Epididymal Protease Inhibitor Protein And Male Infertility

IntroductionMen's infertility is gaining attention.It is realized that in at least half of all cases of infertility,male factors are contributing causes,including low sperm motility,abnormal sperm shape,varicocele,and oligospermia.To find out these causes,therefore,is of benefit to the therapy of infertilty. On the other hand,There is a global need to control the increasing growth rate of the world's population because of its negative impact on environment and economy.Reproductive science has provided a range of contraception methods for the females,however,the choices for male are few and currently limited to condoms and vasectomy.The development of male contraceptives that are effective,safe,and reversible is desired for family planning throughout the world.Eppin(epididymis protease inhibitor),a recently identified and characterized 'male-only' molecule from the testis and epididymis, appears to play an important role in primate fertility.An excellent study had been performed by O'Rand et al and demonstrated that male monkeys immunized with human recombinant Eppin couldn't successfully impregnated female monkeys.The serum anti-Eppin titer was high in these male monkeys.Further studies by Wang Z et al proved that Eppin was bound to semenogelin-I in seminal plasma and on human ejaculate spermatozoa.They suggested that anti-Eppin antibodies probably disrupted the Eppin-semenogelin complex,which resulted in the loss of sperm forward motility and semen coagulum.Eventually these conversions negatively affected the fertilization.Although these studies found that Eppin had a close association with the fertilization,the Eppin's functions in men and its relationships to men's infertility are still little known to us.To reveal the significance and possibility of detection of anti-Eppin antibody in clinical infertilty patients,in the present study,we developed an efficiency method to obtain relatively large quantity of recombinant Eppin protein and verified its biological function by immunological methods.Our results demonstrated that the purified His6-Eppin protein were biologically active and is candidate for clinical infertility diagnosis and male immuno-contraceptive agents development.Aim:To obtain relatively large quantity and biologically active of recombinant Eppin protein,and is candidate for clinical infertility diagnosis and treating.Materials and Metholds:Materials TRIzol Reagent and SuperScriptâ…¡Reverse Transcriptase and pCDNA3.1(+)were purchased from Invitrogen(Carlsbad,CA,USA). Restriction endonucleases,T4 DNA ligase,dNTPs,Restriction endonucleases,agarose Gel Extraction kit and the Plasmid Purification Kit were all from TaKaRa(Dalian,China).Expression vector pET-28a (+),Escherichia coli strain Top10 and BL21(DE3)were obtained from Novagen(Madison,WI,USA).Reagents for polyacrylamide electrophoresis such as acrylamide,bis-acrylamide,ammonium persulfate, and TEMED and Coomassie Brilliant Blue R-250 solution were obtained from Bio-Rad(Hercules,CA).Coomassie Plus protein assay kit was from Pierce(Rockford,IL,USA).Prestained Protein Marker(Broad Range) was obtained from New England Biolabs(Beverly,MA,USA).Most of other reagents such as salt and buffer components were analytical grade and obtained from Sigma(St.Louis,MO,USA).His-Select^TMHC nickel affinity gel was obtained from GE Healthcare(Pharmacia Biotech). Polymerase chain reaction(PCR)primers were synthesized by TaKaRa (Dalian,China).DNA sequencing was performed by TaKaRa(Dalian, China).Metholds:Total RNA was extracted from human epididymis using TRIzol Reagent according to the manufacture's protocol.An Eppin cDNA fragment (GeneBank accession No.BC053369)(nucleotides 70～423)lacking part of the N-terminal secretory sequence was obtained by reverse transcriptase-polymerase chain reaction(RT-PCR)with total RNA.The primers special for Eppin were 5'-GG-GGATCC(BamHI) -ctcttagcgaatgtccagggac-3'and 5'-GGG-CTCGAG(XhoI)-tcagggaaagcgt ttattct-3'.The PCR products were cut with restriction enzymes XhoI and BamHI and agarose gel-purified.The resulting DNA fragment was ligated into the plasmid pET-28a(+)cut with restriction enzymes XhoI and BamHI with T4 DNA polymerase.The generated plasmid named pET28a-His6-Eppin was then transformed into E.coli strain Top10,and the positive clones were