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-Glutamyl-Transpeptidase in Sperm Maturation
From the * Departments of Human Genetics and
Molecular Medicine and Cell and Developmental
Biology, Sackler School of Medicine, Tel-Aviv University, Tel Aviv, Israel;
and the Department of Chemistry and
Biochemistry, University of California, San Diego, La Jolla, California.
| Correspondence to: Dr Nechama S. Kosower, Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel-Aviv University, Tel Aviv 69978, Israel (e-mail: nkosower{at}post.tau.ac.il). |
| Received for publication February 24, 2005; accepted for publication May 6, 2005. |
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Abstract |
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-glutamyl-transpeptidase
(-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 -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 -GT pathway (in the epididymis) provide oxidizing power, leading
to physiologic sperm thiol oxidation.
Key words: Glutathione/glutathione disulfides, cysteine/cysteine disulfides, nonprotein mixed disulfides, nonprotein-protein mixed disulfides, sperm oxidative pathways
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 -glutamyl-transpeptidase (-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 -GT, which initiates the degradation of extracellular GSH, plays a
role in the processes leading to sperm PSH oxidation.
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Materials and Methods |
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Isolation of Spermatozoa and Fluid and Labeling With mBBr
a) Labeling of Sample Thiols—
Epididymal lumen content (fluid plus spermatozoa) was collected from the
caput and cauda by established procedures. Briefly, the outer surface of the
epididymis was cleaned, and the cauda and caput segments obtained from the
same animal were each sliced into 135 mM NaCl/10 mM Tris-HCl buffer, pH 7.4
(TBS), containing 2.5 mM mBBr, in the presence and absence of 0.5 mM of the
-GT inhibitor acivicin, and then agitated for 5 minutes to allow the
release of spermatozoa and the fluid contained within the lumen of the
epididymis. The spermatozoa and fluid were collected in a test tube and kept
in the dark for another 10 minutes, and a sperm count was carried out.
To determine the molar concentrations of NPSH in the EF, the epididymal caudal content was quantitatively obtained by a micropipette. A needle connected to a syringe containing paraffin oil was inserted into the upper segment of the vas deferens. The cauda epididymal content (fluid plus spermatozoa) was displaced by the paraffin oil in the syringe and was collected by a calibrated micropipette through a minute incision in the epididymis at a location where the epididymal duct was relatively wide. The micropipette content was diluted with TBS containing 0.5 mM acivicin and 2.5 mM mBBr for thiol analysis. An aliquot of the whole sample was centrifuged, and the fluid fraction was separated from the spermatozoa. The concentration of NPSH in the whole sample was calculated on the basis of the dilution of the micropipette sample in the buffer. The concentration of NPSH in the fluid fraction was estimated on the basis of a fluid volume of 45% in the whole sample (Levine and Marsh, 1971). For the sperm fraction, the NPSH was based on the sperm number per milliliter of whole sample and on a packed sperm volume of 55%.
b) Labeling of Sample Disulfides— Whole samples (fluid plus spermatozoa), obtained from the cauda epididymis by the micropipette method, and washed caput and cauda spermatozoa obtained by slicing the epididymis were analyzed for NPSSR in relation to NPSH levels. Whole samples (fluid plus spermatozoa), EF, and washed spermatozoa were divided into 2 parts. Aliquots were incubated with 2.5 mM N-ethylmaleimide (NEM) for 30 minutes at 37°C (to block the reactive thiols, allowing the determination of disulfides) or without NEM (for the determination of thiols). All samples were incubated in the presence of 0.5 mM acivicin. Excess NEM was removed by extraction with benzene. To reduce disulfides, samples were incubated with 1 mM DTT for 10 minutes at 37°C and then labeled in the dark with 3 mM mBBr. Under these conditions, the NPSSRs include NPSSRs (symmetrical and unsymmetrical, eg, GSSG, CSSC, GSSC) and nonprotein-protein mixed disulfides (NPSSPs).
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Evaluation of Sperm Thiols by Microscopy and Analysis of Sperm PSHs by Electrophoresis
Labeled spermatozoa were examined with a Leitz fluorescence microscope
equipped with epifluorescence optics and filter A (for ultraviolet [UV]
excitation range of 340-380 nm and emission above 450 nm). Photography was
carried out using identical exposure times. For analysis of PSHs, labeled
spermatozoa were solubilized in 5% sodium dodecyl sulfate (SDS) and 3%
2-mercaptoethanol at 37°C for 10 minutes in the presence of 1 mM EDTA and
0.5 mM phenylmethyl sulfonyl fluoride. Samples were sonicated for 10 seconds
to liquefy the DNA and analyzed by 12% SDS-polyacrylamide gel electrophoresis
(SDS-PAGE). Following electrophoresis, gels were photographed using UV
illumination to detect the fluorescent protein bands and then stained with
Coomassie blue and photographed again.
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Results |
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-glutamylcysteine, cysteinylglycine, and several others
that were not identified). In the caput, the values, expressed as
pmol/106 spermatozoa/mL, were 1750 ± 190 for CSH and 40
± 9 for GSH (mean ± SEM; n = 3 experiments)
(Figure 1A). In the cauda, the
values were 230 ± 50 for CSH and 31 ± 9 for GSH (mean ±
SEM; n = 5 experiments) (Figure
1B). Recovery of CSH and GSH was assessed by the addition of 6
µM CSH or GSH to some aliquots before mBBr labeling. We found that most of
the added CSH was recovered (approximately 80%). In contrast, most of the GSH
was lost (recovery of added GSH was about 5%-15%, with about 60% recovered as
CSH). The results indicated that under these conditions, the apparent higher
content of CSH (than that of GSH) was due to the conversion of GSH to CSH, via
cleavage by -GT, followed by a dipeptidase cleavage
(Kozak and Tate, 1982; Agrawal and Vanha-Perttula,
1988a). To achieve an efficient recovery of GSH when it was added
to the epididymal lumen samples, 0.5 mM of the -GT inhibitor acivicin
was added during sample preparation (85%-100% of GSH recovered). HPLC analysis
of caput and cauda lumen content obtained in the presence of acivicin showed
lower CSH levels and higher GSH levels than those obtained in the absence of
the inhibitor. In the caput, CSH and GSH levels were 940 ± 90 and 610
± 90 pmol/106 spermatozoa/mL, respectively (n = 3)
(Figure 1A); and in the cauda,
CSH and GSH levels were 82 ± 2 and 198 ± 19, respectively (n =
5) (Figure 1B). The combined
levels of CSH and GSH obtained in the presence of acivicin were similar to
those obtained in the absence of acivicin
(Figure 1), indicating that the
GSH was indeed converted to CSH in the absence of the -GT inhibitor. On
the basis of these results, all subsequent sample preparations were carried
out in the presence of 0.5 mM acivicin.
Concentrations of NPSH/NPSSR in the Cauda EF and in the Spermatozoa
To determine the molar concentrations of NPSH/NPSSR in the epididymal
lumen, we used a calibrated micropipette (see "Materials and
Methods"). Only the cauda epididymis was used in this procedure, since
it was adequate for obtaining fluid and spermatozoa from the cauda epididymis
but was not useful for obtaining caput epididymal content without injury to
the wall of the narrow caput epididymal tube. Aliquots were used for sperm
counting and for the determination of NPSHs and NPSSRs, as described in
"Materials and Methods." To assess the recovery of NPSSRs, 2 µM
GSSG was added to aliquots of samples before incubation with NEM. The recovery
of added GSSG was approximately 80%, indicating that the procedure employed
was adequate for the analysis of NPSSRs in these samples. The level of CSH in
the whole samples and in the EF was 24 ± 4 µM and 28 ± 4
µM, respectively (n = 4). Significantly less CSH was found in the
spermatozoa (6.3 ± 1.3 µM, n = 7)
(Figure 2A). The results
indicate that more CSH is present in the EF than in the spermatozoa. Similar
results were obtained for GSH. The GSH concentrations in whole samples and in
EF were 13.5 ± 3.5 µM and 16.9 ± 3.4 µM, respectively (n =
4). Washed spermatozoa contained less GSH than the fluid (8.6 ± 1.6
µM, n = 5). In the whole samples and in the fluid fractions, about half of
the total of CSH plus CSH equivalents in disulfides (CSSR, including CSSC,
nonsymmetrical small disulfides, and mixed CSS proteins) was present as CSH
(Figure 2A). Similarly, about
half of the total GSH plus GSH equivalents in disulfides (GSSR, including
GSSG, nonsymmetrical small disulfides, and mixed GSS proteins) in the fluid
was present as GSH (Figure 2B).
