Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion

Abstract

Summary. Mouse oocytes exposed to 1 \ g=m\ g 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\g=m\g/ml),obscuring the fluorescence of the fertilizing spermatozoa. Spermatozoa added to zona-free mouse oocytes (pre-loaded with 1 or 10 \g=m\gH-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 pg H-33342/ml became labelled. This leakage was not visibly apparent when protease-treated oocytes were exposed to only 1 \g=m\g H\ x=r eq-\ 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.

PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization

“…In vitro fertilization (IVF) was performed using intact oocytes (Hogan et al, 1994) or zona pellucida free eggs stained with 1 µg/ml of Hoechst 33258 (Sigma-Aldrich) (Conover and Gwatkin, 1988). After insemination embryos were cultured in KSOM media (Cell & Molecular Technologies).…”

Tssk6 is required for Izumo relocalization and gamete fusion in the mouse

“…Previously published protocols with slight modifications were used (Conover and Gwatkin, 1988;Wortzman et al, 2006). Briefly, sperm were obtained as above and left to capacitate as described for the IVF assays.…”

Regulation of cellular adhesion molecule expression in murine oocytes, peri-implantation and post-implantation embryos

“…To obtain blastocyst outgrowth freshly isolated blastocysts were cultured in groups of 10 for 96 h [15] on a coverslip (13 mm in diameter, Mediglass, Australia) placed in a 24-well plate (Nunclon 1 43982, Nunc, Denmark) in 2 ml DMEM, pre-equilibrated under standard culture conditions (SCC; i.e., humidified 95% air with 5% CO 2 at 37 ). To obtain hatched blastocysts, freshly isolated blastocysts were cultured under the above conditions for approximately 48 h.…”