| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
-Hydroxysteroid Dehydrogenase
Howard Hughes Medical Institute (R.P.P., S.G., A.H., J.N.I.), Department of Biochemistry (R.P.P., S.G., A.H., J.N.I.), St. Jude Children’s Research Hospital, Memphis, Tennessee 38105; and Faculty of Health Sciences (Y.W.), Department of Microbiology and Immunology, Ben Gurion University of the Negev, Beer Sheva, Israel 84105
Address all correspondence and requests for reprints to: James N. Ihle, Howard Hughes Medical Institute, Department of Biochemistry, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105. E-mail: james.ihle{at}stjude.org.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-hydroxysteroid dehydrogenase (20-HSD) and a decrease in serum progesterone, which normally declines only immediately before parturition. To determine the relative contribution of 20-HSD to progesterone metabolism and Stat5 function during pregnancy and parturition, we created a 20-HSD-deficient strain of mice by gene disruption. Mice deficient for 20-HSD sustain high progesterone levels and display a delay in parturition of several days demonstrating that 20-HSD regulates parturition downstream of the prostaglandin F2 receptor in an essential and nonredundant manner. Moreover, 20-HSD deficiency partially corrected the abortion of pregnancies associated with Stat5b deficiency, supporting the concept that prolactin activation of Stat5b is important in suppressing 20-HSD gene expression and thereby allowing the maintenance of progesterone levels that are required to sustain pregnancy. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The critical targets of Stat5 transcriptional regulation in PRL target tissues are largely unknown. One potential gene target is the progesterone-metabolizing enzyme 20-hydroxysteroid dehydrogenase (20-HSD) (7). The enzyme was initially detected as a progesterone-metabolizing enzyme in the ovary (8) and was subsequently shown to be also highly expressed in thymocytes (9, 10, 11). Based on this observation, its induction in splenocytes was used as an assay for the identification of T cell-derived factors that potentially regulated T cell development (12). This activity was subsequently found to be a function of IL-3 (13). However, the significance or role of the expression of 20-HSD by thymocytes and lymphocytes, as well as myeloid lineage cells, is unknown.
In addition to hematopoietic cells, 20-HSD is highly expressed in the adrenal gland (14) and in granulosa cells of the corpus luteum (15). The potential functions of 20-HSD in the adrenal gland are largely unknown. In contrast, 20-HSD expression by granulosa cells is widely accepted to be involved in the regulation of progesterone levels during the estrous cycle and in pregnancy (8, 16). After oocyte release in response to LH, granulosa cells initiate the formation of a corpus luteum and the cells begin to express 20-HSD. In rodents, in the absence of coital stimulation, the granulosa cells undergo apoptosis and the corpus luteum regresses (17). With coital stimulation, PRL is produced by the anterior pituitary gland (18), and PRL stimulation of granulosa cells suppresses the expression of 20-HSD and apoptosis (7, 19) and allows the development of a secretory corpus luteum producing the progesterone required for the initial stages of implantation and maintenance of pregnancy. At term, the ovarian release of progesterone is terminated, and it has been proposed that 20-HSD contributes to the rapid elimination of progesterone (8, 15). To explore the role of 20-HSD and its regulation by Stat5 in various cell lineages, we have created a strain of mice lacking the enzyme. The results demonstrate that Stat5 plays a critical role in suppressing 20-HSD during pregnancy and that the primary function of 20-HSD is to reduce progesterone levels at term of pregnancy.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-HSD Expression during Pregnancy and Influence of Stat5a/b
, high levels of the progesterone-metabolizing enzyme 20-HSD are initially present during early pregnancy in mouse ovaries, and this activity is due to previous cycling of the ovaries. Thereafter, 20-HSD protein levels drop dramatically, and only low or no protein is detectable throughout mid- to late gestation. At parturition, however, the protein levels dramatically increase. These results were comparable to previous studies showing a stringent regulation of ovarian 20-HSD mRNA expression during rat pregnancy (15). In contrast, the protein levels for 3ß-HSD, an enzyme involved in the last step of progesterone biosynthesis, did not change significantly during the whole course of pregnancy.
