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Nov 2019

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Fluorescence Measurement and Calibration of Intracellular pH in Starfish Oocytes
海星卵母细胞内pH值的荧光测定与校准   

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Abstract

Oocyte maturation is a process wherein an oocyte arrested at prophase of meiosis I resumes meiosis to become a fertilizable egg. In starfish ovaries, a hormone released from follicle cells activates the oocytes, resulting in an increase in their intracellular pH (pHi), which is required for spindle assembly. Herein, we describe a protocol for pHi measurement in living oocytes microinjected with the pH-sensitive dye BCECF. For in vivo BCECF calibration, we treated oocytes with artificial seawater containing CH3COONH4 to clamp pHi, injected pH-standard solutions, and converted the BCECF fluorescence intensity ratios to pHi values. Of note, if the actual pHi is higher or lower than the known pH of injected standard solutions, the BCECF fluorescence intensity ratio will decrease or increase, respectively. On the other hand, the pH of the injected solution displaying no change in fluorescence intensity should be considered the actual pHi. These methods for pHi calibration and clamping are simple and reproducible.

Keywords: Intracellular pH (细胞内pH值), BCECF (2,7-双(2-羧乙基)-5(6)-羧基荧光素), Oocyte (卵母细胞), Microinjection (显微注射), Starfish (海星), Calibration (校准)

Background

Intracellular pH (pHi) measurement is an extremely useful method for the study of cell biology since pHi 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 pHi 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 pHi 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 pHi 6.9 until spawning (Moriwaki et al., 2013).

Fluorescence indicators, including 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) have been used to measure pHi (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 pHi in vivo, BCECF-loaded cells are treated with buffer containing the K+/H+ exchanger nigericin and K+ at high concentration since pHi 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 pHi; 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 pHi 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 pHi 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 pHi. In contrast, CH3COONa dissolved in seawater forms CH3COO- and CH3COOH; thereafter, CH3COOH penetrates the cell membrane, releasing H+ and decreasing pHi (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 pHi by forming NH4+. In contrast, at a lower pH, the seawater-derived intracellular CH3COOH concentration is higher than that of NH3, thus decreasing the pHi (Figure 1). Moreover, the increase in NHE-dependent pHi 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 pHi 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 pHi via NH4+ formation. Conversely, at a lower seawater pH, the intracellular uncharged CH3COOH concentration is higher than that of NH3, thus decreasing the pHi (magenta arrows).

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

Here, to calibrate the pHi of oocytes stimulated with 1-MA in normal SW, we treated immature oocytes with modified ASW, clamping pHi 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 pHi 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 pHi, using this standard linear calibration graph/function-based method (Moriwaki et al., 2013; Hosoda et al., 2019).

Materials and Reagents

  1. Glass capillary for a glass micropipette, Microcap, 50 μl (Drummond Scientific Company, catalog number: 1-000-0500)
    A micropipette was made using the puller PC-100 system, as per the manufacturer’s instructions (Narishige, https://products.narishige-group.com/group1/PC-100/pipette/english.html). The properties (length + outer/inner diameter) of the ideal capillaries are shown in Figure 2A. To control precisely the flow volume out of the glass micropipette, a constriction was made using a handmade loop of platinum wire. Briefly, the micropipette was passed through the loop of the platinum wire. Then, the micropipette (3-5 mm from the end) was heated until a constriction of 3.5-5.0 µm in diameter was formed via the application of an electric current to the loop (see also Hiramoto, 1974).
  2. Cover glasses (Matsunami, 18 mm x 18 mm, thickness 0.12-0.17 mm)
  3. Silicone oil (Shin-Etsu Chemical Co.Ltd. catalog number: KF-96-100CS ) (Figure 2C)
  4. A dextran (10-kD) BCECF conjugate (Fisher Scientific, InvitrogenTM, catalog number: D1878 ) (Figure 2B)


    Figure 2. Materials and equipment used for oocyte manipulation. A. The constriction made in a glass micropipette acts as a brake. B. BCECF solution in a glass capillary. C. BCECF solution in a glass capillary is set in the capillary holding chamber filled with silicone oil. The glass micropipette (A) filled with silicone oil is inserted into the BCECF solution; 20 pL BCECF is aspirated into the micropipette. D. The oocyte holder. E. The manipulation chamber. F. The oocyte holder for 1-MA treatment. G. The manipulation chamber for 1-MA treatment. The chamber has an opening that allows the removal of an ASW volume (25%) and its replacement by the same volume of ASW containing 1-MA. D-G. All measurements are represented in mm unless otherwise indicated.

  5. 1-methyladenine (KANTO CHEMICAL CO. INC., catalog number: 20131-1A )
  6. Potassium aspartate (Tokyo Chemical Industry Co., Ltd., catalog number: A0922 )
  7. HEPES, 2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (DOJINDO, catalog number: 340-01371 )
  8. Pipes, Piperazine-1,4-bis (2-ethanesulfonic acid) (DOJINDO, catalog number: 347-02224 )
  9. KOH (Wako Pure Chemical Industries, Ltd., catalog number: 168-21815)
  10. NP-40 (Nonidet P-40. Nacalai Tesque, catalog number: 252-23 )
  11. NaCl (FUJIFILM Wako Pure Chemical Corporation, catalog number: 191-01665 )
  12. KCl (KANTO CHEMICAL CO. INC., catalog number: 32326-00 )
  13. MgCl2•7H2O (Wako Pure Chemical Industries, Ltd., catalog number: 135-00165 )
  14. MgSO4•7H2O (Wako Pure Chemical Industries, Ltd., catalog number: 131-00405
  15. H3BO3 (Showa Chemical Co. Ltd., catalog number: 0214-6250 )
  16. CH3COONH4 (Wako Pure Chemical Industries, Ltd., catalog number: 019-02835 )
  17. CaCl2•2H2O (Wako Pure Chemical Industries, Ltd., catalog number: 031-00435 )
  18. Calcium-free ASW (see Recipes)
  19. ASW (see Recipes)
  20. Modified ASW pH 6.8 (see Recipes)
  21. Standard pH solutions at pH 6.40-6.75 or 6.80-7.40 (see Recipes)
  22. BCECF solution (see Recipes

