Karen E. Hemmings

Amino Acid Turnover by Bovine Oocytes Provides an Index of Oocyte Developmental Competence In Vitro

ABSTRACT Amino acid profiling has been used to distinguish between human embryos of differing developmental competence. We sought to determine whether amino acid profiling could be used to distinguish between metaphase II (MII) bovine oocytes with different developmental capabilities in vitro. Amino acid turnover was assayed during the final 6 h of in vitro maturation prior to oocytes undergoing individual fertilization in vitro. Following insemination, zygotes were immobilized in groups of 16 on the base of a Petri dish using Cell-Tak tissue adhesive to enable the developmental progress of each to be tracked to the blastocyst stage. Spent droplets of in vitro maturation medium were analyzed by high performance liquid chromatography, which revealed glutamine, arginine, and asparagine were depleted in the greatest quantities. Incompetent MII oocytes that failed to cleave by 72 h postfertilization depleted significantly more glutamine from (P = 0.0006) and released more alanine (P = 0.0001) into the medium than oocytes that cleaved. When cutoff values were selected for the turnover of alanine, arginine, glutamine, leucine, and tryptophan and modeled to predict fertilization and cleavage potential, oocytes that did not exceed the cutoff values for ≥2 of these key amino acids were more likely to cleave. The sensitivity, specificity, accuracy, and positive predictive value of this model were 60.5%, 76.8%, 63.5%, and 92.0%, respectively. Significant differences (P ≤ 0.015) in the consumption/production of alanine and glutamine were also observed when comparing uncleaved oocytes with those that produced blastocysts. The data show that noninvasive amino acid profiling can be used to measure oocyte developmental competence.

Variable imprinting of the MEST gene in human preimplantation embryos

There is evidence that expression and methylation of the imprinted paternally expressed gene 1/mesoderm-specific transcript homologue (PEG1/MEST) gene may be affected by assisted reproductive technologies (ARTs) and infertility. In this study, we sought to assess the imprinting status of the MEST gene in a large cohort of in vitro-derived human preimplantation embryos, in order to characterise potentially adverse effects of ART and infertility on this locus in early human development. Embryonic genomic DNA from morula or blastocyst stage embryos was screened for a transcribed AflIII polymorphism in MEST and imprinting analysis was then performed in cDNA libraries derived from these embryos. In 10 heterozygous embryos, MEST expression was monoallelic in seven embryos, predominantly monoallelic in two embryos, and biallelic in one embryo. Screening of cDNA derived from 61 additional human preimplantation embryos, for which DNA for genotyping was unavailable, identified eight embryos with expression originating from both alleles (biallelic or predominantly monoallelic). In some embryos, therefore, the onset of imprinted MEST expression occurs during late preimplantation development. Variability in MEST imprinting was observed in both in vitro fertilization and intracytoplasmic sperm injection-derived embryos. Biallelic or predominantly monoallelic MEST expression was not associated with any one cause of infertility. Characterisation of the main MEST isoforms revealed that isoform 2 was detected in early development and was itself variably imprinted between embryos. To our knowledge, this report constitutes the largest expression study to date of genomic imprinting in human preimplantation embryos and reveals that for some imprinted genes, contrasting imprinting states exist between embryos.

Combined effects of interleukin-1α and transforming growth factor-β1 on modulation of human cardiac fibroblast function

During cardiac remodeling, cardiac fibroblasts (CF) are influenced by increased levels of interleukin-1α (IL-1α) and transforming growth factor-β1 (TGFβ1). The present study investigated the interaction between these two important cytokines on function of human CF and their differentiation to myofibroblasts (CMF). CF were isolated from human atrial appendage and exposed to IL-1α and/or TGFβ1 (both 0.1 ng/ml). mRNA expression levels of selected genes were determined after 6-24h by real-time RT-PCR, while protein levels were analyzed at 24-48 h by ELISA or western blot. Activation of canonical signaling pathways (NFκB, Smad3, p38 MAPK) was determined by western blotting. Differentiation to CMF was examined by collagen gel contraction assays. Exposure of CF to IL-1α alone enhanced levels of IL-6, IL-8, matrix metalloproteinase-3 (MMP3) and collagen III (COL3A1), but reduced the CMF markers α-smooth muscle actin (αSMA) and connective tissue growth factor (CTGF/CCN2). By contrast, TGFβ1 alone had minor effects on IL-6, IL-8 and MMP3 levels, but significantly increased levels of the CMF markers αSMA, CTGF, COL1A1 and COL3A1. Co-stimulation with both IL-1α and TGFβ1 increased MMP3 expression synergistically. Furthermore, while TGFβ1 had no effect on IL-1α-induced IL-6 or IL-8 levels, co-stimulation inhibited the TGFβ1-induced increase in αSMA and blocked the gel contraction caused by TGFβ1. Combining IL-1α and TGFβ1 had no apparent effect on their canonical signaling pathways. In conclusion, IL-1α and TGFβ1 act synergistically to stimulate MMP3 expression in CF. Moreover, IL-1α has a dominant inhibitory effect on the phenotypic switch of CF to CMF induced by TGFβ1.

