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• W2146502181 abstract "Female CBA/J mice impregnated by male DBA/2J mice (CBA/J×DBA/2J matings) are prone to spontaneous abortion, although the reason for this is unclear. In this study, the stathmin-1 expression pattern was evaluated in uterine natural killer (uNK) cells purified from CBA/J×DBA/2J matings. Results were compared with those in a CBA/J×BALB/c control group that yields successful pregnancies. The mean ± SD percentage of stathmin-1+ cells in the CD49b+ uNK cell population was lower in CBA/J×DBA/2J mice (0.7% ± 0.4%) than in control CBA/J×BALB/c mice (4.9% ± 1.5%, P < 0.01) using flow cytometry, and the intracellular stathmin-1 level in uNK cells was lower in CBA/J×DBA/2J mice than in control mice using Western blot analysis. Co-localization of lectin from Dolichos biflorus agglutinin (DBA-lectin) and stathmin-1 was confirmed using multivision immunohistochemical analysis. The frequency of stathmin-1+DBA-lectin+ cells was lower in CBA/J×DBA/2J mice than in CBA/J×BALB/c mice. A similar trend in the frequency of stathmin-1+CD56+ cells was seen in patients with unexplained spontaneous abortion compared with normal early pregnancy. A neutralizing antibody against stathmin-1 further increased the percentage of embryo loss in CBA/J×DBA/2J matings. These results provide evidence that stathmin-1 expression in uNK cells at the maternal-fetal interface may help modulate uNK cell function and may be beneficial for a successful pregnancy. Female CBA/J mice impregnated by male DBA/2J mice (CBA/J×DBA/2J matings) are prone to spontaneous abortion, although the reason for this is unclear. In this study, the stathmin-1 expression pattern was evaluated in uterine natural killer (uNK) cells purified from CBA/J×DBA/2J matings. Results were compared with those in a CBA/J×BALB/c control group that yields successful pregnancies. The mean ± SD percentage of stathmin-1+ cells in the CD49b+ uNK cell population was lower in CBA/J×DBA/2J mice (0.7% ± 0.4%) than in control CBA/J×BALB/c mice (4.9% ± 1.5%, P < 0.01) using flow cytometry, and the intracellular stathmin-1 level in uNK cells was lower in CBA/J×DBA/2J mice than in control mice using Western blot analysis. Co-localization of lectin from Dolichos biflorus agglutinin (DBA-lectin) and stathmin-1 was confirmed using multivision immunohistochemical analysis. The frequency of stathmin-1+DBA-lectin+ cells was lower in CBA/J×DBA/2J mice than in CBA/J×BALB/c mice. A similar trend in the frequency of stathmin-1+CD56+ cells was seen in patients with unexplained spontaneous abortion compared with normal early pregnancy. A neutralizing antibody against stathmin-1 further increased the percentage of embryo loss in CBA/J×DBA/2J matings. These results provide evidence that stathmin-1 expression in uNK cells at the maternal-fetal interface may help modulate uNK cell function and may be beneficial for a successful pregnancy. Stathmin-1 is a small (19-kDa) regulatory phosphoprotein that integrates diverse intracellular signaling pathways. It is highly conserved among vertebrates and is associated with tubulin binding and microtubule destabilization.1Curmi P.A. Gavet O. Charbaut E. Ozon S. Lachkar-Colmerauer S. Manceau V. Siavoshian S. Maucuer A. Sobel A. Stathmin and its phosphoprotein family: general properties, biochemical and functional interaction with tubulin.Cell Struct Funct. 1999; 24: 345-357Crossref PubMed Scopus (161) Google Scholar, 2Rubin C.I. Atweh G.F. The role of stathmin in the regulation of the cell cycle.J Cell Biochem. 