Ip. Crocker et al., The in-vitro characterization of induced apoptosis in placental cytotrophoblasts and syncytiotrophoblasts, PLACENTA, 22(10), 2001, pp. 822-830
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.