of January 29, 2018.This information is current as

Pregnancy Lossin the Uteroplacental Unit of Mice with

Messenger RNA and Protein ExpressionαTNF-

Savion, Amos Fein, Arkady Torchinsky and Vladimir ToderMarat Gorivodsky, Ilona Zemlyak, Hasida Orenstein, Shoshana

http://www.jimmunol.org/content/160/9/42801998; 160:4280-4288; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1998 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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TNF-a Messenger RNA and Protein Expression in theUteroplacental Unit of Mice with Pregnancy Loss1

Marat Gorivodsky, Ilona Zemlyak, Hasida Orenstein, Shoshana Savion, Amos Fein,Arkady Torchinsky, and Vladimir Toder 2

An elevated expression of TNF-a in embryonic microenvironment was found to be associated with postimplantation loss. In thiswork, we examined the pattern of TNF-a expression at both the mRNA and the protein level as well as the distribution of TNF-areceptor mRNA in the uteroplacental unit of mice with induced (cyclophosphamide-treated) or spontaneous (CBA/J3 DBA/2Jmouse combination) pregnancy loss. RNase protection analysis demonstrated an increase in TNF-a mRNA expression in theplacentae of mice with pregnancy loss compared with that in control mice. TNF-a messages were localized to the uterine epi-thelium and stroma as well as the giant and spongiotrophoblast cells of the placenta. The intensity of the hybridization signal inplacentae of mice with pregnancy loss was substantially higher than that in control mice. The up-regulation of TNF-a mRNA wasaccompanied by an increase in the expression of TNF-a receptor I mRNA in the same cell populations. The elevation of TNF-aproduction was also demonstrated at the protein level. Western blot analysis showed an increased level of the 18- and 26-kDaTNF-a protein species in the uteroplacental unit of mice with pregnancy loss. Immunostaining revealed TNF-a-positive leukocyteslocated in the uterus and placenta. Finally, we found that immunization of mice with cyclophosphamide-induced pregnancy losswhile decreasing the resorption rate in these females resulted in a decline in TNF-a expression at the fetomaternal interface. Thesedata clearly suggest an involvement of TNF-a in pathways leading to both spontaneous and induced placental death.The Journalof Immunology,1998, 160: 4280–4288.

T he presence and normal functioning of cytokine networksat the fetomaternal interface may be important for thecontinued development of pregnancy (1). TNF-a is a mul-

tifunctional cytokine that plays a prominent role in immune andhost defense responses, stimulates angiogenesis, influences tissueremodeling, promotes apoptosis, and takes part in the regulation ofcell proliferation and differentiation (2–7). It has also been re-ported to be instrumental in the regulation of reproductive pro-cesses (8, 9). TNF-a was demonstrated to be produced by bothuterine and placental cells. In humans, TNF-a mRNA and proteinhave been identified in syncytio- and extravillous cytotrophoblast(10–12), and biologically active TNF-a was found in the super-natants of placental and decidual tissue (13, 14). In rodents, TNF-aexpression was demonstrated in the uterine epithelium, decidua,and trophoblast (15, 16). Also, TNF-a mRNA transcripts havebeen identified in murine macrophage-like cells residing in theendometrial stroma and in NK-like cells populating the deciduaand metrial gland (15–17). The expression of TNF-a is tightlyregulated during mouse gestation (18), reaching its maximum atmidgestation and then remaining stable until the end ofpregnancy (15).

The role of uterine and placental TNF-a in pregnancy is poorlyunderstood. It has been suggested that TNF-a may regulate themigration and behavior of uterine leukocytes (19, 20) and affectthe myometrial contractions during labor (13). Furthermore, ma-ternal TNF-a might influence blastocyst growth and implantation(21, 22) due to regulation of trophoblast growth and differentiationin early embryos (23). Recent studies on knockout mice have dem-onstrated that TNF-a is required for normal placental growth andfunction (24). TNF-a binds to one of two distinct cellular recep-tors, TNF-a receptor I (TNFRI)3 (p55) and TNFRII (p75) (25),thereby initiating different cellular responses. Transcripts of bothreceptors have been found in the uterus and placenta of pregnantmice (26).

Abnormal TNF-a production may be associated with pregnancyfailure. The TNF-a level was shown to be significantly elevated inthe amniotic fluid of women with uterine infections, and its in-creased production correlates with the incidence of preterm labor(27). Administration of LPS (an inducer of TNF-a production) orTNF-a itself to pregnant mice results in pregnancy loss (28, 29) orembryo growth retardation (30), whereas treatment with anti-TNF-a Abs or soluble receptors reduces the number of resorptionsin mice with a high rate of immune-mediated pregnancy loss (31,32). Furthermore, enhancement of decidual TNF-a production hasbeen suggested to be one of the mechanisms involved in stress-triggered abortions in mice (33). Also, an increased TNF-a levelhas been demonstrated in supernatants from decidual cell culturesfrom the resorption-prone CBA/J3 DBA/2J mouse comparedwith that in the nonresorption-prone CBA/J3 BALB/c mousecombination (34). In parallel, an elevation in TNF-a was regis-tered at the mRNA level in placentae of CBA/J3 DBA/2J mice(35). Cytokine analysis of supernatants from mixed lymphocyte-placental cell cultures has shown a significantly higher production

Department of Embryology and Teratology, Sackler School of Medicine, Tel AvivUniversity, Ramat Aviv, Tel Aviv, Israel

Received for publication July 31, 1997. Accepted for publication January 6, 1998.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Israel Ministry of Health and the IsraelMinistry of Science and Technology. This work is in partial fulfillment of the re-quirements for the Ph.D. degree of M.G. and the M.Sc. degree of I.Z. from the SacklerSchool of Medicine at Tel Aviv University.2 Address correspondence and reprint requests to Dr. V. Toder, Department of Em-bryology and Teratology, Sackler School of Medicine, Tel Aviv University, RamatAviv, Tel Aviv 69978, Israel. E-mail address: toder@post.tau.ac.il 3 Abbreviations used in this paper: TNFRI, TNF-a receptor I; CP, cyclophosphamide.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00

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of TNF-a in supernatants from CBA/J3 DBA/2J mice comparedwith those from CBA/J3 BALB/c mice (36).

