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Progesterone stimulates prostaglandin synthesis in eggshell gland mucosa of estrogen-primed chickens.

1. Prostaglandins may be involved in calcium translocation in the avian shell gland, since indomethacin, administered at the beginning of shell formation, reduces eggshell thickness as well as 45Ca-uptake and prostaglandin synthesis by a homogenate of eggshell gland mucosa. 2. The stimulus for calcium transport in the shell gland during shell formation remains unknown. 3. The present study was undertaken to investigate the effects of progesterone on prostaglandin formation by the eggshell gland mucosa of the domestic fowl. 4. Progesterone significantly stimulated synthesis of PGF2 alpha, PGE2 and TXB2 by eggshell gland mucosa homogenate. 5. Progesterone treatment also induced the synthesis of the biotin-binding protein, avidin. 6. A microsomal fraction prepared from the eggshell gland mucosa had a high affinity for binding PGE2. 7. Progesterone treatment reduced the KD value of this binding without affecting the maximal number of binding sites. 8. Progesterone did not change the total calcium content of shell gland mucosa. 9. The role progesterone plays in prostaglandin formation and calcium transport in the eggshell gland mucosa is discussed.

Animals

The fabrication and collagenous substructure of the eggshell membrane in the isthmus of the hen oviduct.

The eggshell of the chicken consists of a bi-layered shell membrane overlaid with a thick, calcified shell matrix. The shell membranes and matrix are deposited onto the egg as it passes through the oviduct. To assess the temporal and spatial aspects of the fabrication of type X collagen within the eggshell extracellular matrix, the immunohistochemical localization of type X collagen was studied in three regions of the hen oviduct (magnum, isthmus and uterus), in the membranes of uncalcified eggshells obtained from the oviduct prior to mineral deposition and in eggshell membrane and calcified eggshell matrix. Additionally, immunohistochemical localization of type I and III collagens was done in order to determine possible co-localization of collagen types or to define tissue compartments. None of the collagen epitopes assayed was found in the shell matrix. Type X collagen epitope was immunohistochemically localized only to the epithelial cell layer lining the isthmus region of the oviduct and in the shell membranes of both uncalcified and calcified eggshells. Antitype III collagen monoclonal antibody delineated the inter-tubular gland connective tissue of the oviduct and was negative in the shell layers under conditions which gave strong connective tissue reactivity. Type I collagen epitope was exposed after pepsin treatment of the tissue and co-localizes with the distribution of type III collagen. Type I collagen co-localized with type X collagen in the shell membranes of uncalcified shells. The type I collagen epitope was reactive in the shell membrane of the uncalcified shells, but could only be detected in calcified shells following pepsin digestion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Presclerotized eggshell protein from the liver fluke Fasciola hepatica.

Trematode parasites protect their eggs with a tough tanned eggshell. Eggshell precursor proteins are synthesized and stockpiled within the extensive vitellaria of the animal. A major eggshell precursor protein with an apparent molecular weight of 31,000 and pI of 7.4 was isolated from the vitellaria of Fasciola hepatica. This protein, which represents 6-7% of the total protein in mature Fasciola, is unique in containing rather high levels of the amino acid 3,4-dihydroxyphenylalanine (DOPA), i.e., 110 residues per 1000. Other prominent amino acids are glycine, aspartic acid, and lysine. A prominent DOPA-containing tryptic peptide derived from eggshell precursor protein has the sequence Gly-Gly-Gly-DOPA-Gly-Gly-DOPA-Gly-Lys. DOPA residues disappear during the maturation of the eggshell and by treatment in vitro with mushroom polyphenol oxidase. This disappearance may be related to the formation of cross-links in the eggshell protein.

Amino Acid Sequence

Effects of dietary minerals on acid-base balance and eggshell quality in chickens.