|
-HSD expression during pregnancy has been previously proposed (6). In ovarian tissue the predominant Stat5 protein expressed is Stat5b, and deletion of Stat5b alone is associated with spontaneous abortions during midgestation (5). Correlated with the abortions is a precipitous decline in progesterone concentrations, and administration of progesterone was shown to allow the maintenance of pregnancy (5). In contrast, Stat5a-deficient mice are fertile and carry the embryos to term (4, 6). The levels of 20-HSD protein in ovaries from Stat5b-deficient and Stat5a/b-deficient females at at 14–16 d of pregnancy (P14–P16) are also shown in Fig. 1A
. As illustrated, 20-HSD protein was readily detectable in these tissues at midgestation and, although the protein levels varied among individual females, the range of protein levels observed was comparable between Stat5a/b-deficient and Stat5b-deficient mice. Among a group of Stat5a/b-deficient females examined, there was a direct correlation between the levels of protein detected by Western blotting and the amount of enzyme activity as well as the circulating levels of progesterone (Fig. 1B). Lastly, the levels of 20-HSD protein are regulated at the transcriptional level because, in the absence of Stat5b, the levels of 20-HSD transcripts are dramatically elevated relative to the transcript levels in ovaries of wild-type mice at P12 (Fig. 1C).
The effects of Stat5a/b deficiency on circulating progesterone levels throughout pregnancy are illustrated in Fig. 2. In wild-type mice the circulating concentrations of progesterone increase dramatically from P4–P6 and are maintained at high levels throughout pregnancy (Fig. 2). In contrast, the levels of circulating progesterone show an initial increase in Stat5a/b-deficient mice but drop at P6–P8, although the rates are variable among individual mice. Comparable declines are seen in Stat5b-deficient mice (data not shown) as previously described (5). Together, the results support the hypothesis that 20-HSD expression is suppressed during mid- to late gestation, that this is mediated, in part, through a Stat5b-dependent mechanism, and that this regulation is critical for maintaining the high levels of progesterone required for maintenance of the pregnancy.
|
-HSD Is Disrupted-HSD, a strain of mice was established in which the gene was disrupted by homologous recombination. The targeting vector (Fig. 3A) was designed to delete exons 2 and 3, encoding essential components of the active site of the enzyme (20), and would be predicted to generate a null mutation. Genotyping of F1 mice by PCR (Fig. 3B) revealed a normal frequency of homozygously null mice. Absence of 20-HSD expression in homozygously deleted mice was verified by Western blotting in the adrenal, ovary, and kidney, which normally express high protein amounts (Fig. 3C). In addition, the determination of the enzymatic activity of 20-HSD in vitro, by conversion of radiolabeled progesterone to the biologically inactive form of 20-hydroxyprogesterone (20-OHP), demonstrated that enzymatic activity is absent in 20-HSD-deficient tissues (Fig. 4). The residual activity in the adrenal tissue is due to the activity of another metabolizing enzyme the product of which migrates at the 20-OHP region on the thin layer chromatography plate. In summary, these results demonstrate that the mutated locus creates a 20-HSD protein null phenotype, and that the enzyme is nonredundantly required for all 20-HSD enzyme activity.
