scholarly journals Spindle function in Xenopus oocytes involves possible nanodomain calcium signaling

2016 ◽  
Vol 27 (21) ◽  
pp. 3273-3283 ◽  
Author(s):  
Ruizhen Li ◽  
Julie Leblanc ◽  
Kevin He ◽  
X. Johné Liu
Keyword(s):  
Calcium Signaling ◽  
Intracellular Calcium ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Calcium Transients ◽  
Meiotic Spindle ◽  
First Polar Body ◽  
Spindle Microtubules ◽  
Spindle Function

Intracellular calcium transients are a universal phenomenon at fertilization and are required for egg activation, but the exact role of Ca2+ in second-polar-body emission remains unknown. On the other hand, similar calcium transients have not been demonstrated during oocyte maturation, and yet, manipulating intracellular calcium levels interferes with first-polar-body emission in mice and frogs. To determine the precise role of calcium signaling in polar body formation, we used live-cell imaging coupled with temporally precise intracellular calcium buffering. We found that BAPTA-based calcium chelators cause immediate depolymerization of spindle microtubules in meiosis I and meiosis II. Surprisingly, EGTA at similar or higher intracellular concentrations had no effect on spindle function or polar body emission. Using two calcium probes containing permutated GFP and the calcium sensor calmodulin (Lck-GCaMP3 and GCaMP3), we demonstrated enrichment of the probes at the spindle but failed to detect calcium increase during oocyte maturation at the spindle or elsewhere. Finally, endogenous calmodulin was found to colocalize with spindle microtubules throughout all stages of meiosis. Our results—most important, the different sensitivities of the spindle to BAPTA and EGTA—suggest that meiotic spindle function in frog oocytes requires highly localized, or nanodomain, calcium signaling.

scholarly journals Proline-rich tyrosine kinase2 is involved in F-actin organization during in vitro maturation of rat oocyte

Reproduction ◽  
2006 ◽  
Vol 132 (6) ◽  
pp. 859-867 ◽  
Author(s):  
Xiao-Qian Meng ◽  
Ke-Gang Zheng ◽  
Yong Yang ◽  
Man-Xi Jiang ◽  
Yan-Ling Zhang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Actin Filaments ◽  
Laser Scanning ◽  
Polar Body ◽  
Laser Scanning Microscopy ◽  
Meiotic Spindle ◽  
Confocal Laser ◽  
Cell Cortex ◽  
First Polar Body

Microfilaments (actin filaments) regulate various dynamic events during meiotic maturation. Relatively, little is known about the regulation of microfilament organization in mammalian oocytes. Proline-rich tyrosine kinase2 (Pyk2), a protein tyrosine kinase related to focal adhesion kinase (FAK) is essential in actin filaments organization. The present study was to examine the expression and localization of Pyk2, and in particular, its function during rat oocyte maturation. For the first time, by using Western blot and confocal laser scanning microscopy, we detected the expression of Pyk2 in rat oocytes and found that Pyk2 and Try402 phospho-Pyk2 were localized uniformly at the cell cortex and surrounded the germinal vesicle (GV) or the condensed chromosomes at the GV stage or after GV breakdown. At the metaphase and the beginning of anaphase, Pyk2 distributed asymmetrically both in the ooplasm and the cortex with a marked staining associated with the chromosomes and the region overlying the meiotic spindle. At telophase, Pyk2 was observed in the cleavage furrows in addition to its cortex and cytoplasm localization. The dynamics of Pyk2 were similar to that of F-actin, and this kinase was found to co-localize with microfilaments in several developmental stages during rat oocyte maturation. Microinjection of Pyk2 antibody demolished the microfilaments assembly and also inhibited the first polar body (PB1) emission. These findings suggest an important role of Pyk2 for rat oocyte maturation by regulating the organization of actin filaments.

scholarly journals Effect of taxol on first and second meiotic spindle formation in oocytes of the surf clam, Spisula solidissima

1986 ◽  
Vol 84 (1) ◽  
pp. 153-164
Author(s):  
R. Kuriyama
Keyword(s):  
Untreated Control ◽  
Polar Body ◽  
Immunofluorescence Staining ◽  
Meiotic Spindle ◽  
Spisula Solidissima ◽  
Microtubule Depolymerization ◽  
Spindle Formation ◽  
Surf Clam ◽  
First Polar Body ◽  
Spindle Microtubules

