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Messenger RNA and Protein Expression in the Uteroplacental Unit of Mice with Pregnancy Loss1
Department of Embryology and Teratology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in embryonic microenvironment was
found to be associated with postimplantation loss. In this work, we
examined the pattern of TNF- expression at both the mRNA and the
protein level as well as the distribution of TNF- receptor mRNA in
the uteroplacental unit of mice with induced (cyclophosphamide-treated)
or spontaneous (CBA/J x DBA/2J mouse combination) pregnancy loss.
RNase protection analysis demonstrated an increase in TNF- mRNA
expression in the placentae of mice with pregnancy loss compared with
that in control mice. TNF- messages were localized to the uterine
epithelium and stroma as well as the giant and spongiotrophoblast cells
of the placenta. The intensity of the hybridization signal in placentae
of mice with pregnancy loss was substantially higher than that in
control mice. The up-regulation of TNF- mRNA was accompanied by an
increase in the expression of TNF- receptor I mRNA in the same cell
populations. The elevation of TNF- production was also demonstrated
at the protein level. Western blot analysis showed an increased level
of the 18- and 26-kDa TNF- protein species in the uteroplacental
unit of mice with pregnancy loss. Immunostaining revealed
TNF--positive leukocytes located in the uterus and placenta.
Finally, we found that immunization of mice with
cyclophosphamide-induced pregnancy loss while decreasing the resorption
rate in these females resulted in a decline in TNF- expression at
the fetomaternal interface. These data clearly suggest an involvement
of TNF- in pathways leading to both spontaneous and induced
placental death. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a multifunctional cytokine that plays a
prominent role in immune and host defense responses, stimulates
angiogenesis, influences tissue remodeling, promotes apoptosis, and
takes part in the regulation of cell proliferation and differentiation
(2, 3, 4, 5, 6, 7). It has also been reported to be instrumental in the regulation
of reproductive processes (8, 9). TNF- was demonstrated to be
produced by both uterine and placental cells. In humans, TNF- mRNA
and protein have been identified in syncytio- and extravillous
cytotrophoblast (10, 11, 12), and biologically active TNF- was found in
the supernatants of placental and decidual tissue (13, 14). In rodents,
TNF- expression was demonstrated in the uterine epithelium, decidua,
and trophoblast (15, 16). Also, TNF- mRNA transcripts have been
identified in murine macrophage-like cells residing in the endometrial
stroma and in NK-like cells populating the decidua and metrial gland
(15, 16, 17). The expression of TNF- is tightly regulated during mouse
gestation (18), reaching its maximum at midgestation and then remaining
stable until the end of pregnancy (15).
The role of uterine and placental TNF- in pregnancy is poorly
understood. It has been suggested that TNF- may regulate the
migration and behavior of uterine leukocytes (19, 20) and affect the
myometrial contractions during labor (13). Furthermore, maternal
TNF- might influence blastocyst growth and implantation (21, 22) due
to regulation of trophoblast growth and differentiation in early
embryos (23). Recent studies on knockout mice have demonstrated that
TNF- is required for normal placental growth and function (24).
TNF- binds to one of two distinct cellular receptors, TNF-
receptor I (TNFRI)3
(p55) and TNFRII (p75) (25), thereby initiating different cellular
responses. Transcripts of both receptors have been found in the uterus
and placenta of pregnant mice (26).
Abnormal TNF- production may be associated with pregnancy failure.
The TNF- level was shown to be significantly elevated in the
amniotic fluid of women with uterine infections, and its increased
production correlates with the incidence of preterm labor (27).
Administration of LPS (an inducer of TNF- production) or TNF-
itself to pregnant mice results in pregnancy loss (28, 29) or embryo
growth retardation (30), whereas treatment with anti-TNF- Abs or
soluble receptors reduces the number of resorptions in mice with a high
rate of immune-mediated pregnancy loss (31, 32). Furthermore,
enhancement of decidual TNF- production has been suggested to be one
of the mechanisms involved in stress-triggered abortions in mice (33).
Also, an increased TNF- level has been demonstrated in supernatants
from decidual cell cultures from the resorption-prone CBA/J x
DBA/2J mouse compared with that in the nonresorption-prone CBA/J
x BALB/c mouse combination (34). In parallel, an elevation in TNF-
was registered at the mRNA level in placentae of CBA/J x DBA/2J
mice (35). Cytokine analysis of supernatants from mixed
lymphocyte-placental cell cultures has shown a significantly higher
production of TNF- in supernatants from CBA/J x DBA/2J mice
compared with those from CBA/J x BALB/c mice (36).
The correlation between an elevated level of TNF- and pregnancy
failure raises the possibility that normalization of TNF- expression
at the fetomaternal interface may be associated with improved
reproductive performance in females with pregnancy loss. It has been
widely reported that alloimmunization or nonspecific immune stimulation
may protect the fetus and improve reproductive outcome (37, 38). We
have demonstrated that such immunization may prevent the embryonic
dismorphogenesis induced by extrinsic and intrinsic factors (39, 40).
The protective effect of immunization is generally thought to be due to
modification of the cytokine milieu in embryonic microenvironment
(41).
In this report we present data characterizing the pattern of TNF-
and TNFRI expression at the fetomaternal interface of mice with
spontaneous and induced pregnancy loss and possible changes in this
pattern induced by maternal immunization to determine whether the
protective effect of immunization in females with pregnancy loss is
associated with modulation of TNF- expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to eight-week-old ICR and C57BL/6 mice and Long Evans rats were obtained from the Tel Aviv University breading colonies. CBA/J females and DBA/2J males were obtained from The Jackson Laboratory (Bar Harbor, ME). The animals were maintained on a 14-h light/10-h dark cycle with food and water ad libitum. To obtain pregnancies, females were caged with males overnight, and the presence of a vaginal plug was designated day 1 of pregnancy.
Animal models of pregnancy loss
Two mouse models of induced and spontaneous pregnancy loss were used in this study.
The CBA/J x DBA/2J mouse combination, which is well known for its high level of postimplantation loss, was used as a model of spontaneous abortions (31). Cyclophosphamide (CP)-treated ICR x ICR and CBA/J x C57BL/6 mouse combinations were used as models of induced pregnancy 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 at the time of treatment (42). CBA/J females mated to DBA/2J males were sacrificed on day 12 of gestation, while CP-treated mice were sacrificed on day 15 or 19 of pregnancy. The numbers of implantation sites, resorptions, and live fetuses were recorded, and the incidence of postimplantation loss was 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 before mating as described previously (39, 43). Briefly, spleens were aseptically removed 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 nembutal anesthesia (40 mg/kg) the uterus was identified and injected with 25 to 30 x 106 splenocytes/0.04 ml saline/horn. Mice injected with saline or syngeneic splenocytes served as controls.
Tissue processing
Placentae together with the adjacent uteri were collected from mice with spontaneous resorptions on day 12 and from CP-treated mice on day 15 of pregnancy. The term resorbing placenta refers to a placenta with a pale or visibly destroyed embryo and remnants of extraembryonic tissues but which can still be identified macroscopically as a placenta. The term nonresorbed placenta refers to a macroscopically normal placenta with a live embryo.
For RNase protection and Western blot analysis, placentae and uteri were immediately snap-frozen in liquid nitrogen and stored at -70°C until use. For in situ hybridization or immunohistochemistry techniques, placentae were fixed in 4% paraformaldehyde or in Bouin’s solution, respectively, and embedded in paraffin, and 7-µm sections were further used after histologic examination. Only resorbing placentae containing morphologically unaffected regions were chosen for further analysis.
Probe construction
The 709-bp TNF- and 640-bp TNFRI cDNAs (provided by Prof. D.
Wallach, Weizmann Institute of Science, Rehovot, Israel) were subcloned
into the EcoRI-SacI and
EcoRI-SphI sites of the pBluescript
SK+ vector (Stratagene, La Jolla, CA), respectively. After
linearization with SacI for TNF- and with SphI
for TNFRI cDNA, the DNA template served for generation of
digoxigenin-11-UTP-labeled (Boehringer Mannheim, Mannheim, Germany)
antisense RNA probes using T7 RNA polymerase (Stratagene). RNA probes
for ß-actin (360 bp) and the prokaryotic neo gene (760 bp)
were synthesized as described above. The lengths of the generated RNA
probes were evaluated by comparing their sizes with that of the
digoxigenin-labeled DNA m.w. marker VIII (Boehringer Mannheim) in
denatured 5% polyacrylamide gel.
