Publications by authors named "Martin Anger"

30 Publications

  • Page 1 of 1

Aneuploidy during the onset of mouse embryo development.

Reproduction 2020 11;160(5):773-782

Department of Genetics and Reproduction, Central European Institute of Technology, Veterinary Research Institute, Brno, Czech Republic.

Aneuploidy is the most frequent single cause leading into the termination of early development in human and animal reproduction. Although the mouse is frequently used as a model organism for studying the aneuploidy, we have only incomplete information about the frequency of numerical chromosomal aberrations throughout development, usually limited to a particular stage or assumed from the occurrence of micronuclei. In our study, we systematically scored aneuploidy in in vivo mouse embryos, from zygotes up to 16-cell stage, using kinetochore counting assay. We show here that the frequency of aneuploidy per blastomere remains relatively similar from zygotes until 8-cell embryos and then increases in 16-cell embryos. Due to the accumulation of blastomeres, aneuploidy per embryo increases gradually during this developmental period. Our data also revealed that the aneuploidy from zygotes and 2-cell embryos does not propagate further into later developmental stages, suggesting that embryos suffering from aneuploidy are eliminated at this stage. Experiments with reconstituted live embryos revealed, that hyperploid blastomeres survive early development, although they exhibit slower cell cycle progression and suffer frequently from DNA fragmentation and cell cycle arrest.
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http://dx.doi.org/10.1530/REP-20-0086DOI Listing
November 2020

Cyclin A1 in Oocytes Prevents Chromosome Segregation And Anaphase Entry.

Sci Rep 2020 05 4;10(1):7455. Epub 2020 May 4.

Central European Institute of Technology, Department of Genetics and Reproduction, Veterinary Research Institute, Brno, Czech Republic.

In several species, including Xenopus, mouse and human, two members of cyclin A family were identified. Cyclin A2, which is ubiquitously expressed in dividing cells and plays role in DNA replication, entry into mitosis and spindle assembly, and cyclin A1, whose function is less clear and which is expressed in spermatocytes, leukemia cells and in postmitotic multiciliated cells. Deletion of the gene showed that cyclin A1 is essential for male meiosis, but nonessential for female meiosis. Our results revealed, that the cyclin A1 is not only dispensable in oocytes, we show here that its expression is in fact undesirable in these cells. Our data demonstrate that the APC/C and proteasome in oocytes are unable to target sufficiently cyclin A1 before anaphase, which leads into anaphase arrest and direct inhibition of separase. The cyclin A1-induced cell cycle arrest is oocyte-specific and the presence of cyclin A1 in early embryos has no effect on cell cycle progression or chromosome division. Cyclin A1 is therefore not only an important cell cycle regulator with biased expression in germline, being essential for male and damaging for female meiosis, its persistent expression during anaphase in oocytes shows fundamental differences between APC/C function in oocytes and in early embryos.
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http://dx.doi.org/10.1038/s41598-020-64418-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198627PMC
May 2020

ProTAME Arrest in Mammalian Oocytes and Embryos Does Not Require Spindle Assembly Checkpoint Activity.

Int J Mol Sci 2019 Sep 13;20(18). Epub 2019 Sep 13.

Central European Institute of Technology, Department of Genetics and Reproduction, Veterinary Research Institute, 621 00 Brno, Czech Republic.

In both mitosis and meiosis, metaphase to anaphase transition requires the activity of a ubiquitin ligase known as anaphase promoting complex/cyclosome (APC/C). The activation of APC/C in metaphase is under the control of the checkpoint mechanism, called the spindle assembly checkpoint (SAC), which monitors the correct attachment of all kinetochores to the spindle. It has been shown previously in somatic cells that exposure to a small molecule inhibitor, prodrug tosyl-l-arginine methyl ester (proTAME), resulted in cell cycle arrest in metaphase, with low APC/C activity. Interestingly, some reports have also suggested that the activity of SAC is required for this arrest. We focused on the characterization of proTAME inhibition of cell cycle progression in mammalian oocytes and embryos. Our results show that mammalian oocytes and early cleavage embryos show dose-dependent metaphase arrest after exposure to proTAME. However, in comparison to the somatic cells, we show here that the proTAME-induced arrest in these cells does not require SAC activity. Our results revealed important differences between mammalian oocytes and early embryos and somatic cells in their requirements of SAC for APC/C inhibition. In comparison to the somatic cells, oocytes and embryos show much higher frequency of aneuploidy. Our results are therefore important for understanding chromosome segregation control mechanisms, which might contribute to the premature termination of development or severe developmental and mental disorders of newborns.
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http://dx.doi.org/10.3390/ijms20184537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770151PMC
September 2019

Regulation of the cell cycle in early mammalian embryos and its clinical implications.

