Consumption of hookahs, e-cigarettes, and classic cigarettes and the impact on medically assisted reproduction treatment.

Smoking of classic cigarettes has been well-established as a health risk factor, including cardiovascular, neurological, and pulmonary diseases. Adverse effects on human reproduction have also been shown. Smokers are assumed to have a significantly lower chance of pregnancy, however, the impact of smoking on medically assisted reproduction (MAR) treatment outcomes is controversial. Moreover, smoking habits have changed during the last decades since e-cigarettes and hookahs, or water pipes, have become very popular, yet little is known regarding vaping or hookah-smoking patients undergoing MAR treatments. This prospective study aimed to examine the presence of benzo[a]pyrene, nicotine, and its main metabolite, cotinine, in human follicular fluid (FF) in non-smoking, smoking, and vaping/hookah-smoking patients and to evaluate the impact on female fertility. Human FF samples were collected from 320 women subjected to intracytoplasmic sperm injection (ICSI) cycles due to male subfertility. Gas chromatography combined with mass spectrometry was used to analyse the presence of benzo[a]pyrene, nicotine, and cotinine. A questionnaire was provided to assess patient consumption behaviour and to identify (1) non-smoking patients, (2) patients who consumed cigarettes, and (3) patients with exclusive consumption of e-cigarettes or hookahs. Data were analysed using linear and logistic regression, Fisher's exact test, and the Mann-Whitney U Test. Nicotine was present in 22 (6.8%) and cotinine in 65 (20.3%) of the 320 samples. The nicotine and cotinine concentrations per sample ranged from 0 to 26.3 ng/ml and 0-363.0 ng/ml, respectively. Benzo[a]pyrene was not detectable in any of the samples analysed. Nicotine and cotinine were also present in the FF of patients with exclusive consumption of e-cigarettes or hookahs. The clinical pregnancy rate, fertilization and maturation rates, and number of oocytes per oocyte pick-up were not statistically significantly different between non-smoking, smoking, or vaping/hookah-smoking patients. Smoking and the accumulation of smoking toxins in the FF have no impact on the outcome of MAR treatments-neither the clinical pregnancy rate, maturation and fertilization rates, nor the number of retrieved oocytes were affected. For the first time, nicotine and cotinine were quantified in the FF of patients exclusively vaping e-cigarettes or smoking hookahs. Since vaping liquids and hookah tobaccos contain potentially harmful substances, other adverse effects cannot be excluded.Trial registration ClinicalTrials.gov Identifier: NCT03414567.

Cryopreservation of Ovarian and Testicular Tissue and the Influence on Epigenetic Pattern.

Ovarian tissue cryopreservation (OTC) or testicular tissue cryopreservation (TTC) are effective and often the only options for fertility preservation in female or male patients due to oncological, medical, or social aspects. While TTC and resumption of spermatogenesis, either in vivo or in vitro, has still be considered an experimental approach in humans, OTC and autotransplantation has been applied increasingly to preserve fertility, with more than 200 live births worldwide. However, the cryopreservation of reproductive cells followed by the resumption of gametogenesis, either in vivo or in vitro, may interfere with sensitive and highly regulated cellular processes. In particular, the epigenetic profile, which includes not just reversible modifications of the DNA itself but also post-translational histone modifications, small non-coding RNAs, gene expression and availability, and storage of related proteins or transcripts, have to be considered in this context. Due to complex reprogramming and maintenance mechanisms of the epigenome in germ cells, growing embryos, and offspring, OTC and TTC are carried out at very critical moments early in the life cycle. Given this background, the safety of OTC and TTC, taking into account the epigenetic profile, has to be clarified. Cryopreservation of mature germ cells (including metaphase II oocytes and mature spermatozoa collected via ejaculation or more invasively after testicular biopsy) or embryos has been used successfully for many years in medically assisted reproduction (MAR). However, tissue freezing followed by in vitro or in vivo gametogenesis has become more attractive in the past, while few human studies have analysed the epigenetic effects, with most data deriving from animal studies. In this review, we highlight the potential influence of the cryopreservation of immature germ cells and subsequent in vivo or in vitro growth and differentiation on the epigenetic profile (including DNA methylation, post-translational histone modifications, and the abundance and availability of relevant transcripts and proteins) in humans and animals.

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.