Equipment

  1. CMOS camera (Hamamatsu Photonics K.K., ORCA-Flash2.8, model: C11440-10C )
  2. ECLIPSE Ti-U fluorescent microscope (Nikon Instech)
  3. 4× 0.20 NA CFI super Fluor lens (Nikon Instech)
  4. Navi h pH meter (HORIBA, pH METER, F-52)
  5. Screw-controlled microinjector (Narishige, model: IM-9B )

Software

  1. HCImage U11158-02, 03 (Hamamatsu Photonics K.K., https://hcimage.com/)

Procedure




Figure 3. Flowchart representing the procedure overview (Procedures A to F)


  1. Oocyte preparation (Video 1)

    Video 1. Oocyte Preparation. This video was made at Ochanomizu Univ. according to guidelines from the Ochanomizu Univ. on Animal Care and approved by the Animal Research Ethics Board of Ochanomizu University.

    Maintain starfish (Asterina pectinifera) in laboratory aquariums supplied with circulating seawater at 14 °C (Murabe et al., 2020).
    1. Isolate oocytes or ovaries from female animals and treat them with calcium-free artificial seawater to eliminate follicle cells.
    2. Maintain follicle-free oocytes in artificial seawater (ASW) until use.

  2.  Microinjection of BCECF into oocytes (Video 2)

    Video 2. Microinjection technique

    1. Generate a glass micropipette having a constriction of a few micrometers in diameter to control the flow speed out of the micropipette (Hiramoto, 1974; see also Materials and Reagents 1 and Figure 2A).
    2. Fill the constricted micropipette with silicon oil and connect it to the screw-controlled microinjector ( IM-9B Narishige).
    3. Microscopically aspirate 20 pl BCECF solution into the micropipette (Figures 2A-2C).
    4. Microinject the BCECF solution into a starfish oocyte sandwiched between the two coverslips of an oocyte holder (Chiba et al., 1992) (Figures 2D and 2E).
    5. Incubate oocytes injected with BCECF in ASW for 1 h at 23 °C before measuring the BCECF fluorescence intensity or the pHi.
      Note: Incubation for 1 h is required for diffusion of injected BCECF.

  3. Image acquisition (sequence-imaging) of the oocytes injected with BCECF before and after 1-MA stimulation (Video 3)

    Video 3. pHi measurement using BCFCF

    1. To evaluate the camera “noise”, acquire a dark image (no light is delivered to the CMOS camera controlled by the HCImage acquisition system, which is connected to a fluorescence microscope equipped with differential interference contrast (DIC) optics including a 4× 0.20 NA CFI super Fluor lens).
    2. Set oocytes pre-injected with BCECF (as shown in Procedure B) in a manipulation chamber for 1-MA treatment at 23 °C (Figures 2F and 2G).
    3. Acquire a DIC image of 8-15 oocytes (Figure 4A).
    4. Acquire a fluorescence image of oocytes to configure the region of interest (ROI) (Figure 4B): surround the cytoplasm excluding undiffused BCECF and the GV area in the oocyte image to define the ROI, using the HCImage Analysis software. Most of the injected BCECF is diffusely distributed throughout the cytoplasm 1 h after injection; however, a small, brighter fraction, is usually retained at the site of injection near an oil drop. Importantly, this brighter region should be excluded from the ROIs (Figures 4C and 4D).


      Figure 4. Oocytes’ images acquisition. A. A representative oocytes’ DIC image. B. A fluorescence image of oocytes injected with BCECF. C. The ROI is defined using a green line. The background area is also colored in green. D. An enlarged oocyte image showing the ROI. The GV area is excluded from the ROI since GVBD causes a rapid change in fluorescence intensity.

    5. Select an area without oocytes to get the background fluorescence (Figure 4C).
    6. Before treatment with 1-MA, acquire fluorescence microscopic images of BCECF-injected oocytes, excited every 10 s at 436 nm and 495 nm for more than 5 min. This excitation alternation is obtained from a Xenon lamp using a filter wheel under the computational control of the HCImage acquisition system. Then, the emitted light passes through a dichroic beam splitter at 505 nm and through a 510-560 nm emission filter, and finally through the CMOS camera and is recorded by the HCImage acquisition system.
    7. Pause the CMOS camera, and aspirate 25% of the volume of ASW and subsequently add 25% of the volume of ASW containing 2 µM 1-MA (final concentration: 0.5 µM).
    8. Immediately after addition of 1-MA, turn on (or resume) the CMOS camera to obtain fluorescence images of the oocyte for 2-30 min. Refer to the original study for more detailed information regarding the procedure and prototypical images (Hosoda et al., 2019; Figure S5F, left panel) (see also Video 3).

