Stanislas Haumont

The influence of fixation procedure, embedding medium and section thickness on morphometric data in thyroid gland.

SummaryIn this study, the effects of fixation procedures, embedding medium and section thickness on stereological measurements of normal thyroid were analysed. The following conclusions were drawn:A)the use of a single section for the analysis of a lobe is sufficient if this section is located in the central part of the lobe.B)fixation and embedding with glutaraldehyde-Epon leads to a larger shrinkage than Bouin-paraplast, but the difference between the two procedures is not significant.C)osmium post-fixation reduces the shrinkage induced by glutaraldehyde and lowers the axial deformation produced by sectioning.D)Bouins fixative and paraplast embedding induce considerable shrink-age of the interstitial tissue. The shrinkage obtained with glutaraldehyde-Epon is less. However, it is still not known whether this difference is due to the fixative, or to the embedding procedure or to both.E)only in glutaraldehyde and osmium-fixed material, embedded in Epon, can follicles and colloids be assumed to be spherical in shape without significant errors.

Computer-assisted self-assessment (CASA) in histology

A computer-assisted self-assessment system (CASA) has been developed. It allows students to evaluate their own knowledge and ability in identifying histological structures. It consists of an image digitizing-editing system, an image database shared through a local area network, and teachers- and students-environments. The authoring system allows the teacher to define text frames, multiple-choice and open-ended questions. The students environment includes on-line help and recording of the students identification, answers, scores and messages. The educational impact of the CASA system was tested in 1987 on a single prototype workstation, with a limited number of students. Results showed that the marks in histology at the final examination were significantly increased for the weak students, but not for the good students. Since September 1988, several workstations have been available in the histology classroom and in a microcomputer room located in the Faculty library. In an anonymous questionnaire, nearly 90% of the students answered that they were in favour of this system.

Thymic nurse cells: morphological study during their isolation from murine thymus.

SummaryThymic nurse cells (TNC), which are multicellular complexes composed of epithelial cells and thymocytes, were obtained from C3H-mice thymuses. They were described by means of light and electron microscopy. The morphology of epithelial cells forming isolated TNC compared to that of small tissue fragments obtained by enzymatic digestion revealed that TNC could be derived from all parts of the thymus: cortex, corticomedullary junction and medulla, the cortex being their principal source. This variety of origin, the presence of several epithelial cells inside a single TNC, the presence of non-lymphoid cells, and the various locations of eleaved desmosomes confirmed that their aspect “in vitro” as round and sealed structures can be considered to be an artifact due to the isolation technique used. Indeed, during this procedure, they are formed by a process of wrapping of the epithelial cytoplasm around the tightly associated thymocytes. All three epithelial cell types: cortical reticular cells, medullary reticular cells, and medullary globular cells can form TNC.

Morphological Changes in Mice Thyroid Induced by Iodine Deficiency

SummaryGoitrogenesis induced in mice by iodine deficiency took place in two distinct phases. The first phase lasted four weeks and was characterized histologically by the classic signs of hyperplasia: colloid resorption, increase in the height of the epithelium and enlargement of the capillaries.After the fourth week, the morphological changes in the thyroid were different in males and females. In the male, pluristratified follicles, secondary follicular cavities and papillary projections were observed. In the female, most of the follicles retained their hyperplastic appearance, while papillary projections were observed in very few follicles.

Thymic accessory cell complexes in vitro and in vivo: morphological study.

SummaryMurine thymic macrophages and interdigitating cells, also called thymic accessory cells, were characterized by means of light- and electron microscopy. The cells were studied in suspension, during isolation by enzymatic digestion and in vivo. They were observed as isolated cells or as components of multicellular complexes, some of which were rosettes and were composed of lymphoid cells centered on each type of accessory cell. We also noted other cell complexes including macrophages that resembled classical epithelial nurse cells. We consider that multicellular complexes represent lymphostromal associations already existing in vivo, because we observed them at the periphery of thymic pieces undergoing enzymatic treatment. The heterogeneity of macrophages that we observed in vitro was also noted in vivo. In vivo macrophages were of three types: classical phagocytic cells distributed throughout the gland, cortical elongated cells in close contact with lymphoid blast cells, and atypical nurse cells containing mitotic cells and located in the inner cortex. The morphological aspects of the latter two cell types suggest that cortical macrophages in vivo have other roles: they can be interpreted as images of positive or negative cell selection. We also believe that rosettes are formed by elongated cortical macrophages when they are enzymatically isolated from the thymus.

The thymus of Nu/+ mice.

SummaryAt one month, the thymus weight of NMRI mice heterozygous for the Nu gene was lower than that of the wild type. However, the thymic structure was normal and no significant anomalies at the ultrastructural level could be found either in epithelial cells or in thymocytes. Stereologic data showed that the loss of weight was only due to a decrease of the lymphoid population, whereas the absolute number of epithelial cells was unmodified.Several hypotheses that could explain the reduced number of thymocytes were discussed.

