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Regular Article |
1
a Department of Internal Medicine and Endocrinology, Herlev Hospital, University of Copenhagen, Denmark
b Department of Medical Physiology, The Panum Institute, University of Copenhagen, Denmark
ABSTRACT
In the present study we have shown that the genetic expression of prostaglandin (PG)F2
receptor (R) and cyclooxygenase (COX)-2 increases in laboring rat myometrium. This finding was associated with a relatively weak contractile in vitro response (Emax) of isolated uterine strips when challenged with PGF2. Five days postpartum PGF2-R mRNA values exceeded those during labor while COX-2 mRNA was reduced to preparturient values. Maximal contractility of isolated strips stimulated with PGF2 at this time was enhanced and EC50 decreased. Oxytocin treatment of estrogen-primed nonpregnant rats down-regulated uterine contractile responsiveness to PGF2, leaving mRNA values for this receptor unchanged, whereas oxytocin receptor blockade with atosiban (an oxytocin receptor antagonist) left Emax unaltered. In contrast, atosiban treatment of pregnant rats resulted in a 2.5-fold increase in Emax and a considerably reduced EC50 during labor when compared to untreated delivering rats. The increased contractile ability was associated with a threefold increase in PGF2-R mRNA production, indicating that the regulation by atosiban of the PGF2-induced response is exerted at the genetic level. Based on the present data we suggest that 1) PGF2-R stimulation may not primarily exert a contracting role in the normally delivering myometrium, and 2) the presence of the PGF2-R system in rat myometrium may explain the apparent functional redundancy of the oxytocinergic system during the process of birth in animals lacking oxytocin or where the oxytocin receptor is blocked. In this context PGF2 receptor stimulation may, in the absence of oxytocin receptor stimulation, exert the contractile forces needed for proper propulsion of the fetus.
hormone action, oxytocin, uterus
INTRODUCTION
The transformation of the uterus from a quiescent organ during pregnancy to a highly contractile muscle at delivery is poorly understood. Thus initiation and progression of parturition and the subsequent delivery of the offspring involve several putative receptors. Prostaglandin (PG)F2 induces myometrial contractions through interaction with a plasma membrane-associated G-protein-coupled receptor, and this compound appears to play a pivotal role in parturition. Thus, mice lacking the gene encoding the PGF2receptor (PGF2-R) were unable to deliver normally at term [1], and mice having the gene for phospholipase A2 disrupted similarly failed to deliver their offspring [2]. In both cases, however, a reduction in serum progesterone induced by ovariectomy [1] or the administration of a progesterone receptor antagonist [2] secured normal delivery. The significance of PGF2 in relation to parturition has therefore been suggested to represent primarily the induction of luteolysis with subsequent withdrawal of progesterone and the initiation of birth, rather than being related to labor per se (see McCracken et al. [3] for review).
The activities of cyclooxygenases (COX) represent a rate-limiting step in the formation of PGs from arachidonic acid [4]. Cyclooxygenases exist in two isozyme forms named COX-1 and COX-2. Cyclooxygenase-1 is constitutively expressed in most tissues [5] and is considered a housekeeping enzyme with small variation in synthesis rate [6]. Cyclooxygenase-2, on the other hand, is an inducible enzyme activated by a wide range of ligands [7]. Arslan and Zingg [8] found an increase in uterine COX-2 mRNA from Day 7 of rat pregnancy till parturition. In their study, however, the relative contributions from myometrial tissue and decidua of the transcript were not determined. Furthermore, it was not ascertained at which specific point of gestation the enhanced expression occurred. At the protein level Dong et al. [9] reported an increase in COX-2 expression in laboring rat uterine tissue.
Again, because it is known that stimulation of decidual oxytocin receptors releases PGF2 [10], those observations regarding uterine COX-2 do not extend our knowledge as to whether COX-2 is expressed specifically in rat myometrial cells. As to the importance of COX-1 in the process of delivery, no change in uterine mRNA production of this enzyme was found in rat [8], cow [11], or human [12] pregnancy, leaving it less likely that this substance should play an important role during labor.
Oxytocin has long been regarded as a main mediator of labor. Thus the number of myometrial oxytocin receptors increases dramatically at term [13–15], and the oxytocin antagonist atosiban effectively inhibits contractions in human myometrium in vivo [16] and in vitro [17]. Indeed Soloff et al. [18] have suggested that the rise in oxytocin receptors in itself initiates parturition, a hypothesis that may be supported by the finding that when knockout mice have the gene encoding for oxytocin disrupted they are able to deliver normally [19, 20]. As suggested by Nishimori et al. [19], another plausible explanation of this enigma might be that ligands other than oxytocin replace this hormone at the oxytocin receptor site to ensure proper myometrial contractions. We were, however, not able to detect any change in fetal mortality or gestational length following oxytocin receptor blockade in delivering rats [13].
