Visualization of second polar body chromosomes in fertilized and artificially activated mouse oocytes treated with okadaic acid

1992 ◽  
Vol 9 (6) ◽  
pp. 572-579 ◽  
Author(s):  
A. P. Dyban ◽  
P. De Sutter ◽  
D. Dozortsev ◽  
Y. Verlinsky
Keyword(s):  
Okadaic Acid ◽  
Polar Body ◽  
Mouse Oocytes ◽  
Second Polar Body

scholarly journals Cell cycle modification during the transitions between meiotic M-phases in mouse oocytes

1992 ◽  
Vol 102 (3) ◽  
pp. 457-467 ◽  
Author(s):  
J.Z. Kubiak ◽  
M. Weber ◽  
G. Geraud ◽  
B. Maro
Keyword(s):  
Kinase Activity ◽  
Polar Body ◽  
Metaphase Arrest ◽  
Phosphorylated Proteins ◽  
Mouse Oocytes ◽  
Sequence Of Events ◽  
M Phase ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Centrosomal Proteins

When metaphase II-arrested mouse oocytes (M II) are activated very soon after ovulation, they respond abortively by second polar body extrusion followed by another metaphase arrest (metaphase III, M III; Kubiak, 1989). The M II/M III transition resembles the natural transition between the first and second meiotic metaphases (M I/M II). We observed that a similar sequence of events takes place during these two transitions: after anaphase, a polar body is extruded, the microtubules of the midbody disappear rapidly and a new metaphase spindle forms. The MPM-2 monoclonal antibody (which reacts with phosphorylated proteins associated with the centrosome during M-phase) stains discrete foci of peri-centriolar material only in metaphase arrested oocytes; during both transitional periods, a diffuse staining is observed, suggesting that these centrosomal proteins are dephosphorylated, as in a normal interphase. However, the chromosomes always remain condensed and an interphase network of microtubules is never observed during the transitional periods. Incorporation of 32P into proteins increases specifically during the transitional periods. Pulse-chase experiments, after labeling of the oocytes in M phase with 32P, showed that a 62 kDa phosphoprotein band disappears at the time of polar body extrusion. Histone H1 kinase activity (which reflects the activity of the maturation promoting factor) drops during both transitional periods to the level characteristic of interphase and then increases when the new spindle forms. Both the M I/M II and M II/M III transitions require protein synthesis as demonstrated by the effect of puromycin. These results suggest that the two M-phase/M-phase transitions are probably driven by the same molecular mechanism.

Pleiotropic effect of okadaic acid on maturing mouse oocytes

Development ◽  
1991 ◽  
Vol 112 (4) ◽  
pp. 971-980 ◽  
Author(s):  
H. Alexandre ◽  
A. Van Cauwenberge ◽  
Y. Tsukitani ◽  
J. Mulnard
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatases ◽  
Chromatin Condensation ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Inhibitory Effect ◽  
Negative Control ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes

Okadaic acid (OA), a potent inhibitor of types 1 and 2A protein phosphatases, was shown recently to induce chromatin condensation and germinal vesicle breakdown (GVBD) in mouse oocytes arrested at the dictyate stage by dibutyryl cAMP (dbcAMP), isobutyl methylxanthine (IBMX) and 12,13-phorbol dibutyrate (PDBu). We confirm these results using IBMX and another phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and show that OA also bypasses the inhibitory effect of 6-dimethylaminopurine (6-DMAP). It has been concluded that protein phosphatases 1 and/or 2A (PP1, 2A), involved in the negative control of MPF activation, are thus operating downstream from both the protein kinase A and protein kinase C catalysed phosphorylation steps that prevent the breakdown of GV. Similar enzymatic activities are also able to counteract the general inhibition of protein phosphorylation. However, PP1 and/or PP2A are positively involved in the activation of pericentriolar material (PCM) into microtubule organizing centres (MTOCs). This explains the inhibitory effect of OA on spindle assembly. Finally, OA interferes with the integrity and/or function of actomyosin filaments. This results in a dramatic ruffling of the plasma membrane leading to the internalization of large vacuoles, the inhibition of chromosome centrifugal displacement and, consequently, the prevention of polar body extrusion.

Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring

Development ◽  
1995 ◽  
Vol 121 (8) ◽  
pp. 2397-2405 ◽  
Author(s):  
Y. Kimura ◽  
R. Yanagimachi
Keyword(s):  
Polar Body ◽  
Experimental Conditions ◽  
Male And Female ◽  
Testicular Spermatozoa ◽  
Mouse Oocytes ◽  
Foster Mothers ◽  
Sperm Nuclei ◽  
Second Polar Body ◽  
Normal Offspring ◽  
Gametic Imprinting

Genomic imprinting occurs in both male and female gametes during gametogenesis, but the exact time when imprinting begins and ends is unknown. In the present study we injected nuclei of testicular spermatozoa and round spermatids into mature mouse oocytes to see whether these nuclei are able to participate in syngamy and normal embryonic development. If the injected oocytes develop into normal fertile offspring, imprinting in the male germ cells used must have been completed by the time of injection. Ninety-two percent of mouse oocytes injected with testicular spermatozoa survived and 94% of these were fertilized normally (extrusion of the second polar body and formation of male and female pronuclei). When 44 two-cell embryos so created were transferred to 5 foster mothers, 24 (54.5%) developed into normal offspring. Unlike testicular spermatozoa, round spermatids could not activate the oocytes, and therefore the oocytes had to be activated artificially either before or after spermatid injection. The highest rate (77%) of normal fertilization was obtained when the oocytes were first activated by electric current, then injected individually with a single spermatid nucleus. When 131 two-cell embryos were transferred to 15 foster mothers, 37 (28.2%) reached full term. All but two grew into healthy adults. Thus, it would appear that gametic imprinting in mouse spermatogenic cells is completed before spermiogenesis begins. Under the experimental conditions employed, spermatid nuclei were less efficient than testicular sperm nuclei in producing normal offspring, but perhaps this was due to technical rather than inherent problems.

Activation of p90rsk during meiotic maturation and first mitosis in mouse oocytes and eggs: MAP kinase-independent and -dependent activation

Development ◽  
1996 ◽  
Vol 122 (6) ◽  
pp. 1957-1964 ◽  
Author(s):  
P. Kalab ◽  
J.Z. Kubiak ◽  
M.H. Verlhac ◽  
W.H. Colledge ◽  
B. Maro
Keyword(s):  
Kinase Activity ◽  
Polar Body ◽  
Ribosomal Subunit ◽  
Germinal Vesicle ◽  
Maximum Level ◽  
Meiotic Maturation ◽  
S6 Kinase ◽  
Mouse Oocytes ◽  
M Phase ◽  
Second Polar Body

Mitogen-activated protein kinases (MAPK) become activated during the meiotic maturation of oocytes from many species; however, their molecular targets remain unknown. This led us to characterize the activation of the ribosomal subunit S6 kinase of Mr 82 X 10(3) - 92 X 10(3) (p90rsk; a major substrate of MAPK in somatic cells) in maturing mouse oocytes and during the first cell cycle of the mouse embryo. We assessed the phosphorylation state of p90rsk by examining the electrophoretic mobility shifts on immunoblots and measured the kinase activity of immunoprecipitated p90rsk on a S6-derived peptide. Germinal vesicle stage (GV) oocytes contained a doublet of Mr 82 × 10(3) and 84 × 10(3) with a low S6 peptide kinase activity (12% of the maximum level found in metaphase II oocytes). A band of Mr 86 × 10(3) was first observed 30 minutes after GV breakdown (GVBD) and became prominent within 2 to 3 hours. MAPK was not phosphorylated 1 hour after GVBD, when the p90rsk-specific S6 kinase activity reached 37 % of the M II level. 2 hours after GVBD, MAPK became phosphorylated and p90rsk kinase activity reached 86% of the maximum level. The p90rsk band of Mr 88 × 10(3), present in mature M II oocytes when S6 peptide kinase activity is maximum, appeared when MAPK phosphorylation was nearly complete (2.5 hours after GVBD). In activated eggs, the dephosphorylation of p90rsk to Mr 86 X 10(3) starts about 1 hour after the onset of pronuclei formation and continues very slowly until the beginning of mitosis, when the doublet of Mr 82 X 10(3) and 84 X 10(3) reappears. A role for a M-phase activated kinase (like p34cdc2) in p90rsk activation was suggested by the reappearance of the Mr 86 X 10(3) band during first mitosis and in 1-cell embryos arrested in M phase by nocodazole. The requirement of MAPK for the full activation of p90rsk during meiosis was demonstrated by the absence of the fully active Mr 88 X 10(3) band in maturing c-mos −/− oocytes, where MAPK is not activated. The inhibition of kinase activity in activated eggs by 6-DMAP after second polar body extrusion provided evidence that both MAPK- and p90rsk-specific phosphatases are activated at approximately the same time prior to pronuclei formation.

