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1、183 http:/; | Asian Journal of Andrology npg Correspondence to: Dr Chen Xu, Department of Histology published online 16 February 2009. Keywords: antifertility, gene expression, nuclear autoantigenic sperm protein (NASP), sperm-egg binding, sperm-egg fusion 1 Introduction NASP, a nuclear autoantigen
2、ic sperm protein (GenBank locus ID: 4678; Accession number: P49321), initially described as a highly autoimmunogenic testis- and sperm-specific protein, is present in the nucleus of spermatozoa and spermatogenic cells 1, hence its name. Previous studies have shown that NASP is a histone-binding prot
3、ein that binds H1 linker histones in vivo and has been proposed to transport them into the nucleus of dividing cells 2. Two major forms are encoded by transcript variants of this gene, a testis form NASP (tNASP) and a somatic form NASP (sNASP). The sNASP, expressed in all mitotic cells, is localized
4、 to the nucleus, and is coupled to the cell cycle. The tNASP is expressed in embryonic tissues, tumor cells, and testis. In male germ cells, this protein is localized to the cytoplasm of primary spermatocytes, the nucleus of spermatids, and the periacrosomal region of mature spermatozoa. Mouse NASP
5、(mNASP) somatic protein (Mr 45 751) is identical in amino acid sequence to the testis form (Mr 83 934), except that a region coded by exon 6 has been deleted 2. Human NASP (hNASP) was first described in the testis 3, but also occurs in a somatic form with identical deletion to that found in mouse. A
6、nti-tNASP antibody can result in reproductive failure Min Wang et al. Asian Journal of Andrology | http:/; 184 npg The blood-testis barrier provides an immunological privileged status to the testis, preventing late spermatogenetic cell components from encountering the immune system. Not surprisingl
7、y, many testicular isoenzymes and other proteins are autoantigenic during immunological challenges occurring from testicular injury, infection, or vasectomy 4. In an earlier study 5, it was found that 86% of the vasectomized patients with anti-sperm antibodies had anti-tNASP autoantibodies. Early ep
8、idemiological studies indicated that some infertile men who were infected by Ureaplasma urealyticum 6 had positive antisperm antibodies (ASAs) in their serum and/or semen 7. Recently, we found a pentapeptide identity (IERLT) both in the urease complex component UreG, one of the proteins in Ureaplasm
9、a urealyticum, and in hNASP 8. The pentapeptide, present in tNASP, is located in the region excluded in sNASP. It may explain why infertile men infected with Ureaplasma urealyticum displayed a higher titer of serum and/or semen ASA, but had no symptoms. It has also been shown that the presence of an
10、tinuclear antibody (ANA) significantly reduces pregnancy rates 9. The purpose of this study is to analyze whether the anti-tNASP antibodies affect fertility or not. Mouse tNASP (mtNASP) was cloned and expressed. In vitro fertilization (IVF) assays were performed in the presence of anti-tNASP antibod
11、ies. In addition, we examined the effect of active immunization with recombinant mtNASP (rmtNASP) antigen or a synthesized peptide (containing the pentapeptide IERLT) on the fertile female mice in vivo. 2 Materials and methods 2.1 Subjects and animals This study was approved by the Ethical Review Bo
12、ard at the Shanghai Jiao Tong University School of Medicine. All subjects signed their informed consent before participation in the study. Healthy donors and infertile patients who had ASAs were recruited from Ren Ji Hospital, affiliated to the Shanghai Jiao Tong University School of Medicine (Shang
13、hai, China). BALB/c mice and New Zealand white rabbits were obtained from the Animal Center of the Chinese Academy of Sciences. Animals were housed in specific pathogen-free conditions at the Shanghai Jiao Tong University School of Medicine. All animal work was conducted in accordance with the Shang
14、hai Jiao Tong University School of Medicine Animal Studies Committee. 2.2 Cloning and expression of exon 6 of mtNASP The 16 56517 539-bp cDNA of mNASP was proved to contain exon 6 of mtNASP; therefore, two specific primers were designed by Primer Premier 5.0 (Premier Biosoft International, Palo Alto
15、, CA, USA), which would amplify a 339-bp fragment. The forward primer used was 5-GCGGATCC ATG GAA CTG CTA GGG CAA GA-3 (containing a BamH I site) and the reverse primer was 5-GCAAGCTT TTT GTC TTC AGG TGC TTT-3 (containing a Hind III site). Total RNA was extracted from mouse testis according to the m
16、anual of Qiagen RNAeasy Kit (Qiagen, Hilden, Germany) and quantified with UV absorbance at 260 nm. Reverse transcription was carried out according to the manual of the TaKaRa AMV reverse transcription-polymerase chain reaction (RT-PCR) kit (TaKaRa, Da Lian, China). PCR was carried out 10 in a final
17、volume of 25 L on a PTC-200 Peltier Thermal Cycler (MJ Research, Waltham, MA, USA). The cycling parameters employed were 94C, 2 min; 94C, 30 s; 51C, 1 min; and 72C, 1.5 min, for 35 cycles. PCR reaction products were separated on agarose gels, and a band of 339 bp was isolated and subcloned in pMD-18
18、 vector (TaKaRa) to transform Escherichia coli (strain DH16B) 11. Multiple cDNA clones were sequenced (Sangon, Shanghai, China). Recombinant pET-28a (+) plasmid was propagated in E. coli BL21 (DE3) host cells and the encoded proteins were expressed as IPTG-induced 6 Histidine-tagged fusion proteins
19、12. The molecular size of expressed proteins was verified by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). The rmtNASP was purified 13 on a His-binding Ni2+ chelation affinity resin column by a modification of the manufacturers procedures (Pierce, Rockford, IL, USA). Protein
20、 concentrations were determined by Coomassie Plus-200 using bovine serum albumin (BSA) as a standard. 2.3 Polyclonal antibody production and Western blot analysis Three healthy male New Zealand white rabbits aged 6 months (body weight about 2.5 kg) were housed in the animal facility for at least 34
21、days to acclimate them to the new surroundings. The rabbits were immunized with purified rmtNASP as described 14. At the first and third day, a mixture of antigen and the complete Freunds adjuvant (Sigma, St. Louis, MO, USA) was Anti-tNASP antibody can result in reproductive failure Min Wang et al.
