Figure 1.
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.
Table 1.
Statistics of non-lysis and blastocyst formation rates of mouse embryos with PBS injection using the robotic system.
Figure 2.
Impact of culture medium on developmental competence and BCL-X protein expression of mouse embryos.
(A) HTF culture medium induces 2-cell arrest in a subset of embryos and compromises preimplantation embryo development when compared to KSOM medium (n = 273 embryos/medium). Rates of blastocyst formation at day 4.5 (∼96 hours in culture) as well as total cell number (TCN) are dramatically reduced, while cell death index (CDI) is elevated (n = 54 embryos for KSOM and n = 36 embryos for HTF). Bars indicate mean ± s.e.m. Mann Whitney U-test was used for pairwise comparison. (B) Poor quality of embryos is also reflected by nuclear staining (DAPI), showing smaller blastocysts with multiple apoptotic cells (arrows). (C) Expression of BCL-X protein is decreased in 2-cell embryos cultured for 24 hours in HTF medium. Significant reduction in fluorescent intensity (RFU), generated after immunocytochemical analysis for BCL-X was detected in embryos cultured in HTF (n = 10), when compared to KSOM cultured embryos (n = 9). Control embryos, exposed to no-specific IgG (n = 6), exhibited only very small amount of fluorescence, which was subtracted from the intensity generated by BCL-X antibody. Bars indicate mean ± s.e.m. Student's t-test was used for calculating significance of difference between KSOM and HTF groups.
Figure 3.
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.
Figure 4.
Impact of recBCL-XL (ΔTM) microinjection of embryo metabolism and mitochondrial distribution at 2-cell stage.
(A) Mitochondrial distribution (Mitotracker Red) at 2-cell stage was evaluated by computerized image analysis approach (extracted features; see Text S1) and compared among cultured conditions. RecBCL-XL (ΔTM) protein maintained diffuse mitochondrial pattern (n = 30 embryos), while buffer (n = 30; similar to HTF culture alone), caused preferential clustering of these organelles to subcortical and perinuclear regions (arrows) of 2-cell embryos maintained in culture for 24 hours. (B) Microinjection of recBCL-XL (ΔTM) protein stabilized redox state of 2-cell stage embryos reduced (NAD(P)H and oxidized FAD autofluorescence signal expressed in RFU; n = 15 embryos per condition) and improved Krebs cycle outcome (Citrate/ATP ratio n = 15 embryos per condition). Student's t-test was used for statistical analysis. Bars indicate mean ± s.e.m.
Figure 5.
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.