Int J Dev Biol 2019 ;63(3-4-5):113-122

Central European Institute of Technology, Department of Genetics and Reproduction, Veterinary Research Institute, Brno, Czech Republic.

Early embryonic development is characterized by a plethora of very complex and simultaneously operating processes, which are constantly changing cellular morphology and behaviour. After fertilization, blastomeres of the newly created embryo undergo global epigenetic changes and simultaneously initiate transcription from the zygotic genome and differentiation forming separate cell lineages. Some of these mechanisms were extensively studied during the last several decades and valuable insight was gained into how these processes are regulated at the molecular level. We have, however, a still very limited understanding of how multiple events are coordinated during rapid development of an early mammalian embryo. In this review, we discuss some aspects of early embryonic development in mammals, namely the fidelity of chromosome segregation and occurrence of aneuploidy, as well as the clinical applications of cell cycle monitoring in human embryos.
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http://dx.doi.org/10.1387/ijdb.180400maDOI Listing
January 2020

Increased frequency of chromosome congression defects and aneuploidy in mouse oocytes cultured at lower temperature.

Reprod Fertil Dev 2017 Apr;29(5):968-974

Central European Institute of Technology, Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic.

Optimal culture conditions are essential for successful IVM of mammalian oocytes and for their further development into an embryo. In the present study we used live cell imaging microscopy to assess the effects of suboptimal culture temperature on various aspects of IVM, including duration of meiosis I, dynamics of polar body extrusion, chromosome congression, anaphase-promoting complex/cyclosome (APC/C) activation and aneuploidy. The data showed that even a small deviation from the optimal incubation temperature causes marked changes in the duration and synchronicity of meiosis, APC/C activity and the frequency of chromosome congression and segregation errors. In vitro manipulation and maturation of germ cells is widely used in both human and animal artificial reproduction techniques. Mammalian oocytes are naturally prone to chromosomal segregation errors, which are responsible for severe mental and developmental disorders. The data presented herein demonstrate that exposure of mouse oocytes to suboptimal temperature during manipulation and maturation could further increase the frequency of chromosome segregation defects in these cells.
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http://dx.doi.org/10.1071/RD15306DOI Listing
April 2017

True Nondisjunction of Whole Bivalents in Oocytes with Attachment and Congression Defects.

Cytogenet Genome Res 2017 10;151(1):10-17. Epub 2017 Mar 10.

Veterinary Research Institute, Brno, Czech Republic.

Chromosome segregation in mammalian oocytes is prone to errors causing aneuploidy with consequences such as precocious termination of development or severe developmental disorders. Aneuploidy also represents a serious problem in procedures utilizing mammalian gametes and early embryos in vitro. In our study, we focused on congression defects during meiosis I and observed whole nondisjoined bivalents in meiosis II as a direct consequence, together with a substantially delayed first polar body extrusion. We also show that the congression defects are accompanied by less stable attachments of the kinetochores. Our results describe a process by which congression defects directly contribute to aneuploidy.
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http://dx.doi.org/10.1159/000458513DOI Listing
September 2017

In Vitro Maturation of Mouse Oocytes Increases the Level of Kif11/Eg5 on Meiosis II Spindles.

Biol Reprod 2016 07 4;95(1):18. Epub 2016 May 4.

Veterinary Research Institute, Brno, Czech Republic Faculty of Medicine, Department of Histology and Embryology, Masaryk University, Brno, Czech Republic

Although in vitro maturation (IVM) of oocytes has been used for a relatively long time, during which the culture conditions have improved remarkably, the resulting germ cells are still not fully comparable to the cells obtained from the ovary in many important aspects, namely in fertilization rate and subsequent embryonic development. Some of the differences between IVM and in vivo maturation (IVV) oocytes were already discovered, including variability in spindle assembly and morphology. In this study we focused on a role of molecular motor Kif11 (hereafter referred to as Eg5) in maintaining bipolar spindle structure in IVM and IVV oocytes. Our experiments revealed that in IVM oocytes, Eg5 is abundant on meiosis II spindle, which makes these cells more sensitive to Eg5 inhibition than IVV oocytes. We further demonstrate that this sensitivity is acquired gradually with exposure to the in vitro conditions. This is a remarkable difference in function of spindle apparatus between IVM and IVV oocytes, and we believe our results are important not only for understanding of the chromosome segregation in mammalian oocytes but also because they indicate cells are using alternative pathways to achieve the same function when exposed to different conditions.
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http://dx.doi.org/10.1095/biolreprod.115.133900DOI Listing
July 2016

A Balance between Nuclear and Cytoplasmic Volumes Controls Spindle Length.

PLoS One 2016 17;11(2):e0149535. Epub 2016 Feb 17.

Central European Institute of Technology - Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic.

