6-Dimethylaminopurine (6-DMAP), which is used to produce most cloned animals, is mutagenic in Salmonella typhimurium TA1535

6-Dimethylaminopurine (6-DMAP) and cycloheximide have recently been used for successful production of cloned mammals. We investigated whether 6-DMAP or cycloheximide are mutagenic agents in the Ames test. Whereas cycloheximide showed no differences compared to the negative control in any of the tester strains, 6-DMAP was clearly mutagenic in Salmonella typhimurium strain TA1535. Here, we strongly propose that innocuous chemicals be used in the production of cloned animals.     

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.     

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.     

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.     

The effect of 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and nuclear transfer embryos

The effects of activation by 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and SCNT embryos were investigated. The results revealed that the blastocyst development of parthenogenetic embryos was significantly higher (P < 0.05) in 6-DMAP activated oocytes, compared to those activated with CHX (21.0 +/- 0.9 vs. 14.9 +/- 0.5, respectively). In contrast, the blastocyst frequencies did not significantly differ (P > 0.05) between the two activation treatment groups for SCNT embryos. The 6-DMAP or CHX treatment did not result in any significant difference in the blastocyst total cell number in either parthenote or SCNT embryos. The chromosomal analysis revealed that all the parthenogenetic embryos (100.0%) derived from 6-DMAP treatment, were chromosomally abnormal whereas in CHX-treated embryos, it was significantly lowered (93.6%, P < 0.05). Conversely, the proportions of chromosomally abnormal SCNT embryos did not significantly differ (P > 0.05) among the 6-DMAP and CHX- treated embryo groups (60.0% vs. 56.2%, respectively). This study demonstrated that oocyte activation agents such as DMAP and CHX have differing effects on meiotic or mitotic nuclei. The study also highlighted the feasibility of using bovine X and Y chromosome specific painting probes in sheep embryos.     

Effects of chemical activation and season on birth efficiency of cloned pigs

The effects of chemical activation on birth efficiency of cloned pigs were studied by investigating the developmental process from porcine oocyte activation to birth of cloned pigs. Three different activation methods were used: (i) Electroporation (Ele); (ii) Ele followed by incubation with 6-dimethylaminopurine (6-DMAP); and (iii) Ele followed by a treatment with cycloheximide (CHX). In experiment 1, the rates of cleavage, developmental rates and cell number of porcine parthenogenetic (PA) embryos were investigated in the three treatment groups. In experiment 2, NT embryos produced by the three different activation treatments were compared for the rates of cleavage, development and cell number. Finally, the effects of Ele and Ele+CHX activation methods on birth efficiency of cloned pigs were compared. The activated oocytes treated by combination activation generally showed a higher (P<0.05) blastocyst rate and produced more expanded blastocysts than oocytes activated with Ele. The rates of cleavage and total cell number of parthenotes were not significantly different. Parthenogenetic embryos activated with 6-DMAP developed into blastocyst and expanded blastocyst stages at a significantly (P<0.05) higher rate than those treated with Ele, but the developmental capability was dramatically decreased in NT embryos. With the CHX activation method, the NT embryo blastocyst rate was substantially (P<0.05) increased although the production of expanded blastocysts was not significantly different from that by the other two methods. The birth rate of cloned pigs increased in the CHX group, though the rate was not significantly different from Ele. The effects of season on developmental rate of the porcine PA embryos and birth rate of cloned pigs were also examined in our study. Porcine oocytes collected in the spring had higher developmental capabilities than those collected in the winter. However, no difference in birth rate of the cloned pigs was found between the oocytes collected in the two seasons. The results obtained from PA and NT embryos, following different activation methods, were inconsistent, suggesting that activation mechanisms are dissimilar in PA and NT embryos. Although the chemical activation in our study leads to an elevation of the blastocyst rate, it does not improve the oocyte’s molecular programming and so does not significantly improve the efficiency of producing cloned pig births. Supported by National Key Basic Research and Development Program (China of Grant No. G200000161).     