2004; 93: 242-250Crossref PubMed Scopus (311) Google Scholar Stathmin-1 has a complex phosphorylation pattern in response to various extracellular signals, in particular growth and differentiation factors.3Sobel A. Stathmin: a relay phosphoprotein for multiple signal transduction.Trends Biochem Sci. 1991; 16: 301-305Abstract Full Text PDF PubMed Scopus (250) Google Scholar Moreover, stathmin-1 phosphorylation varies during the cell cycle.4Brattsand G. Marklund U. Nylander K. Roos G. Gullberg M. Cell-cycle-regulated phosphorylation of oncoprotein 18 on ser16, ser25 and ser38.Eur J Biochem. 1994; 220: 359-368Crossref PubMed Scopus (100) Google Scholar It has thus been thought that stathmin-1 can act as a relay integrating the activation of diverse intracellular signaling pathways and mediating the control of cell proliferation, differentiation, and other functions.5Sobel A. Boutterin M.C. Beretta L. Chneiweiss H. Doye V. Peyro-Saint-Paul H. Intracellular substrates for extracellular signaling: characterization of a ubiquitous, neuron-enriched phosphoprotein (stathmin).J Biol Chem. 1989; 264: 3765-3772Abstract Full Text PDF PubMed Google Scholar Stathmin-1 protein and mRNA were previously shown to be expressed in the pregnant uterus and decidualizing endometrial stromal cells in human and murine models.6Tamura K. Yoshie M. Nishi H. Osakabe Y. Isaka K. Hara T. Kogo H. Expression of stathmin in human uterus and decidualizing endometrial stromal cells.Reproduction. 2006; 132: 625-636Crossref PubMed Scopus (20) Google Scholar, 7Yoshie M. Tamura K. Kogo H. Differential localization of decidual stathmin during pregnancy in rats.Placenta. 2004; 25: 449-455Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 8Tamura K. Hara T. Yoshie M. Irie S. Sobel A. Kogo H. Enhanced expression of uterine stathmin during the process of implantation and decidualization in rats.Endocrinology. 2003; 144: 1464-1473Crossref PubMed Scopus (21) Google Scholar Furthermore, stathmin-1 is up-regulated in rodent uteri at the site of embryo implantation and is highly expressed in the decidual zone during the decidualization process.7Yoshie M. Tamura K. Kogo H. Differential localization of decidual stathmin during pregnancy in rats.Placenta. 2004; 25: 449-455Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 8Tamura K. Hara T. Yoshie M. Irie S. Sobel A. Kogo H. Enhanced expression of uterine stathmin during the process of implantation and decidualization in rats.Endocrinology. 2003; 144: 1464-1473Crossref PubMed Scopus (21) Google Scholar These results suggest that stathmin-1 may participate in the modulation of embryo implantation and decidualization. Female CBA/J mice impregnated by male DBA/2J mice (CBA/J×DBA/2J matings) are prone to abortion, in contrast to the major histocompatibility complex–identical CBA/J×BALB/c matings, which are resistant to abortion.9Clark D.A. Chaouat G. Arck P.C. Mittruecker H.W. Levy G.A. Cytokine-dependent abortion in CBA × DBA/2 mice is mediated by the procoagulant fgl2 prothombinase.J Immunol. 1998; 160: 545-549PubMed Google Scholar The underlying mechanisms for these observations are unclear. Clark and colleagues9Clark D.A. Chaouat G. Arck P.C. Mittruecker H.W. Levy G.A. Cytokine-dependent abortion in CBA × DBA/2 mice is mediated by the procoagulant fgl2 prothombinase.J Immunol. 1998; 160: 545-549PubMed Google Scholar suggested that endothelium is the primary effector cell population, and this was supported by a recent work using CBA/J×DBA/2J matings.10Redecha P. van Rooijen N. Torry D. Girardi G. Pravastatin prevents miscarriages in mice: role of tissue factor in placental and fetal injury.Blood. 