Trophoblast apoptosis in human placenta at term as detected by expression of a cytokeratin 18 degradation product of caspase

CONTEXT: Apoptosis occurs in the normal placenta. The monoclonal antibody M30 is directed against a novel epitope of cytokeratin 18 (CK18) that is formed by caspase cleavage early in the apoptotic cascade, and this antibody may therefore be useful for evaluating trophoblast apoptosis. OBJECTIVE: We undertook the present study to evaluate the use of monoclonal antibody M30 to assess trophoblast apoptosis in placenta at term. METHODS: We stained paraffin-embedded placental tissues from 15 deliveries at term with M30. We compared positive M30 staining and CK18 staining (as detected by a monoclonal antibody directed against CK18) of trophoblasts in serial slides. We also compared apoptotic rates as detected by M30 and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) in 7 of the placentas. RESULTS: In fields of villous tissue, most M30-positive cells were CK18-positive syncytiotrophoblasts. Approximately half of M30-positive cells occurred as focal positive staining in the syncytial layer, and half occurred as abundant staining of syncytiotrophoblasts in areas with increased intervillous or perivillous fibrinoid. We found very few M30-positive cells in villous stroma. In decidual/basal plate tissues, most (two thirds) of the M30-positive cells were CK18-positive extravillous trophoblasts, whereas one third were syncytiotrophoblasts of anchoring villi. Since TUNEL detects apoptosis in both epithelial and nonepithelial cells, more cells were positively stained with TUNEL than with M30 in some tissue fields. However, our observations suggest that M30 was more sensitive than TUNEL in recognizing apoptotic trophoblasts and had less nonspecific staining than TUNEL. CONCLUSION: We recommend the use of monoclonal antibody M30 for apoptosis studies in placental tissues. This antibody is easy to handle, the staining obtained seems specific, and the nonspecific staining seems negligible.     

Histamine influence on apoptosis in trophoblast cell cultures

Introduction

It has been demonstrated that histamine plays an important role in the pathogenesis of preeclampsia. Histamine regulates the process of differentiation of trophoblast cells; it also acts as a growth factor in malignant melanoma cells, and prevents monocytic apoptosis. Trophoblast research has shown that in preeclampsia placentas, trophoblast apoptosis is significantly increased.

Aim of the study

The aim of our study was to demonstrate the influence of histamine on the process of apoptosis in human trophoblast cell cultures.

Materials and methods

Placentas were obtained after vaginal delivery. Tissue samples were excised from placentas and, with the use of modified Kliman’s method, trophoblast cell cultures were established. The cultures were incubated with dexamethasone as an apoptosis inducer 48 hours prior to apoptosis detection assays. Along with dexamethasone, selected cell cultures were incubated with histamine (1 μmol/l) or histamine (1 μmol/l) and terfenadine (from 1 to 5 μmol/l), a H₁ receptor antagonist. For apoptotic activity detection, and quantitative analysis, we used an ELISA assay. M30‐Apoptosense ELISA Kit is based on the M30 monoclonal antibody that binds only the caspase‐cleaved cytokeratin 18 formed during apoptosis in trophoblast cells.

Results

Our investigation showed significantly (p < 0.05) increased apoptotic activity in cultures incubated with dexamethasone, histamine and terfenadine (% of reference value, ±SEM): up to 113.1 ± 4.33%. Cell cultures incubated with dexamethasone and histamine only showed significantly lower apoptotic activity 90.2 ± 5.17%. We suggest that histamine may inhibit apoptotic activity in trophoblast cell cultures via H₁ receptor. Thus histamine may regulate the process of trophoblast differentiation (via integrin aV‐b3 expression, as we previously suggested), and influence cell turnover in the placenta.

     

Histamine influence on apoptosis in trophoblast cell cultures

Introduction

It has been demonstrated that histamine plays an important role in the pathogenesis of preeclampsia. Histamine regulates the process of differentiation of trophoblast cells; it also acts as a growth factor in malignant melanoma cells, and prevents monocytic apoptosis. Trophoblast research has shown that in preeclampsia placentas, trophoblast apoptosis is significantly increased.

