Báo cáo Y học: The expression of glutathione reductase in the male reproductive system of rats supports the enzymatic basis of glutathione function in spermatogenesis doc

The expression of glutathione reductase in the male reproductive system of rats supports the enzymatic basis of glutathione function in spermatogenesis Tomoko Kaneko 1,2 , Yoshihito Iuchi 1 , Takashi Kobayashi 1,3 , Tsuneko Fujii 4 , Hidekazu Saito 2 , Hirohisa Kurachi 2 and Junichi Fujii 1 Departments of 1 Biochemistry, 2 Obstetrics and Gynecology, and 3 Urology, Yamagata University School of Medicine, Yamagata, Japan; 4 Cell Recovery Mechanisms, RIKEN Brain Science Institute, Japan Glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) using an NADPH as the electron source. The function of GR in the male genital tract of the rat was examined by meas- uring its enzymatic activity and examining the gene expression and localization of the protein. Levels of GR activity, the protein, and the corresponding mRNA were the highest in epididymis among testes, vas deferens, seminal vesicle, and prostate gland. The l ocalization of GR, as evidenced by immunohistochemical techniques, reveals that it exists at high levels in the epithelia of the genital tract. In testis, GR is mainly localized in Sertoli cells. The enzymatic activity and protein expression of GR in primary cultu red testicular cells confirmed its predom- inant expression in Sertoli cells. Intracellular GSH levels, expressed as mol per mg protein, was higher in sperma- togenic cells than in Serto li cells. As a result of these findings, the effects of buthionine sulfoximine (BSO), an inhibitor f or GSH synthesis, a nd 1,3-bis(2-chlorethyl)-1- nitrosourea (B CNU), a n inhibitor f or GR, on c ultured testicular cells were examined. Sertoli cells were prone to die as the result of BCNU, but not BSO treatment, al- though intracellular levels of GSH declined more severely with BSO treatment. Spermato genic cells were less sensitive to these agents than Sertoli cells, which indicates that the contribution of these enzymes is less significant in sper- matogenic cells. The results herein suggest that the GR system in Sertoli cells is involved in the supplementation of GSH to spermatoge nic cells in which high levels of cysteine are required for protamine synthesis. In turn, the genital tract, the epithelia of which are rich in GR, functions in an antioxidative manner to protect sulfhydryl groups and unsaturated fatty acids in spermatozoa from oxidation during the maturation process and storage. Keywords: glutathione reductase; spermatogenic cell; Sertoli cells; spermatozoa; epididymis. Oxidation affects the spermatozoa in complex ways; either triggering hyperactivation or the suppression of motility largely d epending on conditions [1–3]. The quality of spermatozoa can be evaluated using reagents that distin- guish oxidized thiols from others [4]. Thiol oxidation occurs in the nuclei and the tail during the maturation process in the epididymis. The greater the extent of oxidation in nuclei, the more potent are the spermatozoa. In addition, hyper- activation of spermatozoa is triggered by reactive oxygen species (ROS) [1,5]. On the other hand, oxidation by ROS has been reported to decrease sperm motility [6,7]. Thus, the effects of ROS on spermatozoa are both beneficial and detrimental. In human spermatozoa,  40% by weight of the total fatty acid fraction is composed of polyunsaturated fatty acids, which, in turn, enable spermatozoa t o be more motile [8]. Docosahexaenoic acid comprises more than 60% of the total polyunsaturated fatty acids [9]. As polyunsat- urated fatty acids are vulnerable to peroxidation by ROS, and peroxidized lipids and carbonyl compounds produced by this reaction are toxic to spermatozoa [10,11], protection against oxidative stress is prerequisite for the production of functional sperm. Glutathione has pleiotropic roles, which include the maintenance of cells in a reduced state, serving as an electron donor for certain antioxidative enzymes, and the formation of conjugates with some harmful endogenous and xenobiotic compounds via catalysis of glutathione S-transferase [12]. Levels of the reduced form of glutathione (GSH) are ma intained by two s ystems. One is de novo synthesis from b uilding blocks, glutamate, cysteine, and glycine, via two ATP-con suming s teps involving c-g lut- amylcysteine synthetase ( cGCS) and glutathione synthetase. The other constitutes a recycling system involving GR which reduces oxidized glutathione (GSSG) back to GSH in an NADPH-d ependent manner. In addition to the direct interaction of GSH with ROS, GSH serves a s a n e lectron donor for s ome peroxidases, i ncluding glutathione peroxi- dase [13] and p eroxiredoxins [14,15]. The resulting o xidation Correspondence to J. Fujii, Department of Biochemistry, Yamagata University School of Medicine, 2-2-2 Iida-nishi, Yamagata City, Yamagata 990-9585, Japan. Fax: + 81 23 628 5230, Tel.: + 81 23 628 5227, E-mail: jfujii@med.id.yamagata-u.ac.jp Abbreviations: GR, glutathione reductase; ROS, reactive oxygen species; GSH, glutathione, reduced form; GSSG, glutathione, oxidized form, cGCS, c-glutamylcysteine synthetase; DTNB, 5,5¢-dithiobis (2-nitrobenzoic acid); BSO, buthionine sulfoximine; BCNU, 1,3-bis [2-chlorethyl]-1-nitrosourea. Enzyme: glutathione reductase ( EC 1.6.4.2). (Received 2 October 2001, revised 1 8 January 2002, accepted 23 January 2002) Eur. J. Biochem. 