Delayed Cleavage of Pig Embryos after Intracytoplasmic Sperm Injection (ICSI) Relates to an Ability of Blastocyst Development.

Abstract

We have already reported that electrical stimulation enhances in vitro development of ICSI oocytes to the blastocyst stage in pigs (Nakai et al., 2006). The frequency of development to the blastocyst stage and quality of blastocysts of ICSI oocytes are still lower than those after in vitro fertilization (IVF) even when they are stimulated by electrical stimulation. In our previous experiments, we have investigated an effect of pretreatment (sonication for isolation the sperm heads, freeze-drying and freeze-thawing) for sperm on development to the pronuclear (PN) and blastocyst stages after ICSI without artificial stimulation. Although we obtained significant difference in the rates of PN formation among groups, there was no significant difference in the rates of development to the blastocyst stage. These results suggest that, after the PN stage, there may be a key role(s), which relates to the developmental ability of ICSI embryos. In the present study, we compared a state of development from the PN stage to the blastocyst stage of ICSI embryos with that of IVF embryos. We used in vitro matured pig oocytes and frozen-thawed epididymal sperm for experiments. ICSI was conducted with sperm head and ICSI oocytes were stimulated by electrical stimulation at 1 h after injection. At 10 h after ICSI or IVF, we centrifuged these oocytes and selected only zygotes with visible two PNs (considered as monospermy) under inverted microscope. The oocytes were cultured in vitro and stained with propidium iodide (PI), fluorescein diacetate (FDA) and Hoechst 33342 every 24 h after ICSI or IVF until 168 h to evaluate viability of blastomere, cleavage stage, and abnormality of cleavage. Non-equal cleavage and cellular fragmentation were determined as "abnormal cleavage." Live blastomeres appeared green (FDA positive), and their nuclei were labeled with only Hoechst 33342 (blue). The cytoplasm of dead blastomere was FDA negative, of which nucleus was labeled with PI (red). At the morula and blastocyst stages (96-168 h after insemination or ICSI), all embryos were fixed and stained with aceto-orcein for evaluation of cell number and abnormal cleavage. There was no significant difference between ICSI and IVF in the rates of embryos with abnormal cleavage and/or dead blastomeres at the each culture periods. Furthermore, at 96 h, the rate of IVF embryos (54.5%) with more than 5-cells was higher than that of ICSI embryos (31.9%). At 144 h and 168 h, the rate of development to the blastocyst stage and the mean number of cells per blastocyst in IVF were significantly higher than those of ICSI. IVF embryos showed faster cleavage resulting in enhanced blastocyst development compared with ICSI embryos. These results suggest that abnormal cleavage and degeneration of blastomeres were not observed frequently in ICSI embryos. The delay of cleavage of ICSI embryos is associated with disadvantage of subsequent embryonic development. It may be affected by an external injury (injection pipette, electrical stimulation), intracellular calcium oscillation responses and disturbed actin distribution. Further research is necessary to elucidate the cause of delayed cleavage of ICSI embryos.