Parthenogenetic activation and subsequent development of rat oocytes in vitro.

Studies were undertaken to determine whether electrical stimulation, or ethanol treatment alone or in combination with 6-dimethylaminopurine (6-DMAP) influenced the rate of parthenogenetic activation of rat oocytes. The percentages of activated oocytes with pronuclei (89-91%) and those developed to the two-cell stage (68-72%) were significantly higher after electrical stimulation with direct current (DC) at 100 V/mm, 99 microsec once or twice, than when other DC voltages (75, 150, and 200) were applied or when ethanol or 6-DMAP treatment was given alone. However, none of the activated oocytes developed beyond the four-cell stage. The percentages of activated oocytes with pronuclei (100%) that developed to the two-cell (100%), eight-cell (89%) and blastocyst stages (50%) were significantly higher when electrical stimulation was followed by treatment with 2 mM 6-DMAP for 4 hr than when other combined procedures were applied. In conclusion, the results of the present study clearly showed that combined treatment of electrical stimulation or ethanol with 6-DMAP induces parthenogenetic activation and subsequent development of rat oocytes in vitro.     

Bovine parthenogenetic blastocysts following in vitro maturation and oocyte activation with ethanol

The appropriate in vitro bovine oocyte maturation and ethanol activation conditions for preimplantation bovine embryo parthenogenetic development to the blastocyst stage were investigated. A 7% ethanol concentration significantly enhanced (P<0.05) the proportion of activated, in vitro-matured bovine oocytes (7% ethanol, 83.4 +/- 3.2% versus 0% ethanol, 63.9 +/- 2.0%). The proportion of activated oocytes was significantly higher (P<0.05) by treatment with 7% ethanol for a minimum of 2 minutes (2 minutes, 89.8 +/- 4.0% versus 0.5 minutes 63.4 +/- 4.9%). Oocyte maturation for periods ranging from 30, 34, 38 and 44 hours resulted in a significant increase (P<0.05) in the proportion of activated oocytes, and in oocytes displaying 2 or 3 pronuclei versus oocytes matured for 26 hours. The proportion of cleaved, activated oocytes (2-cell stage), 4 -cell stage and parthenogenetic morula/blastocysts was significantly higher (P<0.05) within the 34-hour oocyte maturation treatment group. Although the 44-hour oocyte maturation treatment group displayed the highest proportion of activated oocytes with 2 pronuclei, it did not display the highest cleavage frequency, possibly due to the effects of postovulatory aging. Several morphologically normal parthenogenetic bovine blastocysts developed from oocytes that were in vitro matured for 34 hours. The ability to produce such parthenogenetic embryos will eventually facilitate investigation into the role(s) of the maternal and paternal genomes during bovine early development.     

Effect of human leukemia inhibitory factor on in vitro development of parthenogenetic bovine morulae

The present study was conducted to investigate the effect of human leukemia inhibitory factor (hLIF) addition to synthetic oviduct fluid medium (SOFM) supplemented with human serum (HS) on the development of in vitro matured and parthenogenetically activated bovine oocytes. The oocytes matured for 30 h were exposured to ethanol (7%, 7 min) and cytochalasin B (5 mug/ml, 5 to 6 h). The treated oocytes were cultured for 5 d in SOFM supplemented with HS, and Day-5 morulae were cultured for 2 d in SOFM supplemented with HS and with or without hLIF (5000 U/ml) to investigate the subsequent in vitro development to the blastocyst stage. Of the 1531 oocytes that were parthenogenetically activated, 592 (37.5%) cleaved to the 2- to 8-cell stage and 174 (13.8%) developed to the morula stage. The addition of hLIF at the morula stage resulted in a significantly (P<0.01) higher rate of development to the blastocyst stage in the medium with hLIF (55.9%) than without hLIF (28.9%). The mean cell number per blastocyst developed in the medium with hLIF was also significantly (P<0.01) higher than that developed in the medium without hLIF. To evaluate the viability, 6 parthenogenetically developed blastocysts were transferred to 3 recipient heifers (2 embryos per heifer), while in 2 other recipient heifers estrus was prolonged after transfer. The plasma progesterone levels of the 2 recipient heifers at the 28th day after transfer were 8.1 ng/ml and 9.0 ng/ml, but pregnancy was not observed by ultrasonic scanning. The present results indicate that the addition of hLIF to in vitro-produced, Day-5 parthenogenetic bovine morulae significantly improves the subsequent development to the blastocysts stage; however, the present method still does not promote for development of parthenogenetic fetuses in cattle.     

