Synergistic effect of ethanol and cycloheximide on activation of freshly matured bovine oocytes

Bovine follicular oocytes were collected from ovarian antral follicles (2 to 7 mm in diameter) from slaughtered cattle. They were matured in vitro (IVM) for 23 to 24 h and then activated. In Experiment 1, 4 concentrations of ethanol were compared. The activation rates of oocytes were 4, 12, 36 and 27%, respectively, following exposure for 7 min to 0, 5, 7 and 10% ethanol. In Experiment 2, 7% ethanol was tested with exposure times of 0, 5, 7.5 and 10 min, and 6, 32, 27 and 33% of the oocytes were activated, respectively. In Experiment 3 the synergistic effect of ethanol and electric pulse was compared within 4 treatments: A) 7% ethanol alone, B) electric pulse alone, C) ethanol first and then electric pulse treatment, and D) electric pulse first followed by ethanol exposure. Of the oocytes activated, 37, 31, 28 and 51%, respectively, were from Treatments A through D. In Experiments 4 and 5 the possible synergistic effect of ethanol and a protein synthesis inhibitor, cycloheximide, was studied within 4 treatments: A) parthenogenetic control with no activation treatment, B) ethanol alone, C) cycloheximide alone, and D) ethanol treatment followed by cycloheximide. The oocyte activation rates in Experiment 4 in Treatments A through D, respectively, were 9, 44, 43 and 84%. Corresponding values for development of oocytes to the 2 to 8-cell stage after culture for 3 d (Experiment 5) were 9, 20, 14 and 45%, respectively (P<0.05). In conclusion, exposure to 7% ethanol for 5 min followed by incubation with cycloheximide was the best activation treatment for bovine IVM oocytes.     

Effects of different activation treatments on fertilization of horse oocytes by intracytoplasmic sperm injection

The effects of four reagents on the activation and subsequent fertilization of equine oocytes, and the development of these after intracytoplasmic sperm injection, were investigated. Cumulus-oocyte complexes collected from equine ovaries obtained from an abattoir were matured in vitro for 40-44 h in TCM199 medium before being injected, when in metaphase II, with an immobilized stallion spermatozoon. The cumulus-oocyte complexes were then subjected to one of five activation treatments: (a) 10 micromol ionomycin l(-1) for 10 min; (b) 7% (v/v) ethanol for 10 min; (c) 100 micromol thimerosal l(-1) for 10 min; (d) 250 micromol inositol 1,4, 5-triphosphate l(-1) injection; and (e) no treatment (control). After 18-20 h further culture, the cumulus-oocyte complexes were assessed for activation by observing whether they had progressed through second anaphase-telophase and had formed a female pronucleus. The proportions of oocytes activated after each treatment were: 16/27 (59%) for ionomycin; 14/25 (56%) for ethanol; 22/28 (79%) for thimerosal; 15/27 (56%) for inositol 1,4,5-triphosphate; and 0/20 (0%) for the untreated controls. Thus, significantly more oocytes (P < 0.05) were activated by treatment with thimerosal than by the other four treatments. The proportions of oocytes that cleaved to the two-cell stage at 24-30 h after sperm injection in the groups treated with ionomycin, ethanol and thimerosal were 7/20 (35%), 5/19 (26%) and 11/23 (48%), respectively. No cleavage was observed in any of the control oocytes or those treated with inositol 1,4, 5-triphosphate. Furthermore, evidence of normal fertilization was observed in 2/7 (29%), 2/5 (40%) and 7/11 (64%) of the oocytes treated with ionomycin, ethanol and thimerosal, respectively. These results demonstrated that: (a) it is possible to activate equine oocytes with the chemical stimulants, ionomycin, ethanol, thimerosal and inositol 1,4,5-triphosphate; (b) thimerosal is more effective than the other three reagents in facilitating both meiotic activation and normal fertilization of equine oocytes; and (c) chemical activation may also stimulate parthenogenetic cleavage of oocytes without concurrent changes in the head of the spermatozoon.     

