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Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development.

Liu X, Fernandes R, Gertsenstein M, Perumalsamy A, Lai I, Chi M, Moley KH, Greenblatt E, Jurisica I, Casper RF, Sun Y, Jurisicova A - PLoS ONE (2011)

Bottom Line: However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos.Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment.This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical and Industrial Engineering and Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.

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Related in: MedlinePlus

Automated robotic microinjection of mouse zygotes.(A) The robotic system employs a glass micro device to immobilize a large number of mouse zygotes into a regular pattern via fine vacuum and micrometer-sized through holes underneath cells. Based on precise position control and microscopy vision feedback, a three-degrees-of-freedom (3-DOF) micromanipulator, a motorized X-Y stage, and an in-house developed rotational stage are automatically controlled by a host computer to control an injection micropipette and position/orient the zygotes, respectively. An inverted microscope mounted with a digital camera is used to provide visual feedback and therefore, guide motions of the micropipette and zygotes to achieve automated microinjection. (B) A mouse zygote with the tip of a micropipette at the cytoplasmic center after material deposition. A droplet of mineral oil, which is easy to observe under a non-fluorescent microscope, was injected for visualization to verify the success of material deposition into the cytoplasmic center. (C) Schematic of the glass micro device for zygote immobilization. Vacuum is applied to each zygote for immobilization via micrometer-sized through holes. (D) Mouse zygotes robotically injected with PBS buffer are developed into blastocysts. (E) Calibration data of deposition volumes as a function of deposition time and pressure. Micropipettes with an opening of 1.2 µm were used, as shown in the inlet. (F) Automated robotic microinjection induced significantly lower lysis rates than manual injection (n = 400 for robotic protein and buffer injection; n = 229 for manual injection). Bars indicate mean ± s.e.m. Kruskal Wallis test followed by Dunn's post test was used for statistical analysis.
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pone-0021687-g001: Automated robotic microinjection of mouse zygotes.(A) The robotic system employs a glass micro device to immobilize a large number of mouse zygotes into a regular pattern via fine vacuum and micrometer-sized through holes underneath cells. Based on precise position control and microscopy vision feedback, a three-degrees-of-freedom (3-DOF) micromanipulator, a motorized X-Y stage, and an in-house developed rotational stage are automatically controlled by a host computer to control an injection micropipette and position/orient the zygotes, respectively. An inverted microscope mounted with a digital camera is used to provide visual feedback and therefore, guide motions of the micropipette and zygotes to achieve automated microinjection. (B) A mouse zygote with the tip of a micropipette at the cytoplasmic center after material deposition. A droplet of mineral oil, which is easy to observe under a non-fluorescent microscope, was injected for visualization to verify the success of material deposition into the cytoplasmic center. (C) Schematic of the glass micro device for zygote immobilization. Vacuum is applied to each zygote for immobilization via micrometer-sized through holes. (D) Mouse zygotes robotically injected with PBS buffer are developed into blastocysts. (E) Calibration data of deposition volumes as a function of deposition time and pressure. Micropipettes with an opening of 1.2 µm were used, as shown in the inlet. (F) Automated robotic microinjection induced significantly lower lysis rates than manual injection (n = 400 for robotic protein and buffer injection; n = 229 for manual injection). Bars indicate mean ± s.e.m. Kruskal Wallis test followed by Dunn's post test was used for statistical analysis.

Mentions: In contrast to conventional manual injection systems, our robotic system (Figure 1A) uses a microfabricated glass cell holding device (Figures 1C and S1A) to immobilize many mouse zygotes into a regular pattern (Figure S1C, Text S1, and Video S1). Switching from one cell to another for injection was greatly simplified and automatically performed via precise position control (Figures S2–S3, Text S1, and Video S2), dramatically enhancing the injection speed. A vision-based cell orientation control technique (Text S1) as well as an in-house developed motorized rotational stage (Figure 1A) was integrated into the robotic system for fast and automated cell orientation. A motorized micromanipulator (i.e., injection microrobot) was automatically controlled to inject mouse zygotes (Figure 1B and Video S2) in a high-speed manner with high repeatability.


Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development.

