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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Authors: | Shubhra Singh Shiv Prasad H P Gupta Sumit Singhal Atul K Gupta Anil Kumar |
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Institution: | (1) Department of Molecular Biology & Genetic Engineering, College of Basic Sciences & Humanities, G.B. Pant University of Agriculture & Technology, Pantnagar, 263145, Uttarakhand, India;(2) Department of Animal Reproduction, Gynaecology & Obstetrics, College of Veterinary and Animal Sciences, G.B. Pant University of Agriculture & Technology, Pantnagar, 263145, Uttarakhand, India; |
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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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