Metaphase Stage Research Articles

Endoplasmic Reticulum Network Formation with Xenopus Egg Extracts.

The endoplasmic reticulum (ER) consists of morphologically distinct domains, including a polygonal network of tubules that is connected by three-way junctions. This network is found in all eukaryotic cells. Extracts from Xenopus laevis eggs contain stockpiles of components that allow the assembly of an ER network in vitro. Here we provide protocols for assembly of ER networks in extracts that are arrested at different stages of the cell cycle. Unfertilized Xenopus laevis eggs contain a cytostatic factor (CSF) that keeps them in the metaphase stage of the cell cycle. Disruption of the eggs by low-speed centrifugation releases calcium and the eggs cycle into interphase. This state can then be maintained by the addition of cycloheximide, which prevents the synthesis of cyclin B. CSF extracts can be also prepared in the presence of a calcium chelator, thus keeping the extract in metaphase. In this protocol, we outline procedures for the assembly of an ER network using either interphase- or metaphase-arrested Xenopus egg extracts. The network assembled is strikingly similar to the network observed in tissue culture cells. The extract allows easy biochemical manipulation, permitting the effects of purified proteins or small molecules, or the depletion of cytosolic components to be tested.

The impact of age and BMI on oocyte maturation and embryo development

Background: Ovarian oogenesis is under the control of gonadotropins. In the human ovary, each fully grown oocyte resumes maturation in response to gonadotrophins. This process is completed after oocytes reach metaphase stage of second meiotic division (MII) stage. The aim of this prospective, clinical trial was to perceive the age and BMI effect on oocytes maturation and embryos development in women undergoing control ovarian hyperstimulation. Method: A total of 40 infertile women undergoing ovarian hyperstimulation their age range between 18-40 y old were included in this study using two type of protocol: long agonist and antagonist. Result: Significant negative correlation was observed between the age and the number of retrieved MII oocytes. Significant negatively effects of the BMI on the number of MII oocytes while significant positive correlation between the BMI and the fertilization rate was noticed. Conclusion: There are negative effects of age and BMI on the oocytes number and quality.

FahamecV1:A Low Cost Automated Metaphase Detection System

In this study, FahamecV1 is introduced and investigated as a low cost and high accuracy solution for metaphase detection. Chromosome analysis is performed at the metaphase stage and high accuracy and automated detection of the metaphase stage plays an active role in decreasing analysis time. FahamecV1 includes an optic microscope, a motorized microscope stage, an electronic control unit, a camera, a computer and a software application. Printing components of the motorized microscope stage (using a 3D printer) is of the main reasons for cost reduction. Operations such as stepper motor calibration, are detection, focusing, scanning, metaphase detection and saving of coordinates into a database are automatically performed. To detect metaphases, a filter named Metafilter is developed and applied. Average scanning time per preparate is 77 sec/cm2. True positive rate is calculated as 95.1%, true negative rate is calculated as 99.0% and accuracy is calculated as 98.8%.

Effect of Recombinant Follicle Stimulating Hormone and Recombinant Luteinizing Hormone on Maturation of Mammalian Oocytes in Vitro

Abstract In vitro maturation (IVM) of oocytes is an important application in assisted reproductive medicine and reproductive biology research. It can provide high quality oocytes from unstimulated ovaries. However, IVM remains a challenge in mammalian species, and has only been achieved with limited degrees of success. This study was carried out to investigate the effect of adding recombinant Follicle Stimulating Hormone (rFSH), recombinant Luteinizing Hormone (rLH) or the combination of both hormones on the IVM of rabbit oocytes, as rabbit is considered an important model system for human reproduction. Unstimulated ovaries of female rabbits were encountered. Oocytes were collected via tissue slice technique. The Cumulus-Oocyte Complexes (COCs), containing immature oocytes at Germinal Vesicle (GV) stage, were counted, washed, and cultured in different concentrations of rFSH, rLH and a combination of both rFSH and rLH. After cultivation, oocytes were examined for maturation. Our study revealed that the addition of hormone combination (rFSH + rLH) with certain concentrations could significantly improve the results of IVM and increase the percentage of oocytes reaching the second Metaphase (MII) stage. Key words: In vitro maturation, Hormones, Rabbit oocytes.