  4. Calculation of the average fluorescence intensity ratios, using the recorded images of BCECF-injected oocytes and the HCImage analysis system
    1. Open an Image Display window showing the fluorescence microscopic image sequence of the oocyte. If oocytes move during recording, new ROIs should be defined according to the procedure in Step C4.
    2. Calculate the fluorescence intensity ratios between 436 nm and 495 nm excitation from 8-15 oocytes, using the HCImage Analysis software.
    3. Average the ROI ratios of 8-15 oocytes at each time point.
    4. After the experiments, convert the average ratios to pHi, using the procedure outlined in Procedure F.

  5. pHi clamping with modified ASW (sodium-free ASW containing CH3COONH4) and calibration after injection of standard solutions with known pH values
    Note: For oocytes treated with modified ASW at pH 6.8, the pHi is expected to be clamped at ~7.0, because the pHi values become ~0.2 higher than the pH values of the modified ASW (Moriwaki et al., 2013; Hosoda et al., 2019).To calibrate accurately the clamped pHi, inject different sets of oocytes with at least three types of pH-standard solutions. When the actual (or real) pHi is higher than the known pH of the injected standard solution, the BCECF fluorescence intensity ratio will decrease after injection since the pHi also decreases upon injection of the standard solution at a lower pH. Conversely, when the actual pHi is lower than the known pH of the injected standard solution, the BCECF fluorescence intensity ratio will increase. Thereafter, the actual pHi should be between the pH values of the two injected standard solutions. When the actual pHi is equal to that of the injected solution, the intensity ratio does not change. Using this method, pHi differences as low as 0.05 can be detected. In this section, taking as an example modified ASW at pH 6.8, the pHi of unstimulated oocytes was clamped, and different sets of clamped oocytes were injected with different pH-standard solutions (pH 7.00, 7.05, 7.10) to estimate the values of clamped pH; readers can expect to see three different outcomes as shown in Figures 5A, 5B and 5C, that allowed the calibration of pHi.

    1. Remove BCECF-injected oocytes from the oocyte holder (Figure 2D) after Procedure B, and recover them in 20 μl ASW, using an adjustable-volume micropipette. Then, transfer them immediately to 1 ml sodium-free ASW containing CH3COONH4 at pH 6.8 (modified ASW pH 6.8). Thereafter, gently agitate the oocytes for 15 s.
    2. Transfer the oocytes in 20 μl modified ASW at pH 6.8 to freshly modified ASW (1 ml) at pH 6.8, and gently agitate the oocytes for 15 s. Repeat this procedure 5 times to completely eliminate Na+ (with modified ASW at pH 6.8).
    3. Sandwich immature oocytes with two cover glasses using the oocyte holder (Figure 2D).
    4. Define the ROI of an oocyte and select an area without oocytes, as background fluorescence–Steps C4 and C5.
      Note: The procedures in Steps E1 to E4 should be completed within 20 min to obtain reproducible data.
    5. Record a fluorescence image sequence of an oocyte for ≥ 100 s.
    6. To inject the pH-standard solution, pause the CMOS camera.
    7. Inject oocytes with 2% of the volume per oocyte of a pH-standard solution at pH 7.00; of note modified ASW at pH 6.8 may clamp pHi at ~7.0 (Moriwaki et al., 2013; Hosoda et al., 2019).
    8. Rapidly turn on the CMOS camera system to record the changes in the fluorescence intensity ratio for at least 20 s.
    9. Measure the average fluorescence intensities inside the ROI and calculate the ratio of the averaged intensities between the 436 nm and 495 nm excitation wavelengths, using the HCImage analysis software. A typical result is shown (Figure 5A). In this particular example, the fluorescence intensity ratio decreased upon injection of the pH-standard solution. Thus, the clamped pHi is estimated to be higher than pH 7.00. Therefore, in the next step, a standard solution with a pH higher than 7.00 should be used, in a way to cause an increase in the fluorescence intensity ratio, and ultimately to determine the real pHi.


      Figure 5. Estimation of the pHi in oocytes treated with modified ASW at pH 6.8. A. The standard solution at pH 7.00 was injected between 100 s and 140 s. Clamped pHi was estimated to be higher than pH 7.00 in this oocyte. B. The standard solution at pH 7.10 was injected between 125 s and 185. Clamped pHi was estimated to be lower than pH 7.10. C. The pH-standard solution at pH 7.05 was injected between 125 s and 220 s. No change in the BCECF fluorescence intensity ratio was observed. Therefore, the clamped pHi is estimated to be pH 7.05.

    10. After the procedure described in Steps E1-E6, inject the oocytes with 2% volume per oocyte of a standard solution at pH 7.10; because pH 7.00 decreased the fluorescence intensity ratio (Figure 5A), pH 7.10 is expected to increase this ratio.
    11. Turn on the CMOS camera system and calculate the fluorescence ratio for the standard solution at pH 7.10, as mentioned in Steps E7-E9.
    12. A typical result is shown (Figure 5B). As expected, the fluorescence intensity ratio increased upon injection of the pH-standard solution. Thus, the clamped pHi is estimated to be lower than pH 7.10 and higher than pH 7.00. Therefore, to find out a standard solution with an adequate pH value causing no change of the fluorescence intensity ratio, standard solutions 7.00 ≤ pH ≤ 7.10 should be considered.
    13. After the procedure described in Steps E1-E6, inject the oocytes with 2% of the volume per oocyte of a standard solution at pH 7.05, and then turn on the CMOS camera system and calculate the respective fluorescence ratio, as mentioned in Steps E7-E9.
    14. A typical result is shown (Figure 5C). As expected, the fluorescence intensity ratio did not change upon injection of the pH-standard solution, indicating that the actual pHi should be equal to that of the injected solution (= pH 7.05).