Cleavage development of bovine oocytes fertilized by sperm injection

Whole in vitro capacitated bovine spermatozoa were microinjected directly into the ooplasm of in vitro matured bovine oocytes in order to determine whether oocytes fertilized by sperm injection could undergo normal pronuclear formation and cleavage development. Immature oocytes recovered from follicles (2-5 mm) of unstimulated ovaries were cultured for 24-25 h in modified TCM 199 medium supplemented with heat-treated day 20 cow serum, luteinizing hormone (LH), and estradiol 17-B. In vitro capacitated, frozen-thawed spermatozoa were injected into the ooplasm, and the injected oocytes were cultured for an additional 24-28 h. Twenty-one percent (21/101) of the sperm-injected oocytes contained a sperm within the ooplasm; however, only 2% (2/101) cleaved. The remaining oocytes either did not contain a sperm or had degenerated. After oocyte activation induced by a 5 min incubation in 1 microM A23187, sperm nuclear decondensation occurred in the A23187-activated, injected oocytes but not in the unactivated, injected controls (37% vs. 0% after 3 h). Those injected, activated oocytes that contained a male pronucleus also exhibited a female pronucleus and second polar body. Furthermore, a significantly higher number (28%, 6/21) of the injected, activated oocytes cleaved to a two- to four-cell stage after 48 h than did the injected, unactivated oocytes (4%). These results indicate that, unlike hamster and rabbit oocytes, bovine oocytes are not sufficiently stimulated by the injection procedure to complete meiosis, but, upon activation by calcium ionophore, they will undergo normal-appearing cleavage development following fertilization by sperm injection.     

Development of pronuclei in pig oocytes activated by a single electric pulse.

Pig oocytes were matured in vitro in a modified M-199 medium for 44 h, subjected to electrical stimulation and scored for activation 6 h later. Sham pulsed oocytes, exposed to electroporation medium and an a.c. field, did not develop the female pronucleus any more frequently than occurs spontaneously (8.3% within 50 h of culture). However, a single d.c. pulse proved extremely efficient in activating pig oocytes. Pulses of 0.75-1.65 kV cm-1 lasting 30 or 100 microseconds activated at least 90% of matured oocytes. The developmental pathway taken by the activated oocytes depended on the parameters of the pulse. The lowest effective stimulation (0.45 and 0.60 kV cm-1 for 30 microseconds) frequently produced oocytes that remained in pre-pronuclear stages of activation (29.4 and 42.3%, respectively). Extrusion of the second polar body and creation of one pronucleus was the most frequent type of activation (in up to 88.2% among the activated oocytes). The strongest stimulations used (1.05-1.65 kV cm-1 for 100 microseconds) often yielded oocytes that failed to extrude the second polar body and formed two or more pronuclei (up to 56.3%). Under optimal stimulation (0.75 kV cm-1), the activated oocytes proceed synchronously to interphase of the first mitotic division. Anaphase II is reached within 30 min and telophase Ii at 1 h after application of the pulse. The second polar body is extruded about 2 h after activation. Well-defined swelling pronuclei were found in oocytes 5-6 h after activation. The relationship between the stage of oocyte maturation and susceptibility to activation was investigated. The period of culture in which the oocytes develop the activation competence (32-36 h of culture) overlapped with the period in which the oocytes complete meiosis (28-38 h). This suggests that ageing in meiotic arrest is not essential for pig oocytes to become activated by electric pulses. Activation of pig oocytes was accompanied by release of cortical granules. In sections of control (metaphase II) oocytes, an average of 7.3 intact cortical granules per 10 microns of overlying cytoplasmic membrane was found. This number dropped to 1.5 in 10 microns within 30 min after the pulse.     

Transformation of sperm nuclei into male pronuclei in nucleate and anucleate fragments of parthenogenetic mouse eggs