The purpose of the present study was 1) to examine whether COX-2 and PGF2-R are variably expressed in rat myometrium across pregnancy, 2) to relate the expression of COX-2 and PGF2-R to myometrial contractility, and 3) to test the hypothesis that the existence of the prostaglandinergic system explains the apparent functional redundancy of oxytocin in the process of parturition. We have measured the concentrations of COX-2 mRNA and PGF2-R mRNA in rat myometrium throughout pregnancy. We have further examined the effects of oxytocin infusions in nonpregnant rats and oxytocin receptor blockade in pregnant rats on myometrial responsiveness to PGF2 and PGF2-R mRNA levels.
MATERIALS AND METHODS
Animals
Animals were maintained under controlled conditions in the Panum Institute Animal House. Food and water were freely available. Both pregnant rats and randomly cycling rats were used for the experiments.
Individual female rats (200–220 g) were placed in a cage with a male rat for 24 h. The appearance of a vaginal plug at the end of the period was taken as a sign that copulation had taken place. Some rats were left untreated and at Days 7, 14, and 21 of pregnancy, as well as during labor or 5 days postpartum when they were anesthetized with CO2 and decapitated. Labor was defined as the point of time following delivery of the first pup [13]. Some rats from the labor group were treated with the oxytocin receptor antagonists atosiban (Ferring Pharmaceuticals, Malmö, Sweden) from Day 18 of pregnancy when they were anesthetized with a mixture of Dormicum (1.25 mg, Hoffman-La-Roche, Basel, Switzerland) and Hypnorm (0.4 ml, Janssen Chimica, Geel, Belgium). An incision in the lower abdominal wall was made and an osmotic minipump (Alzet, Palo Alto, CA) was introduced in the abdominal cavity. The pumps had pumping rates of 1 µl/h and were filled with atosiban at a concentration of 5 mg/ml. This atosiban dosage was chosen because it has previously been shown to affect myometrial oxytocin receptor binding in rats [21]. When corrected for body weight, this dosage was also comparable with the minimal dosage able to totally inhibit human premature uterine contractions [22]. Groups of normally cycling rats were by means of similar osmotic minipumps treated for 5 days with oxytocin (0.5 µg/h, Ferring Pharmaceuticals) or atosiban (5 µg/h). Two days prior to decapitation these nonpregnant rats received an intramuscular injection of 75 µg estradiol-benzoate.
To ensure that the insertion of osmotic minipumps in itself did not influence subsequent myometrial responsiveness to PGF2 a group of pregnant rats had minipumps with saline inserted from Day 18 of pregnancy. On the day of delivery PGF2-induced uterine contractility of those animals was compared with contractile responsiveness from untreated delivering rats. Neither Emax (P = 0.863) nor EC50 (defined below) (P = 0.418) were altered by the operation.
Primers and Construction of Internal mRNA Standard
The primers used for COX-2 mRNA detection were: sense primer: 5' ACG CCA CCC CAA ACA CAG TA 3' (nucleotides 327–346); antisense primer: 5' CCC AGG TCC TCG CTT CTG A 3' (nucleotides 703–721). The primers used for PGF2-R detection were: sense primer: 5' ACG GCG TTT ATC TCC ACA AC 3' (nucleotides 113–122); antisense primer: 5' CCG ATG CAC CTC TCA ATG 3' (nucleotides 506–523).
Basic local alignment search tool (Blast) [23] was used to search all nonredundant databases (GenBank + EMBL + DDBJ + PDB) for sequence homology. No homology with any known product other than the actual receptors was found.
The amplified DNA fragments consisted of 413 base pairs (bp) (COX-2) and 428 bp (PGF2-R). The exact identities of the PCR products were confirmed by sequencing [24].
