristematic activity in primary root tips on the slowly rotating clinostat and their effect on the development of the rapeseed root system.

The morphometry of the root system, the meristematic activity and the level of indole-3-acetic acid (IAA), abscisic acid (ABA) and zeatin in the primary root tips of rapeseed seedlings were analyzed as functions of time on a slowly rotating clinostat (1 rpm) or in the vertical controls (1 rpm). The fresh weight of the root system was 30% higher throughout the growth period (25 days) in clinorotated seedlings. Morphometric analysis showed that the increase in biomass on the clinostat was due to greater primary root growth, earlier initiation and greater elongation of the secondary roots, which could be observed even in 5-day-old seedlings. However, after 15 days, the growth of the primary root slowed on the clinostat, whereas secondary roots still grew faster in clinorotated plants than in the controls. At this time, the secondary roots began to be initiated closer to the root tip on the clinostat than in the control. Analysis of the meristematic activity and determination of the levels in IAA, ABA and zeatin in the primary root tips demonstrated that after 5 days on the clinostat, the increased length of the primary root could be the consequence of higher meristematic activity and coincided with an increase in both IAA and ABA concentrations. After 15 days on the clinostat, a marked increase in IAA, ABA and zeatin, which probably reached supraoptimal levels, seems to cause a progressive disturbance of the meristematic cells, during a decrease of primary root growth between 15 and 25 days. These modifications in the hormonal balance and the perturbation of the meristematic activity on the clinostat were followed by a loss of apical dominance, which was responsible for the early initiation of secondary roots, the greater elongation of the root system and the emergence of the lateral roots near the tip of the primary root.

Ser752 mutation to Pro or Ala in the beta3 integrin subunit differentially affects the kinetics of cell spreading to von Willebrand factor and fibrinogen.

The beta3 cytoplasmic domain of the alpha v beta3 integrin is essential for intracellular signals required for cytoskeletal rearrangements. Expression of beta3Ser752Pro mutation in heterologous cells profoundly affects cell spreading and beta3 localization into focal contacts. However, the beta3Ser752Ala substitution mostly restores normal integrin functions, suggesting that the presence of Pro is responsible for the receptor's loss of function. To further assess the role of the Ser752 of the beta3 cytoplasmic domain in the cytoskeletal organization of adherent cells, we developed a computer-assisted method of image analysis allowing the automatic classification of spread cells according to the quantitative analysis of their cell morphology. We compared adhesion and spreading to von Willebrand factor (vWF) or fibrinogen (Fg) of cells expressing beta3 wild type, beta3Ser752Pro or beta3Ser752Ala mutated integrin subunit as a chimeric alpha v beta3 receptor. The beta3Ser752Ala substitution did not impair the general ability of cells to spread, but resulted in a delayed and reduced spreading on both vWF and Fg. Moreover, the beta3Ser752Ala mutation produced modifications of the morphology of spread cells, suggesting a disorganization of their cytoskeleton. Attachment studies showed that the beta3Ser752Ala mutation did not modify the capacity of cells to attach to the substrate, indicating no change in the ligand binding affinity of the alpha v beta3 integrin. Furthermore, we identified a slight defect of beta3Ser752Pro cell attachment to vWF and Fg, beside their impairment of spreading. Taken together, these results suggest a role of Ser752 of the beta3 cytoplasmic domain in the optimal cytoskeletal organization of adherent cells.

p21WAF-1 reorganizes the nucleus in tumor suppression.

Interphasic nuclear organization has a key function in genome biology. We demonstrate that p21WAF-1, by influencing gene expression and inducing chromosomal repositioning in tumor suppression, plays a major role as a nuclear organizer. Transfection of U937 tumor cells with p21WAF-1 resulted in expression of the HUMSIAH (human seven in absentia homologue), Rb, and Rbr-2 genes and strong suppression of the malignant phenotype. p21(WAF-1) drastically modified the compartmentalization of the nuclear genome. DNase I genome exposure and fluorescence in situ hybridization show, respectively, a displacement of the sensitive sites to the periphery of the nucleus and repositioning of chromosomes 13, 16, 17, and 21. These findings, addressing nuclear architecture modulations, provide potentially significant perspectives for the understanding of tumor suppression.

Ciliary beat frequency in human bronchi and bronchioles.

