Microtubules In Oocytes Research Articles

Coupling of Active Motion and Advection Shapes Intracellular Cargo Transport

Intracellular cargo transport can arise from passive diffusion, active motor-driven transport along cytoskeletal filament networks, and passive advection by fluid flows entrained by such cargo-motor motion. Active and advective transport are thus intrinsically coupled as related, yet different representations of the same underlying network structure. A reaction-advection-diffusion system is used here to show that this coupling affects the transport and localization of a passive tracer in a confined geometry. For sufficiently low diffusion, cargo localization to a target zone is optimized either by low reaction kinetics and decoupling of bound and unbound states, or by a mostly disordered cytoskeletal network with only weak directional bias. These generic results may help to rationalize subtle features of cytoskeletal networks, for example as observed for microtubules in fly oocytes.

Anticancer Peptidylarginine Deiminase (PAD) Inhibitors Regulate the Autophagy Flux and the Mammalian Target of Rapamycin Complex 1 Activity

Tumor suppressor genes are frequently silenced in cancer cells by enzymes catalyzing epigenetic histone modifications. The peptidylarginine deiminase family member PAD4 (also called PADI4) is markedly overexpressed in a majority of human cancers, suggesting that PAD4 is a putative target for cancer treatment. Here, we have generated novel PAD inhibitors with low micromolar IC(50) in PAD activity and cancer cell growth inhibition. The lead compound YW3-56 alters the expression of genes controlling the cell cycle and cell death, including SESN2 that encodes an upstream inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Guided by the gene expression profile analyses with YW3-56, we found that PAD4 functions as a corepressor of p53 to regulate SESN2 expression by histone citrullination in cancer cells. Consistent with the mTORC1 inhibition by SESN2, the phosphorylation of its substrates including p70S6 kinase (p70S6K) and 4E-BP1 was decreased. Furthermore, macroautophagy is perturbed after YW3-56 treatment in cancer cells. In a mouse xenograft model, YW3-56 demonstrates cancer growth inhibition activity with little if any detectable adverse effect to vital organs, whereas a combination of PAD4 and histone deacetylase inhibitors further decreases tumor growth. Taken together, our work found that PAD4 regulates the mTORC1 signaling pathway and that PAD inhibitors are potential anticancer reagents that activate tumor suppressor gene expression alone or in combination with histone deacetylase inhibitors.

A functional link between localized Oskar, dynamic microtubules, and endocytosis

Many cell types including developing oocytes, fibroblasts, epithelia and neurons use mRNA localization as a means to establish polarity. The Drosophila oocyte has served as a useful model in dissecting the mechanism of mRNA localization. The polarity of the oocyte is established by the specific localization of three critical mRNAs—oskar, bicoid and gurken. The localization of these mRNAs requires microtubule integrity, and the activity of microtubule motors. However, the precise organization of the oocyte microtubule cytoskeleton remains an open question. In order to examine the polarity of oocyte microtubules, we visualized the localization of canonical microtubule plus end binding proteins, EB1 and CLIP-190. Both proteins were enriched at the posterior of the oocyte, with additional foci detected within the oocyte cytoplasm and along the cortex. Surprisingly, however, we found that this asymmetric distribution of EB1 and CLIP-190 was not essential for oskar mRNA localization. However, Oskar protein was required for recruiting the plus end binding proteins to the oocyte posterior. Lastly, our results suggest that the enrichment of growing microtubules at the posterior pole functions to promote high levels of endocytosis in this region of the cell. Thus, multiple polarity-determining pathways are functionally linked in the Drosophila oocytes.

Melatonin prevents hypochlorous acid‐induced alterations in microtubule and chromosomal structure in metaphase‐II mouse oocytes