|
|
-HSD-Deficient Mice-HSD has been observed in hematopoietic tissues including the thymus and bone marrow (9, 11), and its expression can be induced by treatment with IL-2 or IL-3 in thymocytes or bone marrow cells, respectively (21, 22). These observations had led to the hypothesis that 20-HSD plays a role in hematopoiesis. However, extensive analysis of hematopoietic functions has failed to detect any phenotypic alterations. For example, single cell suspensions from bone marrow, spleen, and thymus were subjected to fluorescence-activated cell sorting analysis. Analysis of the various subpopulations of the thymus (CD4, CD8), spleen (Thy1.2, B220), and bone marrow (Mac1, Gr1) using specific markers did not reveal any differences between wild-type and homozygously deficient mice in the cellular composition (data not shown). In addition, colony-forming activities in methylcellulose were comparable between 20-HSD-deficient and wild-type bone marrow cells (data not shown). The absence of enzymatic 20-HSD activity in the spleen and thymus and in IL-3-stimulated bone marrow cells was confirmed by enzyme assays (data not shown). In summary, these results indicate that 20-HSD is dispensable for the normal development of hematopoietic cells.
Increased Progesterone Levels and Delayed Parturition in 20-HSD-Deficient Females
To establish the putative role of 20-HSD in reproduction and parturition, the reproductive function of 20-HSD-deficient female mice was addressed. Both homozygously deficient males and females showed a normal fertility, and females maintained pregnancy normally. However, 20-HSD-deficient females failed to induce parturition at the term of pregnancy (d 20) as compared with wild-type or heterozygous animals. Parturition was consistently delayed by 2–3 d (Fig. 5A) leading to the death of pups in utero (data not shown). This phenotypic alteration was directly associated with a failure to completely down-regulate progesterone levels at the end of pregnancy. In wild-type mice, serum progesterone concentrations fell below 10 ng/ml on d 20 of pregnancy, and parturition occurred (Fig. 5B). In contrast, the levels of progesterone in pregnant 20-HSD-deficient females were still at 25–30 ng/ml at P19–P20, and these levels did not decline below 10 ng/ml for the next 2–3 d. These results demonstrate that 20-HSD plays a critical role in the down-regulation of progesterone levels at the end of pregnancy, and that this down-regulation is obligate for the induction of parturition.
|
-HSD deficiency were related solely to the changes in progesterone metabolism, RU486 was administered to pregnant females at P19. Administration of this progesterone antagonist to pregnant 20-HSD females resulted in normal parturition on the next day, although progesterone blood concentrations remained high (data not shown). The pups were born normal and were nursed normally by their 20-HSD-deficient mothers. The results are consistent with the hypothesis that the only role for 20-HSD during pregnancy and parturition is to control the levels of circulating progesterone.
20-HSD Is Necessary for Prostaglandin F2 (PGF2)-Dependent Progesterone Down-Regulation before Parturition
PGF2 signaling induces the down-regulation of blood progesterone concentrations by inhibition of its biosynthesis (23, 24) and by the up-regulation of 20-HSD expression before parturition (25, 26). Comparable to the 20-HSD deficiency, parturition is delayed in females deficient for the PGF2 receptor because blood progesterone levels remain high at the end of pregnancy (27). To determine whether 20-HSD is an essential part in the PGF2-activated pathway toward the induction of parturition, wild-type and 20-HSD-deficient females were injected on day P16–P17 of pregnancy with either 5 µg or 10 µg of cloprostenol, a potent PGF2 receptor agonist. As shown in Fig. 6A, induction of premature births occurred in approximately half of the wild-type females treated with 10 µg cloprostenol as well as in heterozygous mice (data not shown). The induction of parturition was correlated with the levels of progesterone 24 and 48 h after cloprostenol injection (Fig. 6B). In particular, induction of parturition occurred in those females in which progesterone levels were reduced to less than 10–15 ng/ml. In contrast to the induction of parturition in wild-type mice, cloprostenol did not induce parturition in any of the 20-HSD-deficient females treated under the same conditions. Moreover, 20-HSD-deficient animals displayed mean serum progesterone levels of approximately 25–30 ng/ml even 48 h after cloprostenol treatment (Fig. 6B). These findings demonstrate that 20-HSD plays a critical role in PGF2 regulation of parturition.