6—DMAP和胚胎干细胞代数对异种重构卵体外发育的影响（简报）

The development of reconstructed oocytes and the survival rate of cloned animal were affected by many factors during nuclear transfer. The genetic constitution and the genetic state of donor nucleus were proposed to be primary factors, which affected the survival rate of cloned animal. In addition, the survival rate of cloned animal might be influenced by nuclear transfer technique itself and passages of donor cells as well as the activation methods of oocytes. We reconstructed oocytes with outbreeding Kunming albino mouse ES cells and enucleated rabbit oocytes, and analyzed the effects of the passages of ES cells and 6-DMAP on the development of interspecific reconstructed oocytes. The interspecific reconstructed ES-rabbit oocytes were activated either by combined two set electric pulses and 6-DMAP or by two set electric pulses alone. The rate of cleavage was significantly higher for the group (86.2%) treated with 6-DMAP than the group (64.2%, P < 0.05) treated with electric pulses only, and the rate of blastocysts was 17.0% and 13.4% respectively, which were not significantly different between two groups. When ES cells that had been passed for 24 and 14 generations were used as donors, the cleavage rates of the reconstructed oocytes were 88.5% and 82.1%, respectively (P > 0.05), and the rates of blastolation were 16.7% and 15.4%, respectively (P > 0.05). The results show that 6-DMAP increases the cleavage rate of reconstructed oocytes derived from ES cells, and affects slightly the developmental rate of blastocysts. There are no differences when high passage and low passage ES cells are used as nuclear donors.     

6-DMAP和胚胎干细胞代数对异种重构卵体外发育的影响(简报)

核移植技术已经广泛应用于动物克隆,但是克隆动物的成活率仍然很低。许多克隆胚胎死于妊娠期,少部分能发育到期,正常出生,但多数在出生后由于心肺和消化道的问题,很快就夭折,有些克隆动物有异常表型,如出生时体重和胎盘过大等。研究发现,在同种克隆动物实验中用胚胎干细胞(Embryonic stem cell,ES细胞)作为核供体,发育到期的克隆动物比例明显高于体细胞,并且用杂交一代的小鼠ES细胞为核供体,绝大多数克隆仔     

Effect of Different Parthenogenetic Activation Methods on the Developmental Competence of in vitro Matured Porcine Oocytes

The aim of this study was to investigate the effect of electrical pulse, ethanol, and ionomycin combined with cycloheximide (CHX), cytochalasin B (CB), and 6-dimethylaminopurine (6-DMAP) on parthenogenetic developmental competence of in vitro matured porcine oocytes. In experiment 1, oocytes were treated with direct current electrical pulse (DC pulse) and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX, and CB + 6-DMAP for 6 h, respectively. The rate of blastocyst development in DC pulse + CB + 6-DMAP group was significantly higher than those in other groups (42.4% vs 23.9% ～ 35.8%; P < 0.05); however, there were no differences in both of the cleavage rate and the cell number of blastocysts among four groups. In experiment 2, oocytes were treated with NCSU-23 medium containing 20 μM ionomycin for 40 min and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX and CB + 6-DMAP for 6 h, respectively. The rates of cleavage and blastocyst development in ionomycin + 6-DMAP group were higher than those obtained in other groups (66.2% vs 46.3% ～ 57.3%; 22.3% vs 7.4% ～ 16.1%; P < 0.05). In experiment 3, the activation effects of ethanol combined with 6-DMAP, CHX, CB + 6-DMAP and CB + CHX were investigated. The rates of cleavage and blastocyst development in ethanol + CB + 6-DMAP group were significantly higher than those in other groups (55.5% vs 42% ～ 46.2%; 18.0% vs 7.1% ～ 11.9%; P < 0.05). In experiment 4, the optimal activation protocols in each group plus DC pulse + ionomycin + 6-DMAP were compared. The results showed the rates of cleavage in DC pulse + CB + 6-DMAP group and ionomycin + 6-DMAP were higher than those in ethanol + CB + 6-DMAP and DC pulse + ionomycin + 6-DMAP (73.8–74.4% vs 56.5–57.5%; P < 0.05), but the blastocyst development only in DC pulse + CB + 6-DMAP group was significantly higher than that in other groups (34.1% vs 13.4% ～ 22.3%; P < 0.05). Total cell number of blastocysts in the group of DC pulse + ionomycin + 6-DMAP was higher than that in other groups (34.1 vs 25.3–27.2; P < 0.05). In conclusion, DC pulse, ethanol, CB, and 6-DMAP all affected the parthenogenesis of porcine oocytes matured in vitro, but their combination of DC pulse + CB + 6-DMAP showed the best result in both of cleavage and blastocyst development.     

