Preantral follicle culture and oocyte quality.

The formation of high-quality oocytes depends on complex stage-specific interactions between the germ cell and the somatic compartment involving endocrine, paracrine, and autocrine regulation. Cooperativity in bidirectional signalling and metabolism in response to factors in the microenvironment drive growth, proliferation, cell cycle regulation, spindle formation and the establishment of epigenetic marks in oocytes. This is essential to ensure faithful chromosome segregation and to achieve high oocyte quality, with far-reaching consequences for embryo survival, development and the health of offspring. Oocytes reach developmental capacity throughout early meiotic stages up to full growth and acquisition of nuclear and cytoplasmic maturational competence during folliculogenesis. Improved preantral follicle culture in which ideally intimate contacts between oocyte and somatic cells are retained provides unique opportunities to assess the effects of microenvironment, growth factors, hormones, cryopreservation and environmental exposure on folliculogenesis and oocyte quality. An optimised follicle culture can contribute to the generation of high-quality oocytes for use in fertility preservation in cancer patients, animal breeding and the preservation of endangered species. The past decade has brought about major advances in follicle culture from different species. Recent advances in preantral follicle culture are discussed to assess the effects of environment, adverse exposure, cryopreservation and age on oocyte quality.

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.

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.

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.

Elements of transcriptional machinery are compatible among plants and mammals.

In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-κB in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-κB such as its inhibitor IκB isoform α that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-κB in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-κB signaling pathways. The system successfully enabled the controlled manipulation of NF-κB activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e.g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway.