Ribosomal DNA methylation in human and mouse oocytes increases with age

An age-dependent increase in ribosomal DNA (rDNA) methylation has been observed across a broad spectrum of somatic tissues and the male mammalian germline. Bisulfite pyrosequencing (BPS) was used to determine the methylation levels of the rDNA core promoter and the rDNA upstream control element (UCE) along with two oppositely genomically imprinted control genes (PEG3 and GTL2) in individual human germinal vesicle (GV) oocytes from 90 consenting women undergoing fertility treatment because of male infertility. Apart from a few (4%) oocytes with single imprinting defects (in either PEG3 or GTL2), the analyzed GV oocytes displayed correct imprinting patterns. In 95 GV oocytes from 42 younger women (26-32 years), the mean methylation levels of the rDNA core promoter and UCE were 7.4±4.0% and 9.3±6.1%, respectively. In 79 GV oocytes from 48 older women (33-39 years), methylation levels increased to 9.3±5.3% (P = 0.014) and 11.6±7.4% (P = 0.039), respectively. An age-related increase in oocyte rDNA methylation was also observed in 123 mouse GV oocytes from 29 4-16-months-old animals. Similar to the continuously mitotically dividing male germline, ovarian aging is associated with a gain of rDNA methylation in meiotically arrested oocytes. Oocytes from the same woman can exhibit varying rDNA methylation levels and, by extrapolation, different epigenetic ages

Entwicklung, Proteom und chromosomale, epigenetische und mitrochondriale Konstitution von Oozyten nach Vitrifikation im preantralen Follikelstadium und nach chronischer Bisphenol A Behandlung

Trapphoff T. Entwicklung, Proteom und chromosomale, epigenetische und mitrochondriale Konstitution von Oozyten nach Vitrifikation im preantralen Follikelstadium und nach chronischer Bisphenol A Behandlung. Bielefeld; 2014

Induction of Distinct Defense-Associated Protein Patterns in Aphanomyces euteiches (Oomycota)-Elicited and -Inoculated Medicago truncatula Cell-Suspension Cultures: A Proteome and Phosphoproteome Approach

Trapphoff T, Beutner C, Niehaus K, Colditz F. Induction of Distinct Defense-Associated Protein Patterns in Aphanomyces euteiches (Oomycota)-Elicited and -Inoculated Medicago truncatula Cell-Suspension Cultures: A Proteome and Phosphoproteome Approach. Molecular Plant - Microbe Interactions. 2009;22(4):421-436.A comprehensive proteomic approach was applied to investigate molecular events occurring upon inoculation of Medicago truncatula cell-suspension cultures with the oomycete root pathogen Aphanomyces euteiches. Establishment of an inoculation assay in the cell cultures allowed a direct comparison between proteins induced by elicitation with a crude culture extract of the oomycete and by inoculation with A. euteiches zoospores representing the natural infection carrier. Oxidative burst assays revealed responsiveness of the cell cultures for perception of elicitation and inoculation signals. The plant "elicitation proteome" resembles the "inoculation proteome" in early incubation stages and includes proteins induced following initial oxidative burst and defense reactions, but also proteins involved in the antioxidative system. However, approximately 2 days after incubation, the inoculation proteome differs drastically from the proteome of elicited cultures, where a cessation of responses assignable to A. euteiches elicitation occurred. The specific protein induction patterns of zoospore-inoculated cells appeared consistent with the protein induction identified in recent studies for an A. euteiches infection in planta and consist of three functional groups: i) pathogenesis-related proteins, ii) proteins associated with secondary phenylpropanoid or phytoalexin metabolism, and, particularly, iii) proteins assigned to carbohydrate metabolism and energy-related cellular processes. Phosphoproteomic analyses revealed consistent and specific activation of these defense-related pathways already at very early timepoints of inoculation, providing evidence that the identified protein profiles are representative for an established A. euteiches infection of M. truncatula

Preovulatory Aging In Vivo and In Vitro Affects Maturation Rates, Abundance of Selected Proteins, Histone Methylation Pattern and Spindle Integrity in Murine Oocytes

