Localization of Mg++-dependent adenosine triphosphatase and alkaline phosphatase activities in the postimplantation mouse embryos in day 5 and 6

Summary Mg⁺⁺-dependent adenosine triphosphatase (Mg-ATPase) and alkaline phosphatase (ALPase) activities were histo- and cytochemically investigated in postimplantation mouse embryos from day 5 to day 6. In day 5 postimplantation embryos, Mg-ATPase activity was detected in the embryonic ectoderm and weakly in the visceral endoderm. Weak ALPase activity was found in the embryonic ectoderm and visceral endoderm. Parietal endoderm, both in day 5 and in day 6 embryos, had very weak or no Mg-ATPase and ALPase activities. Mg-ATPase activity in day 6 embryos was found with the same localization as that in day 5 embryos. No ALPase activity was observed in their embryonic ectoderm. Extraembryonic ectodermal cell mass had the strongest Mg-ATPase activity in these stage embryos.These results suggest that the localization of both enzyme activities in postimplantation mouse embryos is closely related to the morphogenesis. As regards the proamniotic cavity formation, the fact that Mg-ATPase activity was still observed in the embryonic ectoderm in these stages suggests the involvement of active transport system on the production of nascent proamniotic cavity fluid.     

Morphology and histoenzymology of eosinophilic granulocytes in the circulating blood of the turtle (Chrysemys dorbignih)

The studies on the characterization of eosinophils and neutrophils/heterophils of turtles are contradictory. Some authors have pointed out the existence of two distinct cell types: eosinophils and heterophils. Other authors have proposed that eosinophils and heterophils may be the same cells in different stages of maturation. These interpretations are based only on a morphological analysis. In the blood of the turtle (Chrysemys dorbignih), a South American freshwater species, there are two types of granulocytes with eosinophilic staining pattern: the first with round cytoplasmic granules and the second with ellipsoidal cytoplasmic granules. In the present study by using histoenzymological methods for the analyses of enzymological cellular content, we found that the cells with round cytoplasmic granules were positive for nonspecific esterase and the cells with ellipsoidal granules were positives for acid phosphatase, alkaline phosphatase, nonspecific esterase and peroxidase. The results show that these cells are distinct cells and that the cells with ellipsoidal cytoplasmic granules have the same histoenzymological characteristics as the neutrophils/heterophils of mammalians and other vertebrates.     

Cytochemical localization of tartrate-resistant acid phosphatase,alkaline phosphatase,and nonspecific esterase in perivascular cells of cartilage canals in the developing mouse epiphysis

Cytochemical localization of tartrate-resistant acid phosphatase (TRAP), tartratc-sensitive acid phosphatases (TSAP), alkaline phosphatase, and nonspecific esterase was used to characterize perivascular cells within cartilage canals. In the distal femoral epiphyses of 5- to 7-day-old mice, three stages of canal development can be distinguished, and at each developmental stage different perivascular cells were present with morphological characteristics of degradative cells. Vacuolated cells resembling macrophages, fibroblastic cells, and chondroclasts were present adjacent to the matrix in superficial, intermediate, and deep canals, respectively. In order to characterize these perivascular cells cytochemically, nonspecific esterase and TSAP staining was used to identify macrophages, alkaline phosphatase staining was used to identify fibroblastic cells, and TRAP staining was used to identify chondroclasts. There were no cells present in the canals at any developmental stage that were positive for TSAP or strongly positive for nonspecific esterase, placing doubt on the identity of the vacuolated cells as macrophages. Alkaline phosohatase-positive perivascular cells were present in the intermediate and deep canals adjacant to matrix containing alkaline phosphatase-positive chondrocytes. These alkaline phosphatase-positive cells were found in the same location within canals as the fibroblastic cells. Tartrate-resislant acid phosphatase was localized in chondroclasts at the tips of deep canals but was not confined exclusively to chondroclasts. Except for the very early stage of canal development prior to chondrocyte hypertrophy, TRAP-positive cells were present at the tips of superficial and intermediate canals as well as at the tips of the deep canals. Additionally, the presence of TRAP in chondrocytes with in the growth plate, in chondrocytes within the epiphyseal cartilage near some canals, and in perichondrial cells suggests that TRAP is associated with matrix degradation in the cartilage.     

