Activation of in vitro matured pig oocytes by combined treatment of ethanol and cycloheximide

Activation of meiosis in oocytes by artificial means is important in studies of oocyte function. In pigs, it seems that treatment with ethanol alone is inadequate for efficient activation of oocytes. Data collected in cattle, suggested that addition of a protein synthesis inhibitor increased the effectivness of ethanol for oocyte activation.We investigated the combined effects of exposure to ethanol and to the protein synthesis inhibitor cycloheximide, on activation of in vitro-matured pig oocytes. Treatment with ethanol alone (concentrations 0, 5, 7 and 10 %) for intervals of up to 3 minutes resulted in very limited activation rates (max. 15%). A culture of IVM pig oocytes with cycloheximide alone (10 μg/ml) for 24 hours did not induce oocyte activation either. However, exposure of IVM pig oocytes to 7 and 10 % ethanol followed by culture with cyloheximide substantially increased the activation rate. A maximal activation rate (over 80%) was observed when oocytes were treated with 10% ethanol for 1 min and subsequently cultured with cycloheximide.     

Parthenogenetic development of bovine oocytes matured in vitro for 24 hr and activated by ethanol and cycloheximide

This research was undertaken to improve development of parthenogenetic embryos following various combined treatments of ethanol and cycloheximide. In Experiment 1 in vitro matured oocytes (IVM, 24 hr) were treated with 7% ethanol for 5 min followed by incubation in 10 μg/ml cycloheximide in Medium 199 for 0 (control), 5, 10, and 20 hr. Development to 2–8 cells following culture for 3 days was similar among treated groups (32–41%; P > 0.05), which was higher than that of controls (6%; P < 0.05). Experiment 2 compared pre-ethanol exposures for 0, 1, 2.5, and 5 min, followed by 5 hr cycloheximide treatment on activation development. One- to 5-min groups resulted in 42–44% cleavage contrasted to 1–12% for controls (P < 0.05). Experiment 3 examined the effect on oocyte development of ethanol and different concentrations of cycloheximide (0, 1, 5, and 10 μg/ml). Cleavage to 2–8 cells was similar among the 5 and 10 μg/ml cycloheximide groups (36% and 42%, P > 0.05) but lower (P < 0.05) for the 1 μg/ml group (24%) and the controls (2–13%). When 5 μg/ml cycloheximide was used (Experiment 4), pre-exposure to ethanol (1, 2.5, and 5 min) resulted in more oocytes cleaved (38–41%) than in the cycloheximide alone group (0%) or the control (0%, P < 0.05). Experiment 5 tested blastocyst development of the activated oocytes with or without cytochalasin B treatment. Oocytes developed to blastocyts were 0%, 14%, 3%, and 3% (P < 0.05), respectively, for control, treatment with ethanol and cycloheximide in the presence, or absence of cytochalasin B, or electrical pulse plus cycloheximide. In conclusion, the combined ethanol and cycloheximide treatment supported high rates of parthenogenetic development using 24 hr IVM bovine oocytes. Blastocyst rate was significantly higher when cytochalasin B was added to the combined activation regimen. © 1994 Wiley-Liss, Inc.     

Nuclear dynamics of parthenogenesis of bovine oocytes matured in vitro for 20 and 40 hours and activated with combined ethanol and cycloheximide treatment

Bovine oocytes matured in vitro (IVM) for 20 hr vs. 40 hr were treated for activation with 7% ethanol in Dulbecco's phosphate-buffered saline for 5 min followed by incubation in M199 + 7.5% fetal calf serum containing cycloheximide (10 μg/ml). TreatedIVM oocytes and the controls (no ethanol and cycloheximide exposures) were fixed after 0, 1, 2, 3, 4, 5, 7, 10, and 20 hr of incubation and stained 24 hr later with 1% acetoorcein to examine nuclear events. Different stages of nuclear development of the activated oocytes were identified on the basis of nuclear and chromosomal morphology. Pronuclear development was classified into four stages (PN I, II, III, and IV) according to pronuclear progression in chromatin decondensation, nucleoplasm appearance, and nuclear size. The results demonstrated that the combined activation treatment effectively drove the IVM oocytes, both young (20 hr) and aging (40 hr), out of metaphase arrest. The activation rates for young oocytes examined immediately after 0, 1, 2, 3, 4, 5, 7, 10, and 20 hr of incubation with cycloheximide were, respectively, 7%, 24%, 77%, 96%, 92%, 97%, 98%, 93%, and 98%. For aging oocytes (40 hr) the corresponding activation values at the same time intervals were 6%, 84%, 100%, 100%, 100%, 100%, 98%, 100%, and 100%, respectively. These values were significantly higher than those for the corresponding controls. The activated aging oocytes achieved peak activation response more rapidly than did young oocytes. In addition, nuclear events in aging oocytes proceeded faster than those in young ones. Spontaneous activation rates of the aging oocytes were also higher (6–57%) than those of the young ones (0–14%). © 1994 Wiley-Liss, Inc.     

