Differential Regulation of Copper-Zinc Superoxide Dismutase and Manganese Superoxide Dismutase in the Rat Corpus Luteum: Induction of Manganese Superoxide Dismutase Messenger Ribonucleic Acid by Inflammatory Cytokines¹

^a Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612–7342

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study was undertaken to investigate the regulation of mitochondrial manganese superoxide dismutase (Mn-SOD) and cytosolic copper-zinc SOD (Cu,Zn-SOD) in the corpus luteum by inflammatory cytokines. We first examined the developmental expression of both SOD mRNAs in the rat corpus luteum throughout pregnancy. SOD mRNA levels were determined by semiquantitative reverse transcription-polymerase chain reaction. Whereas Cu,Zn-SOD mRNA levels decreased during late pregnancy, Mn-SOD mRNA levels remained elevated. We secondly examined the effects of inflammatory reaction on luteal SODs. Rats received injections of lipopolysaccharide (LPS; 5 mg, i.p.) on Day 15 of pregnancy, and corpora lutea were removed 2 h later. LPS caused an increase in Mn-SOD mRNA levels in the corpus luteum and a decrease in serum progesterone levels, but neither in levels of Cu,Zn-SOD mRNA. To further study the effects of LPS or LPS-induced cytokines, we incubated either whole corpora lutea obtained on Day 15 of pregnancy or a temperature-sensitive simian virus-40 transformed luteal cell line (GG-CL; derived from large luteal cells of the corpus luteum of pregnant rats) in serum-free medium with LPS, interleukin-1 (IL-1), IL-ß, IL-6, and tumor necrosis factor . LPS and these cytokines induced a remarkable increase in Mn-SOD mRNA levels in both corpora lutea and GG-CL cells but had no effect on Cu,Zn-SOD mRNA expression. In conclusion, Cu,Zn-SOD and Mn-SOD mRNAs are differently expressed and regulated in the corpus luteum of pregnancy. Mn-SOD mRNA, but not Cu,Zn-SOD mRNA, is highly induced by inflammatory cytokines and may play an important role in protecting luteal cells from inflammation-mediated oxidative damage.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Reactive oxygen species including superoxide radicals are well known to cause cell damage. They are increased in the corpus luteum during the regression phase and inhibit progesterone production in rats [1–6]. These findings have strongly suggested the involvement of reactive oxygen species in corpus luteum regression. In contrast, superoxide dismutase (SOD) protects cells by scavenging superoxide radicals. Eukaryotic cells have two types of cellular SOD: copper-zinc SOD (Cu,Zn-SOD) located in the cytosol, and manganese SOD (Mn-SOD) located in the mitochondria. Both belong to a first enzymatic step that protects cells against toxic oxygen radicals. The corpus luteum has both Cu,Zn-SOD and Mn-SOD enzymes [1, 2, 5]. Their activities increase until midpregnancy and gradually decrease thereafter, in a manner similar to the changes in serum progesterone levels in pregnant rats [1], suggesting that these SODs may play important roles in the maintenance of luteal function and that the decrease in SOD activity in the corpus luteum is, at least in part, involved in corpus luteum regression [1–3, 5]. However, the regulation of these SODs at the mRNA level and their individual roles in luteal function have not been clarified yet. It is well known that macrophages are the prominent cell type in the regressing corpus luteum and produce cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF), which initiate an acute-phase inflammatory reaction [7–10]. These cytokines have been also demonstrated to be expressed in the corpus luteum and appear to affect luteal steroidogenesis [8, 9, 11–15]. Interestingly, whereas these cytokines have been reported to cause superoxide radical generation [16, 17], they also protect cells against superoxide radicals by inducing Mn-SOD expression [18, 19]. There is, therefore, a possibility that luteal SODs may be regulated by inflammatory cytokines. However, it is not clear how Cu,Zn-SOD and Mn-SOD respond to inflammatory cytokines in the rat corpus luteum. To better understand the regulation and role of luteal Cu,Zn-SOD and Mn-SOD, we examined the developmental expression of Cu,Zn-SOD and Mn-SOD mRNAs in the corpus luteum throughout pregnancy and the effects of inflammatory cytokines on Cu,Zn-SOD and Mn-SOD mRNA in both the corpus luteum and a cell line derived from rat luteal cells which expresses both antioxidant enzymes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials

McCoy's 5A:Ham's F-12 1:1 medium, D-glucose, mouse recombinant IL-1, human recombinant IL-1ß, human recombinant IL-6, human recombinant TNF, lipopolysaccharide (LPS), actinomycin D, cycloheximide, and phorbol 12-myristate 13-acetate (TPA) were purchased from Sigma Chemical Co. (St. Louis, MO). RPMI-1640 medium, antibiotic-antimycotic solution, nonessential amino acids, and sodium pyruvate were from Mediatech (Washington, DC). [-³²P]Deoxycytidine triphosphate (dCTP) was from Amersham (Arlington Heights, IL). Twenty-four-well tissue culture plates and 25-cm² culture flasks were from Becton Dickinson Co. (Franklin Lakes, NJ). Taq DNA polymerase was from Perkin-Elmer Co. (Foster City, CA).

