Calmodulin inhibitors increase the affinity of Merocyanine 540 for boar sperm membrane under non-capacitating conditions

We aimed to test whether the calmodulin (CaM) inhibitors, calmidazolium (CZ) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), can be used to assess lipid disorder by flow cytometry using Merocyanine 540 (M540). Boar spermatozoa were incubated in non-capacitating conditions for 10 min at...

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Bibliographic Details
Published in:Journal of Reproduction and Development 2018, Vol.64(5), pp.445-449
Main Authors: GONZÁLEZ-FERNÁNDEZ, Lauro, MACÍAS-GARCÍA, Beatriz, CALLE-GUISADO, Violeta, GARCÍA-MARÍN, Luis Jesús, BRAGADO, María Julia
Format: Article
Language:eng ; jpn
Subjects:
Boar spermatozoa
Calcium-binding protein
Calmodulin
Calmodulin (CaM) inhibitors
Cyclic AMP
Flow cytometry
Fluorescence
Merocyanine 540 (M540)
Sperm
Technology Report
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Summary:We aimed to test whether the calmodulin (CaM) inhibitors, calmidazolium (CZ) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), can be used to assess lipid disorder by flow cytometry using Merocyanine 540 (M540). Boar spermatozoa were incubated in non-capacitating conditions for 10 min at room temperature with 1 μM CZ, 200 μM W-7, or 1 mM 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP). Then, sperm were 1) directly evaluated, 2) centrifuged and washed prior to evaluation, or 3) diluted with PBS prior to evaluation. Direct evaluation showed an increase in high M540 fluorescence in spermatozoa treated with the inhibitors (4.7 ± 1.8 [control] vs. 70.4 ± 4.0 [CZ] and 71.4 ± 4.2 [W-7], mean % ± SD, P < 0.001); washing decreased the percentage of sperm showing high M540 fluorescence for W-7 (4.8 ± 2.2, mean % ± SD) but not for CZ (69.4 ± 3.9, mean % ± SD, P < 0.001), and dilution showed an increase in high M540 fluorescence for both CZ and W-7; 8-Br-cAMP did not induce a rise in sperm showing high M540 fluorescence. Therefore, special care must be taken when M540 is used in spermatozoa with CaM inhibitors.
ISSN:0916-8818
1348-4400
DOI:10.1262/jrd.2018-021