Molecular and functional characterization of the SBP-box transcription factor SPL-CNR in tomato fruit ripening and cell death.

SlSPL-CNR, an SBP-box transcription factor (TF) gene residing at the epimutant Colourless non-ripening (Cnr) locus, is involved in tomato ripening. This epimutant provides a unique model to investigate the (epi)genetic basis of fruit ripening. Here we report that SlSPL-CNR is a nucleus-localized protein with a distinct monopartite nuclear localization signal (NLS). It consists of four consecutive residues ' 30KRKR33' at the N-terminus of the protein. Mutation of the NLS abolishes SlSPL-CNR's ability to localize in the nucleus. SlSPL-CNR comprises two zinc-finger motifs (ZFMs) within the C-terminal SBP-box domain. Both ZFMs contribute to zinc-binding activity. SlSPL-CNR can induce cell death in tomato and tobacco, dependent on its nuclear localization. However, the two ZFMs have differential impacts on SlSPL-CNR's induction of severe necrosis or mild necrotic ringspot. NLS and ZFM mutants cannot complement Cnr fruits to ripen. SlSPL-CNR interacts with SlSnRK1. Virus-induced SlSnRK1 silencing leads to reduction in expression of ripening-related genes and inhibits ripening in tomato. We conclude that SlSPL-CNR is a multifunctional protein that consists of a distinct monopartite NLS, binds to zinc, and interacts with SlSnRK1 to affect cell death and tomato fruit ripening.

Identification of uterine pacemaker regions at the myometrial-placental interface in the rat.

KEY POINTS: Coordinated contraction of the uterine smooth muscle is essential to parturition. Histologically and physiologically defined pacemaker structures have not been identified in uterine smooth muscle. Here we report combined electrophysiological and histological evidence of zones associated with pacemaker activity in the rat myometrium. Our method relies crucially on the integration of histological and electrophysiological data in an in silico three-dimensional reconstruction of the rat myometrium at 10 µm resolution. We find that myometrial/placental pacemaking zones are closely related with placental sites and the area of disruptive myometrial remodelling surrounding such sites. If analogues of the myometrial/placental pacemaking zone are present in the human, defining their histology and physiology will be important steps towards treatment of pre-term birth, pre-eclampsia, and postpartum haemorrhage. ABSTRACT: Coordinated uterine contractions are essential for delivering viable offspring in mammals. In contrast to other visceral smooth muscles, it is not known where excitation within the uterus is initiated, and no defined pacemaking region has hitherto been identified. Using multi-electrode array recordings and high-resolution computational reconstruction of the three-dimensional micro-structure of late pregnant rat uterus, we demonstrate that electrical potentials are initiated in distinct structures within the placental bed of individual implantation sites. These previously unidentified structures represent modified smooth muscle bundles that are derived from bridges between the longitudinal and circular layers. Coordinated implantation and encapsulation by invading trophoblast give rise to isolated placental/myometrial interface bundles that directly connect to the overlying longitudinal smooth muscle layer. Taken together, these observations imply that the anatomical structure of the uterus, combined with site-specific implantation, gives rise to emergent patterns of electrical activity that drive effective contractility during parturition.

Reconstruction of Cell Surface Densities of Ion Pumps, Exchangers, and Channels from mRNA Expression, Conductance Kinetics, Whole-Cell Calcium, and Current-Clamp Voltage Recordings, with an Application to Human Uterine Smooth Muscle Cells.

