HDL mediates reverse cholesterol transport from ram spermatozoa and induces hyperactivated motility.

Reverse cholesterol transport or cholesterol efflux is part of an extensive plasma membrane remodeling process in spermatozoa that is imperative for fertilization. For ram spermatozoa, sheep serum is well known to support in vitro fertilization (IVF), but knowledge of its explicit role is limited. Though, it is postulated to elicit cholesterol efflux owing to the presence of high-density lipoproteins (HDLs) that interact with transmembrane cholesterol transporters, such as adenosinetriphosphate (ATP)-binding cassette transporter A1 (ABCA1) and scavenger receptor class B, type I (SR-BI). In this study, we report that both sheep serum and HDLs were able to elicit cholesterol efflux alone by up to 20-40% (as measured by the boron dipyrromethene (BODIPY)-cholesterol assay). Furthermore, when the antagonists glibenclamide and valspodar were used to inhibit the function of ABCA1 and SR-BI or ABCA1 alone, respectively, cholesterol efflux was only marginally reduced (8-15%). Nevertheless, it is likely that in ram spermatozoa, a specific facilitated pathway of cholesterol efflux is involved in the interaction between cholesterol acceptors and transporters. Interestingly, exposure to HDLs also induced hyperactivated motility, another critical event required for successful fertilization. Taken together, this study details the first report of the dual action of HDLs on ram spermatozoa, providing both an insight into the intricacy of events leading up to fertilization in vivo as well as demonstrating the possible application of HDL supplementation in media for IVF.

Synergism between albumin, bicarbonate and cAMP upregulation for cholesterol efflux from ram sperm.

Compared to other mammalian species, ram spermatozoa are difficult to capacitate in vitro. Dibutyryl cAMP (db-cAMP) and the phosphodiesterase (PDE) inhibitors, caffeine and theophylline (cAMP up-regulators), must be added to traditional capacitation media (containing bicarbonate, calcium and BSA) to elicit a capacitation response. In this exploratory study, we assessed whether bicarbonate was still required for ram spermatozoa if cAMP is up-regulated by the addition of db-cAMP and PDE inhibitors and what role BSA plays in cholesterol efflux under these conditions. In this study, the validated BODIPY-cholesterol assay was used for the first time in ram spermatozoa to quantify cholesterol efflux by tracking the loss of BODIPY-cholesterol from the sperm plasma membrane using flow cytometry. The results show that under cAMP up-regulated conditions, an increase in membrane fluidity and tyrosine phosphorylation of sperm proteins remain as bicarbonate-dependent processes. In fact, the supplementation of bicarbonate under these conditions was necessary to further enhance cAMP production in ram spermatozoa, which correlated with the presence of these capacitation-related processes. When BSA was supplemented with cAMP up-regulators (as well as bicarbonate), there was a loss of approximately 20-23% of BODIPY-cholesterol (79.5 ± 30.5% to 76.9 ± 12.3% remaining from 10 min), indicating that BSA is essential for mediating cholesterol efflux in ram spermatozoa as measured by the BODIPY-cholesterol assay. The current study identifies the functional relationship between bicarbonate, BSA and cAMP up-regulators that is required to support capacitation-related processes in ram spermatozoa, specifically cholesterol efflux.