Modulation of Intracellular Glutathione and Cysteine Metabolism in Bovine Oviduct Epithelial Cells Cultured In Vitro¹

^a Toxicology Program, Department of Environmental and Industrial Health, University of Michigan, Ann Arbor, Michigan 48109–2029

	ABSTRACT

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The objective of this study was to determine how alterations in intracellular thiol levels of oviduct epithelium occur in response to chemical or environmental conditions that could result in oxidative stress. Bovine oviducts were classified as follicular (F) or luteal (L) according to the reproductive stage of the ovary. Epithelial cells were harvested from the ampulla (AMP) and isthmus (ISTH) region of each oviduct, suspended in culture medium, and then plated into collagen-coated culture plates and grown to confluency. Basal levels of intracellular cysteine (Cys) and glutathione (GSH) were determined in oviduct epithelial cells and found to range from 0.36 to 0.46 pmol/µg protein for Cys and from 5.3 to 6.4 pmol/µg protein for GSH. Oxidized Cys values ranged from 21% to 39% of total Cys, whereas the oxidized GSH levels observed were from 21% to 28% of total GSH except in luteal ISTH, where they were significantly lower (6%). Confluent cells were exposed to GSH-depleting agents, L-buthionine-S,R-sulfoximine (BSO) or diethyl maleate (DEM), at doses ranging from 10 to 5000 µM. Both compounds depleted GSH in a dose-dependent manner, and 500 µM concentrations were chosen for subsequent studies with each compound. Cys levels in BSO (500 µM)-treated oviduct epithelial cells were transiently elevated over control values during the initial 5-h incubation; there was then a decrease in Cys levels by AMP but not ISTH oviduct epithelial cells. BSO-treated oviduct epithelial cells displayed a continued depletion of GSH over the incubation period and by 24 h were depleted by 38% to 61%. These results demonstrate a difference in GSH turnover in oviduct epithelial cells associated with reproductive stage. Exposure to DEM (500 µM) caused a decline in both Cys and GSH levels, which were partially restored after DEM removal. In general, L-staged oviduct epithelial cells were observed to be more competent at replenishing thiol stores than F-staged oviduct epithelial cells. Results from this study suggest that reproductive stage and region influence intracellular oviduct epithelium thiol status and therefore may affect how this tissue responds to chemicals or environmental conditions leading to oxidative stress.

	INTRODUCTION

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The mammalian oviduct plays a significant role in the physiological events culminating in successful reproduction. The oviduct provides the proper environment for the final preparations gametes undergo prior to and during fertilization, as well as for subsequent preimplantation embryo development [1, 2]. This is accomplished by provision of the milieu in which these events occur: oviduct fluid. Oviduct fluid is a selective transudate of serum and secretory products from the oviduct epithelium [3]. These secretory products are thought to be present in virtually all mammalian species and to function in events associated with fertilization and/or preimplantation embryonic development [4].

The oviduct modulates the luminal environment in response to hormonal cues and to the presence of gametes and/or preimplantation embryos [4]. Hormonal changes that occur during the mammalian reproductive cycle have been found to influence oviduct luminal contents such as protein secretions [3, 5], the volume of oviduct fluid [6], amino acid composition [4], and the electrolyte content, especially calcium, magnesium, phosphate, and bicarbonate ions [4, 7]. It should also be noted that different reproductive events occur between the ampulla and isthmus regions of the oviduct in addition to differences due to hormonal status [8]. These established oviductal environments are the result of oviduct epithelium secretory activity.

