Progesterone reduces erectile dysfunction in sleep-deprived spontaneously hypertensive rats

Background

The rate-limiting step in prostaglandin (PG) biosynthesis is catalyzed by phospholipase A2 (PLA2) enzymes which hydrolyze arachidonic acid from membrane phospholipids. Despite their importance in uterine PG production, little is known concerning the specific PLA2 enzymes that regulate arachidonic acid liberation in the uterine endometrium. The objectives of this study were to evaluate the expression and activities of calcium-independent Group VI and Group IVC PLA2 (PLA2G6 and PLA2G4C) and calcium-dependent Group IVA PLA2 (PLA2G4A) enzymes in the regulation of bovine uterine endometrial epithelial cell PG production.

Methods

Bovine endometrial epithelial cells in culture were treated with oxytocin, interferon-tau and the PLA2G6 inhibitor bromoenol lactone, alone and in combination. Concentrations of PGF2alpha and PGE2 released into the medium were analyzed. Western blot analysis was performed on cellular protein to determine the effects of treatments on expression of PLA2G4A, PLA2G6 and PLA2G4C. Group-specific PLA2 activity assays were performed on cell lysates following treatment with oxytocin, interferon-tau or vehicle (control), alone and in combination. To further evaluate the role of specific PLA2 enzymes in uterine cell PG biosynthesis, cells were transfected with cDNAs encoding human PLA2G6 and PLA24C, treated as described above and PG assays performed.

Results

Constitutive cell production of PGF2alpha was about two-fold higher than PGE2. Oxytocin stimulated production of both PGs but the increase of PGF2alpha was significantly greater. Interferon-tau diminished oxytocin stimulation of both PGs. The PLA2G6 inhibitor, bromoenol lactone, abolished oxytocin-stimulated production of PGF2alpha. Treatments had little effect on PLA2G4A protein expression. In contrast, oxytocin enhanced expression of PLA2G6 and this effect was diminished in the presence of interferon-tau. Expression of PLA2G4C was barely detectable in control and oxytocin treated cells but it was enhanced in cells treated with interferon-tau. Oxytocin stimulated PLA2 activity in assays designed to evaluate PLA2G6 activity and interferon-tau inhibited this response. In assays designed to measure PLA2G4C activity, only interferon-tau was stimulatory. Cells overexpressing PLA2G6 produced similar quantities of the two PGs and these values were significantly higher than PG production by non-transfected cells. Oxytocin stimulated production of both PGs and this response was inhibited by interferon-tau. Bromoenol lactone inhibited oxtocin stimulation of PGF2alpha production but stimulated PGE2 production, both in the absence and presence of oxytocin. Cells over-expressing PLA2G4C produced more PGE2 than PGF2alpha and interferon-tau stimulated PGE2 production.

Conclusion

Results from these studies indicate that oxytocin stimulation of uterine PGF2alpha production is mediated, at least in part, by up-regulation of PLA2G6 expression and activity. In addition to its known inhibitory effect on oxytocin receptor expression, interferon-tau represses oxytocin-stimulated PLA2G6 expression and activity and this contributes to diminished PGF2alpha production. Furthermore, endometrial cell PGE2 biosynthesis was associated with PLA2G4C expression and activity and interferon-tau was stimulatory to this process.     

Effect of oestrous cycle and early pregnancy on uterine production and expression of immune regulatory factors in gilts

