Parthenogenetic development of domestic cat oocytes treated with ionomycin, cycloheximide, roscovitine and strontium

The objective was to evaluate the parthenogenetic activation of domestic cat oocytes. Cumulus-oocyte complexes matured for 36 h were subjected to three protocols of parthenogenetic activation: Group 1 - ionomycin + cycloheximide; Group 2 - ionomycin + roscovitine; and Group 3 - ionomycin + strontium. As a control, a fourth group of oocytes were cultured in the absence of any activation agent. In all groups, embryos were cultured in SOFaa for 72 h after activation and evaluated for activation rate, cleavage, and embryonic development using Hoechst33342. There were no significant differences among the three treated groups for rates of activated oocytes (70.1 ± 4.3, 75.5 ± 4.7, and 61.9 ± 7.2%, for Treatments 1, 2, and 3 respectively; mean ± SEM), or cleavage (48.1 ± 5.9, 47.4 ± 3.8, and 33.3 ± 6.8%). However, activation and cleavage rates were higher (P < 0.05) than those in the control group (35.5 ± 6.4 and 11.8 ± 4.0%). There were no significant differences among treatment groups for proportion of embryos with 2-10 cells, 10-16 cells, and morulas. In the Control group, the embryo production rate was lower (P < 0.05), although the activation rate was high. The authors concluded that all three treatments effectively induced parthenogenetic activation of domestic cat oocytes. However, to optimize the use of strontium and roscovitine, a dose response and the effect of the presence of Ca⁺⁺ in the medium requires further study.     

Parthenogenetic activation of bovine oocytes using bovine and murine phospholipase C zeta

Background  

During natural fertilization, sperm fusion with the oocyte induces long lasting intracellular calcium oscillations which in turn are responsible for oocyte activation. PLCZ1 has been identified as the factor that the sperm delivers into the egg to induce such a response. We tested the hypothesis that PLCZ1 cRNA injection can be used to activate bovine oocytes.     

Effects of duration, concentration, and timing of ionomycin and 6-dimethylaminopurine (6-DMAP) treatment on activation of goat oocytes

The protocol of ionomycin followed by 6-dimethylaminopurine (6-DMAP) is commonly used for activation of oocytes and reconstituted embryos. Since numerous abnormalities and impaired development were observed when oocytes were activated with 6-DMAP, this protocol needs optimization. Effects of concentration and treatment duration of both drugs on activation and development of goat oocytes were examined in this study. The best oocyte activation (87-95%), assessed by pronuclear formation, was obtained when oocytes matured in vitro for 27 hr were treated with 0.625-20 microM ionomycin for 1 min before 6-hr incubation in 2 mM 6-DMAP. Progressional reduction of time for 6-DMAP-exposure showed that the duration of 6-DMAP treatment can be reduced to 1 hr from the second up to the fourth hour after ionomycin, to produce activation rates greater than 85%. Activation rates of oocytes in vitro matured for 27, 30, and 33 hr were higher (P < 0.05) than that of oocytes matured for 24 hr when treated with ionomycin plus 1-hr (the third hour) 6-DMAP, but a 4-hr incubation in 6-DMAP enhanced activation of the 24-hr oocytes. Goat activated oocytes began pronuclear formation at 3 hr and completed it by 5-hr post ionomycin. An extended incubation in 6-DMAP (a) impaired the development of goat parthenotes, (b) quickened both the release from metaphase arrest and the pronuclear formation, and (c) inhibited the chromosome movement at anaphase II (A-II) and telophase II (T-II), leading to the formation of one pronucleus without extrusion of PB2. In conclusion, duration, concentration, and timing of ionomycin and 6-DMAP treatment had marked effects on goat oocyte activation, and to obtain better activation and development, goat oocytes matured in vitro for 27 hr should be activated by 1 min exposure to 2.5 microM ionomycin followed by 2 mM 6-DMAP treatment for the third hour.     