Мейоз, эмбриональное и личиночное развитие черноморского гребешка Flexopecten glaber ponticus (Bucquoy, Dautzenberg & Dollfus, 1889) (Bivalvia, Pectinidae)

Изучены хронология стадий мейоза после оплодотворения, а также эмбриональное и личиночное развитие двустворчатого моллюска гребешка Flexopecten glaber ponticus — эндемика Чёрного моря, сокращающегося в численности и занесённого в Красную книгу Крыма. Размножается в июне — июле. Синхронный гермафродит, способный к самооплодотворению. При стимуляции нереста вымет зрелых яйцеклеток диаметром 54,7 ± 2,11 мкм и оплодотворение происходят на стадии метафазы I мейоза. На метафазной пластинке — 12 бивалентов, размерами от 1,59 до 4,54 мкм. Установлена продолжительность стадий мейоза, эмбрионального развития и развития личинок. Описано строение провинкулума велигера гребешка. Замок правой и левой створок состоит из 4 зубов (по два спереди и сзади на каждой створке). Представлены промеры личинок. Описание морфологии раковины и замка облегчит идентификацию личинок гребешка на стадии велигера, выловленных из планктона. Данные о продолжительности стадий мейоза и эмбрионального и личиночного развития могут быть использованы при разработке биотехники культивирования этого двустворчатого моллюска.

Egg Activation at Fertilization.

Fertilization is the union of gametes to initiate development of a new individual. The female gamete is formed during oogenesis. The process begins when, in the early embryo, primordial germ cells arise and subsequently colonize the genital ridges. They differentiate into oogonia, start meiosis, and become primary oocytes. The cell cycle of the primary oocytes then becomes arrested in mid-meiosis for an extended period of time. Prior to ovulation the oocytes undergo a growth phase and their sizes increase significantly. A hormonal cue then triggers oocyte maturation that involves the resumption of meiosis, the completion of the first meiotic division, and, as a result, the reduction in the diploid chromosome number. The cell cycle then stops again; in vertebrates this arrest occurs at the metaphase stage of the second meiotic division. Meiosis resumes at fertilization, when the sperm activates the egg, i.e., it causes a series of changes that are required for the initiation of embryo development. This is achieved by triggering an elevation in the egg's intracellular free calcium concentration. In response, the fertilized egg completes meiosis and enters the first embryonic cell cycle.

Tight repulsion linkage between Sr36 and Sr39 was revealed by genetic, cytogenetic and molecular analyses.

The shortening of Aegilops speltoides segment did not facilitate recombination between stem rust resistance genes Sr36 and Sr39 . Robustness of marker rwgs28 for marker-assisted selection of Sr39 was demonstrated. Stem rust resistance genes Sr39 and Sr36 were transferred from Aegilops speltoides and Triticum timopheevii, respectively, to chromosome 2B of wheat. Genetic stocks RL6082 and RWG1 carrying Sr39 on a large and a shortened Ae. speltoides segments, respectively, and the Sr36-carrying Australian wheat cultivar Cook were used in this study. This investigation was planned to determine the genetic relationship between these genes. Stem rust tests on F3 populations derived from RL6082/Cook and RWG1/Cook crosses showed tight repulsion linkage between Sr39 and Sr36. The genomic in situ hybridization analysis of heterozygous F3 family from the RWG1/Cook population showed that the translocated segments do not overlap. Meiotic analysis on the F1 plant from RWG1/Cook showed two univalents at the metaphase and anaphase stages in a majority of the cells indicating absence of pairing. Since meiotic pairing has been reported to initiate at the telomere, pairing and recombination may be inhibited due to very little wheat chromatin in the distal end of the chromosome arm 2BS in RWG1. The Sr39-carrying large Ae. speltoides segment transmitted preferentially in the RL6082/Cook F3 population, whereas the Sr36-carrying T. timopheevii segment over-transmitted in the RWG1/Cook cross. Genotyping with the co-dominant Sr39- and Sr36-linked markers rwgs28 and stm773-2, respectively, matched the phenotypic classification of F3 families. The RWG1 allele amplified by rwgs28 was diagnostic for the shortened Ae. speltoides segment and alternate alleles were amplified in 29 Australian cultivars. Marker rwgs28 will be useful in marker-assisted pyramiding of Sr39 with other genes.

