Twist1-IRF9 Interaction Is Necessary for IFN-Stimulated Gene Anti-Zika Viral Infection.

An efficient immune defense against pathogens requires sufficient basal sensing mechanisms that can deliver prompt responses. Type I IFNs are protective against acute viral infections and respond to viral and bacterial infections, but their efficacy depends on constitutive basal activity that promotes the expression of downstream genes known as IFN-stimulated genes (ISGs). Type I IFNs and ISGs are constitutively produced at low quantities and yet exert profound effects essential for numerous physiological processes beyond antiviral and antimicrobial defense, including immunomodulation, cell cycle regulation, cell survival, and cell differentiation. Although the canonical response pathway for type I IFNs has been extensively characterized, less is known regarding the transcriptional regulation of constitutive ISG expression. Zika virus (ZIKV) infection is a major risk for human pregnancy complications and fetal development and depends on an appropriate IFN-ß response. However, it is poorly understood how ZIKV, despite an IFN-ß response, causes miscarriages. We have uncovered a mechanism for this function specifically in the context of the early antiviral response. Our results demonstrate that IFN regulatory factor (IRF9) is critical in the early response to ZIKV infection in human trophoblast. This function is contingent on IRF9 binding to Twist1. In this signaling cascade, Twist1 was not only a required partner that promotes IRF9 binding to the IFN-stimulated response element but also an upstream regulator that controls basal levels of IRF9. The absence of Twist1 renders human trophoblast cells susceptible to ZIKV infection.

Comparative analysis of myometrial and vascular smooth muscle cells to determine optimal cells for use in drug discovery.

Novel therapeutic regulators of uterine contractility are needed to manage preterm labor, induce labor and control postpartum hemorrhage. Therefore, we previously developed a high-throughput assay for large-scale screening of small molecular compounds to regulate calcium-mobilization in primary mouse uterine myometrial cells. The goal of this study was to select the optimal myometrial cells for our high-throughput drug discovery assay, as well as determine the similarity or differences of myometrial cells to vascular smooth muscle cells (VSMCs)-the most common off-target of current myometrial therapeutics. Molecular and pharmacological assays were used to compare myometrial cells from four sources: primary cells isolated from term pregnant human and murine myometrium, immortalized pregnant human myometrial (PHM-1) cells and immortalized non-pregnant human myometrial (hTERT-HM) cells. In addition, myometrial cells were compared to vascular SMCs. We found that the transcriptome profiles of hTERT-HM and PHM1 cells were most similar (r = 0.93 and 0.90, respectively) to human primary myometrial cells. Comparative transcriptome profiling of primary human myometrial transcriptome and VSMCs revealed 498 upregulated (p ≤ 0.01, log2FC≥1) genes, of which 142 can serve as uterine-selective druggable targets. In the high-throughput Ca2+-assay, PHM1 cells had the most similar response to primary human myometrial cells in OT-induced Ca2+-release (Emax = 195% and 143%, EC50 = 30 nM and 120 nM, respectively), while all sources of myometrial cells showed excellent and similar robustness and reproducibility (Z' = 0.52 to 0.77). After testing a panel of 61 compounds, we found that the stimulatory and inhibitory responses of hTERT-HM cells were highly-correlated (r = 0.94 and 0.95, respectively) to human primary cells. Moreover, ten compounds were identified that displayed uterine-selectivity (≥5-fold Emax or EC50 compared to VSMCs). Collectively, this study found that hTERT-HM cells exhibited the most similarity to primary human myometrial cells and, therefore, is an optimal substitute for large-scale screening to identify novel therapeutic regulators of myometrial contractility. Moreover, VSMCs can serve as an important counter-screening tool to assess uterine-selectivity of targets and drugs given the similarity observed in the transcriptome and response to compounds.

Bitter taste receptors as targets for tocolytics in preterm labor therapy.