The secretory epithelium enables the oviduct to establish a microenvironment that optimizes conditions for fertilization and to sustain the developing preimplantation embryo during its transport to the uterus. Therefore, any disruption in oviduct epithelium function due to exposure to chemicals or environmental conditions that cause oxidative stress and lead to altered oviduct function, may result in impaired or failed reproduction. Oxidative stress is a condition that arises when prooxidants are produced in excess of the capacity of antioxidants to remove them, resulting in oxidative damage to cell macromolecules [9]. Prooxidants are oxygen-centered free radicals and their metabolites, such as superoxide anion and hydroxyl radicals that can be products of normal metabolic processes or be derived from exogenous reactions [9, 10]. This result may be characterized by unsuccessful fertilization, preimplantation embryo loss, and/or persistent anatomical or functional birth defects. Unfortunately, very little information is available concerning how the oviduct may respond to oxidative stress and therefore influence events important to reproduction. Of specific interest under these conditions is the status of the tripeptide, glutathione (-glutamylcysteinylglycine; GSH), and the way in which the mammalian oviduct may regulate synthesis, metabolism, and secretion of this important cellular antioxidant. GSH, the predominant low-molecular-weight thiol present in most cell types, can provide the cell with protection against oxidative stress. In addition, GSH regulates several important cellular functions such as maintenance of cell and membrane integrity, redox status regulation of protein and DNA synthesis, modulation of protein folding, microtubule assembly, and participation as a cofactor for various enzyme reactions [11]. Dramatic changes in the amount of GSH available within a cell due to extensive utilization in cellular defense mechanisms may compromise cell viability, as well as alter GSH-mediated cellular functions.

One potentially important aspect of oviduct function related to consequences of chemical exposure is the capacity of this tissue to regulate GSH status through synthesis and other means. Cysteine (Cys) availability has been shown to be a rate-limiting factor in GSH synthesis [12], although the other amino acids (glutamate and glycine) may also be of regulatory importance for this oviduct function. Experimentally, intracellular GSH levels can be modulated by incubation with L-buthionine-S,R-sulfoximine (BSO) or diethyl maleate (DEM). BSO is an inhibitor of GSH synthesis that acts by selectively inhibiting the enzyme, -glutamylcysteine synthetase, responsible for combining glutamate and Cys to form -glutamylcysteine [12–14]. DEM is a compound that depletes intracellular GSH by chemically reacting with GSH to form conjugates [15, 16]. The ability to maintain and regulate thiol levels is a good indicator of how cells react to chemicals or environmental conditions that result in oxidative stress. Recent studies conducted in the pig have shown that precursor amino acids for GSH (glutamate, Cys, and glycine) have been beneficial for pronuclear development [17]. In addition, investigations of the influence of low-molecular-weight thiols on bovine in vitro culture systems show an increase in the percentage of blastocysts and hatched blastocysts derived from in vitro fertilization of in vitro-matured oocytes. This increase was associated with increased intracellular GSH levels in the preimplantation embryo [18, 19]. However, limited information exists concerning the cellular mechanisms functioning in oviduct epithelium to regulate thiols and control antioxidant status in the oviduct.

The availability of GSH and/or precursor amino acids used to synthesize GSH may influence fertilization and preimplantation embryo development. Cell commitment during early embryogenesis can be demonstrated prior to the morula and blastocyst stage of the preimplantation embryo, and recent studies have shown that oxidative stress impairs preimplantation embryo development [20, 21]. Since the oviduct epithelium modulates the embryonic microenvironment during fertilization and preimplantation development, toxicants that cause oxidative stress and alter epithelium function would subsequently affect preimplantation embryo development and lead to death of the embryo or to birth defects. The objective of this study was to measure basal levels of the intracellular thiols Cys and GSH in bovine oviduct epithelium and then to modulate those levels using inhibitors, in order to help predict how the bovine oviduct may respond to situations resulting in oxidative stress.