THE QUANTITY OF CELLS NECESSARY FOR THE ANALYSIS OF CHROMOSOMAL ABERRATIONS AT RESIDENTS OF KUZBASS PATIENTS WITH CANCER OF LUNG

The pilot research executed in 2014 in selection of patients with lung cancer (LC) and healthy residents of Kemerovo region (338 Caucasians: 159 having LC and 179 healthy) has shown statistically significant distinction between groups of level and range of chromosome aberrations in blood lymphocytes. Patients of cancer detection centre (before treatment) had increased chromosome aberration values, both of chromatid and chromosomal types. 250 metaphases were analyzed for each individual. For accuracy test of the received quantitative characteristics of mutational process and definition required and enough number of cells necessary for the analysis, at essential increase in further selection volume, 50 LC patients (selected randomly), comparison of cytogenetic data has been carried out in the analysis of 200 cells, then 400, 600, 800, 1000 and over 1000 metaphases. Totally, in this subgroup 50 000 cells which were at a mitosis metaphase stage have been studied. Statistical data processing was carried out with the use of a software package for Windows Statistica 8.0. Using Mann-Whitney U-criterion it is confirmed that frequency of aberrant metaphases doesn't change significantly for statistics in case of increase in analyzed cells quantity; the analysis of 200 cells gives no less reliable information about the individual CA level, than the analysis of 1000 and more cells. In this regard it is possible to make the conclusion that 200 cells is necessary and enough number of analysed metaphases at the assessment of individual level and chromosomal aberrations' range for Kemerovo region residents who have lung cancer.

Types of cytogenetic abnormalities in apical root meristem of Elodea сanadensis from Yenisei River areas with different types of anthropogenic pollution

Background. The spectrum of cytogenetic abnormalities in the apical root meristem of Elodea сanadensis Michx. (elodea) from the Yenisei River was studied depending on the type of anthropogenic pollution. We compared elodea samples from areas with radiation-contaminated sediments (area affected by Rosatom Mining and Chemical Combine, MCC, Zheleznogorsk) and areas with chemical pollution (Krasnoyarsk). Materials and methods. The apical root meristem of elodea was fixed in acetic alcohol (96 % ethanol and glacial acetic acid, 3 : 1) and stained with 1 % aceto-orcein. The cytogenetic analysis of metaphase and ana-telophase elodea cells was carried out with temporal squashed preparations using an Olympus CX31 microscope. Results. At metaphase stage, the predominant types of abnormalities in elodea cells were disoriented chromosomes (up to 9 % of total metaphase cells) and chromosome agglutination (6 %). In the area affected by the MCC an increased content of ring chromosomes in elodea cells was detected, but in terms of frequency of their occurrence no significant differences were revealed between samples from areas with different types of pollution. Among abnormalities at ana-telophase stage, bridges (to 20% of dividing cells) and disoriented chromosomes (up to 8%) dominated. The following abnormalities were also detected: multipolar and asymmetrical mitoses, agglutination and cells with multiple chromosome abnormalities. It was shown that in areas with the highest content of 137Cs in bottom sediments the frequency of cells with bridges and multiple chromosome abnormalities significantly increased as compared to samples from other areas. Conclusion. In the radioactive contamination area of the Yenisei River the spectrum of abnormalities was dominated by chromosome aberrations (bridges, agglutination, chromosome fragmentation) and multiple abnormalities in one and the same cell. In areas with non-radioactive (chemical) contamination of sediments, mitotic spindle irregularities prevailed.

