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Preterm birth (PTB), when defined as labor before 37 weeks of gestation, affects approximately 1 out of every 10 births in the United States, leading to high rates of mortality. Complete understanding of the mechanism of PTB requires non-invasive, multi-modal techniques that can provide information about the cascade of labor onset. This study compares the cervical remodeling in wild-type term and induced preterm mouse models using Raman spectroscopy. This study demonstrates the potential of Raman spectroscopy as a non-invasive, real-time in-vivo modality to understand cervix remodeling, thus guiding future studies to improve reproductive and neonatal outcomes.

Cyclooxygenase (COX)-derived prostaglandins stimulate uterine contractions and prepare the cervix for parturition. Prior reports suggest Cox-1 knockout (KO) mice exhibit delayed parturition due to impaired luteolysis, yet the mechanism for late-onset delivery remains unclear. Here, we examined key factors for normal onset of parturition to determine whether any could account for the delayed parturition phenotype. Pregnant Cox-1KO mice did not display altered timing of embryo implantation or postimplantation growth. Although messenger RNAs of contraction-associated proteins (CAPs) were differentially expressed between Cox-1KO and wild-type (WT) myometrium, there were no differences in CAP agonist-induced intracellular calcium release, spontaneous or oxytocin (OT)-induced ex vivo uterine contractility, or in vivo uterine contractile pressure. Delayed parturition in Cox-1KO mice persisted despite exogenous OT treatment. Progesterone (P4) withdrawal, by ovariectomy or administration of the P4-antagonist RU486, diminished the delayed parturition phenotype of Cox-1KO mice. Because antepartum P4 levels do not decline in Cox-1KO females, P4-treated WT mice were examined for the effect of this hormone on in vivo uterine contractility and ex vivo cervical dilation. P4-treated WT mice had delayed parturition but normal uterine contractility. Cervical distensibility was decreased in Cox-1KO mice on the day of expected delivery and reduced in WT mice with long-term P4 treatment. Collectively, these findings show that delayed parturition in Cox-1KO mice is the result of impaired luteolysis and cervical dilation, despite the presence of strong uterine contractions.

Use of selective serotonin reuptake inhibitors (SSRIs) is common during pregnancy. Fetal exposure to SSRIs is associated with persistent pulmonary hypertension of the newborn (PPHN); however, a direct link between the two has yet to be established. Conversely, it is well known that PPHN can be caused by premature constriction of the ductus arteriosus (DA), a fetal vessel connecting the pulmonary and systemic circulations. We hypothesized that SSRIs could induce in utero DA constriction. Using isolated vessels and whole-animal models, we sought to determine the effects of two commonly prescribed SSRIs, fluoxetine and sertraline, on the fetal mouse DA. Cannulated vessel myography studies demonstrated that SSRIs caused concentration-dependent DA constriction and made vessels less sensitive to prostaglandin-induced dilation. Moreover, in vivo studies showed that SSRI-exposed mice had inappropriate DA constriction in utero. Taken together, these findings establish that SSRIs promote fetal DA constriction and provide a potential mechanism by which SSRIs could contribute to PPHN.

Background Although studies involving preterm infants ≤34 weeks gestation report a decreased incidence of patent ductus arteriosus after antenatal betamethasone, studies involving younger gestation infants report conflicting results. Methods We used preterm baboons, mice and humans (≤276/7 weeks gestation) to examine betamethasone’s effects on ductus gene expression and constriction both in vitro and in vivo. Results In mice, betamethasone increased the sensitivity of the premature ductus to the contractile effects of oxygen without altering the effects of other contractile or vasodilatory stimuli. Betamethasone’s effects on oxygen sensitivity could be eliminated by inhibiting endogenous prostaglandin/nitric oxide signaling. In mice and baboons, betamethasone increased the expression of several developmentally-regulated genes that mediate oxygen-induced constriction (K+ channels) and inhibit vasodilator signaling (phosphodiesterases). In human infants, betamethasone increased the rate of ductus constriction at all gestational ages. However, in infants born ≤256/7 weeks gestation, betamethasone’s contractile effects were only apparent when prostaglandin signaling was inhibited, whereas, at 26-27 weeks gestation betamethasone’s contractile effects were apparent even in the absence of prostaglandin inhibitors. Conclusions We speculate that betamethasone’s contractile effects may be mediated through genes that are developmentally regulated. This could explain why betamethasone’s effects vary according to the infant’s developmental age at birth.

Bacterial DNA has been reported in the placenta and amniotic fluid by several independent groups of investigators. However, it's taxonomic overlap with fetal and maternal bacterial DNA in different sites has been poorly characterized. Here, we determined the presence of bacterial DNA in the intestines and placentas of fetal mice at gestational day 17 (n = 13). These were compared to newborn intestines (n = 15), maternal sites (mouth, n = 6; vagina, n = 6; colon, n = 7; feces, n = 8), and negative controls to rule out contamination. The V4 region of the bacterial 16S rRNA gene indicated a pattern of bacterial DNA in fetal intestine similar to placenta but with higher phylogenetic diversity than placenta or newborn intestine. Firmicutes were the most frequently assignable phylum. SourceTracker analysis suggested the placenta as the most commonly identifiable origin for fetal bacterial DNA, but also over 75% of fetal gut genera overlapped with maternal oral and vaginal taxa but not with maternal or newborn feces. These data provide evidence for the presence of bacterial DNA in the mouse fetus.

