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Articles |
a Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, Tokyo 113, Japan
b Genetic Division, National Cancer Center Research Institute, Tokyo 104, Japan
c Laboratory of Molecular and Cellular Biology, Equine Research Institute, Japan Racing Association, Tochigi 320, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxysteroid dehydrogenase (20-HSD) catalyzes the conversion of progesterone to an inactive steroid, 20-dihydroprogesterone (20-OHP). Thus, using pseudopregnant rats as a model, physiological cooperation of P450C26 and 20-HSD in the reduction of progesterone release toward the end of the luteal phase was evaluated. Levels of P450C26 and 20-HSD mRNA were examined in corpora lutea from pseudopregnant rats by Northern blot or reverse transcription-polymerase chain reaction or both. P450C26 mRNA was ubiquitously expressed in corpora lutea, and its expression increased toward the end of pseudopregnancy, while 20-HSD was expressed in all corpora lutea on Day 16 (Day 0 = the day of after cervical stimulation) but not detected before Day 10. An inhibitor of 20-HSD, STZ26 (D-homo-16-oxa-4-androstene-3,16-dione), was administered at various doses to rats from Day 12 to 20, effectively suppressing the elevation of 20-OHP in a dose-dependent manner but not the depletion of progesterone completely. The expression of P450C26 mRNA was increased as STZ26 dose increased, which negatively correlated with the progesterone levels. These results strongly suggest that P450C26 cooperated with 20-HSD in the reduction of progesterone release from the rat luteal tissue at the end of the functional luteal phase.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We constructed a subtracted cDNA library and isolated a cDNA clone corresponding to 26-cholesterol hydroxylase (P450C26) [2]. P450C26 is known as an enzyme that catalyzes 26-hydroxylation of cholesterol [3], 25-hydroxylation of vitamin D3 [4], and 4-hydroxylation of retinoic acid [5]. 26-Hydroxycholesterol blocks endogenous cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) synthase [6], HMGCoA reductase, and low-density lipoprotein (LDL) receptor gene expression [7]. Ovarian cholesterol levels remain relatively constant during pseudopregnancy [8]. These facts suggest that P450C26 plays an important role in cholesterol biosynthesis of corpora lutea. The hypothesis underlying this study was that this enzyme is the as-yet-unidentified factor that lowers progesterone levels toward the end of the luteal phase.
In addition to P450C26, 20-hydroxysteroid dehydrogenase (20-HSD) in the luteal tissue of rodents catalyzes the conversion of progesterone to a biologically inactive steroid, 20-hydroxypregn-4-ene-3-one (20-dihydroprogesterone, 20-OHP), and thus reduces the release of progesterone into the circulation [9–14]. An increase in 20-HSD activity occurs in the luteal tissue of rats during the process of functional luteolysis, and thus 20-HSD is considered a regulator of progesterone release from the corpus luteum, acting via enzymatic depletion of progesterone [12–15]. As for the autoregulatory luteotropic function of progesterone [1] mentioned above, the absence of progesterone receptors in rat luteal cells remained to be accounted for, but recently Sugino et al. [16] found that progesterone inhibits the expression of 20-HSD mRNA by means of glucocorticoid receptor. Prolactin is another luteotropic factor in rodents that can down-regulate 20-HSD gene expression [17]. But this pathway operates only in rodents. The regulatory system of luteolysis in other species remains to be elucidated.
Treatment of rats on Days 12 through 18 with D-homo-16-oxa-4-androstene-3,16-dione; STZ26), a novel inhibitor of 20-HSD, inhibited both the elevation of peripheral 20-OHP levels and the concomitant reduction in peripheral progesterone levels at the end of pseudopregnancy [18]. However, although the inhibition of enzymatic activity was complete, peripheral progesterone levels were still partially reduced, suggesting that depletion of progesterone levels toward the end of pseudopregnancy is not solely the result of the conversion to 20-OHP by 20-HSD.
