Flow cytometric detection of transbilayer movement of fluorescent phospholipid analogues across the boar sperm plasma membrane: Elimination of labeling artifacts

Gadella, Bart M.; Miller, Nigel; Colenbrander, B.; Golde, L.M.G. Van; Harrison, R. A. P.

doi:10.1002/(sici)1098-2795(199905)53:1<108::aid-mrd13>3.0.co;2-k

Cited by 59 publications

(18 citation statements)

References 55 publications

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“…[9, 50, 53, 56–63] It has been previously reported that during boar sperm capacitation, the plasma membrane architecture is modified through a translocation of phospholipids to the outer leaflet and that cAMP can trigger this lipid scrambling. [53, 58, 59, 64] We do not know if a similar alteration in lipid architecture occurs in guinea pig sperm. However, it is unlikely that such an increase in lipid disorder would be sufficient to account for the changes in the lateral mobility of ADAM1/ADAM2 observed in this report.…”

Section: Discussionmentioning

confidence: 99%

Cyclic 3′,5′-AMP Causes ADAM1/ADAM2 to Rapidly Diffuse Within the Plasma Membrane of Guinea Pig Sperm1

Hunnicutt

Koppel

Kwitny

et al. 2008

Biology of Reproduction

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Because sperm cannot synthesize new proteins as they journey to the egg, they use multiple mechanisms to modify the activity of existing proteins, including changes in the diffusion coefficient of some membrane proteins. Previously, we showed that during capacitation the guinea pig heterodimeric membrane protein ADAM1/ADAM2 (fertilin) transforms from a stationary state to one of rapid diffusion within the lipid bilayer. The cause for this biophysical change, however, was unknown. In this study we examined whether an increase in cAMP, such as occurs during capacitation, could trigger this change. We incubated guinea pig cauda sperm with the membrane-permeable cAMP analog dibutyryl cAMP (db-cAMP) and the phosphodiesterase inhibitor papaverine and first tested for indications of capacitation. We observed hypermotility and acrosome-reaction competence. We then used fluorescence redistribution after photobleaching (FRAP) to measure the lateral mobility of ADAM1/ADAM2 after the db-cAMP treatment. We observed that db-cAMP caused roughly a 12-fold increase in lateral mobility of ADAM1/ADAM2, yielding diffusion similar to that observed for sperm capacitated in vitro. When we repeated the FRAP on testicular sperm incubated in db-cAMP, we found only a modest increase in lateral mobility of ADAM1/ADAM2, which underwent little redistribution. Interestingly, testicular sperm also cannot be induced to undergo capacitation. Together, the data suggests that the release of ADAM1/ADAM2 from its diffusion constraints results from a cAMP-induced signaling pathway that, like others of capacitation, is established during epididymal sperm maturation.

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Section: Discussionmentioning

confidence: 99%

Cyclic 3′,5′-AMP Causes ADAM1/ADAM2 to Rapidly Diffuse Within the Plasma Membrane of Guinea Pig Sperm1

Hunnicutt

Koppel

Kwitny

et al. 2008

Biology of Reproduction

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“…1995), where phosphatidylserine (PS) and phosphatidylethanolamine are localized in the inner membrane leaflet, whereas sphingomyeline and phosphatidylcholine are localized in the outer leaflet of the membrane (Hammerstedt et al. 1990; Gadella et al. 1999).…”

Section: Introductionmentioning

confidence: 99%

“…Regarding the plasma membrane integrity, the extend of membrane damage may vary from changes in organization, fluidity, permeability and lipid composition of the membrane bilayer to total disruption (Watson 1981). The phospholipids components of the sperm plasma membrane are distributed asymmetrically between the two leaflets of the membrane lipid bilayer (Harrison and Gadella 1995;Nolan et al 1995), where phosphatidylserine (PS) and phosphatidylethanolamine are localized in the inner membrane leaflet, whereas sphingomyeline and phosphatidylcholine are localized in the outer leaflet of the membrane (Hammerstedt et al 1990; Gadella et al 1999). Lateral and transverse arrangement of lipids is important for the regulation of sperm cell function and may be specific for maturation Harrison and Gadella 1995) or destabilization (Gadella and Harrison 2000) steps.…”

