Preparation and mass spectrometric study of egg yolk antibody (IgY) against rabies virus

Rabies virus was used as the antigen to immunize laying chickens. Anti-rabies virus immunoglobulin Y(IgY) was isolated from yolks of the eggs laid by these chickens using a two-step salt precipitation and one-step gel filtration protocol. The purified IgY was reduced with dithiothreitol, and heavy chains (HC) and light chains (LC) were obtained. In addition, the purified IgY was digested with pepsin and the fragment with specific antigen binding properties (Fab) was produced. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS), the average molecular weights of IgY, HC, LC, and Fab were determined as 167 250, 65 105, 18 660, and 45,359 Da, respectively. IgY has two structural differences compared with mammalian IgGs. First, the molecular weight of the heavy chain of IgY is larger than that of its mammalian counterpart, while the molecular weight of the light chain of IgY is smaller. Second, upon pepsin digestion, anti-rabies virus IgY is degraded into Feb, in contrast to mammalian IgG, which has been reported to be degraded into F(ab')(2) under the same conditions. Copyright (C) 2001 John Wiley & Sons, Ltd.

A comparison of the protective action of added egg yolks from five avian species to the cryopreservation of bull sperm

Cryopreservation of domestic animal sperm has been widely used for artificial insemination (AI), and egg yolk is one of the most commonly used cryoprotectants during the freezing-thawing process. The objectives of this study were to compare the effectiven

Cryopreservation of rhesus macaque (Macaca mulatta) spermatozoa and their functional assessment by in vitro fertilization

Although spermatozoa from several species of nonhuman primates have been cryopreserved, there has been no report of success with rhesus macaque spermatozoa as judged by functional assays. Two Tris-egg yolk freezing media. TEST and TTE. which have: been successfully used for cynomolgus macaque (Macaca fascicularis) spermatozoa, were compared for cryopreservation of spermatozoa From four rhesus macaques (Macaca mulatta). The postthaw motility (percentage and duration) of spermatozoa cryopreserved in TTE was much higher than that for spermatozoa cryopreserved in TEST. The function of sperm cryopreserved in TTE was evaluated by in vitro fertilization or oocytes collected from gonadotropin-stimulated prepubertal rhesus macaques. Of the inseminated oocytes. 82 +/- 13% were fertilized and 63 +/- 22 and 39 +/- 21% of the resulting zygotes developed into morulae and blastocysts. respectively. These results indicate that rhesus macaque spermatozoa can be effectively cryopreserved in TTE medium. This finding will facilitate the application of in vivo and in vitro assisted reproductive technologies in this species. (C) 2001 Academic Press.

Characterization and cloning of a novel phospholipase A(2) from the venom of Trimeresurus jerdonii snake

A phospholipase A(2) (PLA(2)) called jerdoxin, was isolated from Trimeresurus jerdonni snake venom and partially characterized. The protein was purified by three chromatographic steps. SDS-polyacrylamide gel electrophoresis in the presence or absence of dithiothreitol showed that it had a molecular mass of 15 kDa. Jerdoxin had an enzymatic activity of 39.4 mumol/min/mg towards egg yolk phosphatidyl choline (PC). It induced edema in the footpads of mice. In addition, jerdoxin exhibited indirect hemolytic activity. About 97% hemolysis was observed when 2 mug/ml enzyme was incubated for 90 min in the presence of PC and Ca2+. No detectable hemolysis was noticed when PC was not added. Ca2+ was necessary for jerdoxin to exert its hemolytic activity, since only 52% hemolysis was seen when Ca2+ was absent in the reaction mixture. Furthermore, jerdoxin inhibited ADP induced rabbit platelet aggregation and the inhibition was dose dependent with an IC50 of 1.0 muM. The complete amino acid sequence of jerdoxin deduced from cDNA sequence shared high homology with other snake venom PLA(2)s, especially the D49 PLA(2)s. Also, the residues concerned to Ca2+ binding were conserved. This is the first report of cDNA sequence of T jerdonii venom PLA(2). (C) 2002 Elsevier Science Ltd. All rights reserved.

Effect of amino acids on cryopreservation of cynomolgus monkey (Macaca fascicularis) sperm

The effects of three amino acids (proline, glutamine, and glycine) added to the freezing medium Tes-Tris-egg yolk (TTE) for cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa were studied. This is the first report on the effects of amino acids on nonhuman primate sperm cryopreservation. The addition of 5 mM proline, 10 mM glutamine, and 10 or 20 mM glycine each significantly improved post-thaw sperm motility and membrane and acrosome integrity compared with the control (TTE alone). However, a significant decrease in motility and membrane/acrosome integrity was observed when amino acid concentrations increased to 60 mM for proline and glutamine, and 80 mM for glycine. The results suggest that adding a limited amount of amino acids to the freezing media is beneficial for freezing cynomolgus monkey sperm.

Cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa in a chemically defined extender

Aim: To establish a method for cynomolgus monkey sperm cryopreservation in a chemically defined extender. Methods: Semen samples were collected by electro-ejaculation from four sexually mature male cynomolgus monkeys. The spermatozoa were frozen in straws by liquid nitrogen vapor using egg-yolk-free Tes-Tris (mTTE) synthetic extender and glycerol as cryoprotectant. The effects of glycerol concentration (1%,3%, 5%, 10% and 15% [v/v]) and its equilibration time (10 min, 30 min, 60 min and 90 min) on post-thaw spermatozoa were examined by sperm motility and sperm head membrane integrity. Results: The post-thaw motility and head membrane integrity of spermatozoa were significantly higher (P < 0.05) for 5% glycerol (42.95 +/- 2.55 and 50.39 +/- 2.42, respectively) than those of the other groups (1%: 19.19 +/- 3.22 and 24.84 +/- 3.64; 3%: 34.23 +/- 3.43 and 41.37 +/- 3.42; 10%: 15.68 +/- 2.36 and 21.39 +/- 3.14; 15%: 7.47 +/- 1.44 and 12.90 +/- 2.18). The parameters for 30 min equilibration (42.95 2.55 and 50.39 2.42) were better (P < 0.05) than those of the other groups (10 min: 31.33 +/- 3.06 and 38. 98 +/- 3.31; 60 min: 32.49 +/- 3.86 and 40.01 +/- 4.18; 90 min: 31.16 +/- 3.66 and 38.30 +/- 3.78). Five percent glycerol and 30 min equilibration yielded the highest post-thaw sperm motility and head membrane integrity. Conclusion: Cynomolgus monkey spermatozoa can be successfully cryopreserved in a chemically defined extender, which is related to the concentration and the equilibration time of glycerol.

Effects of adrenergic agents on the expression of zebrafish (Danio rerio) vitellogenin Ao1

Teleost vitellogenins (VTGs) are large multidomain apolipoproteins, traditionally considered to be estrogen-responsive precursors of the major egg yolk proteins, expressed and synthesized mainly in hepatic tissue. The inducibility of VTGs has made them one of the most frequently used in vivo and in vitro biomarkers of exposure to estrogen-active substances. A significant level of zebrafish vtgAo1, a major estrogen responsive form, has been unexpectedly found in heart tissue in our present studies. Our studies on zebrafish cardiomyopathy, caused by adrenergic agonist treatment, suggest a similar protective function of the cardiac expressed vtgAo1. We hypothesize that its function is to unload surplus intracellular lipids in cardiomyocytes for "reverse triglyceride transportation" similar to that found in lipid transport proteins in mammals. Our results also demonstrated that zebrafish vtgAo1 mRNA expression in heart can be suppressed by both (x-adrenergic agonist, phenylephrine (PE) and beta-adrenergic agonist, isoproterenol (ISO). Furthermore, the strong stimulation of zebrafish vtgAo1 expression in plasma induced by the beta-adrenergic antagonist, MOXIsylyl, was detected by Enzyme-Linked ImmunoSorbent Assay (ELISA). Such stimulation cannot be suppressed by taMOXIfen, an antagonist to estrogen receptors. Thus, Our present data indicate that the production of teleost VTG in vivo can be regulated not only by estrogenic agents, but by adrenergic signals as well. (c) 2009 Elsevier Inc. All rights reserved.

Simultaneous determination of microcystin contaminations in various vertebrates (fish, turtle, duck and water bird) from a large eutrophic Chinese lake, Lake Taihu, with toxic Microcystis blooms

