Measurement of intracellular IP3 during Ca2+ oscillations in mouse eggs with GFP-based FRET probe.
Intracellular Ca2+ oscillations in fertilized mammalian eggs, the key signal that stimulates egg activation and early embryonic development, are regulated by inositol 1,4,5-trisphosphate (IP3) signaling pathway. We investigated temporal changes in intracellular IP3 concentration ([IP3]i) in mouse eggs, using a fluorescent probe based on fluorescence resonance energy transfer between two green fluorescent protein variants, during Ca2+ oscillations induced by fertilization or expression of phospholipase Czeta (PLCzeta), an egg-activating sperm factor candidate. Fluorescence measurements suggested the elevation of [IP3]i in fertilized eggs, and the enhancement of PLCzeta-mediated IP3 production by cytoplasmic Ca2+ was observed during Ca2+ oscillations or in response to CaCl2 microinjection. The results supported the view that PLCzeta is the sperm factor to stimulate IP3 pathway, and suggested that high Ca2+ sensitivity of PLCzeta activity and positive feedback from released Ca2+ are important for triggering and maintaining Ca2+ oscillations.
Shirakawa
Hideki
H
Department of Applied Physics and Chemistry, The University of Electro-Communications, Tokyo 182-8585, Japan. hshrkw@pc.uec.ac.jp
Ito
Masahiko
M
Sato
Moritoshi
M
Umezawa
Yoshio
Y
Miyazaki
Shunichi
S
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000235:genetics / Q000502:physiology*; D005260:Female; D005306:Fertilization / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D015544:Inositol 1,4,5-Trisphosphate / Q000032:analysis / Q000378:metabolism*; D051379:Mice; D010063:Ovum / Q000378:metabolism*; D013997:Time Factors; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism
D049452:Green Fluorescent Proteins; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D002118:Calcium