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The Drosophila female sterile mutation twin peaks is a novel allele of tramtrack and reveals a requirement for Ttk69 in epithelial morphogenesis.

The Drosophila gene tramtrack (ttk) encodes two transcriptional repressors, Ttk69 and Ttk88, which are required for normal embryogenesis and imaginal disc development. Here, we characterize a novel female sterile allele of tramtrack called twin peaks (ttk(twk)) that, unlike othertramtrack alleles, has no effect on viability and produces no obvious morphological defects, except during oogenesis. Females homozygous for twin peaks produce small eggs with thin eggshells and short dorsal respiratory appendages. Complementation analyses, immunolocalization, and rescue data demonstrate that these defects are due to loss of Ttk69, which is expressed in the follicle cells and is required for normal chorion production and dorsal follicle-cell migration. Analyses of phenotypes produced by mutations in other loci that regulate eggshell synthesis suggest that the chorion production and follicle-cell migration defects are independent. We present evidence that twin peaks disrupts a promoter or promoters required for late-stage follicle-cell expression of Ttk69. We hypothesize that loss of Ttk69 in all follicle cells disrupts chorion gene expression and lack of function in dorsal anterior follicle cells inhibits morphogenetic changes required for elongating the dorsal appendages.

Alleles↗

Expression of calcium-sensing receptor gene by avian parathyroid gland in vivo: relationship to plasma calcium.

Calcium-sensing receptor (CaR) gene expression and parathyroid hormone (PTH) content were evaluated in situ in chicken parathyroid glands (PG) in relation to changes in plasma calcium. The CaR gene is expressed by the parathyroid chief cells, the same cells that store and secrete PTH. An increase in plasma calcium, achieved by repletion of vitamin D-deficient chicks with a normal diet, by PTH injection, or during eggshell formation, increased the expression of the CaR gene. Low plasma calcium concentration in vitamin D-deficient chicks or in layers, before or after eggshell formation, was associated with decrease in CaR gene expression in the PG. The level of CaR gene expression was inversely correlated with the PTH content of the PG. The results of this study demonstrate for the first time that, in contrast to mammals, the CaR gene expression in the PG of the chicken is inversely associated with changes in plasma calcium.

Animals↗

The formation of the functional chorion structure of Drosophila virilis involves inercalation of the "middle" and "late" major chorion proteins: a general model for chorion assembly in Drosophilidae.

We have shown by in vitro development of staged follicles in the presence of tritiated proline, as well as by immunoblot analysis, that the Dvs19 and DVS15 chorion proteins of Drosophila virilis are synthesized by the follicular epithelium and are incorporated in the chorion at the terminal stages of choriogenesis. Light microscopy immunolocalization has revealed that these two proteins represent structural components of the endochorion in all the specialized regions of the eggshell, while, contrary to their temporal pattern of secretion, electron microscopic immunolocalization has shown that these two proteins participate in endochorionic structures formed before the onset of their synthesis, thus suggesting an intercalation process during chorion formation. Furthermore, double immunolocalizations demonstrated that cosecreted proteins do not coexist in all secretory vesicles of the follicle cells, indicating that probably the final association of these chorionic components occurs extracellularly. On the basis of these data and those presented in the accompanying paper [Trougakos, I.P., and Margaritis, L.H. (1998) Immunolocalization of the temporally "early" secreted major structural chorion proteins, Dvs38 and Dvs36, in the eggshell layers and regions of Drosophila virilis, J. Struct. Biol. 123, 000-000.], a model for chorion assembly in Drosophilidae is proposed.

Animals↗

Secondary structure of chorion proteins of the teleostean fish Dentex dentex by ATR FT-IR and FT-Raman spectroscopy.

