Comparison of Hoechst 33342 and propidium iodide as fluorescent markers for sperm fusion with hamster oocytes.
Hamster oocytes were loaded with the DNA dyes Hoechst 33342 or propidium iodide. Oocytes incubated in 10 mumol Hoechst 333421(-1) showed intracellular fluorescence within 10-20 s of exposure, as did hamster and guinea-pig spermatozoa. Impaled oocytes to which acrosome-intact hamster spermatozoa were bound before injection of Hoechst 33342 showed dye transfer to adhering spermatozoa within 2 min of injection. Oocytes loaded passively with Hoechst 33342 showed dye transfer to bound, acrosome-intact hamster spermatozoa within 10 min. On ultra-structural examination, no bound, acrosome-intact hamster spermatozoa (n = 311) were found to be fused. By contrast, oocytes incubated with 10 mumol propidium iodide l-1 showed no intracellular fluorescence after 2 h, although in approximately 50% of oocytes, fluorescence developed rapidly in the first polar body. Oocytes injected with propidium iodide showed intracellular fluorescence but no dye transfer to bound, acrosome-intact hamster spermatozoa. Oocytes impaled on pipettes containing propidium iodide showed no dye transfer to unlabelled oocytes with which they were brought into contact, whereas in similar experiments using Hoechst 33342 detectable dye transfer to an adjacent oocyte occurred within 10 min. Oocytes loaded with propidium iodide transferred propidium iodide to fusion-competent guinea-pig spermatozoa during in vitro fertilization. Normally, between 20 and 40 spermatozoa bound per oocyte, and the percentage of spermatozoa showing dye transfer varied between 0 and 41%. Dye transfer occurred within 5-45 min. Only those nuclei that showed propidium iodide transfer subsequently decondensed, suggesting that dye transfer is correlated with fusion. The presence of fused spermatozoa was confirmed by ultrastructural examination of oocytes. In separate experiments, hamster and guinea-pig spermatozoa showed detectable fluorescence from propidium iodide within 20 s of osmotic rupture or membrane stripping by detergent, suggesting the lag in dye transfer to sperm nuclei during fertilization reflects a delay in sperm-oocyte fusion following adhesion. This evidence suggests that Hoechst 33342 could be an unreliable marker for sperm-oocyte fusion in fertilization because of its capacity for passive movement from oocyte to spermatozoon. This problem can be overcome using oocytes injected with propidium iodide. With this technique, it was possible to show that fusion-competent guinea-pig spermatozoa that are held in pipettes will fuse with hamster oocytes when placed mechanically against the oocyte surface.
Green
D P
DP
Department of Pharmacology, University of Otago Medical School, Dunedin, New Zealand.
D000818:Animals; D001562:Benzimidazoles; D006224:Cricetinae; D005260:Female; D005456:Fluorescent Dyes; D006168:Guinea Pigs; D008297:Male; D008854:Microscopy, Electron; D009865:Oocytes / Q000648:ultrastructure; D011419:Propidium; D013084:Sperm-Ovum Interactions; D013194:Staining and Labeling / Q000379:methods*
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D011419:Propidium; C017807:bisbenzimide ethoxide trihydrochloride