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

Abstract

STUDY QUESTIONCould drugs targeting ATP-sensitive K+ (KATP) channels prevent any spontaneous increase in intracellular Ca2+ that may occur in human metaphase II (MII) oocytes under in vitro conditions?SUMMARY ANSWERPinacidil, a KATP channel opener, and glibenclamide, a KATP channel blocker, prevent a spontaneous increase in intracellular Ca2+ in human MII oocytes.WHAT IS KNOWN ALREADYThe 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.STUDY DESIGN, SIZE, DURATIONSupernumerary 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.PARTICIPANTS/MATERIALS/SETTINGS/METHODSOocytes 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.MAIN RESULTS AND THE ROLE OF CHANCEIntracellular 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 µM) 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 µM) 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 µM; n = 9) but not by pinacidil (100 µM; n = 5).LIMITATIONS, REASONS FOR CAUTIONOwing 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, furthermo...

Assessment of Intracellular Calcium and Plasmalemmal Membrane Potential in Cryopreserved Metaphase II Mouse Oocytes

“…Our results are not in agreement with Fernandes et al,ndings (2). In Fernandes et al study, they showed that human oocyte experience intracellular Ca 2+ overloading in vitro (2). To the best of our knowledge, no previous study investigated Ca 2+ dynamics in mouse oocyte before fertilization in vitro.…”

“…In this study, we assessed the intracellular Ca 2+ and plasmalemmal membrane potential trends over time in cryopreserved MII mouse oocytes to see if they show similar changes as those in human oocytes following data from Fernandes et al study (2). Additionally, we investigated the glibenclamide effect on intracellular Ca 2+ , and plasmalemmal membranes in cryopreserved MII mouse oocytes compared to controls.…”

“…Research to date has not yet determined a major causative factor responsible for oocyte impairment in vitro (1). However, many studies have suggested that oocyte quality may be compromised by stimulation regimes, environmental and biological factors among others (1,2). Existing research recognises that oocyte ageing and oxidative stress are the major causes of in vitro oocyte impairment (1).…”

“…Fertilization is one of the most critical events in the oocytes in different maturation stages (14). Recently Fernandes et al have examined the effects of in vitro stress on Ca 2+ homeostasis in human oocytes, and they found that intracellular Ca 2+ was increased in human oocytes when exposed to in vitro conditions (2). To prevent this increase in intracellular Ca 2+ , drugs targeting the ATP sensitive potassium channels (K ATP ) were applied to oocytes to investigate their cytoprotecting effects.…”

“…To prevent this increase in intracellular Ca 2+ , drugs targeting the ATP sensitive potassium channels (K ATP ) were applied to oocytes to investigate their cytoprotecting effects. Glibenclamide, a K ATP channel blocker has been shown to reduce and prevent intracellular Ca 2+ loading and thus provide cytoprotection in human oocytes (2). However, no studies have tested Ca 2+ and plasmalemmal membrane potential dynamics in mouse oocytes.…”