1 Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA and 2 Instituto Valenciano de Infertilidad, Valencia, Spain
3 To whom correspondence should be addressed. e-mail: antoni.duleba{at}yale.edu
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Abstract |
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Key words: antioxidants/oxidative stress/proliferation/theca
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Introduction |
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This study was designed to compare the effects of antioxidants and oxidative stress on proliferation of T-I cells.
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Materials and methods |
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Animals
SpragueDawley female rats were obtained on day 25 of age from Taconic Farms (Germantown, NY) and housed with a 12 h light:12 h dark photoperiod in an air-conditioned environment. Standard rat chow and water were given ad libitum. Starting on day 28 of age, the animals were injected with 17-estradiol (1 mg/0.3 ml sesame oil s.c.) daily for 3 days in order to stimulate ovarian development and development of antral follicles. Approximately 24 h after the last injection (day 31 of age), the animals were anaesthetized with ketamine and xylazine (i.p.) and sacrificed by intracardiac perfusion with 0.9% saline. All treatments and procedures were in accordance with accepted standards of humane animal care as outlined in the NIH Guide for the Care and Use of Laboratory Animals and a protocol approved by the Yale University Animal Care Committee.
Cell culture
Ovarian T-I cells were obtained as follows. Following saline perfusion described above, ovaries were dissected, and T-I cells were purified using discontinuous Percoll gradient centrifugation as described previously (Magoffin and Erickson, 1988; Duleba et al., 1997
). The immunohistochemical purity of this cell preparation has been demonstrated previously (Duleba et al., 1997
). The cells were counted and viability was routinely in the 8595% range. T-I cells were incubated for up to 96 h at 37°C in an atmosphere of 5% CO2 in humidified air, in serum-free McCoys 5a medium (with antibiotics, 0.1% BSA and 2 mmol/l L-glutamine). In cultures carried out for >48 h, media and treatments were replaced at 48 h; in order to minimize detachment of cells, these cultures were carried out on fibronectin-coated plates. The cells were incubated without or with VES (1100 µmol/l), ebselen (0.330 µmol/l), SOD (1001000 U/ml) or HX/XO (1 mmol/l of HX and 11000 µU/ml of XO).
Thymidine incorporation assay
T-I cells were incubated for 48 h in 96-well culture plates with or without individual additives. Assay of DNA synthesis was carried out using the thymidine incorporation assay. Radiolabelled [3H]thymidine (4 µCi/ml) was added to T-I cells during the last 24 h of culture. At the end of the culture period, the cells were harvested using a multiwell cell harvester (PHD Harvester, Model 290; Cambridge Technology, Inc., Watertown, MA). Radioactivity was measured in a liquid scintillation counter, SL 4000 (Intertechnique, Fairfield, NJ). Each treatment was carried out in at least eight replicates.
MTT assay
T-I cells were incubated for up to 96 h with or without individual additives in 96-well fibronectin-coated plates. The media and additives were replaced after 48 h. At the end of the culture period, MTT (125 µg/well) was added for 4 h, then the supernatants were removed and 96 µl of isopropanol + 4 µl of HCl (1 mol/l) was added to each culture well. Optical density at 570 nm was determined.
Cell counting and identification of steroidogenically active cells
T-I cells were cultured in fibronectin-coated 24-well plates for 96 h. Media and treatments were replaced after 48 h of culture. Each treatment was carried out in four replicates. At the end of the cell culture period, the cells were washed with calcium-free and magnesium-free PBS (x1, pH 7.2). Trypsin-EDTA (0.05 and 0.02%, respectively; 0.3 ml/2 cm2) solution was dispensed into culture wells to completely cover the monolayer of cells and the culture dish was placed at 37°C for 23 min. When cells were in suspension and appeared rounded, McCoys 5a medium was added to inhibit trypsin activity. The T-I cells were then washed with PBS (x1, pH 7.2) and fixed in 1% paraformaldehyde for 20 min. Steroidogenically active T-I cells were identified histochemically by detection of 3-hydroxysteroid dehydrogenase (3
-HSD) activity as described by others (Bao et al., 1995
). Briefly, fixed T-I cells were reconstituted in histochemical staining solution containing PBS pH 7.2 supplemented with 0.1% BSA, 1.5 mmol/l
-NAD+, 0.25 mmol/l nitroblue tetrazolium and 0.2 mol/l
-androstane-3
-ol-17-one. The cells were incubated overnight in a shaker at 37°C in the dark, spun down and resuspended in PBS (pH 7.2). The number of stained cells (3
-HSD+; steroidogenically active) and non-stained cells (3
-HSD; steroidogenically inactive) was determined by counting 10 squares from each sample using a haemocytometer. In separate experiments, the viability of T-I cells at the end of cultures and following trypsinization was assessed using the trypan blue exclusion test and was found to be 9498%.
Statistical analysis
Values represent means ± SEM. Statistical analysis was performed using analysis of variance followed by pairwise comparisons using Bonferroni correction.
