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ABSTRACT: Sperm thiol oxidation during sperm maturation is important for sperm component stabilization, the acquisition of sperm motility, and fertilizing ability. A correct degree of oxidation is required, since spermatozoa are very susceptible to oxidative damage. The pathways involved in physiologic sperm thiol oxidation in the epididymis are not completely understood. The nonprotein thiol glutathione (GSH), in addition to playing a major role as an antioxidant and in eliminating toxic compounds, has been implicated in prooxidation processes in various cells, via γ-glutamyl-transpeptidase (γ-GT)-dependent catabolism. Little information is available on the dynamics of nonprotein thiols (NPSHs) and disulfides (NPSSNPs) in spermatozoa and epididymal fluid (EF) during sperm passage in the epididymis. It is not clear whether NPSHs and NPSSNPs are involved in sperm protein thiol (PSH) oxidation or whether GSH catabolism in the epididymis can serve as a pathway for sperm PSH oxidation. In the present study, we used the thiol fluorescence labeling agent monobromobimane to analyze NPSHs and nonprotein disulfides (NPSSRs) (R, nonprotein or protein) in spermatozoa and EF in the rat caput and cauda epididymis. NPSH levels are shown to be significantly higher in the caput than in the cauda (spermatozoa and fluid). GSH in the caput lumen is subject to high γ-GT activity. A marked loss of sperm GSH and a shift to an oxidized state (resulting in a significantly higher concentration of glutathione disulfides [GSSRs] than GSH) occur during the passage of spermatozoa from the caput to the cauda epididymis. Caput EF and extracellular NPSSNPs induce sperm thiol oxidation. The results suggest that epididymal NPSH/NPSSNP participates in sperm PSH oxidation and that some reactions of GSH in the γ-GT pathway (in the epididymis) provide oxidizing power, leading to physiologic sperm thiol oxidation.
Mammalian spermatozoa acquire the ability to fertilize eggs during epididymal maturation while passing from the caput to the cauda epididymis. During maturation, most sperm structures such as the chromatin and tail components become stabilized (Yanagimachi, 1994). Stabilization is achieved mainly through the oxidation of thiol groups (SH) to disulfides (SS) during sperm epididymal maturation (Bedford and Calvin, 1974; Shalgi et al, 1989). An adequate condensation and stability of the chromatin is essential to protect the genome from physical and chemical agents until the spermatozoon reaches the egg. Sperm thiol oxidation is important for the induction of progressive sperm motility (Cornwall et al, 1986) as well as for capacitation, acrosome reaction, egg attachment, and fertilization (Yanagimachi et al, 1983; Saleh and Agarwal, 2002). It is also an important aspect in the protection of spermatozoa during freeze-drying (Kaneko et al, 2003).
The pathways involved in the physiologic sperm thiol oxidation in the epididymis have not been clarified. Factors within the sperm cell itself and/or in the epididymis may control sperm protein thiol (PSH) oxidation. A correct degree of oxidation is required, since spermatozoa are very susceptible to oxidative damage. Excessive activity of oxidative pathways (via reactive oxygen species and lipid peroxidation) leads to damaged spermatozoa and infertility (Maiorino and Ursini, 2002; Saleh and Agarwal, 2002; Baker and Aitken, 2004). Glutathione (GSH), which serves as a major cellular antioxidant (Kosower and Kosower, 1978; Sies, 1999), is considered important in maintaining this redox equilibrium in the mammalian testis and for protecting spermatozoa against oxidative stress. GSH and GSH-related enzymes have been studied in the testes, epididymal tissue, and spermatozoa of various species (Li, 1975; Agrawal and Vanha-Perttula, 1988a; Alvarez and Storey, 1989; Bauche et al, 1994; Lan et al, 1998; Storey et al, 1998; Tramer et al, 1998; Ursini et al, 1999; Lee et al, 2000). GSH-related enzymes have also been studied in epididymal cell cultures (Montiel et al, 2003) and in the aging rat epididymal duct following GSH depletion (Zubkova and Robaire, 2004). In the studies on GSH and GSH-related enzymes, the focus has mostly been on epididymal and sperm GSH function as an antioxidant.
In addition to a major role as an antioxidant and in eliminating toxic compounds, GSH has been implicated in prooxidation processes in various cells, via γ-glutamyl-transpeptidase (γ-GT)-dependent catabolism. GSH catabolism can lead to oxidative modifications of cellular PSHs (Filomeni et al, 2002; Paolicchi et al, 2002). Modulating effects of GSH catabolism have been observed on components of signal transduction pathways, such as those involving cell surface receptors and transcription factors (Accaoui et al, 2000; Filomeni et al, 2002; Paolicchi et al, 2002).
