scholarly journals Effect of genetic background and activating stimulus on the timing of meiotic cell cycle progression in parthenogenetically activated mouse oocytes

Reproduction ◽  
2005 ◽  
Vol 129 (1) ◽  
pp. 27-38 ◽  
Author(s):  
Elena Ibáñez ◽  
David F Albertini ◽  
Eric W Overström
Keyword(s):  
Cell Cycle Progression ◽  
Polar Body ◽  
Ethanol Exposure ◽  
Meiotic Progression ◽  
Spindle Rotation ◽  
Activation Efficiency ◽  
Metaphase Stage ◽  
Nuclear Events ◽  
Second Polar Body ◽  
Activation Rates

With the aim of investigating the effects of oocyte genotype and activating stimulus on the timing of nuclear events after activation, oocytes collected from hybrid B6D2F1, inbred C57BL/6 and outbred CF-1 and immunodeficient nude (NU/+) females were activated using ethanol or strontium and fixed at various time-points. Meiotic status, spindle rotation and second polar body (PB2) extrusion were monitored by fluorescence microscopy using DNA-, microtubule- and microfilament-selective probes. Although activation efficiency was similar in all groups of oocytes, a significant percentage of CF-1 and NU/+ oocytes treated with ethanol and of C57BL/6 oocytes treated either with ethanol or strontium failed to complete activation and became arrested at a new metaphase stage (MIII) after PB2 extrusion. C57BL/6 oocytes also showed slower release from MII arrest but faster progression to telophase (TII) after ethanol exposure, and they exhibited the most rapid exit from TII under both activation treatments. Strontium caused delayed meiotic resumption, spindle rotation and PB2 extrusion, but rapid TII exit, in B6D2F1, CF-1 and NU/+ oocytes when compared with ethanol. Compared with all other strains, NU/+ oocytes were significantly slower in completing spindle rotation and PB2 extrusion, irrespective of the activating stimulus, and a significant decrease in activation rates and pace of meiotic progression was observed after strontium exposure. Thus, our findings demonstrated that the kinetics of meiosis resumption and completion, spindle rotation and PB2 extrusion following parthenogenetic activation depends on both genotype-specific factors and on the activation treatment applied.

Highly effective method of human oocyte activation

Zygote ◽  
1995 ◽  
Vol 3 (2) ◽  
pp. 157-161 ◽  
Author(s):  
Jacob Levron ◽  
Jacques Cohen ◽  
Steen Willadsen
Keyword(s):  
Polar Body ◽  
Human Oocyte ◽  
Isotonic Solution ◽  
Oocyte Activation ◽  
Effective Activation ◽  
Low Concentrations ◽  
Activation Efficiency ◽  
Calcium Magnesium ◽  
Second Polar Body ◽  
Activation Rates

SummaryFresh and aged unfertilised human oocytes were activated by electroporation and by exposure to isotonic solution of mannitol supplemented with low concentrations of calcium magnesium and chloride. Over 95% of the fresh oocytes were activated, all showing formation of one pronucleus and extrusion of the second polar body. Oocytes activated 1 and 2 days post-collection showed activation rates of 66.6% and 64.1%, respectively; however, the proportion of one-pronucleate oocytes in these groups was significantly lower (61.6% and 23.5%, respectively). There was no difference in the activation efficiency between the two activation modes. Twelve activated oocytes from the freshly collected group cleaved when left in culture. It is concluded that, in the human, a brief exposure to isotonic solution of mannitol with low concentrations of calcium, magnesium and chloride is a very effective activation stimulus.

A combination of calcium ionophore and puromycin effectively produces human parthenogenones with one haploid pronucleus

Zygote ◽  
2001 ◽  
Vol 9 (1) ◽  
pp. 83-88 ◽  
Author(s):  
Koji Nakagawa ◽  
Shuji Yamano ◽  
Hisayo Nakasaka ◽  
Kenji Hinokio ◽  
Midori Yoshizawa ◽  
...  
Keyword(s):  
Polar Body ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Control Group ◽  
Ionophore A23187 ◽  
Cleavage Rate ◽  
Parthenogenetic Activation ◽  
Second Polar Body ◽  
Activation Rates ◽  
Pronuclear Formation