2009; 113: 4101-4109Crossref PubMed Scopus (105) Google Scholar Notably, inhibition of natural killer (NK) cells using anti-asialo GM1 antiserum significantly decreased the resorption rate of embryos in CBA/J×DBA/2J matings.9Clark D.A. Chaouat G. Arck P.C. Mittruecker H.W. Levy G.A. Cytokine-dependent abortion in CBA × DBA/2 mice is mediated by the procoagulant fgl2 prothombinase.J Immunol. 1998; 160: 545-549PubMed Google Scholar In the present study, uterine NK (uNK) cells were purified from CBA/J×DBA/2J and CBA/J×BALB/c allogeneic pregnant models using magnetic affinity cell sorting (MACS). The percentage of stathmin-1+ cells in the uNK cell population was determined using flow cytometry, and the stathmin-1 protein expression level in uNK cells was determined using two-dimensional gel electrophoresis (2-DE), mass spectrometry (MS), and Western blot analysis. Multivision immunohistochemical analysis (IHC) was used to examine the distribution patterns of stathmin-1+ cells in the uteri of pregnant female mice and in first-trimester human decidual tissue. In addition, inhibition of stathmin-1 was performed in CBA/J×DBA/2J, CBA/J×BALB/c, and CBA/J×CBA/J mice. From these data, the possible role of stathmin-1 in allogeneic pregnancy tolerance was investigated. Female CBA/J mice and male CBA/J, DBA/2J, and BALB/c mice (8 to 12 weeks old) were purchased from the Model Animal Center of Nanjing University (Nanjing, China) and were housed under specific pathogen-free conditions. Pregnant models of CBA/J×DBA/2J, CBA/J×BALB/c, and CBA/J×CBA/J matings were established by co-caging female CBA/J mice with DBA/2J, BALB/c, and CBA/J males, respectively. Detection of a vaginal plug was chosen to indicate day 0.5 of gestation (E0.5).11Lin Y. Wang H. Wang W. Zeng S. Zhong Y. Li D.-J. Prevention of embryo loss in non-obese diabetic mice using adoptive ITGA2+ISG20+ natural killer-cell transfer.Reproduction. 2009; 137: 943-955Crossref PubMed Scopus (17) Google Scholar, 12Lin Y. Zhong Y. Shen W. Chen Y. Shi J. Di J. Zeng S. Saito S. TSLP-induced placental DC activation and IL-10+ NK cell expansion: comparative study based on BALB/c×C57BL/6 and NOD/SCID×C57BL/6 pregnant models.Clin Immunol. 2008; 126: 104-117Crossref PubMed Scopus (23) Google Scholar Embryonic day E12.5 was chosen as the gestational time to collect uNK cells because the uNK cells are at peak density on day E10 and have not yet begun to decrease in density through apoptosis (which begins on day E13 or E14).13Barber E.M. Pollard J.W. The uterine NK cell population requires IL-15 but these cells are not required for pregnancy nor the resolution of a Listeria monocytogenes infection.J Immunol. 2003; 171: 37-46PubMed Google Scholar Furthermore, we expected that it would be easier to distinguish healthy embryos from resorbing ones on day E12.5 than at an earlier time point. All animal procedures followed the national animal care guidelines, and associated data were approved for publication by the institutional review board of Shanghai Jiaotong University. Cell purification was performed by means of MACS.11Lin Y. Wang H. Wang W. Zeng S. Zhong Y. Li D.-J. Prevention of embryo loss in non-obese diabetic mice using adoptive ITGA2+ISG20+ natural killer-cell transfer.Reproduction. 2009; 137: 943-955Crossref PubMed Scopus (17) Google Scholar, 12Lin Y. Zhong Y. Shen W. Chen Y. Shi J. Di J. Zeng S. Saito S. TSLP-induced placental DC activation and IL-10+ NK cell expansion: comparative study based on BALB/c×C57BL/6 and NOD/SCID×C57BL/6 pregnant models.Clin Immunol. 