ATP-induced apoptosis of human granulosa luteal cells cultured in vitro  Dong-Wook Park, M.Sc., Taesup Cho, M.Sc., Mi Ran Kim, M.D., Young Ah Kim, M.D., Churl K Min, Ph.D., Kyung Joo Hwang, M.D.  Fertility and Sterility  Volume 80, Issue 4, Pages 993-1002 (October 2003) DOI: 10.1016/S0015-0282(03)01118-X

Figure 1 Effect of purinergic ligands on elevating [Ca2+]i in human granulosa luteal cells. Adenosine triphosphatase (ATP) or uridine triphosphatase (UTP) (0.1 mM each) was added at the time point indicated by the arrow for 1 second to the fura-2 acetoxymethylester–loaded human granulosa luteal cells in Ca2+-free solution. [Ca2+]i was presented as a ratio of 340/380 nm from 10 to 20 cells. A representative trace is shown from one of three separate experiments. Park. Apoptosis of granulosa cells.Fertil Steril 2003. Fertility and Sterility 2003 80, 993-1002DOI: (10.1016/S0015-0282(03)01118-X)

Figure 2 Adenosine triphosphatase (ATP)–induced ionic currents in human granulosa luteal cells. Human granulosa luteal cells were bathed in normal Ringer's solution containing 1.0 mM Ca2+ and held at −20 mV, and command pulses were applied from −80 to 80 mV in 20-mV increment at a duration of 400 msec. Representative current traces were generated in the absence (A) and presence (B) of 0.1 mM of ATP. (C), Current traces when 10 mM of tetraethylammonium was added from outside. (D), Current-voltage (I-V) relations of ionic currents measured at the end of voltage pulses (400 msec after initiation of voltage pulses) in the absence of ATP (open circles), in the presence of 0.1 mM ATP (closed circles), and in the presence of 0.1 mM of ATP and 10 mM of tetraethylammonium (closed triangles). Each value shown is the mean of three separate cell preparations. The standard error bars were omitted for the sake of clarity. Park. Apoptosis of granulosa cells.Fertil Steril 2003. Fertility and Sterility 2003 80, 993-1002DOI: (10.1016/S0015-0282(03)01118-X)

Figure 3 Effects of adenosine triphosphatase (ATP), hCG, or both substances on ΔΨm in human granulosa luteal cells, as assessed by staining with JC-1. Human granulosa luteal cells were treated with increasing concentrations of ATP and/or 5 IU/mL of hCG for 24 hours and stained with JC-1 (see Materials and Methods section). (A), A typical confocal microscopic image representing the low or hyperpolarized ΔΨm (left) and the high or depolarized ΔΨm (right). Magnification, ×400. (B), Intensities of the green fluorescence (hatched bars) and the red to orange fluorescence (filled bars) measured at 510 nm and 590 nm, respectively, from cells subjected to different treatments. Each value is the mean of three separate experiments; bars represent SEs. (C), Ratios of the red and orange to green fluorescence intensities, as calculated from the data shown in part B. Data are means (±SE). Data were analyzed by using the Friedmann test for multiple comparisons. *P<.05 compared with the control reaction (no ATP); **P<.05 compared with 0.75 mM of ATP alone. Park. Apoptosis of granulosa cells.Fertil Steril 2003. Fertility and Sterility 2003 80, 993-1002DOI: (10.1016/S0015-0282(03)01118-X)

Figure 4 Detection of adenosine triphosphatase (ATP)–induced apoptosis by using the annexin V binding assay. Fresh human granulosa luteal cells were left untreated (no ATP) in a control reaction or exposed to increasing concentrations of ATP (0.1 to 0.75 mM), 0.75 mM of ATP plus 5 IU/mL of hCG, or 5 IU/mL of hCG for 24 hours. Cells were then doubly stained with annexin V–FITC-conjugated antiannexin V antibody and propidium iodide before being subjected to flow cytometry. Representative flow cytometric results are presented. The X axis (FL1) represents fluorescence intensities from FITC-conjugated antiannexin V antibodies in a logarithmic scale, and the Y axis (FL2) represents fluorescence intensities from propidium iodide in a logarithmic scale. Apoptotic cell populations (annexin V+/propidium iodide−) can be discriminated from normal (annexin V−/propidium iodide−) or necrotic cells (annexin V−/propidium iodide+) on the basis of their bindings. The numbers at the top of each figure are the percentages of apoptotic cell populations in each treatment. More than 10,000 cells were analyzed in each treatment. *P<.05 compared with the control reaction; **P<.05 compared with 0.75 mM of ATP alone (Friedmann test). •••Please Supply Citation Line.•••Fertil Steril 2003. Fertility and Sterility 2003 80, 993-1002DOI: (10.1016/S0015-0282(03)01118-X)

Figure 5 Detection of adenosine triphosphatase (ATP)–induced apoptosis by using the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay. Fresh human granulosa luteal cells were exposed to no ATP (control reaction), 0.75 mM of ATP, 0.75 mM of ATPvS, or 0.75 mM of ATP plus 5 IU/mL of hCG for 24 hours before being subjected to the TUNEL assay and counted under a microscope. (A), Representative microscopic images at ×200 magnification. The apoptotic nuclei were brown after TUNEL staining (arrow). (B), Number of apoptotic cells in three separate images per treatment. Data are means (±SE). *P<.05 compared with the control reaction; **P<.05 compared with 0.75 mM of ATP alone (Friedmann test). Park. Apoptosis of granulosa cells.Fertil Steril 2003. Fertility and Sterility 2003 80, 993-1002DOI: (10.1016/S0015-0282(03)01118-X)