Lipid remodeling in swine spermatozoa: Innovative model for the analysis of oxysterols during capacitation

Capacitation is a vital process that prepares spermatozoa for successful fertilization by inducing significant biochemical and biophysical changes in the plasma membrane. Among these, the role of oxysterols — oxygenated derivatives of cholesterol — has garnered increasing attention due to their influence on membrane dynamics, signaling pathways, and capacitation-related modifications. This study aimed to evaluate the temporal and treatment-dependent changes in oxysterol concentrations during in vitro capacitation of swine spermatozoa. Using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), three key oxysterols were quantified: 7β-hydroxycholesterol (7β-OHC), 7α-hydroxycholesterol (7α-OHC), and 7-ketocholesterol (7-KC). Spermatozoa were subjected to three experimental conditions: a control group (Ctrl), exposure to swine oviductal epithelial cells (SOEC), and SOEC pre-treated with progesterone (SOEC P4), at three time points (t0, t2h, t4h). Findings reveal baseline oxysterol concentrations in non-capacitated spermatozoa and demonstrate condition- and time-dependent dynamics during capacitation. Notably, Principal Component Analysis (PCA) highlighted 7-KC as the oxysterol showing the largest variation across conditions and time points, particularly under the SOEC P4 condition. 7β- and 7α-OHC showed stability as possibly involved in distinct contributions to capacitation processes. These results suggest a potential role of oxysterols in sperm membrane dynamics, especially for 7-KC, and offer insights into their modulation by physiological and biochemical cues during capacitation. This study provides a foundation for understanding the implications of oxysterol dynamics in fertility and reproductive biology.