Preterm birth (PTB) is the leading cause of neonatal mortality and morbidity, with few prevention and treatment options. Uterine contraction is a central feature of PTB, so gaining new insights into the mechanisms of this contraction and consequently identifying novel targets for tocolytics are essential for more successful management of PTB. Here we report that myometrial cells from human and mouse express bitter taste receptors (TAS2Rs) and their canonical signaling components (i.e., G-protein gustducin and phospholipase C ß2). Bitter tastants can completely relax myometrium precontracted by different uterotonics. In isolated single mouse myometrial cells, a phenotypical bitter tastant (chloroquine, ChQ) reverses the rise in intracellular Ca2+ concentration ([Ca2+]i) and cell shortening induced by uterotonics, and this reversal effect is inhibited by pertussis toxin and by genetic deletion of α-gustducin. In human myometrial cells, knockdown of TAS2R14 but not TAS2R10 inhibits ChQ's reversal effect on an oxytocin-induced rise in [Ca2+]i Finally, ChQ prevents mouse PTBs induced by bacterial endotoxin LPS or progesterone receptor antagonist mifepristone more often than current commonly used tocolytics, and this prevention is largely lost in α-gustducin-knockout mice. Collectively, our results reveal that activation of the canonical TAS2R signaling system in myometrial cells produces profound relaxation of myometrium precontracted by a broad spectrum of contractile agonists, and that targeting TAS2Rs is an attractive approach to developing effective tocolytics for PTB management.-Zheng, K., Lu, P., Delpapa, E., Bellve, K., Deng, R., Condon, J. C., Fogarty, K., Lifshitz, L. M., Simas, T. A. M., Shi, F., ZhuGe, R. Bitter taste receptors as targets for tocolytics in preterm labor therapy.

Targeted exome sequencing defines novel and rare variants in complex blood group serology cases for a red blood cell reference laboratory setting.

BACKGROUND: We previously demonstrated that targeted exome sequencing accurately defined blood group genotypes for reference panel samples characterized by serology and single-nucleotide polymorphism (SNP) genotyping. Here we investigate the application of this approach to resolve problematic serology and SNP-typing cases. STUDY DESIGN AND METHODS: The TruSight One sequencing panel and MiSeq platform was used for sequencing. CLC Genomics Workbench software was used for data analysis of the blood group genes implicated in the serology and SNP-typing problem. Sequence variants were compared to public databases listing blood group alleles. The effect of predicted amino acid changes on protein function for novel alleles was assessed using SIFT and PolyPhen-2. RESULTS: Among 29 unresolved samples, sequencing defined SNPs in blood group genes consistent with serologic observation: 22 samples exhibited SNPs associated with varied but known blood group alleles and one sample exhibited a chimeric RH genotype. Three samples showed novel variants in the CROM, LAN, and RH systems, respectively, predicting respective amino acid changes with possible deleterious impact. Two samples harbored rare variants in the RH and FY systems, respectively, not previously associated with a blood group allele or phenotype. A final sample comprised a rare variant within the KLF1 transcription factor gene that may modulate DNA-binding activity. CONCLUSION: Targeted exome sequencing resolved complex serology problems and defined both novel blood group alleles (CD55:c.203G>A, ABCB6:c.1118_1124delCGGATCG, ABCB6:c.1656-1G>A, and RHD:c.452G>A) and rare variants on blood group alleles associated with altered phenotypes. This study illustrates the utility of exome sequencing, in conjunction with serology, as an alternative approach to resolve complex cases.

Uterine endoplasmic reticulum stress-unfolded protein response regulation of gestational length is caspase-3 and -7-dependent.

We previously identified myometrial caspase-3 (CASP3) as a potential regulator of uterine quiescence. We also determined that during pregnancy, the functional activation of uterine CASP3 is likely governed by an integrated endoplasmic reticulum stress response (ERSR) and is consequently limited by an increased unfolded protein response (UPR). The present study examined the functional relevance of uterine UPR-ERSR in maintaining myometrial quiescence and regulating the timing of parturition. In vitro analysis of the human uterine myocyte hTERT-HM cell line revealed that tunicamycin (TM)-induced ERSR modified uterine myocyte contractile responsiveness. Accordingly, alteration of in vivo uterine UPR-ERSR using a pregnant mouse model significantly modified gestational length. We determined that "normal" gestational activation of the ERSR-induced CASP3 and caspase 7 (CASP7) maintains uterine quiescence through previously unidentified proteolytic targeting of the gap junction protein, alpha 1(GJA1); however, surprisingly, TM-induced uterine ERSR triggered an exaggerated UPR that eliminated uterine CASP3 and 7 tocolytic action precociously. These events allowed for a premature increase in myometrial GJA1 levels, elevated contractile responsiveness, and the onset of preterm labor. Importantly, a successful reversal of the magnified ERSR-induced preterm birth phenotype could be achieved by pretreatment with 4-phenylbutrate, a chaperone protein mimic.

