Fertilization Stimulates an Increase in Inositol Trisphosphate and Inositol Lipid Levels inXenopusEggs

Abstract

Previous experiments from our lab have suggested that the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is required for sperm-induced egg activation in Xenopus laevis. Here we measure the endogenous production of both Ins(1,4,5)P3 and PIP2 during the sperm-induced and ionomycin-induced calcium wave in the egg and find that both increase following fertilization. Ins(1,4,5)P3 increases 3.2-fold from an unfertilized egg level of 0.13 pmole per egg (0.29 microM) to a peak of 0.42 pmole per egg (0.93 microM) as the calcium wave reaches the antipode in the fertilized egg. This continuous production of Ins(1,4,5)P3 during the time that the Ca2+ wave is propagating across the egg suggests the involvement of Ins(1,4,5)P3 in wave propagation. This increase in Ins(1,4,5)P3 is smaller in ionomycin-activated eggs than in sperm-activated eggs, suggesting that the sperm-induced production of Ins(1,4,5)P3 involves a PIP2 hydrolysis pathway that is not simply raising intracellular Ca2+. While one might expect PIP2 levels to fall as a result of hydrolysis, we find that PIP2 actually increases 2-fold. The total lipid fraction in unfertilized egg exhibits 0.8 pmole PIP2 per egg and this increases to 1.5 pmole as the calcium wave reaches the antipode. The PIP2 concentration peaks 2 min after the completion of the calcium wave at 1.8 pmole per egg. The amount of PIP2 in the animal and vegetal hemispheres of the egg was also measured by cutting frozen eggs in half. The vegetal hemisphere contained twice the amount of PIP2 as the animal hemisphere but it also contained twice the amount of lipid. Thus, there was an equivalent amount of PIP2 normalized to lipid in each hemisphere. Isolated animal and vegetal hemisphere cortices exhibit similar PIP2 concentrations, suggesting that the 2-fold higher total PIP2 in the vegetal half is not due to a gradient of PIP2 in the plasma membrane, but rather implies that cytoplasmic organelle membranes also contain PIP2.

Lipid levels in sperm, eggs, and during fertilization in Xenopus laevis

“…If this large 48 pmol increase in DAG was produced through PLC hydrolysis of PIP2, one might expect more PIP2 present during Xenopus fertilization (?1 pmol/cell) (45). Further support for the large DAG increase deriving from PC, not PIP2, hydrolysis is the timing: we found that the IP3 (and thus, PIP2-derived DAG) production peak was much earlier (5 min) than that of the larger DAG (?12 min).…”

“…By comparing the time course and size of the increase in DAG mass to the increase in IP3 mass at fertilization [the IP3 increase is about 280 times lower and peaks ?5-10 min before that of DAG; (7,8)], and considering that precursor PIP2 mass is never greater than 2 pmol per Xenopus zygote (45), production of 48 pmol of DAG from PIP2 would be unlikely (9). Because choline mass increases on a magnitude equivalent to the increase in DAG, we suggested that breakdown of PC by phospholipase D (PLD) is the source of DAG (9).…”

“…Activation of this enzyme and production of inositol 1,4,5-trisphosphate (IP3) and sn-1,2 diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (PIP2) may be sufficient to induce all events of fertilization [for Xenopus: (7,8,45,46)]. Much work by Sato and associates (46) suggests that Xenopus sperm activate Src tyrosine kinase, which, in turn, activates PLC-g.…”

“…Although one would expect a decrease in PIP2, two reports show an increase in PIP2 during fertilization. First, PIP2 increases from 0.8 to 1.8 pmol of PIP2 per Xenopus egg/zygote during the first 10 min of fertilization (45). Second, presumably due to stimulation of PI 4 kinase by elevated calcium, there is an increase in PIP2 (as detected by an increased signal from a fluorescent PIP2 binding protein) during mouse fertilization (68).…”

Mammalian Fertilization: From Sperm Factor to Phospholipase Cζ

“…Moreover, the degradation of IP 3 R type 1 that is observed in eggs following sperm entry is an indication of IP 3 production (Brind et al, 2000;Jellerette et al, 2000), since it is well documented in somatic cells that IP 3 R type 1 degradation only occurs as a consequence of IP 3 binding (Bokkala and Joseph, 1997;Oberdorf et al, 1999). In this context, it is worth noting that monitoring of IP 3 production in single mammalian zygotes is still not technically possible, although increases in intracellular IP 3 levels have been detected after fertilization in sea urchin and Xenopus laevis eggs (Stith et al, 1993;Snow et al, 1996;Lee and Shen, 1998;Sato et al, 2000;Kuroda et al, 2001) and after injection of porcine sperm extracts into Xenopus eggs (Wu et al, 2001b (Swann, 1992;Ayabe et al, 1995;Yue et al, 1995;Machaty et al, 1997). However, the physiological significance of RyR-mediated [Ca 2+ ] i oscilla-tions is questionable, since they do not lead to full egg activation (Ayabe et al, 1995;Fissore et al, 1995;Wu et al, 1997), and because inhibition of this receptor does not perturb fertilization-induced egg activation (Ayabe et al, 1995).…”