| IntroductionThe novel gene of human testis sperm binding protein (TSBP) which related to bovine seminal plasma proteins (BSP) is cloned by us in human testis. The protein encoded by this gene is structurally similar to BSP and was estimated to be a sperm binding protein similarly to BSP in function. BSP proteins are the main proteins in bovine seminal plasma whose function has been focused on for many years. It was reported that BSP proteins play an important role in lipid metabolism and also modulate sperm capacitation and acrosome reaction. At present, homogeneous proteins of BSP proteins have been found in the seminal plasma of horse and pig which showed the binding properties similar to BSP proteins. This indicates that BSP proteins and its homogeneous proteins are widespread in mammals. Whereas there was no report about the related protein in human semen.One of the most important manifestations of sperm capacitation is the incresment of protein phosphorylation. In human sperm, the incresment of protein phosphorylation is modulated by cAMP/PKA pathway, so the capacitation of sperm may be relsated to PKA pathway. BSP protein can promote the cholesterol efflux of sperm membrane and the rearrangement of sperm membrane is advantageous for the influx of HCO3-and Ca2+ which can activate AC and the following cAMP/PKA pathway on sperm membrane. Then the protein phosphorylation is promoted via the double modulation of TPK and phosphotyrosine phosphatase and at last capacitation is evoked. To study the relationship between TSBP and PKA, in this study the recombined eukaryotic expressive vector pcDNA3.1/myc-His(-)B/tsbp was transfected to cell line HEK293 and an eukaryotic expressive cell line was established after G418 screening. The activity of PKA in the stablely transfected cell line was detected by the method of autoradiography, and the result showed that PKA could be activated in pcDNA3.1/myc-His(-)B/tsbp transfected cells, which also indicated that TSBP, just like BSP, might modulate sperm capacitation by cAMP/PKA pathway.In the previous study, the novel gene has been expressed in prokaryotic cells. But in order to aquire the recombined protein similarly to the native condition and to further study the influence of this protein on sperm capacitation and acrosome reaction, it is necessary to get the recombined protein expressed by eukaryotic cells via gene recombination. In this study, the Ni2+-NTA metal affinity chromatography was used to purify the aimed protein based on the transfected cell line and the purified protein was used in the the ruction research of this gene.In vivo, only after capacitation and acrosome reaction can the sperm penetrate and fuse with the ovum to complete fertilization. Therefore, the function of sperm can be reflected exactly by the condition of the acrosome and the integrity of sperm membrane which can also be based on to estimate the ability of fertilization of sperm. The typeâ…¡-modules of BSP proteins forms a multimer structure which can bind to the phospholipids on sperm surface and promote sperm capacitation induced by heparin and HDL in bovine epididymis. Morover, BSP proteins can combine with heterogeneous collagen, fibrinogen, heparin, calmoduline, IGF-â…¡and apoA-â… and inhibit the migration of cholesterol on the sperm membrane. TSBP is gene of the novel sperm binding protein related to BSP and is similar to BSP in structure. To further investigate the biological ruction of TSBP in related aspects, we detected the affects of TSBP on sperm fuctions, including capacitation, membrane function, motility and the activity of PKA and PKC in human sperm.Materials and Methods1,Materials and reagentsPlasmid pcDNA3.1/myc-His(-)B, recombinant plasmid pGEX-5X-1/tsbp and HEK293 cells were kept by our lab. E.coli JM109 competent cell, PCR amplification kit, restriction endonuclease BamHâ… and Xhoâ… , plasmid DNA quick-extract kit, DNA gel purification kit, animal RNAout and RT-PCR kit were purchased from Takara. Endotoxin-free ultropure DNA purification kit was purchased from V-Gene. Agarose, pGEM-T Easy system, DNA ligation kit, G418 were from Promega. LB medium, lipofectin TM2000, mouse anti-His6 polyclonal antibody and Ni2+-NTA purification system were from Invitrogen. AP, HRP or FITC-conjungated goat anti mouse IgG, DAB reaction kit were purchased from Beijing Zhongshan. DMEM medium was the product of Hyclone. Primer synthesis and DNA sequencing were completed by Takara. Other agents were prepared according to Molecular Cloning. [γ-32P]ATP was from Beijing Furui Biotech. Other agents used in the assay of kinase activity were from Sigma and of analytical pure.Fresh human semen were provided by Liaoning Family-planning Scientific Research Center. Percoll desity gradient centrifugate and HSA were from Sigma. Typan Blue, Bismark Brown and Rose Red were from Chroma, Schmid H and Fluka, respectively.2,Construction of eukaryotic expressive vector of TSBPUsing the pGEX-5X-1/tsbp cloned with full length TSBP cDNA as template, the PCR reaction introduced the enzyme-incision site of BamHâ… and Xhoâ… into 3'end and 5' end, respectively. And then the PCR product was cloned into pGEM-T Easy. The eukaryotic expressing vector pcDNA3.1/myc-His(-)B that has His6 tag at the-C end was incised simultaneously with pGEM-T Easy/tsbp by BamHâ… and Xhoâ… . After purification and DNA ligation, the recombinant pcDNA3.1/myc-His(-)B/tsbp was acquired and DNA sequencing was applied using universal primers.3,Construction of the stable transfected cell lineHEK293 cell line was transfected with the recombinant pcDNA3.1/myc-His(-)B/tsbp or the vacant vector pcDNA3.1/myc-His(-)B. After G418 screening, the stable