The effect of taxol on first and second meiotic spindle formation was examined in oocytes of the surf clam, Spisula solidissima, by immunofluorescence staining with anti-tubulin antibody. The first meiotic spindle appeared to form as in untreated control cells. However, the spindle did not migrate toward the periphery of taxol-activated oocytes, resulting in blockage of the formation of the first polar body. In spite of inhibited microtubule depolymerization and failure of spindle disappearance, the pole separation in telophase that is typical of this material began at the same time as in untreated cells. Polymerization of the second spindle microtubules onto the spindle persisting from the first meiosis led to the formation of a triple form of spindle connected at the poles of each other. The subsequent emergence of ring-shaped microtubule-containing structures in mature activated eggs was not affected by taxol. The mechanism of meiotic spindle formation thus seemsto be different from that in mitosis, where taxol has been shown to block spindle formation completely.

scholarly journals Relative Position of the Meiotic Spindle and Polar Body as a Marker of Oocyte Maturation Improves the Utilization and Pregnancy Rates

2021 ◽  
Author(s):  
Olga Tepla ◽  
Zinovij Topurko ◽  
Jaromir Masata ◽  
Simona Jirsová ◽  
Martina Moosova ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Pregnancy Rates ◽  
Meiotic Spindle ◽  
Utilization Rate ◽  
Mutual Position ◽  
Control Group ◽  
High Quality ◽  
First Polar Body

Abstract This research demonstrates how a mutual position of the human oocytes meiotic spindle (MS) and the first polar body (PB) correlates with the probability of obtaining high-quality embryos (utilization rates) and high pregnancy rates after intracytoplasmic sperm injection (ICSI). The quality of optically birefringent MS and the angle (α) between MS and PB (evaluated using polarizing microscopy), were used to indicate oocyte maturation and appropriate time for fertilization. In this study, 124 patients undergoing in vitro fertilization (IVF) whose oocytes were evaluated by MS visualization had a significantly higher clinical pregnancy rate (38% vs 26%) and utilization rate (54% vs 38%) when compared to the control group, using one standard IVF cycle without MS visualization. Significantly, in group of 79 patients > 35 years old, 34% became pregnant when α was evaluated and ICSI time adjusted to achieve the full oocyte maturation, compared to only 18% in the control group. The number of high-quality embryos in the MS visualized group was significantly higher compared to the control group, increasing the probability of pregnancy. Based on this research, we propose to incorporate monitoring the mutual position of MS and PB as a valid marker of embryo quality which can significantly improve pregnancy rate.

Platelet-activating factor acetylhydrolase 1B3 (PAFAH1B3) is required for the formation of the meiotic spindle during in vitro oocyte maturation

2018 ◽  
Vol 30 (12) ◽  
pp. 1739 ◽  
Author(s):  
L. T. M. Vandenberghe ◽  
B. Heindryckx ◽  
K. Smits ◽  
K. Szymanska ◽  
N. Ortiz-Escribano ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Close Association ◽  
Polar Body ◽  
Platelet Activating Factor ◽  
Germinal Vesicle ◽  
Meiotic Spindle ◽  
Intracellular Enzyme ◽  
Human Oocytes ◽  
First Polar Body

Platelet-activating factor (PAF) is a well-described autocrine growth factor involved in several reproductive processes and is tightly regulated by its hydrolysing enzyme, PAF acetylhydrolase 1B (PAFAH1B). This intracellular enzyme consists of three subunits: one regulatory, 1B1, and two catalytic, 1B2 and 1B3. PAFAH1B3 has remained uncharacterised until now. Here, we report that PAFAH1B3 is present during the different stages of the first meiotic division in bovine, murine and human oocytes. In these species, the PAFAH1B3 subunit was clearly present in the germinal vesicle, while at metaphase I and II, it localised primarily at the meiotic spindle structure. In cattle, manipulation of the microtubules of the spindle by nocodazole, taxol or cryopreservation revealed a close association with PAFAH1B3. On the other hand, disruption of the enzyme activity either by P11, a selective inhibitor of PAFAH1B3, or by PAFAH1B3 antibody microinjection, caused arrest at the MI stage with defective spindle morphology and consequent failure of first polar body extrusion. In conclusion, our results show that one of the catalytic subunits of PAFAH1B, namely PAFAH1B3, is present in bovine, murine and human oocytes and that it plays a functional role in spindle formation and meiotic progression during bovine oocyte maturation.

