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

STUDY QUESTION: 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)? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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). MATERIAL AND METHODS: 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). MAIN RESULTS AND ROLE OF CHANCE: 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. LIMITATION, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTERESTS: 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.

ESHRE Task Force on Ethics and Law 21: genetic screening of gamete donors: ethical issues.

This Task Force document explores the ethical issues involved in the debate about the scope of genetic screening of gamete donors. Calls for expanded donor screening arise against the background of both occasional findings of serious but rare genetic conditions in donors or donor offspring that were not detected through present screening procedures and the advent of new genomic technologies promising affordable testing of donors for a wide range of conditions. Ethical principles require that all stakeholders' interests are taken into account, including those of candidate donors. The message of the profession should be that avoiding all risks is impossible and that testing should remain proportional.

Environmental hazard in the aetiology of somatic and germ cell aneuploidy.

Sources of environmental exposures to potentially aneugenic agents are many and include occupational and therapeutic exposures, and exposures associated with lifestyle habits. In this present study, some of these agents and exposure scenarios are discussed that involve potentially large population targets and/or seem to affect chromosome segregation by previously unsuspected mechanisms: metals, possibly acting by epigenetic mechanisms; nano-sized particles that might directly interact with subcellular components of the mitotic and meiotic machineries; cytostatic drugs in healthcare occupations; anticancer therapies potentially affecting the genetic integrity of gametes; continuously increasing electromagnetic field exposures with some sparse evidence of aneugenic activity; endocrine disruptors and their seemingly elusive effects in mouse oocytes, including the first evidence that prenatal exposure could affect meiotic nondisjunction in adult life. Hazards are considered for both somatic cells at risk of neoplastic transformation or tumour progression by chromosome loss and gain and germ cells at risk of heritable aneuploidies associated with spontaneous abortions or genetic diseases. Finally, possible synergistic interactions between environmental exposure and ageing or genetic predisposition are considered that could influence ultimate risks.

Age related changes in mitochondrial function and new approaches to study redox regulation in mammalian oocytes in response to age or maturation conditions.

Mammalian oocytes are long-lived cells in the human body. They initiate meiosis already in the embryonic ovary, arrest meiotically for long periods in dictyate stage, and resume meiosis only after extensive growth and a surge of luteinizing hormone which mediates signaling events that overcome meiotic arrest. Few mitochondria are initially present in the primordial germ cells while there are mitogenesis and structural and functional differentiation and stage-specific formation of functionally diverse domains of mitochondria during oogenesis. Mitochondria are most prominent cell organelles in oocytes and their activities appear essential for normal spindle formation and chromosome segregation, and they are one of the most important maternal contributions to early embryogenesis. Dysfunctional mitochondria are discussed as major factor in predisposition to chromosomal nondisjunction during first and second meiotic division and mitotic errors in embryos, and in reduced quality and developmental potential of aged oocytes and embryos. Several lines of evidence suggest that damage by oxidative stress/reactive oxygen species in dependence of age, altered antioxidative defence and/or altered environment and bi-directional signaling between oocyte and the somatic cells in the follicle contribute to reduced quality of oocytes and blocked or aberrant development of embryos after fertilization. The review provides an overview of mitogenesis during oogenesis and some recent data on oxidative defence systems in mammalian oocytes, and on age-related changes as well as novel approaches to study redox regulation in mitochondria and ooplasm. The latter may provide new insights into age-, environment- and cryopreservation-induced stress and mitochondrial dysfunction in oocytes and embryos.

MCAK is present at centromeres, midspindle and chiasmata and involved in silencing of the spindle assembly checkpoint in mammalian oocytes.

