Pregnant Mouse Myometrium Research Articles

Extracellular acidification increases uterine contraction in pregnant mouse by increasing intracellular calcium.

As uterine extracellular pH decreases during the ischemic conditions of labor, but its effects on myometrial contraction are largely unknown, there is a need to elucidate its physiological effects and mechanisms of action. Furthermore, it is not known if any of the effects of extracellular acidification are affected by pregnancy, thus we also determined how gestation affects the response to acidification. Nonpregnant, mid-, and term-pregnant myometrial strips were obtained from humanely killed mice. Contractions were recorded under spontaneous, depolarized, and oxytocin-stimulated conditions. The extracellular pH of the perfusate was changed from 7.4 to 6.9 or 7.9 in HEPES-buffered physiological saline. Intracellular pH was measured using SNARF, and intracellular calcium was measured using Indo-1. Statistical differences were tested using the appropriate t-test. Extracellular acidification significantly increased the frequency and amplitude of spontaneous contractions in pregnant, but not nonpregnant, myometrium, whereas alkalinization decreased contractions. Intracellular acidification, via Na-butyrate, transiently increased force in pregnant tissue. Intracellular pH was gradually acidified when extracellular pH was acidified, but extracellular acidification increased contractility before any significant change in intracellular pH. If myometrial force was driven by oxytocin or high-K depolarization, then extracellular pH did not further increase force. Intracellular calcium changes mirrored those of force in the spontaneously contracting pregnant myometrium, and if calcium entry was prevented by nifedipine, extracellular acidification could not induce a rise in force. Extracellular acidification increases excitability, calcium entry, and thus force in pregnant mouse myometrium, and this may contribute to increasing contractions during labor when ischemic conditions and acidemia occur.

Investigating the role of CFTR in human and mouse myometrium

BackgroundAbnormal cystic fibrosis transmembrane conductance regulator (CFTR) function in cystic fibrosis (CF) has been linked to airway smooth muscle abnormalities including bronchial hyperresponsiveness. However, a role for CFTR in other types of smooth muscle, including myometrium, remains largely unexplored. As CF life expectancy and the number of pregnancies increases, there is a need for an understanding of the potential role of CFTR in myometrial function. MethodsWe investigated the role of CFTR in human and mouse myometrium. We used immunofluorescence to identify CFTR expression, and carried out contractility studies on spontaneously contracting term pregnant and non-pregnant mouse myometrium and term pregnant human myometrial biopsies from caesarean sections. ResultsCFTR was found to be expressed in term pregnant mouse myometrium. Inhibition of CFTR, with the selective inhibitor CFTRinh-172, significantly reduced contractility in pregnant mouse and human myometrium in a concentration-dependent manner (44.89 ± 11.02 term pregnant mouse, 9.23 ± 4.75 term-pregnant human; maximal effect at 60 μM expressed as a percentage of the pre-treatment control period). However, there was no effect of CFTRinh-172 in non-pregnant myometrium. ConclusionThese results demonstrate decreased myometrial function when CFTR is inhibited, which may have implications on pregnancy and labour outcome and therapeutic decisions for labour in CF patients.

Collagen and elastic fiber remodeling in the pregnant mouse myometrium†.

The myometrium undergoes progressive tissue remodeling from early to late pregnancy to support fetal growth and transitions to the contractile phase to deliver a baby at term. Much of our effort has been focused on understanding the functional role of myometrial smooth muscle cells, but the role of extracellular matrix is not clear. This study was aimed to demonstrate the expression profile of sub-sets of genes involved in the synthesis, processing, and assembly of collagen and elastic fibers, their structural remodeling during pregnancy, and hormonal regulation. Myometrial tissues were isolated from non-pregnant and pregnant mice to analyze gene expression and protein levels of components of collagen and elastic fibers. Second harmonic generation imaging was used to examine the morphology of collagen and elastic fibers. Gene and protein expressions of collagen and elastin were induced very early in pregnancy. Further, the gene expressions of some of the factors involved in the synthesis, processing, and assembly of collagen and elastic fibers were differentially expressed in the pregnant mouse myometrium. Our imaging analysis demonstrated that the collagen and elastic fibers undergo structural reorganization from early to late pregnancy. Collagen and elastin were differentially induced in response to estrogen and progesterone in the myometrium of ovariectomized mice. Collagen was induced by both estrogen and progesterone. By contrast, estrogen induced elastin, but progesterone suppressed its expression. The current study suggests progressive extracellular matrix remodeling and its potential role in the myometrial tissue mechanical function during pregnancy and parturition.

