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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

Impact of recBCL-XL (ΔTM) microinjection on early embryo development.(A) Ability of mouse zygotes to progress through the development and form blastocysts in suboptimal HTF medium were significantly increased upon microinjection of recBCL-XL (ΔTM) protein (n = 424) when compared to buffer injected embryos (n = 414). In addition, total cell number (TCN) per embryo was significantly increased and cell death index (CDI) was decreased (n = 71 for buffer injection; n = 110 for protein injection). Nuclear counterstaining (DAPI) images of blastocysts at day 4.5 reflect differences in embryo quality. Mann-Whitney U-test was used for pairwise comparison. (B) Reactive oxygen species (ROS) accumulation, determined by fluorescent measurement of DCHFDA probe fluoresce at 2-cell stage was determined 24 hours after microinjection of either buffer (n = 15) or recBCL-XL (ΔTM) protein (n = 15) and relative fluorescence units (RFU) were used to express fluorescent signal. Injection of recBCL-XL (ΔTM) significantly reduced the accumulation of ROS (student's t-test). (C) Immunocytochemical analysis of total p66SHC or phosphorylated p66SHC on Ser36 was decreased in embryos injected with recBcl-xL (ΔTM) (n = 15/antibody), when compared to buffer injected embryos (n = 15/antibody). In addition, we noticed that Ser10 p66SHC (green) localized to the mitochondria (Mitotracker red), with preferential clustering in subcortical and peri-nuclear regions (yellow overlap; arrows), but this was greatly reduced in recBCL-XL (ΔTM) microinjected embryos. Bars indicate mean ± s.e.m.
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pone-0021687-g003: Impact of recBCL-XL (ΔTM) microinjection on early embryo development.(A) Ability of mouse zygotes to progress through the development and form blastocysts in suboptimal HTF medium were significantly increased upon microinjection of recBCL-XL (ΔTM) protein (n = 424) when compared to buffer injected embryos (n = 414). In addition, total cell number (TCN) per embryo was significantly increased and cell death index (CDI) was decreased (n = 71 for buffer injection; n = 110 for protein injection). Nuclear counterstaining (DAPI) images of blastocysts at day 4.5 reflect differences in embryo quality. Mann-Whitney U-test was used for pairwise comparison. (B) Reactive oxygen species (ROS) accumulation, determined by fluorescent measurement of DCHFDA probe fluoresce at 2-cell stage was determined 24 hours after microinjection of either buffer (n = 15) or recBCL-XL (ΔTM) protein (n = 15) and relative fluorescence units (RFU) were used to express fluorescent signal. Injection of recBCL-XL (ΔTM) significantly reduced the accumulation of ROS (student's t-test). (C) Immunocytochemical analysis of total p66SHC or phosphorylated p66SHC on Ser36 was decreased in embryos injected with recBcl-xL (ΔTM) (n = 15/antibody), when compared to buffer injected embryos (n = 15/antibody). In addition, we noticed that Ser10 p66SHC (green) localized to the mitochondria (Mitotracker red), with preferential clustering in subcortical and peri-nuclear regions (yellow overlap; arrows), but this was greatly reduced in recBCL-XL (ΔTM) microinjected embryos. Bars indicate mean ± s.e.m.

Mentions: Thus, we next attempted to transiently supplement BCL-X levels by microinjecting recBCL-XL (ΔTM) [26] into zygotes and to examine their in vitro developmental potential under an adverse culture condition (HTF). Injection of recBCL-XL (ΔTM) protein significantly improved preimplantation embryo development, when compared to buffer-injected, HTF-cultured embryos (p<0.001; Figure 3A). Rates of blastocyst formation, total cell number (TCN) and cell death index (CDI), which all reflect embryo quality, were restored by recBCL-XL (ΔTM) microinjection to levels comparable with embryos cultured in KSOM medium (Figure 3A and Figure 2). As a negative control, we also injected zygotes with BSA dissolved in microinjection buffer, and this did not significantly improve developmental rates (47%; n = 66) or embryo quality (TCN: 64±5.5%, CDI: 4.2±0.6; n = 13). These results show that microinjection of the recBCL-XL (ΔTM) protein is capable of restoring developmental competence and improving quality of embryos facing conditions of stress. Furthermore, there was no significant difference in studied outcomes if protein was delivered into the cytoplasm or pronucleus (Figure S4). As robotic microinjection resulted in lower lysis rates and a higher degree of consistency, all experiments described below were performed with robotic recBCL-XL (ΔTM) delivery into the cytoplasm.


