ORIGINAL ARTICLE: Effect of Maternal Immunopotentiation on Apoptosis-Associated Molecules Expression in Teratogen-Treated Embryos

ORIGINAL ARTICLE: Effect of Maternal Immunopotentiation on Apoptosis-Associated Molecules Expression in Teratogen-Treated Embryos
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  Effect of Maternal Immunopotentiation on Apoptosis-AssociatedMolecules Expression in Teratogen-Treated Embryos Shoshana Savion*, Ilan Aroch*, Keren Mammon, Hasida Orenstein, Amos Fein, Arkady Torchinsky,Vladimir Toder Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel Introduction It has been apparent for a number of years thatmanipulation of the maternal immune system mightexert a positive effect on fertility in mammals, asprevious studies showed a significant reduction inthe high resorption rate of the CBA   ⁄   JxDBA   ⁄   2Jmouse combination following immunization withpaternal strain leukocytes 1,2 or complete Freundadjuvant. 3 More than that, studies performed in ourlaboratory have shown that potentiation of thematernal immune system (immunopotentiation)might also protect the embryo against teratogenicinsults, when intrauterine administration of alloge-neic (paternal) or xenogeneic splenocytes enhancedthe embryonic tolerance to cyclophosphamide or Keywords Apoptosis, bcl-2, embryo, maternalimmunopotentiation, p53, teratogens Correspondence Shoshana Savion, PhD, Department of Celland Developmental Biology, Sackler School ofMedicine, Tel Aviv University, Ramat Aviv, TelAviv 69978, Israel.E-mail:*Equal contribution.Submitted August 6, 2009;accepted September 1, 2009. Citation Savion S, Aroch I, Mammon K, Orenstein H,Fein A, Torchinsky A, Toder V. Effect ofmaternal immunopotentiation on apoptosis-associated molecules expression in teratogen-treated embryos. Am J Reprod Immunol 2009;62: 400–411doi:10.1111/j.1600-0897.2009.00757.x Problem Potentiation of the maternal immune system was shown by us to affectthe embryonic response to teratogenic insults. In order to understand better the mechanisms underlying that phenomenon, we explored theeffect of maternal immunopotentiation by rat splenocytes on the earlystages of the embryonic response to cyclophosphamide (CP). Method of study Immunopotentiated CP-treated embryos were analysed for cell cyclechanges by flow cytometry, while cell proliferation and apoptosis wereassessed by 5 ¢ -bromo-2 ¢ -deoxyuridine (BrdU) incorporation and terminaldeoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end label-ing (TUNEL) respectively. The expression of the p65 subunit of NF- j B,I j B a , Bax, bcl-2 and p53 was assessed by flow cytometry. Results Exposure to CP resulted in significant growth retardation and in theappearance of cellular damage, a reduction in cell proliferation and theappearance of apoptotic cells, which were all found to be delayed inimmunopotentiated embryos. In parallel, CP-treated embryos demon-strated a reduction in the percentage of p65- or I j B a -positive cells,while the percentage of bcl-2- or p53-positive cells increased initiallyand decreased later. Those changes were normalized by maternal immu-nopotentiation when tested at 24 hrs after exposure to the teratogen. Conclusion Our data implicate maternal immunopotentiation to protect the embryoagainst teratogenic insults, possibly through its effect on the expressionof p65, bcl-2 or p53. ORIGINAL ARTICLE American Journal of Reproductive Immunology  62  (2009) 400–411 400  ª  2009 John Wiley & Sons A/S  maternal diabetes. 