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a Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, Ube 755-8505, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent evidence has shown that reactive oxygen species and their scavenging system play important roles in the regulation of luteal function [2–14]. The corpus luteum has specific enzymes to scavenge superoxide radicals: copper-zinc superoxide dismutase (Cu,Zn-SOD) in the cytosol and manganese SOD (Mn-SOD) in the mitochondria. Both SODs belong to a first enzymatic step that protects cells against toxic oxygen radicals. Our previous data showed that SOD activities in the corpus luteum increased until Day 9 and thereafter decreased in pseudopregnant rats [11]; but in pregnant rats, they further increased until Day 12 of pregnancy in a manner similar to serum progesterone concentrations [7]. Recently, Sugino et al. [15] reported that SOD mRNA expression in luteal cells was up-regulated by rat placental lactogen-I (rPL-I) and rPL-II in vitro. From these observations, the increase in luteal SOD activities during midluteal phase in pregnant rats seems to be regulated by placental luteotropins. It has been also reported that antioxidants including SOD and reactive oxygen species are involved in the regulation of the life span of eukaryotes [16–23]. In addition, SOD acts protectively against superoxide radicals to stimulate progesterone production by the corpus luteum [8,13]. These findings strongly suggest that SOD may play important roles in the rescue of the corpus luteum by placental luteotropins when pregnancy occurs.
To investigate the relationship between maintenance of corpus luteum function caused by placental luteotropins and SOD expression in the corpus luteum, we examined whether treatment with placental luteotropins in vivo increases SOD expression in the corpus luteum and maintains corpus luteum function in pseudopregnant rats. Furthermore, we examined the involvement of testosterone, one of the luteotropic steroid hormones secreted by placentae, in the effects of placental luteotropins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sprague-Dawley rats (Japan SLC, Hamamatsu, Japan), weighing 220–270 g, were housed at 24°C under controlled conditions (lights-on from 0500 to 1900 h) with free access to standard rat chow and water. Vaginal smears were obtained daily, and only rats showing at least two consecutive 4-day estrous cycles were used.
Pseudopregnancy was induced by mechanical stimulation of the uterine cervix for about 1 min with a glass rod at 1800 h on proestrus and at 0900 h on estrus. The last day on which the rat exhibited an estrous smear was designated Day 1 of pseudopregnancy. Vaginal smears from pseudopregnancy were checked every day.
The experiments were reviewed by the Committee for the Ethics on Animal Experiment in Yamaguchi University School of Medicine under the Law (No. 105) and Notification (No. 6) of the Government.
Placental Incubation
To obtain placental luteotropins, placentae removed from rats on Day 12 of pregnancy were incubated as reported previously [24]. In brief, placentae were washed with RPMI-1640 medium (ICN Biomedicals, Costa Mesa, CA) to remove blood and incubated in RPMI-1640 medium (2 placentae per 2 ml/tube) at 37°C for 24 h under an atmosphere of 95% O2 and 5% CO2 in a shaking water bath. After incubation, the supernatant was collected and stored at -80°C. Concentrations of testosterone, progesterone, and estradiol in the placental incubation medium were 107.3 ± 11.1 ng/ml, 438.0 ± 61.5 pg/ml, and 9.6 ± 0.7 pg/ml, respectively.
An aliquot of the placental incubation medium was treated with charcoal (10 mg/ml), and the supernatant was collected and stored at -80°C. Concentrations of testosterone, progesterone, and estradiol in the charcoal-treated placental incubation medium were not detectable. The luteotropic activity of the charcoal-treated placental incubation medium was confirmed in preliminary experiments using incubation of corpora lutea with the charcoal-treated placental incubation medium as reported previously [24].
Experimental Procedures
Experiment 1 A total of 8 ml of placental incubation medium was injected i.p. daily (4 ml/injection, twice daily at 0800 and 1800 h) from Day 9 to Day 12 of pseudopregnancy. The last injection was on the morning of Day 12 of pseudopregnancy. Control rats received the control medium, which was obtained from the incubation without placentae. Our preliminary study with a lower dosage of placental incubation medium (4 ml/day) showed a dose-related response in serum progesterone concentrations (control: 37.4 ± 3.9 ng/ml; 4 ml/day group: 45.7 ± 1.6 ng/ml; 8 ml/day group: 57.8 ± 6.2 ng/ml; mean ± SEM of four animals), in luteal Cu,Zn-SOD activities (control: 135.3 ± 5.3 NU/mg protein; 4 ml/day group: 148.5 ± 2.5 NU/mg protein; 8 ml/day group: 168.0 ± 4.4 NU/mg protein; mean ± SEM of four animals), and in luteal Mn-SOD activities (control: 49.0 ± 4.0 NU/mg protein; 4 ml/day group: 54.1 ± 0.9 NU/mg protein; 8 ml/day group: 66.0 ± 1.6 NU/mg protein; mean ± SEM of four animals). The effect of 8 ml/day was statistically significant, and we decided to use this dosage. The injected medium was taken from the same pool.
