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

Objective: To identify the relative abundance of proteins in pooled reactive oxygen species (ROS)-positive (ROS+) and ROS-negative (ROS-) semen samples with the use of two-dimensional differential in-gel electrophoresis (2D-DIGE).

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

Setting: Tertiary hospital.

Patient(s): 20 donors and 32 infertile men.

Intervention(s): Seminal ejaculates evaluated for semen and proteomic analysis.

Main outcome measure(s): Semen samples from 20 donors and 32 infertile men were pooled, divided into ROS+ and ROS- groups based on the cutoff value of <20 relative light units/s/10(6) sperm and frozen. From each pooled group, spermatozoa were labeled with Cy3/Cy5 fluorescent dye. Duplicate 2D-DIGE gels were run. Image analysis was performed with the use of Decider software. Protein spots exhibiting ≥1.5-fold difference in intensity were excised from the preparatory gel and identified by liquid chromatography-mass spectrometry. Data were analyzed with the use of Sequest and Blast programs.

Result(s): A total of 1,343 protein spots in gel 1 (ROS-) and 1,265 spots in gel 2 (ROS+) were detected. The majority of protein spots had similar expression, with 31 spots were differentially expressed. Six spots were significantly decreased and 25 increased in the ROS- sample compared with the ROS+ sample.

Conclusion(s): Significantly different expression of protective proteins against oxidative stress was found in ROS-compared with ROS+ samples. These differences may explain the role of oxidation species in the pathology of male infertility.