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Original Studies |
,
Charles J. Lockwood,
Linda LaChapelle,
Bruno Dipasquale,
Rita I. Demopoulos,
Asok Kumar and
Seth Guller
Departments of Obstetrics and Gynecology (R.R., C.J.L., L.L., S.G.), Pathology (B.D., R.I.D., A.K.), and Biochemistry (S.G.), New York University Medical Center, New York, New York 10016
Address all correspondence and requests for reprints to: Dr. Seth Guller, Department of Obstetrics and Gynecology, New York University Medical Center, Tisch Hospital Room 531, 550 First Avenue, New York, New York 10016.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fas, a 45-kDa cell surface receptor of the tumor necrosis factor (TNF)/nerve growth factor family, mediates apoptosis of target cells after binding of Fas ligand (FasL) (7). Although Fas/FasL function was originally described in the context of lymphocyte-mediated apoptosis of lymphocytes, recent data indicated that the Fas/FasL signaling system may promote apoptosis of epithelial cells in ovarian follicles (8) and the thyroid gland (9). Local expression of FasL in cells of the testis (10) and the anterior region of the eye (11) was suggested in part to confer immune tolerance by promoting apoptosis of activated Fas-bearing lymphocytes that infiltrate these sites. Our previous results indicated that FasL was expressed in the human placenta and fetal membranes across gestation (12).
The purpose of the present study was to determine, based on biochemical and morphological parameters, whether cells in human fetal membranes undergo apoptosis and express Fas. Based on immunohistochemical, ultrastructural, and biochemical data, we report that apoptosis is a physiological process in human fetal membranes in the third trimester of pregnancy. In addition, our documentation of Fas expression in chorion, amnion, and decidua of fetal membranes at term may suggest a role for Fas/FasL signaling in apoptosis and remodeling of fetal membrane architecture across gestation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fetal membranes and placentas were obtained from 17 preterm
(<37 weeks) and 21 term placentas (
37 weeks). Samples at term were
obtained from women undergoing uncomplicated spontaneous vaginal
delivery or cesarean section with or without labor. Preterm samples
were obtained from pregnancies complicated by preeclampsia, diabetes,
chorioamnionitis, and premature rupture of membranes. All specimens
were obtained with the consent of the surgeon and the pathologist
according to internal review board protocols at New York University
Medical Center.
Terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) and immunohistochemistry
Fetal membrane tissue obtained within 1 h after delivery was dissected from the placental disc at the periplacental edge and cut into strips 1–2 cm wide including the area from the periplacental edge to the rupture site. Tissues were washed in saline, rolled, fixed in 10% formalin, and embedded in paraffin as previously described (13). Embedded tissue sections (5 µm) were applied to poly-L-lysine-treated glass slides (Newcomer Supply, Middleton, WI). Deparaffinization of tissue sections was performed for 2 h at 58 C before dehydration with xylene and rehydration with ethanol. Alternatively, membrane rolls were flash-frozen in OCT (Baxter Scientific Products, Edison, NJ) in dry ice/2-methylbutane (Sigma Chemical Co., St. Louis, MO). Both methods of sample preparation yielded similar patterns of TUNEL and Fas staining.
TUNEL of fetal membranes was performed using the ApopTag kit from Oncor (Gaithersburg, MD). Tissue sections were treated with 20 µg/mL proteinase K for 25 min at room temperature and washed with distilled water, and endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide in 100% methanol for 5 min. Slides were then rinsed with phosphate-buffered saline (PBS) and incubated for 1 h at 37 C in buffer containing digoxigenin-labeled deoxy-UTP and terminal deoxynucleotidyl transferase. Samples were then washed three times with PBS and incubated for 30 min at room temperature with antidigoxigenin antibody-peroxidase conjugate. After rinsing with PBS, slides were incubated at room temperature for 5 min with diaminobenzidine. Slides were counterstained with methyl green (Sigma). Controls were carried out in which terminal deoxynucleotidyl transferase was omitted from the labeling reaction.