Chemical activation of parthenogenetic and nuclear transfer porcine oocytes using ionomycin and strontium chloride

Effective protocols for oocyte activation are crucial for study of parthenogenetic development and to produce nuclear transfer reconstructed embryos. This study investigated the use of ionomycin (ION) and strontium chloride (Sr(2+)) in the activation of parthenogenetic and nuclear transfer porcine oocytes. In-vitro-matured oocytes with a polar body were treated with varying concentrations of ION, Sr(2+) or its combinations, and then fixed or cultured to assess activation and development rates, respectively. Ionomycin concentrations of 10 and 15 microM resulted in more frequent oocyte activation and the 15 microM in advanced development compared to 5 microM (71.8 and 70%vs. 47.5%; P=0.04, and 43.7%vs. 19.3%; P=0.008, respectively). Oocytes treated with 10, 20 or 30 mM of Sr(2+) for 2 or 4h displayed a pronuclear formation rate ranging from 46.7 to 70%. When employed after a 5 min treatment with 10 or 15 microM ION, exposure to 10 mM Sr(2+) for 4 h resulted in higher pronuclear formation than did the 20 mM concentration (82 and 88.6%vs. 63.3 and 73.2%; P=0.03). Nuclear transfer reconstructed oocytes treated with 15 microM/5 min ION followed by 10 mM/4 h Sr(2+) resulted in a higher development to blastocyst stage compared to those treated with 15 microM ION alone (17.7 vs. 11.3%; P=0.06). In conclusion, we inferred that the inclusion of Sr(2+) in the activation protocol can benefit the development of nuclear transfer reconstructed porcine oocytes.     

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 activation of bovine oocytes maturing in vitro]

The effect of hyaluronidase (0.3%) and killed bull spermatozoa on the parthenogenetic activation of cow oocytes matured in vitro until metaphase II was studied. It is shown that hyaluronidase, killed spermatozoa, and both agents in combination activate 3.4, 15.0 and 29.6% oocytes, respectively.     

Effect of 6-dimethylaminopurine on germinal vesicle breakdown of bovine oocytes

The effect of 6-dimethylaminopurine (6-DMAP) on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.8%, 120/124) in control medium, presence of 6-DMAP (2 mM) blocked this process almost completely, irrespective of the presence (98.3% GV, 349/355) or absence (97.1% GV, 165/170) of cumulus cells. When lower concentrations of 6-DMAP were used (100-500 microM), GVBD was observed in 87.9% of oocytes, but their maturation was arrested at late diakinesis-metaphase I stage. The inhibition of GVBD was fully reversible, but most of the metaphase II plates were abnormal (80%). To assess whether the action of 6-DMAP is different from the inhibitors of protein synthesis, metaphase II oocytes were exposed to either cycloheximide or 6-DMAP, respectively. Whereas in cycloheximide-supplemented medium approximately 80% of the oocytes were activated, parthenogenetic activation was much less frequent after incubation in 6-DMAP (14.5%). Fusion studies showed that, even if GVBD occurs in 6-DMAP supplemented medium, the level of the maturation-promoting factor (MPF) is decreased. These experiments may indicate the importance of phosphorylation for GVBD in cattle oocytes.     

Effect of single and combined treatments with MPF or MAPK inhibitors on parthenogenetic haploid activation of bovine oocytes

In bovine, correct oocyte artificial activation is a key step in ICSI and other reproductive biotechnologies, and still needs to be improved. The current study was designed to compare the activating efficiency of ionomycin (Io) followed by: a 4 h time window and ethanol (4h-Et), roscovitine (Rosc), dehydroleucodine (DhL), cycloheximide (CHX) or PD0325901 (PD), each as a single treatment, and then combine them in novel protocols. Parthenogenetic haploid activation was evaluated in terms of pronuclear (PN) formation, second polar body (2PB) extrusion, ploidy of day 2 embryos and in vitro development. Combined treatments with Io-4h-Et-Rosc and Io-Rosc/CHX increased PN formation (92.2% and 96%, respectively) compared with Io-Rosc, Io-CHX or Io-4h-Et, which were equally efficient at inducing PN formation (82–84%) and 2PB extrusion (62.1–70.5%). Oocyte activation with Io-DhL and Io-Rosc/DhL resulted in higher 2PB extrusion rates (90% and 95.9%, respectively) but lower PN formation (49.4–58.8%) and cleavage rates (36–57.9%), as occurred with Io-CHX/DhL (76.4% and 70.4%, respectively). For the first time, results show that Io followed by the MAPK inhibitor PD induces PN formation and 2PB extrusion, but PD combined with Rosc or CHX resulted in low rates of haploid day 2 embryos. In conclusion, DhL strongly induces 2PB extrusion but leads to poor PN formation and embryo development. PD induces bovine oocyte activation but results in low rates of haploid embryos. In contrast, the improved PN formation rates after treatment with combined Io-4h-Et-Rosc and Io-Rosc/CHX suggest they should be further evaluated in ART, aiming to increase success rates in bovine.     

