25 COMPARISON BETWEEN CHEMICALLY ASSISTED, CHEMICALLY INDUCED AND MECHANICAL ENUCLEATION OF MOUSE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 113
Author(s):  
N. Costa-Borges ◽  
S. González ◽  
J. Santaló ◽  
E. Ibáñez
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Cumulus Cell ◽  
Meiotic Spindle ◽  
Immunofluorescence Analysis ◽  
Mouse Oocytes ◽  
Polar Bodies ◽  
Chemically Induced ◽  
Spindle Microtubules ◽  
Bipolar Spindles

Chemically-assisted (AE) and chemically induced (IE) enucleation using demecolcine (DEM) or nocodazole (NOC) have proven to be technically simple procedures to prepare developmentally competent cytoplasts for nuclear transfer (NT) in different species. In this study, we analyzed AE and IE in mouse oocytes in terms of enucleation efficiency, amount of cytoplasmic volume removed and distribution of spindle-associated γ-tubulin after enucleation, and spindle morphology after cytoplast reconstruction by NT. Results were compared to the standard mechanical enucleation (ME) method. Outbred CD-1 and hybrid B6CBAF1 oocytes were collected at 13 to 16 h post-hCG. In AE experiments, oocytes were treated with either 0.4 μg mL–1 DEM or 0.3 μg mL–1 NOC in KSOM for 30 min. Protrusions induced in CD-1 (92.2%, n = 695) and B6CBAF1 (83.3%, n = 370) oocytes were aspirated by piezo-actuated micromanipulation, in H-KSOM with 2.5 μg mL–1 cytochalasin B and 0.05 m sucrose. In IE experiments, oocytes were preactivated with 7% ethanol for 5 min and treated with DEM or NOC in calcium-free KSOM containing 10 mm strontium. At 90 min postactivation (p.a.), completely- and partially-extruded second polar bodies (PBs) were mechanically aspirated. Enucleation efficiencies were higher than 90% both for AE (90.8%, n = 509 CD-1; 90.4%, n = 260 B6CBAF1) and IE methods (90.3%, n = 167 CD-1; 92.9%, n = 197 B6CBAF1), though they were significantly lower than those obtained for ME in nontreated CD-1 (98.4%; n = 126) or B6CBAF1 (100%, n = 498) oocytes. The amount of cytoplasmic volume removed in CD-1 oocytes was smaller in AE than in ME (2.1%, n = 35 and 3.9%, n = 30, respectively). In B6CBAF1 oocytes, used to compare IE (5.4%, n = 60) and ME (4.9%, n = 41), no differences were found. Volumes were calculated using the CellA software on images of cytoplasts and karyoplasts taken after enucleation. Even though both AE and IE methods avoided the removal of the oocyte spindle microtubules, spindle-associated γ-tubulin was eliminated from the cytoplasts generated by all 3 enucleation procedures, as confirmed by immunofluorescence analysis of the cytoplasts and the complementary karyoplasts produced. Finally, spindle morphology was examined in enucleated oocytes reconstructed by NT with a cumulus cell nucleus. Cytoplasts prepared by NOC-AE or NOC-IE displayed morphologically normal bipolar spindles by 2 h post-NT or 18 to 20 h post-activation (hpa), respectively, similar to cytoplasts prepared by ME. However, when DEM was used, microtubule repolymerization was slower and bipolar spindles could not be observed until 4 h post-NT (AE) or 22 to 24 hpa (IE). In conclusion, although enucleation rates are slightly higher for ME, AE and IE protocols allow oocyte enucleation without removal of the meiotic spindle, and a very small cytoplasm volume is eliminated during AE. Treatments with NOC and DEM are reversible, and cytoplasts produced by AE and IE can form morphologically normal spindles after NT, similar to those of cytoplasts produced by ME. MEC BIO 2006-11792; 2005-SGR00437; Portuguese FCT.

scholarly journals Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei

Reproduction ◽  
2001 ◽  
pp. 49-60 ◽  
Author(s):  
T Wakayama ◽  
R Yanagimachi
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Cumulus Cell ◽  
Blastocyst Stage ◽  
Cytochalasin D ◽  
Full Term ◽  
Oocyte Activation ◽  
Cell Nuclei ◽  
Reconstructed Embryos