Combined Treatment with Demecolcine and 6-Dimethylaminopurine during Postactivation Improves Developmental Competence of Somatic Cell Nuclear Transfer Embryos in Pigs

This study determined the effects of postactivation treatment with demecolcine and/or 6-dimethylaminopurine (6-DMAP) on in vivo and in vitro developmental competence of somatic cell nuclear transfer (SCNT) embryos in pigs. SCNT embryos were treated for 4 hours with 0.4?µg/mL demecolcine, 2?mM 6-DMAP, or both after electric activation, then transferred to surrogate pigs or cultured for 7 days. The formation rate of SCNT embryos with a single pronucleus was higher in combined treatment with demecolcine and 6-DMAP (95.2%) than treatment with demecolcine alone (87.1%). Blastocyst formation of SCNT embryos was significantly increased in combined treatment with demecolcine and 6-DMAP (48.7%) compared with demecolcine (22.2%) or 6-DMAP alone (37.3%). Fluctuation of maturation promoting factor activity showed different patterns among various postactivation treatments. Pregnancy was established in 1 of 5 surrogates after transfer of SCNT embryos that were treated with demecolcine and 6-DMAP. The pregnant surrogate delivered one healthy live piglet. The results of our study demonstrated that postactivation treatment with demecolcine and 6-DMAP together improved preimplantation development and supported normal in vivo development of SCNT pig embryos, probably influencing MPF activity and nuclear remodeling, including induction of single pronucleus formation after electric activation.     

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.     

DNA replication initiation by 6-DMAP treatment in maturing oocytes and dividing embryos from marine invertebrates

6-dimethylaminopurine (6-DMAP), a potent protein kinase inhibitor, drives most cells into an interphasic stage. Experiments were undertaken with oocytes from three marine invertebrate species, i.e., Mytilus edulis, Spisula solidissima, and Strongylocentrotus droebachiensis, wherein oocytes were arrested at different phases of meiosis. 6-DMAP induced a continuous DNA synthesis in meiotic cells, whereas it allowed a single round of DNA replication in treated mitotic cells, regardless of species considered. The effects of 6-DMAP were accompanied in all cases by rephosphorylation on tyrosine of the p34^cdc2 homolog, the M-phase promoting factor (MPF) catalytic subunit. The fact that 6-DMAP overcomes the inhibitory control of replication during meiosis suggests that this process depends upon protein phosphorylation, while DNA synthesis regulation in mitotic cells relies on 6-DMAP-insensitive events. © 1996 Wiley-Liss, Inc.     

Activation of Xenopus laevis eggs in the absence of intracellular Ca activity by the protein phosphorylation inhibitor, 6-dimethylaminopurine (6-DMAP).

Xenopus laevis eggs pricked or microinjected with water or saline in medium containing a limited quantity of free Ca (1.0 to 2.0 microM) remain unactivated for at least 6 hr, even after transfer to oocyte medium containing Ca at higher concentrations (0.5-1.0 mM). These injected eggs, when later pricked in oocyte medium or exposed to A23187 or urethane are fully capable of activation. This confirms the observations of Wangh ('89). However, eggs injected in this Ca-limited medium (CaLM) with 6-DMAP as well as those simply exposed to this drug undergo changes characteristic of activation, including cortical contraction, cortical granule breakdown, a loss of MPF and CSF activities, and pronuclear formation. The time required for 6-DMAP to induce egg activation is inversely correlated to its concentration. Interestingly, eggs that have been injected with EGTA, and thus are unable to respond to activation stimuli such as pricking and A23187 or urethane treatment, can also be activated by exposure to 6-DMAP. In contrast, eggs exposed to or injected with a 6-DMAP analogue (6-aminopurine or puromycin) or a protein synthesis inhibitor (cycloheximide or emetine or puromycin) are not activated. As well, eggs injected in CaLM with 6-DMAP simultaneously with a phosphatase inhibitor (NaF or ammonium molybdate) fail to become activated. Although 6-DMAP-activated eggs remain at the pronucleus stage so long as 6-DMAP is present, they resume cell cycle activities after the drug is withdrawn. They form cleavage furrows, disassemble pronuclear envelopes, and recondense chromosomes. Also, MPF activity reappears and cycles at least twice, peaking each time shortly before cleavage furrow formation. These results suggest that activation of Xenopus eggs arrested at metaphase II by inhibition of protein phosphorylation does not require intracellular Ca release and that maintenance of the egg at metaphase II depends upon continuous protein phosphorylation.     

c-Mos proteolysis is independent of the CA(2+) rise induced by 6-DMAP in Xenopus oocytes

