Fig 1
LacZ activation upon conditional Cx43 deletion. (A) A silent lacZ reporter gene (NLS-lacZ) was integrated into the floxed Cx43 allele. After Cre/loxP-mediated rearrangement of the Cx43-coding region, indicated by loxP sites (triangles), the lacZ gene is activated and the-galactosidase protein is expressed. (B) The CreER T2 fusion protein is specifically transcribed under control of the SM22 promoter in smooth muscle cells. In the cytosol, CreER T2 is associated with the heat shock protein 90 (HSP 90). After application of the ligand (tamoxifen) this complex dissociates and CreER T2 is translocated into the nucleus, where deletion of the floxed coding region occurs.
Source publication
Gap junctions are characteristically increased in the myometrium during term and preterm delivery and are thought to be essential for the development of uterine contractions during labour. Expression of connexin43 (Cx43), the major myometrial gap junction protein, is increased during delivery. We have generated a mouse mutant (Cx43fl/fl:SM-CreERT2)...
Contexts in source publication
Context 1
... 2lox/2lox :SM-CreER T2 mice to bypass postnatal lethality of Cx43-null animals and to study the role of Cx43 gap junctions during pregnancy and parturition. Since we found no differences between animals harbouring the Cx43 fl alleles or not carrying the floxed neomycin cassette (Cx43 2lox ), we refer to both groups as Cx43 fl mice. As shown in Fig. 1A, Cre- mediated deletion of the floxed Cx43-coding region resulted in expression of the NLS-lacZ reporter gene under control of the Cx43-specific promoter ( Theis et al., 2001;Eckardt et al., 2004). The Cre recombinase used in this study is a fusion protein of Cre and the tamoxifen-responsive estrogen receptor (ER T2 ; Fig. 1B) ...
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... fl mice. As shown in Fig. 1A, Cre- mediated deletion of the floxed Cx43-coding region resulted in expression of the NLS-lacZ reporter gene under control of the Cx43-specific promoter ( Theis et al., 2001;Eckardt et al., 2004). The Cre recombinase used in this study is a fusion protein of Cre and the tamoxifen-responsive estrogen receptor (ER T2 ; Fig. 1B) regulated by the smooth-muscle-cell-specific SM22 promoter ( Kühbandner et al., 2000). Only after application of tamoxifen, the HSP90-bound SM-CreER T2 fusion protein changed its conformation, separated from HSP90, translocated to the nucleus and mediated the deletion of the Cx43-coding region. To monitor the expression profile of ...
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... generated transgenic Cx43 fl/fl :SM-CreER T2 and Cx43 2lox/2lox :SM-CreER T2 mice to bypass postnatal lethality of Cx43-null animals and to study the role of Cx43 gap junctions during pregnancy and parturition. Since we found no differences between animals harbouring the Cx43 fl alleles or not carrying the floxed neomycin cassette (Cx43 2lox ), we refer to both groups as Cx43 fl mice. As shown in Fig. 1A, Cre- mediated deletion of the floxed Cx43-coding region resulted in expression of the NLS-lacZ reporter gene under control of the Cx43-specific promoter ( Theis et al., 2001;Eckardt et al., 2004). The Cre recombinase used in this study is a fusion protein of Cre and the tamoxifen-responsive estrogen receptor (ER T2 ; Fig. 1B) regulated by the smooth-muscle-cell-specific SM22 promoter ( Kühbandner et al., 2000). Only after application of tamoxifen, the HSP90-bound SM-CreER T2 fusion protein changed its conformation, separated from HSP90, translocated to the nucleus and mediated the deletion of the Cx43-coding region. To monitor the expression profile of Cx43 by means of the lacZ reporter gene, we used Cx43 del/+ mice (i.e. in which one floxed Cx43 allele had been ...