  6. Calibration of pHi in normal seawater
    Note: The procedure in F should be conducted on the same day when the procedure in C is performed.
    1. To conduct a two-point calibration, treat immature oocytes (n = 8-15) with modified ASW to clamp the pHi at higher and lower values, and obtain the respective BCECF high- and low-intensity ratios. Usually, modified SW at pH 6.4 (pHi ~6.6) and that at pH 7.3 (pHi ~7.5) can be used. The actual pHi of oocytes in modified ASW is estimated as described in Procedure E.
    2. Calculate the average values for each point.
    3. Plot fluorescence ratios against pHi 6.6 and pHi 7.5 to obtain a standard linear calibration graph/function. More detailed information regarding this procedure is already provided in the original study (Hosoda et al., 2019; Figure S5D and E). Convert the ratio data from procedure E to pHi values, using the standard linear calibration graph/function. Again, refer to the procedure in the original study for more detailed information (Hosoda et al., 2019; Figure S5F)

Recipes

  1. Calcium-free ASW
    476 mM NaCl
    10 mM KCl
    36 mM MgCl2
    18 mM MgSO4
    20 mM H3BO3, adjusted to pH 8.2
  2. ASW
    462 mM NaCl
    10 mM KCl
    36 mM MgCl2
    18mM MgSO4
    10 mM CaCl2
    20 mM H3BO3, adjusted to pH 8.2
  3. Modified ASW pH 6.8
    480 mM choline chloride
    55 mM MgCl2
    5 mM KCl
    10 mM Pipes
    10 mM HEPES
    20 mM CH3COONH4
    9.2 mM CaCl2
    pH 6.8 is adjusted by KOH
  4. Standard pH solutions at pH 6.40-6.75 or 6.80-7.40
    0.5 M Pipes for pH 6.40-6.75; pH is adjusted by KOH
    0.5 M HEPES for pH 6.80-7.40; pH is adjusted by KOH
  5. BCECF solution
    2 mM dextran (10-kD) conjugate BCECF
    100 mM potassium aspartate
    10 mM HEPES
    0.05% NP-40, pH 7.2
    pH 7.2 is adjusted by KOH

    Note: The pH of the ASW, the modified ASW, and the standard solutions should be adjusted at 23 °C using a Navi h pH meter (HORIBA), which has a resolution of 0.001 pH units. All experiments associated with pHi measurement should be performed at 23 °C.

Acknowledgments

This work was supported by the Japan Society for the Promotion of Science KAKENHI (grant no. 17K07405), the Takeda Science Foundation, and the Cooperative Program provided by the Atmosphere and Ocean Research Institute, University of Tokyo. This protocol was adapted from Moriwaki et al., 2013 and Hosoda et al., 2019 (for pHi calibration).

Competing interests

The authors declare no competing financial interests.

Ethics

Experiments were conducted at Ochanomizu Univ. according to guidelines from the Ochanomizu Univ. on Animal Care and approved by the Animal Research Ethics Board of Ochanomizu University.

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  27. Tilney, L. G., Kiehart, D. P., Sardet, C. and Tilney, M. (1978). Polymerization of actin. IV. Role of Ca++ and H+ in the assembly of actin and in membrane fusion in the acrosomal reaction of echinoderm sperm. J Cell Biol 77(2): 536-550.

简介

[摘要]卵母细胞成熟是在减数分裂I前期停滞的卵母细胞恢复减数分裂成为可受精卵的过程。在海星卵巢中,从卵泡细胞释放的激素激活卵母细胞,导致其胞内pH(pH i )升高,这是纺锤体组装所必需的。在本文中,我们描述了在用pH敏感染料BCECF显微注射的活卵母细胞中测量pH i的方案。对于在体内BCECF校准,我们用含有CH人造海水的卵母细胞3 COONH 4到夹具的pH我 ,注入pH标准溶液,并将BCECF荧光强度比值转换为pH i值。值得注意的是,如果实际pH值我比注入标准溶液的pH值已知更高或更低时,BCECF的荧光强度比将分别减小或增大。另一方面,注入溶液的pH值没有显示出荧光强度的变化,应该认为是实际pH值i 。这些用于pH i校准和钳制的方法简单且可重现。

关键词:细胞内pH,BCECF ,卵母细胞,显微注射,海星,标定



[背景]细胞内pH (pH为我)测量为细胞生物学的研究中是非常有用的方法,因为pH值我起着在多种细胞过程中起重要作用,如配子活化(约翰逊和EPEL ,1976; Shen和斯坦哈特,1978 ;蒂尔尼等,1978 ),细胞分裂(Schuldiner和Rozengurt ,1982; Moolenaar译者等,1983;阿南德和普拉萨德,1989; KARAGIANNIS和Young,2001),和癌细胞存活(格里洛-希尔等人, 2015)。