Proper assembly of the spindle apparatus is crucially important for faithful chromosome segregation during anaphase. Thanks to the effort over the last decades, we have very detailed information about many events leading to spindle assembly and chromosome segregation, however we still do not understand certain aspects, including, for example, spindle length control. When tight regulation of spindle size is lost, chromosome segregation errors emerge. Currently, there are several hypotheses trying to explain the molecular mechanism of spindle length control. The number of kinetochores, activity of molecular rulers, intracellular gradients, cell size, limiting spindle components, and the balance of the spindle forces seem to contribute to spindle size regulation, however some of these mechanisms are likely specific to a particular cell type. In search for a general regulatory mechanism, in our study we focused on the role of cell size and nuclear to cytoplasmic ratio in this process. To this end, we used relatively large cells isolated from 2-cell mouse embryos. Our results showed that the spindle size upper limit is not reached in these cells and suggest that accurate control of spindle length requires balanced ratio between nuclear and cytoplasmic volumes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0149535PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4757572PMC
August 2016

Translation in the mammalian oocyte in space and time.

Cell Tissue Res 2016 Jan 4;363(1):69-84. Epub 2015 Sep 4.

Institute of Animal Physiology and Genetics, ASCR, Rumburska 89, 277 21, Libechov, Czech Republic.

A hallmark of oocyte development in mammals is the dependence on the translation and utilization of stored RNA and proteins rather than the de novo transcription of genes in order to sustain meiotic progression and early embryo development. In the absence of transcription, the completion of meiosis and early embryo development in mammals relies significantly on maternally synthesized RNAs. Post-transcriptional control of gene expression at the translational level has emerged as an important cellular function in normal development. Therefore, the regulation of gene expression in oocytes is controlled almost exclusively at the level of mRNA and protein stabilization and protein synthesis. This current review is focused on the recently emerged findings on RNA distribution related to the temporal and spatial translational control of the meiotic progression of the mammalian oocyte.
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http://dx.doi.org/10.1007/s00441-015-2269-6DOI Listing
January 2016

Temporal and spatial regulation of translation in the mammalian oocyte via the mTOR-eIF4F pathway.

Nat Commun 2015 Jan 28;6:6078. Epub 2015 Jan 28.

Institute of Animal Physiology and Genetics, ASCR, Rumburska 89, 277 21 Libechov, Czech Republic.

The fully grown mammalian oocyte is transcriptionally quiescent and utilizes only transcripts synthesized and stored during early development. However, we find that an abundant RNA population is retained in the oocyte nucleus and contains specific mRNAs important for meiotic progression. Here we show that during the first meiotic division, shortly after nuclear envelope breakdown, translational hotspots develop in the chromosomal area and in a region that was previously surrounded the nucleus. These distinct translational hotspots are separated by endoplasmic reticulum and Lamin, and disappear following polar body extrusion. Chromosomal translational hotspots are controlled by the activity of the mTOR-eIF4F pathway. Here we reveal a mechanism that-following the resumption of meiosis-controls the temporal and spatial translation of a specific set of transcripts required for normal spindle assembly, chromosome alignment and segregation.
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http://dx.doi.org/10.1038/ncomms7078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4317492PMC
January 2015

The frequency of precocious segregation of sister chromatids in mouse female meiosis I is affected by genetic background.

Chromosome Res 2014 Sep 17;22(3):365-73. Epub 2014 Jun 17.

Veterinary Research Institute, Hudcova 70, 621 00, Brno, Czech Republic.

Mammalian female gametes frequently suffer from numerical chromosomal aberrations, the main cause of miscarriages and severe developmental defects. The underlying mechanisms responsible for the development of aneuploidy in oocytes are still not completely understood and remain a subject of extensive research. From studies focused on prevalence of aneuploidy in mouse oocytes, it has become obvious that reported rates of aneuploidy are strongly dependent on the method used for chromosome counting. In addition, it seems likely that differences between mouse strains could influence the frequency of aneuploidy as well; however, up till now, such a comparison has not been available. Therefore, in our study, we measured the levels of aneuploidy which has resulted from missegregation in meiosis I, in oocytes of three commonly used mouse strains-CD-1, C3H/HeJ, and C57BL/6. Our results revealed that, although the overall chromosomal numerical aberration rates were similar in all three strains, a different number of oocytes in each strain contained prematurely segregated sister chromatids (PSSC). This indicates that a predisposition for this type of chromosome segregation error in oocyte meiosis I is dependent on genetic background.
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http://dx.doi.org/10.1007/s10577-014-9428-6DOI Listing
September 2014

Label-free identification of the glioma stem-like cell fraction using Fourier-transform infrared spectroscopy.

Int J Radiat Biol 2014 Aug 25;90(8):710-7. Epub 2014 Mar 25.

Neurosurgery, Carl Gustav Carus University Hospital , Dresden , Germany.

