Title

Voltage Sensitive Phosphoinositide Phosphatases of Xenopus: Their Tissue Distribution and Voltage Dependence

Date of Completion

January 2011

Keywords

Biology, Cell

Degree

Ph.D.

Abstract

The propagation of animal life begins with the generation of a new multicellular organism from the fusion single-celled gametes. In many species, the contents of only one sperm cell may enter the egg cell: a failure to prevent polyspermy results in the eventual failure of the embryo. It is therefore paramount that mechanisms exist to both promote sperm-egg fusion, yet prevent the fusion of multiple sperm with an egg.^ In the frog Xenopus, the prevention of polyspermy is accomplished by a rapid depolarization of the egg cell membrane electrical potential, which has been shown through cross-species fertilization experiments to exert its effect on an unknown 'voltage-sensing' component of the sperm cell. The recent discovery of a voltage-sensitive lipid phosphatase in the sperm of the ascidian Ciona intestinalis (Ci-VSP) has provided a compelling candidate molecule capable of directly effecting a biochemical change in response to a change in membrane electric potential. ^ In the research presented here, I show that the allotetraploid frog Xenopus laevis possesses two VSPs, termed Xl-VSP1 and Xl-VSP2, and that the closely related diploid frog Xenopus tropicalis has only one VSP, termed Xt-VSP. Transcripts for these VSPs were primarily found in the testis and ovary by quantitative RT-PCR. Using a fluorescent PI(4,5)P2 sensor and heterologously expressed Xenopus VSPs, I show that Xl-VSP1 and Xt-VSP activate in the physiological range of voltages known to block polyspermy in Xenopus, but that Xl-VSP2 activates at more positive potentials. Interestingly, the lack of endogenous activity in Xenopus eggs suggests that the transcripts found in the oocyte may be involved in an unknown function during embryonic development. GFP-tagging of Xl-VSP1 has little effect on voltage-dependent enzyme activation, but substitution of glutamine for arginine 152 in the voltage-sensor domain of Xl-VSP1 shifts activation to more negative potentials.^ Furthermore, I describe here the generation and initial characterization of transgenic frogs that express GFP-tagged and R152Q mutant versions of Xl-VSP1 specifically in their sperm for the investigation of the role of VSP in Xenopus sperm physiology. Future research with these frogs may provide important advances in our understanding of this fundamental process of animal reproduction. ^