The Unfolded Protein Response Regulates Uterine Myocyte Antioxidant Responsiveness During Pregnancy.

There is considerable evidence that implicates oxidative stress in the pathophysiology of human pregnancy complications. However, the role and the mechanism of maintaining an antioxidant prosurvival uterine environment during normal pregnancy is largely unresolved. Herein we report that the highly active uterine unfolded protein response plays a key role in promoting antioxidant activity in the uterine myocyte across gestation. The unfolded protein response (UPR) senses the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and activates a signaling network that consists of the transmembrane protein kinase eukaryotic translation initiation factor 2 alpha kinase 3/PKR-like-ER kinase (EIF2AK3), which acts to decrease protein translation levels, allowing for a lowered need for protein folding during periods of ER stress. However, independent of its translational regulatory capacity, EIF2AK3-dependent signals elicit the activation of the transcription factor, nuclear factor erythroid 2-like 2 (NFE2L2) in response to oxidative stress. NFE2L2 binds to antioxidant response elements in the promoters of a variety of antioxidant genes that minimize the opportunities for generation of reactive oxygen intermediates. Our analysis demonstrates that in the absence of EIF2AK3, the uterine myocyte experiences increased levels of reactive oxygen species due to decreased NFE2L2 activation. Elevated levels of intracellular reactive oxygen species were observed in the EIF2AK3 null cells, and this was associated with the onset of apoptotic cell death. These findings confirm the prosurvival and antioxidant role of UPR-mediated EIF2AK3 activation in the context of the human uterine myocyte.

An age-dependent ABO discrepancy between mother and baby reveals a novel A(weak) allele.

BACKGROUND: Discrepancies in ABO grouping arise due to different reasons, posing a threat to patient safety. Underlying causes include mixed-field agglutination after transfusion, chimerism, fetomaternal exchange, or inheritance of unusual alleles resulting in weak A/B antigen expression. Cord blood from the infant of a group A2 B mother typed as group O, H+. Samples were investigated to elucidate this conundrum. STUDY DESIGN AND METHODS: Genomic DNA was analyzed by ABO genotyping and sequencing. Red blood cells (RBCs) were characterized by routine serology and flow cytometry. Glycosyltransferase structure was predicted with 3D-modeling software. RESULTS: The mother genotyped as ABO*A1.01/B.01, and the baby, ABO*A1.01/O.01.01. Sequencing revealed a substitution, 311T>A, in the ABO*A1-like allele, which predicts Ile104Asn. Flow cytometry demonstrated A antigen on the mother's RBCs equivalent to the A2 phenotype while no A was detectable on cord RBCs. However, blood from the 11-month-old child demonstrated markedly increased A expression, likely reflecting initiation of carbohydrate chain branching. CONCLUSION: We unraveled a novel A(weak) allele (ABO*AW.29) in a case of apparent nonmaternity. Residue 104 is far from the catalytic site and may be involved in stabilizing the glycosyltransferase by dimerization. Our data support that the group AB mother's B-transferase stabilizes the altered A-transferase by heterodimerization, exemplifying the allelic enhancement phenomenon.

MicroRNA-200a serves a key role in the decline of progesterone receptor function leading to term and preterm labor.