269, 1570–1578 (2002) Ó FEBS 2002 product, GSSG, is either recycled by GR via electron transfer from NADPH or pumped out of the cells. Thus, GR indirectly participates in the protection o f cells against oxidative stress. The enzymatic activities of GR have been investigated in various tissues under physiologic and pathologic conditions [16], but only few reports have focusedonthelocalizationoftheGRproteinintissues[17]. Significant functions of GSH in spermatogenesis and the reproductive process have been reported [13]. However, the reported values for GSH in spermatozoa are controversial and h ave been reported t o be high in dog, goat, ram, and human spermatozoa [18], but below detectable levels in the frog [19], bull [20,21] and rat [22]. Estimation of GR activity as well as that of glutathione peroxidase has been reported in mammalian s permatozoa and seminal plasma including human [7,23,24]. Although t he pivotal role of GSH and its recycling enzyme in reproductive process is now well recognized [13], no data has yet been presented that shows the localization o f GR, histologically, in the male genital tract. We reported the localization and estrous-cycle-dependent induction of GR in the female genital tract in rats [17] using a specific antibody against rat GR and its cDN A as probe for R NA analysis [25]. In the present report, the potential role of GR is e valuated by biochemical as well as immunohistochemical analyses of the male genital tract in rats. The results suggest a pivotal role for the GSH/GR system in spermatogenesis and the maintenance of sperma- tozoa quality during the maturation process, particularly in the epididymis. EXPERIMENTAL PROCEDURES Materials GSH and GSSG were purchased from Roche (Mannheim, Germany). NADPH and yeast GR were obtained from Oriental Yeast (Tokyo, Japan). 5,5¢-Dithiobis(2-nitroben- zoic acid) (DTNB) was from Kanto Chemicals (Tokyo, Japan). 2-Vinylpyridine was from Wako Pure Chemicals (Osaka, J apan). Buthionine sulfoximine (BSO) and 1,3- bis[2-chlorethyl]-1-nitrosourea (BCNU) were obtained f rom ICN and Sigma, respectively. All other reagents used were of the highest available quality. Animals All experiments were performed under protocols approved by the Animal Research Committee from Yamagata Uni- versity School of Medicine. Wistar rats, purchased from Japan SLC, were m aintained under conventional conditions. Three rats were used for each data point. Tissues, which were obtained under anesthesia with diethyl ether were either fixed immediately in B ouin solution for immunohistochem- ical analysis or frozen under liquid nitrogen and preserved at )80 °C until used for enzyme and mRNA assays. Preparation of tissue homogenates and protein assay Tissues were homogenized in NaCl/P i containing 10 lgÆmL )1 pepstatin, 10 lgÆmL )1 leupeptin, 100 l M phe- nylmethylsulfonyl fluoride, and 1 m M benzamidine with a Physcotron homogenizer (Nichion, Tokyo, Japan). After centrifugation at 10 000 g for 20 min, the supernatant was collected and kept at )20 °C. Protein concentrations were determined using a BCA kit (Pie rce, Rockford, IL). Testicular cell culture Male Wister rats (40–50 days) were killed by diethyl ether anesthesia, and testicular ce lls were isolated as reported previously [26]. Briefly, after decapsulation of the testes, the seminiferous tubules were minced using scissors and incu- bated i n NaCl/P i containing 0.25 % type I collagenase (Wako, Osaka, Japan) at 32.5 °C for 15 min. The semin- iferous tubules were washed with NaCl/P i once and then incubated in NaCl/P i containing 0.25% trypsin (Difco, Detroit, MI, USA) at 32.5 °C for 15 min. After the addition of fetal bovine serum to a concentration of 1 0%, the cell suspension was fi ltered through n ylon mesh to remove aggregates and t issue d ebris. The cells were cultured in an equal mixture of F12-L15 medium s upplemented with 100 U ÆmL )1 penicillin G, 100 lgÆmL )1 streptomycin, 15 m M Hepes (pH 7.3), and 10% fetal bovine serum at 32.5 °Cwith5%CO 2 under humidified conditions. After harvesting the cells with a silicon scraper, they were washed twice with NaCl/P i andsonicatedinanextractionbuffer (25 m M Tris/HCl, pH 7.4, 50 m M NaCl, 10 lgÆmL )1 aprotinin, 10 lgÆmL )1 leupeptin, and 20 l M p-amidi- nophenylmethanesulfonylfluoride hydrochloride). The soluble fractions, after centrifugation at 10 000 g for 20 min at 4 °C, were subjected to protein analyses and enzyme assays. Enzyme assay GR activity was determined spectrophotometrically by measuring the rate of NADPH oxidation at 340 nm [16]. The reaction m ixture consisted of 0.1 M potassium phos- phate,pH7.0,1m M EDTA, 0.1 m M NADPH, 1 m M GSSG, and tissue samples. The decrease in absorbance at 340 nm at room temperature was recorded. As the decrease in absorbance for the control reaction mixture without GSSG or tissue sample was negligible, the contribution o f spontaneous NADPH oxidation and other red uctases in the samples can be ignored. One unit of GR activity was d efined as the amount of enzyme that catalyzes the oxidation of 1 lmol of NADPH per min. All assays were performed on triplicate