4–6 However, the mechanismsunderlying this protective effect of maternal immu-nopotentiation are far from being understood. There-fore, one would need first to explore themechanisms mediating teratogenic activity and pin-point those components that might be affected byimmunopotentiation and thus strengthen the embry-onic response to the teratogenic insult.One such mechanism is believed to be apoptoticcell death, which was shown to be involved in nor-mal embryonic development, 7,8 while being alsoimplicated as one of the mechanisms of action ofvarious teratogens, such as cyclophosphamide(CP), 9–11 heat shock, 12 retinoic acid 13 or irradia-tion. 14 The involvement of the apoptotic process inabnormal embryonic development suggests that anydisturbance in its regulation might affect embryonicsurvival. Indeed, apoptosis is tightly regulated by alarge number molecules and the interplay betweenthem is believed to determine the fate of theembryo. Among those are the nuclear factor- j B(NF- j B) transcription factors, which were shown to be involved in normal embryonic development. 15 More than that, we have demonstrated a reductionin the expression of the p65 subunit of NF- j B 11 aswell as suppression of its DNA-binding activity 16 inaffected organs of CP-treated embryos. Accordingly,suppression of NF- j B DNA-binding activity wasdetected also following exposure to thalidomide orethanol. 17,18 Another group of molecules that wereshown to be involved in the regulation of apoptosisis the bcl-2 family, comprised of pro- and anti-apop-totic members, such as Bax or bcl-2 respectively. 19 Anumber of studies addressed the role of these regula-tors of apoptosis in the embryonic response to tera-togenic insults. Thus, an increase in Bax expressionwas demonstrated in pregnant animals exposed tocadmium, kainic acid or nitric oxide, 20–22 while ourprevious study demonstrated a reduction in bcl-2expression in CP-treated embryos. 23 Another centralmolecule known to exhibit a pro-apoptotic functionis the p53 tumor suppressor gene, which was shownto be involved in normal embryonic develop-ment 24,25 as well as in the embryonic response toteratogenic insults. 26,27 The correlation between the apoptotic process andteratogen-induced anomaly formation might suggesta possible association between its normalization andembryonic survival. Such normalization might beexerted by maternal immunopotentiation, however,the stage of embryonic development at which mightsuch an effect be initiated and its possible targetmolecules are not as yet known. Therefore, in thisstudy we tried to explore the effect of maternalimmunopotentiation by rat splenocytes on the earlystages of the embryonic response to CP, by assessinggrowth retardation and changes in proliferation andapoptotic cell death as well as the expression of thementioned-above molecules, which might beresponsible, at least partially, for the protective effectof maternal immunopotentiation. Materials and methods Animals Six- to 8-week old ICR mice were obtained from theTel Aviv University and kept under standard condi-tions. ICR females were caged with males for 3 hrs(7–10 a.m.) and the presence of a vaginal plug deter-mined the first day of pregnancy. CP Injection and Evaluation of its TeratogenicEffect Cyclophosphamide (CP) (Sigma, Rehovot, Israel)was injected intraperitoneally at 40 mg   ⁄   kg bodyweight in 0.5 mL saline on day 12 of pregnancy.Mice were sacrificed at 6, 24 or 48 hrs after injectionas well as on day 19 of pregnancy. The uteri wereremoved and the percentage of resorptions and liveembryos was recorded. All live embryos wereweighed and examined visually for malformationsand microscopically for morphological changes, usinghematoxylin and eosin-stained tissue sections. Maternal Immunopotentiation Intrauterine administration of rat splenocytes wascarried out 2 weeks before mating. ICR mice wereanesthetized with ketamine and xylazine, 8 mg   ⁄   kgeach and injected with 25–30  ·  10 6 splenocytes   ⁄   0.04 mL phosphate-buffered saline (PBS)   ⁄   horn. Miceinjected accordingly with PBS served as controls. Cell Proliferation Assay Mice from the various