Our objective was to examine the ability of nucleate and anucleate fragments of artificially activated mouse eggs to transform sperm nucleus into male pronucleus. To this end, zona-free oocytes in metaphase II were activated by ethanol and bisected into halves (one with the spindle, the other anucleate) either within 10 to 20 min (series A) or 3 or 5 hr later (series B). In series A, the fragments were inseminated 3,5, and 8 h after activation, and in series B. 3 and 5 h after activation. Both nucleate and anucleate fragments lose the capability of transforming sperm nucleus into fully formed pronucleus sometime between 3 and 5 h after activation. In 8 h old parthenogenetic fragments, the majority of sperm nuclei remain unchanged or begin decondensation but never reach the stage of an early pronucleus. In over 1/3 of anucleate fragments of this age group, sperm nuclei develop defectively: chromatin decondenses inside the persisting nuclear envelope. In other experimental groups, the incidence of these abnormal sperm nuclei varies between 0 and 10%. In general, the anuclcate fragments retain the capability to transform sperm nuclei (fully or partially) longer than their nuclear counterparts. This difference may be accounted for by a different level of substances required for pronuclcar growth (extrachromosomal constituents of the germinal vesicle and nuclear lamins): high and constant in the cytoplasm of anucleate egg halves and low and progressively decreasing in the nucleate halves because of their putative uptake by the female pronucleus. However, the cytoplasmic factors responsible for the initial stages of transformation (nuclear envelope breakdown, chromatin decondensation) become eventually inactivated both in the presence and in the absence of a female pronucleus.     

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

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 microM A23187 for 5 min were treated with 10 microg/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.     

Ethylenediamine-N,N,N',N'-tetraacetic acid induces parthenogenetic activation of porcine oocytes at the germinal vesicle stage, leading to formation of blastocysts

The present study showed that treatment with a cell membrane-impermeable metal ion chelator, EDTA, of porcine oocytes at the germinal vesicle (GV) stage collected from follicles 2-6 mm in diameter induced artificial activation followed by formation of a pronucleus (PN). When the oocytes were cultured for 48 h in medium containing 0.1 to 2 mM EDTA disodium salt (Na-EDTA), they were activated to form PN, and the maximum PN formation rate (63%, n = 68) was achieved in oocytes cultured with 1 mM Na-EDTA. More than 90% of oocytes activated by 1 mM Na-EDTA treatment formed 1 PN without emission of the first and the second polar bodies (PB). This result suggests that EDTA at 1 mM may force the maturing (meiosis I) oocytes to form a PN without chromosome segregation. When oocytes at the GV stage that had been cultured with 1 mM Na-EDTA for 48 h were further cultured in 0.4% BSA-containing NCSU23 medium for 144 h, blastocysts that appeared to be morphologically normal were formed at the rate of 10%, whereas no blastocysts were formed from oocytes that had not been cultured with Na-EDTA. Next we examined the effects of Ca2+, Zn2+, Fe3+, or Cu2+-saturated EDTA (Ca-EDTA, Zn-EDTA, Fe-EDTA, and Cu-EDTA, respectively), and a Ca2+-specific chelator, EGTA, at a concentration of 1 mM. The Ca-EDTA, Fe-EDTA, and Cu-EDTA, but not Zn-EDTA or EGTA, had the ability to activate the oocytes. From these results, it is suggested that extracellular chelation of Zn2+ with EDTA of maturing (meiosis I) porcine oocytes results in parthenogenetic activation of the oocytes, which induces PN formation followed by development to blastocysts.     

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.     

Cytoplasmic maturation for activation of pig follicular oocytes cultured and arrested at metaphase I.

A large population (62-90%) of pig follicular oocytes can mature to metaphase II after culture for 48 h. However, a proportion (6-22%) remain in an immature stage at metaphase I (metaphase I-arrested). The main objective of this study was to determine whether the cytoplasm of metaphase I-arrested pig oocytes is capable of being activated by sperm penetration or parthenogenetic stimulation. After culture for 48 h, oocytes without a polar body (73% were shown to be at metaphase I after staining) and those with a polar body (94% were at metaphase II) were fertilized in vitro. A total of 69% and 62% of the oocytes were activated to form a female pronucleus, respectively, and the rate of polar body extrusion induced by fertilization in the activated oocytes was 90% (the first polar body) and 95% (the second polar body), respectively. When oocytes without and with a polar body were stimulated with an electric pulse, 53% and 81% of the oocytes were activated, respectively. The rate of polar body extrusion in the activated oocytes was 73% (the first polar body) and 79% (the second polar body), respectively. In contrast, young metaphase I oocytes cultured for 24 h had low (6%) or zero activation rate after in vitro fertilization or electric pulse stimulation. However, about one-third of the young metaphase I oocytes penetrated by spermatozoa after in vitro fertilization responded to electric pulse 12 h after insemination, and almost all (93%) were activated when they were stimulated 24 h after insemination. Patterns of polypeptide synthesis and histone H1 kinase activity were similar in metaphase I-arrested and metaphase II oocytes, and were characterized by increase in a 25 kDa polypeptide and by decrease in kinase activity. Although the first step of meiotic division is impaired, these results indicate that metaphase I-arrested oocytes are mature cytoplasmically.     