transfected cell line was constructed. RT-PCR, cell immunofluorescence and Western blot were used to identify the expression of TSBP.4,Purification of the fusion protein by IMACThe native binding buffer provided by the Ni2+-NTA purification kit of Invitrogen was used to collect cells. After repeated freeze thawing, the cell lysate was centrifugated and the supernatant was preserved. Then the purification was carried out according to the directions of the kit. The eluant was preserved in liquid nitrogen after ultrofiltrate concentration and filtration sterilization. The total protein concentration was assayed and the purification result was detected by SDS-PAGE and Western blot.5,Combination of recombinant His6-TSBP with sperm memberaneAfter eliquation of the semen, sperm was separated by Percoll desity gradient centrifugation. Ultrafiltrate was added to the same volume of sperm culture fluid. After 1 hr or 3 hr, the composition of sperm membrane was extracted to detect the combinantion of His6-TSBP by Western blot.6,Assay of the sperm functionHuman sperm was separated after fluidity using Percoll density gradient centrifugation. Each sample was divided into five groups, i.e. the blank, the hybridprotein control, HSA induced capacitated group, 0.01 mg/ml TSBP and 0.1mg/ml TSBP disposed group. After cultivated for 1 hr or 3 hr, capacitation percentage, membrane function and motility of sperm in each group was assayed.7,Activity assay of protein kinases(1) Activity assay of PKAThe lysis buffer was added into the cells transfected by recombinant vector, empty vector and untransfected cells respectively (or the sperm of different groups deccribed in part 6). Then the mixture was centrifugated. The PKA activity of different groups were checked by using kemptide as substrate labeled by [γ-32P] ATP. Kemptide was separated by SDS-PAGE and radioautography was performed. The activity of PKA was showed by checking the band of kemptide in gel analysis system respectively. (2) Activity assay of PKCThe PKC activity of sperm from different groups was detected using the same method as the detection of PKA activity, except for the specific substrate of PKA was substrated by that of PKC, MARCKS.Results1,Construction of eukaryotic expression vectorThe constructed eukaryotic expression vector pcDNA3.1/myc-His(-)B/tsbp was amplificated and incised with Xhoâ… and BamHâ… . The result of agarose electrophoresis showed a 750 bp and a 5.5 kb fragments, which was the same with our expectation. Further DNA sequencing manifested that the insertion element was tsbp sequence with complete reading frame and no frame shift. That is to say, the expression product of the constructed vector has His6 tag and can be used in protein purification and characterization.2,The expression of recombinant TSBPThe result of RT-PCR using TSBP specific primers discovered the expression of this novel gene in pcDNA3.1/myc-His(-)B/tsbp transfected cells and no in untransfected or vacant plasmid transfected cells. The cell immunofluorescence showed the same result with green fluorescence been detected only in pcDNA3.1/myc-His(-)B/tsbp transfected cells. In Western blot analysis, the 30 kDa protein band of the His6 tagged protein was also only detected in the recombinant vector transfected group.3,PKA activity in the recombinant vector transfected cellsThe activity of PKA was detected by using kemptide as substrate labeled by [γ-32P]ATP in cells transfected by recombinant vector, empty vector and in untransfected cells respectively. The result of autoradiography showed that the activity of PKA is significantly higher in the recombinant vector transfected cells than in the other two groups, which manifested that TSBP may promote the activity of PKA. 4,The purification of the recombinant proteinThe sample, effluent and eluant of IMAC was subjected to 12% SDS-PAGE. The result showed that with decrement of pH of the buffers, the affinity between Ni2+-NTA and His6 tag degraded and the fusion protein was eluted. The eluent of pH4.5 could eluted most of the fusion protein. There was an evident protein band at 30kDa which was further confirmed to be the fusion protein tagged with His6 in Western blot analysis.5,Combination of recombinant His6-TSBP with sperm memberaneThe result of Western blot showed that, after cultivated with human sperm for 1 hr or 3 hr, the purified protein could be detected in the membrane ingredient of sperm to different degree.6,The impact of His6-TSBP on sperm function(1) The impact on sperm capacitationTriad colour staining was performed to observe AR rate in each group after treated with prongestone. The result showed that 0.1 mg/ml His6-TSBP treated group could raise the AR rate induced by progesterone significantly (P＜0.05). Whereas for the 0.01 mg/ml His6-TSBP treated group, there was no significant difference.(2) The impact on the membrane function integrity of human spermThe analysis of 22 human semen samples showed that the recombinant TSBP could affect the membrane function of sperm membrane. The swelling rate of sperm tail in the group disposed with high concentration of recombinant TSBP is (90±15)% at 1 hr and (94±18)% at 3 hr, which is significantly higher than that of control groups (P＜0.05). Whereas in the group disposed with lower concentration of recombinant TSBP, the significant difference could be detected only at 16 hr, which indicated that the impact of recombinant TSBP on sperm membrane was concentration-dependent.