Impact of Nonylphenols and Polyhalogenated Compounds in Follicular Fluid on the Outcome of Intracytoplasmic Sperm Injection.

Endocrine-disrupting chemicals (EDCs) interfere with the mammalian hormone system and alter its endo- and paracrine regulation. The goal of the present study was to examine the presence of 14 EDCs, including the technical mixture of nonylphenols and Mirex, in human follicular fluid (FF) and to find a potential correlation between endocrine active substances and a possible impact on female fertility. Furthermore, potential sources of EDC exposition regarding patients' lifestyle and socioeconomic factors were investigated. Human FF was collected from a total of 210 women undergoing intracytoplasmic sperm injection-treatment cycles because of male subfertility. The presence of EDCs was analyzed using gas chromatography coupled with mass spectrometry. Thirteen of the 14 investigated EDCs were present in every FF sample; compounds with the highest concentrations in FF were nonylphenol and Mirex. Nearly all kinds of EDCs led to significantly reduced maturation and fertilization rate. No significant influence of EDC concentration on the clinical pregnancy rate was observed for neither of the analyzed EDCs. Patients who obtained their clothes and textiles at fashion discounters displayed a higher amount of EDCs in their FF. In contrast, patients' residential area, source of food products, and nicotine or caffeine consumed were not associated with EDC accumulation. Clinicaltrials.gov NCT01385605 (11 July 2011).

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

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.

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

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 (EmGSH) 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 intra-oocyte 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 (EmGSH, 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.

Pre- and postovulatory aging of murine oocytes affect the transcript level and poly(A) tail length of maternal effect genes.

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.

Chronic exposure to a low concentration of bisphenol A during follicle culture affects the epigenetic status of germinal vesicles and metaphase II oocytes.

OBJECTIVE: To determine whether exposure to low concentrations of the endocrine disrupting chemical bisphenol A (BPA) during follicle culture and oocyte growth alters the methylation status of differentially methylated regions (DMRs) of imprinted genes and histone posttranslational modification patterns in mammalian oocytes. DESIGN: Comparative and control study. SETTING: Experimental laboratory. ANIMAL(S): C57/Bl6JxCBA/Ca mice. INTERVENTION(S): Exposure of oocytes to 3 nM or 300 nM BPA during follicle culture from preantral to antral stage. MAIN OUTCOME MEASURE(S): Methylation status of DMRs of maternally imprinted (Snrpn, Igf2r, and Mest) and paternally imprinted gene(s) (H19) in mouse germinal vesicle oocytes; trimethylation of histone H3K9, acetylation of histone H4K12, and distance between centromeres of sister chromatids in metaphase II oocytes. RESULT(S): Exposure to 3 nM BPA was associated with slightly accelerated follicle development, statistically significant increases in allele methylation errors in DMRs of maternally imprinted genes, and statistically significant decreases in histone H3K9 trimethylation and interkinetochore distance. CONCLUSION(S): The disturbances in oocyte genomic imprinting and modification of posttranslational histone and centromere architecture provide the first link between low BPA exposures and induction of epigenetic changes that may contribute to chromosome congression failures and meiotic errors, and to altered gene expression that might affect health of the offspring.

Endoplasmic reticulum-derived multilamellar bodies in oocytes of mouse follicle cultures under oxidized low-density lipoprotein treatment.

OBJECTIVE: Multilamellar bodies associated with an organized endoplasmic reticulum (ER) arise in various somatic cell types, and a subtype called multivesicular bodies is described in oocytes. Both entities, so far undetermined in significance, may occur in oocytes of follicles under oxidative stress. In preovulatory follicles, oxidative stress appears to be caused by oxidized low-density lipoprotein (ox-LDL). METHOD: Cultures of preantral mouse follicles were treated with 100 µg/ml ox-LDL or normal LDL (n-LDL) for 12-48 h or for 12 days during antral follicle growth followed by in vitro ovulation and harvest of cumulus oophorus complexes (COCs) with metaphase II (MII) oocytes on day 13. Preantral follicles, COCs, or MII oocytes were immunostained with anti-tubulin antibody or stained with actin-binding phalloidin for confocal microscopy. Ultrathin sections were prepared for electron microscopy. RESULTS: Preantral follicles exposed to n-LDL or ox-LDL developed normally, and MII oocytes in COCs possessed normal spindles with well-aligned chromosomes. In contrast, treated cumulus cells underwent apoptosis. Only the ox-LDL-treated preantral follicle oocytes showed ER-derived multilamellar bodies (EMBs) of type I, consisting of rough ER membranes for the envelope. The MII oocytes of COCs showed type II EMBs consisting of smooth/vesicular ER and were more prominent after ox-LDL than after n-LDL exposure. Degenerating mitochondria were prominent in oocytes of the ox-LDL group and judged as a sign of oxidative stress. CONCLUSION: Oxidative stress presumably induces damage of proteins and organelles in the oocytes. The EMBs might sequester the damaged structures for oocyte survival. Thus, EMBs could represent a novel form of autophagy.