The correlation between an elevated level of TNF-a and preg-nancy failure raises the possibility that normalization of TNF-aexpression at the fetomaternal interface may be associated withimproved reproductive performance in females with pregnancyloss. It has been widely reported that alloimmunization or nonspe-cific immune stimulation may protect the fetus and improve re-productive outcome (37, 38). We have demonstrated that such im-munization may prevent the embryonic dismorphogenesis inducedby extrinsic and intrinsic factors (39, 40). The protective effect ofimmunization is generally thought to be due to modification of thecytokine milieu in embryonic microenvironment (41).

In this report we present data characterizing the pattern ofTNF-a and TNFRI expression at the fetomaternal interface ofmice with spontaneous and induced pregnancy loss and possiblechanges in this pattern induced by maternal immunization to de-termine whether the protective effect of immunization in femaleswith pregnancy loss is associated with modulation of TNF-aexpression.

Materials and MethodsAnimals

Six- to eight-week-old ICR and C57BL/6 mice and Long Evans rats wereobtained from the Tel Aviv University breading colonies. CBA/J femalesand DBA/2J males were obtained from The Jackson Laboratory (Bar Har-bor, ME). The animals were maintained on a 14-h light/10-h dark cyclewith food and water ad libitum. To obtain pregnancies, females were cagedwith males overnight, and the presence of a vaginal plug was designatedday 1 of pregnancy.

Animal models of pregnancy loss

Two mouse models of induced and spontaneous pregnancy loss were usedin this study.

The CBA/J3 DBA/2J mouse combination, which is well known for itshigh level of postimplantation loss, was used as a model of spontaneousabortions (31). Cyclophosphamide (CP)-treated ICR3 ICR and CBA/J3C57BL/6 mouse combinations were used as models of inducedpregnancy loss.

CP was injected i.p. on the morning of day 12 of pregnancy at 40 mg/kg(in 0.5 ml saline/20 g body weight). Dosage was proportional to weight atthe time of treatment (42). CBA/J females mated to DBA/2J males weresacrificed on day 12 of gestation, while CP-treated mice were sacrificed onday 15 or 19 of pregnancy. The numbers of implantation sites, resorptions,and live fetuses were recorded, and the incidence of postimplantation losswas calculated as described previously (42).

Immunization

CBA/J and ICR females were treated with either allogeneic paternal(C57BL/6) or xenogeneic rat splenocytes, respectively, 21 days beforemating as described previously (39, 43). Briefly, spleens were asepticallyremoved and dispersed in RPMI 1640 medium (Biologic Industries, Israel)by pressing them through a stainless steel mesh. The cells were washed,and their viability was assessed by trypan blue staining. Under nembutalanesthesia (40 mg/kg) the uterus was identified and injected with 25 to30 3 106 splenocytes/0.04 ml saline/horn. Mice injected with saline orsyngeneic splenocytes served as controls.

Tissue processing

Placentae together with the adjacent uteri were collected from mice withspontaneous resorptions on day 12 and from CP-treated mice on day 15 ofpregnancy. The term resorbing placenta refers to a placenta with a pale orvisibly destroyed embryo and remnants of extraembryonic tissues butwhich can still be identified macroscopically as a placenta. The term non-resorbed placenta refers to a macroscopically normal placenta with a liveembryo.

For RNase protection and Western blot analysis, placentae and uteriwere immediately snap-frozen in liquid nitrogen and stored at270°C untiluse. For in situ hybridization or immunohistochemistry techniques, pla-centae were fixed in 4% paraformaldehyde or in Bouin’s solution, respec-tively, and embedded in paraffin, and 7-mm sections were further used after

histologic examination. Only resorbing placentae containing morphologi-cally unaffected regions were chosen for further analysis.

Probe construction

The 709-bp TNF-a and 640-bp TNFRI cDNAs (provided by Prof. D.Wallach, Weizmann Institute of Science, Rehovot, Israel) were subclonedinto theEcoRI-SacI andEcoRI-SphI sites of the pBluescript SK1 vector(Stratagene, La Jolla, CA), respectively. After linearization withSacI forTNF-a and with SphI for TNFRI cDNA, the DNA template served forgeneration of digoxigenin-11-UTP-labeled (Boehringer Mannheim, Mann-heim, Germany) antisense RNA probes using T7 RNA polymerase (Strat-agene). RNA probes forb-actin (360 bp) and the prokaryoticneogene (760bp) were synthesized as described above. The lengths of the generatedRNA probes were evaluated by comparing their sizes with that of thedigoxigenin-labeled DNA m.w. marker VIII (Boehringer Mannheim) indenatured 5% polyacrylamide gel.

RNase protection analysis

Total RNA was extracted from placentae and uteri by the method ofChomzynski and Sacchi (44) using the Tri-Reagent (Molecular ResearchCenter, Cincinnati, OH). The RNA concentration was calculated by spec-trophotometry at 260 and 280 nm, and the integrity of the RNA was mon-itored by electrophoresis in 1% agarose/2.2 M formaldehyde gel. The fol-lowing procedures are those described in the protocol of the RNaseProtection Assay System (Promega, Madison, WI). Briefly, 30 to 50mg oftotal RNA were coprecipitated with 30 ng of antisense RNA probe, incu-bated overnight in 20ml of hybridization buffer (80% formamide, 1 mMEDTA, 0.2 M sodium acetate, and 40 mM PIPES, pH 6.4) at 45°C, andthen digested with 16 U of RNase ONE (Promega) for 1 h at room tem-perature. Following RNase inactivation, RNA was precipitated and resus-pended in gel loading buffer, and protected fragments were resolved byelectrophoresis in denatured 5% polyacrylamide/8 M urea gel. The m.w. ofspecific mRNA was calculated using the digoxigenin-labeled DNA m.w.marker VIII. RNA was transferred to Nytran nylon membranes (Schleicher& Schuell, Dassel, Germany), which were rinsed briefly in 63 SSC (150mM sodium chloride and 15 mM sodium citrate, pH 7.0) and exposed toUV light for cross-linking of RNA to the filters. Hybridization bands werevisualized by incubating the blots in alkaline phosphatase-conjugated an-tidigoxigenin Abs at 1/10,000 dilution (Boehringer Mannheim) and thechemiluminescent substrate CSPD (Tropix, Bedford, MA) followed by ex-posure to x-ray film.

As a negative control, tissue RNA was substituted by yeast tRNA. Equiv-alency of RNA loading on the gel was controlled by hybridization of the samequantity of tissue RNA with ab-actin riboprobe. The quantitative character ofthe RNase protection assay was confirmed by titration of tissue RNA with thelabeled riboprobe and generation of a titration curve (data not shown).