Aim of the study

The aim of our study was to demonstrate the influence of histamine on the process of apoptosis in human trophoblast cell cultures.

Materials and methods

Placentas were obtained after vaginal delivery. Tissue samples were excised from placentas and, with the use of modified Kliman’s method, trophoblast cell cultures were established. The cultures were incubated with dexamethasone as an apoptosis inducer 48 hours prior to apoptosis detection assays. Along with dexamethasone, selected cell cultures were incubated with histamine (1 μmol/l) or histamine (1 μmol/l) and terfenadine (from 1 to 5 μmol/l), a H₁ receptor antagonist. For apoptotic activity detection, and quantitative analysis, we used an ELISA assay. M30‐Apoptosense ELISA Kit is based on the M30 monoclonal antibody that binds only the caspase‐cleaved cytokeratin 18 formed during apoptosis in trophoblast cells.

Results

Our investigation showed significantly (p < 0.05) increased apoptotic activity in cultures incubated with dexamethasone, histamine and terfenadine (% of reference value, ±SEM): up to 113.1 ± 4.33%. Cell cultures incubated with dexamethasone and histamine only showed significantly lower apoptotic activity 90.2 ± 5.17%. We suggest that histamine may inhibit apoptotic activity in trophoblast cell cultures via H₁ receptor. Thus histamine may regulate the process of trophoblast differentiation (via integrin aV‐b3 expression, as we previously suggested), and influence cell turnover in the placenta.

     

Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis

A neo-epitope in cytokeratin 18 (CK18) that becomes available at an early caspase cleavage event during apoptosis and is not detectable in vital epithelial cells is characterized. The monoclonal antibody M30, specific for this site, can be utilized specifically to recognize apoptotic cells, which show cytoplasmic cytokeratin filaments and aggregates after immunohistochemistry with M30, while viable and necrotic cells are negative. The number of cells recognized by the antibody increases after induction of apoptosis in exponentially growing epithelial cell lines and immunoreactivity is independent of the phosphorylation state of the cytokeratins. The generation of the M30 neo-epitope occurs early in the apoptotic cascade, before annexin V reactivity or positive DNA nick end labelling. In a flow cytometric assay, the majority of the M30-positive cells appear in the ‘apoptotic’ subG1 peak. Tests with synthetic peptides define positions 387–396 of CK18, with a liberated C-terminus at the caspase cleavage site DALD-S, as the ten-residue epitope of M30. This epitope starts at the end of coil 2 of the predicted CK18 structure, at a probable hinge region, compatible with the sensitivity to proteolytic cleavage. The definition of a specific caspase cleavage site in CK18 as a neo-epitope can be used for quantification of apoptotic epithelial cells with immunocytochemical techniques and is applicable to both fresh and formalin-fixed material. Copyright © 1999 John Wiley & Sons, Ltd.     

X-linked inhibitor of apoptosis (XIAP) confers human trophoblast cell resistance to Fas-mediated apoptosis

Apoptosis occurs in the placenta throughout gestation, with a greater frequency near term in comparison to the first trimester. The Fas/FasL system represents one of the main apoptotic pathways controlling placental apoptosis. Although first trimester trophoblast cells express both Fas and FasL, they are resistant to Fas-induced apoptosis. Therefore, trophoblast resistance to Fas-mediated apoptosis may be due to the inhibition of the pathway downstream of Fas stimulation. Expression levels of X-linked inhibitor of apoptosis (XIAP) were recently shown to decrease in third trimester placentas, correlating with an increase in placental apoptosis. As a potent caspase inhibitor, XIAP prevents the activation of caspase-9 through its BIR3 domain and caspase-3 activation via the linker-BIR2 domain. In the present study, high levels of the active form of XIAP were detected in first trimester trophoblast cells, whereas term placental tissue samples predominantly expressed the inactive form of XIAP. Using a XIAP inhibitor, phenoxodiol, we demonstrate that XIAP inactivation sensitizes trophoblast cells to Fas stimulation, as evidenced by the anti-Fas mAb-induced decrease in trophoblast cell viability and increase in caspase-8, caspase-9 and caspase-3 activation. This suggests a functional role for XIAP in the regulation of the Fas apoptotic cascade in trophoblast cells during pregnancy.     