STUDY OBJECTIVE: This study aimed to determine the differences between ciliary beat frequencies of respiratory ciliated cells from peripheral bronchioles and from proximal bronchi in humans. DESIGN: Measurements were made from resected lungs. Ciliated cells were harvested by brushing the mucosa of each site immediately after surgery. Brushings with a cytology brush were performed on normal areas of the resected cartilaginous bronchus for proximal samplings and through a peripheral bronchiole close to the visceral pleura for peripheral samplings. For each site, at least 12 different measurements were made at 22 degrees C using an image analysis system. RESULTS: A highly significant difference between proximal bronchi (mean, 7.1 Hz; SD, 1.29) and peripheral bronchioles (mean, 4.6 Hz; SD, 1.39) (p < 0.0001) was found. CONCLUSION: Thus, cilia from peripheral bronchioles beat at a 35% lower beat frequency than cilia from proximal bronchi.

TGF beta 1 promotes actin cytoskeleton reorganization and migratory phenotype in epithelial tracheal cells in primary culture.

In the present study we have investigated the effects of transforming growth factor beta (TGF beta 1) on rabbit tracheal epithelial cells in primary culture, with respect to cell proliferation and differentiation. Epithelial tracheal cells derived from an explant plated on an extracellular matrix, formed an outgrowth resulting from cell division and cell migration. TGF beta 1 treatment produced a negative effect on cell proliferation, but in contrast, promoted a marked enhancement of cell migration and increase in outgrowth surface. TGF beta 1 induced marked cell shape changes, including cell spreading and lack of stratification, associated with reduced cell-cell contacts and increased cell-substratum anchorage, as seen by electron microscopic observations. Immunocytological studies demonstrated major TGF beta 1-induced actin cytoskeleton reorganization, corresponding to the development of a basal stress fiber network and decrease of the annular cell border, without affecting the tight junctions. The migratory phenotype was approached by microcinematography which clearly showed that TGF beta 1 triggered a stimulatory effect on migration of epithelial cells, determined using an image analyzing system. Present findings suggest a beneficial role for TGF beta 1 during wound healing in providing the acquisition of a migratory phenotype, with a higher capacity to migrate either on collagen or on different extracellular matrix components including laminin and fibronectin. Conversely, present data are not consistent with a squamous response to TGF beta 1, since metaplastic differentiation did not occur, as characterized by cytokeratin expression and cross-linked envelopes formation.

Epidermal growth factor receptor and c-erbB-2 expression in normal breast tissue during the menstrual cycle.

EGF receptor (EGF-R) and c-erbB-2 are homologous tyrosine kinase transmembrane receptors. They are involved in controlling proliferation, and probably differentiation, of normal breast epithelial cells, and their expression has been linked to the prognosis of breast cancer. Their physiological roles in normal breast tissue remain to be elucidated, as most studies to date have involved breast cancer cell lines. We studied the location of EGF-R and c-erbB-2 in 100 samples of normal breast with standard immunohistochemical methods and double-labelling techniques. EGF-R was mainly expressed on the stroma and myoepithelial cells, whereas c-erbB-2 expression was exclusively epithelial. An image analyser was used to quantitate variations in their expression during the menstrual cycle. EGF-R and c-erbB-2 expression on epithelial cells was stronger during the luteal phase than the follicular phase (p < 0.01 for EGF-R). The pattern of expression was also compared with that in 28 breast cancers and 7 fibroadenomas.

Progress in the measurement of ciliary beat frequency by automated image analysis: application to mammalian tracheal epithelium.

The investigations on the activity of respiratory ciliae in vivo are of considerable interest for the study of physiopathological disorders resulting from chronic infection or inhaled pollutants. We present an image analysis method for the measurement of ciliary beat frequency on mammal or human in vivo samples. Videofilm sequences of samples placed under the microscope were analyzed frame by frame by image analysis. The operator defines on the screen several areas of interest on the cell apex. The variation of grey levels in each areas allows the calculation of ciliary beat parameters. This method permits one to investigate the effects of pharmacological agents or noxious pollutants on ciliae beating and the physiological causes underlying them. Our results on rabbits were in agreement with the data published in literature, showing no significant difference between intratracheal frequencies obtained at medium and lower levels. The upper level near the cracoid cartilage has shown a greater variability than the other two levels with higher beating frequencies, a difference of likely physiological importance.

Zonal variations of types II, IX and XI collagen mRNAs in rat epiphyseal cartilage chondrocytes: quantitative evaluation of in situ hybridization by image analysis of radioautography.

The spatial-temporal distribution of the mRNAs for type IX and type XI collagens were compared to that of type II collagen mRNA in the tibial epiphyseal plate cartilage of normal growing rats. The mRNAs were detected by in situ hybridization with radio-labelled specific probes and visualized by radioautography. The areas covered by the resulting silver grains were quantified by computer assisted image analysis. The areas in chondrocytes of each zone of the epiphyseal plate cartilage, which correspond to the stages of chondrocyte development and function were determined. Types II, IX and XI mRNAs were present to some extent in chondrocytes of all zones. The distributions of type II and type IX collagen mRNAs were similar with the highest concentrations in the proliferative zone, and the lowest in the resting and calcifying zones chondrocytes. In contrast, type XI collagen mRNA had a different distribution, with the lowest concentration in the resting zone chondrocytes and a significant decrease in the calcifying zone chondrocytes. These patterns correlates with the changes in chondrocyte function, and may reflect the roles of the type IX and type XI collagens. The data show that computer assisted image analysis of in situ hybridization radioautographic images is a precise, rapid tool for analysing differences in gene expression.

