Spindle Structure Research Articles

Glimpses on microtubule accessory proteins

Microtubules allow cells to regulate their architecture, movement, and division in a very flexible manner and in concert with the signals and challenges they perceive from the environment. Surprisingly, this versatile tool basically consists of heterodimers of two very similar proteins that are even fairly conserved over evolution. What qualifies these organelles to fulfill so many tasks and functions? Due to the molecular conservation of their building elements, the tubulins, it is generally believed that the functional specificity of microtubules is mediated by associated proteins. In fact, there exists a wealth of such accessory proteins that, during evolution, have diversified considerably. Two contributions in the present issue address functional and evolutionary aspects of such microtubule accessory proteins. The maize mutant brick1 is affected in epidermal morphogenesis as result of altered WAVE/ARP2/3 function. In an attempt to understand the role of the BRICK1 protein in cell division, Panteris et al., in the current issue, follow mitotic actin arrays and observe that, in contrast to the wild type, actin accumulates in the preprophase band of dividing protodermal cells of the mutant. In addition, actin accumulates in mutant spindles after treatment with taxol. Interestingly, this does not affect neither spindle structure nor division activity. This work contributes to the discussion on the role of actomyosin in mitosis (see editorial of the last issue). Based on evolutionary considerations, it is argued that, most likely, cells adopted pre-existing systems of interphase motility to generate the forces required for chromosomal translocation. In fact, there is evidence from insect cells for a role of actomyosin in mitosis. In plant cells, the colocalization of the so-called phragmosome with the preprophase band has been known for a long time. However, the link between microtubules and actin disappears during establishment of the division spindle, but is re-established during late anaphase, when the formation of the phragmoplast is initiated. This shows that, in plant cells as well, mitotic microtubule arrays are acting in concerted action with actin filaments and that BRICK1 is a key regulator for this interaction. On the other hand, the supernumerous actin filaments in preprophase band and spindle seem to have little effect neither for function and organization of mitotic microtubules nor cell division. This, at first glance, would contradict the spindle matrix model. However, it would be naive to assume a homogenous population of actin filaments. The effect of altered BRICK1 function in the mutant is mainly expected for filaments endowed with high nucleation dynamics, whereas functions driven by stable actin filaments (as those participating in myosin-driven movement) should remain largely untouched. The findings reported by Panteris et al. indicate, therefore, that we might be able to dissect the cross-talk between microtubules and actin filaments with respect to functionally and dynamically different subpopulations of actin. The evolutionary origin of microtubule organization has been associated with the basal body. This organizer of the flagellar apparatus is designed to ensure multiple functions such as locomotion, sensory perception, and cell division. In many flagellate species, these basal bodies are tethered by a relatively stiff, noncontractile structure to the plasma membrane of the cell. These socalled striated roots accompany microtubules in green algae and are composed of striated fiber assemblin, an acidic protein structurally related to β-giardin. In addition Protoplasma (2009) 236:1–2 DOI 10.1007/s00709-009-0054-7

The oocyte spindle is preserved by 1,2-propanediol during slow freezing

To investigate the specific changes in oocyte spindle subjected to severe challenges of low temperature, as well as to examine the effect of cryoprotectants in preserving oocyte spindle during cryopreservation. In vitro experimental study. Academic research laboratory. B6D2F1 (C57BL/6 X DBA/2) mice. Mouse oocytes were cryopreserved using a slow freezing method in a sodium-depleted medium with 1.5 mol/l 1,2-propanediol (PROH) and 0.3 M sucrose. To examine the spindle, oocytes were fixed before, during, and after cryopreservation, and oocytes were analyzed by immunocytochemistry and confocal microscopy. The MII spindle was preserved during the slow freezing, because the cryoprotectant PROH was found to support the organization of MII spindle in resisting the subzero temperature. In contrast, the MII spindle was disassembled gradually during the thawing process with or without PROH. Most of the oocytes were able to recover the MII spindle after thawing, but a portion of thawed oocytes could not sustain the meiotic spindle because of parthenogenetic activation. 1,2-Propanediol can support the organization of MII spindle to defy the subphysiologic temperature; however, the PROH cannot sustain oocyte spindle structure after the subsequent thawing process.