Demond H, Trapphoff T, Dankert D, et al. Preovulatory Aging In Vivo and In Vitro Affects Maturation Rates, Abundance of Selected Proteins, Histone Methylation Pattern and Spindle Integrity in Murine Oocytes. PLOS ONE. 2016;11(9): e0162722.Delayed ovulation and delayed fertilization can lead to reduced developmental competence of the oocyte. In contrast to the consequences of postovulatory aging of the oocyte, hardly anything is known about the molecular processes occurring during oocyte maturation if ovulation is delayed (preovulatory aging). We investigated several aspects of oocyte maturation in two models of preovulatory aging: an in vitro follicle culture and an in vivo mouse model in which ovulation was postponed using the GnRH antagonist cetrorelix. Both models showed significantly reduced oocyte maturation rates after aging. Furthermore, in vitro preovulatory aging deregulated the protein abundance of the maternal effect genes Smarca4 and Nlrp5, decreased the levels of histone H3K9 trimethylation and caused major deterioration of chromosome alignment and spindle conformation. Protein abundance of YBX2, an important regulator of mRNA stability, storage and recruitment in the oocyte, was not affected by in vitro aging. In contrast, in vivo preovulatory aging led to reduction in Ybx2 transcript and YBX2 protein abundance. Taken together, preovulatory aging seems to affect various processes in the oocyte, which could explain the low maturation rates and the previously described failures in fertilization and embryonic development

Pre- and Postovulatory Aging of Murine Oocytes Affect the Transcript Level and Poly(A) Tail Length of Maternal Effect Genes

Dankert D, Demond H, Trapphoff T, et al. Pre- and Postovulatory Aging of Murine Oocytes Affect the Transcript Level and Poly(A) Tail Length of Maternal Effect Genes. PLoS ONE. 2014;9(10): e108907.Maternal effect genes code for oocyte proteins that are important for early embryogenesis. Transcription in oocytes does not take place from the onset of meiotic progression until zygotic genome activation. During this period, protein levels are regulated posttranscriptionally, for example by poly(A) tail length. Posttranscriptional regulation may be impaired in preovulatory and postovulatory aged oocytes, caused by delayed ovulation or delayed fertilization, respectively, and may lead to developmental defects. We investigated transcript levels and poly(A) tail length of ten maternal effect genes in in vivo-and in vitro- (follicle culture) grown oocytes after pre- and postovulatory aging. Quantitative RT-PCR was performed using random hexamer-primed cDNA to determine total transcript levels and oligo(dT)(16)-primed cDNA to analyze poly(A) tail length. Transcript levels of in vivo preovulatory-aged oocytes remained stable except for decreases in Brg1 and Tet3. Most genes investigated showed a tendency towards increased poly(A) content. Polyadenylation of in vitro preovulatory-aged oocytes was also increased, along with transcript level declines of Trim28, Nlrp2, Nlrp14 and Zar1. In contrast to preovulatory aging, postovulatory aging of in vivo-and in vitro-grown oocytes led to a shortening of poly(A) tails. Postovulatory aging of in vivo-grown oocytes resulted in deadenylation of Nlrp5 after 12 h, and deadenylation of 4 further genes (Tet3, Trim28, Dnmt1, Oct4) after 24 h. Similarly, transcripts of in vitro-grown oocytes were deadenylated after 12 h of postovulatory aging (Tet3, Trim28, Zfp57, Dnmt1, Nlrp5, Zar1). This impact of aging on poly(A) tail length may affect the timed translation of maternal effect gene transcripts and thereby contribute to developmental defects

Improved cryotolerance and developmental potential of in vitro and in vivo matured mouse oocytes by supplementing with a glutathione donor prior to vitrification

