Cellular analysis by in situ hybridization and immunoperoxidase of alpha-fetoprotein and albumin gene expression in rat liver during the perinatal period.

To analyze at the cellular level the decrease in alpha-fetoprotein (AFP) gene expression during the early postnatal growth, we searched for AFP gene transcripts by in situ hybridization using a specific cDNA probe, and for the corresponding protein by immunocytochemistry, on rat liver sections at various times of the perinatal period. The relative number of mRNA sequences was evaluated by Northern blot analysis. Albumin (ALB) gene expression was studied simultaneously with the same techniques. In 17-19-d-old fetuses all hepatocytes express simultaneously, for both genes, the mRNAs and the corresponding proteins. During the first postnatal weeks, at a time when the global number of AFP mRNA molecules decreases, all hepatocytes still contain cytoplasmic transcripts and protein. A zonal heterogeneity in the level of AFP gene expression develops around the first week, a higher number of gene products being detected in perivenous than in periportal hepatocytes. This heterogeneity persists until the fourth week when AFP mRNA sequences and protein are barely detectable. All hepatocytes express the ALB gene after birth, but at around the second week, a periportal intensification of the in situ hybridization signal and immunostaining becomes apparent. Our data indicate that co-expression of the AFP and ALB genes by all hepatocytes is a normal step in liver ontogeny; the diminution of AFP gene expression after birth is not the result of the disappearance of specialized cell clones; and zonal quantitative differences in the level of AFP and ALB gene expression are observed within the maturing liver lobule.

Pathways of internalization of the hCG/LH receptor: immunoelectron microscopic studies in Leydig cells and transfected L-cells.

Monoclonal anti-receptor antibodies were used to study the cellular traffic of the hCG/LH receptor by immunoelectron microscopy. The LHR38 antibody was shown to bind to the extracellular domain of the receptor but not to interfere with hormone binding, adenylate cyclase activation or with the rate of internalization of the receptor. Pig Leydig cells and a permanent L-cell line expressing the LH receptor were used for the study. Incubation with LHR38-gold complexes showed the LH receptors to be randomly distributed over the cell surface including the clathrin coated pits. The LH receptors were internalized via a route including coated pits, coated vesicles and multivesicular bodies to lysosomes. This route is different from that observed for beta-adrenergic, muscarinic, and yeast mating factor receptors and considered previously as possibly general for G-protein-coupled receptors. The use of [125I]LHR38 allowed precise measurement of the rate of internalization, showing the existence of a constitutive pathway which was increased 11-fold by hormone administration. Double labeling experiments suggested that the hormone (hCG-Au15nm) and the receptor (labeled with LHR38-Au5nm) have similar routes of endocytosis, both of them being degraded in lysosomes. Studies of the reappearance of LHR38-Au5nm on the surface of the cells and the use of monensin indicated that only a very small proportion of the receptor molecules were recycled to the cell surface. The distribution and the intracellular pathways of LH receptors are very similar in Leydig cells and transfected L-cells. This opens the possibility of using the latter to study, by in vitro mutagenesis, the molecular mechanisms involved in the cellular traffic of LH receptors.

Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures.

Our aim was to define optimal conditions for efficient and reproducible albumin mRNA detection in rat liver by in situ hybridization. We used an albumin-specific [3H]-labeled cDNA probe with a specific activity of 6-8.10(6) cpm/microgram DNA. In situ hybridization is as efficient on paraffin sections as on cryostat sections for detecting albumin mRNAs. Perfusion fixation with a 4% paraformaldehyde solution results in homogeneous RNA retention within tissue blocks, in contrast with immersion fixation, which yields heterogeneous RNA preservation. Comparison of immersion fixation with three different fixatives (paraformaldehyde, ethanol-acetic acid, and Bouin's fixative) shows that the highest level of hybridization signal is obtained with paraformaldehyde. Ethanol-acetic acid and Bouin's fixative appear less efficient for albumin mRNA detection. Loss of mRNAs within liver tissue blocks over time is largely although not completely prevented by paraffin embedding.