Uterine smooth muscle cells remain quiescent throughout most of gestation, only generating spontaneous action potentials immediately prior to, and during, labor. This study presents a method that combines transcriptomics with biophysical recordings to characterise the conductance repertoire of these cells, the 'conductance repertoire' being the total complement of ion channels and transporters expressed by an electrically active cell. Transcriptomic analysis provides a set of potential electrogenic entities, of which the conductance repertoire is a subset. Each entity within the conductance repertoire was modeled independently and its gating parameter values were fixed using the available biophysical data. The only remaining free parameters were the surface densities for each entity. We characterise the space of combinations of surface densities (density vectors) consistent with experimentally observed membrane potential and calcium waveforms. This yields insights on the functional redundancy of the system as well as its behavioral versatility. Our approach couples high-throughput transcriptomic data with physiological behaviors in health and disease, and provides a formal method to link genotype to phenotype in excitable systems. We accurately predict current densities and chart functional redundancy. For example, we find that to evoke the observed voltage waveform, the BK channel is functionally redundant whereas hERG is essential. Furthermore, our analysis suggests that activation of calcium-activated chloride conductances by intracellular calcium release is the key factor underlying spontaneous depolarisations.

The location of pacemakers in the uteri of pregnant guinea pigs and rats.

The pregnant uterus is a smooth muscle organ whose pattern of contraction is dictated by the propagation of electrical impulses. Such electrical activity may originate from one or more pacemakers, but the location of these sites has not yet been determined. To detect the location of the pacemaker in the gravid uterus, two approaches were used: 1) determine the site from where the contraction started using isolated uteri from the pregnant guinea pig, and videotape their contractions; and 2) record, in isolated uteri from pregnant term rats, with 240 extracellular electrodes simultaneously, and determine where the electrical bursts started. In both the contractile and electrophysiological experiments, there was not a single, specific pacemaker area. However, most contractions (guinea pig 87%) and bursts (rat 76%) started close to the mesometrial border (mean 2.7 ± 4.0 mm SD in guinea pigs and 1.3 ± 1.4 mm in rats). In addition, in the rat, most sites of initiations were located closer to the ovarial end of the horn (mean distance from the ovarial end 6.0 ± 6.2 mm SD), whereas such an orientation was not seen in the guinea pig. In both guinea pig and rat uteri at term, there is not one specific pacemaker area. Rather, contractile and electrical activity may arise from any site, with the majority starting close to the mesometrial border. Furthermore, in the rat, most activities started at the ovarial end of the horn. This may suggest a slightly different pattern of contraction in both species.

Functional and morphological development of the womb throughout life.

The uterus undergoes changes throughout a woman's life, beginning with her own embryonic development when she is still in the womb, commencing a monthly cycle at the onset of adulthood, and undergoing dramatic changes during pregnancy and parturition. The impact of preterm labour and other perinatal health problems is significant, both in human and financial terms; therefore the study of the physiological and regulatory changes which the uterus undergoes can be of enormous potential benefit. Here we briefly review the current state of knowledge, with an emphasis on the importance of changes in connectivity in the uterine smooth muscle cell network and on recent mathematical modelling work aimed at elucidating the role of spatial heterogeneity in this connected network.

Assessment of myometrial transcriptome changes associated with spontaneous human labour by high-throughput RNA-seq.

The transition of the human uterus from a quiescent to a contractile state takes place over a number of weeks. On such biological time scales, cellular phenotype is modified by changes in the transcriptome, which in turn is under the control of the underlying endocrine, paracrine, and biophysical processes resulting from the ongoing pregnancy. In this study, we characterize the transition of the human myometrial transcriptome at term from not in labour (NIL) to in labour (LAB) using high throughput RNA sequencing (RNA-seq). RNA was isolated from the myometrium of uterine biopsies from patients at term who were not in labour (n = 5) and at term in spontaneous labour (n = 5) without augmentation. A total of 143.6 million separate reads were sequenced, achieving, on average, ∼13 times coverage of the expressed human transcriptome per sample. Principal component analysis indicated that the NIL and LAB transcriptomes could be distinguished as two distinct clusters. A comparison of the NIL and LAB groups, using three different statistical approaches (baySeq, edgeR, and DESeq), demonstrated an overlap of 764 differentially expressed genes. A comparison with currently available microarray data revealed only a partial overlap in differentially expressed genes. We conclude that the described RNA-seq data sets represent the first fully annotated catalogue of expressed mRNAs in human myometrium. When considered together, the full expression repertoire and the differentially expressed gene sets should provide an excellent resource for formulating new hypotheses of physiological function, as well as the discovery of novel therapeutic targets.