2008; 126: 104-117Crossref PubMed Scopus (23) Google Scholar Briefly, hysterolaparotomy was performed on day E12.5 to collect embryo-depleted placentas from CBA/J×DBA/2J and CBA/J×BALB/c matings. The uterine horns were opened longitudinally, and the fetoplacental unit was separated easily from the uterine implantation sites. The whole placental and decidual unit was separated individually from the respective embryo. The pooled placentas and decidua basalis (ie, decidual tissue in implantation sites) were collected into a dish and carefully cut into small pieces, collected in 0.9% NaCl solution, and subsequently filtered through a nylon mesh (50-μm pore size) to obtain a single cell suspension. Mononuclear cells were obtained by centrifuging of the single cell suspension using a Ficoll-Hipaque density column. Any red blood cells that contaminated the single cell suspension were eliminated by incubation with red blood cell lysis buffer (eBioscience Inc., San Diego, CA) two times at 37°C. Subsequently, NK cells were isolated using magnetic bead–conjugated antimouse CD49b monoclonal antibody, and CD49b+ cells were purified by means of Mini MACS columns (Miltenyi Biotec Inc., Auburn, CA),11Lin Y. Wang H. Wang W. Zeng S. Zhong Y. Li D.-J. Prevention of embryo loss in non-obese diabetic mice using adoptive ITGA2+ISG20+ natural killer-cell transfer.Reproduction. 2009; 137: 943-955Crossref PubMed Scopus (17) Google Scholar, 12Lin Y. Zhong Y. Shen W. Chen Y. Shi J. Di J. Zeng S. Saito S. TSLP-induced placental DC activation and IL-10+ NK cell expansion: comparative study based on BALB/c×C57BL/6 and NOD/SCID×C57BL/6 pregnant models.Clin Immunol. 2008; 126: 104-117Crossref PubMed Scopus (23) Google Scholar where CD49b was used as a common marker for murine NK cells.14Arase H. Saito T. Phillips J.H. Lanier L.L. The mouse NK cell-associated antigen recognized by DX5 monoclonal antibody is CD49b (α2 integrin, very late antigen-2).J Immunol. 2001; 167: 1141-1144PubMed Google Scholar The purity of the MACS-isolated NK cells routinely exceeded 95% as determined using flow cytometry.12Lin Y. Zhong Y. Shen W. Chen Y. Shi J. Di J. Zeng S. Saito S. TSLP-induced placental DC activation and IL-10+ NK cell expansion: comparative study based on BALB/c×C57BL/6 and NOD/SCID×C57BL/6 pregnant models.Clin Immunol. 2008; 126: 104-117Crossref PubMed Scopus (23) Google Scholar, 15Lin Y. Zhong Y. Saito S. Chen Y. Shen W. Di J. Zeng S. Characterization of natural killer cells in nonobese diabetic/severely compromised immunodeficient mice during pregnancy.Fertil Steril. 2009; 91: 2676-2686Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Uterine NK cells were stained with phosphatidylethanolamine (PE)-conjugated antimouse CD49b (BioLegend, San Diego, CA) and rabbit anti–stathmin-1 (catalog number ab52906; Abcam, Cambridge, England) antibodies, followed by fluorescein isothiocyanate (FITC)–conjugated antirabbit IgG (Molecular Probes Inc., Eugene, OR). The percentage of stathmin-1+ cells in the CD49b+ NK cell population was determined by using a flow cytometer (FACSAria; BD Biosciences, Franklin Lakes, NJ).11Lin Y. Wang H. Wang W. Zeng S. Zhong Y. Li D.-J. Prevention of embryo loss in non-obese diabetic mice using adoptive ITGA2+ISG20+ natural killer-cell transfer.Reproduction. 2009; 137: 943-955Crossref PubMed Scopus (17) Google Scholar Cells were stained with PE-conjugated antimouse CD49b and FITC-conjugated antimouse CD122 antibodies (both from BioLegend) to determine the percentage of CD122+ cells in the CD49b+ population. Isotype controls were established by using isotype control antibodies (BioLegend) to exclude false-positive cells. All the experiments were independently performed six times.16Lin Y. Liang Z. Chen Y. Zeng Y. TLR3-involved modulation of pregnancy tolerance in double-stranded RNA-stimulated NOD/SCID mice.J Immunol. 2006; 176: 4147-4154PubMed Google Scholar, 17Li C. Xiao Z. Chen Z. Zhang X. Li J. Wu X. Li X. Yi H. Li M. Zhu G. Liang S. Proteome analysis of human lung squamous carcinoma.Proteomics. 