Parthenogenetic activation with various combinations of the calcium ionophore A23187 and protein synthesis or phosphorylation inhibitors was investigated as a means of producing human parthenogenones with one haploid pronucleus. Unfertilised human aged oocytes exposed to 5 μM A23187 for 5 min were treated with 10 μg/ml puromycin (puromycin group, 46 oocytes) or 2 mM 6-dimethylaminopurine (DMAP group, 42 oocytes) for 5 h. Oocytes treated only with A23187 served as a control (control group, 40 oocytes). After washing the oocytes, they were incubated for up to 37 h. Evidence of activation (pronuclear formation) and cleavage was observed 18 h and 42 h after A23187 treatment, respectively. Activation rates in the puromycin and DMAP groups were significantly higher than in the control group (91% (42/46) and 77% (34/44) vs 20% (8/40), p < 0.05, respectively). In the puromycin group, 81% (34/42) of the activated oocytes showed one pronucleus with the second polar body (2ndPB), whereas none (0/34) of the activated oocytes in the DMAP group extruded the 2ndPB. The cleavage rate in the puromycin group was significantly lower than in the DMAP group (38% vs 68%, p < 0.05). The activated oocytes which had one pronucleus with the 2ndPB in the puromycin group showed a haploid set of chromosomes (10/13). In conclusion, the combination of A23187 and puromycin is effective for producing human parthenogenones with one haploid pronucleus.

scholarly journals Mammalian egg activation: from Ca2+ spiking to cell cycle progression

Reproduction ◽  
2005 ◽  
Vol 130 (6) ◽  
pp. 813-823 ◽  
Author(s):  
Keith T Jones
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Polar Body ◽  
Signal Transduction Pathway ◽  
Cyclin B1 ◽  
Egg Activation ◽  
Anaphase Promoting Complex ◽  
Sister Chromatids ◽  
Dependent Protein ◽  
Second Polar Body

Mammalian eggs arrest at metaphase of the second meiotic division (MetII). Sperm break this arrest by inducing a series of Ca2+spikes that last for several hours. During this time cell cycle resumption is induced, sister chromatids undergo anaphase and the second polar body is extruded. This is followed by decondensation of the chromatin and the formation of pronuclei. Ca2+spiking is both the necessary and solely sufficient sperm signal to induce full egg activation. How MetII arrest is established, how the Ca2+spiking is induced and how the signal is transduced into cell cycle resumption are the topics of this review. Although the roles of most components of the signal transduction pathway remain to be fully investigated, here I present a model in which a sperm-specific phospholipase C (PLCζ) generates Ca2+spikes to activate calmodulin-dependent protein kinase II and so switch on the Anaphase-Promoting Complex/Cyclosome (APC/C). APC/C activation leads to securin and cyclin B1 degradation and in so doing allows sister chromatids to be segregated and to decondense.

Intracytoplasmic sperm injection in the rat

Zygote ◽  
1998 ◽  
Vol 6 (2) ◽  
pp. 143-147 ◽  
Author(s):  
D. Dozortsev ◽  
T. Wakaiama ◽  
A. Ermilov ◽  
R. Yanagimachi
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Survival Rates ◽  
Experimental Conditions ◽  
Injection Techniques ◽  
Partial Zona Dissection ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Activation Rates ◽  
Pronuclear Formation

We applied intracytoplasmic sperm injection (ICSI) to the rat comparing three different sperm injection techniques: conventional setup with a sharp needle bearing a spike (method 1), combination of partial zona dissection (PZD) needle and blunt pipette (method 2) and piezo-injection using a blunt pipette (method 3). We also investigated the timing of sperm pronuclear formation after injection. Survival rates after injection were 8%, 24% and 71% for the methods 1, 2 and 3, respectively. All surviving oocytes formed pronuclei by about 6 h after injection. Although the survival and activation rates following sperm injection using piezo-injection were high, the incidence of normal fertilisation, as evidenced by second polar body extrusion and formation of two pronuclei, was only 10%. The vast majority of the zygotes were multinucleated, although most of them subsequently underwent cleavage. Fixation and staining of injected oocytes at different times after injection revealed that replacement of sperm nuclear protamines by histones takes place by 15 min after injection, sperm head swelling occurs within 0.5–1 h after injection and pronuclei become fully developed by 7 h after injection. Although the rate of normal fertilisation in the rat following ICSI was low under the present experimental conditions, the results indicated that direct ICSI using a piezo-driven pipette would be a potentially valuable method of producing rat offspring.