samples, and means ± SD are reported. Measurement of total glutathione and GSSG Total g lutathione and G SSG were determined by the recycling method described by Anderson [27]. Briefly, cultured cells were collec ted and washed t wice with NaCl/ P i . The precipitated cells were sonicated in 5% 5-sulfosul- icylic acid. After centrifugation a t 8000 g for 10 min, the supernatant (25 lL) was applied to a reaction mixture containing 100 m M sodium phosphate buffer, 5 m M EDTA, 200 U ÆmL )1 yeast GR, 0.1 m M NADPH, and 5 m M DTNB. The absorbance at 412 nm was continuously recorded using a spectrophotometer. For GSSG quantifi- cation, free thiols in the samples were derivatized with 2-vinylpyridine and subjected to the recycling assay. The concentrations of total g lutathione and GSSG were calcu- lated from the absorbance change using authentic GSH and Ó FEBS 2002 Glutathione reductase in male reproduction system (Eur. J. Biochem. 269) 1571 GSSG as a standard and the data are expressed as nmolÆmg protein )1 . SDS/PAGE and Western blot analysis Protein samples were subjected to 10% SDS/PAGE [28] and then transferred onto Hybond-P (Amersham Pharmacia, Pscataway, NJ, U SA) under semi-dry conditions by means of a Transfer-blot SD Semi-dry transfer cell ( Bio-Rad, Tokyo, Japan). The membranes were then blocked by incubation with 5% skimmed milk in NaCl/Tris (20 m M Tris/HCl, pH 8.0, and 150 m M NaCl) containing 0.1% Tween-20 for 2 h at room temperature. The membranes were then incubated with a rabbit anti-(rat GR) Ig (1 : 1000 dilution) [25] or a goat anti-(follicle-stimulating hormone receptor) Ig (Santa Cruz Biotechnology, Santa Cruz, CA, USA) fo r 1 2 h a t 4 °C. After washing with NaCl/Tris containing 0.1% Tween-20, the membranes were incubated with 1: 1000 diluted peroxidase-conjugated goat anti-(rabbit IgG) Ig (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. Following the washing, p eroxidase activity w as detected by a chemiluminescence method using an ECL Plus kit (Amersham Pharmacia, Buckinghamshire, UK). Preparation of total RNA Total cellular RNAs were isolated from several rat tissues by homogenization using the guanidine thiocyanate/phenol/ chloroform extraction method [29] using Isogen (Nippon Gene, T okyo, Japan). The final pellet was dissolved in diethylpyrocarbonate-treated H 2 O and was quantified by an absorbance measurement at 260 nm. Northern blot analysis Total RNAs, 5 or 10 lg per lane, were electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde [30]. The size-fractionated RNAs w ere transferred onto a Hybond-N membrane (Amersham P harmacia Biotech) by capillary action. After h ybridization with the 32 P-labeled rat GR cDNA probe [25] at 42 °C in the presence of 50% formamide, the membranes were washed twice for 20 min at 55 °Cin2· NaCl/Cit (1 · NaCl/Cit: 150 m M NaCl and 15 m M sodium citrate, pH 7.5) containing 0.1% SDS and then twice in 0.2 · NaCl/Cit. T he Kodak XAR films were exposed with a n intensifying screen at )80 °C. Immunohistochemistry For the immunohistochemical study, the DAKO Envision System (DAKO Co., Carpinteria, CA, USA) was employed [31]. This system i s based ona horseradish peroxidase-labeled polymer that is conjugated with secondary antibodies. Paraffin-embedded tissue blocks were cut on a microtome at a thickness of 5-lm and the resulting serial sections were mounted on silianized slides. After deparaffinization and rehydration, endogenous peroxidase activity in the sectioned tissues was in activated with 0.1% hydrogen peroxide. The target retrieval procedure involves the immersion of tissue sections in a citrate based buffer solution and heating in an autoclave. Tissue s ections were briefly treated with por cine serum for 10 min to b lock nonspecific binding and then reacted with anti-(rat GR) Ig for 60 m in at a 1 : 200 dilution. The sections were sequentially reacted w ith peroxidase-labeled goat anti- (rabbit IgG) Ig polymer for 3 0 m in, a nd 3,3-diaminobenzi- dine for 1 min. Nonimmune rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a control. Photographs were taken with a digital camera under light microscopy (Olympus BX50, Tokyo, Japan). RESULTS Levels of GR in male reproductive system of the adult rat To identify the cells responsible for GSSG reduction, an attempt was m ade to determine the localization of GR in the male reproductive system of the rat. We fi rst measured GR activity in the cytosolic fractions from testes, epididy- mis, seminal vesicles, vas deferens, and prostate gland in 13-week-old-adult rats (Fig. 1A). The highest activity for GR was f ound in the epididymis, followed by seminal vesicles, prostate gland, vas deferens, and testes. GR activity was negligible in spermatozoa. To evaluate the levels of these enzymes in tissues and spermatozoa, a W estern blot analysis was c arried out (Fig. 1 B) using the anti-(rat GR) Ig. Preincubation of the tissue extract with this antibody completely abolished GR activity (data not shown). The 50-kDa band, corresponding to the GR protein, was observed in a ll tissues except for spermatozoa. A faint GR band c ould be detected in spermatozoa only a fter a longer exposure. Thus, the order of band intensities matched the levels o f enzymatic activity. A Northern blot analysis of total RNAs extracted