Lipid content and cryotolerance of porcine embryos cultured with phenazine ethosulfate.

The addition of phenazine ethosulfate (PES) to culture medium was investigated for its effect on pig embryo development, apoptosis, cytoplasmic lipid content and survival after OPS vitrification. Porcine zygotes were cultured in NCSU-23 medium supplemented with 0 (control) or 0.05 microM PES up to the blastocyst stage and were vitrified using OPS technology. Culture of embryos with PES reduced the cytoplasmic lipid content, as measured by fluorescence of blastocysts stained with Nile Red. The survival rate of vitrified blastocysts was slightly enhanced, although not significantly, in the presence of PES compared to the PES-free group (45.2 and 37.9 percent, respectively). These results showed that culturing porcine embryos in medium with PES increased the proportion of morula and blastocyst formation and reduced the index of DNA fragmentation and the cytoplasmic lipid content of cultured blastocysts. However, the use of PES during in vitro culture had limited effect on porcine blastocyst survival after vitrification.

Changes of lipid composition in non-cultured and cultured porcine embryos.

The objective of the present study was to evaluate the lipid composition of cultured and non-cultured pig embryos during cleavage using histochemical methods. The authors studied pig zygotes as well as 2-to 4-cell embryos, morulae, and blastocysts that were either non-cultured or cultured in NCSU-23 medium. To detect different types of lipids, the authors used the Churukian method with Oil red O, the Sudan black B method, the Cain method with Nile blue sulfate, and the modified osmium tetroxide-ethanol treatment. In the zygotic lipid droplets, diverse classes of unsaturated and saturated lipids were found, with particularly high levels of unsaturated hydrophobic lipids, mainly triglycerides and other esters, free fatty acids, and phospholipids. In the zygotic cytoplasm, the authors observed high levels of fatty acids and phospholipids. The total lipid content remained constant up to the morula stage, decreasing later at the blastocyst stage, but the overall amount of unsaturated lipids declined earlier, at the 2-to 4-cell stage. The amount of free fatty acids and phospholipids decreased during cleavage in both non-cultured and cultured porcine embryos. The main differences between the non-cultured and cultured embryos were the more pronounced reduction in the amount of unsaturated hydrophobic lipids in droplets and the cytoplasmic free fatty acids observed in the cultured morula and the lower content of phospholipids in the cytoplasm of the cultured 2-to 4-cell embryos relative to the non-cultured embryos. The decrease in the unsaturated lipid, free fatty acid, and phospholipid content during in vivo development and the differences in the amount of these types of lipids between developmentally matched cultured and non-cultured pig embryos correlate well with modifications of lipid and carbohydrate metabolism.

Lipid content of non-cultured and cultured pig embryo.

The objectives of the study were: (i) to work out a precise and efficient method for quantitative analysis of lipid content and (ii) to quantitatively determine the lipid content in non-cultured and cultured pig embryos. The experiment was carried out on pig embryos from zygote to late blastocyst stages produced in vivo and embryos collected at the zygote stage and then cultured in vitro up to blastocyst stage. Embryos were fixed, dehydrated, embedded in epoxy resin and cut into semi-thin sections to analyse the quantity of lipids in fat droplets. Stained sections were then analysed with Cavalieri and point counting methods to evaluate the following stereological parameters of the embryo: total embryo volume - V(e), volume density of cytoplasm per unit volume of embryo - Vv(c,e), volume density of lipid droplets per unit volume of embryo cytoplasm - Vv(fat,c) and total volume of lipid droplets per whole embryo - V(fat). Values of Vv(fat,c) and V(fat) remained unchanged up to the morula stage, but decreased significantly at blastocyst and late blastocyst stages both in cultured and non-cultured embryos. Volume density of lipid droplets per unit volume of embryo cytoplasm and total volume of lipid droplets for cultured embryos showed statistically significant differences between late blastocyst and almost all other stages. Comparisons of Vv(fat,c) in embryos at the same stages of development but differing in origin of embryos (non-cultured or cultured) show that statistically significant differences exist for all analysed stages. In conclusion, differences in lipid content observed in pig embryos were dependent on the developmental stage of the embryo as well as the culture conditions (i.e. cultured and non-cultured embryos at the same stage of development).

Hematopoiesis in snakes (Ophidia).

