Palmitoylated GLB1L4 Transfers Via Exosomes To Maintain Sperm Funtion In Rat Epididymis

Mammal sperm are produced in the testis,but they mature under the effect of factors related to the microenvironment of the epididymal lumen.In this process,epididymal protein plays an important role.Previous studies have shown that GLB1L4 protein is specifically expressed in rat epididymal epithelial cells,and it can be secreted into the epididymal lumen.When the epididymis development has completed and sperm enters the epididymal lumen,GLB1L4 protein can bind to the sperm.The GLB1L4 protein may play an important role in epididymal development and sperm maturation,but how it affects sperm maturation and what specific role it plays in sperm maturation is still unclear.This research has obtained the following main results through experiments.1.Tissue localization experiments showed that GLB1L4 protein existed not only in epithelial cells of caput epididymis,but also in sperms of all parts of epididymal lumen,indicating that the principal cells of epididymis could secrete GLB1L4 protein into the lumen fluid of epididymis,and finally transport GLB1L4 protein to sperms of cauda epididymis.2.The expression level of GLB1L4 was knocked down by sh RNA lentivirus injection in vivo,and the hyperactivated motility index and protein tyrosine phosphorylation level of activated sperm were significantly decreased（P<0.05）.The litter size of GLB1L4knockdown group was significantly decreased after mating with normal female rats（P<0.05）.These results indicated that GLB1L4 affected the fertility of rats by affecting sperm capacitability.3.GLB1L4 protein in rat epididymal fluid mainly existed in epididymal exosomes;In addition,the exosomes of rat primary epididymal epithelial cells cultured in vitro carried most of GLB1L4 protein,which could bind to caudal epididymal sperm.These results indicate that GLB1L4 can be transported in the epididymis through exosomes secreted by epithelial cells of caput epididymis,and can reach the cauda epididymis to interact with sperm.4.After the palmitylation inhibitor 2-BP（2-Bromopalmitate,2-BP）was added to the culture medium of rat epididymal caput epithelial cells,the palmitylation degree of GLB1L4protein in cells was significantly reduced,and the content of GLB1L4 protein in exosomes was also significantly reduced.Through online prediction,it was found that the 12^th and 15^thcysteine of GLB1L4 amino acid sequence were the most likely palmitylation modification sites.After mutation of these two amino acids,the localization of GLB1L4 protein in mouse caput epididymal epithelial PC1 cell line changed from punctate distribution to diffuse state.These results indicate that the palmitylation state of GLB1L4 protein exists in epididymal exosomes.In conclusion,palmitylation can make GLB1L4 protein more easily load into epididymal exosomes,so that it can exist stably in the epididymal cavity,and eventually reach caudal epididymis and bind to caudal epididymal sperm,affecting sperm capacity,and thus affecting fertility in rats.