PGF2α induces a pro-labour phenotypical switch in human myometrial cells that can be inhibited with PGF2α receptor antagonists.

Preterm birth is the leading cause of infant morbidity and mortality. There has been an interest in developing prostaglandin F2α (PGF2α) antagonists as a new treatment for preterm birth, although much of the rationale for their use is based on studies in rodents where PGF2α initiates labour by regressing the corpus luteum and reducing systemic progesterone concentrations. How PGF2α antagonism would act in humans who do not have a fall in systemic progesterone remains unclear. One possibility, in addition to an acute stimulation of contractions, is a direct alteration of the myometrial smooth muscle cell state towards a pro-labour phenotype. In this study, we developed an immortalised myometrial cell line, MYLA, derived from myometrial tissue obtained from a pregnant, non-labouring patient, as well as a novel class of PGF2α receptor (FP) antagonist. We verified the functionality of the cell line by stimulation with PGF2α, resulting in Gαq-specific coupling and Ca2+ release, which were inhibited by FP antagonism. Compared to four published FP receptor antagonists, the novel FP antagonist N582707 was the most potent compound [Fmax 7.67 ± 0.63 (IC50 21.26 nM), AUC 7.30 ± 0.32 (IC50 50.43 nM), and frequency of Ca2+ oscillations 7.66 ± 0.41 (IC50 22.15 nM)]. RNA-sequencing of the MYLA cell line at 1, 3, 6, 12, 24, and 48 h post PGF2α treatment revealed a transforming phenotype from a fibroblastic to smooth muscle mRNA profile. PGF2α treatment increased the expression of MYLK, CALD1, and CNN1 as well as the pro-labour genes OXTR, IL6, and IL11, which were inhibited by FP antagonism. Concomitant with the inhibition of a smooth muscle, pro-labour transition, FP antagonism increased the expression of the fibroblast marker genes DCN, FBLN1, and PDGFRA. Our findings suggest that in addition to the well-described acute contractile effect, PGF2α transforms myometrial smooth muscle cells from a myofibroblast to a smooth muscle, pro-labour-like state and that the novel compound N582707 has the potential for prophylactic use in preterm labour management beyond its use as an acute tocolytic drug.

Characterization of the tissue-level Ca2+ signals in spontaneously contracting human myometrium.

In the labouring uterus, millions of myocytes forming the complex geometrical structure of myometrium contract in synchrony to increase intrauterine pressure, dilate the cervix and eventually expel the foetus through the birth canal. The mechanisms underlying the precise coordination of contractions in human myometrium are not completely understood. In the present study, we have characterized the spatio-temporal properties of tissue-level [Ca(2+)](i) transients in thin slices of intact human myometrium. We found that the waveform of [Ca(2+)](i) transients and isotonic contractions recorded from thin slices was similar to the waveform of isometric contractions recorded from the larger strips in traditional organ bath experiments, suggesting that the spatio-temporal information obtained from thin slices is representative of the whole tissue. By comparing the time course of [Ca(2+)](i) transients in individual cells to that recorded from the bundles of myocytes we found that the majority of myocytes produce rapidly propagating long-lasting [Ca(2+)](i) transients accompanied by contractions. We also found a small number of cells showing desynchronized [Ca(2+)](i) oscillations that did not trigger contractions. The [Ca(2+)](i) oscillations in these cells were insensitive to nifedipine, but readily inhibited by the T-type Ca(2+) channel inhibitor NNC55-0396. In conclusion, our data suggest that the spread of [Ca(2+)](i) signals in human myometrium is achieved via propagation of long-lasting action potentials. The propagation was fast when action potentials propagated along bundles of myocytes and slower when propagating between the bundles of uterine myocytes.

Labor and inflammation increase the expression of oxytocin receptor in human amnion.