Three experiments were conducted in hens to determine the effects of high levels of dietary phosphorus (P, 1-1.5%), chloride (Cl, 0.8%) or both on acid-base balance and eggshell quality and to consider whether any adverse effects on eggshell quality can be attributed to alteration of blood acid-base balance. Eggshell quality and values for a blood indicator of acid-base balance were reduced by high levels of dietary P or Cl in all of the experiments. Eggshell quality and values for the blood acid-base indicator were further reduced by high dietary levels of the combination of minerals. The adverse effects associated with high levels of the combination were greater than the sum of adverse effects associated with high levels of each mineral. The effects of P and Cl on plasma levels of these minerals were inconsistent and varied among experiments. The use of pair-fed controls revealed that the adverse effects of combined P and Cl on eggshell quality and acid-base balance were independent of their effects on feed and calcium intake. Calcium excretion was increased by consumption of the high P, high Cl diet. The information obtained from these experiments suggests that the adverse effects of high levels of dietary P and/or Cl on eggshell quality may be due, at least in part, to increased Ca excretion. This, in turn, may be mediated by acidemia resulting from the dietary manipulations, although the present data were insufficient to provide a direct link between acidemia and Ca excretion.

Acid-Base Equilibrium

Interaction of dietary calcium and chloride and the influence of monovalent minerals on eggshell quality.

Two experiments were conducted to determine the influence of dietary sodium and chloride on eggshell quality of leghorn hens. In the first, hens were fed for 4 wk diets containing three levels of calcium (2.0, 2.8, and 3.5%) and two levels of chloride (.25 and .86%) in a factorial arrangement of treatments involving four replicates of five hens/treatment. Neither chloride nor calcium significantly affected hen-day egg production or egg weight (P greater than .05); however, chloride decreased feed consumption and calcium increased body weight gain (P less than .05). Interactions of calcium and chloride were significant for eggshell strength (P less than .06) and eggshell thickness (P less than .05). Chloride decreased these measures of eggshell quality only for hens receiving 2.0% calcium, and calcium improved both measures only for hens receiving .86% chloride. Chloride decreased blood bicarbonate concentration and base excess (P less than .05), but did not affect blood pH and pCO2 (P greater than .05). The effect of dietary sodium and chloride on eggshells of hens receiving 2.0% dietary calcium was determined in a second experiment. Seven diets ranging from .18% sodium and .94% chloride to .76% sodium and .24% chloride were provided for 4 wk to five replicates of five hens/treatment. Dietary sodium and chloride levels did not affect hen-day egg production or egg weight (P greater than .05). Increasing the proportions of sodium relative to chloride decreased food intake but increased eggshell strength and thickness (P less than .01) and increased blood pH (P less than .05), bicarbonate concentration (P less than .01), and base excess (P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

7C female sterile mutants fail to accumulate early eggshell proteins necessary for later chorion morphogenesis in Drosophila.

Seven noncomplementing female sterile mutations that affect eggshell assembly in Drosophila have been mapped to the 7C1-3 region of the X-chromosome. TEM of the mature eggshell of one of the alleles, fs(1)410, shows a lack of organization within the endochorion and an accumulation of electron dense material in the vitelline membrane of stage 14 eggchambers. SDS-PAGE of radiolabeled eggshell proteins shows that two proteins, s67 and s85, fail to accumulate in the fs(1)410 eggshell. In wild-type flies s85 is produced during stage 10 of oogenesis and then processed to s67 in stages 13 and 14. Neither s85 nor an additional stage 10 specific follicle cell protein (s130) are detected in fs(1)410 or four of the mutant alleles. Short-term labeling studies, analyses of in vitro translation products, and the simultaneous occurrence of s85 and s130 as electrophoretic variants in geographic fly strains indicate s85 is derived from s130. Although major biochemical differences appear in stage 10, mutant and wild-type eggshells are morphologically indistinguishable until stages 13-14. These results suggest that follicle cell proteins synthesized during the time of vitelline membrane deposition (stage 10) are important for proper assembly of the chorion layers during stages 13 and 14.

Alleles

Characterization of the eggshell of Haemonchus contortus--I. Structural components.

