Hormonal Regulation of Copper-Zinc Superoxide Dismutase and Manganese Superoxide Dismutase Messenger Ribonucleic Acid in the Rat Corpus Luteum: Induction by Prolactin and Placental Lactogens¹

^a Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612–7342 ^b Laboratory of Cellular Biochemistry, Veterinary Medical Sciences/Animal Resource Sciences, University of Tokyo, 113 Tokyo, Japan

	ABSTRACT

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The corpus luteum expresses two enzymes that scavenge superoxide radicals and protect the cells from their toxic activities: cytosolic copper, zinc-superoxide dismutase (Cu,Zn-SOD) and mitochondrial manganese-SOD (Mn-SOD). The present study was undertaken to investigate whether the mRNA expression of each of these enzymes is regulated by luteotropic hormones. Cu,Zn-SOD and Mn-SOD mRNA levels were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). We first examined the effects of prolactin (PRL) on Cu,Zn-SOD and Mn-SOD mRNA expression in the corpus luteum. Hypophysectomy of Day 3 pregnant rats caused a sharp decline in both Cu,Zn-SOD and Mn-SOD mRNA levels, which was completely reversed by PRL administration. To further examine the effects of PRL and rat placental lactogen (rPL) on the expression of these enzymes, either primary luteinized granulosa cells or temperature-sensitive simian virus-40 transformed luteal cells (GG-CL) were cultured with different doses of PRL or rPL. These hormones induced a remarkable increase in Cu,Zn-SOD and Mn-SOD mRNA levels in both primary luteinized granulosa cells and GG-CL cells. Interestingly, whereas PRL up-regulated the expression of the SOD in luteal cells, other luteotropic hormones such as estradiol and dexamethasone inhibited both SOD mRNA expression while progesterone had no effect. In conclusion, PRL and PRL-like hormones induce a protective ability against toxic oxygen radicals by stimulating the expression of SODs, a phenomenon that may play an important role in maintaining luteal cell integrity and steroidogenic capacity.

	INTRODUCTION

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Ephemerality and prolongation of corpus luteum function have been a matter of concern for many years. However, the control mechanism is complicated and differs among mammalian species. In the rat, the life span of the corpus luteum is too short (2–3 days) to prepare the uterus for implantation, but it is prolonged for approximately 10 days by pituitary prolactin (PRL), which is secreted in surges after mating. The corpus luteum is further maintained by the synergistic action of estradiol and PRL-like hormones of decidual and placental origin (reviewed in [1]). However, the regulation of luteal function also involves several other factors such as progesterone [2, 3] and glucocorticoids [3]. Recent evidence has shown that reactive oxygen species including superoxide radicals, which are well known to cause cell damage, play important roles in the demise of the corpus luteum [4–9]. Reactive oxygen species are generated in the corpus luteum [10–14] and inhibit progesterone production in rats [15–17]. However, 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. Changes in SOD expression in the corpus luteum parallel those of progesterone production in pregnant and pseudopregnant rats [10, 11, 18], suggesting that SODs may play important roles in the maintenance of luteal function. Yet the factors that up-regulate the expression of Cu,Zn-SOD and Mn-SOD in the rat corpus luteum are unknown. Because the main function of luteotropic hormones is to maintain the integrity and steroidogenic activity of the corpus luteum, we became interested in determining whether the tropic activity of these hormones involves regulation of the SOD expression.

	MATERIALS AND METHODS

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Materials

Dulbecco's Modified Eagle medium: Ham's F-12 (DMEM/F12), hCG, HEPES, penicillin, streptomycin, amphotericin B, progesterone, estradiol-17ß, dexamethasone, and D-glucose 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 (sperm-positive is Day 1) 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. To determine the effect of PRL on Cu,Zn-SOD and Mn-SOD mRNA expression in the corpus luteum, pregnant rats were hypophysectomized using a transauricular approach on Day 3 of pregnancy. Surgery was performed under a light ether anesthesia. Completeness of hypophysectomy was evaluated by examination of the pituitary removed at the time of surgery and visualization of the pituitary fossa at autopsy. Hypophysectomized rats received s.c. injections of 125 µg of PRL (NIDDK oPRL-18, 30 IU/mg) or vehicle twice daily in 50% polyvinylpyrrolidone, pH 9.0, for 4 days (from Day 3 to Day 6), and the ovaries were removed on Day 7 of pregnancy. Intact rats on Day 7 of pregnancy were used as controls.