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.

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)

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.

On how the periosteal bone of the delphinid humerus becomes cancellous: ontogeny of a histological specialization.

In cetaceans, the bones of the flippers lack a free medullary cavity and have a cancellous texture, with compact cortices reduced or absent. The present work discusses the ontogenetic basis of these characters in terms of the ontogeny of the structure and textural bone compactness (TBC) of the humeral diaphysis in a growth series of common dolphins (Delphinus delphis). The texture of the primary periosteal deposits is compact; soon after their accretion, the deposits undergo an extensive erosion that turns them into a cancellous tissue. A diffuse endosteal front of resorption expands in parallel with the growth of the cortex and acts as small units scattered within the cortices. Starting soon after birth and continuing throughout the life of the animals, the compactness of the periosteal cortex decreases at both general and local levels. This trend correlates strongly with the increase in size of the diaphyseal section and reflects the fact that relatively more bone is eroded than deposited during growth in the cancellous parts of the cortex. In the broad sense, this is basically an osteoporotic process, which is not identical, however, to senile or disuse osteoporoses.

Respiratory tract epithelium in primary culture: Effects of ciliotoxic compounds.

Acrolein ciliotoxicity was studied on primary cultures from rabbit tracheal epithelium. The inhibition of ciliary beat was chosen as a criterion for ciliotoxicity. The measurement of ciliary beat was accomplished using an original image analysis process.

Comparative quantitative study of Ki-67 antibody staining in 78 B and T cell malignant lymphoma (ML) using two image analyser systems.

Total Ki-67 stained area percentage was studied in 32 B and 46 T malignant lymphomas (ML) using two different image analyser systems (TAS, Leitz; SAMBA TM 2005, TITN) respectively. The total Ki-67 area percentage was highly correlated to the number of Ki-67 positive cellular profiles (B-ML, r = 0.93; T-ML, r = 0.88), indicating that area percentage is a reliable alternative method to the manual cell counting. Image analysis allows quicker measurements, appropriate to large and strictly lymphomatous regions. The cell image processor (SAMBA TM 2005, TITN) linked to a color video camera was more suitable for immunohistochemical sections and allowed more automated and faster measurements than the texture analyser (TAS, Leitz) linked with a black and white camera. Alkaline phosphatase technique with fast red as chromogen was more suitable for the detection of Ki-67 stained area by thresholding than peroxidase technique with aminoethylcarbazol or with diaminobenzidine as chromogens. Significant differences were found between low and high grade in B and T ML according to the Kiel classification (mean values +/- SD of 7.7 +/- 3.8% and 16.6 +/- 6.2% in B-ML and of 10.2 +/- 7.9% and 25.6 +/- 16.3% in T-ML respectively). In follicular B-ML, considering follicular areas only, values were comparable to high grade ML; angioimmunoblastic-lymphadenopathy-like (AILD-type) T-ML belonging to low grade ML showed similar values to pleomorphic T-ML with medium and/or large cells belonging to high grade ML.(ABSTRACT TRUNCATED AT 250 WORDS)

A new automated method of image analysis: comparison of ciliary and flagellar beats.

A new automated method of image analysis of sperm flagellar (human) and cilia (Dunaliella) bends is developed. This method permits an automatic determination of the line characterizing the flagellum. Two dynamic parameters are measured: the wave propagation velocity and the wave curvature radius. The data reveal similar patterns in the propagation of the principal and reverse waves between flagelated and ciliated cells. Conversely, differences are seen in principal wave curvature due perhaps to the presence of periaxonemal structures in the flagellum, absent in cilium. The identical patterns of reverse wave curvaturei in both systems may be linked to axonemal limitations.

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.

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.

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.

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.

Morphometric analysis of the cell outlines of normal and polyomavirus-transformed FR 3T3 fibroblasts.

