Embryotrophic Potential Of Novel And Candidate Oviductal Fluid Proteins Of Buffalo
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Date
2014
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Publisher
Tamil Nadu Veterinary and Animal Sciences University
Abstract
The mammalian oviduct is the site of fertilization and early cleavage
providing an ideal milieu for early embryonic development. The physiological
interaction between gametes or embryos and the oviducts involves intimate and
precise signalling to establish and maintain a healthy conceptus. The present study
was undertaken to investigate the alterations in the oviductal proteins that arise due to
cyclical changes and upon co-incubation of gametes which inturn would lead to
identification of putative embryotrophic factors that could be used to improve the
outcome of in vitro embryo production.
Oviductal epithelial cells (OEC) derived from early, mid and late phase of the
oestrous cycle were used for quantitative RT-PCR and oviductal fluid for protein
analysis by 2D gel electrophoresis. Expression profiling of candidate genes namely
Heat shock protein 70 (Hsp70), Oviduct specific glycoprotein (OVGP) and
Osteopontin (OPN) revealed that the transcripts were differentially regulated by
hormonal influence. Expression of OVGP and OPN mRNA was found to be maximal
during the early phase of the cycle. However, expression of Hsp 70 was not found to
differ much between the different phases of the oestrous cycle.
Co-incubation of OEC with oocytes, sperms and both the gametes resulted in
upregulation of Hsp 70, OVGP and OPN genes when compared to control. However
the upregulation was maximum upto 36-fold for OVGP gene when OEC was
co-incubated with both the gametes instead of either of them.
One of the candidate proteins, Hsp 70, upon supplementation to the embryo
culture medium was found to increase the number of expanded COCs and viable
oocytes to 84.21 ± 1.14 % and 87.77 ± 2.42% when compared to the 74.34 ± 1.50%
and (76.38 ± 2.75%) in the control group. Osmotolerance, acrosome reaction,
viability and sperm-binding rate of Hsp 70 supplemented spermatozoa were found to
be better than the control. The yield of presumptive zygotes (52.34±0.78%) and
morula (36.73±1.07%) were higher in Hsp 70 supplemented group than in the control
(37.71±2.18% and 26.36±2.0%).
Analysis of the oviductal fluid proteins by 2D gel revealed upregulation of 29
proteins and downregulation of 23 proteins in the gamete treated group. A total of 17
differentially expressed proteins were identified by MALDI-TOF/MS out of which
two novel proteins namely thioredoxin and serine threonine protein phosphatase
(PP2A) were analyzed for their functional role by supplementation in in vitro embryo
culture medium. Results of the supplementation studies revealed that both these
oviduct-derived proteins possessed embryotrophic properties.
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Keywords
Buffalo, Candidate gene analysis by qRT-PCR, Oviductal fluid proteins, Co-incubation of gametes, Two dimensional gel electrophoresis, Differential protein expression