Effects of heparin dosage and sperm capacitation time on in vitro fertilization and cleavage of bovine oocytes matured in vitro

The present study was conducted to clarify the effect of heparin dosage and sperm capacitation time on in vitro fertilization (Experiment 1) and cleavage (Experiment 2) rates of bovine oocytes matured in vitro. For in vitro fertilization, seven dosages of heparin (0, 5, 10, 25, 50, 100 and 200 mug/ml) and nine incubation periods (0, 5, 15, 30, 45, 60, 120, 180 and 240 min) in a capacitation medium were examined, using 6,634 oocytes. The mean proportions of fertilized oocytes in 25, 50 and 100 mug/ml of heparin were significantly (P<0.05) higher (53 to 59%) than in the other dosages (3 to 44%). Incubation with heparin for longer than 60 min lowered the frequencies of fertilization (20 to 36%) compared with the shorter incubation periods (38 to 49%). Higher proportions of fertilized oocytes were obtained by 5, 15, 30 or 45 min of incubation (42 to 49%) than by the other time periods (20 to 38%). Cleavage rates were found by using 2,098 oocytes in a factorial study (4 x 4 x 15: dosages -25, 50, 100 and 200 mug/ml; incubation periods -0, 15, 30 and 60 min; and replicates). The incubation periods and replicates resulted in highly significant differences (P<0.001) in development rates to eight-cell stage, but the four dosages of heparin showed no significant differences. The present results indicate that heparin dosage and sperm capacitation time are important factors influencing in vitro fertilization and cleavage rates. Optimal heparin dosages for the capacitation of bull spermatozoa ranged from 25 to 100 mug/ml; optimal incubation periods ranged from 5 to 60 min.     

Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment

The development of nuclear-transfer oocytes and zygotes was tested in the rabbit. Metaphase II oocytes and zygotes in the early pronuclear stage were treated with a cytoskeletal inhibitor (cytochalasin D), enucleated, and subsequently fused either with single blastomeres from eight- and 16-cell stages (oocytes and zygotes) or with pronuclei-containing karyoplasts (zygotes only). Also, nonenucleated zygotes were fused with 1/8 blastomeres. Fusion was performed by means of an electric field. Development of reconstituted embryos was monitored mainly in vitro, but a certain number of embryos developed from oocytes and zygotes receiving nuclei from eight-cell stages were also transferred into pseudopregnant does. Development of nuclear-transfer oocytes was distinctly better than that of nuclear-transfer zygotes, since 16.9% and 9.5% oocytes vs. 8.1% and 3.7% zygotes carrying eight- and 16-cell nuclei, respectively, developed to the blastocyst stage. Two advanced but already dead fetuses were found after transfer of 27 four-cell embryos obtained after fusion of oocytes with 1/8 blastomeres. No implantations were observed after transfer of 25 four-cell embryos developed from enucleated zygotes receiving eight-cell nuclei. These findings indicate that, in the rabbit, some nuclei from 16-cell embryos are still capable of promoting at least preimplantation development. Comparison between the developmental abilities of oocyte- and zygote-derived nuclear-transfer embryos also suggests that the cytoplasmic environment of recipient cell is more crucial for the development of reconstituted embryos than the stage of introduced nuclei (at least up to the 16-cell stage). The majority of pronuclear exchange embryos (69.9%) and 40% of nonenucleated zygotes receiving eight-cell nuclei were able to develop to the blastocyst stage. This latter observation indicates, similarly as with mouse, a supporting role of residual pronuclei for participation of an eight-cell nucleus in the development of reconstituted zygotes.     