Purpose: Vibrational spectroscopy enables the label-free characterization of cells and tissue by probing the biochemical composition. Here, we evaluated these techniques to identify glioblastoma stem cells.

Materials And Methods: The biochemical fingerprints of glioblastoma cells were established in human cell lines with high and low content of CD133 (cluster of differentiation 133)-positive cells using attenuated total reflection Fourier-transform infrared (ATR FT-IR) on vital cells and FT-IR mapping, which delivers spatially resolved spectroscopic datasets. After data preprocessing, unsupervised cluster analysis was applied. CD133 was addressed with flow cytometry and immunohistochemistry and used as a stemness marker.

Results: In all preparations, the algorithm was able to correctly classify the spectra, differentiating CD133-rich and -poor populations. The main spectral differences were found in the region of 1000 cm(- 1) to 1150 cm(- 1) that can be assigned to vibrations of chemical bonds of DNA, RNA, carbohydrates and phospholipids. Interestingly, this spectral region is a key feature to discern glioblastoma from normal brain parenchyma, as FT-IR spectroscopic mapping of experimental brain tumors demonstrated.

Conclusions: We were able to show biochemical differences between glioblastoma cell populations with high and low content of cancer stem cells that are presumably related to changes in the RNA/DNA content.
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http://dx.doi.org/10.3109/09553002.2014.899447DOI Listing
August 2014

Mechanistic basis of infertility of mouse intersubspecific hybrids.

Proc Natl Acad Sci U S A 2013 Feb 17;110(6):E468-77. Epub 2013 Jan 17.

Mouse Molecular Genetics Group, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic.

According to the Dobzhansky-Muller model, hybrid sterility is a consequence of the independent evolution of related taxa resulting in incompatible genomic interactions of their hybrids. The model implies that the incompatibilities evolve randomly, unless a particular gene or nongenic sequence diverges much faster than the rest of the genome. Here we propose that asynapsis of heterospecific chromosomes in meiotic prophase provides a recurrently evolving trigger for the meiotic arrest of interspecific F1 hybrids. We observed extensive asynapsis of chromosomes and disturbance of the sex body in >95% of pachynemas of Mus m. musculus × Mus m. domesticus sterile F1 males. Asynapsis was not preceded by a failure of double-strand break induction, and the rate of meiotic crossing over was not affected in synapsed chromosomes. DNA double-strand break repair was delayed or failed in unsynapsed autosomes, and misexpression of chromosome X and chromosome Y genes was detected in single pachynemas and by genome-wide expression profiling. Oocytes of F1 hybrid females showed the same kind of synaptic problems but with the incidence reduced to half. Most of the oocytes with pachytene asynapsis were eliminated before birth. We propose the heterospecific pairing of homologous chromosomes as a preexisting condition of asynapsis in interspecific hybrids. The asynapsis may represent a universal mechanistic basis of F1 hybrid sterility manifested by pachytene arrest. It is tempting to speculate that a fast-evolving subset of the noncoding genomic sequence important for chromosome pairing and synapsis may be the culprit.
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http://dx.doi.org/10.1073/pnas.1219126110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3568299PMC
February 2013

Lack of response to unaligned chromosomes in mammalian female gametes.

Cell Cycle 2012 Aug 8;11(16):3011-8. Epub 2012 Aug 8.

Institute of Animal Physiology and Genetics, Libechov, Czech Republic.

Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy to the embryo.
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http://dx.doi.org/10.4161/cc.21398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3442912PMC
August 2012

Frequency of aneuploidy related to age in porcine oocytes.

PLoS One 2011 Apr 27;6(4):e18892. Epub 2011 Apr 27.

Veterinary Research Institute, Brno, Czech Republic.

It is generally accepted that mammalian oocytes are frequently suffering from chromosome segregation errors during meiosis I, which have severe consequences, including pregnancy loss, developmental disorders and mental retardation. In a search for physiologically more relevant model than rodent oocytes to study this phenomenon, we have employed comparative genomic hybridization (CGH), combined with whole genome amplification (WGA), to study the frequency of aneuploidy in porcine oocytes, including rare cells obtained from aged animals. Using this method, we were able to analyze segregation pattern of each individual chromosome during meiosis I. In contrast to the previous reports where conventional methods, such as chromosome spreads or FISH, were used to estimate frequency of aneuploidy, our results presented here show, that the frequency of this phenomenon was overestimated in porcine oocytes. Surprisingly, despite the results from human and mouse showing an increase in the frequency of aneuploidy with advanced maternal age, our results obtained by the most accurate method currently available for scoring the aneuploidy in oocytes indicated no increase in the frequency of aneuploidy even in oocytes from animals, whose age was close to the life expectancy of the breed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018892PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083398PMC
April 2011

Kinases involved in Rec8 phosphorylation revealed.