For Fas immunohistochemistry, 5-µm sections of preterm and term fetal membrane rolls (n = 5) were incubated overnight at 4 C with 2 µg/mL rabbit anti-Fas antibody (Santa Cruz Biotechnology, Santa Cruz, CA), with and without 20 µg/mL of a peptide corresponding to amino acids 21–38 of human Fas. After incubation with primary antibody, slides were incubated with antirabbit antibody/peroxidase conjugate at a dilution of 1:500. Color development in peroxidase reactions was carried out using diaminobenzidene supplied with the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA). Samples were counterstained with hematoxylin.
Quantitation of apoptotic index and statistical analysis
For each TUNEL and methyl green-stained tissue section, brown apoptotic nuclei and blue-green healthy nuclei were blindly counted by 2 individuals (R.R. and B.D.) in each of 12 independent microscopic fields for amnion epithelial, chorion trophoblast, and decidua parietalis cell layers using a x40 objective. Between 300-1000 nuclei were counted for each sample. Eighty-one different specimens were employed to calculate interobserver correlation, and intraobserver correlation was carried out in 5 independent specimens. Interobserver (r = 0.884) and intraobserver (r = 0.984; r = 0.980) correlations were high. The apoptotic index (number of apoptotic nuclei per total nuclei x 100) was expressed as the mean ± SE. Statistical analysis was performed using ANOVA to statistically compare the apoptotic indexes of amnion, chorion, and decidua samples, and linear regression analysis was performed to compare the inter- and intraobserver correlations (SigmaStat software, Jandel, San Rafael, CA).
Electron microscopy
Fragments of fetal membranes were fixed in 2.5% glutaraldehyde
in sodium cacodylate buffer, pH 7.2. After overnight fixation by
immersion, tissues were postfixed in 1% osmium tetroxide, dehydrated
through ascending grades of alcohol, embedded in epon, and sectioned on
an ultramicrotome. Thin sections (
60 nm) were stained with saturated
uranyl acetate and aqueous lead citrate for electron microscopy. A
Zeiss 10A transmission electron microscope (Zeiss, New York, NY) was
used to view thin sections and for photography (14).
RT-PCR and Northern blotting for Fas
For RT-PCR analysis of Fas expression, placental and fetal membrane tissue were rinsed with saline, frozen in liquid nitrogen, and stored at -80 C. Frozen tissues were homogenized by Polytron disruption (Brinkmann Instruments, Westbury, NY), and total ribonucleic acid (RNA) was isolated using UltraSpec RNA (Biotecx, Houston, TX). Samples were then extracted with a mixture of phenol-chloroform-isoamyl alcohol (25:24:1) and then with chloroform alone. One microgram of total RNA was primed with 2.5 µmol/L random hexamers and reverse transcribed with 2.5 U/L murine leukemia virus reverse transcriptase (Perkin-Elmer/Cetus, Branchburg, NJ) in a 20-µL reaction mix according to the manufacturer’s protocol. Twenty microliters of complementary DNA (cDNA) were then PCR amplified with 15 pmol Fas cytoplasmic domain-specific primers (sense, 5'-CACTATTGCTGGAGTCATG-3'; antisense, 5'-CTGAGTCACTAGTAATGTCC-3') in a solution containing 200 µmol/L of each deoxy-NTP, 50 mmol/L KCl, 10 mmol/L Tris (pH 8.8), 2.5 U Taq polymerase (buffer A), and 2 mmol/L MgCl2 in a total volume of 100 µL. Primers were synthesized by Genosys (The Woodlands, TX) as previously described (15), and amplification was carried out in a GeneAmp PCR System 9600 (Perkin-Elmer/Cetus). First strand cDNA was denatured at 95 C for 1 min and 45 s. In each subsequent cycle of amplification, DNA was denatured at 95 C for 15 s, annealed at 54 C for 30 s, and polymerized at 72 C for 15 s. After 40 cycles of amplification, polymerization was carried out at 72 C for 7 min, and samples were immediately placed at 4 C.