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 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.     

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.     

Effect on fertilization and development by re-culture after freezing and thawing of bovine oocytes matured in vitro

This study evaluated the effect of 6-h repeat culture before insemination of frozen-thawed, in vitro-matured oocytes on their fertilizability and developmental capacity. Immature oocytes were cultured for 18 h or 24 h and were frozen incrementally. Post-thaw oocytes were repeat cultured for 0 h (control) or 6 h before insemination. With fertilizability, the proportion of enlarged sperm head was significantly (P<0.05) higher in oocytes cultured for 24 h without repeat culture (24:0 h; 51.8%) than those cultured for 24 h with repeat culture (24:6 h; 26.1%) and nonfrozen oocytes (25.9%). However, the proportion of male pronucleus (MPN) in the group of 24:0 h (32.4%) was significantly (P<0.05) lower than that of nonfrozen oocytes (64.3%); the formation of the female pronucleus and MPN were also significantly (P<0.01) lower (17.2%) than that of nonfrozen oocytes (56.1%). Polyspermic oocytes cultured for 18 h with repeat culture for 6 h (18:6 h) were significantly (P<0.05) higher (47.5%) than for nonfrozen oocytes (25.6%). Development to 8-cell stage in the group of 18:6 h was significantly (P<0.05) lower (1.6%) than that of nonfrozen oocytes (18.5%). The cleavage rates in the groups of 24:0 h (16.3%) and 24:6 h (23.4%) were also significantly (P<0.05) lower than for nonfrozen oocytes (63.3%). Development to blastocysts was low (3.9%), but hatched blastocysts were observed in frozen-thawed oocytes cultured for 18:0 h. These results indicate the post-thaw 6-h repeat culture did not greatly improve the fertilizability and embryonic development of oocytes cultured for 18 h or 24 h before freezing. Frozen-thawed oocytes after 24 h in vitro maturation without repeat culture especially had impaired capacities for fertilizability and development.     

Spontaneous and sperm-induced activation of oocytes in LT/Sv strain mice

The oocytes of LT/Sv strain mice are unique in that a high proportion of them (∼40% in this study) are ovulated before reaching metaphase of the second meiotic division (metaphase II). The remaining oocytes of LT/Sv mice are ovulated at metaphase II, as in other strains of mice. When recently ovulated oocytes were cultured in vitro for 11–12 h, those ovulated at metaphase II remained at this stage, whereas those ovulated at metaphase of the first meiotic division (metaphase I) commonly resumed meiosis during in vitro aging. These oocytes extrude the polar body and form a diploid pronucleus. This oocyte activation is not coupled with cortical granule exocytosis. The oocytes ovulated at metaphase II are fully capable of normal fertilization, whereas those ovulated at metaphase I are not. Approximately 50% of metaphase I oocytes penetrated by spermatozoa remain at this stage, and sperm nuclei frequently undergo premature chromosome condensation. Only 13% of spermpenetrated metaphase I oocytes formed a diploid female pronucleus and a haploid male pronucleus by 4 h after insemination. These results demonstrate that the two types of ovulated LT/Sv oocytes have different potentials to undergo either spontaneous or sperm-induced activation.     

A comparative study of parthenogenic activation and in vitro fertilization of bubaline oocytes

