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.
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...
The ultrastructural morphology of the mouse zona pellucida was studied in preovulatory follicles from the ovaries of immature mice treated with exogenous gonadotrophins. The ovaries were fixed in the presence of cetylpyridinium chloride, which precipitates carbohydrates, so that their loss during fixation and processing is substantially reduced. The semi-thin araldite sections obtained from osmicated material were viewed by conventional light microscopy, while the ultra-thin sections were examined by transmission electron microscopy. A parallel series of semi-thin sections of non-osmicated ovaries was deresined and subsequently stained with periodic acid Schiff (PAS). The morphological appearance of the zona pellucida in preovulatory oocytes changed during the final stages of oocyte maturation. A close correlation was observed between the ultrastructural appearance of the zona pellucida and that observed following PAS staining during the period studied. Real differences were observed in the location, density, and distribution of glycoconjugates within the zona pellucida during the final stages of oocyte maturation prior to and immediately following germinal vesicle breakdown. Similar changes in the zona were observed in adult females autopsied during proestrus and oestrus. The changes in the density and distribution of glycoconjugates are likely to have important consequences for fertilization by affecting sperm-zona binding and sperm-egg interactions.
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