For a positive control, 1 µg total RNA was also reverse transcribed as described above, and 20 µL cDNA were PCR amplified in buffer A supplemented with 1.5 mmol/L MgCl2 containing 15 pmol glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primers (sense, 5'-GGTCGGAGTCA-ACGGATTTGGTCG-3'; antisense, 5'-CCTCCGACGCCTGCTTCACCAC-3'; Genosys) as previously described (16). For each cycle of amplification, DNA was denatured at 95 C for 30 s, annealed at 60 C for 30 s, and polymerized at 72 C for 30 s. After 35 cycles of amplification, polymerization was performed at 72 C for 7 min, and samples were immediately placed at 4 C. After amplification, Fas and GAPDH RT-PCR products were visualized by electrophoresis on a 2% agarose gel containing 0.5 µg/mL ethidium bromide.
For Northern blot analysis, 1–4 µg polyadenylated RNA were isolated from approximately 400 mg fresh amnion tissue using the Micro-Fast Track kit (Invitrogen, San Diego, CA). Formaldehyde gel electrophoresis was carried out as previously described (17), and RNA was transferred to a ZetaProbe nylon membrane (Bio-Rad, Richmond, CA). Blots were prehybridized for 2 h at 42 C in buffer containing Denhardt’s solution and SSC (standard saline citrate) as previously described (18). Hybridization was then carried out overnight at 42 C in the same buffer containing 106 cpm/mL of a 32P-labeled 266-bp PCR product corresponding to the cytoplasmic domain of Fas. Levels of GAPDH messenger RNA (mRNA) were analyzed as previously described (19). Scanning and printing of PCR and Northern blot data were carried out using Sigma Scan/Sigma Image software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human fetal membranes consist of amnion, chorion, and decidua
parietalis tissue layers (denoted Am, Ch, and De, respectively, in Fig. 1a
).
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, a, c, and d). Healthy (i.e.
nonfragmented) nuclei retained the blue-green color of the methyl green
counterstain. Under high magnification, amnion epithelial cells (Fig. 1c, upper arrow) and chorionic trophoblasts (Fig. 1d) were
visualized by TUNEL staining. In addition, scattered staining was
observed in the area beneath the basal lamina of the amnion (Fig. 1c, lower arrow), possibly reflecting the presence of apoptotic
bodies within macrophages at this site.
Electron micrographs of human fetal membranes at term revealed
ultrastructural changes in chorionic trophoblasts (Fig. 2, a and b) and amnion epithelial cell
(Fig. 2d) layers consistent with apoptosis, including condensation of
nuclear chromatin along the periphery of the nucleus and shrinkage of
cellular cytoplasm. Peripheral condensation of chromatin, a hallmark of
apoptosis (20), was apparent within highly condensed nuclei (Fig. 2).
Conversely, chromatin remained diffusely and uniformly distributed
within the adjacent healthy nuclei (Fig. 2, b and c).
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To determine whether apoptosis was a progressive process in fetal
membranes in the third trimester of pregnancy, the apoptotic index (the
percentage of TUNEL-positive nuclei per total nuclei) was compared in
amnionic, chorionic, and decidual layers of fetal membrane rolls
obtained from women at 23–30, 31–36, and 37–42 weeks of gestation.
The apoptotic index of chorionic and decidual cell layers was
statistically greater in the 37–42 week group than in the 23–30 week
group (P = 0.01 and P = 0.005 for
chorionic and decidual cell layers, respectively; Fig. 3), suggesting that apoptosis is
up-regulated in chorionic and decidual tissues of fetal membranes at
term. Conversely, the apoptotic index of the amnion epithelial layer
was statistically greater in the 23–30 week group than in the 31–36
week group (P = 0.04), indicating that apoptosis may be
differentially regulated in amnion epithelial, chorionic trophoblast,
and decidual cell layers. Based on comparison of the apoptotic index in
fetal membranes obtained after cesarean section with and without labor
and spontaneous vaginal delivery, we concluded that labor per
se does not affect fetal membrane apoptosis (not shown).