Animals

Pregnant Sprague-Dawley rats (Day 1 = sperm positive) purchased from Sasco Animal Labs (Madison, WI) were housed at 22°C with a 14L:10D cycle (lights-on 0500–1900 h) and allowed free access to Purina rat chow and water. The rat care and handling conformed with the NIH guidelines for animal research. The experimental protocol was approved by the Institutional Animal Care and Use Committee. Rats on Day 15 of pregnancy received i.p. injections of 5 mg of LPS diluted into PBS (0.5 ml), and the ovaries were removed 2 h after injection. Controls received injections of 0.5 ml PBS.

Incubation of Corpora Lutea

Rats were laparotomized under ether anesthesia on Day 15 of pregnancy, and the ovaries were removed. The incubation of corpora lutea was performed as reported previously [20]. In brief, corpora lutea were dissected and cleaned of adhering tissue in a watch glass containing serum-free medium (McCoy's 5A:Ham's F-12 1:1, containing 25 mM HEPES and 2% antibiotic-antimycotic solution). Corpora lutea (5 CL/ml in a 24-well tissue culture plate) were incubated in medium containing LPS (100 µg/ml), IL-1 (100 ng/ml), IL-1ß (100 ng/ml), IL-6 (100 ng/ml), or TNF (100 ng/ml) at 37°C for 4 h under an atmosphere of 100% oxygen in a shaking warmer. After incubation, corpora lutea were immediately frozen in liquid nitrogen and stored at -80°C until RNA isolation.

Cell Culture

The luteal cell line termed GG-CL, which was recently generated in our laboratory [21], was used in this study. Originally, large luteal cells were purified to homogeneity by flow cytometry from corpora lutea of Day 14 pregnant rats as reported previously [22]. Cells were infected with a temperature-sensitive simian virus-40 (SV-40 tsA209) as previously reported [23]. Transformed cells were maintained at the permissive temperature (33°C) until colonies were identified. Several colonies of the transformed cells were isolated and passaged. One clone designated GG-CL cells was extensively characterized and was shown to express many genes characteristic of primary luteal cells, although GG-CL cells lose steroidogenic activity including progesterone production [21]. GG-CL cells were cultured with various doses of LPS (1, 10, and 100 µg/ml), IL-1 (1, 10, and 100 ng/ml), IL-1ß (1, 10, and 100 ng/ml), IL-6 (1, 10, and 100 ng/ml), or TNF (1, 10, and 100 ng/ml) with or without actinomycin D (4 µM) or cycloheximide (50 µM), and with TPA (0.004, 0.04, 0.4, and 4 µM) in 25-cm² flasks in serum-free medium (RPMI-1640 containing double-strength antibiotic-antimycotic solution, single-strength nonessential amino acids, single-strength sodium pyruvate, 0.5% D-glucose) for 4 h at 33°C under an atmosphere consisting of 5% CO₂:95% air. Cells were washed with PBS several times after incubation and stored at -80°C until RNA isolation. Incubations were run in triplicate. LPS and the cytokines used in this study had no effect on cell viability examined by the trypan blue dye exclusion method.

Isolation of Total RNA and Reverse Transcription (RT) Polymerase Chain Reaction (PCR)

Total RNA was isolated from corpora lutea by homogenization in guanidinium thiocyanate and centrifugation through a cesium chloride cushion [24], whereas total RNA from the cultured cells was isolated by the guanidinium-isothiocyanate-phenol-chloroform extraction procedure [25]. For mRNA analysis by RT-PCR, oligonucleotide primers for Cu,Zn-SOD (5'-TTCGAGCAGAAGGCAAGCGGTGAA-3' and 5'-AATCCCAATCACACCACAAGCCAA-3') and for Mn-SOD (5'-ATTAACGCGCAGATCATGCAG-3' and 5'-TTTCAGATAGTCAGGTCTGACGTT-3') were designed on the basis of the rat Cu,Zn-SOD [26] and Mn-SOD cDNA sequences [27]. Each reaction also included two oligonucleotide primers (5'-CGTTCACCTTGATGAGCCCATT-3' and 5'-TCCAAGGGTCCGCTGCAGTC-3') to amplify ribosomal protein S16 as an internal control [28]. The predicted sizes of the PCR-amplified products were 396 base pairs (bp) for Cu,Zn-SOD, 483 bp for Mn-SOD, and 100 bp for S16. Two to three micrograms of total RNA were reverse-transcribed at 42°C in a 20-µl reaction mixture (single-strength PCR buffer, 2.5 mM deoxynucleoside triphosphates, 5 µM random hexamer primers, 1.5 mM MgCl₂, and 200 U Moloney murine leukemia virus reverse transcriptase [Life Technologies, Gaithersburg, MD]). For PCR amplification, a mixture containing the oligonucleotide primers (50 pmol), [-³²P]dCTP (2 µCi at 3000 Ci/mmol), and Taq DNA polymerase (2.5 U) was added to each reaction. The total volume was increased to 90 µl with single-strength PCR buffer, and the samples were overlaid with light mineral oil. Amplification was carried out for 20 cycles using a 65°C annealing temperature in a Perkin-Elmer/Cetus thermal cycler. The conditions were such that the amplification of the product was in the exponential phase and the assay was linear with respect to the amount of input RNA. Reaction products were electrophoresed on an 8% polyacrylamide nondenaturing gel. After autoradiography, data were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