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Results |
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Discussion |
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Importantly, VES exhibited potent inhibitory effects on proliferation at concentrations corresponding to physiological levels of -tocopherol. In a large survey of adults in the USA, the mean concentration of
-tocopherol was 26.8 µmol/l (Ford and Sowell, 1999
).
-Tocopherol is a particularly important natural chain-breaking antioxidant inhibiting lipid peroxidation in membranes by scavenging peroxyl and alkoxyl radicals (Smith et al., 1993
; Ham and Liebler, 1995
).
Proliferation was also inhibited in a dose-dependent fashion by the glutathione peroxidase mimetic, ebselen, and by the scavenging of superoxide anions by SOD. The inhibitory effects of antioxidants occurred under baseline conditions, i.e. in the absence of induction of ROS, thus indicating that the source of ROS resides within T-I cells. Furthermore, our findings suggest that ongoing generation of ROS is required in order to maintain DNA synthesis and that the reduction of the level of ROS inhibits proliferation of T-I cells. The speculation that moderate oxidative stress promotes proliferation is supported by the observation that generation of superoxide radicals using HX/XO results in a significant increase of DNA synthesis (Figure 4). Indeed, growing evidence indicates that ROS may act as important mediators of mitogenic signalling, as demonstrated in several cell types including fibroblasts and smooth muscle (Murrell et al., 1990; Brar et al., 1999
; Clement and Pervaiz, 1999
; Kunsch and Medford, 1999
).
While the findings of this study suggest a role for oxidative stress in the regulation of the proliferation of T-I cells, several important caveats should be noted. Most importantly, the in vitro and in vivo milieu of cells may differ greatly due to endocrine/paracrine effects as well as differences in other parameters such as the amount of available oxygen. In particular, oxygen tension in culture media is significantly greater than that in the theca compartment in vivo; thus, observations in our in vitro system may not accurately reflect effects occurring in vivo. Furthermore, an observation of different rates of DNA synthesis and cell number does not exclude the possibility that changes in oxidative stress may affect the rate of apoptosis. In this study, cell viability at the end of the culture was in the 9698% range even after exposure to the highest concentrations of antioxidants; nevertheless, such findings do not exclude the possibility of changes in the rate of ongoing apoptosis.
The observation in this study of a biphasic effect of the generation of superoxide radicals on proliferation is consistent with the concept that while moderate oxidative stress may induce cell growth, very high levels of ROS are cytotoxic and lead to apoptotic cell death (Roberg and Ollinger, 1998; Takahashi et al., 2002
). However, at moderate concentrations, ROS may act as intra- and intercellular messengers capable of promoting growth responses (Burdon et al., 1995
, 1996; del Bello et al., 1999
).
The above speculations raise interesting questions regarding the mechanisms regulating oxidative stress. It is possible that ROS may be induced by a broad range of agents, including insulin, insulin-like growth factors (IGFs) and TNF-. Indeed, administration of insulin and IGF-I increases low-density lipoprotein (LDL) peroxidation, as measured by the production of thiobarbituric acid-reactive substances (TBARS), and other measures of oxidative stress (Rifici et al., 1994
). Treatment of adipocytes with insulin leads to a rapid increase in hydrogen peroxide (Krieger-Brauer and Kather, 1995
; Krieger-Brauer et al., 1997
). Insulin-induced oxidative stress may explain the observation that insulin decreases circulating vitamin E levels in type II diabetics, as well as in healthy lean and obese subjects; this effect remains significant even after accounting for insulin-induced changes in lipid levels (Krieger-Brauer et al., 1997
). TNF-
may also be involved in oxidative stress. An association between the plasma level of TNF-
and lipid peroxidation has been observed (Chen et al., 1998
). Injection of TNF-
into healthy animals results in changes of plasma lipoprotein lipid composition due to peroxidation (McDonagh et al., 1992
). TNF-
also induces oxidative stress in vitro (Adamson and Billings, 1992
). T-I cells possess receptors to insulin, IGFs and TNF-
, agents that have been shown to induce T-I proliferation (Duleba et al., 1997
, 1998; Spaczynski et al., 1999
). It is, therefore, possible that the actions of insulin, IGFs and TNF-
may be mediated, at least in part, by induction of oxidative stress.
The present findings may also have important clinical relevance in conditions such as PCOS. Recently, Sabuncu et al. (2001) found that women with PCOS have increased oxidative stress and decreased antioxidant reserve. PCOS is also characterized by hyperplasia of ovarian theca and stroma (Hughesdon, 1982
), Thus, it is tempting to speculate that in PCOS, increased oxidative stress and insufficient antioxidant activity contribute to excessive growth of ovarian mesenchyme. At present, the mechanisms involved in the generation of oxidative stress in PCOS remain elusive. However, since PCOS is associated with elevations of insulin, free bioavailable IGF-I and TNF-
, it is possible that these agents act through generation of excessive ROS. Our current studies are directed at evaluating possible interactions between insulin/IGF-I/TNF-
systems and oxidative stress.
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Acknowledgements |
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References |
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Submitted on October 31, 2003; accepted on April 13, 2004.
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