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As the Fas/FasL signaling system has been implicated in mediating
apoptosis (7), we examined levels of Fas (i.e. the receptor
for FasL) in fetal membranes by immunohistochemical staining with
anti-Fas antibodies. As shown in Fig. 4a, Fas expression was localized to the amnion epithelial and chorion
trophoblastic layers, with lower levels of expression observed in the
decidua of fetal membranes at term. Similar patterns of Fas staining
were observed in preterm tissues (data not shown). No Fas staining was
observed in fetal membranes when the anti-Fas antibody was preabsorbed
with a 10-fold excess of Fas antigen (Fig. 4b). Interestingly, Fas
expression was not noted in the periplacental degenerative villi in the
intermediate trophoblast layer of the chorion by immunohistochemical
staining (Fig. 4a).
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). The appearance of the expected
788-bp product corresponding to GAPDH cDNA validated RNA extraction and
RT-PCR protocols (Fig. 5a). Northern blotting using polyadenylated RNA
isolated from an amnion at term revealed a mol wt of approximately 1.9
kb for Fas mRNA (Fig. 5b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Morphological and biochemical data obtained in the current report are consistent with the conclusion that cells of the amnion epithelial, chorionic trophoblast, and decidua parietalis layers in human fetal membranes undergo apoptosis. Immunohistochemical data indicated that between 8–29% of these cell types were TUNEL positive. The observed dispersed pattern of TUNEL staining in human fetal membranes was similar to that noted in other tissues undergoing apoptosis (26, 27). Data obtained by electron microscopy in the present study are consistent with changes noted during apoptosis, including condensation of chromatin along the periphery of the nucleus and, ultimately, shrinkage of the nucleus and the cell itself. Although TUNEL staining is often correlated with apoptosis, it has been suggested that this method also detects fragmented DNA in necrotic cells within a tissue (28). In the present study, the ultrastructural changes described above and the dispersed pattern of TUNEL staining are consistent with apoptosis and not necrosis, in which gross swelling of cells and their organelles occurs in patches within a tissue (5, 6). In light of these results, it is somewhat surprising that extracts of human amnion at term did not reveal an apoptotic DNA ladder pattern after ethidium bromide staining of agarose gels (not shown). This may indicate that internucleosomal cleavage of DNA does not accompany apoptosis in human fetal membranes or simply reflects an inherent difficulty in detecting DNA ladders in samples containing low levels of DNA fragmentation (29). Alternatively, it is well documented that internucleosomal cleavage of DNA to small fragments containing multiples of 180–200 bp does not always occur in cells undergoing apoptosis (30, 31).
Our results also demonstrated that the apoptotic index of chorionic trophoblasts and decidua parietalis cells was higher in term tissue compared to specimens obtained from the preterm (23–30 week) group. In contrast, the apoptotic index of amnion epithelial cells was highest in the 23–30 week group, suggesting that apoptosis in amnion, chorion, and decidua may be differentially regulated. It is of note in the present study that although the use of fetal membranes from pregnancies complicated by infection, ischemia, preeclampsia, diabetes, and premature rupture of membranes may be criticized, it is impossible to obtain preterm fetal membranes from uncomplicated human pregnancies. That these pathological conditions did not induce apoptosis in chorion and decidua is suggested by the similarity in both distribution and quantity of apoptotic cells across each condition. Conversely, high levels of apoptosis in amnion epithelial cells in the 23–30 week group may reflect pathology before term. Therefore, we suggest that apoptosis occurs as part of a program of senescence in chorionic and decidual cells that is not triggered in association with labor. Furthermore, the etiology of amnion apoptosis appears to be more complicated and may be associated with preterm pathology. However, due to the relatively small number of specimens in each group, further studies need to be conducted to associate a particular pathology with apoptosis in the amniotic epithelium and to characterize the progressive nature of apoptosis in the chorion and decidua.