Parthenogenetic development of porcine oocytes treated by ethanol, cycloheximide, cytochalasin B and 6-dimethylaminopurine

This study was carried out to investigate the various concentrations and exposure times of ethanol, one of many intracellular calcium elevating agents, and a sequential combination of ethanol (8%), cycloheximide (CHX, 10 microg/ml), cytochalasin B (CCB, 7.5 microg/ml) and 6-dimethylaminopurine (6-DMAP, 2 mM) to improve parthenogenetic activation and development of in vitro matured porcine oocytes. Cumulus-oocyte complexes (COCs) were matured in tissue culture medium (TCM) 199 for 44 h at 38.5 degrees C, 5% CO2 in air. Cumulus-free oocytes showing first polar body were activated by concentrations of 0, 5, 6, 7, 8, 9 and 10% ethanol for 10 min and exposure times of 0, 5, 8, 10, 12 and 15 min with 8% ethanol in HEPES buffered (25 mM) NCSU-23 medium. Also, oocytes were activated with the NCSU-23 medium containing 8% ethanol for 10 min. After that, oocytes were incubated in the NCSU-23 medium supplemented with CHX, CCB, 6-DMAP, CHX + CCB, CHX + 6-DMAP, CCB + 6-DMAP and CHX + CCB + 6-DMAP for 3h, respectively. Following activation, oocytes were transferred into the NCSU-23 medium containing 0.4% BSA for further culture of 20 and 144 h at 38.5 degrees C, 5% CO2 in air. The activation rates of oocytes were higher in 6, 7 and 8% ethanol concentrations compared with 0, 5, 9 and 10% ethanol concentrations. Significantly, more oocytes (29.3-33.7%) were activated in the exposure for 8, 10, 12 and 15 min than those in the exposure for 0 and 5 min, but there was no difference due to exposure to 8% ethanol for 8-15 min. Oocytes treated by chemical agents (40.5-70.5%) after exposure to ethanol significantly improved the rate of oocyte activation compared with ethanol alone (31.2%). The percentage of cleaved oocytes was higher in the ethanol+CHX+CCB+6-DMAP treatment (66.4%) than in other treatments (24.9-57.6%). Also, the rate of blastocyst formation was higher in the ethanol+CHX+CCB+6-DMAP treatment (25.0%) than in other treatments (0.0-19.3%). In conclusion, the optimal activation treatment of ethanol exposure alone for the in vitro matured porcine oocytes was 8% ethanol for 8-15 min. Oocytes activated by 8% ethanol for 10 min and incubated in the NCSU-23 medium supplemented with CHX, CCB and 6-DMAP for 3 h were more efficient for parthenogenetic development of in vitro matured porcine oocytes.     

Brief exposure to cycloheximide prior to electrical activation improves in vitro blastocyst development of porcine parthenogenetic and reconstructed embryos

To investigate the effects of cycloheximide exposure before electrical activation of in vitro-matured porcine oocytes on the subsequent development of parthenogenetic embryos, cumulus-free mature oocytes were exposed to NCSU-23 medium containing cycloheximide (10 microg/mL) for 0, 5, 10, 20, 30 and 60 min, activated by electrical pulse treatment (1.5 kV/cm, 100 micros) and then cultured in PZM-3 for 7 days. To evaluate the effects of cycloheximide on the activation of nuclear transfer embryos, reconstructed embryos were electrically activated by two DC pulses (1.2 kV/cm, 30 micros) before or after exposure to cycloheximide. The reconstructed embryos were allocated into four groups: electrical pulse treatment alone (Ele); exposure to cycloheximide for 10 min followed by electrical activation (CHX+Ele); electrical activation followed by exposure to cycloheximide for 6h (Ele+CHX); exposure to cycloheximide for 10 min, followed by electrical activation and a further exposure to cycloheximide for 6h (CHX+Ele+CHX). The activated reconstructed embryos were cultured in PZM-3 for 6 days. Oocytes treated with 10 min exposure to cycloheximide followed by electrical activation had a significantly higher percentage of blastocyst formation compared to control oocytes and oocytes exposed for > or =30 min. In the reconstructed embryos, the blastocyst development rates of embryos exposed to cycloheximide (CHX+Ele, Ele+CHX and CHX+Ele+CHX) were significantly higher than those of the control group (Ele). Among the cycloheximide-treated groups, the CHX+Ele group had increased development rate and total blastocyst cell number, though these values were not significantly different from those observed in the other cycloheximide-treated groups. To evaluate the quality of NT embryos treated with cycloheximide, apoptosis in blastocysts was analyzed by TUNEL assay. The 10 min exposure to cycloheximide prior to electrical activation significantly reduced cell death compared with longer exposure to cycloheximide after electrical fusion. In conclusion, brief exposure to cycloheximide prior to electrical activation may increase the subsequent blastocyst development rates in porcine parthenogenetic and reconstructed embryos.     