FT-Raman spectroscopy and ATR-IR spectroscopy were applied to study the secondary structure of the eggshell (chorion) proteins of the teleostean fish Dentex dentex. Raman and IR spectra clearly indicate an abundance of antiparallel beta-pleated sheet conformation in chorion proteins. This finding is further supported by analysis of the vibrational data by regression techniques and deconvolution procedures. Thus, the common morphological characteristics of D. dentex, Salmo gairdneri, and other teleostean fish chorions may be explained on the basis of common secondary structure features of their constituent proteins. A detailed understanding of the interactions that dictate the self-assembly of fish chorion proteins to form the fish eggshell awaits determination of amino acid sequences.

Animals↗

Transgenic birds for the production of recombinant proteins.

Transgenic birds were expected to be an excellent transgenic bioreactor for the production of recombinant pharmaceutical proteins. However, the only successful transgenic bioreactors have been based on mammals. We have developed two key techniques for obtaining transgenic birds. For bird embryo culture, we identified that the low rate of hatchability of cultured embryos is caused by limited oxygen and calcium availability. In quail embryo culture using a chicken eggshell as a culture vessel, hatchability increased to 80% by the supplement of calcium lactate in addition to oxygen aeration. A fully artificial vessel for quail embryo culture using a gas-permeable Teflon membrane was also designed. Although the hatchability was lower than that of cultures using a surrogate eggshell, we succeeded in hatching of bird embryos using a fully artificial vessel. For transgene introduction, a replication-defective pantropic retroviral vector based on Moloney murine leukemia virus (MoMLV) pseudotyped with vesicular stomatitis virus G protein (VSV-G) was injected to laid embryos at the blastodermal stage, and the embryos were hatched in vitro to generate G0 birds. The viral vector sequence was detected in the tissues of all G0 birds. The germ-line transmission efficiency was more than 80%. Plural copies of the transgene were inserted into the genome of G1 transgenic progeny.

Animals↗

Patterns, trends, and toxicological significance of chlorinated hydrocarbon and mercury contaminants in bald eagle eggs from the Pacific coast of Canada, 1990-1994.

Bald eagle (Haliaeetus leucocephalus) eggs were collected during incubation, 1990-1992, from 16 nests near three bleached-kraft pulp mills, from six nests in the Fraser River estuary and from seven nests at a reference site on the Pacific coast of Canada. Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were present in all eggs in a qualitatively similar pattern among sites. Mean concentrations of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) were significantly higher in eggs collected from near three kraft pulp mill sites in the Strait of Georgia (44, 45, 84 ng/kg) than from the reference area in Johnstone Strait (15 ng/kg). There were few differences among sites in mean organochlorine pesticide levels, indicating the diffuse distribution of those chemicals and the domination of atmospheric inputs. Mean concentrations of total polychlorinated biphenyls (PCBs) were highest in eggs from the Strait of Georgia (4.86 mg/kg) and the PCB congener pattern was significantly different between that area and both the lower Fraser valley and Johnstone Strait. Mean mercury concentrations, which were mainly methyl-mercury, were significantly higher in eggs collected from the lower Fraser Valley (0.258 mg/kg) and Johnstone Strait (0.294 mg/kg) compared to the Strait of Georgia (0.188 mg/kg). Individual and regional variation in concentrations of organochlorine pesticides, PCBs and mercury in eagle eggs were thought to be influenced mainly by dietary differences. Toxicologically, in 1990, mean TCDD-toxic equivalents (TEQs) in bald eagle eggs were about two-fold greater than a lowest-observed-effect level, suggested elsewhere for this species, of 210 ng/kg TEQs. In the Strait of Georgia, PCCDs and PCDFs made a greater contribution to TEQs than non-ortho and mono-ortho PCBs, whereas the reverse was true for eggs outside the strait. Mean eggshell thickness was less than the pre-1947 value at all sites, although there was no significant relationship between eggshell thickness and DDE concentrations. Levels of other organochlorine pesticides and mercury were below those considered to be toxic.

Analysis of Variance↗

Water loss from eggs of domestic fowl and calcium status of hatchlings.