from the same tissues showed some inconsistent results between the protein and the mRNA for GR (Fig. 1 C), which appears to be due to different turnover rates of the protein in t hese tissues. Immunohistochemical localization of GR in male reproductive system of the adult rat We employed immunohistochemical a nalyses of G R in order to determine its localization in the male rep roductive system of the adult rat (Fig. 2). Epithelia from the epididymis, vas deferens, seminal vesicles, and prostate glands were strongly stained w ith the GR antibody. In the case of testes, t he immunoreactivity to the anti-GR Ig was strong in Sertoli cells. Concerning the intracellular distribu- tion, positive signals to the an tibody were found mainly in the cytoplasm. However, nuclei of the epithelial cells of the epididymis and vas deferens were also stained, as has also been reported in uteri [17]. Activity and expression of GR in primary cultured spermatogenic and Sertoli cells To identify cells that express GR more clearly, testicular cells were separated under the culture con ditions. As Sertoli cells bec ome attached to conventional plastic plates at 24 h after separation a nd grow, but spermatogenic cells do not, they could be separated by transferring the culture medium containing floating cells [32]. The attached cells were confirmed as Sertoli cells by Western blot analysis using an antibody against the follicle-stimulating hormone recep- tor (data not shown). The attached cells were spread and 1572 T. Kaneko et al. (Eur. J. Biochem. 269) Ó FEBS 2002 amorphous, and, thus, consistent with the shape of Sertoli cells, while the floating cells did not become attached to the dishes and were round and variable in size ( data not shown). Figure 3A shows the GR activity of the separated cells. The GR activity in Sertoli cells was about eightfold higher than that of the spermatogenic cells. A Western blot of proteins from both cells indicated that GR, 50 kDa in size, was expressed in the Sertoli cells, but was below the detectable levels in spermatogenic cells (Fig. 3B). Thus, a high expression of GR in Sertoli cells can b e fully confirmed. Changes of GR expression in testes during sexual maturation To examine relationships between the expression of GR with sexual maturation, Western blot and Northern blot analyses as well as an enzyme assay w ere carried out on testes from premeiotic stage (14 days), meiotic stage (21 d ays), early haploid stage (25 days), and late round/ elongating spermatid stage (30 days) [33] and 13-week-old adult rats (Fig. 4). The highest activity was found in the youngest rats at 14 days of age. GR has complex charac- teristics and has both active and inactive states that are interchangeable by redox conditions. As oxidation of the enzyme makes its activity low, GR may be kept in the oxidized form in the developed rats. Concerning expression in Sertoli c ells, no d ifference was observed during this period by immunohistochemistry (data not shown). As the activity difference was small, the difference i n intensity of the bands for the protein and the mRNA were faint. Effects of inhibitors of cGCS and GR on primary cultured testicular cells To investigate t he contribution of de novo synthesis and the recycling of GSH to the intracellular g lutathione pool, the Fig. 2. Immunohistochemical localization of GR in the male reproduc- tion system of adult rats. Sections of an adult male rat were treated with 1 : 200 dilution of anti-GR Ig. Photographs were taken with a digital camera using light microscopy; 130· magnification: ( A) testis; (C) epididymis, head; (E) epididymis, tail; (F) vas deferens; (G) seminal vesicle; (H) p rostate gland. 650· magnification: (B) testis; ( D) epidi- dymis, head. Fig. 1. GR activities, protein, and mRNA levels in the m ale repro- duction system of adult rats. (A) Enzymatic activities of 90 lg cytosolic proteins from five organs and spermatozoa of 13-week-old- rats were measured in a 1-mL cuvette. Data a re presented as the means ± SD of three rat organ s. (B) Twenty micrograms of soluble protein was subjected to Western blot analyses with the GR antibody ata1:1000dilution.Typicaldatafromseveralexperimentsare shown. Th e a rrowh ead i ndicates the position o f G R (50 kDa). (C) Total RNAs (10 lg) from rat testes at the indicated ages, which were separated on a 1% agarose gel. Ó FEBS 2002 Glutathione reductase in male reproduction system (Eur. J. Biochem. 269) 1573 levels of GSH and GSSG were measured in cultured Sertoli cells and spermatogenic ce lls after treatment with BSO, an inhibitor of c-GCS, and BCNU, an inhibitor of GR (Fig. 5 ). The spermatogenic cells contained GSH levels which were about twice as high as those of Sertoli cells. Treatment of Sertoli cells with 100 or 1000 l M BCNU totally inhibited the GR activity (data not shown). After a 24-h incubation, the total glutathione level in the Sertoli cells was decrease d to 8 and 14% of the control by 10 m M BSO and 1 m M BCNU, respectively (Fig. 5A). However, these agents were less potent to the spermatogenic cells. GSSG levels increased only s lightly after BCNU tre atment. This is likely due to the excretion of the GSSG from the cells by a specific transporter. We then examined changes in total glutathione levels in