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Calcium oscillations in fertilized pig oocytes are associated with repetitive interactions between STIM1 and ORAI1()
The Ca(2+) entry mechanism that sustains the Ca(2+) oscillations in fertilized pig oocytes was investigated. Stromal interaction molecule 1 (STIM1) and ORAI1 proteins tagged with various fluorophores were expressed in the oocytes. In some cells, the Ca(2+) stores were depleted using cyclopiazonic ac...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5905661/ https://www.ncbi.nlm.nih.gov/pubmed/29365044 http://dx.doi.org/10.1093/biolre/ioy016 |
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author | Zhang, Lu Chao, Chi-Hong Jaeger, Laurie A Papp, Agnes Bali Machaty, Zoltan |
author_facet | Zhang, Lu Chao, Chi-Hong Jaeger, Laurie A Papp, Agnes Bali Machaty, Zoltan |
author_sort | Zhang, Lu |
collection | PubMed |
description | The Ca(2+) entry mechanism that sustains the Ca(2+) oscillations in fertilized pig oocytes was investigated. Stromal interaction molecule 1 (STIM1) and ORAI1 proteins tagged with various fluorophores were expressed in the oocytes. In some cells, the Ca(2+) stores were depleted using cyclopiazonic acid (CPA); others were inseminated. Changes in the oocytes’ cytosolic free Ca(2+) concentration were monitored, while interaction between the expressed fusion proteins was investigated using fluorescence resonance energy transfer (FRET). Store depletion led to an increase of the FRET signal in oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, indicating that Ca(2+) release was followed by an interaction between these proteins. A similar FRET increase in response to CPA was also detected in oocytes co-expressing mVenus-STIM1 and mTurquoise2-STIM1, which is consistent with STIM1 forming punctae after store depletion. ML-9, an inhibitor that can interfere with STIM1 puncta formation, blocked store-operated Ca(2+) entry (SOCE) induced by Ca(2+) add-back after a CPA treatment; it also disrupted the Ca(2+) oscillations in fertilized oocytes. In addition, oocytes overexpressing mVenus-STIM1 showed high-frequency Ca(2+) oscillations when fertilized, arguing for an active role of the protein. High-frequency Ca(2+) oscillations were also detected in fertilized oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, and both of these high-frequency Ca(2+) oscillations could be stopped by inhibitors of SOCE. Importantly, in oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, we were also able to detect cyclic increases of the FRET signal indicating repetitive interactions between STIM1 and ORAI1. The results confirm the notion that in pig oocytes, SOCE is involved in the maintenance of the repetitive Ca(2+) transients at fertilization. |
format | Online Article Text |
id | pubmed-5905661 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-59056612018-04-23 Calcium oscillations in fertilized pig oocytes are associated with repetitive interactions between STIM1 and ORAI1() Zhang, Lu Chao, Chi-Hong Jaeger, Laurie A Papp, Agnes Bali Machaty, Zoltan Biol Reprod Research Article The Ca(2+) entry mechanism that sustains the Ca(2+) oscillations in fertilized pig oocytes was investigated. Stromal interaction molecule 1 (STIM1) and ORAI1 proteins tagged with various fluorophores were expressed in the oocytes. In some cells, the Ca(2+) stores were depleted using cyclopiazonic acid (CPA); others were inseminated. Changes in the oocytes’ cytosolic free Ca(2+) concentration were monitored, while interaction between the expressed fusion proteins was investigated using fluorescence resonance energy transfer (FRET). Store depletion led to an increase of the FRET signal in oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, indicating that Ca(2+) release was followed by an interaction between these proteins. A similar FRET increase in response to CPA was also detected in oocytes co-expressing mVenus-STIM1 and mTurquoise2-STIM1, which is consistent with STIM1 forming punctae after store depletion. ML-9, an inhibitor that can interfere with STIM1 puncta formation, blocked store-operated Ca(2+) entry (SOCE) induced by Ca(2+) add-back after a CPA treatment; it also disrupted the Ca(2+) oscillations in fertilized oocytes. In addition, oocytes overexpressing mVenus-STIM1 showed high-frequency Ca(2+) oscillations when fertilized, arguing for an active role of the protein. High-frequency Ca(2+) oscillations were also detected in fertilized oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, and both of these high-frequency Ca(2+) oscillations could be stopped by inhibitors of SOCE. Importantly, in oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, we were also able to detect cyclic increases of the FRET signal indicating repetitive interactions between STIM1 and ORAI1. The results confirm the notion that in pig oocytes, SOCE is involved in the maintenance of the repetitive Ca(2+) transients at fertilization. Oxford University Press 2018-04 2018-01-22 /pmc/articles/PMC5905661/ /pubmed/29365044 http://dx.doi.org/10.1093/biolre/ioy016 Text en © The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Research Article Zhang, Lu Chao, Chi-Hong Jaeger, Laurie A Papp, Agnes Bali Machaty, Zoltan Calcium oscillations in fertilized pig oocytes are associated with repetitive interactions between STIM1 and ORAI1() |
title | Calcium oscillations in fertilized pig oocytes are associated with repetitive interactions between STIM1 and ORAI1() |
title_full | Calcium oscillations in fertilized pig oocytes are associated with repetitive interactions between STIM1 and ORAI1() |
title_fullStr | Calcium oscillations in fertilized pig oocytes are associated with repetitive interactions between STIM1 and ORAI1() |
title_full_unstemmed | Calcium oscillations in fertilized pig oocytes are associated with repetitive interactions between STIM1 and ORAI1() |
title_short | Calcium oscillations in fertilized pig oocytes are associated with repetitive interactions between STIM1 and ORAI1() |
title_sort | calcium oscillations in fertilized pig oocytes are associated with repetitive interactions between stim1 and orai1() |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5905661/ https://www.ncbi.nlm.nih.gov/pubmed/29365044 http://dx.doi.org/10.1093/biolre/ioy016 |
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