Densitometric analysis of films was performed using B.I.S. 202D imagedensitometric system (Bio-Rad, Richmond, CA), and results were analyzedby TINA software (Raytest, Straubenhard, Germany).

In situ hybridization

Tissue sections were deparaffinized and processed as previously described(45). Briefly, the sections were washed and heated for 30 min at 70°C in23 SSC, treated with 10mg/ml proteinase K (IBI, New Haven, CT) for 15min at 37°C, and fixed in 4% ice-cold paraformaldehyde. Prehybridizationwas performed for 1 h at 45°C in 50% formamide, 63SSPE (150 mMsodium chloride, 10 mM sodium phosphate, and 1 mM EDTA, pH 7.4), 53Denhardt’s solution (Sigma, Rehovot, Israel) and 0.5% SDS. The sectionswere overlaid with 30ml of hybridization mixture (50% formamide, 53Denhardt’s solution, 10% dextran sulfate, 63 SSPE, and 0.5% SDS) con-taining 0.5 ng/ml digoxigenin-labeled antisense RNA probe. Hybridizationwas conducted overnight at 45°C in a humidified chamber. The slides werewashed twice for 15 min in 23 SSC, followed by incubation with 20mg/mlRNase A (Sigma) for 30 min at 37°C. High stringency washes were per-formed by incubating the slides twice for 15 min at 50°C in 0.13 SSCfollowed by a 10-min wash in 0.13 SSC at room temperature. The hy-bridization signal was detected by alkaline phosphatase-conjugated anti-digoxigenin Abs followed by incubation in nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate color substrate solution (BoehringerMannheim) containing 1 mM levamisole according to the manufacturer’srecommendations. Finally, sections were lightly counterstained with neu-tral red, and a positive signal was indicated by a deep purple-brownstaining.

As a control for hybridization, a nonhomologic RNA probe synthesizedfrom a prokaryoticneocDNA was substituted for the specific probes. Tis-sue sections pretreated with 100mg/ml RNase A (Sigma) for 30 min at37°C before hybridization served as an additional control.

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Western blot analysis

Placentae and uteri were homogenized in ice-cold buffer containing 100mM Tris-HCl (pH 7.4), 200 mM sodium chloride, 2 mM EDTA, 1 mMPMSF, and 2mg/ml aprotinin. An equal volume of lysing solution (1%desoxycholate, 0.04% Nonidet P-40, and 0.2% SDS) was added then toeach sample, and the resulting homogenates were centrifuged for 10 min at4°C at 10,0003 g, aliquoted, and stored at270°C until use.

Protein concentration was determined by the Bio-Rad protein assaymethod (Bio-Rad). Samples containing 50mg of protein were resolved byelectrophoresis in a 12% SDS-polyacrylamide gel. Prestained m.w. stan-dards (Novex, Rockford, IL) and murine rTNF-a (provided by Prof. D.Wallach, Weizmann Institute of Science) were used as markers. Proteinswere transferred to nitrocellulose membranes (Schleicher and Schuell), andnonspecific binding sites on blots were blocked by incubation in 5% (w/v)low fat dried milk in buffer containing 50 mM Tris-HCl (pH 7.4), 500 mMsodium chloride, and 0.1% SDS (TBST) for 2.5 h at room temperature.Filters were incubated in polyclonal TNF-a rabbit antiserum (Endogen,Cambridge, MA) at 15mg/ml TBST for 30 min at 37°C. Nonimmunerabbit serum, used at the same dilution as the primary Ab, served as anegative control. After intensive washing in TBST, the membranes wereincubated for 1 h at room temperature with biotinylated goat anti-rabbitIgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at 10 ng/ml, washed again, and incubated with streptavidin-conjugated horseradishperoxidase (Zymed, San Francisco, CA) at 0,5mg/ml for 45 min at roomtemperature. After another wash, the membranes were incubated with ECLreagents (Amersham Life Sciences, Arlington Heights, IL) and exposed tox-ray film.

Immunohistochemistry

Tissue sections were deparaffinized, washed briefly in PBS (pH 7.4), andtreated with 1.5 mg/ml hyaluronidase (Sigma) in PBS, pH 6.5, for 1 h at37°C. Ag retrieval was performed by heating the tissue sections in PBS, pH7.4, for 30 min at 80°C. Endogenous peroxidase activity was inhibited byincubating the sections in 3% hydrogen peroxide. Nonspecific binding siteswere blocked by a 20% solution of FCS in PBS/0.05% Tween (PBST) for30 min at 37°C. Sections were stained with rabbit anti-mouse TNF-a-specific Abs diluted to 1/70 in 10% FCS/PBST. Nonimmune rabbit serumused at the same dilution as the primary Ab served as a negative control.Then, slides were washed in PBS and incubated for 30 min at room tem-perature with biotinylated goat anti-rabbit IgG, diluted to 1/1000, followedby incubation in streptavidin-conjugated horseradish peroxidase/PBST at12 mg/ml. Anti-TNF-a Ab-stained cells were visualized by incubating thesections with 0.2 mg/ml diaminobenzidine (Sigma) followed by counter-staining with 0.1% hematoxylin.

Statistical analysis

Each tested sample of total RNA and protein was obtained by combiningfour or five placentae in a tested litter. To evaluate the results of RNaseprotection and Western blot analyses statistically, four or five samples ob-tained from different litters were analyzed and compared by Student’sttest. The two-tailed level of significance of differences wasa 5 0.05. Thereproducibility of RNase protection and Western blot analysis was tested intwo experiments using the same samples.

For in situ hybridization analysis and immunostaining, four or fiveresorbing and/or nonresorbed uteroplacental units collected from four micewere analyzed for each experimental group. To test reproducibility, in situhybridization and immunostaining experiments were repeated three times.In each experiment, four or five tissue sections of each uteroplacental unitwere processed and analyzed by two independent readers. Results charac-terizing signal intensity were averaged.

ResultsPregnancy loss in tested animal models

To estimate the level of postimplantation loss, 15 litters weretested in each animal model.

The rate of resorptions in the CBA/J3 DBA/2J mouse combi-nation evaluated on day 12 of pregnancy was 30.4%. The level ofresorptions in CP-treated ICR mice reached 32. 3% by day 15 ofpregnancy and increased dramatically to approximately 80% onday 19 of pregnancy. The level of resorptions in CBA/J femalesmated to C57BL/6 males and treated with CP was practically iden-tical with that in CP-treated ICR mice.