During pregnancy, uterine quiescence is maintained by increased progesterone receptor (PR) activity, but labor is facilitated by a series of events that impair PR function. Previously, we discovered that miR-200 family members serve as progesterone (P(4))-modulated activators of contraction-associated genes in the pregnant uterus. In this study, we identified a unique role for miR-200a to enhance the local metabolism of P(4) in myometrium and, thus, decrease PR function during the progression toward labor. miR-200a exerts this action by direct repression of STAT5b, a transcriptional repressor of the P(4)-metabolizing enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD). We observed that miR-200a expression increased and STAT5b expression coordinately decreased in myometrium of mice as they progressed to labor and in laboring myometrium from pregnant women. These changes were associated with a dramatic increase in expression and activity of 20α-HSD in laboring myometrium from mouse and human. Notably, overexpression of miR-200a in cultured human myometrial cells (hTERT-HM) suppressed STAT5b and increased 20α-HSD mRNA levels. In uterine tissues of ovariectomized mice injected with P(4), miR-200 expression was significantly decreased, STAT5b expression was up-regulated, and 20α-HSD mRNA was decreased, but in 15 d postcoitum pregnant mice injected with the PR antagonist RU486, preterm labor was associated with increased miR-200a, decreased STAT5b, and enhanced 20α-HSD expression. Taken together, these findings implicate miR-200a as an important regulator of increased local P(4) metabolism in the pregnant uterus near term and provide insight into the importance of miR-200s in the decline in PR function leading to labor.

Implementation of a laborist program and evaluation of the effect upon cesarean delivery.

OBJECTIVE: Laborist programs have expanded throughout the United States in the last decade. Meanwhile, there has been no published research examining their effect on patient outcomes. Cesarean delivery is a key performance metric with maternal health implications and significant financial impact. Our hypothesis is that the initiation of a full-time dedicated laborist staff decreases cesarean delivery. STUDY DESIGN: In a tertiary hospital staffed with private practice physicians, data were retrospectively reviewed for 3 time periods from 2006 through 2011. The first period (16 months) there were no laborists (traditional model), followed by 14 months of continuous in-hospital laborist coverage provided by community staff (community laborist), and finally a 24-month period with full-time laborists providing continuous in-hospital coverage. The primary hypothesis was that full-time laborists would decrease cesarean delivery rates. RESULTS: Data from 6206 term nulliparous patients were retrospectively reviewed. The cesarean delivery rate for no laborist care was 39.2%, for community physician laborist care was 38.7%, and for full-time laborists was 33.2%. With adjustment via logistic regression, full-time laborist presence was associated with a significant reduction in cesarean delivery when contrasted with no laborist (odds ratio, 0.73; 95% confidence interval, 0.64-0.83; P < .0001) or community laborist care (odds ratio, 0.77; 95% confidence interval, 0.67-0.87; P < .001). The community laborist model was not associated with an effect upon cesarean delivery. CONCLUSION: A dedicated full-time laborist staff model is associated with lower rates of cesarean delivery. These findings may be used as part of a strategy to reduce cesarean delivery, lower maternal morbidity and mortality, and decrease health care costs.

Preimplantation apoptotic endometrial caspase-3-mediated phospholipase A2 activation: a potential component in programming uterine receptivity.

OBJECTIVE: To examine the activation and consequence of uterine apoptotic caspase-3 action on 1 day after coitus (dpc) in the pregnant mouse. We have previously demonstrated that in a pregnant uterus, caspase-3 activation from mid to late gestation isolated to the myometrial compartment is largely nonapoptotic and controls uterine quiescence. Additionally, we had demonstrated that apoptotic caspase-3 activation isolated to the endometrial compartment at term regulated endometrial prostaglandin synthesis. DESIGN: Uteri were isolated from pseudopregnant and nonligated controls and unilateral and bilateral ligated uterine horn mouse models at 1, 3, and 6 dpc. Uteri were examined for apoptotic indices, such as caspase-3 activation and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining. Immunohistochemical analysis identified the site of uterine apoptotic caspase-3 activation. The truncated form of phospholipase A2 was examined as a measure of apoptotic caspase-3-mediated calcium independent phospholipase A2 (iPLA2) activation. RESULT(S): We identified the site and impact of uterine apoptotic caspase-3 activation using uteri isolated from nonpregnant control animals at estrous and diestrous and control pregnant mice at 1-19 dpc. Our analysis revealed that apoptotic caspase-3 and iPLA2 activation were limited to the endometrial compartments of the control and unilateral ligated uteri on 1 dpc and were not found in the pseudopregnant or bilateral ligated uterine horn or on 3 or 6 dpc in the control and unilateral ligated uteri. CONCLUSION(S): In this study, we determined that uterine caspase-3 activation on 1 dpc, which is endometrial and apoptotic in nature, may play a potential role in regulating the previously reported preimplantation surge in endometrial PGE2 synthesis through apoptotic caspase-3-mediated iPLA2 activation. Our data indicate that the presence of a conceptus on 1 dpc likely triggers an increase in endometrial apoptotic caspase-3-mediated iPLA2 activation. When activated, iPLA2 causes the hydrolysis of fatty acids, resulting in arachidonic acid release and PGE2 production, which has been demonstrated to act in a leutoprotective manner in early pregnancy, prolonging progesterone synthesis and promoting uterine receptivity.