(3) Impact on motility of human spermThe result of CAS A analysis of human sperm showed that 3hr disposal with 0.1 mg/ml recombinant TSBP could elevate the forward-movement percentage but with no significant impact on the whole activity ratio. After disposed for 3 hr, both the forward-movement percentage and the whole activity ratio were increased. And there was no other significant change among the other groups. The VSL and VCL showed no difference in different groups.7,The influence of recombinant TSBP on protein kinase activity in human sperm(1) The avtivity of PKAThe result of autoradiography showed that in the group treated with 0.1 mg/ml His6-TSBP, the activity of PKA in human sperm was increased while no significant deviation was observed in the group treated with 0.01 mg/ml His6-TSBP.(2) The avtivity of PKCPKC activity in the sperm membrane was a little bit higher in the 0.1 mg/ml His6-TSBP treated group than the control group but with no significant difference.DiscussionThe cDNA sequence of novel gene TSBP (testis sperm binding protein) related to proteins of bovine seminal plasma (BSP) in human testis was found and cloned when we studied the important roles of BSP in fertilization and the development of zygote, named TSBP and registered in Genbank (AF279147). Its ORF encoded a protein of 223 amino acids. There were 7 exons, 6introns and 4 fibronectin typeâ…¡-modules in the amino acid sequence. It is estimated that TSBP is a kind of binding protein whose function and structure are similar to that of BSP. In the previous study, this gene was prokaryotic expressed and located at human 19q1.3 by the method of RH and FISH. The prokaryotic expressing system is efficient and convenient, but it is lack of post-translational modulation. To acquire recombinant protein that similar to the native state, the gene cloning technique should be applied in eukaryotic expression.Fusing His tag at one end of aimed proteins is commonly used these years to express recombinant proteins. Due to the small molecular mass of poly histidine tag, activity of the studied proteins will not be affected and so the purified prtein could be used in function research directly. Here, we used IMAC to purify fusion protein His6-TSBP and Western blot analysis showed that after affinity chromatography and ultrofiltration concentration, the aimed protein was enriched. Besides the His6 tagged fusion protein, there are also some foreign bands in the purification product, which may be due to histidine, glutamine and generous of bovine serum in the culture medium that may disturb the combination of fusion protein to Ni2+-NTA.The change of adipose membrane structure of sperm is one of the most important changes during capacitation. Therefore, detection of acrosome reaction and membrane integrity of sperm can accurately reflect the function of sperm and can be used to predict its potential of fertilizability. In 1999, WHO defined the rate of acrosome reaction and the integrity of sperm membrane as important indexes to evaluate sperm fertilizability? In this study, we detected the role of different concentration of recombinant TSBP on membrane function, capacitation and motility of human sperm. The result showed that, like BSP, recombinant TSBP could also affect sperm fertilizability.Transmembrane cell signaling is correlated with sperm motility. Different signal can regulate sperm motility via different cell signaling pathways. In vitro, protein phosphorylation depends on cholesterol accepters, which implied that there is some relationship between cholesterol efflux and protein phosphorylation. It has been shown that HCO3-is needed in sperm capacitation of hamster and mouse. Though the mechanism of HCO3-transport into sperm is not elucidated yet, it is probable connected with change of the sperm membrane ingredients. HCO3-can not only modulate membrane potential of sperm, but also stimulate AC to synthesize cAMP, activate cAMP/PKA pathway and thereby increase protein phosphorylation and promote acrosome reaction. Considering the relationship between BSP and cholesterol efflux of the sperm membrane and cAMP/PKA pathway, in this study we investigated the action between TSBP and PKA activity. After detected PKA activity of the recombinant pcDNA3.1/myc-His(-)B/tsbp transfected cells via autoradiography, we discovered that TSBP could activate PKA in the transfected cells. PKC has been reported to exist in sperm tail and participate in flagllar movement. Many proteins that related to axial fibril which participate in sperm motiliay were necessary in starting and maintaining of sperm motivation and were found to be substrates o PKC. The activity and concentration of PKC was also correlated with sperm motility. In our study, we discovered that TSBP could activate PKA in the sperm treated with 0.1 mg/ml His6-TSBP, which manifested that TSBP might promote sperm capacitation via this way in vivo.However, we must realize that the fusion protein we got in this study was different from its physiological state in both concentration and action environment, so the protein that purified from tissues should be acquired in order to further determine its biological function.Conclusion1,Stable transfected fusion protein His6-TSBP expressing eukaryotic cell line was constructed.2,PKA activity was discovered to increase in the cell line expressing fusion protein His6-TSBP.3,Fusion protein His6-TSBP was purified by IMAC.4,Recombinant protein His6-TSBP can combine with sperm membrane in vitro.5,Recombinant protein His6-TSBP can influence human sperm function in vitro, including membrane integrity, motility and AR rate induced by progesterone.6,PKA activity was increased in the sperm treated with 0.1 mg/ml His6-TSBP.7,PKC activity of sperm membrane could not be affected under the experimental concentration of His6-TSBP in this study.
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