The oxytocin/oxytocin receptor (OXT/OXTR) system plays an important role in the regulation of parturition. The amnion is a major source of prostaglandins and inflammatory cytokine synthesis, which increase both before and during labor. Amnion is a noncontractile tissue; therefore, the role played by OXT/OXTR in this tissue will be fundamentally different from the role played in myometrial contractions. In the present study, we demonstrate increased OXTR mRNA and protein concentrations in human amnion epithelial cells associated with the onset of labor. We show that incubation of primary human amnion epithelial cells with IL1B results in a rapid, transient up-regulation of OXTR mRNA expression, which peaks in prelabor samples after 6 h. Incubation of prelabor amnion epithelial cells with OXT results in a marked increase of prostaglandin E(2) synthesis, and we demonstrate that OXT activates the extracellular signal-regulated protein kinase signal transduction pathway to stimulate up-regulation of cyclo-oxygenase 2 in human amnion epithelial cells. The increased ability of human amnion to produce prostaglandins in response to OXT treatment suggests a complementary role for the OXT/OXTR system in the activation of human amnion and the onset of labor.

Functionally Selective Inhibition of the Oxytocin Receptor by Retosiban in Human Myometrial Smooth Muscle.

Novel small molecule inhibitors of the oxytocin receptor (OTR) may have distinct pharmacology and mode of action when compared with first-generation oxytocin antagonists when used for the prevention of preterm birth. The aim was to determine the mechanism of action of small molecule OTR antagonists retosiban and epelsiban compared with the currently used peptide-based compound atosiban. Human myometrial samples were obtained at cesarean section and subjected to pharmacological manipulations to establish the effect of antagonist binding to OTR on downstream signaling. Retosiban antagonism of oxytocin action in human myometrium was potent, rapid, and reversible. Inhibition of inositol 1,4,5-trisphosphate (IP3) production followed single-site competitive binding kinetics for epelsiban, retosiban, and atosiban. Retosiban inhibited basal production of IP3 in the absence of oxytocin. Oxytocin and atosiban but not retosiban inhibited forskolin, and calcitonin stimulated 3',5'-cyclic adenosine 5'-mono-phosphate (cAMP) production. Inhibition of cAMP was reversed by pertussis toxin. Oxytocin and atosiban, but not retosiban and epelsiban, stimulated extracellular regulated kinase (ERK)1/2 activity in a time- and concentration-dependent manner. Oxytocin and atosiban stimulated cyclo-oxygenase 2 activity and subsequent production of prostaglandin E2 and F2α. Prostaglandin production was inhibited by rofecoxib, pertussin toxin, and ERK inhibitor U0126. Oxytocin but not retosiban or atosiban stimulated coupling of the OTR to Gα q G-proteins. Oxytocin and atosiban but not retosiban stimulated coupling of the OTR to Gα i G-proteins. Retosiban and epelsiban demonstrate distinct pharmacology when compared with atosiban in human myometrial smooth muscle. Atosiban displays agonist activity at micromolar concentrations leading to stimulation of prostaglandin production.

Computational physiology of uterine smooth muscle.

Pregnancy can be accompanied by serious health risks to mother and child, such as pre-eclampsia, premature birth and postpartum haemorrhage. Understanding of the normal physiology of uterine function is essential to an improved management of such risks. Here we focus on the physiology of the smooth muscle fibres which make up the bulk of the uterine wall and which generate the forceful contractions that accompany parturition. We survey computational methods that integrate mathematical modelling with data analysis and thereby aid the discovery of new therapeutic targets that, according to clinical needs, can be manipulated to either stop contractions or cause the uterine wall muscle to become active.

Myometrial Transcriptional Signatures of Human Parturition.