	MATERIALS AND METHODS

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Bovine Oviduct Epithelium Isolation and Culture

Oviducts and ovaries were recovered from cows at an abattoir after slaughter. Ovaries were used to group oviducts according to reproductive stage, follicular (F) or luteal (L) [22–24]. Once staged, oviductal tissue was immersed in cold D-PBS (Dulbecco's PBS; Sigma Chemical Co., St. Louis, MO) supplemented with 5% antibiotic-antimycotic (ABAM; Sigma) and were kept on ice during transport to the laboratory. Oviducts were trimmed of excess tissue and separated into ampulla (AMP) and isthmus (ISTH) regions. Bovine oviduct epithelial cells were then harvested by the method of Henault and Killian [25]. Briefly, AMP and ISTH regions of F- and L-staged oviducts were filled with an enzyme solution composed of D-PBS containing 2.5% pancreatin (Gibco BRL, Grand Island, NY) and 5.46 mg/ml trypsin 1:250 (Cat. no. T4799; Sigma) with a syringe and blunt 20-gauge needle. The filled oviducts were tied off with cotton thread, submerged in D-PBS supplemented with 2.5% ABAM, and incubated for 2 h at 4°C followed by 1 h at room temperature. After incubation, oviducts were flushed with a warm solution of D-PBS containing 0.4 mg/ml collagenase (type II; Sigma), 10% heat-inactivated fetal calf serum (Gibco BRL), and 1% ABAM. Cell suspensions were incubated in a 37°C shaking water bath (45 cycles/min) for 30 min and then pelleted by centrifugation at 200 x g for 10 min. Harvested cells were washed with D-PBS supplemented with 1% ABAM, centrifuged at 200 x g for 5 min, and resuspended in RDG medium (RPMI 1640 and Dulbecco's Modified Eagle's medium, 1:1; Gibco BRL) supplemented with 10% heat-inactivated fetal calf serum, glucose (3 g/L), L-glutamine (0.292 g/L), sodium bicarbonate (2.85 g/L), and 1% ABAM. Suspended bovine oviduct epithelial cells were pipetted into 24- or 6-well plates that had been previously coated with collagen and were cultured in a humidified, 39°C, 5% CO₂ incubator.

Intracellular Cys and GSH Determinations

Levels of intracellular Cys and reduced GSH were determined by reverse-phase HPLC as described by Fahey and Newton [26] and modified by Harris [14]. The limit of detection for Cys and GSH using this procedure is 20 pmol. In vitro cultures of bovine oviduct epithelial cells from all stages and regions were allowed to come to confluency before harvesting and analysis. Confluent cultures were harvested by trypsin treatment (0.25% trypsin in a calcium- and magnesium-free PBS (CMF-PBS) followed by scraping. Harvested bovine oviduct epithelial cells were washed twice by centrifugation with CMF-PBS at 200 x g for 10 min. The resulting cell pellet was resuspended in 200 µl of 200 mM methanesulfonic acid (Aldrich Chemical Co., Milwaukee, WI), transferred into a 1.5-ml microcentrifuge tube, and promptly frozen in liquid nitrogen. Samples were stored at -75°C until derivitized for HPLC. Thawed samples were homogenized by ultrasonic cell disruption and held on ice. To precipitate proteins, 200 µl 4 M sodium methanesulfonate was added to the homogenized samples, which were then vortexed and centrifuged at 4°C for 10 min at 13 000 x g. The supernatant was removed and pipetted into new microcentrifuge tubes, held on ice, to which 160 µl Hepes buffer (1 M Hepes, 5 mM diethylenetriaminepentaacetic acid; pH 8.5) and 20 µl monobromobimane (Calbiochem, LaJolla, CA; 1.5 mg dissolved in 1 ml acetonitrile) had been added. This and succeeding steps were carried out in the dark. Samples were vortexed, and the reaction was allowed to proceed for 20 min at room temperature. To stop the reaction, 380 µl of 400 mM methanesulfonic acid was added to the tubes and vortexed. Derivitized samples were stored at -75°C until assayed by HPLC. HPLC analysis was conducted as described by Harris [14]. Resulting protein pellets (proteins extracted from bovine oviduct epithelial cells) from the derivitization procedure were held on ice until addition of 0.25 N NaOH, to dissolve cellular protein, and then assayed for total protein content using the method of Bradford [27], modified for use with a microtiter plate spectrophotometer [28]. BSA (fraction V; Sigma) was used to prepare the standard curve. For total thiol determinations, bovine oviduct epithelial cells were reduced by the addition of 1 mM dithiothreitol in Hepes buffer and incubated in a 37°C water bath for 30 min. Monobromobimane was then added and derivitization proceeded as described above.

