Ocean acidification impacts sperm swimming performance and pHi in the New Zealand sea urchin Evechinus chloroticus.

In sea urchins, spermatozoa are stored in the gonads in hypercapnic conditions (pH<7.0). During spawning, sperm are diluted in seawater of pH>8.0, and there is an alkalinization of the sperm's internal pH (pHi) through the release of CO2 and H+. Previous research has shown that when pHi is above 7.2-7.3, the dynein ATPase flagellar motors are activated, and the sperm become motile. It has been hypothesized that ocean acidification (OA), which decreases the pH of seawater, may have a narcotic effect on sea urchin sperm by impairing the ability to regulate pHi, resulting in decreased motility and swimming speed. Here, we used data collected from the same individuals to test the relationship between pHi and sperm motility/performance in the New Zealand sea urchin Evechinus chloroticus under near-future (2100) and far-future (2150) atmospheric PCO2 conditions (RCP 8.5: pH 7.77, 7.51). Decreasing seawater pH significantly negatively impacted the proportion of motile sperm, and four of the six computer-assisted sperm analysis (CASA) sperm performance measures. In control conditions, sperm had an activated pHi of 7.52. Evechinus chloroticus sperm could not defend pHi in future OA conditions; there was a stepped decrease in the pHi at pH 7.77, with no significant difference in mean pHi between pH 7.77 and 7.51. Paired measurements in the same males showed a positive relationship between pHi and sperm motility, but with a significant difference in the response between males. Differences in motility and sperm performance in OA conditions may impact fertilization success in a future ocean.

Measuring Phagosomal pH by Fluorescence Microscopy.

Dual-wavelength and dual-fluorophore ratiometric imaging has become a powerful tool for the study of pH in intracellular compartments. It allows for the dynamic imaging of live cells while accounting for changes in the focal plane, differential loading of the fluorescent probe, and photobleaching caused by repeated image acquisitions. Ratiometric microscopic imaging has the added advantage over whole-population methods of being able to resolve individual cells and even individual organelles. In this chapter, we provide a detailed discussion of the basic principles of ratiometric imaging and its application to the measurement of phagosomal pH, including probe selection, the necessary instrumentation, and calibration methods.

Studying Proton Gradients Across the Nuclear Envelope.

The existence of nuclear pore complexes in the nuclear envelope has led to the assumption that ions move freely from the cytosol into the nucleus, and that the molecular mechanisms at the plasma membrane that regulate cytosolic pH also regulate nuclear pH. Furthermore, studies to measure pH in the nucleus have produced contradictory results, since it has been found that the nuclear pH is either similar to the cytosol or more alkaline than the cytosol. However, most studies of nuclear pH have lacked the rigor needed to understand pH regulation in the nucleus. A major problem has been the lack of in situ titrations in the nucleus and cytosol, since the intracellular environment is different in the cytosol and nucleus and the behavior of fluorescent pH probes is different in these environments. Here we present a method that uses the fluorescence of SNARF-1 that labels both cytosol and nucleus. Using ratio imaging microscopy, regions of interest corresponding to the nucleus and cytosol to perform steady-state pH measurements followed by in situ titrations, to correctly assign pH in those cellular domains.

Mitochondrial matrix pH acidifies during anoxia and is maintained by the F1Fo-ATPase in anoxia-tolerant painted turtle cortical neurons.

The western painted turtle (Chrysemys picta bellii) can survive extended periods of anoxia via a series of mechanisms that serve to reduce its energetic needs. Central to these mechanisms is the response of mitochondria, which depolarize in response to anoxia in turtle pyramidal neurons due to an influx of K+. It is currently unknown how mitochondrial matrix pH is affected by this response and we hypothesized that matrix pH acidifies during anoxia due to increased K+/H+ exchanger activity. Inhibition of K+/H+ exchange via quinine led to a collapse of mitochondrial membrane potential (Ψm) during oxygenated conditions in turtle cortical neurons, as indicated by rhodamine-123 fluorescence, and this occurred twice as quickly during anoxia which indicates an elevation in K+ conductance. Mitochondrial matrix pH acidified during anoxia, as indicated by SNARF-1 fluorescence imaged via confocal microscopy, and further acidification occurred during anoxia when the F1Fo-ATPase was inhibited with oligomycin-A, indicating that ΔpH collapse is prevented during anoxic conditions. Collectively, these results indicate that the mitochondrial proton electrochemical gradient is actively preserved during anoxia to prevent a collapse of Ψm and ΔpH.

Imaging of Mitochondrial pH Using SNARF-1.

Laser scanning confocal microscopy provides the ability to image submicron sections in living cells and tissues. In conjunction with pH-indicating fluorescent probes, confocal microscopy can be used to visualize the distribution of pH inside living cells. Here we describe a confocal microscopic technique to image intracellular pH in living cells using carboxyseminaphthorhodafluor-1 (SNARF-1), a ratiometric pH-indicating fluorescent probe. SNARF-1 is ester-loaded into the cytosol and mitochondria of adult cardiac myocytes or other cell type. Using 568-nm excitation, emitted fluorescence longer and shorter than 595-nm is imaged and then ratioed after background subtraction. Ratio values for each pixel are converted to values of pH using a standard curve (lookup table). Images of the intracellular distribution of pH show cytosolic and nuclear areas to have a pH of ~7.1, but in regions corresponding to mitochondria, pH is 8.0, giving a mitochondrial ΔpH of 0.9. During hypoxia, mitochondrial pH decreases to cytosolic values, signifying the collapse of ΔpH. These results illustrate the ability of laser scanning confocal microscopy to image the intracellular distribution of pH in living cells and to determine mitochondrial ΔpH.

Measuring Phagosomal pH by Fluorescence Microscopy.

Dual wavelength ratiometric imaging has become a powerful tool for the study of pH in intracellular compartments. It allows for the dynamic imaging of live cells while accounting for changes in the focal plane, differential loading of the fluorescent probe, and photobleaching caused by repeated image acquisitions. Ratiometric microscopic imaging has the added advantage over whole population methods of being able to resolve individual cells and even individual organelles. In this chapter we provide a detailed discussion of the basic principles of ratiometric imaging and its application to the measurement of phagosomal pH, including probe selection, the necessary instrumentation, and calibration methods.

Confocal ratio imaging of cytoplasmic pH during germ tube growth and appressorium induction by Magnaporthe grisea.

The role of cytosolic pH (pHc ) in growing germ tubes of the filamentous fungus Magnaporthe grisea was analysed by confocal ratio imaging of the pH-sensitive fluorescent dye 5(6)-carboxyseminaphthorhodafluor-1 (SNARF-1). The cytosol of these cells was successfully loaded with the acetoxymethyl ester of the dye and the pHc was visualized and quantified during conidium germination, germ tube growth and appressorium induction by simultaneous dual-emission confocal ratio imaging. Calibrations of the free acid in vitro and calibrations in vivo produced results indicating a similar dynamic response in the pH range 6.0-8.0 for both methods. The pHc in growing germ tubes was consistently pH 7.2±0.1 during all developmental stages analysed. Only slight changes in pHc (<0.1 pH unit) were found in response to alkaline external pH (pH 8.0). No changes in pHc occurred in response to an acidic extracellular pH (pH 6.0) or to the presence of nutrients. There was no observation of either pronounced gradients or changes in pHc in growing germ tubes accompanying conidium germination, germ tube growth or early appressorium formation.