Improvement in development of porcine embryos reconstituted with cells from blastocyst-derived cell lines and enucleated oocytes by optimization of reconstruction methods

The present study was conducted to establish the most suitable system for producing porcine reconstructed embryos by transferring cells from blastocyst-derived cell lines into enucleated oocytes. When the cells were fused to preactivated metaphase II oocytes, or the cells and arrested metaphase II oocytes were fused in medium without CaCl(2) and MgSO(4), the percentages (43-53%) of fused embryos were significantly lower than those (72-79%) produced by fusing the cells to arrested metaphase II oocytes in medium containing CaCl(2) and MgSO(4). High productive efficiency (7%) of blastocysts was obtained when reconstituted embryos produced by the last method were activated again at 3 hours after fusion (F/A --> Activation). Pronuclear formation was observed in 80-91% of the reconstructed embryos produced by F/A --> Activation, with no significant differences between different culture periods in the medium containing cytochalasin B. When cultured in the medium containing cytochalasin B for 0-1 h, almost all (83-85%) the embryos had one pronucleus and one polar body. However, the number of embryos with two pronuclei and no polar bodies was increased significantly by culturing in the medium containing cytochalasin B for 2-4 h. The cleavage rate (34-48%) of reconstructed embryos was not affected by the presence of cytochalasin B for 2 h after activation. However, the percentage of embryos that developed to the blastocyst stage was significantly higher in the presence (23%) than absence (5%) of cytochalasin B. The results indicate that F/A --> Activation and cytochalasin B treatment are effective for the production of porcine embryos reconstituted with cells from blastocyst-derived cell lines and enucleated 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.     

Developmental ability of enucleated bovine oocytes matured in vitro after fusion with single blastomeres of eight-cell embryos matured and fertilized in vitro

Single blastomeres from eight-cell stage bovine embryos matured and fertilized in vitro were electrically fused with enucleated oocytes matured in vitro. In experiment 1, The percentage of these reconstituted embryos developed to the two- to eight-cell stage 48 hr after electrofusion was increased when both the eight-cell embryos and the enucleated oocytes were derived from oocytes cultured with granulosa cells (14% vs. 38%). In experiment 2, the relationship between activation of oocytes and developmental ability of reconstituted embryos was examined. Although both ethanol and electrical stimulation efficiently induced parthenogenetic activation of oocytes matured in vitro for 26–28 hr (ethanol, 89%; electrical stimulation, 73%), the ratio of the second polarbody extrusion differed (80% vs. 22%). Ethanol-treated enucleated oocytes, however, were not significantly different from the early cleavage of the reconstituted embryos 48 hr after electrofusion (nontreated, 38%; treated, 43%). In experiment 3, reconstituted embryos at the two- to eight-cell stage 48 hr after the electrofusion were cocultured with granulosa cells for 6–7 days. Of 69 embryos, one developed to a morula and three developed to blastocysts. © 1993 Wiley-Liss, Inc.     

Oocyte activation and parthenogenetic development of bovine oocytes following intracytoplasmic sperm injection.

Development of bovine oocytes after intracytoplasmic sperm injection (ICSI) was investigated. Oocytes were matured for 24-26 h in vitro and injected with isolated sperm heads. When treated with 7% ethanol (v/v) for 5 min, 71.7% of ICSI oocytes were activated as shown by the resumption of meiosis and the formation of female pronuclei. However, 41.5% of injected sperm heads remained condensed at 18-20 h after injection into the ooplasm. The incidence of decondensing sperm and that of male pronuclei at this stage were 15.1% and 26.4%, respectively. A total of 55.5% of oocytes reached the 2-cell stage following sperm head injection and 54.7% after sham-ICSI; these percentages were not significantly different from those following in vitro fertilisation (IVF) (73.1%). The percentage of 2-cell embryos reaching the 8-cell stage following ICSI was 37.5%, and 27.6% after sham-ICSI, which were significantly lower (p < 0.01) than the equivalent percentage following IVF (62.4%). The percentages of parthenogenetic embryos reaching the 2-cell, 4-cell and 8-cell stages following ICSI were 56.4%, 48.9% and 30.0%, respectively. These results indicate that the low rate of normal embryonic development of bovine oocytes following ICSI is largely due to the parthenogenetic activation of the oocytes.     

