A Novel Signaling Network Essential for Regulating Pseudomonas aeruginosa Biofilm Development

The important human pathogen Pseudomonas aeruginosa has been linked to numerous biofilm-related chronic infections. Here, we demonstrate that biofilm formation following the transition to the surface attached lifestyle is regulated by three previously undescribed two-component systems: BfiSR (PA4196-4197) harboring an RpoD-like domain, an OmpR-like BfmSR (PA4101-4102), and MifSR (PA5511-5512) belonging to the family of NtrC-like transcriptional regulators. These two-component systems become sequentially phosphorylated during biofilm formation. Inactivation of bfiS, bfmR, and mifR arrested biofilm formation at the transition to the irreversible attachment, maturation-1 and -2 stages, respectively, as indicated by analyses of biofilm architecture, and protein and phosphoprotein patterns. Moreover, discontinuation of bfiS, bfmR, and mifR expression in established biofilms resulted in the collapse of biofilms to an earlier developmental stage, indicating a requirement for these regulatory systems for the development and maintenance of normal biofilm architecture. Interestingly, inactivation did not affect planktonic growth, motility, polysaccharide production, or initial attachment. Further, we demonstrate the interdependency of this two-component systems network with GacS (PA0928), which was found to play a dual role in biofilm formation. This work describes a novel signal transduction network regulating committed biofilm developmental steps following attachment, in which phosphorelays and two sigma factor-dependent response regulators appear to be key components of the regulatory machinery that coordinates gene expression during P. aeruginosa biofilm development in response to environmental cues

P–528 rDNA methylation of human oocytes of women undergoing intracytoplasmic sperm injection (ICSI) increases with maternal age

Abstract Study question Is there a correlation between the age of women undergoing ICSI and methylation pattern of rDNA core promoter and upstream control element in immature human oocytes? Summary answer Methylation levels of the upstream control element and the rDNA core promoter in immature human oocytes increase with age of women undergoing ICSI. What is known already Methylation of ribosomal DNA (rDNA) in germ cells regulates temporary and spatially highly coordinated nucleolar activity, cellular metabolism, and thus developmental potential of the early embryo. Alterations of methylation pattern may therefore cause dysregulation of genes and signal cascades resulting in limited fertility. It has been shown that the methylation of sperm rDNA increases with the donor’s age. The positive correlation between sperm rDNA methylation and age has been conserved among mammals during evolution including humans and mice. In contrast to sperm, little is known about the methylome of human oocytes and its role in human reproduction. Study design, size, duration Consecutive women undergoing ICSI because of male subfertility were included. Patients with endometriosis, polycystic ovary syndrome, ovarian, uterine or breast cancer, as well as patients with an anti-Mullerian hormone level &amp;lt;1ng/ml were excluded. Immature oocytes (germinal vesicle; GV) collected during oocyte pick-up at the Fertility Centre Dortmund between 2018 and 2020 were examined. Participants/materials, setting, methods Cumulus-free GV oocytes which were not usable for ICSI were rinsed in phosphate buffer and stored at –20 °C until further investigation. Multiplex-PCR followed by singleplex-PCRs were carried out on the rDNA core promoter and upstream control element. Methylation levels were quantified by bisulphite pyrosequencing. Two oppositely imprinted genes (hPEG3 and hGTL2) were used as controls to ensure correct amplification and bisulphite conversion. Spearman’s-rank-order-correlation and Mann-Whitney-U-Test were used for statistical analysis. Main results and the role of chance For each GV oocyte, nine different Cytosine-phosphate-Guanine dinucleotides (CpGs) were quantified by bisulphite pyrosequencing for the rDNA core promoter and 26 different CpGs for the upstream control element (UCE). 120 human single oocytes from 60 women were analyzed. Connected statistical analysis was used if one patient had more than one oocyte. The age of the included women ranged from 26 to 40 years (mean±SD 33.5±3.2). Only oocytes which showed a correct methylation pattern for at least one imprinting control gene (hPEG3 and hGTL2) were considered for analysis. Mean methylation level ranged from 2–31% (mean±SD 8.7±5.5) of the analyzed CpGs for the rDNA core promoter and from 3–36% (mean±SD 11.4±7.1) CpGs for UCE. Spearman’s correlation analysis revealed that the methylation levels of the human oocyte rDNA core promoter and rDNA UCE significantly increased with the age of the donor (p &amp;lt; 0.05). Correlation coefficient for rDNA core promoter was r = 0.22 and for upstream control element r = 0.21. It is also interesting to note that different oocytes from the same donors can display enormous methylation variation. Regarding clinical parameters, no correlation was observed between the methylation pattern of the rDNA core promoter or UCE and the body mass index or smoking status, respectively. Limitations, reasons for caution Limitations of this study include difficulties in extrapolating the findings to the general population, because no data of women not undergoing ICSI are available. Only GV-oocytes were analyzed. Additional research is needed to clarify the effect of different methylation pattern with increasing female age and its role in human reproduction. Wider implications of the findings: We propose that the increase of rDNA methylation in male and female germ cells with advanced age directly or indirectly influences the regulation of nucleolar activity, cellular metabolism, and thus the developmental potential of the early embryo. This age-dependent epigenetic effect may result in decreased human fertility. Trial registration number NCT03565107 </jats:sec