Chromosomes in a genome-wise order: evidence for metaphase architecture.

BackgroundOne fundamental finding of the last decade is that, besides the primary DNA sequence information there are several epigenetic “information-layers” like DNA-and histone modifications, chromatin packaging and, last but not least, the position of genes in the nucleus.ResultsWe postulate that the functional genomic architecture is not restricted to the interphase of the cell cycle but can also be observed in the metaphase stage, when chromosomes are most condensed and microscopically visible. If so, it offers the unique opportunity to directly analyze the functional aspects of genomic architecture in different cells, species and diseases. Another aspect not directly accessible by molecular techniques is the genome merged from two different haploid parental genomes represented by the homologous chromosome sets. Our results show that there is not only a well-known and defined nuclear architecture in interphase but also in metaphase leading to a bilateral organization of the two haploid sets of chromosomes. Moreover, evidence is provided for the parental origin of the haploid grouping.ConclusionsFrom our findings we postulate an additional epigenetic information layer within the genome including the organization of homologous chromosomes and their parental origin which may now substantially change the landscape of genetics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-016-0243-y) contains supplementary material, which is available to authorized users.

Chromosomal studies on six species of crop aphids from Himachal Pradesh, India

Chromosomes of six species of aphids infesting different crops from Shimla region of Himachal Pradesh have been studied. These aphids belong to genus Aphis, Hysteroneura, Macrosiphum, Phorodon and Rhopalosiphum. Chromosomes were studied using squash preparation. Diploid chromosome numbers in these species ranged from 2n = 8 to 2n = 12. The actual lengths of chromosomes were measured at metaphase stage and total complement length (TCL) was calculated for each species. Relative lengths of chromosomes were calculated and the idiograms thus constructed are presented.

Karyotype study on 15 populations of Eremostachys laciniata Bunge in Iran

A karyotype study was performed on 15 populations of Eremostachys laciniata Bunge from Iran in order to quantify the extent of cytological variation for use in breeding programmes. Ten mitotic root tip cells at the metaphase stage were prepared from each population by the squash method. The chromosomes of suitable mitotic cells were counted and various parameters, including long and short arm lengths, chromosome lengths, total length of the haploid chromosome complement, arm ratios, and the centromeric index, were measured. All 15 populations were diploid with 2n = 2x = 22 and were differentiated by their karyotypic parameters. All 15 E. laciniata populations occupied Class 1A of Stebbins’ karyotype classification, indicating the presence of a primitive symmetrical karyotype. The size of the mitotic chromosomes was medium, and mean chromosome lengths ranged from 3.84 to 4.77 μm. The shortest chromosome lengths were observed in the Ajabshir population and the longest in the Heydarabad population. The Heydarabad population also had the longest total chromosome length and the Ajabshir population had the shortest total chromosome length. The centromeric indices of the chromosome complements varied from 39.3% to 43.7%. Metacentric chromosomes were the most common in all populations, whereas submetacentric chromosomes were rare. The A1 index varied from 0.215 (Areshtanab) to 0.337 (Til). The A2 index ranged from 0.116 (Til) to 0.143 (Kaleybar), and the karyotype asymmetry index varied between 0.051 and 0.097. The results of a principal component analysis (PCA) showed that the main elements of the first three main principal components accounted for 97% of the total variance. Cluster analysis by Ward’s method indicated that all 15 populations could be classified into three clusters. Finally, the results of the karyotypic parameters showed that the Til, Heydarabad, and Marand populations had higher levels of karyotypic heterogeneity and may be useful when developing breeding programmes.

