Molecular Mechanism Of Heparin Regulating Boar Sperm Capacitation Through Olfactory Receptor OR2C1

Sperm capacitation is a necessary for the insemination,and is the key to improving the fertility of boars and the success rate of artificial insemination techniques.Heparin is a glycosaminoglycan（GAG）class substance,which is often used to induce sperm capacitation,but the specific mechanism is still unclear.Therefore,it is important to reveal the mechanisms by which heparin-induced capacitation improves the sperm capacitation rate and the success rate of artificial insemination.Differentially expressed mRNA was found to be highly enriched in the olfactory transduction pathway in heparin-capacitated boar sperm.The olfactory receptor（ORs）family is one of the largest membrane protein families in mammals and is believed to be involved in the sperm chemotaxis and capacitation.However,the molecular mechanism by which ORs mediate capacitation in mammalian sperm remains unclear.Therefore,boar sperm was selected for this study,and techniques such as qRT-PCR,double fluorescence staining,homology modeling,molecular docking,and molecular dynamics simulations were used to detect differentially expressed olfactory receptor genes in sperm treated with different capacitation factors.Subsequently,receptor ligand pairing and function validation was performed using dual-glo luciferase assay,olfactory agonist and inhibitor,and siRNA knockdown to explore the mechanisms of olfactory receptor involvement in sperm capacitation.The main results are as follows:1)The capacitation rate of boar sperm was significantly increased after treated with heparin,hyaluronic acid,BSA,CaCl₂and NaHCO₃for 1h-4h.The expression levels of 14 olfactory receptor genes in boar sperm were detected.The results showed that the mRNA expression levels of 11 olfactory receptor genes after treated with heparin were significantly up-regulated in 1h-4h（p<0.01）.Meanwhile,olfactory marker protein（OMP）localization in sperm was altered after treated with heparin.2)Three-dimensional models of 14 olfactory receptor proteins were constructed,and molecular docking with heparin was performed,respectively.The results showed that the OR2C1 receptor was most likely to bind to heparin.Therefore,we further simulated the dynamics between the OR2C1 receptor and heparin and found that the heparin is mainly bound to a hydrophobic cavity composed of Arg3,Ala6,Thr7,Asn171,Arg172,Arg173 and Pro287 in the OR2C1 protein.Pro287 exists in the transmembrane helical region 7（TMH7）of OR2C1.The dual-glo luciferase assay further confirms the binding of OR2C1 to heparin.3)OR2C1 mRNA expression was significantly up-regulated in sperm treated with heparin and olfactory receptor agonist,but better results were found in the heparin-treated group.Compared with fresh sperm,OR2C1 mRNA expression of sperm treated with olfactory receptor inhibitor was significantly decreased at 1h-4h（p<0.01）.When olfactory receptor agonists or inhibitors were added to heparin-treated sperm,OR2C1 mRNA expression was significantly decreased compared with heparin-treated sperm alone（p<0.01）,but was significantly higher than that in heparin-treated sperm alone（p<0.01）.4)After transfection of OR2C1-targeting siRNA,the expression level of OR2C1mRNA in sperm transfected with siOR2C1 was significantly decreased compared with that in siNC group（p<0.01）.When heparin was added,the OR2C1 mRNA expression and capacitation rate of sperm in siOR2C1 transfected group were significantly lower than those in siNC group（p<0.01）.In summary,heparin is a novel olfactory receptor agonist that modulates boar sperm capacitation by binding to the olfactory receptor OR2C1.In addition,we reveal that OR2C1 binds with heparin via a hydrophobic cavity comprises of Arg3,Ala6,Thr7,Asn171,Arg172,Arg173,and Pro287.This study is important for clarifying the molecular mechanism of olfactory receptor in mammalian sperm,and also provide scientific reference for revealing the role of olfactory receptors in boar sperm capacitation,further understanding the molecular regulation mechanism of sperm capacitation and improving sperm capacitation.