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

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

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,

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

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

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

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

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-

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

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

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

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
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