RNase protection analysis
Total RNA was extracted from placentae and uteri by the method of Chomzynski and Sacchi (44) using the Tri-Reagent (Molecular Research Center, Cincinnati, OH). The RNA concentration was calculated by spectrophotometry at 260 and 280 nm, and the integrity of the RNA was monitored by electrophoresis in 1% agarose/2.2 M formaldehyde gel. The following procedures are those described in the protocol of the RNase Protection Assay System (Promega, Madison, WI). Briefly, 30 to 50 µg of total RNA were coprecipitated with 30 ng of antisense RNA probe, incubated overnight in 20 µl of hybridization buffer (80% formamide, 1 mM EDTA, 0.2 M sodium acetate, and 40 mM PIPES, pH 6.4) at 45°C, and then digested with 16 U of RNase ONE (Promega) for 1 h at room temperature. Following RNase inactivation, RNA was precipitated and resuspended in gel loading buffer, and protected fragments were resolved by electrophoresis in denatured 5% polyacrylamide/8 M urea gel. The m.w. of specific 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 6x SSC (150 mM sodium chloride and 15 mM sodium citrate, pH 7.0) and exposed to UV light for cross-linking of RNA to the filters. Hybridization bands were visualized by incubating the blots in alkaline phosphatase-conjugated antidigoxigenin Abs at 1/10,000 dilution (Boehringer Mannheim) and the chemiluminescent substrate CSPD (Tropix, Bedford, MA) followed by exposure to x-ray film.
As a negative control, tissue RNA was substituted by yeast tRNA. Equivalency of RNA loading on the gel was controlled by hybridization of the same quantity of tissue RNA with a ß-actin riboprobe. The quantitative character of the RNase protection assay was confirmed by titration of tissue RNA with the labeled riboprobe and generation of a titration curve (data not shown).
Densitometric analysis of films was performed using B.I.S. 202D image densitometric system (Bio-Rad, Richmond, CA), and results were analyzed by 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 in 2x SSC, treated with 10 µg/ml proteinase K (IBI, New Haven, CT) for 15 min at 37°C, and fixed in 4% ice-cold paraformaldehyde. Prehybridization was performed for 1 h at 45°C in 50% formamide, 6x SSPE (150 mM sodium chloride, 10 mM sodium phosphate, and 1 mM EDTA, pH 7.4), 5x Denhardt’s solution (Sigma, Rehovot, Israel) and 0.5% SDS. The sections were overlaid with 30 µl of hybridization mixture (50% formamide, 5x Denhardt’s solution, 10% dextran sulfate, 6x SSPE, and 0.5% SDS) containing 0.5 ng/µl digoxigenin-labeled antisense RNA probe. Hybridization was conducted overnight at 45°C in a humidified chamber. The slides were washed twice for 15 min in 2x SSC, followed by incubation with 20 µg/ml RNase A (Sigma) for 30 min at 37°C. High stringency washes were performed by incubating the slides twice for 15 min at 50°C in 0.1x SSC followed by a 10-min wash in 0.1x SSC at room temperature. The hybridization signal was detected by alkaline phosphatase-conjugated antidigoxigenin Abs followed by incubation in nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate color substrate solution (Boehringer Mannheim) containing 1 mM levamisole according to the manufacturer’s recommendations. Finally, sections were lightly counterstained with neutral red, and a positive signal was indicated by a deep purple-brown staining.
As a control for hybridization, a nonhomologic RNA probe synthesized from a prokaryotic neo cDNA was substituted for the specific probes. Tissue sections pretreated with 100 µg/ml RNase A (Sigma) for 30 min at 37°C before hybridization served as an additional control.
Western blot analysis
Placentae and uteri were homogenized in ice-cold buffer containing 100 mM Tris-HCl (pH 7.4), 200 mM sodium chloride, 2 mM EDTA, 1 mM PMSF, and 2 µg/ml aprotinin. An equal volume of lysing solution (1% desoxycholate, 0.04% Nonidet P-40, and 0.2% SDS) was added then to each sample, and the resulting homogenates were centrifuged for 10 min at 4°C at 10,000 x g, aliquoted, and stored at -70°C until use.