Sepsis is strongly associated with patency of the ductus arteriosus (PDA) in critically ill newborns. Inflammation and the aminoglycoside antibiotics used to treat neonatal sepsis cause smooth muscle relaxation, but their contribution to PDA is unknown. We examined whether: 1) lipopolysaccharide (LPS) or inflammatory cytokines cause relaxation of the ex vivo mouse DA; 2) the aminoglycosides gentamicin, tobramycin, or amikacin causes DA relaxation; and 3) newborn infants treated with aminoglycosides have an increased risk of symptomatic PDA (sPDA). Changes in fetal mouse DA tone were measured by pressure myography in response to LPS, TNF-α, IFN-γ, macrophage-inflammatory protein 2, IL-15, IL-13, CXC chemokine ligand 12, or three aminoglycosides. A clinical database of inborn patients of all gestations was analyzed for association between sPDA and aminoglycoside treatment. Contrary to expectation, neither LPS nor any of the inflammatory mediators caused DA relaxation. However, each of the aminoglycosides caused concentration-dependent vasodilation in term and preterm mouse DAs. Pretreatment with indomethacin and N-(G)-nitro-L-arginine methyl ester did not prevent gentamicin-induced DA relaxation. Gentamicin-exposed DAs developed less oxygen-induced constriction than unexposed DAs. Among 488,349 infants who met the study criteria, 40,472 (8.3%) had sPDA. Confounder-adjusted odds of sPDA were higher in gentamicin-exposed infants, 32 wk. Together, these findings suggest that factors other than inflammation contribute to PDA. Aminoglycoside-induced vasorelaxation and inhibition of oxygen-induced DA constriction support the paradox that antibiotic treatment of sepsis may contribute to DA relaxation. This association was also found in newborn infants, suggesting that antibiotic selection may be an important consideration in efforts to reduce sepsis-associated PDA.

Failure of the ductus arteriosus (DA) to close at birth can lead to serious complications. Conversely, certain profound congenital cardiac malformations require the DA to be patent until corrective surgery can be performed. In each instance, clinicians have a very limited repertoire of therapeutic options at their disposal - indomethacin or ibuprofen to close a patent DA (PDA) and prostaglandin E1 to maintain patency of the DA. Neither treatment is specific to the DA and both may have deleterious off-target effects. Therefore, more therapeutic options specifically targeted to the DA should be considered. We hypothesized the DA possesses a unique genetic signature that would set it apart from other vessels. A microarray was used to compare the genetic profiles of the murine DA and ascending aorta (AO). Over 4,000 genes were differentially expressed between these vessels including a subset of ion channel-related genes. Specifically, the alpha and beta subunits of large-conductance calcium-activated potassium (BKCa) channels are enriched in the DA. Gain- and loss-of-function studies showed inhibition of BKCachannels caused the DA to constrict, while activation caused DA relaxation even in the presence of O2. This study identifies subsets of genes that are enriched in the DA that may be used to develop DA-specific drugs. Ion channels that regulate DA tone, including BKCachannels, are promising targets. Specifically, BKCachannel agonists like NS1619 maintain DA patency even in the presence of O2and may be clinically useful.

Alkaline phosphatase (AP) activity has been demonstrated in the uterus of several species, but its importance in the uterus, in general and during pregnancy, is yet to be revealed. In this study, we focused on identifying AP isozyme types and their hormonal regulation, cell type, and event-specific expression and possible functions in the hamster uterus during the cycle and early pregnancy. Our RT-PCR andin situhybridization studies demonstrated that among the knownAkp2,Akp3,Akp5, andAkp6murine AP isozyme genes, hamster uteri express onlyAkp2andAkp6; both genes are co-expressed in luminal epithelial cells. Studies in cyclic and ovariectomized hamsters established that while progesterone (P4) is the major uterineAkp2inducer, both P4and estrogen are strongAkp6regulators. Studies in preimplantation uteri showed induction of both genes and the activity of their encoded isozymes in luminal epithelial cells during uterine receptivity. However, at the beginning of implantation,Akp2showed reduced expression in luminal epithelial cells surrounding the implanted embryo. By contrast, expression ofAkp6and its isozyme was maintained in luminal epithelial cells adjacent to, but not away from, the implanted embryo. Following implantation, stromal transformation to decidua was associated with induced expressions of onlyAkp2and its isozyme. We next demonstrated that uterine APs dephosphorylate and detoxify endotoxin lipopolysaccharide at their sites of production and activity. Taken together, our findings suggest that uterine APs contribute to uterine receptivity, implantation, and decidualization in addition to their role in protection of the uterus and pregnancy against bacterial infection.

