270 PRETREATMENTS OF PORCINE SPERM BEFORE INTRACYTOPLASMIC SPERM INJECTION AFFECT QUANTITY OF PLCζ;

Abstract

In pigs, the intracytoplasmic sperm injection (ICSI) procedure alone is insufficient to induce oocyte activation for embryonic development. Artificial activation can be accomplished, such as by electrical pulse-enhanced in vitro development to the blastocyst stage (Nakai et al. 2006). It is well known that the sperm factor (phospholipase Cζ; PLCζ) in spermatozoa, which triggers oocyte activation, is diffused into ooplasm when sperm fuse with oocytes. Our previous study showed that the activation rate of porcine oocytes injected with one sonicated sperm head was significantly lower than that of oocytes injected with a whole spermatozoon or with 3 sonicated sperm heads (Nakai et al. IETS 2007). These results suggest that the sonication treatment per se may affect the quantity of PLCζ in sperm. Furthermore, various pretreatments of sperm besides sonication have been conducted (e.g. removal of the sperm membrane) to increase the efficacy of ICSI. In this study, we investigated the effect of pretreatments (sonication, Triton X-100, and repeated cycles of freezing–thawing without cryoprotectant) on the quantity of PLCζ in porcine sperm. Cryopreserved-thawed boar-ejaculated sperm were used for 3 experimental groups: (1) sperm were sonicated for 10 s in pig-fertilization medium (pig-FM; Suzuki et al. 2002; Soni group), (2) freezing–thawing was repeated 3 times in pig-FM without cryoprotectant (3-F/T group), or (3) sperm were incubated in pig-FM supplemented with 0.1 or 1% Triton X-100 at 37°C for 1 min (0.1 and 1% Triton X-100 groups, respectively). Cryopreserved-thawed whole sperm without any treatment was used as a control. Results from staining with fluorescein diacetate and propidium iodide showed that almost all sperm were propidium iodide positive (dead sperm) immediately after the each treatment. In the control group, approximately 40% of sperm were fluorescein diacetate positive (live sperm) after thawing. The presence of PLCζ (72 kDa) was examined by Western blotting using the antibody against the N-terminal 19-mer sequence of porcine PLCζ (Kurokawa et al. 2005). A band corresponding to porcine PLCζ was not detected in any treatment group in any culture period (from 0 to 135 min). In contrast, PLCζ was detected in the control group and in all culture periods. These results strongly suggest that PLCζ in porcine sperm was lost immediately after the pretreatments, such as by sonication, incubation with 0.1 or 1% Triton X-100, and repeated cycles of freezing–thawing. The decrease in PLCζ protein by pretreatment may be one of the causes of incomplete activation of oocytes in porcine ICSI.

This work was supported by a Grant-in-Aid for JSPS Fellows.