Title

Signal transduction in frog and ascidian eggs at fertilization

Date of Completion

January 2000

Keywords

Biology, Cell|Biology, Animal Physiology

Degree

Ph.D.

Abstract

At fertilization, the egg must undergo a rise in intracellular Ca 2+ so that embryogenesis may begin. How the sperm signals the egg to release Ca2+ is not well understood. This dissertation examines how the sperm stimulates Ca2+ release in frog and ascidian eggs at fertilization. ^ One secondary messenger used by somatic cells to elicit intracellular Ca2+ release is inositol trisphosphate (IP3). Results from experiments examining Xenopus oocyte maturation indicate that frog oocytes become more sensitive to IP3 as they mature into fertilizable eggs. Because this evidence suggested that IP3 may be important for Xenopus fertilization, frog eggs were injected with an antibody against the IP3 receptor and then inseminated. The inhibitory effect of this antibody on Ca2+ release after insemination indicates that the Ca2+ rise at fertilization in frog eggs requires IP3. ^ Because IP3 is generated by phospholipase C (PLC) enzymes, the role played by PLCs during Xenopus fertilization was investigated. The PLC family contains three subtypes: β, γ, and δ. Xenopus eggs injected with an antibody against Gq family G-proteins (to inhibit PLCβ activation) do not release Ca2+ in response to serotonin but do release Ca2+ at fertilization. Injecting Xenopus eggs with recombinant Src homology 2 (SH2) domains of PLCγ (to inhibit PLCγ) blocks platelet derived growth factor-induced Ca2+ release but not the Ca2+ rise at fertilization. These results indicate that the Ca2+ rise at fertilization in Xenopus eggs requires neither G q-mediated activation of PLCβ nor SH2-mediated activation of PLCγ. ^ In contrast to the Xenopus results, injecting eggs of an ascidian (an invertebrate chordate) with PLCγ SH2 domains (to inhibit PLCγ) or with Fyn SH2 domains (to inhibit Src family kinases) inhibits the Ca2+ rise at fertilization. That these SH2 domains also inhibit the Ca2+ rise in response to injecting ascidian eggs with an extract of sperm indicates that both sperm extract and fertilization stimulate Ca2+ release using a pathway requiring PLCγ and a Src family kinase. These results support the hypothesis that, during sperm-egg fusion in ascidians, the sperm signals the egg to release Ca 2+ by introducing a protein that activates the Src/PLCγ pathway in the egg. ^