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Biology of Reproduction 61, 1133-1138 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Suppression of Intracellular Superoxide Dismutase Activity by Antisense Oligonucleotides Causes Inhibition of Progesterone Production by Rat Luteal Cells1

Norihiro Sugino2,a, Shuji Takiguchia, Shiro Kashidaa, Hisako Takayamaa, Yoshiaki Yamagataa, Yasuhiko Nakamuraa, and Hiroshi Katoa

a Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, Ube 755-8505, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Superoxide radicals are known to inhibit progesterone production by luteal cells and have also been reported to cause apoptosis in various cells. The corpus luteum has an antioxidant enzyme to scavenge superoxide radicals: copper-zinc superoxide dismutase (Cu,Zn-SOD). However, it remains unknown how the decrease in intracellular Cu,Zn-SOD activity influences luteal function. This study was therefore undertaken to investigate whether suppression of intracellular Cu,Zn-SOD activity inhibits progesterone production by rat luteal cells and causes apoptosis. To suppress intracellular Cu,Zn-SOD activity, dispersed rat luteal cells were incubated with Cu,Zn-SOD antisense oligonucleotides. The 48-h treatment with antisense oligonucleotides (10 µM) inhibited Cu,Zn-SOD activity by 50% and Cu,Zn-SOD mRNA level by 30%, whereas sense oligonucleotides used as the control had no effect. Progesterone concentration in the medium was significantly decreased by the 48-h treatment with antisense oligonucleotides in the presence of hCG, and this inhibitory effect was completely blocked by the simultaneous addition of N-acetyl-L-cysteine, an antioxidant. Treatment with antisense oligonucleotides caused no significant change in the percentage of apoptotic cells as morphologically evaluated by the nuclear staining with Hoechst dye. In conclusion, the decrease in intracellular Cu,Zn-SOD activities inhibits progesterone production by rat luteal cells, which may be mediated by superoxide radicals, suggesting that intracellular Cu,Zn-SOD plays important roles in the regulation of luteal function.


   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 [1–16], suggesting the involvement of reactive oxygen species in corpus luteum regression. In contrast, the corpus luteum has specific enzymes to scavenge superoxide radicals: copper-zinc superoxide dismutase (Cu,Zn-SOD), located in the cytosol, and manganese SOD (Mn-SOD), located in the mitochondria. Both SODs belong to a first enzymatic step that protects cells against toxic oxygen radicals. It is thought that Cu,Zn-SOD is a constitutive type and Mn-SOD is an inducible type that can be responsive to inflammatory reaction or cytokines [17]. Recent evidence from studies using knock-out mice has shown that Cu,Zn-SOD is important in reproduction [18, 19]. In rats, Cu,Zn-SOD forms 80% of total SOD in the corpus luteum [7, 13]. Cu,Zn-SOD activity in the corpus luteum increases until the mid-luteal phase and gradually decreases thereafter, in a manner similar to the change in serum progesterone levels in pregnant and pseudopregnant rats [7, 13]. In addition, luteotropic hormones such as prolactin and rat placental lactogens up-regulate the Cu,Zn-SOD mRNA expression in rat luteal cells [20]. These findings strongly suggest that Cu,Zn-SOD may play an important role in the maintenance of luteal function. However, it remains unknown whether a decrease in intracellular Cu,Zn-SOD activities influences progesterone production by luteal cells.

Recently, much attention has been focused on the involvement of apoptosis in corpus luteum regression [21–23]. Recent evidence has shown that accumulation of superoxide radicals and a decrease in SOD levels are involved in apoptotic cell death, whereas antioxidants including SOD can inhibit apoptosis [24–31]. However, it remains unknown whether a decrease in SOD activities induces apoptosis of luteal cells. Therefore, the present study was undertaken to investigate the effect of the decrease in intracellular Cu,Zn-SOD activities on progesterone production by luteal cells, and to determine whether the decrease in Cu,Zn-SOD activities causes apoptosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Sprague-Dawley immature (23- to 25-day-old) rats were housed under controlled conditions (lights-on 0500–1900 h) with free access to standard rat chow and water. The experimental protocol was reviewed and approved by the Committee for the Ethics on Animal Experiments in the Yamaguchi University School of Medicine. The rat care and handling conformed with the Guideline for Animal Experiments in the Yamaguchi University School of Medicine and the Law (No 105) and Notification (No 6) of the Government.