The process of parturition involves the transformation of the quiescent myometrium (uterine smooth muscle) to the highly contractile laboring state. This is thought to be driven by changes in gene expression in myometrial cells. Despite the existence of multiple myometrial gene expression studies, the transcriptional programs that initiate labor are not known. Here, we integrated three transcriptome datasets, one novel (NCBI Gene Expression Ominibus: GSE80172) and two existing, to characterize the gene expression changes in myometrium associated with the onset of labor at term. Computational analyses including classification, singular value decomposition, pathway enrichment, and network inference were applied to individual and combined datasets. Outcomes across studies were integrated with multiple protein and pathway databases to build a myometrial parturition signaling network. A high-confidence (significant across all studies) set of 126 labor genes were identified and machine learning models exhibited high reproducibility between studies. Labor signatures included both known (interleukins, cytokines) and unknown (apoptosis, MYC, cell proliferation/differentiation) pathways while cyclic AMP signaling and muscle relaxation were associated with non-labor. These signatures accurately classified and characterized the stages of labor. The data-derived parturition signaling networks provide new genes/signaling interactions to understand phenotype-specific processes and aid in future studies of parturition.

Corrigendum: Myometrial Transcriptional Signatures of Human Parturition.

[This corrects the article DOI: 10.3389/fgene.2019.00185.].

Orexigen-sensitive NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus.

The hypothalamic arcuate nucleus (ARC) integrates and responds to satiety and hunger signals and forms the origins of the central neural response to perturbations in energy balance. Here we show that rat ARC neurons containing neuropeptide Y (NPY) and agouti-related protein (AgRP), which are conditional pacemakers, are activated by orexigens and inhibited by the anorexigen leptin. We propose a neuron-specific signaling mechanism through which central and peripheral signals engage the central neural anabolic drive.

Anoctamin Channels in Human Myometrium: A Novel Target for Tocolysis.

BACKGROUND: Spontaneous preterm labor leading to preterm birth is a significant obstetric problem leading to neonatal morbidity and mortality. Current tocolytics are not completely effective and novel targets may afford a therapeutic benefit. OBJECTIVE: To determine whether the anoctamin (ANO) family, including the calcium-activated chloride channel ANO1, is present in pregnant human uterine smooth muscle (USM) and whether pharmacological and genetic modulation of ANO1 modulates USM contraction. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR, and immunohistochemical staining were done to determine which members of the ANO family are expressed in human USM. Uterine smooth muscle strips were studied in an organ bath to determine whether ANO1 antagonists inhibit oxytocin-induced USM contractions. Anoctamin 1 small interfering RNA (siRNA) knockdown was performed to determine its effect on filamentous-/globular (F/G)-actin ratio, a measurement of actin polymerization's role in promoting smooth muscle contraction. RESULTS: Messenger RNA (mRNA) encoding all members of the ANO family (except ANO7) are expressed in pregnant USM tissue. Anoctamin 1 mRNA expression was decreased 15.2-fold in pregnant USM compared to nonpregnant. Anoctamin 1 protein is expressed in pregnant human USM tissue. Functional organ bath studies with pregnant human USM tissue demonstrated that the ANO1 antagonist benzbromarone attenuates the force and frequency of oxytocin-induced contractions. In human USM cells, siRNA knockdown of ANO1 decreases F-/G-actin ratios. CONCLUSION: Multiple members of the ANO family, including the calcium-activated chloride channel ANO1, are expressed in human USM. Antagonism of ANO1 by pharmacological inhibition and genetic knockdown leads to an attenuation of contraction in pregnant human USM. Anoctamin 1 is a potentially novel target for tocolysis.

Control of uterine Ca2+ by membrane voltage: toward understanding the excitation-contraction coupling in human myometrium.

Myometrial contractility is a complex and dynamic physiological process that changes substantially during pregnancy and culminates in childbirth. Uterine contractions are initiated by transient rises in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), which in turn are triggered and controlled by myometrial action potentials. The sequence of events between the action potential generation and the contraction initiation is referred to as excitation-contraction coupling. Hormones and other physiologically active substances affect myometrial contractility by modulating different steps in the excitation-contraction coupling process. It is therefore imperative that we understand that process to understand the regulation of myometrial contractility. The complex action potentials generated by human myometrium result from the activity of many ion channels, transporters, and pumps. Two types of myometrial action potential waveform have been described in the literature: a plateau type and a spike type. Parameters of the myometrial [Ca(2+)](i) transients and contractions differ depending on the type of action potential that triggers them. Some aspects of the excitation-contraction coupling are unique to human myometrium and cannot be found in animal models; some others are common between many species. This article reviews the current state and discusses future directions of physiological research on human myometrial excitation-contraction coupling.