Taken together, these results suggest the possibility that the reduction of progesterone release toward the end of pseudopregnancy in rodents is the result of concomitant activation of 20-HSD and P450C26. In the present investigation, therefore, the expression of P450C26 mRNA was evaluated in corpora lutea from pseudopregnant rats. In addition, the levels of P450C26 mRNA were examined when pseudopregnant rats were treated with STZ26.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two-month-old female Wistar-Imamichi rats were purchased from Imamichi Institute for Animal Reproduction (Tsuchiura, Japan) and housed under controlled lighting conditions of 14L:10D (lights-on at 0500 h). Food and water were available ad libitum.
Vaginal cytology was monitored every day, and only animals that had shown a regular 4-day estrous cycle for more than 2 cycles were used. They were rendered pseudopregnant by cervical stimulation between 1800 and 1900 h on the day of proestrus, and the day following cervical stimulation was designated Day 0. Ovarian samples were collected only from animals that had maintained the leukocytic diestrous stage by the day of collection of blood or ovarian samples.
Rats were killed by decapitation between 1200 and 1300 h on Days 6, 10, 16, and 18 of pseudopregnancy. Ovaries were collected, frozen immediately in liquid nitrogen, and stored at -80°C. In some cases, only newly formed corpora lutea were enucleated; these were stored separately from the rest of the ovarian tissues. Newly formed corpora lutea were recognized by their color and location in the ovarian tissue. There were some corpora lutea of ambiguous appearance, which were excluded from sampling.
STZ26, an inhibitor of 20-HSD [18], was given s.c. at various doses to a group of pseudopregnant animals at 1300 h from Day 12 to 20. STZ26 was dissolved in ethanol and then in sesame oil and was injected into rats after the ethanol had evaporated. Blood samples were collected every day, allowed to clot at 4°C for 2 h, centrifuged at 1500 x g for 15 min, and stored at -20°C until the determination of progesterone and 20-OHP by RIAs. No cross-reactivities were detected among progesterone, 20-OHP, and STZ26 in progesterone and 20-OHP RIAs.
RNA Preparations
Total RNAs from ovaries were isolated by guanidinium/cesium chloride ultracentrifugation [19], and total RNAs from corpora lutea were extracted using the guanidinium thiocyanate/phenol extraction method (Trizol reagent; Gibco BRL, Gaithersburg, MD). Poly(A)+ RNAs were obtained using oligo(dT)+-cellulose chromatography.
Construction and Screening of Subtracted Library
A subtracted cDNA library was constructed by a modification of the method reported by Tanaka et al. [20]. Poly(A)+ RNA (5 µg) extracted from rat ovaries on Day 16 was dissolved in water. First and second cDNA strands were synthesized using a cDNA synthesis system (Amersham, Buckinghamshire, England). The size-fractionated cDNA obtained was ligated to an EcoRI-NotI-BamHI adaptor (Takara, Kyoto, Japan) and digested with BamHI. The digested cDNA was ligated to the second adaptor containing BamHI and EcoRI sites. All ligated cDNAs were amplified by polymerase chain reaction (PCR), using the oligomer within the second adaptor, and were used as tester DNAs.
Size-fractionated cDNA from rat ovaries on Day 6 was ligated to 
ZAPII (Stratagene, La Jolla, CA), and then inserted into phage particles with Gigapack II Gold Packaging Extract (Stratagene). The resulting phage clones (3 x 106) were converted to plasmid clones according to the excision protocol recommended by the manufacturer. Whole-plasmid DNAs were purified by ultracentrifugation in cesium chloride ethidium bromide density gradients, digested with restriction enzyme ApaI, and used as competitor DNAs.
PCR-amplified tester DNAs were hybridized with the ApaI-digested competitor DNAs in a hybridization buffer, and single-stranded DNAs that failed to hybridize with the competitors were separated by hydroxylapatite chromatography (Bio-Rad Laboratories, Hercules, CA). The single-stranded cDNAs thus obtained were amplified by PCR using the oligomer with an EcoRI site within the second adaptor. Amplified DNAs were purified, digested with EcoRI, and ligated into a gt10 vector to produce a subtracted library. Duplicated filters were prepared from the subtracted library and were differentially screened with the subtracted cDNA probes and competitor cDNA probes, respectively. Only clones that gave strong hybridization signals to the subtracted DNA probes, but not to the competitor cDNA probes, were isolated. The probes were labeled with [-32P]dCTP using multiprime labeling kit (Amersham).