182 EFFECTS OF IGF-1 OR IGF-1-LongR3 ON CELLULAR AND MOLECULAR ASPECTS OF CUMULUS-OOCYTE COMPLEXES DURING IN VITRO OOCYTE MATURATION IN CATTLE

The insulin-like growth factor-1 recombinant -3 (IGF-1-LongR3), a synthetic analogue of IGF-1 with increased bioavailability has not yet been used in vitro maturation (IVM) medium of bovine oocytes. Therefore, the aim of this study was to evaluate and compare the addition effects of IGF-1-LongR3 or IGF-1 in IVM bovine oocytes on meiotic progression, apoptosis, and profile of oocytes genes (GDF9, BMP15, BAX, BCL2, OOSP1, IGFBP2, IGFBP4 and IGFBP5) and genes in cumulus cells (AREG, EGFR, FSHR, COX2, BAX, BCL2, IGFBP2, IGFBP4 and IGFBP5). Bovine ovaries were collected in slaughterhouses, and 739 oocytes with grades 1 or 2 were selected after aspiration of 2- to 8-mm follicles. IVM was carried out in TCM199 with FSH, LH, and antibiotics (BM) supplemented with 100 ng mL–1 IGF-1 or 100 ng mL–1 LongR3-IGF-1. Control oocytes were matured in BM supplemented with 0.1% polyvinyl alcohol (PVA) or 10% FCS. For all groups, maturation was performed during 22–24 h in an incubator at 38.5°C and 5% CO2 in air. Subsequently oocytes were denuded and analysed for apoptosis, nuclear maturation, and gene expression by TUNEL assay, staining Hoechst 33342, and RT-qPCR, respectively. Statistical analysis was performed using a linear mixed effects model, which correlated the change in metaphase stage 1 to 2 and the absence of apoptosis among the experimental groups. ANOVA and Tukey tests were used to analyse the results obtained by RT-qPCR. After 10 replicates of IVM, 339 oocytes were evaluated for meiotic progression and apoptosis and 400 oocytes for gene expression. There was no statistical difference between the experimental groups with respect to meiotic progression and apoptosis. BCL2 and IGFBP4 gene were less expressed in oocytes matured with IGF-1 and LongR3-IGF-1 compared with control groups. GFBP4 was also less expressed in cumulus cell of oocytes from the experimental groups. Moreover COX2 expression was statistically elevated in cumulus cells matured in the presence of IGF-1 and LongR3-IGF-1 It was possible to perform IVM of bovine oocytes in the presence of LongR3-IGF-1, allowing its use in replacement of IGF-1 and FCS. The results of this study will provide more information on the interaction of IGF with the IGFBP and its importance for oocyte maturation.

FISH and GISH: molecular cytogenetic tools and their applications in ornamental plants.

The innovations in chromosome engineering have improved the efficiency of interrogation breeding, and the identification and transfer of resistance genes from alien to native species. Recent advances in molecular biology and cytogenetics have brought revolutionary, conceptual developments in mitosis and meiosis research, chromosome structure and manipulation, gene expression and regulation, and gene silencing. Cytogenetic studies offer integrative tools for imaging, genetics, epigenetics, and cytological information that can be employed to enhance chromosome and molecular genomic research in plant taxa. In situ hybridization techniques, such as fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH), can identify chromosome morphologies and sequences, amount and distribution of various types of chromatin in chromosomes, and genome organization during the metaphase stage of meiosis. Over the past few decades, various new molecular cytogenetic applications have been developed. The FISH and GISH techniques present an authentic model for analyzing the individual chromosome, chromosomal segments, or the genomes of natural and artificial hybrid plants. These have become the most reliable techniques for studying allopolyploids, because most cultivated plants have been developed through hybridization or polyploidization. Moreover, introgression of the genes and chromatin from the wild types into cultivated species can also be analyzed. Since hybrid derivatives may have variable alien chromosome numbers or chromosome arms, the use of these approaches opens new avenues for accurately identifying genome differences.

