Establishment Of Progesterone-based Sperm Selection Method And The Relevant Regulatory Mechanism

Objective:To establish a progesterone-based sperm selection method and to elaborate the relevant regulatory mechanism.Method:A long-distance channel device mimicking human female reproductive system was designed and fabricated.The different concentrations of progesterone and their different positions at the device were set for the preparation for sperm selection.The sperm selection conditions were validated by the measurements of sperm concentration at different positions on the different time points.To evaluate the effectiveness of the established sperm selection method,Diff-quik staining,chromatin diffusion method and annexin V-fluorescein isothiocyanate fluorescence/propidium iodide staining were used to examine the morphology,DNA fragments and apoptotic status of sperm before and after selection,respectively.Human sperm induced with progesterone were treated by geldanamycin,a heat shock protein 90(Hsp90)specific inhibitor,during capacitation.Then sperm capacitation was evaluated by chlorotetracycline staining and the sperm motility parameters and the percentage of hyperactivation were analyzed by CASA.The co-localization of Hsp90 with cell division cycle protein Cdc37(Cdc37)and endothelial nitric oxide synthase(eNOS)was detected by indirect immunofluorescence staining,respectively.The interaction between Hsp90 and its co-chaperone Cdc37 was detected by co-immunoprecipitation.Western blotting was used to explore the Hsp90 expression in human sperm and the effect of geldanamycin on it during capacitation.Western blotting was also used to analyze the effect of H-89,a protein kinase A(PKA)inhibitor,and Go6983,a protein kinase C(PKC)inhibitor on the phosphorylation of serine,threonine and tyrosine of Hsp90,the effect of 17-AAG(Hsp90 inhibitor)on phosphorylation of Y416,the activity site of tyrosine protein kinase Src,and the effect of 17-AAG and geldanamycin on protein threonine phosphorylation in sperm during capacitation.The tyrosine,serine and threonine phosphorylation of Hsp90 were examined by immunoprecipitation.Protein palmitoylation in sperm was detected by the acyl-biotinyl exchange method;palmitoylated sites of mouse Hsp90 were predicted by CSS-Palm 4.0 software,and the palmitoylated Hsp90 was further confirmed by the acyl-biotinyl exchange method combining immunoprecipitation.Result:The experimental conditions of progesterone-based sperm selection method were validated.After the selection by the established method,the proportion of sperm with normal morphology and DNA integrity increased,and sperm apoptotic status did not significantly change.Geldanamycin decreased progesterone-induced sperm capacitation in a dose-dependent manner,motility parameters including VSL,VAP,STR,BCF and the percentage of hyperactivation.Hsp90 co-localized with eNOS and co-chaperone Cdc37,interacted with co-chaperone Cdc37.Hsp90 expression increased and was inhibited by geldanamycin during human sperm capacitation.During human sperm capacitation,the phosphorylation levels of serine,threonine and tyrosine of Hsp90 were inhibited by H-89 and Go6983;17-AAG and geldanamycin altered threonine phosphorylation level of part proteins and 17-AAG inhibited the phosphorylation level of Y416 in tyrosine protein kinase Src.Various proteins in mouse sperm were palmitoylated.Five sites in mouse Hsp90 that would be palmitoylated were predicted.Hsp90 palmitoylation was exist in mouse epididymal caput and cauda sperm.The Hsp90 palmitoylation level of sperm in caput epididymis was lower than that in cauda epididymis.While sperm in cauda epididymis were incubated under capacitated conditions,the palmitoylation level increased.Conclusion:The progesterone-based sperm selection method has been successfully established and improves sperm quality.Progesterone regulates sperm function via Hsp90 mediation,while Hsp90 interacts with Cdc37;the phosphorylation of serine,threonine and tyrosine in Hsp90 is controlled by PKA and PKC;Hsp90 is palmitoylated and regulates the Src activity during sperm capacitation.