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 ).     

Parthenogenetic development and protein patterns of newly matured bovine oocytes after chemical activation

Development of an effective activation protocol is of great importance for studying oocyte competence and embryo cloning. Experiments were designed to examine effects of intracellular calcium elevating agents such as calcium ionophore A23187 (CaA) and ethanol, or protein synthesis and phosphorylation inhibitors such as cycloheximide (CH) and 6-dimethylaminopurine (6-DMAP), or a sequential combination of these agents on both parthenogenetic development and protein patterns of newly matured bovine oocytes. Oocytes were matured for 24 hr in M-199 supplemented with follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol at 39°C in humidified air. They were then activated by various treatments and cultured in KSOM. Protein patterns at 15 hr after treatment were determined on 8–15% gradient SDS-PAGE and silver stained. Results demonstrated that none of the chemical agents—CaA, ethanol, 6-DMAP, or cycloheximide—could effectively induce parthenogenetic development of young bovine oocytes. When compared with the single treatments, sequentially combined treatments of CaA with 6-DMAP or with cycloheximide plus cytochalasin D (CD) significantly increased the rates of cleavage (78–82% versus 3–13%) and blastocyst development (31–40% versus 0%), which were comparable with those of IVF group (80% and 35%, respectively; P > 0.05). Supplementation with CD to the combined CaA and CH treatment improved rates of cleavage and blastocyst development versus without CD supplementation (31% versus 7%; P < 0.05). Fluorescent microscopy revealed that 95% (n = 40) of oocytes treated with CaA plus 6-DMAP had one pronucleus (PN) and one polar body (PB), while 88% (n = 40) in the CaA plus cycloheximide–treated group had one PN and two PBs and 85% (n = 40) in CaA plus cycloheximide and CD group had two PNs and one PB. Treatment by CaA alone resulted in 73% of oocytes (n = 40) arrested at a metaphase stage with two PBs (named as metaphase III or MIII). Protein patterns were similar for chemically activated and in vitro–fertilized (IVF) oocytes in that the 138- and 133-kDa proteins, whose functions are not yet known, were present in the metaphase-stage (MII 24 hr, MII 40 hr, and MIII) oocytes but were absent in PN-stage oocytes regardless of treatment. Therefore, these proteins seem to be metaphase-associated proteins. Taken together, we conclude that optimal parthenogenetic development of newly matured bovine oocytes can be obtained by calcium ionophore treatment followed by incubation in either 6-DMAP or cycloheximide plus cytochalasin D and that the reduction of the 138- and 133-kDa proteins might be necessary for the full activation of bovine oocytes. Mol. Reprod. Dev. 49:298–307, 1998. © 1998 Wiley-Liss, Inc.     

Parthenogenetic activation of mouse oocytes by strontium chloride: a search for the best conditions