H1foo is essential for in vitro meiotic maturation of bovine oocytes

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 416-425 ◽  
Author(s):  
Yan Yun ◽  
Peng An ◽  
Jing Ning ◽  
Gui-Ming Zhao ◽  
Wen-Lin Yang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Linker Histone ◽  
Meiotic Maturation ◽  
Control Group ◽  
Bovine Oocyte ◽  
First Polar Body ◽  
Effective Sirnas ◽  
Bovine Oocytes

SummaryOocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

Maturation promoting factor in ascidian oocytes is regulated by different intracellular signals at meiosis I and II

Development ◽  
1996 ◽  
Vol 122 (7) ◽  
pp. 1995-2003 ◽  
Author(s):  
G.L. Russo ◽  
K. Kyozuka ◽  
L. Antonazzo ◽  
E. Tosti ◽  
B. Dale
Keyword(s):  
Kinase Activity ◽  
Phase 1 ◽  
Polar Body ◽  
Calcium Transients ◽  
Phase 2 ◽  
Phase 3 ◽  
First Polar Body ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Maturation Promoting Factor

Using the fluorescent dye Calcium Green-dextran, we measured intracellular Ca2+ in oocytes of the ascidian Ciona intestinalis at fertilization and during progression through meiosis. The relative fluorescence intensity increased shortly after insemination in a single transient, the activation peak, and this was followed by several smaller oscillations that lasted for approximately 5 minutes (phase 1). The first polar body was extruded after the completion of the phase 1 transients, about 9 minutes after insemination, and then the intracellular calcium level remained at baseline for a period of 5 minutes (phase 2). At 14 minutes postinsemination a second series of oscillations was initiated that lasted 11 minutes (phase 3) and terminated at the time of second polar body extrusion. Phases 1 and 3 were inhibited by preloading oocytes with 5 mM heparin. Simultaneous measurements of membrane currents, in the whole-cell clamp configuration, showed that the 1–2 nA inward fertilization current correlated temporally with the activation peak, while a series of smaller oscillations of 0.1-0.3 nA amplitude were generated at the time of the phase 3 oscillations. Biochemical characterization of Maturation Promoting Factor (MPF) in ascidian oocytes led to the identification of a Cdc2-like kinase activity. Using p13suc1-sepharose as a reagent to precipitate the MPF complex, a 67 kDa (67 × 10(3) Mr) protein was identified as cyclin B. Histone H1 kinase activity was high at metaphase I and decreased within 5 minutes of insemination reaching a minimum level during phase 2, corresponding to telophase I. During phase 3, H1 kinase activity increased and then decayed again during telophase II. Oocytes preloaded with BAPTA and subsequently inseminated did not generate any calcium transients, nonetheless H1 kinase activity decreased 5 minutes after insemination, as in the controls, and remained low for at least 30 minutes. Injection of BAPTA during phase 2 suppressed the phase 3 calcium transients, and inhibited both the increase in H1 kinase activity normally encountered at metaphase II and second polar body extrusion.

scholarly journals The roles of distinct Ca2+ signaling mediated by Piezo and inositol triphosphate receptor (IP3R) in the remodeling of E-cadherin during cell dissemination

2021 ◽  
Author(s):  
Alejandra J.H. Cabrera ◽  
Barry M Gumbiner ◽  
Young V Kwon
Keyword(s):  
Calcium Signaling ◽  
Intracellular Calcium ◽  
Cell Invasion ◽  
Adherens Junctions ◽  
Transformed Cells ◽  
Inositol Triphosphate ◽  
Intracellular Calcium Signaling ◽  
Cell Dissemination