RIA

Progesterone concentrations in the serum and the medium were measured using a commercially obtained kit (Diagnostic Products Corporation, Los Angeles, CA). The sensitivity of the assay was 0.02 ng/ml, and the inter- and intraassay coefficients of variation were 5% and 6%, respectively.

Statistical Analysis

Data were examined by ANOVA and Duncan's new multiple-range test. Where appropriate, Student's t-test was employed. Differences were considered to be significant if p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In order to determine whether Cu,Zn-SOD mRNA and Mn-SOD mRNA are expressed in the rat corpus luteum, we performed RT-PCR to specifically detect Cu,Zn-SOD and Mn-SOD transcripts. As shown in Figure 1, a linear RT-PCR amplification of Cu,Zn-SOD, Mn-SOD, and S16 mRNAs was obtained using up to 25 cycles of PCR and 2 µg of total RNA. Therefore, subsequent experiments were performed with 20 cycles of amplification for PCR.

View larger version (22K): [in this window] [in a new window]   FIG. 1. Relationship between cycle numbers and RT-PCR amplification of Cu,Zn-SOD, Mn-SOD and S16 mRNAs. RT-PCR was performed with a fixed RNA concentration (2 µg) of Day 15 pregnant rat corpus luteum as described in Materials and Methods. An autoradiogram of a polyacrylamide gel on which the products of an RT-PCR have been separated, and its quantification data are shown.

To investigate the developmental changes in Cu,Zn-SOD and Mn-SOD mRNA, corpora lutea were isolated on different days of pregnancy, the day of parturition, or the day after parturition. Total RNA was isolated and subjected to RT-PCR with S16 as an internal control. The results shown in Figure 2 indicate that Cu,Zn-SOD mRNA in the corpus luteum increased from early to mid pregnancy, decreased thereafter, and remained at low levels at the end of pregnancy and after parturition. Mn-SOD mRNA changed in a manner similar to that of Cu,Zn-SOD mRNA during early to mid pregnancy but remained elevated during late pregnancy and after parturition, suggesting that Mn-SOD and Cu,Zn-SOD mRNA may be regulated differently during the regression phase of the corpus luteum.

View larger version (41K): [in this window] [in a new window]   FIG. 2. Developmental expression of Mn-SOD (A) and Cu,Zn-SOD (B) mRNA in the corpus luteum throughout pregnancy and after parturition (mean ± SEM of three experiments). Total RNA isolated from corpora lutea throughout pregnancy and after parturition were subjected to RT-PCR. RNA samples were collected from more than three animals on each day in each experiment. P0 and P1, postpartum Day 0 and Day 1, respectively. Lowercase letters indicate significant differences. For A: a, p < 0.01 vs. Days 15, 17, 19, 20, and 21; b, p < 0.05 vs. Days 12, 18, 22, P0, and P1; c, p < 0.01 vs. Days 15, 17, 19, 20, 21, and 22; d, p < 0.05 vs. Days 12 and 18, P0, and P1. For B: a, p < 0.01 vs. Day 17; b, p < 0.05 vs. Day 17; c, p < 0.01 vs. Day 18; d, p < 0.05 vs. Day 18; e, p < 0.05 vs. Days 4 and 6.

To induce an acute inflammatory reaction in the corpus luteum, rats received injections in vivo of LPS on Day 15 of pregnancy, and corpora lutea were isolated 2 h after injection. As shown in Figure 3A, LPS administered in vivo caused a remarkable increase in Mn-SOD mRNA levels in the corpus luteum but not in levels of Cu,Zn-SOD mRNA (Fig. 3B). Serum progesterone concentrations in rats receiving LPS were significantly (p < 0.05) lower than those in controls (71.9 ± 17.4 ng/ml vs. 132.2 ± 9.7 ng/ml, n = 5).

