| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Department of Biochemistry and
b Department of Obstetrics and Gynecology, Yamagata University School of Medicine, Yamagata, Yamagata 990-9585, Japan
c Department of Developmental Neurobiology, Graduate School of Medicine, Tohoku University, Sendai, Miyagi 980-8575, Japan
ABSTRACT
The enzyme glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) in an NADPH-dependent manner. A specific antibody raised against recombinant rat GR was used to localize the protein in the female reproductive organs during the estrous cycle in the rat. In the ovary, the strongest reactivity to the antibody was observed in oocytes, followed by granulosa cells, corpus luteum, and interstitial cells. A strongly positive reaction was also observed mainly in the oviduct epithelia, uterine epithelia, and endometrial gland in the reproductive tract. Oviducts contained the highest GR activity. The GR activity of uterus during metestrus was about twice as high as that for other stages of the cycle. The levels of GR proteins in the tissues roughly matched the activities. The expression of the GR mRNA was highest during metestrus. Because GSH is known to increase gamete viability and the efficiency of fertility, GR, which is expressed in these tissues, is predicted to play a pivotal role in the reproduction process as a source of GSH.
corpus luteum, fallopian tubes, follicle, granulosa cells, oviduct, ovum, uterus
INTRODUCTION
Glutathione in its reduced form (GSH) has pleiotropic roles, which include maintaining cells in a reduced state, serving as an electron donor for some antioxidative enzymes, and forming conjugates with some harmful endogenous and xenobiotic compounds via catalysis of glutathione S-transferase [1]. GSH also has important functions in the reproductive system. For example, GSH synthesis is involved in oocyte maturation and male pronucleus formation after fertilization [2, 3]. In addition, the depletion of GSH by oxidation with diamide to the oxidized form of glutathione (GSSG) alters microtubule function and affects pronucleus development [4]. GSH content decreases about 10-fold from the unfertilized oocyte to the blastocyst [5]. GSH in reproductive tract secretion appears to improve mouse embryo development [6]. A decreased ratio of GSH:GSSG is hypothesized to be a major contributing factor to the detrimental effects of maternal aging and postovulatory oocyte aging [7, 8].
Because GSH is a small molecule and is commonly distributed in organisms, it is not possible to directly specify its location in situ. Thus, information on the localization of enzymes involved in GSH production would be beneficial in evaluating the physiologic role of GSH. Intracellular GSH levels are maintained in a constant physiologic state by two enzymatic systems [1]. One involves de novo synthesis from three amino acids, glutamate, cysteine, and glycine, via catalysis by 
-glutamylcysteine synthetase and glutathione synthetase with the consumption of ATP. The other involves the reduction of GSSG back to GSH via catalysis by glutathione reductase (GR) with the consumption of NADPH. Because the latter system is more convenient than the former from an energetic standpoint, it is the predominant system in most tissues.
By generating GSH, GR indirectly participates in the protection of cells against oxidative stress and cytotoxic compounds and is deeply involved in the maintenance of the redox status of cells. The biochemical characteristics of the GR protein, such as the structure and reaction mechanism, have been extensively studied. The enzymatic activities of GR have also been investigated in various tissues under physiologic and pathologic conditions [9]. Although the recycle system of GSSG, comprising GR and NADPH, is the predominant one, few studies have demonstrated the localization of the GR protein. It is present in tissues such as lung cancer cells [10] and brain cells [11, 12].
A specific antibody against rat GR has recently been established using the rat recombinant GR protein produced by a baculovirus/insect cell system [13]. In the present experiment, the potential physiologic significance of GR was evaluated by immunohistochemical and biochemical analyses of the female rat reproductive system. The results suggest a pivotal role of GR in the reproductive process.
MATERIALS AND METHODS
Materials
GSSG was purchased from Boehringer Mannheim (Mannheim, Germany), and NADPH was obtained from Oriental Yeast Co. (Tokyo, Japan). 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 University School of Medicine. Wistar rats were maintained under conventional conditions. Seven-week-old female Wistar rats were housed under 12L:12D conditions, with lights-on at 0600 h and at a temperature of 21–23°C. Vaginal smears were collected daily, and only rats with consistent 4-day cycles were used in the experiment. 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 immunohistochemical analysis or frozen under liquid nitrogen and preserved at -80°C until used for protein and mRNA assays.
