A spontaneous increase in intracellular Ca2+ in metaphase II human oocytes in vitro can be prevented by drugs targeting ATP-sensitive K+ channels

Goncalo Fernandes, Navin Dasai, Natalia Kozlova, Albaraa Mojadadi, Mandy Gall, Ellen Drew, Evelyn Barratt, Oladipo A. Madamidola, Sean G. Brown, Alison M. Milne, Sarah J. Martins Da Silva, Katherine M. Whalley, Christopher L. R. Barratt, Aleksandar Jovanovic (Lead / Corresponding author)

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Abstract

STUDY QUESTION: Could drugs targeting ATP-sensitive K + (K ATP ) channels prevent any spontaneous increase in intracellular Ca 2+ that may occur in human metaphase II (MII) oocytes under in vitro conditions? SUMMARY ANSWER: Pinacidil, a K ATP channel opener, and glibenclamide, a K ATP channel blocker, prevent a spontaneous increase in intracellular Ca 2+ in human MII oocytes. WHAT IS KNOWN ALREADY: The quality of the oocyte and maintenance of this quality during in vitro processing in the assisted reproductive technology (ART) laboratory is of critical importance to successful embryo development and a healthy live birth. Maintenance of Ca 2+ homeostasis is crucial for cell wellbeing and increased intracellular Ca 2+ levels is a well established indicator of cell stress. STUDY DESIGN, SIZE, DURATION: Supernumerary human oocytes (n=102) collected during IVF / ICSI treatment that failed to fertilize were used from October 2013 to July 2015. All experiments were performed on mature (MII) oocytes. Dynamics of intracellular Ca 2+ levels were monitored in oocytes in the following experimental groups: 1) Control, 2) Dimethyl sulfoxide (DMSO; used to dissolve pinacidil, glibenclamide and 2,4-Dinitrophenol (DNP)), 3) Pinacidil, 4) Glibenclamide, 5) DNP: an inhibitor of oxidative phosphorylation), 6) Pinacidil and DNP and 7) Glibenclamide and DNP. PARTICIPANTS/MATERIALS/SETTINGS/METHODS: Oocytes were collected under sedation as part of routine treatment at an assisted conception unit from healthy women (mean±SD) age 34.1±0.6 years, n=41). Those surplus to clinical use were donated for research. Oocytes were loaded with Fluo-3 Ca 2+ -sensitive dye, and monitored by laser confocal microscopy for 2 hours at 10 min intervals. Time between oocyte collection and start of Ca 2+ monitoring was 80.4±2.1 h 26 MAIN RESULTS AND THE ROLE OF CHANCE: Intracellular levels of Ca 2+ increased under in vitro conditions with no deliberate challenge, as shown by Fluo-3 fluorescence increasing from 61.0±11.8 AU (AU=arbitrary units; n=23) to 91.8±14.0 AU (n=19; P<0.001) after 2 hours of monitoring. Pinacidil (100µM) inhibited this increase in Ca 2+ (85.3±12.3 AU at the beginning of the experiment, 81.7±11.0 AU at the end of the experiment; n=13; P=0.616). Glibenclamide (100µM) also inhibited the increase in Ca 2+ (74.7±10.6 AU at the beginning and 71.8±10.9 AU at the end of the experiment; n=13; P=0.851. DNP (100 mM) induced an increase in intracellular Ca 2+ that was inhibited by glibenclamide (100µM; n=9) but not by pinacidil (100µM; n=5). LIMITATIONS, REASONS FOR CAUTION: Owing to clinical and ethical considerations it was not possible to monitor Ca 2+ in MII oocytes immediately after retrieval. MII oocytes were available for our experimentation only after unsuccessful IVF or ICSI, which was, on average, 80.4±2.1 h (n=102 oocytes) after the moment of retrieval. As the MII oocytes used here were those that were not successfully fertilised, it is possible that they may have been abnormal with impaired Ca 2+ homeostasis and, furthermore, the altered Ca 2+ homeostasis might have been associated solely with the protracted incubation. WIDER IMPLICATIONS OF THE FINDINGS: These results show that maintenance of oocytes under in vitro conditions is associated with intracellular increase in Ca 2+ , which can be counteracted by drugs targeting K ATP channels. As Ca 2+ homeostasis is crucial for contributing to a successful outcome of ART, these results suggest that K ATP channel openers and blockers should be tested as drugs for improving success rates of ART.
Original languageEnglish
Pages (from-to)287-297
Number of pages11
JournalHuman Reproduction
Volume31
Issue number2
Early online date18 Dec 2015
DOIs
Publication statusPublished - Feb 2016