Luteinized Granulosa Cell Culture

Maturation of preovulatory follicles was stimulated by treatment of immature rats (Sasco Animal Lab) at Day 28 of age with 0.15 IU hCG s.c. twice daily for two days [19]. Luteinization of these preovulatory follicles was subsequently achieved by an ovulatory dose (10 IU) of hCG via the tail vein on the third day. Ovaries were harvested 7 h after the intravenous injection of hCG and incubated in DMEM/F12 (1:1), containing 6 mM EGTA and 0.5 M sucrose. Granulosa cells were harvested by needle-pricking the follicles. The cells were plated in 60-mm culture dishes at 8 x 10⁵ cells/ml and incubated in DMEM/F12 containing 15 mM HEPES, 1% fetal bovine serum (FBS), 100 IU/ml penicillin G, 100 µg/ml streptomycin, and 0.25 mg/ml amphotericin B. After 72 h of incubation, the medium was changed, and the cells were treated for 12 h with various doses of PRL (NIDDK oPRL-20, 31 IU/mg), purified rat placental lactogen-I (rPL-I) or recombinant rPL-II kindly provided by Dr. M. Robertson (Department of Physiology, University of Manitoba, Winnipeg, MB, Canada) at 37°C in the absence of serum under an atmosphere of 5% CO₂:95% air. Cells were washed with PBS several times after treatment and were stored at -80°C until RNA isolation.

GG-CL Cell Culture

The luteal cell line termed GG-CL, which was recently generated in our laboratory [20], 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 [21]. Cells were infected with a temperature-sensitive simian virus-40 (SV-40 tsA209) as previously reported [22]. 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 used in this study. The GG-CL cells were cultured in a 25- or 75-cm² flask with the incubation medium (RPMI-1640 containing double-strength antibiotic-antimycotic solution, single-strength nonessential amino acids, single-strength sodium pyruvate, 0.5% D-glucose, and 10% FBS) at permissive (33°C) and nonpermissive (39°C) temperatures under an atmosphere of 5% CO₂:95% air. These cells show a morphologically normal, differentiated phenotype similar to that of primary luteal cells at 39°C and express key genes encoding enzymes and receptors inherent in primary luteal cells although they do not produce progesterone [20].

Transfection of GG-CL Cells with the PRL Receptor

For the stable transfection of GG-CL cells with the long form of the PRL receptor, GG-CL cells were transfected using the Lipofectin technique described by Felgner et al. [23] and reported previously [20]. The cells were transfected with 10 µg of the expression vector (pMT2poly containing the long form of the PRL receptor cDNA) and with pSV2neo vector, both generously provided by Dr. Daniel Linzer (Northwestern University, Chicago, IL). After transfection, the medium was replaced by growth medium containing 5% FBS and antibiotics, and the cells were incubated for 48 h. After 48 h, the medium was again replaced by fresh growth medium and treated with 100 µg/ml of G418 sulfate. The G418 sulfate was added every other day until G418 sulfate-resistant colonies were identified. These colonies were selected and cultured in the growth medium containing 5% FBS until the cells were confluent. To identify successful stable transfection with the PRL receptor, cells were grown and passaged several times. PRL receptor mRNA expression in these cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) using PRL receptor-specific primers as reported previously [20].

Treatment of GG-CL Cells

To examine the effects of estradiol, progesterone, and dexamethasone, GG-CL cells were cultured at 33°C until 50% confluent and shifted to 39°C for 2 days. Cells were then treated with either estradiol-17ß, progesterone, or dexamethasone. To examine the effects of PRL or rPL in GG-CL cells, cells transfected with the long form of the PRL receptor were cultured at 33°C until 50% confluent and shifted to 39°C for 2 days. Cells were then treated with either ovine PRL (NIDDK oPRL-20, 31 IU/mg) or recombinant rPL-I mosaic, which has more than 90% homology with rPL-I and also has PRL-like activities [24, 25]. Cells were cultured in the presence of 1% FBS during the treatment at 39°C because they require serum to survive at 39°C. After culture, the cells were washed with PBS several times and stored at -80°C for RNA isolation.

