R Ya Smith1, D Morgan2, L Sharma1, V V Cherny2, N Tidswell1, M W Molo1, T E DeCoursey2. 1. Rush Center for Advanced Reproductive Care, Department of Obstetrics and Gynecology, Rush University Medical Center, Chicago, IL 60612, USA. 2. Department of Physiology & Biophysics, Rush University, Chicago, IL 60612, USA.
Abstract
STUDY QUESTION: Do human oocytes express voltage-gated proton channels? SUMMARY ANSWER: Human oocytes exhibit voltage-gated proton currents. WHAT IS KNOWN ALREADY: Voltage-gated proton currents have been reported in human sperm, where they contribute to capacitation and motility. No such studies of human oocytes exist. STUDY DESIGN, SIZE, DURATION: Voltage-clamp studies were undertaken using entire oocytes and vesicles derived from oocytes and in excised patches of membrane from oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Frozen, thawed human metaphase II oocytes were obtained from material donated to the gamete repository at the Rush Center for Advanced Reproductive Care. Prior to patch clamping, oocytes were warmed and equilibrated. Formation of an electrically tight seal requires exposing bare oolemma. Sections of the zona pellucida (ZP) were removed using a laser, followed by repeated pipetting, to further separate the oocyte from the ZP. Patch-clamp studies were performed using the whole-cell configuration on oocytes or vesicles derived from oocytes, and using inside-out patches of membrane, under conditions optimized to detect voltage-gated proton currents. MAIN RESULTS AND THE ROLE OF CHANCE: Proton currents are 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 that codes for the voltage-gated proton channel. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Human oocytes are large cells, which limits our ability to control the intracellular solution. Subtle effects of cryopreservation by vitrification and subsequent warming on properties of HVCN1, the HVCN1 gene product, cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS: Possible functions for voltage-gated proton channels in human oocytes may now be contemplated. STUDY FUNDING/COMPETING INTEREST(S): NIH R35GM126902 (TED), Bears Care (DM). No competing interests. TRIAL REGISTRATION NUMBER: N/A.
STUDY QUESTION: Do human oocytes express voltage-gated proton channels? SUMMARY ANSWER: Human oocytes exhibit voltage-gated proton currents. WHAT IS KNOWN ALREADY: Voltage-gated proton currents have been reported in human sperm, where they contribute to capacitation and motility. No such studies of human oocytes exist. STUDY DESIGN, SIZE, DURATION: Voltage-clamp studies were undertaken using entire oocytes and vesicles derived from oocytes and in excised patches of membrane from oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Frozen, thawed human metaphase II oocytes were obtained from material donated to the gamete repository at the Rush Center for Advanced Reproductive Care. Prior to patch clamping, oocytes were warmed and equilibrated. Formation of an electrically tight seal requires exposing bare oolemma. Sections of the zona pellucida (ZP) were removed using a laser, followed by repeated pipetting, to further separate the oocyte from the ZP. Patch-clamp studies were performed using the whole-cell configuration on oocytes or vesicles derived from oocytes, and using inside-out patches of membrane, under conditions optimized to detect voltage-gated proton currents. MAIN RESULTS AND THE ROLE OF CHANCE: Proton currents are 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 that codes for the voltage-gated proton channel. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Human oocytes are large cells, which limits our ability to control the intracellular solution. Subtle effects of cryopreservation by vitrification and subsequent warming on properties of HVCN1, the HVCN1 gene product, cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS: Possible functions for voltage-gated proton channels in human oocytes may now be contemplated. STUDY FUNDING/COMPETING INTEREST(S): NIH R35GM126902 (TED), Bears Care (DM). No competing interests. TRIAL REGISTRATION NUMBER: N/A.
Keywords:
HV1; HVCN1; IVF; assisted reproduction; gamete repository; ion channels; metaphase II human oocytes; patch clamp; vitrified oocytes; voltage clamp
Authors: Thomas K Berger; David M Fußhöller; Normann Goodwin; Wolfgang Bönigk; Astrid Müller; Nasim Dokani Khesroshahi; Christoph Brenker; Dagmar Wachten; Eberhard Krause; U Benjamin Kaupp; Timo Strünker Journal: J Physiol Date: 2017-01-15 Impact factor: 5.182