Mitotic centromere-associated kinesin (MCAK) is an ATP-dependent microtubule (MT) depolymerase regulated by Aurora kinase (AURK) phosphorylation and implicated in resolution of improper MT attachments in mitosis. Distribution of MCAK was studied in oocyte maturation by anti-MCAK antibody, anti-tubulin antibody, anti-AURKB antibody and anti-centromere antibody (ACA) and by the expression of MCAK-enhanced green fluorescent protein fusion protein in maturing mouse oocytes. Function was assessed by knockdown of MCAK and Mad2, by inhibiting AURK or the proteasome, by live imaging with polarization microscope and by chromosomal analysis. The results show that MCAK is transiently recruited to the nucleus and transits to spindle poles, ACA-positive domains and chiasmata at prometaphase I. At metaphase I and II, it is present at centrosomes and centromeres next to AURKB and checkpoint proteins Mad2 and BubR1. It is retained at centromeres at telophase I and also at the midbody. Knockdown of MCAK causes a delay in chromosome congression but does not prevent bipolar spindle assembly. MCAK knockdown also induces a meiosis I arrest, which is overcome by knockdown of Mad2 resulting in chiasma resolution, chromosome separation, formation of aberrant meiosis II spindles and increased hypoploidy. In conclusion, MCAK appears to possess a unique distribution and function in oocyte maturation. It is required for meiotic progression from meiosis I to meiosis II associated with silencing of the spindle assembly checkpoint. Alterations in abundance and activity of MCAK, as implicated in aged oocytes, may therefore contribute to the loss of control of cell cycle and chromosome behaviour, thus increasing risk for errors in chromosome segregation and aneuploidy.

Aurora kinase B, epigenetic state of centromeric heterochromatin and chiasma resolution in oocytes.

Aurora kinases comprise a family of phosphoproteins performing multiple functions in mitosis and meiosis. Because Aurora kinase B (AURKB) expression is altered in aged oocytes and there is only limited information on its function in meiosis, it was decided to study the spatial distribution and co-localization of AURKB with other regulatory proteins at centromeres during mouse oocyte maturation. AURKB associates with chromosomes after germinal vesicle breakdown, is enriched at centromeres from prometaphase I and transits to the spindle midzone at late anaphase I. Preferential inhibition of AURKB by low concentrations of ZM 447439 inhibitor prevents polar body formation and affects spindle formation and chromosome congression at meiosis I, associated with expression of BubR1 checkpoint protein at kinetochores. Release of cohesion between sister chromatids appears inhibited resulting in failure of chiasma resolution in oocytes progressing to anaphase I. Concomitantly, the inhibitor reduces histone H3 lysine 9 trimethylation at centromeric heterochromatin and affects chromosome condensation. The cytokinesis arrest protects young, healthy oocytes from errors in chromosome segregation although increasing polyploidy. This study shows that changes in activity of AURKB may increase risks for chromosome non-disjunction and aneuploidy in mammalian oocytes, irrespective of age.

Evaluation of aneugenic effects of bisphenol A in somatic and germ cells of the mouse.

Bisphenol A (BPA) is a synthetic monomer widely used to polymerize polycarbonate plastics and resins. It is shown in vitro to interfere with microtubules, producing aberations in mitotic and meiotic spindles. An increase of meiotic abnormalities in untreated female mice from an experimental colony was temporally correlated with the accidental release of BPA from polycarbonate cages and bottles damaged by inadvertent treatment with harsh alkaline detergents [P.A. Hunt, K.E. Koehler, M. Susiarjo, C.A. Hodges, A. Ilagan, R.C. Voigt, S. Thomas, B.F. Thomas, T.J. Hassold, Bisphenol A exposure causes meiotic aneuploidy in the female mouse, Curr. Biol. 13 (2003) 546-553]. In the present study, potential aneugenic effects of BPA on mouse male and female germ cells and bone marrow cells have been evaluated after acute, sub-chronic or chronic in vivo exposure. Female mice were orally treated with a single BPA dose, with 7 daily administrations or exposed for 7 weeks to BPA in drinking water. No significant induction of hyperploidy or polyploidy was observed in oocytes and zygotes at any treatment condition. The only detectable effect was a significant increase of metaphase II oocytes with prematurely separated chromatids after chronic exposure; this effect, however, had no irreversible consequence upon the fidelity of chromosome segregation during the second meiotic division, as demonstrated by the normal chromosome constitution of zygotes under the same exposure condition. With male mice, no delay of meiotic divisions was found after six daily oral doses of BPA with the BrdU assay. Similarly, no induction of hyperploidy and polyploidy was shown in epydidimal sperm hybrized with probes for chromosomes 8, X and Y, 22 days after six daily oral BPA doses. Finally, two daily oral BPA doses did not induce any increase of micronucleus frequencies in polychromatic erythrocytes of mouse bone marrow. In conclusion, our results do not add evidence to the suspected aneugenic activity of BPA and suggest that other factors or co-factors should be considered to explain the unexpected burst of meiotic abnormalities previously attributed to accidental BPA exposure.