Authors:
Martin Anger

Cell Cycle 2010 Jul 15;9(14):2708. Epub 2010 Jul 15.

Institute of Animal Physiology and Genetics, Czech Republic.

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July 2010

Structure and function of the PP2A-shugoshin interaction.

Mol Cell 2009 Aug;35(4):426-41

Department of Biological Structure, University of Washington, Seattle, WA 98195, USA.

Accurate chromosome segregation during mitosis and meiosis depends on shugoshin proteins that prevent precocious dissociation of cohesin from centromeres. Shugoshins associate with PP2A, which is thought to dephosphorylate cohesin and thereby prevent cleavage by separase during meiosis I. A crystal structure of a complex between a fragment of human Sgo1 and an AB'C PP2A holoenzyme reveals that Sgo1 forms a homodimeric parallel coiled coil that docks simultaneously onto PP2A's C and B' subunits. Sgo1 homodimerization is a prerequisite for PP2A binding. While hSgo1 interacts only with the AB'C holoenzymes, its relative, Sgo2, interacts with all PP2A forms and may thus lead to dephosphorylation of distinct substrates. Mutant shugoshin proteins defective in the binding of PP2A cannot protect centromeric cohesin from separase during meiosis I or support the spindle assembly checkpoint in yeast. Finally, we provide evidence that PP2A's recruitment to chromosomes may be sufficient to protect cohesin from separase in mammalian oocytes.
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http://dx.doi.org/10.1016/j.molcel.2009.06.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2749713PMC
August 2009

Role of cleavage by separase of the Rec8 kleisin subunit of cohesin during mammalian meiosis I.

J Cell Sci 2009 Aug;122(Pt 15):2686-98

Research Institute of Molecular Pathology, A-1030 Vienna, Austria.

Proteolytic activity of separase is required for chiasma resolution during meiosis I in mouse oocytes. Rec8, the meiosis-specific alpha-kleisin subunit of cohesin, is a key target of separase in yeast. Is the equivalent protein also a target in mammals? We show here that separase cleaves mouse Rec8 at three positions in vitro but only when the latter is hyper-phosphorylated. Expression of a Rec8 variant (Rec8-N) that cannot be cleaved in vitro at these sites causes sterility in male mice. Their seminiferous tubules lack a normal complement of 2 C secondary spermatocytes and 1 C spermatids and contain instead a high proportion of cells with enlarged nuclei. Chromosome spreads reveal that Rec8-N expression has no effect in primary spermatocytes but produces secondary spermatocytes and spermatids with a 4 C DNA content, suggesting that the first and possibly also the second meiotic division is abolished. Expression of Rec8-N in oocytes causes chromosome segregation to be asynchronous and delays its completion by 2-3 hours during anaphase I, probably due to inefficient proteolysis of Rec8-N by separase. Despite this effect, chromosome segregation must be quite accurate as Rec8-N does not greatly reduce female fertility. Our data is consistent with the notion that Rec8 cleavage is important and probably crucial for the resolution of chiasmata in males and females.
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http://dx.doi.org/10.1242/jcs.035287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909317PMC
August 2009

Regulation of APC/C activity in oocytes by a Bub1-dependent spindle assembly checkpoint.

Curr Biol 2009 Mar 26;19(5):369-80. Epub 2009 Feb 26.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.

Background: Missegregation of chromosomes during meiosis in human females causes aneuploidy, including trisomy 21, and is thought also to be the major cause of age-related infertility. Most errors are thought to occur at the first meiotic division. The high frequency of errors raises questions as to whether the surveillance mechanism known as the spindle assembly checkpoint (SAC) that controls the anaphase-promoting complex or cyclosome (APC/C) operates effectively in oocytes. Experimental approaches hitherto used to inactivate the SAC in oocytes suffer from a number of drawbacks.

Results: Bub1 protein was depleted specifically in oocytes with a Zp3-Cre transgene to delete exons 7 and 8 from a floxed BUB1(F) allele. Loss of Bub1 greatly accelerates resolution of chiasmata and extrusion of polar bodies. It also causes defective biorientation of bivalents, massive chromosome missegregation at meiosis I, and precocious loss of cohesion between sister centromeres. By using a quantitative assay for APC/C-mediated securin destruction, we show that the APC/C is activated in an exponential fashion, with activity peaking 12-13 hr after GVBD, and that this process is advanced by 5 hr in oocytes lacking Bub1. Importantly, premature chiasmata resolution does not occur in Bub1-deficient oocytes also lacking either the APC/C's Apc2 subunit or separase. Finally, we show that Bub1's kinase domain is not required to delay APC/C activation.

Conclusions: We conclude that far from being absent or ineffective, the SAC largely determines the timing of APC/C and hence separase activation in oocytes, delaying it for about 5 hr.
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http://dx.doi.org/10.1016/j.cub.2009.01.064DOI Listing
March 2009

Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes.