confirmed by DNA sequencing.The same primers were also designed for Eppin eukaryotic expression.In a similar manner to pET28a-His6-Eppin plasmid construction,pCDNA-Eppin was generated by ligation plasmid pCDNA3.1(+)with Eppin cDNA.The E. coli strain BL21(DE3)was transformed with pET28a-His6-Eppin.The successfully transformed E.coli was picked up from a single colony and was grown overnight at 37℃in Luria Bertani(LB)medium(0.5%yeast extract,1%Tryptone,1%NaCl),supplemented with kanamycin (50mg/L).The culture mixture was then inoculated to fresh LB medium (1:50 dilution)containing kanamycin and grown at 37℃under continuous shaking until the absorbance at 600 nm reached 0.6～0.8.To optimize the culture conditions,expression was induced in different conditions and the bacteria were incubated for a period of 3 h respectively. The degree of expression was evaluated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE). His-Select^TMHC nickel affinity gel was used to purify recombinant Eppin. Prior to purification,the induced bacteria were harvested by centrifugation at 6,000g for 20 min at 4℃.The bacterial pellets were resuspended in 50mL lysis buffer(50mM sodium phosphate,0.3M NaCl, pH 8.0)and lysed by ultrasonication then centrifuged at 12,000g for 20min at 4℃.The supematant was loaded onto Ni²⁺affinity column equilibrated with equilibration buffer(50mM sodium phosphate,0.3M NaCl,10mM imidazole,pH 8.0).Nonspecific binding proteins were washed with washing buffer(50mM sodium phosphate,0.3M NaCl,10～50 mM imidazole,pH 8.0).Recombinant proteins were eluted with elution buffer(50mM sodium phosphate,0.3M NaCl,50～250mM imidazole,pH 8.0).Finally,imidazole in the eluted fraction was removed by dialyzing against 20 mM Tris-HCl(pH8.0)containing 200 mM NaCl at 4℃overnight.Protein concentration was determined by using the Coomassie Plus protein assay kit according to manufacturer's instructions. The assay is based on the method of Bradford(Bradford,1976),and bovine serum albumin was used as a reference standard.The expressed and purified proteins were analyzed with SDS-PAGE.The recombinant E. coli extracts or the purified proteins were resuspended in SDS-PAGE loading buffer(50mM Tris-HCl,pH 6.8,2%SDS,5% 2-mercaptoethanol,10%sucrose,0.02%bromophenol blue),and fractionated on a 12%discontinuous SDS-PAGE after being denatured. The proteins were stained with 0.1%Coomassie Brilliant Blue R-250 solution.Electrophoresis was performed in a Bio-Rad's Mini-PROTEAN 3 Cell with standard molecular mass markers from Bio-Rad.Six female 8-week-old Balb/c mice were purchased from SLAC(Shanghai,China). All animal procedures were approved by the ethical committee for animal care of Nanjing Medical University.Freund was as the adjuvant and injections were made at intervals of two weeks.1st injection:30μg His6-Eppin in 100 ul PBS add to 100μl adjuvant,injected subcutaneously. 2nd injection:2 weeks later,boost with 60μg of His6-Eppin adjuvant.8 days later bleed the mouse and test serum using the assay which will be used for screening.Serum Preparation:After collecting,blood was allowed to clot for 60 min at 37℃,and then spin at 10,000 g for 10 min at 4℃to separate the serum.Antibody purification was accomplished using protein A agarose according to the manufacture's protocol.Purified polyclonal antibody was stored at -20℃after adding Glycerol to 50%.The ELISA plate(Nunc,Roskilde,Denmark)was coated with purified His6-Eppin in carbonate coating buffer,pH 9.6,overnight at 4℃, followed by five washes with PBS,containing 0.05%(v/v)Tween 20. Wells were blocked by incubation for 1 h with 2%BSA in PBS.After washing,mice sera diluted serially which was added in triplicate and incubated for 1 h at 37℃.The plates were washed and HRP-conjugated goat anti-mouse IgG(Sigma)was added at a dilution of 1:2,000.Plates were washed and TMB substrate was added(Sigma).Optical density was determined using an ELISA reader at 450 nm.Total protein of human epididymal extracts were prepared as described,using mouse polyclonal antibody against His6-Eppin as primary antibody(1:500)and peroxidase conjugated goat anti-mice Ig G(Sigma)as secondary antibody(1:5,000). 