Evidence for multiple cell surface chicken fetal-leukemic antigens (CFA) in the developing chick and other avian species.

Chicken fetal antigen (CFA), a membrane antigen present on fetal chicken red blood cells is lost with chicken development, and reappears on the red blood cells of leukemic chickens. Seven avian species were found to possess CFA. A species hierarchy comparing the quantitative expression of CFA has been established. The levels of CFA expression with development are compared in the chicken and Japanese quail. Specific adsorptions of R-anti-CFA with avian red blood cells revealed the existence of multiple CFAs. Four groups of antigenic determinants (CFA a,b,c,d) have been characterized and defined by their expression among avian species. Multiple CFA determinants are discussed with regard to possible membrane alterations and gene function.     

Cell surface acetylcholinesterase molecules on multinucleated myotubes are clustered over the nucleus of origin

Multinucleated skeletal muscle fibers are compartmentalized with respect to the expression and organization of several intracellular and cell surface proteins including acetylcholinesterase (AChE). Mosaic muscle fibers formed from homozygous myoblasts expressing two allelic variants of AChE preferentially translate and assemble the polypeptides in the vicinity of the nucleus encoding the mRNA (Rotundo, R. L. 1990. J. Cell Biol. 110:715-719). To determine whether the locally synthesized AChE molecules are targeted to specific regions of the myotube surface, primary quail myoblasts were mixed with mononucleated cells of the mouse muscle C2/C12 cell line and allowed to fuse, forming heterospecific mosaic myotubes. Cell surface enzyme was localized by immunofluorescence using an avian AChE-specific monoclonal antibody. HOECHST 33342 was used to distinguish between quail and mouse nuclei in myotubes. Over 80% of the quail nuclei exhibited clusters of cell surface AChE in mosaic quail-mouse myotubes, whereas only 4% of the mouse nuclei had adjacent quail AChE-positive regions of membrane, all of which were located next to a quail nucleus. In contrast, membrane proteins such as Na+/K+ ATPase, which are not restricted to specific regions of the myotube surface, are free to diffuse over the entire length of the fiber. These studies indicate that the AChE molecules expressed in multinucleated muscle fibers are preferentially transported and localized to regions of surface membrane overlying the nucleus of origin. This targeting could play an important role in establishing and maintaining specialized cell surface domains such as the neuromuscular and myotendinous junctions.     

Improved hatching for in vitro quail embryo culture using surrogate eggshell and artificial vessel

The establishment of avian embryonic culture is important both for the analysis of the developmental process and the establishment of transgenic chickens that produce useful biological materials in eggs. However, the hatchability of cultured embryos has been ∼50%. We identified that the low rate of hatchability of cultured embryos was caused by limited oxygen and calcium availability. In quail embryo culture using chicken eggshell as a culture vessel,viability in the middle stage of culture was improved and 30% of embryos were hatched by oxygen enrichment. Furthermore, hatchability increased to 80% by supplementation with calcium lactate in addition to oxygen aeration. In the present study, a fully artificial vessel for quail embryo culture was designed using a gas-permeable Teflon membrane. By the addition of fine eggshell powder and calcium lactate, quail embryos grew and developed normally, and 43% of embryos hatched. Although the hatchability was lower than that of cultures using a surrogate eggshell, we achieved in hatching an avian embryo using a fully artificial vessel.     

Development of the skeleton in Japanese quail embryos

In the research fields of experimental embryology, teratological testing, and developmental engineering in avian species, a knowledge of normal embryonic development is necessary so that research may be performed efficiently and precisely. A series of normal stages based on external appearance has been established in both chicken and quail embryos. Those based on skeletal features, however, have not been elucidated. The present study newly established a series of normal stages for the development of the Japanese quail embryo skeleton. This series is composed of 15 stages determined by observing the timing of chondrification and calcification of the skeleton every 24 h, from 3 to 17 days of incubation. Cartilage and ossified bones were stained blue and red with Alcian blue 8GX and alizarin red S, respectively. These skeletogenous stages of the Japanese quail embryo will be useful as a normal control not only in studies of experimental embryology, teratological testing, and developmental engineering, but also in the analysis of mutant embryos with skeletal abnormalities.     