TNF-a mRNA expression

TNF-a mRNA expression was evaluated using RNase protectionanalysis. In placentae of control mice, two species of mRNA cor-responding to 320 and 283 bp were detected (Fig. 1). The densi-tometric analysis revealed that in nonresorbed placentae of micewith induced pregnancy loss, the expression of both fragments was2.4-fold higher than that in placentae of control mice, while inresorbing placentae of these animals this increase was less prom-inent. A third fragment, corresponding to 363 bp, was detectedonly in the placentae of CP-treated mice, whether resorbing ornonresorbed, but not in control mice.

In the CBA/J 3 DBA/2J mouse model of spontaneous abor-tions, one fragment of RNA corresponding to 220 bp was detected(Fig. 2). Densitometric analysis revealed a 20% increase in thelevel of TNF-a mRNA expression in the resorbing vs the non-resorbed placenta.

Localization of TNF-a mRNA

Data from in situ hybridization analysis characterizing the cellularlocalization and intensity of the hybridization signal are summa-rized in Table I and Figure 3.

FIGURE 1. RNase protection analysis of TNF-a mRNA in the placentaof control and CP-treated mice.Top, Hybridization with TNF-a-specificantisense RNA probe:Lane 1, yeast tRNA;lane 2, undigested TNF-ariboprobe; lane 3, nonresorbed placenta from CP-treated mice;lane 4,placenta from control mice;lane 5, resorbing placenta from CP-treatedmice; lane 6, nonresorbed placenta from immunized mice;lane 7, non-resorbed placenta from immunized and CP-treated mice.Middle, Hybrid-ization withb-actin-specific riboprobe (a 250-bp protected fragment).Bot-tom, Densitometric analysis of the protected fragment corresponding to 283bp. CP, Nonresorbed placentae from CP-treated mice; IM, nonresorbedplacentae from immunized mice; IM1 CP, nonresorbed placentae fromimmunized CP-treated mice. Bars represent the percentage of mRNA(6SE) in the experimental groups (CP and IM1 CP) relative to the mRNAlevel in the control group (100%). The level of TNF-a mRNA expressionin placentae of CP-treated mice was significantly higher (p , 0.05) thanthat in control mice. Also, TNF-a mRNA expression in placentae fromimmunized CP-treated mice was significantly lower (p , 0.05) than that inplacentae of nonimmunized CP-treated mice.

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The distributions of cells expressing TNF-a mRNA in placentaeand uteri were similar in control and CP-treated mice. In theuterus, TNF-a mRNA expression was demonstrated in cells of

luminal epithelium and stroma (Fig. 3,c andd, and Table I). In theplacenta, giant and spongiotrophoblast were the dominant cell pop-ulations containing specific messages (Fig. 3,e and f ), while lab-yrinthine trophoblast cells were negative (data not shown). Also,leukocytes containing TNF-a mRNA were detected in placentalblood lacunae (Fig. 3h).

In the resorbing placenta of CP-treated mice, trophoblastcells demonstrated a loss of TNF-a transcripts, in contrast tometrial gland cells and uterine stroma, which were positive(Table I).

The intensity of the hybridization signal was elevated in theuterine epithelium as well as in trophoblast cells of nonresorbedplacentae of CP-treated compared with control mice (Table I andFig. 3, a–f). The resorbing placentae (vs nonresorbed placentae)showed a clear induction of TNF-a mRNA expression in metrialgland cells and an enhanced hybridization signal in uterine stroma.In parallel, the signal was weaker in the uterine epithelium of theseplacentae (Table I).

The cellular pattern of TNF-a mRNA expression in the utero-placental unit of the CBA/J3 DBA/2J mouse combination wasbasically similar to that in CP-treated mice, except for metrialgland cells, which demonstrated a positive signal (Table I).

In resorbing placentae of mice with spontaneous pregnancy loss,the specific signal was more intensive than in nonresorbed placen-tae (Table I). As expected, numerous leukocytes containing TNF-a

mRNA were found to infiltrate the tissue areas of the resorbingplacenta undergoing necrosis (data not shown).

Hybridization with nonhomologic prokaryotic RNA probe (Fig.3g) as well as hybridization of tissue sections pretreated withRNase before hybridization with specific riboprobes (data notshown) demonstrated no signal.

Localization of TNFRI mRNA

In control mice, the expression of TNFRI mRNA was observedbasically in the same cells that expressed TNF-a mRNA (Tables Iand II).

In mice with spontaneous and induced abortions, TNFRI mRNAtranscripts were found in the uterine epithelium (Table II) as wellas in giant and spongiotrophoblast cells (Fig. 4 and Table II). Aweak signal was also detected in metrial gland cells of CP-treatedmice compared with that in control mice (Table II).

FIGURE 2. RNase protection analysis of TNF-a mRNA in placentae fromCBA/J females (CBA/J3 DBA/2J mouse combination).Top, Hybridizationwith TNF-a-specific antisense RNA probe:lane 1, yeast tRNA;lane 2, undi-gested TNF-a riboprobe;lane 3, nonresorbed placenta;lane 4, resorbing pla-centa.Middle, Hybridization with ab-actin-specific riboprobe (a 250-bp pro-tected fragment).Bottom, Densitometric analysis of RNA blots. Bars representthe percentage of mRNA (6SE) in resorbing placentae relative to the mRNAlevel in nonresorbed placenta (100%). The levels of TNF-a mRNA expressionin resorbing and nonresorbed placentae were significantly different (p , 0.05).

Table I. Tissue distribution of TNF-a mRNA in the uteroplacental unita

Groups of Animals andTypes of Tested

Uteroplacental Unitsb SpongiotrophoblastGiant

TrophoblastUterine

EpitheliumMetrialGland

UterineStroma

Nontreated mice (control)Nonresorbed 11 1 1 2 1

CP-treated miceNonresorbed 11/111 11 11/111 2 1Resorbing 2 2 6 11 111

Immunized CP-treated miceNonresorbed 11 1 1 2 1

CBA/J 3 DBA/2JNonresorbed 1 6 1/11 1 11Resorbing 11 11 11/111 11 111

a In situ hybridization signals were scored by two independent readers as follows:2, none detectable;6 , detectable butpatchy;1, weak signal;11, moderate signal;111, high intensity signal.

b Four to five uteroplacental units were tested in each group, and four to five tissue sections per unit were analyzed by readersin each experiment.

