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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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Expression of BCL-X in human oocytes.(A) Distribution of human oocytes obtained form 43 patients based on their BCL-X expression in either germinal vesicle stage (GV - left) or meiosis I stage (MI - right). Arrows point to groups of oocytes with insufficient endowment of BCL-X transcript. (B) Visualization of protein interaction network that connects BCL-X (BC2L1) with other targets known to be deregulated in arrested human embryos. Node shape, represented by triangles, indicates trends of expression. Shape of triangles pointing up corresponds to genes up-regulated and triangles pointing down correspond to genes down-regulated in arrested human embryos; circles represent direct interacting partners that link BCL-X (BCL2L1) to up- and down-regulated targets. Red highlight on nodes represents the set of cross-linked proteins. Node color is based on gene ontology as per legend. To reduce network complexity, all other nodes and edges are made partially transparent.
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pone-0021687-g005: Expression of BCL-X in human oocytes.(A) Distribution of human oocytes obtained form 43 patients based on their BCL-X expression in either germinal vesicle stage (GV - left) or meiosis I stage (MI - right). Arrows point to groups of oocytes with insufficient endowment of BCL-X transcript. (B) Visualization of protein interaction network that connects BCL-X (BC2L1) with other targets known to be deregulated in arrested human embryos. Node shape, represented by triangles, indicates trends of expression. Shape of triangles pointing up corresponds to genes up-regulated and triangles pointing down correspond to genes down-regulated in arrested human embryos; circles represent direct interacting partners that link BCL-X (BCL2L1) to up- and down-regulated targets. Red highlight on nodes represents the set of cross-linked proteins. Node color is based on gene ontology as per legend. To reduce network complexity, all other nodes and edges are made partially transparent.

Mentions: While experiments described above dealt with in vitro induced phenotype, they have clinical relevance to human IVF, where embryos are always maintained in culture for at least 3 days. In addition, it is also possible that some oocytes may lack sufficient storage of maternally derived Bcl-x gene products. In order to determine if variability in the endowment of maternally accumulated BCL-X transcripts could contribute to poor quality of human embryos in patients undergoing IVF, we analyzed its expression in human oocytes. As we have previously observed that Bcl-x transcript expression peaks in germinal vesicle (GV) stage of mouse oocytes, followed by dramatic decline in metaphase II stage [23], we used only human GV and metaphase I (MI) arrested oocytes. The GV and MI oocytes were analyzed separately. Within the cohort of oocytes obtained from patients undergoing infertility treatment, 6/72 GV and 14/65 MI oocytes failed to express detectable levels of BCL-X transcripts. An additional sub-group of oocytes (8 at GV stage) expressed reduced levels (less than mean) of BCL-X transcripts (Figure 5A). These data indicate that ∼20% of human growing oocytes obtained from infertile patients either completely lack or possess diminished endowment of BCL-X transcripts.


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)

Expression of BCL-X in human oocytes.(A) Distribution of human oocytes obtained form 43 patients based on their BCL-X expression in either germinal vesicle stage (GV - left) or meiosis I stage (MI - right). Arrows point to groups of oocytes with insufficient endowment of BCL-X transcript. (B) Visualization of protein interaction network that connects BCL-X (BC2L1) with other targets known to be deregulated in arrested human embryos. Node shape, represented by triangles, indicates trends of expression. Shape of triangles pointing up corresponds to genes up-regulated and triangles pointing down correspond to genes down-regulated in arrested human embryos; circles represent direct interacting partners that link BCL-X (BCL2L1) to up- and down-regulated targets. Red highlight on nodes represents the set of cross-linked proteins. Node color is based on gene ontology as per legend. To reduce network complexity, all other nodes and edges are made partially transparent.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0021687-g005: Expression of BCL-X in human oocytes.(A) Distribution of human oocytes obtained form 43 patients based on their BCL-X expression in either germinal vesicle stage (GV - left) or meiosis I stage (MI - right). Arrows point to groups of oocytes with insufficient endowment of BCL-X transcript. (B) Visualization of protein interaction network that connects BCL-X (BC2L1) with other targets known to be deregulated in arrested human embryos. Node shape, represented by triangles, indicates trends of expression. Shape of triangles pointing up corresponds to genes up-regulated and triangles pointing down correspond to genes down-regulated in arrested human embryos; circles represent direct interacting partners that link BCL-X (BCL2L1) to up- and down-regulated targets. Red highlight on nodes represents the set of cross-linked proteins. Node color is based on gene ontology as per legend. To reduce network complexity, all other nodes and edges are made partially transparent.
Mentions: While experiments described above dealt with in vitro induced phenotype, they have clinical relevance to human IVF, where embryos are always maintained in culture for at least 3 days. In addition, it is also possible that some oocytes may lack sufficient storage of maternally derived Bcl-x gene products. In order to determine if variability in the endowment of maternally accumulated BCL-X transcripts could contribute to poor quality of human embryos in patients undergoing IVF, we analyzed its expression in human oocytes. As we have previously observed that Bcl-x transcript expression peaks in germinal vesicle (GV) stage of mouse oocytes, followed by dramatic decline in metaphase II stage [23], we used only human GV and metaphase I (MI) arrested oocytes. The GV and MI oocytes were analyzed separately. Within the cohort of oocytes obtained from patients undergoing infertility treatment, 6/72 GV and 14/65 MI oocytes failed to express detectable levels of BCL-X transcripts. An additional sub-group of oocytes (8 at GV stage) expressed reduced levels (less than mean) of BCL-X transcripts (Figure 5A). These data indicate that ∼20% of human growing oocytes obtained from infertile patients either completely lack or possess diminished endowment of BCL-X transcripts.

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