Oligonucleotides

Antisense and sense oligonucleotides for Cu,Zn-SOD cDNA were synthesized on the basis of the rat Cu,Zn-SOD cDNA sequence [32]. The antisense sequence 5'-GCCTTCATCGCCATGCTTCCC-3' extends from 7 bases 5' to 11 bases 3' of the start codon. The sense sequence is 5'-GGGAAGCATGGCGATGAAGGC-3' and was used as a control for the antisense oligonucleotide. These oligonucleotides were synthesized by Iwaki Co., Ltd. (Chiba, Japan).

Cell Dispersion and Culture

Cell dispersion from luteinized ovaries was performed according to the procedure reported by Sawada and Carlson [14] with a slight modification. Rats were superovulated with s.c. injections of eCG (40 IU; Sigma Chemical Co., St. Louis, MO), and hCG (40 IU; Sigma) 65 h later. The rats were killed under ether anesthesia 7 days after hCG injection, and the ovaries were removed. After the removal of fat and nonluteal tissue from the ovary, corpora lutea were minced in the medium (Dulbecco's modified Eagle's Medium: Ham's nutrient mixture F-12 1:1, containing 30 µg/ml glutamine [DMEM/F12]) and incubated in DMEM/F12 containing 0.2% collagenase (type I; Sigma), 0.3% hyaluronidase (Sigma), and 25 U/ml deoxyribonuclease (DNase; Sigma) for 90 min at 37°C in a shaking water bath. The medium, then, was filtered through a 70-µm nylon mesh (Nippon Becton Dickinson Co., Tokyo, Japan) to remove debris, and the filtrate was centrifuged at 220 x g for 10 min. Cell pellets were washed twice with the medium, and the number of cells was adjusted to 300–350 x 105/ml viable cells with incubation medium (DMEM/F12 containing 100 µg/ml streptomycin and 50 U/ml penicillin). The steroidogenic profile of the dispersed cells by this method has been reported to be consistent with previous studies using isolated rat luteal cells [33]. Serum was not added to avoid rapid degradation of the oligonucleotide by serum endonucleases. Cell number and viability was tested by the trypan blue exclusion method. Cell viability was more than 90%, and the few apoptotic cells were evaluated in freshly prepared cells as described below. The cell suspension (100 µl) was distributed into each well of a Falcon 24-well tissue plate (Nippon Becton Dickinson). Then, Cu,Zn-SOD antisense oligonucleotides or sense oligonucleotides in 900 µl incubation medium were added into each well to obtain final concentrations of 5 µM or 10 µM, and incubation was carried out in the absence of hCG for 24 h or 48 h at 37°C in an atmosphere of 5% CO2 and 95% air. In the second experiment, cells were incubated with Cu,Zn-SOD antisense oligonucleotides (10 µM) with or without N-acetyl-L-cysteine (Nac, 100 mM; Sigma), an antioxidant, in the presence of hCG (2 IU/ml) for 48 h under the same conditions as described above. The incubation was run in triplicate and repeated three times. There was no difference in cellular morphology under a microscope between the start of incubation and the end of incubation. After incubation, cell viability was checked (hCG [-]: 56.3 ± 1.3%, hCG [+]: 52.8 ± 1.5%, hCG+antisense: 52.7 ± 4.3%, hCG+antisense+Nac: 63.4 ± 4.0%, hCG+Nac: 67.5 ± 3.1%; mean ± SEM of three different experiments), and actual cell number ranged from 13 x 105 to 19 x 105 cells per well.