2006; 6: 547-558Crossref PubMed Scopus (139) Google Scholar, 18Shen J. Pavone A. Mikulec C. Hensley S.C. Traner A. Chang T.K. Person M.D. Fischer S.M. Protein expression profiles in the epidermis of cyclooxygenase-2 transgenic mice by 2-dimensional gel electrophoresis and mass spectrometry.J Proteome Res. 2007; 6: 273-286Crossref PubMed Scopus (16) Google Scholar Uterine NK cells were suspended in a modified radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% Triton X-100 (Roche Diagnostics GmbH, Mannheim, Germany), 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 0.66 μg/ml of aprotinin, 0.5 μg/ml of leupeptin, 1 μg/ml of pepstatin, 1 mmol/L Na3VO4, and 1 mmol/L NaF] and were sonicated three times for 5 seconds each. The cell lysates were centrifuged at 14,000 × g for 15 minutes at 4°C. The supernatants were collected, and their protein concentrations were measured by using the Bradford assay (Bio-Rad Laboratories, Hercules, CA).19DiGiovanni J. Bol D.K. Wilker E. Beltran L. Carbajal S. Moats S. Ramirez A. Jorcano J. Kiguchi K. Constitutive expression of insulin-like growth factor-1 in epidermal basal cells of transgenic mice leads to spontaneous tumor promotion.Cancer Res. 2000; 60: 1561-1570PubMed Google Scholar, 20Li C. Wang W. Wang H. Zhong Y. Di J. Lin Y. Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild-type and NOD mice.J Cell Biochem. 2009; 108: 447-457Crossref PubMed Scopus (17) Google Scholar The 2-DE was performed according to the manufacturer's instructions. Samples were loaded onto linear immobilized pH gradient (IPG) strips (IPGstrip, pH 4–7 L, 180 × 3 × 0.5 mm; Amersham Biosciences, Piscataway, NJ). Briefly, 1-mg protein samples were diluted to 350 μL with a rehydration solution [7 mol/L urea, 2 mol/L thiourea, 2% 3-([3-Cholamidopropyl]dimethylammonio)-l propanesulfonate, 18 mmol/L dithiothreitol, 0.5% (v/v) pH 4–7 IPG buffer, and trace bromophenol blue] and were applied to the IPG strips with 14 hours of rehydration at 30 V. The proteins were successively focused for 1 hour at 500 V, 1 hour at 1000 V, and 5 hours at 8000 V for a total of 41,920 V hours on an IPGphor (Amersham Biosciences). The focused IPG strips were equilibrated for 15 minutes in solution (6 mol/L urea, 2% SDS, 30% glycerol, 50 mmol/L Tris-HCl, pH 8.8, and 1% dithiothreitol) and then for an additional 15 minutes in the same solution containing 2.5% iodoacetamide instead of dithiothreitol. After equilibration, SDS–polyacrylamide gel electrophoresis was performed at 10°C on 10% SDS slab gels using the Ettan DALT II system (Amersham Biosciences) with the IPG strips sealed on the top of the gels with 0.5% agarose. An SDS–polyacrylamide gel electrophoresis was performed at a constant power of 2W/gel for 30 minutes and then switched to 12 W/gel until the bromophenol blue marker reached the bottom of the gel. Finally, the blue silver staining method (a modified Neuhoff's colloidal Coomassie Blue G-250 stain) was used to visualize the protein spots in the 2-DE gels.17Li C. Xiao Z. Chen Z. Zhang X. Li J. Wu X. Li X. Yi H. Li M. Zhu G. Liang S. Proteome analysis of human lung squamous carcinoma.Proteomics. 2006; 6: 547-558Crossref PubMed Scopus (139) Google Scholar, 20Li C. Wang W. Wang H. Zhong Y. Di J. Lin Y. Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild-type and NOD mice.J Cell Biochem. 