We have previously analyzed sperm PSHs and disulfides (Shalgi et al, 1989; Seligman et al, 1997), based on the use of the fluorescent thiol labeling agent monobromobimane (mBBr) (Kosower and Kosower, 1987). Labeling by mBBr can be carried out in the intact sperm before fractionation, thus avoiding possible thiol modification and loss during subsequent cell fractionation and analysis. These procedures allowed the morphologic evaluation and quantitative determination of SH and SS (after the reduction of SS by dithiothreitol [DTT]) in whole spermatozoa and subcellular fractions and the analysis of PSH status by electrophoretic separation of labeled sperm proteins (Kosower and Kosower, 1987).
Little information is available on the dynamics of GSH and glutathione disulfide (GSSG) and other nonprotein thiols (NPSHs) and disulfides in spermatozoa and epididymal fluid (EF) during sperm passage in the epididymis. It is not clear whether the NPSHs and disulfides (NPSSNPs) are involved in sperm PSH oxidation or whether GSH catabolism in the epididymis can serve as a pathway for sperm PSH oxidation. Identification and quantitative analysis of mBBr-labeled NPSHs at the picomole levels can be carried out by high-performance liquid chromatographic (HPLC) methods (Fahey and Newton, 1987). In the present study, we used mBBr to analyze NPSHs and NPSSRs (R, nonprotein or protein) in the spermatozoa and EF of the rat caput and cauda epididymis and examined whether small thiols and disulfides such as GSH/glutathione disulfides (GSSRs), cysteine (CSH), and cysteine disulfides (CSSRs) are involved in sperm PSH oxidation during epididymal maturation. Our results suggest that epididymal NPSH/NPSSNP participates in sperm PSH oxidation and that γ-GT, which initiates the degradation of extracellular GSH, plays a role in the processes leading to sperm PSH oxidation.
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Oxidation-reduction (redox) reactions are important to the regulation of metabolic functions of the cell and in the protection of cellular components against oxidative damage (Sies, 1991). In the case of spermatozoa, a shift of the redox status of PSHs to disulfides occurs as the spermatozoa undergo maturation during their passage through the epididymis (Maiorino and Ursini, 2002). The epididymal region in which this change occurs differs among species (Kosower et al, 1992). In the rat, spermatozoa isolated from the caput epididymis contain high levels of thiols, while cauda epididymis spermatozoa contain mostly disulfides (Shalgi et al, 1989). An accurate degree of thiol oxidation is important for acquisition of sperm motility and fertilizing ability (Cornwall et al, 1986; Seligman et al, 1991, 1992). In addition to participation in structural changes, sperm PSH oxidation promotes tyrosine phosphorylation, which is important for normal sperm function (Aitken et al, 1995; Baker and Aitken, 2004; Seligman et al, 2004).
The mechanisms and factors responsible for the physiologic oxidation of sperm PSHs to disulfides during epididymal sperm maturation are not completely understood. Several pathways leading to sperm PSH oxidation have been considered. The selenoprotein phospholipid hydroperoxide GSH peroxidase (PHGPx) uses PSHs as substrates for the reduction of hydroperoxides when GSH levels are low (Flohé et al, 2002). PHGPx is present in testicular spermatids and is assumed to protect them from oxidative damage (Roveri et al, 1992; Godeas et al, 1997; Flohé et al, 2002). In the late stages of sperm maturation in the epididymis, when sperm GSH is low, PHGPx oxidizes PSHs. At this stage, it is involved in the stabilization of the mitochondrial capsule, resulting in mature spermatozoa, when PHGPx is present as oxidatively cross-linked, enzymatically inactive, insoluble protein (Ursini et al, 1999; Flohé et al, 2002; Maiorino and Ursini, 2002). PHGPx may also play a role in sperm chromatin condensation (Godeas et al, 1997; Maiorino and Ursini, 2002).