SOD Assay

After incubation, cells were collected by pipetting carefully and were centrifuged at 220 x g for 10 min, and the supernatant was stored at -20°C until progesterone assay. The pelleted cells were washed twice with PBS, resuspended with Tris-HCl buffer (0.1 M, pH 7.4), and sonicated. Cu,Zn-SOD activity in the sonicated samples was determined as reported previously [7].

Reverse Transcription (RT)-Polymerase Chain Reaction (PCR)

After incubation, total RNA was isolated from cells with Isogen (Wako Pure Chemical Industries Ltd., Osaka, Japan) according to the manufacturer's method. For mRNA analysis, RT-PCR was performed as reported previously [17], with oligonucleotide primers for Cu,Zn-SOD (5'-TTCGAGCAGAAGGCAAGCGGTGAA-3' and 5'-AATCCCAATCACACCACAAGCCAA-3') designed on the basis of the rat Cu,Zn-SOD cDNA sequence [32]. In brief, 3 µg of total RNA was reverse-transcribed at 42°C in a reaction mixture containing 200 U Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer, Roche Molecular Systems Inc., Branchburg, NJ). For PCR amplification, a mixture containing the oligonucleotide primers [{alpha}-32P]dCTP (2 µCi at 3000 Ci/mmol) and 2.5 U Taq DNA polymerase (Perkin-Elmer) was added to each reaction. Each reaction also included two oligonucleotide primers (5'-CGTTCACCTTGATGAGCCCATT-3' and 5'-TCCAAGGGTCCGCTGCAGTC-3') to amplify ribosomal protein S16 as an internal control [34]. Amplification was carried out for 20 cycles using a 65°C annealing temperature in a program temperature control system PC-800 (ASTEC, Fukuoka, Japan). The predicted sizes of the PCR-amplified products were 396 base pairs (bp) for Cu,Zn-SOD and 100 bp for S16. Reaction products were electrophoresed on an 8% polyacrylamide nondenaturing gel. After autoradiography, data were quantified using a bioimaging analyzer BAS2000 (Fuji Photo Film Co., Tokyo, Japan).

Detection of Apoptotic Cells

Apoptotic cells were evaluated according to the method reported previously [35]. After incubation, cells were collected by pipetting carefully and centrifuged at 220 x g for 10 min, and the pelleted cells were fixed with 1% glutaraldehyde for 30 min, washed with PBS, and incubated with Hoechst 33342 dye (167 mM) for 1 min. The samples were mounted and examined under a fluorescence microscope. According to the morphological analysis of the nucleus, apoptotic cells were characterized as having condensed chromatin or nuclear fragmentation. The percentage of apoptotic cells in total cells was counted at 200x on ten randomly chosen areas. Counting was performed independently by three observers. An observer-related mean was calculated for each slide, and the mean of the three observer-related means was used as a single observation. The incubation was repeated three times.

Progesterone Assay

Progesterone concentrations in the medium were determined by a specific RIA, reported previously [36]. The sensitivity of the assay was 100 pg/ml, and the intra- and interassay coefficients of variation were 7.0% and 14.4%, respectively.

Statistical Analysis

Data were examined by ANOVA and Duncan's new multiple-range test. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cu,Zn-SOD activity in dispersed cells of corpora lutea was significantly suppressed by the 48-h treatment with Cu,Zn-SOD antisense oligonucleotides (10 µM), whereas sense oligonucleotides used as the control for the antisense oligonucleotide caused no significant change in Cu,Zn-SOD activities (Fig. 1A). The mRNA expression of Cu,Zn-SOD was also significantly inhibited by antisense oligonucleotides (10 µM), but its inhibitory effect was smaller than that on Cu,Zn-SOD activities (Fig. 1B).



