Voltage-Gated Proton Channels Exist in the Plasma Membrane of Human Oocytes

Abstract

Voltage gated proton channels (HV1) are highly expressed in human (but not mouse) sperm where they contribute to capacitation and motility (Lishko et al. 2010. Cell. 140:327; Musset et al. 2012. J. Biol. Chem. 287:9376). We investigated whether HV1 are also present in human oocytes. Frozen, thawed human metaphase II oocytes were obtained from material donated to the gamete repository at the Rush Center for Advanced Reproductive Care. Oocytes were retrieved by transvaginal ovarian follicle aspiration following treatment with recombinant follicle stimulating hormone and purified urinary gonadotropins with GnRH antagonist for controlled ovarian hyperstimulation. After sufficient 18-20 mm follicles were visualized by transvaginal ultrasound, human chorionic gonadotropin was administered to initiate the ovulatory cascade. Transvaginal aspiration of the follicles under ultrasound guidance was performed 36 h later. Human oocytes are large ∼110 μm diameter spheres surrounded by a thick extracellular glycoprotein matrix (the zona pellucida). Their size and zona pellucida make oocytes difficult to study by conventional patch-clamp techniques. To obtain an electrically tight seal we exposed bare oolemma by removing sections of the zona pellucida using a laser, followed by repeated pipetting to further separate the oocyte from the zona pellucida. Patch-clamp studies were performed using whole-cell configuration on oocytes, vesicles derived from oocytes, or inside-out patches of membrane, under conditions optimized for voltage-gated proton currents. Proton currents were present at significant levels in human oocytes where they exhibit properties similar to those reported in other human cells, as well as those in heterologous expression systems transfected with the HVCN1 gene. The gating kinetics, voltage dependence, and selectivity were indistinguishable from those of hHV1 in heterologous expression systems. Possible functions for voltage gated proton channels in human oocytes, most likely involving pH regulation, may now be contemplated.