Effective activation method with A23187 and puromycin to produce haploid parthenogenones from freshly ovulated mouse oocytes

Zygote ◽  
2000 ◽  
Vol 8 (3) ◽  
pp. 203-208 ◽  
Author(s):  
Hisayo Nakasaka ◽  
Shuji Yamano ◽  
Kenji Hinokio ◽  
Koji Nakagawa ◽  
Midori Yoshizawa ◽  
...  
Keyword(s):  
Polar Body ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Effective Activation ◽  
Control Groups ◽  
Mouse Oocytes ◽  
Activation Rate ◽  
Second Polar Body ◽  
And Control

Freshly ovulated mouse oocytes exposed to 5 mM calcium ionophore A23187 for 5 min and controls (not exposed) were cultured in TYH medium with 10 μg/ml puromycin (the puromycin group) or 2 mM 6-dimethylaminopurine (DMAP; the DMAP group) for 4 h. Among the controls, few oocytes were activated even if they were treated with DMAP or puromycin. In the oocytes exposed to A23187, in contrast, the activation rate, i.e. the rate of oocytes showing at least one pronucleus (PN) after the treatment, was 46.2% (48/104) in the DMAP group and 90.0% (118/131) in the puromycin group. Activation rate in the puromycin group was significantly higher than in the DMAP and control groups (p < 0.0001, respectively). Furthermore, 82.4% (108/131) of the activated oocytes in the puromycin group showed one PN with extrusion of the second polar body (PB). In the puromycin group, the DNA content of the PN of parthenogenones with 1PN2PB was half that of a set of metaphase II chromosomes. Chromosomal analysis was possible in 14 parthenogenones with 1PN2PB in the puromycin group. The parthenogenones possessed a normal set (n = 20) of haploid chromosomes. The combination of A23187 and puromycin proved to be an effective method of producing haploid parthenogenones.

33 ENUCLEATION OF PRE-ACTIVATED MOUSE OOCYTES INDUCED BY DEMECOLCINE, NOCODAZOLE, AND VINBLASTINE

2007 ◽  
Vol 19 (1) ◽  
pp. 135
Author(s):  
N. Costa-Borges ◽  
J. Santaló ◽  
E. Ibàñez
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Significant Proportion ◽  
Chi Square ◽  
Microtubule Stabilization ◽  
Mouse Oocytes ◽  
Antimitotic Drugs ◽  
Ultraviolet Illumination ◽  
Second Polar Body ◽  
Activation Rates

Demecolcine-induced enucleation has been previously used to prepare developmentally competent enucleated mouse and bovine cytoplasts for nuclear transfer (Gasparrini et al. 2003 Biol. Reprod. 68, 1259–1266; Fischer-Russell et al. 2005 Mol. Reprod. Dev. 72, 161–170). The approach is technically simple, but the proportion of pre-activated oocytes that extrude all of the chromatin within the second polar body (PB) after exposure to demecolcine is relatively low, especially in the mouse (20%). This study was designed to explore the potential of other antimitotic drugs (nocodazole and vinblastine), besides demecolcine, to induce enucleation of mouse oocytes and to characterize the morphological progression of the treated oocytes after drug removal. Metaphase II (MII) oocytes were collected from cytochalasin D-1 (CD-1) females (6–12 weeks old) at 16 h post-hCG, activated in 7% ethanol (for a fast release from MII arrest) for 5 min and immediately treated for 15, 30, or 60 min with demecolcine (DEM, 0.4 �g mL-1), nocodazole (NOC, 0.3 �g mL-1), or vinblastine (VIN, 0.1 �g mL-1), prepared in calcium-free KSOM containing 10 mM strontium. Then, the oocytes were cultured in drug-free medium for up to 2 h, 6 h, or 20 h post-activation (p.a.) and fixed in a microtubule stabilization buffer-extraction fixative. A triple-labelling protocol for microtubules, microfilaments, and chromatin was used to analyze oocytes (approximately 60 per treatment) by fluorescence microscopy. Results were statistically analyzed by chi-square. At 2 h p.a., the highest rates of enucleation were achieved when pre-activated oocytes were treated with VIN (63.8%) or NOC (41.9%) for 15 min or with DEM (66.1%) for 30 min. Although antimitotic treatments did not affect activation rates (91.8–100%), a significant proportion of DEM- (19.6%) and of VIN-treated (15.5%) oocytes failed to complete second PB extrusion when compared to control (0%) or NOC-treated (4.8%) oocytes. From the total of the enucleated oocytes, 11.5%, 24.3%, and 29.7% had an incomplete second PB extrusion in NOC, VIN, and DEM groups, respectively, and therefore were classified as partially enucleated. Further culture of oocytes after drug withdrawal resulted in 100% of activated oocytes having a completely extruded second PB in all groups by 6 h p.a. and resulted in a significant and similar decrease in enucleation rates for all treatments by 6 h (20.3–34.9%) and 20 h p.a. (10.2–16.1%). This decrease might be caused by the reintegration of the chromosomes into the oocyte after incomplete second PB extrusion, or by re-fusion of second PBs to enucleated oocytes. Thus, our results show that both VIN and NOC, in addition to DEM, can be successfully applied to produce enucleated mouse cytoplasts, omitting the potentially harmful step (staining and ultraviolet illumination) of the traditional enucleation method. However, removal of the second PB at 2 h p.a. is recommended in order to achieve an irreversible oocyte enucleation. It remains to be demonstrated if the cytoplasts prepared with VIN or NOC are as competent as those prepared by DEM to support embryo development to term after being reconstructed by nuclear transfer.