Hypochlorous acid (HOCl) is generated by myeloperoxidase, using chloride and hydrogen peroxide as substrates. Here we demonstrate that HOCl alters metaphase-II mouse oocyte microtubules and chromosomal (CH) alignment which can be prevented by melatonin. Metaphase-II mouse oocytes, obtained commercially, were grouped as: control, melatonin (150, 200nmol/mL), HOCl (10, 20, 50, and 100nmol/mL), and HOCl (50nmol/mL) pretreated with 150 and 200 nmol/mL of melatonin. Microtubule and CH alignment was studied utilizing an indirect immunofluorescence technique and scored by two observers. Pearson chi-square test and Fisher's exact test were used to compare outcomes between controls and treated groups and also among each group. Poor scores for the spindle and chromosomes increased significantly at 50nmol/mL of HOCl (P<0.001). Oocytes treated with melatonin only at 150 and 200 nmol/mL showed no changes; significant differences (P<0.001) were observed when oocytes exposed to 50nmol/mL of HOCl were compared to oocytes pretreated with 200 nmol/mL melatonin. Fifty percent of the oocytes demonstrated good scores, both in microtubule and CH alterations, when pretreated with melatonin at 150 nmol/mL compared to 0% in the HOCl-only group. HOCl alters the metaphase-II mouse oocyte spindle and CH alignment in a dose-dependant manner, which might be a potential cause of poor oocyte quality (e.g., in patients with endometriosis). Melatonin prevented the HOCl-mediated spindle and CH damage, and therefore, may be an attractive therapeutic option to prevent oocyte damage in endometriosis or inflammatory diseases where HOCl levels are known to be elevated.

Role of melatonin in preventing hypochlorous acid induced alterations in microtubule and chromosomal structure in metaphase-II mouse oocytes in vitro

Recently, we have shown that melatonin, a pineal gland hormone, can prevent hypochlorous acid (HOCl) mediated protein aggregation and hemeprotein destruction. We have demonstrated that HOCl may alter metaphase-II oocyte microtubule and chromosomal alignment. The objective of the study was to test whether melatonin prevents the impairment of oocyte quality induced by HOCl in vitro.

Regulation of mouse oocyte microtubule and organelle dynamics by PADI6 and the cytoplasmic lattices

Organelle positioning and movement in oocytes is largely mediated by microtubules (MTs) and their associated motor proteins. While yet to be studied in germ cells, cargo trafficking in somatic cells is also facilitated by specific recognition of acetylated MTs by motor proteins. We have previously shown that oocyte-restricted PADI6 is essential for formation of a novel oocyte-restricted fibrous structure, the cytoplasmic lattices (CPLs). Here, we show that α-tubulin appears to be associated with the PADI6/CPL complex. Next, we demonstrate that organelle positioning and redistribution is defective in PADI6-null oocytes and that alteration of MT polymerization or MT motor activity does not induce organelle redistribution in these oocytes. Finally, we report that levels of acetylated microtubules are dramatically suppressed in the cytoplasm of PADI6-null oocytes, suggesting that the observed organelle redistribution failure is due to defects in stable cytoplasmic MTs. These results demonstrate that the PADI6/CPL superstructure plays a key role in regulating MT-mediated organelle positioning and movement.

MCAK regulates chromosome alignment but is not necessary for preventing aneuploidy in mouse oocyte meiosis I

Errors in chromosome segregation in mammalian oocytes lead to aneuploid eggs that are developmentally compromised. In mitotic cells, mitotic centromere associated kinesin (MCAK; KIF2C) prevents chromosome segregation errors by detaching incorrect microtubule-kinetochore interactions. Here, we examine whether MCAK is involved in spindle function in mouse oocyte meiosis I, and whether MCAK is necessary to prevent chromosome segregation errors. We find that MCAK is recruited to centromeres, kinetochores and chromosome arms in mid-meiosis I, and that MCAK depletion, or inhibition using a dominant-negative construct, causes chromosome misalignment. However, the majority of oocytes complete meiosis I and the resulting eggs retain the correct number of chromosomes. Moreover, MCAK-depleted oocytes can recover from mono-orientation of homologous kinetochores in mid-meiosis I to segregate chromosomes correctly. Thus, MCAK contributes to chromosome alignment in meiosis I, but is not necessary for preventing chromosome segregation errors. Although other correction mechanisms may function in mammalian meiosis I, we speculate that late establishment of kinetochore microtubules in oocytes reduces the likelihood of incorrect microtubule-kinetochore interactions, bypassing the requirement for error correction.