A method is described for the analysis of cell shape, using an image analyzer connected to a computer to assess the cell outline. A series of parameters to assess the contribution of large cytoplasmic expansions to cell morphology and to cell spreading on a planar substratum were used to quantify the visual morphologic differences between normal (nontransformed; N.3T3) and polyomavirus-transformed (Py.3T3) Fisher rat 3T3 fibroblasts. The results show that the Py.3T3 fibroblasts are more spherical than are the N.3T3 fibroblasts and that the cytoplasmic expansions of the Py.3T3 fibroblasts are smaller than those of N.3T3, with the spreading of these two cell strains being different. These differences can be explained by the difference in cell-substratum affinity between these two cell strains.

Statistical analysis of morphometrically analyzed outlines of cells in culture. Comparison of populations of normal and polyomavirus-transformed FR 3T3 fibroblasts.

The correspondence analysis method was used to statistically characterize the morphologies of populations of normal (nontransformed; N.3T3) and polyomavirus-transformed (Py.3T3) Fisher rat 3T3 fibroblasts in culture, based on morphologic parameters calculated according to a previously described morphometric method. With this statistical method, each cell is considered as a vector in a space defined by an arrangement of the calculated morphologic parameters. The N.3T3 cells and the Py.3T3 have two distinct morphologic aspects in culture: they have either a smooth or a multipolar outline. The normal cell population contained twice as many cells with smooth outlines (46%) as did the transformed one (23%). Moreover, the cells with smooth outlines in the two strains could be classified into three homologous subpopulations that were present in significantly different proportions in the N.3T3 cells versus the Py.3T3 cells. In addition, morphologic differences were observed among the cells with multipolar outlines in these two strains, due to differences in the morphologies, size, number and distribution of the cytoplasmic expansions along the cell outline.

Dynamic image analysis applied to the study of ciliary beat on cultured ciliated epithelial cells from rabbit trachea.

We have developed an automated image analysis method to study the ciliary beat frequency of ciliated cells of the primary culture from rabbit trachea. The ciliated outgrowth image is digitized and the variation in optical density is automatically calculated for each selected area of interest. 32 measurements of ciliary beat frequency are, in this way, calculated simultaneously in 6 min. With this reliable device, some studies on baseline frequency of control culture have been carried out. There was no variation in the mean frequencies of ciliated cells of the primary culture of different tracheas in our culture conditions. Moreover, the values of ciliary beat frequency at the starting point of the outgrowth were similar to those at the periphery of the outgrowth. There is nevertheless a slow decrease in frequencies versus the duration of culture. We have also established that the frequency of ciliary beat of some cells fluctuates in a periodic pattern whereas the majority of the ciliated population beat in a stable way. The image analysis process allows us to perform a cartography of frequencies on the video display. It also allows us to have access to the frequency of one cilium. Our method therefore seems to be reliable and furthermore simple in the evaluation of the potential effect of inhaled toxic compounds on ciliated cells of mammalian respiratory tract.

Comparative study of automated morphometric and semiquantitative estimations of alcoholic liver steatosis.

Needle biopsy of the liver is of great value in appreciating the intensity, type and topography of the steatosis commonly observed during chronic alcoholic diseases. The usual semiquantitative optical analysis is very inaccurate and depends on the subjectivity and training of the pathologist. We therefore performed an automated analysis of liver steatosis using a QTM 720 image analyzer connected to a PDP 11/34 minicomputer. Visual control of the results of the automated analysis showed it to give good results: 94% of the droplets were detected and only 10% of the patterns automatically selected were not droplets. Eight normal biopsies and 37 biopsies showing alcoholic liver steatosis were analyzed. The automated morphometric analysis calculated the mean density (percentage) of steatosis and the size distribution of the droplets. Statistical comparison of these results with those of the semiquantitative optical analysis performed independently by two pathologists showed a significant correlation between their calculations of the density/degree of steatosis but significant differences for their evaluation of the type of steatosis. The pathologists constantly overestimated the ratio of macrodroplets to microdroplets.

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.