Using a PCR-MIMIC construction kit (Clontech, Palo Alto, CA), a 240-bp internal standard DNA was constructed [25]. The internal standard RNA was constructed mainly as described by Faure et al. [26]. A composite primer, comprising 37 nucleotides of bacteriophage T7 RNA-polymerase promoter followed by the sequence of our usual sense primer, was used for polymerase chain reaction (PCR) amplification of the 240-bp internal standard DNA PCR product. The resulting 277-bp product was reamplified using our antisense primer and a primer consisting of the initial 23 nucleotides of the T7 RNA-polymerase promoter region. Following HPLC purification, the reamplified product was used for production of RNA by in vitro transcription (Riboprobe, Promega, Madison, WI). The resulting RNA standard was quantitated by UV detection at 260 nM (Gene-quant, Pharmacia, Stockholm, Sweden). Subsequently, the RNA standard underwent reverse transcription in order to verify that the resulting product was indistinguishable from the internal DNA standard.
Reverse Transcription Polymerase Chain Reaction
Polyadenylated mRNA from homogenized rat myometrium was isolated using the PolyATract system 1000 (Promega, Madison, WI) [25]. Reverse transcription (RT) was performed in a mixture consisting of 250 µM dNTP, 40 U mouse mammary leukemia virus RT (Promega), 31.2 U RNA-guard, 200 pmol antisense primer, and 5 µl internal standard RNA in Promega RT buffer. Incubations were carried out for 60 min at 37°C and the resulting cDNA used immediately or stored at -80°C.
Polymerase chain reaction was carried out with 5 µl cDNA, 37.5 µM of each dNTP, 1.0 U Taq polymerase (Pharmacia), and 40 pmol of both sense primer and antisense primer in PCR buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, pH 9.0). Amplification took place in a Perkin Elmer model 460 thermocycler. Cycling parameters were 95°C for 2 min followed by 27 cycles consisting of 1.5 min at 94°C, 56°C for 45 sec, and 70°C for 2 min. The PCR products were used immediately or stored at -80°C.
Quantitation of PCR products was carried out by means of HPLC using a TSK DEAE-NPR column [25]. Following chromatography, PCR products were UV-detected at 254 nm. The areas of the 240-bp PCR products of the internal standards represented 0.011 (COX-2) and 0.130 (PGF2-R) amol, respectively. Hence, the amount of the PCR products could be quantitated relative to these standards and they were finally related to tissue wet weight. The average value of the uterine horns of each animal was calculated to represent the amount of specific mRNA.
In Vitro Examination of the Contractile Force of Myometrial Strips
One uterine horn was opened longitudinally. A 5-mm-long middle segment covering the full circumference of the horn was isolated, and the segment was subsequently mounted in an isometric myograph connected to a Grass force transducer. The resting tension was adjusted to 1.5 g. This level was found empirically and secured such that the tissue was sufficiently stretched during the time of the experiment, and at the same time overstretching was avoided. The strip was placed in an organ bath containing 7 ml of Krebs-Ringer buffer (NaCl 119 mM, KCl 4.6 mM, NaHCO3 20 mM, CaCl2 1.5 mM, NaH2PO4 1.2 mM, glucose 11 mM, pH 7.4) and allowed to rest for 30 min. Every 10 min during this period the buffer was refreshed. Before stimulation with PGF2 the specimen was further washed five times. The temperature was kept at 30°C and the solution constantly aerated with 5% CO2 in O2. The choice of a temperature lower than the normal physiological temperature of 37°C was chosen to eliminate spontaneous contractions.
Contractile responsiveness to PGF2 (Sigma Chemical Co., St. Louis, MO) was measured using doses over the range of 5.7 x 10-10–4.7 x 10-5 M. The addition of PGF2 was done in a cumulative manner in order to obtain increasing concentrations. The response to each dose was allowed to reach a plateau before the addition of the next. Ratios of connective tissue:smooth muscle within the specimens and the thickness of the strips vary across pregnancy and between individual rats of the same gestational ages. Therefore different strips may contain different quantities of smooth muscle, which in itself may alter the contractile response upon agonist stimulation. To overcome this problem all responses were finally expressed as a percentage of the contraction induced by modified Krebs-Ringer buffer containing 50 mM KCl (and 73 mM NaCl) and were plotted against the logarithm to the agonist concentration.
Data Analysis
A computer program (Fig. P., Biosoft, Cambridge, UK) was used for data analysis of myometrial responsiveness to PGF2. A four-parameter nonlinear curve-fitting model was used to evaluate maximal contraction (Emax), and the PGF2 concentration giving half this effect (EC50) using the equation: E = Emin + (Emax- Emin)/{1 + [([PGF2]/EC50)-P]}.