Spindle Fusion Requires Dynein-Mediated Sliding of Oppositely Oriented Microtubules

Bipolar spindle assembly is critical for achieving accurate segregation of chromosomes. In the absence of centrosomes, meiotic spindles achieve bipolarity by a combination of chromosome-initiated microtubule nucleation and stabilization and motor-driven organization of microtubules. Once assembled, the spindle structure is maintained on a relatively long time scale despite the high turnover of the microtubules that comprise it. To study the underlying mechanisms responsible for spindle assembly and steady-state maintenance, we used microneedle manipulation of preassembled spindles in Xenopus egg extracts. When two meiotic spindles were brought close enough together, they interacted, creating an interconnected microtubule structure with supernumerary poles. Without exception, the perturbed system eventually re-established bipolarity, forming a single spindle of normal shape and size. Bipolar spindle fusion was blocked when cytoplasmic dynein function was perturbed, suggesting a critical role for the motor in this process. The fusion of Eg5-inhibited monopoles also required dynein function but only occurred if the initial interpolar separation was less than twice the microtubule radius of a typical monopole. Our experiments uniquely illustrate the architectural plasticity of the spindle and reveal a robust ability of the system to attain a bipolar morphology. We hypothesize that a major mechanism driving spindle fusion is dynein-mediated sliding of oppositely oriented microtubules, a novel function for the motor, and posit that this same mechanism might also be involved in normal spindle assembly and homeostasis.

The Homotetrameric Kinesin-5 KLP61F Preferentially Crosslinks Microtubules into Antiparallel Orientations

The segregation of genetic material during mitosis is coordinated by the mitotic spindle, whose action depends upon the polarity patterns of its microtubules (MTs). Homotetrameric mitotic kinesin-5 motors can crosslink and slide adjacent spindle MTs, but it is unknown whether they or other motors contribute to establishing these MT polarity patterns. Here, we explored whether the Drosophila embryo kinesin-5 KLP61F, which plausibly crosslinks both parallel and antiparallel MTs, displays a preference for parallel or antiparallel MT orientation. In motility assays, KLP61F was observed to crosslink and slide adjacent MTs, as predicted. Remarkably, KLP61F displayed a 3-fold higher preference for crosslinking MTs in the antiparallel orientation. This polarity preference was observed in the presence of ADP or ATP plus AMPPNP, but not AMPPNP alone, which induces instantaneous rigor binding. Also, a purified motorless tetramer containing the C-terminal tail domains displayed an antiparallel orientation preference, confirming that motor activity is not required. The results suggest that, during morphogenesis of the Drosophila embryo mitotic spindle, KLP61F's crosslinking and sliding activities could facilitate the gradual accumulation of KLP61F within antiparallel interpolar MTs at the equator, where the motor could generate force to drive poleward flux and pole-pole separation.

The Coordination of Centromere Replication, Spindle Formation, and Kinetochore–Microtubule Interaction in Budding Yeast

The kinetochore is a protein complex that assembles on centromeric DNA to mediate chromosome–microtubule interaction. Most eukaryotic cells form the spindle and establish kinetochore–microtubule interaction during mitosis, but budding yeast cells finish these processes in S-phase. It has long been noticed that the S-phase spindle in budding yeast is shorter than that in metaphase, but the biological significance of this short S-phase spindle structure remains unclear. We addressed this issue by using ask1-3, a temperature-sensitive kinetochore mutant that exhibits partially elongated spindles at permissive temperature in the presence of hydroxyurea (HU), a DNA synthesis inhibitor. After exposure to and removal of HU, ask1-3 cells show a delayed anaphase entry. This delay depends on the spindle checkpoint, which monitors kinetochore–microtubule interaction defects. Overproduction of microtubule-associated protein Ase1 or Cin8 also induces spindle elongation in HU-arrested cells. The spindle checkpoint-dependent anaphase entry delay is also observed after ASE1 or CIN8 overexpression in HU-arrested cells. Therefore, the shorter spindle in S-phase cells is likely to facilitate proper chromosome–microtubule interaction.