spermatogenic cells up to 3 days after the addition of these agents (Fig. 5B). Intracellular glutathione levels decreased spontaneously during the culture period. The presence of BSO accelerated this decline, but BCNU was not as effec tive as BSO. The rate of reduction in glutathione levels with BCNU did not appear to be different from that of the spontaneous decrease and corresponded to the low level of GR content in t he spermatogenic cells. W hen morphology of the cells were observed by a light microscope, a marked effect was observed only for BCNU-treated Sertoli cells (Fig. 6 ). Thus, BCNU exerted a more toxic effect on Sertoli cells than on spermatogenic cells. DISCUSSION The findings in t his study show that GR is highly expressed in epithelial cells of the m ale genital tract, especially in the Fig. 4. Decrease in the levels of GR in testes around pubertal stages. (A) Enzymatic activities o f 90 lg cytosolic p roteins from pre- and post- pubertal as well as adult rat testes were measured. Data are presented as the means + S D for three rat organs. (B) Ten micrograms of pro- teins from testes were subjected to Western blot analysis with a 1 : 3000 d ilution of th e anti-GR Ig. (C) Total RNAs (5 lg) from rat testes at the indicated ages w ere separated on 1% agarose ge l. The blotted membrane was hybridized with the rat GR cDNA probe. Fig. 3. GR activity and the protein in primary cultured testicular cells. Testicular cells were primary cultured from a 5-week-old rat. After isolation from the seminiferous tubules, the cells were plated on a conventional 9-cm dishes. After 24 h, unattached spermatogenic cells were separated from the attached Sertoli cells and cultured for 24 h at 32.5 °C. The Sertoli cells were grown for a further 3 days. Cytosolic proteins were extracted from the harvested cells. (A) The GR activity in floated spermatogenic cells (left) and the attached Sertoli cells (right). (B) Expression of GR was analyzed by Western blot for 10 lg proteins with the anti-GR Ig. 1574 T. Kaneko et al. (Eur. J. Biochem. 269) Ó FEBS 2002 epididymis, and Sertoli cells in the seminiferous tubules in the rat (Figs 2 and 3). The specific activity of GR decreased during sexual maturation in testes, while the total activity increased (Fig. 4). This can be attributed to the proliferation of spermatogenic cells around the pubertal stage, and the reduction in the population of Sertoli cells relative to the spermatogenic c ells. Even though the GR content was very low in spermatogenic cells, their GSH levels were somewhat higher than in the Sertoli cells (Figs 3 and 5). According to Fig. 5A, the increase in the G SSG level i n BCNU-treated Sertoli cells did not correspond to the decrease in levels o f total glutathione. Thus the excretio n of GSSG is only mechanism for Sertoli cells. M uch weaker effects of BSO and BCNU on spermatogenic cells suggest that they are resistant to these reagents with unknown mechanism. Although GR, glutathione peoxidase and superoxide dismutase are all present in seminal plasma, with their origin thought to be the prostate [34], the activity of GR was low in prostate tissues (Fig. 1 ). It is well known that the matur- ation of spermatozoa proceeds via oxidation and reduction processes in the male genital tract, especially in the epididymis. Disulfide bond formation in p rotamine, a counter part of histone in sperm nuclei, is required for packaging the DNA into the small head space a nd to Fig. 6. Sertoli cell death by BCNU treatment. Morphology of the Sertoli c ells in Fig. 5 is s hown at 6 h after the addition of BSO or BCNU. Magnification ¼ 50·. Fig. 5. Effects of BSO and BCNU on intracellular glutathione levels in the spermatogenic and Sertoli cells. After isolation from seminiferous tubules, the cells were cultured fo r 24 h at 32.5 °C. Spermatogenic cells floating in the culture media were transferred to dishes and were treated with BSO (10 m M )orBCNU(1 m M ). Sertoli cells were treated with the same reagents after incubation for 4 d ays. (A) The cells were harvestedafter24hincubationwiththereagentsandassayedfortotal glutathione and GSSG. SPG, spermatogenic ells; T, total glutathione (GSH + GSSG); O, oxid ized glutathione (GSSG). (B) Total g luta- thione levels were measured for s permatogenic cells a fter separation from Sertoli cells for 3 days. Ó FEBS 2002 Glutathione reductase in male reproduction system (Eur. J. Biochem. 269) 1575 maintain sperm motility during the fertilization process. In contrast, ROS, which generally mediates sulfhydryl oxida- tion, causes ma le infertility [ 35]. Spermatozoa show a high vulnerability to oxidative stress [1], which would lead to the peroxidation of polyunsaturated fatty acids, which are present at high levels in the plasma membrane. Thus, oxidation should o ccur in a regulated way in the m ale genital tract. Uncontrolled oxidation occurs during heat stress [26] and inflammation in the testes [6] and causes apoptosis of spermatogenic cells. Reducing power, on the other hand, is essential for male pronuclear formation, which appears to be related to the reduction of disulfide bonds in th e nucleus [36–38]. As GSH is a major source of reducing power in the oocyte [36,39], a high content of G R would be responsible for this [17]. Among the many changes