The computer program SigmaStat for windows version 2.0 (SPSS Inc., Chicago, IL) was used for statistical analysis. One-way analysis of variance was used to compare multiple groups. In case of unequal variances between groups data were transformed by means of the square root function to obtain equality. Significant effects among individual means were subsequently separated using posthoc tests for multiple comparison (Student Neuman-Keuls method). P < 0.05 was considered statistically significant. Results are presented as means ± SEM unless otherwise stated.
RESULTS
Messenger RNA
The expression of the PGF2-R was highest in laboring rats pretreated with atosiban (Fig. 1, P < 0.001, one-way ANOVA). Values from nontreated animals in labor showed a fourfold increase compared to Day 21 of pregnancy (0.373 ± 0.087 vs. 0.085 ± 0.044 amol/mg wet tissue, P < 0.05, Student Neuman-Keul). Five days postpartum, the level of PGF2-R mRNA was further increased twofold when compared to labor (0.790 ± 0.065 amol/mg wet tissue, P < 0.05, Student Neuman-Keul). No differences between Days 7, 14, and 21 of gestation were found. When rats in labor were pretreated with atosiban from Day 18 of pregnancy, PGF2-R mRNA was increased threefold as compared to nontreated rats in labor (1.009 ± 0.337 amol/mg wet tissue, P < 0.05, Student Neuman-Keul).
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The PGF2-R mRNA values from nonpregnant rats were in general of a magnitude comparable to preparturient levels (Fig. 1). No statistically significant difference was found between differently treated groups (P = 0.222, one-way ANOVA).
The highest concentration of COX-2 mRNA was found during labor (0.207 ± 0.048 amol/mg wet tissue, Fig. 2, P < 0.001, one-way ANOVA). This concentration was 12-fold higher than on Day 21 of pregnancy (0.017 ± 0.006 amol/mg wet tissue, P < 0.05, Student Neuman-Keul) and 14-fold higher than 5 days postpartum (0.015 ± 0.001 amol/mg wet tissue, P < 0.05, Student Neuman-Keul). Prepartum concentrations were not different from the 5 days postpartum value.
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Contractile Responsiveness of Isolated Uterine Strips Challenged with PGF2
Stimulation of isolated pregnant uterine strips with PGF2 revealed sigmoid dose-response curves (Fig. 3). The Emax-values varied across pregnancy (Table 1, P < 0.001, one-way ANOVA), with the lowest value obtained during labor and the highest 5 days postpartum. A 27% decrease during labor was seen when compared to Day 21 of pregnancy (P < 0.05, Student Neuman-Keul, Table 1). Five days postpartum Emax increased 66% when compared to the labor value (P < 0.05, Student Neuman-Keul, Table 1). When animals in the labor group were pretreated with atosiban from Day 18 of pregnancy, Emax increased 2.5-fold in comparison with control rats in labor (Table 1, P < 0.05, Student Neuman-Keul). The EC50-values were significantly different across pregnancy (Table 1, P = 0.016, one-way ANOVA). A ninefold increase in this parameter was seen when rats in labor were compared to rats at Day 21 of pregnancy, indicating a considerable decrease in uterine sensitivity to PGF2 during labor (Table 1, P < 0.05, Student Neuman-Keul). Five days postpartum EC50 decreased more than a thousandfold compared to the labor value (Table 1, P < 0.05, Student Neuman-Keul). In addition to enhancing the maximal contractile ability, atosiban treatment augmented uterine sensitivity upon challenge with PGF2 as indicated by a 10-fold decrease in EC50 (Table 1, P < 0.05, Student Neuman-Keul).
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Stimulation of isolated nonpregnant uterine strips with PGF2 revealed sigmoid dose-response curves with significantly different Emax-values (Fig. 4, Table 2, P < 0.001, one-way ANOVA). Posthoc analysis revealed a significant decrease in Emax of uteri from oxytocin-treated rats compared to those treated with estrogen alone and those treated with atosiban (Table 2, P < 0.05, Student Neuman-Keul). The EC50 values were not statistically different between the three groups.
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DISCUSSION
In the present study we have shown that myometrial COX-2 and PGF2-R mRNA increase during rat pregnancy, indicating that the myometrium is under significant influence of PGF2-R stimulation at term labor. Five days postpartum PGF2-R mRNA increased further while COX-2 mRNA was reduced to preparturient values. In spite of the increasing mRNA levels, PGF2-induced myometrial contractility was curbed during labor. However, when pregnant rats were pretreated with atosiban, the delivering myometrium became more sensitive to PGF2 stimulation as shown by an increased Emax and a decreased EC50. This effect of atosiban was accompanied by an augmented genetic expression of PGF2-R. Finally oxytocin treatment decreased PGF2-induced maximal contractility in nonpregnant estrogen-primed rats, whereas oxytocin receptor blockade with atosiban left Emax unaltered.