Second Meiotic Spindle Integrity Requires MEK/MAP Kinase Activity in Mouse Eggs

ERK-type MAP kinase activity is required for normal first meiotic (MI) metaphase spindle dynamics and first polar body formation at the MI/MII transition, and for MII arrest until egg activation. MEK and MAPK, however, remain active until meiosis is completed and pronuclei form, but whether MEK/MAPK activity affects MII spindle function during egg activation has been unknown. Polarized light microscopy revealed that the MII spindle rapidly (within approximately 15 min) lost birefringence upon treatment of the egg with U0126, indicating decreased organization at the molecular level upon MEK inhibition. In contrast, birefringence rapidly increased when MPF was inhibited with roscovitine, and this was similar to the increased birefringence previously shown after fertilization or parthenogenetic activation with Sr(2+). Confocal microscopy indicated that many spindles in U0126-activated eggs had failed to rotate or were dissociated from the egg cortex. Subsequently, abnormally-located midbodies were evident in U0126-induced parthenogenotes. Thus, MEK/MAPK activity is required to maintain the ordered structure of the MII spindle and for normal spindle dynamics during second polar body formation.

Protein-Nanocrystal Conjugates Support a Single Filament Polymerization Model in R1 Plasmid Segregation

To ensure inheritance by daughter cells, many low-copy number bacterial plasmids, including the R1 drug-resistance plasmid, encode their own DNA segregation systems. The par operon of plasmid R1 directs construction of a simple spindle structure that converts free energy of polymerization of an actin-like protein, ParM, into work required to move sister plasmids to opposite poles of rod-shaped cells. The structures of individual components have been solved, but little is known about the ultrastructure of the R1 spindle. To determine the number of ParM filaments in a minimal R1 spindle, we used DNA-gold nanocrystal conjugates as mimics of the R1 plasmid. We found that each end of a single polar ParM filament binds to a single ParR/parC-gold complex, consistent with the idea that ParM filaments bind in the hollow core of the ParR/parC ring complex. Our results further suggest that multifilament spindles observed in vivo are associated with clusters of plasmids segregating as a unit.

Meiotic spindle recovery is faster in vitrification of human oocytes compared to slow freezing

To investigate spindle behavior during and after slow freezing at room temperature (RT) and vitrification at different temperatures. Randomized, comparative study. University hospital. Patients undergoing IVF treatment volunteered for the study and donated part of their supernumerary oocytes. Metaphase II oocytes were divided into group A: slow freezing RT /thawing RT; group B: vitrification RT/warming RT; group C: vitrification RT/warming 37 degrees C; and group D: vitrification 37 degrees C/warming 37 degrees C. Spindle presence was evaluated at each step of the four procedures and in culture. Cumulative spindle recovery rate comparing warming phase of the three vitrification groups and culture phase among the four groups. During warming, the three vitrification groups showed a significantly fast spindle recovery rate compared to the thawing of the slow freezing group. A progressively significant fast cumulative recovery rate was observed in the three vitrification groups by increasing the number of phases at physiological temperature (hazard rate = 2.68; 95% confidence interval 1.71-4.02). The present study demonstrates that spindle recovery is faster in vitrification than in slow freezing. These data support a possible protective effect of vitrification/warming at 37 degrees C on the meiotic spindle structure and, therefore, on the subsequent clinical outcome of the procedure, although comparative clinical studies are needed.

Inhibition of Aurora-A suppresses epithelial–mesenchymal transition and invasion by downregulating MAPK in nasopharyngeal carcinoma cells

Mitotic serine/threonine kinase Aurora-A (Aur-A) plays a critical role in regulating centrosome segregation and spindle assemble. Aur-A overexpression causes excessive centrosome duplication and abnormal spindle structure, leading to tumor malignant progression. Here, we investigated Aur-A expression in nasopharyngeal carcinoma (NPC) and the association between Aur-A and NPC invasiveness. We showed that overexpression of Aur-A in tumor tissues was correlated with cranial bone invasion and clinical stage in NPC patients. Suppression of Aur-A by either selective Aurora inhibitory VX-680 or small-interfering RNA caused G(2)/M arrest and apoptotic cell death in NPC CNE-2 cells. Significantly, inhibition of Aur-A suppressed CNE-2 cell invasion and restored membrane expression of epithelial markers, E-cadherin and beta-catenin, suggesting a reversed epithelial-mesenchymal transition process in cancer cells. In addition, we found that Aur-A-regulated epithelial-mesenchymal transition and invasion were mediated by mitogen-activated protein kinase (MAPK) phosphorylation. Moreover, suppression of MAP kinase by small-interfering RNA or its upstream MEK1/2-selective inhibitor U0126 abrogated cell invasion enhanced by Aur-A overexpression. On the other hand, forced overexpression of constitutively active form of MEK1/2, MEK2DD, in CNE-2 cancer cells rescued cell invasive ability suppressed by VX-680-imposed Aur-A inhibition. Our results indicated that Aur-A acted through a downstream MAP kinase pathway to promote epithelial-mesenchymal transition and invasiveness in nasopharyngeal tumorigenesis. Small chemical inhibitor VX-680 may offer as a promising molecular targeting agent in human NPC.

