Fertilization induces a transient exposure of phosphatidylserine in mouse eggs.
Phosphatidylserine (PS) is normally localized to the inner leaflet of the plasma membrane and the requirement of PS translocation to the outer leaflet in cellular processes other than apoptosis has been demonstrated recently. In this work we investigated the occurrence of PS mobilization in mouse eggs, which express flippase Atp8a1 and scramblases Plscr1 and 3, as determined by RT-PCR; these enzyme are responsible for PS distribution in cell membranes. We find a dramatic increase in binding of flouresceinated-Annexin-V, which specifically binds to PS, following fertilization or parthenogenetic activation induced by SrCl2 treatment. This increase was not observed when eggs were first treated with BAPTA-AM, indicating that an increase in intracellular Ca(2+) concentration was required for PS exposure. Fluorescence was observed over the entire egg surface with the exception of the regions overlying the meiotic spindle and sperm entry site. PS exposure was also observed in activated eggs obtained from CaMKIIγ null females, which are unable to exit metaphase II arrest despite displaying Ca(2+) spikes. In contrast, PS exposure was not observed in TPEN-activated eggs, which exit metaphase II arrest in the absence of Ca(2+) release. PS exposure was also observed when eggs were activated with ethanol but not with a Ca(2+) ionophore, suggesting that the Ca(2+) source and concentration are relevant for PS exposure. Last, treatment with cytochalasin D, which disrupts microfilaments, or jasplakinolide, which stabilizes microfilaments, prior to egg activation showed that PS externalization is an actin-dependent process. Thus, the Ca(2+) rise during egg activation results in a transient exposure of PS in fertilized eggs that is not associated with apoptosis.
Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científico y Técnicas, Buenos Aires, Argentina.
Claudio A
CA
Curia
Juan I
JI
Ernesto
Paula
P
Stein
Dolores
D
Busso
Richard M
RM
Schultz
Patricia S
PS
Cuasnicu
Débora J
DJ
Cohen
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000172:deficiency / Q000235:genetics; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D005306:Fertilization / Q000502:physiology*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D015870:Gene Expression; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D010063:Ovum / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D010718:Phosphatidylserines / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology; D015053:Zygote / Q000378:metabolism
D017304:Annexin A5; D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; C493426:Plscr1 protein, mouse; C493428:Plscr3 protein, mouse; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D015638:Cytochalasin D; D004533:Egtazic Acid; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium