Effects of activation methods and culture conditions on development of parthenogenetic porcine embryos.

The effects of different activation methods and culture conditions on early development of porcine parthenotes were examined. Three different activation methods were tested: (1) electroporation; (2) electroporation followed by incubation in the presence of butyrolactone I, an inhibitor of cdc2 and cdk2 kinases; and (3) electroporation followed by a treatment with cycloheximide, a blocker of protein synthesis. The activated oocytes were cultured in two different media, NCSU-23 and PZM-3 under 5% CO2 in air. In a separate experiment, the effects of high (approximately 20%) or low (5%) O2 tension on early embryo development were also evaluated. The average pronuclear formation was less (p<0.05) in the electroporated oocytes (83.9+/-1.7%) compared with those activated by electroporation and butyrolactone I or electroporation plus cycloheximide (92.8+/-0.8 and 93.0+/-1.0%). In PZM-3 medium, the average frequencies of blastocyst formation (59.7+/-3.6%) and hatching (10.6+/-1.3%) were greater than those in NCSU-23 medium (39.9+/-3.1% blastocyst formation, p<0.05; and 0.2+/-0.2% hatching; p<0.001). Furthermore, the average nuclear number was also greater (p<0.001) in blastocysts developed in PZM-3 (50.2+/-1.3) than in those developed in NCSU-23 (35.3+/-1.1). Blastocyst formation was similar (p>0.10) among the three activation procedures when parthenotes were cultured in NCSU-23, while in PZM-3 more (p<0.05) parthenotes produced by electroporation plus butyrolactone or electroporation plus cycloheximide developed into blastocysts compared to electroporation alone (64.9+/-5.2 and 68.6+/-3.5% compared with 45.6+/-4.7%). Incidences of apoptotic nuclei were similar (p>0.10) among all treatments. No difference in development was found between parthenotes that developed under high versus low O2 tension (p>0.10). These results demonstrate that activation methods targeting the calcium signaling pathway at several points trigger embryonic development more efficiently than electroporation alone. The data also imply that the PZM-3 medium provides for enhanced culture conditions for the early development of parthenogenetic porcine embryos than NCSU-23.

Nánássy L ，Lee K ，Jávor A ，Macháty Z ... -  《ANIMAL REPRODUCTION SCIENCE》

In vitro development of preimplantation porcine nuclear transfer embryos cultured in different media and gas atmospheres.

This study investigated the effect of culture media and gas atmospheres on the development of porcine nuclear transfer embryos. Oocytes derived from a local abattoir were matured for 42-44 h and enucleated. Fetal fibroblasts were prepared from a Day 35 porcine fetus. Confluent stage fetal fibroblasts were introduced into the perivitelline space of enucleated oocytes. Fusion and activation were induced simultaneously with two direct current (1.2 kV/cm for 30 micros) in 0.3 M mannitol medium. For parthenogenetic activation, the same pulses were used. In Experiment 1, parthenogenetically activated oocytes were cultured in North Carolina State University-23 (NCSU-23), Porcine Zygote Medium-3 (PZM-3), or Beltsville Embryo Culture Medium-3 (BECM-3). Parthenogenetically activated oocytes cultured in PZM-3 had a higher (P < 0.05) developmental rate to the blastocyst stage (15.2% versus 3.7-9.6%) as compared to BECM-3 or NCSU-23. The number of nuclei in Day 6 blastocysts was higher (P < 0.05) in PZM-3 (23.6) and NCSU-23 (21.4) than BECM-3 (14.2). In Experiment 2, parthenogenetically activated oocytes were cultured in NCSU-23 under a gas atmosphere of 5% CO(2) in air for 6 days (T1), 5% CO(2), 5% O(2), 90% N(2) for 6 days (T2), 5% CO(2) in air for 3 days, then 5% CO(2), 5% O(2), 90% N(2) for 3 days (T3), or 5% CO(2), 5% O(2), 90% N(2) for 3 days, then 5% CO(2) in air for 3 days (T4). Blastocyst formation rates were not different among treatments (12.9 =/-3.6 %, 13.5 +/- 4.2%, 10.8+/-2.4%, and 12.6+/-2.7%, respectively). However, T2 (36.7+/-2.9) and T3 (33.8+/-3.0) resulted in more nuclei per blastocyst than T1 (23.2+/-2.1) or T4 (26.0+/-2.1 ). In Experiment 3, reconstructed porcine nuclear transfer (NT) embryos were cultured in NCSU-23 or PZM-3 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2). Developmental rates to blastocyst stage for porcine NT embryos cultured in NCSU-23 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2) were 7.2+/-1.4% and 12.3+/-1.4%, and the number of nuclei was 12.2=/-0.8% and 19.4+/-1.0, respectively. NT embryos cultured in PZM-3 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2) had developmental rates to blastocyst stage of 18.8+/-1.9 %, and 17.8+/-3.8% the nuclei number was 20.9 +/- 1.9 and 21.9+/-3.3, respectively. NT embryos cultured in NCSU-23 had a higher developmental rate to the blastocyst stage in 5% CO(2), 5% O(2), 90% N(2) than in 5% CO(2) in air (P < 0.05). Regardless of gas atmospheres, NT embryos cultured in PZM-3 had a higher developmental rate (18.3 =/- 1.7% versus 16.9 +/- 1.2%) and nuclei number (21.4 +/-1.8 versus 16.9 +/- 1.2) than in NCSU-23 (P < 0.05). In conclusion, a gas atmosphere of 5% CO(2), 5% O(2), 90% N(2) supported a higher development rate of porcine NT embryos than 5% CO(2) in air when the porcine NT embryos were cultured in NCSU-23. Furthermore, regardless of atmosphere, PZM-3 supported a higher development rate of porcine nuclear transfer embryos than NCSU-23.

Im GS ，Lai L ，Liu Z ，Hao Y ，Wax D ，Bonk A ，Prather RS ... -  《THERIOGENOLOGY》

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

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

Naruse K ，Quan YS ，Kim BC ，Lee JH ，Park CS ，Jin DI ... -  《THERIOGENOLOGY》

Changes in MPF and MAPK activities in porcine oocytes activated by different methods.

The effect of different oocyte activation methods on the dynamics of M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity in porcine oocytes were examined. Three activativation methods were tested: (1) electroporation (EP); (2) electroporation combined with butyrolactone I (BL), an inhibitor of cdc2 and cdk2 kinases; (3) electroporation followed by a treatment with cycloheximide (CHX), a protein synthesis blocker. The activity of cdc2 in MII oocytes was 0.067+/-0.011pmol/oocyte/min (mean+/-S.E.M.), which by 1h decreased in every treatment group (P<0.05) and stayed at low levels until 6h post-activation, approximately the time of pronuclear formation. The initial MAPK activity (0.123+/-0.017pmol/oocyte/min) also decreased 1h after each type of activation treatment (P<0.005). However, in the electroporation only group, activity reached its lowest level at 3h; thereafter, it started to recover and at later time points, MAPK activity did not differ from that in non-treated oocytes (P>0.1). In contrast, oocytes where electroporation was followed by protein kinase or protein synthesis inhibition had low MAPK activity by the time pronuclei were to be formed. Pronuclear formation in these groups (86.3+/-3.3% for EP+BL and 87.6+/-3.7% for EP+CHX) was higher compared to that found in the EP-only oocytes (69.4+/-3.3%; P<0.05). These findings demonstrated that electroporation alone efficiently triggered the inactivation of MPF but not that of MAPK. In order to achieve low MAPK activity to allow high frequency of pronuclear formation, electroporation should be followed by a treatment that inhibits protein synthesis or specific protein kinases. The combined activation methods provided stimuli that efficiently induced both MPF and MAPK inactivation and triggered pronuclear formation with high frequencies.

Nanassy L ，Lee K ，Javor A ，Machaty Z ... -  《THERIOGENOLOGY》

Incidence of apoptosis in parthenogenetic porcine embryos generated by using protein kinase or protein synthesis inhibitors.

In mammals, embryonic development can be artificially initiated by activating the oocyte using a number of methods. In the present research, we investigated whether butyrolactone I and cycloheximide, two chemicals frequently used in combined oocyte activation protocols, have any detrimental effect on programmed cell death in the developing porcine embryo. Parthenogenetic porcine blastocysts were generated by the following methods: (1) electroporation followed by blocking the activity of specific protein kinases with butyrolactone I; (2) electroporation followed by inhibition of protein synthesis using cycloheximide; and (3) electroporation only (control). The viability of the embryos was evaluated by monitoring the frequency of cells with early and late signs of programmed cell death. There was no difference between the embryos in terms of blastocyst formation (ranging from 27.5+/-3.8 and 33.3+/-3.5%) and total nuclear number (ranging from 23.5+/-1.1 and 31.8+/-4.4). In addition, the occurrence of apoptosis was also similar in the three experimental groups. The proportion of cells with active caspase-9 (a sign of early apoptosis) in the blastocysts produced by the different activation methods was between 19.4+/-1.9 and 23.0+/-2.4%. The annexin V assay revealed that phosphatidylserine flip (another early apoptotic event) took place in 24.4+/-2.1 to 32.3+/-2.4% of the blastomeres. Finally DNA fragmentation, a sign of late stage apoptosis determined by the TUNEL assay, occurred in 8.5+/-2.4 to 10.1+/-3.0% of the cells. The results indicate that temporary inhibition of specific protein kinases or protein synthesis does not increase the onset of apoptosis in parthenogenetic porcine blastocysts.

Lee K ，Hyslop JM ，Nanassy L ，Machaty Z ... -  《-》