In mammals, repetitive increases of the intracellular Ca2+ level, known as Ca2+ oscillations, are observed in oocytes immediately after sperm-oocyte fusion, which is a prerequisite event for oocyte activation. Previous studies indicate that phospholipase C zeta (PLCζ), a strong candidate sperm factor for triggering Ca2+ oscillations, is localized in the sperm head of several mammalian species. We have reported that the rate of pronucleus formation in oocytes injected with a sperm head is lower than that for oocytes injected with a whole spermatozoon (Nakai et al. 2009 IETS). This has given rise to a hypothesis that not only the sperm head but also the tail play a role in inducing oocyte activation in pigs. In this study, we attempted to detect the localization of PLCζ in the pig sperm tail and also its ability to activate porcine oocytes after injection. To clarify the localization of PLCζ in pig sperm, frozen-thawed ejaculated pig sperm were immunostained using an anti-PLCζ antibody that has been reported previously (Kurokawa et al. 2005). Western blotting was also carried out to examine whether PLCζ (72 kDa) was present in the sperm tail. Sperm tails were detached from the head by sonication and then collected after centrifugation in a Percoll density gradient. We also confirmed whether the sperm tail itself had the ability to trigger oocyte activation using the following 4 injection groups: (1)1 sperm head (Head), (2) 1 sperm tail (Tail), (3) 1 sperm head and 1 tail (Head + Tail), and (4) Sham. The nuclear status of the injected oocyte was evaluated at 10 h after injection. In the present study, we used 3 sperm samples that were prepared from different boars. In pig sperm, the acrosome, tail, and post-acrosomal regions were stained by the PLCζ antibody. The signals in both the post-acrosomal and tail regions disappeared after pretreatment with antigenic peptide, but that in the acrosome region was retained. Furthermore, we confirmed the presence of a band of approximately 72 kDa from the sperm tail and also confirmed its disappearance upon pretreatment with antigenic peptide. The rates of oocytes released from metaphase-II arrest in the Head, Tail, and Head+Tail groups were significantly higher than that in the Sham group (P < 0.05 by ANOVA andTukey test). However, most of the oocytes in the Tail group failed to form pronuclei and showed other meiotic stages (anaphase-II, telophase-II, or metaphase-III). In conclusion, we have shown that PLCζ is expressed in the post-acrosomal and tail region of pig sperm. It is suggested that, in the pig, the sperm tail participates in the triggering of oocyte activation.
The authors thankRafaelA. Fissore (Department ofVeterinary and Animal Sciences, University of Massachusetts Amherst) for providing the antigenic peptide for PLCζ. This study was supported in part by JSPS Fellowship (71310042 to M.N.) from the Japanese Society for Promotion of Science (JSPS).
Mammalian Oocyte Activation by the Synergistic Action of Discrete Sperm Head Components: Induction of Calcium Transients and Involvement of Proteolysis