Modulation of Cellular Thiols

A dose-response study was conducted to determine maximal thiol depletion by adding BSO (Sigma) or DEM (Aldrich Chemical Co.; dissolved in dimethyl sulfoxide) directly to the culture medium, which was then pipetted onto confluent bovine oviduct epithelial monolayers. Concentrations used for the dose-response study ranged from 10 to 5000 µM. These concentrations were chosen to encompass values found to modulate thiol content in other cell types. Control bovine oviduct epithelium monolayers were given dimethyl sulfoxide at concentrations equal to the amount added with the highest concentration of thiol modulator. Treated bovine oviduct epithelium monolayers were incubated with various levels of BSO for 5 h and DEM for 1 h. Maximum depletion of thiols was observed at 500 µM concentration levels for BSO and DEM treatment while maintaining 90% or greater viability.

To determine GSH turnover in bovine oviduct epithelium monolayers, 500 µM BSO was added to culture, and samples were taken at 0, 1, 2, 3, 5, and 24 h. Estimation of GSH synthesis capacity was accomplished by incubating bovine oviduct epithelium monolayers for 1 h in the presence of 500 µM DEM, to deplete GSH stores, followed by a wash and addition of fresh RDG medium to allow GSH to return to normal levels. Samples were taken prior to (-1 h) and after (0 h) a 1-h DEM exposure, and subsequently at 1, 3, 5, and 12 h post-DEM exposure.

Statistical Analysis

Data were analyzed using the Statistical Analysis System for personal computers [29]. Statistical differences in thiol levels—Cys and GSH—present in bovine oviduct epithelium monolayers from F-AMP, F-ISTH, L-AMP, and L-ISTH reproductive stage and region were tested using a nonparametric test (Kruskal-Wallis rank test), and pair-wise comparisons were tested by tabulating confidence intervals. Cys and GSH levels in bovine oviduct epithelium monolayers from F-AMP, F-ISTH, L-AMP, and L-ISTH samples treated with BSO or DEM were analyzed using the General Linear Models procedure to generate least square means and assess calculated probability values to find significance between sample means. A significance level of p < 0.05 was used for statistical testing.

	RESULTS

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Basal Thiol Levels in Bovine Oviduct Epithelial Cells

Basal levels of Cys and GSH in bovine oviduct epithelial cells cultured under optimal conditions did not differ significantly between reproductive stage (F or L) and region (AMP or ISTH). Cys concentrations ranged between 0.36 and 0.46 pmol/µg protein (Fig. 1A), whereas concentrations of GSH ranged from 5.3 to 6.4 pmol GSH/µg protein in oviduct epithelium of the various stages and regions (Fig. 1B). In addition, the percentage of total thiol in the oxidized form was determined for in vitro-cultured bovine oviduct epithelial cells (Table 1). Total GSH present in the oxidized form was significantly lower in the L-ISTH than in other stages and regions, whereas the F-AMP displayed the highest level.

View larger version (43K): [in this window] [in a new window]   FIG. 1. Basal levels of Cys (A) and reduced GSH (B) determined in bovine oviduct epithelial cells cultured in vitro. Bovine oviduct epithelial cells were cultured and maintained in a humidified, 39°C, 5% CO2 incubator until confluency (5–7 days). Thiol levels were measured from the AMP and ISTH regions of the oviduct from F- and L-staged cows. Each value represents the mean ± SE of eight separate determinations from three different cell isolations.