Augmentation of endogenous cannabinoid (eCB) signaling represents an emerging approach to the treatment of affective disorders. Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid to form prostaglandins, but also inactivates eCBs in vitro. However, the viability of COX-2 as a therapeutic target for in vivo eCB augmentation has not been explored. Here we utilized medicinal chemistry and in vivo analytical and behavioral pharmacological approaches to demonstrate a key role for COX-2 in the regulation of endocannabinoid (eCB) levels in vivo. A novel pharmacological strategy involving “substrate-selective” inhibition of COX-2 was used to augment eCB signaling without affecting related non-eCB lipids or prostaglandin synthesis. Behaviorally, substrate-selective inhibition of COX-2reducedanxiety-like behaviors in mice via increasede CB signaling. These data elucidate a key role for COX-2 in the regulation of eCB signaling and suggest substrate-selective pharmacology represents a viable approach for eCB augmentation with broad therapeutic potential.

To examine the underlying processes of an innovative mind-body practice, Mindful Body Awareness, this exploratory study involved four case studies analyzed phenomenologically using the dialogal method. Mindful Body Awareness combines manual (touch-based) and verbal processing, and is focused on facilitation of client body awareness. Four individuals were recruited to receive weekly 1.25 hour sessions over four weeks. The Helpfulness Aspects of Therapy (HAT) form was administered immediately after each session to access participants' perceptions of the therapy experience. In addition, the Scale of Body Connection was used to examine pre- and post-body awareness and bodily dissociation. Analysis involved phenomenology and descriptive statistics. The overall perceived helpfulness of the intervention was evident in the four themes that emerged from the analysis. These themes were gaining interoceptive awareness, personal agency, therapist trust and conceptual framing, and transformation. The participants' responses were also used to investigate the therapy process across time. A pattern of increased interoceptive depth was apparent, as was a concomitant progression in embodied sense of self. Improvements in body awareness and bodily dissociation were evident for two of the four participants. These findings help to identify primary components of Mindful Body Awareness and suggest the role of these components in the embodiment process.

Cytosolic phospholipase A2 (cPLA2, PLA2G4A) catalyzes the release of arachidonic acid for prostaglandin synthesis by cyclooxygenase 1 (PTGS1) and cyclooxygenase 2 (PTGS2). Mice with Pla2g4a deficiency have parturition delay and other reproductive deficits, including deferred onset of implantation, crowding of implantation sites, and small litters. In this study, we examined the contribution of PLA2G4A to parturition in mice. Pla2g4a mRNA and protein expression were discretely localized in the term and preterm uterine luminal epithelium and colocalized with Ptgs1, but not Ptgs2, expression. The levels of PGE2, PGF2alpha, 6-keto-PGF1alpha, and TxB2 were significantly decreased in Pla2g4a-null uterine tissues, similar to Ptgs1-null uteri, consistent with predominance of PLA2G4A-PTGS1-mediated prostaglandin synthesis in preparation for murine parturition. Litter size was strongly associated with the timing of parturition in Pla2g4a-null mice but could not fully account for the parturition delay. Pla2g4a-null females that received PGE2 + carbaprostacyclin at the time of implantation delivered earlier (20.5 +/- 0.2 days vs. 21.6 +/- 0.2 days, P < 0.01), although litter size was not improved (4.6 vs. 4.4 pups per litter, P = 0.6). After correction for small litter size, multivariate analysis indicated that Pla2g4a-null mice given prostaglandin treatment to improve implantation timing had gestational length that was similar to wild-type and Pla2g4a heterozygous mice. These results indicate that, despite specific Pla2g4a expression and function in term gestation uteri, the delayed parturition phenotype in Pla2g4a-null mice is primarily due to deferral of implantation. The role of PLA2G4A in timely parturition appears to be critically related to its actions in early pregnancy.

It is unknown whether or not tight junction formation plays any role in morula to blastocyst transformation that is associated with development of polarized trophoblast cells and fluid accumulation. Tight junctions are a hallmark of polarized epithelial cells and zonula occludens-1 (ZO-1) is a known key regulator of tight junction formation. Here we show that ZO-1 protein is first expressed during compaction of 8-cell embryos. This stage-specific appearance of ZO-1 suggests its participation in morula to blastocyst transition. Consistent with this idea, we demonstrate that ZO-1 siRNA delivery inside the blastomeres of zona-weakened embryos using electroporation not only knocks down ZO-1 gene and protein expressions, but also inhibits morula to blastocyst transformation in a concentration-dependent manner. In addition, ZO-1 inactivation reduced the expression of Cdx2 and Oct-4, but not ZO-2 and F-actin. These results provide the first evidence that ZO-1 is involved in blastocyst formation from the morula by regulating accumulation of fluid and differentiation of nonpolar blastomeres to polar trophoblast cells.