22、http:/; | Asian Journal of Andrology 185 npg injected subcutaneously on the back and proximal limbs of the rabbits. On the 28th day, a mixture of antigen and the incomplete Freunds adjuvant (Sigma) was injected in the same way. The target antigen (200300 g) was injected into each rabbit at each tim
23、e. On the 35th day, the titer of the antiserum was checked with enzyme- linked immunosorbent assay (ELISA). The control sera were obtained from animals immunized with only the adjuvant. The blood was collected and the serum was removed and purified with Protein A affinity chromatography according to
24、 the manual (Millipore, Billerica, MA, USA). The specificity of the antiserum was judged by (a) the reactivity of the antiserum to rmtNASP, (b) the inability of the antiserum to cross- react with any other protein from mouse total sperm extracts on Western blot, (c) the ability of antigen pre- adsor
25、ption to abolish immunorecognition, and (d) reproducibility of the results with antiserum from different animals. rmtNASP, mouse sperm, testis, and epididymis extracts were subjected to 1D SDS-PAGE 15. Proteins were then blotted to nitrocellulose. All blots were blocked with 5% BSA in TBS with 0.05%
26、 Tween 20 (TBS-T) for 30 min at room temperature. Immunoblotting was tested using different antisera as primary antibodies 16. The blots were then incubated with 1:4 000 dilution of peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody for 1 h. The blots were then develope
27、d with ECL reagent (Amersham Corp., Buckinghamshire, UK). 2.4 Sperm-egg binding and fusion assays Female 8-week-old BALB/c mice were primed with 10 IU of pregnant mares serum gonadotropin (PMSG, Sigma) and then induced to ovulate with 10 IU of hCG (Sigma) 4856 h later 17. Cumulus-enclosed egg comple
28、xes were collected from the oviductal ampullae 15 h after hCG administration, and treated with 0.1% hyaluronidase (Sigma) in M16 medium (Sigma) containing 10% fetal calf serum to disperse the cumulus cells. The ZP was removed with acidic (pH 2.5) Tyrodes solution (Sigma) 18. The eggs were washed thr
29、ice with M16 medium. Spermatozoa were obtained from male BALB/c mice (810 weeks old) by placing the cauda epididymides and vasa deferentia in 900 L Tyrodes solution (containing 3 mg mL1 BSA). Spermatozoa were allowed to swim out for 10 min, and then the tissue was removed from the solution and 5 mol
30、 L1 calcium ionophore A23187 (Sigma) was added for another 60 min for the acrosome reaction 19. Eggs were placed in 50-L drops of M16 medium. Capacitated spermatozoa were pretreated with different concentrations of affinity-purified IgG from immune serum against rmtNASP or IgG from adjuvant control
31、serum for 1 h 20. The treated spermatozoa were added to a final concentration of 1.0 106 per mL and incubated for 30 min in a 5% CO2, 37C incubator 21. After three washes, eggs were stained with 10 g mL 1 Hoechst 33342 for 15 min, and then washed three times in fresh medium 22. About 1020 eggs were
32、analyzed for each treatment, and the average number of spermatozoa-bound per egg was determined. The treated spermatozoa were added to a final concentration of 1.0 106 mL 1 and incubated with eggs for 3 h in a 5% CO2, 37C incubator 23. Eggs were then washed and mounted on glass slides for analysis f
33、or evidence of sperm penetration under phase-contrast microscopy. Eggs were considered to be penetrated if a decondensing sperm head or two pronuclei and at least a sperm tail were present in the ooplasma 24. The fusion rate (percentage of eggs fertilized) was determined. 2.5 Synthesized peptide and
34、 competitive ELISA The peptide human tNASP (htNASP) 393408 (EPQTSIERLTETKDGS), corresponding to amino acids 393408 of htNASP, was synthesized. The peptide was coupled to keyhole limpet hemocyanin (KLH) 25 by the manufacturer (Sangon). The polyclonal antiserum against the peptide was made as describe
35、d 14. Antiserum against rmtNASP at a nonsaturating dilution was incubated with various concentrations (0.11 mmol L 1 ) of competing synthesized peptides at room temperature for 2 h 26. This mixture of peptide and antiserum was then transferred to a plate coated with rmtNASP and further incubated for