Fingerprint

Drug Delivery Systems
Metaphase
Oocytes
Adenosine Triphosphate
Pinacidil
Glyburide
2,4-Dinitrophenol
Assisted Reproductive Techniques
Homeostasis
Intracytoplasmic Sperm Injections
Dimethyl Sulfoxide
Confocal Microscopy
In Vitro Techniques
Maintenance
Dye Lasers
Oocyte Retrieval
Oxidative Phosphorylation
Live Birth
Embryonic Development
Fluorescence

Keywords

  • Oocyte quality
  • Assisted reproduction
  • Oxidative stress
  • Ca2+
  • ATP-sensitive K+ channels
  • Pinacidil
  • Glibenclamide

Cite this

Fernandes, Goncalo ; Dasai, Navin ; Kozlova, Natalia ; Mojadadi, Albaraa ; Gall, Mandy ; Drew, Ellen ; Barratt, Evelyn ; Madamidola, Oladipo A. ; Brown, Sean G. ; Milne, Alison M. ; Martins Da Silva, Sarah J. ; Whalley, Katherine M. ; Barratt, Christopher L. R. ; Jovanovic, Aleksandar. / A spontaneous increase in intracellular Ca2+ in metaphase II human oocytes in vitro can be prevented by drugs targeting ATP-sensitive K+ channels. In: Human Reproduction. 2016 ; Vol. 31, No. 2. pp. 287-297.
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abstract = "STUDY QUESTION: Could drugs targeting ATP-sensitive K + (K ATP ) channels prevent any spontaneous increase in intracellular Ca 2+ that may occur in human metaphase II (MII) oocytes under in vitro conditions? SUMMARY ANSWER: Pinacidil, a K ATP channel opener, and glibenclamide, a K ATP channel blocker, prevent a spontaneous increase in intracellular Ca 2+ in human MII oocytes. WHAT IS KNOWN ALREADY: The quality of the oocyte and maintenance of this quality during in vitro processing in the assisted reproductive technology (ART) laboratory is of critical importance to successful embryo development and a healthy live birth. Maintenance of Ca 2+ homeostasis is crucial for cell wellbeing and increased intracellular Ca 2+ levels is a well established indicator of cell stress. STUDY DESIGN, SIZE, DURATION: Supernumerary human oocytes (n=102) collected during IVF / ICSI treatment that failed to fertilize were used from October 2013 to July 2015. All experiments were performed on mature (MII) oocytes. Dynamics of intracellular Ca 2+ levels were monitored in oocytes in the following experimental groups: 1) Control, 2) Dimethyl sulfoxide (DMSO; used to dissolve pinacidil, glibenclamide and 2,4-Dinitrophenol (DNP)), 3) Pinacidil, 4) Glibenclamide, 5) DNP: an inhibitor of oxidative phosphorylation), 6) Pinacidil and DNP and 7) Glibenclamide and DNP. PARTICIPANTS/MATERIALS/SETTINGS/METHODS: Oocytes were collected under sedation as part of routine treatment at an assisted conception unit from healthy women (mean±SD) age 34.1±0.6 years, n=41). Those surplus to clinical use were donated for research. Oocytes were loaded with Fluo-3 Ca 2+ -sensitive dye, and monitored by laser confocal microscopy for 2 hours at 10 min intervals. Time between oocyte collection and start of Ca 2+ monitoring was 80.4±2.1 h 26 MAIN RESULTS AND THE ROLE OF CHANCE: Intracellular levels of Ca 2+ increased under in vitro conditions with no deliberate challenge, as shown by Fluo-3 fluorescence increasing from 61.0±11.8 AU (AU=arbitrary units; n=23) to 91.8±14.0 AU (n=19; P<0.001) after 2 hours of monitoring. Pinacidil (100µM) inhibited this increase in Ca 2+ (85.3±12.3 AU at the beginning of the experiment, 81.7±11.0 AU at the end of the experiment; n=13; P=0.616). Glibenclamide (100µM) also inhibited the increase in Ca 2+ (74.7±10.6 AU at the beginning and 71.8±10.9 AU at the end of the experiment; n=13; P=0.851. DNP (100 mM) induced an increase in intracellular Ca 2+ that was inhibited by glibenclamide (100µM; n=9) but not by pinacidil (100µM; n=5). LIMITATIONS, REASONS FOR CAUTION: Owing to clinical and ethical considerations it was not possible to monitor Ca 2+ in MII oocytes immediately after retrieval. MII oocytes were available for our experimentation only after unsuccessful IVF or ICSI, which was, on average, 80.4±2.1 h (n=102 oocytes) after the moment of retrieval. As the MII oocytes used here were those that were not successfully fertilised, it is possible that they may have been abnormal with impaired Ca 2+ homeostasis and, furthermore, the altered Ca 2+ homeostasis might have been associated solely with the protracted incubation. WIDER IMPLICATIONS OF THE FINDINGS: These results show that maintenance of oocytes under in vitro conditions is associated with intracellular increase in Ca 2+ , which can be counteracted by drugs targeting K ATP channels. As Ca 2+ homeostasis is crucial for contributing to a successful outcome of ART, these results suggest that K ATP channel openers and blockers should be tested as drugs for improving success rates of ART.",
keywords = "Oocyte quality, Assisted reproduction, Oxidative stress, Ca2+, ATP-sensitive K+ channels, Pinacidil, Glibenclamide",
author = "Goncalo Fernandes and Navin Dasai and Natalia Kozlova and Albaraa Mojadadi and Mandy Gall and Ellen Drew and Evelyn Barratt and Madamidola, {Oladipo A.} and Brown, {Sean G.} and Milne, {Alison M.} and {Martins Da Silva}, {Sarah J.} and Whalley, {Katherine M.} and Barratt, {Christopher L. R.} and Aleksandar Jovanovic",
note = "University of Dundee, MRC (MR/K013343/1, MR/012492/1), NHS Tayside, Funding NHS fellowship (Dr Sarah Martins da Silva), NHS Scotland. Funding to pay the Open Access publication charges for this article was provided by MRC.",
year = "2016",
month = "2",
doi = "10.1093/humrep/dev300",
language = "English",
volume = "31",
pages = "287--297",
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Fernandes, G, Dasai, N, Kozlova, N, Mojadadi, A, Gall, M, Drew, E, Barratt, E, Madamidola, OA, Brown, SG, Milne, AM, Martins Da Silva, SJ, Whalley, KM, Barratt, CLR & Jovanovic, A 2016, 'A spontaneous increase in intracellular Ca2+ in metaphase II human oocytes in vitro can be prevented by drugs targeting ATP-sensitive K+ channels', Human Reproduction, vol. 31, no. 2, pp. 287-297. https://doi.org/10.1093/humrep/dev300

