Heparin enhances protamine disulfide bond reduction during in vitro decondensation of human spermatozoa
Background Human sperm nuclear decondensation in vivo involves protamine disulfide bond reduction by glutathione (GSH) and protamine/histone exchange, presumably with heparan sulfate (HS) as the protamine acceptor. The aim of the present study was to test the hypothesis that these two events occur s...
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2012
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_02681161_v27_n7_p1930_Julianelli http://hdl.handle.net/20.500.12110/paper_02681161_v27_n7_p1930_Julianelli |
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paper:paper_02681161_v27_n7_p1930_Julianelli2023-06-08T15:24:09Z Heparin enhances protamine disulfide bond reduction during in vitro decondensation of human spermatozoa Julianelli, Vanina Laura Romanato, Marina Calvo, Juan Carlos glutathione heparan sulfate human oocyte protamine disulfide reduction sperm nuclear decondensation acridine orange bromobimane dithiothreitol DNA glutathione heparin protamine reagent reducing agent thiol article cell nucleus chemical reaction kinetics chromatin controlled study decondensation disulfide bond DNA denaturation human human cell immunocytochemistry in vitro study male metaphase oocyte oocyte maturation phase contrast microscopy semen analysis spermatogenesis spermatozoon staining Background Human sperm nuclear decondensation in vivo involves protamine disulfide bond reduction by glutathione (GSH) and protamine/histone exchange, presumably with heparan sulfate (HS) as the protamine acceptor. The aim of the present study was to test the hypothesis that these two events occur simultaneously rather than sequentially, as has been hitherto accepted, and to test for the presence of HS in the human oocyte. Methods Spermatozoa and isolated sperm nuclei obtained from normal volunteers were exposed in vitro to heparin, the functional analogue of HS and either GSH or dithiothreitol (DTT) as the disulfide reducing agent. Decondensing reagents were added either simultaneously or sequentially. Percentage sperm nuclear decondensation was assayed by phase contrast microscopy. Thiol reduced status of isolated sperm nuclei was evaluated both indirectly [acridine orange (AO) staining of acid-denatured DNA] and directly [monobromobimane (mBBr) staining of protamine-free thiols]. The presence of HS in mature metaphase II (MII) human oocytes was analyzed by immunocytochemistry. Results Sequential addition of reagents always resulted in significantly lower decondensation if GSH was used as the disulfide bond reducer (P < 0.05 for sperm and P < 0.001 for nuclei), but only when heparin was used first, when DTT was the disulfide reducing agent (P < 0.05 for sperm and P < 0.01 for nuclei). Both AO staining of DNA and mBBr staining of protamines revealed that the addition of heparin to GSH but not to DTT significantly increased the thiol reduced status of sperm chromatin. HS was detected in the ooplasm of zona-free MII human oocytes. Conclusions The results presented in this paper clearly show that heparin enhances the sperm chromatin thiol reducing activity of GSH in vitro, suggesting that in vivo thiol reduction and protamine/histone exchange could occur as simultaneous, rather than sequential, events. We also demonstrate for the first time the presence of HS in the human oocyte. © The Author 2012. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. Fil:Julianelli, V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Romanato, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Calvo, J.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 2012 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_02681161_v27_n7_p1930_Julianelli http://hdl.handle.net/20.500.12110/paper_02681161_v27_n7_p1930_Julianelli |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
glutathione heparan sulfate human oocyte protamine disulfide reduction sperm nuclear decondensation acridine orange bromobimane dithiothreitol DNA glutathione heparin protamine reagent reducing agent thiol article cell nucleus chemical reaction kinetics chromatin controlled study decondensation disulfide bond DNA denaturation human human cell immunocytochemistry in vitro study male metaphase oocyte oocyte maturation phase contrast microscopy semen analysis spermatogenesis spermatozoon staining |
| spellingShingle |