Mixed micropapillary and trophoblastic carcinoma of bladder: report of a first case with new immunohistochemical evidence of urothelial origin

The micropapillary variant of urothelial carcinoma has a reported incidence of 0.7%. Trophoblastic urinary carcinoma is very rare, with roughly 30 cases reported during the last century. This is the first report of mixed micropapillary and trophoblastic bladder carcinoma. A 45-year-old man presented with gross hematuria. His tumor contained choriocarcinomatoid areas with syncytiotrophoblasts, classic micropapillary carcinoma, conventional high-grade urothelial carcinoma, and flat carcinoma in situ. He underwent radical surgery; tumor stage was T4N2M0. Despite postoperative combination chemotherapy, he developed pulmonary and retroperitoneal metastases and died 20 months after presentation. The tumor was immunopositive for human chorionic gonadotropin and human placental lactogen in trophoblast and for cytokeratin 20 and high-molecular-weight cytokeratin in all tumor components. Because high-molecular-weight cytokeratin is expressed by urothelium but is rarely found in placental trophoblast or germ-cell choriocarcinoma, its presence in trophoblastic bladder carcinoma is new evidence that the latter is a transformed neoplasm of urothelial origin.     

Antibodies to cytokeratins bind to epitopes in human uterine smooth muscle cells in normal and pathological pregnancies

Cytokeratin antibodies have been widely used for the identification of trophoblast cells in the placental bed, following their invasion from the developing conceptus. Their identification centres upon the expression of cytokeratin in epithelial cells, from which trophoblast cells are derived. Our recent observations indicate that this strict relationship may be more complex than was thought. Cryostat and paraffin sections of human decidua and myometrium, taken from the placental bed and the uterotomy cut, were examined immunocytochemically for cytokeratins using ten antibody clones selected to identify different cytokeratin proteins and antigenic epitopes. Biopsy specimens were obtained from normal and pathological pregnancies (pre-eclampsia, fetal retardation, amnioninfection, hysterorrhexis, placenta praevia) at the time of caesarean section (26–41 weeks of pregnancy). Antibodies against nine clones, CAM 5.2, MNF 116, AE1/AE3, CK5, KS-B17.2, CY-90, M20, E3, and 34βE12 identified, as expected, syncytial giant cells and mononuclear trophoblasts within the placental bed and glandular epithelial cells throughout the uterus. In addition, they stained numerous fusiform cells that were classified by established criteria to represent smooth muscle cells, both within blood vessels and myometrium. No staining differences were observed between normal and pathological disorders. These results indicate that cytokeratin antibodies CAM 5.2, MNF 116 and AE1/AE3, and other antibodies targeting proteins 8 and 18, cross-react with epitopes expressed in cells other than giant trophoblastic cells and mononuclear trophoblasts in the uterus and, thus, caution has to be used when such antibodies are used for the diagnostic characterization of tissues related to the placental bed.     

Histochemical and morphological examination of proliferation and apoptosis in human first trimester villous trophoblast