Isolation of Total RNA and RT-PCR

Total RNA was isolated from corpora lutea by homogenization in guanidinium thiocyanate and centrifugation through a cesium chloride cushion [26], whereas total RNA from the cultured cells was isolated by the guanidinium-isothiocyanate-phenol-chloroform extraction procedure [27]. 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 [28] and Mn-SOD cDNA sequences [29]. Each reaction also included two oligonucleotide primers (5'-CGTTCACCTTGATGAGCCCATT-3' and 5'-TCCAAGGGTCCGCTGCAGTC-3') to amplify ribosomal protein S16 as an internal control [30]. 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 3 µg 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 [18]. Reaction products were electrophoresed on an 8% polyacrylamide nondenaturing gel. After autoradiography, data were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Statistical Analysis

Data were examined by ANOVA and Duncan's new multiple-range test. Differences were considered to be significant if p < 0.05.

	RESULTS

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PRL Regulation of Luteal Cu,Zn-SOD and Mn-SOD mRNA Expression In Vivo

Since the corpus luteum is exposed to high levels of pituitary PRL in early pregnancy, and to even higher concentrations of placental PRL-related hormones later on in gestation [1], we examined the effect of PRL in vivo on luteal Cu,Zn-SOD and Mn-SOD mRNA expression. Rats were hypophysectomized on Day 3 of pregnancy and treated with either PRL or vehicle. As shown in Figure 1, hypophysectomy caused a marked decrease in the expression of both Cu,Zn-SOD and Mn-SOD mRNA (Figure 1, HYPOX). These inhibitory effects induced by hypophysectomy were totally reversed by treatment with PRL (Fig. 1, HYPOX+PRL).

View larger version (44K): [in this window] [in a new window]   FIG. 1. Effects of PRL on Cu,Zn-SOD (A) and Mn-SOD (B) mRNA expression in the corpus luteum. Pregnant rats were hypophysectomized (HYPOX) on Day 3 of pregnancy and received s.c. injections of 125 µg PRL or vehicle twice daily for 4 days. Intact rats on Day 7 of pregnancy were used as controls. Corpora lutea were isolated, and total RNA was prepared and subjected to RT-PCR. RNA samples were collected from more than three animals in each group and in each experiment. The quantification data (the ratio of SOD to S16) are mean ± SEM of three different experiments. a, p < 0.01; b, p < 0.05 vs. HYPOX.

Regulation of Cu,Zn-SOD and Mn-SOD mRNA Expression by PRL and rPL in Cultured Luteinized Granulosa Cells

To further examine the regulation of Cu,Zn-SOD and Mn-SOD mRNA expression by PRL, we used highly luteinized granulosa cells in culture. These cells express both the long form and the short form of the PRL receptor [31]. Since PRL ceases to be secreted at mid-pregnancy in the rat whereas the placenta sequentially produces PRL-like hormones termed rPL-I and rPL-II [1], we also examined the effect of these hormones on Cu,Zn-SOD and Mn-SOD mRNA expression. As shown in Figures 2 and 3, PRL, rPL-I, and rPL-II stimulated both Cu,Zn-SOD and Mn-SOD mRNA expression in a dose-dependent manner.

View larger version (59K): [in this window] [in a new window]   FIG. 2. Effects of PRL on Cu,Zn-SOD (A) and Mn-SOD (B) mRNA expression in luteinized granulosa cells, prepared as described in Materials and Methods. Cells were incubated with PRL (0.01, 0.1, and 1 µg/ml) for 12 h at 37°C in the absence of serum. Total RNA was isolated and subjected to RT-PCR. The quantification data are mean ± SEM of three different experiments. a, p < 0.01; b, p < 0.05 vs. control (C).

View larger version (53K): [in this window] [in a new window]   FIG. 3. Effects of rPL-I and rPL-II on Cu,Zn-SOD (A and C) and Mn-SOD (B and D) mRNA expression in luteinized granulosa cells, prepared as described in Materials and Methods. Cells were incubated with either rPL-I or rPL-II (0.01, 0.1, and 1 µg/ml) for 12 h at 37°C in the absence of serum. Total RNA was isolated and subjected to RT-PCR. The quantification data are mean ± SEM of three different experiments. a, p < 0.01; b, p < 0.05 vs. control (C).