Postovulatory aging affects dynamics of mRNA, expression and localization of maternal effect proteins, spindle integrity and pericentromeric proteins in mouse oocytes

Trapphoff T, Heiligentag M, Dankert D, et al. Postovulatory aging affects dynamics of mRNA, expression and localization of maternal effect proteins, spindle integrity and pericentromeric proteins in mouse oocytes. HUMAN REPRODUCTION. 2016;31(1):133-149.Is the postovulatory aging-dependent differential decrease of mRNAs and polyadenylation of mRNAs coded by maternal effect genes associated with altered abundance and distribution of maternal effect and RNA-binding proteins (MSY2)? Postovulatory aging results in differential reduction in abundance of maternal effect proteins, loss of RNA-binding proteins from specific cytoplasmic domains and critical alterations of pericentromeric proteins without globally affecting protein abundance. Oocyte postovulatory aging is associated with differential alteration in polyadenylation and reduction in abundance of mRNAs coded by selected maternal effect genes. RNA-binding and -processing proteins are involved in storage, polyadenylation and degradation of mRNAs thus regulating stage-specific recruitment of maternal mRNAs, while chromosomal proteins that are stage-specifically expressed at pericentromeres, contribute to control of chromosome segregation and regulation of gene expression in the zygote. Germinal vesicle (GV) and metaphase II (MII) oocytes from sexually mature C57B1/6J female mice were investigated. Denuded in vivo or in vitro matured MII oocytes were postovulatory aged and analyzed by semiquantitative confocal microscopy for abundance and localization of polyadenylated RNAs, proteins of maternal effect genes (transcription activator BRG1 also known as ATP-dependent helicase SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (SMARCA4) and NOD-like receptor family pyrin domain containing 5 (NLRP5) also known as MATER), RNA-binding proteins (MSY2 also known as germ cell-specific Y-box-binding protein, YBX2), and post-transcriptionally modified histones (trimethylated histone H3K9 and acetylated histone H4K12), as well as pericentromeric ATRX (alpha thalassemia/mental retardation syndrome X-linked, also termed ATP-dependent helicase ATRX or X-linked nuclear protein (XNP)). For proteome analysis five replicates of 30 mouse oocytes were analyzed by selected reaction monitoring (SRM). GV and MII oocytes were obtained from large antral follicles or ampullae of sexually mature mice, respectively. Denuded MII oocytes were aged for 24 h post ovulation. For analysis of distribution and abundance of polyadenylated RNAs fixed oocytes were in situ hybridized to Cy5 labeled oligo(dT)(20) nucleotides. Absolute quantification of protein concentration per oocyte of selected proteins was done by SRM proteome analysis. Relative abundance of ATRX was assessed by confocal laser scanning microscopy (CLSM) of whole mount formaldehyde fixed oocytes or after removal of zona and spreading. MSY2 protein distribution and abundance was studied in MII oocytes prior to, during and after exposure to nocodazole, or after aging for 2 h in presence of H2O2 or for 24 h in presence of a glutathione donor, glutathione ethylester (GEE). The significant reduction in abundance of proteins (P < 0.001) translated from maternal mRNAs was independent of polyadenylation status, while their protein localization was not significantly changed by aging. Most of other proteins quantified by SRM analysis did not significantly change in abundance upon aging except MSY2 and GTSF1. MSY2 was enriched in the subcortical RNP domain (SCRD) and in the spindle chromosome complex (SCC) in a distinct pattern, right and left to the chromosomes. There was a significant loss of MSY2 from