Differential molecular mechanisms of heparin, BSA, and Ca²⁺-Induced boar sperm capacitation revealed by...

Sperm capacitation is a prerequisite for successful fertilization. In vitro, this process can be induced by various agents such as heparin, bovine serum albumin (BSA), and calcium ions (Ca²⁺), yet their underlying regulatory mechanisms remain incompletely understood. This study employed RNA sequencing (RNA-seq) to comparatively investigate the transcriptomic profiles and regulatory mechanisms during sperm capacitation induced by heparin, BSA, or Ca²⁺. Phenotypic analysis confirmed that all three inducers significantly promoted capacitation, albeit with differential effects on motility parameters and ATP levels. Transcriptome analysis revealed both shared and unique differentially expressed genes (DEGs) among the treatment groups. Common pathways enriched across all groups included PI3K-Akt, MAPK, calcium signaling, and lipid metabolism, indicating core regulatory networks in capacitation. Specifically, heparin-induced capacitation was predominantly associated with chemokine signaling and steroid biosynthesis pathways. We identified and validated the chemokine receptor CCR1 as a novel mediator of heparin's action, with its knockdown significantly impairing heparin-induced capacitation. BSA treatment uniquely enriched pathways related to autophagy, apoptosis, AMPK signaling, and peroxisome function, suggesting a role in modulating energy metabolism and membrane remodeling. Ca²⁺ treatment was primarily linked to ABC transporters and glycerolipid metabolism. This study provides the first comprehensive comparative transcriptomic landscape of boar sperm capacitation induced by heparin, BSA, and Ca²⁺, revealing distinct molecular signatures. The identification of CCR1 as a key player in heparin-induced capacitation offers a new target for optimizing in vitro capacitation systems and improving artificial insemination efficiency.