This study was undertaken to characterize uterine immune factors involved in the establishment of pregnancy in gilts. Thirty crossbred Yorkshire-Landrace gilts of similar age and weight were observed twice a day for oestrous behaviour with intact boars. On the day of first standing oestrus (Day 0) and 12h later, 15 gilts were inseminated with pooled semen from Duroc boars of proven fertility. Pregnant gilts were slaughtered either on Days 10, 15 or 25 of gestation (n=5 per day). The other 15 gilts were not inseminated and were slaughtered on either Days 0, 10 or 15 of the oestrous cycle (n=5 per day). Immediately after slaughter, endometrial tissue samples from the mesometrial side were removed for gene expression using RNase protection assay and in situ hybridization methodologies. The other uterine horn was flushed with 20 ml of PBS to collect the uterine fluid. In pregnant gilts, endometrial interleukin (IL)-6 mRNA expression was higher on Day 15 than on Days 10 and 25 (P<0.01 and P<0.1, respectively). On Day 15, IL-6 expression was also significantly higher (P<0.01) in pregnant gilts than in cyclic gilts. In both pregnant and cyclic gilts, transforming growth factor (TGF)-beta2 in uterine fluid was significantly higher (P<0.0001) on Day 15 than on Day 10. At the gene expression level, TGF-beta2 also increased between Days 10 and 15 in both cyclic and pregnant gilts but differences were not significant. On Day 15, concentrations of interferon-gamma and prostaglandin E(2) (PGE(2)) in uterine fluid were markedly higher (P<0.001) in pregnant gilts than in cyclic gilts, whereas the total amount of TGF-beta2 in uterine fluid and its endometrial expression were approximately 70% higher although this increase was not significant. Finally, tumour-necrosis factor-alpha and granulocyte-macrophage/colony-stimulating factor mRNA expressions were undetectable in all endometrial samples. In conclusion, production and/or expression of uterine TGF-beta2, IL-6 and PGE(2) increased during the embryonic attachment period and are coincidental with embryonic interferon-gamma production.     

Use of a novel and highly selective oxytocin receptor antagonist to characterize uterine contractions in the rat

Spontaneous and induced uterine contractions in the rat were found to be inhibited by a novel and selective oxytocin receptor antagonist GSK221149A (3R,6R)-3-Indan-2-yl-1-[(1R)-1-(2-methyl-1,3-oxazol-4-yl)-2-morpholin-4-yl-2-oxoethyl]-6-[(1S)-1-methylpropyl]-2,5-piperazinedione. GSK221149A displayed nanomolar affinity (K(i) = 0.65 nM) for human recombinant oxytocin receptors with >1,400-fold selectivity over human V1a, V1b, and V2 receptors. GSK221149A had similar affinity (K(i) = 4.1 nM) and selectivity for native oxytocin receptors from rat and produced a functional, competitive block of oxytocin-induced contractions in isolated rat myometrial strips with a pA(2) value of 8.18. Intravenous administration of GSK221149A produced a dose-dependent decrease in oxytocin-induced uterine contractions in anesthetized rats with an ID(50) = 0.27 +/- 0.60 mg/kg (corresponding plasma concentrations were 88 ng/ml). Oral administration of GSK221149A (5 mg/kg) was effective in inhibiting oxytocin-induced uterine contractions after single and multiple (4-day) dosing. Spontaneous uterine contractions in late-term pregnant rats (19-21 days gestation) were significantly reduced by intravenous administration of 0.3 mg/kg of GSK221149A. These results provide further evidence that selective oxytocin receptor antagonism may offer an effective treatment for preterm labor.     

Placental Transfer of a Fully Human IgG2 Monoclonal Antibody in the Cynomolgus Monkey,Rat, and Rabbit: A Comparative Assessment from during Organogenesis to Late Gestation

Understanding species differences in the placental transfer of monoclonal antibodies is important to inform species selection for nonclinical safety assessment, interpret embryo‐fetal changes observed in these studies, and extrapolate their human relevance. Data presented here for a fully human immunoglobulin G2 monoclonal antibody (IgG2X) revealed that, during organogenesis, in both the cynomolgus monkey (gestation day 35 [gd35]) and the rat (gd10) the extent of IgG2X placental transfer (approximately 0.5% maternal plasma concentration, MPC) was similar to the limited published human data for endogenous IgG. At this early gestational stage, IgG2X placental transfer was approximately 6‐fold higher in the rabbit (gd10). By the end of organogenesis, rat embryonic plasma concentrations (gd16) exceeded those in the cynomolgus monkey (gd50) by approximately 3‐fold. These data suggest that relative to the cynomolgus monkey, the rabbit (and to a lesser extent the rat) may overestimate potential harmful effects to the human embryo during this critical period of development. Beyond organogenesis, fetal IgG2X plasma concentrations increased approximately 10‐fold early in the second trimester (gd50–70) in the cynomolgus monkey and remained relatively unchanged thereafter (at approximately 5% MPC). Late gestational assessment was precluded in rabbits due to immunogenicity, but in rats, fetal IgG2X plasma concentrations increased more than 6‐fold from gd16 to gd21 (reaching approximately 15% MPC). In rats, maternal exposure consistent with that achieved by ICH S6(R1) high‐dose selection criteria resulted in embryonic plasma concentrations, reaching pharmacologically relevant levels during organogenesis. Furthermore, dose proportional exposure in both mothers and embryos indicated that this was unlikely to occur at the lower therapeutic dose levels used in humans     