The Circadian Clock Gene Bmal1 Modulates Myometrium Contractile Function in Pregnant Mice

Caesarian section should be avoided unless medically required. Caesarian section is often a result of failed labor enhancement. While most natural births occur at night, labor enhancement is often scheduled during the day. We propose that the disparity between the commonly used timing of labor enhancement in the clinic, from the timing of natural birth, could contribute to the high rate of labor induction failure. Oxytocin receptor agonists are the most used labor enhancing agents. In pregnant non-human primates, oxytocin enhances uterine contractions more efficiently at night than during the day, suggesting a daily change in uterine sensitivity to oxytocin in pregnancy. To identify the molecular mechanisms generating daily changes in uterine function, we here explore the role of the molecular clock gene, Bmal1 (Brain and muscle ARNTL1-like), in the pregnant mouse myometrium. BMAL1 is a transcription factor required to generated circadian rhythms at the cellular level. We hypothesize that Bmal1 in uterine myometrial cells generates circadian rhythms and establishes the daily change in uterine contractile response to oxytocin. To evaluate circadian rhythms ex vivo, we collected myometrium samples from the validated circadian Per2:luciferase reporter mice at gestation day 17-18. We found that the pregnant mouse myometrium possesses circadian rhythms, which are generated by the molecular clock, as triple transgenic Per2:luciferase mice with Bmal1 conditionally deleted in the myometrium (cKO) do not have rhythmic expression of the Per2:luciferase reporter. To determine if BMAL1 is required to establish uterine contractions, we used a myograph to measure ex vivo uterine contractions at gestation day 17-18. In controls, uterine contraction force was significantly higher at ZT15 (3h after lights OFF) versus ZT3 (3h after lights ON). Interestingly, our preliminary data show increased basal contractile force at ZT15 in cKO as compared to controls. In addition, the cKO uterus contracted stronger to oxytocin than controls. Our findings identify Bmal1 as a clock gene modulating basal contractions in the mouse uterus and indicate Bmal1 might be a regulator of uterine sensitivity to oxytocin. Future work will focus on identifying the molecular mechanisms driven by BMAL1 to regulate uterine function in pregnancy. This work has the potential to provide insights into how we can improve labor enhancing treatment strategies in the clinic in the future.

Adiponectin links maternal metabolism to uterine contractility.

Adiponectin is secreted by adipose tissue and promotes insulin sensitivity. Low circulating adiponectin is associated with increased risk for preterm labor, but the influence of adiponectin on uterine myometrial physiology is unknown. We hypothesized that adiponectin receptors (AdipoRs) decrease myometrial contractility via AMPK to promote uterine quiescence in pregnancy. Using quantitative RT-PCR, we found that nonpregnant or pregnant human and mouse myometrium express AdipoR1 and AdipoR2 mRNAs. We confirmed AdipoR2 protein expression in human and mouse myometrium, with increased abundance in late mouse pregnancy. Both recombinant adiponectin and a pharmacologic AdipoR agonist, AdipoRon, potently inhibited uterine myometrial strip contractions in physiologic organ bath. The relaxation was independent of contractile stimulus (oxytocin, KCl, U46619). AdipoR agonists increased AMPK phosphorylation in pregnant mouse myometrium, and the direct AMPK activator A769662 also relaxed myometrial strips. However, the AMPK inhibitor dorsomorphin (compound C) blocked AMPK phosphorylation but did not abolish relaxation with either AdipoRon or A769662. In summary, adiponectin inhibits myometrial contractility consistent with the possibility that it is a previously unrecognized link between maternal metabolism and pregnancy maintenance. We also identify a separate role for AMPK regulating myometrial contractions that may influence labor onset.-Vyas, V., Guerra, D. D., Bok, R., Powell, T., Jansson, T., Hurt, K. J. Adiponectin links maternal metabolism to uterine contractility.