View larger version (36K): [in this window] [in a new window]   FIG. 3. Effects of LPS administration on Mn-SOD (A) and Cu,Zn-SOD (B) mRNA levels in the corpus luteum (mean ± SEM of four experiments). Rats received i.p. injections of 5 mg of LPS, and total RNA was isolated from corpora lutea 2 h after injection and subjected to RT-PCR. RNA samples of corpora lutea were collected from more than three animals in each group in each experiment. C, Control. a, p < 0.01 vs. C (Student's t-test).

To further examine the effects of LPS on luteal SOD expressions, whole corpora lutea from Day 15 of pregnancy were incubated with either LPS or LPS-induced inflammatory cytokines IL-1, IL-1ß, IL-6, or TNF for 4 h in the absence of serum. All treatments induced an increase in Mn-SOD mRNA levels (Fig. 4A), but had no effect on Cu,Zn-SOD mRNA levels (Fig. 4B). There were no significant changes in progesterone concentrations in the medium (data not shown).

View larger version (71K): [in this window] [in a new window]   FIG. 4. Effects of LPS, IL-1, IL-1ß, IL-6, and TNF on Mn-SOD (A) and Cu,Zn-SOD (B) mRNA levels in the corpus luteum in vitro (mean ± SEM of four experiments). Corpora lutea obtained on Day 15 of pregnancy were incubated with LPS (100 µg/ml), IL-1 (100 ng/ml), IL-1ß (100 ng/ml), IL-6 (100 ng/ml), or TNF (100 ng/ml) for 4 h in the absence of serum. Total RNA was isolated and subjected to RT-PCR. a, p < 0.01 vs. the other groups.

The corpus luteum is formed by many different cell types—endothelial cells, fibroblasts, and leukocytes, in addition to luteal cells. These cells have both SODs, and macrophages can respond to LPS and cytokines with increased cytokine expression. To examine whether the effects of LPS and cytokines on Mn-SOD mRNA expression occur in luteal cells, we used the SV-40 transformed luteal cell line (GG-CL) derived from large luteal cells of the corpus luteum of pregnant rats and recently developed in our laboratory [21]. We first established that these cells expressed both Mn-SOD and Cu,Zn-SOD mRNA (Fig. 5, A and B, and lane 1) and then examined the dose-related effects of LPS and of inflammatory cytokines on the expression of both SOD mRNAs in cells incubated in the absence of serum. As shown in Figure 5, LPS added to the luteal cells in culture caused a remarkable dose-related increase in Mn-SOD mRNA expression (Fig. 5A) but had no effect on Cu,Zn-SOD mRNA expression (Fig. 5B). Similar dose-related up-regulation of Mn-SOD was obtained with IL-1 (Fig. 6), IL-1ß (Fig. 7) and TNF (Fig. 8). IL-6, however, was less potent at up-regulating Mn-SOD mRNA (Fig. 9A), and an increase in mRNA levels was observed at the highest dose only. These findings in GG-CL cells were similar to those in the corpus luteum incubation, suggesting that the different regulation of Mn-SOD and Cu,Zn-SOD mRNAs by cytokines in GG-CL cells is not due to cell transformation.

View larger version (49K): [in this window] [in a new window]   FIG. 5. Effects of LPS on Mn-SOD (A) and Cu,Zn-SOD (B) mRNA levels in GG-CL cells (mean ± SEM of three experiments). GG-CL cells were incubated for 4 h with LPS (1, 10, and 100 µg/ml) in the absence of serum. Total RNA was isolated and subjected to RT-PCR. b, p < 0.05 vs. control (C).

View larger version (53K): [in this window] [in a new window]   FIG. 6. Effects of IL-1 on Mn-SOD (A) and Cu,Zn-SOD (B) mRNA levels in GG-CL cells (mean ± SEM of three experiments). GG-CL cells were incubated for 4 h with IL-1 (1, 10, and 100 ng/ml) in the absence of serum. Total RNA was isolated and subjected to RT-PCR. a, p < 0.01 vs. control (C).

View larger version (54K): [in this window] [in a new window]   FIG. 7. Effects of IL-1ß on Mn-SOD (A) and Cu,Zn-SOD (B) mRNA levels in GG-CL cells (mean ± SEM of three experiments). GG-CL cells were incubated for 4 h with IL-1ß (1, 10, and 100 ng/ml) in the absence of serum. Total RNA was isolated and subjected to RT-PCR. a, p < 0.01 vs. control (C).

View larger version (49K): [in this window] [in a new window]   FIG. 8. Effects of TNF on Mn-SOD (A) and Cu,Zn-SOD (B) mRNA levels in GG-CL cells (mean ± SEM of three experiments). GG-CL cells were incubated for 4 h with TNF (1, 10, and 100 ng/ml) in the absence of serum. Total RNA was isolated and subjected to RT-PCR. a, p < 0.01, and b, p < 0.05, vs. control (C).

