Chemiluminescence and the Antioxidant Properties of Seminal Plasma

Another variant on the chemiluminescence theme is to use this technology to monitor both ROS generation by the ejaculate and the antioxidant potential of the seminal plasma, with a view to generating a readout reflecting the balance between these two opposing forces. There can be no doubt that seminal plasma is a very powerful anti-oxidant medium that serves to protect spermatozoa during their perilous journey from the male to the female reproductive tract. This fluid is rich in antioxidant enzymes such as SOD, glutathione peroxidase, and some catalase as well as small molecular mass scavengers, including ascorbate, pyruvate, urate, alpha tocopherol, glutathione, albumin, beta carotene, hypotaurine, and ubiquinol [10, 60, 61] . The ability of this fluid to protect spermatozoa from the emission of free radicals by contaminating leukocytes has been clearly demonstrated, as have the adverse consequences of removing this antioxidant protection when leukocytes are present in the sperm suspension [18, 28]. Significantly, seminal plasma has been shown to protect spermatozoa from DNA damage inflicted as a consequence of leukocytic infiltration [62],

Although the antioxidant properties of semen can be assessed by measuring the individual constituents of the system such as SOD [63] alpha-tocopherol or ascorbate [64], a more convenient approach is to assess the sum total of all antioxidant activities in the semen using a TRAP assay. The latter measures the ability of seminal plasma to extinguish a free radical signal typically generated by a compound such as 2,2-azobis-(2-amidinopropane) (ABAP), which generates alkylperoxyl radicals at 37°C. In the presence of luminol, a steady state chemiluminescent signal is generated with ABAP and the ability of seminal plasma to inhibit this output gives an indication of total antioxidant capacity. Using a related compound [2,2' Azino-to(3-ethylbenzothiazoline-6-sulfonic acid)] as the radical source in a postaddition assay, Rhemrev et al. [65] demonstrated that the total TRAP activity of seminal plasma is about ten times higher than that of blood plasma. These authors also differentiated (1) a fast TRAP activity detectable in 10 s, 37% of which could be attributed to vitamin C, uric acid, and tyrosine, while proteins and polyphenolic compounds contributed a further 57% and (2) a slow TRAP activity detectable in 300 s attributable to vitamin C (1%), uric acid (2%), tyrosine (15%), and to proteins and polyphenolic compounds (33%). It was not possible to account for the remaining 49%.

An alternative ROS source (H.O2 in the presence of horse radish peroxidase linked to immunoglobulin) has also been used in combination with luminol as the basis for an alternative TRAP assay [66]. This approach would focus such antioxidant assessments on H2O2-scavenging activity. Whether this is an advantage is not known at the present time because the relationship between the results of this assay and conventional ABAP-based TRAP assays has never been assessed. Indeed, there are several variants on the TRAP assay that use a variety of free radical/oxidant sources and detection systems. Systematic evaluation of these assays for their relative ability to detect differences in the antioxidant activity of human seminal plasma would be a worthwhile exercise.

Many authors have found an inverse relationship between ROS generation and seminal antioxidant activity in semen [62, 66] . This inverse relationship probably reflects the fact that quantitatively, a majority of the free radicals detected in human semen samples are generated by contaminating leukocytes. Since the leukocytes release ROS into the extracellular space, they will be rapidly intercepted by the free radical scavengers in seminal plasma leading to the observed negative correlation between ROS generation and TRAP values. Viewed in this light, the loss of antioxidant activity might be considered a consequence of excess ROS generation by leukocytes entering the seminal fluid at the moment of ejaculation. Alternatively, antioxidant activity in seminal plasma may be a reflection of the redox status of the individual and be a cause of oxidative stress in the germ line. Thus, the oxidative DNA damage seen in the spermatozoa of heavy smokers is thought to reflect a systemic loss of antioxidant protection precipitated by the free radicals present in cigarette smoke [67]. Under such circumstances, it is not clear how a loss of antioxidant protection could induce an increase in ROS generation by spermatozoa, unless oxidative stress itself triggers ROS production by these cells [68].

Simultaneous chemiluminescence assessments of ROS generation and anti-oxidant protection are potentially valuable and have the possibility of being expressed as a ratio of activities, thereby circumventing the difficulties inherent in calibrating chemiluminescence assays. However, interpretation of the results requires a knowledge of the leukocytic contribution to the ROS signal, which could be readily acquired using leukocyte-specific agonists (opsonized zymosan) as part of the chemiluminescence protocol possibly in combination with sperm isolation protocols that effectively remove contaminating leukocytes such as swim-up or electrophoresis.

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