Water balance in eggs of domestic fowl was manipulated by drilling holes (each 3 mm in diameter) through the calcareous layer into the air cell on day 10 of incubation. Water loss between days 0 and 18 averaged 6 g for eggs in the control group (no hole) but increased to 8, 12, and 15 g for eggs with 1, 2, or 3 holes, respectively. Hatching success was 79-87% for eggs with 0-2 holes through the eggshell but only 43% for eggs with three holes. Live mass of hatchlings declined as the number of holes drilled in the eggshell increased, but dry mass of carcasses was unaffected by the treatments. The quantity of Ca2+, Mg2+, and phosphorus in residual yolks and yolk-free carcasses of hatchlings was not influenced by the amount of water lost from eggs. Plasma Ca2+ and Mg2+ were elevated in hatchlings from eggs with high rates of water loss. The inability to regulate plasma Ca2+ may be a negative consequence of excessive water loss and could contribute to increased mortality of embryos.

Animals↗

Formation by the uterus of a peripheral layer of the sheath in microfilariae of Litomosoides carinii and Brugia malayi.

The eggshells of young developmental stages in the uterus are rather thin and homogenous. In the brezel stage of Brugia malayi they are 35 nm thick and 20 nm in Litomosoides carinii. In young developmental stages up to brezel stages the eggshells bind the lectins WGA, DBA and PNA labelled with colloidal gold. This shows that GlcNAc, GalNAc and Gal residues are present at the surface of the sheath. In intrauterine microfilariae of B. malayi the original sheath is reduced to a thickness of 7 nm. It is reinforced by secretions from a specialized area of the epithelium of the uterus which do not appear as a homogeneous layer but look like a string of pearls. This layer may be called the "uterine layer". It has a thickness of 40-80 nm. In the microfilaria of L. carinii, the thickness of the original sheath is reduced to 2-3 nm and the uterine layer has a thickness of 7 nm. The uterine layer does not react with any of the lectins, which shows that the surface lacks N-acetylglucosamine, N-acetylgalactosamine and galactose residues. The uterine layer appears to be an ancestral (plesiomorphic) feature which is present in free-living nematodes and the highly specialized bloodforms of filariae. The uterine layer seems to protect and disguise the original sheath against the immune reactions of the host.

Animals↗

Immunoelectron microscopical localization of a circulating antigen in the excretory system of Schistosoma mansoni. Ultrastructural localization studies of the excretory system of S. mansoni.

In this study the excretory system of Schistosoma mansoni was ultrastructurally examined with a recently described monoclonal antibody (mAb) against a circulating antigen. In previous studies this mAb was found to have affinity for the excretory system. Strong immunoreactivity was found on the flagella of the flame cells and of the collecting ducts throughout the worm. The eggshell and the space between the miracidium and the eggshell showed strong reactivity with a declining gradient towards the exterior, suggesting a secretion process. In cercariae, immunoreactivity was restricted to the tegument, whereas in schistosomula the labeling pattern resembled that of the adult worm, demonstrating positive reactivity of the flame cells and no immunostaining of the tegument. This stage-dependent differential expression of different antigens in the excretory system and in the tegument could suggest a maturation process of the excretory system.

Animals↗

Lead in tissues of mallard ducks dosed with two types of lead shot.

Mallard ducks (Anas platyrhynchos) were sacrificed one month after ingesting one number 4 all-lead shot or one number 4 lead-iron shot. Livers, kidneys, blood, wingbones, and eggs were analyzed for lead by atomic absorption. Necropsy of sacrificed ducks failed to reveal any of the tissue lesions usually associated with lead poisoning in waterfowl. Lead levels in ducks given all-lead shot averaged about twice those in ducks given lead-iron shot, reflecting the amount of lead in the two types of shot. Lead in the blood of ducks dosed with all-lead shot averaged 0.64 ppm, and 0.28 ppm in ducks given lead-iron shot. Lead residues in livers and kidneys of females given all-lead shot were significantly higher than in males. In both dosed groups, lead levels in wingbones of females were about 10 times those in males, and were significantly correlated with the number of eggs laid after dosage. Lead levels in contents and shells of eggs laid by hens dosed with all-lead shot were about twice those in eggs laid by hens dosed with lead-iron shot. Eggshells were found to best reflect levels of lead in the blood. Our results indicate that mallards maintained on a balanced diet and dosed with one lead shot may not accumulate extremely high lead levels in the liver and kidney. However, extremely high lead deposition may result in the bone of laying hens after ingesting sublethal amounts of lead shot as a result of mobilization of calcium from the bone during eggshell formation.