Section: Introductionmentioning

confidence: 99%

Assessment of Sperm Apoptosis in Cryopreserved Bull Semen After Swim‐up Treatment: A Flow Cytometric Study

Chaveiro

Santos

Silva

2007

Reprod Domestic Animals

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The techniques used to prepare bovine spermatozoa for in vitro fertilization, to enhance the percentage of motile sperm cells include the swim-up (SU) method, among others. The objective of the present study was to evaluate the phosphatidylserine (PS) translocation and plasma membrane integrity as the indicator of apoptosis and necrosis in post-thaw bull sperm after SU treatment using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay. A flow cytometric method was employed to measure apoptosis levels on frozen-thawed bull spermatozoa. The assay detects PS translocation across the plasma membrane using a fluorescein-labelled annexin-V and PI. By using the annexin V/PI assay four different subpopulations of sperm were observed: (i) a population of apoptotic sperm, labelled with annexin V-FITC but not with PI; (ii) a population of early necrotic spermatozoa, sperm labelled with annexin-FITC and PI; (iii) a population of necrotic sperm, labelled with PI but not with annexin-FITC; and (iv) a population of fully viable sperm cells, sperm not labelled with annexin V-FITC and without PI. Results clearly indicated that SU technique itself could have an adverse effect on the spermatozoa membrane stability. It has also been found, significant differences between bulls in the levels of apoptotic sperm, after SU treatment.

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“…Aminophospholipid translocase assay APLT activity is measured using NBD-PS (Avanti Polar Lipids, Alabaster, AL) by modifying reported procedures [20][21][22][23]. Cells (10 6 ) were washed and resuspended in 1 ml ice-cold incubation buffer (136 mM NaCl, 2.7 mM KCl, 2 mM MgCl 2 , 5 mM glucose, 500 μM PMSF, 10 mM HEPES, pH 7.5).…”

Section: Methodsmentioning

confidence: 99%

A genetic strategy involving a glycosyltransferase promoter and a lipid translocating enzyme to eliminate cancer cells

et al. 2009

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The most common therapeutic strategy for the treatment of cancer uses antimetabolites, which block uncontrolled division of cancer cells and kill them. However, such antimetabolites also kill normal cells, thus yielding detrimental side effects. This emphasizes the need for an alternative therapy, which would have little or no side effects. Our approach involves designing genetic means to alter surface lipid determinants that induce phagocytosis of cancer cells. The specific target of this strategy has been the enzyme activity termed aminophospholipid translocase (APLT) or flippase that causes translocation of phosphatidylserine (PS) from the outer to the inner leaflet of the plasma membrane in viable cells. Efforts to identify the enigmatic, plasma membrane APLT of mammalian cells have led investigators to some P-type ATPases, which have often proven to be the APLT of internal membranes rather than the plasma membrane. By measuring kinetic parameters for the plasma membrane APLT activity, we have shown that the P-type ATPase Atp8a1 is the plasma membrane APLT of the tumorigenic N18 cells, but not the non-tumorigenic HN2 (hippocampal neuron x N18) cells. Targeted knockdown of this enzyme causes PS externalization in the N18 cells, which would trigger phagocytic removal of these cells. But how would we specifically express the mutants or antisense Atp8a1 in the cancer cells? This has brought us to a glycosyltransferase, GnT-V, which is highly expressed in the transformed cells. By using the GnT-V promoter to drive a luciferase reporter gene we have demonstrated a dramatic increase in luciferase expression selectively in tumor cells. The described strategy could be tested for the removal of cancer cells without the use of antimetabolites that often kill normal cells.

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Flow cytometric detection of transbilayer movement of fluorescent phospholipid analogues across the boar sperm plasma membrane: Elimination of labeling artifacts

Cited by 59 publications

References 55 publications

Cyclic 3′,5′-AMP Causes ADAM1/ADAM2 to Rapidly Diffuse Within the Plasma Membrane of Guinea Pig Sperm1

Cyclic 3′,5′-AMP Causes ADAM1/ADAM2 to Rapidly Diffuse Within the Plasma Membrane of Guinea Pig Sperm1

Assessment of Sperm Apoptosis in Cryopreserved Bull Semen After Swim‐up Treatment: A Flow Cytometric Study

A genetic strategy involving a glycosyltransferase promoter and a lipid translocating enzyme to eliminate cancer cells

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