Effects of different activation protocols on preimplantation development, apoptosis and ploidy of bovine parthenogenetic embryos

The objective of this study was to optimize the protocols for bovine oocytes activation through comparing the effectiveness of different treatments on the activation and subsequent development of oocytes and examining the effects of two combined activation treatments on the blastocyst apoptosis and ploidy. Cumulus-oocyte complexes (COCs) were recovered from abattoir-derived ovaries and matured in vitro. After maturation, cumulus-free oocytes were activated according to the experiment designs. Activated oocytes were cultured in vitro in modified synthetic oviductal fluid (mSOF) medium and assessed for pronuclear formation (15-16 h), cleavage (46-48 h) and development to the blastocyst stage. In Experiment 1, the matured oocytes were treated with single activation agents, including ionomycin (5 microM for 5 min), ethanol (7% for 7 min), calcium ionophore A23187 (5 microM for 5 min) or strontium (10mM for 5h). The pronuclear formation and cleavage rate were higher significantly in ionomycin (39.0 and 30.7%) and ethanol (41.5 and 28.1%) treatment alone compared to other treatments (9.7-25.2 and 11.3-23.7%, respectively, P<0.05). Very low blastocyst rates (3.9-5.3%) resulted which were not significantly different among treatments (P>0.05). For the combined activation treatment (Experiment 2), the same concentrations of ionomycin and ethanol as in Experiment 1 were used in combination with either 6-dimethylaminopurine (6-DMAP, 2.0 mM for 3 h) or cycloheximide (CHX)+cytochalasin B (CB, 10 microg/ml for 3 h). The pronuclear formation, cleavage rate, blastocyst rate and cell number of blastocyst were higher significantly (P<0.05) in ionomycin+6-DMAP treatment (67.1, 69.2, 28.0 and 91.3%, respectively) and ethanol+CHX+CB treatment (68.9, 70.2, 25.5 and 89.3%, respectively) compared to other treatments (11.7-58.1, 10.2-47.1, 1.5-24.2 and 34.2-62.7%, respectively). In Experiment 3, the parthenogenetic blastocysts produced by activation with ionomycin+6-DAMP and ethanol+CHX+CB and in vitro fertilized blastocysts (control group) were examined for apoptosis using a terminal deoxynucleotidyl transferase mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. The ethanol+CHX+CB treatment (7.0%) showed significantly lower blastocyst apoptosis index compared to ionomycin+6-DAMP treatment (9.1%, P<0.05). Furthermore, the chromosomal composition in the parthenotes embryos differed (P<0.05) among treatments. The percentage of haploid parthenotes was higher in ionomycin+6-DMAP treatment than ethanol+CHX+CB treatment. These results suggested that ethanol+CHX+CB treatment was more favorable protocol for parthenogenesis of bovine oocytes.     

Improved parthenogenetic development of vitrified-warmed bovine oocytes activated with 9% ethanol plus 6-DMAP

The objective was to compare various activation protocols on developmental potential of vitrified bovine oocytes. Bovine oocytes matured in vitro for 23 h were vitrified with EDFSF30 in open pulled straws. After warming, they were cultured in vitro for 1 h, followed by parthenogenetic activation. Vitrified-warmed oocytes had a morphologically normal rate similar to that of controls (nonvitrified oocytes cultured in vitro for 24 h; 98.6% vs. 100%, P > 0.05). When vitrified-warmed oocytes were first activated with 7% ethanol for 5 min and then incubated in 6-dimethylaminopurin (6-DMAP) for 4 h, cleavage and blastocyst rates were 41.2% and 23.2%, respectively, which were lower than those of controls (77.5% and 42.0%, P < 0.05). Subsequently, we varied the ethanol concentration to increase the effectiveness of parthenogenetic activation. When either 5%, 6%, 7%, 8%, 9%, 10%, or 11% ethanol alone (for 5 min) or in combination with 6-DMAP (4 h) was used to activate vitrified-warmed oocytes, cleavage rates ranged from 22.3% to 61.1% and blastocyst rates ranged from 1.1% to 30.6%. These rates were optimized when oocytes were treated with 9% ethanol plus 6-DMAP; this was verified in experiments evaluating other activation protocols with 9% ethanol, calcium ionophore A23187, or ionomycin alone, or in combination with DMAP or cycloheximide (CHX). In conclusion, the oocyte activation protocol affected developmental capacity of vitrified bovine oocytes; 9% ethanol (5 min) followed by 6-DMAP (4 h) promoted optimal parthenogenetic activation.     

