Mostrar el registro sencillo del ítem
dc.contributor.author
Matamoros Volante, Arturo
dc.contributor.author
Moreno, Ayelen
![Se ha confirmado la validez de este valor de autoridad por un usuario](/themes/CONICETDigital/images/authority_control/invisible.gif)
dc.contributor.author
Torres Rodriguez, Paulina
dc.contributor.author
Giojalas, Laura Cecilia
![Se ha confirmado la validez de este valor de autoridad por un usuario](/themes/CONICETDigital/images/authority_control/invisible.gif)
dc.contributor.author
Gervasi, Maria Gracia
![Se ha confirmado la validez de este valor de autoridad por un usuario](/themes/CONICETDigital/images/authority_control/invisible.gif)
dc.contributor.author
Visconti, Pablo E.
dc.contributor.author
Treviño, Claudia L.
dc.date.available
2019-10-18T14:40:29Z
dc.date.issued
2018-02
dc.identifier.citation
Matamoros Volante, Arturo; Moreno, Ayelen; Torres Rodriguez, Paulina; Giojalas, Laura Cecilia; Gervasi, Maria Gracia; et al.; Semi-automatized segmentation method using image-based flow cytometry to study sperm physiology: The case of capacitation-induced tyrosine phosphorylation; Oxford University Press; Molecular Human Reproduction; 24; 2; 2-2018; 64-73
dc.identifier.issn
1360-9947
dc.identifier.uri
http://hdl.handle.net/11336/86324
dc.description.abstract
Is image-based flow cytometry a useful tool to study intracellular events in human sperm such as protein tyrosine phosphorylation or signaling processes? Image-based flow cytometry is a powerful tool to study intracellular events in a relevant number of sperm cells, which enables a robust statistical analysis providing spatial resolution in terms of the specific subcellular localization of the labeling. Sperm capacitation is required for fertilization. During this process, spermatozoa undergo numerous physiological changes, via activation of different signaling pathways, which are not completely understood. Classical approaches for studying sperm physiology include conventional microscopy, flow cytometry and Western blotting. These techniques present disadvantages for obtaining detailed subcellular information of signaling pathways in a relevant number of cells. This work describes a new semi-automatized analysis using image-based flow cytometry which enables the study, at the subcellular and population levels, of different sperm parameters associated with signaling. The increase in protein tyrosine phosphorylation during capacitation is presented as an example. Sperm cells were isolated from seminal plasma by the swim-up technique. We evaluated the intensity and distribution of protein tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h under different experimental conditions. We used an antibody against FER kinase and pharmacological inhibitors in an attempt to identify the kinases involved in protein tyrosine phosphorylation during human sperm capacitation. Semen samples from normospermic donors were obtained by masturbation after 2-3 days of sexual abstinence. We used the innovative technique image-based flow cytometry and image analysis tools to segment individual images of spermatozoa. We evaluated and quantified the regions of sperm where protein tyrosine phosphorylation takes place at the subcellular level in a large number of cells. We also used immunocytochemistry and Western blot analysis. Independent experiments were performed with semen samples from seven different donors. Using image analysis tools, we developed a completely novel semi-automatic strategy useful for segmenting thousands of individual cell images obtained using image-based flow cytometry. Contrary to immunofluorescence which relies on the analysis of a limited sperm population and also on the observer, image-based flow cytometry allows for unbiased quantification and simultaneous localization of post-translational changes in an extended sperm population. Interestingly, important data can be independently analyzed by looking to the frame of interest. As an example, we evaluated the capacitation-associated increase in tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h. As previously reported, protein tyrosine phosphorylation increases in a time-depending manner, but our method revealed that this increase occurs differentially among distinct sperm segments. FER kinase is reported to be the enzyme responsible for the increase in protein tyrosine phosphorylation in mouse sperm. Our Western blot analysis revealed for the first time the presence of this enzyme in human sperm. Using our segmentation strategy, we aimed to quantify the effect of pharmacological inhibition of FER kinase and found a marked reduction of protein tyrosine phosphorylation only in the flagellum, which corresponded to the physical localization of FER in human sperm. Our method provides an alternative strategy to study signaling markers associated with capacitation, such as protein tyrosine phosphorylation, in a fast and quantitative manner. LARGE SCALE DATA: None. LIMITATIONS REASONS FOR CAUTION: This is an in vitro study performed under controlled conditions. Chemical inhibitors are not completely specific for the intended target; the possibility of side effects cannot be discarded. Our results demonstrate that the use of image-based flow cytometry is a very powerful tool to study sperm physiology. A large number of cells can be easily analyzed and information at the subcellular level can be obtained. As the segmentation process works with bright-field images, it can be extended to study expression of other proteins of interest using different antibodies or it can be used in living sperm to study intracellular parameters that can be followed using fluorescent dyes sensitive to the parameter of interest (e.g. pH, Ca2+). Therefore, this a versatile method that can be exploited to study several aspects of sperm physiology.