Amino acid turnover by human oocytes is influenced by gamete developmental competence, patient characteristics and gonadotrophin treatment

STUDY QUESTION Can amino acid profiling differentiate between human oocytes with differing competence to mature to metaphase II (MII) in vitro? SUMMARY ANSWER Oocytes which remained arrested at the germinal vesicle (GV) stage after 24 h of in vitro maturation (IVM) displayed differences in the depletion/appearance of amino acids compared with oocytes which progressed to MII and patient age, infertile diagnosis and ovarian stimulation regime significantly affected oocyte amino acid turnover during IVM. WHAT IS KNOWN ALREADY Amino acid profiling has been proposed as a technique which can distinguish between human pronucleate zygotes and cleavage stage embryos with the potential to develop to the blastocyst stage and implant to produce a pregnancy and those that arrest. Most recently, the amino acid turnover by individual bovine oocytes has been shown to be predictive of oocyte developmental competence as indicated by the gametes capacity to undergo fertilization and early cleavage divisions in vitro. STUDY DESIGN, SIZE, DURATION The study was conducted between March 2005 and March 2010. A total of 216 oocytes which were at the GV or metaphase I (MI) stages at the time of ICSI were donated by 67 patients. PARTICIPANTS/MATERIALS, SETTINGS, METHODS The research was conducted in university research laboratories affiliated to a hospital-based infertility clinic. Oocytes were cultured for 24 h and the depletion/appearance of amino acids was measured during the final 6 h of IVM. Amino acid turnover was analysed in relation to oocyte meiotic progression, patient age, disease aetiology and controlled ovarian stimulation regime. MAIN RESULTS AND THE ROLE OF CHANCE The depletion/appearance of key amino acids was linked to the maturation potential of human oocytes in vitro. Oocytes which arrested at the GV stage (n = 9) depleted significantly more valine and isoleucine than those which progressed to MI (n = 32) or MII (n = 107) (P < 0.05). Glutamate, glutamine, arginine and valine depletion or appearance differed in MII versus degenerating oocytes (n = 20) (P < 0.05). Glutamine, arginine, methionine, phenylalanine, total depletion and total turnover all differed in oocytes from patients aged < 35 years versus patients ≥35 years (P < 0.05). MII oocytes obtained following ovarian stimulation with recombinant FSH depleted more isoleucine (P < 0.05) and more alanine and lysine (P < 0.05) appeared than oocytes from hMG-stimulated cycles. MII oocytes from patients with a polycystic ovary (PCO) morphology (n = 33) depleted more serine (P < 0.05) than oocytes from women with normal ovaries (n = 61). LIMITATIONS, REASONS FOR CAUTION Immature oocytes collected at the time of ICSI were used as the model for human oocyte maturation. These oocytes have therefore failed to respond to the ovulatory hCG trigger in vivo (they are meiotically incompetent), and have limited capacity to support embryo development in vitro. The lack of cumulus cells and stress of the conditions in vitro may have influenced turnover of amino acids, and owing to the small sample sizes further studies are required to confirm these findings. WIDER IMPLICATIONS OF THE FINDINGS The findings provide support for the hypothesis that oocyte metabolism reflects oocyte quality. Longitudinal studies are required to link these functional metabolic indices of human oocyte quality with embryo developmental competence. Oocyte amino acid profiling may be a useful tool to quantify the impact of new assisted reproduction technologies (ART) on oocyte quality. STUDY FUNDING/COMPETING INTERESTS This project was funded by the UK Biology and Biotechnology Research Council (BB/C007395/1) and the Medical Research Council (G 0800250). K.E.H was in receipt of a British Fertility Society/Merck Serono studentship. H.J.L. is a shareholder in Novocellus Ltd, a company which seeks to devise a non-invasive biochemical test of embryo health.

Quantitative analysis of DNA methylation of imprinted genes in single human blastocysts by pyrosequencing

We report the first quantitative assessment of DNA methylation for any gene in the human preimplantation embryo to reveal that imprints exist at KvDMR1, RB1, SNRPN, and GRB10 in the human blastocyst. For comparison, in two human embryonic stem cell lines, imprints were also observed at KvDMR1, SNRPN, GRB10, and other imprinted loci, whereas RB1 and MEG3 were hypermethylated.

