Dynamic quantification of the tyrosine phosphorylation of the sperm surface proteins during capacitation.
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TLDR
Spermatozoa acquire active fertilizing competence only after deposition in the female tract and subsequent capacitation, and a calcium influx seems to be required for the completion of tyrosine phosphorylation in some species.Abstract:
Background:
Spermatozoa acquire active fertilizing competence only after deposition in the female tract and subsequent capacitation. Recent studies on the cellular location of major sperm phosphoproteins suggest that capacitation is associated with tyrosine phosphorylation of proteins exposed on the sperm surface. However, these changes have not yet been quantified objectively. A calcium influx seems to be required for the completion of tyrosine phosphorylation in some species; however, the exact temporal coordination between these processes is still poorly understood.
Methods:
Flow cytometry was used to quantify the degree of phosphorylation of the sperm surface proteins by probing with fluorescein isothiocyanate-conjugated anti-phosphotyrosine (pY) antibody raised in mouse. Dynamic changes in other sperm parameters (calcium influx, membrane integrity, and spontaneous acrosome reaction) were assessed to analyze their temporal coordination.
Results:
The changes in specific phosphotyrosine (pY) fluorescence signal detected in live, nonpermeabilized boar cell suspensions were biphasic during incubation under capacitating conditions. After 120 min of incubation, the degree of pY fluorescence increased threefold, indicating the changes in proteins exposed on sperm surface. At the same time there was a gradual increase in cytosolic calcium ion levels with the maximal rate at 60 min of incubation. This rate slowed immediately before the onset of the massive rise in tyrosine phosphorylation and decreased by 90% after its completion. The integrity of plasma and acrosome membranes decreased only slowly, illustrating that the changes observed were not due to the process of spontaneous acrosome reaction.
Conclusions:
These data provide quantitative evidence for the appearance of tyrosine-phosphorylated proteins on the surface of live boar spermatozoa during capacitation. An exact temporal coordination exists between cytosolic calcium ion content and protein tyrosine phosphorylation under these conditions. This novel approach has the advantage of making possible a precise quantification and kinetic comparison of molecular processes in different cell subpopulations. © 2006 International Society for Analytical Cytologyread more
Citations
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In vitro evaluation of sperm quality.
E. Mocé,James K. Graham +1 more
TL;DR: Current and past data, correlating laboratory assay data with sperm fertility are presented in an effort to determine which types of assays are important to conduct and when to conduct them.
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Probes and techniques for sperm evaluation by flow cytometry.
Felipe Martínez-Pastor,M. Mata-Campuzano,Manuel Alvarez-Rodriguez,Mercedes Alvarez,Luis Anel,P. de Paz +5 more
TL;DR: The literature is revised, describing fluorescent probes that have been used for analysing spermatozoa by flow cytometry, regarding viability, acrosomal status, capacitation, mitochondrial status, apoptotic markers, oxidative stress markers, DNA damage, sperm counting and sperm sizing.
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Determinants of sperm quality and fertility in domestic species
TL;DR: A range of in vitro tests has been developed to monitor crucial aspects of sperm function: their ability to adapt to changing osmotic conditions, to bind to the oviductal epithelium, and to undergo capacitation in an appropriate and timely manner.
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Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art.
Sharoare Hossain,Anders Johannisson,Margareta Wallgren,Szabolcs Nagy,Amanda Pimenta Siqueira,Heriberto Rodriguez-Martinez +5 more
TL;DR: The present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting are critically evaluated.
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New insights into the molecular mechanisms of sperm-egg interaction
TL;DR: Two models indicating the plethora of highly orchestrated molecular interactions underlying successful sperm zona binding and sperm oocyte fusion are described.
References
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Journal ArticleDOI
Fertilizing Capacity of Spermatozoa deposited into the Fallopian Tubes
TL;DR: The following experiment demonstrates that such a period of time in the female tract is required for the spermatozoa to acquire their fertilizing capacity.
Journal ArticleDOI
Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation.
Pablo E. Visconti,Janice L. Bailey,Grace D. Moore,Dieyun Pan,Patricia Olds-Clarke,Gregory S. Kopf +5 more
TL;DR: Caput epididymal sperm, which lack the ability to undergo capacitation in vitro, do not display this capacitation-dependent subset of tyrosine phosphorylated proteins in complete media even after extended incubation periods, and do not fertilize metaphase II-arrested eggs in vitro.
Journal ArticleDOI
Observations on the penetration of the sperm in the mammalian egg.
TL;DR: It is considered that the evidence so far put forward for the fertilization in vitro of mammalian eggs is inconclusive, and attempts made to effect the fertilizing of the mammalian egg in vitro are terminated.
Journal ArticleDOI
Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway.
Pablo E. Visconti,Grace D. Moore,Janice L. Bailey,P. Leclerc,Stephanie A. Connors,Dieyun Pan,Patricia Olds-Clarke,Gregory S. Kopf +7 more
TL;DR: Up-regulation of protein tyrosine phosphorylation by cAMP/PKA in sperm is, to the authors' knowledge, the first demonstration of such an interrelationship between tyrosin kinase/phosphatase and PKA signaling pathways.
Journal ArticleDOI
Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa
TL;DR: The technique appeared to provide more reliable estimations of the percentage of functional cells than did motility estimations or assessments of acrosomal integrity, and demonstrated the sensitivity of the sperm plasma membrane to cold shock: virtually all cells rapidly became permeable to the stains after such stress.