A spontaneous increase in intracellular Ca2+ in metaphase II human oocytes in vitro can be prevented by drugs targeting ATP-sensitive K+ channels. / Fernandes, Goncalo; Dasai, Navin; Kozlova, Natalia; Mojadadi, Albaraa; Gall, Mandy; Drew, Ellen; Barratt, Evelyn; Madamidola, Oladipo A.; Brown, Sean G.; Milne, Alison M.; Martins Da Silva, Sarah J.; Whalley, Katherine M.; Barratt, Christopher L. R.; Jovanovic, Aleksandar (Lead / Corresponding author).

In: Human Reproduction, Vol. 31, No. 2, 02.2016, p. 287-297.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A spontaneous increase in intracellular Ca2+ in metaphase II human oocytes in vitro can be prevented by drugs targeting ATP-sensitive K+ channels

AU - Fernandes, Goncalo

AU - Dasai, Navin

AU - Kozlova, Natalia

AU - Mojadadi, Albaraa

AU - Gall, Mandy

AU - Drew, Ellen

AU - Barratt, Evelyn

AU - Madamidola, Oladipo A.

AU - Brown, Sean G.

AU - Milne, Alison M.

AU - Martins Da Silva, Sarah J.

AU - Whalley, Katherine M.

AU - Barratt, Christopher L. R.

