| 摘要: | During sperm capacitation, proteins are phosphorylated and lead to the induction of signal transduction which were hampered by excess iron (EI) and therefore affect sperm motility. However, phosphoproteomic analysis of mouse sperm capacitation still not fully understood. In addition, Lycium barbarum polysaccharides (LBP), a traditional Chinese medicine, was known to process iron chelation. In this study, we aimed to identify various phosphorylation events in proteins, signaling cascades, interacting proteins network and molecular function in mouse sperm capacitation and then evaluate how excess iron induced changes on AKAP4 and GSK-3 phosphorylations of capacitated mouse sperms. The protective effects of LBP and its active fraction on excess iron-induces changes were then determined by label-free quantification (LFQ) phosphoproteomics using TiO2 (titanium dioxide) enrichment integrated with bioinformatics analysis using ingenuity pathway analysis (IPA). LBPs were purified by enzymatic reactions, gel filtration, and then GC/MS was applied to determine monosaccharides composition. The active LBP fraction (2, 4 mg/mL) possessing iron chelating activity were then incubated with mouse sperm exposure to EI (40 M). Changes of sperm motility and AKAP4 and GSK-3 phosphorylations during sperm capacitation were determined by western blot analysis. Results showed that (I) LFQ phosphoproteomics analysis identified 3177 phosphopeptides corresponding to 943 proteins and there were 1632 total phosphosites in serine, threonine, and tyrosine. There were 1050 novel phosphosites corresponding to 402 proteins which have not identified before. Gene ontology analysis for cellular component determined cytoplasm as the major location of the proteins. In the category molecular and biological function, ion binding and regulation of biological function were the most represented terms. IPA canonical pathway analysis found GP6 signaling pathway as a novel pathway associated with sperm capacitation, which involved GSK-3 phosphorylation expression changes. IPA network analysis of cell signaling, cellular movement, and reproductive system development and function revealed the upregulated of AKAP4 and downregulated of GSK-3 while the activity was increased. (II) LBP crude extracts were separated into LBP1, LBP2 and LBP3 and only LBP3 showed concentration dependent iron chelate activity. LBP3 treatment (4 mg/mL) significantly ameliorated EI induced sperm motility reduction at capacitation 60 minutes. GC/MS analysis indicated that LBP3 was composed of galactose (Gal), glucose (Glc), arabinose (Ara), rhamnose (Rha), and mannose (Man). (III) The greater increase in tyrosine phosphorylation of GSK-3 and fewer changes of GSK-3 serine phosphorylation of western blot analysis supporting the increasing of GSK-3α predicted by IPA of capacitation 60 minutes dataset. Iron insult decreased GSK-3 serine phosphorylation in both sites (pGSK-3α Ser21 and pGSK-3β Ser9). The presence of LBP3 itself increased pGSK-3α Ser21 but decreased pGSK-3β Ser9. EI incubated with LBP3 2 or 4 mg/mL increased pGSK-3β Tyr279 and pGSK-3α Tyr216, respectively. In conclusion, phosphorylation changes of GSK-3 and AKAP4 play an important role during sperm capacitation and also EI insults induced reductions of pAKAP4 and phosphoserine GSK-3 (pGSK-3α Ser21 and pGSK-3β Ser9) during sperm capacitation. Incubated with LBP3 at 4 mg/mL can reserve the motility against iron insults and the mechanism might be associated with the increasing of AKAP4 and GSK-3α in serine phosphorylation (pGSK-3α Ser21) and tyrosine phosphorylation (pGSK-3α Tyr216). |