The Relationship Between Transcript Expression Levels of Nuclear Encoded (TFAM, NRF1) and Mitochondrial Encoded (MT-CO1) Genes in Single Human Oocytes During Oocyte Maturation

In some cases of infertility in women, human oocytes fail to mature when they reach the metaphase II (MII) stage. Mitochondria plays an important role in oocyte maturation. A large number of mitochondrial DNA (mtDNA), copied in oocytes, is essential for providing adenosine triphosphate (ATP) during oocyte maturation. The purpose of this study was to identify the relationship between transcript expression levels of the mitochondrial encoded gene (MT-CO1) and two nuclear encoded genes, nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) in various stages of human oocyte maturation. Nine consenting patients, age 21–35 years old, with male factors were selected for ovarian stimulation and intracytoplasmic sperm injection (ICSI) procedures. mRNA levels of mitochondrial-related genes were performed by singlecell TaqMan® quantitative real-time polymerase chain reaction (qRT-PCR). There was no significant relationship between the relative expression levels in germinal vesicle (GV) stage oocytes (p = 0.62). On the contrary, a significant relationship was seen between the relative expression levels of TFAM and NRF1 and the MT-CO1 genes at the stages of metaphase I (MI) and MII (p = 0.03 and p = 0.002). A relationship exists between the transcript expression levels of TFAM and NRF1, and MT-CO1 genes in various stages of human oocyte maturation.

A conversation with Susan Band Horwitz.

Susan Band Horwitz is a Distinguished Professor and holds the Falkenstein Chair in Cancer Research at Albert Einstein College of Medicine in New York. She is co-chair of the Department of Molecular Pharmacology and associate director for therapeutics at the Albert Einstein Cancer Center. After graduating from Bryn Mawr College, Dr. Horwitz received her PhD in biochemistry from Brandeis University. She has had a continuing interest in natural products as a source of new drugs for the treatment of cancer. Her most seminal research contribution has been in the development of Taxol(®). Dr. Horwitz and her colleagues made the discovery that Taxol had a unique mechanism of action and suggested that it was a prototype for a new class of antitumor drugs. Although Taxol was an antimitotic agent blocking cells in the metaphase stage of the cell cycle, Dr. Horwitz recognized that Taxol was blocking mitosis in a way different from that of other known agents. Her group demonstrated that the binding site for Taxol was on the β-tubulin subunit. The interaction of Taxol with the β-tubulin subunit resulted in stabilized microtubules, essentially paralyzing the cytoskeleton, thereby preventing cell division. Dr. Horwitz served as president (2002-2003) of the American Association for Cancer Research (AACR). She is a member of the National Academy of Sciences, the Institute of Medicine, the American Academy of Arts and Sciences, and the American Philosophical Society. She has received numerous honors and awards, including the C. Chester Stock Award from Memorial Sloan Kettering Cancer Center, the Warren Alpert Foundation Prize from Harvard Medical School, the Bristol-Myers Squibb Award for Distinguished Achievement in Cancer Research, the American Cancer Society's Medal of Honor, and the AACR Award for Lifetime Achievement in Cancer Research. The following interview was conducted on January 23, 2014.

Studia nad kariologią i rozwojem pyłku kilku odmian Medicago saliva L. Cz. I [Studies on karyology and pollen development in several varieties of Medicago sativa L. Part I

At the stage of diakinensis and metaphase of the first meiotic division, conjugating chromosomes in the form of bivalents were always observed. At the pre-meiotic stage some of .the mother pollen cells underwent degeneration (in 'Australian' var. 17% in 'Warminska' 20%, in 'Kleszczewska' 23%, in 'Grimma' 30% and in 'Miechowska' 40%). In the degenerating mother pollen cells no meiotic division were observed. In the surviving mother pollen cells, meiotic division were found to be normal. The mature pollen grains were trinucleate.

