Figure 2.
Figure 2. Extracellular ATP stimulates spermatogonia. (A) Original current traces from representative whole-cell patch-clamp recordings (Vhold = −80 mV; S1, S5) from a cultured mouse spermatogonium (DIV7) challenged with elevated [ATP]ex (30 µM) at increasing durations (0.3, 1.0, 3.3 s; horizontal bars). Prolonged stimulation (≥1 s) reveals current desensitization. (Inset) Phase-contrast micrograph depicting putative Asingle, Apaired, and Aaligned spermatogonia on a Sertoli cell feeder layer. The single spermatogonium is targeted by a patch pipette (pp). (B) Quantification of recordings as shown in A. Bar chart depicting peak current density measurements (mean ± SEM). Numbers of experiments are indicated above bars. Asterisk (*) denotes statistical significance, P = 0.01 (paired t test). (C) Representative whole-cell voltage-clamp traces (inset) and normalized mean peak response data (mean ± SEM) from multi-pulse stimulation experiments (Vhold = −80 mV; [ATP]ex = 100 µM/200 ms; n = 8). ISIs are progressively reduced from 90 s to 5.1 s. (D and E) Dose–response analysis of ATP-mediated whole-cell currents (Vhold = −80 mV; [ATP]ex = 0.03 µM to 10 mM; n = 5–50). Original traces from two recordings (D) and quantified data (E) reveal a double sigmoid curve on a logarithmic [ATP]ex scale. Individual data points in E are means ± SEM. Data are fit by the Hill equation. Dashed lines indicate the stimulus concentration that induces half-maximal activation of the high-affinity response (EC50 = 13.5 ± 1 µM; Hill coefficient η = 1.6 ± 0.2). Peak current amplitudes are normalized to each cell’s response to 100 µM ATP. Note the discontinuous ordinate (//). (F) Representative traces illustrating a cell’s ATP response profile (30 µM; 5 s; ISI = 180 s) at different Vhold (−80 to 60 mV; 20-mV increment). Note the biphasic response (arrows and open rectangles) consisting of an instantaneous current that is only observed at negative Vhold and a delayed outward current that increases with depolarization. (G) Current–voltage relationship of both the instantaneous (Iinst; black) and the delayed (Idel; gray) current. Individual data points depict mean current densities ± SEM (n = 18). Peak currents were measured during the boxed periods in F.

Extracellular ATP stimulates spermatogonia. (A) Original current traces from representative whole-cell patch-clamp recordings (Vhold = −80 mV; S1, S5) from a cultured mouse spermatogonium (DIV7) challenged with elevated [ATP]ex (30 µM) at increasing durations (0.3, 1.0, 3.3 s; horizontal bars). Prolonged stimulation (≥1 s) reveals current desensitization. (Inset) Phase-contrast micrograph depicting putative Asingle, Apaired, and Aaligned spermatogonia on a Sertoli cell feeder layer. The single spermatogonium is targeted by a patch pipette (pp). (B) Quantification of recordings as shown in A. Bar chart depicting peak current density measurements (mean ± SEM). Numbers of experiments are indicated above bars. Asterisk (*) denotes statistical significance, P = 0.01 (paired t test). (C) Representative whole-cell voltage-clamp traces (inset) and normalized mean peak response data (mean ± SEM) from multi-pulse stimulation experiments (Vhold = −80 mV; [ATP]ex = 100 µM/200 ms; n = 8). ISIs are progressively reduced from 90 s to 5.1 s. (D and E) Dose–response analysis of ATP-mediated whole-cell currents (Vhold = −80 mV; [ATP]ex = 0.03 µM to 10 mM; n = 5–50). Original traces from two recordings (D) and quantified data (E) reveal a double sigmoid curve on a logarithmic [ATP]ex scale. Individual data points in E are means ± SEM. Data are fit by the Hill equation. Dashed lines indicate the stimulus concentration that induces half-maximal activation of the high-affinity response (EC50 = 13.5 ± 1 µM; Hill coefficient η = 1.6 ± 0.2). Peak current amplitudes are normalized to each cell’s response to 100 µM ATP. Note the discontinuous ordinate (//). (F) Representative traces illustrating a cell’s ATP response profile (30 µM; 5 s; ISI = 180 s) at different Vhold (−80 to 60 mV; 20-mV increment). Note the biphasic response (arrows and open rectangles) consisting of an instantaneous current that is only observed at negative Vhold and a delayed outward current that increases with depolarization. (G) Current–voltage relationship of both the instantaneous (Iinst; black) and the delayed (Idel; gray) current. Individual data points depict mean current densities ± SEM (n = 18). Peak currents were measured during the boxed periods in F.

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