Two-dimensional differential in-gel electrophoresis–based proteomics of male gametes in relation to oxidative stress

Proteomic analysis shows that differences in protein expression in spermatozoa producing higher levels of ROS may play a role in the pathophysiology of male infertility.


Alaa Hamada, M.D., Rakesh Sharma, Ph.D., Stefan S. Du Plessis, Ph.D., Belinda Willard, Ph.D., Satya Yadav, Ph.D., Edmund Sabanegh, M.D., Ashok Agarwal, Ph.D.

Volume 99, Issue 5, Pages 1216-1226.e2, April 2013



To identify the relative abundance of proteins in pooled ROS positive (ROS+ve) and ROS negative (ROS- ve) semen samples using two dimensional differential in-gel electrophoresis (2D-DIGE).


Spermatozoa suspensions from ROS+ve and ROS-ve groups by 2D-DIGE. analysis.


Tertiary hospital.


Twenty donors and 32 infertile men.


Seminal ejaculates evaluated for semen and proteomic analysis.

Main Outcome Measures:

Semen samples from 20 donors and 32 infertile men were pooled, divided into ROS+ve and ROS- ve groups based on the cut-off value of <20 RLU/sec/106 12 sperm and frozen. From each pooled group, spermatozoa were labeled with Cy3/ Cy5 fluorescent Clyde. Duplicate 2D-DIGE gels were run. Image analysis was performed using Decider™ software. Protein spots exhibiting at least a 1.5-fold difference in intensity were excised from the preparatory gel and identified by LC-MS16 MS. Data were analyzed using SyQuest and Blast programs. Results:

A total of 1343 protein spots in gel 1 (ROS- ve) and 1265 spots in gel 2 (ROS+ ve) were detected. The majority of protein spots had similar expression with 31 spots differentially expressed. Six spots were significantly decreased and 25 increased in the ROS- ve sample compared to the ROS+ ve sample.


Significantly different expression of protective proteins against oxidative stress (OS) was found in ROS- ve compared to ROS+ ve samples. These differences may explain the role of OS in the pathology of male infertility.

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