In Xenopus oocytes, metaphase II arrest is due to a cytostatic factor (CSF) that involves c-Mos, maintaining a high MPF (cdk1/cyclin B) activity in the cell. At fertilization, a rise in intracellular calcium triggers the proteolysis of both cyclin B and c-Mos. The kinase inhibitor 6-dimethylaminopurine (6-DMAP) is also able to release matured Xenopus oocytes from metaphase II block. This is characterized by c-Mos proteolysis without degradation of cyclin B. We hypothesized that 6-DMAP induced an increase in intracellular calcium. Using the calcium-sensitive fluorescent dye Fura-2, we observed a systematic increase in intracellular calcium following 6-DMAP application. In matured oocytes previously microinjected with the calcium chelator BAPTA, no calcium changes occurred after 6-DMAP addition; however, c-Mos was still proteolysed. In oocytes at the GVBD stage, c-Mos proteolysis occurred in response to 6-DMAP but not to calcium ionophore treatment. We suggest that c-Mos proteolysis is rather controlled by a phosphorylation-dependent process.     

Embryo development,oocyte morphology,and kinetics of meiotic maturation in bovine oocytes exposed to 6-dimethylaminopurine prior to in vitro maturation

The developmental competence of bovine follicular oocytes that had been meiotically arrested with the phosphokinase inhibitor 6-dimethylaminopurine (6-DMAP) was studied. After 24 h in vitro culture with 2 mM 6-DMAP, 85 ± 12% of the oocytes were at the germinal vesicle stage compared to 97 ± 3% at the start of culture (P > 0.05). After release of the 6-DMAP inhibition, followed by 24 h IVM, 82 ± 18% were at MII stage, compared with 93 ± 7% in the control group (P > 0.05). The 6-DMAP oocytes displayed a much higher frequency of abnormal MII configurations than the control oocytes (67% vs 23%; P < 0.0001). In addition spontaneous oocyte activation was more frequent than among control oocytes (5% vs 0.3%; P 0.0006). After IVF of 6-DMAP oocytes, normal fertilization was lower (76 ± 8% vs 89 ± 7%; P < 0.01), oocyte activation higher (11 ± 5% vs 2 ± 2%; P < 0.01), and polyspermy slightly but not significantly higher (8 ± 7% vs 4 ± 4%; P > 0.05), compared with the control group. Cleavage was lower (61 ± 13% vs 81 ± 6%; P < 0.001), as well as day 8 blastocyst formation (17 ± 7% vs 36 ± 8%; P < 0.001). The MII kinetics was different for 6-DMAP and control oocytes. Maximum MII levels were reached at 22 h IVM in both groups, but 50% MII was reached at 17 h in 6-DMAP oocytes, compared to 20 h in control oocytes. Ultrastructure of MII oocytes was similar in the two groups, but in 6-DMAP oocytes the ooplasmic vesicle pattern at GV was at a more advanced stage than in control oocytes. In conclusion, 6-DMAP exposure of GV oocytes prior to IVM induce asynchronous cytoplasmic maturation, leading to aberrant MII kinetics. Thus, at the time of insemination a smaller cohort of oocytes will be at the optimal stage for normal fertilization and subsequent blastocyst development. Mol. Reprod. Dev. 50:334–344, 1998. © 1998 Wiley-Liss, Inc.     

Replication of Nuclei from Cycling and Quiescent Mammalian Cells in 6-DMAP-TreatedXenopusEgg Extract

Nuclear membrane permeabilization is required for replication of quiescent (G0) cell nuclei inXenopusegg extract. We now demonstrate that establishment of replication competence in G0 nuclei is dependent upon a positive activity present in the soluble egg extract. Our hypothesis is that G0 nuclei lose the license to replicate following growth arrest and that this positive activity is required for relicensing DNA for replication. To determine if G0 nuclei contain licensed DNA, we used the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), to prepare egg extracts that are devoid of licensing activity. Intact nuclei, isolated from mammalian cells synchronized in G1-phase (licensed), G2-phase (unlicensed), and G0 were permeabilized and assayed for replication in 6-DMAP-treated and untreated extracts supplemented with [α-³²P]dATP or biotinylated-dUTP. Very little radioactivity was incorporated into nascent DNA in each nuclear population; however, nearly all nuclei in each population incorporated biotin in 6-DMAP extract. The pattern of biotin incorporation within these nuclei was strikingly similar to the punctate pattern observed within nuclei incubated in aphidicolin-treated extract, suggesting that initiation events occur within most replication factories in 6-DMAP extract. However, density substitution and alkaline gel analyses indicate that the incorporated biotin within these nuclei arises from a small number of active origins which escape 6-DMAP inhibition. We conclude that 6-DMAP-treated egg extract cannot differentiate licensed from unlicensed mammalian somatic cell nuclei and, therefore, cannot be used to determine the “licensed state” of G0 nuclei using the assays described here.     

