Intracytoplasmic sperm injection (ICSI) is a technique commonly used in clinical and research settings. In mouse oocytes, conventional ICSI has a poor survival rate caused by a high level of lysis. Cytochalasin B (CB) is a toxic microfilament-inhibiting agent that is known to relax the cytoskeleton and enhance the flexibility of oocytes. CB has been used widely in nuclear transfer experiments to improve the success rate of the micromanipulation, however information describing the use of CB in ICSI is limited. Here, we demonstrated that the addition of 5 μg/ml CB to the manipulation medium of ICSI procedure significantly improved the survival rate of the ICSI embryos (80.74% vs. 89.50%, p < 0.05), and that there was no harm for the in vitro or in vivo development. The birth rates and birth weights were not significantly different between the CB-treated and -untreated groups. Interestingly, the microfilaments of the ICSI embryos were almost undetectable immediately after CB treatment; however, they gradually re-appeared and had fully recovered to the normal level 2 h later. Moreover, CB did not disturb spindle rotation, second polar body formation or pronuclei migration, and had no effect on the microtubules. We thus conclude that ICSI manipulation in CB-containing medium results in significantly improved survival rate of mouse ICSI embryos, and that short-term treatment with CB during ICSI manipulation does not have adverse effects on the development of ICSI embryos.

Tests were conducted to highlight the effects of donor cell treatment (bovine G0- and non-G0 phase somatic cells refrigerated for 1–5 or 12–14 days at 4°C), fusion solution (with or without cytochalasin B) and age of the rabbit (young, 3–5-months old; or multiparous, 10–12 months old) as oocyte donor on the fusion and mortality rates of bovine–rabbit interspecies reconstructed eggs. Results showed that there were no significant differences (P>0.05) in fusion and mortality rates of eggs reconstructed with G0 cells or non-G0 cells as nuclear donor somatic cells. The same was also true when nuclear donor somatic cells (G0 cells or non-G0 cells) were treated at low temperature (4°C) for 1–5 or 12–14 days. The fusion rate of reconstructed eggs did not change significantly (P>0.05) when adding cytochalasin B into the fusion solution, but the mortality rate increased significantly (P<0.05). No significant effect (P>0.05) was found in fusion rate of the eggs reconstructed with young rabbits' and multiparous rabbits' oocytes as recipient, but the mortality rate decreased significantly (P<0.01) for young rabbit oocytes compared with multiparous rabbit ones.

The organization of F-actin in vegetative cells of the brown alga Sphacelaria rigidula was studied after staining with a modified rhodamine-phalloidin (Rh-Ph) protocol. The interphase vegetative cells display a well-organized F-actin cytoskeleton, consisting of cortical, endoplasmic and perinuclear arrays of actin filaments (AFs). The organization of these AFs changes slightly during mitosis, while they almost disappear at cytokinesis. The perinuclear AF population becomes more obvious during prophase, especially at the poles. At metaphase, an actin spindle is organized, co-localized with the microtubule spindle, while at anaphase an interzonal AF population appears, which persists at early telophase. At advanced telophase the image changes to a rather diffuse actin meshwork in the mid-area between the daughter nuclei. During post-telophase-early cytokinesis this AF system becomes gradually disassembled and a conspicuous actin plate is formed on the cytokinetic plane. This plate becomes more compact with the progress of cytokinesis, and seems to overcoat the patches of the forming diaphragm, and finally the daughter plasmalemmata. Treatment with cytochalasin B disturbs mitosis, not allowing the cells to proceed to anaphase, and prevents cytokinesis. These observations show that AFs are ubiquitous cytoskeletal elements of the vegetative cells of brown algae. The probable role of AFs during the cell cycle and particularly the unique actin configuration involved in cytokinesis are discussed.

Hatching has been suggested to occur as a result of protease-mediated lysis and the blastocoele tension. However, even if rupturing is initiated at multiple sites, interestingly only a single site is used for escape. This implies that there are several mechanisms involved in hatching. In this study, the involvement of actin filaments in mouse embryo hatching was examined. We treated mouse embryos with cytochalasin B for 12 h or 24 h at the morula, middle blastocyst, expanded blastocyst, lobe-formed blastocyst and hatching blastocyst stages, and measured the amount and distribution of actin filaments using a confocal microscope. At morula, middle blastocyst, lobe-formed blastocyst and hatching blastocyst stages embryonic development was completely arrested by cytochalasin B. However, when transferred to cytochalasin-B-free medium, the embryos resumed development and escaped the zona pellucida. In the expanded blastocysts development was almost completely inhibited by cytochalasin B, but rupturing occurred in some embryos. However, development stopped completely at the ruptured stage. Distribution of actin filaments was prominent at rupturing and hatching sites regardless of cytochalasin B treatment. The amount of actin filaments was prominent at hatching embryos compared with other developmental stages of embryos. These actin filaments were distributed intensively between the trophectodermal cells, and formed locomotion patterns. Taken together, these results suggest that not only tension and lytic enzymes are required to rupture, but the activity of actin filaments may have a crucial role in the process of hatching.

Tetraploidy was induced in embryos of Ruditapes philippinarum by treating the eggs with cytochalasin B (CB). Two treatment times were found to be effective in inducing tetraploidy: 0 to 10 minutes after insemination (before formation of the first polar body) and 45 minutes after insemination (just before the first cleavage). Treatments, started 5 and 45 minutes after insemination, produced $64.4\pm7.8{\%}$ and $28.3\pm2.3{\%}$ tetraploid embryos, respectively, after 6 hours (on the average over 3 experiments). However, no tetraploids were detected in 4-month-old spat. Sperm entry delayed by the CB could be involved in induction of tetraploidy by early treatment.

An effective treatment using cytochalasin B (1 mg CB/ml dimethylsulfoxyde in 1 litre of sea water) was developed to induce triploidy in the Manila clam Ruditapes philippinarum (Adams and Reeve). The percentage of induced triploidy was assessed by karyological examination of embryos and spat. The treatment was found to be optimal beween 20 to 35 minutes post-insemination exposure to CB, at 25 °C, and produced 75.8 ± 5.7% triploid embryos on the average over four experiments. Growth and survival of larvae to metamorphosis were recorded for two different rearings each split into two replicates. Both larval rearings showed similar survival up to settlement: 19.3 and 27.0%, on average over two replicates, in the treated batches relative to the controls. Mean shell length of treated larvae was significantly smaller than that of the controls in one of the larval rearings, where mortality of triploids relative to diploids within the treated group was low (21.0%). No significant differences in shell length were observed in the other trial where a high rate of differential mortality (64.5%) was recorded.