Ovo Treatment Research Articles

Effects of polyamine limitation on nucleolar development and morphology in early chick embryos

Inhibition of polyamine synthesis in early chick embryos by in ovo treatment with dl-α-difluoromethylomithine (DFMO), injected beneath the blastoderm after 5 h of incubation, permanently blocks post-gastrular development. After the first day of polyamine limitation, the embryos possess a thickened primitive streak. Further morphogenesis is blocked and the ectoderm and mesoderm are condensed around the streak. There is obvious suppression of nucleolar formation in 24 h DFMO-treated embryos. In the mesoderm obliquely in front of Hensen's node the frequency of nucleolus-possessing cells is only a few percent lower in DFMO-treated than in control embryos. However, in the same area the frequency of mesoderm cells possessing multiple nucleoli is about 50% lower in the polyamine-depleted embryos. At the ultrastructural level, mesoderm cells from 24 h DFMO-treated embryos show a reduction of the fibrillar component of the nucleolus with a resulting segregation of the nucleolar material. Our data indicate that stimulation of polyamine synthesis is an obligatory step in the differentiation of epiblast cells into mesoderm cells.

The Synthesis of Hyaluronic Acid by Ectoderm During Early Organogenesis in the Chick Embryo

This study demonstrates that the dorsal ectoderm of the stage 14 chick embryo synthesizes hyaluronic acid. About 49 to 52% of the H3 glucosamine-labeled glycosaminoglycan that is synthesized by explanted ectoderm can be identified as hyaluronic acid on the basis of its susceptibility to Streptomyces hyaluronidase or isolation of chondroitinase ABC digestion products. In addition, autoradiographic evidence shows that the ectoderm, unlike adjacent tissues like epithelial somites or neural tube, incorporates glucosamine into hyaluronidase-sensitive material which becomes largely extracellular and localized in the subectodermal cell-free space. Ultrastructural evidence shows that there is a fine fibrillar matrix between the ectodermal cells and in the subectodermal spaces when tannic acid is included in the primary fixative. This material resembles authentic hyaluronate, similarly fixed, and is absent when tannic acid is omitted from the fixative or when embryos have been previously treated in ovo with Streptomyces hyaluronidase. The concomitant reduction in the intercellular and subectodermal cell-free spaces after in ovo treatment with Streptomyces hyaluronidase supports the hypothesis that the dorsal ectoderm plays a morphogenetic role by contributing hyaluronate to the forming extracellular spaces. It is proposed that ectodermally derived hyaluronate might influence the morphogenesis of subjacent tissues such as the dermatome and neural crest.

Alkaline Phosphatase in the Developing Bursa of Fabricius

The ontogeny of alkaline phosphatase in the bursa of Fabricius was studied by histochemical and biochemical methods. According to the quantitative determinations, the activity of alkaline phosphatase increased from the 11th to 17th day of incubation--that is, during the time of the lymphoid follicle formation in the developing bursa. The activity was localized in the mesenchymal tissue surrounding the lymphoid follicles. Testosterone given in ovo prevented the appearance of alkaline phosphatase in the bursal mesenchyme but had no effect on the activity of the embryonic liver. In contrast, in ovo treatment with cyclophosphamide had no effect on the alkaline phosphatase in the bursa. By using transplantation of embryonic bursal stem cells, it was further shown that, in contrast to cyclophosphamide, testosterone destroys the capacity of the bursa to serve as a differentiation site for the B-cell lineage. The results indicate that testosterone affects the stromal cells of the bursa, whereas cyclophosphamide destroys only the lymphoid population undergoing differentiation and leaves the bursal stroma intact.

Effect of in ovo treatment with cyclophosphamide on lymphoid system in chicken

To develop a model for studies on the embryonic lymphoid cell maturation in chicken, cyclophosphamide (CY) was administered intravenously to chick embryos on three consecutive days, starting either on Day 12, 14, or 16 of incubation. To evaluate the effects of CY, survival and growth patterns, antibody-forming capacity, lymphocyte responses to phytohemagglutinin (PHA) and concanavalin A (Con A), and weight and microscopic morphology of bursa of Fabricius, thymus, and spleen were assessed. Like CY treatment in the newly hatched period, CY given in ovo caused a long-lasting and deep humoral immunodeficiency, when administered on Days 14–16 or on Days 16–18 of incubation. In microscopic examination at the age of 44 days, only rudimentary follicles and increased interfollicular connective tissue were found in the bursa. Thymus weights and lymphocyte responses to PHA and Con A were significantly reduced following in ovo treatment with CY. In reconstitution studies, this model of embryonic immunodeficiency will provide a possibility for a match according to the maturity of transplanted cells and of the microenvironment they require for further development.