Activation of pig oocytes by intracytoplasmic injection of strontium and barium

Ovulated mouse oocytes are activated by exposure to culture medium containing Sr2+ or Ba2+ or by intracytoplasmic injection of the divalent cations. It is known that in vitro matured pig oocytes are activated by the intracytoplasmic injection of Ca2+. In this study, we examined the effect of exposure and of intracytoplasmic injection of Sr2+ or Ba2+ on in vitro matured pig oocytes (MII-oocytes). When MII-oocytes were exposed to the medium containing divalent cations, no oocytes were activated. However, in the case of oocytes that were injected with Sr2+, Ba2+ and Ca2+, at 6 h after injection, 64%, 71% and 86% of the oocytes had been released from MII-arrest, and 51%, 67% and 84% formed female pronuclei, respectively. The initial transient in intracellular Ca2+ concentration ([Ca2+]i) was measured by the Ca2+ indicator dye fluo-4 dextran. Microinjection of Sr2+, Ba2+ or Ca2+ induced a rapid elevation of [Ca2+]i. The exocytosis of cortical granules was examined by staining with fluorescein isothiocyanate (FITC)-labelled peanut agglutinin. After an injection of divalent cations, a release of cortical granules was observed in the oocytes. Maturation promoting factor (MPF) activity declined to a low level after 6 h in all the oocytes injected with divalent cations. To test their developmental ability, injected oocytes were treated with cytochalasin B and then cultured for 168 h in NCSU23 medium. After 168 h, 29% (Sr2+), 29% (Ba2+) and 51% (Ca2+) of the oocytes had developed to the blastocyst stage. These results indicate that intracytoplasmic injection of Sr2+ and Ba2+, like that of Ca2+, induces in vitro matured pig oocytes to be released from MII-arrest and leads them into a series of events related to oocyte activation.     

Fertilizability and subsequent developmental ability of bovine oocytes matured in medium containing epidermal growth factor (EGF)

We have previously shown that epidermal growth factor (EGF) is capable of promoting maturation of bovine cumulus-oocyte complexes in chemically defined serum-free medium. In this study, fertilizability and subsequent developmental capacity of bovine oocytes matured in EGF-containing medium were evaluated. Fetal bovine serum (FBS, 10%) and EGF at 10 ng/ml in Dulbecco's modified Eagles medium with Ham's nutrient mixture F-12 (DME F12 ) significantly increased the rate of formation of two pronuclei compared with the rate obtained from DME-F12 alone (P<0.05). Early embryonic development was assessed during 48 h in culture. Data were evaluated in terms of cleavage and four- to eight-cell formation. Oocytes matured in 10 ng/ml EGF showed significantly higher rates of cleavage (P<0.01) and four- to eight-cell formation than did oocytes matured in control medium (P<0.05). Bovine oocytes matured in the presence of EGF can be normally fertilized and can cleave and develop in vitro up to the eight-cell stage.     

Parthenogenetic activation by electric stimulus of bovine oocytes matured in vitro

The purpose of this study was to determine the optimal conditions for parthenogenetic activation of in vitro-matured bovine oocytes by electric stimulus in vitro. Oocytes were assigned to a factorial treatment structure with direct current ranging from 0.5 to 1 KW/cm for 25 to 100musec and single or double pulses. The optimal conditions for activation were found to be direct current pulses of 1 KV for 25 musec x 2, under which 84% of stimulated oocytes formed one (70%), two (13%) or three (2%) pronuclei. When the stimulated oocytes were incubated in a culture medium containing cytochalasin B, 80% of the oocytes formed two pronuclei. A proportion of the parthenogenetic oocytes developed to the two-cell stage or higher (27%, 83 312 ) in vitro; however, this was significantly (P<0.001) lower than that of the oocytes fertilized in vitro (46%, 736 1608 ).     