Spindle formation, chromosome segregation and the spindle checkpoint in mammalian oocytes and susceptibility to meiotic error.

The spindle assembly checkpoint (SAC) monitors attachment to microtubules and tension on chromosomes in mitosis and meiosis. It represents a surveillance mechanism that halts cells in M-phase in the presence of unattached chromosomes, associated with accumulation of checkpoint components, in particular, Mad2, at the kinetochores. A complex between the anaphase promoting factor/cylosome (APC/C), its accessory protein Cdc20 and proteins of the SAC renders APC/C inactive, usually until all chromosomes are properly assembled at the spindle equator (chromosome congression) and under tension from spindle fibres. Upon release from the SAC the APC/C can target proteins like cyclin B and securin for degradation by the proteasome. Securin degradation causes activation of separase proteolytic enzyme, and in mitosis cleavage of cohesin proteins at the centromeres and arms of sister chromatids. In meiosis I only the cohesin proteins at the sister chromatid arms are cleaved. This requires meiosis specific components and tight regulation by kinase and phosphatase activities. There is no S-phase between meiotic divisions. Second meiosis resembles mitosis. Mammalian oocytes arrest constitutively at metaphase II in presence of aligned chromosomes, which is due to the activity of the cytostatic factor (CSF). The SAC has been identified in spermatogenesis and oogenesis, but gender-differences may contribute to sex-specific differential responses to aneugens. The age-related reduction in expression of components of the SAC in mammalian oocytes may act synergistically with spindle and other cell organelles' dysfunction, and a partial loss of cohesion between sister chromatids to predispose oocytes to errors in chromosome segregation. This might affect dose-response to aneugens. In view of the tendency to have children at advanced maternal ages it appears relevant to pursue studies on consequences of ageing on the susceptibility of human oocytes to the induction of meiotic error by aneugens and establish models to assess risks to human health by environmental exposures.

Gender effects on the incidence of aneuploidy in mammalian germ cells.

Aneuploidy occurs in 0.3% of newborns, 4% of stillbirths, and more than 35% of all human spontaneous abortions. Human gametogenesis is uniquely and gender-specific susceptible to errors in chromosome segregation. Overall, between 1% and 4% of sperm and as many as 20% of human oocytes have been estimated by molecular cytogenetic analysis to be aneuploid. Maternal age remains the paramount aetiological factor associated with human aneuploidy. The majority of extra chromosomes in trisomic offspring appears to be of maternal origin resulting from nondisjunction of homologous chromosomes during the first meiotic division. Differences in the recombination patterns between male and female meiosis may partly account for the striking gender- and chromosome-specific differences in the genesis of human aneuploidy, especially in aged oocytes. Nondisjunction of entire chromosomes during meiosis I as well as premature separation of sister chromatids or homologues prior to meiotic anaphase can contribute to aneuploidy. During meiosis, checkpoints at meiotic prophase and the spindle checkpoint at M-phase can induce meiotic arrest and/or cell death in case of disturbances in pairing/recombination or spindle attachment of chromosomes. It has been suggested that gender differences in aneuploidy may result from more permissive checkpoints in females than males. Furthermore, age-related loss of chromosome cohesion in oocytes as a cause of aneuploidy may be female-specific. Comparative data about the susceptibility of human male and female germ cells to aneuploidy-causing chemicals is lacking. Increases of aneuploidy frequency in sperm have been shown after exposure to therapeutic drugs, occupational agents and lifestyle factors. Conversely, data on oocyte aneuploidy caused by exogenous agents is limited because of the small numbers of oocytes available for analysis combined with potential maternal age effects. The vast majority of animal studies on aneuploidy induction in germ cells represent cause and effect data. Specific studies designed to evaluate possible gender differences in induction of germ cell aneuploidy have not been found. However, the comparison of rodent data available from different laboratories suggests that oocytes are more sensitive than male germ cells when exposed to chemicals that effect the meiotic spindle. Only recently, in vitro experiments, analyses of transgenic animals and knockdown of expression of meiotic genes have started to address the molecular mechanisms underlying chromosome missegregation in mammalian germ cells whereby striking differences between genders could be shown. Such information is needed to clarify the extent and the mechanisms of gender effects, including possible differential susceptibility to environmental agents.