Interleukin-1 has opposing effects on connective tissue growth factor and tenascin-C expression in human cardiac fibroblasts

Cardiac fibroblasts (CF) play a central role in the repair and remodeling of the heart following injury and are important regulators of inflammation and extracellular matrix (ECM) turnover. ECM-regulatory matricellular proteins are synthesized by several myocardial cell types including CF. We investigated the effects of pro-inflammatory cytokines on matricellular protein expression in cultured human CF. cDNA array analysis of matricellular proteins revealed that interleukin-1α (IL-1α, 10ng/ml, 6h) down-regulated connective tissue growth factor (CTGF/CCN2) mRNA by 80% and up-regulated tenascin-C (TNC) mRNA levels by 10-fold in human CF, without affecting expression of thrombospondins 1-3, osteonectin or osteopontin. Western blotting confirmed these changes at the protein level. In contrast, tumor necrosis factor α (TNFα) did not modulate CCN2 expression and had only a modest stimulatory effect on TNC levels. Signaling pathway inhibitor studies suggested an important role for the p38 MAPK pathway in suppressing CCN2 expression in response to IL-1α. In contrast, multiple signaling pathways (p38, JNK, PI3K/Akt and NFκB) contributed to IL-1α-induced TNC expression. In conclusion, IL-1α reduced CCN2 expression and increased TNC expression in human CF. These observations are of potential value for understanding how inflammation and ECM regulation are linked at the level of the CF.

Cardiac fibroblast-specific p38α MAP kinase promotes cardiac hypertrophy via a putative paracrine interleukin-6 signaling mechanism

Recent studies suggest that cardiac fibroblast‐specific p38α MAPK contributes to the development of cardiac hypertrophy, but the underlying mechanism is unknown. Our study used a novel fibroblast‐specific, tamoxifen‐inducible p38α knockout (KO) mouse line to characterize the role of fibroblast p38α in modulating cardiac hypertrophy, and we elucidated the mechanism. Myocardial injury was induced in tamoxifen‐treated Cre‐positive p38α KO mice or control littermates via chronic infusion of the β‐adrenergic receptor agonist isoproterenol. Cardiac function was assessed by pressure‐volume conductance catheter analysis and was evaluated for cardiac hypertrophy at tissue, cellular, and molecular levels. Isoproterenol infusion in control mice promoted overt cardiac hypertrophy and dysfunction (reduced ejection fraction, increased end systolic volume, increased cardiac weight index, increased cardiomyocyte area, increased fibrosis, and up‐regulation of myocyte fetal genes and hypertrophy‐associated microRNAs). Fibroblast‐specific p38α KO mice exhibited marked protection against myocardial injury, with isoproterenol‐induced alterations in cardiac function, histology, and molecular markers all being attenuated. In vitro mechanistic studies determined that cardiac fibroblasts responded to damaged myocardium by secreting several paracrine factors known to induce cardiomyocyte hypertrophy, including IL‐6, whose secretion was dependent upon p38α activity. In conclusion, cardiac fibroblast p38α contributes to cardiomyocyte hypertrophy and cardiac dysfunction, potentially via a mechanism involving paracrine fibroblast‐to‐myocyte IL‐6 signaling.—Bageghni, S.A., Hemmings, K.E., Zava, N., Denton, C. P., Porter, K. E., Ainscough, J. F. X., Drinkhill, M. J., Turner, N. A. Cardiac fibroblast‐specific p38α MAP kinase promotes cardiac hypertrophy via a putative paracrine interleukin‐6 signaling mechanism. FASEB J. 32, 4941–4954 (2018). www.fasebj.org

Expression and function of TLR4- induced B1R bradykinin receptor on cardiac fibroblasts.

ABSTRACT Cardiac fibroblasts (CF) are key cells for maintaining extracellular matrix (ECM) protein homeostasis in the heart, and for cardiac repair through CF‐to‐cardiac myofibroblast (CMF) differentiation. Additionally, CF play an important role in the inflammatory process after cardiac injury, and they express Toll like receptor 4 (TLR4), B1 and B2 bradykinin receptors (B1R and B2R) which are important in the inflammatory response. B1R and B2R are induced by proinflammatory cytokines and their activation by bradykinin (BK: B2R agonist) or des‐arg‐kallidin (DAKD: B1R agonist), induces NO and PGI2 production which is key for reducing collagen I levels. However, whether TLR4 activation regulates bradykinin receptor expression remains unknown. CF were isolated from human, neonatal rat and adult mouse heart. B1R mRNA expression was evaluated by qRT‐PCR, whereas B1R, collagen, COX‐2 and iNOS protein levels were evaluated by Western Blot. NO and PGI2 were evaluated by commercial kits. We report here that in CF, TLR4 activation increased B1R mRNA and protein levels, as well as COX‐2 and iNOS levels. B1R mRNA levels were also induced by interleukin‐1&agr; via its cognate receptor IL‐1R1. In LPS‐pretreated CF the DAKD treatment induced higher responses with respect to those observed in non LPS‐pretreated CF, increasing PGI2 secretion and NO production; and reducing collagen I protein levels in CF. In conclusion, no significant response to DAKD was observed (due to very low expression of B1R in CF) – but pre‐activation of TLR4 in CF, conditions that significantly enhanced B1R expression, led to an additional response of DAKD. HIGHLIGHTSIn cardiac fibroblast TLR4 activation increased B1R mRNA and protein levels, COX‐2 and iNOS levels.In cardiac fibroblast B1R mRNA levels were induced by interleukin‐1&agr; via its cognate receptor IL‐1R1.In TLR4‐activated CF, DAKD induced a higher PGI2 secretion, NO production and reducition of collagen I levels.

