Ultrastructural analysis of apoptosis and autophagy in the midgut epithelium of Piscicola geometra (Annelida, Hirudinida) after blood feeding.

Cell death in the endodermal region of the digestive tract of the blood-feeding leech Piscicola geometra was analyzed using light and transmission electron microscopes and the fluorescence method. Sexually mature specimens of P. geometra were bred under laboratory conditions and fed on Danio rerio. After copulation, the specimens laid cocoons. The material for our studies were non-feeding juveniles collected just after hatching, non-feeding adult specimens, and leeches that had been fed with fish blood (D. rerio) only once ad libitum. The fed leeches were prepared for our studies during feeding and after 1, 3, 7, and 14 days (not sexually mature specimens) and some weeks after feeding (the sexually mature). Autophagy in all regions of the endodermal part of the digestive system, including the esophagus, the crop, the posterior crop caecum (PCC), and the intestine was observed in the adult non-feeding and feeding specimens. In fed specimens, autophagy occurred at very high levels--in 80 to 90 % of epithelial cells in all four regions. In contrast, in adult specimens that did not feed, this process occurred at much lower levels--about 10 % (esophagus and intestine) and about 30 % (crop and PCC) of the midgut epithelial cells. Apoptosis occurred in the feeding adult specimens but only in the crop and PCC. However, it was absent in the non-feeding adult specimens and the specimens that were collected during feeding. Moreover, neither autophagy nor apoptosis were observed in the juvenile, non-feeding specimens. The appearance of autophagy and apoptosis was connected with feeding on toxic blood. We concluded that autophagy played the role of a survival factor and was involved in the protection of the epithelium against the products of blood digestion. Quantitative analysis was prepared to determine the number of autophagic and apoptotic cells.

Cellular organisation of the mature testes and stages of spermiogenesis in Danio rerio (Cyprinidae; Teleostei)--structural and ultrastructural studies.

The male gonads of Danio rerio occupy a position typical of the Teleostei species. The structure of the testes corresponds to the anastomosing tubular type with unrestricted spermatogonia and represents a cystic type of spermatogenesis. The results of this study indicate that four distinct stages of cell differentiation can be identified during spermiogenesis. These stages are characterised by chromatin condensation, the development of flagellum, nuclear rotation, the formation of nuclear fossa and the elimination of excess cytoplasm. A round head and the absence of an acrosome characterise the differentiated sperm. The midpiece is short and large, and C-shaped mitochondria form a ring surrounding the initial region of the flagellum. The axoneme shows a 9+2 pattern. In the D. rerio spermatozoa the flagellar axis is at an angle of 110° to the nucleus diameter running through the centriole.

Early development of the adrenal glands in the grass snake Natrix natrix L. (Lepidosauria, Serpentes).

The aim of the study was to investigate the development and differentiation of the adrenal glands in the grass snake (Natrix natrix L.) during the early stages of ontogenesis, i.e., from egg-laying to hatching of the first specimens. The material used for the studies consisted of a collection of embryos of the grass snake. The Natrix eggs were incubated in the laboratory at a constant temperature of 30 degrees C and 100% relative humidity. Embryos were isolated in a regular sequence of time from egg-laying to hatching. The age of the embryos was qualified according to normal tables for this species. For histological and histochemical investigations, the smallest embryos were fixed in toto. From the oldest embryos, the medial region with the mesonephros and adrenal primordium were resected. Depending on the requirements of histochemical methods, the material was fixed in various fixatives, namely, 10% formalin solution, Bouin, Wood and Millonig fluid, embedded in paraffin and sectioned into serial transversal, sagittal and longitudinal sections. The sections for review were stained with H&E and azan. For detection of adrenaline and noradrenaline in chromaffin tissue, the Wood and Honoré methods were used. SGC cells were detected with the silver stain method after Bodian. For electron microscopic studies, the adrenal gland was fixed in 2.5% glutaraldehyde and 2.0% paraformaldehyde 1:1 in 0.1 M phosphate buffer at pH 7.4 and post-fixed in 1.5% osmic acid in the same buffer. The fixed sections of the adrenal glands were embedded in Epon 812. Semithin and ultrathin sections were cut on ultramicrotome ultratome IV. Semithin sections were stained with methylene blue and ultrathin sections were routinely contrasted with uranyl acetate and lead citrate, then examined and photographed with the JEM JEOL 1220 electron microscope. According to morphological and metrical observation in the course of the grass snake embryo development, one can distinguish 12 stages of development. The primordia of the adrenal cortex appear at the first trimester of egg incubation as two asymmetrical strands between the mesonephros and aorta dorsalis. They are made of dense mesenchymal cells. At the second trimester of development, primordia are penetrated by chromaffinoblasts and capillaries. The mesenchymal cells differentiate into interrenal cells, while chromaffinoblasts are arranged dorsally of the gland. The glands are enclosed by the capsule which separates them from the mesonephros. At the third trimester of the eggs incubation, only noradrenaline appears in a chromaffin tissue. At the moment of snake hatching, the adrenal glands are completely differentiated, both in their structure and their function. The primordia of the interrenal tissue differentiate from mesenchymal cells similarly to mammals. During the development of the snake interrenal tissue, several types of cells can be recognized, varying in the degree of differentiation and in ultrastructural features: 1. Undifferentiated cells with features of mesenchymal cells 2. Differentiating mesenchymal cells 3. Transitional cells with features of mesenchymal and steroidogenic cells 4. Differentiating interrenal cells with pleomorphic mitochondria and numerous lipid droplets 5. Embryonic interrenal cells containing circular lipid droplets and underdeveloped smooth endoplasmic reticulum 6. Transitional interrenal cells containing mitochondria with tubular and vesicular cristae, large lipid droplets, numerous myelin structures, and well-developed smooth endoplasmic reticulum 7. Degenerating cells of embryonic interrenal tissue 8. Differentiating mesenchymal cells with features of fibroblasts The above classification is very schematic and presumptive. In developing adrenal glands at the first trimester of egg incubation type 1 and 2 cells predominate. Type 3 and 4 cells were observed at the second trimester of the adrenal primordia development. At the third trimester of egg incubation, embryonic adrenal glands were composed of the type 5 cells. At the moment of snake hatching, interrenal tissue contained type 5 and 6 cells. In the next days of the adrenal gland development, at the border between the cortex and in medulla as under the capsule, numerous cells were degenerated. During the entire development period the adrenal capsule was built from type 7 cells. The chromaffin tissue of the adrenal glands is derived from the neural crest. These findings agree with the findings of all embryologists. The first chromaffinoblasts infiltrated the adrenal cortex primordium around stage IV of development. They were mixed with interrenal cells and just at hatching they were localized dorsally of the gland. The chromaffinoblasts differentiated gradually from neuron-like cells to typical chromaffinocytes. All the chromaffinoblasts contained the chromaffin granules. The size and numerical density of the chromaffin granules increased with development. Just before hatching, the cells of the chromaffin tissue contained only noradrenaline. Differentiation chromaffinoblasts into chromaffin cells are probably stimulated and controlled by the influence of hormones, which are produced by the cells of the interrenal tissue. According to morphological, histochemical and ultrastructural observation in the course of adrenal differentiation and development in the grass snake, six morphological phases can be distinguished.