Inhibition of the cholesterol transporter ABCA1 by probucol decreases capacitation and tyrosine phosphorylation of dog spermatozoa, and is dose dependent

The ATP binding cassette (ABC) transporter molecule ABCA1 participates in the cholesterol transport within and through cell membranes. We recently demonstrated that in dog spermatozoa, capacitation could be decreased with probucol (PRO), an ABCA1 specific antagonist. In this study, a dose-effect relationship of PRO on dog sperm capacitation, tyrosine phosphorylation and cholesterol efflux from the sperm plasma membrane was investigated. A total of 16 ejaculates from dogs of different breeds, aged 2-4 years were used. Sperm motility and membrane integrity in the main fraction was determined by CASA. Samples were stained with a boron dipyrromethene difluoride (BODIPY) fluorophore (P9672, Sigma-Aldrich, A) diluted in DMSO at a final concentration of 0.4 mu M. All samples were divided into 5 aliquots, with 0,100, 250, 500 and 1000 mu M of PRO. After incubation at 37 degrees C for 2 h, PI was added and flow cytometry performed. All aliquots were examined for capacitation and acrosome reaction by using the CTC assay and tyrosine phosphorylation (TP). Membrane integrity was measured in all aliquots to investigate the effect of PRO on cell membranes. Membrane integrity did not differ between controls (0 mu M), and 100, 250 and 500 mu M PRO, but decreased with 1000 mu M PRO (p < 0.05). Increasing PRO concentration decreased the percentage alive cells with cholesterol efflux per PRO group (0 mu M: 77.8 +/- 10.6%, 100 mu M: 63.7 +/- 11.7%, 250 mu M: 52.1 +/- 12.9%, 500 mu M: 37.7 +/- 11.6%, 1000 mu M: 33.1 +/- 14.4%; p < 0.05), decreased head and entire tail phosphorylated cells (0 mu M: 34.6%, 1000 mu M: 5.1% p < 0.05); and decreased the percentage capacitated cells (maximum with PRO 500 mu M: capacitated vs. control: 54.2 +/- 17% vs 25 +/- 7.7%, p < 0.05). Conclusion: PRO decreased the cholesterol efflux, and decreased tyrosine phosphorylation and capacitation in a dose-dependent manner. This suggests a strong involvement of the ABCA1 transporter in different functional aspects of sperm capacitation in dogs.(c) 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Automated summary

This study investigated the dose-effect relationship of ABC transporter molecule ABCA1 on sperm capacitation in dogs. The results showed that PRO decreased cholesterol efflux, tyrosine phosphorylation, and the percentage of capacitated cells.