T4 Cardiac fibroblast P38α map kinase is essential for cardiomyocyte hypertrophy and cardiac dysfunction following myocardial injury

Introduction Cardiac fibroblasts represent the majority of non-myocyte cell population in the heart and play an essential role in cardiac remodelling. Following myocardial damage, cardiac fibroblasts can respond to damage-associated molecular patterns (DAMPs) and pro-inflammatory cytokines that stimulate specific intracellular signalling cascades, including p38 MAPK, to regulate cardiac fibroblast function. We aimed to understand the role of p38 MAPK signalling in the cardiac fibroblast during cardiac remodelling. Methods A novel transgenic mouse model was developed to enable inducible and conditional knockout of p38α in fibroblasts in vivo using a Cre-loxP approach. Spatial and temporal deletion of p38α in fibroblasts was achieved by intra-peritoneal injection of tamoxifen. Adult tamoxifen-injected male mice (Cre-negative control and Cre-positive knockout) were subjected to myocardial injury by subcutaneous infusion of isoproterenol (ISO) or saline for 2 weeks, followed by removal of pumps and recovery for 1 week. Cardiac function was assessed by Millar conductance PV catheter. Heart samples were weighed and analysed by RT-PCR for mRNA and microRNA expression. Results Isoproterenol infusion of control mice caused overt cardiac hypertrophy and dysfunction compared with saline-infused mice. This was indicated by deterioration of a number of cardiac parameters, including ejection fraction and end systolic volume, and up-regulation of cardiac hypertrophy markers (ANF, β-MHC) and hypertrophy-associated microRNAs. Fibroblast-specific p38α knockout mice exhibited remarkable protection against myocardial injury with improved cardiac function and normalisation of hypertrophy markers. Conclusion p38α in cardiac fibroblasts plays a key role in driving cardiomyocyte hypertrophy and cardiac dysfunction and its deletion in fibroblasts is protective.

33 Developing a transgenic mouse model with inducible cardiac fibroblast-selective deletion of the interleukin-1 receptor, IL1R1

Rationale Interleukin-1a (IL-1a) released from necrotic cardiomyocytes is hypothesised to stimulate inflammatory responses in cardiac fibroblasts (CF) and contribute to inflammation post-myocardial infarction. Here we describe a murine model with tamoxifen-inducible fibroblast-specific deletion of the IL-1 receptor (IL1R1) for assessing the importance of the IL-1/CF axis in vivo. Methods IL1R1-/- mice and IL1R1flx/flx mice, co-expressing tamoxifen-inducible Cre-recombinase under the control of a fibroblast-specific promoter (Col1a2-CreER(T)), were crossed to produce hemizygous offspring (IL1R1flx/-). Cre-positive IL1R1flx/- (experimental) and Cre-negative IL1R1flx/- (control) mice aged 2–3 weeks were injected with tamoxifen (100mg/kg, 5 days) and hearts isolated 4–8 weeks later to establish primary CF cultures. The functional effect of IL1R1 deletion was determined by measuring cytokine and MMP expression (qRT-PCR, ELISA) in response to IL-1a or TNFa. Results Without tamoxifen administration, IL-1a responses in IL1R1flx/flx and IL1R1flx/- CF were identical despite 50% reduction of IL1R1 mRNA in IL1R1flx/- cells. IL1R1-/- CF showed no response to IL-1a. Monoallelic deletion of IL1R1 therefore had no functional impact. With tamoxifen treatment, a further 75% reduction in basal IL1R1 mRNA levels was observed in Cre-positive IL1R1flx/- CF vs. Cre-negative cells. IL-1a-induced MMP-3, MMP-9 and IL-6 mRNA was reduced by 65–75% in CF from tamoxifen-injected Cre-positive IL1R1flx/- mice vs. control. Similar results were obtained for protein secretion. TNFa responses were unaffected by IL1R1 deletion. Conclusions We have developed a fibroblast-selective IL1R1 knockout mouse in which functional deletion of IL1R1 can be induced in ~75% of CF. The impact of this will be studied in an experimental myocardial infarction model.