Context 4
... generated transgenic Cx43 fl/fl :SM-CreER T2 and Cx43 2lox/2lox :SM-CreER T2 mice to bypass postnatal lethality of Cx43-null animals and to study the role of Cx43 gap junctions during pregnancy and parturition. Since we found no differences between animals harbouring the Cx43 fl alleles or not carrying the floxed neomycin cassette (Cx43 2lox ), we refer to both groups as Cx43 fl mice. As shown in Fig. 1A, Cre- mediated deletion of the floxed Cx43-coding region resulted in expression of the NLS-lacZ reporter gene under control of the Cx43-specific promoter ( Theis et al., 2001;Eckardt et al., 2004). The Cre recombinase used in this study is a fusion protein of Cre and the tamoxifen-responsive estrogen receptor (ER T2 ; Fig. 1B) regulated by the smooth-muscle-cell-specific SM22 promoter ( Kühbandner et al., 2000). Only after application of tamoxifen, the HSP90-bound SM-CreER T2 fusion protein changed its conformation, separated from HSP90, translocated to the nucleus and mediated the deletion of the Cx43-coding region. To monitor the expression profile of Cx43 by means of the lacZ reporter gene, we used Cx43 del/+ mice (i.e. in which one floxed Cx43 allele had been ...
Citations
... RNA-seq data from the murine myometrium demonstrates that Sox4, Sox7, and Sox9 are upregulated whereas Sox8 is downregulated at labor onset compared to late pregnancy [3]. Furthermore, the promoter of Gja1, a gene used as a proxy for transcription at labor, owing to its elevated expression in the laboring myometrium, [3,[13][14][15] contains several SOX motifs. Interestingly, SOX4, SOX8, and SOX9 were also found to cooperate with cJUN, a member of the AP-1 family, in activating the expression of Gja1 in Sertoli cells [16]. ...
The uterine muscular layer, or myometrium, undergoes profound changes in global gene expression during its progression from a quiescent state during pregnancy to a contractile state at the onset of labor. In this study, we investigate the role of SOX family transcription factors in myometrial cells and provide evidence for the role of SOX4 in regulating labor-associated genes. We show that Sox4 has elevated expression in the murine myometrium during a term laboring process and in two mouse models of preterm labor. Additionally, SOX4 differentially affects labor-associated gene promoter activity in cooperation with activator protein 1 (AP-1) dimers. SOX4 exerted no effect on the Gja1 promoter; a JUND-specific activation effect at the Fos promoter; a positive activation effect on the Mmp11 promoter with the AP-1 dimers; and surprisingly, we noted that the reporter expression of the Ptgs2 promoter in the presence of JUND and FOSL2 was repressed by the addition of SOX4. Our data indicate SOX4 may play a diverse role in regulating gene expression in the laboring myometrium in cooperation with AP-1 factors. This study enhances our current understanding of the regulatory network that governs the transcriptional changes associated with the onset of labor and highlights a new molecular player that may contribute to the labor transcriptional program.
... With a well-established rat model of maternal obesity that exhibits prolonged labor, the potential mechanisms can now be unraveled to improve labor outcomes in obese human pregnancies. Ablation of murine uterine gene expression of cx43 by Tamoxifen has been observed to prolong labor by compromising synchronization of myometrial contractions (Döring et al., 2006). This finding matches the significant decrease in uterine expression of cx43 in the laboring uteri of HFHC rats (Elmes et al., 2011;Muir et al., 2016) that also exhibit asynchronous myometrial contractions ex vivo (Muir et al., 2016). ...
Maternal obesity is associated with increased risk of prolonged and dysfunctional labor and emergency caesarean section. To elucidate the mechanisms behind the associated uterine dystocia, a translational animal model is required. Our previous work identified that exposure to a high-fat, high-cholesterol (HFHC) diet to induce obesity down-regulates uterine contractile associated protein expression and causes asynchronous contractions ex vivo. This study aims to investigate the impact of maternal obesity on uterine contractile function in vivo using intrauterine telemetry surgery. Virgin female Wistar rats were fed either a control (CON, n = 6) or HFHC (n = 6) diet for 6 weeks prior to conception, and throughout pregnancy. On Day 9 of gestation, a pressure-sensitive catheter was surgically implanted aseptically within the gravid uterus. Following 5 days recovery, intrauterine pressure (IUP) was recorded continuously until delivery of the 5th pup (Day 22). HFHC induced obesity led to a significant 1.5-fold increase in IUP (p = 0.026) and fivefold increase in frequency of contractions (p = 0.013) relative to CON. Determination of the time of labor onset identified that HFHC rats IUP (p = 0.046) increased significantly 8 h prior to 5th pup delivery, which contrasts to CON with no significant increase. Myometrial contractile frequency in HFHC rats significantly increased 12 h prior to delivery of the 5th pup (p = 0.023) compared to only 3 h in CON, providing evidence that labor in HFHC rats was prolonged by 9 h. In conclusion, we have established a translational rat model that will allow us to unravel the mechanism behind uterine dystocia associated with maternal obesity.