Parthenogenetic activation of porcine oocytes by electric pulse and/or butyrolactone I treatment

The goal of the present research was to study the parthenogenetic activation of porcine oocytes following treatment with the specific cyclin-dependent kinase inhibitor butyrolactone I (BL I). In Experiment I, the effective dose of BL I was determined by the rates of the subsequent pronuclear formation in oocytes after the activation. In Experiment II, BL I was further tested alone or in combination with an electric pulse. The efficiency of the various treatments to induce activation and parthenogenetic development was examined. In Experiment III parthenogenetic development of activated oocytes in two different media was compared. Cleavage and blastocyst developmental rates were examined, and number of cells in the blastocysts was determined. Our results indicate that, in pig, the optimal activation dose for BL I was 150 microM; a combined electrical and BL I treatment resulted in superior cleavage rates compared to an electric pulse, 150 microM of BL I, or 200 microM of BL I alone (74%, 60%, 41%, and 42%, respectively; P < 0.05); and the rate of parthenogenetic development of activated oocytes to the blastocyst stage in mNCSU37 medium was significantly higher than that in Whitten's medium (59% vs. 5%, P < 0.05) and the resulting day-6 blastocysts had higher cell numbers (35.5 +/- 14.1 vs. 19.5 +/- 2.5). This activation protocol might be useful in porcine nuclear transfer experiments and for the generation of parthenogenetic fetuses.     

Differential effects of protein synthesis inhibitors on porcine oocyte activation

This study was designed to evaluate the effects of cycloheximide and puromycin on activation and protein synthesis of porcine oocytes. When matured oocytes were electrostimulated, then cultured in the presence of cycloheximide (5 μ/ml) for 6 or 24 hr, 92% of oocytes were activated as indicated by pronuclear formation, vs. 2.8% for untreated oocytes, 5.3% for oocytes not electrostimulated but cultured with cycloheximide, and 60.0% for those only electrostimulated. When cultured with L-[³⁵S]methionine in the presence of cycloheximide, puromycin (100 μg/ml), or no protein synthesis inhibitor for 24 hr, oocytes had mean radiolabeled incorporation rates of 36.5, 2.21, and 32.0 fmol/4 hr/oocyte, respectively. Thus, cycloheximide had little effect on protein synthesis after 24 hr of culture. A 1D-SDS PAGE showed that oocytes cultured with puromycin or cycloheximide are not activated, while electrostimulated oocytes are activated, as characterized by the conversion of a 25-kDa polypeptide to a 22-kDa polypeptide. The radiolabeling experiment was repeated, except that oocytes were cultured for 4 or 24 hr. At 4 hr, mean incorporation rates were lower in the cycloheximide group (2.34 fmol/4 hr/oocyte), but similar in the puromycin (15.7 fmol/4 hr/oocyte) and control groups (18.9 fmol/4 hr/oocyte). At 24 hr, the puromycin group (5.73 fmol/4 hr/oocyte) had a lower rate of incorporation, while the cycloheximide (22.6 fmol/4 hr/oocyte) and control (26.0 fmol/4 hr/oocyte) groups were similar. Cycloheximide was more effective earlier during culture, while puromycin was more effective later. When combined with ES, puromycin did have a higher rate (P = 0.10) of activation (87.8%) than with electrostimulation alone (73.0%). A final experiment evaluated the development to blastocyst after transfer to a ligated oviduct. Cycloheximide treatment in conjunction with an electric pulse did not increase the rate of compact morula or blastocyst formation. In conclusion, puromycin and cycloheximide have differential effects on protein synthesis, and although cycloheximide alone will not induce activation in porcine oocytes, it is very effective in generating activated oocytes in combination with electrostimulation. © 1995 Wiley-Liss, Inc.     