Lewis et al. [27] previously showed that acetylsalicylic acid delays parturition, and subsequent studies have unequivocally proven that prostaglandins are essential mediators of parturition. Thus Lim et al. [28] showed that COX-2 deficiency in mice produces multiple female reproductive failures and Sugimoto et al. [29] demonstrated failure of parturition in mice lacking the PGF2-R. Parturition was, however, secured if ovariectomy leading to a subsequent fall in plasma progesterone was performed and the reproductive defect therefore may occur at the ovarian level, indicating that PGF2 may not primarily control labor per se. The present study supports this suggestion because PGF2-induced myometrial contractility was actually more pronounced before than during labor. In view of the high level of myometrial PGF2-R and COX-2 mRNA the relatively weak in vitro response to PGF2 is surprising. Nevertheless, the finding is in accordance with similar observations in human pregnant myometrium [30]. The formation of gap junctions between the myometrial cells seems essential for the synchronization and progression of myometrial activity [31]. Mackenzie and Garfield [32] found estrogen to increase the number of myometrial gap junctions. Concomitant administration of the COX inhibitor indomethacin enhanced this effect, suggesting that PGs may suppress gap junction formation and in this way contribute to the feeble contractile response upon PGF2 challenge. Because indomethacin is a nonspecific inhibitor of COX with only modest influence on COX-2, this effect may, however, primarily be due to COX-1 inhibition.
The apparent discrepancy between Emax and PGF2-R mRNA during labor suggests that the transcript is not processed further upstream to produce functional receptors. This phenomenon may be related to translational or post-translational processes, or it may reflect the presence of uncoupled receptors. The PGF2-R is functionally coupled to the activation of phospholipase C initiating the formation of inositol triphosphate with a subsequent mobilization of intracellular calcium [33]. The pertussis insensitive G-protein Gq bridges the receptor and phospholipase C [34]. The upregulation of Gq in term myometrium [35, 36] suggests a strong coupling between PGF2-R and its intracellular signaling cascade at this point of gestation. It is therefore more likely that translational or post-translational receptor modifications explain the discrepancy. In general prolonged receptor occupancy by a given agonist is known to reduce the response to subsequent stimulation [37], a process known as desensitization. The myometrium in labor may alternatively be in a desensitized state due to in vivo exposure of a high level of PGF2 [38], and in this case the enhanced mRNA level may express a constant regeneration of the receptor population. Actually, in rat corpus luteum PGF2-R mRNA increased after 48 h of PGF2 challenge, indicating, in accordance with the present data, that in these cells desensitization does not appear at the level of genetic expression [39].
In the present study we suggest that the myometrial PGF2-R play a substituting role for another contractile hormone system, namely the oxytocinergic. Thus, in recent years an apparent contradiction regarding the oxytocin receptor system has evolved. Despite the concordantly described [13–15, 40] increase in myometrial oxytocin receptors at delivery, mice unable to synthesize oxytocin [19, 20] and rats having oxytocin receptors blocked by atosiban [13] deliver normally. Our present results show that oxytocin receptor blockade upregulates the uterine contractile response to PGF2. This is indicated both by the augmented maximal contractile ability of PGF2 under these circumstances and by the fact that a much lower concentration of PGF2 is needed to elicit a given response as indicated by the reduced EC50. The effect of oxytocin receptor blockade appears to be mediated by long-term exposure to atosiban since Fejgin et al. [41] found a maintained uterine response to PGF2 following short-term treatment with their antagonist. As implied by the increased PGF2-R mRNA production following atosiban exposure, the regulation appears partly to take place at the genetic level.
Stimulation of cAMP-coupled receptors such as hCG and FSH receptors induces PGF2-R mRNA production [42]. Oxytocin receptor stimulation does, however, lead to inositol phosphate formation [43] and does not in itself alter the intracellular cAMP level. Despite this fact, it is possible that oxytocin receptor blockade by atosiban shifts the balance between cAMP- and inositol phosphate-promoting receptors, thereby inducing PGF2-R expression. Because both the PGF2-R and the oxytocin receptor are linked to phospholipase C-dependent second messengers, it is alternatively suggested that oxytocin receptor blockade make available a more substantial part of their common intracellular signaling cascade to the PGF2-R.