Endoplasmic reticulum stress response and the regulation of endometrial interferon-beta production.

OBJECTIVE: To gain an understanding of the potential role of endoplasmic reticulum (ER) stress in the endometrial compartment during early pregnancy, a highly understudied area. DESIGN: This study examined the regulation of interferon-ß (IFNß) in response to ER stress in human decidualized and nondecidualized endometrial cells (human endometrial stromal cells [HESCs]) in vitro. In vivo, we examined ER stress and the IFNß levels locally in the mouse endometrium before and after implantation at embryonic day (E)1, E3, and E6. SETTING: The study was performed in a reproductive sciences laboratory for Human Growth and Development. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Quantitative polymerase chain reaction, Western blotting, and immunohistochemical analysis allowed us to test the action of endogenous ER stress activation in the endometrial compartment likely triggered by implantation and its ability to increase the endometrial IFNß levels. RESULT(S): In vitro, we observed a significant difference in the IFNß levels in HESCs, in response to ER stress activation, where decidualized HESCs exhibited a threefold increase in the IFNß levels compared with nondecidualized HESCs. Apoptotic caspase-3 activation was also isolated to the decidualized cells as a result of ER stress-dependent suppression of nuclear factor-kappa beta-regulated antiapoptotic factors, XIAP and MCL-1. In vivo, mouse endometrial IFNß was present in F4/80-positive macrophages at all time points examined. After implantation (E6), the mouse luminal epithelial cells robustly coexpressed both IFNß and the ER stress marker immunoglobulin heavy chain binding protein (BiP). CONCLUSION(S): These analyses demonstrate that both in vivo and in vitro, differentiated and decidualized endometrial cells undergoing ER stress have the capacity to produce increased IFNß levels; therefore, ER stress activation in the endometrial compartment may play a vital role in promoting successful implantation events.

A Clinician's Guide to Running Custom Machine-Learning Models in an Electronic Health Record Environment.

We recently brought an internally developed machine-learning model for predicting which patients in the emergency department would require hospital admission into the live electronic health record environment. Doing so involved navigating several engineering challenges that required the expertise of multiple parties across our institution. Our team of physician data scientists developed, validated, and implemented the model. We recognize a broad interest and need to adopt machine-learning models into clinical practice and seek to share our experience to enable other clinician-led initiatives. This Brief Report covers the entire model deployment process, starting once a team has trained and validated a model they wish to deploy in live clinical operations.

Elevated levels of uterine anti-apoptotic signaling may activate NFKB and potentially confer resistance to caspase 3-mediated apoptotic cell death during pregnancy in mice.

Preserving the uterus in a state of relative quiescence is vital to the maintenance of a successful pregnancy. Elevated cytoplasmic levels of uterine caspase 3 during pregnancy have been proposed as a potential regulator of uterine quiescence through direct targeting and disabling of the uterine contractile architecture. However, despite highly elevated levels of uterine caspase 3 during pregnancy, there is minimal evidence of apoptosis. This current study defines the mechanism whereby the pregnant uterine myocyte may harness the tocolytic activity of active caspases while avoiding apoptotic cell death. Using the pregnant mouse model, we have analyzed the uterus for changes in pro- and antiapoptotic signaling patterns associated with the advancing stages of pregnancy. Briefly, we have found that members of the IAP family, such as SURVIVIN and XIAP, and the Bcl2 family members, such as MCL1, are elevated in the uterine myocyte during late gestation. The IAP family members are the only endogenous inhibitors of active caspase 3, and MCL1 limits activation of caspase 3 by suppressing proapoptotic signaling. Elevated XIAP levels partner with SURVIVIN, resulting in increased levels of the antiapoptotic MCL1 via NFKB activation; these together have the potential to limit both the activity and level of active caspase 3 in the pregnant uterus as term approaches. We propose that modification of these antiapoptotic signaling partners allows the pregnant uterus to escape the apoptotic action of elevated active caspase 3 levels but also functions to limit the levels of active uterine caspase 3 near term.

