1997 Molecular Biology of the Cell Article
Vol. 8, pages 755-765, 1997
sn-1,2-DIACYLGLYCEROL AND CHOLINE INCREASE AFTER FERTILIZATION IN XENOPUS LAEVIS.
Bradley J. Stith*, Keith Woronoff, Ronald Espinoza, Tanya Smart
SUMMARY
After fertilization of Xenopus laevis eggs, sn 1,2-diacylglycerol (DAG) mass increased from 62 to 110 pmol/cell at 10-15 min after insemination. This can be compared with our published work showing that the inositol 1,4,5-trisphosphate (IP3) increase is 173 fmol (282-fold smaller) and that IP3 peaks by 5 min after insemination. Choline mass (a measure of phosphatidylcholine-specific phospholipase D) peaked before DAG (at 5 min) and the choline increase (134 pmol/cell) was greater than that of DAG. There was no detectable change in phosphocholine mass (a measure of phosphatidylcholine-specific phospholipase C). During first cleavage, DAG decreased and choline increased and peaked (whereas published work shows an increase in IP3). Artificial elevation of intracellular calcium ([Ca2+]i) increased DAG, choline but not phosphocholine mass. However, prevention of the [Ca2+]i increase after insemination reduced but did not prevent an increase in DAG or choline. Sperm stimulate production of DAG and choline through [Ca++]i-independent and [Ca2+]i-dependent paths.
INTRODUCTION
The events of fertilization may be directly or indirectly due to phosphatidylinositol 4,5-bisphosphate (PIP2)1 breakdown to inositol 1,4,5-trisphosphate (IP3) and sn-1,2-diacylglycerol (DAG) (Whitaker and Irvine, 1984; Turner et al., 1984; Kamel et al., 1985; LePeuch et al., 1985; Ciapa et al., 1992, Stith et al., 1993; Ciapa et al., 1994). A sperm-induced increase in IP3 and intracellular calcium ([Ca2+]i) are thought to be responsible for fertilization events such as membrane depolarization, cortical granule breakdown, intracellular pH increase and the induction of other developmental events (for Xenopus, Busa et al., 1985; Kline, 1988; Grandin and Charbonneau, 1992).
Role of DAG and Protein Kinase C after fertilization
The other second messengers, DAG, may also play a role in fertilization. Acting as an activator of protein kinase C (PKC) (Nishizuka, 1992), DAG may induce resumption of endocytosis (Vasilets et al., 1990; Khan et al., 1991), cortical contraction, cortical granule exocytosis, chromosome decondensation, nuclear envelope and Golgi reformation, and cleavage furrow formation in Xenopus laevis (Bement and Capco, 1989, 1990, 1991a, b). DAG may act independently of PKC since DAG (but not the 1,3 isomer of DAG) regulates actin polymerization (Shariff and Luna, 1992). DAG may be needed for sperm-egg fusion in the sea urchin as neomycin (which presumably inhibits DAG production) inhibits sperm-egg fusion whereas calcium chelators do not (Swann et al., 1987). DAG may also act as a fusogen during cortical granule-plasma membrane fusion (Whitaker, 1987).
Counter to a positive role for PKC, Grandin and Charbonneau (1991) find that PKC inhibitors actually induce Xenopus egg activation and suggest that a decrease in PKC activity is necessary for resumption of the cell cycle (see Bement, 1992, for a possible explanation for
the discrepancy). There is other evidence for a negative role for PKC at fertilization; phorbol esters (which activate PKC) inhibit the fertilization [Ca2+]i increase (Bement and Capco, 1990) and the [Ca2+]i and intracellular pH increase induced by ionophore (Grandin and Charbonneau, 1991). PKC may terminate the fertilization [Ca2+]i increase in hamsters (Swann et al., 1989) but not sea urchin eggs (Ciapa et al., 1988). The extent of a negative role for PKC at fertilization is still under debate (see Bement, 1992).
Using label turnover, Alonso et al. (1986) suggested that diacylglycerol does not change for an hour after fertilization in toads. Yet Ciapa and Whitaker (1986) report a 44% diacylglycerol turnover increase by 21 seconds after insemination with sea urchin gametes, a decrease at 40-60 seconds, and no pronounced second peak. Eckberg and Szuts (1993) found a 15% increase in DAG mass within 5 min of fertilization of Cheatopterus eggs (in these eggs, maturation of the oocyte to the egg is coupled to fertilization; vertebrates such as Xenopus uncouple maturation and fertilization).
Due to methodology concerns, the conflicting published turnover measurements and the possibility of DAG acting independently of PKC, DAG mass measurement is important for an understanding of the events of fertilization. As opposed to many turnover experiments, the DAG mass assay used in the present report records only the active sn-1,2-DAG isomer. The mass assays do not require prelabeling and record the result of all synthetic and degradation pathways. Label turnover measurements may not label all, possibly unknown, precursor pools and the specific activity of the labeled pool may decline during stimulation. Finally, mass assays record an actual amount (not a relative change in turnover rate) that allows direct comparison with changes in the mass of other second messengers.
With mass assays, we report that DAG and choline levels increase after fertilization in Xenopus laevis zygotes (even if elevation of [Ca++]i is prevented) whereas there is no detectable phosphocholine change. During first cleavage, DAG declines whereas choline increases. We also compare these mass changes with those for IP3 (Stith et al., 1993, 1994) and after artificial activation by ionophore or poking.
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1.Abbreviations used: 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid, BAPTA; [Ca2+]i, cytosolic free calcium ion concentration; inositol 1,4,5-trisphosphate, IP3; phosphatidylinositol 4,5-bisphosphate, PIP2; sn-1,2-diacylglycerol, DAG; phosphatidic acid, PA; phosphatidylcholine, PC; protein kinase C, PKC.