An avian embryo culture system for embryogenesis using an artificial vessel: possible conservation benefits in the rescue and management of endangered avian species

The development of artificial means to conserve some endangered avian species seems urgently needed. We devised an eggshell‐less embryo culture system for embryogenesis using chicken or quail embryos as a model system. As a result, 41/56 (73%) chicken embryos and 52/62 (83%) quail embryos with their own thick albumen developed as usual for 72 and 55 hr, respectively, using a Teflon membrane (Milliwrap) and an egg‐shaped plastic vessel. Furthermore, this study revealed that the most suitable vessel for culture would have the same size as the intact original eggshell. This technique opens the way for the rescue of endangered avian species even if their eggshells are abnormal or cracked. This new procedure also facilitates investigation of developmental events in many unknown birds. We discuss various aspects of the embryological techniques applicable to endangered avian species. Zoo Biol 00:000–000, 2005. © 2005 Wiley‐Liss, Inc.     

Neurosteroid biosynthesis in the quail brain: a review

The brain traditionally has been considered to be a target site of peripheral steroid hormones. In contrast to this classical concept, new findings over the past decade have shown that the brain itself also has the capability of forming steroids de novo, the so-called "neurosteroids". De novo neurosteroidogenesis in the brain from cholesterol is a conserved property of vertebrates. Our studies using the quail, as an excellent animal model, have demonstrated that the avian brain possesses cytochrome P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/c17,20-lyase (P450(17alpha,lyase)), 17beta-HSD, etc., and produces pregnenolone, progesterone, 3beta, 5beta-tetrahydroprogesterone, androstenedione, testosterone and estradiol from cholesterol. However, the biosynthetic pathway of neurosteroids in the avian brain from cholesterol may be still incomplete, because we recently found that the quail brain actively produces 7alpha-hydroxypregnenolone, a previously undescribed avian neurosteroid. This paper summarize the advances made in our understanding of biosynthesis of neurosteroids in the avian brain.     

Proteolytic activity in the yolk sac membrane of quail eggs.

Extraembryonal degradation of yolk protein is necessary to provide the avian embryo with required free amino acids during early embryogenesis. Screening of proteolytic activity in different compartments of quail eggs revealed an increasing activity in the yolk sac membrane during the first week of embryogenesis. In this tissue, the occurrence of cathepsin B, a lysosomal cysteine proteinase, and cathepsin D, a lysosomal aspartic proteinase, has been described recently (Gerhartz et al., Comp Biochem Physiol, 118B:159-166, 1997). Determination of cathepsin B-like and cathepsin D-like proteolytic activity in the yolk sac membrane indicated a significant correlation between growth of the yolk sac membrane and proteolytic activity, shown by an almost constant specific activity. Both proteinases could be localized in the endodermal cells, which are in direct contact to the yolk. The concentration of proteinases in the endodermal cells appears to be almost unaltered in the investigated early stage of quail development, whereas the amount of endodermal cells increases rapidly, seen by a complicated folding of the yolk sac membrane. In the same cells quail cystatin, a potent inhibitor of quail cathepsin B (Ki 0.6 nM), has been localized at day 8 of embryonic development. Approximately at this stage of development, the quail embryo stops metabolizing yolk. In conclusion, it is strongly indicated that the amount of available free amino acids, produced by proteolytic degradation and supporting embryonic growth, is regulated by the growth of the yolk sac membrane.     

Avian alcohol dehydrogenase. Characterization of the quail enzyme, functional interpretations, and relationships to the different classes of mammalian alcohol dehydrogenase

The primary structure of the major quail liver alcohol dehydrogenase was determined. It is a long-chain, zinc-containing alcohol dehydrogenase of the type occurring also in mammals and hence allows judgement of the gene duplications giving rise to the classes of the human alcohol dehydrogenase system. The avian form is most closely related to the class I mammalian enzyme (72-75% residue identity), least related to class II (60% identity), and intermediately related to class III (64-65% identity). This pattern distinguishes the mammalian enzyme classes and separates classes I and II in particular. In addition to the generally larger similarities with class I, the avian enzyme exhibits certain residue patterns otherwise typical of the other classes, including an extra Trp residue, present in both class II and III but not in class I, with a corresponding increase in the UV absorbance. The avian enzyme further shows that a Gly residue at position 260 previously considered strictly conserved in alcohol dehydrogenases can be exchanged with Lys. However, zinc-binding residues, coenzyme-binding residues, and to a large extent substrate-binding residues are unchanged in the avian enzyme, suggesting its functional properties to be related to those of the class I mammalian alcohol dehydrogenases. In contrast, the areas of subunit interactions in the dimers differ substantially. These results show that (a) the vertebrate enzyme classes are of distant origin, (b) the submammalian enzyme exhibits partly mixed properties in relation to the classes, and (c) the three mammalian enzyme classes are not as equidistantly related as initially apparent but suggest origins from two sublevels.     