Influence of follicular fluid meiosis-activating sterol on aneuploidy rate and precocious chromatid segregation in aged mouse oocytes.

BACKGROUND: Follicular fluid meiosis-activating sterol (FF-MAS) protects young oocytes from precocious chromatid separation (predivision). Reduced expression of cohesion and checkpoint proteins and predivision has been hypothesized to occur in age-related aneuploidy in oocytes. METHODS: To know whether FF-MAS also protects aged oocytes from predivision and from age-related non-disjunction, we analysed chromosome constitution in mouse oocytes matured spontaneously with or without 10 microM FF-MAS and in hypoxanthine (HX)-arrested young and aged oocytes induced to resume maturation by FF-MAS. Messenger RNA for checkpoint protein MAD2 and cohesion protein SMC1beta was compared between oocytes matured with or without FF-MAS. RESULTS: Aged oocytes possessed many bivalents with single distal chiasma at meiosis I. Predivision was especially high in aged oocytes cultured sub-optimally to metaphase II in alpha-minimum essential medium (alpha-MEM). FF-MAS reduced predivision significantly (P < 0.001) but neither reduced non-disjunction nor induced aneuploidy in aged oocytes. Polyploidy was high in FF-MAS-stimulated maturation, in particular in the aged oocytes (P > 0.001). Relative levels of Smc1beta mRNA appeared increased by maturation in FF-MAS, and mitochondrial clustering was restored. CONCLUSIONS: Sister chromatids of aged oocytes appear to be highly susceptible to precocious chromatid separation, especially when maturation is under sub-optimal conditions, e.g. in the absence of cumulus and FF-MAS. This may relate to some loss of chromatid cohesion during ageing. FF-MAS protects aged oocytes from predivision during maturation, possibly by supporting Smc1beta expression, thus reducing risks of meiotic errors, but it cannot prevent age-related non-disjunction. Aged oocytes appear prone to loss of co-ordination between nuclear maturation and cytokinesis suggesting age-related relaxed cell cycle control.

Light retardance by human oocyte spindle is positively related to pronuclear score after ICSI.

Disturbed spindle assembly increases risks of chromosome mal-segregation. Non-invasive polarization microscopy (PolScope) was employed in two centres to assess spindle integrity for the first time quantitatively in human oocytes from consenting patients undergoing intracytoplasmic sperm injection (ICSI) with respect to pronuclear (PN) score after fertilization. In one centre oocytes were selected before ICSI, in another selection was after ICSI according to PN score. In both centres, mean retardance of light by birefringent spindles in oocytes forming a pre-embryo with good PN score after ICSI was significantly higher compared with spindles in oocytes developing into a lower PN score pre-embryo with limited developmental potential (P < 0.001). Transfers involving oocytes with high retardance and at least one good PN score embryo resulted more frequently in a conception than transfers from oocytes with spindles of lower mean retardance and lower PN score embryos. There was a trend for an inverse relationship between age and magnitude of retardance in a small oocyte cohort. The study suggests that quantitative evaluation of mean retardance of light by the oocyte spindle predicts oocyte health, is related to PN score of the embryo and may be especially useful to assess oocyte quality in countries with legal restrictions to select after fertilization.

Mouse genetic models for aneuploidy induction in germ cells.

Rodents have been successfully used as models to identify risks of chemical exposures or age to aneuploidy induction in germ cells, which may be transmitted to the progeny. For this administration in vivo as well as exposures to in vitro maturing germ cells have been useful. Genetic models involving mice with structural chromosomal rearrangements and transgenic animals have the potential to model conditions predisposing to aneuploidy in one or both sexes, and in this way to identify potential targets for aneugens and gender-effects. The review provides an overview of mouse genetic models for aneuploidy induction in mammalian germ cells and discusses perspectives for combining genetic with experimental approaches in aneuploidy research.

High magnitude of light retardation by the zona pellucida is associated with conception cycles.