Modulation of Thiol Levels by BSO

Bovine oviduct epithelium cultured in the presence of 500 µM BSO (Fig. 2A) responded with a transient elevation of Cys concentrations. Cys levels were observed to peak at 3–5 h of culture, with the largest increases observed in the AMP (F, 167%; L, 178%). Bovine oviduct epithelium from the F-AMP, however, reached a maximum level by 3 h, followed by a decline in Cys content that was significantly lower at 5 h when compared to the L-AMP level. Interestingly, Cys content of BSO-treated oviduct epithelium derived from the AMP increased during the first hour of incubation and then declined to below control values after 24 h in each region. In contrast, the Cys content of ISTH oviduct epithelium remained elevated throughout the treatment period. Exposure to 500 µM BSO caused a significant depletion in GSH levels in bovine oviduct epithelium cultured over a 24-h incubation period (F-AMP, 38%; F-ISTH, 58%; L-AMP, 58%; L-ISTH, 61%; Fig. 3). This depletion was first observed to occur within the first 5 h of culture. These results suggest that in AMP-derived oviduct epithelium there is a higher degree of GSH turnover in L- compared to F-staged regions. These differences related to reproductive stage were not observed in the ISTH. Overall, the information obtained from exposing bovine oviduct epithelium to BSO suggests that the F-AMP appears to be more sensitive to environmental conditions than the other reproductive regions and stages owing to lower GSH turnover and elevated loss of Cys.

View larger version (23K): [in this window] [in a new window]   FIG. 2. Influence of BSO (500 µM) on Cys levels in confluent bovine oviduct epithelium cultures. Cys levels were measured in bovine oviduct epithelial cells from the A) AMP and B) ISTH region of the oviduct from F- and L-staged cows. Data are expressed as the percentage of control cell values observed in treated cells. Each value represents the mean ± SE of three separate determinations from two different cell isolations. An asterisk denotes significance at p < 0.05 between reproductive stage at designated time point.

View larger version (23K): [in this window] [in a new window]   FIG. 3. Influence of BSO (500 µM) on GSH levels in confluent bovine oviduct epithelium cultures. GSH levels were measured in bovine oviduct epithelial cells from the A) AMP and B) ISTH region of the oviduct from F- and L-staged cows. Data are expressed as the percentage of control cell values observed in treated cells. Each value represents the mean ± SE of three separate determinations from two different cell isolations. An asterisk denotes significance at p < 0.05 between reproductive stage at designated time point.

Influence of DEM Exposure on Thiol Levels and Recovery

Bovine oviduct epithelium was subjected to a 1-h exposure to 500 M DEM and then allowed to recover for 12 h to assess GSH synthesis in this culture system. Cys levels were observed to decrease in cultured bovine oviduct epithelium up to 52% during the 1-h exposure to 500 µM DEM (Fig. 4). Cys depletion was nearly identical between L- and F-staged oviduct epithelium in the AMP up to 1 h after DEM exposure. In the ISTH, Cys depletion was significantly greater in the L-ISTH (52%) as compared to the corresponding F-ISTH (26%). After DEM removal, Cys level recovery during the next 12 h of culture appeared to be dependent on reproductive stage. Cys recovery was greatest in L-staged oviduct epithelial cells of either region; however, values never returned to initial observed levels. In bovine oviduct epithelium from the F stages, a depletion in Cys levels was observed after DEM removal for the first 5 h of incubation; but during the remaining recovery period, low levels were maintained (Fig. 4, A and B). GSH levels in cultured bovine oviduct epithelium were depleted up to 60% during the 1-h exposure to 500 µM DEM (F-AMP, 49%; F-ISTH, 37%; L-AMP, 60%; L-ISTH, 52%; Fig. 5). After DEM removal, bovine oviduct epithelial cells were able to replenish GSH from 52% (F-ISTH) to 75% (L-AMP) of the control value. The reestablishment of normal GSH levels appeared to occur more efficiently in L-staged oviduct epithelium tissue than in F-staged samples. These results suggest that reproductive stage appears to influence the synthesis of GSH in bovine oviduct epithelial cells cultured in vitro, where L-staged bovine oviduct epithelium synthesizes or restores GSH faster than F-staged bovine oviduct epithelial cells, regardless of region.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Influence of a 1-h exposure of DEM (500 µM) on Cys levels in confluent bovine oviduct epithelium cultures. Cys levels were measured in bovine oviduct epithelial cells from the A) AMP and B) ISTH region of the oviduct from F- and L-staged cows. Data are expressed as the percentage of control cell values observed in treated cells. Each value represents the mean ± SE of three separate determinations from two different cell isolations. An asterisk denotes significance at p < 0.05 between reproductive stage at designated time point.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Influence of a 1-h exposure of DEM (500 µM) on GSH levels in confluent bovine oviduct epithelium cultures. GSH levels were measured in bovine oviduct epithelial cells from the A) AMP and B) ISTH region of the oviduct from F- and L-staged cows. Data are expressed as the percentage of control cell values observed in treated cells. Each value represents the mean ± SE of three separate determinations from two different cell isolations. An asterisk denotes significance at p < 0.05 between reproductive stage at designated time point.