experimental groups wereinjected intraperitoneally with 5 ¢ -bromo-2 ¢ -deoxyuri-dine (BrdU) (Sigma, Rehovot, Israel) at 200 mg   ⁄   kg body weight in 0.5 mL PBS. Embryos were collected IMMUNOPOTENTIATION-MEDIATED EMBRYONIC RESPONSE TO CP American Journal of Reproductive Immunology  62  (2009) 400–411 ª  2009 John Wiley & Sons A/S  401  6 hrs later, fixed in ice-cold 4% paraformaldehyde inPBS and embedded in paraffin and 7- l m  thick sec-tions were deparaffinized and rehydrated in gradedalcohols. Sections were treated with 3% H 2 O 2  andincubated with 1.5N HCl for 20 min to hydrolysenuclear proteins, followed by treatment with 0.1  m sodium borate for 10 min, to neutralize the acid.After blocking with PBS containing 5% fetal calfserum (FCS) and 0.1% Triton-X-100, sections wereincubated overnight with mouse anti-mouse BrdU(1  l g   ⁄   mL; Beckton and Dickinson, Franklin Lakes,NJ, USA). The next day, sections were incubatedwith a biotinylated goat anti-mouse secondary anti- body at 0.5  l g   ⁄   mL for 30 min, followed by Strepta-vidin-Horseraddish peroxidase (HRP, 1.25  l g   ⁄   mL;Zymed Laboratories, South San Francisco, CA, USA)in blocking solution for 30 min. BrdU-positive nucleiwere detected by incubation with diaminobenzidine(DAB Substrate Kit, Zymed Laboratories, South SanFrancisco, CA, USA) in the presence of H 2 O 2  andquantified by the Image Pro Plus software (MediaCybernetics, Bethesda, MD, USA). Cell Cycle Analysis Cyclophosphamide (CP)-induced changes in the per-centage of cells in the various stages of the cell cycleas well as the percentage of apoptotic cells wereevaluated by fluorescence-activated cell sorter(FACS) analysis. Thus, embryonic heads from allexperimental groups were teased mechanically intosingle-cell suspensions in 20% FCS in PBS. Nucleiwere isolated by resuspending the cells in a hypo-tonic fluorochrome solution containing 50  l g   ⁄   mLpropidium iodide (PI; Sigma, Rehovot, Israel), 0.1%sodium citrate and 0.1% Triton-X-100. Ten thousandnuclei   ⁄   sample were analysed by a FacSort (BectonDickinson FACS Systems, Mountain View, CA, USA)and the percentage of cells in the various stages ofthe cell cycle was determined by the WinMDI 2.1software (Scripps Institute, La Jolla, CA, USA). Localization of Apoptotic Cells by the TUNELMethod Detection of apoptotic cells in tissue sections ofembryos from the various experimental groups wasperformed by the terminal deoxynucleotidyl trans-ferase (TdT)-mediated dUTP-biotin nick-end labeling(TUNEL) method. Embryos were collected at 6, 24or 48 hrs after CP injection and processed asdescribed above. Sections were treated with pro-teinase K (20  l g   ⁄   mL; Ameresco Inc., Solob, OH,USA) to hydrolyse nuclear proteins and then with2% H 2 O 2  and 0.05% Tween-20, to inactivateendogenous HRP. For nick-end labeling, cells werecovered with reaction buffer (140 m m  sodium caco-dylate, 1 m m  cobalt-chloride and 30 m m  Tris, pH7.2) containing 60 U   ⁄   mL TdT (Promega, Madison,WI, USA) and 5  l m  biotinylated-dUTP (ClontechLaboratories, Palo Alto, CA, USA) and incubated for1.5 hrs at 37  C in a humidified incubator. Sectionsprocessed in the absence of biotinylated-dUTPserved as negative controls. Sections were blockedwith 3% low-fat milk and 0.05% Tween-20 in PBSand incubated with 1.25  l g   ⁄   mL Streptavidin-HRPin blocking solution for 20 min. TUNEL-positivenuclei were detected by incubation with DAB asdescribed earlier and quantified by the Image ProPlus software. Confocal Microscopy Samples of cells from the various experimentalgroups that were prepared for cell cycle analysis asdescribed earlier, were mounted onto slides andexamined under a LSM 410 Inverted Laser ScanMicroscope (Zeiss, Germany). Flow Cytometric Analysis of the Expression of Apoptosis-associated Molecules Cyclophosphamide (CP)-induced