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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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Validation of intracytoplasmic injection technique. a, b, c: Sequence of injection. a) Pipette loaded with Texas Red dextran just before injection. b) Pipette advanced into the oocyte; cytoplasm is aspirated to break the plasma membrane. c) Aspirated cytoplasm and Texas Red dextran are injected into the oocyte. d) Schematic representation of the microscope reticulum used as guide to control the injected volume. The oocyte is represented in yellow and the pipette in blue. The red lines indicate the volume introduced into the oocyte which, calculated measuring the pipette internal diameters at both ends, is 5.9 pL. e) An oil drop of the same size as the injected volume is shown next to an oocyte. f) Oocytes injected using Texas Red dextran. g) From left to right, oocyte injected 2X and 1X the normal volume of Texas Red dextran. h) Fluorescent intensity profile of the line shown in f. i) Fluorescent intensity profile of the line shown in g. j) Developmental rates of injected and uninjected bovine oocytes after activation using ionomycin/DMAP.
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Figure 1: Validation of intracytoplasmic injection technique. a, b, c: Sequence of injection. a) Pipette loaded with Texas Red dextran just before injection. b) Pipette advanced into the oocyte; cytoplasm is aspirated to break the plasma membrane. c) Aspirated cytoplasm and Texas Red dextran are injected into the oocyte. d) Schematic representation of the microscope reticulum used as guide to control the injected volume. The oocyte is represented in yellow and the pipette in blue. The red lines indicate the volume introduced into the oocyte which, calculated measuring the pipette internal diameters at both ends, is 5.9 pL. e) An oil drop of the same size as the injected volume is shown next to an oocyte. f) Oocytes injected using Texas Red dextran. g) From left to right, oocyte injected 2X and 1X the normal volume of Texas Red dextran. h) Fluorescent intensity profile of the line shown in f. i) Fluorescent intensity profile of the line shown in g. j) Developmental rates of injected and uninjected bovine oocytes after activation using ionomycin/DMAP.

Mentions: Intracytoplasmic injections into bovine oocytes represent a challenge because of the high elasticity of the plasma membrane and the opacity and darkness of these oocytes. In this study, we adapted the technique used for ICSI to inject consistent volumes of PLCZ1 cRNA into bovine MII oocytes. Using this technique, we were able to inject a precise amount of media, confident of having penetrated the plasma membrane. To achieve this, a determined volume of media was loaded into a Fluorinert-filled pipette using a hydraulic microinjector. Then, the pipette was advanced into the oocyte up to about three-quarters of its diameter. By applying negative pressure, the oocyte cytoplasm was slowly aspirated. A well-defined meniscus was observed at the interface of the oocyte cytoplasm and the media when the plasma membrane was intact. When the plasma membrane was broken, the meniscus disappeared, and the flow of cytoplasm into the pipette was faster as a consequence of lower resistance. These indicators were used to determine that the membrane had been penetrated. Then, applying positive pressure, the cytoplasm was injected back into the oocyte, followed by the media containing cRNA (Figure 1a–c). The volume of media injected was controlled by observing the meniscus at the interface of media and Fluorinert, guided by the reticulum present in the microscope's field of view (Figure 1d). According to our measurements of the internal diameter of the pipette and the length of the injected column of media, we calculated that the injection volume would be ~6 pL.


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)

Validation of intracytoplasmic injection technique. a, b, c: Sequence of injection. a) Pipette loaded with Texas Red dextran just before injection. b) Pipette advanced into the oocyte; cytoplasm is aspirated to break the plasma membrane. c) Aspirated cytoplasm and Texas Red dextran are injected into the oocyte. d) Schematic representation of the microscope reticulum used as guide to control the injected volume. The oocyte is represented in yellow and the pipette in blue. The red lines indicate the volume introduced into the oocyte which, calculated measuring the pipette internal diameters at both ends, is 5.9 pL. e) An oil drop of the same size as the injected volume is shown next to an oocyte. f) Oocytes injected using Texas Red dextran. g) From left to right, oocyte injected 2X and 1X the normal volume of Texas Red dextran. h) Fluorescent intensity profile of the line shown in f. i) Fluorescent intensity profile of the line shown in g. j) Developmental rates of injected and uninjected bovine oocytes after activation using ionomycin/DMAP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Validation of intracytoplasmic injection technique. a, b, c: Sequence of injection. a) Pipette loaded with Texas Red dextran just before injection. b) Pipette advanced into the oocyte; cytoplasm is aspirated to break the plasma membrane. c) Aspirated cytoplasm and Texas Red dextran are injected into the oocyte. d) Schematic representation of the microscope reticulum used as guide to control the injected volume. The oocyte is represented in yellow and the pipette in blue. The red lines indicate the volume introduced into the oocyte which, calculated measuring the pipette internal diameters at both ends, is 5.9 pL. e) An oil drop of the same size as the injected volume is shown next to an oocyte. f) Oocytes injected using Texas Red dextran. g) From left to right, oocyte injected 2X and 1X the normal volume of Texas Red dextran. h) Fluorescent intensity profile of the line shown in f. i) Fluorescent intensity profile of the line shown in g. j) Developmental rates of injected and uninjected bovine oocytes after activation using ionomycin/DMAP.
Mentions: Intracytoplasmic injections into bovine oocytes represent a challenge because of the high elasticity of the plasma membrane and the opacity and darkness of these oocytes. In this study, we adapted the technique used for ICSI to inject consistent volumes of PLCZ1 cRNA into bovine MII oocytes. Using this technique, we were able to inject a precise amount of media, confident of having penetrated the plasma membrane. To achieve this, a determined volume of media was loaded into a Fluorinert-filled pipette using a hydraulic microinjector. Then, the pipette was advanced into the oocyte up to about three-quarters of its diameter. By applying negative pressure, the oocyte cytoplasm was slowly aspirated. A well-defined meniscus was observed at the interface of the oocyte cytoplasm and the media when the plasma membrane was intact. When the plasma membrane was broken, the meniscus disappeared, and the flow of cytoplasm into the pipette was faster as a consequence of lower resistance. These indicators were used to determine that the membrane had been penetrated. Then, applying positive pressure, the cytoplasm was injected back into the oocyte, followed by the media containing cRNA (Figure 1a–c). The volume of media injected was controlled by observing the meniscus at the interface of media and Fluorinert, guided by the reticulum present in the microscope's field of view (Figure 1d). According to our measurements of the internal diameter of the pipette and the length of the injected column of media, we calculated that the injection volume would be ~6 pL.

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