Scanning electron microscopy of Echinostoma revolutum (Trematoda) during development in the chick embryo and the domestic chick

Fried B. and Fujino T. 1984. Scanning electron microscopy of Echinostoma revolutum (Trematoda) during development in the chick embryo and the domestic chick. International Journal for Parasitology14: 75–81. Scanning electron microscopy (SEM) was used to study the development of chemically excysted metacercariae of Echinostoma revolutum on the chick chorioallantois. SEM studies were also made on preovigerous adults of E. revolutum grown in the domestic chick. During worm development on the chorioallantois the tegument changed from smooth to granular and sensory papillae on the suckers became well-defined. As worms developed on the chorioallantois the cephalic collar spines became thicker and more curved and the tegumentary spines showed marked changes in shape, size and distribution on both ventral and dorsal aspects of the body. Changes in the surface ultrastructure of worms grown on the chorioallantois were essentially similar to those observed in preovigerous worms from chicks.     

Ultrastructural differentiation of glandular stomach (proventriculus) in chick embryo

Cytochemical characterization of mucosubstances of chick glanular stomach (proventriculus) changes from 15 days of development to postnatal and adult stages was studied. To corroborate these data cytochemical, ultrastructural and ultracytochemical study of chick embryo proventriculus from 7 to 20 days of development was performed. At the 7th day several layers of undifferentiated cells formed an epithelium which covered the walls of the glandular stomach. Mocosubstances were not found. Between the 9th and 5th day a single layer of cylindrical cells was encountered forming invaginations which originated deep glands. Three types of cells were separated from the above mentioned layer, dark, clear and undifferentiated. The dark cells had organelles which are involved in protein synthesis and the clear ones were rich in mitochondria. Argentaffine cells appeared at 15th day instead mucosubstances formed a thin coat on the epithelium at 9th day which increased at the end of development in the apical cytoplasm and gland cells. These observations demonstrate that proventriculus of chick embryo has ultrastructurally differentiated cells involved with enzymatic and hydrochloric acid secretion after the 9th day. These progressive events are correlated with the digestion process of yolk during embryogenesis. At the end of development the proventriculus has completely organized the glandular layer.     

Scanning electron microscopy (SEM) of the chick embryo primitive streak

The structure of the cells forming the primitive streak was examined by SEM in a series of embryos at Hamburger and Hamilton's stages 2--5. Specimens were prepared by stripping the endoderm from fresh embryos in New Culture and by fracturing whole fixed embryos along and at right angles to the primitive streak. At all stages of examination the SEM appearance of cells within the privitive streak was quite different from that of ectodermal, endodermal or mesodermal cells away from the streak. Streak cells were closely packed, lay with their long axes directed from ectoderm to endoderm and possessed many flat leaf-like processes. By contrast the ectoderm formed a columnar epithelium, the endoderm a flat epithelium and the mesoderm was a layer of loosely arrangedcells with long. thin processes. Within the streak SEM did not show any differences between cells that could identify them specifically as future endoderm or mesoderm cells. It was concluded that during gastrulation all the cells migrating through the primitive streak have the same appearance regardless of their eventual destination in the embryo. This structure may be attributable to the type of movement made by cells during invagination.     

Erythropoiesis in the developing chick embryo

The types of erythroid cells of chick embryos developing in ovo have been correlated with the hemoglobins of the embryos. Prior to 5 days, when primitive cells constitute the only erythroid cells, two hemoglobins can be resolved by polyacrylamide gel electrophoresis. The two adult hemoglobins and a minor hemoglobin found only in embryos and young chicks first appear simultaneously with initiation of definitive erythropoiesis.     

Vasculogenesis of the bursa cloacalis (bursa of Fabricius) of the chick embryo

Vasculogenesis of the bursa cloacalis (bursa of Fabricius) was examined in 10- to 21-day chick embryos and in chicks during the first 5 days post-hatching. The entire circulatory system was injected with India ink, and the bursae were then removed and either cleared for examination in toto or sectioned serially. The bursa was supplied by three pairs of extrinsic blood vessels. At 10 and 11 days of incubation, most intrinsic vessels were arranged in a superficial, hexagonal network. In regions of developing plicae, the hexagonal plexus extended into the core of each plica, forming middle plical vessels. The latter were interconnected across interplical areas by cross-connecting vessels. The middle plical vessels gave rise to small capillary offshoots, which soon increased in complexity, forming delicate loops. Branches extended from these loops through the subepithelial lamina propria to incipient epithelial buds by 12 days of incubation. All epithelial buds were supplied by at least one such branch, and similar branches extended to the basal aspect of the epithelium in areas where epithelial buds had not yet formed. This observation is consistent with the hypothesis that blood vessels induce formation of epithelial buds. At about 15 days of incubation, the cortex and medulla of each developing lymphatic follicle were defined clearly, and an intricate, web-like, capillary network coursed throughout the follicular cortex. The medulla appeared to be devoid of capillaries. The diameters of all intrinsic and extrinsic bursal blood vessels gradually increased throughout development. During post-hatching stages, the diameters of the extrinsic vessels continued to increase, whereas those of the intrinsic vessels were markedly decreased from late pre-hatching stages.     