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The hybridization signal was more intense in both uteri andplacentae of females with CP-induced pregnancy loss than in con-trols (Fig. 4,a andb, and Table II).

In the CBA/2 3 DBA/2J mouse combination, an elevation ofTNFRI mRNA expression was observed in giant trophoblast cellsof the resorbing placentae compared with that in the nonresorbedplacenta (Table II and Fig. 4,c andd).

TNF-a protein expression

Results of Western blot analysis of homogenates from placentaeof control and CP-treated mice are presented in Figure 5. Prob-ing the blots with TNF-a antiserum revealed multiple immu-noreactive proteins with molecular masses of 18, 19, 26, 30,32, 36, and 38 kDa. These proteins were not detected after

FIGURE 3. Distribution of TNF-a mRNA in placentae and uteri of control and CP- treated ICR mice (day 15 of pregnancy).a andb, Low magnificationof the uteroplacental unit of control (a) and CP-treated (b) mice hybridized with TNF-a-specific probes (e, epithelium; m, myometrium; mg, metrial gland;d, decidua; tr, trophoblast; magnification,315).c andd, Expression of TNF-a mRNA in the uterine epithelium (arrowheads) of control (c) and CP-treated(d) mice (3100).eandf, Expression of TNF-a mRNA in giant (arrowheads) and spongiotrophoblast (sp) cells in the placenta of control (e) and CP-treated( f) mice (magnification,3100).g, Hybridization of placental tissue from control mice with a nonhomologic probe (magnification,3100).h, Leukocytescontaining TNF-a mRNA (arrowheads) in placental blood lacunae of control mice (magnification,3280).

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incubation of the blots with nonimmune rabbit serum (data notshown).

Analysis of TNF-a-immunoreactive proteins showed that the18- and 26-kDa forms were expressed at a low level in controlplacentae, while their expression in nonresorbed placentae ofanimals with pregnancy loss was increased (Fig. 5). The im-munoreactive forms corresponding to 30- and 32-kDa TNF-awere highly expressed in resorbing placentae, while their ex-pression in control placentae was weak (Fig. 5). Also, the 18-and 19-kDa immunospecific proteins were not detected inresorbing placentae (Fig. 5).

Immunolocalization of the TNF-a protein

In tissue sections of placentae and uteri of control mice, TNF-a-positive leukocytes were identified in placental lacunae located

between decidua and trophoblast (Fig. 6b). A weak positive stain-ing was also detected in placental giant cells, and the intensity ofstaining was not changed following CP treatment (data not shown).

The cellular patterns of TNF-a protein expression in the utero-placental units of control mice (Fig. 6b) and CP-treated mice (Fig.6c) were similar. Also, the distribution of TNF-a-positive cells inthe uteroplacental unit of the CBA/J3 DBA/2J mouse combina-tion was basically the same as that in CP-treated mice (data notshown). No staining was observed in the tissue sections incubatedwith nonimmune rabbit serum (Fig. 6a).

Effect of immunization on TNF-a expression at the fetomaternalinterface

Since our previous works (39, 43) demonstrated that.80% ofnonresorbed day 15 placentae in CP-treated mice are destined to be

Table II. Tissue distribution of TNFRI mRNA in the uteroplacental unita

Groups of Animalsand Types of TestedUteroplacental Unitsb Spongiotrophoblast

GiantTrophoblast

UterineEpithelium

MetrialGlands

Non-treated mice (control)Nonresorbed 11 11 11 2

CP-treated miceNonresorbed 111 111 111 1Resorbing 111 111 11/111 1

Immunized CP-treated miceNonresorbed 11/111 11/111 11/111 2

CBA/J 3 DBA/2JNonresorbed 11 1 11 2Resorbing 11 11/111 11 2

a Signals were scored as described in Table I.b Four to five uteroplacental units were tested in each group, and four to five tissue sections per unit were analyzed by readers

in each experiment.

FIGURE 4. Expression of TNFRI mRNA in control placenta (a) and nonresorbed placenta of CP-treated ICR mice (day 15 of pregnancy;b) and innonresorbed (c) and resorbing (d) placenta of the CBA/J3 DBA/2J mouse combination (day 12 of pregnancy). sp, spongiotrophoblast cells. Arrowheadsindicate giant trophoblast cells (magnification,3100).

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resorbed by the end of pregnancy, we used still nonresorbed 15-day-old placentae from immunized and nonimmunized micetreated with CP to evaluate the effect of immunization on TNF-aexpression in the uteroplacental unit.

In immunized CP-treated mice, in situ hybridization analysisrevealed a decreased intensity of the hybridization signal in theuterine epithelium and trophoblast cells (Table I).

Results of RNase protection analysis also showed a clear de-crease in placental TNF-a mRNA expression following immuni-zation. Thus, the expression of the 283-bp fragment was lower inplacentae of immunized CP-treated (Fig. 1,lane 7) than in those ofnonimmunized CP-treated mice (Fig. 1,lane 3).

Finally, immunization resulted in a clear decrease in TNF-aprotein expression in the uteroplacental unit of CP-treated mice(Fig. 5). The proportion of leukocytes expressing the TNF-a pro-tein in placentae of mice with pregnancy loss was also decreasedfollowing immunization (Fig. 6,c andd).

No major differences were found in TNFRI mRNA expressionin placentae of CP-treated mice following immunization, exceptfor metrial gland cells, which showed a loss of TNFRI mRNAtranscripts (Table II).

DiscussionIt was shown earlier that the pattern of TNF-a expression in com-bined nonresorbed and resorbing placentae obtained from miceexhibiting a high rate of resorptions differs significantly from thatobserved in nonresorption-prone mice (35). It might be supposed,however, that resorbing and nonresorbed placentae have differentpatterns of TNF-a expression. Therefore, in the present work,TNF-a expression was tested separately in nonresorbed andresorbing placentae.

The quantitative analysis of mRNA expression revealed that inmice with CP-induced pregnancy loss, TNF-a mRNA expressionwas higher in nonresorbed placentae compared with that in controluntreated animals. Also, in placentae of mice with CP-inducedpregnancy loss, three forms of TNF-a mRNA corresponding to theprotected fragments of 363, 320, and 283 bp were revealed, whilein placentae of control mice only the 283- and 320-bp TNF-a

mRNA forms were detected. The physiologic role of the proteinsencoded by these transcripts remains to be elucidated. It cannot beexcluded that the 363-bp TNF-a mRNA variant detected in des-tined to be resorbed placentae may encode a TNF-a form contrib-uting to signals mediating cell death.

