Sperm factor induces intracellular free calcium oscillations by stimulating the phosphoinositide pathway.
Injection of a porcine cytosolic sperm factor (SF) or of a porcine testicular extract into mammalian eggs triggers oscillations of intracellular free calcium ([Ca(2+)](i)) similar to those initiated by fertilization. To elucidate whether SF activates the phosphoinositide (PI) pathway, mouse eggs or SF were incubated with U73122, an inhibitor of events leading to phospholipase C (PLC) activation and/or of PLC itself. In both cases, U73122 blocked the ability of SF to induce [Ca(2+)](i) oscillations, although it did not inhibit Ca(2+) release caused by injection of inositol 1,4,5-triphosphate (IP(3)). The inactive analogue, U73343, had no effect on SF-induced Ca(2+) responses. To determine at the single cell level whether SF triggers IP(3) production concomitantly with a [Ca(2+)](i) rise, SF was injected into Xenopus oocytes and IP(3) concentration was determined using a biological detector cell combined with capillary electrophoresis. Injection of SF induced a significant increase in [Ca(2+)](i) and IP(3) production in these oocytes. Using ammonium sulfate precipitation, chromatographic fractionation, and Western blotting, we determined whether PLCgamma1, PLCgamma2, or PLCdelta4 and/or its splice variants, which are present in sperm and testis, are responsible for the Ca(2+) activity in the extracts. Our results revealed that active fractions do not contain PLCgamma1, PLCgamma2, or PLCdelta4 and/or its splice variants, which were present in inactive fractions. We also tested whether IP(3) could be the sensitizing stimulus of the Ca(2+)-induced Ca(2+) release mechanism, which is an important feature of fertilized and SF-injected eggs. Eggs injected with adenophostin A, an IP(3) receptor agonist, showed enhanced Ca(2+) responses to CaCl(2) injections. Thus, SF, and probably sperm, induces [Ca(2+)](i) rises by persistently stimulating IP(3) production, which in turn results in long-lasting sensitization of Ca(2+)-induced Ca(2+) release. Whether SF is itself a PLC or whether it acts upstream of the egg's PLCs remains to be elucidated.
Wu
H
H
Molecular and Cellular Biology Program and Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst 01003, USA.
Smyth
J
J
Luzzi
V
V
Fukami
K
K
Takenawa
T
T
Black
S L
SL
Allbritton
N L
NL
Fissore
R A
RA
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D017398:Alternative Splicing; D000818:Animals; D001685:Biological Factors / Q000494:pharmacology*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005260:Female; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D008845:Microinjections; D009865:Oocytes / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology; D012661:Semen / Q000737:chemistry; D013552:Swine; D013737:Testis / Q000737:chemistry; D014020:Tissue Extracts / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014981:Xenopus
D001685:Biological Factors; D004791:Enzyme Inhibitors; D004963:Estrenes; D007527:Isoenzymes; D010716:Phosphatidylinositols; D011760:Pyrrolidinones; D014020:Tissue Extracts; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C085029:adenophostin A; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D000241:Adenosine; D002122:Calcium Chloride; D002118:Calcium