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

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: (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/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 h at 10 min intervals. Time between oocyte collection and start of Ca(2+) monitoring was 80.4 ± 2.1 h. 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 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). 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 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. 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. STUDY FUNDING/COMPETING INTERESTS: 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.

The clinical significance of calcium-signalling pathways mediating human sperm hyperactivation.

STUDY QUESTION: What is the prevalence of defects in the Ca(2+)-signalling pathways mediating hyperactivation (calcium influx and store mobilization) among donors and sub-fertile patients and are they functionally significant, i.e. related to fertilization success at IVF? SUMMARY ANSWER: This study identifies, for the first time, the prevalence of Ca(2+) store defects in sperm from research donors, IVF and ICSI patients. It highlights the biological role and importance of Ca(2+) signalling (Ca(2+) store mobilization) for fertilization at IVF. WHAT IS KNOWN ALREADY: Sperm motility and hyperactivation (HA) are important for fertility, mice with sperm incapable of HA are sterile. Recently, there has been significant progress in our knowledge of the factors controlling these events, in particular the generation and regulation of calcium signals. Both pH-regulated membrane Ca(2+) channels (CatSper) and Ca(2+) stores (potentially activating store-operated Ca(2+) channels) have been implicated in controlling HA. STUDY DESIGN, SIZE, AND DURATION: This was a prospective study examining a panel of 68 donors and 181 sub-fertile patients attending the Assisted Conception Unit, Ninewells Hospital Dundee for IVF and ICSI. Twenty-five of the donors gave a second sample (∼4 weeks later) to confirm consistency/reliability of the recorded responses. Ca(2+) signalling was manipulated using three agonists, NH4Cl (activates CatSper via pH), progesterone (direct activation of CatSper channels, potentially enhancing mobilization of stored Ca(2+) by CICR) and 4-aminopyridine (4-AP) (effect on pH equivalent to NH4Cl and mobilizes stored Ca(2+)). The broad-spectrum phosphodiesterase inhibitor 3-isobutyl-1-methyxanthine (IBMX), a potent activator of HA was also used for comparison. For patient samples, an aliquot surplus to requirements for IVF/ICSI treatment was examined, allowing direct comparison of Ca(2+) signalling and motility data with functional competence of the sperm. MATERIALS, SETTING, METHODS: The donors and sub-fertile patients were screened for HA (using CASA) and changes in intracellular Ca(2+) were assessed by loading with Fura-2 and measuring fluorescence using a plate reader (FluoStar). MAIN RESULTS AND THE ROLE OF CHANCE: The relative efficacy of the stimuli in inducing HA was 4-AP >> IBMX > progesterone. NH4Cl increased [Ca(2+)]i similarly to 4-AP and progesterone but did not induce a significant increase in HA. Failure of samples to generate HA (no significant increase in response to stimulation with 4-AP) was seen in just 2% of research donors but occurred in 10% of IVF patients (P = 0.025). All donor samples generated a significant [Ca(2+)]i increase when stimulated with 4-AP but 3.3% of IVF and 28.6% of ICSI patients failed to respond. Amplitudes of HA and [Ca(2+)]i responses to 4-AP were correlated with fertilization rate at IVF (P= 0.029; P = 0.031, respectively). Progesterone reliably induced [Ca(2+)]i responses (97% of donors, 100% of IVF patients) but was significantly less effective than 4-AP in inducing HA. Twenty seven per cent of ICSI patients failed to generate a [Ca(2+)]i response to progesterone (P= 0.035). Progesterone-induced [Ca(2+)]i responses were correlated with fertilization rate at IVF (P= 0.037) but induction of HA was not. In donor samples examined on more than one occasion consistent responses for 4-AP-induced [Ca(2+)]i (R(2) = 0.97) and HA (R(2) = 0.579) were obtained. In summary, the data indicate that defects in Ca(2+) signalling leading to poor HA do occur and that ability to undergo Ca(2+) -induced HA affects IVF fertilizing capacity. The data also confirm that release of stored Ca(2+) is the crucial component of Ca(2+) signals leading to HA and that Ca(2+) store defects may therefore underlie HA failure. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study of sperm function. While the repeatability of the [Ca(2+)]i and HA responses in samples from the same donor were confirmed, data for patients were from 1 assessment and thus the robustness of the failed responses in patients' needs to be established. The focus of this study was on using 4AP, which mobilizes stored Ca(2+) and is a potent inducer of HA. The n values for other agonists, especially calcium assessments, are smaller. WIDER IMPLICATIONS OF THE FINDINGS: Previous studies have shown a significant relationship between basal levels of HA, calcium responses to progesterone and IVF fertilization rates. Here, we have systematically investigated the ability/failure of human sperm to generate Ca(2+) signals and HA in response to targeted pharmacological challenge and, related defects in these responses to IVF success. [Ca(2+)]i signalling is fundamental for sperm motility and data from this study will lead to assessment of the nature of these defects using techniques such as single-cell imaging and patch clamping. STUDY FUNDING/COMPETING INTEREST(S): Resources from a Wellcome Trust Project Grant (#086470, Publicover and Barratt PI) primarily funded the study. The authors have no competing interests.

Human oocytes express ATP-sensitive K(+) channels.

BACKGROUND: ATP-sensitive K(+) (K(ATP)) channels link intracellular metabolism with membrane excitability and play crucial roles in cellular physiology and protection. The K(ATP) channel protein complex is composed of pore forming, Kir6.x (Kir6.1 or Kir6.2) and regulatory, SURx (SUR2A, SUR2B or SUR1), subunits that associate in different combinations. The objective of this study was to determine whether mammalian oocytes (human, bovine, porcine) express K(ATP) channels. METHODS: Supernumerary human oocytes at different stages of maturation were obtained from patients undergoing assisted conception treatments. Bovine and porcine oocytes in the germinal vesicle (GV) stage were obtained by aspirating antral follicles from abattoir-derived ovaries. The presence of mRNA for K(ATP) channel subunits was determined using real-time RT-PCR with primers specific for Kir6.2, Kir6.1, SUR1, SUR2A and SUR2B. To assess whether functional K(ATP) channels are present in human oocytes, traditional and perforated patch whole cell electrophysiology and immunoprecipitation/western blotting were used. RESULTS: Real-time PCR revealed that mRNA for Kir6.1, Kir6.2, SUR2A and SUR2B, but not SUR1, were present in human oocytes of different stages. Only SUR2B and Kir6.2 mRNAs were detected in GV stage bovine and porcine oocytes. Immunoprecipitation with SUR2 antibody and western blotting with Kir6.1 antibody identified bands corresponding to these subunits in human oocytes. In human oocytes, 2,4-dinitrophenol (400 µM), a metabolic inhibitor known to decrease intracellular ATP and activate K(ATP) channels, increased whole cell K(+) current. On the other hand, K(+) current induced by low intracellular ATP was inhibited by extracellular glibenclamide (30 µM), an oral antidiabetic known to block the opening of K(ATP) channels. CONCLUSIONS: In conclusion, mammalian oocytes express K(ATP) channels. This opens a new avenue of research into the complex relationship between metabolism and membrane excitability in oocytes under different conditions, including conception.