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Developmentally regulated cell cycle dependence of swelling-activated anion channel activity in the mouse embryo

Ottawa Health Research Institute, Hormones, Growth and Development Unit and Departments of Obstetrics and Gynecology (Division of Reproductive Medicine), and Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, K1Y 4E9 Canada

View larger version (35K): [in a new window]   Fig. 1. ICl,swell activity in mouse oocytes and embryos. (A) Magnitudes (mean ± s.e.m.) of whole-cell currents measured at +50 mV at various developmental stages of oocytes and embryos first in isotonic (240 mOsM; white bars, see key), then in hypotonic (180 mOsM; black bars), and then in hypotonic solution after the introduction of DIDS (180+DIDS; gray bars). Data are shown for oocytes in prophase of first meiosis (GV) and metaphase of second meiosis (O), as well as for 1-cell (1c), 2-cell (2c), 4-cell (4c), and 8-cell (8c) cleavage-stage embryos, morulae (M) and blastocysts (B). The time since ovulation was induced is indicated below as hours post-hCG (GV oocytes were obtained before hCG would have been administered; for embryos, fertilization occurred at about 12 hours post-hCG). Mean increase in total oocyte/embryo diameter in 180 mOsM medium at the time of the reported measurements (black bars) was 17-20%, except for oocytes and 1-cell embryos where it was 13-15%. Number of replicates (number of oocytes or embryos) is indicated above bars. Significant difference in current before swelling or after DIDS introduction relative to current at maximal swelling is indicated by asterisks (***P<0.001; **P<0.01; ns, P>0.05; ND, not determined). (B) Examples of currents elicited by swelling in ovulated (second meiotic metaphase) oocyte, 1-cell embryo, and 8-cell embryo (as indicated at bottom). At the top are traces of current over time, with each spike representing currents elicited by a voltage ramp (see text). The superimposed solid line shows total oocyte or embryo diameter relative to initial diameter before swelling (axis at right). The variable lag time between introduction of hypotonic solution and the initiation of cell swelling evident here was routinely observed. This probably arises from the time needed for the hypotonic solution to wash out the more dense and viscous 240 mOsM solution. External solutions are indicated by bars with same shadings as in the key in A. The 8-cell embryo shown here was one of the few to exhibit any significant ICl,swell. At the bottom are current versus voltage plots for the same oocyte/embryos (solid line, before swelling; dotted line, after swelling; dashed line, after DIDS). Voltage has been corrected for 10 mV liquid junction potential (LJP).

View larger version (79K): [in a new window]   Fig. 2. Nuclear morphology of 2-cell embryos at the G2/M border. (A) One cell of a 2-cell embryo in interphase, showing prominent nucleoli (n). Part of the zona pellucida (z) surrounding the embryo is visible on the right. The position of the nuclear membrane is marked by the transition between the clear area surrounding the nucleoli and the more granular cytoplasm. (B) Cell just before nuclear envelope breakdown, exhibiting small, more dispersed nucleoli indicative of prophase. (C) Cell in metaphase, showing absence of nucleus or nucleoli, and an even distribution of granular cytoplasm throughout. Bar, 5 µm.

View larger version (30K): [in a new window]   Fig. 3. Changes in ICl,swell at the G2/M border. (A) Mean (±s.e.m.) currents in 2-cell stage embryos in late G2 (2c late G2), in prophase (2c proph), and in metaphase (2c meta), as determined by nuclear morphology (Fig. 2), and in 4-cell embryos 1 hour and 5-6 hours after cytokinesis, as indicated. Data for 1-cell embryos in metaphase (1c meta) are shown at right. Annotations are as in Fig. 1 (in addition, *P<0.05). The mean increase in diameter upon swelling was 16, 17 and 19% for late G2, prophase, and metaphase 2-cell embryos respectively, 20 and 17% for 1 hour and 5-6 hours 4-cell embryos respectively, and 14% for 1-cell metaphase embryos. The inset shows currents for individual 2-cell embryos during late G2, prophase, or metaphase, shown above the corresponding bars, with data obtained from the same embryo before swelling, after swelling, and then after introduction of DIDS connected. (B) Examples of currents in individual embryos in late G2 of the 2-cell stage, and metaphase of the 1-cell and 2-cell stages (see Fig. 1 legend for description).

View larger version (10K): [in a new window]   Fig. 4. ICl,swell at the G2/M border measured by nystatin-permeablized patch recordings. See Fig. 1 and Fig. 3 legends for descriptions.

View larger version (15K): [in a new window]   Fig. 5. Currents in embryos arrested at different points in the cell cycle. 2-cell embryos were arrested in late G2 using cycloheximide (cyclohex); 1-cell and 2-cell embryos were arrested in metaphase using monastrol (monastr) or demecolcine (deme); 2-cell embryos were arrested in prophase using roscovitine (rosc) introduced either about 3 hours or within 1 hour before expected entry into metaphase, as indicated. Annotations are as in the legends to Fig. 1 and Fig. 3.

View larger version (17K): [in a new window]   Fig. 6. Permeability to glycine induced by hypotonic swelling in embryos. Glycine permeability was determined by exposing 1-cell (1c), 2-cell (2c), or 4-cell (4c) embryos to [3H]glycine for 15 minutes, and then measuring total accumulated glycine (see text). Tonicity of the solution is specified in the key. Data are normalized to the mean glycine accumulation at 285 mOsM under each condition, arbitrarily set to one. DIDS completely blocked the swelling-induced component of glycine permeability in interphase 2-cell embryos as shown by the labeled bar. Two cell embryos in late G2 were either arrested by cycloheximide (cyclohex) or measured in embryos obtained as close to entry into prophase as possible. 1-cell and 2-cell embryos in metaphase were either arrested with demecolcine (deme) or measurements made during metaphase. Significant differences in glycine permeability between hypotonically swollen embryos versus embryos in 285 mOsM medium are indicated by letters (a, a': P<0.0001; b: P<0.0005; c: P<0.001; d: P<0.005; e: P=0.02; NS: P=0.56; a' indicates significant difference in the presence of DIDS versus without DIDS). The number of replicates (each replicate a group of 5-10 embryos) was 7-10 for each treatment, except only 5 replicates of 2-cell embryos in metaphase were obtained. Interphase 2-cell embryos were included in several separate experiments (total of 21 replicates at 285 mOsM and 29 at 185 mOsM); the data obtained in separate experiments were not significantly different and thus are pooled for presentation. However, statistical significance as shown was determined for differences between groups within the same experiment (e.g., for 185 mOsM versus 185+DIDS).