ERK-mediated uterine artery contraction: role of thick and thin filament regulatory pathways

We have demonstrated that extracellular signal-regulated kinase (ERK) plays an important role in the regulation of uterine artery contraction. The present study tested the hypothesis that ERK regulates thick and thin filament regulatory pathways in the uterine artery. Isometric tension, intracellular free Ca2+ concentration ([Ca2+]i), and 20-kDa myosin light chain (LC20) phosphorylation were measured simultaneously in uterine arteries isolated from near-term (140 days gestation) pregnant sheep. Phenylephrine produced time-dependent increases in [Ca2+]i and LC20 phosphorylation that preceded the contraction, which were inhibited by the MEK (ERK) inhibitor PD-098059. In addition, PD-098059 decreased the intercept of the regression line of LC20 phosphorylation vs. [Ca2+]i but increased the rate of tension development vs. LC20 phosphorylation. In contrast to phenylephrine, phorbol 12,13-bibutyrate (PDBu) produced contractions without changing [Ca2+]i or LC20 phosphorylation. PD-098059 potentiated PDBu-induced contractions without affecting [Ca2+]i and LC20 phosphorylation. PDBu produced time-dependent increases in phosphorylation of p42 and p44 ERK and ERK-dependent phosphorylation of caldesmon at Ser789 in the uterine artery. PD-098059 blocked PDBu-mediated phosphorylation of p42 and p44 ERK and caldesmon. The results indicate that ERK may regulate force by a dual regulation of thick and thin filaments in uterine artery smooth muscle. ERK potentiates the thick filament regulatory pathway by enhancing LC20 phosphorylation via increases in [Ca2+]i and Ca2+ sensitivity of LC20 phosphorylation. In contrast, ERK attenuates the thin filament regulatory pathway and suppresses contractions independent of changes in LC20 phosphorylation in the uterine artery.     

Adaptation of uterine artery thick- and thin-filament regulatory pathways to pregnancy

Little is known about the adaptation of uterine artery smooth muscle contractile mechanisms to pregnancy. The present study tested the hypothesis that pregnancy differentially regulates thick- and thin-filament regulatory pathways in uterine arteries. Isometric tension, intracellular free Ca(2+) concentration, and phosphorylation of 20-kDa myosin light chain (MLC(20)) were measured simultaneously in uterine arteries isolated from nonpregnant and near-term (140 days gestation) pregnant sheep. Phenylephrine-mediated intracellular free Ca(2+) concentration, MLC(20) phosphorylation, and contraction tension were significantly increased in uterine arteries of pregnant compared with nonpregnant animals. In contrast, phenylephrine-mediated Ca(2+) sensitivity of MLC(20) phosphorylation was decreased in the uterine arteries of pregnant sheep. Simultaneous measurement of phenylephrine-stimulated tension and MLC(20) phosphorylation in the same tissue indicated a decrease in MLC(20) phosphorylation-independent contractions in the uterine arteries of pregnant sheep. In addition, activation of PKC produced significantly lower sustained contractions in uterine arteries of pregnant compared with nonpregnant animals in the absence of changes in MLC(20) phosphorylation levels in either vessels. In uterine arteries of nonpregnant sheep, the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase inhibitor PD-098059 significantly increased phenylephrine-mediated, MLC(20) phosphorylation-independent contractions. The results suggest that in uterine arteries, pregnancy upregulates alpha(1)-adrenoceptor-mediated Ca(2+) mobilization and MLC(20) phosphorylation. In contrast, pregnancy downregulates the Ca(2+) sensitivity of myofilaments, which is mediated by both thick- and thin-filament pathways.     