Animals↗

Effect of the substratum on the growth of CFU-c in continuous marrow culture.

The effect of the substratum on the maintenance of granulocyte-macrophage progenitors (CFU-c) was studied in continuous mouse marrow culture. Glass and decalcified eggshell membrane were found preferable to plastic, providing better adherence of stromal cells. Eggshell membrane consists of a meshwork of branching fibers suitable for the adherence of stromal cells. The glass surface with its high surface pH may provide an electrostatic attraction for the negatively charged surface of stromal cells.

Animals↗

The effect of mirex on reproduction of Japanesequail and on characteristics of eggs from Japanese quail and chickens.

Laying White Leghorn chickens were fed mirex at 0, 5, 10, 20, 40, 80, and 160 ppm for 12 weeks, and laying Japanese quail were fed mirex at 0, 5, 40, and 80 ppm for 12weeks. The data suggest that dietary mirex at these levels did not affect egg production, egg weight, shell thickness, shell calcium, the proportion of broken eggs, or the proportion of soft-shelled eggs of either chickens or quail. Statistical significance (P greater than 0.05) associated with dietary mirex was detected in the analysis of eggshell weight for chickens; however, because a dose-response relationship of shell weight vs. level of mirex fed was not evident, this observation was attributed to chance. The data suggest that dietary mirex did not affect eggshell weight, fertility, or hatchability of quail. Mirex accumulation in eggs and carcasses of both species was proportional to dose and was slightly higher in quail than in chickens.

Analysis of Variance↗

Inductive specificity of mineralized bone matrix in ectopic osteoclast differentiation.

The present report describes the first in a series of studies designed to identify the factor or factors responsible for eliciting osteoclast differentiation. Particles of mineralized and demineralized bone, hydroxyapatite (HA), and eggshell were grafted onto the chorioallantoic membranes (CAMs) of chick embryos. After 3 of 6 days, portions of CAMs with associated grafts were harvested, processed for light and electron microscopy, and examined for the presence of multinucleated giant cells with the morphological characteristics of osteoclasts. Light microscopic examination revealed that, within only 3 days, many particles of mineralized materials had become surrounded or engulfed by multinucleated giant cells. Ultrastructurally, all such cells possessed a vacuolated and mitochondria-enriched cytoplasm, but they differed in the nature of the contacts formed at the cell-particle interface. With eggshell, the cells developed filopodia but lacked clear zones and ruffled membranes. With HA, clear zones were evident but cytoplasmic extensions and membrane ruffling were absent. Implants of mineralized bone, however, elicited the formation of giant cells with prominent clear zones and ruffling of the plasma membrane like that observed in bonafide osteoclasts. In contrast, grafts of demineralized bone did not evoke giant cell formation but rather recruited two cell types morphologically akin to either fibroblasts or macrophages. We conclude that the factor(s) responsible for osteoclast differentiation resides specifically within bone matrix and is intimately associated with the mineral phase. Further, in response to such a factor(s), osteoclast differentiation can occur ectopically, outside of the developing vertebrate body.

Allantois↗

Avian transgenerational reproductive toxicity test with in ovo exposure.

