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

Ross PJ, Beyhan Z, Iager AE, Yoon SY, Malcuit C, Schellander K, Fissore RA, Cibelli JB - BMC Dev. Biol. (2008)

Bottom Line: At their most effective concentrations, PLCZ1 induced parthenogenetic development at rates similar to those observed using other activation stimuli such as Ionomycin/CHX and Ionomycin/DMAP.Injection of bovine and mouse PLCZ1 cRNA also induced IP3R-1 degradation, although bovine PLCZ1 cRNA evoked greater receptor degradation than its mouse counterpart.Importantly, the high rate of aneuploidy encountered in parthenogenetic embryos activated by certain chemical means was not observed in PLCZ1 activated embryos.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cellular Reprogramming Laboratory, Department of Animal Science, Michigan State University, East Lansing, MI 48824, USA. rosspabl@msu.edu

ABSTRACT

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.

Results: Mouse and bovine PLCZ1 cRNAs were injected into matured bovine oocytes at different concentrations. Within the concentrations tested, mouse PLCZ1 injection activated bovine oocytes at a maximum rate when the pipette concentration of cRNA ranged from 0.25 to 1 mug/muL, while bovine PLCZ1 was optimal at 0.1 mug/muL. At their most effective concentrations, PLCZ1 induced parthenogenetic development at rates similar to those observed using other activation stimuli such as Ionomycin/CHX and Ionomycin/DMAP. Injection of mouse and bovine PLCZ1 cRNA induced dose-dependent sperm-like calcium oscillations whose frequency increased over time. Injection of bovine and mouse PLCZ1 cRNA also induced IP3R-1 degradation, although bovine PLCZ1 cRNA evoked greater receptor degradation than its mouse counterpart.

Conclusion: Injection of PLCZ1 cRNA efficiently activated bovine oocytes by inducing a sperm-like calcium oscillatory pattern. Importantly, the high rate of aneuploidy encountered in parthenogenetic embryos activated by certain chemical means was not observed in PLCZ1 activated embryos.

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Representative chromosomal spreads from 8-cell stage bovine embryos (1000×). a) Diploid cell, b) triploid cell, and c) tetraploid cell.
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Figure 5: Representative chromosomal spreads from 8-cell stage bovine embryos (1000×). a) Diploid cell, b) triploid cell, and c) tetraploid cell.

Mentions: Activation of development in oocytes of large domestic species in the absence of fertilization requires the successive application of a Ca2+ ionophore followed by incubation for a few hours with a protein kinase or protein synthesis inhibitor [21]. While these treatments are highly effective at inducing pre-implantation embryo development, they cause high rates of chromosomal abnormalities [25-27]. Given that we have shown that injection of PLCZ1 cRNAs dose-dependently induce high rates of parthenogenetic pre-implantation development, we asked whether a higher proportion of these embryos showed normal chromosomal composition. Accordingly, we compared the chromosomal composition of eight-cell parthenogenetic embryos generated by injection of PLCZ1 cRNAs, versus that of embryos activated by two common chemical activation procedures, as well as of IVF-derived embryos (Table 4; Figure 5). Embryos generated using ionomycin/DMAP showed the highest proportion of abnormal ploidy (70%), while embryos activated using ionomycin/cycloheximide showed a modest amount of aneuploidy (33%) and IVF-derived embryos the least (6%). PLCZ1 cRNA-activated embryos exhibited the lowest percentage of aneuploid embryos (25%) among the parthenogenetic treatments, although it was not significantly different than CHX-activated embryos. These results demonstrate that injection of PLCZ1 cRNA is effective at inducing development of parthenogenetic embryos with high rates of normal chromosomal composition.


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

Ross PJ, Beyhan Z, Iager AE, Yoon SY, Malcuit C, Schellander K, Fissore RA, Cibelli JB - BMC Dev. Biol. (2008)

Representative chromosomal spreads from 8-cell stage bovine embryos (1000×). a) Diploid cell, b) triploid cell, and c) tetraploid cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266721&req=5

Figure 5: Representative chromosomal spreads from 8-cell stage bovine embryos (1000×). a) Diploid cell, b) triploid cell, and c) tetraploid cell.
Mentions: Activation of development in oocytes of large domestic species in the absence of fertilization requires the successive application of a Ca2+ ionophore followed by incubation for a few hours with a protein kinase or protein synthesis inhibitor [21]. While these treatments are highly effective at inducing pre-implantation embryo development, they cause high rates of chromosomal abnormalities [25-27]. Given that we have shown that injection of PLCZ1 cRNAs dose-dependently induce high rates of parthenogenetic pre-implantation development, we asked whether a higher proportion of these embryos showed normal chromosomal composition. Accordingly, we compared the chromosomal composition of eight-cell parthenogenetic embryos generated by injection of PLCZ1 cRNAs, versus that of embryos activated by two common chemical activation procedures, as well as of IVF-derived embryos (Table 4; Figure 5). Embryos generated using ionomycin/DMAP showed the highest proportion of abnormal ploidy (70%), while embryos activated using ionomycin/cycloheximide showed a modest amount of aneuploidy (33%) and IVF-derived embryos the least (6%). PLCZ1 cRNA-activated embryos exhibited the lowest percentage of aneuploid embryos (25%) among the parthenogenetic treatments, although it was not significantly different than CHX-activated embryos. These results demonstrate that injection of PLCZ1 cRNA is effective at inducing development of parthenogenetic embryos with high rates of normal chromosomal composition.

Bottom Line: At their most effective concentrations, PLCZ1 induced parthenogenetic development at rates similar to those observed using other activation stimuli such as Ionomycin/CHX and Ionomycin/DMAP.Injection of bovine and mouse PLCZ1 cRNA also induced IP3R-1 degradation, although bovine PLCZ1 cRNA evoked greater receptor degradation than its mouse counterpart.Importantly, the high rate of aneuploidy encountered in parthenogenetic embryos activated by certain chemical means was not observed in PLCZ1 activated embryos.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cellular Reprogramming Laboratory, Department of Animal Science, Michigan State University, East Lansing, MI 48824, USA. rosspabl@msu.edu

ABSTRACT

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.

Results: Mouse and bovine PLCZ1 cRNAs were injected into matured bovine oocytes at different concentrations. Within the concentrations tested, mouse PLCZ1 injection activated bovine oocytes at a maximum rate when the pipette concentration of cRNA ranged from 0.25 to 1 mug/muL, while bovine PLCZ1 was optimal at 0.1 mug/muL. At their most effective concentrations, PLCZ1 induced parthenogenetic development at rates similar to those observed using other activation stimuli such as Ionomycin/CHX and Ionomycin/DMAP. Injection of mouse and bovine PLCZ1 cRNA induced dose-dependent sperm-like calcium oscillations whose frequency increased over time. Injection of bovine and mouse PLCZ1 cRNA also induced IP3R-1 degradation, although bovine PLCZ1 cRNA evoked greater receptor degradation than its mouse counterpart.

Conclusion: Injection of PLCZ1 cRNA efficiently activated bovine oocytes by inducing a sperm-like calcium oscillatory pattern. Importantly, the high rate of aneuploidy encountered in parthenogenetic embryos activated by certain chemical means was not observed in PLCZ1 activated embryos.

Show MeSH
Related in: MedlinePlus