Vitrification at the pre-antral stage transiently alters inner mitochondrial membrane potential but proteome of in vitro grown and matured mouse oocytes appears unaffected.

BACKGROUND: Vitrification is a fast and effective method to cryopreserve ovarian tissue, but it might influence mitochondrial activity and affect gene expression to cause persistent alterations in the proteome of oocytes that grow and mature following cryopreservation. METHODS: In part one of the study, the inner mitochondrial membrane potential (Ψ(mit)) of JC-1 stained oocytes from control and CryoTop vitrified pre-antral follicles was analyzed by confocal microscopy at Day 0, or after culture of follicles for 1 or 12 days. In part two, proteins of in vivo grown germinal vesicle (GV) oocytes were subjected to proteome analysis by SDS polyacrylamide gel electrophoresis, tryptic in-gel digestion of gel slices, and one-dimensional-nano-liquid chromatography of peptides on a multi-dimensional-nano-liquid chromatography system followed by mass spectrometry (LC-MS/MS) and Uniprot Gene Ontology (GO) analysis. In part three, samples containing the protein amount of 40 GV and metaphase II (MII) oocytes, respectively, from control and vitrified pre-antral follicles cultured for 12 or 13 days were subjected to 2D DIGE saturation labeling and separated by isoelectric focusing and SDS gel electrophoresis (2D DIGE), followed by DeCyder(Tm) analysis of spot patterns in three independent biological replicates. Statistical and hierarchical cluster analysis was employed to compare control and vitrified groups. RESULTS: (i) Mitochondrial inner membrane potential differs significantly between control and vitrified GV oocytes at Day 0 and Day 1, but is similar at Day 12 of culture. (ii) LC-MS/MS analysis of SDS gel fractionated protein lysates of 988 mouse GV oocytes revealed identification of 1123 different proteins with a false discovery rate of <1%. GO analysis assigned 811 proteins to the 'biological process' subset. Thirty-five percent of the proteins corresponded to metabolic processes, about 15% to mitochondrion and transport, each, and close to 8% to oxidation-reduction processes. (iii) From the 2D-saturation DIGE analysis 1891 matched spots for GV-stage and 1718 for MII oocyte proteins were detected and the related protein abundances in vitrified and control oocytes were quantified. None of the spots was significantly altered in intensity, and hierarchical cluster analysis as well as histograms of p and q values suggest that vitrification at the pre-antral stage does not significantly alter the proteome of GV or MII oocytes compared with controls. CONCLUSIONS: Vitrification appears to be associated with a significant transient increase in Ψ(mit) in oocyte mitochondria, which disappears when oocyte/cumulus cell apposition is restored upon development to the antral stage. The nano-LC-MS/MS analysis of low numbers of oocytes is useful to obtain information on relevant biological signaling pathways based on protein identifications. For quantitative comparisons, saturation 2D DIGE analysis is superior to LC-MS/MS due to its high sensitivity in cases where the biological material is very limited. Genetic background, age of the female, and/or stimulation protocol appear to influence the proteome pattern. However, the quantitative 2D DIGE approach provides evidence that vitrification does not affect the oocyte proteome after recovery from transient loss of cell-cell interactions, in vitro growth and in vitro maturation under tested conditions. Therefore, transient changes in mitochondrial activity by vitrification do not appear causal to persistent alterations in the mitochondrial or overall oocyte proteome.

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.

Chromosomal and cytoplasmic context determines predisposition to maternal age-related aneuploidy: brief overview and update on MCAK in mammalian oocytes.