... Inhibition of the contractile associated protein Cx43 has been suggested as a potential tocolytic strategy [23]. Cx43 gap junctions increase intercellular communications to play a critical role in propagating contractions in the myometrium [24]. At the time of labor, Cx43 gene expression increases [25]. ...
Preterm labor, delivery prior to 37 completed weeks of gestation, is the leading cause of infant morbidity and mortality. β3 adrenergic receptor protein expression is increased in the myometrium during pregnancy, and the agonist, mirabegron, relaxes the myometrium making the β3 adrenergic receptor a potential therapeutic target in PTL. β3 adrenergic receptor has been shown to activate the tyrosine kinase, Src, which can down regulate connexin 43, a contractile associated protein which promotes the formation of gap junctions that create an electrical syncytium. We hypothesize that mirabegron downregulates connexin 43, imparting quiescence effects on the myometrium. Employing contractile studies, we demonstrate that Src is involved in the mirabegron-induced relaxation of contracting pregnant human myometrial tissue strips. Western blot analysis demonstrates that Src kinase expression is decreased in both preterm and term laboring myometrial tissue. Imaging revealed that mirabegron stimulation of the β3 adrenergic receptor phosphorylates tyrosine at position Y265 on connexin 43 in pregnant human uterine myocytes. Western blot analysis and immunofluorescent imaging indicate that mirabegron decreases the expression of connexin 43 and mediates relaxation over a 24-h exposure period, suggesting that mirabegron has long lasting quiescent effects on the human myometrium. The relationship between the β3 adrenergic receptor and down regulation of the contractile associated protein connexin 43 through activation of Src kinase suggests that mirabegron may be useful in combination tocolysis.
... Lastly, the identical parturition timing of untreated B6 and Il33 À/À mice (Figure 1D) meant that the extended delay of DMPA-injected Il33 À/À mice could not be explained by intrinsic defects in myometrial contractility. As exemplified by mice with defects in connexin 43 expression by SMCs, 14 which prevents these cells from achieving the electrical coupling necessary for synchronous contraction, such defects would be expected to delay luteolysis-driven parturition onset. Together, these results suggest that maternal IL-33 plays a critical role in labor onset timing in mice when parturition occurs through uterus-intrinsic pathways. ...
Although mice normally enter labor when their ovaries stop producing progesterone (luteolysis), parturition can also be triggered in this species through uterus-intrinsic pathways potentially analogous to the ones that trigger parturition in humans. Such pathways, however, remain largely undefined in both species. Here, we report that mice deficient in innate type 2 immunity experienced profound parturition delays when manipulated endocrinologically to circumvent luteolysis, thus obliging them to enter labor through uterus-intrinsic pathways. We found that these pathways were in part driven by the alarmin IL-33 produced by uterine interstitial fibroblasts. We also implicated important roles for uterine group 2 innate lymphoid cells, which demonstrated IL-33-dependent activation prior to labor onset, and eosinophils, which displayed evidence of elevated turnover in the prepartum uterus. These findings reveal a role for innate type 2 immunity in controlling the timing of labor onset through a cascade potentially relevant to human parturition.
... More specifically, many contraction-driving genes undergo increased primary transcript synthesis at labor onset [8]. For instance, the widely studied Connexin 43 (encoded by the GJA1 gene), a vital contraction-associated protein [9,10], is expressed at low levels in the late gestational quiescent stages, and sharply induced at term labor onset in part due to the spike in active transcription at its coding gene [8]. But the mechanism by which these transcriptional changes are conferred on genes like GJA1 during the SMC quiescence-to-contractility transition is not well understood. ...