Diagnostic and predictive accuracy of the Clinical Frailty Scale among hospitalised older medical patients: a systematic review and meta-analysis protocol.

INTRODUCTION: Frailty is a common condition affecting older adults and is associated with increased mortality and adverse outcomes. Identification of older adults at risk of adverse outcomes is central to subsequent resource planning and targeted interventions. This systematic review and meta-analysis will examine the: (1) diagnostic accuracy of the Clinical Frailty Scale (CFS) in identifying hospitalised adults ≥65 years with frailty and a medical diagnosis compared with the reference standard Frailty Index or Frailty Phenotype and (2) predictive value of the CFS in determining those at increased risk of subsequent adverse outcomes. METHODS AND ANALYSIS: We will include cross-sectional, retrospective and prospective cohort studies, and randomised controlled trials that assess either the diagnostic accuracy of the CFS when compared with the reference standard Frailty Index/Frailty Phenotype or the predictive validity of the CFS to predict subsequent adverse outcomes in hospitalised adults over 65 years with medical complaints. Adverse outcomes include falls, functional decline, unplanned Emergency Department attendance, emergency rehospitalisation, nursing home admission or death. A systematic search will be conducted in Embase, AMED, MEDLINE (Ebsco, Ovid, Pubmed), CINAHL, PsycINFO, Cochrane Library. Studies will be limited to those published from 2005 to 30 October 2019. Two independent reviewers will screen all titles and abstracts to identify relevant studies. The methodological quality of studies will be independently assessed using the Quality Assessment of Diagnostic Accuracy Studies-2. A CFS score of >4 will be used to identify frailty. We will construct 2×2 tables and determine true positives, true negatives, false positives and false negatives for each study when compared with the reference standard and for each adverse outcome. A bivariate random effects model will be applied to generate pooled summary estimates of sensitivity and specificity. ETHICS AND DISSEMINATION: Ethical approval is not required for this systematic review. We will disseminate our findings through a peer-reviewed journal.

Estrogen receptor alpha isoform ERdelta7 in myometrium modulates uterine quiescence during pregnancy.

BACKGROUND: Circulating estrogen (E2) levels are high throughout pregnancy and increase towards term, however its local tissue specific actions vary across gestation. For example, myometrial E2 regulated uterotonic action is disabled until term, whereas it's proliferative function is maintained in the breast. We have identified gestationally regulated splicing events, mediated by hnRNPG and modulated by E2 that generate alternatively spliced estrogen receptor alpha (ERα) variants (ERΔ7 and ERα46) in the myometrium. These variants allow for differential, gestationally regulated, modulation of the uterotonic action of E2. METHODS: Human myometrium isolated from preterm and term non-laboring and laboring pregnant women were analyzed for ERα isoforms and splice factor levels. Lentiviral mediated shRNA knockdown of hnRNPG and overexpression of ERΔ7 were performed in human myometrial (hTERT-HM) cells. Functional 3D collagen contraction assays were executed. FINDINGS: ERΔ7 acts as a dominant negative repressor of the uterotonic action of ERα66 and ERα46 isoforms through the regulation of the myometrial gap junction protein GJA1. Elimination of hnRNPG inhibits the generation of ERΔ7 while overexpression of ERΔ7 inhibited GJA1 expression. Moreover in vivo human myometrial hnRNPG levels decline at term in an E2 dependent manner resulting in a withdrawal of ERΔ7 levels and its tocolytic action at term. INTERPRETATION: Our findings implicate the unique role of ERΔ7 as a modulator of myometrial quiescence and define the mechanism of ERΔ7 generation, through hormonally regulated splicing events. FUND: This study was supported by NIH OPRU U01 supplement (HD047905), University of Pittsburgh and Wayne State University Perinatal Research Initiative (USA).

Steroidogenic Factor 1 (Nr5a1) is Required for Sertoli Cell Survival Post Sex Determination.