glutathione heparan sulfate human oocyte protamine disulfide reduction sperm nuclear decondensation acridine orange bromobimane dithiothreitol DNA glutathione heparin protamine reagent reducing agent thiol article cell nucleus chemical reaction kinetics chromatin controlled study decondensation disulfide bond DNA denaturation human human cell immunocytochemistry in vitro study male metaphase oocyte oocyte maturation phase contrast microscopy semen analysis spermatogenesis spermatozoon staining Julianelli, Vanina Laura Romanato, Marina Calvo, Juan Carlos Heparin enhances protamine disulfide bond reduction during in vitro decondensation of human spermatozoa |
| topic_facet |
glutathione heparan sulfate human oocyte protamine disulfide reduction sperm nuclear decondensation acridine orange bromobimane dithiothreitol DNA glutathione heparin protamine reagent reducing agent thiol article cell nucleus chemical reaction kinetics chromatin controlled study decondensation disulfide bond DNA denaturation human human cell immunocytochemistry in vitro study male metaphase oocyte oocyte maturation phase contrast microscopy semen analysis spermatogenesis spermatozoon staining |
| description |
Background Human sperm nuclear decondensation in vivo involves protamine disulfide bond reduction by glutathione (GSH) and protamine/histone exchange, presumably with heparan sulfate (HS) as the protamine acceptor. The aim of the present study was to test the hypothesis that these two events occur simultaneously rather than sequentially, as has been hitherto accepted, and to test for the presence of HS in the human oocyte. Methods Spermatozoa and isolated sperm nuclei obtained from normal volunteers were exposed in vitro to heparin, the functional analogue of HS and either GSH or dithiothreitol (DTT) as the disulfide reducing agent. Decondensing reagents were added either simultaneously or sequentially. Percentage sperm nuclear decondensation was assayed by phase contrast microscopy. Thiol reduced status of isolated sperm nuclei was evaluated both indirectly [acridine orange (AO) staining of acid-denatured DNA] and directly [monobromobimane (mBBr) staining of protamine-free thiols]. The presence of HS in mature metaphase II (MII) human oocytes was analyzed by immunocytochemistry. Results Sequential addition of reagents always resulted in significantly lower decondensation if GSH was used as the disulfide bond reducer (P < 0.05 for sperm and P < 0.001 for nuclei), but only when heparin was used first, when DTT was the disulfide reducing agent (P < 0.05 for sperm and P < 0.01 for nuclei). Both AO staining of DNA and mBBr staining of protamines revealed that the addition of heparin to GSH but not to DTT significantly increased the thiol reduced status of sperm chromatin. HS was detected in the ooplasm of zona-free MII human oocytes. Conclusions The results presented in this paper clearly show that heparin enhances the sperm chromatin thiol reducing activity of GSH in vitro, suggesting that in vivo thiol reduction and protamine/histone exchange could occur as simultaneous, rather than sequential, events. We also demonstrate for the first time the presence of HS in the human oocyte. © The Author 2012. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. |
| author |
Julianelli, Vanina Laura Romanato, Marina Calvo, Juan Carlos |
| author_facet |
Julianelli, Vanina Laura Romanato, Marina Calvo, Juan Carlos |
| author_sort |
Julianelli, Vanina Laura |
| title |
Heparin enhances protamine disulfide bond reduction during in vitro decondensation of human spermatozoa |
| title_short |
Heparin enhances protamine disulfide bond reduction during in vitro decondensation of human spermatozoa |
| title_full |
Heparin enhances protamine disulfide bond reduction during in vitro decondensation of human spermatozoa |
| title_fullStr |
Heparin enhances protamine disulfide bond reduction during in vitro decondensation of human spermatozoa |
| title_full_unstemmed |
Heparin enhances protamine disulfide bond reduction during in vitro decondensation of human spermatozoa |
| title_sort |
heparin enhances protamine disulfide bond reduction during in vitro decondensation of human spermatozoa |
| publishDate |
2012 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_02681161_v27_n7_p1930_Julianelli http://hdl.handle.net/20.500.12110/paper_02681161_v27_n7_p1930_Julianelli |
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AT julianellivaninalaura heparinenhancesprotaminedisulfidebondreductionduringinvitrodecondensationofhumanspermatozoa AT romanatomarina heparinenhancesprotaminedisulfidebondreductionduringinvitrodecondensationofhumanspermatozoa AT calvojuancarlos heparinenhancesprotaminedisulfidebondreductionduringinvitrodecondensationofhumanspermatozoa |
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1768542985394323456 |