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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 (Oxford, England) (2015-12-20)
Gonçalo 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 Jovanović
ABSTRACT

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? 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. 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. 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: (i) Control, (ii) Dimethyl sulfoxide (DMSO; used to dissolve pinacidil, glibenclamide and 2,4-Dinitrophenol (DNP)), (iii) Pinacidil, (iv) Glibenclamide, (v) DNP: an inhibitor of oxidative phosphorylation, (vi) Pinacidil and DNP and (vii) Glibenclamide and DNP. 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 h at 10 min intervals. Time between oocyte collection and start of Ca(2+) monitoring was 80.4 ± 2.1 h. 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 h of monitoring. Pinacidil (100 µM) 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 (100 µM) 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 (100 µM; n = 9) but not by pinacidil (100 µM; n = 5). 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 fertilized, 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. 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. University of Dundee, MRC (MR/K013343/1, MR/012492/1), NHS Tayside. Funding NHS fellowship (Dr Sarah Martins da Silva), NHS Scotland. The authors declare no conflicts of interest.

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Medium 199, With Hanks′ salts and L-glutamine, without sodium bicarbonate, powder, suitable for cell culture