View larger version (60K): [in this window] [in a new window]   FIG. 9. Effects of IL-6 on Mn-SOD (A) and Cu,Zn-SOD (B) mRNA levels in GG-CL cells (mean ± SEM of three experiments). GG-CL cells were incubated for 4 h with IL-6 (1, 10, and 100 ng/ml) in the absence of serum. Total RNA was isolated and subjected to RT-PCR. b, p < 0.05 vs. control (C).

The increase in Mn-SOD mRNA levels induced by cytokines could reflect either increased transcription or increased stability of the message. To investigate whether the induction of Mn-SOD mRNA by cytokines is dependent on gene transcription and to determine whether de novo protein synthesis is required for these effects, GG-CL cells were incubated with IL-1, IL-1ß, IL-6, or TNF in the presence or absence of either actinomycin D, a potent RNA synthesis inhibitor, or cycloheximide, a protein synthesis inhibitor. Cells were also treated with the inhibitors alone. As shown in Figure 10, actinomycin D completely abolished the stimulatory effect of IL-1 (panel A, lanes 2 and 3), IL-1ß (panel B, lanes 2 and 3), IL-6 (panel C, lanes 2 and 3), and TNF (panel D, lanes 2 and 3). This inhibitor itself, however, had no effect (lane 5 in all panels). In sharp contrast to results obtained with actinomycin D, cycloheximide had no effect on stimulation of Mn-SOD mRNA mediated by IL-1 (panel A, lanes 2 and 4), IL-1ß (panel B, lanes 2 and 4), IL-6 (panel C, lanes 2 and 4), or TNF (panel D, lanes 2 and 4). Nor did this inhibitor affect the basal expression of Mn-SOD mRNA (lane 6 in all panels).

View larger version (36K): [in this window] [in a new window]   FIG. 10. Effects of actinomycin D and cycloheximide on the Mn-SOD mRNA induction by IL-1 (A), IL-1ß (B), IL-6 (C), and TNF (D) (mean ± SEM of three experiments). GG-CL cells were incubated with IL-1 (100 ng/ml), IL-1ß (100 ng/ml), IL-6 (100 ng/ml), or TNF (100 ng/ml) for 4 h in the presence or absence of actinomycin D (AD; 4 µM) or cycloheximide (CHX; 50 µM). Total RNA was isolated and subjected to RT-PCR. a, p < 0.01 and b, p < 0.05, vs. control or cytokine+AD.

Since it has been reported that protein kinase C (PKC)-dependent phosphorylation is involved in the Mn-SOD induction by cytokines [29,30], we examined the effects of PKC activation on SOD mRNA levels in GG-CL cells. To activate PKC, GG-CL cells were incubated with TPA at concentrations that have been reported to be effective. TPA had no effect on either Mn-SOD or Cu,Zn-SOD mRNA expression (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
To our knowledge, this is the first report showing the changes in luteal mRNA levels of Cu,Zn-SOD and Mn-SOD throughout pregnancy and the different regulation of both SODs by cytokines in the rat corpus luteum. Whereas Cu,Zn-SOD mRNA levels in the corpus luteum decreased and remained at low levels during the regression phase, Mn-SOD mRNA levels did not decrease and remained at relatively high levels. This may be due, at least in part, to the selective induction of Mn-SOD mRNA by inflammatory reactions or cytokines, because the regressing corpus luteum is presumed to be under a cytokine-rich environment [14, 15]. In fact, these cytokines have been demonstrated to be expressed in the corpus luteum [9, 11–13], and increased TNF levels in luteal tissue around the time of luteolysis have also been reported in several species [9, 31,32]. In addition, since progesterone can inhibit superoxide radical production by the macrophages in the rat corpus luteum [7] as well as their metabolic activities [33], a decrease in progesterone levels may allow macrophages to enhance cytokine production in the corpus luteum during the regression phase. In other words, the present results may also suggest that the expression of inflammatory cytokines is increased in the corpus luteum during the regression phase in rats.

The present study indicates that LPS injection decreases the serum progesterone level, suggesting that an inflammatory reaction may inhibit luteal function. LPS may stimulate macrophages to secrete cytokines, which in turn inhibit ovarian function [34]. There is, however, evidence to suggest a direct effect of LPS [35]. Since inflammation and cytokines cause the generation of superoxide radicals, which cause cell damage [16, 17], the inhibitory effect of LPS on serum progesterone levels may be due, at least in part, to increased superoxide radicals. However, LPS and cytokines had no significant effect on progesterone production by the corpus luteum in vitro in this study, perhaps because the incubation time used in this study (4 h) was too short to affect progesterone production by the corpus luteum.