In parallel, a substantial increase in the level of TNF-a mRNAexpression in resorbing placentae of CBA females mated toDBA/2 males compared with that in nonresorbed placentae ofthese females was observed. However, unlike ICR mice, in pla-centae of the CBA/J3 DBA/2J mouse combination we detected

FIGURE 5. Western blot analysis of TNF-a in placentae of micetreated with CP. Proteins were probed with TNF-a-specific Abs.Lane 1,placentae of control mice;lane 2, nonresorbed placentae of CP-treatedmice;lanes 3and4, placentae of immunized only or immunized CP-treatedmice, respectively;lane 5, resorbing placentae of CP-treated mice.

FIGURE 6. TNF-a-positive leukocytes in placental lacunae of CP-treated ICR mice (day 15 of pregnancy).a, Placental tissue sections incubated withnonimmune rabbit antiserum.b throughd, TNF-a-positive leukocytes (arrowheads) in control (b), CP-treated (c), and immunized CP-treated (d) mice.Magnification,3220.

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only a 220-bp TNF-a mRNA variant. This difference may be at-tributed to the TNF-a gene polymorphism demonstrated in differ-ent mouse strains (46). It has been shown that different sensitivitiesof various mouse strains to infections and some physical factorsmay be associated with TNF-a gene polymorphism (47–49).Whether the differences in TNF-a transcripts found in placentae ofICR and CBA mice have some functional significance remains tobe elucidated.

An increased expression of TNF-a in placentae of mice with ahigh rate of pregnancy loss was observed not only at the mRNAbut also at the protein level. Western blot analysis of proteins fromplacentae of mice with induced abortions revealed, besides theearlier described 18-kDa secreted and the 26-kDa membrane forms(50), multiple variants of 26, 30, 32, 36, and 38 kDa of the TNF-aprotein. It is possible that the 36- and 38-kDa species are dimers ofthe 18- and 19-kDa TNF-a forms, respectively. The 30- and 32-kDa immunoreactive forms of TNF-a, were highly expressed inthe resorbing placenta of mice with pregnancy loss. Such a findingmay suggest that TNF-a gene expression may be differentiallyregulated at the post-transcriptional and/or post-translational levelat different stages of the placental death process. Further studiesare needed for understanding the biologic functions of theseTNF-a forms.

The uterine epithelium and stroma as well as placental giant andspongiotrophoblast cells were found to express TNF-a mRNA.This cellular pattern of TNF-a mRNA expression is practicallyidentical with that observed in the pioneer works of Hunt et al.performed in nonresorption-prone Swiss and C57BL/6 mice (18,15). Additionally, as expected, tissue areas in resorbing placentaeundergoing necrosis were found to be infiltrated with numerousleukocytes containing the TNF-a mRNA transcripts.

Earlier studies in the CBA/J3 DBA/2J mouse combination inwhich combined nonresorbed and resorbing placentae were testedraised the question of whether the elevation in placental TNF-aexpression is an upstream event or, the opposite, a consequence ofplacental death (35). The results of the present study may clarifythis point.

Indeed, our studies revealed an increased expression of TNF-anot only in resorbing placentae of the CBA/J3 DBA/2J mousecombination, but also in the nonresorbed placenta of mice withCP-induced pregnancy loss. In this model, the level of resorptionreaches;30% up to day 15 of pregnancy and exceeds 80% by theend of pregnancy. This fact allows us to suppose that most ofnonresorbed uteroplacental units tested on day 15 of pregnancy inthis model are destined to be resorbed by the end of pregnancy.Thus, the elevation in TNF-a expression demonstrated in non-resorbed placentae of CP-treated mice is an event that precedesplacental death.

The involvement of TNF-a in mechanisms underlying preg-nancy loss was additionally confirmed by the analysis of its ex-pression in mice with reproductive failure after immunization. Itwas reported earlier that maternal alloimmunization with BALB/clymphocytes significantly decreased the level of pregnancy loss inthe CBA/J3 DBA/2J mouse combination (37). It was also dem-onstrated that nonspecific maternal immunization with CFA mayimprove the reproductive performance of CBA/J females mated toDBA/2J males (42). Finally, the level of CP-induced pregnancyloss in CBA/J3 C57BL/6 or ICR 3 ICR mouse models wasshown to be decreased by specific maternal immunization withallogeneic paternal splenocytes or nonspecific immunization withrat splenocytes, respectively (39, 43). In this study we have clearlydemonstrated that the decrease in the rate of induced resorptionscaused by maternal immunization is accompanied by a decline inthe TNF-a mRNA level and by a decrease in the levels of all

immunoreactive forms of the TNF-a protein at the fetomaternalinterface.

Finally, an increased expression of TNFRI mRNA transcriptswas demonstrated in placentae and uteri of mice with pregnancyloss. This finding seems to implicate the existence of a TNF-a-associated signaling pathway leading to placental death. Indeed,since the level of TNFRI mRNA in the placenta was constantthroughout pregnancy (51), it is reasonable to suppose that its in-creased expression may lead to an alteration of TNF-a signaling inthe placenta. One of the cellular responses to TNF-a is suggestedto be associated with apoptotic cell death (6). It has recently beenshown that TNF-a may promote apoptosis in trophoblast cells fol-lowing binding to TNFRI (52). This ligand-receptor interactionwas found to be critical, since cells lacking the TNFRI did notshow a detectable level of apoptosis (25).

In conclusion, the results of the present study clearly demon-strate that up-regulation of TNF-a expression in the embryonicmicroenvironment may contribute to spontaneous and induced pla-cental death. Furthermore, down-regulation of TNF-a expressionby maternal immunization might play an important role in mech-anisms underlying its beneficial effect on reproductiveperformance.

AcknowledgmentsWe are grateful to Dr. J. Zaretsky for critical reading of the article and toProf. D. Wallach for providing us with murine rTNF-a and cDNAs ofmurine TNF-a and TNFRI. We appreciate the help of Dr. Z. Zaslavsky inperforming the densitometric analysis and of Mr. A. Pinchasov for prep-aration of the photomicrographs.

References1. Robertson, S. A., R. F. Seamark, L. J. Guilbert, and T. G. Wegmann. 1994. The

role of cytokines in gestation.Crit. Rev. Immunol. 14:239.2. Vassali, P. 1992. The pathophysiology of tumor necrosis factors.Annu. Rev.