that occur in spermatozoa during their migration through the epididymis, the oxida- tion of sulfhydryls is related to the acquisition of motility [4,5]. During epididymial migration, sulfhydryls in the nucleus and t he tail of spermato zoa are oxidized to disulfides. Levels of antioxidative enzyme a ctivities alter during this process [40]. Thus, it would be expected that the reductant level inside the epididymis would be l ow. How- ever, we found a high level of GR in the epithelia of the epididymis (Fig. 1 ). As the epididymis is rich in sulfoxidase, the oxidation of sulfhydryls in the sperm head and tail would be mediated by this enzyme in a specific manner. The predominant expression of GR suggests that epididymal fluid as well as epithelia is protected from nonspecific oxidation. Materials extracted from the epididymis c ontain numerous proteins [41,42]. Several antioxidative enzymes are present in this tissue, as well as the ductal fluids. These include selenium-containing GPX3 and GPX4, which are commonly found in other tissues, as well as epididymis- specific GPX [43]. T hese enzymes m ay be responsible for the reduction of coincidently produced toxic p roducts, such as lipid hydroperoxides. Hence, GR would be responsive, in terms of providing redox equivalents to GPX through GSH from NADPH. Many metabolites, including steroid hormones, glycation reaction intermediates, and lipid peroxidation products, are produced and are largely detoxified by certain aldo-keto reductases. Srivastava et al. [44] demonstrated that gluta- thione conjugates of 4-hydroxy-2-nonenal actually serve as a substrate for aldose reductase. A high level of expression of aldose reductase was also detected in the epithelia of the male genital tract. In addition, glutathione S-transferase is present i n the male re productive system [45]. Thus, the abundant expression of GR in cooperation with glutathione S-transferase could facilitate the detoxification function o f aldose reductase by catalyzing the formation of glutathione conjugates. The enzymatic activity of GR in pachytene spermatocytes and round spermatotids is low compared to Sertoli cells, and the GR activities and GSH contents are below the detectable level in the spermatozoa of the rat [22]. However, the spermatogenic cells contain quite high levels of GSH (Fig. 5 ). If the reducing power is too low, this would render spermatogenic cells prone to apopotosis by ROS and other stimuli [46,47]. Given the reported data here and our collective observations, some simple questions can be raised. What is the origin of the h igh level of GSH in spermatogeinc cells? If GSH is important for the protection of spermatozoa from oxidative damage, why is the GR content so low in spermatogenic cells? This inappropriate distribution, at first glance, may be attributed to the unique protein metabolism in these cells. Histone is rich in the basic amino acids, lysine and arginine, but contains no cysteine. Protamine, which is replaced for hitstone via transit proteins during spermio- genesis, is rich in both arginine and cysteine. The cysteine sulfhydryls in protamine must form disulfide bond to package DNA into small sperm head during the maturation process. Thus, while cysteine, a building b lock of GSH, is required for spermatozoa, the presence of GSH presents somewhat of an obstacle due to its reducing power. As t he biosynthetic rate of protamine production is very high during the spermiogenic process, large a mounts of cysteine are required for these cells. Sertoli cells are known to provide a variety of nutrients to spermatogenic cells, and GSH synthesis in these cells is regulated by i nteractions with Sertoli cells [19]. Such circumstantial evidence suggests that Sertoli cells may provide GSH as a source for c ysteine, as well as reducing power to the spermatogenic cells. The significant role of GSH from Sertoli cells in the supply of cysteine is also supported by data on c-glutamyl transpeptidase-deficient mice [48]. This knockout mouse has a reduced size of testis and s eminal vesicle and is severely oligospermic and infertile. The administration of GSH or N-acetylcysteine, a mem- brane permeable pre cursor of cysteine, totally restores the testis and seminal sizes to values which are comparable to those of wild-type mice a nd which render the mutant mice fertile. T his indicates t hat the c-glutamyl transpeptidase present in the cell surface metabolizes extracellular GSH to individual amino acids, which a re then incorporated into the spermatogenic cells and can be used for spermatogenesis. The administration of GSH or its equivalent to patients has been actually performed for therapeutic purposes [49–51]. The systemic supplementation of reduced GSH t o patients with dyspermia due to varicocele or a germ-free genital trac t infection resulted in improved sperm parameters and cell membrane characteristics [50]. Thus GSH and enzymes that increase GSH levels appear to be target for the therapeutic purpose of male infertility. Some anticancer agents, such as BCNU, would primarily impair Sertoli cells, as shown in Fig.6,andresultinmaleinfertility.Insuchacase,GSH may also be an effective therapeutic. The findings here demonstrates a high expression of GR in epithelial tissues of the male genital