1. By transmission electron microscopy, the eggshell of Haemonchus contortus was seen to be similar to previously studied nematodes, with an outer vitelline layer bounded by a trilaminate membrane, a broad medial region, containing chitin, and an electron dense basal region, containing lipid and protein. 2. Exposure of Haemonchus contortus eggs to proteases resulted in disruption of the shell with removal of components of the outer, medial and basal regions. Exposure to chitinase depleted fibrillar components of the medial region of the shell, while collagenase had no effect. 3. Chloroform/methanol extraction of fresh eggshells caused a minor condensation of the outer, vitelline layer and some depletion of the basal layer. 4. After normal hatching, shells appeared similar to those treated with protease and chitinase, but also lacked the basal, lipid layer. 5. Extracts of isolated unhatched eggshells and hatched eggshells, and extracts of biotin-labelled whole fresh eggs showed three major protein bands when run on sodium dodecyl sulphate-polyacrylamide gels indicating that these three proteins are most likely structural in nature and do not participate in the release of the larva from the eggshell. 6. Biotin-labelled protein bands were degraded by proteases and chitinase, but not collagenase or lipase.

Animals

A note on eggshell porosity, nest humidity and the effects of DDE in the grey heron (Ardea cinerea).

Water vapour conductance (GH2O) was determined for 25 grey heron Ardea cinerea eggs in the laboratory, and in nests during natural incubation at two Scottish colonies. The mean GH2O of eggs measured in the nest which successfully hatched was 9.0 mgH2O/mmHg/day and the mean water vapour pressure gradient between egg and nest (delta PH2O), measured using "calibrated" duck eggs, averaged at 31 mmHg (4.13 kPa). Based on eggshell porosity results, from the eggs which hatched, such a gradient would result in a loss of water from the eggs during incubation equivalent to 11% of their fresh weight. Shell thickness, the number of pores/cm2 of eggshell and DDE content were also determined for the 25 eggs measured in the laboratory. Eggs containing high levels of DDE had thinner shells, more pores in the eggshell and a higher overall eggshell porosity. The main problem posed by a high level of DDE would appear, however, not to be an excessive water loss from the egg during incubation, but rather eggshell thinning leading to a loss of the egg due to breakage in the nest.

Animals

Soluble protein constituents of the domestic fowl's eggshell.

1. The protein components of the domestic fowl's eggshell are believed to influence appreciably the mechanical properties of the shell and/or its biomineralisation. The purpose of this study was to compare the protein species composing the eggshell matrix in different parts of the shell structure, by SDS-PAGE and chromatography, utilising eggshell cleaned by different methodologies. 2. Protein species were identified whose absence was associated with the removal of the mammillary knobs. In particular, a prominent 81 kDa protein, as well as 38 and 54 kDa calcium-binding proteins, were concentrated within the mammillary layer, as was a 129 kDa insoluble protein. By contrast, soluble proteins of 54, 33, 22, and 14 kDa were enriched in the palisade layer. 3. Our results demonstrate that the mineralised layers of the fowl's eggshell possess a complex array of distinct proteins. The different proteins which have been detected in the mammillary and palisade layers may be related to the distinct crystallisation patterns of calcium carbonate in these zones of the eggshell.

Animals

Use of ascorbic acid to prevent the decline in eggshell quality observed with saline drinking water.

Three experiments were carried out to determine the value of ascorbic acid in overcoming the poor eggshell quality problem associated with the use of saline drinking water. Laying hens were supplied with either town water or town water supplemented with NaCl, ascorbic acid, or a combination of NaCl and ascorbic acid. Supplementing the drinking water lf laying hens with NaCl (2 g/L) significantly increased the incidence of eggshell defects and significantly decreased eggshell quality. Simultaneous supplementation of the saline drinking water with ascorbic acid (1 g/L) prevented these detrimental effects. This response to ascorbic acid was dependent upon concentration. Ascorbic acid acted as a preventive rather than a remedial treatment: hens producing eggs with defective shells as a result of receiving saline drinking water failed to show any improvement in eggshell quality or any reduction in the incidence of eggshell defects when the saline water was supplemented with ascorbic acid.

Animals

Calcium-45 uptake by shell gland, oviduct, plasma and eggshell of DDT-dosed ducks and chickens.