Differential Regulation of Human and Mouse Myometrial Contractile Activity by FSH as a Function of FSH Receptor Density.

Previous studies from our laboratory revealed that the follicle-stimulating hormone receptor (FSHR) is expressed at low levels in nonpregnant human myometrium and that it is up-regulated in pregnant term nonlaboring myometrium; however, the physiological relevance of these findings was unknown. Herein, we examined signaling pathways stimulated by FSH in immortalized uterine myocytes expressing recombinant FSHR at different densities and showed that cAMP accumulation is stimulated in all cases but that inositol phosphate accumulation is stimulated only at high FSHR densities. Because an increase in cAMP quiets myometrial contractile activity but an increase in 1,4,5-triphosphoinositol stimulates contractile activity, we hypothesized that FSHR density dictates whether FSH quiets or stimulates myometrial contractility. Indeed, in human and mouse nonpregnant myometrium, which express low levels of FSHR, application of FSH resulted in a quieting of contractile activity. In contrast, in pregnant term nonlaboring myometrium, which expresses higher levels of FSHR, application of FSH resulted in increased contractile activity. Examination of pregnant mouse myometrium from different stages of gestation revealed that FSHR levels remained low throughout most of pregnancy. Accordingly, through mid-gestation, the application of FSH resulted in a quieting of contractile activity. At Pregnancy Day (PD) 16.5, FSHR was up-regulated, although not yet sufficiently to mediate stimulation of contractility in response to FSH. This outcome was not observed until PD 19.5, when FSHR was further up-regulated. Our studies describe a novel FSHR signaling pathway that regulates myometrial contractility, and suggest that myometrial FSHR levels dictate the quieting vs. stimulation of uterine contractility in response to FSH.

Uterine endoplasmic reticulum stress and its unfolded protein response may regulate caspase 3 activation in the pregnant mouse uterus.

We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine caspase-3 at the transcriptional and translational level. Our study revealed robust activation of the uterine myocyte endoplasmic reticulum stress response and its adaptive unfolded protein response during pregnancy coinciding respectively with increased uterine caspase-3 activity and its withdrawal to term. In contrast the intrinsic and extrinsic apoptotic signaling pathways remained inactive across gestation. We speculate that physiological stimuli experienced by the pregnant uterus likely potentiates the uterine myocyte endoplasmic reticulum stress response resulting in elevated caspase-3 activation, which is isolated to the pregnant mouse myometrium. However as term approaches, activation of an elevated adaptive unfolded protein response acts to limit the endoplasmic reticulum stress response inhibiting caspase-3 resulting in its decline towards term. We speculate that these events have the capacity to regulate gestational length in a caspase-3 dependent manner.

The role of voltage-gated potassium channels in the regulation of mouse uterine contractility