Marker rescue of endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase.

Endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase was studied, using a marker rescue assay to detect biological activity of subgenomic fragments of virus-related DNAs of uninfected avian cells. Recipient cultures of chicken embryo fibroblasts were treated with sonicated DNA fragments and were infected with a temperature-sensitive mutant of Rous sarcoma virus that encoded a thermolabile DNA polymerase. Wild-type progeny viruses were isolated by marker rescue with fragments of DNA of uninfected chicken, pheasant, quail, and turkey cells. The DNAs of these uninfected avian cells, therefore, appeared to contain endogenous genetic information related to the avian leukosis virus DNA polymerase gene.     

Autoradiographic evidence for the sliding of the upper layer over the basement membrane in chicken blastoderms during gastrulation.

An upper layer (epiblast) fragment taken laterally from the Anlage fields of neural plate or chordamesoderm of a quail blastoderm, labelled with 3H-glucosamine, was grafted isotopically (in a similar region), isochronically (at the similar stage of development) and isotropically (with the same caudocranial and dorsoventral polarity) in the epiblast of a mesoblast free area of a chicken blastoderm (St 4-5 Vakaet, 1970: full grown primitive streak). On the autoradiographs of the sections through such cultured blastoderms with fully integrated quail grafts, we observed a labelling of the basement membrane laterally and slightly cranially from the labelled graft in its final position. Since only the epiblast and its basement membrane are involved, the pattern of the observed labelling indicates that the grafted and integrated quail epiblast fragment glides in toto over the mediocaudally localized basement membrane, leaving behind a track of radioactivity. Sliding of whole groups of epiblast cells over the basement membrane seems thus to be a normal phenomenon during avian gastrulation.     

Uptake of Yolk Very Low Density Lipoprotein by Chicken and Quail Embryos Is Not Mediated by a Homologue of the Oocyte Vitellogenesis Receptor

In chicken (Gallus domesticus) embryos, a limited amount of yolk engulfment occurs via coated invaginations at the yolk sac membrane apical surface. Because the presence of these so-called “coated pits” is associated with receptor-mediated endocytosis, the purpose of the present study was to demonstrate the existence on the yolk sac membrane of receptor sites for the interaction with very low density lipoprotein (VLDL), the major component of egg yolk. Ligand blotting experiments revealed the presence of a VLDL-binding protein (M_r ∼95 kDa) in yolk sac membranes of both chicken and Japanese quail (Coturnix coturnix japonica) embryos 8 days of age and older. However, these VLDL-binding proteins were present in very low abundance relative to that of another apolipoprotein B receptor that is found in the plasma membrane of chicken and quail oocytes (the so-called oocyte vitellogenesis receptor [OVR]; M_r 95 kDa). Furthermore, no signals were detected when chicken and quail yolk sac membrane proteins were probed with a rabbit polyclonal antibody raised against the 14 C-terminal amino acids of the chicken OVR. It was concluded that chicken and quail yolk sac membrane VLDL-binding proteins were structurally different from the chicken OVR and that receptor-mediated endocytosis plays a minor role in the uptake of yolk VLDL by developing avian embryos.     

Initial expression of the genes for fructose 1,6-diphosphatase,malic enzyme,and aspartate aminotransferase in Japanese quail and chicken-quail hybrid embryos

The initial appearance of a number of enzymes involved in gluconeogenesis was investigated in the early embryogenesis of the Japanese quail (Coturnix coturnix japonica), the domestic chicken (Gallus gallus domesticus), and chicken-quail hybrids. Starch gel electrophoresis and enzyme-specific stains revealed genetic differences between muscle and liver fructose 1,6-diphosphatase (FDPase) as well as malic enzyme (ME) and mitochondrial aspartate aminotransferase (AAT) isozymes of the two species. ME and AAT were present in unfertilized unincubated eggs, indicating maternal storage of these enzymes. The initial expression of the paternally inherited genes in the hybrid occurred before oviposition in the case of ME, and between 12 and 18 hr incubation in the case of AAT. Initial expression of both parental sets of genes for FDPase occurred synchronously between 16 and 24 hr in the hybrid, corresponding to the time of initial appearance of this enzyme in the quail and chicken. Glucose 6-phosphate administration at 0 hr was found to cause no prevention or delay of initial enzyme activation. These results are interpreted in terms of early patterns of enzyme activation regulation and nutrition in the avian embryo.     

