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Isolation and Characterization of Oviduct-specific Glycoproteins from Ampulla and Isthmus Parts of Cyclic and Acyclic Buffalo for Studying Differential Microenvironment

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Abstract

The present study characterized the glycoproteins synthesized by buffalo oviduct. Scanning electron microscopy analyses of the ampullary and isthmic segments of cyclic and acyclic buffaloes showed ultrastructural variations in ciliated and nonciliated cells. Mucosal proteins were extracted by scrapping of different segments of oviduct and, after centrifugation, the remainder tissues were subjected to establish primary cell culture system of oviduct epithelial cells and conditioned media were prepared. Time- and concentration-dependent effects of trypsinization on the establishment of primary monolayer culture showed that 0.25% trypsin for 1–2 min at 37 °C were the optimal conditions. Total protein content in oviductal tissues and conditioned media was quantified and analyzed by SDS-PAGE which showed marked variation in different segments of the oviduct. Western blot analysis revealed five major oviduct-specific glycoproteins (OGPs) in cyclic oviduct (ampulla and isthmus) with Mw 180, 95, 75, 66 and 35 kDa in the oviduct extract and two glycoproteins with Mw 95 and 66 kDa in conditioned media. However, in acyclic oviduct (ampulla and isthmus), three glycoproteins were immunostained with Mw 180, 95 and 66 kDa in the oviduct extract and one glycoprotein with Mw 66 kDa in conditioned media. Indirect Enzyme-Linked Immuno Sorbent Assay (ELISA) results showed significant differences of OGPs in different segments of cyclic and acyclic buffaloes and, thus, indicative of segmental variation in the synthesis and secretion of glycoproteins. Oviductal extract secretes more amounts of OGPs as compared to the conditioned medium. The role of these OGPs may be defined and exploited for influencing the fertilization process and/or subsequent embryonic development.

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Acknowledgment

The authors would like to thank Dean, College of Basic Sciences and Humanities and Dean, College of Postgraduate Studies, G.B. Pant University of Agriculture and Technology, Pantnagar for providing necessary facilities.

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  1. Department of Molecular Biology & Genetic Engineering, College of Basic Sciences & Humanities, G.B. Pant University of Agriculture & Technology, Pantnagar, 263145, Uttarakhand, India

    Shubhra Singh, Atul K. Gupta & Anil Kumar

  2. Department of Animal Reproduction, Gynaecology & Obstetrics, College of Veterinary and Animal Sciences, G.B. Pant University of Agriculture & Technology, Pantnagar, 263145, Uttarakhand, India

    Shiv Prasad, H. P. Gupta & Sumit Singhal

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  1. Shubhra Singh
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Correspondence to Anil Kumar.

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Singh, S., Prasad, S., Gupta, H.P. et al. Isolation and Characterization of Oviduct-specific Glycoproteins from Ampulla and Isthmus Parts of Cyclic and Acyclic Buffalo for Studying Differential Microenvironment. Appl Biochem Biotechnol 166, 1814–1830 (2012). https://doi.org/10.1007/s12010-012-9599-6

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