Role of phospholipase Cgamma at fertilization and during mitosis in sea urchin eggs and embryos.
It is well known that stimulation of egg metabolism after fertilization is due to a rise in intracellular free calcium concentration. In sea urchin eggs, this first calcium signal is followed by other calcium transients that allow progression through mitotic control points of the cell cycle of the early embryo. How sperm induces these calcium transients is still far from being understood. In sea urchin eggs, both InsP3 and ryanodine receptors contribute to generate the fertilization calcium transient, while the InsP3 receptor generates the subsequent mitotic calcium transients. The identity of the mechanisms that generate InsP3 after fertilization remains an enigma. In order to determine whether PLCgamma might be the origin of the peaks of InsP3 production that punctuate the first mitotic cell cycles of the fertilized sea urchin egg, we have amplified by RT-PCR several fragments of sea urchin PLCgamma containing the two SH2 domains. The sequence shares similarities with SH2 domains of PLCgamma from mammals. One fragment was subcloned into a bacterial expression plasmid and a GST-fusion protein was produced and purified. Antibodies raised to the GST fusion protein demonstrate the presence of PLCgamma protein in eggs. Microinjection of the fragment into embryos interferes with mitosis. A related construct made from bovine PLCgamma also delayed or prevented entry into mitosis and blocked or prolonged metaphase. The bovine construct also blocked the calcium transient at fertilization, in contrast to a tandem SH2 control construct which did not inhibit either fertilization or mitosis. Our data indicate that PLCgamma plays a key role during fertilization and early development.
Shearer
J
J
Department of Physiological Sciences, Medical School, University of Newcastle upon Tyne, Framlington Place, NE2 4HH, UK.
De Nadai
C
C
Emily-Fenouil
F
F
Gache
C
C
Whitaker
M
M
Ciapa
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D018076:DNA, Complementary; D005260:Female; D005306:Fertilization / Q000502:physiology*; D005982:Glutathione Transferase / Q000235:genetics; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D008297:Male; D008938:Mitosis / Q000502:physiology*; D008969:Molecular Sequence Data; D010063:Ovum / Q000201:enzymology*; D051966:Phospholipase C gamma; D011233:Precipitin Tests; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D012617:Sea Urchins; D013084:Sperm-Ovum Interactions / Q000502:physiology; D010738:Type C Phospholipases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D018909:src Homology Domains
D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D010738:Type C Phospholipases; D051966:Phospholipase C gamma