The uterine myometrium (UT-myo) is a therapeutic target for preterm labor, labor induction, and postpartum hemorrhage. Stimulation of intracellular Ca2+-release in UT-myo cells by oxytocin is a final pathway controlling myometrial contractions. The goal of this study was to develop a dual-addition assay for high-throughput screening of small molecular compounds, which could regulate Ca2+-mobilization in UT-myo cells, and hence, myometrial contractions. Primary murine UT-myo cells in 384-well plates were loaded with a Ca2+-sensitive fluorescent probe, and then screened for inducers of Ca2+-mobilization and inhibitors of oxytocin-induced Ca2+-mobilization. The assay exhibited robust screening statistics (Z´ = 0.73), DMSO-tolerance, and was validated for high-throughput screening against 2,727 small molecules from the Spectrum, NIH Clinical I and II collections of well-annotated compounds. The screen revealed a hit-rate of 1.80% for agonist and 1.39% for antagonist compounds. Concentration-dependent responses of hit-compounds demonstrated an EC50 less than 10μM for 21 hit-antagonist compounds, compared to only 7 hit-agonist compounds. Subsequent studies focused on hit-antagonist compounds. Based on the percent inhibition and functional annotation analyses, we selected 4 confirmed hit-antagonist compounds (benzbromarone, dipyridamole, fenoterol hydrobromide and nisoldipine) for further analysis. Using an ex vivo isometric contractility assay, each compound significantly inhibited uterine contractility, at different potencies (IC50). Overall, these results demonstrate for the first time that high-throughput small-molecules screening of myometrial Ca2+-mobilization is an ideal primary approach for discovering modulators of uterine contractility.

Use of cyclooxygenase (COX) inhibitors to delay preterm birth is complicated by in utero constriction of the ductus arteriosus and delayed postnatal closure. Delayed postnatal closure has been attributed to loss of vasa vasorum flow and ductus wall ischemia resulting from constriction in utero. We used the murine ductus (which does not depend on vasa vasorum flow) to determine whether delayed postnatal closure may be because of mechanisms independent of in utero constriction. Acute inhibition of both COX isoforms constricted the fetal ductus on days 18 and 19 (term) but not earlier in gestation; COX-2 inhibition constricted the fetal ductus more than COX-1 inhibition. In contrast, mice exposed to prolonged inhibition of COX-1, COX-2, or both COX isoforms (starting on day 15, when the ductus does not respond to the inhibitors) had no contractile response to the inhibitors on days 18 or 19. Newborn mice closed their ductus within 4 h of birth. Prolonged COX inhibition on days 11–14 of gestation had no effect on newborn ductal closure; however, prolonged COX inhibition on days 15–19 resulted in delayed ductus closure despite exposure to 80% oxygen after birth. Similarly, targeted deletion of COX-2 alone, or COX-1/COX-2 together, impaired postnatal ductus closure. Nitric oxide inhibition did not prevent the delay in ductus closure. These data show that impaired postnatal ductus closure is not the result of in utero ductus constriction or upregulation of nitric oxide synthesis. They are consistent with a novel role for prostaglandins in ductus arteriosus contractile development.

Patency of the fetal ductus arteriosus (DA) is maintained in an environment of low relative oxygen tension and a preponderance of vasodilating forces. In addition to prostaglandins, nitric oxide (NO), a potent vasodilator in the pulmonary and systemic vasculatures, has been implicated in regulation of the fetal DA. To further define the contribution of NO to DA patency, the expression and function of NO synthase (NOS) isoforms were examined in the mouse DA on days 17–19 of pregnancy and after birth. Our results show that endothelial NOS (eNOS) is the predominant isoform expressed in the mouse DA and is localized in the DA endothelium by in situ hybridization. Despite rapid constriction of the DA after birth, eNOS expression levels were unchanged throughout the fetal and postnatal period. Pharmacological inhibition of prostaglandin vs. NO synthesis in vivo showed that the preterm fetal DA on day 16 is more sensitive to NOS inhibition than the mature fetal DA on day 19, whereas prostaglandin inhibition results in marked DA constriction on day 19 but minimal effects on the day 16 DA. Combined prostaglandin and NO inhibition caused additional DA constriction on day 16. The contribution of vasa vasorum to DA regulation was also examined. Immunoreactive platelet endothelial cell adhesion molecule and lacZ tagged FLK1 localized to DA endothelial cells but revealed the absence of vasa vasorum within the DA wall. Similarly, there was no evidence of vasa vasorum by vascular casting. These studies indicate that eNOS is the primary source of NO in the mouse DA and that vasomotor tone of the preterm fetal mouse DA is regulated by eNOS-derived NO and is potentiated by prostaglandins. In contrast to other species, mechanisms for DA patency and closure appear to be independent of any contribution of the vasa vasorum.

