Oxidative Stress in the Seminiferous Tubules and Epididymis

As previously indicated, oxygen radical-induced DNA damage may take place in germ cells during the process of spermatogenesis leading to sperm DNA fragmentation and telomere damage. This may be the result of either an increase in oxygen radical production, a decrease in the expression of antioxidant enzymes or both. Although in most mammalian species the process of spermatogenesis results in the release of mature sperm into the seminiferous tubules, in some species, including the human, a significant proportion of the spermatozoa released have not completed their process of maturation and are therefore considered immature. This process of incomplete maturation that has been designated as defective spermiogenesis is characterized by the release into the seminiferous tubules of immature spermatozoa with proximal cytoplasm retention that produce very high levels of oxygen radicals [23, 24]. During spermiogenesis, round spermatids experiment a process of cell remodelling that involves the release of excess membranes from the midpiece and loss of docosahexaenoic acid (DHA) from the plasma membrane [25]. Those stages, at which elongating spermatids with retention of excess cytoplasm and high content of phospholipid-bound DHA are found in the seminiferous tubules epithelium, entail a high risk for the occurrence of intratesticular oxygen radical-induced germ cell DNA damage. During the normal process of spermiogenesis, there are several stages at which some specific elongating spermatids, given its particular content in NADPH, NADPH oxidase, NADH oxidase, superoxide dismutase (SOD), catalase, mitochondria, DHA and high rate of production of oxygen radicals, become an oxidative liability to the seminiferous tubules epithelium [26] . In fact, it has been shown in the rat model that during those stages at which these elongating sperma-tids with this particular enzymatic profile and high DHA content are found (stages III-VI in the rat), there is a significant increase in the expression of SOD mRNA in the Sertoli cell, most likely in an effort to protect the seminiferous tubules epithelium against the potential oxidative damage that could be produced during these stages [27]. However, in species such as the human, a significant proportion of these elongating spermatids does not complete the process of maturation and are released prematurely into the lumen of the seminiferous tubules. Should that occur, co-migration of oxygen radical-producing immature spermatozoa with mature sperm through the epididymis may lead to DNA damage in mature sperm. Since (1) sperm are highly packed in the epididymis and therefore immature sperm are in close contact with mature sperm and (2) the lifespan of oxygen radicals in the extracellular medium is of the order of nano- to microseconds, the close contact between mature and immature spermatozoa in the epididymis facilitates the occurrence of oxygen radical-induced DNA damage.

Recent studies have shown that the sperm membrane and DNA damage observed in ejaculated spermatozoa is produced, for the most part, in the epididymis [1,2]. This is supported by the fact that in 90% of the cases, the levels of DNA damage observed in ejaculated spermatozoa are significantly higher than those observed in testicular sperm [1, 28-30]. This DNA damage may be induced directly through the impact of the free radicals on the DNA strands or indirectly through the activation of sperm caspases and/or endonucleases [2]. Direct DNA damage induced by free radicals may take place through three main mechanisms: (1) DNA damage induced by oxygen radicals produced by immature sperm. As previously indicated, immature sperm, especially those with proximal cytoplasm retention in the midpiece, produce very high levels of ROS and may produce cross cell damage of mature sperm [23, 24, 26]; (2) DNA damage induced by oxygen radicals utilized in the oxidation of disulphide bridges of protamines in the sperm chromatin. In the final segment of the caput epididymis, a recycling mechanism of oxygen radicals utilized in the oxidation of disulphide bridges has been described [31], Alteration in the normal balance of this recycling with leakage of oxygen radicals could lead to oxidative damage of sperm DNA; and (3) DNA damage induced by oxygen radicals produced by the epithelial cells of the epididymis. Another mechanism that could lead to sperm DNA damage in the epididymis would be that related to the production of free radicals by epithelial cells from the epididymis coupled to the low levels of antioxidant enzymes in both the epithelium and the lumen of the epididymis [31]. Antioxidant enzymes such as the different isozymes of glutathione peroxidase (GPX) play a central role in the protection of both the epididymal epithelium and spermatozoa during their passage through the epididymis [31] , It has been shown that this damage progressively increases from the caput to the cauda epididymis and is related to a decrease in the levels of the isozyme GPX-5 [31],

In a study carried out in infertility patients, it was found that co-incubation of sperm suspensions with phytohaemagglutinin-activated polymorphonuclear leukocytes in vitro produces a significant increase in the production of oxygen radicals by immature sperm [32]. The authors of this study conclude that proinflammatory factors produced by activated leukocytes amplify oxygen radical production by immature sperm by 2-3 orders of magnitude. Therefore, in patients in whom there may be an increase in the levels of proinflammatory factors in the epididymis, e.g. inflammatory or infectious processes [33], an increase in DNA damage may take place. In addition, since it has been suggested that the epididymis is in a chronic pseudoinflammatory state [31], proinflammatory factors may play an important role in the pathophysiology of oxygen radical-induced sperm DNA damage in the epididymis. This explains, at least in part, the beneficial effects of antibiotics in patients with high levels of sperm DNA damage in ejaculated sperm [33] .

a

Degraded sperm

f

b

A

% #

• »

f

Normal sperm

ë

c

«

Fragmented sperm

Fig. 11.2 Different patterns of sperm DNA damage in a patient with varicocele. Samples were assessed for sperm DNA damage using the Halosperm test. (a) General view of a sample processed using the Halosperm test and stained with Gel Red. Note the different protein/DNA removal on sperm containing fragmented DNA (small halo or no halo) when compared with a normal sperm head (large halo of chromatin dispersion). (b) Standard spermatozoa presenting fragmented DNA; (c) Sperm with "degraded" chromatin

Fig. 11.2 Different patterns of sperm DNA damage in a patient with varicocele. Samples were assessed for sperm DNA damage using the Halosperm test. (a) General view of a sample processed using the Halosperm test and stained with Gel Red. Note the different protein/DNA removal on sperm containing fragmented DNA (small halo or no halo) when compared with a normal sperm head (large halo of chromatin dispersion). (b) Standard spermatozoa presenting fragmented DNA; (c) Sperm with "degraded" chromatin

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