Effects of mepacrine on uterine contractile responses

Mepacrine is a potent inhibitor of uterine contractile responses in vitro. Pretreatment of isolated rat uterine horns with mepacrine (1.3 X 10(-4)M) for periods of time ranging from 15 s to 5 min prior to the addition of carbachol (1.0 X 10(-4)M) showed that mepacrine could significantly reduce carbachol-induced uterine contractile responses within 15 s of exposure. The maximal inhibitory effects of mepacrine on uterine contractile responses were observed within 2 min of mepacrine treatment. A dose-response study related to the effect of increasing concentrations of mepacrine (7.5 X 10(-6) to 1.3 X 10(-4)M) on carbachol-induced (1 X 10(-4)M) uterine contractions revealed that a dose of 3.1 X 10(-5)M mepacrine reduced the carbachol-induced contraction by 50%. A dose of 7.8 X 10(-5)M mepacrine produced the maximal inhibitory effect on the carbachol-induced uterine contractions. Two doses of mepacrine (3.1 X 10(-5) and 1.3 X 10(-4)M) significantly reduced maximal contractile responses and shifted contractile dose-response curves of carbachol, oxytocin, prostaglandin F2 alpha, and BaCl2 to the right. Based on the nonselective inhibition by mepacrine of contractile responses induced by different uterotonic agents, these results suggest that mepacrine cannot be used to characterize the role of phospholipase in regulating the actions of hormones in uterine tissue.     

Expression level of ABCG2 in the placenta decreases from the mid stage to the end of gestation

The aim of this study was to elucidate the expression pattern of ABCG2 in the placenta from the mid stage to the end of gestation. rABCG2 expression was investigated in rats on the 14th gestation day (gd) and the 20th gd. Expression of the rABCG2 gene and expression of rABCG2 protein in the placenta were detected on gd 14 by RT-PCR and Western blot analysis respectively. The expression level of rABCG2 on gd 20 was less than that on gd 14. We investigated whether progesterone, secreted from the placenta, regulates the expression of ABCG2 in BeWo cells. Expression levels of the ABCG2 gene and protein in BeWo cells were decreased by progesterone treatment. We conclude that progesterone plays a role in reduction in the expression level of ABCG2 in the placenta with the advance of gestation from the mid stage to the end of gestation.     

Delayed uterine fluid clearance and reduced uterine perfusion in bitches with endometrial hyperplasia and clinical management with postmating antibiotic

In many species a transient uterine inflammatory response follows mating and is proposed to remove excess spermatozoa, bacteria, and other contaminants from the uterus. Similar events have been documented in the bitch involving increased uterine contractions, polymorphonuclear neutrophil influx and uterine artery vasodilation. Some healthy bitches with endometrial hyperplasia have increased numbers of uterine luminal polymorphonuclear neutrophils after mating and reduced fertility; it is purported that this represents a presumed postmating endometritis. This study used B-mode and Doppler ultrasonography at the time of mating to measure uterine contractions, clearance of ejaculated fluid, and uterine artery velocity in normal bitches and those with endometrial hyperplasia. Mating resulted in an increase in the number of uterine contractions, although fewer mating-induced contractions were noted in bitches with endometrial hyperplasia. Interestingly, uterine fluid cleared significantly more slowly after mating from the bitches with endometrial hyperplasia than the normal bitches (P = 0.01). In a further study, Doppler ultrasonography showed that in normal bitches there was a significant increase in uterine artery blood velocity (P = 0.04) and a decrease in the resistance index after mating (P = 0.04), indicating vasodilation. In bitches with endometrial hyperplasia the baseline resistance index was significantly higher than normal bitches (P = 0.05), and furthermore, although there was a significant decrease in resistance index after mating, in the bitches with endometrial hyperplasia this was of a smaller magnitude that in normal bitches. These findings indicate lower baseline uterine perfusion, and a blunted vasodilation response to mating in bitches with endometrial hyperplasia. Short-duration postmating administration of systemic antibiotic increased pregnancy rates in bitches with endometrial hyperplasia (P < 0.01). Litter sizes in bitches with endometrial hyperplasia were lower than those of normal bitches both before and after treatment with postmating antibiotic (P = 0.04 and < 0.01, respectively). Mating-induced endometritis in bitches with endometrial hyperplasia appears to affect fertility by reducing the uterine vasodilatory response to mating and delaying clearance of uterine fluid as a result of decreased uterine contractions but the effect can be ameliorated in part by the postmating administration of antibiotic.     