|
also affects the levels of progesterone by inhibition of its synthesis, and the decreases that were observed in 20-HSD-deficient mice could be speculated to be due to this component of regulation. One important regulatory component of the progesterone synthetic pathway that is affected by PGF2 is the protein StAR (steriodogenic acute regulatory protein) (28, 29). In the sequence of events leading to the reduction of progesterone levels, the decrease in StAR, a protein required for translocation of intracellular cholesterol in progesterone synthesis, occurs rapidly and is followed by the induction of 20-HSD enzyme activity. We therefore examined the affects of 20-HSD deficiency on the initial reductions in progesterone levels. As illustrated in Fig. 7A, cloprostenol induced a decrease in progesterone levels of approximately 40% by 3 h in wild-type mice when injected at P17–P18. This reduction occurred in the absence of detectable 20-HSD protein that became detectable only at 24 h (Fig. 7B). As with wild-type mice, cloprostenol induced a decrease in progesterone levels of approximately 40% in 20-HSD-deficient mice. The results are therefore consistent with the hypothesis that PGF2 induces a loss of progesterone by initially affecting its synthesis, independent of 20-HSD, and subsequently inducing the expression of 20-HSD.
|
-HSD Deficiency-HSD. If correct, it would be predicted that a deficiency of 20-HSD should correct the abortion of embryos during midgestation. We therefore crossed the 20-HSD deficiency onto the Stat5b deficiency and examined the status of the pregnancies at P18.5–P19.5. The numbers of viable or dead/resorbed embryos among the individual females is illustrated in Fig. 8. Among five females that were Stat5b deficient and wild-type or heterozygous for the loss of 20-HSD, only one female had maintained pregnancy as defined by more than 80% of the embryos present remaining viable and among 41 embryos that could be detected, only nine were viable. In contrast, six of seven females that were 20-HSD deficient and Stat5b deficient had maintained pregnancy resulting in 62 visible embryos of which 54 were viable. In contrast to the ability to rescue the defects of Stat5b-deficient mice with 20-HSD deficiency, the defects associated with Stat5a/b deficiency could not be rescued (data not shown).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-HSD in vivo and determining whether Stat5 regulation of its expression contributed to the physiological functions associated with this transcription factor. Although 20-HSD has been studied in the context of a variety of cell lineages and organs, the consequences of deleting the gene are remarkably specific. In particular, in contrast to the predictions of a variety of studies of hematopoietic lineage cells, a deficiency of 20-HSD had no impact on the parameters of differentiation and function that we have examined. The lack of any phenotypic changes is unlikely to be due to the presence of redundant enzyme activities because, as demonstrated, the deletion of the 20-HSD gene completely eliminates the ability to convert progesterone to 20-hydroxyprogesterone in all the tissues examined. It is possible that 20-HSD is important in stress conditions associated with high sustained levels of progesterone. Indeed, it has been shown that progesterone can affect thymic cellularity and various immunological responses. Consistent with this, female mice lacking the progesterone receptor are resistant to estrogen/progesterone-mediated thymic involution that occurs during pregnancy (30). Based on these observations, it will be important to examine very defined physiological and pathophysiological states for potential roles for 20-HSD.
In the context of reproductive physiology, it is notable that 20-HSD deficiency did not affect the mating behavior of females, the regulation of the estrous cycle, or the onset of pregnancy. Indeed, the primary, nonredundant function of 20-HSD established from the gene deficiency is to contribute to the reduction of progesterone levels that must occur at term to allow parturition to take place. It is important to note, however, that significant reduction of progesterone levels does occur in the absence of 20-HSD (Fig. 5B), and thus the overall process leading to parturition involves multiple components.