在减数分裂I的前期卵母细胞处于静止状态,直到激素刺激恢复减数分裂。海星卵母细胞的研究报告说,激素1-甲基腺嘌呤(1-MA)与卵母细胞质膜上的一个未知受体结合(Tadenuma et al。,1992),使异源三聚体GTP结合蛋白(G)α亚基解离(G α )从βγ亚基(ģ βγ),其激活磷脂酰肌醇-3激酶(PI3K) (千叶等人,1993 ; Sadler的和Ruderman,1998) 。之后,血清和糖皮质激素调节激酶(SGK)被雷帕霉素复合物2(TORC2)和3-磷酸肌醇依赖性蛋白激酶1(PDK1)的靶磷酸化并激活(Hiraoka等人,2019; Hosoda等人, 2019)。依赖于Na + / H +交换子(NHE)的pH i在卵巢卵母细胞中从〜6.7增加到〜6.9时需要海星SGK (Harada等,2010; Moriwaki等,2013; Hosoda等,2019)。同时,SGK使Cdc25和Myt1磷酸化,诱导细胞周期蛋白B–Cdk1的去磷酸化和激活,导致生小泡破裂(GVBD)(Hiraoka et al。,2019)。重要的是,pH i升高和GVBD都需要在中期I进行卵母细胞纺锤体组装(Harada等,2003; Hosoda等,2019),随后将MI抑制在pH i 6.9直至产卵(Moriwaki等。,2013)。

荧光指示剂,包括2',7'-双(羧乙基)-5,6-羧基荧光素(BCECF)已用于测量pH i (Rink等,1982; Bassil等,2013; Behbahan等, 2014; Mortera等,2015)。BCECF是一种pH敏感的比例染料,需要双重激发。为了在体内校准pH i ,将BCECF加载的细胞用含有K + / H +交换剂尼日利亚和K +的高浓度缓冲液处理,因为预计pH i会等于细胞外pH,并在存在的情况下调整为已知值如果细胞外K +等于细胞内K + (尼古丁)(Thomas等,1979; Rink等,1982 )。但是,当细胞内K +浓度未知时,很难使用该方法。在另一项研究,在体外BCECF校准无细胞进行估计的pH我; 然而,细胞内BCECF的pKa有时大于体外溶液的pKa (Boyarsky等,1996)。用洋地黄皂苷或Triton-X-100透化细胞膜是另一种用于pH i校准的方法(Rink等,1982)。然而,由于细胞裂解,荧光比率通常不稳定(Harada等,2003;Moriwaki等,2013)。

弱碱和酸可以直接影响pH值我因为所有的膜是可渗透的不带电荷的分子(鲁斯和硼,1981) 。例如,海水中的NH 4 Cl形成NH 4 +和NH 3 ,不带电荷的NH 3穿透细胞膜并与细胞内H +结合,从而增加pH i 。与此相反,CH 3 COONa溶解在海水形式CH 3 COO -和CH 3 COOH; 之后,CH 3 COOH穿透细胞膜,释放H +并降低pH i (Hamaguchi et al。,1997)。同样,CH 3 COONH 4溶解在海水中形成NH 4 + ,NH 3 ,CH 3 COO - ,和CH 3 COOH。NH 3和CH 3 COOH容易穿透细胞膜,并分别结合或释放H + 。值得注意的是,在较高pH的海水中,NH 3的浓度大于CH 3 COOH的浓度,从而导致较高的NH 3浓度,并通过形成NH 4 +增加pH i 。与此相反,在较低的pH下,海水衍生的细胞内CH 3 COOH浓度比NH的更高3 ,因此降低了pH值我(图URE 1)。此外,在不含钠的人造海水中,NHE依赖性pH i的升高受到抑制。实际上,使用包含各种pH值的CH 3 COONH 4 (改良的ASW)的无钠人工海水(ASW),我们以前能够将海星卵母细胞的pH i固定在所需的pH值(Moriwaki等,2013)。 ; Hosoda等人,2019)。





图1.图表显示了海水中基于CH 3 COONH 4的平衡及其对细胞内pH的影响。在具有较高的pH海水中,NH 3浓度比CH更大3 COOH(蓝色箭头)由于平衡移动,从而导致更高的细胞内的NH 3浓度在卵母细胞,可渗透NH的不带电荷的形式3 。于是,卵母细胞中的细胞内NH 3通过NH 4 +的形成增加pH i 。相反,在较低的海水pH值下,细胞内不带电的CH 3 COOH浓度高于NH 3 ,从而降低了pH i (洋红色箭头)。



此外,我们可以通过注入标准溶液来估计改良ASW中卵母细胞的实际pH i 。当实际(或实际)pH i高于注入的标准溶液的已知pH时,由于在较低pH值下注入标准溶液时pH i也降低,因此BCECF荧光强度比降低。另一方面,当实际pH i低于注入的标准溶液的已知pH值时,上述比率增加。因此,实际的pH i应该在这两个pH值之间。实际上,基于此前提,我们能够选择实际pH i下的注射液,从而在给药后强度比没有变化(Moriwaki等,2013;Hosoda等,2019)。

在这里,为了校准在正常SW中1-MA刺激的卵母细胞的pH i ,我们用修饰的ASW处理未成熟的卵母细胞,将pH i固定在较高和较低的值,从而产生高和低的BCECF荧光强度比。之后,我们向这些卵母细胞注射标准溶液以估计实际的pH i值。使用这些参考,我们进行了两点校准,形成了跨越两点的直线。尽管可以使用≥3个点进行校准,但我们确认两点的校准图是线性的 –根据使用三个以上点获得的校准数据(Moriwaki等,2013;Hosoda等,2019)。因此,我们可以成熟卵母细胞的荧光比转换在正常海水的pH我,使用该标准的线性校准曲线/基于函数的方法(森胁等人,2013;细田等人,2019) 。

