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 enzymatic activity and protein expression of GR in primary cultured testicular cells confirmed its predom-inant expression in Sertoli cells.. The results herein suggest that the GR s

The expression of glutathione reductase in the male reproductive system of rats supports the enzymatic basis of glutathione function

in spermatogenesis

Tomoko Kaneko1,2, Yoshihito Iuchi1, Takashi Kobayashi1,3, Tsuneko Fujii4, Hidekazu Saito2,

Hirohisa Kurachi2and Junichi Fujii1

Departments of1Biochemistry,2Obstetrics and Gynecology, and3Urology, Yamagata University School of Medicine, Yamagata, Japan;4Cell 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 localization 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 cultured 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 Sertoli cells As a result of these

findings, the effects of buthionine sulfoximine (BSO), an

inhibitor for GSH synthesis, and

1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU), an inhibitor for GR, on cultured 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 Spermatogenic 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 spermatogenic 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 depending 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 to 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 maintained by two systems One is de novo synthesis from building blocks, glutamate, cysteine, and glycine, via two ATP-consuming steps involving c-glut-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-dependent manner In addition to the direct interaction of GSH with ROS, GSH serves as an electron donor for some peroxidases, including glutathione peroxi-dase [13] and peroxiredoxins [14,15] The resulting oxidation

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 18 January 2002, accepted 23

January 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 of 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

focused on the localization of the GR protein in tissues [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 have been reported to 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 spermatozoa and seminal plasma including

human [7,23,24] Although the 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 of 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 cDNA as probe

for RNA analysis [25] In the present report, the potential

role of GR is evaluated 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

E X P E R I M E N T A L P R O C E D U R E S

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, Japan) Buthionine sulfoximine (BSO) and

1,3-bis[2-chlorethyl]-1-nitrosourea (BCNU) were obtained from

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 maintained 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 Bouin 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/Pi containing

10 lgÆmL)1pepstatin, 10 lgÆmL)1leupeptin, 100 lM

phe-nylmethylsulfonyl fluoride, and 1 mMbenzamidine 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 (Pierce, Rockford, IL) Testicular cell culture

Male Wister rats (40–50 days) were killed by diethyl ether anesthesia, and testicular cells were isolated as reported previously [26] Briefly, after decapsulation of the testes, the seminiferous tubules were minced using scissors and incu-bated in NaCl/Pi containing 0.25% type I collagenase (Wako, Osaka, Japan) at 32.5°C for 15 min The semin-iferous tubules were washed with NaCl/Pi once and then incubated in NaCl/Pi 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 10%, the cell suspension was filtered through nylon mesh to remove aggregates and tissue debris The cells were cultured in an equal mixture of F12-L15 medium supplemented with

100 UÆmL)1 penicillin G, 100 lgÆmL)1 streptomycin,

15 mM Hepes (pH 7.3), and 10% fetal bovine serum at 32.5°C with 5% CO2under humidified conditions After harvesting the cells with a silicon scraper, they were washed twice with NaCl/Pi and sonicated in an extraction buffer (25 mM Tris/HCl, pH 7.4, 50 mM NaCl, 10 lgÆmL)1 aprotinin, 10 lgÆmL)1 leupeptin, and 20 lM 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 mixture consisted of 0.1M potassium phos-phate, pH 7.0, 1 mM EDTA, 0.1 mM NADPH, 1 mM 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 of spontaneous NADPH oxidation and other reductases in the samples can be ignored One unit of GR activity was defined

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 glutathione and GSSG were determined by the recycling method described by Anderson [27] Briefly, cultured cells were collected and washed twice with NaCl/

Pi The precipitated cells were sonicated in 5% 5-sulfosul-icylic acid After centrifugation at 8000 g for 10 min, the supernatant (25 lL) was applied to a reaction mixture containing 100 mMsodium phosphate buffer, 5 mMEDTA,

200 UÆmL)1 yeast GR, 0.1 mM NADPH, and 5 mM 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 glutathione and GSSG were calcu-lated from the absorbance change using authentic GSH and

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, USA) 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 mM

Tris/HCl, pH 8.0, and 150 mM 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) for 12 h at 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, peroxidase activity was

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, Tokyo, Japan) The final pellet was dissolved in

diethylpyrocarbonate-treated H2O 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 2.2M formaldehyde [30] The

size-fractionated RNAs were transferred onto a Hybond-N

membrane (Amersham Pharmacia Biotech) by capillary

action After hybridization with the 32P-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°C in 2 · NaCl/Cit (1 · NaCl/Cit: 150 mMNaCl and

15 mM sodium citrate, pH 7.5) containing 0.1% SDS and

then twice in 0.2· NaCl/Cit The Kodak XAR films were

exposed with an intensifying screen at)80 °C

Immunohistochemistry

For the immunohistochemical study, the DAKO Envision

System (DAKO Co., Carpinteria, CA, USA) was employed

[31] This system is based on a 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 inactivated 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 sections were

briefly treated with porcine serum for 10 min to block

nonspecific binding and then reacted with anti-(rat GR) Ig

for 60 min at a 1 : 200 dilution The sections were sequentially reacted with peroxidase-labeled goat anti-(rabbit IgG) Ig polymer for 30 min, and 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)