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  		FIG. 1. Effects of Cu,Zn-SOD antisense oligonucleotide treatment on activities (A) and mRNA levels (B) of Cu,Zn-SOD in dispersed luteal cells. A) Cells prepared as described in Materials and Methods were incubated with Cu,Zn-SOD antisense oligonucleotides or sense oligonucleotides (5 µM and 10 µM) for 48 h in the absence of hCG. B) Cells were incubated with antisense oligonucleotides or sense oligonucleotides (10 µM) for 48 h in the absence of hCG. Total RNA was isolated and subjected to RT-PCR. The data are mean ± SEM of three different experiments. a, P < 0.01 and b, P < 0.05 vs. control

Cu,Zn-SOD antisense oligonucleotides caused no significant change in progesterone concentrations in the medium in the absence of hCG (Fig. 2A). To examine the apoptotic change in dispersed cells of corpora lutea by the suppression of intracellular Cu,Zn-SOD activity, cells were stained with Hoechst dye, and nuclear morphology was examined using a fluorescence microscope. Figure 2B shows a microphotograph of apoptotic cells with nuclear fragmentation. There was no significant change in the percentage of apoptotic cells by the 48-h treatment with Cu,Zn-SOD antisense oligonucleotides (Fig. 2C). The 24-h treatment with antisense oligonucleotides also caused no significant change in intracellular Cu,Zn-SOD activities, progesterone concentrations in the medium (data not shown), and the percentage of apoptotic cells (control: 13.2 ± 2.3%, antisense 5 µM: 14.0 ± 2.5%, antisense 10 µM: 15.3 ± 3.0%, sense 5 µM: 13.4 ± 4.0%, sense 10 µM: 12.0 ± 3.8%; mean ± SEM of three different experiments).



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  		FIG. 2. Effects of Cu,Zn-SOD antisense oligonucleotide treatment on progesterone concentrations in the medium (A) and apoptosis of dispersed luteal cells (B and C). Cells prepared as described in Materials and Methods were incubated with Cu,Zn-SOD antisense oligonucleotides or sense oligonucleotides (5 µM and 10 µM) for 48 h in the absence of hCG. Nuclear morphology was examined by fluorescence microscopy in dispersed luteal cells stained with Hoechst dye. B) Microphotograph of apoptotic cells after the treatment with antisense oligonucleotides (10 µM). The fragmented nuclei, indicated by arrows, show apoptotic cells (magnification x300). The percentage of the apoptotic cells in total cells was counted at x200 (C). The data are mean ± SEM of three different experiments

In a second experiment, we examined the effects of Cu,Zn-SOD antisense oligonucleotides (10 µM) in the presence of hCG. Cu,Zn-SOD activity in dispersed cells of corpora lutea was significantly suppressed by the 48-h treatment with Cu,Zn-SOD antisense oligonucleotides in the presence of hCG as well as in the absence of hCG (Fig. 1A and Fig. 3A). Progesterone concentrations in the medium were significantly decreased by the Cu,Zn-SOD antisense oligonucleotide treatment (Fig. 3B). To determine whether the decrease in progesterone concentrations caused by the antisense oligonucleotides was mediated by superoxide radicals, dispersed cells of corpora lutea were treated with Nac, an antioxidant, in the presence of Cu,Zn-SOD antisense oligonucleotides. The inhibitory effect of the antisense oligonucleotides on progesterone concentrations was completely reversed by the simultaneous addition of Nac, whereas Nac alone had no effect on progesterone concentrations in the medium (Fig. 3B). To study the possibility that superoxide radicals inhibited progesterone production through apoptosis, changes in the number of apoptotic cells by the treatment with antisense oligonucleotides were examined. There was no significant difference in the percentage of apoptotic cells between the control group and the antisense group (Fig. 3C). Interestingly, Nac remarkably decreased the percentage of apoptotic cells compared with the control group and the hCG (-) group (Fig. 3C). Since the treatment with Nac significantly inhibited apoptosis, there was a possibility that Nac had blocked the inhibitory effect of antisense oligonucleotides on progesterone concentrations in the medium through the inhibition of apoptosis. Therefore, progesterone concentrations in the medium were expressed on the basis of the number of viable cells (Fig. 4). Antisense oligonucleotides significantly inhibited progesterone production by viable cells, and this inhibitory effect was significantly blocked by the simultaneous addition of Nac, whereas Nac alone had no effect on progesterone production.