Effect of co-culture with theca interna on nuclear maturation of horse oocytes with low meiotic competence,and subsequent fusion and activation rates after nuclear transfer

We conducted this study to examine whether or not co-culture with theca cells improves the maturation rate of horse oocytes with compact cumuli and to evaluate the cytoplasmic competence of oocytes after maturation by assessing fusion, activation and cleavage rates after nuclear transfer. We collected oocytes by scraping follicles from slaughterhouse-derived ovaries and classified them as having an expanded or a compact cumulus. Expanded oocytes were matured in M199 supplemented with 10% FBS and 5 microU/ml FSH for 24 h: compact oocytes were cultured in the same medium, or they were co-cultured in the same medium with theca interna explants, for 24 or 42 h. Oocytes were held with or without 10 microg/ml cytochalasin B, before washing and micromanipulation. and they were fused with donor fibroblasts by electrical pulse. Fused oocytes were activated with Ca ionophore/cycloheximide, cultured for 5 days, and stained with Hoechst to assess nuclear development. We considered oocytes with an enlarged nucleus, or having cleavage with multiple nuclei, to be activated. There was no significant difference in overall maturation rate between compact oocytes cultured with theca and compact controls. When these two groups were combined, there was a significant increase in the proportion of oocytes in MII between 24 and 42 h (P < 0.05). Expanded oocytes had a significantly higher rate of maturation than did compact oocytes (64% versus 25-30%; P < 0.001). There were no significant differences in rates of successful enucleation, fusion, activation or cleavage between compact control and compact + theca oocytes, nor between compact and expanded oocytes; however, expanded oocytes treated with cytochalasin B had a significantly higher survival rate after enucleation than did untreated expanded oocytes (P < 0.05). Three embryos developed from recombined oocytes, with maximum cleavage to 10 cells. The results of this study indicate that co-culture with theca cells does not increase either nuclear or cytoplasmic maturation of compact oocytes. Cytochalasin B is helpful in increasing survival of horse oocytes during enucleation. In vitro matured equine oocytes have the potential to develop into embryos after nuclear transfer; this is the first full report of production of cloned embryos in this species.     

A simple method for producing tetraploid porcine parthenogenetic embryos

The objective was to produce porcine tetraploid parthenogenetic embryos using cytochalasin B, which inhibits polar body extrusion. Porcine cumulus-enclosed oocytes aspirated from antral follicles were cultured for 51 h, and treated with cytochalasin B from 35 h to 42 h after the start of culture. After maturation culture, 74.7% (2074/2775) of oocytes treated with cytochalasin B did not extrude a polar body (0PB oocytes). In contrast, 80.4% (1931/2403) of control oocytes extruded a polar body (1PB oocytes). The 0PB oocytes were electrically stimulated, treated with cytochalasin B again for 3 h, and then cultured without cytochalasin B. Six days after electrical stimulation, 49.8% (321/644) reached the blastocyst stage. The number of cells in these blastocysts derived from 0PB oocytes was significantly lower than that from 1PB oocytes (0PB: 24.9 ± 10.6; 1PB: 43.0 ± 17.1; mean ± SD). A porcine chromosome 1-specific sequence was detected in parthenogenetic 0PB embryos by fluorescence in situ hybridization (FISH) analysis. Typical pronucleus-stage samples derived from 0PB embryos had two pronuclei, each with two signals. In two-cell and blastocyst-stage embryos, four signals were detected in each nucleus derived from 0PB embryos. We inferred that 0PB oocytes, which had a tetraploid number of chromosomes, started to develop as tetraploid parthenotes after electrical stimulation, and that tetraploid status was stably maintained during early embryonic development, at least until the blastocyst stage.     

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.     

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.     

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.     