scholarly journals Effects of EDTA saturated with Ca2+ (Ca-EDTA) on pig, bovine and mouse oocytes at the germinal vesicle stage during maturation culture and the involvement of chelation of Zn2+ in pronuclear formation induction by Ca-EDTA

Reproduction ◽  
2002 ◽  
pp. 235-240 ◽  
Author(s):  
T Azuma ◽  
T Kondo ◽  
S Ikeda ◽  
H Imai ◽  
M Yamada
Keyword(s):  
Polar Body ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown ◽  
Parthenogenetic Activation ◽  
Mouse Oocytes ◽  
Maturation Medium ◽  
Second Polar Body ◽  
Pronuclear Formation ◽  
Bovine Oocytes

EDTA saturated with Ca(2+), Fe(3+) or Cu(2+) can induce parthenogenetic activation of pig oocytes at the germinal vesicle stage, whereas EDTA saturated with Zn(2+), which is unable to chelate Zn(2+), does not, indicating that chelation of Zn(2+) with EDTA saturated with Ca(2+) (Ca-EDTA) in maturing pig oocytes plays a pivotal role in the induction of parthenogenetic activation of oocytes. In the present study, the involvement of Zn(2+) chelation in the induction of parthenogenetic activation of pig oocytes at the germinal vesicle stage was confirmed first by examining the effects of concomitant addition of Zn(2+), Cu(2+) or Ni(2+) at various concentrations together with 1 mmol Ca-EDTA l(-1) to the maturation medium. The titration experiments revealed that the pronuclear formation induced by 1 mmol Ca-EDTA l(-1) was completely inhibited by the addition of > 30 micromol Zn(2+) l(-1) to the medium, but not by the addition of Cu(2+) and Ni(2+) at any concentration examined. Second, bovine and mouse oocytes at the germinal vesicle stage were cultured in medium with or without 1 mmol Ca-EDTA l(-1) for 48 h to examine the effects of Ca-EDTA treatment on these oocytes during maturation culture. Most (70-86%) of the bovine oocytes that underwent germinal vesicle breakdown matured to the MII stage via the MI phase, regardless of whether Ca-EDTA was present for the first 24 h of culture. However, 61% of oocytes that had been cultured with Ca-EDTA for 48 h formed a pronucleus without a second polar body, whereas oocytes cultured in the absence of Ca-EDTA were not observed to form a pronucleus at any time during culture. However, even when mouse oocytes at the germinal vesicle stage were cultured for up to 48 h in maturation medium containing Ca-EDTA, pronuclear formation was not observed. Finally, when bovine oocytes that had been cultured with 1 mmol Ca-EDTA l(-1) for 48 h from the germinal vesicle stage were cultured further in medium without Ca-EDTA that was supplemented with 5% fetal calf serum, only 26% of the oocytes developed to the cleaved stage, and none could develop further.

scholarly journals PAR-1 and the microtubule-associated proteins CLASP2 and dynactin-p50 have specific localisation on mouse meiotic and first mitotic spindles

Reproduction ◽  
2005 ◽  
Vol 130 (3) ◽  
pp. 311-320 ◽  
Author(s):  
Catherine A Moore ◽  
Magdalena Zernicka-Goetz
Keyword(s):  
Polar Body ◽  
Model Systems ◽  
Regulatory Proteins ◽  
Meiotic Spindle ◽  
Mitotic Spindles ◽  
Microtubule Associated Proteins ◽  
Mouse Oocytes ◽  
Polar Bodies ◽  
Associated Proteins ◽  
Second Polar Body