Glucocorticoid-induced changes in poly(ADP-ribose) synthetase activity from chick embryo liver

Glucocorticoid treatment produced changes in poly(ADP-ribose) synthetase activities in subcellular fractions from chick embryo liver. The reduction of poly(ADP-ribose) synthetase activity in the nuclear fraction with this hormone treatment was accompanied by a concomitant increase in the postnuclear enzyme activity, particularly in the microsomal fraction. The reduced enzyme activities found in the nuclei were restored within a few days after the injection of 10 μg hydrocortisone into the fertilized egg incubated for 11 days. However, these restorations were not observed during the period tested, when over 100 μg of the hormone was given. The changes in the poly(ADP-ribose) formation by glucocorticoid treatment were due to alteration of a number of acceptor sites, but not to chain elongation, for the molecule. With regard to decrease in the nuclear poly(ADP-ribose) synthetase activity and increase in the postnuclear enzyme activity, possible mechanisms by which glucocorticoid induces these changes are discussed.     

Hypophysis and estradiol secretion in the chick embryo

Ovaries from 10- to 18-day-old chick embryos hypophysectomized by partial decapitation were cultured in vitro and their estradiol secretion was compared to that of ovaries from control embryos. The production of estradiol was not less in the decapitated than in the control embryos at 15-18 days, neither per ovary nor on the basis of ovarian weight. However, the difference was significant at 10-11 days. These results suggest that the hypophysis controls estradiol secretion by the chick embryo ovary in the early stages, but not in the later ones.     

Immunohistochemical detection of prolactin in the hypophysis of the chick and chick embryo

The appearance and localization of immunoreactive prolactin in the adenohypophysis of chick embryos and chickens was studied by antisera to the bovine prolactin. Immunoreactive prolactin was found in the chick embryos from the 15th day of development on using the methods of indirect immunofluorescence and of unlabelled antibodies with a complex PAP. In the chick embryos and in chickens during the first 10 days of life, the prolactin-containing cells were distributed, mainly, in the cephalic part of adenohypophysis; in the chickens, scarce cells were also found in the caudal part. These results suggest that in the domestic fowl the immunoreactive prolactin, similar by immunochemical specificity with the mammalian prolactin, is a late appearing marker of the adenohypophysis differentiation.     

Theophylline reduces poly(ADP-ribose) synthetase from chick embryo liver nuclei

The effect of theophylline on poly(ADP-ribosyl)ation was investigated. The poly(ADP-ribose) synthetase activity in vitro was markedly reduced in the liver nuclei prepared from theophylline-treated chick embryo. This reduction was not due to the enzyme inhibition by theophylline contamination in the nuclear fraction. The hydroxyapatite column chromatographic analysis of [³H]adenosine-labelled poly(ADP-ribose) molecules formed in vivo revealed that the in vivo formation of poly(ADP-ribose) molecules was also decreased by theophylline administration. The theophylline-induced reduction of poly(ADP-ribose) synthesis was not due to either low NAD levels or to a decrease in the chain length of the poly(ADP-ribose) molecule, rather this reduction was derived from a decrease in the number of poly(ADP-ribose) molecules. Possible mechanisms related to reduction of poly(ADP-ribose) synthesis in vivo are discussed.     

Glycosaminoglycans in early chick embryo

The developmental profile of glycosaminoglycans (GAGs) were examined by cellulose acetate electrophoresis and high performance liquid chromatography in the early chick embryo from late blastula (stage XIII+) to early somite developmental stages (stage HH7-9). Sulphated GAGs were present from the earliest stages. They were more abundant than the non-sulphated forms and showed stage-related changes. Chondroitin sulphate and especially dermatan sulphate appeared to be the predominant GAGs in embryos at stage XIII+. Dermatan sulphate was about three times as abundant as chondroitin sulphate at stage XII+. In contrast, embryos at the definitive streak stage (stage HH4) produced about twice as much chondroitin sulphate as dermatan sulphate. At the head process stage (stage HH5), the level of chondroitin sulphate was reduced and its relative content in the embryo was about the same as dermatan sulphate. Levels of dermatan sulphate were more than five times those of heparan sulphate from stage XIII through to stage HH5 and three times more at stage HH7-9. The 4- and 6- sulphation of chondroitin sulphate increased 14- and 10-fold respectively, from stage XIII+ to stage HH 7-9. The sulphation pattern of chondroitin sulphate had a delta(di)-4S:delta(di)-6S molar ratio ranging from 4 to 8:1 and a delta(di)-4S:delta(di)-OS molar ratio ranging from 9 to 16:1 and was developmentally regulated. Thus, chondroitin sulphate in the early chick embryo was sulphated predominately in the 4-position in all stages studied. The presence of both 4- and 6-sulphated disaccharides in chondroitin sulphate indicated that both 4 and 6 sulfotransferases were active in the early embryo. Hyaluronate and sulphated GAG content increased markedly at gastrulation when the first major cellular migrations and tissue interactions begin.     

Theophylline reduces poly (ADP-ribose) synthetase from chick embryo liver nuclei

The effect of theophylline on poly(ADP-ribosyl)ation was investigated. The poly(ADP-ribose) synthetase activity in vitro was markedly reduced in the liver nuclei prepared from theophylline-treated chick embryo. This reduction was not due to the enzyme inhibition by theophylline contamination in the nuclear fraction. The hydroxyapatite column chromatographic analysis of [3H]adenosine-labelled poly(ADP-ribose)molecules formed in vivo revealed that the in vivo formation of poly(ADP-ribose)molecules was also decreased by theophylline administration. The theophylline-induced reduction of poly(ADP-ribose) synthesis was not due to either low NAD levels or to a decrease in the chain length of the poly(ADP-ribose) molecule, rather this reduction was derived from a decrease in the number of poly(ADP-ribose) molecule. Possible mechanisms related to reduction of poly(ADP-ribose) synthesis in vivo are discussed.