Imbalance between ROS production and elimination results in apoptosis induction in primary smooth chorion trophoblast cells prepared from human fetal membrane tissues
Introduction
The rupture of fetal membrane (FM) is an essential part of the delivery process, accompanied by the onset of uterine contractions (Challis, 2000). Although it has been hypothesized that some biochemical events, including degradation of the connective tissue (Stegemann and Stalder, 1967), reduced elasticity (Artal et al., 1976) and decreased thickness (Helmig et al., 1991) of FM, which contribute to the rupture at the normal delivery, the mechanisms involved in the FM rupture during the birth process are little known. In our previous studies, we proposed that apoptosis induction in the chorion tissues is one of the mechanisms of FM rupture associated with labor (Ohyama et al., 1998, Ohyama et al., 2001, Yuan et al., 2006). The induction of apoptosis was observed in the smooth chorion trophoblast layer of human FM prepared at the term. The extent of apoptosis induction in the trophoblast layer progressed rapidly during in vitro incubation of the tissues (Ohyama et al., 1998). We also demonstrated that the progression of apoptosis was suppressed by the presence of antioxidative reagents, suggesting that an intracellular oxidative stress played critical roles in the induction of apoptosis observed in the trophoblast layer at the stage of parturition (Ohyama et al., 2001). Furthermore, we demonstrated that the expression levels of Bax, Bak and Bad mRNAs increased, while those of Bcl-2 mRNA decreased; the expression levels of pro-caspase-3 and -9, proform-poly (ADP-ribose) polymerase decreased; and a leakage of cytochrome c from mitochondria in the chorion tissues was observed during the in vitro incubation. These results suggested that the apoptosis induction observed in the chorion trophoblasts was induced by oxidative stress resulting in mitochondria damage (Ohyama et al., 2001, Yuan et al., 2006).
Moreover, we demonstrated that an inducible nitric oxide synthase (iNOS) inhibitor, a general and a selective cyclooxygenase (Cox)-2 inhibitors suppressed in vitro progression of the apoptosis, and that an NO donor reagent induced apoptosis in primary cultured chorion cells. These results suggested for the first time that iNOS and Cox-2, both of which are known to play a central role in reactive oxygen species (ROS) production (Payne et al., 1995), participated in the induction of chorion cells apoptosis through production of ROS (Yuan et al., 2006). Oxidative stress, as a result of an imbalance between the production and elimination of ROS within the cells (Margaret and Amanda, 1996), is known to induce apoptosis in various cell types (Payne et al., 1995). Therefore, we have been investigating on the contribution of ROS production system (e.g., oxidant enzymes like iNOS and Cox-2) to the trophoblasts apoptosis in the chorion tissues. However, the study concerning a possible contribution of ROS elimination system (e.g., antioxidant enzymes like glutathione peroxidase (GPx), catalase) to the apoptosis induction in the chorion trophoblasts has not yet performed at cellular level.
In this study, in order to reveal the relationship between ROS elimination system and the chorion trophoblasts apoptosis induction, we investigated the contribution of antioxidant enzymes, such as GPx and catalase, to the apoptosis induction in the primary cultured chorion cells, using known inhibitors for GPx, such as mercaptosuccinic acid (MS) and for catalase, such as 3-amino-1,2,4-triazole (ATZ) (Shiba and Shimamoto, 1999, Ishihara et al., 2005).
Section snippets
Materials
MS and ATZ were purchased from Sigma Ltd. (St. Louis, MO, USA). Agarose X for DNA fragmentation analysis was purchased from Nippon Gene (Tokyo, Japan). GPx assay kit and catalase assay kit were purchased from Cayman Chemical (Ann Arbor, MI, USA).
Preparation of primary cultured cells from human fetal membrane
Fetal membranes were prepared aseptically from placenta by Cesarean section at the month of normal parturition as described (Ohyama et al., 1998). Primary culture chorion and amnion cells were prepared from freshly prepared fetal membrane tissues
Effects of MS (GPx inhibitor) and ATZ (catalase inhibitor) on intracellular GPx and catalase activity in primary culture chorion and amnion cells
Primary culture chorion and amnion cells were treated with MS and ATZ at final concentrations of 14 mM and 40 mM, respectively, either for 24 or 48 h. GPx activity observed in untreated chorion cells (ca. 1200 units/mg of protein) was similar to that in untreated amnion cells (Fig. 1A and B). In the chorion cells, GPx activity after 24-h incubation with MS alone or in combination with ATZ decreased to 700 units/mg of protein, and the activity was not changed after 48-h incubation (Fig. 1A). In
Discussion
We have previously demonstrated that the apoptosis induction of trophoblasts in smooth chorion tissues of human fetal membranes (FM) was mediated through the intracellular oxidative stress, resulting in mitochondria damage at the term (Ohyama et al., 1998, Ohyama et al., 2001, Yuan et al., 2006). We have also demonstrated that an inducible nitric oxide synthase (iNOS) inhibitor, a general and a selective cyclooxygenase (Cox)-2 inhibitors suppressed in vitro progression of the trophoblasts
Conclusions
We proposed that inability of ROS elimination system as a result of reducing antioxidant enzyme activities induced apoptosis only in the chorion trophoblast layer when exposed to oxidative stress. Our results suggest that the threshold levels of stress tolerance in the amnion cells are much higher than those in the chorion cells, which results in more vulnerable to apoptosis induction in the chorion cells by oxidative stress.
Acknowledgments
This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology and by the Promotion and Mutual Aid Corporation for Private Schools of Japan.
References (30)
- et al.
The mechanical properties of prematurely and non-prematurely ruptured membranes. Methods and preliminary results
American Journal of Obstetrics and Gynecology
(1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding
Analytical Biochemistry
(1976)- et al.
Improved microfluorometric DNA determination in biological material using 33258 Hoechst
Analytical Biochemistry
(1979) - et al.
IL-1 beta is a better inducer of apoptosis in human fetal membranes than IL-6
Placenta
(2003) - et al.
Increased generation of reactive oxygen species in embryos cultured in vitro
Free Radical Biology and Medicine
(1993) - et al.
Role of free radicals and catalytic metal ions in human disease
Methods in Enzymology
(1990) - et al.
Changes in enzymatic antioxidant activity in pregnant rats exposed to hyperoxia or hypoxia
Comparative Biochemistry and Physiology. Part C, Pharmacology, Toxicology and Endocrinology
(1997) - et al.
Attenuation of endogenous oxidative stress-induced cell death by cytochrome P450 inhibitors in primary cultures of rat hepatocytes
Free Radical Biology and Medicine
(1999) - et al.
Determination of hydroxyproline
Clinica Chimica Acta
(1967) - et al.
Microsomal heme oxygenase
Characterization of the enzyme. The Journal of Biological Chemistry
(1969)
Antioxidant systems in normal pregnancy and in pregnancy-induced hypertension
American Journal of Obstetrics and Gynecology
Contribution of inducible nitric oxide synthase and cyclooxygenase-2 to apoptosis induction in smooth chorion trophoblast cells of human fetal membrane tissues
Biochemical and Biophysical Research Communications
Measurement of tumor necrosis factor α mRNA in small numbers of cells by quantitative polymerase chain reaction
Regional Immunology
Glutathione peroxidase–catalase cooperativity is required for resistance to hydrogen peroxide by mature rat oligodendrocytes
The Journal of Neuroscience
Mechanism of parturition and preterm labor
Obstetrical and Gynecological Survey
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