36、 2 h at room temperature to measure antibody unabsorbed by the synthesized peptide with ELISA 27. 2.6 Sperm motility analysis Spermatozoa were obtained from male BALB/c mice (810 weeks old) by placing the cauda epididymides and vasa deferentia in 900 L Tyrodes solution (containing 3 mg mL-1 BSA). Th
37、e cells were incubated with 100 g mL-1 of purified adjuvant control IgG or purified anti-rmtNASP IgG or anti-NASP393408 IgG at 37C for 1 h. Computer-assisted sperm motility Anti-tNASP antibody can result in reproductive failure Min Wang et al. Asian Journal of Andrology | http:/; 186 npg analysis w
38、as performed by using a semen autoanalyser (Hamilton Thorne, Beverly, MA, USA). Motile percentage, curvilinear velocity (VCL), and straight- line velocity (VSL) were measured. 2.7 Binding of ASAs with rmtNASP or htNASP393408 Sera were obtained from 22 infertile men who had ASAs and 25 men with norma
39、l fertility. The study groups were matched by age (31.0 3.5 years and 29.4 3.4 years, respectively). Each infertile mans wife was found to be healthy, lacking any detectable reproductive- system anomalies or other known clinical disorders. Additionally, the wives had not been pregnant for at least 1
40、 year. Polystyrene 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated with rmtNASP or htNASP393408 KLH at a volume of 100 L per well (0.5 g per well). Using sera from the infertile men as primary antibodies, ELISA was performed as described previously 28. Sera from the fertile men were
41、used as control. 2.8 Fertility tests Female BALB/c mice of proven fertility were immunized against purified rmtNASP or synthesized peptides htNASP393408 by i.m. and s.c. routes as described previously. Each animal received a total of three injections. Each injection consisted of 100 L of phosphate b
42、uffered saline (PBS) containing 50 g rmtNASP protein or the synthesized peptide htNASP393408 emulsified with 100 L Freunds adjuvant. Control animals were injected with KLH in Freunds adjuvant. One week after the last immunization, male animals, proven to be fertile, were individually and continuousl
43、y caged with experimental and control female mice at a ratio of 1:2 (eight animals per group) 29. After 3 weeks, pregnant females were counted. The animals were kept for a longer time, up to 240 days, to examine the effect of disappearance of antibody titers on the regain of fertility. 2.9 Statistic
44、al analysis Experimental and control group averages were reported as mean SD (Table 1) or mean SEM (Table 2, Figure 1). The results were analyzed by upaired t-test. Correlation between the antibody titer and fertility was analyzed by linear regression. Results were considered statistically significa
45、nt at P 0.05. 3 Results 3.1 Characteristics of the testicular-specific exon 6 of NASP The exon 6 of the NASP gene is selectively transcribed in the testis. Some infertile men displayed high titers of serum anti-NASP antibody, but they did not show any Table 1. Effect of the antibody against rmtNASP
46、and anti-NASP393408 antibody on sperm motility. BALB/c mouse spermatozoa were incubated with 100 mg mL-1 of purified adjuvant control IgG or the same concentration of purified anti-rmtNASP IgG or anti- NASP393408 IgG at 37C for 1 h. Motile percentage, VCL and VSL were measured. All results were expr
47、essed as mean SD. Group Motile (%) VCL (mm/s) VSL (mm/s) Control group 67 12 272.5 68.0 145.2 76.0 Anti-rtNASP IgG group 58 15 262.0 72.0 127.3 62.0 Anti-tNASP393408 IgG group 62 17 255.0 66.0 146.6 75.0 Abbreviations: VCL, curvilinear velocity; VSL, straight line velocity. Table 2. Effects of rmtNA
48、SP and the synthesized peptide immunization on fertility of female mice. Female BALB/c mice of proven fertility were immunized with purified rmtNASP or the synthesized peptide htNASP393408 coupled to KLH. The control animals were immunized with keyhole limpet hemocyanin (KLH) emulsified in phosphate buffered saline (PBS) and Freunds adjuvant. The animals were then housed with males of proven fertility. The number of babies delivered by each mated female was counted. No. of pregnancies Pups born (mean SEM) Group No. of animals Housed for 2130 Housed for 150180 Housed for 2130 House