Ecological risk assessment of environmental pollutants requires effective laboratory assays and extrapolation of the resultant data to wild species. Because avian reproductive disorder and accumulation of persistent compounds in wild birds and their eggs have long been observed in polluted regions, we have developed an assay for investigating whether pollutants accumulated in eggs impair the reproduction of the exposed birds and the survival of the next generation using the Japanese quail. A typical estrogenic compound, diethylstilbestrol (DES), dissolved in olive oil was injected into the air-chamber of fertilized eggs on day 10 of incubation. After sexual maturation of hatched chicks, we mated pairs of male and female quails following an observation period of egg production and collected their eggs. The collected eggs were incubated and checked for the fertility and hatchability, and then the hatchlings were raised and observed in growth for 3 weeks. A dosage of 5 ng/g per egg of DES caused eggshell thinning in eggs laid by exposed females and reduction in eggshell strength. DES also induced shortening of the left oviduct and unexpected development of the right oviduct, while testis weight was reduced symmetrically. The ability of quail pairs to produce offspring was significantly diminished by exposure of females to DES independently of exposure of males, which mainly arose from production of abnormal and inviable eggs. Fertility of normal-shelled eggs and hatchability of fertilized eggs were unchanged regardless of treatments. External morphological abnormalities, which were mostly unopened toes of the foot, were frequently observed in hatchlings from exposed males independently of exposure of females. Additionally, we attempted to extrapolate the experimental results to the northern bobwhite and to predict population trends for quails in a polluted habitat using a population projection model composed of a combination of a Leslie matrix and the logistic equation. In the event of accumulation of an estrogenic compound equivalent to a dosage of 5 ng/g DES in quail eggs, the average population size was predicted to decrease by 20.2% after 1 year, to approximately half after 4 years, and to a fifth after 14 years. When observed weakening of individuals and the risk of egg breakage are taken into consideration, the decline in population was further accelerated. The proposed assay appears to be suitable not only for assessing adverse effects of chemicals on avian reproduction but for population projection of affected wild birds.

Abnormalities, Drug-Induced↗

Disrupted carbonic anhydrase distribution in the avian shell gland following in ovo exposure to estrogen.

Eggshell thinning among wild birds has been an environmental concern for almost half a century and the underlying mechanisms are still not fully understood. Previously we showed that exposure of quail embryos to ethynylestradiol (EE2) caused disorganization of the tubular glands in the shell gland of adult birds. In this study, we have examined the effect of in ovo exposure to EE2 on carbonic anhydrase (CA) localization, especially in the shell gland, because CA is required for shell formation. In the control birds, CA was localized in the cell membranes of the tubular gland cells of the shell gland, whereas the surface epithelium was always devoid of CA. In ovo treatment with 20ng EE2/g egg resulted in a loss of CA activity in the tubular glands while the surface epithelium showed strong induction of both membrane bound and cytoplasmic CA activity in 49+/-1% of the cells. The dose 2ng EE2/g egg resulted in partial loss of tubular gland CA and strong induction of CA activity in 2.5+/-0.5% of the surface epithelial cells and weaker induction in 22+/-2% of the epithelial cells. In conclusion, this study shows that embryonic exposure to a xenoestrogen disrupts CA distribution in the adult shell gland. We propose that eggshell thinning in avian wildlife could reflect a functional malformation in the shell gland, already induced by xenoestrogen during embryonic development rather than being caused solely by exposure of the adult bird.

Animals↗

Heavy-metal concentrations in female laying great tits (Parus major) and their clutches.

Egg laying may be an additional excretory pathway for heavy metals available to laying female birds. In this study, we examined the relationships between tissue concentrations (both internal organs and feathers) of 10 breeding female great tits (Parus major) and the concentrations in their eggs. We also investigated differences in metal concentrations (Ag, Al, As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Zn) among eggs as a result of the laying order of the eggs. Heavy-metal concentrations in internal tissues were highest in bone (Cr, Ni, Pb, Zn), kidney (Cd), liver (Cu), blood (Al, As, Co, Hg), and intestine (Ag, Mn). In the egg contents, relatively high concentrations of the essential elements Cr, Co, Cu, Mn, Ni, and Zn were found. In eggshells, concentrations of Ag, Al, As, Co, Cr, and Ni were high compared with internal tissue concentrations. Metal concentrations in the egg contents and eggshells were poorly correlated with metal concentrations in internal tissues and feathers; significant positive correlations were found only for cadmium and lead. We found few significant differences in metal concentrations among eggs, and none were according to the laying order of the eggs. The egg volume tended to decrease significantly as egg laying progressed. Our results seem to suggest that no special sampling strategies (e.g., sampling the first egg or pooling eggs from one clutch) are required for most metals.