Trapphoff T, Heiligentag M, Simon J, et al. Improved cryotolerance and developmental potential of in vitro and in vivo matured mouse oocytes by supplementing with a glutathione donor prior to vitrification. MOLECULAR HUMAN REPRODUCTION. 2016;22(12):867-881.STUDY QUESTION: Can supplementation of media with a glutathione (GSH) donor, glutathione ethyl ester (GEE), prior to vitrification protect the mouse oocyte from oxidative damage and critical changes in redox homeostasis, and thereby improve cryotolerance? SUMMARY ANSWER: GEE supplementation supported redox regulation, rapid recovery of spindle and chromosome alignment after vitrification/warming and improved preimplantation development of mouse metaphase II (MII) oocytes. WHAT IS KNOWN ALREADY: Cryopreservation may affect mitochondrial functionality, induce oxidative stress, and thereby affect spindle integrity, chromosome segregation and the quality of mammalian oocytes. GEE is a membrane permeable GSH donor that promoted fertilization and early embryonic development of macaque and bovine oocytes after IVM. STUDY DESIGN, SIZE, DURATION: Two experimental groups consisted of (i) denuded mouse germinal vesicle (GV) oocytes that were matured in vitro in the presence or absence of 1 mM GEE (IVM group 1) and (ii) in vivo ovulated (IVO) MII oocytes that were isolated from the ampullae and exposed to 1 mM GEE for 1 h prior to vitrification (IVO group 2). Recovery of oocytes from both groups was followed after CryoTop vitrification/warming for up to 2 h and parthenogenetic activation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Reactive oxygen species (ROS), spindle morphology and chromosome alignment were analyzed by confocal laser scanning microscopy (CLSM) and polarization microscopy in control and GEE-supplemented MII oocytes. The relative overall intra-oocyte GSH content was assessed by analysis of monochlorobimane (MBC)-GSH adduct fluorescence in IVM MII oocytes. The GSH-dependent intra-mitochondrial redox potential (Em GSH) of IVM MII oocytes was determined after microinjection with specific mRNA at the GV stage to express a redox-sensitive probe within mitochondria (mito-Grx1-roGFP2). The absolute negative redox capacity (in millivolts) was determined by analysis of fluorescence of the oxidized versus the reduced form of sensor by CLSM and quantification according to Nernst equation. Proteome analysis was performed by quantitative 2D saturation gel electrophoresis (2D DIGE). Since microinjection and expression of redox sensor mRNA required removal of cumulus cells, and IVM of denuded mouse oocytes in group 1 induces zona hardening, the development to blastocysts was not assessed after IVF but instead after parthenogenetic activation of vitrified/warmed MII oocytes from both experimental groups. MAIN RESULTS AND ROLE OF CHANCE: IVM of denuded mouse oocytes in the presence of 1 mM GEE significantly increased intraoocyte GSH content. ROS was not increased by CryoTop vitrification but was significantly lower in the IVM GEE group compared to IVM without GEE before vitrification and after recovery from vitrification/warming (P < 0.001). Vitrification alone significantly increased the GSH-dependent intra-mitochondrial redox capacity after warming (E-GSH(m), P < 0.001) in IVM oocytes, presumably by diffusion/uptake of cytoplasmic GSH into mitochondria. The presence of 1 mM GEE during IVM increased the redox capacity before vitrification and there was no further increase after vitrification/warming. None of the reproducibly detected 1492 spots of 2D DIGE separated proteins were significantly altered by vitrification or GEE supplementation. However, IVM of denuded oocytes significantly affected spindle integrity and chromosome alignment right after warming from vitrification (0 h) in group 1 and spindle integrity in group 2 (P < 0.05). GEE improved recovery in IVM group as numbers of oocytes with unaligned chromosomes and aberrant spindles was not significantly increased compared to unvitrified controls. The supplementation with GEE for 1 h before vitrification also supported more rapid recovery of spindle birefringence. GEE improved significantly development to the 2-cell stage for MII oocytes that were activated directly after vitrification/warming in both experimental groups, and also the blastocyst rate in the IVO GEE-supplemented group compared to the controls (P < 0.05). LARGE SCALE DATA: None LIMITATIONS, REASONS FOR CAUTION: The studies were carried out in a mouse model, in IVM denuded rather than cumulus-enclosed oocytes, and in activated rather than IVF MII oocytes. Whether the increased GSH-dependent intra-mitochondrial redox capacity also improves male pronuclear formation needs to be studied further experimentally. The influence of GEE supplementation requires also further examination and optimization in human oocytes before it can be considered for clinical ART. WIDER IMPLICATIONS OF THE FINDINGS: Although GEE supplementation did not alter the proteome at MII, the GSH donor may support cellular homeostasis and redox regulation and, thus, increase developmental competence. While human MII oocyte vitrification is an established procedure, GEE might be particularly beneficial for oocytes that suffer from oxidative stress and reduced redox capacity (e.g. aged oocytes) or possess low GSH due to a reduced supply of GSH from cumulus. It might also be of relevance for immature human oocytes that develop without cumulus to MII in vitro (e.g. in ICSI cycles) for ART. STUDY FUNDING AND COMPETING INTERESTS: The study has been supported by the German Research Foundation (DFG FOR 1041; EI 199/3-2). There are no conflict of interests