Experiments were conducted with ducks and chickens to determine the effects of acute doses of DDT and DDE on calcium-45 balance and calcium-45 distribution among plasma, femur, tibia, oviduct, shell gland, and eggshell. Species differences in distribution of calcium-45 were evident between control ducks and control chickens. Ducks contained more radiocalcium in plasma, shell glands, tibias, and femurs than chickens and eliminated less radiocalcium in their droppings than chickens. The percentage of radiocalcium deposited in eggshells by control birds of either species was about equal. Ducks and chickens treated with DDT or DDE contained more radiocalcium in their shell glands than their respective controls. Treatment with DDT or DDE caused eggshell thinning in ducks but not in chickens; however, treatment with DDT or DDE did not alter significantly the percentage of radiocalcium that appeared in eggshells of either species.

Animals

Nematodirus battus: permeability changes, calcium binding, and phosphorylation of the eggshell during hatching.

Eggshells of Nematodirus battus leaked trehalose 4 hr after being stimulated to hatch, and became permeable to trypan blue at their poles; 80% of eggs were stained blue 24 hr later. Exogenous application of ruthenium red significantly inhibited chill- and sodium fluoride-stimulated hatching, 50% hatch inhibition occurring in 44.67 +/- 2.2 and 8.5 +/- 1.5 microM, respectively. Lanthanum chloride, however, was not as inhibitory as ruthenium red on fluoride-stimulated hatching, 50% occurring at 31.60 +/- 1.25 microM. A Scatchard plot of the competitive binding of ruthenium red to eggshells demonstrated a high-affinity binding site for calcium, KCa' = 1.92 microM and a second, low-affinity site, KCa" = 1169.60 microM. Ruthenium red binding was significantly reduced by several enzymes, e.g., EGTA-buffered trypsin reduced binding by 73%. Radioiodinated concanavalin A also bound competitively to the eggshells in the presence of alpha-D-glucosyl-alpha-D-glucopyranoside and alpha-methyl-D-mannopyranoside. Eggshells incorporated phosphorus-32 from ATP after chilling or on exposure to sodium fluoride; gel filtration of solubilized homogenates of these samples showed that two proteins were radiolabelled with molecular weights of 38 X 10(3) and 8 X 10(3) Da, respectively. This phosphorylation was inhibited by N-ethylmaleimide, which also prevented hatching.

Adenosine Triphosphate

Laser-Raman and infrared spectroscopic studies of protein conformation in the eggshell of the fish Salmo gairdneri.

Laser-Raman and infrared spectroscopic studies reveal abundant beta-pleated sheet conformation in the eggshell proteins of the fish Salmo gairdneri. This secondary structure is the underlying molecular conformation, dictating the formation of the helicoidal architecture of the eggshell. Disulphide bonds crosslink the eggshell proteins of the fertilized eggs and are apparently found in g-g-g (gauche-gauche-gauche), g-g-t (gauche-gauche-trans) and t-g-t (trans-gauche-trans) conformation. There is no evidence for the existence of free sulphydryls. The tyrosines appear to act as hydrogen-bond acceptors, whereas the aromatic residues phenylalanine and tryptophan are also eggshell protein constituents.

Animals

Effects of suppression of eggshell calcification and of 1,25(OH)2D3 on Mg2+, Ca2+ and Mg2+HCO-3 ATPase, alkaline phosphatase, carbonic anhydrase and CaBP levels--II. The laying hen intestine.

Activity of a HCO-3 stimulated Mg2+ dependent ATPase is demonstrated in mitochondrial fractions of the avian duodenum. Suppression of eggshell calcification resulted in a slight reduction in Mg2+, Ca2+ and Mg2+HCO-3 ATPase activities. Duodenal carbonic anhydrase activity was lower in birds laying soft-shelled eggs than in birds laying normal eggs. Alkaline phosphatase and calcium binding protein levels both decreased along the length of the small intestine, but the effect was more pronounced for alkaline phosphatase. Suppression of eggshell calcification and treatment of shell-less laying hens with 1,25(OH)2D3 influenced alkaline phosphatase activity only in the duodenal mucosa. Suppression of eggshell calcification reduced CaBP levels in all sections of the intestine. Treatment with 1,25(OH)2D3 restored CaBP levels. Regulation of intestinal CaBP levels by 1,25(OH)2D3 would therefore, seem to be controlled more directly by calcium requirements associated with eggshell calcification than by gonadal hormones.

