Intracellular pH regulation and sperm motility in the European eel
Introduction
pH is an important environmental factor in seawater chemistry and in cell physiology. Seawater acidification induced by climate change mainly affects calcium shell organisms, but it can also have important effects in other living organisms or cells, including unicellular gametes [1]. It is known that maintaining intracellular pH (pHi) within physiological limits is key for cell function, as most cellular processes are influenced and operate within a narrow pH range. pHi regulation is crucial for motility initiation and chemotaxis in sea urchin sperm, and for sperm capacitation and hyperactivation in mammalian sperm [1].
In fish, final sperm maturation (including motility acquisition) is mediated by an increase in seminal plasma pH in the sperm duct, at least in Anguilla japonica [2] and two salmonid species [3]. In those species, sperm obtained from the testis without passing by the sperm duct cannot be activated, but after incubation in a high pH and HCO3−, it acquires the capacity to become motile when activated by seawater (eel case) or freshwater (salmonid case).
Sperm pHi and their changes upon activation have been studied in some fish. In demembranated pufferfish (Takifugu niphobles) sperm, the initiation and termination of motility were caused by the high and low pH of the activating solution, respectively, suggesting pHi contributes to the regulation of flagellar motility [4]. In both pufferfish and flounder (Kaireus bicoloratus) a transient increase in pHi during hyperosmotic activation of sperm motility was observed [5]. These authors indicated that intracellular alkalinization by NH4Cl was able to induce sperm motility even in isosmotic conditions in both species, and suggested that an internal alkalinization (in response to the osmotic shock) could be responsible for the initiation of sperm motility. However, other authors [6] suggested the opposite for another marine fish, the Japanese eel (Anguilla japonica), with an H+ uptake (then an internal acidification) triggering sperm motility. Our group observed a decrease in sperm pHi post-activation in a closed species, the European eel (Anguilla anguilla; [7]) which was later confirmed [8]. In terms of freshwater fish species, hypoosmotic shock was accompanied by a fast alkalinization of the sperm cells in common carp [9]. In contrast, it was found an acidification of pHi (of 0.2 pH units) when rainbow trout (Oncorhynchus mykiss) sperm was activated [10].
Thus, there is no consensus regarding how pHi changes are related to sperm motility in fish. For that reason, one of the objectives of this study was to further our understanding of sperm pHi regulation and the pHi changes related to motility activation in the European eel. This species is a useful model for sperm studies, as it is possible to obtain good quality sperm in any season by well standardized hormonal treatments [[11], [12], [13]]. Eels are ancient teleosts, with a simple flagellum. Thus, studying the pH regulation in the sperm of this species could provide a better understanding of the primitive regulatory functions of pH in the sperm cells of early vertebrates.
Section snippets
Chemicals and solutions
Recombinant hCG (hCGrec, Ovitrelle) was purchased from Merck Serono, Madrid. Bovine Serum Albumin, (BSA) from Sigma-Aldrich (St. Louis, MO, USA) and Nigericin Free Acid, from Invitrogen™. The fluorophore SNARF-5F AM (pH indicator dye), and Pluronic® F-127 20% in DMSO were purchased from Molecular Probes (Life Technologies, Madrid, Spain). The salts were of reagent grade.
Stock solution 2 mM SNARF-AM was prepared in Pluronic 20% in DMSO, aliquoted and maintained at −20 °C until use. For the
Quantification of intracellular pH by nigericin method
Calibration of SNARF-5F AM fluorescence vs pHi was repeated five times, using individual sperm samples with high motility. Results are shown in Table 1. According to the formula indicated by Molecular Probes (1) the data should yield a linear plot with a slope of 1 and an intercept equal to the pKA.
Calibrations 1 and 2 (Table 1) showed a slope different to 1 (0.71, 0.78). However, the other 3 calibrations showed a slope of 1 and intercepts of 7.48, 7.64 and
pH calibration with nigericin and null point
pKa for fluorochrome SNARF-5F AM, an indicator of pH, has been calculated to be 7.5–7.6 in the present conditions. This is different from pKa = 7.2 indicated in Molecular Probes Instructions, and also different from the pKa calculated by our group [8] for SNARF-5F AM in European eel sperm, which was 7.16. In that case, the pH calibration was performed in only one sperm sample, while in the present experiment the calibration was repeated 5 times, using 5 different samples. So, the present
Acknowledgements
Funded by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement Nº 642893 (IMPRESS). Víctor Gallego has a postdoc grant from the MICIU (Juan de la Cierva- Incorporacion; IJCI-2017-34200).
We would like to thank the technical support of PhD Juan German Herranz-Jusdado.
References (28)
- et al.
Intracellular pH in sperm physiology
Biochem Biophys Res Commun
(2014) - et al.
Intracellular changes in Ca2+ , K+ and pH after sperm motility activation in the European eel (Anguilla anguilla): Preliminary results
Aquaculture
(2014) - et al.
Role of potassium and pH on the initiation of sperm motility in the European eel
Comp Biochem Physiol Mol Integr Physiol
(2017) - et al.
Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software
Theriogenology
(2013) - et al.
Hormonal manipulations for the enhancement of sperm production in cultured fish and evaluation of sperm quality
Aquaculture
(2017) - et al.
Role of ion channels and membrane potential in the initiation of carp sperm motility
Aquat Living Resour
(2003) - et al.
Sperm biology of Merluccius australis: sperm structure, semen characteristics and effects of pH, temperature and osmolality on sperm motility
Aquaculture
(2013) - et al.
Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm
Dev Biol
(1983) - et al.
Independent elevation of cytosolic [Ca2+] and pH of mammalian sperm by voltage-dependent and pH-sensitive mechanisms
J Biol Chem
(1987) - et al.
Increases in concentrations of potassium and bicarbonate ions promote acquisition of motility in vitro by Japanese eel spermatozoa
J Exp Zool
(1997)
Induction of potential for sperm motility by bicarbonate and pH in rainbow trout and chum salmon
J Exp Biol
Change in intracellular K+ concentration caused by external osmolality change regulates sperm motility of marine and freshwater teleosts
J Cell Sci
Morisawa M Rises of intracellular Ca2+ and pH mediate the initiation of sperm motility by hyperosmolality in marine teleosts
Cell Motil Cytoskelet
Role of sodium bicarbonate on the initiation of sperm motility in the Japanese eel
Fish Sci
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