Protein concentration was determined by the Bio-Rad protein assay
method (Bio-Rad). Samples containing 50 µg of protein were resolved
by electrophoresis in a 12% SDS-polyacrylamide gel. Prestained m.w.
standards (Novex, Rockford, IL) and murine rTNF- (provided by Prof.
D. Wallach, Weizmann Institute of Science) were used as markers.
Proteins were transferred to nitrocellulose membranes (Schleicher and
Schuell), and nonspecific 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 mM sodium chloride, and 0.1% SDS (TBST) for
2.5 h at room temperature. Filters were incubated in polyclonal
TNF- rabbit antiserum (Endogen, Cambridge, MA) at 15 µg/ml TBST
for 30 min at 37°C. Nonimmune rabbit serum, used at the same dilution
as the primary Ab, served as a negative control. After intensive
washing in TBST, the membranes were incubated for 1 h at room
temperature with biotinylated goat anti-rabbit IgG (Jackson
ImmunoResearch Laboratories, West Grove, PA) at 10 ng/ml, washed again,
and incubated with streptavidin-conjugated horseradish peroxidase
(Zymed, San Francisco, CA) at 0,5 µg/ml for 45 min at room
temperature. After another wash, the membranes were incubated with ECL
reagents (Amersham Life Sciences, Arlington Heights, IL) and exposed to
x-ray film.
Immunohistochemistry
Tissue sections were deparaffinized, washed briefly in PBS (pH
7.4), and treated with 1.5 mg/ml hyaluronidase (Sigma) in PBS, pH 6.5,
for 1 h at 37°C. Ag retrieval was performed by heating the
tissue sections in PBS, pH 7.4, for 30 min at 80°C. Endogenous
peroxidase activity was inhibited by incubating the sections in 3%
hydrogen peroxide. Nonspecific binding sites were blocked by a 20%
solution of FCS in PBS/0.05% Tween (PBST) for 30 min at 37°C.
Sections were stained with rabbit anti-mouse TNF--specific Abs
diluted to 1/70 in 10% FCS/PBST. Nonimmune rabbit serum used 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 temperature
with biotinylated goat anti-rabbit IgG, diluted to 1/1000, followed
by incubation in streptavidin-conjugated horseradish peroxidase/PBST at
12 µg/ml. Anti-TNF- Ab-stained cells were visualized by incubating
the sections with 0.2 mg/ml diaminobenzidine (Sigma) followed by
counterstaining with 0.1% hematoxylin.
Statistical analysis
Each tested sample of total RNA and protein was obtained by
combining four or five placentae in a tested litter. To evaluate the
results of RNase protection and Western blot analyses statistically,
four or five samples obtained from different litters were analyzed and
compared by Student’s t test. The two-tailed level of
significance of differences was = 0.05. The reproducibility of
RNase protection and Western blot analysis was tested in two
experiments using the same samples.
For in situ hybridization analysis and immunostaining, four or five resorbing and/or nonresorbed uteroplacental units collected from four mice were analyzed for each experimental group. To test reproducibility, in situ hybridization and immunostaining experiments were repeated three times. In each experiment, four or five tissue sections of each uteroplacental unit were processed and analyzed by two independent readers. Results characterizing signal intensity were averaged.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To estimate the level of postimplantation loss, 15 litters were tested in each animal model.
The rate of resorptions in the CBA/J x DBA/2J mouse combination evaluated on day 12 of pregnancy was 30.4%. The level of resorptions in CP-treated ICR mice reached 32. 3% by day 15 of pregnancy and increased dramatically to approximately 80% on day 19 of pregnancy. The level of resorptions in CBA/J females mated to C57BL/6 males and treated with CP was practically identical with that in CP-treated ICR mice.
