On methods for the detection of reactive oxygen species generation by human spermatozoa: analysis of the cellular responses to catechol oestrogen, lipid aldehyde, menadione and arachidonic acid

• Published in: Andrology (Oxford) Vol. 1; no. 2; pp. 192 - 205
• Main Authors: Aitken, R. J., Smith, T. B., Lord, T., Kuczera, L., Koppers, A. J., Naumovski, N., Connaughton, H., Baker, M. A., Iuliis, G. N.
• Format: Journal Article
• Language: English
• Published: Schaumburg, IL American Society of Andrology 01.03.2013; Wiley Subscription Services, Inc
• Subjects: Aldehydes - analysis; Aldehydes - chemistry; Aldehydes - metabolism; Arachidonic Acid - analysis; Arachidonic Acid - chemistry; Arachidonic Acid - metabolism; Biological and medical sciences; Estradiol - analogs & derivatives; Estradiol - chemistry; Estrogens, Catechol - metabolism; Ethidium - analogs & derivatives; Ethidium - chemistry; Flow Cytometry - methods; Fluoresceins - chemistry; Fundamental and applied biological sciences. Psychology; Gynecology. Andrology. Obstetrics; Humans; Leukocytes - metabolism; Luminescence; Luminescent Measurements - methods; Luminol; Male; Male genital diseases; Mammalian male genital system; Medical sciences; Methods; Mitochondria - metabolism; Oxidative Stress; oxydative stress; Oxygen; Phenanthridines - chemistry; Reactive Oxygen Species - metabolism; Sensitivity and Specificity; Spectrophotometry - methods; Sperm; sperm function; sperm motility; sperm quality parameters; Spermatozoa - metabolism; Vertebrates: reproduction; Vitamin K 3 - analysis; Vitamin K 3 - chemistry; Vitamin K 3 - metabolism; Human; Reactive oxygen species; Spermatozoa; sperm motility; Menadione; Estrogen; Polyunsaturated fatty acid; Lipids; oxydative stress; Germinal cell; Arachidonic acid; Ovarian hormone; Stress; sperm function; n-6 fatty acid; Unsaturated fatty acid; sperm quality parameters; Sex steroid hormone; Vitamin K
• ISSN: 2047-2919; 2047-2927
• DOI: 10.1111/j.2047-2927.2012.00056.x

Summary Oxidative stress is known to have a major impact on human sperm function and, as a result, there is a need to develop sensitive methods for measuring reactive oxygen species (ROS) generation by these cells. A variety of techniques have been developed for this purpose including chemiluminescence (luminol and lucigenin), flow cytometry (MitoSOX Red, dihydroethidium, 4,5‐diaminofluorescein diacetate and 2′,7′‐dichlorodihydrofluorescein diacetate) and spectrophotometry (nitroblue tetrazolium). The relative sensitivity of these assays and their comparative ability to detect ROS generated in different subcellular compartments of human spermatozoa, have not previously been investigated. To address this issue, we have compared the performance of these assays when ROS generation was triggered with a variety of reagents including 2‐hydroxyestradiol, menadione, 4‐hydroxynonenal and arachidonic acid. The results revealed that menadione predominantly induced release of ROS into the extracellular space where these metabolites could be readily detected by luminol‐peroxidase and, to a lesser extent, 2′,7′‐dichlorodihydrofluorescein. However, such sensitivity to extracellular ROS meant that these assays were particularly vulnerable to interference by leucocytes. The remaining reagents predominantly elicited ROS generation by the sperm mitochondria and could be optimally detected by MitoSOX Red and DHE. Examination of spontaneous ROS generation by defective human spermatozoa revealed that MitoSOX Red was the most effective indicator of oxidative stress, thereby emphasizing the general importance of mitochondrial dysregulation in the aetiology of defective sperm function.