AU - Jovanovic, Aleksandar

N1 - University of Dundee, MRC (MR/K013343/1, MR/012492/1), NHS Tayside, Funding NHS fellowship (Dr Sarah Martins da Silva), NHS Scotland. Funding to pay the Open Access publication charges for this article was provided by MRC.

PY - 2016/2

Y1 - 2016/2

N2 - STUDY QUESTION: Could drugs targeting ATP-sensitive K + (K ATP ) channels prevent any spontaneous increase in intracellular Ca 2+ that may occur in human metaphase II (MII) oocytes under in vitro conditions? SUMMARY ANSWER: Pinacidil, a K ATP channel opener, and glibenclamide, a K ATP channel blocker, prevent a spontaneous increase in intracellular Ca 2+ in human MII oocytes. WHAT IS KNOWN ALREADY: The quality of the oocyte and maintenance of this quality during in vitro processing in the assisted reproductive technology (ART) laboratory is of critical importance to successful embryo development and a healthy live birth. Maintenance of Ca 2+ homeostasis is crucial for cell wellbeing and increased intracellular Ca 2+ levels is a well established indicator of cell stress. STUDY DESIGN, SIZE, DURATION: Supernumerary human oocytes (n=102) collected during IVF / ICSI treatment that failed to fertilize were used from October 2013 to July 2015. All experiments were performed on mature (MII) oocytes. Dynamics of intracellular Ca 2+ levels were monitored in oocytes in the following experimental groups: 1) Control, 2) Dimethyl sulfoxide (DMSO; used to dissolve pinacidil, glibenclamide and 2,4-Dinitrophenol (DNP)), 3) Pinacidil, 4) Glibenclamide, 5) DNP: an inhibitor of oxidative phosphorylation), 6) Pinacidil and DNP and 7) Glibenclamide and DNP. PARTICIPANTS/MATERIALS/SETTINGS/METHODS: Oocytes were collected under sedation as part of routine treatment at an assisted conception unit from healthy women (mean±SD) age 34.1±0.6 years, n=41). Those surplus to clinical use were donated for research. Oocytes were loaded with Fluo-3 Ca 2+ -sensitive dye, and monitored by laser confocal microscopy for 2 hours at 10 min intervals. Time between oocyte collection and start of Ca 2+ monitoring was 80.4±2.1 h 26 MAIN RESULTS AND THE ROLE OF CHANCE: Intracellular levels of Ca 2+ increased under in vitro conditions with no deliberate challenge, as shown by Fluo-3 fluorescence increasing from 61.0±11.8 AU (AU=arbitrary units; n=23) to 91.8±14.0 AU (n=19; P<0.001) after 2 hours of monitoring. Pinacidil (100µM) inhibited this increase in Ca 2+ (85.3±12.3 AU at the beginning of the experiment, 81.7±11.0 AU at the end of the experiment; n=13; P=0.616). Glibenclamide (100µM) also inhibited the increase in Ca 2+ (74.7±10.6 AU at the beginning and 71.8±10.9 AU at the end of the experiment; n=13; P=0.851. DNP (100 mM) induced an increase in intracellular Ca 2+ that was inhibited by glibenclamide (100µM; n=9) but not by pinacidil (100µM; n=5). LIMITATIONS, REASONS FOR CAUTION: Owing to clinical and ethical considerations it was not possible to monitor Ca 2+ in MII oocytes immediately after retrieval. MII oocytes were available for our experimentation only after unsuccessful IVF or ICSI, which was, on average, 80.4±2.1 h (n=102 oocytes) after the moment of retrieval. As the MII oocytes used here were those that were not successfully fertilised, it is possible that they may have been abnormal with impaired Ca 2+ homeostasis and, furthermore, the altered Ca 2+ homeostasis might have been associated solely with the protracted incubation. WIDER IMPLICATIONS OF THE FINDINGS: These results show that maintenance of oocytes under in vitro conditions is associated with intracellular increase in Ca 2+ , which can be counteracted by drugs targeting K ATP channels. As Ca 2+ homeostasis is crucial for contributing to a successful outcome of ART, these results suggest that K ATP channel openers and blockers should be tested as drugs for improving success rates of ART.