The site of second meiotic division, marked by the second polar body, is an important reference point in the early mouse embryo. To study its formation, we look at the highly asymmetric meiotic divisions. For extrusion of the small polar bodies during meiosis, the spindles must be located cortically. The positioning of meiotic spindles is known to involve the actin cytoskeleton, but whether microtubules are also involved is not clear. In this study we investigated the patterns of localisation of microtubule regulatory proteins in mouse oocytes. PAR-1 is a member of the PAR (partitioning-defective) family with known roles in regulation of microtubule stability and spindle positioning in other model systems. Here we show its specific localisation on mouse meiotic and first mitotic spindles. In addition, the microtubule-associated proteins CLASP2 (a CLIP associating protein) and dynactin-p50 are found on kinetochores and a subset of microtubule-organising centres. Thus we show specific localisation of microtubule regulatory proteins in mouse oocytes, which could indicate roles in meiotic spindle organisation.

scholarly journals Ral GTPase is essential for actin dynamics and Golgi apparatus distribution in mouse oocyte maturation

Cell Division ◽  
2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Ming-Hong Sun ◽  
Lin-Lin Hu ◽  
Chao-Ying Zhao ◽  
Xiang Lu ◽  
Yan-Ping Ren ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Mouse Oocyte ◽  
Actin Dynamics ◽  
Mouse Oocytes ◽  
Oocyte Meiosis ◽  
Ral Gtpase ◽  
Polar Body Extrusion ◽  
Cytoskeleton Dynamics

Abstract Background Ral family is a member of Ras-like GTPase superfamily, which includes RalA and RalB. RalA/B play important roles in many cell biological functions, including cytoskeleton dynamics, cell division, membrane transport, gene expression and signal transduction. However, whether RalA/B involve into the mammalian oocyte meiosis is still unclear. This study aimed to explore the roles of RalA/B during mouse oocyte maturation. Results Our results showed that RalA/B expressed at all stages of oocyte maturation, and they were enriched at the spindle periphery area after meiosis resumption. The injection of RalA/B siRNAs into the oocytes significantly disturbed the polar body extrusion, indicating the essential roles of RalA/B for oocyte maturation. We observed that in the RalA/B knockdown oocytes the actin filament fluorescence intensity was significantly increased at the both cortex and cytoplasm, and the chromosomes were failed to locate near the cortex, indicating that RalA/B regulate actin dynamics for spindle migration in mouse oocytes. Moreover, we also found that the Golgi apparatus distribution at the spindle periphery was disturbed after RalA/B depletion. Conclusions In summary, our results indicated that RalA/B affect actin dynamics for chromosome positioning and Golgi apparatus distribution in mouse oocytes.

Triphenyltin chloride induces spindle microtubule depolymerisation and inhibits meiotic maturation in mouse oocytes

2014 ◽  
Vol 26 (8) ◽  
pp. 1084 ◽  
Author(s):  
Yu-Ting Shen ◽  
Yue-Qiang Song ◽  
Xiao-Qin He ◽  
Fei Zhang ◽  
Xin Huang ◽  
...  
Keyword(s):  
Polar Body ◽  
Meiotic Maturation ◽  
Dependent Manner ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes ◽  
First Polar Body ◽  
Triphenyltin Chloride ◽  
Dose Dependent

Meiosis produces haploid gametes for sexual reproduction. Triphenyltin chloride (TPTCL) is a highly bioaccumulated and toxic environmental oestrogen; however, its effect on oocyte meiosis remains unknown. We examined the effect of TPTCL on mouse oocyte meiotic maturation in vitro and in vivo. In vitro, TPTCL inhibited germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) in a dose-dependent manner. The spindle microtubules completely disassembled and the chromosomes condensed after oocytes were exposed to 5 or 10 μg mL–1 TPTCL. γ-Tubulin protein was abnormally localised near chromosomes rather than on the spindle poles. In vivo, mice received TPTCL by oral gavage for 10 days. The general condition of the mice deteriorated and the ovary coefficient was reduced (P < 0.05). The number of secondary and mature ovarian follicles was significantly reduced by 10 mg kg–1 TPTCL (P < 0.05). GVBD decreased in a non-significant, dose-dependent manner (P > 0.05). PBE was inhibited with 10 mg kg–1 TPTCL (P < 0.05). The spindles of in vitro and in vivo metaphase II oocytes were disassembled with 10 mg kg–1 TPTCL. These results suggest that TPTCL seriously affects meiotic maturation by disturbing cell-cycle progression, disturbing the microtubule cytoskeleton and inhibiting follicle development in mouse oocytes.

Sign in / Sign up

Export Citation Format

Share Document