Abstract
In Xenopus laevis, sperm-egg interaction promotes partial proteolysis and/or tyrosine phosphorylation of uroplakin III (UPIII) and the tyrosine kinase Src, which both localize to the cholesterol-enriched egg membrane microdomains (MDs). Here we show that sperm promote proteolysis and/or tyrosine phosphorylation of UPIII and Src in MDs isolated from ovulated and unfertilized eggs (UF-MDs). An antibody against the extracellular domain of UPIII interferes with these events. Inhibition of fertilization by anti-UPIII antibody is rescued by co-incubation with UF-MDs. This suggests that, like MDs in intact eggs, the isolated UF-MDs are capable of interacting with sperm, an interaction that does not interfere with normal fertilization but rather augments the ability of sperm to fertilize eggs pretreated with anti-UPIII antibody. This unexpected effect of UF-MDs on sperm requires UPIII function in UF-MDs and protein kinase activity in sperm. MDs isolated from progesterone-treated mature oocytes, but not ovarian immature oocytes, are similarly functional as UF-MDs. The anti-UPIII extracellular domain antibody binds more effectively to the surface of mature than immature ovarian oocytes. We propose that the structural and functional competency of the UPIII-Src signaling system in MDs is strictly regulated during oocyte maturation and subsequently in sperm-mediated egg activation and fertilization. The fertilization-related signaling properties seen in UF-MDs can be partially reconstituted in MDs of human embryonic kidney 293 cells (293-MDs) expressing UPIII, Src and uroplakin Ib. However, 293-MDs expressing a proteolysis-resistant mutant of UPIII are less functional, suggesting that the availability of UPIII to protease action is important for MD function.
Highlights
IntroductionWe demonstrated that uroplakin III (UPIII) is a substrate of the egg cytoplasmic tyrosine kinase Src (Sakakibara et al, 2005), the activation of which is required for sperm-induced activation of phospholipase Cγ and transient increase in intracellular calcium concentration (Sato et al, 1999, 2000, 2001)
We previously showed that uroplakin III (UPIII), a singletransmembrane-domain protein that is expressed in the unfertilized egg of the frog Xenopus laevis, serves as a target of sperm-derived protease known to be essential for successful fertilization, and that pretreatment of unfertilized Xenopus eggs with an antibody that recognizes the extracellular sequence of UPIII is inhibitory to fertilization (Mahbub Hasan et al, 2005; Sakakibara et al, 2005)
Sperm-dependent tyrosine phosphorylation of UPIII and Src can be reconstituted in MDs isolated from unfertilized eggs To examine fertilization-related signaling function of MDs in vitro, we isolated MDs from ovulated, unfertilized Xenopus eggs and performed an in vitro kinase assay in the absence or presence of either sperm or cathepsin B, a tryptic protease that can promote the parthenogenetic activation of Xenopus eggs (Fig. 1D)
Summary
We demonstrated that UPIII is a substrate of the egg cytoplasmic tyrosine kinase Src (Sakakibara et al, 2005), the activation of which is required for sperm-induced activation of phospholipase Cγ and transient increase in intracellular calcium concentration (Sato et al, 1999, 2000, 2001). We showed that activation of Src could be reconstituted in vitro by the use of MDs that are isolated from unfertilized eggs (Sato et al, 2003) These results suggest that the MD-associated UPIII-Src system plays a pivotal role in gamete adhesion/fusion and subsequent developmental activation (Mahbub Hasan et al, 2011)
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