Protein kinase C delta (PKCδ) interacts with microtubule organizing center (MTOC)-associated proteins and participates in meiotic spindle organization

Defects in meiotic spindle structure can lead to chromosome segregation errors and genomic instability. In this study the potential role of protein kinase C delta (PKCδ) on meiotic spindle organization was evaluated in mouse oocytes. PKCδ was previously shown to be phosphorylated during meiotic maturation and concentrate on the meiotic spindle during metaphases I and II. Currently we show that when phosphorylated on Threonine 505 (pPKCδ Thr505), within the activation loop of its C4 domain, PKCδ expression was restricted to the meiotic spindle poles and a few specific cytoplasmic foci. In addition, pPKCδ Thr505 co-localized with two key microtubule organizing center (MTOC)-associated proteins, pericentrin and γ-tubulin. An interaction between pPKCδ Thr505 and pericentrin as well as γ-tubulin was confirmed by co-immunoprecipitation analysis using both fetal fibroblast cells and oocytes. Notably, targeted knockdown of PKCδ expression in oocytes using short interfering RNAs effectively reduced pPKCδ Thr505 protein expression at MTOCs and leads to a significant ( P < 0.05) disruption of meiotic spindle organization and chromosome alignment during MI and MII. Moreover, both γ-tubulin and pericentrin expression at MTOCs were decreased in pPKCδ Thr505-depleted oocytes . In sum, these results indicate that pPKCδ Thr505 interacts with MTOC-associated proteins and plays a role in meiotic spindle organization in mammalian oocytes.

ECRG2 Disruption Leads to Centrosome Amplification and Spindle Checkpoint Defects Contributing Chromosome Instability

Cancer cells contain an abnormal number of chromosomes (aneuploidy), which is a prevalent form of genetic instability in human cancers. Abnormal amplification of centrosomes and defects of spindle assembly checkpoint are the major causes of chromosome instability in cancer cells. Here we present biochemical evidence to suggest a role of ECRG2, a novel tumor suppressor gene, in maintaining chromosome stability. ECRG2 localized to centrosomes during interphase and kinetochores during mitosis. Further analysis revealed that ECRG2 participates in centrosome amplification in a p53-dependent manner. Depletion of ECRG2 not only destabilized p53, down-regulated p21, and increased the cyclin E/CDK2 activity, thus initiating centrosome amplification, but also abolished the ability of p53 localize to centrosomes. Overexpression of ECRG2 restored the p53-dependent suppression of centrosome duplication. Furthermore, ECRG2-depleted cells show severely disrupted spindle phenotype but fail to maintain the mitotic arrest due to minimal BUBR1 protein levels. Taken together, our results indicate that ECRG2 is important for ensuring centrosome duplication, spindle assembly checkpoint, and accurate chromosome segregation, and its depletion may contribute to chromosome instability and aneuploidy in human cancers.

Cytokinetic furrowing in toroidal, binucleate and anucleate cells inC. elegansembryos

Classical experimental studies on echinoderm zygotes concluded that the juxtaposition of two astral microtubule arrays localizes the stimulus for cytokinetic furrowing. However, recent experimental and genetic studies in Caenorhabditis elegans, Drosophila and mammalian cultured cells implicate microtubules of the central spindle, and regulatory proteins associated with this structure, suggesting that the essential conditions for furrow induction may differ from one animal cell to another. We used micromanipulation and laser microsurgery to create, in three ways, the juxtaposition of astral microtubules in C. elegans embryonic cells. In toroidal cells we observe that furrows initiate both where astral microtubule arrays are juxtaposed, and where the cortex most closely approaches the central spindle. We find that binucleate cells successfully furrow not only across the spindles, but also between unconnected spindle poles. Finally, we find that anucleate cells containing only a pair of centrosomes nevertheless attempt to cleave. Therefore, in C. elegans embryonic cells, as in echinoderms, juxtaposition of two asters suffices to induce furrowing, and neither the chromatin nor the physical structure of the central spindle are indispensable for furrow initiation. However, furrows that cross a central spindle are more likely to complete than those that do not.