tract whose roles appear to supply reducing equivalents to spermatozoa for protection against R OS and for supplying reducing equiv- alents to GSH-dependent detoxifying enzymes. In sperma- togenic cells, cysteine rather than GSH is directly required for spermiogenesis, and, hence, the participation of GR is small. However, supplementation of GSH from Sertoli cells would be required for the spermatogenic cells both as protection from ROS and as an amino-acid source for spermatogenesis. ACKNOWLEDGEMENTS We wish to thank the staff from the Laboratory Animal Center, Yamagata University School of Medicine, for taking care of the rats. Supported in part by a Gran t-in-Aid for Scientific Research (C) (no. 13670111) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan and by Japan Organon, Co. Ltd. 1576 T. Kaneko et al. (Eur. J. Biochem. 269) Ó FEBS 2002 REFERENCES 1. Kim, J.G. & Parthasarathy, S. (1998) Oxidation and the sper- matozoa. Semin. Reprod. Endocrinol. 16, 235–239. 2. Aitken, R.J., Clarkson, J.S. & Fishel, S. (1989) Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol. R eprod. 41, 183–197. 3. Aitken, R.J. ( 2000) Pos sible redox regulation of sperm motility activation. J. Androl. 21, 491–496. 4. Shalgi, R., Seligman, J. & Kosower, N.S. (1989) Dynamics of the thiol status of rat spermatozoa during maturation: analysis with the fluorescent labeling agent mon obromobimane . Biol. Reprod. 40, 1037–1045. 5. Cornwall, G.A., Vindivich, D., Tillman, S. & Chang, T.S. (1988) The effect of sulfhydryl oxidation on the morphology of immature hamster epididymal spermatozoa induced to acquire motility in vitro. Biol. Reprod. 39, 141–155. 6. Baker, H.W., Brindle, J., Irvine, D.S. & Aitken, R.J. (1996) Protective effect of antioxidants on the impairment of sperm motility by a ctivated polymorphonuclear leukocytes. Fertil. St eril. 65, 411–419. 7. Storey, B.T. (1997) Biochemistry of the induction and prevention of lipoperoxidative damage in human spermatozoa. Mol. Hum. Reprod. 3, 203–237. 8. Poulos, A., Darin-Bennett, A. & Wh ite, I.G. (1973) The phospholipid-bound fatty acids and aldehydes of mammalian spermatozoa. Comp.Biochem.Physiol.B.46, 541–549. 9. Zalata, A.A., Christophe, A.B., Depuydt, C.E., Schoonjans, F. & Comhaire, F.H. (1998) The fatt y acid composition of phospholi- pids of spermatozoa from infertile patients. Mol. Hum. Reprod. 4, 111–118. 10. Jones, R. & Mann, T. (1976) Lipid peroxides in spermatozoa; formation, role of plasmalogen, and physiological significance. Proc.R.Soc.Lond.B.Biol.Sci.193, 317–333. 11. Alvarez, J.G. & Storey, B .T. (1984) Lipid peroxidation and the reactions of superoxide and hydrogen peroxide in mouse spermatozoa. Biol. Reprod. 30, 833–841. 12. Meister, A. & Anderson, M.E. (1983) Glutathione. Annu. R ev. Biochem. 52, 711–760. 13. Knapen, M.F., Zuste rzeel, P.L ., Peters, W.H. & S teegers, E.A. (1999) Glutathione and glutathio ne-related e nzymes in reproduction. A review. Eur. J. Obstet. Gynecol. Reprod. Biol. 82, 171–184. 14. Okado-Matsumoto, A., Matsum oto, A., Fujii, J. & T aniguch i, N. (2000) Peroxiredoxin IV is a secretable protein with heparin- binding properties under reduced conditions. J. Bioche m. 127, 493–501. 15. Fujii, T., Fujii, J. & Taniguchi, N. (2001) Augmented expression of peroxiredoxinVI in rat lung and kidney after birth implies an antioxidative role. Eur. J. Biochem. 268, 218–224. 16. Carlberg, I . & M annervik, B. ( 1985) Glutathione reductase. Methods Enzymol. 113, 484–490. 17. Kaneko, T., Iuchi, Y., Kawachiya, S., Fujii, T., Saito, H., Kurachi, H. & Fujii, J. (2001) Alteration of glutathione reductase expression in the female r eproductive o rgans during the estrous cycle. Biol. Reprod. 65, 1410–1416. 18. Li, T.K. (1975) The g lutathione and thiol content of mammalian spermatozoa and seminal plasma. Biol. Reprod. 12, 641–646. 19. Li, L.Y., Seddon, A.P., Meister, A. & Risley, M .S. (1989) Spermatogenic cell–somatic cell i nteractions are required for maintenance of spermatogenic cell glutathione. Biol. Reprod. 40, 317–331. 20. Lindemann, C.B., O’Brien, J.A. & Giblin, F.J. (1988) An investigation of the e ffectiveness of certain antioxidants in preserving the motility of reactivated bu ll sperm mod els. Biol. Reprod. 38, 114–120. 21. Agrawal, Y.P. & Vanha-Perttula, T. ( 1988) Glutathione, L -glu- tamic acid and c-glutamyl transpeptidase in the bull reproductive tissues. Int. J. Androl. 11, 123–131. 22. Bauche, F., Fouchard, M.H. & Jegou, B. (1994) Antioxidant system in rat testicular cells. FEBS Lett. 349, 392–396. 23. Alvarez, J.G., Touchstone, J.C., Blasco, L. & Storey, B.T. (1987) Spontaneous lipid peroxidation and production of h ydroge n peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity. J. Androl. 8, 338–348. 24. Polak, B. & Daunter, B. (1989) Seminal plasma biochemistry. IV: Enzymes involved in the liquefaction of human seminal plasma. Int. J. Androl. 12, 187–194. 25. Fujii, T., Hamaoka, R., Fujii, J. & Taniguchi, N. (2000) Redox capacity of cells affects inactivation of glutathione reductase b y nitrosative stress. Arch. Biochem. Biophys. 378, 123–130. 26. Ikeda, M., Kodama, H ., Fukuda, J., Shimizu, Y., Murata, M., Kumagai, J. & Tanaka, T. (1999) Role of radical oxygen species in rat testicular germ cell apoptosis induced by heat stress. Biol. Reprod. 