R E S U L T S

Levels of GR in male reproductive system of the adult rat

To identify the cells responsible for GSSG reduction, an attempt was made to determine the localization of GR in the male reproductive system of the rat We first 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 found 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 Western blot analysis was carried out (Fig 1B) 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 all tissues except for spermatozoa A faint GR band could be detected in spermatozoa only after a longer exposure Thus, the order

of band intensities matched the levels of 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 1C), which appears to

be due to different turnover rates of the protein in these tissues

Immunohistochemical localization of GR

in male reproductive system of the adult rat

We employed immunohistochemical analyses of GR in order to determine its localization in the male reproductive system of the adult rat (Fig 2) Epithelia from the epididymis, vas deferens, seminal vesicles, and prostate glands were strongly stained with the GR antibody In the case of testes, the immunoreactivity to the anti-GR Ig was strong in Sertoli cells Concerning the intracellular distribu-tion, positive signals to the antibody 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 conditions As Sertoli cells become attached to conventional plastic plates at 24 h after separation and 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

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 be 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 were carried out on

testes from premeiotic stage (14 days), meiotic stage (21 days), 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 cells, no difference was observed during this period

by immunohistochemistry (data not shown) As the activity difference was small, the difference in 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 the contribution of de novo synthesis and the recycling of GSH to the intracellular glutathione pool, the

Fig 2 Immunohistochemical localization of GR in the male reproduc-tion system of adult rats Secreproduc-tions 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) prostate gland 650· magnification: (B) testis; (D) epidi-dymis, head.

Fig 1 GR activities, protein, and mRNA levels in the male

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 are presented as the

means ± SD of three rat organs (B) Twenty micrograms of soluble

protein was subjected to Western blot analyses with the GR antibody

at a 1 : 1000 dilution Typical data from several experiments are

shown The arrowhead indicates the position of GR (50 kDa).

(C) Total RNAs (10 lg) from rat testes at the indicated ages, which

were separated on a 1% agarose gel.

levels of GSH and GSSG were measured in cultured Sertoli

cells and spermatogenic cells 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 lM BCNU

totally inhibited the GR activity (data not shown) After a

24-h incubation, the total glutathione level in the Sertoli cells

was decreased to 8 and 14% of the control by 10 mMBSO

and 1 mM BCNU, respectively (Fig 5A) However, these

agents were less potent to the spermatogenic cells GSSG

levels increased only slightly after BCNU treatment 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 effective

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 the spermatogenic cells When 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

D I S C U S S I O N

The findings in this study show that GR is highly expressed

in epithelial cells of the male genital tract, especially in the

Fig 4 Decrease in the levels of GR in testes around pubertal stages (A) Enzymatic activities of 90 lg cytosolic proteins from pre- and post-pubertal as well as adult rat testes were measured Data are presented

as the means + SD for three rat organs (B) Ten micrograms of pro-teins from testes were subjected to Western blot analysis with a

1 : 3000 dilution of the anti-GR Ig (C) Total RNAs (5 lg) from rat testes at the indicated ages were separated on 1% agarose gel 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.

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 cells 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 GSSG level in BCNU-treated Sertoli cells did not correspond to the decrease in levels of total glutathione Thus the excretion of GSSG is only mechanism for Sertoli cells Much 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 oxidmatur-ation and reduction processes in the male genital tract, especially in the epididymis Disulfide bond formation in protamine, a counter part of histone in sperm nuclei, is required for packaging the DNA into the small head space and to

Fig 6 Sertoli cell death by BCNU treatment Morphology of the Sertoli cells in Fig 5 is shown 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 for 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 ) or BCNU (1 m M ) Sertoli cells were treated

with the same reagents after incubation for 4 days (A) The cells were

harvested after 24 h incubation with the reagents and assayed for total

glutathione and GSSG SPG, spermatogenic ells; T, total glutathione

(GSH + GSSG); O, oxidized glutathione (GSSG) (B) Total

gluta-thione levels were measured for spermatogenic cells after separation

from Sertoli cells for 3 days.

maintain sperm motility during the fertilization process In

contrast, ROS, which generally mediates sulfhydryl

oxida-tion, causes male 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 occur in a regulated way in the male

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 the nucleus [36–38] As GSH is a major source of

reducing power in the oocyte [36,39], a high content of GR

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 acquisioxida-tion of motility

[4,5] During epididymial migration, sulfhydryls in the

nucleus and the tail of spermatozoa are oxidized to

disulfides Levels of antioxidative enzyme activities alter

during this process [40] Thus, it would be expected that the

reductant level inside the epididymis would be low

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 contain

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] These enzymes may be responsible for the

reduction of coincidently produced toxic products, 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 in the male reproductive system [45] Thus, the

abundant expression of GR in cooperation with glutathione

S-transferase could facilitate the detoxification function of

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 high 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 block of GSH, is required for spermatozoa, the presence of GSH presents somewhat of an obstacle due to its reducing power As the biosynthetic rate of protamine production is very high during the spermiogenic process, large amounts 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 interactions with Sertoli cells [19] Such circumstantial evidence suggests that Sertoli cells may provide GSH as a source for cysteine, 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 seminal vesicle and is severely oligospermic and infertile The administration of GSH or N-acetylcysteine, a mem-brane permeable precursor of cysteine, totally restores the testis and seminal sizes to values which are comparable to those of wild-type mice and which render the mutant mice fertile This indicates that the c-glutamyl transpeptidase present in the cell surface metabolizes extracellular GSH to individual amino acids, which are 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 to patients with dyspermia due to varicocele or a germ-free genital tract 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, and result in male infertility In such a case, 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 ROS 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

A C K N O W L E D G E M E N T S

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 Grant-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.

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