Mn-SOD and Cu,Zn-SOD mRNA responded differently to endotoxin and cytokines, suggesting that they may be regulated differently in the corpus luteum and play different roles in regulating luteal function. The induction of Mn-SOD mRNA expression by endotoxin and cytokines suggests a protective effect of this SOD against inflammatory- and cytokine-mediated oxidative stress, since cytotoxic effects of cytokines can be reduced by increased levels of Mn-SOD [17, 18]. Sugino et al. [1] showed that the enzyme activity of Mn-SOD gradually decreased in the corpus luteum during the regression phase, although mRNA levels of Mn-SOD did not decrease in the present study. The increased mRNA levels and the decreased enzyme activity of Mn-SOD may indicate the enhancement of superoxide radical-associated metabolic activity [36]. Interestingly, this finding is also observed in the rat ovary during the ovulatory process [36], which has been compared to an inflammatory reaction. Recently, Rueda et al. [37] reported that the levels of Mn-SOD mRNA in regressed corpora lutea were much lower than those in young corpora lutea in cattle, suggesting that the corpus luteum undergoing structural luteolysis has lost a protective ability against oxidative stress. In contrast, the present study may suggest that the corpus luteum undergoing functional luteolysis still has a protective ability against oxidative stress, and this may be involved in the survival of luteal cells. Recent studies have shown that accumulation of superoxide radicals is involved in apoptosis and that antioxidants including SOD can inhibit apoptosis [38, 39]. It will be of interest to investigate the relation between cytokine-induced Mn-SOD expression and apoptosis in the luteolytic process.

In contrast to the mRNA levels of the inducible Mn-SOD, those of the constitutive Cu,Zn-SOD in the corpus luteum declined during the regression phase in a manner similar to that of serum progesterone levels. This change in Cu,Zn-SOD mRNA levels is consistent with that in the enzyme activity reported previously [1]. Reactive oxygen species are increased in the corpus luteum during the regression phase as Cu,Zn-SOD decreases [1], and Cu,Zn-SOD forms 80% of total SOD [1, 5], suggesting that Cu,Zn-SOD also plays an important role in the prevention of superoxide radical accumulation in addition to the acute response of Mn-SOD. However, further studies are needed regarding the role and regulation of Cu,Zn-SOD.

The corpus luteum and luteal cells respond to endotoxin and to acute inflammatory cytokines with a selective increase in Mn-SOD mRNA levels. This result is in good agreement with the previous reports showing that LPS, IL-1, IL-1ß, IL-6, and TNF induce Mn-SOD expression in normal cells and in various cell lines [18, 29, 30, 40–42]. The cytokine-induced increase in Mn-SOD mRNA levels was completely abolished by actinomycin D, an RNA synthesis inhibitor, whereas cycloheximide, a protein synthesis inhibitor, had no effect, suggesting that this increase in Mn-SOD mRNA levels in the corpus luteum may be dependent on transcription and that de novo protein synthesis is not required. Although the mechanisms by which cytokines enhance Mn-SOD expression are still unknown, several possibilities have been suggested. Taniguchi and coworkers [29, 30] have reported that PKC-dependent phosphorylation is in part involved in the Mn-SOD induction by TNF and IL-1. In the present study, PKC is unlikely to have been involved in the induction of Mn-SOD in GG-CL cells because PKC activation did not cause an increase in Mn-SOD expression. The biological action of IL-1 and TNF has been shown to be mediated by the nuclear factor-B (NF-B) [41, 43, 44], a transcriptional regulatory factor. Since NF-B can regulate Mn-SOD gene expression [43, 44], these cytokines may directly activate transcriptional factors that increase Mn-SOD expression. However, we cannot neglect the possibility that superoxide radicals generated by cytokines induce Mn-SOD expression [41, 42]. Further studies are needed regarding the mechanisms of transcription and the involvement of transcription factors in cytokine-induced Mn-SOD mRNA expression in the corpus luteum.

	ACKNOWLEDGMENTS

 
We thank Linda Alaniz for her photographic work, Rosemary Clepper for animal care, and Vivian Rogala for assistance in the manuscript preparation.

	FOOTNOTES

 
¹ This work was supported by NIH HD–11119 and FIC 1F05TW05241. G.G. is the recipient of an NIH Merit Award (HD11119).

² Correspondence: Geula Gibori, Department of Physiology and Biophysics (M/C 901), University of Illinois at Chicago, 901 South Wolcott Avenue, Chicago, IL 60612–7342. FAX: (312) 413–0159; ggibori{at}uic.edu

Accepted: March 9, 1998.