10-micron sections of frozen human epididymis were fixed on gelatin-coated slides with acetone for 15 to 30 minutes.293T were cultured in Dulbecco's modified Eagle's medium containing 10%(v/v) fetal bovine serum,4 mM glutamine,100 U/ml penicillin,and 0.1 mg/ml streptomycin under humidified air containing 5%CO₂ at 37℃and seeded onto cover slips.Transient transfections were performed using Lipofectamine 2000(Invitrogen)according to the manufacturer's instructions.Immunofluorescence was carried out with mice polyclonal anti-Eppin antibody(above purified)(1:100)as the primary antibody and Texas Red conjugated gate anti-mouse IgG(1010-07 SouthemBiotech, USA)as the secondary antibody.Results:1 In order to express the recombinant Eppin in E.coli,we amplified the Eppin gene from human epididymis by RT-PCR and produced a single amplified 360 bp DNA fragment,coding for a mature Eppin protein of 120 amino acids.Transformants were screened on LB plates containing 50μg/ml of kanamycin and validated by PCR-based selection,restriction digestion and following DNA sequencing.DNA sequencing and BLAST algorithm revealed that the cDNA was exactly identical to the reported sequence(GeneBank accession No.BC053369).Subsequently,the recombinant expression vectors were transformed into BL21(DE3) competent cells to generate the desired gengineering bacteria.The recombinant plasmid named pET28a-His6-Eppin is as shown in Figl.A. The DNA fragment of Eppin identified was cloned into pCDNA3.1(+)to yield a mammalian expression plasmid,recombinant plasmid named pCDNA-Eppin.2 After induction with 1mM IPTG at 37℃for 4h,E.coli BL21(DE3) transformed with pET28a-His6-Eppin produced a fusion protein of approximately 19 kDa,which contained a His-tagged Eppin.The size of the fusion protein matched well with its theoretical molecular weight. However,most of His6-Eppin existed in inclusion bodies.To improve the production of soluble,biologically active recombinant His6-Eppin, low-IPTG and low-temperature were employed.As shown in Figure 2.A, when the concentration of IPTG decreased from 1mM to 0.5mM,the soluable His6-Eppin increased from 8%to 18%;lower temperture to 30℃,cannot remarkbly improve the solubility.However,decrease temperature to 26℃,40%of His6-Eppin appeared in the soluable fraction result in about 5 fold more yield than that expressed at 37℃.These results were validated by both SDS-PAGE and dinstitometric quantification analysis.We concluded that lower temperature and lower IPTG were required to significantly improve the yield of soluable, biologiaclly active His6-Eppin.So we chose 0.5 mM IPTG at 26℃for large scale His6-Eppin expression.3 To purify the recombinant His6-Eppin,one step Ni²⁺affinity chromatography was employed.From 1-L flask culture,about 18.33 mg His6-Eppin was obtained and validated with SDS-PAGE.4 The polyclonal antibody was developed in mouse and had a titer of 1:2 with His6-Eppin showed by ELISA.The polyclonal antibody has been successfully purified using protein A agarose.5 Western blot analysis of human epididymis using affinity purified anti-recombinant human Eppin antibody revealed strong reactivity to a band at nearly 14kDa protein(matched well with its theoretical molecular weight)and weaker reactivity to a band at nearly 12 kDa.6 To further confirm the specificity of polyclonal antibody of His6-Eppin in the human epididymal tissue,tissue sections of human epididymal were probed with affinity purified anti-His6-Eppin antibodies.The principle cells of the epididymidis show staining within the apical cytoplasm and luminal staining on the long microvilli.No fluorescence was seen in control slides using non-specific mouse IgG as primary antibody.293T cells transiently transfected with eukaryotic expression plasmid pCDNA-Eppin were also analysed by immunofluorescent assay as done with epididymal tissue.Eppin proteins were found to localize predominately in the cytoplasm.While fluorescence was not seen in the cells transfected with pCDNA3.1(negative control).These results indicated that the Eppin eukaryotic expression vector was successfully consctructed,and the protein was recognized by our purified polyclonal anti-His6-Eppin antibody.Discussion:we have expressed,purified and characterized a human recombinant biological active Eppin protein using a pET-28a vector and Ni²⁺affinity chromatography procedures,which will be beneficial for clinical diagnosis and treatment of infertility patients and the possibility of male immuno-contraceptive agents development. IntroductionEppin(epididymal protease