Bazooka is required for polarisation of the Drosophila anterior-posterior axis

The Drosophila anterior-posterior (AP) axis is determined by the polarisation of the stage 9 oocyte and the subsequent localisation of bicoid and oskar mRNAs to opposite poles of the cell. Oocyte polarity has been proposed to depend on the same PAR proteins that generate AP polarity in C. elegans, with a complex of Bazooka (Baz; Par-3), Par-6 and aPKC marking the anterior and lateral cortex, and Par-1 defining the posterior. The function of the Baz complex in oocyte polarity has remained unclear, however, because although baz-null mutants block oocyte determination, egg chambers that escape this early arrest usually develop normal polarity at stage 9. Here, we characterise a baz allele that produces a penetrant polarity phenotype at stage 9 without affecting oocyte determination, demonstrating that Baz is essential for axis formation. The dynamics of Baz, Par-6 and Par-1 localisation in the oocyte indicate that the axis is not polarised by a cortical contraction as in C. elegans, and instead suggest that repolarisation of the oocyte is triggered by posterior inactivation of aPKC or activation of Par-1. This initial asymmetry is then reinforced by mutual inhibition between the anterior Baz complex and posterior Par-1 and Lgl. Finally, we show that mutation of the aPKC phosphorylation site in Par-1 results in the uniform cortical localisation of Par-1 and the loss of cortical microtubules. Since non-phosphorylatable Par-1 is epistatic to uninhibitable Baz, Par-1 seems to function downstream of the other PAR proteins to polarize the oocyte microtubule cytoskeleton.

Bazooka regulates microtubule organization and spatial restriction of germ plasm assembly in the Drosophila oocyte

Localization of the germ plasm to the posterior of the Drosophila oocyte is required for anteroposterior patterning and germ cell development during embryogenesis. While mechanisms governing the localization of individual germ plasm components have been elucidated, the process by which germ plasm assembly is restricted to the posterior pole is poorly understood. In this study, we identify a novel allele of bazooka (baz), the Drosophila homolog of Par-3, which has allowed the analysis of baz function throughout oogenesis. We demonstrate that baz is required for spatial restriction of the germ plasm and axis patterning, and we uncover multiple requirements for baz in regulating the organization of the oocyte microtubule cytoskeleton. Our results suggest that distinct cortical domains established by Par proteins polarize the oocyte through differential effects on microtubule organization. We further show that microtubule plus-end enrichment is sufficient to drive germ plasm assembly even at a distance from the oocyte cortex, suggesting that control of microtubule organization is critical not only for the localization of germ plasm components to the posterior of the oocyte but also for the restriction of germ plasm assembly to the posterior pole.

Endometriosis-induced alterations in mouse metaphase II oocyte microtubules and chromosomal alignment: a possible cause of infertility

To examine the effect of peritoneal fluid (PF) of patients with endometriosis on the cytoskeleton of metaphase II oocytes and correlate the results with the stage of endometriosis and the duration of infertility. Prospective-controlled study. Center for reproductive medicine at a tertiary-care hospital. Women with endometriosis (n=23) and tubal ligation/reversal (n=15). Peritoneal fluid obtained from 38 women (23 with endometriosis and 15 tubal ligation/reversal) after laparoscopy. Four hundred metaphase II oocytes were used: 165 frozen metaphase II oocytes were incubated in the PF of patients with endometriosis, 135 oocytes incubated in the PF of nonendometriosis patients (control subjects) and 100 oocytes incubated in human tubal fluid (HTF) media. Spindle abnormalities (microtubule and chromosomal) were evaluated by confocal imaging. In the endometriosis group, the cytoskeleton had a higher frequency of abnormal meiotic spindle and chromosomal misalignment (score ≥3), indicating severe damage compared with the control groups. The proportions of abnormalities in microtubule and chromosome alterations in endometriosis (67.9% and 63.6%, respectively) were significantly higher than for oocytes incubated with PF of the nonendometriosis group (24.4% and 14.8%) as well as the HTF group (13% and 13%). Oocyte cytoskeleton damage positively correlated with the duration of infertility and the stage of endometriosis. Alteration of oocyte cytoskeleton might be one of the causes of poor oocyte quality in patients with endometriosis.