TUNEL and ultrastructural results from other laboratories suggested
that first trimester syncytiotrophoblasts in first trimester human
placental villi undergo extensive apoptosis, whereas significantly less
apoptosis was observed in term placental villi (28). TNF
and
interferon-
were demonstrated to induce apoptosis of
cytotrophoblasts isolated from human term placentas (32).
A report by Paavola et al. documented an apoptotic program in rat amnion cells before parturition characterized by degradation of type I collagen by interstitial collagenase (33). There is precedent for apoptosis and necrosis occurring concomitantly in placental as well as cardiac tissue (28, 34). Our previous data suggested that glucocorticoids may alter the integrity of fetal membranes by reducing the synthesis of collagen III and fibronectin by amnion epithelial cells (35).
Fas (CD95), a cell surface receptor, is a member of the TNF receptor and nerve growth factor receptor family (7). It has been established that Fas and TNF receptor modulate the immune response by triggering apoptosis of lymphocytes after binding of FasL and TNF, their respective ligands (7). Recent data indicated that the apoptotic program induced by FasL and TNF required the interaction of common interleukin-converting enzyme and interleukin-converting enzyme-like proteases with receptor complexes (36, 37). It is interesting to note that although Fas-mediated apoptosis was originally described in the context of autoregulation of T lymphocyte proliferation (7), recent studies suggested that production of FasL by cells of the testis (10), the anterior region of the eye (11), the brain (38) and tumors (39, 40) may also serve an immunoprotective function by promoting apoptosis of Fas-bearing lymphocytes that infiltrate these sites. Fas-mediated apoptosis has also been implicated in the regulation of ovarian and thyroid function (8, 9).
We reported that cytotrophoblasts in human placenta and fetal membranes express FasL across gestation (12). Therefore, we hypothesized that the presence of FasL in human placenta and fetal membranes serves to protect the fetus against activated Fas-bearing maternal lymphocytes at maternal-fetal interfaces (12). Based on the involvement of Fas in mediating apoptosis (7) and our demonstration of FasL expression in human placenta and fetal membranes (12), in the present study we determined whether Fas was expressed in human fetal membranes. We documented by immunohistochemistry that Fas was present at high levels in amnion epithelial cells, chorion trophoblasts, and decidua parietalis cells of second and third trimester human fetal membranes. In addition, RT-PCR and Northern blotting techniques demonstrated the expression of Fas in term amnion, chorion, decidual, and placental tissue. These results do not provide relative levels of expression of Fas in these tissues since quantitative procedures were not used. The finding that chorionic cytotrophoblasts and amnion epithelial cells express Fas and FasL in term fetal membranes suggests that these cells may self-regulate apoptosis at this site. However, expression of Fas alone does not guarantee activation of Fas-mediated apoptosis. It is clear that other factors, including the level of expression of FasL, will determine whether the Fas-FasL apoptotic pathway is activated (7). In addition, it is of note that although human colon carcinoma and leukemia cells concomitantly express functional Fas and FasL, they do not undergo "suicide apoptosis" (39, 40).
In conclusion, our results document for the first time the expression of apoptosis and Fas in human fetal membranes. Future studies will elucidate the role that Fas/FasL signaling may play in physiologically and pathologically triggering apoptosis in fetal membranes in association with human parturition.
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Acknowledgments |
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Footnotes |
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Received July 14, 1997.
Revised October 28, 1997.
Accepted November 7, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R, Lockwood CJ, Ma Y, DiPasquale B, Guller
S. 1996 Expression of Fas ligand by human cytotrophoblasts:
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D. R Balkundi, J. A Ziegler, J. F Watchko, C. Craven, and M. Trucco Regulation of FasL/Fas in Human Trophoblasts: Possible Implications for Chorioamnionitis Biol Reprod, August 1, 2003; 69(2): 718 - 724. [Abstract] [Full Text] [PDF]
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x 174 DNA standards
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