关键字:细胞内pH值, 2,7-双(2-羧乙基)-5(6)-羧基荧光素, 卵母细胞, 显微注射, 海星, 校准

材料和试剂
1.用于玻璃微量移液器的玻璃毛细管,Microcap,50μl (Drummond Scientific Company,目录号:1-000-0500 )      
按照制造商的说明(Narishige ,https://products.narishige-group.com/group1/PC-100/pipette/english.html),使用拉拔器PC-100系统制作微量移液器。理想毛细管的特性(长度+外/内径)示于图URE 2A。为了精确控制从玻璃微量移液器流出的流量,使用手工制作的铂金线环进行了收缩。简要地,使微量移液器穿过铂丝的环。然后,将微量(3 -从端部5毫米)的混合物加热,直到3.5的收缩- 5.0微米直径经由电流的施加到环路形成(也参见平本,1974)。
2.盖玻片(Matsunami ,18 mm x 18 mm,厚度0.12-0.17 mm)      
3.硅油(Shin-Etsu化学Co.Ltd 。目录号:KF-96-100CS )(图URE 2C)      
4.甲葡聚糖(10-kD)的BCECF缀合物(Fisher Scientific公司,Invitrogen公司TM ,目录号:D1878 )(图URE 2B )      
图2.用于卵母细胞操作的材料和设备。A.用玻璃微量移液器制成的收缩物起制动器的作用。B. BCECF在玻璃毛细管中的溶液。C.将玻璃毛细管中的BCECF溶液放入装有硅油的毛细管容纳腔中。将装有硅油的玻璃微量移液器(A)插入BCECF溶液中;20 PL BCECF被吸入到微量。D.卵母细胞支架。E.操纵室。F.用于1-MA治疗的卵母细胞支架。G.用于1-MA治疗的操纵室。腔室有一个开口,可以取出ASW体积(25%),并用包含1-MA的相同体积的ASW代替。DG 除非另有说明,否则所有测量值均以毫米表示。
5. 1-甲基腺嘌呤(关东化学株式会社,目录编号:20131-1A )      
6.天门冬氨酸钾(东京化学工业株式会社,目录号:A0922)      
7. HEPES,2- [4-(2-羟乙基)-1-哌嗪基]乙磺酸(DOJINDO,目录号:340-01371)      
8.管,哌嗪-1,4-双(2-乙磺酸)(DOJINDO,目录号:347-02224)      
9. KOH(和光纯药工业株式会社,目录号:168-21815)      
10. NP-40 (的Nonidet P-40ナTesque公司,目录号:252-23)   
11. NaCl (FUJIFILM Wako Pure Chemical Corporation,目录号:191-01665)   
12. KCl (KANTO CHEMICAL CO。INC 。,目录号:32326-00)   
13. MgCl 2 ·7H 2 O (和光纯药工业株式会社,目录号:135-00165)   
14. MgSO 4 ·7H 2 O (和光纯药工业株式会社,目录号:131-00405)   
15. H 3 BO 3 (昭和化学株式会社,目录号:0214-6250)   
16. CH 3 COONH 4 (和光纯药工业株式会社,目录号:019-02835)   
17.氯化钙2 ·2H 2 Ô (和光纯药工业株式会社,目录号码:031-00435)   
18.无钙ASW(请参阅食谱)   
19. ASW(请参阅食谱)   
20.改良的ASW pH 6.8(请参阅食谱)   
21. pH为6.40-6.75或6.80-7.40的标准pH溶液(请参见配方)   
22. BCECF解决方案(请参阅食谱)   
 
设备
 
CMOS相机(滨松光子学株式会社,ORCA-Flash2.8,米Odel等:C11440-10C)
ECLIPSE Ti -U荧光显微镜(Nikon Instech )
4×0.20 NA CFI超级荧光镜片(Niko n Instech )
导航pH计(HORIBA,pH METER,F-52)
螺杆控制的微型注射器(成茂,型号:IM-9B )
 
软件
 
HCImage U11158-02,03(Hamamatsu Photonics KK,https: //hcimage.com/ )

程序
 

图3.表示过程概述w的流程图(过程A至F)
 
卵母细胞制备(视频1)
 

视频1.卵母细胞制备。该视频是在御茶之水大学制作的。根据御茶水大学的指导。并由御茶之水大学动物研究伦理委员会批准。
 
在提供有循环海水且温度为14°C的实验室水族馆中维持海星(Asterina pectinifera )(Murabe et al。,2020)。
从雌性动物中分离卵母细胞或卵巢,并用无钙人工海水处理以消除卵泡细胞。
使用前,请在人造海水(ASW)中保持无卵泡的卵母细胞。
 
将BCECF显微注射到卵母细胞中(视频2)
 

视频2.显微注射技术
 
生成具有玻璃微量几的收缩微米直径,以控制流速出微量的(平本,1974年;还参见材料与试剂1和图URE 2A) 。
用硅油填充收缩的微量移液器,并将其连接到螺杆控制的微量进样器(IM-9B Narishige )。
镜下抽吸20 PL乙CECF溶液引入微量(图URES 2A -2 ℃)。
Microinject的BCECF溶液进入夹在卵母细胞保持器的两个盖玻片之间的卵母细胞海星(千叶等人,1992) (图URE小号2 d和2 E)。
在测量BCECF荧光强度或pH i之前,将注射BCECF的卵母细胞在ASW中于23° C孵育1 h 。
注意:注入的BCECF的扩散需要孵育1小时。
 
1-MA刺激前后注射BCECF的卵母细胞的图像采集(序列成像)(视频3)
 

