Male infertility markers: Proteins influenced by reactive oxygen species

Radicals and infertility

There are a number of factors behind male infertility in humans but one of the principle causes is the presence of excess reactive oxygen species in the semen, accounting for about 40% of male infertility problems. Small amounts of ROS are needed for sperm to function normally but their levels are controlled by natural antioxidants. When this balance is disturbed, oxidative stress occurs and the sperm become damaged and unable to function correctly.

Other factors that contribute to oxidative stress include lifestyle choices such as cigarette smoking, drinking alcohol, and poor diet, as well as less controllable issues like infection and disease.

A team of scientists has been studying the effects of high levels of ROS on the proteins in spermatozoa and seminal plasma to try and gain an understanding of the molecular processes associated with male infertility. Now, the same team has extended that work to look at the effects of low, medium and high ROS levels on spermatozoic proteins and compared them to those in the sperm of normal fertile men.

Ashok Agarwal colleagues from the Glickman Urological & Kidney Institute, Cleveland Clinic, Ohio and the Department of Urology, Hamad Hospital, Doha, Qatar, published their findings in the open access journal Clinical Proteomics.

Altered protein abundances

Seminal ejaculates were collected from 17 fertile men and 42 infertile men with different levels of oxidative stress. The mean levels of ROS in the fertile men were measured by a luminal-based chemiluminescence assay to be 27.7 relative light units/sec x 10⁶ sperm and those for the men with low, medium and high ROS levels were 0, 189 and 2003 RLU/sec x 10⁶ sperm.

The proteins from each patient category were extracted and separated by one-dimensional gel electrophoresis and analysed by mass spectrometry after digestion with the enzyme trypsin. The peptides were identified by searching the peptide fragments against a human reference database and they were quantified by the spectral counting method.

This led to a total of 1035 identified proteins and 305 had different abundances between the groups. From those a considerable number were found to be unique to each group, with 51, 47 and 104 of the differentially expressed proteins found only in the low, medium and high ROS patients. Some of these had increased abundances compared with the fertile men and some were less abundant. Other differentially expressed proteins were common to more than one of the groups.

Six potential biomarkers of elevated reactive oxygen species

The differentially expressed proteins were associated with many different functions, including protein metabolism, the immune system, disease, transmembrane transport, extracellular matrix organization, signal transduction, post-translational modifications, cellular response to stress, lipid metabolism, small molecule biochemistry, and nucleic acid metabolism.

From that large group, the researchers homed in on six proteins that are known to be involved in reproduction and sperm production. The first of these was calmegin, which was over-expressed in infertile men, possibly leading to the prevention of spermatozoa to bind with the membrane around the egg known as the zona pellucida. Tripeptidyl peptidase II was also more abundant than in fertile men and might modify the phosphorylation levels of tyrosine groups in proteins, thereby inhibiting several key processes.

Early endosome antigen 1 was uniquely expressed in the men with ROS above normal physiological levels, which could lead to the failure of the spermatozoa head to develop the cap-like structure called the acrosome.

The remaining three proteins dynein intermediate chain 2, heat shock 70kDa protein 4L and plasma serine protease inhibitor all had reduced abundances and their potential effects on sperm motility and viability were also discussed.

The researchers suggested that this panel of six could be used as markers of the effects of oxidative stress on male fertility because their altered abundances represent the change in normal function due to the presence of ROS. However, more work is required to understand their roles more fully and to validate the results with a larger number of subjects.

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