2009; 108: 447-457Crossref PubMed Scopus (17) Google Scholar Stained 2-DE gels were scanned using LabScan software and ImageScanner (Amersham Biosciences) at a solution of 300 dpi. Spot-intensity calibration, spot detection, matching, 1-D calibration, and establishment of an average gel were performed using the PDQuest System (Bio-Rad Laboratories). The theoretical molecular weight and pI value of the identified protein spots were calculated according to algorithms included in the PDQuest analysis software package. Significant differences in the protein expression levels were determined using the t-test, with significance defined at P < 0.05.17Li C. Xiao Z. Chen Z. Zhang X. Li J. Wu X. Li X. Yi H. Li M. Zhu G. Liang S. Proteome analysis of human lung squamous carcinoma.Proteomics. 2006; 6: 547-558Crossref PubMed Scopus (139) Google Scholar, 20Li C. Wang W. Wang H. Zhong Y. Di J. Lin Y. Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild-type and NOD mice.J Cell Biochem. 2009; 108: 447-457Crossref PubMed Scopus (17) Google Scholar Protein spots were excised from the preparative gels and were placed into a 96-well microtiter plate. Proteins were digested in gel as previously described.17Li C. Xiao Z. Chen Z. Zhang X. Li J. Wu X. Li X. Yi H. Li M. Zhu G. Liang S. Proteome analysis of human lung squamous carcinoma.Proteomics. 2006; 6: 547-558Crossref PubMed Scopus (139) Google Scholar, 20Li C. Wang W. Wang H. Zhong Y. Di J. Lin Y. Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild-type and NOD mice.J Cell Biochem. 2009; 108: 447-457Crossref PubMed Scopus (17) Google Scholar The gel spots were destained using destaining solution [200 mmol/L NH4HCO3 and 100% acetonitrile (1:1)] for 20 minutes at room temperature. Gel spots were washed twice with deionized water, shrunk by dehydration in acetonitrile solution, and dried in a vacuum centrifuge. Samples were then swollen in a digestion buffer (20 mmol/L ammonium bicarbonate and 12.5 ng/μL of trypsin) (Sigma-Aldrich, St. Louis, MO) for 30 minutes at 4°C. The gels were then digested for 12 hours at 37°C. Tryptic peptides were extracted twice from the gel slices by sonication for 15 minutes in a 0.1% trifluoroacetic acid/50% acetonitrile solution. The supernatants were collected and dried to a pellet in a high-purity nitrogen flow. Peptides were eluted with 0.7 μL of α-cyano-4-hydroxycinnamic acid matrix solution and were loaded onto a stainless steel target with 192 wells (Applied Biosystems, Framingham, MA).17Li C. Xiao Z. Chen Z. Zhang X. Li J. Wu X. Li X. Yi H. Li M. Zhu G. Liang S. Proteome analysis of human lung squamous carcinoma.Proteomics. 2006; 6: 547-558Crossref PubMed Scopus (139) Google Scholar, 20Li C. Wang W. Wang H. Zhong Y. Di J. Lin Y. Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild-type and NOD mice.J Cell Biochem. 2009; 108: 447-457Crossref PubMed Scopus (17) Google Scholar Samples were air-dried and then were analyzed by using the Voyager System 4700 matrix-assisted laser desorption/ionization–time of flight–time of flight mass spectrometer (Applied Biosystems).17Li C. Xiao Z. Chen Z. Zhang X. Li J. Wu X. Li X. Yi H. Li M. Zhu G. Liang S. Proteome analysis of human lung squamous carcinoma.Proteomics. 2006; 6: 547-558Crossref PubMed Scopus (139) Google Scholar, 20Li C. Wang W. Wang H. Zhong Y. Di J. Lin Y. Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild-type and NOD mice.J Cell Biochem. 2009; 108: 447-457Crossref PubMed Scopus (17) Google Scholar Known contaminating peaks (eg, keratin and autoproteolysis peaks) were removed before the database search. Spectra were processed and analyzed using a GPS Explorer (Applied Biosystems). Mascot software (Matrix Science, London, England) was used to search for peptide mass fingerprints and MS/MS data in the NCBInr database. Protein scores by Mascot search analysis that were >63 were considered significant (P < 0.05).17Li C. Xiao Z. Chen Z. Zhang X. Li J. Wu X. Li X. Yi H. Li M. Zhu G. Liang S. Proteome analysis of human lung squamous carcinoma.Proteomics. 