... Prior studies had observed elevated reporter gene expression levels under the control of the Gja1 promoter in Syrian hamster myometrial (SHM) cells in the presence of AP-1 heterodimers but not AP-1 homodimers [11,12]. As previously outlined, Gja1 is necessary for the initiation of contractions [9,10] and the output of this gene promoter has often been used as a proxy for the laboring transcriptional program phenotype. We therefore generated a luciferase construct with a reporter gene under the control of the endogenous murine Gja1 promoter to test the capacity of MYB and ELF3 in activating this promoter, either on their own or in the presence of AP-1 factors. ...
Spontaneous uterine contractions are initiated when smooth muscle cells (SMCs) within the uterine muscle, or myometrium, transition from a functionally dormant to an actively contractile phenotype at the end of the pregnancy period. We know that this process is accompanied by gestational time point-specific differences in the SMC transcriptome, which can be modulated by the activator protein 1 (AP-1), nuclear factor kappa beta (NF-κβ), estrogen receptor (ER), and progesterone receptor (PR) transcription factors. Less is known, however, about the additional proteins that might assist these factors in conferring the transcriptional changes observed at labor onset. Here, we present functional evidence for the roles of two proteins previously understudied in the SMC context-MYB and ELF3-which can contribute to the regulation of labor-driving gene transcription. We show that the MYB and ELF3 genes exhibit elevated transcript expression levels in mouse and human myometrial tissues during spontaneous term labor. The expression of both genes was also significantly increased in mouse myometrium during preterm labor induced by the progesterone antagonist mifepristone (RU486), but not during infection-simulating preterm labor induced by intrauterine infusion of lipopolysaccharide (LPS). Furthermore, both MYB and ELF3 proteins affect labor-driving gene promoter activity, although in surprisingly opposing ways: Gja1 and Fos promoter activation increases in the presence of MYB and decreases in the presence of ELF3. Collectively, our study adds to the current understanding of the transcription factor network that defines the transcriptomes of SMCs during late gestation and implicates two new players in the control of labor timing.
... 4.6.1. Connexin 43 fl/fl :SM-CreER T2 mice: Sm-CreER T2 KO Delayed delivery was observed in 82% of mice [19]. Uterine contractions were not directly measured. ...
... Uterine contractions were not directly measured. The author reported that the cause of the abnormal delivery was insufficient contraction due to the loss of coordinated contraction of the uterine muscle [19]. 4.6.2. ...
In humans, the incidence of post-term delivery is 1%–10%. Post-term delivery significantly increases the risk of cesarean section or neonatal intensive care unit (NICU) admission. Despite these serious challenges, the cause of prolonged delivery remains unclear. Several common factors of delayed parturition between mice and humans will help elucidate the mechanisms of pregnancy and labor. At present, gene modification techniques are rapidly developing; however, there are limited reviews available describing the mouse phenotype analysis as a human model for post-term delivery. We classified the delayed-labor mice into nine types according to their causes. In mice, progesterone (P₄) maintains pregnancy, and the most common cause of delayed labor is luteolysis failure. Other contributing factors include humoral molecules in the fetus/placenta, uterine contractile dysfunction, poor cervical ripening, and delayed implantation. The etiology of delayed parturition is overexpression of the pregnancy maintenance mechanism or suppression of the labor induction mechanism. Here, we describe how to investigated their causes using mouse genetic analysis. In addition, we generated a list to identify the causes. Our review will help understand the findings obtained using the mouse model, providing a foundation for conducting more systematic research on delayed delivery.
... As aforementioned, oxytocin and Oxtr knockout mice have no apparent parturition defects, despite an absence of oxytocin-induced uterine contractions [40,41]. Almost 20% of mice lacking connexin 43, the major myometrial gap junction protein, still deliver on time [66]. These examples are consistent with the idea that redundancies and adaptations make labor resilient to the removal of any one player in the process, and suggest that the labor dysfunction phenotype of the Lepr cKO is biologically significant, even though it occurred in less than half the dams. ...