In contrast, the spermatozoa contained little CSH and GSH but large amounts of
the disulfides (Figure 2A and
B).
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In Vitro Effects of EF, NPSHs, and Disulfides on Sperm Thiol Status
We have previously shown that in the rat, mBBr-labeled caput spermatozoa
exhibit high fluorescence, indicative of high thiol levels, whereas cauda
spermatozoa contain low thiol levels, and mBBr-labeled cauda sperm heads are
almost totally dark (Shalgi et al,
1989). To examine the effects of EF on sperm thiol status,
unwashed caput spermatozoa (ie, samples that retained the caput EF) and washed
sperm samples (freed of EF), were incubated for 3 days at 4°C and labeled
with mBBr, and the thiol status of spermatozoa was assessed by fluorescence
microscopy. As can be seen in Figure
4, caput spermatozoa that were washed and incubated in TBS
exhibited high fluorescence in heads and tails, similar to the fluorescence
intensity shown by spermatozoa that were labeled without incubation
(Figure 4A and B). In contrast,
a diminution in fluorescence was observed in unwashed caput spermatozoa kept
for 3 days in the presence of EF. Diminution in fluorescence was especially
noted in sperm heads, which were almost totally dark, with some diminution
also observed in sperm tails (Figure
4C). Cauda sperm exhibited low fluorescence when kept in either EF
or TBS, especially in sperm heads, similar to the low fluorescence shown by
nonincubated spermatozoa (data not shown). Thus, the heads of caput
spermatozoa that were kept in the caput EF resembled cauda sperm heads.
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To examine the effects of small thiols and disulfides on sperm thiol status, washed and unwashed caput and cauda spermatozoa were treated with or without GSH, GSSG, CSH, and CSSC. As shown in the Table, the washed caput spermatozoa (incubated in the absence of EF) exhibited a high fluorescence intensity when either untreated or treated with GSH or CSH (Figure 4B; Table). Treatment of washed caput spermatozoa with the disulfides GSSG and CSSC led to a striking diminution in head fluorescence, with some diminution observed in tail fluorescence. Unwashed caput spermatozoa (incubated in the presence of EF) exhibited a low fluorescence when compared to washed spermatozoa. This effect was especially noted in sperm heads, which became dark and resembled cauda sperm heads (Figure 4C; Table). Treatment of the unwashed caput spermatozoa with GSH or CSH led to the appearance of some fluorescence, mainly noted in the sperm heads. Spermatozoa incubated with CSH exhibited somewhat a higher fluorescence intensity than those incubated with GSH. Heads of washed cauda spermatozoa, which were almost totally dark, exhibited a high fluorescence intensity when treated with GSH or CSH (Table), with a slight increase in fluorescence of the sperm tails. Treatment of unwashed cauda spermatozoa with GSH and CSH had no effect on these spermatozoa, with heads remaining dark. The results presented indicate that EF has an oxidizing effect on sperm thiols (most easily observed in sperm heads of caput spermatozoa kept in caput EF). In addition, the results demonstrate that extracellular NPSHs and NPSSRs can interact with spermatozoa and alter the thiol status of caput and cauda spermatozoa. Thus, GSSG and CSSC lead to the oxidation of sperm thiols, especially noted in the caput sperm heads. Conversely, the thiols in the buffer (GSH, CSH) lead to an increase in sperm thiols, noted in the cauda sperm heads.
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Discussion |
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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 -GT
pathway is an oxidant source for sperm PSH oxidation. -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). -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). -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
-GT-catalyzed GSH cleavage
(Dominici et al, 1999;
Filomeni et al, 2002;
Paolicchi et al, 2002), and
thus, -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 -GT inhibition, most NPSH is
present as CSH, with very little GSH present. In contrast, in the presence of
a -GT inhibitor, the GSH level is significantly higher and is
especially notable in the caput (Figure
1). The results point to -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 -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 -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
-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.
-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
-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
-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 -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
-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 -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.
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In conclusion, the results presented, along with the published data, point
to GSH catabolism via -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.
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,
high activity; 
, 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.