Symmetry Breaking During Drosophila Oogenesis

The orthogonal axes of Drosophila are established during oogenesis through a hierarchical series of symmetry-breaking steps, most of which can be traced back to asymmetries inherent in the architecture of the ovary. Oogenesis begins with the formation of a germline cyst of 16 cells connected by ring canals. Two of these 16 cells have four ring canals, whereas the others have fewer. The first symmetry-breaking step is the selection of one of these two cells to become the oocyte. Subsequently, the germline cyst becomes surrounded by somatic follicle cells to generate individual egg chambers. The second symmetry-breaking step is the posterior positioning of the oocyte within the egg chamber, a process mediated by adhesive interactions with a special group of somatic cells. Posterior oocyte positioning is accompanied by a par gene-dependent repolarization of the microtubule network, which establishes the posterior cortex of the oocyte. The next two steps of symmetry breaking occur during midoogenesis after the volume of the oocyte has increased about 10-fold. First, a signal from the oocyte specifies posterior follicle cells, polarizing a symmetric prepattern present within the follicular epithelium. Second, the posterior follicle cells send a signal back to the oocyte, which leads to a second repolarization of the oocyte microtubule network and the asymmetric migration of the oocyte nucleus. This process again requires the par genes. The repolarization of the microtubule network results in the transport of bicoid and oskar mRNAs, the anterior and posterior determinants, respectively, of the embryonic axis, to opposite poles of the oocyte. The asymmetric positioning of the oocyte nucleus defines a cortical region of the oocyte where gurken mRNA is localized, thus breaking the dorsal-ventral symmetry of the egg and embryo.

254 DYNAMICS OF GOLGI APPARATUS DURING BOVINE OOCYTE IN VITRO MATURATION: EFFECTS OF INHIBITORS ON CDC2A AND CYTOPLASMIC-DYNEIN ATPase ACTIVITY

The process of maturation encompasses a complex series of molecular and structural events. Completion of the nuclear changes to produce a metaphase II (MII) oocyte does not reflect the molecular and structural maturity of an oocyte, which is sometimes termed cytoplasmic maturation. The Golgi apparatus phosphorylates, fragments, and changes the localization during oocyte maturation. GM130 and phospho-GM130 are used as markers for the Golgi apparatus and phosphorylated Golgi apparatus, respectively. The goal of this study was to analyze the dynamics of the Golgi apparatus and its association with microtubules in bovine oocytes at different stages of in vitro maturation [IVM; i.e. germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and MII]. The roles of CDC2A kinase (also known as p34cdc2) and cytoplasmic-dynein ATPase on Golgi dynamics were studied by using specific inhibitors. The distribution of the markers was assessed by immunocytochemistry and laser confocal microscopy. To unravel the role of CDC2A and cytoplasmic dynein ATPase on the dynamics of the Golgi apparatus, the inhibitors roscovitine (ROS) and sodium-orthovanadate (SOV), respectively, were used. In the first experiment, the nuclear maturation rate was checked in the presence of the inhibitors at different times and for different incubation times to explore whether oocytes were able to reach the MII stage. At the GV and GVBD stages, the Golgi apparatus is observed as fragments named mini-Golgies and at the MI and MII stages as punctate foci throughout the cytoplasm. Our results showed 2 well-defined movements of the Golgi apparatus toward opposite directions, depending on the maturation stage. The first movement was observed between 5 and 9 h of IVM (i.e. the GVBD stage), when the Golgi apparatus relocalized from the ooplasm to the periphery. The second movement was observed between 9 and 15 h of IVM (i.e. the MI stage), when the Golgi apparatus moved from the cortex to throughout the cytoplasm and remained there up to the MII stage. The use of inhibitors on CDC2A and cytoplasmic-dynein ATPase at selected time points revealed that CDC2A played a crucial role on the distribution of this organelle during the first movement, whereas the final localization at the GVBD stage was dependent on cytoplasmic-dynein transport. The second movement of the Golgi apparatus was disturbed by the SOV treatment, but not by the use of ROS, suggesting a role of cytoplasmic-dynein-dependent transport during the distribution and organization of the punctate foci at the MI stage. The phosphorylation status of Golgi was not affected at the different incubation times with inhibitors, except in those oocytes incubated with ROS for 24 h, suggesting a role of CDC2A. In conclusion, we describe the involvement of CDC2A during the first movement of the Golgi apparatus and the importance of cytoplasmic-dynein ATPase activity in the first and second relocalization of Golgi during bovine oocyte maturation. DAAD.