AB - STUDY QUESTION: Could drugs targeting ATP-sensitive K + (K ATP ) channels prevent any spontaneous increase in intracellular Ca 2+ that may occur in human metaphase II (MII) oocytes under in vitro conditions? SUMMARY ANSWER: Pinacidil, a K ATP channel opener, and glibenclamide, a K ATP channel blocker, prevent a spontaneous increase in intracellular Ca 2+ in human MII oocytes. WHAT IS KNOWN ALREADY: The quality of the oocyte and maintenance of this quality during in vitro processing in the assisted reproductive technology (ART) laboratory is of critical importance to successful embryo development and a healthy live birth. Maintenance of Ca 2+ homeostasis is crucial for cell wellbeing and increased intracellular Ca 2+ levels is a well established indicator of cell stress. STUDY DESIGN, SIZE, DURATION: Supernumerary human oocytes (n=102) collected during IVF / ICSI treatment that failed to fertilize were used from October 2013 to July 2015. All experiments were performed on mature (MII) oocytes. Dynamics of intracellular Ca 2+ levels were monitored in oocytes in the following experimental groups: 1) Control, 2) Dimethyl sulfoxide (DMSO; used to dissolve pinacidil, glibenclamide and 2,4-Dinitrophenol (DNP)), 3) Pinacidil, 4) Glibenclamide, 5) DNP: an inhibitor of oxidative phosphorylation), 6) Pinacidil and DNP and 7) Glibenclamide and DNP. PARTICIPANTS/MATERIALS/SETTINGS/METHODS: Oocytes were collected under sedation as part of routine treatment at an assisted conception unit from healthy women (mean±SD) age 34.1±0.6 years, n=41). Those surplus to clinical use were donated for research. Oocytes were loaded with Fluo-3 Ca 2+ -sensitive dye, and monitored by laser confocal microscopy for 2 hours at 10 min intervals. Time between oocyte collection and start of Ca 2+ monitoring was 80.4±2.1 h 26 MAIN RESULTS AND THE ROLE OF CHANCE: Intracellular levels of Ca 2+ increased under in vitro conditions with no deliberate challenge, as shown by Fluo-3 fluorescence increasing from 61.0±11.8 AU (AU=arbitrary units; n=23) to 91.8±14.0 AU (n=19; P<0.001) after 2 hours of monitoring. Pinacidil (100µM) inhibited this increase in Ca 2+ (85.3±12.3 AU at the beginning of the experiment, 81.7±11.0 AU at the end of the experiment; n=13; P=0.616). Glibenclamide (100µM) also inhibited the increase in Ca 2+ (74.7±10.6 AU at the beginning and 71.8±10.9 AU at the end of the experiment; n=13; P=0.851. DNP (100 mM) induced an increase in intracellular Ca 2+ that was inhibited by glibenclamide (100µM; n=9) but not by pinacidil (100µM; n=5). LIMITATIONS, REASONS FOR CAUTION: Owing to clinical and ethical considerations it was not possible to monitor Ca 2+ in MII oocytes immediately after retrieval. MII oocytes were available for our experimentation only after unsuccessful IVF or ICSI, which was, on average, 80.4±2.1 h (n=102 oocytes) after the moment of retrieval. As the MII oocytes used here were those that were not successfully fertilised, it is possible that they may have been abnormal with impaired Ca 2+ homeostasis and, furthermore, the altered Ca 2+ homeostasis might have been associated solely with the protracted incubation. WIDER IMPLICATIONS OF THE FINDINGS: These results show that maintenance of oocytes under in vitro conditions is associated with intracellular increase in Ca 2+ , which can be counteracted by drugs targeting K ATP channels. As Ca 2+ homeostasis is crucial for contributing to a successful outcome of ART, these results suggest that K ATP channel openers and blockers should be tested as drugs for improving success rates of ART.

KW - Oocyte quality

KW - Assisted reproduction

KW - Oxidative stress

KW - Ca2+

KW - ATP-sensitive K+ channels

KW - Pinacidil

KW - Glibenclamide

U2 - 10.1093/humrep/dev300

DO - 10.1093/humrep/dev300

M3 - Article

VL - 31

SP - 287

EP - 297

JO - Human Reproduction

JF - Human Reproduction

SN - 0268-1161

IS - 2

ER -