Experiment 2 To study the involvement of testosterone in the placental incubation medium, a total of 8 ml of the charcoal-treated placental incubation medium was injected i.p. daily as described above, with or without the simultaneous s.c. injection of 1.0 µg/day (0.5 µg in 250 µl sesame oil per injection, twice daily) of testosterone (Sigma Chemical Co., St. Louis, MO) or dihydrotestosterone (DHT: a nonaromatizable testosterone; Sigma). This dosage of testosterone was almost the same as that contained in the placental incubation medium (0.86 µg/day). Testosterone or DHT was also tested alone. Control rats received the same volume of sesame oil (s.c.) and control medium (i.p.).
Rats were laparotomized under light ether anesthesia between 1600 and 1800 h on Day 12 of pseudopregnancy, and blood samples were obtained from the abdominal vein. The ovaries were perfused with saline via the abdominal vein during draining of the inferior vena cava to remove the blood, as described previously [7], and removed. Corpora lutea were dissected and cleaned of adhering tissue in a watch glass. For SOD activity assay, corpora lutea were homogenized in Tris-HCl buffer (0.01 M, pH 7.4) and centrifuged at 800 x g for 10 min at 4°C, and the supernatant was stored at -80°C. For RNA isolation, corpora lutea were immediately frozen in liquid nitrogen and stored at -80°C. Corpora lutea were pooled from each animal for SOD activity assay or RNA isolation. Serum samples from each animal were stored at -20°C until progesterone and PRL assay.
SOD Assay
Cu,Zn-SOD activity and Mn-SOD activity in the corpus luteum were measured as reported previously [7]. The amount of protein required for 50% inhibition in the absorbance at 550 nm was defined as one nitrite unit (NU) of SOD activity. All data were expressed in NU of SOD activity per milligram protein. Protein concentration was determined by the method described by Lowry et al. [25]. The intra- and interassay coefficients of variation were, respectively, 3.8% and 9.6% for the Cu,Zn-SOD assay, and 4.7% and 6.4% for the Mn-SOD assay.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from corpora lutea with Isogen (Wako Pure Chemical Industries, Ltd., Osaka, Japan) by the method provided by the manufacturer. For mRNA analysis, RT-PCR was performed as reported previously [26], with oligonucleotide primers for Cu,Zn-SOD (5'-TTCGAGCAGAAGGCAAGCGGTGAA-3' and 5'-AATCCCAATCACACCACAAGCCAA-3') and for Mn-SOD (5'-ATTAACGCGCAGATCATGCAG-3' and 5'-TTTCAGATAGTCAGGTCTGACGTT-3') designed on the basis of the rat Cu,Zn-SOD and Mn-SOD cDNA sequences [27,28]. 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 (Gibco BRL Life Technologies, Rockville, MD). For PCR amplification, a mixture containing the oligonucleotide primers, AmpliTaq DNA polymerase (2.5 U) (Perkin-Elmer, Irvine, CA), and [
-32P]deoxycytidine triphosphate (2 µCi of 3000 Ci/mmol) (Amersham, Buckinghamshire, UK) 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 [29]. Amplification was carried out for 20 cycles using 95°C (1 min) for denaturing, 65°C (1 min) for annealing, and 72°C (1 min) for extension 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, 483 bp for Mn-SOD, and 100 bp for S16. 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 [26]. 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).
To validate that the amplified cDNAs were Cu,Zn-SOD and Mn-SOD, the PCR products were cloned with the TA cloning kit (Invitrogen Co., San Diego, CA). Then, direct sequence analyses of the PCR products were performed. The cDNA sequences of the amplified cDNA with primer sets for Cu,Zn-SOD and Mn-SOD were consistent with the previously reported sequences of rat Cu,Zn-SOD and Mn-SOD [27,28].
Hormone Assay
Progesterone, testosterone, and estradiol concentrations were determined by a specific RIA as described previously [24,30]. The sensitivities of the assay were 100 pg/tube for the progesterone assay and 2.7 pg/tube for both the testosterone and estradiol assays. The intra- and interassay coefficients of variation were, respectively, 7.0% and 14.4% for the progesterone assay, 13.2% and 11.8% for the testosterone assay, and 10.0% and 10.4% for the estradiol assay.