Surface ultrastructural alterations of bovine oocytes after parthenogenetic activation

Oocyte activation is a critical component of the current animal cloning scheme. This study was designed to examine surface characteristics of bovine oocytes by scanning electron microscopy (SEM) after activation by calcium ionophore A23187 (A23187) and electric pulse combined with cycloheximide (CHX) or 6-dimethylaminopurine (6-DMAP) treatments. In vitro matured (IVM) oocytes were activated then harvested at 0 to 19 hours after the onset of treatments for SEM processing. The zona pellucida (ZP) of untreated IVM oocytes exhibited an open mesh structure. The ZP surface showed little changes after A23187 alone, but dramatically changed to a less porous surface 3 hours after combined treatments with CHX or 6-DMAP. The vitelline membrane of IVM oocytes was covered with well-developed microvilli (MV). The MV became shorter (0.83 vs. 1.35 microm, p < 0.01) 8 hours after A23187 treatment alone. The vitelline membrane was altered in all oocytes examined 3 hours after incubation with A23187 plus CHX or 6-DMAP. A 1.5-fold increase in the diameter of MV in the CHX group and a higher incidence of large cytoplasmic protrusions (more than 1 microm width) in the 6-DMAP group were observed. After removal of inhibitors, the surface morphologies of the ZP and vitelline membrane were returned nearly to those of untreated IVM oocytes in both groups. The present study clearly showed that surface characteristics of the bovine oocyte were more profoundly changed by a combination of agents for parthenogenetic stimulation, and that the ultrastructural effects were reversible.     

Parthenogenetic activation of domestic cat oocytes using ethanol, calcium ionophore, cycloheximide and a magnetic field

The objective of this study was to evaluate parthenogenetic activation of domestic cat oocytes after being exposed to either ethanol, magnetic field, calcium ionophore A23187, or cycloheximide and a combination of these agents. We also wished to evaluate the usefulness of the magnetic field for oocyte activation. In vitro matured oocytes subjected to artificial activation were randomly assigned into eight groups according to activating agents: (1) 10% ethanol; (2) the magnetic field (slow-changing, homogenous magnetic field with low values of induction); (3) 10% ethanol plus magnetic field; (4) 10 microM calcium ionophore A23187; (5) 10 microM calcium ionophore A23187 plus magnetic field; (6) 10% ethanol and 10 microg/mL of cycloheximide; (7) 10% ethanol and 10 microg/mL of cycloheximide plus magnetic field; (8) oocytes were not exposed to any of the activating agents. After activation oocytes were stained with Hoechst 33258 and parthenogenetic activation was defined as oocytes containing pronuclei and second polar bodies or two to four or six nuclei (embryonic cleavage). The total activation rate by using different activation treatments was 40%. The addition of the magnetic field to ethanol or calcium ionophore treatments resulted in increased parthenogenetic activation rates from 47% to 75%, and from 19% to 48%, respectively (P<0.001). Instead, when the magnetic field was added to ethanol and cycloheximide treatment, activation rate decreased from 48% to 30%. Oocytes activated with magnetic field only gave the lowest activation rate (12%). We concluded that a magnetic field can be used as an activating agent, and the combination of ethanol and magnetic field is an effective method for domestic cat oocyte activation.     

In vitro development of vitrified buffalo oocytes following parthenogenetic activation and intracytoplasmic sperm injection

The objective of this study was to investigate the potential of swamp buffalo oocytes vitrified-warmed at the metaphase of the second meiotic cell division (M-II) stage to develop to the blastocyst stage after parthenogenetic activation (PA) or intracytoplasmic sperm injection (ICSI). In Experiment 1, we examined the effects of exposure time of oocytes to cryoprotectants (CPA) on their in vitro development after PA. In vitro matured (IVM) oocytes were placed in 10% dimethylsulfoxide (DMSO) + 10% ethylene glycol (EG) for 1 min and then exposed to 20% DMSO + 20% EG + 0.5 M sucrose for 30 s, 45 s or 60 s (1 min + 30 s, 1 min + 45 s and 1 min + 60 s groups, respectively). The oocytes were then exposed to warming solution (TCM199 HEPES + 20% FBS and 0.5M sucrose) for 5 min and then washed in TCM199 HEPES + 20% FBS for 5 min. IVM oocytes without CPA treatments served as a control group. The viability assessed by fluorescein diacetate (FDA) staining was 100% in all groups. The developmental rates after PA to the blastocyst stage between 1min+30s (16%) and control (26%) groups did not differ significantly, but they were significantly higher than those in 1 min + 45 s (10%) and 1 min + 60 s (2%) groups. In Experiment 2, we examined the effect of two CPA exposure times, 1 min + 30 s and 1 min + 45 s on the in vitro development after PA of oocytes vitrified by the microdrop method. The viabilities in vitrified 1 min + 30 s, 1 min + 45 s and the control (without CPA treatments) groups were not different (97%, 95% and 100%, respectively). The development of surviving oocytes to the blastocyst stage in the vitrified 1 min + 30 s group (8%) was significantly higher than that in the vitrified 1 min + 45 s group (4%) and significantly lower than those in control group (26%). In Experiment 3, we examined the effect of two CPA exposure times, 1 min + 30 s and 1 min + 45 s on in vitro development after ICSI of vitrified oocytes. Viabilities in vitrified oocytes among 1 min + 30 s, 1 min + 45 s and control groups were not different (96%, 91% and 100%, respectively). After ICSI, vitrified-warmed oocytes were activated and oocytes with the second polar body were cultured for 7 days. The development of ICSI oocytes to the blastocyst stage in the vitrified 1 min + 30 s group (11%) was significantly higher than that in the vitrified 1 min + 45 s (7%) group and significantly lower than those in control group (23%). In conclusion, our study demonstrated that the 1 min + 30 s CPA treatment regimen could yield the highest blastocyst formation rates after PA and ICSI for oocytes vitrified by the microdrop method.     