Northern Blot Analysis
Total RNAs from ovaries and corpora lutea were separated in formaldehyde-denaturing agarose gel and transferred to nylon membrane (Paul, Glen Cove, NY). Hybridization was carried out with [-32P]dCTP-labeled 20-HSD or P450C26 cDNA. Hybridization and washing conditions were those described by Sambrook et al. [19]. The membranes were exposed to x-ray film (XAR5; Eastman Kodak, Rochester, NY), and autoradiographs were obtained.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis
Levels of P450C26 mRNA expression were determined by RT-PCR. Total RNA (1 µg) was reverse transcribed at 42°C using avian myeloblastosis virus reverse transcriptase and oligo(dT)+ primer in a total volume of 20 µl. PCR was performed on synthesized cDNAs in a total volume of 100 µl containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.01% gelatin, 1 mM dNTPs, and 2.5 U of LA Taq polymerase (Takara). After samples were overlaid with light mineral oil, PCR amplification was carried out for 28 cycles in a Perkin-Elmer/Cetus thermal cycler (Norwalk, CT), each cycle consisting of 30 sec at 94°C (denaturation), 30 sec at 58°C (annealing), and 1 min at 74°C (elongation). PCR products were analyzed by agarose gel electrophoresis on 1.0% gels and visualized with UV light after ethidium bromide staining. Sets of oligonucleotides used for P450C26 cDNA amplification (5'-GCTTCTCAGACACGATCTATG-3' and 5'-TTGAATCTGGCATCGTGACAT-3') and for rat ribosomal protein L19 mRNA (5'-TCAAAGATGGCCTGATCATC-3' and 5'-TGGACAGAGTCTTGATGATC-3') were designed from rat P450C26 [21] and ribosomal protein L19 cDNA sequence [22], respectively. The primers used for PCR amplification gave PCR products at sizes of 1716 base pairs (bp) and 434 bp for P450C26 and ribosomal protein L19, respectively. Band intensities of RT-PCR products were determined with NIH Image analyzer (Bethesda, MD), and data were normalized to the intensities of ribosomal protein L19 cDNA.
DNA Sequencing
The cDNA clones isolated from the subtracted library and PCR products subcloned into pUC118 plasmid vector were sequenced by the double-strand dideoxy-chain termination protocol, using T7 DNA polymerase (Pharmacia Biotech, Piscataway, NJ). A computer-assisted homology search was performed in the nucleic acid databases of DDBJ (DNA Data Bank of Japan, National Institute of Genetics, Mishima, Japan).
Statistical Analysis
Data were analyzed by ANOVA followed by Duncan's multiple range test. Differences at p < 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The strategy for constructing the subtracted cDNA library was to select a gene expressed specifically or more abundantly at the luteolytic stage (tester cDNAs from ovaries on Day 16) than during the functional luteal stage (competitor cDNAs from ovaries on Day 6). A subtracted cDNA library consisting of 1.7 x 105 recombinant phages was obtained, and 3.0 x 104 clones were screened. After differential screening with the subtracted and the competitor cDNA probes, 100 candidate clones matching the strategy were isolated. Among these, 5 clones were arbitrarily chosen at a time and used as probes for Southern blot hybridization on all 100 candidates. These procedures were repeated several times, and 10 independent clones were isolated (data not shown). Partial cDNA sequencings were performed on these 10 clones and referred to the DDBJ database. One of these clones represented a nucleotide sequence between nucleotides 623 and 1426 of rat P450C26 cDNA [2].
Expression of P450C26 in Ovaries and Corpora Lutea
Using the partial sequence of P450C26 cDNA selected from the subtracted cDNA library, Northern blot analysis was performed with poly(A)+ RNA isolated from ovaries of pseudopregnant rats on Days 6 and 16. The results indicated that the expression level of P450C26 mRNA was slightly but certainly higher in ovaries on Day 16 than on Day 6 (data not shown), which was in agreement with the subtracting strategy.
To further analyze changes in the expression of P450C26 in the ovary during pseudopregnancy, another series of Northern blot analyses was done on total RNA extracted from ovaries on Days 6, 10, and 16. The results showed that the expression of P450C26 was much higher in the later stage of pseudopregnancy (data not shown).