PRL concentrations were determined by a Biotrak rat PRL enzyme immunoassay system (Amersham). The sensitivity of the assay was 0.44 ng/ml. The intra- and interassay coefficients of variation were 9.3% and 6.0%, respectively.
Statistical Analyses
Data were examined by ANOVA and Duncan's New Multiple Range test. Differences were considered to be significant if P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Charcoal treatments of the placental incubation medium significantly inhibited the stimulatory effects of placental incubation medium on serum progesterone concentrations and luteal Mn-SOD activities, but not on luteal Cu,Zn-SOD activities (Table 1). Expression of Cu,Zn-SOD and Mn-SOD mRNA in the corpus luteum was also increased by the administration of placental incubation medium (Figs. 1 and 2), and the charcoal treatment significantly inhibited the stimulatory effect of placental incubation medium on luteal Mn-SOD mRNA levels, but not on luteal Cu,Zn-SOD mRNA levels (Figs. 1 and 2).
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Testosterone, one of the steroid hormones secreted by placentae, is well known to act luteotropically in the corpus luteum through conversion to estrogen. There are, however, several reports showing that testosterone itself has a direct luteotropic effect, not mediated through conversion to estrogen, in luteal cells [31,32]. Also, it has been reported that estrogen administered exogenously causes a luteolytic effect in the presence of the uterus and hypothalamus-pituitary system [33–35]. Therefore, we used testosterone to examine whether the effects abolished by the charcoal treatment are due to the removal of testosterone and whether testosterone acts directly in the corpus luteum. As shown in Figure 3, the inhibitory effect of the charcoal treatment on luteal Mn-SOD activities was reversed by supplementation with testosterone or DHT, but serum progesterone concentrations were recovered only by DHT. As shown in Figure 2, the inhibitory effect of the charcoal treatment on Mn-SOD mRNA expression in the corpus luteum was also reversed by supplementation with either testosterone or DHT. Testosterone and DHT alone had no effect on serum progesterone concentrations or luteal Mn-SOD activities and its mRNA levels (Figs. 2 and 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There may be a possibility that treatment with placental incubation medium stimulates PRL production by the pituitary, which in turn maintains luteal function. However, the present study showed that treatment with placental incubation medium did not influence serum PRL concentrations. Therefore, it is unlikely that placental incubation medium influences luteal function through PRL production by the pituitary.
Charcoal treatments of the placental incubation medium decreased luteal Mn-SOD expression, but not Cu,Zn-SOD expression, suggesting that they are regulated differently as noted previously [26]. These different types of regulation may also suggest that Cu,Zn-SOD and Mn-SOD play different roles in regulating corpus luteum function, but this remains unknown. We recently found that the suppression of intracellular Cu,Zn-SOD activity by antisense oligonucleotides to Cu,Zn-SOD cDNA inhibited progesterone production by rat luteal cells [37]. It has been also reported that Mn-SOD scavenges superoxide radicals generated in mitochondria to maintain progesterone production by the corpus luteum, as oxygen radicals inhibit cholesterol transport into mitochondria during steroidogenesis [5,38]. These findings suggest that both Cu,Zn-SOD and Mn-SOD have protective roles against toxic oxygen radicals for progesterone production.
This is also, to our knowledge, the first report demonstrating in vivo that androgen, without conversion to estrogen, has a luteotropic effect in the presence of other placental luteotropins in pseudopregnant rats, because supplementation with DHT completely reversed the inhibitory effects of charcoal treatments on serum progesterone concentrations and luteal Mn-SOD expression. Increasing evidence indicates that conversion to estrogen is not required for the action of androgens in the ovaries [32,39–40]. A recent report showing that androgens directly stimulated progesterone production by mouse luteal cells [31] also supports our results. Sugino et al. [15] reported that Cu,Zn-SOD and Mn-SOD mRNA expression was down-regulated by estrogen in a rat luteal cell line, which may support our results showing that testosterone, but not estrogen, interacts with other placental luteotropins to stimulate luteal Mn-SOD expression. It is not clear at present how androgen exerts its effect on progesterone production and Mn-SOD expression. Thordarson et al. [31] reported the non-genomic direct effect of androgen, not acting through nuclear androgen receptor, to produce progesterone by mouse luteal cells. Non-genomic steroid activities may be mediated through changes in the cell membrane fluidity or through intracellular second messenger systems [41]. The present results suggest that the interaction between testosterone and peptide hormones is required for the stimulation of progesterone production and luteal Mn-SOD expression. Thordarson et al. [31] reported that androgens acted synergistically to stimulate progesterone production by mouse luteal cells when administered together with placental lactogens. Gibori and her colleagues also have reported the synergistic actions of estradiol and PRL-like hormones [42,43].