Thymic nurse cells in culture: morphological and antigenic characterization.

Epithelial monolayers were derived from thymic nurse cells (TNC), and were seeded onto collagen-coated dishes immediately after their isolation from young adult C3H-murine thymuses. Different media and supplements were tested in order to obtain cultures that were as pure as possible. Primary cultures were enriched in epithelial cells but always contained non-epithelial components among which fibroblasts predominated. Immunodetection of keratins, and repeated light- and electron-microscopic observations established the epithelial nature of the elongated cells derived from TNC; these elongated cells were cortical reticular cells, and were different from medullary globular cells that immediately adopted a mosaic pattern in vitro. At the beginning of the culture, the necrosis of cortical lymphocytes appeared to be toxic for epithelial cells; when epithelial cells survived, they showed a temporary lipid accumulation. After a 5-day culture, they still synthesized DNA but lost this capacity thereafter and dedifferentiated. The lympho-epithelial symbiosis appeared to be necessary to maintain some epithelial characteristics of the cultured cells, such as the clear vesicles and the expression of la antigens. In sub-cultures, the monolayers were almost purely epithelial in nature but growth was no longer observed. The cells remained reticular in shape, as they were in vivo, but their cytoplasm and their nucleus became larger and numerous cells were multinucleated. Confluence was not obtained with classical media even after mitogenic stimulation. The frequent observation of strongly keratinized areas suggested a process of terminal differentiation; this could not be avoided by using low serum concentration.

Morphometry of nuclear pore complexes in thyroid cells during hyperplasia and involution.

Nuclear pore complexes were analyzed in freeze-fractured replicas of thyroid follicular cells of C3H mice in different physiological states. Thyroid stimulation induced a rapid and simultaneous increase of the nuclear surface and volume and of the total number of pore complexes. The numerical density (Na) of pore complexes increased at the 6th day of stimulation, but after that time the proportion of cells with an increased Na was always higher than the proportion of 3H-labelled nuclei. During thyroid involution, all the nuclear parameters, including the Na, returned to normal values. These results indicate that the total number of pore complexes and their Na are correlated with the cellular activity rather than with the cell cycle. They also suggest that 2 different mechanisms are involved in the generation of pore complexes: first, an addition of new membranes with a low density of pore complexes; later, a formation of new pore complexes in preexisting membranes. However, during involution, parts of the nuclear membranes and pore complexes in the remaining parts disappear synchronously. In freeze-fractured thyroid nuclear membranes, 2 neighbouring pore complexes were always separated by a distance of 105 nm. Clusters of pore complexes were not observed. A comparison of the distances between pore complexes and between randomly generated points never showed any significant differences indicating that pore complexes were randomly distributed.

Changes of relaxin concentrations determined by immunodensitometry in ovaries of NMRI mice during pregnancy.

Relaxin has been localized in corpora lutea (CL) of pregnant NMRI mice using the avidin-biotin complex immunocytochemical procedure and an antiserum against highly purified porcine relaxin. The immunostaining was measured by immunodensitometry. Relaxin immunostaining was first observed in luteal cells of type I gestational CL on day 11.5 (D11.5). For each investigated day, all CL were identically stained, and immunostaining was evenly dispersed all over the CL. Seventy-five percent of cells were stained at D11.5, and nearly all cells were stained between D13.5 and D18.5. The staining intensity increased throughout the last half of pregnancy, reaching a maximum at D18. A few hours before parturition, at D18.5, relaxin immunostaining decreased dramatically and reached the background level shortly after delivery. From our results we may conclude that, in murine CL, the number of relaxin-secreting cells and the intracellular storage of the peptide increase during pregnancy. The disappearance of relaxin with the cells occurs rapidly +/- 12 h before parturition.

Evolution of NMRI mice ovaries and corpora lutea during pregnancy: morphologic and morphometric study

During pregnancy in mice, three groups of corpora lutea (CL) originating from 4 successive ovulatory cycles could be distinguished taking into account their size, cellular structure and stain affinity. Type I CL originated at the onset of pregnancy, type II CL originated during the 2 previous estrous cycles and type III CL were produced during a cycle preceding the two others. CL volume of type I increased 10-fold between day 0 and day 18.5, a 2.5-fold increase in volume occurred in type II CL between day 0 and day 3, although they derived from cycles preceding pregnancy. The volume of type III remained unchanged. This fact suggested that types I and II CL probably play a functional role in the ongoing pregnancy. After day 10 of pregnancy a high correlation coefficient (r = 0.933) is observed between type I CL and the number of embryos in the corresponding uterine horn.