The mouse model has greatly contributed to understanding molecular mechanisms involved in the regulation of progesterone (P4) plus estrogen (E)-dependent blastocyst implantation process. However, little is known about contributory molecular mechanisms of the P4-only-dependent blastocyst implantation process that occurs in species such as hamsters, guineapigs, rabbits, pigs, rhesus monkeys, and perhaps humans. We used the hamster as a model of P4-only-dependent blastocyst implantation and carried out cross-species microarray (CSM) analyses to reveal differentially expressed genes at the blastocyst implantation site (BIS), in order to advance the understanding of molecular mechanisms of implantation. Upregulation of 112 genes and downregulation of 77 genes at the BIS were identified using a mouse microarray platform, while use of the human microarray revealed 62 up- and 38 down-regulated genes at the BIS. Excitingly, a sizable number of genes (30 up- and 11 down-regulated genes) were identified as a shared pool by both CSMs. Real-time RT-PCR andin situhybridization validated the expression patterns of several up- and down-regulated genes identified by both CSMs at the hamster and mouse BIS to demonstrate the merit of CSM findings across species, in addition to revealing genes specific to hamsters. Functional annotation analysis found that genes involved in the spliceosome, proteasome, and ubiquination pathways are enriched at the hamster BIS, while genes associated with tight junction, SAPK/JNK signaling, and PPARα/RXRα signalings are repressed at the BIS. Overall, this study provides a pool of genes and evidence of their participation in up- and down-regulated cellular functions/pathways at the hamster BIS.

We evaluated the clinical effectiveness of variable courses of paracetamol on patent ductus arteriosus (PDA) closure and examined its effect on the in vitro term and preterm murine ductus arteriosus (DA).Neonates received one of the following three paracetamol regimens: short course of oral paracetamol (SCOP), long course of oral paracetamol (LCOP), and intravenous paracetamol (IVP) for 2-6 d. Pressure myography was used to examine changes in vasomotor tone of the preterm and term mouse DA in response to paracetamol or indomethacin. Their effect on prostaglandin synthesis by DA explants was measured by mass spectroscopy.Twenty-one preterm infants were included. No changes in PDA hemodynamics were seen in SCOP infants (n = 5). The PDA became less significant and eventually closed in six LCOP infants (n = 7). PDA closure was achieved in eight IVP infants (n = 9). On pressure myograph, paracetamol induced a concentration-dependent constriction of the term mouse DA, up to 30% of baseline (P0.01), but required1 µmol/l. Indomethacin induced greater DA constriction and suppression of prostaglandin synthesis (P0.05).The clinical efficacy of paracetamol on PDA closure may depend on the duration of treatment and the mode of administration. Paracetamol is less potent than indomethacin for constriction of the mouse DA in vitro.

PRL and its homologs accomplish their biological effects through the PRL receptor (PRLR). We evaluated the expression and function of PRLR in the embryo and uterus during the periimplantation period because PRLR deficiency results in implantation failure. In wild-type mice, PRLR expression was localized to undecidualized stromal cells in the antimesometrial border on days 6-8 of pregnancy. A small population of PRLR-expressing cells was observed adjacent to the ectoplacental cone in the mesometrial stroma. Low levels of PRLR expression were also detected in the developing embryo on days 6-8. To determine the significance of PRLR expression in this distribution, we examined implantation and decidualization in PRLR-/- mice. Progesterone (P4) administration rescued infertility in PRLR-/- mice from the periimplantation period to midgestation. Artificially induced decidualization was absent in pseudopregnant PRLR-/- mice but was identical to wild-type in P4-treated PRLR-/- mice. Furthermore, wild-type and P4-treated PRLR-/- mice had similar expression of the implantation-specific genes, LIF, amphiregulin, HB-EGF, COX-1, COX-2, PPARdelta, Hoxa-10, cyclin-D3, VEGF, and its receptors, Flk-1 and neuropilin-1. Together, these results show that luteal P4 production via ovarian PRLR signaling is required for implantation and early pregnancy. The function of uterine PRLR remains unclear. However, the eventual loss of pregnancy in P4-treated PRLR-/- mice suggests that uterine PRLR may be essential for the support of late gestation.

Complex cellular interactions occur between the blastocyst and the uterus during implantation. The expression of various polypeptide growth factors and their receptors in the uterus and/or blastocyst during the periimplantation period suggest that growth factors participate in the implantation process. Neu differentiation factor (NDF) is a member of the epidermal growth factor (EGF) family of growth factors and is represented by multiple conserved isoforms. The expression of several EGF-like ligands in the periimplantation uterus has been characterized, including EGF, heparin binding-EGF, transforming growth factor alpha, amphiregulin, betacellulin, and epiregulin. We analyzed the expression pattern of NDF in the periimplantation mouse uterus because of its mitogenic and differentiation-promoting effects. By using Northern analysis and isoform-specific polymerase chain reaction, we found that multiple isoforms are expressed in the periimplantation uterus. NDF displays a highly restricted temporal and spatial expression, with autoradiographic signals localized to the uterine stroma immediately surrounding the implanting blastocyst. NDF expression was absent in mice with delayed implantation but briefly reappeared with the same restricted distribution after termination of the delay by an injection of estrogen. Taken together, these results suggest that an activated blastocyst is required for the expression of NDF and that multiple isoforms may be involved in the complex network of cell-signaling events between the implanting blastocyst and the receptive uterus.

