Cargando…
High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding
The exclusive expression of CatSper in sperm and its critical role in sperm function makes this channel an attractive target for contraception. The strategy of blocking CatSper as a male, non-hormonal contraceptive has not been fully explored due to the lack of robust screening methods to discover n...
Autores principales: | , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9892719/ https://www.ncbi.nlm.nih.gov/pubmed/36743410 http://dx.doi.org/10.3389/fcell.2023.1010306 |
_version_ | 1784881380993269760 |
---|---|
author | Luque, Guillermina M. Schiavi-Ehrenhaus, Liza J. Jabloñski, Martina Balestrini, Paula A. Novero, Analia G. Torres, Nicolás I. Osycka-Salut, Claudia E. Darszon, Alberto Krapf, Dario Buffone, Mariano G. |
author_facet | Luque, Guillermina M. Schiavi-Ehrenhaus, Liza J. Jabloñski, Martina Balestrini, Paula A. Novero, Analia G. Torres, Nicolás I. Osycka-Salut, Claudia E. Darszon, Alberto Krapf, Dario Buffone, Mariano G. |
author_sort | Luque, Guillermina M. |
collection | PubMed |
description | The exclusive expression of CatSper in sperm and its critical role in sperm function makes this channel an attractive target for contraception. The strategy of blocking CatSper as a male, non-hormonal contraceptive has not been fully explored due to the lack of robust screening methods to discover novel and specific inhibitors. The reason for this lack of appropriate methodology is the structural and functional complexity of this channel. We have developed a high-throughput method to screen drugs with the capacity to block CatSper in mammalian sperm. The assay is based on removing external free divalent cations by chelation, inducing CatSper to efficiently conduct monovalent cations. Since Na(+) is highly concentrated in the extracellular milieu, a sudden influx depolarizes the cell. Using CatSper1 KO sperm we demonstrated that this depolarization depends on CatSper function. A membrane potential (Em) assay was combined with fluorescent cell barcoding (FCB), enabling higher throughput flow cytometry based on unique fluorescent signatures of different sperm samples. These differentially labeled samples incubated in distinct experimental conditions can be combined into one tube for simultaneous acquisition. In this way, acquisition times are highly reduced, which is essential to perform larger screening experiments for drug discovery using live cells. Altogether, a simple strategy for assessing CatSper was validated, and this assay was used to develop a high-throughput drug screening for new CatSper blockers. |
format | Online Article Text |
id | pubmed-9892719 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-98927192023-02-03 High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding Luque, Guillermina M. Schiavi-Ehrenhaus, Liza J. Jabloñski, Martina Balestrini, Paula A. Novero, Analia G. Torres, Nicolás I. Osycka-Salut, Claudia E. Darszon, Alberto Krapf, Dario Buffone, Mariano G. Front Cell Dev Biol Cell and Developmental Biology The exclusive expression of CatSper in sperm and its critical role in sperm function makes this channel an attractive target for contraception. The strategy of blocking CatSper as a male, non-hormonal contraceptive has not been fully explored due to the lack of robust screening methods to discover novel and specific inhibitors. The reason for this lack of appropriate methodology is the structural and functional complexity of this channel. We have developed a high-throughput method to screen drugs with the capacity to block CatSper in mammalian sperm. The assay is based on removing external free divalent cations by chelation, inducing CatSper to efficiently conduct monovalent cations. Since Na(+) is highly concentrated in the extracellular milieu, a sudden influx depolarizes the cell. Using CatSper1 KO sperm we demonstrated that this depolarization depends on CatSper function. A membrane potential (Em) assay was combined with fluorescent cell barcoding (FCB), enabling higher throughput flow cytometry based on unique fluorescent signatures of different sperm samples. These differentially labeled samples incubated in distinct experimental conditions can be combined into one tube for simultaneous acquisition. In this way, acquisition times are highly reduced, which is essential to perform larger screening experiments for drug discovery using live cells. Altogether, a simple strategy for assessing CatSper was validated, and this assay was used to develop a high-throughput drug screening for new CatSper blockers. Frontiers Media S.A. 2023-01-19 /pmc/articles/PMC9892719/ /pubmed/36743410 http://dx.doi.org/10.3389/fcell.2023.1010306 Text en Copyright © 2023 Luque, Schiavi-Ehrenhaus, Jabloñski, Balestrini, Novero, Torres, Osycka-Salut, Darszon, Krapf and Buffone. https://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 Luque, Guillermina M. Schiavi-Ehrenhaus, Liza J. Jabloñski, Martina Balestrini, Paula A. Novero, Analia G. Torres, Nicolás I. Osycka-Salut, Claudia E. Darszon, Alberto Krapf, Dario Buffone, Mariano G. High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding |
title | High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding |
title_full | High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding |
title_fullStr | High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding |
title_full_unstemmed | High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding |
title_short | High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding |
title_sort | high-throughput screening method for discovering catsper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding |
topic | Cell and Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9892719/ https://www.ncbi.nlm.nih.gov/pubmed/36743410 http://dx.doi.org/10.3389/fcell.2023.1010306 |
work_keys_str_mv | AT luqueguillerminam highthroughputscreeningmethodfordiscoveringcatsperinhibitorsusingmembranedepolarizationcausedbyexternalcalciumchelationandfluorescentcellbarcoding AT schiaviehrenhauslizaj highthroughputscreeningmethodfordiscoveringcatsperinhibitorsusingmembranedepolarizationcausedbyexternalcalciumchelationandfluorescentcellbarcoding AT jablonskimartina highthroughputscreeningmethodfordiscoveringcatsperinhibitorsusingmembranedepolarizationcausedbyexternalcalciumchelationandfluorescentcellbarcoding AT balestrinipaulaa highthroughputscreeningmethodfordiscoveringcatsperinhibitorsusingmembranedepolarizationcausedbyexternalcalciumchelationandfluorescentcellbarcoding AT noveroanaliag highthroughputscreeningmethodfordiscoveringcatsperinhibitorsusingmembranedepolarizationcausedbyexternalcalciumchelationandfluorescentcellbarcoding AT torresnicolasi highthroughputscreeningmethodfordiscoveringcatsperinhibitorsusingmembranedepolarizationcausedbyexternalcalciumchelationandfluorescentcellbarcoding AT osyckasalutclaudiae highthroughputscreeningmethodfordiscoveringcatsperinhibitorsusingmembranedepolarizationcausedbyexternalcalciumchelationandfluorescentcellbarcoding AT darszonalberto highthroughputscreeningmethodfordiscoveringcatsperinhibitorsusingmembranedepolarizationcausedbyexternalcalciumchelationandfluorescentcellbarcoding AT krapfdario highthroughputscreeningmethodfordiscoveringcatsperinhibitorsusingmembranedepolarizationcausedbyexternalcalciumchelationandfluorescentcellbarcoding AT buffonemarianog highthroughputscreeningmethodfordiscoveringcatsperinhibitorsusingmembranedepolarizationcausedbyexternalcalciumchelationandfluorescentcellbarcoding |