inhibitor;SPINLW1,serine peptidase inhibitor-like with Kunitz and WAP domains 1)coats the surface of human ejaculate spermatozoa and originates from Sertoli and epididymal epithelial cells.Two isoforms of the Eppin protein are expressed:one is secreted and one lacks a signal sequence.The secreted form of Eppin (CAB37635)has a theoretical pI of 8.52 and a calculated molecular weight of 15,283.72.Analysis of native Eppin by SDS-PAGE indicates that the monomer form has an apparent molecular weight of 16-18 kDa and the dimer an apparent molecular weight of 33-36 kDa.Higher multimer forms of Eppin are often seen in seminal plasma and analysis of recombinant human Eppin indicates that multimer forms(2×,3×,4×), which are stable to boiling in SDS in the presence of reducing agents, easily form in vitro.Eppin was identified and characterized 'male-only' molecule from the testis and epididymis,appears to play an important role in primate fertility.O'Rand et al demonstrated that male monkeys immunized with human recombinant Eppin couldn't successfully impregnated female monkeys.The serum anti-Eppin titer was high in these male monkeys.Although Eppin had a close association with the fertilization,the anti-Eppin antibody in the semen and its correlation to men's infertility remains unknown.In the present study,we developed an efficiency method to obtain relatively large quantity of recombinant Eppin protein and determined the presence of anti-Eppin antibodies in the semen of a population of patients with infertility.Aim:To reveal the presence of anti-Eppin antibody in the semen of clinical infertile cases by recombinant human Eppin.Methods:25 infertile patients were included in this study.Eligibility criteria:Failure to conceive after 2 years of unprotected sexual intercourse,patients and their wives excluded organic disease and AsAb-positive.As comparison,we studied 25 age-matched healthy fertile men.AsAb-positive group mean age is(28.51 +3.38)y,healthy fertile group mean age is(29.74±5.36)y.There are 4 cases of Chronic Prostatitits,1 case of epididymitis and 2 cases of nongonococcal urethritis(NGU)in AsAb-positive group,no in healthy fertile group.Hospital Ethical Committee approval and informed consent were obtained for all subjects in this study.Then with the recombinant His6-Eppin as the antigen,the ELISA method and Western blot were used to detect the positive rate of antibody in the semen of twenty-five infertile patients.ELISA The ELISA plate(Nunc,Roskilde,Denmark)was coated with purified His6-Eppin in carbonate coating buffer,pH 9.6,overnight at 4℃,followed by five washes with PBS,containing 0.05%(v/v)Tween 20.Wells were blocked by incubation with 2%BSA in PBS.After washing,the semen of infertile patients diluted serially was added in triplicate and incubated for 1 h at 37℃.The plates were washed and HRP-conjugated goat anti-human IgG(Sigma)was added at a dilution of 1:2,000.Plates were washed and TMB substrate was added(Sigma). Optical density(OD)was determined using an ELISA reader at 450 nm. Western-blot The Purified His6-Eppin were prepared using RIPA buffer (50 mM Tris-HCl(pH 8.0),150 mM NaCl,0.1%SDS,1%NP40,1 mM phenylmethylsulfonyl fluoride,5μg/ml aprotinin,and 5μg/ml leupeptin.), using the diluted semen of infertile patients(perhaps contains anti-Eppin antibody)as primary antibody(1:500)and peroxidase conjugated goat anti-human Ig G(Sigma)as secondary antibody(1:5,000). Immunoreactive protein bands were detected with an Odyssey Scanning System(LI-COR inc.,U.S.A.).Results:We selected 25 infertile patients and 25 normal men.The positive rate of anti-Eppin antibodies in the semen of patients with infertility was 28%(7/25,No.3,4,9,14,17,19 and 22 specimens).The positive samples were confirmed by Western blot.The blots indicated the presence of anti-Eppin antibody in the semen of the infertile patients(No. 3,4,14,19,22 specimens).There were five semen specimens contained anti-Eppin antibodies among the seven positive casesConclusion:This work proved for the first time that anti-Eppin antibody was present in the semen from a population of infertile men.The anti-Eppin antibody may be one of the key anti-sperm antibodies that partially account for clinical male infertility,which can be taken as auxiliary diagnostic index of infertility.Eppin is a promising potential candidate immunocontraception vaccine.