Animals↗

Heavy metal distribution in tissues and eggs of Chinese alligator (Alligator sinensis).

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. Concentrations of heavy metals (As, Fe, Mn, Cu, Pb, Cd, Cr, Zn, and Hg) were examined in the tissues of Chinese alligators to elucidate the background distribution of these metals in the alligator body. Generally, within the body compartments, metal concentrations were high in liver, kidney, and heart, and low in pancreas and gonad. Study of heavy metal levels in the feces and eggs of Chinese alligator suggested that Chinese alligators could reduce body burden of toxic substances by excreting them to feces and/or sequestrating them into eggs to a lesser extent. In addition, to test whether eggshell or egg membrane could be used as surrogates to measure heavy metal load in egg contents, the correlation of metal concentrations between three egg compartments was determined. Of the nine elements analyzed, concentrations of iron, copper, and zinc in the shell membrane were highly correlated with the levels in egg contents, whereas no metal was significantly correlated between eggshell and egg contents. This suggested that the shell membrane could be a useful bioindicator for Fe, Cu, and Zn contaminations in the eggs of Chinese alligator. In a comparison of metal contents in the eggs of individuals from the Anhui captive population, the wild population in Anhui Province, and those of the Changxing captive population, higher Cu, Zn, and Cd levels and a lower Pb level were found in the Changxing individuals, indicative of specific pollutants in different areas. In addition, the majority of metal elements in the muscles of Chinese alligators and American alligators are in the same ranges. As a result of the data found in the eggs of the two alligator species, the Chinese alligators may be exposed to a higher level of metal pollutants. The study provided measurement of the heavy metal distribution in the endangered Chinese alligator for the first time and could serve as the background for the monitoring of possible heavy metal contaminations in the alligator habitats.

Alligators and Crocodiles↗

Effect of various copper supplements to feed of laying hens on cu content in eggs, liver, excreta, soil, and herbage.

Copper is often added to poultry diets as an antimicrobial agent at doses greatly exceeding the nutritional requirement. In this study, the basal diet of laying hens containing 9.2 mg Cu/kg was supplemented with CuSO(4) x 5H(2)O at 0, 25, 65, 115, and 240 mg Cu/kg dry matter (DM). At Cu dietary concentration just below the level permitted by the European Union (35 mg/kg), the Cu content in the egg yolk was significantly (p < 0.05) increased by 26%, and nonsignificantly by 4.1, 9.1, and 7.9% in the egg white, eggshell, and liver, respectively. When Cu concentration in the diet was doubled, the effect of Cu on Cu content in eggshell and liver was statistically significant as well. In no liver sample was the hygienic limit of Cu content (80 mg/kg) exceeded. Supplementation of diets with Cu increased Cu concentration in excreta linearly from 25.3 to 396.8 mg/kg DM. Dried excreta were used for fertilization of grassland at 21 g N/m(2). Three months later, soil and herbage were sampled and analyzed. The Cu concentration in soil increased from 25.3 to only 46.4 mg/kg DM when dietary Cu concentration rose from 9.2 to 243.7 mg Cu/kg DM. Corresponding Cu concentrations in herbage were 6.8 and 19.2 mg/kg DM. It can be concluded that the deposition of Cu in eggs and liver of hens fed Cu-supplemented diets does not represent a hygienic risk. The accumulation of Cu in soil fertilized with excreta of Cu-fed hens and in herbage was limited.

Animals↗