2006; 6: 547-558Crossref PubMed Scopus (139) Google Scholar, 20Li C. Wang W. Wang H. Zhong Y. Di J. Lin Y. Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild-type and NOD mice.J Cell Biochem. 2009; 108: 447-457Crossref PubMed Scopus (17) Google Scholar Tissue aliquots were homogenized to powder using liquid nitrogen and then were dissolved in lysis buffer [150 mmol/L NaCl, 50 mmol/L Tris-Cl, pH 8.0, 0.1% Nonidet P-40 (Caledon Laboratories Ltd., Georgetown, Ontario, Canada), 1 mmol/L phenylmethylsulfonyl fluoride, 25 μg/ml of aprotinin, and 25 μg/ml of leupeptin], vortexed, and incubated at room temperature for 2 hours. The mixture was centrifuged at 20,644 × g for 30 minutes at 4°C, and the supernatant was used as the total protein solution. The lysate concentration was assayed using the Bradford assay. Western blotting analysis was performed as previously described.17Li C. Xiao Z. Chen Z. Zhang X. Li J. Wu X. Li X. Yi H. Li M. Zhu G. Liang S. Proteome analysis of human lung squamous carcinoma.Proteomics. 2006; 6: 547-558Crossref PubMed Scopus (139) Google Scholar, 20Li C. Wang W. Wang H. Zhong Y. Di J. Lin Y. Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild-type and NOD mice.J Cell Biochem. 2009; 108: 447-457Crossref PubMed Scopus (17) Google Scholar Briefly, 100 μg of total protein was separated on a 12% SDS–polyacrylamide gel electrophoresis gel before being transferred onto a nitrocellulose membrane. After blocking with 5% milk in Tris-buffered saline/0.2% Tween 20 for 1 hour at room temperature, the membrane was incubated with rabbit antimouse stathmin-1 antibody (1:100 dilution) (Abcam) for 1 hour at room temperature, followed by incubation with horseradish peroxidase–conjugated goat antirabbit IgG secondary antibody (1:10,000 dilution; Amersham Biosciences) for 1 hour at room temperature. Detection of NADPH was used as a loading control. Reactions were visualized using an enhanced chemiluminescence detection system (ECL; Amersham Biosciences). Signals on the blots were visualized using autography. Placentas together with decidua basalis harvested on day E10.5 from CBA/J×DBA/2J and CBA/J×BALB/c matings (none of which were analyzed by means of 2-DE) were used to measure the distribution of stathmin-1 protein in lectin from Dolichos biflorus agglutinin–positive (DBA-lectin+) cells using a multivision IHC procedure. Paraffin-embedded tissue blocks were cut into 4-μm-thick sections, which were then deparaffinized in xylene and rehydrated in graded alcohol concentrations. Nonspecific binding was further blocked by preincubation with blocking solution for 5 minutes, followed by incubation for 1 hour at 4°C with rabbit antimouse stathmin-1 (1:200 dilution) (Cell Signaling Technology Inc., Beverly, MA). Meanwhile, FITC-conjugated DBA-lectin (1:200 dilution) (Sigma-Aldrich) was added onto the section in the dark for 1 hour. The sections were then washed three times with PBS for 5 minutes each and incubated with PE-conjugated antirabbit IgG (1:200 dilution) (Alpha Diagnostic International, San Antonio, TX) for 30 minutes at room temperature in the dark. Then, 4′,6-diamidino-2-phenylindole (Invitrogen, San Diego, CA) was used to stain nuclei for 10 minutes in the dark. Negative controls were established using rabbit Ig of the isotype identical to the rabbit antimouse primary antibody in place of the specific primary antibody (Cell Signaling Technology Inc.).21Lin Y. Zeng Y. Di J. Zeng S. Murine CD200+CK7+ trophoblasts in a poly (I: c)-induced embryo resorption model.Reproduction. 