Preparation of Tissue Homogenates and Protein Assay
Tissues were homogenized with a polytron homogenizer in four volumes of PBS containing 10 µg/ml pepstatin, 10 µg/ml leupeptin, 100 µM phenylmethylsulfonyl fluoride, and 1 mM benzamidine. After centrifugation at 10 000 x g for 20 min, the supernatant was collected and kept at -20°C. Protein concentrations were determined using a BCA kit (Pierce, Rockford, IL) with BSA as the standard.
Enzyme Assay
The GR activity was determined spectrophotometrically by measuring the rate of NADPH oxidation at 340 nm [9]. The reaction mixture consisted of 0.1 M potassium phosphate, pH 7.0, 1 mM EDTA, 0.1 mM NADPH, 1 mM GSSG, and tissue samples. The decrease in absorbance at 340 nm at 30°C was recorded. Because the decrease of absorbance of the control reaction mixture without GSSG was less than 0.002, contribution of spontaneous NADPH oxidation and other reductases in the samples was ignored. One unit of GR activity was defined as the amount of enzyme that catalyzes the oxidation of 1 µmol of NADPH per minute. All assays were performed in triplicate, and means + SDs are reported.
SDS-PAGE and Western Blot Analysis
Protein samples were subjected to 10% SDS-PAGE [14] and then transferred onto Hybond-P membranes (Amersham Pharmacia, Piscataway, NJ) under semidry conditions with the use of a Transfer-Blot SD Semi-Dry transfer cell (Bio-Rad, Tokyo, Japan). The membranes were then blocked by incubation with 5% skim milk in Tris-buffered saline with Tween 20 (TBST; 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween 20) for 2 h at room temperature. The membranes were then incubated with the rabbit antibody to rat GR (1:1000 dilution) [13] for 12 h at 4°C. After washing with TBST, the membranes were incubated with 1:1000 peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. Following the washing, the peroxidase activity was determined by a chemiluminescence method using an ECL Plus kit (Amersham Pharmacia, Buckinghamshire, UK).
Preparation of Total RNA
Total cellular RNA was isolated from several rat tissues by homogenization with the guanidine thiocyanate/phenol/chloroform extraction method [15] using ISOGEN (Nippon Gene, Tokyo, Japan). The final pellet was dissolved in diethylpyrocarbonate-treated water and quantified by measuring the absorbance at 260 nm.
Northern Blot Analysis
Rat GR cDNA [13], excised with HindIII and EcoRI, was subcloned into pBluescript SK (-) vector. The digoxigenin (DIG)-labeled antisense GR RNA probe was produced by in vitro transcription with T7 RNA polymerase in a DIG labeling mixure (Boehringer Mannheim). For Northern blot analysis, 10 µg of total RNA from each tissue was separated by electrophoresis on a 1% agarose gel under denaturing conditions and subsequently transferred onto a Hybond-N+ membrane (Amersham Pharmacia). The amount of RNA in the sample was confirmed by staining the ribosomal RNA in the agarose gel with ethidium bromide. The RNA was then fixed to the membrane by ultraviolet (UV) crosslinking using a UV crosslinker (Amersham Pharmacia). After prehybridization for 2 h in a prehybridization solution (5x SSPE, 0.5% SDS, 5% Irish Cream liquor, 50% formamide, and 100 µg/ml salmon sperm DNA), the membrane was incubated with a DIG-labeled RNA probe for GR at 60°C for 12 h. The membranes were washed twice at room temperature with 1x standard saline citrate (SSC; 150 mM NaCl, 15 mM sodium citrate, pH 7.5) containing 0.1% SDS for 20 min. Membranes then were washed twice with 0.5x SSC containing 0.1% SDS. The hybridized membranes were reacted with the DIG luminescent detection kit using standard protocols (Roche Diagnostics Co., Tokyo, Japan) and then exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY).