Strontium has been successfully used to induce activation of mouse oocytes in nuclear transfer and other experiments, but the optimum treatment conditions have not been studied systematically. When cumulus-free oocytes were treated with 10mM SrCl(2) for 0.5-5h, activation rates (88.4+/-4.1 to 91.2+/-2.7%) did not differ (mean+/-S.E.; P>0.2), but rate of blastulation (57.3+/-3.5%) and cell number per blastocyst (45.0+/-2.4) were the highest after treatment for 2.5h. When treated with 1-20mM SrCl(2) for 2.5h, the activation rate and cell number per blastocyst were higher (P<0.02) after 10mM SrCl(2) treatment than other treatments. The best activation and development were obtained with Ca(2+)-free Sr(2+) medium, but the activation rate was low (37.7+/-1.6%) in Ca(2+)-containing medium. Activation rates were the same, regardless of the presence or absence of cytochalasin B (CB) in the activating medium, but the blastulation rate was higher (P<0.001) in the presence of CB. Only 70% of the cumulus-enclosed oocytes were activated and 10% blastulated after a 10 min exposure to 1.6mM SrCl(2), and many lysed, with increased intensity of Sr(2+) treatment. The presence of CB in SrCl(2) medium markedly reduced lysis of cumulus-enclosed oocytes. Media M16 and CZB did not differ when used as activating media. Only 10.5% of the oocytes collected 13 h post hCG were activated by Sr(2+) treatment alone, with 34% blastulating, but rates of activation and blastulation increased (P<0.001) to 94 and 60%, respectively, when they were further treated with 6-dimethylaminopurine (6-DMAP). The total and ICM cell numbers were less (P<0.001) in parthenotes than in the in vivo fertilized embryos. In conclusion, the concentration and duration of SrCl(2) treatment and the presence or absence of CB in activating medium and cumulus cells had marked effects on mouse oocyte activation and development. To obtain the best activation and development, cumulus-free oocytes collected 18 h post hCG should be treated for 2.5h with 10mM SrCl(2) in Ca(2+)-free medium supplemented with 5 microg/mL of CB.     

Developmental capacity of bovine oocytes following inhibition of meiotic resumption by cycloheximide or 6-dimethylaminopurine

This study was conducted to assess the fertilizability and developmental capacity of bovine oocytes which had been maintained in meiotic arrest by either a protein synthesis inhibitor, cycloheximide (CHX), or an inhibitor of serine/threonine protein kinases, 6-dimethylaminopurine (6-DMAP). Both CHX and 6-DMAP reversibly prevented nuclear maturation of nearly all oocytes for 24 h. After the reversal of arrest, CHX-treated oocytes could be successfully matured and fertilized. They developed to the blastocyst stage at slightly lower rates than oocytes cultured without inhibition for 22 h prior to sperm addition but at higher rates than those incubated in a medium containing no inhibitors for 46 h prior to fertilization. Oocytes inhibited by CHX for 48 h matured and fertilized normally but failed to develop into blastocysts. Even though 6-DMAP-treated oocytes completed meiosis I after removal from the drug, the rates of fertilization and blastocyst formation were lower than for untreated oocytes or CHX-treated oocytes. Effects of adding FSH and/or estradiol-17 beta (E(2)) during CHX-inhibition for 24 h were also examined. Embryos from oocytes treated with CHX and E(2) or with CHX and FSH + E(2) developed into blastocysts at similar rates as the controls. Further development of inhibited oocytes was examined by transferring blastocysts derived from oocytes inhibited by CHX with FSH and E(2) for 24 h to recipient heifers. Two calves were obtained following transfer. These results indicate that CHX-inhibited oocytes retain developmental competence, while 6-DMAP-inhibited oocytes after the reversal of arrest have reduced capacities for fertilization and further development. The addition of FSH and E(2) during CHX-inhibition improves development to the blastocyst stage of the oocytes that are capable of initiating and maintaining pregnancy after embryo transfer to recipient animals.     

6—DMAP对小鼠卵母细胞减数分裂启动及孤雌发育作用

小鼠卵泡卵母细胞体外培养过程中加入２ｍｍｏｌ／Ｌ６－ＤＭＡＰ可抑制卵母细胞自发的染色持浓缩和生发泡破裂（ＧＶＢＤ）。源自超排的ＭⅡ期卵母细胞则能为６－ＤＭＡＰ所激活。ｈＣＧ注射后１８－１９ｈ的卵母细胞置于２ｍｍｏｌ／Ｌ６－ＤＭＡＰ的ＣＺＢ溶液中培养０．５ｈ、１ｈ、２ｈ、３ｈ,卵母细胞的激活率分别为２６．１％、７５．２％、７５．８％、７７．３％、卵裂率分别为８８．２％、７３．２％、６７．０％、５８．     