Nature 2008 May 10;453(7194):534-8. Epub 2008 Apr 10.

Cold Spring Harbor Laboratory, Watson School of Biological Sciences and Howard Hughes Medical Institute, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.

Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways. Here we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from protein-coding genes to antisense transcripts from homologous pseudogenes. An inverted repeat pseudogene can also generate abundant small RNAs directly. A second class of endo-siRNAs may enforce repression of mobile genetic elements, acting together with Piwi-interacting RNAs. Loss of Dicer, a protein integral to small RNA production, increases expression of endo-siRNA targets, demonstrating their regulatory activity. Our findings indicate a function for pseudogenes in regulating gene expression by means of the RNA interference pathway and may, in part, explain the evolutionary pressure to conserve argonaute-mediated catalysis in mammals.
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http://dx.doi.org/10.1038/nature06904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2981145PMC
May 2008

Implication of nucleolar protein SURF6 in ribosome biogenesis and preimplantation mouse development.

Biol Reprod 2006 Nov 19;75(5):690-6. Epub 2006 Jul 19.

Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018, USA.

The step-wise assembly of a functional nucleolus, which occurs over the first few cell cycles during preimplantation development, is poorly understood. In this study, we examined the function of the evolutionary conserved nucleolar protein SURF6 in preimplantation mouse embryo development. Immunocytochemical analyses revealed that the localization of SURF6 was similar but not identical to those of fibrillarin and B23/nucleophosmin 1, which are involved in rRNA processing and ribosome biogenesis in mammalian somatic cells. Surf6 mRNA, which is expressed in oocytes and maternally inherited in the zygote, reached a peak level of expression during the 8-cell stage of embryo development, at which time rDNA is highly transcribed. Knock-down of Surf6 mRNA by RNAi led to a decrease in both the mRNA and protein levels, and resulted in developmental arrest at the 8-cell/morula stage, as well as a decrease in the level of 18S rRNA. These results suggest that Surf6 is essential for mouse preimplantation development, presumably by regulating ribosome biogenesis.
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http://dx.doi.org/10.1095/biolreprod.106.054072DOI Listing
November 2006

Resolution of chiasmata in oocytes requires separase-mediated proteolysis.

Cell 2006 Jul;126(1):135-46

Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria.

In yeast, resolution of chiasmata in meiosis I requires proteolytic cleavage along chromosome arms of cohesin's Rec8 subunit by separase. Since activation of separase by the anaphase-promoting complex (APC/C) is supposedly not required for meiosis I in Xenopus oocytes, it has been suggested that animal cells might resolve chiasmata by a separase-independent mechanism related to the so-called "prophase pathway" that removes cohesin from chromosome arms during mitosis. By expressing Cre recombinase from a zona pellucida promoter, we have deleted a floxed allele of separase specifically in mouse oocytes. This prevents removal of Rec8 from chromosome arms and resolution of chiasmata. It also hinders extrusion of the first polar body (PBE) and causes female sterility. mRNA encoding wild-type but not catalytically inactive separase restores chiasma resolution. Both types of mRNA restore PBE. Proteolytic activity of separase is therefore essential for Rec8's removal from chromosome arms and for chiasma resolution but not for PBE.
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http://dx.doi.org/10.1016/j.cell.2006.05.033DOI Listing
July 2006

CDC6 requirement for spindle formation during maturation of mouse oocytes.

Biol Reprod 2005 Jan 22;72(1):188-94. Epub 2004 Sep 22.

Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018, USA.

A master regulator of DNA replication, CDC6 also functions in the DNA-replication checkpoint by preventing DNA rereplication. Cyclin-dependent kinases (CDKs) regulate the amount and localization of CDC6 throughout the cell cycle; CDC6 phosphorylation after DNA replication initiation leads to its proteolysis in yeast or translocation to the cytoplasm in mammals. Overexpression of CDC6 during the late S phase prevents entry into the M phase by activating CHEK1 kinase that then inactivates CDK1/cyclin B, which is essential for the G2/M-phase transition. We analyzed the role of CDC6 during resumption of meiosis in mouse oocytes, which are arrested in the first meiotic prophase with low CDK1/cyclin B activity; this is similar to somatic cells at the G2/M-phase border. Overexpression of CDC6 in mouse oocytes does not prevent resumption of meiosis. The RNA interference-mediated knockdown of CDC6, however, reveals a new and unexpected function for CDC6; namely, it is essential for spindle formation in mouse oocytes.
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http://dx.doi.org/10.1095/biolreprod.104.035451DOI Listing
January 2005

Timing of Plk1 and MPF activation during porcine oocyte maturation.

