Sperm-egg fusion is the prelude to the initial Ca2+ increase at fertilization in the mouse.
Fusion of sperm and egg plasma membranes is an early and essential event at fertilization but it is not known if it plays a part in the signal transduction mechanism that leads to the oscillations in the cytoplasmic free Ca2+ concentration ([Ca2+]i) that accompany mammalian egg activation. We have used two independent fluorescence methods and confocal microscopy to show that cytoplasmic continuity of egg and sperm precedes the onset of the first [Ca2+]i increase in mouse eggs. The Ca2+ indicator dye Ca2+-green dextran was microinjected and its transfer from egg to sperm was monitored. We found that it occurred before, and without a requirement for, any detectable [Ca2+]i increase in the egg. In separate experiments [Ca2+]i changes were recorded in populations of eggs, using fura red, and the eggs fixed at various times after some of the eggs had shown a [Ca2+]i transient. Fusion of the sperm and egg was then assessed by Hoechst dye transfer. All eggs that showed a [Ca2+]i increase had a fused sperm but more than half of the eggs contained a sperm but had not undergone a [Ca2+]i increase. These data indicate that sperm-egg fusion precedes [Ca2+]i changes and we estimate that the elapsed time between sperm-egg fusion and the onset of the [Ca2+li oscillations is 1-3 minutes. Finally, sperm-egg fusion was prevented by using low pH medium which reversibly prevented [Ca2+]i oscillations in eggs that had been inseminated. This was not due to disruption of signalling mechanisms, since [Ca2+]i changes still occurred if low pH was applied after the onset of oscillations at fertilization. [Ca2+]i changes also occurred in eggs in low pH in response to the muscarinic agonist carbachol. These data are consistent with the idea that the [Ca2+]i signals that occur in mammalian eggs at fertilization are initiated by events that are closely coupled to the fusion of the sperm and egg membranes.
Lawrence
Y
Y
Department of Anatomy and Developmental Biology, University College London, UK.
Whitaker
M
M
Swann
K
K
D000177:Acrosome / Q000502:physiology; D000818:Animals; D001562:Benzimidazoles; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D002459:Cell Fusion; D002462:Cell Membrane / Q000502:physiology; D003593:Cytoplasm / Q000502:physiology; D005260:Female; D005456:Fluorescent Dyes; D007093:Imidazoles; D007700:Kinetics; D008297:Male; D008561:Membrane Fusion; D051379:Mice; D008815:Mice, Inbred Strains; D018613:Microscopy, Confocal; D009991:Oscillometry; D010063:Ovum / Q000166:cytology / Q000502:physiology*; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology*; D013997:Time Factors
D001562:Benzimidazoles; D001572:Benzofurans; D005456:Fluorescent Dyes; D007093:Imidazoles; C082877:fura red; C017807:bisbenzimide ethoxide trihydrochloride; D002118:Calcium