BACKGROUND: Our present knowledge about trophoblast turnover in human first trimester placental villi based on multiparametric examination of proliferation and apoptosis is limited. METHODS: Human villous placentae collected during 6, 7 and 8 weeks (n = 10/each group) of gestation were examined for trophoblast proliferation and apoptosis based on quantitative analyses of immunopositive Fas, tumor necrosis factor receptor 1 (TNFR1), cytokeratin 18 fragment (18f), number of proliferating cell nuclear antigen (PCNA), Ki67 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positive nuclei, scores of mitotic and apoptotic indices and ultrastructural characteristics. RESULTS: Mitotic index in cytotrophoblast higher (P < 0.05) at 6 week compared with 7 and 8 weeks of gestation showed significant (P < 0.05) negative correlation between its prevalence and gestational age. Syncytiotrophoblast exhibited higher number of TUNEL positive nuclei (P < 0.01), TUNEL positive apoptotic nuclei (P < 0.05) and apoptotic index (P < 0.05) compared with cytotrophoblast at same gestational age. Positive correlations found between cytokeratin 18f and apoptotic index (P < 0.01), Fas and apoptotic index (P < 0.01), TUNEL positive nuclei and apoptotic index (P < 0.05), cytokeratin 18f and Fas (P < 0.01), whereas cytokeratin 18f (P < 0.05) and Fas (P < 0.05) showed positive correlation only with TUNEL positive apoptotic nuclear data. Phalangeal intrusions of syncytiotrophoblast between transitional cytotrophoblasts showed apposed plasma membranes bearing thickened membrane leaflets, inter-membranous gaps enclosing membranous invaginations, liposome-like particles; patches of membrane seen to be dissolved resulting in cytoplasmic continuity typical of syncytial formation. CONCLUSION: Cellular remodeling of first trimester villous placenta requires a complex homeodynamics involving proliferation in cytotrophoblast, development-associated syncytialization and apoptosis in syncytiotrophoblast.     

Are human placental bed giant cells merely aggregates of small mononuclear trophoblast cells? An ultrastructural and immunocytochemical study

The ultrastructure of placental bed giant cells in early human pregnancies of 7-12 weeks gestational age is described. Their nature and function was further characterized by confocal immunofluorescence microscopy of paraffin sections labelled for cytokeratin, gap junction connexins (CX) 32 or 43, and placental hormones, alpha-human chorionic gonadotrophin (alpha-HCG) and human placental lactogen (HPL). Placental bed giant cells were observed with two phenotypes; as single large trophoblast cells containing one or more nuclear profiles in a voluminous cytoplasm, and as cell aggregates comprising mononuclear trophoblast cells in close apposition separated by narrow intercellular spaces. Cells within the aggregates are attached to one another by desmosomes, and also possess gap junctions as shown by immunolabelling for CX32 and CX43. By contrast, gap junctions were absent in the true multinucleated giant cells. Organelles present within the cytoplasm of the giant cells and their immunoreactivity for HPL and alpha-HCG suggest protein synthesis.     

Evaluation of trophoblast HLA-G antigen with a specific monoclonal antibody

A monoclonal antibody to HLA-G has been generated by immunizing HLA-A2.1/human β²-microglobulin (β²m) double transgenic mice with murine L cells transfected with both human β²m and HLA-G. This monoclonal antibody, designated as G233, has been found not to cross-react with other HLA class I antigens when tested on numerous cell lines by flow cytometry. With immunohistology, all populations of extravillous trophoblast (cell columns, interstitial trophoblast, endovascular trophoblast, placental bed giant cells) were stained. An extensive range of adult and fetal tissues was also tested but none reacted with monoclonal antibody G233, including those previously reported to express HLA-G mRNA, indicating that the protein has a highly restricted distribution. Failure to detect HLA-G in the fetal thymus raises the question as to how T-cell tolerance to this antigen is induced. Immunoprecipitation of trophoblast surface proteins with monoclonal antibody G233 revealed a heavy chain of 39 kDa and a light chain of 12 kDa, indicating that HLA-G expressed on the surface of trophoblast is complexed with p2m. However, sequential immunoprecipitation with monoclonal antibody W6/32 followed by monoclonal antibody G233 continued to detect a residual band of 39 kDa, suggesting that trophoblast surface HLA-G may also occur as free heavy chains not associated with p2m. Immunoprecipitation followed by two dimensional gel electrophoresis showed that monoclonal antibody G233 recognizes several iso-forms of HLA-G from trophoblast similar to the characteristic spot array previously described for HLA-G. This monoclonal antibody G233 will be highly useful in future experiments to elucidate the function of HLA-G.     

Cell surface antigen expression of first trimester chorionic villus samples

First trimester chorionic villi obtained by chorionic villus sampling at approximately 9 weeks of gestation were investigated by indirect immunofluorescence to demonstrate trophoblast cell surface antigen expression. Villous trophoblast expressing the trophoblast specific markers transferrin receptor, human placental lactogen, and cytokeratin was also found to express a monomorphic major histocompatibility complex class I determinant recognized by the monoclonal antibody W6/32. W6/32 positive regions included sparsely scattered regions of villous trophoblast and fanning outgrowths of trophoblast. The class I antigenic determinant expressed by first trimester trophoblast was found to be recognized exclusively by W6/32 when assayed with a panel of anti-class I determinant monoclonal antibodies. Trophoblast W6/32 determinant expression was not increased after 24 hour organ culture in the presence of 200 U of interferon gamma. Exposure to interferon gamma resulted in increased class I antigen expression by mesenchyme and low level de novo mesenchyme class II antigen expression. These data suggest that early gestational stage villous trophoblast express non-classical class I antigens which do not seem to be subject to the regulatory effects of interferon gamma.     

Hypoxic switch in mitochondrial myeloid cell leukemia factor-1/Mtd apoptotic rheostat contributes to human trophoblast cell death in preeclampsia

Preeclampsia, a disorder of pregnancy, is characterized by increased trophoblast cell death and altered trophoblast-mediated remodeling of myometrial spiral arteries resulting in reduced uteroplacental perfusion. Mitochondria-associated Bcl-2 family members are important regulators of programed cell death. The mechanism whereby hypoxia alters the mitochondrial apoptotic rheostat is essential to our understanding of placental disease. Herein, myeloid cell leukemia factor-1 (Mcl-1) isoform expression was examined in physiological/pathological models of placental hypoxia. Preeclamptic placentae were characterized by caspase-dependent cleavage of death-suppressing Mcl-1L and switch toward cell death-inducing Mcl-1S. In vitro, Mcl-1L cleavage was induced by hypoxia-reoxygenation in villous explants, whereas Mcl-1L overexpression under hypoxia-reoxygenation rescued trophoblast cells from undergoing apoptosis. Cleavage was mediated by caspase-3/-7 because pharmacological caspase inhibition prevented this process. Altitude-induced chronic hypoxia was characterized by expression of Mcl-1L; resulting in a reduction of apoptotic markers (cleaved caspase-3/-8 and p85 poly-ADP-ribose polymerase). Moreover, in both physiological (explants and high altitude) and pathological (preeclampsia) placental hypoxia, decreased trophoblast syncytin expression was observed. Hence, although both pathological and physiological placental hypoxia are associated with slowed trophoblast differentiation, trophoblast apoptosis is only up-regulated in preeclampsia, because of a hypoxia-reoxygenation-induced switch in generation of proapoptotic Mcl-1 isoforms.     

Apoptosis and expression of bcl-2 protein in invasive ductal adenocarcinoma of the pancreas

Apoptosis plays a central role in the development and/or progression of cancer. There are several methods for detection of apoptotic cells in tissue sections including light and electron microscopy, in situ nick end-labeling (ISEL), TdT-mediated dUTP nick-end labeling (TUNEL) and immunohistochemical detection of proteins associated with apoptosis. Apoptosis was assessed by the monoclonal antibody M30 CytoDEATH (M30), which is specific for neo-epitope in cytokeratin 18 that becomes available at an early caspase cleavage during apoptosis. Expression of bcl-2 protein was evaluated, because bcl-2 protein plays an important role in the regulation of apoptosis. Twenty-six invasive ductal adenocarcinomas of the pancreas were studied immunohistochemically with antibodies M30 and bcl-2. The mean apoptotic index (AI, the percentage of apoptotic cells of the total tumor cells number) was 2.75%. High AI (> 10%) was observed in 4 cases of the 26 pancreatic carcinomas (15%). Protein bcl-2 was expressed in 3 cases (11.5%). The AI did not correlate with the expression of protein bcl-2. In conclusion, the detection of neo-epitope in cytokeratin 18 by monoclonal antibody M30 can be used for quantification of apoptotic cells with immunohistochemical techniques in tissue sections. It is a new approach to evaluate apoptosis in pancreatic carcinomas. The low positivity of bcl-2 expression in pancreatic adenocarcinomas suggests that bcl-2 protein does not play a central role in pancreatic tumorigenesis and cancer progression.     

Apoptosis in the early human placental bed and its discrimination from necrosis using the in-situ DNA ligation technique

Extensive areas of necrosis are present in the early human placental bed. Our aim was to determine whether apoptosis is also a feature. A method was therefore required to differentiate unequivocally necrosis and apoptosis. Initially, terminal deoxynucleotide transferase-mediated dUTP nick-end labelling was used to visualize apoptotic cells. However, non-specific labelling, probably due to free DNA released by necrotic cells, was excessive; thus, in-situ DNA ligation was employed. In this technique, two DNA fragments with single-base 3' overhangs and blunt- ends were labelled with a fluorochrome and then ligated to the DNA breaks on the sections. Immunolabelling for cytokeratin or leukocyte common antigen was performed to determine the phenotype of apoptotic cells identified by the in-situ DNA ligation technique. A proportion of the dying cells was confirmed to be trophoblasts. No co-localization with leukocyte common antigen was found in this region, suggesting that maternal macrophages and natural killer cells (CD56+) were not dying by apoptosis in significant numbers. In conclusion, in-situ DNA ligation in association with immunocytochemistry can readily distinguish apoptosis from necrosis in the placental bed. The results suggest that a proportion of invading trophoblast cells are eliminated by apoptosis in early pregnancy.      

Serine protease HtrA1 is developmentally regulated in trophoblast and uterine decidual cells during placental formation in the mouse.

Development of a hemochorial placenta involves trophoblast proliferation, differentiation, and invasion into the uterus to promote blood flow to the embryo. Trophoblast invasion is tightly controlled by expression of specific proteases in the trophoblast and highly coordinated activities in the uterus. One uterine event essential for placentation is the developmentally regulated formation and regression of the decidua. In mice, decidual regression takes place in a temporal- and spatial-specific manner that is coordinated with placental development. In this study, we identified that the serine protease HtrA1 (high temperature requirement factor A1) was specifically expressed in differentiated trophoblast cells, especially the giant cells, during the early stages of placental development. A high level of HtrA1 expression was also detected in decidua capsularis specifically at the decidual-trophoblast interface where active involution occurs. Thus, we have identified a previously unknown role for HtrA1 as a protease potentially important for trophoblast differentiation/invasion and uterine decidual regression during placental development.     

The adhesion molecule CEACAM1 (CD66a, C-CAM, BGP) is specifically expressed by the extravillous intermediate trophoblast

CEACAM1 (CD66a, C-CAM, BGP) is an adhesion molecule of the carcinoembryonic antigen family which has been shown to be normally expressed at the apical pole of epithelial cells, including the apical pole of endometrial surface and glandular epithelia. The purpose of the present study was to investigate its expression pattern at the maternal-fetal interface, and thus to determine whether CEACAM1 could be implicated in the human implantation process. For this purpose, we performed immunohistochemistry using the 4D1/C2 monoclonal antibody (mAb) as well as flow cytometry and Western blot on isolated trophoblast populations. On the maternal side of the maternal-fetal interface, CEACAM1 was present in epithelial cells of pregnancy endometrium as well as in small endometrial vessels, whereas it was absent from decidual cells. On the fetal side, CEACAM1 was strongly expressed by the extravillous (intermediate) trophoblast at the implantation site, as well as by extravillous trophoblast cells with invasive phenotype in primary culture, as shown by flow cytometry and Western blot. Expression was also observed in placental villous core vessels but was absent from both villous cyto- and syncytiotrophoblasts throughout the pregnancy. We conclude that, given its specific expression pattern, CEACAM1 can be a useful marker for extravillous intermediate trophoblast and might be functionally implicated in mediating trophoblast/endometrial and/or trophoblast/endothelial interactions during the trophoblastic invasion of the endometrium.     

