Objective To reveal the mechanisms of immunological infertility,the method of coimmunoprecipitation(CO-IP) and liquid chromatogram mass/mass (LC-MS/MS) was used to screen sperm membrane proteins which interacting with antisperm antibodies (ASA).Methods This study was designed as a case-control.The disease group including 56 serum samples from 521 cryptogenic infertile patients were screened ASA positive by ELISA and conformed with mixed antiglobulin reaction(MAR).The controls were 31 serum samples which ASA is negative and already possessed healthy offspring.All subjects were enrolled from September 2015 to December 2015 in China PLA General Hospital.Spermatozoa samples from 48donors with normal sperm parameters were from January 2016 to April 2016 in China PLA General Hospital.The purified human sperm membrane proteins were then mixed with serum from disease group (positive for ASA) and control group (not containing ASA).The binding proteins of antisperm antibodies were enriched using CO-IP assay.The immunoprecipitates were separated on sodium dodecyl sulfate-polyactylamide gel (SDS-PAGE),then the binding proteins were cut from the gel and analyzed by LC-MS/MS after the enzymolysis.These proteins could bc idcntified as definition,biological function (s) and subccllular localization with Uniprot database.Results The serum samples from infertile persons (39 females and 17males) were screened ASA positive by ELISA and conformed with MAR.The healthy controls (17 females and 14 males) were ASA-negative in ELISA.Forty proteins that interact with ASA were obtained from the study and these could be divided into three groups:11 antigens detected by control serum samples only,14antigens recognised by both infertile patients and control sera,and 15 antigens specific for patients with ASA.These 15 proteins are Sperm Cation channcl protein 1,Sperm Cation channel protein 3,Sperm Cation channel protein 4,Sperm associated antigen 9,Apolipoprotein A-I,Dynein heavy chain 14,Cylicin-2,Izumo sperm-egg fusion protein 4,Thioredoxin domain-containing protein 2,IQ domain-containing protein H,IQ domain-containing protein F1,Spermatogenesis-associated protein 5,Spermatogenesis-associated protein 5-like protein 1,Sperm acrosome membrane-associated protein 1,E3 ubiquitin-protein ligase RNF 114.Conclusion Fifteen proteins discovered with CO-IP technology and LC-MS/MS analysis could be referred as male immunoinfertility-related antigens and they may hold the great importance in revealing the secret of immunological infertility.

Objective To observe the effect of Bushen Huoxue Decoction(BSHX)on PI3K/Akt/mTOR signaling pathway and explore its possible mechanism in improving synaptic plasticity in a vascular dementia(VD)rat model.Methods Sprague-Dawley rats were randomly divided into sham operation group(Sham group),model group(VD group),Bushenhuoxue decoction group(BSHXD group),nimodipine group(NMDP group),with 10 rats in each group.The VD model of rats was established by two-vessel(2-VO)occlusion method.Rats in BSHXD group were given BSHXD at a weight of 10.14 g·kg-1,while rats in the NMDP group were given nimodipine decoction at 11 mg·kg-1.The SHAM group and the VD group were given saline at a weight of 10 mL·kg-1 once a day for 4 weeks.Morris water maze was used to observe the spatial learning and memory ability of rats in each group.Nissl staining was used to observe the damage of Nissl bodies and neurons in CA1 area of hippocampus of rats.The expression of synaptophysin(SYN)and postsynaptic density protein 95(PSD-95)in hippocampal CA1 region was detected by immunohistochemistry.Golgi-Cox staining method was used to observe the number changes of dendritic branches and spines of hippocampal neurons.Transmission electron microscopy(TEM)observed the ultrastructural change of synapses.The protein and mRNA expressions of phosphatidylinositol 3-kinase(PI3K),serine-threonine kinase(AKT)and mammalian target of rapamycin(mTOR)in rat hippocampus were detected by Western blot and Reverse transcription quantitative PCR(RT-qPCR).Results Compared with the control group,the learning and memory ability of VD rats decreased.These rats showed abnormal synaptic ultrastructure of hippocampal neurons and neuronal cell damage,and this was accompanied by a decrease in the density of dendrite branches and dendritic spines of neurons.The expression of both SYN and PSD-95 proteins in the hippocampus decreased(P<0.05),and synaptic plasticity was damaged.Both mRNA and protein expression of PI3K,Akt,and mTOR in the hippocampus decreased in the VD rats(P<0.05).Also observed in VD rats was that administration of BHSX mitigated the learning and memory impairment observed in these animals,improved the morphology and synaptic ultrastructure of hippocampal neurons,increased the mRNA and protein expression levels of PI3K,Akt,mTOR,and increased the protein levels of SYN and PSD-95(P<0.05).Conclusion BSHX can alleviate the learning and memory impairment of VD rats and increase the protein expression levels of synapse-related proteins.These effects may be related to the promotion of synaptic plasticity by BSHX through activation of PI3K/Akt/mTOR signaling.