Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) has a significant importance for spermatozoa’s physiology. The expression of CFTR in human spermatozoa has been associated with motility and morphology, whereas CFTR inhibition prevents capacitation. We investigated whether the use of egg L-α-phosphatidylcholine (ePC)/cholesterol (Chol) proteoliposomes was able to deliver functional CFTR to human spermatozoa. CFTR with an enhanced green fluorescence protein (EGFP) FLAG epitope-bearing cell membrane fragments (CMF) were integrated into proteoliposomes using the thin-film hydration method. We evaluated two distinct ePC/Chol lipid mixtures, 72:28 and 80:20 (in molar). Overall, 72:28 proteoliposomes exhibited higher CFTR delivery efficiency to spermatozoa after 1 h of incubation. Further, the incubation of human spermatozoa with 72:28 proteoliposomes was shown to promote a significant increase in Cl ^- influx, with no apparent adverse effects on sperm vitality and motility. By comparing the intracellular [Cl⁻] accumulation in proteoliposome-treated spermatozoa with and without CFTR function inhibition, we found that 43 % of the intracellular [Cl⁻] increase in proteoliposome-treated spermatozoa could be attributed to the activity of newly integrated CFTR channels. We observed by fluorescence microscopy that CFTR-GFP integration occurred mainly in the sperm head, where CFTR is naturally expressed. Although this approach requires further investigation, we also observed that CFTR-carrying proteoliposomes promote the restoration of total and progressive motility in human spermatozoa with compromised CFTR function via inhibition with CFTR _Inh -172.

Our findings suggest that CFTR delivery via proteoliposomes could represent a novel strategy to enhance the in vitro quality of spermatozoa with reduced CFTR expression/function.