2005; 130: 529-537Crossref PubMed Scopus (33) Google Scholar First-trimester human decidual tissue was obtained from five normal pregnancies (free of spontaneous abortion history; mean ± SD age, 27.5 ± 2.2 years; mean ± SD gestational age at sampling, 8.2 ± 1.1 weeks, terminated for nonmedical reasons) and five miscarriages [maternal history of more than three unexplained recurrent spontaneous abortions (RSAs); mean ± SD age, 32.4 ± 3.9 years; mean ± SD gestational age at sampling, 8.5 ± 2.8 weeks], which were classified as unexplained after the exclusion of maternal anatomical or hormonal abnormalities and paternal or maternal chromosomal abnormalities. All the samples were obtained from Renji Hospital, Shanghai Jiaotong University, with written informed consent from the patients and permission from the research ethics committee of Shanghai Jiaotong University. To confirm the existence and define the distribution pattern of CD56+stathmin-1− and CD56+stathmin-1+ cells in human decidual tissue, paraffin sections were stained with rabbit antihuman stathmin-1 (Abcam) and mouse antihuman CD56 (Lab Vision/NeoMarkers, Fremont, CA) monoclonal antibodies, followed by staining with multivision antirabbit/horseradish peroxidase (horseradish peroxidase/diaminobenzidine) plus antimouse/alkaline phosphatase polymers (Biolab Science, Beijing, China), according to the manufacturers' instructions. Using this multivision polymer detection system, stathmin-1+ cells were stained brown, CD56+ cells were stained red, and double-positive cells were double colored. Nuclei were lightly stained with hematoxylin. Inhibition of stathmin-1 was performed in CBA/J×DBA/2J, CBA/J×BALB/c, and CBA/J×CBA/J matings by i.p. injection of anti–stathmin-1 antibody (GenScript USA Inc., Piscataway, NJ) on days E4.5, E5.5, and E6.5 (20 μg in 0.2 ml of PBS) once a day. Mice injected with the same volume of rabbit IgG isotype control antibody were used as controls for each group. The percentage of embryo resorption was detected on day E12.5 by using the method described previously herein (n = 8 per group). Flow cytometry data were analyzed by using Quad statistics.16Lin Y. Liang Z. Chen Y. Zeng Y. TLR3-involved modulation of pregnancy tolerance in double-stranded RNA-stimulated NOD/SCID mice.J Immunol. 2006; 176: 4147-4154PubMed Google Scholar The resorption rate was compared using the χ2 test, and the cell percentage was compared using the independent-samples t-test. Cell percentage results are presented as mean ± SD.17Li C. Xiao Z. Chen Z. Zhang X. Li J. Wu X. Li X. Yi H. Li M. Zhu G. Liang S. Proteome analysis of human lung squamous carcinoma.Proteomics. 2006; 6: 547-558Crossref PubMed Scopus (139) Google Scholar, 18Shen J. Pavone A. Mikulec C. Hensley S.C. Traner A. Chang T.K. Person M.D. Fischer S.M. Protein expression profiles in the epidermis of cyclooxygenase-2 transgenic mice by 2-dimensional gel electrophoresis and mass spectrometry.J Proteome Res. 2007; 6: 273-286Crossref PubMed Scopus (16) Google Scholar Significance was defined at P < 0.05. The percentage of spontaneously resorbed embryos on day E12.5 was 22.6% (35 of 155; n = 16) in CBA/J×DBA/2J matings, 7.6% (13 of 170; n = 18) in CBA/J×BALB/c matings, and 7.2% (8 of 111; n = 12) in CBA/J×CBA/J matings. There was no significant difference between CBA/J×BALB/c and CBA/J×CBA/J matings in the percentage of embryo loss, whereas the percentage of embryo resorption in CBA/J×DBA/2J matings was significantly higher than that in CBA/J×BALB/c and CBA/J×CBA/J matings (P < 0.01 for both). The increased resorption rate of CBA/J×DBA/2J matings supports the hypothesis that these mice are prone to spontaneous embryo loss. The mean ± SD percentage of stathmin-1+ cells in the CD49b+ NK cell population was approximately six-fold higher in CBA/J×BALB/c matings (" @default.
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