The suppression of fragmentation by stabilization of actin filament in porcine enucleated oocytes

A thorough understanding of the mechanism underlying fragmentation would contribute to the improvement of the developmental ability of reconstructed embryos after nuclear transfer. We conducted the present study to elucidate the influence of the nuclear transfer method on fragmentation of enucleated oocytes and the relationship between change in actin filament distribution and fragmentation. In Experiment 1, we examined activation rates of in vitro matured oocytes. These were 12.9% in maturation alone, 75.7% in electrical stimulation, and 57.9% in ethanol/cycloheximide treatment. In Experiment 2, we observed a higher rate of fragmentation (P < 0.05) in cultured oocytes that had been enucleated and electrically stimulated than in oocytes subjected to the other treatments (maturation alone, enucleation alone and enucleation plus ethanol/cycloheximide activation). In Experiment 3, we stained enucleated and electrically stimulated oocytes with rhodamine/phalloidin dye to show discontinuous distributions in the ooplasm of treated oocytes; oocytes in the other treatment groups showed homogenous distributions of actin filaments (AFs). In Experiment 4, we added cytochalasin B, an inhibitor of AF polymerization, to the culture medium, which prevented fragmentation of enucleated plus electrically stimulated oocytes (cytochalasin B, [+] 0.0%, [-] 60.7% at 24 h after treatment, P < 0.05). In Experiment 5, we investigated the relationship between fragmentation and alteration in AF distribution in enucleated plus electrically stimulated oocytes. At 0 h of culture, enucleated plus electrically stimulated oocytes showed discontinuous distributions of AFs, while nontreated oocytes showed homogenous AF distributions. At 24 and 48 h of culture, fragmentation proceeded in enucleated plus electrically stimulated oocytes and the discontinuous AF distribution diminished with time. In Experiment 6, we added hyaluronic acid (HA) to the culture medium, which suppressed fragmentation of enucleated plus electrically stimulated oocytes (HA, [+] 28.5%, [-] 66.4% at 24 h after treatment, P < 0.05). The results suggest that electrical stimulation induces a change in the AF distribution of oocytes, resulting in fragmentation, and that the addition of HA to the culture media is effective for the suppression of fragmentation.     

A separate procedure of fusion and activation in an ear fibroblast nuclear transfer program improves preimplantation development of bovine reconstituted oocytes

This study was conducted to examine whether preimplantation development of bovine ("HanWoo," Bos Taurus corcanae) oocytes reconstituted with ear fibroblasts could be improved by a modified procedure of fusion and activation. In Experiment 1, enucleated oocytes were reconstituted with ear fibroblast by a combined procedure of electric fusion and activation at either 24 or 28 hours after IVM. The 28 hours reconstitution yielded more blastocysts (4% vs. 21%, P = 0.0025) and higher ratio of blastocysts per 2-cell embryos (0.05 vs. 0.25, P = 0.003) than the 24 hours. In Experiment 2, enucleated oocytes were reconstituted by one of the three fusion and activation protocols; 1) a combined procedure of electric fusion and activation at 28 hours after IVM, 2) a combined procedure of electric fusion and chemical activation at 28 hours, and 3) a separate procedure of electric fusion at 24 hours and chemical activation at 28 hours. When compared two combined procedures, chemical activation with 5 microM ionomycin for 4 minutes did not promote embryo development and significantly reduced the fusion rate (42% vs. 53%, P = 0.0395). However, significant (P < 0.0113) increases in the development to 2-cell (90% vs. 70 to 74%) and blastocyst (47% vs. 7 to 13%) stages and in the ratio of blastocysts per 2-cell embryos (0.52 vs. 0.11 to 0.18) were obtained by a separate procedure of electric fusion and chemical activation than by the combined procedures. This separate protocol did not reduce the fusion rate compared with the combined procedures (58%). In conclusion, improved development of oocytes reconstituted with ear fibroblasts was achieved by undergoing a separate procedure of electric fusion and chemical activation 4 hours apart.     