As mentioned, myometrial oxytocin receptors increase at term and an additional rise in rat [44] and human [45] plasma oxytocin occurs during labor. These observations indicate that a high oxytocinergic tone is naturally imposed on the myometrium during delivery. In this context, the above effect of oxytocin receptor blockade on the PGF2-induced contractility suggests that oxytocin receptor stimulation by oxytocin may blunt myometrial contractile responsiveness to PGF2. Due to the naturally occurring high concentration of oxytocin during labor, it is unlikely that any experimental oxytocin treatment of pregnant rats would reveal an additional effect on the PGF2 system. As a consequence, we have instead shown that oxytocin treatment downregulates the uterine PGF2 responsiveness in nonpregnant estrogen-primed rats known to have only trace amounts of circulating oxytocin [46]. The high estrogen level obtained following an injection with this particular compound characterizes term pregnancy [47]. Gordan et al. [48] showed that estradiol in itself attenuated nonpregnant uterine response to PGF2. However, in our study, all nonpregnant rats received estradiol treatment, and we were thus able to identify an attenuation specifically mediated by oxytocin treatment. The fact that atosiban treatment of nonpregnant estrogen-primed rats was unable to alter myometrial contractility when challenged with PGF2 can be explained by the low levels of plasma oxytocin in these animals and therefore further adds to the hypothesis that oxytocin moderates PGF2-R function. The fact that mRNA values in these experiments were unaltered despite reduced contractile responses suggests that the heterologous desensitization of the PGF2-R system upon oxytocin exposure in nonpregnant rats occurs above the level of genetic expression.
The high level of PGF2-R mRNA compared to that of COX-2 mRNA 5 days postpartum indicates that the influence of PGF2 on the myometrium at this time is controlled by the amount of receptors rather than by the level of ligand. Furthermore, the associated immense increase in maximal PGF2-induced contractility at this point of reproduction suggests a particular role in controlling postpartum hemorrhage as shown by Takagi et al. [49]. Metabolites of PGF2 are low postpartum [50], and thus the forceful contractile ability may reflect sensitization of the myometrium to PGF2 as opposed to the circumstance during labor.
In summary we have in the present study shown that a high level of PGF2-R and COX-2 mRNA is present in rat myometrium in labor, although this finding is associated with a relatively weak in vitro contractile response upon PGF2 challenge. Therefore, PGF2-R stimulation appears not to maintain a primarily contracting role in normal delivery. We have further shown that oxytocin receptor stimulation in nonpregnant animals decreases the contractile response to PGF2, whereas this response is considerably increased following oxytocin receptor blockade in parturient rats. We therefore suggest that the presence of the prostaglandinergic system may explain the apparent functional redundancy of the oxytocinergic system in the process of birth in animals lacking oxytocin or when the oxytocin receptor is blocked.
ACKNOWLEDGMENTS
We thank technicians Gurli Habekost and Jakob Utzon-Frank for their helpful assistance.
FOOTNOTES
First decision: 28 February 2000.
1 The present study was supported by The Danish Biotechnology Program, The Danish Medical Research Council, The Novo Nordisk Foundation, Jacob Madsen & Hustru Olga Madsens Foundation, The Danish Medical Association Research Fund, Foundation Idella, The Dagmar Marshall Foundation, Direktør E. Danielsen og Hustrus Foundation, and Ove Villiam Buhl Olesen & ægtefælle Edith Buhl Olesens Foundation. 
2 Correspondence: Thomas Engstrøm, Department of Medical Physiology 12.2.39, The Panum Institute, University of Copenhagen, Copenhagen, Denmark. FAX: 45 35327537; engstrom{at}mfi.ku.dk
Accepted: June 15, 2000.
Received: January 19, 2000.
REFERENCES
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  A. Mueller, T. Maltaris, J. Siemer, H. Binder, I. Hoffmann, M. W. Beckmann, and R. Dittrich Uterine contractility in response to different prostaglandins: results from extracorporeally perfused non-pregnant swine uteri Hum. Reprod., August 1, 2006; 21(8): 2000 - 2005. [Abstract] [Full Text] [PDF]
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O.N. Richter, C. Bartz, J. Dowaji, M. Kupka, J. Reinsberg, U. Ulrich, and W. Rath Contractile reactivity of human myometrium in isolated non-pregnant uteri Hum. Reprod., January 1, 2006; 21(1): 36 - 45. [Abstract] [Full Text] [PDF]
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