The regulation of the expression of 20-HSD in granulosa cells involves different factors at different stages of function. Early in the development of the corpus luteum, 20-HSD expression is induced, and the factors controlling this expression are unknown. After coital stimulation, the production of PRL and its activation of Stat5 in granulosa cells are responsible for the down-regulation of expression as established by the persistent expression of 20-HSD in ovaries of pregnant Stat5-deficient females and consistent with previous studies. Interestingly, PRL has been shown to be responsible for both the induction and repression of genes in ovaries of pregnant rodents. The mechanisms by which Stat5 represses 20-HSD gene expression are not known and, in particular, it is not known whether Stat5 represses gene expression directly or whether it induces a transcriptional repressor. It should be noted, however, that the promoter region of the gene does not contain a consensus Stat5-binding site.
The ability of the 20-HSD deficiency to rescue the premature abortions that occur in Stat5b-deficient mice demonstrates the importance of the function of Stat5 to suppress enzyme expression. This result is consistent with the observation that progesterone administration could similarly rescue the pregnancies of Stat5b-deficient mice (5). However, 20-HSD deficiency was unable to rescue the pregnancies of Stat5a/b-deficient mice, and again consistent with this is the observation that progesterone administration was not able to rescue the pregnancies (data not shown). These results suggest that Stat5 proteins have additional functions in ovarian activity that are critical for maintenance of pregnancy. Thus removing Stat5b, which is the predominant protein expressed in the ovary, impairs suppression of 20-HSD expression sufficient to affect progesterone levels, but the residual Stat5a activity is sufficient for the other critical functions. In particular, we have shown previously that p27 expression in the ovary is dependent upon the presence of both Stat5a and Stat5b (6). The importance of this expression is shown by the loss of ovarian function in p27-deficient mice (31).
The results are consistent with the hypothesis that Stat5a/b play a critical role in the physiological responses to PRL. However, it is important to note that the deficiency of either PRL or the PRL receptor results in lack of implantation at an earlier stage than that seen in Stat5a/b-deficient mice. This would suggest that there are additional, Stat5a/b-independent functions provided by signaling through the PRL receptor.
Immediately before term, 20-HSD expression is strongly induced at the transcriptional level. Based on a number of observations, this induction is dependent upon PGF2. In particular, failure to reduce progesterone levels at term and to deliver is a phenotype that is also seen in PGF2 receptor-deficient mice (27). However, the overall reduction of progesterone levels is clearly dependent upon additional regulation beyond degradation. In particular, mice lacking the enzymes required for its synthesis including phospholipase A2 (32, 33) and cyclooxygenase 1 (34) have a similar phenotype. This is consistent with the observation that PGF2 induces the rapid decline in the expression of the sterol carrier protein-2 (35) and StAR, the steroidogenic acute regulatory protein (28, 29). The inability of the PGF2 agonist, cloprostenol, to induce parturition in 20-HSD-deficient pregnant females emphasizes the importance of 20-HSD induction in the process. Together the studies support a model in which both the synthesis of progesterone is affected at term as well as its degradation and these result in sufficient reductions in progesterone levels to allow parturition.
Mechanistically, PGF2 may either induce transcription of 20-HSD in a manner that overrides the suppression due to Stat5 or may function to remove the Stat5-dependent suppression at various stages of signal transduction. In this regard it is important to note that PGF2 has been shown to induce SOCS3 (36), which could potentially inhibit PRL signaling. It has also been proposed that PGF2 may inhibit the expression of the PRL receptor (37). Alternatively it has been proposed that PGF2-induced expression of 20-HSD is mediated via the transcription factor NUR77 (26). However, the lack of reproductive defects in NUR77-deficient mice (38) would indicate that there are other, perhaps redundant, pathways. Nevertheless, the studies presented here demonstrate that the regulation of 20-HSD expression is an essential function for normal reproductive activity.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-HSD were raised in rabbits using a C-terminal peptide (amino acids 294–313, KILDGLDRNLRYFPADMFKA) of murine 20-HSD (324 amino acids) coupled to keyhole limpet hemocyanin. Total cell extracts were prepared in lysis buffer containing 0.5% Nonidet P-40 (39). Antibodies against human 3ß-HSD (type I) were a generous gift of Dr. F. Labrie (40) Proteins were separated by SDS-10% PAGE and transferred to nitrocellulose. Membranes were probed by using the 20-HSD antiserum (1:1000) or anti-ß-actin antibodies (clone C4, 1:5000, ICN Biomedicals, Inc., Aurora, OH) and visualized with the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Arlington Heights, IL).