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)

Impact of recBCL-XL (ΔTM) microinjection on early embryo development.(A) Ability of mouse zygotes to progress through the development and form blastocysts in suboptimal HTF medium were significantly increased upon microinjection of recBCL-XL (ΔTM) protein (n = 424) when compared to buffer injected embryos (n = 414). In addition, total cell number (TCN) per embryo was significantly increased and cell death index (CDI) was decreased (n = 71 for buffer injection; n = 110 for protein injection). Nuclear counterstaining (DAPI) images of blastocysts at day 4.5 reflect differences in embryo quality. Mann-Whitney U-test was used for pairwise comparison. (B) Reactive oxygen species (ROS) accumulation, determined by fluorescent measurement of DCHFDA probe fluoresce at 2-cell stage was determined 24 hours after microinjection of either buffer (n = 15) or recBCL-XL (ΔTM) protein (n = 15) and relative fluorescence units (RFU) were used to express fluorescent signal. Injection of recBCL-XL (ΔTM) significantly reduced the accumulation of ROS (student's t-test). (C) Immunocytochemical analysis of total p66SHC or phosphorylated p66SHC on Ser36 was decreased in embryos injected with recBcl-xL (ΔTM) (n = 15/antibody), when compared to buffer injected embryos (n = 15/antibody). In addition, we noticed that Ser10 p66SHC (green) localized to the mitochondria (Mitotracker red), with preferential clustering in subcortical and peri-nuclear regions (yellow overlap; arrows), but this was greatly reduced in recBCL-XL (ΔTM) microinjected embryos. Bars indicate mean ± s.e.m.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140481&req=5

pone-0021687-g003: Impact of recBCL-XL (ΔTM) microinjection on early embryo development.(A) Ability of mouse zygotes to progress through the development and form blastocysts in suboptimal HTF medium were significantly increased upon microinjection of recBCL-XL (ΔTM) protein (n = 424) when compared to buffer injected embryos (n = 414). In addition, total cell number (TCN) per embryo was significantly increased and cell death index (CDI) was decreased (n = 71 for buffer injection; n = 110 for protein injection). Nuclear counterstaining (DAPI) images of blastocysts at day 4.5 reflect differences in embryo quality. Mann-Whitney U-test was used for pairwise comparison. (B) Reactive oxygen species (ROS) accumulation, determined by fluorescent measurement of DCHFDA probe fluoresce at 2-cell stage was determined 24 hours after microinjection of either buffer (n = 15) or recBCL-XL (ΔTM) protein (n = 15) and relative fluorescence units (RFU) were used to express fluorescent signal. Injection of recBCL-XL (ΔTM) significantly reduced the accumulation of ROS (student's t-test). (C) Immunocytochemical analysis of total p66SHC or phosphorylated p66SHC on Ser36 was decreased in embryos injected with recBcl-xL (ΔTM) (n = 15/antibody), when compared to buffer injected embryos (n = 15/antibody). In addition, we noticed that Ser10 p66SHC (green) localized to the mitochondria (Mitotracker red), with preferential clustering in subcortical and peri-nuclear regions (yellow overlap; arrows), but this was greatly reduced in recBCL-XL (ΔTM) microinjected embryos. Bars indicate mean ± s.e.m.
Mentions: Thus, we next attempted to transiently supplement BCL-X levels by microinjecting recBCL-XL (ΔTM) [26] into zygotes and to examine their in vitro developmental potential under an adverse culture condition (HTF). Injection of recBCL-XL (ΔTM) protein significantly improved preimplantation embryo development, when compared to buffer-injected, HTF-cultured embryos (p<0.001; Figure 3A). Rates of blastocyst formation, total cell number (TCN) and cell death index (CDI), which all reflect embryo quality, were restored by recBCL-XL (ΔTM) microinjection to levels comparable with embryos cultured in KSOM medium (Figure 3A and Figure 2). As a negative control, we also injected zygotes with BSA dissolved in microinjection buffer, and this did not significantly improve developmental rates (47%; n = 66) or embryo quality (TCN: 64±5.5%, CDI: 4.2±0.6; n = 13). These results show that microinjection of the recBCL-XL (ΔTM) protein is capable of restoring developmental competence and improving quality of embryos facing conditions of stress. Furthermore, there was no significant difference in studied outcomes if protein was delivered into the cytoplasm or pronucleus (Figure S4). As robotic microinjection resulted in lower lysis rates and a higher degree of consistency, all experiments described below were performed with robotic recBCL-XL (ΔTM) delivery into the cytoplasm.

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