Mol Reprod Dev 2004 Sep;69(1):11-6

Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA.

A Polo-like kinase 1 (Plk1) appears involved in an autocatalytic loop between CDC25C phosphatase and M phase promoting factor (MPF) in Xenopus oocytes and leads to activation of MPF that is required for germinal vesicle breakdown (GVBD). Although similar evidence for such a role of Plk1 in MPF activation during maturation of mammalian oocytes is absent, changes in Plk1 enzyme activity correlate with MPF activation, Plk1 co-localizes with MPF, and microinjection of antibodies neutralizing Plk1 delays GVBD. In this study, we exploited the prolonged time required for maturation of porcine oocytes to define precisely the timing of Plk1 and MPF activation during maturation. GVBD typically occurs between 24 and 26 hr of culture in vitro and meiotic maturation is completed after 40-44-hr culture. We find that Plk1 is activated before MPF, which is consistent with its role in activating MPF in mammalian oocytes.
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http://dx.doi.org/10.1002/mrd.20151DOI Listing
September 2004

RNAi and expression of retrotransposons MuERV-L and IAP in preimplantation mouse embryos.

Dev Biol 2004 May;269(1):276-85

Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA.

Both murine endogenous retrovirus-L (MuERV-L) and intracisternal A particle (IAP), two autonomous long terminal repeat (LTR) retrotransposons, are activated during genome activation in the preimplantation mouse embryo, and both sense and antisense transcripts are detected in 2-cell and 8-cell stage embryos. Because RNA interference (RNAi) functions in the preimplantation mouse embryo, we analyzed the relationship between RNAi and MuERV-L and IAP expression by inhibiting RNAi and measuring relative changes of the levels of these transcripts. We inhibited the initial step in the RNAi pathway by injecting 1-cell embryos with mDicer siRNA or long mDicer dsRNA and analyzed MuERV-L and IAP expression at the 8-cell stage. This approach resulted in the targeted destruction of mDicer mRNA, but not Hdac1 mRNA, inhibited the RNAi pathway, and resulted in a 50% increase in IAP and MuERV-L transcript abundance. These results suggest that RNAi constrains expression of repetitive parasitic sequences in preimplantation embryos, and thereby contributes to preserving genomic integrity at a stage of development when the organism consists of only a few cells.
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http://dx.doi.org/10.1016/j.ydbio.2004.01.028DOI Listing
May 2004

Cell cycle dependent expression of Plk1 in synchronized porcine fetal fibroblasts.

Mol Reprod Dev 2003 Jul;65(3):245-53

Institute of Animal Physiology and Genetics, Libechov, Czech Republic.

Enzymes of the Polo-like kinase (Plk) family are active in the pathways controlling mitosis in several species. We have cloned cDNA fragments of the porcine homologues of Plk1, Plk2, and Plk3 employing fetal fibroblasts as source. All three partial cDNAs showed high sequence homology with their mouse and human counterparts and contained the Polo box, a domain characteristic for all Polo kinases. The expression levels of Plk1 mRNA at various points of the cell cycle in synchronized porcine fetal fibroblasts were analyzed by both RT-PCR and the ribonuclease protection assay. Plk1 mRNA was barely detectable in G0 and G1, increased during S phase and peaked after the G2/M transition. A monoclonal antibody was generated against an in vitro expressed porcine Plk1-protein fragment and used to detect changes in Plk1 expression at the protein level. Plk1 protein was first detected by immunoblotting at the beginning of S phase and was highest after the G2/M transition. In summary, the Plk1 expression pattern in the pig is similar to that reported for other species. The absence of Plk1 mRNA and protein appears to be a good marker for G0/G1 and thus for the selection of donor cells for nuclear transfer based somatic cloning.
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http://dx.doi.org/10.1002/mrd.10289DOI Listing
July 2003

The appearance of truncated cyclin A2 correlates with differentiation of mouse embryonic stem cells.

Biochem Biophys Res Commun 2003 Mar;302(4):825-30

Department of Biology, University of Pennsylvania, USA.

The presence of a form of cyclin A2 with an N-terminal truncation has recently been reported in various murine cell lines and tissues. The truncated cyclin A2 binds to and activates the cyclin-dependent kinase 2 (CDK2). However, CDK2 bound by the truncated cyclin A2 is located in the cytoplasm in contrast to CDK2 bound to full-length cyclin A2, which is in the nucleus. Here, we show that proliferating mouse embryonic stem cells (ES cells) contain very little truncated cyclin A2 but as the cells are induced to differentiate the amount of truncated cyclin A2 increases. The expression pattern of truncated cyclin A2 was the same in p27(Kip1) -/- differentiating ES cells as in the differentiating wild-type cells. We conclude that p27(Kip1) is not necessary for the proteolytic cleavage that gives rise to the truncated form of cyclin A2 in differentiating ES cells and that this post-translational modification is not a function of the cell density but is correlated with differentiation.
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http://dx.doi.org/10.1016/s0006-291x(03)00270-5DOI Listing
March 2003

RNAi: mammalian oocytes do it without RNA-dependent RNA polymerase.

