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.