Effects of PRL and rPL on Cu,Zn-SOD and Mn-SOD mRNA Expression in GG-CL Cells

To examine whether PRL signaling to both SODs occurs through the long form of the PRL receptor, we used the SV-40 transformed luteal cell line (GG-CL) derived from the large luteal cell of the corpus luteum of pregnant rats, which was recently developed and characterized in our laboratory [20]. These cells express both Mn-SOD and Cu,Zn-SOD mRNA [18], but not the PRL receptor mRNA [20]. We stably transfected GG-CL cells with the long form of the PRL receptor as reported previously [20] and cultured them with either PRL or rPL-I mosaic for 8 h. As shown in Figure 4, PRL and rPL-I mosaic stimulated both Cu,Zn-SOD and Mn-SOD mRNA expression in the GG-CL cells expressing only the long form of the PRL receptor in a manner similar to that in the primary luteinized granulosa cell culture.

View larger version (52K): [in this window] [in a new window]   FIG. 4. Effects of PRL and rPL-I mosaic (rPL-Im) on Cu,Zn-SOD (A and C) and Mn-SOD (B and D) mRNA expression in GG-CL cells. GG-CL cells transfected with the long form of the PRL receptor were incubated with either PRL or rPL-Im (0.01, 0.1, and 1 µg/ml) for 8 h at 39°C. Total RNA was isolated and subjected to RT-PCR. The quantification data are mean ± SEM of three different experiments. a, p < 0.01; b, p < 0.05 vs. control (C).

Effects of Estradiol, Dexamethasone, and Progesterone on Cu,Zn-SOD and Mn-SOD mRNA Expression in GG-CL Cells

We recently reported that GG-CL cells respond to progesterone and glucocorticoid through the glucocorticoid receptor [3], and to estradiol through estrogen receptor-ß [20, 32]. GG-CL cells were incubated with either estradiol, dexamethasone, or progesterone. As shown in Figures 5 and 6d, estradiol and dexamethasone inhibited both Cu,Zn-SOD and Mn-SOD mRNA levels in a dose-dependent manner, whereas progesterone had no effect (Fig. 7).

View larger version (60K): [in this window] [in a new window]   FIG. 5. Effects of estradiol on Cu,Zn-SOD (A) and Mn-SOD (B) mRNA expression in GG-CL cells. GG-CL cells were incubated with estradiol-17ß (10-8, 10-7, and 10-6 M) for 8 h at 39°C. Total RNA was isolated and subjected to RT-PCR. The quantification data are mean ± SEM of three different experiments. a, p < 0.01; b, p<0.05 vs. control (C).

View larger version (60K): [in this window] [in a new window]   FIG. 6. Effects of dexamethasone on Cu,Zn-SOD (A) and Mn-SOD (B) mRNA expression in GG-CL cells. GG-CL cells were incubated with dexamethasone (10-7, 10-6, and 10-5 M) for 24 h at 39°C. Total RNA was isolated and subjected to RT-PCR. The quantification data are mean ± SEM of three different experiments. a, p < 0.01 vs. control (C).

View larger version (62K): [in this window] [in a new window]   FIG. 7. Effects of progesterone on Cu,Zn-SOD (A) and Mn-SOD (B) mRNA expression in GG-CL cells. GG-CL cells were incubated with progesterone (10-7, 10-6, and 10-5 M) for 24 h at 39°C. Total RNA was isolated and subjected to RT-PCR. The quantification data are mean ± SEM of three different experiments.