Changes in the response to adrenergic drugs on mouse uterine contractions during pregnancy

The effect of isoproterenol (ISO), norepinephrine (NE) and phenylephrine (PHE) on electrically-induced contractions of mice uterine horns was studied during pregnancy. At the different times of gestation adrenergic agonists always inhibited uterine contractions in the following rank order of potency: ISO greater than NE greater than PHE. Cumulative dose-response curves constructed for the effect of these amines during diestrous, and at days 3-7, 10-15, 17-21 of gestation, showed that EC50 values increased gradually as term approached, which could imply a lower capacity of the uterus to respond to adrenergic drugs. Some likely explanations for this phenomenon are proposed. It is suggested that this lower response to catecholamines at the end of pregnancy could be a cause for the reduced success of beta 2-adrenergic drugs to stop premature labor.     

Osteopontin is synthesized by uterine glands and a 45-kDa cleavage fragment is localized at the uterine-placental interface throughout ovine pregnancy

Osteopontin (OPN) is a phosphorylated and glycosylated, secreted protein that is present in various epithelial cells and biological fluids. On freezing and thawing or treatment with proteases, the native 70-kDa protein gives rise to 45- and 24-kDa fragments. Secreted OPN functions as an extracellular matrix (ECM) protein that binds cell surface receptors to mediate cell-cell adhesion, cell-ECM communication, and cell migration. In sheep and humans, OPN is proposed to be a secretory product of uterine glandular epithelium (GE) that binds to uterine luminal epithelium (LE) and conceptus trophectoderm to mediate conceptus attachment, which is essential to maintain pregnancy through the peri-implantation period. Cell-cell adhesion, communication, and migration likely are important at the interface between uterus and placenta throughout pregnancy, but to our knowledge, endometrial and/or placental expression of OPN beyond the peri-implantation period has not been documented in sheep. Therefore, the present study determined temporal and spatial alterations in OPN mRNA and protein expression in the ovine uterus between Days 25 and 120 of pregnancy. The OPN mRNA in total ovine endometrium increased 30-fold between Days 40 and 80 of gestation. In situ hybridization and immunofluorescence analyses revealed that the predominant source of OPN mRNA and protein throughout pregnancy was the uterine GE. Interestingly, the 45-kDa form of OPN was detected exclusively, continuously, and abundantly along the apical surface of LE, on conceptus trophectoderm, and along the uterine-placental interface of both interplacentomal and placentomal regions through Day 120 of pregnancy. The 45-kDa OPN is a proteolytic cleavage fragment of the native 70-kDa OPN, and it is the most abundant form in uterine flushes during early pregnancy. The 45-kDa OPN is more stimulatory to cell attachment and cell migration than the native 70-kDa protein. Collectively, the present results support the hypothesis that ovine OPN is a component of histotroph secreted by the uterine GE that accumulates at the uterine-placental interface to influence maternal-fetal interactions throughout gestation in sheep.     