视频3.使用BCFCF测量pH i
 
要评估相机的“噪点”,请获取暗图像(没有光传输到由HCImage采集系统控制的CMOS相机,该系统连接到配备有差分干涉对比(DIC)光学器件的荧光显微镜,该光学器件包括4×0.20 NA) CFI超级Fluor镜头)。
组卵母细胞预先注射无线个BCECF(如在所示的P rocedure B)在操作腔室1-MA治疗在23℃ (图2F和2G) 。
获取DIC的卵母细胞8-15图像(图URE 4A)。
获得的卵母细胞的荧光图像来配置关注区域(ROI)(图1的区域URE 4B):包围不含未扩散的BCECF和卵母细胞的图像中的区域GV来定义ROI细胞质中,使用HCI法师分析软件。注射后1 h,大多数注射的BCECF散布在整个细胞质中。但是,通常在喷油点附近油滴处保留一小部分较亮的部分。重要的是,这更亮区域应被排除在感兴趣区(图URES 4C和4 d)。
 

图4.卵母细胞的图像采集。一。代表性卵母细胞的DIC图像。乙。注射BCECF的卵母细胞的荧光图像。Ç 。ROI用绿线定义。背景区域也用绿色上色。d 。放大的卵母细胞图像显示ROI。由于GVBD导致荧光强度快速变化,因此GV区域不包括在ROI中。
 
选择一个区域,而不卵母细胞,以获得背景荧光(图URE 4C)。
在用1-MA处理之前,获取BCECF注射的卵母细胞的荧光显微图像,在436 nm和495 nm下每10 s激发5分钟以上。在HCImage采集系统的计算控制下,使用滤光轮从氙气灯获得这种激发交替。然后,在505nm处,并通过510通过一个二向色分束器出射的光穿过- 560nm的发射滤光器,最后通过CMOS摄像头,并通过记录HCImage采集系统。
暂停CMOS相机,吸出25%的ASW,然后再添加25%的ASW,其中包含2 µM 1-MA(最终浓度:0.5 µM)。
立即加入1-MA后,打开(或重新开始)的CMOS摄像头获得卵母细胞的荧光图像2 - 30分钟。有关程序和原型图像的更多详细信息,请参阅原始研究(Hosoda等人,2019年;图S5F,左图)(另请参见视频3)。
 
使用BCECF注射的卵母细胞和HCImage分析系统记录的图像计算平均荧光强度比
打开“图像显示”窗口,显示卵母细胞的荧光显微图像序列。如果卵母细胞在记录过程中移动,则应根据步骤C4中的步骤定义新的ROI 。
从8计算436 nm和495 nm激发之间的荧光强度比- 15个卵母细胞,使用HCImage分析软件。
平均每个时间点8-15个卵母细胞的ROI比率。
实验之后,使用程序F中概述的程序将平均比率转换为pH i 。
 
用改良的ASW(含CH 3 COONH 4的无钠ASW)进行pH i钳制,并在注入已知pH值的标准溶液后进行校准
注意:对于卵母细胞请客编在pH 6.8具有经修饰ASW中,pH值我预计在〜7.0至待夹持的,因为pH值我值变得〜除改性ASW的pH值高0.2 (森胁等人,2013; Hosoda et al 。,2019 )。要准确地校准钳制的pH i ,请使用至少三种类型的pH标准溶液注入不同组的卵母细胞。当实际(或实数)的pH我比的已知pH值更高标准注射溶液,该BCECF的荧光强度比将注入后降低,因为在pH值我还取决于在较低的标准溶液的注射降低pH值。相反,当实际的pH我比所注入的标准溶液的pH值已知较低时,BCECF的荧光强度比将增大。其后,实际的pH值我应为两个注入标准溶液的pH值之间。当实际pH值我等于注入的溶液中,其强度比不发生变化。使用此方法,可以检测到pH i差异低至0.05。在本节中,以pH值为6.8的改良ASW为例,将未刺激的卵母细胞的pH i固定,并向不同组的卵母细胞中注入不同的pH标准溶液(pH 7.00、7.05、7.10)以估算固定pH值 读者可以期望看到如图5A,5 B和5 C所示的三种不同结果,可以对pH i进行校准。
 
除去BCECF-注入OOC从卵母细胞保持器(图ytes URE 2D)后程序B ,并且在20恢复它们微升ASW,使用可调整容积微量。然后,将它们立即转移到1毫升的无钠ASW中,其中含有pH 6.8的CH 3 COONH 4 (改性ASW pH 6.8)。之后,轻轻搅动卵母细胞15 s。
转移卵母细胞20微升pH 6.8的改性ASW在pH 6.8至新鲜改性ASW(1毫升)中,并轻轻搅动卵母细胞15秒。重复此过程5次以完全消除Na + (在pH 6.8时使用改良的ASW)。
三明治未成熟卵母细胞使用卵母细胞保持器(图两个覆盖眼镜URE 2D)。
定义卵母细胞的ROI并选择没有卵母细胞的区域作为背景荧光–步骤C4和C5 。
注意:步骤E1至E4中的过程应在20分钟内完成,以获得可复制的数据。
记录卵母细胞的荧光图像序列≥100 s。
要注入pH标准溶液,请暂停CMOS相机。
注入卵母细胞,其卵母细胞的体积为pH 7.00的pH标准溶液的2%;音符的改性ASW在pH6.8可以夹紧pH值我在〜7.0(森胁等人,2013;细田等人,2019)。
快速打开CMOS相机系统,以记录荧光强度比的变化至少20 s。
使用HCImage分析软件,测量ROI内部的平均荧光强度,并计算436 nm和495 nm激发波长之间的平均强度之比。典型的结果示(图URE 5A)。在该特定示例中,荧光强度比在注入pH标准溶液时降低。因此,估计钳制的pH i高于pH 7.00。因此,在下一步中,应使用pH值高于7.00的标准溶液,以引起荧光强度比增加,并最终确定实际pH i 。