NADPH oxidase, present in the male genital tract, has been proposed to be an important oxidant source responsible for the production of reactive oxygen species (ROS), leading to PSH oxidation. Spermatozoa have been considered a source for NADPH oxidase, in addition to the oxidase activity in leukocytes present in the male genital tract (Griveau and Le Lannou, 1997; Maiorino and Ursini, 2002; Baker and Aitken, 2004). However, mammalian spermatozoa may not possess significant NADPH oxidase activity, as indicated by recent studies (Richer and Ford, 2001; Baker et al, 2004). Thus, ROS may mainly be generated by sources external to spermatozoa. Electron leakage from the mitochondria and nitric oxide (NO) have been proposed as additional oxidant sources (Maiorino and Ursini, 2002). The ROS generated would serve dual roles. Depending on the nature, on the location in the male genital tract, and on the level of ROS, the outcome would be a physiologic or pathologic oxidation of sperm components (Griveau and Le Lannou, 1997; Maiorino and Ursini, 2002; Baker and Aitken, 2004).
The results presented suggest that GSH catabolism via the γ-GT pathway is an oxidant source for sperm PSH oxidation. γ-GT is a ubiquitous enzyme present in many tissues, including the testis, epididymis, and seminal vesicle. The enzyme is associated with the apical surface of the epididymal epithelium and is also present within the epididymal luminal fluid in vesicles or in solubilized form (Agrawal and Vanha-Perttula, 1988a; Lan et al, 1998). γ-GT acts to cleave the gamma bond between glutamate and CSH in GSH to yield cysteinylglycine (Cys-Gly) and glutamate (Kozak and Tate, 1982; Lan et al, 1998). Cys-Gly is then cleaved by dipeptidase (Kozak and Tate, 1982). γ-GT has been shown to be important in the development of male reproductive organs and their functions (Agrawal and Vanha-Perttula, 1988b; Lee et al, 2000) and is assumed to play a role in protecting spermatozoa from oxidative stress (Lan et al, 1998). However, ROS are produced as a by-product of γ-GT-catalyzed GSH cleavage (Dominici et al, 1999; Filomeni et al, 2002; Paolicchi et al, 2002), and thus, γ-GT activity can serve as an oxidant source.
We found that the total GSH and CSH level in the caput epididymis lumen (fluid and spermatozoa) is significantly higher than that in the cauda epididymis. We also found that without γ-GT inhibition, most NPSH is present as CSH, with very little GSH present. In contrast, in the presence of a γ-GT inhibitor, the GSH level is significantly higher and is especially notable in the caput (Figure 1). The results point to γ-GT activity, especially significant in the caput. The high level of CSH observed in the samples, when these are processed in the absence of the γ-GT inhibitor, is thus a result of GSH cleavage. Our results are consistent with other published observations showing higher levels of GSH in the caput epididymis than in the cauda (Agrawal and Vanha-Perttula, 1988a; Zubkova and Robaire, 2004). In addition, amino acid analysis of EF has shown the presence of CSH with significantly higher levels present in the caput than in the cauda (Hinton, 1990).
Analysis of NPSH/NPSSR levels in the spermatozoa themselves showed significant differences in GSH/GSSR concentrations between caput and cauda spermatozoa. Caput spermatozoa contain GSH in concentrations about 3 times higher than the GSSR level. Cauda spermatozoa contain about 10 times less GSH than caput spermatozoa, whereas GSSR in cauda spermatozoa is at a level similar to that of caput spermatozoa (Figure 3). These results indicate a loss of intracellular GSH during the passage of spermatozoa through the epididymis and a shift to the oxidized state. Our results are consistent with previous observations, showing that caput epididymal spermatozoa have more GSH than cauda spermatozoa or ejaculated spermatozoa (Agrawal and Vanha-Perttula, 1988a) and that spermatozoa also contain an appreciable amount of GSSG (Storey et al, 1998). The results may also explain the lack of GSH under certain assay conditions, such as sperm collected from an excised epididymis in the absence of γ-GT inhibition (Bauche et al, 1994).