Given the role of E-cadherin (E-cad) in holding epithelial cells together, the inverse relationship between E-cad levels and cell invasion has been perceived as a principle underlying the invasiveness of tumor cells. In contrast, our study employing the Drosophila model of cell dissemination demonstrates that E-cad is necessary for the invasiveness of RasV12-transformed cells in vivo. Drosophila E-cad/β-catenin disassembles at adherens junctions and assembles at invasive protrusions—the actin- and cortactin-rich invadopodia-like protrusions associated with breach of the extracellular matrix (ECM)—during cell dissemination. Loss of E-cad attenuates dissemination of RasV12-transformed cells by impairing their ability to compromise the ECM. Strikingly, the remodeling of E-cad/β-catenin subcellular distribution is controlled by two discrete intracellular calcium signaling pathways: Ca2+ release from endoplasmic reticulum via the inositol triphosphate receptor (IP3R) disassembles E-cad at adherens junctions while Ca2+ entry via the mechanosensitive channel Piezo assembles E-cad at invasive protrusions. Thus, our study provides molecular insights into the unconventional role of E-cad in cell invasion during cell dissemination in vivo and describes the discrete roles of intracellular calcium signaling in the remodeling of E-cad/β-catenin subcellular localization.

Role of microtubules and microtubule organizing centers on meiotic chromosome elimination in Sciara ocellaris

1997 ◽  
Vol 110 (6) ◽  
pp. 721-730 ◽  
Author(s):  
M.R. Esteban ◽  
M.C. Campos ◽  
A.L. Perondini ◽  
C. Goday
Keyword(s):  
Meiotic Chromosome ◽  
Chromosome Elimination ◽  
Male Meiosis ◽  
Meiotic Spindle ◽  
Monopolar Spindle ◽  
Meiotic Spindles ◽  
Cytoplasmic Microtubules ◽  
Spindle Microtubules ◽  
Meiosis I

Spindle formation and chromosome elimination during male meiosis in Sciara ocellaris (Diptera, Sciaridae) has been studied by immunofluorescence techniques. During meiosis I a monopolar spindle is formed from a single polar complex (centrosome-like structure). This single centrosomal structure persists during meiosis II and is responsible for the non-disjunction of the maternal X chromatids. During meiosis I and II non-spindle microtubules are assembled in the cytoplasmic bud regions of the spermatocytes. The chromosomes undergoing elimination during both meiotic divisions are segregated to the bud region where they associate with bundles of microtubules. The presence and distribution of centrosomal antigens in S. ocellaris meiotic spindles and bud regions has been investigated using different antibodies. gamma-Tubulin and centrin are present in the bud as well as in the single polar complex of first meiotic spindle. The results suggest that spermatocyte bud regions contain microtubule-organizing centres (MTOCs) that nucleate cytoplasmic microtubules that are involved in capturing chromosomes in the bud regions. The distribution of actin and myosin in the spermatocytes during meiosis is also reported.

Antioxidants Stimulate Germinal Vesicle Breakdown, But Inhibit Chromosome Formation And Spindle Assembly In Porcine Oocytes

2000 ◽  
Vol 6 (S2) ◽  
pp. 964-965
Author(s):  
Qing-Yuan Sun ◽  
Randall S. Prather ◽  
Heide Schatten
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Spindle Assembly ◽  
Mouse Oocyte ◽  
Germinal Vesicle Breakdown ◽  
Oocyte Meiosis ◽  
First Polar Body ◽  
Porcine Oocyte ◽  
Chromosome Formation

Mammalian oocytes are arrested at the diplotene stage of the first meiotic division. Release of oocytes from their follicles induces meiotic resumption characterized by germinal vesicle breakdown (GVBD), followed by the chromosome formation and metaphase I spindle organization and finally the extrusion the first polar body. Recently it was shown that cellpermeant antioxidants significantly inhibit spontaneous resumption of meiosis in mouse oocytes, which may indicate a role of oxygen radicals in oocyte maturation. The regulation of mouse oocyte meiosis resumption is different from that of large domestic animals in that GVBD is independent of Ca2+ and protein synthesis. The present study investigated the influence of two cell-permeant antioxidants, 2(3)-ter-butyl-4-hydroxyanisole (BHA) and nordihydroguaiaretic acid (NDGA), on porcine oocyte meiosis resumption, chromatin behavior and spindle assembly. Our findings revealed a different role of antioxidants in porcine oocyte meiosis resumption than in mouse oocyte maturation.

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