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Bakst M.R.,Animal Biosciences and Biotechnology Laboratory | Bauchan G.,U.S. Department of Agriculture
Poultry Science

Mammalian sperm bind to terminal carbohydrates associated with glycoconjugates on the apical surface of oviduct epithelial cells in the caudal region of the oviduct and undergo cellular and molecular modifications associated with capacitation prior to ovulation. In contrast, chicken sperm are stored for up to 23 d in sperm-storage tubules (SST) localized in the uterovaginal junction (UVJ). Little is known of the cellular and molecular mechanisms that regulate sperm storage in and release from the SST. The purpose of this study was to identify glycoconjugates associated with the SST epithelial cell surface using lectins. Virgin hens and hens of higher and lower fertility in egg production for 6 to 16 wk were used in this study. Sections of UVJ mucosa containing SST were stained with fluorescent conjugated lectins and examined by confocal microscopy. Carbohydrate moieties associated with the UVJ and SST epithelia differed in their lectin binding patterns. No differences in the lectin binding patterns within the 2 epithelia were discernible between the virgin and younger and older hens. Minor differences were observed between the higher and lower fertility hens. Only lectins specific for galactose and N-acetylgalactosamine moieties were localized to the luminal surface of the SST. While resident sperm may be closely apposed to the SST epithelial cell apical microvilli, it is unlikely that sperm binding to the microvilli via terminal carbohydrates associated with glycoconjugates is a requisite for prolonged storage. However, the possibility of SST epithelial cell communication with resident sperm via shedding microvillous vesicles characterized by surface glycoconjugates with terminal galactose and N-acetylgalactosamine moieties is currently being investigated. © 2016 Published by Oxford University Press. Source

Chen X.,Nanjing Agricultural University | MacDonald M.H.,U.S. Department of Agriculture | Khan F.,U.S. Department of Agriculture | Garrett W.M.,Animal Biosciences and Biotechnology Laboratory | And 2 more authors.
Current Proteomics

To gain new insights into the mechanism of soybean (Glycine max) interaction with the soybean cyst nematode (Heterodera glycines), we compared protein abundance in soybean roots infected by the soybean cyst nematode at different time intervals. Proteins were extracted from roots of 3 and 8 days post-inoculation (dpi) by soybean cyst nematode Heterodera glycines and separated by 2-DE. A difference in abundance was found in a total of 42 protein spots between the susceptible and resistance responses. After in-gel digestion with trypsin, 39 spots representing 33 proteins were subsequently identified by LC-MS/MS. The proteins showing 1.5-fold changes in intensities are related to biochemical processes that may be differentially altered after soybean cyst nematode challenge. The identified proteins belong to the categories of metabolism, energy, cell growth and division, transcription, protein synthesis, protein destination and storage, signal transduction, disease/defense, and secondary metabolism. Taken together, our study provides important information on the use of proteomic methods for studying protein regulation during soybean-soybean cyst nematode interactions. © 2013 Bentham Science Publishers. Source

Chen X.,U.S. Department of Agriculture | MacDonald M.H.,U.S. Department of Agriculture | Garrett W.M.,Animal Biosciences and Biotechnology Laboratory | Matthews B.F.,U.S. Department of Agriculture | Natarajan S.S.,U.S. Department of Agriculture

Soybean cyst nematode (Heterodera glycines, SCN) is the most destructive pathogen of soybean (Glycine max (L.) Merr.) worldwide. In this study, three different protein extraction methods including phenol/ammonium acetate (phenol method), thiourea/urea solublization (lysis method) and trichloroacetic acid/acetone (TCA method) were evaluated to determine their efficacy in separating Heterodera glycines proteins by two-dimensional polyacrylamide gel electrophoresis (2-DE). In all three methods, nematode proteins were well separated with minor variations in the intensity of the protein spots. The phenol method showed higher protein resolution and spot intensity of all proteins compared with the other two methods. In addition, in the high-pI region, proteins were clearly resolved and strongly detected using the phenol method. Protein spots obtained from the phenol method were subjected to further analysis to test their suitability for identification by mass spectrometry. Twenty protein spots were randomly selected, digested with trypsin, and analyzed using Matrixassisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) or liquid chromatography mass spectrometry (LC-MS/MS). While these results suggest that phenol method and the direct lysis method are efficient and reliable for the 2D separation of Heterodera glycines proteins, the phenol extraction procedure is superior for the alkaline proteins. Source

Chen X.,U.S. Department of Agriculture | MacDonald M.H.,U.S. Department of Agriculture | Khan F.,U.S. Department of Agriculture | Garrett W.M.,Animal Biosciences and Biotechnology Laboratory | And 2 more authors.

2-DE reference maps of Heterodera glycines were constructed. After in-gel digestion with trypsin, 803 spots representing 426 proteins were subsequently identified by LC-MS/MS. Proteins with annotated function were further categorized by Gene Ontology. The results showed that proteins involved in metabolic, developmental and biological regulation processes were the most abundant. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

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