Persistent patency of the ductus arteriosus (PDA) is a common problem in preterm infants. The antacid cimetidine is a potent antagonist of the H2 histamine receptor but it also inhibits certain cytochrome P450 enzymes (CYPs), which may affect DA patency. We examined whether cimetidine contributes to PDA and is mediated by CYP inhibition rather than H2 blockade. Analysis of a clinical trial to prevent lung injury in premature infants revealed a significant association between cimetidine treatment and PDA. Cimetidine and ranitidine, both CYP inhibitors as well as H2 blockers, caused relaxation of the term and preterm mouse DA. CYP enzymes that are inhibited by cimetidine were expressed in DA subendothelial smooth muscle. The selective CYP3A inhibitor ketoconazole induced greater DA relaxation than cimetidine, whereas famotidine and other H2 antagonists with less CYP inhibitory effects caused less dilation. Histamine receptors were developmentally regulated and localized in DA smooth muscle. However, cimetidine caused DA relaxation in histamine-deficient mice, consistent with CYP inhibition, not H2 antagonism, as the mechanism for PDA. Oxygen-induced DA constriction was inhibited by both cimetidine and famotidine. These studies show that antacids and other compounds with CYP inhibitory properties pose a significant and previously unrecognized risk for PDA in critically ill newborn infants.

Increased oxygen tension at birth regulates physiologic events that are essential to postnatal survival, but the accompanying oxidative stress may also generate isoprostanes. We hypothesized that isoprostanes regulate ductus arteriosus (DA) function during postnatal vascular transition.Isoprostanes were measured by gas chromatography-mass spectrometry. DA tone was assessed by pressure myography. Gene expression was measured by quantitative PCR.Oxygen exposure was associated with increased 8-iso-prostaglandin (PG)F2α in newborn mouse lungs. Both 8-iso-PGE2 and 8-iso-PGF2α induced concentration-dependent constriction of the isolated term DA, which was reversed by the thromboxane A2 (TxA2) receptor antagonist SQ29548. SQ29548 pretreatment unmasked an isoprostane-induced DA dilation mediated by the EP4 PG receptor. Exposure of the preterm DA to 8-iso-PGE2 caused unexpected DA relaxation that was reversed by EP4 antagonism. In contrast, exposure to 8-iso-PGF2α caused preterm DA constriction via TxA2 receptor activation. Further investigation revealed the predominance of the TxA2 receptor at term, whereas the EP4 receptor was expressed and functionally active from mid-gestation onward.This study identifies a novel physiological role for isoprostanes during postnatal vascular transition and provide evidence that oxidative stress may act on membrane lipids to produce vasoactive mediators that stimulate physiological DA closure at birth or induce pathological patency of the preterm DA.

Scents and Sperm Once sperm enter the female reproductive tract, they have an arduous task to find an egg at a distant, often concealed, location. McKnight et al. (p. 754 ) show that Caenorhabditis elegans make this task more or less difficult, depending on pheromones in the external environment. Pheromones perceived by female sensory neurons modulate the synthesis of ovarian prostaglandins, which provide sperm positional information. Thus, environmental cues can indirectly impact sperm function even when the sperm themselves are not directly exposed.

The objective was to study the expression of zonula occludens-2, a tight junction protein, during preimplantation hamster embryonic development, to predict its possible localization, source, and roles in trophectoderm differentiation and blastocyst formation in this species. Comparison of zonula occludens-2 expression pattern between the hamster and mouse preimplantation embryos from the zygote up to the blastocyst stage was also an objective of this study. Zonula occludens-2 localization was noted in nuclei of blastomeres in all stages of hamster and mouse embryonic development. Compared to mice, where zonula occludens-2 was first localized in the interblastomere membrane at the morula stage, hamster embryos had membranous zonula occludens-2 localization from the 2-cell stage onwards. Based on combined results of immunolocalization study in parthenogenic embryos and ovarian and epididymal sections, and quantitative PCR done in oocytes and all developmental stages of preimplantation embryos, perhaps there was a carry-over of zonula occludens-2 proteins or mRNA from the dam to the embryo. Based on these findings, we inferred that maternally derived zonula occludens-2 was involved in nuclear functions, as well as differentiation of blastomeres and blastocoel formation during preimplantation embryonic development in the hamster.

Although prostaglandin E2 (PGE2) vasodilates the ductus arteriosus, tocolysis with cyclooxygenase (COX) inhibitors delays postnatal ductus arteriosus closure. We used fetal mice and sheep to determine if PGE2 has a role in the development of ductus contractility that is distinct from its function as a vasodilator. Prolonged exposure of fetal ductus to PGE2, in vitro, increased expression of CaL- and K+-channel genes (CaLα1c, CaLβ2, Kir6.1, Kv1.5) (which regulate oxygen-induced constriction) without affecting genes that regulate Rho-kinase-mediated calcium sensitization. Conversely, chronic exposure to cyclooxygenase inhibitors in utero decreased expression of CaL- and K+-channel genes, without affecting Rho-kinase-associated genes. Chronic cyclooxygenase inhibition in utero decreased the ductus’ in vitro contractile response to stimuli that utilize CaL- and K+-channels (like O2 and K+), whereas the response to stimuli that act through Rho-kinase–mediated pathways (like U46619) was not significantly affected. Phosphodiesterase expression, which decreases the ductus’ sensitivity to cAMP/cGMP-dependent vasodilators, was increased by PGE2 exposure and decreased by cyclooxygenase inhibition, respectively. These studies identify potential downstream effectors of a PGE2-mediated, developmental program, regulating oxygen-induced ductus closure. Alterations in these effectors may explain the increased risk of patent ductus arteriosus (PDA) after in utero cyclooxygenase inhibition.