changes in thepercentage of cells expressing the various apoptosis-associated molecules were evaluated by FACSanalysis. Thus, embryonic heads from all experi-mental groups were teased mechanically into single-cell suspensions in 20% FCS in PBS and the cellswere fixed in 2% paraformaldehyde for 15 min andthen in 70% ice-cold methanol for 1 hr. Cells wereincubated with antibodies against the p65 subunit ofNF- j B, I j B a , Bax, bcl-2 or p53 (0.05–0.8  l g   ⁄   mL;Santa Cruz Biotechnology, Santa Cruz, CA, USA) for30 min. Omission of the primary antibody or its pre-incubation with appropriate blocking peptides servedas negative controls. Cells expressing the variousmolecules were detected by incubation with fluores-cein isothiocyanate (FITC)-conjugated secondaryantibodies (50 ng   ⁄   mL; Santa Cruz Biotechnology,Santa Cruz, CA, USA) for 30 min in the dark. Tenthousand cells per sample were analysed by FACSand the percentage of cells expressing the various SAVION ET AL. American Journal of Reproductive Immunology  62  (2009) 400–411 402  ª  2009 John Wiley & Sons A/S  molecules was determined by the level of fluores-cence, using the WinMDI 2.1 software. Statistical Analysis Statistical analysis of the reproductive performancewas performed on a litter basis using the GT2method for multiple comparisons. 28 Statistical analy-sis of the percentage of apoptotic cells as well as thepercentage of cells expressing the various apoptosis-associated molecules was performed by Student’s t  -test. The two-tailed level of significance of differ-ence was 0.05. For BrdU incorporation and TUNELanalysis, three mid sagittal sections collected fromembryos from the various experimental groups werechosen randomly. The number of BrdU- or TUNEL-positive nuclei was determined by the Image ProPlus software and presented as average ± standarderror of eight fields (1.5  l m 2 each)   ⁄   section. Results Early Embryonic Response to CP inImmunopotentiated Embryos Maternal immunopotentiation with rat splenocyteswas shown by us previously to protect the embryofrom the teratogenic insult exerted by its exposure toCP, demonstrated by an increase in embryonic weightand a reduction in the resorption rate and the propor-tion of malformed embryos when tested on day 19 ofpregnancy. 23 In order to understand better the mech-anisms underlying the normalizing effect of maternalimmunopotentiation on the reproductive perfor-mance of CP-treated mice, we tried in this study toanalyse the early embryonic response to the terato-gen, which is manifested mainly by growth retarda-tion, by evaluating its activity in immunopotentiatedembryos at several time-points shortly after exposure.As can be seen in Table I and Fig. 1, exposure to CPresulted in a significant reduction in embryonicweight, which was detected in non-immunopotenti-ated embryos already at 24 hrs after exposure, whileat 48 hrs and on day 19 of pregnancy it was visiblein both non-immunopotentiated and immunopoten-tiated embryos. Immunopotentiation itself had noinfluence on the embryonic weight (Table I). Interest-ingly, hematoxylin- and eosin-stained brain sections(Fig. 2) revealed the appearance of cellular damageonly in non-immunopotentiated CP-treated embryos,again as early as 24 hrs after exposure (Fig. 2e), whileat 48 hrs after treatment it was noticed in the brainof both non-immunopotentiated and immunopoten-tiated embryos (Fig. 2g,h). Cyclophosphamide-induced Changes in CellProliferation in Immunopotentiated Embryos Cyclophosphamide (CP)-induced changes in cellproliferation were analysed by BrdU incorporation Table I  Effect of maternal immunopotentiation on CP-induced changes in embryonic weight Average embryonic weight (g) ± S.E.6 Hrs 24 Hrs 48 Hrs Day 19IM )  IM+ IM )  IM+ IM )  IM+ IM )  IM+Non-treated 0.039 ± 0.004 0.039 ± 0.002 0.084 ± 0.007 0.087 ± 0.013 0.148 ± 0.020 0.155 ± 0.001 1.280 ± 0.009 1.270 ± 0.03340 mg   ⁄   kg (CP) 0.039 ± 0.003 0.024 ± 0.004 0.060* ± 0.003 0.069 ± 0.004 0.075* ± 0.005 0.066* ± 0.004 0.306* ± 0.011 0 392** ± 0.032IM, immunopotentiation; g, gram; S.E., standard error.