Adenosine Triphosphatases

Lysine derived cross-links are present in a non-elastin, proline-rich protein fraction of Iguana iguana eggshell.

1. Eggshell of Iguana iguana contains a variety of lysine-derived cross-linking amino acids apparently identical with some that occur in elastin. 2. Quantification of desmosine by amino acid analysis and radioimmunoassay gave estimates of 217 ng per mg and 109 ng per mg (dry weights), respectively. 3. [3H]NaBH4 reduction of eggshell protein established presence of lysine-derived aldehydes, allysine and allysine-aldol. 4. Enzymatic and chemical treatments indicate these cross-links occur in a non-elastin eggshell protein. 5. Iguana eggshell protein is unusually proline-rich, containing almost 440 proline residues per thousand.

Amino Acids

Histochemical studies on eggshell formation in Paramphistomum cervi (Digenea: Paramphistomatidae).

The formation of the egg mass in the ootype, containing the oocyte, vitelline cells, numerous spermatozoa and Mehlis' gland secretion marks the beginning of eggshell formation. Mehlis' gland cells pour their lipoprotein secretion into the ootype; this secretion forms a thin template around the egg mass and upon this template the shell globules are deposited. Eggshell formation begins in the proximal uterus and is completed in the middle uterus. The eggshells are devoid of phenols and phenolase indicating that hardening is not by quinone tanning. The eggshells contain keratin and are stabilized by disulphide linkages.

Animals

Ootaxonomy and eggshell ultrastructure of Phlebotomus sandflies.

The eggshell structure of four sandfly species: Phlebotomus perniciosus Newstead, P.perfiliewi Parrot, P.papatasi Scopoli and P.duboscqi Neveu-Lemaire, was examined by scanning and transmission electron microscopy (SEM and TEM). At the TEM level, the eggshell appears to have a homogeneous vitelline envelope and a thick chorion. At SEM level, the eggshell of all species is characterized by the outer chorion forming a series of fifteen to twenty longitudinal sinuous ridges, cross-linked in places to form a pattern of polygons, each line of the chorion consisting of columns arranged in a palisade. The aeropyle region of the egg is described for the first time in phlebotomine sandflies. Specific characters of the eggshell topography are described for distinguishing between these and other species of Phlebotomus.

Animals

Small gene family encoding an eggshell (chorion) protein of the human parasite Schistosoma mansoni.

We have isolated six independent genomic clones encoding schistosome chorion or eggshell proteins from a Schistosoma mansoni genomic library. A linkage may of five of the clones spanning 35 kilobase pair (kbp) of the S. mansoni genome was constructed. The region contained two eggshell protein genes closely linked, separated by 7.5 kbp of intergenic DNA. The two genes of the cluster were arranged in the same orientation, that is, they were transcribed from the same strand. The sixth clone probably represents a third copy of the eggshell gene that is not contained within the 35-kbp region. The 5' end of the mRNA transcribed from these genes was defined by primer extension directly off the RNA. The ATCAT cap site sequence was homologous to a silkmoth chorion PuTCATT cap site sequence, where Pu indicates any purine. DNA sequence analysis showed that there were no introns in these genes. The DNA sequences of the three genes were very homologous to each other and to a cDNA clone, pSMf61-46, differing only in three or four nucleotides. A multiple TATA box was located at positions -23 to -31, and a CAAAT sequence was located at -52 upstream of the eggshell transcription unit. Comparison of sequences in regions further upstream with silkmoth and Drosophila sequences revealed several very short elements that were shared. One such element, TCACGT, recently shown to be an essential cis-regulatory element for silkmoth chorion gene promoter function, was found at a similar position in all three organisms.

Amino Acid Sequence