dc.format
application/pdf
dc.language.iso
eng
dc.publisher
Oxford University Press
![Se ha confirmado la validez de este valor de autoridad por un usuario](/themes/CONICETDigital/images/authority_control/invisible.gif)
dc.rights
info:eu-repo/semantics/openAccess
dc.rights.uri
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.subject
FER KINASE
dc.subject
IMAGE-BASED FLOW CYTOMETRY
dc.subject
PROTEIN TYROSINE PHOSPHORYLATION
dc.subject
PYK2
dc.subject
SPERM CAPACITATION
dc.subject
SPERM IMAGE SEGMENTATION
dc.subject.classification
Biología Celular, Microbiología
![Se ha confirmado la validez de este valor de autoridad por un usuario](/themes/CONICETDigital/images/authority_control/invisible.gif)
dc.subject.classification
Ciencias Biológicas
![Se ha confirmado la validez de este valor de autoridad por un usuario](/themes/CONICETDigital/images/authority_control/invisible.gif)
dc.subject.classification
CIENCIAS NATURALES Y EXACTAS
![Se ha confirmado la validez de este valor de autoridad por un usuario](/themes/CONICETDigital/images/authority_control/invisible.gif)
dc.title
Semi-automatized segmentation method using image-based flow cytometry to study sperm physiology: The case of capacitation-induced tyrosine phosphorylation
dc.type
info:eu-repo/semantics/article
dc.type
info:ar-repo/semantics/artículo
dc.type
info:eu-repo/semantics/publishedVersion
dc.date.updated
2019-08-09T15:04:55Z
dc.identifier.eissn
1460-2407
dc.journal.volume
24
dc.journal.number
2
dc.journal.pagination
64-73
dc.journal.pais
Reino Unido
![Se ha confirmado la validez de este valor de autoridad por un usuario](/themes/CONICETDigital/images/authority_control/invisible.gif)
dc.journal.ciudad
Oxford
dc.description.fil
Fil: Matamoros Volante, Arturo. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México
dc.description.fil
Fil: Moreno, Ayelen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Biología Celular y Molecular; Argentina
dc.description.fil
Fil: Torres Rodriguez, Paulina. Universidad Nacional Autónoma de México; México
dc.description.fil
Fil: Giojalas, Laura Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Biología Celular y Molecular; Argentina
dc.description.fil
Fil: Gervasi, Maria Gracia. University of Massachussets; Estados Unidos
dc.description.fil
Fil: Visconti, Pablo E.. University of Massachussets; Estados Unidos
dc.description.fil
Fil: Treviño, Claudia L.. Universidad Nacional Autónoma de México; México
dc.journal.title
Molecular Human Reproduction
![Se ha confirmado la validez de este valor de autoridad por un usuario](/themes/CONICETDigital/images/authority_control/invisible.gif)
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/url/https://academic.oup.com/molehr/article/24/2/64/4658825
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/doi/http://dx.doi.org/10.1093/molehr/gax062
Archivos asociados