Northern Blot Analysis
Total RNA from ovaries was isolated using RNAzol (Tel-Test, Friendswood, TX) and separated on 1% formaldehyde agarose gels. A cDNA fragment of murine 20-HSD was labeled using [-32P]dCTP and a random priming labeling kit (Amersham, Arlington Heights, IL) and used as probe. Membranes were hybridized using a rapid hybridization solution (Amersham) followed by final stringent washes in 0.5x saline sodium citrate/0.2% SDS at 65 C.
Construction of 20-HSD Targeting Vector and Generation of 20-HSD-Deficient Mice
The 20-HSD gene was isolated from a 129/SVJ mouse embryonic cell (ES) cell genomic phage library (Stratagene, La Jolla, CA) using a full-length 20-HSD cDNA expressed sequence tag clone from a mouse macrophage library (GenBank accession no. AA105098). A 14-kb SalI phage insert, identified by Southern blotting, was subcloned into pBluescriptIISK(+). For the 20-HSD targeting vector a 1.9-kb BamHI/EcoRI fragment containing exons 2 and 3 was replaced with a neomycin resistance cassette previously described (41). The sizes of the flanking genomic regions were 1.8 and 5 kb, 5' and 3' of the neo gene, respectively. A herpes simplex thymidine kinase cassette mediating negative selection was inserted in the 5'-end of the 20-HSD-neo construct. Twenty micrograms of the NotI linearized targeting vector (20 µg) were electroporated into E14 (129/Ola mouse strain) ES cells, and cells were grown under double selection as described (42). Clones were picked and expanded 7–9 d after electroporation. Correctly targeted ES-clones were identified by genomic PCR (data not shown), and a karyotypically normal clone was injected. Conditions for blastocyst injection of ES cells and breeding to generate mice homozygous for the mutated 20-HSD gene were followed as described elsewhere (43).
Genotyping of 20-HSD-Deficient Mice by PCR
Genomic DNA was amplified in 50-µl reactions using 2.5 U of AmpliTaq Gold (Perkin-Elmer Corp.) in PCR buffer with a final concentration of deoxynucleotide triphosphates at 0.2 mM and MgCl2 at 2 mM. The PCR primer consisted of P1 primer (5'-TGCAAAATCTGACAACTACTC-3') at 0.6 µM, P3 primer (5'-TCTGCTTGGAGTAATGGATC-3') at 0.2 µM, and the neomycin primer P2 (5'-AGTATCCATCATGGCTGATGC-3') at 0.6 µM. The PCR cycle profile was as follows: 1 cycle at 94 C for 10 min followed by 35 cycles at 94 C for 1 min, 55 C for 1 min, 72 C for 1 min with 5-sec autoextension in every cycle, and finally one cycle at 72 C for 10 min. PCR products were analyzed on 2% agarose gels. A 250-bp fragment indicates the presence of the wild-type allele, whereas a 800-bp fragment is amplified from the mutated allele.