Immunohistochemistry
For the immunohistochemical study, Dako Envision System (Dako Co., Carpinteria, CA) was employed. 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 5 µm thickness, and the resulting serial sections were mounted on silianized slides. After deparaffinization and rehydration, tissue sections were quenched with 0.1% hydrogen peroxide. The target retrieval procedure involved immersion of tissue sections in a citrate-based buffer solution and then heating in an autoclave. Tissue sections were briefly treated with porcine serum for 10 min to block nonspecific binding and then reacted with an antibody specific for rat GR for 60 min at a 1:200 dilution. The sections were sequentially reacted with peroxidase-labeled goat anti-rabbit IgG polymer for 30 min and 3,3-diaminobenzidine substrate for 1 min. Nonimmune rabbit IgG (Santa Cruz Biotechnology) was used as a control. Photographs were taken with a digital camera under light microscopy (Olympus BX50, Tokyo, Japan).
Statistics
The activity of GR during the estrous cycles was analyzed with a Kruskal-Wallis test, and the activity of GR in porcine oviducts was analyzed by the Wilcoxon signed ranks test.
RESULTS
Changes of GR Activity During the Estrous Cycle
The effects of the administration of estrogen, progesterone, or both on GR activity were investigated only in the uterus of ovariectomized rats [16]. To evaluate the role of GR in the female reproductive system, GR activity was measured in the ovary, oviducts, and uterus during the natural estrous cycle (Fig. 1). The highest activity was obtained in the oviducts. However, the levels of GR activity in the ovaries and oviducts remained unchanged during the estrous cycle. In the uterus, GR activity was significantly higher at the metestrous stage than at other stages (P < 0.05).
|
Expression of GR Protein During the Estrous Cycle
Levels of GR protein were also examined by immunoblotting using the GR antibody. Figure 2 shows the results of an immunoblot analysis for the same samples that were used for the GR activity assay. Only a 50-kDa band corresponding to molecular size of rat GR was detected on the blot. The levels of GR proteins in the tissues roughly matched the activities. The highest level of GR protein was observed in the oviduct, followed by the uterus and ovaries. GR protein was transiently increased at the metestrous stage in the uterus. However, no significant difference was detected in ovaries and oviducts during the estrous cycle.
|
Expression of GR mRNA During the Estrous Cycle
Northern blot analysis showed that GR mRNA was expressed in the ovaries, the oviducts, and the uterus throughout the cycles (Fig. 3). The amount of mRNA in the ovaries and the oviducts remained unchanged. However, there were cyclic changes in mRNA in the uterus. The level of GR mRNA was the highest at the metestrous stage among the estrous cycles in the uterus. Oviducts showed higher GR activity and protein level but expressed less mRNA than did other tissues.
|
Immunohistochemical Localization of GR in the Female Reproductive System
Although the presence of GSH in the reproductive tract has been established, the cells responsible for GSH production have not yet been identified. The polyclonal antibody against rat GR was employed to localize GR in the female reproductive system (ovaries, oviducts, and uterus) during the estrous cycle (Figs. 4–6). Numerous cells in the ovary were positively stained with this antibody. However, no staining was seen with nonimmune rabbit IgG (data not shown), indicating that the positive signals, including nuclei, were specific. The strongest staining was observed in the oocytes in the ovary throughout the maturation process (Fig. 4). The granulosa cells and some of the lutein and interstitial cells were also stained. In the oviducts, only epithelial cells showed strong staining, as did the oocytes (Fig. 5). Most nuclei in the epithelial cells were also stained. Both surface epithelia and endometrial glands, which proliferated at the metestrous stage, were more strongly stained in the uterus, and the intensity appeared to be higher during metestrus (Fig. 6).
|
|
|
DISCUSSION
The maturation of spermatozoa proceeds via an oxidative process in the male accessory organ. Disulfide bond formation in the protein is required for spermatozoa to maintain their high viability during the fertilization process. Because reactive oxygen species (ROS) cause male infertility [17], regulated oxidation in the male accessory organ is critical. However, reducing conditions are essential for male pronuclear formation, probably relating to the reduction of disulfide bonds in the nucleus [2], and GSH is a major source for reducing power in the oocyte [18–20].
