Bovine oocyte plasma membrane binding sites for sperm plasma membrane during in vitro oocyte maturation and fertilisation.

Abstract

The experimental objective was to determine whether the capability of bovine oocyte plasma membrane to bind sperm changes during in vitro oocyte maturation and fertilisation. Binding was quantified by the intensity of tetramethylrhodamine isothiocyanate (TRITC) fluorescence at the periphery of oocytes following incubation with biotinylated sperm plasma membrane proteins and subsequent incubation with TRITC-avidin. Bovine oocytes were matured in vitro. Sample groups were removed after 0,6 and 22 h, or inseminated and further cultured for 24 or 48 h. Oocytes were denuded of cumulus cells and zona pellucida and co-incubated with 56 micrograms biotinylated bovine sperm plasma membrane protein for 45 min in 150 microliters drops of saline-BSA. Controls were incubated for the same time period in the absence of sperm plasma membrane proteins. All oocytes were rinsed, incubated with TRITC-avidin and subsequently fixed and transferred to mounting medium. Oocytes were scanned with a confocal microscope and analysed using ImageQuant software. The binding of sperm plasma membrane was quantified by integrated fluorescent intensity in standardised ellipses spaced around the plasma membrane of the oocyte. Values are expressed as mean intensity units per 320 pixel ellipse. Binding of sperm plasma membrane continued to increase throughout in vitro oocyte maturation and fertilisation (9051, 24318 and 49953 for 0 and 22 h in vitro matured oocytes and fertilised oocytes, respectively; p = 0.0001). A dramatic decrease in sperm plasma membrane binding to the oocyte plasma membrane was observed in 2-cell embryos (mean intensity = 24477, p = 0.0001). The observed binding was primarily due to the binding of sperm plasma membrane proteins, as control oocytes incubated with TRITC-avidin only were barely visible (integrated fluorescence intensity values ranged from 8 to 3757.