Cytophysiological differences between the embryoblast and trophoblast of rat embryos as revealed by vital staining

Summary The cytophysiological method was used to study the early stages of rat development in vitro. Along with the formation of granules de novo in the cytoplasm of embryonic cells neutral red stained some pre-existing structures. The type of intravital staining and the intensity of granule formation differed at various developmental stages. In the early preimplantation stages (from the second to the fifth day) the intensity of granule formation in the embryoblast cells was greater than in the trophoblast cells. Conversely, immediately after the implantation (the seventh-tenth days) the extraembryonic formations (yolk entoderm, ectoplacental cone, giant cells of the trophoblast) stain more intensely; embryonic and intestinal entoderm stain much more weakly. The character of granule dis tribution in the tissues of rat embryos at various developmental periods is explained by the peculiarities of growth and differentiation of individual anlagen of mammals at various stages of ontogenesis.Presented by Active Member AMN SSSR, N. N. Anichkov Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 57, No. 1, pp. 98–103, January, 1964     

Acid and alkaline phosphatase activity in migrating primordial germ cells of the early chick embryo

Little information is available concerning enzyme activity in primordial germ cells (PGCs) of the early chick embryo. The present study is designed to examine the disposition of alkaline and acid phosphatase activity in the PGCs during their migration into the developing gonads of the early chick embryo. White Leghorn chick embryos were sacrificed at daily intervals from 1 to 6 days of incubation. Following sacrifice the embryos were fixed, dehydrated, and embedded in glycol methacrylate (GMA). Alkaline and acid phosphatases were demonstrated by the simultaneous diazo-coupling method. The embryonic tissues at the different ages were examined for PGCs and the histochemical reactions for alkaline and acid phosphatases in these cells evaluated. Acid phosphatase activity did not appear within PGCs until 3 days of incubation, and then in only a few PGCs in the active phase of their migration in the dorsal mesentery, suggesting that there is no large wave of degeneration of these cells during migration. Alkaline phosphatase activity was observed as early as 2 days of incubation in PGCs during the passive phase of their migration in extraembryonic blood vessels. Alkaline phosphatase-positive PGCs in the active phase of migration were also found in the dorsal mesentery; however, the cellular localization of this enzyme differed from that observed in the passively migrating PGCs, indicating that there are alterations in the metabolic activities of these cells during the active and passive phases of migration.     

Cytochemistry of Reed-Sternberg cells in lymph node imprints

Cytochemical reactions were examined in lymph node imprints from a group of 53 previously untreated patients with histologically proven Hodgkin's disease. In 40 of 51 cases investigated, Reed-Sternberg (R-S) cells, irrespective of the cytologic appearances and the histologic types, showed moderate to strong reactions with acid phosphatase (ACP). In 12 cases ACP activity was present in more than 25% of the R-S cells. The reaction consisted of formation of small- to medium-sized granules, which were located close to the nuclei on a diffusely positive background or irregularly distributed throughout the cytoplasm. In three cases, a coarse granular reaction product with periodic acid-Schiff was present. R-S cells were positive to the naphthol-AS acetate esterase and beta-glucuronidase reactions in four and two cases, respectively. Alkaline phosphatase and naphthol-AS-D-chloroacetate esterase reactions were completely negative. Our results have revealed a pattern of staining in the diagnostic R-S cells similar to that in its morphologic variants; this supports the view that these cells may derive from a common primitive cell. Moreover, the quality and quantity of the ACP reaction product shows that R-S cells differ from both neoplastic histiocytes of malignant histiocytosis and neoplastic lymphocytes of T-cell lymphomas. This study confirms that R-S cells lack definite cytochemical characteristics of each of supposed progenitor cells: histiocytes and T-lymphocytes.     