Locations of the hematopoietic tissue have been described in the following ophidian species: Bothrops jararaca, Bothrops jararacusu, Waglerophis merremii, Elaphe teniura teniura, Boa constrictor, and Python reticulatus. Studies were carried out on perfusion fixed vertebrae, ribs, spleen, liver, thymus, and kidney. Routine histological technique was applied using both light and electron microscopy. Hematopoietic tissue was found in the following locations of the vertebrae: neural spine, neural arch, postzygophysis processes, hypapophysis, vertebral centre. Moreover, intense hematopoiesis was found inside the ribs. In the spleen and thymus, only lymphopoiesis was found. Hematopoietic islets in the spleen were sporadically found only in young specimens. No hematopoiesis was observed in the liver and kidney. In the studied species, there were no differences in the location of hematopoietic tissue. A new model of mature and immature blood cell release to the lumen of marrow sinuses different from that known to operate in higher vertebrates is proposed.

Utilization of yolk platelets during early embryonic development of Rana temporaria and Bufo bufo.

Utilization of yolk platelets in cleaving embryos of Rana temporaria and Bufo bufo was studied by different methods. Morphological observations of yolk platelets of R. temporaria embryos at tail bud stage by transmission electron microscopy indicated four initial phases of platelet degradation. The pattern of these events is similar to that found in embryos of B. bufo. The morphological observations were confirmed by energy-dispersive X-ray microanalysis of the elemental content of platelets and by selected-area electron diffraction of platelet cores. Covalently bound sulphur content decreased during cleavage and the content of different inorganic ions changed, whereas the structure of crystalline core remained constant. Morphological changes found in the amorphous cortex of yolk platelets were due to their utilization. Stereological measurements indicated that utilization during cleavage increased, but only the initial phases of yolk platelet degradation were seen. The volume of the cortex did not decrease and the crystalline core did not fragment.

The corpus luteum of the pig. Scanning electron microscopic study of surface features at different times of incubation.

The surface ultrastructure of porcine early corpus luteum cells (days 1-3 of the luteal phase) was studied in SEM and correlated with progesterone secretion. Luteal cells were divided into 2 groups: small cells (10-20 microns) and large cells (20-30 microns) and their surface features were observed after 1, 3, and 5 h of incubation in the control medium and in a medium supplemented with prolactin (PRL). The surface morphology of control cells was characterized by numerous smooth blebs and the presence or absence of thin microvilli. Small and large cells showed a tendency to adhere to the glass during the experiment, but on the large cells the number of thin adhesive filopodia was greater. After the 1st and 3rd h of incubation with PRL the number of microvilli and numerous filopodia on the small cells increased substantially. Nodular blebs were scattered and appeared to protrude from the cell surface. Many small cells adhered to the glass by thick, layered and thin thread-like cytoplasmic processes. After the 5th h distinct smoothing of the surface of the small cells was seen. The number of microvilli seen on the PRL stimulated surface of the large cells was smaller and in some cases even entirely absent. After the 1st and 3rd h of the experiment the large cell surface was ruffled with minute folds. Numerous nodular blebs protruded from the cell surface. The number of adhesive filopodia attaching the cells to the glass decreased or vanished during the experiment. After the 5 h of incubation most of the cells had smooth surface with smooth blebs. Progesterone secretion was measured by radioimmunoassay. The cells in the medium without exogenous hormone (control) secreted relatively low levels of progesterone throughout 1-5 h of the incubation period. After addition of PRL to the medium the amount of secreted progesterone increased.

Cytochemical localization of adenylate cyclase activity in rat bone marrow.

The localization of adenylate cyclase (AC) in rat bone marrow was studied at the ultrastructural level using a cytochemical technique with adenosine triphosphate (ATP) as substrate. The strongest precipitate occurred around cytoplasmic membranes of cells of granulocytic lineage. Around cells of erythrocytic lineage, as well as megakaryocytes, lymphocytes and the plasma cells, the granules resulting from the cytochemical reaction revealed various degrees of density. The region within the bone marrow tissue where the cells manifested an increased reaction product was called 'reaction fields'. Most often the 'reaction fields' were formed by maturing cells of the granulocytic and erythrocytic lineages, surrounding the reticular cells. The reaction precipitate was also apparent around the advential and endothelial cells of the sinuses. Mature cells migrating from the bone marrow space to the sinus lumen also showed an intensified reaction product. The causes of such localization of AC were discussed.