Cloning methods are now well described and in almost routine use. However, the frequencies of production of live offspring from activated oocytes remain at < 3% and little is known about the factors that affect these frequencies. The effects of cytokinesis inhibitors, dimethylsulphoxide (DMSO) and the cell cycle of recipient cytoplasm on the cloning of mice were examined. Reconstructed oocytes, which were activated immediately after nucleus injection and cultured without cytochalasin B, developed into blastocysts at a frequency of 30--54% and into live cloned offspring at a frequency of 2--3%. Activated zygotes did not support development to full term after nuclear transfer. Reconstructed oocytes were activated 1--3 h after nuclear transfer and were exposed separately to three inhibitors of cytokinesis (cytochalasin B, cytochalasin D or nocodazole) to examine the toxicity of these inhibitors on cloning. All of the oocytes exposed to nocodazole-containing media formed many small pseudo-pronuclei, whereas with cytochalasin-containing media most of the activated oocytes formed only two pseudo-pronuclei. Despite such differences, 42--61% of reconstructed embryos developed to the morula-blastocyst stage and 1--3% developed to full term in all groups. Addition of 1% (v/v) DMSO to the activation medium significantly improved the frequency of development to the blastocyst stage and full term; however, this improvement did not lead to a higher success rate in the generation of live cloned offspring. These results show that activated mouse oocytes/zygotes are not effective cytoplasmic recipients with the methods described and that the lack of success of cloning is not due to inhibition of cytokinesis.

scholarly journals Specific deletion of Cdc42 does not affect meiotic spindle organization/migration and homologous chromosome segregation but disrupts polarity establishment and cytokinesis in mouse oocytes

2013 ◽  
Vol 24 (24) ◽  
pp. 3832-3841 ◽  
Author(s):  
Zhen-Bo Wang ◽  
Zong-Zhe Jiang ◽  
Qing-Hua Zhang ◽  
Meng-Wen Hu ◽  
Lin Huang ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Homologous Chromosome ◽  
Polar Body ◽  
Conditional Knockout ◽  
Meiotic Spindle ◽  
Spindle Positioning ◽  
Mouse Oocytes ◽  
Polar Bodies ◽  
And Migration ◽  
Specific Deletion

Mammalian oocyte maturation is distinguished by highly asymmetric meiotic divisions during which a haploid female gamete is produced and almost all the cytoplasm is maintained in the egg for embryo development. Actin-dependent meiosis I spindle positioning to the cortex induces the formation of a polarized actin cap and oocyte polarity, and it determines asymmetric divisions resulting in two polar bodies. Here we investigate the functions of Cdc42 in oocyte meiotic maturation by oocyte-specific deletion of Cdc42 through Cre-loxP conditional knockout technology. We find that Cdc42 deletion causes female infertility in mice. Cdc42 deletion has little effect on meiotic spindle organization and migration to the cortex but inhibits polar body emission, although homologous chromosome segregation occurs. The failure of cytokinesis is due to the loss of polarized Arp2/3 accumulation and actin cap formation; thus the defective contract ring. In addition, we correlate active Cdc42 dynamics with its function during polar body emission and find a relationship between Cdc42 and polarity, as well as polar body emission, in mouse oocytes.

F-actin is required for spindle anchoring and rotation in Xenopus oocytes: a re-examination of the effects of cytochalasin B on oocyte maturation

Zygote ◽  
1995 ◽  
Vol 3 (1) ◽  
pp. 17-26 ◽  
Author(s):  
David Lynn Gard ◽  
Byeong-Jik Cha ◽  
Amy Diane Roeder
Keyword(s):  
Cytochalasin B ◽  
Meiotic Spindle ◽  
The Novel ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Rhodamine Phalloidin ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Meiotic Spindles ◽  
Bipolar Spindles