It has been known for more than half a century that the risk of conceiving a child with trisomy increases with advanced maternal age. However, the origin of the high susceptibility to nondisjunction of whole chromosomes and precocious separation of sister chromatids, leading to aneuploidy in aged oocytes and embryos derived from them, cannot be traced back to a single disturbance and mechanism. Instead, analysis of recombination patterns of meiotic chromosomes of spread oocytes from embryonal ovary, and of origins and exchange patterns of extra chromosomes in trisomies, as well as morphological and molecular studies of oocytes and somatic cells from young and aged females, show chromosome-specific risk patterns and cellular aberrations related to the chronological age of the female. In addition, analysis of the function of meiotic- and cell-cycle-regulating genes in oogenesis, and the study of the spindle and chromosomal status of maturing oocytes, suggest that several events contribute synergistically to errors in chromosome segregation in aged oocytes in a chromosome-specific fashion. For instance, loss of cohesion may differentially predispose chromosomes with distal or pericentromeric chiasmata to nondisjunction. Studies on expression in young and aged oocytes from human or model organisms, like the mouse, indicate that the presence and functionality/activity of gene products involved in cell-cycle regulation, spindle formation and organelle integrity may be altered in aged oocytes, thus contributing to a high risk of error in chromosome segregation in meiosis I and II. Genes that are often altered in aged mouse oocytes include MCAK (mitotic-centromere-associated protein), a microtubule depolymerase, and AURKB (Aurora kinase B), a protein of the chromosomal passenger complex that has many targets and can also phosphorylate and regulate MCAK localization and activity. Therefore we explored the role of MCAK in maturing mouse oocytes by immunofluorescence, overexpression of a MCAK-EGFP (enhanced green fluorescent protein) fusion protein, knockdown of MCAK by RNAi (RNA interference) and inhibition of AURKB. The observations suggest that MCAK is involved in spindle regulation, chromosome congression and cell-cycle control, and that reductions in mRNA and protein in a context of permissive SAC (spindle assembly checkpoint) predispose to aneuploidy. Failure to recruit MCAK to centromeres and low expression patterns, as well as disturbances in regulation of enzyme localization and activity, e.g. due to alterations in activity of AURKB, may therefore contribute to maternal age-related rises in aneuploidy in mammalian oocytes.

DNA integrity, growth pattern, spindle formation, chromosomal constitution and imprinting patterns of mouse oocytes from vitrified pre-antral follicles.

BACKGROUND: Cryopreservation of follicles for culture and oocyte growth and maturation in vitro provides an option to increase the number of fertilizable oocytes and restore fertility in cases where transplantation of ovarian tissue poses a risk for malignant cell contamination. Vitrification for cryopreservation is fast and avoids ice crystal formation. However, the influences of exposure to high concentrations of cryoprotectants on follicle development, oocyte growth and maturation, and particularly, on the DNA integrity and methylation imprinting has not been studied systematically. METHODS: Follicle survival and development, DNA damage, oocyte growth patterns, maturation, spindle formation and chromosomal constitution were studied after Cryo-Top vitrification of mouse pre-antral follicles cultured to the antral stage and induced to ovulate in vitro. Methylation of differentially methylated regions (DMRs) of two maternally (Snrpn and Igf2r) and one paternally (H19) imprinted genes was studied by bisulfite pyrosequencing. RESULTS: Vitrification results in partial or total loss of oocyte-granulosa cell apposition and actin-rich transzonal projections, a transient increase in DNA breaks and a delay in follicle development. However, the oocyte growth pattern, maturation, spindle and chromosomal constitution are not significantly different between the vitrified and the control groups. Vitrification is not associated with elevated levels of imprinting mutations (aberrant methylation of the entire DMR), although the distribution of sporadic CpG methylation errors in the Snrpn DMR appears to differ slightly between control and vitrified oocytes. CONCLUSIONS: DNA breaks appear to be rapidly repaired and vitrification of oocytes inside pre-antral follicles by the Cryo-Top method does not appear to increase risks of abnormal imprinting or disturbances in spindle formation and chromosome segregation.

Induction of distinct defense-associated protein patterns in Aphanomyces euteiches (Oomycota)-elicited and -inoculated Medicago truncatula cell-suspension cultures: a proteome and phosphoproteome approach.

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.