	DISCUSSION

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This is the first study to address the potential inherent protective capacity that intracellular thiols, such as GSH, may provide in oviduct epithelium. The goal was to determine the status of thiols (Cys, GSH) in cultured bovine oviduct epithelium; to modulate that status using inhibitors, with the purpose of simulating changes that are elicited by oviduct epithelium in response to oxidants; and to define thiol regulation in bovine oviduct epithelium cultured in vitro. The ability of oviduct epithelium to maintain normal physiological functions may be dependent on its ability to regulate GSH stores, which can be lost through turnover, detoxification, and oxidative reactions. Results from this study provide evidence suggesting that reproductive stage (F or L) and region (AMP or ISTH) are important in assessing how this tissue may respond to chemical and environmental conditions that result in oxidative stress.

BSO, a selective inhibitor of GSH synthesis, was used to investigate the GSH turnover capacity of oviduct epithelium cultured under in vitro conditions. BSO does not interact directly with GSH; it alters GSH status only as a function of the normal rates of GSH turnover in a particular cell type after synthesis has ceased. At the concentration of BSO used in this study (500 µM), it was shown to deplete GSH (38–61%) but not influence viability; and we observed that GSH levels were depleted in L-staged oviduct epithelial cells more readily than in F-staged samples. In addition, a greater percentage of depletion was observed in the ISTH compared to the AMP region of the oviduct. These findings suggest that turnover is higher in cultured cells from L-staged tissue and the ISTH region of the oviduct. It is interesting to note that early stages of preimplantation embryo development occur in the ISTH region of the oviduct [1]. In addition, since reproductive stage was found to influence GSH turnover, one may infer that the hormonal environment and/or the estrogen-to-progesterone ratio may influence the ability of oviduct epithelial cells to modulate intracellular thiol status.

In comparison, Cys levels in BSO-exposed oviduct epithelium were found to be elevated in cells harvested from the AMP and ISTH, compared to controls, during the initial 3–5 h of culture. It should be noted that elevated Cys levels may be expected during BSO exposure if Cys is being taken up by the cell but not being incorporated into new GSH. However, further culture under these conditions revealed that the ISTH maintained elevated levels of Cys whereas the AMP displayed a decline in cellular Cys over time. This result suggests that Cys may be regulated differently depending upon oviduct region and demands of reproductive processes. Reproductive stage did not appear to influence cellular Cys content. This may have reproductive importance, because Cys, although essential for GSH synthesis, may also be required for proper preimplantation embryo development. Cys has been shown to be beneficial in preimplantation embryo development in some species. In the rabbit, Cys was shown to be one of the amino acids required for second-stage cleavage [30]. This sulfur-containing amino acid has also been shown to be important in male pronuclear formation in in vitro-fertilized pig oocytes [17]. However, it should also be mentioned that in the hamster, 0.5 mM concentrations of Cys were demonstrated to be inhibitory to 1-cell embryo development up to the blastocyst stage [31], yet a lower concentration (0.05 mM) of this amino acid was found to sustain normal development.

