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

Capsule:
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.

Authors:
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

Abstract:

Objective:
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).

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

Setting:
Tertiary hospital.

Patients:
Twenty donors and 32 infertile men.

Interventions:
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.

Conclusions:
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.

Translate »