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Quantitative Intracellular pH Determinations in Single Live Mammalian Spermatozoa Using the Ratiometric Dye SNARF-5F
Intracellular pH (pH(i)) plays a crucial role in mammalian sperm physiology. However, it is a challenging task to acquire quantitative single sperm pH(i) images due to their small size and beating flagella. In this study, we established a robust pH(i) imaging system using the dual-emission ratiometr...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978660/ https://www.ncbi.nlm.nih.gov/pubmed/32010689 http://dx.doi.org/10.3389/fcell.2019.00366 |
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author | Chávez, Julio C. Darszon, Alberto Treviño, Claudia L. Nishigaki, Takuya |
author_facet | Chávez, Julio C. Darszon, Alberto Treviño, Claudia L. Nishigaki, Takuya |
author_sort | Chávez, Julio C. |
collection | PubMed |
description | Intracellular pH (pH(i)) plays a crucial role in mammalian sperm physiology. However, it is a challenging task to acquire quantitative single sperm pH(i) images due to their small size and beating flagella. In this study, we established a robust pH(i) imaging system using the dual-emission ratiometric pH indicator, SNARF-5F. Simultaneous good signal/noise ratio fluorescence signals were obtained exciting with a green high-power LED (532 nm) and acquiring with an EM-CCD camera through an image splitter with two band-pass filters (550–600 nm, channel 1; 630–650 nm, channel 2). After in vivo calibration, we established an imaging system that allows determination of absolute pH(i) values in spermatozoa, minimizing cell movement artifacts. Using this system, we determined that bicarbonate increases non-capacitated human pH(i) with slower kinetics than in mouse spermatozoa. This difference suggests that distinct ionic transporters might be involved in the bicarbonate influx into human and mouse spermatozoa. Alternatively, pH(i) regulation downstream bicarbonate influx into spermatozoa could be different between the two species. |
format | Online Article Text |
id | pubmed-6978660 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-69786602020-02-01 Quantitative Intracellular pH Determinations in Single Live Mammalian Spermatozoa Using the Ratiometric Dye SNARF-5F Chávez, Julio C. Darszon, Alberto Treviño, Claudia L. Nishigaki, Takuya Front Cell Dev Biol Cell and Developmental Biology Intracellular pH (pH(i)) plays a crucial role in mammalian sperm physiology. However, it is a challenging task to acquire quantitative single sperm pH(i) images due to their small size and beating flagella. In this study, we established a robust pH(i) imaging system using the dual-emission ratiometric pH indicator, SNARF-5F. Simultaneous good signal/noise ratio fluorescence signals were obtained exciting with a green high-power LED (532 nm) and acquiring with an EM-CCD camera through an image splitter with two band-pass filters (550–600 nm, channel 1; 630–650 nm, channel 2). After in vivo calibration, we established an imaging system that allows determination of absolute pH(i) values in spermatozoa, minimizing cell movement artifacts. Using this system, we determined that bicarbonate increases non-capacitated human pH(i) with slower kinetics than in mouse spermatozoa. This difference suggests that distinct ionic transporters might be involved in the bicarbonate influx into human and mouse spermatozoa. Alternatively, pH(i) regulation downstream bicarbonate influx into spermatozoa could be different between the two species. Frontiers Media S.A. 2020-01-17 /pmc/articles/PMC6978660/ /pubmed/32010689 http://dx.doi.org/10.3389/fcell.2019.00366 Text en Copyright © 2020 Chávez, Darszon, Treviño and Nishigaki. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Cell and Developmental Biology Chávez, Julio C. Darszon, Alberto Treviño, Claudia L. Nishigaki, Takuya Quantitative Intracellular pH Determinations in Single Live Mammalian Spermatozoa Using the Ratiometric Dye SNARF-5F |
title | Quantitative Intracellular pH Determinations in Single Live Mammalian Spermatozoa Using the Ratiometric Dye SNARF-5F |
title_full | Quantitative Intracellular pH Determinations in Single Live Mammalian Spermatozoa Using the Ratiometric Dye SNARF-5F |
title_fullStr | Quantitative Intracellular pH Determinations in Single Live Mammalian Spermatozoa Using the Ratiometric Dye SNARF-5F |
title_full_unstemmed | Quantitative Intracellular pH Determinations in Single Live Mammalian Spermatozoa Using the Ratiometric Dye SNARF-5F |
title_short | Quantitative Intracellular pH Determinations in Single Live Mammalian Spermatozoa Using the Ratiometric Dye SNARF-5F |
title_sort | quantitative intracellular ph determinations in single live mammalian spermatozoa using the ratiometric dye snarf-5f |
topic | Cell and Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978660/ https://www.ncbi.nlm.nih.gov/pubmed/32010689 http://dx.doi.org/10.3389/fcell.2019.00366 |
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