Cytogenetic Characterization of the Genome of Interspecies Hybrids (Alopex-Vulpes)

Abstract Creation of interspecific hybrids is widely common among plants and animals in order to improve economically important traits for humans. The studied material consisted of chromosomal preparations in the metaphase stage obtained from interspecies hybrids of arctic foxes (Alopex lagopus) and red foxes (Vulpes vulpes). The aim of the study was to analyze the karyotype of the interspecific hybrids taking into account the number of chromosomes of sets A and B. With the use of techniques of classical cytogenetics (C bands, AgNOR bands) and molecular cytogenetics (FISH, PRINS) we carried out a genome analysis of Alopex-Vulpes hybrids. The results of this study showed that chromosomal markers of the interspecies hybrids are inherited from the parent species and are a result of combination of their two genomes. However, intraindividual differences are also observed which may result from aberrations of chromosome segregation during embryonic development. This may lead to the formation of different cell lines with different karyotypes (mosaicism). Moreover, chromosomes of the interspecies hybrids showed telomeric signals at the ends, in the centromers, as well as short chromosome arm rich in heterochromatin. The use of PRINS method led to identification of nucleolus organizer regions on 12 chromosomes of the interspecies hybrids. The hybridization signals obtained were characterized by different size and intensity. In addition, single copies of rDNA in the centromeric regions of several metacentric chromosomes were identified.

Different fates of oocytes with DNA double-strand breaks in vitro and in vivo

In female mice, despite the presence of slight DNA double-strand breaks (DSBs), fully grown oocytes are able to undergo meiosis resumption as indicated by germinal vesicle breakdown (GVBD); however, severe DNA DSBs do reduce and delay entry into M phase through activation of the DNA damage checkpoint. But little is known about the effect of severe DNA DSBs on the spindle assembly checkpoint (SAC) during oocyte maturation. We showed that nearly no first polar body (PB1) was extruded at 12 h of in vitro maturation (IVM) in severe DNA DSBs oocytes, and the limited number of oocytes with PB1 were actually at telophase. However, about 60% of the severe DNA DSBs oocytes which underwent GVBD at 2 h of IVM released a PB1 at 18 h of IVM and these oocytes did reach the second metaphase (MII) stage. Chromosome spread at MI and MII stages showed that chromosomes fragmented after GVBD in severe DNA DSBs oocytes. The delayed PB1 extrusion was due to the disrupted attachment of microtubules to kinetochores and activation of the SAC. At the same time, misaligned chromosome fragments became obvious at the first metaphase (MI) in severe DNA DSBs oocytes. These data implied that the inactivation of SAC during the metaphase-anaphase transition of first meiosis was independent of chromosome integrity. Next, we induced DNA DSBs in vivo, and found that the number of superovulated oocytes per mouse was significantly reduced; moreover, this treatment increased the percentage of apoptotic oocytes. These results suggest that DNA DSBs oocytes undergo apoptosis in vivo.

Merotelic attachments allow alignment and stabilization of chromatids in meiosis II oocytes.

The chromosome segregation process in human oocytes is highly error-prone, generating meiosis II (MII) oocytes with unbalanced chromatids that contribute to aneuploidy in offspring. This raises questions regarding the mechanism for transmission of chromatids and how chromatids evade the error correction mechanisms in MII oocytes. Here, we analyse the behaviour of chromatids in mouse MII oocytes. We find that chromatids align at the spindle equator at the metaphase stage of MII and that their presence does not obstruct entry into the anaphase stage. The alignment process is mediated by merotelic (bi-directional) microtubule-kinetochore attachments, revealing a multi-domain organization of the kinetochore of mammalian meiotic chromosomes. Our results suggest that biorientation of chromatids stabilize microtubule attachments at the kinetochores in a tension-dependent manner. Our results also suggest that merotelic attachments contribute to chromosome mis-segregation in wild-type MII oocytes. Thus, merotely is an important promoter of aneuploidy in mammalian oocytes.