Cysteamine, glutathione and ionomycin treatments improve in vitro fertilization of prepubertal goat oocytes

The aim of this study was to improve in vitro embryo development of prepubertal goat oocytes by studying the effect of adding cysteamine to in vitro maturation medium, glutathione (GSH) to in vitro fertilization medium and ionomycin to the sperm capacitation medium. In experiment 1, we analysed the effect of 1 mM GSH added to fertilization medium of oocytes matured with 400 microM cysteamine. The control group were oocytes without cysteamine and GSH. In experiment 2, oocytes matured and fertilized in the presence of 400 microM cysteamine and 1 mM GSH, respectively, were inseminated with spermatozoa treated with ionomycin or heparin. In experiment 1, the percentages of total and normal fertilized oocytes were significantly higher for oocytes supplemented with cysteamine and GSH (40.26% and 30.20%, respectively) than for oocytes from the control group (16.66%, and 10.61%, respectively). The percentage of total embryos obtained after 7 days of culture was significantly higher in the group supplemented with cysteamine and GSH (30.62%) than in the control group (8.09%). In experiment 2, percentages of total and normal fertilized oocytes were significantly higher for the group of spermatozoa capacitated with ionomycin (52.21% and 37.17%, respectively) than with heparin (38.62% and 28.35%, respectively). After 7 days of culture, total embryo rate was significantly higher in the group of sperm capacitated with ionomycin (44.91%) than with heparin (38.69%). However, the percentage of embryos developed to the blastocyst stage was not affected by any of the treatments studied.     

Parthenogenetic activation of mouse oocytes by exposure to strontium as a source of cytoplasts for nuclear transfer

Cell-cycle phase of the donor and recipient cells at the moment of nuclear transfer influences subsequent development of the reconstituted embryo. In order to study this effect, the precise cell-cycle phase of the recipient oocyte at the time of fusion must be known and this depends on reliable activation of oocytes in a protocol that has a low incidence of spontaneous activation. Mouse oocytes recovered before (8-10 hours post-human chorionic gonadotropin [hCG]) and after ovulation (14 and 18 hours post-hCG) were exposed to strontium ions in calcium magnesium-free M16 culture medium. The effect on development of haploid parthenotes of post-hCG age of the oocyte, the duration of exposure, and strontium concentration in the medium was determined. These experiments established a reliable method of parthogenetic activation of recently ovulated mouse oocytes, involving the culture of oocytes for 60 minutes in 25 mM strontium in a calcium magnesium-free M16 medium. This method of activation was also able to induce activation of preovulatory oocytes after a preincubation period in vitro. Only a low incidence of spontaneous activation was observed if oocytes were recovered before or immediately after ovulation (14 hours after hCG).     

Parthenogenetic activation of porcine oocytes after nuclear transfer

Mature porcine oocytes are arrested at metaphase II of meiosis. At fertilization, like all mammalian oocytes they exhibit a low frequency Ca(2+) oscillation lasting several hours. This oscillation is thought to be the signal that triggers resumption of meiosis and activates the developmental program of the oocyte. The signal transduction mechanism of the sperm-induced Ca(2+) signal is not known in detail, and attempts to generate the oscillation artificially have met with little success. Nevertheless, artificial activation of the oocyte is a crucial step during nuclear transfer. Methods are available to induce a transient elevation in the intracellular free Ca(2+) concentration to surpass the meiotic arrest and induce development of the constructed embryo. Further studies concentrating on the mechanism of Ca(2+) signaling during fertilization will help to improve the efficiency of the procedures used for parthenogenetic activation of the oocyte.     

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.     