Cytochemical staining and ultrastructural characteristics of peripheral blood leucocytes from the yellow rat snake (Elaphe obsoleta quadrivitatta)

Cytochemical staining and ultrastructural characteristics of peripheral blood leucocytes from the yellow rat snake are described. A panel of cytochemical stains, including demonstration of myeloperoxidase, acid phosphatase, naphthol AS-D chloracetate esterase, alpha-naphthol butyrate esterase and alkaline phosphatase activities; and periodic acid-Schiff and Sudan Black-B staining was performed. Snake heterophils lacked peroxidase, alkaline phosphatase and acid phosphatase activity. Azurophils stained positively for all stains except alkaline phosphatase activity. Lymphocytes showed positive acid phosphatase activity. Differentiation of thrombocytes from lymphocytes was very difficult even with cytochemical staining. Only a minor staining difference was observed with periodic acid-Schiff stain. Thrombocytes exhibited coarse, dark, purple stippling usually located in the polar area of the cytoplasm, whereas lymphocyte staining varied from none to very fine, pale pink granules dispersed throughout the cytoplasm. Ultrastructural characteristics were similar to those of mammalian leucocytes with the exception that the snake basophil granules have no crystalline matrix, and heterophil granules appeared as large, elongate, membrane bound structures of varying density with no distinct core or matrix.     

Cytochemical staining and ultrastructural characteristics of peripheral blood leucocytes from the yellow rat snake (Elaphe obsoleta quadrivitatta)

Cytochemical staining and ultrastructural characteristics of peripheral blood leucocytes from the yellow rat snake are described. A panel of cytochemical stains, including demonstration of myeloperoxidase, acid phosphatase, naphthol AS-D chloracetate esterase, alpha-naphthol butyrate esterase and alkaline phosphatase activities; and periodic acid-Schiff and Sudan Black-B staining was performed. Snake heterophils lacked peroxidase, alkaline phosphatase and acid phosphatase activity. Azurophils stained positively for all stains except alkaline phosphatase activity. Lymphocytes showed positive acid phosphatase activity. Differentiation of thrombocytes from lymphocytes was very difficult even with cytochemical staining. Only a minor staining difference was observed with periodic acid-Schiff stain. Thrombocytes exhibited coarse, dark, purple stippling usually located in the polar area of the cytoplasm, whereas lymphocyte staining varied from none to very fine, pale pink granules dispersed throughout the cytoplasm. Ultrastructural characteristics were similar to those of mammalian leucocytes with the exception that the snake basophil granules have no crystalline matrix, and heterophil granules appeared as large, elongate, membrane bound structures of varying density with no distinct core or matrix.     

Effects of disruption of the embryonic alkaline phosphatase gene on preimplantation development of the mouse.

Embryonic alkaline phosphatase (EAP) is expressed during the preimplantation period of mouse development; however, its function is unknown. To determine whether the absence of an EAP gene affects development of preimplantation embryos, we studied mice homozygous for the disrupted EAP gene (EAP.ko mice). Time to reach morphologically definedpreimplantation stages, preimplantation loss, cell count, gestation length, and litter size were monitored, and it was found that EAP.ko embryos have slower development and higher rates of degeneration during in vitro preimplantation development. In vivo, EAP.ko mice had a longergestation, smaller litter size, and fewer cells at 93 hr after human chorionic gonadotropin injection. Furthermore, there was no compensation for the absence of EAP gene in EAP.ko embryos by other isozymes of alkaline phosphatase. We conclude that the presence of an active EAP gene is beneficial for preimplantation development of the mouse embryo, and its absence leads to fewer blastocysts in vitro, delayed parturition, and reduced litter size in vivo.