To determine time-specific expression of P450C26 mRNA on Days 6, 10, 16, and 18, total RNA from corpora lutea that could be identified macroscopically as newly formed was extracted and also subjected to Northern blot analysis. As shown in Figure 1A, P450C26 mRNA was expressed in corpora lutea at all stages, but the expression level increased as pseudopregnancy progressed. The progressive changes of P450C26 mRNA were examined by RT-PCR (Fig. 1B). Densitometric measurement of PCR products revealed that the signal on Day 18 was 1.6-fold as much as on Day 6 (Fig. 1C). It should be noted that the expression of P450C26 mRNA was also detected on Day 6 when peripheral progesterone was at a high level [12–14].
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Expression of 20-HSD in Ovaries and Corpora Lutea
Northern blot analysis was carried out on total RNA extracted from ovaries on Days 6, 10, and 16 using the rat 20-HSD cDNA probe [23]. The results showed that expression of mRNA corresponding to 20-HSD was low on Day 10 but high on Days 6 and 16 (Fig. 2A).
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To determine time-specific expression of 20-HSD in the corpora lutea, Northern blot analysis using total RNA extracted only from newly formed corpora lutea on Days 6, 10, 16, and 18 was performed. The results indicated that expression of 20-HSD mRNA in corpora lutea was lower on Days 8 and 10 than on Days 16 and 18 (Fig. 2B). A relatively high expression of ovarian 20-HSD on Day 6 (Fig. 2A) may be attributable to expression in the corpora lutea remaining from previous luteal phases, because the expression was limited in the newly formed corpora lutea on Day 6 (Fig. 2B).
Progestin Levels and Expression of P450C26 mRNA in the Rat Ovary Treated with STZ26
Between Days 6 and 16, 20-OHP levels rose and progesterone levels decreased in pseudopregnant rats without STZ26 treatment (Fig. 3). When STZ26 was administered from Day 12 to 20, the majority of those animals were maintained at the diestrous stage until the end of the treatment. 20-OHP levels on Day 20 in pseudopregnant rats treated with STZ26 were suppressed dose-dependently, and the level in the animals treated with the highest dose of STZ26 on Day 20 was comparable to that observed in pseudopregnant rats on Day 12 without the treatment. Progesterone levels, however, were comparable to that observed at the end of pseudopregnancy (Day 16) (Fig. 3). Thus, decrease in progesterone levels was not prevented by the STZ26 treatment. All pseudopregnant rats without STZ26 treatment entered the vaginal proestrous stage on Day 17.
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Northern blot and RT-PCR analysis on total RNAs revealed that the expression of P450C26 mRNA in ovaries of rats treated with STZ26 between Days 12 and 20 was intensified dose-dependently (Fig. 4, A and B). Densitometric measurement indicated that the expression level in ovaries obtained from pseudopregnant rats treated with STZ26 at 30 mg/kg BW was 1.4-fold of that in vehicle-treated rats (Fig. 4C). Expression of 20-HSD mRNA from the same specimen was not altered (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using the cloned cDNA as a probe for P450C26, Northern blot analysis was done on poly(A)+ RNA prepared from pseudopregnant rat ovaries on Day 6 or 16, both of which had been used for constructing a subtracted cDNA library. Results indicated that ovarian P450C26 mRNA was higher on Day 16 than on Day 6 (data not shown). An additional Northern blot analysis on total RNA extracted from pseudopregnant rat ovaries on Days 6, 10, and 16 also indicated a marked increase in the expression of P450C26 mRNA between Days 6 and 16 (data not shown). This observation corresponded to the start of a decline in peripheral progesterone levels on Day 8 ([11, 15] and Fig. 3). Furthermore, in addition to follicles and interstitial tissues, the ovarian tissue consists of not only a crop of newly formed corpora lutea but also old corpora lutea formed after previous ovulations. Therefore, total RNA was isolated only from newly formed corpora lutea and subjected to Northern blot and RT-PCR analysis. The results indicated that P450C26 mRNA was expressed mainly in corpora lutea and that expression gradually increased from Day 6 to 16/18 (Fig. 1). The increase in the expression of P450C26 mRNA in corpora lutea corresponded negatively to the continuous decrease in peripheral progesterone levels during this period.