*Significantly different ( P  < 0.05) from non-treated embryos; **Significantly different ( P  < 0.05) from IM-, CP-treated embryos. Non treatedimmunopotentiatedCPNon - immunopotentiatedCPImmunopotentiated Fig. 1  CP-induced changes in growth retardation of non-treated(immunopotentiated) or CP-treated (non-immunopotentiated or immu-nopotentiated; 40 mg   ⁄   kg) embryos, tested on day 19 of pregnancy( · 1.5). IMMUNOPOTENTIATION-MEDIATED EMBRYONIC RESPONSE TO CP American Journal of Reproductive Immunology  62  (2009) 400–411 ª  2009 John Wiley & Sons A/S  403   by the embryonic cells. As can be seen in Fig. 3,exposure to CP resulted in a clear time-dependentreduction in the number of BrdU-positive cells,which started to be visible at 24 hrs after treat-ment only in the brain of non-immunopotentiatedembryos, as compared with their immunopotentiatedcounterparts (Fig. 3e versus f, 23.7 ± 1.2 versus34.3 ± 0.9 BrdU-positive nuclei) and became moreprominent at 48 hrs after treatment, however,similarly in the brain of both non-immunopotentiat-ed and immunopotentiated embryos (Fig. 3g versush, 11.0 ± 2.0 versus 11.7 ± 0.5 BrdU-positivenuclei). It is important to point out, that the level ofcell proliferation in non-treated embryos did notchange at the different time-points tested (data notshown). Cell Cycle Changes in ImmunopotentiatedCP-treated Embryos Cyclophosphamide (CP)-induced changes in the per-centage of cells in the various stages of the cell cyclewere examined by flow cytometry. As can be seenin Fig. 4, a significant time-dependent increase inthe percentage of cells in the Sub-G 1  area of the cellcycle, indicating the presence of cells undergoingapoptotic cell death, was noticed in the heads of both non-immunopotentiated and immunopotenti-ated embryos, as compared with non-treatedembryos, starting at 6 hrs after treatment andincreasing at later time-points (Fig. 4a,b). Interest-ingly, the increase in the percentage of cells in theSub-G 1  area of the cell cycle detected at 6 hrs afterexposure to CP was found to be significantly lowerin immunopotentiated embryos, while at later time-points it was similar to that revealed in their non-immunopotentiated counterparts (Fig. 4b). Identification of Apoptotic Cells by TUNEL andConfocal Microscopy in ImmunopotentiatedCP-treated Embryos Examination of TUNEL-stained tissue sectionsrevealed the appearance of a time-dependent apop-totic process in the brain of both non-immunopoten-tiated and immunopotentiated CP-treated embryos(Fig. 5), in the same areas that exhibited cellulardamage as described previously (Fig. 2g,h). Thus, CP-treated, 6 hrNon treatedCP-treated, 24 hrCP-treated, 48 hr (a) (c) (e)(b) (d) (f) (h)(g) Fig. 2  CP-induced cellular damage (seearrows) in hematoxylin- and eosin-stainedbrain sections of non-immunopotentiated(a, c, e, and g) or immunopotentiated (b, d, f,and h) embryos. a. and b., Non-treated; c. andd., CP-treated, 6 hrs; e. and f., CP-treated,24 hrs; g. and h., CP-treated, 48 hrs ( · 80). Non treatedCP-treated, 6 hrCP-treated, 48 hrNon-immunopotentiatedImmunopotentiatedCP-treated, 24 hr (a) (c) (e) (g)(b) (d) (f) (h) Fig. 3  CP-induced changes in BrdU incorpora-tion in the brain of non-immunopotentiated (a,c, e and g) or immunopotentiated (b, d, f, andh) embryos. a. and b., Non-treated; c. and d.,CP-treated, 6 hrs; E. and F., CP-treated,24 hrs; G. and H., CP-treated, 48 hrs ( · 80). SAVION ET AL. American Journal of Reproductive Immunology  62  (2009) 400–411 404  ª  2009 John Wiley & Sons A/S
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