Received: December 29, 1997.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Sugino N, Nakamura Y, Takeda O, Ishimatsu M, Kato H. Changes in activities of superoxide dismutase and lipid peroxide in corpus luteum during pregnancy in rats. J Reprod Fertil 1993; 97:347–351.[Abstract]
2. Sugino N, Nakamura Y, Nagato O, Ishimatsu M, Teyama T, Kato H. Effect of ovarian ischemia-reperfusion on luteal function in pregnant rats. Biol Reprod 1993; 49:354–358.[Abstract]
3. Sugino N, Kato H. The role of ischemia-reperfusion injuries in generating reactive oxygen species during luteolysis. Adv Contracept Delivery Syst 1994; 10:95–106.
4. Musicki B, Aten RF, Behrman HR. Inhibition of protein synthesis and hormone-sensitive steroidogenesis in response to hydrogen peroxide in rat luteal cells. Endocrinology 1994; 134:588–595.[Abstract]
5. Shimamura K, Sugino N, Yoshida Y, Nakamura Y, Ogino K, Kato H. Changes in lipid peroxide and antioxidant enzyme activities in corpora lutea during pseudopregnancy in rats. J Reprod Fertil 1995; 105:253–257.[Abstract]
6. Sawada M, Carlson JC. Intracellular regulation of progesterone secretion by the superoxide radical in the rat corpus luteum. Endocrinology 1996; 137:1580–1584.[Abstract]
7. Sugino N, Shimamura K, Tamura H, Ono M, Nakamura Y, Ogino K, Kato H. Progesterone inhibits superoxide radical production by mononuclear phagocytes in pseudopregnant rats. Endocrinology 1996; 137:749–754.[Abstract]
8. Adashi EY. The potential relevance of cytokines to ovarian physiology: the emerging role of resident ovarian cells of the white blood cell series. Endocr Rev 1990; 11:454–464.[Medline]
9. Bagavandoss P, Wiggins RC, Kunkel SL, Remick DG, Keyes PL. Tumor necrosis factor production and accumulation of inflammatory cells in the corpus luteum of pseudopregnancy and pregnancy in rabbits. Biol Reprod 1990; 42:367–376.[Abstract]
10. Brannstrom M, Giesecke L, Moore IC, Van Den Heuvel CJ, Robertson SA. Leukocyte subpopulations in the rat corpus luteum during pregnancy and pseudopregnancy. Biol Reprod 1994; 50:1161–1167.[Abstract]
11. Simon C, Frances A, Piquette G, Polan ML. Immunohistochemical localization of the interleukin-1 system in the mouse ovary during follicular growth, ovulation, and luteinization. Biol Reprod 1994; 50:449–457.[Abstract]
12. Telleria CM, Ou J, Gibori G. Interleukin-6 (IL-6) and IL-6 receptor are expressed in the rat corpus luteum and are negatively regulated by progesterone and prolactin respectively. In: Program of the 79th annual meeting of The Endocrine Society; 1997; Minneapolis, MN. Abstract 384.
13. Roby KF, Weed J, Lyles R, Terranova PF. Immunological evidence for a human ovarian tumor necrosis factor-. J Clin Endocrinol & Metab 1990; 71:1096–1102.[Abstract]
14. Behrman HR, Endo T, Aten RF, Musicki B. Corpus luteum function and regression. Reprod Med Rev 1993; 2:153–180.
15. Terranova PF, Rice VM. Review: cytokine involvement in ovarian processes. Am J Reprod Immunol 1997; 37:50–63.
16. Matsubara T, Ziff M. Increased superoxide anion release from human endothelial cells in response to cytokines. J Immunol 1996; 137:3295–3298.[Abstract]
17. Meier J, Radeke HH, Selle S, Younes M, Sies H, Resch K, Habermehl GG. Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumor necrosis factor-. Biochem J 1989; 263:539–545.[Medline]
18. Wong GHW, Goeddel DV. Induction of manganous superoxide dismutase by tumor necrosis factor: possible protective mechanism. Science 1988; 242:941–944.[Abstract/Free Full Text]
19. Wong GHW, Elwell JH, Oberley LW, Goeddel DV. Manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor. Cell 1989; 58:923–931.[CrossRef][Medline]
20. Sugino N, Telleria CM, Gibori G. Progesterone inhibits 20-hydroxysteroid dehydrogenase expression in the rat corpus luteum through the glucocorticoid receptor. Endocrinology 1997; 138:4497–4500.[Abstract/Free Full Text]
21. Sugino N, Zilberstein M, Srivastava RK, Telleria CM, Nelson SE, Risk M, Chou JY, Gibori G. Establishment and characterization of a simian virus 40-transformed temperature sensitive rat luteal cell line. Endocrinology 1998; 139:1936–1942.[Abstract/Free Full Text]