Culture of oviduct epithelial cells in the presence of DEM, which depletes GSH by rapidly forming an adduct with this tripeptide, provides insight into the capacity of the oviduct epithelium to synthesize and replenish GSH stores because GSH biosynthesis is not inhibited. Results from this study suggest that L-staged oviduct epithelium is able to synthesize GSH more readily than oviduct epithelium cultured from F-staged oviducts. Also, the AMP region appeared to be more successful at restoring GSH levels than the ISTH, especially the F-ISTH, which achieved only 50% of control levels by 12 h post-DEM exposure in culture. However, it should be noted that the greatest percentage of recovery for GSH was only 75%, which was observed in the L-AMP. This information suggests that GSH synthesis is slow in oviduct epithelial cells compared to other cell types. For example, in the rat postimplantation conceptus, GSH pools were shown to be replenished at a rate of 273 pmol/conceptus per hour after a 45-min exposure to DEM [14]. This would calculate into a 4.5- to 5.0-h recovery period, after DEM depletion, to restore GSH to control levels in this tissue. The decreased ability to restore GSH levels in the oviduct epithelium would again suggest that this tissue may be sensitive to chemicals or environmental conditions that could lead to oxidative stress. This could result in a decrease in oviduct function, subsequently leading to a decrease in fertility or preimplantation embryo development.

Maintenance of Cys stores after DEM exposure also appears to be influenced by reproductive stage and oviduct region. As observed with GSH levels, L-staged cells can restore Cys levels more rapidly than cells from F-staged tissue. The ISTH again appears to be able to maintain Cys levels better than the AMP, especially when compared to the F-AMP—as observed in the previous study using BSO in which the ISTH was shown to maintain elevated levels of Cys. The ability to regulate Cys could play an important role in the ability of oviduct epithelial cells to synthesize GSH, since this amino acid is considered rate-limiting in this process.

Information obtained from this study strongly supports the hypothesis that hormonal stage, as well as the oviduct region, is important in the regulation of thiol status in this tissue. The hormonal environment has been observed to regulate uterine tissue physiology. Estrogen was shown not only to increase uterine weight in vivo but also to increase GSH levels in exposed tissue [32]. In addition, GSH status in rat hepatocytes has been shown to be regulated by various hormones [33]. Hirayama et al. [34] have reported that gonadal steroid hormones, specifically estradiol and testosterone, appear to regulate hepato-renal GSH metabolism. Gonadal steroid hormones have been shown to regulate enzymes important in maintaining GSH status and function, such as GSH-S-transferase, -glutamyltranspeptidase, and GSH peroxidase [35, 36]. This information provides evidence that GSH regulation in oviduct epithelial cells may possibly be dependent on the hormonal environment.

Regulation of thiol status by the oviduct epithelium, in both the cellular (epithelium) and extracellular (oviduct fluid) compartments, may be instrumental in successful reproduction. The ability of the oviduct epithelium to regulate thiol levels not only may be essential for proper cellular function but also is important for modulation of the oviduct environment. Events of fertilization and preimplantation embryo development are dependent on proper oviduct function. This study provides initial evidence that the oviduct may be sensitive to chemicals or environmental conditions leading to oxidative stress. Results from this study warrant further investigation into thiol regulation in the oviduct and the manner in which this may influence reproduction.

	FOOTNOTES

 
¹ Research supported by NIH grants ES05235 and ES07062.

² Correspondence: Craig Harris, Toxicology Program, Department of Environmental and Industrial Health, University of Michigan, 1420 Washington Heights, Ann Arbor, MI 48109–2029. FAX: (734) 763–8095.

³ Current address: Tamara L. McNutt-Scott, Biology Department, 2500 Walton Way, Augusta State University, Augusta, GA 30904–2200.

Accepted: March 18, 1998.

Received: September 22, 1997.

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