61, 393–399. 27. Anderson, M.E. (1985) Determination of glutathione and g luta- thione disulfide in biologic al samples. Methods Enzymol. 113, 548– 555. 28. Laemmli, U .K. (1970) Cle avage o f structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. 29. Chomczynski, P. & Sacchi, N. (1987) Single-step method of RNA isolation by aci d guanid inium thioc yanate-p henol-chloroform extraction. Anal. Biochem. 162, 156–159. 30. Sasagawa, I., Matsuki, S., Suzuki, Y., Iuchi, Y., Tohya, K., Kimura, M., Nakada, T. & Fujii, J. (2001) Possible involvement of the membrane-bound form of peroxiredoxin 4 in acrosome for- mation during spe rmiogenesis of rats. Eur. J. Biochem. 26 8, 3053– 3061. 31. Iuchi, Y., Kobayashi, T., Kane ko, T ., T akahara, M., Ogino, T. & Fujii, J. (2001) The expression of a Y-box protein, YB2/RYB-a, precedes protamine 2 during spermatogenesis i n rodents. Mol. Hum. Reprod. 7, 1023–1031. 32. Dorrington, J.H., Roller, N.F. & Fritz, I.B. (1975) Effects of fol- licle-stimulating hormone on cultures of Sertoli cell preparations. Mol. Cell. Endocrinol. 3, 57–70. 33. Malkov. M., Fisher, Y. & D on, J. (1998) Developmental schedule of the postn atal rat testis determined by flow cyto metry. Biol. Reprod. 59, 84–92. 34.Yeung,C.H.,Cooper,T.G.,DeGeyter,M.,DeGeyter,C., Rolf, C., Kamischke, A . & Nieschlag, E. (1998) Studies on the origin of redox enzymes in seminal plasma and their relationship with results of in-vitro fertilization. Mol. Hum. Reprod. 4, 835–839. 35. Sharma, R.K. & Agarwal, A. (1996) Role of reactive oxygen species in male infertility. Urology 48, 835–850. 36. Perreault, S.D., W olff, R .A. & Zirkin, B.R. (1984) The r ole o f disulfide bond reduction during mammalian sperm nuclear decondensation in vivo. Dev. Biol. 101, 160–167. 37. Yoshida, M., Ishigaki, K., Nagai, T., Chikyu, M. & Pursel, V.G. (1993) Glutathione concentration durin g maturation and after fertilization in pig oocytes: relevance to the ability of oocytes to form male pronucleus. Biol. Reprod. 49, 89–94. 38. Sutovsky, P. & Schatten, G. (1997) Depletion of glutathione during bovine oocyte maturatio n reversibly blo cks the deco n- densation of the male pronucleus and pronuclear apposition during fertilization. Biol. Reprod. 56, 1503–1512. 39. Perreault, S.D., Barbee, R.R. & Slott, V.L. (1988) Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in matu ring hamster oocytes. Dev. Biol. 125, 181–186. Ó FEBS 2002 Glutathione reductase in male reproduction system (Eur. J. Biochem. 269) 1577 40. Tramer, F., Rocco, F., Micali, F., Sandri, G. & Panfili, E. (1998) Antioxidant systems in rat epididymal spermatozoa. Biol. Reprod. 59, 753–758. 41. Ghyselinck, N .B., Jimenez, C. & Dufaure, J.P. (1991) Sequence homology of androgen-regulated epid idymal proteins with glu- tathione peroxidase in mice. J. Reprod. Fertil. 93, 461–466. 42. Godeas, C ., Tramer, F., Micali, F ., Soranzo, M., Sandri, G . & Panfili, E. (1997) Distribution and p ossib le n ovel role of phos- pholipid hydroperoxide g lutathione peroxidase in rat epididymal spermatozoa. Biol. Reprod. 57, 1502–1508. 43. Syntin, P., Dacheux, F., Druart, X., Gatti, J.L., Okamura, N. & Dacheux, J.L. (1996) Characterization and identification of pro- teins secreted in the various regions of the adult boar epididymis. Biol. Reprod. 55, 956–974. 44. Srivastava, S., Chandra, A., Ansari, N.H., Srivastava, S.K. & Bhatnagar, A. (1998) Identification o f cardiac oxidoreductase(s) involved in the metabolism of the lipid peroxidation-derived aldehyde-4-hydroxynonenal. Biochem. J. 329, 4 69–475. 45. Gandy, J., Primiano, T., Novak, R.F., Kelce, W.R. & York, J.L. (1996) Differential expression of glutathione S-transferase iso- forms in compartments of the testis and segments of the epididy- mis of the rat. Drug Metab. Dispos. 24, 725–733. 46. Twigg,J.,Fulton,N.,Gomez,E.,Irvine,D.S.&Aitken,R.J. (1998) Analysis of the impact of intracellular reactive oxygen species generation o n the structural and f unction al integrity of human spermatozoa: lipid peroxid ation, DNA fragmen- tation and effectiveness of antioxidants. Hum. Reprod. 13, 1429– 1436. 47. Aitken, R.J., Gordon, E., Harkiss, D., Twigg, J.P., Milne, P., Jennings, Z. & Irvine, D.S. (1998) Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol. Reprod. 59, 1037–1046. 48. Kumar, T.R., Wiseman, A.L., Kala, G., Kala, S.V., Matzuk, M.M. & Lieberman, M.W. (2000) Reproductive defects in gamma-glutamyl transpeptidase-deficient mice. Endocrinology 141, 4270–4277. 49. Irvine, D.S. (1996) Glutathione as a treatment for male infertility. Rev. Reprod. 1, 6–12. 50. Lenzi, A., P icardo, M., Gandini, L., Lombardo, F., Terminali, O., Passi, S. & Dondero, F. (1994) Glutathione treatment of dysper- mia: effect on the lipoperoxidation process. Hum. Reprod. 9, 2044–2050. 51. Lenzi, A., Gandini, L. & Picardo, M. (1998) A rationale for glutathione therapy. Hum Reprod. 13, 1419–1422. 1578 T. Kaneko et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . The expression of glutathione reductase in the male reproductive system of rats supports the enzymatic basis of glutathione function in spermatogenesis Tomoko. of GR in the male genital tract of the rat was examined by meas- uring its enzymatic activity and examining the gene expression and localization of the protein.