Liu X, Fernandes R, Gertsenstein M, Perumalsamy A, Lai I, Chi M, Moley KH, Greenblatt E, Jurisica I, Casper RF, Sun Y, Jurisicova A - PLoS ONE (2011)

Automated robotic microinjection of mouse zygotes.(A) The robotic system employs a glass micro device to immobilize a large number of mouse zygotes into a regular pattern via fine vacuum and micrometer-sized through holes underneath cells. Based on precise position control and microscopy vision feedback, a three-degrees-of-freedom (3-DOF) micromanipulator, a motorized X-Y stage, and an in-house developed rotational stage are automatically controlled by a host computer to control an injection micropipette and position/orient the zygotes, respectively. An inverted microscope mounted with a digital camera is used to provide visual feedback and therefore, guide motions of the micropipette and zygotes to achieve automated microinjection. (B) A mouse zygote with the tip of a micropipette at the cytoplasmic center after material deposition. A droplet of mineral oil, which is easy to observe under a non-fluorescent microscope, was injected for visualization to verify the success of material deposition into the cytoplasmic center. (C) Schematic of the glass micro device for zygote immobilization. Vacuum is applied to each zygote for immobilization via micrometer-sized through holes. (D) Mouse zygotes robotically injected with PBS buffer are developed into blastocysts. (E) Calibration data of deposition volumes as a function of deposition time and pressure. Micropipettes with an opening of 1.2 µm were used, as shown in the inlet. (F) Automated robotic microinjection induced significantly lower lysis rates than manual injection (n = 400 for robotic protein and buffer injection; n = 229 for manual injection). Bars indicate mean ± s.e.m. Kruskal Wallis test followed by Dunn's post test was used for statistical analysis.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3140481&req=5

pone-0021687-g001: Automated robotic microinjection of mouse zygotes.(A) The robotic system employs a glass micro device to immobilize a large number of mouse zygotes into a regular pattern via fine vacuum and micrometer-sized through holes underneath cells. Based on precise position control and microscopy vision feedback, a three-degrees-of-freedom (3-DOF) micromanipulator, a motorized X-Y stage, and an in-house developed rotational stage are automatically controlled by a host computer to control an injection micropipette and position/orient the zygotes, respectively. An inverted microscope mounted with a digital camera is used to provide visual feedback and therefore, guide motions of the micropipette and zygotes to achieve automated microinjection. (B) A mouse zygote with the tip of a micropipette at the cytoplasmic center after material deposition. A droplet of mineral oil, which is easy to observe under a non-fluorescent microscope, was injected for visualization to verify the success of material deposition into the cytoplasmic center. (C) Schematic of the glass micro device for zygote immobilization. Vacuum is applied to each zygote for immobilization via micrometer-sized through holes. (D) Mouse zygotes robotically injected with PBS buffer are developed into blastocysts. (E) Calibration data of deposition volumes as a function of deposition time and pressure. Micropipettes with an opening of 1.2 µm were used, as shown in the inlet. (F) Automated robotic microinjection induced significantly lower lysis rates than manual injection (n = 400 for robotic protein and buffer injection; n = 229 for manual injection). Bars indicate mean ± s.e.m. Kruskal Wallis test followed by Dunn's post test was used for statistical analysis.
Mentions: In contrast to conventional manual injection systems, our robotic system (Figure 1A) uses a microfabricated glass cell holding device (Figures 1C and S1A) to immobilize many mouse zygotes into a regular pattern (Figure S1C, Text S1, and Video S1). Switching from one cell to another for injection was greatly simplified and automatically performed via precise position control (Figures S2–S3, Text S1, and Video S2), dramatically enhancing the injection speed. A vision-based cell orientation control technique (Text S1) as well as an in-house developed motorized rotational stage (Figure 1A) was integrated into the robotic system for fast and automated cell orientation. A motorized micromanipulator (i.e., injection microrobot) was automatically controlled to inject mouse zygotes (Figure 1B and Video S2) in a high-speed manner with high repeatability.

Bottom Line: However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos.Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment.This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical and Industrial Engineering and Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.

Show MeSH
Related in: MedlinePlus