A new marker for identifying quail cells in embryonic avian chimeras: a quail-specific antiserum

The characterization of cell behavior in quail chick chimeras has greatly increased our knowledge of the ontogeny of embryonic cell populations and the role of cell-cell interactions in development. We sought to extend the value of avian chimeras by producing a marker that would recognize cell surface components and that could be used instead of the traditional nuclear marker to identify quail cells within chimeras. We describe here a quail-specific antiserum produced by injecting chickens with a membrane fraction of 6-10-day quail embryos. By use of peroxidase coupling of a second antibody, serum reactivity was tested in tissue sections of normal quail and chick embryos and of somitic mesoderm and neural tube chimeras. The primary time period examined was 6-10 days of development. At these stages, the antiserum recognizes only quail cells and stains both plasma membrane-associated and cytoplasmic cell components. The latter characteristics allow the identification of quail axons in chimeras and facilitate visualization of quail cells at low magnification. We show that antiserum staining can also be used to identify quail cells in culture and can be combined with orthograde HRP labeling of neurons.     

Species-related variations in tissue antioxidant status--I. Differences in antioxidant enzyme profiles.

1. Antioxidant enzyme activity profiles in red cells of man, rabbit, quail, pig and rat have been investigated and found to exhibit striking differences. 2. No direct correlations between activities of "functionally coupled" enzymes (superoxide dismutase/catalase and glutathione peroxidase/glutathione reductase) were apparent, suggesting their independent regulation. 3. However, activities of red cell catalase and glutathione peroxidase in the various species studied were inversely correlated. 4. This was most evident in quail red cells, which showed negligible catalase activity but the highest levels of glutathione peroxidase of all the species examined. 5. A significant positive correlation between catalase and glutathione reductase activities was also demonstrated. 6. This may be relevant to the suggestion that the binding of NADPH to catalase may serve to decrease the intracellular inactivation of this reducing cofactor which may be limiting in the glutathione reductase reaction. 7. Basal levels of glutathione, which have been claimed to be limiting for the glutathione peroxidase reaction, were found to correlate positively with the activity of this enzyme in red cells. 8. Myocardial tissues also exhibited species-related differences in antioxidant enzyme profiles but these did not bear any obvious relationship to patterns observed in the corresponding red cells.     

Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail

Several continuous tissue culture cell lines were established from methylcholanthrene-induced fibrosarcomas of Japanese quail. The lines consist either of fibroblastic elements, round refractile cells or polygonal cells. They show transformed characteristics in agar colony formation and hexose uptake, and most are tumorigenic. Their cloning efficiency in plastic dishes is not increased over that of normal quail embryo fibroblasts. The quail tumor cell lines do not produce endogenous avian oncoviruses and fail to complement the Bryan high titer strain of Rous sarcoma virus; those tested lack the p27 protein of avian oncoviruses. Most of the cell lines are susceptible to subgroup A avian sarcoma viruses, but are relatively resistant to viruses of subgroups C, E and F as compared to normal quail embryo fibroblasts.     

Origin of 33 kDa protein of vitelline membrane of quail egg: Immunological studies

The inner layer of vitelline membrane is an investment of avian ovum at the time of ovulation, but its formation is poorly understood. In order to elucidate the origin of the inner layer of vitelline membrane, a 33 kDa protein, one of the components of the inner layer, was purified from quail eggs and polyclonal antibody was raised against this protein. The tissue distribution of protein interacted with the antibody was studied by Western blotting technique. No immunoreactive component could be observed in extracts of liver, kidney, heart, lung, small intestine, brain, infundibulum, albumen-secreting region of oviduct, uterus, and wall of small white follicles. The intensive band was detected in the granulosa layer, which was isolated from the large preovulatory follicles as a monolayer of granulosa cells sandwiched between the inner layer of vitelline membrane and the basal lamina. The granulosa cells isolated from the granulosa layer also reacted with this antibody. Theca layer had no immunoreactive components. The position of the band of the 33 kDa protein on SDS-PAGE was sifted to higher molecular weight in follicular tissues as compared with that in the laid eggs, indicating that the structural change of the protein occurs after ovulation. These studies indicate that the material reactive to the antibody raised against a 33 kDa protein of quail vitelline membrane is synthesized by the granulosa cells.