In this study, the location and activity of GR were investigated in the ovary, oviduct, and uterus to evaluate the physiologic role of GR and GSH in the female rat reproductive organs. Although both GR activity and the level of GR protein were higher in the oviducts than in the ovaries and uterus, the GR mRNA expression in the oviducts was lower than that in other organs. This inconsistency can be explained by tissue turnover. Although continuous folliculogenesis, ovulation, corpus luteum formation, and collapse occur in the ovary and cyclic proliferation and exfoliation of endometrium occur in the uterus, only morphologic conversion from ciliated to secreting epithelium rather than actual cell proliferation is seen in the oviducts. Thus, the half-life of the GR protein may be longer in the oviducts and a low level of transcription would be sufficient to maintain a high level of protein expression.
The oocyte contains high levels of GSH, 9–10 mM [5]. The presence of the highest level of GR protein in ovary, as demonstrated in this study, would explain at least in part the high GSH content in oocytes. Because oocytes from the glutathione synthetase transgenic mice are more resistant to diamide toxicity [21], GSH would be expected to have a role in the protection of oocytes from oxidative stress and in male pronuclear formation. Because the oocyte must store energy for subsequent developmental processes, a GSH regenerating system catalyzed by GR seems to be more beneficial than de novo synthesis. Glycolytic activity and the hexose monophosphate shunt, a regenerating system of NADPH, are enhanced during spermatozoa penetration into the oocyte [22].
Granulosa cells and lutein cells also expressed relatively high levels of GR. GSH synthesis by the cumulus cells occurs during in vitro oocyte maturation in cows [23] and during in vivo meiotic maturation in hamsters [4]. Because the corpus luteum produces much of the progesterone in conjunction with the reaction of P450s by consuming molecular oxygen and, hence, produces ROS as a byproduct, damage could be inflicted by ROS. The detoxification of the produced ROS by GSH in conjunction with antioxidative enzymes would be particularly important for the corpus luteum and surrounding cells.
ROS play multiple roles during ovulation and reproduction processes. Those such as superoxide appear to be essential in the mechanical or chemical process of ovulation. ROS are thought to be released in connection with follicle rupture. Inhibition of ROS actually hinders ovulation [24]. Gardiner et al. [6] reported the presence of GSH in oviductal fluid. The high concentrations of GR in the epithelia of the oviducts could be responsible for this finding. The pivotal role of GR in the oviductal epithelia was supported in the present study by high expression of GR in different species, including the rat and pig. The secreted GSH could protect the oocyte against ROS excessively produced during the ovulation, thus maintaining fertilization potency. Gardiner and Reed [25] showed that buthionine sulfoximine decreased GSH levels to a greater extent in the blastocyst than in the two-cell embryo. Because buthionine sulfoximine depletes intracellular GSH by inhibiting -glutamylcysteine synthetase, these results suggest that the contribution of the de novo synthesis of GSH is much lower during the two-cell stage than during the blastocyst stage. Hence, the recycling of GSSG must play an important role in the maintenance of the intracellular GSH level from the oocyte to the two-cell stage.
It is commonly believed that GR exists both in cytosol and in mitochondria. The mitochondrial form was biochemically indistinguishable from the cytosolic form [13]. The immunohistochemical data clearly showed the nuclear localization of GR in some tissues. Some redox proteins, such as thioredoxin [26] and peroxiredoxin I [27], are known to be translocated from cytosol to nuclei. Although the precise mechanism is unknown at present, GR may also be translocated to nuclei by certain stimuli.
The detoxification of the oxidants present in reproductive tract fluids also would be advantageous to the oocytes and embryos at their early developmental stage in oviducts and uterus. Steroid metabolites such as isocorticosteroids and progesterone [28], the glycation reaction intermediates such as methylglyoxal and 3-deoxyglucosone [29, 30], and lipid peroxidation products such as 4-hydroxynonenal and acrolein [31, 32] are all aldose reductase substrates. Recently, Srivastava et al. [33] demonstrated that glutathione conjugates of 4-hydroxy-2-nonenal actually served as the substrates for aldose reductase. Glutathione conjugates and xenobiotic aldehydes [34], therefore, could efficiently bind to aldose reductase. Our findings show that aldose reductase and aldehyde reductase, the closest family members of aldoketo reductase [35, 36], were highly expressed in the epithelia of oviducts and in the uterus (unpublished observations). Glutathione S-transferase is known to be present in the reproductive system [37]. Thus, the abundant expression of GR in cooperation with glutathione S-transferase could facilitate the detoxification function of aldose reductase by producing glutathione conjugates.