The effect of chemical activation protocols on in vitro-matured oocytes were compared to results with IVF (natural activation). Buffalo ovaries were collected in normal saline and transported to the laboratory within 2 h of slaughter. Good quality oocytes, collected by aspiration from 3 to 10 mm follicles, were matured for 22-24 h. Matured oocytes were subjected to either IVF (control) or chemical activation (treatment). For IVF, in vitro-matured oocytes were co-incubated with in vitro-capacitated approximately 1x10(6) frozen/thawed sperm of a Murrah bull and fertilized in modified synthetic oviductal fluid (mSOF) medium. Chemicals for oocytes activation comprised (a) 7% ethanol (ET) for 7 min+2.5 mM 6-dimethyl amino purine (6-DMAP) for 4h, (b) 7% ET for 7 min+10 microg/ml cycloheximide (CHX) for 6h and (c) 7% ET for 7 min+2.5 mM 6-DMAP+10 microg/ml CHX for 6 h. To study embryo development, fertilized and chemically activated oocytes were cultured in mSOF medium for up to 8 days. In this study, a mean of 1.9+/-0.02 maturable oocytes/ovary were recovered and 90.4% matured. Cleavage rate was significantly higher following ET+DMAP, ET+CHX and ET+CHX+DMAP activation (52.5%, 52.5% and 44.4%, respectively) compared to IVF (36.5%, 23.4% and 26.8%, respectively). Blastocyst development (30.9% versus 15.2%) was also significantly higher following ET+CHX+DMAP activation than IVF. The results of parthenogenesis reveal that buffalo oocytes had better inherent developmental competence and that the poor cleavage and embryo development following IVF may be due partly to the poor quality of frozen/thawed sperm, improper sperm capacitation and/or fertilization.     

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.     

The kinetics of oocyte activation and dynamics of polar body formation in Calomys musculinus and Calomys laucha (Rodentia-Sigmodontinae)

The objective of this study was to determine whether Calomys laucha and Calomys musculinus superovulated oocytes undergo parthenogenetic activation following activation stimuli. Cumulus-intact or denuded oocytes were treated with medium containing ethanol (7%), medium containing strontium chloride, or medium alone. They were then incubated for 6-8 h to allow for activation. A group of oocytes was fixed immediately after maturation to serve as a control. The nuclear status of the oocytes was examined after staining with Hoechst 33342, to determine the timing of pronuclear progression from metaphase II to anaphase II or telophase II or to the pronuclear stage. The proportion of oocytes that underwent activation was higher for oocytes treated with ethanol or strontium chloride than in those incubated in medium alone, for the two species studied (p < 0.001). There was little evidence of spontaneous activation occurring in oocytes during the treatments. Most of the activated oocytes contained a single haploid pronucleus, but it was possible to find immediate cleavage and two pronuclei. The different classes of activated oocytes were cultured for 5 days. The type of activating treatment had a marked effect on the ability of the resulting C. musculinus and C. laucha parthenogenetic embryos to develop to the preimplantation stages. Incubation with ethanol produced only 8-cell embryos while the embryos induced with strontium chloride reached the blastocyst stage. This is the first report of parthenogenesis in C. musculinus and C. laucha. The ability of strontium ions to induce matured secondary oocytes to initiate parthenogenesis and obtain further development of Calomys provides opportunities to use Calomys oocytes in vitro and, therefore, to study the genetics, cell biology and virology of development.     

Cytoskeletal alteration in aged porcine oocytes and parthenogenesis

The objective of this study was to examine the changes in microtubule and microfilament assembly in aged porcine oocytes and to determine their developmental pattern after parthenogenetic activation. Porcine oocytes were cultured in Whitten's medium containing 10% follicular fluid with hormonal supplements (eCG and hCG) for 22 hr and 40 hr additional culture without hormonal supplements. At 40, 50, and 60 hr of culture, the oocytes were fixed for immunocytochemistry or activated by electrical pulse. In metaphase II stage oocytes, microtubules were detected only in the meiotic spindle. Two microfilament domains existed in the egg cortex, a thick and a thin microfilament domain. In aged oocytes (50 and 60 hr of culture), the incidence of metaphase II plates observed outside of the thick microfilament domain was higher (P < 0.05) than in young oocytes (40 hr of culture). After activation, a polar body was usually emitted from the chromatin at the microfilament rich domain or two pronuclei were formed outside of the microfilament rich domain. The percentage of activated oocytes with one female pronucleus was higher (P < 0.05) in oocytes at 40 hr of maturation than at 50 and 60 hr of culture. At 24 and 30 hr after stimulation the incidence of cleavage to the 3- to 4-cell stage was higher (P < 0.05) in aged oocytes (50 and 60 hr of maturation) than that in oocytes at 40 hr of culture. These results suggested that a role of microfilaments is to retain the chromatin at the proper position in the oocyte cortex, and that aging results in a disruption of the microfilaments such that atypical development results after parthenogenetic activation. © 1996 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.     

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。