Immunol. 10:411.3. Beutler, B., and A. Cerami. 1989. The biology of cachectin/TNF-a primary me-

diator of the host response.Annu. Rev. Immunol. 7:625.4. Frater-Schroder, M., W. Riasu, R. Hallmann, P. Gautschi, and P. Bohlein. 1987.

Tumor necrosis factor typea, a potent inhibitor of endothelial cell growth invitro, is angiogenic in vivo.Proc. Natl. Acad. Sci. USA 84:5277.

5. Dayer, J. M., B. Beutler, and A. Cerami. 1985. Cachectin/tumor necrosis factor(TNF) stimulates collagenase and PGE2 production by human synovial cells anddermal fibroblasts.J. Exp. Med. 162:2163.

6. Robaye, B., R. Mosselmans, W. Fiers, J. E. Dumont, and P. Galand. 1991. Tumornecrosis factor induces apoptosis (programmed cell death) in normal endothelialcells in vitro.Am. J. Pathol. 138:447.

7. Witsell, A. L., and L. B. Schook. 1992. Tumor necrosis factora is an autocrinegrowth regulator during macrophage differentiation.Proc. Natl. Acad. Sci. USA89:4754.

8. Terranova, P. F., V. J. Hunter, K. F. Roby, and J. S. Hunt. 1995. Tumor necrosisfactor-a in the female reproductive tract.Proc. Soc. Exp. Biol. Med. 209:325.

9. Hunt, J. S., and K. F. Roby. 1995. Tumor necrosis factor-a and immunity in thefemale reproductive tract. In Immunology of Human Reproduction.M. Kurpisz and N. Fernandez, eds. BIOS Scientific Publishers, Oxford, U.K.,p. 251.

10. Yang, Y., K. K. Yelavarthi, H.-L. Chen, J. L. Pace, P. F. Terranova, andJ. S. Hunt. 1993. Molecular, biochemical and functional characteristics of tumornecrosis factor-a produced by human placental cytotrophoblastic cells.J. Immu-nol. 150:5614.

11. Chen, J. L., Y. Yang, X.-L. Xu, K. K. Yelavarthi, J. L. Fishback, and J. S. Hunt.1991. Tumor necrosis factor alpha mRNA and protein are present in humanplacental and uterine cells at early and late stages of gestation.Am. J. Pathol.139:327.

12. Vince, G., S. Shorter, P. Starkey, J. Humphreys, L. Clover, T. Wilkins, I. Sargent,and C. Redman. 1992. Localization of tumor necrosis factor production in cellsat the materno/fetal interface in human pregnancy.Clin. Exp. Immunol. 88:174.

13. Casey, M. L., S. M. Cox, B. Beutler, L. Milewich, and P. C. MacDonald. 1989.Cachectin/tumor necrosis factor-alpha formation in human decidua: potential roleof cytokines in infection-induced preterm labor.J. Clin. Invest. 83:430.

14. Jaattela, M., P. Kuusela, and E. Saksela. 1988. Demonstration of tumor necrosisfactor in human amniotic fluid and supernatants of placental and decidual tissues.Lab. Invest. 58:48.

15. Hunt, J. S., H.-L. Chen, X.-L. Hu, and J. W. Pollard. 1993. Normal distributionof tumor necrosis factor-a messenger ribonucleic acid and protein in virgin andpregnant osteopetrotic (op/op) mice.Biol. Reprod. 49:441.

4287The Journal of Immunology

by guest on January 29, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

16. Yelavarthi, K. K., H.-L. Chen, Y. Yang, J. L. Fishback, B. Courley, Jr., andJ. S. Hunt. 1991. Tumor necrosis factor-a mRNA and protein in rat uterine andplacental cells.J. Immunol. 146:3840.

17. Croy, B. A., L. J. Guilbert, M. A. Browne, N. M. Gough, D. T. Stinchcomb,N. Reed, and T. G. Wegmann. 1991. Characterization of cytokine production bythe metrial gland and granulated metrial gland cells.J. Reprod. Immunol. 19:149.

18. Roby, K. F., and J. S. Hunt. 1994. Mouse endometrial tumor necrosis factor-amRNA and protein localization and regulation by estradiol and progesterone.Endocrinology 135:2780.

19. Hunt, J. S., and S. A. Robertson. 1996. Uterine macrophages and environmentalprogramming for pregnancy success.J. Reprod. Immunol. 32:1.

20. Tabibzadeh, S., Q. F. Kong, and X. Z. Sun. 1993. Regulatory role of TNF-a intransepithelial migration of leukocytes and epithelial dyscohesion.Endocr. J.1:417.

21. Pampfer, S., Y.-D. Wuu, I. Vanderheyden, and R. De Hertogh. 1994. Expressionof tumor necrosis factor-a receptors and selective effect of TNFa on the inner cellmass in mouse blastocysts.Endocrinology 134:206.

22. Tartakovsky, B., and E. Ben-Yair. 1991. Cytokines modulate preimplantationdevelopment and pregnancy.Dev. Biol. 146:345.

23. Hunt, J. S., R. A. Atherton, and J. L. Pace. 1990. Differential responses of rattrophoblast cells and embryonic fibroblast cells to cytokines that regulate prolif-eration and class I MHC antigen expression.J. Immunol. 145:184.

24. Hunt, J. S. 1996. Functional aspects of the tumor necrosis factor gene family inpregnancy. InProceedings of the 13th Rochester Trophoblast Conference, Banff,Canada.Banff Conferences Publishing, Banff, Alberta, Canada, abstr. 38.

25. Vandenabeele, P., W. Declercq, R. Beyaert, and W. Fiers. 1995. Two tumornecrosis factor receptors: structure and function.Trends Cell Biol. 5:392.

26. Roby, K. F., N. Laham, H. Kroning, P. F. Terranova, and J. S. Hunt. 1995.Expression and localization of messenger RNA for tumor necrosis factor receptor(TNF-R) I and TNF-RII in pregnant mouse uterus and placenta.Endocrine 3:557.

27. Romero, R., K. H. Manogue, M. D. Mitchell, Y. K. Wu, E. Oyarzun,J. C. Hobbins, and A. Cerami. 1989. Infection and labor. IV. Cachectin-tumornecrosis factor in the amniotic fluid of women with intraamniotic infection andpreterm labor.Am. J. Obstet. Gynecol. 161:336.