Mitotic checkpoint defects in human cancers and their implications to chemotherapy

The mitotic checkpoint, also known as spindle assembly checkpoint, is to ensure accurate chromosome segregation by inducing mitotic arrest when errors occur in the spindle structure or in the alignment of the chromosomes on the spindle. Loss of mitotic checkpoint control is a common event in human cancer cells, which is thought to be responsible for chromosome instability frequently observed in cancer cells. Several reports have shown that cells with a defective mitotic checkpoint are more resistant to several types of anticancer drugs from microtubule disruptors to DNA damaging agents. In addition, inactivation of key mitotic checkpoint proteins such as BUB (budding uninhibited by benzimidazole) and MAD (mitotic arrest deficient ) is influential in drug resistance in mitotic checkpoint defective cancer cells. The mitotic checkpoint has also been linked to DNA damage response and a defective mitotic checkpoint confers cancer cells resistance to certain DNA damaging anticancer drugs. This review presents recent evidence on mitotic checkpoint defects in human cancers and their association with resistance to anticancer drugs. In addition, the clinical importance and potential therapeutic implications of targeting the mitotic checkpoint to reverse drug resistance in cancer cells are also discussed.

A theoretical model of mitotic spindle elongation under experimental constraints

Mol Syst Biol. 4: 194 During the division of eukaryotic cells, the duplicated set of chromosomes is separated by the mitotic spindle, a large multicomponent assembly consisting of several hundred proteins in human cells (Sauer et al , 2005). Researchers have started to move from putting together the parts list of spindle components and from generating an atlas of their localizations toward trying to understand at a systems level the dynamic interplay between these components that ultimately translates into spindle function. This requires, in part, shifting the focus from biochemistry to mechanics. It is clear now that the mechanical properties of microtubules and molecular motors are crucial for spindle structure and function. But how they work together is still a mystery and there is a need for modeling this interplay, because our intuition reaches its limits when trying to understand it. The paper by Wollman et al (2008) in this issue reports on the next step of the development of a model that allows one to describe one aspect of spindle behavior for which good quantitative experimental data exist, namely spindle elongation during the period between prophase and anaphase B in Drosophila embryos. As their model is based on the mechanical properties of major elements of the spindle, the quantitative comparison with available data allows the authors to make predictions about how the concerted action of the different mechanical elements leads to spindle elongation. Among others, there are two major challenges when trying to model the spindle (Karsenti et al , 2006). (1) What is the right level of description? This means what is the minimal set of molecular activities that needs to be considered and …

Tissue engineering intrafusal fibers: Dose- and time-dependent differentiation of nuclear bag fibers in a defined in vitro system using neuregulin 1- β-1

While much is known about muscle spindle structure, innervation and function, relatively few factors have been identified that regulate intrafusal fiber differentiation and spindle development. Identification of these factors will be a crucial step in tissue engineering functional muscle systems. In this study, we investigated the role of the growth factor, neuregulin 1- β-1 (Nrg 1- β-1) EGF, for its ability to influence myotube fate specification in a defined culture system utilizing the non-biological substrate N-1[3-(trimethoxysilyl)propyl]-diethylenetriamine (DETA). Based on morphological and immunocytochemical criteria, Nrg 1- β-1 treatment of developing myotubes increases the ratio of nuclear bag fibers to total myotubes from 0.019 to 0.100, approximately a five-fold increase. The myotube cultures were evaluated for expression of the intrafusal fiber-specific alpha cardiac-like myosin heavy chain and for the expression of the non-specific slow myosin heavy chain. Additionally, the expression of ErbB2 receptors on all myotubes was observed, while phosphorylated ErbB2 receptors were only observed in Nrg 1- β-1-treated intrafusal fibers. After Nrg 1- β-1 treatment, we were able to observe the expression of the intrafusal fiber-specific transcription factor Egr3 only in fibers exhibiting the nuclear bag phenotype. Finally, nuclear bag fibers were characterized electrophysiologically for the first time in vitro. This data shows conclusively, in a serum-free system, that Nrg 1- β-1 is necessary to drive specification of forming myotubes to the nuclear bag phenotype.