Differential effects of inducers of syncytialization and apoptosis on BeWo and JEG-3 choriocarcinoma cells

BACKGROUND: The interactions of trophoblasts with the cytokine network at the fetomaternal interface determine the pathway the cell undertakes, e.g. proliferation, differentiation and apoptosis. METHODS: We used cultures of fusigenic BeWo and non-fusigenic JEG-3 choriocarcinoma cells to study the effects of inducers of syncytialisation (forskolin) and apoptosis [tumour necrosis factor-alpha (TNFalpha)] on differentiation, viability, proliferation and apoptosis. RESULTS: E-cadherin immunostaining showed that syncytium formation was confined to BeWo and not JEG-3 cells, while secretion of hCG was promoted by forskolin in both cell types implying a 'dissociation' between morphological and biochemical differentiation. Forskolin also had differential effects on cell viability (MTT reduction test) and proliferation (Ki67 immunostaining with MIB-1 monoclonal antibody), both decreasing in BeWo and increasing in JEG-3 cells. TNFalpha increased apoptosis (cytokeratin neo-epitope immunostaining with M30 monoclonal antibody) in both cell types, an effect which was blocked by epidermal growth factor selectively in JEG-3 cells. CONCLUSION: Our results suggest that the differential responses of BeWo and JEG-3 cells to inducers of syncytialization and apoptosis might be related to their fusigenic capacity. Caution is needed when extrapolating results obtained by these models to normal trophoblast populations. However, we speculate that these models can help identify key factors involved in trophoblast differentiation at the placental bed.     

1141180772Activated NK cells in spontaneous abortion cases induce apoptosis on extravillous trophoblast in a granulysin dependent manner

Extravillous trophoblast (EVT) is well known to undergo apoptosis in spontaneous abortion cases although the apoptotic mechanism is still unclear. Granulysin (Gr) is a cytotoxic granule protein of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) with tumoricidal activities. We have reported that the expression of granulysin, which was mainly produced by decidual NK cells, is significantly higher in the deciduas of spontaneous abortion cases than early normal pregnancy. We then have attempted to investigate the molecular mechanism of apoptosis associated with Gr. We also assessed apoptosis in the HTR8/SV40 cell, extravillous trophoblast (EVT) cell line, which were transfected with GFP-fused Gr. Anti-cleaved cytokeratin 18 antibody and Annexin-V were used for detecting apoptosis by IS and FC. We detected the apoptosis of the EVT in miscarriage cases. In those EVT, we detected nuclear staining of Gr. We next examined the effect of Gr with the target trophoblast cells by using GFP-fused Gr vector. Exogenously expressed GFP-fused Gr preferentially accumulated in nucleus from cytoplasm, resulting in apoptosis. Similar findings were obtained by native granulysin produced by IL-2 stimulated decidual lymphocytes. Our findings suggest that the granulysin-positive NK cells might attack EVT and they release granulysin into the intracellular space, resulting in apoptosis of EVT. So far, other groups have reported that Fas-FasL pathway and and/or TNF-α pathway is associated with apoptosis of EVT. In additiont, we propose granulysin-positive NK cells-induced apoptosis as a new mechanism for EVT cell death.     

Lack of correlation between placenta and offspring size in mouse interspecific crosses

The placenta plays a pivotal role in fetal growth control and is considered a major site of genetic conflict between maternal and paternal genomes within the conceptus and, in addition, the genome of the mother. Accordingly, placental development is a strictly controlled process, and both placental and fetal weights do not vary much in intraspecific crosses of laboratory mice (Mus musculus). In mouse interspecific crosses and backcrosses [(M. musculus x M. spretus) x M. musculus], tremendous variation of placental, but not of fetal weight was observed. We have studied trophoblast cell type distribution and differentiation, and their effect on the associated placentas and fetuses in such backcrosses. Differentiation of spongious trophoblast, but not size of materno-fetal interface, correlated with fetal weight. Giant fetuses were observed only if less than one third of the spongiotrophoblast was formed by glycogen cells. Thus, placental efficiency was inversely related to the amount of glycogen cells. This influence of a trophoblast-derived cell type on fetal growth was not anticipated. We conclude that: (1) glycogen cells are able to negatively modulate fetal growth by an as yet unidentified mechanism; (2) correlation between fetal and placental weights is weak or absent in interspecific hybrids; (3) impaired control over placental and embryonic development in hybrids may contribute to post-mating isolation of species.