Rapid visual detection of sperm-egg fusion using the DNA-specific fluorochrome Hoechst 33342.
When unfertilized sea urchin eggs are pretreated with the bisbenzimide DNA-specific fluorochrome Hoechst 33342, then washed and fertilized, a single sperm bound to the egg surface becomes intensely fluorescent. The location of the fluorescent sperm on the egg surface coincides exactly with the epicenter of the cortical reaction and the site at which the insemination cone subsequently appears. These observations, coupled with studies of eggs treated with quercetin to prevent fusion, as well as eggs made polyspermic by halothane exposure, indicate that the sperm acquires fluorescence as a consequence of fusion with the fluorochrome preloaded egg. Using a modification of this technique, we have found that cytoplasmic continuity between the sperm and egg is established at 4-8 sec after the onset of the sperm-induced conductance increase in the egg.
D000818:Animals; D001562:Benzimidazoles; D005260:Female; D005306:Fertilization / Q000187:drug effects; D006221:Halothane / Q000494:pharmacology; D008297:Male; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence / Q000379:methods; D011794:Quercetin / Q000494:pharmacology; D012617:Sea Urchins / Q000196:embryology; D013084:Sperm-Ovum Interactions / Q000187:drug effects; D013997:Time Factors
D001562:Benzimidazoles; D011794:Quercetin; C017807:bisbenzimide ethoxide trihydrochloride; D006221:Halothane