A Multi-Fluorescent MDA435 Cell Line for Mitosis Inhibitor Studies: Simultaneous Visualization of Chromatin, Microtubules, and Nuclear Envelope in Living Cells

As a part of our efforts to create a pre-made multi-fluorescent cell library for various cytological assays, we made a triple-fluorescent cell line in which microtubules, chromosomes, and nuclear envelopes were visualized for simultaneous observation of spindle structure and chromosome distribution in living cells. Pilot experiments with microtubule-disturbing drugs showed the advantages of this cell line in mitosis inhibitor studies.

Structure of the meiotic spindle

![Graphic][1] Fluorescently labeled tubulin monomers within meiotic spindle filaments are seen as speckles whose motion can be tracked. KAPOOR/MACMILLAN The meiotic spindle is made up of shorter microtubules than previously believed, suggest results from Ge Yang, Gaudenz Danuser (

Effect of Temperature Decline on the Cytoskeletal Organization of the Porcine Oocyte

The purpose of this study was to evaluate effect of cooling on the cytoskeletal organization affecting the distribution and structure of the meiotic spindle in the porcine metaphase II (MII) oocyte. Fluorescence staining was done for visualization of microtubules, microfilaments and chromosomes. In vitro-matured oocytes were either maintained at 37 °C (for controls) or cooled abruptly to 5 or 18°C. Microtubules were preferentially labeled at the meiotic spindle and microfilaments were mainly detected at the cortex of the MII oocyte. The majority of controls (87%, n=97) had a barrel-shaped spindle, whereas the remaining 13% showed a broad, box-shaped spindle. After exposure to 18 °C for 30 min, 94% of oocytes (n=48) possessed either a box-shaped spindle (63%) with a shortened interpolar distance (7.4 ± 0.4 μm vs. 9.8 ± 0.2 μm for controls P<0.05), or no spindle (31%). Exposure of oocytes to 5 °C for 30 min caused complete disassembly of the spindle in 81% of the oocytes (n=103). Such spindle-disassembled oocytes had increased intensity of microtubule signaling in the cytoplasm and irregular staining of cortical microfilaments. Cortical microfilament staining decreased in intensity in 64% (n=33) and 89% (n=74) of the oocytes cooled to 18 and 5 °C, respectively. When oocytes were rewarmed at 37 °C after exposure to 5 °C for 30 min, the spindles were not restored to normal in any oocyte (n=138) regardless of the rewarming duration (0, 1, 10, 20 or 60 min), although microtubules and microfilaments were reassembled. However, more oocytes contained dispersed chromosomes with no spindle and collapsed microfilament architecture, as rewarming time increased beyond 20 min. These results suggest that in porcine MII oocytes both microtubular spindle and cortical microfilaments are irreversibly affected by a 5 °C cold shock, and that the polar microtubule-organizing centers malfunction even after rewarming.

Response of immature and mature mouse cytoskeleton to endometriosis – role of oxidative stress