Effect of 6-dimethylaminopurine on electrically activated in vitro matured porcine oocytes

The effect of the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), on the maturation promoting factor (MPF) activity, pronuclear formation, and parthenogenetic development of electrically activated in vitro matured (IVM) porcine oocytes was investigated. Oocytes were activated by exposure to two DC pulses, each of 1.5 kV/cm field strength and 60 microsec duration, applied 1 sec apart. In the first experiment, subsequent incubation with 2 or 5 mM 6-DMAP for 3 hr increased the incidence of blastocyst formation compared with no treatment, whereas incubation with 2 or 5 mM 6-DMAP for 5 hr did not. In the proceeding experiments, oocytes exposed to 6-DMAP were incubated with 2 mM of the reagent for 3 hr. Assaying histone H1 kinase activity in the second experiment revealed that the levels of active MPF in electrically activated oocytes treated with 6-DMAP were depleted more rapidly and remained depleted for longer compared with electrical activation alone. The kinetics of MPF activity following 6-DMAP treatment were similar to that found in inseminated oocytes in the third experiment. The effect of 6-DMAP was correlated with an increased incidence of parthenogenetic blastocyst formation. A fourth experiment was undertaken to examine the diploidizing effect of 6-DMAP. Electrically activated oocytes treated with 6-DMAP and cytochalasin B, either alone or in combination, displayed a higher incidence of second polar body retention compared with those that were untreated or treated with cycloheximide alone. After 6 days of culture in vitro, parthenotes exposed to 6-DMAP, either alone or in combination with cytochalasin B, formed blastocysts at a greater rate compared with those exposed to cytochalasin B alone, cycloheximide alone or no treatment. The combined 6-DMAP and cytochalasin B treatment induced the highest rate of blastocyst formation (47%), but the numbers of trophectoderm and total cells in these blastocysts were lower compared with those obtained following exposure to 6-DMAP alone. These results suggest that the increased developmental potential of 6-DMAP-treated parthenotes may be attributable to the MPF-inactivating effect of 6-DMAP, rather than the diploidizing effect of 6-DMAP.     

乙醇及6-DMAP对小鼠卵母细胞孤雌激活的研究

实验研究了乙醇、6-DMAP以及二者联合使用时对注射hCG后18小时采集的小鼠卵母细胞孤雌激活的效果。结果证明:(1)用5％的乙醇分别作用5和10分钟及10％的乙醇分别作用5和10分钟,小鼠卵母细胞的孤雌激活率分别为41.3％、63.7％、57.9％和85.6％。说明在一定范围内,随着乙醇浓度和作用时间的增加,小鼠卵母细胞孤雌激活率有上升的趋势。(2)用2mM 6-DMAP作用2、4和6小时,小鼠卵母细胞的孤雌激活率分别为 12.0％、25.0％和40.0％。说明随着6-DMAP作用时间的增加,小鼠卵母细胞的孤雌激活率有所升高。(3)用5％乙醇作用5分钟,再用含有2mmol/L 6-DMAP的培养液培养6小时,小鼠卵母细胞的孤雌激活率可达65.5％,明显高于单独使用5％乙醇作用5分钟或单独使用2mmol/L 6-DMAP作用6小时卵母细胞的孤雌激活率。(4)用10％的乙醇作用5分钟,再用含有2mmol/L 6-DMAP的培养液培养6小时,小鼠卵母细胞的孤雌激活率达到100％,远远高于单独使用10％乙醇作用5分钟或单独使用2mmol/L 6-DMAP作用6小时卵母细胞的孤雌激活率。(5)在单独使用乙醇刺激时,激活卵母细胞中直接卵裂(2-细胞)的比率随乙醇作用强度的增加而增加,最高达62.5％;但6-DMAP则抑制激活卵母细胞的直接卵裂,增加二原核卵的比例。     

Chemical activation of buffalo (Bubalus bubalis) oocytes by different methods: effects of aging on post-parthenogenetic development

