The in-vitro characterization of induced apoptosis in placental cytotrophoblasts and syncytiotrophoblasts.
Placental trophoblasts undergo apoptosis as part of normal epithelial turnover and placental ageing. Classically, the induction of apoptosis in in vitro preparations has utilized the cytokines TNFalpha and IFNgamma and has been measured using the TUNEL technique. The aim of this study was to compare apoptotic susceptibility of mononucleated and differentiated trophoblasts using a range of cytotoxic agents. To achieve this, an in vitro model of syncytialization was used, along with isolated placental cytotrophoblasts and an extravillous cytotrophoblast derived cell line (SGHPL-4). Cytotrophoblasts from term placentae (n=12), syncytiotrophoblasts (n=12) and SGHPL-4s (n=8) were cultured under reduced oxygen or with TNFalpha/IFNgamma, dexamethasone or staurosporine. Apoptosis assessments were made using TUNEL, Annexin V binding, fluorescence microscopy and ATP/ADP measurements. Each cytotoxic agent increased apoptosis in all three cell populations. For untreated cells, cytotrophoblasts showed the greatest levels of apoptosis in culture. With stimulation, these levels were significantly elevated using dexamethasone, TNFalpha/IFNgamma and staurosporine and further raised under hypoxic conditions. SGHPL-4 cells showed similar trends to those of cytotrophoblasts, however the syncytiotrophoblasts, although responsive to dexamethasone and TNFalpha/IFNgamma, showed lower levels of apoptosis with staurosporine and hypoxia. ADP : ATP measurements gave similar results to the other techniques and ratios of less than 1.0 were correlated with Annexin V measurements on the flow cytometer (P< 0.001). The typical morphological features of apoptosis i.e. chromatin margination, membrane blebbing and apoptotic body formation were detected in cytotrophoblasts and SGHPL-4 cells. However, only chromatin condensation could be recognized in syncytiotrophoblast preparations. Necrotic cell numbers were also increased under all cytotoxic conditions. Although elevated with dexamethasone, staurosporine and hypoxia, these levels were markedly raised in cytotrophoblasts and SGHPL-4 cells following incubations with TNFalpha/IFNgamma. These observations show variations in apoptosis between mononuclear trophoblasts and differentiated multinucleated syncytiotrophoblasts. Differential effects of stimuli may suggest disparate apoptotic pathways. These variations may reflect functional differences between placental cellular and syncytial components and may highlight the importance of exogenous stimulation in various stages of placental development.
Crocker
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IP
School of Human Development, University of Nottingham, City Hospital, Nottingham, UK. Ian.Crocker@nottingham.ac.uk
Barratt
S
S
Kaur
M
M
Baker
P N
PN
D000165:Acridine Orange; D000244:Adenosine Diphosphate / Q000032:analysis; D000255:Adenosine Triphosphate / Q000032:analysis; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004996:Ethidium; D005260:Female; D005456:Fluorescent Dyes; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D020287:In Situ Nick-End Labeling; D007202:Indicators and Reagents; D007371:Interferon-gamma / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010100:Oxygen / Q000008:administration & dosage; D011247:Pregnancy; D011419:Propidium; D019311:Staurosporine / Q000494:pharmacology; D014327:Trophoblasts / Q000166:cytology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D017304:Annexin A5; D005456:Fluorescent Dyes; D005938:Glucocorticoids; D007202:Indicators and Reagents; D014409:Tumor Necrosis Factor-alpha; D011419:Propidium; D000244:Adenosine Diphosphate; D004815:Epidermal Growth Factor; D003907:Dexamethasone; D007371:Interferon-gamma; D000255:Adenosine Triphosphate; D004996:Ethidium; D000165:Acridine Orange; D019311:Staurosporine; D010100:Oxygen