ObjectiveMature and immature oocytes are differentiated by the presence of the first polar body. Oxidative stress is involved in the pathophysiology of endometriosis. The goal of our study was to examine the effect of peritoneal fluid from patients with endometriosis on the microtubule and chromosomal alterations in both mature and immature oocytes.DesignProspective in vitro study.Materials and methods50 oocytes were divided into 2 groups according to the presence or absence of the polar body. Group I: 29 mature oocytes with polar body. Group II: 21 immature oocytes and no polar body. Both groups were incubated in peritoneal fluid of patients with endometriosis + HTF (50:50 vol./vol.) for one hour. Oocytes were fixed, and immunohistochemical staining was done to evaluate the alterations in the microtubules. Propidium iodide was used for chromosomal staining. Microtubules and chromosomes were examined by fluorescent and confocal microscopy. Modified scoring system was used (Choi et al. 2007). For microtubules a scores of 1 and 2 = normal; score 3 = slightly abnormal, and score 4 = missing. For the chromosomal alterations: Scores 1 and 2 = normal; score 3 = slightly abnormal and score 4 = totally abnormal.ResultsTableAlterations in microtubule and chromosomes in both mature and immature oocytes following exposure to peritoneal fluid from women with endometriosis Open table in a new tab ConclusionsSpindle structure (microtubules and chromosomes) in immature oocytes is more susceptible to oxidative stress induced damage compared to mature oocytes. Mature oocytes appear to be more resistant to oxidative stress following exposure to peritoneal fluid. It is critical to minimize the exposure of immature oocytes to oxidative stress during in vitro maturation of the oocytes. ObjectiveMature and immature oocytes are differentiated by the presence of the first polar body. Oxidative stress is involved in the pathophysiology of endometriosis. The goal of our study was to examine the effect of peritoneal fluid from patients with endometriosis on the microtubule and chromosomal alterations in both mature and immature oocytes. Mature and immature oocytes are differentiated by the presence of the first polar body. Oxidative stress is involved in the pathophysiology of endometriosis. The goal of our study was to examine the effect of peritoneal fluid from patients with endometriosis on the microtubule and chromosomal alterations in both mature and immature oocytes. DesignProspective in vitro study. Prospective in vitro study. Materials and methods50 oocytes were divided into 2 groups according to the presence or absence of the polar body. Group I: 29 mature oocytes with polar body. Group II: 21 immature oocytes and no polar body. Both groups were incubated in peritoneal fluid of patients with endometriosis + HTF (50:50 vol./vol.) for one hour. Oocytes were fixed, and immunohistochemical staining was done to evaluate the alterations in the microtubules. Propidium iodide was used for chromosomal staining. Microtubules and chromosomes were examined by fluorescent and confocal microscopy. Modified scoring system was used (Choi et al. 2007). For microtubules a scores of 1 and 2 = normal; score 3 = slightly abnormal, and score 4 = missing. For the chromosomal alterations: Scores 1 and 2 = normal; score 3 = slightly abnormal and score 4 = totally abnormal. 50 oocytes were divided into 2 groups according to the presence or absence of the polar body. Group I: 29 mature oocytes with polar body. Group II: 21 immature oocytes and no polar body. Both groups were incubated in peritoneal fluid of patients with endometriosis + HTF (50:50 vol./vol.) for one hour. Oocytes were fixed, and immunohistochemical staining was done to evaluate the alterations in the microtubules. Propidium iodide was used for chromosomal staining. Microtubules and chromosomes were examined by fluorescent and confocal microscopy. Modified scoring system was used (Choi et al. 2007). For microtubules a scores of 1 and 2 = normal; score 3 = slightly abnormal, and score 4 = missing. For the chromosomal alterations: Scores 1 and 2 = normal; score 3 = slightly abnormal and score 4 = totally abnormal. ResultsTableAlterations in microtubule and chromosomes in both mature and immature oocytes following exposure to peritoneal fluid from women with endometriosis Open table in a new tab ConclusionsSpindle structure (microtubules and chromosomes) in immature oocytes is more susceptible to oxidative stress induced damage compared to mature oocytes. Mature oocytes appear to be more resistant to oxidative stress following exposure to peritoneal fluid. It is critical to minimize the exposure of immature oocytes to oxidative stress during in vitro maturation of the oocytes. Spindle structure (microtubules and chromosomes) in immature oocytes is more susceptible to oxidative stress induced damage compared to mature oocytes. Mature oocytes appear to be more resistant to oxidative stress following exposure to peritoneal fluid. It is critical to minimize the exposure of immature oocytes to oxidative stress during in vitro maturation of the oocytes.

Identification of PARP homologues in human ejaculated sperm

(Poly(ADP-ribose) polymerases consist of 18 protein family members (PARP-1 to PARP 16). These proteins are involved in DNA repair and maintenance of genomic stability (PARP 1, 2), transcriptional regulation (PARP 4), centromere function (PARP 1, 2, 3), mitotic spindle formation, structure and function of vault particles (PARP 4), telomere dynamics (Tankyrase 1–2), trafficking of endosomal vesicles (Tankyrase), apoptosis and necrosis (PARP 1). Our goal was to identify these PARP homologues using peptide mass finger printing tool.