TNF- mRNA expression
TNF- mRNA expression was evaluated using RNase protection
analysis. In placentae of control mice, two species of mRNA
corresponding to 320 and 283 bp were detected (Fig. 1
). The densitometric analysis revealed
that in nonresorbed placentae of mice with induced pregnancy loss, the
expression of both fragments was 2.4-fold higher than that in placentae
of control mice, while in resorbing placentae of these animals this
increase was less prominent. A third fragment, corresponding to 363 bp,
was detected only in the placentae of CP-treated mice, whether
resorbing or nonresorbed, but not in control mice.
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). Densitometric analysis revealed a
20% increase in the level of TNF- mRNA expression in the resorbing
vs the nonresorbed placenta.
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mRNA
Data from in situ hybridization analysis characterizing the
cellular localization and intensity of the hybridization signal are
summarized in Table I and Figure 3.
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mRNA in placentae and
uteri were similar in control and CP-treated mice. In the uterus,
TNF- mRNA expression was demonstrated in cells of luminal epithelium
and stroma (Fig. 3
, c and d, and Table I). In
the placenta, giant and spongiotrophoblast were the dominant cell
populations containing specific messages (Fig. 3, e and
f), while labyrinthine trophoblast cells were
negative (data not shown). Also, leukocytes containing TNF- mRNA
were detected in placental blood lacunae (Fig. 3h).
In the resorbing placenta of CP-treated mice, trophoblast cells
demonstrated a loss of TNF- transcripts, in contrast to metrial
gland cells and uterine stroma, which were positive (Table I).
The intensity of the hybridization signal was elevated in the uterine
epithelium as well as in trophoblast cells of nonresorbed placentae of
CP-treated compared with control mice (Table I and Fig. 3, a–f). The resorbing placentae (vs nonresorbed
placentae) showed a clear induction of TNF- mRNA expression in
metrial gland cells and an enhanced hybridization signal in uterine
stroma. In parallel, the signal was weaker in the uterine epithelium of
these placentae (Table I).
The cellular pattern of TNF- mRNA expression in the
uteroplacental unit of the CBA/J x DBA/2J mouse combination was
basically similar to that in CP-treated mice, except for metrial gland
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 placentae (Table I). As expected, numerous leukocytes containing TNF- mRNA were found
to infiltrate the tissue areas of the resorbing placenta undergoing
necrosis (data not shown).
Hybridization with nonhomologic prokaryotic RNA probe (Fig. 3g) as well as hybridization of tissue sections
pretreated with RNase before hybridization with specific riboprobes
(data not shown) demonstrated no signal.
Localization of TNFRI mRNA
In control mice, the expression of TNFRI mRNA was observed
basically in the same cells that expressed TNF- mRNA (Tables I
and II).
In mice with spontaneous and induced abortions, TNFRI mRNA transcripts
were found in the uterine epithelium (Table II) as well as in giant and
spongiotrophoblast cells (Fig. 4 and
Table II). A weak signal was also detected in metrial gland cells of
CP-treated mice compared with that in control mice (Table II).
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, a and b, and Table II).
In the CBA/2 x DBA/2J mouse combination, an elevation of TNFRI
mRNA expression was observed in giant trophoblast cells of the
resorbing placentae compared with that in the nonresorbed placenta
(Table II and Fig. 4, c and d).
TNF- protein expression
Results of Western blot analysis of homogenates from
placentae of control and CP-treated mice are presented in Figure 5. Probing the blots with TNF-
antiserum revealed multiple immunoreactive proteins with molecular
masses of 18, 19, 26, 30, 32, 36, and 38 kDa. These proteins
were not detected after incubation of the blots with
nonimmune rabbit serum (data not shown).
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-immunoreactive proteins showed that the 18-
and 26-kDa forms were expressed at a low level in control placentae,
while their expression in nonresorbed placentae of animals
with pregnancy loss was increased (Fig. 5). The immunoreactive forms
corresponding to 30- and 32-kDa TNF- were highly expressed in
resorbing placentae, while their expression in control placentae was
weak (Fig. 5). Also, the 18- and 19-kDa immunospecific proteins were
not detected in resorbing placentae (Fig. 5).
Immunolocalization of the TNF- protein
In tissue sections of placentae and uteri of control mice,
TNF--positive leukocytes were identified in placental lacunae
located between decidua and trophoblast (Fig. 6b). A weak positive
staining was also detected in placental giant cells, and the intensity
of staining was not changed following CP treatment (data not
shown).