Regulation of baseline Ca2+ sensitivity in permeabilized uterine arteries: effect of pregnancy

The adaptation of contractile mechanisms of the uterine artery to pregnancy is not fully understood. The present study examined the effect of pregnancy on the uterine artery baseline Ca2+ sensitivity. In beta-escin-permeabilized arterial preparations, Ca2+ -induced concentration-dependent contractions were significantly decreased in uterine arteries from pregnant animals compared with those of nonpregnant animals. Time-course studies showed that Ca2+ increased phosphorylation of 20-kDa myosin light chain (MLC20), which preceded the tension development in vessels from both pregnant and nonpregnant animals. When compared with vessels from nonpregnant animals, there was a significant increase in the protein level of MLC20 and an accordance increase in the level of Ca2+ -induced phosphorylated MLC20 (MLC20-P) in uterine arteries during pregnancy. Simultaneous measurements of MCL20-P levels and contractions stimulated with Ca2+ in the same tissues demonstrated a significant attenuation in the tension-to-MLC20-P ratio in uterine arteries during pregnancy. Activation of PKC with phorbol 12,13-dibutyrate (PDBu) potentiated Ca2+ -induced contractions in uterine arteries from nonpregnant but not pregnant animals. Accordingly, inhibition of PKC attenuated Ca2+ -induced contractions in uterine arteries from nonpregnant but not pregnant animals. PDBu produced contractions in the presence or absence of Ca2+ in the beta-escin-permeabilized arteries, which were significantly decreased in uterine arteries from pregnant compared with nonpregnant animals. The results suggest that pregnancy upregulates the thick-filament regulatory pathway by increasing MLC20 phosphorylation but downregulates the thin-filament regulatory pathway by decreasing the contractile sensitivity of MLC20-P, resulting in attenuated baseline Ca2+ sensitivity in the uterine artery. In addition, PKC plays an important role in the regulation of basal Ca2+ sensitivity, which is downregulated during pregnancy.     

CyPPA, a positive modulator of small-conductance Ca(2+)-activated K(+) channels, inhibits phasic uterine contractions and delays preterm birth in mice

Organized uterine contractions, including those necessary for parturition, are dependent on calcium entry through voltage-gated calcium channels in myometrial smooth muscle cells. Recent evidence suggests that small-conductance Ca(2+)-activated potassium channels (K(Ca)2), specifically isoforms K(Ca)2.2 and 2.3, may control these contractions through negative feedback regulation of Ca(2+) entry. We tested whether selective pharmacologic activation of K(Ca)2.2/2.3 channels might depress uterine contractions, providing a new strategy for preterm labor intervention. Western blot analysis and immunofluorescence microscopy revealed expression of both K(Ca)2.2 and K(Ca)2.3 in the myometrium of nonpregnant (NP) and pregnant (gestation day 10 and 16; D10 and D16, respectively) mice. Spontaneous phasic contractions of isolated NP, D10, and D16 uterine strips were all suppressed by the K(Ca)2.2/2.3-selective activator CyPPA in a concentration-dependent manner. This effect was antagonized by the selective K(Ca)2 inhibitor apamin. Whereas CyPPA sensitivity was reduced in D10 and D16 versus NP strips (pIC(50) 5.33 ± 0.09, 4.64 ± 0.03, 4.72 ± 0.10, respectively), all contractions were abolished between 30 and 60 μM. Blunted contractions were associated with CyPPA depression of spontaneous Ca(2+) events in myometrial smooth muscle bundles. Augmentation of uterine contractions with oxytocin or prostaglandin F(2α) did not reduce CyPPA sensitivity or efficacy. Finally, in an RU486-induced preterm labor model, CyPPA significantly delayed time to delivery by 3.4 h and caused a 2.5-fold increase in pup retention. These data indicate that pharmacologic stimulation of myometrial K(Ca)2.2/2.3 channels effectively suppresses Ca(2+)-mediated uterine contractions and delays preterm birth in mice, supporting the potential utility of this approach in tocolytic therapies.     

Calcitonin down-regulates E-cadherin expression in rodent uterine epithelium during implantation