22. Nelson SE, McLean MP, Jayatilak PG, Gibori G. Isolation, characterization, and culture of cell subpopulations forming the pregnant rat corpus luteum. Endocrinology 1992; 130:954–966.[Abstract]
23. Chou JY. Establishment of rat liver lines and characterization of their metabolic and hormonal properties: use of temperature sensitive SV–40 virus. Methods Enzymol 1985; 109:385–396.[Medline]
24. Chirgwin JJ, Przbyla AE, MacDonalds RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 1979; 18:5294–5299.[CrossRef][Medline]
25. Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156–159.[Medline]
26. Ho YS, Crapo JD. cDNA and deduced amino acid sequence of rat copper-zinc-containing superoxide dismutase. Nucleic Acids Res 1987; 15:6746.[Free Full Text]
27. Ho YS, Crapo JD. Nucleotide sequence of cDNAs coding for rat manganese-containing superoxide dismutase. Nucleic Acids Res 1987; 15:10070.[Free Full Text]
28. Batra SK, Metzgar RS, Hollingsworth MA. Molecular cloning and sequence analysis of human ribosomal protein S16. J Biol Chem 1991; 266:6830–6833.[Abstract/Free Full Text]
29. Suzuki K, Tatsumi H, Satoh S, Senda T, Nakata T, Fujii J, Taniguchi N. Manganese-superoxide dismutase in endothelial cells: localization and mechanism of induction. Am J Physiol 1993; 265:H1173–H1178.
30. Fujii J, Taniguchi N. Phorbol ester induces manganese-superoxide dismutase in tumor necrosis factor-resistant cells. J Biol Chem 1991; 266:23142–23146.[Abstract/Free Full Text]
31. Ji I, Slanghter RG, Ellis JA, Ji TH, Murdoch WJ. Analyses of ovine corpora lutea for tumor necrosis factor mRNA and bioactivity during prostaglandin-induced luteolysis. Mol Cell Endocrinol 1991; 51:77–80.
32. Shaw D, Britt J. Concentrations of tumor necrosis factor- and progesterone within the bovine corpus luteum sampled by continuous-flow microdialysis during luteolysis in vivo. Biol Reprod 1995; 53:847–854.[Abstract]
33. Stites DP, Siiteri PK. Steroids as immunosuppressants in pregnancy. Immunol Rev 1983; 75:117–138.[CrossRef][Medline]
34. Sancho-Tello M, Tash JS, Roby KF, Terranova PF. Effects of lipopolysaccharide on ovarian function in the pregnant mare serum gonadotropin-treated immature rats. Endocr J 1993; 1:503–512.
35. Sancho-Tello M, Chen TY, Clinton TK, Lyles R, Moreno RF, Tilzer L, Imakawa K, Terranova PF. Evidence for lipopolysaccharide binding in human granulosa-luteal cells. J Endocrinol 1992; 135:571–578.[Abstract]
36. Sato EF, Kobuchi H, Edashige K, Takahashi M, Yoshida T, Utsumi K, Inoue M. Dynamic aspects of ovarian superoxide dismutase isozymes during the ovulatory process in the rat. FEBS Lett 1992; 303:121–125.[CrossRef][Medline]
37. Rueda BR, Tilly KI, Hansen TR, Hoyer PB, Tilly JL. Expression of superoxide dismutase, catalase and glutathione peroxidase in the bovine corpus luteum: evidence supporting a role for oxidative stress in luteolysis. Endocrine 1995; 3:227–232.
38. Hockenbery DM, Oltvai ZN, Yin X-M, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 1993; 75:241–251.[CrossRef][Medline]
39. Tilly JL, Tilly KI. Inhibitors of oxidative stress mimic the ability of follicle-stimulating hormone to suppress apoptosis in cultured rat ovarian follicles. Endocrinology 1995; 136:242–252.[Abstract]
40. Dougall WC, Nick HS. Manganese superoxide dismutase: a hepatic acute phase protein regulated by interleukin-6 and glucocorticoids. Endocrinology 1991; 129:2376–2384.[Abstract]
41. Warner BB, Stuart L, Gebb S, Wispe JR. Redox regulation of manganese superoxide dismutase. Am J Physiol 1996; 271:L150–L158.
42. Mitchell J, Jiang H, Berry L, Meyrick B. Effect of antioxidants on lipopolysaccharide-stimulated induction of mangano superoxide dismutase mRNA in bovine pulmonary artery endothelial cells. J Cell Physiol 1996; 169:333–340.[CrossRef][Medline]
43. Antras-Ferry J, Maheo K, Morel F, Guillouzo A, Cillard P, Cillard J. Dexamethasone differently modulates TNF-- and IL-1ß-induced transcription of the hepatic Mn-superoxide dismutase gene. FEBS Lett 1997; 403:100–104.[CrossRef][Medline]
44. Das KC, Lewis-Molock Y, White CW. Thiol modulation of TNF and IL-1 induced MnSOD gene expression and activation of NF-B. Mol Cell Biochem 1995; 148:45–57.[CrossRef][Medline]