This is the first to conduct Simultaneous determination of microcystin (MC) contaminations in multi-groups of vertebrates (fish, turtle, duck and water bird) from Lake Taihu with Microcystis blooms. MCs (-RR, -YR, -LR) in Microcystis scum was 328 mu g g(-1) DW. MCs reached 235 mu g g(-1) DW in intestinal contents of phytoplanktivorous silver carp, but never exceeded 0.1 mu g g(-1) DW in intestinal contents of other animals. The highest MC content in liver of fish was in Carassius auratus (150 ng g(-1) DW), followed by silver carp and Culter ilishaeformis, whereas the lowest was in common carp (3 ng g(-1) DW). In livers of turtle, duck and water bird, MC content ranged from 18 to 30 ng g(-1) DW. High MC level was found in the gonad, egg yolk and egg white of Nycticorax nycticorax and Anas platyrhynchos, suggesting the potential effect of MCs on water bird and duck embryos. High MC contents were identified for the first time in the spleens of N. nycticorax and A. platyrhynchos (6.850 and 9.462 ng g(-1) DW, respectively), indicating a different organotropism of MCs in birds. Lakes with deaths of turtles or water birds in the literatures had a considerably higher MC content in both cyanobacteria and wildlife than Lake Taihu, indicating that toxicity of cyanobacteria may determine accumulation level of MCs and consequently fates of aquatic wildlife. (C) 2009 Elsevier B.V. All rights reserved.

Effects of adrenergic agonists on the extrahepatic expression of vitellogenin Ao1 in heart and brain of the Chinese rare minnow (Gobiocypris rarus)

Teleost vitellogenins (VTGs) are large multidomain apolipoproteins and traditionally considered as the estrogen responsive precursors of the major egg yolk proteins. We identified five clones encoding VTGs, about 16% of the random EST clones from our constructed cDNA library from Chinese rare minnow liver tissue treated with 17 beta-estradiol (E2). Full-length vtgAo1 has been obtained based on the sequence information of four partial cDNA inserts by RACE. The inducibility of the vtgAo1 expression in liver by E2 was confirmed by RT-PCR. The presence of vtgAo1 transcripts have been observed primarily in liver. However. a significant level of the vtgAo1 was found in an unexpected location, heart, particularly in atrial cells by RT-PCR and whole mount in situ hybridization analyses. The vtgAo1 mRNA expression in heart and liver tissue could be suppressed by both alpha-adrenergic agonist, phenylephrine (PE) and beta-adrenergic agonist, isoproterenol (ISO). The expression of VTG in the heart observed in the present studies suggested it may provide protection from surplus intracellular lipids in fish cardiomyocytes as triglyceride transport proteins do in mammals. The results also indicated that the production of teleost vtg in vivo can be regulated by riot only estrogenic agents, but adrenergic signals as well. (c) 2008 Elsevier B.V. All rights reserved.

A novel disintegrin, jerdonatin, inhibits platelet aggregation and sperm-egg binding

A novel disintegrin, jerdonatin, was purified to homogeneity from Trimeresurus jerdonii venom by gel filtration and reversed-phase high-pressure liquid chromatography. We isolated the cDNA encoding jerdonatin from the snake venom gland. Jerdonatin cDNA precursor,;encoded pre-peptide, metalloprotease and disintegrin domain. Jerdonatin is composed of 72 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonatin was determined to be 8011 Da by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Jerdonatin inhibited ADP- and collagen-induced human platelet aggregation with IC50 of 123 and 135 nM, respectively. We also investigated the effect of jerdonatin on the binding of B6D2F1 hybrid mice spermatozoa to mice zona-free eggs and their subsequent fusion. Jerdonatin significantly inhibited sperm-egg binding in a concentration-dependent manner, but had no effect on the fusion of sperm-egg. These results indicate that integrins on the egg play a role in mammalian fertilization. (C) 2004 Elsevier Inc. All rights reserved.

The giant panda (Ailurapoda melanoleuca) somatic nucleus can dedifferentiate in rabbit ooplasm and support early development of the reconstructed egg

The giant panda skeletal muscle cells, uterus epithelial cells and mammary gland cells from an adult individual were cultured and used as nucleus donor for the construction of interspecies embryos by transferring them into enucleated rabbit eggs. All the three kinds of somatic cells were able to reprogram in rabbit ooplasm and support early embryo development, of which mammary gland cells were proven to be the Lest, followed by uterus epithelial cells and skeletal muscle cells. The experiments showed that direct injection of mammary gland cell into enucleated rabbit ooplasm, combined with in vivo development in ligated rabbit oviduct, achieved higher blastocyst development than in vitro culture after the somatic cell was injected into the perivitelline space and fused with the enucleated egg by electrical stimulation. The chromosome analysis demonstrated that the genetic materials in reconstructed blastocyst cells were the same as that in panda somatic cells. In addition, giant panda mitochondrial DNA (mtDNA) was shown to exist in the interspecies reconstructed blastocyst. The data suggest that (i) the ability of ooplasm to dedifferentiate somatic cells is not species-specific; (ii) there is compatibility between interspecies somatic nucleus and ooplasm during early development of the reconstructed egg.