SummaryWe used confocal immunofluorescence microscopy to examine spindle migration, morphology and orientation during the maturation of Xenopus oocytes, in the presence or absence of cytochalasin B (CB), an inhibitor of actin assembly. Treatment with CB during maturation (10–50 μg/ml beginning 0–3h prior to addition of progesterone) disrupted the normal organisation of the novel MTOC and transient microtubule array (MTOC-TMA complex) that serves as the immediate precursor of the first meiotic spindle, suggesting that F-actin plays an important role in the assembly or maintenance of this complex. However, CB treatment did not block translocation of the MTOC-TMA complex to the oocyte cortex, suggesting that MTOC-TMA translocation is not dependent on an actin-based mechanism. Bipolar spindles were observed in CB-treated oocytes fixed during both M1 and M2. However, rotation of the M1 and M2 spindles into an orientation orthogonal to the oocyte surface was inhibited by CB. Rhodamine-phalloidin revealed a concentration of F-actin at the site of M1 spindle attachment, further suggesting that cortical actin is required for anchoring and rotation of the meiotic spindles. Finally, the incidence of M1 monasters was significantly increased in CB-treated oocytes, suggesting that interactions between the nascent M1 spindle and cortex are dependent on F-actin.

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 Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs

Animals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 1034
Author(s):  
Joohyeong Lee ◽  
Eunhye Kim ◽  
Seon-Ung Hwang ◽  
Lian Cai ◽  
Mirae Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Glucuronic Acid ◽  
Cumulus Cell ◽  
Cortical Granule ◽  
Oocyte Diameter

This study aimed to examine the effects of treatment with glucuronic acid (GA) and N-acetyl-D-glucosamine (AG), which are components of hyaluronic acid (HA), during porcine oocyte in vitro maturation (IVM). We measured the diameter of the oocyte, the thickness of the perivitelline space (PVS), the reactive oxygen species (ROS) level, and the expression of cumulus cell expansion and ROS-related genes and examined the cortical granule (CG) reaction of oocytes. The addition of 0.05 mM GA and 0.05 mM AG during the first 22 h of oocyte IVM significantly increased oocyte diameter and PVS size compared with the control (non-treatment). The addition of GA and AG reduced the intra-oocyte ROS content and improved the CG of the oocyte. GA and AG treatment increased the expression of CD44 and CX43 in cumulus cells and PRDX1 and TXN2 in oocytes. In both the chemically defined and the complex medium (Medium-199 + porcine follicular fluid), oocytes derived from the GA and AG treatments presented significantly higher blastocyst rates than the control after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In conclusion, the addition of GA and AG during IVM in pig oocytes has beneficial effects on oocyte IVM and early embryonic development after PA and SCNT.

scholarly journals Cytochalasin B-induced triploidy in mouse oocytes fertilized in vitro

Reproduction ◽  
1976 ◽  
Vol 48 (2) ◽  
pp. 279-284 ◽  
Author(s):  
A. Niemierko ◽  
A. Komar
Keyword(s):  
Cytochalasin B ◽  
Mouse Oocytes

scholarly journals Kinetochore Fibers Are Not Involved in the Formation of the First Meiotic Spindle in Mouse Oocytes, but Control the Exit from the First Meiotic M Phase

1999 ◽  
Vol 146 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Stéphane Brunet ◽  
Angélica Santa Maria ◽  
Philippe Guillaud ◽  
Denis Dujardin ◽  
Jacek Z. Kubiak ◽  
...  
Keyword(s):  
Polar Body ◽  
Meiotic Spindle ◽  
Homologous Chromosomes ◽  
Mouse Oocytes ◽  
Sister Chromatids ◽  
M Phase ◽  
Continuous Presence ◽  
Set Up ◽  
Polar Body Extrusion ◽  
Reductional Division

During meiosis, two successive divisions occur without any intermediate S phase to produce haploid gametes. The first meiotic division is unique in that homologous chromosomes are segregated while the cohesion between sister chromatids is maintained, resulting in a reductional division. Moreover, the duration of the first meiotic M phase is usually prolonged when compared with mitotic M phases lasting 8 h in mouse oocytes. We investigated the spindle assembly pathway and its role in the progression of the first meiotic M phase in mouse oocytes. During the first 4 h, a bipolar spindle forms and the chromosomes congress near the equatorial plane of the spindle without stable kinetochore– microtubule end interactions. This late prometaphase spindle is then maintained for 4 h with chromosomes oscillating in the central region of the spindle. The kinetochore–microtubule end interactions are set up at the end of the first meiotic M phase (8 h after entry into M phase). This event allows the final alignment of the chromosomes and exit from metaphase. The continuous presence of the prometaphase spindle is not required for progression of the first meiotic M phase. Finally, the ability of kinetochores to interact with microtubules is acquired at the end of the first meiotic M phase and determines the timing of polar body extrusion.

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