Mass Spectrometry Reveals New Insights into the Production of Superoxide Anions and 4‐Hydroxynonenal Adducted Proteins in Human Sperm

Abstract

The free-radical theory of male infertility suggests that reactive oxygen species produced by the spermatozoa themselves are a leading cause of sperm dysfunction, including loss of sperm motility. However, the field is overshadowed on several fronts, primarily because: i) the probes used to measure reactive oxygen species (ROS) are imprecise; and ii) many reports suggesting that oxygen radicals are detrimental to sperm function add an exogenous source of ROS. Herein, a more reliable approach to measure superoxide anion production by human spermatozoa based on MS analysis is used. Furthermore, the formation of the lipid-peroxidation product 4-hydroxynonenal (4-HNE) during in vitro incubation using proteomics is also investigated. The data demonstrate that neither superoxide anion nor other free radicals that cause 4-HNE production are related to the loss of sperm motility during incubation. Interestingly, it appears that many of the 4-HNE adducted proteins, found within spermatozoa, originate from the prostate. A quantitative SWATH analysis demonstrate that these proteins transiently bind to sperm and are then shed during in vitro incubation. These proteomics-based findings propose a revised understanding of oxidative stress within the male reproductive tract.

The Role of Genetics and Oxidative Stress in the Etiology of Male Infertility—A Unifying Hypothesis?

“…In a recent study of human spermatozoa incubated over a 5 day period, the loss of virtually all motility was not accompanied by a corresponding increase in 4-HNE levels by Western blot (102). In light of these data, we have to conclude that there may be multiple FIGURE 3 | Importance of PI3 kinase in the maintenance of sperm viability.…”

“…It should also be recognized that a vast majority of the probes used with flow cytometry are redox active agents that do not measure ROS directly but rather, oxidative activity. Nevertheless, by using dihydroethidium as a probe and separating out the reaction product specifically generated by superoxide anion, 2-hydroxyethidium, it has been possible to generate evidence of superoxide production by mammalian spermatozoa (102,103). Moreover, these data have been confirmed with definitive techniques such as electron paramagnetic resonance spectroscopy (104)(105)(106).…”

Genetic mutation ofCep76results in male infertility due to abnormal sperm tail composition

“…Sperm were washed three times in 1× Tris-buffered saline, pelleted, and then stored at −80°C. Sperm pellets were then dried in a speed vacuum, prepared for liquid chromatography–tandem mass spectrometry, and run on a SCIEX QTRAP6500 as described previously ( 77 ).…”

CEP76 is a critical regulator of male germ cell transition zone function and tail composition

“…Sperm were washed three times in 1 x Tris buffered saline, pelleted, and then stored at -80°C. Sperm pellets were then dried in a speed vacuum and prepared for liquid chromatography tandem mass spectrometry and run on a SCIEX QTRAP6500 as described previously [70].…”