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J Reprod Dev. 2018 Oct 12;64(5):445-449. doi: 10.1262/jrd.2018-021. Epub 2018 Jun 10.

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

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Research Group of Intracellular Signalling and Technology of Reproduction (SINTREP), Institute of Biotechnology in Agriculture and Livestock (INBIO G+C), University of Extremadura, 10003 Cáceres, Spain.
Jesús Usón Minimally Invasive Surgery Centre (CCMIJU), Assisted Reproduction Unit, 10071 Cáceres, Spain.


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.


Boar spermatozoa; Calmodulin (CaM) inhibitors; Merocyanine 540 (M540)

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