	DISCUSSION

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This is, to our knowledge, the first report showing marked stimulatory effects of PRL and rPLs on both Cu,Zn-SOD and Mn-SOD mRNA expression in rat luteal cells. Pituitary PRL and placental PRL-like hormones such as rPLs are essential for the maintenance of luteal function in the rat. These hormones, in combination with estradiol, stimulate overall protein synthesis and steroidogenic capacity of the luteal cell [1]. This increase in metabolism stimulates the generation of superoxide radicals since superoxide radicals are normally generated during normal metabolic activity including steroidogenesis [33, 34]. The present study shows both in vivo and in vitro that PRL and rPLs up-regulate both Cu,Zn-SOD and Mn-SOD mRNA expression in luteal cells, suggesting that one facet of the luteotropic effects of PRL and rPLs is to induce the expression of molecules that scavenge and protect the luteal cells from toxic oxygen radicals. Recent evidence has shown that accumulation of superoxide radicals and a decline in SOD levels are involved in apoptotic cell death and aging [35–38], whereas antioxidants including SODs can inhibit apoptosis [39–41]. PRL has been also reported to inhibit apoptosis in lymphoma cells [42]. It will be of interest to investigate the relationship between PRL- and PRL-like hormone-stimulated SOD expression and apoptosis in the luteotropic process.

The mechanism through which PRL and rPLs signal to stimulate expression of Cu,Zn-SOD and Mn-SOD remains to be determined. The rat corpus luteum and the luteinized granulosa cells express both the short and the long form of the PRL receptor [31]. However, it is clear from the results obtained with GG-CL cells that stimulation of both SOD mRNAs involves signaling through the long form of the PRL receptor since the GG-CL cells used in this study express the long but not the short form of the PRL receptor.

Although results of the present investigation clearly demonstrate a PRL-mediated up-regulation of the two types of oxygen radical scavenger, they do not support the contention that this action is generalized to all tropic hormones. In fact, some hormones have no effect on Cu,Zn-SOD and Mn-SOD mRNA expression whereas others have inhibitory action. Estradiol down-regulates the mRNA expression of both SODs despite the fact that it has tropic action in the rat corpus luteum. We recently studied the developmental changes in SOD mRNA levels in the corpus luteum during pregnancy and found that both SOD mRNA levels were increased during mid-pregnancy when estradiol production is high in the corpus luteum [18]. It is possible that the inhibitory action of estradiol may be overwhelmed by the stimulatory effect of rPL in the corpus luteum during mid-pregnancy. It is difficult to explain the physiological roles of the inhibitory effects of estradiol on SOD mRNA expression; however, estradiol may contribute to restricting the capacity of the corpus luteum to express SODs.

Progesterone was recently shown to act as a tropic hormone and to stimulate its own production [3]. In addition, luteal expression of both Cu,Zn-SOD and Mn-SOD changes in a manner similar to that of serum progesterone levels [10, 11, 18]. However, results obtained with progesterone treatment indicate that this steroid does not affect the expression of SODs. GG-CL cells are highly responsive to progesterone, which inhibits the expression of 20-hydroxysteroid dehydrogenase in a dose-related manner [3]. This inhibition appears to be selective and not to involve SODs.

Dexamethasone significantly reduced luteal mRNA levels of both Cu,Zn-SOD and Mn-SOD, whereas progesterone had no effect although it acts through the glucocorticoid receptor [3]. This may be due to the higher affinity of the synthetic glucocorticoid for the glucocorticoid receptor [3]. Investigators have previously shown that dexamethasone can reduce basal or cytokine-stimulated Mn-SOD mRNA expression, suggesting that the Mn-SOD gene is under glucocorticoid regulation [43–45]. However, to our knowledge, this is the first report showing that dexamethasone can also inhibit Cu,Zn-SOD mRNA expression. Whether this effect is due to the high concentration of dexamethasone used in this study or whether glucocorticoid inhibition is specific to the corpus luteum remains to be investigated.

In conclusion, the expression of both Cu,Zn-SOD and Mn-SOD mRNA is under multi-hormonal regulation in the rat corpus luteum. Among the tropic hormones in the rat, only PRL and placental lactogens up-regulate the expression of the two types of SODs that can scavenge superoxide radicals and protect the corpus luteum against toxic oxygen radicals. This PRL effect may play an important role in the prolongation of the life span of the corpus luteum.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. M.C. Robertson for the purified rat placental lactogen-I and the recombinant rat placental lactogen-II, to Dr. D.I.H. Linzer for the PRL receptor expression vector, and to the NIDDK and National Hormone and Pituitary Program (NIH) for the ovine PRL. We also 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: April 23, 1998.

Received: March 20, 1998.

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