BackgroundUterine smooth muscle cells exhibit ionic currents that appear to be important in the control of uterine contractility, but how these currents might produce the changes in contractile activity seen in pregnant myometrium has not been established. There are conflicting reports concerning the role of voltage-gated potassium (Kv) channels and large-conductance, calcium-activated potassium (BK) channels in the regulation of uterine contractility. In this study we provide molecular and functional evidence for a role for Kv channels in the regulation of spontaneous contractile activity in mouse myometrium, and also demonstrate a change in Kv channel regulation of contractility in pregnant mouse myometrium.MethodsFunctional assays which evaluated the effects of channel blockers and various contractile agonists were accomplished by quantifying contractility of isolated uterine smooth muscle obtained from nonpregnant mice as well as mice at various stages of pregnancy. Expression of Kv channel proteins in isolated uterine smooth muscle was evaluated by Western blots.ResultsThe Kv channel blocker 4-aminopyridine (4-AP) caused contractions in nonpregnant mouse myometrium (EC50 = 54 micromolar, maximal effect at 300 micromolar) but this effect disappeared in pregnant mice; similarly, the Kv4.2/Kv4.3 blocker phrixotoxin-2 caused contractions in nonpregnant, but not pregnant, myometrium. Contractile responses to 4-AP were not dependent upon nerves, as neither tetrodotoxin nor storage of tissues at room temperature significantly altered these responses, nor were responses dependent upon the presence of the endometrium. Spontaneous contractions and contractions in response to 4-AP did not appear to be mediated by BK, as the BK channel-selective blockers iberiotoxin, verruculogen, or tetraethylammonium failed to affect either spontaneous contractions or 4-AP-elicited responses. A number of different Kv channel alpha subunit proteins were found in isolated myometrium from both nonpregnant and term-pregnant mice, and one of these proteins – Kv4.3 – was found to disappear in term-pregnant tissues.ConclusionThese findings suggest a role for Kv channels in the regulation of uterine contractility, and that changes in the expression and/or function of specific Kv channels may account for the functional changes seen in pregnant myometrium.

Role of RYR3 splice variants in calcium signaling in mouse nonpregnant and pregnant myometrium

Alternative splicing of ryanodine receptor subtype 3 (RYR3) may generate a short isoform (RYR3S) without channel function and a functional full-length isoform (RYR3L). The RYR3S isoform has been shown to negatively regulate the native RYR2 subtype in smooth muscle cells as well as the RYR3L isoform when both isoforms were coexpressed in HEK-293 cells. Mouse myometrium expresses only the RYR3 subtype, but the role of RYR3 isoforms obtained by alternative splicing and their activation by cADP-ribose during pregnancy have never been investigated. Here, we show that both RYR3S and RYR3L isoforms are differentially expressed in nonpregnant and pregnant mouse myometrium. The use of antisense oligonucleotides directed against each isoform indicated that only RYR3L was activated by caffeine and cADP-ribose in nonpregnant myometrium. These RYR3L-mediated Ca(2+) releases were negatively regulated by RYR3S expression. At the end of pregnancy, the relative expression of RYR3L versus RYR3S and its ability to respond to cADP-ribose were increased. Therefore, our results suggest that physiological regulation of RYR3 alternative splicing may play an essential role at the end of pregnancy.

CME Test Questions Advances in cardiometabolic health

In the non-pregnant mouse myometrium, both arginine vasopressin and oxytocin induced contractions (pD2=8.55±0.13 and 9.23±0.09, respectively). The effect of oxytocin was the most potent, while the maximum contractions induced by these two peptides were almost of the same magnitude. Both vasopressin- and oxytocin-induced contractions were strongly inhibited by an oxytocin receptor antagonist, CL-12-42 (d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]OVT), and weakly inhibited by a vasopressin V1a receptor antagonist, SR49059 ((2S)1-[(2R,3S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide). Similar results were obtained in the pregnant mouse myometrium. These results suggest that not only oxytocin- but also vasopressin-induced contraction is mediated by the activation of oxytocin receptors in the mouse myometrium. A reverse transcription polymerase chain reaction study failed to reveal mRNA of the vasopressin V1a receptor in the mouse myometrium. In contrast, in the non-pregnant human myometrium, vasopressin-induced contraction was inhibited by SR49059. Oxytocin showed no effect on the myometrium. These results suggest that there are significant differences in the functional receptors and contractile responses to vasopressin and oxytocin in the human and mouse uteri.