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  		FIG. 3. Effects of Cu,Zn-SOD antisense oligonucleotide treatment on Cu,Zn-SOD activity (A), progesterone concentrations in the medium (B), and apoptosis in dispersed luteal cells (C) in the presence of hCG. Cells, prepared as described in Materials and Methods, were incubated with Cu,Zn-SOD antisense oligonucleotides (10 µM), with or without N-acetyl-L-cysteine (Nac; 100 mM) for 48 h in the presence of hCG (2 IU/ml). The data are mean ± SEM of three different experiments. a, P < 0.01 vs. control; b, P < 0.01 vs. antisense; c, P < 0.01 vs. hCG (-), control or antisense



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  		FIG. 4. Effects of Cu,Zn-SOD antisense oligonucleotide treatment on progesterone production by viable cells in the presence of hCG. The progesterone concentration in the medium was expressed on the basis of the number of viable cells after incubation. a, P < 0.01 vs. control; b, P < 0.05 vs. control and antisense + Nac


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The influence of SOD or antioxidants administered exogenously on progesterone production by rat luteal cells has been reported [8, 14, 33]. However, it remained unknown how a decrease in intracellular Cu,Zn-SOD affects luteal progesterone production. The present study shows that a decrease in intracellular Cu,Zn-SOD activities inhibited the hCG-stimulated progesterone production by luteal cells and that this inhibition may be mediated by superoxide radicals. The action of superoxide radicals is unlikely to be mediated through apoptosis of luteal cells because the decreased Cu,Zn-SOD activity did not cause a significant increase in number of apoptotic cells. It has been reported that superoxide radicals cause membrane damage of luteal cells [5, 6, 14] and inhibit the cholesterol transport to mitochondria in luteal cells [4, 10]. Thus, decreased Cu,Zn-SOD activity results in the loss of luteal function, which may be mediated by superoxide radicals.

Regarding the action of antisense oligonucleotides, it has been reported that they can cause inhibition of protein synthesis by actions on several possible sites, e.g., by inhibition of transcription or translation [37]. Since the treatment with antisense oligonucleotides inhibited the mRNA expression in the present study, it is suggested that they act, at least in part, at the transcriptional level. However, antisense oligonucleotides are also likely to act after mRNA formation, e.g., inhibition of translation, because the inhibitory effect on mRNA levels was smaller than that on activities.

It is of interest to note that the decrease in intracellular Cu,Zn-SOD activities inhibited the progesterone production by luteal cells only in the presence of hCG, but not in the absence of hCG. Behrman and his colleagues [3] and we [7] previously reported that reactive oxygen species inhibited progesterone production by luteal cells in the presence of LH or hCG but not in their absence. The present data are consistent with these findings and may suggest the possibilities that superoxide radicals specifically inhibited the hCG-stimulated intracellular signaling pathway [4, 10, 33], and superoxide radical generation was increased by hCG stimulation [14, 33].

We previously reported that Cu,Zn-SOD activity in the corpus luteum increases until the midluteal phase and gradually decreases thereafter, in a manner similar to the change in serum progesterone levels in pregnant and pseudopregnant rats [7, 13]. Changes in Cu,Zn-SOD mRNA expression in the corpus luteum also parallel those of serum progesterone levels in pregnant rats [17], and luteotropic hormones such as prolactin and rat placental lactogens up-regulate the Cu,Zn-SOD mRNA expression in rat luteal cells [20]. The present study also showed that Cu,Zn-SOD plays important roles in progesterone production by rat luteal cells. These findings strongly suggest that Cu,Zn-SOD is involved in the regulation of luteal function. Interestingly, Sawada and Carlson [14] reported that the insertion of SOD into luteal cells by electroporation interfered not only with the inhibition of progesterone production caused by high levels of superoxide radicals but also with the stimulation of progesterone production induced by low levels of superoxide radicals, suggesting that progesterone production by luteal cells may depend on the level of superoxide radicals achieved. However, the level of SOD activity achieved by the insertion of SOD into luteal cells is unclear. In our previous data, the levels of Cu,Zn-SOD activity in the corpus luteum on Day 21 of pregnancy and on Day 13 of pseudopregnancy were 60% of those on Day 12 of pregnancy and on Day 9 of pseudopregnancy [7, 13]. The reduction in Cu,Zn-SOD activity by 50% by antisense oligonucleotides is approximately the same change as the Cu,Zn-SOD activity loss in the corpus luteum undergoing functional luteolysis, suggesting that the effect of antisense oligonucleotides on Cu,Zn-SOD activity shown in the present study may be within a physiological range.

