PURPOSE: Our goal was to study the activation current in physiologically competent metaphase II human oocytes, i.e., not previously exposed to spermatozoa or aged in vitro, and, in particular, to determine whether a soluble sperm factor triggers a fertilization current comparable to that observed with intact spermatozoa and to characterize the current involved. METHODS: The whole-cell voltage-clamp technique was used on spare metaphase II oocytes, obtained with patient consent from IVF programs. In this configuration a soluble fraction from human spermatozoa was microinjected, and the current recorded. RESULTS: Metaphase II human oocytes generate bell-shaped outward currents of 400-1000 pA (X = 706 +/- 322; n = 10), following injection of a cytosolic extract from human spermatozoa. The amount of sperm extract injected was less than 10% of the total oocyte volume and was equivalent to 1-10 spermatozoa. A similar current was generated following exposure to 20 microM of the calcium ionophore A23187 (n = 10). The steady-state conductance of the oocyte increased from 10 to 19.8 nS (n = 10) following injection of the sperm factor and from 5.3 to 27.7 nS following ionophore exposure. Both sperm factor- and ionophore-induced currents were reduced in amplitude when the unfertilized oocyte was preexposed to 25-75 microM iberiotoxin (n = 8) and eliminated at a concentration of 100 microM iberiotoxin. CONCLUSIONS: The data support the hypothesis of a soluble sperm factor involved in the activation of human oocytes and shows that the initial activation response in the human oocyte is the gating of Ca(2+)-activated K+ channels.
PURPOSE: Our goal was to study the activation current in physiologically competent metaphase II human oocytes, i.e., not previously exposed to spermatozoa or aged in vitro, and, in particular, to determine whether a soluble sperm factor triggers a fertilization current comparable to that observed with intact spermatozoa and to characterize the current involved. METHODS: The whole-cell voltage-clamp technique was used on spare metaphase II oocytes, obtained with patient consent from IVF programs. In this configuration a soluble fraction from human spermatozoa was microinjected, and the current recorded. RESULTS: Metaphase II human oocytes generate bell-shaped outward currents of 400-1000 pA (X = 706 +/- 322; n = 10), following injection of a cytosolic extract from human spermatozoa. The amount of sperm extract injected was less than 10% of the total oocyte volume and was equivalent to 1-10 spermatozoa. A similar current was generated following exposure to 20 microM of the calcium ionophore A23187 (n = 10). The steady-state conductance of the oocyte increased from 10 to 19.8 nS (n = 10) following injection of the sperm factor and from 5.3 to 27.7 nS following ionophore exposure. Both sperm factor- and ionophore-induced currents were reduced in amplitude when the unfertilized oocyte was preexposed to 25-75 microM iberiotoxin (n = 8) and eliminated at a concentration of 100 microM iberiotoxin. CONCLUSIONS: The data support the hypothesis of a soluble sperm factor involved in the activation of human oocytes and shows that the initial activation response in the human oocyte is the gating of Ca(2+)-activated K+ channels.
Authors: Qingyou Du; Sofija Jovanović; Andriy Sukhodub; Evelyn Barratt; Ellen Drew; Katherine M Whalley; Vanessa Kay; Marie McLaughlin; Evelyn E Telfer; Christopher L R Barratt; Aleksandar Jovanović Journal: Hum Reprod Date: 2010-09-16 Impact factor: 6.918