The elevated level of Steroidogenic Factor 1 (Nr5a1, Sf-1) expression in the male gonadal development pathway, post sex determination, implies a vital role in testis gonadal differentiation. In this study we generated Sertoli cell-specific Nr5a1 KO mice (SC-SF-1-/-) at E14.5, which coincides with testis development post sex determination, using the Amh-Cre mouse model. Analysis of SC-SF-1-/- (Sertoli cell specific Nr5a1 knockout) testes demonstrated apoptosis as early as E15. Further analysis revealed that SC-SF-1-/- gonads displayed lower MDM2 levels resulting in elevated TP53 levels, which we believe may lead to apoptosis of the Sertoli cell population, inferring the possibility that NR5A1 directly regulates MDM2 expression. By E15.5, the Sertoli cell and germ cell population declined in SC-SF-1-/- mice resulting in the disruption of seminiferous cords with limited cord structure remaining at E18.5. Due to the loss of Sertoli and germ cells, the testis weights of SC-SF-1-/- mice at 6-weeks were much reduced; however, SC-SF-1-/- seminal vesicles weights were comparable suggesting intact Leydig cell androgen production. We conclude that NR5A1 regulates the TP53 pathway during development, is essential for fetal Sertoli cell survival and controls the cell cycle of Sertoli cells during differentiation.

Preconditioning the uterine unfolded protein response maintains non-apoptotic Caspase 3-dependent quiescence during pregnancy.

The prevention of apoptotic caspase 3 activation through biological preconditioning, mediated through the modulation of the unfolded protein response has been demonstrated to ameliorate multiple pathophysiologies. The maintenance of non-apoptotic caspase 3 activity by the unfolded protein response within the pregnant uterus has previously been proven to be critical in inhibiting uterine myocyte contractility during pregnancy. Here we report that the pregnant uterus utilizes an unfolded protein response-preconditioning paradigm to conserve myometrial caspase 3 in a non-apoptotic state in order to effectively inhibit uterine contractility thereby preventing the onset of preterm labor. In the absence of appropriate endogenous preconditioning during pregnancy, uterine caspase 3 is transformed from a non-apoptotic to an apoptotic phenotype. Apoptotic caspase 3 activation results in the precocious triggering of local uterine inflammatory signaling and prostaglandin production, consequently resulting in an increased incidence of preterm birth. These findings represent a paradigm shift in our understanding of how preconditioning promotes the maintenance of uterine non-apoptotic caspase 3 action during pregnancy preventing the onset of premature uterine contraction and therefore defining the timing of the onset of labor.

Up-regulation of the progesterone receptor (PR)-C isoform in laboring myometrium by activation of nuclear factor-kappaB may contribute to the onset of labor through inhibition of PR function.

Progesterone acting via the progesterone receptor (PR) plays a critical role in maintaining uterine quiescence during pregnancy. In the present study, we tested the hypothesis that the transactivating capability of the PR is down-regulated in the myometrium at term by a change in uterine PR isoform ratio resulting from local activation of the nuclear factor (NF)-kappaB pathway. Overexpression of the truncated PR-C isoform in human myometrial cells inhibited PR-B transactivation. Expression of PR isoforms, PR-A, PR-B, and PR-C, was characterized by immunoblotting and quantitative PCR (Q-PCR) in fundal and lower uterine segment myometrium from pregnant women in labor and not in labor and in the pregnant mouse uterus during late gestation. We observed a marked increase in levels of PR-C and transcriptionally active PR-B specifically in fundal myometrium of women in labor. In pregnant mouse uterus, levels of PR-B and PR-C also increased between 15 days post coitum and term, whereas expression of PR-A was dramatically up-regulated at 19 days post coitum. In studies of uterine tissues of mice injected intraamniotically with surfactant protein A and of human myometrial and T47D breast cancer cells in culture, up-regulation of PR isoform expression was observed in response to activation of the NF-kappaB pathway. Chromatin immunoprecipitation analysis revealed IL-1beta induced binding of NF-kappaB to the PR promoter. Collectively, these findings suggest that up-regulation of inhibitory PR isoform expression by NF-kappaB activation in both laboring human fundus and pregnant mouse uterus near term may inhibit PR transactivation and thereby lead to a loss of uterine quiescence and the onset of labor.