The possibility of artificially inducing activation of MII buffalo oocytes may allow us to evaluate indirectly the quality of oocytes after in vitro maturation. The aim of this work was to compare buffalo embryo development after IVF and after chemical activation by two different agents. A further goal was to evaluate the effects of aging of oocytes on post-parthenogenetic and post-fertilization development. In Experiment 1 cumulus-oocyte complexes (COCs) were recovered from abattoir-derived ovaries and matured in vitro. After IVM the COCs were either fertilized in vitro (positive control) or activated with ethanol and ionomycin, both followed by immediate exposure to 6-diethylaminopurine (6-DMAP) for 4 h. In vitro culture (IVC) was carried out up to the blastocyst stage. In Experiment 2 COCs were matured in vitro for 18, 21, 24, 27 and 30 h before activation was triggered with ethanol, followed by 6-DMAP. In Experiment 3 COCs were fertilized in vitro at 18, 21, 24, 27 and 30 h post-maturation. Ethanol activation gave better results than the IVF control group, with higher cleavage rate (71.4 +/- 7.8 versus 55.8 +/- 5.8, respectively; P < 0.05) and a higher proportion of oocytes developing into morulae-blastocysts (32.6 +/- 6.5 versus 22.9 +/- 7.5, respectively; P < 0.05). Within the activation groups, ethanol supported the highest development in terms of cleavage (71.4 +/- 7.8 versus 59.4 +/- 10.7; P < 0.05) and morulae-blastocysts rate (32.6 +/- 6.5 versus 25.7 +/- 8.3; n.s.). It was also demonstrated that aging negatively affects post-parthenogenetic and post-fertilization development.     

Chemical activation of in vitro matured dromedary camel (Camelus dromedarius) oocytes: optimization of protocols

Experiments were conducted to study the efficiency of sequential treatments of ionomycine and ethanol combined with phosphorylation inhibitor (6-dimethylaminopurine) or the specific maturation promoting factor inhibitor (roscovitine) in inducing artificial activation in dromedary M-II oocytes. Cumulus oocyte complexes (COCs), collected from slaughterhouse ovaries were cultured at 38.5 degrees C in an atmosphere of 5% CO2 in air for 24-48 h. In experiment 1, the COCs were either fertilized in vitro or activated with 5 microM ionomycine for 5 min or 7% ethanol for 7 min, both followed by exposure to 6-diethylaminopurine or roscovitine for 4h. After 14-15 h of in vitro culture, the oocytes were fixed and stained with 1% aceto-orcein to evaluate their nuclear status. In experiment 2, the oocytes were activated in the same manner as in experiment 1 but were cultured for 7 days to evaluate their post-parthenogenetic development. In experiment 3, oocytes were exposed to the ionomycine for 2, 3, 4 or 5 min to evaluate the better exposure time while as in experiment 4, the oocytes matured for 28-48 h were activated to see the effect of aging on post-parthenogenetic development. Higher proportion (P<0.01) of oocytes was activated in ionomycine/6-DMAP and ionomycine/roscovitine groups when compared with ethanol/6-DMAP, ethanol/roscovitine and in vitro fertilized groups. However, there was no difference (P>0.05) in the proportion of oocytes activated with ethanol when compared with in vitro fertilized group. No significant difference was seen on the proportion of morula on day 7 of culture, however the development to blastocyst stage was higher (P<0.01) in ionomycine/6-DMAP and ionomycine/roscovitine when compared with ethanol/6-DMAP and ethanol/roscovitine treated oocytes. A higher proportion of oocytes reached blastocyst stage when they were exposed to ionomycine for 3 min but they were not significantly different from the others (P>0.05). The proportion of blastocysts obtained was higher (P<0.05) in oocytes activated after 28 h of maturation when compared with oocytes activated after 32, 36, 40, 44 and 48 h of maturation. In conclusion, a protocol for chemical activation of dromedary camel oocytes with ionomycine/6-DMAP is demonstrated and optimized in the present study for further use in the development of assisted reproductive techniques in this species.     

Activation of pig oocytes using calcium ionophore: effect of protein synthesis inhibitor cycloheximide

In vitro matured pig oocytes were activated using a combined treatment of calcium ionophore A 23187 with cycloheximide. The oocytes were exposed to ionophore (10, 25 or 50 microM) for 0.5, 1, 3, 5 or 7 min and then cultured with cycloheximide (0 or 10 microg/ml) for 6 h. Cycloheximide treatment significantly increased the activation rate of oocytes and the percentage of oocytes that were able to develop after activation. The highest activation rate was observed after treatment with 50 microM ionophore. The highest percentage of developing eggs was observed after combined treatment of ionophore (25 microM) with cycloheximide. The percentage of oocytes developing up to the morula and blastocyst stage was not significantly increased after cycloheximide treatment.