In silico molecular characterization of cholesterol binding to species-specific and non-specific albumins and their effects on in vitro quality parameters of ram spermatozoa

Abstract

In the present study, the interactions of cholesterol (CHOL) with bovine (BSA), human (HSA), ovine (OSA) and caprine (CSA) albumins were analyzed using in silico approaches (docking, molecular dynamics, and quantum biochemical calculations). A sequential, in vitro experiment evaluated the effects of species-specific (OSA) and non-species-specific (BSA, HSA and CSA) albumins on ram sperm. In this case, semen collected from 10 rams were mixed with extender, aliquots were subjected to cooling for 3 (15 °C) and 24 hours (5 °C), and incubated with TALP medium and BSA, HSA, OSA or CSA (5 mg/mL). Overall, OSA combines energetically favorable poses with stable complexes for CHOL interaction, especially at FA1-FA9 and FA1 sites. FA1 was present in three of the four albumins, supporting its relevance as a CHOL binding site. Although molecular docking ranked HSA highest and molecular dynamics simulations highlighted CSA’s rigidity at FA6/NS5 sites, OSA was a more consistent CHOL binder after evaluations with all methods. In 3-hour cooled samples, OSA decreased total sperm motility (44 ± 2.4 %; p < 0.05) compared to the control, BSA, HSA, and CSA groups (76.1 ± 3.8, 81.3 ± 4.1, 72.8 ± 3.4, and 70.3 ± 4.2 %). OSA reduced sperm viability (28.8 ± 5.3 %; p < 0.05) compared to control (50.1 ± 4.2 %), with intermediate values in BSA, HSA, and CSA groups. Apoptosis or mitochondrial functionality were not affected by treatments. Acrosome-reacted spermatozoa were higher in OSA (40.2 ± 3.5 %; p < 0.05) compared to control and BSA (24.4 ± 1.6 and 23.5 ± 1.4 %), with HSA and CSA showing intermediate results. In 24-hour cooled samples, OSA reduced sperm motility (48.8 ± 3.8 %; p < 0.05) in comparison with BSA (72.2 ± 4.5 %). OSA decreased sperm viability (21.9 ± 1.6 %; p < 0.05) compared to control and BSA (40.3 ± 2.4 %), while HSA and CSA groups had intermediate values. Apoptosis was higher in OSA and mitochondrial functionality was enhanced by BSA and HSA compared to OSA. Treatments with HSA and OSA had greater acrosome reaction rates in ram spermatozoa (35.1 ± 2.3 and 30.8 ± 1.4 %; p < 0.05) than in control (19.2 ± 4.2 %), while BSA and CSA showed intermediate results. Thus, capacitation-related changes were pronounced with species-specific albumin, partially explained by energetically favorable OSA–CHOL binding (shown by in silico analyses). OSA significant effects on sperm acrosome reaction compromised sperm motility, viability and mitochondrial function, especially after prolonged cooling. Within the ovine model, OSA represents a promising alternative to BSA in capacitation systems. However, OSA concentrations and incubation parameters need to be optimized to balance capacitation induction with sperm preservation