RNA 2003 Feb;9(2):187-92

Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA 19104-6018, USA.

Studies in mutant organisms deficient in RNA interference (RNAi) and related post-transcriptional gene silencing implicated a role for a single class of RNA-dependent RNA polymerases (RdRp). Nevertheless, sequence homologs to these RdRps have not been found in coelomate organisms such as Drosophila or mammals. This lack of homologous sequences does not exclude that an RdRp functions in RNAi in these organisms because an RdRp could be acquired by horizontal transfer from an RNA virus. In fact, such a sequence is found in mice (Aquarius) and we observe that it is expressed in mouse oocytes and early embryos, which exhibit RNAi. We report here that cordycepin, an inhibitor of RNA synthesis, does not prevent Mos double-strand RNA (dsRNA) to target endogenous Mos mRNA in mouse oocytes and that targeting a chimeric Mos-EGFP mRNA with dsRNA to EGFP does not reduce the endogenous Mos mRNA, but does target the chimeric mRNA. These results indicate that an RdRp is not involved in dsRNA-mediated mRNA degradation in mammalian oocytes, and possibly in mammals in general, and therefore that only homologous sequences to the dsRNA are targeted for degradation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1370384PMC
http://dx.doi.org/10.1261/rna.2860603DOI Listing
February 2003

Chromosome condensation in pig oocytes: lack of a requirement for either cdc2 kinase or MAP kinase activity.

Mol Reprod Dev 2002 Sep;63(1):110-8

Department of Physiology of Reproduction, Institute of Animal Physiology and Genetics, Libechov, Czech Republic.

In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases (cdk), is shown to inhibit germinal vesicle breakdown (GVBD) in pig oocytes. Oocytes treated with 100 microM BL I were arrested in the germinal vesicle (GV)-stage and displayed low activity of cdc2 kinase and MAP kinase. Nevertheless, chromosome condensation occurred and highly condensed bivalents were seen within an intact GV after a 24-hr culture in the presence of BL I. The inhibitory effect of BL I on MAP kinase activation during culture was likely mediated through a cdk-dependent pathway, since MAP kinase activity present in extracts derived from metaphase II eggs was not inhibited by BL I. The block of GVBD could be released by treating oocytes with okadaic acid (OA), an inhibitor of type 1 and 2A phosphatases; 82% of the oocytes treated with the combination of OA/BL I underwent GVBD, and MAP kinase became activated, while cdc2 kinase remained inhibited. These results suggest that both chromosome condensation and GVBD could occur without activation of cdc2 kinase, whereas an increase in MAP kinase activity may be a requisite for GVBD in pig oocytes in conditions when cdc2 kinase activation is blocked by BL I.
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http://dx.doi.org/10.1002/mrd.10176DOI Listing
September 2002

Activation of pig and cattle oocytes by butyrolactone I: morphological and biochemical study.

Zygote 2002 Feb;10(1):47-57

Institute of Animal Physiology and Genetics, Libĕchov, Czech Republic.

In this study a specific inhibitor of cyclin-dependent kinases (cdks), butyrolactone I (BL I), was used for activation of pig and cattle metaphase II (MII) oocytes. BL I at a concentration of 100 microM was able to induce activation of both pig and cattle MII oocytes in a manner dependent on exposure time; however, precise timing of BL I exposure was required for the best activation results. The optimum activation rates were obtained when cattle MII oocytes were treated for 5 h with BL I and subsequently for 3-11 h in control medium, and pig MII oocytes for 8 h in BL I and then for 8-16 h in control medium; the percentage of activated oocytes after such treatment varied between 55% and 74% and between 53% and 81% for cattle and pig oocytes, respectively. Shorter exposures to BL I led to re-entry of the oocytes to the metaphase state in 35-50% of oocytes, the remaining oocytes forming a pronuclear stage; longer exposure to BL I led to increased numbers of oocytes being abnormal or degenerated. The behaviour of histone H1 kinase and mitogen activated protein (MAP) kinase, also measured during the experiment, reflected the morphological changes in the oocytes: both were inactivated after BL I treatment, though the inactivation of histone H1 kinase occurred 2 h ahead of that of MAP kinase. However, in the oocytes treated for a shorter time with BL I, with the reoccurrence of condensed chromatin in proportion of the oocytes cultured in control medium after BL I treatment, both kinases became reactivated. Taken together, these results suggest the possibility of using BL I for activation and cloning experiments in both species.
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http://dx.doi.org/10.1017/s0967199402002071DOI Listing
February 2002