Intracellular pH regulation and sperm motility in the European eel

doi:10.1016/j.theriogenology.2020.01.026

Theriogenology

Volume 145, 15 March 2020, Pages 48-58

https://doi.org/10.1016/j.theriogenology.2020.01.026 Get rights and content

Highlights

•
A gradient of potassium between the eel sperm cell and the seminal plasma has been demonstrated for the first time.
•
The intracellular pH of eel sperm cells oscillates between 7.4 and 8.0 in physiological conditions.
•
The intracellular pH of spermatozoa is linearly dependent on the extracellular pH.
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The change in the intracellular pH of sperm cells at activation is dependent on the pH of the diluent medium.

Abstract

Sperm activation involves ion fluxes as well as a previous maturation in the seminal plasma, something which has not been studied in depth in marine fish species. pH and potassium are probably involved in sperm maturation and motility in the European eel, as indicated in previous studies. In this work, the absolute intracellular concentration of potassium in European eel sperm has been determined for the first time. In addition, the intracellular pH (pH_i) of quiescent eel spermatozoa was determined by two methods (nigericin and null point) that gave similar results, 7.4–7.6. The natural pH_i range of sperm samples in the quiescent stage was 7.4–8.0, with no evident relationship with sperm motility. However, a linear correlation was seen between sperm motility and the pH of the diluent or extracellular pH (pH_e), as well as between the pH_i and the pH of the diluent. The pH_i change post-activation in seawater (ASW) depended on the initial pH_e of the diluent medium. Activation with ASW induced an internal alkalinization of the cells when the sample had previously been diluted in a pH_e < 8.0; an acidification when pH_e > 8, and no pH_i variation when pH_e was 8.0. These experiments indicated that a careful selection of the diluents should be performed before measuring natural pH_i changes in sperm cells. Thus, studies on the specific seminal plasma composition of marine fish species are necessary before studying their physiology. Furthermore, our study indicates that intracellular alkalinization is not a universal fact during sperm activation.

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 (pH_i) within physiological limits is key for cell function, as most cellular processes are influenced and operate within a narrow pH range. pH_i 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 pH_i 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 pH_i contributes to the regulation of flagellar motility [4]. In both pufferfish and flounder (Kaireus bicoloratus) a transient increase in pH_i 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 pH_i 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 pH_i (of 0.2 pH units) when rainbow trout (Oncorhynchus mykiss) sperm was activated [10].

Thus, there is no consensus regarding how pH_i 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 pH_i regulation and the pH_i 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 pH_i 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 pK_A. $p H = p K_{A} - log [\frac{R - R_{B}}{R_{A} - R} X \frac{F_{B} (λ_{2})}{F_{A} (λ_{2})}]$

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.

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The European eel is a critically endangered teleost fish with very poor success rate for captive breeding and artificial reproduction. Therefore, to support its conservation, new strategies are needed to ensure fertilization. Objective analysis of sperm motility may be critical as it potentially represents one of the most important reproductive quality parameters. Spermatozoa acquire motility once in contact with hyperosmotic solutions as saltwater, yet the exact mechanisms and the role of temperature are still to be clarified. The main aim of the study was to assess the effects of 3 activating media (artificial sea water, tank water and commercial Actifish®) at 4 and 20 °C on sperm motility, by means of computer assisted sperm analysis. Secondary aim was to test 2 different concentrations of Actifish® mimicking sea water pH/osmolality, at 4 °C. The results suggested how both temperature and activating media have effects on spermatozoa motility and kinematics, with temperature mainly acting upon interaction with the media type. The samples activated with tank water at 20 °C showed the poorest motility outcomes (mean 38.1%), while the ones activated with Actifish® diluted 1:4 and artificial sea water, at 4 °C, the highest (means 51.8 and 51.5% respectively). Additionally, diluting Actifish® to reach same pH and osmolality of seawater led to worse motility outcomes, suggesting that composition may be the critical factor for activation rather than osmolality itself.
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Citation Excerpt :
The pH of the pufferfish seminal plasma has been measured as 8.0 (this study), and whole sperm as pH = 7.5. These facts suggest that a pH gradient exists in the quiescent sperm, with a more acidic pHi than the extracellular pH. In other fish species intracellular pH is more or less similar, as in the common carp (Cyprinus carpio), with a pH of 7.15 in quiescent stage and 7.4 in activated stage (Krasznai et al., 2003b), or in European eel, where pHi is 7.6 in quiescent conditions (Pérez et al., 2020). Seminal plasma of fish species, including the pufferfish, contains magnesium (Alavi and Cosson, 2006; Morisawa, 1985), which suggests that this ion plays a role in sperm physiology.
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Intracellular pH regulation and sperm motility in the European eel

Highlights

Abstract

Introduction

Section snippets

Chemicals and solutions

Quantification of intracellular pH by nigericin method

pH calibration with nigericin and null point

Acknowledgements

Biochem Biophys Res Commun

Aquaculture

Comp Biochem Physiol Mol Integr Physiol

Theriogenology

Aquaculture

Aquat Living Resour

Aquaculture

Dev Biol

J Biol Chem

Increases in concentrations of potassium and bicarbonate ions promote acquisition of motility in vitro by Japanese eel spermatozoa

J Exp Zool

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

Change in intracellular K⁺ concentration caused by external osmolality change regulates sperm motility of marine and freshwater teleosts

Morisawa M Rises of intracellular Ca²⁺ and pH mediate the initiation of sperm motility by hyperosmolality in marine teleosts