Fluorescence Measurement and Calibration of Intracellular pH in Starfish Oocytes

Background

Intracellular pH (pH_i) measurement is an extremely useful method for the study of cell biology since pH_i plays important roles in a variety of cell processes, such as gametes’ activation (Johnson and Epel, 1976; Shen and Steinhardt, 1978; Tilney et al., 1978), cell division (Schuldiner and Rozengurt, 1982; Moolenaar et al., 1983; Anand and Prasad, 1989; Karagiannis and Young, 2001), and cancer cell survival (Grillo-Hill et al., 2015).

An oocyte at prophase, during meiosis I, is quiescent until hormonal stimulation resume meiosis. Studies on starfish oocytes have reported that the hormone 1-methyladenine (1-MA) binds to an unidentified receptor on the plasma membrane of oocytes (Tadenuma et al., 1992), dissociating the heterotrimeric GTP-binding protein (G) α-subunit (Gα) from the βγ subunit (Gβγ), which activates phosphatidylinositol-3 kinase (PI3K) (Chiba et al., 1993; Sadler and Ruderman, 1998). Thereafter, serum- and glucocorticoid-regulated kinase (SGK) is phosphorylated and activated by the target of rapamycin complex2 (TORC2) and 3-phosphoinositide-dependent protein kinase 1 (PDK1) (Hiraoka et al., 2019; Hosoda et al., 2019). Starfish SGK is required for Na⁺/H⁺ exchanger (NHE) dependent pH_i increase from ~6.7 to ~6.9 in the ovarian oocytes (Harada et al., 2010; Moriwaki et al., 2013; Hosoda et al., 2019). Simultaneously, SGK phosphorylates Cdc25 and Myt1, inducing the de-phosphorylation and activation of cyclin B–Cdk1, causing germinal vesicle breakdown (GVBD) (Hiraoka et al., 2019). Importantly, both pH_i increase and GVBD are required for spindle assembly of ovarian oocytes at metaphase I (Harada et al., 2003; Hosoda et al., 2019), followed by MI arrest at pH_i 6.9 until spawning (Moriwaki et al., 2013).

Fluorescence indicators, including 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) have been used to measure pH_i (Rink et al., 1982; Bassil et al., 2013; Behbahan et al., 2014; Mortera et al., 2015). BCECF is a pH-sensitive and ratiometric dye requiring dual-excitation. To calibrate pH_i in vivo, BCECF-loaded cells are treated with buffer containing the K⁺/H⁺ exchanger nigericin and K⁺ at high concentration since pH_i is expected to become equal to the extracellular pH, adjusted to known values in the presence of nigericin, if extracellular K⁺ is equal to intracellular K⁺ (Thomas et al., 1979; Rink et al., 1982). However, it is difficult to use this method when the intracellular K⁺ concentration is unknown. In another study, in vitro BCECF calibration without cells was performed to estimate pH_i; however, the pKa of intracellular BCECF is sometimes greater than that of the in vitro solution (Boyarsky et al., 1996). Permeabilization of the cell membrane with digitonin or Triton-X-100 is another method used for pH_i calibration (Rink et al., 1982); however, the fluorescence ratio is often unstable owing to cell lysis (Harada et al., 2003; Moriwaki et al., 2013).

Weak bases and acids can directly affect pH_i since all membranes are permeable to uncharged molecules (Roos and Boron, 1981). For example, NH₄Cl in seawater forms NH₄⁺ and NH₃, and the uncharged NH3 penetrates the cell membrane and binds to intracellular H⁺, thus increasing the pH_i. In contrast, CH₃COONa dissolved in seawater forms CH₃COO^- and CH₃COOH; thereafter, CH₃COOH penetrates the cell membrane, releasing H⁺ and decreasing pH_i (Hamaguchi et al., 1997). Similarly, CH₃COONH₄ dissolved in seawater forms NH4⁺, NH₃, CH₃COO^-, and CH₃COOH. NH₃ and CH₃COOH easily penetrate the cell membrane, and bind to or release H⁺, respectively. Of note, in seawater at a higher pH, the NH₃ concentration is greater than that of CH₃COOH, resulting in a higher NH₃ concentration and an increase in pH_i by forming NH₄⁺. In contrast, at a lower pH, the seawater-derived intracellular CH₃COOH concentration is higher than that of NH₃, thus decreasing the pH_i (Figure 1). Moreover, the increase in NHE-dependent pH_i is inhibited in sodium-free artificial seawater. In fact, using sodium-free artificial seawater (ASW) containing CH₃COONH₄ (modified ASW) at various pH values, we were previously able to clamp the pH_i of starfish oocytes at desired pH values (Moriwaki et al., 2013; Hosoda et al., 2019).

Figure 1. Diagram showing the CH₃COONH₄-based equilibrium in seawater and its effect on intracellular pH. In seawater with a higher pH, the NH₃ concentration is greater than that of CH₃COOH (blue arrows) due to equilibrium shift, resulting in a higher intracellular NH₃ concentration in oocytes, permeable to the uncharged form of NH₃. Then, intracellular NH₃ in oocytes increases pH_i via NH₄⁺ formation. Conversely, at a lower seawater pH, the intracellular uncharged CH₃COOH concentration is higher than that of NH₃, thus decreasing the pH_i (magenta arrows).

Furthermore, we could estimate the actual pH_i of oocytes in modified ASW via the injection of standard solutions. When the actual (or real) pH_i is higher than the known pH of the injected standard solution, the BCECF fluorescence intensity ratio decreases since the pH_i is also decreased upon injection of standard solutions at lower pH values. On the other hand, when the actual pH_i is lower than the known pH of the injected standard solutions, the abovementioned ratio increases. Thus, the actual pH_i should be between these two pH values. Indeed, based on this premise, we were able to select the injection solutions at the actual pH_i, resulting in no change in the intensity ratio upon their administration (Moriwaki et al., 2013; Hosoda et al., 2019).

Here, to calibrate the pH_i of oocytes stimulated with 1-MA in normal SW, we treated immature oocytes with modified ASW, clamping pH_i at higher and lower values, and thus yielding high and low BCECF fluorescence intensity ratios. Thereafter, we injected these oocytes with the standard solutions to estimate the actual pH_i values. Using these references, we conducted a two-point calibration, forming a straight line crossing the two points. Although a calibration using ≥ 3 points is achievable, we confirmed that the two-points’ calibration graph was linear–as per the calibration data obtained using more than three points (Moriwaki et al., 2013; Hosoda et al., 2019). Thus, we could convert the fluorescence ratios of maturing oocytes in normal seawater to pH_i, using this standard linear calibration graph/function-based method (Moriwaki et al., 2013; Hosoda et al., 2019).