Multiple Kinesin Motors Coordinate Cytoplasmic RNA Transport on a Subpopulation of Microtubules in Xenopus Oocytes

RNA localization is a widely conserved mechanism for generating cellular asymmetry. In Xenopus oocytes, microtubule-dependent transport of RNAs to the vegetal cortex underlies germ layer patterning. Although kinesin motors have been implicated in this process, the apparent polarity of the microtubule cytoskeleton has pointed instead to roles for minus-end-directed motors. To resolve this issue, we have analyzed participation of kinesin motors in vegetal RNA transport and identified a direct role for Xenopus kinesin-1. Moreover, in vivo interference and biochemical experiments reveal a key function for multiple motors, specifically kinesin-1 and kinesin-2, and suggest that these motors may interact during transport. Critically, we have discovered a subpopulation of microtubules with plus ends at the vegetal cortex, supporting roles for these kinesin motors in vegetal RNA transport. These results provide a new mechanistic basis for understanding directed RNA transport within the cytoplasm.

L-carnitine supplementation reduces oocyte cytoskeleton damage and embryo apoptosis induced by incubation in peritoneal fluid from patients with endometriosis

To investigate the protective effect of L-carnitine (LC) against deleterious substances present in the peritoneal fluid (PF) of patients with endometriosis, which may affect the oocyte cytoskeleton and embryogenesis. Experimental study. Research embryology laboratory at an academic hospital. Frozen metaphase II mouse oocytes and embryos. One hundred metaphase II mouse oocytes were divided into five groups and incubated: PF from endometriosis patients; PF from endometriosis patients + LC; PF from tubal ligation patients (patient control); LC only; and human tubal fluid (HTF) alone. A total of 180 eight-cell mouse embryos were divided into: endometriosis only; tubal ligation only; endometriosis + LC; LC alone; and HTF alone. Protective effect of LC on oocytes and embryos. Incubation of the oocytes and the embryos with PF from patients with endometriosis statistically significantly damaged the oocyte microtubules and chromosomes and increased embryo apoptosis compared with controls. Incubation with LC (0.6 mg/mL) statistically significantly improved microtubule and chromosome structure and decreased the level of embryo apoptosis. We propose the use of LC as a supplement in patients with endometriosis, a novel approach that may help improve in vitro fertilization outcome in these patients.

Ultrastructure of bovine oocytes exposed to Taxol prior to OPS vitrification

Our objective was to document potential subcellular consequences of treatment with the microtubule stabilizer Taxol with or without subsequent vitrification of cow and calf oocytes by the open pulled straw (OPS) method. Oocytes were divided into four experimental groups for cows and four groups for calves: (1) a control group fixed immediately after maturation; (2) an OPS group cryopreserved by conventional OPS; (3) a Taxol/CPA group exposed to 1 microM Taxol and cryoprotective agents (CPAs); and (4) a Taxol/OPS group vitrified by OPS including 1 microM Taxol to the vitrification solution. All oocytes were processed for light and transmission electron microscopy. The main injuries were observed on the metaphase plate and the spindle. In control oocytes, the metaphase appeared as condensed chromosomes arranged in a well-organized metaphase plate and the spindle showed well organized microtubules in both cow and calf oocytes. However, in cow OPS oocytes, the metaphase plate was disorganized into scattered chromosomes or the chromosomes were condensed into a single block of chromatin. In addition, microtubules were not organized as typical spindles. In contrast, cow Taxol/OPS oocytes as well as both cow and calf Taxol/CPAs oocytes showed well-organized metaphase plates and normal spindle morphology. All calf OPS and calf Taxol/OPS oocytes displayed a single block of chromatin and no microtubules could be observed around the chromosomes. In conclusion, treatment with 1 microM Taxol before and during vitrification did not induce adverse changes in the oocyte cytoplasm or metaphase spindles in adult bovine oocytes, but stabilized the metaphase and spindle morphology.

Protein kinase B (PKB/Akt) is required for the completion of meiosis in mouse oocytes

Akt, also known as protein kinase B, is implicated in many cellular processes. Akt is phosphorylated at two residues, Thr308 and Ser473. Thr308-phosphorylated Akt is present in pericentriolar materials, while localization of Ser473-phosphorylated Akt was similar to that of microtubules in metaphase oocytes. Spindles were shorter and aberrant in oocytes injected with Thr308- or Ser473-phosphorylated Akt antibodies. Specifically, Thr308- and Ser473-phosphorylated Akts function individually and are both necessary to assemble the metaphase II (MII) spindle. Moreover, the functions of Thr308- and Ser473-phosphorylated Akts differ in MII oocytes. Although oocytes exhibited second polar body (PB2) emission after the injection of a peptide for Thr308, the chromosomal alignment and microtubular organization were aberrant. In contrast, the injection of a peptide for Ser473 caused a failure of PB2 emission. These results suggest that Thr308- and Ser473-phosphorylated Akts are individually involved in fertilization to complete meiosis, including different roles (i.e., Ser473-phosphorylated Akts are involved in PB2 emission, whereas Thr308-phosphorylated Akts regulate the organization of microtubules).