Interestingly, the suppression of intracellular Cu,Zn-SOD by antisense oligonucleotides did not cause apoptosis whereas Nac alone added into the medium remarkably inhibited apoptosis. Rothstein et. al. [26] reported that a 40% decrease in Cu,Zn-SOD activities by the antisense oligonucleotide treatment did not cause apoptosis, but a 60% decrease caused apoptosis in nerve cells. Troy and Shelanski [27] also reported that a 50% decrease in Cu,Zn-SOD activities did not cause apoptosis in neuronal cells. These findings suggest that apoptosis may depend on the level of Cu,Zn-SOD activity achieved. The decrease in intracellular Cu,Zn-SOD activities by the antisense oligonucleotide treatment shown in the present study may have been insufficient to induce apoptosis, although the level of intracellular Cu,Zn-SOD activity achieved may well reflect the level of such activity in the corpus luteum undergoing luteolysis. The inhibitory effect of Nac on apoptosis may occur because Nac has scavenged not only intracellular superoxide radicals but also superoxide radicals generated in the medium. Tilly et al. [38] showed the possibility that superoxide radicals generated in the medium could cause apoptosis, because follicle apoptosis was inhibited by the addition of SOD into the medium, and SOD in the medium cannot enter the cell. We cannot, however, neglect the suggestion that Nac may have an anti-apoptotic effect in addition to scavenging superoxide radicals. The present study also showed that Nac inhibited luteal cell apoptosis but did not change progesterone concentrations in the medium. It is hard to explain this discrepancy. It may be suggested that progesterone concentrations in the medium are not always correlated with the frequency of apoptosis when the percentage of apoptotic cells is small.

The present study demonstrated for the first time that a decrease in intracellular Cu,Zn-SOD activities inhibited hCG-stimulated progesterone production by rat luteal cells, mediated by superoxide radicals, suggesting that superoxide radicals and Cu,Zn-SOD play important roles in regulating luteal function.


   FOOTNOTES
 
1 This work was supported in part by a grant from the UBE Foundation and Grant-in-Aid 11671623 from the Ministry of Education, Science, and Culture, Japan. Back

2 Correspondence: Norihiro Sugino, Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, Minamikogushi 1–1–1, Ube 755–8505, Japan. FAX: 836 22 2287. Back

Accepted: June 3, 1999.

Received: February 5, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone withdrawal in human endometrial stromal cells
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Induction of manganese superoxide dismutase by tumour necrosis factor-{alpha} in human endometrial stromal cells
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Dual effects of nitric oxide in functional and regressing rat corpus luteum
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Regulation and Role of Vascular Endothelial Growth Factor in the Corpus Luteum During Mid-Pregnancy in Rats
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Decreased superoxide dismutase expression and increased concentrations of lipid peroxide and prostaglandin F2{alpha} in the decidua of failed pregnancy
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N. Sugino, S. Kashida, S. Takiguchi, Y. Nakamura, and H. Kato
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Rescue of the Corpus Luteum and an Increase in Luteal Superoxide Dismutase Expression Induced by Placental Luteotropins in the Rat: Action of Testosterone Without Conversion to Estrogen
Biol Reprod, February 1, 2000; 62(2): 398 - 403.
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Superoxide dismutase expression in the human corpus luteum during the menstrual cycle and in early pregnancy
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