Tolerance of zygotes to photobleaching.

<p>(A) The effects of stronger and/or longer photobleaching on preimplantation development were investigated. Preimplantation development of the embryos subjected to 3-fold stronger (300 μW) and/or 6-fold longer (30 s) bleaching than the standard conditions (110 μW; 5 s). Confocal images of embryos during FRAP analysis. ROIs are indicated by red rectangles. Scale bar = 10 μm. The blastocysts shown on the right were derived from the FRAP-analyzed zygotes shown on the left. (Scale bar = 100 μm). (B) Rate of development of embryos subjected to standard (110 μW, 5 s) and stronger and longer (300 μW, 30 s) bleaching: unbleached control, white; standard, gray; and stronger/longer bleaching, black. Three independent experiments were performed in which at least 42 embryos were examined in total. Two-cell, 4–8 cell, morula (Mor), and blastocyst (Blast) stages were observed at 24, 48, 72, and 96 hpi, respectively. There is no significant difference between 110 μW, 5 s and 300 μW, 30 s; <i>P</i> value by Fisher’s exact test is <i>P</i> < 0.9999 (2C-Mor.) and <i>P</i> = 0.7136 (Blast.).</p

Healthy pups derived from FRAP-analyzed zygotes.

<p>Photographs of the pups obtained from FRAP-analyzed zygotes (A). Normal growth was observed during nursing of the FRAP-analyzed pups (B) and chart indicating the weights of pups derived from intact embryos (blue), unbleached control embryos (red), and FRAP-analyzed embryos (green). Body weights of 29 pups derived from intact embryos, 24 from no-FRAP embryos, and 15 from FRAP-analyzed embryos over an 8-week period. (D) PCR genotyping of 15 pups derived from FRAP-analyzed embryos. DNA was extracted from tails and purified for use as PCR templates. Mr, indicates molecular marker. P.C (positive control) was prepared with using a GFP-expressing transgenic ICR mouse. A mouse derived from intact zygotes was used as an N.C (negative control).</p

Optimization of fluorescence recovery after photobleaching (FRAP).

<p>(A) mRNA encoding eGFP-H2B was injected into the cytoplasm of 1-cell-stage embryos 1 h post-insemination (hpi). The eGFP-H2B–expressing zygotes were collected at 8–12 hpi and subjected to FRAP analysis. Representative photographs of FRAP analysis were taken before bleaching (A-1), soon after bleaching (A-2), and after recovery (A-3). Regions of interests (ROIs) are indicated by red rectangles. The nuclear membranes (white dotted lines) and nucleoli (asterisks) are indicated. Scale bar = 10 μm. (B) Recovery curves obtained from zygotes injected with eGFP-H2B mRNA at 50, 100, and 250 ng/μL. Curves indicate fluorescence recovery in male (blue) and female (red) pronuclei. Three independent experiments were performed in which 31, 30, and 21 embryos were examined in total, respectively. (C) Recovery curve obtained using newer confocal laser scanning microscope (FV1200). Data shown are from three independent experiments, examining 58 embryos in total. (D, E) Recovery curves and mobile fraction obtained from zygotes prepared by using spermatozoa from ICR, BDF1, B6N, and C3H. At least three experiments were performed in which 45, 35, 27 and 31 embryos were used, respectively. Single dots indicate individual pronuclei; blue and red dots indicate the mobile fraction score obtained from each male and female pronuclei, respectively. Asterisks indicate significant differences between male and female pronuclei (<i>P</i> < 0.05, by paired <i>t</i> test) (F) Preimplantation development of FRAP-analyzed embryos with eGFP-H2B mRNA at 250 ng/μL. Representative images of FRAP-analyzed preimplantation developing embryos are shown. Two-cell, 4–8 cell, and blastocyst stage embryos at 24, 48, and 96 hpi, respectively. Yellow circle indicates the well-developed blastocyst. This blastocyst is enlarged and shown on the right panel. Scale bar = 100 μm. (G) Bar graph of developmental rates of FRAP-analyzed embryos. Bars indicate FRAP-analyzed (FRAP) embryos (black) and controls embryos (white) injected with mRNA but no FRAP analysis (No FRAP). Data shown are from three independent experiments, examining at least 57 embryos in total. Two-cell, 4–8 cell, morula (Mo), and blastocyst (Bl) stages were observed at 24, 48, 72, and 96 hpi, respectively.</p