Previous studies indicated that calcitonin (CT), a peptide hormone involved in calcium homeostasis, is transiently expressed in the receptive rat and human endometrial epithelia within the window of implantation. Attenuation of uterine CT expression using antisense methods severely impaired implantation in the rat. The molecular pathway of CT in the pregnant uterus, however, remains unknown. In the present study, we investigated the cellular events following the binding of CT to its membrane receptors in human endometrial epithelial cell line Ishikawa. We observed that CT treatment triggers a transient rise in intracellular calcium in these cells. Most interestingly, CT treatment also led to the disappearance of E-cadherin, a critical cell adhesion molecule, from cell-cell contact sites. Blockade of intracellular calcium release by BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl) prevented the CT-induced disappearance of E-cadherin. Our studies further revealed that CT treatment markedly down-regulates the level of E-cadherin mRNA in Ishikawa cells. We also examined whether CT influences the expression of E-cadherin mRNA in intact rat uterine tissue during implantation. In pregnant rats, high levels of E-cadherin mRNA were expressed during the first 3 days of gestation when the CT mRNA in uterine epithelial cells is undetectable. Concomitant with a transient burst of CT expression during days 4-5 of pregnancy, the level of E-cadherin mRNA declined sharply. Furthermore, administration of exogenous CT to animals on day 2 of pregnancy led to a premature suppression of E-cadherin mRNA level on day 3, indicating a direct link between elevated levels of uterine CT and the down-regulation of E-cadherin expression in the surface epithelium. Collectively, our results are consistent with the hypothesis that CT-induced reduction in E-cadherin expression may remodel the adherens junctions between epithelial cells, and this change in epithelial cell phenotype might be a critical event during the implantation of the blastocyst.     

Genistein and daidzein modulate in vitro rat uterine contractile activity

The present study investigated the effect of genistein, daidzein and estradiol on in vitro rat uterine responsiveness to oxytocin (OT) and PGF(2)alpha or luprostiol (L). In a first experiment, animals were either sham-operated (SH; n=5), or ovariectomized (OVX; n=20) and orally treated for three months with either genistein (G; n=5; 10 microg/g BW/d) or daidzein (D; n=5; 10 microg/g BW/d) or 17 alpha-ethinylestradiol (E; n=5; 23 microg/kg BW/d) or untreated (OVX; n=5). At necropsy, the basal uterine tension was lower in OVX, G and D than in SH, the highest value being measured in E. Oxytocin (10(-12); 10(-11) M) or PGF(2)alpha (10(-12); 10(-9) M) induced an increase in SH, but not in OVX, E and G. In D, only the highest doses were efficient. In a second experiment, 20 intact animals were s.c. injected with either genistein (G; n=5; 10 microg/g BW) or daidzein (D; n=5; 10 microg/g BW) or estradiol benzoate (E; n=5; 23 microg/kg BW) or vehicle (C: controls; n=5), and killed 24 h later. In C and E, OT (10(-15) to 10(-10) M) or L (10(-12) to 10(-7) M) stimulated uterine contractile activity in a dose-dependent manner until a maximal level. On the opposite, in G and D, contractile agents (except the highest luprostiol doses) did not stimulate myometrium contractions. Moreover, radioligand binding assays showed that genistein or daidzein inhibited the specific binding of [(3)H] estradiol to the calf uterus estrogen receptor (ER). Therefore, it could be postulated that both genistein and daidzein might bind to the rat uterus ER, inducing either anti-estrogenic or very weak estrogenic effects (depending on the experimental conditions) on in vitro uterine responsiveness to OT and PGF(2)alpha or luprostiol.     

Beta 2-agonist treatment enhances uterine oxytocin receptor mRNA expression in pregnant rats

The objective of this study was to disclose an interaction between Beta(2)-adrenergic (Beta(2)-ARs) and oxytocin (OT) receptors (OTRs) in the late-pregnant rat uterus. We investigated the level of uterine OTR mRNA expression after the administration of Beta(2)-AR agonists fenoterol and hexoprenaline to rats from day 18 to 22 of pregnancy, and also tested the effect of fenoterol on uterine explants. Hexoprenaline induced a maximum 24% increase of OTR mRNA. Fenoterol in vivo elicited a maximum 125% increase of OTR mRNA, in vitro produced a maximum fourfold increase in OTR mRNA. In fenoterol-treated rats the maximal contractility increasing effect of OT on isolated uterine rings was significantly higher than in intact term pregnant rats, but the EC50 values were not statistically different. It was concluded that the enhanced expression of OTR mRNA induced by Beta(2)-agonists in the late-pregnant rat uterus may be a possible drawback to effective therapy of preterm uterine contractions with Beta(2)-agonists.