The present study showed that supplementation of charcoal-treated placental incubation medium with DHT reversed the inhibitory effect of the charcoal treatment on progesterone production, but supplementation with testosterone could not. It is difficult to explain clearly the different effects of testosterone and DHT on progesterone production. Since estrogen has a luteolytic effect in the presence of the uterus and hypothalamus-pituitary system [33–35], estrogen converted from testosterone may have indirectly affected the progesterone production. Another possibility is that there is some factor in the placental incubation medium, removed by the charcoal treatment, that modifies the action of testosterone on progesterone production. However, further studies are needed to clarify these possibilities.
In conclusion, the present study showed that placental luteotropins stimulated SOD expression in the corpus luteum and increased progesterone production in pseudopregnant rats. In addition, we indicated that testosterone, not estrogen, acts in the corpus luteum to stimulate progesterone production and Mn-SOD expression synergistically with other placental luteotropins.
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FOOTNOTES |
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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. 
2 Correspondence: Hiroshi Kato, Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube 755-8505, Japan. FAX: 836 22 2287; hkato{at}po.cc.yamaguchi-u.ac.jp
Accepted: September 16, 1999.
Received: April 26, 1999.
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REFERENCES |
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and ß in the rat corpus luteum of pregnancy: regulation by prolactin and placental lactogens. Endocrinology 1998; 139:2432–2442.This article has been cited by other articles:
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 K H Al-Gubory, S Camous, G Germain, P Bolifraud, A Nicole, and I Ceballos-Picot Reconsideration of the proposed luteotropic and luteoprotective actions of ovine placental lactogen in sheep: in vivo and in vitro studies. J. Endocrinol., March 1, 2006; 188(3): 559 - 568. [Abstract] [Full Text] [PDF]
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 A. Matsuoka-Sakata, H. Tamura, H. Asada, I. Miwa, T. Taketani, Y. Yamagata, and N. Sugino Changes in vascular leakage and expression of angiopoietins in the corpus luteum during pregnancy in rats Reproduction, February 1, 2006; 131(2): 351 - 360. [Abstract] [Full Text] [PDF]
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A Makker, F W Bansode, V M L Srivastava, and M M Singh Antioxidant defense system during endometrial receptivity in the guinea pig: effect of ormeloxifene, a selective estrogen receptor modulator J. Endocrinol., January 1, 2006; 188(1): 121 - 134. [Abstract] [Full Text] [PDF]
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 S. Takiguchi, N. Sugino, K. Esato, A. Karube-Harada, A. Sakata, Y. Nakamura, H. Ishikawa, and H. Kato Differential Regulation of Apoptosis in the Corpus Luteum of Pregnancy and Newly Formed Corpus Luteum after Parturition in Rats Biol Reprod, February 1, 2004; 70(2): 313 - 318. [Abstract] [Full Text] [PDF]
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 N. Sugino, A. Karube-Harada, A. Sakata, S. Takiguchi, and H. Kato Different mechanisms for the induction of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone in human endometrial stromal cells Hum. Reprod., July 1, 2002; 17(7): 1709 - 1714. [Abstract] [Full Text] [PDF]
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S. Kashida, N. Sugino, S. Takiguchi, A. Karube, H. Takayama, Y. Yamagata, Y. Nakamura, and H. Kato Regulation and Role of Vascular Endothelial Growth Factor in the Corpus Luteum During Mid-Pregnancy in Rats Biol Reprod, January 1, 2001; 64(1): 317 - 323. [Abstract] [Full Text]
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 N. Sugino, T. Suzuki, S. Kashida, A. Karube, S. Takiguchi, and H. Kato Expression of Bcl-2 and Bax in the Human Corpus Luteum during the Menstrual Cycle and in Early Pregnancy: Regulation by Human Chorionic Gonadotropin J. Clin. Endocrinol. Metab., November 1, 2000; 85(11): 4379 - 4386. [Abstract] [Full Text]
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N. Sugino, S. Kashida, S. Takiguchi, A. Karube, and H. Kato Expression of Vascular Endothelial Growth Factor and Its Receptors in the Human Corpus Luteum during the Menstrual Cycle and in Early Pregnancy J. Clin. Endocrinol. Metab., October 1, 2000; 85(10): 3919 - 3924. [Abstract] [Full Text]
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 N. Sugino, M. Nakata, S. Kashida, A. Karube, S. Takiguchi, and H. Kato Decreased superoxide dismutase expression and increased concentrations of lipid peroxide and prostaglandin F2{alpha} in the decidua of failed pregnancy Mol. Hum. Reprod., July 1, 2000; 6(7): 642 - 647. [Abstract] [Full Text] [PDF]
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