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.     

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.     

Effect of cycloheximide, 6-DMAP,roscovitine and butyrolactone I on resumption of meiosis in porcine oocytes

Improvement of the ability to maintain germinal vesicle stage oocytes in vitro is important for the acquisition of developmental competence. Maintaining oocytes at this stage without damaging their quality would allow synchronization of maturation and homogenization of the oocytes population. More investigations are needed to better understand how the oocyte cell cycle is blocked without consequences to future developmental competence. This study tested the efficacy of pharmacological inhibitors of the G2/M cell cycle transition in keeping porcine oocytes at the germinal vesicle (GV) stage and the reversibility of this inhibition. Porcine cumulus-oocyte complexes (COCs) were thus incubated without any hormones for 24 h in the presence or absence of tested inhibitors: 6-DMAP (protein kinase inhibitor, 2 mM), cycloheximide (protein synthesis inhibitor, 2 microg/ml), roscovitine (cyclin-dependent kinase inhibitor, 50 microM) and butyrolactone I (cyclin-dependent kinase inhibitor, 50 microM). Cumulus-oocyte complexes cultured with any of the inhibitors were significantly blocked at the GV stage. The inhibitory effect varied according to the products, with cycloheximide being the most efficient. Reversibility of the pharmacological inhibitors was assessed by culturing COCs an additional 24 h in inhibitor-free culture medium. Examination of oocytes revealed that the inhibitory effect was fully reversible. This study suggests that 6-DMAP, cycloheximide, roscovitine and butyrolactone I can be use to block meiotic resumption in porcine oocytes in NCSU culture medium.     

Effect of different parthenogenetic activation methods on the developmental competence of in vitro matured porcine oocytes

The aim of this study was to investigate the effect of electrical pulse, ethanol, and ionomycin combined with cycloheximide (CHX), cytochalasin B (CB), and 6-dimethylaminopurine (6-DMAP) on parthenogenetic developmental competence of in vitro matured porcine oocytes. In experiment 1, oocytes were treated with direct current electrical pulse (DC pulse) and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX, and CB + 6-DMAP for 6 h, respectively. The rate of blastocyst development in DC pulse + CB + 6-DMAP group was significantly higher than those in other groups (42.4% vs 23.9% approximately 35.8%; P < 0.05); however, there were no differences in both of the cleavage rate and the cell number of blastocysts among four groups. In experiment 2, oocytes were treated with NCSU-23 medium containing 20 muM ionomycin for 40 min and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX and CB + 6-DMAP for 6 h, respectively. The rates of cleavage and blastocyst development in ionomycin + 6-DMAP group were higher than those obtained in other groups (66.2% vs 46.3% approximately 57.3%; 22.3% vs 7.4% approximately 16.1%; P < 0.05). In experiment 3, the activation effects of ethanol combined with 6-DMAP, CHX, CB + 6-DMAP and CB + CHX were investigated. The rates of cleavage and blastocyst development in ethanol + CB + 6-DMAP group were significantly higher than those in other groups (55.5% vs 42% approximately 46.2%; 18.0% vs 7.1% approximately 11.9%; P < 0.05). In experiment 4, the optimal activation protocols in each group plus DC pulse + ionomycin + 6-DMAP were compared. The results showed the rates of cleavage in DC pulse + CB + 6-DMAP group and ionomycin + 6-DMAP were higher than those in ethanol + CB + 6-DMAP and DC pulse + ionomycin + 6-DMAP (73.8-74.4% vs 56.5-57.5%; P < 0.05), but the blastocyst development only in DC pulse + CB + 6-DMAP group was significantly higher than that in other groups (34.1% vs 13.4% approximately 22.3%; P < 0.05). Total cell number of blastocysts in the group of DC pulse + ionomycin + 6-DMAP was higher than that in other groups (34.1 vs 25.3-27.2; P < 0.05). In conclusion, DC pulse, ethanol, CB, and 6-DMAP all affected the parthenogenesis of porcine oocytes matured in vitro, but their combination of DC pulse + CB + 6-DMAP showed the best result in both of cleavage and blastocyst development.