Leptin is required for fertility, including initiation of estrous cycles. It is therefore challenging to assess the role of leptin signaling during pregnancy. While neuron-specific transgene approaches suggest that leptin signaling in the central nervous system is most important, experiments with pharmacologic inhibition of leptin in the uterus or global replacement of leptin during pregnancy suggest leptin signaling in the reproductive tract may be required. Here, conditional leptin receptor knockout (Lepr cKO) with a progesterone receptor-driven Cre recombinase was used to examine the importance of leptin signaling in pregnancy. Lepr cKO mice have almost no leptin receptor in uterus or cervix, and slightly reduced leptin receptor levels in corpus luteum. Estrous cycles and progesterone concentrations were not affected by Lepr cKO. Numbers of viable embryos did not differ between primiparous control and Lepr cKO dams on days 6.5 and 17.5 of pregnancy, despite a slight reduction in the ratio of embryos to corpora lutea, showing that uterine leptin receptor signaling is not required for embryo implantation. Placentas of Lepr cKO dams had normal weight and structure. However, over four parities, Lepr cKO mice produced 22% fewer live pups than controls, and took more time from pairing to delivery by their fourth parity. Abnormal birth outcomes of either dystocia or dead pups occurred in 33% of Lepr cKO deliveries but zero control deliveries, and the average time to deliver each pup after crouching was significantly increased. Thus, leptin receptor signaling in the reproductive tract is required for normal labor and delivery.
Summary sentence.
Mice lacking leptin receptor in the reproductive tract produce fewer live pups and have more adverse labor outcomes than controls, but normal numbers of embryos near term, showing that leptin receptor signaling is required for normal parturition.
... Research by Takayanagi et al. revealed that Oxtr-deficient mice are still able to perform normal parturition [58]. Interestingly, a delayed delivery occurred in connexin43-deficient mice in the study of Döring et al [59]. Coincidentally, the results here also illustrate that blockade of Gja1 and Ptgs2 suppresses the enhanced myometrial contractility under hypoxia more than the inhibition of Oxtr. ...
... Research by Takayanagi et al. revealed that Oxtr-deficient mice are still able to perform normal parturition [58]. Interestingly, a delayed delivery occurred in connexin43-deficient mice in the study of Döring et al [59]. Coincidentally, the results here also illustrate that blockade of Gja1 and Ptgs2 suppresses the enhanced myometrial contractility under hypoxia more than the inhibition of Oxtr. ...
... To this end, we provide here a consolidated overview of research in broader areas, covering not only machine-learning based EHG signal analysis and applications, but also on clinical studies, biological experiments and theoretical physics, in the hope of inspiring new directions for research on EHG-based preterm birth diagnosis methods. However, research on the effect of medical interventions are not included, as medications should only be given to pregnant women diagnosed as being at high risk of preterm delivery [15][16][17]. ...
Preterm birth is the leading cause of neonatal morbidity and mortality. Early identification of high-risk deliveries, combined with appropriate medication appears as the way to treat the problem, although effective early diagnostic methods remain elusive. Since the strong contractions that drive the fetus out of the uterus can be traced back to the electrical activity of uterine muscle cells, the external recording of these activities in the form of electrohysterogram (EHG) opens a new research direction for the development of preterm diagnostic methods. This review fo- cuses on EHG signal analysis and its application to preterm birth diagnostic methods, and in particular on the analysis of such signals using machine learning techniques. We introduce the publicly available databases of EHG recordings, used as a testing ground for this approach, as well as the methods to extract meaningful information from the raw data. We stress the problem of imbalance, due to the comparatively small number of patients suffering from preterm births, and discuss techniques to mitigate these effects. Last, we discuss the possibility to identify and characterize contractions directly from EHG signals. We conclude by discussing new research directions, in particular based on a biophysical description of uterus contraction towards the end of pregnancy, and their possible applications to extract more effective features and for better dealing with the shortage of training examples.
