Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization

Abstract

We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC- γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca]i). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 minute after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca]i and other fertilization events. As compared to 14 other lipids, PA strongly bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca]i, PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca]i release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca]i release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization.

Phospholipase C and D regulation of Src, calcium release and membrane fusion during Xenopus laevis development

“…In Xenopus fertilization, sperm activate phospholipase D1b (PLD1b) to produce PA which then binds and activates Src tyrosine kinase (Bates et al, 2014). A new model for Src activation through PA binding to the SH4, SH3 and a unique domain is presented.…”

“…As two different PLD inhibitors fully inhibited the increase of PA and Src activation at fertilization, but only partially inhibited the increase in IP 3 mass and [Ca +2 ] i release (the latter, by ~87%), sperm activate a different pathway that induces a delayed, weak [Ca +2 ] i release (Bates et al, 2014). In addition, multiple studies with tyrosine kinase inhibitors or inhibition of PLCγ activation with SH2 peptides do not eliminate the [Ca +2 ] i increase induced by sperm (Runft et al, 1999).…”

Extracellular Ca2+ Is Required for Fertilization in the African Clawed Frog, Xenopus laevis

“…laevis [10–13], here we present several pieces of evidence documenting that it is absolutely required. Primarily, we report that both BAPTA and EGTA effectively prevented fertilization and embryonic development in X .…”

“…Moreover, it is a widely held belief in the field of reproductive physiology that Ca 2+ is unessential for fertilization in X . laevis [10–13]. …”

Dynamic analysis of phospholipid metabolism of mouse macrophages treated with common non-steroidal anti-inflammatory drugs

“…, thereby reducing the endoplasmic reticulum stress caused by calcium and affecting macrophage endocytosis [4]. Moreover, PA substances can activate protein kinase C and other related pathways, thus affecting the cell cycle arrest and apoptosis [5].…”