Measurement of 20-HSD Enzymatic Activity
The enzymatic activity of 20-HSD was determined essentially as described previously (9, 10). Tissues were disrupted in glass Teflon homogenizers in PBS, the homogenate was centrifuged at 5000 rpm for 5 min, and the supernatants were analyzed for 20-HSD activity (conversion of progesterone as substrate to 20-OHP). The activity assay was performed in glass tubes, containing 1–50 µg protein in a 0.25-ml reaction mixture consisting of 0.01 M sodium phosphate, pH 7.4; 0.1 M sucrose; 1.0 mM MgCl2; 10–5 M progesterone; 0.02 µCi 14C-marked progesterone (PerkinElmer Life and Analytical Sciences, Inc., Boston, MA); and a reduced nicotinamide adenine dinucleotide phosphate regenerating system consisting of 1.0 mM reduced nicotinamide adenine dinucleotide phosphate, 10 mM glucose-6-phosphate, and 1.5 U glucose-6-phosphate dehydrogenase (all reagents from Sigma Chemical Co., St. Louis, MO). After an incubation period of 30 min at 37 C, reactions were stopped by adding 3 ml diethyl ether containing 1 µg progesterone and 1 µg 20-OHP/ml as carriers. The tubes were vigorously vortexed to extract the steroids into the organic phase that was subsequently separated and evaporated overnight in a chemical hood. The extracted steroids were separated on fluorescent silica gel 60 F 254 plates (Merck, Darmstadt, Germany) in a diethyl ether/chloroform 3:10 (vol/vol) system. Radioactive progesterone and 20-OHP were visualized and quantitated by phosphoimaging on a Storm 860 (Molecular Dynamics, Inc., Sunnyvale, CA). Enzymatic activities were calculated as the amount of picomoles 20-OHP generated/h/µg protein.
Treatment of Pregnant Mice with Cloprostenol (PGF2 Agonist) or Mifepristone (RU486, Progesterone Receptor Antagonist)
Pregnant mice (P15–P17) were injected sc in the upper back with 5–10 µg cloprostenol (Sigma) freshly dissolved in 200 µl PBS (two doses of 100 µl at 1000 h and 1400 h, or one dose at 1000 h). Control mice received PBS alone. Premature birth was observed usually 24 h after the second injection, but all mice were monitored for another 24 h. Mifepristone/RU486 (Sigma, 0.2 mg in 100 µl olive oil) was injected sc into pregnant mice at d 19 of gestation. Parturition occurred 24 h later.
Measurement of Serum Progesterone Levels by RIA
Serum progesterone levels were measured using a solid-phase 125I RIA kit (Diagnostic Products Corp., Los Angeles, CA). Female mice were anesthetized and bled into tubes containing 20 µl Heparin (Elkins-Sinn, Inc., Cherry Hill, NJ). Sera were recovered after centrifugation at 3500 rpm for 5 min and frozen at –80 C. Serum samples were either diluted with calibrator serum (containing no progesterone) or analyzed directly in the RIA procedure exactly as described by the manufacturer. Radioactivity counts were determined in a 
-counter (1-min counting time), and the progesterone concentrations were calculated from a standard curve (0–40 ng/ml progesterone) that was run in parallel.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Current address for R.P.P.: Institute for Biochemistry and Molecular Biology II, Heinrich-Heine-University, Duesseldorf D40225, Germany.
First Published Online October 7, 2004
1 R.P.P., S.G., and A.H. contributed equally to this work and should all be considered first authors. 
Abbreviations: ES cell, Embryonic stem cell; 20-HSD, 20-hydroxysteriod dehydrogenase; 20-OHP, 20-hydroxyprogesterone; P14, d 14 of pregnancy; PGF2, prostaglandin F2; PRL, prolactin; StAR, steroidogenic acute regulatory protein; Stat, no. 5, signal transducer and activator of transcription, no. 5.