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.

Preservation of sperm within the mouse cauda epididymidis in salt or sugars at room temperature

Zygote ◽  
2010 ◽  
Vol 18 (3) ◽  
pp. 245-256 ◽  
Author(s):  
Tetsuo Ono ◽  
Eiji Mizutani ◽  
Chong Li ◽  
Teruhiko Wakayama
Keyword(s):  
Room Temperature ◽  
Polar Body ◽  
Methylation Status ◽  
Cost Effective ◽  
Oocyte Activation ◽  
Male Gametes ◽  
Effective Manner ◽  
Cell Embryo ◽  
Cauda Epididymidis ◽  
Second Polar Body

SummaryThe development of preservation techniques for male gametes at room temperature might allow us to store them in a simple and cost-effective manner. In this study, we studied the use of pure salt or sugar to preserve the whole cauda epididymidis, because it is known that food can be preserved in this way at room temperature for long periods. Mouse epididymides were placed directly in powdered salt (NaCl) or sugars (glucose or raffinose) for 1 day to 1 year at room temperature. Spermatozoa were recovered from the preserved organs after being rehydrated with medium and then isolated sperm heads were microinjected into fresh oocytes. Importantly, the oocyte activation capacity of spermatozoa was maintained after epididymal storage in NaCl for 1 year, whereas most untreated spermatozoa failed to activate oocytes within 1 month of storage. Pronuclear morphology, the rate of extrusion of a second polar body and the methylation status of histone H3 lysine 9 (H3K9me3) in those zygotes were similar to those of zygotes fertilized with fresh spermatozoa. However, the developmental ability of the zygotes decreased within 1 day of sperm storage. This effect led to nuclear fragmentation at the 2-cell embryo stage, irrespective of the storage method used. Thus, although the preserved sperm failed to allow embryo development, their oocyte activation factors were maintained by salt storage of the epididymis for up to 1 year at room temperature.

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.

The early development of haploid and aneuploid parthenogenetic embryos

Development ◽  
1975 ◽  
Vol 34 (3) ◽  
pp. 645-655
Author(s):  
Matthew H. Kaufman ◽  
Leo Sachs
Keyword(s):  
Early Development ◽  
Polar Body ◽  
Chromosome Counts ◽  
Cell Stage ◽  
Haploid Chromosome Number ◽  
Stage Of Development ◽  
Morula Stage ◽  
Second Polar Body ◽  
Similar Development ◽  
Parthenogenetic Embryos

The early development of parthenogenetically activated oocytes has been studied in C57BL × CBA-T6T6 (F1T6) translocation heterozygote mice and C57BL × CBA-LAC (F1LAC) mice. All F1T6 oocytes had either a quadrivalent or a univalent-trivalent configuration at meiosis I; no such chromosome configurations were observed in the F1LAC oocytes. At ovulation 36·5 % of the F1T6 oocytes had 19 or 21 chromosomes, whereas 97 % of the F1LAC had the normal haploid chromosome number of 20. After parthenogenetic activation, chromosome counts at metaphase of the first cleavage mitosis were made of the eggs with a single pronucleus following extrusion of the second polar body. These activated eggs had similar frequencies of 19, 20 and 21 chromosomes as had the oocytes at ovulation. The activated 1-cell eggs were transferred to the oviducts of pseudopregnant recipients and the embryos recovered 3 days later. At this stage of development, most of the F1T6 embryos with 19 chromosomes were no longer found, but the frequency of 21-chromosome embryos was similar to the frequency of 21-chromosome oocytes and activated eggs. There was a similar mean number of cells in the embryos with 20 and 21 chromosomes. The results indicate that nearly all the embryos with 19 chromosomes failed to develop, probably beyond the 2-cell stage, whereas oocytes with 21 chromosomes had a similar development to oocytes with 20 chromosomes up to the morula stage.

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