BACKGROUND: Failures in expression of zona proteins correlate to subfertility in animals. Low expression of the zona proteins by the growing human oocyte may indicate reduced developmental potential. Therefore, we non-invasively analysed the thickness and the structure of the zona pellucida (ZP) of human oocytes with respect to embryo fate after ICSI. METHODS: Retardance magnitude and thickness of the inner, middle and outer layers of the ZP were quantitatively analysed by a Polscope in 166 oocytes selected for transfer after ICSI (63 patients; 32.8 +/- 4.4 years) on the basis of pronuclear score at day 1. Blastomere number was determined at day 2. Data were compared between conception cycles (CC; 65 oocytes/23 patients) and non-conception cycles (NCC; 101 oocytes/40 patients) and with respect to maternal age. RESULTS: The thickness was slightly elevated (P < 0.001), and the mean magnitude of light retardance was nearly 30% higher (P < 0.001) in the inner layer of the zona pellucida of oocytes contributing to CC compared to NCC. Embryos in the CC group tended to develop faster. CONCLUSIONS: The magnitude of light retardance by the zona pellucida inner layer appears to present a unique non-invasive marker for oocyte developmental potential.

Spindles, mitochondria and redox potential in ageing oocytes.

Studies of human oocytes obtained from women of advanced reproductive age revealed that spindles are frequently aberrant, with chromosomes sometimes failing to align properly at the equator during meiosis I and II. Chromosomal analyses of donated and spare human oocytes and cytogenetic and molecular studies on the origin of trisomies collectively suggest that errors in chromosome segregation during oogenesis increase with advancing maternal age and as the menopause approaches. Disturbances in the fidelity of chromosome segregation, especially at anaphase I, leading to aneuploidy are prime causes of reduced developmental competence of embryos in assisted reproduction, as well as being responsible for the genesis of genetic disease. This review provides an overview of spindle formation and chromosome behaviour in mammalian oocytes. Evidence of a link between abnormal mitochondrial function in oocytes and somatic follicular cells, and finally disturbances in chromosome cohesion and segregation, and cell cycle control in aged mammalian oocytes, are also discussed.

Meiosis-activating sterol protects oocytes from precocious chromosome segregation.

BACKGROUND: Follicular fluid meiosis-activating sterol (FF-MAS) overcomes hypoxanthine (HX)-mediated meiotic arrest in mammalian oocytes. METHODS: In order to determine whether chromosome segregation was normal in oocytes matured in FF-MAS, the development, chromosomal constitution and chromosome alignment was analysed in spontaneously matured as well as HX-arrested mouse oocytes cultured in the absence or presence of FF-MAS. RESULTS: FF-MAS-induced meiotic maturation was significantly less effective compared with spontaneous maturation in supporting cytokinesis ( approximately 40 and approximately 90% polar body formation respectively). The majority of oocytes stimulated by FF-MAS to overcome the HX block developed to metaphase II (MII), but 23.4% of meiosis II oocytes were diploid. Chromosomes were well aligned on the spindle, and hyperploidy was low in spontaneously matured oocytes and HX-arrested oocytes cultured with or without FF-MAS. Unexpectedly, almost 40% of spontaneously matured MII oocytes contained chromatids/monads. Precocious loss of chromatid cohesion was significantly reduced in spontaneously matured as well as HX-arrested oocytes cultured in the presence of FF-MAS but not lanosterol. CONCLUSIONS: FF-MAS induces full nuclear maturation to MII, and chromosomes segregate with high fidelity. However, in delayed FF-MAS-stimulated meiotic maturation, anaphase I may occur in the absence of cytokinesis. FF-MAS appears to protect mammalian oocytes from precocious chromatid segregation.

Reproductive semi-cloning respecting biparental origin. Reconstitution of gametes for assisted reproduction.

There are basically two major problems in the genesis of "cloned" gametes in mammals, which have not been addressed in the original debate article. There is no adequate discussion on the mechanisms providing for high fidelity of chromosome segregation in mitosis and meiosis, and for proper imprinting in construction of "reconstituted gametes". The original debate article is uncritical with respect to the currently insufficient database and the incomplete documentation of results.

