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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 IP3 R-1 downregulation. a) Immunoblot. Five bovine oocytes were used per lane; samples were collected 12 hours after cRNA injection. MII oocytes were collected at the time of cRNA injection. Aged MII are noninjected oocytes that were left in culture the same amount of time as the injected ones. bPLCZ1 = bovine PLCZ1; mPLCZ1 = mouse PLCZ1. b) Quantification of relative abundance of IP3R-1 versus the levels observed in MII oocytes. The number in the bars indicates relative IP3R-1 mass. Data represented as mean ± SEM of two replications. Different letters indicate P < 0.06.
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Figure 4: PLCZ1 cRNA injection induces IP3 R-1 downregulation. a) Immunoblot. Five bovine oocytes were used per lane; samples were collected 12 hours after cRNA injection. MII oocytes were collected at the time of cRNA injection. Aged MII are noninjected oocytes that were left in culture the same amount of time as the injected ones. bPLCZ1 = bovine PLCZ1; mPLCZ1 = mouse PLCZ1. b) Quantification of relative abundance of IP3R-1 versus the levels observed in MII oocytes. The number in the bars indicates relative IP3R-1 mass. Data represented as mean ± SEM of two replications. Different letters indicate P < 0.06.

Mentions: Fertilization-associated [Ca2+]i oscillations are thought to be underlined by steady production of IP3 [15] that leads to IP3R-1 down-regulation [17]. Accordingly, we evaluated whether injection of PLCZ1 cRNA resulted in IP3R-1 degradation, and whether IP3R-1 down regulation was associated with PLCZ1 cRNA concentration and species-of-origin. We found that injections of 0.1 μg/μL bPLCZ1 or 1 μg/μL mPLCZ1 cRNA induced fertilization-like IP3R-1 degradation (Figure 4), whereas injection of 1 μg/μL bPLCZ1 cRNA depleted IP3R-1 from oocytes (Figure 4),


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 IP3 R-1 downregulation. a) Immunoblot. Five bovine oocytes were used per lane; samples were collected 12 hours after cRNA injection. MII oocytes were collected at the time of cRNA injection. Aged MII are noninjected oocytes that were left in culture the same amount of time as the injected ones. bPLCZ1 = bovine PLCZ1; mPLCZ1 = mouse PLCZ1. b) Quantification of relative abundance of IP3R-1 versus the levels observed in MII oocytes. The number in the bars indicates relative IP3R-1 mass. Data represented as mean ± SEM of two replications. Different letters indicate P < 0.06.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: PLCZ1 cRNA injection induces IP3 R-1 downregulation. a) Immunoblot. Five bovine oocytes were used per lane; samples were collected 12 hours after cRNA injection. MII oocytes were collected at the time of cRNA injection. Aged MII are noninjected oocytes that were left in culture the same amount of time as the injected ones. bPLCZ1 = bovine PLCZ1; mPLCZ1 = mouse PLCZ1. b) Quantification of relative abundance of IP3R-1 versus the levels observed in MII oocytes. The number in the bars indicates relative IP3R-1 mass. Data represented as mean ± SEM of two replications. Different letters indicate P < 0.06.
Mentions: Fertilization-associated [Ca2+]i oscillations are thought to be underlined by steady production of IP3 [15] that leads to IP3R-1 down-regulation [17]. Accordingly, we evaluated whether injection of PLCZ1 cRNA resulted in IP3R-1 degradation, and whether IP3R-1 down regulation was associated with PLCZ1 cRNA concentration and species-of-origin. We found that injections of 0.1 μg/μL bPLCZ1 or 1 μg/μL mPLCZ1 cRNA induced fertilization-like IP3R-1 degradation (Figure 4), whereas injection of 1 μg/μL bPLCZ1 cRNA depleted IP3R-1 from oocytes (Figure 4),

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
Related in: MedlinePlus