Preovulatory Aging <i>In Vivo</i> and <i>In Vitro</i> Affects Maturation Rates, Abundance of Selected Proteins, Histone Methylation Pattern and Spindle Integrity in Murine Oocytes - Fig 3

<p><b><i>Ybx2</i> mRNA levels (A) and YBX2 protein localization and abundance in control and preovulatory-aged (PreOA) MII oocytes grown <i>in vitro</i> (B-D) or <i>in vivo</i> (E-G).</b> SCC: Spindle chromosome complex; SCMC: Subcortical maternal complex. Arrowheads in <b>B</b>’, <b>C’</b>, and <b>E’</b>: spindle domain right and left of chromosomes that is enriched for YBX2. Scale bar in <b>F</b> = 20 μm and also applies to <b>B, C</b> and <b>E</b>. Scale bar in <b>F</b>’ = 10 μm and applies to <b>B’, C’</b> and <b>E’</b>. Significant difference to control: * <i>p</i> < 0.05, *** <i>p <</i> 0.001.</p

Spindle abnormalities and chromosome alignment in <i>in vitro</i> control and preovulatory-aged (PreOA) MII oocytes.

<p>Spindles (green) and chromosomes (red) in control <b>(A)</b> and PreOA MII oocytes <b>(B)</b> and the percentage of oocytes with spindle and chromosome abnormalities <b>(C)</b>. Scale bar in <b>B</b> = 10 μm and also applies to <b>A</b>. Significant differences between groups: ** <i>p <</i> 0.01; *** <i>p <</i> 0.001.</p

Follicle morphology, morphokinetics, and hormone concentrations in conditioned medium of preantral follicle culture.

<p>A) Follicle characteristics, culture survival and maturation of control (<i>n</i> = 1230), preovulatory-aged (PreOA; <i>n</i> = 411) and postovulatory-aged (PostOA; <i>n</i> = 613) oocytes. B-D) Antral stage follicle grown in vitro for 12 d (B) and cumulus-oocyte complexes on day 13 after in vitro ovulation in control oocytes (C) and after postovulatory aging (D) for 12 h. E, F) Altered granulosa cell characteristics after preovulatory aging at day 15 of culture; follicles with an increased accumulation of mural granulosa cells and an apparent follicle compaction (E), and a degenerating follicle with dispersed granulosa cells and a nearly denuded oocyte from day 15 of culture (F). G) Estrogen and (H) progesterone levels (mean ± SEM) in conditioned culture medium prior to and past hormonal stimulation by rhCG/rEGF in the different experimental groups (* <i>P</i><0.05, ** <i>P</i><0.01).</p

Quantification of poly(A) tail length for <i>Dnmt1</i> and <i>Zar1</i> by ePAT.

<p>24 h postovulatory-aged, in vivo-grown oocytes were analyzed by extension poly(A) test (ePAT). A) Gel electrophoresis of the product shows a decrease of poly(A) tail length for <i>Dnmt1</i> in aged oocytes compared to controls, whereas poly(A) tail length of <i>Zar1</i> remains widely stable. These results were quantified by capillary electrophoresis for <i>Zar1</i> (B) and <i>Dnmt1</i> (C). Indicated is the fluorescence intensity (FU) of amplicon lengths (in base pairs) for aged oocytes (red line) and controls (blue line).</p