Received for publication July 27, 2004. Accepted for publication September 27, 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-hydroxysteroid dehydrogenase: coordinate regulation of its activity, protein content, and messenger ribonucleic acid expression. Endocrinology 134:2453–2460[Abstract]
-hydroxypregn-4-en-3-one during parturition in the rat. Endocrinology 83:1181–1184[Medline]
-Hydroxysteroid dehydrogenase: a T lymphocyte-associated enzyme. J Immunol 119:1223–1229-hydroxysteroid dehydrogenase in rat thymus. Endocr J 48:557–563[Medline]
hydroxysteroid dehydrogenase in splenic lymphocytes from athymic mice by a unique lymphokine. J Immunol 126:2184–2189[Abstract]
-Hydroxysteroid dehydrogenase and reductive pathways in mouse adrenal glands in vitro. Endocrinology 82:527–534[Medline]
-hydroxysteroid dehydrogenase enzymes in immature, mature, and pregnant rats. Endocrinology 138:2886–2892-hydroxysteroid dehydrogenase: differential responses to IL-1 and IL-2. Clin Exp Immunol 70:578–584[Medline]
-hydroxysteroid dehydrogenase-positive cells. J Immunol 135:1864–1871[Abstract]
-hydroxysteroid dehydrogenase in rat corpora lutea by pharmacological blockade of pituitary prolactin secretion. Biochim Biophys Acta 187:133–143[Medline]
-induced expression of 20-hydroxysteroid dehydrogenase involves the transcription factor NUR77. J Biol Chem 275:37202–37211) induces a rapid decline in progesterone production and steroidogenic acute regulatory protein expression in isolated rat corpus luteum without altering messenger ribonucleic acid expression. Biol Reprod 61:643–650 as revealed by gene-targeted mice. Prostaglandins Other Lipid Mediat 68–69:59–69
mediates ovarian sterol carrier protein-2 expression during luteolysis. Endocrinology 136:4963–4972[Abstract]
analog induces suppressors of cytokine signaling-3 expression in the corpus luteum of the pregnant rat: a potential new mechanism in luteolysis. Endocrinology 143:3984–3993 (PGF2) and prolactin signaling: PGF2-mediated inhibition of prolactin receptor expression in the corpus luteum. Endocrinology 144:3301–3305NURSA Molecule Pages Link:
This article has been cited by other articles:
 |
|
 |
|
  R. Gonzalez-Fernandez, E. Martinez-Galisteo, F. Gaytan, J. A. Barcena, and J. E. Sanchez-Criado Changes in the Proteome of Functional and Regressing Corpus Luteum During Pregnancy and Lactation in the Rat Biol Reprod, July 1, 2008; 79(1): 100 - 114. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Roizen, M. Asada, M. Tong, H.-H. Tai, and L. J. Muglia Preterm Birth without Progesterone Withdrawal in 15-Hydroxyprostaglandin Dehydrogenase Hypomorphic Mice Mol. Endocrinol., January 1, 2008; 22(1): 105 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Hershkovitz, F. Beuschlein, S. Klammer, M. Krup, and Y. Weinstein Adrenal 20{alpha}-Hydroxysteroid Dehydrogenase in the Mouse Catabolizes Progesterone and 11-Deoxycorticosterone and Is Restricted to the X-Zone Endocrinology, March 1, 2007; 148(3): 976 - 988. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 M. B. Hapon, A. B Motta, M. Ezquer, M. Bonafede, and G. A Jahn Hypothyroidism prolongs corpus luteum function in the pregnant rat Reproduction, January 1, 2007; 133(1): 197 - 205. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Roizen and L. J. Muglia Understanding the Timing of Birth: The Continuing Challenge to Prevent Prematurity NeoReviews, March 1, 2006; 7(3): e151 - e159. [Full Text] [PDF]
|
|
|
|
|
|
R. A. Word, C. P. Landrum, B. C. Timmons, S. G. Young, and M. S. Mahendroo Transgene Insertion on Mouse Chromosome 6 Impairs Function of the Uterine Cervix and Causes Failure of Parturition Biol Reprod, November 1, 2005; 73(5): 1046 - 1056. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kawana, Y. Kawana, and D. J. Schust Female Steroid Hormones Use Signal Transducers and Activators of Transcription Protein-Mediated Pathways to Modulate the Expression of T-bet in Epithelial Cells: A Mechanism for Local Immune Regulation in the Human Reproductive Tract Mol. Endocrinol., August 1, 2005; 19(8): 2047 - 2059. [Abstract] [Full Text] [PDF]
|
|
| ||||||
