The in-vitro characterization of induced apoptosis in placental cytotrophoblasts and syncytiotrophoblasts

Citation
Ip. Crocker et al., The in-vitro characterization of induced apoptosis in placental cytotrophoblasts and syncytiotrophoblasts, PLACENTA, 22(10), 2001, pp. 822-830
Citations number
36
Categorie Soggetti
Reproductive Medicine","da verificare
Journal title
PLACENTA
ISSN journal
01434004 → ACNP
Volume
22
Issue
10
Year of publication
2001
Pages
822 - 830
Database
ISI
SICI code
0143-4004(200111)22:10<822:TICOIA>2.0.ZU;2-2
Abstract
Placental trophoblasts undergo apoptosis as part of normal epithelial turno ver and placental ageing. Classically, the induction of apoptosis in in vit ro preparations has utilized the cytokines TNF alpha and IFN gamma and has been measured using the TUNEL technique. The aim of this study was to compa re apoptotic susceptibility of mononucleated and differentiated trophoblast s using a range of cytotoxic agents. To achieve this, an in vitro model of syncytialization was used, along with isolated placental cytotrophoblasts a nd an extravillous cytotrophoblast derived cell line (SGHPL-4). Cytotrophob lasts from term placentae (n = 12), syncytiotrophoblasts (n = 12) and SGHPL -4s (n = 8) were cultured under reduced oxygen or with TNF alpha /IFN gamma , dexamethasone or staurosporine. Apoptosis assessments were made using TUN EL, 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 usin g dexamethasone, TNF alpha /IFN gamma and staurosporine and further raised under hypoxic conditions. SGHPL-4 cells showed similar trends to those of c ytotrophoblasts, however the syncytiotrophoblasts, although responsive to d examethasone and TNF alpha /IFN gamma, showed lower levels of apoptosis wit h staurosporine and hypoxia. ADP : ATP measurements gave similar results to the other techniques and ratios of less than 1.0 were correlated with Anne xin V measurements on the flow cytometer (P<0.001). The typical morphologic al features of apoptosis i.e. chromatin margination, membrane blebbing and apoptotic body formation were detected in cytotrophoblasts and SGHPL-4 cell s. However, only chromatin condensation could be recognized in syncytiotrop hoblast preparations. Necrotic cell numbers were also increased under all c ytotoxic conditions. Although elevated with dexamethasone, staurosporine an d hypoxia, these levels were markedly raised in cytotrophoblasts and SGHPL- 4 cells following incubations with TNF<alpha>/IFN gamma. These observations show variations in apoptosis between mononuclear trophob lasts and differentiated multinucleated syncytiotrophoblasts. Differential effects of stimuli may suggest disparate apoptotic pathways. These variatio ns may reflect functional differences between placental cellular and syncyt ial components and may highlight the importance of exogenous stimulation in various stages of placental development. (C) 2001 Harcourt Publishers Ltd.