Biochemical changes of cervical connective tissue, including progressive disorganization of the collagen network and increased water content, occur during gestation to allow for cervical dilatation during labor, but the mechanisms that regulate cervical fluid balance are not fully understood. We examined whether aquaporins (AQPs), a family of membrane channel proteins that facilitate water transport, help mediate fluid balance in the mouse cervix during parturition. Of the 13 known murine AQPs, AQP0–2, 6, 7, 9, 11, and 12 were absent or at the limits of detection. By Northern blot and real-time PCR, AQP3 expression was low in nongravid and mid-pregnancy cervices with peak expression on d 19 and postpartum d 1 (PP1). AQP4 expression was generally low throughout pregnancy but showed a small upward trend at the time of parturition. AQP5 and AQP8 expression were significantly increased on d 12–15 but fell to nongravid/baseline by d 19 and PP1. By in situ hybridization and immunohistochemistry, AQP3 was preferentially expressed in basal cell layers of the cervical epithelium, whereas AQP4, 5, and 8 were primarily expressed in apical cell layers. Females with LPS-induced preterm labor had similar trends in AQP4, 5, and 8 expression to mice with natural labor at term gestation. Mice with delayed cervical remodeling due to deletion of the steroid 5α-reductase type 1 gene showed significant reduction in the levels of AQP3, 4, and 8 on d 19 or PP1. Together, these studies suggest that AQPs 3, 4, 5, and 8 regulate distinct aspects of cervical water balance during pregnancy and parturition.

Neuregulins (NRGs) are cell-signaling molecules with recognized roles in cancer and development, but little is known about their role in embryo implantation. Among representative NRG-1 isoforms, neu differentiation factor (NDF, type I) is expressed in the female reproductive tract and is localized to the implantation site. Here, we show that sensory and motor neuron-derived factor (SMDF, type III) is expressed in the uterine subepithelial stroma around the blastocyst and is only upregulated at the time of implantation. The cellular distribution of SMDF is similar to that of NDF and requires an implantation-competent blastocyst. The glial growth factor (GGF, type II) isoform of NRG-1 and the NRG-2 and NRG-3 genes were not expressed in the peri-implantation uterus, as determined by reverse transcription-polymerase chain reaction or in situ hybridization. In contrast to the cellular expression pattern of NDF and SMDF, NRG-4 was present in the luminal and glandular epithelium throughout the uterus during the preimplantation period. Expression of NRG-4 declined in the uterine luminal epithelium during implantation but persisted in the glandular epithelium through Day 8 of pregnancy. Studies in ovariectomized mice showed that NRG-4 is a progesterone-regulated gene, with partial augmentation by estrogen. We also observed upregulation of the erbB2 and erbB3 receptors at the blastocyst stage of embryo development. Together, these findings suggest that a distinct subset of NRGs participates in the signaling network that directs embryo implantation. Upregulation of embryonic erbB2/ erbB3 in the blastocyst trophectoderm and induction of certain NRG-1 isoforms with blastocyst activation help to define additional aspects of the embryo-uterine cross-talk that underlies the implantation process.

The periimplantation period is marked by edematous changes in the uterus. In the mouse, increased uterine vascular permeability occurs in response to estrogen and certain vasoactive mediators, but the mechanisms that regulate fluid transport during implantation are not fully understood. Aquaporins (AQPs) are a family of membrane channel proteins that facilitate bulk water transport. To assess their role in implantation, we examined the expression of AQPs 0-9 in the mouse uterus on d 1-8 of pregnancy. Our results show distinct uterine expression patterns for AQP1, AQP4, and AQP5. AQP1 is localized to the inner circular myometrium throughout the periimplantation period. AQP4 is highly expressed in the luminal epithelium on d 1 of pregnancy but barely detectable at the time of implantation. AQP5 is expressed at low levels in the glandular epithelium during early pregnancy but is markedly increased on d 5. By immunohistochemistry, AQP5 is localized in the basolateral region of the uterine glands. Treatment of adult ovariectomized mice with replacement steroids demonstrates an estrogen-induced shift in AQP1 signals from the myometrium to the uterine stromal vasculature, suggesting a role in uterine fluid imbibition. In contrast, AQP5 is induced only in estrogen-treated, progesterone-primed uteri. We also observed expression of AQP8 in the inner-cell mass and AQP9 in the mural trophectoderm of the implanting blastocyst. Collectively, these results suggest that members of the AQP family are involved in embryo and uterine fluid homeostasis during implantation.

