科研之友
微信
新浪微博
Facebook
分享链接
摘要
Could drugs targeting ATP-sensitive K+ (K-ATP) channels prevent any spontaneous increase in intracellular Ca2+ 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 Ca2+ 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 Ca2+ homeostasis is crucial for cell wellbeing and increased intracellular Ca2+ 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 Ca2+ 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 Ca2+-sensitive dye, and monitored by laser confocal microscopy for 2 h at 10 min intervals. Time between oocyte collection and start of Ca2+ monitoring was 80.4 +/- 2.1 h. Intracellular levels of Ca2+ 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 A mu M) inhibited this increase in Ca2+ (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 A mu M) also inhibited the increase in Ca2+ (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 Ca2+ that was inhibited by glibenclamide (100 A mu M; n = 9) but not by pinacidil (100 A mu M; n = 5). Owing to clinical and ethical considerations, it was not possible to monitor Ca2+ 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 Ca2+ homeostasis and, furthermore, the altered Ca2+ 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 Ca2+, which can be counteracted by drugs targeting K-ATP channels. As Ca2+ 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.
- 出版日期2016-2
