Introduction

In 1943, MacLeod [1] first noted that spermatozoa incubated under high oxygen tensions rapidly lost their motility. He suggested that such loss was due to the cellular production of hydrogen peroxide (H2O2) because addition of catalase (a hydrogen peroxide scavenger) under the same conditions restored sperm movement. The notion that actively respiring spermatozoa can produce factors that inhibit cell metabolism can be traced back to studies on the phenomenon of allelostasis in populations of sea urchin spermatozoa [2]. Allelostasis refers to the increase in oxygen uptake that accompanies the dilution of sperm populations which, it was thought, might have been due to the "autointoxication" of spermatozoa as a result of oxygen metabolites generated in densely packed populations of these cells. The first clear demonstration that spermatozoa can in fact generate reactive oxygen species (ROS) was presented in a paper published in Nature by Tosic and Walton [3]. These authors identified H2O2 as an inhibitor of bovine sperm respiration and used a very simple benzidine-peroxidase reaction to quantify the generation of this powerful oxidant. In 1950, Tosic and Walton [4] produced a second more extensive paper describing the autointoxication of bovine sperm populations, which seemed to generate a potent inhibitor of respiration and motility when incubated under aerobic conditions. The inhibition was reversed by catalase and peroxidase and was chemically identified as H2O2. The particular toxicity of this oxidant towards spermatozoa was not only demonstrated in this paper, but has been subsequently rediscovered many times in several different species including man, bull, dog, fowl, ram, mouse, rabbit, and rat [5-9]. Thus, the importance of cellular ROS generation is well established in the sperm biology literature and predates the discovery of this activity in phago-cytic leukocytes by several decades.

The vulnerability of spermatozoa to ROS exposure stems from several important, and even unique, attributes of these cells. For example, spermatozoa are very well endowed with substrates for free radical attack, particularly in the form of polyunsaturated fatty acids, notably docosahexaenoic acid (22:6), which readily undergoes lipid peroxidation [10, 11]. Spermatozoa also possess additional targets for oxidative stress in the form of proteins, which can be modified by ROS to generate a wide variety of products. For example, ROS may oxidize amino acid side chains into ketone or aldehyde derivatives or induce the formation of nitrotyrosine residues, advanced glycation end products, or protein adducts via their interaction with the electrophilic products of lipid peroxidation, such as 4 hydroxynonenal [12-14]. Of course, another major target for oxidative stress in the male germ line is the DNA in the sperm nucleus and mitochondria [15]. Where and how such oxidative attacks arise is the subject of some speculation; however, the consequences of this stress, in terms of the functionality of the spermatozoa and their capacity to support normal embryonic development, are beyond reasonable doubt [16, 17]. In light of these considerations, it is clearly important that we develop reliable, robust methods for detecting the generation of ROS by mammalian spermatozoa. The issues that have to be addressed in developing such diagnostic tests vary somewhat between species, so at the outset it is important to define the context in which such diagnostic procedures will be applied—before discussing their technical idiosyncrasies. For the purpose of this discussion, we shall focus exclusively on the diagnosis of ROS generation by human spermatozoa.

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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