BOVINE OOCYTE PLASMA-MEMBRANE BINDING-SITES FOR SPERM PLASMA-MEMBRANEDURING IN-VITRO OOCYTE MATURATION AND FERTILIZATION

The experimental objective was to determine whether the capability of bovine oocyte plasma membrane to bind sperm changes during in vitro oo cyte maturation and fertilisation. Binding was quantified by the inten sity of tetramethylrhodamine isothiocyanate (TRITC) fluorescence at th e periphery of oocytes following incubation with biotinylated sperm pl asma membrane proteins and subsequent incubation with TRITC-avidin. Bo vine 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. Oocy tes were denuded of cumulus cells and zona pellucida and co-incubated with 56 mu g biotinylated bovine sperm plasma membrane protein for 45 min in 150 mu l drops of saline-BSA. Controls were incubated for the s ame time period in the absence of sperm plasma membrane proteins. All oocytes were rinsed, incubated with TRITC-avidin and subsequently fixe d and transferred to mounting medium. Oocytes were scanned with a conf ocal microscope and analysed using ImageQuant software. The binding of sperm plasma membrane was quantified by integrated fluorescent intens ity in standardised ellipses spaced around the plasma membrane of the oocyte. Values are expressed as mean intensity units per 320 pixel ell ipse. Binding of sperm plasma membrane continued to increase throughou t in vitro oocyte maturation and fertilisation (9051, 24 318 and 49 95 3 for 0 and 22 h in vitro matured oocytes and fertilised oocytes, resp ectively; p = 0.0001). A dramatic decrease in sperm plasma membrane bi nding to the oocyte plasma membrane was observed in 2-cell embryos (me an intensity = 24 477, p = 0.0001). The observed binding was primarily due to the binding of sperm plasma membrane proteins, as control oocy tes incubated with TRITC-avidin only were barely visible (integrated f luorescence intensity values ranged from 8 to 3757).