O-15: Reduced Fertilization After ICSI and Abnormal Phospholipase C Zeta Presence in Spermatozoa from the Wobbler Mouse

Background: Failed fertilization after intracytoplasmic sperm injection (ICSI) can be due to a reduced oocyte-activation capacity caused by reduced concentrations and abnormal localization of the oocyte-activation factor phospholipase C (PLC) zeta. Patients with this condition can be helped to conceive by artificial activation of oocytes after ICSI with calcium ionophore (assisted oocyte activation; AOA). However some concern still exists about this approach. Mouse models could help to identify potential oocyteactivation strategies and evaluate their safety. Materials and Methods: In this study, the fertilizing capacity of wobbler sperm cells was tested and the efficiency of AOA with two exposures to ionomycin to restore fertilization and embryo development was studied. The quality of the obtained blastocysts was assessed and embryo transfer was performed to evaluate post-implantation development. The presence of PLCzeta in the spermatozoa and testis of the wobbler mouse was evaluated by PLCzeta immunostaining and quantitative reverse-transcription polymerase chain reaction. Results: A significant down-regulation of Plcz1 mRNA was seen in wobbler mouse testis, but also down-regulation of protamine 1, a marker for post meiotic spermatids, while the expression of the pre-meiotic germ-cell marker gene VASA (Mvh/Ddx4) was not altered. Sperm cells from wobbler mice had reduced fertilizing capacity and abnormalities in PLCzeta localization, but not in its expression. Artificially activating the oocytes restored fertilization and embryo development. The AOA pups did not show any abnormalities and their postnatal growth was normal in comparison to in vivo control pups. The AOA mice successfully mated and healthy, normal pups were born. Conclusion: The wobbler mouse can be a model for failed fertilization after ICSI to study PLCzeta dynamics and aid in optimization of the AOA method.