the SCRD (P < 0.001) and the spindle after postovulatory aging. Microtubule de- and repolymerization caused reversible loss of MSY2 spindle-association whereas H2O2 stress did not significantly decrease MSY2 abundance. Aging in presence of GEE decreased significantly (P < 0.05) the aging-related overall and cytoplasmic loss of MSY2. Postovulatory aging increased significantly spindle abnormalities, unaligned chromosomes, and abundance of acetylated histone H4K12, and decreased pericentromeric trimethylated histone H3K9 (all P < 0.001). Spreading revealed a highly significant increase in pericentromeric ATRX (P < 0.001) upon ageing. Thus, the significantly reduced abundance of MSY2 protein, especially at the SCRD and the spindle may disturb the spatial control and timely recruitment, deadenylation and degradation of developmentally important RNAs. An autonomous program of degradation appears to exist which transiently and specifically induces the loss and displacement of transcripts and specific maternal proteins independent of fertilization in aging oocytes and thereby can critically affect chromosome segregation and gene expression in the embryo after fertilization. We used the mouse oocyte to study processes associated with postovulatory aging, which may not entirely reflect processes in aging human oocytes. However, increases in spindle abnormalities, unaligned chromosomes and H4K12 acetylated histones, as well as in mRNA abundance and polyadenylation have been observed also in aged human oocytes suggesting conserved processes in aging. Postovulatory aging precociously induces alterations in expression and epigenetic modifications of chromatin by ATRX and in histone pattern in MII oocytes that normally occur after fertilization, possibly contributing to disturbances in the oocyte-to-embryo transition (OET) and the zygotic gene activation (ZGA). These observations in mouse oocytes are also relevant to explain disturbances and reduced developmental potential of aged human oocytes and caution to prevent oocyte aging in vivo and in vitro. The study has been supported by the German Research Foundation (DFG) (EI 199/7-1 | GR 1138/12-1 | HO 949/21-1 and FOR 1041). There is no competing interest

Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes

To detect rare epigenetic effects associated with assisted reproduction, it is necessary to monitor methylation patterns of developmentally important genes in a few germ cells and individual embryos. Bisulfite treatment degrades DNA and reduces its complexity, rendering methylation analysis from small amounts of DNA extremely challenging. Here we describe a simple approach that allows determining the parent-specific methylation patterns of multiple genes in individual early embryos. Limiting dilution (LD) of bisulfite-treated DNA is combined with independent multiplex PCRs of single DNA target molecules to avoid amplification bias. Using this approach, we compared the methylation status of three imprinted (H19, Snrpn and Igf2r) and one pluripotency-related gene (Oct4) in three different groups of single mouse two-cell embryos. Standard in vitro fertilization of superovulated oocytes and the use of in vitro matured oocytes were not associated with significantly increased rates of stochastic single CpG methylation errors and epimutations (allele methylation errors), when compared with the in vivo produced controls. Similarly, we compared the methylation patterns of two imprinted genes (H19 and Snrpn) in individual mouse 16-cell embryos produced in vivo from superovulated and non-superovulated oocytes and did not observe major between-group differences. Using bovine oocytes and polar bodies as a model, we demonstrate that LD even allows the methylation analysis of multiple genes in single cells