Regulation of oxytocin receptors and oxytocin receptor signaling.

The oxytocin (OT) -oxytocin receptor (OTR) system plays a key role in many aspects of mammalian reproduction as well as several other physiological processes such as bond pairing and cardiovascular homeostasis. To manifest these diverse physiological roles, the transcription and expression of the OTR is tightly regulated within reproductive, cardiovascular, and neuronal tissues. The expression of the OTR within the mammalian uterus is regulated during gestation with a peak at the day of delivery. The control of this dramatic increase in expression is mediated in rodent species by a combination of stretch, classical steroid hormone stimulation, and repression. In the human uterus events are less clear, although a prominent role for inflammatory-related rapid-response genes and novel transcription factors such as hMafF (human homologue of chicken musculoaponeurotic fibrosarcoma oncogene family protein F) have been put forward. In the uterus the potent uterotonic actions of OT are mediated by the OTR through G-protein activation to stimulate phospholipase C activity. The activated OTR increases contraction frequency and increases force by sensitizing the contractile apparatus of the myocytes to calcium. This review summarizes the current knowledge of the complex regulation of OTR transcription in the myometrium and the intracellular signaling mechanisms through which OT mediates its potent stimulatory effects.

Preterm labour. Myometrial function in prematurity.

The primary function of the uterus during gestation is to harbour the growing conceptus in a largely quiescent environment. Upon maturation of the fetus to a point sufficient for extrauterine survival, the uterus must remodel itself sufficiently to generate forceful contractions during labour. During preterm delivery, the process of remodelling of the myometrium occurs early due to a number of different causes, although the underlying basis for myometrial contraction remains the same. This review summarises the anatomical, physiological and molecular basis for contraction. We describe the fibre structure of the human uterus and how this relates to the spread of electrical excitation during a contraction. The process of excitation within a single myometrial cell is described, as well as how this relates to contraction. We then focus on how excitation-contraction coupling is modulated by intercellular communication, pharmacomechanical-coupling and hormonal milieu. Lastly, we consider the actions of the commonly accepted uterine agonists oxytocin, prostaglandin F(2alpha), and prostaglandin E(2), and the tocolytic ritodrine.

A computational method for three-dimensional reconstruction of the microarchitecture of myometrial smooth muscle from histological sections.

BACKGROUND: The fibrous structure of the myometrium has previously been characterised at high resolutions in small tissue samples (< 100 mm3) and at low resolutions (∼500 µm per voxel edge) in whole-organ reconstructions. However, no high-resolution visualisation of the myometrium at the organ level has previously been attained. METHODS AND RESULTS: We have developed a technique to reconstruct the whole myometrium from serial histological slides, at a resolution of approximately 50 µm per voxel edge. Reconstructions of samples taken from human and rat uteri are presented here, along with histological verification of the reconstructions and detailed investigation of the fibrous structure of these uteri, using a range of tools specifically developed for this analysis. These reconstruction techniques enable the high-resolution rendering of global structure previously observed at lower resolution. Moreover, structures observed previously in small portions of the myometrium can be observed in the context of the whole organ. The reconstructions are in direct correspondence with the original histological slides, which allows the inspection of the anatomical context of any features identified in the three-dimensional reconstructions. CONCLUSIONS AND SIGNIFICANCE: The methods presented here have been used to generate a faithful representation of myometrial smooth muscle at a resolution of ∼50 µm per voxel edge. Characterisation of the smooth muscle structure of the myometrium by means of this technique revealed a detailed view of previously identified global structures in addition to a global view of the microarchitecture. A suite of visualisation tools allows researchers to interrogate the histological microarchitecture. These methods will be applicable to other smooth muscle tissues to analyse fibrous microarchitecture.