Microtubule anchoring by cortical actin bundles prevents streaming of the oocyte cytoplasm

The localisation of the determinants of the body axis during Drosophila oogenesis is dependent on the microtubule (MT) cytoskeleton. Mutations in the actin binding proteins Profilin, Cappuccino (Capu) and Spire result in premature streaming of the cytoplasm and a reorganisation of the oocyte MT network. As a consequence, the localisation of axis determinants is abolished in these mutants. It is unclear how actin regulates the organisation of the MTs, or what the spatial relationship between these two cytoskeletal elements is. Here, we report a careful analysis of the oocyte cytoskeleton. We identify thick actin bundles at the oocyte cortex, in which the minus ends of the MTs are embedded. Disruption of these bundles results in cortical release of the MT minus ends, and premature onset of cytoplasmic streaming. Thus, our data indicate that the actin bundles anchor the MTs minus ends at the oocyte cortex, and thereby prevent streaming of the cytoplasm. We further show that actin bundle formation requires Profilin but not Capu and Spire. Thus, our results support a model in which Profilin acts in actin bundle nucleation, while Capu and Spire link the bundles to MTs. Finally, our data indicate how cytoplasmic streaming contributes to the reorganisation of the MT cytoskeleton. We show that the release of the MT minus ends from the cortex occurs independently of streaming, while the formation of MT bundles is streaming dependent.

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.

Rab6 mediates membrane organization and determinant localization duringDrosophilaoogenesis

The Drosophila melanogaster body axes are defined by the precise localization and the restriction of molecular determinants in the oocyte. Polarization of the oocyte during oogenesis is vital for this process. The directed traffic of membranes and proteins is a crucial component of polarity establishment in various cell types and organisms. Here, we investigate the role of the small GTPase Rab6 in the organization of the egg chamber and in asymmetric determinant localization during oogenesis. We show that exocytosis is affected in rab6-null egg chambers, which display a loss of nurse cell plasma membranes. Rab6 is also required for the polarization of the oocyte microtubule cytoskeleton and for the posterior localization of oskar mRNA. We show that, in vivo, Rab6 is found in a complex with Bicaudal-D, and that Rab6 and Bicaudal-D cooperate in oskar mRNA localization. Thus, during Drosophila oogenesis, Rab6-dependent membrane trafficking is doubly required; first, for the general organization and growth of the egg chamber, and second, more specifically, for the polarization of the microtubule cytoskeleton and localization of oskar mRNA. These findings highlight the central role of vesicular trafficking in the establishment of polarity and in determinant localization in Drosophila.

Major sperm protein signaling promotes oocyte microtubule reorganization prior to fertilization in Caenorhabditis elegans

In most animals, female meiotic spindles assemble in the absence of centrosomes; instead, microtubule nucleation by chromatin, motor activity, and microtubule dynamics drive the self-organization of a bipolar meiotic spindle. Meiotic spindle assembly commences when microtubules gain access to chromatin after nuclear envelope breakdown (NEBD) during meiotic maturation. Although many studies have addressed the chromatin-based mechanism of female meiotic spindle assembly, it is less clear how signaling influences microtubule localization and dynamics prior to NEBD. Here we analyze microtubule behavior in Caenorhabditis elegans oocytes at early stages of the meiotic maturation process using confocal microscopy and live-cell imaging. In C. elegans, sperm trigger oocyte meiotic maturation and ovulation using the major sperm protein (MSP) as an extracellular signaling molecule. We show that MSP signaling reorganizes oocyte microtubules prior to NEBD and fertilization by affecting their localization and dynamics. We present evidence that MSP signaling reorganizes oocyte microtubules through a signaling network involving antagonistic Gα o/i and Gα s pathways and gap-junctional communication with somatic cells of the gonad. We propose that MSP-dependent microtubule reorganization promotes meiotic spindle assembly by facilitating the search and capture of microtubules by meiotic chromatin following NEBD.