Offspring from Mouse Embryos Developed Using a Simple Incubator-Free Culture System with a Deoxidizing Agent

<div><p>To culture preimplantation embryos <em>in vitro</em>, water-jacketed CO₂ incubators are used widely for maintaining an optimal culture environment in terms of gas phase, temperature and humidity. We investigated the possibility of mouse embryo culture in a plastic bag kept at 37°C. Zygotes derived from <em>in vitro</em> fertilization or collected from naturally mated B6D2F1 female mice were put in a drop of medium on a plastic culture dish and then placed in a commercially available plastic bag. When these were placed in an oven under air at 37°C for 96 h, the rate of blastocyst development and the cell numbers of embryos decreased. However, when the concentration of O₂ was reduced to 5% using a deoxidizing agent and a small oxygen meter, most zygotes developed into blastocysts. These blastocysts were judged normal according to their cell number, Oct3/4 and Cdx2 gene expression levels, the apoptosis rate and the potential for full-term development after embryo transfer to pseudopregnant recipients. Furthermore, using this system, normal offspring were obtained simply by keeping the bag on a warming plate. This culture method was applied successfully to both hybrid and inbred strains. In addition, because the developing embryos could be observed through the transparent wall of the bag, it was possible to capture time-lapse images of live embryos until the blastocyst stage without needing an expensive microscope-based incubation chamber. These results suggest that mouse zygotes are more resilient to their environment than generally believed. This method might prove useful in economical culture systems or for the international shipment of embryos.</p> </div

Abnormalities in placentae derived from cloned embryos.

<p>(A) Placental weights from ICSI-derived control embryos and CB- or LatA-treated cloned embryos. Error bars indicate SD. Asterisks indicate significant difference at p < 0.05. (B) Hematoxylin and eosin staining of placentae derived from ICSI-derived control and CB- or LatA-treated cloned embryos. Abnormal distortion of the boundary between the spongiotrophoblast and labyrinth layers was observed in placentas derived from both CB- and LatA-treated cloned embryos. Bar = 500 μm.</p

Full-term development of embryos cultured in the warm box.

<p>Key: Gas perm. film, gas-permeable film; M/B, Morula/Blastocyst; Frag, fragmentation; implant., implantation</p><p>Full-term development of embryos cultured in the warm box.</p

Full-term development of embryos cultured in a plastic bag using a warming plate.

*<p>5% CO₂, 5% O₂ and 90% N_2.</p

Enucleation of MII chromosomes from oocytes with fluorescence observation using halogen lamp.

<p>The MII chromosomes of bovine oocytes are normally invisible by conventional microscopy because of dark lipid droplets in the ooplasm (A). After labeling with an H3S10ph antibody–phycoerythrin conjugate, the MII chromosomes could be recognized clearly and this allowed us to remove them along with minimal cytoplasm (B). MII chromosomes in porcine oocytes were detected by the same method (C). Some chromosomes (arrow) were left occasionally inside the cytoplasm of mouse oocytes after enucleation (D). Live and healthy cloned mice (brown) were obtained after enucleation using this method without any significant decrease in the success rate (E). Enucleation of the MII spindle from mouse oocytes with this system. (F) Before, (G) during and (H) after enucleation.</p

Production of cloned mice using phycoerythrin and antibody injected oocyte.

<p>Production of cloned mice using phycoerythrin and antibody injected oocyte.</p

Experimental procedure and culture systems.

<p>(A) Schematic diagram of the experimental procedure and each culture system. Air-Oven experiment: the bag was filled with air and kept in an oven at 37°C. DeO₂-Oven experiment: the deoxidizing agent was put into the bag, which was then kept in the oven. MixG-Oven experiment: the bag was filled with a gas mixture (see methodology) and kept in the oven. DeO₂-WP experiment: the deoxidizing agent was put into the bag, which was then placed on a warming plate at 37°C. (B) The hermetically sealed plastic bag with a deoxidizing agent is indicated by an arrow. (C) The O₂ concentration was regulated by the deoxidizing agent and measured using the oxygen meter (arrowhead). (D) After removal of the deoxidizing agent from the plastic bag.</p