Development of porcine blastocysts from in vitro- matured and activated haploid and diploid oocytes

The purpose of this study was to determine the developmental capacity of electro-activated porcine oocytes. Follicular oocytes collected from gilts at local slaughterhouses were matured for 48 h and were then subjected to a single square pulse of direct current for 100 rhojusec at 1,500 V/cm for activation. To obtain activated diploid oocytes, some were treated with 5.0 micro/ml cytochalasin B for 4 h immediately after electro-activation. The frequency of activation ranged from 96 to 100%. While 91% of activated oocytes that had not been treated with cytochalasin B had 2 polar bodies and a nucleus (haploids), 92% of the oocytes treated with cytochalasin B had only the first polar body and 2 nuclei (diploids). Haploids and diploids were further cultured in TCM-199 medium that contained 10% (v/v) heat- treated fetal calf serum (FCS) and 0.1 mg/ml sodium pyruvate (mTCM) or in Whittenk medium plus 0.4% (w/v) bovine serum albumin (BSA). The frequency of abnormal oocytes was significantly higher in mTCM (83%) than in Whitten's medium (65%) 96 h after the electro-activation (P < 0.01), suggesting that Whitten's medium supported the development of activated oocytes beyond the morula stage. In all cases, several oocytes developed to the blastocyst stage 144 h after electro- activation (1 to 12%). The frequency was significantly higher in the case of diploids cultured in Whitten's medium (12%) (P < 0.01) than in the case of haploids cultured in Whitten's medium (4%), or in the case of haploids cultured in mTCM (1%). The mean number of nuclei per blastocyst was significantly lower in mTCM (haploids, 15; diploids, 16.1) than in Whitten's medium (haploids, 35.7; diploids, 40.1; P < 0.01), suggesting that the number of nuclei in blastocysts was affected by the culture medium.     

Bovine parthenogenesis is characterized by abnormal chromosomal complements: Implications for maternal and paternal co-dependence during early bovine development

The present study was conducted to examine the karyotypes of parthenogenetic bovine embryos arising from the application of standard oocyte activation and diploidization methods. Bovine cumulus–oocyte complexes were collected and matured in vitro for 24 hr prior to oocyte activation with either 5 μM ionomycin or 7% ethanol for 5 min. Groups of activated oocytes were further treated with 5 μg/ml cytochalasin D or 1.9 mM 6-dimethylaminopurine (DMAP) for 6 hr. Cleavage varied significantly (P < .05) among the treatment groups with 68.0% of the ethanol- and DMAP-treated oocytes dividing. Blastocyst development did not vary with 18.4 ± 2.5% of all treated oocytes progressing to this stage. Blastocyst development did not occur in groups subjected to oocyte activation alone. Blastocysts displayed haploid (2.3%), diploid (11.4%), tetraploid (40.9%), octaploid (4.5%), and mixoploid chromosomal complements (40.9%). Two-cell stage parthenogenotes resulting from ethanol or ionomycin treatment alone displayed haploid (66.7%), diploid (16.7%), tetraploid (4.2%), and mixoploid (12.5%) complements. Our results demonstrate that diploid bovine parthenogenotes arising from these procedures are a minority, with the majority of parthenogenotes displaying polyploid and mixoploid chromosomal complements. The events contributing to these abnormal chromosomal complements occur as early as completion of the first cell cycle, possibly linking these events with the absence of a paternally supplied centrosome. Dev. Genet. 21:160–166, 1997. © 1997 Wiley-Liss, Inc.     

Effect of Actin Polymerization Inhibitor During Oocyte Maturation on Parthenogenetic Embryo Development and Ploidy in Capra hircus

This study was designed to observe the effect of cytochalasin B (CCB) concentrations on ploidy and early development of parthenogenetic embryos in a caprine species. Caprine oocytes were matured in the presence of different concentrations of CCB (5, 10, 15, and 20 μg/ml) and activated by 7% ethanol followed by incubation with 2 mM DMAP. For embryos fertilized in vitro, oocytes were matured in maturation medium without CCB. The cleavage rate and further embryo development were significantly higher (P < 0.05) when oocytes were treated in this way. The percentage of embryos showed higher diploid values in 15 μg/ml CCB (83.66 ± 1.13), followed by 20 (72.22 ± 1.22), 10 (68.57 ± 1.17), and 5 μg/ml (62.00 ± 2.48). These results indicate that CCB with a concentration of 15 μg/ml in maturation medium can be used for the production of diploid parthenogenetic embryos in the caprine species.