We observed the highest activity at metestrus, about 36 h after peaking of the hormone levels during the natural estrous cycle. However, Diaz-Flores et al. [16] administered estradiol or progesterone or both to ovariectomized rats after 15 days, when effects of intrinsic hormones were abolished, and then observed the highest activity after 24 h. Thus, there was inconsistency in the time between the two experiments. Because ovariectomized rats are kept under hormone-deficient conditions for a long time, cells may become more sensitive to hormonal stimuli. In addition, Diaz-Flores et al. administered high amounts of hormones, about two orders of magnitude higher than the normal levels. These high doses may have caused inconsistencies in the data. Promoter assays of the GR gene with regard to steroid hormone responsiveness should be carried out to clarify this point. However, no significant changes were found in the other tissues. Because GR activity was elevated in endometrial tissue samples during metestrus and during concomitant endometrial gland progress, the activity change could have been caused by the proliferation of cells that express GR. Progesterone, whose level is kept high by mating, would cause elevation of GR activity in the uterus. The GR activity participates in the reduction of GSSG to GSH, which can protect gametes from oxidative damage. Thus, the elevation of GR activity appears to be responsible for maintaining gamete viability and should result in an increase in the efficiency of fertility.
GR appears to play a crucial role in the female reproductive system by recycling GSSG. The resultant GSH appears to have multiple functions, such as the maintenance of oocyte potency, the formation of the male pronucleus, and the protection of embryos against oxidative stress via antioxidative and redox enzymes.
ACKNOWLEDGMENTS
We thank the staff from the Laboratory Animal Center, Yamagata University School of Medicine, for taking care of the rats.
FOOTNOTES
1 Supported in part by the Naito Foundation. 
2 Correspondence: Junichi 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;jfujii{at}med.id.yamagata-u.ac.jp
Accepted: June 22, 2001.
Received: March 7, 2001.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  K. H. Al-Gubory, P. Bolifraud, and C. Garrel Regulation of Key Antioxidant Enzymatic Systems in the Sheep Endometrium by Ovarian Steroids Endocrinology, September 1, 2008; 149(9): 4428 - 4434. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Makker, F W Bansode, V M L Srivastava, and M M Singh Antioxidant defense system during endometrial receptivity in the guinea pig: effect of ormeloxifene, a selective estrogen receptor modulator J. Endocrinol., January 1, 2006; 188(1): 121 - 134. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. Salmen, F. Skufca, A. Matt, G. Gushansky, A. Mason, and C. S. Gardiner Role of Glutathione in Reproductive Tract Secretions on Mouse Preimplantation Embryo Development Biol Reprod, August 1, 2005; 73(2): 308 - 314. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R Gonzalez-Fernandez, F Gaytan, E Martinez-Galisteo, P Porras, C A Padilla, J E Sanchez Criado, and J A Barcena Expression of glutaredoxin (thioltransferase) in the rat ovary during the oestrous cycle and postnatal development J. Mol. Endocrinol., June 1, 2005; 34(3): 625 - 635. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.C.L. Ford Regulation of sperm function by reactive oxygen species Hum. Reprod. Update, September 1, 2004; 10(5): 387 - 399. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Chen, P. H. Chow, S. K. Cheng, A. L. M. Cheung, L. Y. L. Cheng, and W.-S. O Male Genital Tract Antioxidant Enzymes: Their Source, Function in the Female, and Ability to Preserve Sperm DNA Integrity in the Golden Hamster J Androl, September 1, 2003; 24(5): 704 - 711. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kobayashi, T. Kaneko, Y. Iuchi, S. Matsuki, M. Takahashi, I. Sasagawa, T. Nakada, and J. Fujii Localization and Physiological Implication of Aldose Reductase and Sorbitol Dehydrogenase in Reproductive Tracts and Spermatozoa of Male Rats J Androl, September 1, 2002; 23(5): 674 - 683. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