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protein expression in the
uteroplacental units of control mice (Fig. 6b) and
CP-treated mice (Fig. 6c) were similar. Also, the
distribution of TNF--positive cells in the uteroplacental unit of
the CBA/J x DBA/2J mouse combination was basically the same as
that in CP-treated mice (data not shown). No staining was observed in
the tissue sections incubated with nonimmune rabbit serum (Fig. 6a).
Effect of immunization on TNF- expression at the fetomaternal
interface
Since our previous works (39, 43) demonstrated that >80% of
nonresorbed day 15 placentae in CP-treated mice are destined to be
resorbed by the end of pregnancy, we used still nonresorbed 15-day-old
placentae from immunized and nonimmunized mice treated with CP to
evaluate the effect of immunization on TNF- expression in the
uteroplacental unit.
In immunized CP-treated mice, in situ hybridization analysis revealed a
decreased intensity of the hybridization signal in the uterine
epithelium and trophoblast cells (Table I).
Results of RNase protection analysis also showed a clear decrease in
placental TNF- mRNA expression following immunization. Thus, the
expression of the 283-bp fragment was lower in placentae of immunized
CP-treated (Fig. 1, lane 7) than in those of
nonimmunized CP-treated mice (Fig. 1, lane 3).
Finally, immunization resulted in a clear decrease in TNF- protein
expression in the uteroplacental unit of CP-treated mice (Fig. 5). The
proportion of leukocytes expressing the TNF- protein in placentae of
mice with pregnancy loss was also decreased following immunization
(Fig. 6, c and d).
No major differences were found in TNFRI mRNA expression in placentae
of CP-treated mice following immunization, except for metrial gland
cells, which showed a loss of TNFRI mRNA transcripts (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression in
combined nonresorbed and resorbing placentae obtained from mice
exhibiting a high rate of resorptions differs significantly from that
observed in nonresorption-prone mice (35). It might be supposed,
however, that resorbing and nonresorbed placentae have different
patterns of TNF- expression. Therefore, in the present work, TNF-
expression was tested separately in nonresorbed and resorbing
placentae.
The quantitative analysis of mRNA expression revealed that in mice with
CP-induced pregnancy loss, TNF- mRNA expression was higher in
nonresorbed placentae compared with that in control untreated animals.
Also, in placentae of mice with CP-induced pregnancy loss, three forms
of TNF- mRNA corresponding to the protected fragments of 363, 320,
and 283 bp were revealed, while in placentae of control mice only the
283- and 320-bp TNF- mRNA forms were detected. The physiologic role
of the proteins encoded by these transcripts remains to be elucidated.
It cannot be excluded that the 363-bp TNF- mRNA variant detected in
destined to be resorbed placentae may encode a TNF- form
contributing to signals mediating cell death.
In parallel, a substantial increase in the level of TNF- mRNA
expression in resorbing placentae of CBA females mated to DBA/2 males
compared with that in nonresorbed placentae of these females was
observed. However, unlike ICR mice, in placentae of the CBA/J x
DBA/2J mouse combination we detected only a 220-bp TNF- mRNA
variant. This difference may be attributed to the TNF- gene
polymorphism demonstrated in different mouse strains (46). It has been
shown that different sensitivities of various mouse strains to
infections and some physical factors may be associated with TNF-
gene polymorphism (47, 48, 49). Whether the differences in TNF-
transcripts found in placentae of ICR and CBA mice have some functional
significance remains to be elucidated.
An increased expression of TNF- in placentae of mice with a high
rate of pregnancy loss was observed not only at the mRNA but also at
the protein level. Western blot analysis of proteins from placentae of
mice with induced abortions revealed, besides the earlier described
18-kDa secreted and the 26-kDa membrane forms (50), multiple variants
of 26, 30, 32, 36, and 38 kDa of the TNF- protein. It is possible
that the 36- and 38-kDa species are dimers of the 18- and 19-kDa
TNF- forms, respectively. The 30- and 32-kDa immunoreactive forms of
TNF-, were highly expressed in the resorbing placenta of mice with
pregnancy loss. Such a finding may suggest that TNF- gene expression
may be differentially regulated at the post-transcriptional and/or
post-translational level at different stages of the placental death
process. Further studies are needed for understanding the biologic
functions of these TNF- forms.