Expression in in-vivo and in-vitro growing and maturing oocytes: focus on regulation of expression at the translational level.

Studies of expression in in-vivo and in-vitro maturing oocytes have the potential to elucidate signalling pathways involved in the intricate crosstalk between the oocyte and its somatic compartment during differentiation and morphogenetic processes, and the origin of disturbances in oocyte maturation possibly involved in reduced fertility. This review summarizes data on expression studies with focus on regulation of expression at the translational level in the maturing oocyte. The regulation of gene expression at the translational level as analysed in in-vitro maturing oocytes is complex and highly conserved between different species. It is characterized by differential degradation, and by storage and recruitment of distinct maternal mRNAs involving conserved consensus sequences in the 5' or 3' untranslated regions (UTRs) of mRNAs. Proteins interacting with such sequences affect the temporal 3' polyadenylation, and bring the 5' and 3' UTRs of mRNAs into close proximity for efficient initiation of translation. Post-translational modifications of mRNA-associated proteins contribute to maturation- and developmentally controlled and to cell cycle-dependent expression. New methodologies for analysis of ovary-specific gene expression and function of genes in oogenesis are also reviewed, e.g. RT-PCR, SAGE-PCR, real-time rapid cycle fluorescence monitored RT-PCR, differential display techniques, and microinjection of anti-sense RNA, double-stranded RNAs or mRNAs expressing green fluorescent protein-tagged proteins into maturing oocytes.

Maternal versus paternal expression of a LacZ transgene in preimplantation mouse embryos: effects of genetic background and 2-cell block.

The expression of a transgene NI-ROSA LacZ (LacZtg) trapped into the genes for two presumably untranslated, ubiquitously expressed RNAs, was studied in preimplantation mouse embryos with respect to penetrance (fraction of expressing embryos) and to localisation of beta-galactosidase activity. With maternal origin in NMRI mice beta-galactosidase was first detected within one dot in the cytoplasm of zygotes at 30 h post-hCG. The staining pattern progressed to small clusters and to dense, homogeneous staining of the entire cytoplasm during further development. Within the NMRI background, penetrance in utero was delayed by at least 6 h when the transgene was of paternal as compared with maternal origin. Paternal transgene expression increased marginally during culture to 50 h after explantation of embryos at 30-48 h post-hCG and remained low or decreased in the '2-cell block'. Expression of a paternal transgene in preimplantation embryos developing in utero was further delayed in the maternal MF1 as compared with the NMRI background. In contrast to NMRI x NMRI embryos with paternally derived transgene, expression increased with time during the 2-cell block in MF1 x NMRI embryos. Thus, in the earliest phase of mammalian development expression of this LacZtg is influenced by parental origin, maternal genetic background and environment. The spatial distribution of the gene product is developmentally controlled.

Differential chromosome behaviour in mammalian oocytes exposed to the tranquilizer diazepam in vitro.

There are several reports demonstrating that aneugens may preferentially affect segregation of particular chromosomes in somatic cells. Much less is known on specific susceptibility of individual chromosomes to non-disjunction in mammalian meiosis in response to chemical exposures. To explore possible chromosome-specific behaviour and susceptibility to errors in chromosome segregation in mammalian oogenesis we employed spindle immunofluoresecence in combination with FISH with chromosome-specific probes to analyse congression of chromosomes X, 8 and 16 in diazepam (DZ)-treated, meiotically delayed meiosis I oocytes of the mouse. Concomitantly, we assessed the susceptibility of homologues to precociously segregate prior to anaphase I during DZ-induced meiotic arrest. About 50% of all oocytes exposed to 25 microg/ml DZ became meiotically delayed. Chromosomes failed to congress at the spindle equator in one-third of these meiosis I oocytes. The X chromosome was significantly more often located away from the spindle equator as compared with the expected random behaviour. Concomitantly, DZ exposure induced untimely segregation of homologous chromosomes of the gonosome and the autosomes in meiosis I. This occurred with similar frequencies. The observations confirm that DZ perturbs cell cycle progression, interferes with chromosome alignment, causes predivision and thus may predispose mammalian oocytes to errors in chromosome segregation. For the first time, chromosome-specific behaviour is reported in female meiosis in response to exposure to an aneugenic chemical.