Parthenogenetic development of activated in vitro matured bovine oocytes

Bovine oocytes matured in vitro for 26 hours were electrically stimulated 1) by a single pulse (Treatment A); 2) by 3 pulses 30 minutes apart (Treatment B); 3) by a single pulse followed by 5 minutes of incubation in the stimulation medium (Treatment C); or 4) by a single pulse at 27 hours of maturation (Treatment D). The oocytes were then cultured for up to 8 days to assess parthenogenetic activation and development. Each electrical stimulation consisted of a 60-mus square wave pulse of 2.5 or 3.6 kV/cm. Treatment A was less effective than the other treatments (P<0.05), activating 47 or 59% of oocytes at 2.5 or 3.6 kV/cm, respectively. However, there were no differences due to voltage nor among the other treatments, which activated 64 to 78% of the oocytes. The cleavage rate, 28 to 38%, was not affected by the activation treatment, but development to the 8-cell stage or beyond was greater after activation with the higher voltage. While the numbers of morulae or blastocysts resulting from any given treatment were too small to support meaningful statistical comparison, the results indicate that bovine parthenogenotes produced in vitro are capable of development to the blastocyst stage.     

Effects of post-treatment with 6-dimethylaminopurine (6-DMAP) on ethanol activation of mouse oocytes at different ages

To study the effect of post-treatment with 6-Dimethylaminopurine (6-DMAP) on oocyte activation and development, mouse oocytes collected at different times post human chorion gonadotropin (hCG) injection were incubated in 6-DMAP-containing Chatot-Ziomek-Bavister (CZB) medium for different periods after ethanol exposure, and activation and development were observed. When oocytes were cultured in 6-DMAP without prior ethanol exposure, the highest activation rate was only 40%. Incubation in 6-DMAP for 6 h following ethanol exposure significantly (P < 0.05) increased the activation rate in oocytes recovered 15 and 18 h post hCG, but this effect was not significant in the 21 h oocytes. When oocytes were incubated in 6-DMAP for 1 h at different time points after ethanol, a 6-DMAP susceptible temporal window was found to be located from the second to the fifth h in the 18 h oocytes and from the fourth to the fifth h in the 15 h oocytes, and within the window, the duration of 6-DMAP incubation can be reduced to 0.5 h with more than 80% activation. With the 13 h oocytes, however, 6-DMAP-incubation can only be shortened to 3 h and no specific temporal window was identified. Oocytes that were incubated in 6-DMAP for 1 or 2 h after ethanol exposure developed to morula/blastocyst stages at significantly (P < 0.05) higher rates than those incubated in 6-DMAP for 6 h. Our results suggested that (i) long duration of 6-DMAP incubation impaired the development of mouse parthenogenotes; (ii) the effect of 6-DMAP alone was limited without prior ethanol exposure; (iii) the egg age affected both the timing of 6-DMAP susceptibility and the duration of exposure required to obtain a maximal activating effect; (iv) the most effective activating protocols varied for oocytes of different ages.     

Parthenogenetic activation of rhesus monkey oocytes and reconstructed embryos.

This study determines the efficiency of sequential calcium treatments (electroporation or ionomycin) combined with protein synthesis (cycloheximide) or phosphorylation inhibitors (6-dimethylaminopurine) or the specific maturation promoting factor (MPF) inhibitor, roscovitine, in inducing artificial activation and development of rhesus macaque parthenotes or nuclear transfer embryos. Exposure of oocytes arrested at metaphase II (MII) to ionomycin followed by 6-dimethylaminopurine or to electroporation followed by cycloheximide and cytochalasin B induced pronuclear formation and development to the blastocyst stage at a rate similar to control embryos produced by intracytoplasmic sperm injection. Parthenotes did not complete meiosis or extrude a second polar body, consistent with their presumed diploid status. In contrast, oocytes treated sequentially with ionomycin and roscovitine extruded the second polar body and formed a pronucleus at a rate higher than that observed in controls. Following reconstruction by nuclear transfer, activation with ionomycin/6-dimethylaminopurine resulted in embryos that contained a single pronucleus and no polar bodies. All nuclear transfer embryos activated with ionomycin/roscovitine contained one large pronucleus. However, a third of these embryos emitted one or two polar bodies, clearly containing chromatin material. In summary, we have identified simple yet effective methods of oocyte or cytoplast activation in the monkey, ionomycin/6-dimethylaminopurine, electroporation/cycloheximide/cytochalasin B, and ionomycin/roscovitine, which are applicable to parthenote or nuclear transfer embryo production.