Vasopressin-induced contraction of uterus is mediated solely by the oxytocin receptor in mice, but not in humans

In the non-pregnant mouse myometrium, both arginine vasopressin and oxytocin induced contractions (p D 2=8.55±0.13 and 9.23±0.09, respectively). The effect of oxytocin was the most potent, while the maximum contractions induced by these two peptides were almost of the same magnitude. Both vasopressin- and oxytocin-induced contractions were strongly inhibited by an oxytocin receptor antagonist, CL-12-42 (d(CH 2) 5[Tyr(Me) 2,Thr 4,Tyr-NH 2 9]OVT), and weakly inhibited by a vasopressin V 1a receptor antagonist, SR49059 ((2 S)1-[(2 R,3 S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1 H-indole-2-carbonyl]-pyrrolidine-2-carboxamide). Similar results were obtained in the pregnant mouse myometrium. These results suggest that not only oxytocin- but also vasopressin-induced contraction is mediated by the activation of oxytocin receptors in the mouse myometrium. A reverse transcription polymerase chain reaction study failed to reveal mRNA of the vasopressin V 1a receptor in the mouse myometrium. In contrast, in the non-pregnant human myometrium, vasopressin-induced contraction was inhibited by SR49059. Oxytocin showed no effect on the myometrium. These results suggest that there are significant differences in the functional receptors and contractile responses to vasopressin and oxytocin in the human and mouse uteri.

Catecholamines are not linked to myometrial phospholipase C and uterine contraction in late pregnant and parturient mouse.

1. We investigated whether catecholamines through activation of alpha(1)-adrenergic receptors (alpha(1)-AR) are involved in mouse uterine contraction at parturition. Myometrial phospholipase C (PLC) activity and uterine contraction were measured in response to noradrenaline (NA), the specific alpha(1)-AR agonist phenylephrine (Phe) and oxytocin (OT). 2. Using the reverse transcription-polymerase chain reaction RT-PCR, we detected the alpha(1a)-AR subtype in late pregnant mouse myometrium. We also detected, by immunoblotting studies, PLCbeta(1), PLCbeta(3) and different alpha-subunits of pertussis toxin-insensitive (Galpha(q/11)) and -sensitive G proteins (Galpha(o/i3), Galpha(i1/2)). 3. Phenylephrine and NA did not alter the myometrial inositol phosphate (InsP) production of late pregnant or parturient mouse. In similar conditions, OT increased InsP production in a dose-dependent manner. Consistent with these results, only OT (10 microM) recruited PLCbeta(1) and PLCbeta(3) to myometrial plasma membranes. The OT-induced InsP response was not altered by pertussis toxin (300 ng ml(-1), 2 h pretreatment), suggesting the involvement of a member of the Galpha(q) family. 4. Noradrenaline and Phe failed to increase uterine contraction at late pregnancy and at parturition. In contrast, OT induced uterine contraction in a dose-dependent manner with maximal increase (400 %) at a concentration of 1 microM. 5. The results indicate that OT receptors (OTR) but not alpha(1)-AR are linked to myometrial PLC activation and uterine contraction in late pregnant and parturient mouse. This discrepancy between mouse and other mammals could be attributed to the alpha(1)-AR subtype expressed in myometrium at this time.

Excitatory action of synthetic prostaglandin E2 on the electrical activity of pregnant mouse myometrium in relation to temperature changes and external sodium and calcium concentrations.

Influences of ions and temperature on the excitatory action of prostaglandin E2 (PGE) in the pregnant mouse myometrium were studied. In the normal Locke solution (35°C), low concentrations of PGE (10-9-10-8g/ml) did not depolarize the membrane, but increased the frequency of the burst spike discharges. High concentrations (10-6-10-5) caused depolarization by about 15-25 mV, and the muscle underwent contracture. The effects of low doses of PGE were potentiated to produce a measurable depolarization in low Ca media or at lower temperature, whereas the depolarizing action of PGE of high doses was depressed in excess Ca or in low Na media. Depolarization by PGE was restored in low Na solution when the external Ca concentration was reduced or when the tissue was exposed to lower temperature, suggesting that a possible shift of ENa alone was not the limiting factor in determining the amount of depolarization. It is argued from the above results that the excitatory effect of PGE varies, depending on the ratio of [Ca] 0/[Na] 0, in such a way that the relative dominance of [Ca] 0 tends to depress the drug action and vice versa. The effect of low temperatures on PGE action appears to resemble that of low Ca.