28. Chaouat, G., E. Menu, J. Szeerkeres-Bartho, C. Rebut-Bonneton, P. Bustany,R. Kinsky, and D. A. Clark. 1991. Immunological and endocrinological factorsthat contribute to a successful pregnancy. InMolecular and Cellular Immunologyof the Fetal-Maternal Interface.T. G. Wegmann, T. G. Gill, andR. Nisbet-Brown, eds. Oxford University Press, New York, p. 277.

29. Gendron, R. L., F. P. Nestel, W. S. Lapp, and M. G. Baines. 1990. Lipopolysac-charide-induced fetal resorption in mice is associated with the intrauterine pro-duction of tumor necrosis factor-alpha.J. Reprod. Fertil. 90:395.

30. Heyborne, K. D., S. S. Witkin, and J. A. McGregor. 1992. Tumor necrosis fac-tor-a in midtrimester amniotic fluid is associated with impaired intrauterine fetalgrowth.Am. J. Obstet. Gynecol. 167:920.

31. Chaouat, G., E. Menu, D. A. Clark, M. Minkowsky, M. Dy, and T. G. Wegmann.1990. Control of fetal survival in CBA3 DBA/2 mice by lymphokine therapy.J. Reprod. Fertil. 89:447.

32. Arck, P. C., A. B. Troutt, and D. A. Clark. 1997. Soluble receptors neutralizingTNF-a and IL-1 block stress-triggered murine abortion.Am. J. Reprod. Immunol.37:262.

33. Arck, P. C., F. S. Meroli, J. Manuel, G. Chaouat, and D. A. Clark. 1995. Stress-triggered abortion: inhibition of protective suppression and promotion of tumornecrosis factor-a (TNF-a) release as a mechanism triggering resorptions in mice.Am. J. Reprod. Immunol. 33:74.

34. Chaouat, G., E. Menu, J. Szeerkeres-Bartho, C. Rebut-Bonneton, P. Bustany,R. Kinsky, D. A. Clark, and T. G. Wegmann. 1989. Lymphokines, steroids,

placental factors and trophoblast intrinsic resistance to immune cell-mediatedlysis are involved in pregnancy success or immunologically mediated pregnancyfailure. In Molecular Biology of the Feto-Maternal Interface.T. G. Wegmann and T. G. Gill, eds. Oxford University Press, New York, p. 213.

35. Tangri, S., and R. Raghupathy. 1993. Expression of cytokines in placentas ofmice undergoing immunologically mediated spontaneous fetal resorptions.Biol.Reprod. 49:850.

36. Tangri, S., T. G. Wegmann, H. Lin, and R. Raghupathy. 1994. Maternal anti-placental reactivity in natural, immunologically mediated fetal resorptions.J. Im-munol. 152:4903.

37. Chaouat, G., N. Kiger, and T. G. Wegmann. 1983. Vaccination against sponta-neous abortion in mice.J. Reprod. Immunol. 5:389.

38. Toder, V., D. Strassburger, H. Carp, Y. Irlin, S. Lurie, M. Pecht, and N. Trainin.1988. Immunopotentiation and pregnancy loss.J. Reprod. Fertil. 37(Suppl.):1.

39. Torchinsky, A., A. Fein, H. J. A. Carp, and V. Toder. 1994. MHC-associatedimmunopotentiation affects the embryo response to teratogens.Clin. Exp. Immu-nol. 98:513.

40. Torchinsky, A., V. Toder, S. Savion, J. Shepshelovich, H. Orenstein, and A. Fein.1997. Immunostimulation increases the resistance of mouse embryos to the ter-atogenic effect of diabetes mellitus.Diabetologia 40:635.

41. Wegmann, T. G. 1990. The cytokine basis for cross-talk between the maternalimmune and reproductive systems.Curr. Opin. Immunol. 2:765.

42. Toder, V., D. Strassburger, Y. Irlin, H. Carp, M. Pecht, and N. Trainin. 1990.Nonspecific immunopotentiators and pregnancy loss: complete Freund adjuvantreverses high fetal resorption rate in CBA3 DBA/2 mouse combination.Am. J. Reprod. Immunol. 24:63.

43. Toder, V., S. Savion, M. Gorivodsky, J. Shepshelovich, Z. Zaslavsky, A. Fein,and Torchinsky. 1996. Teratogen-induced apoptosis may be affected by immu-nopotentiation.J. Reprod. Immunol. 30:173.

44. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation byacid guanidinum thiocyanate-phenol-chloroform extraction.Anal. Biochem. 162:156.

45. Sasson, D., and N. Rosenthal. 1993. Detection of messenger RNA by in situhybridization.1993.Methods Enzymol. 225:384.

46. Beutler, B., and T. Brown. 1993. Polymorphism of the mouse TNF-a locus:sequence studies of the 39-untranslated region and first intron.Gene 129:279.

47. Freund, Y. R., G. Sgarlato, C. O. Jacob, Y. Suzuki, and J. S. Remington. 1996.Polymorphism in the tumor necrosis factor-a (TNF-a) gene correlates with mu-rine resistance to development of toxoplasmic encephalitis and with levels ofTNF-a mRNA in injected brain tissue.J. Exp. Med. 175:683.

48. Cabrera, M., M. A. Shaw, C. Sharples, H. Williams, M. Castes, J. Convit, andJ. M. Blackwell. 1995. Polymorphism in tumor necrosis factor genes associatedwith mucocutaneous leishmaniasis.J. Exp. Med. 182:1259.

49. Vincek, V., I. Kurimoto, J. P. Medema, E. Prieto, and J. W. Streilein. 1993.Tumor necrosis factor alpha polymorphism correlates with deleterious effects ofultraviolet B light on cutaneous immunity.Cancer Res. 53:728.

50. Cseh, K., and B. Beutler. 1989. Alternative cleavage of the cachectin/tumor ne-crosis factor propeptide results in a larger inactive form of secreted protein.J. Biol. Chem. 264:16256.

51. Roby, K. F., N. Laham, H. Kroning, P. F. Terranova, and J. S. Hunt. 1995.Expression and localization of messenger RNA for tumor necrosis factor receptor(TNF-R) I and TNF-RII in pregnant mouse uterus and placenta.Endocrine 3:557.

52. Yui, J., M. Garcia-Lloret, T. G. Wegmann, and L. J. Guilbert. 1994. Cytotoxicityof tumor necrosis factor-alpha and gamma-interferon against primary human pla-cental trophoblast.Placenta 15:819.

4288 TNF-a EXPRESSION IN UTEROPLACENTAL UNIT OF MICE WITH PREGNANCY LOSS

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