Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion.
Mouse oocytes exposed to 1 microgram Hoechst 33342 (H-33342)/ml and then fertilized in vitro developed normally into blastocysts and blastocyst outgrowths. After penetration of the zona, the fertilizing spermatozoon showed intense fluorescence upon fusion with the vitelline membrane. Due to fluorochrome leakage from the perivitelline space a faint fluorescence was detected in zona-bound spermatozoa. This fluorescence of zona-bound spermatozoa intensified with increased fluorochrome concentration (10 micrograms/ml), obscuring the fluorescence of the fertilizing spermatozoa. Spermatozoa added to zona-free mouse oocytes (pre-loaded with 1 or 10 micrograms H-33342/ml) fluoresced within 10 min of insemination, provided the zonae were removed mechanically. Removal by protease digestion induced leakage of fluorochrome, so that all spermatozoa in the vicinity of an oocyte pre-loaded with 10 micrograms H-33342/ml became labelled. This leakage was not visibly apparent when protease-treated oocytes were exposed to only 1 microgram H-33342/ml. The technique could not be applied to zona-free hamster oocytes under our conditions, since the fluorochrome leaked freely from the oocytes whether the zona was removed mechanically or enzymically.
Conover
J C
JC
Research Institute and Gynecology, Cleveland Clinic Foundation, OH 44106.
Gwatkin
R B
RB
D000818:Animals; D001562:Benzimidazoles; D002118:Calcium / Q000494:pharmacology; D006224:Cricetinae; D005260:Female; D005307:Fertilization in Vitro; D005456:Fluorescent Dyes; D008297:Male; D051379:Mice; D008808:Mice, Inbred CBA; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D009865:Oocytes / Q000187:drug effects; D013084:Sperm-Ovum Interactions
D001562:Benzimidazoles; D005456:Fluorescent Dyes; C017807:bisbenzimide ethoxide trihydrochloride; D002118:Calcium