图5.在pH 6.8下用修饰的ASW处理的卵母细胞中pH i的估算。一。pH值为7.00的标准溶液在100 s至140 s之间注入。在该卵母细胞中,夹紧的pH i估计高于pH 7.00。乙。在125 s和185之间注入pH为7.10的标准溶液。据估计,钳制的pH i低于pH 7.10。Ç 。钍在pH 7.05的pHË标准溶液125秒和220秒之间注入。没有观察到BCECF荧光强度比的变化。因此,钳制的pH i估计为pH 7.05。
 
在步骤E1- E 6中描述的步骤之后,向卵母细胞注入每个卵母细胞2%的pH值为7.10的标准溶液。因为pH值下降7.00的荧光强度比(图URE 5A),pH值7.10预计将增加该比值。
打开CMOS相机系统,并计算标准溶液在pH 7.10下的荧光比,如步骤E7 -E 9所述。
典型的结果示(图URE 5B)。如预期的那样,在注入pH标准溶液后,荧光强度比增加。因此,CLA地跳过pH值我被估计为低于pH值7.10的pH大于7.00和更高。因此,要找出pH值适当且不会引起荧光强度比变化的标准溶液,应考虑标准溶液7.00≤pH≤7.10。
在手术后DESCR在IBED步骤E1-E6 ,注射用pH 7.05每一个标准溶液的卵母细胞体积的2%的卵母细胞,然后打开CMOS摄像系统上,并计算各自的荧光比率,如在所提到的步骤E7 -E9 。
典型的结果示(图URE 5C)。正如预期的那样,荧光强度比未在所述pH值标准溶液注射改变,这表明实际的pH值我应该等于所注射溶液(=的pH值7.05)。
 
正常海水中pH i的校准
注意:F中的步骤应在执行C中的步骤的同一天进行。
要进行两点校准,请使用改良的ASW处理未成熟的卵母细胞(n = 8-15),以将pH i固定在较高和较低的值,并获得各自的BCECF高强度和低强度比。通常,修饰的SW在pH 6.4(pH值我〜6.6)和在pH 7.3(pH值我〜7.5)可被使用。实际pH值我在改性ASW卵母细胞所描述的i的估计Ñ P rocedure E.
计算每个点的平均值s。
绘制相对于pH i 6.6和pH i 7.5的荧光比率,以获得标准线性校准图/函数。原始研究已经提供了有关此程序的更多详细信息(Hosoda等,2019;图S5D和E)。使用标准的线性校准图/函数,将过程E的比率数据转换为pH i值。再次,请参阅原始研究中的步骤以获取更多详细信息(Hosoda等,2019;图S5F)
 
菜谱
 
无钙ASW
476 mM的NAC升
10毫米氯化钾
36毫米MgCl 2
18毫米硫酸镁4
20 mM H 3 BO 3 ,调节至pH 8.2
反潜战
462毫米氯化钠
10毫米氯化钾
36毫米MgCl 2
18mM硫酸镁4
10毫米CaCl 2
20 mM H 3 BO 3 ,调节至pH 8.2
修改后的ASW pH 6.8
480 mM氯化胆碱
55毫米MgCl 2
5毫米氯化钾
10 mM管道
10毫米HEPES
20毫米CH 3 COONH 4
9.2毫米CaCl 2
用KOH调节pH 6.8
pH 6.40-6.75或6.80-7.40的标准pH溶液
0.5 M pH值为6.40-6.75的管道; 用KOH调节pH
pH值为6.80-7.40的0.5 M HEPES ;用KOH调节pH
BCECF解决方案
2 mM右旋糖酐(10-kD)共轭BCECF
100 mM天门冬氨酸钾
10毫米HEPES
0.05%NP-40,pH 7.2
用KOH调节pH 7.2
注意:应使用Navi h pH计(HORIBA)将ASW,改良的ASW和标准溶液的pH调节至23°C ,其分辨率为0.001 pH单位。与pH i测量相关的所有实验均应在23 °C下进行。
 
致谢
 
这项工作得到了日本科学促进协会KAKENHI(批准号为17K07405),武田科学基金会以及东京大学大气与海洋研究所提供的合作计划的支持。该协议改编自Moriwaki等。,2013年和Hosoda等人。,2019(用于pH i校准)。
 
利益争夺
 
作者宣称没有相互竞争的经济利益。
 
伦理
 
实验是在御茶之水大学进行的。根据御茶水大学的指导。并由御茶之水大学动物研究伦理委员会批准。

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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Hosoda, E. and Chiba, K. (2020). Fluorescence Measurement and Calibration of Intracellular pH in Starfish Oocytes. Bio-protocol 10(19): e3778. DOI: 10.21769/BioProtoc.3778.
  2. Hosoda, E., Hiraoka, D., Hirohashi, N., Omi, S., Kishimoto, T. and Chiba, K. (2019). SGK regulates pH increase and cyclin B-Cdk1 activation to resume meiosis in starfish ovarian oocytes. J Cell Biol 218(11): 3612-3629. 
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