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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 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.

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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PLCZ1 cRNA injection induces bovine parthenogenetic development at similar rates to common chemical activation protocols. a) Mean cleavage (open bars) and blastocyst (black bars) rates after IVF or parthenogenetic activation using different methods. Error bar represents SEM. a, b or y, z: different superscripts represent P < 0.05. b) Cleavage of embryos after IVF or parthenogenetic activation using different methods. Iono = ionomycin; CHX = cycloheximide.
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Figure 2: PLCZ1 cRNA injection induces bovine parthenogenetic development at similar rates to common chemical activation protocols. a) Mean cleavage (open bars) and blastocyst (black bars) rates after IVF or parthenogenetic activation using different methods. Error bar represents SEM. a, b or y, z: different superscripts represent P < 0.05. b) Cleavage of embryos after IVF or parthenogenetic activation using different methods. Iono = ionomycin; CHX = cycloheximide.

Mentions: With the optimal concentrations of m and bPLCZ1 cRNAs determined, we investigated whether PLCZ1 cRNAs induced pre-implantation embryo development to the blastocyst stage at rates comparable to those induced by IVF and by frequently used parthenogenetic procedures. Cleavage and blastocyst rates were similar among parthenogenetic embryos regardless of the activation method (Figure 2a), and were higher than those achieved by IVF-derived embryos (P < 0.05). Also, parthenogenetically activated zygotes consistently cleaved to the two-cell stage at earlier times than IVF embryos (Figure 2b). Among parthenotes, a higher proportion of DMAP-activated embryos had cleaved by 18 hours postactivation, but no differences were observed thereafter (Figure 2b). Collectively, our results show that injection of PLCZ1 cRNAs induces high rates of pre-implantation bovine embryo development.


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)

PLCZ1 cRNA injection induces bovine parthenogenetic development at similar rates to common chemical activation protocols. a) Mean cleavage (open bars) and blastocyst (black bars) rates after IVF or parthenogenetic activation using different methods. Error bar represents SEM. a, b or y, z: different superscripts represent P < 0.05. b) Cleavage of embryos after IVF or parthenogenetic activation using different methods. Iono = ionomycin; CHX = cycloheximide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: PLCZ1 cRNA injection induces bovine parthenogenetic development at similar rates to common chemical activation protocols. a) Mean cleavage (open bars) and blastocyst (black bars) rates after IVF or parthenogenetic activation using different methods. Error bar represents SEM. a, b or y, z: different superscripts represent P < 0.05. b) Cleavage of embryos after IVF or parthenogenetic activation using different methods. Iono = ionomycin; CHX = cycloheximide.
Mentions: With the optimal concentrations of m and bPLCZ1 cRNAs determined, we investigated whether PLCZ1 cRNAs induced pre-implantation embryo development to the blastocyst stage at rates comparable to those induced by IVF and by frequently used parthenogenetic procedures. Cleavage and blastocyst rates were similar among parthenogenetic embryos regardless of the activation method (Figure 2a), and were higher than those achieved by IVF-derived embryos (P < 0.05). Also, parthenogenetically activated zygotes consistently cleaved to the two-cell stage at earlier times than IVF embryos (Figure 2b). Among parthenotes, a higher proportion of DMAP-activated embryos had cleaved by 18 hours postactivation, but no differences were observed thereafter (Figure 2b). Collectively, our results show that injection of PLCZ1 cRNAs induces high rates of pre-implantation bovine embryo development.

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 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.

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