Cyclooxygenase (COX)-derived prostaglandins are critical in female reproduction. Gene targeting studies show that ovulation, fertilization, implantation, and decidualization are defective in COX-2 deficient mice. We used genetic and pharmacologic approaches to perturb COX function and examine the differential and synergistic effects of inhibition of COX-1, COX-2, or of both isoforms on reproductive outcomes during early pregnancy in mice. The results demonstrate that simultaneous inhibition of COX-1 and COX-2 produces more severe effects on early pregnancy events than inhibition of either isoform alone. The effects of pharmacological inhibition of COX-2 on female reproductive functions were less severe than the null mutation of the COX-2 gene. A combined approach showed that COX-2 inhibition in COX-1−/− mice induced complete reproductive failure, suggesting a lack of alternative sources of prostaglandin synthesis. This investigation raises caution regarding the indiscriminate use of COX inhibitors and shows for the first time the distinct and overlapping pathways of the cyclooxygenase systems in female reproduction.

Cyclooxygenase (COX)-derived prostaglandins (PGs) regulate numerous maternal–fetal interactions during pregnancy. PGs stimulate uterine contractions and prepare the cervix for parturition, whereas in the fetus, PGs maintain patency of the ductus arteriosus (DA), a vascular shunt that transmits oxygenated placental blood to the fetal systemic circulation. However, the origin and site of action of these PGs remain undefined. To address this, we analyzed mice lacking COX-1 (null mutation) or COX-2 (pharmacologic inhibition) or pups with a double null mutation. Our results show that COX-1 in the uterine epithelium is the major source of PGs during labor and that COX-1 −/− females experience parturition failure that is reversible by exogenous PGs. Using embryo transfer experiments, we also show that successful delivery occurs in COX-1 −/− recipient mothers carrying wild-type pups, establishing the sufficiency of fetal PGs for parturition. Although patency of the DA is PG dependent, neither COX-1 nor COX-2 expression was detected in the fetal or postnatal DA, and offspring with a double null mutation died shortly after birth with open DAs. These results suggest that DA patency depends on circulating PGs acting on specific PG receptors within the DA. Collectively, these findings demonstrate the coordinated regulation of fetal and maternal PGs at the time of birth but raise concern regarding the use of selective COX inhibitors for the management of preterm labor.

Between the ages of 3 and 23 months, the cerebella of NIH Fischer 344 rats lose 30% of the thickness of the molecular layer, 60% of the length of parallel fibers, and 80% of the synaptic varicosities along parallel fibers. Nearly 60% of these synaptic varicosities disappear between 3 and 9 months. Thus, the loss of cerebellar synapses is unusual, may have begun early in life, and continue into old age, causing profound synaptic losses in older rats. In addition to serious implications to functional losses, we speculate that these synaptic losses may trigger other age-related cellular losses in the cerebellum.

Prostaglandins (PGs) produced by cyclooxygenase (COX) participate in many aspects of female reproduction. The two isoforms of cyclooxygenase, COX-1 and COX-2, have distinct expression patterns in the mouse uterus during the peri-implantation period and suggest their independent contribution to uterine PGs. Using wild type and COX-1(-/-) mice, we examined the role of COX-1-derived PGs on day 4 of pregnancy, when its expression is maximal. Uterine vascular permeability was measured by 125I-labeled bovine serum albumin (BSA) uptake, and PG content was measured by gas chromatography-mass spectrometry. Vascular permeability and PG concentrations were reduced in COX-1(-/-) mice, but by less than the expected amount. After ovariectomy, uterine vascular permeability declined in both groups, but returned to baseline in wild type and was exaggerated in COX-1(-/-) females after treatment with ovarian steroids. Most importantly, COX-1(-/-) uteri displayed COX-2 expression on the morning of day 4, when COX-2 is normally absent. This hybridization pattern resembles the native expression of COX-1, and may partially offset the loss of COX-1-derived PGs. These data indicate that COX-1-derived PGs are important during uterine preparation for implantation, and that COX-2 compensation occurs in the absence of COX-1.

Erratum to ‘‘COX-2 compensation in the uterus of COX-1 deficient mice during the pre-implantation period’’ [Mol. Cell. Endocrinol. 150 (1999) 23–31] Jeff Reese , Naoko Brown , Bibhash C. Paria , Jason Morrow , Sudhansu K. Dey a a Department of Pediatrics, Uni6ersity of Kansas Medical Center, Kansas City, KS 66160-7338, USA b Molecular and Integrati6e Physiology, Uni6ersity of Kansas Medical Center, Kansas City, KS 66160-7338, USA c Department of Medicine and Pharmacology, Vanderbilt Uni6ersity Medical Center, Nash6ille, TN 37232-6602, USA www.elsevier.com/locate/mce

Isoform-Specific Expression of Neuregulin Family Members in the Mouse Uterus During Embryo Implantation 299

Reduced Uterine Vascular Permeability During the Pre-Implantation Period in Cox-1 Deficient Mice. † 300

Temporal and Spatial Expression of neu Differentiation Factor (NDF) in the Mouse Uterus during the Periimplantation Period. • 286