The uterine epithelium and stroma as well as placental giant and
spongiotrophoblast cells were found to express TNF- mRNA. This
cellular pattern of TNF- mRNA expression is practically identical
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 placentae undergoing necrosis were
found to be infiltrated with numerous leukocytes containing the TNF-
mRNA transcripts.
Earlier studies in the CBA/J x DBA/2J mouse combination in which
combined nonresorbed and resorbing placentae were tested raised the
question of whether the elevation in placental TNF- expression is an
upstream event or, the opposite, a consequence of placental death (35).
The results of the present study may clarify this point.
Indeed, our studies revealed an increased expression of TNF- not
only in resorbing placentae of the CBA/J x DBA/2J mouse
combination, but also in the nonresorbed placenta of mice with
CP-induced pregnancy loss. In this model, the level of resorption
reaches 
30% up to day 15 of pregnancy and exceeds 80% by the end
of pregnancy. This fact allows us to suppose that most of nonresorbed
uteroplacental units tested on day 15 of pregnancy in this model are
destined to be resorbed by the end of pregnancy. Thus, the elevation in
TNF- expression demonstrated in nonresorbed placentae of CP-treated
mice is an event that precedes placental death.
The involvement of TNF- in mechanisms underlying pregnancy loss was
additionally confirmed by the analysis of its expression in mice with
reproductive failure after immunization. It was reported earlier that
maternal alloimmunization with BALB/c lymphocytes significantly
decreased the level of pregnancy loss in the CBA/J x DBA/2J mouse
combination (37). It was also demonstrated that nonspecific maternal
immunization with CFA may improve the reproductive performance of CBA/J
females mated to DBA/2J males (42). Finally, the level of CP-induced
pregnancy loss in CBA/J x C57BL/6 or ICR x ICR mouse models
was shown to be decreased by specific maternal immunization with
allogeneic paternal splenocytes or nonspecific immunization with rat
splenocytes, respectively (39, 43). In this study we have clearly
demonstrated that the decrease in the rate of induced resorptions
caused by maternal immunization is accompanied by a decline in the
TNF- mRNA level and by a decrease in the levels of all
immunoreactive forms of the TNF- protein at the fetomaternal
interface.
Finally, an increased expression of TNFRI mRNA transcripts was
demonstrated in placentae and uteri of mice with pregnancy loss. This
finding seems to implicate the existence of a TNF--associated
signaling pathway leading to placental death. Indeed, since the level
of TNFRI mRNA in the placenta was constant throughout pregnancy (51),
it is reasonable to suppose that its increased expression may lead to
an alteration of TNF- signaling in the placenta. One of the cellular
responses to TNF- is suggested to be associated with apoptotic cell
death (6). It has recently been shown that TNF- may promote
apoptosis in trophoblast cells following binding to TNFRI (52). This
ligand-receptor interaction was found to be critical, since cells
lacking the TNFRI did not show a detectable level of apoptosis
(25).
In conclusion, the results of the present study clearly demonstrate
that up-regulation of TNF- expression in the embryonic
microenvironment may contribute to spontaneous and induced placental
death. Furthermore, down-regulation of TNF- expression by maternal
immunization might play an important role in mechanisms underlying its
beneficial effect on reproductive performance.
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Acknowledgments |
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and cDNAs of murine TNF- and TNFRI. We
appreciate the help of Dr. Z. Zaslavsky in performing the
densitometric analysis and of Mr. A. Pinchasov for preparation of the
photomicrographs. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. V. Toder, Department of Embryology and Teratology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel. E-mail address:
3 Abbreviations used in this paper: TNFRI, TNF- receptor I; CP, cyclophosphamide.
Received for publication July 31, 1997. Accepted for publication January 6, 1998.
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(TNF-) gene correlates with murine resistance to development of toxoplasmic encephalitis and with levels of TNF- mRNA in injected brain tissue. J. Exp. Med. 175:683.This article has been cited by other articles:
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