In vitro quantification by image analysis of inotropic and chronotropic effects of drugs on cultures of cardiac myocytes.

Quantitative image analysis is used to measure the inotropic and chronotropic effects of drugs on cultured heart cells maintained at 37 degrees C on the stage of an inverted light microscope, and sequentially superfused with control and treatment media. The beating of the cardiac myocytes is evaluated by simultaneously selecting up to eight areas, including cell edges, from digitized video image. The sizes and positions of these areas are controlled by the operator. To analyze the motion of cell edges in each area, the computer measures the shift of the mass center of pixels' grey levels. Finally, a few parameters are calculated for the eight areas and displayed graphically. In order to assess treatment effects, appropriate statistical tests are performed on the data. Image analysis is an efficient screening test for evaluating the pharmacologic or toxic effects of a substance on isolated or cultured cardiac myocytes from various species.

Calmodulin during human sperm incorporation into hamster oocyte: an immunogold electron microscope study.

In the present study, immunogold labeling of ultrathin sections of human sperm, before and after incorporation into hamster oocyte, was used to obtain insight into the ultrastructural localization and possible function of calmodulin during fertilization. In heads of ejaculated, capacitated, and acrosome-reacted fixed human sperm, calmodulin was mainly found in two compartments, the subacrosomal layer and the postacrosome. After sperm-egg fusion, the subacrosomal calmodulin was unaltered and surrounded by the fertilization cone in which actin was abundant. There was no co-localization of calmodulin and actin. In contrast, postacrosomal calmodulin disappeared as soon as the sperm head was incorporated into egg cytoplasm. These unique localizations and redistributions are in agreement with the concept of a calmodulin targeting from acrosome toward postacrosome through the subacrosomal layer during spermatogenesis (Weinman et al., 1986b: J Histochem Cytochem 34:118). Moreover, they strongly suggest a role for calmodulin both in sperm-egg fusion and in the initial pulse of Ca2+ occurring during fertilization.

Densitometric analysis of nuclear DNA content in lentil roots grown in space.

Lentil seedlings were grown for 28 h in space, on board Spacelab (IML 1 Mission) and growth of the primary root was analysed. The length of cortical cells was less in near weightlessness than on the 1 g centrifuge (flight control) and mitotic index was lower but there was no apparent perturbation in the mitosis. To further investigate which phase of cell cycle was modified, densitometric analysis of nuclear DNA content in cortical cells was carried out by the mean of an image processing system (SAMBA). In microgravity there was a decrease in DNA synthesis and a promotion of the arrest in the G2 phase of cell cycle. These results, and other ones obtained elsewhere on a slowly rotating clinostat, led us to think that in microgravity the perturbation of the gravisensing cells and/or the absence of convection could account for the modification of cell growth registered in the primary root.

The nuclear status of human sperm cells by TEM image cytometry: nuclear shape and chromatin texture in semen samples from fertile and infertile men.

Changes in nuclear size, shape, and chromatin texture during spermiogenesis and epididymal transport of human sperm were recently analyzed using transmission electron microscopy (TEM) image cytometry followed by multivariate statistical analysis of data. In the present study, this same methodology was used to investigate the nuclear morphology of spermatozoa in semen samples from fertile and infertile men. Analysis was carried out on a large series of micrographs of sections of sperm nuclei from a donor group with proven fertility and from a patient group with a mean infertility duration of 10 years with no obvious male or female infertility factors (only a slight decrease in the proportion of sperm heads with normal morphology was noted in routine semen tests). For the patient group, it was found that nuclei had a significantly less flattened shape (i.e., increased roundness as a consequence of increased thickness and decreased length). Furthermore, significant differences between donor and patient groups were found for most parameters of chromatin texture. In the patient group, chromatin was less condensed, and there was more homogeneous distribution of the different degrees of chromatin condensation. In addition, the organization of chromatin condensation and distribution along the major axis of the nucleus was found to be significantly different in the two groups. Stepwise linear discriminant analysis indicated a good classification rate of only 66% for nuclei of patients when using the eight major nuclear parameters, thus indicating the striking heterogeneity of nuclear morphology for both patient and donor groups.(ABSTRACT TRUNCATED AT 250 WORDS)