Modification of the mechanical response of the smooth muscles of pregnant mouse myometrium and guinea pig ileum by cadmium and manganese ions.

Effects of divalent cations on pregnant mouse myometrium and guinea pig ileum were investigated by recording electrical and mechanical activities.1. Application of transition metal cations (Mn, Cd, Co, Ni, and Zn) inhibited the spontaneous activities of mouse myometrium. Except for Mn, treatment with these cations in a concentration of 1mM and for 1-2min caused an irreversible suppression of the mechanical response.2. Myometrial membrane was hyperpolarized with 0.5mM Cd decreasing membrane resistance. During application, contractures induced by excess K or carbachol was inhibited. After treatment with 0.5-1mM Cd for 1-2min, electrical and mechanical activities dissociated, i. e. spike activity resumed but mechanical activity was suppressed. The membrane was depolarized by carbachol to the control value, but contraction did not developed.3. By contrast, contracture of mouse myometrium induced by excess K or carbachol application were potentiated after treatment with Mn.4. The effects of divalent cations on guinea pig ileum were essentially the same as with mouse myometrium, i. e. treatment with Mn potentiated mechanical response whereas other ions caused irreversible suppression.

Effects of external Na and Ca on the mouse myometrium in relation to the effects of oxytocin and carbachol.

Depolarizing action of 10-3U/ml oxytocin and 10-4g/ml carbachol on pregnant mouse myometrium in ionic solutions containing various concentrations of external Na and Ca was studied.1) Excess Ca (10mM) caused hyperpolarization in normal and in low Na (15.5mM) solutions, and decreased membrane conductance.2) The membrane was depolarized by about 10 mV in low Na solutions (15.5mM) and membrane conductance was decreased. Reduction of external Ca to 0.25 mM caused further depolarization and an increase membrane conductance.3) Depolarization caused by carbachol was reduced in solutions containing low Na or excess Ca. The depolarizing action was restored by reducing external Ca to 0.25mM in low Na solutions. The peak depolarization produced by oxytocin was little affected in any of the above ionic solutions.4) In Na-free solutions, where the membrane was depolarized to -25 mV and contracture was produced, neither oxytocin nor carbachol was effective in producing changes in membrane potential or in tension development.

Effects of catecholamines on electrical and mechanical activities of the pregnant mouse myometrium.

1. The effect of catecholamines (adrenaline, noradrenaline, and isoprenaline) in combination with α- and β-blocking agents (phentolamine and propranolol) on pregnant mouse myometrium was investigated. The catecholamines had an inhibitory action in this tissue, the minimal dose required to produce inhibition being 3×10-9 (g/ml) for isoprenaline, <10-7 for adrenaline, and <10-6 for noradrenaline. While β-adrenergic blocking agent antagonized the inhibitory action of the catecholamines, α-blocking agent did not.2. Catecholamines produced a hyperpolarization of the membrane and hence spontaneous activity was inhibited. External current stimulation produced a spike potential with an amplitude comparable to that in Krebs solution. During hyperpolarization, there was no statistically significant change in membrane resistance, nor was there a difference between the effects of noradrenaline and isoprenaline.3. When spontaneous activity was present in normal or low-Na (about 10mM) solution, isoprenaline (10-8g/ml) abolished it. Depolarization and contracture evoked in zero Na (replacement with Tris or sucrose) were restored to normal by isoprenaline, and a spike potential was evoked which was bigger than that in Krebs solution. The existence of external Ca was essential to production of the repolarization.4. From the above results, a tentative hypothesis on the mechanism of action of catecholamines on pregnant mouse uterus was made as follows:(i) Hyperpolarization, which is responsible for the inhibitory action, is due either to an acceleration of electrogenic Na pumping or to a change in the interaction between the membrane and extracellular, as well as intracellular, Ca.(ii) Isoprenaline reduces [Ca] i, hence abolishing the contracture in zero Na. This may also lead to the production of an action potential in Na-free solution.