It has been suggested that GSH is transported to the lumen from epithelial cells and sperm cells (Agrawal and Vanha-Perttula, 1988a). There are no actual data regarding the efflux of GSH from sperm cells in addition to its being exported from the epididymal epithelial cells. GSH synthesized within the sperm cells (from γ-glutamylcysteine) and/or converted from GSSR (by exchange reactions) may be exported into the lumen. On reaching the cauda, rat spermatozoa contain mostly disulfides (NPSSRs). Low levels of GSH and CSH are present in the cauda spermatozoa (with the major part present as GSSR and CSSR), whereas the cauda EF contains about 50% GSH (of total GSH/GSSR), with similar values for CSH/CSSR (Figure 2). These results indicate that in the cauda epididymis, the intrasperm milieu is at a more oxidized state than the surrounding fluid. The sperm cell is thus unique among other cells, since under physiologic conditions, in most tissues, the intracellular GSH level is higher than the GSSR level, and extracellular GSH levels are very low. These results are consistent with the notion that during the passage of spermatozoa in the epididymis, the activities of the reactions leading to sperm thiol oxidation and stabilization of sperm components diminish. Thus, GSH cleavage would occur mostly in the caput epididymis. γ-GT is known to be much more active in the caput epididymis than in the distal epididymal regions (Kozak and Tate, 1982; Agrawal and Vanha-Perttula, 1988b; Lan et al, 1998). Dipeptidase is also more active in the caput than in the cauda epididymis (Kozak and Tate, 1982). As a result, GSH in the cauda EF is not subjected to extensive cleavage, does not serve as a source for ROS, and may then act as an antioxidant, allowing the maintenance of reducing power in the EF in the distal parts of the epididymis. This reducing power may play a role in protecting the mature spermatozoa from excess oxidative damage to membrane components (Storey et al, 1998; Saleh and Agarwal, 2002; Baker and Aitken, 2004).
The results presented thus suggest the involvement of epididymal GSH and γ-GT in sperm PSH oxidation during sperm maturation. It is of interest to note that following testosterone withdrawal by castration, the level of cauda sperm thiols increases, indicating that testosterone withdrawal leads to inhibition of sperm thiol oxidation (Seligman et al, 1997). Since γ-GT is a testosterone-dependent enzyme (Hatier et al, 1994), testosterone withdrawal could influence PSH levels via effects on GSH catabolism.
The proposed steps involving γ-GT, GSH, CSH, GSSG, CSSC, and GSSC are summarized in the following scheme (Figure 6): GSH, present in the caput EF and/or exported from the epididymal epithelial cells, is cleaved by the very active caput epididymis γ-GT. The product Cys-Gly is further cleaved by dipeptidase to free CSH and glycine (Kozak and Tate, 1982; Lan et al, 1998). GSH and CSH are oxidized by the thiol oxidase present in the EF (Chang and Morton, 1975; Chang and Zirkin, 1978). Thiol oxidation may also be achieved via nonenzymatic chemical reactions catalyzed by metal ions (Held and Biaglow, 1994; Dominici et al, 1999; Paolicchi et al, 2002). Metal ions such as copper and iron are present in the EF (Gaur et al, 2000). CSH has been shown to autoxidize significantly faster than GSH at a pH greater than 7.0 in the presence of catalytic amounts of copper (Held and Biaglow, 1994). This autoxidation would be expected to be similarly fast with Cys-Gly. Such autoxidation has been shown to produce hydrogen peroxide (Held and Biaglow, 1994; Dominici et al, 1999; Paolicchi et al, 2002). Thus, the initial cleavage of GSH by γ-GT to Cys-Gly and CSH in the EF may generate a rapidly autoxidizing environment, giving the observed NPSSNP (ie, GSSG, CSSC, GSSC). Additional studies are required to clarify the role played by enzymatic vs nonenzymatic reactions in NPSH oxidation. The disulfides (formed by enzymatic or nonenzymatic reactions) then participate in sperm PSH oxidation (as shown in the Table). This conclusion is strengthened by the finding that sperm thiols are oxidized when spermatozoa are exposed to caput EF (Figures 4 and 5; Table). The NPSSNP may enter the spermatozoa or interact with surface thiols, initiating further thiol-disulfide interchange reactions within the cell. The NPSSNP-induced sperm PSH oxidation may also involve cross-talk between GSH/GSSG and factors such as thioredoxin (Casagrande et al, 2002), sperm-specific forms of which have been identified in the sperm tail (Jiménez et al, 2002). Possible regulation of sperm thioredoxin by GSH/GSSG requires further study.
Figure 6. . Scheme presenting the proposed steps that involve γ-glutamyl-transpeptidase (γ-GT), glutathione (GSH), cysteine (CSH), and glutathione and cysteine disulfides (GSSG, CSSC, and GSSC) in sperm thiol oxidation during sperm maturation in the epididymis. γ-GT ( low activity); Glu, glutamate; Cys-Gly, cysteine-glycine; Oxidase/Fe2+, Cu2+, oxidase and/or metal ions; PSH, protein thiols; PSSPs, protein disulfides; and NPSSPs, nonprotein-protein mixed disulfides.
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In conclusion, the results presented, along with the published data, point to GSH catabolism via γ-GT as a source for sperm thiol oxidation, in addition to other pathways thought to be involved (NADPH oxidase, NO, and PHGPx). The quantitative parts played by each remain to be studied.