Ovarian 20-HSD catalyzes the conversion of progesterone to a biologically inactive steroid, 20-OHP [9–11]. During pregnancy or pseudopregnancy in the rat, the secretory rate of progesterone is inversely correlated with that of 20-OHP [11–15]; thus it has been generally believed that increase in 20-HSD level is negatively associated with progesterone output from murine luteal tissue. An inhibitor of 20-HSD developed recently in our laboratory [18] could not prevent the precipitous depletion of progesterone levels occurring at the end of pseudopregnancy, although it could considerably inhibit the simultaneous elevation of 20-OHP levels. This finding strongly suggests that elevation of 20-HSD activity, namely an enhancement of progesterone catabolism, is not solely responsible for the depletion of progesterone levels at the end of the murine luteal phase.
Northern blot analysis of either P450C26 or 20-HSD mRNA, and RT-PCR analysis of P450C26 mRNA, were performed on total RNA extracted from ovaries of pseudopregnant rats that had been treated with graded doses of STZ26, an inhibitor of 20-HSD, from Days 12 to 20 after cervical stimulation. This treatment extended the state of pseudopregnancy to Day 20 and enhanced P450C26 mRNA expression in a dose-dependent manner. But this was not the case with 20-HSD mRNA. These results suggest that there could be an unidentified mechanism by which P450C26 mRNA expression is continuously enhanced when progesterone concentration is maintained above a certain threshold level.
In summary, the present study demonstrates that P450C26 is expressed in corpora lutea and that its expression is enhanced as pseudopregnancy progresses. Thus, not only 20-HSD but P450C26 may negatively regulate progesterone secretion from corpora lutea at the end of the functional luteal phase in rats.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Michio Takahashi, Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113, Japan. FAX: 81 3815 4266; amtaka{at}hongo.ecc.u-tokyo.ac.jp
3 Current address: Laboratory of Experimental Animal Science, Kitasato University, School of Veterinary Medicine and Animal Science, 35–1 Higashi-23bancho, Towada, Aomori 034, Japan.
Accepted: March 31, 1999.
Received: August 27, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ol-3-one by rat ovarian tissue in vitro. J Biol Chem 1959; 234:3115–3121.-hydroxysteroid dehydrogenase. Steroids 1966; 7:395–413.[CrossRef][Medline]-ol-3-one in ovarian venous blood during various reproductive states in the rat. Endocrinology 1968; 82:333–341.[Medline]-hydroxysteroid dehydrogenase in luteotrophic and luteolytic processes during rat pseudopregnancy. J Reprod Fertil 1990; 88:467–474.[Abstract]-hydroxysteroid dehydrogenase isozymes in rat corpora lutea. J Reprod Fertil 1992; 96:573–580.[Abstract]-hydroxysteroid dehydrogenase expression in the rat corpus luteum through the glucocorticoid receptor. Endocrinology 1997; 138:4497–4500.-hydroxysteroid dehydrogenase isozymes (HSD1). Biol Reprod 1997; 57:1433–1437.[Abstract]-hydroxysteroid dehydrogenase (HSD1). Biochem J 1994; 299:561–567.This article has been cited by other articles:
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  C. Stocco, C. Telleria, and G. Gibori The Molecular Control of Corpus Luteum Formation, Function, and Regression Endocr. Rev., February 1, 2007; 28(1): 117 - 149. [Abstract] [Full Text] [PDF]
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 N. Tinfo and C. Komar Potential role for peroxisome proliferator-activated receptor {gamma} in regulating luteal lifespan in the rat Reproduction, January 1, 2007; 133(1): 187 - 196. [Abstract] [Full Text] [PDF]
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 C. M. Komar and T. E. Curry Jr Localization and Expression of Messenger RNAs for the Peroxisome Proliferator-Activated Receptors in Ovarian Tissue from Naturally Cycling and Pseudopregnant Rats Biol Reprod, May 1, 2002; 66(5): 1531 - 1539. [Abstract] [Full Text]
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B. D. Murphy Models of Luteinization Biol Reprod, July 1, 2000; 63(1): 2 - 11. [Abstract] [Full Text]
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